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.

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.

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.

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.

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.

Optimization of rice-washed water rinsing process technology for Atractylodis Macrocephalae Rhizoma by central composite design-response surface methodology / 中草药

Objective: To optimize the processing technology of Atractylodis Macrocephalae Rhizoma prepared by rice-washed water rinsing, and provide a scientific basis for producing specification in processing. Methods: Design the processing with central composite design-response surface methodology and take the factors of volume of rice-washed water, rinsing time, and rinsing temperature as independent variables. The contents of atractylenolide I, II, and III were determined by HPLC and the comprehensive scores of the three components were regarded as the response index or OD. By analyzing with Design Expert, the best processing parasite for the experiment could be induced. Results: The best processing conditions were 9-time volume of rice-washed water, 55 h for rinsing, and at the temperature of 26℃. On the selected condition, the value of OD was at 0.960. Conclusion: The rice-washed water rinsing processing technology for Atractylodis Macrocephalae Rhizoma is stable and feasible under the condition selected, which can be used as reference for its production and quality control.

Simultaneous determination of 10 components in Shendan Sanjie Capsule by HPLC-DAD / 中草药

Objective: To establish an HPLC-DAD method for the simultaneous determination of 10 components in Shendan Sanjie Capsule including tanshinone IIA, magnolol, honokiol, naringin, neohesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, calycosin 7-O-β-D-glucopyranoside, and atractylenolide I. Methods: The chromatographic separation was performed on a Hypersil BDS column (150 mm × 4.6 mm, 3.5 μm) with acetonitrile-merhanol-0.1% phosphate acid solution as mobile phase at the flow rate of 1.0 mL/min for gradient elution and the column temperature was 40 ℃. The detection wavelength was set at 270 nm for tanshinone IIA, 294 nm for magnolol and honokiol, 283 nm for naringin and neohesperidin, 203 nm for ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1, 260 nm for calycosin 7-O-β-D-glucopyranoside, and 220 nm for atractylenolide I. The volume of sample injection was 10 μL. Results: Ten compounds were well separated under the determined chromatographic conditions. The RSD values of precision and repeatability experiment were all less than 2% and the sample solution was stable during 10 h. All the compounds had a wide linear range and good linearity: the linear range of tanshinone IIA, magnolol, honokiol, naringin, neohesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, calycosin 7-O-β-D-glucopyranoside, and atractylenolide I were 112-560 μg/mL (r = 0.999 6), 64-320 μg/mL (r = 0.999 1), 48-240 μg/mL (r = 0.999 3), 80-400 μg/mL(r = 0.999 4), 80-400 μg/mL (r = 0.999 5), 16-80 μg/mL (r = 0.999 2), 16-80 μg/mL (r = 0.999 1), 16-80 μg/mL (r = 0.999 1), 40-200 μg/mL (r = 0.999 2), and 56-280 μg/mL (r = 0.999 3), respectively. The average recoveries were in the range of 98.43%-101.52% and the RSD values were all less than 2.0%. The content ranges of tanshinone IIA, magnolol, honokiol, naringin, neohesperidin, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, calycosin 7-O-β-D-glucopyranoside, and atractylenolide I in six batches of Shendan Sanjie Capsule were 0.829-0.840 mg/g, 0.538-0.548 mg/g, 0.360-0.369 mg/g, 0.210-0.219 mg/g, 0.111-0.118 mg/g, 0.081-0.089 mg/g, 0.070-0.078 mg/g, 0.111-0.117 mg/g, 0.072-0.080 mg/g, and 0.130-0.137 mg/g, respectively. Conclusion: The method is simple and convenient, the methodology validation shows that determination result of the method is accurate and reliable and it can be an effective approach for the quality control of Shendan Sanjie Capsule.

HPLC fingerprint of Ermiao Pills and chemical constituents analysis by UPLC-LTQ-Orbitrap / 中草药

Objective To establish a quality assessment method for Ermiao Pills (EP) based on HPLC fingerprint and qualitatively analyze the chemical constituents. Methods The chromatographic column Shiseido C18 (250 mm × 4.6 mm, 5 μm) was used, acetonitrile-0.1% formic acid as mobile phase with gradient elution at the flow rate of 1.0 mL/min, and the detection wavelength was 330 nm. The standard chromatographic fingerprint was synthesized from chromatogram of the mixed standard herbs of Phellodendron Rupr. and Atractylodes DC, and the similarity evaluation of Ermiao Pills samples was carried out by the Similarity Evaluation System for Chromatographic Fingerprints of TCM (Chinese Pharmacopoeia Commission, version 2012A). Ultra high-performance liquid chromatography coupled with linear ion trap-Orbitrap Elite mass spectrometer (UPLC-LTQ-Orbitrap) was used to characterize the chemical constituents of Ermiao Pills. A Thermo Scientific Syncronis C18 (100 mm × 2.1 mm, 1.9 μm) column and a gradient elution of acetonitrile-0.1% formic acid were used for UPLC separation. The combination of ESI-LTQ-Orbitrap mass analyzer with a linear ion trap was applied for high resolution mass spectrometry and collision-induced dissociation (CID). Results The chromatographic fingerprints were generated with 10 common peaks. The similarity scores of 20 samples between each material batch and the reference fingerprint ranged from 0.869-0.992. Twenty-one components were identified via referring to reference components and literatures and analyzing MS data, they were neo-chlorogenic acid, magnocurarine, xanthoplanine, magnoflorine, 3-O-feruloylquinic acid, menisperine, demethyleneberberine, oxyberberine, columbamine, jatrorrhizine, berberubine, palmatine, berberine, syringic acid, caffeic acid, (E)-4-(3-hydroxyprop-1-en1yl)-2-methoxyphenol, atractylenolide II, acetosyringone, atractylenolide I, selina-4(14), 7(11)-dien-8-one, and atractylodin. Conclusion The established method of fingerprint is specific, combined with LC-MS qualitative analysis, can be used for the quality evaluation of Ermiao Pills, giving support to quality control comprehensively.

Simultaneous determination of 12 chemical components in Tiaojing Pills by RP-HPLC under variable wavelengths / 中草药

Objective: To establish a RP-HPLC method to determine the contents of paeoniflorin, acteoside, ferulic acid, leonurine hydrochloride, hesperidin, paeonol, baicalin, asperosaponin VI, limonin, atractylenolide I, atractylenolide III, and pachymic acid in Tiaojing Pills. Methods: The determination was performed on a Venusil MP-C18 column (250 mm × 4.6 mm, 5.0 μm) with ethanol-acetonitrile (40∶60, A) and 0.2% phosphoric acid (B) as mobile phases for gradient elution, at the flow rate of 0.8 mL/min; The column temperature was 45 ℃. Results: The nine components were well separated and showed good linearity, such as paeoniflorin 0.5-50.0 mg/L (r = 0.999 5), acteoside 0.1-10.0 mg/L (r = 0.999 1), ferulic acid 0.2-20.0 mg/L (r = 0.999 2), leonurine hydrochloride 0.3-30.0 mg/L (r = 0.999 3), hesperidin 4.0-400.0 mg/L (r = 0.999 8), paeonol 0.2-20.0 mg/L (r = 0.999 1), baicalin 0.6-60.0 mg/L (r = 0.999 4), asperosaponin VI 1.5-150.0 mg/L (r = 0.999 8), limonin 7.0-700. 0 mg/L (r = 0.999 9), atractylenolide I 0.5-50. 0 mg/L (r = 0.999 3), atractylenolide III 0.5-50. 0 mg/L (r = 0.999 4), and pachymic acid 1.0-100. 0 mg/L (r = 0.999 6). The precision was good, RSD ≤ 0.97%, the repeatability was good in terms of RSD ≤ 1.25% and the recovery rate was 98.5%-103.5% (RSD ≤ 1.24%). Test solution was stable at room temperature within 24 h. The contents of twelve batches of the paeoniflorin, acteoside, ferulic acid, leonurine hydrochloride, hesperidin, paeonol, baicalin, asperosaponin VI, limonin, atractylenolide I, atractylenolide III and pachymic acid were 4.328-4.688, 0.033-0.054, 0.073-0.091, 0.177-0.199, 0.243-0.283, 0.043-0.069, 1.144-1.173, 0.037-0.061, 0.094-0.126, 0.127-0.157, 0.155-0.179, and 0.285-0.327 mg/g, respectively. Conclusion: The method is rapid and has high sensitivity, high accuracy, and good specificity, It can be applied to the quality control of Tiaojing Pills.

Qualitative evaluation of Atractylodis Macrocephalae Rhizoma from different habitats by HPLC-PDA fingerprint combined with UFLC-Q-TOF/MS qualitative identification / 中草药

Objective: To establish and identify the HPLC-PDA fingerprint of Atractylodis Macrocephalae Rhizoma (AMR) and provide a reference for the comprehensive control of the quality of AMR. Methods: AMR was extracted with 70% methanol by sonicating for 60 min. The analysis of AMR extract was performed on Inertsil® ODS-SP column (150 mm × 4.6 mm, 5 μm), column temperature was maintained at 40 ℃, flow rate was 1.0 mL/min, and detector was Waters 2998 UV detector with detection wavelength 235 nm. Mobile phase was acetonitrile (B)-water (A) with the elution gradient 0 -10 min, 30%-45% B, 10-25 min, 45% B, 25-50 min, 45%-70% B, 50-55 min, 70% B, 55-62 min, 70%-30% B, 62-75 min, 30% B. Time-of-flight mass spectrometer (TOF/MS) and electro-spray ion (ESI) source were used for the qualitative analysis in a positive ion mode, and mass scan range was m/z 50-1 500. Results: Comparing and fitting the peaks of AMR from different habitats (Zhejiang, Anhui, and Hunan Provinces), the HPLC-PDA fingerprint was set up with six common peaks, and they were identified by UFLC-Q-TOF/MS as 5-(hydroxymethyl)-2-furaldehyde, atractylenolide III, atractylenolide I, atractylenolide II, atractylenolide VI, and biatractylenolide. System suitability, extraction, and chromatographic conditions of AMR were optimized. RSD of accuracy, stability and repeatability was all less than 2%. Measuring ten batches and fitting fingerprint similarity, the values were all greater than 0.95. Conclusion: The HPLC fingerprint can be used as standard uniformity and stability of quality control methods for AMR slice.

Optimization of processing technology for Atractylodis Macrocephalae Rhizoma fried with honey rice chaff in Jian Chang Bang / 中草药

To optimize the processing technology of Atractylodis Macrocephalae Rhizoma fried with honey rice chaff in Jian Chang Bang. With the contents of atractylenolide I, atractylenolide II, atractylenolide III, atractylone, and alcohol extract as indexes, which were determined by HPLC and alcohol extract method in Pharmacopoeia 2010 edition, L9 (34) orthogonal test was used to determine the best processing technology. The optimum processing technology of Atractylodis Macrocephalae Rhizoma fried with honey rice chaff was as following: the amount of honey rice chaff 50%, frying temperature 200℃, and frying time 5 min. The optimum processing technology is stable and feasible by verification.

Identification of an anticancer compound against HT-29 cells from Phellinus linteus grown on germinated brown rice

Objective:To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate (EtOAc) extract of Phellinus linteus grown on germinated brown rice (PB). Methods: EtOAc extract of PB was partitioned with n-hexane, EtOAc, and water-saturated n-butanol. Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR, respectively. Cytotoxicity against HT-29 cells was tested by SRB assay. Results: The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line. Atractylenolide I, a eudesmane-type sesquiterpene lactone, a major anticancer substance of PB, was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC. This structure was elucidated by one-and two-dimensional NMR spectroscopic data. Atractylenolide I has not been reported in mushrooms or rice as of yet. The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells. Conclusions:Atractylenolide I might contribute to the anticancer effect of PB.

Study on extraction technology of Zhixuan Granula / 中草药

Object To optimize the preparation procedure for Zhixuan Granula (ZXG). Methods The optimum extracting conditions of ZXG were selected by orthogonal test with the active components: 23-acetate alisol B, atractylenolide I, and dried extract as the index, it mice sedation of ZXG was clarified by pharmacodynamics. Results The optimum preparation procedure was as follows: Rhizoma Alismatis and Rhizoma Atractylodis Macrocephalae were extracted with alcohol first, adding 12-fold 70% alcohol by refluxing, extracting twice, 2 h once, then extracted with water, adding 14-fold water, extracting twice, 2 h once. The extract showed the obvious effect on sedation of mice. Conclusion The optimum preparation procedure is reliable, with higher extracting ratio of the active components.