South African Journal of Botany 86 (2013) 111–115

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South African Journal of Botany

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Antigonococcal activity of caffra Sond. () and identification of the active principle

J.J. Nair a, R.B. Mulaudzi a, J.C. Chukwujekwu a, F.R. Van Heerden b, J. Van Staden a,⁎ a Research Centre for Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, b Department of Chemistry, School of Chemistry and Physics, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa article info abstract

Article history: The “large sour plum” tree Ximenia caffra Sond. (Olacaceae) is the only African representative of the genus Received 11 December 2012 Ximenia L., with a distribution covering East Africa, from in the north to South Africa, as well as Received in revised form 14 February 2013 parts of and Namibia. It has many uses in traditional medicine, including treatment of wounds Accepted 18 February 2013 and infections as well as diarrhea, which have been verified through various pharmacological studies. Fur- Available online 25 March 2013 thermore, the plant is also known for its nutritional and cosmetic value. Its edible fruits are known to be “ ” Edited by AK Jäger high in protein, potassium and vitamin C, while the seed oil, also referred to as Ximenia oil is rich in long chain unsaturated fatty acids and finds use as a domestic biofuel, with characteristics amenable to cosmetic Keywords: development as topical applicants. Extracts of the are also widely used for treating sexually transmit- Antigonococcal ted infections (STIs), which has been verified through ethnobotanical surveys and pharmacological screens. Neisseria gonorrhoeae Based on these observations, X. caffra var. caffra was examined for its phytochemical composition and activity Olacaceae against the gonorrheal pathogen Neisseria gonorrhoeae. Initially, the leaf ethanolic extract was seen to be ac- Vomifoliol tive (43.9% inhibition). The DCM partition of this extract (with 78.8% inhibition) was subjected to bioassay Ximenia caffra guided fractionation leading to the identification of the bisnorsesquiterpene vomifoliol which exhibited sig- nificant activity (63.1%) against N. gonorrhoeae. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction and creamy-green flowers which appear in single-stem clusters from August to October. These are replaced by white-spotted, The genus Ximenia L. belongs to the family Olacaceae and com- deep-red, oval fruits edible by birds, animals and humans. The fruits prises eight known species which inhabit tropical and subtropical re- are high in vitamin C, protein and potassium value and are used for gions of the globe (Heywood, 1993). Of these, the “yellow plum” making preserves such as jam and jelly (Venter and Venter, 1996; Ximenia americanum Linn. is the most common and although indige- Van Wyk and Gericke, 2000). The seed kernels are also edible and nous to Australia and Asia, it has been naturalized in some locations known for their high oil content (~65%), hence their use as a domestic outside this territory, including parts of South America and Africa biofuel (Venter and Venter, 1996; Van Wyk and Gericke, 2000). The (Heywood, 1993). By contrast, Ximenia caffra Sond. (also referred to Khoi-San use the oil as a skin conditioner, and to treat chapped as “large sour plum”) is African in origin, with a distribution along hands and feet (Van Wyk et al., 1997). The oil extract has been East Africa, from Tanzania in the north to the KwaZulu-Natal province shown to contain tannins, hence its use in softening clothing leather of South Africa, and stretching across into parts of Botswana and Na- such as leather boots and skirts (Van Wyk et al., 1997). Based on mibia (Coates Palgrave, 2003). Two varieties of the plant are found in these properties, Ximenia L. has garnered widespread support for South Africa: var. caffra which occurs in the northern and central re- commercial development. Commonly referred to as “Ximenia oil”, gions of Limpopo, and var. natalensis which extends further east and seed oil from both species of Ximenia is rich in unsaturated fatty south into Mpumalanga and KwaZulu-Natal (Coates Palgrave, 2003). acids (~92%), and therefore has a considerable nutritional value It is a deciduous tree of around 6 m in height with clustered leaves (Van Wyk et al., 1997). Furthermore, its long chain fatty acids provide good substantivity to the oil, which is said to be nourishing and mois- turizing, capable of preserving the integrity of the cell wall (Van Wyk Abbreviations: CDCl3, deuterochloroform; COSY, correlation spectroscopy; DCM, dichloromethane; DEPT, distortionless enhancement by polarization transfer; EA, et al., 1997). Consequently, several branded formulas of the oil as ethyl acetate; EIMS, electron impact mass spectrometry; EtOH, ethanol; HMBC, restructuring and anti-aging agents are available commercially for heteronuclear multiple bond correlation; HRMS, high resolution mass spectrometry; cosmetic purposes (PhytoTrade Africa). HSQC, heteronuclear single quantum coherence; MeOH, methanol; NMR, nuclear X. caffra is well known for its many applications in traditional magnetic resonance; nH, hexane; STI, sexually transmitted infection. fl ⁎ Corresponding author. Tel.: +27 332605130; fax: +27 332605897. medicine. Leaf decoctions are used as a wash to soothe in amed E-mail address: [email protected] (J. Van Staden). eyes, as a gargle for tonsillitis and are also applied as a vermifuge

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.02.170 112 J.J. Nair et al. / South African Journal of Botany 86 (2013) 111–115

(Mulaudzi et al., 2011; De Wet et al., 2012). In addition, powdered dried leaves are taken orally for fever and infertility (Mulaudzi et al., 2011; De Wet et al., 2012). Apart from this, root infusions are used for dysentery and diarrhea and together with the leaves are taken for abdominal pain and bilharzia (Mulaudzi et al., 2011; De Wet et al., 2012). Furthermore, powdered roots are applied to wounds and infections to facilitate healing and used in soups and beer as an aphrodisiac (Mulaudzi et al., 2011; De Wet et al., 2012). Several studies have sought to clarify the pharmacological basis to some of these traditional uses, both in South Africa (Steenkamp, 2003; Samie et al., 2005; Mathabe et al., 2006; Steenkamp et al., 2007; Corrigan et al., 2011; Mulaudzi et al., 2011; De Wet et al., 2012; Semenya and Maroyi, 2012) and on the African continent (Fabry et al., 1998; Geyid et al., 2005; Ndhlala et al., 2008). Most of these studies evolved out of the common usage of the plant for wounds and infections, as well as for diarrhea-related ailments. For example, MeOH root extracts of X. caffra originating in Limpopo province were tested via the microdilution assay on selected diarrhea causative microorganisms and shown to be markedly active against Scheme 1. Structurally similar natural norisoprenoids, including vomifoliol isolated Vibrio cholerae and Shigella flexneri (both with MICs of 0.156 mg/mL) during the present investigation of Ximenia caffra. (Mathabe et al., 2006). Not long after this, Steenkamp et al. (2007) showed that the MeOH root extract of X. caffra was active against be active against N. gonorrhoeae (63.1% inhibition), the common Staphylococcus epidermidis (MIC 1.42 mg/mL) and Staphylococcus gonorrhoeal pathogen, thus accounting for the traditional medicinal aureus (MIC 5.66 mg/mL), in contrast to the H2O extract which usage of the plant for venereal diseases and allied STIs. exhibited MICs of 10.30 and 1.29 mg/mL for the two pathogens, re- spectively. Further to this, work by us on Venda medicinal plants re- 2. Materials and methods vealed that EtOH root extracts of X. caffra var. caffra were notably active against Bacillus subtilis and S. aureus (both with MICs of 2.1. General 0.195 mg/mL) (Mulaudzi et al., 2011), which might explain the com- mon usage of the plant in this region to treat diarrhea-related ail- Melting points (uncorrected) were measured on a Gallenkamp ments and diverse infections. Interestingly, in a screen against the melting point apparatus. Optical rotations were determined on a tubercular pathogen Mycobacterium tuberculosis, X. caffra acetone Perkin-Elmer 241 polarimeter installed with a λ589 sodium lamp. IR root extracts were weakly active with an MIC > 100 μg/mL (Green spectra were measured on a Bio-Rad FTS-40 series spectrometer in et al., 2010). dry film. EIMS was run on a Micromass Quattro Ultima spectrometer Amongst its diverse medicinal applications, X. caffra stands out as fitted with a direct injection probe (DIP) with ionization energy set at a common traditional remedy for venereal diseases, which has been 70 eV and HRMS (EI) was performed with a Micromass Q-Tof Ultima verified by two independent studies at separate geographical loca- spectrometer. 1H, 13CNMR, DEPT, COSY, HSQC and HMBC spectra tions within South Africa. Firstly, we showed that amongst a collec- were recorded on a Bruker AV400 spectrometer in CDCl3, chemical tion of twelve plants used commonly for sexually-transmitted shifts are reported in units of δ (ppm) and coupling constants (J) infections (STIs) in Venda traditional medicine, X. caffra var. caffra are expressed in Hz. Silica gel Merck KGaA (70–230 mesh) was used was prominent for its activity against the gonococcal pathogen for CC and TLC silica gel 60 F254 for analytical and preparative TLC N. gonorrhoeae (44 to 87% inhibition for various extracts) (Mulaudzi (both Merck KGaA). Spots on chromatograms were detected under et al., 2011). Furthermore, De Wet et al. (2012) recently detailed UV light (254 and 365 nm) and by anisaldehyde reagent stain com- the use of X. caffra var. caffra for STIs in a survey covering the northern prising anisaldehyde–H2SO4–methanol (1:2:97). Maputaland region of KwaZulu-Natal, where thirty three different plant taxa are known to be used for such purposes. Apart from this, 2.2. Plant material Geyid et al. (2005) showed that various extracts of X. americanum exhibited significant activity against N. gonorrhoeae when tested at Leaves of X. caffra var. caffra were collected in August 2010 at concentrations as low as 250 μg/mL. However, no chemical entities Mandiwana (22°85′34.57˝ S, 30°141′65.6˝ E) along the Tshiendeulu were linked to this strong activity despite the plant reportedly mountains of Venda (Limpopo province, South Africa). A voucher spec- containing several classes of phytochemical constituents, including imen (Mulaudzi_24_NU) was authenticated by Dr. Christina Potgieter isoprenoids, fatty acids, sesquiterpenes, triterpenes and steroids and deposited at the Herbarium of the University of KwaZulu-Natal, (Geyid et al., 2005). Pietermaritzburg. Motivated by these observations, we became interested in elucidat- ing the chemical and pharmacological basis to the ethnic usage of 2.3. Extraction and isolation of vomifoliol X. caffra var. caffra for STIs in South Africa. As the first phytochemical in- vestigation of the plant, bioassay-guided fractionation of the EtOH leaf Powdered dried leaves (500 g) were extracted by stirring for 48 h extract here led to the identification of the known bisnorsesquiterpene at room temperature with 80% EtOH. After this time, the mixture was vomifoliol 1 (also referred to as a megastigmane) whose structure was filtered and solvent removed under reduced pressure. The residue unambiguously assigned via physical and spectroscopic means, includ- (54 g) was re-suspended in 500 mL H2O/MeOH (9:1), and extracted ing high field 2D NMR and mass spectrometric techniques. It comprises consecutively with 2 × 500 mL each of nH, DCM and EA. Based on of a polyfunctionalized, α,β-unsaturated hexyl ring system together its antigonococcal activity, the DCM fraction (15 g) was further frac- with a 1,3-disubstituted butylidene side chain (Scheme 1). To the best tionated by column chromatography on silica gel using step gradients of our knowledge, this finding represents the first description of of nH-EA and EA-MeOH. In this way, and aided by TLC analysis, 12 vomifoliol from the genus Ximenia and is thus of chemotaxonomic fractions were generated, of which only fraction 8 exhibited signifi- significance. Moreover, vomifoliol is shown here for the first time to cant antigonococcal activity against N. gonorrhoeae. Fraction 8 was J.J. Nair et al. / South African Journal of Botany 86 (2013) 111–115 113 subsequently purified via preparative TLC (Merck glass plates, of hydroxyl and carbonyl functional groups, respectively. Unambigu-

20 × 20 cm, silica gel F254, and 0. 25 cm thickness) using nH-EA ous assignment of the structure of vomifoliol was arrived at by 1D and (1:1) as developing solvent to give vomifoliol 1 (5.2 mg) as an off- 2D high field NMR. To this extent, its 1H NMR spectrum had the white, semi-crystalline solid. downfield-shifted olefinic proton signals resonant in the region δ 5.77 to 5.91, the most deshielded of which was assignable to H-4 (δ 2.4. Physcial and spectroscopic data for vomifoliol 5.91, bt, J = 1.4, 1.2 Hz) which had distinctive COSY contours to both H-2 eq (δ 2.24, dd, J = 17.1, 1.2 Hz) and H-13 (δ 1.89, 3H, d, 25 −1 M.pt. 100–102 °C. [α]D +36.0° (c 0.16 in CHCl3). IR νMAX/cm J = 1.4 Hz), indicative of homoallylic coupling. As expected, H-13 (dry film): 3502 (OH), 2900 (C\H), 1683 (C_O), 1355 (C\O). was the most deshielded of the four methyl group protons resonant

HRMS (EI): calcd. 224.1413 for C13H20O3, found 224.1421. LRMS (EI) for vomifoliol due to its vinylic substitution. The corresponding car- 70 eV, m/z (rel. int.): 224 [M]+ (5), 168 (30), 150 (25), 135 (26), bon resonances were assignable at δ 50.09 (t, C-2), 127.31 (d, C-4) 1 124 (100), 107 (20). HNMR (400 MHz, CDCl3): δ 1.01 (3H, s, and 19.20 (q, C-13) taking into account HSCQ connectivities. The H-11), 1.08 (3H, s, H-12), 1.30 (3H, dd, J = 6.4, 1.68 Hz, H-10), 1.89 remaining two olefinic proton signals at δ 5.79 (dd, J = 15.7, (3H, d, J = 1.4 Hz, H-13), 2.24 (1H, dd, J = 17.1, 1.2 Hz, H-2 eq), 0.56 Hz) and 5.87 (ddd, J = 15.7, 5.16, 1.68 Hz) were attributable 2.45 (1H, d, J = 17.1 Hz, H-2ax), 4.41 (1H, m, J = 11.8, 6.4, 5.20, to H-7 and H-8, respectively, with their accompanying carbon signals 0.56 Hz, H-9), 5.79 (1H, dd, J = 15.7, 0.56 Hz, H-7), 5.87 (1H, ddd, resonant at δ 129.36 (d, C-7) and 136.12 (d, C-8) as shown by HSQC J = 15.7, 5.16, 1.68 Hz, H-8), 5.91 (1H, bt, J = 1.4, 1.2 Hz, H-4). contour correlation, in accordance with the trans-geometry previous- 13 CNMR (100 MHz, CDCl3): δ 19.20 (q, C-13), 23.26 (q, C-12), 24.13 ly shown for vomifoliol (Siddiqui et al., 2003; Hammami et al., 2004). (q, C-10), 24.41 (q, C-11), 41.51 (s, C-1), 50.09 (t, C-2), 68.36 (d, Again, homoallylic coupling was indicated for both H-7 and H-8 due C-9), 79.41 (s, C-6), 127.31 (d, C-4), 129.36 (d, C-7), 136.12 (d, C-8), to the small value of their coupling constants to H-9 (J = 0.56 Hz) 162.87 (s, C-5), 198.19 (s, C-3). and H-10 (J = 1.68 Hz), respectively. Further upfield, the oxygen- related H-9 proton was located at δ 4.41 (m, J = 11.8, 6.4, 5.2, 2.5. Antigonococcal activity 0.56 Hz) and linked via COSY contours to both H-8 (δ 5.87, ddd, J = 15.7, 5.16, 1.68 Hz) and H-10 (δ 1.30 (dd, J = 6.4, 1.68 Hz), and Antigonococcal activity of crude extracts and pure compounds directly via HSQC to the carbon doublet signal at δ 68.36, shown to was determined by the disc diffusion method as previously reported be the C-9 methine carbon. The H-2ax proton resonance (δ 2.45, d, (Ringertz et al., 1991; Putnam et al., 1992; Shokeen et al., 2005). Ac- J = 17.1 Hz) was found adjacent to its diastereomeric partner cordingly, extracts were dissolved in 80% EtOH to a final concentra- H-2 eq (δ 2.24, dd, J = 17.1, 1.2 Hz) and that these were geminally tion of 2 mg/mL. 5 μL of each extract was impregnated into sterile substituted was indicated by a single HSQC cross peak for both pro- Whatman No. 1 filter paper discs (6 mm diameter) and dried under tons to C-2 (δ 50.09, t). The three-proton doublet resonance at δ a stream of sterile air. The bacterial pathogen N. gonorrhoeae (ATCC 1.30 (dd, J = 6.4, 1.68 Hz), whose chemical shift, multiplicity and 49226 from ATCC as Kwik-STK™ plus microorganism) with viable splitting pattern was characteristic for a secondary methyl group, counts reaching 108 CFU/mL was then streaked with sterile swabs was ascribable to H-10, located at the terminus of the 1,3-disubstituted onto Petri dishes containing chocolate (GC) agar base medium butylidene side chain, with COSY correlations to both H-9 (vicinal) and (CMO367, Oxoid Ltd., England) supplemented with 2% (w/v) hemo- H-8 (homoallylic) and an HSQC connection to the carbon signal at δ globin and 1% (v/v) Vitox and the plates dried for 5 min at room 24.13 (q, C-10). The geminal dimethyl groups (H-11 and H-12) were temperature. The procedure was done in triplicate. Ciprofloxacin assignable due to mutual two and three bond HMBC connectivities to

(1 μg/disc) was used as a positive control, while H2O and 80% EtOH C-1 (δ 41.51, s), C-2 (δ 50.09, t) and C-6 (δ 79.41, s), respectively. Fur- were used as negative and solvent controls, respectively. Discs were thermore, COSY contours established links for H-11 (δ 1.01, s) with applied to the dry surface of a Petri dish containing GC agar base me- H-2 eq, and H-12 (δ 1.08, s) with H-2ax, indicating that they shared dium and bacteria. The plates were inverted and incubated at 37 °C in planar relationships. The remaining carbon signal was ascribable to

5% CO2 for 24 h. Activity against N. gonorrhoeae was indicated by clear C-3, with a chemical shift (δ 198.19, s) characteristic of an zones of inhibition. Percentage inhibition was determined from zonal α,β-unsaturated ketone, with two bond HMBC cross linkages to both diameters as measured for the sample and positive control respec- C-2 (δ 50.09, t) and C-4 (δ 127.31, d). This collectively accounted for tively, according to the equation below: the thirteen carbon skeleton of vomifoliol, which was further resolved to 4 quartets, 4 doublets, 4 singlets and a triplet by DEPT anal- Diameterof thesample %Inhibition ¼ 100: ysis. The C-6 and C-9 stereogenic centers in vomifoliol were previously Diameterof thepositivecontrol shown to be (S)- and (R)-configured, respectively (Siddiqui et al., 2003; Hammami et al., 2004). Previous studies on X. caffra focused on the quantitative estima- 3. Results and discussion tion of constituents such as fatty acids, triacylglycerols, tocopherols and phenols (Khumalo et al., 2002; Řezanka and Sigler, 2007; Vomifoliol 1 was isolated according to the fractionation sequence Chivandi et al., 2008; Mitei et al., 2008, 2009; Ndhlala et al., 2008), described in the experimental section (Section 2.3.). Its physical and due mainly to its cosmetic and nutritive value in traditional medicine. spectroscopic data closely matched those that have been previously In this regard, the present identification of vomifoliol 1 in X. caffra var. reported (Siddiqui et al., 2003; Hammami et al., 2004). EIMS showed caffra is significant. The compound has been found in several other the molecular ion peak at m/z 224 (with a 5% relative intensity), cor- plant families, including the Annonaceae, Apocynaceae, Boraginaceae, rect for the molecular formula C13H20O3, with diagnostic fragment ion Euphorbiaceae, Lythraceae and Podocarpaceae, mainly as glycoconjugates signals detected at m/z 168, 150, 135, 124 and 107. HRMS indicated a (Siddiqui et al., 2003; Hammami et al., 2004). It has a close structural mass of 224.1421 g/mol for the compound, correct for its molecular similarity to natural norisoprenoids such as the ionones (α-ionone 2 is formula and theoretical mass of 224.1413 g/mol. Furthermore, dou- shown in Scheme 1) and the damascones (α-damascone 3 is shown in ble bond equivalence manipulation of the molecular formula sug- Scheme 1) known for their potent odorant properties (such as flowery gested four degrees of unsaturation for its structure, two of which and fruity odors) and widely exploited in viticulture (Naves, 1971). could be justified by the α,β-unsaturated ketone and the remaining These compounds are biochemically derived from carotenoids via the ox- two by the C-7/C-8 double bond and the hexyl ring system. IR absorp- idative pathway involving dioxygenase enzyme systems (Naves, 1971). tion bands were found at (inter alia) 3502 and 1683 cm−1, indicative Of greater significance, is its close resemblance to the common plant 114 J.J. Nair et al. / South African Journal of Botany 86 (2013) 111–115 hormone abscisic acid (ABA) 4 known for its significant role in several the bisnorsesquiterpene vomifoliol which is here uncovered as the plant development processes, including bud dormancy, leaf abscission, component responsible for its antigonococcal activity, and thus sub- as well as effects pertaining to environmental stress and plant patho- stantiates the ethnic medicinal usage of the plant for such purposes. genesis (Nambara and Marion-Poll, 2005). Not surprisingly, vomifoliol has been shown to exhibit similar physiological effects, such as, amongst others, stomatal closure in Eichhornia crassipes epidermal Acknowledgements strips (Coke et al., 1975). Structurally, ABA differs from vomifoliol by virtue of a two carbon homologation leading to the carboxylic acid func- Financial assistance from the University of KwaZulu-Natal is gratefully tionalized side chain. The two compounds are also thought to be closely acknowledged. related biosynthetically, as the enzyme vomifoliol dehydrogenase iso- lated from a species of Corynebacterium was capable of metabolizing ABA into dehydrovomifoliol, the C-9 keto analogue of vomifoliol References (Hasegawa et al., 1984). Chivandi, E., Davidson, B.C., Erlwanger, K.H., 2008. A comparison of the lipid and fatty Based on its widespread traditional usage for STIs (Ndubani and acid profiles from the kernels of the fruit (nuts) of Ximenia caffra and Ricinodendron Höjer, 1999; Kambizi and Afolayan, 2001; Maroyi, 2011), we next rautanenii from . Industrial Crops and Products 27, 29–32. turned our attention to ascertaining the activity of X. caffra var. caffra Coates Palgrave, K., 2003. Trees of Southern Africa, third ed. Struik, Cape Town. Coke, L.B., Stuart, K.L., Whittle, Y.G., 1975. Further effects of vomifoliol on stomatal ap- against the common gonococcal pathogen N. gonorrhoeae. Table 1 erture and on the germination of lettuce and the growth of cucumber seedlings. shows the inhibitory activities of the X. caffra EtOH leaf extract, as Planta 127, 21–25. well as various fractions obtained by solvent partitioning and column Corrigan, B.M., Van Wyk, B.-E., Geldenhuys, C.J., Jardine, J.M., 2011. Ethnobotanical plant uses in the KwaNibela Peninsula, St Lucia, South Africa. South African Journal chromatography. Of these, the DCM fraction obtained after partition of Botany 77, 346–359. was the most potent (at 10 μg/disc) with 78.8% inhibition of De Wet, H., Nzama, V.N., Van Vuuren, S.F., 2012. Medicinal plants used for the treat- N. gonorrhoeae. Bioassay-guided fractionation of the DCM fraction ment of sexually transmitted infections by lay people in northern Maputaland, KwaZulu-Natal Province, South Africa. South African Journal of Botany 78, 12–20. then led to the active fraction 8 (59.1% inhibition) and subsequent Fabry, W., Okemo, P.O., Ansorg, R., 1998. Antibacterial activity of East African medicinal isolation of vomifoliol 1 which was identified as the active component plants. Journal of Ethnopharmacology 60, 79–84. with 63.1% inhibition. The marked decrease in inhibition in going Geyid, A., Abebe, D., Debella, A., Makkonen, Z., Aberra, F., Teka, F., Kebede, T., Urga, K., from the DCM fraction to the sub-fraction (fraction 8) indicates that Yersaw, K., Biza, T., Mariam, B.H., Guta, M., 2005. Screening of medicinal plants of Ethiopia for their antimicrobial properties and chemical profiles. Journal of there may be other components in the solvent partitioned fraction Ethnopharmacology 97, 421–427. which could synergistically or additively enhance activity. Although Green, E.C., 1992. Sexually transmitted disease, ethnomedicine and health policy in – there are more than 30 bacterial, viral and parasitic pathogens Africa. Social Science & Medicine 35, 121 130. Green, E., Samie, A., Obi, C.L., Bessong, P.O., Ndip, R.N., 2010. Inhibitory properties of se- which are sexually transmissible, Treponema pallidum (syphilis), lected South African medicinal plants against Mycobacterium tuberculosis. Journal N. gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis are of Ethnopharmacology 130, 151–157. responsible for most sexually transmitted infections (World Health Hammami, S., Jannet, H.B., Bergaoui, A., Ciavatta, L., Cimino, G., Mighri, Z., 2004. Isola- tion and structure elucidation of a flavanone, a flavanone glycoside and vomifoliol Organization, 2007). Gonorrrhea is the most commonly known vene- from Echiochilon fruticosum growing in Tunisia. Molecules 9, 602–608. real disease caused by the bacterium N. gonorrhoeae. Most STIs are Hasegawa, S., Poling, S.M., Maier, V.P., Bennett, R.D., 1984. Metabolism of abscisic acid: curable by appropriate antimicrobial measures; however, socio- bacterial conversion to dehydrovomifoliol and vomifoliol dehydrogenase activity. Phytochemistry 23, 2769–2771. economic and other exacerbating factors render these services Heywood, V.H. (Ed.), 1993. Flowering Plants of the World. Oxford University Press, unattainable by many, especially in rural communities (De Wet et New York. al., 2012). Consequently, the use of traditional medicines for such ail- Kambizi, L., Afolayan, A.J., 2001. An ethnobotanical study of plants used for the treat- ment of sexually transmitted diseases (njovhera) in Guruve district, Zimbabwe. ments is thus a frontline regimen for the vast majority of sufferers (De Journal of Ethnopharmacology 77, 5–9. Wet et al., 2012). Not surprisingly, it has been shown that most suf- Khumalo, L.W., Majoko, L., Read, J.S., Ncube, I., 2002. Characterisation of some ferers of STIs in Sub-Saharan Africa resort to traditional cures, as op- underutilized vegetable oils and their evaluation as starting materials for lipase- posed to modern therapeutic cures (Green, 1992). It is within this catalysed production of cocoa butter equivalents. Industrial Crops and Products 16, 237–244. context that the isolation of vomifoliol from X. caffra var. caffra, a tra- Maroyi, A., 2011. An ethnobotanical survey of medicinal plants used by the people in ditional herbal remedy for STIs, together with its identification as a Nhema communal area, Zimbabwe. Journal of Ethnopharmacology 136, 347–354. novel antigonococcal agent, is significant. Mathabe, M.C., Nikolova, R.V., Lall, N., Nyazema, N.Z., 2006. Antibacterial activities of medicinal plants used for the treatment of diarrhoea in Limpopo Province, South In summary, based on the widespread usage of X. caffra in the tra- Africa. Journal of Ethnopharmacology 105, 286–293. ditional remediation of STIs in South Africa, EtOH leaf extracts were Mitei, Y.C., Ngila, J.C., Yeboah, S.O., Wessjohann, L., Schmidt, J., 2008. 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b Naves, Y.R., 1971. Some developments in the chemistry of ionones and their deriva- EtOH extract 43.9 (±2.4) — – b tives a subject review. Journal of the Society of Cosmetic Chemists 22, 439 456. nH fraction 73.4 (±4.3) Ndhlala, A.R., Muchuweti, M., Mupure, C., Chitindingu, K., Murenje, T., Kasiyamhuru, A., b DCM fraction 78.8 (±3.2) Benhura, M.A., 2008. Phenolic content and profiles of selected wild fruits of Zimba- b EA fraction 52.3 (±0.9) bwe: Ximenia caffra, Artobotrys brachypetalus and Syzygium cordatum. International Fraction 8b,c 59.1 (±0.7) Journal of Food Science and Technology 43, 1333–1337. Vomifoliolb,d 63.1 (±4.0) Ndubani, P., Höjer, B., 1999. Traditional healers and the treatment of sexually transmit- Ciprofloxacine 100 (±0.0) ted illnesses in rural . Journal of Ethnopharmacology 67, 15–25. PhytoTrade Africa, http://www.phytotradeafrica.com/products/XimeniaOil.html (accessed a Values are means of three experiments, standard error is given in parentheses. on 08 February 2013). b μ Carried out at 10 g/disc. Putnam, S.D., Lavin, B.S., Stone, J.R., Oldfield, E.C., Hooper, D.G., 1992. Evaluation of the c Obtained via column chromatography of the DCM fraction. standardized disk diffusion and agar dilution antibiotic susceptibility test methods d Obtained via PTLC of fraction 8. by using strains of Neisseria gonorrhoeae from the United States and Southeast Asia. e Used as a positive control and run at 1 μg/disc. Journal of Clinical Microbiology 30, 974–980. J.J. Nair et al. / South African Journal of Botany 86 (2013) 111–115 115

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