Beatrice Irungu

Anti-parasitic activity and cytotoxicity of selected medicinal plants from Kenya.

Indigenous rural communities in the tropics manage parasitic diseases, like malaria and leishmaniasis, using herbal drugs. The efficacy, dosage, safety and active principles of most of the herbal preparations are not known. Extracts from 6 selected plant species, used as medicinal plants by indigenous local communities in Kenya, were screened for in vitro anti-plasmodial and anti-leishmanial activity, against 2 laboratory-adapted Plasmodium falciparum isolates (D6, CQ-sensitive and W2, CQ-resistant) and Leishmania major (IDU/KE/83=NLB-144 strain), respectively. The methanol extract of Suregada zanzibariensis leaves exhibited good anti-plasmodial activity (IC(50) 4.66+/-0.22 and 1.82+/-0.07 microg/ml for D6 and W2, respectively). Similarly, the methanol extracts of Albizia coriaria (IC(50) 37.83+/-2.11 microg/ml for D6) and Aspergillus racemosus (32.63+/-2.68 and 33.95+/-2.05 microg/ml for D6 and W2, respectively) had moderate anti-plasmodial activity. Acacia tortilis (IC(50) 85.73+/-3.36 microg/ml for W2) and Albizia coriaria (IC(50) 71.17+/-3.58 microg/ml for W2) methanol extracts and Aloe nyeriensis var kedongensis (IC(50) 87.70+/-2.98 and 67.84+/-2.12 microg/ml for D6 and W2, respectively) water extract exhibited mild anti-plasmodial activity. The rest of the extracts did not exhibit any anti-plasmodial activity. Although the leishmanicidal activity of extracts were lower than for pentosam (80%), reasonable activity was observed for Aloe nyeriensis methanol (68.4+/-6.3%), Albizia coriara water (66.7+/-5.0%), Maytenus putterlickoides methanol (60.0+/-6.23%), Asparagus racemosus methanol and water (58.3+/-8.22 and 56.8+/-6.58%, respectively), Aloe nyeriensis water (53.3+/-5.1%) and Acacia tortilis water (52.9+/-6.55%) extracts at 1000 microg/ml. Leishmania major infected macrophages treated with methanol extracts of Suregada zanzibariensis and Aloe nyeriensis var kedongensis and pentostam had infection rates of 28+/-2.11, 30+/-1.22 and 40+/-3.69%, respectively at 1000 microg/ml, indicating better anti-leishmanial activity for the extracts. The methanol extract of Albizia coriara (44.0+/-3.69%) and aqueous extracts of Asparagus racemosus (42+/-3.84%) and Acacia tortilis (44+/-5.59%) had similar activity to pentosam. Multiplication indices for Leishmania major amastigotes treated with methanol extracts of Albizia coriaria, Suregada zanzibariensis and Aloe nyeriensis var kedongensis, aqueous extract of Acacia tortilis and pentosam were 28.5+/-1.43, 29.4+/-2.15, 31.1+/-2.22, 35.9+/-3.49 and 44.0+/-3.27%, respectively, at 1000 microg/ml, confirming better anti-leishmanial activity for the extracts. Aqueous extracts of Aloe nyeriensis (46.7+/-3.28%) and Albizia coriaria (47.5+/-3.21%) had similar activity level to pentosam. The plant extracts have better inhibitory activity while pentosam has better leishmanicidal activity. All extracts exhibited very low cytotoxicity (CC(50) > 500 microg/ml) against human embryonic lung fibroblast (HELF) cells. The investigations demonstrated the efficacy and safety of some extracts of plants that are used by rural indigenous communities for the treatment of parasitic diseases.

The antiplasmodial and radical scavenging activities of flavonoids of Erythrina burttii

The acetone extract of the root bark of Erythrina burttii showed in vitro antiplasmodial activity against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum with IC(50) values of 0.97 ± 0.2 and 1.73 ± 0.5 μg/ml respectively. The extract also had radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with an EC(50) value of 12.0 μg/ml. The isoflav-3-enes burttinol-A and burttinol-C, and the 2-arylbenzofuran derivative burttinol-D were identified as the most active antiplasmodial (IC(50)<10 μM) and free radical scavenging (EC(50)ca. 10 μM) principles. The acetone extract of E. burttii at 800 mg/kg/day, in a 4-day Plasmodium berghei ANKA suppressive test, showed in vivo antimalarial activity with 52% chemosuppression. In the same in vivo test, marginal activities were also observed for the extracts of the root and stem bark of Erythrina abyssinica and the root bark of Erythrina sacleuxii.

Plasmodium berghei ANKA: selection of resistance to piperaquine and lumefantrine in a mouse model.

We have selected piperaquine (PQ) and lumefantrine (LM) resistant Plasmodium berghei ANKA parasite lines in mice by drug pressure. Effective doses that reduce parasitaemia by 90% (ED90) of PQ and LM against the parent line were 3.52 and 3.93 mg/kg, respectively. After drug pressure (more than 27 passages), the selected parasite lines had PQ and LM resistance indexes (I90) [ED90 of resistant line/ED90 of parent line] of 68.86 and 63.55, respectively. After growing them in the absence of drug for 10 passages and cryo-preserving them at −80 °C for at least 2 months, the resistance phenotypes remained stable. Cross-resistance studies showed that the PQ-resistant line was highly resistant to LM, while the LM-resistant line remained sensitive to PQ. Thus, if the mechanism of resistance is similar in P. berghei and Plasmodium falciparum, the use of LM (as part of Coartem®) should not select for PQ resistance.

Constituents of the Roots and Leaves of Ekebergia capensis and Their Potential Antiplasmodial and Cytotoxic Activities

A new triterpenoid, 3-oxo-12β-hydroxy-oleanan-28,13β-olide (1), and six known triterpenoids 2–7 were isolated from the root bark of Ekebergia capensis, an African medicinal plant. A limonoid 8 and two glycoflavonoids 9–10 were found in its leaves. The metabolites were identified by NMR and MS analyses, and their cytotoxicity was evaluated against the mammalian African monkey kidney (vero), mouse breast cancer (4T1), human larynx carcinoma (HEp2) and human breast cancer (MDA-MB-231) cell lines. Out of the isolates, oleanonic acid (2) showed the highest cytotoxicity, i.e., IC50’s of 1.4 and 13.3 µM against the HEp2 and 4T1 cells, respectively. Motivated by the higher cytotoxicity of the crude bark extract as compared to the isolates, the interactions of oleanonic acid (2) with five triterpenoids 3–7 were evaluated on vero cells. In an antiplasmodial assay, seven of the metabolites were observed to possess moderate activity against the D6 and W2 strains of P. falciparum (IC50 27.1–97.1 µM), however with a low selectivity index (IC50(vero)/IC50(P. falciparum-D6) < 10). The observed moderate antiplasmodial activity may be due to general cytotoxicity of the isolated triterpenoids.

Antiplasmodial potential of traditional phytotherapy of some remedies used in treatment of malaria in Meru-Tharaka Nithi County of Kenya

ETHNOPHARMACOLOGICAL RELEVANCE Medicinal plants play a major role in many communities across the world, in the treatment and prevention of disease and the promotion of general health. The aim of the study was to escalate documentation from an earlier study of medicinal plants, traditionally used to combat malaria by the Ameru community of Imenti Forest area and Gatunga in Eastern Region of Kenya, and validate their ethnopharmacological claims by evaluating their antiplasmodial efficacies. MATERIALS AND METHODS The study was carried out in Meru County at Imenti Forest Game Reserve and in Tharaka Nithi County at Gatunga. Traditional health practitioners (THP) were interviewed with a standard questionnaire to obtain information on medicinal plants traditionally used for management of malaria. Group interviews were also held among THPs and members of the community. The antiplasmodial activities of the crude extracts against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum were determined using the semi-automated micro-dilution technique that measures the ability of the extracts to inhibit the incorporation of (G-3H) hypoxanthine into the malaria parasite. RESULTS Ninety nine (99) species in eighty one (81) genera and forty five (45) families were documented and evaluated for in vitro antiplasmodial activity. Compositae, Fabaceae, Meliceae, Rubiaceae, Rutaceae and Verbenaceae had the highest number of species mentioned in treatment of malaria in Meru/Tharaka Nithi study area. Twenty four (24.2%) species showed antiplasmodial efficacy of IC50 ≤ 5 µg/ml and were considered to have potential for isolation of antimalarial compounds. Eight plant (8) species with moderate antiplasmodial activity namely; Cordia africana, Commiphora africana, Elaeodendron buchananii, Gomphocarpus semilunatus, Tarena graveolens, Plectranthus igniarius, Acacia senegal and Ziziphus abyssinica were documented from this region for the first time for the treatment of malaria. The antiplasmodial activity of MeOH root bark extract of Maytenus obtusifolia was very promising (IC50 < 1.9 µg/ml) and this is the first report on traditional use of M. obtusifolia for treatment of malaria and antimalarial activity. CONCLUSIONS The results seem to indicate that ethnopharmacological inquiry used in search for new herbal remedies as predictive and could be used as the basis for search of new active principles. Eight plant (8) species are documented from this region for the first time for the treatment of malaria. This is the first report on traditional use of M. obtusifolia for treatment of malaria and evaluation of its antiplasmodial activity.

Antiplasmodial and cytotoxic activities of the constituents of Turraea robusta and Turraea nilotica.

Ethnopharmacological relevance Turraea robusta and Turraea nilotica are African medicinal plants used for the treatment of a wide variety of diseases, including malaria. The genus Turraea is rich in limonoids and other triterpenoids known to possess various biological activities. Materials and methods From the stem bark of T. robusta six compounds, and from various parts of T. nilotica eleven compounds were isolated by the use of a combination of chromatographic techniques. The structures of the isolated compounds were elucidated using NMR and MS, whilst the relative configuration of one of the isolated compounds, toonapubesin F, was established by X-ray crystallography. The antiplasmodial activities of the crude extracts and the isolated constituents against the D6 and W2 strains of Plasmodium falciparum were determined using the semiautomated micro dilution technique that measures the ability of the extracts to inhibit the incorporation of (G-3H, where G is guanine) hypoxanthine into the malaria parasite. The cytotoxicity of the crude extracts and their isolated constituents was evaluated against the mammalian cell lines African monkey kidney (vero), mouse breast cancer (4T1) and human larynx carcinoma (HEp2). Results The extracts showed good to moderate antiplasmodial activities, where the extract of the stem bark of T. robusta was also cytotoxic against the 4T1 and the HEp2 cells (IC50<10 μg/ml). The compounds isolated from these extracts were characterized as limonoids, protolimonoids and phytosterol glucosides. These compounds showed good to moderate activities with the most active one being azadironolide, IC50 2.4±0.03 μM and 1.1±0.01 μM against the D6 and W2 strains of Plasmodium falciparum, respectively; all other compounds possessed IC50 14.4–40.5 μM. None of the compounds showed significant cytotoxicity against vero cells, yet four of them were toxic against the 4T1 and HEp2 cancer cell lines with piscidinol A having IC50 8.0±0.03 and 8.4 ±0.01 μM against the 4T1 and HEp2 cells, respectively. Diacetylation of piscidinol A resulted in reduced cytotoxicity. Conclusion From the medicinal plants T. robusta and T. nilotica, twelve compounds were isolated and characterized; two of the isolated compounds, namely 11-epi-toonacilin and azadironolide showed good antiplasmodial activity with the highest selectivity indices.

Antiplasmodial activity of compounds from Drypetes gerrardii

0009-3130/12/4802-0339 2012 Springer Science+Business Media, Inc. 1) Department of Chemistry, Kenyatta University, P.O. Box 43844-00100, Nairobi, Kenya; 2) Department of Chemistry, Universitat Paderborn, Warburger Street 100, 33098 Paderborn, Germany, e-mail: Hidayat110@gmail.com; 3) Department of Biological Sciences and Chemistry, College of Arts and Sciences University of Nizwa, Birkat Al-Mouz, PC 616, Oman; 4) Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute, P. O. Box 54840-00200, Nairobi, Kenya; 5) NUST Center of Virology and Immunology, National University of Science and Technology, H-12, Islamabad, Pakistan. Published in Khimiya Prirodnykh Soedinenii, No. 2, March–April, 2012, pp. 304–305. Original article submitted January 4, 2011. Chemistry of Natural Compounds, Vol. 48, No. 2, May, 2012 [Russian original No. 2, March–April, 2012]

Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.

Antiplasmodial potential of traditional antimalarial phytotherapy remedies used by the Kwale community of the Kenyan coast

ETHNOPHARMACOLOGICAL RELEVANCE In Kenya, 22 million people are at risk of malaria, 70% of them are in rural areas and most of these people use traditional plant based medicines to treat malaria. The aim of the study was to escalate documentation, from an earlier study of medicinal plants, traditionally used to treat malaria by the Digo community of Kwale County, taking cognizance of their pharmacological information by evaluating their antiplasmodial efficacies. MATERIALS AND METHODS The study was carried out in Kwale County at Shimba Hills Game Reserve and adjoining part of Kinango. Traditional health practitioners (THP) were interviewed with a standard questionnaire to obtain information on medicinal plants traditionally used for management of malaria. Group interviews were also held among THPs and members of the community. The plant samples collected were tested for antiplasmodial activity against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum using the ability of extracts, prepared from the plant species, to inhibit the incorporation of [G-3H] hypoxanthine into the malaria parasites. RESULTS Fifty seven (57) species in forty eight (48) genera and thirty (30) families were documented and evaluated for in vitro antiplasmodial activity. Apocynaceae, Euphorbiaceae, and Rubiaceae families had each about 12% of the plant species reported as antimalarial remedy and represented the species that are most commonly used. Twelve species (21.1%) showed antiplasmodial efficacy of IC50<5µg/ml and these were Boscia salicifolia, Cissampelos mucronata, Clerodendrum myricoides, Commiphora schimperi, Flueggea virosa, Maytenus undata, Maytenus senegalensis, Maytenus putterlickioides, Vernonia amygdalina, Warburgia stuhlmannii, Zanthoxylum chalybeum and Tabernaemontana pachysiphon. CONCLUSIONS These results seem to indicate that ethnopharmacological inquiry used in search for new herbal remedies as predictive and could form the basis of an ethnopharmacopoeia and search for new active principles. This is the first report on traditional use of T. pachysiphon for malaria and its antiplasmodial activity.

Piperaquine and Lumefantrine resistance in Plasmodium berghei ANKA associated with increased expression of Ca2+/H+ antiporter and glutathione associated enzymes

We investigated the mechanisms of resistance of two antimalarial drugs piperaquine (PQ) and lumefantrine (LM) using the rodent parasite Plasmodium berghei as a surrogate of the human parasite, Plasmodium falciparum. We analyzed the whole coding sequence of Plasmodium berghei chloroquine resistance transporter (Pbcrt) and Plasmodium berghei multidrug resistance gene 1(Pbmdr-1) for polymorphisms. These genes are associated with quinoline resistance in Plasmodium falciparum. No polymorphic changes were detected in the coding sequences of Pbcrt and Pbmdr1 or in the mRNA transcript levels of Pbmdr1. However, our data demonstrated that PQ and LM resistance is achieved by multiple mechanisms that include elevated mRNA transcript levels of V-type H(+) pumping pyrophosphatase (vp2), Ca(2+)/H(+) antiporter (vcx1), gamma glutamylcysteine synthetase (ggcs) and glutathione-S-transferase (gst) genes, mechanisms also known to contribute to chloroquine resistance in P. falciparum and rodent malaria parasites. The increase in ggcs and gst transcript levels was accompanied by high glutathione (GSH) levels and elevated activity of glutathione-S-transferase (GST) enzyme. Taken together, these results demonstrate that Pbcrt and Pbmdr1 are not associated with PQ and LM resistance in P. berghei ANKA, while vp2, vcx1, ggcs and gst may mediate resistance directly or modulate functional mutations in other unknown genes.