Bangladesh Journal of Pharmacology Volume: 13; Number 4; Year 2018 Cite this article as: Deepika S, Selvaraj CI, Anbalagan M. Phytochemical characterization and cancer cell line cyto- toxicity of Clitoria ternatea. Bangladesh J Pharmacol. 2018; 13: 349-52. Phytochemical characterization and cancer cell line cytotoxicity of Clitoria ternatea Sir, Ayurveda, a prominent traditional system, is using seed, leaf and root of Clitoria ternatea for antidiabetic, anti-inflammatory, anti-depressant, cardiovascular disease, urinary problems, hepatopathy, ulcer; as local anesthesia and also to act against laryngeal, colon and breast carcinoma (Jain and Shukla, 2011). C. ternatea is an indigenous plant to the South East Asia. Its phyto- constituents having bioactivities are previously repor- ted as alkaloids (ethyl D-galactopyranoside), triterpe- noids like taraxerol and taraxerone from leaf (Singh and Tiwari, 2010); phenols like 3,5,7,4-tetrahydroxyflavone- 3-rhamoglycoside, syringic, epicatechin, gallic acid; flavonoids like flavonol glycosides (rutin), kaempferol glycosides (kaempferol-3-neohesperidoside), p-couma- ric, rosmarinic, chlorogenic, apigenin, caffeic, ferulic, cinnamic acids and hydroxycinnamic acid derivatives; phytosterol such as β-sitosterol and campesterol from seed are considered valuable. Likewise, the hydrophilic seed extract cumulatively having these compounds were evaluated to show 92.8% cytotoxic to Hep2 laryngeal carcinoma cell line at 250 μg (Hamedi et al., 2014). The fresh, tender, healthy leaf and fruit of C. ternatea were screened for the anti-oxidant, antibacterial and cytotoxic efficiency by various solvent extractions. The collected sample were authenticated and provided by Dr. N. M. Ganesh Babu, Foundation of Revitalization of Local Health Traditions, Bangalore. The plant metabo- lites were extracted using five different polarity-based solvents by soxhlet method to derive all types of available compounds in these particular plant parts. In the phytochemicals investigation, the primary and secondary metabolites were recognized (Table I). An UV analysis was carried out from 800-200 nm using A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2018; 13: 349-352 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v13i4.38612 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor Table I Identification of phytochemicals from Clitoria ternatea extracts by various standard tests Petroleum ether Chloroform Ethyl acetate Methanol Aqueous Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Leaf Fruit Carbohydrate Fehling's test + + + + + + + + + + Molisch's test - + + + - - + + + - Protein Ninhydrin - + - - - + - + - + Biuret - - + + - - - - - + Phenols Ferric chloride - - - - + - - - - + Lead acetate - + - - - - - + + + Flavonoid - + + + + - - + + - Glycoside Salkowski's test + + - - + + - - - - Keller Kilani test - + - + - + + + + + Steroid - - + - - + - + + + Terpenoid - + - - - - - + - - Alkaloid Mayer's - + - + - - + - + + Wagner's - - - + - - - + - + Hager's + - - + - - - + + + “+” means present; “-” means absent Jasco V-670 (Japan) spectrophotometer to detect a specific wavelength of the available phytochemicals (data not shown). The peaks in the subsequent ranges 300-450, 510, 540, 605 nm were seen. The compounds obtained at 330-420 nm are due to the oxidation of polyphenols like phenolic acids, flavonoids and their derivatives like flavones, flavonols, phenylpropenes and quinines. Generally, 510 nm belongs to antho- cyanins, the more discussed anthocyanin from Clitoria flower is delphinidin (Andersen and Markham, 2005); peak of chlorophylls are developed at 600-660 nm. Through gas chromatography-mass spectroscopy ana- lysis, the following compounds were predicted (Table II) using their fragmented mass and retention time. Apart from that a compound 13-octadecenal (z), which had a earlier support from a plant Cassia auriculata in the same fabaceae family (Majumder and Paridhavi, 2013) was identified. Another compound lanosterol had prior report in the Lablab purpureus plant (Khare, 2008). An acyclic olefins compound, 1-pentadecene was also found in the leaf extract. Finally, an anti-cancer compound 2-methyl-z,z-3,13-octadecadienol was re- vealed (Hase et al., 2017). In Fourier Transform-Infra Red analysis, a peak at 3277 cm-1 refers to be the presence of –NH or –OH stretching bond that corresponding alcohol and phenol group were noticed. A peak at 2922 cm-1 was assigned as alkane asymmetric C-H stretching vibration of methyl and methylene group. The peak 2852 cm-1 represented to sp2 and sp3 stretching of C-H bonds of aldehyde and methyl group was predicted (Easmin et al., 2017). The C –O valence vibration of primary alcohols and polysaccharide were observed at 1066 cm-1 (Mak et al., 2013). The frequency 1587 cm-1 was denoted to be aromatic C–C skeletal vibrations respectively (Bonde et al., 2012). The peak at 1049 was distinguished as C–N stretch, an aliphatic amines and C-N band was found at 1396 cm-1. A DPPH scavenging assay was demonstrated for fruit and leaf extract were found to be 65% and 12.3% and their IC50 value was 75 μg/mL for the fruit extract, in case of flower extract 195.5 μg/mL (Zakaria et al., 2018) and 480 μg/mL for leaf were mentioned before. From an antibacterial activity, the well diffusion method was performed and found to be 0.5 cm for fruit extract against a pathogen E. coli and 0.2 cm against Staphylo- coccus aureus at a concentration of 200 μg. In case of leaf extract, 0.6 cm bacterial growth inhibition were formed at 150 μg. By the positive control ampicillin, the bacterial sensitivity effect showed nearly 1.2 cm. A protein called finotin having antimicrobial property was isolated and reported formerly from the seed extract of C. ternatea. The cytotoxic effect of methanolic fruit extract may be due to its enriched phytoconstituents were found to be 54 and 42% against the cervical cancer, HeLa. It was done at the concentration of 100 and 75 μg of extract by MTT colorimetric assay which can be used to measure the cell metabolic activity with the contribution of NADPH-dependent cellular oxidoreductases that redu- ces tetrazolium dye. A standard anti-cancer drug, cisplatin showed 84% cytotoxicity. As shown in the Figure 1A-D, a moderate anti-cancer activity was observed due to the change in their morphology that may lead to apoptosis. Similar results were reported previously and the formation of few formazan crystals might be due to the presence of lesser viable cells according to the drug dosages. The existing cyclic proteins like cyclotides from C. ternatea causes abnormal cells death by disrupting their cell membrane integrity i.e. membrane permeabilization. Cycloviolacin O2, the strongest cyclotide which was along with psyle cyclotides were proven to hold effective anticancer activity against doxorubicin-resistant MCF-7/ADR. The efficacy of doxorubicin was enhanced, when they were loaded along with that. Thus, they inhibit the proliferation of breast cancer cell lines (Sen et al., 2013). This study explains that the C. ternatea extract having prospective cytotoxicity can be incorporated as an anti- cancer drug apart from its antibacterial and anti- oxidant property. To the best of our knowledge, there was no previous data stating the major bioactivities of fruit extract so far referring to the others attributes on remaining parts of C. ternatea elevates/highlights fruit extract selected as successful or powerful natural medicine. Additionally, the predicted compounds can 350 Bangladesh J Pharmacol 2018; 13: 349-352 Figure 1: Human HeLa cervical carcinoma cell without treat- ment (A), at 75 μg (B), at 100 μg (C) of Clitoria ternatea extract and cisplatin-positive control (D) A B C D Table II Identification of various compounds available in crude extract Retention time (RT) Peak area (%) Fragmented mass (m/z) Compound structure 18.0 and 20.1 31.1 185.3, 213.3, 227.3 and 256.4 19.7 20.0 123.3, 138.3, 207.2, 235.3 and 264.3 19.9 and 21.2 10.7 281, 264, 207, 190, 135, 129 and 108 30.0 8.0 281.2, 313.4, 355.2 and 424.6 16.57 7.95 137.3, 138.3, 179.3 and 207.2 30.41 4.52 207 2-Methyl-z,z-3,13-octadecadienol 29.6 6.2 281, 267, 207, 191, 176, 103 24.9 11.8 281, 207,163 and 133 2,6-Lutidine 3,5-dichloro-4-dodecylthio Trimethyl[4-(2-methyl-4-oxo-2-phenoxy]silane L-Pentadecene Oleic acid 13-Octadecenal (z) N-Hexadecanoic acid Lanosterol Bangladesh J Pharmacol 2018; 13: 349-352 351 be isolated and further can be used as a lead molecule in cancer drug development as sometime the syner- gistic effect of the extract may not support or they prevent the toxicity towards cancer cells. We would like to thank Vellore Institute of Technology, Vellore for providing facilities and instrumentation required for our experiments such as UV Spec, GC–MS and FT-IR analysis. Subramanyam Deepika1, Chinnadurai Imma- nuel Selvaraj2 and Moorthy Anbalagan3 1Department of Biotechnology, School of Biosciences and Technology, Vellore Institute of Technology, Vellore 632014, India; 2Vellore Institute of Technology Center for Agricultural Innovations and Advanced Learning, School of Biosciences and Technology, Vellore Institute of Technology, Vellore 632014, India; 3Department of Integrative Biotechnology, School of Biosciences and Technology, Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India. Corresponding author: email: immanuelselvaraj@vit.ac.in References Andersen OM, Markham KR. Flavonoids: Chemistry, bio- chemistry and applications. CRC Press, 2005. Bonde SR, Rathod DP, Ingle AP, Ade RB, Gade AK, Rai MK. 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In vitro protective effects of an aqueous extract of Clitoria ternatea L. flower against hydrogen peroxide฀ induced cytotoxicity and UV฀induced mtDNA damage in human keratinocytes. Phytother Res. 2018; 2018. 352 Bangladesh J Pharmacol 2018; 13: 349-352 DatePrinted: This article was downloaded by you on: Nov 28, 2018