Phytomedicine Vol. 4/1995, pp. 299-302 © 1995 by Gustav Fischer Verlag, Stuttgart· jena . New York Biological investigations on Harrisonia abyssinica A. M. BALDE1, L. PIETERS2, T. DE BRUYNE2, S. GEERTS 3 , D. VANDEN BERGHE2 and A. VLlETINCK2 Faculte de Medecine-Pharrnacie, Universite de Conakry, Guinee-Conakry (present address ) Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium 3 Prince Leopold Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium 1 2 Summary Extracts of the bark and the root of Harrisonia abyssinica exhibited in vitro antiviral, antifungal, antibacterial and molluscicidal activities. Bioassay-guided fractionation resulted in the isolation of the active compounds. Introduction Harrisonia abyssinica Olivo (Simaroubaceae) is widely used in various African folk remedies. The root and the stem-bark are used in the treatment of gonorrhoea, dysentery, skin diseases, tuberculosis, bilharzia infections and as an ascaricide throughout Africa (Balde et aI., 1989; Bouquet and Debray, 1974; Chhabra et aI., 1984; Khan et aI., 1980; Sofowora, 1982). Previous investigations on the plant reported an antifeedant activity of the root bark against the most common East African crop pests, the monophageous Spodoptera exempta (African army worm) and S. eridania (Southern army worm), an antibacterial activity against gram-positive microorganisms, as well as cytotoxic and plant growth inhibitory activities (Kubo et aI., 1976; Liu et aI., 1982). Although some early results did not support the antimicrobial use of the plant in traditional medicine, other investigators found that the ether extract of the root showed antimicrobial activity against Trichophyton mentagrophytes and Neisseria gonorrhoeae (Sofowora, 1982). The root bark and twigs also showed a potent antibacterial activity against Staphylococcus aureus (Khan et aI., 1980). Isolation of bioactive principles guided by antifeedant and antibacterial tests led to the limonoid harrisonin (the ultimate activity being 20 ppm for its antifeedant activity and 5 ppm for its activity against Bacillus subtilis). Monitoring the fractionation of the extract of the air-dried root bark by insect antifeedant bioassays against S. eridania led to the isolation of another antifeedant Iimonoid, ace- toxyharrisonin (Liu et aI., 1982). On the other hand, we have previously reported the in vivo activity of an ethanol extract of the plant on experimental Schistosoma mansoni infection in mice (Balde et aI., 1989). Chemical investigation of the active extract provided steroids, limonoids and chromones (Baldc, 1990; Baldc et aI., 1988). This paper describes the antibacterial, antiviral and molluscicidal properties of extracts of H. abyssinica. Material and Methods Plant material- The root and stem-bark of H. abyssinica were collected in Seredou (Guinea-Conakry) in ]uly 1987; the plant was taxonomically identified at the Department of Botany of the Research Center of Medicinal Plants Seredou, Voucher specimens have been deposited at the herbarium of the Center and the herbarium of the National Botanical Garden of Belgium at Meise. Preparation of the extracts - Each extract was prepared from 50 g of the dried powdered root bark (HR) or stembark (HT) of H. abyssinica and evaporated to dryness in vacuo below 40 "C. Fractions HRI and HTI were obtained by percolation with 80 % ethanol and yielded 17.3 % and 16.7% w/w dry weight, respectively. HR2 and HT2 were obtained by percolation with chloroform and yielded 6.4 % and 4.7% w/w dry weight, respectively. HR3 and HT3 were obtained by maceration with distilled water (1500 ml) at room temperature for 24 hours and yielded 300 A. M. Balde et al. 0.8 % and 0.7% w/w dry weight, respectively. HR4 and HT4 were obtained by percolation of the marc resulting from the cloroform extraction with 70 % methanol and yielded 9.6 % and 8.1 % w/w dry weight, respectively. Fractionation - Powdered root bark (500 g) or stem-bark (500 g) were percolated with clorofrom at room temperature. The organic extract was concentrated in vacuo and yielded a thick yellow oil (30 g and 21 g for root and stem bark, respectively) which was applied to a silica gel column and eluted with chloroform and a chloroform-methanol gradient. The fractionation of the oil was guided by an antibacterial bioassay against Neisseria gonorrhoeae, an antifungal bioassay using Aspergillus niger and Microsporum canis, and a molluscicidal bioassay using the snail Biomphalaria glabrata. The marc resulting from the above treatment of the stem bark with chloroform was percolated with 70 % methanol at room temperature. The extract was concentrated in vacuo and was subjected to a combination of CC, droplet counter current chromatography (DCCC) (Balde et al., 1990) and Sephadex LH-20. The fractionation was guided by the antiviral activity against herpes simplex type 1 virus. Preparation of samples for antimicrobial testing Antimicrobial activity - Aliquots of the polar fractions (HR1 and HT1; HR3 and HT3) were dissolved in a mixture of dimethylsulfoxide (DMSO) and physiological tris budder (pH 7.4) (1: 9). The lipophilic fractions (HR2 and HT2) were dissolved in a mixture of polyethyleneglycol 400 (PEG 400) and physiological tris buffer (pH 7.4) (1: 9). All extracts were tested at a concentration of 1 mg/rnl, Test organisms were exposed to maximum 1 % DMSO or PEG 400. Antiviral activity - An aliquot of each sample was diluted with DMSO (0.1 ml) and adjusted with tissue culture medium M-2 to pH 7.2 in order to obtain a concentration of 1 mg/rnl, Test organisms were exposed to a maximum of 1 % DMSO or PEG 400. Chemotherapeutic evaluation - (Balde et al., 1990). The following microorganisms were used: gram positive cocci including Staphylococcus aureus ATCC 25923, Streptococcus viridans, Str. pneumoniae; gram negative cocci including Neisseria gonorrhoeae; gram negative enteric bacilli such as Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella type B, Serratia marcescens, Shigella flexneri; acid-fast bacilli including Mycobacterium fortuitum; yeasts including Candida albicans, C. tropicalis; and fungi such as Aspergillus flavus, A. fumigatus, A. niger, Microsporum canis and Trichophyton mentagrophytes. Test viruses included herpes simplex type 1, coxsackie B1, Semliki forest and vesicular stomatitis virus (VSV). Molluscicidal activity - Groups of 10 healthy and uniform Biomphalaria glabrata snails (6-8 mm) were placed into beakers containing 1 000 ml of demineralized water. Test samples were dissolved in the beakers to mark concentrations of 1 000,500 and 100 ppm. The snails were put in a small perforated plastic beaker, and covered with gauze. They remained in contact with the drugs for 4 days, under laboratory conditions, in a poorly lighted room, at room temperature. During the experiment, the dead snails were removed. Following the 4 days of exposure the snails were placed in 1 000 ml of demineralized water. After 24 hours the dead snails were counted. The criteria of death were inactivity and discoloration. The procedure is that recommended by the World Health Organization (WHO) (WHO, 1987). Results Antimicrobial activity - The antimicrobial activity of H. abyssinica extracts is given in Table 1. At a concentration of 1 mg/ml none of the extracts showed any antimicrobial activity against Candida albicans, C. tropicalis, Escherichia coli, Streptococcus viridans, Salmonelle type B, Serratia marcescens, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa and Mycobacterium fortuitum. While only the ethanol extracts showed a moderate activity against Staphylococcus aureus, both the ethanol and chloroform extracts exhibited a pronounced antibacterial effect against Neisseria gonorrhoeae. All the extracts moderately inhibited the growth of Streptococcus pneumoniae. The antibacterial spectra and activities of extracts of the root and the stem bark of H. abyssinica were not significantly different. Antifungal activity - The ethanol and aqueous extracts of both the root and stem bark of H. abyssinica were inactive against Aspergillus niger, A. fumigatus, A. flavus, Microsporum canis and Trichophyton mentagrophytes. The chlo- Table 1. Antibacterial activity! of the extracts (1 mg/ml) of H. abyssinica. Extract ethanol (HTl) ethanol (HR1) chloroform (HTl) chloroform (HR1) aqueous (HTl) aqueous (HR1) PEG 1 % DMSO 1 % neomycin (500 ug/rnl) penicillin (50 ug/ml) Inhibition zone width (mm) S. aureus S. pneuN. gonor8 12 o o o o o o moniae rhoeae 10 15 13 10 8 13 16 16 18 18 13 10 o o o o 40 20 30 30 ! The antibacterial activity is expressed as the zone of inhibition in mm, - not determined. Biological Investigations on Harrisonia Abyssinica 301 Table 2. Antiviral activity! of the extracts of H. abyssinica Extract Cone. (ug/rnl) herpes simplex coxsackie B2 Semliki forest poliomyelitis vesicular stomatitis HRI 50 25 12.5 6.25 3.12 50 25 12.5 6.25 3.12 50 25 12.5 6.25 3.12 50 25 12.5 6.25 3.12 50 25 12.5 6.25 3.12 50 25 12.5 6.25 50 25 12.5 6.25 3.12 50 25 12.5 6.25 3.12 T2 T T 10 2 1 1 T T T 10 2 1 T T 10 10 10 T T 10 3{T/4) 102 T 10 3(T/2) 10 2(T/4) 10 1 T T 10(T/2) 1 10 3(T/2) 102(T/4) 10 10 1 103(T/2) 102(T/4) 10 1 10 1 T T 10(T/2) 1(T/4) 1 T 10(T/2) 10 T T 1 T 10(TI2) 10 4(T/2) 10 1 10 3 10 1 T T 1 HR2 HR3 HR4 HTl HT2 HT3 HT4 T T 10(T/4) 1 10 4(T/2) 10 4(T/4) 102 10 1 T 10 4(T/2) 104 102 10 T T 1 T T 10 4(T/4) 104 103 T T 1 T 10 4(T/4) 102 102 10 1 T l(TI2) T T T 1 T T 1 104(T/2) T T 10 4(T/4) 102(T/2) T 1(TI2) 10 4 102 10 104 10 3 10 2 1 10 1 T T 1 T T 1(T/2) 102(T/4) 10 1 T T 1(T/4) T T T 1(T/2) ! The antiviral activity is expressed as the reduction factor of the viral titer (ratio of viral titer in the absence and presence of the samples). A reduction factor of 1 means no antiviral activity. 2 T, T/2, T/4 =decreasing cytotoxicity of the samples; T =complete degeneration of the cells; T/2 =heavily affected cells, which are still attached to the monolayer; T/4 =moderately affected cells. roform extract of the root (HR2) was only active against A niger, whereas the c1oroform extract of the stem bark (HT2) inhibited the growth of A. niger, A. [umigatus, A. flavus, M. canis and T. mentagrophytes. The extracts of the stem bark were more antifungal than those of the root. Antiviral activity - The antiviral activities of the extracts of H. abyssinica are given in Table 2. Significant antiviral properties, which correspond to reduction of the viral titer of 10 3 or more, were found against herpes simplex type 1 (HT1, HT3, HT4 and HR4 extracts), coxsackie B2 (HT1, HT3, HT4 and HR3 extracts), Semliki forest (HR1, HR2 and HR3 extracts) and vesicular stomatitis (HT2 extract) viruses in concentrations varying from 50 to 6.25l!g/mI. Molluscicidal activity - As shown in Table 3, all extracts were toxic to snails at a concentration equivalent to 500 ppm. At 100 ppm, the aqueous extracts of both the stem bark (HT3) and the root (HR3) were devoid of any moluscicidal effect, while the ethanol extracts showed a weak potency. On the contrary, the c1oroform extracts of both stem bark (HT2) and root (HR2) exhibited a pronounced toxicity at 100 ppm leading to 70 % and 80 % lethality against snails, respectively. 302 A. M . Balde et al. Table 3. Mortal ity of Biomphalaria glabrata exposed to H. abyssinica extracts (number of tested snails: 10). Number of dead snails Concentration (ppm) 1000 500 100 Periode of exposure (hour) Extract 24 48 24 48 24 48 HR l HR2 HR3 HTl HTl HT3 7 10 3 5 10 1 10 10 10 10 10 10 4 10 1 3 10 1 8 10 5 7 10 5 0 6 0 0 4 0 3 8 0 2 7 0 herpetic activity the two princip al antiv irally active compounds were isolated from th e etha no l extract (HT4). The reduction factor of the viral tit er was equivalent to 10 3 a t a concentration of 251lg/ml. Isol ated biologically active compounds were found to contain severa l ca r bony l functi on alities exhibiting keto/enol equilibria , and isoprenyl groups. Although the carbon skeleto n appeared to be qu ite d ifferent, they sho w so me simi larity to th e hop bitter acids from Humulus lupulus L. The stru ctur e eluc idation of the isolated bioactive compunds will be the subject of a separate publication. These results not only give support for the traditional use of H. abyssinica against gonococcal infection s, but also reveal antifungal, antiviral and molluscicidal properties. To the best of our knowledge, th e antivir al and molluscicidal activities of H. abyssinica are reported for the first time. Discussion The extracts of H . abyssinica ob viously exhibit antibacterial, antiviral and molluscicidal properties. The activities of th e extract s against N. gon orrhoeae and S. aureus were in agreement w ith those reported in the literature (Kahn et aI., 19 80 ; Sofowora, 1982). However, th e activity against S. aureus (o nly show n by the etha nol extracts) as well as that against S. pneumoniae was rather moderate. Both the ethanol and chlorofo rm extracts showe d an important antigenococcal potenc y. These results provide support for the traditional use of the plant against gonoc occal infections. W ith regard to the antiviral activity, the extracts of the stem bark were found to be more effective than those of th e ro ot ag ai nst herpes simplex type 1, coxsacki B2 and vesic ular sto matitis. However, only th e extracts of the root bark showed a pronounced antiviral act ivity against Semliki forest . When testing against herpes simplex type 1 and coxsackie B2, the polar extracts of both the root and stem bark (HR3 an d HR4; HT3 and HT4) were less cytotoxic and mo re active than the corresponding ethanol and chloroform extracts. Becau se of the molluscicidal acti vity observed, and in view of the fact that H . abyssinica is also used as a n antifeedant against Spodoptera exe mpta a nd S. eridania (Liu et aI., 1982), multiple uses of the plant for agricult ural pest and vecto r control purposes favour an ecologic approach to integrated pest management programs, as ad vised by Kloos and McCullough (19 87) . Based on these results, the cloroform extract of the ste m bark (H T 2) was subjected to bioassay guided fractionation. The antibacterial bioassays against Neisseria gonorrhoeae led to the isolation of the principal biologicall y active compound with a min imal inhibitory concentr ation of less than 6.25Ilg/mi. The molluscicidal bioa ssa ys resulted in the isolation of th e ma in active constituent, which was 100 % lethal to Biomphalaria glabrata at a concentration of less than 5 ppm. Guided by the anti- Acknowledgements This research was supported by grant No. 92/9 4.09 (concerted action) of the Flemish government (Belgium). T. De Bruyne is a research assistant of the National Fund for Scientific Research (NFWO, Belgium). A. M. Bald e received a grant from the General Agency for Development and Cooperation (ABOS, Belgium). References Balde, A. M., Vanhaelen, M. and Daloze, D.: Phytochemistry 27, 942, 1988. Balde, A. M., Van Marek, E., Kestens, L., Gigase, P. L. and VIietinck, A. J.: Planta Med. 55, 41, 1989. Balde, A. M.: Biological and Phytochemical Investigations on Thr ee Plants Widely Used in Guinean Traditional Medic ine, Ph. D. Thesis, University of Antwerp, part 5, 1990. Balde, A. M., Van Hoof, L., Pieters, L., Vanden Berghe,D. A. and Vlietinck, A. J.: Phytother. Res. 4,1 82,1990. Bouquet, A. and Debray, M.: Plantes Med icinales de Cote d'Ivoire, Travaux et Documents de I'ORSTOM , 32, 1974. Chhabra, S. c., Uiso, F. C. and Mshiu, E. N.: t. Ethnopharmacol. 11,1 57,1 984. Khan, M. R., Ndaalid, G., Nku nya, M. H. H., Wevers, H. and Sawhney, A. N.: Planta Med. 38, suppl., 91, 1980. Kloos, H . and McCullough, F. S.: Plant Molluscicides, Ed. Mott K. E., Wiley, Chichester, p. 45,1 987. Kubo, 1., Tanis, S. P., Lee, Y. M., Miura, 1., Nakanishi , K. and Cha pya , A.: Heterocycles 5. 485, 1976. Liu, H. W., Kubo, 1. and Nakanishi, K.: Heterocycles 17, 67, 1982. Sofowora, A.: Medicinal Plants and Traditional Medicine in Africa, Wiley, Chichester, p. 223, 1982. WHO : Parasitic Diseases Programm e. Atlas of the Global Distribution of Schistosomiasis, 7, 95, 1987. Address L. Pieters, Department of Pharmacentical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp/Belgium