The Study On The Quality Standard And Antioxidant Bioactive Constituents Of Callicarpa Kwangtungensis Chun

ObjectiveTo make a series of studies about chromatographic fingerprint, content determination, antioxidant activity and bioactive constituents of Callicarpa kwangtungensis Chun (CK) It may provide scientific basis for the quality standard and rational use in order to obtain safe, effective TCM preparation containing CK.Method1. Chromatographic fingerprint of Callicarpa kwangtungensis Chun and its application in the identification of the crude drugs of Callicarpa speciesTo develop a RP-HPLC-DAD method and optimize the extracting condition,16batches of CK were detected and HPLC fingerprint of CK was established. Their characteristic peaks were confirmed. Clustering analysis and similarity evaluation were used to evaluate quality and authenticity of CK.2. Determination of three benzene glycosides of Callicarpa kwangtungensis Chun by external standard method and multi-components by single-markerTo develop a RP-HPLC-DAD method, the values of relative correlation factors of poliumoside to forsythiaside B and verbascoside were established, the determination of three benzene glycosides in the leaves and stems of CK from different habitats with external standard method (ESM) and multi-components by single-marker (QAMS), and the results were compared by this two methods.3. Qualitative study on chemical constituents of Callicarpa kwangtungensis ChunA HPLC-DAD-ESI-Trap MS identification method was develop for components of CK. 4. Correlation of the contents of total phenols and total flavonoids and antioxidant activities of Callicarpa kwangtungensis ChunThe antioxidant activities of CK were determined by seven antioxidant methods, including·O2-scavenging, DPPH· scavenging, ABTS+· scavenging,·OH scavenging, Cu2+-chelating, and Fe3+reducing assays, anti-lipidperoxidation. The correlations between antioxidant activity from seven methods and known contents were counted respectively. A mice study and a cell test were carried out to verify the antioxidant activity of n-butanol fraction.5. Screening of the antioxidant bioactive constituents of Callicarpa kwangtungensis ChunWith RP-HPLC-DAD method, comparing the peak area of every identified constituent in previous and later chromatograms, some identified compounds can be rapidly screened as antioxidants.Result1. Chromatographic fingerprint of Callicarpa kwangtungensis Chun and its application in the identification of the crude drugs of Callicarpa speciesThe methodological evaluation results of Chromatographic fingerprint conformed to the requirements, RSD<5%. There were8characteristic peaks to be confirmed. With the clustering analysis and similarity evaluation,16batches of CK were classified into3groups and except L-2(0.879),L-8(0.843),all the CK similarity were over0.900. The similarities between10batches of Callicarpa species and HPLC fingerprints were less than0.200.2. Determination of three benzene glycosides of Callicarpa kwangtungensis Chun by external standard method and multi-components by single-markerThe methodological evaluation results of ESM and QAMS conformed to the requirements, RSD<5%. Within the linear range, the values of relative correlation factors at330nm of poliumoside to forsythiaside B and verbascoside were1.226,1.010, respectively. There was no significant difference between calculated value of multi-components by single-marker and measured value of external standard method and the relative correlation factors was credible. The contents of three benzene glycosides of CK in various parts and habitats were very different.3. Qualitative study on chemical constituents of Callicarpa kwangtungensis ChunCK contained at least11kind of benzene glycosides and2kinds of flavones including β-OH-forsythoside B, β-OH-poliumoside, Forsythoside B, Poliumoside, Acteoside, Alyssonoside,2’-forsythoside B, Brandioside,2’-acetylacteoside,2’,7"-diacetoxylacteoside,3,4-dimethoxy,β-OH-2’-acetyacteoside, Acacetin-7-O-diglucuronide and Rhamnazin.4. Correlation of the contents of total phenols and total flavonoids and antioxidant activities of Callicarpa kwangtungensis ChunThere were significant positive correlations between DPPH· scavenging activity (0.840), ABTS+· scavenging activity (0.905),·O2-scavenging activity (0.974), Cu2+-chelating activity (0.998), antioxidant effect on anti-lipidperoxidation (0.987b) and total phenolic content in various fractions of extract while non-significant correlation was observed between·OH scavenging activity, Fe3+reducing assays and total phenolic content. Non-significant correlations were present between various antioxidant activities and total flavonoid content in this study. An n-butanol fraction of CK showed a protective effect on CCl4-induced acute liver injury mice and TBHP-induced HepG2cells.5. Screening of the antioxidant bioactive constituents of Callicarpa kwangtungensis ChunApart from2’,7"-diacetoxylacteoside,3,4-dimethoxy,β-OH-2’-acetyacteoside, Acacetin-7-O-diglucuronide, some constituents including β-OH-forsythoside B, β-OH-poliumoside, Forsythoside B, Poliumoside, Acteoside, Alyssonoside,2’-forsythoside B, Brandioside,2’-acetylacteoside and Rhamnazin showed a good antioxidant activity.ConclusionChromatographic fingerprint, ESM and QAMS can be used as comprehensive assessment method of CK. The polyphenolic constituents in n-butanol fraction were the antioxidant bioactive constituents of CK. In this fraction, there were at least9kinds of benzene glycosides and2kinds of flavones which showed a good antioxidant activity. The study not only provided references to completely quality control of CK, but also provided research foundation for the study on pharmacological activity of CK.