- Email: [email protected]
Hepatoprotective and antibacterial effects of extracts from Trichiliaemetica Vahl. (Meliaceae)
Journal of Ethnopharmacology 96 (2005) 227–232
Hepatoprotective and antibacterial effects of extracts from Trichilia emetica Vahl. (Meliaceae) M.P. German`oa,∗ , V. D’Angeloa , R. Sanogob , S. Cataniac , R. Almaa , R. De Pasqualea , G. Bisignanoa a c
Pharmaco-Biological Department, School of Pharmacy, University of Messina, Vill. SS. Annunziata, 98168 Messina, Italy b Traditional Medicine Department—B.P. 1746, Bamako, Mali Interdipartimental Center for Experimental Toxicology (CITSAL), School of Medicine, University of Messina, Messina, Italy Received 17 February 2004; received in revised form 15 July 2004; accepted 6 September 2004 Available online 22 October 2004
Abstract Trichilia emetica Vahl. (Meliaceae) is a tree widely distributed in Tropical Africa. It has been used in Mali folk medicine for the treatment of various illnesses. The aim of this work was to study the hepatoprotective and antibacterial effects of a crude aqueous extract from Trichilia emetica root. An ethyl ether fraction from the aqueous extract was also prepared and studied. We have examined the hepatoprotective activity of the extracts on CCl4 -induced damage in rat hepatocytes, their toxicity using the brine shrimp bioassay and their antibacterial activity against clinical isolated bacterial strains, which are commonly responsible for respiratory infections. A preliminary phytochemical analysis showed a high polyphenolic content in the aqueous extract and the presence of limonoids in the ethyl ether fraction. These latter compounds may be considered responsible for the good activity against the bacterial strains tested. Trichilia emetica extracts exerted also a significant (P < 0.05) hepatoprotective effect at a dose of 1000 g/ml both on plasma membrane and mitochondrial function as compared to silymarin used as a positive control. These activities may be a result of the presence of either polyphenols or limonoids. Finally, both the aqueous extract and its ethyl ether fraction did not show toxicity (LC50 > 1000 g/ml) in the brine shrimp bioassay. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Trichilia emetica; Biological activity; CCl4 -induced liver damage in rat hepatocytes; Brine shrimp bioassay; Polyphenols; Limonoids; Antibacterial activity
1. Introduction Medicinal plants have always played an important role in African society. Traditions of plant-collecting and plantbased medications have been handed down from generation to generation (von Maydell, 1996). McGaw et al. (1997) reported that about 80% of the African population consults traditional healers and uses folk medicine for the treatment of various diseases. A scientific evaluation of the plants used by healers is essential before tra∗ Corresponding author. Tel.: +39 090 6766549/3533112; fax: +39 090 3533112. E-mail address: [email protected] (M.P. German`o).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.09.011
ditional medicine can be incorporated into the officinal health care system of Africa. Trichilia emetica Vahl. syn. Trichilia roka Chiov. (Meliaceae) (Oliver-Bever, 1986; Iwu, 1993), commonly known with the name of “Sulafinzan” in the language of Bambara, is a tree widely distributed in tropical areas and in WestAfrica lands. The plant is employed for the treatment of various disorders in folk medicine of Mali: it is used in hepatic diseases, as a purgative, antiepileptic, antipyretic and antimalarial agent (Iwu, 1993). A decoction of the root is taken as a remedy for colds and bronchial inflammation (Kokwaro, 1976; Malgras, 1992). A small glass of root decoction is generally consumed daily for 3 days against intestinal worms and to treat jaundice (Ak´e Assi and Guinko, 1991). In Senegal,
228
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
Trichilia emetica is also used in skin diseases (Oliver-Bever, 1986). Previous phytochemical studies have led to the isolation of several types of limonoids from an ethyl ether extract of Trichilia emetica root (Nakatani et al., 1981, 1985); these compounds are known for their biological activity (Champagne et al., 1992). McGaw et al. (1997) reported that an ethanolic extract of Trichilia emetica leaves has an inhibitory activity against cyclooxygenase; moreover, Gunatilaka et al. (1998) showed selective toxicity of some constituents of Trichilia emetica stem bark against DNA-repair deficient yeast. Most of the plants belonging to the family of Meliaceae, including Trichilia emetica, showed high antiplasmodial (El-Tahir et al., 1999; Traor´e-K´eita et al., 2000) and antischistosomiasis activities (Sparg et al., 2000). Diallo et al. (2003) reported the complement fixation ability of polysaccharides isolated from an aqueous extract of Trichilia emetica leaves. The effect of a decoction of the root on CCl4 -induced acute liver damage in the rat was evaluated by German`o et al. (2001). The antipyretic activity of a dried aqueous extract of Trichilia emetica root was compared with indomethacin treatment (Sanogo et al., 2001). In the present study, the hepatoprotective and antibacterial effects of an aqueous extract and its ethyl ether fraction from the root of Trichilia emetica were evaluated. We have examined the toxicity using the brine shrimp bioassay, the hepatoprotective activity on CCl4 -induced liver damage in vitro using rat hepatocytes and the antibacterial activity against clinical isolated bacterial strains which are commonly responsible for respiratory infections.
2. Materials and methods 2.1. Plant material Plant material was collected in the belt of Bamako (Mali) and identified by comparison with a voucher specimen deposited at the Herbarium of Traditional Medicine Division of Bamako. 2.2. Plant extracts Trichilia emetica root was air dried and ground using a laboratory mill. A crude extract was prepared by decoction of 100 g in 1000 ml of water for 30 min. The obtained extract was then filtered and concentrated to dryness under vacuum (yield 13.64%). An aliquot part of this extract (20 g) was dissolved in water (200 ml) and then extracted with ethyl ether (100 ml × 2). The organic fractions were recovered, combined, filtered and dried under reduced pressure (yield 0.15 g). The presence of limonoids was tested dissolving some milligrams of the ethyl ether fraction and then adding the same volume of hexane to give a precipitate (Nakatani et al., 1994).
2.3. Chemicals Bovine serum albumin Fr. V, foetal bovine serum, collagenase type IA (449 IU/mg), dexamethasone, (N[2-hydroxyethyl]piperazine-N -[2-ethanesulphonic acid]) (HEPES), insulin, 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyl tetrazolium bromide (MTT), RPMI-1640 medium, and sodium bicarbonate were Sigma; CCl4 > 99.5% purity, dimethylsulphoxide (DMSO), ethylene glycol-O-O -bis (2-aminoethyl)-N,N,N N -tetracetic acid (EGTA) were from Fluka; Hank’s Ca2+ and Mg2+ -free balanced salt solution was from Euro-Clone, isopropanol from Lab-Scan, Triton X-100 from Bio-Rad, and Trypan Blue from Flow Laboratories. 2.4. Determination of total phenolic compounds The level of total phenolic compounds was determined in the aqueous extract. An aliquot of extract (0.1 ml of 10 mg/ml) was mixed with 0.2 ml of Folin-Ciocalteau reagent (Analyticals, Carlo Erba), 2 ml of H2 O and 1 ml of 15% Na2 CO3 . The absorbance was measured at 765 nm with a Shimadzu UV-1601 spectrophotometer after incubation for 2 h at room temperature in the dark. Quantification was based on a standard curve using gallic acid. Total phenols (mean ± S.D.) were expressed as milligrams of gallic acid equivalents per gram of aqueous extract. 2.5. The brine shrimp lethality biossay The brine shrimp lethality biossay was done according to the method of Meyer et al. (1982). Brine shrimp eggs (Artemia salina Leach) were hatched in artificial sea water (33 g/l of sea salt, Sigma Chemicals) and then they were incubated at room temperature for 48 h. With the help of a light source, the larvae (nauplii) were attracted to one side of the vessel and easily collected for the assay. The aqueous extract and its ethyl ether fraction were dissolved in dimethyl sulphoxide (DMSO) at a maximum concentration not exceeding 0.05% and then diluted with sea water to be tested at final concentrations of 10, 100 and 1000 g/ml. Each dose was tested in triplicate. Ten nauplii were used to test each dose of extracts. Nauplii were counted under a magnifying glass after 24 h of incubation. The number of dead nauplii were recorded and it was used for calculate LC50 concentration by the Finney Probit analysis program. LC50 values greater than 1000 ppm were considered inactive. 2.6. CCl4 -induced hepatocyte injury in vitro Hepatocytes were isolated from rats (Male Wistar rats weighing 150–200 g, Morini, Italy) by the procedure previously described by German`o et al. (1999). After a time preculture (30 min), the cells were exposed to fresh culture medium (RPMI-1640 medium supplemented with 10% inactivated foetal bovine serum, penicillin 100 IU/ml, strepto-
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
mycin 100 g/ml, dexamethasone 10 mM and insulin 1 mM) containing 10 mM CCl4 with or without test samples in DMSO (0.01 ml) (Xiong et al., 1999). The final concentration of DMSO did not exceed 0.05%. For the test, the aqueous extract was added to the vials to give a final concentration of 10, 100, or 1000 g/ml. The ethyl ether fraction was added to give a final concentration corresponding to 10, 100, or 1000 g/ml of aqueous extract. Silymarin was tested as positive control at doses of 10, 100 and 1000 g/ml. Each dose was tested in triplicate. Control cultures contained the same amount of DMSO. 2.7. Assessment of hepatotoxic activity in vitro AST and LDH activities in the medium were measured 60 min after CCl4 challenge. After incubation, culture medium from each vials was collected and centrifuged at 50 × g at 4 ◦ C for 2 min. The supernatant was used to measure AST and LDH levels using AST/UV-Autom and LDH/UV Liquid test kits (Sentinel) with a bioanalyser (ARCO PC). It is generally accepted that experiments are valid only if the background activity of control cells is ≤25% of total available intercellular activity. So, for control cells, the total intracellular enzyme content was determined after treatment of cells with Triton X-100 detergent to induce 100% lysis (Tyson et al., 1983). 2.8. MTT reduction assay The cell survival rate was assessed by the 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay according to Mosmann (1983) with slight modifications. Briefly, MTT (0.3 mg/ml) was dissolved in phosphate buffered saline (PBS, pH 7.4) and a MTT solution (200 l/ml) was added to each vial and incubated for 2 h at 37 ◦ C with 5% CO2 . After incubation, the cell suspension with formazan crystals was transferred into Eppendorf tubes and centrifuged at 5000 × g for 3 min. Supernatant was discarded and 1 ml of a lysis-solubilization solution (10% Triton X-100 in isopropanol, pH 2) was added to each tube. The pellet was dissolved by sonication and cell debris was collected by brief centrifugation. The absorbance of the purple solution was measured at 565 nm with a spectrophotometer (Shimadzu UV-1601). The cell survival rate was expressed as percentage formazan production by treated samples compared with control samples.
229
to use; it was then dissolved in DMSO and finally diluted in standard buffer at concentrations ranging from 0.015 to 500 g/ml. 2.10. Bacterial strains The bacterial strains used in this study were clinical isolated samples (Pediatric Section, School of Medicine, University of Messina): Haemophilus influenzae (12); Staphylococcus aureus (8); Streptococcus pneumoniae (4); Streptococcus pyogenes (8); and Moraxella catarrhalis (8). They were isolated from specimens obtained from oropharyngeal, nasopharyngeal or ear swabs of patients with acute infections (laryngitis, sinusitis, otitis) and identified by conventional procedures (confirmed by API system, Bio Merieux). Each bacterial strain cells were grown in Triptic Soy broth (Oxoid), 3 ml, for 18–24 h at 37 ◦ C. Before use the overnights cultures were diluted 1:10 with nutrient broth. To ensure that density of diluted cultures were all within the range 107 –108 colony forming units ml−1 , serial plate counts were performed for each culture. 2.11. Antibacterial assay The disc-diffusion method (Bauer et al., 1966) was used to test the activity. The minimum inhibitory concentration (MIC) of the extracts was measured using an agar dilution method (Barry, 1991). Each extract was incorporated into a liquefied M¨ueller–Hinton agar medium, which was then mixed, poured into Petri plates, and allowed to solidify. A series of Petri plates was prepared to obtain the desired concentrations within 0.95 and 500 g/ml. A standard 1 × 105 to 5 × 105 colony forming units ml−1 inoculum was tested for each bacteria strain, using a Steer’s replicator. The plates were incubated at 37 ◦ C for 18–24 h. The MIC was defined as the lowest concentration of the plant extracts that completely inhibited the visible growth. All antibacterial assays were performed in triplicate. 2.12. Statistical analysis Values are given as means ± S.D. Statistical analysis was done by Student’s t-test.
3. Results
2.9. Antibacterial agents
3.1. Total phenolic compounds
The dried aqueous extract and ethyl ether fraction were initially dissolved in DMSO (10 mg/ml) and subsequently diluted in sterile phosphate buffer (pH 7.2) for antibacterial test. The same amount of DMSO was added in control test. Ampicillin, used as positive control, was obtained from the Sigma Chemical Co. It was kept at 4 ◦ C in dessicator prior
The level of total phenolic compounds of Trichilia emetica aqueous extract as determined by the Folin-Ciocalteau reagent was 169.152 ± 2.34 mg expressed as gallic acid equivalents per gram of extract. The presence of limonoids in the ethyl ether fraction was confirmed using a simple screening described by Nakatani et al. (1994).
230
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
Table 1 Antibacterial activity of Trichilia emetica extracts: MIC g/ml Strains
Ethyl ether fraction
Aqueous extract
Ampicillin
15.60–31.25 15.60–62.50 7.80–125.00 7.80–31.25 125.00–125.00
>500 >500 >500 >500 >500
15.60–125.00 0.12–1.95 0.12–0.97 0.97–3.90 3.90–250.00
(8)a
Staphylococcus aureus Streptococcus pyogenes (8)a Streptococcus pneumoniae (4)a Moraxella catarrhalis (8)a Haemophilus influenzae (12)a a
Values in parentheses indicate number of strains tested.
3.2. Toxicity testing against the brine shrimp On the brine shrimp bio-assay Trichilia emetica aqueous extract and its ethyl ether fraction did not show cytotoxicity (LC50 > 1000 g/ml). 3.3. Effect on CCl4 -induced liver injury in vitro The hepatoprotective effects of Trichilia emetica extracts on CCl4 -induced liver injury are shown in Figs. 1 and 2. It is evident that the aqueous extract at the concentration of 1000 g/ml significantly prevented both AST (P < 0.05) and LDH (P < 0.01) release from the cells as compared to CCl4 group. The ethyl ether fraction did not produce any protective effects on the release of cytosolic enzymes. Silymarin was able to reduce AST release at concentration of 1000 g/ml (P < 0.05), while on LDH release exhibited protective effect both at 100 g/ml (P < 0.05) and 1000 g/ml
Fig. 2. Effect of Trichilia emetica extracts and reference drug silymarin on the survival rate of rat hepatocytes after CCl4 exposure. Bars are mean ± S.D.; ◦ P < 0.05 significantly different from control; * P < 0.05 significantly different from CCl4 -treated group.
(P < 0.01) (Fig. 1A and B). In the MTT assay, a significant reduction on cell viability was observed in CCl4 -treated groups as shown in Fig. 2. The aqueous extract (100 and 1000 g/ml) and the ethyl ether fraction (1000 g/ml) were able to elevate cell viability at a level of control group. 3.4. Antibacterial screening Antibacterial activity of Trichilia emetica extracts is reported in Table 1. The aqueous extract had a low antibacterial activity (MIC > 500 g/ml). The ethyl ether fraction showed a good activity against Streptococcus pneumoniae and Moraxella catarrhalis with range-MIC between 7.80 and 125.00 g/ml, Staphylococcus aureus and Streptococcus pyogenes with range-MIC between 15.60 and 62.50, while it resulted less active against Haemophilus influenzae (MIC 125 g/ml).
4. Discussion
Fig. 1. Effect of Trichilia emetica extracts and reference drug silymarin on AST (A) and LDH (B) activities of rat hepatocytes after CCl4 exposure. Bars are mean ± S.D.; ◦ P < 0.05 significantly different from control; * P < 0.05 and ** P < 0.01 significantly different from CCl -treated group. 4
The present investigation reports the hepatoprotective and antibacterial effects of Trichilia emetica giving a support for its traditional usage in folklore medicine. Previous phytochemical studies have reported the presence of tannins in the root and stem bark (Burkill, 1997). Our investigation on the aqueous extract confirms a high polyphenolic content which was evaluated as 169.152 mg/g of extract.
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
In addition, several types of limonoids, named “trichilins”, have been isolated from the root bark of Trichilia emetica (Nakatani et al., 1981, 1985). These latter compounds have attracted much attention for their wide range of biological activities including insect antifeedant, growth regulating properties, antifungal, bactericidal and antiviral activity (Champagne et al., 1992). In this study, an aqueous extract by decoction of root has been prepared on the basis of the traditional usage of this drug by healers. Moreover, in order to verify whether the therapeutic potential of Trichilia emetica root may be a result of the presence of either polyphenols or limonoids, an ethyl ether fraction from the aqueous extract was also prepared and studied. Results of the antibacterial screening showed that the ethyl ether fraction has a good activity against clinical isolated bacterial strains, which are commonly responsible for respiratory infections. It is noteworthy that this fraction was effective at the same concentration as ampicillin against some strains of Staphylococcus aureus. Moreover, on Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae, this fraction evidenced a good activity against most of the bacterial strains tested, except for some strains which proved to be particularly resistant reaching a MIC value of 125 g/ml. The aqueous extract showed a very low antibacterial activity with MICs over 500 g/ml. In accordance with these results, it may be hypothesized that limonoids, which are present in the ethyl ether fraction, could be considered responsible for the antibacterial activity. In the rat in vivo model, we previously reported that treatment with Trichilia emetica extracts was effective in protecting against CCl4 -induced liver damage (German`o et al., 2001). To examine the effectiveness at cell level, this study was performed with an in vitro model using a suspension of rat hepatocytes. It is well known that hepatocytes are damaged by CCl4 , and cytosolic enzymes in the injured hepatocytes are leaked out of the cells due to an increase in cell permeability, membrane damage, and cell necrosis (Tezuka et al., 1995). The aqueous extract at the highest dose (1000 g/ml) exhibited hepatoprotective effects against CCl4 -intoxication as evidenced from the significantly reduced AST and LDH release into the medium as compared to CCl4 -treated cells, achieving the same protective potency as 1000 g/ml of silymarin. Moreover, in MTT assay the extract was able to reduce cell death induced by CCl4 both at 100 and 1000 g/ml. Although the ethyl ether fraction did not reduce the release of AST and LDH in the medium, even a high cell survival rate was still achieved at the dose of 1000 g/ml in MTT reaction. The release of cytosolic enzymes mainly reflects loss of integrity of plasma membrane, while MTT reaction estimates mitochondrial function (Rodriguez and Acosta, 1997). It may thus be hypothesized that the aqueous extract exerts a protection both on plasma membrane and mitochondria, while the ethyl ether fraction affords hepatoprotective effects only on
231
mitochondria. The protective effects of the aqueous extract against CCl4 -induced liver damage, both in vivo and in vitro could be related to the presence of phenolic compounds. As well known, phenolic antioxidants, such as flavonoids and tannins, are considered promising therapeutic agents for free radical pathologies due to their scavenging ability with ROS (Halliwell and Gutteridge, 1984). The good tolerability of Trichilia emetica extracts was also confirmed by the brine shrimps toxicity study (CL50 > 1000 g/ml). In conclusion, this study underlines the therapeutic potential of Trichilia emetica.
References Ak´e Assi, L., Guinko, S., 1991. Plants Used in Traditional Medicine in West Africa. Editions Roche, Basel, Switzerland, p. 90. Barry, A.L., 1991. Antibiotics in Laboratory Medicine, third ed. Editor Lorian, New York, pp. 1–16. Bauer, S.W., Kirby, W.M., Sherris, J.C., Thurck, M., 1966. Antibiotic susceptibility testing by standardized single disc method. American Journal of Pathology 45, 493–496. Burkill, H.M., 1997. The Useful Plants of West Tropical Africa, vol. 4, second ed. Royal Botanic Gardens Kew, pp. 88–134. Champagne, D.E., Koul, O., Isman, M.B., Scudder, G.G.E., Towers, G.H.N., 1992. Biological activity of limonoids from the Rutales. Phytochemistry 31, 377–394. Diallo, D., Paulsen, B.S., Liljeb¨ack, T.H.A., Michaelsen, T.E., 2003. The malian medicinal plant Trichilia emetica; studies on polysaccharides with complement fixing ability. Journal of Ethopharmacology 84, 279–287. El-Tahir, A., Satti, G.M.H., Khalid, S.A., 1999. Antiplasmodial activity of selected sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell. Journal of Ethopharmacology 64, 227– 233. German`o, M.P., Sanogo, R., Costa, C., Fulco, R., D’Angelo, V., Torre, E.A., Viscomi, M.G., De Pasquale, R., 1999. Hepatoprotective properties in the rat of Mitracarpus scaber (Rubiaceae). Journal of Pharmacy and Pharmacology 51, 729–734. German`o, M.P., D’Angelo, V., Sanogo, R., Morabito, A., Pergolizzi, S., De Pasquale, R., 2001. Hepatoprotective activity of Trichilia roka on carbon tetrachloride-induced liver damage in rats. Journal of Pharmacy and Pharmacology 53, 1569–1574. Gunatilaka, A.A.L., Bolzani, V., da, S., Dagne, E., Hofmann, G.A., Johnson, R.K., McCabe, F.L., Mattern, M.R., Kingston, D.G.I., 1998. Limonoids showing selective toxicity to DNA repair-deficient yeast and other constituents of Trichilia emetica. Journal of Natural Products 61, 179–184. Halliwell, B., Gutteridge, J.M., 1984. Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1, 1396–1397. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, Inc, pp. 252–253. Kokwaro, J.O., 1976. Medicinal Plants of East Africa. East Africa Literature Bureau, Kampala Nairobi Kenya, pp. 157–158. Malgras, D., 1992. Arbres et arbustes gu´erisseurs des savanes maliannes. Edition KARTHALA et ACCT, Paris, pp. 330–331. McGaw, L.J., J¨ager, A.K., van Staden, J., 1997. Prostaglandin synthesis inhibitory activity in Zulu, Xhosa and Sotho medicinal plants. Phytotherapy Research 11, 113–117. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., McLaughlin, J.L., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica 45, 31–34.
232
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival. Application to proliferation and cytotoxicity assay. Journal of Immunology Methods 65, 55–63. Nakatani, M., James, J.C., Nakanishi, K., 1981. Isolation and structures of trichilins, antifeedants against the Southern Army Worm. Journal of American Chemical Society 103, 1228–1230. Nakatani, M., Iwashita, T., Naoki, H., Hase, T., 1985. Structure of limonoid antifeedant from Trichilia roka. Phytochemistry 24, 195–196. Nakatani, M., Huang, R.C., Okamura, H., Naoki, H., Iwagawa, T., 1994. Limonoid antifeedants from Chinese Melia azedarach. Phytochemistry 36, 39–41. Oliver-Bever, B., 1986. Anti-infective activity of higher plants. In: Medicinal Plants in Tropical West Africa. Cambridge University Press, Cambridge, pp. 164–165. Rodriguez, R.J., Acosta Jr., D., 1997. N-Deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes. Toxicology 117, 123–131. Sanogo, R., German`o, M.P., D’Angelo, V., Forestieri, A.M., Ragusa, S., Rapisarda, A., 2001. Trichilia roka Chiov. (Meliaceae): pharmacognostic researches. Il Farmaco 56, 357–360.
Sparg, S.G., van Staden, J., J¨ager, A.K., 2000. Efficiency of traditionally used South African plants against schistosomiasis. Journal of Ethopharmacology 73, 209–214. Tezuka, M., Sadanobu, S., Gomi, K., Tachikawa, M., Sawamura, R., 1995. In vitro effect of chromiunm and other trace metals on mouse hepatotoxicity induced by carbon tetrachloride exposure. Biological Pharmaceutical Bulletin 18, 256–261. Traor´e-K´eita, F., Gasquet, M., Di Giorgio, C., Ollivier, E., Delmas, F., K´eita, A., Doumbo, O., Balansard, G., Timon-David, P., 2000. Antimalarial activity of four plants used in traditional medicine in Mali. Phytotherapy Research 14, 45–47. Tyson, C.A., Hawk-Prather, K., Story, D.L., Gould, D.H., 1983. Correlation of in vitro and in vivo hepatotoxicity for five haloalkanes. Toxicology and Applied Pharmacology 70, 289–302. von Maydell, H.J., 1996. Trees and Shrubs of the Sahel. Verlag Josef Margraf, Weikersheim. Xiong, Q., Fan, W., Tezuka, Y., Adnyana, I.K., Stampoulis, P., Hattori, M., Namba, T., Kadota, S., 1999. Hepatoprotective effect of Apocynum venetum and its active constituents. Planta Medica 66, 127– 133.
Hepatoprotective and antibacterial effects of extracts from Trichilia emetica Vahl. (Meliaceae) M.P. German`oa,∗ , V. D’Angeloa , R. Sanogob , S. Cataniac , R. Almaa , R. De Pasqualea , G. Bisignanoa a c
Pharmaco-Biological Department, School of Pharmacy, University of Messina, Vill. SS. Annunziata, 98168 Messina, Italy b Traditional Medicine Department—B.P. 1746, Bamako, Mali Interdipartimental Center for Experimental Toxicology (CITSAL), School of Medicine, University of Messina, Messina, Italy Received 17 February 2004; received in revised form 15 July 2004; accepted 6 September 2004 Available online 22 October 2004
Abstract Trichilia emetica Vahl. (Meliaceae) is a tree widely distributed in Tropical Africa. It has been used in Mali folk medicine for the treatment of various illnesses. The aim of this work was to study the hepatoprotective and antibacterial effects of a crude aqueous extract from Trichilia emetica root. An ethyl ether fraction from the aqueous extract was also prepared and studied. We have examined the hepatoprotective activity of the extracts on CCl4 -induced damage in rat hepatocytes, their toxicity using the brine shrimp bioassay and their antibacterial activity against clinical isolated bacterial strains, which are commonly responsible for respiratory infections. A preliminary phytochemical analysis showed a high polyphenolic content in the aqueous extract and the presence of limonoids in the ethyl ether fraction. These latter compounds may be considered responsible for the good activity against the bacterial strains tested. Trichilia emetica extracts exerted also a significant (P < 0.05) hepatoprotective effect at a dose of 1000 g/ml both on plasma membrane and mitochondrial function as compared to silymarin used as a positive control. These activities may be a result of the presence of either polyphenols or limonoids. Finally, both the aqueous extract and its ethyl ether fraction did not show toxicity (LC50 > 1000 g/ml) in the brine shrimp bioassay. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Trichilia emetica; Biological activity; CCl4 -induced liver damage in rat hepatocytes; Brine shrimp bioassay; Polyphenols; Limonoids; Antibacterial activity
1. Introduction Medicinal plants have always played an important role in African society. Traditions of plant-collecting and plantbased medications have been handed down from generation to generation (von Maydell, 1996). McGaw et al. (1997) reported that about 80% of the African population consults traditional healers and uses folk medicine for the treatment of various diseases. A scientific evaluation of the plants used by healers is essential before tra∗ Corresponding author. Tel.: +39 090 6766549/3533112; fax: +39 090 3533112. E-mail address: [email protected] (M.P. German`o).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.09.011
ditional medicine can be incorporated into the officinal health care system of Africa. Trichilia emetica Vahl. syn. Trichilia roka Chiov. (Meliaceae) (Oliver-Bever, 1986; Iwu, 1993), commonly known with the name of “Sulafinzan” in the language of Bambara, is a tree widely distributed in tropical areas and in WestAfrica lands. The plant is employed for the treatment of various disorders in folk medicine of Mali: it is used in hepatic diseases, as a purgative, antiepileptic, antipyretic and antimalarial agent (Iwu, 1993). A decoction of the root is taken as a remedy for colds and bronchial inflammation (Kokwaro, 1976; Malgras, 1992). A small glass of root decoction is generally consumed daily for 3 days against intestinal worms and to treat jaundice (Ak´e Assi and Guinko, 1991). In Senegal,
228
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
Trichilia emetica is also used in skin diseases (Oliver-Bever, 1986). Previous phytochemical studies have led to the isolation of several types of limonoids from an ethyl ether extract of Trichilia emetica root (Nakatani et al., 1981, 1985); these compounds are known for their biological activity (Champagne et al., 1992). McGaw et al. (1997) reported that an ethanolic extract of Trichilia emetica leaves has an inhibitory activity against cyclooxygenase; moreover, Gunatilaka et al. (1998) showed selective toxicity of some constituents of Trichilia emetica stem bark against DNA-repair deficient yeast. Most of the plants belonging to the family of Meliaceae, including Trichilia emetica, showed high antiplasmodial (El-Tahir et al., 1999; Traor´e-K´eita et al., 2000) and antischistosomiasis activities (Sparg et al., 2000). Diallo et al. (2003) reported the complement fixation ability of polysaccharides isolated from an aqueous extract of Trichilia emetica leaves. The effect of a decoction of the root on CCl4 -induced acute liver damage in the rat was evaluated by German`o et al. (2001). The antipyretic activity of a dried aqueous extract of Trichilia emetica root was compared with indomethacin treatment (Sanogo et al., 2001). In the present study, the hepatoprotective and antibacterial effects of an aqueous extract and its ethyl ether fraction from the root of Trichilia emetica were evaluated. We have examined the toxicity using the brine shrimp bioassay, the hepatoprotective activity on CCl4 -induced liver damage in vitro using rat hepatocytes and the antibacterial activity against clinical isolated bacterial strains which are commonly responsible for respiratory infections.
2. Materials and methods 2.1. Plant material Plant material was collected in the belt of Bamako (Mali) and identified by comparison with a voucher specimen deposited at the Herbarium of Traditional Medicine Division of Bamako. 2.2. Plant extracts Trichilia emetica root was air dried and ground using a laboratory mill. A crude extract was prepared by decoction of 100 g in 1000 ml of water for 30 min. The obtained extract was then filtered and concentrated to dryness under vacuum (yield 13.64%). An aliquot part of this extract (20 g) was dissolved in water (200 ml) and then extracted with ethyl ether (100 ml × 2). The organic fractions were recovered, combined, filtered and dried under reduced pressure (yield 0.15 g). The presence of limonoids was tested dissolving some milligrams of the ethyl ether fraction and then adding the same volume of hexane to give a precipitate (Nakatani et al., 1994).
2.3. Chemicals Bovine serum albumin Fr. V, foetal bovine serum, collagenase type IA (449 IU/mg), dexamethasone, (N[2-hydroxyethyl]piperazine-N -[2-ethanesulphonic acid]) (HEPES), insulin, 3-(4,5-dimethylthiazole-2-yl)-2,5diphenyl tetrazolium bromide (MTT), RPMI-1640 medium, and sodium bicarbonate were Sigma; CCl4 > 99.5% purity, dimethylsulphoxide (DMSO), ethylene glycol-O-O -bis (2-aminoethyl)-N,N,N N -tetracetic acid (EGTA) were from Fluka; Hank’s Ca2+ and Mg2+ -free balanced salt solution was from Euro-Clone, isopropanol from Lab-Scan, Triton X-100 from Bio-Rad, and Trypan Blue from Flow Laboratories. 2.4. Determination of total phenolic compounds The level of total phenolic compounds was determined in the aqueous extract. An aliquot of extract (0.1 ml of 10 mg/ml) was mixed with 0.2 ml of Folin-Ciocalteau reagent (Analyticals, Carlo Erba), 2 ml of H2 O and 1 ml of 15% Na2 CO3 . The absorbance was measured at 765 nm with a Shimadzu UV-1601 spectrophotometer after incubation for 2 h at room temperature in the dark. Quantification was based on a standard curve using gallic acid. Total phenols (mean ± S.D.) were expressed as milligrams of gallic acid equivalents per gram of aqueous extract. 2.5. The brine shrimp lethality biossay The brine shrimp lethality biossay was done according to the method of Meyer et al. (1982). Brine shrimp eggs (Artemia salina Leach) were hatched in artificial sea water (33 g/l of sea salt, Sigma Chemicals) and then they were incubated at room temperature for 48 h. With the help of a light source, the larvae (nauplii) were attracted to one side of the vessel and easily collected for the assay. The aqueous extract and its ethyl ether fraction were dissolved in dimethyl sulphoxide (DMSO) at a maximum concentration not exceeding 0.05% and then diluted with sea water to be tested at final concentrations of 10, 100 and 1000 g/ml. Each dose was tested in triplicate. Ten nauplii were used to test each dose of extracts. Nauplii were counted under a magnifying glass after 24 h of incubation. The number of dead nauplii were recorded and it was used for calculate LC50 concentration by the Finney Probit analysis program. LC50 values greater than 1000 ppm were considered inactive. 2.6. CCl4 -induced hepatocyte injury in vitro Hepatocytes were isolated from rats (Male Wistar rats weighing 150–200 g, Morini, Italy) by the procedure previously described by German`o et al. (1999). After a time preculture (30 min), the cells were exposed to fresh culture medium (RPMI-1640 medium supplemented with 10% inactivated foetal bovine serum, penicillin 100 IU/ml, strepto-
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
mycin 100 g/ml, dexamethasone 10 mM and insulin 1 mM) containing 10 mM CCl4 with or without test samples in DMSO (0.01 ml) (Xiong et al., 1999). The final concentration of DMSO did not exceed 0.05%. For the test, the aqueous extract was added to the vials to give a final concentration of 10, 100, or 1000 g/ml. The ethyl ether fraction was added to give a final concentration corresponding to 10, 100, or 1000 g/ml of aqueous extract. Silymarin was tested as positive control at doses of 10, 100 and 1000 g/ml. Each dose was tested in triplicate. Control cultures contained the same amount of DMSO. 2.7. Assessment of hepatotoxic activity in vitro AST and LDH activities in the medium were measured 60 min after CCl4 challenge. After incubation, culture medium from each vials was collected and centrifuged at 50 × g at 4 ◦ C for 2 min. The supernatant was used to measure AST and LDH levels using AST/UV-Autom and LDH/UV Liquid test kits (Sentinel) with a bioanalyser (ARCO PC). It is generally accepted that experiments are valid only if the background activity of control cells is ≤25% of total available intercellular activity. So, for control cells, the total intracellular enzyme content was determined after treatment of cells with Triton X-100 detergent to induce 100% lysis (Tyson et al., 1983). 2.8. MTT reduction assay The cell survival rate was assessed by the 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay according to Mosmann (1983) with slight modifications. Briefly, MTT (0.3 mg/ml) was dissolved in phosphate buffered saline (PBS, pH 7.4) and a MTT solution (200 l/ml) was added to each vial and incubated for 2 h at 37 ◦ C with 5% CO2 . After incubation, the cell suspension with formazan crystals was transferred into Eppendorf tubes and centrifuged at 5000 × g for 3 min. Supernatant was discarded and 1 ml of a lysis-solubilization solution (10% Triton X-100 in isopropanol, pH 2) was added to each tube. The pellet was dissolved by sonication and cell debris was collected by brief centrifugation. The absorbance of the purple solution was measured at 565 nm with a spectrophotometer (Shimadzu UV-1601). The cell survival rate was expressed as percentage formazan production by treated samples compared with control samples.
229
to use; it was then dissolved in DMSO and finally diluted in standard buffer at concentrations ranging from 0.015 to 500 g/ml. 2.10. Bacterial strains The bacterial strains used in this study were clinical isolated samples (Pediatric Section, School of Medicine, University of Messina): Haemophilus influenzae (12); Staphylococcus aureus (8); Streptococcus pneumoniae (4); Streptococcus pyogenes (8); and Moraxella catarrhalis (8). They were isolated from specimens obtained from oropharyngeal, nasopharyngeal or ear swabs of patients with acute infections (laryngitis, sinusitis, otitis) and identified by conventional procedures (confirmed by API system, Bio Merieux). Each bacterial strain cells were grown in Triptic Soy broth (Oxoid), 3 ml, for 18–24 h at 37 ◦ C. Before use the overnights cultures were diluted 1:10 with nutrient broth. To ensure that density of diluted cultures were all within the range 107 –108 colony forming units ml−1 , serial plate counts were performed for each culture. 2.11. Antibacterial assay The disc-diffusion method (Bauer et al., 1966) was used to test the activity. The minimum inhibitory concentration (MIC) of the extracts was measured using an agar dilution method (Barry, 1991). Each extract was incorporated into a liquefied M¨ueller–Hinton agar medium, which was then mixed, poured into Petri plates, and allowed to solidify. A series of Petri plates was prepared to obtain the desired concentrations within 0.95 and 500 g/ml. A standard 1 × 105 to 5 × 105 colony forming units ml−1 inoculum was tested for each bacteria strain, using a Steer’s replicator. The plates were incubated at 37 ◦ C for 18–24 h. The MIC was defined as the lowest concentration of the plant extracts that completely inhibited the visible growth. All antibacterial assays were performed in triplicate. 2.12. Statistical analysis Values are given as means ± S.D. Statistical analysis was done by Student’s t-test.
3. Results
2.9. Antibacterial agents
3.1. Total phenolic compounds
The dried aqueous extract and ethyl ether fraction were initially dissolved in DMSO (10 mg/ml) and subsequently diluted in sterile phosphate buffer (pH 7.2) for antibacterial test. The same amount of DMSO was added in control test. Ampicillin, used as positive control, was obtained from the Sigma Chemical Co. It was kept at 4 ◦ C in dessicator prior
The level of total phenolic compounds of Trichilia emetica aqueous extract as determined by the Folin-Ciocalteau reagent was 169.152 ± 2.34 mg expressed as gallic acid equivalents per gram of extract. The presence of limonoids in the ethyl ether fraction was confirmed using a simple screening described by Nakatani et al. (1994).
230
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
Table 1 Antibacterial activity of Trichilia emetica extracts: MIC g/ml Strains
Ethyl ether fraction
Aqueous extract
Ampicillin
15.60–31.25 15.60–62.50 7.80–125.00 7.80–31.25 125.00–125.00
>500 >500 >500 >500 >500
15.60–125.00 0.12–1.95 0.12–0.97 0.97–3.90 3.90–250.00
(8)a
Staphylococcus aureus Streptococcus pyogenes (8)a Streptococcus pneumoniae (4)a Moraxella catarrhalis (8)a Haemophilus influenzae (12)a a
Values in parentheses indicate number of strains tested.
3.2. Toxicity testing against the brine shrimp On the brine shrimp bio-assay Trichilia emetica aqueous extract and its ethyl ether fraction did not show cytotoxicity (LC50 > 1000 g/ml). 3.3. Effect on CCl4 -induced liver injury in vitro The hepatoprotective effects of Trichilia emetica extracts on CCl4 -induced liver injury are shown in Figs. 1 and 2. It is evident that the aqueous extract at the concentration of 1000 g/ml significantly prevented both AST (P < 0.05) and LDH (P < 0.01) release from the cells as compared to CCl4 group. The ethyl ether fraction did not produce any protective effects on the release of cytosolic enzymes. Silymarin was able to reduce AST release at concentration of 1000 g/ml (P < 0.05), while on LDH release exhibited protective effect both at 100 g/ml (P < 0.05) and 1000 g/ml
Fig. 2. Effect of Trichilia emetica extracts and reference drug silymarin on the survival rate of rat hepatocytes after CCl4 exposure. Bars are mean ± S.D.; ◦ P < 0.05 significantly different from control; * P < 0.05 significantly different from CCl4 -treated group.
(P < 0.01) (Fig. 1A and B). In the MTT assay, a significant reduction on cell viability was observed in CCl4 -treated groups as shown in Fig. 2. The aqueous extract (100 and 1000 g/ml) and the ethyl ether fraction (1000 g/ml) were able to elevate cell viability at a level of control group. 3.4. Antibacterial screening Antibacterial activity of Trichilia emetica extracts is reported in Table 1. The aqueous extract had a low antibacterial activity (MIC > 500 g/ml). The ethyl ether fraction showed a good activity against Streptococcus pneumoniae and Moraxella catarrhalis with range-MIC between 7.80 and 125.00 g/ml, Staphylococcus aureus and Streptococcus pyogenes with range-MIC between 15.60 and 62.50, while it resulted less active against Haemophilus influenzae (MIC 125 g/ml).
4. Discussion
Fig. 1. Effect of Trichilia emetica extracts and reference drug silymarin on AST (A) and LDH (B) activities of rat hepatocytes after CCl4 exposure. Bars are mean ± S.D.; ◦ P < 0.05 significantly different from control; * P < 0.05 and ** P < 0.01 significantly different from CCl -treated group. 4
The present investigation reports the hepatoprotective and antibacterial effects of Trichilia emetica giving a support for its traditional usage in folklore medicine. Previous phytochemical studies have reported the presence of tannins in the root and stem bark (Burkill, 1997). Our investigation on the aqueous extract confirms a high polyphenolic content which was evaluated as 169.152 mg/g of extract.
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
In addition, several types of limonoids, named “trichilins”, have been isolated from the root bark of Trichilia emetica (Nakatani et al., 1981, 1985). These latter compounds have attracted much attention for their wide range of biological activities including insect antifeedant, growth regulating properties, antifungal, bactericidal and antiviral activity (Champagne et al., 1992). In this study, an aqueous extract by decoction of root has been prepared on the basis of the traditional usage of this drug by healers. Moreover, in order to verify whether the therapeutic potential of Trichilia emetica root may be a result of the presence of either polyphenols or limonoids, an ethyl ether fraction from the aqueous extract was also prepared and studied. Results of the antibacterial screening showed that the ethyl ether fraction has a good activity against clinical isolated bacterial strains, which are commonly responsible for respiratory infections. It is noteworthy that this fraction was effective at the same concentration as ampicillin against some strains of Staphylococcus aureus. Moreover, on Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae, this fraction evidenced a good activity against most of the bacterial strains tested, except for some strains which proved to be particularly resistant reaching a MIC value of 125 g/ml. The aqueous extract showed a very low antibacterial activity with MICs over 500 g/ml. In accordance with these results, it may be hypothesized that limonoids, which are present in the ethyl ether fraction, could be considered responsible for the antibacterial activity. In the rat in vivo model, we previously reported that treatment with Trichilia emetica extracts was effective in protecting against CCl4 -induced liver damage (German`o et al., 2001). To examine the effectiveness at cell level, this study was performed with an in vitro model using a suspension of rat hepatocytes. It is well known that hepatocytes are damaged by CCl4 , and cytosolic enzymes in the injured hepatocytes are leaked out of the cells due to an increase in cell permeability, membrane damage, and cell necrosis (Tezuka et al., 1995). The aqueous extract at the highest dose (1000 g/ml) exhibited hepatoprotective effects against CCl4 -intoxication as evidenced from the significantly reduced AST and LDH release into the medium as compared to CCl4 -treated cells, achieving the same protective potency as 1000 g/ml of silymarin. Moreover, in MTT assay the extract was able to reduce cell death induced by CCl4 both at 100 and 1000 g/ml. Although the ethyl ether fraction did not reduce the release of AST and LDH in the medium, even a high cell survival rate was still achieved at the dose of 1000 g/ml in MTT reaction. The release of cytosolic enzymes mainly reflects loss of integrity of plasma membrane, while MTT reaction estimates mitochondrial function (Rodriguez and Acosta, 1997). It may thus be hypothesized that the aqueous extract exerts a protection both on plasma membrane and mitochondria, while the ethyl ether fraction affords hepatoprotective effects only on
231
mitochondria. The protective effects of the aqueous extract against CCl4 -induced liver damage, both in vivo and in vitro could be related to the presence of phenolic compounds. As well known, phenolic antioxidants, such as flavonoids and tannins, are considered promising therapeutic agents for free radical pathologies due to their scavenging ability with ROS (Halliwell and Gutteridge, 1984). The good tolerability of Trichilia emetica extracts was also confirmed by the brine shrimps toxicity study (CL50 > 1000 g/ml). In conclusion, this study underlines the therapeutic potential of Trichilia emetica.
References Ak´e Assi, L., Guinko, S., 1991. Plants Used in Traditional Medicine in West Africa. Editions Roche, Basel, Switzerland, p. 90. Barry, A.L., 1991. Antibiotics in Laboratory Medicine, third ed. Editor Lorian, New York, pp. 1–16. Bauer, S.W., Kirby, W.M., Sherris, J.C., Thurck, M., 1966. Antibiotic susceptibility testing by standardized single disc method. American Journal of Pathology 45, 493–496. Burkill, H.M., 1997. The Useful Plants of West Tropical Africa, vol. 4, second ed. Royal Botanic Gardens Kew, pp. 88–134. Champagne, D.E., Koul, O., Isman, M.B., Scudder, G.G.E., Towers, G.H.N., 1992. Biological activity of limonoids from the Rutales. Phytochemistry 31, 377–394. Diallo, D., Paulsen, B.S., Liljeb¨ack, T.H.A., Michaelsen, T.E., 2003. The malian medicinal plant Trichilia emetica; studies on polysaccharides with complement fixing ability. Journal of Ethopharmacology 84, 279–287. El-Tahir, A., Satti, G.M.H., Khalid, S.A., 1999. Antiplasmodial activity of selected sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell. Journal of Ethopharmacology 64, 227– 233. German`o, M.P., Sanogo, R., Costa, C., Fulco, R., D’Angelo, V., Torre, E.A., Viscomi, M.G., De Pasquale, R., 1999. Hepatoprotective properties in the rat of Mitracarpus scaber (Rubiaceae). Journal of Pharmacy and Pharmacology 51, 729–734. German`o, M.P., D’Angelo, V., Sanogo, R., Morabito, A., Pergolizzi, S., De Pasquale, R., 2001. Hepatoprotective activity of Trichilia roka on carbon tetrachloride-induced liver damage in rats. Journal of Pharmacy and Pharmacology 53, 1569–1574. Gunatilaka, A.A.L., Bolzani, V., da, S., Dagne, E., Hofmann, G.A., Johnson, R.K., McCabe, F.L., Mattern, M.R., Kingston, D.G.I., 1998. Limonoids showing selective toxicity to DNA repair-deficient yeast and other constituents of Trichilia emetica. Journal of Natural Products 61, 179–184. Halliwell, B., Gutteridge, J.M., 1984. Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1, 1396–1397. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press, Inc, pp. 252–253. Kokwaro, J.O., 1976. Medicinal Plants of East Africa. East Africa Literature Bureau, Kampala Nairobi Kenya, pp. 157–158. Malgras, D., 1992. Arbres et arbustes gu´erisseurs des savanes maliannes. Edition KARTHALA et ACCT, Paris, pp. 330–331. McGaw, L.J., J¨ager, A.K., van Staden, J., 1997. Prostaglandin synthesis inhibitory activity in Zulu, Xhosa and Sotho medicinal plants. Phytotherapy Research 11, 113–117. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., McLaughlin, J.L., 1982. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica 45, 31–34.
232
M.P. German`o et al. / Journal of Ethnopharmacology 96 (2005) 227–232
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival. Application to proliferation and cytotoxicity assay. Journal of Immunology Methods 65, 55–63. Nakatani, M., James, J.C., Nakanishi, K., 1981. Isolation and structures of trichilins, antifeedants against the Southern Army Worm. Journal of American Chemical Society 103, 1228–1230. Nakatani, M., Iwashita, T., Naoki, H., Hase, T., 1985. Structure of limonoid antifeedant from Trichilia roka. Phytochemistry 24, 195–196. Nakatani, M., Huang, R.C., Okamura, H., Naoki, H., Iwagawa, T., 1994. Limonoid antifeedants from Chinese Melia azedarach. Phytochemistry 36, 39–41. Oliver-Bever, B., 1986. Anti-infective activity of higher plants. In: Medicinal Plants in Tropical West Africa. Cambridge University Press, Cambridge, pp. 164–165. Rodriguez, R.J., Acosta Jr., D., 1997. N-Deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes. Toxicology 117, 123–131. Sanogo, R., German`o, M.P., D’Angelo, V., Forestieri, A.M., Ragusa, S., Rapisarda, A., 2001. Trichilia roka Chiov. (Meliaceae): pharmacognostic researches. Il Farmaco 56, 357–360.
Sparg, S.G., van Staden, J., J¨ager, A.K., 2000. Efficiency of traditionally used South African plants against schistosomiasis. Journal of Ethopharmacology 73, 209–214. Tezuka, M., Sadanobu, S., Gomi, K., Tachikawa, M., Sawamura, R., 1995. In vitro effect of chromiunm and other trace metals on mouse hepatotoxicity induced by carbon tetrachloride exposure. Biological Pharmaceutical Bulletin 18, 256–261. Traor´e-K´eita, F., Gasquet, M., Di Giorgio, C., Ollivier, E., Delmas, F., K´eita, A., Doumbo, O., Balansard, G., Timon-David, P., 2000. Antimalarial activity of four plants used in traditional medicine in Mali. Phytotherapy Research 14, 45–47. Tyson, C.A., Hawk-Prather, K., Story, D.L., Gould, D.H., 1983. Correlation of in vitro and in vivo hepatotoxicity for five haloalkanes. Toxicology and Applied Pharmacology 70, 289–302. von Maydell, H.J., 1996. Trees and Shrubs of the Sahel. Verlag Josef Margraf, Weikersheim. Xiong, Q., Fan, W., Tezuka, Y., Adnyana, I.K., Stampoulis, P., Hattori, M., Namba, T., Kadota, S., 1999. Hepatoprotective effect of Apocynum venetum and its active constituents. Planta Medica 66, 127– 133.