Phytochemical Composition, Antiviral Activity and Toxicity of Dichrocephala Integrifolia Kuntze. Crude Extracts

Abstract

Human herpes simplex viruses are among the world’s most ubiquitous human infections. Generally, there are two types of Human Herpex simplex viruses (HSV); HSV-1 and HSV-2. Worldwide over 90% of people are infected by either one or both. HSV-1 is a viral disease known to cause genital and oral lesions. Currently, a major antiviral drug is acyclovir that is used for the treatment of HSV infections has proven unsatisfactory as resistance to this drug has been frequently reported. Furthermore, severe side effects have been witnessed in pregnant mothers and infants. High prevalence of HSV, lack of vaccines and limited treatments options warrants an urgent need for more effective anti-HSV agents. The aim of this study was to investigate phytochemical composition, in vitro anti-HSV, in vivo and in vitro toxicity of D. integrifolia crude extracts. Leaves, roots, flowers, and stem of D. integrifolia were collected from the plants’ natural habitat in Mabariri village in Nyamira County. The identification of the plants was done by a botanist in the school of biological sciences, University of Nairobi. The materials were extracted with methanol and water using standard methods. Qualitative chemical tests were carried out to determine the presence or absence of alkaloids, flavonoids, saponins, tannins, glycosides, and terpenoids. The MTT assay was used to investigate the in vitro cytotoxic activity of the crude extracts using Vero cell lines obtained from Kenya Medical Research Institute (KEMRI) in the center for viral research. Antiviral activity was assessed based on the ability of the extract to protect normal cells (Vero cell lines) from HSV attack. In vivo toxic effects of the crude extracts were evaluated in female Swiss albino mice using standard oral acute toxicity protocols described by Organization for Economic Co-operation and Development. The qualitative phytochemical analysis results showed that the test extracts contained tannins, flavonoids, alkaloids, terpenoids, phenols, glycosides, and saponins. The extracts were not xvi cytotoxic to Vero cells except the methanolic extract of the flower which had CC50 value of 71.31± 2.65μg/ml. The extracts demonstrated interference in the adsorption step of HSV-1 may be by blocking the epitopes for the virus on the cell's membrane. Methanolic extracts of the stem, leaves and aqueous extracts of leaves inhibited the ability of HSV-1 virus to cause a cytopathic effect when pre-treatment was done, with IC50 values of 63.95±5.36 μg/ml, 54.45±3.45 μg/ml, 86.20±7.56 μg/ml respectively. The methanolic extract of flower, aqueous extract of the root and the methanolic extract of leaves showed virucidal activity with IC50 values of 45.27±2.41 μg/ml, 0.333±1.23 μg/ml, and 30.53±4.51 μg/ml respectively. Oral administration of the extracts to mice at both 300mg/kg and 2000mg/kg did not result in any toxic effects or mortality. In all the groups, no major behavioral or appearance changes were observed. Generally, the presence of pharmacologically important phytochemicals such as flavones, phenols, terpenoids, and tannins that have been implicated in antimicrobial action support the use of this plant in the management of these pathologies. Results on cytotoxicity, efficacy, and acute oral toxicity depict that the plant has no major toxicity. Therefore, the preparation of antiviral herbal remedy from the plant parts may be safe for use in patients. However further research is required to elucidate the mechanisms of actions of the extracts and isolate the bioactive agents for the treatment of viral infections. It is recommended that the toxic effects of the use of the plant's products to be studied on formulated products for pharmacological aspects. Keywords: Dichrocephala integrifolia, antiviral, phytochemical, cytotoxicity, Herpes Virus simplex, In vivo safety