The structures of two new isoflavonoids from the combined ethyl acetate/methanolic extracts of the stem bark of Bolusanthus speciosus have been established as 4,7,2' -trihydroxy-4' -methoxyisoflavanol (1) and 5,7,3',4' -tetrahydroxy-5' -(2-epoxy-3-methylbutyl)isoflavanone (2). Five other known isoflavonoids, 5,7,3' -trihydroxy-4' -methoxy-5' -γγ; -dimethylallylisoflavanone, 5,7,2' -trihydroxy-4' -methoxy-6,5' -di)(γγ -dimethylallyl)isoflavanone, 5,7,2' ,4'-tetrahydroxy-8,5'2; -di(γγ -dimethylallyl)flavanone, 5,7,2',4' -tetrahydroxy-8,3' -di(γγ -dimethylallyl)-isoflavanone, and derrone were also isolated. Compound 2 showed moderate activity against gram positive bacteria and weak activity against gram negative bacteria, while compound 1 was weakly active against both organisms in a TLC bioautography assay

Cornelius C.W. Wanjala

Gomotsang Bojase

Runner R.T. Majinda

Bulletin of the Chemical Society of Ethiopia

. The structures of two new isoflavonoids from the combined ethyl acetate/methanolic extracts of the stem bark of Bolusanthus speciosus have been established as 4,7,2' -trihydroxy-4' -methoxyisoflavanol (1) and 5,7,3' ,4' -tetrahydroxy-5' -(2-epoxy-3-methylbutyl)isoflavanone (2). Five other known isoflavonoids, 5,7,3' -trihydroxy-4' -methoxy-5' -&gamma;&gamma;; -dimethylallylisoflavanone, 5,7,2' -trihydroxy-4' -methoxy-6,5' -di)(&gamma;&gamma; -dimethylallyl)isoflavanone, 5,7,2' ,4'-tetrahydroxy-8,5'2; -di(&gamma;&gamma; -dimethylallyl)flavanone, 5,7,2' ,4' -tetrahydroxy-8,3' -di(&gamma;&gamma; -dimethylallyl)-isoflavanone, and derrone were also isolated. Compound 2 showed moderate activity against gram positive bacteria and weak activity against gram negative bacteria, while compound 1 was weakly active against both organisms in a TLC bioautography assay. 
 
(Received March 22, 2001; revised August 13 2001)
Bull.Chem.Soc.Ethiop. 2001, 15(2), 131-13

Bojase, Gomotsang

Wanjala, Cornelius C.W.

Majinda, Runner R.T.

AJOL - African Journals Online

Bu ll. Chem . Soc. E th iop. 2001 , 15(2), 131-136.                                                           ISSN  1011-3924P r in ted in  E thiopia                                                                       ã 2001 Ch em ica l Society of E th iopia__________*Correspondin g a uthor . E -m ail: m a jinda r@m opipi.ub.bwTWO NEW ISOFLAVANOIDS FROM BOLUSANTHUS SPECIOSUSGomotsang Bojase, Cornelius C.W. Wanjala and Runner R.T. Majinda*Department of Chemistry, University of Botswana, Private Bag 00704, Gaborone, Botswana(Received March 22, 2001; revised August 13 2001)AB STR ACT. Th e st ru ct ures  of two new isoflavonoids from th e com bin ed et h yl acet a te/m et h an olicext r a ct s of the st em  bark  of Bolusa n thu s speciosus h ave been  es tablished  as 4,7,2¢-t r ih ydroxy-4¢-m et h oxyisofla van ol (1) a nd 5,7,3¢,4¢-t et r ah ydroxy-5¢-(2-epoxy-3-m et h ylbutyl)isoflavanone (2). Fiveoth er  known  isoflavonoids, 5,7,3¢-t r ih ydroxy-4¢-m et hoxy-5¢-g,g-dim et h yla llylisoflavanone, 5,7,2¢-t r ihydroxy-4¢-m et h oxy-6,5¢-di(g,g-dim et h yla llyl)isoflavanone, 5,7,2¢,4¢-t et r ah ydroxy-8,5¢-di(g,g-dim et hylallyl)flavanone, 5,7,2¢,4¢-t et r ah ydroxy-8,3¢-di(g,g-dim et h yla llyl)-isoflavanone, a nd der ronewer e a lso isola ted. Compound 2  showed m oder a te act ivity aga in st  gra m  posit ive bact er ia  a ndwea k a ct ivity a gainst  gra m  nega t ive bact er ia , while com poun d 1  wa s wea kly a ct ive a gainst  bothorganism s in  a  TLC bioautogra phy assa y.KEY WOR D S: Bolusa n thu s speciosus, 4,7,2¢-Tr ihydroxy-4¢-m et h oxyisofla vanol, 5,7,3¢,4¢-Tet ra h ydroxy-5¢-(2-epoxy-3-met h ylbutyl)isoflavan one, An t ibact er ia l act ivityINTRODUCTIONBolusanthus speciosus Harms (Fabaceae), otherwise called tree wisteria, is a monotypic andendemic tree in subtropical Southern Africa [1, 2].  It occurs in open areas on granite basaltic orlimestone soils [1].  B. speciosus is normally used as an ornamental tree in gardens and parks,because of its beauty [3]. The root infusion of this plant is used by some communities as anemetic while the dried inner bark is used to relieve abdominal pains [4]. Previous work on B.speciosus revealed the presence of alkaloids [5] and isoflavonoids which were identified asgenestein, orobol, 3¢-methoxyorobol, pratensein, 3¢-O-methylpratensein and 3,5,7,3¢-tetrahydroxy-4 ¢-methoxyisoflavone (bolusanthin) [6].RESULTS AND DISCUSSIONThe ethyl acetate and methanolic extracts from the stem bark of B. speciosus were worked up asdescribed in the experimental section to give two new isoflavonoids which were identified usingUV, IR, 1D and 2D NMR, EIMS and HRTOFMS spectroscopic methods.The HRTOFEIMS of 1 showed the ion peak at 270.0892 corresponding to molecularformula C16H14O4 [M-H2O]. The low resolution EIMS also gave the highest molecular ion at m/z270 which formed the base peak. This loss of 18 mass units from the expected molecular ion,[C16H16O5]+., is due to loss of water molecule, a common phenomenon in 4-hydroxyisoflavanols[7]. The EIMS gave other major mass fragments at m/z 137, 161 due to retro-Diels-Alderfragmentation. The fragment at m/z 137 confirms a flavonoid moiety with only one hydroxylsubstituent on ring A [8]. The 1H NMR spectra showed two proton dd’s (J = 9.8, 10.2 Hz) at dH3.59 (H-2a) and (J = 5.9, 9.8 Hz) at dH 4.28 (H-2b), one proton ddd (J = 5.7, 6.1, 10.2 Hz) at dH3.61 (H-3) and one proton d (J = 5.7 Hz) at dH 5.52 (H-4). The DEPT spectrum also showedmethine carbons at dC 39.9 and 79.1, a strong indication that compound 1 was an isoflavan-4-ol.Gomotsa ng Bojase et al.Bull. Ch em . Soc. E thiop. 2001, 15(2)132The 13C NMR spectra showed sixteen carbon signals (Table 1) and the HMQC together withDEPT spectra confirmed that ten of these were protonated. The DEPT spectrum showed thepresence of one methylene carbon, eight methine carbons and one methoxyl. The nature andidentity of the isoflavan-4-ol was evident from the 1H NMR which showed the following salientfeatures: two 1,3-dioxyphenyl groups at dH = 6.57 (dd, J = 2.4, 8.4 Hz, 1H); 7.34, (d, J = 8.4 Hz,1H); 6.36, (d, J = 2.4 Hz, 1H) and dH = 6.46 (dd, J = 2.3, 8.4 Hz, 1H); 7.24, (d, J = 8.1 Hz, 1H);6.39, (d, J =2.3 Hz, 1H). The methoxyl group could be placed at either C-2¢ or C-4¢ of theisoflavanol moiety since both these carbons have resonances which showed they were oxygenbearing. The connectivity of the methoxyl (dH 3.76) group to this moiety was evident from theHMBC correlation with carbon at dC 161.7 (C-4¢). The proton ddd at dH 3.61 (H-3) showedHMBC correlations with carbon signals dC 161.3 (C-2¢) and dC 125.4 (C-6¢) further confirmingthat the carbon atom resonating at dC 161.7 is indeed C-4¢. The data available thus enable theidentification of compound 1 as 4,7,2¢-trihydroxy-4 ¢-methoxyisoflavanol, named bolusanthol D,and it is being reported for the first time. The problem that remains unresolved is the absolutestereochemistry at C-3 and C-4. We were able to determine, from our data, the trans relationshipbetween the two H-3 and H-4 and hence structure 1 as drawn shows only the relativestereochemistry at C-3 and C-4.The HRTOFEIMS of 2 gave the ion peak at 372.1203 consistent with molecular formulaC20H20O7, with the low resolution EIMS also giving the molecular ion peak at m/z 372. The massfragments shown at m/z 220 and 153 were consistent with the retro-Diels–Alder fragmentation,with former ion indicating substituent on ring B of the flavonoid moiety [8]. The 1H NMR data(Table 1) shows the presence of the 2-epoxy-3-methybutyl substituent from the followingfeatures: two methyl singlets at dH 1.34 (3H) and 1.27 (3H); an aliphatic proton dd (J = 5.1, 7.3Hz) at dH 3.70 and the non-equivalent methylene protons at dH 2.69 (dd, J = 7.3, 16.8 Hz) and2.98 (dd, J = 5.1, 16.8 Hz). The presence of the 2-epoxy-3-methylbutyl substituent was alsoevident from 13C NMR data which showed two methyl carbons at dC 20.0 and 24.7, a quaternarycarbon at dC 77.6, a methine carbon at dC 69.6 and a methylene carbon at dC  31.1. The presenceof three one-proton dd’s at dH 3.8 (H-3) (J = 5.6, 9.0 Hz), dH 4.50 (H-2a) (J = 5.6, 11.4 Hz) anddH 4.52 (H-2b) (J = 9.0, 11.4 Hz) in the proton NMR spectrum together with a methine carbon atdC 50.7 and a methylene carbon at dC 71.6, in the 13C NMR, indicated that compound 2 was anisoflavanone. The nature of the isoflavanone was evident from the 1H NMR data (Table 1)which showed the following salient features: two shielded equivalent meta-coupled protons atdH 5.92 (d, J = 2.1 Hz, 2H) and another set of meta-coupled protons at dH 6.60 (1H, d, J = 2.1Hz) and dH 6.53 (1H, d, J = 2.1 Hz). The 13C NMR showed 20 carbon signals (Table 1), and theHMQC and DEPT confirmed that ten of these were protonated. The DEPT spectrum furthershowed the presence of six methines, two methylenes and two methyl carbons. The massfragment at m/z 220 showed that the 2-epoxy-3-methylbutyl group was in the B-ring of theisoflavonoid moiety. The connectivity of the 2-epoxy-3-methylbutyl group to the isoflavanone                     1OHOOHHO OCH3Two n ew isoflavanoids from Bolusanthus speciosusBull. Ch em . Soc. E thiop. 2001, 15(2)133moiety was evident from the following HMBC correlations: the methylene protons at dH 2.69and 2.98 showed correlations with carbons at dC 120.5 (C-6’), 121.1 (C-5’), 140.8 (C-4’), 69.5(C- 2’’) and 77.6 (C-3’’), while the dd at dH 3.70 showed correlations with carbons at dC 121.1 (C-5’), 77.6 (C- 3’’), 20.0 (C- 4’’) and 24.7 (C-5’’), thus establishing the position of attachment of the2-epoxy-3-methylbutyl group to the isoflavonoid moiety as C-5¢. Consequently compound 2was identified as 5,7,3’,4’-tetrahydroxy-5’-(2-epoxy-3-methylbutyl)isoflavanone, namedbolusanthol E, and is also reported for the first time. Reported work [9,10] showed (+)-isoflavanones to have 3R configuration and  (-)-isoflavanones to have 3S configuration. Since 2is an  (+)-isoflavanone, it probably has 3R configuration at C-3.Table 1. 1H and 13C NMR data for Compounds 1 and 2 in acetone-d 6.1 2Position dH  dC dH dC2a  b3.59, dd, (9.8, 10.2)4.28, dd, (6.1, 9.8)  66.5  (t) 4.50, dd, (5.6, 11.4)4.52, dd (9.0, 11.4)71.6   (t)3 3.61ddd,(5.7, 6.1, 10.2) 39.9    (d) 3.8, dd, (5.6, 9.0) 50.7   (d)4 5.52, d, (5.7) 79.1    (d) 197.0  (s)5 7.34, d, (8.4) 132.2  (d) 164.9  (s)6 6.57, dd, (2.4, 8.4) 110.0  (d) 5.92, d, (2.1) 95.0    (d)7 159.1  (s) 167.5  (s)8 6.36, d, (2.4) 103.4  (d) 5.92, d, (2.1) 96.2    (d)9 157.0  (s) 163.8  (s)10 111.9  (s) 102.4  (s)1’ 119.8  (s) 127.8  (s)2’ 161.3  (s) 6.60, d, (2.1) 113.4  (d)3’ 6.39, d, (2.3) 96.7    (d) 146.0  (s)4’ 161.7  (s) 140.8  (s)5’ 6.46, dd, (2.3, 8.4) 106.4  (d) 121.1  (s)6’ 7.24, d, (8.4) 125.4  (d) 6.53, d, (2.1) 120.5  (d)1’’ 2.98, dd, (5.1, 16.8) 2.69,dd, (7.3, 16.8)31.1    (t)2’’ 3.70, dd, (5.1, 7.3) 69.5    (d)3’’ 77.6    (s)4’’ 1.27, s 20.0    (q)5’’ 1,34, s 24.7    (q)-OCH3 3.76, s 54.9  (q)Assignments were confirmed by COSY, HMQC, HMBC and DEPT.                                       2OHOOH OOHOHOGomotsa ng Bojase et al.Bull. Ch em . Soc. E thiop. 2001, 15(2)134The structures of the known compounds 5,7,3¢-trihydroxy-4 ¢-methoxy-5¢-g,g-dimethylallylisoflavanone [11], 5,7,2¢-trihydroxy-4 ¢-methoxy-6,5¢-di(g,g-dimethylallyl)-isoflavanone [12], 5,7,2¢,4¢-tetrahydroxy-8,3 ¢-di(g,g-dimethylallyl)-isoflavanone [13] and5,7,2¢,4¢-tetrahydroxy-8,5 ¢-di(g,g-dimethylallyl)- flavanone [10] and derrone [14] weredetermined from spectroscopic data and by comparison of our spectral data with those reportedin literature.Compounds 1 and 2 were tested for antibacterial activity using the TLC bioautographytechnique [15, 16]. The results are shown in Table 2. Compound 2 seems to be consistentlymore active than 1 against the organism tested. The results seem to support the traditional use ofthe plant in treatment of abdominal pains, which mostly are due to bacterial infections.Table 2. Antibacterial activity of compounds 1 and 2.Loading (mg)Microorganism1 2 ChloramphenicolBacillus subtilis  (NCTC 3610) 100 10 0.001Staphylococcus aureus 10 1 0.001Escherichia coli (NCTC 09001) 100 10 0.01EXPERIMENTALGeneral experimental procedures. Melting point: Stuart Scientific (SMP1) melting pointapparatus; specific rotation [a]D: Polatronic-D (Schimdt + Haensch) polarimeter; UV: ShimadzuUV-2101PC spectrophotometer;  IR: Perkin Elmer 2000 FT-IR spectrometer. The 1D {1H (300MHz), 13C (75.5 MHz), DEPT} and 2D (COSY, HMQC, HMBC) spectra acquired on BrukerAvance DPX 300 spectrometer and referenced to residual solvent signal. MS: HREIMS wasdone on autospec TOF spectrometer. EI on Finnigan MAT SSQ 700 single quadrupoleinstrument. Column chromatography-silica gel 60 particle size 0.040-0.063 mm for columnchromatography (Merck); vacuum liquid chromatography: silica gel HF254 5-15 mm mesh(Merck); Sephadex LH-20 (Sigma); preparative TLC: silica gel 60 PF254 + 366 for preparative layerchromatography (Merck); analytical TLC: silica gel 60-F254 precoated alumina sheets (Merck),and visualized using UV (254 and 366 nm) and vanillin-sulfuric acid spray.Plant material. The stem bark of B. speciosus was collected from Mapoka, North East District,Botswana in August 1997, identified by Dr. L. M. Turton and a voucher specimen (B 0897) wasdeposited at the University of Botswana Herbarium.Extraction and isolation. The air dried stem bark was ground to fine powder (1.5 kg) and wassequentially extracted in MeOH-CHCl3 and aqueous MeOH. The solvents were then removed togive 150 g of brown residue. The residue was then subjected to vacuum liquid chromatographyby elution with hexane, ethyl acetate and methanol in order of increasing polarity. The ethylacetate-methanol fractions were combined and applied to Sephadex LH-20 column and theelution was carried out with CHCl3-MeOH (1:1), to give fractions A and B. Fraction A was thensubjected to column chromatography silica gel 60 and eluted with toluene-EtOAc-HOAc(45:4:1) to obtain fractions A1 and A2. Concentrated fraction A1 was separated by multipledevelopment on preparative TLC using solvent systems toluene-EtOAc-HOAc (35:14:1) andCHCl3-EtOAc (6:1) to give 5,7,3¢-trihydroxy-4 ¢-methoxy-5¢-g,g-dimethylallylisoflavanone (10Two n ew isoflavanoids from Bolusanthus speciosusBull. Ch em . Soc. E thiop. 2001, 15(2)135mg) [11], 5,7,2¢-trihydroxy-4 ¢-methoxy-6,5¢-di(g,g-dimethylallyl)isoflavanone (60 mg) [12],5,7,2¢,4¢-tetrahydroxy-8,5 -di(g,g-dimethylallyl)flavanone (15 mg) [13] and 5,7,2¢,4¢-tetrahydroxy-8,3 ¢-di(g,g-dimethylallyl)-isoflavanone (15 mg) [10]. Fraction A2 was alsosubjected to multiple development preparative TLC with toluene-EtOAC-HOAc (45:4:1) toobtain 4,7,2¢-trihydroxy-4 ¢-methoxyisoflavanol (1) (30 mg) and derrone [14] (18 mg). FractionB was applied to column chromatography silica gel 60 and eluted with CHCl3-MeOH (14:1),followed by preparative TLC in the same solvent system to give 5,7,3¢,4¢-tetrahydroxy-5 ¢-(2-epoxy-3-methylbutyl)isoflavanone (2) (75 mg).4,7,2¢-Trihydroxy-4 ¢-methoxyisoflavanol (1) was obtained as brown syrup: [a]25D 21.6 (c0.019, MeOH); UV (MeOH) lmax (log e) 290 (3.9), + AlCl3 376, 312, +AlCl3/HCl 376, 310,+NaOAc 326, 248, +NaOAc/H3BO3 291, 248 nm; IR (KBr) nmax 3401, 2979, 2928, 1638, 1497,1378 cm-1; 1H (300 MHz) and 13C (75.5 MHz) NMR (in CD3COCD3), (see Table 1); EIMS m/z270 [M-H2O]+ (100), 255 (42), 161 (30) 137 (15); HRTOFEIMS m/z 270.0890 (calcd forC16H14O4 , 270.0892).5,7,3¢,4¢-Tetrahydroxy-5 ¢-(2-epoxy-3-methylbutyl)isoflavanone (2) was obtained as yellowcrystals (CHCl3/MeOH 1:1): m.p. 102-104 °C; [a]25D 6.0 (c 0.025, MeOH); UV (MeOH) lmax(log e) 286 nm (4.2), +NaOMe 290, 248, +AlCl3 286, 248, +AlCl3/HCl 286,248, +NaOAc 286,+NaOAc/H3BO3 286 nm; IR (KBr) nmax 3393. 1621, 1499, 944, 839 cm-1; 1H (300 MHz) and 13C(75.5 MHz) NMR (in CD3COCD3), (see Table 1); EIMS m/z 153, [M]+ (100), 372 (70), 220(65), 149 (25); HRTOFEIMS m/z 372.1203 (calcd for C20H20O7, 372.1209).The isolated compounds 1 and 2 were tested against Bacillus subtilis (NCTC 3610),Staphylococcus aureus (NCTC 8532) and Escherichia coli (NCTC 09001) using a standardprocedure [15, 16].ACKOWLEDGEMENTSG.B. thanks the University of Botswana for the scholarship and the study leave. C.C.W.W.thanks the UNESCO-ROSTA through Deutscher Akademischer Austauschdienst (DAAD),Germany for a Scholarship and Jomo Kenyatta University of Agriculture and Technology,Kenya for a study leave. R.R.T.M. thanks IFS for a research grant. Dr. Turton (retiredbotanist/chemist, University of Botswana) is acknowledged for identifying the plant.REFERENCES1. Allen, O.N.; Allen, E.K. The Leguminoseae, Macmillan Publishers: London; 1981; p 101.2. Fabian, A.; Germishhuizen, G. Wild Flowers of Northern South Africa, Fernwood Press:Vlaeberg; 1997; pp 12, 166.3. Van-Wyk, B.; Van-Wyk, P. Field Guide to Trees of Southern Africa, Struik Publishers:Cape Town; 1998; pp 25, 452.4. Venter, F.; Julye, A. Making most of Indigenous Trees, Briza Publications: Pretoria; 1996; p238.5. Asres, K.; Phillipson, D.J.; Mascagani, P. Phytochemistry 1986, 25, 1449.6. Asres, K.; Mascagini, P.; O’neil, M.J.; Philipson, J.D. Z. Naturforsch 1985, 40C, 617.7. Porter, Q.N.; Baldas, J. Mass Spectroscopy of Heterocyclic Compounds , Taylor, E.C.;Weissberger, A. (Eds.); John Wiley: New York; 1985; p 108.Gomotsa ng Bojase et al.Bull. Ch em . Soc. E thiop. 2001, 15(2)1368. Mabry, T.J.; Markham, K.R. The Flavanoids , Part 1, Harborne, J.B.; Mabry, T.J.; Mabry, H.(Eds); Academic Press: London; 1975; p 81.9. Osawa, K.; Yasuda, H.; Maruyama, T.; Morita, H.; Takeya, K.; Itokawa, H. Chem. Pharm.Bull. 1992, 40, 2970.10. Fukai, T.; Zeng, L.; Nishizawa, J.; Wang, Y.; Nomura, T. Phytochemistry 1994, 36, 233.11. Maillard, M.; Hamburger, M.; Gupta, M.P.; Hostettmann, K. Planta Medica 1989, 55, 281.12. Komatsu, M.; Yokoe, I.; Shirataki, Y. Chem Pharm. Bull. 1981, 29, 532.13. O’Neil, M.J.; Adesanya, S.A.; Roberts, M.F.; Pantry, I.R. Phytochemistry 1986, 25, 315.14. Chibber, S.S. Phytochemistry 1980, 19, 1857.15. Holmans, A.L.; Fuchs, J. J. Chromatogr . 1970, 51, 327.16. Rahalison, L.; Hamburger, M.; Hostettmann, K.; Monod, M.; Frenk, E. Phytochem. Anal.1991, 2, 199.

Two new isoflavanoids from <i>bolusanthus speciosus</i>

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