Research On The Quality Evaluation Of Baiji Medicinal Materials

Background Bletillae rhizoma is the dry tuber of Bletilla striata(Thunb.)Reichb.f.The 2020 edition of "Chinese Pharmacopoeia" records that it can converge and stop bleeding,reduce swelling and grow muscles,and be used for the treatment of hemoptysis,vomiting,traumatic bleeding,sore swelling,and skin chapped.Modern research shows that it has anti-bacterial,anti-tumor,anti-oxidant and anti-viral effects.Bletillae rhizoma is widely planted inYangtze River Basin and other provinces to the south,such as Guizhou,Hubei,Yunnan and other places.With the extensive planting of Bletillae rhizoma,whether Bletillae rhizoma have uneven quality problems requires more research.Regarding the quality evaluation standard of Bletillae rhizoma,"Chinese Pharmacopoeia" 2015 edition only has qualitative identification and impurity inspection;"Chinese Pharmacopoeia" 2020 edition and "Hong Kong Standards for Medicinal Materials Ninth" also adds militarine content determination indicator.Based on the in-depth research on the chemical composition and pharmacology of Bletillae rhizoma,the quality evaluation indicators of the Bletillae rhizoma needs to be further improved.Objective quality evaluation indicators and methods of Bletillae rhizoma are revised and improved to evaluate and analyze the quality of Bletillae rhizoma in different origins and grades.And provide a basis for a more comprehensive understanding of the quality of different origins and different grades of Bletillae rhizoma.Methods(1)Collected 36 batches of Bletillae rhizoma from different origins,and evaluated the quality of Bletillae rhizoma through thin layer chromatography,polysaccharide content and UPLC fingerprint.(2)Collected 74 batches of Bletillae rhizoma samples from Guizhou,Yunnan,and Hubei,and divided them into three grades due to their size.Through the determination of the UPLC fingerprint and polysaccharide content,and combined with similarity analysis,cluster analysis,heat map analysis,and Spearman correlation analysis,a comparative study of Bletillae rhizoma from Guizhou,Yunnan and Hubei were carried out,and the relationship between grade,origin and internal components were analyzed.(3)Through the UPLC-Q-TOF MS/MS qualitative analysis method and comparison of the reference substance,the common peaks of Bletillae rhizoma fingerprint were initially identified.(4)Based on the research on the quality evaluation of Bletillae rhizoma,an updated draft of the quality standard for Bletillae rhizoma was initially formulated and drafting instructions were formulated.Results(1)The TLC method of the Pharmacopoeia was optimized,and the UPLC fingerprint spectrum with catechin as the internal standard was established,and 14 common peaks were obtained.The similarity of different batches of samples was above 0.8.The range of polysaccharide content was(25.16±9.17)%.(2)When comparing samples from the same origin,Except for the common peaks of No.3,5,6,10,and 13 in the fingerprints of the Yunnan and Guizhou,the chemical composition content of the other common peaks were negatively correlated with the size of Bletillae rhizoma(P<0.05,0.4<r ≤0.8),among which peaks 7,8,and 9 have a strong correlation with the size of Bletillae rhizoma(r>0.7);polysaccharide content of Bletillae rhizoma was positively correlated.The content of chemical components in different sizes of Bletillae rhizoma from Hubei origin tended to be uniform.When comparing samples from different origins,the spectrum of Bletillae rhizoma from Yunnan lacked peaks 15 and 16;and the content of common peaks’chemical components in samples of the same grade was Guizhou>Yunnan.>Hubei,polysaccharide content of rhizoma from Yunnan(26.98±8.56)%and Hubei(28.73±5.20)%>Guizhou(14.50±4.67)%.(3)Through the UPLC-Q-TOF MS/MS analysis and comparison of reference substances,the common peaks of fingerprint were qualitatively analyzed.A total of 10 common peaks were identified.They were dactylorhin E or isomer(peak 3);dactylorhin A(peak 4),gymnosideⅢ(peak 6),militarine(peak 7),habenarioside or gymnoside Ⅷ(peak 8),gymnosideⅧ or habenarioside(peak 10),gymnoside Ⅷ or habenarioside(peak 11),1-(p-hydroxybenzyl)-4-methoxy-9,10-dibydrophenanthrene(peak 13),1-(p-hydroxy benzyl)-4-methxoyphenanthrene-2,7-diol(peak 14).(4)Based on the above experimental results and the existing quality standards for Bletillae rhizoma,it was planned to increase the content determination and fingerprinting as quality evaluation indicators.Conclusion Thin-layer chromatography and UPLC fingerprint spectroscopy could effectively identify adulterant of Bletillae rhizoma.Using polysaccharide content and UPLC fingerprint to analyze the chemical composition differences of different grades of Bletillae rhizoma from different origins,It was found that compared with the quality of Bletillae rhizoma in different grades,the influence of the origin on the quality of Bletillae rhizoma was more obvious.It was recommended to further investigate the possibility of using the origin to divide the specifications of Bletillae rhizoma.The polysaccharide content and UPLC fingerprint analysis could provide a reference for the evaluation of Bletillae rhizoma quality.