Study On Antioxidation, Anti-hypertension And Hematopoiesis Of Taihe Black-bone Silky Fowl (gallus Gallus Domesticus Brission) Bioactive Peptides

Taihe Black-bone silky fowl (TBSF)(Gallus gallus domesticus brisson), an important food and drug resource in Jiangxi province, has a long history and it is famous at home and abroad. It is not only a kind of food with abundant nutrition but also one of chicken breeds with unique medicinal value, and it is used in clinical practice as genuine medicinal materials by physicians during past generations in China. According to a large majority of material medica and literatures, Black-bone silky fowl has health functions of supplementing liver and kidney, supplementing blood gas, clearing deficiency heat, regulating menstruation and arresting leukorrhea of women, etc., and can be used to cure various diseases, such as abdominal pain, wasting, collapse in the vaginal discharge, emissions, diabetes, lingering dysentery, bone fracture, sore waist and skelalgia, hemorrhage diseases, purpura, hepatitis, etc. Therefore, bioactive peptides produced using TBSF as raw materials are predicted of great research value. In consideration of this fact, the present study focused on TBSF bioactive peptides. Comprehensively applying a series of technical means and methods, such as food science, modern separation technology, natural products chemistry, modern instrumental analysis, cell and animal experiments, the present study systematically investigated TBSF bioactive peptides'activity of antioxidant, anti-hypertension and hematopoiesis. The main conclusions are as follows:1. TBSF bioactive peptides were produced by enzymatic hydrolysis of papain using the method of uniform design. The resulting TBSF bioactive peptides exhibited strong O2'-,OH, DPPH and ABTS'+ scavenging activity and reducing power, and displayed good Fe2+ and Cu2+ chelating activity and lipids peroxidation inhibition activity. Using ultrapure water as mobile phase, preparative RP-HPLC fractionated TBSF bioactive peptides into 4 parts:fractionⅠ,Ⅱ,Ⅲand IV. Among these fractions, fraction II showed the strongest O2·- and DPPH'scavenging activity and reducing power, and displayed strong ABTS·+ scavenging activity. DPPH' and ABTS'+ scavenging activity and reducing power of fractionⅡwere stronger than carnosine at the same concentration. Fraction I had the strongest·OH scavenging activity. TBSF bioactive peptides contained abundant amino acids of Glu, Asp, Gly, Ala and Leu, etc., and a considerable amount of the small-size bioactive peptides with the molecular weight (MW) of 291-6070 Da. These factors were mainly responsible for strong antioxidant activity of TBSF bioactive peptides.2.<6 kDa TBSF bioactive peptides obtained by ultrafiltration exhibited strong O2'- and ABTS'+ scavenging activity and reducing power, and had good OH and DPPH' scavenging activity, and displayed good Fe2+ and Cu2+ chelating activity and lipids peroxidation inhibition activity.0.01 M phosphate (pH 6.8) was selected as mobile phase for the best separation of<6 kDa TBSF bioactive peptides. Preparative RP-HPLC fractionated the<6 kDa TBSF bioactive peptides into 13 fractions, which were then desalted by the strong acid cation exchange resin connected in series with the strong basic anion exchange resin, with good results. Fraction 4 had the strongest reducing power; Fraction 7 showed the strongest lipids peroxidation inhibition activity; Fraction 8 displayed powerful O2'- and ABTS'+ scavenging activity and reducing power; Fraction 13 exhibited the strongest DPPH' scavenging activity. Fraction 4 and 8 were then gradiently eluted by analytical RP-HPLC using ultrawater-acetonitrile as mobile phase, and 4 subfractions were obtained respectively. Results of antioxidant activity determination showed that subfraction 4-3 had the strongest reducing power, and subfraction 8-3 had the strongest O2'- and ABTS'+ scavenging activity and reducing power. O2'- and ABTS'+ scavenging activity and reducing power of subfraction 8-3 were stronger than carnosine at the same concentration. Subfraction 8-3 displayed a high purity and was identified by HPLC-ESI+-MS/MS as Glu-Pro-Asp-Arg-Tyr.3. ACE which extracted from pork lung had an activity of 27 U/mL. The determination method of ACE inhibitory activity of TBSF bioactive peptides was established using analytical RP-HPLC combined with mobile phase of ultrawater (0.1% formic acid)-acetonitrile. In vitro simulated gastrointestinal digestion was employed to hydrolyze TBSF meat. As increase of digestion time, the hydrolysis degree and content of small-size bioactive peptides of digests at different stages increased, and the ACE inhibitory rate maintained up to 57.8%. Ultrafiltration was used in the beginning to separate 360 min intestinal digest, and<3 kDa TBSF bioactive peptides were obtained. The IC50 value of<3 kDa TBSF bioactive peptides was 0.56 mg/mL. Secondly, preparative RP-HPLC fractionated the<3 kDa TBSF bioactive peptides into 3 fractions, among which F2 had the strongest ACE inhibitory activity, with the IC50 value of 0.21 mg/mL. Finally, gel column was used to separate F2 into 2 subfractions, of which F2-2 displayed the strongest ACE inhibitory activity, with the IC50 value of 0.04 mg/mL. HPLC/Q-TOF MS was employed to identify F2-2, and a novel peptide named Glu-Pro-Leu-Tyr-Tyr was found.4. Cell and animal experiments were used to evaluate the hematopoietic activity of TBSF bioactive peptides and their fractions. TBSF bioactive peptides had the effect of stimulating proliferation of normal mice bone marrow nucleated cells. Fraction 7,8, 9 and 10 exhibited significantly strong activity (P<0.01) in stimulating normal mice bone marrow nucleated cells, and the proliferation rate could be up to 130%. The amino acid composition of these four fractions was determined by analytical RP-HPLC precolumn derivation with 2,4-dinitrofluorobenzene method, and the result showed that they mainly contained Tyr, Gly, Glu, Asp and Ala. Hence we speculated that these five amino acids are the material base of active component in stimulating proliferation of normal mice bone marrow nucleated cells. TBSF bioactive peptides elevated the thymus and spleen index of blood-deficiency anemia mice, indicating enhancement of the immunologic functions of anemia mice, and consequent acceleration of quick hematopoiesis. TBSF bioactive peptides significantly increased RBC of blood-deficiency anemia mice (P＜0.05), and rapidly recovered RBC, HGB and HCT of 5-fluorouracil induced anemia mice to normal levels, and significantly elevated RBC of blood-deficiency and 5-fluorouracil induced anemia mice (P<0.05), and rapidly recovered HGB of blood-deficiency and 5-fluorouracil induced anemia mice to normal levels. Fraction 8 of TBSF bioactive peptides rapidly recovered HGB of blood-deficiency and 5-fluorouracil induced anemia mice to normal levels, and rapidly enhanced RBC and HCT. The animal experiment demonstrated that TBSF bioactive peptides and their fractions had a certain hematopoietic effect.