The New Resourse And Research Of Anti-thrombus Effects Of The α-linolenic Acid

Theα-linolenic acid (ALA) as an essential fatty acid has important physiological activities to keep health of human body. A lot of fundamental researches , epidemiologic surveys , animal experiments and clinical observations in international medical science and alimentology group have indicated that ALA has many aspects of physiological functions as below,such as preservation of cardiovascular and cerebrovascular disease,inhibition of cancer generation and metastasis , inhibition of allergic response ,anti-inflammatory effect, inhibition of ageing,enhancement of intelligence and protection of sight etc.. Consequently,ALA is an important biological nutrition ingredient. Respecting of analysis of domestic and overseas research progress about ALA,the amount of Pericarpium Zanthoxyli seed (bunge pricklyash seed,BPS) has reached more than 4000 tons only in hancheng city of shannxi province,and 120,000 tons in China. Owing to the fact that BPS is the off-fall of Pericarpium Zanthoxyli,therefore,purification of high purity ALA from BPS is a technology on utilization of waste material and it also helps to benefit the nation and the people. The reports on purification of high purity ALA from BPS were not found in domestic and external documents.To explore new type ligneous cerolein resource of ALA from BPS and research effect features of antithrombotics the ALA and KO-BPS to human body,We have finished as follow research.ObjectiveTo extract KO-BPS(to be near essential unsaturated fatty acid) from BPS and cleansing ALA from KO-BPS,make the contents reach more than 95%. To testified the anti- thrombus activity of high purity ALA and mix fatty acid with certain ALA content. To compare the relationship between the content and curative effect of ALA one step further to supply feasible data for exploitation of new resources.Methods1 Technology research of extraction KO-BPS from BPS1.1 Extracting KO-BPS by expression technology Expression technology was adopted to extract KO-BPS from BPS. The extraction yield was determined. The content of ALA was determined by HPLC.1.2 Extracting KO-BPS by chemical method Chemical method was adopted to extract KO-BPS from BPS according to ALA polarity. The extraction yield was determined. The content of ALA was determined by HPLC.1.3 Extracting KO-BPS by SFE-C02 method SFE-C02 method was adopted to extract KO-BPS from BPS. The extraction yield was determined.The content of ALA was determined by HPLC.1.4 Comparison and analysis of three kinds of technologies Compare the extraction efficiency by three kinds of technologies and the content of ALA in the KO-BPS extracted from BPS. Consider the feasibility of industrial production and select the optimized technology.2 Extracting high purity ALA from KO-BPS We have used four steps of extracting high purity ALA from KO-BPS according to physico-chemical property of ALA and KO-BPS and make the content of the ALA reached more than 95%.2.1 Saponifying and acid hydrolysis Transform ALA from glyceride state to free state using Saponifying and acid hydrolysis. 2.2 Gradient freezing Gradient freezing method was used to separate out subtotal saturated fatty acid gradually according to principle of different dissolubility under the same temperature between saturated fatty acid and unsaturated fatty acid.2.3 Urea clathration According to the principle that barrel-shaped Urea molecule can inclus saturated fatty acid and part monounsaturated fatty acid,one step further to separate and remove saturated fatty acid and part monounsaturated fatty acid.2.4 Silver nitrate complexation According to the principle that Silver nitrate molecule can complex with molecule with delta,and the more delta the better complexing power,as an triene acid,ALA can complex with Silver nitrate to reach to goal of separation and purification.3 Activity research of Anti- thrombus on the ALA3.1 Determination of bleeding and clotting time After mice were continuous intragastric gived with different dosage of the ALA and admixture unsaturated fatty acid(ALA:LA=1:1)for 10 days,the bleeding and clotting time was determined by cuted off the tail of mice.3.2 Thrombotest in vivo induced by CP-AD After mice were continuous intragastric gived with different dosage of the ALA and admixture unsaturated fatty acid(ALA:LA=1:1)for 10 days, they were used by CP-AD to induce thrombogenesis to cause mice in vivo pulmonary embolism. Persistence of every group of mice was determined.3.3 Thrombotest of artery-vein alternative pathway Rats mould of artery-vein alternative pathway were builded by operation and humid weight of the thrombus were weighed, after they were continuous intragastric gived with different dosage of the ALA and admixture unsaturated fatty acid(ALA:LA=1:1)for 10 days.3.4 Platelet agglutination test with nephelometry in vitro We have determined the platelet aggregation of blood in rate with nephelometry in vitro, after they were continuous intragastric gived with different dosage of the ALA and admixture unsaturated fatty acid(ALA:LA=1:1)for 10 days. Results1 Extraction of BPS-oil from BPS According to aggregate analysis and comparison of three methods of extraction of KO-BPS from BPS, extraction efficiency,the content of ALA, color and luster of the obtained oil, and clarity etc. were investigated. Moreover, we considered the productive feasibility of industrialization and finally chose the traditional squeeze method to extract the KO-BPS from BPS.2 Extraction of high purity ALA from KO-BPS BPS was the free mixed fatty acid to obtain by saponifying acid hydrolyzed. High purity ALA was obtained by gradient freezing, urea inclusion and silver nitrate complexation. The content of ALA detected by HPLC reached up 95.6%.3 Anti- thrombus activity research on ALA 50mg/kg,100mg/kg and 250mg/kg of high purity ALA and mixed unsaturated fatty acid (ALA:LA=1:1) were intragastric administrated in mice for 10 days. The results revealed that bleeding and clotting time was significantly prolonged(P<0.01),the death rate of mice pulmonary embolism induced by collagen protein Adrenaline was obviously cut down(P<0.01),35mg/kg,70mg/kg and 175mg/kg high purity ALA and mixed unsaturated fatty acid were intragastric administrated in mice for 10 days. The deposition of platelet in artery and vein alternative pathway chorda serica chirurgicalis and platelet aggregation in vitro of rats were obviously inhibited.Conclusion1 The BPS could be used as a new woody lipid resource of extracting the ALASqueeze technology is an ideal industrialized productive method. The content of ALA and other unsaturated fatty acid in lipid can reach higher than 80%. It has an important practical significance in one step further to exploit new edible constitutional vegetable oil, especially ALA and linoleic acid (LA) as essential nutrient and develop health protective or therapeutical new drugs.2 The best condition of winterization of extracting the ALA The best condition is the mixed fatty acid:95% alcoh=1:1, 24 hours in -10℃,Duplicate test of the optimized winterization Method,the content of ALA and the yield of oil can reach 39.90% and 70% respectively after winterization.3 The best condition of Urea clathrationThe best condition of Urea clathration is the mixed fatty acid:95% alcoh:urea=1:2:1, 16 hours in -12℃; Duplicate test of the optimized condition, the content of ALA and the yield of oil can reach 47.37% and 59.92% respectively after clathration.4 Silver nitrate complexation is key techniqueFor increase the content of ALA,the content of ALA can reached 90% and the yield of oil is 20.18% after Silver nitrate complexation, and silver nitrate can use repeatly after reclaiming.5 Detection and new ideasWe first time procure the pure ALA with 98.6% as an unsaturated fatty acid extracted from BPS, and the technique for extracting and depurating the pure ALA had be patented by SIPO in PRC(No. ZL200510096193.6). Second,discover anti-platelet aggregation and thrombolytic pharmacologic action of the pure ALA and admixture unsaturated fatty acid (ALA:LA=1:1) extracted from BPS and presence dose-effect relationship. Third,Discovered when ratio of the ALA:LA in admixture unsaturated fatty acid is 1:1,and anti-thromb pharmaco-activity surpass the same dosage of purified ALA.