The Study On The Processing Mechanism Of Fengyan Pieces For Relieving Dryness

1 ObjectiveFructus Aurantii is a commonly used medicine for regulating qi,and its pieces are generally produced by cutting into irregular arc strips according to the 2015 edition of the Chinese Pharmacopoeia method(the following is called Fructus Aurantii pieces).The Fengyan Fructus Aurantii pieces is the local characteristic pieces which is processed by Zhangbang method with unique preparation technique(the following is called Fengyan pieces).Compared with Fructus Aurantii pieces,Fengyan pieces is more moderate in its dryness which proves to be advantageous over those processed with pharmacopoeia method.In this experiment,the chemical components of Fengyan pieces and Fructus Aurantii pieces,and the differences of dryness and functions of invigorating spleen have been studied,and the method of molecular biology is used to discuss the mechanism of the main components which affect the dryness so as to identify the material basis and principle of the dryness relieving effect of the Fengyan pieces processing.2 Methods2.1 Ultra high performance liquid chromatography coupled with time-of-flight mass spectrometry to identify the chemical components of ethanol extracts from Fructus AurantiiThe components of 70% ethanol extract of Fructus Aurantii pieces were qualitatively analyzed by UHPLC-Q-TOF-MS/MS,and identified compounds in positive ion mode.2.2 Study on the different chemical components of the ethanol extracts of Fengyan pieces and Fructus Aurantii piecesThe HPLC fingerprints of Fructus Aurantii were established by HPLC and common peaks were selected as the characteristic fingerprint peaks.Compared with the Fructus Aurantii pieces.UPLC-Q-TOF-MS/MS wasfurther used to analyze the components of the Fructus Aurantii pieces and Fengyan pieces.The datas were analyzed by Markerview and SIMCA-P14.1software,using PCA,OPLS-DA analysis to discover the different components between Fengyan pieces and Fructus Aurantii pieces.2.3 Comparison of the effects of the ethanol extracts of Fengyan pieces and Fructus Aurantii pieces on improving gastrointestinal functionThe functional dyspepsia rats were selected as the animal model.The weight,food intake,intestinal propulsive rate,gastric residual rate,serum GAS,serum VIP,plasma MTL,and pathological changes of gastric tissue were taken as the indexes to study the effects of 70% ethanol extract of Fengyan pieces and Fructus Aurantii pieces on improving gastrointestinal function of model rats.2.4 Comparison of the effects of ethanol extracts and its extractions of Fengyan pieces and Fructus Aurantii pieces on the fluids indexIn this study,normal mice were selected as the study object,and water intake,whole blood viscosity,and submandibular gland AQP5 content were used as indexes to compare the effects of 70% ethanol extract of the Fengyan pieces and Fructus Aurantii pieces on fluids index.The functional dyspepsia rats were selected as model animals.The salivary flow rate and submandibular gland AQP5 content were used as indicators to compare the effects of 70%ethanol extract of the Fengyan pieces and Fructus Aurantii pieces on fluids index.The normal mice were selected as the study subjects.The whole blood viscosity and submandibular gland AQP5 were used as indicators to compare the effects of the different extractions of the Fengyan pieces and Fructus Aurantii pieces on fluids index.2.5 Analysis of the relationship between AQP5 and the different components of ethanol extracts of Fengyan pieces and Fructus Aurantii piecesOrthogonal partial least squares method was used to find the different component closely associated with AQP5 from Fengyan pieces and Fructus Aurantii pieces,and the content of this compound was negatively correlated with AQP5 content,The HPLC method was used to verify the mainly fraction that differential components belonged to.2.6 The effect of extractions and compounds of Fengyan pieces and Fructus Aurantii pieces on AQP5 content of HSG cellsThe human submandibular gland cell HSG was used as the research object,and the expression of AQP5 mRNA in HSG cells was used as an index to study the differences of the effects of methylene chloride,ethyl acetate,n-butanol,and water extraction of Fengyan pieces and Fructus Aurantii pieces in inhibiting of AQP5 mRNA expression,and the expression of AQP5 protein in HSG cells was used as an index to study the differences of the effects of ethyl acetate of Fructus Aurantii pieces,3',4',3,5,6,7,8-heptamethoxyflavone and other compounds in inhibiting of AQP5 protein expression.3 Results3.1 Ultra high performance liquid chromatography coupled with time-of-flight mass spectrometry to identify the chemical components of ethanol extracts from Fructus Aurantii46 compounds were identified in positive ion mode by UHPLC-Q-TOFMS/MS.There were 33 flavonoids,such as lonicerin-7-rhamnoglucoside,neoeriocitrin,eriocitrin,naringin,narirutin,naringenin,naringenin-7-O-glucoside and so on;10 coumarins,such as umbelliferone,eriodictyol,meranzin,meranzinhydrate,xanthotoxo I,prangenin and so on.Besides,limonin,nomilin belongs to lomonins and ferulic acid belongs to total phenolic acids.The base peak map and two fragments of ion data were combined to infer mass spectrometry cleavage rules of naringin,5-demethylnobiletin,3',4',3,5,6,7,8-heptamethoxyflavone,and so on.3.2 Study on the different chemical components of the ethanol extracts of Fengyan pieces and Fructus Aurantii piecesThe HPLC fingerprints of Fructus Aurantii were established by HPLC and 11 common peaks were selected as the characteristic fingerprint peaks.Compared with the Fructus Aurantii pieces,the similarity of the Fengyan pieces was between 0.793 and 0.886,showing greater differences.UPLC-Q-TOF-MS/MS was further used to analyze the components of the Fructus Aurantii pieces and Fengyan pieces.Discover the different components between Fengyan pieces and Fructus Aurantii pieces,such as umbelliferone,naringenin-7-O-glucoside and 3',4',3,5,6,7,8-heptamethoxyflavone,limonin.3.3 Comparison of the effects of the ethanol extracts of Fengyan pieces and Fructus Aurantii pieces on improving gastrointestinal functionThe results showed that the high,medium and low dose groups of Fructus Aurantii pieces and Fengyan pieces can increase the weight(P<0.01)and food intake of model rats.There were no differences in the body weight of the low dose group and the food intake of the high,medium and low dose groups(P>0.05).The high,medium and low dose groups of Fructus Aurantii pieces,the high dose group of Fengyan pieces can increase small intestine advancing rate of model rats and reduce gastric residual rate,and the differences were statistically significant(P<0.01 or P<0.05).The high,medium and low dose groups of Fructus Aurantii pieces and Fengyan pieces can increase GAS content of model rats(P<0.01).The GAS content of medium dose group of Fengyan pieces was higher than that of Fructus Aurantii pieces.The high,medium and low dose groups of two kinds of pieces can significantly increase the model rats' MTL content(P<0.01).All dose groups of the two kinds of pieces can reduce the VIP value in model rats(P<0.01);this effect was weakened after being fried with wheat bran.The high,medium andlow dose groups of Fengyan pieces were lower than that of Fructus Aurantii pieces in VIP value,and closed to the nomal group.The results of pathological sections showed that there was mild inflammation in gastric tissue of rats in the model group,but no obvious pathological changes were observed in the administration group.3.4 Comparison of the effects of ethanol extracts and its extractions of Fengyan pieces and Fructus Aurantii pieces on the fluids indexThe result showed that the water intake of high and low dose groups of Fructus Aurantii pieces was higher than that of the normal group,and the difference was statistically significant(P<0.01).The water intake of high and low dose groups of Fengyan pieces was lower than normal group,and the difference was statistically significant(P<0.01).The high and low dose groups of the ethanol extracts of two kinds of pieces can improve the whole blood viscosity of normal mice.Compared with the normal group,the difference in whole blood viscosity of the high and low groups of Fructus Aurantii pieces was significant(P<0.01),and the whole blood viscosity value of Fructus Aurantii pieces was higher than Fengyan pieces.Normal mice submandibular gland AQP5 content can be reduced by the high and low dose groups of two kinds of pieces.Compared with the normal group of submandibular gland AQP5 content,there was significant difference in the high group of Fructus Aurantii pieces(P<0.05),no difference was found in the high and low group of Fengyan pieces(P>0.05).According to the results,the high,medium,low dose groups of two kinds of pieces can increase saliva flow rate(P<0.01)and submandibular gland AQP5 content in functional dyspeptic rats.The group of Fengyan pieces was higher than group of Fructus Aurantii pieces in saliva flow rate and content of submandibular gland AQP5.And saliva flow rate was statistically significant(P<0.01).According to the result,the stronger extractions of increasing the whole blood viscosity and Down-regulating AQP5 content were the ethyl acetate extraction and methylene chloride extraction.3.5 Analysis of the relationship between AQP5 and the different components of ethanol extracts of Fengyan pieces and Fructus Aurantii piecesAccording to the results,the normalized regression coefficients obtained from the orthogonal partial least squares analysis showed that 3',4',3,5,6,7,8-heptamethoxyflavone was negatively correlated with AQP5,and 3',4',3,5,6,7,8-heptamethoxyflavone contributed the most in the negative correlation components.And naringin-7-O-glucoside,limonin and other conponents were positively correlated with AQP5.The HPLC method was used to verify that3',4',3,5,6,7,8-heptamethoxyflavone was mainly present in the dichloromethane extraction,followed by the ethyl acetate extraction.3.6 The effect of extractions and compounds of Fengyan pieces and Fructus Aurantii pieces on AQP5 content of HSG cellsAccording to the result,Fructus Aurantii pieces had stronger inhibitory effect than Fengyan pieces,and the ethyl acetate extraction of Fructus Aurantii pieces had stronger inhibitory effect than its other extractions.Using AQP5 mRNA expression and AQP5 protein expression as indicators,the effects of3',4',3,5,6,7,8-heptamethoxyflavone and ethyl extraction on AQP5 were studied.Western blot analysis showed that 3',4',3,5,6,7,8-heptamethoxyflavone significantly inhibited AQP5 protein expression.(P<0.05).The effects of ethyl acetate extraction and hesperidin were not significant(P>0.05).4 Conclusion4.1 Ultra high performance liquid chromatography coupled with time-of-flight mass spectrometry to identify the chemical components of ethanol extracts from raw Fructus Aurantii pieces processed withpharmacopoeia methodsA total of 46 compounds were identified in the UHPLC-Q-TOF-MS/MS positive ion mode.About these ingredients,mainly flavonoids,followed by coumarins,limonoids and total phenolic compounds.Among them,the compounds of eriodictyol,naringenin,neohesperidin,5-demethylnobiletin,3',4',3,5,6,7,8-heptamethoxyflavone,auraptene and umbelliferone were identified by the reference substances.The mass spectrometry cleavage of some of these compounds was deduced.4.2 Study on the different chemical components of the ethanol extracts of Fengyan pieces and Fructus Aurantii piecesFour different components of the alcohol extract of “Fengyan pieces” and Fructus Aurantii pieces were found and identified by UHPLC-Q-TOF-MS/MS technology,Markerview and SIMCA-P14.1 software,including umbelliferone,3',4',3,5,6,7,8-heptamethoxyflavone,naringenin-7-O-glucoside,limonin.4.3 Comparison of the effects of the ethanol extracts of Fengyan pieces and Fructus Aurantii pieces on improving gastrointestinal functionThe ethanol extracts of Fengyan pieces and Fructus Aurantii pieces can improve the body weight,food intake,small intestine advancing rate,GAS,MTL,VIP levels and mild inflammation of stomach tissue in functional dyspepsia rats.The small intestine advancing rate of Fructus Aurantii pieces group was higher than that of Fengyan group,and there was no significant difference between high and low dose groups.The gastric residual rate of Fructus Aurantii pieces group was lower than that of Fengyan group,and there was no significant difference in medium dose group.The GAS value of Fructus Aurantii pieces group was higher than that of Fengyan pieces,and there was no significant difference in low dose group.The MTL value of Fructus Aurantii pieces group was significantly higher than that of Fengyan pieces group.The VIP value of Fengyan pieces group waslower than Fructus Aurantii pieces group and closer to the normal value.In general,the Fengyan pieces were equivalent to or better than the Fructus Aurantii pieces in some indicators.4.4 Comparison of the effects of ethanol extracts and its extractions of Fengyan pieces and Fructus Aurantii pieces on the fluids indexEffect of the ethanol extracts of Fructus Aurantii pieces was stronger than that of Fengyan pieces in improving the blood viscosity and water intake of normal mice and reducing the content of AQP5 in submandibular glands of normal mice.The effect of the Fructus Aurantii pieces ethanol extracts on the saliva flow rate and AQP5 content of model rats was weaker than that of Fengyan pieces ethanol extracts,and the Fructus Aurantii pieces ethanol extracts showed stronger dryness.The main extractions for increasing the whole blood viscosity and lowering the AQP5 content in the submandibular gland were dichloromethane extraction and ethyl acetate extraction.4.5 Analysis of the relationship between AQP5 and the different components of ethanol extracts of Fengyan pieces and Fructus Aurantii piecesThe analysis showed that the component with the greatest negative correlation contribution to AQP5 was 3',4',3,5,6,7,8-heptamethoxyflavone,and the positively related components was naringin-7-O-glucoside,limonin,etc.From this,it was inferred that the dryness relieving effect of Fengyan pieces might be related to reducing the content of 3',4',3,5,6,7,8-heptamethoxyflavone and other components,and increasing the content of naringin-7-O-glucoside,limonin etc.4.6 The effect of extractions and compounds of Fengyan pieces and Fructus Aurantii pieces on AQP5 content of HSG cellsIn terms of the effect of inhibiting AQP5 mRNA expression in human submandibular gland cells,Fructus Aurantii pieces was stronger than Fengyanpieces,and the ethyl acetate extraction of the Fructus Aurantii pieces fried with wheat was stronger than its other extractions.The effect of3',4',3,5,6,7,8-heptamethoxyflavone,hesperidin on inhibiting the expression of AQP5 mRNA in human submandibular gland cells was stronger than neohesperidin,naringin and the mixture of neohsperidin and naringin.3',4',3,5,6,7,8-heptamethoxyflavone can significantly inhibit the expression of AQP5 protein in HSG cells,but the inhibitory effect of hesperidin was not obvious.It was initially determined that 3',4',3,5,6,7,8-heptamethoxyflavone was one of the dry ingredients of Fructus Aurantii.The dryness relieving effect of the Fengyan pieces might be related to reducing the content of the substances dry in nature such as 3',4',3,5,6,7,8-heptamethoxyflavone.In summary,the comparison of results of invigorating spleen effect showed that the efficacy of Fengyan pieces and Fructus Aurantii pieces was similar;the comparison of dryness results showed that the Fengyan pieces was moderate than the Fructus Aurantii pieces,and the process of Fengyan pieces can relieve dryness.This project preliminarily clarified the main material basis and the action mechanism of key components for the dryness relieving effect of Fengyan pieces processed with Zhangbang technology.The results can provide a scientific basis for the dryness relieving effect of the Fengyan pieces processing and references for further optimization of Fengyan pieces processing technology.