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Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell.
Journal of Ethnopharmacology 64 (1999) 227 – 233
Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell. Ahmed El Tahir a,c, Gwiria M.H. Satti a,*, Sami A. Khalid b a
Department of Biochemistry, Faculty of Medicine, Uni6ersity of Khartoum, P.O.Box 102, Khartoum, Sudan b Department of Pharmacognosy, Faculty of Pharmacy, Uni6ersity of Khartoum, Khartoum, Sudan c Department of Biochemistry, Faculty of Medicine, Uni6ersity of Gezira, Gezira, Sudan Received 15 December 1997; received in revised form 8 July 1998; accepted 21 July 1998
Abstract The antiplasmodial activity of plant extracts related to four families was tested on chloroquine sensitive strain 3D7 and chloroquine resistant strain Dd2 of Plasmodium falciparum. The methanolic extract of Harrisonia abyssinica (Simaroubaceae) inhibited Dd2 with IC50 value of 4.7 mg/ml, while in 3D7, the IC50 value was 10 mg/ml. Most of the plants from the family Meliaceae showed highly potent antiplasmodial activity against the two tested strains. Khaya senegalensis, Azadirachta indica and Trichilia emetica showed IC50 values less than 5 mg/ml. The methanolic extract of Annona squamosa (Annonaceae) leaves showed high antiplasmodial activity with IC50 values of 2 and 30 mg/ml on 3D7 and Dd2, respectively. While stem bark showed moderate activity with IC50 values of 8.5 and 120 mg/ml on Dd2. Maytenus senegalensis (Celastraceae) possessed IC50 values of 3.9 on 3D7, 10 mg/ml on Dd2 and had no effect on lymphocyte proliferation even at the highest tested concentration; the IC50 was greater than 100 mg/ml. Liquid-liquid separation of the methanolic extract of M. senegalensis revealed that the dichloromethane extract possessed an IC50 value of only 2.1 mg/ml. Column fractionation of dichloromethane extract gave four fractions and fraction two showed an IC50 value of 0.5 mg/ml. Preliminary phytochemical analysis of dichloromethane fraction revealed terpenoids and traces of phenolic principles but no alkaloid, tannins or flavonoids were detected. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: In vitro antiplasmodial activity; Inhibition of lymphocytes; Medicinal plants; Sudan
1. Introduction
* Corresponding author.
Malaria is one of the most prevalent diseases in the world. It affects about 300–500 million each year, mostly from sub-Saharan Africa and causes
0378-8741/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-8741(98)00129-9
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
228
Table 1 The antiplasmodial activity of plants from the family Simaroubaceae tested on (3D7) and (Dd2) Plasmodium falciparum strains Plant species
Weight of plant part (g)
H. abyssinica leaves
600
H. abyssinica stem bark
144
A. excelsa leaves
124
A. excelsa stem bark
74
Yield of extract
L.I.a IC50 (mg/ml)
Strain
3D7 Dd2 3D7 Dd2
(g)
(%)
45
7.5
95
5.6
N.T.c
8.3 20 3.1
16 4.2
N.T. \100
Chloroquine diphospate
3D7 Dd2 3D7 Dd2 3D7 Dd2
ICb50 (mg/ml)
60 50 10 4.7 16 180 27 50 0.23 0.8
a
L.I., lymphocyte inhibition. IC50, the concentration of plant extract that caused 50% inhibtion of the cell growth c N.T., not tested. b
about 2.3 million deaths a year (World Health Organization, 1996). The problems of the resistance of the vector mosquitoes to insecticides and of the parasite to most of the commercially available antimalarials seriously weaken the control approaches (Wernsdorfer and Trigg, 1988). The success of quinine in the treatment of malaria for many decades, and later of artemisinin and its derivatives for treatment of cerebral malaria, has turned attention to plants as potential sources of antimalarial drugs (Wright and Phillipson, 1990). Sudan is polyethnic, with a diverse flora, and most of the people in rural areas rely on traditional medicine for the treatment of many infectious diseases. They commonly treated the recurrent fever typical of malaria with plant extracts. It is not clear whether those plants contain ingredients with antimalarial activity or that they exert their actions through other mechanisms, such as immuno-modulation (Luetting et al., 1989). The present study was carried out to screen the antiplasmodial activity of the plants used in traditional medicine and this may provide significant medical and economic benefit.
2. Experimental
2.1. Phytochemical methods 2.1.1. Plant material The plants studied, namely Harrisonia abyssinica Oliv., Ailanthus excelsa Roxb. (Simaroubaceae), Khaya senegalensis (Desr.) A. Juss., Azadirachta indica A. Juss., Trichilia emetica Vahl, Pseudocederla kotosifyi (Schweinf.) Harms. (Meliaceae) and Maytenus senegalensis (Lam.) Exell. (Celastraceae) were collected from the wild plants of the Sudan on the basis of ethanomedical information, whereas Annona squamosa L. (Annonaceae) was collected according to the botanical classification. Authentication was achieved by comparison with herbarium specimens by taxonomists and a voucher specimen from each plant was deposited in the Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum. 2.1.2. Preparation of plant materials Samples from the leaves and stems of the above mentioned plants were dried under the shade and coarsely powdered. The powdered materials were
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
229
Table 2 Antiplasmodlal activity of medicinal plants from the family Meliaceae screened for their activity on (3D7) and (Dd2) Plasmodium falciparum strains Plant species
Weight of plant part Yield of extract L.I.a IC50 (mg/ (g) ml) (g)
(%)
25
5
\100 \100
K. senegalensis leaves
500
K. senegalensis seeds
50
2.2
4.4
K. senegalensis seed water extract
50
0.5
5
K. senegalensis stem bark chloroform extract
30
3.1
4.2
K. senegalensis stem bark methanolic extract K. senegalensis butanolic extract K. senegalensis aqueous extract
30
10
50 \100
Strain
3D7 Dd2 3D7 Dd2 3D7 Dd2 3D7
ICb50 (mg/ml)
47 5.5 4.3 38 30 31.5 11.5
9
N.T.c
Dd2 3D7
25 150
N.T. N.T.
Dd2 Dd2
500 100
30 30
0.9
3.9
A. indica leaves
100
3.1
3.1
\100
A. indica stem bark
250
8.7
3.5
80
T. emetica leaves
300
29
9.6
T. emetica stem bark
215
18.9
8.8
\100
P. kotosifye leaves
395
21
5.3
\100
P. kotosifye stem bark
345
16
4.6
2.5
N.T.
3D7 Dd2 3D7 Dd2
5.8 1.7 8.5 40
3D7 Dd2 3D7 Dd2
17.5 2.5 8.5 200
3D7 Dd2 3D7 Dd2
15 50 40 45
a
L.I., lymphocyte inhibition. IC50, the concentration of plant extract that caused 50% inhibition of the cell growth. c NT, not tested. b
extracted by maceration in conical flask for 24 h at 37°C using 80% methanol and continuous shaking. Some of the methanolic extracts of M. senegalensis and K. senegalensis were partitioned between dichioromethane, ethyl acetate, chloroform and water. The aqueous plant extracts were dried whilst the organic solvents were removed by evaporation under reduced pressure. The dichloromethane extracts of M. senegalensis (1 g) were chromatographed over Silica gel 60 (particle size 0.063–0.2 mm and 70 – 230 mesh ASTM) eluted with a chloroform – methanol gradient. Each 10 ml of the solvent were collected in a tube according to the TLC pattern. The fractions giv-
ing similar chromatograms were pooled and dried. The final dry extracts obtained were stored at − 20°C until used. The weights of plant material used in preparing the extract and the yield obtained are shown in Tables 1–4.
2.2. Parasite culti6ation and in 6itro testing The chloroquine and pyrimethamine sensitive 3D7 strain and chloroquine resistant and pyrimethamine sensitive Dd2 strain were used in these experiments for in vitro assessment according to the method of Thaithong et al. (1983). Dried extracts of plant material were dissolved or
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
230
Table 3 Antiplasmodial activity of Annona squamosa (Annonaceae) on Plasmodium falciparum strains Plant species
A. squamosa leaves A. squamosa stem bark
Weight of plant parts (g)
Yield of extract
L.I.a IC50 (mg/ml)
Strain
ICb50 (mg/ml)
3D7 Dd2 3D7 Dd2
2 30 8.5 120
(g)
(%)
35
1.06
3.1%
N.T.c
125
3.91
2.8%
\100
a
L.I., lymphocyte inhibition. IC50, the concentration of plant extract that caused 50% inhibition of the cell growth. c N.T., not tested. b
micronized in RPMI 1640 medium. Four concentrations (0.5, 5, 50 and 500 mg/ml) were prepared in complete medium (RPMI 1640+10% human serum). Each extract was evaluated in triplicate and tested three times. After incubation at 38°C for 48 h, [3H]hypoxanthine was added to each well and the incubation continued for a further 18 h. The culture was harvested on filter paper by a cell harvester and the cells were counted in a scintillation countier.The IC50 value (concentration of drug or extract at which inhibition of parasite growth represents 50%) was calculated using Probit analysis. Chloroquine was tested concomitantly on each occasion.
received 20 ml of medium alone. PHA stimulated cells without plant extract were always included as positive control. The cultures were incubated at 37°C in a humidified atmosphere (5% CO2) for 24 h and then 20 ml of [3H]thymidine were added into each well and incubated for another 24 h. The PBM cells were harvested using a Skatron harvester and [3H]thymidine incorporation was measured as cpm in a liquid scintillation counter. The effect of crude extracts on human PBM Iymphocyte was estimated as previously calculated by Kemp et al. (1991).
3. Results and discussion
2.3. Lymphoproliferation assay for toxicity Human peripheral blood mononuclear cells (PBMC) from heparinized blood were isolated using lymphoprep solution (Nyegaard, Oslo, Norway). They were then washed three times in RPMI 1640 medium (Gibco) supplemented with 15% pooled human serum (complete medium) and penicillin (20 IU) plus streptomycin (20 mg/ml) according to Kemp et al. (1991). Each cell of 96-well round bottomed microtitre plates received 0.33× 106/ml PBMC in 150 ml of RPMI 1640. The plant extracts were diluted in the complete medium to make final test concentrations of 0.5, 5, 50 and 100 mg/ml. Each extract (20 ml) was added to PBMC culture, except the positive and negative controls, then 20 ml of phyto-hemagglutinin (47 mg/ml), (Difco) were added into all microplate wells, except the negative control which
Table 1 presents the antiplasmodial activity of the plants from the family Simaroubaceae. H. abyssinica stem bark inhibited the resistant strain (Dd2) more efficiently than the sensitive strain (3D7) with IC50 values of 4.79 0.113 (S.E.M.) and 1090.114 mg/ml, respectively. Whereas chloroquine diphosphate exhibited an IC50 equal to 0.89 0.112 and 0.239 0.160 mg/ml on Dd2 and 3D7, respectively. A. excelsa stem bark showed moderate activity with IC50 value of 27 mg/ml at 5 mg/ml. Certain species of this family has been used in traditional medicine throughout the tropical world in order to combat various diseases. The activity was attributed to their quassinoid content (Trager and Polonsky 1981; Bray et al., 1987). Table 2 shows the antiplasmodial activity of plants selected from the family Meliaceae and screened for their biological activity against P.
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
231
Table 4 Antiplasmodial activity of Maytenus senegalensis on Plasmodium falciparium strains Plant species
M. senegalensis leaves M. senegalensis stem bark
Weight of plant parts (g)
339
Yield of extract L.I.a IC50 (mg/ml) (g)
(%)
45
7.5
N.T.c
5.6
\100
2235
8.3
Strain
ICb50 (mg/ml)
3D7 Dd2 3D7 Dd2
65 5.1 3.9 10
a
L.I., lymphocyte inhibition IC50, the concentration of plant extract that caused 50% inhibition of the cell growth. c N.T., not tested. b
falciparum strains. Most plants from this family showed highly potent antiplasmodial activity against the two tested strains. K. senegalensis seed, A. indica leaves and T. emetica leaves showed IC50 values less than 5 mg/ml while the chloroform extract of K. senegalensis stem bark showed moderate activity on Dd2 with IC50 value of 25 mg/ml, while the methanolic extract had no activity at this concentration. Members of this family were subjected to intensive antimalarial screening and they were found to contain highly oxygenated terpenoids called limonoids which are biosynthetically related to the quassinoids (Connolly, 1983). The presence of limonoids such as methyl angolensate, Khayasin and Khivorin were reported in several Khaya species (Adesida et al., 1967; Adesogan and Taylor, 1968). A. indica was tested against the malaria parasite and the ethanolic extract of leaves showed an IC50 value of 5 mg/ml (Bray et al., 1985) which is comparable with our finding. Gedunin isolated from A. indica proved to be the most active compound of a series of 21 natural chemical products obtained from Sudanese traditional plants used as antimalarials
(Khalid et al., 1986). The T. emetica leaves exert high activity on P. falciparum strains (IC50 value less than 17.5 mg/ml) and at this concentration haemolysis of the red blood cells was also observed. However, parasite death may be secondary to haemolysis. This plant was also shown to inhibit Iymphocyte proliferation at low concentration (IC50 value 2.25 mg/ml), so its use as antimalarial plant is not recommended so far. A. squamosa leaves, family Annonaceae showed antiplasmodail activity with an IC50 value of 2 mg/ml on 3D7 strain and the stem bark showed moderate activity with an IC50 value of 8.5 mg/ml on 3D7 and 120 mg/ml on Dd2 (Table 3). In a study carried out by Bories et al. (1991) the methanolic extract of Annona species showed antiparasitic activity. The alkaloid isolated from A. squamosa showed larvicidal growth regulation and chemosterilant activities against Anopheles stephensis (Saxena et al., 1993). Table 4 presents the antiplasmodial activity of M. senegalensis, family Celastraceae on P. falciparum. M. senegalensis stem bark and leaves possessed IC50 values of 3.9 and 5.1 mg/ml on 3D7
Table 5 In vitro antiplasmodial activity of Maytenus senegalensis stem bark prepared using liquid–liquid partition on (3D7)
Table 6 Activity of fractions of dichloromethane extract of Maytenus senegalensis on Plasmodium falciparum (3D7)
Liquid fractions
IC50 (mg/ml)
Column fractions
IC50 (mg/ml)
Petroleum ether Dichloromethane Ethylacetate Aqueous extract
\500 2.1 \500 \500
F1 F2 F3 F4
35 0.5 150 500
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
232
Table 7 Phytochemical analysis of Maytenus senegalensis Principles
Alkaloids Phenolic Tannins Flavonoids Saponin Terpenoids
Test applied
Dragendorff reagent Ferric chloride Ferric chloride test Aluminum chloride RBCs haemolysis Vanillin-sulphuric acid and anisaldehyde
Extract MeOHa
P etherb
CH2CLc2
E acetated
+ + + + − ++
− − − − + −
− + − − − ++
+ + + + − +
a
MeOH, methanol. P ether, petroleum ether. c CH2CL2, dichloromethane. d E acetate, ethylacetate. b
and 10 and 65 mg/ml on Dd2, respectively. Liquid – liquid separations of the methanolic extract showed that the dichloromethane extract possessed an IC50 value of only 2.1 mg/ml (Table 5). Dichloromethane extract was purified using column chromatography, fraction 2 showed an IC50 value of 0.5 mg/ml and the potent antiplasmodial activity was concentrated at the high non-polar fraction (Table 6). Preliminary phytochemical analysis of the dichloromethane fraction of M. senegalensis revealed terpenoids and traces of phenolic principles but no alkaloid, tannins or flavonoids were detected (Table 7). M. senegalensis is used traditionally as an antimalarial in Tanzania and Senegal (Von Sengbusch, 1980) and as remedy for the treatment of fever in Madagascar (Rasoanaivo et al., 1992). Many bioactive compounds such as the maytansinoid (with antitumour activity), triterpenes and sesquiterepene polyesters have so far been isolated (Abraham et al., 1971; Wang et al., 1981). It can be concluded that some Sudanese plants used in traditional medicine possess a potent antiplasmodial activity with minor effects on lymphocyte proliferation and hence we propose that those plants should be further investigated.
References Abraham, D.J., Trojanek, J., Munzing, H.P., Fong, H.H., Farnswoth, N.R., 1971. Structure elucidation of
maytenonic acid a new triterpene from Maytenus senegalensis. Journal of Pharmaceutical Sciences 60 (7), 1085 – 1087. Adesida, G.A., Adesogan, E.K., Taylor, D.A.H. 1967. Extractive from Khaya senegalensis A. Juss. Journal of the Chemical Society of Chemists Communication, pp. 790. Adesogan, E.K., Taylor, D.A.H., 1968. Extractive from the seed of Khaya senegalensis (Desr.) A. Juss. Journal of the Chemical Society C 16, 1974 – 1981. Bories, C., Loiseau, P., Cortes, D., Myint, S.H., Hocquemiller, R., Gayral, P., Cave, A., Laurens, A., 1991. Antiparasitic activity of Annona muricata and Annona cherimolia seeds. Planta Medica 57 (5), 434 – 436. Bray, D.H., Connolly, J. D., Peters, W., Phillipson, J. D., Robinson, B.L., Tella, A., Thebtarananth, Y., Warhurst, D.C., Yutharong, Y., 1985. Antimalarial activity of some limonoids. Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 426. Bray, D.H., Boardman, P., O’Neill, M.J, Chan, K.L., Phillipson, J. D., Warhurst, D.C., Suffness, M., 1987. Plants as sources of antimalarial drugs 5: activities of Ailanthus altissima stem constituents and of some related quassinoids. Phytotherapy Research 1, 22 – 24. Connolly, D.L. 1983. Chemistry of limonoids of the Meliaceae and Cneoraceae. In: Waterman, P.G., Grundon, M.F. (Eds.), Chemistry and Chemical Taxonomy of the Rutales. Academic Press, London. Kemp, M., Theander, T.G., Handman, E., Hey, A.S., Kurtzhals, J.A.L., Hviid, L., Sorensen, A.L., Were, J.O.B., Koech, P.K., Kharazmi, A., 1991. Activation of human T Iymphocytes by leishmania liopphosphoglycan. Scandinavian Journal of Immunology 33, 219 – 224. Khalid, S.A., Farouk, A., Geary, T.G., Jensen, J.B., 1986. Potential antimalarial candidates from African plants: an in vitro approach using Plasmodium falciparum. Journal of Ethnopharmacology 15, 201 – 209.
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233 Luetting, P., Steinmuller, C., Gifford, G. E., Wagner, H., Lohmann-Matthes, M. L., 1989. Macrophage activation by polysaccharide arabinogalacton isolated from plant cell cultures of Echinaceae purpurea. Journal of the National Cancer Institute 9, 669–675. Rasoanaivo, P., Petitjean, A., Ratsimamanga-Urverg, S., Pakoto-Ratsimamanga, A., 1992. Medicinal plants used to treat malaria in Madagascar. Journal of Ethnopharmacology 37, 117. Saxena, R.C., Harshan, V., Saxena, A., Sukumaran, P., Sharma, M.C., Kumar, M. L., 1993. Larvicidal and Chemosterilant activity of Annona squamosa alkaloids against Anopheles stephensi. Journal of the American Mosquito Control Association 9 (1), 84–87. Thaithong, S., Beale, G.H., Chutmongkonkul, M., 1983. Susceptibility of Plasmodium falciparum to 5 drugs: an in vitro study of isolates mainly from Thailand. Transactions of the Royal Society of Tropical Medicine and Hygiene 77 (2), 228 – 231. Trager, W., Polonsky, J., 1981. Antimalarial activity of quassi-
.
233
noids against chloroquine -resistant Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 30, 531 – 537. Von Sengbusch, V. 1980. Das Entwicklungspatential Afrikanischer Heilpflanzen. IFB, Mockmuhl, pp. 1 – 365. Wang, X.F., Chen, J.Y., Wei, R.F., Jiang, D.Q., 1981. Studies on antitumor constituents of Maytenus confertiflora luo et chen II Isolation and characterization of maytansine and maytanprine from the stems (abstract). Yao Hsueh-HuschPao 16 (8), 628 – 630. Wernsdorfer, W.H., Trigg, P.I. 1988. Recent progress of malaria research: chemotherapy. In: Wernsdorfer, W.H., McGregor, Sir I. (Eds.), Malaria Principle and Practice of Malariology. Vol. 2. Churchill Livingstone, London, pp. 1569 – 1674. World Health Organization, 1996. Malaria distribution. A weekly epidemiological record. 71 (3), 17 – 24. Wright, C.W., Phillipson, J.D., 1990. Natural products and the development of selective antiprotozoal drugs. Phytotherapy Research 4, 127 – 139.
Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Maytenus senegalensis (Lam.) Exell. Ahmed El Tahir a,c, Gwiria M.H. Satti a,*, Sami A. Khalid b a
Department of Biochemistry, Faculty of Medicine, Uni6ersity of Khartoum, P.O.Box 102, Khartoum, Sudan b Department of Pharmacognosy, Faculty of Pharmacy, Uni6ersity of Khartoum, Khartoum, Sudan c Department of Biochemistry, Faculty of Medicine, Uni6ersity of Gezira, Gezira, Sudan Received 15 December 1997; received in revised form 8 July 1998; accepted 21 July 1998
Abstract The antiplasmodial activity of plant extracts related to four families was tested on chloroquine sensitive strain 3D7 and chloroquine resistant strain Dd2 of Plasmodium falciparum. The methanolic extract of Harrisonia abyssinica (Simaroubaceae) inhibited Dd2 with IC50 value of 4.7 mg/ml, while in 3D7, the IC50 value was 10 mg/ml. Most of the plants from the family Meliaceae showed highly potent antiplasmodial activity against the two tested strains. Khaya senegalensis, Azadirachta indica and Trichilia emetica showed IC50 values less than 5 mg/ml. The methanolic extract of Annona squamosa (Annonaceae) leaves showed high antiplasmodial activity with IC50 values of 2 and 30 mg/ml on 3D7 and Dd2, respectively. While stem bark showed moderate activity with IC50 values of 8.5 and 120 mg/ml on Dd2. Maytenus senegalensis (Celastraceae) possessed IC50 values of 3.9 on 3D7, 10 mg/ml on Dd2 and had no effect on lymphocyte proliferation even at the highest tested concentration; the IC50 was greater than 100 mg/ml. Liquid-liquid separation of the methanolic extract of M. senegalensis revealed that the dichloromethane extract possessed an IC50 value of only 2.1 mg/ml. Column fractionation of dichloromethane extract gave four fractions and fraction two showed an IC50 value of 0.5 mg/ml. Preliminary phytochemical analysis of dichloromethane fraction revealed terpenoids and traces of phenolic principles but no alkaloid, tannins or flavonoids were detected. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: In vitro antiplasmodial activity; Inhibition of lymphocytes; Medicinal plants; Sudan
1. Introduction
* Corresponding author.
Malaria is one of the most prevalent diseases in the world. It affects about 300–500 million each year, mostly from sub-Saharan Africa and causes
0378-8741/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-8741(98)00129-9
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
228
Table 1 The antiplasmodial activity of plants from the family Simaroubaceae tested on (3D7) and (Dd2) Plasmodium falciparum strains Plant species
Weight of plant part (g)
H. abyssinica leaves
600
H. abyssinica stem bark
144
A. excelsa leaves
124
A. excelsa stem bark
74
Yield of extract
L.I.a IC50 (mg/ml)
Strain
3D7 Dd2 3D7 Dd2
(g)
(%)
45
7.5
95
5.6
N.T.c
8.3 20 3.1
16 4.2
N.T. \100
Chloroquine diphospate
3D7 Dd2 3D7 Dd2 3D7 Dd2
ICb50 (mg/ml)
60 50 10 4.7 16 180 27 50 0.23 0.8
a
L.I., lymphocyte inhibition. IC50, the concentration of plant extract that caused 50% inhibtion of the cell growth c N.T., not tested. b
about 2.3 million deaths a year (World Health Organization, 1996). The problems of the resistance of the vector mosquitoes to insecticides and of the parasite to most of the commercially available antimalarials seriously weaken the control approaches (Wernsdorfer and Trigg, 1988). The success of quinine in the treatment of malaria for many decades, and later of artemisinin and its derivatives for treatment of cerebral malaria, has turned attention to plants as potential sources of antimalarial drugs (Wright and Phillipson, 1990). Sudan is polyethnic, with a diverse flora, and most of the people in rural areas rely on traditional medicine for the treatment of many infectious diseases. They commonly treated the recurrent fever typical of malaria with plant extracts. It is not clear whether those plants contain ingredients with antimalarial activity or that they exert their actions through other mechanisms, such as immuno-modulation (Luetting et al., 1989). The present study was carried out to screen the antiplasmodial activity of the plants used in traditional medicine and this may provide significant medical and economic benefit.
2. Experimental
2.1. Phytochemical methods 2.1.1. Plant material The plants studied, namely Harrisonia abyssinica Oliv., Ailanthus excelsa Roxb. (Simaroubaceae), Khaya senegalensis (Desr.) A. Juss., Azadirachta indica A. Juss., Trichilia emetica Vahl, Pseudocederla kotosifyi (Schweinf.) Harms. (Meliaceae) and Maytenus senegalensis (Lam.) Exell. (Celastraceae) were collected from the wild plants of the Sudan on the basis of ethanomedical information, whereas Annona squamosa L. (Annonaceae) was collected according to the botanical classification. Authentication was achieved by comparison with herbarium specimens by taxonomists and a voucher specimen from each plant was deposited in the Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum. 2.1.2. Preparation of plant materials Samples from the leaves and stems of the above mentioned plants were dried under the shade and coarsely powdered. The powdered materials were
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
229
Table 2 Antiplasmodlal activity of medicinal plants from the family Meliaceae screened for their activity on (3D7) and (Dd2) Plasmodium falciparum strains Plant species
Weight of plant part Yield of extract L.I.a IC50 (mg/ (g) ml) (g)
(%)
25
5
\100 \100
K. senegalensis leaves
500
K. senegalensis seeds
50
2.2
4.4
K. senegalensis seed water extract
50
0.5
5
K. senegalensis stem bark chloroform extract
30
3.1
4.2
K. senegalensis stem bark methanolic extract K. senegalensis butanolic extract K. senegalensis aqueous extract
30
10
50 \100
Strain
3D7 Dd2 3D7 Dd2 3D7 Dd2 3D7
ICb50 (mg/ml)
47 5.5 4.3 38 30 31.5 11.5
9
N.T.c
Dd2 3D7
25 150
N.T. N.T.
Dd2 Dd2
500 100
30 30
0.9
3.9
A. indica leaves
100
3.1
3.1
\100
A. indica stem bark
250
8.7
3.5
80
T. emetica leaves
300
29
9.6
T. emetica stem bark
215
18.9
8.8
\100
P. kotosifye leaves
395
21
5.3
\100
P. kotosifye stem bark
345
16
4.6
2.5
N.T.
3D7 Dd2 3D7 Dd2
5.8 1.7 8.5 40
3D7 Dd2 3D7 Dd2
17.5 2.5 8.5 200
3D7 Dd2 3D7 Dd2
15 50 40 45
a
L.I., lymphocyte inhibition. IC50, the concentration of plant extract that caused 50% inhibition of the cell growth. c NT, not tested. b
extracted by maceration in conical flask for 24 h at 37°C using 80% methanol and continuous shaking. Some of the methanolic extracts of M. senegalensis and K. senegalensis were partitioned between dichioromethane, ethyl acetate, chloroform and water. The aqueous plant extracts were dried whilst the organic solvents were removed by evaporation under reduced pressure. The dichloromethane extracts of M. senegalensis (1 g) were chromatographed over Silica gel 60 (particle size 0.063–0.2 mm and 70 – 230 mesh ASTM) eluted with a chloroform – methanol gradient. Each 10 ml of the solvent were collected in a tube according to the TLC pattern. The fractions giv-
ing similar chromatograms were pooled and dried. The final dry extracts obtained were stored at − 20°C until used. The weights of plant material used in preparing the extract and the yield obtained are shown in Tables 1–4.
2.2. Parasite culti6ation and in 6itro testing The chloroquine and pyrimethamine sensitive 3D7 strain and chloroquine resistant and pyrimethamine sensitive Dd2 strain were used in these experiments for in vitro assessment according to the method of Thaithong et al. (1983). Dried extracts of plant material were dissolved or
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
230
Table 3 Antiplasmodial activity of Annona squamosa (Annonaceae) on Plasmodium falciparum strains Plant species
A. squamosa leaves A. squamosa stem bark
Weight of plant parts (g)
Yield of extract
L.I.a IC50 (mg/ml)
Strain
ICb50 (mg/ml)
3D7 Dd2 3D7 Dd2
2 30 8.5 120
(g)
(%)
35
1.06
3.1%
N.T.c
125
3.91
2.8%
\100
a
L.I., lymphocyte inhibition. IC50, the concentration of plant extract that caused 50% inhibition of the cell growth. c N.T., not tested. b
micronized in RPMI 1640 medium. Four concentrations (0.5, 5, 50 and 500 mg/ml) were prepared in complete medium (RPMI 1640+10% human serum). Each extract was evaluated in triplicate and tested three times. After incubation at 38°C for 48 h, [3H]hypoxanthine was added to each well and the incubation continued for a further 18 h. The culture was harvested on filter paper by a cell harvester and the cells were counted in a scintillation countier.The IC50 value (concentration of drug or extract at which inhibition of parasite growth represents 50%) was calculated using Probit analysis. Chloroquine was tested concomitantly on each occasion.
received 20 ml of medium alone. PHA stimulated cells without plant extract were always included as positive control. The cultures were incubated at 37°C in a humidified atmosphere (5% CO2) for 24 h and then 20 ml of [3H]thymidine were added into each well and incubated for another 24 h. The PBM cells were harvested using a Skatron harvester and [3H]thymidine incorporation was measured as cpm in a liquid scintillation counter. The effect of crude extracts on human PBM Iymphocyte was estimated as previously calculated by Kemp et al. (1991).
3. Results and discussion
2.3. Lymphoproliferation assay for toxicity Human peripheral blood mononuclear cells (PBMC) from heparinized blood were isolated using lymphoprep solution (Nyegaard, Oslo, Norway). They were then washed three times in RPMI 1640 medium (Gibco) supplemented with 15% pooled human serum (complete medium) and penicillin (20 IU) plus streptomycin (20 mg/ml) according to Kemp et al. (1991). Each cell of 96-well round bottomed microtitre plates received 0.33× 106/ml PBMC in 150 ml of RPMI 1640. The plant extracts were diluted in the complete medium to make final test concentrations of 0.5, 5, 50 and 100 mg/ml. Each extract (20 ml) was added to PBMC culture, except the positive and negative controls, then 20 ml of phyto-hemagglutinin (47 mg/ml), (Difco) were added into all microplate wells, except the negative control which
Table 1 presents the antiplasmodial activity of the plants from the family Simaroubaceae. H. abyssinica stem bark inhibited the resistant strain (Dd2) more efficiently than the sensitive strain (3D7) with IC50 values of 4.79 0.113 (S.E.M.) and 1090.114 mg/ml, respectively. Whereas chloroquine diphosphate exhibited an IC50 equal to 0.89 0.112 and 0.239 0.160 mg/ml on Dd2 and 3D7, respectively. A. excelsa stem bark showed moderate activity with IC50 value of 27 mg/ml at 5 mg/ml. Certain species of this family has been used in traditional medicine throughout the tropical world in order to combat various diseases. The activity was attributed to their quassinoid content (Trager and Polonsky 1981; Bray et al., 1987). Table 2 shows the antiplasmodial activity of plants selected from the family Meliaceae and screened for their biological activity against P.
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231
Table 4 Antiplasmodial activity of Maytenus senegalensis on Plasmodium falciparium strains Plant species
M. senegalensis leaves M. senegalensis stem bark
Weight of plant parts (g)
339
Yield of extract L.I.a IC50 (mg/ml) (g)
(%)
45
7.5
N.T.c
5.6
\100
2235
8.3
Strain
ICb50 (mg/ml)
3D7 Dd2 3D7 Dd2
65 5.1 3.9 10
a
L.I., lymphocyte inhibition IC50, the concentration of plant extract that caused 50% inhibition of the cell growth. c N.T., not tested. b
falciparum strains. Most plants from this family showed highly potent antiplasmodial activity against the two tested strains. K. senegalensis seed, A. indica leaves and T. emetica leaves showed IC50 values less than 5 mg/ml while the chloroform extract of K. senegalensis stem bark showed moderate activity on Dd2 with IC50 value of 25 mg/ml, while the methanolic extract had no activity at this concentration. Members of this family were subjected to intensive antimalarial screening and they were found to contain highly oxygenated terpenoids called limonoids which are biosynthetically related to the quassinoids (Connolly, 1983). The presence of limonoids such as methyl angolensate, Khayasin and Khivorin were reported in several Khaya species (Adesida et al., 1967; Adesogan and Taylor, 1968). A. indica was tested against the malaria parasite and the ethanolic extract of leaves showed an IC50 value of 5 mg/ml (Bray et al., 1985) which is comparable with our finding. Gedunin isolated from A. indica proved to be the most active compound of a series of 21 natural chemical products obtained from Sudanese traditional plants used as antimalarials
(Khalid et al., 1986). The T. emetica leaves exert high activity on P. falciparum strains (IC50 value less than 17.5 mg/ml) and at this concentration haemolysis of the red blood cells was also observed. However, parasite death may be secondary to haemolysis. This plant was also shown to inhibit Iymphocyte proliferation at low concentration (IC50 value 2.25 mg/ml), so its use as antimalarial plant is not recommended so far. A. squamosa leaves, family Annonaceae showed antiplasmodail activity with an IC50 value of 2 mg/ml on 3D7 strain and the stem bark showed moderate activity with an IC50 value of 8.5 mg/ml on 3D7 and 120 mg/ml on Dd2 (Table 3). In a study carried out by Bories et al. (1991) the methanolic extract of Annona species showed antiparasitic activity. The alkaloid isolated from A. squamosa showed larvicidal growth regulation and chemosterilant activities against Anopheles stephensis (Saxena et al., 1993). Table 4 presents the antiplasmodial activity of M. senegalensis, family Celastraceae on P. falciparum. M. senegalensis stem bark and leaves possessed IC50 values of 3.9 and 5.1 mg/ml on 3D7
Table 5 In vitro antiplasmodial activity of Maytenus senegalensis stem bark prepared using liquid–liquid partition on (3D7)
Table 6 Activity of fractions of dichloromethane extract of Maytenus senegalensis on Plasmodium falciparum (3D7)
Liquid fractions
IC50 (mg/ml)
Column fractions
IC50 (mg/ml)
Petroleum ether Dichloromethane Ethylacetate Aqueous extract
\500 2.1 \500 \500
F1 F2 F3 F4
35 0.5 150 500
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233
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Table 7 Phytochemical analysis of Maytenus senegalensis Principles
Alkaloids Phenolic Tannins Flavonoids Saponin Terpenoids
Test applied
Dragendorff reagent Ferric chloride Ferric chloride test Aluminum chloride RBCs haemolysis Vanillin-sulphuric acid and anisaldehyde
Extract MeOHa
P etherb
CH2CLc2
E acetated
+ + + + − ++
− − − − + −
− + − − − ++
+ + + + − +
a
MeOH, methanol. P ether, petroleum ether. c CH2CL2, dichloromethane. d E acetate, ethylacetate. b
and 10 and 65 mg/ml on Dd2, respectively. Liquid – liquid separations of the methanolic extract showed that the dichloromethane extract possessed an IC50 value of only 2.1 mg/ml (Table 5). Dichloromethane extract was purified using column chromatography, fraction 2 showed an IC50 value of 0.5 mg/ml and the potent antiplasmodial activity was concentrated at the high non-polar fraction (Table 6). Preliminary phytochemical analysis of the dichloromethane fraction of M. senegalensis revealed terpenoids and traces of phenolic principles but no alkaloid, tannins or flavonoids were detected (Table 7). M. senegalensis is used traditionally as an antimalarial in Tanzania and Senegal (Von Sengbusch, 1980) and as remedy for the treatment of fever in Madagascar (Rasoanaivo et al., 1992). Many bioactive compounds such as the maytansinoid (with antitumour activity), triterpenes and sesquiterepene polyesters have so far been isolated (Abraham et al., 1971; Wang et al., 1981). It can be concluded that some Sudanese plants used in traditional medicine possess a potent antiplasmodial activity with minor effects on lymphocyte proliferation and hence we propose that those plants should be further investigated.
References Abraham, D.J., Trojanek, J., Munzing, H.P., Fong, H.H., Farnswoth, N.R., 1971. Structure elucidation of
maytenonic acid a new triterpene from Maytenus senegalensis. Journal of Pharmaceutical Sciences 60 (7), 1085 – 1087. Adesida, G.A., Adesogan, E.K., Taylor, D.A.H. 1967. Extractive from Khaya senegalensis A. Juss. Journal of the Chemical Society of Chemists Communication, pp. 790. Adesogan, E.K., Taylor, D.A.H., 1968. Extractive from the seed of Khaya senegalensis (Desr.) A. Juss. Journal of the Chemical Society C 16, 1974 – 1981. Bories, C., Loiseau, P., Cortes, D., Myint, S.H., Hocquemiller, R., Gayral, P., Cave, A., Laurens, A., 1991. Antiparasitic activity of Annona muricata and Annona cherimolia seeds. Planta Medica 57 (5), 434 – 436. Bray, D.H., Connolly, J. D., Peters, W., Phillipson, J. D., Robinson, B.L., Tella, A., Thebtarananth, Y., Warhurst, D.C., Yutharong, Y., 1985. Antimalarial activity of some limonoids. Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 426. Bray, D.H., Boardman, P., O’Neill, M.J, Chan, K.L., Phillipson, J. D., Warhurst, D.C., Suffness, M., 1987. Plants as sources of antimalarial drugs 5: activities of Ailanthus altissima stem constituents and of some related quassinoids. Phytotherapy Research 1, 22 – 24. Connolly, D.L. 1983. Chemistry of limonoids of the Meliaceae and Cneoraceae. In: Waterman, P.G., Grundon, M.F. (Eds.), Chemistry and Chemical Taxonomy of the Rutales. Academic Press, London. Kemp, M., Theander, T.G., Handman, E., Hey, A.S., Kurtzhals, J.A.L., Hviid, L., Sorensen, A.L., Were, J.O.B., Koech, P.K., Kharazmi, A., 1991. Activation of human T Iymphocytes by leishmania liopphosphoglycan. Scandinavian Journal of Immunology 33, 219 – 224. Khalid, S.A., Farouk, A., Geary, T.G., Jensen, J.B., 1986. Potential antimalarial candidates from African plants: an in vitro approach using Plasmodium falciparum. Journal of Ethnopharmacology 15, 201 – 209.
A. El Tahir et al. / Journal of Ethnopharmacology 64 (1999) 227–233 Luetting, P., Steinmuller, C., Gifford, G. E., Wagner, H., Lohmann-Matthes, M. L., 1989. Macrophage activation by polysaccharide arabinogalacton isolated from plant cell cultures of Echinaceae purpurea. Journal of the National Cancer Institute 9, 669–675. Rasoanaivo, P., Petitjean, A., Ratsimamanga-Urverg, S., Pakoto-Ratsimamanga, A., 1992. Medicinal plants used to treat malaria in Madagascar. Journal of Ethnopharmacology 37, 117. Saxena, R.C., Harshan, V., Saxena, A., Sukumaran, P., Sharma, M.C., Kumar, M. L., 1993. Larvicidal and Chemosterilant activity of Annona squamosa alkaloids against Anopheles stephensi. Journal of the American Mosquito Control Association 9 (1), 84–87. Thaithong, S., Beale, G.H., Chutmongkonkul, M., 1983. Susceptibility of Plasmodium falciparum to 5 drugs: an in vitro study of isolates mainly from Thailand. Transactions of the Royal Society of Tropical Medicine and Hygiene 77 (2), 228 – 231. Trager, W., Polonsky, J., 1981. Antimalarial activity of quassi-
.
233
noids against chloroquine -resistant Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 30, 531 – 537. Von Sengbusch, V. 1980. Das Entwicklungspatential Afrikanischer Heilpflanzen. IFB, Mockmuhl, pp. 1 – 365. Wang, X.F., Chen, J.Y., Wei, R.F., Jiang, D.Q., 1981. Studies on antitumor constituents of Maytenus confertiflora luo et chen II Isolation and characterization of maytansine and maytanprine from the stems (abstract). Yao Hsueh-HuschPao 16 (8), 628 – 630. Wernsdorfer, W.H., Trigg, P.I. 1988. Recent progress of malaria research: chemotherapy. In: Wernsdorfer, W.H., McGregor, Sir I. (Eds.), Malaria Principle and Practice of Malariology. Vol. 2. Churchill Livingstone, London, pp. 1569 – 1674. World Health Organization, 1996. Malaria distribution. A weekly epidemiological record. 71 (3), 17 – 24. Wright, C.W., Phillipson, J.D., 1990. Natural products and the development of selective antiprotozoal drugs. Phytotherapy Research 4, 127 – 139.