Commercial Samples Investigation And Quality Evaluation Of Cicadae Periostracum

Cicadae Periostracum(CP),which is derived from the slough of Cieadidae insects,is a commonly used crude drug in traditional Chinese medicine.Cryptotympana atrata is the only official origin of the CP speciafied in Chinese Pharmacopoeia(2015 Edition).In recent years,commercial demand of CP has gradually expanded,so other insects,such as Auritibicenflammatus,C.mandrina,Lyristes pekinensis,Oncotympana maculaticollis and Platypleura icaempferi,are also used as the origins of CP.However,there is little scientific information about the chemical profiles,quality consistence and quality control method of different origins of CP.In present study,we systemically investigated the origins of CP samples from various regions of China,separated and identified major constituents of CP,evaluated quality consistency among different origins,revealed absorbtion and metabolic metabolism of CP,improved quality standard of CP.The paper is divied into following sections:1.Review on research progress of CPThe research progress of CP,including historical application evolution,material resources and quality evalution were summarized.The limitations of the current research and the prospections on CP exploration were also discussed.2.Commodity market survey of CPFor clarifing the origins,distribution and specifications of CP,we investigated main Chinese medicinal materials markets and collected 56 batches of CP samples.It was found that the slough of other six species,including Auritibicen flammatus,C.mandrina,Platypleura kaempferi,Lyristes pekinensis,Oncotympana maculaiicollis,Tibicen flammatus,were also used as CP in China,which accounted for a large market share.The distribution of CP was mainly in Shandong,Henan,Hunan,Hubei,Anhui,Zhejiang and Sichuan provinces.3.Target separation and identificaiton of constituents in CPRecent studies have shown that N-acetyldopamine dimers and their derivers in CP have significant antitumor,anti-inflammatory and antioxidant activities.N-acetyldopamine polymers may be the bioactive components that contribute to the efficacy of CP.Isolation and purification of main N-acetyldopamine polymers in CP may provide material basis for quality evaluation studies.CP(derivded from C.atrata)was extracted by refluxing with 95%ethanol,and the extract was repeatedly separated and purified by silica gel column and ODS column chromatography.Then,the target fraction was subjected to chiral resolution column based semi,preparation liquid chromatography.UPLC-Q-TOF-MS/MS technology was used to monitor the target compounds in real-time during the whole separation process.Finally,four N-acetyldopamine dimers were structurally identified by 1H-NMR,13C-NMR,UPLC-Q-TOF-MS/MS and UV.4.Quality consistency evaluation on seven origins of CPFor exploring the quality consistency of seven different CP orgins,we established an UPLC-Q-TOF-MS/MS method for qualitative and semi-quantitative analysis.UPLC-Q-TOF-MS/MS analysis was established using two purified N-acetyldopamine dimers as reference substance,and their characteristic fragments pathways were summarized.Totally,34 acetyldopamine polymers were identified or tentatively assigned,including 4 dimers,11 trimers,10 tetramers and 9 pentamers from CP.Then,the established UPLC-Q-TOF-MS/MS analysis method was employed to quantify the two N-acetyldopamine dimers in 56 batches of CP.And semi-quantitative analysis was carried out for the whole N-acetyldopamine polymers.Our results showed that the N-acetyldopamine polymers contents and the chemical profiles of Tibicen flammatus,Auritibicen flammatus,Cryptotympana mandrina were similar to Cryptotympana atrata,which indicated that these three species may be the potential resources of CP.5.Absorbtion and metabolism of CP in vivoFor clarifing whether the chemical constituents of CP can be absorbed and metabolized or not,UPLC-Q-TOF-MS/MS technology was employed to identify metabolites in plasma,urine,feces and bile samples of rats after oral administration of CP extract.The possible chemical structure of metabolites was assigned by comparing chromatographic information and mass spectrometry with that of compounds in CP.As a result,a total of 34 prototypes and I5 metabIites were identified or tentatively characterized in rat biological samples,including 5 letablites in plasma,3 prototypes and 9 metablites in urine,34 prototypes and 8 metablites in feces,2 metablites in bile.N-acetyldopamine dimer and acetyl-benzodioxane underwent phase Ⅰ and phase Ⅱreactions,such as oxidation,hydrogenation,methylation,sulfation,and glucuronidation.Among four different bio-samples,urine contained the most abundant metabolites.Apart from xenobiotics absorbed in blood,it was inferred that abundant prototypes in feces may be also the potential effective substances,for example through regulating the dyversity of gut microbiotato exert the pharmacological activities of CP.6.Improvement of CP quality control standardsPrevious metabolic results have shown that metabolites of N-acetyldopamine dimers can be absorbed in blood,which is the important potential pharmacological substance basis of CP.However,there is no quantitative marker for CP in current Chinese Pharmacopoeia.Therefore,we established an HPLC-UV quantitative analysis method using N-acetyldopamine dimer as index for improving the quality standard of CP.The extraction and liquid chromatography conditions were systematically investigated and optimized.The results showed that the method was stable and reliable.Based on the established quantitative method,we drawn up the quality standard of CP(draft),in which a quantitative method was employed for improving the quality standard of CP in Chinese Pharmacopoeia.