| Pig placenta is a kind of animal medicine which contains a lot of bioactive componets. Pig placenta has the function of endocrine regulation, enhancing immunity, prevention and treatment of Alzheimer’s disease, anti-oxidation, enhancing memory and so on. However, pig placenta is often abandoned and there are few reports about pig placenta. In this experiment, the preparation of the pig placenta antioxidant peptides by tissue mashing, freezing and thawing was investigated. The preparation process of freeze-dried powder was optimized, and its function was evaluated in order to provide bases for the full use of pig placenta. The main conclusions are listed as below.1. Extraction and analysis of pig placenta peptidePig placenta extract was prepared by tissue mashing, freezing and thawing. The physical and chemical properties of pig placenta extract were identified. The pig placenta peptide was identified by SDS-PAGE electrophoresis and a method of RP-HPLC was established. The results as follows:firstly the pig placenta extract is slightly yellow transparent liquid. Besides, its pH was 6.70. The electrophoresis result had shown that the pig placenta extract includes the molecular peptide class material and its molecular weight was 74.4 kDa,61.16 kDa,43.39 kDa, 14.52 kDa and its peptide content was 6.050 mg·ml-1. It had an absorption peak at 260.5 nm between 200 nm and 600 nm. The column was Symmetry C18 (5μm,4.6×250 mm). The mobile phase was acetonitrile-water. The eluting sequence was gradient elution:acetonitrile:0～15 min 10%～20%,15～20 min 20%～25%. The column temperature was 25℃. The flow rate was 0.5 ml·min-1. The detection wavelength was 260.5 nm and the injection volume was 10μl. There were 12 peaks by RP-HPLC. Pig placenta peptides may contain albumin, immunoglobulin, interferon which was similar to sheep placenta peptides.2. Preparation of pig placenta peptides lyophilized power and the stabilization inspectionSingle factor experiments were carried out to determine that sucrose was the best kind of lyoprotectant. We determined the best drying process by using a pig placenta extract arance, hydration speed, polypeptide content as test materials. The results showed that the best vacuum freeze-drying process was protective agent sucrose with 4% content,30 h drying time,3 ml solution. Design-Response Surface Analysis was carried out using polypeptide content as indexes and there were three factors:content of lyoprotectant, drying time and volume of solution. The results showed that the best vacuum freeze-drying process was protective agent sucrose with 4.35% content,30 h,3.41 mm. And the order of influence degree to polypeptide content was protective volume of solution> agent sucrose> drying time. The stability of the pig placenta peptides lyophilized powder was detected by putting them under high temperature, humidity and illumination. It also showed that the storage conditions of pig placenta peptides were required to be dry, sealing, lucifyge and low temperature.3. Efficacy inspection of pig placenta peptidesThe scavenging activity of pig placenta peptides on the DPPH·, superoxide anion radical and hydroxyl radical was measured. The results showed that the DPPH·, O2-, ·OH- were inhibited by pig placenta peptides. The scavenging ability of pig placenta peptides was equvivalent to the scavenging ability of vitamin C.Aging mice induced by D(+)Galactose were given pig placenta peptides. After the administration, the body weight was measured at weekend. Morris water maze was used to test learning and memory ability of mice at the end of week 5. At the end of week 6, we measured moisture content of the skins and the SOD, MDA, Hyp content and observe the pathological sections. Results:Compared with the NC group, the latency of the model group was longer (p<0.05), time of staying in platform quadrant and frequency of crossing the platform were decreased (p<0.05). The body weight, moisture content of skins, SOD and Hyp content were significantly lower (P<0.01, P<0.05) and MDA content was higher (P<0.01, P<0.05). Pathological examination revealed that the epidermal of the skin was irregular and the thickness of the dermal was decreased significantly, the collagen fibers were thinner and looser in the model group. After the intervention with pig placenta extract and compared with the model group, latency was shortened in the pig placenta extract high, middle dose group (P<0.05), time and percentage of staying in platform quadrant and frequency of crossing the platform were increased in the pig placenta extract high dose group (p<0.05). The body weight, moisture content of skins, SOD and Hyp content were significantly higher and MDA content was significantly lower in the high, middle dose group (P<0.01, P<0.05). In the pig placenta extract high and middle dose groups, the structure of the skins were complete and the epidermal of the skins were regular and the thickness of the dermal of the skins were increased and the collagen fibers were arranged in order. |
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