Ginseng Protein Activity

Ginseng(Panax ginseng C. A. Meyer.) is a traditional Chinese herbal medicines and it has high medicinal value and wide range of pharmacological effects. In this study, we conducted experiments on macromolecules of which had varied activities while not being further studied. The biological activity of these macromolecules-namely ginseng proteins and peptides, had been tested via modern separation and purification technology, analysis and identification technology and detection of activity in vitro and in vivo technology in order to provide scientific evidences and technical support for optimizing the process of preparing ginseng, exposing the clinical material base of ginseng and developing a series of ginseng products including innovative drug and health foods etc.1,Using in-gel activity staining method of PAGE electrophoresis to study the activity of 22 kinds of isozyme among 5 different enzyme systems consisting of oxidoreductase system, hydrolase system, transferase system, catenase system and isomerase system in wild ginseng, ginseng in forest and cultivated ginseng. Results show clear isoenzymes bands of 13 kinds, which participate in 11 kinds of fresh ginseng, 8 kinds of dry ginseng, 9 kinds of wild ginseng, 8 kinds of ginseng in forest. The peroxydase(POD), catalase(CAT), malic dehydrogenase (MDH),Superoxide dismutase (SOD), Esterase (EST), acid phosphatase (ACP) and Amylase (AMY) in all ginsengs; lactate dehydrogenase (LDH), cytochrome oxidase (CYT) and glutamic oxaloacetic transaminase (GOT) only in fresh ginseng; phosphoglucomutase (PGM) and glucose-6-phosphate isomerase (GPI) only in wild ginseng; 6-phosphogluconate dehydrogenase (PGD) in fresh and dry ginseng. Differences in enzyme activity of four kinds of ginseng suggest that there are some quality differences in ginseng because of differences of cultivation methods, processing methods, which play a certain reference of the selection of species of ginseng and clinical application.2,Using colorimetric technology of activity detection to test the activity of POD, CAT, SOD, MDH(oxidoreductase system) and EST, ACP, AMY (hydrolase system) which of them appeared to be high in activity in ginseng. Then to make comparison on enzyme activity of ginseng from different sources(10 sources in Jilin province ), with different parts (rhizome,taproot and fibrous root) and of different ages (4 years and 5 years) , respectively. It turned out that ginseng from different sources and with different parts did show a great difference in enzyme activity among each group. And ginseng of different ages had but not so much difference in enzyme ability. Differences in enzyme activity of ginseng from different origins suggest that there are some quality differences because of cultivation regional difference, which provide certain scientific reference for ginseng standardized planting; Differences of the enzyme activity in ginseng different parts show that various enzymes in the body of the distribution are different ,and the role of reed head, main root and fibrous roots can not be ignored, which has laid a theoretical foundation for medicine and clinical screening of the ginsengs; Differences of enzyme activity in 4 and 5 years old ginseng, show that ginseng can be harvested in 4 years or 5years.3,Using method of neutral buffer extraction and ammonium sulfate fractionation to extract crude enzyme from ginseng and then using isoelectric point precipitation, SP ion-exchange chromatography, DEAE ion-exchange chromatography, G-75 gel filtration column chromatography, high performance gel filtration chromatography(HPGFC) and matrix-asisted laser desorption ionization/time of flight mass spectrometry(MALDI-TOF-MS) to further isolate and purify to get a homodimer of SOD with molecule weight of 31 kDa. The activity of the enzyme tested by improved Pyrogallol autoxidation method appeared to be 9480.43 U/mg, inferring that the purification factor was 308.51. The enzyme was sensitive to detergent as H2O2,CHCl3-EtOH,Urea,NaN3 etc and exhibited high thermo stability (70℃) over the pH range from 4.0 to 9.0. Its maximum absorption wavelength was 278 nm and it was sensitive to hydrogen peroxide. The studies of purification and characterization of ginseng, play a guiding role for clinical medicinal research of ginseng antiaging.4,Ginseng protein which was extracted by neutral pH buffer and hollow fiber membrane filtration method had been isolated and purified via acidic buffer(pH 5.0) dialysis,hollow fiber membrane ultrafiltration(10 kDa and 2 kDa) and sephadex G-75 gel-filtration chromatography. Then conducting the determination of molecule weight and identification of purification on SDS-PAGE,HPGFC and MALDI-TOF-MS, resulting in a polypeptide(ginseng growth peptide) with molecule weight of 8000Da. Comparing the functional activity on mouse embryonic fibroblasts cells (3T3),human embryo lung diploid cells (2BS) and human laryngeal carcinoma cells (Hep-2) of this polypeptide with other ginseng proteins (proteinⅠ,Ⅱ, andⅢ), leading to the result that this polypeptide did best in promoting the growth of normal cells. The purification of ginseng growth peptide and significantly promoting the proliferation of 3T3 cells, suggest that the role of ginseng in maintaining the organizational structure and participation in damage repair and prevent or treat heart disease can not be ignored.5,To conduct toxicology tests of ginseng protein on ICR male mouse of SPF level. The results of adverse reaction test suggested that though having certain stimulating effects on liver, ginseng protein either had no obvious influences on the weight and visceral organs of juvenile male mice or promote the growth of Male genitals. Furthermore acute toxicity test showed that mice of maximum dose acted like normal mice without poisoning, indicating that ginseng protein was safe and non-toxicity. The safety experiments of ginseng protein provided a scientific basis for product development of ginseng protein, and layed an experimental foundation of the functional studies of ginseng protein.6,To conduct function tests of ginseng protein on Kunming mice of cleaning level. The results of dinitrofluorobenzene (DNFB) induced delayed-type hypersensitivity (DTH) in mice and mice carbon clearance experiment suggested that the ear swelling degree and phagocytic index had largely increased, informing of the immune enhancing function of ginseng protein; Swimming test at room temperature and hypoxia tolerance test showed that ginseng protein had an ability in enhancing hypoxia function and alleviating physical fatigue because of the prolongation of swimming and hypoxia tolerant time. The radiation of 60Co-γray on mice greatly increased the number of peripheral blood leucocyte and sharply decreased the number of bone marrow micronucleus in mice, informing of anti-radiation function of ginseng. The experiment of carrageenan-induced non-specific acute inflammation in mice showed decreasing trend in mouse toe swelling degree with no statistical significance, indicating that ginseng protein had no obvious anti-inflammatory effects. Acetic acid-induced writhing test and pain threshold measuring test by hot-plate in mice showed that the writhing time of mice had been greatly prolonged and the number of paw-licking had decreased, indicating that ginseng protein had certain effect on easing pain. In addition, our studies also found that after being fed with ginseng protein, normal mice and the radiated ones showed higher SOD activity on the contrary to lower MDA concentration, proving that ginseng protein had the ability in promoting antioxidant function.In summary, The research focuses on bioactive macromolecules of ginseng protein. According to compare with the activity of isozyme from different sources of ginsengs, provides enzymology basis for germplasm resource identification of ginseng. According to function and toxicology testing of ginseng protein, provide a pharmacological basis for the development and applications of ginseng protein drugs. In addition, this research systematically isolated and purified SOD and growth peptide from ginseng, and carried out physical and chemical properties and active research respectively, which layed a theoretical and experimental foundation for purification and identification of the biological activity protein of ginseng.