Fitoterapia 70 Ž1999. 412]416 A new flavan from Elephantorrhiza goetzei Fortune Moyo a , Berhanu A. Gashe b, Runner R.T. Majindaa,U a Department of Chemistry, Uni¨ ersity of Botswana, Pri¨ ate Bag 0022, Gaborone, Botswana Department of Biological Sciences, Uni¨ ersity of Botswana, Pri¨ ate Bag 0022, Gaborone, Botswana b Received 17 February 1999; accepted 11 March 1999 Abstract A new compound, 3,39,49,5,6,7,8-heptahydroxyflavan Ž1., was isolated from the roots of Elephantorrhiza goetzei. The crude extract, and its n-butanol and residual water fractions showed antimicrobial activity. Semi-purified extracts showed higher activity and of the purified compounds only methyl gallate gave activity below 100 mg of sample. Q 1999 Elsevier Science B.V. All rights reserved. Keywords: Elephantorrhiza goetzei; Flavonoids; 3,39,49,5,6,7,8-heptahydroxyflavan; Antibacterial activity; Antifungal activity 1. Introduction The genus Elephantorrhiza is made up of shrubs, small trees, or low bushes springing from underground rhizomes w1x. In Southern Africa four species are known, namely E. burkei, E. elephantina, E. goetzei and E. suffruticosa w2x. The first three are indigenous to Botswana while the last one is reported only in Zimbabwe and Namibia w3x. E. goetzei ŽHarms. Harms ŽLeguminosae. is found mainly in North Eastern Botswana. The plant has a variety of medicinal uses such as a U Corresponding author. E-mail address: majindar@noka.ub.bw ŽR.R. Majinda. 0367-326Xr99r$ - see front matter Q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 9 9 . 0 0 0 6 8 - 4 F. Moyo et al. r Fitoterapia 70 (1999) 412]416 413 remedy for sores of the penis and vulva, womb problems, irregular menstruation, and for cleansing the womb after abortion w2,4x. Plants of the genus Elephantorrhiza were found to contain flavonoids, gallic acid derivatives, esterified sugars and simple phenolic derivatives w5x. E. goetzei root, previously not investigated for its constituents, has now been found to contain a new flavan 1 ŽScheme 1.. Its extractives also showed weak to moderate activity against gram positive and gram negative bacteria and fungi. 2. Experimental Plant material. E. goetzei roots were collected from Mapoka North East District, Botswana, in August 1997 and identified by Dr L.M. Turton. A voucher specimen ŽNo. 3393. was deposited at the University of Botswana Herbarium. Extraction and isolation. Air-dried and powdered root Ž2 kg. was extracted with CH 2 Cl 2-EtOAc-MeOH-H 2 O 0.9:1:6:1 Ž10 l.. The extract was concentrated, the residue was dissolved in water and partitioned successively between CH 2 Cl 2 , EtOAc and n-BuOH. Solvents were removed to give the respective crude fractions Ž5, 7 and 22 g, respectively.. The residual water yielded 65 g of crude extract upon removal of solvent. The CH 2 Cl 2 and EtOAc fractions, giving similar TLC profiles ŽC 6 H 6-EtOAc-MeOH 6:3:1, 3:6:1. were combined, to give a mixture of phytosterols which was not investigated further. The n-BuOH fraction was subjected to VLC using CH 2 Cl 2 with increasing amounts of methanol to yield fractions S1 Ž3.1 g., S2 Ž1.3 g., S3 Ž2.8 g., and S4 Ž1.8 g.. Fraction S1, following gel filtration with Sephadex LH-20 ŽCHCl 3-MeOH 1:1. and purification with PTLC ŽC 6 H 6-EtOAcMeOH 6:3:1. yielded Žq.-catechin Ž25 mg.. CC Si-gel ŽEtOAc-MeOH. followed by gel filtration Ž Sephadex LH-20 . on fraction S2 yielded 2- Ž 39,49dihydroxyphenyl.ethanol Ž5 mg.. CC Si-gel ŽCH 2 Cl 2-MeOH. followed by PTLC on fraction S3 yielded methyl gallate Ž6 mg.. CC Si-gel ŽCH 2 Cl 2-MeOH. followed by Sephadex LH-20 on fraction S4 yielded compound 1 Ž3.5 mg.. 3,39,49,5,6,7,8-Heptahydroxyfla¨ an (1). Yellow granules, mp 2608C dec. R f s 0.55 ŽCHCl 3-MeOH 1:1., w a x D 12.9 Žc 0.001, MeOH.. UV ŽMeOH.: 226, 284 nm; IRŽKBr.: 3438, 2926, 1620, 1452, 1360 cmy1 ; EI-MS mrz Žrel. int..: 322 Ž5., 304 Ž2., 290 Ž7., 271 Ž12., 170 Ž100., 152 Ž24., 126 Ž22., 109 Ž40.; 1 H and 13 C-NMR: see Table 1. 3. Results and discussion Compound 1 was obtained as yellow granules which decomposed before melting. The EIMS gave a molecular ion wMqx at mrz 322 consistent with a molecular formula C 15 H 14 O 8 . The proton spectrum of 1 compares well with that of catechin ŽTable 1., except that the signals at d H 5.84 and 5.92, attributable to H-6 and H-8 F. Moyo et al. r Fitoterapia 70 (1999) 412]416 414 Table 1 1 H Ž300 MHz. and Carbon 13 C-NMR Ž75 MHz. chemical shifts for compound 1a and catechin in CD3 OD Catechin 1 1 d H Ž J value in parentheses . 2 3 4 4.56, 1H, 3.97, 1H, 2.53, 1H, 2.82, 1H, 5 6 7 8 9 10 11 21 31 41 51 61 d Ž7.1. m Ž4.7, 7.8. dd Ž8.4, 16.1. dd Ž5.5,16.1. 6.83, 1H, d Ž2.0. 6.76, 1H, d Ž8.2. 6.72, 1H, dd Ž2.0, 8.2. d 13 C 82.8 Ž d . 68.8 Ž d . 28.5 Ž d . 148.6 Ž s . 138.6 Ž s . 151.8 Ž s . 132.7b Ž s . 147.6 Ž s . 100.9 Ž s . 132.2b Ž s . 116.1 Ž d . 146.2 Ž s . 146.4 Ž s . 115.3 Ž d . 120.1 Ž d . d 1H Ž J value in parentheses . 4.53, 1H, 3.99, 1H, 2.51, 1H, 2.90, 1H, d, Ž7.5. m, Ž4.7, 7.8. dd Ž8.4, 16.1. dd Ž5.5,16.1. 5.84, 1H, d Ž2.2. 5.92, 1H, d Ž2.2. 6.85, 1H, d Ž2.0. 6.80, 1H, d Ž8.4. 6.70, 1H, dd Ž2.0, 8.4. d 13 C 82.7 Ž d . 68.6 Ž d . 28.6 Ž d . 157.2 Ž s . 95.2 Ž d . 157.7 Ž s . 96.3 Ž d . 156.9 Ž s . 100.6 Ž s . 132.1 Ž s . 114.9 Ž d . 145.7 Ž s . 146.2 Ž s . 115.5 Ž d . 119.9 Ž d . a Assignments were confirmed by DEPT, HETCOR and COSY. Assignment may be reversed. b of catechin, respectively, are missing. This means that ring A was fully oxygenated. The nature of the substituents, namely all hydroxy, was clear from the 1 H-NMR spectrum which showed no other signals. This was confirmed by the 13 C-NMR which showed absence of signals at d C 95.2 ŽC-6. and 96.3 ŽC-8., replaced by signals at d C 135.6 ŽC-6. and 132.7 ŽC-8.. The latter chemical shifts are normal and expected for those two positions in a fully oxygenated system w6x. The MS data gave a base peak at mrz 170 consistent with the cleavage ŽScheme 1. of ring C at C2rC3 bond Žfragment A. and C2rheteroatom 1 bond. The other fragment from the above cleavage gave an ion at mrz 152 Žfragment B.. Thus compound 1, named elephantorrhizol, was identified as being 3,39,49,5,6,7,8-heptahydroxyflavan, reported for the first time. The crude extract, fractions, and isolated compounds were tested for antibacterial and antifungal activity using the TLC bioautography technique w7,8x. Though the activity went up with purification, isolated compounds showed either no or reduced Žonly methyl gallate at a loading of 100 mg. activity ŽTable 2.. Therefore, the data obtained also seem to support some folkloric use of the plant. Acknowledgements F.M would like to thank DAAD for a scholarship. R.R.T.M. thanks IFS for a research grant. F. Moyo et al. r Fitoterapia 70 (1999) 412]416 415 Table 2 Antimicrobial activity of Elephantorrhiza goetzei extractives a Microorganism B subtilis ŽNCTC 3610. S. aureus ŽNCTC 8582. P. mirabilis ŽNCTC11938. E. coli ŽNCTC 09001. P. aeruginosa ŽNCTC10332. C. mycoderma ŽSM 428. Loading Žmg. A B C E S1 S2 S3 S4 Mg Chlo Mic 100 100 100 100 100 100 ] ] ] ] ] ] 100 ] ] ] ] ] 100 100 100 100 100 100 15 15 25 50 15 15 15 15 25 50 15 15 15 15 25 50 15 15 15 15 15 15 15 15 72 70 72 72 70 72 0.01 0.01 0.01 0.01 0.01 ) 50 0.50 0.50 1.0 ) 50 ) 50 0.01 a A s crude extract, B s dichloromethane fraction, C s ethyl acetate fraction, D s n-butanol fraction, E s residual water extract, S1]S4 s semi-purified fractions from the n-butanol fraction, Mg s methyl gallate, Chlo s chloramphenicol, and Mic s miconazole. Scheme 1. Major fragments in 1. References w1x Palgrave KC. Trees of southern Africa. Cape Town: Struik Publishers, 1995:258. w2x Watt MJ, Breyer-Brandwijk MG. Medicinal and poisonous plants of southern and eastern Africa, 2 ed. Edinburgh and London: E. and S. Livingstone Ltd, 1962:535. 416 F. Moyo et al. r Fitoterapia 70 (1999) 412]416 w3x Brenan JPM. Flora Zambesiaca, Part 1. Whitestable, Kent: Whitestable Litho Printers Ltd, 1970:24, 3. w4x Hedberg I, Staugard F. Traditional medicinal plants. Gaborone: Ipelegeng Publishers, 1989: 120,307. w5x Aaku E, Office M, Dharani SP, Motswaledi MS, Majinda RRT. Fitoterapia 1998;69:464. w6x Adinolfi M, Aquila T, Barone G, Lanzetta R, Parrilli M. Phytochemistry 1989;28:3444. w7x Holmans AL, Fuchs AJ. J Chromatogr 1970;51:327. w8x Rahalison L, Hamburger M, Hostettmann K, Monod M, Frenk E. Phytochem Anal 1991;2:199.