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Aim To investigate the anti-inflammatory effects of Aesculetin from Viola tianshanica Maxim in LPS-stimulated RAW 264.7 cells and the underlying mechanism.Methods RAW 264.7 cells were divided into control group, model group( LPS), 0.16, 0.8, 4, 20 μmol·L-1 AESN groups( different concentrations of AESN + LPS)and positive control group(10 μmol·L-1 Indomethacin+LPS).LPS(1 mg·L-1)was used to stimulate RAW 264.7 cells for 24 h to establish inflammatory model.MTS assay was used to detemine cytotoxicity of Aesculetin in RAW 264.7 cells.Griess method was used to detect NO secretion in LPS-stimulated RAW 264.7 cells.ELISA was applied to determine the contents of TNF-α, IL-6 and IL-1β in cell culture supernatant.qRT-PCR was employed to detect the mRNA expression levels of TNF-α, IL-6, IL-1β and iNOS.Immunofluorescence assay was used to evaluated the protein expressions of iNOS, p-NF-κB p65, IκBα, p-p38 and p-ERK1/2.Enzyme assay was used to detect the inhibition activity of Aesculetin on cyclooxygenase 1/2(COX 1/2).Results Aesculetin significantly inhibited the secretion of inflammatory mediator NO, mRNA and protein expression of iNOS in LPS-induced RAW 264.7 at 0.16, 0.8, 4 and 20 μmol·L-1.The contents of TNF-α, IL-6 and IL-1β in supernatant significantly decreased, and the mRNA expression levels of TNF-α, IL-6 and IL-1β were also reduced by Aesculetin.Aesculetin also obviously inhibited the protein degradation of IκBα and inhibited the nuclear translocation of p-NF-κB p65, p-p38, p-ERK1/2.In addition, Aesculetin had significant inhibitory activities on COX-1 and COX-2, and the IC50 was 28.1 μmol·L-1, 2.3 μmol·L-1, respectively.Conclusions AESN has good anti-inflammatory effect, and its mechanism is closely related to the inhibition of NF-κB and MAPK signaling pathways
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Objective:To optimize the decoction process of Digda-4 decoction(DGD-4D), and provide reference for the standardization study of decoction of Mongolian medicine decoction. Method:Taking DGD-4D as model drug, different decoction methods of Mongolian medicine were compared, HPLC was used to determine contents of aesculetin, geniposide, picroside Ⅰ and picroside Ⅱ.On the basis of single factor tests, central composite design-response surface methodology was adopted to optimize the decoction process of DGD-4D with transfer rates of 4 components and dry extract rate as indexes, regression model fitting was carried out by Design-Expert 8.0.6 software, prediction model of process parameters was established, and the optimal process was verified. Result:The optimal decoction condition of DGD-4D was determined to be adding 40 times the amount of water and decocting for 17 min, decocting once.Transfer rates of aesculetin, geniposide, picroside Ⅰ, picroside Ⅱ and dry extract rate were 70.01%, 94.11%, 61.23%, 92.32%, 32.89%, respectively. Conclusion:The optimum decoction process of DGD-4D is established, it has important reference significance for excavating, sorting, improving the level of Mongolian medicine preparations and ensuring the consistency of their clinical efficacy.
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Objective:To establish an HPLC method for the simultaneous determination of chlorogenic acid , aesculetin, rutin, acacetin-7-O-β-D-glucoside, quercetin and luteolin in the extract of Viola yedoensis Makino.Methods:The HPLC analysis was carried out on a Hypersil ODS C18 column (250 mm ×4.6 mm, 5μm) with 0.1％phosphoric acid (A)-methanol (B) as the mobile phase with gradient elution at the flow rate of 1.0 ml· min-1 .The detection wavelength was 345 nm and the column temperature was 35℃. Results:Good linear relationship was found within the range of 4.3175-172.7000 mg· L-1 for chlorogenic acid, 2.7350-109.4000 mg· L1 for aesculetin, 6.9800-279.2000 mg · L-1 for rutin, 3.7200-148.8000 mg · L-1 for acacetin-7-O-β-D-glucoside, 4.1350-165.4000 mg· L-1 for quercetin, and 3.3950-135.8000 mg · L-1 for luteolin.The average recovery was 99.06％, 98.84％, 98.77％, 99.40％, 98.53％and 98.71％, respectively.The content of chlorogenic acid , aesculetin, rutin, acacetin-7-O-β-D-glucoside, quercetin and luteolin in the extract of Viola yedoensis Makino was 1.6290, 1.1910, 4.5850, 2.2810, 3.1790 and 1.9710 mg· g-1, respectively.Conclusion:The method is accurate and reliable with good reproducibility , which can be used for the quality control of the extract of Viola yedoensis Makino.
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Objective: To establish the quantitative analysis of multi-components by single marker (QAMS) method to determine the content of chlorogenic acid, aesculetin, isochlorogenic acid B, and isochlorogenic acid A in Cichorii Herba (chicory). Methods: Selecting aesculetin as the internal reference substance, the relative correction factors (RCF) of chlorogenic acid, isochlorogenic acid B and isochlorogenic acid A were established respectively, and then the contents of the three constituents were calculated by RCF, to achieve the quality of chicory through QAMS. At the same time, the external standard method was used to determine the content of four constituents in chicory and compare the difference between calculated values and measured values, so as to verify the construction method for accuracy, applicability, and repeatability. Results: No significant difference was observed in chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A from eight batches of chicory in the quantitative results by these two methods. The validated QAMS method had good precision, reproducibility, and reliability. Conclusion: The established QAMS method is suitable and feasible for the quality control of chicory.
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Objective To develop a HPLC method for determination of aesculin, aesculetin,baicalin and ellagic acid in Xielining tablets. Methods The hypersil C18 column was used with the flow rate of 1. 1 mL·min-1 . The mobile phase A consisted of methanol-acetonitrile(4∶1),the mobile phase B consisted of 0. 1% phosphoric acid solution; The detection wave-lengths were λ1 =334 nm(aesculin and aesculetin),λ2 = 280 nm(baicalin),and λ3 = 254 nm(ellagic acid). Results There was a good linear relationship between the peak area values and concentrations of aesculin,aesculetin,baicalin and ellagic acid. The quantitation range of aesculin,aesculetin,baicalin and ellagic acid was 0. 058 6 -1. 172 0 μg( r = 0. 999 2), 0. 015 4 -0. 308 0 μg(r=0. 999 8),0. 447 2-8. 944 0 μg(r=0. 999 6),and 0. 072 6-1. 452 0 μg(r=0. 999 5), respectively. The aver-age recovery was 97. 24% (RSD=0. 78% ),97. 76% (RSD=1. 11% ),98. 43% (RSD=0. 93% ) and 96. 89% (RSD=0. 62% ), respectively. Conclusion The method is convenient,accurate,sensitive,reproducible and may be used in the determination of aesculin, aesculetin,baicalin and ellagic acid in Xielining tablets.
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Objective To optimize the extraction technology of Zhiqian Capsule;To provide evidence for researches on preparation into new Chinese medicine. Methods The main influential factors of extraction technology included the quantity of water, extraction time and immersion time. The extraction effect was evaluated with the content of aesculetin and the extract yield as indexes. The optimal extraction technology of Zhiqian Capsule was selected by single factor experiment and central composite design-response surface method. Results The best extraction conditions were as follows:reflux extraction for twice, the first time adding 10-fold of water and extraction for 2.5 h, the second time adding 8-fold of water and extraction for 2 h. Conclusion The optimal extraction technology of Zhiqian Capsule is efficient for extracting aesculetin, as well as economical, reasonable, easy and feasible.
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Objective: To optimize the purification process of total coumarin from Fraxini Cortex using macroporous resin. Methods: The single factor methods have been used to investigate the choice of type, adsorption performance, and desorption performance and the purification of the total coumarin from Fraxini Cortex by macroporous resin. The adsorption rate, resolution, resolution rate, and transfer rate of the total coumarin from Fraxini Cortex, four kinds of coumarin constituents, such as aesculin, aesculetin, fraxin, and fraxetin were used as examining indexes. Results: The ADS-5 was the most suitable type for the purification of total coumarin from Fraxini Cortex among the seven kinds of macroporous resin. Adsorption parameters: Crude drug-the resin was 0.8 g/g which was the sample amount; The concentration of sample solution was 0.75 g crude drug/mL; The pH value of sample liquid was 4.0-4.3 (liquid sample); The speed of the sample through the resin column was 2-4 BV/h. Elution parameters: The volume of the water cleaning fluid impurities was 1 BV; The elution solvent was 25% ethanol; The elution speed was 2 BV/h; The elution volume was 3 BV. After the purification, the transfer rate of total coumarin from Fraxini Cortex was 74.27%, the transfer rate of four kinds of coumarin was 83.06%, the extract rate of total coumarin was 7.35%, the removal of impurities was 14.00%, among which the content of total coumarin was 54.72%, the contents of the four kinds of components were 36.01%, the total coumarin extraction yield was 4.02%. Conclusion: This method to purify the total coumarin from Fraxini Cortex using ADS-5 can get better purification effect, the purification process is also stable.
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Objective: To optimize the purifying process of water-extraction solution of Xiaoyan Tuire Granules Oral Liquid by flocculation clarifying method, reserve more active components while less impurities, and improve the clarity of oral liquid. Methods: After clearing out the impurities composition of water-extraction solution of Xiaoyan Tuire Granules Oral Liquid, the flocculant of chitosan chosen by screening test was used to analyze the flocculation effect. The retention rate of aesculetin, the removal rate of impurity, and supernatant turbidity were set as mainly indexes to estimate the influence of the dosage of flocculant, temperature, fast mixing speed, and fast mixing time. Results: The optimal flocculating process conditions which were analyzed by experiments were as follows: the dosage of chitosan was 1.25 g/L, the flocculation temperature was 40 ℃, the fast mixing speed was 350 r/min, and the fast mixing time was 3 min. Under these conditions, the retention rate of aesculetin was 88.92%, the removal rates of protein and tanning were 62.96% and 67.19%, the supernatant turbidity was 5.0 NTU after the oral liquid was flocculated for 24 h. Conclusion: Using chitosan as flocculant in the water-extraction solution of Xiaoyan Tuire Granules Oral Liquid, it could improve the removal rate of impurities effeitively as well as keep higher active components and enhance the clarity of oral liquid.
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Objective: To study the chemical constituents from Lepidogrammitis drymoglossoides. Methods: The constituents were isolated and purified by various column chromatographies, and their structures were identified on the basis of chemical evidences and spectroscopic analysis including MS, 1H-NMR, and 13C-NMR. Results: Fourteen compounds were isolated and identified as β-ecdysterone (1), physcion (2), emodin (3), umbelliferone (4), scoparone (5), aesculetin (6), caffeic acid (7), chlorogenic acid (8), protocatechuic acid (9), pyrocatechualdehyde (10), gallic acid (11), 4-hydroxybenzoic acid methyl ester (12), docosanyl tetracosanoate (13), and hexadecanoic acid (14). Conclusion: Compounds 3-5, 8, and 11-13 are isolated from the plants of genus Lepidogrammitis Ching for the first time.
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OBJECTIVE: To establish a method for the determination of aesculin and aesculetin in Jiuxiang zhixie tablets.METHODS: HPLC method was developed to quantitative determination.Thermo C18(250 mm?4.60 mm,5?m) column was adopted.The mobile phase consisted of acetonitrile-0.1%H3PO4(12 ∶ 88)with flow rate of 1.0 mL?min-1 and the detection wavelength of 334 nm.The column tempreture was set at 30℃.RESULTS: The linear range of aesculin was 80～800 ng(r=0.999 8).The average recovery was 100.1%(RSD=1.89%,n=9).The linear range of aesculetin was 32.96～329.6 ng(r=0.999 5).The average recovery of aesculetin was 101.5%(RSD=2.42%,n=9).CONCLUSION: The method is simple,accurate for the content determination of Jiuxiang zhixie tablet.
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Objective: To determine the contents of aesculin and aesculetin in periderms and leaves of Cortex Fraxini among differnet provenances. Methods: HPLC was used with acetonitrile-water(15∶85) as the mobile phase and detected wavelength at 348nm. C 18 column was adopted. Results: The calibration curves were linear. The value of correlation coefficient were 0.9992 and 0.9994, respectively. The average recovery of aesculin was 98.2% and aesculetin was 99.2%. RSD were 2.24% and 2.15%, respectively. The quality differences of Cortex Fraxini among different provenances were remarked. The quality of Cortex Fraxini from Shanxi province was the best. Conclusion: The method is applied in determination and analytics of content of Cortex Fraxini and is rapid, simple and easy to carry out. The method is with the feature of accuracy, repetition and stability.