Study On Quality Evaluation Of Amomum Villosum Based On Visualization Of Appearance Traits,Component Analysis And Pharmacodynamics

ObjectiveBeginning with traits and microscopic identification,combined with image processing technology,the objective and quantitative methods of Amomum villosum were explored to solve the limitation of subjectivity of traditional experience identification and inconsistent standards.At the same time,the study on the non-volatile components of Amomum villosum,including determination of total phenolic content and it's anti-oxidative activity of Amomum villosum in different producing areas,establishment of HPLC fingerprints of Amomum villosum in different producing areas and establishment of UPLC-ESI-QTOF-MS qualitative analysis of Amomum villosum of non-volatile components,are aimed to evaluate the differences in composition of Amomum villosum from different regions.In addition,compared the differences in pharmacodynamics of Amomum villosum with different qualities by the experiments of gastrointestinal motility and diarrhea in mice.A variety of quality control models for Amomum villosum were established from the aspects of quantification of traits,composition analysis,and efficacy comparison.Exploring rapid,efficient,and accurate quality evaluation of Amomum villosum to evaluate the quality of Amomum villosum in circulation.At the same time,we explored the proximity of Amomum villosum introduced in Yunnan in terms of traits,composition and curative effect to the authentic Amomum villosum.It provides a theoretical basis for the cultivation and comprehensive development of Amomum villosum in China.Methods1.Quantitative Study of Amomum villosum based on characterization and microscopic identificationCombining with the image processing technology,the peels of Amomum villosum were embedded in polyethylene glycol respectively,then the microtome pieces were sliced and photographed after enlargement.The thickness of peels in different areas were measured by Image pro plus?IPP?.In addition,Amomum villosum samples were taken randomly by a quartering method and Amomum villosum pictures were taken with a single-lens reflex camera.The operating programs were combined and writed based on the operating functions provided by the Matlab 2014a software and the characteristics of the image.Then calculated the parameters of shape,color and texture of Amomum villosum through the use of the same program.And finally statistical methods were used to analyze the differences between Amomum villosum from different habitats.2.Quality evaluation of Amomum villosum based on non-volatile constituentsWith the content of total phenols as index,solid-liquid ratio,extraction time and times on extraction technology of total phenols were investigated by L₉?3⁴?orthogonal test,in order to optimize the extraction techonology.Folin-Ciocalteu Colorimetry was adopted to determine the content of total phenols in Fructus Amomi by taking vanillic acid as a reference.DPPH assay,ABTS assay and FRAP assay were employed to analyze the antioxidant activities.And the statistical analysis was used to determine the correlation between the total phenolics content and antioxidant activity of Amomum villosum.The HPLC fingerprints of Amomum villosum in different habitats were established and the quality of Amomum villosum in different habitats was evaluated through the methods of similarity evaluation,cluster analysis and PCA.The UPLC-ESI-QTOF-MS method was established for the determination of the non-volatile components of Amomum villosum.Combined with the information of secondary fragment ions and reported in the literature,we speculate the possible constituents and identify the non-volatile constituents of Amomum villosum by standard comparison.3.The Pharmacodynamic study on different quality Amomum3.1 Experimental study on the effect of Amomum villosum aqueous extract on Gastrointestinal MotilityThe samples of Amomum villosum from three different habitats were Yunnan,Yangchun and Burma.Sixty-four Kunming mice were randomly divided into 8 groups,each of which was set high and low dose groups,positive drug?Mosapride citrate?and blank control group.The modified phenol red paste method was adopted to measure the food gastric emptying,the gastric residual of phenol red and the propulsion of small intestine in each experimental group.The purpose is to compare the different origin of Amomum villosum on gastric emptying and intestinal propulsion.3.2 Antidiarrheal effect of volatile oil of Amomum villosum on mice induced by sennaSeventy-two male Kunming mice were randomly divided into 9 groups.The Amomum villosum were set high and low dose groups,the other group was positive?berberine hydrochloride?group,model group and blank group.The diarrhea index was calculated on wet diarrhea mice model induced by senna and analyzed statistically.Results1.Quantitative Study of Amomum villosum based on characterization and microscopic identification?1?The thickness of peel of 22 batches of Amomum villosum from different habitats were measured and arranged in ascending order of magnitude:Amomum villosum from Yunnan,Amomum villosum,fake Amomum,A.villosum Var.xanthioides,imported Amomum.The 22 batches of Amomum can be divided into two categories according to the cluster analysis.The 5 batches of imported Amomum and 2 batches of Alpinia zerumbet were classified as one group,and the other 17 Amomum villosums were classified as another group.It can be seen that this method can distinguish the imported Amomum and Alpinia zerumbet from the other Amomum Fructus and provide reference for the quality evaluation of Amomum villosum.?2?The parameters of shape?aspect ratio,squareness,circularity?,color?hue,saturation?and texture?grayscale texture,hue texture,saturation texture?of 22 different areas Amomum villosum were calculated.In combination with the cluster analysis method,the parameters of shape can be used to distinguish the Alpinia zerumbet,long strips of imported Amomum from the elliptic or oval-shaped Amomum.The color characteristics can clearly identify the differences between Alpinia zerumbet,some fake Amomum and other samples of Amomum villosum.The texture features can clearly distinguish the imported Amomum,the fake Amomum,and the Alpinia zerumbet from the Amomum villosum Lour.and the A.villosum Var.xanthioides.This helps to identify pharmacopoeia Amomum and non pharmacopoeia Amomum.The principal component analysis of the three characteristics?shape,color,and texture?of Amomum villosum to evaluate the Amomum villosum samples was more accurate for identification of the Amomum villosum Lour.,A.villosum Var.xanthioides.and counterfeit goods non collected by the Pharmacopoeia,and the results are consistent with the results of cluster analysis using texture feature parameters.2.Quality evaluation of Amomum villosum based on non-volatile constituents?1?The concentration of vanillic acid had a good linear relationship in range of0.0020⁰.0119 mg.mL^-1.The average recovery was 96.6%?RSD=3.3%,n=6?.The antioxidant activities determined by three different methods were all significantly and positively correlated with total phenol?P<0.01?.The correlation coefficients were 0.964,0.965and 0.958,respectively.?2?The established HPLC fingerprints of Amomum villosum had 29 same peaks,and the similarity of 22 Amomum villosum samples from different areas was between 0.548 and0.944.Through the cluster analysis method,the differences between Alpinia zerumbet,imported Amomum and Amomum villosum Lour.,A.villosum Var.xanthioides.can be distinguished.However,the differences between Amomum villosum produced in Yunnan cannot be completely separated from Alpinia zerumbet and imported Amomum.?3?A UPLC-ESI-QTOF-MS method was developed for the determination of non-volatile components of Amomum villosum,and 12 non-volatile components were identified.It identifies the differences between Alpinia zerumbet,as well as distinguishes Amomum villosum in different regions to some extent.3.The Pharmacodynamic study on different quality Amomum3.1 Experimental study on the effect of Amomum villosum aqueous extract on Gastrointestinal MotilityMosapride citrate,Yunnan and Amomum villosum aqueous extract can promote gastric emptying in normal mice.Water extract of Amomum villosum in Yunnan and Yangchun can promote gastric emptying better than Mosapride citrate.Mosapride citrate,aqueous extract of Amomum villosum in Yunnan and Yangchun can promote the small intestine propulsion with the same effect and have no significant difference between them.However,the aqueous extract of Amomum villosum from Burma had no significant effect on gastric emptying,there was no difference with the blank mice.And the phenol red was promoted to small intestine?segment.It had significant difference compared with the blank group.3.2 Antidiarrheal effect of volatile oil of Amomum villosum on mice induced by sennaBerberine hydrochloride,high-dose volatile oil of Amomum villosum from Yangchun and Yunnan can effectively reduce the diarrhea index of mice with diarrhea,andit have statistical significance.However,the volatile oil of Amomum villosum in Burma also reduced the diarrhea index,but there was no significant difference compared with the model group.In addition,there is no significant difference in the anti-diarrheal effect between volatile oil of Amomum villosum from Yangchun and Yunnan.The results showed that the essential oil of Amomum villosum in Yunnan and Yangchun had anti-diarrhea effects on the mice induced by Senna.ConclusionBased on the character and microscopic identification of Amomum villosum and combined with image processing technology,the data measurement of appearance characteristics of Amomum villosum was achieved.The peel thickness distinguishes the imported Amomum and the Alpinia zerumbet from the other Amomum samples.The shape,color or texture feature parameters can be used to identify the differences between Alpinia zerumbet,imported Amomum and other samples.However,the method of combining texture feature parameters or combining three kinds of feature parameters has a better effect on the identification of Amomum villosum Lour.,A.villosum Var.xanthioides contained in the pharmacopoeia and the non-pharmacopoeia contained counterfeit goods?imported Amomum,fake Amomum,and Alpinia zerumbet?.The established HPLC fingerprint and UPLC-ESI-QTOF-MS analysis method can clearly identify the differences of Alpinia zerumbet,and it can be used to distinguish different varieties of Amomum villosum to some extent.The total phenolic content of Amomum villosum from different origins has significant differences and has certain antioxidant activity.It needs to be further controlled and developed.The Amomum villosum from Yangchun and Yunnan both have a good effect on gastrointestinal motility and antidiarrhea and there is no significant difference,and it has a good two-way adjustment effect on the gastrointestinal tract.The efficacy of Amomum from Burma is relatively poor.The established method can effectively distinguish Amomum villosum and reflect the quality of amomum.The imports of Amomum villosum from Southeast Asia are quite different from Amomum villosum from Yangchun.Coupled with factors such as chaotic varieties and low quality qualification rate,it is suggested to strengthen market supervision and strictly control the source of imported Amomum villosum.The Amomum villosum from Yunnan is very close to authentic Amomum villosum in terms of traits,composition and efficacy.Therefore,we should actively promote the development of the Amomum villosum in Yunnan to meet more needs.