Preparation And Separation Of Velvet Antlers Polypeptide And Study On The Antioxidant Activity

Deer velvet antler is the young antler which is still not ossified and full of velvet of the male deer Cervus Nippon Temminck or Cervus Elaphus Linnaeus. Many studies demonstrated that the deer velvet antler have many bio-activities, which are closely related to amino acid compositions, such as promoting metabolism activity, immunostimulating activity, antioxidative activity. Velvet as a special cartilage, is a rich variety of natural factors of the cytokines in their process of self-growth ,bone, blood, nerve growth, cells or epithelial cell growth in tandem with a deer of the most active growth point in his store before the ossification of a large number of growth factors, which are velvet clinical biochemistry role of the foundation of modern medical research showed that velvet with a wide range of pharmacological activities. Velvet is all mammals in the most rapid growth of renewable organs, prompt the growth and development of bone, blood vessels, skin, hair and nerves and other organs. Velvet can promote growth, promot metabolism, anti-inflammatory and immune regulation, brain puzzle, anti-aging, anti-whiplash,antistress, health blood, and promote the healing of wounds and fractures pharmacological effects, etc.Velvet antler contains numerous regulatory substances, chemicals components. Recently, most researches are focus on protein and polypeptides, amino acids ( Including the essential amino acids--10 kinds of phospholipid compositions and nine kinds of fatty acids, which human body can not synthetic. The contents of the highest biological activities are oleic acid, linoleic acid and linolenic acid, which are at a high level ), lipid, polysaccharide, trace elements, hormone, mineral compositions, etc.Under normal circumstances, it is a dynamic equilibrium for producing and scavenging free radicals. In the pathological conditions, free radicals generated by physical factors and xenobiotics directly or indirectly cannot be scavenged in time, or the equilibrium of producing and scavenging endogenous free radicals were broken, then the body are injured by free radicals. Once producing too much oxygen free radicals or the failure of anti-oxidation system have broken, which will result in the imbalance of oxygen free radicals in vivo metabolism, which we called it oxidative stress, then it will lead to serious diseases, such as cell damage, heart disease, cancer and aging, etc. In this situation, it is essential to add some anti-oxidants to help the body maintain the balance of oxygen metabolism for preventing diseases and maintaining the health of the body.In our experiments, velvet antler which prepared from the velvet antler of sika deer (Cervus Nippon Temminck) contains 81.94 percents of water content in fresh deer velvet, and dry matter protein content is 64.1%, Velvet antler ( Cervus Nippon Temminck ) using a combination of gel filtration chromatography and RP-C18 chromatography technology. The chemistry nature and structure of those protein and polypeptides were discussed by the spectrum technologies of HPLC-MS.With the use of an industrial alkaline protease Alcalase, at 50℃, pH8.0, and the 10 percents degree of hydrolysis conditions for hydrolysis of orthogonal test design,and optimize the enzyme concentration, substrate concentration and reaction time. We have selected the best conditions, with the enzyme concentration (substrate protein : enzyme / g:g) is 1:150, substrate concentration (dry substrate matter:water / g:g) is 1:13 and time conditions for 60 min. we used such methods as distillment of neutral buffer and thiamine hierarchical-deposit to separate velvet antler polypeptide protein. After hydrolysis followed by centrifugation and filtration, and then by Sephadex G-25, We get the strongest antioxidant activity peptide components (VAP-B). Furthermore, with preparative high performance liquid chromatography separated and purified, We get peptide components (VAP-B1)which molecular weight was less than 800U , and finally through vacuum freeze-drying, the bioactive peptide velvet protein (VAP-B1) content is 70.45%, and examine its physical and chemical composition analysis. We detected pilose antioxidant peptides, including the removal of superoxide anion experiments, the removal of hydroxyl radical experiments, lipid peroxidation experiments, DPPH free radical scavenging and reducing power test experiments. The results show that the velvet antlers peptide(VAP-B1)in different detection system performance can be different degrees of antioxidant capacity. When VAP-B1's concentration is 20 mg/ml, the inhibition ratio to pyrogallic acid autoxidation reach to 91.9%, having a strong ability to scavage superoxide anion; when VAP-B1's concentration is 10 mg/ml, the clearance rate to hydroxyl radical is 100%; the VAP-B1 can inhibit the degree of lipid peroxidation and the reduce power when concentration is 10mg/ml; the effects exist in dose-dependent manners.At the same time, we also found that VAP-B1 can not scavage DPPH radical effectlyOn the basis of above content, velvet antler indeed contains a very strong biological antioxidant activity factors - pilose peptides, We get peptides which molecular weight was less than 800 Da , the velvet antler peptides can be regarded as one kind of extensive antioxidant, which can scavage superoxide anion and hydroxyl radical effectly and inhibit the degree of lipid peroxidation and the reduce power within some concentration degrees. All above studies provided an important experimental basis for the further in-depth research in the Pilose anti-peptide.