Quality Evaluation Of Amomum Villosum Based On Metabolic Absorption Of Gastrointestinal Tract In Decoction

[Research Background]As fructus amomi is a valuable southern medicine and traditional bulk medicine,the problem of its varietal complexity has always been there.In recent years,the fact that the fructus amomi has been in a state of short supply provides an opportunity for the substitutes or adulterants of fructus amomi with the similar species to flow into the market,which will undoubtedly affect the medicinal quality of fructus amomi and its safe and effective use.At present,the research on the quality standards of fructus amomi is mainly concentrated on the volatile oils,while the study of non-volatile components in fructus amomi is rare.In the 2015 edition of the Chinese Pharmacopoeia,the content of Amomum villosum was determined by the total amount of volatile oil and a single component of bornyl acetate.However,most of the substitutes or adulterants come from similar species of fructus amomi,such as Amomum aurantiacum,and both of them contain bornyl acetate,and the content of bornyl acetate meets the requirements of the Chinese Pharmacopoeia.In this way,it is difficult to distinguish authenticity.Meanwhile,identification the true or false in nature and good or bad in quality of fructus amomi relying on only one indicator component not only is exclusive,but also ignores the integrity and efficacy of fructus amomi as a traditional Chinese medicine.The common clinical application of Amomum villosum is "put in water after boiling," in which"water decoction" emphasizes the non-volatile oil components that can be decocted in fructus amomi.It has been shown in the literature that some of these compounds existing in fructus amomi have similar efficacy and all can be absorbed into the blood,so it is necessary to study the composition of decoction.At present,comprehensive evaluation method which effectively combine pharmacodynamic components according to in-vivo drug metabolism and quality standards has been accepted by more and more scholars.Of course,the in vivo metabolism of fructus amomi has not seen reported.The metabolism and absorption of the gastrointestinal tract is the first gateway in which the drug enters the body and produces the curative effect.Only the drugs are absorbed into the blood and participate in the circulation of blood to produce pharmacological action.Therefore,based on previous literature,this research is intended to use gastrointestinal metabolism to find the components in decoction of fructus amomi that can be absorbed into the blood to improve the existing standard of fructus amomi.[Purpose and Significance]1.The metabolism and absorption of the gastrointestinal tract is the first gateway where drugs enter the body and produce a therapeutic effect.The drugs can only be absorbed into the blood and participate in the blood circulation to produce pharmacological effects.According to the literature,some of the components of each type of compound in fructus amomi have similar efficacy with fructus amomi and all can be absorbed into the blood and work.In general,structurally similar components have similar physical and chemical properties and pharmacological effects.Therefore,the search for medicinal substances of fructus amomi's decoction ingredients can utilize the gastrointestinal metabolism method.In the study,Single-pass intestinal perfusion method was used to find out the components of decoction of Amomum villosum that could be absorbed by gastrointestinal metabolism.The absorbed components were used as medicinal components and the contents of medicinal materials were determined.The results were combined with traditional traits data and the gas component content specified in the standard,and these three sets of data are used for correlation analysis to determine the rationality of the components found as quality standards.According to the characteristics of clinical application of Amomum villosum to study the quality standards of Amomum jasminoides,it is beneficial to improve the quality evaluation of amomum.2.To a certain extent,this research method is in line with the actual practice of Amomum medicine,compared with the current evaluation methods of "content theory".It is more conducive to the improvement of the quality evaluation of Amomum villosum,and it provides reference for the study of traditional Chinese medicine on the premise of the practical application of traditional Chinese Medicine.3.In this study,a grey relational analysis was performed to calculate the correlation between the found components and the traditional traits.It is proposed that the components with greater relevance to the traditional traits be used as indicator components for quality control of medicinal materials to improve the existing quality better.[Content and Method]1.Sample collection and basic data measurementCollects Amomum villosum with different varieties in the markets and describes the traits according to the "Chinese Pharmacopoeia"(15th edition)to determine the species initially.According to the description of "Great,solid,full and strong smelling",we measure the weight of 100 fruits,longitudinal diameter,transverse diameter,longitudinal diameter/transverse diameter and electronic nose odor value;2.Identifying varieties using DNA molecular identification techniques;DNA samples were extracted from Amomum villosum seeds using a plant DNA extraction kit and sent to Sangko(Shenggong Bioengineering Co.,Ltd.)for sequencing.ContigExpress was used to sequence the measured sequences to obtain a mosaic sequence,the mosaic sequence was inserted into NCBI to determine the original.3.Determination of the liquid phase conditions of fructus amomi's decoction;4.Metabolism of Gastrointestinal tract digestion fluid in fructus amomi's decoction;The human digestive fluid was simulated with artificial gastric fluid and artificial intestinal fluid.The composition peak area of the ingredients in the decoction were measured at 0,1,2,and 4 h in gastric fluid,and at 0,2,4,and 6 h in the intestinal fluid respectively.Calculate the rate of change f = Abs(Ai-Ao)/Ai*100%.When a rate of change>10%,it means that the drug has absorption.5.Single-pass intestinal perfusion method to study the metabolism and absorption of fructus amomi's decoction in the intestine.Single-pass intestinal perfusion was used to perfuse the duodenum,jejunum and ileum of rats.The perfusion time was 3 hours.The liquid began to collect at the beginning of 45 minutes,and the liquid was collected every 15 minutes.Measure the peak area in liquid phase and calculate the apparent absorption coefficient(Papp).6.Analyse correlation between absorbed components and measured indicators,and analyse the rationality of using absorbable decoction components as quality control standards.Determine the 13 batches of purchased Amomum villosum medicinal materials' peak areas in HPLC,and calculate contents and relative contents.The results were combined with traditional traits data and the gas component content specified in the standard,and these three sets of data are used for correlation analysis to determine the rationality of the components found as quality standards.[Result]1.In combination with the traditional trait identification and DNA molecular identification methods,the species of fuctus amomi was finally identified.No.1,2,7,8,9,10,and 13 were Amomum villosum Lour.,No.3,4,5,6,11 and 12 were Amomum longiligulare T.L.Wu.2.In the decoction of fructus amomi,19 components were calibrated.These 19 components were studied for the stability of artificial gastric fluid for 4 hours and artificial intestinal fluid for 6 hours.The results showed that they were stable in both simulated digestive fluids.3.In Single-pass intestinal perfusion experiments,19 calibration components in the decoction of fructus amomi showed different absorption in different intestinal segments.Components that exhibit strong absorption characteristics in the duodenum are C1,C2,C3.C4,C5,C6,C7,C8,C11,C12,C13,C14,C16,C17,C18,C19;C10 in the duodenum showed moderate absorption characteristics;while C9 and C15 were less absorptive in the duodenum,less than 0.03×10-4 cm·s-1,which could be considered as no absorption.The absorption characteristics of 19 components in the jejunum:C1,C2,C3,C5,C6,C7,C8,C11,C12,C13,C16,C17 showed strong absorption,and C4,C14 were moderate absorption,while C9,C10,C15,C18,C19 have no absorption.In the ileum,all 19 components were found to be absorptive,except that the C10 component exhibited moderate absorption characteristics,and the other components were strongly absorptive.The absorption of types of fructus amomi's components in the three small intestine segments was compared:ileum>duodenum>jejunum.The absorption capacity of components C3,C5,C7,C11,C17 in the small intestine was duodenum>jejunum>ileum.Absorption capacity of components C2,C4,C10,C13,C14,C16,C18,C19 in the small intestine was ileum>duodenum>jejunum.Absorption capacity of components C1,C8 in the small intestine was duodenum>ileum>jejunum.For C6,intestinal absorption capacity from strong to weak is jejunum>ileum>duodenum.For C12,intestinal absorption capacity from strong to weak is jejunum>duodenum>ileum,while components C9 and C15 are only absorbed in the duodenum and are strongly absorbed.In a comprehensive way,it is true that there are indeed components closely related to the traits and efficacy of fructus amomi in water decoction of fructus amomi.19 target water soluble components are selected through the absorption of the intestines.According to the literature and the traditional efficacy of fructus amomi,it is proposed to use it as one of the index components of the quality evaluation of fructus amomi.In addition,the chemical formulas of the six components were determined by the standards:C4(catechin),C7(vanillic acid),C8(epicatechin),C11(polydatin),C13(isoquercitrin),C14(quercitrin).4.Compared with the HPLC spectrum of 13 batches of fructus amomi,Amomum villosum Lour.'s spectral peak diagram was distincently different from Amomum longiligulare T.L.Wu.after 45 min.In Amomum villosum Lour.,there were four components of C15,C16,C18 and C19 more than Amomum longiligulare T.L.Wu.,and these components could be absorbed in the intestinal perfusion experiment.The reason why Amomum villosum Lour.'s quality is better than that of Amomum longiligulare T.L.Wu.might be there are more kinds of components in the former.Of course,we can also use these four types of ingredients to distinguish between different cultivars of fructus amomi.5.The results of grey relational analysis from chemical composition and character identification showed that the correlation degree was in descending order:borneol(r=0.7511)>quercetin(r=0.7446)>camphor(r=0.7107)>limonene(r=0.6817)>C1(r=0.5684)>bomyl acetate(r=0.5398)>C17(r=0.5054).It can be seen that the borneol has the greatest influence on the characters,followed by quercetin,camphor and limonene,while the correlation between borneol acetate and characters is not very high,accounting for the penultimate position among the seven components tested.[Conclusion]1.In this study,the Gray Relational Analysis of the intrinsic and traditional traits of fructus amomi was carried out.Finally,the partial components that can be absorbed by the gastrointestinal tract and have pharmacological activities in the decoction were identified,and their correlation with the traits was greater than the existing content determination index of bomeol acetate.The results showed that there are indeed components closely related to the properties and efficacy of fructus amomi in water decoction of Amomum villosum.Speaking from the data and talking with the facts,it is fully verified the necessity and feasibility for more in-depth research on the components and pharmacological effects of fructus amomi 's decoction.It is indeed scientific and feasible to establish quality standards for Chinese herbal medicines from the clinical use characteristics of Chinese herbal medicines,and introduce selected components of water decoction into fructus amomi's quality standards.This study finally only completed the correlation analysis of the partial components of the water decoction,and the correlation of these components showed that the improvement of quality standards of Amomum villosum can be divided into two aspects:one is to perfect the volatile oil on the basis of "decocted later",and can add three indexes with high relative content,clear pharmacological action and stronger correlation with traditional characters,which includes borneol,camphor and limonene.the other is non-volatile components on the basis of "decoct with water" can be added,namely the quercitrin and C1 components in this paper.2.To guide the establishment of quality standards for Chinese herbal medicines on the basis of the clinical use characteristics of Chinese herbal medicines,our study use the in-vivo metabolic methods to study the metabolic status of traditional Chinese medicine ingredients in the body,and use the ingredients that can enter the body and produce therapeutic effects as one of the criteria for the establishment of Chinese herbal medicine standards,which provides new research ideas on how to improve the current quality standards.