In vitro anthelmintic activity of Albizia gummifera, Crotalaria axillaris, Manilkara discolor, Teclea trichocarpa and Zanthoxylum usambarense extracts

Abstract

Helminthiasis affects more than one and a half billion people in the world most of them living in low income countries. Majority of these people rely on traditional remedies mostly derived from plants for their health needs. Some of the plants used in Africa, including Kenya are Albizia gummifera, Crotalaria axillaris, Manilkara discolor, Teclea trichocarpa, and Zanthoxylum usambarense. Various parts of A. gummifera have been used to treat stomach pains, malaria, diarrhoea, pain and sleeping sickness. Crotalaria axillaris has been used to treat ophthalmic and kidney problems and Manilkara discolor stem bark infusion is used to treat stomach disorders and as an astringent. Teclea trichocarpa has been traditionally used by the Kamba community in Kenya to treat malaria, helminth infections and fever. Zanthoxylum usambarense is used to treat rheumatism, backache, painful joints, fever, sore throat, tonsillitis, chest pains, malaria, abscesses and wounds. Although the above plants have been used to treat helminth infections and/or stomach problems, no studies have been done to confirm their anthelmintic activity. The objective of the work was to evaluate the in vitro anthelmintic activity of dichloromethanemethanol extracts of A. gummifera, C. axillar is, M discolor, T trichocarpa and Z. usambarense using nematode egg hatch assay and larval development assay. The plants were collected in May 2012 from Ngong Hills forest, Nairobi, Kenya and identified. Voucher specimens of the plants were prepared and deposited at the Department of Botany Herbarium, University of Nairobi under the voucher numbers BMDK01, BMDK02, BMDK03, BMDK04 and BMDK05, respectively. Egg hatch assay was conducted 2n dichloromethane-methanol extracts using Heligmosomoides polygyrus (a mice parasite) and nematode eggs from naturally infected sheep containing mainly

Trichostrongylus spp., Oesophagastomum spp. and Haemonchus contortus. The eggs were incubated together with the extracts at concentrations ranging from 78 ug/ml to 10,000 ug/ml in 96-well plates for 48 h at 27°C. Larval development assay was carried out using nematode eggs from sheep by incubating the first stage larvae in 96-well plates together with the extracts for five days at 27°C. Extracts concentration range was as in the egg hatch assay. A negative control consisting of 3 % Tween- 80 solution was treated in a similar manner and six replicates were done for the two tests. In the egg hatch assay using H polygyrus eggs, the most active extracts were C. axillaris twigs (ICso 180 ± 71 ug/ml) and A. gummifera root bark (ICso 181 ± 60 ug/ml). Extracts of Z. usambarense root bark and stem bark; A. gummifera root and stem bark; T trichocarpa root, twigs and stem bark; and M discolor root bark had ICso in the range 200-500 ug/rnl. The extracts ofM discolor stem bark and A. gummifera pods were the least active (ICso over 500 ug/ml). In the egg hatch assay using nematode eggs from sheep, the most active extracts were A. gummifera root bark (ICso 219 ± 94 ug/ml) and Z. usambarense stem bark (ICso 297 ± 122 ug/ml). The extracts of Z. usambarense root bark, C. axillar is twigs, A. gummifera root, stem bark and pods; T trichocarpa root and stem bark had ICso between 300-500 ug/ml. The least active extracts were M discolor root bark and stem bark; and T trichocarpa twigs with ICso > 500llg/ml. In the larval development assay using sheep nematode eggs, the most active extracts were A. gummifera root (ICso 154 ± 41 ug/ml), Z. usambarense root bark (ICso 160 ± 41 11g/ml) , A. - gummifera root bark (ICso 187 ± 83 ug/ml), Z. usambarense stem bark (ICso 196 ± 51 ug/ml) and A. gummifera stem bark (ICso 197 ± 59 ug/ml). The extracts of C. axillar is twigs, A. gummifera

pods, T trichocarpa root, twigs and stem bark had ICso in the range 200-500 ug/rnl, The least active extracts with ICso > 500 ug/ml were M discolor root bark and stem bark. ICsovalues obtained from egg hatch assay using different species showed varying responsiveness of individual test helminths to different extracts. In addition, the ICso from egg hatch assay and larval development assay showed that nematodes' larvae are generally more sensitive to extracts than the nematodes' eggs (eleven out of twelve) although the difference was statistically significant in a quarter of the cases only (three out of twelve). Albizia gummifera, C. axillar is, M discolor, T trichocarpa, and Z. usambarense have varying degrees of in vitro anthelmintic activity. Albizia gummifera root bark and Z. usambarense root bark were the most active extracts in the H polygyrus egg hatching inhibition assay and sheep nematodes larval development inhibition assay. Further work to confirm in vivo activity and establish safety profile as well as bio-assay guided fractionation and identification of the biomolecules responsible for their in vitro anthelmintic activity is recommended.