Hindawi Publishing Corporation

Evidence-Based Complementary and Alternative Medicine

Volume 2013, Article ID 519174, 6 pages
http://dx.doi.org/10.1155/2013/519174

Research Article
Isolation and Chemical Structural Characterisation of
a Compound with Antioxidant Activity from the Roots of
Senna italica

Matlou Phineas Mokgotho,1 Stanley Sechene Gololo,1 Peter Masoko,1

Ladislaus Kakore Mdee,2 Vusi Mbazima,1 Leshwene Jeremiah Shai,3
Victor Patrick Bagla,1 Jacobus Nicolaas Eloff,4 and Leseilane Mampuru1
1
Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Turfloop Campus, Private Bag X1106,
Sovenga 0727, South Africa
2
Department of Pharmacy, University of Limpopo, Turfloop Campus, Private Bag X1106, Sovenga 0727, South Africa
3
Department of Biomedical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680,
Pretoria 0001, South Africa
4
Faculty of Veterinary Science, Department of Paraclinical Science, Phytomedicine Programme, University of Pretoria,
Onderstepoort, Pretoria 0001, South Africa

Correspondence should be addressed to Matlou Phineas Mokgotho; [email protected]

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

Academic Editor: Mohamed Eddouks

Copyright © 2013 Matlou Phineas Mokgotho et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Senna italica, a member of the Fabaceae family (subfamily Caesalpiniaceae), is widely used in South African traditional medicine
to treat a number of disease conditions. Aqueous extracts of the plant are mainly used to treat sexually transmitted infections
and intestinal complications. The roots of S. italica were ground to a fine powder and sequentially extracted with n-hexane,
dichloromethane, acetone, and methanol using serial exhaustive extraction (SEE) method. Thin layer chromatography was used
to analyse the phytochemical composition of the extracts and DPPH radical scavenging method to detect the presence of
antioxidant compounds. The bioassay guided fractionation of the acetone fraction afforded an antioxidant compound with free
radical scavenging activity. The isolated compound was subsequently identified as 3,4󸀠 ,5-trihydroxystilbene (resveratrol). This study
represents the first report of the stilbene resveratrol in S. italica.

1. Introduction agents [2]. Other examples include anticancer agents derived

from Catharanthus roseus as well as laxative agents from
Plants have formed the basis of sophisticated traditional me- Cassia species [1].
dicinal systems that have been in existence for thousands of The current advancement in science has made it possi-
years, and they continue to provide humanity with new reme- ble for the isolation of compounds of medical importance
dies [1]. The importance of plants in human and animal health from plants that are used in traditional medicinal practices.
is evident in the increasing presence of natural product drugs Different parts of plants such as leaves, roots, stems barks
in modern medicine. Indeed, natural products and their de- and rhizomes are often extracted using different solvents.
rivatives represent more than 50% of all the drugs in clinical In most cases, extracts have been shown to be biologically
use in the world today. During the last 40 years, at least a active both in the in vitro and in vivo test systems [3]. Some
dozen potent drugs have been derived from flowering plants. plant-derived compounds are effective in combination with
One example is that of diosgenin, a drug template derived others, while others are active as single entities [4]. How-
from Dioscorea species and used to synthesise contraceptive ever, the isolation of compounds remains a challenging and
2 Evidence-Based Complementary and Alternative Medicine

a mammoth task. Conventionally, the isolation of bioactive chloroform/ethyl acetate/formic acid (CEF) (5 : 4 : 1) (inter-
compounds is preceded by the determination of the presence mediate polarity/acidic); benzene/ethanol/ammonium hy-
of such compounds within plant extracts through a number droxide (BEA) (90 : 10 : 1) (nonpolar/basic). For the determi-
of bioassays. nation of antioxidant activity, the plates were sprayed with
Senna italica, a member of the Fabaceae family (subfamily 0.2% 2,2-diphenyl-1-picryl-hydrazyl (DPPH) (Sigma) in meth-
Caesalpinaceae), is known for its therapeutic properties in anol as an indicator. A positive reaction is indicated by the
African folk medicine. Some Senna species from Venda in appearance of a yellow spot against a purple background.
South Africa are used to treat sexually transmitted infections
(STIs) while others are considered to possess significant anti- 2.3. Fractionation. The acetone extract had constituents with
bacterial activities [5]. In the northern parts of the Limpopo antioxidant activity and was thus chosen for further frac-
province of South Africa, S. italica is indicated for the treat- tionation with CHCl3 : MeOH of increasing gradient polarity,
ment of STIs [6]. A literature survey on the chemical constit- starting with 100% CHCl3 to 100% MeOH. A 460 mg sample
uents of the genus Senna revealed the presence of alkaloids, of acetone extract was fractionated (Figure 1) into seven frac-
quinines, and anthraquinones. These types of compounds tions of CHCl3 : MeOH in the following ratios: 1 : 0, 9 : 1, 8 : 2,
have been isolated from heartwood, seeds, root bark, roots, 7 : 3, 1 : 1, 3 : 7, and 0 : 1 (v/v) using column chromatography
and leaves of the genus Senna [7]. Other studies have report- packed with silica gel 60 (63–200 𝜇m). Following elution, the
ed the isolation of many other compounds including beta- antioxidant constituents of the resultant fractions were qual-
sitosterol, stigmasterol, and amyrin from the aerial parts of itatively evaluated using the TLC-DPPH method described
S. italica [8]. We have previously reported the antibacterial above. The fraction that eluted with 9 : 1 (CHCl3 : MeOH
effect of the acetone extract of S. italica on selected bacterial (9 : 1)) was found to contain the major compound with anti-
strains, total phenolic content as well as its effect on cancerous oxidant activity. Hence, the fraction was chosen for further
Jurkat T cell [9]. Based on these findings, S. italica was further purification of the target compound.
investigated with the aim of identifying the compound(s)
that exerted the observed biological activities. Antioxidant 2.4. Elution and Structural Characterisation of the Antioxidant
compounds are important in the prevention or treatment of Compound. Several mobile phases were tested and fraction
various human diseases. Undeniably, reports abound that F9 : 1 was better resolved with CHCl3 : EtAOC (1 : 1). The 9 : 1
link free radical scavenging potential and other biological (125 mg) was subjected to a silica gel 60 column chromatog-
benefits of antioxidant compounds [10]. We here report the raphy using CHCl3 : MeOH (4 : 1) as the eluent. Fractions 13–
isolation of resveratrol, a compound with antioxidant activity, 18 were subjected to chromatography using CHCl3 : MeOH
from the acetone root extract of Senna italica through bioas- (9 : 1) as eluent. After recrystallisation, a 13 mg of the pure
say-guided fractionation. compound was obtained. The compound was dissolved either
in methanol, chloroform, or dimethyl sulfoxide depending on
2. Materials and Method its solubility for analysis. The structure of the isolated com-
pound was elucidated from the data obtained from 1 H- and
13
2.1. Extraction. The roots of Senna italica were collected from C-NMR spectra.
Bolahlakgomo village (Zebediela subregion, Limpopo prov-
ince, South Africa). A voucher specimen (UNIN11129) has 3. Results
been deposited in the Larry Leach herbarium of the Univer-
sity of Limpopo. The roots were dried at room temperature Different solvents, depending on their polarity, extract vary-
and subsequently milled to a fine powder using a grinder (ML ing quantities of components in crude plant material that
90L4, Monitoring and Control Laboratories (Pty) Ltd., RSA). may be beneficial or harmful to biological systems. 𝑛-Hexane,
The milled material (700 g) was serial exhaustively extracted for instance, extracts waxes, fats, and fixed oils while acetone
using 𝑛-hexane, dichloromethane (DCM), acetone and meth- extracts alkaloids, aglycones, and glycosides. On the other
anol (MeOH) as extractants. The extraction process was re- hand, methanol extracts sugars, amino acids, and glycosides
peated three times with 2 litres of each solvent. The extracts while DCM will commonly extract alkaloids, aglycones, and
were filtered through a Whatman no. 3 filter paper and the fil- volatile oils. For these reasons, plant material was extracted
trates concentrated using a rotary evaporator (Büchi Labotec using solvent of varying polarity, namely, hexane, DCM, ace-
rotavapor model R-205, Germany). The concentrated extracts tone, and methanol. An increase in the extracted material was
were then transferred into preweighed beakers, dried under observed as the polarity of the extracting solvent increased
a stream of cold air, and weighed to determine the resultant (Table 1). Methanol extract had the highest percent yield
mass. followed by DCM, hexane and acetone yielded the least.
Extracted materials were spotted on TLC plates and elut-
2.2. Free Radical Scavenging Activity. The presence of anti- ed in BEA, EMW, and CEF and thereafter sprayed with
oxidant constituents in the various extracts was determined vanillin-sulphuric acid. The free radical scavenging activity of
using the method of [11]. Thin layer chromatographic plates the extracts was determined using the DPPH assay. The yield
were prepared and spotted with the different extracts and of the methanol extract did not correlate with the presence
eluted in solvents systems of varying polarity, namely, ethyl of antioxidant constituents in the extract. Although acetone
acetate/methanol/water (EMW) (40 : 5.4 : 5) (polar/neutral); extracts yielded the least, more active constituents were
Evidence-Based Complementary and Alternative Medicine 3

Senna roots, 700 g

Serial exhaustive extraction

DCM fraction Acetone Methanol fraction

n-Hexane fraction
(6.2 g) fraction (4.6 g) (32.3 g)
(4.8 g)

CHCl3 : MeOH (v/v) fractionation

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

CHCl3 : EtoAc (1 : 1, v/v) elution

Tubes 13–18 pooled together

CHCl3 : EtoAc (1 : 1, v/v) reelution

Compound I
NMR
13 mg

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

present in these extracts with strong antioxidant activity. followed by fraction 9 : 1 (125 mg), with fraction 0 : 1 giving
This may possibly be related to the ability of acetone to extract the lowest yield (14 mg) as shown (Table 2). Fractions 8 : 2
both polar and nonpolar constituents. Since acetone extract and 9 : 1 contained appreciable amounts of vanillin-reacting
demonstrated a superior free radical scavenging activity, it compounds than the other fractions, with fraction 9 : 1 show-
was thus chosen for further purification. The extract was ing prominent antioxidant activity than the rest (data not
fractionated with the gradient solutions of CHCl3 : MeOH of included). Thus, fraction 9 : 1 was selected for further purifi-
different ratios. Fraction 8 : 2 gave the highest yield (237 mg) 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 CHCl3 : EtOAc (1 : 1, v/v) 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- Table 2: Fractionation of the acetone root extract (700 mg) with
rial of Senna italica with 𝑛-hexane, DCM, acetone, and MeOH. CHCl3 : MeOH different ratios on silica gel chromatography.
Extract solvent Yield (g) Total yield (g) Yield (%)
Systems CHCl3 MeOH Solvent quantity (mL) Yield (mg)
Hexane 1 2.8 (%) (%)
Hexane 2 1.3 4.8 0.69 A 100 — 500 58
Hexane 3 0.7
B 90 10 700 125
DCM 1 3.5
DCM 2 1.5 6.2 0.89 C 80 20 1200 237
DCM 3 1.2 D 70 30 500 30
Acetone 1 2.3 E 50 50 600 25
Acetone 2 1.4 4.6 0.66 F 500 16
30 70
Acetone 3 0.9
G — 100 600 14
MeOH 1 17.9
MeOH 2 8.5 32.3 4.6
MeOH 3 5.9

A suitable mobile phase for elution of the bioactive com- obtained from 1 H- and 13 C-NMR spectra and those obtained
pound was obtained by comparison of the separated com- from the literature [12, 13] (Table 3).
pounds attained from the various ratios of CHCl3 : MeOH Compound 1 was obtained as a white powder that was
solution used. The CHCl3 : MeOH ratio of 1 : 1 (v/v) gave a positive to FeCl3 test which indicates the presence of phenolic
better separation of the active compound. The 9 : 1 fraction group. In the 1H-NMR spectrum, the coupling patterns
of the acetone extract was then subsequently eluted with between 𝛿 7.43 (2H, d, J = 9.0 Hz) and 6.86 (2H, d, J = 9.0 Hz,)
CHCl3 : MeOH (1 : 1, v/v) through a silica gel column. Tubes and 𝛿 6.55 (2H, brs) and 6.16 (1H, brs) showed the typical
containing similar compounds noted on TLC fingerprint resveratrol skeleton [14]. The coupling constant (J = 18.0 Hz)
were pooled together and reeluted with CHCl3 : MeOH (1 : 1, between H-7 (𝛿 6.89, 1H, d) and H-8 (𝛿 7.03, 1H, d) implied
v/v) through a silica gel column. The reelution of the fraction that the configuration of compound 1 was in a trans form
obtained from the pooled tubes gave yield to a pure com- configuration. In the 13 C-NMR spectrum, three oxygenated
pound with a positive free radical scavenging activity evident aromatic carbons (𝛿 159.6 (×2) and 158.2) and eleven sp2
by a yellow spot against a purple background on TLC fol- carbons (102.7∼140.9) were shown. From these data and those
lowing spraying with DPPH (Figure 2). The structure of the presented in Table 3, compound 1 was identified as trans-
isolated compound was elucidated (Figure 3) from the data resveratrol [12, 13].
Evidence-Based Complementary and Alternative Medicine 5

Table 3: 13 C NMR data of isolated compound (300 MHz, acetone- A literature survey on the chemical constituents of the
d6 ). genus Senna revealed the presence of alkaloids, quinines, and
anthraquinones that are mainly polar-based compounds [17].
C number Literature data Isolated compound data
Resveratrol is a natural polyphenolic compound found in a
1 141.3 140.9 number of edible plants, especially grapes and peanuts, and
2 105.8 105.7 has been found to exist in both the cis and trans isomeric
3 159.7 159.6 forms. Although the nutritional value and biological activ-
4 102.7 102.7 ity of resveratrol have been well documented [18], its oli-
5 159.7 159.6 gomers are commonly reported to occur in plants belonging
6 105.8 105.7 to families such as Dipterocarpaceae, Vitaceae, Cyperaceae,
7 129.4 129.1 Gnetaceae, Welwitschiaceae, Umbelliferae, and Leguminosae
8 127.0 126.9 [19, 20]. This study represents the first isolation of trans res-
1󸀠 130.4 130.0 veratrol from the roots of Senna italica belonging to the
2󸀠 128.8 128.7 family Fabaceae. The biological effects of resveratrol together
3󸀠 116.5 116.4 with its well-known antioxidant properties are beneficial for
4󸀠 158.4 158.2
the prevention of several diseases. Findings in our previous
study [9] showed the presence of substances in the acetone
5󸀠 116.5 116.4
root extract of S. italica with antioxidant, antibacterial, and
6󸀠 128.8 128.7
antiproliferative activities while other studies have also con-
firmed the antiproliferative effect of resveratrol [21]. The pres-
ence of resveratrol may possibly explain the observed effect
3󳰀 of the acetone crude extracts on cancerous Jurkat T cells in
󳰀 OH
2 󳰀
our previous studies [9] and may be the major contributors
4
to biological activity observed in that study.
2 7
1󳰀 Structural activity relationships of the antioxidant po-
HO 3 1 5󳰀
tential of resveratrol from other studies suggest that the hy-
6󳰀
8 droxyl group in 4󸀠 position is not the sole determinant for
4 6 antioxidant activity. It has also been suggested that the pres-
5 ence of 4󸀠 -OH together with its stereoisomer in the trans-
conformation (4󸀠 -hydroxystyryl moiety), is a significant pre-
OH requisite in its ability to inhibit cell proliferation [21]. The
Figure 3: The compound identified as 3,4󸀠 ,5-trihydroxystilbene presence of resveratrol in S. italica and the observed antioxi-
(C14 H12 O3 ) from 1 H- and 13 C-NMR spectra peak assignment. dant effect could possibly give more credence to its traditional
use in treating various ailments in the Limpopo Province of
South Africa [5, 6]. Further studies will focus on the antidia-
betic properties of the isolated compound.
4. Discussion and Conclusion
Conflict of Interests
Plant parts such as roots, leaves, stems, and rhizomes possess
a myriad of chemical constituents that are biologically active The authors declare no conflict of interests.
against various disease conditions [3]. In this study, solvents
of varying polarity were used in the extraction procedure Acknowledgments
in an attempt to accommodate the range of polarities of
compounds obtained from the roots of S. italica. The National Research Foundation (NRF) South Africa
Methanol extract had the highest yield. The high extract (Gun no. N710), the Department of Biochemistry, Microbi-
yield obtained with methanol extract could be related to ology and Biotechnology, University of Limpopo, and Phy-
the ability of the solvent to extract compounds of varying tomedicine Programme, University of Pretoria, are acknowl-
polarity [15]. All solvents extracted vanillin-reactive com- edged for the financial and technical support. M. P. Mokgotho
pounds from the roots of S. italica. The phytochemical con- is a recipient of NRF funding under the Thuthuka-CSPG.
stituents of the extracts were best separated in the intermedi-
ate/polar mobile phase, ethylacetate/methanol/water (EMW: References
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