Research Of Active Substance Recognition Of Periploca Forrestii Schltr. By Spectrum-effect Relationship

Periploca forrestii Schltr.,which belongs to Asclepiadaceae family,a widely distributed shrub found in southwest China and the roots of which have been used as traditional Chinese medicines for the treatment of rheumatoid arthritis and wounds.Identification of efficient constituents,is always considered as a difficult problem because of the limitation of techniques and methods.To establish a new pattern to solve the problem,spectrum-effect relationships were applied to investigate the active substance recognition of P.forrestii Schltr in this study through the support of Natural Science Foundation of China and our group research on 80% ethonal extract from P.forrestii Schltr.Fractions were separated by macroporous resin and then analyzed carefully to locate the corresponding position in the HPLC-DAD fingerprint based on the establishment of spectrum-effect relationships and chemical fingerprints.After the comparative studies of biological activities of these fractions prepared were performed,to further find the fraction with better biological activities,as a result,the flavoniods were recognized as the active substances.Thus,flavonoids extracted from P.forrestii Schltr(TFPF).attracted much interest.In this study,Microwave-assisted extraction with ultrasonic pretreatment(UAE+MAE)was used to investigate the extraction yield of flavonoids from Periploca forrestii Schltr.and the experimental conditions were optimized by response surface methodology(RSM).Furthermore,the extraction yield of TFPF obtained by UAE+MAE,Microwave-assisted extraction(MAE),ultrasonic-assisted extraction(UAE),as well as the anti-allergic activities of TFPF obtained by UAE+MAE,MAE,UAE after purification were evaluated.The main results were as follows: 1.Preparative separation of fractions by macroporous resinIn order to preparate the active fractions isolated from P.forrestii Schltr,the study of screening separation material was performed,D101 macroporous resin was chosen.Six fractions of P.forrestii Schltr were separated by macroporous resin using 5%-95% ethonal as eluent,which were A(18 g),B(8 g),C(5 g),D(10 g),E(10 g),F(10 g),respectively.Then they were analyzed by HPLC-DAD to locate the responding characteristic peaks in the chemical fingerprints,which was marked with characteristic peaks 1-8,they were belonged to A(1,2),B(3),C(4,5),D(6),E(7,8),F(compounds in the time of 30-35min).2.optimization study on biological activities of fractionsAccording to the traditional efficacy of P.forrestii Schltr and the research progress of pharmacological activities,the anti-allergic activity,DPPH·scavenging activity,ABTS·+scavenging activity,AChE inhibitor,cell cytotoxicity of six group of active fractions(A-F)prepared and 80% ethonal crude extract(G)from Periploca forrestii Schltr.were performed in this study,the anti-allergic ability of the fractions is as follows: E>A>C>F>B>D>G,the order of DPPH· radical scavenging ability of these samples was: E>F>D>C>A>B>G,the order of ABTS·radical scavenging ability of these samples was:E>D>B>C>A>F>G,AChE inhibitor ability of the samples is as follows: E>D>F>B>A>C>G,cell cytotoxicity ability of these samples was:E>A>B>C>D>G>F.Fraction A,E were confirmed as the main effective constituents in the evaluations of anti-allergic activity and cell cytotoxicity.Besides,fraction E revealed better DPPH·scavenging ability,ABTS·+ scavenging ability,AChE inhibiting ability.3.The study of active substance recognitionThe structures of active fraction E and reference substances were analyzed by ultraviolet spectroscopy and HPLC-DAD.5 characteristic peaks were marked in chemical fingerprints of fraction E,two peaks were recognized,quercetin-3-O-?-L-arabinopyranoside and quercetin-3-O-?-D-glucopyranoside were confirmed by comparison with their standard substances.4.The extraction process study of PFTFFlavonoids were recognized in the previous study,providing science references for further researching of P.forrestii Schltr.Microwave extraction combined ultrasonic pretreatment of flavonoids from P.forrestii Schltr was investigated in this study,extraction process was first performed in an ultrasonic cleaner,then treated by microwave irradiation.The optimum ultrasonic time of 25 min was selected by single-factor experiments.A response surface methodology has been used to obtain a mathematical model that describes the process and analyzes the significant parameters ethanol concentration 59.92%,liquid to raw materials ratio 21.24 mL·g-1,microwave radiation time 209.53 s,and microwave power 274.14 W.In these optimum conditions,the yield of flavonoids from P.forrestii Schltr could be up to 9.11 ± 0.08%,which was increased by 14.30 and 19.86% compared microwave extraction and ultrasonic extraction,respectively.In vitro suppress hyaluronidase experimentation showed that TFPF extracted by UAE+MAE and purified using polyamide exhibited better anti-hyaluronidase ability with IC50 value of 1.033 mg·mL-1 compared to that of UAE(IC50 = 12.913 mg·m L-1)and MAE(IC50 =1.762 mg·mL-1).