CYTOTOXIC AND PHYTOCHEMICAL ANALYSIS OF SELECTED MEDICINAL PLANTS FROM SALT RANGE, PAKISTAN

Abstract

Medicinal plants are providing raw material to herbal and pharmaceutical industry. These plants are endowed with variety of phytochemicals commonly known as secondary metabolites. A large number of secondary metabolites are being extracted and utilized against various disorders including cancer. Medicinal plants are fairly distributed in Salt Range, Pakistan and are traditionally used by local herbalists (Hakeems) to treat various noxious diseases including cancer. This is a malignant disease and is increasing at rapid pace. Cancer was responsible for over 8.8 million death in 2015, i.e. one out of six deaths globally. It is the second leading cause of death over the world and its incidence rate will increase by 70% over the next two decades. Keeping this into consideration, a study was carried out to record folk knowledge of medicinal plants, cytotoxic activity as well as phytochemical analysis of some selected plants from the studied area. A total of 71 plants species belonging to 61 genera and 35 families were documented in using various allied complaints by the natives of the area. Based on fidelity level (FL %), some species such as Artemisia scoparia Waldst. & Kitam., Fagonia indica Burm. f., Moringa oleifera Lam., Otostegia limbata (Benth.) Boiss., Rhazya stricta Decne., Physorrhynchus burhaicus Hook.f. and Withania coagulans (Stocks) Dunal were selected for screening cytotoxic activity against cancer cell lines viz., human breast cancer (MCF-7), human cervical cancer (HeLa), human skin cancer (RD), rat pancreatic tumor (INS-1) and rat brain tumor (RG2). The plant materials of the said species were dried and ground to make powder for extracts preparation by using methanol. 2 All plant extracts showed cytotoxic effects against the selected cell lines, however, the inhibitory activity was found specific to plant extracts versus cell lines. In the case of Artemisia scoparia, except HeLa cell, the whole plant extract showed 80% cytotoxicity against all cell lines. The whole plant extracts of Fagonia indica showed 80% cytotoxic effect on three cell lines such as MCF-7, INS-1 and RG2. The fruit extract of Moringa oleifera had leading effects (> 80%) on all cell lines. Besides, leaves extract also inhibited 80% cells of MCF-7 and INS-1. The whole plant extract of Otostegia limbata showed maximum cytotoxic effect (80%) against all cell lines, except HeLa cell. From the Rhazya stricta, root extract showed highest cytotoxic effect (> 90%) for RG2 and HeLa cell lines and 80% for MCF-7 and INS-1. The whole plant and leaves extract of Physorhynchus brahuicus showed maximum cytotoxic effect (80%) on three cell lines viz., MCF-7, INS-1 and RG2 and root extract induced 80% RD cells. The leaves stalk and fruit extract of Withania coagulans had highest cytotoxic effect on all cell lines in which MCF-7, RG2, RD cells and HeLa cells were inhibited at 90%, while INS-1 was inhibited up to 80%. In order to examine the behavior of all these extracts, IC50 values were calculated against the selected cell lines. It was observed that the activity was very strong to moderate ranging from 0.99 to 23.15μgmL-1 after incubation of 48 hours. The same trend was observed after 72 hours of application, however the magnitude of activity was very strong to strong that ranged from 0.86 to16.78 μgmL-1. Physorhynchus brahuicus was subjected to bioactivity guided isolation of compounds through column chromatography (CC) which may be responsible for 3 activity. This species was selected due to higher ethnobotanical use (FL %), previous reported activities/biochemistry, preliminary cytotoxic screening and range of IC50 values. Six column chromatography (CC) fractions of Physorhynchus brahuicus (P. brahuicus) were subjected for cytotoxicity test against the five cell lines at the dose of 20μgmL-1 with Methotrexate (MTX) as comparator. All the fractions were found effective against the selected cell lines and the IC50 values were within the range of 0.38±0.08 to 12.86±0.10 μgmL-1. Except CCU, rest of fractions revealed very strong to strong inhibitory effects in terms of IC50 value. Among them, CCO fraction was found highly effective which resulted IC50 ranging from 0.68±0.07 to 6.74±0.07 μgmL-1. Out of 21 column fractions (CCFs), six fractions were selected based on their best performance. These were subjected for in depth evaluation of their IC50 values. According to the National Cancer Institute (NCI), USA, plant screening program, the plants extract and fractions can be considered active/putative if their IC50 value is less than 20 μgmL-1against cancer cell lines. The IC50 value was recorded between 0.38±0.08 to 12.86±0.10 μgmL-1 revealing very strong to strong inhibition against all the selected cell lines. Bioassay guided isolation of compounds was carried out to identify the compound (s) responsible for the activities. The chemical structures were established with the aid of extensive LC-MS spectroscopic, mass-spectral analyses and published data. Eight compounds viz., N,N-dipropyl propan-1-amine, 4- acetamidophenyl) 2-(diethylamino) acetate, 3-Methyl benzo furan-2-carboxylic 4 acid, [2-(Diphenylphosphoryl) ethyl] (methyl) oxo(phenyl) phosphine, 2-(3- Amino-1H-1, 2, 4-triazol-1-yl) acetohydrazide, 3-Methyl-N-(3-methylbutyl)-1- butanamin, 1, 3-Benzenedimethanol, α1-[[(1, 1-dimethylethyl) amino] methyl]-4- hydroxy and 3-Chloro-6-hydrazinopyridazine were identified from six column chromatographic fractions. This study provided detailed scientific information about the cytotoxic activity and phytochemistry of P. brahuicus that would serve as benchmark towards anticancer drug development. Structural elucidation and in vivo activity is therefore suggested to test potential toxicity of the fractions that may be used in the cancer drug development programme.