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.

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.

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.

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.

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.

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.

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.

<b>Effects of Rikkunshito on Cisplatin-induced Delay </b><b>in Gastric Emptying in Rats </b> / 日本東洋医学雑誌

Rikkunshito is comprised of 8 crude drugs and is used for the treatment of gastrointestinal dysfunctions such as anorexia and heavy stomach feeling. These symptoms are often caused by delay in gastric emptying. Cisplatin is a representative cancer chemotherapeutic drug with severe adverse effects such as anorexia and nausea, that gives rise to a delay in gastric emptying. However, it is still unknown whether rikkunshito has effects on improving the delayed gastric emptying induced by cisplatin. In the present study, we examined the effects of rikkunshito (an Atractylodis rhizoma-containing formula) on cisplatin-induced delay in gastric emptying in the rat. Rikkunshito improved this. Among the crude drugs that comprise rikkunshito, Atractylodis rhizoma, Ginseng radix, Poria and Aurantii nobilis pericarpium individually improved the delay in gastric emptying, suggesting that they all contribute to the action of rikkunshito. Moreover, the effects of these 4 crude drugs in combinations were also examined, and as a result, tended to be stronger when Atractylodis rhizoma was included. On the other hand, when Atractylodis rhizoma was excluded from rikkunshito, the effects were weaker. Meanwhile, atractylenolide III, a specific chemical constituent of Atractylodis rhizoma, improved delay in gastric emptying in a manner similar to that of rikkunshito with Atractylodis rhizoma. These results, taken together, suggest that Atractylodis rhizoma likely contributes greatly to the improving effect of rikkunshito on cisplatin-induced delay in gastric emptying.

Study on targeted separation of atractylenolide IQ from traditional Chinese herbs by molecularly imprinted solid phase extraction / 中国药学杂志

OBJECTIVE: To establish a novel and efficient method for enriching low content active components from traditional Chinese herbs. METHODS: The enrichment of atractylenolide III was taken as an example. First, molecular imprinting polymers (MIPs) of atractylenolide III were prepared by precipitation polymerization using 1-Vinylimidazole as functional monomer. Second, the atractylenolide III MIPs were packed into solid-phase column to separate atractylenolide III and its analogues from the exracts of Herba Atractylodes Macrocephaia and Codonopsis pilosula. Moreover, the adsorption performance of MIPs for the target components was also investigated. RESULTS: Enrichment factor (EF) of MIP-SPE and C18-SPE column were 78.90 and 51.56 μg · g-1 respectively, suggesting that MIPs had better adsorption property than C18; the precision and accuracy of the developed method were satisfactory with recoveries of 102.2% and LOD of 0.36 μg · mL-1. CONCLUSION These results demonstrate the feasibility of molecularly imprinted solid phase extraction for enriching low content active components in traditional Chinese herbs.