Evaluation Method Of Hypolipidemic Efficacy Component Of The Biological Effects Of Citrus Aurantium

1. ObjectivesThe research is about the active components alignment (ACA) of Fructus Aurantii Immaturus reducing hyperlipoidemia, Fructus Aurantii Immaturus and its ACA's biological effects of reducing hyperlipoidemia, meanwhile, compared with Fructus Ponciri; and also about establishing the method of biological-effect evaluation about reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri, discussing the biologic quality standard of Fructus Aurantii Immaturus ACA for settling the foundation of new quality evaluation system on Chinese Medicine (CM) ACA.2. MethodsThe reducing hyperlipodemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri were analyzed by analyzing the concentration according to HPLC and determination of biological effect about reducing hyperlipoidemia.The reducing hyperlipoidemia effect was validated by using hyperlipoidemia model of SD rat induced by high-fat food and Triton WR-1339. By combining HPLC technique and determination of biological effects on Fructus Aurantii Immaturus ACA and Fructus Ponciri ACA the reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri were found. The in vitro indexes about hyperlipoidemia were chosen to study the effect on reducing hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs. That is choosing HUVEC model induced by Ox-LDL to study the effect on ICAM-1 expression and NO concentration in supernate of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs. The effects of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs on activity of LDH in supernate, concentration of MDA, T-AOC, activity of Cu-Zn-SOD, activity of GSH-Px, activity of CAT in HepG2 induced by H2O2 and t-BOOH were studied. The effects of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs about MMP of HepG2 induced by H2O2 and t-BOOH were researched by flow cytometry(FCM). The protective effect of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs to DNA of HepG2 induced by H2O2 and t-BOOH was studied by using SCGE.3. Results3.1 Reducing Hyperlipoidemia ACA of Fructus Aurantii Immaturus (1) ACA of Fructus Aurantii ImmaturusExtraction of 70%EtOH which was recorded in pharmacopoeia was the researchful object and its ACA (CJSE ACA) is Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46:1); And the ACA of its water fraction (CJSW ACA) is Naringin- Hesperidin- Neohesperidin- Naringenin(178.51:15.18:257.67:1); The ACA of its chloroform fraction (CJSC ACA) is Hesperidin- Hesperetin- Naringenin(2.92:1:2.99).(2) ACA of Fructus PonciriACA of Extraction of 70%EtOH (KJSE ACA) is Naringin-Hesperidin-Poncirin- Naringenin- Hesperetin(514.55:16.37:2454.23:20:1); And the ACA of its water fraction (KJSW ACA) is Naringin- Hesperidin- Poncirin(28.78:1:144.24); The ACA of its chloroform fraction (KJSC ACA) is Naringenin- Hesperetin(19.29:1)3.2 Reducing Hyperlipoidemia Effect of Fructus Aurantii Immaturus in vivo3.2.1 Fructus Aurantii ImmaturusThe contents of TG, TC, LDL-C in serum of hyperlipoidemia model SD rat were decreased while the content of HDL-C in serum of hyperlipoidemia model SD rat was increased after being cured by Fructus Aurantii Immaturus, when the activity of AST can be reduced and the activity of ALT and the content of glucose can not be affected.3.2.2 Fructus PonciriThe contents of TG, TC, LDL-C in serum of hyperlipoidemia model SD rat were decreased while the content of HDL-C in serum of hyperlipoidemia model SD rat was increased after being cured by Fructus Ponciri, when activity of ALT, AST and the content of glucose in serum of hyperlipoidemia model SD rat can not be affected.The reducing hyperlipoidemia effect of Fructus Ponciri is a little better than that of Fructus Aurantii Immaturus in the hyperlipoidemia model SD rat induced by high-fat diet.3.3 Biological Effect about Reducing Hyperlipoidemia in vitro of Fructus Aurantii Immaturus and Its ACA3.3.1 Fructus Aurantii Immaturus(1) Decoction Pieces①ICAM-1 expression of HUVEC induced by Ox-LDL was inhibited.②The content of NO in supernate medium of HUVEC induced by Ox-LDL can be regulated to normal level;③The viability of HepG2 induced by H2O2 can be elevated.④Leakage of LDH in HepG2 induced by t-BOOH can be inhibited and leakage of LDH in HepG2 induced by H2O2 can be inhibited by CJSC.⑤The content of MDA in HepG2 induced by H2O2 and t-BOOH can be decreased.⑥The T-AOC of HepG2 induced by H2O2 and t-BOOH can be elevated.⑦Activity of Cu-Zn-SOD in HepG2 induced by H2O2 can be increased by CJSC. Activity of Cu-Zn-SOD in HepG2 induced by H2O2 and t-BOOH can be increased.⑧Activity of GSH-Px in HepG2 induced by H2O2 and t-BOOH can be increased.⑨Activity of CAT in HepG2 induced by H2O2 and t-BOOH can be increased.⑩MMP of HepG2 induced by H2O2 and t-BOOH can be reduced.○11 The oxidative damage on DNA of HepG2 which was induced by H2O2 and t-BOOH can be protected(2)ACA①ICAM-1 expression of HUVEC induced by Ox-LDL was inhibited.②The content of MDA in HepG2 induced by H2O2 and t-BOOH can be decreased.③The T-AOC of HepG2 induced by H2O2 and t-BOOH can be elevated.④Activity of Cu-Zn-SOD in HepG2 induced by t-BOOH can be increased.⑤Activity of GSH-Px in HepG2 induced by H2O2 and t-BOOH can be increased.⑥Activity of CAT in HepG2 induced by H2O2 and t-BOOH can be increased.⑦MMP of HepG2 induced by H2O2 and t-BOOH can be reduced.3.3.2 Fructus Ponciri(1) Decoction PiecesThe biological effects about ICAM-1 expression of HUVEC induced by Ox-LDL, the viability of HepG2 induced by H2O2, the content of MDA, T-AOC, activity of GSH-Px and CAT in HepG2 induced by H2O2 and t-BOOH, activity of Cu-Zn-SOD in HepG2 induced by t-BOOH, MMP of HepG2 induced by H2O2 and t-BOOH are the same with those of Fructus Aurantii Immaturus decoction pieces.The content of NO in supernate medium of HUVEC induced by Ox-LDL can be increased to the level which was higher than normal level. Leakage of LDH in HepG2 induced by H2O2 and t-BOOH can be inhibited by KJSC.3.4 Establishment of Biological-Effect Evaluation Method about Reducing Hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri(1) Index of biological effects: BERs of Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA about the biological effect of reducing hyperlipoidemia are between 0.9 and 1.1. And only the difference between the biological effect of activity of GSH-Px from KJSC ACA and that from KJSC is significant (t-test). The differences between the biological effect from Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA are not significant (t-test). The standard biological effects which are about reducing hyperlipoidemia of ACA are as a standard of effect and then the biological-effect evaluation method was established.(2) Index of ACA: Fructus Aurantii Immaturus: Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46 : 1 ) ;Fructus Ponciri: Naringin-Hesperidin-Poncirin- Naringenin- Hesperetin(514.55:16.37:2454.23:20:1).4. Conclusions4.1 The effects of reducing hyperlipoidemia of Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA are equivalent. The difference between the two is not significant according to t-test. It shows Fructus Aurantii Immaturus ACA can represent the quality characteristics about reducing hyperlipoidemia of Fructus Aurantii Immaturus.4.2 Biological-Effect Evaluation Method of Fructus Aurantii Immaturus(1) Biological effect indexes about reducing hyperlipoidemia①Antioxidant Index: The contents of MDA, T-AOC, activity of GSH-Px and CAT, the change of MMP in HepG2 induced by H2O2 and t-BOOH; The activity of Cu-Zn-SOD in HepG2 induced by t-BOOH;②Expression of HUVEC ICAM-1(2) Establishment of Biological-Effect Evaluation Method①Method of Antioxidant index: BERs about content of MDA in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 0.9879 and 1.0258 respectively. BERs of T-AOC in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 1.00558 and 0.99777 respectively; BERs of GSH-Px in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 1.026 and 1.044; BERs of CAT in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 0.991 and 1.023; BERs of reducing MMP of HepG2 induced by H2O2 and t-BOOH. BER of Cu-Zn-SOD activity is 1.01546.②Method of ICAM-1 Expression: BER of inhibition to ICAM-1 expression induced by Ox-LDL is 0.9781.The BERs of Fructus Aurantii Immaturus and its ACA are all between 0.9 and 1.1. At the same time the difference between the biological effect from Fructus Aurantii Immaturus and its ACA is not significant according to mathematical statistics calculation. So the standard biological effects of ACAs of Fructus Aurantii Immaturus can be as standards of effect.4.3 Reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus The reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus could be one of indexes for evaluating the qulity of Fructus Aurantii Immaturus.4.4 Suggestions of Quality Standards to the Effects about Reducing Hyperlipoidemia of Fructus Aurantii Immaturus4.4.1 Method of Antioxidant Index and Expression of ICAM-1①The biological effects of the sample should be checked by using the same method which was used to checked the biological effect of ACA.②BER should be calculated and Statistical Test should be done.③Suggestions about Standard of Biological-Effect Evaluation Method Eligible Sample: 1≥BERS≥0.9 and the difference is not significant between the biological effect and standards of effect.High Quality Sample: BERS>1 and the difference is not significant between the biological effect and standards of effect.Unqualified Sample: BERS<0.9 or the difference is significant between the biological effect and standards of effect.4.4.2 Analysis of ACAHPLC is used in the analysis of ACA.Ratio and concentration of ACA of samples should be in the range which benchmark is Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46:1)5. Innovations(1) Reducing hyperlipoidemia effect (Xue Zhuo Zheng)of Fructus Aurantii Immaturus and Fructus Ponciri were validated by modern medical experiment first.(2) ACAs of Fructus Aurantii Immaturus and Fructus Ponciri were determined.(3) Biological-effect evaluation method about reducing hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri was established.(4) Formula of calculating BER was established: BERsample=Xsample/XACA...