In vitro assessment of effect on genetic material (genotoxicity) of Caesalpinia crista plant seed extract on cultured human peripheral blood lymphocytes using Chromosomal Aberration Assay and Cytokinesis Blocked Micronuclei Assay

Abstract

Medicinal plants continue to be an important therapeutic aid for alleviating the ailments of human kind. Caesalpinia Crista of family Fabaceae is a moderately sized deciduous tree, growing wild throughout India especially in the coastal area, forests, and hills. The extracts of various other parts of Caesalpinia crista are reported to have anthelmintic, anti-amyloidogenic, immunomodulatory, analgesic, antipyretic, anti-inflammatory, memory enhancing, antidiabetic activity and also antitumor and antioxidant activity. The plant parts have been widely used in medicines for its unique and diverse pharmacological properties. The literature has revealed that seeds and leaves of Caesalpinia crista contain around fourteen compounds. The isolated compounds are cassane-and norcassane-type diterpenes and other flavonoids and triterpenoides. Chemical investigation on Caesalpinia crista revealed two diterpenoids (6b-cinnamoyloxy-7b-acetoxyvouacapen-5a-ol and 6b,7b-dibenzoyloxyvouacapen-5a-ol), which have been reported to be cytotoxic to two different cancer cell lines. The phytoconstituents of the plant and its protective effect as antioxidant and antitumor activity in various pathologies triggered our interest to investigate the genotoxic effect of the methanolic extract of Caesalpinia crista seeds on cultured human peripheral blood lymphocytes. Our literature survey revealed that, there is little information about genotoxicity of this plant extract. This was undertaken for two aspects: 1) to check if the reported anti-tumor activity of this extract is due to DNA-affecting activity or not, which can be evident if increased DNA-damage is observed in terms of genotoxicity parameters. This is envisaged due to the fact that most of the anti-cancer compounds are DNA-damaging agents. 2) Any effect on chromosome aberration levels following treatment with extract would indicate its mode of action i.e. clastogenic (reflected as chromosome aberration per cell frequency) or aneugenic (reflected as CBMN frequency). The study was carried out in the presence and absence of metabolic activation system to check if the extract contains a direct acting DNA damaging compound(s) or liver enzyme activation dependent compound(s). 7 The assessment of genotoxic potential of Caesalpinia crista plant seed extract was done using in vitro genotoxicity endpoints, in terms of frequency of chromosome aberrations (CA), and %age of Cytokinesis blocked binucleated cells with micronucleus (CBMN) assay. This study was carried out in compliance with the Test Guidelines of the Organization for Economic Cooperation and Development (OECD, 1997a, b, 2004). Experiments were done by treating the in vitro short-term cultured human blood lymphocytes at various concentrations of plant extract dissolved in DMSO (vehicle) in the absence and presence of metabolic activation system for long term and short term exposure time respectively. Results demonstrated significant induction of structural chromosomal aberrations. The methanolic extract of Caesalpinia crista plant seeds significantly decreased the mitotic index value at the highest tested concentration indicating its cytotoxicity. The results of chromosomal aberrations indicated that methanolic extract of Caesalpinia crista plant seeds had clastogenic activity. Whereas, the results of CBMN assay following treatment with extract did not reveal significant aneugenic activity. The effect of incubation with S9 fraction on the experiment revealed that the methanolic extract of Caesalpinia crista plant seeds contains direct acting DNA-damaging compound(s), the activity of which is not significantly affected following metabolic activation. The present study describes a novel approach for the study of mechanism of action of a complex mixture with potential anti-cancer activity employing endpoints of in vitro genotoxicity assessment in order to assess the DNA-damaging activity.