The Effects And Regulation Of Flavonoids And Iridoid Extracts On Growth,antioxidant,lipid Metabolism And Flesh Quality Of Grass Carp (Ctenopharyngodon Idellus)

The quality and safety of aquatic products has always been the focus of consumers.In recent years,more and more Chinese herbal medicines and plant extracts have been applied in aquaculture.Flavonoids and iridoid extracts have been shown the improving effects on growth performance and muscle quality of aquatic animals.However,the studies regarding flavonoids and iridoid are limited,and the acting mechanism is still unclear.Therefore,grass carp（Ctenopharyngodon idella）was used as the experimental animal to investigate the effects and mechanism of flavonoids（rutin,quercetin,baicalein,catechins,kaempferol）and iridoid（aucubin）on growth,antioxidant,lipid metabolism and flesh quality at the biochemical,cellular and molecular levels through feeding trial and cell culture experiment combining metabolomics technology.These results will provide theoretical basis for the research and application of plant extracts in aquatic diets and for the development of green aquaculture.The main research contents of this paper were as follows:1.Dietary rutin promoted the growth,serum antioxidant response and flesh quality of grass carp（Ctenopharyngodon idella）To investigate the effects of dietary rutin on growth,serum antioxidant response and flesh quality of grass carp,six diets were prepared with the rutin inclusion at 0（control diet）,0.2,0.4,0.6,0.8 and 1.0 g/kg,respectively.Grass carp with initial body weight of 18.5±0.2 g were fed with the six diets for 60 days.The weight gain was increased from 348.5%（the control）to 364.4%,366.5%,and feed conversion ratio was decreased from 1.56（the control）to 1.49,1.48,respectively,by the supplementation of 0.8 and 1.0 g/kg rutin in diets.Serum superoxide dismutase（SOD）activity was increased by the addition of 0.2-1.0 g/kg rutin,and serum malondialdehyde（MDA）content was decreased by the addition of 0.4-1.0 g/kg rutin.The inclusion of 1.0 g/kg rutin and 0.6-1.0 g/kg rutin significantly increased the collagen content and the total free amino acids in flesh,respectively.The content of n-3 polyunsaturated fatty acids（n-3 PUFA）in flesh was also promoted by the inclusion of 0.2 and 0.6 g/kg rutin.However,dietary rutin did not significantly affect the flesh texture and water-holding capacity.In conclusion,dietary rutin could improve the growth,antioxidant response ability and flesh quality of grass carp,and the recommended supplemental level of rutin was 0.8-1.0 g/kg.2.Flavonoid-enriched diets improved the growth and flesh quality of grass carp（Ctenopharyngodon idellus）based on metabolomicsThis study investigated the effects of four flavonoids on growth and flesh quality of grass carp.Five diets were designed with the supplementation of quercetin,rutin,baicalein and catechin in basal diet（control）,and were fed to grass carp（59.7±0.2 g）for 60 days.The fish fed quercetin and rutin diets,rather than baicalein and catechin diets,showed higher weight gain（+4.0%,+3.9%）and lower feed conversion ratio（-0.07,-0.06）than the control fish.The content of total collagen,heat-soluble collagen,total free amino acids（TFAA）and delicious amino acids（DAA）in flesh was significantly increased by the supplementation of quercetin and rutin in diets.The SOD activity and the content of C22:5,C22:6 and n-3 PUFA were significantly increased,and the MDA and protein carbonyl（PC）contents were significantly decreased in the quercetin,rutin and catechin groups.In the metabolomics profiling of muscle,102 and 68 named differential metabolites were identified in the quercetin and rutin groups,and these metabolites were found to be involved in the second-grade pathways of“lipid metabolism”and“amino acid metabolism".In addition,the m RNA expression of IGF-1,PI3K,AKT and FAS in flesh was significantly up-regulated in quercetin and rutin groups.In summary,dietary quercetin and rutin promoted the growth and flesh quality of grass carp which might be associated with IGF-1/TOR,Nrf2,TGF-β/Smad4 and PPAR signaling pathway.3.Dietary effects of kaempferol on growth,lipid metabolism and flesh quality of grass carp（Ctenopharyngodon idellus）based on metabolomicsTo investigate the effects of dietary kaempferol on growth,lipid metabolism and flesh quality of juvenile grass carp,five diets with kaempferol inclusion at 0（control diet）,0.2,0.4,0.6 and 0.8 g/kg（Kae-0.2,Kae-0.4,Kae-0.6,Kae-0.8）,then fed to fish with initial body weight of 17.0±0.2 g for 60 days.The results indicated that dietary supplementation of 0.6 and 0.8 g/kg kaempferol increased weight gain by 4.9%,5.6%and decreased feed conversion ratio by 0.08,0.08,respectively.Compared to the control flesh,the supplementation of 0.2-0.8 g/kg kaempferol increased adhesiveness,SOD activity and TFAA content,and the hardness,chewiness,resilience and DAA content were also promoted by the kaempferol supplementation of 0.4-0.8 g/kg,while the catalase（CAT）and glutathione peroxidase（GPx）activities were enhanced only by dietary supplementation of 0.6-0.8 g/kg kaempferol.Moreover,intraperitoneal fat ratio and MDA content in flesh and triacylglycerol（TG）and cholesterol（CHO）in serum and liver were decreased in Kae-0.8 group.In the metabolomics profiling of flesh,102 and 133 named differential metabolites were identified in Kae-0.4 and Kae-0.8 groups when compared to the control,and these metabolites were involved in the second-grade pathways of“lipid metabolism”and“amino acid metabolism”.In gene expression,the m RNA level of m TOR,CAT,PPARαin flesh as well as PPARαin liver was up-regulated,and FAS gene expression in liver was down-regulated by dietary supplementation of 0.4 and 0.8 g/kg kaempferol.In summary,dietary kaempferol improved the growth,lipid metabolism and flesh quality of juvenile grass carp,which might be associated with the IGF-1/m TOR,PPAR and Nrf2 pathways.The recommended supplementation level of kaempferol in diet was 0.8 g/kg for juvenile grass carp.4.Dietary supplementation of aucubin improved the growth and flesh quality of grass carp（Ctenopharyngodon idellus）based on metabolomicsThis study aimed to investigate the effects of dietary aucubin on growth and flesh quality of grass carp.Five diets were designed with the aucubin inclusion at 0（control diet）,0.2,0.4,0.6 and 0.8 g/kg（Auc-0.2,Auc-0.4,Auc-0.6,Auc-0.8）,then fed to grass carp with initial body weight of 17.0±0.2 g for 60 days.The results indicated that dietary aucubin did not significantly affect the growth performance of grass carp.Compared to the control,the dietary supplementation of 0.2-0.8 g/kg aucubin increased flesh hardness,chewness,the activity of SOD,CAT and GPx and the content of TFAA and n-3 PUFA of flesh.The content of MDA and LD in flesh was significantly decreased by the addition of 0.4-0.6 g/kg aucubin and by the addition of 0.6-0.8 g/kg aucubin,respectively,while the content of delicious amino acids（DAA）also was significantly enhanced by the addition of 0.4-0.8 g/kg aucubin.Moreover,the content of collagen and C22:6n3（DHA）in flesh of Auc-0.8 group was significantly higher than those of control.In the metabolomics profiling of muscle,133 and 135 named differential metabolites were identified in Auc-0.4 and Auc-0.8groups when compared to the control,respectively,and these metabolites were found to be involved in the second-grade pathways of“lipid metabolism”and“amino acid metabolism”.In gene expression,the m RNA level of Cu Zn-SOD,CAT,COL1A1,COL1A2 and FAS of flesh was up-regulatedin in Auc-0.4 and Auc-0.8 groups.In summary,dietary aucubin did not promote the growth,but improved the flesh quality of grass carp,which might be associated with TGF-β/Smads and Nrf2 pathways.The recommended supplementation level of aucubin in the diet of grass carp was 0.6-0.8g/kg.5.Effects of six flavonoids and iridoid extracts on lipid metabolism,collegn metabolism and anti-oxidation of grass carp cellsGrass carp hepatocytes,fibroblasts and muscle cells were cultured and treated with six flavonoids（rutin,kaempferol,quercetin,baicalein and catechin）and iridoid（aucubin）at concentrations of 5,10,20,40,80,and 160μg/m L,to investigate the effects of flavonoids and iridoid on lipid metabolism of hepatocytes,collegn metabolism of fibroblasts and anti-oxidation of muscle cells.The results were as follows:The supplementation of 40μg/m L rutin and 10μg/m L baicalein significantly increased the TG content in hepatocytes,but did not affect the CHO content.40μg/m L kaempferol also significantly reduced the content of TG and CHO.In genes expression,40μg/m L rutin significantly increased the expression of FAS and PPARγm RNA and reduced CPT-1 m RNA level.40μg/m L kaempferol increased PPARαm RNA expression and decreased the expression of FAS and PPARγm RNA.10μg/m L baicalein also significantly decreased CPT-1 m RNA level.In summary,kaempferol and rutin might regulate the expression of lipid metabolism-related genes by regulating the PPAR signaling pathway,thereby affecting the lipid accumulation of grass carp hepatocytes.Baicalein increased the triglyceride content in hepatocytes by up-regulating the level of CPT-1 m RNA.Except for baicalein and kaempferol,the other four compounds significantly increased the collagen content of fibroblasts.After silencing Smad4 gene,the collagen content and collagen gene（COL1A1 and COL1A2）levels in cells were all significantly decreased.When 20μg/m L quercetin or 40μg/m L rutin were added to Smad4-silencing fibroblasts（si RNA+quercetin group and si RNA+rutin group）,the collagen content and collagen gene expression levels showed no difference with the si RNA group,and both were significantly lower than the quercetin group and the rutin group,respectively.After silencing Smad4,the supplementation 20μg/m L catechin or40μg/m L aucubin（si RNA+catechin group and si RNA+aucubin group）,the collagen content and collagen gene levels were significantly higher than the si RNA group,and were significantly lower than the catechin group and the aucubin group（except for the collagen content of the si RNA+catechin group）,respectively.In summary,quercetin,rutin,catechin and aucubin could promote the synthesis of collagen in intramuscular fibroblasts.Smad4 played a key role in regulating the collagen metabolism,and it was also a key gene for quercetin and rutin to regulate collagen metabolism in fibroblasts of grass carp.Baicalein and aucubin might regulate the metabolism of collagen in fibroblasts through other pathways.A oxidative injury model of grass carp muscle cells was established with 400μmol/L H₂O₂treatment for 2 hours.H₂O₂injury significantly reduced the activity of SOD,GPx,the level of Cu Zn-SOD,CAT,Keap 1a m RNA,and increased MDA content in muscle cell.All the six extracts increased the activity of SOD,CAT,the level of Cu Zn SOD,CAT m RNA,and reduced the content of MDA in muscle cells of the normal group and H₂O₂injury group.In normal cells,the supplementation of 40μg/m L rutin or 10μg/m L baicalein increased the GPx activity.In H₂O₂injury cells,the extracts significantly increased the GPx activity except kaempferol.In both normal cells and H₂O₂injured cells,the extracts significantly increased the expression of Nrf2 gene except catechins.Quercetin,rutin and kaempferol significantly increased the level of keap 1a and HO-1 m RNA in both normal cells and H₂O₂injured cells.In summary,baicalein,catechin,rutin,kaempferol,quercetin and aucubin increased the activity of SOD,CAT and GPx and reduced MDA content in muscle cells,thereby protect grass carp muscle cells from oxidative injury induced by H₂O₂.Except catechin,the improved anti-oxidation of muscle cells by plant extracts might be associated with Nrf2 pathway.