PHD-Department of Biochemistry and Biotechnology

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Banana Rhizosphere Microbial Diversity, Low-Cost Tissue Culture Bananas (Musa spp.) Micropropagation, Genetic Fidelity and Acclimatization Using Arbuscular Mycorrhizal Fungi
(Kenyatta University, 2024-05) Wahome, Caroline Nyawira
Banana production in Kenya predominantly involves smallholder farmers, yet current yields fall short of meeting local and international market demands due to constraints faced by these farmers among them being soil health and lack of clean planting materials. To address this issue, the study assessed banana cultivation practices in Kisii, Nyamira, and Embu counties, aiming to understand strengths and challenges within these regions. In this study, rhizospheric soil physicochemical properties and the diversity of microbial communities was assessed, shedding light on soil health status. Furthermore, the efficacy of a cost-effective micro-propagation technique for local banana cultivars was determined, considering contamination susceptibility and acclimatization efficacy of arbuscular mycorrhizal fungi on micropropagated banana plantlets. The study revealed a reliance on ineffective traditional propagation methods, with over 30% of the smallholder farmers facing a banana seedlings shortage. The soil physic-chemical parameters indicated signs of degradation, with low pH and nutrient deficiencies raising concerns with Embu County having the lowest pH measured (4.43). Soil metagenomic studies by NGS showed that proteobacteria and basidiomycota were the two most prevalent bacterial and fungus divisions, respectively. Based on the Shannon diversity indices, the highest bacterial and fungal community diversity was in soil samples obtained from Embu County and Kisii County respectively. To evaluate efficacy using low cost tissue culture technology, selected banana cultivars were utilized and results indicated the highest survival rate of 100 % in Kiganda, Muraru and Kienyeji banana cultivars established in BAP and IAA. Ng’ombe cultivar best responded to low cost micropropagation with the highest shoot establishment (80.5 %) and shoot emergence rates (75.5 %) producing the highest number of shoots per culture (10.61). To investigate clonal fidelity of the tissue culture plantlets Inter Simpler Sequence Repeats (ISSR) and the research demonstrated genetic homogeneity in banana seedlings generated via low cost tissue culture to the mother plant. During the micropropagation process the presence of bacterial and fungal contaminants in the tissue culture laboratory was noted with the most predominant bacterial and fungal contaminants being Microccoccus luteus and Cladosporium sp respectively. Additionally, broad-spectrum antimicrobial agents nalidixic acid (30 µg) and carbendazim+ triadimefom (0.3 µg and 0.6 µg) exhibited 100% inhibition rates against these contaminants. To determine the response of the micropropagated plantlets to AMF acclimatization, it was observed that Kiganda cultivar responded best to Rhizophagus irregularis which showed the highest recorded biomass at 0.97. There is need to emphasize collaborative efforts among various stakeholders in establishing sustainable practices. Additionally, low-cost TC is a viable tool for producing disease-free seedlings and intergrating antimicrobial agents is recommended to minimize contamination risks. Finally, the use of AMF treatment in acclimatization with further research needed for validation across different agroecological conditions. These recommendations aim to enhance productivity, sustainability, and resilience in banana farming systems.
Diversity of Moulds, Sources of Contamination, Moisture Content and Aflatoxin Levels in Fish and Fish Feeds in Kisii County, Kenya
(Kenyatta University, 2024-02) Nyamwaka, Irene Seila
Aflatoxins are toxic substances produced by some members of Aspergillus species which commonly invade foodstuffs as well as animal feeds. When consumed in quantities exceeding 10 µg/kg, they cause nephrotoxic and hepatotoxic effects in humans and other animals. Data on the levels of aflatoxins in fish and fish feeds sold in Kisii County is not available. This study was carried out to assess the diversity of moulds associated with fresh, sundried fish and fish feeds sold in selected markets in Kisii County, determine aflatoxin levels in sundried fish and fish feeds, evaluate the relationship between aflatoxin levels and moisture content in sundried fish and fish feeds and assess sources of fungal contamination of sundried and fresh fish sold in selected markets in Kisii County. Samples of sundried and fresh fish from retailers in four main markets in Kisii County were obtained by random sampling. Samples from fish farmers in Kisii County and shores of Lake Victoria were obtained to serve as control. Commercial and home-made fish feeds were purposively obtained from five main outlets and three self-help groups respectively. Moisture content of all samples was determined by oven drying and the levels of aflatoxin in sundried fish and fish feeds determined by ELISA. Isolated moulds were identified using macroscopic and microscopic characteristics and Sanger sequencing technique. Sources of fish contamination were determined using a structured questionnaire and level of contamination expressed as cfu/g. Data on aflatoxin levels and colony forming units in different types of fish and fish feed samples was analysed using one-way analysis of variance. The means that were significantly different were separated using Tukey’s HSD test. Two-way analysis of variance was used to analyse the interactions between markets and type of fungi, types of fish feeds and fungal species, markets and types of fish. Data from molecular work was analysed using MEGA version 4. Relationship between moisture content and aflatoxin levels in fish and fish feed samples was analysed using regression analysis. Thirty fungal species from fourteen genera namely; Acremonium, Alternaria, Aspergillus, Candida, Cladophialophora, Cladosporium, Cunninghamella, Fusarium, Microporum, Mucor, Penicillium, Phoma, Trichoderma and Verticillium species were isolated from fresh and sundried fish samples. Eighteen fungal species were isolated from commercial and home made fish feeds. They comprised of eleven genera; Alternaria, Aspergillus, Aureobasidium, Cladosporium, Eurotium, Fusarium, Mucor, Penicillium, Stachybotrys, Trichoderma and Rhizopus species. Aflatoxin levels in sundried fish ranged from 20-840 ng/kg and 80-500 ng/kg in fish feeds. The cfus/g from control samples were significantly low compared to those from fresh and dried fish samples (p < 0.05). Transportation, handling and storage were found to be sources of fish contamination. The relationship between moisture content and fish, moisture content and fish feed was statistically significant (R2 = 0.942, p = 0.000) and (R2 = 0.797, p = 0.000) respectively. The findings of this study offer new insights into the levels of aflatoxins and associated fungi in fish and fish feeds sold in Kisii County. Therefore, there is need for frequent surveillance on aflatoxin levels in dried fish and fish feeds sold in this County.
Relationship between Arterial and Venous Biochemistry and Acid-Base Values in Patients with Diabetic Ketoacidosis and Chronic Kidney Disease
(Kenyatta University, 2024-06) Gatua, Wilfred Kimani
Two of the complications of diabetes mellitus include diabetic ketoacidosis (DKA) and chronic kidney disease (CKD) and both conditions may lead to metabolic acidosis. In DKA there is excessive oxidation of fatty acids with subsequent accumulation of acetoacetate and β-hydroxybutyric acid while in patients with kidney dysfunction, the renal tubules lose the ability to regenerate bicarbonates effectively with resultant reduction of bicarbonate in blood. Clinical management of metabolic acidosis requires frequent arterial blood gas analysis (BGA) to determine the levels of acid-base parameters. Other biochemistry parameters tested to monitor the progress of management include: liver function tests, kidney function tests and pancreatic function tests. Arterial puncture is a painful and traumatic procedure which may lead to the formation of hematomas and sepsis and requires special skills to perform. There is therefore the need to substitute arterial blood with venous blood which is easy to obtain and is less painful to patients and has less complications. However, venous blood as a substitute for arterial blood has not been evaluated in the country to ascertain the level of relationship and agreement with arterial blood. The objective of this study was to evaluate levels of acid-base and routine biochemistry parameters in both arterial and venous blood in patients with diabetic ketoacidosis and chronic kidney disease and also determine the relationship and agreement between the two types of blood samples. A cross-sectional study composed of 200 patients with CKD and 150 patients sampled at Kenyatta National Hospital were uesd. Arterial and venous blood were collected from each study subject, and acid-base parameters, liver, kidney, and pancreatic function tests were carried out. Laboratory methods included measurements of acid-base parameters using RapidLab 348 blood gas analyzer. Biolis Clinical Chemistry Autoanalyser was used for the measurements of biochemistry parameters. The data generated was tested for its normality using Kolmogorov-Smirnov test. The results were expressed as mean ± SD and t-test used for statistical comparison of two means for two data sets. For relationships between measured arterial and venous blood parameters, correlation coefficient, simple and multiple linear regressions was used. For agreement between measured arterial and venous blood parameters, Bland-Altman bias plot was used. The key findings of this study were that patients with DKA and CKD have significant variations in the levels of both acid-base and biochemistry parameters. The generated bootstrapped simple linear regression equations between the measured arterial and venous blood analytes demonstrated strong correlations and agreements. This study therefore validates the use of venous blood for the measurement of blood gas and biochemistry parameters in the management and monitoring of acid-base metabolic disorders in patients with chronic kidney disease and diabetic ketoacidosis. This will hopefully minimize the need for arterial sampling which is hazardous and traumatic to the patients.
Development of Rapid Diagnostic Tool and Characterization of Rift Valley Fever Virus Infection in Kenya
(Kenyatta University, 2024-06) Wekesa, Francis Chaka
Rift Valley fever (RVF) is a high-priority zoonotic disease characterized by massive loss of livestock within a short period of its outbreak. The disease is endemic in sub-Saharan Africa and mainly spread via infected female Aedes and Culex genera of mosquitoes. This study aimed to develop a rapid diagnostic tool and spatio-temporal characterization of RVF in Kenya. During this study, a calorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of Rift Valley Fever Virus was developed and validated. In addition, RVF infection was characterized through generation of the current RVFV transmission risk maps of Kenya by computation of the basic reproduction number (Ro) based on temperature change, presence of vector and host, and the host recovery rate. The risk maps were validated using a serological study that was carried out on 615 blood samples collected from Siaya, Busia, and Kisumu counties in the year 2018. The RVFV IgG and IgM antibodies were screened and the results confirmed using either serum viral neutralization test and/or real time polymerase chain reaction (RT-qPCR). The novel diagnostic tool that was developed detected the presence of the virus within 30 minutes at a constant temperature of 65oC with a sensitivity of 98.36% and a specificity of 96.49%. The RVFV transmission risk maps showed the likelihood of having outbreaks in seventeen counties of Kenya. Most of these counties were from arid and semi-arid lands (ASALS) whose livelihood greatly depends on livestock. However, the possibility of the disease to occur outside ASAL areas was demonstrated by the presence of IgG and IgM antibodies in counties previously perceived as RVFV-free zones. The average seropositivity rate of RVFV IgM antibodies in the three counties was estimated to be 10.33% with Siaya county leading with 24% (95% CI (2.269-8.009) OR 4.26) followed by Busia County 7% (95% CI (0.532-1.88) OR 4.26). Kisumu County recorded Zero RVFV positivity rate. The overall seropositivity rate for RVFV IgG antibodies from the three counties was at 14% with Siaya County leading at a positivity rate of 22% (95% CI 0.805 -2.140) OR 1.31), followed by Busia 18% (95% CI 0.349 -2.867) OR 1) and Kisumu 2% (95% CI (0.032-0.265) OR 0.09). The colorimetric-RVFV-UDG-RT LAMP assay was ten-fold more sensitive compared to the RT–qPCR. This is attributed to many start points for amplification in the LAMP technology as opposed to single start point in real time polymerase chain reaction (RT–qPCR). The RVF spatio-temporal model correctly predicted regions and months that the disease is likely to occur. The presence of undetected circulation of RVFV IgG and IgM antibodies in Siaya, Busia, and Kisumu counties, points to a possible impending future outbreak of the disease in these areas as a result of the paradigm shift in weather conditions due to climate change. Siaya county showed highest prevalence of the RVFV followed by Busia county due to their proximity to Uganda that recently experienced RVF infection outbreak in one of the districts. Therefore, the kit can be adopted and deployed for rapid screening of RVFV in Kenya.This creates the need to focus and invest in the RVF infection control measures. The findings of this study calls for increased disease surveillance to cover wider western borderline counties and proper emergency preparedness in case of RVF outbreak so that the disease is managed on time to minimize losses during outbreaks. More importantly, RVF risk maps could contribute to data-driven decision-making for disease management when deploying vector control interventions.
Determination of Wilt Disease Resistance, Performance and Environmental Safety of Transgenic Bananas Expressing Hrap-Pflp and Espflp Genes under Field Conditions
(Kenyatta University, 2024-04) Muchira, Dancun King’ang’i
Banana Xanthomonas wilt (BXW) disease is caused by the bacterium Xanthomonas campestris pv musaceae. Transgenic banana expressing the genes hypersensitive reactive associated protein (Hrap), plant ferredoxins-like protein (Pflp), and extracellular secreted plant ferredoxin-like protein (EsPflp) have been developed and shown to control BXW in glasshouse in Uganda. However, the transformed banana lines have not been evaluated in Kenya for BXW disease resistance, field performance, and environmental safety. BXW disease causes over 50% losses of production in farmer’s fields. Banana production is essential to Kenya and the region as it provides food, fodder, and income. In Uganda, these transgenes have been used to transform banana cultivars of Sukali Ndiizi (AB) and found to be resistant to BXW under glasshouse and field trials. Non-target effect studies of the transgenes have also been conducted in Uganda for non-target soil bacteria colonization. As demonstrated in the trials, the technology has potential, so conducting field trials for the transgenic banana events in Kenya is important to generate data on disease resistance, performance and environmental safety before they are incorporated into agricultural production. With respect to this, a confined field trial was established in Busia County at Kenya agricultural and livestock research organization (KALRO) Alupe research station. Randomized complete block design (RCBD) with 30 banana events replicated 12 times was used. Bananas were planted at a spacing of 3m by 3m from each other and a hole size of 2.5 cubic feet. During planting, 20 kg of well-composted manure and 100 g of double ammonium phosphate (DAP) were used for each banana stool. At 5 months after planting, molecular characterization of the plants was carried out. The plants were then physically infected with the wilt-causing cultures. Disease resistance was carried out by scouting for disease symptoms post-infection. Morphological/Agronomical data was collected on traits such as height, girth, and leaf total area. Soil studies were also conducted by analyzing plant litter decomposition rate and carbon readings. Environmental studies were conducted by analyzing the numbers of soil nematodes, flying insects, and banana weevils. All the data were analyzed using one-way ANOVA, and significance levels were read at α=0.05. In the BXW disease resistance evaluation and agronomy, the effects of the transgenes could not be determined, as the study ended prematurely because both transgenic and non-transgenic plants died within 12 months. The differences between transgenic and non-transgenic control in soil qualities, soil nematodes, flying insects, and banana weevils did not differ significantly (p˃0.05). The study implies that Hrap-Pflp and EsPflp are environmentally safe as they did not affect non-target organisms and soil quality. The transgenes can, therefore, be selected for multilocation assessment to collect more data on safety.
Plant regeneration from leaf mesophyll-derived protoplasts and gene editing of cassava for enhancement of root shelf-life
(Kenyatta University, 2024-05) Njeru, Asunta Mukami
Manihot esculenta (Crantz) a starchy, multipurpose root crop is grown by millions of smallholder farmers in tropical and subtropical countries. Despite many benefits, physiological degradation of storage roots which happens within 24–72 hours post-harvest makes them unpleasant and unmarketable, severely constraining its production. The storage root experiences an oxidative burst and a buildup of secondary metabolites, with scopoletin being the most prevalent, as a result of the inevitable damage that occurs to the roots during harvest. Blue-black discoloration and vascular streaking observed during deterioration occurs due to over-accumulation of scopoletin whose biosynthesis involves key enzymes including feruloyl CoA 6′-hydroxylase coded by Manihot esculenta feruloyl CoA 6′-hydroxylase1, 2 and 3 genes. Although plants' genomes have been edited using tools such as zinc finger and transcription activator-like effector nucleases, these tools are time and labour consuming. Clustered regularly interspaced palindromic repeats (CRISPR)-Cas technology has provided an effective and successful gene-editing method for crop improvement since it is quicker, less expensive, and better at multiplexing than other technologies. CRISPR-Cas9 targeted mutagenesis of feruloyl CoA 6′-hydroxylase genes (MeF6'H1, MeF6'H1-2 and MeF6'H3) were used in this study to delay postharvest physiological deterioration in cassava roots. Three cassava varieties Tms 60444, Muchericheri and Karibuni were used. A reproducible cassava protoplast regeneration protocol was established. Although cassava protoplast regeneration was not successfully established in a solid culture system, regeneration was successful using a suspension culture system. Evaluation of optimum cell density for cassava protoplast regeneration showed callus induction in all cell densities employed (1, 2, and 3×105 p/mL). The study established 2×105 p/mL as the best protoplast cell density in the suspension culture system for somatic embryogenesis. To generate cassava plants with increased root shelf-life, CRISPR-Cas9 cassettes with Cas9 gene and sgRNA targeting the three genes were introduced into cassava varieties using Agrobacterium-based gene delivery method and transgenic plants developed through somatic embryogenesis. The gene constructs altered to incorporate neomycin, hygromycin and basta resistant genes were transformed into Agrobacterium strain GV3101 facilitating the screening of transgenic events. Polymerase chain reaction (PCR) confirmed integration of transgene in putative transgenic plants while targeted Sanger sequencing revealed CRISPR-Cas9 induced mutations with substitution and deletions being observed above and below the protospacer adjacent motif of target genes. The amounts of scopoletin in mutant cassava were evaluated using high performance liquid chromatography (HPLC). Plant targeted for edition of dual genes exhibited significant reduction of scopoletin below HPLC detectable levels and had improved shelf-life compared to one gene knockout events and wildtype plants. Evaluation of phenotypic variations between mutant and wildtype cassava plants revealed mutationevent associated traits including stem and petiole depigmentation, auxiliary budding, clawlike leaf appearance, loss of apical dominance and leaf chlorosis. This study shows a practical and viable method to improve cassava storage root shelf-life. Production of cassava with enhanced shelf-life will stabilize yield production, prevent post-harvest wastage, and improve farmers’ income.
Genetic Mechanisms of Pre-Attachment Striga hermonthica (Del.) Benth., Resistance in Sorghum Revealed by Ribonucleic Acid Sequencing
(Kenyatta University, 2024-03) Gilles, Irafasha
Striga hermonthica is an obligate root parasitic plant that has a significant negative impact on the yield of major crops in sub-Saharan Africa, causing devastating yield loss to already struggling economies. This parasite has purple flowers with tinny dusty seeds of 0.2 to 0.5 mm that are easily dispersed. Within the soil, they can stay dormant for up to a decade waiting for ideal conditions to align, to ensure successful germination near a potential host. S. hermonthica seeds germination is a decision mediated by endogenous hormones in response to different environmental cues, of which the most important are different strigolactones which are found within a complex blend of many secondary metabolites exuding from nearby plant roots. Although S. hermonthica seeds depend on strigolactones to germinate, little is known about how it produces or uses hormones that play important roles in the germination of its seeds and how they react to differences in root exudate composites from potential hosts as they germinate. This study presents a transcriptomics analysis of how hormones regulate S. hermonthica seeds germination upon exposure to root exudates from both susceptible and resistant sorghum genotypes, and a synthetic Strigolactone GR24. The study investigated the expression patterns of genes involved in the biosynthesis of major hormones during S. hermonthica seed germination at different time points (6, 12, and 18 hours) upon germination induction and used preconditioned but untreated seeds as control. Resulted transcripts entail that Violaxanthin de-epoxidase (VDE) inhibits the ABA biosynthesis a hormone that promotes seeds dormancy. The biosynthesis of Gibberellic acid (GA1 and GA4) promotes seed germination and its biosynthesis was highly enriched at 18 hours in seeds treated with SRN39 root exudates, suggesting that the pre-attachment resistance associated with this genotype is due to delayed germination in contrast to other resistant genotypes (IS27146 and IS 41724), ABA and Auxin were highly upregulated in seed that showed the least germination metrics. Overall, GA and ABA were the main hormones that controlled germination and GR24-treated seeds showed a relatively different germination mechanism from what was observed in seeds treated with sorghum root exudates. This study shows how S. hermonthica germination program and rate varies according to different germination stimulants from root exudates. Therefore, profiling of these root exudates is recommended. Findings in this study are necessary for future Striga resistant sorghum breeding programs.
Cancer Risks Associated with Exposure to Background Ionizing Radiation in Human Habitat, Soil, and Food in Kenya
(kenyatta university, 2023-10) Ogola, Phillip Einstein; Richard Okoth Oduor; Mathew Piero Ngugi; Siphila Mumenya Wanjiku
Cancer is described as uncontrollable cell proliferation in the body. By spreading outside their borders and infiltrating neighbouring tissue, such cells can invade to other organs. Cancer is the third leading cause of mortality in Kenya, trailing only infectious and cardiovascular diseases, with an annual incidence increasing from 37,000 new cases in 2012 to 48,000 new cases in 2018. Similarly, annual cancer mortality increased from around 28,000 to 33,000 within the same time span. Cervical, breast, colorectal, prostate, and esophageal cancers are the most prevalent types of cancer in Kenya. According to the National Cancer Control Strategy 2011-2016, cancer arises from abnormal alterations resulting from interactions between genetic and environmental elements, which encompass physical carcinogens such as ionizing radiation. The GLOBOCAN 2020 estimates reported that in Kenya, there were 42,116 new cancer cases and 27,092 deaths attributed to cancer. Types of cancer linked to high radiation dose exposure include leukemia, breast, bladder, colon, liver, lung, esophagus, ovarian, multiple myeloma, and stomach cancers. Naturally Occurring Radioactive Materials (NORMs) are found in trace amounts in soil, water, food, and the atmosphere. While natural radioactivity is formed from primordial radionuclides spread throughout the earth's crust, human activities such as nuclear power generation and mining contribute to the release of additional radionuclides into the environment. Human exposure to radiation comes through external sources as well as consuming radionuclides through water and food ingestion or breathing of radioactive vapors. This research focused on establishing the cancer risks associated with exposure to background ionizing radiation in quarries, human premises, soils from agricultural zones, and tuber crops (cassava, sweet potatoes, and Irish potatoes) in Kisii, Kakamega and Busia Counties in Kenya. Ionizing radiation levels were detected using handheld radiation detectors in quarry sites and buildings, whereas 232Th, 40K, and 238U activity concentrations in soil and tuber crops were determined using high purity germanium detectors. In quarry sites, the calculated average annual effective dose (AED) was 1.702 mSv in Kisii County, 2.083 mSv in Kakamega County, and 2.016 mSv in Busia County. Furthermore, the mean excess lifetime cancer risk (ELCR) due to occupational exposure in quarry sites were 5.702×10-3 in Kisii County, 6.979×10-3 in Kakamega County, and 6.754×10- 3 in Busia County. The computed AED values in the premises were 1.369 mSv in Kisii County, 1.317 mSv in Kakamega County and 1.268 mSv in Busia County. Also, the lifetime cancer risk attributable to indoor background ionizing radiation exposure were 4.586×10-3 in Kisii County, 4.411×10-3 in Kakamega County and 4.247×10-3 in Busia County. From the findings, cancer risk associated with lifetime exposure to ionizing radiation in quarry sites and premises sampled herein was low. Moreover, the mean activity concentrations of 232Th, 238U, and 40K in soil samples were 83.58 Bq/kg, 28.6 Bq/kg, and 483.40 Bq/kg respectively in Kakamega County, 65.36 Bq/kg, 22.88 Bq/kg, and 176.72 Bq/kg respectively in Busia County and 106.6 Bq/kg, 46.08 Bq/kg, and 153.42 Bq/kg respectively in Kisii County. Additionaly, the mean activity concentrations of 232Th, 238U, and 40K in tuber crops sampled in this study were below the global average values. Furthermore, the estimated mean annual ingestion effective dose (AIED) values in tuber crops ranged from 0.049 mSv to 0.062 mSv for cassava, 0.027 mSv to 0.064 mSv for sweet potatoes, and 0.057 mSv to 0.153 mSv for Irish potato. Similarly, the computed radiation hazard indices in soil samples and tuber crops yielded values below the acceptable limits, indicating not only insignificant cancer risk associated with the general public's exposure to ionizing radiation from the soil samples but also safety in consumption of these tuber crops
Genome Wide Association Studies Of Striga Hermonthica (Del.) Benth. Resistance in the Sorghum Diversity Panel
(kenyatta university, 2023) Kavuluko, Jacinta Muthini; Steven Runo; Mark Wamalwa; Titus Magomere
Enhancing parasite resistance in sorghum (Sorghum bicolor (L.) Moench.), a crucial cereal for the vast majority of inhabitants of sub-Saharan Africa (SSA), is vital to safeguarding food security. Striga hermonthica (Delile) Benth. being the most widespread and destructive hemiparasitic root weed of the family Orobanchaceae, is of economic concern. Developing crops that are resistant to Striga is one of the most efficient and economical methods to manage this parasite. Many researchers in the past have identified Striga resistant sorghum which have through the years been incorporated into existing breeding programs in SSA. However, the resistance has not been durable and disintegrates when new Striga ecotypes emerge. The current study sought to screen a diverse collection from the sorghum diversity panel to identify various sources of post- attachment resistance against S. hermonthica (Kibos ecotype). Subsequently, the laboratory resistance of twenty-one accessions was validated in Kibos (Western Kenya) for two rainy seasons in 2018-2019. The study further elucidated the associated genomic regions using Genome Wide Association Studies. To determine the resistance response of 151 sorghum accessions against S. hermonthica, root observation chambers (rhizotrons) were used. Further, the extent of parasitism was studied by dissecting small sections of parasite-host root attachments from sorghum roots nine days after infection. To determine the field response of 21 sorghum accessions with varying post-attachment resistance to S. hermonthica, the average maximum number of emerged Striga plants and the Area Under Striga Number Progress Curve were used as measures to indicate the level of Striga resistance. Relative grain yield loss due to Striga infestation was used as a measure of tolerance. Population stratification of the sorghum diversity panel was evaluated using the ADMIXTURE software, principal component analysis (PCA), and neighbour joining (NJ) phylogeny. Association scans were performed using Fixed and random model Circulating Probability Unification algorithm implemented in the Genomic Association and Prediction Integrated Tool. There were significant differences in resistance responses of sorghum accessions to Striga infection with regard to numbers, size and biomass of attached Striga. A total of ten accessions were resistant or highly resistant by the three metrics. Three diverse resistant mechanisms were observed ranging from hypersensitive reaction, resistance due to mechanical barriers and resistance attributed to the parasites inability to differentiate. The sorghum accessions displayed varied levels of Striga resistance in the field. The yields of the accessions differed significantly between the Striga-infested and non-Striga fields in both seasons. The varied Striga resistance responses exhibited in the laboratory was validated in the natural Striga environments in Kibos for most sorghum accessions. The NJ tree showed that the population structure of most accessions followed the pattern of morphological type within geographic origin. This was further supported by ADMIXTURE and PCA biplot analyses. Association Mapping identified several single nucleotide polymorphism (SNP) markers significantly associated with genes involved in cell wall modifications, innate immunity, systemic acquired immunity, and pathogenesis-related proteins. The newly identified high-yielding Striga-resistant sorghum landraces have great potential for immediate integration into breeding programs. Furthermore, in future breeding programs, the identified multiple genes can be combined in a single farmer-preferred sorghum cultivar to establish a more durable and broad-spectrum Striga resistance.
Reference Ranges for Triage Room, Biochemical, Hematological and Tumour Marker Parameters for Taita-Taveta County, Kenya
(Kenyatta University, 2023) Gitimu, Richard; Eliud NM Njagi; Joseph K Gikunju
Medical diagnosis is based on information from findings such as the medical history of the client, physical examination, self-report and interviews with the patient’s relative or both clinical reports from laboratory tests and radiologic studies. To assess one’s health status, different parameters are analysed progressively, and the generated reports are used to make a medical decision on the health status of the patients. Thus vital signs and laboratory report test results must be reported along with reference ranges. Little has been done to establish reference ranges for different populations in Kenya. Laboratory interpretation of test results with appropriate precision and accuracy for diagnostic purposes requires reference ranges or cutoff values. This study was a comprehensive determination of reference ranges for vital signs, hematology, clinical chemistry; cancer markers apparently from a healthy volunteer coastal population; vital parameters (for ages 4-93 years were 191 males and 317 females), body mass index (125 males and 127 females) pregnant women (296), infants, children, adolescents (males 129 and 132 females) and the geriatric (males 153 and 177 females), random blood sugar (131 males and 175 females), thyroid profile (124 males and 120 females) from which a total of 2390 subjects were recruited. Biochemistry analysis for liver, kidney function tests, lipid profile and electrolytes were analyzed using intergra 400 chemistry autoanalyzer. Tumour marker samples for the elderly population were analyzed using Chemwell auto analyzer (Biomerieux, Lyon, France), a closed system machine used to analyze tumor makers. The glucometer (On-call Plus, glucose meter from Roche Diagnostics GmbH, Mannheim, Germany) was used for random blood glucose analysis. All data were expressed as median and 2.5-97.5 %interval. Subsequently, independent-sample t-tests were performed to identify significant differences between the data of the males and the females. An analysis of variance (ANOVA) was performed for clinical chemistry, hematology and special chemistry parameters, with sex as a factor and age as covariate. Statistical significance was reached in the case of ρ < 0.05. The results were expressed as mean ± SD. Statistical Package for Social Science version 20 statistical software was used for data analysis. The study established combined male and female reference ranges for vital signs, biochemical, and hematological for children, adolescents, pregnant mothers and the geriatric population of the Taita Taveta community. The study reported statically differences in EOS (%), NEU (x109/L), BAS (x109/L), HB (g/dL), MCV (fL), PLT and CA for infants, children and adolescents by gender. ALP (U/L), D-BIL, CREAT, NA, UA and PVC for pregnant mothers per trimester. For geriatric population, the difference was recorded by gender in NEU (%), LMP (%), ALT (U/L), T-BIL, CREAT, CA19-9 and CEA. The study also compared established reference ranges by age and what is published in medical books and similar research works where differences were reported in ALP (U/L), AST (U/L), HB (g/dL), PVC (%) and MCV (fL) by age. At the same time, PSA, CA15-3 and CA 125 were recorded by gender. The present study was the first to report hematology analysis, serum biochemistry, tumor/ cancer markers and vital signs reference ranges for selected categories in Kenya and one of very few such studies in Africa different from those in use in the local health facility. Since there was a statistical difference in gender and age, both the sex and age of a patient must be considered when analyzing different patient samples. This will help improve on management and can be adapted for caregiving to the people of Taita-Taveta County in addition to setting a bench for future research works
Phytochemical Profiles and Bioefficacy of Methanol Extracts of Cissampelos Pareira L., Lantana Camara L., and Ocimum Gratissimum L. Against Inflammation, Pain And Fever
(Kenyatta University, 2023) Nyawira, Kimuni Susan; Mathew Piero Ngugi; Eliud Mwaniki Njagi
Herbal medicine is an effective therapy and safe for treating and managing various illnesses. In Kenya, the Embu and Mbeere communities that reside in Embu County use, Cissampelos pareira (Menispermaceae), Lantana camara (Santalaceae) and Ocimum gratissimum (Asteraceae) in managing inflammation, pyrexia, and other diseases. Nevertheless, their scientific data is yet to be validated. The current study aspired to investigate the quantitative phytochemical profiles, in vivo antinociceptive, anti-inflammatory and antipyretic characteristics of C. pareira, L. camara and O. gratissimum methanol extracts. The local herbalist assisted in identifying and collecting plant samples from Mbeere North Sub-County, Embu County. The collected specimens were packed in khaki bags then taken to Kenyatta University, where extraction was carried out using methanol. Samples of four grams each of the three studied plants were used to evaluate quantitative phytochemical composition using LC-MS and GC-MS. Antipyretic activity of the extracts was bioscreening in Wistar rats while antinociceptive and anti-inflammatory activities were screened in Swiss albino mice. Eight groups constituting five test animals for each experimental group were used: Normal, positive, negative control, and five extract groups. Carrageenan, Formalin and Turpentine were used to induce inflammation, pain and fever, respectively. Diclofenac, Paracetamol, and Aspirin were used as reference drugs for inflammation, pain, and pyrexia. The plants were tested for anti-inflammatory, antipyretic, and analgesic properties at extract concentrations of 50, 100, 150, 200 and 250mg/kg body weight. Phytochemical analysis revealed 25, 30 and 38 phytocompounds for C. pareira, L. camara and O. gratissimum, respectively. The antipyretic, antinociceptive, and anti-inflammatory assays in animal models revealed that the three-leaf extracts have potential anti-inflammatory, antipyretic, and analgesic activities attributed to individual phytochemicals that included limonene, quercetin, apigenin, alpha and beta-pinene, among others. Additionally, the potent antipyretic, antinociceptive and anti-inflammatory effects showed by the three studied plant extracts were comparable to the standard drugs (Aspirin, Diclofenac and Paracetamol, respectively). Compared to the orally administered extracts, the intraperitoneally administered plant extracts demonstrated the greater antipyretic, anti-inflammatory and antinociceptive activities. The extract concentrations (200 and 250 mg/kg bw) administered both orally and intraperitoneally were appropriate in animal models' bioassay of the three effects (anti-inflammatory, antipyretic and analgesic). This study further, demonstrated that C. pareira, L. camara and O. gratissimum at the tested doses of 250 and 500 mg/kg body weights caused no alterations to the vital organs of mice. However, C. pareira and L. camara extracts at the dose of 1000 mg/kg bw, recorded significant changes in mice's organ weights and biochemical parameters. The research rooted the forkloric use of the three plants in curing various diseases, including inflammation, pain, and fever. Therefore, this study supports and confirms studied plant extracts as alternative and complementary remedies against fever, inflammation and pain.
Phytochemical Composition, Anti-Rheumatoid Arthritis Potential and Toxicity Profiles of Methanol Extracts of Zanthoxylum Usambarense Engl. And Rhamnus Prinoides L’hér
(Kenyatta University, 2023) James, Kamau Kimani; Mathew Piero Ngugi; Joseph J.N. Ngeranwa
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune ailment marked by synovial joint edema, stiffness and pain resulting in severe disability and sometimes premature fatalities. A combination of glucocorticoids, non-steroidal anti-inflammatory drugs and disease-modifying anti-rheumatic drugs are often prescribed to treat RA. However, these medications are linked to severe effects, necessitating alternative medical interventions. Herbal medicines are regarded as alternative therapeutics as they are considered efficacious, easily biodegradable and have arguably minimal severe effects. In Kenya, the Maasai Community uses the root bark of Zanthoxylum usamberense and the stem bark of Rhamnus prinoides to treat RA. However, there is a paucity of scientific evidence on quantitative phytochemical screening, anti-RA potential and mechanistic effects, as well as toxicity profiles of the two acclaimed medicinal plants. The current study objectives were to determine the qualitative and quantitative phytochemical composition, anti-RA potential, expressions of selected RA biomarker genes as well as acute and subacute toxicities profiles of MeOH extracts of the two medicinal plants. Plant samples were collected in Narok County, Kenya. The two medicinal plant samples were identified by a taxonomist, prepared, and extracted using MeOH. Liquid chromatography-mass spectrometry (LC-MS) and GC-MS (Gas chromatography-mass spectrometry) were used in qualitative and quantitative phytochemical analysis. In the anti-RA assay, rats were assigned 6 groups of 5 rats each: normal control, disease control, positive control (methotrexate), and 3 extract-treated groups (100, 200 and 300mg/kg body weight (bw)). RA was induced using complete Freud’s adjuvant (CFA). Methotrexate (weekly) and extracts (daily) at the three doses were orally administered from day 8 post-induction of RA. The experimental rats were euthanized, and blood was drawn for hematological and biochemical indices on the 29th day. The liver and tissue around the ankle joint were also detached and used for antioxidant and gene expression assays, respectively. Selected RA biomarker genes were assayed using quantitative Real-Time Polymerase Chain Reaction assay. In vivo acute and sub-acute toxicological studies were performed according to the OECD guidelines 425 and 407, respectively. The LC-MS analysis identified phytochemicals of flavonoids, phenolic acids, tannins, stilbenes, phytosterols, anthraquinones, and coumarins classes. Fatty acid methyl ester, fatty acid, diterpenes, steroids, lignans, benzofuran, pyranone and triterpene were also detected using GC-MS. The two extracts significantly attenuated body weight loss, alleviated ankle joint edema, reduced arthritis scores, ameliorated increased thymus and spleen indices, attenuated aberrant hematological and biochemical parameters, reduced levels of malonaldehyde and enhanced enzymatic antioxidant activities following CFA-induced RA in rats. The two extracts also significantly downregulated expression of STAT-3, MMP-3, NF-κB, RANKL, COX-2, TNF-α, IL-1β and IL-6 and genes, as well as upregulated expression of IL-4, IL-10 and I-κBα genes following CFA-induced RA in rats. Zanthoxylum usambarense had better anti-RA effects than R. prinoides. At the highest dosage of 2000mg/kg bw, the acute toxicity tests of the two extracts neither caused toxicity signs nor fatalities in rats. The subacute toxicity study of the two extracts did not adversely affect body weights, relative organ weights, hematological parameters, liver function and renal function biomarkers, lipid profiles and blood glucose levels, except for AST levels in rats administered with R. prinoides extract dose of 900mg/kg bw. In conclusion, the two extracts possess phytochemicals with anti-RA potential and can be used as potential leads in developing anti-RA agents with minimal side effects. The selected RA biomarker genes can also be used as targets in developing anti-RA agents. Therefore, the current study scientifically validates the folkloric use of Z. usambarense and R. prinoides in managing RA. However, the classes of phytocompounds with anti-RA activity should be isolated and their effects determined.
Nutritional Analysis, In Vitro Antioxidant Activity and Hepatoprotective Potential of Solanum Villosum Linn. and Solanum Scabrum Mill. Methanol Leaves Extracts
(Kenyatta University, 2023) Onyango, Osano Kenneth; Mathew Piero Ngugi; Alex King’ori Machocho
Drug induced liver injury (DILI) is caused by prescribed drugs, herbs and various xenobiotics resulting in liver malfunctions. It remains a principal challenge associated with prescribed drugs and a leading basis for withdrawal of approved medicinal products from usage. Presently, a narrow range of drugs are available for treatment of DILI, hence continued search for safe, effective and affordable treatment alternatives such as from plants. The current study evaluated quantitative nutritional profiles, in vitro antioxidant activities and hepatoprotective activities of methanol leaves extracts of Solanum villosum and Solanum scabrum in acetaminophen induced mice models. Vegetables samples were randomly collected from farmers in Uasin Gishu County. Quantitative nutritional analysis was performed based on Association of Official Agricultural Chemist (AOAC) protocols; while phytochemicals analysis was done by LC-MS. Animals were given extract at 30, 100, 300 and 500 mg/Kgbw orally for 21 days. Hepatotoxicity was induced by acetaminophen 300 mg/Kgbw, intraperitoneally on 18th day. Silymarin 25 mg/Kgbw was the positive control. Full hemogram and liver function tests were performed by automated chemical analyzers using blood samples collected from the mice on 22nd day. Superoxide dismutase, catalase and glutathione peroxidase activities were assessed using liver homogenates of euthanized mice. We found out that S. villosum and S. scabrum are endowed with substantial quantities of vitamins A, B1, B2, B3, B6, C and E, in addition to sufficient quantities of the elements Na, K, Mg, Ca, Zn, Fe, Cu, Mn and P. Macromolecules including simple sugars, polysaccharides, crude lipids and crude proteins were also established in the powdered leaves. Similarly, phytochemicals analysis of the methanol extracts revealed the presence of phenolic acids and flavonoids, which demonstrated in vitro antioxidant activities, linked to the reported medicinal potential. Both extracts enhanced lymphocytes synthesis in dose dependent manner, but inhibited proliferation of granulocytes in mice induced with APAP hepatotoxicity. Further, both extracts demonstrated hepatoprotective activities by marked reduction in plasma ALT, AST, ALP and γ-GGT concentrations in dose dependent manner, following elevations in mice induced with APAP hepatotoxicity. Overall, S. villosum extracts showed higher hepatoprotective activities than S. scabrum extracts at the same dosages. The extracts induced glutathione peroxidase, superoxide dismutase synthesis, and anti-inflammatory activities associated with hepatoprotection. Nutritionally, the antioxidant vitamins C and E, microelements Mn, Zn and Cu that are cofactors to various antioxidant enzymes, and vitamin B6, a coenzyme for transaminases present in the vegetable‘s leaves could further enhance hepatoprotection and promote good health. The present study thus, demonstrated scientifically that S. villosum and S. scabrum are highly nutritious and have hepatoprotective activity against drug induced liver injury therefore, may be classified as functional foods. Their production, consumption or commercialization could be encouraged to deal with the challenge of undernutrition, diet diversification and possible processing as hepatoprotective agents, thus promoting universal healthcare.
Cognitive Enhancing Effects and Antioxidant Activities of Crude Methanol Extracts of Prunus Africana in Scopolamine-Induced Alzheimer’s Disease Mice
(Kenyatta University, 2023) Ngai, David Nyaga; Mathew Piero Ngugi; Cromwell Mwiti Kibiti
Alzheimer’s disease (AD) is a type of dementia marked by gradual progressive neurodegeneration caused by excessive neurofibrillary tangles and amyloid beta (Aβ) build-up in hippocampus and other parts of the brain. Ultimately, this reduces the number of neurons, synapses and pyramidal nerve connections resulting in compromised cognitive functions and memory loss. The immune system clears senile plaques and neurofibrilary tangles oxidatively and in the process, there is excessive generation of reactive nitrogen and oxygen species that cause the chronic oxidative stress observed in AD. Conventionally, cholinergic agonists, particularly inhibitors of acetylcholinesterase including rivastigmine, donepezil and galantamine are the most commonly used drugs in AD therapy. Unfortunately, their efficacy is low and they have short half lives hence higher and more frequent dose requirement which is limited by their side effects that are sometimes unbearable. Fortunately, there are phytochemicals with AD ameliorating effects. These include phenolic compounds that clear aggregated amyloid proteins and have antioxidant and anti-inflammatory properties, steroidal alkaloids that have anti-cholinesterase activity while ascorbic acid, terpenoids and rivastigmine are not only free radical scavengers but also stabilizers of muscarinic receptors. Antioxidant activity is a vital requirement for plant extracts that are used with great success to manage degenerative diseases like AD since such diseases are usually accompanied by oxidative stress. In the current study, in vivo cognitive advancing and antioxidant effects of crude methanol extract of stem bark and leaf of Prunus africana were investigated in scopolamine-induced AD mice. Morris water maze and Passive avoidance task were used to measure spatial memory and cognitive enhancing effects of the two plant extracts, respectively. Scopolamine butylbromide (5mg/kg bw) was administered intraperitoneally to induce AD during the study. Donepezil (DNP) was used as the standard drug in the current study because of its ability to improve and maintain high global cognitive potentials and high torelability. The drug substance, scopolamine, is the most commonly used substance in biosreening for AD drugs. It is known to lower transcription of brain derived neurotrophic factor responsible for memory elasticity and development and also interferes with functioning of muscarinic receptors. The study revealed phytocompounds with anticholinesterase activity including chlorogenic acid, pCoumaric acid, vitexin, isovitexin, sitostenone and campesterol. Phytocompounds with enzymatic antioxidant activity including ursolic acid, campesterol and sitostenone were also revealed. The two extracts also proved to be rich in phytochemicals with antioxidant and anti-inflammatory activities which are important neuroprotective properties. The two extracts significantly improved cognition and spatial memory in the two behavioural experiments. This was indicated by significant increase in escape latencies in passive avoidance task and decrease in escape latencies in Morris water maze tasks in a dose related pattern. These behavioural results are in collaboration with the anticholisterase, antioxidant and anti-inflammatory activities of compounds revealed in the two extracts. Generally, the stem bark extract had significantly higher efficacy than leaf extract in most of the tests carried out. The disparity in efficacies of the two extracts can be explained in terms of differences in diversity and proportions of phytochemicals present. From the current study, it is concluded that crude methanol extracts of stem bark and leaf of P. africana have several phytocompounds with cognitive and spatial memory enhancing effects, anticholinesterase and antioxidant activities in scopolamine induced-Alzheimer’s disease mice.
Taxonomy, Distribution and Host Range of Cuscuta Species Across Kenyan Ecosystems
(Kenyatta University, 2022) Masanga, Joel Okoyo; Richard Oduor; Steven Runo; Mathew Ngugi; Amos Alakonya
A largely unreported and ever-expanding infestation of African ecosystems by dodders (Cuscuta spp.) portends for severe threats to food security and economic livelihoods on the African continent. Dodders are noxious parasitic weeds that colonize numerous host plants, and entirely depend on them for support and nourishment. This parasitism is detrimental to host plants, since they are deprived of water, nutrients, and photosynthates. Over 200 dodder species have been identified worldwide, some natively endemic, while others are invasive in new localities. The successful spread of dodder is attributed to its ability to parasitize an array of angiosperms under different environmental conditions. In Africa, little is known or reported regarding the diversity of dodder species, their host range and whether these parasites can affect essential crops. Moreover, distribution patterns of these weeds under current and future environmental conditions are unknown. Therefore, this study hypothesized that different dodder species have invaded Kenyan ecosystems and that their geographical distribution and success in colonizing plants is influenced by a combination of climatic and environmental factors, coupled with high genetic variability and distinct population structures. Using a combination of taxonomic and genomics tools, dodder accessions collected across Kenya were characterized. Further, integrated phylogeographic and population genetics analyses, targeting genes from the chloroplast and nuclear genomes, were used to determine genetic variability and population structure of the dodder accessions to reveal factors that have shaped their successful colonization of current localities. These analyses revealed presence of 3 dodder species, namely Cuscuta kilimanjari, Cuscuta campestris, and Cuscuta reflexa, in Kenya. In addition, population genetics profiling for C. reflexa and C. campestris revealed that these species had high genetic variation, evidenced by high numbers of haplotypes, gene diversities and heterozygosity (0.2-0.66 and 0-0.6 in C. campestris and C. reflexa, respectively). Moreover, these species had distinct lineages, corresponding to Kenya's eastern and western regions, with evidence of isolation by distance. A comprehensive list of host plants, from sampling efforts and literature review, was compiled to reveal dodder's host range. Additionally, species distribution modelling (SDM), based on the Maximum Entropy (MaxEnt) algorithm, was used to determine dodder's distribution patterns targeting climatic and vegetation variables. Results revealed highly suitable dodder habitats across Eastern Africa. Crucially, some of the localities identified were important locations for high-value commercial agriculture. Histological analysis of the host-parasite interface was performed to determine the ability of C. reflexa to parasitize three economically-important crops, namely mango (Mangifera indica) coffee (Coffea arabica), and tea (Camellia sinensis). Experimental results proved that C. reflexa could parasitize all three crops. Interestingly, mango exhibited post-attachment resistance response to dodder, which occurred through secretion of a sap-like substance that arrested parasitism. These results are applicable in policy development among governments and related agencies and will guide targeted approaches aimed at eradicating current dodder populations and prevent new introductions.
Phytochemical Quantification, Anthelmintic, Antioxidant and Acute Toxicity Properties of Dichloromethane Extracts of Maytenus Senegalensis and Dalbergia Melanoxylon
(2022) Mwangi, Boniface Maina; Mathew Piero Ngugi; John Mwonjoria
Maytenus senegalensis was traditionally used as a therapeutic option against chest pains and anthelmintic while Dalbelgia melanoxylon was used to treat anthelmintic. However, their anthelmintic potentials, antioxidant potentials and safety have not been scientifically validated. The aim of this study therefore, was to determine in vitro anthelminthic properties, quantitative phytochemical properties and in vivo sub-acute toxicity of Dicholoromethane plants extracts in mice. Seventy five worms were grouped into 5 groups in each of 3 petri plates. Group I was treated with distilled water (50ml). Group II were treated with albendazole at dose of 25mg/ml. Groups III, IV, V were treated with plants extracts of 12.5, 25 and 50mg/ml, respectively. Results were interpreted as time taken for paralysis and death of earthworms. The extracts manifested paralytic effects on worms after exposure periods ranging between 03.07min and 11.25min for M. senegalensis and 02.13min and 07.24min for D. melanoxylon. The extracts showed mortality effects on worms after periods ranging between 04.45min and 13.29min for M. senegalensis and 03.36min and 08.76min for D. melanoxylon. The antioxidant potential of the extracts were assessed against I,I-diphenyl-2-picrylhydrazyl, hydroxyl, hydrogen peroxide, iron chelating and ferric-reducing power. Phytochemical analysis was conducted using gas chromatography linked to mass spectrophotometry. Results revealed the extracts exhibited scavenging activities against all radicals. The extracts exhibited iron chelating and ferric reducing abilities. The extracts indicated a higher half inhibitory concentration value than the standard used. For instance, the extracts of M. senegalensis and D. melanoxylon exhibited 50% of the formation of 2, 2-diphenyl-i- picrylhydrazine of at concentration of 1.31±0.40 and1.31±0.04μg/ml and standard was 0.50±0.04μg/ml. Similarly, the extract scavenged 50% of hydroxyl radical of M. senegalensis at 1.24±0.02 and D. melanoxylon at 1.24±0.03 and the standard (citric acid) at 0.04±0.05μg/ml. Further, stem barks of D. melanoxylon exhibited significantly higher phenolic content than the extract of M. senegalensis at all the tested concentrations. For sub-acute toxicity test, the mice were orally administered with different doses of plants extracts. They were weighed on the first day and after 7 days during treatment with the extracts. After 28 days, the mice were sacrificed and blood samples taken for full hemogram, renal and liver function tests. The extracts had no lethal effects on body organ weights as well as on the haematological and biochemical parameters in normal mice. The study concluded that the plant extracts have phytochemicals safely associated with anthelmintic and antioxidant activities. Further, the plants extracts have in vitro anthelmintic activities in earthworms at dose level of 50 mg/ml. The plants extracts studied have no in vivo toxicity in normal mice at dose level of 100 and 300 mg/kg body weight. The study recommends the undertaking of in vivo antioxidant studies on plants extracts. Also there is need to carry out bioassay guided fractionation and structure elucidation of phytochemicals in the plants extracts. Further there is need for efficient conservation strategies for these medicinal plants in Kenya.
Phytochemical Profiles, Antiproliferative and Antioxidant Activities of Carissa Edulis (Forssk.) Vahl) and Pappea Capensis (L.) Extracts
(2022) Muruthi, W. Carolyn; Mathew Piero Ngugi; Steven Maina Runo
Cancer mortality is a global concern. In Kenya, cancer is ranked third cause of death. Incidence rates for breast and prostate cancers exceed those of other forms of cancer. Reactive species are implicated in all aspect of cancer, that is, initiation, promotion progreesion. With the increased changes in lifestyle and environmental pollutants; the burden caused by cancer will continue to have an upward trajectory. The current therapeutic approaches despite showing efficacy are characterized by several limitations such as adverse effects, prohibitive costs and sturdy resistance. This drives the need to explore alternative anticancer drugs. Several studies have evaluated anticancer and antioxidant effects of diverse plant extracts. In addition, toxicity effect of herbal plants has been evaluated using various assays including brine shrimp (Artemia salina) lethality assay. C. edulis and P. capensis are used as traditional therapy among the African communities to manage cancer and oxidative stress-related ailments. However, there is limited research on the antiproliferative and antioxidant effects of these medicinal plants. The current study, therefore, sought to evaluate the phytochemical profile, antiproliferative, antioxidant and in vivo cytotoxicity activities of C. edulis and P. capensis extracts. Samples were collected from Embu County, Kenya. Phytochemical profiles were determined using Liquid Chromatography Mass Spectrophotometer (LCMS) and Gas Chromatography Mass Spectrophotometer (GC-MS) analyses. Antiproliferative activity of the plant extracts against breast cancer (HCC 1395), prostate cancer (DU145) and Vero (normal) cell lines was determined using MTT assay. Effect of the extracts on expression of apoptotic genes (p53, bax and HOXB13) was evaluated using quantitative Real Time Polymerase Chain Reaction (qRTPCR). In vitro antioxidant properties of the extracts were evaluated through ferric reduction, Iron chelating, hydroxyl radical, and I, I-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. Activities of catalase, superoxide dismutase and glutathione reductases of the extracts were further determined. Toxicity of the extracts against was determined using the brine shrimp lethality test (BSLA). The results indicated that ethyl acetate extracts of C. edulis and P. capensis had the highest activity against cancer cells with IC50 values of 2.12±0.02, and 6.57±0.03μg/ml on HCC 1395 and 2.92±0.17 and 5.00±0.17μg/ml on DU145, respectively. Moreover, all the extracts exhibited relatively less cytotoxic activities against Vero cell lines (IC50>20μg/ml). The extracts also exhibited selectivity againt the cancer cells (SI>3). Further, mRNA expression of p53 in the treated HCC 1395 was increased by 7 and 3-fold, whereas by 3 and 2-fold in DU145 cells, upon treatment with ethyl acetate extracts of C. edulis and P. capensis, respectively. Similarly, severalfold increases were observed in the number of transcripts of Bax in HCC 1395 and HOXB13 in DU145 cells. The extracts had antioxidant activities whereby IC50 values were less than 50mg/ml, indicating strong scavenging activities. BSLA assay indicated that the extracts were non-toxic (LC50≥1000μg/ml) and had low toxicity (LC50≥500- 1000μg/ml) effects against A. salina. Phytochemical analyses revealed presence of compounds which are associated with antiproliferative and antioxidant activities including flavonoids, phenolics, tocopherols and terpenoids. The findings provide a scientific validation for the folklore use of C. edulis and P. capensis in management of cancer and oxidative stress. Nevertheless, there is a need for further purification and characterization of phytochemicals associated with anticancer and antioxidant activities. In addition, further toxicity assays should be conducted to explore effects of extracts on organs weight, body weight, haematological and biochemical parameters.
Somatic Embryogenesis and Genetic Engineering of Banana with Elongation Factor Receptor Gene for Resistance to Banana Xanthomonas Wilt
(2022) Ochieng, Mark Adero; Richard Oduor,; Leena Tripathi
Banana is among the top ten most important food crops worldwide in production and consumption. However, its production is threatened by several phytopathogens. Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pathovar (pv.) musacearum (Xcm) is one of the biggest threats to banana production in the African Great Lakes region. Crop breeding via conventional means is key to overcoming yield losses to phytopathogens. However, conventional breeding of clonally propagated crops like banana is limited by the lack of diversity and important traits in the gene pool. At present, only Musa balbisiana (banana progenitor species) is resistant to BXW, but breeders do not prefer it because it harbors banana streak virus (BSV) sequences in its genome, which get activated during abiotic stress, leading banana streak disease (BSD). Thus, genetic engineering serves as a viable alternative and complement to conventional breeding for banana improvement. Interfamily transfer of pattern recognition receptor genes, such as elongation factor receptor (EFR), through transgenic approach can enhance plant resistance to bacterial diseases of diverse genera. An efficient regeneration system based on embryogenic cell suspensions (ECS) is a prerequisite for efficient genetic transformation of bananas. Till now, limited progress has been made on establishing protocols for somatic embryogenesis and transformation of most East African highland banana (EAHB) cultivars. This study aimed to establish somatic embryogenesis, cell suspension cultures, and transformation protocol for EAHB cultivars ‘Ngombe’ and ‘Kisansa’ and transform banana with elongation factor receptor (AtEFR) gene from Arabidopsis thaliana for resistance to BXW. Various media and plant growth regulators (PGRs) were tested for their ability to induce somatic embryogenesis and plant regeneration or generation of cell suspension cultures. Embryogenic calli were transformed with Agrobacterium strain EHA 105 containing a binary vector pCAMBIA2301 harboring a reporter gene (β-glucuronidase, gusA), followed by selection and plant regeneration. Putative transformants were subjected to GUS assay and molecular characterization. For the development of transgenic bananas expressing AtEFR, ECS of two banana cultivars, ‘Sukari Ndizi’ and ‘Dwarf Cavendish’ were transformed with Agrobacterium strain EHA 105 containing a binary vector pBIN19g-35S:: EFR and the generated transgenic events were evaluated for BXW resistance in the greenhouse. Among the medium and PGRs tested for callus induction and somatic embryogenesis, half-strength Murashige and Skoog (MS) medium containing 2 mg/l picloram or dicamba was optimum for both varieties. The highest frequency of plant regeneration from embryogenic calli (35%) was achieved in MS medium containing 1 mg/l 6-benzylaminopurine (BAP). In total, 257 putatively transformed events expressing AtEFR were regenerated and characterized using polymerase chain reaction (PCR), Southern blot, reverse transcription (RT)-PCR, and quantitative RT-PCR analyses. Among the 48 transgenic events evaluated for BXW resistance in the greenhouse, three showed complete resistance, 23 partial resistance, and 22 were susceptible. The transgenic events also showed responsiveness to the N-terminal acetylated peptide elf18 of bacterial elongation factor thermal unstable (EFTu), suggesting that the function of the AtEFR gene was retained in banana. Overall, this study indicates that EAHBs are amenable to somatic embryogenesis and genetic transformation and further confirms that adaptors of EFR are conserved across a diverse range of plant families and can potentially enhance the resistance of bananas to BXW under field conditions.
Taxonomy, Distribution and Host Range of Cuscuta Species Across Kenyan Ecosystems
(2022) Masanga, Joel Okoyo; Steven Runo; Mathew Ngugi
A largely unreported and ever-expanding infestation of African ecosystems by dodders (Cuscuta spp.) portends for severe threats to food security and economic livelihoods on the African continent. Dodders are noxious parasitic weeds that colonize numerous host plants, and entirely depend on them for support and nourishment. This parasitism is detrimental to host plants, since they are deprived of water, nutrients, and photosynthates. Over 200 dodder species have been identified worldwide, some natively endemic, while others are invasive in new localities. The successful spread of dodder is attributed to its ability to parasitize an array of angiosperms under different environmental conditions. In Africa, little is known or reported regarding the diversity of dodder species, their host range and whether these parasites can affect essential crops. Moreover, distribution patterns of these weeds under current and future environmental conditions are unknown. Therefore, this study hypothesized that different dodder species have invaded Kenyan ecosystems and that their geographical distribution and success in colonizing plants is influenced by a combination of climatic and environmental factors, coupled with high genetic variability and distinct population structures. Using a combination of taxonomic and genomics tools, dodder accessions collected across Kenya were characterized. Further, integrated phylogeographic and population genetics analyses, targeting genes from the chloroplast and nuclear genomes, were used to determine genetic variability and population structure of the dodder accessions to reveal factors that have shaped their successful colonization of current localities. These analyses revealed presence of 3 dodder species, namely Cuscuta kilimanjari, Cuscuta campestris, and Cuscuta reflexa, in Kenya. In addition, population genetics profiling for C. reflexa and C. campestris revealed that these species had high genetic variation, evidenced by high numbers of haplotypes, gene diversities and heterozygosity (0.2-0.66 and 0-0.6 in C. campestris and C. reflexa, respectively). Moreover, these species had distinct lineages, corresponding to Kenya's eastern and western regions, with evidence of isolation by distance. A comprehensive list of host plants, from sampling efforts and literature review, was compiled to reveal dodder's host range. Additionally, species distribution modelling (SDM), based on the Maximum Entropy (MaxEnt) algorithm, was used to determine dodder's distribution patterns targeting climatic and vegetation variables. Results revealed highly suitable dodder habitats across Eastern Africa. Crucially, some of the localities identified were important locations for high-value commercial agriculture. Histological analysis of the host-parasite interface was performed to determine the ability of C. reflexa to parasitize three economically-important crops, namely mango (Mangifera indica) coffee (Coffea arabica), and tea (Camellia sinensis). Experimental results proved that C. reflexa could parasitize all three crops. Interestingly, mango exhibited post-attachment resistance response to dodder, which occurred through secretion of a sap-like substance that arrested parasitism. These results are applicable in policy development among governments and related agencies and will guide targeted approaches aimed at eradicating current dodder populations and prevent new introductions.
Phytochemical, Anti-Inflammatory, Antinociceptive, Antipyretic and Toxicity Analysis of Dichloromethane and Methanol Extracts of Five Selected Plants Using Animal Model
(Kenyatta University, 2022) Mutuma, Gitonga Godfrey; Joseph J. N. Ngeranwa; Alex Machocho; Silas Kiruki
Inflammation results from irritants causing body injury. Pain is discomfort associated with illness or injury while pyrexia can be defined as elevated body temperature. Common conventional anti-inflammatory, analgesic and antipyretic agents are expensive and have severe adverse effects. Traditional medicines are regarded by various communities as safe, efficacious with little or no adverse side effects. The study aim was to evaluate phytochemical profile, in vivo anti-inflammatory, antinociceptive, antipyretic activities as well as safety associated with Senna didymobotyra, Eucalyptus saligna, Bidens pilosa, Mangifera indica and Prunus africana. Plant samples were collected from Kanjagi sub - location, Kirima – Itune location, Giaki division, Meru County in Kenya. Extractions of phytochemicals were carried out using dichloromethane and methanol. Edema, pain and fever were induced in test animals using 1% carrageenan (0.5 ml), 2.5% v/v formalin (0.05 ml) and 20% v/v turpentine respectively. Wistar rats and albino mice were used in this study. The animal models were grouped into normal, negative, positive and experimental test groups I - III. Experimental test groups I, II and III were treated with 50, 100 and 150 mg/kg of the plant extract respectively. Anti-inflammatory and antinociceptive activities associated with extracts were evaluated against the standard anti-inflammatory and antinociceptive drug (Diclofenac), while antipyretic activities were established against aspirin. The herbal extract of Senna didymobotyra, Eucalyptus saligna, Bidens pilosa, Mangifera indica and Prunus africana and the reference drugs indicated some in vivo anti- inflammatory, antinociceptive and antipyretic effects. For anti-inflammatory activity, the extracts were associated with reduced hind paw diameter in relation to what was observed in control groups. The inhibitory rates observed in paw edema ranged from 1.59 - 11.05%, Diclofenac edema inhibition was ranging between 0.1 and 8.78%. For the analgesic study, the Senna didymobotyra, Eucalyptus saligna, Bidens pilosa, Mangifera indica and Prunus africana extract reduced paw licking time by 1.38-48.26% during early phase and between 28.45-83.90% during late phase while diclofenac pain inhibitory rates ranged between 12.20-80.20% in both phases. The herbal extract of Senna didymobotyra, Eucalyptus saligna, Bidens pilosa, Mangifera indica and Prunus africana were associated with 0.16 and 3.95% antipyretic activity while aspirin was associated with 1.52 and 3.60% antipyretic activities. The qualitative phytochemical evaluation indicated positive results for alkaloids, cardiac glycosides, flavonoid, saponins, steroids, tannins terpenoids and phenolics compound which are associated with anti-inflammatory, antinociceptive and antipyretic activities. The herbal extract of Senna didymobotyra, Eucalyptus saligna, Bidens pilosa, Mangifera indica and Prunus africana were not associated with any significant repeated dose toxicity at p<0.05. The study, confirms the role of Senna didymobotyra, Eucalyptus saligna, Bidens pilosa, Mangifera indica and Prunus africana extract by Meru community in disease management associated with inflammation, pain and fever. The results support the need to preserve or protect the biodiversity of the Senna didymobotyra, Eucalyptus saligna, Bidens pilosa, Mangifera indica and Prunus africana

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