| Anisodus tanguticus(AT)(Maxim.)Pascher(Solanaceae)is an important resource plant of extracting tropane alkaloids and also considered as analgesic and sedative herb in Tibetan folk.Roots or seeds are the medicinal parts of AT in Tibetan medicine.The wild resource of AT roots in Sichuan is extremely abundant,and they are mainly distributed in Ganzi and Aba prefecture.AT roots is only considered as crude drug by local Tibetan doctors at present,and so numerous resource of AT roots is not used fully.Ethnic drugs are characterized by distinctively regional and ethnic characteristics.Although many achievements have been obtained from investigation of ethnic drugs in China,the holistic level in investigation of ethnic drugs are low,which seriously hinders industrial development of ethnic drugs.The basic elements of core competitiveness of ethnic drugs are their products consist of systematic varieties systematics,quality research,scientific and reasonable quality standards.Meanwhile,they also are importantly technical guarantee to develop industry of ethnic drugs.Based on research and application status of Tibetan medicine AT roots and basic theory of Tibetan medicine,this work aimed to investigate pharmacodynamic material basis and quality evaluation of AT roots by modern pharmacology and chemical separation or analysis techniques.Then,the quality standard(draft)of AT roots was draw up to solve its the "non-standard" status.This work provided important references for enhancing quality control of Tibetan medicine,guaranteeing safety and effectivity of clinical application of Tibetan medicine,promoting resources exploitation and utilization of Tibetan medicine.The methods,results and conclusion of this work are as follows.1 Investigation of pharmacodynamic material basis of AT roots1.1 Investigation of chemical compositionsSeven compounds were separated from Tibetan medicine AT roots by separation and purification technologies such as extraction,concentration,chromatography, enrichment of macroporous adsorption resin,preparative chromatography.Seven compounds were separately identified as 4 tropane alkaloids(scopolamine,anisodine,atropine and anisodamine),1 amide compound(caffeoylputrescine)and 2 coumarin glycosides compounds(fabiatrin and scopolin)by identification of reference substances,nuclear magnetic resonance spectrometer and mass spectrometer.Water-soluble constituents(caffeoylputrescine,fabiatrin and scopolin)are firstly separated from AT roots.This study further expounded chemical material basis of AT roots,and also established foundation to illuminate their pharmacodynamic material basis and quality evaluation.1.2 Investigation of acute toxicity,analgesic effect and sedative effectThe acute toxicity of Tibetan medicine AT roots was investigated by maximum tolerance dose assay.The results indicated that the maximum administration dosage of AT roots for mouse was 24.00 g/kg within a day,which amounts to 480 times of human dosage.Therefore,the recommendatory dosage in relative works is safe for humans.The analgesic and sedative effects of AT roots were investigated by writhing,hot plate and chloral hydrate hypnosis tests in mice.The results showed that AT roots(0.25,0.50,1.00 or 2.00 g/kg)could prolong incubation period of writhing responses and sleep time,reduce frequency of writhing responses,increase pain threshold and shorten incubation period of sleep in mice,indicating that they showed obviously analgesic and sedative effects.1.3 Investigation of spectrum-effect relationships1.3.1 Pharmacodynamics studyThe analgesic and sedative effects of 18 batches of AT roots were investigated by writhing and chloral hydrate hypnosis tests in mice.The results indicated that 18 batches of AT roots(2.00 g/kg)could reduce frequency of writhing responses and prolong sleep time,indicating that they showed obviously analgesic and sedative effects.1.3.2 HPLC-DAD analysisHPLC-DAD fingerprint analytical method of above pharmacodynamics sampleswas developed.After above pharmacodynamics samples were analyzed by this method,fingerprint including 16 common peaks was established.Peaks 1,2,6,7,8,9 and 12 were identified as caffeoylputrescine,anisodine,fabiatrin,scopolin,scopolamine,anisodamine and atropine,respectively.HPLC-DAD method used to determine caffeoylputrescine,anisodine,fabiatrin,scopolin,scopolamine,anisodamine and atropine contents of above pharmacodynamics samples was developed.Then,the 7 compounds contents in above pharmacodynamics samples were determined by this method.The results indicated that everyday caffeoylputrescine,anisodine,fabiatrin,scopolin,scopolamine,anisodamine and atropine intragastric contents in mice were 0.31 ~ 5.17 mg/kg,0.66 ~ 8.60 mg/kg,3.26 ~ 15.19 mg/kg,0.46 ~ 7.21 mg/kg,0.09 ~ 6.44 mg/kg,0.15 ~ 3.86 mg/kg and 0.51 ~ 19.44 mg/kg.1.3.3 Correlation analysisBivariate correlation analysis between inhibitory rates of above pharmacodynamics samples against writhing responses,or prolonged rates of above pharmacodynamics samples on sleep time and 16 common peaks areas,or intragastric contents of test constituents in mice were performed with Pearson's correlation coefficient on SPSS 21.0.The results suggested that areas of common peaks 3,14 and 16 were negatively related to inhibitory rates of writhing responses,and areas of common peaks 3,5,14 and 16 were negatively related to prolonged rates of sleep time.Other correlations were positive correlations.Namely,test constituents(caffeoylputrescine,anisodine,fabiatrin,scopolin,scopolamine,anisodamine and atropine)possessed contribution degree to analgesic and sedative effects of AT roots,suggesting that the above 7 compounds may be the pharmacodynamic material basis of Tibetan medicine AT roots.This study provided scientific justification for the indices constituents selection of following quality evaluation of Tibetan medicine AT roots.This study has been published by Tropical Journal of Pharmaceutical Research.2 Investigation of quality evaluation of AT roots2.1 Resource distribution and producing area investigationResource distribution,varieties and usage status of Tibetan medicine AT roots in Sichuan province were clearly investigated by investigating related references and main producing areas in Sichuan of Tibetan medicine AT roots.The chief variety of AT roots in Sichuan province is A.tanguticus(AT)(Maxim.)Pascher(Solanaceae),which is rich in resources.The crude drug of AT roots in Sichuan province is wild,and local Tibetan doctors have the custom to obtain crude drug nearby for treating patients.At roots are considered as analgesic and sedative herbs in Tibetan folk at present.The fundamental research of AT roots is lacking,and so their resource is not used fully.2.2 Investigation of morphology and histologyThe mainly identified characteristics of primary plant,character of crude drug and microstructure of AT roots were studied by investigating morphology and histology of AT roots using pharmacognosy routine method.The mainly identified characteristics of primary plant of AT roots were leaf margin shape,corolla color,clothing hair of calyx,fruit upright,etc.The mainly identified characteristics of character of crude drug of AT roots were shape,surface characteristics,sectional concentric annular striation,smell,etc.The mainly identified characteristics of microstructure of AT roots were anomalous structure(interxylary phloem)of xylem in cross section or roots,and starch grain,calcium oxalate sand crystals,ducts,etc in powder.2.3 Investigation of physiochemical identification2.3.1 Thin-layer chromatography(TLC)TLC identification methods for alkaloids constituents(anisodine,scopolamine and atropine)and coumarin glycosides constituents(fabiatrin and scopolin)in AT roots were developed and used to identify 18 batches of AT roots samples.The results indicated that these methods possessed good adaptability and reproducibility.Based on the research results and operability,the authors suggested that atropine was selected as reference substance to identify alkaloids constituents of AT roots,and fabiatrin and scopolin was selected as reference substance to identify coumarin glycosides constituents of AT roots.2.3.2 HPLC-DAD fingerprintHPLC-DAD fingerprint analytical method of AT roots was developed.The method possessed good stability,reliability and repeatability,and could be used to analyze the quality of AT roots by similarity between HPLC chromatogram of AT roots sample and HPLC chromatogram of reference standard fingerprint.According to the research results,16 chromatographic peaks should be observed in HPLC chromatogram of AT roots,and chromatographic peak 6(fabiatrin)was selected as reference chromatographic peak.The relative retention time of 16 chromatographic peaks were 0.49(Peak 1),0.58(Peak 2),0.62(Peak 3),0.72(Peak 4),0.86(Peak 5),1.00(Peak 6),1.07(Peak 7),1.14(Peak 8),1.23(Peak 9),1.38(Peak 10),1.56(Peak 11),1.74(Peak 12),2.14(Peak 13),2.34(Peak 14),2.49(Peak 15)and 2.72(Peak 16),respectively.The allowable fluctuation ranges of relative retention time of all peaks were within ±5%.2.4 Investigation of routine examination,heavy metals and harmful elements detection,and pesticide residues screening2.4.1 Determination of moisture and ash contentsThe contents of moisture and asd(total ash and acid-insoluble ash)in 18 batches of AT roots samples were determined according to the related methods in the Chinese Pharmacopoeia(2015 edition).The contents of moisture,total ash and acid-insoluble ash in 18 batches of AT roots were 5.2 ~ 14.1% with a mean of 7.7%,3.7 ~ 12.6% with a mean of 8.3%,0.2 ~ 2.8% with a mean of 1.2%,respectively.Based on test data and production practice,the authors suggested that the contents of moisture,total ash and acid-insoluble ash in AT roots should be lower than 10.0%,12.0% and 3.0%,respectively.2.4.2 Heavy metals and harmful elements detectionICP-MS method for determining contents of lead(Pb),cadmium(Cd),mercury(Hg),copper(Cu)and arsenic(As)in AT roots was developed and used to determine the contents of Pb,Cd,Hg,Cu and As in 18 batches of AT roots samples.The results indicated that the contents of Pb,Cd,Hg,Cu and As in 18 batches of AT roots were 0.20 ~ 1.70 mg/kg,0.05 ~ 0.26 mg/kg,0.02 ~ 0.19 mg/kg,2.98 ~ 13.94 mg/kg and0.10 ~ 0.94 mg/kg,respectively,which meet the related stipulation in the Chinese Pharmacopoeia(2015 edition).Based on test data and production practice,the authors suggested that it was not necessary to enact limit stipulation for the contents of Pb,Cd,Hg,Cu and As in AT roots.2.4.3 Pesticide residues screeningPesticide residues situations of 18 batches of AT roots samples were investigated by GC-MS/MS.The results showed that pesticides,such as ?-BHC,carbofuran,clomazone,mirex or phosalone,were detected in some batches of AT roots.Since AT roots in Sichuan province are wild,the results indicated that human activities could bring pesticide pollution into wild crude drugs,and the related scholars and government departments should pay attention to the situations.But considering production practice,the authors suggested that tentatively,it was not necessary to enact limit stipulation for related pesticides contents in AT roots.Moreover,an optimizing method for selecting multiple reaction monitoring(MRM)transition of pesticides was developed,and this method could be used to optimizing for selecting MRM transition of all compounds.This study has been published by Food Control and Journal of Chromatography B.2.5 Determination of extractum and active ingredients contents2.5.1 Determination of extractum contentThe solvent and method for determining extractum contents of AT roots were confirmed as 95% ethanol and hot dipping,respectively,by trial test.Then,the extractum contents in 18 batches of AT roots samples were determined.The results showed that the extractum contents in 18 batches of AT roots were 13.4 ~ 22.8% with a mean of 19.1%.Based on test data and production practice,the authors suggested that the extractum contents in AT roots should be higher than 10.0%.2.5.2 Determination of total alkaloids contentsA spectrophotometry for determining total alkaloids contents in AT roots was developed and used to determine the total alkaloids contents in 18 batches of AT roots samples.The results showed that the total alkaloids contents in 18 batches of AT roots were 1.58 ~ 5.16% with a mean of 3.45%.Based on test data and production practice,the authors suggested that the total alkaloids contents(atropine)in AT roots should be higher than 2.0%.2.5.3 Determination of anisodine,anisodamine,scopolamine and atropine contentsA HPLC-DAD method for determining anisodine,anisodamine,scopolamine and atropine contents in AT roots was developed and used to determining anisodine,anisodamine,scopolamine and atropine contents in 18 batches of AT roots samples.The results showed that the anisodine,anisodamine,scopolamine and atropine contents in 18 batches of AT roots were 0.050 ~ 0.538%,0.003 ~ 0.118%,0.004 ~ 0.422% and 0.015 ~ 0.824%,respectively,and the total contents of the 4 alkaloids were 0.145 ~ 1.172%.Based on test data,production practice and the similarity of 4 alkaloids(tropane alkaloids),the authors suggested that the total contents of the 4 alkaloids in AT roots should be higher than 0.30%.2.5.4 Determination of caffeoylputrescine,scopolin and fabiatrin contentsA HPLC-DAD method for determining caffeoylputrescine,scopolin and fabiatrin contents in AT roots was developed and used to determining caffeoylputrescine,scopolin and fabiatrin contents in 18 batches of AT roots samples.The results showed that the caffeoylputrescine,scopolin and fabiatrin contents in 18 batches of AT roots were 0.025 ~ 0.585%,0.056 ~ 0.549% and 0.362 ~ 1.710%,respectively,and the total contents of scopolin and fabiatrin were 0.446 ~ 2.229%.Based on test data,production practice and the similarity of scopolin and fabiatrin(coumarin glycosides),the authors suggested that the caffeoylputrescine content and the total contents of scopolin and fabiatrin in AT roots should be higher than 0.050% and 0.70%,respectively.2.6 Quality standard(draft)The quality standard(draft)of Tibetan medicine AT roots from Sichuan province was draw up according to the investigation of their pharmacodynamic material basis and quality evaluation,production practice and operability.The part contents of this quality standard(draft)has been published in the Sichuan Tibetan medicinal standard(2014 edition).In conclusion,this work resolved the "non-standard" status of Tibetan medicine AT roots from Sichuan province.Meanwhile,this work provided important references for enhancing quality control of Tibetan medicine,guaranteeing safety and effectivity of clinical application of Tibetan medicine,promoting resources exploitation and utilization of Tibetan medicine. |
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