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This study explored the protective effect of atractylenolide Ⅰ(AO-Ⅰ) against acetaminophen(APAP)-induced acute liver injury(ALI) in mice and its underlying mechanism. C57 BL/6 J mice were randomly divided into a control group, an APAP group(500 mg·kg~(-1)), a low-dose combination group(500 mg·kg~(-1) APAP + 60 mg·kg~(-1) AO-Ⅰ), and a high-dose combination group(500 mg·kg~(-1) APAP + 120 mg·kg~(-1) AO-Ⅰ). ALI was induced by intraperitoneal injection of APAP(500 mg·kg~(-1)). AO-Ⅰ by intragastric administration was performed 2 hours before APAP treatment, and the control group received the same dose of solvent by intragastric administration or intraperitoneal injection. The protective effect of AO-Ⅰ against APAP-induced ALI was evaluated by detecting alanine aminotransferase(ALT) and aspartate aminotransferase(AST) levels in the plasma and H&E staining in liver tissues of mice. The malondialdehyde(MDA) and glutathione(GSH) content and catalase(CAT) activity in mouse liver tissues were detected to evaluate the effect of AO-Ⅰ on APAP-induced oxidative stress in the liver. The proteins in the liver p38 mitogen-activated protein kinase(p38 MAPK), c-jun N-terminal kinase(JNK), and nuclear factor kappa-B p65(NF-κB p65) signaling pathways were measured by Western blot, and the liver inflammatory cytokines interleukin-1β(IL-1β) and interleukin-6(IL-6) were detected by real-time PCR. Compared with the APAP group, the combination groups showed reduced APAP-induced ALT level and liver MDA content, potentiated liver CAT activity, and elevated GSH content. Mechanistically, AO-Ⅰ treatment significantly inhibited APAP-up-regulated MAPK phosphorylation and NF-κB p65, and significantly reduced the transcriptional activities of IL-1β and IL-6, downstream targets of NF-κB p65. AO-Ⅰ can improve APAP-induced ALI and the underlying mechanism is related to the inhibition of the MAPK/NF-κB p65 signaling pathway in APAP-challenged mice.
Subject(s)
Animals , Mice , Acetaminophen/adverse effects , Chemical and Drug Induced Liver Injury/drug therapy , Lactones , NF-kappa B/metabolism , Sesquiterpenes , Signal TransductionABSTRACT
OBJECTIVE：To establish a method for simultaneous determination of the contents of codonopatin ，syringin， atractylenolide Ⅰ，atractylenolide Ⅱ and atractylenolide Ⅲ，and to compare the contents of above 5 components in different varieties and harvesting periods of Codonopsis Radix. METHODS ：HPLC method was used. The column was Inertsil ODS- 3 with mobile phase consisted of acetonitrile-water （gradient elution ）at the flow rate of 0.8 mL/min. The detection wavelengths were 210 nm （codonopatin），220 nm （syringin，atractylenolide Ⅱ ，atractylenolide Ⅲ），276 nm （atractylenolide Ⅰ）. The column temperature was set at 30 ℃ ，and the sample size was 20 μ L. RESULTS：The linear range of codonopatin ，syringin， atractylenolide Ⅰ，atractylenolide Ⅱ and atractylenolide Ⅲ were 44.30-886.00 μg/mL（r＝0.999 7），6.50-130.03 μg/mL（r＝0.999 6）， 4.47-89.46 μg/mL（r＝0.999 5），2.53-50.50 μg/mL（r＝0.999 4），5.64-112.80 μg/mL（r＝0.999 5）；the limits of quantification were 2.446 0，0.168 0，0.248 1，0.065 7，0.099 8 μg/mL，and detection limits were 1.352 0，0.067 2，0.005 4，0.006 3，0.007 3 μ g/mL；RSDs of precision ，stability（24 h），repeatability and durability tests were all less than 2％；the recoveries were 98.87％-100.62％（RSD＝0.73％，n＝6），98.46％-101.54％ （RSD＝1.15％，n＝6），98.32％-101.12％（RSD＝1.19％，n＝ 96.83％-104.16％（RSD＝2.62％，n＝6），97.87％-100.99％ （RSD＝1.07％，n＝6）. The average contents were 33.78-431.82， 0-20.60，0.44-3.68，0-10.83，0.27-73.40 μ g/g. The content of 1271985629@qq.com codonopatin was in descending order was as follows as Codonopsis pilosula ＞C. tangshen ＞C. pilosula Nannf. var. modesta （Nannf.） L. T. Shen ＞ecotypic variety of C.·1677· tangshen. The content of syringin in descending order was C. pilosula ＞C. pilosula Nannf. var. modesta（Nannf.）L. T. Shen ＞C. tangshen，but it was not detected in ecotypic variety of C. tangshen . The content of atractylenolide Ⅰ in descending order was C. pilosula Nannf. var. modesta（Namf.）L. T. Shen ＞ecotypic variety of C. tangshen ＞C. pilosula ＞C. tangshen . The content of atractylenolide Ⅱ in C. pilosula was higher than C. pilosula Nannf. var. modesta（Nannf.）L. T. Shen ，but was no detected in C. tangshen and ecotypic variety of C. tangshen . The content of atractylenolide Ⅲ in descending order was C. pilosula ＞C. pilosula Nannf. var. modesta（Nannf.）L. T. Shen ＞ecotypic variety of C. tangshen ＞C. tangshen . In Codonopsis Radix collected from Jul. to Oct. ，the content of codonopatin was the highest ；the content of atractylenolide Ⅰ was lower in sample collected from Jun. to Oct.；atractylenolide Ⅱ was not detected in sample collected in Aug. ；the contents of atractylenolide Ⅰ and atractylenolide Ⅱ were the lower in sample collected in Sept. ，and syringin and atractylenolide Ⅱ were not detected in some samples. CONCLUSIONS ： The established HPLC method is simple ，accurate，highly sensitive and reproducible. It can be used to simultaneously determine 5 active ingredients contents of Codonopsis Radix ；there are great difference in contents of 5 active ingredients in different varieties and harvesting periods of Codonopsis Radix.
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Objective To investigate the mechanisms and proliferation inhibitory effects of atractylenolide Ⅰ on SK-OV-3 and OVCAR-3 ovarian cancer cell.Methods SK-OV-3 and OVCAR-3 cells were treated with atractylenolide I with various concentrations at 24 hours,48 hours and 72 hours,and the changes in proliferation were detected by MTT assay.The cell cycles were measured by PI staining and flow cytometry,and the expressions of cyclin D1 and CDK1 were detected by ELISA assay.Western blot was then applied to investigate the effects of atractylenolide Ⅰ on PI3K/AKT signaling pathway in SK-OV-3 and OVCAR-3 cells.Results Atractylenolide I could significantly inhibit the proliferation of SK-OV-3 and OVCAR-3 cells,and its inhibitory effects were concentration and time dependent.In addition,atractylenolide I could also significantly reduce the proportion of cells in S phase and increase the proportion of cells in G2/M phase,and these effects were associated with the down-regulation of CDK1.The results of Western blot indicated that PI3K/AKT signaling pathway was involved in the inhibitory effects of atractylenolide Ⅰ on proliferation and cell cycle.Conclusion Atractylenolide I can down-regulate the expression of CDK1 in ovarian cancer SK-OV-3 and OVCAR-3 cells through PI3K/AKT pathway,which led to cell cycle arrest in G2/M phase,and played an important role in proliferation inhibition of tumor cells.
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OBJECTIVE@#To investigate whether atractylenolide Ⅰ (ATL-Ⅰ) has protective effect on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in vivo and in vitro, and explore whether NF-κB signaling pathway is involved in ATL-Ⅰ treatment.@*METHODS@#New Zealand white rabbits were injected with LPS through marginal ear vein over a period of 6 h at a rate of 600 μg/kg (10 mL/h). Similarly, in the treatment groups, 1.0, 2.0, or 5.0 mg/kg ATL-Ⅰ were given. Both survival rate and organ function were tested, including the level of alanine aminotransferase (ALT), blood urine nitrogen (BUN), and TNF-α were examined by ELISA. Also hemostatic and fibrinolytic parameters in serum were measured. RAW 264.7 macrophage cells were administered with control, LPS, LPS + ATL-Ⅰ and ATL-Ⅰ alone, and TNF-α, phosphorylation (P)-IκBα, phosphorylation (P)-NF-κB (P65) and NF-κB (P65) were determined by Western blot.@*RESULTS@#The administration of LPS resulted in 73.3% mortality rate, and the increase of serum TNF-α, BUN and ALT levels. When ATL-Ⅰ treatment significantly increased the survival rate of LPS-induced DIC model, also improved the function of blood coagulation. And protein analysis indicated that ATL-I remarkably protected liver and renal as decreasing TNF-α expression. In vitro, ATL-I obviously decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65), with the decrease of P-IκBα.@*CONCLUSIONS@#ATL-Ⅰ has protective effect on LPS-induced DIC, which can elevate the survival rate, reduce organ damage, improve the function of blood coagulation and suppress TNF-α expression by inhibiting the activation of NF-κB signaling pathway.
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Objective To establish a multi-index quality evaluation method of Atracylodis Macrocephalae Rhizoma based on principal component analysis (PCA) model.Methods The contents of atractylenolideⅠ and atractylenolideⅢ were determined by HPLC; atractylodes macrocephalaon polysaccharide was determined by phenol-sulfuric acid method; ethanol soluble extractum was determined by hot soak in general rule of Chinese Pharmacopoeia (2015 Edition); PCA was used to establish a multi-index quality evaluation model. Results AtractylenolideⅠ and atractylenolideⅢ showed good linear correlations (r=0.999 9), and the average recoveries were 100.9% and 102.1%, respectively. PCA showed that the Atracylodis Macrocephalae Rhizoma from Panan County, Yuqian Town, Jinyun County, Xinchang County of Zhejiang Province had the best quality, from Bozhou City and Fuyang City of Anhui Province had good quality.Conclusion The established multi-index quality evaluation method based on PCA model can be applied to objectively evaluate the quality of Atracylodis Macrocephalae Rhizoma from different producing areas.
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Objective: To establish a method for the determination of atractylenolide Ⅰ in Atractylodes macrocephala by HPCE. Methods:The separation conditions of HPCE were as follows:50 mmol·L-1 borate buffer was used as the running buffer, the UV de-tection was set at 210 nm, the separation voltage was 20 kV, the column temperature was 25℃ and the pressure injection was 50 mbar × 5 sec. Results:The lower detection limit was 0. 5 μg·ml-1 . The concentration of atractylenolide Ⅰ showed a linear plot within the range of 2-100 μg·ml-1(r=0. 996 0). The average recovery was 97. 1%, and the intra-and inter-day RSD was 1. 3% and 2. 5%, respectively. Conclusion:The established method is simple, sensitive and economic. The method is suitable for the quality control of atractylodes macrocephala.
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Objective To establish a RP-HPLC method for the determination of drug release in vitro of atractylenolide Ⅰ liposomes. Methods The release behavior of the drug from liposomes was studied by the third method for dissolution. ZORBAX C18 column (4.6 mm × 250 mm, 5 μm) was used with a mobile phase of Methanol-Acetonitrile-0.2% from liposomes in vitro fitted the log-normal distribution equation and had a property of sustained release. Conclusion The method is simple, fast and selective. It is suitable for the determination of release profile in vitro of atractylenolide Ⅰ liposomes.
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OBJECTIVE:To study the change in grain size and in vitro dissolution ratio of Atractylodes macrocephala after ultramicronization. METHODS: The particle size before and after ultramicronization was analyzed using particle size analyzer. The content of the sample was determined by HPLC using atractylenolide Ⅲ and atractylenolide Ⅰ as indexes to reflect the dissolution ratio. RESULTS: After ultramicronization, the particle size of the sample became thinner obviously, about 30% that of the common fine powder, and the content increased by 27% as compared with the common fine powder. CONCLUSION: The ultramicronization can significantly decrease the particle size, increase specific surface area and contribute to the dissolution of atractylenolide Ⅲ and atractylenolide I from Atractylodes macrocephala.
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Objective To develop a method for simultaneous determination of 4 compounds in Zhizhu Pills by HPLC.Method Reversed-phase HPLC was used to determine the content of the compounds such as hesperidin,naringin,neohesperidin and atractylenolideⅠ.The column was symmetry C18(250 mm?4.6 mm,5 ?m) with temperature of 30 ℃.Methanol-0.095 %phosphoric acid solution served as the mobile phase by gradient elution.The wavelength of detection was set at 283 nm and 224 nm.The injection volume was 10 ?L and the flow rate was 1.0 mL/min.Results Hesperidin,naringin,neohesperidin and atractylenolideⅠin Zhizhu Pills can be isolated completely.Three batches of samples were determined and the recoveries of naringin,hesperidin,neohesperidin and atractylenolideⅠwere 98.81 %(RSD=1.11 %,n=6),100.63 %(RSD=1.90 %,n=6),99.32 %(RSD=1.44 %,n=6) and 99.65 %(RSD=1.53 %,n=6) respectively.Conclusion This method is accurate,reliable and with good reproducibility.It can be used for the quality control of Zhizhu Pills.
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Objective To investigate the extraction technique for atractylenolide Ⅰ in Atractylodes macrocephala by supercritical CO_2 fluid extraction and develop a method used for determining the content of atractylenolide Ⅰ in the extract by HPLC. Methods The effects of seven facters, such as the extracting pressure, resolving pressure etc, to the extraction rate of atractylenolide Ⅰ in A. macrocephala by supercritical CO_2 extraction were investigated. RP-HPLC was used to determine the content of atractylenolide Ⅰ in extraction of A. macrocephala. The separation was performed on Hypersil ODS2 column with methanol-water (70∶30) as mobile phase. The flow rate was 1.0 mL/min and the wavelength of UV detector was 220 nm. Results The optimal extracting conditions: taking 10% alcohol as entraiter, the particle size of medicinal substances was 60 screen meshes, extracting pressure 25 MPa, resolving pressure 5 MPa, extracting temperature 40 ℃, resolving temperature 30 ℃, and the extracting time 4 h. Conclusion Supercritical extraction is time-shorter and efficient in extracting atractylenolide Ⅰ from A. macrocephala. It is suitable to both trial and industrialized production. The method established to determine the content of atractylenolide Ⅰ of A. macrocephala by supercritical extraction is simple, sensitive, and reliable.
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Objective: To develop a method to determine the content of a atractylenolideⅠ in Xiangshayangwei Pills. Methods: Reverse Phase HPLC was used to determine the content of atractylenolideⅠ in Xiangshayangwei Pills. The separation was performed on YWG ODS column with methol water (60∶40) as a mobile phase and the wavelength of UV detector was 220nm. Results: The linearity of this method was well with the average recovery of 99.54%. Conclusion: The method is simple, reliable, and sensitive. It also shows good separating degree. It can be used in quality controll of Xiangshayangwei Pills.
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OBJECTIVE: To develop a method for determination of atractylenolide Ⅰ and atractylone in Chang’ankang granule by HPLC. METHODS: The determination was performed on Yilite ODS column (250 mm?4.6 mm,5 ?m). The mobile phase contained of acetonitrile-water (gradient elution) with flow rate of 1 mL?min-1 and detection wavelength of 220 nm. RESULTS: The linear range of atractylenolide Ⅰwere 0.1～2.0 ?g(r=0.999 9) with mean recovery of 98.84%(RSD=0.76%,n=6).The linear range of atractylone were 0.5～10.0 ?g with mean recovery of 98.43%(RSD=1.14%,n=6). CONCLUSION: This method is simple, convenience and accurate for quality control of Chang’ankang granule.