Large scale isolation and purification of some bio active compounds from Cleistanthus collinus by liquid liquid partition chromatography and study of their effect on isolated Wistar rat heart

Abstract

We have developed a method for extraction of fluorescent compounds from aqueous extracts of leaves of C.collinus by liquid-liquid partition chromatography using chloroform. Various types of aqueous extracts of leaves were made to assess the nature of fluorescent active principles present and to quantify them so as to identify a method for large scale isolation of the active compounds. We focused on the fluorescent principles, because we found that the fluorescent fraction caused death in rats. and the non-fluorescent fraction was non-toxic. There were only 4 major fluorescent principles present in any type of aqueous extract. 2 of these compounds were identified as Cleistanthins A and C. Compounds | and 3 are yet to be purified to confirm identity with NMR. Recommendations on the best method to isolate each of the above compounds has been made. The major achievement in this study is that we now hold authenticated stocks of the two major toxic constituents of the aqueous extract of C.collinus, namely. Cleistanthin A and C. Though Cleistanthin B has been repeatedly stated as the toxin in many manuscripts, we were not able to identify Cleistanthin B in any of the aqueous extracts. In animal study, the effect of Cleistanthin-A and Cleistanthin-C (10µM concentration) was tested on rat hearts perfused in Langendorff mode to see its effects. The result indicates the isolated active compounds do not produce any significant effect on heart rate. FUTURE STUDY: The toxicity of isolated principles Cleistanthin A and Cleistanthin C have been determined in other experiments. The mechanism of action of the toxin is yet to be identified. Once the mechanism of action has been established, attempts must be made to develop treatment options for poisoning in the clinical setting. Compound 1 and 3 is yet to be isolated in sufficient quantities. So as to perform purification and further structural elucidation. The toxicity of these compounds is yet to be confirmed. Authentic sources of compounds were not available during our search.