Antiviral activity of Rwandan medicinal plants against human immunodeficiency virus type-1 (HIV-1).
Selected plants used in Rwandan traditional medicine for the treatment of infections and/or rheumatoid diseases were investigated for antiviral activity in vitro against human immunodeficiency virus type-1 (HIV-1). Of the 38 tested 80% ethanolic extracts, belonging to plants of 21 different families only the extracts from the leaves of Aspilia pluriseta (Asteraceae) and Rumex bequaertii (Polygonaceae) had interesting selectivity indices (SI = ratio of the 50% cytotoxic concentration to the 50% effective antiviral concentration) higher than 1.
Further fractionation of the initially antivirally inactive ethanolic extract of Tithonia diversifolia, however, led to an aqueous fraction with a high anti-HI V-1 activity (SI > 461), indicating that the cytotoxicity of some plant components may mask the antiviral properties of the active plant substances in total plant extracts.
Key words: African medicinal plants, antiviral, HIV-1, MTT, Rwanda
Acquired immunodeficiency syndrome (AIDS) is a pandemic immunosuppressive disease that results in life-threatening opportunistic infections and malignancies. AIDS is caused by the human immunodeficiency virus (HIV), a lentivirus within the family Retroviridae. From the two known types of HIV HIV-1 is the most pathogenic. Due to the high number of HIV-infected people and the rapid emergence of drug-resistant strains, the demand for new antiviral therapeutics against HIV-1 is great. However, the standard antiviral therapies are too expensive for most of the Africans. In order to manage the AIDS epidemic in Africa, alternative treatments are clearly needed. One of the possible approaches is the screening of plants based on their ethnomedicinal data (Locher et al., 1996; Vlietinck et al., 1998). In this study, Rwandan plants were selected on base of their ethnomedicinal use against infections and rheumatic diseases (Baerts and Lehmann, 1989; Rwangabo, 1993; Van Puyvelde et al., 1977). Ethanolic extracts of the se lected plants were tested for their activity against HIV-1.
* Materials and Methods
All plants were collected by J. Mvukiyumwami in the district of Butare in Rwanda at a mean altitude of 1700 m. Voucher specimens were deposited in the herbarium of the Institut de la Recherche Scientifique et Technique (IRST) at Butare, Rwanda.
The air-dried plant material was ground and extracted with 80% ethanol by maceration. The macerate was filtered and the marc was percolated with 80% ethanol until exhaustion. The filtrate and percolate were combined and concentrated under reduced pressure at 40 [degrees]C. The residue was taken up in 60% methanol and defatted with petroleum ether (non-polar fraction). The residual polar fraction was lyophilized and tested for anti-HIV-1 activity. In the case of Tithonia diversifolia, the residual fraction was further extracted with ethylacetate and the resulting ethylacetate and water fractions were tested for anti-HIV-1 activity.
HIV inhibition assays
The antiviral screening was determined by evaluating cell death caused by plant extract cytotoxicity and viral cytopathic effect as previously described (Pauwels et al., 1988).
The MT-4 cells were grown in RPMI 1640 DM ("Dutch modification") medium, supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) and 20[micro]g/ml gentamicin. The cells were maintained at 37 [degrees]C in a humidified atmosphere containing 5% [CO.sub.2]. Every 3-4 days, cells were spun down and seeded in new culture flasks at a density of 2 x [10.sup.5] cells/ml.
HIV (strain HTLV-[III.sub.B]/LAI) was obtained from the culture supernatant of HIV-infected HUT-78 cell line. The virus titer of the supernatant was determined in MT-4 cells. The virus stock was stored at -70[degrees]C until used.
Flat bottom, 96-well microtiter plates were filled with 100 [micro]l of complete medium using a Titertek multidrop dispenser (Flow Laboratories). Subsequently, stock solutions (10 x final test concentration) of plant extracts were added in 25 [micro]l volumes to two series of triplicate wells to allow simultaneous evaluation of their effects on HIV- and mock-infected cells. Serial five-fold dilutions of plant extracts were made directly in the microtiter plates using a Biornek 1000 robot (Beckman, Fullerton, California, USA). Thus each plant extract had final concentrations of 200, 40, 8, 1.6 and 0.32 [micro]g/ml in each experiment.
Fifty [mu]l of HIV-1 stock at 100 [TCID.sub.50] (Tissue Culture Infectious Dose) or medium was added to either the HIV- or mock-infected part of the microtiter plate. Exponentially growing MT-4 cells were centrifuged for 5 minutes at 140 g and the supernatant was discarded. The MT-4 cells were resuspended at 6 x [10.sup.5] cells/ml in a flask which was connected with an autoclavable dispensing cassette of a Titertek multidrop dispenser. Under slight magnetic stirring 50 [mu]l volumes were then transferred to the microtiter tray wells. The outer row wells were filled with 200 [mu]l of medium. The cells were incubated at 37 [degrees]C in a humidified atmosphere containing 5% [CO.sub.2]. Five days after infection the viability of mock- and HIV-infected cells was examined spectrophotometrically by the MTT-method.
The MTT assay is based on the reduction of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, Missouri, USA) by mitochondrial dehydrogenases of metabolically active cells to a blue formazan which can be measured spectrophotometrically.
To each well, 20 [mu]l of a 7.5 mg/ml MTT solution in phosphate-buffered saline was added using the Titertek multidrop. After incubation for one hour at 37 [degrees]C, a fixed volume of 150 [mu]l medium was removed from each well using an M96 washer (ICN flow). Solubilization of the formazan crystals was achieved by addition of 100 [mu]l 10% (v/v) Triton X-100 in acidified isopropanol (2 ml concentrated HCl per 500 ml solvent). Complete dissolution of the formazan crystals was obtained by shaking the plates for 10 minutes on a plate shaker (ICN flow). Finally, the optical density of each well was measured using an automatic plate reader (Multiscan MCC, ICN Flow) with a test wavelength of 540 nm and a reference wavelength of 690 nm. Blanking was carried out with wells containing all reagents except MT-4 cells, virus, and plant extract.
The 50% cytotoxic concentration ([CC.sub.50]) was defined as the concentration of the test compound that reduces the absorbance ([OD.sub.540]) of the mock-infected control sample by 50%. The percent protection achieved by the test compound in HIV-infected cells was calculated by the following formula:
[([OD.sub.t]).sub.HIV] - [([OD.sub.c]).sub.HIV] / [([OD.sub.c]).sub.mock] - [([OD.sub.c]).sub.HIV] x 100 (expressed in %)
where [([OD.sub.t]).sub.HIV] is the optical density measured with a given concentration of the test compound in HIV-infected cells; [([OD.sub.c]).sub.HIV] is the optical density measured for the control untreated HIV-infected cells; and [([OD.sub.c]).sub.mock] is the optical density measured for control untreated mock-infected cells. The concentration achieving 50% protection according to the formula above was defined as the 50% effective concentration ([EC.sub.50]). The selectivity index was defined as [CC.sub.50]/[EC.sub.50].
Results and Discussion
A total of 38 different plant species from 21 families were investigated on their antiviral activity against HIV-1 (Table 1). The anti-HIV-1 activity was measured using a tetrazolium-based colorimetric assay in infected MT-4 cells. The results of the screening are shown in Table 2. Antiviral activity was found for the ethanolic extracts from the leaves of Aspilia pluriseta and Rumex bequaertii with selectivity index (SI) values higher than 12 and 11, respectively. The ethanolic extract of Aspilia pluriseta showed a complete cell protection against HIV-induced cytopathic effect compared to cell controls, while the ethanolic extract of Rumex bequaertii exhibited 89% protection. A previous study reported the isolation of the photosensitiser thiarubrine-A, a dithiacyclohexadiene polyacetylene, from the leaves of Aspilia pluriseta (Rodriguez et al., 1985). It was shown that thiarubrine-A had a phototoxic activity against cytomegalovirus and Sindbis virus, both of which are enveloped viruses (Hudson et al., 1986).
The ethanolic extract of Tithonia diversifolia showed a high cytotoxicity (Table 2). The ethanolic residue was suspended in 60% methanol and further extracted with petroleum ether and ethylacetate, consecutively. The residual aqueous fraction showed a pronounced anti-HIV-1 activity with a SI higher than 461 (Table 3).
The genus Tithonia (Asteraceae) comprising 11 species and 13 taxa, ranges from the south-western United States to Panama, although one species, Tithonia diversifolia (Hemsl.) A. Gray, has been escaped to Africa, Australia, Asia, and North America where it is locally abundant. Several studies reported the isolation of sesquiterpene lactones from T. diversifolia (Kuo and Chen, 1998; Pereira et al., 1997). In one study, three sesquiterpene lactones, i.e. diversifolin, diversifolin methyl ether, and tirotundin, were isolated from the leaves of T. diversifolia Rungeler et al., 1998). They inhibited the DNA binding activity of the transcription factor NF-[kappa]B, resulting in a decreased production of inflammatory mediators, such as cytokines and chemokines. In another study, a new artemisinic acid analogue compound was isolated from mature stems of T. diversifolia (Bordoloi et al., 1996). Artemisinic acid, is the biogenetic precursor of artemisinin, a potent antimalarial drug isolated from Artemisia annua. In ad dition, several semi-synthetic derivatives of artemisinin, such as 12-n-butyldeoxoartemisinin, showed an anti-HIV activity (Jung et al., 2000). It must be emphasized that the anti-HIV-1 activity of T. diversifolia was found in the polar layer, indicating that the active ingredients are hydrophilic compounds. Various sulfated polysaccharides have been found to be the anti-HIV active substances of many antivirally active plant extracts (Vlietinck et al., 1998). These compounds are targeted at the interaction between the viral envelope glycoprotein gp120 and the [CD.sub.4], receptor, and as a consequence, they inhibit not only virus adsorption to the cells but also virus-induced syncytium (giant cell) formation. In addition, sulfated polysaccharides may also directly interfere with the binding of HIV particles to the heparin sulfate proteoglycan of the cell surface (Vlietinck et al., 1998). Alternatively, polyphenolic compounds such as hydrolysable tannins show anti-HIV activity, which is partly mediated by inhi bition of HIV adsorption to the cells. Experiments are underway to determine if polysaccharides or polyphenolic compounds are responsible for the observed antiviral activity of the aqueous extract of T. diversifolia.
Plant extracts are complex mixtures of many compounds. This study clearly shows that the cytotoxicity and the antiviral activity of a total extract are not necessary due to the same compound(s), and that the cytotoxicity of some plant compounds may mask the antiviral properties of other plant substances. Our results suggest that the separation of apolar from polar components can increase the chance to find highly active antiviral compounds with low cytotoxicity. The two other active plants are currently investigated and the results of these studies will be published in due course.
Table 1 List of Rwandan medicinal plants studied (Baerts and Lehmann, 1989; Rwangabo, 1993). Family and species Vernacular name ACANTHACEAE Monechma subsessile Umubazi (Oliv.) C.B. Clarke AMARANTHACEAE Cyathula uncinulata Igifashi (Schrad.) Schinz APIACEAE Caucalis incognita Akaturambisiti (Norman) Heywood et Jury ASTERACEAE Aspilia pluriseta Schweinf. Icyumwa cy'agasozi Berkkeya spekeana Oliv. Ikigwarara Bidens pilosa L. Inyabarasanya Guizotia scabra (Vis.) Chiov. Igishikashike Guthenbergia cordifolia Umweza Benth. Ex Oliv Microglossa pyrifolia Umuhe (Lam.) Kuntze Senecio maranguensis O. Hoffm. Imbatura Spilanthes mauritiana Gashegenyura (Rich. et Pers.) DC. Tagetes minuta L. Nyiramunukanabi Tithonia diversifolia Lkicamahirwe (Hemsl.) A. Gray Vernonia amygdalina Del. Umubilizi Vernonia miombicola Wild. Idoma CAESALPINIACEAE Cassia didymobotrya Fresen. Umubagabaga CHENOPODIACEAE Chenopodium ugandae Umugombe (Aellen) Aellen CRASSULACEAE Kalanchoe crenata Igitenetene (Andr.) Haw. EUPHORBIACEAE Clutia abyssinica Jaub. et Spach. Umutarishonga Macaranga kilimandscharica Pax Umusekera FABACEAE Eriosema montanum Baker f. Umugfunyantoke Indigofera arrecta Umusororo Hochst. ex A. Rich HYPERICACEAE Hypericum revolutum Vaml. Ikinyamucucu LAMIACEAE Coleus kilimandschari Gurke Igicunshu Leonotis nepetaefolia R.Br. Igicumucumu MALVACEAE Hibiscus fuscus Garcke Umutozo MELASTOMATACEAE Dissotis throthae Gilg Icyeba MIMOSACEAE Entada abyssinica Umusange Steud ex A. Rich. PEDALIACEAE Sesamum angolense Welw. Delele, Sope PHYTOLACCACEAE Phytolacca dodecandra Umuhoko l'Herit. POLYGONACEAE Rumex bequaertii De Wild Nyiramuko RANUNCULACEAE Clematis hirsuta Per. et Guill. Umunkamba var. hirsuta RUBIACEAE Pavetta ternifolia (Oliv.) Hiern. Umumenamabuye Pentas longiflora Oliver Isagara Virectaria major (Schumann) verdc. Umukilyi TILIACEAE Triumfetta rhomboidea Jacq. Umushyigura VERBENACEAE Clerodendrum myricoides (Hochst.) Umukuzanyana R.Br. ex Vatke Lantana trifolia L. Umuhengeri Family and species Traditional Uses ACANTHACEAE Monechma subsessile Acne, cough, dehydration (Oliv.) C.B. Clarke AMARANTHACEAE Cyathula uncinulata Dehydration, diarrhoea, wounds (Schrad.) Schinz APIACEAE Caucalis incognita (Norman) Heywood et Jury ASTERACEAE Aspilia pluriseta Schweinf. Kwashiorkor, worms, wounds Berkkeya spekeana Oliv. Anthrax, cough Bidens pilosa L. Furuncle, hepatitis, otitis, wounds Guizotia scabra (Vis.) Chiov. Agalactia, gonorrhoea, hepatitis Guthenbergia cordifolia East coast fever Benth. Ex Oliv Microglossa pyrifolia Cough, elephantiasis, wounds (Lam.) Kuntze Senecio maranguensis O. Hoffm. Cough, otitis, wounds Spilanthes mauritiana Malaria, pneumonia, tonsillitis (Rich. et Pers.) DC. Tagetes minuta L. Impotence, toothache Tithonia diversifolia Ascariasis, diarrhoea (Hemsl.) A. Gray Vernonia amygdalina Del. Ascariasis, hepatitis, malaria Vernonia miombicola Wild. Gonorrhoea, malaria, worms CAESALPINIACEAE Cassia didymobotrya Fresen. Ascariasis, neuropsychopathy CHENOPODIACEAE Chenopodium ugandae Eczema, hepatitis, snake bite (Aellen) Aellen CRASSULACEAE Kalanchoe crenata Otitis, skin diseases (Andr.) Haw. EUPHORBIACEAE Clutia abyssinica Jaub. et Spach. Abortifacient, gonorrhoea Macaranga kilimandscharica Pax Afterpains FABACEAE Eriosema montanum Baker f. Conjunctivitis, cough, snake bite Indigofera arrecta Emetic, furuncle, scabies Hochst. ex A. Rich HYPERICACEAE Hypericum revolutum Vaml. Cough, respiratory diseases LAMIACEAE Coleus kilimandschari Gurke Cough, worms Leonotis nepetaefolia R.Br. Hepatitis, pneumonia, wounds MALVACEAE Hibiscus fuscus Garcke Diarrhoea, pneumonia, sprain MELASTOMATACEAE Dissotis throthae Gilg Diarrhoea, wounds MIMOSACEAE Entada abyssinica Ascariasis, fever Steud ex A. Rich. PEDALIACEAE Sesamum angolense Welw. Skin diseases PHYTOLACCACEAE Phytolacca dodecandra Emetic, otitis, pneumonia l'Herit. POLYGONACEAE Rumex bequaertii De Wild Furuncle, kwashiorkor, worms RANUNCULACEAE Clematis hirsuta Per. et Guill. Abortifacient, urinary var. hirsuta diseases RUBIACEAE Pavetta ternifolia (Oliv.) Hiern. Skin diseases, worms Pentas longiflora Oliver Fever, worms Virectaria major (Schumann) verdc. Eye diseases, wounds TILIACEAE Triumfetta rhomboidea Jacq. Abortifacient, snake bite VERBENACEAE Clerodendrum myricoides (Hochst.) Constipation, hepatitis, R.Br. ex Vatke syphilis Lantana trifolia L. Gonorrhoea, heart failure Table 2 Anti-HIV-1 activity of Rwandan plant extracts. Family and species Plant part (a) [EC.sub.50] ([mu]g/ml) (b) ACANTHACEAE Monechma subsessile LF > 103.77 AMARANTHACEAE Cyathula uncinulata LF > 134.52 Cyathula uncinulata RT > 168.92 APIACEAE Caucalis incognita LF > 10.1 ASTERACEAE Aspilia pluriseta LF 16.13 Berkheya spekeana LF > 200.00 Bidens pilosa LF > 153.79 Guizotia scabra LF > 141.37 Guthenbergia cordifolia LF > 29.18 Microglossa pyrifolia LF > 140.10 Microglossa pyrifolia ST > 125.41 Senecio maranguensis LF > 28.01 Spilanthes mauritiana LF > 140.72 Tagetes minuta LF > 143.38 Tithonia diversifolia LF > 1.60 Vernonia amygdalina LF > 19.66 Vernonia miombicola LF > 14.62 CAESALPINIACEAE Cassia didymobotrya LF > 133.54 CHENOPODIACEAE Chenopodium ugandae LF > 91.92 CRASSULACEAE Kalanchoe crenata LF > 13.86 Kalanchoe crenata ST > 11.1 EUPHORBIACEAE Clutia abyssinica LF > 128.01 Macaranga kilimandscharica LF > 5.23 Macaranga kilimandscharica ST > 8.2 FABACEAE Eriosema montanum LF > 166.03 Indigofera arrecta LF > 280.1 Indigofera arrecta ST > 20.0 HYPERICACEAE Hypericum revolutum LF > 131.13 Hypericum revolutum RT > 11.9 LAMIACEAE Coleus kilimandschari LF > 158.45 Leonotis nepetaefolia LF > 140.33 MALVACEAE Hibiscus fuscus LF > 144.74 Hibiscus fuscus RT > 1.19 MELASTOMATACEAE Dissotis throthae LF > 174.36 MIMOSACEAE Entada abyssinica LF > 56.68 Entada abyssinica ST > 89.65 PEDALIACEAE Sesamum angolense LF > 50.52 PHYTOLACCACEAE Phytolacca dodecandra LF > 62.82 POLYGONACEAE Rumex bequaertii LF 17.695 RANUNCULACEAE Clematis hirsuta LF > 23.98 RUBIACEAE Pavetta ternifolia LF > 133.43 Pavetta ternifolia RT > 346.71 Pentas longiflora LF > 20.79 Pentas 1ongiflora RT > 125.40 Virectaria major LF > 186.23 TILIACEAE Triumfetta rhomboidea LF > 0.03 VERBENACEAE Clerodendrum myricoides LF > 114.19 Lantana trifolia > 4.01 Family and species [CC.sub.50]([mu]g/ml) (c) SI (d) ACANTHACEAE Monechma subsessile 103.77 < 1 AMARANTHACEAE Cyathula uncinulata 134.52 < 1 Cyathula uncinulata 168.92 < 1 APIACEAE Caucalis incognita 10.1 < 1 ASTERACEAE Aspilia pluriseta > 200.00 > 12 Berkheya spekeana > 200.00 > < 1 Bidens pilosa 153.79 < 1 Guizotia scabra 141.37 < 1 Guthenbergia cordifolia 29.18 < 1 Microglossa pyrifolia 140.10 < 1 Microglossa pyrifolia 125.41 < 1 Senecio maranguensis 28.01 < 1 Spilanthes mauritiana 140.72 < 1 Tagetes minuta 143.38 < 1 Tithonia diversifolia < 1.60 < 1 Vernonia amygdalina 19.66 < 1 Vernonia miombicola 14.62 < 1 CAESALPINIACEAE Cassia didymobotrya 133.54 < 1 CHENOPODIACEAE Chenopodium ugandae 91.92 < 1 CRASSULACEAE Kalanchoe crenata 13.86 < 1 Kalanchoe crenata 11.1 < 1 EUPHORBIACEAE Clutia abyssinica 128.01 < 1 Macaranga kilimandscharica 5.23 < 1 Macaranga kilimandscharica 8.2 < 1 FABACEAE Eriosema montanum 166.03 < 1 Indigofera arrecta 280.1 < 1 Indigofera arrecta > 20.0 > < 1 HYPERICACEAE Hypericum revolutum 131.13 < 1 Hypericum revolutum 11.9 < 1 LAMIACEAE Coleus kilimandschari 158.45 < 1 Leonotis nepetaefolia 140.33 < 1 MALVACEAE Hibiscus fuscus 144.74 < 1 Hibiscus fuscus 1.19 < 1 MELASTOMATACEAE Dissotis throthae 174.36 < 1 MIMOSACEAE Entada abyssinica 56.68 < 1 Entada abyssinica 89.65 < 1 PEDALIACEAE Sesamum angolense 50.52 < 1 PHYTOLACCACEAE Phytolacca dodecandra 62.82 < 1 POLYGONACEAE Rumex bequaertii > 200.00 > 11 RANUNCULACEAE Clematis hirsuta 23.98 < 1 RUBIACEAE Pavetta ternifolia 133.43 < 1 Pavetta ternifolia 346.71 < 1 Pentas longiflora 20.79 < 1 Pentas 1ongiflora 125.40 < 1 Virectaria major 186.23 < 1 TILIACEAE Triumfetta rhomboidea 0.03 < 1 VERBENACEAE Clerodendrum myricoides 114.19 < 1 Lantana trifolia 4.01 < 1 Family and species % protection (e) ACANTHACEAE Monechma subsessile 2 AMARANTHACEAE Cyathula uncinulata 2 Cyathula uncinulata 4 APIACEAE Caucalis incognita 6 ASTERACEAE Aspilia pluriseta 103 Berkheya spekeana 12 Bidens pilosa 2 Guizotia scabra 9 Guthenbergia cordifolia 5 Microglossa pyrifolia 1 Microglossa pyrifolia -5 Senecio maranguensis 11 Spilanthes mauritiana 8 Tagetes minuta 0 Tithonia diversifolia -26 Vernonia amygdalina -7 Vernonia miombicola 0 CAESALPINIACEAE Cassia didymobotrya -2 CHENOPODIACEAE Chenopodium ugandae 1 CRASSULACEAE Kalanchoe crenata 9 Kalanchoe crenata -5 EUPHORBIACEAE Clutia abyssinica 0 Macaranga kilimandscharica 7 Macaranga kilimandscharica 7 FABACEAE Eriosema montanum -4 Indigofera arrecta 11 Indigofera arrecta 26 HYPERICACEAE Hypericum revolutum -4 Hypericum revolutum 21 LAMIACEAE Coleus kilimandschari 5 Leonotis nepetaefolia -5 MALVACEAE Hibiscus fuscus 2 Hibiscus fuscus 21 MELASTOMATACEAE Dissotis throthae -10 MIMOSACEAE Entada abyssinica 1 Entada abyssinica 22 PEDALIACEAE Sesamum angolense 0 PHYTOLACCACEAE Phytolacca dodecandra 3 POLYGONACEAE Rumex bequaertii 89 RANUNCULACEAE Clematis hirsuta -1 RUBIACEAE Pavetta ternifolia 5 Pavetta ternifolia 1 Pentas longiflora 5 Pentas 1ongiflora 2 Virectaria major -5 TILIACEAE Triumfetta rhomboidea 26 VERBENACEAE Clerodendrum myricoides -2 Lantana trifolia 1 (a) LF - leaves RT - roots ST - stem (b) [EC.sub.50] = 50% Effective inhibitory concentration (c) [CC.sub.50] = 50% Cytotoxic concentration (d) SI = Selectivity Index ([CC.sub.50]/[EC.sub.50]) (e) Precent protection of viral cytopathic effect compared to control wells Table 3 Anti-HIV-1 activity of subfractions of Tithonia diversifolia. Tithonia [EC.sub.50] [CC.sub.50] SI (c) % pro- diversifolia ([mu]g/ml) (a) ([mu]g/ml) (b) tection (d) Petroleum ether > 18.3 18.3 < 1 -20 Ethylacetate > 0.2 < 0.2 < 1 -2 Water 0.04 > 20.0 > 461 104 (a) [EC.sub.50] = 50% Effectiv inhibitory concentration (b) [CC.sub.50] = 50% Cytotoxi concentration (c) SI = Selectivity Index ([CC.sub.50]/[EC.sub.50]) (d) Percent protection of vira cytopathic effect compared to control wells
This work was supported by grant no. 92/94-09 of the Flemish Government. T. De Bruyne and S. Apers are postdoctoral researchers of the Fund for Scientific Research -- Flanders (Belgium).
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P. Cos (1), N. Hermans (1), T. De Bruyne (1), S. Apers (1), J. B. Sindambiwe (1), M. Witvrouw (2), E. De Clercq (2), D. Vanden Berghe (1), L. Pieters (1), A. J. Vlietinck (1)
(1.) Faculty of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium
(2.) Department of Microbiology and Immunology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuyen, Belgium
A.J. Vlietinck, Faculty of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B - 2610 Antwerp, Belgium Tel: ++32-3-820 27 33; Fax: ++32-3-820 27 09; e-mail: firstname.lastname@example.org