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Separation and Purification of Alliinase and Alliin from Garlic (Allium Sativum)

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Separation and Purification of Alliinase and Alliin from Garlic (Allium Sativum) Journal of Academia and Industrial Research (JAIR) Volume 2, Issue 11 April 2014 599 ISSN: 2278-5213 RESEARCH ARTICLE Separation and Purification of Alliinase and Alliin from Garlic (Allium sativum) Thapa Mallika1,2, Elshareif Omer1,2 and Zhang Lianfu1,2,3* 1School of Food Science and Technology; 2National Engineering Research Center for Functional Food; 3State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China [email protected]*; Tel: +8651085917081; Fax: +8651085917081 ______________________________________________________________________________________________ Abstract Two bioactive components, Alliinase and Alliin from garlic were successfully separated in the present investigation. Alliin was separated using microwave-assisted extraction followed by purification of garlic enzyme (Alliinase). This approach for extraction for the substrate (Alliin) illustrated higher activity when brought in contact with alliinase. The result obtained at optimum extraction conditions were analyzed by HPLC. The extraction efficiency of Alliin was 89.46±0.65%. Subsequently, Alliinase was extracted using different buffers at 4°C. The sodium phosphate buffer with the addition of EDTA, NaCl and Glycerol had highest stability and considerable activity. The total percentage of 25% (w/v) Polyethylene glycol 6000 (PEG 6000) was found to have higher activity with the addition of dual frequency 15% (w/v) followed by 10% (w/v). The freeze dried Alliinase was found to have recovery rate of >60% at optimum temperature of 31°C at optimum pH of 6.1 buffer system with natural substrate. The stability the garlic enzyme increased 10% by freeze drying. However, as the temperature increased its stability declined. The garlic bioactive compounds obtained in this study have a great potential in pharmaceutical industry when used in the form of tablets or capsules. Keywords: Allium sativum, garlic, alliin, alliinase, polyethylene glycol, enzyme stability, bioactive compounds. Introduction as well as in cell culture and callus tissues (Lancaster Garlic (Allium sativum) is a widely distributed plant used and Collin, 1991). Alliinase is extremely abundant in throughout the world not only as a spice and a food, but garlic tissue, consisting of at least 10% of the total clove also as a folk-medicine. Previous studies show that many protein (Van Damme et al., 1992; Ellmore and Feldberg, Allium plants other than A. Sativum and A. cepaare of 1994). The enzyme from bulb tissues is a glycoprotein great importance due to their uses as flavoring agents, containing 6% carbohydrate and exists as a dimer of antioxidants, fragrance and therapeutics (Stajner et al., two subunits of MW 51.5 kDa each (Shimon et al., 2007). 2006). The antioxidant activity of Allium species is due to The reaction catalyzed by alliinase, which requires a variety of sulphur-containing compounds and their pyridoxal phosphate (PLP) as a cofactor, is categorized precursors, but it is also related to other bioactive as a â-elimination-deamination reaction involving an compounds such as polyphenols, dietary fiber and aminoacryl intermediate bound to PLP (Tomofumi et al., microelements (Nencini et al., 2007). Other studies show 1998). that allicin prevents the development of the atherosclerotic process, reduces serum cholesterol, Alliinase is the enzyme that initiates the conversion of the normalizes lipoprotein balance, decreases blood alkyl cysteine sulphoxide flavour (ACSO) precursor pressure and has antithrombotic and antiinflammatory (alliin) to allicin and its derivatives. Alliinase is located in activities (Sela et al., 2004; Miron et al., 2006). Studies the vacuoles of vascular bundle sheath cells which are indicated that garlic is effective in protecting against located around the phloem (Ellmore and Feldberg, numerous diseases because of its cholesterol 1994), rather than the abundant storage mesophyll lowering, lipid lowering, antihypertensive, antidiabetic cells that contain the CSOs (Koch and Lawson, 1996) and antihyperhomocysteinemic characteristics. Other which may be linked to the dynamic re-mobilization of biological properties of garlic include antioxidant, CSOs during development. This contrasts with onion antimicrobial, anticarcinogenic and antimutagenic where both alliinase and CSOs are present in all activities (Reutar, 1995; Koch and Lawson, 1996; cells, respectively in the cell vacuoles and cytoplasmic Kuettner et al., 2002). The general composition of the vesicles (Lancaster and Shaw, 2000). Garlic alliinase fresh garlic bulbs is found to be 84.09% H2O, 13.38% more rapidly hydrolyzes (+)-alliin, a naturally organic matter and 1.53% inorganic matter. Studies on occurring substrate for the enzymatic synthesis of allicin, the distribution of cysteine sulfoxides (CSOs) in Alliums than (-)-alliin (Stoll and Seebeck, 1951; Lancaster show that they are found in a range of tissues, including and Collin, 1981; Kuettner et al., 2002; Shimon et al., leaf, bulb, root and flower scapes (Briggs et al., 2002) 2007). ©Youth Education and Research Trust (YERT) jairjp.com Thapa Mallika et al., 2014 Journal of Academia and Industrial Research (JAIR) Volume 2, Issue 11 April 2014 600 Alliinase (Cys sulfoxide lyase, alliin lyase, C-S lyase; Alliin preparation EC 4.4.1.4) from garlic (Allium sativum) is an enzyme Microwave-assisted method: Alliin was prepared that uses pyridoxal-5-phosphate (PLP) as a cofactor to according to the method described by Li et al. (2011) catalyze the conversion of a non-protein amino acid alliin with slight modifications. About 50 g of freshly peeled (þ) (Sallylcysteine sulfoxide) to allicin (diallyl garlic were taken and were microwaved for 90 sec, thiosulfinate), pyruvate and ammonia as shown in the 750 W for permanent deactivation of the enzyme. following scheme: In addition of 80 mL ethyl acetate for the removal of fat soluble components, they were blended for 5 min with the lab blender. The so formed homogenate was left still for an hour and then was centrifuged 8000 rpm for 5 min to separate the layers of organic solvent mixed along with the crude garlic oil. As the supernatant was discarded, the precipitate is then taken for extraction. Water was used as extracting solvent. Vacuum filtering is necessary to get the final product. Chemical method (MCW): Alliin was prepared according to the method described by Yanhui et al. (2008) with slight modifications. The composition of 50 mL MCW Alliin and alliinase precursors must remain separated (Methanol, chloroform and water) was prepared in the prior to consumption to preserve the allicin potential. ratio of 12:5:3. About 20 g of garlic was immersed in the The S-alk (en) yl-L-cysteine sulfoxides (ACSO) MCW solution for 12, 24, and 48 h respectively for substrates are located in the cytoplasm, while alliinase is preparation of 3 different samples. With the addition of compartmentalized in the vacuole until cell rupture 4.5 mL chloroform and 5.5 mL water per 10 mL of MCW (Lancaster and Collin, 1981). The production of allicin solution, the samples were left at stationery position until from alliin and alliinase is hampered by the instability of the sharp demarcation was seen in separator funnel. both enzyme and allicin and their tendency to aggregate The water solution was extracted and moved to rotary (Miron et al., 2006). Most of the supplements are either evaporator. The final solution was then diluted to garlic powder or the allicin derived from garlic or garlic 100 mL. extracts. However, allicin is not suitable during the intake due to its high instability. But for the pharmaceutical Hot water method: Alliin was prepared with slight utilization, deriving of pure garlic compounds could be of modification as mentioned by Wei and Luo (2012). higher aptitude. The objective of this study was to isolate Pre-cooled garlic cloves were used and were boiled for two bioactive compounds primarily for pharmaceutical 15 min. The crushed garlic was then filtered through four purpose. Alliin was isolated using various methods from layers of cheese cloths. Acetone was added with the the fresh garlic. The method with higher activity and ratio of 1:4 to the initial volume. The homogenate was lesser toxicity was considered as the potential substrate centrifuged at the speed of 8000 rpm for 15 min. in enzyme purification procedure. Additionally, the The precipitate was discarded and the supernatant process of protein sedimentation using PEG 6000 was obtained was concentrated to a certain volume. studied for the enhancing refinement of alliinase followed by dialysis and freeze drying. Sonication: The preparation of this natural substrate was done using 20 g of pre-cooled garlic cloves. They were Materials and methods immersed in 100 mL of methanol for 30 min. The slurry Chemicals: Alliin standard (Shanxi Ciyuan Biotech), was then prepared by multiple sonications and Pyridoxine 5'-phosphate (PLP), Sodium Pyruvate, Bovine centrifuged at 8000 rpm for 15 min. The supernatant serum albumin (Standard, Ourpharm, Shanghai), Ethyl obtained was used as natural substrate. acetate, Methanol and EDTA. All the chemicals and solvents unless mentioned were bought from Sino Pharm Preparation of substrate (Alliin): About 50 g of freshly chemical reagent and were of analytical grade. peeled garlic were taken and microwaved for 90 sec, 750 W for permanent deactivation of the enzyme. Plant material: Garlic bulbs were purchased from local To remove fat soluble components, 80 mL ethyl acetate markets in Wuxi, Jiangsu, China. The bulbs were stored was added and blended for 5 min with the lab blender. in the refrigerator at 4°C before use. The so formed homogenate was left still for an hour and then was centrifuged at 8000 rpm for 5 min to separate Equipments: WD750B Microwave oven, UV-Vis3000 the layers of organic solvent mixed along with the crude spectrophotometer, Vortex, KJ-300 Ultrasonic bath, and garlic oil. As the supernatant was discarded, the Waters 2695 HPLC system connected to Waters UV-Vis precipitate is then taken for extraction. The extracting detector and (Nacalai Tesque Inc., Japan), R-205 Rotary solvent is water in the solid-liquid ratio1:5, at 35°C for evaporator.
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