Chemosystematics, Antimicrobial and Antiproliferative Activities of the Kenyan Zanthoxylum Species and Dodonaea Viscosa Populations

Abstract

The aim of this study was to establish the relationship in Zanthoxylum species, and Dodonaea viscosa populations based on the metabolite profiles. The study also included the evaluation of the crude extracts and pure compounds obtained from Zanthoxylum and Dodonaea species for antimicrobial activity and cytotoxicity. The chemosystematics study was carried out using metabolomics approach for Zanthoxylum species and D. viscosa populations in Kenya. Through phytochemical investigations some compounds were isolated and characterized with eight compounds from Z. paracanthum (myristic acid (1), stigmasterol (2), sesamin (3), 8-acetonyldihydrochelerythrine (4), arnottianamide (5), 10-methoxycanthin-6-one (6), canthin-6-one (7) and 8-oxochelerythrine (8)), from D. viscosa subsp viscosa three compounds (dodonic acid (9), 5,7,4',5'-tetrahydroxy-3,6,2'-trimethoxyflavone (10) and hautriwaic acid lactone (11)) and from D. viscosa subsp angustifolia one compound (catechin (12)) were isolated by the use of a combination of chromatographic techinques. These compounds were evaluated for antimicrobial activity and cytotoxicity along with crude extracts. Agar well and broth dilution were used to test antimicrobial activity of root, stem and leaf extracts of five Zanthoxylum species (Z. chalybeum, Z. gilletii, Z. holtzianum, Z. paracanthum and Z. usambarense) as well as leaf extracts of ten populations of D. viscosa (Coastal, Makueni, Machakos, Karura, Nanyuki, Maimahiu, Narok, Loita Forest, Marigat and Homa bay) were tested against six microbes (Bacillus cereus Methicilin Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Escherichia coli, Candida albicans, Aspergillus flavus). Eight compounds isolated from Z. paracanthum as well as the four compounds isolated from D. viscosa were evaluated for antimicrobial activity against methicilin resistant Staphylococcus aureus (MRSA), typed Staphylococcus aureus, Escherichia coli and Candida albicans. The positive controls were omacilin 1000μg/ml for bacteria and fluconazole 1000μg/ml for fungi. The antiproliferative activity of the eight compounds isolated from Z. paracanthum, and three compounds from D. viscosa as well as the crude extracts (Z. paracanthum roots, D. viscosa subsp viscosa and D. viscosa subsp angustifolia leaves) was determined using MTT assay against kidney normal cell line from African green monkey (E6 vero), human breast cancer cell line (HCC 1395) and human prostate cancer cell line (DU 145). Chemometric analysis of relatedness of Zanthoxylum group of plants conformed to the current classification of Zanthoxylum species in Kenya established on morphology. Based on phytochemical production, the five Zanthoxylum species segregated into two clusters; cluster one was composed of Z. gilletii Z. usambarense and Z. paracanthum while Z. chalybeum and Z. holtzianum constituted cluster two. On the other hand, analysis of D. viscosa populations showed that Nanyuki, Machakos and coastal collections were found to be closely related while Narok and Nairobi populations were found to be related. This is in contrast with the existing morphological classification of D. viscosa where the coastal population is believed to be composed of D. viscosa subsp viscosa and the Kenyan inland populations are composed of D. viscosa subsp angustifolia. The antimicrobial activity results of Zanthoxylum species showed that the root bark extracts were most active followed by leaf and stem bark extracts respectively. The extracts from the root and stem bark of Z. paracanthum were the most active compared to root and stem bark extracts of other species while leaf extract of Z. usambarense was the most active compared to leaf extracts from other Zanthoxylum species. On the other hand, Nanyuki population recorded the highest antimicrobial activity overall followed by Coastal population while Homabay, Makueni and xv Machakos populations followed in that order. Leaf extract from Karura population was the least in terms of antimicrobial activity. The eight compounds isolated from Z. paracanthum roots, canthin-6-one (6) was the most active followed by 10-methoxycanthin-6-one (7) while myristic acid (1) and arnottianamide (5) did not show any activity against the tested microbes. Hautriwaic acid lactone (11) was the most active compound from D. viscosa Subsp viscosa while 5,7,4',5'-tetrahydroxy-3,6,2'-trimethoxyflavone (10) did not show any activity against the tested microbes. Catechin (12) on the other hand exhibited good activity against the tested microbes. In the order listed below, stigmasterol, Sesamin, 8-oxochelerythrine, canthin-6-one, 8-acetonyldihydrochelerythrine, hautriwaic acid lactone, root bark extract of Z. paracanthum and 10-methoxycanthin-6-one showed antiproliferative potential against HCC 1395. While antiproliferative potential against DU 145 was highest in 10-methoxycanthin-6-one followed by canthin-6-one, 8-oxochelerythrine and leaf extract of D. viscosa subsp angustifolia. Chemosystematics of Zanthoxylum spp conformed to the morphological classification while that of D. viscosa disagreed. This study reports isolation of myristic acid, stigmasterol, 8-acetonyldihydrochelerythrine, arnottianamide, 10-methoxycanthin-6-one and 8-oxochelerythrine from Z. paracanthum for the first time while 5,7,4',5'-tetrahydroxy-3,6,2'-trimethoxyflavone is reported for the first time in D. viscosa. Canthin-6-one, 10-methoxycanthin-6-one and 8-oxochelerythrine were all broad spectrum in terms of antimicrobial and antiproliferative activities. This study provides scientific evidence for the popularity of Zanthoxylum spp and D. viscosa as traditional medicine plants. This study recommends invivo studies of the most active compounds (canthin-6-one, 10-methoxycanthin-6-one, hautriwaic acid lactone and catechin) isolated in this study as well as increased scope to cover other microbes of economic importance and other cancer cell lines. Additionally, revision of the current classification of D. viscosa using modern taxonomy tools such as molecular phylogenetics and chemosystematics is recommended.