Studies On Analysis Of Flavonoids Of Flowers, Stems And Leaves Of Abelmoschus Manihot(L.)Medic

Objectives:To establish a method for the determination of main constituents and HPLC fingerprint of Abelmoschus corolla in order to optimize the quality evaluation system; To analyze the volatile constituents from Flos Abelmoschus manihot in order to provide reference for the development and utilization of Flos Abelmoschus manihot; To study the chemical constituents of stems and leaves of Abelmoschus manihot and establish a method for the determination of main constituents of stems and leaves of Abelmoschus manihot, and reveal the effective constituents of stems and leaves of Abelmoschus manihot, which was greatly contributed to the study of quality control and pharmacodynamics.Methods:(1) To establish a method for the determination of total flavonoids of flowers, stems and leaves of Abelmoschus manihot by UV-VIS spectrophotometry.(2) To establish a method for simultaneous determination of hyperin, isoquercitrin, myricetin and quercetin in the flowers of Abelmoschus corolla from different areas by HPLC, and compare their content differences.(3) To establish HPLC fingerprint of Abelmoschus corolla, and the data was processed on the similarity of fingerprint software and the SPSS analysis software for cluster analysis.(4) To analyze the volatile constituents from Flos Abelmoschus manihot by GC-MS method.(5) To analyze the flavonoids in stems and leaves of Abelmoschus manihot by HPLC-ITMS.(6) To study the chemical constituents of stems and leaves of Abelmoschus manihot by using several separation and purification methods and determination technology (such as 1H-NMR,13C-NMR).(7) To establish a method for simultaneous determination of rutin, hyperin and isoquercitrin in the stems and leaves of Abelmoschus manihot from different areas by HPLC, and compare their differences.Results:(1) Established the content determination method of total flavonoids, the linearity of flavonoids was fine in 12～72μg/ml, average recovery rate 97.96%, RSD 2.63%(n=6). The method has satisfactory accuracy, stability and reproducibility.(2) Established the simultaneous determination method of hyperin, isoquercitrin, myricetin and quercetin, the regression equation:Y= 1489889X-14982(r=0.9999); Y=2183245X-23801(r=1); Y=2830492X-44686(r=0.9990); Y=3135211X-25706(r=0.9994). The method has satisfactory accuracy, stability and reproducibility.(3) Seventeen common peaks in HPLC fingerprint of Abelmoschus corolla were separated from the 10 batches of samples, the common pattern was established, and the similarity of Abelmoschus corolla from different areas was above 90%.(4) A total of 47 volatile components from Flos Abelmoschus manihot were successfully identified by GC-MS, which were mainly composed of hydroxy compounds, acyclic alkane compounds and other aromatic compounds. And the eight components with the highest relative content fraction (>2%) are hexadecane,2,6,10,14-tetramethyl-(37.83%), pentacosane (13.49%), heptadecane,9-octyl-(9.78%), cedrol (3.07%), 9,12,15-octadecatrienoic acid,methyl ester, (Z,Z,Z)-(2.79%), tetratetracontane (2.78%), cyclotetracosane (2.61%) and hexadecanoic acid, methyl ester (2.32%).(5) 5 flavonol glycosides from stems and leaves of Abelmoschus manihot were successfully identified by HPLC-ITMS, including:quercetin-3-O-glucosyl-arabinoside, quercetin-3-O-galactorhamnsoide, rutin, hyperin and isoquercitrin, which are also riched in Flos Abelmoschus manihot.(6) 9 compounds were isolated from stems and leaves of Abelmoschus manihot, and 6 compounds were identified, including 3 flavonoids (hyperin, isoquercitrin and rutin) which are also riched in Flos Abelmoschus manihot and 3 other compounds (n-Hexacosanoic acid,10-Eicosenoic acid and ethyl heptadecanoate).(7) Established the simultaneous determination method of rutin, hyperin and isoquercitrin, the regression equation:Y= 1522361X+4585(r= 0.9999); Y= 1511417X-273 (r= 1); Y= 2286106X-434 (r=0.9999). The method has satisfactory accuracy, stability, reproducibility and has laid the foundation for the control of the inherent quality in the stems and leaves of Abelmoschus manihot.Conclusions:The method established for quality evaluation has excellent specificity and reproducibility for the quality control of Abelmoschus corolla. The qualitative analysis of volatile constituents from Flos Abelmoschus manihot laid the foundation for the development and utilization of Flos Abelmoschus manihot. This study revealed the effective constituents of stems and leaves of Abelmoschus manihot, and the method established for content determination has satisfactory accuracy, stability, reproducibility and has laid the foundation for the control of the inherent quality in the stems and leaves of Abelmoschus manihot.