Study On Extraction And Separation Of Active Components From Antler Velvet And The Antioxidant Properties

Antler velvet, as a kind of traditional Chinese medicine, is unossified horn cut from male deer, which belongs to the Cervidae, and has long been recognized as one of the most effective and powerful invigorants for strengthening the kidney, sexual-reinforcing and pro-longing life. The pharmaceutical analysis shows that there are many important active components in antler velvet. Among these compositions, estradiol and progesterone have effectiveness in the prevention and treatment of female climacteric syndrome; hypoxanthine, uracil and uridine have inhibitory effect on monoamine oxidase; insulin-like growth factor-1 (IGF-1) is a sort of somatomedin, which have biological functions in vivo or in cells and tissues cultured in vitro. IGF-1 has special effect on diabetes mellitus and its complications.The traditional processing methods may lose some active components and can not make use of the active components effectively. Therefore, it is significant of utilizing advanced technology to extract, separate and analysis the effective components from antler velvet for scientifically utilizing the precious antler velvet materials, enhancing their subjoin value and promoting the modernization of Chinese traditional medicine.The analysis methods have been established for separately determining the sex hormones (i.e. estradiol and progesterone) and biological bases (i.e. uracil, hypoxanthine and uridine) in antler velvet. The refluxing extraction method was optimized with methanol as extraction solvent, and the radioimmunoassay method was utilized to determine the content of estradiol and progesterone in antler velvet. Meanwhile, the HPLC method was used to simultaneously and rapidly determine the contents of biological base components in antler velvet. The experimental results indicate that the uracil, hypoxanthine and uridine were well separated under the optimum HPLC conditions. The method not only presents good resolution, but also shows good precision and reproducibility. The average recoveries of the three biological base components were found in the range of 98.39%-103.4%. Based on the established methods the contents of both sex hormones and the biological bases in antler velvet were determined, which provided the basic data for getting the recovery of the components.Supercritical CO2 extraction in the presence of cosolvent was used to extract the active sex hormones from antler velvet. The effects of ethanol concentration, flow rate of co-solvent, extraction pressure, temperature, and extraction time on the extraction yield and content of estradiol and progesterone were studied, and the optimal conditions of supercritical CO2 and cosolvent extraction were obtained. Then, ultrasonic technology was used to further extract IGF-1 from antler velvet residue. The effects of pH value of solvent, the volume of solvent, the extraction temperature and extraction time on the activity remaining of IGF-1 were investigated, and the effect of supercritical CO2 and cosolvent extraction conditions on the activity remaining of IGF-1 was discussed. Finally, the optimal extraction conditions were determined. The experimental results show that the recovery of estradiol and progesterone was above 88 % when 75 % ethanol was used as co-solvent under the conditions of extraction pressure of 30 MPa, temperature of 35°C, extraction time of 30 min and CO2 consumption of 15 L/g at the flow rate of 2.0 L/min. The recovery of estradiol by SFE was 1.9 times of that by refluxing extraction. The recovery of progesterone by SFE was 1.5 times of that by refluxing extraction. The consumption of solvent was only 1/6 times of that by refluxing extraction. A high activity remaining of 93.68 % of IGF-1 in residue was obtained. Additionally,the contents of protein,amino acids and polysaocharides in the extracts obtained from antler velvet residue with ultrasonic extraction methods were determined.The extracts obtained from antler velvet with above SFE method contain not only the active components, such as sex hormones, biological bases, p-hydroxyl- benzaldehyde and phospholipid, but also quite amounts of cholesterol, which could have side effect on the human health if it existed in the extracts as health products or drugs. Therefore, the investigation of cholesterol removal from the extracts of antler velvet using saturated water solution ofβ-cyclodextrin was confirmed using single factor experiment method. And the ratio ofβ-cyclodextrin to the extracts, the inclusion temperature, inclusion time and the concentration of extract were investigated. The experimental results indicate that the removal rate of cholesterol was up to 91.64 % under the optimum conditions, while the retention rate of three biological bases were 77.31 %, 82.58 %, 85.19 % respectively. The retention rate of estradiol and progesterone were 90.30 % and 46.69 %.In order to investigate the in vitro antioxidant activity of antler velvet extract prepared by supercritical extraction, the antioxidant properties (including hydroxyl radical-scavenging by inhibition of lipid peroxidation from lipoprotein induced by iron, phenanthroline-Fe(II) oxidation and the ability of the extract to protect 2-deoxy-D-ribose against hydroxyl radical-mediated degradation) were presented in this study. The results were compared with that of Vc solution, 75 % ethanol refluxing extract and methanol refluxing extract. It is showed that the antler velvet extracts demonstrated activity in the three antioxidant assays in a concentration-dependent fashion. The supercritical extract technology showed obvious advantage, and the antioxidant activity of SFE extract was obviously higher than that of traditional refluxing extracts and Vc solution. Therefore, it may provide a foundational data to discuss the anti-aging function mechanism of antler velvet.Uracil is an important component in antler velvet, which has inhibitory effect on monoamine oxidase. The solubility of uracil in supercritical carbon dioxide was determined with dynamic method at temperatures (308.15 to 348.15) K and various pressures in the range of (15 to 40) MPa. The effects of extraction temperature and pressure on solubility were studied. The experimental data were also correlated. The experimental results indicate that the crossover point was observed around a pressure of 15 MPa. The experimental data were correlated by empirical equation (i.e. Gordillo equation and Abolghasem equation), M-S-T equation based diluted solution theory, modified Ziger-Eckert equation, respectively. The results show that Gordillo equation with five parameters has the lowest correlation error of AARD of 9.8 % among the four equations. However, the modified Ziger-Eckert equation with three parameters has higher error because the solubility parameters in it need to be estimated,. Moreover, the solubility data of uracil were collected by the Chrastil equation, modified Chrastil equation and solvent-solute clusters equation which were derived from the solvation concept. The results show that the collection result of modified Chrastil equation with AARD of 10.1% was better than that of Chrastil equation. But the solvent-solute clusters equation has a high correlation error. The dimensionless dimension was introduced, and new collection equation which we named as dimensionless new association equation was established, which has seven parameters. The lowest collection error of AARD of 9.2 % was obtained when it was used to correlate the solubility data of uracil in supercritical carbon dioxide. The above research can supply the basic data for the theoretic research on supercritical fluid technology.