In Vitro Antiproliferative Effects and Phytochemical Characterization of Carissa edulis ((Forssk) Vahl) and Pappea capensis (Eckyl and Zeyh) Extracts.

Cancer mortality is a global concern. The current therapeutic approaches despite showing efficacy are characterized by several limitations. Search for alternatives has led to the use of herbal plants including C. edulis and P. capensis. However, there is limited research on antiproliferative effects of these medicinal plants. The study sought to evaluate antiproliferative effects of the plants against human breast and prostate cancers using cell viability, and gene expression assays to determine modulation of apoptotic genes. Further, Liquid Chromatography Mass Spectrophotometer (LC-MS) and Gas Chromatography Mass Spectrophotometer (GC-MS) analyses were performed to confirm phytocompounds in the extracts. The results indicated that ethylacetate 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 ± 0.03 µg/ml on HCC 1395 and 2.92 ± 0.17 and 5.00 ± 0.17 µg/ml on DU145, respectively. Moreover, the plants extracts exhibited relatively less cytotoxic activities against Vero cell lines (IC50 > 20 µg/ml). The extracts also exhibit selectivity against 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 ethylacetate extracts of C. edulis and P. capensis, respectively. Similarly, several-fold increases were observed in the number of transcripts of Bax in HCC 1395 and HOXB13 in DU145 cells. Phytochemical analyses detected presence of phytocompounds including flavonoids, phenolics, tocopherols and terpenoids which are associated with anticancer activity. Findings from this study provide a scientific validation for the folklore use of these plants in management of cancer.

Prevalence of mutations in the cysteine desulfurase IscS (Pfnfs1) gene in recurrent Plasmodium falciparum infections following artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) treatment in Matayos, Western Kenya.

BACKGROUND: Malaria remains a public health concern globally. Resistance to anti-malarial drugs has consistently threatened the gains in controlling the malaria parasites. Currently, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) are the treatment regimens against Plasmodium falciparum infections in many African countries, including Kenya. Recurrent infections have been reported in patients treated with AL or DP, suggesting the possibility of reinfection or parasite recrudescence associated with the development of resistance against the two therapies. The Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) K65 selection marker has previously been associated with decreased lumefantrine susceptibility. This study evaluated the frequency of the Pfnfs1 K65 resistance marker and associated K65Q resistant allele in recurrent infections collected from P. falciparum-infected individuals living in Matayos, Busia County, in western Kenya. METHODS: Archived dried blood spots (DBS) of patients with recurrent malaria infection on clinical follow-up days after treatment with either AL or DP were used in the study. After extraction of genomic DNA, PCR amplification and sequencing analysis were employed to determine the frequencies of the Pfnfs1 K65 resistance marker and K65Q mutant allele in the recurrent infections. Plasmodium falciparum msp1 and P. falciparum msp2 genetic markers were used to distinguish recrudescent infections from new infections. RESULTS: The K65 wild-type allele was detected at a frequency of 41% while the K65Q mutant allele was detected at a frequency of 22% in the recurrent samples. 58% of the samples containing the K65 wild-type allele were AL treated samples and while 42% were DP treated samples. 79% of the samples with the K65Q mutation were AL treated samples and 21% were DP treated samples. The K65 wild-type allele was detected in three recrudescent infections (100%) identified from the AL treated samples. The K65 wild-type allele was detected in two recrudescent DP treated samples (67%) while the K65Q mutant allele was identified in one DP treated (33%) recrudescent sample. CONCLUSIONS: The data demonstrate a higher frequency of the K65 resistance marker in patients with recurrent infection during the study period. The study underscores the need for consistent monitoring of molecular markers of resistance in regions of high malaria transmission.

In vitro antioxidant activities of Carissa edulis ((Forssk) Vahl) and Pappea capensis (Eckyl. & Zeyh) extracts.

Herbal medications are gaining popularity due to their long history of use in traditional medicine. They serve as a reservoir for a diverse array of phytocompounds linked to amelioration of oxidative stress. Oxidative stress is a disturbance in the balance between generation and elimination of reactive species in human body. Moreover, reactive species are implicated in the onset and progression of chronic disorders. The current therapeutic approaches despite showing efficacy are characterized by several limitations such as adverse effects and prohibitive costs. This drives the need to explore alternatives that can inhibit, ameliorate or reverse conditions caused by oxidative stress. Several studies have evaluated antioxidant effects of diverse plant extracts. C. edulis and P. capensis are used as traditional therapy among the African communities to manage oxidative stress-related ailments. However, there is limited research on the antioxidant effects of these medicinal plants. The current study, therefore, sought to evaluate the antioxidant and phytochemical profile, of C. edulis and P. capensis extracts. Samples were collected from Embu County, Kenya. In vitro antioxidant properties of the extracts were evaluated through ferric reduction, Iron chelating, hydroxyl radical, and DPPH radical scavenging activities. Activities of catalase, superoxide dismutase and glutathione reductases of the extracts were further determined. Phytochemical profiles were determined using Liquid Chromatography Mass Spectrophotometer (LC-MS) and Gas Chromatography Mass Spectrophotometer (GC-MS) analyses. The extracts displayed concentration dependent antioxidant activities. Phytochemical analyses revealed presence compounds which are associated with 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 oxidative stress. Nevertheless, there is a need for further purification and characterization of phytochemicals associated with antioxidant activities.

Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA.

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kß), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. Methods: We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kß, and CDPK1. Using qPCR analysis, we finally measured the mRNA amount of PANK1, DAGK, and PI4Kß at trophozoites and schizonts stages. Results: We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kß, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kß possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His while DAGK contained K270R and K292R mutations. PI4Kß had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kß in the trophozoites but upregulation at the schizonts stages in the AQr parasites. Conclusions: The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and the impact on drug responses require further investigations in malaria parasites.

Evaluation of the antimicrobial activity and safety of Rhus vulgaris (Anacardiaceae) extracts.

BACKGROUND: Medicinal plants have been used in the treatment of various ailments in most developing countries. Oral infections are the most prevalent diseases in man. The Rhus family has been found to have antimicrobial, antimalarial, and anti-inflammatory properties. Few studies have been done on Rhus vulgaris Meikle. A study was conducted to determine the effect of Rhus vulgaris Meikle stem bark extracts against selected oral pathogenic microorganisms and the safety of the extracts in vitro and in vivo. METHODS: Methanol:dichloromethane (1:1), methanol and aqueous extracts were tested for bacteriostatic and bactericidal effects against Methicillin Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Streptococcus mutans and Candida albicans. Cytotoxicity of the active extracts was determined using Vero E6 cell lines while safety was evaluated in mice and rats. Phytochemical screening was performed on the methanol extracts. One-way ANOVA and Tukey's multiple comparisons tests were performed using IBM SPSS statistics 20.0 for antimicrobial assay and acute toxicity testing. One-way ANOVA and Dunnett's multiple comparison tests were conducted using GraphPad Prism 8.0 for cytotoxicity assay. RESULTS: Methanol extract of Rhus vulgaris showed significant antimicrobial activity against MRSA (12.00 ± 0.00 mm; p-value of < 0.005; Minimum Inhibitory Concentration of 0.391 mg/ml; Minimum Bactericidal Concentration of 1.563 mg/ml). The extract were not cytotoxic at 100 µg/ml which was the highest tested concentration. In acute dermal irritation testing, the methanol extract resulted in mild irritation with erythema and flaking that cleared within 8 days. There were no observable adverse effects from oral administration of the extracts (acute oral toxicity testing) at concentrations of 50 mg/kg, 300 mg/kg and 2000 mg/kg. Tannins, saponins, flavonoids, terpenoids, glycosides, alkaloids and phenols were detected in the methanol extract. CONCLUSIONS: Antimicrobial activity of R. vulgaris extracts supports its traditional use as a toothbrush. Cytotoxicity demonstrated by the extracts as well as the mild skin irritation warrants further study before R. vulgaris can be recommended for the development of effective and safe mouthwashes.

Ten new glycosides, carissaedulosides A-J from the root barks of Carissa edulis and their cytotoxicities.

Ten previously undescribed glycosides, carissaedulosides A-J (1-10) referring to six apiosylated phenylpropanoids (1-6), one coumarin-secoiridoid hybrid (7), and three furofuran lignans (8-10) were isolated from the root barks of Carissa edulis, together with 13 known analogues (11-23). Their structures were elucidated by spectroscopic analysis, ECD computational methods, and chemical derivations for configurations of sugar moieties. The new lignan bisdesmoside, 10, exhibited significant cytotoxicity against A549 (IC50 = 3.87 ± 0.03 µM) and MCF-7 (IC50 = 9.231 ± 0.290 µM) cell lines, while the known lignan monodesmoside, 12, showed impressive cytotoxic efficacy (IC50 = 5.68 ± 0.180 µM) against only MCF-7 cell line. It is noted that a known cardenolide, 11, displayed strong cytotoxic potency against HL-60, A549, MCF-7 and SW480 cell lines with IC50 values ranging from 0.023 to 0.137 µM. Moreover, compound 11 induced dose-dependent apoptosis on SW480 cell, but not explicit dose-dependent apoptosis on HL-60 cells.

Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei.

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4_KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7_KO and PM8_KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs.

Secondary bacterial infections and antibiotic resistance among tungiasis patients in Western, Kenya.

Tungiasis or jigger infestation is a parasitic disease caused by the female sand flea Tunga penetrans. Secondary infection of the lesions caused by this flea is common in endemic communities. This study sought to shed light on the bacterial pathogens causing secondary infections in tungiasis lesions and their susceptibility profiles to commonly prescribed antibiotics. Participants were recruited with the help of Community Health Workers. Swabs were taken from lesions which showed signs of secondary infection. Identification of suspected bacteria colonies was done by colony morphology, Gram staining, and biochemical tests. The Kirby Bauer disc diffusion test was used to determine the drug susceptibility profiles. Out of 37 participants, from whom swabs were collected, specimen were positive in 29 and 8 had no growth. From these, 10 different strains of bacteria were isolated. Two were Gram positive bacteria and they were, Staphylococcus epidermidis (38.3%) and Staphylococcus aureus (21.3%). Eight were Gram negative namely Enterobacter cloacae (8.5%), Proteus species (8.5%), Klebsiellla species (6.4%), Aeromonas sobria (4.3%), Citrobacter species (4.3%), Proteus mirabillis(4.3%), Enterobacter amnigenus (2.1%) and Klebsiella pneumoniae (2.1%). The methicillin resistant S. aureus (MRSA) isolated were also resistant to clindamycin, kanamycin, erythromycin, nalidixic acid, trimethorprim sulfamethoxazole and tetracycline. All the Gram negative and Gram positive bacteria isolates were sensitive to gentamicin and norfloxacin drugs. Results from this study confirms the presence of resistant bacteria in tungiasis lesions hence highlighting the significance of secondary infection of the lesions in endemic communties. This therefore suggests that antimicrobial susceptibility testing may be considered to guide in identification of appropriate antibiotics and treatment therapy among tungiasis patients.

Immunomodulatory and anticancer potential of Gan cao (Glycyrrhiza uralensis Fisch.) polysaccharides by CT-26 colon carcinoma cell growth inhibition and cytokine IL-7 upregulation in vitro.

BACKGROUND: Chinese licorice, (Glycyrrhiza uralensis Fisch.) is one of the commonly prescribed herbs in Traditional Chinese Medicine (TCM). Gancao, as commonly known in China, is associated with immune-modulating and anti-tumor potential though the mechanism of action is not well known. In this study, we investigated the in vitro immunomodulatory and antitumor potential of Glycyrrhiza uralensis polysaccharides fractions of high molecular weight (fraction A), low molecular weight (fraction B) and crude extract (fraction C). METHODS: Cell proliferation and cytotoxicity was investigated using Cell Counting kit 8 (CCK-8) on Intestinal epithelial cell line (IEC-6) and Colon carcinoma cell line (CT-26). IL-7 gene expression relative to GAPDH was analysed using Real time PCR. The stimulation and viability of T lymphocytes was determined by Trypan blue exclusion assay. RESULTS: G.uralensis polysaccharides did not inhibit proliferation of IEC-6 cells even at high concentration. The ED50 was found to be 100 µg/ml. On the other hand, the polysaccharides inhibited the proliferation of cancer cells (CT-26) at a concentration of ≤50 µg/ml. Within 72 h of treatment with the polysaccharides, expression of IL-7 gene was up-regulated over 2 times. It was also noted that, IEC-6 cells secrete IL-7 cytokine into media when treated with G.uralensis polysaccharides. The secreted IL-7 stimulated proliferation of freshly isolated T lymphocytes within 6 h. The effect of the polysaccharides were found to be molecular weight depended, with low molecular weight having a profound effect compared to high molecular weight and total crude extract. CONCLUSION: Our findings indicate that G.uralensis polysaccharides especially those of low molecular weight have a potential as anticancer agents. Of great importance, is the ability of the polysaccharides to up-regulate anticancer cytokine IL-7, which is important in proliferation and maturation of immune cells and it is associated with better prognosis in cancer. Therefore, immunomodulation is a possible mode of action of the polysaccharides in cancer therapy.

Antimicrobial activity and probable mechanisms of action of medicinal plants of Kenya: Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus.

Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus are used traditionally in Kenya for treatment of microbial infections and cancer. Information on their use is available, but scientific data on their bioactivity, safety and mechanisms of action is still scanty. A study was conducted on the effect of organic extracts of these plants on both bacterial and fungal strains, and their mechanisms of action. Extracts were evaluated through the disc diffusion assay. Bacteria and yeast test strains were cultured on Mueller-Hinton agar and on Sabouraud dextrose agar for the filamentous fungi. A 0.5 McFarland standard suspension was prepared. Sterile paper discs 6 mm in diameter impregnated with 10 µl of the test extract (100 mg/ml) were aseptically placed onto the surface of the inoculated media. Chloramphenicol (30 µg) and fluconazole (25 µg) were used as standards. Discs impregnated with dissolution medium were used as controls. Activity of the extracts was expressed according to zone of inhibition diameter. MIC was determined at 0.78-100 mg/ml. Safety studies were carried using Cell Counting Kit 8 cell proliferation assay protocol. To evaluate extracts mechanisms of action, IEC-6 cells and RT-PCR technique was employed in vitro to evaluate Interleukin 7 cytokine. Investigated plants extracts have both bactericidal and fungicidal activity. W. ugandensis is cytotoxic at IC50<50 µg/ml with MIC values of less than 0.78 mg/ml. Prunus africana shuts down expression of IL 7 mRNA at 50 µg/ml. W. somnifera has the best antimicrobial (1.5625 mg/ml), immunopotentiation (2 times IL 7 mRNA expression) and safety level (IC50>200 µg/ml). Fractions from W. ugandensis and W. somnifera too demonstrated antimicrobial activity. Mechanisms of action can largely be attributed to cytotoxicity, Gene silencing and immunopotentiation. Use of medicinal plants in traditional medicine has been justified and possible mechanisms of action demonstrated. Studies to isolate and characterize the bioactive constituents continue.