| Selaginella doederleinii Hieron (SD), belongs to the family Selaginellaceae. It has been used as a traditional Chinese medicine and has attracted great interest among researchers because of good pharmaceutical, in recent years. A series of original creative investigations about biflavonoids of SD have been accomplished and some significant outcomes obtained through the by multi-spectroscopic method.1. Applied the central composite design combined with response surface methodology to optimize the parameter of ultrasonic-assisted extraction the total flavonoids from SD. The results indicate that the maximum extract of total flavonoids was 4.4 mg/g by using 70% ethanol as solvent, extract 50 min at 65?. Antioxidant activity of extracts, determined by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay, IC50 ranged from 20.22 to 46.64 mg/ml of raw SD. The optimization condition for antioxidant activity was similar to extract of total flavonoids. Yield increase of the total flavonoids depends on the cooperative effect of three selected parameters in the process of ultrasonic-assisted extract. Antioxidant activity more reckon on the ethanol concentration. Moreover, it was also found that there was significant correlation between the bioactivities and content of total flavonoids.2. Basing on natural medicine chemistry, utilizing physical and chemical methods, two bi-flavonoids, amentoflavone (AM) and Heveaflavone (HE) were purified from the ethanol extraction of SD. Meanwhile, the stearic acid,?-sitosterol and stigmasterol were also isolated. The structures of them were identified by NMR, ESI-MS spectroscopies.3. Direct identification of constituent of SD by gas chromatography with mass spectrometry (GC-MS) and high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSn) technique was established. There were 59 kinds chemical compounds have been analyzed. Including 42 components of essential oil, such as n-octacosane,9-octyl-hexacosane,3-phenyl-2-butanol. There were 17 components of bi-flavonoids, which linkage by different C-C and C-O-C. AM and HE were the principal ingredient of SD. A reversed-phase high performance liquid chromatography (RP-HPLC) procedure was developed for simultaneous determination of AM and HE compounds. The variation in precision and reproducibility in peak area was<1.6 and <1.8%, respectively. The correlation coefficients, r, of calibration curves of the 2 compounds were>0.99. The spike recovery of the method was> 95%. That indicated the method could be used to quantify the quality of SD. A fingerprint profile method was developed using a HPLC method which can identify the distributions of major active components. According to the separation system of optimality conditions,15 samples from 5 regions of GuangXi province were analyzed. A standardization fingerprint profile of SD can be obtained by similarity calculation for traditional Chinese medicines fingerprints software. The correlation coefficients, r, of 15 peaks of active compounds were>0.9 by the software calculation. There are 14 peaks were the owned by all samples, which can be identified as the characteristic of SD.4. Amentoflavone and robustaflavone were investigated by negative ion electrospray ionization tandem mass spectrometry (ESI-MS). The relationship between their structural features(C3'-C8" or C3'-C6")and the corresponding characteristic ions fragmentation behavior was discussed. The conclusions were obtained about the fragmentation behavior of bi-flavonoid, which linkage by different position.(1) Bi-flavonoid, belongs to the family Selaginellaceae, have the same fragmentation pathways. Under the condition of mass spactrum, the bond of C-ring broken at 0,4 positions, and than gave the principal fragment ion at m/z=375 by lossing of neutral species. For the bi-flavonoid of C-O-C linkage, there was a fragmentation pathway that the bond of C-O broken by balanced method, and than would bring to the principal fragment ion at m/z=271. All of these ions may be the characteristic ions.(2) It was very important that the stereostructure of bi-flavonoid effects on the fragmentation pathway. Compare to the C3'-C8", bi-flavonoid of C3'-C6" and C2'-C8", dehydrating will be easy to produce cyclization process, and than gave the principal fragment ion at m/z=519, m/z=309 or m/z=401. For C3'-C3"', may produce lactones, and than gave the principal fragment ion at m/z=371. As far as the bi-flavonoid of C-O-C linkage, the fragmentation ions m/z=285 has stranger abundance value because the bond of C-O broken by unbalanced method. There was p-phenolic group on the fragmentation ions, which will be easy broken. It may bring the fragment ion at m/z=133,199 or 257. The rules may be used to distinguish the bi-flavones of SD. There were 17 constituents of bi-flavones were quality analyzed.5. Apigenin, Amentoflavone and Heveaflavone were selected to investigate the binding to bovine serum albumin. Systemic study the interaction mechanisms under different temperature and concentration. The conclusion is that:(1) It was found that two different quenching mode, static mechanism for apigenin, and the dynamic mechanism for amentoflavone, although they conformed Stern-Volmer equation.(2) The relationship between molecule structure and the BSA binding to flavonoids.A:It was found that the affinity of amentoflavone binding to BSA increases 3-20 times than apigenin, increasing 8-10 times than heveaflavone. Results show that the hydroxyl groups on the melcules might play a major role in binding flavonoids to BSA. The binding distance between apigenin and BSA was smallest. The binding distance between heveaflavone and BSA was largest. It indicated that the binding distance not only was affected by affinity, but also the molecule size.B:under the experiments conditions, the effect of AP on the submain conformational of BSA was stronger than AM, which may be affected by the molecule size. |
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