- Email: [email protected]
Bioassay-guided isolation of a diastereoisomer of kolavenol from Entada abyssinica active on Trypanosoma brucei rhodesiense
Journal of Ethnopharmacology 61 (1998) 179 – 183
Bioassay-guided isolation of a diastereoisomer of kolavenol from Entada abyssinica active on Trypanosoma brucei rhodesiense F. Freiburghaus a,b, A. Steck a, H. Pfander a,*, R. Brun b a
Uni6ersity of Berne, Department of Chemistry and Biochemistry, Freiestrasse 3, Berne CH-3012, Switzerland b Swiss Tropical Institute, P.O. Box, Basel CH-4002, Switzerland Received 21 January 1998; received in revised form 24 February 1998; accepted 3 March 1998
Abstract Bioassay-guided fractionation of the dichloromethane rootbark extract of Entada abyssinica (Leguminosae), a plant used by traditional healers in Uganda for the treatment of sleeping sickness, led to the isolation of a diastereoisomer of the clerodane type diterpene kolavenol. This is the first report on this compound. It showed a trypanocidal activity with an IC50 value of 2.5 mg/ml (8.6 mM) against Trypanosoma brucei rhodesiense, the causing agent of the acute form of human African trypanosomiasis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Entada abyssinica; Trypanosoma brucei rhodesiense; Diterpene; Sleeping sickness; Natural compound; Kolavenol
1. Introduction Human African trypanosomiasis (sleeping sickness) is caused by the flagellated parasites Trypanosoma brucei gambiense and T. b. rhodesiense, respectively, transmitted by tsetse flies (Glossina sp.) and occurring in 36 African countries. The estimated prevalence in 1993 was 250000 – 300000 (WHO, 1994) with a high mortality since the * Corresponding author. Fax: +41 31 6313425; e-mail: [email protected]
disease is fatal if untreated. The existing drugs for treatment of sleeping sickness are far from ideal: the duration of treatment lasts several weeks, administration needs to be parenteral and severe side-effects have to be endured (Kuzoe, 1993; Pepin and Milord, 1994). Thus, there is a great need for new drugs to combat this disease. With the aim of identifying lead compounds for the development of new chemotherapeutic agents, several plant species documented to be used in African traditional medicine for the treatment of sleeping sickness have been screened against T. b.
0378-8741/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-8741(98)00035-X
180
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183
rhodesiense bloodstream forms (Freiburghaus et al., 1996). The dichloromethane extract of the rootbark of Entada abyssinica showed promising trypanocidal activity and was selected for identification of the active principle. In this contribution we report on the bioassay-guided fractionation of the Entada abyssinica extract and on the first isolation of a new diastereoisomer of the previously isolated diterpene kolavenol (Misra et al., 1964; Anthonsen and McCrindle, 1969).
2. Materials and methods
2.1. Instrumentation and chromatography material Neutral alumina (507-C-1, CAMAG, Switzerland) and silica gel (60 – 200 m, Uetikon, Switzerland) were used for all column chromatography and solvents were distillated prior to use. High performance liquid chromatography (HPLC) was performed by using a preparative (250× 10 i.d. mm, 3-mm particle size) Nucleosil (Macherey Nagel, Switzerland) endcapped reversed phase (C18) column and HPLC purity solvents. Electron impact mass spectra were obtained with a MS9 (AE, UK) apparatus equipped with a ZAB console (VG, UK) and data system SS300 (Finnigan, Germany). NMR spectra (1H, COSY, TOCSY, T-ROESY, 13 C, DEPT-135, inverse HMQC and gradient-selective inverse HMBC experiment) were recorded on a BRUKER DRX-400 spectrometer with inverse-detection equipment (1H: 400.13 MHz, 13C: 100.62 MHz) and a BRUKER DRX-500 spectrometer with inverse-detection and z-gradient equipment (1H: 500.13 MHz, 13C: 125.77 MHz) at 23°C in CDCl3 (99.98% D). Chemical shifts (d) are related to the residual solvent signal.
2.2. Plant material Rootbark of Entada abyssinica Steud. ex A. Rich (Leguminosae) was collected in December 1994 in Tanzania (identification by Mr. L.B. Mwasumbi) and voucher specimens were de-
posited at the herbaria of the Universities of Dar Es Salaam (Tanzania) and Basel (Switzerland).
2.3. Trypanocidal drugs The following commercial drugs for the treatment of sleeping sickness were used to give reference values: Suramin (Germanin) from Bayer (Leverkusen, Germany) and melarsoprol (Arsobal) from Specia (Paris, France). Stock solutions were freshly prepared in sterile distilled water and diluted in complete culture medium.
2.4. Trypanosome stocks Trypanosoma brucei rhodesiense STIB 900 (Swiss Tropical Institute, Basel) was isolated in 1982 from a male patient in Ifakara, Tanzania. After several passages in Swiss ICR mice and preparation of multiple clones, one bloodstream form clone was finally adapted for axenic culture. Culture stabilates were prepared containing 10% (v/v) glycerol and stored in liquid N2. After thawing, the trypanosome population was kept in culture for up to 4 months.
2.5. Culture medium The culture medium for the axenic cultivation of the bloodstream forms consisted of minimum essential medium with Earle’s salts (MEM; Gibco-BRL072-1100 powder) supplemented with 1 g glucose/l, 25 mM HEPES (Calbiochem), 2.2 g NaHCO3/l and 10 ml MEM non-essential amino acids/l ( ×100). In addition, 10% (v/v) heat-inactivated (56°C, 30 min) horse serum was added, as well as 0.2 mM 2-mercaptoethanol, 16 mM thymidine, 1 mM sodium pyruvate and 0.1 mM hypoxanthine (Baltz et al., 1985).
2.6. Extraction and isolation For removing fat from the dried and powdered rootbark (900 g), the material was first extracted with petroleum ether followed by extraction with dichloromethane, three times for 8 h at room temperature. Tenfold quantities of dichloromethane in relation to plant material were
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183
used. The dichloromethane extracts were combined, filtered through a filter paper (Schleicher and Schuell, Germany) and the filtrates concentrated on a rotary evaporator (Bu¨chi, Switzerland) at 30° under reduced pressure. The residue (5 g) was applied to a neutral alumina column (grade III, 800 g, 60×4 cm) and eluted with mixtures of ligroin - t-butyl methyl ether (tBME) - ethyl acetate (EtOAc) of increasing polarity [ligroin - t-BME, 95:5, 2.5 l; 9:1, 1 l; 7:3, 1 l;), ligroin - t-BME - EtOAc (7:3:1, 0.5 l), tBME - EtOAc (4:3, 0.5 l) and EtOAc (0.3 l)] to give eight fractions (I – VIII). The trypanocidal activity tested by the procedure described in Section 2.7 was present only in fraction VI (990 mg) eluted with ligroin - t-BME - EtOAc (7:3:1). Further separation of fraction VI on a flash column (neutral alumina grade III, 2 bar, 100 g, 35× 1.5 cm) using mixtures of hexane - EtOAc (95:5, 2.5 l to 9:1, 0.6 l) yielded four fractions (VI.1.–VI.4.). Only fraction VI.3. (850 mg), eluted with hexane - EtOAc (95:5) showed trypanocidal activity. This active fraction VI.3. was applied to a silica gel column (35 g, 25× 1 cm) and eluted with mixtures of ligroin - t-BME EtOAc (9:1:1, 0.5 l; 8:2:1, 0.3 l) to give three fractions (VI.3.1.–VI.3.3.). The fraction VI.3.2. (410 mg) eluted with ligroin - t-BME - EtOAc (9:1:1) exhibited trypanocidal activity. Thus, fraction VI.3.2. was applied to a further chromatographic silica gel column (30 g, 25×1 cm) and eluted with toluene - acetonitrile (95:5, 0.4 l; 9:1, 0.4 l) to give two fractions (VI.3.2.1. and VI.3.2.2.). Both these fractions showed trypanocidal activity, but only fraction VI.3.2.1. (355 mg), eluted with toluene-acetonitrile (95:5), was further investigated since the yield of fraction VI.3.2.2. was too small (4 mg) for the performance of biological assays after further separation. The isolation of the diastereoisomer of kolavenol (1) from fraction VI.3.2.1. was achieved by HPLC using a preparative column with a mixture of acetonitrile - H2O (8:2) as eluent at a flow rate of 3 ml/min. For detection, a Waters photodiode array system 991 was used at a wavelength of 210 nm. From the six fractions collected by this method, fraction 5 (110
181
mg of the diastereoisomer of kolavenol) revealed trypanocidal activity.
2.7. Bioassay for plant extract fractions and commercial drugs All fractions obtained in the course of the separation procedure of the crude extract were tested for their in vitro activity against T. b. rhodesiense. Two methods were applied to assess growth inhibition of trypanosomes after 66 h exposure to an extract or drug. Firstly, the MIC (minimum inhibitory concentration) was determined microscopically with an inverted microscope (× 200) according to Brun and Lun (1994). The MIC was defined as the lowest concentration of plant extract fraction which completely inhibited growth of trypanosomes. Secondly, a modified version of the fluorescence assay described by Obexer et al. (1995), was performed. The fractions were suspended in 10% DMSO. After serial dilution with complete culture medium, the highest concentration of DMSO was 0.8%. Each fraction was tested at least twice in duplicate in 96-well microtiter plates (Costar, USA) in threefold serial dilutions ranging from 500 to 0.07 mg/ml. A parasite cell suspension was added to each well to (50 ml) to give a final density of 2× 102/well. Control wells without plant fractions were included, as well as with solvent. After incubation at 37°C for 66 hours in a humidified incubator containing 5% CO2, the MIC were determined. For all fractions which showed MIC values 5 56 mg/ml, 100 ml of a solution containing 4 mM BCECF-AM (2%,7%-bis (-carboxyethyl)-5(6)-carbofluorescein-penta-acetoxy-methylester) (Calbiochem, Switzerland) was added to each well. After incubation for another 45 min, fluorescence units were read by a fluorescence plate reader (Cytofluor 2300, Millipore, Bedford, MA) at 485 nm ex. and 530 nm em. wavelength. The IC50 (concentration of plant extract that inhibited growth of trypanosomes by 50%) was calculated according to Hills et al. (1986). For reference, tests with commercial drugs (suramin and melarsoprol) were performed.
182
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183
3. Results and discussion The antitrypanosomal activity of the isolated compound 1 and some fractions of the dichloromethane rootbark extract of Entada abyssinica is shown in Table 1. With an IC50 value of 2.5 mg/ml, compound 1 was moderately active whereas fraction VI.3.2.2. showed a promising trypanocidal activity with an IC50 value of 0.2 mg/ml. For comparison purposes, the clinically used antitrypanosomal agents suramin and melarsoprol were tested. As shown in Table 1, trypanocidal activity of 1 cannot compete with the one of the standard drugs. Moreover, the IC50 value of 1 was less favourable than the activity value (1.8 mg/ml) of the fraction from which it
Table 1 In vitro 50% inhibitory concentrations (IC50) against T. b. rhodesiense Compound
IC50 (mg/ml)
Dichloromethane extract Fraction VI.3.2.1. Fraction VI.3.2.2. Compound 1 Suraminb Melarsoprolb
MICa = 56 mg/ml 1.89 0.2 0.29 0.03 2.59 0.2 0.0190.002 0.00790.001
a Minimum inhibitory concentration (concentration of extract that completely inhibited growth of trypanosomes). b Commercially available drugs for treatment of sleeping sickness.
was isolated. This finding suggests that the antitrypanosomal effect may be due to a synergistic mode of action of several compounds present in the fraction. Terpenes are well known to be active against protozoan parasites (Phillipson and Wright, 1991; Phillipson et al., 1995). Nevertheless, it is unlikely that 1 itself will serve as a novel drug for the treatment of sleeping sickness considering its modest in vitro activity. Based on the interesting result obtained with fraction VI.3.2.2., it is planned to repeat the fractionation, starting off with larger quantities of crude extract. In the mass spectrum of 1, a signal was observed at m/z (rel. int.) 291 [M+ H]+ (35) which corresponds to a molecular formula of C20H34O. The constitution of 1 was established by NMR spectroscopy and was found to be identical with kolavenol. Kolavenol has previously been isolated from natural sources (Misra et al., 1964; Anthonsen and McCrindle, 1969). However, several observations indicated that 1 has a different relative stereostructure compared to kolavenol. An indication is the [a]25 D value (CHCl3; c= 0.0113; + 40° for 1, [a]30 = − 42° for kolavenol). In addition, D kolavenol was reported to be an oily liquid at room temperature, whereas 1 formed colourless crystals melting at 89.5°C. The final proof is provided by the results of a T-ROESY NMR experiment (Hwang and Shaka, 1992) which supported the fact that 1 is a diastereomer of kolavenol considering the stereochemical arrangement of 1. Reports on NMR and MS data of kolavenol have been poor and did not allow detailed comparison with the isolated compound 1. Therefore, the structure elucidation of 1 by means of highresolution NMR spectroscopy is discussed briefly and relevant 1H and 13C NMR data given in Table 2. The constitution was established by 1H, 13 C, DEPT-135, COSY, TOCSY (total correlation spectroscopy), inverse HMQC (heteronuclear multiple-quantum coherence) (Bax and Subramanian, 1986) and gradient selective inverse HMBC (heteronuclear multiple bond correlation) experiments. Well-resolved proton-proton coupling interactions H3C(17) l H-C(8), H3C(19) l H-C(3) allowed to define the positions of H3C(17) and H3C(19) at C(8) and C(4); the 13C resonance
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183 Table 2 H and 13C NMR data of compound 1
1
C atom
d 1H [ppm], (J [Hz])a
d
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.65 2.03 5.16 (1.0 ) — — 1.43 1.28 (14.3 ); 1.94 (14.3 ) 1.59 (7.1 ) — 1.41 1.95 1.13; 1.53 — 5.43 (7.0, 1.3 ) 4.15 (7.0, 7.0 ) 1.69 (1.3) 0.94 1.05 1.58 0.92 (0.8 )
17.86 26.89 120.25 144.62 38.34 30.22 25.61 35.15 37.47 45.19 32.75 37.86 141.33 122.70 59.50 16.59 14.86 20.61 18.07 20.42
13
C [ppm]
a
Only coupling constant values well-resolved and relevant for signal assignment are listed.
for C(5), identified to be that of a quaternary carbon in the DEPT-135 spectrum and significantly low-field shifted compared to C(10), indicated the position of H3C(18). A small coupling interaction between H3C(20) and one of the H2C(11) protons proved the geminal arrangement of this methyl group and the side chain, and their connection to C(9) was justified by a distinct dipolar interaction (ROE) between H3C(18) and H3C(20) visible in the T-ROESY spectrum (transverse rotating-frame Overhauser effect spectroscopy) (Hwang and Shaka, 1992). The ROE crosspeaks between H3C(18) and H3C(20) and between H3C(20) and H-C(9) proved these groups to be in a syn arrangement, while the lack of an ROE between H3C(18) and H-C(10) indicated their anti arrangement.
Acknowledgements We express our sincere thanks to L.B. Mwasumbi for the identification of the plant, to C. Schmid and Y. Grether-Bu¨hler for technical assis-
183
tance and to T. Schneeberger for mass spectrometry data. The financial support from the Rudolf-Geigy-Foundation is gratefully acknowledged.
References Anthonsen, T., McCrindle, R., 1969. The constitution of diterpenoids from Solidago elongata Nutt. Acta Chemica Scandinavica 23 (3), 1068 – 1070. Baltz, T., Baltz, D., Giroud, Ch., Crockett, J., 1985. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. eqiperdum, T. e6ansi, T. rhodesiense and T. gambiense. EMBO Journal 4, 1273 – 1277. Bax, A., Subramanian, J., 1986. Sensitivity-enhanced two-dimensional heteronuclear shift correlation NMR spectroscopy. Journal of Magnetic Resonance 67, 565 – 569. Brun, R., Lun, Z.-R., 1994. Drug sensitivity of Chinese Trypanosoma e6ansi and Trypanosoma equiperdum isolates. Veterinary Parasitology 52, 37 – 46. Freiburghaus, F., Ogwal, E.N., Nkunya, M.H.H., Kaminsky, R., Brun, R., 1996. In vitro antitrypanosomal activity of African plants used in traditional medicine in Uganda to treat sleeping sickness. Tropical Medicine and International Health 1 (6), 765 – 771. Hills, M., Hudson, C., Smith, P.G., 1986. Global monitoring of the resistance of malarial parasites to drugs: statistical treatment of micro-data. Working paper No. 2.8.5 for the informal consultation on the epidemiology of drug resistance of malarial parasites. World Health Organisation, Geneva. Hwang, T.L., Shaka, A.J., 1992. Cross relaxation without TOCSY: transverse rotating frame Overhauser effect spectroscopy. Journal of the American Chemical Society 114, 3157 – 3159. Kuzoe, F.A.S., 1993. Current situation of African trypanosomiasis. Acta Tropica 54, 153 – 162. Misra, R., Pandey, R.C., Sukh, D., 1964. The chemistry of the oleo resin from Hardwickia pinnata: a series of new diterpenoids. Tetrahedron Letters 49, 3751 – 3759. Obexer, W., Schmid, C., Brun, R., 1995. A novel in vitro screening assay for trypanocidal activity using the fluorescent dye BCECF-AM. Tropical Medicine and Parasitology 46, 45 – 48. Pepin, J., Milord, F., 1994. The treatment of human African trypanosomiasis. Advances in Parasitology 33, 1 – 47. Phillipson, J.D., Wright, C.W., 1991. Antiprotozoal agents from plant sources. Planta Medica 57, 53 – 59. Phillipson, J.D., Wright, C.W., Kirby, G.C., Warhust, D.C., 1995. Phytochemistry of some plants used in traditional medicine for the treatment of protozoal diseases. In: Hostettmann, K., Marston, A., Maillard, M., Hamburger, M. (Eds.), Phytochemistry of Plants used in Traditional Medicine. Proceedings of the Phytochemical Society of Europe. Clarendon Press, Oxford, pp. 95 – 135. WHO (1994) Control of tropical diseases: sleeping sickness. Brochure of World Health Organization, Geneva.
Bioassay-guided isolation of a diastereoisomer of kolavenol from Entada abyssinica active on Trypanosoma brucei rhodesiense F. Freiburghaus a,b, A. Steck a, H. Pfander a,*, R. Brun b a
Uni6ersity of Berne, Department of Chemistry and Biochemistry, Freiestrasse 3, Berne CH-3012, Switzerland b Swiss Tropical Institute, P.O. Box, Basel CH-4002, Switzerland Received 21 January 1998; received in revised form 24 February 1998; accepted 3 March 1998
Abstract Bioassay-guided fractionation of the dichloromethane rootbark extract of Entada abyssinica (Leguminosae), a plant used by traditional healers in Uganda for the treatment of sleeping sickness, led to the isolation of a diastereoisomer of the clerodane type diterpene kolavenol. This is the first report on this compound. It showed a trypanocidal activity with an IC50 value of 2.5 mg/ml (8.6 mM) against Trypanosoma brucei rhodesiense, the causing agent of the acute form of human African trypanosomiasis. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Entada abyssinica; Trypanosoma brucei rhodesiense; Diterpene; Sleeping sickness; Natural compound; Kolavenol
1. Introduction Human African trypanosomiasis (sleeping sickness) is caused by the flagellated parasites Trypanosoma brucei gambiense and T. b. rhodesiense, respectively, transmitted by tsetse flies (Glossina sp.) and occurring in 36 African countries. The estimated prevalence in 1993 was 250000 – 300000 (WHO, 1994) with a high mortality since the * Corresponding author. Fax: +41 31 6313425; e-mail: [email protected]
disease is fatal if untreated. The existing drugs for treatment of sleeping sickness are far from ideal: the duration of treatment lasts several weeks, administration needs to be parenteral and severe side-effects have to be endured (Kuzoe, 1993; Pepin and Milord, 1994). Thus, there is a great need for new drugs to combat this disease. With the aim of identifying lead compounds for the development of new chemotherapeutic agents, several plant species documented to be used in African traditional medicine for the treatment of sleeping sickness have been screened against T. b.
0378-8741/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-8741(98)00035-X
180
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183
rhodesiense bloodstream forms (Freiburghaus et al., 1996). The dichloromethane extract of the rootbark of Entada abyssinica showed promising trypanocidal activity and was selected for identification of the active principle. In this contribution we report on the bioassay-guided fractionation of the Entada abyssinica extract and on the first isolation of a new diastereoisomer of the previously isolated diterpene kolavenol (Misra et al., 1964; Anthonsen and McCrindle, 1969).
2. Materials and methods
2.1. Instrumentation and chromatography material Neutral alumina (507-C-1, CAMAG, Switzerland) and silica gel (60 – 200 m, Uetikon, Switzerland) were used for all column chromatography and solvents were distillated prior to use. High performance liquid chromatography (HPLC) was performed by using a preparative (250× 10 i.d. mm, 3-mm particle size) Nucleosil (Macherey Nagel, Switzerland) endcapped reversed phase (C18) column and HPLC purity solvents. Electron impact mass spectra were obtained with a MS9 (AE, UK) apparatus equipped with a ZAB console (VG, UK) and data system SS300 (Finnigan, Germany). NMR spectra (1H, COSY, TOCSY, T-ROESY, 13 C, DEPT-135, inverse HMQC and gradient-selective inverse HMBC experiment) were recorded on a BRUKER DRX-400 spectrometer with inverse-detection equipment (1H: 400.13 MHz, 13C: 100.62 MHz) and a BRUKER DRX-500 spectrometer with inverse-detection and z-gradient equipment (1H: 500.13 MHz, 13C: 125.77 MHz) at 23°C in CDCl3 (99.98% D). Chemical shifts (d) are related to the residual solvent signal.
2.2. Plant material Rootbark of Entada abyssinica Steud. ex A. Rich (Leguminosae) was collected in December 1994 in Tanzania (identification by Mr. L.B. Mwasumbi) and voucher specimens were de-
posited at the herbaria of the Universities of Dar Es Salaam (Tanzania) and Basel (Switzerland).
2.3. Trypanocidal drugs The following commercial drugs for the treatment of sleeping sickness were used to give reference values: Suramin (Germanin) from Bayer (Leverkusen, Germany) and melarsoprol (Arsobal) from Specia (Paris, France). Stock solutions were freshly prepared in sterile distilled water and diluted in complete culture medium.
2.4. Trypanosome stocks Trypanosoma brucei rhodesiense STIB 900 (Swiss Tropical Institute, Basel) was isolated in 1982 from a male patient in Ifakara, Tanzania. After several passages in Swiss ICR mice and preparation of multiple clones, one bloodstream form clone was finally adapted for axenic culture. Culture stabilates were prepared containing 10% (v/v) glycerol and stored in liquid N2. After thawing, the trypanosome population was kept in culture for up to 4 months.
2.5. Culture medium The culture medium for the axenic cultivation of the bloodstream forms consisted of minimum essential medium with Earle’s salts (MEM; Gibco-BRL072-1100 powder) supplemented with 1 g glucose/l, 25 mM HEPES (Calbiochem), 2.2 g NaHCO3/l and 10 ml MEM non-essential amino acids/l ( ×100). In addition, 10% (v/v) heat-inactivated (56°C, 30 min) horse serum was added, as well as 0.2 mM 2-mercaptoethanol, 16 mM thymidine, 1 mM sodium pyruvate and 0.1 mM hypoxanthine (Baltz et al., 1985).
2.6. Extraction and isolation For removing fat from the dried and powdered rootbark (900 g), the material was first extracted with petroleum ether followed by extraction with dichloromethane, three times for 8 h at room temperature. Tenfold quantities of dichloromethane in relation to plant material were
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183
used. The dichloromethane extracts were combined, filtered through a filter paper (Schleicher and Schuell, Germany) and the filtrates concentrated on a rotary evaporator (Bu¨chi, Switzerland) at 30° under reduced pressure. The residue (5 g) was applied to a neutral alumina column (grade III, 800 g, 60×4 cm) and eluted with mixtures of ligroin - t-butyl methyl ether (tBME) - ethyl acetate (EtOAc) of increasing polarity [ligroin - t-BME, 95:5, 2.5 l; 9:1, 1 l; 7:3, 1 l;), ligroin - t-BME - EtOAc (7:3:1, 0.5 l), tBME - EtOAc (4:3, 0.5 l) and EtOAc (0.3 l)] to give eight fractions (I – VIII). The trypanocidal activity tested by the procedure described in Section 2.7 was present only in fraction VI (990 mg) eluted with ligroin - t-BME - EtOAc (7:3:1). Further separation of fraction VI on a flash column (neutral alumina grade III, 2 bar, 100 g, 35× 1.5 cm) using mixtures of hexane - EtOAc (95:5, 2.5 l to 9:1, 0.6 l) yielded four fractions (VI.1.–VI.4.). Only fraction VI.3. (850 mg), eluted with hexane - EtOAc (95:5) showed trypanocidal activity. This active fraction VI.3. was applied to a silica gel column (35 g, 25× 1 cm) and eluted with mixtures of ligroin - t-BME EtOAc (9:1:1, 0.5 l; 8:2:1, 0.3 l) to give three fractions (VI.3.1.–VI.3.3.). The fraction VI.3.2. (410 mg) eluted with ligroin - t-BME - EtOAc (9:1:1) exhibited trypanocidal activity. Thus, fraction VI.3.2. was applied to a further chromatographic silica gel column (30 g, 25×1 cm) and eluted with toluene - acetonitrile (95:5, 0.4 l; 9:1, 0.4 l) to give two fractions (VI.3.2.1. and VI.3.2.2.). Both these fractions showed trypanocidal activity, but only fraction VI.3.2.1. (355 mg), eluted with toluene-acetonitrile (95:5), was further investigated since the yield of fraction VI.3.2.2. was too small (4 mg) for the performance of biological assays after further separation. The isolation of the diastereoisomer of kolavenol (1) from fraction VI.3.2.1. was achieved by HPLC using a preparative column with a mixture of acetonitrile - H2O (8:2) as eluent at a flow rate of 3 ml/min. For detection, a Waters photodiode array system 991 was used at a wavelength of 210 nm. From the six fractions collected by this method, fraction 5 (110
181
mg of the diastereoisomer of kolavenol) revealed trypanocidal activity.
2.7. Bioassay for plant extract fractions and commercial drugs All fractions obtained in the course of the separation procedure of the crude extract were tested for their in vitro activity against T. b. rhodesiense. Two methods were applied to assess growth inhibition of trypanosomes after 66 h exposure to an extract or drug. Firstly, the MIC (minimum inhibitory concentration) was determined microscopically with an inverted microscope (× 200) according to Brun and Lun (1994). The MIC was defined as the lowest concentration of plant extract fraction which completely inhibited growth of trypanosomes. Secondly, a modified version of the fluorescence assay described by Obexer et al. (1995), was performed. The fractions were suspended in 10% DMSO. After serial dilution with complete culture medium, the highest concentration of DMSO was 0.8%. Each fraction was tested at least twice in duplicate in 96-well microtiter plates (Costar, USA) in threefold serial dilutions ranging from 500 to 0.07 mg/ml. A parasite cell suspension was added to each well to (50 ml) to give a final density of 2× 102/well. Control wells without plant fractions were included, as well as with solvent. After incubation at 37°C for 66 hours in a humidified incubator containing 5% CO2, the MIC were determined. For all fractions which showed MIC values 5 56 mg/ml, 100 ml of a solution containing 4 mM BCECF-AM (2%,7%-bis (-carboxyethyl)-5(6)-carbofluorescein-penta-acetoxy-methylester) (Calbiochem, Switzerland) was added to each well. After incubation for another 45 min, fluorescence units were read by a fluorescence plate reader (Cytofluor 2300, Millipore, Bedford, MA) at 485 nm ex. and 530 nm em. wavelength. The IC50 (concentration of plant extract that inhibited growth of trypanosomes by 50%) was calculated according to Hills et al. (1986). For reference, tests with commercial drugs (suramin and melarsoprol) were performed.
182
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183
3. Results and discussion The antitrypanosomal activity of the isolated compound 1 and some fractions of the dichloromethane rootbark extract of Entada abyssinica is shown in Table 1. With an IC50 value of 2.5 mg/ml, compound 1 was moderately active whereas fraction VI.3.2.2. showed a promising trypanocidal activity with an IC50 value of 0.2 mg/ml. For comparison purposes, the clinically used antitrypanosomal agents suramin and melarsoprol were tested. As shown in Table 1, trypanocidal activity of 1 cannot compete with the one of the standard drugs. Moreover, the IC50 value of 1 was less favourable than the activity value (1.8 mg/ml) of the fraction from which it
Table 1 In vitro 50% inhibitory concentrations (IC50) against T. b. rhodesiense Compound
IC50 (mg/ml)
Dichloromethane extract Fraction VI.3.2.1. Fraction VI.3.2.2. Compound 1 Suraminb Melarsoprolb
MICa = 56 mg/ml 1.89 0.2 0.29 0.03 2.59 0.2 0.0190.002 0.00790.001
a Minimum inhibitory concentration (concentration of extract that completely inhibited growth of trypanosomes). b Commercially available drugs for treatment of sleeping sickness.
was isolated. This finding suggests that the antitrypanosomal effect may be due to a synergistic mode of action of several compounds present in the fraction. Terpenes are well known to be active against protozoan parasites (Phillipson and Wright, 1991; Phillipson et al., 1995). Nevertheless, it is unlikely that 1 itself will serve as a novel drug for the treatment of sleeping sickness considering its modest in vitro activity. Based on the interesting result obtained with fraction VI.3.2.2., it is planned to repeat the fractionation, starting off with larger quantities of crude extract. In the mass spectrum of 1, a signal was observed at m/z (rel. int.) 291 [M+ H]+ (35) which corresponds to a molecular formula of C20H34O. The constitution of 1 was established by NMR spectroscopy and was found to be identical with kolavenol. Kolavenol has previously been isolated from natural sources (Misra et al., 1964; Anthonsen and McCrindle, 1969). However, several observations indicated that 1 has a different relative stereostructure compared to kolavenol. An indication is the [a]25 D value (CHCl3; c= 0.0113; + 40° for 1, [a]30 = − 42° for kolavenol). In addition, D kolavenol was reported to be an oily liquid at room temperature, whereas 1 formed colourless crystals melting at 89.5°C. The final proof is provided by the results of a T-ROESY NMR experiment (Hwang and Shaka, 1992) which supported the fact that 1 is a diastereomer of kolavenol considering the stereochemical arrangement of 1. Reports on NMR and MS data of kolavenol have been poor and did not allow detailed comparison with the isolated compound 1. Therefore, the structure elucidation of 1 by means of highresolution NMR spectroscopy is discussed briefly and relevant 1H and 13C NMR data given in Table 2. The constitution was established by 1H, 13 C, DEPT-135, COSY, TOCSY (total correlation spectroscopy), inverse HMQC (heteronuclear multiple-quantum coherence) (Bax and Subramanian, 1986) and gradient selective inverse HMBC (heteronuclear multiple bond correlation) experiments. Well-resolved proton-proton coupling interactions H3C(17) l H-C(8), H3C(19) l H-C(3) allowed to define the positions of H3C(17) and H3C(19) at C(8) and C(4); the 13C resonance
F. Freiburghaus et al. / Journal of Ethnopharmacology 61 (1998) 179–183 Table 2 H and 13C NMR data of compound 1
1
C atom
d 1H [ppm], (J [Hz])a
d
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.65 2.03 5.16 (1.0 ) — — 1.43 1.28 (14.3 ); 1.94 (14.3 ) 1.59 (7.1 ) — 1.41 1.95 1.13; 1.53 — 5.43 (7.0, 1.3 ) 4.15 (7.0, 7.0 ) 1.69 (1.3) 0.94 1.05 1.58 0.92 (0.8 )
17.86 26.89 120.25 144.62 38.34 30.22 25.61 35.15 37.47 45.19 32.75 37.86 141.33 122.70 59.50 16.59 14.86 20.61 18.07 20.42
13
C [ppm]
a
Only coupling constant values well-resolved and relevant for signal assignment are listed.
for C(5), identified to be that of a quaternary carbon in the DEPT-135 spectrum and significantly low-field shifted compared to C(10), indicated the position of H3C(18). A small coupling interaction between H3C(20) and one of the H2C(11) protons proved the geminal arrangement of this methyl group and the side chain, and their connection to C(9) was justified by a distinct dipolar interaction (ROE) between H3C(18) and H3C(20) visible in the T-ROESY spectrum (transverse rotating-frame Overhauser effect spectroscopy) (Hwang and Shaka, 1992). The ROE crosspeaks between H3C(18) and H3C(20) and between H3C(20) and H-C(9) proved these groups to be in a syn arrangement, while the lack of an ROE between H3C(18) and H-C(10) indicated their anti arrangement.
Acknowledgements We express our sincere thanks to L.B. Mwasumbi for the identification of the plant, to C. Schmid and Y. Grether-Bu¨hler for technical assis-
183
tance and to T. Schneeberger for mass spectrometry data. The financial support from the Rudolf-Geigy-Foundation is gratefully acknowledged.
References Anthonsen, T., McCrindle, R., 1969. The constitution of diterpenoids from Solidago elongata Nutt. Acta Chemica Scandinavica 23 (3), 1068 – 1070. Baltz, T., Baltz, D., Giroud, Ch., Crockett, J., 1985. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. eqiperdum, T. e6ansi, T. rhodesiense and T. gambiense. EMBO Journal 4, 1273 – 1277. Bax, A., Subramanian, J., 1986. Sensitivity-enhanced two-dimensional heteronuclear shift correlation NMR spectroscopy. Journal of Magnetic Resonance 67, 565 – 569. Brun, R., Lun, Z.-R., 1994. Drug sensitivity of Chinese Trypanosoma e6ansi and Trypanosoma equiperdum isolates. Veterinary Parasitology 52, 37 – 46. Freiburghaus, F., Ogwal, E.N., Nkunya, M.H.H., Kaminsky, R., Brun, R., 1996. In vitro antitrypanosomal activity of African plants used in traditional medicine in Uganda to treat sleeping sickness. Tropical Medicine and International Health 1 (6), 765 – 771. Hills, M., Hudson, C., Smith, P.G., 1986. Global monitoring of the resistance of malarial parasites to drugs: statistical treatment of micro-data. Working paper No. 2.8.5 for the informal consultation on the epidemiology of drug resistance of malarial parasites. World Health Organisation, Geneva. Hwang, T.L., Shaka, A.J., 1992. Cross relaxation without TOCSY: transverse rotating frame Overhauser effect spectroscopy. Journal of the American Chemical Society 114, 3157 – 3159. Kuzoe, F.A.S., 1993. Current situation of African trypanosomiasis. Acta Tropica 54, 153 – 162. Misra, R., Pandey, R.C., Sukh, D., 1964. The chemistry of the oleo resin from Hardwickia pinnata: a series of new diterpenoids. Tetrahedron Letters 49, 3751 – 3759. Obexer, W., Schmid, C., Brun, R., 1995. A novel in vitro screening assay for trypanocidal activity using the fluorescent dye BCECF-AM. Tropical Medicine and Parasitology 46, 45 – 48. Pepin, J., Milord, F., 1994. The treatment of human African trypanosomiasis. Advances in Parasitology 33, 1 – 47. Phillipson, J.D., Wright, C.W., 1991. Antiprotozoal agents from plant sources. Planta Medica 57, 53 – 59. Phillipson, J.D., Wright, C.W., Kirby, G.C., Warhust, D.C., 1995. Phytochemistry of some plants used in traditional medicine for the treatment of protozoal diseases. In: Hostettmann, K., Marston, A., Maillard, M., Hamburger, M. (Eds.), Phytochemistry of Plants used in Traditional Medicine. Proceedings of the Phytochemical Society of Europe. Clarendon Press, Oxford, pp. 95 – 135. WHO (1994) Control of tropical diseases: sleeping sickness. Brochure of World Health Organization, Geneva.