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Anti-inflammatory activity of compounds isolated from the aerial parts of Abrus precatorius (Fabaceae)
Phytomedicine, Vol. 8(1), pp. 24–27 © Urban & Fischer Verlag 2001 http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Anti-inflammatory activity of compounds isolated from the aerial parts of Abrus precatorius (Fabaceae) E. M. Anam Chemistry Department, University of Calabar, Calabar, CRS, Nigeria
Summary Two triterpenoid saponins 1 and 2 isolated from the aerial parts of Abrus precatorius and their acetates derivatives, 3 and 4 have been tested for anti-inflammatory activity using the croton oil ear model. All the compounds exhibited anti-inflammatory activity but the acetates showed greater inhibition than the parent compounds. Key words: Anti-inflammatory activity, croton oil ear model, triterpenoid saponins, oleanen-triol (tetrol) tetraglycosides
j Introduction Some triterpenes have been reported to have anti-inflammatory activity (Mukherjee et al., 1997; Recio et al., 1995; Takagi et al., 1980). Several anti-inflammatory substances have been known to inhibit the action of tumour promoters (Tokuda et al., 1991). Several bioassays have shown many saponins to be cytotoxic, anti-tumoral and chemopreventive. Thirteen triterpene-saponins from Calendura arvensis were assayed for antimutagenicity against the known promutagen benzopyrene (BaP) and a mutagenic urine concentrate from a smoker (SU) and all the saponins were found to be non-toxic and non-mutagenic at doses of 400 µg. Saponins isolated from about 50 plants showed antiinflammatory activity against several experimental models of inflammation in mice and rats (LacailleDubois and Wagner, 1996). Among these, the most frequently used are the carragenin-induced hand paw edema in mice or in rats and the mouse ear edema induced by several irritants (12-O-tetradecanoyl-13-acetate [TPA], arachidonic acid [AA]). At a dose of 1 mg/ear, in the (TPA)-mouse ear model, saikosaponins were shown to significantly inhibit swelling and were as potent as the reference drug indomethacin (Bermejko et al., 1998). Plants belonging to the genus Abrus are well-known drugs in African folk medicine. The 0944-7113/01/08/01-024 $ 15.00/0
isolation and structure elucidation of two triterpenoid saponins and their acetates, along with their anti-inflammatory activity, are reported here.
j Materials and Methods Croton oil
This was acquired from Sigma Company, St. Louis, Mo, U.S.A. Compounds under test were isolated from the crude extract of the aerial parts of Abrus precatorius. Analytical-grade solvents and reagents Diphenylamine, Ethanol, Dichloromethane, Methanol, Vanillin, Phosphoric acid and acetonitrile were obtained from Merck Co. Animals
Male albino rats, (28–30 g body wt each) were acquired from Switzerland and from Charles River (Italian imprint). Male albino rats were preferred to female in order to avert the complications of hormonal fluctuations connected with the female estrous cycle. All animals were maintained at constant room temperature and moisture and in an artificially illuminated environment for a period of one week prior to the experiment.
Anti-inflammatory activity of compounds of Abrus precatorius
1 2 3 4
R1
R2
H OH H OAc
H H Ac Ac
The inflammatory reaction was produced at the same time of day (11.00 a.m.–1.00 p.m.) in order to avoid changes in edematic connection caused by fluctuations in the amount of Cortico-steroid endogen, a hormone which affects histological activities. Anti-inflammatory agent
Two different concentrations were prepared. The first contained 600 µg each of compounds 1–4 whereas the second contained 300 µg each of compounds 1–4 [3 and 4 being the acetylated products of 1 and 2] in croton oil dissolved in acetone. Inflammatory agent
25
4.5 mg of 1 and 3.0 mg of 2 1H NMR spectra were recorded at 500 MHz. 13C NMR spectra were obtained at 125 MHz. The negative-ion mass spectra were recorded on an electron-attachment mass spectrograph (Finnigan MAT 8500). The matrix for the liquid SIMS was glycerine. CC was carried out over silica gel 60 (0.063–0.2 mm), TLC over silica sheets (0.3 mm, HF254) and prep. TLC over silica gel sheets (1 mm, PF254). The spots were detected using 'triterpene reagent' (1% solution of Vanillin in 50% H3PO4), which indicated the aglycone moiety, and using 'sugar reagent' (4% ethanolic aniline – 4% ethanolic diphenylamine-H3PO4, 5:5:1), which indicated the carbohydrate moiety.
Pure Croton oil was used as an inflammatory agent. Plant material
The plants were collected from Eniong Abatim and authenticated by the Botany Division of the Biological Sciences Department of the University of Calabar in February, 1998. A voucher specimen documenting the collection of this plant has been deposited in the University of Calabar herbarium. Extraction and Isolation
The dried and powdered aerial parts of the plant (1.5 kg) were extracted with MeOH. The extract was concentrated in vacuo. Compounds 1 and 2 could be detected in the crude extract by HPLC on reversed phase silica gel (RP-8) eluting with MeCN-H2O (1:5). The crude saponin fraction (1.50 g) was obtained by repeated CC on silica gel eluting with CHCl2-MeOH mixtures. This fraction was first chromatographed using prep. TLC with CHCl2-MeOH-H2O, 74:23:3 as eluent and then subjected to HPLC on reversed phase silica gel (TP-8) eluted with MeCN-H2O (1:5) to yield
j Results and Discussion The methanolic extract of the dried aerial parts of the plant was subjected to TLC And CC over silica gel followed by HPLC separation to yield compounds 1 and 2. Their structures were determined by physical methods and by comparison with authentic samples as 3-O-1{[β-D-glucopyranosyl-(1xx4)-β-D-glucopyranosyl-(1xx3)]-[β-D-glucopyranosyl-(1xx2)]-β-D-fucopyranosyl}-Olean-12-ene-3β, 23, 28-triol [1] and 3-O-{[β- D -glucopyranosyl-(1xx4)-β- D -glucopyranosyl-(1xx3)]-[β-D-glucopyranosyl-(1xx2)-β-D-fucopyranosyl}-Olean-12-ene-3β, 16β, 23, 28-tetrol [2] (Seifert et al., 1991; Hartleb and Seifert, 1994; and Shimizu et al., 1985). Acetylation of Compounds 1 and 2
Each compound (200 mg) in pyridine (3 ml) was treated with AC2O for 24 h. Ice flakes were added and de-
26
E. M. Anam
posited crystals were filtered, washed several times and tested for homogeneity. The appearance of single spots on TLC indicated their purity. Anti-inflammatory activity The rats were divided into eight groups of 14 each for the experiment while 24 rats formed the control group. 75 µg of anti-inflammatory agent (concentration 1) were applied to the inner surfaces of the right ears of rats in groups 1, 3, 5 and 7, whereas 75 µg of anti-inflammatory agent (concentration 2) were applied to the inner surfaces of the right ears of rats in groups 2, 4, 6 and 8. 75 µg of pure croton oil (Tubaro et al., 1985) are applied to the inner surfaces of the right ears of the 24 rats who formed the control group. The experiment was repeated twice for each group. All the animals were anesthetized with chloral hydrate (150 mg/kg body wt.) administered through interperitoneum for a few minutes before the application of both the anti-inflammatory and the inflammatory agents. All the animals were killed by separation of their cervical nerves after six h. induction of the inflammation. Finally tissue pieces of about 6 mm in diameter were cut off from both tested ears of all the rats under experiment and those of the 24 rats who formed the control group. The cut-off pieces were all weighed. Table 1 provides a comprehensive overview of the results of the experiment. By comparing the tissue portions of the ears of the rats treated with anti-inflammatory agents and those of the control group treated with inflammatory agent, a light inflammation was observed in the ears of the rats under test, whereas inflammation was markedly observed in the ears of the rats under control. Table 1. Anti-inflammatory effect of compounds 1, 2, 3 and 4 on the ear tissue of white rats. Compound
Concentration (µg)
Edema mg ± S.Ea
% Reduction
1
600 300 600 300 600 300 600 300
0.46c ± 0.2 0.93c ± 0.3 0.45c ± 0.1 0.90c ± 0.2 0.22c ± 0.3 0.44c ± 0.3 0.20c ± 0.3 0.32c ± 0.1
92.6 85.0 92.7 85.5 98.5 94.9 98.7 95.2
2 3 4
a means standard error % Reduction = b – c/b×100 where b = average inflammation weight of the ears of rats in the control group. c average inflammation weight of the ears of the rats under experiment.
In inflammation, small blood vessels become dilated and fluid and proteins leak into the interstitial spaces to produce the characteristic swelling (edema). Many polymorphonuclear leukocytes migrate into the inflamed area, engulfing dead tissue and bacteria. In this process, lysosomes of the leukocytes release phospholipase A (Metzler, 1977) which, in turn, hydrolyzes phospholipids and releases polyunsaturated fatty acids, the precursors of prostaglandins. Prostaglandins, notably PGE2, have been implicated in both the induction of inflammation and its relief. Prostaglandins could be produced at a local site as a result of injury, burns or noxious stimulus, such as stimulus produced by the application of croton oil to the ears of the white rats, and they act as mediators of inflammatory response. The ability of aspirin or other drugs to inhibit the inflammatory response is due to their inhibitory action on prostaglandin synthase (White et al., 1978). The conversion of arachidonic acid to PGG2 is blocked by aspirin which inhibits fatty acid cyclooxygenase by transacetylation from the aspirin molecule to the active site of the enzyme with inactivation of the enzyme (White et al., 1978). This may explain the excellent anti-inflammatory activity of compounds 3 and 4, the acetylated products of 1 and 2 respectively, as compared to that of compounds 1 and 2. Cortisol and other synthetic steroids prevent inflammatory response due, in part, to inhibition of arachidonic acid release from cellular phosphoglycerides, thus limiting formation of the inflammatory prostaglandins. They also inhibit the influx of polymorphonuclear leukocytes into the inflamed tissue and reduce the localized destruction of fibroblasts. Cortisol and Cortisone are polynuclear dihydroxyketones and compounds 1–4 have structural similarity to the steroids, the two sides being built up from the isoprene units, and their antiinflammatory action may be attributed to similarity in structure. Substituents such as the methyl group in cortisol have been known to demonstrate, anti-inflammatory activity ten times more powerful than that of Cortisol itself (White et al., 1978). It could, therefore, be inferred that methoxylated or acetylated products of compounds 1 and 2 may demonstrate more potency than the already known anti-inflammatory drugs. Synthesis of compounds 1 and 2 and their methoxylated and acetylated derivatives is being undertaken. Success in the synthesis will enable us to determine their bioactivity with life samples. Acknowledgement
The author is thankful to TWAS, Trieste, Italy for providing funds for this research and grateful to the Chemistry Department of the University of London for the spectral measurements of the compounds.
Anti-inflammatory activity of compounds of Abrus precatorius
j References Bermejo, B., Abad Martinez, M.I., Silvan, Sen A.M., San Gomez, A., Fernandenz Matellano, L., Sanchez Contreras, S., Diaz Lanza, a.M.: In vivo and in vitro antiinflammatory activity of saikosaponins. Life Sci. 63: 1147–1156, 1998. Hartleb, I., Seifert, K.: Songarosaponin D-A. Triterpenoid Saponin from Verbascum songaricum. Phytochemistry 35: 1009–1013, 1994. Lacaille-Dubois, M.A., Wagner, H.: A review of the Biological and pharmacological activities of saponins. Phytomedicine 2(4): 363–386, 1996. Metzler, D.E.: Biochemistry (The chemical reactions of living cells). Academic Press, New York: 707–719, 1977. Mukherjee, P.K., Saha, K., Das, J., Pal, M., Saha, B.P.: Studies on the Anti-Inflammatory Activity of Rhizomes of Nelumbo nucifera. Planta Med. 63: 367–369, 1997. Recio, M. del C., Giner, R.M., Manex, S., Rios, J.L.: Structural Requirements for the Anti-Inflammatory Activity of Natural Triterpenoids. Planta Med. 61: 182–185, 1995. Seifert, K., Preiss, A., Johne, S., Schmidt, J., Lien, N.T., Lavaud, C., Massiot, G.: Triterpene saponins from Verbascum songaricum. Phytochemistry 30: 3395–3398, 1991.
27
Shimizu, K., Amagaya, S., Ogihara, Y.: New Derivatives of Saiko saponins. Chem. Pharm. Bull. 33: 3349–3352, 1985. Takagi, K., Park, E.H., Kato, H.: Anti-inflammatory Activities of Hederagenin and Crude Saponin isolated from Sapindus mukorossi (GAERTN). Chem. Pharm. Bull. 28: 1183–1188, 1980. Tokuda, H., Konoshima, T., Kozuka, M., Kimura, R.: Antitumour Activities of Triterpene Saponins from Verbascum songaricum. Oncology 48: 77–80, 1991. Tubaro, A., Dri, P., DelBellow, G., Zilli, C., DellaLoggia, R.: The Croton oil ear test revisited. Agents and Actions 17: 3–5, 1985. White, A., Handler, P., Smith, E.L., Hill, R.L., Lehman, I.R.: Principles of Biochemistry. McGraw-Hill Inc., U.S.A.: 645: 1258–1263T, 1978.
j Address E.M. Anam, Chemistry Department, University of Calabar, P.M.B. 1115, Calabar, Cross River State, Nigeria
Phytomedicine
Anti-inflammatory activity of compounds isolated from the aerial parts of Abrus precatorius (Fabaceae) E. M. Anam Chemistry Department, University of Calabar, Calabar, CRS, Nigeria
Summary Two triterpenoid saponins 1 and 2 isolated from the aerial parts of Abrus precatorius and their acetates derivatives, 3 and 4 have been tested for anti-inflammatory activity using the croton oil ear model. All the compounds exhibited anti-inflammatory activity but the acetates showed greater inhibition than the parent compounds. Key words: Anti-inflammatory activity, croton oil ear model, triterpenoid saponins, oleanen-triol (tetrol) tetraglycosides
j Introduction Some triterpenes have been reported to have anti-inflammatory activity (Mukherjee et al., 1997; Recio et al., 1995; Takagi et al., 1980). Several anti-inflammatory substances have been known to inhibit the action of tumour promoters (Tokuda et al., 1991). Several bioassays have shown many saponins to be cytotoxic, anti-tumoral and chemopreventive. Thirteen triterpene-saponins from Calendura arvensis were assayed for antimutagenicity against the known promutagen benzopyrene (BaP) and a mutagenic urine concentrate from a smoker (SU) and all the saponins were found to be non-toxic and non-mutagenic at doses of 400 µg. Saponins isolated from about 50 plants showed antiinflammatory activity against several experimental models of inflammation in mice and rats (LacailleDubois and Wagner, 1996). Among these, the most frequently used are the carragenin-induced hand paw edema in mice or in rats and the mouse ear edema induced by several irritants (12-O-tetradecanoyl-13-acetate [TPA], arachidonic acid [AA]). At a dose of 1 mg/ear, in the (TPA)-mouse ear model, saikosaponins were shown to significantly inhibit swelling and were as potent as the reference drug indomethacin (Bermejko et al., 1998). Plants belonging to the genus Abrus are well-known drugs in African folk medicine. The 0944-7113/01/08/01-024 $ 15.00/0
isolation and structure elucidation of two triterpenoid saponins and their acetates, along with their anti-inflammatory activity, are reported here.
j Materials and Methods Croton oil
This was acquired from Sigma Company, St. Louis, Mo, U.S.A. Compounds under test were isolated from the crude extract of the aerial parts of Abrus precatorius. Analytical-grade solvents and reagents Diphenylamine, Ethanol, Dichloromethane, Methanol, Vanillin, Phosphoric acid and acetonitrile were obtained from Merck Co. Animals
Male albino rats, (28–30 g body wt each) were acquired from Switzerland and from Charles River (Italian imprint). Male albino rats were preferred to female in order to avert the complications of hormonal fluctuations connected with the female estrous cycle. All animals were maintained at constant room temperature and moisture and in an artificially illuminated environment for a period of one week prior to the experiment.
Anti-inflammatory activity of compounds of Abrus precatorius
1 2 3 4
R1
R2
H OH H OAc
H H Ac Ac
The inflammatory reaction was produced at the same time of day (11.00 a.m.–1.00 p.m.) in order to avoid changes in edematic connection caused by fluctuations in the amount of Cortico-steroid endogen, a hormone which affects histological activities. Anti-inflammatory agent
Two different concentrations were prepared. The first contained 600 µg each of compounds 1–4 whereas the second contained 300 µg each of compounds 1–4 [3 and 4 being the acetylated products of 1 and 2] in croton oil dissolved in acetone. Inflammatory agent
25
4.5 mg of 1 and 3.0 mg of 2 1H NMR spectra were recorded at 500 MHz. 13C NMR spectra were obtained at 125 MHz. The negative-ion mass spectra were recorded on an electron-attachment mass spectrograph (Finnigan MAT 8500). The matrix for the liquid SIMS was glycerine. CC was carried out over silica gel 60 (0.063–0.2 mm), TLC over silica sheets (0.3 mm, HF254) and prep. TLC over silica gel sheets (1 mm, PF254). The spots were detected using 'triterpene reagent' (1% solution of Vanillin in 50% H3PO4), which indicated the aglycone moiety, and using 'sugar reagent' (4% ethanolic aniline – 4% ethanolic diphenylamine-H3PO4, 5:5:1), which indicated the carbohydrate moiety.
Pure Croton oil was used as an inflammatory agent. Plant material
The plants were collected from Eniong Abatim and authenticated by the Botany Division of the Biological Sciences Department of the University of Calabar in February, 1998. A voucher specimen documenting the collection of this plant has been deposited in the University of Calabar herbarium. Extraction and Isolation
The dried and powdered aerial parts of the plant (1.5 kg) were extracted with MeOH. The extract was concentrated in vacuo. Compounds 1 and 2 could be detected in the crude extract by HPLC on reversed phase silica gel (RP-8) eluting with MeCN-H2O (1:5). The crude saponin fraction (1.50 g) was obtained by repeated CC on silica gel eluting with CHCl2-MeOH mixtures. This fraction was first chromatographed using prep. TLC with CHCl2-MeOH-H2O, 74:23:3 as eluent and then subjected to HPLC on reversed phase silica gel (TP-8) eluted with MeCN-H2O (1:5) to yield
j Results and Discussion The methanolic extract of the dried aerial parts of the plant was subjected to TLC And CC over silica gel followed by HPLC separation to yield compounds 1 and 2. Their structures were determined by physical methods and by comparison with authentic samples as 3-O-1{[β-D-glucopyranosyl-(1xx4)-β-D-glucopyranosyl-(1xx3)]-[β-D-glucopyranosyl-(1xx2)]-β-D-fucopyranosyl}-Olean-12-ene-3β, 23, 28-triol [1] and 3-O-{[β- D -glucopyranosyl-(1xx4)-β- D -glucopyranosyl-(1xx3)]-[β-D-glucopyranosyl-(1xx2)-β-D-fucopyranosyl}-Olean-12-ene-3β, 16β, 23, 28-tetrol [2] (Seifert et al., 1991; Hartleb and Seifert, 1994; and Shimizu et al., 1985). Acetylation of Compounds 1 and 2
Each compound (200 mg) in pyridine (3 ml) was treated with AC2O for 24 h. Ice flakes were added and de-
26
E. M. Anam
posited crystals were filtered, washed several times and tested for homogeneity. The appearance of single spots on TLC indicated their purity. Anti-inflammatory activity The rats were divided into eight groups of 14 each for the experiment while 24 rats formed the control group. 75 µg of anti-inflammatory agent (concentration 1) were applied to the inner surfaces of the right ears of rats in groups 1, 3, 5 and 7, whereas 75 µg of anti-inflammatory agent (concentration 2) were applied to the inner surfaces of the right ears of rats in groups 2, 4, 6 and 8. 75 µg of pure croton oil (Tubaro et al., 1985) are applied to the inner surfaces of the right ears of the 24 rats who formed the control group. The experiment was repeated twice for each group. All the animals were anesthetized with chloral hydrate (150 mg/kg body wt.) administered through interperitoneum for a few minutes before the application of both the anti-inflammatory and the inflammatory agents. All the animals were killed by separation of their cervical nerves after six h. induction of the inflammation. Finally tissue pieces of about 6 mm in diameter were cut off from both tested ears of all the rats under experiment and those of the 24 rats who formed the control group. The cut-off pieces were all weighed. Table 1 provides a comprehensive overview of the results of the experiment. By comparing the tissue portions of the ears of the rats treated with anti-inflammatory agents and those of the control group treated with inflammatory agent, a light inflammation was observed in the ears of the rats under test, whereas inflammation was markedly observed in the ears of the rats under control. Table 1. Anti-inflammatory effect of compounds 1, 2, 3 and 4 on the ear tissue of white rats. Compound
Concentration (µg)
Edema mg ± S.Ea
% Reduction
1
600 300 600 300 600 300 600 300
0.46c ± 0.2 0.93c ± 0.3 0.45c ± 0.1 0.90c ± 0.2 0.22c ± 0.3 0.44c ± 0.3 0.20c ± 0.3 0.32c ± 0.1
92.6 85.0 92.7 85.5 98.5 94.9 98.7 95.2
2 3 4
a means standard error % Reduction = b – c/b×100 where b = average inflammation weight of the ears of rats in the control group. c average inflammation weight of the ears of the rats under experiment.
In inflammation, small blood vessels become dilated and fluid and proteins leak into the interstitial spaces to produce the characteristic swelling (edema). Many polymorphonuclear leukocytes migrate into the inflamed area, engulfing dead tissue and bacteria. In this process, lysosomes of the leukocytes release phospholipase A (Metzler, 1977) which, in turn, hydrolyzes phospholipids and releases polyunsaturated fatty acids, the precursors of prostaglandins. Prostaglandins, notably PGE2, have been implicated in both the induction of inflammation and its relief. Prostaglandins could be produced at a local site as a result of injury, burns or noxious stimulus, such as stimulus produced by the application of croton oil to the ears of the white rats, and they act as mediators of inflammatory response. The ability of aspirin or other drugs to inhibit the inflammatory response is due to their inhibitory action on prostaglandin synthase (White et al., 1978). The conversion of arachidonic acid to PGG2 is blocked by aspirin which inhibits fatty acid cyclooxygenase by transacetylation from the aspirin molecule to the active site of the enzyme with inactivation of the enzyme (White et al., 1978). This may explain the excellent anti-inflammatory activity of compounds 3 and 4, the acetylated products of 1 and 2 respectively, as compared to that of compounds 1 and 2. Cortisol and other synthetic steroids prevent inflammatory response due, in part, to inhibition of arachidonic acid release from cellular phosphoglycerides, thus limiting formation of the inflammatory prostaglandins. They also inhibit the influx of polymorphonuclear leukocytes into the inflamed tissue and reduce the localized destruction of fibroblasts. Cortisol and Cortisone are polynuclear dihydroxyketones and compounds 1–4 have structural similarity to the steroids, the two sides being built up from the isoprene units, and their antiinflammatory action may be attributed to similarity in structure. Substituents such as the methyl group in cortisol have been known to demonstrate, anti-inflammatory activity ten times more powerful than that of Cortisol itself (White et al., 1978). It could, therefore, be inferred that methoxylated or acetylated products of compounds 1 and 2 may demonstrate more potency than the already known anti-inflammatory drugs. Synthesis of compounds 1 and 2 and their methoxylated and acetylated derivatives is being undertaken. Success in the synthesis will enable us to determine their bioactivity with life samples. Acknowledgement
The author is thankful to TWAS, Trieste, Italy for providing funds for this research and grateful to the Chemistry Department of the University of London for the spectral measurements of the compounds.
Anti-inflammatory activity of compounds of Abrus precatorius
j References Bermejo, B., Abad Martinez, M.I., Silvan, Sen A.M., San Gomez, A., Fernandenz Matellano, L., Sanchez Contreras, S., Diaz Lanza, a.M.: In vivo and in vitro antiinflammatory activity of saikosaponins. Life Sci. 63: 1147–1156, 1998. Hartleb, I., Seifert, K.: Songarosaponin D-A. Triterpenoid Saponin from Verbascum songaricum. Phytochemistry 35: 1009–1013, 1994. Lacaille-Dubois, M.A., Wagner, H.: A review of the Biological and pharmacological activities of saponins. Phytomedicine 2(4): 363–386, 1996. Metzler, D.E.: Biochemistry (The chemical reactions of living cells). Academic Press, New York: 707–719, 1977. Mukherjee, P.K., Saha, K., Das, J., Pal, M., Saha, B.P.: Studies on the Anti-Inflammatory Activity of Rhizomes of Nelumbo nucifera. Planta Med. 63: 367–369, 1997. Recio, M. del C., Giner, R.M., Manex, S., Rios, J.L.: Structural Requirements for the Anti-Inflammatory Activity of Natural Triterpenoids. Planta Med. 61: 182–185, 1995. Seifert, K., Preiss, A., Johne, S., Schmidt, J., Lien, N.T., Lavaud, C., Massiot, G.: Triterpene saponins from Verbascum songaricum. Phytochemistry 30: 3395–3398, 1991.
27
Shimizu, K., Amagaya, S., Ogihara, Y.: New Derivatives of Saiko saponins. Chem. Pharm. Bull. 33: 3349–3352, 1985. Takagi, K., Park, E.H., Kato, H.: Anti-inflammatory Activities of Hederagenin and Crude Saponin isolated from Sapindus mukorossi (GAERTN). Chem. Pharm. Bull. 28: 1183–1188, 1980. Tokuda, H., Konoshima, T., Kozuka, M., Kimura, R.: Antitumour Activities of Triterpene Saponins from Verbascum songaricum. Oncology 48: 77–80, 1991. Tubaro, A., Dri, P., DelBellow, G., Zilli, C., DellaLoggia, R.: The Croton oil ear test revisited. Agents and Actions 17: 3–5, 1985. White, A., Handler, P., Smith, E.L., Hill, R.L., Lehman, I.R.: Principles of Biochemistry. McGraw-Hill Inc., U.S.A.: 645: 1258–1263T, 1978.
j Address E.M. Anam, Chemistry Department, University of Calabar, P.M.B. 1115, Calabar, Cross River State, Nigeria