| ObjectiveTortoise shells(Testudinis Carapax et Plastrum)rooted in upper and lower shells of turtles(Chinemys reevesii(Gray)),have a long history of application.They usually are used as shell pieces,vinegar shells,tortoise-shell glues and other forms of medicines,having a high demand.Tortoise-shell glues(Testudinis Carapacis et Plastri Colla)made by decocting tortoiseshells and concentration,have tonic effect and belong to Chinese unique medicines.In order to control their adulterations' occurrence,in order to compensate for the lack of existing identification methods of tortoise shells,tortoise-shell glues,the paper systematically introduces the sample difference on character identification,microscopic identification and TLC identification.Using pre-column derivatization HPLC method analyses the sorts,content,ratio of amino acids.And using electronic nose analyses their smells.The amino acid ratio evaluation index and electronic nose discrimination are suitable for the identification of fragments and powder and have guiding significance to the quality of tortoise shells and tortoise-shell glues.Also,them provide reference for perfecting their quality evaluation system about tortoise shells and tortoise-shell glues.Method 1 Sample collection,character identification,and microscopic identificationThis study collected 12 species of tortoises totally,and obtained 44 batches of shell samples,consisting of 22 batches of genuine tortoise shells and 22 batches of adulterants.In addition,collect 19 batches of commercially tortoise shells and 24 batches of commercially tortoise-shell glues.These samples were identified by character identification and microscopic identification,and their characteristics were described and photographed.2 Samples of tortoise shells' TLC identificationAccording to the 2015 edition of "Chinese Pharmacopoeia" method,using cholesterol as control sample,12 species of tortoise shells were identified by thin layer chromatography(TLC)to test their differences.These samples also were separately upper and lower shells.Then,these commercially tortoise shells identify by TLC.3 Determination of amino acids in samples by AQC pre-column derivatization HPLCThe hydrolysis time,dephosphorization conditions,decalcification conditions and calculation of amino acid content were investigated.Using principal component analysis(PCA),cluster analysis and other multivariate statistical method,respectively analysis their different amino acid contents or ratios about genuine tortoise shells and adulterants,commercially tortoise shells and commercially tortoise-shell glues.4 Establish tortoise shells and tortoise-shell glues discriminant models by electronic noseRespectively test 5 single factors of tortoise shells and tortoise-shell glues,includingsample weight,sample volume,incubation temperature,incubation time,particle size.And conduct orthogonal test to determine the optimum conditions.Verified by the test set and training set,establish tortoise shells model of discriminant factor analysis(DFA),and use the model to evaluate the quality of commercially tortoise shells.In addition,using the method evaluated the quality of commercially tortoise-shell glues.Result1 There were obvious differences between tortoise shells and most of their adulterants in character,mainly in the color of upper and lower shells,scutellum characteristics and etc.The characters of commercially tortoise-shell glues mainly in color,texture,cross section,but their differences were not obvious.Microscopic characteristics of all the samples had bone fragments and horny fragments,and their differences were small.It was difficult to accurately used for identification of tortoise species.2 TLC results showed that each sample were similar with the control spot in the Rf value of about 0.50,so tortoise shells of 12 species contain cholesterol;in addition,in the Rf value 0.20,the upper and lower shells of mauremysmuticacantor appeared two blue spots,but other samples appeared one or not obvious,which could identify shells of mauremysmuticacantor.In the TLC detection of commercially tortoise shells,they showed the same color spots with the reference substance and there were not shells of mauremysmuticacantor.3 Pretreatment of samples for amino acids:tortoise shells powder of 0.1 g hydrolysis 8h,20mL of 70%ethanol to dephosphorization,8mL of 0.1 mol·L-1 to decalcify;tortoise-shell glues powder of 0.05g hydrolysis 8h,20mL of 70%ethanol to dephosphorization,6mL of 0.1 mol·L-1 to decalcify.Through comprehensively analyzing amino acids and contents,it found differences between upper and lower tortoise shells.The PCA analysis could distinguish them.In addition to shells of mauremysmuticacantor,ratios of Gly/Pro and Gly/Thr could distinguish genuine tortoise shells and their adulterants.The range of Gly/Pro is 1.763?1.854,Gly/Thr 5.372?7.729.And the identification rate was 90.91%.Using this method to identify commercially tortoise shells,genuine tortoise shells accounted for 73.68%.For tortoise-shell glues,contents of Hyp,Gly,Ala,Pro almost fitted with the provisions of 2015 edition of "Chinese Pharmacopoeia".24 batches of commercially tortoise-shell glues were mainly divided into two categories through cluster analysis.Choosing one of Gly/Arg,Gly/Ala,Gly/Pro with Gly/Hyp agreed with the clustering analysis.The range of Gly/Arg is 2.78?2.88,Gly/Ala 2.28?2.39,Gly/Pro 1.84?1.90,Gly/Hyp 2.62?2.85.It showed that the method of amino acid ratios analysis was also applicable to the tortoise-shell glues.4 The optimum conditions of tortoise shells is sample weight of 0.3g,incubation temperature of 55?,sample volume of 2000?L,particle size of No.3 sieve,incubation time of 300s and tortoise-shell glues is sample weight of 0.6g,sample volume of 2500?L,incubation temperature of 70?,particle size of No.3 sieve,incubation time of 1200s.Through the validation of test set and training set,the DFA model established by genuine tortoise shells and their adulterants had a good identification effect.The genuine group of tortoise-shell glues could be separated from the pseudo group using their DFA model.These showed the electronic nose odor recognition technology had certain discriminatory to tortoise shells and their adulterants,commercially tortoise-shell glues.Conclusion1 TLC method can identify shells of mauremysmuticacantor in all of tortoise shells adulterants.2 The method of AQC pre-column derivatization HPLC can determine varieties and contents of amino acids in tortoise shells and tortoise-shell glues.The amino acid ratio method can identify tortoise shells and their adulterants and preliminarily evaluate the quality of tortoise-shell glues.3 Electronic nose odor recognition technology can recognize smells of tortoise shells and their adulterants and preliminarily evaluate the quality of tortoise-shell glues.InnovationThe method of AQC pre-column derivatization HPLC is the first time to determine contents of 16 amino acids in tortoise shells and their adulterants.These obtained samples are stable and the detection efficiency is high.For the first time the method of amino acid ratio is used to identify tortoise shells and their adulterants,tortoise-shell glues.This method and TLC method can effectively identify tortoise shells and other 11 species of their adulterants.It is the first time using electronic nose odor recognition technology to identify tortoise shells and their adulterants and this method is effective. |
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