J. of university of anbar for pure science : Vol.4:NO.2 : 2010 ISSN: 1991-8941 Determination of Alliin and Allicin in different types Garlic using High Performance Liquid Chromatography Mohammad J. Abdul Ghani University of Sallah Aldin - Collage of Basic Education Received: 10/1/2010 Accepted: 28/4/2010 Abstract:Alliin and Allicin products were measured and determined by ion-pair reversed-phase liquid chromatography (RP-LC) with UV detection at 210 nm. These two Compounds were extracted from various types of garlic with methanol / ethyl acetate and chromatographed on octadecyl silane column [ODS C18 (250 x 4.6 mm id)] with gradient elution from 0.01M phosphate buffer (PH=2.5) with 5M heptanslfonic acid (mobile phaseA) to 0.01M phosphate buffer (PH=2.5) acetonitrile(1:1) (mobile phase B). Allicin was eluted after Alliin. The results observed show that the concentration differs between the different types of garlic. The aqueous Iraqi garlic extract has the highest concentration of Alliin and Allicin (17.9 ppm, 0.9%),(23.94 ppm, 1.2%) respectively. But the lowest concentration of allicin was found in French garlic extract (0.56 ppm, 0.03%) while the lowest level of Alliin was (4.3 ppm, 0.22%) in Chinese garlic extract. Key words: Determination .Alliin . Allicin . Garlic . HPLC Introduction S Garlic (Allium Sativum), like othter plants, H2C S has an exquisite defense system composed of as CH2 many different components boosting human immune system. In order to protect itself from O insects and fungi, garlic enzymatically produces Allicin when injured. Allicin Allin (S-allylcysteine sulfoxide, percent Allicin was discovered in 1944 by Cavallito et composition: C 40.66%, H 6.26%, N 7.90%, O al., who first noted its potent antimicrobial 27.08% and S 18.09%) is constructed of an allyl activity [4]. Allicin is produced by an enzymatic group, a sulfoxide group, and the amino acid reaction when raw garlic is either crushed or cysteine ( contains SH rather than S=0). Alliin is injured. The enzyme allinase combined with biosynthesized from its parent ompound, S-Allyl Alliin and produces Allicin[5]. cysteine (deoxyalliin). [1] O H 2C Allin is quite stable in the absence of active S A lliinase OH alliinase, and it can also be found in cooked +H 2O garlic. It has been demonstrated as antioxidant O NH 2 activity. [2] A lliin Allicin is known as 2-propene-1-sulfinothioic acid S-2-propenyl ester, thio-2-propene-1- O sulfinic acid S-allyl ester, diellyl disulfide-oxide, + - S 2N H 4 +2 H 3C COO + H 2C diallyl thiosulfinate [3] percent composition: C S CH 2 44.4%, H 6.21%, 0 9.86%, and S 39.52%. P yrovate O A llicin Allicin has been reported to possess numerous J. of university of anbar for pure science : Vol.4:NO.2 : 2010 biological and biochemical activities. They ethanol:ethylether (1:1) into blender, and include beside antibectrial effects[4], reduction extracted twice with (10)ml of cool mixture of serum cholesterol and triglycerides[6], about {10 min) for each extraction. The extracts inhibition of platet aggregation[7]. were dried over anhydrous sodium sulfate and Because of its instability, allicin is not filtered. The extract was immediately subjected commercially available and can be conveniently to HPLC. synthesized by oxidation of diallyl disulfide with 4. Simultaneous qualitative and quantitative acidic hydrogen peroxide and purified using Si- determination of Alliin and Allicin by HPLC TLC[8]. [14]: Allicin can also be isolated from The isolated components were analyzed by ion- dichloromethane extract of garlic homogenated pair reversed- phase liquid chromatography with by C18-TLC. Asimple and sensitive method for UV detection, using an octadecyl silane column quantitation of this compound is still not with gradient elution.The operation conditions available. Indirect quantitation of allicin by are listed in table (1). conversion of either dially disulfide or allyl Results and discussion mercaptan followed by gas chromatographic Determination of Allicin In aqueous garlic (GC) analysis[9]. These GC and HPLC methods extract: all require allicin as an external standard [8,9]. Asimple and rapid HPLC method suitable for Han J.et al [10]described a spectrophotometric routine analysis of Alliin and Allicin, was method for quantitative determination of Allcin developed by Arnault l. et al.[15] using eluent [11,12] based on, that one molecule of Allicin containing an ion-pairing reagent (Heptane reacts rapidly with two molecules of cysteine to sulfonate) and a (150*3) mm column. Allicin form two molecules of S-allyl was eluted after Alliin and the synthetic mercaptocysteine(SAMC). The mechanism of reference compounds were characterized by the this reaction is not known. same chromatographic method using diod-array Unlike GC and HPLC methods[8,9], Han J. et al UV detector. [10] method does not require an allicin standard Addition of hydrochloric acid solution to garlic to quantify allicin and can be conveniently used extract will ihibit the formation of Allicin, and in to measure the total concentration of addition to this, adding of sulfite can be thiosulfinates in garlic extract determined, without interference of Allicin by Experimental reversed phase ion-pairing liquid 1. Adjustment of ODS column: chromatography with post-column detection For any chromatographic column to be [16]. maximally effective at retarding a sample Mochizuki E.N. et al.[17] reported, that Allicin molecule and more stability may be adjustment. and Alliin in garlic were determined Therefore, they are flushing with methanol or simultaneously by ion-pair reversed liquid acetonitrile once aweek under 0.1ml/min. Any chromatography with diod array UV detection. non polar compounds, which remain on the In these articles Alliin is extacted from garlic and reversed phase column, are easily removed by applies as external standard after purification by flushing with methanol or acetonitrile once a ion-exchange chromatography. day. Columns should not be back flushed unless The method that consists of using an octadecyl indicated in the column manual, nor should they silane (OSD) column with gradient elution from be stored in buffer, such as phosphate buffer, that 0.01M phosphate buffer (pH 2.5) with 5mM promotes microbial growth. heptane sulphonic acid (A) to 0.01M phosphate 2. Sample preparation: buffer (pH 2.5)-acetonitrile [(1:1),(B)] can be Samples rarely come in a form that can be used to analyze fresh garlic preparations, and injected directly into the instrument; some form health foods. of sample preparation usually is required. The limits of detection were between 1.7 and In this research, sample preparation includes any 9.40 ng for allyl methyl sulfide and dimethyl manipulation of the sample prior to analysis, disulfide, respectively, and percentage recovery involving techniques such as weighing, dilution, rates of aqueous garlic extracts ranged from concentration, filtration, centrifugation, 74.4% for the first to 90.3% for dipropyl derivatization, and chromatography. disulphide, using GC and MS [18]. 3. Sample preparation for seperationof Allicin By Applying adeveloped liquid chromatography extract [13]: technique based on florescent detection of 9- Frozen fresh garlic cloves (20 g) of each sample fluorenyl methyl chloroformate derivatives, were pleaded, chopped, blended with absolute Methyl-L-Cystein sulfoxide and 2-propyl-L- J. of university of anbar for pure science : Vol.4:NO.2 : 2010 Cystein sulfoxide were determined in garlic with J. Nutr., 131(3S), 951S- detection limits less down to 2.5mg/100g fresh 4S(2001). weight [19]. 2. Rabinkov et al., Biochim Biophys Acta, The major sulphoxide component that is found in 1379(2):233-234, Kourounaskis, P.N., garlic was (+)-S-allyl-Cystein sulphoxide Rekka, E.A. Res Commun Chem (>95%) can be determined by HPLC on two Pathol Pharmacol, 74(2:249-52(1991). spherisorb columns (OSD) in series with elution 3. Allicin. The Merk Index. 1999 by Merk&& of extracted garlic, allinase was inhibited by Co Inc, Whitehouse Station, NJ, USA, addition of 10mM of hydroxyl amine during Published on CD-ROM by extraction and eluted with 2M ammonium Chapman&& Hall/CRC. hydroxide through an Amberlite IR-120 anion 4. Cavallito C.J. and Bbailey J.H., Allicin, the exchange column [20]. antibacterial principle of Allium Kasuga S. et al.[21] found, in Japanese garlic, sativum.I. isolation physical properties that raw garlic juice contained 0.162% allicin but and antibacterial action, J. Am. no Alliin, heated garlic juice contained 0.266% Chem.Soc., 66, 1950-1(1944). Alliin and 0.001% Allicin, dehydrated garlic 5. Freeman F. and Kkodera Y. Garlic chemistry: juice contained 0.462% Alliin but no Allicin and stability of S-(2-propenyl)-2-propene-1- aged garlic extract contained 0.003% alliin but sulphinothioate(Allicin) in blood, no Allicin. solvents, and simulated physiological In our work we applied Mochizuki E.N. et al. fluids,J. Agric. Food Chem., 43, 2332- [17] method to determine simultaneous Alliin 8(1995). and Allicin by ion-pair reversed liquid 6. Augusti K. and Mathew P.T., Lipid lowering chromatography using UV detection under the effect of Allicin (diallyldisulfide-oxide) conditions listed in table (1). on long term feeding on normal rats, In table(2), the retention times, area, and Experienta, 30, 468-70(1994), Han J.C. concentration of Alliin and Allicin in standard, Lawson L.D., Han G., and Han P., Iraqi, Iranian, Lebanes, French, and Chinese Biochem., 157-60(1995). garlic extracts are listed. 7. Lawson L.D., Ransom D.K., and Huges B.G., It is seen that Iraqi aqueous garlic extract is high Inhibition of whole blood platelet- in Alliin (17.9 ppm, 0.9%), and Allicin(23.94 aggregation by compounds in garlic ppm, 1.2%) concentrations.