Research ArticleUPLC-ESI-MSMS Profile and Antioxidant CytotoxicAntidiabetic and Antiobesity Activities of the Aqueous Extracts ofThree Different Hibiscus Species

Hanan M Al-Yousef 1 Wafaa H B Hassan2 Sahar Abdelaziz2 Musarat Amina 1

Rasha Adel2 and May A El-Sayed2

1Department of Pharmacognosy College of Pharmacy King Saud University Riyadh Saudi Arabia2Department of Pharmacognosy Faculty of Pharmacy Zagazig University Zagazig Egypt

Correspondence should be addressed to Hanan M Al-Yousef halyousefksuedusa

Received 8 February 2020 Revised 20 May 2020 Accepted 3 June 2020 Published 25 June 2020

Academic Editor Casimiro Mantell

Copyright copy 2020 Hanan M Al-Yousef et al )is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

)e aqueous extracts of Hibiscus calyphyllus (HcA) Hibiscus micranthus (HmA) and Hibiscus deflersii (HdA) growing in SaudiArabia did not receive enough attention in phytochemical and biological studies )is inspired the authors to investigate thephytochemicals of these extracts for the first time using UPLC-ESI-MSMS in negative and positive ionizationmodes)e analysisafforded the tentative identification of 103 compounds including phenolic compounds flavonoids and anthocyanins Moreoverin vitro evaluations of their cytotoxic antioxidant antidiabetic and antiobesity activities were carried out)e results showed thataqueous extract of Hibiscus calyphyllus had the highest activity as an antioxidant agent (SC50 = 111plusmn 15 μgmL) compared withascorbic acid (SC50 = 142plusmn 05 μgmL) MTTassay was used to evaluate cytotoxic activity compared to cisplatinHibiscus deflersiishowed the most potent cytotoxic effect against A-549 (human lung carcinoma) with IC50 = 50plusmn 51 μgmL and Hibiscusmicranthus showed a close effect with IC50 = 604plusmn 17 μgmL Hibiscus micranthus showed the most potent effect on HCT-116(human colon carcinoma) with IC50 = 56plusmn 19 μgmL compared with cisplatin (IC50 = 753plusmn 38 μgmL) HcA and HdA extractsshowed weak cytotoxic activity against A-549 and HCT-116 cell lines compared to the other extracts EventuallyHibiscus deflersiishowed astonishing antidiabetic (IC50 = 56plusmn 19 μgmL) and antiobesity (IC50 = 9545plusmn 19 μgmL) activities using in vitroα-amylase inhibitory assay (compared with acarbose (IC50 = 3471plusmn 07 μgmL)) and pancreatic lipase inhibitory assay (comparedwith orlistat (IC50 = 238plusmn 07 μgmL)) respectively In conclusion these findings are regarded as the first vision of the phy-tochemical constituents and biological activities of differentHibiscus aqueous extractsHibiscus deflersii aqueous extract might bea hopeful origin of functional constituents with anticancer (on A-549 cell line) antidiabetic and antiobesity activities It might bea natural alternative remedy and nutritional policy for diabetes and obesity treatment without negative side effects Isolation of thebioactive phytochemicals from the aqueous extracts of aerial parts of Hibiscus calyphyllus Hibiscus micranthus and Hibiscusdeflersii and estimation of their biological effects are recommended in further studies

1 Introduction

Hibiscus (Malvaceae) consists of approximately 200 specieswidely distributed in tropical and subtropical regions of theworld [1] Hibiscus is a genus of herbs shrubs and trees [2]Phytochemical investigation of Hibiscus has been reportedto contain many classes of secondary metabolites includinganthocyanins flavonoids steroids terpenoids alkaloidsquinones and sesquiterpene [2]

Many Hibiscus species are valued as ornamentalplants and are cultivated in gardens [2] Fruits of somespecies are used as food a soft drink is provided fromflowers of some species (H sabdariffa L) and also used infood industry for example in cakes wines syrupsjellies puddings and cold or hot beverages and as acolorant for herbal teas [1] Since ancient times Hibiscushas been used in traditional folk medicine for differentdisorders

HindawiJournal of ChemistryVolume 2020 Article ID 6749176 17 pageshttpsdoiorg10115520206749176

Various pharmacological effects have also been shownfor Hibiscus and its components such as antihypertensiveantiatherosclerotic antioxidant antihypercholesterolemichypolipidemic antinociceptive anti-inflammatory antipy-retic analgesic antifungal antibacterial antifertility anti-diabetic anticancer antimutagenic chemopreventiveanthelminthic and anticonvulsant activities [1 3 4]

Hibiscus deflersii (HdA) which is native to Ethiopia andgrown as ornamental plant worldwide is 1m high erectperennial or annual leafy untidy shrub of bright greennarrow dentate leaves surrounding bright crimson-redflowers Many interesting pharmacological activities of HdAhad been reported its leaves extract is used to treat cardiacdisorders and diabetes However its flower infusion andextract are used as demulcent and emollient while its de-coction is used for the treatment of bronchial catarrh [5]

Hibiscus micranthus (HmA) which is commonly dis-tributed from south to western part of Saudi Arabia is a45 cm shrub with heavy leaves white flowers and shortpedicels with very distinctive capsules of pea-size fruits andis distributed vastly in Saudi Arabia India tropical Africaand Ceylon Its flowers and fruits exhibited antidiabetic andlaxative activities when used orally and when appliedtopically it is used as antidandruff agent )e plant alsoshowed anti-inflammatory antipyretic antitumor antimi-crobial and antiviral activities Literature detects a widerange of phytochemicals in HmA as flavonoids phenolicacids fatty alcohols fatty acids β-sitosterol and alkanes [5]

Hibiscus calyphyllus (HcA) is 1m high leafy shrubcharacterized by bright yellow flower with dark red centersurrounded by simple wide serrate leaves It is commonlyfound in Jazan south-western region of Saudi Arabia )eethyl acetate fraction of this plant showed potent antioxidantactivity [5]

Hibiscus aerial parts are considered as food crops con-sumed as hot or cold beverages (aqueous extract) Numerousscientific papers have been published discussing thechemical contents of different fractions of HcA HdA andHmA which showed their high biological effectivenesshaving antioxidant antidiabetic antiobesity and cytotoxicactivities For our knowledge the previous literature onHibiscus species suffers from insufficiency of detailed in-formation on the phytoconstituents of aqueous extracts ofSaudi HdA HcA and HmA and their biological activities)erefore the aims of the present research were (i) toperform direct analysis of aqueous extracts which relies onUPLC coupled with ESI-MSMS detection (ii) to detect theantioxidant antidiabetic antiobesity and anticancer activ-ities of aqueous extracts of HdA HcA and HmA and (iii) toanticipate the components responsible for antioxidantantidiabetic antiobesity and anticancer activities

2 Materials and Methods

21 Plant Material Aerial parts of three different species ofgenus Hibiscus were collected from As-Sarawat mountainsJabal As-Sahlarsquo and Aseer province in Saudi Arabia(2556m height above sea level) during March 2009 Tax-onomical authentication of plant samples was performed by

Prof Dr Mohamed Yousef from the Pharmacognosy De-partment College of Pharmacy of King Saud University andvoucher specimens (H calyphyllus no HA-234 H deflersiino HA-567 and H micranthus no HA-16240) were kept atthe herbarium of the department

22 Preparation of Extracts Air-dried powder aerial parts ofselected plant samples (600 g) were individually extractedwith distilled water at 100degC with continuously shaking for3 hrs )e marc of each plant material was extracted thriceunder similar conditions by repeating the above-mentionedprocedure )e aqueous extracts were then filtered bycentrifugation and the filtrates were pooled )e obtainedfiltrates were freed from the solvent by freeze-drying to getdark brown solid masses)e weight of resulted residues was3402 2625 and 3641 g forH calyphyllus (HcA)H deflersii(HdA) and H micranthus (HmA) respectively

23 Chemicals and Reagents Sigma Aldrich (HamburgGermany) provided all the chemical and reagents usedthroughout the experiments including HPLC grade meth-anol (ge999) acetonitrile water for ESI-MS analyses re-agent grade formic acid (ge95) 22-diphenyl-1-picrylhydrazyl (DPPH) (freshly dissolved in methanol at aconcentration of 0004) ascorbic acid (99) DulbeccorsquosModified Eaglersquos Medium (DMEM) L-glutamine dini-trosalicylic acid (DNS colour reagent) p-nitrophenyl bu-tyrate (NPB ge98) and potassium phosphate buffer (pH60) 3-(45-Dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT) cisplatin DMSO porcine pancreatic lipaseand α-amylase enzyme acarbose (ge95) and orlistat(ge98) were used as control Human lung carcinoma (A-549) and human colon carcinoma (HCT-116) cell lines wereobtained from the American Type Culture Collection(ATCC Rockville MD)

24 UPLC-ESI-MSMS Ultra-performance liquid chroma-tography with electrospray ionization quadrupole-linear iontrap-tandem mass spectrometry analysis performed on ESI-MS positive and negative ion acquisition mode was carriedout on a XEVO TQD triple quadruple instrument Methodin a multiple-reaction monitoring (MRM) mode wasemployed for the quantitative determination of phyto-chemicals )e crude Hibiscus extracts were analyzed byUPLC in order to obtain chromatographic profiles of themore polar portions of the extracts which contain phenolicand flavonoid compounds )e samples were dissolved inHPLC grade methanol filtered through 02 μm membranedisc filter and resulting solution concentrations were in therange of 02 to 05mgmL depending on each crude extract

)e UPLC system was a mass spectrometer WatersCorporation Milford USA )e reverse-phase separationswere performed (ACQUITY UPLC BEH C18 Column17 μmndash21times 50mm 50mmtimes 12mm inner diameter17 μm particle size) at 02mmL flow rate A previouslyreported gradient program was applied for the analysis [6])emobile phase comprised acidified water containing 01

2 Journal of Chemistry

formic acid (A) and acidified methanol containing 01formic acid (B) )e employed elution conditions were asfollows 0ndash2min isocratic elution at 10 B 2ndash5min lineargradient from 10 to 30 B 5ndash15min linear gradient from30 to 70 B 15ndash22min linear gradient from 70 to 90B and 22ndash25min isocratic elution at 90 B finally washingand reconditioning of column were done Electrosprayionization (ESI) was performed in both negative and positiveion modes to obtain more data )e parameters for analysiswere set using negative ion mode as follows source tem-perature 150degC cone voltage 30 eV capillary voltage3 kV desolvation temperature 440degC cone gas flow 50 Lhand desolvation gas flow 900 Lhr Mass spectra were de-tected in the ESI between mz 100 and 1000 atomic massunits Chemical constituents were identified by their ESIndashQqQLITndashMSMS spectra and fragmentation patterns )epeaks and spectra were processed using the MassLynx 41software and tentatively identified by comparing their re-tention time (Rt) and mass spectrum with reported data andlibrary search (such as FooDB (httpwwwFoodbca))

25AntioxidantActivity )e antioxidant activity of extractswas determined at the Regional Center for Mycology andBiotechnology (RCMB) at Al-Azhar University by theDPPH free radical scavenging assay in triplicate and averagevalues were considered

251 DPPH Radical Scavenging Activity [7] Freshly pre-pared (01mM) solution of 22-diphenyl-1-picrylhydrazyl(DPPH) and different tested extracts prepared at 5 10 2040 80 160 and 320 μgmL in methanol were vigorouslymixed and allowed to stand for 30min at room temperaturein the dark [8] )e absorbance values of the resulting so-lution were recorded with a UV-visible spectrophotometer(Milton Roy Spectronic 1201) at λmax of 517 nm againstDPPH radical without antioxidant (control) and the refer-ence compound ascorbic acid (5 10 20 40 80 160 and320 μgmL) All the determinations were performed in threereplicates and averaged )e percentage inhibition of theDPPH radical was calculated according to the followingformula

DPPH radical minus scavenging (AC minus AS)

AC1113896 1113897 times 1001113890 1113891

(1)

where AC is the absorbance of the control solution and AS isthe absorbance of the sample in DPPH solution

)e percentage of DPPH radical scavenging was plottedagainst each extract concentration and ascorbic acid (μgmL) to determine scavenging capacity (SC50) which is theconcentration required to scavenge DPPH by 50 (ieconcentration giving 50 reduction in the absorbance of aDPPH solution from its initial absorbance)

26 Evaluation of Cytotoxicity HdA HmA and HcA weretested for their cytotoxic activity against human lung car-cinoma (A-549) and human colon carcinoma (HCT-116)

cell lines using 3-(45-dimethylthiazol-2-yl)-25-diphe-nyltetrazolium bromide (MTT) against DMSO and cisplatinas negative and positive controls respectively )esemammalian cell lines were obtained from American TypeCulture Collection (ATCC Rockville MD) )e cells werepropagated on Dulbeccorsquos Modified Eaglersquos Medium(DMEM) supplemented with 10 heat-inactivated fetalbovine serum 1 L-glutamine HEPES buffer and 50 μgmLgentamycin )e cells were maintained at 37degC in a hu-midified atmosphere with 5CO2 and were subcultured twoto three times a week

For antitumor assay the tumor cell lines were suspendedin medium at concentration of 5times104 cellwell in Corningreg96-well tissue culture plates and then incubated for 24 hrs)e tested extracts were then added to 96-well plates (sixreplicates) to achieve eight concentrations for each extractranging from 1 μgmL to 500 μgmL Six vehicle controlswith media or 05DMSOwere run for each 96-well plate asa control After incubation for 24 h the numbers of viablecells were determined by the MTT test Briefly the mediawere removed from the 96-well plates and replaced with100 μL of fresh culture DMEM without phenol red then10 μL of the 12mM MTT stock solution (5mg of MTT in1mL of phosphate buffered saline (PBS)) was added to eachwell including the untreated controls )e 96-well plateswere then incubated at 37degC and 5 CO2 for 4 hrs An 85 μlaliquot of the media was removed from the wells and 50 μLof DMSOwas added to each well mixed thoroughly with thepipette and incubated at 37degC for 10min )en the opticaldensity was measured at 590 nm with the microplate reader(Sunrise Tecan Inc USA) to determine the number ofviable cells and the percentage of viability was calculated

cell viability 1 minusODt

ODc1113874 11138751113890 1113891 times 100 (2)

where ODt is the mean optical density of wells treated withthe tested sample and ODc is the mean optical density ofuntreated cells

)e relation between surviving cells and each extractconcentration (1ndash500 μgmL) was plotted to get the survivalcurve of each tumor cell line after treatment with the testedextract )e 50 inhibitory concentration (IC50) the con-centration required to cause toxic effects in 50 of intactcells was estimated from graphic plots of the dose-responsecurve (log extract concentration on x-axis vs percentageviability from untreated cells on y-axis) for each concen-tration through nonlinear regression analysis (dose-re-sponse inhibition log inhibitor vs normalized response-variable slop) using GraphPad Prism 5 software (GraphPadSoftware San Diego California) [9 10] All experimentswere repeated at least three times Results are reported asmeansplusmn SD

27 In Vitro Antidiabetic Assay

271 α-Amylase Inhibition Method In a-amylase inhibitionmethod the enzyme solution was prepared by dissolvingα-amylase in 20mM phosphate buffer (69) at a

Journal of Chemistry 3

formic acid (A) and acidified methanol containing 01formic acid (B) )e employed elution conditions were asfollows 0ndash2min isocratic elution at 10 B 2ndash5min lineargradient from 10 to 30 B 5ndash15min linear gradient from30 to 70 B 15ndash22min linear gradient from 70 to 90B and 22ndash25min isocratic elution at 90 B finally washingand reconditioning of column were done Electrosprayionization (ESI) was performed in both negative and positiveion modes to obtain more data )e parameters for analysiswere set using negative ion mode as follows source tem-perature 150degC cone voltage 30 eV capillary voltage3 kV desolvation temperature 440degC cone gas flow 50 Lhand desolvation gas flow 900 Lhr Mass spectra were de-tected in the ESI between mz 100 and 1000 atomic massunits Chemical constituents were identified by their ESIndashQqQLITndashMSMS spectra and fragmentation patterns )epeaks and spectra were processed using the MassLynx 41software and tentatively identified by comparing their re-tention time (Rt) and mass spectrum with reported data andlibrary search (such as FooDB (httpwwwFoodbca))

25AntioxidantActivity )e antioxidant activity of extractswas determined at the Regional Center for Mycology andBiotechnology (RCMB) at Al-Azhar University by theDPPH free radical scavenging assay in triplicate and averagevalues were considered

251 DPPH Radical Scavenging Activity [7] Freshly pre-pared (01mM) solution of 22-diphenyl-1-picrylhydrazyl(DPPH) and different tested extracts prepared at 5 10 2040 80 160 and 320 μgmL in methanol were vigorouslymixed and allowed to stand for 30min at room temperaturein the dark [8] )e absorbance values of the resulting so-lution were recorded with a UV-visible spectrophotometer(Milton Roy Spectronic 1201) at λmax of 517 nm againstDPPH radical without antioxidant (control) and the refer-ence compound ascorbic acid (5 10 20 40 80 160 and320 μgmL) All the determinations were performed in threereplicates and averaged )e percentage inhibition of theDPPH radical was calculated according to the followingformula

DPPH radical minus scavenging (AC minus AS)

AC1113896 1113897 times 1001113890 1113891

(1)

where AC is the absorbance of the control solution and AS isthe absorbance of the sample in DPPH solution

)e percentage of DPPH radical scavenging was plottedagainst each extract concentration and ascorbic acid (μgmL) to determine scavenging capacity (SC50) which is theconcentration required to scavenge DPPH by 50 (ieconcentration giving 50 reduction in the absorbance of aDPPH solution from its initial absorbance)

26 Evaluation of Cytotoxicity HdA HmA and HcA weretested for their cytotoxic activity against human lung car-cinoma (A-549) and human colon carcinoma (HCT-116)

cell lines using 3-(45-dimethylthiazol-2-yl)-25-diphe-nyltetrazolium bromide (MTT) against DMSO and cisplatinas negative and positive controls respectively )esemammalian cell lines were obtained from American TypeCulture Collection (ATCC Rockville MD) )e cells werepropagated on Dulbeccorsquos Modified Eaglersquos Medium(DMEM) supplemented with 10 heat-inactivated fetalbovine serum 1 L-glutamine HEPES buffer and 50 μgmLgentamycin )e cells were maintained at 37degC in a hu-midified atmosphere with 5CO2 and were subcultured twoto three times a week

For antitumor assay the tumor cell lines were suspendedin medium at concentration of 5times104 cellwell in Corningreg96-well tissue culture plates and then incubated for 24 hrs)e tested extracts were then added to 96-well plates (sixreplicates) to achieve eight concentrations for each extractranging from 1 μgmL to 500 μgmL Six vehicle controlswith media or 05DMSOwere run for each 96-well plate asa control After incubation for 24 h the numbers of viablecells were determined by the MTT test Briefly the mediawere removed from the 96-well plates and replaced with100 μL of fresh culture DMEM without phenol red then10 μL of the 12mM MTT stock solution (5mg of MTT in1mL of phosphate buffered saline (PBS)) was added to eachwell including the untreated controls )e 96-well plateswere then incubated at 37degC and 5 CO2 for 4 hrs An 85 μlaliquot of the media was removed from the wells and 50 μLof DMSOwas added to each well mixed thoroughly with thepipette and incubated at 37degC for 10min )en the opticaldensity was measured at 590 nm with the microplate reader(Sunrise Tecan Inc USA) to determine the number ofviable cells and the percentage of viability was calculated

cell viability 1 minusODt

ODc1113874 11138751113890 1113891 times 100 (2)

where ODt is the mean optical density of wells treated withthe tested sample and ODc is the mean optical density ofuntreated cells

)e relation between surviving cells and each extractconcentration (1ndash500 μgmL) was plotted to get the survivalcurve of each tumor cell line after treatment with the testedextract )e 50 inhibitory concentration (IC50) the con-centration required to cause toxic effects in 50 of intactcells was estimated from graphic plots of the dose-responsecurve (log extract concentration on x-axis vs percentageviability from untreated cells on y-axis) for each concen-tration through nonlinear regression analysis (dose-re-sponse inhibition log inhibitor vs normalized response-variable slop) using GraphPad Prism 5 software (GraphPadSoftware San Diego California) [9 10] All experimentswere repeated at least three times Results are reported asmeansplusmn SD

27 In Vitro Antidiabetic Assay

271 α-Amylase Inhibition Method In a-amylase inhibitionmethod the enzyme solution was prepared by dissolvingα-amylase in 20mM phosphate buffer (69) at a

Journal of Chemistry 3

concentration of 05mgmL One mL of the extract ofvarious concentrations (781ndash1000 μgmL) and 1mL ofenzyme solution were mixed together and incubated at 25degCfor 10min After incubation 1mL of starch (05) solutionwas added to the mixture and further incubated at 25degC for10min )e reaction was then stopped by adding 2mL ofdinitrosalicylic acid (DNS colour reagent) heating the re-action mixture in a boiling water bath (5min) After coolingthe absorbance was measured calorimetrically at 565 nm)e inhibition percentage was calculated using the followingformula inhibition (1minusAsAc)times 100 where Ac is theabsorbance of control and As is the absorbance of testedextracts Acarbose was used as a control [11] )e IC50 valuewas defined as the concentration of α-amylase inhibitorneeded to inhibit 50 of its activity under the assayconditions

Nonlinear regression analysis using GraphPad Prism 5software (GraphPad Software San Diego California) wasconducted to calculate IC50 from graphic plots of the dose-response curve for each applied concentration Each ex-periment was performed in triplicate and all values arerepresented as meansplusmn SD

28 In Vitro Antiobesity Using Pancreatic Lipase InhibitoryAssay )e lipase inhibition activity of plant extract wasdetermined by the method in [12] In this assay the porcinepancreatic lipase activity was measured using p-nitrophenylbutyrate (NPB) as a substrate Lipase solution (100 μgmL)was prepared in a 01mM potassium phosphate buffer (pH60) Samples with different concentrations (781ndash1000 μgmL) were preincubated with 100 μgmL of lipase for 10minat 37degC )e reaction was then started by adding 01mL NPBsubstrate After incubation at 37degC for 15min p-nitrophenolamount released in the reaction was measured using mul-tiplate reader Orlistat was used with the same concentra-tions as a control )e results were expressed as percentageinhibition which was calculated using the following for-mula inhibitory activity () (1minusAsAc)times 100 where As isthe absorbance in the presence of test substance and Ac is theabsorbance of control )e IC50 value was defined as theconcentration of pancreatic lipase inhibitor required toinhibit 50 of its activity under the assay conditions Es-timation of IC50 was done from dose-response curve graphicplots for each concentration by nonlinear regression analysisusing GraphPad Prism 5 software Each experiment wasperformed in triplicate and all values are represented asmeansplusmn SD of triplicates

3 Results and Discussion

31 UPLC-ESI-MSMS Identification of the chemicalcomposition of the aqueous extract of the HdA HmA andHcA was carried out by UPLC-ESI-MSMS in negative andpositive ion modes Totally 103 secondary metabolitesarranged according to retention time (Rt) were identifieddepending on their MS2 information given by the precursorionrsquos mass their fragments known fragmentation patternsfor the given classes of compounds and neutral mass loss as

well as comparison with the available literature andsearching in an online database [13] as shown in Table 1Figure 1 shows the base peak chromatograms of the threeaqueous extracts

311 Phenolic Compounds Phenolic acid derivatives aremostly glycosides their fragmentation stage started with thecleavage of the glycosidic linkage to provide the mz of thephenolic acid and the corresponding neutral mass loss ofsugar molecules (minus162Da) and then neutral mass losses ofhydroxyl (minus18Da) methyl (minus15Da) or carboxylic (minus44Da)groups were helpful in identification of the specific phenolicacid Methyl gallate (72) [17] and its derivative (85) [39] andsyringic acid derivative (76) [33] were identified Compound33 and its isomer (89) were tentatively identified as 4-hydroxybenzoic acid while compound 55 and its isomer (59)were tentatively identified as 3-hydroxybenzoic acid [13]

Tyrosol (4) and its isomers (26 67 and 94) werecharacterized by two fragments mz 77 corresponding tothe aromatic ring mz 93 corresponding to the phenolgroup respectively [15] Tyrosol precursor ion at mz 121does not refer to the [M+H]+ ion but to the [M+H-H2O]+according to [15] this may be due to in-source fragmen-tation even under mild ionization conditions

312 Flavone C-Glycosides In negative ionization modethe presence of [MminusH-90]minus and [MminusH-120]minus confirmedthat the compounds are mono-C-hexosylated flavonoids)e sugar on position 8 can be detected by investigation ofMS2 spectrum (ie the absence of the fragment peak at mz[MminusH-18]minus) as in compound 27 which was identified asorientin (luteolin-8-C-glucoside) [25 27 31 32] andcompound 98 which was tentatively identified as kaemp-ferol-8C-glucoside

)e substitution of the two C-glucosides in positions 6and 8 in compound 20 and its isomer (41) can be confirmedby the characteristic fragments at mz 383 corresponding to[MminusH-120-90]minus and mz 353 corresponding to [MminusH-120-120]minus in MSMS spectrum )e compound was iden-tified as vicenin (apigenin 68-di-C-glucoside) [26 27]Luteolin C-hexoside-C-pentoside (30) and its isomer (50)with [MminusH]minus at mz 579 showed ion fragments at mz 489[MminusH-90]minus mz 459 [MminusH-120]minus mz 429 [MminusH-150]minusmz 369 [MminusH-120-90]minus and mz 339 [MminusH-120-120]minus[25]

Compound 8 and its isomers (19 36 43) exhibitedcharacteristic fragments at mz 443 corresponding to[MminusH-120]minus mz 431 corresponding to [MminusH-132]minus mz353 corresponding to [MminusH-120-90]minus and mz 341 cor-responding to [MminusH-132-90]minus in MS2 spectrum thatconfirm the mono-C-hexoside-C-pentoside substitution inpositions 6 and 8 )e compound was identified as apigeninC-hexoside-C-pentoside [18 25 40]

Schaftoside (apigenin-6-C-glucoside-8-C-riboside) (32)and its isomer (51) showed a pseudomolecular ion peak[M+H]+ at mz 565 and the typical fragmentation pathwayof C-glycosylated flavonoids resulted in the formation ofions atmz 475 [M+H-90]+ corresponding to the loss of an

4 Journal of Chemistry

Table 1 Metabolites identified in the aqueous extracts of Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA) usingUPLCminusESIminusMS in negative and positive ionization modes

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

1 L-ascorbic acid 030 177 133 129 127 113 103 101 57 radic radic radic 12 Oleuropein 082 539 377 341 307 215 179 radic radic 23 Succinic acidlowast 086 119 101 radic mdash4 Tyrosol 093 121 103 97 93 89 79 77 73 65 45 radic 3

5 Sucrose 095 341 179 (MminusH-162)minus 161 131 119 117113 103 101 89 87 71 59 radic 4

6 Hydroxycitric acidderivative 136 593 209 radic 1

7 Butein 143 273 163 143 137 radic 5

8 Apigenin C-hexoside-C-pentoside 153 563

545 (MminusH-18)minus 443 (MminusH-120)minus431 (MminusH-132)minus 353 (MminusH-120-90)minus 341 (MminusH-132-90)minus 311

(MminusH-132-120)minusradic 6

9 L-ascorbic acid isomer 181 177 133 129 127 113 103 101 57 radic 110 N-feruloyltyramine 332 314 235 181 177 145 121 103 93 45 radic 111 Cyanidin 3-O-galactoside 357 449 287 137 radic 7 812 L-ascorbic acid isomer 452 177 133 129 127 113 103 101 57 radic 1

13 Patuletin (6-methoxyquercetin) 497 333 318 301 169 155 radic 9

14 Succinic acid isomer 605 117 99 (MminusH-18)minus radic 10

15Diosmetin-7-O-glucuronide-3prime-O-

pentoside631 607 475 (MminusH-132)minus 299 (MminusH-132-

176)minus 179 radic 11

16 L-ascorbic acid isomer 632 177 133 129 127 113 103 101 57 radic 117 Succinic acid isomer 649 117 117 99 radic 10

18 Cyanidin 3-O-sambubioside 676 579 339 285 radic 12

19 Apigenin C-hexoside-C-pentoside isomer 702 563

545 (MminusH-18)minus 443 (MminusH-120)minus431 (MminusH-132)minus 341 (MminusH-132-

90)minus 311 (MminusH-132-120)minusradic 13

20 Vicenin (apigenin 68-di-C-glucoside) 710 593 473 (MminusH-120)minus 383 (MminusH-120-

90)minus 285 radic 14 15

21Kaempferol-3-O-

rutinosideluteolin-7-O-rutinoside

725 593 285 (MminusH-Rut)minus radic 1617

22 Apiin (apigenin-7-apiosylglucoside) 729 563 443 413 269 (MminusH-132-162)minus radic 5 18

23 Apiin isomer 731 565 433 (M+H-132)+ 413 271(M+H-162- 132)+ radic 5

24 Cyanidin 3-O-glucoside 737 494 287 (M+H-162)+ 137 radic 3

25 Diosmetin C-glucosideC-pentosidelowast 737 593 413 (MminusH-90-90)minus 383 (MminusH-90-

120)minus radic mdash

26 Tyrosol isomer 746 121 93 77 radic 3

27 Orientin (luteolin 8-C-glucoside) 746 447 357 (MminusH-90)minus 327 (MminusH-120)minus

297 (MminusH-150)minus 285(MminusH-162)minus radic13 15 19

20

28 Delphinidin malonylglucuronidelowast 759 565 303 (M+H-176-86)+ radic mdash

29 Methyl apigeninderivativelowast 781 799 285 radic mdash

30 Luteolin C-hexoside-C-pentoside 782 579

489 (MminusH-90minus) 459 (MminusH-120)minus429 (MminusH-150)minus 411 399 369(MminusH-120-90)minus 365 353 339

(MminusH -120-120)minus 299

radic 13

31 Luteolin derivative 798 737 285 radic 18 21

32Schaftoside (apigenin 6-

C-glucoside 8-C-riboside)

799 565529 511 475 457 445 427

415(M+H-150)+ 409 391 379 361349 337 325 307 295 273

radic 1 22

Journal of Chemistry 5

Table 1 Continued

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

33 4-Hydroxybenzoic acid 799 139 121 111 105 97 93 79 radic 1

34 Peonidin-3-(p-coumaroyl-glucoside) 827 609 301(M+H-146-162)+ radic 23

35 Kaempferol-3-(p-coumaryl-glucoside) 827 593 593 447(MminusH-146)minus 327

285(MminusH-146-162)minus radic 24

36 Apigenin C-hexoside-C-pentoside isomer 832 563

545 (MminusH-18)minus 473 (MminusH-90)minus443 (MminusH-120)minus 431 (MminusH-132)minus 353 (MminusH-120-90)minus 341(MminusH-132-90)minus 311 (MminusH-132-

120)minus

radic radic 13

37 Kaempferol-3-O-glucoside 841 447 285 radic 6 8

38 Gamma-eudesmolrhamnoside derivative 847 577 439 397 379 367 349 322 293 249

197 127 radic 1

39 L-ascorbic acid isomer 855 177 133 129 127 113 103 101 57 radic 1

40Peonidin-3-(p-

coumaroyl-glucoside)isomer

869 609 301(M+H-146-162)+ radic radic 23

41 Vicenin isomer 889 593 473 (MminusH-120)minus 395 383 (M-120-90)minus 338 327 298 radic 15 25

42 Apiin isomer 898 565 433 (M+H-132)+ 413 271(M+H-162-132)+ radic radic 5

43 Apigenin C-hexoside-C-pentoside isomer 898 563

545 (MminusH-18)minus 443 (MminusH-120)minus431 (MminusH-132)minus 353 (MminusH-120-90)minus 341 (MminusH-132-90)minus 311

(MminusH-132-120)minusradic 6 13

44 Unknown 904 319 239 204 195 97 radic mdash

45Kaempferol-3-(p-

coumaryl-glucoside)isomer

912 593 447 (MminusH-146)minus 327 285(MminusH-146-162)minus radic 24

46Peonidin-3-(p-

coumaroyl-glucoside)isomer

937 609 609 579 463 (M+H-146)+ 301(peonidin) (M+H-146-162)+ radic radic radic 23

47Methyl apigenin-C-rhamnoside-O-glucosidelowast

938 593 327 (M+H-162-104)+ 297 (M+H-162-134)+ 285 radic mdash

48 Diosmetin-7-O-rutinoside 950 607 607 577 461 (MminusH-146)minus 299

(MminusH-146-162)minus radic radic 26

49 Luteolin hexoside 955 449 287 (M+H-162)+ radic 5

50 Luteolin C-hexoside-C-pentoside isomer 963 579 369 (MminusH-120-90)minus 339 (MminusH-

120-120)minus 322 281 259 124 radic 13

51 Schaftoside isomer 994 565529 511 475 457 445 427

415(M+H-150)+ 409 391 379 361349 337 325 307 295 273

radic 1 22

52 Succinic acid isomer 1001 117 99 radic 1053 L-ascorbic acid isomer 1036 177 133 129 127 113 103 101 57 radic 154 Peonidin dirhamnosidelowast 1037 593 301 (M+H-146-146)+ radic mdash55 3-Hydroxybenzoic acid 1049 139 105 97 93 79 radic 1

56 Acacetin-rhamnoglucoside 1061 591 283 (MminusH-Rut)minus 103 58 radic radic 18

57 Kaempferol dimethylether dipentosidelowast 1068 579 579 315 (M+H-132-132)+ radic mdash

58 Peonidin glucosideferuloyl glucuronide 1082 815 463 (M+H-352 feruloyl

glucuronide)+ radic 7

59 3-Hydroxybenzoic acidisomer 1094 139 105 97 93 79 radic 1

60 N-feruloyltyramineisomer 1096 314 235 218 181 177 145 121 103 93

45 radic 1

61 Unknown 1107 877 877 813 783 557 radic mdash62 Apigenin-O-dihexoside 1138 593 269 (MminusH-162-162)minus radic 9

6 Journal of Chemistry

Table 1 Continued

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

63 Isorhamnetin-3-O-rutinoside 1159 623 315 (MminusH-Rut)minus 300 271 radic 27

64 Peonidin dirhamnosideisomerlowast 1169 593 447 (M+H-146)+ 301 (M+H-146-

146)+ radic radic mdash

65 Acacetin-rhamnoglucoside isomer 1169 591 283 (MminusH-Rut)minus radic radic 18

66 Succinic acid isomer 1178 117 99 radic 1067 Tyrosol isomer 1205 121 93 77 radic 3

68 Diosmetin rhamnosideferuloyl glucuronidelowast 1246 797 445 (MminusH-176-176)minus 299 (MminusH-

176-176-146)minus 237 205 radic mdash

69 Apiin isomer 1287 563 269 radic 1870 Succinic acid isomer 1290 117 99 radic 10

71 Peonidin dipentosidelowast 1313 565 565 301(M+H-132-132)+ 336 265195 135 91 45 radic mdash

72 Methyl gallate 1325 185 168 124 radic 573 Succinic acid isomer 1365 117 99 radic 10

74 Delphinidin-3-arabinoside derivative 1540 799 435 303 (M+H-364-132)+ radic 7 23

75Hydroxy-

octadecadienoic acidderivative

1557 593295 (MminusH-298)minus 277 (MminusH-298-18)minus 251(MminusH-298-18-CO2)minus 195

171radic 28

76 Syringic acid derivative 1583 377 197 radic 2177 22-Dehydrocholesterol 1600 393 393 273 173 171 130 125 radic 178 Malvidin derivativelowast 1672 565 331 147 radic mdash

79 Kaempferide derivativeisomer 1740 623 299 (MminusH-2 Glc)minus 163 radic 29

80 Unknown 1742 274 274 256 (M+H-18)+ 210 (M+H-18-46)+ 111 105 102 88 71 radic radic mdash

81 Unknown 1754 399 267 253 227 radic mdash82 L-ascorbic acid isomer 1782 177 133 129 127 113 103 101 57 radic 183 Succinic acid isomer 2149 117 117 99 radic 10

84 Apiin isomer 2189 565 433 (M+H-132)+ 413 271 (M+H-162-132)+ radic 5

85 Methyl gallate derivative 2306 325 183 (MminusH-142)minus radic 2986 Gallocatechin derivative 2448 561 305 radic 2187 Succinic acid isomer 2496 117 99 radic 1088 Unknown 2501 515 515 353 331 313 239 radic mdash

89 4-Hydroxybenzoic acidisomer 2528 139 121 111 105 97 93 79 radic radic 1

90 Succinic acid isomer 2611 117 117 101 99 radic radic 10

91 Unknown 2666 805 615 606 598 413 391 279 167 149113 radic mdash

92 Luteolin-7-glucuronide-3acute4prime-pentoside 2677 593 461 (MminusH-132)minus 285 (MminusH-132-

176)minus 169 radic 11

93 Peonidin dipentosideisomerlowast 2745 565 547 301 (M+H-132-132)+ 259 219

133 113 85 45 radic radic mdash

94 Tyrosol isomer 2755 121 93 77 radic 395 L-ascorbic acid isomer 2761 177 133 129 127 113 103 101 57 radic 1

96Delphinidin-3-(p-

coumaroyl-glucoside)derivative

2836 799 611 303 (M+H-188-308)+ radic 23

97 Epicatechin derivative 2891 678 289 radic 6

98 Kaempferol-8C-glucosidelowast 2940 449 329 (M+H-120)+ 299 (M+H-150)+ radic mdash

99 Malvidin 3-O-glucosidederivative 2983 871 493 331 radic 8

100 Unknown 3000 871 593 552 369 260 105 radic mdash101 L-ascorbic acid isomer 3041 177 133 129 127 113 103 101 57 radic radic 1102 Peonidin derivativelowast 3065 799 648 (M+H-galloyl)+ 301 (peonidin) radic mdash103 Succinic acid isomer 3152 117 117 99 radic 101 [13] 2 [14] 3 [15] 4 [16] 5 [17] 6 [18] 7 [19] 8 [20] 9 [21] 10 [22] 11 [23] 12 [24] 13 [25] 14 [26] 15 [27] 16 [28] 17 [29] 18 [30] 19 [31] 20[32] 21 [33] 22 [34] 23 [35] 24 [28] 25 [26] 26 [36] 27 [37] 28 [38] 29 [39] lowastTentative identified Rt retention time Rut rutinose Glc glucose HdAHibiscus deflersii HcA H calyphyllus HmA H micranthus

Journal of Chemistry 7

arabinose unit (riboside) and at mz 445 [M+H-120]+corresponding to the loss of a glucose unit Further frag-mentations of the sugar moieties were observed to generate[M+H-186]+ ions at mz 379 [M+H-240]+ ions (formingthe base peaks) atmz 325 and [M+H-270]+ ions atmz 295[34]

Diosmetin C-glucoside C-pentoside (25) showed apseudomolecular ion peak [MminusH]minus at mz 593 )e typicalfragmentation pathway ofC-glycosylated flavonoids resultedin the formation of ions at mz 413 [MminusH-90-90]minus cor-responding to the loss of an pentosidemoiety and atmz 383[MminusH-120-90]minus corresponding to the loss of a glucosemoiety

313 Flavonoid OC-Glycosides Flavonoid OC-glycosidesare distinguished by the lack of the aglycone ion Only theprecursor ion [MminusH]minus is detected in addition to the ions

resulting from the interglycosidic linkage cleavage includingthe key fragmentation ions at [MminusH-120]minus and [MminusH-120-162]minus or [MminusH-90- 146]minus with or without [MminusH-18]minusOn the basis of these rules compound 47 (Rt 938min) witha [M+H]+ ion at mz 593 was tentatively identified asmethyl apigenin-C-rhamnoside-O-glucoside as it showedMS2 fragments atmz 327 [M+H-162-104]+ correspondingto loss of O-glucoside moiety (minus162Da) mz 297 [M+H-162-134]+ corresponding to additional loss of C-rhamnosylmoiety (minus134Da) and mz 285 (methyl apigenin)

314 Flavonoid O-Glycosides Fragmentation pattern offlavonoid O-glycosides is characterized by the loss of thesugar moiety [41 42] and as a result a deprotonatedaglycone ion is yielded in MS2 Compound 21 (Rt 725min)with [MminusH]minus ion peak at mz 593 was tentatively identifiedas kaempferol-3-O-rutinosideluteolin-7-O-rutinoside

HdA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 2 scan Es-

2021

1914

45

6365

695670

68

73 75 79

8586 103

43

4827

2

36

(a)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 1 scan Es+

HdA(+)

1

3 7

1213

23 24 40

4239

29

4649

58

64

5471

7478

81

89

91

93

99

101

(b)

HcA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20182 scan Es-

18

2225 37 52

62

61 70

76 8387

90

3850

66

318

(c)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20181 scan Es+

HcA(+) 102

10198

9697

89 94

828072

67

6459

53

5551

404647

3334

2628

16111

6

(d)

HmA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

92

15

2

30

36 41

48

56443517

HMA12-Jun-2018 2 scan Es-

(e)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HMA12-Jun-2018 2 scan Es+

HmA(+)

110 32

9

46 57 60 77 80 84

889395

100

(f )

Figure 1 UPLS-ESI-MS chromatograms of aqueous extracts of aerial parts of H deflersii (HdA) H calyphyllus (HcA) and H micranthus(HmA) in negative (minus) and positive (+) ionization modes

8 Journal of Chemistry

based on the ESI mass data and the 308Da neutral lossleaving fragment ion at mz 285 corresponding to kaemp-ferolluteolin aglycones [29 37] Diosmetin-7-O-glucuro-nide-3prime-O-pentoside (15) (Rt 631min) produced a [MminusH]minusion atmz 607 with MS2 fragments atmz 475 [MminusH-132]minuscorresponding to loss of pentosyl moiety and 299 [MminusH-132-176]minus corresponding to loss of additional glucuronylmoiety [23]

Apigenin-7-O-apiosylglucoside (apiin) (22) and its iso-mer (69) were tentatively identified from the MS profile ofpeak at Rt of 729 and 1287min with [MminusH]minus at mz 563and MSMS base peak fragment ion at mz 269 which gavethe loss of 294Da (162 + 132Da) (apiosylglucoside moiety)[17 30] Furthermore other three apiin isomers (23 42 and84) were tentatively identified from the MS profile of peaksat retention time of 731 898 and 2189min with [M+H]+at mz 565 and MS2 base peak fragment ion at mz 433[M+H-132]+ corresponding to apiosyl moiety loss and atmz 271 which indicated the loss of 294Da (162 + 132Da)(apiosylglucoside moiety) [17] Compounds 35 and 45 with[MminusH]minus ion at mz 593 were tentatively identified askaempferol-3-O-(p-coumaryl glucoside) )e fragmentationpattern showed MS2 fragments at mz 447 [MminusH-146]minuscorresponding to loss of rhamnose (minus146Da) moiety and atmz 285 [MminusH-146-162]minus corresponding to additional lossof glucose moiety (minus162Da) )ey both gave the product ionat mz 285 corresponding to the kaempferol aglycone [28]Compound 37 (Rt 841min) with [MminusH]minus at mz 447 wastentatively identified as kaempferol-3-O-glucoside fromESIminus mass data and the neutral loss of 162Da for glucosylmoiety [18 20] By the same manner compound 49 (Rt955min) was identified as luteolin hexoside ([M+H]+ ionat mz 449 and MS2 ion at mz 287 [M+H-glc]+))eprecursor ion of compound 48 was detected at mz 607[MminusH]minus and its characteristic MS2 fragment ion at mz 299[MminusH-rut]minus related to deprotonated diosmetin andconsequently it was tentatively identified as diosmetin-7-O-rutinoside [36] Additionally the precursor ion of com-pounds 56 and 65 was detected at mz 591 [MminusH]minus and itscharacteristic MS2 fragment ion at mz 283 [MminusH-rut]minusrelated to deprotonated acacetin and consequently it wastentatively identified as acacetin-rhamnoglucoside and itspositional isomers [30] Compound 57 was tentativelyidentified as kaempferol dimethyl ether dipentoside (Rt1068min) as it produced a [M+H]+ ion at mz 579 withMS2 fragments atmz 315 [M+H-132-132]+ correspondingto loss of dipentosyl moieties Compound 62 with a[MminusH]minus at mz 593 and MS2 ions at mz 269 [MminusH-162-162]minus was tentatively identified as apigenin-O-dihexoside[21] Compound 63 [MminusH]minus ion at mz 623 was tentativelyidentified as isorhamnetin-3-O-rutinoside )e fragmenta-tion patterns showed MS2 fragments at mz 315 [MminusH-308]minus (isorhamnetin) corresponding to loss of rutinose(minus308Da) moiety [37] Compound 68 with [MminusH]minus at mz797 was tentatively identified as diosmetin rhamnosideferuloyl glucuronide as it showed MS2 fragments at mz 445[MminusH-176-176]minus corresponding to loss of feruloyl(minus176Da) and glucuronide (-176Da) moieties and 299[MminusH-176-176-146]minus corresponding to additional loss of

rhamnose moiety (minus146Da) According to [39] kaempferidederivative (79) was tentatively identified by its molecular ion[MminusH]minusat mz 623 and fragmentation pattern containingspecific ion at mz 299 [MminusH-162-162]minus corresponding toloss of two glucose moieties and ion at mz 163 Accordingto [23] the diglycosylated flavonoid (92) was detected as itpossessed a pseudomolecular ion [MminusH]minus at mz 593 withMS2 fragment ions at mz 461 [MminusH-132]minus correspondingto pentosyl moiety loss as well as other less abundant ion atmz 285 [MminusH- 132-176]minus resulting from the loss of glu-curonyl moiety (minus176Da) )is compound was identified asluteolin-7-glucuronide-3rsquo4rsquo-pentoside

315 Flavonoid Aglycones Patuletin (6-methoxy quercetin)(13) was identified by comparing its MS2 fragmentationpattern with the previously reported data [21] Compound31 with [MminusH]ndash ion atmz 737 andMS2 fragment ion atmz285 was determined to be luteolin derivative [30 33] whilecompound 29 which gave a [M+H]+ ion at mz 799 andMS2 fragment ion at mz 285 was identified tentatively asmethyl apigenin derivative Gallocatechin derivative (86)[33] and epicatechin derivative (97) [18] were recognized bycomparing their MS2 fragmentation pattern with the pre-viously published data

316 Anthocyanins A total of 9 anthocyanin derivativeshave been detected in Hibiscus Compounds 34 40 46 5458 64 71 93 and 102 were tentatively identified as peonidinderivatives )us mass data of compound 34 and its isomers(40 and 46) were proposed to be peonidin-3-(p-coumaroyl-glucoside) as they showed a molecular ion peak [M+H]+ atmz 609 and MS2 fragment ions at mz 301 [M+H-162-146]+ )ese were indicative of a peonidin with glucose(162Da) and coumaroyl (146Da) moieties [35]

Compounds 71 and 93 produced a [M+H]+ ion at mz565 with MS2 fragments atmz 301 [M+H-132-132]+)esewere indicative of a peonidin with two pentoside moieties(264Da) and were tentatively identified as peonidindipentoside while compounds 54 and 64 produced [M+H]+ion at mz 593 with MS2 fragments at mz 301 [M+H-146-146]+ (peonidin))ese were indicative of peonidin with tworhamnoside moieties (292Da) and were tentatively identi-fied as peonidin dirhamnoside Compound 58 was tenta-tively identified as peonidin glucoside feruloyl glucuronideas it produced a [M+H]+ ion atmz 815 with MS2 fragmentions atmz 463 [M+H-176-176]+ corresponding to the lossof a feruloyl (minus176Da) and glucuronide (minus176Da) moietiesand at mz 301 [M+H-176-176-162]+ (peonidin) corre-sponding to the loss of a glucose unit Compound 102 wastentatively identified as peonidin derivative as it showed a[M+H]+ ion at mz 799 with MS2 fragment ions at mz 648[M+H-151]+ corresponding to the loss of a galloyl(minus151Da) moiety and at mz 301 (peonidin)

)e mass spectra of compounds 11 and 24 showed theirprotonated aglycon ions [M+H]+ to be mz 287 corre-sponding to cyanidin )ese protonated aglycon ions wereall formed by loss of a sugar moiety with 162 units from their[M+H]+ indicating that they are anthocyanidin

Journal of Chemistry 9

monoglucosides or monogalactosides )is suggests thepresence of cyanidin 3-O-galactoside (11) and cyanidin 3-O-glucoside (24) )e mz values of compound 18 detected atmz 579 in the negative ion mode are similar to those ofcyanidin 3-O-sambubioside [24]

Compounds 28 74 and 96 were tentatively identified asdelphinidin derivatives Compound 28 showed a molecularion peak [M+H]+ at mz 565 and MS2 fragment ions at mz303 [M+H-176-86]+ )ese were indicative of a delphinidinwith glucuronide (176Da) and malonyl (86Da) moieties)is compound was suggested to be delphinidin malonylglucuronide

Compound 74 showed a molecular ion peak [M+H]+ atmz 799 and MS2 fragment ions at mz 435 [M+H-364]+(delphinidin-3-arabinoside) andmz 303 [M+H-364-132]+corresponding to loss of arabinose moiety )ese were in-dicative of a delphinidin-3-arabinoside derivative [19 35]Compound 96 showed a molecular ion peak [M+H]+ atmz799 and MS2 fragment ions at mz 611 [M+H-188]+ and atmz 303 [M+H-188-308]+ )ese were indicative of a del-phinidin with coumaroyl-glucoside moieties (minus308Da) [43])is compound was proposed to be delphinidin-3-(p-cou-maroyl-glucoside) derivative [35]

Compound 78 was tentatively identified as malvidinderivative as it showed a protonated aglycone ion peak atmz 331 corresponding to malvidin [43] Compound 99 pro-duced a [M+H]+ ion at mz 871 with MS2 fragments at mz493 corresponding to malvidin-3-O-glucoside [20] and atmz 331 [M+H-378-162]+ corresponding to malvidin [43]with one glucose moiety (162Da) and was identified asmalvidin 3-O-glucoside derivative [20]

317 Fatty Acid Derivatives For compound 75 a pseu-domolecular ion peak [MminusH]minus at mz 593 was observedwith MS2 fragment ions at mz 295 [MminusH-298]minus 277[MminusH-298-H2O]minus 251 [MminusH-298-CO2]minus 195 and 171were detected suggesting the presence of hydroxy-octade-cadienoic acids derivative [38]

318 Miscellaneous Compounds For L-ascorbic acid (1) andits isomers (9 12 16 39 53 82 95 and 101) a protonatedpseudomolecular ion was observed at mz 177 and MS2 ionat mz 133 (M+H-44)+ [13] Moreover compound 5 wassuggested to be sucrose (MS1 atmz 341 [MminusH]minus MS2 atmz 179 [MminusH-glc]minus 161 131) [16] It was previously re-ported that L-ascorbic acid and sucrose were identified inHibiscus species as H sabdariffa contains higher amount ofascorbic acid compared to orange and mango [44ndash46]

Oleuropein (2) showed a deprotonated pseudomolecularion at mz 539 (MS2 at mz 377 [MminusH-glc]minus and 307[MminusH-glc-C4H6O]minus) [14] It was previously reported thatoleuropein was identified inHibiscus [47] Succinic acid (14)and its isomers (17 52 66 70 73 83 87 90 and 103) wererecognized by comparing their MS2 fragmentation patternwith the reported data [22] )ey showed a deprotonatedmolecular ion atmz 117 and an intense fragment atmz 99attributed to the loss of water molecule [22] while com-pound 3 showed a protonated molecular ion atmz 119 with

MS2 intense fragment ion atmz 101 [M+H-18]+ attributedto the loss of water molecule (minus18Da) thus it was tentativelyidentified as succinic acid

Hydroxycitric acid derivative (6) and butein chalcone (7)were identified by comparison with published data [13 17]respectively Hydroxycitric acid is the principal organic acidfound in the calyces ofHibiscus according to [48] Moreovercompound 38 was identified as a sesquiterpenoid derivativegamma-eudesmol rhamnoside derivative (MS1 at mz 577[MminusH]minus MS2 at mz 439 [MminusH-138]minus corresponding togamma-eudesmol 293 [MminusH-138-146]minus corresponding toloss of rhamnose moiety (minus146Da)) [13] N-feruloyltyr-amine (10) its isomer (60) and 22-dehydrocholesterol (77)were recognized by comparing their MSMS fragmentationpattern with the reported data [13]

319 Unidentified Compounds Finally seven compounds(44 61 80 81 88 91 and 100) with the pseudomolecularions [MminusH]minus at mz 319 and 877 and [M+H]+ at mz 274274 399 515 805 and 871 respectively could not beidentified Furthermore of the 103 compounds identified 7compounds are unidentified 46 compounds in HdA 42compounds in HcA and 25 compounds in HmA have beenreported in the present study (Table 1) In conclusioncombination of accurate mass measurement and LC abilityto separate isomeric compounds can be considered apowerful tool in the identification of polyphenol diversity inthree species of the Hibiscus genus even in the absence ofstandards but the stereochemical differentiation betweenthe large number of isomers that were found in our speciesfor example isomers of luteolin C-hexoside-C-pentosideapigenin C-hexoside-C-pentoside and cyanidin rutinosidewas not possible with our methodology

32AntioxidantActivity It is well known that plant phenolsand flavonoids in general are highly effective free radicalscavengers and antioxidants )us they are used for theprevention and cure of various disorders which are mainlyassociated with free radicals Series of concentrations rangedfrom 5 to 320 μgmL in methanol were used )e DPPHscavenging percentage of different extracts as well asascorbic acid and SC50 values (the concentration required toscavenge DPPH by 50) are shown in Figures 2 and 3respectively HcA exhibited the highest antioxidant activityas indicated by its high DPPH scavenging percentage (65)at 320 μgmL and low SC50 values (111plusmn 15 μgmL) Itsactivity can be attributed to its contents of polyphenoliccompounds such as phenolic acids flavonoids and antho-cyanins (ie apigenin C-hexoside-C-pentoside luteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoicacid tyrosol and peonidin derivative and succinic acid)Unfortunately both HdA and HmA displayed moderateantioxidant activities with SC50 1376plusmn 03 and135plusmn 05 μgmL respectively with ascorbic acidSC50 142plusmn 05 μgmL as standard

During this work many major anthocyanins were de-tected in LC-MS analysis of HcA (such as peonidin dir-hamnoside peonidin derivative and peonidin-3-(p-

10 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

Table 1 Metabolites identified in the aqueous extracts of Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA) usingUPLCminusESIminusMS in negative and positive ionization modes

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

1 L-ascorbic acid 030 177 133 129 127 113 103 101 57 radic radic radic 12 Oleuropein 082 539 377 341 307 215 179 radic radic 23 Succinic acidlowast 086 119 101 radic mdash4 Tyrosol 093 121 103 97 93 89 79 77 73 65 45 radic 3

5 Sucrose 095 341 179 (MminusH-162)minus 161 131 119 117113 103 101 89 87 71 59 radic 4

6 Hydroxycitric acidderivative 136 593 209 radic 1

7 Butein 143 273 163 143 137 radic 5

8 Apigenin C-hexoside-C-pentoside 153 563

545 (MminusH-18)minus 443 (MminusH-120)minus431 (MminusH-132)minus 353 (MminusH-120-90)minus 341 (MminusH-132-90)minus 311

(MminusH-132-120)minusradic 6

9 L-ascorbic acid isomer 181 177 133 129 127 113 103 101 57 radic 110 N-feruloyltyramine 332 314 235 181 177 145 121 103 93 45 radic 111 Cyanidin 3-O-galactoside 357 449 287 137 radic 7 812 L-ascorbic acid isomer 452 177 133 129 127 113 103 101 57 radic 1

13 Patuletin (6-methoxyquercetin) 497 333 318 301 169 155 radic 9

14 Succinic acid isomer 605 117 99 (MminusH-18)minus radic 10

15Diosmetin-7-O-glucuronide-3prime-O-

pentoside631 607 475 (MminusH-132)minus 299 (MminusH-132-

176)minus 179 radic 11

16 L-ascorbic acid isomer 632 177 133 129 127 113 103 101 57 radic 117 Succinic acid isomer 649 117 117 99 radic 10

18 Cyanidin 3-O-sambubioside 676 579 339 285 radic 12

19 Apigenin C-hexoside-C-pentoside isomer 702 563

545 (MminusH-18)minus 443 (MminusH-120)minus431 (MminusH-132)minus 341 (MminusH-132-

90)minus 311 (MminusH-132-120)minusradic 13

20 Vicenin (apigenin 68-di-C-glucoside) 710 593 473 (MminusH-120)minus 383 (MminusH-120-

90)minus 285 radic 14 15

21Kaempferol-3-O-

rutinosideluteolin-7-O-rutinoside

725 593 285 (MminusH-Rut)minus radic 1617

22 Apiin (apigenin-7-apiosylglucoside) 729 563 443 413 269 (MminusH-132-162)minus radic 5 18

23 Apiin isomer 731 565 433 (M+H-132)+ 413 271(M+H-162- 132)+ radic 5

24 Cyanidin 3-O-glucoside 737 494 287 (M+H-162)+ 137 radic 3

25 Diosmetin C-glucosideC-pentosidelowast 737 593 413 (MminusH-90-90)minus 383 (MminusH-90-

120)minus radic mdash

26 Tyrosol isomer 746 121 93 77 radic 3

27 Orientin (luteolin 8-C-glucoside) 746 447 357 (MminusH-90)minus 327 (MminusH-120)minus

297 (MminusH-150)minus 285(MminusH-162)minus radic13 15 19

20

28 Delphinidin malonylglucuronidelowast 759 565 303 (M+H-176-86)+ radic mdash

29 Methyl apigeninderivativelowast 781 799 285 radic mdash

30 Luteolin C-hexoside-C-pentoside 782 579

489 (MminusH-90minus) 459 (MminusH-120)minus429 (MminusH-150)minus 411 399 369(MminusH-120-90)minus 365 353 339

(MminusH -120-120)minus 299

radic 13

31 Luteolin derivative 798 737 285 radic 18 21

32Schaftoside (apigenin 6-

C-glucoside 8-C-riboside)

799 565529 511 475 457 445 427

415(M+H-150)+ 409 391 379 361349 337 325 307 295 273

radic 1 22

Journal of Chemistry 5

Table 1 Continued

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

33 4-Hydroxybenzoic acid 799 139 121 111 105 97 93 79 radic 1

34 Peonidin-3-(p-coumaroyl-glucoside) 827 609 301(M+H-146-162)+ radic 23

35 Kaempferol-3-(p-coumaryl-glucoside) 827 593 593 447(MminusH-146)minus 327

285(MminusH-146-162)minus radic 24

36 Apigenin C-hexoside-C-pentoside isomer 832 563

545 (MminusH-18)minus 473 (MminusH-90)minus443 (MminusH-120)minus 431 (MminusH-132)minus 353 (MminusH-120-90)minus 341(MminusH-132-90)minus 311 (MminusH-132-

120)minus

radic radic 13

37 Kaempferol-3-O-glucoside 841 447 285 radic 6 8

38 Gamma-eudesmolrhamnoside derivative 847 577 439 397 379 367 349 322 293 249

197 127 radic 1

39 L-ascorbic acid isomer 855 177 133 129 127 113 103 101 57 radic 1

40Peonidin-3-(p-

coumaroyl-glucoside)isomer

869 609 301(M+H-146-162)+ radic radic 23

41 Vicenin isomer 889 593 473 (MminusH-120)minus 395 383 (M-120-90)minus 338 327 298 radic 15 25

42 Apiin isomer 898 565 433 (M+H-132)+ 413 271(M+H-162-132)+ radic radic 5

43 Apigenin C-hexoside-C-pentoside isomer 898 563

545 (MminusH-18)minus 443 (MminusH-120)minus431 (MminusH-132)minus 353 (MminusH-120-90)minus 341 (MminusH-132-90)minus 311

(MminusH-132-120)minusradic 6 13

44 Unknown 904 319 239 204 195 97 radic mdash

45Kaempferol-3-(p-

coumaryl-glucoside)isomer

912 593 447 (MminusH-146)minus 327 285(MminusH-146-162)minus radic 24

46Peonidin-3-(p-

coumaroyl-glucoside)isomer

937 609 609 579 463 (M+H-146)+ 301(peonidin) (M+H-146-162)+ radic radic radic 23

47Methyl apigenin-C-rhamnoside-O-glucosidelowast

938 593 327 (M+H-162-104)+ 297 (M+H-162-134)+ 285 radic mdash

48 Diosmetin-7-O-rutinoside 950 607 607 577 461 (MminusH-146)minus 299

(MminusH-146-162)minus radic radic 26

49 Luteolin hexoside 955 449 287 (M+H-162)+ radic 5

50 Luteolin C-hexoside-C-pentoside isomer 963 579 369 (MminusH-120-90)minus 339 (MminusH-

120-120)minus 322 281 259 124 radic 13

51 Schaftoside isomer 994 565529 511 475 457 445 427

415(M+H-150)+ 409 391 379 361349 337 325 307 295 273

radic 1 22

52 Succinic acid isomer 1001 117 99 radic 1053 L-ascorbic acid isomer 1036 177 133 129 127 113 103 101 57 radic 154 Peonidin dirhamnosidelowast 1037 593 301 (M+H-146-146)+ radic mdash55 3-Hydroxybenzoic acid 1049 139 105 97 93 79 radic 1

56 Acacetin-rhamnoglucoside 1061 591 283 (MminusH-Rut)minus 103 58 radic radic 18

57 Kaempferol dimethylether dipentosidelowast 1068 579 579 315 (M+H-132-132)+ radic mdash

58 Peonidin glucosideferuloyl glucuronide 1082 815 463 (M+H-352 feruloyl

glucuronide)+ radic 7

59 3-Hydroxybenzoic acidisomer 1094 139 105 97 93 79 radic 1

60 N-feruloyltyramineisomer 1096 314 235 218 181 177 145 121 103 93

45 radic 1

61 Unknown 1107 877 877 813 783 557 radic mdash62 Apigenin-O-dihexoside 1138 593 269 (MminusH-162-162)minus radic 9

6 Journal of Chemistry

Table 1 Continued

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

63 Isorhamnetin-3-O-rutinoside 1159 623 315 (MminusH-Rut)minus 300 271 radic 27

64 Peonidin dirhamnosideisomerlowast 1169 593 447 (M+H-146)+ 301 (M+H-146-

146)+ radic radic mdash

65 Acacetin-rhamnoglucoside isomer 1169 591 283 (MminusH-Rut)minus radic radic 18

66 Succinic acid isomer 1178 117 99 radic 1067 Tyrosol isomer 1205 121 93 77 radic 3

68 Diosmetin rhamnosideferuloyl glucuronidelowast 1246 797 445 (MminusH-176-176)minus 299 (MminusH-

176-176-146)minus 237 205 radic mdash

69 Apiin isomer 1287 563 269 radic 1870 Succinic acid isomer 1290 117 99 radic 10

71 Peonidin dipentosidelowast 1313 565 565 301(M+H-132-132)+ 336 265195 135 91 45 radic mdash

72 Methyl gallate 1325 185 168 124 radic 573 Succinic acid isomer 1365 117 99 radic 10

74 Delphinidin-3-arabinoside derivative 1540 799 435 303 (M+H-364-132)+ radic 7 23

75Hydroxy-

octadecadienoic acidderivative

1557 593295 (MminusH-298)minus 277 (MminusH-298-18)minus 251(MminusH-298-18-CO2)minus 195

171radic 28

76 Syringic acid derivative 1583 377 197 radic 2177 22-Dehydrocholesterol 1600 393 393 273 173 171 130 125 radic 178 Malvidin derivativelowast 1672 565 331 147 radic mdash

79 Kaempferide derivativeisomer 1740 623 299 (MminusH-2 Glc)minus 163 radic 29

80 Unknown 1742 274 274 256 (M+H-18)+ 210 (M+H-18-46)+ 111 105 102 88 71 radic radic mdash

81 Unknown 1754 399 267 253 227 radic mdash82 L-ascorbic acid isomer 1782 177 133 129 127 113 103 101 57 radic 183 Succinic acid isomer 2149 117 117 99 radic 10

84 Apiin isomer 2189 565 433 (M+H-132)+ 413 271 (M+H-162-132)+ radic 5

85 Methyl gallate derivative 2306 325 183 (MminusH-142)minus radic 2986 Gallocatechin derivative 2448 561 305 radic 2187 Succinic acid isomer 2496 117 99 radic 1088 Unknown 2501 515 515 353 331 313 239 radic mdash

89 4-Hydroxybenzoic acidisomer 2528 139 121 111 105 97 93 79 radic radic 1

90 Succinic acid isomer 2611 117 117 101 99 radic radic 10

91 Unknown 2666 805 615 606 598 413 391 279 167 149113 radic mdash

92 Luteolin-7-glucuronide-3acute4prime-pentoside 2677 593 461 (MminusH-132)minus 285 (MminusH-132-

176)minus 169 radic 11

93 Peonidin dipentosideisomerlowast 2745 565 547 301 (M+H-132-132)+ 259 219

133 113 85 45 radic radic mdash

94 Tyrosol isomer 2755 121 93 77 radic 395 L-ascorbic acid isomer 2761 177 133 129 127 113 103 101 57 radic 1

96Delphinidin-3-(p-

coumaroyl-glucoside)derivative

2836 799 611 303 (M+H-188-308)+ radic 23

97 Epicatechin derivative 2891 678 289 radic 6

98 Kaempferol-8C-glucosidelowast 2940 449 329 (M+H-120)+ 299 (M+H-150)+ radic mdash

99 Malvidin 3-O-glucosidederivative 2983 871 493 331 radic 8

100 Unknown 3000 871 593 552 369 260 105 radic mdash101 L-ascorbic acid isomer 3041 177 133 129 127 113 103 101 57 radic radic 1102 Peonidin derivativelowast 3065 799 648 (M+H-galloyl)+ 301 (peonidin) radic mdash103 Succinic acid isomer 3152 117 117 99 radic 101 [13] 2 [14] 3 [15] 4 [16] 5 [17] 6 [18] 7 [19] 8 [20] 9 [21] 10 [22] 11 [23] 12 [24] 13 [25] 14 [26] 15 [27] 16 [28] 17 [29] 18 [30] 19 [31] 20[32] 21 [33] 22 [34] 23 [35] 24 [28] 25 [26] 26 [36] 27 [37] 28 [38] 29 [39] lowastTentative identified Rt retention time Rut rutinose Glc glucose HdAHibiscus deflersii HcA H calyphyllus HmA H micranthus

Journal of Chemistry 7

arabinose unit (riboside) and at mz 445 [M+H-120]+corresponding to the loss of a glucose unit Further frag-mentations of the sugar moieties were observed to generate[M+H-186]+ ions at mz 379 [M+H-240]+ ions (formingthe base peaks) atmz 325 and [M+H-270]+ ions atmz 295[34]

Diosmetin C-glucoside C-pentoside (25) showed apseudomolecular ion peak [MminusH]minus at mz 593 )e typicalfragmentation pathway ofC-glycosylated flavonoids resultedin the formation of ions at mz 413 [MminusH-90-90]minus cor-responding to the loss of an pentosidemoiety and atmz 383[MminusH-120-90]minus corresponding to the loss of a glucosemoiety

313 Flavonoid OC-Glycosides Flavonoid OC-glycosidesare distinguished by the lack of the aglycone ion Only theprecursor ion [MminusH]minus is detected in addition to the ions

resulting from the interglycosidic linkage cleavage includingthe key fragmentation ions at [MminusH-120]minus and [MminusH-120-162]minus or [MminusH-90- 146]minus with or without [MminusH-18]minusOn the basis of these rules compound 47 (Rt 938min) witha [M+H]+ ion at mz 593 was tentatively identified asmethyl apigenin-C-rhamnoside-O-glucoside as it showedMS2 fragments atmz 327 [M+H-162-104]+ correspondingto loss of O-glucoside moiety (minus162Da) mz 297 [M+H-162-134]+ corresponding to additional loss of C-rhamnosylmoiety (minus134Da) and mz 285 (methyl apigenin)

314 Flavonoid O-Glycosides Fragmentation pattern offlavonoid O-glycosides is characterized by the loss of thesugar moiety [41 42] and as a result a deprotonatedaglycone ion is yielded in MS2 Compound 21 (Rt 725min)with [MminusH]minus ion peak at mz 593 was tentatively identifiedas kaempferol-3-O-rutinosideluteolin-7-O-rutinoside

HdA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 2 scan Es-

2021

1914

45

6365

695670

68

73 75 79

8586 103

43

4827

2

36

(a)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 1 scan Es+

HdA(+)

1

3 7

1213

23 24 40

4239

29

4649

58

64

5471

7478

81

89

91

93

99

101

(b)

HcA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20182 scan Es-

18

2225 37 52

62

61 70

76 8387

90

3850

66

318

(c)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20181 scan Es+

HcA(+) 102

10198

9697

89 94

828072

67

6459

53

5551

404647

3334

2628

16111

6

(d)

HmA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

92

15

2

30

36 41

48

56443517

HMA12-Jun-2018 2 scan Es-

(e)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HMA12-Jun-2018 2 scan Es+

HmA(+)

110 32

9

46 57 60 77 80 84

889395

100

(f )

Figure 1 UPLS-ESI-MS chromatograms of aqueous extracts of aerial parts of H deflersii (HdA) H calyphyllus (HcA) and H micranthus(HmA) in negative (minus) and positive (+) ionization modes

8 Journal of Chemistry

based on the ESI mass data and the 308Da neutral lossleaving fragment ion at mz 285 corresponding to kaemp-ferolluteolin aglycones [29 37] Diosmetin-7-O-glucuro-nide-3prime-O-pentoside (15) (Rt 631min) produced a [MminusH]minusion atmz 607 with MS2 fragments atmz 475 [MminusH-132]minuscorresponding to loss of pentosyl moiety and 299 [MminusH-132-176]minus corresponding to loss of additional glucuronylmoiety [23]

Apigenin-7-O-apiosylglucoside (apiin) (22) and its iso-mer (69) were tentatively identified from the MS profile ofpeak at Rt of 729 and 1287min with [MminusH]minus at mz 563and MSMS base peak fragment ion at mz 269 which gavethe loss of 294Da (162 + 132Da) (apiosylglucoside moiety)[17 30] Furthermore other three apiin isomers (23 42 and84) were tentatively identified from the MS profile of peaksat retention time of 731 898 and 2189min with [M+H]+at mz 565 and MS2 base peak fragment ion at mz 433[M+H-132]+ corresponding to apiosyl moiety loss and atmz 271 which indicated the loss of 294Da (162 + 132Da)(apiosylglucoside moiety) [17] Compounds 35 and 45 with[MminusH]minus ion at mz 593 were tentatively identified askaempferol-3-O-(p-coumaryl glucoside) )e fragmentationpattern showed MS2 fragments at mz 447 [MminusH-146]minuscorresponding to loss of rhamnose (minus146Da) moiety and atmz 285 [MminusH-146-162]minus corresponding to additional lossof glucose moiety (minus162Da) )ey both gave the product ionat mz 285 corresponding to the kaempferol aglycone [28]Compound 37 (Rt 841min) with [MminusH]minus at mz 447 wastentatively identified as kaempferol-3-O-glucoside fromESIminus mass data and the neutral loss of 162Da for glucosylmoiety [18 20] By the same manner compound 49 (Rt955min) was identified as luteolin hexoside ([M+H]+ ionat mz 449 and MS2 ion at mz 287 [M+H-glc]+))eprecursor ion of compound 48 was detected at mz 607[MminusH]minus and its characteristic MS2 fragment ion at mz 299[MminusH-rut]minus related to deprotonated diosmetin andconsequently it was tentatively identified as diosmetin-7-O-rutinoside [36] Additionally the precursor ion of com-pounds 56 and 65 was detected at mz 591 [MminusH]minus and itscharacteristic MS2 fragment ion at mz 283 [MminusH-rut]minusrelated to deprotonated acacetin and consequently it wastentatively identified as acacetin-rhamnoglucoside and itspositional isomers [30] Compound 57 was tentativelyidentified as kaempferol dimethyl ether dipentoside (Rt1068min) as it produced a [M+H]+ ion at mz 579 withMS2 fragments atmz 315 [M+H-132-132]+ correspondingto loss of dipentosyl moieties Compound 62 with a[MminusH]minus at mz 593 and MS2 ions at mz 269 [MminusH-162-162]minus was tentatively identified as apigenin-O-dihexoside[21] Compound 63 [MminusH]minus ion at mz 623 was tentativelyidentified as isorhamnetin-3-O-rutinoside )e fragmenta-tion patterns showed MS2 fragments at mz 315 [MminusH-308]minus (isorhamnetin) corresponding to loss of rutinose(minus308Da) moiety [37] Compound 68 with [MminusH]minus at mz797 was tentatively identified as diosmetin rhamnosideferuloyl glucuronide as it showed MS2 fragments at mz 445[MminusH-176-176]minus corresponding to loss of feruloyl(minus176Da) and glucuronide (-176Da) moieties and 299[MminusH-176-176-146]minus corresponding to additional loss of

rhamnose moiety (minus146Da) According to [39] kaempferidederivative (79) was tentatively identified by its molecular ion[MminusH]minusat mz 623 and fragmentation pattern containingspecific ion at mz 299 [MminusH-162-162]minus corresponding toloss of two glucose moieties and ion at mz 163 Accordingto [23] the diglycosylated flavonoid (92) was detected as itpossessed a pseudomolecular ion [MminusH]minus at mz 593 withMS2 fragment ions at mz 461 [MminusH-132]minus correspondingto pentosyl moiety loss as well as other less abundant ion atmz 285 [MminusH- 132-176]minus resulting from the loss of glu-curonyl moiety (minus176Da) )is compound was identified asluteolin-7-glucuronide-3rsquo4rsquo-pentoside

315 Flavonoid Aglycones Patuletin (6-methoxy quercetin)(13) was identified by comparing its MS2 fragmentationpattern with the previously reported data [21] Compound31 with [MminusH]ndash ion atmz 737 andMS2 fragment ion atmz285 was determined to be luteolin derivative [30 33] whilecompound 29 which gave a [M+H]+ ion at mz 799 andMS2 fragment ion at mz 285 was identified tentatively asmethyl apigenin derivative Gallocatechin derivative (86)[33] and epicatechin derivative (97) [18] were recognized bycomparing their MS2 fragmentation pattern with the pre-viously published data

316 Anthocyanins A total of 9 anthocyanin derivativeshave been detected in Hibiscus Compounds 34 40 46 5458 64 71 93 and 102 were tentatively identified as peonidinderivatives )us mass data of compound 34 and its isomers(40 and 46) were proposed to be peonidin-3-(p-coumaroyl-glucoside) as they showed a molecular ion peak [M+H]+ atmz 609 and MS2 fragment ions at mz 301 [M+H-162-146]+ )ese were indicative of a peonidin with glucose(162Da) and coumaroyl (146Da) moieties [35]

Compounds 71 and 93 produced a [M+H]+ ion at mz565 with MS2 fragments atmz 301 [M+H-132-132]+)esewere indicative of a peonidin with two pentoside moieties(264Da) and were tentatively identified as peonidindipentoside while compounds 54 and 64 produced [M+H]+ion at mz 593 with MS2 fragments at mz 301 [M+H-146-146]+ (peonidin))ese were indicative of peonidin with tworhamnoside moieties (292Da) and were tentatively identi-fied as peonidin dirhamnoside Compound 58 was tenta-tively identified as peonidin glucoside feruloyl glucuronideas it produced a [M+H]+ ion atmz 815 with MS2 fragmentions atmz 463 [M+H-176-176]+ corresponding to the lossof a feruloyl (minus176Da) and glucuronide (minus176Da) moietiesand at mz 301 [M+H-176-176-162]+ (peonidin) corre-sponding to the loss of a glucose unit Compound 102 wastentatively identified as peonidin derivative as it showed a[M+H]+ ion at mz 799 with MS2 fragment ions at mz 648[M+H-151]+ corresponding to the loss of a galloyl(minus151Da) moiety and at mz 301 (peonidin)

)e mass spectra of compounds 11 and 24 showed theirprotonated aglycon ions [M+H]+ to be mz 287 corre-sponding to cyanidin )ese protonated aglycon ions wereall formed by loss of a sugar moiety with 162 units from their[M+H]+ indicating that they are anthocyanidin

Journal of Chemistry 9

monoglucosides or monogalactosides )is suggests thepresence of cyanidin 3-O-galactoside (11) and cyanidin 3-O-glucoside (24) )e mz values of compound 18 detected atmz 579 in the negative ion mode are similar to those ofcyanidin 3-O-sambubioside [24]

Compounds 28 74 and 96 were tentatively identified asdelphinidin derivatives Compound 28 showed a molecularion peak [M+H]+ at mz 565 and MS2 fragment ions at mz303 [M+H-176-86]+ )ese were indicative of a delphinidinwith glucuronide (176Da) and malonyl (86Da) moieties)is compound was suggested to be delphinidin malonylglucuronide

Compound 74 showed a molecular ion peak [M+H]+ atmz 799 and MS2 fragment ions at mz 435 [M+H-364]+(delphinidin-3-arabinoside) andmz 303 [M+H-364-132]+corresponding to loss of arabinose moiety )ese were in-dicative of a delphinidin-3-arabinoside derivative [19 35]Compound 96 showed a molecular ion peak [M+H]+ atmz799 and MS2 fragment ions at mz 611 [M+H-188]+ and atmz 303 [M+H-188-308]+ )ese were indicative of a del-phinidin with coumaroyl-glucoside moieties (minus308Da) [43])is compound was proposed to be delphinidin-3-(p-cou-maroyl-glucoside) derivative [35]

Compound 78 was tentatively identified as malvidinderivative as it showed a protonated aglycone ion peak atmz 331 corresponding to malvidin [43] Compound 99 pro-duced a [M+H]+ ion at mz 871 with MS2 fragments at mz493 corresponding to malvidin-3-O-glucoside [20] and atmz 331 [M+H-378-162]+ corresponding to malvidin [43]with one glucose moiety (162Da) and was identified asmalvidin 3-O-glucoside derivative [20]

317 Fatty Acid Derivatives For compound 75 a pseu-domolecular ion peak [MminusH]minus at mz 593 was observedwith MS2 fragment ions at mz 295 [MminusH-298]minus 277[MminusH-298-H2O]minus 251 [MminusH-298-CO2]minus 195 and 171were detected suggesting the presence of hydroxy-octade-cadienoic acids derivative [38]

318 Miscellaneous Compounds For L-ascorbic acid (1) andits isomers (9 12 16 39 53 82 95 and 101) a protonatedpseudomolecular ion was observed at mz 177 and MS2 ionat mz 133 (M+H-44)+ [13] Moreover compound 5 wassuggested to be sucrose (MS1 atmz 341 [MminusH]minus MS2 atmz 179 [MminusH-glc]minus 161 131) [16] It was previously re-ported that L-ascorbic acid and sucrose were identified inHibiscus species as H sabdariffa contains higher amount ofascorbic acid compared to orange and mango [44ndash46]

Oleuropein (2) showed a deprotonated pseudomolecularion at mz 539 (MS2 at mz 377 [MminusH-glc]minus and 307[MminusH-glc-C4H6O]minus) [14] It was previously reported thatoleuropein was identified inHibiscus [47] Succinic acid (14)and its isomers (17 52 66 70 73 83 87 90 and 103) wererecognized by comparing their MS2 fragmentation patternwith the reported data [22] )ey showed a deprotonatedmolecular ion atmz 117 and an intense fragment atmz 99attributed to the loss of water molecule [22] while com-pound 3 showed a protonated molecular ion atmz 119 with

MS2 intense fragment ion atmz 101 [M+H-18]+ attributedto the loss of water molecule (minus18Da) thus it was tentativelyidentified as succinic acid

Hydroxycitric acid derivative (6) and butein chalcone (7)were identified by comparison with published data [13 17]respectively Hydroxycitric acid is the principal organic acidfound in the calyces ofHibiscus according to [48] Moreovercompound 38 was identified as a sesquiterpenoid derivativegamma-eudesmol rhamnoside derivative (MS1 at mz 577[MminusH]minus MS2 at mz 439 [MminusH-138]minus corresponding togamma-eudesmol 293 [MminusH-138-146]minus corresponding toloss of rhamnose moiety (minus146Da)) [13] N-feruloyltyr-amine (10) its isomer (60) and 22-dehydrocholesterol (77)were recognized by comparing their MSMS fragmentationpattern with the reported data [13]

319 Unidentified Compounds Finally seven compounds(44 61 80 81 88 91 and 100) with the pseudomolecularions [MminusH]minus at mz 319 and 877 and [M+H]+ at mz 274274 399 515 805 and 871 respectively could not beidentified Furthermore of the 103 compounds identified 7compounds are unidentified 46 compounds in HdA 42compounds in HcA and 25 compounds in HmA have beenreported in the present study (Table 1) In conclusioncombination of accurate mass measurement and LC abilityto separate isomeric compounds can be considered apowerful tool in the identification of polyphenol diversity inthree species of the Hibiscus genus even in the absence ofstandards but the stereochemical differentiation betweenthe large number of isomers that were found in our speciesfor example isomers of luteolin C-hexoside-C-pentosideapigenin C-hexoside-C-pentoside and cyanidin rutinosidewas not possible with our methodology

32AntioxidantActivity It is well known that plant phenolsand flavonoids in general are highly effective free radicalscavengers and antioxidants )us they are used for theprevention and cure of various disorders which are mainlyassociated with free radicals Series of concentrations rangedfrom 5 to 320 μgmL in methanol were used )e DPPHscavenging percentage of different extracts as well asascorbic acid and SC50 values (the concentration required toscavenge DPPH by 50) are shown in Figures 2 and 3respectively HcA exhibited the highest antioxidant activityas indicated by its high DPPH scavenging percentage (65)at 320 μgmL and low SC50 values (111plusmn 15 μgmL) Itsactivity can be attributed to its contents of polyphenoliccompounds such as phenolic acids flavonoids and antho-cyanins (ie apigenin C-hexoside-C-pentoside luteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoicacid tyrosol and peonidin derivative and succinic acid)Unfortunately both HdA and HmA displayed moderateantioxidant activities with SC50 1376plusmn 03 and135plusmn 05 μgmL respectively with ascorbic acidSC50 142plusmn 05 μgmL as standard

During this work many major anthocyanins were de-tected in LC-MS analysis of HcA (such as peonidin dir-hamnoside peonidin derivative and peonidin-3-(p-

10 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

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60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

Table 1 Continued

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

33 4-Hydroxybenzoic acid 799 139 121 111 105 97 93 79 radic 1

34 Peonidin-3-(p-coumaroyl-glucoside) 827 609 301(M+H-146-162)+ radic 23

35 Kaempferol-3-(p-coumaryl-glucoside) 827 593 593 447(MminusH-146)minus 327

285(MminusH-146-162)minus radic 24

36 Apigenin C-hexoside-C-pentoside isomer 832 563

545 (MminusH-18)minus 473 (MminusH-90)minus443 (MminusH-120)minus 431 (MminusH-132)minus 353 (MminusH-120-90)minus 341(MminusH-132-90)minus 311 (MminusH-132-

120)minus

radic radic 13

37 Kaempferol-3-O-glucoside 841 447 285 radic 6 8

38 Gamma-eudesmolrhamnoside derivative 847 577 439 397 379 367 349 322 293 249

197 127 radic 1

39 L-ascorbic acid isomer 855 177 133 129 127 113 103 101 57 radic 1

40Peonidin-3-(p-

coumaroyl-glucoside)isomer

869 609 301(M+H-146-162)+ radic radic 23

41 Vicenin isomer 889 593 473 (MminusH-120)minus 395 383 (M-120-90)minus 338 327 298 radic 15 25

42 Apiin isomer 898 565 433 (M+H-132)+ 413 271(M+H-162-132)+ radic radic 5

43 Apigenin C-hexoside-C-pentoside isomer 898 563

545 (MminusH-18)minus 443 (MminusH-120)minus431 (MminusH-132)minus 353 (MminusH-120-90)minus 341 (MminusH-132-90)minus 311

(MminusH-132-120)minusradic 6 13

44 Unknown 904 319 239 204 195 97 radic mdash

45Kaempferol-3-(p-

coumaryl-glucoside)isomer

912 593 447 (MminusH-146)minus 327 285(MminusH-146-162)minus radic 24

46Peonidin-3-(p-

coumaroyl-glucoside)isomer

937 609 609 579 463 (M+H-146)+ 301(peonidin) (M+H-146-162)+ radic radic radic 23

47Methyl apigenin-C-rhamnoside-O-glucosidelowast

938 593 327 (M+H-162-104)+ 297 (M+H-162-134)+ 285 radic mdash

48 Diosmetin-7-O-rutinoside 950 607 607 577 461 (MminusH-146)minus 299

(MminusH-146-162)minus radic radic 26

49 Luteolin hexoside 955 449 287 (M+H-162)+ radic 5

50 Luteolin C-hexoside-C-pentoside isomer 963 579 369 (MminusH-120-90)minus 339 (MminusH-

120-120)minus 322 281 259 124 radic 13

51 Schaftoside isomer 994 565529 511 475 457 445 427

415(M+H-150)+ 409 391 379 361349 337 325 307 295 273

radic 1 22

52 Succinic acid isomer 1001 117 99 radic 1053 L-ascorbic acid isomer 1036 177 133 129 127 113 103 101 57 radic 154 Peonidin dirhamnosidelowast 1037 593 301 (M+H-146-146)+ radic mdash55 3-Hydroxybenzoic acid 1049 139 105 97 93 79 radic 1

56 Acacetin-rhamnoglucoside 1061 591 283 (MminusH-Rut)minus 103 58 radic radic 18

57 Kaempferol dimethylether dipentosidelowast 1068 579 579 315 (M+H-132-132)+ radic mdash

58 Peonidin glucosideferuloyl glucuronide 1082 815 463 (M+H-352 feruloyl

glucuronide)+ radic 7

59 3-Hydroxybenzoic acidisomer 1094 139 105 97 93 79 radic 1

60 N-feruloyltyramineisomer 1096 314 235 218 181 177 145 121 103 93

45 radic 1

61 Unknown 1107 877 877 813 783 557 radic mdash62 Apigenin-O-dihexoside 1138 593 269 (MminusH-162-162)minus radic 9

6 Journal of Chemistry

Table 1 Continued

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

63 Isorhamnetin-3-O-rutinoside 1159 623 315 (MminusH-Rut)minus 300 271 radic 27

64 Peonidin dirhamnosideisomerlowast 1169 593 447 (M+H-146)+ 301 (M+H-146-

146)+ radic radic mdash

65 Acacetin-rhamnoglucoside isomer 1169 591 283 (MminusH-Rut)minus radic radic 18

66 Succinic acid isomer 1178 117 99 radic 1067 Tyrosol isomer 1205 121 93 77 radic 3

68 Diosmetin rhamnosideferuloyl glucuronidelowast 1246 797 445 (MminusH-176-176)minus 299 (MminusH-

176-176-146)minus 237 205 radic mdash

69 Apiin isomer 1287 563 269 radic 1870 Succinic acid isomer 1290 117 99 radic 10

71 Peonidin dipentosidelowast 1313 565 565 301(M+H-132-132)+ 336 265195 135 91 45 radic mdash

72 Methyl gallate 1325 185 168 124 radic 573 Succinic acid isomer 1365 117 99 radic 10

74 Delphinidin-3-arabinoside derivative 1540 799 435 303 (M+H-364-132)+ radic 7 23

75Hydroxy-

octadecadienoic acidderivative

1557 593295 (MminusH-298)minus 277 (MminusH-298-18)minus 251(MminusH-298-18-CO2)minus 195

171radic 28

76 Syringic acid derivative 1583 377 197 radic 2177 22-Dehydrocholesterol 1600 393 393 273 173 171 130 125 radic 178 Malvidin derivativelowast 1672 565 331 147 radic mdash

79 Kaempferide derivativeisomer 1740 623 299 (MminusH-2 Glc)minus 163 radic 29

80 Unknown 1742 274 274 256 (M+H-18)+ 210 (M+H-18-46)+ 111 105 102 88 71 radic radic mdash

81 Unknown 1754 399 267 253 227 radic mdash82 L-ascorbic acid isomer 1782 177 133 129 127 113 103 101 57 radic 183 Succinic acid isomer 2149 117 117 99 radic 10

84 Apiin isomer 2189 565 433 (M+H-132)+ 413 271 (M+H-162-132)+ radic 5

85 Methyl gallate derivative 2306 325 183 (MminusH-142)minus radic 2986 Gallocatechin derivative 2448 561 305 radic 2187 Succinic acid isomer 2496 117 99 radic 1088 Unknown 2501 515 515 353 331 313 239 radic mdash

89 4-Hydroxybenzoic acidisomer 2528 139 121 111 105 97 93 79 radic radic 1

90 Succinic acid isomer 2611 117 117 101 99 radic radic 10

91 Unknown 2666 805 615 606 598 413 391 279 167 149113 radic mdash

92 Luteolin-7-glucuronide-3acute4prime-pentoside 2677 593 461 (MminusH-132)minus 285 (MminusH-132-

176)minus 169 radic 11

93 Peonidin dipentosideisomerlowast 2745 565 547 301 (M+H-132-132)+ 259 219

133 113 85 45 radic radic mdash

94 Tyrosol isomer 2755 121 93 77 radic 395 L-ascorbic acid isomer 2761 177 133 129 127 113 103 101 57 radic 1

96Delphinidin-3-(p-

coumaroyl-glucoside)derivative

2836 799 611 303 (M+H-188-308)+ radic 23

97 Epicatechin derivative 2891 678 289 radic 6

98 Kaempferol-8C-glucosidelowast 2940 449 329 (M+H-120)+ 299 (M+H-150)+ radic mdash

99 Malvidin 3-O-glucosidederivative 2983 871 493 331 radic 8

100 Unknown 3000 871 593 552 369 260 105 radic mdash101 L-ascorbic acid isomer 3041 177 133 129 127 113 103 101 57 radic radic 1102 Peonidin derivativelowast 3065 799 648 (M+H-galloyl)+ 301 (peonidin) radic mdash103 Succinic acid isomer 3152 117 117 99 radic 101 [13] 2 [14] 3 [15] 4 [16] 5 [17] 6 [18] 7 [19] 8 [20] 9 [21] 10 [22] 11 [23] 12 [24] 13 [25] 14 [26] 15 [27] 16 [28] 17 [29] 18 [30] 19 [31] 20[32] 21 [33] 22 [34] 23 [35] 24 [28] 25 [26] 26 [36] 27 [37] 28 [38] 29 [39] lowastTentative identified Rt retention time Rut rutinose Glc glucose HdAHibiscus deflersii HcA H calyphyllus HmA H micranthus

Journal of Chemistry 7

arabinose unit (riboside) and at mz 445 [M+H-120]+corresponding to the loss of a glucose unit Further frag-mentations of the sugar moieties were observed to generate[M+H-186]+ ions at mz 379 [M+H-240]+ ions (formingthe base peaks) atmz 325 and [M+H-270]+ ions atmz 295[34]

Diosmetin C-glucoside C-pentoside (25) showed apseudomolecular ion peak [MminusH]minus at mz 593 )e typicalfragmentation pathway ofC-glycosylated flavonoids resultedin the formation of ions at mz 413 [MminusH-90-90]minus cor-responding to the loss of an pentosidemoiety and atmz 383[MminusH-120-90]minus corresponding to the loss of a glucosemoiety

313 Flavonoid OC-Glycosides Flavonoid OC-glycosidesare distinguished by the lack of the aglycone ion Only theprecursor ion [MminusH]minus is detected in addition to the ions

resulting from the interglycosidic linkage cleavage includingthe key fragmentation ions at [MminusH-120]minus and [MminusH-120-162]minus or [MminusH-90- 146]minus with or without [MminusH-18]minusOn the basis of these rules compound 47 (Rt 938min) witha [M+H]+ ion at mz 593 was tentatively identified asmethyl apigenin-C-rhamnoside-O-glucoside as it showedMS2 fragments atmz 327 [M+H-162-104]+ correspondingto loss of O-glucoside moiety (minus162Da) mz 297 [M+H-162-134]+ corresponding to additional loss of C-rhamnosylmoiety (minus134Da) and mz 285 (methyl apigenin)

314 Flavonoid O-Glycosides Fragmentation pattern offlavonoid O-glycosides is characterized by the loss of thesugar moiety [41 42] and as a result a deprotonatedaglycone ion is yielded in MS2 Compound 21 (Rt 725min)with [MminusH]minus ion peak at mz 593 was tentatively identifiedas kaempferol-3-O-rutinosideluteolin-7-O-rutinoside

HdA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 2 scan Es-

2021

1914

45

6365

695670

68

73 75 79

8586 103

43

4827

2

36

(a)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 1 scan Es+

HdA(+)

1

3 7

1213

23 24 40

4239

29

4649

58

64

5471

7478

81

89

91

93

99

101

(b)

HcA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20182 scan Es-

18

2225 37 52

62

61 70

76 8387

90

3850

66

318

(c)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20181 scan Es+

HcA(+) 102

10198

9697

89 94

828072

67

6459

53

5551

404647

3334

2628

16111

6

(d)

HmA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

92

15

2

30

36 41

48

56443517

HMA12-Jun-2018 2 scan Es-

(e)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HMA12-Jun-2018 2 scan Es+

HmA(+)

110 32

9

46 57 60 77 80 84

889395

100

(f )

Figure 1 UPLS-ESI-MS chromatograms of aqueous extracts of aerial parts of H deflersii (HdA) H calyphyllus (HcA) and H micranthus(HmA) in negative (minus) and positive (+) ionization modes

8 Journal of Chemistry

based on the ESI mass data and the 308Da neutral lossleaving fragment ion at mz 285 corresponding to kaemp-ferolluteolin aglycones [29 37] Diosmetin-7-O-glucuro-nide-3prime-O-pentoside (15) (Rt 631min) produced a [MminusH]minusion atmz 607 with MS2 fragments atmz 475 [MminusH-132]minuscorresponding to loss of pentosyl moiety and 299 [MminusH-132-176]minus corresponding to loss of additional glucuronylmoiety [23]

Apigenin-7-O-apiosylglucoside (apiin) (22) and its iso-mer (69) were tentatively identified from the MS profile ofpeak at Rt of 729 and 1287min with [MminusH]minus at mz 563and MSMS base peak fragment ion at mz 269 which gavethe loss of 294Da (162 + 132Da) (apiosylglucoside moiety)[17 30] Furthermore other three apiin isomers (23 42 and84) were tentatively identified from the MS profile of peaksat retention time of 731 898 and 2189min with [M+H]+at mz 565 and MS2 base peak fragment ion at mz 433[M+H-132]+ corresponding to apiosyl moiety loss and atmz 271 which indicated the loss of 294Da (162 + 132Da)(apiosylglucoside moiety) [17] Compounds 35 and 45 with[MminusH]minus ion at mz 593 were tentatively identified askaempferol-3-O-(p-coumaryl glucoside) )e fragmentationpattern showed MS2 fragments at mz 447 [MminusH-146]minuscorresponding to loss of rhamnose (minus146Da) moiety and atmz 285 [MminusH-146-162]minus corresponding to additional lossof glucose moiety (minus162Da) )ey both gave the product ionat mz 285 corresponding to the kaempferol aglycone [28]Compound 37 (Rt 841min) with [MminusH]minus at mz 447 wastentatively identified as kaempferol-3-O-glucoside fromESIminus mass data and the neutral loss of 162Da for glucosylmoiety [18 20] By the same manner compound 49 (Rt955min) was identified as luteolin hexoside ([M+H]+ ionat mz 449 and MS2 ion at mz 287 [M+H-glc]+))eprecursor ion of compound 48 was detected at mz 607[MminusH]minus and its characteristic MS2 fragment ion at mz 299[MminusH-rut]minus related to deprotonated diosmetin andconsequently it was tentatively identified as diosmetin-7-O-rutinoside [36] Additionally the precursor ion of com-pounds 56 and 65 was detected at mz 591 [MminusH]minus and itscharacteristic MS2 fragment ion at mz 283 [MminusH-rut]minusrelated to deprotonated acacetin and consequently it wastentatively identified as acacetin-rhamnoglucoside and itspositional isomers [30] Compound 57 was tentativelyidentified as kaempferol dimethyl ether dipentoside (Rt1068min) as it produced a [M+H]+ ion at mz 579 withMS2 fragments atmz 315 [M+H-132-132]+ correspondingto loss of dipentosyl moieties Compound 62 with a[MminusH]minus at mz 593 and MS2 ions at mz 269 [MminusH-162-162]minus was tentatively identified as apigenin-O-dihexoside[21] Compound 63 [MminusH]minus ion at mz 623 was tentativelyidentified as isorhamnetin-3-O-rutinoside )e fragmenta-tion patterns showed MS2 fragments at mz 315 [MminusH-308]minus (isorhamnetin) corresponding to loss of rutinose(minus308Da) moiety [37] Compound 68 with [MminusH]minus at mz797 was tentatively identified as diosmetin rhamnosideferuloyl glucuronide as it showed MS2 fragments at mz 445[MminusH-176-176]minus corresponding to loss of feruloyl(minus176Da) and glucuronide (-176Da) moieties and 299[MminusH-176-176-146]minus corresponding to additional loss of

rhamnose moiety (minus146Da) According to [39] kaempferidederivative (79) was tentatively identified by its molecular ion[MminusH]minusat mz 623 and fragmentation pattern containingspecific ion at mz 299 [MminusH-162-162]minus corresponding toloss of two glucose moieties and ion at mz 163 Accordingto [23] the diglycosylated flavonoid (92) was detected as itpossessed a pseudomolecular ion [MminusH]minus at mz 593 withMS2 fragment ions at mz 461 [MminusH-132]minus correspondingto pentosyl moiety loss as well as other less abundant ion atmz 285 [MminusH- 132-176]minus resulting from the loss of glu-curonyl moiety (minus176Da) )is compound was identified asluteolin-7-glucuronide-3rsquo4rsquo-pentoside

315 Flavonoid Aglycones Patuletin (6-methoxy quercetin)(13) was identified by comparing its MS2 fragmentationpattern with the previously reported data [21] Compound31 with [MminusH]ndash ion atmz 737 andMS2 fragment ion atmz285 was determined to be luteolin derivative [30 33] whilecompound 29 which gave a [M+H]+ ion at mz 799 andMS2 fragment ion at mz 285 was identified tentatively asmethyl apigenin derivative Gallocatechin derivative (86)[33] and epicatechin derivative (97) [18] were recognized bycomparing their MS2 fragmentation pattern with the pre-viously published data

316 Anthocyanins A total of 9 anthocyanin derivativeshave been detected in Hibiscus Compounds 34 40 46 5458 64 71 93 and 102 were tentatively identified as peonidinderivatives )us mass data of compound 34 and its isomers(40 and 46) were proposed to be peonidin-3-(p-coumaroyl-glucoside) as they showed a molecular ion peak [M+H]+ atmz 609 and MS2 fragment ions at mz 301 [M+H-162-146]+ )ese were indicative of a peonidin with glucose(162Da) and coumaroyl (146Da) moieties [35]

Compounds 71 and 93 produced a [M+H]+ ion at mz565 with MS2 fragments atmz 301 [M+H-132-132]+)esewere indicative of a peonidin with two pentoside moieties(264Da) and were tentatively identified as peonidindipentoside while compounds 54 and 64 produced [M+H]+ion at mz 593 with MS2 fragments at mz 301 [M+H-146-146]+ (peonidin))ese were indicative of peonidin with tworhamnoside moieties (292Da) and were tentatively identi-fied as peonidin dirhamnoside Compound 58 was tenta-tively identified as peonidin glucoside feruloyl glucuronideas it produced a [M+H]+ ion atmz 815 with MS2 fragmentions atmz 463 [M+H-176-176]+ corresponding to the lossof a feruloyl (minus176Da) and glucuronide (minus176Da) moietiesand at mz 301 [M+H-176-176-162]+ (peonidin) corre-sponding to the loss of a glucose unit Compound 102 wastentatively identified as peonidin derivative as it showed a[M+H]+ ion at mz 799 with MS2 fragment ions at mz 648[M+H-151]+ corresponding to the loss of a galloyl(minus151Da) moiety and at mz 301 (peonidin)

)e mass spectra of compounds 11 and 24 showed theirprotonated aglycon ions [M+H]+ to be mz 287 corre-sponding to cyanidin )ese protonated aglycon ions wereall formed by loss of a sugar moiety with 162 units from their[M+H]+ indicating that they are anthocyanidin

Journal of Chemistry 9

monoglucosides or monogalactosides )is suggests thepresence of cyanidin 3-O-galactoside (11) and cyanidin 3-O-glucoside (24) )e mz values of compound 18 detected atmz 579 in the negative ion mode are similar to those ofcyanidin 3-O-sambubioside [24]

Compounds 28 74 and 96 were tentatively identified asdelphinidin derivatives Compound 28 showed a molecularion peak [M+H]+ at mz 565 and MS2 fragment ions at mz303 [M+H-176-86]+ )ese were indicative of a delphinidinwith glucuronide (176Da) and malonyl (86Da) moieties)is compound was suggested to be delphinidin malonylglucuronide

Compound 74 showed a molecular ion peak [M+H]+ atmz 799 and MS2 fragment ions at mz 435 [M+H-364]+(delphinidin-3-arabinoside) andmz 303 [M+H-364-132]+corresponding to loss of arabinose moiety )ese were in-dicative of a delphinidin-3-arabinoside derivative [19 35]Compound 96 showed a molecular ion peak [M+H]+ atmz799 and MS2 fragment ions at mz 611 [M+H-188]+ and atmz 303 [M+H-188-308]+ )ese were indicative of a del-phinidin with coumaroyl-glucoside moieties (minus308Da) [43])is compound was proposed to be delphinidin-3-(p-cou-maroyl-glucoside) derivative [35]

Compound 78 was tentatively identified as malvidinderivative as it showed a protonated aglycone ion peak atmz 331 corresponding to malvidin [43] Compound 99 pro-duced a [M+H]+ ion at mz 871 with MS2 fragments at mz493 corresponding to malvidin-3-O-glucoside [20] and atmz 331 [M+H-378-162]+ corresponding to malvidin [43]with one glucose moiety (162Da) and was identified asmalvidin 3-O-glucoside derivative [20]

317 Fatty Acid Derivatives For compound 75 a pseu-domolecular ion peak [MminusH]minus at mz 593 was observedwith MS2 fragment ions at mz 295 [MminusH-298]minus 277[MminusH-298-H2O]minus 251 [MminusH-298-CO2]minus 195 and 171were detected suggesting the presence of hydroxy-octade-cadienoic acids derivative [38]

318 Miscellaneous Compounds For L-ascorbic acid (1) andits isomers (9 12 16 39 53 82 95 and 101) a protonatedpseudomolecular ion was observed at mz 177 and MS2 ionat mz 133 (M+H-44)+ [13] Moreover compound 5 wassuggested to be sucrose (MS1 atmz 341 [MminusH]minus MS2 atmz 179 [MminusH-glc]minus 161 131) [16] It was previously re-ported that L-ascorbic acid and sucrose were identified inHibiscus species as H sabdariffa contains higher amount ofascorbic acid compared to orange and mango [44ndash46]

Oleuropein (2) showed a deprotonated pseudomolecularion at mz 539 (MS2 at mz 377 [MminusH-glc]minus and 307[MminusH-glc-C4H6O]minus) [14] It was previously reported thatoleuropein was identified inHibiscus [47] Succinic acid (14)and its isomers (17 52 66 70 73 83 87 90 and 103) wererecognized by comparing their MS2 fragmentation patternwith the reported data [22] )ey showed a deprotonatedmolecular ion atmz 117 and an intense fragment atmz 99attributed to the loss of water molecule [22] while com-pound 3 showed a protonated molecular ion atmz 119 with

MS2 intense fragment ion atmz 101 [M+H-18]+ attributedto the loss of water molecule (minus18Da) thus it was tentativelyidentified as succinic acid

Hydroxycitric acid derivative (6) and butein chalcone (7)were identified by comparison with published data [13 17]respectively Hydroxycitric acid is the principal organic acidfound in the calyces ofHibiscus according to [48] Moreovercompound 38 was identified as a sesquiterpenoid derivativegamma-eudesmol rhamnoside derivative (MS1 at mz 577[MminusH]minus MS2 at mz 439 [MminusH-138]minus corresponding togamma-eudesmol 293 [MminusH-138-146]minus corresponding toloss of rhamnose moiety (minus146Da)) [13] N-feruloyltyr-amine (10) its isomer (60) and 22-dehydrocholesterol (77)were recognized by comparing their MSMS fragmentationpattern with the reported data [13]

319 Unidentified Compounds Finally seven compounds(44 61 80 81 88 91 and 100) with the pseudomolecularions [MminusH]minus at mz 319 and 877 and [M+H]+ at mz 274274 399 515 805 and 871 respectively could not beidentified Furthermore of the 103 compounds identified 7compounds are unidentified 46 compounds in HdA 42compounds in HcA and 25 compounds in HmA have beenreported in the present study (Table 1) In conclusioncombination of accurate mass measurement and LC abilityto separate isomeric compounds can be considered apowerful tool in the identification of polyphenol diversity inthree species of the Hibiscus genus even in the absence ofstandards but the stereochemical differentiation betweenthe large number of isomers that were found in our speciesfor example isomers of luteolin C-hexoside-C-pentosideapigenin C-hexoside-C-pentoside and cyanidin rutinosidewas not possible with our methodology

32AntioxidantActivity It is well known that plant phenolsand flavonoids in general are highly effective free radicalscavengers and antioxidants )us they are used for theprevention and cure of various disorders which are mainlyassociated with free radicals Series of concentrations rangedfrom 5 to 320 μgmL in methanol were used )e DPPHscavenging percentage of different extracts as well asascorbic acid and SC50 values (the concentration required toscavenge DPPH by 50) are shown in Figures 2 and 3respectively HcA exhibited the highest antioxidant activityas indicated by its high DPPH scavenging percentage (65)at 320 μgmL and low SC50 values (111plusmn 15 μgmL) Itsactivity can be attributed to its contents of polyphenoliccompounds such as phenolic acids flavonoids and antho-cyanins (ie apigenin C-hexoside-C-pentoside luteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoicacid tyrosol and peonidin derivative and succinic acid)Unfortunately both HdA and HmA displayed moderateantioxidant activities with SC50 1376plusmn 03 and135plusmn 05 μgmL respectively with ascorbic acidSC50 142plusmn 05 μgmL as standard

During this work many major anthocyanins were de-tected in LC-MS analysis of HcA (such as peonidin dir-hamnoside peonidin derivative and peonidin-3-(p-

10 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

Table 1 Continued

Compno Compound name Rt

(min)

[Mminus

H]minus

(mz)

[M+H]+

(mz) MS2 fragments (mz) HdA HcA HmA References

63 Isorhamnetin-3-O-rutinoside 1159 623 315 (MminusH-Rut)minus 300 271 radic 27

64 Peonidin dirhamnosideisomerlowast 1169 593 447 (M+H-146)+ 301 (M+H-146-

146)+ radic radic mdash

65 Acacetin-rhamnoglucoside isomer 1169 591 283 (MminusH-Rut)minus radic radic 18

66 Succinic acid isomer 1178 117 99 radic 1067 Tyrosol isomer 1205 121 93 77 radic 3

68 Diosmetin rhamnosideferuloyl glucuronidelowast 1246 797 445 (MminusH-176-176)minus 299 (MminusH-

176-176-146)minus 237 205 radic mdash

69 Apiin isomer 1287 563 269 radic 1870 Succinic acid isomer 1290 117 99 radic 10

71 Peonidin dipentosidelowast 1313 565 565 301(M+H-132-132)+ 336 265195 135 91 45 radic mdash

72 Methyl gallate 1325 185 168 124 radic 573 Succinic acid isomer 1365 117 99 radic 10

74 Delphinidin-3-arabinoside derivative 1540 799 435 303 (M+H-364-132)+ radic 7 23

75Hydroxy-

octadecadienoic acidderivative

1557 593295 (MminusH-298)minus 277 (MminusH-298-18)minus 251(MminusH-298-18-CO2)minus 195

171radic 28

76 Syringic acid derivative 1583 377 197 radic 2177 22-Dehydrocholesterol 1600 393 393 273 173 171 130 125 radic 178 Malvidin derivativelowast 1672 565 331 147 radic mdash

79 Kaempferide derivativeisomer 1740 623 299 (MminusH-2 Glc)minus 163 radic 29

80 Unknown 1742 274 274 256 (M+H-18)+ 210 (M+H-18-46)+ 111 105 102 88 71 radic radic mdash

81 Unknown 1754 399 267 253 227 radic mdash82 L-ascorbic acid isomer 1782 177 133 129 127 113 103 101 57 radic 183 Succinic acid isomer 2149 117 117 99 radic 10

84 Apiin isomer 2189 565 433 (M+H-132)+ 413 271 (M+H-162-132)+ radic 5

85 Methyl gallate derivative 2306 325 183 (MminusH-142)minus radic 2986 Gallocatechin derivative 2448 561 305 radic 2187 Succinic acid isomer 2496 117 99 radic 1088 Unknown 2501 515 515 353 331 313 239 radic mdash

89 4-Hydroxybenzoic acidisomer 2528 139 121 111 105 97 93 79 radic radic 1

90 Succinic acid isomer 2611 117 117 101 99 radic radic 10

91 Unknown 2666 805 615 606 598 413 391 279 167 149113 radic mdash

92 Luteolin-7-glucuronide-3acute4prime-pentoside 2677 593 461 (MminusH-132)minus 285 (MminusH-132-

176)minus 169 radic 11

93 Peonidin dipentosideisomerlowast 2745 565 547 301 (M+H-132-132)+ 259 219

133 113 85 45 radic radic mdash

94 Tyrosol isomer 2755 121 93 77 radic 395 L-ascorbic acid isomer 2761 177 133 129 127 113 103 101 57 radic 1

96Delphinidin-3-(p-

coumaroyl-glucoside)derivative

2836 799 611 303 (M+H-188-308)+ radic 23

97 Epicatechin derivative 2891 678 289 radic 6

98 Kaempferol-8C-glucosidelowast 2940 449 329 (M+H-120)+ 299 (M+H-150)+ radic mdash

99 Malvidin 3-O-glucosidederivative 2983 871 493 331 radic 8

100 Unknown 3000 871 593 552 369 260 105 radic mdash101 L-ascorbic acid isomer 3041 177 133 129 127 113 103 101 57 radic radic 1102 Peonidin derivativelowast 3065 799 648 (M+H-galloyl)+ 301 (peonidin) radic mdash103 Succinic acid isomer 3152 117 117 99 radic 101 [13] 2 [14] 3 [15] 4 [16] 5 [17] 6 [18] 7 [19] 8 [20] 9 [21] 10 [22] 11 [23] 12 [24] 13 [25] 14 [26] 15 [27] 16 [28] 17 [29] 18 [30] 19 [31] 20[32] 21 [33] 22 [34] 23 [35] 24 [28] 25 [26] 26 [36] 27 [37] 28 [38] 29 [39] lowastTentative identified Rt retention time Rut rutinose Glc glucose HdAHibiscus deflersii HcA H calyphyllus HmA H micranthus

Journal of Chemistry 7

arabinose unit (riboside) and at mz 445 [M+H-120]+corresponding to the loss of a glucose unit Further frag-mentations of the sugar moieties were observed to generate[M+H-186]+ ions at mz 379 [M+H-240]+ ions (formingthe base peaks) atmz 325 and [M+H-270]+ ions atmz 295[34]

Diosmetin C-glucoside C-pentoside (25) showed apseudomolecular ion peak [MminusH]minus at mz 593 )e typicalfragmentation pathway ofC-glycosylated flavonoids resultedin the formation of ions at mz 413 [MminusH-90-90]minus cor-responding to the loss of an pentosidemoiety and atmz 383[MminusH-120-90]minus corresponding to the loss of a glucosemoiety

313 Flavonoid OC-Glycosides Flavonoid OC-glycosidesare distinguished by the lack of the aglycone ion Only theprecursor ion [MminusH]minus is detected in addition to the ions

resulting from the interglycosidic linkage cleavage includingthe key fragmentation ions at [MminusH-120]minus and [MminusH-120-162]minus or [MminusH-90- 146]minus with or without [MminusH-18]minusOn the basis of these rules compound 47 (Rt 938min) witha [M+H]+ ion at mz 593 was tentatively identified asmethyl apigenin-C-rhamnoside-O-glucoside as it showedMS2 fragments atmz 327 [M+H-162-104]+ correspondingto loss of O-glucoside moiety (minus162Da) mz 297 [M+H-162-134]+ corresponding to additional loss of C-rhamnosylmoiety (minus134Da) and mz 285 (methyl apigenin)

314 Flavonoid O-Glycosides Fragmentation pattern offlavonoid O-glycosides is characterized by the loss of thesugar moiety [41 42] and as a result a deprotonatedaglycone ion is yielded in MS2 Compound 21 (Rt 725min)with [MminusH]minus ion peak at mz 593 was tentatively identifiedas kaempferol-3-O-rutinosideluteolin-7-O-rutinoside

HdA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 2 scan Es-

2021

1914

45

6365

695670

68

73 75 79

8586 103

43

4827

2

36

(a)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 1 scan Es+

HdA(+)

1

3 7

1213

23 24 40

4239

29

4649

58

64

5471

7478

81

89

91

93

99

101

(b)

HcA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20182 scan Es-

18

2225 37 52

62

61 70

76 8387

90

3850

66

318

(c)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20181 scan Es+

HcA(+) 102

10198

9697

89 94

828072

67

6459

53

5551

404647

3334

2628

16111

6

(d)

HmA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

92

15

2

30

36 41

48

56443517

HMA12-Jun-2018 2 scan Es-

(e)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HMA12-Jun-2018 2 scan Es+

HmA(+)

110 32

9

46 57 60 77 80 84

889395

100

(f )

Figure 1 UPLS-ESI-MS chromatograms of aqueous extracts of aerial parts of H deflersii (HdA) H calyphyllus (HcA) and H micranthus(HmA) in negative (minus) and positive (+) ionization modes

8 Journal of Chemistry

based on the ESI mass data and the 308Da neutral lossleaving fragment ion at mz 285 corresponding to kaemp-ferolluteolin aglycones [29 37] Diosmetin-7-O-glucuro-nide-3prime-O-pentoside (15) (Rt 631min) produced a [MminusH]minusion atmz 607 with MS2 fragments atmz 475 [MminusH-132]minuscorresponding to loss of pentosyl moiety and 299 [MminusH-132-176]minus corresponding to loss of additional glucuronylmoiety [23]

Apigenin-7-O-apiosylglucoside (apiin) (22) and its iso-mer (69) were tentatively identified from the MS profile ofpeak at Rt of 729 and 1287min with [MminusH]minus at mz 563and MSMS base peak fragment ion at mz 269 which gavethe loss of 294Da (162 + 132Da) (apiosylglucoside moiety)[17 30] Furthermore other three apiin isomers (23 42 and84) were tentatively identified from the MS profile of peaksat retention time of 731 898 and 2189min with [M+H]+at mz 565 and MS2 base peak fragment ion at mz 433[M+H-132]+ corresponding to apiosyl moiety loss and atmz 271 which indicated the loss of 294Da (162 + 132Da)(apiosylglucoside moiety) [17] Compounds 35 and 45 with[MminusH]minus ion at mz 593 were tentatively identified askaempferol-3-O-(p-coumaryl glucoside) )e fragmentationpattern showed MS2 fragments at mz 447 [MminusH-146]minuscorresponding to loss of rhamnose (minus146Da) moiety and atmz 285 [MminusH-146-162]minus corresponding to additional lossof glucose moiety (minus162Da) )ey both gave the product ionat mz 285 corresponding to the kaempferol aglycone [28]Compound 37 (Rt 841min) with [MminusH]minus at mz 447 wastentatively identified as kaempferol-3-O-glucoside fromESIminus mass data and the neutral loss of 162Da for glucosylmoiety [18 20] By the same manner compound 49 (Rt955min) was identified as luteolin hexoside ([M+H]+ ionat mz 449 and MS2 ion at mz 287 [M+H-glc]+))eprecursor ion of compound 48 was detected at mz 607[MminusH]minus and its characteristic MS2 fragment ion at mz 299[MminusH-rut]minus related to deprotonated diosmetin andconsequently it was tentatively identified as diosmetin-7-O-rutinoside [36] Additionally the precursor ion of com-pounds 56 and 65 was detected at mz 591 [MminusH]minus and itscharacteristic MS2 fragment ion at mz 283 [MminusH-rut]minusrelated to deprotonated acacetin and consequently it wastentatively identified as acacetin-rhamnoglucoside and itspositional isomers [30] Compound 57 was tentativelyidentified as kaempferol dimethyl ether dipentoside (Rt1068min) as it produced a [M+H]+ ion at mz 579 withMS2 fragments atmz 315 [M+H-132-132]+ correspondingto loss of dipentosyl moieties Compound 62 with a[MminusH]minus at mz 593 and MS2 ions at mz 269 [MminusH-162-162]minus was tentatively identified as apigenin-O-dihexoside[21] Compound 63 [MminusH]minus ion at mz 623 was tentativelyidentified as isorhamnetin-3-O-rutinoside )e fragmenta-tion patterns showed MS2 fragments at mz 315 [MminusH-308]minus (isorhamnetin) corresponding to loss of rutinose(minus308Da) moiety [37] Compound 68 with [MminusH]minus at mz797 was tentatively identified as diosmetin rhamnosideferuloyl glucuronide as it showed MS2 fragments at mz 445[MminusH-176-176]minus corresponding to loss of feruloyl(minus176Da) and glucuronide (-176Da) moieties and 299[MminusH-176-176-146]minus corresponding to additional loss of

rhamnose moiety (minus146Da) According to [39] kaempferidederivative (79) was tentatively identified by its molecular ion[MminusH]minusat mz 623 and fragmentation pattern containingspecific ion at mz 299 [MminusH-162-162]minus corresponding toloss of two glucose moieties and ion at mz 163 Accordingto [23] the diglycosylated flavonoid (92) was detected as itpossessed a pseudomolecular ion [MminusH]minus at mz 593 withMS2 fragment ions at mz 461 [MminusH-132]minus correspondingto pentosyl moiety loss as well as other less abundant ion atmz 285 [MminusH- 132-176]minus resulting from the loss of glu-curonyl moiety (minus176Da) )is compound was identified asluteolin-7-glucuronide-3rsquo4rsquo-pentoside

315 Flavonoid Aglycones Patuletin (6-methoxy quercetin)(13) was identified by comparing its MS2 fragmentationpattern with the previously reported data [21] Compound31 with [MminusH]ndash ion atmz 737 andMS2 fragment ion atmz285 was determined to be luteolin derivative [30 33] whilecompound 29 which gave a [M+H]+ ion at mz 799 andMS2 fragment ion at mz 285 was identified tentatively asmethyl apigenin derivative Gallocatechin derivative (86)[33] and epicatechin derivative (97) [18] were recognized bycomparing their MS2 fragmentation pattern with the pre-viously published data

316 Anthocyanins A total of 9 anthocyanin derivativeshave been detected in Hibiscus Compounds 34 40 46 5458 64 71 93 and 102 were tentatively identified as peonidinderivatives )us mass data of compound 34 and its isomers(40 and 46) were proposed to be peonidin-3-(p-coumaroyl-glucoside) as they showed a molecular ion peak [M+H]+ atmz 609 and MS2 fragment ions at mz 301 [M+H-162-146]+ )ese were indicative of a peonidin with glucose(162Da) and coumaroyl (146Da) moieties [35]

Compounds 71 and 93 produced a [M+H]+ ion at mz565 with MS2 fragments atmz 301 [M+H-132-132]+)esewere indicative of a peonidin with two pentoside moieties(264Da) and were tentatively identified as peonidindipentoside while compounds 54 and 64 produced [M+H]+ion at mz 593 with MS2 fragments at mz 301 [M+H-146-146]+ (peonidin))ese were indicative of peonidin with tworhamnoside moieties (292Da) and were tentatively identi-fied as peonidin dirhamnoside Compound 58 was tenta-tively identified as peonidin glucoside feruloyl glucuronideas it produced a [M+H]+ ion atmz 815 with MS2 fragmentions atmz 463 [M+H-176-176]+ corresponding to the lossof a feruloyl (minus176Da) and glucuronide (minus176Da) moietiesand at mz 301 [M+H-176-176-162]+ (peonidin) corre-sponding to the loss of a glucose unit Compound 102 wastentatively identified as peonidin derivative as it showed a[M+H]+ ion at mz 799 with MS2 fragment ions at mz 648[M+H-151]+ corresponding to the loss of a galloyl(minus151Da) moiety and at mz 301 (peonidin)

)e mass spectra of compounds 11 and 24 showed theirprotonated aglycon ions [M+H]+ to be mz 287 corre-sponding to cyanidin )ese protonated aglycon ions wereall formed by loss of a sugar moiety with 162 units from their[M+H]+ indicating that they are anthocyanidin

Journal of Chemistry 9

monoglucosides or monogalactosides )is suggests thepresence of cyanidin 3-O-galactoside (11) and cyanidin 3-O-glucoside (24) )e mz values of compound 18 detected atmz 579 in the negative ion mode are similar to those ofcyanidin 3-O-sambubioside [24]

Compounds 28 74 and 96 were tentatively identified asdelphinidin derivatives Compound 28 showed a molecularion peak [M+H]+ at mz 565 and MS2 fragment ions at mz303 [M+H-176-86]+ )ese were indicative of a delphinidinwith glucuronide (176Da) and malonyl (86Da) moieties)is compound was suggested to be delphinidin malonylglucuronide

Compound 74 showed a molecular ion peak [M+H]+ atmz 799 and MS2 fragment ions at mz 435 [M+H-364]+(delphinidin-3-arabinoside) andmz 303 [M+H-364-132]+corresponding to loss of arabinose moiety )ese were in-dicative of a delphinidin-3-arabinoside derivative [19 35]Compound 96 showed a molecular ion peak [M+H]+ atmz799 and MS2 fragment ions at mz 611 [M+H-188]+ and atmz 303 [M+H-188-308]+ )ese were indicative of a del-phinidin with coumaroyl-glucoside moieties (minus308Da) [43])is compound was proposed to be delphinidin-3-(p-cou-maroyl-glucoside) derivative [35]

Compound 78 was tentatively identified as malvidinderivative as it showed a protonated aglycone ion peak atmz 331 corresponding to malvidin [43] Compound 99 pro-duced a [M+H]+ ion at mz 871 with MS2 fragments at mz493 corresponding to malvidin-3-O-glucoside [20] and atmz 331 [M+H-378-162]+ corresponding to malvidin [43]with one glucose moiety (162Da) and was identified asmalvidin 3-O-glucoside derivative [20]

317 Fatty Acid Derivatives For compound 75 a pseu-domolecular ion peak [MminusH]minus at mz 593 was observedwith MS2 fragment ions at mz 295 [MminusH-298]minus 277[MminusH-298-H2O]minus 251 [MminusH-298-CO2]minus 195 and 171were detected suggesting the presence of hydroxy-octade-cadienoic acids derivative [38]

318 Miscellaneous Compounds For L-ascorbic acid (1) andits isomers (9 12 16 39 53 82 95 and 101) a protonatedpseudomolecular ion was observed at mz 177 and MS2 ionat mz 133 (M+H-44)+ [13] Moreover compound 5 wassuggested to be sucrose (MS1 atmz 341 [MminusH]minus MS2 atmz 179 [MminusH-glc]minus 161 131) [16] It was previously re-ported that L-ascorbic acid and sucrose were identified inHibiscus species as H sabdariffa contains higher amount ofascorbic acid compared to orange and mango [44ndash46]

Oleuropein (2) showed a deprotonated pseudomolecularion at mz 539 (MS2 at mz 377 [MminusH-glc]minus and 307[MminusH-glc-C4H6O]minus) [14] It was previously reported thatoleuropein was identified inHibiscus [47] Succinic acid (14)and its isomers (17 52 66 70 73 83 87 90 and 103) wererecognized by comparing their MS2 fragmentation patternwith the reported data [22] )ey showed a deprotonatedmolecular ion atmz 117 and an intense fragment atmz 99attributed to the loss of water molecule [22] while com-pound 3 showed a protonated molecular ion atmz 119 with

MS2 intense fragment ion atmz 101 [M+H-18]+ attributedto the loss of water molecule (minus18Da) thus it was tentativelyidentified as succinic acid

Hydroxycitric acid derivative (6) and butein chalcone (7)were identified by comparison with published data [13 17]respectively Hydroxycitric acid is the principal organic acidfound in the calyces ofHibiscus according to [48] Moreovercompound 38 was identified as a sesquiterpenoid derivativegamma-eudesmol rhamnoside derivative (MS1 at mz 577[MminusH]minus MS2 at mz 439 [MminusH-138]minus corresponding togamma-eudesmol 293 [MminusH-138-146]minus corresponding toloss of rhamnose moiety (minus146Da)) [13] N-feruloyltyr-amine (10) its isomer (60) and 22-dehydrocholesterol (77)were recognized by comparing their MSMS fragmentationpattern with the reported data [13]

319 Unidentified Compounds Finally seven compounds(44 61 80 81 88 91 and 100) with the pseudomolecularions [MminusH]minus at mz 319 and 877 and [M+H]+ at mz 274274 399 515 805 and 871 respectively could not beidentified Furthermore of the 103 compounds identified 7compounds are unidentified 46 compounds in HdA 42compounds in HcA and 25 compounds in HmA have beenreported in the present study (Table 1) In conclusioncombination of accurate mass measurement and LC abilityto separate isomeric compounds can be considered apowerful tool in the identification of polyphenol diversity inthree species of the Hibiscus genus even in the absence ofstandards but the stereochemical differentiation betweenthe large number of isomers that were found in our speciesfor example isomers of luteolin C-hexoside-C-pentosideapigenin C-hexoside-C-pentoside and cyanidin rutinosidewas not possible with our methodology

32AntioxidantActivity It is well known that plant phenolsand flavonoids in general are highly effective free radicalscavengers and antioxidants )us they are used for theprevention and cure of various disorders which are mainlyassociated with free radicals Series of concentrations rangedfrom 5 to 320 μgmL in methanol were used )e DPPHscavenging percentage of different extracts as well asascorbic acid and SC50 values (the concentration required toscavenge DPPH by 50) are shown in Figures 2 and 3respectively HcA exhibited the highest antioxidant activityas indicated by its high DPPH scavenging percentage (65)at 320 μgmL and low SC50 values (111plusmn 15 μgmL) Itsactivity can be attributed to its contents of polyphenoliccompounds such as phenolic acids flavonoids and antho-cyanins (ie apigenin C-hexoside-C-pentoside luteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoicacid tyrosol and peonidin derivative and succinic acid)Unfortunately both HdA and HmA displayed moderateantioxidant activities with SC50 1376plusmn 03 and135plusmn 05 μgmL respectively with ascorbic acidSC50 142plusmn 05 μgmL as standard

During this work many major anthocyanins were de-tected in LC-MS analysis of HcA (such as peonidin dir-hamnoside peonidin derivative and peonidin-3-(p-

10 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

arabinose unit (riboside) and at mz 445 [M+H-120]+corresponding to the loss of a glucose unit Further frag-mentations of the sugar moieties were observed to generate[M+H-186]+ ions at mz 379 [M+H-240]+ ions (formingthe base peaks) atmz 325 and [M+H-270]+ ions atmz 295[34]

Diosmetin C-glucoside C-pentoside (25) showed apseudomolecular ion peak [MminusH]minus at mz 593 )e typicalfragmentation pathway ofC-glycosylated flavonoids resultedin the formation of ions at mz 413 [MminusH-90-90]minus cor-responding to the loss of an pentosidemoiety and atmz 383[MminusH-120-90]minus corresponding to the loss of a glucosemoiety

313 Flavonoid OC-Glycosides Flavonoid OC-glycosidesare distinguished by the lack of the aglycone ion Only theprecursor ion [MminusH]minus is detected in addition to the ions

resulting from the interglycosidic linkage cleavage includingthe key fragmentation ions at [MminusH-120]minus and [MminusH-120-162]minus or [MminusH-90- 146]minus with or without [MminusH-18]minusOn the basis of these rules compound 47 (Rt 938min) witha [M+H]+ ion at mz 593 was tentatively identified asmethyl apigenin-C-rhamnoside-O-glucoside as it showedMS2 fragments atmz 327 [M+H-162-104]+ correspondingto loss of O-glucoside moiety (minus162Da) mz 297 [M+H-162-134]+ corresponding to additional loss of C-rhamnosylmoiety (minus134Da) and mz 285 (methyl apigenin)

314 Flavonoid O-Glycosides Fragmentation pattern offlavonoid O-glycosides is characterized by the loss of thesugar moiety [41 42] and as a result a deprotonatedaglycone ion is yielded in MS2 Compound 21 (Rt 725min)with [MminusH]minus ion peak at mz 593 was tentatively identifiedas kaempferol-3-O-rutinosideluteolin-7-O-rutinoside

HdA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 2 scan Es-

2021

1914

45

6365

695670

68

73 75 79

8586 103

43

4827

2

36

(a)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HDA12-Jun-2018 1 scan Es+

HdA(+)

1

3 7

1213

23 24 40

4239

29

4649

58

64

5471

7478

81

89

91

93

99

101

(b)

HcA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20182 scan Es-

18

2225 37 52

62

61 70

76 8387

90

3850

66

318

(c)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HCA12-Jun-20181 scan Es+

HcA(+) 102

10198

9697

89 94

828072

67

6459

53

5551

404647

3334

2628

16111

6

(d)

HmA(ndash)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

92

15

2

30

36 41

48

56443517

HMA12-Jun-2018 2 scan Es-

(e)

100

0

()

250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000Time

HMA12-Jun-2018 2 scan Es+

HmA(+)

110 32

9

46 57 60 77 80 84

889395

100

(f )

Figure 1 UPLS-ESI-MS chromatograms of aqueous extracts of aerial parts of H deflersii (HdA) H calyphyllus (HcA) and H micranthus(HmA) in negative (minus) and positive (+) ionization modes

8 Journal of Chemistry

based on the ESI mass data and the 308Da neutral lossleaving fragment ion at mz 285 corresponding to kaemp-ferolluteolin aglycones [29 37] Diosmetin-7-O-glucuro-nide-3prime-O-pentoside (15) (Rt 631min) produced a [MminusH]minusion atmz 607 with MS2 fragments atmz 475 [MminusH-132]minuscorresponding to loss of pentosyl moiety and 299 [MminusH-132-176]minus corresponding to loss of additional glucuronylmoiety [23]

Apigenin-7-O-apiosylglucoside (apiin) (22) and its iso-mer (69) were tentatively identified from the MS profile ofpeak at Rt of 729 and 1287min with [MminusH]minus at mz 563and MSMS base peak fragment ion at mz 269 which gavethe loss of 294Da (162 + 132Da) (apiosylglucoside moiety)[17 30] Furthermore other three apiin isomers (23 42 and84) were tentatively identified from the MS profile of peaksat retention time of 731 898 and 2189min with [M+H]+at mz 565 and MS2 base peak fragment ion at mz 433[M+H-132]+ corresponding to apiosyl moiety loss and atmz 271 which indicated the loss of 294Da (162 + 132Da)(apiosylglucoside moiety) [17] Compounds 35 and 45 with[MminusH]minus ion at mz 593 were tentatively identified askaempferol-3-O-(p-coumaryl glucoside) )e fragmentationpattern showed MS2 fragments at mz 447 [MminusH-146]minuscorresponding to loss of rhamnose (minus146Da) moiety and atmz 285 [MminusH-146-162]minus corresponding to additional lossof glucose moiety (minus162Da) )ey both gave the product ionat mz 285 corresponding to the kaempferol aglycone [28]Compound 37 (Rt 841min) with [MminusH]minus at mz 447 wastentatively identified as kaempferol-3-O-glucoside fromESIminus mass data and the neutral loss of 162Da for glucosylmoiety [18 20] By the same manner compound 49 (Rt955min) was identified as luteolin hexoside ([M+H]+ ionat mz 449 and MS2 ion at mz 287 [M+H-glc]+))eprecursor ion of compound 48 was detected at mz 607[MminusH]minus and its characteristic MS2 fragment ion at mz 299[MminusH-rut]minus related to deprotonated diosmetin andconsequently it was tentatively identified as diosmetin-7-O-rutinoside [36] Additionally the precursor ion of com-pounds 56 and 65 was detected at mz 591 [MminusH]minus and itscharacteristic MS2 fragment ion at mz 283 [MminusH-rut]minusrelated to deprotonated acacetin and consequently it wastentatively identified as acacetin-rhamnoglucoside and itspositional isomers [30] Compound 57 was tentativelyidentified as kaempferol dimethyl ether dipentoside (Rt1068min) as it produced a [M+H]+ ion at mz 579 withMS2 fragments atmz 315 [M+H-132-132]+ correspondingto loss of dipentosyl moieties Compound 62 with a[MminusH]minus at mz 593 and MS2 ions at mz 269 [MminusH-162-162]minus was tentatively identified as apigenin-O-dihexoside[21] Compound 63 [MminusH]minus ion at mz 623 was tentativelyidentified as isorhamnetin-3-O-rutinoside )e fragmenta-tion patterns showed MS2 fragments at mz 315 [MminusH-308]minus (isorhamnetin) corresponding to loss of rutinose(minus308Da) moiety [37] Compound 68 with [MminusH]minus at mz797 was tentatively identified as diosmetin rhamnosideferuloyl glucuronide as it showed MS2 fragments at mz 445[MminusH-176-176]minus corresponding to loss of feruloyl(minus176Da) and glucuronide (-176Da) moieties and 299[MminusH-176-176-146]minus corresponding to additional loss of

rhamnose moiety (minus146Da) According to [39] kaempferidederivative (79) was tentatively identified by its molecular ion[MminusH]minusat mz 623 and fragmentation pattern containingspecific ion at mz 299 [MminusH-162-162]minus corresponding toloss of two glucose moieties and ion at mz 163 Accordingto [23] the diglycosylated flavonoid (92) was detected as itpossessed a pseudomolecular ion [MminusH]minus at mz 593 withMS2 fragment ions at mz 461 [MminusH-132]minus correspondingto pentosyl moiety loss as well as other less abundant ion atmz 285 [MminusH- 132-176]minus resulting from the loss of glu-curonyl moiety (minus176Da) )is compound was identified asluteolin-7-glucuronide-3rsquo4rsquo-pentoside

315 Flavonoid Aglycones Patuletin (6-methoxy quercetin)(13) was identified by comparing its MS2 fragmentationpattern with the previously reported data [21] Compound31 with [MminusH]ndash ion atmz 737 andMS2 fragment ion atmz285 was determined to be luteolin derivative [30 33] whilecompound 29 which gave a [M+H]+ ion at mz 799 andMS2 fragment ion at mz 285 was identified tentatively asmethyl apigenin derivative Gallocatechin derivative (86)[33] and epicatechin derivative (97) [18] were recognized bycomparing their MS2 fragmentation pattern with the pre-viously published data

316 Anthocyanins A total of 9 anthocyanin derivativeshave been detected in Hibiscus Compounds 34 40 46 5458 64 71 93 and 102 were tentatively identified as peonidinderivatives )us mass data of compound 34 and its isomers(40 and 46) were proposed to be peonidin-3-(p-coumaroyl-glucoside) as they showed a molecular ion peak [M+H]+ atmz 609 and MS2 fragment ions at mz 301 [M+H-162-146]+ )ese were indicative of a peonidin with glucose(162Da) and coumaroyl (146Da) moieties [35]

Compounds 71 and 93 produced a [M+H]+ ion at mz565 with MS2 fragments atmz 301 [M+H-132-132]+)esewere indicative of a peonidin with two pentoside moieties(264Da) and were tentatively identified as peonidindipentoside while compounds 54 and 64 produced [M+H]+ion at mz 593 with MS2 fragments at mz 301 [M+H-146-146]+ (peonidin))ese were indicative of peonidin with tworhamnoside moieties (292Da) and were tentatively identi-fied as peonidin dirhamnoside Compound 58 was tenta-tively identified as peonidin glucoside feruloyl glucuronideas it produced a [M+H]+ ion atmz 815 with MS2 fragmentions atmz 463 [M+H-176-176]+ corresponding to the lossof a feruloyl (minus176Da) and glucuronide (minus176Da) moietiesand at mz 301 [M+H-176-176-162]+ (peonidin) corre-sponding to the loss of a glucose unit Compound 102 wastentatively identified as peonidin derivative as it showed a[M+H]+ ion at mz 799 with MS2 fragment ions at mz 648[M+H-151]+ corresponding to the loss of a galloyl(minus151Da) moiety and at mz 301 (peonidin)

)e mass spectra of compounds 11 and 24 showed theirprotonated aglycon ions [M+H]+ to be mz 287 corre-sponding to cyanidin )ese protonated aglycon ions wereall formed by loss of a sugar moiety with 162 units from their[M+H]+ indicating that they are anthocyanidin

Journal of Chemistry 9

monoglucosides or monogalactosides )is suggests thepresence of cyanidin 3-O-galactoside (11) and cyanidin 3-O-glucoside (24) )e mz values of compound 18 detected atmz 579 in the negative ion mode are similar to those ofcyanidin 3-O-sambubioside [24]

Compounds 28 74 and 96 were tentatively identified asdelphinidin derivatives Compound 28 showed a molecularion peak [M+H]+ at mz 565 and MS2 fragment ions at mz303 [M+H-176-86]+ )ese were indicative of a delphinidinwith glucuronide (176Da) and malonyl (86Da) moieties)is compound was suggested to be delphinidin malonylglucuronide

Compound 74 showed a molecular ion peak [M+H]+ atmz 799 and MS2 fragment ions at mz 435 [M+H-364]+(delphinidin-3-arabinoside) andmz 303 [M+H-364-132]+corresponding to loss of arabinose moiety )ese were in-dicative of a delphinidin-3-arabinoside derivative [19 35]Compound 96 showed a molecular ion peak [M+H]+ atmz799 and MS2 fragment ions at mz 611 [M+H-188]+ and atmz 303 [M+H-188-308]+ )ese were indicative of a del-phinidin with coumaroyl-glucoside moieties (minus308Da) [43])is compound was proposed to be delphinidin-3-(p-cou-maroyl-glucoside) derivative [35]

Compound 78 was tentatively identified as malvidinderivative as it showed a protonated aglycone ion peak atmz 331 corresponding to malvidin [43] Compound 99 pro-duced a [M+H]+ ion at mz 871 with MS2 fragments at mz493 corresponding to malvidin-3-O-glucoside [20] and atmz 331 [M+H-378-162]+ corresponding to malvidin [43]with one glucose moiety (162Da) and was identified asmalvidin 3-O-glucoside derivative [20]

317 Fatty Acid Derivatives For compound 75 a pseu-domolecular ion peak [MminusH]minus at mz 593 was observedwith MS2 fragment ions at mz 295 [MminusH-298]minus 277[MminusH-298-H2O]minus 251 [MminusH-298-CO2]minus 195 and 171were detected suggesting the presence of hydroxy-octade-cadienoic acids derivative [38]

318 Miscellaneous Compounds For L-ascorbic acid (1) andits isomers (9 12 16 39 53 82 95 and 101) a protonatedpseudomolecular ion was observed at mz 177 and MS2 ionat mz 133 (M+H-44)+ [13] Moreover compound 5 wassuggested to be sucrose (MS1 atmz 341 [MminusH]minus MS2 atmz 179 [MminusH-glc]minus 161 131) [16] It was previously re-ported that L-ascorbic acid and sucrose were identified inHibiscus species as H sabdariffa contains higher amount ofascorbic acid compared to orange and mango [44ndash46]

Oleuropein (2) showed a deprotonated pseudomolecularion at mz 539 (MS2 at mz 377 [MminusH-glc]minus and 307[MminusH-glc-C4H6O]minus) [14] It was previously reported thatoleuropein was identified inHibiscus [47] Succinic acid (14)and its isomers (17 52 66 70 73 83 87 90 and 103) wererecognized by comparing their MS2 fragmentation patternwith the reported data [22] )ey showed a deprotonatedmolecular ion atmz 117 and an intense fragment atmz 99attributed to the loss of water molecule [22] while com-pound 3 showed a protonated molecular ion atmz 119 with

MS2 intense fragment ion atmz 101 [M+H-18]+ attributedto the loss of water molecule (minus18Da) thus it was tentativelyidentified as succinic acid

Hydroxycitric acid derivative (6) and butein chalcone (7)were identified by comparison with published data [13 17]respectively Hydroxycitric acid is the principal organic acidfound in the calyces ofHibiscus according to [48] Moreovercompound 38 was identified as a sesquiterpenoid derivativegamma-eudesmol rhamnoside derivative (MS1 at mz 577[MminusH]minus MS2 at mz 439 [MminusH-138]minus corresponding togamma-eudesmol 293 [MminusH-138-146]minus corresponding toloss of rhamnose moiety (minus146Da)) [13] N-feruloyltyr-amine (10) its isomer (60) and 22-dehydrocholesterol (77)were recognized by comparing their MSMS fragmentationpattern with the reported data [13]

319 Unidentified Compounds Finally seven compounds(44 61 80 81 88 91 and 100) with the pseudomolecularions [MminusH]minus at mz 319 and 877 and [M+H]+ at mz 274274 399 515 805 and 871 respectively could not beidentified Furthermore of the 103 compounds identified 7compounds are unidentified 46 compounds in HdA 42compounds in HcA and 25 compounds in HmA have beenreported in the present study (Table 1) In conclusioncombination of accurate mass measurement and LC abilityto separate isomeric compounds can be considered apowerful tool in the identification of polyphenol diversity inthree species of the Hibiscus genus even in the absence ofstandards but the stereochemical differentiation betweenthe large number of isomers that were found in our speciesfor example isomers of luteolin C-hexoside-C-pentosideapigenin C-hexoside-C-pentoside and cyanidin rutinosidewas not possible with our methodology

32AntioxidantActivity It is well known that plant phenolsand flavonoids in general are highly effective free radicalscavengers and antioxidants )us they are used for theprevention and cure of various disorders which are mainlyassociated with free radicals Series of concentrations rangedfrom 5 to 320 μgmL in methanol were used )e DPPHscavenging percentage of different extracts as well asascorbic acid and SC50 values (the concentration required toscavenge DPPH by 50) are shown in Figures 2 and 3respectively HcA exhibited the highest antioxidant activityas indicated by its high DPPH scavenging percentage (65)at 320 μgmL and low SC50 values (111plusmn 15 μgmL) Itsactivity can be attributed to its contents of polyphenoliccompounds such as phenolic acids flavonoids and antho-cyanins (ie apigenin C-hexoside-C-pentoside luteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoicacid tyrosol and peonidin derivative and succinic acid)Unfortunately both HdA and HmA displayed moderateantioxidant activities with SC50 1376plusmn 03 and135plusmn 05 μgmL respectively with ascorbic acidSC50 142plusmn 05 μgmL as standard

During this work many major anthocyanins were de-tected in LC-MS analysis of HcA (such as peonidin dir-hamnoside peonidin derivative and peonidin-3-(p-

10 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

based on the ESI mass data and the 308Da neutral lossleaving fragment ion at mz 285 corresponding to kaemp-ferolluteolin aglycones [29 37] Diosmetin-7-O-glucuro-nide-3prime-O-pentoside (15) (Rt 631min) produced a [MminusH]minusion atmz 607 with MS2 fragments atmz 475 [MminusH-132]minuscorresponding to loss of pentosyl moiety and 299 [MminusH-132-176]minus corresponding to loss of additional glucuronylmoiety [23]

Apigenin-7-O-apiosylglucoside (apiin) (22) and its iso-mer (69) were tentatively identified from the MS profile ofpeak at Rt of 729 and 1287min with [MminusH]minus at mz 563and MSMS base peak fragment ion at mz 269 which gavethe loss of 294Da (162 + 132Da) (apiosylglucoside moiety)[17 30] Furthermore other three apiin isomers (23 42 and84) were tentatively identified from the MS profile of peaksat retention time of 731 898 and 2189min with [M+H]+at mz 565 and MS2 base peak fragment ion at mz 433[M+H-132]+ corresponding to apiosyl moiety loss and atmz 271 which indicated the loss of 294Da (162 + 132Da)(apiosylglucoside moiety) [17] Compounds 35 and 45 with[MminusH]minus ion at mz 593 were tentatively identified askaempferol-3-O-(p-coumaryl glucoside) )e fragmentationpattern showed MS2 fragments at mz 447 [MminusH-146]minuscorresponding to loss of rhamnose (minus146Da) moiety and atmz 285 [MminusH-146-162]minus corresponding to additional lossof glucose moiety (minus162Da) )ey both gave the product ionat mz 285 corresponding to the kaempferol aglycone [28]Compound 37 (Rt 841min) with [MminusH]minus at mz 447 wastentatively identified as kaempferol-3-O-glucoside fromESIminus mass data and the neutral loss of 162Da for glucosylmoiety [18 20] By the same manner compound 49 (Rt955min) was identified as luteolin hexoside ([M+H]+ ionat mz 449 and MS2 ion at mz 287 [M+H-glc]+))eprecursor ion of compound 48 was detected at mz 607[MminusH]minus and its characteristic MS2 fragment ion at mz 299[MminusH-rut]minus related to deprotonated diosmetin andconsequently it was tentatively identified as diosmetin-7-O-rutinoside [36] Additionally the precursor ion of com-pounds 56 and 65 was detected at mz 591 [MminusH]minus and itscharacteristic MS2 fragment ion at mz 283 [MminusH-rut]minusrelated to deprotonated acacetin and consequently it wastentatively identified as acacetin-rhamnoglucoside and itspositional isomers [30] Compound 57 was tentativelyidentified as kaempferol dimethyl ether dipentoside (Rt1068min) as it produced a [M+H]+ ion at mz 579 withMS2 fragments atmz 315 [M+H-132-132]+ correspondingto loss of dipentosyl moieties Compound 62 with a[MminusH]minus at mz 593 and MS2 ions at mz 269 [MminusH-162-162]minus was tentatively identified as apigenin-O-dihexoside[21] Compound 63 [MminusH]minus ion at mz 623 was tentativelyidentified as isorhamnetin-3-O-rutinoside )e fragmenta-tion patterns showed MS2 fragments at mz 315 [MminusH-308]minus (isorhamnetin) corresponding to loss of rutinose(minus308Da) moiety [37] Compound 68 with [MminusH]minus at mz797 was tentatively identified as diosmetin rhamnosideferuloyl glucuronide as it showed MS2 fragments at mz 445[MminusH-176-176]minus corresponding to loss of feruloyl(minus176Da) and glucuronide (-176Da) moieties and 299[MminusH-176-176-146]minus corresponding to additional loss of

rhamnose moiety (minus146Da) According to [39] kaempferidederivative (79) was tentatively identified by its molecular ion[MminusH]minusat mz 623 and fragmentation pattern containingspecific ion at mz 299 [MminusH-162-162]minus corresponding toloss of two glucose moieties and ion at mz 163 Accordingto [23] the diglycosylated flavonoid (92) was detected as itpossessed a pseudomolecular ion [MminusH]minus at mz 593 withMS2 fragment ions at mz 461 [MminusH-132]minus correspondingto pentosyl moiety loss as well as other less abundant ion atmz 285 [MminusH- 132-176]minus resulting from the loss of glu-curonyl moiety (minus176Da) )is compound was identified asluteolin-7-glucuronide-3rsquo4rsquo-pentoside

315 Flavonoid Aglycones Patuletin (6-methoxy quercetin)(13) was identified by comparing its MS2 fragmentationpattern with the previously reported data [21] Compound31 with [MminusH]ndash ion atmz 737 andMS2 fragment ion atmz285 was determined to be luteolin derivative [30 33] whilecompound 29 which gave a [M+H]+ ion at mz 799 andMS2 fragment ion at mz 285 was identified tentatively asmethyl apigenin derivative Gallocatechin derivative (86)[33] and epicatechin derivative (97) [18] were recognized bycomparing their MS2 fragmentation pattern with the pre-viously published data

316 Anthocyanins A total of 9 anthocyanin derivativeshave been detected in Hibiscus Compounds 34 40 46 5458 64 71 93 and 102 were tentatively identified as peonidinderivatives )us mass data of compound 34 and its isomers(40 and 46) were proposed to be peonidin-3-(p-coumaroyl-glucoside) as they showed a molecular ion peak [M+H]+ atmz 609 and MS2 fragment ions at mz 301 [M+H-162-146]+ )ese were indicative of a peonidin with glucose(162Da) and coumaroyl (146Da) moieties [35]

Compounds 71 and 93 produced a [M+H]+ ion at mz565 with MS2 fragments atmz 301 [M+H-132-132]+)esewere indicative of a peonidin with two pentoside moieties(264Da) and were tentatively identified as peonidindipentoside while compounds 54 and 64 produced [M+H]+ion at mz 593 with MS2 fragments at mz 301 [M+H-146-146]+ (peonidin))ese were indicative of peonidin with tworhamnoside moieties (292Da) and were tentatively identi-fied as peonidin dirhamnoside Compound 58 was tenta-tively identified as peonidin glucoside feruloyl glucuronideas it produced a [M+H]+ ion atmz 815 with MS2 fragmentions atmz 463 [M+H-176-176]+ corresponding to the lossof a feruloyl (minus176Da) and glucuronide (minus176Da) moietiesand at mz 301 [M+H-176-176-162]+ (peonidin) corre-sponding to the loss of a glucose unit Compound 102 wastentatively identified as peonidin derivative as it showed a[M+H]+ ion at mz 799 with MS2 fragment ions at mz 648[M+H-151]+ corresponding to the loss of a galloyl(minus151Da) moiety and at mz 301 (peonidin)

)e mass spectra of compounds 11 and 24 showed theirprotonated aglycon ions [M+H]+ to be mz 287 corre-sponding to cyanidin )ese protonated aglycon ions wereall formed by loss of a sugar moiety with 162 units from their[M+H]+ indicating that they are anthocyanidin

Journal of Chemistry 9

monoglucosides or monogalactosides )is suggests thepresence of cyanidin 3-O-galactoside (11) and cyanidin 3-O-glucoside (24) )e mz values of compound 18 detected atmz 579 in the negative ion mode are similar to those ofcyanidin 3-O-sambubioside [24]

Compounds 28 74 and 96 were tentatively identified asdelphinidin derivatives Compound 28 showed a molecularion peak [M+H]+ at mz 565 and MS2 fragment ions at mz303 [M+H-176-86]+ )ese were indicative of a delphinidinwith glucuronide (176Da) and malonyl (86Da) moieties)is compound was suggested to be delphinidin malonylglucuronide

Compound 74 showed a molecular ion peak [M+H]+ atmz 799 and MS2 fragment ions at mz 435 [M+H-364]+(delphinidin-3-arabinoside) andmz 303 [M+H-364-132]+corresponding to loss of arabinose moiety )ese were in-dicative of a delphinidin-3-arabinoside derivative [19 35]Compound 96 showed a molecular ion peak [M+H]+ atmz799 and MS2 fragment ions at mz 611 [M+H-188]+ and atmz 303 [M+H-188-308]+ )ese were indicative of a del-phinidin with coumaroyl-glucoside moieties (minus308Da) [43])is compound was proposed to be delphinidin-3-(p-cou-maroyl-glucoside) derivative [35]

Compound 78 was tentatively identified as malvidinderivative as it showed a protonated aglycone ion peak atmz 331 corresponding to malvidin [43] Compound 99 pro-duced a [M+H]+ ion at mz 871 with MS2 fragments at mz493 corresponding to malvidin-3-O-glucoside [20] and atmz 331 [M+H-378-162]+ corresponding to malvidin [43]with one glucose moiety (162Da) and was identified asmalvidin 3-O-glucoside derivative [20]

317 Fatty Acid Derivatives For compound 75 a pseu-domolecular ion peak [MminusH]minus at mz 593 was observedwith MS2 fragment ions at mz 295 [MminusH-298]minus 277[MminusH-298-H2O]minus 251 [MminusH-298-CO2]minus 195 and 171were detected suggesting the presence of hydroxy-octade-cadienoic acids derivative [38]

318 Miscellaneous Compounds For L-ascorbic acid (1) andits isomers (9 12 16 39 53 82 95 and 101) a protonatedpseudomolecular ion was observed at mz 177 and MS2 ionat mz 133 (M+H-44)+ [13] Moreover compound 5 wassuggested to be sucrose (MS1 atmz 341 [MminusH]minus MS2 atmz 179 [MminusH-glc]minus 161 131) [16] It was previously re-ported that L-ascorbic acid and sucrose were identified inHibiscus species as H sabdariffa contains higher amount ofascorbic acid compared to orange and mango [44ndash46]

Oleuropein (2) showed a deprotonated pseudomolecularion at mz 539 (MS2 at mz 377 [MminusH-glc]minus and 307[MminusH-glc-C4H6O]minus) [14] It was previously reported thatoleuropein was identified inHibiscus [47] Succinic acid (14)and its isomers (17 52 66 70 73 83 87 90 and 103) wererecognized by comparing their MS2 fragmentation patternwith the reported data [22] )ey showed a deprotonatedmolecular ion atmz 117 and an intense fragment atmz 99attributed to the loss of water molecule [22] while com-pound 3 showed a protonated molecular ion atmz 119 with

MS2 intense fragment ion atmz 101 [M+H-18]+ attributedto the loss of water molecule (minus18Da) thus it was tentativelyidentified as succinic acid

Hydroxycitric acid derivative (6) and butein chalcone (7)were identified by comparison with published data [13 17]respectively Hydroxycitric acid is the principal organic acidfound in the calyces ofHibiscus according to [48] Moreovercompound 38 was identified as a sesquiterpenoid derivativegamma-eudesmol rhamnoside derivative (MS1 at mz 577[MminusH]minus MS2 at mz 439 [MminusH-138]minus corresponding togamma-eudesmol 293 [MminusH-138-146]minus corresponding toloss of rhamnose moiety (minus146Da)) [13] N-feruloyltyr-amine (10) its isomer (60) and 22-dehydrocholesterol (77)were recognized by comparing their MSMS fragmentationpattern with the reported data [13]

319 Unidentified Compounds Finally seven compounds(44 61 80 81 88 91 and 100) with the pseudomolecularions [MminusH]minus at mz 319 and 877 and [M+H]+ at mz 274274 399 515 805 and 871 respectively could not beidentified Furthermore of the 103 compounds identified 7compounds are unidentified 46 compounds in HdA 42compounds in HcA and 25 compounds in HmA have beenreported in the present study (Table 1) In conclusioncombination of accurate mass measurement and LC abilityto separate isomeric compounds can be considered apowerful tool in the identification of polyphenol diversity inthree species of the Hibiscus genus even in the absence ofstandards but the stereochemical differentiation betweenthe large number of isomers that were found in our speciesfor example isomers of luteolin C-hexoside-C-pentosideapigenin C-hexoside-C-pentoside and cyanidin rutinosidewas not possible with our methodology

32AntioxidantActivity It is well known that plant phenolsand flavonoids in general are highly effective free radicalscavengers and antioxidants )us they are used for theprevention and cure of various disorders which are mainlyassociated with free radicals Series of concentrations rangedfrom 5 to 320 μgmL in methanol were used )e DPPHscavenging percentage of different extracts as well asascorbic acid and SC50 values (the concentration required toscavenge DPPH by 50) are shown in Figures 2 and 3respectively HcA exhibited the highest antioxidant activityas indicated by its high DPPH scavenging percentage (65)at 320 μgmL and low SC50 values (111plusmn 15 μgmL) Itsactivity can be attributed to its contents of polyphenoliccompounds such as phenolic acids flavonoids and antho-cyanins (ie apigenin C-hexoside-C-pentoside luteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoicacid tyrosol and peonidin derivative and succinic acid)Unfortunately both HdA and HmA displayed moderateantioxidant activities with SC50 1376plusmn 03 and135plusmn 05 μgmL respectively with ascorbic acidSC50 142plusmn 05 μgmL as standard

During this work many major anthocyanins were de-tected in LC-MS analysis of HcA (such as peonidin dir-hamnoside peonidin derivative and peonidin-3-(p-

10 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

monoglucosides or monogalactosides )is suggests thepresence of cyanidin 3-O-galactoside (11) and cyanidin 3-O-glucoside (24) )e mz values of compound 18 detected atmz 579 in the negative ion mode are similar to those ofcyanidin 3-O-sambubioside [24]

Compounds 28 74 and 96 were tentatively identified asdelphinidin derivatives Compound 28 showed a molecularion peak [M+H]+ at mz 565 and MS2 fragment ions at mz303 [M+H-176-86]+ )ese were indicative of a delphinidinwith glucuronide (176Da) and malonyl (86Da) moieties)is compound was suggested to be delphinidin malonylglucuronide

Compound 74 showed a molecular ion peak [M+H]+ atmz 799 and MS2 fragment ions at mz 435 [M+H-364]+(delphinidin-3-arabinoside) andmz 303 [M+H-364-132]+corresponding to loss of arabinose moiety )ese were in-dicative of a delphinidin-3-arabinoside derivative [19 35]Compound 96 showed a molecular ion peak [M+H]+ atmz799 and MS2 fragment ions at mz 611 [M+H-188]+ and atmz 303 [M+H-188-308]+ )ese were indicative of a del-phinidin with coumaroyl-glucoside moieties (minus308Da) [43])is compound was proposed to be delphinidin-3-(p-cou-maroyl-glucoside) derivative [35]

Compound 78 was tentatively identified as malvidinderivative as it showed a protonated aglycone ion peak atmz 331 corresponding to malvidin [43] Compound 99 pro-duced a [M+H]+ ion at mz 871 with MS2 fragments at mz493 corresponding to malvidin-3-O-glucoside [20] and atmz 331 [M+H-378-162]+ corresponding to malvidin [43]with one glucose moiety (162Da) and was identified asmalvidin 3-O-glucoside derivative [20]

317 Fatty Acid Derivatives For compound 75 a pseu-domolecular ion peak [MminusH]minus at mz 593 was observedwith MS2 fragment ions at mz 295 [MminusH-298]minus 277[MminusH-298-H2O]minus 251 [MminusH-298-CO2]minus 195 and 171were detected suggesting the presence of hydroxy-octade-cadienoic acids derivative [38]

318 Miscellaneous Compounds For L-ascorbic acid (1) andits isomers (9 12 16 39 53 82 95 and 101) a protonatedpseudomolecular ion was observed at mz 177 and MS2 ionat mz 133 (M+H-44)+ [13] Moreover compound 5 wassuggested to be sucrose (MS1 atmz 341 [MminusH]minus MS2 atmz 179 [MminusH-glc]minus 161 131) [16] It was previously re-ported that L-ascorbic acid and sucrose were identified inHibiscus species as H sabdariffa contains higher amount ofascorbic acid compared to orange and mango [44ndash46]

Oleuropein (2) showed a deprotonated pseudomolecularion at mz 539 (MS2 at mz 377 [MminusH-glc]minus and 307[MminusH-glc-C4H6O]minus) [14] It was previously reported thatoleuropein was identified inHibiscus [47] Succinic acid (14)and its isomers (17 52 66 70 73 83 87 90 and 103) wererecognized by comparing their MS2 fragmentation patternwith the reported data [22] )ey showed a deprotonatedmolecular ion atmz 117 and an intense fragment atmz 99attributed to the loss of water molecule [22] while com-pound 3 showed a protonated molecular ion atmz 119 with

MS2 intense fragment ion atmz 101 [M+H-18]+ attributedto the loss of water molecule (minus18Da) thus it was tentativelyidentified as succinic acid

Hydroxycitric acid derivative (6) and butein chalcone (7)were identified by comparison with published data [13 17]respectively Hydroxycitric acid is the principal organic acidfound in the calyces ofHibiscus according to [48] Moreovercompound 38 was identified as a sesquiterpenoid derivativegamma-eudesmol rhamnoside derivative (MS1 at mz 577[MminusH]minus MS2 at mz 439 [MminusH-138]minus corresponding togamma-eudesmol 293 [MminusH-138-146]minus corresponding toloss of rhamnose moiety (minus146Da)) [13] N-feruloyltyr-amine (10) its isomer (60) and 22-dehydrocholesterol (77)were recognized by comparing their MSMS fragmentationpattern with the reported data [13]

319 Unidentified Compounds Finally seven compounds(44 61 80 81 88 91 and 100) with the pseudomolecularions [MminusH]minus at mz 319 and 877 and [M+H]+ at mz 274274 399 515 805 and 871 respectively could not beidentified Furthermore of the 103 compounds identified 7compounds are unidentified 46 compounds in HdA 42compounds in HcA and 25 compounds in HmA have beenreported in the present study (Table 1) In conclusioncombination of accurate mass measurement and LC abilityto separate isomeric compounds can be considered apowerful tool in the identification of polyphenol diversity inthree species of the Hibiscus genus even in the absence ofstandards but the stereochemical differentiation betweenthe large number of isomers that were found in our speciesfor example isomers of luteolin C-hexoside-C-pentosideapigenin C-hexoside-C-pentoside and cyanidin rutinosidewas not possible with our methodology

32AntioxidantActivity It is well known that plant phenolsand flavonoids in general are highly effective free radicalscavengers and antioxidants )us they are used for theprevention and cure of various disorders which are mainlyassociated with free radicals Series of concentrations rangedfrom 5 to 320 μgmL in methanol were used )e DPPHscavenging percentage of different extracts as well asascorbic acid and SC50 values (the concentration required toscavenge DPPH by 50) are shown in Figures 2 and 3respectively HcA exhibited the highest antioxidant activityas indicated by its high DPPH scavenging percentage (65)at 320 μgmL and low SC50 values (111plusmn 15 μgmL) Itsactivity can be attributed to its contents of polyphenoliccompounds such as phenolic acids flavonoids and antho-cyanins (ie apigenin C-hexoside-C-pentoside luteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoicacid tyrosol and peonidin derivative and succinic acid)Unfortunately both HdA and HmA displayed moderateantioxidant activities with SC50 1376plusmn 03 and135plusmn 05 μgmL respectively with ascorbic acidSC50 142plusmn 05 μgmL as standard

During this work many major anthocyanins were de-tected in LC-MS analysis of HcA (such as peonidin dir-hamnoside peonidin derivative and peonidin-3-(p-

10 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

coumaroyl-glucoside)) According to [49] anthocyaninrsquoshigh antioxidant activity was evidenced and is related to itsstructure including the type number and position of thesubstituents in the flavylium cation 3prime-Hydroxyl group incyanidin forms a catechol structure in the B ring stabilizesthe semiquinone radical and forms a stable quinone thatinhibits free radicals such as DPPH while 3prime5prime-dihydroxylgroup of delphinidin forms a pyrogallol structure in the Bring that has more delocalized electrons to stabilize the freeradical generated in the medium Ethyl acetate fraction ofHcA in a previous study showed more significant anti-oxidant activity (SC50 176plusmn 18 μgmL) than the otherextracts of HdA and HmA [5] Many potential pathways forphenolic compounds to act as antioxidants were listed suchas inhibiting free radical formation peroxide decomposi-tion oxygen radical absorbance free radical scavengingsuppression of singlet oxygen increasing the levels of en-dogenous defenses chelating of metal ions and enzymaticinhibition [50 51])ere is a direct relation between the totalpolyphenolic content and the antioxidant activity [52] aspolyphenolic compounds such as phenolic acids flavonoidsand anthocyanins may be responsible for the antioxidantactivity on a large proportion [51]

Correlation between radical scavenging ability SC50values and the identified phenolic acids in LC-MS analysis isconsiderable as extracts with higher flavonoids andorphenolics contents showed higher antioxidant activity andlower SC50 value Salem et al [53] stated in a previous studythat flowers and leaves ofH rosa-sinensisH sabdariffa calyxextract and H platanifolius leaves extract possessed anti-oxidant activity that may be attributed to anthocyaninsflavonoids and ascorbic acid content

33 Cytotoxicity Assay About 70 of death in low- andmiddle-income countries is caused by cancer [54] For neweffective anticancer drug discovery screening of the cyto-toxic activity of the plant extracts and natural products isnecessary [55] Edible plants are excellent resources of an-ticancer agents [56]

In our study in vitro cytotoxic activity of the appliedsamples against tested cell lines using MTT assay and cis-platin as a positive standard showed a decrease in cell vi-ability in dose-dependent manner as illustrated in Figure 4and Table 2 Evaluation was based on IC50 values as followsIC50le 20 μgmL highly active IC50 21ndash200 μgmLmoder-ately active IC50 201ndash500 μgmLweakly active andIC50gt 501 μgmL inactive which is in a good accordancewith the American National Cancer Institute protocol [57]

In case of A-549 cell line the cytotoxicity of the appliedextracts was arranged as follows HdAgtHmAgtHcA Un-fortunately HcA exhibited the weakest cytotoxic activityagainst A-549 cell line with IC50 of 113plusmn 34 μgmL whencompared to cisplatin 753plusmn 38 μgmL )e higher activityof HdA (IC50 = 50plusmn 51 μgmL) as a strong antioxidant maybe attributed to the presence of major compounds such asbutein flavonoid [58] and peonidin dipentoside anthocyaninaccording to Mahadevan et al [44 49 59] HdA is morecytotoxic to A-549 cells (IC50 = 50plusmn 51 μgmL) than HmA(IC50 = 604plusmn 17 μgmL) although both of them have nearlysimilar common major compounds oleuropein and peo-nidin dipentoside

As indicated by IC50 values the cytotoxicity of testedsamples against HCT-116 cell is arranged as followHmAgtHcAgtHdA A close cytotoxic effect on HCT-116cell line was shown by HdA and HcA (IC50 96plusmn 32 and929plusmn 41 μgmL respectively))e cytotoxic activity ofHmA may be attributed to N-feruloyltyramine as it wasreported as a cytotoxic agent in a previous study [60] andthis cytotoxic activity may be enhanced by the presence ofascorbic acid according to [61 62]

Our results are in agreement with those reported for thecytotoxicity of flavonoids phenolic acids and terpenescontent which are major constituents identified in HmA inthat study [63ndash66] In a previous study different extracts(ethyl acetate chloroform petroleum ether) of HcA HdAandHmA showed strong anticancer property against humanhepatocellular carcinoma (HepG2) and human breast car-cinoma (MCF-7) cell lines [63] Leaves calyx and stemextracts of other species ofHibiscus (such asH sabdariffa Hrosa-sinensis H micranthus H vitifolius and H syriacus)have shown promising cytotoxic activity against many

0

20

40

60

80

100

0 100 200 300 400

DPP

H sc

aven

ging

Concentration (microgmL)

HdAHmA

HcAAscorbic acid

Figure 2 DPPH scavenging capacity of aqueous extracts of Hi-biscus deflersii (HdA) H micranthus (HmA) and H calyphyllus(HcA)

1376

111

1357

142

0

50

100

150

200

HdA HcA HmA Ascorbic acid

SC50

(microg

mL)

Extracts

Figure 3 SC50 of antioxidant activity of Hibiscus deflersii (HdA)H micranthus (HmA) and H calyphyllus (HcA)

Journal of Chemistry 11

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

cancer cells such as breast lung and human leukemia cells(HL-60) and liver cancer cell lines and they showed potentcytotoxic activity against human lung cancer cell line (A-549) that may be attributed to the presence of flavonoidstannins triterpenes phenols steroids [67 68] polyphenoliccompounds such as protocatechuic acid anthocyanins suchas delphinidin-3-sambubioside and myristic acid anduncarinic acid A [45 48 52]

34 Antidiabetic Assay Diabetes mellitus (DM) is a per-sistent disorder that is incurable due to the deficiency ofinsulin that affects 10 of the population It is expected toextend the number of diabetic individuals to 230 million in2025 )ere are many side effects for drugs currently used inDM treatment so herbal medicines are highly recom-mended for the treatment of diabetes instead of othersynthetic drugs [3] Since ancient times DM has beentreated orally using folklore medicine with several medicinalplants or their extracts [65]

In the present study in vitro α-amylase inhibitory ac-tivity of the applied samples evaluated using different doses(781ndash1000 μgmL) showed significant inhibition of car-bohydrate hydrolyzing enzymes (α-amylase) in dose-de-pendent manner as illustrated in Figures 5 and 6 HdAexhibited higher α-amylase inhibitory activity (7885plusmn 18

IC50 5622plusmn 19 μgmL) than that of HcA (680plusmn 04IC50 1039plusmn 15 μgmL) and HmA (6358plusmn 19 IC50

14907plusmn 21 μgmL) against acarbose standard with IC50

3471plusmn 07 μgmL )e higher activity of HdA (IC50 5622plusmn19μgmL) may be attributed to the presence of oleur-opein Jemai et al [69] previously reported that oleur-opein prevents some metabolic diseases related tooxidative stress such as diabetes hypercholesterolemiaassociated with diabetes and cardiovascular complica-tions which are very predominant in diabetics due to itshypoglycemic activity as it enhances peripheral glucoseuptake or insulin release and stimulates the synthesis ofliver glycogen through its antioxidant power

In agreement with our results other studies have re-ported that aerial parts of HdA were used as potential an-tidiabetics due to the presence of flavonoids [70])e flowersand fruits of HmA were found effective in diabetes [71] )ereported hypoglycemic activity of methanol leaf extract ofHsabdariffa and H rosa-sinensis and flowers extract of Hvitifolius and H tiliaceus may refer to the presence of fla-vonoids phenols tannins alkaloids and saponins[48 65 72]

35AntiobesityActivity Overweight and obesity are chronicdisorders that are considered as a growing issue influencingboth adults and children Obesity is defined as irregular orexcessive fat accumulation caused by the imbalance betweenenergy intake and expenditure )e vast majority of meta-bolic disorders such as cardiovascular disease dyslipidemiahypertension and diabetes may be due to obesity or over-weight [45 73] )e inhibition of the digestion and ab-sorption of dietary fats is a promising remedy for obesityNatural products are preferable to obesity drugs such asorlistat which have many side effects (ie development ofcardiovascular problems restlessness sleeping disorder andstomach pain) [73]

Results of the antiobesity activity of the three Hibiscusspecies aqueous extracts grown in Saudi Arabia using in vitro

0

20

40

60

80

100

120

0 100 200 300 400 500 600

A 5

49 ce

ll vi

abili

ty

Extract conc (microgmL)

HdAHmA

HcACisplatin

(a)

0

20

40

60

80

100

120

0 100 200 300 400 500 600

HCT

-116

cell

viab

ility

Extract conc (microgmL)

HdAHmA

HcACisplatin

(b)

Figure 4 In vitro cytotoxic activity of aqueous extracts of tested Hibiscus deflersii (HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 (a) and HCT-116 (b) cell lines

Table 2 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against A-549 andHTC-116 cell lines

IC50 (μgmL)Tested extracts A-549 cell line HCT-116 cell lineHdA 50plusmn 51 96plusmn 32HcA 113plusmn 34 929plusmn 28HmA 604plusmn 17 56plusmn 19Cisplatin 753plusmn 38 243plusmn 41)ese are the means of three determinations )e data are presented asμgmL

12 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

pancreatic lipase inhibitory assay are shown in Figure 7 andTable 3 HdA exhibited higher inhibitory activity than theHmA and HcA with IC50 of 9545plusmn 19 1077plusmn 15 andgt1000 μgmL respectively comparable with orlistat(IC50 238plusmn 07 μgmL) as standard Lipase inhibitory ac-tivity of HdA may be attributed to the presence of antho-cyanins (peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside peonidin glucoside feruloyl glucuronidepeonidin dipentoside malvidin derivative and malvidin-3-O-glucoside derivative) and organic acids (such as succinicascorbic and 4-hydroxybenzoic acid) It was reported thatpolyphenol compounds such as anthocyanins [74] and or-ganic acids [75] are responsible for the antiobesity activityDa-Costa-Rocha et al [48] stated that Hibiscus extract (ortea) may help in weight loss as antiobesity agent due to itseffects on fat absorption-excretion inhibition of the activityof α-amylase starch absorption and blocking sugarsMoreover aqueous extract of Hibiscus species showed apowerful inhibition of triglyceride accumulation as wholeextract was more active than isolated polyphenols

In a previous study aqueous extract of H sabdariffa(with anthocyanins being major compounds) exhibitedmany potential antiobesity mechanisms including

antihyperglycemic activity reduction in plasma cholesterollevel inhibition of gastric and pancreatic lipase enzymesthermogenesis stimulation inhibition of lipid droplet ac-cumulation in fat cells and fatty acid synthase inhibition[76] Drinking a cup of Hibiscus tea after meals can reducethe absorption of dietary carbohydrates and assist in weightloss [45]

4 Conclusion

Phenolic compounds flavonoids and anthocyanins wereidentified in three different Hibiscus species using UPLC-ESI-MSMS analysis HcA showed the highest in vitro an-tioxidant activity compared with other tested extracts andthis activity can be attributed to its contents of polyphenoliccompounds such as apigenin C-hexoside-C-pentosideluteolin C-hexoside-C-pentoside luteolin derivative 4-hydroxybenzoic acid and tyrosol and peonidin derivative inaddition to presence of anthocyanin contents such aspeonidin dirhamnoside peonidin derivative and peonidin-3-(p-coumaroyl-glucoside) HdA showed the most potenteffect on human lung carcinoma (A-549) cell line whichmay be attributed to the presence of major compounds suchas butein flavonoid and peonidin dirhamnoside and peo-nidin dipentoside anthocyanins with the highest activities asantidiabetic (due to oleuropein presence) and as antiobesity(may be attributed to the presence of major anthocyanins

0

20

40

60

80

100

0 200 400 600 800 1000 1200

Lipa

se in

hibi

tory

Extract conc (microgmL)

HcAHmA

HdAOrlistat

Figure 7 In vitro lipase inhibitory activity of aqueous extracts oftested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA) in comparison with orlistat standard

Table 3 IC50 of tested aqueous extracts ofHibiscus deflersii (HdA)H micranthus (HmA) andH calyphyllus (HcA) against pancreaticlipase enzyme

IC50 (μgmL)Tested extractsHdA 9545plusmn 19HcA gt1000HmA 1077plusmn 15Orlistat standard 238plusmn 07)ese are the means of three determinations )e data are presented asμgmL

0

20

40

60

80

100

0 200 400 600 800 1000 1200

α-A

myl

ase i

nhib

itory

Extract conc (μgmL)

HcAHmA

HdAAcarbose

Figure 5 In vitro α-amylase inhibitory activity of aqueous extractsof tested Hibiscus deflersii (HdA) H micranthus (HmA) and Hcalyphyllus (HcA)

5622

1039

14907

3471

0

40

80

120

160

HdA HcA HmA Acarbose

IC50

(microg

mL)

Extracts

Figure 6 IC50 of α-amylase inhibitory activity of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA) aqueousextracts and standard acarbose

Journal of Chemistry 13

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

such as peonidin-3-(p-coumaroyl-glucoside) peonidindirhamnoside and peonidin glucoside feruloyl glucuronidein addition to organic acids (such as succinic ascorbic and4-hydroxybenzoic acid)) )e results recommend that HdAneed further studies for the possible use as anticancer an-tidiabetic and antiobesity agent as it might be a naturalalternative remedy and nutritional policy for diabetes andobesity treatment without negative side effects Isolation ofthe bioactive phytochemical from the HcA HmA and HdAand estimation of their biological effects are recommendedin further studies

Data Availability

)e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

)e authors declare that they have no conflicts of interest

Authorsrsquo Contributions

All authors made considerable contributions to the manu-script HA ME MA RA and SA designed the study MEWH HA SA MA and RA performed the experiments MESA HA and WH interpreted the results ME HA and SAwrote the manuscript All authors revised the manuscriptand approved it for publication

Acknowledgments

)e authors thank Prof Dr Mohamed Yousef Pharma-cognosy Department College of Pharmacy King SaudUniversity (KSU) for the plant identification )is researchwas carried out with personal funds from the authors

Supplementary Materials

Table S1 metabolites identified in the aqueous extract ofthree different Hibiscus species Hibiscus deflersii (HdA) Hmicranthus (HmA) and H calyphyllus (HcA) using UPLC-ESI-MS in negative and positive ionization modes Table S2IC50 of tested aqueous Hibiscus extracts of Hibiscus deflersii(HdA) H micranthus (HmA) and H calyphyllus (HcA)against A-549 and HTC-116 cell lines )e data are pre-sented as μgmL Table S3 IC50 of tested aqueous Hibiscusextracts of Hibiscus deflersii (HdA) H micranthus (HmA)and H calyphyllus (HcA) against pancreatic lipase enzyme)e data are presented as μgmL (Supplementary Materials)

References

[1] C S Kılıccedil S Aslan and M Kartal MandCoskun ldquoFatty acidcomposition of Hibiscus trionum L (Malvaceae)rdquo Journal ofNatural Products vol 5 pp 65ndash69 2011

[2] N Vasudeva and S K Sharma ldquoBiologically active com-pounds from the Genus Hibiscusrdquo Pharmaceutical Biologyvol 46 no 3 pp 145ndash153 2008

[3] S Venkatesh J )ilagavathi and D Shyam sundar ldquoAnti-diabetic activity of flowers of Hibiscus rosasinensisrdquo Fitoter-apia vol 79 no 2 pp 79ndash81 2008

[4] L B Vinh N T M Nguyet C D )anh et al ldquoChemicalconstituents of Vietnamese mangrove Hibiscus tiliaceus withantioxidant and alpha-glucosidase inhibitory activityrdquo Nat-ural Product Research pp 1ndash6 2019

[5] N A Siddiqui H M Al-Yousef T A Alhowiriny et alldquoConcurrent analysis of bioactive triterpenes oleanolic acidand β-amyrin in antioxidant active fractions of Hibiscuscalyphyllus Hibiscus deflersii and Hibiscus micranthus grownin Saudi Arabia by applying validated HPTLCmethodrdquo SaudiPharmaceutical Journal vol 26 no 2 pp 266ndash273 2018

[6] W H B Hassan S Abdelaziz and H M Yousef ldquoChemicalcomposition and biological activities of the aqueous fractionof Parkinsonea aculeata L growing in Saudi Arabiardquo ArabianJournal of Chemistry vol 12 no 3 pp 377ndash387 2019

[7] G C Yen and P D Duh ldquoScavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygenspeciesrdquo Journal of Agricultural and Food Chemistry vol 42no 3 pp 629ndash632 1994

[8] I Gulccedilin Oufre Kufrevioǧlu M Oktay andM E Buyukokuroǧlu ldquoAntioxidant antimicrobial antiulcerand analgesic activities of nettle (Urtica dioica L)rdquo Journal ofEthnopharmacology vol 90 no 2-3 pp 205ndash215 2004

[9] S M Gomha T A Salah and A O Abdelhamid ldquoSynthesischaracterization and pharmacological evaluation of somenovel thiadiazoles and thiazoles incorporating pyrazolemoiety as anticancer agentsrdquo Monatshefte fur Chemie -Chemical Monthly vol 146 no 1 pp 149ndash158 2015

[10] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983

[11] M B Narkhede P V Ajimire A E Wagh M Mohan andA T Shivashanmugam ldquoIn vitro antidiabetic activity ofCaesalpina digyna (R) methanol root extractrdquo Asian Journalof Plant Science and Research vol 1 no 2 pp 101ndash106 2011

[12] Y S Kim Y M Lee H Kim J Kim D S Jang and J H KimldquoAnti-obesity effect of Morus bombycis root extract anti-li-pase activity and lipolytic effectrdquo Journal of Ethno-pharmacology vol 130 no 3 pp 621ndash624 2010

[13] FooDB ldquo)e Metabolomics Innovation Centrerdquo 2019httpsfoodbca

[14] H Zemmouri S Ammar A Boumendjel M Messarah andA El Feki ldquoChemical composition and antioxidant activity ofBorago officinalis L leaf extract growing in Algeriardquo ArabianJournal of Chemistry vol 12 no 8 pp 1954ndash1963 2019

[15] M Lambert E Meudec A Verbaere et al ldquoA high-throughput UHPLC-QqQ-MS method for polyphenol pro-filing in rose winesrdquo Molecules vol 20 no 5 pp 7890ndash79142015

[16] S Moco Metabolomics Technologies Applied to the Identifi-cation of Compounds in Plants A Liquid Chromatography-Mass Spectrometry-Nuclear Magnetic Resonance Perspectiveover the Tomato Fruit PhD thesis Wageningen UniversityWageningen )e Netherlands 2007

[17] I M Shi M S Ali-Shtayeh R M Jamous and D Arraez-Roman ldquoHPLC-DAD-ESI-MSMS screening of bioactivecomponents from Rhus coriaria L (Sumac) fruitsrdquo FoodChemistry vol 166 pp 179ndash191 2015

[18] P Efferth A Vallverdu-Queralt M Martınez-Huelamo et alldquoA comprehensive characterisation of beer polyphenols by

14 Journal of Chemistry

high resolution mass spectrometry (LC-ESI-LTQ-Orbitrap-MS)rdquo Food Chemistry vol 169 pp 336ndash343 2015

[19] E Emile Nicoue ldquoIdentification des anthocyanes de deuxespeces de bleuets sauvages du Quebec ldquoVaccinium angus-tifoliumrdquo et ldquoVaccinium myrtilloıdesrdquo et evaluation de leurpotentiel antioxydantrdquo Universite Laval Quebec CanadaDoctoral dissertation 2010

[20] Q Chang and Y S Wong ldquoIdentification of flavonoids inhakmeitau beans (Vigna sinensis) by high-performance liquidchromatographyminuselectrospray mass spectrometry (LC-ESIMS)rdquo Journal of Agricultural and Food Chemistry vol 52no 22 pp 6694ndash6699 2004

[21] R O Bakr S A E H Mohamed and N Ayoub ldquoPhenolicprofile of Centaurea aegyptiaca L growing in Egypt and itscytotoxic and antiviral activitiesrdquo African Journal of Tradi-tional Complementary and Alternative Medicines vol 13no 6 pp 135ndash143 2016

[22] O Al Kadhi A Melchini R Mithen and S Saha ldquoDevel-opment of a LC-MSMS method for the simultaneous de-tection of tricarboxylic acid cycle intermediates in a range ofbiological matricesrdquo Journal of Analytical Methods inChemistry vol 2017 Article ID 5391832 12 pages 2017

[23] F Ferreres C Grosso A Gil-Izquierdo P Valentatildeo andC Azevedo ldquoHPLC-DAD-ESIMSn analysis of phenoliccompounds for quality control of Grindelia robusta Nutt andbioactivitiesrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 94 pp 163ndash172 2014

[24] A Sinela N Rawat C Mertz N Achir and M FulcrandHandDornier ldquoAnthocyanins degradation during storage ofHibiscus sabdariffa extract and evolution of its degradationproductsrdquo Food Chemistry vol 214 pp 234ndash241 2017

[25] S Ammar M Del Mar Contreras O Belguith-HadrichA Segura-Carretero and M Bouaziz ldquoAssessment of thedistribution of phenolic compounds and contribution to theantioxidant activity in Tunisian fig leaves fruits skins andpulps using mass spectrometry-based analysisrdquo Food andFunction vol 6 no 12 pp 3663ndash3677 2015

[26] D Barreca E Bellocco C Caristi and U Leuzzi ldquoFlavonoidprofile and radical-scavenging activity ofMediterranean sweetlemon (Citrus limetta risso) juicerdquo Food Chemistry vol 129no 2 pp 417ndash422 2011

[27] R M IbrahimLin A M El-Halawany D O Saleh et alldquoHPLC-DAD-MSMS profiling of phenolics from Securigerasecuridaca flowers and its anti-hyperglycemic and anti-hyperlipidemic activitiesrdquo Revista Brasileira de Farm-acognosia vol 25 no 2 pp 134ndash141 2015

[28] I C Rodrıguez-Medina R Beltran-Debon V M Molinaet al ldquoDirect characterization of aqueous extract of Hibiscussabdariffa using HPLC with diode array detection coupled toESI and ion trap MSrdquo Journal of Separation Science vol 32no 20 pp 3441ndash3448 2009

[29] M Simirgiotis J Benites and C Areche ldquoAntioxidant ca-pacities and analysis of phenolic compounds in three endemicnolana species by HPLC-PDA-ESI-MSrdquo Molecules vol 20no 6 pp 11490ndash11507 2015

[30] U Shin ldquoNegative atmospheric pressure chemical ionisationlow-energy collision activation mass spectrometry for thecharacterisation of flavonoids in extracts of fresh herbsrdquoJournal of Chromatography A vol 902 no 2 pp 369ndash3792000

[31] V Spınola J Pinto and P C Castilho ldquoIdentification andquantification of phenolic compounds of selected fruits fromMadeira Island by HPLC-DAD-ESI-MSn and screening for

their antioxidant activityrdquo Food Chemistry vol 173 pp 14ndash30 2015

[32] C A Ledesma-Escobar F Priego-Capote and M D LuqueDe Castro ldquoCharacterization of lemon (Citrus limon) polarextract by liquid chromatography-tandem mass spectrometryin high resolution moderdquo Journal of Mass Spectrometryvol 50 no 11 pp 1196ndash1205 2015

[33] M B Hossain D K Rai N P Brunton A B Martin-Dianaand B R Catherine ldquoCharacterization of phenolic compo-sition in lamiaceae spices by LC-ESI-MSMSrdquo Journal ofAgricultural and Food Chemistry vol 58 no 19 pp 10576ndash10581 2010

[34] R Colombo J H Yariwake and M McCullagh ldquoStudy ofC-and O-glycosylflavones in sugar cane extracts using liquidchromatography-exact mass measurement mass spectrome-tryrdquo Journal of the Brazilian Chemical Society vol 19 no 3pp 483ndash490 2008

[35] R Stein-Chisholm J Beaulieu and C Grimm ldquoLC-MSMSand UPLC-UV evaluation of anthocyanins and anthocyani-dins during rabbiteye blueberry juice processingrdquo Beveragesvol 3 no 4 p 56 2017

[36] A Brito A Gattuso J Ramirez C Areche B Sepulveda andM Simirgiotis ldquoHPLC-UV-MS profiles of phenolic com-pounds and antioxidant activity of fruits from three citrusspecies consumed in northern Chilerdquo Molecules vol 19no 11 pp 17400ndash17421 2014

[37] M P Rodrıguez-Rivera E Lugo-Cervantes andP Winterhalter ldquoMetabolite profiling of polyphenols in peelsof Citrus limetta Risso by combination of preparative high-speed countercurrent chromatography and LC-ESI-MSMSrdquoFood Chemistry vol 158 pp 139ndash152 2014

[38] M A Farag S T Sakna N M El-Fiky M M Shabana andL A Wessjohann ldquoPhytochemical antioxidant and antidi-abetic evaluation of eight Bauhinia L species from Egyptusing UHPLC-PDA-qTOF-MS and chemometricsrdquo Phyto-chemistry vol 119 pp 41ndash50 2015

[39] A A Chernonosov E A Karpova and E M LyakhldquoIdentification of phenolic compounds inMyricaria bracteataleaves by high-performance liquid chromatography with adiode array detector and liquid chromatography with tandemmass spectrometryrdquo Revista Brasileira de Farmacognosiavol 27 no 5 pp 576ndash579 2017

[40] W Y Sayadi J S Jin Y A Cho et al ldquoDetermination ofpolyphenols in three Capsicum annuum L (bell pepper)varieties using high-performance liquid chromatography-tandem mass spectrometry their contribution to overallantioxidant and anticancer activityrdquo Journal of SeparationScience vol 34 no 21 pp 2967ndash2974 2011

[41] K Ablajan Z Abliz X-Y Shang J-M He and R-P ZhangldquoStructural characterization of flavonol 37-di-O-glycosidesand determination of the glycosylation position by usingnegative ion electrospray ionization tandem mass spec-trometryrdquo Journal of Mass Spectrometry vol 41 no 3pp 352ndash360 2006

[42] R Wang M Ye H Guo K Bi and D-a Guo ldquoLiquidchromatographyelectrospray ionization mass spectrometryfor the characterization of twenty-three flavonoids in theextract of Dalbergia odoriferardquo Rapid Communications inMass Spectrometry vol 19 no 11 pp 1557ndash1565 2005

[43] A Andrade R Heydari Z Talebpour et al ldquoStudy of newextraction methods for separation of anthocyanins from redgrape skins analysis by HPLC and LC-MSMSrdquo Journal ofLiquid Chromatography amp Related Technologies vol 31no 17 pp 2686ndash2703 2008

Journal of Chemistry 15

[44] N Mahadevan and P Kamboj ldquoHibiscus sabdariffa Linnndashanoverviewrdquo Natural Product Radiance vol 8 no 1 pp 77ndash832009

[45] P Singh M Khan and H Hailemariam ldquoNutritional andhealth importance of Hibiscus sabdariffa a review and in-dication for research needsrdquo Journal of Nutritional Health ampFood Engineering vol 6 no 5 Article ID 00212 2017

[46] P K Wong S Yusof H M Ghazali and Y B Che ManldquoPhysico-chemical characteristics of roselle (Hibiscus sab-dariffa L)rdquoNutrition amp Food Science vol 32 no 2 pp 68ndash732002

[47] J Bouaziz X Zhang J Zhang M Li and T Chen ldquoMinorcompounds of the high purity salvianolic acid B freeze-driedpowder from Salvia miltiorrhiza and antibacterial activityassessmentrdquo Natural Product Research vol 32 no 10pp 1198ndash1202 2018

[48] I Da-Costa-Rocha B Bonnlaender H Sievers I Pischel andM Heinrich Hibiscus sabdariffa LmdashA Phytochemical andPharmacological Review Vol 165 Elsevier Ltd AmsterdamNetherlands 2014

[49] L G Maciel M A V do Carmo L Azevedo et al ldquoHibiscussabdariffa anthocyanins-rich extract chemical stability invitro antioxidant and antiproliferative activitiesrdquo Food andChemical Toxicology vol 113 pp 187ndash197 2018

[50] M N Moraes G L Zabot J M Prado andM A A MeirelesldquoObtaining antioxidants from botanic matrices applyingnovel extraction techniquesrdquo Food and Public Health vol 3no 4 pp 195ndash214 2013

[51] A Obouayeba N Djyh S Diabate et al ldquoPhytochemical andantioxidant activity of roselle (Hibiscus sabdariffa L) petalextractsrdquo Research Journal of Pharmaceutical Biological andChemical Sciences vol 5 no 2 pp 1453ndash1465 2014

[52] S Abdelhamid O Martı n-Belloso Y-S Park et al ldquoCom-parison of some biochemical characteristics of different citrusfruitsrdquo Food Chemistry vol 74 no 3 pp 309ndash315 2001

[53] M Z M Salem J Olivares-Perez and A Z M Salem ldquoStudieson biological activities and phytochemicals composition ofHibiscus species-a reviewrdquo Life Science Journal vol 11 no 5pp 1ndash8 2014

[54] V Kim F W Fokou O Karaosmanoglu V P Beng andH Sivas ldquoCytotoxicity of the methanol extracts of Ele-phantopus mollis Kalanchoe crenata and 4 other Camer-oonian medicinal plants towards human carcinoma cellsrdquoBMC Complementary and Alternative Medicine vol 17 no 12017

[55] L KWasiman J OMidiwo VMMasila et al ldquoCytotoxicityof 91 Kenyan indigenous medicinal plants towards humanCCRF-CEM leukemia cellsrdquo Journal of Ethnopharmacologyvol 179 pp 177ndash196 2016

[56] H-H Qian H-P Huang C-C Huang and J-H ChenldquoHibiscus polyphenol-rich extract induces apoptosis in hu-man gastric carcinoma cells via p53 phosphorylation and p38MAPKFasL cascade pathwayrdquo Molecular Carcinogenesisvol 43 no 2 pp 86ndash99 2005

[57] T Srisawat P Chumkaew W Heed-Chim Y Sukpondmaand K Kanokwiroon ldquoPhytochemical screening and cyto-toxicity of crude extracts of Vatica diospyroides symingtontype LSrdquo Tropical Journal of Pharmaceutical Research vol 12no 1 pp 71ndash76 2013

[58] Y Luque de Castro C Ma M Qian Z Wen and H JingldquoButein induces cell apoptosis and inhibition of cyclo-oxygenase-2 expression in A549 lung cancer cellsrdquoMolecularMedicine Reports vol 9 no 2 pp 763ndash767 2014

[59] C-H Che Man C-C Huang C-H Hung F-Y YaoC-J Wang and Y-C Chang ldquoDelphinidin-rich extracts ofHibiscus sabdariffa L trigger mitochondria-derived auto-phagy and necrosis through reactive oxygen species in humanbreast cancer cellsrdquo Journal of Functional Foods vol 25pp 279ndash290 2016

[60] J-J Wong S-Y Huang C-Y Duh I-S Chen T-C Wangand H-Y Fang ldquoA new cytotoxic amide from the stem woodof Hibiscus tiliaceusrdquo Planta Medica vol 72 no 10pp 935ndash938 2006

[61] C M Narsu U Wagnera B Kolsterb P E AndreotticD Krebsa and H W Brucknerd ldquoAscorbic acid (vitamin C)improves the antineoplastic activity of doxorubicin cispiatinand paclitaxel in human breast carcinoma cells in vitrordquoCancer Letters vol 103 no 2 pp 183ndash189 1996

[62] H Sakagami K Satoh H Ohata et al ldquoRelationship betweenascorbyl radical intensity and apoptosis-inducing activityrdquoAnticancer Res vol 16 no 5 pp 2635ndash2644 1996

[63] P Segura-Carretero H M Al-Yousef N A Siddiqui et alldquoAnticancer activity and concurrent analysis of ursolic acidβ-sitosterol and lupeol in three different Hibiscus species(aerial parts) by validated HPTLC methodrdquo Saudi Pharma-ceutical Journal vol 26 no 7 pp 1060ndash1067 2018

[64] R Jain R Arora and S Jain ldquoChemical constituents andbioactivity studies of Hibiscus Mucranthus Linnrdquo IndianJournal of Pharmaceutical Sciences vol 59 no 2 p 91 1997

[65] K A Kumar S R Setty and L Narsu ldquoPharmacognostic andphytochemical investigations of stems of Hibiscus micranthusLinnrdquo Pharmacognosy Journal vol 2 no 15 pp 21ndash30 2010

[66] R M Rosa M I S Melecchi and R D C HalmenschlagerJ Saffi and A L L D Ramos Antioxidant and antimutagenicproperties of Hibiscus tiliaceus L methanolic extractrdquo Journalof Agricultural and Food Chemistry vol 54 no 19pp 7324ndash7330 2006

[67] T Lamuela-Raventos ldquoComparative pharmacological studyof aerial parts and roots of ethanolic extract of Hibiscusmicranthus Linnrdquo Journal of Medical Pharmaceutical andallied Sciences vol 1 pp 581ndash587 2017

[68] L-S Shi C-H Wu T-C Yang C-W Yao H-C Lin andW-L Chang ldquoCytotoxic effect of triterpenoids from the rootbark of Hibiscus syriacusrdquo Fitoterapia vol 97 pp 184ndash1912014

[69] H Jemai A E L Feki and S Sayadi ldquoAntidiabetic andantioxidant effects of hydroxytyrosol and oleuropein fromolive leaves in alloxan-diabetic ratsrdquo Journal of Agriculturaland Food Chemistry vol 57 no 19 pp 8798ndash8804 2009

[70] S Lakshman A Jyothi V Mounica A R KumarS Rathinam and K Rajesh ldquoAntidiabetic activity ofmethonolic extract of in streptozotocin induced diabetic ratsHibiscus deflersiirdquo International Research Journal of Phar-maceutical and Applied Sciences vol 4 no 1 pp 1ndash3 2014

[71] H Kakrani B H Kakrani and A K Saluja ldquoTraditionaltreatment of diabetes through herbs in Kutch district Gujratstaterdquo Planta Indica vol 1 no 1 pp 16ndash21 2005

[72] T A Ndarubu O S Chiamaka S Alfa et al ldquoPhytochem-icals hypoglycemic and hypolipidemic effects of methanolleaf extract of Hibiscus sabdariffa in alloxan induced diabeticratsrdquo GSC Biological and Pharmaceutical Sciences vol 8no 3 pp 70ndash78 2019

[73] M H Omar N Shamsahal H Muhammad W A N WanAhmad and M I Wasiman ldquoAnti-obesity and haemato-logical effects of Malaysia Hibiscus sabdariffa L aqueousextract on obese sprague dawley ratsrdquo Functional Foods inHealth and Disease vol 8 no 6 p 340 2018

16 Journal of Chemistry

[74] T-W Huang C-L Chang and E-S Kao ldquoEffect ofHibiscussabdariffaextract on high fat diet-induced obesity and liverdamage in hamstersrdquo Food amp Nutrition Research vol 59no 1 Article ID 29018 2015

[75] D Abdalla I F Perez-Ramırez J Perez-Jimenez andG M R Nava ldquoComparison of the bioactive potential ofroselle (Hibiscus sabdariffa L) calyx and its by-productphenolic characterization by UPLC-QTOFMS and their anti-obesity effect in vivordquo Food Research International vol 126Article ID 108589 2019

[76] J W Yun ldquoPossible anti-obesity therapeutics from nature - areviewrdquo Phytochemistry vol 71 no 14-15 pp 1625ndash16412010

Journal of Chemistry 17

[44] N Mahadevan and P Kamboj ldquoHibiscus sabdariffa Linnndashanoverviewrdquo Natural Product Radiance vol 8 no 1 pp 77ndash832009

[45] P Singh M Khan and H Hailemariam ldquoNutritional andhealth importance of Hibiscus sabdariffa a review and in-dication for research needsrdquo Journal of Nutritional Health ampFood Engineering vol 6 no 5 Article ID 00212 2017

[46] P K Wong S Yusof H M Ghazali and Y B Che ManldquoPhysico-chemical characteristics of roselle (Hibiscus sab-dariffa L)rdquoNutrition amp Food Science vol 32 no 2 pp 68ndash732002

[47] J Bouaziz X Zhang J Zhang M Li and T Chen ldquoMinorcompounds of the high purity salvianolic acid B freeze-driedpowder from Salvia miltiorrhiza and antibacterial activityassessmentrdquo Natural Product Research vol 32 no 10pp 1198ndash1202 2018

[48] I Da-Costa-Rocha B Bonnlaender H Sievers I Pischel andM Heinrich Hibiscus sabdariffa LmdashA Phytochemical andPharmacological Review Vol 165 Elsevier Ltd AmsterdamNetherlands 2014

[49] L G Maciel M A V do Carmo L Azevedo et al ldquoHibiscussabdariffa anthocyanins-rich extract chemical stability invitro antioxidant and antiproliferative activitiesrdquo Food andChemical Toxicology vol 113 pp 187ndash197 2018

[50] M N Moraes G L Zabot J M Prado andM A A MeirelesldquoObtaining antioxidants from botanic matrices applyingnovel extraction techniquesrdquo Food and Public Health vol 3no 4 pp 195ndash214 2013

[51] A Obouayeba N Djyh S Diabate et al ldquoPhytochemical andantioxidant activity of roselle (Hibiscus sabdariffa L) petalextractsrdquo Research Journal of Pharmaceutical Biological andChemical Sciences vol 5 no 2 pp 1453ndash1465 2014

[52] S Abdelhamid O Martı n-Belloso Y-S Park et al ldquoCom-parison of some biochemical characteristics of different citrusfruitsrdquo Food Chemistry vol 74 no 3 pp 309ndash315 2001

[53] M Z M Salem J Olivares-Perez and A Z M Salem ldquoStudieson biological activities and phytochemicals composition ofHibiscus species-a reviewrdquo Life Science Journal vol 11 no 5pp 1ndash8 2014

[54] V Kim F W Fokou O Karaosmanoglu V P Beng andH Sivas ldquoCytotoxicity of the methanol extracts of Ele-phantopus mollis Kalanchoe crenata and 4 other Camer-oonian medicinal plants towards human carcinoma cellsrdquoBMC Complementary and Alternative Medicine vol 17 no 12017

[55] L KWasiman J OMidiwo VMMasila et al ldquoCytotoxicityof 91 Kenyan indigenous medicinal plants towards humanCCRF-CEM leukemia cellsrdquo Journal of Ethnopharmacologyvol 179 pp 177ndash196 2016

[56] H-H Qian H-P Huang C-C Huang and J-H ChenldquoHibiscus polyphenol-rich extract induces apoptosis in hu-man gastric carcinoma cells via p53 phosphorylation and p38MAPKFasL cascade pathwayrdquo Molecular Carcinogenesisvol 43 no 2 pp 86ndash99 2005

[57] T Srisawat P Chumkaew W Heed-Chim Y Sukpondmaand K Kanokwiroon ldquoPhytochemical screening and cyto-toxicity of crude extracts of Vatica diospyroides symingtontype LSrdquo Tropical Journal of Pharmaceutical Research vol 12no 1 pp 71ndash76 2013

[58] Y Luque de Castro C Ma M Qian Z Wen and H JingldquoButein induces cell apoptosis and inhibition of cyclo-oxygenase-2 expression in A549 lung cancer cellsrdquoMolecularMedicine Reports vol 9 no 2 pp 763ndash767 2014

[59] C-H Che Man C-C Huang C-H Hung F-Y YaoC-J Wang and Y-C Chang ldquoDelphinidin-rich extracts ofHibiscus sabdariffa L trigger mitochondria-derived auto-phagy and necrosis through reactive oxygen species in humanbreast cancer cellsrdquo Journal of Functional Foods vol 25pp 279ndash290 2016

[60] J-J Wong S-Y Huang C-Y Duh I-S Chen T-C Wangand H-Y Fang ldquoA new cytotoxic amide from the stem woodof Hibiscus tiliaceusrdquo Planta Medica vol 72 no 10pp 935ndash938 2006

[61] C M Narsu U Wagnera B Kolsterb P E AndreotticD Krebsa and H W Brucknerd ldquoAscorbic acid (vitamin C)improves the antineoplastic activity of doxorubicin cispiatinand paclitaxel in human breast carcinoma cells in vitrordquoCancer Letters vol 103 no 2 pp 183ndash189 1996

[62] H Sakagami K Satoh H Ohata et al ldquoRelationship betweenascorbyl radical intensity and apoptosis-inducing activityrdquoAnticancer Res vol 16 no 5 pp 2635ndash2644 1996

[63] P Segura-Carretero H M Al-Yousef N A Siddiqui et alldquoAnticancer activity and concurrent analysis of ursolic acidβ-sitosterol and lupeol in three different Hibiscus species(aerial parts) by validated HPTLC methodrdquo Saudi Pharma-ceutical Journal vol 26 no 7 pp 1060ndash1067 2018

[64] R Jain R Arora and S Jain ldquoChemical constituents andbioactivity studies of Hibiscus Mucranthus Linnrdquo IndianJournal of Pharmaceutical Sciences vol 59 no 2 p 91 1997

[65] K A Kumar S R Setty and L Narsu ldquoPharmacognostic andphytochemical investigations of stems of Hibiscus micranthusLinnrdquo Pharmacognosy Journal vol 2 no 15 pp 21ndash30 2010

[66] R M Rosa M I S Melecchi and R D C HalmenschlagerJ Saffi and A L L D Ramos Antioxidant and antimutagenicproperties of Hibiscus tiliaceus L methanolic extractrdquo Journalof Agricultural and Food Chemistry vol 54 no 19pp 7324ndash7330 2006

[67] T Lamuela-Raventos ldquoComparative pharmacological studyof aerial parts and roots of ethanolic extract of Hibiscusmicranthus Linnrdquo Journal of Medical Pharmaceutical andallied Sciences vol 1 pp 581ndash587 2017

[68] L-S Shi C-H Wu T-C Yang C-W Yao H-C Lin andW-L Chang ldquoCytotoxic effect of triterpenoids from the rootbark of Hibiscus syriacusrdquo Fitoterapia vol 97 pp 184ndash1912014

[69] H Jemai A E L Feki and S Sayadi ldquoAntidiabetic andantioxidant effects of hydroxytyrosol and oleuropein fromolive leaves in alloxan-diabetic ratsrdquo Journal of Agriculturaland Food Chemistry vol 57 no 19 pp 8798ndash8804 2009

[70] S Lakshman A Jyothi V Mounica A R KumarS Rathinam and K Rajesh ldquoAntidiabetic activity ofmethonolic extract of in streptozotocin induced diabetic ratsHibiscus deflersiirdquo International Research Journal of Phar-maceutical and Applied Sciences vol 4 no 1 pp 1ndash3 2014

[71] H Kakrani B H Kakrani and A K Saluja ldquoTraditionaltreatment of diabetes through herbs in Kutch district Gujratstaterdquo Planta Indica vol 1 no 1 pp 16ndash21 2005

[72] T A Ndarubu O S Chiamaka S Alfa et al ldquoPhytochem-icals hypoglycemic and hypolipidemic effects of methanolleaf extract of Hibiscus sabdariffa in alloxan induced diabeticratsrdquo GSC Biological and Pharmaceutical Sciences vol 8no 3 pp 70ndash78 2019

[73] M H Omar N Shamsahal H Muhammad W A N WanAhmad and M I Wasiman ldquoAnti-obesity and haemato-logical effects of Malaysia Hibiscus sabdariffa L aqueousextract on obese sprague dawley ratsrdquo Functional Foods inHealth and Disease vol 8 no 6 p 340 2018

16 Journal of Chemistry

[74] T-W Huang C-L Chang and E-S Kao ldquoEffect ofHibiscussabdariffaextract on high fat diet-induced obesity and liverdamage in hamstersrdquo Food amp Nutrition Research vol 59no 1 Article ID 29018 2015

[75] D Abdalla I F Perez-Ramırez J Perez-Jimenez andG M R Nava ldquoComparison of the bioactive potential ofroselle (Hibiscus sabdariffa L) calyx and its by-productphenolic characterization by UPLC-QTOFMS and their anti-obesity effect in vivordquo Food Research International vol 126Article ID 108589 2019

[76] J W Yun ldquoPossible anti-obesity therapeutics from nature - areviewrdquo Phytochemistry vol 71 no 14-15 pp 1625ndash16412010

Journal of Chemistry 17

[74] T-W Huang C-L Chang and E-S Kao ldquoEffect ofHibiscussabdariffaextract on high fat diet-induced obesity and liverdamage in hamstersrdquo Food amp Nutrition Research vol 59no 1 Article ID 29018 2015

[75] D Abdalla I F Perez-Ramırez J Perez-Jimenez andG M R Nava ldquoComparison of the bioactive potential ofroselle (Hibiscus sabdariffa L) calyx and its by-productphenolic characterization by UPLC-QTOFMS and their anti-obesity effect in vivordquo Food Research International vol 126Article ID 108589 2019

[76] J W Yun ldquoPossible anti-obesity therapeutics from nature - areviewrdquo Phytochemistry vol 71 no 14-15 pp 1625ndash16412010

Journal of Chemistry 17