US007 179632B2 (12) United States Patent (10) Patent No.: US 7,179,632 B2 Williams et al. (45) Date of Patent: Feb. 20, 2007 (54) PROCESS FOR THE PRODUCTION OF (56) References Cited ALLCIN U.S. PATENT DOCUMENTS (75) Inventors: David Michael Williams, Cardiff (GB); 5,741,932 A * 4/1998 Dressnandt et al. .......... 568.21 Chandra Mohan Pant, Cardiff (GB) 5,788,758. A * 8/1998 Sawada et al. ............. 106,493 (73) Assignee: Neem Biotech Ltd., Cardiff (GB) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this WO WO97.39115 10, 1997 patent is extended or adjusted under 35 WO WO 9739 115 A1 * 10, 1997 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 10/483,016 “Garlic: The Science and Therapeutic Application of Allium sativum L. and Related Species'. Second Edition, H. P. Koch and L. D. (22) PCT Fed: Jul. 4, 2002 Lawson, eds., Lippincott Williams & Wilkins, Philadephia, 1996, pp. 56-57.* (86) PCT No.: PCT/GBO2/O3O83 Kubec et al., "Sulfur-containing volatiles arising by thermal deg radation of alliin and deoxyallin.” J Agric Food Chem 45(9):3580 S 371 (c)(1), 3585, 1997.* (2), (4) Date: Jun. 21, 2004 The Merck Index, 10th Ed., M. Windholz et al., eds., Merck & Co., Inc., Rahway, NJ, 1983, p. 40.* (87) PCT Pub. No.: WOO3/OO4668 * cited by examiner PCT Pub. Date: Jan. 16, 2003 Primary Examiner Jon Weber Assistant Examiner—Rosanne Kosson (65) Prior Publication Data (74) Attorney, Agent, or Firm—Williams, Morgan & US 2004/O247711 A1 Dec. 9, 2004 Amerson, P.C. (30) Foreign Application Priority Data (57) ABSTRACT Jul. 4, 2001 (GB) ................................. O116361.7 There is disclosed a method for preparing allicin which (51) Int. C. comprises the following steps: (a) mechanically treating a CI2N 9/88 (2006.01) natural source of alliinase to release alliinase therefrom; (b) A6 IK 36/8962 (2006.01) contacting the mechanically treated allinase source with an C07C323/22 (2006.01) aqueous solution of allicin containing allin at a concentra C07C 7/10 (2006.01) tion greater than that found in raw garlic, whereby the alliin C07C 2/76 (2006.01) is enzymatically converted to allicin by the allinase released (52) U.S. Cl. ........................ 435/232; 424/754:568/22: from the alliinase source; and optionally (c) extracting the 585/601:585/833 resultant allicin into a low boiling point non-polar organic (58) Field of Classification Search ................ 435/232; solvent. 424/754; 568/22; 585/601, 833 See application file for complete search history. 19 Claims, 4 Drawing Sheets All to Acireactor d PyruvicAcid x Air o 0.20 ,4 3. Time ours U.S. Patent Feb. 20, 2007 Sheet 1 of 4 US 7,179,632 B2 C O s co Off 02 SLOW teaWared U.S. Patent Feb. 20, 2007 Sheet 2 of 4 US 7,179,632 B2 e S. S. s 3. 0. | | | | | | | S. \ || || 3 TNT 3 \ g HN o 9 8 uddug upoly U.S. Patent Feb. 20, 2007 Sheet 3 of 4 US 7,179,632 B2 0009 Q004. 0009 000; 000€ tudd up unv U.S. Patent Feb. 20, 2007 Sheet 4 of 4 US 7,179,632 B2 ra s 92 s is St S ao O)y s es s a 9. Z 2 s Wh S. S 3. e s o Spuesno UW US 7,179,632 B2 1. 2 PROCESS FOR THE PRODUCTION OF The use of an enzymatic method for producing allicin has ALLCN been described as seeming to be “attractive', see WO97/ 39115. However, the so-called "suicidal' nature of the This application is the U.S. national phase of International enzyme, namely that it is rapidly and irreversibly rendered Application No. PCT/GB02/03083, filed on Jul. 4, 2002, inactive by its own reaction product allicin, led the inventors which claims priority to United Kingdom Application No. of WO97/39115 to propose the use of an immobilized form GB 0116361.7, filed on Jul. 4, 2001. of alliinase which is not inactivated by allicin. The present invention relates to a process for the produc The present invention offers an alternative method for the tion of allicin from allin, and a method for the preparation production of allicin, which is simple and inexpensive. By of pure allicin. 10 the use of the process of the invention it is possible eco Garlic and onions are members of the lily family. Many nomically to synthesis pure pharmaceutical grade allicin. medical properties have been ascribed to garlic and onions The present invention also provides a method for the volume and they have been used in folk medicine for thousands of production of the ajoenes and dithins as a consequence of years. being able to volume produce allicin. Previous methods A large spectrum of medical properties has been ascribed 15 have relied on the production of synthetic allicin. to garlic, Allium Sativum, (Block E 1985) Sci. Am. 252 (3): According to a first aspect of the present invention, there 114-119). In modern times the interest in the therapeutic is provided a method for preparing allicin which comprises properties of garlic has been revived, and it is the object of the following steps: an increasing number of biochemical and clinical studies. (a) mechanically treating a natural source of alliinase to An extensive review of the beneficial properties of garlic release alliinase therefrom; extracts may be found in WO99/21008. (b) contacting the mechanically treated allinase source Garlic preparations are commercially available in the with an aqueous Solution of alliin containing alliin at a form of garlic oil, extracts, pills or tablets. Usually the concentration greater than that found in raw garlic, whereby preparation procedures of Such garlic preparations are the alliin is enzymatically converted to allicin by the allii unknown, and the composition and amount of their active 25 nase released from the alliinase source; and optionally ingredients are not defined. (c) extracting the resultant allicin into a low boiling point The active principles present in garlic have been found to non-polar organic solvent. be Sulphur-containing compounds. The principal component of a colourless oil obtained from steam distillates of garlic DESCRIPTION OF THE DRAWINGS extracts was shown to be allicin, an unusual Sulphur com 30 pound of formula CHS2O (thio-2-propene-1-sulfinic acid The following drawings form part of the present specifi S-allyl ester) (Cavallito et al., (1944) J. Am. Chem. Soc. 66, cation and are included to demonstrate certain aspects of the 1944–1954). Allicin was found to be a chemically unstable, present invention. The invention may be better understood colourless to straw coloured liquid. This liquid is thought to by reference to one or more of these drawings in combina be responsible for both the odour and much of the biological 35 tion with the detailed description of specific embodiments activity of garlic. presented herein. Although allicin is responsible for the Smell of garlic, a FIG. 1 shows the reduction in the reaction rate of alliinase garlic bulb exhibits little or no odour until it is cut or in Solutions containing high levels of allicin. crushed. The intact garlic clove does not contain allicin but FIG. 2 shows the formation of allicin for a given amount rather its odourless precursor alliin (+)(S-allyl-L-cysteine 40 of allin with increasing addition of garlic. Sulfoxide). This is converted to allicin, pyruvate and ammo FIG. 3 shows the formation of pyruvic acid during the nia by a C-S-lyase present in the garlic plant termed alliin conversion of alliin to allicin. lyase or alliinase (Stoll and Seebeck, 1949 Helv Chim Acta FIG. 4 shows a series of traces showing the growth of the 32: 197-205). Alliin and alliinase are found in different allicin peak and the concurrent disappearance of the alliin compartments of the garlic clove and the cutting or crushing 45 peak. of the clove releases the enzyme allowing it to come into Alliin exists in raw garlic at a concentration of around 0.4 contact with the precursor of allicin. to 0.9%, and varies significantly depending on the growth Allicin is unstable and breaks down into a number of conditions of the garlic bulbs, which can vary from year to different compounds some of which are thought to be of year. pharmaceutical use. A list of the various compounds present 50 Preferably, allin is used in the method of the present in garlic and their reported activity is given in Dr. Duke's invention at a minimum concentration of 1%, more prefer Phytochemical and Ethnobotanical Database for Allium sati ably of 5%, more preferably still of 10% and most preferably vum. The main compounds of interest, in addition to allin at a minimum concentration of 15%. and allicin, are the (E/Z)Ajoenes and the various dithins. Alliin is freely soluble in water and can therefore be used Despite the impressive effect of garlic, studies have been 55 in the method of the present invention at high concentra limited by several factors such as lack of controlled methods tions. Preferably, alliin is used at a maximum concentration and suitable double blind studies, and use of preparations of 50%, more preferably of 40%, more preferably still of with unknown amounts and chemical identification of the 30% and most preferably at a maximum concentration of active agent. Allicin has been shown to exhibit the beneficial 25%. properties ascribed to garlic and thus it would be useful to 60 Preferably, allin is used in the method of the present be able to produce allicin in controlled and known amounts invention at a concentration of between 10% and 30%, more for use as the active ingredient of pharmaceutical composi preferably at a concentration of between 15% and 25%.