Herbal Textual Research And Study On Quality Evaluation Of Chinese Materia Medica Of ’The Longer,The Better’

Research background There is a record in Shennong’s Classic of Materia Medica that ’Dry in the shade or in the air,the true and the false,and storage for long-term or short-term,has their own methods’.In ’A Variorum of Medicinal Properties of Plants(also called Bencaojingjizhu)’ written by Tao Hongjing in Liang dynasty,a view of ’the longer the better’ was pointed out for six Chinese Materia Medicas(CMMs),those are Ephedrae Herba(Mahuang),Citri Reticulatae Pericarpium(Chenpi),Aurantii Fructus(Zhiqiao),Pinelliae Rhizoma(Banxia),Euphorbiae Ebracteolatae Radix(Langdu),Euodiae Fructus(Wuzhuyu),also called ’LiuDaChenYao’.Since the 1990s,researchers have carried out researches and discussion on the theory of ’the longer,the better’.However,there are few literatures,and there is no report on the systematic herbal textual research of this theory from the perspective of the herbal literatures.In addition,there is no literatures on the actual production of CMMs in genuine producing areas.As for the research on the quality evaluation of aged CMMs,there are more researches on ’LiuDaChenYao’.The researches on Citri Reticulatae Pericarpium(Chenpi)are the most.The color,smell,volafewtile components,flavonoids and pharmacological activities of Chenpi have been studied.However,there are some problems such as unclear research varieties and unsystematic research contents.researches on other aged CMMs are few,and there are some problems,such as unclear research varieties and single evaluation index.Therefore,this present study carried out the systematic herbal textual research on the theory of ’the longer,the better’ firstly and chosen Citri Reticulatae Pericarpium(Chenpi),Citri Reticulatae Pericarpium Viride(Qingpi),and Aurantii Fructus(Zhiqiao)as the research objects for field investigation.A variety of analytical techniques were used to determine the physical and chemical properties of the three CMMs,and the quality evaluation was carried out to clarify the value and mechanism of aged CMMs,and to lay a theoretical and data foundation for the further research of CMMs of ’the longer,the better’.Research contents Part 1 Herbal textual research and field investigation of CMMs of ’the longer,the better’ The study methods were found and read the Literature of Materia Medica by website,references,e-books,and ancient books,summarized records about ’the longer,the better’ according to the order of dynasties,and analyzed these records.According to the results of literatures,the field survey on Citri Reticulatae Pericarpium(Chenpi),Aurantii Fructus(Zhiqiao),Pinelliae Rhizoma(Banxia),Euodiae Fructus(Wuzhuyu),Artemisiae Argyi Folium(Aiye)were carried out in Xinhui County of Guangdong Province,Zhangshu City of Jiangxi Province,Qichun County of Hubei Province,Anguo county of Hebei Province,etc.Part 2 Study on Quality Evaluation of CMMs of ’the longer,the better’1 Multi-directional evaluation of the quality of Chenpi stored for different years by multiple analysis techniques A total of 12 batches of Chenpi stored from 1 to 23 years were collected as the research sample.A colorimeter,E-nose and E-tongue were used to detect the changes of color,odor and taste.The high-performance liquid chromatography(HPLC)was used for comparative study of fingerprints.The untargeted metabolomics based on ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)combined multivariate statistical analysis was used to determined and screened differential compounds.An HPLC method was established and used to detect the contents of hesperidin,nobiletin and tangerine were detected.The content of total phenolics(TPs),total flavonoids(TFs),the antioxidant activity based on 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine(DPPH)and ferric-reducing antioxidant potential(FRAP)methods were determined and evaluated by ultraviolet spectrophotometry.The headspace solid-phase microextraction gas chromatographymass spectrometry(HS-SPME-GC-MS)were used to detect the types and relative contents of volatile components.In addition,the correlation between color,odor,and taste were calculated with bivariate correlation analysis.2 Dynamic evaluation of the quality of Qingpi stored for different years Qingpi was stored for 5 years.The color and odor were detected by a colorimeter and E-nose.The contents of hesperidin,nobiletin and tangerine were determined by HPLC method.The content of TPs and TFs were determined by ultraviolet spectrophotometry.The untargeted metabolomics based on UPLC-QTOF-MS combined with multivariate statistical analysis was used to screed differential compounds.The HS-SPME-GC-MS was used to determine the volatile components.The antioxidant activity was determined and evaluated based on DPPH and FRAP methods.3 Dynamic evaluation of the quality of Zhiqiao stored for different time in different storage environment Three kinds of storage environment were established:low temperature and medium humidity(LTMH),room temperature and room humidity(RTRH),and high temperature and high humidity(HTHH).The physical and chemical properties were determined dynamically in the 0,1,3,5,and 9 months.The color change was determined by a colorimeter.The contents of narirutin,naringin,neohesperidin,tangeretin,and nobiletin were determined by an HPLC method.The contents of TPs and TFs were determined with ultraviolet spectrophotometry as well as the antioxidant activity based on the DPPH and FRAP methods.The one-way analysis of variance(ANOVA)was used to compared the significance between data.The bivariate correlation analysis was used to evaluate the correlation between data.Results Part 1 Herbal textual research and field investigation of CMMs of ’the longer,the better’ The results showed that there were almost 90 CMMs were recorded in Literature of Materia Medica which included the six Chinese Materia Medica,commonly used CMMs such as Genkwa Flos(Yuanhua),Trachycarpi Petiolus(Zonglv),Chaenomelis Fructus(Mugua),Arisaematis Rhizoma(Tiannanxing),zymotic CMMs such as vinegar,Medicated Leaven(Shenqu),etc.’Compendium of Materia Medica’ written by Li Shizhen in Ming dynasty recorded 29 CMMs by entry that need to be stored for a long term.’Ben Cao Cong Xin’ in Qing dynasty recorded 40 CMMs concentratively that need to be stored.The reason to stored CMMs for long term were removing and reducing toxicity and side effects,enhancing efficacy and choosing specific efficacy.The results of field survey showed that the Chenpi and Qingpi produced in Xinhui County of Guangdong Province had to be stored for at least 3 years.The warehoused included large-scaled warehouses,medium-sized warehouses,and small-scaled warehouses for farmer.Zhiqiao produced in Zhangshu Jiangxi Province and Jiangjin Chongqing Ctity was not stored for long term.The main reasons for this phenomenon are as follows:the concept of producers in production areas is weak,and do not know the traditional view of ’the longer,the better’.The deteriorations during storage impeded its development.Because of the high demand,Zhiqiao has become a fastselling product,which was harvested and sold after drying in the producing area.There is a lack of the modern researches.Part 2 Study on Quality Evaluation of CMMs of’the longer,the better’1 Multi-directional evaluation of the quality of Chenpi stored for different years by multiple analysis techniques The results showed that the color of Chenpi stored for 1～2 years was orange and darkened to brown of Chenpi stored for 3～23 years.Accompanied by the increased values of L*,b*,c*,and ΔE*,which clarified the changes of color parameters with changed color of Chenpi stored for different years.The changes of odor were not obvious.The taste of aqueous solution of Chenpi were mainly performed as bitterness,umami and sweetness and the bitterness increased with the storage time.The HPLC fingerprints showed that 14 common peaks were calibrated.The similarity of common peak area and cluster analysis were performed and the results showed that Chenpi stored for 1～2 years and stored for 3～23 years were well divided into two categories.The untargeted metabolomics based on UPLC-QTOF-MS coupled with multivariate statistical analysis including principal component analysis(PCA)and orthogonal partial least squares discrimination analysis(OPLS-DA)showed that both could discriminated chenpi stored for 1～2 years and 3～23 years.A total of 26 compounds were screened based on conditions of variable importance for the projection(VIP),Splot,ANOVA p,and max fold change.Nine among them were identified by comparing MS spectra with reference standard and literatures.The contents of chrysoeriol 6,8-diC-glucoside,glutamic acid,5-methyluridine,picraquassioside A,and limonin in Chenpi stored for 1～2 years were significantly decreased compared to that of Chenpi stored for 3～23 years.The contents of Rhoifolin,Hesperetin,Monohydroxypentamethoxyflavanone,and Monohydroxy-tetramethoxyflavanone in Chenpi stored for 3～23 years were significantly higher than that in Chenpi stored for 1～2 years.The contents of hesperidin,nobiletin,and tangerine increased during storage for 1～14 years,and then decreased gradually.The content of TPs decreased with storage time.The content of TFs fluctuated from 1 to 14 years,and decreased obviously in 17～23 years.The antioxidant activity decreased from 14th years and was correlated with the contents of TPs and TFs.The results of HS-SPME-GC-MS showed that the contents of butyl 2butoxyacetate,3-methyl-4-isopropylphenol,methyl 2-(methylamino)-benzoate,and(Z,E)-3,7,11-trimethyl-1,3,6,10-dodecatetraene decreased with the extension of time.the contents of 1-methyl-3-(1-methylethenyl)-benzene,cis-2-methyl-5-(1-methylethenyl)2-Cyclohexen-1-ol,D-carvone,and 3-methyl-6-(1-methylethenyl)-(S)-2-Cyclohexen1-one increased with the extension of storage time.The contents of aromatics decreased obviously in Chenpi stored for 14～23 years.The changes of color,odor and taste were correlated with the changes of internal chemical composition.2 Dynamic evaluation of the quality of Qingpi stored for different years The results showed that the color of Qingpi deepened from dark green to dark brown,which reflected in color parameters changes were the significantly decreased values of L*,b*,c*,and h*and increased value of ΔE*.The contents of hesperidin,nobiletin and tangerine decreased significantly with the extension of storage.years.The contents of TPs,TFs,and the antioxidant activity were decreased significantly.The untargeted metabolomics combined with partial least squares discrimination analysis(PLS-DA)screed 21 compounds and 9 among them were identified as narirutin,diosmetin 6,8-diC-glucoside,melitidin,picraquassioside A,5-methyluridine,glutamic acid,rhamnetin3-O-glucoside,trihydroxy-dimethoxyflavone,and diosmin/neodiosmin.The contents of narirutin,diosmin,diosmetin 6,8-di-C-glucoside,and rhamnetin-3-O-glucoside increased significantly in Qingpi stored for 4～5 years,which were inferred as the material basis of ’the longer the better’ of Qingpi.The contents of 5-methyluridine and glutamic acid were decreased.The results of HS-SPME-GC-MS showed that the relative contents of butyl 2-butoxyacetate and esters decreased with the extension of storage years.The relative contents of alkenes and alkanes increased.In addition,the results of bivariate correlation analysis showed that the changes of color and odor were correlated with internal chemical composition.3 Dynamic evaluation of the quality of Zhiqiao stored for different time in different storage environment The results showed that in LTMH storage condition,the contents of naringin and neohesperidin increased in 0～3 months and then kept stable.The contents of narirutin,tangeretin,and nobiletin kept stable from 0～1 month and increased obviously in 3～9 months.The contents of TPs decreased gradually,and the content of TFs increased gradually.The antioxidant activity of Zhiqiao based on DPPH and FRAP enhanced during storage.In RTRH condition,the contents of naringin and neohesperidin increased in 0～3 months and then kept stable.The content of narirutin changed slightly.The content of nobiletin increased gradually.The content of tangeretin kept stable in 0～3 months and increased significantly in 5～9 months.The content of TPs decreased while the content of TFs increased.The results of DPPH and FRAP indicated that the antioxidant activity of Zhiqiao during storage enhanced.In HTHH condition,the contents of naringin and neohesperidin increased in 0～3 months and then kept stable.The content of narirutin kept stable in 0～1 month and then increased in 3～9 months.The contents of nobiletin and tangeretin kept stable in 0～3 months and then increased in 5～9 months.The content of TPs decreased while that of TFs increased.The antioxidant activity value based on DPPH and FRAP methods increased and they increased more than that in other two storage conditions.The color changed from yellowish green to yellowish brown under HTHH condition and change slightly in other two conditions,and the color parameters were significantly correlated with the content of chemical components.Conclusion There are approximately 90 CMMs that needed to be stored for long term to get a better quality according to records in Literature of Materia Medica.In actual production,only small amount CMMs are stored for years such as Chenpi and Aiye.Other CMMs,which needed to be stored according to Literature of Materia Medica are not to be stored for a long term in actual production.The color of Chenpi deepened from orange to brown with the extension of storage years.HPLC fingerprint could well discriminated Chenpi stored for 1～2 years and 3～23 years.Nine compounds were identified,which could be the marker of Chenpi stored for different years.The contents of hesperidin,nobiletin,tangerine,TPs,and TFs were decreased or increased.The antioxidant activity was affected by the contents of TPs and TFs.The deepened color was correlated with areas of common peaks.The odor change was correlated with the types and relative contents of the volatile components.Taste change was correlated with contents of volatile components and TFs.It is considered that it is necessary to store Chenpi for years.The material basis of ’the longer the better’ may be the increase of the contents of rhoifolin,hesperetin,nobiletin,tangerine,TFs,and D-carvone,etc.In addition,combined with the change trend of each detection index,it is considered that the longer the storage year of Chenpi is not the better,and the recommended storage time is 3～14 years.Narirutin,diosmin,diosmetin 6,8-di-C-glucoside,and rhamnetin-3-O-glucoside may be the material basis of ’the longer the better’ of Qingpi.However,the contents of main components(hesperidin,tangerine peel and hesperidin)decreased significantly from the third year of storage.Therefore,it was considered that whether Qingpi need to be stored for years was still inconclusive and need further research.Three kinds of storage conditions including LTMH,RTRH,and HTHH were established and the quality was evaluated for the first time.It was considered that the aging occurred during the storage and then the content of main flavonoids in Zhiqiao increased and finally improved the quality.Appropriate high temperature and humidity are conducive to the aging of Zhiqiao.This research confirmed the traditional theory of’the longer,the better’ of Zhiqiao.Chenpi,Qingpi,and Zhiqiao are all of Citrus of Rutaceae and aged CMMs.During storage,the color of them deepen to brown or dark brown.The change of antioxidant activity was correlated with the change of the contents of flavonoids.The contents of flavonoids were increased.It is considered that the flavonoids were the material basis of ’the longer,the better’ of these three CMMs.It is suggested that Qingpi and Zhiqiao should also be stored for a certain period of time before using.