Seasonal Effect on the Biological Activities of Litsea ...downloads.hindawi.com/journals/ecam/2018/2738489.pdf · ResearchArticle Seasonal Effect on the Biological Activities of Litsea

Research ArticleSeasonal Effect on the Biological Activities of Litsea glaucescensKunth Extracts

Julio Ceacutesar Loacutepez-Romero1 Humberto Gonzaacutelez-Riacuteos1 Aida Pentildea-Ramos1

Carlos Velazquez2 Moises Navarro2 Ramoacuten Robles-Zepeda 2

Evelin Martiacutenez-Benavidez3 Inocencio Higuera-Ciapara3 Claudia Virueacutes4

Joseacute Luis Olivares5 Zaira Domiacutenguez 6 and Javier Hernaacutendez 6

1Centro de Investigacion en Alimentacion y Desarrollo (CIAD) 83000 Hermosillo SON Mexico2Departamento de Ciencias Quımico Biologicas Universidad de Sonora Rosales y Luis Encinas 83000 Hermosillo SON Mexico3Centro de Investigacion y Asistencia en Tecnologıa y Diseno del Estado de Jalisco AC CONSORCIO-ADESURAv Normalistas 800 Colinas de la Normal 44270 Guadalajara JAL Mexico4Centro de Investigacion y Asistencia en Tecnologıa y Diseno del Estado de Jalisco AC Cluster Cientıfico y Tecnologico BiomimicCarretera Antigua a Coatepec No 351 Colonia El Haya 91070 Xalapa VER Mexico5Red de Estudios Moleculares Avanzados Cluster Biomimic Instituto de Ecologıa A C Carretera Antigua a Coatepec No 351Colonia El Haya 91070 Xalapa VER Mexico6Unidad de Servicios de Apoyo en Resolucion Analıtica Universidad Veracruzana 575 Xalapa VER Mexico

Correspondence should be addressed to Zaira Domınguez zdominguezuvmx and Javier Hernandez javmartinezuvmx

Received 14 October 2017 Revised 14 January 2018 Accepted 22 January 2018 Published 20 February 2018

Academic Editor Letizia Angiolella

Copyright copy 2018 Julio Cesar Lopez-Romero et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

This study shows the seasonal effect on the antioxidant antiproliferative and antimicrobial activities of L glaucescens Kunth (LG)leaves extracts Their antioxidant activity was evaluated through the DPPH FRAP and ORAC assays Their phenolic content (PC)was determined by means of the Folin-Ciocalteu method and the main phenolic compounds were identified through a HPLC-DAD analysis Antiproliferative activity was determined by MTT assay against HeLa LS 180 M12C3F6 and ARPE cell linesAntimicrobial potential was evaluated against Staphylococcus aureus and Escherichia coli using a microdilution method All theLG extracts presented high antioxidant activity and PC with quercitrin and epicatechin being the most abundant Antioxidantactivity andPCwere affected by the season particularly autumn (ALGE) and summer (SULGE) extracts exhibited the highest values(119901 lt 005) All extracts presentedmoderate antiproliferative activity against the cell lines evaluatedHeLa being themost susceptibleof themHowever ALGE and SULGEwere themost active too About antimicrobial activity SULGE (MIC90 lt 800 120583gmLMIC50 lt400 120583gmL) and SLGE (MIC50 lt 1000120583gmL) showed a moderate inhibitory effect against S aureus These findings provide newinformation about the seasonal effect on the PC and biological properties of LG extracts Clearly antioxidant activity was the mostimportant with respect to the other two

1 Introduction

Nowadays diseases related to oxidative stress and to antimi-crobial resistance are considered the main public healthconcern leading to the highest mortality rates worldwide[1 2] Oxidative stress has been explained in terms of the over-production of intracellular reactive oxygen species whichmay produce damage to biomolecules such as DNA RNA

lipids and proteins [3] Therefore the cellular damage wouldeventually result in the development of chronic diseaseslike cancer atherosclerosis rheumatoid arthritis diabeteschronic inflammation and cardiovascular ills among others[4]

On the other hand antimicrobial resistance is the resultof antibiotic misuse which conduces to stronger infectionswith complicated clinical treatments like respiratory tract

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 2738489 11 pageshttpsdoiorg10115520182738489

2 Evidence-Based Complementary and Alternative Medicine

infections rhinosinusitis otitis media cystic fibrosis lunginfection dental caries and chronic wounds among others[5 6] These complications reduce the conventional antibi-otics efficacy and length of the hospitalization stays andincrease the medical treatment costs associated with theresearch and application of broad spectrum antibiotics [7]Each year around 2 million people are infected by antibioticresistant bacteria in USA and thousands die due to infectionswith clinical complications [8] In this context natural agentsemerge as a safe alternative to reduce the problem of theoxidative stress and antimicrobial diseases

Plants are traditionally used in folk medicine to treatdifferent illnesses and nearly 80 of worldwide populationhad used them with this purpose especially for beinga natural source easily available for the communities [910] Their positive health benefits are associated with thepresence of chemical compounds derived from secondarymetabolism such as phenolic compounds essential oilsterpenes saponins alkaloids and polypeptides which areused by plants as part of their defense mechanisms [11 12] Inaddition these compounds had shown a broad spectrum ofbiological activities demonstrating the potential of plants asalternative drugs [13 14] However the content of bioactivecompounds depends on biotic and abiotic factors such asthe presence of microorganisms and competitor speciesaround the plant temperature light intensity UV radiationhumidity water minerals and environmental contamination[15 16] These factors regulate the production of secondarymetabolites and subsequently the potential use of medicinalplants [17] In this sense the study of the effect that thedifferent seasons have on the chemical composition andbiological properties of plants can contribute to their optimaluse in the folk medicine [18 19]

Litsea glaucescens Kunth is a native plant from CentralAmerica and Mexico mainly distributed in the states ofChiapas Nayarit and Veracruz where it is known as ldquolaurelrdquo[20] Its leaves have been traditionally used as food seasoningas well as remedy in folk medicine against central nervoussystem illness depression colic pain vomit and diarrhea[21] These activities are mainly related to the presence ofdifferent compounds such as terpenes and phenolic com-pounds [22 23]The goal of the present study was to evaluatethe seasonal effect on the antioxidant antimicrobial andthe antiproliferative activities of L glaucescens Kunth leavesextracts as well as on their content and profile of phenoliccompounds since to the best of our knowledge this is thefirst effort to describe at this level the biological propertiesand chemical composition of ldquolaurelrdquo commonly used as aremedy by the communities from the mountainous region ofVeracruz Mexico

2 Materials and Methods

21 Reagents Folin-Ciocalteursquos phenol reagent sodiumcarbonate (Na2CO3) 11-diphenyl-2-picrylhydrazyl (DPPH)6-hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) 246-tri(2-pyridyl)-striazine (TPTZ) iron(III) chloride hexa-hydrate sodium acetate trihydrate

(C2H3NaO2sdot3H2O) hydrochloric acid 221015840-azobis(2-amidi-

nopropane) dihydrochloride (AAPH) gentamicin sodiumchloride (NaCl) Dulbeccorsquos Modified Eaglersquos Medium(DMEM) high glucose dimethyl sulfoxide (DMSO)isopropyl alcohol and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) as well as authenticstandards of epicatechin quercitrin gallic acid chlorogenicacid caffeic acid trans-cinnamic acid scopoletin hesperidinrosmarinic acid myricetin genistein luteolin and apigeninwere purchased from Sigma-Aldrich (St Louis MO USA)Standards of naringenin hesperetin chrysin galangin andacacetin were purchased from INDOFINE Chemical CoInc (Hillsborough NJ USA) Mueller-Hinton broth (MHB)andMueller-Hinton agar (MHA) were obtained from BectonDickinson (Cockeysville MD USA) HPLC-grade water(18mΩ) was performed by a Milli-Q50 purified system(Millipore Corp Bedford MA USA)

22 PlantMaterial and Preparation of Extracts L glaucescensleaves were collected during autumn (November 2015)winter (February 2016) spring (May 2016) and summer(September 2016) in Xico Veracruz Mexico L glaucescensleaves were identified in the Herbarium of Instituto de Inves-tigaciones Biologicas of Universidad Veracruzana MexicoCollected leaves were washed and dried Dried leaves wereextracted withmethanol (96) during 4 days with occasionalstirring (2-3 times per day) The extracts were filtered usingfilter paper (Whitman grade number 4) and the solvent wasevaporated to dryness under reduced pressure at 40∘C in arotary evaporatorThe obtained extracts were stored at minus20∘Cand identified as L glaucescens autumn winter spring andsummer extracts (ALGE WLGE SLGE and SULGE resp)

23 Total Phenolic Content Total phenolic concentrationwasdetermined with Folin-Ciocalteu reagent according to themethod described by Velazquez et al [24] Briefly 10 120583Lof extracts (1mgmL) was mixed with 80 120583L of distilledwater 40 120583L of Folin-Ciocalteu reagent 025N 60 120583L sodiumcarbonate (5 in distilled water) and 80 120583L of distilled waterThe mixtures were incubated at room temperature (1 h) Theabsorbance of the samples was measured at 750 nm on a Flu-ostar Omega microplate reader (BMG Labtech OrtenbergGermany) and the results were expressed as milligrams ofgallic acid equivalent (GAE)gram of dry weight (d w)

24 HPLC-DAD Analysis Analytical HPLC-DAD analysiswas carried out on an Agilent 1220 Infinity DAD LC (Wald-bronn Germany) equipped with a Zorbax SB-C18 column(250 times 46mm Oslash 35 120583m Agilent USA) The mobilephase consisted of 5 formic acid in water (solvent A)and methanol (solvent B) The elution was accomplishedwith a solvent flow rate of 1mLmin using a gradientprogram as follows 5 B (0ndash5min) 10 B (5ndash10min) 15B (10ndash18min) 25 B (18ndash28min) 30 B (28ndash40min) 40B (40ndash45min) 45 B (45ndash55min) 60 B (55ndash60min) 80B (60ndash65min) 100 B (65ndash76min) and 30 B (76ndash86min)Flavonoids were monitored at 280 and 340 nm Identificationof phenolic compounds was carried out by comparison of theretention times and spectra with those of authentic standards

Evidence-Based Complementary and Alternative Medicine 3

Quantification of both compounds was performed throughcalibration curves Results were expressed as mg of eachcompound100mg of dw

25 DPPH Assay Free-radical scavenging activity was mea-sured following the modified method reported by Usia et al[25] L glaucescens extracts (100 120583L) were mixed with a 300120583M DPPH solution (100 120583L) Samples were kept in the darkfor 30min Afterward absorbance at 517 nm was measuredon a microplate reader (Fluostar Omega microplate readerBMG Labtech Ortenberg Germany) L glaucescens extractswere tested at different concentrations (0 to 100 120583gmL)Results were expressed as 120583M of trolox equivalents (TE)g ofdw and IC50 IC50 values were calculated throughout linearregression analysis using Microsoft Excel software

26 FRAP Assay Ferric reducing ability was evaluatedaccording to themethodology described by Benzie and Strain[26] Working FRAP reagent was elaborated reacting 10volumes of 300mM acetate buffer (pH 36) 1 volume of40mM TPTZ (dissolved in 40mM HCl) and 1 volume of20mM ferric chloride (dissolved in water) Subsequently 280120583L of FRAP reagent was mixed with 20 120583L (05mgmL) of Lglaucescens extracts and the absorbancewas read at 630 nmatamicroplate reader (FluostarOmegamicroplate reader BMGLabtech Ortenberg Germany) after 30min of storage in thedark Results were reported as 120583M of Fe(II)g of dw

27 ORAC Assay Oxygen radical absorbance capacity assaywas carried out using a modified method described by Ouet al [27] AAPH reagent was used as peroxyl radical gen-erator and fluorescein as the fluorescent indicator Reactionmixture contained 150 120583L of fluorescein (10 nM) 25 120583L ofphosphate buffer (75mM pH 74) as blank and 25 120583L (50120583gmL) of extracts Reaction was started by the additionof AAPH (240mM) Samples were preincubated at 37∘C(15min) and the fluorescence was monitored every 90 s for15 h at 485ndash520 nm on a microplate reader (Fluostar Omegamicroplate reader BMG Labtech Ortenberg Germany)Results were expressed as 120583M TEg dw

28 Bacterial Strains and Growth Conditions Escherichia coliATCC 25922 and Staphylococcus aureus ATCC 25923 wereemployed in the experiments These strains were maintainedat minus70∘C in cryovials containing glycerol (10) broth andsubculture in Mueller-Hinton broth at 37∘C during 24 hbefore testing

29 Antibacterial Assay Antibacterial activity of extracts wasevaluated by the modified microdilution broth method [24]Briefly after overnight growth at 37∘C in Mueller-Hintonagar 15 120583L (15 times 106 CFU) of a suspension of a logarith-mic phase bacterial culture [108 CFUmlminus1 the turbidity ofthis bacterial suspension matching the turbidity of a 05McFarland standard] was inoculated into each well of a flat96-well microplate (Costar Corning NY USA) containing200 120583L of different concentrations (100ndash1000 120583gmL) of Lglaucescens The extracts were dissolved previously in DMSO

and subsequently diluted in sterile MHB The percentage ofDMSO did not exceed 2 (vv) of the total volume per well(215 120583L) Gentamicin (12 120583gmL) was used as positive controlof bacterial growth inhibition Plates were incubated for 48 hat 37∘C and read later at 620 nm on a microplate reader(Multiskan EXThermoLab System) at 6 12 24 and 48 hTheminimal inhibitory concentration was defined as the lowestextracts concentration that inhibited at least 50 (MIC50) or90 (MIC90) of the bacterial growth after incubation (37

∘C times24 h) MICs values were calculated from the Optical Density(OD620 nm) data using the following equations

MIC50

(OD620 nm untreated bacteria minusOD620 nm test concentration)(OD620 nm untreated bacteria)

times 100 ge 50

MIC90


times 100 ge 90

(1)

210 Cell Lines Cell lines LS 180 (human colonic adeno-carcinoma) HeLa (human cervix carcinoma) and ARPE-19(human retinal pigmented epithelium) were obtained fromthe American Type Culture Collection (ATCC RockvilleMD USA) The cell line M12C3F6 (murine B-cell lym-phoma) was provided by Dr Emil R Unanue (Departmentof Pathology and Immunology Washington University in StLouis MO USA) Cells were cultured in DMEM supple-mented with 5 FBS (Sigma St Louis MO USA)

211 Antiproliferative Assay Cell proliferation was evaluatedthrough the MTT assay [28] modified by Hernandez et al[29] Briefly 50 120583L (1 times 104 cells) was placed in each well ofa flat 96-well plate and incubated for 24 h (37∘C 5 of CO2atmosphere) Then 50 120583L of medium containing differentconcentrations of extracts was added and the cell cultureswere incubated for 48 h Extracts were previously dissolvedin DMSO DMSO did not exceed 05 of the total volumeper well (preliminary studies showed that DMSO at thisconcentration does not cause damage of cell) Caffeic acidphenethyl ester (CAPE) was used as a positive control in theantiproliferative assay In the last 4 h of the LS 180 HeLaand ARPE cell line cultures each well was washed with PBSand refilled with new fresh culture medium Subsequently10 120583L of a MTT solution (5mgmL) was added to each well(in the case of the M12C3F6 cell line culture only MTTsolution (5mgmL) was added) Metabolically active cellsreduced tetrazolium salt to colored formazan crystals whichwere dissolved with acidic isopropyl alcohol Microplateswere read at 570 and 650 nm (Multiskan EX ThermoLabSystem) Resultswere expressed as IC50 values (IC50 is definedas the required concentration to inhibit 50 of the cellproliferation) Graphic of living cells () versus extractsconcentrations was traced IC50 values were calculated usingnonlinear regression analysis in Microsoft Excel software


ALGE WLGE

a

b

c c

SLGE SULGE0

20

40

60

80

100

120

140

160

(mg

GA

Eg

dw

)

Figure 1 Phenolic content of L glaucescens extracts andashcBars withdifferent superscript indicate statistical differences (119901 lt 005)(ALGE autumn L glaucescens extract WLGE winter L glaucescensextract SLGE spring L glaucescens extract SULGE summer Lglaucescens extract)

212 Statistical Analysis Data analysis was performed usingthe NCCS 2007 statistical software One-way ANOVA wasused and mean comparisons were performed using theTukey-Kramer test Significance level in Type I error was119901 le 005 Pearson correlation between phenolic content andDPPH FRAP and ORAC values were estimated too

3 Results and Discussion

31 Phenolic Compounds The phenolic content of the Lglaucescens extracts ranged from 929 plusmn 44 to 1382 plusmn 67mgGAEg dw The highest concentrations (119901 lt 005) werefound in ALGE and SULGE followed by SLGE and WLGE(Figure 1) These data agree with those reported by Iqbaland Bhanger [30] Brahmi et al [31] and Sivaci and Duman[32] who evaluated the seasonal effect of phenolic content ofmoringa olive and almond leaves extracts respectively Inthe three studies they found that autumn extracts presentedthe highest phenolic concentrations in comparison with thesamples of the other seasons

To identify the main phenolic compounds of the extractsof L glaucescens a HPLC-DAD analysis was performed Thechromatographic profiles of the four seasonal extracts areshown in Figure 2 As can be observed the evident differenceamong them is the height of the chromatographic peaks(related to the concentration of the phenolics) Comparisonof the retention times and spectra with those from a set ofcommercial standards allowed us to identify two of the mainphenolic compounds present in L glaucescens extracts epi-catechin and quercitrin which present quantitative variationin L glaucescens throughout the year (Figure 2) Quercitrinwas the most abundant phenolic compound in the fourextracts and to the best of our knowledge this is the first timethat it is reported as a component of L glaucescens SULGEpresented the highest amount (119901 lt 005) of quercitrinfollowed by SLGE ALGE and WLGE (Table 1) Epicatechinthe second-major flavonoid found in the extracts has been

ALGE

SLGE

WLGE

SULGEQR

QR

QR

QREP

EP

EP

EP

00

10 20

20

30 40

40

50 60

60

70 80

80100120

(min)(m

AU)

020406080

100120

(mAU

)

020406080

100120

(mAU

)

020406080

100120

(mAU

)

Figure 2 HPLC chromatogram of L glaucescens extracts (recordedat 280 nm) (EP epicatechin QR quercitrin) (ALGE autumn Lglaucescens extract WLGE winter L glaucescens extract SLGEspring L glaucescens extract SULGE summer L glaucescensextract)

reported before by Gamboa-Gomez et al [33] as a secondarymetabolite of L glaucescens In this work the ALGE extractpresented the highest (119901 lt 005) epicatechin content while asimilar amount (119901 gt 005) of this compound was observed inthe other extracts

Phenolic composition of plants is mainly affected bybiotic and abiotic factors In normal conditions abioticfactors such as thermal stress play an important role in thebiosynthesis of phenolic compounds in plants because theyinduce the phenylalanine ammonia-lyase (PAL) activationwhich is the main enzyme involved in the biosynthesis ofphenylpropanoid [34 35] In addition the increase in theenzymatic activity of PAL is related to an adaptation of theplant to stress [36]Therefore it is possible to hypothesize thatL glaucescenswas subjected to a higher thermal stress duringsummer and autumn compared with spring and winterresulting in an increase of phenolic compounds duringthese seasons On the other hand phenolic compoundsare associated with a wide range of biological activities Tocontribute to the biological characterization of this plantwe evaluated its potential as antioxidant antimicrobial andantiproliferative agent

32 Antioxidant Activity Different assays are available andhave been used to evaluate the antioxidant activity of plantextracts Most of them are based on scavenging specificradicals such asDPPHand peroxyl radicals ormetal reducingpotential such as the FRAP assay In the present study weevaluated the antioxidant activity of L glaucescens extracts


Table 1 Concentration of major phenolic compounds identified in L glaucescens extracts

Compound L glaucescens extracts (mg100mg dw)ALGE WLGE SLGE SULGE

Epicatechin 156 plusmn 019b 088 plusmn 0009a 073 plusmn 002a 068 plusmn 001a

Quercitrin 211 plusmn 005b 139 plusmn 017a 301 plusmn 016c 389 plusmn 032d

andashdMeans with different superscript within the same row indicate statistical differences (119901 lt 005) All values represent mean plusmn standard deviation (ALGEautumn L glaucescens extract WLGE winter L glaucescens extract SLGE spring L glaucescens extract SULGE summer L glaucescens extract)

Table 2 Antioxidant activity of L glaucescens extracts

Extract

Antioxidant assayDPPH

(120583M TEg ofdw)

DPPH(IC50 120583gmL)

FRAP(120583M Fe (II)g of

dw)

ORAC(120583M TEg of

dw)ALGE 12645 plusmn 185c 147 plusmn 007c 26143 plusmn 1831c 36733 plusmn 611b

WLGE 6681 plusmn 199a 272 plusmn 08a 14664 plusmn 1476a 34133 plusmn 461a

SLGE 8411 plusmn 259b 243 plusmn 09b 19997 plusmn 424b 36933 plusmn 461b

SULGE 12219 plusmn 326c 152 plusmn 03c 25734 plusmn 1389c 37003 plusmn 529b

andashcMeans with different superscript within the same column indicate statistical differences (119901 lt 005) All values represent mean plusmn standard deviation (ALGEautumn L glaucescens extract WLGE winter L glaucescens extract SLGE spring L glaucescens extract SULGE summer L glaucescens extract)

throughout three chemicals assays DPPH FRAP and ORAC(Table 2) Results obtained through the DPPH methodshowed variations among the seasonal extracts From theIC50 values it is possible to observe that ALGE and SULGEwere the most active samples (119901 lt 005) against the DPPHradical in comparison with WLGE and SLGE In additionbased onBlois [37] andFidrianny et al [38] classifications theantioxidant capacity of the four extracts must be categorizedas very strong since all of them had IC50 values lower than50 120583gmL These results agree with previous studies relatedto plants from Litsea genus such as Litsea glutinosa Litseafloribunda and Litsea japonica where IC50 values rangedfrom 968 to 6692 120583gmL [39ndash41]

On the other hand ferric reducing power of the Lglaucescens extracts was evaluated through their ability toreduce the ferric complex Fe3+-tripyridyltriazine to Fe2+-tripyridyltriazine The corresponding values are shown inTable 2 and as can be observed there are significant dif-ferences among them (119901 lt 005) Particularly ALGE andSULGE exhibited the stronger power whereasWLGEhad thelowest activity These values are higher than those reportedbefore for other Litsea species (14ndash638 120583M Fe(II)g of dw)[40 42 43] In addition according to the classification ofmedicinal plants performed by Wong et al [44] the Lglaucescens extracts had an extremely high ferric reductionpower since the obtained values were higher than 500 120583MFe(II)g of dw

The capacity of L glaucescens extracts to scavenge theAAPH-derived peroxyl radical was evaluated through theORAC assay and the results are shown in Table 2 similarvalues were obtained for SULGE SLGE and ALGE whilethe lowest one was registered for WLGE Although all theextracts had strong antioxidant activity the results presentedhere demonstrated that during the winter the capacity of Lglaucescens to react with peroxyl radical decreased

The three types of tests performed in this study providedevidences about the high ability of the four extracts to transferelectron and hydrogen atoms to stabilize free radicals andreduce metals related to their strong antioxidant activity Inaddition the significant effect that the seasons had on theantioxidant capacity of L glaucescens extracts is notoriousParticularly ALGE and SULGE showed a higher activity withrespect to SLGE and WLGE Additionally the four extractsexhibited an interesting ability to act as preventive and chain-breaking antioxidants with activity against biological andsynthetic radicals These facts suggest that they have thepotential to stabilize biological radicals and to inhibit thegeneration of reactive oxygen species which could contributeto reducing the oxidative stress caused by them and thereforeto avoiding the DNA damage

It is well known that the antioxidant activity of naturalproducts is strongly related to the content of phenolic com-pounds that they have and the results obtained in this workagree with that fact In order to demonstrate the correlationbetween both parameters a series of plots of the three datapieces obtained through the DPPH FRAP and ORAC assaysfor each L glaucescens extract (the samples were evaluated intriplicate) against the corresponding averaged concentrationof phenolic compound (CPC) were performed (Figure 3)The regression coefficients (119903) of the linear correlations foreach series are presented in Figure 3 As can be observedpositive slopes were obtained in all the cases On the otherhand the lowest regression coefficient was obtained for thecorrelation between the data of the ORAC test versus CPChowever the value is still into an acceptable range As wehypothesized it seems that phenolic compounds were themain compounds responsible for the antioxidant activity ofthe four L glaucescens extracts Although the evaluated sam-ples have other phenolic constituents not identified in thiswork it is possible that epicatechin and quercitrin could play


r = 092

90

100

110

120

130

140

150CP

C

700 800 900 1000 1100 1200 1300 1400600DPPH

(a)

r = 093

1500 2000 2500 30001000FRAP

90

100

110

120

130

140

150

CPC

(b)

r = 080

90

100

110

120

130

140

150

CPC

3400 3500 3600 3700 38003300ORAC

(c)

Figure 3 Plots of the data obtained through the DPPH FRAP and ORAC assays against the concentration of phenolic compound (CPC) inthe four L glaucescens extracts The correlation coefficients (119903) are shown in each graph

O

OH

OH

HO

OH

1

2

3

456

78

A

B

C

OH

O

OH

HO

OH

1

2

3

456

78

A

B

C

O

OH

O-Rhamnose

Epicatechin Quercitrin

1

2

3

4

5

6

1

2

3

4

5

6

Figure 4 Structure of identified phenolic compounds in L glaucescens extracts

an important role in the high antioxidant activity of ALGEand SULGE since different studies have demonstrated thatboth phenolics are considered among the most antioxidantphenolic compounds [45 46] and that capacity has beenattributed to the catechol and chromane moieties that theyhave (Figure 4) Particularly the presence of hydroxyl groupsin 31015840- and 41015840-position of ring B hydroxyl group 3 of ring Cand double bond betweenC2 andC3 enhance the antioxidantactivity of these phenolic compounds since they can transferelectrons and protons to stabilize free radicals or to reduceand chelate metals These structural features confer to bothcompounds a greater stability compared with those thatlack them [13 47] In addition these facts determine also

the redox potential and therefore the antioxidant activity ofphenolic compounds [48]

33 Antiproliferative Activity The results of the antiprolifera-tive activity evaluation of L glaucescens extracts againstHeLaLS 180 M12C3F6 and ARPE cells are shown in Table 3Although all the extracts inhibited the proliferation of humanand murine cells lines their effect was moderate As can beobserved in Table 3 HeLa was the more sensitive cell lineto the L glaucescens extracts particularly to the SULGE andALGE ones which exhibited the highest activity (119901 lt 005)against its proliferation Regarding LS 180 ALGE and SULGEshowed the stronger activity too (119901 lt 005) whereas in the


Table 3 Antiproliferative activity of L glaucescens extracts

Cell lineL glaucescens extracts IC50 (120583gmL)

ALGE WLGE SLGE SULGE CAPE(120583gmL120583M)

HeLa 487 plusmn 18b 539 plusmn 26bc 59 plusmn 78c 458 plusmn 16b 97 plusmn 007a341 plusmn 02

LS 180 531 plusmn 12a 852 plusmn 35c 675 plusmn 39b 556 plusmn 15a 178 plusmn 02a626 plusmn 07

M12C3F6 719 plusmn 62b 706 plusmn 21b 732 plusmn 25b 681 plusmn 13b 058 plusmn 004a204 plusmn 01

ARPE 621 plusmn 36b 1661 plusmn 49d 1019 plusmn 56c 1022 plusmn 19c 102 plusmn 018a359 plusmn 06andashdMeans with different superscript within the same row indicate statistical differences (119901 lt 005) All values represent mean plusmn standard deviation (ALGEautumn L glaucescens extract WLGE winter L glaucescens extract SLGE spring L glaucescens extract SULGE summer L glaucescens extract)

case of the cancerous murine cell line (M12C3F6) the fourextracts had a similar antiproliferative effect (119901 gt 005) Inaddition ALGE showed the lower IC50 value (119901 lt 005)to inhibit the proliferation of the noncancerous ARPE cellline however much higher concentrations of SLGE SULGEand WLGE were required Even more these last three valuesare the highest of Table 3 and constitute an evidence ofthe selectiveness of the L glaucescens extracts to inhibit theproliferation of cancerous cell lines with respect to those non-cancerous ones These findings are consistent with previousstudies from Litsea plants For example in a study performedby Herrera-Carrera et al [49] it was demonstrated that aherbal infusion obtained from L glaucescens was able toinhibit the proliferation of human colon cancer cell line (HT-29) In the same way Ndi et al [50] observed inhibitionon HT-29 (IC50 = 379 120583gmL) and melanoma (SK-MEL-28)(IC50 gt 100 120583gmL) cells treated with of L glutinosa extractSubarnas et al [51] evaluated the antiproliferative activity ofL mappaceae extracts against human breast cancer (MCF-7)and reported that 200 120583gmL of plant extract was required toinhibit the 50 of cell proliferation Although in all the casesthe positive control CAPE was several times more active thanthe L glaucescens extracts (see Table 3) it should be kept inmind that the purity of the CAPE used in the assays (above95) is much higher than those of the active compoundspresent in the extracts and this fact could contribute to thedifferences observed in the IC50 values

The results reported in this work are an evidence of theantiproliferative effect of L glaucescens against cancerouscell lines in comparison with those noncancerous ones Inaddition the influence of seasons on its antiproliferativeactivity was significatively different in three of the four celllines studied here However in some cases the registereddifferences were still modest ALGE and SULGE were themore active extracts which could be related to their highcontent of phenolic compounds Nevertheless the structuralfeatures of these phenolics are important too such as thosedescribed above as enhancer of the antioxidant activity of theL mappaceae extracts [52] In this sense Kinjo et al [53] andNagarajan et al [54] proposed that epicatechins one of themost abundant phenolics of the four extracts reported herepossess potent antiproliferative activity against cancerouscells lines which was related to an arrest of the cell cyclein the G2 phase On the other hand previous studies havereported that phenolic compounds exhibit different mode of

actions against cancerous cell lines for example the induc-tion of apoptosis the cell cycle arrest and the preventionof carcinogen metabolic activation with the subsequent celldeath [55 56]

34 Antimicrobial Activity Results of antibacterial activityof L glaucescens extracts are summarized in Table 4 andFigure 5 As can be observed from Table 4 SULGE andSLGE showed the strongest activity against S aureus sincethe MIC50 values obtained for both are below the maximumconcentration evaluated (1000 120583gmL) which constitutes anevidence of the important seasonal effect on the biologicalproperties of L glaucescens In contrast no antimicrobialactivity of any of the four extracts was observed againstE coliThe fact that S aureus (Gram-positive) was less resistant thanE coli (Gram-negative) to L glaucescens could be attributedto the cell structure and composition of both types ofmicroorganisms In this regard it has been proposed by otherauthors that the outer membrane of Gram-negative bac-teria constituted by phospholipid and lipopolysaccharidesprovides resistance to antimicrobial treatments [57] On theother hand the porins in outer membrane could regulatetoo the penetration of hydrophilic substances and reduce thefluidity of the lipopolysaccharides layer decreasing the rateof transmembrane diffusion [58] According to those studiesthe Gram-positive bacteria would have lesser resistance toantimicrobial treatments

In addition the dose-depend relationships of the activeextracts (SULGE and SLGE) against S aureus were exploredand the corresponding plots are shown in Figure 5 As can beobserved there is a clear effect of the concentration of both ontheir antimicrobial activity SULGE the most potent extractwas able to inhibit 100 of bacteria growth at the highesttested concentration (1000 120583gmL) 98 at 800 120583gmL 70at 600 120583gmL and up to 63 at 400 120583gmL In additionhigher concentrations such as 1000 and 800 120583gmL had asimilar activity with respect to gentamicin (gt98 inhibition)Although in a minor proportion than SULGE SLGE alsoexhibited antimicrobial effect depending on concentrationagainst S aureus As can be observed from Figure 5 a con-centration of 1000 120583gmL inhibited 51 of bacteria growthwhile other concentrations provoked an inhibition lower than50 Phytochemicals are classified as antimicrobials basedon susceptibility test achieving inhibitory concentrations inthe range of 100 to 1000 120583gmL Thus extracts with MIC


Table 4 Growth-inhibitory activity of L glaucescens extracts against S aureus and E coli

StrainL glaucescens extracts (120583gmL)

ALGE WLGE SLGE SULGEMIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90

S aureus gt1000 gt1000 gt1000 gt1000 lt1000 gt1000 lt400 lt800E coli gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000 gt1000

SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)

Figure 5 Antibacterial activity of L glaucescens extracts against Staphylococcus aureus (SLGE spring L glaucescens extract SULGE summerL glaucescens extract) Bacterial cell cultures were treated with different concentrations of L glaucescens extracts during 48 hX 1000 120583gmL998787 800 120583gmL 998771 600120583gmL ◼ 400120583gmL e 0 120583gmL times gentamicin Control bacterial cultures were incubated with DMSO (08ndash2)Gentamicin (12 120583gmL) was used as positive control All values represent mean plusmn standard deviation

values equal to or lower than 100 120583gmL are consideredeffective antimicrobial agents while those with MIC valuesbetween 100 and 500 120583gmL and between 500 and 1000120583gmL are known as moderate and low antimicrobial agentsrespectively Extracts with MIC values above 1000 120583gmL donot have antimicrobial activity [59 60] In this sense SULGEand SLGE showed moderate to low antimicrobial activityagainst S aureus while the other extracts were consideredinactive against this bacterium

On the other hand the antibacterial activity of Lglaucescens extracts evaluated in the present research isconsistent with previous works about the Litsea genus Forexample in a study performed by Ali-Ahmmad et al [61]L monopetala extracts presented inhibitory effect againstS aureus and E coli at concentrations of 625 and 250120583gmL respectively Similarly Areekul et al [62] evaluatedthe antimicrobial activity of a L glutinosa extract at 697(wv) against S aureus and E coli Although lower incomparison to the effect of chloramphenicol which was usedas positive control the L glutinosa extract showed activityagainst S aureus (2134mm and 878mmof inhibition resp)Nevertheless it was not able to inhibit the growth of E coli Inaddition Pradeepa et al [63] reported a behavior close to theresults reported here since they observed that the L glutinosaextracts presented greater antimicrobial activity against Saureus (MIC = 25mgmL) compared with E coli (MIC =5mgmL)

Antimicrobial effect of active L glaucescens extracts couldalso be related to the high phenolic compounds content

In this regard Borges et al [64] and Andrade et al [65]demonstrated that phenolic compounds induced alteration ofmembrane properties producing changes in the hydropho-bicity surface charge and membrane integrity with thesubsequent leakage of essential intracellular constituents ofGram-positive and Gram-negative bacteria On the otherhand Cushnie and Lamb [66 67] have concluded thatphenolic compounds have different mechanism of action asantimicrobial such as inhibitors of nucleic acid synthesisof the energy metabolism or of the cytoplasmic membranefunction of the microorganisms

4 Conclusions

This research provides information about seasonal effect onthe concentration of phenolic compounds (particularly epi-catechin and quercitrin) present in L glaucescens extracts aswell as on a series of biological activities that they have Fromthe results reported here the antioxidant activity was thestrongest one and clearly dependent on the seasonThereforethe L glaucescens extracts could be a promising alternativeto avoid oxidative stress On the other hand antiproliferativeand antimicrobial activities were moderate The first of themincreased slightly during the autumn and summer which isrelated to the major concentration of phenolic compoundsproduced by the plant as response to the environmentalconditions In addition L glaucescens extracts collectedduring summer and spring showed moderate antimicrobialactivity against S aureus which may be related to the high


quercitrin concentration produced by the plant during thoseseasons Despite the interesting properties that this Mexicanplant has subsequent studies are required to support itseffectiveness and safety doses for human applications

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

Thanks are due to Q B Monica Villegas for her helpwith the ORAC assay and M C Lucila Rascon for hertechnical assistance with the cell culture and the antipro-liferative assays Julio Cesar Lopez-Romero acknowledgesCONACYT-Mexico for a PhD scholarship

References

[1] I RocaMAkova F Baquero et al ldquoThe global threat of antimi-crobial resistance Science for interventionrdquo New Microbes andNew Infections vol 6 pp 22ndash29 2015

[2] Y Kayama U Raaz A Jagger et al ldquoDiabetic cardiovasculardisease induced by oxidative stressrdquo International Journal ofMolecular Sciences vol 16 no 10 pp 25234ndash25263 2015

[3] A M Pisoschi and A Pop ldquoThe role of antioxidants in thechemistry of oxidative stress a reviewrdquo European Journal ofMedicinal Chemistry vol 97 pp 55ndash74 2015

[4] A H Bhat K B Dar S Anees et al ldquoOxidative stressmitochondrial dysfunction and neurodegenerative diseases amechanistic insightrdquo Biomedicine amp Pharmacotherapy vol 74pp 101ndash110 2015

[5] J M A Blair M A Webber A J Baylay D O Ogbolu and LJ V Piddock ldquoMolecular mechanisms of antibiotic resistancerdquoNature Reviews Microbiology vol 13 no 1 pp 42ndash51 2015

[6] M Wilkins L Hall-Stoodley R N Allan and S N FaustldquoNew approaches to the treatment of biofilm-related infectionsrdquoInfection vol 69 no 1 pp S47ndashS52 2014

[7] S E Cosgrove ldquoThe relationship between antimicrobial resis-tance and patient outcomes Mortality length of hospital stayand health care costsrdquo Clinical Infectious Diseases vol 42 no 2pp S82ndashS89 2006

[8] CDC (Center forDiseasesControl andPrevention) ldquoAntibioticAntimicrobial Resistancerdquo 2018 httpswwwcdcgovdrugresist-ance

[9] H-S Kim ldquoDo not put too much value on conventionalmedicinesrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp37ndash39 2005

[10] M Asadi-Samani N Kafash-Farkhad N Azimi A Fasihi EAlinia-Ahandani and M Rafieian-Kopaei ldquoMedicinal plantswith hepatoprotective activity in Iranian folk medicinerdquo AsianPacific Journal of Tropical Biomedicine vol 5 no 2 pp 146ndash1572015

[11] J C Lopez-Romero R Ansorena G A Gonzalez-AguilarH Gonzalez-Rios J F Ayala-Zavala and M W SiddiquildquoChapter 5 Applications of plant secondary metabolites in foodsystemsrdquo in Plant SecondaryMetabolites Volume 2 StimulationExtraction and Utilization pp 195ndash232 Apple Academic Press2016

[12] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medicinalplants associated with anticancerrdquo Life Sciences vol 74 no 17pp 2157ndash2184 2004

[13] N Balasundram K Sundram and S Samman ldquoPhenolic com-pounds in plants and agri-industrial by-products antioxidantactivity occurrence and potential usesrdquo Food Chemistry vol99 no 1 pp 191ndash203 2006

[14] F Bakkali S Averbeck D Averbeck and M Idaomar ldquoBio-logical effects of essential oilsmdasha reviewrdquo Food and ChemicalToxicology vol 46 no 2 pp 446ndash475 2008

[15] A Ramakrishna and G A Ravishankar ldquoInfluence of abioticstress signals on secondary metabolites in plantsrdquo Plant Signal-ing and Behavior vol 6 no 11 pp 1720ndash1731 2011

[16] N J Atkinson and P E Urwin ldquoThe interaction of plantbiotic and abiotic stresses From genes to the fieldrdquo Journal ofExperimental Botany vol 63 no 10 pp 3523ndash3544 2012

[17] L G Korkina ldquoPhenylpropanoids as naturally occurringantioxidants from plant defense to human healthrdquo Cellular andMolecular Biology vol 53 no 1 pp 15ndash26 2007

[18] S M Alencar T L Oldoni and M L Castro ldquoChemicalcomposition and biological activity of a new type of Brazilianpropolis red propolisrdquo Journal of Ethnopharmacology vol 113no 2 pp 278ndash283 2007

[19] V Cechinel Filho ldquoChemical composition and biological poten-tial of plants from the genus Bauhiniardquo Phytotherapy Researchvol 23 no 10 pp 1347ndash1354 2009

[20] A O Tucker M J Maciarello and M Hill ldquoLitsea glaucescensHumb Bonpl amp Kunth var Glaucescens (Lauraceae) A Mexi-can bayrdquo Economic Botany vol 46 no 1 pp 21ndash24 1992

[21] D N C Del Jimenez-Perez F G Lorea-Hernandez C KJankowski and R Reyes-Chilpa ldquoEssential oils inMexican bays(Litsea spp Lauraceae) Taxonomic assorment and ethnob-otanical implicationsrdquo Economic Botany vol 65 no 2 pp 178ndash189 2011

[22] J A Lopez W Barillas J Gomez-Laurito et al ldquoFlavonoids ofLitsea glaucescensrdquo Planta Medica vol 61 no 2 p 198 1995

[23] S L Guzman-Gutierrez R Gomez-Cansino J C Garcıa-Zebadua N C Jimenez-Perez and R Reyes-Chilpa ldquoAntide-pressant activity of Litsea glaucescens essential oil Identifica-tion of 120573-pinene and linalool as active principlesrdquo Journal ofEthnopharmacology vol 143 no 2 pp 673ndash679 2012

[24] C Velazquez M Navarro A Acosta et al ldquoAntibacterial andfree-radical scavenging activities of Sonoran propolisrdquo Journalof Applied Microbiology vol 103 no 5 pp 1747ndash1756 2007

[25] T Usia A H Banskota Y Tezuka K Midorikawa K Mat-sushige and S Kadota ldquoConstituents of Chinese propolis andtheir antiproliferative activitiesrdquo Journal of Natural Productsvol 65 no 5 pp 673ndash676 2002

[26] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of rsquoantioxidant powerrsquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[27] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal ofAgricultural and Food Chemistry vol 49 no 10 pp 4619ndash46262001

[28] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983


[29] J Hernandez F M Goycoolea J Quintero et al ldquoSonoranpropolis Chemical composition and antiproliferative activityon cancer cell linesrdquo Planta Medica vol 73 no 14 pp 1469ndash1474 2007

[30] S Iqbal and M I Bhanger ldquoEffect of season and productionlocation on antioxidant activity ofMoringa oleifera leaves grownin Pakistanrdquo Journal of Food Composition and Analysis vol 19no 6-7 pp 544ndash551 2006

[31] F Brahmi B Mechri S Dabbou M Dhibi and M HammamildquoThe efficacy of phenolics compounds with different polaritiesas antioxidants from olive leaves depending on seasonal varia-tionsrdquo Industrial Crops and Products vol 38 no 1 pp 146ndash1522012

[32] A Sivaci and S Duman ldquoEvaluation of seasonal antioxidantactivity and total phenolic compounds in stems and leavesof some almond (Prunus amygdalus L) varietiesrdquo BiologicalResearch vol 47 no 1 article no 9 2014

[33] C I Gamboa-Gomez R F Gonzalez-Laredo J A Gallegos-Infante et al ldquoAntioxidant and angiotensin-converting enzymeinhibitory activity of Eucalyptus camaldulensis and Litseaglaucescens infusions fermented with kombucha consortiumrdquoood Technology and Biotechnology vol 54 no 3 pp 367ndash3732016

[34] V Cheynier G Comte K M Davies V Lattanzio and SMartens ldquoPlant phenolics Recent advances on their biosynthe-sis genetics andecophysiologyrdquo Plant Physiology and Biochem-istry vol 72 pp 1ndash20 2013

[35] R M Rivero J M Ruiz P C Garcıa L R Lopez-LefebreE Sanchez and L Romero ldquoResistance to cold and heatstress Accumulation of phenolic compounds in tomato andwatermelon plantsrdquo Journal of Plant Sciences vol 160 no 2 pp315ndash321 2001

[36] A Kacperska ldquoWater potential alteration a prerequisite or atriggering stimulus for the development of freezing tolerancein overwintering herbaceous plantsrdquo in Advances in Plant ColdHardiness P H Li and L Christerson Eds pp 73ndash81 CRCPress Boca Raton FL USA 1992

[37] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199-1200 1958

[38] I Fidrianny T Aristya and R Hartati ldquoAntioxidant capacitiesof various leaves extracts from three species of legumes andcorrelation with total flavonoid phenolic carotenoid contentrdquoInternational Journal of Pharmacognosy and PhytochemicalResearch vol 7 no 3 pp 628ndash634 2015

[39] R Bhowmick M S Sarwar S M R Dewan et al ldquoCharacter-ization of chemical groups and investigation of cytotoxic andantioxidant activity of Litsea glutinosa leavesrdquo Journal of PlantSciences vol 2 no 6-1 pp 24ndash29 2014

[40] M Devika H Joshi and M S Nalini ldquoPhytochemicalsantioxidative and in vivo hepatoprotective potentials of Litseafloribunda (BL)Gamble (lauraceae) - an endemic tree species ofthe SouthernWestern Ghats Indiardquo Jordan Journal of BiologicalSciences vol 9 no 3 pp 163ndash171 2016

[41] W-J Yoon S C Kang Y-M Ham et al ldquoAntioxidative andanti-inflammatory activities of Litsea japonica leavesrdquo Journalof the Korean Society for Applied Biological Chemistry vol 53no 1 pp 27ndash32 2010

[42] L Fu B-T Xu X-R Xu X-S Qin R-Y Gan and H-B LildquoAntioxidant capacities and total phenolic contents of 56 wildfruits from South Chinardquo Molecules vol 15 no 12 pp 8602ndash8617 2010

[43] N Jain S Goyal andKG Ramawat ldquoEvaluation of antioxidantproperties and total phenolic content of medicinal plants usedin diet therapy during postpartum healthcare in RajasthanrdquoInternational Journal of Pharmacy and Pharmaceutical Sciencesvol 3 no 3 pp 248ndash253 2011

[44] C-C Wong H-B Li K-W Cheng and F Chen ldquoA systematicsurvey of antioxidant activity of 30 Chinese medicinal plantsusing the ferric reducing antioxidant power assayrdquo Food Chem-istry vol 97 no 4 pp 705ndash711 2006

[45] M S Hasan M I Ahmed S Mondal et al ldquoAntioxidantantinociceptive activity and general toxicity study ofDendroph-thoe falcata and isolation of quercitrin as themajor componentrdquoOriental Pharmacy and Experimental Medicine vol 6 no 4 pp355ndash360 2006

[46] D Villano M S Fernandez-Pachon M L Moya A M Tron-coso and M C Garcıa-Parrilla ldquoRadical scavenging ability ofpolyphenolic compounds towards DPPH free radicalrdquo Talantavol 71 no 1 pp 230ndash235 2007

[47] C A Rice-Evans N J Miller and G Paganga ldquoStructure-antioxidant activity relationships of flavonoids and phenolicacidsrdquo Free Radical Biology amp Medicine vol 20 no 7 pp 933ndash956 1996

[48] E J Lien S Ren H H Bui and R Wang ldquoQuantitativestructure- activity relationship analysis of phenolic antioxi-dantsrdquo Free Radical BiologyampMedicine vol 26 no 3-4 pp 285-94 1999

[49] E Herrera-Carrera M R Moreno-Jimenez N E Rocha-Guzman et al ldquoPhenolic composition of selected herbal infu-sions and their anti-inflammatory effect on a colonic model invitro in HT-29 cellsrdquo Cogent Food and Agriculture vol 1 no 1Article ID 1059033 2015

[50] C PNdiM J SykesD J Claudie RAMcKinnon S J Sempleand B S Simpson ldquoAntiproliferative Aporphine Alkaloids fromLitsea glutinosa and Ethnopharmacological Relevance to KuukuIrsquoyu Traditional MedicinerdquoAustralian Journal of Chemistry vol69 no 2 pp 145ndash151 2016

[51] A Subarnas A Diantini R Abdulah et al ldquoApoptosis-mediated antiproliferative activity of friedolanostane triter-penoid isolated from the leaves of Garcinia celebica againstMCF-7 human breast cancer cell linesrdquo Biomedical Reports vol4 no 1 pp 79ndash82 2016

[52] C Martınez J Yanez V Vicente et al ldquoEffects of several poly-hydroxylated flavonoids on the growth of B16F10 melanomaand Melan-a melanocyte cell lines Influence of the sequentialoxidation state of the flavonoid skeletonrdquo Melanoma Researchvol 13 no 1 pp 3ndash9 2003

[53] J Kinjo T Nagao T Tanaka et al ldquoActivity-guided fractiona-tion of green tea extract with antiproliferative activity againsthuman stomach cancer cellsrdquo Biological amp PharmaceuticalBulletin vol 25 no 9 pp 1238ndash1240 2002

[54] S Nagarajan R Nagarajan S J Braunhut et al ldquoBiocatalyt-ically oligomerized epicatechin with potent and specific anti-proliferative activity for human breast cancer cellsrdquo Moleculesvol 13 no 11 pp 2704ndash2716 2008

[55] W Ren Z Qiao H Wang L Zhu and L Zhang ldquoFlavonoidspromising anticancer agentsrdquo Medicinal Research Reviews vol23 no 4 pp 519ndash534 2003

[56] E Alday D Valencia A L Carreno et al ldquoApoptotic inductionby pinobanksin and some of its ester derivatives from Sonoranpropolis in a B-cell lymphoma cell linerdquo Chemico-BiologicalInteractions vol 242 pp 35ndash44 2015


[57] F Nazzaro F Fratianni L De Martino R Coppola andV De Feo ldquoEffect of essential oils on pathogenic bacteriardquoPharmaceuticals vol 6 no 12 pp 1451ndash1474 2013

[58] P Plesiat and H Nikaido ldquoOuter membranes of Gram-negativebacteria are permeable to steroid probesrdquoMolecular Microbiol-ogy vol 6 no 10 pp 1323ndash1333 1992

[59] M Simoes R N Bennett and E A S Rosa ldquoUnderstandingantimicrobial activities of phytochemicals against multidrugresistant bacteria and biofilmsrdquoNatural Product Reports vol 26no 6 pp 746ndash757 2009

[60] G Morales A Paredes P Sierra and L A Loyola ldquoAntimicro-bial activity of three Baccharis species used in the traditionalmedicine of Northern ChilerdquoMolecules vol 13 no 4 pp 790ndash794 2008

[61] M Ali-Ahmmad T Islam I Sultana et al ldquoPharmacologi-cal and phytochemical screening of ethanol extract of Litseamonopetala (Roxb) Persrdquo IOSR Journal of Pharmacy vol 2 no3 pp 398ndash402 2012

[62] V Areekul P Jiapiyasakul and A Chandrapatya ldquoIn vitroantimicrobial screening of selected traditionalThai plantsrdquoThaiJournal of Agricultural Science vol 42 no 2 pp 81ndash89 2009

[63] K Pradeepa V Krishna Venkatesh K Girish Kumar B VThirumalesh and K J Naveen Kumar ldquoAntibacterial screeningof the stem bark and leaf extracts of Litsea glutinosa (Lour)CB Rob - An ethnomedicinally important tree of the WesternGhatsrdquo Pharmacognosy Journal vol 3 no 21 pp 72ndash76 2011

[64] A Borges C Ferreira M J Saavedra andM Simoes ldquoAntibac-terial activity and mode of action of ferulic and gallic acidsagainst pathogenic bacteriardquoMicrobial Drug Resistance vol 19no 4 pp 256ndash265 2013

[65] M Andrade S Benfeito P Soares et al ldquoFine-tuning of thehydrophobicity of caffeic acid studies on the antimicrobialactivity against Staphylococcus aureus and Escherichia colirdquo RSCAdvances vol 5 no 66 pp 53915ndash53925 2015

[66] T P Cushnie and A J Lamb ldquoAntimicrobial activity offlavonoidsrdquo International Journal of Antimicrobial Agents vol26 no 5 pp 343ndash356 2005

[67] T P T Cushnie and A J Lamb ldquoRecent advances in under-standing the antibacterial properties of flavonoidsrdquo Interna-tional Journal of Antimicrobial Agents vol 38 no 2 pp 99ndash1072011

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


infections rhinosinusitis otitis media cystic fibrosis lunginfection dental caries and chronic wounds among others[5 6] These complications reduce the conventional antibi-otics efficacy and length of the hospitalization stays andincrease the medical treatment costs associated with theresearch and application of broad spectrum antibiotics [7]Each year around 2 million people are infected by antibioticresistant bacteria in USA and thousands die due to infectionswith clinical complications [8] In this context natural agentsemerge as a safe alternative to reduce the problem of theoxidative stress and antimicrobial diseases

Plants are traditionally used in folk medicine to treatdifferent illnesses and nearly 80 of worldwide populationhad used them with this purpose especially for beinga natural source easily available for the communities [910] Their positive health benefits are associated with thepresence of chemical compounds derived from secondarymetabolism such as phenolic compounds essential oilsterpenes saponins alkaloids and polypeptides which areused by plants as part of their defense mechanisms [11 12] Inaddition these compounds had shown a broad spectrum ofbiological activities demonstrating the potential of plants asalternative drugs [13 14] However the content of bioactivecompounds depends on biotic and abiotic factors such asthe presence of microorganisms and competitor speciesaround the plant temperature light intensity UV radiationhumidity water minerals and environmental contamination[15 16] These factors regulate the production of secondarymetabolites and subsequently the potential use of medicinalplants [17] In this sense the study of the effect that thedifferent seasons have on the chemical composition andbiological properties of plants can contribute to their optimaluse in the folk medicine [18 19]

Litsea glaucescens Kunth is a native plant from CentralAmerica and Mexico mainly distributed in the states ofChiapas Nayarit and Veracruz where it is known as ldquolaurelrdquo[20] Its leaves have been traditionally used as food seasoningas well as remedy in folk medicine against central nervoussystem illness depression colic pain vomit and diarrhea[21] These activities are mainly related to the presence ofdifferent compounds such as terpenes and phenolic com-pounds [22 23]The goal of the present study was to evaluatethe seasonal effect on the antioxidant antimicrobial andthe antiproliferative activities of L glaucescens Kunth leavesextracts as well as on their content and profile of phenoliccompounds since to the best of our knowledge this is thefirst effort to describe at this level the biological propertiesand chemical composition of ldquolaurelrdquo commonly used as aremedy by the communities from the mountainous region ofVeracruz Mexico

2 Materials and Methods

21 Reagents Folin-Ciocalteursquos phenol reagent sodiumcarbonate (Na2CO3) 11-diphenyl-2-picrylhydrazyl (DPPH)6-hydroxy-2578-tetramethylchroman-2-carboxylic acid(Trolox) 246-tri(2-pyridyl)-striazine (TPTZ) iron(III) chloride hexa-hydrate sodium acetate trihydrate

(C2H3NaO2sdot3H2O) hydrochloric acid 221015840-azobis(2-amidi-

nopropane) dihydrochloride (AAPH) gentamicin sodiumchloride (NaCl) Dulbeccorsquos Modified Eaglersquos Medium(DMEM) high glucose dimethyl sulfoxide (DMSO)isopropyl alcohol and 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT) as well as authenticstandards of epicatechin quercitrin gallic acid chlorogenicacid caffeic acid trans-cinnamic acid scopoletin hesperidinrosmarinic acid myricetin genistein luteolin and apigeninwere purchased from Sigma-Aldrich (St Louis MO USA)Standards of naringenin hesperetin chrysin galangin andacacetin were purchased from INDOFINE Chemical CoInc (Hillsborough NJ USA) Mueller-Hinton broth (MHB)andMueller-Hinton agar (MHA) were obtained from BectonDickinson (Cockeysville MD USA) HPLC-grade water(18mΩ) was performed by a Milli-Q50 purified system(Millipore Corp Bedford MA USA)

22 PlantMaterial and Preparation of Extracts L glaucescensleaves were collected during autumn (November 2015)winter (February 2016) spring (May 2016) and summer(September 2016) in Xico Veracruz Mexico L glaucescensleaves were identified in the Herbarium of Instituto de Inves-tigaciones Biologicas of Universidad Veracruzana MexicoCollected leaves were washed and dried Dried leaves wereextracted withmethanol (96) during 4 days with occasionalstirring (2-3 times per day) The extracts were filtered usingfilter paper (Whitman grade number 4) and the solvent wasevaporated to dryness under reduced pressure at 40∘C in arotary evaporatorThe obtained extracts were stored at minus20∘Cand identified as L glaucescens autumn winter spring andsummer extracts (ALGE WLGE SLGE and SULGE resp)

23 Total Phenolic Content Total phenolic concentrationwasdetermined with Folin-Ciocalteu reagent according to themethod described by Velazquez et al [24] Briefly 10 120583Lof extracts (1mgmL) was mixed with 80 120583L of distilledwater 40 120583L of Folin-Ciocalteu reagent 025N 60 120583L sodiumcarbonate (5 in distilled water) and 80 120583L of distilled waterThe mixtures were incubated at room temperature (1 h) Theabsorbance of the samples was measured at 750 nm on a Flu-ostar Omega microplate reader (BMG Labtech OrtenbergGermany) and the results were expressed as milligrams ofgallic acid equivalent (GAE)gram of dry weight (d w)

24 HPLC-DAD Analysis Analytical HPLC-DAD analysiswas carried out on an Agilent 1220 Infinity DAD LC (Wald-bronn Germany) equipped with a Zorbax SB-C18 column(250 times 46mm Oslash 35 120583m Agilent USA) The mobilephase consisted of 5 formic acid in water (solvent A)and methanol (solvent B) The elution was accomplishedwith a solvent flow rate of 1mLmin using a gradientprogram as follows 5 B (0ndash5min) 10 B (5ndash10min) 15B (10ndash18min) 25 B (18ndash28min) 30 B (28ndash40min) 40B (40ndash45min) 45 B (45ndash55min) 60 B (55ndash60min) 80B (60ndash65min) 100 B (65ndash76min) and 30 B (76ndash86min)Flavonoids were monitored at 280 and 340 nm Identificationof phenolic compounds was carried out by comparison of theretention times and spectra with those of authentic standards










MIC50


times 100 ge 50

MIC90


times 100 ge 90

(1)




ALGE WLGE

a

b

c c

SLGE SULGE0

20

40

60

80

100

120

140

160

(mg

GA

Eg

dw

)






ALGE

SLGE

WLGE

SULGEQR

QR

QR

QREP

EP

EP

EP

00

10 20

20

30 40

40

50 60

60

70 80

80100120

(min)(m

AU)

020406080

100120

(mAU

)

020406080

100120

(mAU

)

020406080

100120

(mAU

)












Extract


(120583M TEg ofdw)



dw)

ORAC(120583M TEg of












r = 092

90

100

110

120

130

140

150CP

C

700 800 900 1000 1100 1200 1300 1400600DPPH

(a)

r = 093

1500 2000 2500 30001000FRAP

90

100

110

120

130

140

150

CPC

(b)

r = 080

90

100

110

120

130

140

150

CPC

3400 3500 3600 3700 38003300ORAC

(c)


O

OH

OH

HO

OH

1

2

3

456

78

A

B

C

OH

O

OH

HO

OH

1

2

3

456

78

A

B

C

O

OH

O-Rhamnose


1

2

3

4

5

6

1

2

3

4

5

6























SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)






4 Conclusions






Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




























MIC50


times 100 ge 50

MIC90


times 100 ge 90

(1)




ALGE WLGE

a

b

c c

SLGE SULGE0

20

40

60

80

100

120

140

160

(mg

GA

Eg

dw

)






ALGE

SLGE

WLGE

SULGEQR

QR

QR

QREP

EP

EP

EP

00

10 20

20

30 40

40

50 60

60

70 80

80100120

(min)(m

AU)

020406080

100120

(mAU

)

020406080

100120

(mAU

)

020406080

100120

(mAU

)












Extract


(120583M TEg ofdw)



dw)

ORAC(120583M TEg of












r = 092

90

100

110

120

130

140

150CP

C

700 800 900 1000 1100 1200 1300 1400600DPPH

(a)

r = 093

1500 2000 2500 30001000FRAP

90

100

110

120

130

140

150

CPC

(b)

r = 080

90

100

110

120

130

140

150

CPC

3400 3500 3600 3700 38003300ORAC

(c)


O

OH

OH

HO

OH

1

2

3

456

78

A

B

C

OH

O

OH

HO

OH

1

2

3

456

78

A

B

C

O

OH

O-Rhamnose


1

2

3

4

5

6

1

2

3

4

5

6























SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)






4 Conclusions






Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















ALGE WLGE

a

b

c c

SLGE SULGE0

20

40

60

80

100

120

140

160

(mg

GA

Eg

dw

)






ALGE

SLGE

WLGE

SULGEQR

QR

QR

QREP

EP

EP

EP

00

10 20

20

30 40

40

50 60

60

70 80

80100120

(min)(m

AU)

020406080

100120

(mAU

)

020406080

100120

(mAU

)

020406080

100120

(mAU

)












Extract


(120583M TEg ofdw)



dw)

ORAC(120583M TEg of












r = 092

90

100

110

120

130

140

150CP

C

700 800 900 1000 1100 1200 1300 1400600DPPH

(a)

r = 093

1500 2000 2500 30001000FRAP

90

100

110

120

130

140

150

CPC

(b)

r = 080

90

100

110

120

130

140

150

CPC

3400 3500 3600 3700 38003300ORAC

(c)


O

OH

OH

HO

OH

1

2

3

456

78

A

B

C

OH

O

OH

HO

OH

1

2

3

456

78

A

B

C

O

OH

O-Rhamnose


1

2

3

4

5

6

1

2

3

4

5

6























SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)






4 Conclusions






Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























Extract


(120583M TEg ofdw)



dw)

ORAC(120583M TEg of












r = 092

90

100

110

120

130

140

150CP

C

700 800 900 1000 1100 1200 1300 1400600DPPH

(a)

r = 093

1500 2000 2500 30001000FRAP

90

100

110

120

130

140

150

CPC

(b)

r = 080

90

100

110

120

130

140

150

CPC

3400 3500 3600 3700 38003300ORAC

(c)


O

OH

OH

HO

OH

1

2

3

456

78

A

B

C

OH

O

OH

HO

OH

1

2

3

456

78

A

B

C

O

OH

O-Rhamnose


1

2

3

4

5

6

1

2

3

4

5

6























SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)






4 Conclusions






Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















r = 092

90

100

110

120

130

140

150CP

C

700 800 900 1000 1100 1200 1300 1400600DPPH

(a)

r = 093

1500 2000 2500 30001000FRAP

90

100

110

120

130

140

150

CPC

(b)

r = 080

90

100

110

120

130

140

150

CPC

3400 3500 3600 3700 38003300ORAC

(c)


O

OH

OH

HO

OH

1

2

3

456

78

A

B

C

OH

O

OH

HO

OH

1

2

3

456

78

A

B

C

O

OH

O-Rhamnose


1

2

3

4

5

6

1

2

3

4

5

6























SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)






4 Conclusions






Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





































SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)






4 Conclusions






Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























SLGE SULGE

00

02

04

06

08

6 12 18 24 30 36 42 480Time (h)

00

02

04

06

08

＄

620

＄

620

6 12 18 24 30 36 42 480Time (h)






4 Conclusions






Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























Acknowledgments


References










































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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Seasonal Effect on the Biological Activities of Litsea ...downloads.hindawi.com/journals/ecam/2018/2738489.pdf · ResearchArticle Seasonal Effect on the Biological Activities of Litsea