Isolation and identification of bioactive compounds from chloroform fraction of methanolic extract of Carissa opaca roots

Abstract Carissa opaca is a shrub known for its variety of medicinal applications. This study reports isolation and identification of four chemical compounds from its roots for the first time. The methanolic extract of the roots was fractionated into various solvents with increasing polarity. Chloroform fraction was subjected to column and thin layer chromatography to ultimately yield 2H-cyclopropanaphthalene-2-one, 7-hydroxy-6-methoxy-2H-1-benzopyran-2-one, 3-(4-methoxyphenyl)-2,6-dimethylbenzofuran and 5(1H)-azulenone, 2,4,6,7,8,8a-hexahydro-3,8-dimethyl-4-(1-methylethylidene)-,(8S-cis). They were identified by GC–MS analysis. The compounds exhibited considerable antimicrobial activities against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger with zones of inhibition ranging from 10 to 13 mm as compared to the standard drug amoxicillin with zones of inhibition 13–17 mm under the similar conditions. In conclusion, the roots of C. opaca can provide new leads for future antimicrobial drugs.

. As the current medicines are facing serious safety, efficacy and/or cost problems, better alternatives are required. The problem is exponentially compounded with the emergence of multidrug resistant strains of pathogens, necessitating search for more potent new antibiotics (Kanafani & Perfect 2008;Savoia 2012;Martins et al. 2015).
The objective of this study was to isolate and identity chemical constituents from chloroform fraction of methanolic extract of the roots of C. opaca, and to determine antimicrobial activities of the compounds obtained in sufficient quantity. This study is being reported here for the first time.

Results and discussion
Methanolic extract of powdered roots of C. opaca, obtained by cold maceration, was fractionated into solvents with increasing polarity (Hossain & Ismail 2013). Chloroform fraction, which was of good quantity, was selected for phytochemical investigation.
Antimicrobial activities were determined against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger. Results are shown in Table S4. COC1 and COC4 showed highest activity (ZOI 13 mm) against E. coli. Quiet interestingly, COC3 did not show any activity against P. aeruginosa and C. albicans. COC2 too was inactive against C. albicans. This fungus is, in fact, one of the pathogens posing serious challenge to the current antimicrobial therapies (Tsai et al. 2013). The zones of inhibition ranged from 10-13 mm as compared to the standard drug amoxicillin with zones of inhibition 13-17 mm under the similar conditions. The findings are in agreement with those of other studies on the extracts (Awasthi et al. 2013). The activity provides scientific basis for the use of the plant as antiseptic (Handoo 2006). Compounds COC1 and COC4 are terpenoids, which, as part of many essential oils, are known to have diverse medicinal properties (Thoppil & Bishayee 2011). COC2 is also known as scopoletin, buxuletin or scopoletol. It belongs to coumarins, which constitute an important class of pharmacologically active phytochemicals (Walasek et al. 2015). It also has a phenolic group that may render it with good antioxidant activities.
In conclusion, the next logical step should be to isolate these compounds in sufficient amounts to investigate antimicrobial activities in further detail followed by clinical trials.

Conclusion
The chloroform fraction of the roots of C. opaca yielded four major bioactive compounds upon extensive chromatography. They were identified by GC-MS, and found to show, in general, considerable antimicrobial activity against the test microbes. Further pharmacological studies of these compounds may provide leads for future antibiotics.

Supplemental material
experimental details and data related to this article are available online.