Study On The Extraction, Separation And Purification Of The Essential Oil, Fat-Soluble Antioxidants And Water-Soluble Antioxidants From Rosmarinus Officinalis L.

In recent years, the planting range and area of Rosmarinus officinalis L. is larger and larger with the enhancement of introduction in China. Compared with the increasing resource quantity of Rosmarinus officinalis L., the overall level of the processing industry is low. Because of the few number, the small scale of the specialized processing enterprise, and the simple and traditional processing technology, high-quality product is rare and the productive profit is low. Thus, the possibility to produce jointly variety of effective substances from Rosmarinus officinalis L. is small, the application of the products originated from Rosmarinus officinalis L. in some high-end fields is difficult.Under this background, the extraction, separation, purification and antioxidant activities of the essential oil, fat-soluble antioxidants and water-soluble antioxidants originated from the dry leaves of Rosmarinus officinalis L. is studied in this dissertation. Moreover, the study on the annual yield and the chemistry composition of the essential oil extracted from the fresh leaves of Rosmarinus officinalis L. gathered from Pingjiang county, Yueyang city, Hunan province is included in it. The main aim of this study is to structure the joint processing technology system of extrcting and separating the 3 kinds of effective substances exist in Rosmarinus officinalis L., i.e. the essential oil, the fat-soluble antioxidants and water-soluble antioxidants, directed by the content of the potential antioxidant ingredients. The study can provide basis and foundation for the high effective process of Rosmarinus officinalis L.. The main research results of the dissertation are as follows: 1. The essential oil is extracted by the Steam Distillation method (SD) from the fresh leaves of Rosmarinus officinalis L., and its chemistry composition is determined with GC-MS technology. (1) The yield of the essential oil. The yield of the same type of fresh leaf is different in different months, shows positive correlation with the atmospheric temperature and less correlation with the rainfall, and the highest yield appears in July.The yield of the essential oil of the narrow-type leaf is higher than which of the broad-type in the same month.(2) The chemistry composition of the essential oil. The ingredients in the essential oil from the same type of fresh leaf produced in different months are almost same, but the content of each ingredient is different. The ingredients in the essential oil from different type of fresh leaf are different even they are gathered in the same time.Moreover, there are 33 same ingredients between the two kinds of essential oil, but their respective content is different. The main ingredients in the two kinds of essential oil are a-pinene, camphene,1,8-borneol, borneol, camphor, verbenone, borneol acetate, terpineol and β-linalool (geraniol in the essential oil from the narrow-type leaf), and the total content of which is stable in different months and accounts for over 70% of the essential oil. The essential oil from Rosmarinus officinalis L. cultivated in Yueyang county belongs to the Tunisian/Morocco Type.2. Simultaneous Distillation Extraction method (SDE) was used for the first time to extract the essential oil from the dry leaves of Rosmarinus officinalis L., compared with the SD method. The extraction technology under the two kinds of extraction method is optimized, the effect of microwave to enhance the extraction under the SD method is studied, and the chemistry composition of the essential oil is determined with GC-MS technology.(1) The optimal extration conditions of the essential oil from the dry leaves of Rosmarinus officinalis L. by the SDE method and the SD method are as follows: granularity 120-140 meshes, solid to liquid ratio 1:10 g/mL, extractng time 2.5 hrs. Under the optimal extration condition mentioned above, the yield of the essential oil is 4.108%(dry basis) by SDE and 1.32%(dry basis) by the SD.(2) Under the same technology conditions, the extracting time can be shorted for 0.5 hr and the yield of the essential oil can increase from 1.32% to 1.45% when the extraction process by SD method is enhanced by microwave.(3) The ingredients in the essential oil extracted by SDE method are more than in the one extracted by SD. The main ingredients are almost same but their respective content is different in the two kinds of essential oil.3. The joint extraction and separation of the essential oil, fat-soluble antioxidants and water-soluble antioxidants originated from the dry leaves of Rosmarinus officinalis L. is studied, directed by the content of the potential antioxidant ingredients.(1) The SD method is adopted for the first time to extract the essential oil and the water-soluble antioxidants at the same time. The yield of the water-soluble antioxidants is up to 13.78%(dried basis) and the content of rosmarinic acid in it is 6.57%.(2) Guided by the content of rosmarinic acid, the water-soluble antioxidants is separated by the combined technology consisting of extraction with acetone and adsorption with macroporous resin.The impurities are removed effectively and the content of rosmarinic acid is raised from 6.57%to 9.42% when the water-soluble antioxidants are extracted with a gradient solvent system including of petroleum ether, ethyl acetate and acetone.The optimal technology conditions to separate and purify rosmarinic acid with NKA-Ⅱ-type macroporous resin from the acetone extract is obtained as follows:the content of the acetone extract in the adsorption solution 2.5%(equivalent to concentration of rosmarinic acid 1.09 mg/mL), pH value of the adsorption solution 3.0, adsorption temperature 20℃～30℃, the velocity of the adsorption solution in the column 2 BV/h, the dosage of the adsorption solution≤6 BV, the desorption solvent is 80%(V/V) ethanol-water solution, the desorption temperature 40℃～50℃, the velocity of the desorption solvent 1 BV/h, the dosage of the desorption solvent 3.5 BV. Under the optimal technology mentioned above, the end purified product is good water-soluble, odorless and yellowish powder with the content of rosmarinic acid of 44.397%(HPLC). The properties of the end product can meet the market requirements, and the purity has reached and even exceeded the domestic similar products.The optimal technology conditions to extract the fat-soluble antioxidants with organic solvent from the solid remainder after having removed the essential oil and the water-soluble antioxidants is obtained as follows:the concentration of ethanol 95%(V/V), the extraction temperature 80℃, the extraction time 1.5 h for one time and extracting twice, the solid to liquid ratio 1:13 g/mL, the granularity of the raw material 140-160 meshes. Under the optimal technology conditions mentioned above, the yield of the fat-soluble antioxidants is up to 19.2%(wet basis) or 22.0% (dry basis), and the contents of carnosic acid and carnosol are 6.302% and 9.547% (HPLC), respectively.(4) Guided by the content of carnosol or carnosic acid, carnosol and carnosic acid in the fat-soluble antioxidants are effectively separated by the combined technology consisting of the adsorption with macroporous resin and pH control method.The optimal technology conditions to separate and enrich carnosol with NKA-Ⅱ-type macroporous resin from the ethanol-water extract is obtained as follows:the content of the ethanol-water extract in the adsorption solution 1.5%(equivalent to concentration of carnosol 0.936 mg/mL), pH value of the adsorption solution 3.0～4.0, adsorption temperature 20℃～30℃, the velocity of the adsorption solution in the column 3 BV/h, the dosage of the adsorption solution<7.5 BV, the desorption solvent is 80%(V/V) ethanol-water solution, the desorption temperature 20℃～30℃, the velocity of the desorption solvent 3 BV/h, the dosage of the desorption solvent 13.5 BV. Under the optimal technology mentioned above, the end purified product is yellowish powder with the content of carnosol of 35.784% (HPLC).The content of carnosic acid is increased from 7.34%(HPLC) in the remaining solution after the adsorption to 20.24%(HPLC) in the end solid product(HPLC) by pH control method.(5) The content of carnosic acid is increased from 6.302%(HPLC) in the ethanol-water extract to 37.156%(HPLC) in the end solid product(HPLC) by pH control method.4. The antioxidative activities of 12 kinds of homemade extract originated from the leaves of Rosmarinus officinalis L. and 3 kinds of commonly used synthetic antioxidants, i.e. BHT, PG and Vc, are evaluated based on the reduction ability, the superoxide anion radical scavenging activity, the ABTS+ free radical scavenging activity and inhibition ability of lipid peroxidation in linoleic acid.In some respects, the homemade 12 kinds of extract originated from the leaves of Rosmarinus officinalis L. show good antioxidant activities, the activities of some of which even close to or exceed the activities of the 3 kinds of commonly used synthetic antioxidants. The content of the potential active substances in some kind of extract show positive correlation with the antioxidant ability of which.