PROFILING AND QUANTIFICATION OF COSMOS CAUDATUS KUNTH AND CENTELLA ASIATICA LINN. AND IN VITRO ANTI CANCER ACTIVITY OF COSMOS CAUDATUS
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Date
2014-09
Authors
MOHD AMIN SHARIFULDIN, MUNIRA
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Abstract
This study aims to check the quality of raw materials, develop and validate analytical methods for chemical profiling and standardization of C. asiatica and C. caudatus extracts from 3 different locations. The plants were extracted with 3 different solvents and were studied for antioxidant activity. C. caudatus leaves extracts were also screened for in vitro anti-cancer properties.
In this study, the quality of the raw materials have been determined based on several parameters such as total ash, total insoluble ash, total moisture content, heavy metal test, microbial limit test and extractive values. The results showed acceptable values compared to the reference limits.
Extraction was done using 96%, 75%, 50% and 25% ethanol and water and the extracts were analyzed qualitatively using Fourier Transform Infrared (FTIR) and Ultraviolet/visible (UV/Vis) spectroscopy. The spectra collected from FTIR analyses were subjected to principal component analysis which showed that the extracts were clustered based on the same functional group and chemical constituents.
The extracts were also analysed for the primary and secondary metabolites content. For C. caudatus, the highest total glycosaponins, total protein, polysaccharides and phenolics content was 46.12%, 57.36%, 7.54% and 4.42%, respectively, while the highest total glycosaponins, total proteins, polysaccharides and phenolics content for C. asiatica was 54.60%, 24.58%, 11.98% and 6.82%, respectively. The DPPH scavenging activity showed moderate activity in all extracts.
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Qualitative analysis of the extracts by high performance thin layer chromatography (HPTLC) was done using 1 marker compound in C. caudatus which was quercitrin (Rf: 0.21) and 4 marker compounds in C. asiatica, namely madecassoside (Rf: 0.31), asiaticoside (Rf: 0.39), madecassic acid (Rf: 0.87) and asiatic acid (Rf: 0.96). Marker compounds were chosen based on the uniqueness and amount in that herb. High performance liquid chromatography (HPLC) was done to quantify 3 marker compounds in C. caudatus and 4 marker compounds in C. asiatica. The percentage of rutin, quercitrin and quercetin in C. caudatus was 0.13 - 0.94%, 1.51 - 13.78% and 0.18 - 0.92%, respectively. The percentage of madecassoside, asiaticoside, madecassic acid and asiatic acid in C. asiatica extracts was in the range 0.10 - 9.86%, 0.14 - 7.74%, 0.41 - 3.32% and 0.15 - 1.44%, respectively.
For anti cancer activity, the fractions obtained from C. caudatus extract were evaluated for cytotoxicity, apoptosis and antitumorgenicity on human colorectal carcinoma cell HCT 116. The F2, F7 and F8 fractions showed potent dose dependent cytotoxicity with IC50 value of 15.53 ± 0.4, 32.72 ± 0.3 and 34.16 ± 1.4 μg/mL, respectively. From the observation on mitochondrial membrane potential, chromatin structure and nuclear morphology, it showed apoptotic on the treatment cells. Assay on cell migration, cell invasion and clonogenicity showed that the fractions inhibit these three pathways that are required for metastasis and cancer cell proliferation.
In conclusion, the new development of HPLC method can help in determining the quality of the raw materials and product for standardization process as well as routine works. C. caudatus can be considered as a source for anti cancer agent as it perturbs the carcinogenesis pathway.
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PROFILING AND QUANTIFICATION OF COSMOS CAUDATUS KUNTH AND CENTELLA ASIATICA LINN