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OBJECTIVE To investigate the effect and mechanism of gracillin from Reineckia carnea on autophagy in non- small cell lung cancer A549 cells. METHODS Using A549 cells as subjects， the effects of different concentrations of gracillin （0.25， 0.5， 1， 2， 4 μmol/L） on the proliferation of cells were detected by CCK-8 after being treated for different time （12， 24， 48 h）. Compared with the control group without medication， the effect of gracillin （2 μmol/L） on the formation of autophagosomes in cells was observed by transmission electron microscope after 24 h of exposure. The aggregation of GFP-LC3 on autophagosome membrane was detected by GFP-LC3 plasmid transfection after being treated with gracillin （0.25， 0.5， 1， 2 μmol/L） for 24 h. Quantitative real-time PCR and Western blot assay were used to detect the mRNA and protein expressions of family with sequence similarity 102 member A（FAM102A）， the expressions of autophagy-related proteins [p62， Beclin-1， microtubule-associated protein 1 light chain 3B （LC3B）]， and the expressions of phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway-related proteins in A549 cells after being treated with gracillin （0.25， 0.5， 1 and 2 μmol/L） for 24 h. RESULTS Gracillin significantly inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. The IC50 was 2.55 μmol/L at 24 h. After 24 h of gracillin treatment， autophagosomes with bilayer membrane structure were found in the cell cytoplasm， and GFP-LC3 green fluorescent spots on autophagosome membrane were obvious， representing an increasing trend as drug concentration. Compared with the control group， mRNA and protein expressions of FAM102A （0.5， 1， 2 μmol/L groups）， protein expression of Beclin-1 （1， 2 μmol/L groups） and LC3B-Ⅱ/LC3B-Ⅰ ratio （2 μmol/L group） were significantly increased in different concentrations of gracillin groups， while the protein expression of p62 （1， 2 μmol/L groups）， and the protein phosphorylations of Akt （1， 2 μmol/L groups） and PI3K （2 μmol/L group） were all decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Gracillin can promote excessive autophagy in A549 cells by up-regulating mRNA and protein expressions of FAM102A and inhibiting PI3K/Akt signaling pathway， thus inhibiting cell proliferation.
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Objective: To study the biotransformation of gracillin by Penicillium lilacinumACC 31890,to isolate and to identify the structures of metabolites and investigate the pharmacological activities.Methods: The conversion products were isolated and purified by silica gel column chromatography and semi-preparative reversed phase liquid chromatography.Their structures were identified by MS and NMR, and the anti-inflammatory activity of the conversion products was investigated as well.Results: Three metabolites were isolated and purified, and identified as 5R-spirost-5-ene-3-ol-O-β-D-Glucopyranoside-(1→3)-β-D-glucopyranosyl (1), trillin (2) and diosgenin (3) with the conversion rate of 1%, 1% and 45%, respectively.In vitro study showed that the three products showed certain degrees of activity to inhibit the production of NO, IL-6 and MCP-1 in LPS-primed RAW264.7 macrophages.Moreover, the anti-inflammatory activity of the bioconversion products increased along with the hydrolyzation of carbohydrate chain.Diosgenin, the final product, showed the strongest anti-inflammatory activity among the three products.Conclusion: The biotransformation of gracillin by Penicillium lilacinum has a high productivity of diosgenin.The amount of glycosyls has notable influence on the anti-inflammatory activity of steroid sapoinin.
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Objective: To develop an HPLC-DVD wavelength switching combined with gradient elution method for the determination of the contents of hydroxysafflor yellow A, protodioscin, dioscin, methylprotodioscin, pseudoprotodioscin, and gracillin in Dieda Zhitong San (DZS) simultaneously. Methods: The chromatographic separation was achieved on a Diamonsil C18 (250 mm × 4.6 mm, 5 μm) with acetonitrile (A)-0.4% formic acid solution (B) as mobile phase at the flow rate of 0.9 mL/min for gradient elution; Hydroxysafflor yellow A was detected at 403 nm; Protodioscin, dioscin, methylprotodioscin, pseudoprotodioscin, and gracillin were detected at 203 nm; Sample quantity was 20 μL. Results: The six active components were well separated and showed good linearity, such as hydroxysafflor yellow A 3.46-69.20 μg/mL (r = 0.999 4), protodioscin 9.52-190.40 μg/mL (r = 0.999 7), dioscin 8.74-174.80 μg/mL (r = 0.999 6), methylprotodioscin 4.45-89.00 μg/mL (r = 0.999 9), pseudoprotodioscin 2.64-52.80 μg/mL (r = 0.999 5), and gracillin 3.28-65.60 μg/mL (r = 0.999 8). The precision and repeatability were good, and RSD values were less than 2.0%. The stability was good in 12 h. The average recoveries and the corresponding RSD values were 98.57% (1.59%), 97.64% (1.28%), 99.43% (1.07%), 97.98% (1.64%), 98.57% (1.16%), and 97.17% (1.37%), respectively. Conclusion: An HPLC wavelength switching combined with gradient elution method has been successfully established for simultaneous determination of six components in DZS. The method is simple, quick, accurate, and helpful for the quality control of DZS.
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Objective: To establish a quantitative analysis method of seven steroidal saponins in Paridis Rhizoma and its polygerm varieties from different regions in Yunnan province, to simultaneously determine the contents of seven steroidal saponins, to establish the fingerprint for the polygerm varieties in Gaoligong Mountain by UPLC-ELSD, and to evaluate the qualities. Methods: It was detected with an Acquity UPLC® BEH C18 (2.1 mm × 50 mm, 1.7 μm) column, and gradually diluted with acetonitrile-water at a flow rate of 0.2 mL/min, drift tube temperature of 55℃, nitrogen pressure 275.8 kPa, and gain 500 by UPLC-ELSD. Results: The 23 samples could be separated and analyzed of seven steroids within 23 min. All the indexes of the methodological investigation met the requirements. The results showed that the contents of saponins from 19 samples met the requirement in Chinese Pharmacopoeia 2015. The average content of four saponins was 1.42% and among them the content from the sample in Gaoligong Mounta was up to 1.660%. The similarity was lower than 0.9 in the samples of Gaoligong Mountain, and there were eight common peaks in the fingerprints, which were identified four characteristic peaks. The type and content of seven saponins in the samples could not be clearly classified by PCA analysis and system cluster analysis. Conclusion: The method is convenient, accurate, and suitable for the quantitative analysis of Paridis Rhizoma and polygerm varieties. There is no obvious difference between Paridis Rhizoma and polygerm varieties in chemical components and contents. Paridis Rhizoma (polygerm varieties) in Gaoligong Mountain is a variety worth popularization and in-depth researches.
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Objective To control the quality of the species in Dioscorea L. better. Methods An HPLC-ELSD method was developed for the first time to simultaneously determine four bioactive ingredients:dioscin gracillin,protoneodioscin, and protoneogracillin in 31 samples belonging to seven species of Dioscorea L. from different areas. The column was an Inertsil HILIC (250mmx4.6 mm,5pm). The separation was carried out with a gradient program. The mobile phase was acetonitrile-water at a flow rate of 0.8 mL/min. Results The standard curve was rectilinear in the range of 0.464-12.97 gg (r=0.9969) for dioscin, 0.310-7.09 ltg (r = 0.9953) for gracillin, 0.469-11.66 gg (r=0.9970) for protoneodioscin, and 0.276-6.87 gg (r＝0.9992) for protoneogracillin. The recoveries of the markers were 98.1%, 100.1%, 97.2%, and 96.4%, respectively. The contents of the four components were quite different among the seven species of Dioscorea L. Conclusion The proposed HPLC-ELSD method is convenient,fast, accurate, and applicable for simultaneous analysis of multiple bioactive components of species in Dioscorea L.for quality control, which could facilitate discovering new natural resources of steroidal saponin.