Study On The Preparation And Pharmacology And Toxicology Of Polygonum Hydropiper Flavonoids Extract

Penaeus vannamei is the fastest growing and most productive crustacean breed in China.But its growth is susceptible to many pathogenic factors.Its incidence rate and mortality rate are very high,and there is a great risk of breeding.This restricts the development of industry.In this study,the alcohol extraction technology of Polygonum hydropiper flavonoid extract was optimized.The effects of Polygonum hydropiper flavonoid extract on the immune regulation of blood lymphocytes,immune function and growth performance of Penaeus vannamei were observed.The protective effects of Polygonum hydropiper flavonoid extract on Penaeus vannamei under ammonia nitrogen stress were studied.The toxicological tests of Polygonum hydropiper flavonoid extract were carried out to evaluate its safety.（1）Study on the preparation technology of Polygonum hydropiper flavonoids extractThe ethanol solution was used as solvent to extract Polygonum hydropiper by heating and reflux.The contents of rutin,quercetin and quercetin in the extract were determined by high performance liquid chromatography,and the best orthogonal technology was explored.Then,water-soluble silica was added as excipient to obtain the flavonoid extract of Polygonum hydropiper.The results showed that the best extraction process of Polygonum hydropiper flavonoids was A3 B3 C3 D3,that is,the ratio of material to liquid was 1:20,the concentration of ethanol was 80%,the extraction was heated for 3 times,and each time for 2hours.The content of Polygonum hydropiper flavone in crude extract of Polygonum hydropiper was 65.37 mg/g,in which rutin content was 12.91 mg/g,quercetin content was 52.46 mg/g,dry extract yield was 8.23%,and the content of flavone in the final Polygonum hydropiper flavone extract was 7.51%.（2）Immunomodulatory effect of Polygonum hydropiper flavone extract on hemocytes of Penaeus vannameiIn order to screen the safe concentration of Polygonum hydropiper flavonoid extract and evaluate the immunomodulatory effect of Polygonum hydropiper flavonoid extract on blood lymphocytes of Penaeus vannamei.CCK8 method was used to detect the effects of Polygonum hydropiper flavonoid（12.5μg/m L～1600μg/m L）,Astragalus polysaccharide（12.5μg/m L～1600μg/m L）and 0.5%DMSO on the lymphocyte activity of Penaeus vannamei.The results showed that when the concentration of Polygonum hydropiper flavonoid extract was more than 400μg/m L,it would significantly reduce the cell activity（P<0.05）,when the concentration of Astragalus polysaccharide was more than 400μg/m L,it would significantly reduce the cell activity（P<0.01）;when 0.5%DMSO solution was used as solvent to dissolve Polygonum hydropiper flavonoid and Astragalus polysaccharide,it would not affect the cell activity of Penaeus vannamei.Astragalus polysaccharide（100μg/m L）was used as the control drug,and Polygonum hydropiper flavonoid（12.5μg/m L～100μg/m L）was added into the blood lymphocyte culture system of Penaeus vannamei for 4-24 hours.The blood lymphocytes were collected and the activities of ACP,AKP,PO,SOD and other immune related enzymes were detected.The results showed that compared with the blank group,there was no significant difference in ACP activity between 12.5μg/m L～100μg/m L（P>0.05）and Polygonum hydropiper flavonoid at concentration from 12.5μg/m Lto 100μg/m L respectivelyμg/m L of extract could significantly enhance the activity of SOD in cells after 24 hours of culture（P<0.01）.The results showed that the flavonoid extract of Polygonum hydropiper had a certain enhancing effect on the immune function of blood lymphocytes of Penaeus vannamei.（3）Effect of Polygonum hydropiper flavonoid extract on the growth and immune function of Penaeus vannameiIn order to investigate the effect of Polygonum hydropiper flavonoid extract on the immune function and growth performance of Penaeus vannamei,five experimental groups were set up,including blank control group,Astragalus polysaccharide（0.2%）positive drug group and Polygonum hydropiper flavonoid extracts at low,medium and high（0.2%,0.4%,0.8%）dose group,and corresponding doses of drugs were added to prawn feed.Blood samples were collected on the 7th,14th,21st and 28th day of administration to measure the activities of ACP,AKP,AST,ALT,PO and SOD in the plasma,and the body length and weight of Penaeus vannamei were measured to calculate the weight gain rate and growth rate.The results showed that Polygonum hydropiper flavonoid at high dose could promote the growth of Penaeus vannamei,and the low dose significantly enhanced the SOD activity of Penaeus vannamei after 28days of administration（P<0.01）,The activities of ALT,PO and SOD were significantly increased in the middle dose group（P<0.01）,and ACP,AKP,ALT,PO and SOD were significantly increased in the high dose group（P<0.01）.The results showed that the flavonoid extract of Polygonum hydropiper could effectively promote the growth of Penaeus vannamei and enhance the immunity of Penaeus vannamei.（4）Effect of Polygonum hydropiper flavonoid extract on the survival rate and immunity of Penaeus vannamei under ammonia nitrogen stressIn order to study the effect of Polygonum hydropiper flavonoid extract on the immune function and growth performance of Penaeus vannamei,the blank control group,ammonia nitrogen stress model group,Astragalus polysaccharide（0.2%）control drug group and Polygonum hydropiper flavonoid extract were set up with low,medium and high（0.2%,0.4%,0.8%）.In addition to the blank control group and the ammonia nitrogen stress model group,the corresponding doses of drugs were added to the prawn feed of the other four experimental groups.After 28 days of feeding,72 h ammonia nitrogen stress test was carried out at120.00 mg/L.After 72 hours of ammonia nitrogen stress,the death of different dose groups after 72 hours of ammonia stress was observed,and some surviving Penaeus vannamei were randomly selected for sampling to detect the activities of ACP,AKP,AST,ALT,PO and SOD in their plasma.The results showed that after 72 hours of ammonia nitrogen stress,the mortality of model group was30.00%,that of control group and low dose group was 13.33%,that of medium dose group and high dose group was 10.00%,and that of high dose group was significantly higher than that of model group（P<0.01）The activity of ACP,AKP and PO in the high dose group was significantly higher than that in the blank control group（P<0.01）.The results showed that adding a certain dose of Polygonum hydropiper flavonoid extract to feed could regulate the activity of related non-specific immune enzymes in the plasma of Penaeus vannamei and improve the protective effect of Penaeus vannamei against ammonia nitrogen stress.（5）Toxicological study on the flavonoid extract of Polygonum hydropiperThe acute toxicity test of Kunming mice was carried out by gavage,and LD₅₀and maximum dosage of Polygonum hydropiper flavonoid extract were determined.In the subchronic toxicity test,Polygonum hydropiper flavonoid extract was added to the diet of SD rats for 30 days to observe the physiological,biochemical indexes and histopathology of rats.The results showed that in the acute toxicity test,there was no poisoning symptom or death,and LD₅₀ was not measured.The maximum dose of the test drug was 30 g/kg·BW.In the 30 day feeding experiment of rats,the high,middle and low doses of Polygonum hydropiper flavonoid extract had no toxic reaction to SD rats for one month,and had no adverse effect on the body weight,hematology index,blood biochemical index,etc.,and had no obvious pathological damage to the main organs such as heart,liver,kidney,spleen,gastrointestinal tract,testis and egg nest.The results showed that the flavonoid extract of Polygonum hydropiper had no side effects and good safety.