Study On Identification And Differentiation Of Bio-active Constituents In Chinese Medicine By Selective Knock-out

In order to provide theory basis and scientific data for clinical applications of traditional Chinese medicines (TCMs) and promote the development of TCMs, a variety of approaches for the screening and analysis of bioactive compounds have been utilized and improved during the past decades. However, these approaches are time consuming, arduous, and unsuitable for the direct screening of TCM bioactive compounds because the therapeutic effect of TCM does not depend on a single active compound, the selection of individual bioactive compounds neglects the interactions of multiple compounds. Therefore, all bioactive components of TCM must be tested as an indivisible whole using holistic approaches and integrated evaluation models. In this study, our aim was to develop a generally applicable approach and methodology for investigating and differentiating bioactive components in herbal medicines by using preparative high performance liquid chromatography (prep-HPLC) and selective oriental knock-out strategy on the basis of holism.This work was supported by National Key Technology R&D Program named as "Study on key technologies for bio-active constituents in traditional Chinese medicine".This dissertation is divided into two major parts. In the first part, a systemic review was provided for the research overview of thoughts and methods to study on bio-active constituents in traditional Chinese medicine.There are three chapters in second part, specific research content and correlated conclusion as follows:The first chapter was to explore a method for studying the difference on the role of the same compound in different environment by preparative HPLC and to reveal the significance by investigating bio-active constituents in traditional Chinese medicine (TCM) on the basis of holism. In this study, target component Ferulic acid (FA) eliminated by using preparative high performance liquid chromatography (prep-HPLC) followed by antioxidant activity test was applied to investigate the roles of FA in Angelicae Sinensis Radix (DG), Chuanxiong Rhizoma (CX) and their combination (GX).There are two segments in this chapter. In first segment, FA was knocked out from samples by prep-HPLC and the results were analysised by HPLC. The resulted showed FA was successfully and exclusively depleted from DQ CX and GX, respectively.In the second segment, the antioxidant activity was performed by DPPH radical-scavenging activity test. The effect of the recovery samples ((DG-FA)+FA’,(CX-FA)+FA’and (GX-FA)+FA), in which the content of FA was the same as the original sample (DQ CX and GX), had no obvious difference with their original sample at certain more doses. The results revealed that samples after eliminating the FA reached the demand on the antioxidant activity evaluation, so the eliminated samples could be used for evaluating the roles of FA in DQ CX and GX on antioxidant activity. Comparing the effects of all samples, the result found that FA was one of the main antioxidant constituents in DG, CX and GX, and the roles of FA were DG>CX>GX. Furthermore, the roles of FA in DG, CX and GX on antioxidant activity had an apparent dose-dependent manner, and their dose-effect relationship curves had the same trend at the tested concentrations, which demonstrated that FA could produce a significant effect on antioxidant activity in DQ CX and GX. The results also showed that the roles of FA were DG> CX-GX, which also further demonstrated that the role of FA in DG was greater than that in CX and GX.This chapter provided a reliable and effective approach to clarify the contribution of same compound in different TCMs to their bio-activities.The second chapter was to explore a method for studying bio-active constituents in TCM on the basis of holism. In this study, based on the strategy of selective knock-out, elimination of main components by using prep-HPLC followed by bio-activity evaluation was applied to investigate the roles of major components in Honghua. Then, the main bio-active components and the contribution of these constitutes in Honghua can be found by comparing their different bio-activities.There are three segments in the second chapter. First segment, mian constituents in honghua were knocked out by prep-HPLC and the results were analysised by HPLC. The resulted showed13major components were eliminated by this method and26samples including samples after depleting the target component (HH1-HH13) and depleted components (C1-C13) were obtained. Moreover, components were indentified with UV data, MS data, reference compound comparison and literature comparison. There were Cytidine (1), Adenosine (2), hydroxysafflor yellow A (3), unknown (4),6-hydroxykaempferol-3,6-di-O-β-glucoside-7-O-β-glucuronide (5),6-hydroxykaempferol-3,6,7-tri-O-β-glucoside (6),6-hydroxyapigenin-6-O-glucoside-7-O-glucuronide (7), the mixture of6-hydroxykaempferol-3,6-di-O-β-glucoside and6-hydroxykaempferol-3-O-β-rutinoside-6-O-β-glucoside (8), anhydrosafflor yell-ow B (9), Keampferol-3-O-β-rutinoside (10) and6-hydroxykaempferol-3-O-rutinoside (11),4,12and13haven’t been identificated.In the second segment, the activity on activating blood circulation (such as platelet aggregation and anticoagulation in vitro), antioxidant (such as DPPH, ABTS and OH radical-scavenging activity test, Fe3+reducing prower test) and promotion (such as activity on mouse uterine contraction in vitro and on rats ovary granulosa cell proliferation) were investigated to optimize proper indexes and concentration for further study. According to the result, samples at the concertration of0.5g/mL (crude drug dosage) were used for activity tests on ADP-induced platelet aggregation and APTT, and0.7g/mL were for activity tests on PAF-induced platelet aggregation, TT and PT. The sample concentration used for DPPH radical-scavenging activity test, ABTS radical-scavenging activity test and Fe3+reducing prower test was5mg/mL,0.156mg/mL and5mg/mL respectively. Activity on rat ovary granulosa cell proliferation was investigated with samples at final concentration of200μg/mL. Effect on OH radical-scavenging activity and mouse uterine contraction in vitro was too low to be used as activity indexes for further investigation.In the third segment, activities on the activity on platelet aggregation and anticoagulation in vitro, antioxidant and rats ovary granulosa cell proliferation were investigated on proper condition to explore the main bio-active components and evaluate the contribution of these bio-active constitutes in Honghua.The result indicated C5, C6, C8, C10, C12and C13were considered as main bio-active constituents on ADP-induced platelet aggregation in honghua, contribution of these bio-active constituents in honghua was C8>C10>C6>C5;C3,C8, C9and C10were major bio-active constituents on PAF-induced platelet aggregation in honghua as compared with original samples HH, the contribution was C8>C9>C3> C10; C3, C4, C6, C9, C10and C11were considered as the main bio-active constituents on PT, the strength was C3>C10>C11>C6>C4>C9; C3,C9and C10were major bio-active constituents to extend APTT, the strength was C10>C9> C3; C1,C3,C4, C6, C10and C11could influence TT directly or indirectly when they existed in honhua, the strength was C9>C10>C3>C7. Finally, based on the bio-active constituents of single and total effect index, four components were considered as main bio-active constituents on total activating blood circulation in honghua. There were C3(hydroxysafflor yellow A), C8(6-hydroxykaempferol-3,6-di-O-β-glucoside and6-hydroxykaempferol-3-O-p-rutinoside-6-O-β-glucoside), C9(anhydrosafflor yellow B), C10(6-hydroxykaempferol-3-O-rutinoside), the contritution value was C10>C9>C3>C8.Result on antioxidant activitie showed that six of the target depleted constituents were found as main contribution for antioxidant property. They were1(Cytidine)、4(unknown),8(mixture of6-hydroxykaempferol-3,6-di-O-β-glucoside and6-hydroxykaempferol-3-O-β-rutinoside-6-O-β-glucoside),9(anhydrosafflor yellow B),10(6-hydroxykaempferol-3-O-rutinoside),11(kaempferol-3-O-rutinoside) and12(unknown), the strength was C10>C8>C4>C1>C9>C11>C12.The effect on rats ovary granulosa cell proliferation showed all eliminated components except C3and C5were main bio-active constituents on this activity, the strength of the contributin value was C7>C6>C4>C11>C1>C8>C10>C9> C12>C13>C2.However, more indexes are needed to clarify the bio-active constituents on promotion.This chapter suggested a novel and effective approach for studying complex bio-active constituents of TCM with selective knock-out followed by bio-activity evaluation. The direct and indirect role of the target components in different TCMs can be demonstrated by using this method, which can clarify their bio-active constituents more clearly. The mechanism also can be clarified simultaneously.The third chapter was to explore a method for studying the change on the contribution of the main bio-active constituents in TCM pairs on the basis of holism. In this study, based on the strategy of selective knock-out, depletion of main components by using prep-HPLC followed by bio-activity evaluation was applied to investigate the roles of target components in Honghua pairs.There are two segments in the third chapter. First segment, mian bio-active constituents hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (ASYB) in honghua were knocked out by prep-HPLC from different Honghua pairs such as honghua-taoren (HT), honghua-gancao (HG), honghua-hongshen (HS), honghua-danggui (HD), honghua-sumu (HM), honghua-danshen (DH), honghua-huangqi (HQ) pairs and honghua (HH), and the results were analysised by HPLC. The resulted showed18samples obtained after depleting the target component HSYA and ASYB, there were HSYA and8depleted samples after depleted HSYA (HT-1, HG-1, HS-1, HD-1, HM-1, DH-1, HQ-1and HH-1), ASYB and8depleted samples after depleted ASYB (HT-2, HG-2, HS-2, HD-2, HM-2, DH-2, HQ-2and HH-1).In the second segment, activities on the activity on platelet aggregation and anticoagulation in vitro were investigated on proper condition to explore the main bio-active components and evaluate the contribution of this two constitutes in Honghua pairs. The result indicated HSYA and ASYB were considered as main bio-active constituents on activating blood circulation in honghua and its pairs, contribution of HSYA in these honghua pairs was HS> HM> HD> HQ> HT> HG> DH> HH; contribution of ASYB in these honghua pairs was HG> HT> HD> HM> DH> HQ> HS> HH. The contribution of these two components showed difference in different compatibility and activity index.This chapter suggested a novel and effective approach for studying the comparison of bio-active constituents in Chinese medicine pairs (CMP) with selective knock-out followed by bio-activity evaluation, and the change of CMP can be clarified.This dissertation established an identification and proof technology by selective knock-out for bio-active constituents in traditional Chinese medicine with investigating the contribution of FA in Angelicae Sinensis Radix, Chuanxiong Rhizoma and their combination, main bio-active constituent on different activities in honghua, and the contribution of HSYA and ASYB in honghua pairs. Furthermore, this dissertation also suggested the potential utilization of preparative HPLC in the characterization for the roles of multi-ingredients in TCM, and suggested a novel and effective approach for the study of TCM.