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A Comparative Study on Phyto-Chemical Investigation and Pharmacological Screening of Allium Sativum from Different Geographical Locations in India.”

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A Comparative Study on Phyto-Chemical Investigation and Pharmacological Screening of Allium Sativum from Different Geographical Locations in India.” International Journal of Research ISSN NO:2236-6124 “A Comparative study on phyto-chemical investigation and Pharmacological Screening of Allium sativum from different geographical locations in India.” Atul Kumar Singh1, Shriram Haribhau Kulkarni2, Arun Bhargav Jadhav3, Manisha Prashant Gajendragadkar4* 1) Postgraduate Scholar, Department of Homoeopathic Pharmacy, Bharati Vidyapeeth (Deemed to be) University, Homoeopathic Medical College & Hospital, Katraj – Dhankawadi, Pune, India. 2) Branch Head Vimta labs ltd, Bhakti Genesis, Wakad, Mulshi Dist.Pune - 411 057, Maharashtra, India. 3) Principal, Head of Institute Bharati Vidyapeeth (Deemed to be) University, Homoeopathic Medical College and Hospital, Katraj - Dhankawadi, Pune, India. 4) Professor Head of Department of Homoeopathic Pharmacy, Bharati Vidyapeeth (Deemed to be) University, Homoeopathic Medical College & Hospital, Katraj – Dhankawadi, Pune, India. Email id- [email protected] , [email protected], [email protected], [email protected] Abstract: Allium sativum is used as a medicine in complementary and alternative medicine. The raw garlic is collected from three geographical locations of India (Bihar, Maharashtra and Kashmir). A comparative study of Allium sativum having Allicin as a phytoconstituent was analyzed. LCMS/MS was used to analyse the Allium sativum samples and compared with the standard of Allicin SAC (S-allyl- cysteine) and the evaluation was presented with m/z value and retention time of standard compared with 3 samples of Allium sativum. The maximum content of Allicin was obtained in the sample of Maharashtra i.e. (1614.430ppm) when compared with standard Allicin at 2.5 minutes peak followed by sample of Bihar i.e. (1084.579ppm); sample of Kashmir i.e. (295.642ppm). With the obtained results, Allicin is comparatively found in more concentration in sample of Maharashtra and is therapeutically potent then the other samples of Allium sativum. Keywords: Allicin, Allium Sativum, LCMS-MS, m/z, Retention time. 1. Introduction Garlic is one of the most important bulb crop grown in the world. [1] The name garlic comes from an old Anglo – saxon word ‘gerleac’ which means ‘Spear leek’. [2, 3] Garlic is harvested in 1,437,690 area throughout the world and its annual production is 24,255,303 tonnes of dry bulb according to FAO, 2013. [1] It is a member of Alliaceae or Liliaceae family and botanical name is Allium Sativum. [4] It was first utilize as medicine by the sumerians. [2] Garlic has an apomictic nature which leads to the existence of extensive somatic mutation. [5] Garlic contains Enzymes, Vitamins, Flavonoids and certain Minerals. [2, 6] It is rich source of sugar, protein, fat, calcium, potassium, phosphorous, sulphur, iodine fiber and silicon. [6] The bulb contains a colorless, odorless and water soluble compound called Allicin. It is a major biologically active organosulfer compound of garlic which is formed by cutting or crushing the garlic clove, hence the potential of this plant has been assumed to be due to Allicin. [2, 6, 7, 8, 9] Allicin is effective towards most gram – Positive and gram – Negative bacteria. [10] Allicin can iduce apoptosis in cancer cells through oxidative modification of cellular sulfhydryl (thiol) groups. [8, 10] Allicin is a flavour component of Garlic. [11] Allicin were identified and quantified by using Liquid Chromatography and Mass Spectrography (LC/MS). [10, 12] Garlic contains about 0.1% volatile oil. Volume 7, Issue XI, November/2018 Page No:787 International Journal of Research ISSN NO:2236-6124 The oil is rich in sulphur, but contains no oxygen. Its constituents are diallyl disulphide (60%) diallyl- trisulphide (20%) allyl propyl disulphide (6%) with small quantity of diesel disulphide and diallyl polysulphide. [2,7] Garlic bulb comprises of 84.09% of water & 13.38% organic matter.[2] There are 20 types of sulphide compounds (eg allicin, methyl allyl trisulphide and diallyl trisulphide) reported in garlic. Seven organosulphur compounds like Alliin, soallin, methiin, cycloalliin, and gamma-1-glutanyl-s-methyl-l-cyseine, were also determined in it. [2, 7] In traditional medicinal systems of India i.e. Tibbi, Unani and Ayurveda garlic is recommended due to its healing properties. [5] In Charaka Smhita (1000 – 800 BCE) Garlic is recommended for treatment of heart diseases and arthritis. [5] Garlic has a great antioxidant activity. [13] It has anticancer, antiviral, anti-inflammatory, anti-diabetic, antimicrobial, cardio protective and immunomodulatory type of medicinal properties. [1, 2, 14] Garlic has Pharmaceutical effects and used to cure various diseases such as heart disease, arthritis, pulmonary complaints, diarrhoea, and worm infections. [5, 6, 9] It is used to prevent coronary thrombosis, atherosclerosis and stroke. [9] Garlic can reduce serum cholesterol level and inhibit cancer cell growth. [2, 9, 14] Garlic is a wonderful remedy for treating fever, cough, headache, stomach pain, haemorrhoids, asthma, bronchitis, both low and high blood sugar, hypertension and snake bites. [2] Garlic commonly used as spices and flavouring ager for foods and added taste of food. [6] The Aim of the study is to evaluate the concentration of Allicin collected from different geographical states i.e. Maharashtra, Bihar, and Kashmir by LC-MS method. 2. Materials and Methods 2.2 Samples collection: Samples of Garlic is collected from local farmers of that particular area from Bihar (gopalganj). Maharashtra (solapur), Kashmir. 2.3 Laboratory: Vimta labs Limited, Bhakti Genesis, Fifth Floor,S No. 245 H. No. 2B, Situated at Wakad, Tal Mulshi Dist.Pune - 411 057, Maharashtra, India. 2.4 Chemicals : Type A Water, Methanol 2.5 Standard- Standard of Allicin was procured from Sigma Aldrich. Reference standard of United state Pharmacopeia. 2.6 Glassware & Consumables: 100 ml glass beaker, Mortar and pestle, 20-200 µl Micropipette, 100-1000 µl Micropipette, 2ml Glass vials, 50ml tarson tubes 100 ml volumetric flask, C-18 HPLC column, 0.22µ nylon filters 2.7 Instruments Analytical Balance Vortex Mixer Centrifuge machine LCMS-8040 instrument Volume 7, Issue XI, November/2018 Page No:788 International Journal of Research ISSN NO:2236-6124 2.8 Preparation of Standard Stock Solution: Weighed 10.0 mg of standard and transfer into10.0mL individual volumetric flask, containing 2.0mL of methanol solvent and make up the volume to the mark with the same. Concentration by considering its purity and label it with obtained concentration and store at -20ºC. 2.9 Preparation of Calibration Standards: Stock Volume of Volume of Final Final conc. Label Conc.(µg/L Stock (µL) Dilutent(µL) Volume (µL) (µg/L) ) 1000 500 500 1000 500 CC4 500 500 500 1000 250 CC3 1000 100 900 1000 100 CC2 500 100 900 1000 50 CC1 2.10 Methods of Extraction- 1) Weighed the 10 gram of fresh garlic bulb using analytical balance then transfer into mortal and pestle and chopped in to a small cubes then add 50 ml of distilled water and Vortex for 5 min using vortex mixer, then transfer the all samples into 100 ml volumetric flask and make up the volume with type A water. 2) Transfer the 25ml extract into 50ml tarson tube and centrifuge for 10min at 10°C temperature, then filter through 0.22µ nylon filters and inject on LC-MS/MS. 2.11 Identification of garlic water soluble constituents by Liquid Chromatography Mass Spectrometry (LCMS/MS) LC-MS/MS Shimdazu LC 8040 system with an electrospray ionization (ESI) source was used to identify the individual compound natural source based on polarity and involatile compounds. Therefore this study provided LC-MS/MS [10, 12, 13, 15, 16] for identifying several constituents in Garlic water soluble. 3. Result Quantitative LCMS analysis of Allicin from 3 samples of Allium Sativum. Quantitative method:- External standard Id 1 m/z 130.65>41.05 Name Allicin- I Quantitative Method External standard Function F(x)=224.899*x-2377.96 Fit type Linear Zero through Not through Weighted Regression None Rr1=0.999659 RF SD: 1.548795e+001 RF%RSD: 7.32540 Volume 7, Issue XI, November/2018 Page No:789 International Journal of Research ISSN NO:2236-6124 ID#1Compound Name: Allicin-I m/z: 130.65>41.05 Sample Sampl Ret. Area m/z Height Conc. Name e ID Time Allicin Std 2.055 9218 130.65>41.0 512 51.563 Std50ppm 5 Allicin Std Std 2.036 20822 130.65>41.0 1089 103.156 100ppm 5 Allicin Std Std 2.038 52079 130.65>41.0 3078 242.138 250ppm 5 Allicin Std Std 2.042 110778 130.65>41.0 6156 503.144 500ppm 5 Figure:1 (A) The presence of Allicin standard at 50ppm with (m/z=130.65>41.05, tR=2.055min. Figure:1 (B) The presence of Allicin standard at 100ppm with (m/z=130.65>41.05, tR=2.036min. Figure:1 (C) The presence of Allicin standard at 250ppm with (m/z=130.65>41.05, tR=2.038min. Volume 7, Issue XI, November/2018 Page No:790 International Journal of Research ISSN NO:2236-6124 Figure:1 (D) The presence of Allicin standard at 500ppm with (m/z=130.65>41.05, tR=2.042min Linearity Graph:- Mass Spectrometry Quantitative Table Ret. Sample Name Sample ID Area m/z Act % Conc. Time I-130.65>89.20 Allicin Garlic 18-00358- II- 55.84 2.093 22014 1084.579 Bihar 001 130.65>69.20 8.35 I-130.65>89.20 Allicin Garlic 18-00358- 59.23 2.094 33930 II- 1614.430 Maharashtra 002 8.59 130.65>69.20 I-130.65>89.20 Allicin Garlic 18-00358- 59.19 2.086 4271 II- 295.642 Kashmir 003 0.00 130.65>69.20 Volume 7, Issue XI, November/2018 Page No:791 International Journal of Research ISSN NO:2236-6124 Figure: 2 (A) The presence of Garlic water soluble constituents in LC-MS were observed in full LC-MS chromatogram with (m/z) = 130.65>89.20, 130.65>69.20, for 5min Volume 7, Issue XI, November/2018 Page No:792 International Journal of Research ISSN NO:2236-6124 Figure: 2 (B) The presence of Garlic water soluble constituents in LC-MS were observed in full LC-MS chromatogram with (m/z)= 130.65>89.20, 130.65>69.20 for 5min.
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