Preparation And Quality Research Of Alliin Reference Substance

Garlic is the underground bulb of plants in the onion family Liliaceae. (Allium Sativum L.). More than 5,000 years ago, people started using garlic to prevent disease. Much research had shown that garlic is effective in lowering lipid level, preventing arteriosclerosis, coronary heart disease and cerebral thrombosis,anti-inflammatory, disinfection, fighting cancer and slowing aging process.Alliin (S-allyl-L-cysteine sulfoxide, alliin) is the most abundant organic sulfides in garlic. It exists in the cytoplasm of garlic cell and is very stable. When garlic cell is damaged, Alliinase enzyme is released from cell sap into cytoplasm. When Alliin comes into contact with Alliinase enzyme, it is quickly turned into a series of new compounds: Allicin and its similar substance. Allicin (double Allylic-thiosulfinates, allicin) is considered the most important active ingredient of garlic.However, it is quite unstable and is easily reacted. Therefore,it is possible to create real"high efficiency garlic agent"by extracting relatively stable Alliinase enzyme and Alliin from fresh garlic, creating appropriate drug formula using high purity of such raw material and releasing Allicin and its effective ingredient after releasing it into human body. As regulated by"Regulation of Drug Registration and Management"and"Principles of Chemical Drug Research Technology", it is required to identify the chemical structure of reference substance of effective ingredient and carry out research on its quality in order to register a new drug. Reference substance is standard material that is used for identification, examination and quantification. The quality of reference substance directly affects the result of analysis. Therefore, there is higher standard in terms of systematic and scientific requirement of reference substance research. In China, reference substance is examined and distributed by the Chinese Bio-Pharmaceutical Institution. Currently, the research on Allicin is becoming more and more popular both domestically and internationally. However, there is no supply of reference substance from legal channel domestically. Pharmacopoeia reference substance is available in the United States at the price of $1649 per 25 mg of L-(+) Alliin. It is expensive and inconvenient to purchase. Since Alliin reference substance is required as reference in pharmaceutical research and development of garlic agents, this research topic is thus created.Object: The object is to optimize the purification process of raw material that contains 90% of Alliin, determine the optimal purification condition and investigate the linear relationship between the melting point and content percentage of Alliin. It is also to investigate the process to use second cation exchange resin to purify raw material that contains 90% Alliin, compare it with the re-crystallization method and select the optimal purification methods. In the end, to produce high purity Alliin reference substance and carry out its quality research. Methods: The raw material contains 30% ~ 68% Alliin is purified to 90% Alliin by re-crystallization in this experiment. Using crystallization yield and crystallization purity as indicator, the impact on re-crystallization of crystallization solvent, solvent volume,crystallization temperature,pH value of crystallization and drying conditions are investigated respectively to determine the optimal crystallization condition. The Ninhydrin method is used to investigate the absorption time of ion exchange resins,the degree to which Alliin is eluted. Using Alliin melting point and yielding ratio as indicator, it is compared with the re-crystallization process.The content of amino acid of Alliin raw material is quantitatively measured using L-cysteine instrument. The high purity Alliin reference substance is produced using solvent system 1 and solvent system 2 respectively. UV and IR,MS,NMR Spectroscopy are used to verify its structure. Polarimeter is used to measure its specific rotation.Its three-dimensional structure is determined by combining data from reference literature. Its microstructure is observed by using transmission electron microscope. Its purity is measured using the HPLC method. Results:The re-crystallization method is determined to be the method of choice for raw material that contains 90% Alliin. The best purification conditions are 70% ethanol, pH 4.8 and slow cooling to 30℃for crystallization.The drying condition for crystallization is 50℃vacuum drying until constant weight.The melting point and content is proportional when Alliin content is 85% or greater.8.6g high purity Alliin reference substance with purity higher than 98% had been produced using the two solvent systems.After observing the stability of preparation process, solvent system 2 is chosen to prepare Alliin reference substance. It is eventually confirmed to be Alliin reference substance after verification of its physical and chemical properties and structure. Conclusion: Using 30% ~ 68% Alliin as preparation raw material, Alliin of 90% purity can be obtained after a step re-crystallization, and can be declared classⅠdrug raw materials as active ingredients. Using 90% Alliin as preparation raw material, optimization of two solvent system, high purity Alliin reference substance after two steps of re-crystallization. Solvent system1 reference substance by alliin content 99%, crystallization yield≥20%; Solvent system2 reference substance by alliin content≥99%, crystallization yield≥25%.The manufacturing process of Alliin reference substance is stable and feasible, achieved mass production, this manufacturing process of Alliin reference substance can be used in drug research and pilot production of qualitative and quantitative determination of the reference substance.