Study On The Quality Standard Of Agkistrodon And Its Processing Products

ObjectiveAgkistrodon is the dry bulk of Agkistrodon acutus(Güenther).Agkistrodon is warm,sweet in nature.It has the functions of removing wind,clearing collaterals and relieving spasm.Indications:rheumatism,numbness,clonus,apoplexy,facialdistortion,hemiplegia,convulsion,tetanus,leprosy,scabies.Agkistrodon is a valuable medicine,and its adulteration with counterfeit is common in the market.The standards recorded in the Chinese pharmacopoeia are not perfect enough.In the Chinese pharmacopoeia,the standards only control the quality of Agkistrodon decoctions,characteristics and extract,and use polymerase chain reaction to identify the authenticity.It is difficult to accurately evaluate the authenticity of Agkistrodon and its decoctions.On the basis of the authenticity identification of Agkistrodon,this paper aims to improve the quality standards of Agkistrodon and its decoctions,so as to provide a scientific basis for the quality evaluation of Agkistrodon and its decoctions.Methods1.IdentificationIn this study,Agkistrodon in 42 batches was described and its length,maximum diameter of abdominal extension and body weight were measured.The characteristics of Agkistrodon's powder were described by means of electron microscope.Thin layer identification of Agkistrodon and other snakes was carried out under different chromatographic conditions.The DNA of Agkistrodon was identified according to the polymerase chain reaction method under the first term of the Chinese pharmacopoeia(the 2015th edition).2.HPLC fingerprintWater extracts from 10 batches of Agkistrodon and 18 batches of Agkistrodon~'processing products were determined by HPLC.Methanol(B)-0.1%formic acid(A)was used as the mobile phase,and DAD detector was used to determine at 254 nm,and the similarity analysis of the common peaks was carried out by using the similarity evaluation system of Chinese medicine fingerprint 2004.3.Assay of nucleosides and amino acids of Agkistrodon acctus and its processing productsThe nucleosides and amino acids of 11 batches of Agkistrodon and their three processed products were determined by HPLC.The changes of Agkistrodon before and after processing were compared to provide scientific basis for the standardization of processing technology.4.Agkistrodon,grade and its decoctions,quality standardsIn this study,the medicinal materials of 42 batches of Agkistrodon were graded and the quality standards of 47 batches of Agkistrodon,decoctions which including 19batches of Agkistrodon(AG),16 batches of Agkistrodon Praeparata Cum Vino(APCV)and 12 batches of Agkistrodon Musculus Praeparata Cum Vino(AMPCV)were studied.In the study,on the grade of Agkistrodon,the characteristics,alcohol soluble extract and nucleoside content of Agkistrodon were used as the classification indicators.In the study,on quality standard of Agkistrodon,processing products,the characteristics,moisture content,total ash content and alcohol soluble extract of the slices were taken as quality standard indexes.Results1.IdentificationCharacteristics of Agkistrodon were identified according to the following characteristics:upturned nose,tubular fangs,“Prismatic pattern on the back”(fang sheng wen),condyloma,knife blade protrusion,and“Buddha nails”at the end of tail.The microscopic features of Agkistrodon acutus or its decoctions powder include the papillary and imbricated scales.The scaly spots on the back are light brown or white.Striated muscle fibers,bone fragments and fat oil droplets were found under microscopy.According to the Thin-Layer Chromatography analysis of Agkistrodon,six species of snakes were selected to distinguish from Agkistrodon,there came from Daboia russelii siamensis,Bungarus multicinctus,Xenopeltis unicolor,Naja naja and Zaocys dhumnades.In the molecular identification of Agkistrodon and its decoctions,there were single bands in all the medicinal materials and decoctions of Agkistrodon at 300~400bp.2.HPLC fingerprintUsing pure water as the extraction solvent and high performance liquid chromatography(HPLC),nine common peaks were calibrated as the common peaks of HPLC fingerprint.The evaluation results showed that the medicinal herbs of Agkistrodon and its decoctions pieces had a good similarity,with the similarity over0.926 and the common peak area accounting for more than 90%of the total peak area.The third peak was uracil,the sixth peak was hypoxanthine,the seventh peak was xanthine,the eighth peak was inosine,and the ninth peak was guanosine.3.Assay of nucleosides and amino acids of Agkistrodon and its processing productsNucleosides composition research of Agkistrodon and its processing products showed that four main class components,respectively was the uracil,hypoxanthine,xanthine and inosine.In the study,on the components of Agkistrodon before and after processing,the total amount of four nucleoside components of the three types of the preprocessing Agkistrodon and the three types of the processing were respectively 2.963 mg/g of Agkistrodon's medicinal materials,2.971 mg/g of AG,2.834 mg/g of APCV and 3.370 mg/g of AMCV,After processing,all of Agkistrodon,processing products,nucleosides were decreased actually while the yield factors enter in consideration.The total contents of 17 hydrolyzed amino acids and the total essential amino acid of Agkistrodon acutus were 469.79 mg/g and 179.80 mg/g,while 570.02mg/g and 216.07 mg/g of AG,625.72 mg/g and 221.28 mg/g of APCV,871.94 mg/g and 330.59 mg/g of AMPCV.After the processing,the contents of total 17 amino acids and essential amino acid in 3 kinds of processing product were increased.4.Agkistrodon,grade and its decoctions,quality standards The grade of Agkistrodon was defined and classified according to the five indexes of traits,maximum width of abdominal extension,alcohol soluble extract,and content of nucleoside components.The results showed that the first grade accounted for 38.1%,the second grade for 57.1%,and the unqualified grade for 4.8%.Studing on quality standards of Agkistrodon,decoctions the characteristics and identification of agkistrodon slices were defined,and the measured values of water content,ash content and alcohol soluble extract were defined.ConclusionThe identification of Agkistrodon has great significance to distinguish Agkistrodon from other counterfeits,and the research on its characters is also helpful to the classification of Agkistrodon.The fingerprint analysis method established by Agkistrodon and its decoctions has good reproducibility and stability,which provides reference for evaluating the internal quality of Agkistrodon and its decoctions.Research on the components of Agkistrodon and its processing products provides reference basis for exploring the active components of Agkistrodon,and for evaluating the internal quality of Agkistrodon and its processing products.The changes of Agkistrodon components before and after processing provide scientific basis for improving the processing technology and for clarifying the changes of the effects of Agkistrodon after processing.The classification standard of Agkistrodon was established for the first time.The study on the quality standard of Agkistrodonand its decoctions has clarified the direction for regulating the production of its decoctions.