Chemical and biological studies on traditional anti-malarial plants from Meru and Kilifi districts
Kirira, peter Gakio
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More than half of the world's population live in areas where they are at risk of malaria infection. Africa contributes 90% of > 2 million deaths and > 500 million clinical cases annually. Malaria accounts for 10% of the continental disease burden. Pregnant women and children under 5 years are the main risk groups in the endemic areas. Malaria control is becoming more difficult due to the spread of resistance of anopheline mosquitoes and the parasites to insecticides and drugs, respectively. The absence of an effective commercial anti-malarial vaccine that offers full protection despite several decades of intensive research is a dangerous situation. Emergence of resistant malaria parasites has caused a crisis in the use of anti-malarial drugs for prophylaxis and therapy. Chemotherapy being the primary strategy of malaria control in the developing world, new anti-malarial compounds with different modes of action are urgently required. Plants are used widely in traditional health systems to treat a variety of diseases. Ethnobotanical information has previously provided potent anti-malarial compounds like quinine, artemisinin, cryptolepine and nitidine that led to the development of synthetic drugs such as chloroquine and artemether. It is therefore necessary to evaluate the traditional anti-malarial plants with the aim of incorporating them into the national health care systems or discovering new potent anti-plasmodial molecules which may be developed into drugs or used as templates for the development of new more potent synthetic analogues. In the search for new anti-malarial principles, we carried out bio-evaluation of 10 plants used to treat malaria in Meru and Kilifi Districts. Preliminary activity and cytotoxicity studies of extracts were carried out using brine shrimp tests. Only Neoboutonia macrocalyx, Azadirachta indica, Fagaropsis angolensis and Harrisonia abyssinica showed toxicity (LD5o 41.69 f 0.9, 101.26 f 3.7, 173.48 f 0.6 and 234.71 f 11.5 µg/ml, respectively) for aqueous extracts. However, methanol extracts from N. macrocalyx, F. angolensis, A. indica, Zanthoxylum usambarense, Strychnos heningsii, Carissa edulis, Withania somnifera and H. abyssinica (LD5o 21.04 f 1.8, 57.09 f 1.4, 61.43 f 2.9, 97.66 f 3.6, 101.22 f 3.2, 186.71 f 6.9, 207.27 f 0.7 and 217.34 f 7.2 µg/ml, respectively) exhibited toxicity against brine shrimp nauplii. Extracts were screened against CQsusceptible and CQ-resistant strain of Plasmodium falciparum (NF54 & ENT30, respectively). The order of anti-plasmodial activity was as follows: F. angolensis (IC50 10.65 f 1.23), Z. usambarense (IC5o 14.33 ± 4.22), S. heningsii (IC50 73.39 f 9,75), Myrica salicifolia (IC50 85.97 ± 5.48), H. abyssinica (IC50 89.74 f 8.12), N. macrocalyx (IC50 92.85 f 7.65), C. edulis (IC50 > 250), A. indica (IC5o > 250), Acacia nilotica (IC50> 250) and W somnifera (IC5o > 250 µg/ml) for aqueous extracts against ENT30 and: F. angolensis (IC5o 5.04 f 0.68), Z. usambarense (IC5o 5.54 ± 1.70), M. salicifolia (IC5o 55.89 f 2.00), A. nilotica (IC5o 73.59 ± 2.87), N. macrocalyx (IC5o 78.44 ±2.89), H. abyssinica (IC5o 79.50 f 3.31), W somnifera (IC5o 145.86 f 2.23), S. heningsii (IC5o 190.0 f 16.85), C. edulis (IC5o > 250 ), A. indica (IC5o > 250 µg/ml) for methanol extracts against the same isolate. Two plants, F. angolensis and Z. usambarense, showed good in vitro anti-plasmodial activity (IC50 5.0-11 µg/ml) against CQ-resistant strain. For NF54, the order was: Z. usambarense (IC50 5.25 ± 0.27), F. angolensis (ICso 6.13 ± 1.15), M. salicifolia (ICso 66.84 ± 2.88), S. heningsii (ICso 67.16 ± 8.78), N. macrocalyx (IC50 84.56 ± 8.93), H. abyssinica (IC50 86.56 ± 3.21), C. edulis (IC50 148.53 ± 12.65), A. nilotica (ICso 153.79 ± 15.79), A. indica (IC50 > 250 ), W. somnifera (ICso > 250 Ng/ml) for the aqueous extracts and: Z. usambarense (IC50 3.20 ± 0.45), F. angolensis (IC50 4.68 ± 0.09), M. salicifolia (ICso 51.07 ± 1.70), A. nilotica (IC50 70.33 ± 1.89), H. abyssinica (IC50 72.66 ± 1.39), N. macrocalyx (IC5078.40 ± 4.68), W. somnifera. (IC50 125.59 ± 1.30), S. heningsii (IC5o 157.91 ± 10.03), C. edulis (IC50> 250 ), A. indica (IC50 > 250 µg/ml) for the methanol extracts. Two plants, F. angolensis and Z. usambarense, showed good in vitro anti-plasmodial activity (ICso 3-6 µg/ml) against CQsensitive strain. Extracts of F. angolensis and N. macrocalyx were subjected to bioassay guided fractionation to avail 9 compounds. 6,7-Epoxy-4,5,9-trihydroxy-13-hexadecanoate-20dodecanoate-l-tiglien-1-3-one (140), 6,7-epoxy-4,5,9,20-tetrahydroxy-13-tetradecanoate1-tiglien-3-one (141), xadecanoate-13-dedecan20-hexadecanoate-13-dodecanoate-1,6-tigliadien-3-one (142), 12-deoxyphorbol-13-pentadecanoate (145) and methyl 3heptaeicosanoyloxyoleanoate (143) are being reported for the first time while stigmasterol (126) and montanin-20-palmitate (79) are known compounds. A further two compounds, P3 and K1, were also isolated but not characterized. The order of in vitro anti-plasmodial activity of some of the isolated compounds is: 4,9dihydroxy-20-hexadecanoate-l3-dodecanoate-1,6-tigliadien-3-one and montanin-20palmitate mixture (IC50 241.39±4.73), P3 (IC50 241.63±18.24), 4,9-dihydroxy-20hexadecanoate-l3-dodecanoate-1,6-tigliadien-3 -one (>250), stigmasterol (IC50>250) and Kl (IC50 >250 gg/ml) against ENT30. Similarly, only P3 had mild anti-plasmodial activity (IC50 237.47±11.23 gg/ml) against NF54. P3 had high cytotoxicity levels (LD50 18.36 µg/ml) as compared to the other compounds (LD50> 100 µg/Ml)
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