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Objective To explore the effect of timosaponin B-II ( TB-II) on the differentiation of neural stem cells (NSCs) into tyrosine hydroxylase (TH) positive neurons in neonatal rats. Methods The biological functions of self-proliferation and multi-differentiation of NSCs were identified by primary culture, cell proliferation counting,morphological observation and immunology. NSCs of SD rats were cultured in vitro and treated with different concentrations of TB-II (10 μg/ml,30 μg/ml ,100 μg/ml) for 7 days. Immuno-histochemistry was used to detect the effect of TB-II on the differentiation of NSCs into TH-positive neurons, and Western blot was used to detect the expression of TH protein in neurons. Results ( 1) The cultured cells had the ability to self-proliferation,expressed nestin protein and differentiated into neurons and glial cells. So the cultured cells were conformed to the biological function of neural stem cells. (2)Compared with the control group,the TH positive cell ratio of TB-II 30 μg/ml group and TB-II 100 μg/ml group increased ((10. 03± 1. 36)%),( 20. 01± 3. 37)%),(31. 32± 3. 98)%) ,the difference was significant ( t=6. 15, 16. 54,both P<0. 05). There was no significant difference between TB-II 10 μg/ml group and control group (P>0. 05). (3)Western results showed that the relative expression of TH protein in TB-II 30 g/ml group and TB-II 100 μg/ml group was higher than that in control group,the difference was statistically significant (con-trol group: (1. 02±0. 24),TB-II 30μg/ml group: (3. 64±1. 78),TB-II 100 μg/ml group: (5. 88±2. 34);t=12. 58,9. 15,both P<0. 05). There was no significant difference between TB-II 10 μg/ml group and con-trol group (P>0. 05). Conclusion TB-II can promote the differentiation of NSCs into TH-positive neurons.
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Objective: To provide the HPLC-ELSD fingerprint analysis method of Anemarrhenae Rhizoma, and to reflect and comprehensively control the quality of Anemarrhenae Rhizoma. Methods: The HPLC-ELSD separation was performed on a C8 analytical column (4.6 mm × 250 mm, 5 μm) gradient eluted with a mixture consisting of acetonitrile (A) and 0.2% acetic acid-water (B) at a flow rate of 1 mL/min with ELSD detector. Using a linear gradient of 0 min, 5% A; 5 min, 7% A; 5-15 min, 15% A; 15-36 min, 22% A; 36-43 min, 35% A; 43 min, 40% A; 51 min, 50% A; 51-60 min, 100% A. The temperature of column was 25 ℃. The flow rate of ELSD detector atomizer (air) was 2.6 mL/min. The temperature of the drift tube was 100 ℃ and the injection volume was 20 μL. Results: The fingerprint with better separation effect by using gradient elution method within 60 min was established, and also the 12 peak which are common peak were determined. The most of chemical components of Anemarrhenae Rhizoma were chromatographic separated in this HPLC-ELSD fingerprint. Conclusion: The method is simple and reliable, and in this way for the steroidal saponins and flavonoids of Anemarrhenae Rhizoma can be identified at the same time. Moreover, it can fully reflect the characteristics of the chemical composition of Anemarrhenae Rhizoma, therefore it can be used as an effective quality control method for Anemarrhenae Rhizoma.
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Objective: To establish a UPLC-PDA-ELSD method for the fingerprint of Xiaokeqing Granules (XG) and to determine the contents of neomangiferin, mangiferin, timosaponin BII, palmatine, and berberine so as to provide the basis for the quality control of XG. Methods: The chromatographic fingerprints were determined by UPLC-PDA-ELSD using mangiferin and timosaponin BII peaks as the reference peaks, 23 PDA and 10 ELSD common peaks were selected, and the digital quantitative fingerprint of XG was established. Using the third systematic quantified fingerprint method, the quality levels of 28 batches of XG were assessed. The contents of neomangiferin, mangiferin, timosaponin BII, palmatine, and berberine in the 28 batches of samples were determined using external st andard method. Results: Twenty batches of XG were identified as qualified and eight batches were unqualified, in which there were differences in the five main consitituents. The quantitative results were consistent with the fingerprint. Conclusion: The method is accurate, stable, and simple, which could be applied to the quality control of XG.
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Objective: To explore the advantages of decompression extraction applied in the preparation of Sansheng Hehuang Decoction (SHD). Methods: The process parameters of decompression extraction of SHD were optimized using synephrine and magnolol as indexes, the active components content, extract yield, extract powder characteristics, and volatile components by decompression extraction and atmospheric extraction were compared, and the microcosmic structures of synephrine and magnolol were observed. Results: The optimum extraction parameters were as follows: vacuum degree of 0.08 MPa (60°C), extracting twice for 30 min each time, and solid-liquid ratio of 1:10. Compared with the extraction at normal pressure by water, the content of synephrine increased by 13%, the extracting rate of magnolol increased by 10% while the extracting rate of timosaponin BII was almost the same, and the yield of the dried extract decreased by about 33%. The kinds of volatile components by decompression extraction were almost the same as those by atmospheric extraction, but the yield was lower than that by atmospheric extraction. Besides, the powder characteristics including flowability and hygroscopicity of the extract prepared by decompression extraction were similar to those by water extraction and alcohol precipitation at normal pressure. Scanning electron microscopy showed that the decompression extraction did not break the cell wall, and the cells presented arrangement disorder and shrinkage, while the cell integrity was better than that extracted at normal pressure. Conclusion: In regard to energy consumption, extraction effectness, and convenience to the subsequent preparation, the decompression extraction method is superior to the traditional atmospheric extraction method, and it could be applied in the extraction process for Chinese materia medica preparations.