Study On The Partition Rationality Of Grades And Genetic Relationship In Asparagi Radix

Objective Study on the quality of grades in Asparagi Radix by detection of characteristics of appearance,chemistry quality evaluation indexes and pharmacodynamics index of Asparagi Radix in different grades,the correlation between them was analyzed,which to analysis rationality of grading method of Asparagi Radix and provide experimental evidence for the standardization of industry.To establish molecular marker method of RAPD,then,analyze genetic diversity of Asparagi Radix and provide the base for seed selection and breeding.Methods?1?Study on characteristics of appearance.Referring to?76 kinds of medicinal materials commodity specification standard?requirement,Asparagi Radix were classified into three grades.The diameter and length of samples in different grades were measured.?2?To determine chemistry quality evaluation indexes contents.According to the Chinese Pharmacopoeia?2015 edition,I?herbal identification method,the moisture,total ash,ulfur dioxide residue and alcohol-soluble extractive contents of Asparagi Radix in different grades were determined.Ultraviolet visible spectrophotometry was used to determine saponins and polysaccharides in different grades samples.The correlation between grades and quality evaluation indexes was analyzed by SPSS 17.0.The principal component analysis was used to evaluate the quality of Asparagi Radix in different grades.The multinomial logistic regression model was established to classify Asparagi Radix grades.?3?To explore antitussive and expectorant effects of Asparagi Radix in different grades in mice.Ammonia water stronger nebulization was used to analyze the effect of antitussive,and tracheal phenol red excretion was studied by mice model of phenol test,which was used to analyze the expectorant effect.The SPSS 17.0 was used to analyze correlation among grades with the effects of antitussive and expectorant.?4?RAPD analyze of Asparagi Radix.The suitable RAPD-PCR reaction system was established by optimizing the concentration of DNA template,primers and 2×Taq PCR Master Mix in this study.?5?Study on the quality standard.The Asparagi Radix were examined according to China pharmacopeia?2015 edition,I?.UV was used to determine the content of saponin that was the active component of principal drug.Then the quality standard of preparation was established.Results?1?Study on characteristics of appearance.25 batches samples were divided into three grades?the first grade,the second grade and the third grade?.The diameter existed significant difference in different grades of samples.The length was different significantly?P<0.05?between the first and others grades,but the difference was not significant?P>0.05?between the second and the third grades.?2?To determine chemistry quality evaluation indexes contents.The moisture,total ashes,alcohol-soluble extractive and ulfur dioxide residues were 8.8%¹7.2%,1.4%⁴.8%,63.16%⁹4.43%and 0³86.92?g·g^-1respectively.The saponins and polysaccharides contents in Asparagi Radix were 5.67%²9.38%and 0.19%⁰.71%respectively.The contents of total ashes,alcohol-soluble extractive,polysaccharides and saponins existed significant difference?P<0.05?in different grades of samples,but the difference was not significant in moisture and ulfur dioxide residues.?3?The correlation analysis indicated that grades showed extremely significant positive correlation with alcohol extractive and saponins?P<0.01?,significantly correlation with polysaccharides?P<0.05?.The principle component analysis extracted three principle components which cumulative contribution rate reaching76.173%,the saponin,alcohol-soluble extractive,polysaccharide,total ash and ulfur dioxide residues effect the quality of Asparagi Radix deeply according to the load feature vectors.On this basis,multinomial logistic regression models were established,whichwereLogisticP1=1.14x₁+1.98x₂-2.85x₃+4.65x₄+4.25x₅+4.89x₆-3.021and LogisticP2=-0.41x₁+1.03x₂-1.30x₃+1.50x₄+1.21x₅+1.37x₆+1.243.Theregression model was used to classify the grade of Asparagi Radix and the discriminant rate?83.8%?of synthesis was obtained.?4?To explore antitussive and expectorant effects of Asparagi Radix in different grades in mice.Compared with the normal saline group,Asparagi Radix apozeme could significant prolonged the latent period?P<0.05?and extremely significant decreased the cough frequency per 3 minutes induced by ammonia water?P<0.01?.The concentration of phenol red excretion of mice trachea in middle dose group was significantly increased.The cough frequency per 3 minutes and the concentration of phenol red excretion of mice were significantly difference between the first and the third grade?P<0.05?,but the difference was not significant among others grades?P>0.05?.The correlation analysis indicated that grades showed significant positive correlation with the cough frequency per 3 minutes,significant negative correlation with the concentration of phenol red excretion of mice.?5?RAPD analysis of Asparagi Radix.The suitable RAPD-PCR reaction system included 2×Taq PCR MasterMix 10.5?L,DNA template 48ng,primer 1.2?mol·25?L^-1,use ddH₂O completed to 25?L.With the DNA extracted from 18 samples as template,183 bands were amplified from 20 random primers which were screened out from 60primers,164 bands were polymorphic,polymorphic rate was 89.62%.The genetic similarity coefficient were 0.4590⁰.7486.?6?Study on the quality standard.The results of examination items of Asparagi Radix were consistent with the correlated regulation.The diosgenin showed good linearity in the range of 0.0048⁰.0240mg·mL^-1?r=0.9998?and the average recovery was97.34%?RSD=2.5%?.The content of saponin in Asparagi Radix could not be less than 0.34%.Conclusion?1?The contents of alcohol-soluble extractive,saponins and effects of antitussive and expectorant in Asparagi Radix were consistent with the grades classification,which indicated that the grading method of Asparagi Radix still with rationality.Except for the contents of alcohol-soluble extractive prescribed in the Pharmacopoeia,the content of saponin and polysaccharide in Asparagi Radix could be considered as the indicators of quality evaluation.?2?Asparagi Radix has a high level of genetic diversity that from different habits.The RAPD-PCR reaction system could be used as analysis of Asparagi Radix diversity.?3?The established quality standard could be used to control the quality of Asparagi Radix effectively.The research could provide a feasible criterion to control quality of Asparagi Radix and also supply a gap of quality criterion about Asparagi Radix.