Detection of novel metaboities in garlic (Allium sativum L.) through in silico analysis and its validation
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Date
2016
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Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara
Abstract
Garlic belonging to Alliaceae family, is an important spice crop used from long back in Ayurveda and other forms of medicine for treatment of various ailments such as cancer, diabetes, cardiovascular diseases etc. The medicinal properties of garlic are mainly due to the presence of organosulfur compounds and several polyphenolic compounds. The major organosulfur compounds in garlic include alliin, allicin, l-γ-glutamyl-s-allyl-l-cysteine, s-allyl cysteine, s-allyl mercapto cysteine, allyl mercaptan, diallyl disulfide, diallyl trisulfide, allyl propyl disulfide, vinyldithiin etc. L-γ-Glutamyl-S-allyl-L-cysteine and alliin are the precursor molecules of all other compounds, the breakdown of which produce other compounds by several reactions with the help of enzyme allinase. Polyphenols like apigenin, quercetin, phloroglucinol, p-coumaric-acid, ferulic- acid, sinapic acid etc. also contribute to the medicinal effects of garlic. Though the medical literature for garlic is well established, the exact compounds from garlic against various diseases is not much experimented. The study entitled “Detection of novel metabolites in garlic (Allium sativum L.) through in silico analysis and its validation” was taken up to analyze the medicinal effects of important compounds in garlic by inhibiting the targets involved in lifestyle diseases such as cancerous, cardiovascular, arthritic, hypercholesterol and diabetic complications through in silico molecular docking analysis and validation through wet lab analysis. The experiment was performed using a commercial software Discovery Studio version 4.0 at Distributed Information Centre (DIC), CPBMB, KAU. For the study, 32 protein targets involved in different diseases were selected. Wet lab experiments such as in vitro cell line cytotoxicity test was conducted at Amala Cancer Research Centre, Thrissur and at CPBMB, KAU. Gene expression studies were conducted for validating the anti- cancerous properties of garlic compounds through RT-qPCR. Seventeen compounds from garlic both of allyl nature and polyphenols exhibited positive interaction with the targets selected for the lifestyle diseases. The important targets most inhibited by garlic phytocompounds were DNAtopoisomerase, Epidermal growth factor receptor (EGFR), Phosphoinositide 3- kinase Gamma (PI3Kγ) and Thymidilate synthase for cancer; Glucokinase (GK) and Dipeptidyl peptidase (DPP4) for diabetes; Cyclooxygenase 2 (COX2) and Nitric Oxide Synthase (NOS) for arthritis and inflammation; and Angiotensin Converting Enzyme (ACE) and HMG CoA reductase for blood pressure and cholesterol. It was found from the dock scores obtained, that s-allyl cysteine (SAC), alliin/ s-allyl cysteine sulfoxide (SACS), ferulic acid (FA) and p-coumaric acid (pCA) were found superior to other compounds and were found to inhibit most of the targets. These compounds (SAC, SACS, FA and pCA) which gave positive interaction in docking studies were procured in their pure form (upto 98% HPLC grade) from Sigma Aldrich and used for cell line studies. The three cancer cell lines HCT-15, Raw264.7 and L929 exhibited dose dependent cytotoxicity for all the four test compounds from garlic. The IC 50 value for true Allium compounds such as SAC and SACS were found in the range of 71-100 μg/ml, whereas for polyphenolic compounds such as FA and pCA, was found in the range of 145-222 μg/ml. HCT-15, the human colon cancer cell line showed more sensitivity to all the compounds and so it was forwarded for gene expression studies. EGFR was the gene selected for the study, which is most commonly expressed in human colon cancer. All the compounds from garlic showed down regulation of the EGFR gene dose dependently, except for SACS. Overall results from both in silico and wet lab studies indicated the medicinal effects of different compounds in garlic on various lifestyle diseases. Thus the present study gives a strong scientific background to highlight the health benefits of garlic.
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