Study On The Inhibitory Effect Of Camellia Nitidissima Chi Flower Extract On Pepsin And Trypsin In Vitro

Flavonoids,polyphenols,polysaccharides,and other active substances are abundant in Camellia nitidissima Chi flower,which has potential medicinal value in hypertension,obesity,and,hangover respects.Based on previous studies,C.nitidissima Chi flower extract have inhibitory effect on the activities of carbohydrate hydrolase and lipohydrolase,which can effectively reduce postprandial blood glucose and the intake of fat.Protein,carbohydrate,and fat are interactional in vivo.The excess energy will be converted into triglycerides,leading to obesity and other diseases,when the intake of productive substances is higher than that of body’s consumption.At present,the high protein diet has gradually become one of the dietary strategies to reduce weight,the obesity caused by it was always ignored.Therefore,it is of great significance to explore the interaction between C.nitidissima Chi flower extract and digestive protease to reduce the absorption of excess protein,in order to provide theoretical support for the development of C.nitidissima Chi flower weight loss products.This study uses C.nitidissima Chi flower as the raw material.Firstly,it is optimized to the extraction process of alcohol extraction and aqueous extraction of flavonoids from C.nitidissima Chi flower.Secondly,it is investigated to the effects of alcohol and aqueous extract of C.nitidissima Chi flower on the activity of digestive protease and its mechanism.Finally,it is stuied to the effects of C.nitidissima Chi flower extract on protein digestion by digestion experiments in vitro.The main results are as follows:（1）The determination of the components of the extract of C.nitidissima Chi flower,that can inhibit the digestive protease activity,and its extraction process optimized.The results showed that total flavonoids（34.677±0.867 and 23.967±0.705 g rutin（RE）/100 g）,polyphenols（10.148±0.116 and 7.559±0.206 g gallic acid（GAE）/100 g）,polysaccharides（34.752±0.570 and 45.185±0.098 g glucose/100 g）,and other physiologically active substances were bundant in the crude ethanol and aqueous extracts of C.nitidissima Chi flower.The inhibitory effect of flavonoids,that extracted from C.nitidissima Chi flower in the early stage,on digestive protease was better than that of crude polysaccharides,that extracted from C.nitidissima Chi flower.All the different sampling method can inhibit the activity of digestive protease in some extent,and the way of the preferential interaction between extracts and enzymes was more significant.The extraction processes of C.nitidissima Chi flower was optimized by single factor and orthogonal experiments:The yield of C.nitidissima Chi flower ethanol extract（CNEE）was 19.62%,when temperature was55℃,the ratio of material to liquid was 1:25 g/m L,extraction time was 5 h,and ethanol concentration was 50%.The yield of C.nitidissima Chi flower water extract（CNAE）was 16.56%,when temperature was 90℃,the material-to-liquid ratio was1:30 g/m L,and extraction time was 2 h.（2）It is investigated to the effects of ethanol extract（CNEE）and aqueous extract（CNAE）of C.nitidissima Chi flower on digestive protease activity and its mechanism.The results showed that CNEE and CNAE significantly inhibited pepsin(IC₅₀ of3.194 and 3.330 mg/m L,respectively)and trypsin(IC₅₀ of 29.131 and 56.534 mg/m L,respectively)activity,and the type of inhibition was mixed type.CNEE and CNAE can reduce the endogenous fluorescence of stomach protease（quenching constants were 1.522 and 1.205 m L/mg,respectively）and trypsin（quenching constants were2.175 and 1.392 m L/mg,respectively）,which cause the quenching phenomenon,leading to the change of eneyme conformation.Stern-Volmer quenching constants of pepsin affected by CNEE and CNAE decreased with the increase of temperature,the quenching type is static quencher.The result of ultraviple-visible absorption spectroscopy indicated that the interaction between CNEE or CNAE and digestive protease change the wavenumber and absorption strength of characteristic absorption peak of pepsin.The result of circular dichroism showed that CNEE and CNAE can change the secondary structure of pepsin fromα-helix and random coiling transform toβ-folding andβ-turning,the extract has little effect on the secondary structure of trypsin in the same concentration.（3）The effects of CNEE and CNAE on the hydrolysis of bovine hemoglobin,soy protein isolate and whey protein by digestive protease.In the initial stage of gastrointestinal digestion,compared with the blank group,the high molecular weight bands of the all proteins are more and high density in CNEE and CNAE groups.In the late stage of gastrointestinal digestion,compared with the CNEE and CNAE group,the number of the high and low molecular weight bands of the all proteins obviously decrease and increase in blank group,respectively,by SDS-PAGE.The results of the free amino groups content in the supernatant showed that the free amino content of bovine hemoglobin,soy protein isolate,and whey protein respectively fell by 33.61%,24.29%and 25.45%in the CNEE group.However,the free amino content of bovine hemoglobin,soy protein isolate,and whey protein content respectively fell by 20.17%,23.85%and 19.01%in the CNAE group,indicating that CNEE is storonger than CNAE to inhibit hydrolysis of the substrate protein.In this study,Using C.nitidissima Chi flower as raw material,the aimed is to investigate the effects of C.nitidissima Chi flower extracts on protease activity and proteolysis.It was proved that C.nitidissima Chi flower extracts had the properties of inhibiting protease activity in digestion and reducing redundant proteolysis.This not only provides a theoretical basis for the development of C.nitidissima Chi flower weight-loss products,but also promotes the development of C.nitidissima Chi flower industry towards high precision and deep processing.