Two new antioxidant flavones from the twigs of Eriosema robustum (Fabaceae)

Phytochemistry Letters 6 (2013) 62–66

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Two new antioxidant flavones from the twigs of Eriosema robustum (Fabaceae) Maurice D. Awouafack a,b, Pierre Tane b,*, Jacobus N. Eloff a,** a b

Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa Laboratory of Natural Products Chemistry, Department of Chemistry, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 August 2012 Received in revised form 22 October 2012 Accepted 25 October 2012 Available online 10 November 2012

Phytochemical study of the ethanol extract of the twigs of Eriosema robustum, a Cameroonian medicinal plant resulted to the isolation of two new flavones, 20 ,30 ,50 ,5,7-pentahydroxy-3,40 -dimethoxyflavone (1) and 20 ,3,50 ,5,7-pentahydroxy-40 -methoxyflavone (2), along with five known compounds: 6-prenylpinocembrin (3), 1-O-heptatriacontanoyl glycerol (4), b-sitosterol (5), stigmasterol (6) and 3-O-b-Dglucopyranoside of sitosterol (7). The structure of the isolated compounds were elucidated on the basis of their NMR, UV and MS data, and by comparison with those reported in the literature. The ethanol crude extract, fractions and some isolated compounds (1–4) were evaluated for their radical scavenging capacity using 2,2-diphenyl-1-picryhydrazyl (DPPH). The crude extract, fraction II, the new compounds namely robusflavones A (1) and B (2) exhibited significant antioxidant activity. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Eriosema robustum Fabaceae Flavone Robusflavone A Robusflavone B Antioxidant activity

1. Introduction

2. Results and discussion

Eriosema robustum a member of Fabaceae family is a perennial non-climbing shrub with yellow flowers native from Burundi, Ethiopia, Kenya, Rwanda, Tanzania, Uganda, Democratic Republic of Congo and Cameroon (Gillett et al., 1971). It is used traditionally for the treatment of coughs in East Africa (Kokwaro, 2009) and skin diseases in Central Africa. To the best of our knowledge, no phytochemical work has been done on the constituents of this species. The investigation carried out on some species of the genus Eriosema led mainly to the isolation of polyphenols (Ma et al., 1995, 1999), chromones (Ma et al., 1996a,b) and flavonoids (Awouafack et al., 2008; Ma et al., 1998; Drewes et al., 2002, 2004; Ojewole et al., 2006; Sutthivaiyakit et al., 2009). We report herein the isolation, the structure elucidation and the antioxidant activity of two new flavones (1–2) along with five known compounds (3–7) from the twigs of E. robustum, as part of our continuing phytochemical and biological investigation of Cameroonian medicinal plant of Fabaceae family (Awouafack et al., 2008, 2011). The isolation of 6-prenylpinocembrin (3) from the genus Eriosema and its antioxidant activity are also reported herein for the first time. The structures of the new compounds were established using their NMR (1 and 2D), UV and MS data.

The ethanol crude extract of the twigs of E. robustum was subjected to repeated silica gel column chromatography followed by preparative TLC and Sephadex LH-20 to afford robusflavones A (1) and B (2) along with five known compounds including 6prenylpinocembrin (3) (Caffaratti et al., 1994), 1-O-heptatriacontanoyl glycerol (4) (Qi et al., 2004), b-sitosterol (5) (Al-Oqail et al., 2012), stigmasterol (6) (Forgo and Ko¨ve´r, 2004) and 3-O-b-Dglucopyranoside of sitosterol (7) (Al-Oqail et al., 2012) (Fig. 1). The isolation of 6-prenylpinocembrin (3) from the genus Eriosema is also reported here for the first time as well as its antioxidant activity. The known compounds were identified by comparison of their spectroscopic data with those reported in the literature. Robusflavone A (1) was obtained as a yellowish amorphous compound. The molecular formula C17H15O9 was obtained from its HR-ESI-TOF-MS spectrum, which showed a pseudo-molecular ion peak [M+H]+ at m/z 363.2416. The bands exhibited at nmax 3437, 1650 and 1128 cm1 and the maximum absorption bands observed at lmax 343, 300 and 245 on its IR and UV spectra, respectively, were suggestive for a flavone skeleton (Gao et al., 2010). The 1H NMR spectrum (Table 1) displayed signals of two aromatic doublets at d 6.29 (1H, J = 2.0 Hz) and 6.55 (1H, J = 2.0 Hz) assignable, respectively, to protons H-6 and H-8, and characteristic for A-ring of flavones with the oxygenation at positions 5 and 7 (Gao et al., 2010; Queiroz et al., 2005). Moreover, the 1H NMR spectrum (Table 1) exhibited signals of singlets at d 6.96 (1H, s), 3.72 (3H, s), 3.85 (3H, s) and 12.89 (1H, brs) corresponding, respectively, to the aromatic proton H-60 of B-ring, the methoxyl

* Corresponding author. ** Corresponding author. Tel.: +27 012 529 8525; fax: +27 012 529 8525. E-mail address: [email protected] (J.N. Eloff).

1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.10.017

M.D. Awouafack et al. / Phytochemistry Letters 6 (2013) 62–66

63

R2 3'

HO 2' 8

HO

9 7

A

O

2

5

OH

B

C

OCH3

HO

B

O

5'

1'

A

OH

6'

C

3

6 10

4'

OR1

4

OH

O

R1

O

3

R2 O

1: CH3 OH 2: H H

CH3

O

HO

23

OH

R

O

4

R 5: H 7: Glc

6

HO

Fig. 1. Compounds (1–7) isolated from Eriosema robustum.

group protons 3-OCH3 and 40 -OCH3, and the chelated phenolic hydroxyl group proton 5-OH. The 13C NMR spectrum (Table 1) displayed characteristic signals at d 178.5 (C-4), 60.7 (3-OCH3) and 56.1 (40 -OCH3), due, respectively, to one carbonyl and two methoxyl groups of flavones (Gao et al., 2010). Further signals were also observed on the 13C NMR spectrum (Table 1) and imputable to other carbons: at d 152.1 (C-2), 135.7 (C-3), 162.1 (C5), 164.7 (C-7), 154.7 (C-9), 103.0 (C-10), 115.8 (C-10 ), 136.7 (C-30 ), 152.1 (C-40 ) and 133.0 (C-50 ). The correlations observed from the

Table 1 1 H (500 MHz) and Position

13

C (125 MHz) NMR data of robusflavones A (1) and B (2) in DMSO-d6 [d (ppm), J (Hz)]. Robusflavone A (1)

dC 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 3-OCH3 40 -OCH3 5-OH OHa OHa

HMBC spectrum (Fig. 2) between the proton at d 3.72 and the carbon at d 135.7 (C-3), and also between the proton at d 3.85 and the carbon at d 152.1 (C-40 ) assigned these methoxyl groups to be sited, respectively, at positions C-3 and C-40 . Moreover, the HMBC correlations (Fig. 2) between the proton at d 6.96 (H-60 ) and the carbons at d 152.1 (C-2), 133.0 (C-50 ) and 152.1 (C-40 ) confirmed that this proton should be located at position C-60 of B-ring which in turn linked to C-ring. Furthermore, the HMBC correlations (Fig. 2) between the proton at d 6.29 (H-6) and the carbon at d 103.0

152.1 135.7 178.5 162.1 99.7 164.7 95.0 154.7 103.0 115.8 ND 136.7 152.1 133.0 93.5 60.7 56.1

ND: not detected. a Interchangeable for 7-OH, 20 -OH, 30 -OH and 50 -OH. b Interchangeable for 7-OH, 20 -OH and 50 -OH.

Position

dH

dC 145.8 137.8 178.1 162.1 99.6 163.5 94.9 154.6 102.9 112.5 164.1 99.3 146.7 143.3 102.7

6.29, d (2.0) 6.55, d (2.0)

6.96, 3.72, 3.85, 12.89, 10.96, 10.22,

Robusflavone B (2)

s s s brs brs brs

56.2 OHb OHb

dH

6.27, d (2.0) 6.52, d (2.0)

7.15, brs

7.34, brs 3.89, 12.87, 10.93, 9.51,

s brs brs brs

64

M.D. Awouafack et al. / Phytochemistry Letters 6 (2013) 62–66

OH HO HO

H OCH3

O

OH

H OCH3

H OH

HO HO

O

H

O

OCH3

OH OH

OH

H

O

Fig. 2. Key HMBC correlations of robusflavones A (1) and B (2).

(C-10) as well as the 1H–1H COSY coupling (J = 2.0 Hz) between this proton and the one at d 6.55 (H-8) were substantiated for these protons to be located on A-ring. The assignment of carbons at positions C-10 , 30 , 40 , 50 , 60 and 40 -OMe were achieved by HMBC correlations and/or the values of their chemical shifts in comparison with the literature data of similar flavones with tetrasubstituted Bring (Gao et al., 2010; Queiroz et al., 2005). The hydroxyl group was assigned to be at position C-20 , where no carbon signal was detected on the 13C NMR spectrum, by taking in consideration the number of aromatic protons [6.29 (H-6), 6.55 (H-8) and 6.96 (H-60 )] observed on the 1H NMR spectrum and the molecular formula of 1. However, the ortho-position of 20 -OH with 30 -OH was confirmed by the relative bathochromic shifts for band I (37, 27 and 28 nm) on the UV spectra when using UV-shift reagents (AlCl3, NaOAc + H3BO3 and AlCl3 + HCl, respectively) (Fathiazad et al., 2006; Krenn et al., 2003; Ducrey et al., 1995). Its ESI-MS/MS spectrum had prominent fragments at m/z 344 [M+HH2O]+, 335 [M+HCO]+, 332 [M+HOCH3]+, 320 [M+HCH3CO]+, 318 [M+HCOH2O]+, 301 [M+H2OCH3]+ and 273 [M+H2OCH3CO]+, in agreement with the fragmentation pattern of the proposed structure for compound 1. The pentasubstitution pattern of ring B for robsuflavone A (1) was therefore assigned based on the 1H, 13C NMR data and the HMBC correlations (dos Santos et al., 1995) as well as the UV-shift reagents and the MS data. From the NMR, UV and MS data above and by comparison to those previously reported for flavones (Gao et al., 2010; Fathiazad et al., 2006; Queiroz et al., 2005; Krenn et al., 2003; Ducrey et al., 1995), the structure of the novel compound was assigned as 20 ,30 ,50 ,5,7-pentahydroxy-3,40 -dimethoxyflavone to which the trivial name Robusflavone A (1) was given. Robusflavone B (2) was obtained as a yellowish amorphous compound. Its HR-ESI-TOF-MS spectrum had a pseudo-molecular ion peak [M+H]+ at m/z 333.1924 corresponding to a molecular formula C16H13O8, which is 30 a.m.u. lower than that of robusflavone A (1) and consistent with 11 double-bond equivalents. It showed similar IR (nmax 3425, 1653 and 1123 cm1) and UV (lmax 342, 314 and 263 nm) data compared to those of robusflavone A (1), and characteristic of a flavone skeleton (Gao et al., 2010). The 1 H NMR spectrum (Table 1) had similar signals of two aromatic doublets at d 6.27 (1H, J = 2.0 Hz) and 6.52 (1H, J = 2.0 Hz) due to protons H-6 and H-8 characteristic of a 5,7-dihydroxyl A-ring (Gao et al., 2010; Queiroz et al., 2005). Two broad singlet signals were observed on the 1H NMR spectrum (Table 1) at d 7.15 (1H, brs) and 7.34 (1H, brs) corresponding to protons H-30 and H-60 of B-ring, and characteristic for a downfield para-protons of a tetrasubstituted aromatic ring (Gao et al., 2010; Queiroz et al., 2005). Moreover the 1 H NMR spectrum (Table 1) displayed signals at d 3.89 (3H, s) and 12.87 (1H, brs) owing, respectively, to the protons of one methoxyl (40 -OCH3) and one chelated hydroxyl (5-OH) groups. The signal at d 56.2 (40 -OCH3) exhibited by the 13C NMR spectrum (Table 1) confirmed the presence of a methoxyl group that is substantiated by the fragments observed on the ESI-MS/MS spectrum at m/z 269 [M+HOCH3]+. The HMBC correlation (Fig. 2) between the proton

at d 3.89 (40 -OCH3) and carbon at d 146.7 (C-40 ) was indicative for the methoxyl group to be attached at position C-40 . This was further confirmed by the presence of a characteristic fragment, observed on the ESI-MS/MS spectrum, at m/z 318 [M+H15]+ corresponding to the loss of CH3 (a radical) at position C-40 of Bring that occurred by the same fragmentation mechanism given for flavonoid with methoxyl at C-6 or C-8 (Markham, 1982). Further HMBC correlations (Fig. 2) observed between the proton at d 7.15 and carbons at d 112.5 (C-10 ), 146.7 (C-40 ) and 143.3 (C-50 ), and between the proton at d 7.34 and carbons at d 145.8 (C-2) and 143.3 (C-50 ) were substantiated for these protons to be sited at the B-ring. The ion fragments in agreement with the fragmentation pattern of the proposed structure for robusflavone B (2) were observed on its ESI-MS/MS at m/z 315 [M+HH2O]+, 305 [M+HM+HCO]+, 301 [M+HOCH3]+, 291 [M+HCOCH3]+, 269 [M+HOCH32OH]+. Bathochromic shift of 36 nm was observed in band I with AlCl3 and no shift was materialized on the same band after addition of AlCl3/ HCl. This suggested the formation of a hydroxyl-keto complex at positions C-4 and C-5, and was indicative for no ortho-hydroxylation in B-ring (Edewor and Olajire, 2011). Apart of the lack of one hydroxyl group on the B-ring and the substitution of methoxyl with hydroxyl group on C-ring, the NMR data of robusflavone B (2) were similar to those of robusflavone A (1). From the NMR, UV and MS data above, and by comparison with those of reported compounds (Edewor and Olajire, 2011; Queiroz et al., 2005; Tachakittirungrod et al., 2007), the structure of robusflavone B (2) was assigned as 20 ,3,50 ,5,7-pentahydroxy-40 -methoxyflavone (2). This compound which was only been synthesized in 1975, was isolated and described here as new naturally occurring for the first time (Ro¨sler, 1975). The antioxidant activities of the ethanol crude extract, fractions, and some isolated compounds (1–4) from the twigs of E. robustum have been evaluated using the DPPH method and the results are given in terms of the concentration of the sample decreasing 50% of free radical scavenging (IC50) (Table 2). All the tested samples had

Table 2 Concentration of extracts and compounds (1–4) from E. robustum reducing 50% of free radical DPPH (IC50). Samples

IC50 (mg/mL)

Extracts Crude extract Fraction I Fraction II

1.84  0.01 10.81  0.02 1.54  0.00

Isolated compounds 1 2 3 4

1.13  0.01 1.19  0.01 37.45  0.01 21.84  0.02

Reference standard L-Ascorbic acid

1.00  0.02

M.D. Awouafack et al. / Phytochemistry Letters 6 (2013) 62–66

the radical scavenging activity at different concentrations with compounds 1 and 2 as most active followed by fraction II, the crude extract, fraction I, compounds 3 and 4, compared to the activity of the standard reference, ascorbic acid. The potential radical scavenging activities of the novel compounds, robusflavones A (1) and B (2), could be justified by the activity exhibited by the crude extract and fraction II from where they were isolated. This activity could be assigned to the numerous hydroxyl groups present on their structures (Fig. 1). Nevertheless, compound 2 was less active than 1. Interestingly, the highest activity exhibited by robusflavone A (1) could be explained by the number of the phenolic hydroxyl on its structures (Fig. 1); the presence in B-ring of ortho- and parahydroxyl, and ortho-methoxyl substitutions which are enhancers of antioxidant efficiency in phenolic compounds (Brand-Williams et al., 1995; Lien et al., 1999; Krafczyk et al., 2009). The antioxidant activity of these two new flavones, robusflavones A (1) and B (2) assigned to some functional groups mentioned above were obviously confirmed by the lower radical scavenging activity exhibited by 6-prenylpinocembrin (3) which is a flavonone without the hydroxyl substitution on B-ring. This is the first report of the antioxidant activity of 6-prenylpinocembrin (3) which was found to be the major constituent of fraction I and this result was in agreement with the lower radical scavenging capacity of this fraction. Flavonoids and polyphenols have been reported to have antioxidant activity which is more potent with the hydroxylation at position C-3 of C-ring, a double bond at C-2, C-3 along with a carbonyl group at C-4, in addition to the ortho- and the paradihydroxylations as well as the ortho-methylation of B-ring (Brand-Williams et al., 1995; Lien et al., 1999; Krafczyk et al., 2009). The antioxidant activity of the new flavones namely robusflavones A (1) and B (2), the crude extract and fraction II (IC50 between 1.13 and 1.84 mg/mL) was not strong enough. However, this could be considered significantly active when comparing their results to that of the reference standard ascorbic acid (IC50 of 1.00 mg/mL). The antioxidant activity of robusflavones A (1) and B (2) isolated from the twigs of E. robustum are reported here for the first time, and were in agreement with reported results on radical scavenging properties of flavonoids. 3. Materials and methods 3.1. General experimental procedures IR spectra were recorded on a Bruker Alpha FT-IR spectrometer (Optik GmbH, Germany). UV spectra were recorded on Thermo Electron Helios spectrophotometer (UVB 120726, England). AlCl3 (Sigma, Germany), NaOAc (Seelze-Hannover, Germany), H3BO3 (Merck, South Africa), HCl and NaOH were used as UV-shift reagents. The HR-TOF-MS and ESI-MS/MS were obtained on Waters Synapt HDMS spectrometer in positive mode using BEH C18 column 1.7 mm (4.6 mm  100 mm) (Waters Acquity) with a water (+0.1% HCOOH) (A)–acetonitrile (+0.1% HCOOH) (B) gradient. The 1H and 13C NMR spectra were recorded with a Bruker spectrometer at 500 MHz and Varian spectrometer at 400 MHz. Chemical shifts (d) are quoted in parts per million (ppm) from internal standard tetramethylsilane (TMS). Column chromatography was performed on MN silica gel 60 (0.063–0.2 mm/70–230) mesh. Preparative TLC was performed using high-purity grade powder silica gel (60 A, 2–25 mm; Sigma-Aldrich, Germany). Precoated plates of TLC silica gel 60 F254 (Merck, Germany) were used for monitoring fractions and spots were detected with UV light (254 and 365 nm) and then sprayed with 30% H2SO4 followed by heating up to 110 8C. Spectrophotometric data were recorded using Multi-Mode Microplate Reader (BioTek Instruments serial

65

number 250428, USA) at 570 nm with computer controlled software (BioTek GenTM). A 96 wells microplate (Bioster, Spain) was used for visible absorbance measurements. 3.2. Plant material The twigs of E. robustum were collected in Dschang, Western Region of Cameroon, on December 2011 and identified after deposition of the specimen (voucher no 35291/HNC) at the Cameroon National Herbarium in Yaounde´. 3.3. Extraction and isolation The dried and powdered twigs of E. robustum (2 kg) was extracted for three days in ethanol (10 L  3 times) to yield the crude extract (115 g) after filtration and evaporation in vacuo. This extract was subjected to a silica gel column chromatography and eluted with an increasing polarity of n-hexane, chloroform, ethyl acetate and methanol to afford 52 fractions of 600 mL each which were combined using the co-TLC into 4 fractions: I [15 g, Hex– CHCl3 (100:0, 80:20, 60:40, 20:80) and CHCl3–EtOAc (100:0, 80:20)], II [28 g, CHCl3–EtOAc (80:20, 60:40, 20:80)], III [33 g, CHCl3–EtOAc (20:80) and EtOAc–MeOH (100:0, 30:70)] and IV [1.75 g, MeOH (100:0)]. Fraction II was subjected to a purification silica gel column chromatography eluted with n-hexane, acetone and methanol in increasing polarity to yield 180 fractions of 300 mL each which were combined in subfractions after monitoring with Co-TLC. Subfractions F25–26 and F27–30 eluted with n-hexane:acetone (17:3) gave 2 (25 mg) and 6 (12 mg). Subfractions F63–86 eluted with nhexane:acetone (7:3, 3:2) and F87–138 eluted with n-hexane:acetone (11:9, 1:9) were similarly subjected to another silica gel column chromatographies eluted with n-hexane, ethyl acetate, methanol in gradient polarity followed by preparative TLC and Sephadex LH-20 to yield 1 (6 mg), 4 (12 mg) and 7 (30 mg). Fraction I was subjected to Sephadex LH-20 to remove chlorophyll and the resultant fraction was separated using similar silica gel column technique as described above for fraction II to give mainly 3 (50 mg), 5 (15 mg) and 6 (8 mg). 3.3.1. 20 ,30 ,50 ,5,7-Pentahydroxy-3,40 -dimethoxyflavone or robusflavone A (1) Yellowish amorphous; molecular formula C17H14O9; UV (DMSO) lmax (log e) 343 (2.15), 300 (2.12), 245 (1.52) nm; UV (DMSO + reagents) lmax + AlCl3 380, 300, 256, +NaOAc 378, 350, 302; +(NaOAc + H3BO3) 370, 333, 299; +(AlCl3 + HCl) 371, 301, 255, +NaOH 389, 341, 301 nm; IR nmax 3437, 3393, 2946, 1650, 1595, 1367, 1128, 1102 and 827 cm1; 1H (DMSO-d6, 500 MHz) and 13C (DMSO-d6, 125 MHz) NMR (Table 1); ESI-MS/MS (rel. Int.) m/z 344 ([M+HH2O1]+, 16), 335 ([M+HCO]+, 53), 332 ([M+HOCH3]+, 25), 320 ([M+HCH3CO]+, 51), 318 ([M+HCO–H2O]+, 48), 301 ([M+H2OCH3]+, 100), 273 ([M+H2OCH3CO]+, 42); HR-ESITOF-MS m/z 363.2416 ([M+H]+), calcd. 363.2383 for C17H15O9. 3.3.2. 20 ,3,50 ,5,7-Pentahydroxy-40 -methoxyflavone or robusflavone B (2) Yellowish amorphous; molecular formula C16H12O8; UV (DMSO) lmax (log e) 342 (2.27), 314 (1.51), 263 (0.93) nm; UV (DMSO + reagents) lmax + AlCl3 378, 371, 305, +(AlCl3 + HCl) 378, 298, 248 nm; IR nmax 3425, 3206, 3079, 2960, 1653, 1598, 1513, 1364, 1123, 1031, 817 cm1; 1H (DMSO-d6, 500 MHz) and 13C (DMSO-d6, 125 MHz) NMR (Table 1); ESI-MS/MS (rel. Int.) m/z 318 ([M+H–CH3]+, 5), 315 ([M+HH2O]+, 8), 305 ([M+HCO]+, 4), 301 ([M+HOCH3]+, 8), 291 ([M+HCOCH3]+, 100), 269 ([M+HOCH32OH]+, 23), HR-ESI-TOF-MS m/z 333.1924 ([M+H]+), calcd. 333.1913 for C16H13O8.

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3.4. Antioxidant assay The HPLC-grade methanol was used to prepare the 2,2diphenyl-1-picryhydrazyl (DPPH) (Sigma-Aldrich, Germany) at the concentration of 3.7 mg/100 mL. L-Ascorbic acid (Sigma, Germany) was used as standard reference. The antioxidant activity was performed using the method described by Shikanga et al. (2010) with slight modifications. Briefly, 200 mL of DPPH solution was introduced in each well. An initial concentration of 1 mg/mL of sample and ascorbic acid was two-fold serially diluted to a final concentration of 0.063 mg/mL and 50 mL of each sample and its dilutions were introduced to the well. Prior to the measurement of absorbance, the microtitre plates containing samples were kept in the dark for 30 min. The radical scavenging capacity was expressed in terms of the concentrations of the crude extract, fractions and compounds required to decrease the initial absorbance of DPPH at 570 nm. The free radical scavenging activity of each sample and the reference standard were determined as percent of the inhibition obtained from the absorbance of the sample (Abs) and that of the negative control (Ab, DPPH and MeOH) using the following formula: radical scavenging capacity = [100  (Abs/Ab)  100]. The concentration of samples reducing 50% of free radical DPPH was determined by plotting the percentage of inhibition against the sample concentration. The assay was replicated three times and results were expressed as mean  standard deviation. The investigations of other biological activities of the isolated compounds from several Eriosema species are currently in process. Acknowledgements The authors are grateful to the University of Pretoria for the Postdoctoral Fellowship awarded to one of us (MDA) to work at the Faculty of Veterinary Science, Department of Paraclinical Sciences, Phytomedicine Programme in 2012. Funding was provided by the National Research Foundation of South Africa and the Organization for the Prohibition of Chemical Weapons (OPCW). References Al-Oqail, M., Hassan, W.H.B., Ahmad, M.S., Al-Rehaily, A.J., 2012. Phytochemical and biological studies of Solanum schimperianum Hochst. Saudi Pharm. J. 20, 371– 379. Awouafack, M.D., Spiteller, P., Lamsho¨ft, M., Kusari, S., Ivanova, B., Tane, P., Spiteller, M., 2011. Antimicrobial isopropenyl-dihydrofuranoisoflavones from Crotalaria lachnophora. J. Nat. Prod. 74, 272–278. Awouafack, M.D., Kouam, S.F., Hussain, H., Ngamga, D., Tane, P., Schulz, B., Green, I.R., Krohn, K., 2008. Antimicrobial prenylated dihydrochalcones from Eriosema glomerata. Planta Med. 74, 50–54. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci. Technol. 28, 25–30. Caffaratti, M., Ortega, M.G., Scarafia, M.E., Ariza Espinar, L., Juliani, H.R., 1994. Prenylated flavanones from Dalea elegans. Phytochemistry 36, 1082–1084.

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