The Study On The Diffrernce Of Chemical Components And Sedative-hypnotic Effects Of Alcohol Extracts From Hybrid Gastrodia Elata And Its Parents

Objective:A preliminary comparative study of hybrid Gastrodia elata and its parents based on the chemical composition and sedative-hypnotic pharmacological effects of ethanol extract was conducted to analyze the differences between Zhaotong G.elata Bl.f.glauca S.Chow and traditional Jinzhai G.elata Bl.f.elata and their first-generation（F₁）hybrids,to analyze the advantages of the first-generation（F₁）hybrids of G.elata and to provide a basis for the rational use of the first-generation（F₁）hybrids of G.elata in production.Methods:1 The experimental protocol for the determination of the ethanol extract content of G.elata was determined by reference to the method in the Chinese Pharmacopoeia（2020 edition）under the 2201 extract determination method,combined with the requirements under the Pharmacopoeia for the examination of G.elata,to determine the ethanol extract content of G.elata.2 Analysis of the overall chemical composition species of G.elata based on UPLC-Q-Exactive-Orbitrap-MS metabolomics technique.3 A C₁₈column（4.6 mm×250 mm,5μm）with methanol（B）-0.1%phosphoric acid solution（A）as mobile phase,detection wavelength 270 nm,column temperature35℃,injection volume 10μL,volume flow rate 1.0 m L/min was used to establish the characteristic fingerprints of G.elata using the software of the"Chromatographic Fingerprint Evaluation System for Traditional Chinese Medicine（2012 version）".The quality of G.elata was evaluated comprehensively by cluster analysis and principal component（PCA）analysis.4 The content of the six phenolic components of G.elata was further determined by using a high performance liquid chromatography instrument to analyze the variability of the content of the main phenolic components of G.elata by detecting the phenolic components..5 The sedative-hypnotic effect in mice was selected as the pharmacological evaluation index,and the preliminary study on the sedative-hypnotic effect of G.elata was carried out through the experiments on autonomic activity,subvalent dose and supravalent dose of pentobarbital sodium synergistic effect,combined with the determination of neurotransmitter content in isolated brain tissue of mice.Results1 The same results were observed for the variability between the extract contents of the three G.elata when dilute ethanol-75%ethanol-pure ethanol was used as the extracting solvent,respectively.The extract content of hybrid G.elata and its parental G.elata showed that:Although the F₁generation of hybrid G.elata was not as good as that of G.elata Bl.f.glauca S.Chow,there was no significant difference（P>0.05）compared with G.elata Bl.f.glauca S.Chow,showing consistency in the content of extract with that of G.elata Bl.f.glauca S.Chow in the main production area of Zhaotong.The F₁generation of hybrid G.elata showed a significant increase（P<0.05）in the extract content compared with the parental G.elata Bl.f.elata.2 A total of 38 chemical constituents was identified from G.elata,including eight flavonoids,four phenols,seven alkaloids,nine phenylpropanoids,four terpenoids,four amino acid derivatives,one organic acid and derivatives,and one aliphatic acyl.It was found that the hybrid G.elata and its parents showed consistency in the overall metabolite,and the identified chemical composition species were completely consistent with the parents in the F₁generation.3 The HPLC fingerprints of G.elata was established by using Chinese medicine fingerprinting software,and cluster analysis and principal component analysis were performed.Among them,when different concentrations of ethanol were selected as the extracting solvent,the three G.elata were clustered into two categories,which could be divided into a large category and a small category according to the quantity share.In the category with a large volume share,the hybrid F₁-generation and its parental G.elata Bl.f.elata and G.elata Bl.f.glauca S.Chow were included,and there was no obvious classification boundary among the three G.elata.In the principal component analysis,when dilute ethanol was used as the extract solvent,hybrid G.elata（S4）and G.elata Bl.f.elata（S11）were of better quality and ranked first and second,respectively;when 75%ethanol was used as the extract solvent,hybrid G.elata（S3）and G.elata Bl.f.elata（S8）were of better quality and ranked first and second,respectively;When pure ethanol was used as the solvent for the extract,G.elata Bl.f.glauca S.Chow（S19 and S20）had a better overall evaluation and ranked first and second.4 The content of the six phenolic components of G.elata was determined,and when dilute ethanol was used as the solvent of the extract,there was a difference in the content of p-hydroxybenzyl alcohol between hybrid G.elata and G.elata Bl.f.glauca S.Chow,G.elata Bl.f.elata and G.elata Bl.f.glauca S.Chow（P<0.05）.The three varieties of G.elata,differed from each other in the content of parishin A（P<0.05）,with G.elata Bl.f.elata>hybrid G.elata>G.elata Bl.f.glauca S.Chow in the content of parishin A.When 75%ethanol was used as the solvent for the extracts,hybrid G.elata differed from G.elata Bl.f.glauca S.Chow,G.elata Bl.f.elata and G.elata Bl.f.glauca S.Chow in the content of p-hydroxybenzyl alcohol（P<0.05）.G.elata Bl.f.elata and G.elata Bl.f.glauca S.Chow differed from each other（P<0.05）in parishin A content.When pure ethanol was used as the solvent for the extracts,there was a difference in the content of gastrodin and parishin B between hybrid G.elata and G.elata Bl.f.glauca S.Chow（P<0.05）,and the three varieties of G.elata differed from each other in the content of parishin A（P<0.05）,with hybrid G.elata>G.elata Bl.f.elata>G.elata Bl.f.glauca S.Chow.In terms of some phenolic components,the first-generation（F₁）hybrids of G.elata showed a superior advantage over both parents.5 Compared with the blank group,dilute ethanol and 75%ethanol G.elata alcoholic extract not only significantly shortened the number of mice’s voluntary activity standing and increased the rate of sleep induced by sub-threshold dose of sodium pentobarbital,but also synergized with sodium pentobarbital to shorten the time to sleep,prolong the duration of sleep and increase the content of GABA and5-HT in mice’s brain tissues.It has obvious sedative and hypnotic effects.Moreover,for different varieties of G.elata,hybrid F1 generation and its parent G.elata have similar sedative and hypnotic effects.Conclusion:Experimental studies on the chemical composition and sedative-hypnotic properties of hybrid G.elata and its parents revealed that there was consistency and variability between the intrinsic qualities of the three varieties of G.elata.In particular,the consistency was that the different varieties of G.elata did not distinguish well between the three types of G.elata in terms of chemical composition,cluster and principal component analysis and sedative-hypnotic experimental results.The F₁generation of hybrid G.elata showed similar experimental results to the parents,and the F₁generation of hybrid G.elata and its parental G.elata showed a high degree of consistency.In contrast,the determination of the content of extracts and major phenolics of G.elata showed significant variability between the t three varieties of G.elata.The differences were that the F1 generation of hybrid G.elata was significantly higher in extract content than the G.elata Bl.f.elata,as was the case with G.elata Bl.f.glauca S.Chow.In the partial phenolic content of the first-generation（F₁）hybrids of G.elata showed a superior hybrid advantage over both parents.In terms of extract content and partial phenolic content,the F₁generation was significantly better than the parent G.elata Bl.f.elata,with a higher consistency with G.elata Bl.f.glauca S.Chow,the first-generation（F₁）hybrids can be used as a mainstream cultivar in Jinzhai.