PHYTOCHEMICALS AND ANTIMICROBIAL ACTIVITIES OF PRUNUS AFRICANA STEM BARK

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PHYTOCHEMICALS AND ANTIMICROBIAL ACTIVITIES OF PRUNUS AFRICANA STEM BARK

ABSTRACT

This research work is aimed at determine the phytochemicals and antimicrobialactivity of Prunus African . The designof the study was experimental laboratory-based study. The methodology involve the in vitro assays involved determination of the cytotoxic concentration levels (CC50) of the plant extracts on Vero cells as well as calculating the inhibitory concentration (IC50) of the plant extracts on breast and colon cancer cell lines. The drugs with the highest selectivity have low IC50 in the breast and colon cancer cell lines and high CC50 in Vero cells were used in the in vivo assays which

involved acute oral toxicity studies, conducted on 8 weeks old Swiss albino mice to calculate the median lethal dose, LD50. The results shows safest and effective drugs were methanol extracts of leaves from Prunus Africana whose results showed an average IC50 of 164.64±4.14μg/ml in the breast cancer cell lines and 21.33±0.5μg/ml in the colon cancer cell lines, as well as the stem bark water extracts from Warburgia stuhlmannii, whose results showed an average IC50 of 332.79±7.53μg/ml in the breast cancer cell lines and 107.20±2.50μg/ml in the colon cancer cell lines. Both extracts had an average CC50 of >1000μg/ml in Vero cells. Based on positive cytotoxicity results on the two extracts, acute oral toxicity studies were conducted on 8 weeks old female Swiss albino mice. This revealed no signs of acute toxicity after drug administration with LD50 of >5000mg/kg body weight, therefore the extracts were considered to be safe. The conclusionwas that methanol extract from the leaves of Prunus Africana and the water extracts from the stem bark of Maytenus senegalensis were safe for use in the murine model. These extracts also showed a level of anti-proliferative activity in both breast and colon cancer cells without being toxic to Vero cells. This information forms a basis for the development of the extracts as safer alternative therapies for the management of cancer.

CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Prunus Africana is an evergreen tree with a height of more than 40 meters and a stem diameter of up to 1 meter (Gachie et al., 2012). It has shining foliage, blackish-brown bark and greenish or white flowers. P. africana is a geographically widespread species restricted to underlying islands like Sao Tome’ and Madagascar and Afromontane forest islands in Africa (Somalia, South Africa, Kenya, Tanzania, Sudan, Uganda, Malawi, Ethiopia, Zaire, Cameroon, Zimbabwe, Angola and Malawi) which are above 1500 meters altitude (Hall et al., 2000). The species grows in the humid midlands and in humid and semi-humid highlands.

In Kenya, it grows in the slopes of Mt. Elgon, Mt. Kenya, Cherangani Hills, Tugen Hills, Aberdares Range, Mau range and Kakamega, Nandi and Timboroa forests (Gachie et al., 2012). P. africana tree is used for timber and also in traditional and modern medicine in Africa. Bark extracts of P. africana are used to treat benign prostate cancer hyperplasia (Stewart, 2003). Prostafx, Tadenan and Pygenil are the herbal preparations of P. africana available in the market. Extracts from stem and root barks contain phytochemicals with anticancer, anti-inflammatory and antiviral effects (Vinceti et al., 2013). Bark extracts improve urologic symptoms in prostate cancer patients as they have apoptotic and anti-proliferative effect on the prostate (Kadu et al., 2012).

The pharmacological efficacy of the wild tree bark extracts is thought to be due to synergistic effect of various compounds. The main compounds are; pentacyclic triterpenoids (ursolic and oleanolic acids) which inhibit glucosyl-transferase activity and have anti-edematous activity (Donovan et al., 1998), phytosterols mainly β-sitosterol and β-sitostenone have anti-inflammatory effect as they suppress the production of prostaglandins and thus prevent swelling of the prostate (Carbin et al., 1990). The bark also has ferulic acid esters (n-tetracosanol and n-docosanol) and their derivatives which have antitumor and hypocholesterolemic activity on the prostate (Kampa et al., 2004). The three compounds work synergistically to counteract the biochemical and structural changes associated with benign prostatic hyperplasia (BPH). Benign Prostate Hyperplasia is a non-cancerous urologic condition that leads to enlargement of the prostate gland. The bark extracts from P. africana also inhibit bladder hyperactivity. The use of the bark in traditional medicine includes the treatment of chest pain, urinary and bladder infections, stomach aches, kidney disease and malaria. The bark is either chewed or crushed into powder and drunk as tea (Stewart, 2003).

Chemical composition varies between different plant species due to environmental differences in their habitats, reflecting defined geographical patterns. Knowledge of variation of the chemical constituents of P. africana is important to optimize the use and ensure sustainable conservation of the tree. Studies carried out in Cameroon, Madagascar and Zaire showed that the chemical composition of the extracts of P. africana depends on the habitat of the species (Gachie et al, 2012). Studies based on nuclear and chloroplasts DNA markers in P. africana show five distinct regions throughout Africa that reflect divergent population across the continent (Kadu et al., 2011). In “High Africa” extending from 34°S to 12°N, the species is confined in volcanic highlands and mountains. In equatorial Africa, the species is found at elevation ranges from 1000 meters to 3500 meters with annual rainfall of over 3000 mm in low altitudes and between 500 to 700 mm in high altitudes (Kadu et al., 2011). The species occurrence below the montane zone is mainly at drainage lines and rocky areas (Hall et al., 2000). In southern Africa, the species occurs in elevation ranges between 600 and 1000 meters above sea level (Geldenhuys, 1993). Geological barriers and environmental changes have effects on the ecosystem, occurrence of species, their genetic diversity and evolutionary processes.

The high demand of the bark extracts of P. africana has caused serious damage to the wild population (Cunningham et al., 1997). More than 3000 tons of bark oand bark extracts are exported to Europe per year for the production of herbal preparations for BPH treatment (Cunningham et al., 1997). This high demand causes devastating effect to the wild population of P. africana which is the main source of the bark. Attempts at cultivation of P. africana are underway in Kenya and other countries. The bark of P. africana can regenerate if bark removal does not interfere with the vascular cambium and thus harvesting is sustainable (Cunningham & Mbenkum, 1993).

Despite the resilience of P. africana to debarking, in dry areas, bark re-growth is limited and large scale debarking stresses the tree even when complete re-growth occurs. Limited distribution of the species only in the afromontane islands and increased clearing for agriculture increases the threat to P. africana. The species was included as endangered species in Appendix II of the Convention of International Trade in Endangered Species (CITES) at the ninth conference due to the increasing international demand (Betti, 2008). The species has also been assigned a vulnerable conservation status on the IUCN Red List. Import of the bark from Cameroon into the European Union was banned from November 2007 to December 2010 when CITES declared that the ban be lifted but with reduced quota of 150,000 kg for 2010 and 2011. In some African countries, policies have been established aiming at ensuring sustainable management of forests that contain P. africana species.

However, control problems and enforcement issues persist and there is need to identify and implement sustainable management options including conservation and domestication measures. In order to optimize sustainable conservation of the vulnerable African cherry, knowledge of the regional variation is essential. World Agroforestry Centre established a P. africana stand at Muguga for determination of the growth performance and phytochemical yield. The seeds for this stand were obtained from a wild stand at Kobujoi, Nandi County.

1.2 STATEMENT OF THE PROBLEM

Prunus Africana bark extracts are used in the treatment of benign prostate hyperplasia (BPH), a common medical condition in older men. The bark extracts that are currently used for the herbal medicine are mainly from the natural forests. Incidence of BPH increases by 10% per decade and thus demand for the bark continues to increase (Iran et al., 2003). In addition, forest clearing continues for urban development, human settlement and farming. Limited distribution of the species only to the Afromontane regions means the demand for bark is focused on a limited area of the forests. The high demand of P. africana for medicine and other uses leads to uncontrolled exploitation and illegal harvesting of natural populations (Stewart, 2003).

The demand is projected to increase due to growing confidence in herbal remedies in consumer markets (Cunningham et al., 1997). Although P. africana bark demand for commercial processing of drugs for cure of benign prostate hyperplasia has been on the rise, bark harvesting from the wild is unsustainable. Most people believe that bark extracts from wild populations are more potent than those from cultivated plants (Canuto et al., 2012). This is as a result of lack of information on phytochemical yields and profiles of domesticated P. africana. Before this study, there was no study that evaluated the growth performance of on-farm P. africana.

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