Protective Effect of Combined Metoprolol and Atractylenolide I in Rats with Acute Myocardial Infarction via Modulation of the SIRT3/ß-CATENIN/PPAR-γ Signaling Pathway

ABSTRACT Herein, we examined the protective effect of metoprolol combined with atractylenolide I (Atr I) in acute myocardial infarction (AMI) by regulating the SIRT3 (silent information regulator 3)/ß-catenin/peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling pathway. Briefly, 50 rats were randomly divided into the sham operation, model, metoprolol, Atr I, and combination metoprolol with Atr I groups (combined treatment group). The AMI model was established by ligating the left anterior descending coronary artery. After treatment, infarct size, histopathological changes, and cell apoptosis were examined using 2,3,5-triphenyltetrazolium chloride staining, hematoxylin-eosin staining, and the TUNEL assay. The left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS), and left ventricular mass index (LVMI) were detected by echocardiography. Endothelin-1 (ET-1), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) levels were detected using enzyme-linked immunosorbent assays. Furthermore, we measured lactate dehydrogenase (LDH), creatine kinase (CK) isoenzyme (CK-MB), and CK levels. Western blotting was performed to determine the expression of SIRT3, ß-catenin, and PPAR-γ. Herein, the combined treatment group exhibited increased levels of LVEF, LVFS, and NO, whereas LVMI, ET-1, TNF-α, IL-6, LDH, CK-MB, and CK levels were decreased. Importantly, the underlying mechanism may afford protection against AMI by increasing the expression levels of SIRT3, ß-catenin, and PPAR-γ

Atractylenolide-I covalently binds to CYP11B2, selectively inhibits aldosterone synthesis, and improves hyperaldosteronism

Hyperaldosteronism is a common disease that is closely related to endocrine hypertension and other cardiovascular diseases. Cytochrome P450 11B2 (CYP11B2), an important enzyme in aldosterone (ALD) synthesis, is a promising target for the treatment of hyperaldosteronism. However, selective inhibitors targeting CYP11B2 are still lacking due to the high similarity with CYP11B1. In this study, atractylenolide-I (AT-I) was found to significantly reduce the production of ALD but had no effect on cortisol synthesis, which is catalyzed by CYP11B1. Chemical biology studies revealed that due to the presence of Ala320, AT-I is selectively bound to the catalytic pocket of CYP11B2, and the C8/C9 double bond of AT-I can be epoxidized, which then undergoes nucleophilic addition with the sulfhydryl group of Cys450 in CYP11B2. The covalent binding of AT-I disrupts the interaction between heme and CYP11B2 and inactivates CYP11B2, leading to the suppression of ALD synthesis; AT-I shows a significant therapeutic effect for improving hyperaldosteronism.

Atractylenolide Ⅰ improves acetaminophen-induced acute liver injury in mice by inhibiting MAPK/NF-κB signaling pathway / 中国中药杂志

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.

Atractylenolide III alleviates sepsis-mediated lung injury via inhibition of FoxO1 and VNN1 protein

ABSTRACT Purpose: To evaluate the influence of atractylenolide (Atr) III on sepsis-induced lung damage. Methods: We constructed a mouse sepsis model through cecal ligation and puncture. These mice were allocated to the normal, sepsis, sepsis + Atr III-L (2 mg/kg), as well as Atr III-H (8 mg/kg) group. Lung injury and pulmonary fibrosis were accessed via hematoxylin-eosin (HE) and Masson's staining. We used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and flow cytometry for detecting sepsis-induced lung cell apoptosis. The contents of the inflammatory cytokines in lung tissue were measured via enzyme-linked immunosorbent assay (ELISA). Results: Atr III-H did not only reduce sepsis-induced lung injury and apoptosis level, but also curbed the secretion of inflammatory factors. Atr III-H substantially ameliorated lung function and raised Bcl-2 expression. Atr III-H eased the pulmonary fibrosis damage and Bax, caspase-3, Vanin-1 (VNN1), as well as Forkhead Box Protein O1 (FoxO1) expression. Conclusions: Atr III alleviates sepsis-mediated lung injury via inhibition of FoxO1 and VNN1 protein.

Effect of Zhenwu Decoction on chronic heart failure rats based on RAAS/NF-κB/ inflammatory factor cascade reaction / 中草药

Objective: To investigate the therapeutic effect and potential mechanism of Zhenwu Decoction (ZWD) on chronic heart failure (CHF) rats. Methods: HPLC fingerprint of ZWD was established. All male SD rats were randomly divided into the sham operation group, the model group, the low, medium and high dose ZWD group (2.187 5, 4.375, and 8.75 g/kg) and the captopril group (10 mg/kg). Except for the sham operation group, the rest of rats were all established into the CHF model rats by ligating the left anterior descending branch of the coronary artery, after 8 weeks, all rats were ig administration for 4 weeks. The hemodynamic, viscera index, HE dyeing test were conducted at the end of experiments. Serum angiotensin II (Ang II), aldosterone (ALD), nuclear factor kappa B (NF-κB), amino terminal brain natriuretic peptide (NT-proBNP), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) were determinated by ELISA, and myocardial NF-κB protein expression was detected by Western blotting. Results: Higenamine, paeoniflorin, atractylenolide III, 6-gingerol and dehydrotumulosic acid, the five constituents of Zhenwu Decoction, were identified by HPLC chart. Compared with the model group, the administration of the ZWD significantly improved the hemodynamic parameters (P < 0.05), reduced the organ index (P < 0.05) and improved myocardial injury, reduced the serum Ang II, ALD, NF-κB, NT-proBNP, TNF-α and IL-6 levels and the myocardial NF-κB protein expression (P < 0.05). Conclusion: HPLC results provided an evidence for the quality control and pharmacodynamic substance of ZWD. ZWD can ameliorate CHF, which may be related to the inhibition of renin- angiotensin-aldosterone system (RAAS)/NF-κB/inflammatory factor cascade reaction.

Effect of particle design on pharmacokinetics of Shenling Baizhu Pulvis in rats / 中草药

Objective: Using LC-MS to explore the pharmacokinetic process in rats of Shenling Baizhu Pulvis (SBP), which was modified by particle design technology. Methods: Particle design powder of SBP was prepared by particle design technology. A scientific and feasible LC-MS analysis method was established to determine the blood concentration of index compounds such as ginsenoside Re (GI-Re), ginsenoside Rb1 (GI-Rb1), ginsenoside Rg1 (GI-Rg1), atractylenolide I (AT-I), atractylenolide II (AT-II) and pachymic acid (PA) in rats at different time points after administration. DAS 3.2.8 pharmacokinetic software was adopted to analyze the data, which related to blood concentration of index compounds, and the pharmacokinetics parameters were calculated by the non-compartmental model. Results: LC-MS analysis method was established, which has a good linear relationship and specificity for the index compounds in rats, and the RSD of precision, accuracy, extraction recovery and stability were all less than 5% or 10%. Compared with ordinary powder, the particle design powder displayed increased Cmax and AUC0-∞ after administration, and the AUC0-∞ of GI-Re, GI-Rb1, GI-Rg1, AT-I, AT-II and PA were increased to 1.52, 2.02, 1.22, 1.41, 1.13 and 1.43 times, respectively. Conclusion: The LC-MS analysis method meet the requirements of biological sample analysis in Pharmacopoeia of the People's Republic of China. After particle design and modification, the absorption speed of SBP in vivo become faster and the bioavailability is improved significantly.

Quality evaluation of Shirebi Tablets based on combinative methods of HPLC fingerprint, quantitative analysis of multi-components and chemometrics analysis / 中草药

Objective: To establish a quality evaluation method of Shirebi Tablet based on HPLC fingerprints, quantitative analysis of multi-components and chemometrics analysis. Methods: The chromatographic column was Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm), and the column temperature was set at 30 ℃. The detection wavelength was set at 303 nm (for mulberroside A, mulberroside F and moracin M) and 270 nm (for forsythoside B, forsythoside A, forsythin, atractylodinol, atractylenolide II, and atractylodin). The mobile phase was composed of acetonitrile-0.2% phosphate acid solution in gradient elution manner at a flow rate of 1.0 mL/min. The HPLC fingerprint of Shirebi Tablet was established, the common peaks were determined by similarity evaluation system for chromatographic fingerprint of TCM (Version 2012.130723), and the similarity was calculated. The content determination methods for mulberroside A, mulberroside F, moracin M, forsythoside B, forsythoside A, forsythin, atractylodinol, atractylenolide II, and atractylodin were validated. The chemometrics methods such as cluster analysis and principal component analysis were used to evaluate the quality of Shirebi Tablet from different batches based on the results of fingerprint common peak area. Results: The fingerprint of Shirebi Tablet was established. Sixteen common peaks were identified. The similarity of fingerprints of 10 batches of Shirebi Tablet was more than 0.95. Nine components had good linear relationship in the range of mass concentration (r2 ≥ 0.999 1), and the average recoveries were 98.87%, 97.44%, 97.94%, 98.39%, 100.13%, 99.06%, 96.80%, 98.44% and 99.15% with the RSDs of 1.42%, 1.17%, 1.30%, 0.91%, 0.86%, 1.23%, 1.08%, 1.37% and 0.79%, respectively. The concentrations of mulberroside A, mulberroside F, moracin M, forsythoside B, forsythoside A, forsythin, atractylodinol, atractylenolide II, and atractylodin in 10 batches were 0.192—0.289, 0.057—0.095, 0.113—0.158, 0.309—0.375, 1.537—1.916, 0.478—0.596, 0.049—0.072, 0.279—0.354, and 0.629—0.759 mg/g, respectively. The results of cluster analysis showed that 10 batches of Shirebi Tablet were clustered into two groups, and the results of principal component analysis showed that the principal components 1—6 were the main factor affecting the quality evaluation of Shirebi Tablets. Conclusion: The method is simple, accurate and reproducible, which can be used for the quality control and evaluation of Shirebi Tablet.

Simultaneous determination of 10 active ingredients in Yiganning Granules by UPLC / 中草药

Objective: To establish a UPLC method to simultaneously determine 10 active ingredients in Yiganning Granules (YG) and provide scientific basis for the quality control, evaluation and standard revision of YG preparations. Methods: A UPLC method was used with an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm). The mobile phase was acetonitrile-mehanol-0.15% phosphoric acid solution with gradient elution. The flow rate was 0.3 mL/min. The column temperature was 45 ℃. The injection volume was 2 μL. Results: Ten active ingredients (chlorogenic acid, atractylenolide I, paeoniflorin, calycosin 7-O-β-D- glucopyranoside, stilbene glycoside, caffeic acid, toosendanin, kaempferol, paeonol and tanshinone ⅡA) in YG were simultaneously determined. The linearity was good (r ≥ 0.999 0), the limit of detection and quantification were 0.006-0.017 μg/mL and 0.017-0.510 μg/mL. The average recoveries were 98.8%-102.5% with RSDs of 1.13%-5.37%. Through the determination of 16 batches of samples, the average content of the above 10 ingredients was in turn (5.724 ± 0.017), (0.273 ± 0.003), (0.854 ± 0.005), (1.228 ± 0.004), (0.496 ± 0.003), (1.287 ± 0.004), (0.137 ± 0.004), (3.624 ± 0.014), (7.366 ± 0.032) and (1.754 ± 0.005) mg/g, respectively. Conclusion The established UPLC method is simple, specific, sensitive, stable, precise, accurate, and reproducible, which can be used for quality control and evaluation of YG.

HPLC-ELSD fingerprint of Zhenwu Decoction and simultaneous determination of 13 components / 中草药

Objective: To establish HPLC-ELSD fingerprint of Zhenwu Decoction(ZWD), screen out the signature components of ZWD through chemical pattern recognition, so as to establish the content determination method of ZWD based on this index. Methods: The fingerprint of 16 batches of ZWD was established by HPLC-ELSD method. The similarity evaluation system of traditional Chinese medicine chromatographic fingerprint (2012 Version) was used for similarity evaluation to determine the common peaks and its attribution. Cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to select the index components of ZWD. Results: The fingerprint of ZWD was established, 38 common peaks were confirmed, and the similarity was > 0.95. The results of CA, PCA and OPLS-DA were consistent and the samples were divided into three categories. Benzoylmesaconine, benzoylaconitine, benzoylhypacoitine, polyporenic acid C, pachymic acid, atractylenolide II, atractylenolide III, oxypaeoniflorin, albiflorin, paeoniflorin and benzoylpaeoniflorin were identified as the 11 index components with significant difference contribution in different batches of ZWD samples. 6-Gingerol and 6-shogaol were the main active components of ginger, so the above 13 components were taken as the index components of ZWD. The chromatographic peak separation degree and linear relationship were good. The average recovery rate was 96.46%-99.80%, RSD ≤ 3.15%. The mass fraction range of benzoylmesaconine, benzoylaconitine, benzoylhypacoitine, polyporenic acid C, pachymic acid, atractylenolide II, atractylenolide III, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin, 6-gingerol, 6-shogaol in 16 batches were 283.93-576.86, 25.05-147.39, 62.96-303.37, 31.24-131.27, 9.76-44.04, 32.15-83.55, 76.55-333.13, 17.48-146.61, 456.58-1554.14, 3 322.48-5 590.01, 158.21-556.50, 525.85-582.92 and 68.52-74.73 mg/g, respectively. Conclusion: The fingerprint combined with PCA, CA and OPLS-DA can comprehensively evaluate the quality of ZWD. This method is stable and reliable, providing reference for the quality evaluation.

Observation on Effect of Atractylodesin Ⅱ on Gastric Cancer Cells Based on Macrophage Polarization / 中国实验方剂学杂志

Objective:To investigate the anti-tumor effect mechanism of atractylenolide Ⅱ by studying its effect on macrophage polarization. Method:Phorbol myristate acetate （PMA） was used to induce THP-1 cells differentiation into macrophages， and methylthiazolyldiphenyl-tetrazolium bromide（MTT）colorimetric assay was used to detect the effect of different concentrations of atractylenolide Ⅱ on macrophage growth at different time points to screen out the safe concentration of atractylenolide Ⅱ. The macrophages were treated with different concentrations of atractylenolide Ⅱ for 24 hours and then were co-cultured with gastric cancer cells. The survival of the two types of cells was observed under light microscope. The proliferation of gastric cancer cells was detected by MTT assay to determine the effective administration concentrations of atractylenolide Ⅱ. Cells were divided into blank group， model group， atractylenolide Ⅱ high dose group （200 mg·L-1）， atractylenolide Ⅱ medium dose group （100 mg·L-1）， and atractylenolide Ⅱ low dose group（50 mg·L-1）. Wound healing assay was carried out to observe the effects of different concentrations of atractylenolide Ⅱ on the migration and morphology of gastric cancer cells. The expression levels of M1 and M2 macrophage surface markers CD86 and CD206 were detected by flow cytometry analysis（FCM）. Quantitative polymerase chain reaction（Real-time PCR）and Western blot were used to detect M1， M2 macrophage-associated tumor necrosis factor （TNF） -α， human leukocyte antigen 2 （HLA-DRA）， CD80， transforming growth factor （TGF）-β， interleukin （IL） -10 and IL-6 genes and protein expression. Western blot was used to detect intracellular phosphatidyl inositol kinase （PI3K） and p-PI3K protein expression in macrophages. Result:When the concentration of atractylenolide Ⅱ was 1， 10， 50， 100， 200 mg·L-1， it showed no inhibition on macrophage growth. As compared with the model group， macrophages treated with 50， 100， 200 mg·L-1 atractylenolide Ⅱ significantly inhibited tumor cell proliferation （P<0.01）. As compared with the model group， the migration rate of tumor cells in the atractylenolide Ⅱ （200，100 mg·L-1） groups decreased （P<0.05）. The expression levels of CD86 on M1 macrophage surfacen in the atractylenolide Ⅱ （200，100，50 mg·L-1） groups were increased（P<0.05，P<0.01）， and the expression levels of CD206 on M2 macrophagen in the atractylenolide Ⅱ （200 mg·L-1） group were decreased （P<0.05）. The expression levels of M1 macrophage-associated cytokines TNF-α， HLA-DRA， CD80 mRNA in the atractylenolide Ⅱ （200，100 mg·L-1） groups were increased（P<0.05，P<0.01）， and TNF-α protein expression in the atractylenolide Ⅱ （200 mg·L-1） group was increased （P<0.05）， M2 type macrophage-associated cytokine TGF-β mRNA expression levels in the atractylenolide Ⅱ （50 mg·L-1） group were decreased， and IL-10， IL-6 protein expression levels in the atractylenolide Ⅱ （200 mg·L-1） group were decreased （P<0.05，P<0.01）. The expression levels of p-PI3K protein in the atractylenolide Ⅱ （200，100 mg·L-1） groups were also decreased（P<0.05，P<0.01）. Conclusion:Atractylenolide Ⅱ could induce the polarization of macrophages to M1 type by reducing the expression of p-PI3K in macrophages and inhibiting the proliferation and migration of gastric cancer cells.

Comparative Study on Contents of 5 Active Ingredients in Different Varieties and Harvesting Periods of Codo- nopsis Radix / 中国药房

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.

Involvement of mitochondrial apoptotic pathway and MAPKs/NF-κ B inflammatory pathway in the neuroprotective effect of atractylenolide III in corticosterone-induced PC12 cells / 中国天然药物

Atractylenolide III (ATL-III), a sesquiterpene compound isolated from Rhizoma Atractylodis Macrocephalae, has revealed a number of pharmacological properties including anti-inflammatory, anti-cancer activity, and neuroprotective effect. This study aimed to evaluate the cytoprotective efficiency and potential mechanisms of ATL-III on corticosterone injured rat phaeochromocytoma (PC12) cells. Our results demonstrate that ATL-III increases cell viability and reduces the release of lactate dehydrogenase (LDH). The results suggest that ATL-III protects PC12 cells from corticosterone-induced injury by inhibiting the intracellular Ca overloading, inhibiting the mitochondrial apoptotic pathway and modulating the MAPK/NF-ΚB inflammatory pathways. These findings provide a novel insight into the molecular mechanism by which ATL-III protected the PC12 cells against corticosterone-induced injury for the first time. Our results provide the evidence that ATL-III may serve as a therapeutic agent in the treatment of depression.

Effect of Different Processing Methods on Types and Contents of Chemical Constituents in Codonopsis Radix from Shanxi / 中国实验方剂学杂志

Objective:To establish the HPLC fingerprint of raw products,rice-processed products and honey-processed products of Codonopsis Radix from Shanxi,and establish determination of their chemical constituents,which was used to analyze the changes of types and contents of chemical constituents in Codonopsis Radix from Shanxi before and after processing. Method:Agilent ZORBAX SB-C18 column(4.6 mm×250 mm,5 μm) was adopted,the separation process was carried out using a binary gradient elution system composed of 0.5% phosphoric acid aqueous solution and acetonitrile,the column temperature was 30℃ and the detection wavelength was 220 nm. Result:Compared with the corresponding reference fingerprint,the similarities of HPLC fingerprint of 10 batches of raw products and processed products were >0.90.In raw products,rice-processed products and honey-processed products of Codonopsis Radix from Shanxi,the contents of lobetyolin were (0.33±0.049),(0.24±0.034),(0.18±0.047) mg·g-1,the contents of atractylenolide Ⅲ were (0.20±0.046),(0.40±0.046),(0.31±0.060) mg·g-1,the contents of 5-hydroxymethylfurfural(5-HMF) were (0.74±0.16),(1.45±0.19),(1.54±0.12) mg·g-1,respectively. Conclusion:Different processing methods have little effect on types of chemical constituents in Codonopsis Radix from Shanxi,but have great effect on the contents of some chemical constituents.

Effects of atractylenolide I, II, and III against rotavirus in vitro and in vivo / 中草药

Objective To study the effects of atractylodes I, II, and III against rotavirus in vitro and in vivo. Methods An in vitro study model was established using Caco-2 cells. The cytopathic effect (CPE) and MTT staining were used to determine the toxicity of atractylenolide I, II, and III to cells for the inhibition of rotavirus biosynthesis, direct inactivation of rotavirus, and antiviral adsorption, with ribavirin as a positive drug. With half of the therapeutic concentration (EC50) and half of the cytotoxic concentration (TC50), the treatment index TI value was obtained and used as the evaluation index. An RV-infected model of suckling diarrhea was established in vivo to observe the signs and symptoms of the suckling mice, and the in vivo anti-rotavirus effect was preliminarily determined according to the diarrhea score and the weight gain. Results In vitro studies found that atractylenolide III had the direct inactivation effect on rotavirus with TI value of 8; atractylodes III medium-dose group has the best anti-rotavirus effect in vivo. Conclusion Atractylodes III, the main active component of Atractylodes macrocephala, has significant anti-rotavirus effect in vitro and in vivo; Atractylenolide III mainly works by directly inactivating rotavirus in vitro.

Huangqi Baizhu decoction prevents CAC process by inhibiting MIR-31-5p/STAT3 positive circle / 中国药理学通报

To explore the role of miR-31-5p/STAT3 in the pathogenesis of colitis-assosiated cancer (CAC) and the intervention mechanism of Huangqi Baizhu decoction. Methods The CAC model of C57BL/6 mice was established by AOM/DSS method. The differential expression of MicroRNA in control and CAC mice was detected. qPCR was used to screen differential MicroRNAs; Western blot was used to detect the expression levels of STAT3 , p-STAT3 and IL-6Ra. Results AOM was injected into abdominal cavity once, combined with 3% DSS free drinking for three cycles to establish model. After eight weeks, moderate and/or severe dysplasia of colonic mucosa appeared in model mice. After detection, the expression of miR-31-5p and STAT3 and p-STAT3 proteins significantly increased. After intervention with Huangqi Baizhu decoction, the expressions of miR-31-5p and STAT3 and p-STAT3 proteins were down-regulated. In vitro, miR-31-5p over-expression in RAW264. 7 by transfect method, and the expression of STAT3 protein increased significantly compared with that of control. On the other hand, stimulated by IL-6 or TNF-a for 24 h, miR-31-5p levels were up-regulated significantly, suggesting that inflammatory factors could cause a positive correlation between miR-31-5p/STAT3. At the same time, ATRII + AST (atractylodesin II + astragaloside) mixture significantly down-regulated miR-31-5p, STAT3, IL-6Ra increase induced by IL-6 stimulation. Conclusions miR-31-5p/STAT3 forms a positive feedback loop, which plays an important role in the pathogenesis of CAC. miR-31-5p/STAT3 may continue to amplify the inflammation effect and eventually lead to the dysplasia of colonic epithelium and even tumorigenesis.

Comparative analysis on Zhizhu Pills and Zhizhu Granules by multiple components / 中草药

Objective To comparative analyze the contents of 20 active ingredients in two dosage forms of Zhizhu Pills (ZP) and Zhizhu Granules (ZG). Methods Three batches of raw materials were used for the preparation of ZP and ZG according to the preparation process of China Pharmacopoeia (2015 Editon). Twenty active ingredients in the two dosage forms were detected simultaneously using high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QqQ-MS). The separation was performed on a Poroshell 120 SB-C18 (100 mm × 4.6 mm, 2.7 μm) column with a flow rate of 0.5 mL/min. Mobile phase consisted of 0.1% formic acid in water (A)-0.1% formic acid in acetonitrile (B). The column temperature was set at 30 ℃. The gradient elution conditions: 0-7 min, 23% B; 7-17 min, 23%-80% B; 17-20 min, 80%-100% B, 20-30 min, 100% B; flow rate was 0.5 mL/min. Results The contents of 20 active ingredients in the pills were 1.48-13.37 times that of the granules, the total difference of them perday were 493.02-615.08 mg. Conclusion There were significant differences in the content of active components in the two dosage forms of ZP and ZG, especially those components with poor solubility in water and thermal instability.

Quantitative Analysis of Multi-Components by Single Marker for Determination of Three Atractylenolides in Atractylodis Macrocephalae Rhizoma / 中国药学杂志

OBJECTIVE: To establish a quantitative analysis of multi-components by single marker(QAMS) for the determination of three atractylenolides in Atractylodes macrocephala Koidz. METHODS: UPLC method was applied to determine atractylenolide Ⅰ, Ⅱ and Ⅲ by external standard method (ESM) first. With this established UPLC method, atractylenolide Ⅲ was used as the internal standard (IS) to determine the correction factors (RCFs) of the two other atractylenolides, and their contents in all the samples were calculated by their RCFs at the same time. By comparing the contents determined by the ESM and QAMS methods, the feasibility and accuracy of QAMS method were verified. RESULTS: Within a certain range(atractylenolideⅠ: 3.7 - 59.3 μg•mL-1, atractylenolide Ⅱ: 3.9 - 63.5 μg•mL-1, atractylenolide Ⅲ: 7.3 - 116.1 μg•mL-1), the RCFs of atractylenolide Ⅰ, Ⅱ to atractylenolide Ⅲ were 0.633 and 1.895, respectively, which had good repeatability in different experimental conditions. There was no significant difference between the QAMS method and ESM method(P > 0.05). CONCLUSION: The QAMS method is feasible and accurate for the determination of the three atractylenolides and is beneficial to the quality control of Atractylodes macrocephala Koidz..

Mechanisms and proliferation inhibitory effects of atractylenolide Ⅰ on SK-OV-3 and OVCAR-3 ovarian cancer cell / 局解手术学杂志

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.

Optimization of the Soxhlet Extraction Technology of Atractylodes macrocephala Formula Granules by Or-thogonal Test / 中国药房

OBJECTIVE:To optimize the soxhlet extraction technology of Atractylodis macrocephalae,and to provide evi-dence for research and preparation of its formula granules. METHODS:Using the contents of atractylenolide Ⅰ,Ⅱ,Ⅲ as index, based on single factor test,the Soxhlet extraction technology of A. macrocephalae formula granules was optimized and verified by L9(34)orthogonal test with extraction time,solid-liquid ratio,extraction times as factors,and then compared with other technolo-gies (normal temperature extraction method,ultrasonic extraction method,reflux extraction method). RESULTS:The optimal ex-traction technology was as follows as 6-fold ethanol,extracting for 3 times,lasting for 8 h. Results of validation test showed that the extraction amounts of atractylenolide were 0.769,0.752,0.781 mg/g (RSD=1.99%,n=3) for 3 times,which were higher than the extraction amounts of other 3 methods(0.683,0.489,0.693 mg/g). CONCLUSIONS:The optimized extraction technolo-gy possesses high extraction rates of atractylenolide Ⅰ,Ⅱ,Ⅲ,and can be used for the extraction of internal ether from A. macro-cephalae formula granules.

Atractylenolide protects against lipopolysaccharide-induced disseminated intravascular coagulation by anti-inflammatory and anticoagulation effect

Objective To investigate whether atractylenolide (ATL-) 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- 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- 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- and ATL- 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- 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- 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.