Drug Standards of Quality Control ofLonicerae Japonicae Flos14402020林程Objectives1.Master the microscopic identification of LJF.2.Master the description of LJF and learn the difference of description between LJF and LF.3.Master the TLC identification of LJF and LF.4.Master the method for the determination of marker constituents in crude drug.5.Study the application of HPLC in determination of chlorogenic acid and luteolin-7-O-glucoside in LJF.Methods(I).Identifications1.DescriptionBreed LFJ LFCharacter Clavate,stout in upper part andtapered downwards,slightly curvedClavate,slightly curvedSurface color Yellow white or green white Green Brown to yellow whiteSize2~3cm long,upper diameter is3mm,downwards diameter is1.5mm 3~4.5cm long,upper diameter is2mm, downwards diameter is1mmWistiti Densely Less2.Microscopic identificationi).Preparation of slides of corolla surface.Tear LJF corolla by tweezers————→Place LJF corolla on the slide————→Add1-2 drops of glycerine alcohol mixture and put the cover on the slideii).Preparation of slides of LJF powderPlace small amount of powder on the slide————→Add1-3drops of chloral hydrate————→Heat and stir by alcohol lamp————→Absorb the excess liquid by paper————→Use forceps to put the cover on the slide by one side————→Add1-2drops of glycerine alcohol mixtureiii).Observe characteristics of LJFGlandular hairs·The head of glandular hairs are multicellular with subrounded or slightly oblate.·The stalks of glandular hairs are unicellular or multicellular.Non-glandular hairs•Numerous.•Thick walls,unicellular,with fine verrucae on the surface,some have corneous spiral.•Thin walls,slender,curved or shrinkage,with fine verrucae on the surface.Pollen grains·Pollen grains are spherical,with3germinal pores.3.TLC identification of LJF and LFi).Differences of morphological between LFJ and LFBreed LFJ LFOrganic acids+Chlorogenic acid+ Chlorogenic acidFlavonoids++ Iridoids++ Saponins-+Macranthoidin BDipsacoside Bii).Experiment Materials•TLC silica gel plate(10cm×5cm),chromatography cylinder,capillary tube•Developing solvent:mixture of n-butanol,formic acid and water(4:1:5)•Reference substances：Macranthoidin B(5mg/ml),Dipsacoside B(5mg/ml),Chlorogenic acid(1mg/ml)•Chromogenic agent:10%sulfuric acid in ethanol(saponins chromogenic agent)•Samples：powder of LJF and LFTLC procedures•Sample solution preparation:Add1g powder of LJF and LF into10ml methanol, sonicated for about10minutes,filter,and get supernatant as sample solution.•Chromatography cylinder saturation:Add developing solvent into the chromatography cylinder,saturated.•Spotting:Add reference substances(chlorogenic acid,dipsacoside B,macranthoidin B),LJF, LF.Put the plate into the chromatography cylinder,cover the lid.•Developing Distance：5~6cm•Examination:1.Observe the spot under UV light at365nm(chlorogenic acid).2.Spray with10%sulfuric acid in ethanol,heat at105ºC for1~2min and observe under daylight (saponins).iii).Ideal result1:chlorogenic acid2:Macranthoidin B3:Dipsacoside B4:LF sample solution5:LFJ sample solution(II).Tests1.Water(toluene method)Experimental equipment:i).Notes:A:Short necked round bottomed flask B:Water determination pipe C:Straight condenser tube All the instruments should be cleaned and dried in the oven before used.ii).Procedures：Take LFJ samples in right amount(about contain1~4ml water),smash them and weigh accurately.Then put them into bottom A and add toluene about200ml.Connect equipments,add toluene from top of condenser tube until thin tube in pipe B is full of toluene.Heat bottom A until toluene is boiled.Then adjust the heating temperature until the flow rate is about2d/s.After water distilled completely,wash the condenser tube inside by toluene.Then push all toluene into bottom by other method.Distill5min.Cool down to room temperature.Push water into bottom by Copper wire with toluene if there is water in bottom B inside.Place it there until water is completely separated from toluene.check and calculate the yield of water in LFJ.iii).Requirement:No more than12.0%.2.Total ashi).Procedures：Smash LFJ samples,then take the samples about2~3g.Put them into crucible burned until constant weight.Weigh accurate to0.01g,heat slowly and avoid burning until samples change into carbon completely.Increase the temperature gradually to500~600℃and make samples ashing to constant weight.Calculate the total ash content(%)of the sample according to the weight ofthe residue.ii).Requirement:No more than12.0%.3.Acid-insoluble ashi).Procedures：Take some ash from total ash,add about10ml Dilute hydrochloric acid Carefully.Cover crucible by watch glass.Heat it in water bath10min.Then wash watch glass by hot water into crucible.Filter the wash water by filter paper without ash.The residue in crucible should be washed and combined into the filter paper.Then wash them until they don’t show chloride reaction.Put the ash with filter paper into crucible,dry and heat to constant weight.Calculate the Acid-insoluble ash content(%)of the sample according to the weight of the residue.ii).Requirement:No more than3.0%.4.Heavy metal and hazardous elements(Inductively Coupled Plasma-Mass Spectrometry)Procedures：i).Preparation of standard stock solutionMeasure accurately standard solution of lead,arsenic,cadmium,mercury and copper,dilute to1ug,0.5ug,1ug,1ug,10ug per ml respectively by10%nitric acid solution.ii).Preparation of standard solutionMeasure accurately standard stock lead,arsenic,cadmium,and copper solution in appropriate amount.Dilute to containing lead and arsenic0ng,1ng,5ng,10ng and20ng per ml by 10%nitric acid solution.Dilute to containing cadmium0ng,0.5ng,2.5ng,5ng and10ng per ml by 10%nitric acid solution.Dilute to containing copper0ng,50ng,100ng,200ng and500ng per ml by 10%nitric acid solution.Then measure Measure accurately standard stock mercury solution in appropriate amount.Dilute to containing mercury0ng,0.2ng,0.5ng,1ng,2ng and5ng per ml by 10%nitric acid solution.This solution should be prepared when using.iii).Preparation of internal standard solutionMeasure accurately germanium,indium and bismuth standard solution in appropriate amount.Dilute to1ug/ml mixture solution by water.iv).Preparation of test solutionTake samples and dry them in60℃2h.Smash them and take about0.5g,weigh accurately.Put them into high pressure and temperature resistant microwave digestion tank.Add 5~10ml nitric acid.Seal and start digestion following with the steps of requirements of microwave digestion instrument.After digestion completely,cool temperature under60℃.Take out digestion tank and cool down.Transfer digestion solution to50ml volumetric flask.Wash the digestion tank by little water three times and combine the wash solution into volumetric flask.Add standard solution of gold single element(1ug/ml)200ul.Add water to the mark.Shake.Prepare blank solution by the same method expect adding standard solution of gold single element.v).DeterminationTreat72Ge as63Cu and75As’s internal standard element.Treat115In as114Cd’s internal standard element.Treat209Bi as202Hg and208Pb’s internal standard element.Correcting elements to be measured with the requirements of instrument.The internal standard sampling tube is always inserted in Internal standard solution in analysis process.Insert the sampling tubes of instrument in different concentration of standard solutions respectively(concentration is increasing).Treat the measurements as ordinate and the concentration as abscissa.Then make standard curve.Insert the sample solutions of instrument into test solutions.Take the average of the three readings.Calculate the concentration of heavy metals from standard curve.vi).Requirement:Lead should not more than5mg/kg.Cadmium should not more than 0.3mg/kg.Arsenic should not more than2mg/kg.Mercury should not more than0.2mg/kg.Copper should not more than20mg/kg.(III).Assay1.Chlorogenic acidi).Chromatographic conditions·Instruments:Shimadzu DGU-LC-20AT HPLC；·Colume:Shimadzu Inertsil ODS-SP(4.6×150mm,5um)；·Temperature：30℃；·Flow rate：1ml/min；·Detection：327nm；·Mobile phase：acetonitrile(B phase)-0.1%phosphoric acid solution(A phase)·0min-8min:14%-19%B phase·8min-10min:19%-19%B phase·The theoretical stage number of Chlorogenic acid should bigger than1000ii).Preparation of the standard solutionThe reference standard of chlorogenic acid is precisely weighted,and dissolved in50% methanol to a final concentration of40ug/ml.iii).Preparation of the sample solution0.1g of dried powder of LJF was precisely weighed and added into the stopper conical flask. Then,50mL of50%methanol is added into the flask,weighted,and ultrasonically extracted at 100Hz for30min.After compensating for the lost weight with50%methanol,the extracted solution was well shaked,filtered,and the filtrate was subjected to HPLC for analysis.iv).CalculateInject10ul of reference solution and the sample solution into HPLC for analysis, respectively.Record the procedure and result.Calculate the content of chlorogenic acid in Lonicerae Japonica Flos.v).Requirement:It should contain not less than1.5%chlorogenic acid.2.Luteolin-7-O-glucosidei).Chromatographic conditions·Instruments:Shimadzu DGU-LC-20AT HPLC·Colume:Phenyl silane bonding adhesive(4.6×150mm,5um)；·Temperature：RT；·Flow rate：1ml/min；·Detection：350nm；·Mobile phase：0.5%Glacial acetic acid(B phase)-acetonitrile(A phase)·The theoretical stage number of luteolin-7-O-glucoside should bigger than20000·Gradient elution followed with this tableTime(min)Mobile phase A(%)Mobile phase B(%)0~1510→2090→8015~30208030~4020→3080→70 ii).Preparation of the standard solutionThe reference standard of luteolin-7-O-glucoside is precisely weighted,and dissolved in70% ethanol to a final concentration of40ug/ml.iii).Preparation of the sample solution2g of dried powder of LJF was precisely weighed and added into the stopper conical flask. Then,50mL of70%ethanol is added into the flask,weighted,and ultrasonically extracted at100 Hz for30min.After compensating for the lost weight with70%ethanol,the extracted solution was well shaked,filtered.Weigh accurately10ml of the filtrate and wait it dry.Dissolution the residue by70%ethanol and transfer it to5ml volumetric flask.Then add70%ethanol to the mark.Shake.iv).CalculateInject10ul of reference solution and the sample solution into HPLC for analysis, respectively.Record the procedure and result.Calculate the content of luteolin-7-O-glucoside in Lonicerae Japonica Flos.v).Requirement:It should contain not less than0.050%luteolin-7-O-glucoside.Functions of curingHeat-clearing and detoxifying.Dispelling wind and heat.Curing furuncle,carbuncle, pharyngitis,erysipelas,toxic heat,bloody flux,wind-heat type common cold and fever with warm disease.References:[1].国家药典委员会.中国药典.2015版第一部.中国医药科技出版社[Z].2015-06-05.221[2].国家药典委委会.中国药典.2015版第四部.中国医药科技出版社[Z].2015-06-05.101-105，204-207。