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Research ArticleAntioxidant Activities and Phytochemicals of Leaf Extracts from10 Native Rhododendron Species in Taiwan

Chi-Yang Lin1 Lei-Chen Lin2 Shang-Tse Ho1 Yu-Tang Tung3

Yen-Hsueh Tseng1 and Jyh-Horng Wu1

1 Department of Forestry National Chung Hsing University Taichung 402 Taiwan2Department of Forestry and Natural Resources National Chiayi University Chiayi 600 Taiwan3Department of Life Sciences National Chung Hsing University Taichung 402 Taiwan

Correspondence should be addressed to Jyh-Horng Wu ericnchuedutw

Received 12 February 2014 Revised 8 April 2014 Accepted 8 May 2014 Published 2 June 2014

Academic Editor Virginia S Martino

Copyright copy 2014 Chi-Yang Lin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Rhododendron one of the most famous ornamental plants in the world is traditionally a medicinal plant However the potentialbioactivities of native Rhododendron in Taiwan have not been completely studied In this study the results revealed thatRhododendron pseudochrysanthum exhibited the best antioxidant activities among 10 native Rhododendron species in TaiwanFurthermore based on a bioactivity-guided isolation principle nine specific phytochemicals were isolated and identified as(2R3S)-catechin (1) (2R3R)-epicatechin (11015840) (2R3R)-dihydromyricetin 3-O-120573-l-arabinopyranoside (2) (2S3S)-taxifolin 3-O-120573-l-arabinopyranoside (21015840) (2R3R)-taxifolin 3-O-120573-l-arabinopyranoside (3) myricetin 3-O-120573-d-glucopyranoside (31015840) rutin (4)hyperoside (5) and quercitrin (6) Of these compounds 2 and 3 were found to be major bioactive compounds and theirconcentrations in the n-butanol (BuOH) fraction were determined to be 520 and 673mg per gram respectively These resultsdemonstrated that methanolic extracts of Rhododendron pseudochrysanthum leaves have excellent antioxidant activities and greatpotential as a source for natural health products

1 Introduction

Reactive oxygen species (ROS) including superoxide radicalhydroxyl radical singlet oxygen and hydrogen peroxideplay an important role in the initiation and progression ofchronic and age-related diseases such as aging inflammatoryinjury neural disorders diabetes atherosclerosis cancer andcardiovascular disease [1] Thus a potential ROS scavengermay serve a role in the prevention of chronic and age-relateddiseases induced by ROS [2] It has been demonstrated thatthe antioxidant effects of plants can be mainly attributedto phenolic compounds such as flavonoids phenolic acidstannins and phenolic diterpenes [3 4] Therefore the intakeof dietary antioxidants that act as ROS scavengers is expectedto be effective in preventing many chronic and age-relateddiseases [5 6]

The genus Rhododendron which contains over 1000 spec-ies includes flowering plants widely distributed throughout

the world (with the exception of Africa and South America)[7] Many species of the genus Rhododendron contain a largenumber of phenolic compounds and antioxidant activitiesthat could be developed into pharmaceutical products [8]In addition some members of the genus are already used intraditional medicine for several ailments especially arthritisacute and chronic bronchitis asthma pain inflammationrheumatism hypertension and muscle and metabolic dis-eases [9ndash13] In Taiwan Rhododendron species are foundfrom the lowlands to 3950m in elevation However to thebest of our knowledge there is no prior report on theantioxidative phytochemicals of leaf extracts from nativeRhododendron species in Taiwan Thus in this study anumber of in vitro assays were performed to evaluate theantioxidant activities of methanolic extracts from the leavesof 10 native Rhododendron species In addition the charac-teristics of bioactive phytochemicals were addressed in thisstudy

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 283938 9 pageshttpdxdoiorg1011552014283938

2 Evidence-Based Complementary and Alternative Medicine

2 Materials and Methods

21 Extraction and Fractionation of 10 Native RhododendronSpecies The leaves of 10 native Rhododendron species inTaiwan were collected at the end of April 2011 The collectionlocations of these specimens including R breviperulatumHayata (voucher number 1) R ellipticum Maxim (vouchernumber 2) R formosanum Hemsl (voucher number 3) RkanehiraiWilson (voucher number 4)RmariesiiHemsl andWilson (voucher number 5) R oldhamii Maxim (vouchernumber 6) R pseudochrysanthum Hayata (voucher number7) R rubropilosum Hayata var rubropilosum (voucher num-ber 8)R rubropilosumHayata var taiwanalpinum (Ohwi) LuYang and Tseng (voucher number 9) and R simsii Planch(voucher number 10) are presented in Figure 1(a) All ofthe voucher specimens were deposited at the herbarium ofthe Department of Forestry and Natural Resources NationalChiayi University (NCYU) Taiwan The species were iden-tified by Dr Lei-Chen Lin (NCYU) After identification thesamples were cleaned with tap water and dried Then theywere extracted twice with methanol by ultrasound-assistedextraction for 30min at room temperature The leaf extractsof 10 native Rhododendron species were decanted filteredunder vacuum and either concentrated in a rotator evapo-rator or lyophilized Furthermore the resulting methanoliccrude extracts of R pseudochrysanthum were fractionatedsuccessively with n-hexane ethyl acetate (EtOAc) n-butanol(BuOH) and water to yield soluble fractions of hexaneEtOAc BuOH and water All the extracts were stored in anairtight container at minus40∘C until further use

22 11-Diphenyl-2-picrylhydrazyl (DPPH) Assay The DPPHradical scavenging activity of the test extracts was examinedaccording to the method reported by Ho et al [14] Tenmicroliters of each test sample in methanol yielding a seriesof extract concentrations of 1 5 10 50 and 100120583gmLrespectively in each reaction was mixed with 200120583L of01mM DPPH-ethanol solution and 90120583L of 50mM Tris-HCl buffer (pH 74) Methanol (10 120583L) alone was used asthe control for this experiment After 30min of incubationat room temperature the reduction in DPPH radicals wasmeasured by reading the absorbance at 517 nm using aThermo Scientific Multiskan GO microplate reader (VantaaFinland) (+)-Catechin was used as the positive control Theinhibition ratio was calculated using the following equation inhibition = [(absorbance of control minus absorbance of testsample)absorbance of control] times 100

23 Superoxide Radical Scavenging Assay (NBT Assay) Themeasurement of superoxide radical scavenging activity wasconducted according to the method of Tung et al [15] First20120583L of 15mM Na

2EDTA in buffer (50mM KH

2PO4KOH

pH 74) 50 120583L of 06mM NBT (Nitro blue tetrazolium)in buffer 30 120583L of 3mM hypoxanthine in 50mM KOH5 120583L of the test samples in methanol (final concentrationswere 1 5 10 50 and 100 120583gmL resp) and 145 120583L of thebuffer were mixed in 96-well microplates The reaction wasstarted by adding 50 120583L of xanthine oxidase solution in buffer

(1 unit in 10mL buffer) to the mixture The reaction mixturewas incubated at room temperature and the absorbance at570 nm was determined every 1min (up to 9min) using amicroplate reader The control was 5120583L of methanol insteadof the sample solution and (+)-catechin was used as thepositive controlThe inhibition ratio was calculated using thefollowing equation inhibition = [(rate of control reactionminus rate of sample reaction)rate of control reaction] times 100

24 Ferrous Ion Chelating Assay The ferrous ion chelatingpotential of the test samples was evaluated according tothe method of Tung et al [15] Briefly 200120583L of the testsample in methanol (final concentrations were 125 250 5001000 and 2000120583gmL resp) and 740 120583L of methanol wereadded to 20 120583L of 2mM FeCl

2 The reaction was initiated

by adding 40 120583L of 5mM ferrozine The mixture was shakenvigorously and permitted to rest at room temperature for10min The absorbance of the solution was then measuredat 562 nm (+)-Catechin was used as the positive control Thepercent inhibition of Fe2+-ferrozine complex formation wascalculated according to the following equation inhibition=[(absorbance of control minus absorbance of sample)absorbanceof control] times 100

25 Reducing Power Assay This assay was determinedaccording to the method reported by Tung et al [15] using(+)-catechin as the standard Briefly 1mL of a reaction mix-ture containing 500120583L of 880 120583gmL test sample in 500120583L ofphosphate buffer (02M pH 66) was incubated with 500120583Lof potassium ferricyanide (1 wv) at 50∘C for 20min Thereaction was terminated by adding 500 120583L of trichloroaceticacid (10 wv) and the mixture was then centrifuged at12000 g for 10min The supernatant solution (500120583L) wasmixed with distilled water (500 120583L) and 100 120583L of the ferricchloride (01 wv) solution and the optical density (OD)was then measured at 700 nm The reducing power abilitywas expressed as milligrams of (+)-catechin equivalents (mgCE) per gram of sample or as micromolar of (+)-catechinequivalents (120583MCE) per millimolar of compound

26 Determination of Total Phenolics Total phenolic contentswere determined according to the Folin-Ciocalteu method[15] using gallic acid as the standard The test samples (5mg)were dissolved in 5mL of methanolwater (50 50 vv) andthe extract solution (500120583L) was mixed with 500120583L of 50Folin-Ciocalteu reagent The mixture was incubated at roomtemperature for 5min which was followed by the additionof 10mL of 20 Na

2CO3 After an additional 10min of

incubation at room temperature themixture was centrifugedfor 8min (12000 g) and the supernatant absorbancewasmea-sured at 730 nmThe total phenolic content was expressed asmilligrams of gallic acid equivalents (mg GAE) per gram ofsample

27 OnlineHPLC-DPPHMethod TheBuOH soluble fractionof R pseudochrysanthum was further analyzed by the onlineHPLC-DPPH method The instrumental setup is depicted inFigure 1(b) The BuOH soluble fraction (stock concentration

Evidence-Based Complementary and Alternative Medicine 3

N

Tropic of cancer

25∘N

24∘N

23∘N

22∘N

122∘E120∘E 121∘E

3952

M

3000

M

2000

M

1000

M

8 9

22

37

1 5

9

6

4 104 10

1 5

37

8

6

(a)

280517

MeOHPump Pump

Injector

Extract

Mixer

Detector

DPPH solution Online HPLC-DPPH chromatograms

RP-amidecolumn

H2O

(b)

Figure 1 (a) The locations of 10 native Rhododendron species in Taiwan (b) The instrumental setup for the HPLC-DPPH online detectionof radical scavenging phytochemicals

= 20mgmL) was monitored by HPLC on a Jasco PU-2080instrument (Tokyo Japan) with a 250 times 46mm id anda 5 120583m Supelco RP-amide column (Bellefonte PA USA)The mobile phase consisted of solvent A 100 MeOH andsolvent B ultrapure water The elution conditions were 0ndash5min of 30ndash36 A and 5ndash50min of 36ndash72 A at a flowrate of 075mLmin using a Jasco MD-2010 photodiodearray detector (Tokyo Japan) at 280 nm wavelength Forthe online DPPH radical scavenging analysis the flow ofDPPH reagent (50mgL inMeOH)was 075mLmin and theinduced bleaching was detected photometrically as a negativepeak at 517 nm

28 Isolation and Identification of Bioactive PhytochemicalsBased on the bioactivity-guided isolation principle theBuOH-soluble fraction from R pseudochrysanthum had anexcellent antioxidant activity Therefore it was separatedand purified by semipreparative HPLC using a PU-2080pump equipped with a MD-2010 multiwavelength detectorand a 250 times 100mm id 5 120583m Supelco RP-amide column(Bellefonte PA USA) The mobile phase consisted of solventA 100 MeOH and solvent B ultrapure water The elutionconditions were 0ndash5min of 30ndash36 A and 5ndash50min of 36ndash72 A at a flow rate of 2mLmin for isolation of the BuOHfractionThe structures of the phytochemicals were identifiedby electrospray ionization mass (ESIMS) nuclear magneticresonance (NMR) and circular dichroism (CD) spectrom-eters and all spectral data (as shown in supplementary

data see S1ndashS9 in Supplementary Material available online athttpdxdoiorg1011552014283938) were consistent withthe literature [16ndash24] ESIMS data were collected using aFinnigan MAT-95S mass spectrometer (San Jose CA USA)NMR spectra were recorded using a Bruker Avance 500MHzFTNMR spectrometer (Rheinstetten Germany) and CDspectra were obtained on a Jasco J-815 spectropolarimeter(Tokyo Japan)

29 Statistical Analysis Significant differences were calcu-lated by Scheffersquos test and results with 119875 lt 005 were con-sidered statistically significant Comparisons of total phenoliccontents and various antioxidant activities were carried outusing Pearsonrsquos correlation test

3 Results and Discussion

31 The Methanolic Extract Yields of 10 Native RhododendronSpecies in Taiwan The leaves of 10 native Rhododendronspecies in Taiwan yielded methanolic extracts from 68 to334 (ww) based on dry weight (Table 1) The yields of 7species were higher than 15 including R ellipticum (334)R oldhamii (291) R kanehirai (225) R breviperulatum(184) R formosanum (182) R mariesii (180) and Rrubropilosum var taiwanalpinum (167) In addition theyields among different species showed great variability For


example the yield of R ellipticum (334) was fivefold higherthan that of R pseudochrysanthum (68)

32 Antioxidant Activities of Methanolic Extracts of 10 NativeRhododendron Species in Taiwan An approach using mul-tiple assays for screening antioxidant properties is highlyadvisable [25 26] Thus the extracts were subjected to fourdifferent antioxidant assays employing DPPH NBT andferrous ion chelating and reducing power methods In regardto the inhibitory effects of the leaf extracts from 10 nativeRhododendron species in Taiwan on DPPH radicals Table 1shows that most extracts revealed good DPPH radical scav-enging activity The concentrations required to inhibit 50of the radical scavenging effect (IC

50) were determined from

the results of a series of tested concentrationsThe IC50values

of crude extracts increased in the following order R pseu-dochrysanthum (75 120583gmL)R oldhamii (75 120583gmL)R kane-hirai (77 120583gmL) R breviperulatum (88 120583gmL) R rubropi-losum var taiwanalpinum (104 120583gmL) R formosanum(107 120583gmL) R simsii (118 120583gmL) R rubropilosum varrubropilosum (121 120583gmL) R ellipticum (142 120583gmL) andR mariesii (147 120583gmL) In comparison with a well-knownantioxidant (+)-catechin (IC

50= 21 120583gmL) the crude

extracts of theRhododendron speciesmentioned above exhib-ited a good DPPH radical scavenging activity Furthermorethe crude extract of green tea showed an excellent DPPHradical scavenging activity with an IC

50value of 5 120583gmL

[5] These results indicate that the leaf extracts of R pseu-dochrysanthum and R oldhamii would be excellent sourcesof natural antioxidants and merit further investigation

Additionally the superoxide radical scavenging activitiesof the leaf extracts from 10 native Rhododendron speciesin Taiwan were determined by the hypoxanthine-xanthineoxidase system The inhibitory activities of 10 species wereobserved in a dose-dependent manner (Table 1) and the leafextracts ofR pseudochrysanthum exhibited the highest super-oxide radical scavenging activity among all of the speciesTheIC50values of (+)-catechin R breviperulatum R ellipticum

R formosanum R kanehirai R mariesii R oldhamii Rpseudochrysanthum R rubropilosum var rubropilosum Rrubropilosum var taiwanalpinum and R simsii were 166243 157 201 339 290 196 124 245 340 and 316120583gmLrespectively

On the other hand Table 1 shows the reducing powerand ferrous ion chelating activity of the leaf extracts ofthe native Rhododendron species The reducing powersof the different species occurred in decreasing order asfollows R pseudochrysanthum (387mg CEg) R kanehirai(328mg CEg) R oldhamii (326mg CEg) R breviperulatum(320mg CEg) R ellipticum (298mg CEg) R formosanum(282mg CEg) R simsii (277mg CEg) R rubropilosumvar rubropilosum (263mg CEg) R rubropilosum vartaiwanalpinum (243mg CEg) and R mariesii (225mgCEg) As for the chelating effects of the methanolic extractson ferrous ions the results revealed that the IC

50values of

chelating effect for leaf extracts were in increasing order asfollowsR kanehirai (4291 120583gmL)Rmariesii (5153 120583gmL)R breviperulatum (6653 120583gmL) R oldhamii (7141 120583gmL)

R rubropilosum var taiwanalpinum (7741 120583gmL) R rubro-pilosum var rubropilosum (8049 120583gmL) R formosanum(9288 120583gmL) R simsii (9612 120583gmL) R ellipticum(10959 120583gmL) and R pseudochrysanthum (13444 120583gmL)Comparison of the aforementioned results indicated that theferrous ion chelating effects of the methanolic extracts didnot correlate with the results from the DPPH and reducingpower assays Similar results were reported by Chua et al[27] and Tung et al [28] It has been reported that an effectivechelating agent can stabilize the oxidized form of the metalion through formation of 120590 bonds with the metal leading toreduce the redox potential [29] In contrast the free radicalscavenging activities of the test samples are mainly due totheir hydrogen-donating ability Accordingly in the presentstudy the discrepancy of ion chelating assay and otherantioxidant assays may be due to the different mechanismsinvolved in different assays

33 Total Phenolics of Methanolic Extracts of 10 NativeRhododendron Species in Taiwan Table 1 shows that thecontents of total phenolics in crude extracts were determinedspectrometrically according to the Folin-Ciocalteu methodand calculated as gallic acid equivalents (GAE) Accordinglythe total phenolic contents of the 10 native Rhododendronspecies in Taiwan were ranked in decreasing order as followsR pseudochrysanthum (319mg GAEg) R breviperulatum(265mg GAEg) R oldhamii (264mg GAEg) R kanehirai(238mg GAEg) R formosanum (222mg GAEg) R ellip-ticum (221mg GAEg) R simsii (219mg GAEg) R rubropi-losum var taiwanalpinum (198mg GAEg) R rubropilosumvar rubropilosum (193mg GAEg) and R mariesii (165mgGAEg) This result revealed that the leaf extracts of Rpseudochrysanthum had the highest phenolic contents amongall species of Rhododendron

34 Correlation Coefficients among DPPH Radical ScavengingActivity Superoxide Radical Scavenging Activity Ferrous IonChelatingAbility Reducing Power andTotal Phenolic Contentsin Extracts Phenolic compounds had been widely evaluatedfor their bioactivities especially in antioxidant activitiesThus the correlation between the total phenolic contentsand antioxidant activities has been studied in different plants[14] In this study correlation coefficients for total phenoliccontents with the DPPH NBT ferrous ion chelating andreducing power assays are shown in Table 2 These resultsshowed that strong correlations were obtained between totalphenolic contents and DPPH assay as well as total phenoliccontents and reducing power with 1198772 values of 0787 (119875 lt005) and 0966 (119875 lt 001) respectively However the corre-lations between total phenolic contents andNBT assay as wellas total phenolic contents and ferrous ion chelating assaywerenot significant (119875 gt 005) Accordingly antioxidant activitiesof extracts especially in DPPH radical scavenging ability andreducing power are correlated to their phenolic contentsFurthermore from the results obtained from theDPPHNBTreducing power and total phenolic content assays it is clearthat effective antioxidants can be obtained from methanolicextracts of R pseudochrysanthum leaves and proposed that


Table 1 Antioxidant activities of methanolic extracts from leaves of 10 native Rhododendron species

Specimens Yield(wt)

IC50 (120583gmL) Reducingpower

(mgCEg)

Total phenoliccontent

(mgGAEg)DPPH radicalscavenging

Superoxide radicalscavenging

Ferrous ionchelating

R breviperulatum 184 88 plusmn 16CD 243 plusmn 24ABC 6653 plusmn 98F 320 plusmn 18BC 265 plusmn 2B

R ellipticum 334 142 plusmn 09AB 157 plusmn 12CD 10959 plusmn 313B 298 plusmn 15BCD 221 plusmn 16CD

R formosanum 182 107 plusmn 10BCD 201 plusmn 13BCD 9288 plusmn 381CD 282 plusmn 5CDE 222 plusmn 16CD

R kanehirai 225 77 plusmn 04D 339 plusmn 27A 4291 plusmn 148G 328 plusmn 8B 238 plusmn 5BC

R mariesii 180 147 plusmn 04A 290 plusmn 28AB 5153 plusmn 48G 225 plusmn 11F 165 plusmn 7E

R oldhamii 291 75 plusmn 09D 196 plusmn 10BCD 7141 plusmn 600EF 326 plusmn 19B 264 plusmn 9B

R pseudochrysanthum 68 75 plusmn 07D 124 plusmn 08D 13444 plusmn 185A 387 plusmn 9A 319 plusmn 17A

R rubropilosum var rubropilosum 114 121 plusmn 15ABC 245 plusmn 12ABC 8049 plusmn 220DE 263 plusmn 9DEF 193 plusmn 7DE

R rubropilosum var taiwanalpinum 167 104 plusmn 12BCD 340 plusmn 12A 7741 plusmn 319EF 243 plusmn 6EF 198 plusmn 5DE

R simsii 92 118 plusmn 10ABC 316 plusmn 29A 9612 plusmn 633C 277 plusmn 15CDE 219 plusmn 4CD

(+)-Catechin mdash 21 plusmn 03E 166 plusmn 09CD gt2000 mdash mdashThe results are presented as means plusmn SD (n =3) Different letters within a column indicate significant difference at the P lt 005 level according to Scheffersquos testThe reducing power was calculated as (+)-catechin equivalents (CE) The total phenolic content was calculated as gallic acid equivalents (GAE)

Table 2 Correlation coefficients amongDPPH radical scavenging activity (DPPH assay) superoxide radical scavenging activity (NBT assay)ferrous ion chelating ability (Chelating assay) reducing power assay (Reducing power) and total phenolic contents (TPC) in extracts

DPPH assay NBT assay Chelating assay Reducing power TPCDPPH assay mdash 0103 0004 0752lowast 0787lowast

NBT assay 0103 mdash 0711lowast 0577 0581Chelating assay 0004 0711lowast mdash 0424 0480Reducing power 0752lowast 0577 0424 mdash 0966lowastlowast

TPC 0787lowastlowast 0581 0480 0966lowastlowast mdashlowastP lt 005 lowastlowastP lt 001

phenolic compounds from the methanolic extracts may playan important role in antioxidant activities

35 Antioxidant Activities of the Crude Extracts and TheirDerived Soluble Fractions from R pseudochrysanthum LeavesBased on the bioactivity-guided isolation principle themethanolic crude extracts of R pseudochrysanthum leaveswere fractionated to yield soluble fractions of hexane EtOAcBuOH and water As shown in Table 3 the DPPH radicalscavenging superoxide radical scavenging and ferrous ionchelating activities of methanolic extracts and their derivedsoluble fractions fromR pseudochrysanthum leaves increasedwith the increasing concentration of the test sampleThe IC

50

values of the crude extract hexane fraction EtOAc fractionBuOH fraction and water fraction of R pseudochrysanthumleaves were 75 717 37 33 and 154 120583gmL for the DPPHassay 124 gt100 142 74 and 193 120583gmL for the NBTassay and 13444 gt2000 19078 gt2000 and 2817 120583gmL forthe ferrous ion chelating ability respectively Additionallythe reducing powers of the fractions (Figure 2) were indecreasing order those of the BuOH fraction (599mg CEg)gt EtOAc fraction (475mg CEg) gt water fraction (208mgCEg) gt hexane fraction (12mg CEg) Furthermore thetotal phenolic contents of the crude extract hexane fractionEtOAc fraction BuOH fraction and water fraction were319 22 395 438 and 159mg GAEg respectively (Figure 2)

Accordingly except for the ferrous ion chelating effect theantioxidant activities of R pseudochrysanthum leaves can beeffectively enriched in the BuOH fraction Chelating agentsare effective as secondary antioxidants because they reducethe redox potential thereby stabilizing the oxidized formsof metal ions [30] Therefore the BuOH-soluble fractionfrom R pseudochrysanthum leaves was not a good secondaryantioxidant because of its poor capacity formetal ion bindingbut it was an excellent primary antioxidant (or free radicalscavenger) These results revealed that the BuOH-solublefraction from theR pseudochrysanthum leaves has a powerfulantioxidant activity and might be a good candidate fordevelopment as a novel natural antioxidant

36 Screening Quantification andDetermination of theMajorAntioxidants from the BuOH Fraction of R pseudochry-santhum Leaves The online HPLC-DPPH method is arapid assessment for detecting pure antioxidants in com-plex mixtures particularly plant extracts [31 32] The morethe absorbance decreases the greater the potential antiox-idant activity of the compound (in terms of hydrogen-donating ability) is [33] Figure 3(a) shows combinedUV (positive signals) and DPPH radical-quenching (neg-ative signals) chromatograms under the elution conditionsof the BuOH fraction from the R pseudochrysanthumleaves Nine phytochemicals that is (2R3S)-catechin (1)


Table 3 Antioxidant activities of methanolic extracts and their derived soluble fractions from the leaves of R pseudochrysanthum

Extracts IC50 (120583gmL)DPPH radical scavenging Superoxide radical scavenging Ferrous ion chelating

Crude extract 75 plusmn 07BC 124 plusmn 08C 13444 plusmn 185B

Hexane fraction 717 plusmn 74A gt100 gt2000EtOAc fraction 37 plusmn 02C 142 plusmn 14BC 19078 plusmn 2125A

BuOH fraction 33 plusmn 02C 74 plusmn 06D gt2000Water fraction 154 plusmn 02B 193 plusmn 15A 2817 plusmn 185C

(+)-Catechin 21 plusmn 03C 166 plusmn 09AB gt2000The results are presented as means plusmn SD (n = 3) Different letters within a column indicate significant difference at the P lt 005 level according to Scheffersquos test

Table 4 Antioxidant activities and contents of major phytochemicals of the BuOH fraction from leaves of R pseudochrysanthum

PhytochemicalsContents

(mgg of BuOH fraction)IC50 (120583M) Reducing power

(120583MCEmM )DPPH radicalscavenging


1 132 plusmn 14 68 plusmn 05CD 454 plusmn 03B 1000 plusmn 34AB

2 520 plusmn 28 116 plusmn 03A 86 plusmn 03E 727 plusmn 76D

3 673 plusmn 64 112 plusmn 03A 456 plusmn 17B 1074 plusmn 35A

4 134 plusmn 08 61 plusmn 03D 1075 plusmn 24A 917 plusmn 68ABC

5 238 plusmn 16 78 plusmn 07C 369 plusmn 01C 834 plusmn 14BCD

6 111 plusmn 09 96 plusmn 04B 249 plusmn 13D 753 plusmn 59CD

The results are presented as means plusmn SD (n = 3) Different letters within a column indicate a significant difference at the P lt 005 level according to Scheffersquostest The reducing power was calculated as (+)-catechin (1) equivalents (CE)

C

E

B

A

D

c

e

ba

d

0

200

400

600

800

0

200

400

600

800

Fractions

Crudeextract

n-Hexane EtOAc BuOH Water

Redu

cing

pow

er (m

g CE

g)

Reducing power (mg CEg)Total phenolic content (mg GAEg)

Tota

l phe

nolic

cont

ent (

mg

GA

Eg)

Figure 2 The reducing powers and total phenolic contents ofmethanolic extracts and their derived soluble fractions from Rpseudochrysanthum leaves The results are presented as means plusmnSD (119899 = 3) The bars marked by different letters are significantlydifferent at the 119875 lt 005 level according to Scheffersquos test

(2R3R)-epicatechin (11015840) (2R3R)-dihydromyricetin 3-O-120573-l-arabinopyranoside (2) (2S3S)-taxifolin 3-O-120573-l-arabino-pyranoside (21015840) (2R3R)-taxifolin 3-O-120573-l-arabinopyran-oside (3) myricetin 3-O-120573-d-glucopyranoside (31015840) rutin (4)hyperoside (5) and quercitrin (6) were eluted and identified(Figure 3(b)) Among them phytochemicals 1ndash6 showed

significant hydrogen-donating capacity (negative peak) andtheir concentrations in the BuOH fraction were determinedto be 132plusmn14 520plusmn28 673plusmn64 134plusmn08 238plusmn16 and111 plusmn 09mg per gram respectively (Table 4) Accordinglythe results indicated that these 6 specific phytochemicalswere the major antioxidants in the leaf extracts of R pseu-dochrysanthum The online HPLC-DPPH method can beapplied for a rapid screening of antioxidants and it is nolonger necessary to isolate nonantioxidant phytochemicals

Moreover to determine the antioxidant activities ofphytochemicals 1ndash6 DPPH NBT ferrous ion chelatingand reducing power assays were performed As shown inTable 4 the effectiveness in DPPH radical scavenging activitywas in increasing order that of 4 1 5 6 3 and 2 whilethe IC

50values of these compounds were 61 68 78 96

112 and 116 120583M respectively This result demonstratedthat compounds 1 and 4 exhibited greater DPPH radicalscavenging activity In addition the decreasing superoxideradical scavenging activity and order of phytochemicals inthe NBT assay can be ranked as 2 6 5 1 3 and 4 As forthe reducing power that of compound 3 was the greatestfollowed by those of compounds 1 4 5 6 and 2 Accordinglythe structure-activity relationships of flavonoid glycosides 45 and 6 were also investigated in this study In the DPPHand reducing power assays compound 4 a flavonoid withan 120572-l-rhamnopyranosyl-(1rarr 6)-120573-d-glucopyranosyl groupin the C ring exhibited better activities than compounds 5and 6 However in the NBT assay compound 6 exhibitedthe best superoxide radical scavenging activity among these3 flavonoid glycosides According to the above results the


minus250

0

250

500

750

10 20 30 40 50

Abso

rban

ce (m

AU)

Retention time (min)

12

3

5

6439984001 9984002998400

280nm

517nm

(a)

O

OH

OHOH

HO

O

2346

910

O

OH

HO

OHOH

OO Ara

2346

910

OOH

HOOH

OHO

OH

OHOHO

HO

OH

OHOHO

OH

OHOHO

OH

O

O

OH

HOOH

OH

OO

2346

910

OHOHO

OH

O

OH

HOOH

OH

OH

2346

910

1998400

1998400

19984001998400

1R1 = H2R2 = HR3 = H2 R1 = OR2 = OHR3 = ara3 R1 = OR2 = HR3 = ara

1998400

2998400

3998400 R1 = gluR2 = OH4 R1 = rutR2 = H5 R1 = galR2 = H

R1 R3

R2

R1

R2

6 R1 = rhamR2 = H

CH3

CH3CH2OH

CH2OH

Ara = 120573-l-arabinopyranosyl Glu = 120573-d-glucopyranosyl

Gal = 120573-d-galactopyranosyl Rham = 120572-l-rhamnopyranosyl

Rut = 120572-l-rhamnopyranosyl-(1rarr6)-120573-d-glucopyranosyl

(b)

Figure 3 (a) The online HPLC-DPPH chromatograms of the BuOH fraction from leaves of R pseudochrysanthum (b) Isolated andidentified phytochemicals (2R3S)-catechin (1) (2R3R)-epicatechin (11015840) (2R3R)-dihydromyricetin 3-O-120573-l-arabinopyranoside (2) (2S3S)-taxifolin 3-O-120573-l-arabinopyranoside (21015840) (2R3R)-taxifolin 3-O-120573-l-arabinopyranoside (3) myricetin 3-O-120573-d-glucopyranoside (31015840) rutin(4) hyperoside (5) and quercitrin (6)

sugar moiety on a flavonoid plays an important role in itsantioxidant activities The effect of the sugar moiety wascompletely different in the DPPH and NBT assays andthis phenomenon warrants further examination in futurestudies

4 Conclusions

In the present study the leaf extracts of different nativeRhododendron species inTaiwanwere assayed to explore theirantioxidant activities The results indicate that a number of


extracts exhibit significant antioxidant activities Among 10Rhododendron species the leaf extracts of R pseudochrysan-thum exhibited the strongest antioxidant activity especially inthe BuOH-soluble fraction In total 6 specific and excellentantioxidants were purified and identifiedThese results implythat the methanolic extracts of R pseudochrysanthum leavesor the phytochemicals derived from these extracts couldbe used to prevent diseases caused by the overproductionof radicals and might also be suitable for the treatment ofdegenerative diseases

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was financially supported by a research grant fromthe National Chung Hsing University

References

[1] B Halliwell A Zentella E O Gomez and D KershenobichldquoAntioxidants and human disease a general introductionrdquoNutrition Reviews vol 55 no 1 pp S44ndashS52 1997

[2] W-C Hou R-D Lin K-T Cheng et al ldquoFree radical-scavenging activity of Taiwanese native plantsrdquo Phytomedicinevol 10 no 2-3 pp 170ndash175 2003

[3] C-L Si J-K Kim Y-S Bae and S-M Li ldquoPhenolic com-pounds in the leaves ofPopulus ussuriensis and their antioxidantactivitiesrdquo Planta Medica vol 75 no 10 pp 1165ndash1167 2009

[4] P Black A Saleem A Dunford et al ldquoSeasonal variationof phenolic constituents and medicinal activities of northernlabrador tea Rhododendron tomentosum ssp subarcticum anInuit and cree first nations traditionalmedicinerdquo PlantaMedicavol 77 no 14 pp 1655ndash1662 2011

[5] A Buyukbalci and S N El ldquoDetermination of in vitro antidia-betic effects antioxidant activities and phenol contents of someherbal teasrdquo Plant Foods for Human Nutrition vol 63 no 1 pp27ndash33 2008

[6] M-H Oak J El Bedoui and V B Schini-Kerth ldquoAntiangio-genic properties of natural polyphenols from red wine andgreen teardquo Journal of Nutritional Biochemistry vol 16 no 1 pp1ndash8 2005

[7] J-DChung T-P Lin Y-L Chen Y-P Cheng and S-YHwangldquoPhylogeographic study reveals the origin and evolutionary his-tory of a Rhododendron species complex in Taiwanrdquo MolecularPhylogenetics and Evolution vol 42 no 1 pp 14ndash24 2007

[8] Y Qiang B Zhou and K Gao ldquoChemical constituents of plantsfrom the genus Rhododendronrdquo Chemistry and Biodiversity vol8 no 5 pp 792ndash815 2011

[9] R Popescu and B Kopp ldquoThe genus Rhododendron anethnopharmacological and toxicological reviewrdquo Journal ofEthnopharmacology vol 147 no 1 pp 42ndash62 2013

[10] N Iwata N Wang X Yao and S Kitanaka ldquoStructuresand histamine release inhibitory effects of prenylated orcinolderivatives from Rhododendron dauricumrdquo Journal of NaturalProducts vol 67 no 7 pp 1106ndash1109 2004

[11] Q-Y Li L Chen Y-H Zhu M Zhang Y-P Wang andM-W Wang ldquoInvolvement of estrogen receptor-Β in farrerolinhibition of rat thoracic aorta vascular smooth muscle cellproliferationrdquo Acta Pharmacologica Sinica vol 32 no 4 pp433ndash440 2011

[12] Z Y Wu and R T Li ldquoChemical constituents of the roots ofRhododendron spiciferumrdquo Natural Product Research amp Devel-opment vol 23 no 2 pp 253ndash257 2011

[13] Y Fu L Zhang and G Chen ldquoFar infrared-assisted extractionfollowed by MEKC for the simultaneous determination offlavones and phenolic acids in the leaves of Rhododendronmucronulatum TurczrdquoThe Journal of Separation Science vol 35no 3 pp 468ndash475 2012

[14] S-T Ho Y-T Tung Y-L Chen Y-Y Zhao M-J Chungand J-H Wu ldquoAntioxidant activities and phytochemical studyof leaf extracts from 18 indigenous tree species in TaiwanrdquoEvidence-based Complementary and Alternative Medicine vol2012 Article ID 215959 2012

[15] Y-T Tung K-C Cheng S-T Ho et al ldquoComparison andcharacterization of the antioxidant potential of three wildgrapesmdashVitis thunbergii V flexuosa and V kelungeusisrdquo Journalof Food Science vol 76 no 5 pp C701ndashC706 2011

[16] K Ali F Maltese R Toepfer Y H Choi and R VerpoorteldquoMetabolic characterization of Palatinate German white winesaccording to sensory attributes varieties and vintages usingNMR spectroscopy and multivariate data analysesrdquo Journal ofBiomolecular NMR vol 49 no 3-4 pp 255ndash266 2011

[17] F Khallouki R Haubner W E Hull et al ldquoIsolation purifica-tion and identification of ellagic acid derivatives catechins andprocyanidins from the root bark of Anisophyllea dichostyla RBrrdquo Food and Chemical Toxicology vol 45 no 3 pp 472ndash4852007

[18] P Kiatgrajai J D Wellons L Gollob and J D White ldquoKineticsof epimerization of (+)-catechin and its rearrangement tocatechinic acidrdquo The Journal of Organic Chemistry vol 47 no15 pp 2910ndash2912 1982

[19] A Louis F Petereit M Lechtenberg A Deters and A HenselldquoPhytochemical characterization of Rhododendron ferrugineumand in vitro assessment of an aqueous extract on cell toxicityrdquoPlanta Medica vol 76 no 14 pp 1550ndash1557 2010

[20] S Hosoi E Shimizu K Ohno et al ldquoStructural studies ofzoospore attractants from Trachelospermum jasminoides varpubescens Taxifolin 3-O-glycosidesrdquo Phytochemical Analysisvol 17 no 1 pp 20ndash24 2006

[21] K Kazuma N Noda and M Suzuki ldquoMalonylated flavonolglycosides from the petals of Clitoria ternateardquo Phytochemistryvol 62 no 2 pp 229ndash237 2003

[22] G K Jayaprakasha M Ohnishi-Kameyama H Ono MYoshida and L J Rao ldquoPhenolic constituents in the fruitsof Cinnamomum zeylanicum and their antioxidant activityrdquoJournal of Agricultural and Food Chemistry vol 54 no 5 pp1672ndash1679 2006

[23] H D Chludil G B Corbino and S R Leicach ldquoSoil qualityeffects on Chenopodium album flavonoid content and antioxi-dant potentialrdquo Journal of Agricultural and Food Chemistry vol56 no 13 pp 5050ndash5056 2008

[24] D Y Lee H N Lyu H Y Kwak et al ldquoIsolation of flavonoidsfrom the fruits of Cornus kousa Burgrdquo Journal of The KoreanSociety For Applied Biological vol 50 no 3 pp 144ndash147 2007

[25] O I Aruoma ldquoMethodological considerations for character-izing potential antioxidant actions of bioactive components in


plant foodsrdquo Mutation Research - Fundamental and MolecularMechanisms of Mutagenesis vol 523-524 pp 9ndash20 2003

[26] K Schlesier M Harwat V Bohm and R Bitsch ldquoAssessmentof antioxidant activity by using different in vitromethodsrdquo FreeRadical Research vol 36 no 2 pp 177ndash187 2002

[27] M-T Chua Y-T Tung and S-T Chang ldquoAntioxidant activ-ities of ethanolic extracts from the twigs of Cinnamomumosmophloeumrdquo Bioresource Technology vol 99 no 6 pp 1918ndash1925 2008

[28] Y-T Tung J-H Wu C-Y Huang Y-H Kuo and S-T ChangldquoAntioxidant activities and phytochemical characteristics ofextracts from Acacia confusa barkrdquo Bioresource Technology vol100 no 1 pp 509ndash514 2009

[29] S Kumar G Chashoo A K Saxena and A K Pandey ldquoParthe-nium hysterophorus a probable source of anticancer antioxidantand anti-HIV agentsrdquo BioMed Research International vol 2013Article ID 810734 11 pages 2013

[30] M H Gordon ldquoThe mechanism of the antioxidant action invitrordquo inFoodAntioxidants B J FHudsonNewYorkNYUSA1990

[31] J-H Wu C-Y Huang Y-T Tung and S-T Chang ldquoOnlineRP-HPLC-DPPH screening method for detection of radical-scavenging phytochemicals from flowers of Acacia confusardquoJournal of Agricultural and Food Chemistry vol 56 no 2 pp328ndash332 2008

[32] I I Koleva H A G Niederlander and T A Van BeekldquoAn on-line HPLC method for detection of radical scavengingcompounds in complexmixturesrdquoAnalytical Chemistry vol 72no 10 pp 2323ndash2328 2000

[33] A Von Gadow E Joubert and C F Hansmann ldquoComparisonof the antioxidant activity of aspalathin with that of other plantphenols of rooibos tea (Aspalathus linearise) 120572-tocopherolBHT andBHArdquo Journal of Agricultural and FoodChemistry vol45 no 3 pp 632ndash638 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


2 Materials and Methods

21 Extraction and Fractionation of 10 Native RhododendronSpecies The leaves of 10 native Rhododendron species inTaiwan were collected at the end of April 2011 The collectionlocations of these specimens including R breviperulatumHayata (voucher number 1) R ellipticum Maxim (vouchernumber 2) R formosanum Hemsl (voucher number 3) RkanehiraiWilson (voucher number 4)RmariesiiHemsl andWilson (voucher number 5) R oldhamii Maxim (vouchernumber 6) R pseudochrysanthum Hayata (voucher number7) R rubropilosum Hayata var rubropilosum (voucher num-ber 8)R rubropilosumHayata var taiwanalpinum (Ohwi) LuYang and Tseng (voucher number 9) and R simsii Planch(voucher number 10) are presented in Figure 1(a) All ofthe voucher specimens were deposited at the herbarium ofthe Department of Forestry and Natural Resources NationalChiayi University (NCYU) Taiwan The species were iden-tified by Dr Lei-Chen Lin (NCYU) After identification thesamples were cleaned with tap water and dried Then theywere extracted twice with methanol by ultrasound-assistedextraction for 30min at room temperature The leaf extractsof 10 native Rhododendron species were decanted filteredunder vacuum and either concentrated in a rotator evapo-rator or lyophilized Furthermore the resulting methanoliccrude extracts of R pseudochrysanthum were fractionatedsuccessively with n-hexane ethyl acetate (EtOAc) n-butanol(BuOH) and water to yield soluble fractions of hexaneEtOAc BuOH and water All the extracts were stored in anairtight container at minus40∘C until further use

22 11-Diphenyl-2-picrylhydrazyl (DPPH) Assay The DPPHradical scavenging activity of the test extracts was examinedaccording to the method reported by Ho et al [14] Tenmicroliters of each test sample in methanol yielding a seriesof extract concentrations of 1 5 10 50 and 100120583gmLrespectively in each reaction was mixed with 200120583L of01mM DPPH-ethanol solution and 90120583L of 50mM Tris-HCl buffer (pH 74) Methanol (10 120583L) alone was used asthe control for this experiment After 30min of incubationat room temperature the reduction in DPPH radicals wasmeasured by reading the absorbance at 517 nm using aThermo Scientific Multiskan GO microplate reader (VantaaFinland) (+)-Catechin was used as the positive control Theinhibition ratio was calculated using the following equation inhibition = [(absorbance of control minus absorbance of testsample)absorbance of control] times 100

23 Superoxide Radical Scavenging Assay (NBT Assay) Themeasurement of superoxide radical scavenging activity wasconducted according to the method of Tung et al [15] First20120583L of 15mM Na

2EDTA in buffer (50mM KH

2PO4KOH

pH 74) 50 120583L of 06mM NBT (Nitro blue tetrazolium)in buffer 30 120583L of 3mM hypoxanthine in 50mM KOH5 120583L of the test samples in methanol (final concentrationswere 1 5 10 50 and 100 120583gmL resp) and 145 120583L of thebuffer were mixed in 96-well microplates The reaction wasstarted by adding 50 120583L of xanthine oxidase solution in buffer

(1 unit in 10mL buffer) to the mixture The reaction mixturewas incubated at room temperature and the absorbance at570 nm was determined every 1min (up to 9min) using amicroplate reader The control was 5120583L of methanol insteadof the sample solution and (+)-catechin was used as thepositive controlThe inhibition ratio was calculated using thefollowing equation inhibition = [(rate of control reactionminus rate of sample reaction)rate of control reaction] times 100

24 Ferrous Ion Chelating Assay The ferrous ion chelatingpotential of the test samples was evaluated according tothe method of Tung et al [15] Briefly 200120583L of the testsample in methanol (final concentrations were 125 250 5001000 and 2000120583gmL resp) and 740 120583L of methanol wereadded to 20 120583L of 2mM FeCl

2 The reaction was initiated

by adding 40 120583L of 5mM ferrozine The mixture was shakenvigorously and permitted to rest at room temperature for10min The absorbance of the solution was then measuredat 562 nm (+)-Catechin was used as the positive control Thepercent inhibition of Fe2+-ferrozine complex formation wascalculated according to the following equation inhibition=[(absorbance of control minus absorbance of sample)absorbanceof control] times 100

25 Reducing Power Assay This assay was determinedaccording to the method reported by Tung et al [15] using(+)-catechin as the standard Briefly 1mL of a reaction mix-ture containing 500120583L of 880 120583gmL test sample in 500120583L ofphosphate buffer (02M pH 66) was incubated with 500120583Lof potassium ferricyanide (1 wv) at 50∘C for 20min Thereaction was terminated by adding 500 120583L of trichloroaceticacid (10 wv) and the mixture was then centrifuged at12000 g for 10min The supernatant solution (500120583L) wasmixed with distilled water (500 120583L) and 100 120583L of the ferricchloride (01 wv) solution and the optical density (OD)was then measured at 700 nm The reducing power abilitywas expressed as milligrams of (+)-catechin equivalents (mgCE) per gram of sample or as micromolar of (+)-catechinequivalents (120583MCE) per millimolar of compound

26 Determination of Total Phenolics Total phenolic contentswere determined according to the Folin-Ciocalteu method[15] using gallic acid as the standard The test samples (5mg)were dissolved in 5mL of methanolwater (50 50 vv) andthe extract solution (500120583L) was mixed with 500120583L of 50Folin-Ciocalteu reagent The mixture was incubated at roomtemperature for 5min which was followed by the additionof 10mL of 20 Na

2CO3 After an additional 10min of

incubation at room temperature themixture was centrifugedfor 8min (12000 g) and the supernatant absorbancewasmea-sured at 730 nmThe total phenolic content was expressed asmilligrams of gallic acid equivalents (mg GAE) per gram ofsample

27 OnlineHPLC-DPPHMethod TheBuOH soluble fractionof R pseudochrysanthum was further analyzed by the onlineHPLC-DPPH method The instrumental setup is depicted inFigure 1(b) The BuOH soluble fraction (stock concentration


N

Tropic of cancer

25∘N

24∘N

23∘N

22∘N

122∘E120∘E 121∘E

3952

M

3000

M

2000

M

1000

M

8 9

22

37

1 5

9

6

4 104 10

1 5

37

8

6

(a)

280517

MeOHPump Pump

Injector

Extract

Mixer

Detector


RP-amidecolumn

H2O

(b)





















50values of







Specimens Yield(wt)


(mgCEg)






















50























C

E

B

A

D

c

e

ba

d

0

200

400

600

800

0

200

400

600

800

Fractions

Crudeextract


Redu

cing

pow

er (m

g CE

g)


Tota

l phe

nolic

cont

ent (

mg

GA

Eg)








minus250

0

250

500

750

10 20 30 40 50

Abso

rban

ce (m

AU)


12

3

5

6439984001 9984002998400

280nm

517nm

(a)

O

OH

OHOH

HO

O

2346

910

O

OH

HO

OHOH

OO Ara

2346

910

OOH

HOOH

OHO

OH

OHOHO

HO

OH

OHOHO

OH

OHOHO

OH

O

O

OH

HOOH

OH

OO

2346

910

OHOHO

OH

O

OH

HOOH

OH

OH

2346

910

1998400

1998400

19984001998400


1998400

2998400


R1 R3

R2

R1

R2

6 R1 = rhamR2 = H

CH3

CH3CH2OH

CH2OH




(b)



4 Conclusions






Acknowledgment


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N

Tropic of cancer

25∘N

24∘N

23∘N

22∘N

122∘E120∘E 121∘E

3952

M

3000

M

2000

M

1000

M

8 9

22

37

1 5

9

6

4 104 10

1 5

37

8

6

(a)

280517

MeOHPump Pump

Injector

Extract

Mixer

Detector


RP-amidecolumn

H2O

(b)





















50values of







Specimens Yield(wt)


(mgCEg)






















50























C

E

B

A

D

c

e

ba

d

0

200

400

600

800

0

200

400

600

800

Fractions

Crudeextract


Redu

cing

pow

er (m

g CE

g)


Tota

l phe

nolic

cont

ent (

mg

GA

Eg)








minus250

0

250

500

750

10 20 30 40 50

Abso

rban

ce (m

AU)


12

3

5

6439984001 9984002998400

280nm

517nm

(a)

O

OH

OHOH

HO

O

2346

910

O

OH

HO

OHOH

OO Ara

2346

910

OOH

HOOH

OHO

OH

OHOHO

HO

OH

OHOHO

OH

OHOHO

OH

O

O

OH

HOOH

OH

OO

2346

910

OHOHO

OH

O

OH

HOOH

OH

OH

2346

910

1998400

1998400

19984001998400


1998400

2998400


R1 R3

R2

R1

R2

6 R1 = rhamR2 = H

CH3

CH3CH2OH

CH2OH




(b)



4 Conclusions






Acknowledgment


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























50values of







Specimens Yield(wt)


(mgCEg)






















50























C

E

B

A

D

c

e

ba

d

0

200

400

600

800

0

200

400

600

800

Fractions

Crudeextract


Redu

cing

pow

er (m

g CE

g)


Tota

l phe

nolic

cont

ent (

mg

GA

Eg)








minus250

0

250

500

750

10 20 30 40 50

Abso

rban

ce (m

AU)


12

3

5

6439984001 9984002998400

280nm

517nm

(a)

O

OH

OHOH

HO

O

2346

910

O

OH

HO

OHOH

OO Ara

2346

910

OOH

HOOH

OHO

OH

OHOHO

HO

OH

OHOHO

OH

OHOHO

OH

O

O

OH

HOOH

OH

OO

2346

910

OHOHO

OH

O

OH

HOOH

OH

OH

2346

910

1998400

1998400

19984001998400


1998400

2998400


R1 R3

R2

R1

R2

6 R1 = rhamR2 = H

CH3

CH3CH2OH

CH2OH




(b)



4 Conclusions






Acknowledgment


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Specimens Yield(wt)


(mgCEg)






















50























C

E

B

A

D

c

e

ba

d

0

200

400

600

800

0

200

400

600

800

Fractions

Crudeextract


Redu

cing

pow

er (m

g CE

g)


Tota

l phe

nolic

cont

ent (

mg

GA

Eg)








minus250

0

250

500

750

10 20 30 40 50

Abso

rban

ce (m

AU)


12

3

5

6439984001 9984002998400

280nm

517nm

(a)

O

OH

OHOH

HO

O

2346

910

O

OH

HO

OHOH

OO Ara

2346

910

OOH

HOOH

OHO

OH

OHOHO

HO

OH

OHOHO

OH

OHOHO

OH

O

O

OH

HOOH

OH

OO

2346

910

OHOHO

OH

O

OH

HOOH

OH

OH

2346

910

1998400

1998400

19984001998400


1998400

2998400


R1 R3

R2

R1

R2

6 R1 = rhamR2 = H

CH3

CH3CH2OH

CH2OH




(b)



4 Conclusions






Acknowledgment


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































C

E

B

A

D

c

e

ba

d

0

200

400

600

800

0

200

400

600

800

Fractions

Crudeextract


Redu

cing

pow

er (m

g CE

g)


Tota

l phe

nolic

cont

ent (

mg

GA

Eg)








minus250

0

250

500

750

10 20 30 40 50

Abso

rban

ce (m

AU)


12

3

5

6439984001 9984002998400

280nm

517nm

(a)

O

OH

OHOH

HO

O

2346

910

O

OH

HO

OHOH

OO Ara

2346

910

OOH

HOOH

OHO

OH

OHOHO

HO

OH

OHOHO

OH

OHOHO

OH

O

O

OH

HOOH

OH

OO

2346

910

OHOHO

OH

O

OH

HOOH

OH

OH

2346

910

1998400

1998400

19984001998400


1998400

2998400


R1 R3

R2

R1

R2

6 R1 = rhamR2 = H

CH3

CH3CH2OH

CH2OH




(b)



4 Conclusions






Acknowledgment


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus250

0

250

500

750

10 20 30 40 50

Abso

rban

ce (m

AU)


12

3

5

6439984001 9984002998400

280nm

517nm

(a)

O

OH

OHOH

HO

O

2346

910

O

OH

HO

OHOH

OO Ara

2346

910

OOH

HOOH

OHO

OH

OHOHO

HO

OH

OHOHO

OH

OHOHO

OH

O

O

OH

HOOH

OH

OO

2346

910

OHOHO

OH

O

OH

HOOH

OH

OH

2346

910

1998400

1998400

19984001998400


1998400

2998400


R1 R3

R2

R1

R2

6 R1 = rhamR2 = H

CH3

CH3CH2OH

CH2OH




(b)



4 Conclusions






Acknowledgment


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgment


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Antioxidant Activities and Phytochemicals of …downloads.hindawi.com/journals/ecam/2014/283938.pdf · Research Article Antioxidant Activities and Phytochemicals