MST-Department of Biochemistry and Biotechnology

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Abundance, genetic diversity and symbiotic potential of common bean (phaseolus vulgaris l.) nodule associated bacteria in Western Kenya soils
(Kenyatta University, 2016-07) Simiyu, Wekesa Clabe
Plant growth-promoting rhizobacteria (PGPR) are beneficial native soil bacteria that colonize plant roots and result in increased plant growth. Those that colonise the nodules of legumes are known as nodule associated bacteria (NAB). The aim of this study was to determine the distribution and genetic diversity of NAB that colonize Phaseolus vulgaris, their abundance, and symbiotic efficiency when coinoculated with Phaseolus vulgaris in Western Kenya soils. The soil samples were collected from cultivated lands in Kisumu near Lake Victoria, slopes of Mt. Elgon and Kakamega. In each of these regions, the soil samples were collected from four regions. 1ml of soil solution at 10 fold dilution for seven dilution steps (10-1 to 10-7) and three replications for each dilution was used to inoculate common bean seedling in Leonard jars. They were harvested after four weeks to determine abundance of NAB using most probable number method. Common bean nodules were also collected directly from the farmers’ farms in the above three regions. Harvested nodules and those collected from the field were cleaned and surface sterilized, crushed and exudates streaked on YEM agar growth media. Pure colonies were further cultured in YEM broth at 280C for three days and the genomic DNA isolated from the bacteria using Qiagen DNA extraction kit. 16SrRNA gene was amplified by 27F and 1492R primers and PCR products resolved by agarose gel electrophoresis and sequenced. 16SrRNA gene analysis revealed that NAB that nodulate with common beans are genetically diverse as they formed clusters on the phylogenetic tree and their distribution depends on chemical characteristics of the soil. BLASTn showered that isolated strains belonged to the genus Pseudomonas, Providencia, Rhizobia, Klebsiella, Sphingobacterium, Enterobacter, Delfitia, Acinetobacter and one strain did not have sequence homology at the GenBank. Mt. Elgon region had the highest population of NAB (120000 cells per gram of the soil), followed by Kisumu (1290 cells per gram of the soil) and Kakamega region had the lowest (17 cells per gram of the soil). The effect of PGPR on the yield of common beans was significantly higher (p < 0.001) when co-inoculated with Rhizobia compared to the yield of Rhizobia inoculated alone or control (not inoculated) (p < 0.05). This study therefore provides knowledge on the type of NAB that nodulates with common beans and factors that favour their distribution necessary for production of PGPR inoculants suitable to the soils of Western Kenya.
Adaptation of an enzyme-linked immunosorbent assay for determination of diminazene aceturate in goat serum and tissue residues of animals
(2014-11-27) Karanja, Wycliff
The importance of food safety through the reduction of residues in our food supply cannot be overemphasized. Food safety remains a major challenge confronting contemporary society. Analytical methods are needed to generate the data on which dietary exposure assessments are based and to enforce statutory maximum residue limits (MRLs) that are set. Diminazene aceturate is one of the few drugs used for animal trypanosomosis. Because of it's wide use in livestock, the risk of unwanted residues in edible products may exist. A competitive enzyme-linked immunosorbent assay (ELISA) for determination of diminazene residues in edible animal tissues after extraction in 0.1 M borax at pH 9.7 was investigated. The assay used rabbit anti-diminazene polyclonal antibody on the solid phase support. Horseradish peroxidase-labeled diminazene was incubated with sample overnight at 4°C. After five washes with buffer enzyme activity was determined by adding tetramethyl-benzidine and hydrogen peroxide as substrate. The resulting blue colour whose intensity was inversely proportional to the drug concentration changed to yellow when the reaction was stopped by addition of 0.1 M orthophosphoric acid. The assay was optimized and validated for determination of diminazene in tissues. The assay exhibited high specificity (99.997%) for diminazene recognizing only isometamidium at 0.003% and this may be contributed by the amidinophenyl that is common in both drugs. Recoveries from spiked tissues were above 77% while Dilutional parallelism experiments demonstrated a recovery of 96.0% ± 9.5%. The limit of detection (LaD) for the assay was 2.4ng/g for muscle, 2.5ng/g for liver and 2.2ng/g for kidney while limits of quantification (LOQ) were 5.51ng/g, 4.11 ng/g and 3.74 ng/g respectively. The LaDs are 4.4x103 to 3.5x103 lower than the MRLs that are 500llg/kg, 12,000Ilg/kg and 6,000Ilg/kg of muscle liver and kidney respectively: Assay precision was characterized by a within assay coefficient of variation (CV) of 2.4% and between assays CV of 15.5%. When diminazene was administered intramuscularly at 3.5mg/kg to five goats that were sacrificed seven days later, the mean diminazene residue levels were 0.75Ilg/g±0.14Ilg/g for skeletal muscle, 32.05Ilg/g±5.7Ilg/g for liver and 4.29Ilg/g±0.66Ilg/g for kidney. The analysis of tissue samples collected from slaughterhouses around Nairobi showed that out of 35 muscle samples, only one was positive and had a diminazene concentration of 0.039 ug/g. Four out of 32 kidney samples were positive for diminazene with levels of 0.63, 1.66,2.61 and 3.96Ilg/g. From ten liver samples two were positive with levels of 1.07 and 1.74 ug/g. From this analysis none of the positive samples had levels above the MRL values. This study has demonstrated that competitive ELISA can be employed for the determination of diminazene residues. The results of this study are relevant to food scientists, toxicologists and analysts working in the area of detection and safety assessment of food residues, companies developing veterinary drugs, regulatory bodies involved in safety assessment of veterinary drugs and residue monitoring and to regulatory bodies responsible for veterinary drugs registration.
Agrobacterium mediated transformation of selected maize inbred lines with ppzp200 towards enhancment of lysine and methionine content
(2012-03-26) Limbua, Purity Gacheri; Machuka, Jesse; Ombori, O.
Maize (Zea mays (L.) is one of the most important cereals used both for human and animal consumption in the world. Despite its importance, maize is not a suitable single source of nutrition because it does not provide the essential amino acids lysine and methionine in sufficient quantities to meet the nutritional needs of humans and other animals. Lysine is a necessary building block for protein in the body while methionine is the body's primary source of sulphur. Strategies to improve the nutritional quality of maize for high lysine and methionine have involved both genetic engineering (GE) and non-genetic engineering approaches such as marker assisted selection. Breeding is however laborious, lengthy and carries along undesired alleles. The objective of this work was to manipulate maize inbred lines towards enhancement of lysine and methionine content in the endosperms through Agrobacterium mediated transformation. Maize kernels mainly store proteins as a, {3, y and 0 zeins. The immature embryos of three tropical maize inbred lines (TL IS, CML216 and CMLI44) and a temperate line (AlSS) were transformed using Agrobacterium tumejaciens strain EHAI01 carrying an expression cassette designed to upregulate the Z I 0 protein for methionine enhancement as well as down-regulate the a. zein storage protein by RNAi. The T -DNA also contained P-zp22/6 as the promoter and the phosphinothricin acetyltransfarase gene (bar) used for selection of transformed tissue. Putative transformants were tested for presence of the transgene by PCR designed to amplify the P-zp22/6 promoter sequence. Calli survival frequencies were calculated as a percentage number of surviving calli in relation to the total number of embryos infected. These ranged from 2.S9 % forTLIS to 9.11 % for AISS. This data did not detect any significant difference (p>0.05) among the genotypes on the percentage of calli which survived. Transformation efficiency was calculated as a percentage of the number of PCR positive plants divided by the total number of embryos infected. This ranged from 0% for TLIS to I.S3% for AISS. The data suggest the possibility of manipulating storage proteins and regenerating normal transgenic maize with normal kernels. Further work should involve gene expression assays for accumulation of {3, y and 0 prolamins in the kernels and southern blot analysis to confirm stable integration and the copy numbers ofPzp22/6 gene in the PCR positive plants.
Agrobacterium tumefaciens mediated transformation of Sudan maize genotypes using NPKI gene for enhancing drought stress tolerance
(2011-11-29) Abdalla, Rasha Adam Omer; Jesse Machuka; Abdelbagi M. Ali
Drought is one of the most important abiotic factor affecting maize production worldwide. Agrobacterium-mediated gene transfer technique has been established as a versatile way of improving important crops for tolerance to biotic and abiotic factors. Through this technique, the drought tolerance gene, NPKI, has been used in the transformation of temperate maize after its isolation and characterization from tobacco. Recovered transgenic events were observed to have enhanced tolerance to water stress. The accelerated adoption of the transformation technique in Africa, and indeed in Sudan, will depend on the ease with which transgenes of agronomic importance can be integrated into appropriate germplasms. This study aimed at screening important Sudanese maize inbred lines and open pollinated varieties (OPVs) for transformability via the integration of the NPKI gene. Eight inbred lines and three OPVs were evaluated. A188 was used as the standard inbred line check while KAT was used as the local OPV check. Freshly isolated immature embryos of maize were inoculated with Agrobacterium strain EHA101 harbouring the plasmid pSHX004 in LS infection media for 5 minutes and then co-cultivated on LS cocultivation media for 3 days. Embryos were then transferred to selection media supplemented with 250mg/l cefotaxime and 1.5mg/L bialaphos. After two weeks on this media, calli were subcultured on selection media containing 3.0 mg/L bialaphos for 4 weeks. Bialaphos resistant callus events were then transferred to maturation media supplemented 3mg/L bialaphos for 2 weeks before transferring to shooting media. Shoots were then transferred to rooting media. Plantlets with well-formed root system were transferred from the in vitro environment to green house for hardening. Hardened plantlets were transplanted to soil in the greenhouse and maintained till they set seeds. To confirm the presence of the transgene, PCR analysis was done on putative transgenic plants using the Bar primers. Out of a total of 4401 immature embryos from the 13 genotypes infected, 327 survived selection in bialaphos. Bialaphos resistant calli emerged 3-4 weeks after selection. IL3, IL15, Hudiba-2, IL1, IL38, Hudiba-1, A188 and KAT produced compact calli from their scutella surfaces while IL28, IL42, IL43, Mojtamaa-45 and IL16 established watery nonembryogenic calli. Statistically significant differences (p<0.05) were observed between the genotypes with respect to transformation frequency (TF). IL3 was identified as the most amenable to transformation with a TF of 31.7% and proved to be superior to A188, which recorded a TF of 5.82%. Hudiba-2 was identified as the most transformable OPV with a TF of 8.7% compared to that of 7.3% for KAT. ILI and Mojatamaa-45 proved to be poor responders to transformation with TFs of 2.5% and 1.7%, respectively. Putative transgenics were recovered from IL3, IL 15, Hudiba-2, ILI, IL38, Mojatamaa-45, A188 and KAT. The frequency of regeneration of bialaphos resistant shoots varied from 6.9% for IL38 to 100% for Mojtamaa-45. PCR analysis indicated a 540bp fragment in the DNA extracts from transgenic R, plants. Transformation efficiency (TE) was found to depend on the genotype used. The highest TE was observed for IL3 (3.7%), while the lowest TE of 0.0% was observed in IL42 IL43, IL16 and 11,28. Various abnormalities were observed in putative transformants including dwarfism, tussel seed and lack of ear. However, plants grew to maturity and were able to establish seeds in spite of these abnormalities. In conclusion, the inbred line IL3 and the OPV Hudiba-2 proved to be the most amenable Sudanese genotypes to A. tumefaciens-mediated transformation. Future research in maize improvement through biotechnologies such as tissue culture and genetic transformation should be focussed on these good responders.
Agrobacterium tumefaciens-mediated transformation of three groundnut (Arachis hypogae L.) genotypes from Southern and Eastern Africa
(2012-03-26) Kahariri, Esther Wanja
Groundnut or peanut (Arachis hypogae L.) is one of the principal economic oilseed legumes and "is largely cultivated in tropical, subtropical and warm temperate regions of the world. It is an . upright or prostrate annual plant. Groundnut contributes significantly to household food security and cash income through the sale of the seeds and also provides a valuable source of proteins, .. fats, energy and minerals. Developing countries account for nearly 9S% of the world production .Groundnut production in African countries has been fluctuating greatly over the last decade. This has been attributed to biotic and abiotic constraints. Pests and diseases that are major biotic factors can lead to yield losses as high as 100% resulting in total crop failure. Traditional plant breeding methods used to improve the crop are time consuming, expensive and involve transfer of unwanted traits along with the desired ones. Besides they are limited to the existing narrow gene pool within compatible groundnut genotypes. Recent advances in biotechnology offer alternative tools such as genetic engineering through which genes that confer some of these traits can be isolated, cloned and introduced into important crops. Genetic transformation protocols are both genotype and species dependent and specific protocols need to be developed for every plant species and sometimes even each genotype. Development ofa good transformation protocol for African groundnuts will provide a platform for further genetic improvement for traits such as drought, pest and disease resistance and biofortification. Transformability of three groundnut genotypes; ICGV90704, ICGV12991 and JL24 was assessed using cotyledon explants from mature seeds infected with four Agrobacterium tumefaciens strains AGLO, EHA lOS, CS8 and LBA 4404 containing a standard binary vector with a GUS reporter gene. The transformation efficiency (TE) expressed as a % of PCR positive shoots out of the total number of shoots infected, of groundnut variety ICGV90704 was 1.33% with strain EHAI0S, 0.81% with strain AGLO and 0.39% with strain LBA4404. Variety ICGV12991 recorded a TE of 1.11% with strain AGLO, 0.S9% with strain CS8 and 0.53% with strain LBA4404. Variety JL24 had a TE of 1.03% with strain AGLO and 0.99% with strain CS8. ANOV A between and within the three varieties at the explant producing shoots, total shoots in S 1, total shoots in S2 and total shoots in RIM showed strain LBA4404to be more efficient followed by AGLO and EHAI0S. The least efficient strain wasCS8. Groundnut variety ICGV 90704 was found to be the most amenable to transformation. It is concluded that cotyledons can be used as explants in the transformation of African groundnuts.
Agrobacterium-Mediated Transformation of Elite Kenyan Maize Germplasm with CRY3A Gene for the Control of the Larger Grain Borer and Maize Weevil
(2013-10-18) Taracha, Catherine Ongecha
In Kenya maize (Zea mays L.) is the most important staple food. Owing to its predominant role, food security in the future cannot be achieved without major increases in cereal production. An efficient in vitro regeneration and transformation system holds a great potential for genetic improvement of maize against production constraints. This study was conducted with the objective of assessing the regenerative capacity, genetic transformation of Kenyan maize genotypes and efficacy of transformed maize in controlling maize pests. Six Kenyan inbred lines and four CIMMYT lines and their single crosses were evaluated for their in vitro response on three different media (MS, N6 and N6CL). The embryogenic callus induction and regeneration capacity was higher on MS than on N6 basal salts. Plant regeneration was influenced by genotype. Transformation experiments were carried out using Agrobacterium tumefaciens strain EHAI01 containing pTF 102 binary vector harbouring a GUS gene. The transformation frequency was highest in 104 (15.2%) and lowest in QPM. The highest transformation efficiency was recorded in inbred T04, 104 and their crosses . with CML 216 and ranged 2.4% to 3.0%. The transgene was detected in all the maize genotypes using GUS assays, and PCR. Maize genotypes were transformed using a Bacillus thuringiensis tenebrionis gene Cry3A. The highest transformation frequency was recorded in three inbred lines, H04, T04 and 104 (9.3%, 12.2% and 13.4% respectively). The transformation efficiency ranged between 0.5% to 2.5%. PCR and RT-PCR amplification of the Cry3A gene, and the l)AS-Elisa confirmed the presence of the gene To,T1 and T2 generations. 'Insect bioassays established that transgenic maize provided protection against the larger grain borer and maize weevil This study established a reproducible regeneration and transformation system for tropical maize, which can be used in a pest management programme
Ameliorative Effects of Coadministered Coenzyme Q10 And Dimercaptosuccinic Acid on Arsenic-Induced Toxicity in a Mouse Model
(Kenyatta University, 2021-04) Mwaeni Kiwasi, Victoria; George Isanda Omwenga; Alfred Isaac Orina; Mathew Piero Ngugi
Exposure to arsenic is a growing human health threat to millions of people globally. Dimercaptosuccinic Acid (DMSA), the current treatment drug, chelates extracellularly distributed arsenic, allowing the intracellular arsenic to persist and get metabolized to toxic by-products. Previous studies on Coenzyme Q10 (CoQ10) have demonstrated remarkable protective roles against organic forms of arsenic. So far, no studies have determined if coadministered CoQ10 and DMSA can protect from inorganic forms of arsenic. Therefore, this study was conducted to evaluate the ameliorative effect of coadministered DMSA and Coenzyme Q10 on inorganic arsenite-induced toxicity in male Swiss albino mice. Male mice were exposed to sodium arsenite (15mg/kg) then either maintained on drinking water or treated. Treated mice received 50mg/kg DMSA (5 days) and 200mg/kg CoQ10 (30 days) either singly or in combination. Mice were subjected to weekly weight measurement and Rapid Murine Behavioral Coma scoring tests (RMBC). After 30 days’ post treatment mice were sacrificed and samples obtained for analysis. Further hematological and biochemical parameters as well as inflammatory and histopathological profiles were determined. Data collected was analyzed using One-Way ANOVA with Tukey’s post hoc test for pairwise comparisons. Coadministered CoQ10 and DMSA significantly protected mice from arsenite-induced weight loss. Further, exposure to arsenite resulted in a significant reduction of Hematocrit, Red blood cells concentration, and hemoglobin. Treatment with CoQ10 and DMSA either alone or in combination prevented the suppression of the Hematocrit, Red blood cells and hemoglobin levels following arsenite exposure. Further, arsenite caused an increase in White blood cell (WBC) count and basophils with a decrease in the neutrophils, monocytes, and eosinophils. Coadministered CoQ10 and DMSA prevented arsenite-induced changes in WBC and differential count. Mice exposed to arsenite had high cholesterol, triglyceride, and high density lipoprotein levels. Coenzyme Q10, and DMSA, significantly protected mice from arsenite-induced alterations of the lipid profiles. This study demonstrated arsenic-driven oxidative stress in the liver, brain, lungs, spleen, and heart indicated by drastic depletion of (Reduced Glutathione) GSH. Administered CoQ10 protected mice from arsenic-induced depletion of GSH. Elevation of liver (Aspartate aminotransferase, Alanine aminotransferase, Gamma glutamyl transferase, bilirubin) and kidney (Creatinine) markers further demonstrated the possible injury due to arsenite exposure. Investigations of liver, kidney, and brain tissue sections also confirmed this finding. It was noted that CoQ10 blocked those adverse events more efficiently than DMSA. Moreover, arsenic-induced inflammation was evident by the elevation of Interferon- gamma (INF- and Tumor necrosis factor-alpha (TNF-, with concomitant depletion of anti-inflammatory marker Interleukin -10 (IL-10). The remarkable finding from this study is that treatment with CoQ10 and DMSA was effective in countering oxidative stress and inflammation than DMSA alone that showed no protection against organ injury. Findings from this study provide an alternative avenue for developing new strategies to improve the treatment and management of arsenic-induced toxicity.
Analgesic and Anti-Inflammatory Activities of Methanol Extracts OF Pistacia aethiopica (Kokwaro) and Warbugia ugandensis (Sprague) in Mice Models
(Kenyatta University, 2019) Ireri, Moses Munene
Inflammation and pain are symptoms associated with many pathological conditions. These symptoms cause distress to the victims. Management of pain and inflammation is done by conventional drugs such as non-steroidal anti-inflammatory drugs (NSAIDs) which may be expensive, not easily available and cause adverse effects. Traditional medicines provide viable alternatives in the management of pain and inflammation. Traditional medicines are easily accessible, cheap with minimal side effects. Warbugia ugandensis Sprague and Pistacia aethiopica Kokwaro are medicinal plants, which have for long been used by people in Embu County as analgesic and anti-inflammatory drugs. In spite being in use for long, no scientific study has validated their use. This study was therefore designed to establish the claimed analgesic and anti-inflammatory effects of these plants. Fresh leaf samples of Warbugia ugandensis and fresh bark samples of Pistacia aethiopica were collected in Embu County, Kenya. The plant samples were then air dried after which they were transported to the Department of Biochemistry, Microbiology, and Biotechnology, Kenyatta University. The dry plant materials were then pulverized by electric mill. Crude methanol extracts were prepared using 1 litre methanol per 200g powder. Male albino mice were divided into six groups of 5 animals each; normal control, negative control, positive control and three experimental groups for extract dose levels of 50, 100 and 150mg/kg body weight. Analgesic studies used formalin model while anti-inflammation studies used carrageenan-induced acute edema model. Diclofenac was used as the positive control in analgesic and anti-inflammatory studies. Stem bark extracts of P. aethiopica inhibited paw licking in mice by between 47.24-55.13% in the early phase and by between 30.69-52.12% in the late phase. W. ugandensis leaf extracts inhibited paw licking by between 38.45-51.85% in the early phase and by between 43.48-65.61% in the late phase. Diclofenac inhibited paw licking by between 30.33-30.36% in the early phase and by between 62.93-77.08% in the late phase. For anti-inflammatory effects P. aethiopica extract suppressed carrageenan induced paw edema by between 4.6-7.6% while W. ugandensis suppressed paw edema by between 6.08-7.59%. Diclofenac suppressed carrageenan induced paw edema by between 8.86-9.57%. Qualitative phytoconstituents screening revealed presence of phenols, saponins, flavonoids alkaloids and terpenoids which have been previously linked to analgesic and anti-inflammatory effects. Therefore the current study has validated folkloric use of P. aethiopica and W. ugandensis as remedies for pain and inflammation.
Analgesic, antipyretic and anti-inflammatory potential of dichloromethanolic root extract of clutia abyssinica jaub and spach in rats and mice models
(Kenyatta University, 2017-07) Koech, Samson Cheruiyot
Various diseases and injuries are always presented with pain, fever and inflammation. These are considered as symptoms associated with various pathological processes in an animal body. Drugs that are used to alleviate pain, fever and inflammation such as non-steroidal anti-inflammatory drugs exhibit adverse effects for example cardiac abnormalities, peptic ulcers, liver toxicity and kidney failure. Therefore, there is need to come up with alternative remedies. Herbal medicines are deemed to be safe, have good efficacy and have fewer side effects. Clutia abyssinica is a shrub that is found in East, Central, and South Africa and it has been used traditionally to cure several ailments including malaria, chest pain, gonorrhea, fever, infertility, pain, inflammation, skin diseases and cancer. Roots of this medicinal plant have been used traditionally to prepare decoctions. The aim of the project was to evaluate the analgesic, antipyretic and anti-inflammatory potential of dichloromethanolic root extract of Clutia abyssinica in animal models. Plant sample material was collected from Kaptebee village, Turbo sub-county in Uasin Gishu County Kenya, and the active components extracted using dichloromethane. Pain, fever and inflammation were induced Swiss albino mice and Wistar albino rats using acetic acid, turpentine and carrageenan respectively. Swiss albino mice and Wistar albino rats were grouped into normal control, negative control, positive control and 3 experimental groups. Extracted root extracts were administered intraperitoneally to Swiss albino mice and Wistar albino rats at predetermined doses (50, 100, and 150 mg/kg body weight). The analgesic and anti-inflammatory activities of the plant root extract were compared to diclofenac the (reference drug) while the antipyretic activity was compared to aspirin. The dichloromethanolic root extract of C. abyssinica demonstrated significant analgesic, antipyretic and anti-inflammatory activities. Number of abdominal writhing was reduced between 33.95-49.51% by dichloromethanolic root extract while the diclofenac (reference drug) reduced abdominal writhing by 46.51%. Reduction in number of abdominal writhings by the extract indicates the plant posse‟s analgesic properties. The elevated temperature was reduced between 0.68-3.34% by the dichloromethanolic root extract while Aspirin the (reference drug) reduced elevated temperature between 3.32-4.96%. Edema was reduced between 0.88-5.34% by the plant extract while diclofenac reduced edema between 2.21-5.35% respectively. Rectal temperature and the size of the edema was reduced more in the third and fourth hours signifying better blockage of mediators responsible for fever and inflammation. Data was analyzed using one way analysis of variance followed by turkey‟s test. Qualitative phytochemical analysis revealed the presence of alkaloids, flavonoids, terpenoids, steroids, saponins and cardiac glycosides. The extract from Clutia abyssinica may be used as an alternative bioresource in development of analgesic, antipyretic and anti-inflammatory agent. The study therefore, confirms the folklore use of the medicinal plant by Kalenjin community of Kenya to manage pain, fever and inflammation.
Analysis of Expresion Patterns of zmrcp1 and camv35s in Plantains in Association with Nematodes Infection
(2013-08-14) Onyango, Stephen Odhiambo
Plantains are among staple foods in Africa. Worldwide, Musa spp. have an annual production of 88 million tonnes. Nematodes cause significant annual yield losses to these crops. The losses are estimated at about 20% worldwide. The most disastrous nematodes are Radopholus similis and Platylenchus goodeyi. Genetic improvement of the crops by conventional breeding has proved to be laborious and time consuming. Transgenic technology can offer sustainable solutions to the problem of controlling plant parasitic nematodes. To date, only constitutive heterologous promoters such as maize ubiquitin 1, rice actin 1, viral promoter (CaMV35S) have been used for the production of transgenic banana plants. This study defined the potential of Zea mays root cap specific promoter-1 (ZmRCP1) to drive biosynthesis of an anti-nematodes effector in plantain roots. Transgenic plants used in this study were regenerated from embryogenic cell suspension (ECS) of Gonja manjaya transformed by Agrobacterium tumefaciens strain EHA105 harboring the binary vectors pBI121 and pBI-ZmRCP:GUS. The study was designed to establish the difference in expression patterns of gusA gene under Cauliflower Mosaic Virus 35S (CaMV35S) constitutive promoter and ZmRCP1 and to assess the impact of R.similis infection on plantains at various stages of growth. To achieve the objectives, the study involved confirmation of the presence of gusA gene in plantain genomic DNA, inoculation of the plantains with nematodes and analysis of gusA gene expression pattern in the infected plants. Nematodes infection was achieved with R. similis. PCR amplicons were observed in all transgenic lines when DNA amplification was done using gusA specific primers. A histochemical GUS assay showed root cap expression of gusA in RCP1 lines. R.similis infection had no influence on ZmRCP1 expression pattern. The effect of R. similis infection on total leaf area (TLA) and total plant biomass (TPB) during infection period was analyzed. Consequently, a two-tailed t-test performed at 95% confidence level revealed a significant difference (P<0.05) between TLA and TPB of infected and uninfected plants. Infected lines showed lower TLA than uninfected lines. Nematodes infection reduced TPB of infected clones. The decline in TLA and TPB shows minimal or negligible natural resistance from G.manjaya against R. similis. As nematode population increased in roots, gusA expression at the root tip of RCP1 plants intensified. ZmRCP1 restricts gene expression at the root cap hence making it favourable for use in delivering a chemodisruptive peptide that can be released into rhizosphere with the root exudates. A lethal peptide can be used to achieve a complete resistance status in Musa spp.
Analysis of Genetic Diversity and Population Structure of Wild Loquat (Uapaca Kirkiana (Müell) Arg.)) Using Dartseq-Generated Single Nucleotide Polymorphisms
(Kenyatta University, 2022) Gati, Jane Maurine; Steven M. Runo; Alice Muchugi
Uapaca kirkiana (Müell) Arg, is a popular fruit tree that grows in the wild and is majorly found in the Miombo Woodland. It is popularly known as sugar plum or the wild loquat by the English name. It is a species of plant in the Euphorbiaceae family. U. kirkiana has been found to grow naturally south of the equator in Mozambique, Tanzania, Burundi, Zambia, Malawi, Zimbabwe, Burundi, Angola and Democratic Republic of Congo. There are 60 known species of the genus Uapaca. Increased consumption and utilization of U. kirkiana has led to high demand for the fruit and tree. Increased population and human activities have led to high pressure on land. As a result, forest reserves and national parks have been cleared to create space for the growing demand leading to loss of biodiversity. The domestication of U. kirkiana is a more significant step towards the management and conservation of biodiversity. Information on the amount as well as the distribution of genetic diversity is essential in effective management of germplasm resources. However, minimal molecular genetic evaluation on U. kirkiana has been carried out. The objectives of the research were to assess the genetic diversity and population genetic parameters, genetic relationships and population structure in U. kirkiana sampled from International Centre for Research in Agroforestry gene bank locations. Leaf material from 500 samples of U. kirkiana were collected, air-dried and well-preserved using silica gel then kept at –20 C till the extraction of DNA. The extraction of genomic DNA was done using the Cetyl Trimethyl Ammonium Bromide method with variations. Samples were then loaded onto 96 well plates and were sequenced at the Diversity Arrays Technology Pty. Ltd Australia. Data analysis was conducted through R, PHYLIP, and iTOL applications. The populations were divided into four groups by discriminant analysis of principal components and in the Neighbor joining analysis where cluster 1 had a total of 3 individuals, cluster 2 with 47, cluster 3 with 2 and cluster 4 with 289 individuals. However, the grouping pattern did not correspond to the geographical distribution of the plant. The overall genetic diversity was low with a value of Ht=0.1040. Analysis of molecular variance results indicated a high genetic density of 93.4% within samples and a lower genetic density of 1.3% between populations. Since the population was divided into four clusters, it would be economical to select a representative sample of each cluster to be preserved for germplasm conservation. The genetic diversity was low across the populations which may have been a result of the tree conservation strategy. The Germplasm conservation unit at International Centre for Research in Agroforestry may want to use populations that are genetically distant to increase diversity and enhance the long-term existence of the fruit tree. Genetic information obtained from this study will be beneficial in the domestication program and the genetic resources unit at the International Centre for Research in Agroforestry. Further analysis of U. kirkiana accessions for sex markers will lead to identification of the sex-specific markers at the molecular level and this information will be helpful in selection of the most desirable.
Analysis of genetic diversity in Eucalyptus Grandis (Hillex Maiden) seed sources using ISSR molecular markers
(2012-01-10) Okun, D. O.; Muluvi, G. M.; Kenya, E.U.; Oballa, P. O.; Odee, David W.
Eucalyptus grandis (Hill ex Maiden) is an economically important tree species that is native to the Australian continent and its northern neighbours, where it is grown primarily for its hard wood timber and pulp for paper industries. The species was introduced in the African continent as a railroad fuel wood and pulp wood source. It is widely grown in tropical countries such as South Africa, Kenya, Angola, Ghana, and Zimbabwe. Improved varieties of E. grandis can enhance production and help meet the local demands of its wood products. Similarly, planting in agroforestry systems can provide revenue for small-scale farmers. Although knowledge of genetic variation is vital for the sustainable management of the species under cultivation, little information on genetic diversity of the seed sources is currently available. Low genetic variation may lead to unsustainable production through inbreeding depression or inability to adapt to changing environmental conditions. In order to preserve and exploit the valuable genetic resources of tropical trees such as E. grandis, a systematic assessment of the available genetic variability within the existing seed sources is necessary. Inter-Simple Sequence Repeats (ISSR) marker analysis was used to assess the genetic variation between and within existing local seed sources of the species currently being used in Kenya. Improved E. grandis seeds currently being planted by farmers as introduction from S. Africa were included in the study. In total, 180 individuals from six seed source were assessed at 41 ISSR loci. Out of the 41 bands generated, 27(65.9%) of them were polymorphic. Cluster analysis (dendrogram) generated by UPGMA showed grouping together of the Western Kenya seed sources except for the seed source from Kaimosi. Variation also occurred among the South African seed source, which clustered with the Western Kenya seed sources and closely to the Zimbabwean seed source. The results therefore indicate that both international and national based strategies for conservation of the species are crucial. Thus, ISSR-PCR technology is a reliable, rapid (high throughput), and cost effective marker system that can be used to study genetic variation and genetic relationships among E. grandis seed sources. The highest variation was attributed to variation within (73.6%) than between seed sources, suggesting that most of genetic variation within species can be captured by sampling within a single seed source.
Anti-acetylcholinesterase activities of leaf extracts of carphalea glaucescens and gnidia glauca from Mbeere North Subcounty, Kenya on chilo partellus larvae
(Kenyatta University, 2016-07) Njoroge, Anne W.
The stem borer (Chilo partellus) is one of the major constraints in maize and sorghum production worldwide. Control of Chilo partellus is mainly done through synthetic insecticides which are very expensive and have negative effects to environment and non-target organisms. Farmers in Mbeere use Carphalea glaucescens and Gnidia glauca in control of Chilo partellus because of the high cost of conventional insecticides. However, there is lack of scientific information on the their mode of action is not known. This study was designed to gather preliminary data that can be used to develop a bio-insecticide to control the Chilo partellus. Aqueous and dichloromethane leaf extracts from Carphalea glaucescens and Gnidia glauca were assayed in vitro for their activities against Chilo partellus anti-acetylcholinesterase activity. Acetylcholinesterase is one of the most efficient enzymes of nervous system which is concentrated at the cholinergic synapses and at neuromuscular synapses where it rapidly hydrolyses the neurotransmitter acetylcholine. Acetylcholinesterase plays a critical role in terminating synaptic transmission so that the next nerve impulse can be transmitted across the synapse. Plants were collected from Siakago, Mbeere North sub-county in Embu County, Kenya. Chilo partellus were obtained from KALRO (Katumani) and the crude enzyme acetlycholinesterase extracted through homogenizination. Activity of the isolated crude enzyme was determined as described by Ellman et al. (1961). Acetylthiocholine iodide was used as a substrate in the assay. The optical density (OD) was measured at 412 nm by spectrophotometer. The experiments were done in triplicates. This study bioassay six extracts concentrations for both aqueous and DCM extracts of C. glaucescens and G. glauca. Cyclone was used as the standard drug and normal control lacked the inhibitor. This design was followed for aqueous and dichloromethane of the two plants. The aqueous leaf extracts of C. glaucescens percent enzyme inhibition was between 86.67% – 47.57% while DCM extracts of C. glaucescens percent inhibition was between 73.64% - 34.54%. Aqueous extracts of G. glauca percent inhibition was between 90.00% - 33.63% and DCM extracts of G. glauca percent inhibition was between 96.97% - 35.09%. Cyclone percent inhibition was 96.97%. Results also showed that the extracts had tannins, phenols, flavonoids, terpenoids, saponins, alkaloids, cardiac glycosides and steroids which have been associated with AChE inhibition activity. Therefore, the study has revealed that aqueous and DCM leaf extracts from C. glaucescens and G. glauca have the potential of anti- acetlycholinesterase activity. Hence the studied extracts can further be purified and developed into plant- derived biopesticides.
Anti-Nociceptive and Anti-Inflammatory Potential of Dichloromethane: Methanolic Extract of Caesalpinia Volkensii and May Tenus Obscura Cuf. in Animal Models.
(Kenyatta University, 2015-12) Mwangi, Boniface Maina.
The purpose of physiological pain is protection. Pain is associated with high morbidity and socioeconomic burden. On the other hand, inflammation is the reaction to injury of the living tissues. Conventional medication of pain and inflammation are expensive and arguably associated with various side effects hence the need to develop alternative agents. C. volkensii and M obscura are plants that grow in Mbeere County of Eastern region of Kenya. This study was designed to evaluate the antinociceptive, anti-inflammatory activity and to determine the qualitative phytochemical composition of C. volkensii and Mobscura plants. Each sample, 200g of powder was weighed, soaked separately in a cold 1:1mixture of methanol and DCM and stirred for six hours to obtain the extract. Experimental animals were divided in to four groups; normal group, diseased negative control group, diseased reference group and diseased experimental groups. Pain was induced into rats using formalin while inflammation was induced into the mice using carrageenan. The experimental groups were treated with leaf extracts of the plants at concentration of 50mg/kg, 100mg/kg and 150mg/kg. Anti-nociceptive and anti inflammatory activities in rats were compared with diclofenac (l5mg/kg) as the standard conventional drug. The leaf extracts of C. volkensii reduced the paw edema by between 6.50 - 13.42% while the extracts of M obscura reduced it by between 4.94 - 22.36%. Diclofenac reduced the paw edema by between 4.11% - 10.47%. For antinociceptive, the leaf extract C. volkensii reduced pain by between 6.82 - 15.24% in the early phase while the leaf extracts of M obscura reduced it by between 12.39- 34.81% in the early phase, and between 6.4 -12.4% in the late phase. Diclofenac reduced pain by between 7.58- 9.66%, in the early phase, and by 69.87% in the late phase. Phytochemical bioscreening showed that the extracts of C. volkensii had flavonoids, steroids and phenolics while the leaf extracts M obscura had phenolics, terpenoids, and saponins. Flavonoids, saponins and phenolics have been associated with anti-nociceptive and anti- inflammatory activities. Therefore, the study has established that the DCM: methanolic leaf extracts of Caesalpinia volkensii and May tenus obscura are effective in management of pain and inflammation. The study recommends that in antinociceptive studies using rat model, C.volkensii and M obscura at a dose levels of 100, and 150 mg/kg body weight, are effective in the early phase.The study futher recommends the use of dose levels of 150, and 50mg/kg body weight as an anti-inflammatory control.
Antibacterial Antioxidant, Phytochemical Profile and Toxicological Efffects of Aqueous extract of Rhaphiolepis Bibas (Lour.)
(Kenyatta University, 2023-05) Kariuki Ibrahim Waweru
Abstract
Antibacterial Activities of Dichloromethane: Methanolic Leaf and Stem Bark Extracts of Psidium Guajava Linn against Selected Bacteria
(Kenyatta University, 2019) Ayienda, Carol Kerubo
Antibiotics have been very effective in the management of microbial infections. However they are expensive and have many side effects. Additionally, the menace of antimicrobial resistance has resulted to the inactivity of various conventional antibiotics. Medicinal plants are used in herbal medicine to control microbial infections since they are considered cost effective and have shown less side effects. Plant extracts with medicinal value have been used to treat many diseases that can either be bacterial, fungal or parasitic among many others. Plants with medicinal value produce certain chemical elements known as phytochemicals that have antimicrobial activity. They include secondary metabolites such as flavonoids, tannins, and saponins which are the main chemical structural classes. Psidium guajava Linn is a plant which has been used in traditional medicine to manage various conditions including toothache, malaria, gastroenteritis and vomiting. This research focused determining the antibacterial activities of dichloromethane: Methanolic extracts of Psidium guajava stem bark and leaf extracts against selected microorganisms Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella typhi ATCC 19430 and Bacillus cereus clinical isolate. These microbes are normally associated with illnesses that are characterized with vomiting, enteric fevers, diarrhea and abdominal pain. The plant’s leaf and stem bark were collected from Bonchari Sub-county of Kisii County and transported to Kenyatta University. The samples were extracted using 1:1 mixture of DCM and methanol. The plant extracts antibacterial activities was established by taking measurements of the zones of inhibition in millimeters, MIC and MBC. The collected data was then analyzed using Minitab 17 statistical software, Student’s Test (T-test), one way ANOVA with P ≤ 0.05 being considered significant, followed by Tukey’s post hoc test. Both plant parts analyzed had antimicrobial activity against all the tested microorganisms. However the extracts did not exhibit any bactericidal activity (MBC) on all the tested microorganisms. The leaf extracts had MIC levels of 25mg/ml, 100mg/ml, 25mg/ml and 100mg/ml against Staphylococcus aureus, Escherichia coli, Salmonella typhi and Bacillus cereus respectively. The stem bark showed an MIC dose of 50mg/ml against all the tested bacteria. The phytochemical analysis showed the presence of all the phytochemicals tested; saponins, alkaloids, terpenoids, flavonoids, steroids and phenolic in the stem bark, while steroids were not present in the leaf extract. The results provides an enlightment concerning the antimicrobial activities of the plant extracts and their use in the treatment of several bacterial infections.
Antidiabetic activity and safety of aloe volkensii, acacia nilotica, euclea divinorum, rhoicissus tridentata, cynanchum viminale and urtica dioica in mice.
(Kenyatta University, 2015-11) Mwangi, John Mukundi
Diabetes is increasingly affecting a growing number of patients and seriously reducing their quality of life. Use of conventional drugs in diabetes management is expensive, thus, unaffordable to most patients. Furthermore most of these conventional drugs are associated with undesirable side effects. Incorporation of herbal medicine into conventional healthcare system may significantly improve the overall management of Diabetes. Evaluation of efficacy and safety by scientific method is necessary to validate herbal medicine utilization. In most cases even where efficacy of the plants has been established the standard dosage required to bring about healing is not clear. This study evaluated six plants for their efficacy and toxicity using aqueous extracts in alloxan induced diabetic mice. Both the Intraperitoneal and oral routes were used in the administration of the extracts. Stem bark extracts of Euclea divinorum (Olkinyei) and Acacia nilotica (Olkiloriti), whole stem extracts of Rhoicissus tridentata (Orkilenyai) and Cynanchum viminale (Oleilei), leaf extracts of Aloe volkensii (Osuguroi) and Urtica dioica (Entamejoi) were used. In the evaluation for efficacy, mice were divided into seven groups, diabetes was induced using alloxan and each group was treated with a specified dose of the plant extract. The results were compared to those obtained from the group treated with conventional drug Insulin through the intraperitoneal route and glibeclamide administered by using the oral route. Standard procedures were used in determination of phytochemical content of the plant extracts, while TXRF and AAS methods were used to determine the minerals present in the extacts. Toxicity studies involved administration of a high dose of the extracts at 1000mg/kgbw through the intraperitoneal and oral routes. Phytochemical analysis was done by use of standard procedures. Results revealed antidiabetic activity of all the plants at varyng levels, Urtica dioica was the most effective with blood sugar lowering ability being comparable to that of conventional drugs. Cynanchum viminale and Euclea divinorum showed the lowest hypoglycemic activity. Intraperitoneal route showed greater hypoglycemic activity than oral route. Toxicity studies confirmed the safety of most of the plant extracts in both intraperitoneal and oral routes. Quantitative and qualitative phytochemical analysis revealed the presence of Alkaloids, Tannins, Saponins, Phenols, Flavonoids and Phylobatanins. Determination of the levels of mineral constituents demonstrated the present of K+, Ca2+, Cr6+, Cd2+, V3+, Mn2+, Fe2+, Cu2+, Zn2+, Mg2+, K+, Na+, Se2+, Ni2+, As5+, Cl-, Rb2+, Br2+ and Pb+. In conclusion, the results showed that all the six plant extracts except Euclea divinorum and Cynanchum viminale were effective in reducing blood sugar levels. Euclea divinorum was the only plant extract that showed high levels of toxicity the other plant extracts did not reveal significant toxicity effects.These results validates the use of the plant extracts in phytomedicine. Consideration should be made to carry out similar studies using higher animal models for confirmatory purposes.
Antidiabetic activity and safety of piper capense, berberis holstii, sonchus asper, vernonia lasiopus and galinsoga parviflora in alloxan-induced diabetic albino mice
(2017-09) Kimani, Lucy Njeri
In Kenya, diabetes mellitus is of health concern to the public, because it causes substantial morbidity, mortality, and long-term complications. Synthetic drugs used in the management of diabetes are unavailable, have numerous side effects and are expensive. Many plants such as Piper capense, Berberis holstii, Sonchus asper, Vernonia lasiopus and Galinsoga parviflora used traditionally to manage many diseases including diabetes mellitus but their efficacy and safety after long-term use are not scientifically validated. This study aimed to determine in vivo antidiabetic activity and safety of aqueous plant extracts of Piper capense, Berberis holstii, Sonchus asper, Vernonia lasiopus and Galinsoga parviflora in male albino mice. Aqueous plant extracts were screened for antidiabetic activity in diabetic mice using the intraperitoneal and the oral routes. In the study, albino mice were assigned into eight groups of five mice each. For this purpose, reduction in blood glucose relative to their initial values was determined after oral and intraperitoneal administration of 25, 48.4, 93.5, 180.9, and 350 mg of aqueous extracts/kg body weight. 1IU/kg body weight dose of insulin and 4.6 mg of glibenclamide (200 mg/kg body weight) were used as a standard hypoglycemic agent to compare the results. Glucose levels were estimated at the beginning of the experiment and repeated after 2, 4, 6, 8, 10 and 24 hours after administering the drugs. Significant reduction in blood glucose relative to their initial values was determined for all treated non-diabetic and diabetic groups at the end of experiment. Mineral composition of the aqueous plant extracts was determined using TRXF (total reflection X-ray fluorescence system) while the quantities and types of phytochemicals present were determined using standard procedures. Toxicity of the aqueous plant extracts to normal mice was studied by orally and intraperitoneally administering them with 450, 670 and 1000 mg/kg body weight daily for 28 days and kept under close observation. Changes in body and organ weight, hematological and biochemical parameters were also determined. After the 28th day, mice were sacrificed and pieces of pancreas, lungs, brain, testis, heart, kidney, spleen and livers were removed for weight change evaluation. Aqueous extracts orally and intraperitoneally administered at 25, 48.4, 93.5, 180.9, and 350 mg/kg body weight showed antidiabetic activity through either route. Oral and intraperitoneal dose of 450, 670 and 1000 mg/kg body weight of the plant extracts significantly reduced the body weight gain. The same oral and intraperitoneal dose of Piper capense, Berberis holstii, Sonchus asper, Vernonia lasiopus and Galinsoga parviflora altered the hemoglobin levels, mean cell hemoglobin concentration, platelets, red blood cell count, packed cell volume, mean cell volume, white blood cell count and their differential counts. The dose also altered activities of aspartate and alanine aminotransferases, alkaline phosphatase, α-amylase and lactate dehydrogenase. The plants extract contained phenols, tannins, saponins, flavonoids, and alkaloids. Minerals present were potassium, calcium, titanium, bromine, iron, zinc, copper, chromium, manganese, vanadium, rhubidium, strontium, and heavy metal lead. The observed antidiabetic activity toxicity observed in the plants extracts could be due to the phytochemicals and minerals present in the plants extracts. The study recommends use of safe plants with antidiabetic activity as herbal remedies. Comprehensive safety studied on the plants and organic solvent extraction for comparison of the activities of both organic and aqueous extracts
Antigenic and molecular characterization of influenza viruses isolated from patients with respiratory tract infections in Nairobi
(2011-11-29) Maina, George Gachara; Ngeranwa, J.J.N.; Makumi, J. N.
Influenza virus infection is a highly contagious respiratory disease that can spread easily and that is responsible for considerable morbidity and mortality each year. There are three types of influenza viruses, designated A, B and C all which cause the disease characteristically recognized as influenza. Types A and B influenza viruses can cause a wide spectrum of illness, including lower respiratory tract disease, pneumonia and even, in the case of type A, encephalopathy and encephalitis. On the other hand type C infections are limited to the upper respiratory tract. While antivirals are available against the virus, vaccination remains the most important measure for reducing this sizeable public health burden. I Iowever, as a result of the antigenic variation of its surface glycoproteins, the influenza virus is an ever-changing target. Influenza vaccines therefore present special challenges because each year the vaccine strains must be updated in order to retain optimal protective efficiency. In the face of a looming pandemic, there is need to step up surveillance and characterize local strains inorder to identify vaccine strain variants and a potential pandemic strain. This study is the first attempt to characterize local influenza strains. Between January and August 2005, 660 throat swab samples were collected from patients in eleven sentinel sites around Nairobi that are involved in influenza surveillance. The samples were processed for immunofluorescence. stained using SimulfluorTm Flu A/Flu B reagent and examined under a fluorescent microscope. 252 (38.2%) samples were found to be influenza B while none was positive for influenza A. The positive samples were inoculated and cultured onto MDCK cell monolayers in tissue culture tubes. A `blind' passage was performed and the samples frozen at -85°C. Haemagglutination (HA) using 1 % guinea pig red blood cells was performed on recovered isolates to determine haemagglutinating isolates and 135 (54.4%) samples gave a positive HA result. These were confirmed to be influenza viruses and subtyped by the Haemagglutination inhibition test (HAI) using the 2005 reference reagents for influenza virus diagnosis prepared by the WHO Collaborating Center for Reference and Research on Influenza, Melbourne, Australia. 134 (99.25%) isolates were characterized as influenza B%Shanghai/361i2002-like strains while 1 (0.75%) isolate was identified as an influenza B/Hong Kung/330/2001-like strain. RT-PCR was performed on the HAI positive isolates that had different titres from the vaccine strain and some which had similar titres with the vaccine strain. This was followed by a sequencing PCR and sequencing of the HAI domain of the HA gene carried out in an automated sequencer ABI 310. 8 isolates were successfully sequenced on the basis of the HAI titres observed in HAI and a BLAST analysis used to identify similar strains. All the sequences that were generated in this study were analyzed phylogenetically. Multiple sequence alignments and phylogenetic analysis of the nucleotide sequences obtained was carried out by ClustalW software inorder to generate phylogenetic trees. Influenza A was not isolated in this study, it's presence in tropical African countries is usually associated with epidemics and none was experienced in the study period. At least 3 variants of the vaccine strain were identified indicating that the local strains are drifting away from the vaccine strain. The nucleotide sequences were translated using the EMBOSS software and the resultant amino acid sequences used to generate phylogenetic trees. Based on the protein sequences, all the isolates were found to be closely related showing that antigenic drift observed is slow. Findings in this study and others globally have led to the WHO changing the influenza B vaccine component for the 2006 season. This study reveals that local strains are progressively undergoing antigenic drift and recommends wider continued active surveillance.
Antimalarial activity of some plants traditionally used in treatment of Malaria in Kwale and Meru districts
(2011-11-29) Muthaura, C. N.
Plasmodium falciparum, the commonest etiological agent for human malaria is becoming increasingly resistant to commonly used drugs like chloroquine (CQ), which was efficacious, available, affordable and of low toxicity. Therapeutic regimens for prevention and treatment, of CQ resistant P. falciparum are associated with higher costs and side effects compared to CQ. Efforts are being directed towards obtaining new drugs with different structural features. The success of artemisinin, a sesquiterpene lactone from Chinese traditional medicine which has a different chemical structure from quinine has stimulated the search for new antimalarial drugs from traditional antimalarial remedies. Since many modern drugs originated from plants, the investigation of chemical components of traditional medicinal plants could lead to development of new effective, affordable and safe antimalarial drugs. Kenya, with its rich floral resources and ethnobotanical history is an ideal place to document and screen plants for antiplasmodial activities. Sixty four medicinal plants with a traditional reputation for their use against malaria were collected and documented from two study sites at Meru and Kwale districts. 15 plant species were selected for antimalarial assays. 15 methanolic and 15 water extracts from these plants were screened for their in vitro antiplasmodial activities by inhibition of radio labeled uptake of hypoxanthine by CQ sensitive (D6) and resistant (W2) P. falciparum clones. They were also screened for their antimalarial activities in vivo in a 4- Day suppressive test using mice infected with P. berghei strain ANKA. Animal acute toxicity and cell cytotoxicity using Vero E6 cells was carried out on all the extracts. Of the 15 methanolic extracts tested for antiplasmodial activity, 10 (67%) had IC5O<20mg/ml for W2 and D6 sensitivity. The water extracts were less active. For the same 15 methanolic extracts, 10 (67%) had a chemo suppression >50% while 6 (40%) water extracts had chemo suppression >50%. Half of the water extracts were inactive in vitro, but active in vivo suggesting a prodrug effect. Four methanol and four water extracts gave survival times which were similar to that of mice treated with CQ (p>0.05). The CQ sensitive clone was more susceptible to the extracts than the CQ resistance clone. The ratio between the two, for some of the extracts, was less than that of CQ reference drug suggesting no cross-resistance to CQ. Almost all the extracts tested for acute toxicity were safe with LD5O>5000 mg/kg body weight and the selectivity index (SI) of the extracts was high indicative of non-toxicity. These findings seem to confirm the validity of these plants for traditional treatment of malaria and a possibility of activity-guided isolation of active principles from the bioactive extracts.

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