Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 519174 6 pageshttpdxdoiorg1011552013519174

Research ArticleIsolation and Chemical Structural Characterisation ofa Compound with Antioxidant Activity from the Roots ofSenna italica

Matlou Phineas Mokgotho1 Stanley Sechene Gololo1 Peter Masoko1

Ladislaus Kakore Mdee2 Vusi Mbazima1 Leshwene Jeremiah Shai3

Victor Patrick Bagla1 Jacobus Nicolaas Eloff4 and Leseilane Mampuru1

1 Department of Biochemistry Microbiology and Biotechnology University of Limpopo Turfloop Campus Private Bag X1106Sovenga 0727 South Africa

2 Department of Pharmacy University of Limpopo Turfloop Campus Private Bag X1106 Sovenga 0727 South Africa3 Department of Biomedical Sciences Faculty of Science Tshwane University of Technology Private Bag X680Pretoria 0001 South Africa

4 Faculty of Veterinary Science Department of Paraclinical Science Phytomedicine Programme University of PretoriaOnderstepoort Pretoria 0001 South Africa

Correspondence should be addressed to Matlou Phineas Mokgotho matloumokgothoulacza

Received 3 April 2013 Revised 22 May 2013 Accepted 23 May 2013

Academic Editor Mohamed Eddouks

Copyright copy 2013 Matlou Phineas Mokgotho et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Senna italica a member of the Fabaceae family (subfamily Caesalpiniaceae) is widely used in South African traditional medicineto treat a number of disease conditions Aqueous extracts of the plant are mainly used to treat sexually transmitted infectionsand intestinal complications The roots of S italica were ground to a fine powder and sequentially extracted with n-hexanedichloromethane acetone and methanol using serial exhaustive extraction (SEE) method Thin layer chromatography was usedto analyse the phytochemical composition of the extracts and DPPH radical scavenging method to detect the presence ofantioxidant compounds The bioassay guided fractionation of the acetone fraction afforded an antioxidant compound with freeradical scavenging activityThe isolated compoundwas subsequently identified as 3410158405-trihydroxystilbene (resveratrol)This studyrepresents the first report of the stilbene resveratrol in S italica

1 Introduction

Plants have formed the basis of sophisticated traditional me-dicinal systems that have been in existence for thousands ofyears and they continue to provide humanity with new reme-dies [1]The importance of plants in human and animal healthis evident in the increasing presence of natural product drugsin modern medicine Indeed natural products and their de-rivatives represent more than 50 of all the drugs in clinicaluse in the world today During the last 40 years at least adozen potent drugs have been derived from flowering plantsOne example is that of diosgenin a drug template derivedfrom Dioscorea species and used to synthesise contraceptive

agents [2] Other examples include anticancer agents derivedfrom Catharanthus roseus as well as laxative agents fromCassia species [1]

The current advancement in science has made it possi-ble for the isolation of compounds of medical importancefrom plants that are used in traditional medicinal practicesDifferent parts of plants such as leaves roots stems barksand rhizomes are often extracted using different solventsIn most cases extracts have been shown to be biologicallyactive both in the in vitro and in vivo test systems [3] Someplant-derived compounds are effective in combination withothers while others are active as single entities [4] How-ever the isolation of compounds remains a challenging and

2 Evidence-Based Complementary and Alternative Medicine

a mammoth task Conventionally the isolation of bioactivecompounds is preceded by the determination of the presenceof such compounds within plant extracts through a numberof bioassays

Senna italica amember of the Fabaceae family (subfamilyCaesalpinaceae) is known for its therapeutic properties inAfrican folk medicine Some Senna species from Venda inSouth Africa are used to treat sexually transmitted infections(STIs) while others are considered to possess significant anti-bacterial activities [5] In the northern parts of the Limpopoprovince of South Africa S italica is indicated for the treat-ment of STIs [6] A literature survey on the chemical constit-uents of the genus Senna revealed the presence of alkaloidsquinines and anthraquinones These types of compoundshave been isolated from heartwood seeds root bark rootsand leaves of the genus Senna [7] Other studies have report-ed the isolation of many other compounds including beta-sitosterol stigmasterol and amyrin from the aerial parts ofS italica [8] We have previously reported the antibacterialeffect of the acetone extract of S italica on selected bacterialstrains total phenolic content as well as its effect on cancerousJurkat T cell [9] Based on these findings S italicawas furtherinvestigated with the aim of identifying the compound(s)that exerted the observed biological activities Antioxidantcompounds are important in the prevention or treatment ofvarious human diseases Undeniably reports abound thatlink free radical scavenging potential and other biologicalbenefits of antioxidant compounds [10] We here report theisolation of resveratrol a compoundwith antioxidant activityfrom the acetone root extract of Senna italica through bioas-say-guided fractionation

2 Materials and Method

21 Extraction The roots of Senna italicawere collected fromBolahlakgomo village (Zebediela subregion Limpopo prov-ince South Africa) A voucher specimen (UNIN11129) hasbeen deposited in the Larry Leach herbarium of the Univer-sity of Limpopo The roots were dried at room temperatureand subsequentlymilled to a fine powder using a grinder (ML90L4 Monitoring and Control Laboratories (Pty) Ltd RSA)The milled material (700 g) was serial exhaustively extractedusing 119899-hexane dichloromethane (DCM) acetone andmeth-anol (MeOH) as extractants The extraction process was re-peated three times with 2 litres of each solvent The extractswere filtered through aWhatman no 3 filter paper and the fil-trates concentrated using a rotary evaporator (Buchi Labotecrotavapormodel R-205 Germany)The concentrated extractswere then transferred into preweighed beakers dried undera stream of cold air and weighed to determine the resultantmass

22 Free Radical Scavenging Activity The presence of anti-oxidant constituents in the various extracts was determinedusing the method of [11] Thin layer chromatographic plateswere prepared and spotted with the different extracts andeluted in solvents systems of varying polarity namely ethylacetatemethanolwater (EMW) (40 54 5) (polarneutral)

chloroformethyl acetateformic acid (CEF) (5 4 1) (inter-mediate polarityacidic) benzeneethanolammonium hy-droxide (BEA) (90 10 1) (nonpolarbasic) For the determi-nation of antioxidant activity the plates were sprayed with0222-diphenyl-1-picryl-hydrazyl (DPPH)(Sigma)inmeth-anol as an indicator A positive reaction is indicated by theappearance of a yellow spot against a purple background

23 Fractionation The acetone extract had constituents withantioxidant activity and was thus chosen for further frac-tionationwith CHCl

3MeOHof increasing gradient polarity

starting with 100 CHCl3to 100 MeOH A 460mg sample

of acetone extract was fractionated (Figure 1) into seven frac-tions of CHCl

3MeOH in the following ratios 1 0 9 1 8 2

7 3 1 1 3 7 and 0 1 (vv) using column chromatographypacked with silica gel 60 (63ndash200120583m) Following elution theantioxidant constituents of the resultant fractions were qual-itatively evaluated using the TLC-DPPH method describedabove The fraction that eluted with 9 1 (CHCl

3MeOH

(9 1)) was found to contain the major compound with anti-oxidant activity Hence the fraction was chosen for furtherpurification of the target compound

24 Elution and Structural Characterisation of the AntioxidantCompound Several mobile phases were tested and fractionF9 1 was better resolved with CHCl

3 EtAOC (1 1) The 9 1

(125mg) was subjected to a silica gel 60 column chromatog-raphy using CHCl

3MeOH (4 1) as the eluent Fractions 13ndash

18 were subjected to chromatography using CHCl3MeOH

(9 1) as eluent After recrystallisation a 13mg of the purecompoundwas obtainedThe compoundwas dissolved eitherinmethanol chloroform or dimethyl sulfoxide depending onits solubility for analysis The structure of the isolated com-pound was elucidated from the data obtained from 1H- and13C-NMR spectra

3 Results

Different solvents depending on their polarity extract vary-ing quantities of components in crude plant material thatmay be beneficial or harmful to biological systems 119899-Hexanefor instance extracts waxes fats and fixed oils while acetoneextracts alkaloids aglycones and glycosides On the otherhand methanol extracts sugars amino acids and glycosideswhile DCM will commonly extract alkaloids aglycones andvolatile oils For these reasons plant material was extractedusing solvent of varying polarity namely hexane DCM ace-tone andmethanol An increase in the extractedmaterial wasobserved as the polarity of the extracting solvent increased(Table 1) Methanol extract had the highest percent yieldfollowed by DCM hexane and acetone yielded the least

Extracted materials were spotted on TLC plates and elut-ed in BEA EMW and CEF and thereafter sprayed withvanillin-sulphuric acidThe free radical scavenging activity ofthe extracts was determined using the DPPH assay The yieldof the methanol extract did not correlate with the presenceof antioxidant constituents in the extract Although acetoneextracts yielded the least more active constituents were

Evidence-Based Complementary and Alternative Medicine 3

NMR

Serial exhaustive extraction

Acetone DCM fraction Methanol fraction

Senna roots 700 g

n-Hexane fraction(48 g) fraction (46 g)(62 g) (323 g)

CHCl3 MeOH (vv) fractionation

9 1 8 2 7 3 6 4 1 1 0 11 0

CHCl3 EtoAc (1 1 vv) elution

Tubes 13ndash18 pooled together

CHCl3 EtoAc (1 1 vv) reelution

Compound I13mg

Figure 1 Schematic representation of fractionation of acetone extracts of S italica

present in these extracts with strong antioxidant activityThismay possibly be related to the ability of acetone to extractboth polar and nonpolar constituents Since acetone extractdemonstrated a superior free radical scavenging activity itwas thus chosen for further purification The extract wasfractionated with the gradient solutions of CHCl

3MeOH of

different ratios Fraction 8 2 gave the highest yield (237mg)

followed by fraction 9 1 (125mg) with fraction 0 1 givingthe lowest yield (14mg) as shown (Table 2) Fractions 8 2and 9 1 contained appreciable amounts of vanillin-reactingcompounds than the other fractions with fraction 9 1 show-ing prominent antioxidant activity than the rest (data notincluded) Thus fraction 9 1 was selected for further purifi-cation

4 Evidence-Based Complementary and Alternative Medicine

(a) (b)

Figure 2 TLC plate of isolated compound sprayed with DPPH (a) and isolated compound sprayed with vanillin-sulphuric acid reagent (b)The plates were developed in CHCl

3 EtOAc (1 1 vv) as the mobile phase A positive reaction is indicated by the appearance of a yellow spot

against a purple background

Table 1 Yields following serial exhaustive extraction of root mate-rial of Senna italica with 119899-hexane DCM acetone and MeOH

Extract solvent Yield (g) Total yield (g) Yield ()Hexane 1 28Hexane 2 13 48 069Hexane 3 07DCM 1 35DCM 2 15 62 089DCM 3 12Acetone 1 23Acetone 2 14 46 066Acetone 3 09MeOH 1 179MeOH 2 85 323 46MeOH 3 59

A suitable mobile phase for elution of the bioactive com-pound was obtained by comparison of the separated com-pounds attained from the various ratios of CHCl

3MeOH

solution used The CHCl3MeOH ratio of 1 1 (vv) gave a

better separation of the active compound The 9 1 fractionof the acetone extract was then subsequently eluted withCHCl

3MeOH (1 1 vv) through a silica gel column Tubes

containing similar compounds noted on TLC fingerprintwere pooled together and reeluted with CHCl

3MeOH (1 1

vv) through a silica gel columnThe reelution of the fractionobtained from the pooled tubes gave yield to a pure com-pound with a positive free radical scavenging activity evidentby a yellow spot against a purple background on TLC fol-lowing spraying with DPPH (Figure 2) The structure of theisolated compound was elucidated (Figure 3) from the data

Table 2 Fractionation of the acetone root extract (700mg) withCHCl3 MeOH different ratios on silica gel chromatography

Systems CHCl3()

MeOH()

Solvent quantity (mL) Yield (mg)

A 100 mdash 500 58B 90 10 700 125C 80 20 1200 237D 70 30 500 30E 50 50 600 25F 30 70 500 16G mdash 100 600 14

obtained from 1H- and 13C-NMR spectra and those obtainedfrom the literature [12 13] (Table 3)

Compound 1 was obtained as a white powder that waspositive to FeCl

3test which indicates the presence of phenolic

group In the 1H-NMR spectrum the coupling patternsbetween 120575 743 (2H d J = 90Hz) and 686 (2H d J = 90Hz)and 120575 655 (2H brs) and 616 (1H brs) showed the typicalresveratrol skeleton [14] The coupling constant (J = 180Hz)between H-7 (120575 689 1H d) and H-8 (120575 703 1H d) impliedthat the configuration of compound 1 was in a trans formconfiguration In the 13C-NMR spectrum three oxygenatedaromatic carbons (120575 1596 (times2) and 1582) and eleven sp2carbons (1027sim1409) were shown From these data and thosepresented in Table 3 compound 1 was identified as trans-resveratrol [12 13]

4 Evidence-Based Complementary and Alternative Medicine

(a) (b)

Figure 2 TLC plate of isolated compound sprayed with DPPH (a) and isolated compound sprayed with vanillin-sulphuric acid reagent (b)The plates were developed in CHCl

3 EtOAc (1 1 vv) as the mobile phase A positive reaction is indicated by the appearance of a yellow spot

against a purple background

Table 1 Yields following serial exhaustive extraction of root mate-rial of Senna italica with 119899-hexane DCM acetone and MeOH

Extract solvent Yield (g) Total yield (g) Yield ()Hexane 1 28Hexane 2 13 48 069Hexane 3 07DCM 1 35DCM 2 15 62 089DCM 3 12Acetone 1 23Acetone 2 14 46 066Acetone 3 09MeOH 1 179MeOH 2 85 323 46MeOH 3 59

A suitable mobile phase for elution of the bioactive com-pound was obtained by comparison of the separated com-pounds attained from the various ratios of CHCl

3MeOH

solution used The CHCl3MeOH ratio of 1 1 (vv) gave a

better separation of the active compound The 9 1 fractionof the acetone extract was then subsequently eluted withCHCl

3MeOH (1 1 vv) through a silica gel column Tubes

containing similar compounds noted on TLC fingerprintwere pooled together and reeluted with CHCl

3MeOH (1 1

vv) through a silica gel columnThe reelution of the fractionobtained from the pooled tubes gave yield to a pure com-pound with a positive free radical scavenging activity evidentby a yellow spot against a purple background on TLC fol-lowing spraying with DPPH (Figure 2) The structure of theisolated compound was elucidated (Figure 3) from the data

Table 2 Fractionation of the acetone root extract (700mg) withCHCl3 MeOH different ratios on silica gel chromatography

Systems CHCl3()

MeOH()

Solvent quantity (mL) Yield (mg)

A 100 mdash 500 58B 90 10 700 125C 80 20 1200 237D 70 30 500 30E 50 50 600 25F 30 70 500 16G mdash 100 600 14

obtained from 1H- and 13C-NMR spectra and those obtainedfrom the literature [12 13] (Table 3)

Compound 1 was obtained as a white powder that waspositive to FeCl

3test which indicates the presence of phenolic

group In the 1H-NMR spectrum the coupling patternsbetween 120575 743 (2H d J = 90Hz) and 686 (2H d J = 90Hz)and 120575 655 (2H brs) and 616 (1H brs) showed the typicalresveratrol skeleton [14] The coupling constant (J = 180Hz)between H-7 (120575 689 1H d) and H-8 (120575 703 1H d) impliedthat the configuration of compound 1 was in a trans formconfiguration In the 13C-NMR spectrum three oxygenatedaromatic carbons (120575 1596 (times2) and 1582) and eleven sp2carbons (1027sim1409) were shown From these data and thosepresented in Table 3 compound 1 was identified as trans-resveratrol [12 13]

Evidence-Based Complementary and Alternative Medicine 5

Table 3 13C NMR data of isolated compound (300MHz acetone-d6)

C number Literature data Isolated compound data1 1413 14092 1058 10573 1597 15964 1027 10275 1597 15966 1058 10577 1294 12918 1270 126911015840 1304 130021015840 1288 128731015840 1165 116441015840 1584 158251015840 1165 116461015840 1288 1287

12

3

4

56

7

8

OH

OH

HO1998400

2998400

3998400

4998400

5998400

6998400

Figure 3 The compound identified as 3410158405-trihydroxystilbene(C14H12O3) from 1H- and 13C-NMR spectra peak assignment

4 Discussion and Conclusion

Plant parts such as roots leaves stems and rhizomes possessa myriad of chemical constituents that are biologically activeagainst various disease conditions [3] In this study solventsof varying polarity were used in the extraction procedurein an attempt to accommodate the range of polarities ofcompounds obtained from the roots of S italica

Methanol extract had the highest yield The high extractyield obtained with methanol extract could be related tothe ability of the solvent to extract compounds of varyingpolarity [15] All solvents extracted vanillin-reactive com-pounds from the roots of S italica The phytochemical con-stituents of the extracts were best separated in the intermedi-atepolar mobile phase ethylacetatemethanolwater (EMW40 54 5) which separates polar and neutral compoundsAcetone extracts showed the presence of antioxidant com-pounds which were better separated in the intermediate mo-bile phase (CEF)The free radical scavenging activity demon-strates the hydrogen donating ability on reaction to the stablefree radical which results in the decolouration of the DPPHfree radical from purple to yellow [16]

A literature survey on the chemical constituents of thegenus Senna revealed the presence of alkaloids quinines andanthraquinones that are mainly polar-based compounds [17]Resveratrol is a natural polyphenolic compound found in anumber of edible plants especially grapes and peanuts andhas been found to exist in both the cis and trans isomericforms Although the nutritional value and biological activ-ity of resveratrol have been well documented [18] its oli-gomers are commonly reported to occur in plants belongingto families such as Dipterocarpaceae Vitaceae CyperaceaeGnetaceaeWelwitschiaceae Umbelliferae and Leguminosae[19 20] This study represents the first isolation of trans res-veratrol from the roots of Senna italica belonging to thefamily Fabaceae The biological effects of resveratrol togetherwith its well-known antioxidant properties are beneficial forthe prevention of several diseases Findings in our previousstudy [9] showed the presence of substances in the acetoneroot extract of S italica with antioxidant antibacterial andantiproliferative activities while other studies have also con-firmed the antiproliferative effect of resveratrol [21]The pres-ence of resveratrol may possibly explain the observed effectof the acetone crude extracts on cancerous Jurkat T cells inour previous studies [9] and may be the major contributorsto biological activity observed in that study

Structural activity relationships of the antioxidant po-tential of resveratrol from other studies suggest that the hy-droxyl group in 41015840 position is not the sole determinant forantioxidant activity It has also been suggested that the pres-ence of 41015840-OH together with its stereoisomer in the trans-conformation (41015840-hydroxystyryl moiety) is a significant pre-requisite in its ability to inhibit cell proliferation [21] Thepresence of resveratrol in S italica and the observed antioxi-dant effect could possibly givemore credence to its traditionaluse in treating various ailments in the Limpopo Province ofSouth Africa [5 6] Further studies will focus on the antidia-betic properties of the isolated compound

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The National Research Foundation (NRF) South Africa(Gun no N710) the Department of Biochemistry Microbi-ology and Biotechnology University of Limpopo and Phy-tomedicine Programme University of Pretoria are acknowl-edged for the financial and technical supportM PMokgothois a recipient of NRF funding under theThuthuka-CSPG

References

[1] A Gurib-Fakim ldquoMedicinal plants traditions of yesterday anddrugs of tomorrowrdquo Molecular Aspects of Medicine vol 27 no1 pp 1ndash93 2006

[2] A Sala M Del Carmen Recio R M Giner et al ldquoAnti-inflam-matory and antioxidant properties of Helichrysum italicumrdquoJournal of Pharmacy and Pharmacology vol 54 no 3 pp 365ndash371 2002

6 Evidence-Based Complementary and Alternative Medicine

[3] B Van Wyk and M WinkMedicinal Plants of the World BrizaPublications 2004

[4] M J Balunas and A D Kinghorn ldquoDrug discovery from me-dicinal plantsrdquo Life Sciences vol 78 no 5 pp 431ndash441 2005

[5] T E Tshikalange J J MMeyer and A A Hussein ldquoAntimicro-bial activity toxicity and the isolation of a bioactive compoundfrom plants used to treat sexually transmitted diseasesrdquo Journalof Ethnopharmacology vol 96 no 3 pp 515ndash519 2005

[6] L J Shai PMasokoM PMokgotho et al ldquoYeast alpha glucosi-dase inhibitory and antioxidant activities of sixmedicinal plantscollected in Phalaborwa South Africardquo South African Journal ofBotany vol 76 no 3 pp 465ndash470 2010

[7] F G Barbosa M D C F De Oliveira R Braz-Filho and ER Silveira ldquoAnthraquinones and naphthopyrones from Sennarugosardquo Biochemical Systematics and Ecology vol 32 no 3 pp363ndash365 2004

[8] N H El-Sayed A M A Dooh S A M El-Khrisy and T JMabry ldquoFlavonoids of Cassia italicardquoPhytochemistry vol 31 no6 p 2187 1992

[9] P Masoko S S Gololo M P Mokgotho J N Eloff R LHoward and L J Mampuru ldquoEvaluation of the antioxidantantibacterial and antiproliferative activities of the acetoneextract of the roots of Senna italica (Fabaceae)rdquo African Journalof Traditional Complementary and Alternative Medicines vol 7no 2 pp 138ndash148 2010

[10] Y Li andM A Trush ldquoReactive oxygen-dependent DNA dam-age resulting from the oxidation of phenolic compounds by acopper-redox cycle mechanismrdquo Cancer Research vol 54 no 7pp 1895ndash1898 1994

[11] C Deby and G Magotteaux ldquoRelationship between essentialfatty acids and tissue antioxidant levels in micerdquo Comptes Ren-dus des Seances de la Societe de Biologie et de Ses Filiales vol 164no 12 pp 2675ndash2681 1970

[12] M Guiso C Marra and A Farina ldquoA new efficient resveratrolsynthesisrdquo Tetrahedron Letters vol 43 no 4 pp 597ndash5982002

[13] C Roat N Kumar and K G Ramawat ldquoOccurrence of stil-benes in unorganized cultures of Cayratia trifolia (L) DominrdquoJournal of HerbalMedicine and Toxicology vol 2 no 1 pp 11ndash152008

[14] Y Lee S Kwon H Kim et al ldquoIsolation of oleanane triterpenesand trans-resveratrol from the root of peanut (Arachis hypogae-a)rdquo Journal of the Korean Society For Applied Biological Chemis-try vol 52 no 1 pp 40ndash44 2009

[15] J N Eloff ldquoWhich extractant should be used for the screeningand isolation of antimicrobial components from plantsrdquo Jour-nal of Ethnopharmacology vol 60 no 1 pp 1ndash8 1998

[16] N-E Es-Safi A Kollmann S Khlifi and P-H Ducrot ldquoAntiox-idative effect of compounds isolated from Globularia alypumL structure-activity relationshiprdquo Food Science and Technologyvol 40 no 7 pp 1246ndash1252 2007

[17] L Fremont ldquoMinireview biological effects of resveratrolrdquo LifeSciences vol 66 no 8 pp 663ndash673 2000

[18] J Kawabata S Ichikawa H Kurihara J Mizutani and AKobophenol ldquoA unique tetrastilbene from Carex KobomugiOhwi (Cyperaceae)rdquo Tetrahedron Letters vol 30 no 29 pp3785ndash3788 1989

[19] S Sotheeswaran and V Pasupathy ldquoDistribution of resveratrololigomers in plantsrdquo Phytochemistry vol 32 no 5 pp 1083ndash1092 1993

[20] O P Mgbonyebi J Russo and I H Russo ldquoAntiproliferativeeffect of synthetic resveratrol on human breast epithelial cellsrdquoInternational Journal of Oncology vol 12 no 4 pp 865ndash8691998

[21] L A Stivala M Savio F Carafoli et al ldquoSpecific structuraldeterminants are responsible for the antioxidant activity and thecell cycle effects of resveratrolrdquo Journal of Biological Chemistryvol 276 no 25 pp 22586ndash22594 2001

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