Study On The Preparation Of Peanut Skin Proanthocyanidins And Its Anti-intestinal Inflammation Mechanism

Gut Health is vital to the farming industry.The plant-derived active ingredients are highly valued for their physiological functions of multi-component,multi-function and multi-target regulation with the continuous pursuit of "Quality"and "Safety" in the field of animal husbandry.The research and application of plantderived active ingredients is the basis for promoting the healthy development of ’a new era of antibiotic-free breeding’,and also is an important way to expand the field of veterinary drug research and improve the international competitiveness of veterinary drug products of China.Proanthocyanidins,especially grape seed proanthocyanidins(GSPC),are widely used because of their strong antioxidant and immune-enhancing effects.At present,some studies have found that peanut skin proanthocyanidins(PSPC)have higher bioavailability,but its preventive and therapeutic effects on animal intestinal inflammation,especially diet allergic intestinal inflammation,are still unclear.Therefore,this paper was based on the study of the extraction and purification process of peanut skin proanthocyanidins and the identification of its component structure.Then the therapeutic effect of peanut proanthocyanidins on intestinal inflammation in DSS-induced colitis mice was studied.The intervention mechanism of peanut proanthocyanidins on allergic enteritis was discussed by constructing a cell model of allergic enteritis.The main research contents were as follows:1.Study on the ultrasonic-microwave synergistic extraction process and the purification process of AB-8 macroporous resin of proanthocyanidins from peanut skin.On the basis of single factor experiment and Plackett-Burman design,the optimum extraction process of proanthocyanidins was established by Box-Behnken central experimental design:ultrasonic 160 W,10 min,microwave 240 W,90 s,solidliquid ratio 1:40,70%ethanol 50℃ extraction 20 min.A good extraction model Y=184.20-7.67A+0.8838B+7.47C-1.33AB+4.41AC-1.41BC-5.85A2-12.62B2+2.35C2 was established.The sample concentration was 1.2 mg/mL,pH 3 flow rate was 1.0 mg/mL,and the purification process of AB-8 macroporous resin was determined under the conditions of adsorption 180 min,desorption 40 min and desorption flow rate 1.0 mg/mL.It provided the basis for the application research of peanut red skin procyanidins in breeding industry.2.Preliminary efficacy evaluation and component identification of PSPC.The antioxidant capacity of PSPC was preliminarily evaluated by antioxidant evaluation system in vitro,which showed that it was superior to grape seed proanthocyanidins in hydroxyl radical scavenging,DPPH radical scavenging and FRAP value antioxidant capacity.Ultraviolet/visible spectrum analysis showed that the purified product belonged to PC class.Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopy analysis showed that procyanidin was the main structural unit;UPLCQTOF-MS/MS analysis showed that it contained catechin,protocatechuic acid,A-type PC dimer,trimer and tetramer,and B-type PC tetramer.These results showed that the PSPC had good antioxidant capacity and was rich in type A PC oligomers.3.The alleviation effect of peanut procyanidins on intestinal inflammation in DSS-induced colitis mice,Dextran Sulfate Sodium Salt(DSS)-induced colitis mouse model was used to evaluate the therapeutic effect of PSPE and PSPC on animal intestinal inflammation.It was found that PSPC and PSPE could reduce the production and expression of inflammatory factors TNF-α,IL-6 and IL-1β in DSS-induced colitis mice,and inhibit the production of oxidative stress marker MDA and the rapid expression of COX-2 mRNA and iNOS mRNA,thereby alleviating intestinal inflammation.PSPC and PSPE could significantly increase the number of GC and promote the secretion of MUC-2,significantly increase the production and expression of Claudins-1 protein,and increase the expression of Occludin mRNA and ZO-1 mRNA,which were helpful for the recovery of intestinal mucosal barrier protection.The symptoms of the disease showed that under the intervention of PSPE and PSPC,the intestinal DAI score,weight loss,colon shortening,hematochezia and other symptoms of colitis mice were alleviated.The results demonstrated that PSPC and PSPE had a certain therapeutic effect on intestinal inflammation in DSS-induced colitis mice.4.Effect of peanut skin proanthocyanidins on Microflora GUT in DSSinduced colitis mice.Diversity,abundance,similarity,function and differential genus of Microflora GUT in DSS-induced colitis mice were studied by Microflora GUT16S rRNA sequencing analysis.The production of SCFAs was determined by gas chromatography.The deeptools plot Corrlation data analysis technique was used to analyze the correlation between intestinal inflammation and Microflora GUT characteristics,and to explore the regulatory effects of PSPC and PSPE on Microflora GUT in colitis mice.It was found that PSPC and PSPE significantly affected the diversity,abundance and composition of Microflora GUT,while significantly interferred the expression of oxidative stress markers and inflammatory factors,and enhanced the production of microbial metabolites SCFAs.The results showed that in colitis mice,PSPC and PSPE act on the relief and treatment of intestinal inflammation through excessive targets:not only by affecting the oxidative stress response and inflammatory factors of intestinal inflammation,but also by remodeling Microflora GUT and enhancing the production of SCFAs to achieve the purpose of treating intestinal inflammation.This suggested that PSPE and PSPC might be safe and efficient natural drugs for the treatment of intestinal inflammation.5.Study on intervention mechanism of peanut coat procyanidins on diet allergic enteritis cell model,Caco-2 cells were induced by Gluten protein digest(DPG)to construct a cell model of allergic enteritis,and to explore the intervention mechanism of PSPC on allergic enteritis.It was found that DPG induced oxidative stress in cells and promoted the occurrence of inflammatory response by inhibiting antioxidant defense molecules SIRT1 and Nrf2,and activating NF-κB signaling pathway.At the same time,it induced TGM2 up-regulation and increased immune response,damaged mitochondrial membrane integrity,caused abnormal expression of apoptotic proteins BAX and Caspsae-3,and promoted apoptosis.This process was significantly reversed by PSPC.This process was significantly reversed by PSPC.The results showed that PSPC blocked the oxidative stress and inflammatory response induced by DPG through the regulation of SIRT1/NF-κB/Nrf2 signaling pathway,increased the expression of Occludin and repaired the damaged monolayer.The study revealed the molecular mechanism of PSPC intervention in the treatment of allergic enteritis at the cellular level.