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Hindawi Publishing CorporationISRN ChromatographyVolume 2013 Article ID 283462 11 pageshttpdxdoiorg1011552013283462

Research ArticleProfiling of Phytochemicals in Tissues from Sclerocarya birreaby HPLC-MS and Their Link with Antioxidant Activity

Daniela Russo12 Owen Kenny1 Thomas J Smyth1 Luigi Milella2 Mohammad B Hossain1

Moussoukhoye Sissokho Diop3 Dilip K Rai1 and Nigel P Brunton4

1 Department of Food Biosciences Teagasc Food Research Centre Ashtown Dublin 15 Ireland2Department of Biology University of Basilicata Vle dellrsquoAteneo Lucano 85100 Potenza Italy3 Faculte des Sciences et Techniques Universite Cheikh Anta Diop de Dakar BP 5005 Dakar-Fann Senegal4 School of Agriculture and Food Science University College Dublin Dublin 4 Ireland

Correspondence should be addressed to Dilip K Rai dilipraiteagascie

Received 27 March 2013 Accepted 29 April 2013

Academic Editors R F D Nascimento T Oi Y Qiu and Y Uesawa

Copyright copy 2013 Daniela Russo 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

High performance liquid chromatography-tandemmass spectrometry (HPLC-MSMS) was employed to investigate the differencesin phytochemicals in roots bark and leaf of Sclerocarya birrea (marula) for methanol and water extracts that exhibited the bestantioxidant activities Asmany as 36 compoundswere observed in the extracts of these tissues ofwhich 27 phenolic compoundsweretentatively identified The HPLC-MSMS results showed flavonoid glycosides were prominent in leaf extracts while the galloylatedtannins were largely in bark and root extracts Four flavonoid glycosides that were reported for the first time in the marula leaf havebeen identifiedTheHPLC-MSMS studies also illustrated different degrees (highest degree = 3) of oligomerisation and galloylationof tannins in the bark and root extracts

1 Introduction

Sclerocarya birrea (A Rich) Hochst more commonly knownas marula is taxonomically derived from the Anacardiaceaeplant family It is an indigenous fruit-bearing tree of sub-Saharan Africa [1] It grows mostly at low altitudes andcan reach up to 20m in height and 12m in diameter [2]Traditionally marula has multiple uses the fruits are eaten orprocessed tomake beer and jam the kernels are eaten or theiroils extracted the leaves are used as forage for livestock andthe wood is carved into utilitarian items such as spoons andplates [2] The marula tree has been the subject of numerouschemical biological and environmental investigations since1906 [3] and has been identified as one of five fruit tree speciesthat should be integrated in the domestication process inAfrican farming system [4 5] This is due to its use as sourceof food and medicine in rural communities and its potentialto generate income through the sale of its derivates Thebark leaves and roots of Sclerocarya birrea (S birrea)haveattracted attention because they have been traditionally used

to treat an assortment of human ailments such as dysenteryfeversmalaria diarrhea stomach ailments rheumatism soreeyes gangrenous rectitis infertility headaches toothacheand body pains [6 7] As a result extracts of this planthave been reported to possess antioxidant antibacterial anti-fungal astringent anticonvulsant [8ndash10] antihyperglycemicanti-inflammatory [11] and antiatherogenic properties [12]Several of these properties could be attributed to the highcontent of polyphenols and its antioxidant activity [13ndash16]As a result of their high antioxidant activities extracts fromS birrea could also be used to control theoxidation ofunsaturated lipids in foods which not only lead to flavour [17]and colour deterioration [18] but can also lower nutritionalvalue and is associated with negative health effects suchas increased risk of heart disease and cancer in humans[19] Previous studies involving S birrea have detected thepresence of 11 phenolic compounds in leaf extracts usingHPLC-UV and HPLC-ESIMS [13] and also in pulp extractsby HPLC-UVVis detection [15] Only one previous studyhas investigated the phenolic composition of the bark where

2 ISRN Chromatography

NMR analysis identified the presence of a catechin derivate[20] In addition it has been reported that bark from Sbirrea contains a significant amount of highmolecular weighttannins [16 21] Condensed tannins such as procyanidinshave attracted increasing attention in the fields of nutritionand medicine due to their potential health benefits observedin vitro and in vivo For instance procyanidin oligomershave been shown to have potent antioxidant activity andthe ability to scavenge reactive oxygen and nitrogen species[22 23] However no study to date has attempted to quantifythe relative contribution of high and low molecular weightpolyphenols to the total in vitro antioxidant activity of Sbirrea In the present study we investigated a polyphenolrich crude extract that has been fractionated into high andlow molecular weight components The relative phenoliccontent and antioxidant activity of various crude and dialysedextracts from three different parts of S birrea bark leaf androot were determined Antioxidant extracts were selected forqualitative analysis by means of HPLC-ESI-MSMS basedon their relative in vitro antioxidant activities

2 Materials and Methods

21 Samples and Chemicals Fresh leaves bark and rootsof S birrea were collected from Dakar region in Senegalin September 2010 For the extraction analytical graden-hexane chloroform (CHCl

3) methanol (MeOH) ethyl-

acetate and water were acquired from Carlo Erba Reagents(Milan Italy) while 22-diphenyl-1-picrylhydrazyl (DPPH)ferric chloride (FeCl

3lowast6H2O) hydrochloric acid (HCl)

246-Tris(2-pyridil)-striazine (TPTZ) sodiumacetate Folin-Ciocalteu phenol reagent sodium carbonate Trolox HPLCgrade acetonitrile methanol formic acid (HCOOH) andwater along with leucine-enkephalin gallic acid catechinepicatechin epicatechin-3-O-gallate epigallocatechin-3-O-gallate quercetin-3-O-arabinoside quercetin-3-O-rhamno-side quercetin-3-O-glucoside and procyanidin B2 were pur-chased from Sigma-Aldrich (Arklow Co Wicklow Ireland)

22 Extraction and Fractionation Based on Polarity andMolecular Size The different parts of the plant which werecarefully cleaned removing foreign particles were cut intosmall pieces and air-dried at room temperature Leaves werepowdered using a pestle and mortar while the bark androots of S birrea were further broken up using a pestleand mortar Samples of S birrea bark leaves and rootsweighing 215 150 and 160 g respectively were defatted using5 volumes (vw) of n-hexane and extracted with CHCl

3

CHCl3MeOH (9 1) and MeOH at room temperature in

amber bottles over 6 days The bottles were periodicallyshaken and the solvent replenished at 48 hr intervals Eachsolvent extract was filtered through cellulose filter paper (17ndash25 120583m) and concentrated using a rotary evaporator underreduced pressure at 37∘C All extracts were weighed andstored in the dark at 4∘C

Each methanol extract was further partitioned intohydrophobic and hydrophilic fractions using water and ethy-lacetate exhaustively using a separating funnel The water

fractions were lyophilized with the aid of a freeze dryerwhile the ethylacetate fractions were concentrated using arotary evaporator The ethylacetate fractions were weighedand stored in the dark at 4∘C Dialysis tubing with amolecular weight cut-off of 35 kDa (Fisher Scientific LtdLoughborough Leicestershire UK) was used to separatemetabolites present in each water fraction into lowmolecularweight (lt35 kDa) and high molecular weight (gt35 kDa)extracts Each of the lt35 kDa and gt35 kDa dialysed extractswas lyophilized to dryness The weights of the lyophilisedextracts were recorded and stored in the dark at 4∘C (Table 1)

23 Free Radical Scavenging Activity by DPPH Method Amodified version of the DPPH assay with Trolox as astandard was used to measure antioxidant activity [24]Briefly 100120583L of various concentrations (stock solution of2mgmL) of the extract or Trolox were added to 100 120583L ofa methanol solution of DPPH (00476mgmL) in a 96-wellplateTheplatewas incubated at room temperature for 30minin the dark The absorbance of the mixture was measuredat 515 nm against the blank (MeOH) using a plate reader(FLUOstar Omega Ortenberg Germany)

24 Ferric Reducing Antioxidant Power (FRAP) Assay TheFRAP assay was carried out as described by Stratil et al withslight modifications [25] The FRAP reagent was prepared bymixing 38mM sodium acetate anhydrous buffer in distilledwater pH 36 with 20mM FeCl

3lowast6H2O in distilled water

and 10mM TPTZ in 40mM HCl in a ratio of 10 1 1 A20120583L of appropriately diluted sample extract and 180120583L ofFRAP reagent were mixed in a 96-well plate and incubatedat 37∘C for 40min in the dark In the case of the blank 20120583Lmethanol was added to 180120583L FRAP reagentThe absorbanceof the resulting solution was measured at 593 nm using aplate reader Trolox at concentrations ranging from 01mMndash04mM was used as a reference antioxidant standard Eachsample was performed in triplicate

25 Total Phenolic Content (TPC) The TPC was deter-mined using the Folin-Ciocalteu method as described bySingleton et al [26] Methanolic gallic acid solutions (10ndash200mgL) were used as standards In each replicate 100 120583Lof the appropriately diluted sample extract 100 120583L methanol100 120583LFolin-Ciocalteu reagent and 700120583L sodiumcarbonate(20wv) were added together and vortexed The mixturewas incubated for 20min in the dark at room temperatureAfter incubation the mixture was centrifuged at 13000 rpmfor 3minThe absorbance of the supernatant wasmeasured at735 nm using a UV-Vis spectrophotometer The experimentwas carried out in triplicate for all extract samples andstandard dilutions

26 HPLC-ESI-MSMS The analysis was carried out usingan Alliance 2695 HPLC system (Waters Corp Milford MA)coupled to aQ-Tof Premiermass spectrometer (Waters CorpMicromass MS Technologies Manchester UK) The Q-Tof

ISRN Chromatography 3

Table 1 Dry weight of the extracts and their fractions

Name of theplant

Parts of theplant

Extraction solvent

Hexane Chloroform Methanol Solvent based partitioning of themethanol extract

Membrane dialysisof the water extract

Marula

Bark 300mg 100mg 3600mg Water 3480mg lt35 kDa 1090mgEthylacetate 121mg gt35 kDa 1560mg

Leaf 350mg 500mg 900mg Water 263mg lt35 kDa 110mgEthylacetate 182mg gt35 kDa 20mg

Root 300mg 150mg 3000mg Water 2780mg lt35 kDa 340mgEthylacetate 196mg gt35 kDa 1220mg

was equipped with a lockspray source with an internal refer-ence compound (leucine-enkephalin) for accuratemassmea-surements An Atlantis T3 C18 column (waters Corp Mil-fordMA 21times 100mm)was used to separate the compoundsA binary mobile phase consisting of 05 HCOOH (solventA) and 05 HCOOH in 50 50 vv acetonitrilemethanol(solvent B) was used The gradient program was as follows0ndash15B in 1min 60ndash40B in 5min 50ndash50B in 2min30ndash70B in 6min 20ndash80B in 4min and 80ndash20Bin 7min The flow rate was 02mLmin Electrospray massspectra data were recorded in the negative ionization modefor a mass range from mz 100 to 1000 The capillary andcone voltage were set at 3 kV and 30V respectively and thedata were acquired using MassLynx version 41 The collisionenergy used for the MSMS experiments ranged from 8 eV to40 eV depending on the size of the molecular mass selectedfor the collision induced dissociation (CID)

27 Statistical Analysis Analysis of variance (ANOVA one-way) was used to find differences between the extractsstudied Means were compared by least significant difference(LSD) test at a significance level 119875 = 005 using theStatgraphics software (version 21 Statistical Graphics CoRockville USA)

3 Results and Discussion

31 Antioxidant Activity and Total Phenolic Content Theresults of the two antioxidant assays for the 15 differentextracts were broadly in agreement This was reflected bythe high Pearsonrsquos correlation coefficient value (119903 = 0940)between both In relation to each plant tissue under inves-tigation a common trend emerged which showed that thedialysed and water extracts of the leaf root and bark hadthe highest antioxidant activities in each case (Table 2)Most notably the root lt35 kDa dialysed extract exhibitedthe highest FRAP (22041 plusmn 4655 Trolox equivalent (TE)g100 g dry weight sample (DWS)) value The DPPH radicalscavenging activity of this extract also had a high value(137974 plusmn 6790 TE g100 g DWS) though the highest activityreported for the DPPH assay was seen in the bark water

crude extract (183973 plusmn 7029 TE g100 g DWS) The hexaneand ethyl acetate extracts of the leaf root and bark recordedgenerally lower antioxidant activities than those of the waterand methanolic extracts in this study This indicated that theantioxidant compounds inmarula were predominantly polarA comparison of DPPH IC

50values between the lowest and

highest antioxidant activity confirmed that the leaf hexanebark ethyl acetate and root hexane extracts had a significantly(119875 lt 005) lower antioxidant potential than the leaf lt35 kDaand water extracts of both the bark and roots

In line with their high antioxidant activities these Sbirrea polar extracts also possessed a high total phenoliccontent (TPC) The TPC data of these extracts revealed ahigh degree of positive correlation with the results of boththe DPPH (Pearsonrsquos correlation coefficient 119903 = 0905) andFRAP (Pearsonrsquos correlation coefficient 119903 = 0855) assaysMoyo et al also reported that leaf young stem and operculaextracts from S birrea had the highest antioxidant activityand possessed the highest phenolic content thus indicating astrong relationship between phenolic content and antioxidantactivity [27] The root gt35 kDa dialysed extract had thehighest TPC value (10339 plusmn 0006 gallic acid equivalent(GAE) g100 g DWS) followed by the root lt35 kDa dialysedextract (101586 plusmn 0009 GAE g100 g DWS) This could bedue to the presence of high molecular weight polymericpolyphenols in these extracts [16 21]The leaf hexane extractas expected from the low antioxidant activity values had thelowest TPC value (1000 plusmn 001 GAE g100 g DWS) In thecase of leaf extracts the hydrophilic lt35 kDa fractions hadsignificantly higher TPC (119875 lt 005) than the fractions ofgt35 kDa This might be due to greater relative abundance oflow molecular weight unbound polar phenolic compoundsin the leaf compared to the bark and root Other authorshave also reported that when a high level of structuralpolysaccharides are present in food which could be the casefor both the bark and the root extracts a high proportion ofpolyphenols will be bound to these polysaccharides [28]

To date the majority of studies on S birrea have con-centrated on the fruit and leaves [12 13] Mariod et alinvestigated the phenolic content and antioxidant activity of60 aqueous methanol extracts from leaf bark and root ofS birrea and reported that the bark and root extracts hadhigher phenolic content than the leaf extract [15] which is


Table 2 Total phenol FRAP and DPPH radical-scavenging activities for various leaf bark and root crude and dialysed extracts of marulatree (S birrea) Values are expressed as means plusmn standard deviations (119899 = 3)1

Extract TPC (GAE g100 g DWS) FRAP (TE g100 g DWS) DPPH IC50 (120583gmL) DPPH (TE g100 g DWS)Leaf hexane 1000 plusmn 001a 008 plusmn 003a 17657 plusmn 142e 427 plusmn 034a

Leaf ethylacetate 1829 plusmn 002b 802 plusmn 076b 7663 plusmn 531d 982 plusmn 069a

Leaf lt35 kDa 5388 plusmn 002e 7284 plusmn 188e 842 plusmn 036ab 8920 plusmn 395e

Leaf gt35 kDa 4453 plusmn 001d 5510 plusmn 223d 1388 plusmn 040b 5407 plusmn 180c

Leaf Water 4535 plusmn 0001d 5610 plusmn 220d 1223 plusmn 024b 6136 plusmn 119c

Bark hexane 4843 plusmn 002de 8812 plusmn 809f 991 plusmn 059ab 7585 plusmn 459d

Bark ethylacetate 2302 plusmn 001bc 2333 plusmn 119c 2612 plusmn 309c 2830 plusmn 320b

Bark lt35 kDa 10017 plusmn 001i 21664 plusmn 840k 511 plusmn 010a 14686 plusmn 272i

Bark gt35 kDa 6966 plusmn 0003f 10246 plusmn 549g 595 plusmn 050ab 12664 plusmn 1077g

Bark water 9033 plusmn 003h 19734 plusmn 558j 408 plusmn 016a 18397 plusmn 703j

Root hexane 2653 plusmn 0005c 2663 plusmn 349c 2965 plusmn 021c 2539 plusmn 185b

Root ethylacetate 7864 plusmn 002g 11459 plusmn 531h 623 plusmn 020ab 12038 plusmn 401g

Root lt35 kDa 10159 plusmn 001i 22041 plusmn 465k 544 plusmn 027a 13797 plusmn 679h

Root gt35 kDa 10340 plusmn 001i 12071 plusmn 344h 705 plusmn 048ab 10666 plusmn 705f

Root Water 7253 plusmn 002fg 18479 plusmn 1105i 542 plusmn 006a 13845 plusmn 162h1For all the values within a column different letter superscripts mean significant differences (119875 lt 005)

also the case in the present study The antioxidant activityof methanolic extract of marula leaf has been reported to behigher or equal that of butylated hydroxytoluene [27 29]Thepresent study has shown that the bark and root extracts hadeven higher antioxidant activity than the leaf extracts

32 Analyses of Polyphenols by HPLC-ESI-MSMS Thelt35 kDa dialysed extracts that possessed the best antioxi-dant activity and phenolic content were selected for char-acterisation by HPLC-MSMS A total of 36 compoundswere observed with 27 phenolic compounds being ten-tatively or fully identified a disaccharide four deriva-tives of either sugar or galloyl derivatives and four com-pounds could not be identified (Table 3) Structural char-acterisation of each compound was achieved throughaccurate mass measurement and interpretation of CIDmass spectrum Of the 36 compounds the identitiesof 9 phenolic compounds namely gallic acid catechinepicatechin epicatechin-3-O-gallate epigallocatechin-3-O-gallate quercetin-3-O-arabinoside quercetin-3-O-rhamno-side quercetin-3-O-glucoside and procyanidin B2 were con-firmed by the comparison with retention times of the stan-dards

The phenolic compounds present in the leaf bark androot of S birrea differed from those reported in the fruitFor example Ndhlala et al reported the presence of caffeicacid vanillic acid ferulic acid and p-coumaric acid in thepeel and pulp of the fruit none of which were detectedin extracts from the plant tissues examined in the presentstudy [16] Galloyl derivatives of flavonoid glycosides andprocyanidins were the common phenolic compounds inthe marula plant The flavonoid glycosides were exclusivelyfound in the leaf extracts while the galloylated tannins werepredominant in the bark extracts (Table 3 andFigure 1)Three

previously unreported phenolic glycosides for this speciesnamely dihydrobenzoic acid-O-pentoside quercetin-3-O-arabinoside kaempferol-O-pentoside and hydroxyl benzoylkaempferol-O-hexoside were identified (Figure 2) in the leafextract in addition to the eight compounds (peaks 12ndash1517ndash19 21) that had been previously reported [13] Shown inthe inserts of Figure 2 are the proposed chemical structurescleavages during CID experiment and the expected exactmz values for the fragment ions of four compounds newto the marula plant The accurate mass measurement ofmz 2851 (peak 4) produced the elemental composition ofthe compound as C

12H14O8 Subsequent MSMS studies

on the ions mz 2851 showed the major product ions mz1530 tentatively assigned as deprotonated dihydroxyben-zoic acid (DHBA) from the loss of dehydrated pentose(132Da) and a further loss of CO

2generating the minor

product ions mz 1080 Accurate mass measurements onthe product ions mz 1530 and mz 1080 fitted well withthe empirical formula for DHBA and the decarboxylatedDHBA respectively (Figure 2(a)) The mz 4331 (peak 16)was initially assigned as quercetin-O-pentoside based onthe accurate mass measurement and the loss of a dehy-drated pentoside residue which produced the product ionmz 3011 (deprotonated quercetin) in the MSMS spectrum(Figure 2(b)) the identification was further supported bythe identical retention time of the standard quercetin-3-O-arabinoside Similarly the identification of kaempferol-O-pentoside was reached for the [MminusH]minus ion at mz 4171(peak 20) and upon its fragmentation produced the production mz of 2851 corresponding to kaempferol (or luteolin)via the loss of a dehydrated pentoside residue (Figure 2(c))A hydroxyl benzoyl kaempferol-O-hexoside was assignedfor peak 22 based on the accurate mass measurement andMSMS fragmentation pattern (Figure 2(d)) It must be noted


Table 3 Phytochemicals observed in HPLC-MS analysis of various parts of S birrea

Peaknnmber RT (min) Proposed compound ID Empirical formula Calculated119898119911 Observed119898119911 MSMS ions

1 154 Disaccharide C12H21O11minus 3411084 3411075 1790 1431 1010

2 202 Gallic acidlowastlowastlowast C7H5O5minus 1690137 1690126 1250

3 274 Procyanidin dimer B2 C30H25O12minus 5771346 5771326 4071 2890 1690 1250

4lowastlowast 296 Dihydroxy benzoic acidpentoside C12H13O8

minus 2850610 2850605 15211531 10811091

5 348Monogalloylatedprocyanidin dimer B(P2G1)

C37H29O16minus 7291456 7291442 5771 4071 2891 1690

6 388 Benzyl alcoholhexoside-pentoside C18H25O10

minus 4011448 4011454 3010 2691 1611 1010

7 457 Hexose derivative C20H31O10minus 4311917 4311924 269 1531

8 466 Digalloylated procyanidinB (P2G2) C44H33O20

minus 8811565 8811535 7292 5596 4071 1690

9 560 Megastigmane hexoside C26H35O12minus 5392129 5392111 4912 3292 1651


11 734 Epicatechin 3-O-gallate(P1G1)lowastlowastlowast C22H17O10

minus 4410822 441083 2891 1690

12lowast 820 Quercetin 3-O-120573-D-(610158401015840-galloyl)glucopyranoside C28H23O16

minus 6150986 6150982 4632 3011

13lowast 857 Quercetin 3-O-120573-D-(610158401015840-galloyl)galactopyranoside C28H23O16

minus 6150986 6150982 4632 3011

14lowast 937 Myricetin 3-O-120572-L-rhamnopyranoside C21H19O12

minus 4630877 4630877 3011

15lowast 963 Quercetin 3-O-120573-D-glucopyranosidelowastlowastlowast C21H19O12

minus 4630877 4630887 3011

16lowastlowast 1011 Quercetin3-O-arabinosidelowastlowastlowast C20H17O11

minus 4330771 4330776 3011

17lowast 1053 Kaempferol 3-O-120573-D-(610158401015840-galloyl)glucopyranoside C28H23O15

minus 5991037 5991041 4471 3131 2851 1690

18lowast 1103 Quercetin 3-O-120572-L-rhamnopyranosidelowastlowastlowast C21H19O11

minus 4470928 4470941 30113000 28512841

19lowast 1136 Quercetin 3-O-120572-(510158401015840-galloyl)arabinofuranoside C27H21O15

minus 5850881 5850876 4331 3011

20lowastlowast 1160 Kaempferol pentoside C20H17O10minus 4170822 4170838 28512840

21lowast 1236 Kaempferol 3-O-120572-L-rhamnopyranoside C21H19O10

minus 4310978 4310975 2851

22lowastlowast 1261 Hydroxy benzoylkaempferol-O-hexoside C28H23O13

minus 5671139 5671148 4471 2850 2550 1370

23 1299 Galloyl hexoside derivative C26H35O11minus 5232179 5232172 3612 3131 1690

24 238 Pentoside derivative C20H29O12minus 4611659 4611653 3291 1911 1791 1491

25 279 Catechinlowastlowastlowast C15H13O6minus 2890712 2890700

26 312Galloylepicatechin-epigallocatechin-3-O-gallate

C44H33O21minus 8971514 8971510 4491 4071 2891 1770 1251

27 335 Epigallocatechin3-O-gallatelowastlowastlowast C22H17O11

minus 4570771 4570763 3051

28 375 Epicatechinlowastlowastlowast C15H13O6minus 2890712 2890700

29 495 Trigalloylated procyanidintrimer (P3G3) C66H50O30

minus 13212309 1321237 11693 10173 8811 5771 4070 1690

30 547 Unknown C21H19O17minus 5430622 5430618

31 804 Epicatechin-O-gallate(P1G1) C22H17O10

minus 4410822 441083 2891 1690


Table 3 Continued



33 1049 Digalloylated procyanidindimer B (P2G2) C44H33O20

minus 8811565 8811577 7292 5596 4071 1690



36 356 Galloyl derivative C20H21O12minus 4531033 453104 3130 1690

lowastIdentified based on previous report by Braca et al 2003 [13]lowastlowastPreviously unreported in Sclerocarya birrealowastlowastlowastIdentification by comparison with standards

100 TOF MS ES-BPI

1

23

46

78 9 1011 12

13

14

15

16

17

1819

20

21

22

23

5

()

0

SB leaf

2171198903

2 4 6 8 10 12 14 16 18

(a)

0

100 TOF MS ES-BPI 709

1

2

8

27511

2832

2425

30 3331

34

26 29

35

()

8

SB bark

2 4 6 8 10 12 14 16 18

(b)

Time

0

100 TOF MS ES-BPI 1

2

11

8

27536

26

2 4 6 8 10 12 14 16 18

()

SB root

1491198903

(c)

Figure 1 HPLC chromatograms showing the phytochemicals in (a) leaf extract (b) bark extract and (c) root extract of S birrea

that kaempferol has an identical chemical formula to that ofluteolin but they are structurally different and hence producesdifferent fragment ions [30] Kaempferol unlike luteolin hasbeen shown to lose 30Da neutral molecule resulting in aproduct ion mz 255 which was also seen in the MSMSspectra of peak 20 and peak 22 (Figures 2(c) and 2(d))thus confirming these two peaks were kaempferol derivativesThis approach was taken to identification of megastigmane

hexosides (peaks 9 and 10) and benzyl-alcohol hexoside-pentoside (peak 6) Our findings are in congruent with thereports in the past where benzyl derived phytochemicals hadbeen commonly seen in the marula fruit pulp and the wholefruit [31]

Procyanidins a group of flavonoids ubiquitous in theplant kingdom are a mixture of flavan-3-ol monomers (epi-catechin andor catechin) commonly bonded throughC4ndashC8


100 150 200 250 300 350 400 450 500 550 6000

1003 TOF MSMS 28507ES-

2850629

1530196

1080229

HO

HOHO

OH

OH

OH

OH

OH

O

O

O

O

O()

SB leaf 32 (2504)

Dihydroxy benzoicacid pentoside

Dihydroxy benzoate Hydroxy benzoquinone radical

minus∙

1651198904

119898119911 1530188119898119911 1080211

119898119911

119898119911

285061

O

(a)

100 150 200 250 300 350 400 450 500 550 6000

100

TOF MSMS 43309ES- 145

4330749

3010359

O

O

OO

()

SB leaf 021 (0017)

Quercetin-3-O-arabinoside

OHOH

OH

OHOH

OH

119898119911

119898119911

4330771

O

(b)

100 150 200 250 300 350 400 450 500 550 6000

100

6 TOF MSMS 41709ES-

2840348

255032

41708422850414

28604564180873

OOO

O

O

O

O

OO(

)

SB leaf 39 (9711)

Kaempferol-3-O-pentoside

Kaempferol

OH

OHOH

OH

OH

OH

OH OH

OH

1311198904

119898119911 4170822

119898119911 2850399

119898119911

119898119911

2550293

O O

O

(c)

Figure 2 Continued


100 150 200 250 300 350 400 450 500 550 6000

100 2840345

25503311370248

2850422

28604585671123

4470893287047 5681173

O

O

O O

O Glu

Kaempferol-O-hexoside-

()

SB leaf 31 (10755)

Hydroxy benzoylkaempferol hexoside

Hydroxy-benzoate

OH

OHHO

O

3051198903

119898119911 5671139

119898119911

119898119911

1370239

7 TOF MSMS 5671ES-

O

O

Kaempferol

(d)

Figure 2 ESI-MSMS spectra of four polyphenolic glycosides reported for the first time in the S birrea (a) dihydroxybenzoic acid-O-pentoside (peak 4) (b) quercetin-3-O-arabinoside (peak 16) (c) kaempferol-O-pentoside (peak 20) and (d) hydroxyl benzoyl kaempferol-O-hexoside (peak 22)

linkages The procyanidins were predominantly found in thebark of S birrea (Figure 1) Epicatechin-3-O-gallate (ECG)in this study was found in the water part of the partitionedof the methanolic extract in contrast to previous report onits occurrence in the ethylacetate portion of a methanolicextract from themarula bark [20] Another compound (peak31) with identical molecular mass and fragment ions as thatof ECG but in less abundance was observed in the barkand root extracts this compound was assigned as an isomerof epicatechin-3-O-gallate Most of the procyanidins wereesterified with gallic acid residues and produced polymersof varying molecular mass (Table 3) The largest galloylatedprocyanidin polymer was found with a molecular mass of1322Da (peak 29) with a degree of galloylation of 3 andwas identified as trigalloylated procyanidin trimer (P3G3)Lower degrees of galloylated procyanidins such as P3G2 atmz 8812 (peaks 8 33) P2G1 at mz 7292 (peak 5) werealso found largely in the bark and root extracts FurtherHPLC-ESI-MSMS data supported the diagnosis where thesequential loss of neutral molecules due to the loss of thedehydrated gallic acid residue (152Da) and the eliminationof an epicatechin unit (288Da) produced the major productions from the galloylated procyanidins as shown in Figure 3Although several previous studies have shown the presenceof procyanidins and their galloyl derivatives in marula thisreport profiles the presence of an additional 6 procyanidins(Table 3) A variety of these types of procyanidin derivativeshave also been reported previously in both grape seeds andpomace [32 33] In many cases a range of different structuresand isomers of procyanidins would be present that currentlywould not be detectable using HPLC-MS due to inability toseparate and elute them fromanHPLC column anddue to the

lack of sensitivity based on the small quantities that would bepresent for each particular structure

As evident from this study flavonoid glycosides andgalloylated procyanidins constituted the bulk of phenoliccompounds in the lt35 kDa fractions The only previousreport on the phenolic composition of the bark identified oneepicatechin derivative The present study is also the first toreport on the phenolic composition of root extracts from thiscommercially important tree and they contain a mixture ofmostly epicatechin and gallic acid derivatives Epicatechin-3-O-gallate has been shown to illicit secretagogue activity whenderived from the bark of the plant [20] The procyanidinswere mainly found in roots and barks with high phenoliccontents and were thus responsible for the high antioxidantactivity Previous studies have shown that the procyanidinsalso possess antiviral and anticancer properties but limitedantimicrobial activity [34]

4 Conclusion

Application of HPLC-MSMS technique provided usefulinformation to characterize 27 phenolic compounds in theextracts of marula The accurate mass measurements andfragments produced during CID are the diagnostic featuresof these compounds which could be used to identify themin different extracts Four phenolic glycosides and 6 pro-cyanidins have been reported for the first time in marulaFurther spectroscopic characterization specifically NMRwill be required to establish the identity of the glycoside andunderpin the position of its linkage to the flavonoid ringHowever due to small amount of sample material this couldnot be carried out in this present study The antioxidant


()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)

Figure 3 ESI-MSMS spectra of various oligomers of galloylated procyanidins (a)monogalloylated procyanidin or P1G1 (peaks 11 and 31) (b)monogalloylated procyanidin dimer B or P2G1 (peak 5) (c) digalloylated procyanidin dimer B or P2G2 (peaks 8 and 33) and (d) trigalloylatedprocyanidin trimer C or P3G3 (peak 29) The loss of galloyl residue (152Da) is illustrated with dashed arrow while the elimination of theepicatechin unit (288Da) is represented with dotted arrow

activities of the extracts were linked to the occurrence ofmoderate to highly polar polyphenols particularly flavonoidglycosides

Conflict of Interests

There is no conflict of interests

Acknowledgments

The authors thank Professor Valeria Costantino of the Uni-versity of Napoli Italy for her support and encouragementtowards this project They have been most grateful forthe financial support from the Irish Phytochemical FoodNetwork (IPFN) which is funded under Food InstitutionalResearch Measure (FIRM) by the Irish Department of Agri-culture Food and Marine

References

[1] C R Peter ldquoNotes on the distribution and relative abundanceof Sclerocarya birrea (A Rich)Hochst (Anacardiaceae)rdquoMono-graphs in Systematic Botany of the Missouri Botanical Gardensvol 25 pp 403ndash410 1988

[2] G N Gouwakinnou A M Lykke A E Assogbadjo andB Sinsin ldquoLocal knowledge pattern and diversity of use ofSclerocarya birreardquo Journal of Ethnobiology and Ethnomedicinevol 7 article 8 2011

[3] R Wynberg J Cribbins R Leakey et al ldquoKnowledge onSclerocarya birrea subsp caffra with emphasis on its importanceas a non-timber forest product in South and southern Africa asummary Part 2 commercial use tenure and policy domestica-tion intellectual property rights and benefit-sharingrdquo SouthernAfrican Forestry Journal no 196 pp 67ndash77 2002

[4] B A Jama A M Mohamed J Mulatya and A N NjuildquoComparing the ldquoBig Fiverdquo a framework for the sustainablemanagement of indigenous fruit trees in the drylands of East


and Central Africardquo Ecological Indicators vol 8 no 2 pp 170ndash179 2008

[5] N C Mokgolodi Y-F Ding M P Setshogo C Ma and Y JLiu ldquoThe importance of an indigenous tree to southern Africancommunities with specific relevance to its domestication andcommercialization a case of the marula treerdquo Forestry Studiesin China vol 13 no 1 pp 36ndash44 2011

[6] M Gelfand The Traditional Medical Practitioner in ZimbabweHis Principles of Practice and Pharmacopoeia Mambo Press1985

[7] J A O Ojewole T Mawoza W D H Chiwororo and P MO Owira ldquoSclerocarya birrea (A Rich) Hochst [ldquoMarulardquo](anacardiaceae) a review of its phytochemistry pharmacologyand toxicology and its ethnomedicinal usesrdquo PhytotherapyResearch vol 24 no 5 pp 633ndash639 2010

[8] J N Eloff ldquoAntibacterial activity of Marula (Sclerocarya birrea(A rich) Hochst subsp Caffra (Sond) Kokwaro) (Anacar-diaceae) bark and leavesrdquo Journal of Ethnopharmacology vol 76no 3 pp 305ndash308 2001

[9] P Masoko T J Mmushi M M Mogashoa M P MokgothoL J Mampuru and R L Howard ldquoIn vitro evaluation ofthe antifungal activity of Sclerocarya birrea extracts againstpathogenic yeastsrdquo African Journal of Biotechnology vol 7 no20 pp 3521ndash3526 2008

[10] L J McGaw D Van der Merwe and J N Eloff ldquoIn vitroanthelmintic antibacterial and cytotoxic effects of extractsfrom plants used in South African ethnoveterinary medicinerdquoVeterinary Journal vol 173 no 2 pp 366ndash372 2007

[11] I G M Ndifossap F Frigerio M Casimir et al ldquoSclerocaryabirrea (Anacardiaceae) stem-bark extract corrects glycaemiain diabetic rats and acts on 120573-cells by enhancing glucose-stimulated insulin secretionrdquo Journal of Endocrinology vol 205no 1 pp 79ndash86 2010

[12] H Borochov-Neori S Judeinstein A Greenberg et al ldquoPheno-lic antioxidants and antiatherogenic effects ofmarula (Sclerocar-rya birrea subsp caffra) fruit juice in healthy humansrdquo Journal ofAgricultural and Food Chemistry vol 56 no 21 pp 9884ndash98912008

[13] A Braca M Politi R Sanogo et al ldquoChemical compositionand antioxidant activity of phenolic compounds from wild andcultivated Sclerocarya birrea (Anacardiaceae) leavesrdquo Journal ofAgricultural and Food Chemistry vol 51 no 23 pp 6689ndash66952003

[14] A Lamien-Meda C E Lamien M M Y Compaore et alldquoPolyphenol content and antioxidant activity of fourteen wildedible fruits from Burkina Fasordquo Molecules vol 13 no 3 pp581ndash594 2008

[15] A A Mariod B Matthaus and I H Hussein ldquoAntioxi-dant properties of methanolic extracts from different parts ofSclerocarya birreardquo International Journal of Food Science andTechnology vol 43 no 5 pp 921ndash926 2008

[16] A R Ndhlala A Kasiyamhuru C Mupure K ChitindinguM A Benhura and M Muchuweti ldquoPhenolic composition ofFlacourtia indica Opuntia megacantha and Sclerocarya birreardquoFood Chemistry vol 103 no 1 pp 82ndash87 2007

[17] N P Brunton D A Cronin and F J Monahan ldquoVolatilecomponents associated with freshly cooked and oxidized off-flavours in Turkey breast meatrdquo Flavour and Fragrance Journalvol 17 no 5 pp 327ndash334 2002

[18] M N OrsquoGrady F J Monahan and N P Brunton ldquoOxymyo-globin oxidation and lipid oxidation in bovine musclemdashmechanistic studiesrdquo Journal of Food Science vol 66 no 3 pp386ndash392 2001

[19] P B Addis ldquoOccurrence of lipid oxidation products in foodsrdquoFood and Chemical Toxicology vol 24 no 10-11 pp 1021ndash10301986

[20] J Galvez Peralta A Zarzuelo R Busson C Cobbaert andP De Witte ldquo(-)-Epicatechin-3-galloyl ester a secretagoguecompound from the bark of Sclerocarya birreardquo Planta Medicavol 58 no 2 pp 174ndash175 1992

[21] A A Aganga and K W Mosase ldquoTannin content nutritivevalue and dry matter digestibility of Lonchocarpus capassaZizyphus mucronata Sclerocarya birrea Kirkia acuminata andRhus lancea seedsrdquo Animal Feed Science and Technology vol 91no 1-2 pp 107ndash113 2001

[22] T Ariga and M Hamano ldquoRadical scavenging action andits mode in Procyanidins B-1 and B-3 from azuki beans toperoxyl radicals (FoodampNutrition)rdquoAgricultural andBiologicalChemistry vol 54 no 10 pp 2499ndash2504 1990

[23] G E Arteel and H Sies ldquoProtection against peroxynitrite bycocoa polyphenol oligomersrdquo FEBS Letters vol 462 no 1-2 pp167ndash170 1999

[24] P GoupyMHugues P Boivin andM J p Amiot ldquoAntioxidantcomposition and activity of barley (Hordeum vulgare) and maltextracts and of isolated phenolic compoundsrdquo Journal of theScience of Food and Agriculture vol 79 no 12 pp 1625ndash16341999

[25] P Stratil B Klejdus and V Kuban ldquoDetermination of totalcontent of phenolic compounds and their antioxidant activity invegetablesmdashevaluation of spectrophotometric methodsrdquo Jour-nal of Agricultural and Food Chemistry vol 54 no 3 pp 607ndash616 2006

[26] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998

[27] M Moyo A R Ndhlala J F Finnie and J Van StadenldquoPhenolic composition antioxidant and acetylcholinesteraseinhibitory activities of Sclerocarya birrea and Harpephyllumcaffrum (Anacardiaceae) extractsrdquo Food Chemistry vol 123 no1 pp 69ndash76 2010

[28] J Perez-Jimenez and F Saura-Calixto ldquoLiterature data mayunderestimate the actual antioxidant capacity of cerealsrdquo Jour-nal of Agricultural and Food Chemistry vol 53 no 12 pp 5036ndash5040 2005

[29] P Olsen O Meyer N Bille and G Wurtzen ldquoCarcinogenic-ity study on butylated hydroxytoluene (BHT) in Wistar ratsexposed in uterordquo Food and Chemical Toxicology vol 24 no1 pp 1ndash12 1986

[30] M Ye Y Yan and D A Guo ldquoCharacterization of phenoliccompounds in the Chinese herbal drug Tu-Si-Zi by liquidchromatography coupled to electrospray ionization mass spec-trometryrdquo Rapid Communications in Mass Spectrometry vol 19no 11 pp 1469ndash1484 2005

[31] AMViljoen G P P Kamatou andKH C Baser ldquoHead-spacevolatiles of marula (Sclerocarya birrea subsp caffra)rdquo SouthAfrican Journal of Botany vol 74 no 2 pp 325ndash326 2008

[32] C P Passos SMCardosoMRMDomingues PDominguesC M Silva andM A Coimbra ldquoEvidence for galloylated type-A procyanidins in grape seedsrdquo Food Chemistry vol 105 no 4pp 1457ndash1467 2007


[33] I I Rockenbach E Jungfer C Ritter B Thiele R Fett andR Galensa ldquoCharacterization of flavan-3-ols in seeds of grapepomace by CE HPLC-DAD-MS and LC-ESI-FTICR-MSrdquo FoodResearch International vol 48 pp 848ndash855 2012

[34] T De Bruyne L Pieters M Witvrouw E De Clercq D VBerghe and A J Vlietinck ldquoBiological evaluation of proantho-cyanidin dimers and related polyphenolsrdquo Journal of NaturalProducts vol 62 no 7 pp 954ndash958 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


NMR analysis identified the presence of a catechin derivate[20] In addition it has been reported that bark from Sbirrea contains a significant amount of highmolecular weighttannins [16 21] Condensed tannins such as procyanidinshave attracted increasing attention in the fields of nutritionand medicine due to their potential health benefits observedin vitro and in vivo For instance procyanidin oligomershave been shown to have potent antioxidant activity andthe ability to scavenge reactive oxygen and nitrogen species[22 23] However no study to date has attempted to quantifythe relative contribution of high and low molecular weightpolyphenols to the total in vitro antioxidant activity of Sbirrea In the present study we investigated a polyphenolrich crude extract that has been fractionated into high andlow molecular weight components The relative phenoliccontent and antioxidant activity of various crude and dialysedextracts from three different parts of S birrea bark leaf androot were determined Antioxidant extracts were selected forqualitative analysis by means of HPLC-ESI-MSMS basedon their relative in vitro antioxidant activities

2 Materials and Methods

21 Samples and Chemicals Fresh leaves bark and rootsof S birrea were collected from Dakar region in Senegalin September 2010 For the extraction analytical graden-hexane chloroform (CHCl

3) methanol (MeOH) ethyl-

acetate and water were acquired from Carlo Erba Reagents(Milan Italy) while 22-diphenyl-1-picrylhydrazyl (DPPH)ferric chloride (FeCl

3lowast6H2O) hydrochloric acid (HCl)

246-Tris(2-pyridil)-striazine (TPTZ) sodiumacetate Folin-Ciocalteu phenol reagent sodium carbonate Trolox HPLCgrade acetonitrile methanol formic acid (HCOOH) andwater along with leucine-enkephalin gallic acid catechinepicatechin epicatechin-3-O-gallate epigallocatechin-3-O-gallate quercetin-3-O-arabinoside quercetin-3-O-rhamno-side quercetin-3-O-glucoside and procyanidin B2 were pur-chased from Sigma-Aldrich (Arklow Co Wicklow Ireland)

22 Extraction and Fractionation Based on Polarity andMolecular Size The different parts of the plant which werecarefully cleaned removing foreign particles were cut intosmall pieces and air-dried at room temperature Leaves werepowdered using a pestle and mortar while the bark androots of S birrea were further broken up using a pestleand mortar Samples of S birrea bark leaves and rootsweighing 215 150 and 160 g respectively were defatted using5 volumes (vw) of n-hexane and extracted with CHCl

3

CHCl3MeOH (9 1) and MeOH at room temperature in

amber bottles over 6 days The bottles were periodicallyshaken and the solvent replenished at 48 hr intervals Eachsolvent extract was filtered through cellulose filter paper (17ndash25 120583m) and concentrated using a rotary evaporator underreduced pressure at 37∘C All extracts were weighed andstored in the dark at 4∘C

Each methanol extract was further partitioned intohydrophobic and hydrophilic fractions using water and ethy-lacetate exhaustively using a separating funnel The water

fractions were lyophilized with the aid of a freeze dryerwhile the ethylacetate fractions were concentrated using arotary evaporator The ethylacetate fractions were weighedand stored in the dark at 4∘C Dialysis tubing with amolecular weight cut-off of 35 kDa (Fisher Scientific LtdLoughborough Leicestershire UK) was used to separatemetabolites present in each water fraction into lowmolecularweight (lt35 kDa) and high molecular weight (gt35 kDa)extracts Each of the lt35 kDa and gt35 kDa dialysed extractswas lyophilized to dryness The weights of the lyophilisedextracts were recorded and stored in the dark at 4∘C (Table 1)

23 Free Radical Scavenging Activity by DPPH Method Amodified version of the DPPH assay with Trolox as astandard was used to measure antioxidant activity [24]Briefly 100120583L of various concentrations (stock solution of2mgmL) of the extract or Trolox were added to 100 120583L ofa methanol solution of DPPH (00476mgmL) in a 96-wellplateTheplatewas incubated at room temperature for 30minin the dark The absorbance of the mixture was measuredat 515 nm against the blank (MeOH) using a plate reader(FLUOstar Omega Ortenberg Germany)

24 Ferric Reducing Antioxidant Power (FRAP) Assay TheFRAP assay was carried out as described by Stratil et al withslight modifications [25] The FRAP reagent was prepared bymixing 38mM sodium acetate anhydrous buffer in distilledwater pH 36 with 20mM FeCl

3lowast6H2O in distilled water

and 10mM TPTZ in 40mM HCl in a ratio of 10 1 1 A20120583L of appropriately diluted sample extract and 180120583L ofFRAP reagent were mixed in a 96-well plate and incubatedat 37∘C for 40min in the dark In the case of the blank 20120583Lmethanol was added to 180120583L FRAP reagentThe absorbanceof the resulting solution was measured at 593 nm using aplate reader Trolox at concentrations ranging from 01mMndash04mM was used as a reference antioxidant standard Eachsample was performed in triplicate

25 Total Phenolic Content (TPC) The TPC was deter-mined using the Folin-Ciocalteu method as described bySingleton et al [26] Methanolic gallic acid solutions (10ndash200mgL) were used as standards In each replicate 100 120583Lof the appropriately diluted sample extract 100 120583L methanol100 120583LFolin-Ciocalteu reagent and 700120583L sodiumcarbonate(20wv) were added together and vortexed The mixturewas incubated for 20min in the dark at room temperatureAfter incubation the mixture was centrifuged at 13000 rpmfor 3minThe absorbance of the supernatant wasmeasured at735 nm using a UV-Vis spectrophotometer The experimentwas carried out in triplicate for all extract samples andstandard dilutions

26 HPLC-ESI-MSMS The analysis was carried out usingan Alliance 2695 HPLC system (Waters Corp Milford MA)coupled to aQ-Tof Premiermass spectrometer (Waters CorpMicromass MS Technologies Manchester UK) The Q-Tof



Name of theplant

Parts of theplant

Extraction solvent



Marula













































minus 2850610 2850605 15211531 10811091


C37H29O16minus 7291456 7291442 5771 4071 2891 1690


minus 4011448 4011454 3010 2691 1611 1010



minus 8811565 8811535 7292 5596 4071 1690




minus 4410822 441083 2891 1690


minus 6150986 6150982 4632 3011


minus 6150986 6150982 4632 3011


minus 4630877 4630877 3011


minus 4630877 4630887 3011


minus 4330771 4330776 3011


minus 5991037 5991041 4471 3131 2851 1690


minus 4470928 4470941 30113000 28512841


minus 5850881 5850876 4331 3011



minus 4310978 4310975 2851


minus 5671139 5671148 4471 2850 2550 1370





C44H33O21minus 8971514 8971510 4491 4071 2891 1770 1251


minus 4570771 4570763 3051



minus 13212309 1321237 11693 10173 8811 5771 4070 1690



minus 4410822 441083 2891 1690


Table 3 Continued




minus 8811565 8811577 7292 5596 4071 1690





100 TOF MS ES-BPI

1

23

46

78 9 1011 12

13

14

15

16

17

1819

20

21

22

23

5

()

0

SB leaf

2171198903

2 4 6 8 10 12 14 16 18

(a)

0


1

2

8

27511

2832

2425

30 3331

34

26 29

35

()

8

SB bark

2 4 6 8 10 12 14 16 18

(b)

Time

0

100 TOF MS ES-BPI 1

2

11

8

27536

26

2 4 6 8 10 12 14 16 18

()

SB root

1491198903

(c)






100 150 200 250 300 350 400 450 500 550 6000


2850629

1530196

1080229

HO

HOHO

OH

OH

OH

OH

OH

O

O

O

O

O()

SB leaf 32 (2504)



minus∙

1651198904

119898119911 1530188119898119911 1080211

119898119911

119898119911

285061

O

(a)

100 150 200 250 300 350 400 450 500 550 6000

100


4330749

3010359

O

O

OO

()

SB leaf 021 (0017)


OHOH

OH

OHOH

OH

119898119911

119898119911

4330771

O

(b)

100 150 200 250 300 350 400 450 500 550 6000

100

6 TOF MSMS 41709ES-

2840348

255032

41708422850414

28604564180873

OOO

O

O

O

O

OO(

)

SB leaf 39 (9711)


Kaempferol

OH

OHOH

OH

OH

OH

OH OH

OH

1311198904

119898119911 4170822

119898119911 2850399

119898119911

119898119911

2550293

O O

O

(c)

Figure 2 Continued


100 150 200 250 300 350 400 450 500 550 6000

100 2840345

25503311370248

2850422

28604585671123

4470893287047 5681173

O

O

O O

O Glu


()

SB leaf 31 (10755)


Hydroxy-benzoate

OH

OHHO

O

3051198903

119898119911 5671139

119898119911

119898119911

1370239

7 TOF MSMS 5671ES-

O

O

Kaempferol

(d)





4 Conclusion



()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)





Acknowledgments


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Name of theplant

Parts of theplant

Extraction solvent



Marula













































minus 2850610 2850605 15211531 10811091


C37H29O16minus 7291456 7291442 5771 4071 2891 1690


minus 4011448 4011454 3010 2691 1611 1010



minus 8811565 8811535 7292 5596 4071 1690




minus 4410822 441083 2891 1690


minus 6150986 6150982 4632 3011


minus 6150986 6150982 4632 3011


minus 4630877 4630877 3011


minus 4630877 4630887 3011


minus 4330771 4330776 3011


minus 5991037 5991041 4471 3131 2851 1690


minus 4470928 4470941 30113000 28512841


minus 5850881 5850876 4331 3011



minus 4310978 4310975 2851


minus 5671139 5671148 4471 2850 2550 1370





C44H33O21minus 8971514 8971510 4491 4071 2891 1770 1251


minus 4570771 4570763 3051



minus 13212309 1321237 11693 10173 8811 5771 4070 1690



minus 4410822 441083 2891 1690


Table 3 Continued




minus 8811565 8811577 7292 5596 4071 1690





100 TOF MS ES-BPI

1

23

46

78 9 1011 12

13

14

15

16

17

1819

20

21

22

23

5

()

0

SB leaf

2171198903

2 4 6 8 10 12 14 16 18

(a)

0


1

2

8

27511

2832

2425

30 3331

34

26 29

35

()

8

SB bark

2 4 6 8 10 12 14 16 18

(b)

Time

0

100 TOF MS ES-BPI 1

2

11

8

27536

26

2 4 6 8 10 12 14 16 18

()

SB root

1491198903

(c)






100 150 200 250 300 350 400 450 500 550 6000


2850629

1530196

1080229

HO

HOHO

OH

OH

OH

OH

OH

O

O

O

O

O()

SB leaf 32 (2504)



minus∙

1651198904

119898119911 1530188119898119911 1080211

119898119911

119898119911

285061

O

(a)

100 150 200 250 300 350 400 450 500 550 6000

100


4330749

3010359

O

O

OO

()

SB leaf 021 (0017)


OHOH

OH

OHOH

OH

119898119911

119898119911

4330771

O

(b)

100 150 200 250 300 350 400 450 500 550 6000

100

6 TOF MSMS 41709ES-

2840348

255032

41708422850414

28604564180873

OOO

O

O

O

O

OO(

)

SB leaf 39 (9711)


Kaempferol

OH

OHOH

OH

OH

OH

OH OH

OH

1311198904

119898119911 4170822

119898119911 2850399

119898119911

119898119911

2550293

O O

O

(c)

Figure 2 Continued


100 150 200 250 300 350 400 450 500 550 6000

100 2840345

25503311370248

2850422

28604585671123

4470893287047 5681173

O

O

O O

O Glu


()

SB leaf 31 (10755)


Hydroxy-benzoate

OH

OHHO

O

3051198903

119898119911 5671139

119898119911

119898119911

1370239

7 TOF MSMS 5671ES-

O

O

Kaempferol

(d)





4 Conclusion



()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)





Acknowledgments


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

































minus 2850610 2850605 15211531 10811091


C37H29O16minus 7291456 7291442 5771 4071 2891 1690


minus 4011448 4011454 3010 2691 1611 1010



minus 8811565 8811535 7292 5596 4071 1690




minus 4410822 441083 2891 1690


minus 6150986 6150982 4632 3011


minus 6150986 6150982 4632 3011


minus 4630877 4630877 3011


minus 4630877 4630887 3011


minus 4330771 4330776 3011


minus 5991037 5991041 4471 3131 2851 1690


minus 4470928 4470941 30113000 28512841


minus 5850881 5850876 4331 3011



minus 4310978 4310975 2851


minus 5671139 5671148 4471 2850 2550 1370





C44H33O21minus 8971514 8971510 4491 4071 2891 1770 1251


minus 4570771 4570763 3051



minus 13212309 1321237 11693 10173 8811 5771 4070 1690



minus 4410822 441083 2891 1690


Table 3 Continued




minus 8811565 8811577 7292 5596 4071 1690





100 TOF MS ES-BPI

1

23

46

78 9 1011 12

13

14

15

16

17

1819

20

21

22

23

5

()

0

SB leaf

2171198903

2 4 6 8 10 12 14 16 18

(a)

0


1

2

8

27511

2832

2425

30 3331

34

26 29

35

()

8

SB bark

2 4 6 8 10 12 14 16 18

(b)

Time

0

100 TOF MS ES-BPI 1

2

11

8

27536

26

2 4 6 8 10 12 14 16 18

()

SB root

1491198903

(c)






100 150 200 250 300 350 400 450 500 550 6000


2850629

1530196

1080229

HO

HOHO

OH

OH

OH

OH

OH

O

O

O

O

O()

SB leaf 32 (2504)



minus∙

1651198904

119898119911 1530188119898119911 1080211

119898119911

119898119911

285061

O

(a)

100 150 200 250 300 350 400 450 500 550 6000

100


4330749

3010359

O

O

OO

()

SB leaf 021 (0017)


OHOH

OH

OHOH

OH

119898119911

119898119911

4330771

O

(b)

100 150 200 250 300 350 400 450 500 550 6000

100

6 TOF MSMS 41709ES-

2840348

255032

41708422850414

28604564180873

OOO

O

O

O

O

OO(

)

SB leaf 39 (9711)


Kaempferol

OH

OHOH

OH

OH

OH

OH OH

OH

1311198904

119898119911 4170822

119898119911 2850399

119898119911

119898119911

2550293

O O

O

(c)

Figure 2 Continued


100 150 200 250 300 350 400 450 500 550 6000

100 2840345

25503311370248

2850422

28604585671123

4470893287047 5681173

O

O

O O

O Glu


()

SB leaf 31 (10755)


Hydroxy-benzoate

OH

OHHO

O

3051198903

119898119911 5671139

119898119911

119898119911

1370239

7 TOF MSMS 5671ES-

O

O

Kaempferol

(d)





4 Conclusion



()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)





Acknowledgments


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Table 3 Continued




minus 8811565 8811577 7292 5596 4071 1690





100 TOF MS ES-BPI

1

23

46

78 9 1011 12

13

14

15

16

17

1819

20

21

22

23

5

()

0

SB leaf

2171198903

2 4 6 8 10 12 14 16 18

(a)

0


1

2

8

27511

2832

2425

30 3331

34

26 29

35

()

8

SB bark

2 4 6 8 10 12 14 16 18

(b)

Time

0

100 TOF MS ES-BPI 1

2

11

8

27536

26

2 4 6 8 10 12 14 16 18

()

SB root

1491198903

(c)






100 150 200 250 300 350 400 450 500 550 6000


2850629

1530196

1080229

HO

HOHO

OH

OH

OH

OH

OH

O

O

O

O

O()

SB leaf 32 (2504)



minus∙

1651198904

119898119911 1530188119898119911 1080211

119898119911

119898119911

285061

O

(a)

100 150 200 250 300 350 400 450 500 550 6000

100


4330749

3010359

O

O

OO

()

SB leaf 021 (0017)


OHOH

OH

OHOH

OH

119898119911

119898119911

4330771

O

(b)

100 150 200 250 300 350 400 450 500 550 6000

100

6 TOF MSMS 41709ES-

2840348

255032

41708422850414

28604564180873

OOO

O

O

O

O

OO(

)

SB leaf 39 (9711)


Kaempferol

OH

OHOH

OH

OH

OH

OH OH

OH

1311198904

119898119911 4170822

119898119911 2850399

119898119911

119898119911

2550293

O O

O

(c)

Figure 2 Continued


100 150 200 250 300 350 400 450 500 550 6000

100 2840345

25503311370248

2850422

28604585671123

4470893287047 5681173

O

O

O O

O Glu


()

SB leaf 31 (10755)


Hydroxy-benzoate

OH

OHHO

O

3051198903

119898119911 5671139

119898119911

119898119911

1370239

7 TOF MSMS 5671ES-

O

O

Kaempferol

(d)





4 Conclusion



()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)





Acknowledgments


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


100 150 200 250 300 350 400 450 500 550 6000


2850629

1530196

1080229

HO

HOHO

OH

OH

OH

OH

OH

O

O

O

O

O()

SB leaf 32 (2504)



minus∙

1651198904

119898119911 1530188119898119911 1080211

119898119911

119898119911

285061

O

(a)

100 150 200 250 300 350 400 450 500 550 6000

100


4330749

3010359

O

O

OO

()

SB leaf 021 (0017)


OHOH

OH

OHOH

OH

119898119911

119898119911

4330771

O

(b)

100 150 200 250 300 350 400 450 500 550 6000

100

6 TOF MSMS 41709ES-

2840348

255032

41708422850414

28604564180873

OOO

O

O

O

O

OO(

)

SB leaf 39 (9711)


Kaempferol

OH

OHOH

OH

OH

OH

OH OH

OH

1311198904

119898119911 4170822

119898119911 2850399

119898119911

119898119911

2550293

O O

O

(c)

Figure 2 Continued


100 150 200 250 300 350 400 450 500 550 6000

100 2840345

25503311370248

2850422

28604585671123

4470893287047 5681173

O

O

O O

O Glu


()

SB leaf 31 (10755)


Hydroxy-benzoate

OH

OHHO

O

3051198903

119898119911 5671139

119898119911

119898119911

1370239

7 TOF MSMS 5671ES-

O

O

Kaempferol

(d)





4 Conclusion



()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)





Acknowledgments


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


100 150 200 250 300 350 400 450 500 550 6000

100 2840345

25503311370248

2850422

28604585671123

4470893287047 5681173

O

O

O O

O Glu


()

SB leaf 31 (10755)


Hydroxy-benzoate

OH

OHHO

O

3051198903

119898119911 5671139

119898119911

119898119911

1370239

7 TOF MSMS 5671ES-

O

O

Kaempferol

(d)





4 Conclusion



()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)





Acknowledgments


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


()

0

100TOF MSMS 4411ES-

1894411

169 2891P1G1

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark04 52 (0889)

mz

(a)

0

100

()

TOF MSMS 72914ES- 1347292

40712891169

5771P2G14411

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark03 113 (1933)

mz

(b)

0

100

()

TOF MSMS 88117ES- 4818812

169929 40712891 72925591 6492

P2G2

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300

SB bark02 3 (0051)

mz

(c)

100 200 300 400 500 600 700 800 900 1000 1100 1200 13000

100

()


13212116928812169

1252871 729155714071

9992P3G3SB bark0149 (0838)

mz

(d)





Acknowledgments


References















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of











































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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