Detoxification Mechanism of Combined Chebulae Fructus， Glycyrrhizae Radix et Rhizoma and Prepared Aconiti Kusnezoffii Radix Cocta in Regulating Cardiac Metabolic Enzyme CYP2J3 / 中国实验方剂学杂志

ObjectiveTo investigate the detoxification mechanism of Chebulae Fructus， Glycyrrhizae Radix et Rhizoma and Prepared Aconiti Kusnezoffii Radix Cocta， and their effective components ellagic acid， liquiritin and aconitine based on cardiac cytochrome P450 （CYP450） system. MethodIn in vivo experiments， rats were randomly divided into control group， prepared Aconiti Kusnezoffii Radix Cocta group （0.25 g·kg-1）， Chebulae Fructus group （0.252 g·kg-1）， Glycyrrhizae Radix et Rhizoma group （0.25 g·kg-1） and combination group （0.25 g·kg-1 Chebulae Fructus+0.25 g·kg-1 Glycyrrhizae Radix et Rhizoma+0.25 g·kg-1 prepared Aconiti Kusnezoffii Radix Cocta， with prepared Aconiti Kusnezoffii Radix Cocta as standard）. After 8 days of administration， creatine kinase （CK） and lactate dehydrogenase （LDH） in rats were detected to observe the pathological changes of heart tissue. Real-time PCR and Western blot were performed to detect the mRNA and protein expressions of CYP2J3， respectively. In in vitro experiments， control group， aconitine group， ellagic acid group， liquiritin group and combination group （aconitine+ellagic acid+liquiritin） were set， and their effects on cell number， DNA content， reactive oxygen species （ROS） and mitochondrial membrane potential （MMP） were detected by high content analysis. The changes in the mRNA and protein expressions of CYP2J3 were also observed. ResultIn vivo experiments， compared with the control group， the prepared Aconiti Kusnezoffii Radix Cocta group had increased CK and LDH in serum （P<0.05， P<0.01）， while the combination group had decreased activities of CK and LDH. Additionally， pathological staining results showed that Chebulae Fructus and Glycyrrhizae Radix et Rhizoma reduced the cardiac toxicity caused by prepared Aconiti Kusnezoffii Radix Cocta. Real-time PCR found that compared with the control group， prepared Aconiti Kusnezoffii Radix Cocta down-regulated the mRNA level of CYP2J3 （P<0.05）， while up-regulated that expression when used in combination with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma （P<0.01）. The protein and mRNA translation levels were basically consistent. In vitro experiments， high content analysis revealed that there was a decrease in the cell number， DNA content and MMP fluorescence value of the aconitine group （P<0.01） and the combination group （P<0.05， P<0.01）， and the fluorescence value of the combination group was higher than that of the aconitine group. Moreover， aconitine down-regulated the mRNA level of CYP2J3 （P<0.05）， but the down-regulating ability of aconitine was reversed in the combination group （P<0.05）. ConclusionThe detoxification mechanism of combined Chebulae Fructus， Glycyrrhizae Radix et Rhizoma and prepared Aconiti Kusnezoffii Radix Cocta is mainly that the combination of ellagic acid， liquiritin and aconitine can up-regulate the expression of CYP2J3， and promote the metabolism of arachidonic acid （AA） to produce epoxyeicosatrienoic acids （EETs）， thus reducing the cardiac toxicity， and this effect may start from the transcriptional link.

Study on regulation of CYP450 enzyme system to reduce liver toxicity through compatibility of Aconiti Kusnezoffii Radix Cocta with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma / 中国中药杂志

Aconiti Kusnezoffii Radix Cocta is one of the most commonly used medicinal materials in Mongolian medicine. Due to the strong toxicity of Aconiti Kusnezoffii Radix Cocta, Mongolian medicine often uses Chebulae Fructus, Glycyrrhizae Radix et Rhizoma to reduce the toxicity, so as to ensure the curative effect of Aconiti Kusnezoffii Radix Cocta while ensuring its clinical curative effect, but the mechanism is not clear. The aim of this study was to investigate the effects of Chebulae Fructus, Glycyrrhizae Radix et Rhizoma and Aconiti Kusnezoffii Radix Cocta on the mRNA transcription and protein translation of cytochrome P450(CYP450) in the liver of normal rats. Male SD rats were randomly divided into negative control(NC) group, phenobarbital(PB) group(0.08 g·kg~(-1)·d~(-1)), Chebulae Fructus group(0.254 2 g·kg~(-1)·d~(-1)), Glycyrrhizae Radix et Rhizoma group(0.254 2 g·kg~(-1)·d~(-1)), Aconiti Kusnezoffii Radix Cocta group(0.254 2 g·kg~(-1)·d~(-1))and compatibility group(0.254 2 g·kg~(-1)·d~(-1),taking Aconiti Kusnezoffii Radix Cocta as the standard). After continuous administration for 8 days, the activities of total bile acid(TBA), alkaline phosphatase(ALP), amino-transferase(ALT) and aspartate aminotransferase(AST)in serum were detected, the pathological changes of liver tissue were observed, and the mRNA and protein expression levels of CYP1 A2, CYP2 C11 and CYP3 A1 were observed. Compared with the NC group, the serum ALP, ALT and AST activities in the Aconiti Kusnezoffii Radix Cocta group were significantly increased, and the ALP, ALT and AST activities were decreased after compatibility. At the same time, compatibility could reduce the liver injury caused by Aconiti Kusnezoffii Radix Cocta. The results showed that Aconiti Kusnezoffii Radix Cocta could inhibit the expression of CYP1 A2, CYP2 C11 and CYP3 A1, and could up-regulate the expression of CYP1 A2, CYP2 C11 and CYP3 A1 when combined with Chebulae Fructus and Glycyrrhizae Radix et Rhizoma. The level of translation was consistent with that of transcription. The compatibility of Chebulae Fructus and Glycyrrhizae Radix et Rhizoma with Aconiti Kusnezoffii Radix Cocta could up-regulate the expression of CYP450 enzyme, reduce the accumulation time of aconitine in vivo, and play a role in reducing toxicity, and this effect may start from gene transcription.

Mechanism of Aconiti Kusnezoffii Radix Processed with Chebulae Fructus Against H9c2 Cardiomyocyte Toxicity Based on TRPV1 Channel / 中国实验方剂学杂志

ObjectiveTo explore the role of transient receptor potential vanilloid 1 （TRPV1） channel in reducing cardiomyocyte toxicity of Aconiti Kusnezoffii Radix processed with Chebulae Fructus. MethodH9c2 cardiomyocytes cultured in vitro were used as a model to assess cell viability by methyl thiazolyl tetrazolium （MTT） assay， the expression of TRPV1 mRNA was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and the leakage rate of lactate dehydrogenase （LDH）， the changes of nucleus， reactive oxygen species （ROS）， mitochondrial membrane potential and Ca2+ contents were detected by enzyme linked immunosorbent assay （ELISA）. ResultCompared with the blank group， when the concentration was ≥0.5 g·L-1， the cell viability was significantly decreased （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ were increased， the mitochondrial membrane potential was decreased， and the nucleus was pyknosis or even broken in raw Aconiti Kusnezoffii Radix and Aconiti Kusnezoffii Radix processed with Chebulae Fructus groups. When the concentration was ≥0.5 g·L-1， compared with the same mass concentration of raw Aconiti Kusnezoffii Radix group， the cell viability increased significantly （P<0.01）， the leakage rate of LDH， the release of ROS and Ca2+ decreased， the mitochondrial membrane potential increased， and the nuclear morphology improved in Aconiti Kusnezoffii Radix processed with Chebulae Fructus group. Application of the same mass concentration of raw Aconiti Kusnezoffii Radix to H9c2 cardiomyocytes pretreated with the TRPV1 inhibitor BCTC significantly increased cell viability， decreased leakage rate of LDH， ROS and Ca2+ release， increased mitochondrial membrane potential and improved nuclear pyknosis compared with untreated H9c2 cardiomyocytes. Application of the same mass concentration of Aconiti Kusnezoffii Radix processed with Chebulae Fructus to H9c2 cardiomyocytes pretreated with BCTC decreased cell viability， increased LDH leakage rate， ROS and Ca2+ release， reduced mitochondrial membrane potential compared with untreated H9c2 cardiomyocytes. Real-time PCR results showed that both raw Aconiti Kusnezoffii Radix and Chebulae Fructus decoction could increase the expression of TRPV1 mRNA in cardiomyocytes in a concentration dependent manner. ConclusionRaw Aconiti Kusnezoffii Radix can induce cardiomyocyte apoptosis and cardiotoxicity by activating TRPV1 channel， while Aconiti Kusnezoffii Radix processed with Chebulae Fructus can attenuate the toxicity through TRPV1 channel， which may be related to the synergistic effect of acid components in Chebulae Fructus and alkaloids in Aconiti Kusnezoffii Radix on TRPV1 channel.

Chemical variation in Aconti Kusnezoffii Radix before and after processing based on UPLC-Orbitrap-MS / 中国中药杂志

Some Chinese herbal medicine needs to be processed before it can be used as medicine, especially toxic Chinese medicine. Highly toxic Aconti Kusnezoffii Radix(Caowu in Chinese) is widely used in traditional Chinese medicine and Mongolian medicine. In traditional Chinese medicine, Caowu is usually processed by boiling with water(CW) until no white part inside and being tasted without tongue-numbing. In Mongolian medicine, it is usually soaked in Chebulae Fructus(Hezi in Chinese) decoction for several days(CH). Both methods could reduce toxicity according to reports. The biggest difference between CW and CH is that CW needs to be heated for 4-6 h, while CH needs Hezi as processing adjuvants. To explore the toxicity reduction mechanism of CW and CH, we studied the contents of various compounds in Caowu processed by two methods by UPLC-Orbitrap-MS. The results indicated that CW had 14 new ingredients, such as 14-O-anisoylneoline and dehydro-mesaconitine, while N-demethyl-mesaconitine and aconitine disappeared. At the same time, it could significantly decrease the content of diester diterpenoid alkaloids and increase the contents of monoester diterpenoid alkaloids and amine-diterpenoid alkaloids. CH had 9 new ingredients from Hezi, like gallic acid, chebulic acid and shikimic acid. Neither the kinds nor the contents of compositions from Caowu in CH changed little. This suggested that the processing mechanism of CW reduced highly toxic components(diester diterpenoid alkaloids) and increased the content of lowly toxic components(monoester diterpenoid alkaloids and amine-diterpenoid alkaloids). Attenuated principle of CH may be related to the components of Hezi. In this experiment, the conclusion shows that the chemical constituents of CW and CH are essentially different, and the two methods have different toxicity reduction principles.

Comparison of Component Differences in Chebulae Fructus and Chebulae Fructus Immaturus Based on Chemical Pattern Recognition and Multi-index Quantitative Analysis / 中国药房

OBJECTIVE：To provide reference for the identification of Chebulae Fructus and Chebulae Fructus Immaturus . METHODS：UPLC method was adopted. The determination was performed on Waters Cortecs T 3 C18 column with mobile phase consisted of acetonitrile- 0.2％ phosphoric acid solution （gradient elution ）at the flow rate of 0.35 mL/min. The column temperature was 30 ℃，and the detection wavelength was set at 270 nm. The sample size was 1 μL. Using gallic acid as reference，UPLC fingerprints of 17 batches of Chebulae Fructus and 14 batches of Chebulae Fructus Immaturus were established and their similarity was evaluated by TCM Chromatographic Fingerprint Similarity Evaluation System （2012 edition）. By comparing substance control ， UV absorption spectrum and related literaturs ，common peaks were identified. PCA and PLS-DA were performed by using SPSS 20.0 and SIMCA 14.1 software. The contents of main difference components in Chebulae Fructus and Chebulae Fructus Immaturus were determined by above UPLC method and compared. RESULTS ：There were 8 common peaks in UPLC fingerprint of Chebulae Fructus and Chebulae Fructus Immaturus ，i.e. chebulic acid （peak 1），gallic acid （peak 2），punicalagin A （peak 3），punicalagin B （peak 4），corilagin（peak 6），chebulagic acid （peak 7）and chebulinic acid （peak 8）. The similarities of 17 batches of Chebulae Fructus were from 0.92 to 0.99，while 14 batches of Chebulae Fructus Immaturus were all above 0.99. The similarity of control fingerprint between Chebulae Fructus and Chebulae Fructus Immaturus was 0.909. PCA demonstrated the differences between Chebulae Fructus and Chebulae Fructus Immaturus . The results of PLS-DA were consistent with those of PCA ，and the variable importance in projection （VIP）values of peak 5，4，7，3 and 2 were above 1 in the PLS-DA model. In 31 batches of samples ，the contents of gallic acid （peak 2），punicalagin A（peak 3），punicalagin B （peak 4）and chebulagic acid （peak 7）were 2.63-10.31， 5.37-44.63，8.02-60.77，44.07-162.98 mg/g；RSDs were 40.14％， 47.91％ ，53.97％ ，36.22％（n＝31）. There was statistical significance in the differences of the mentioned 4 components between Chebulae Fructus and Chebulae Fructus Immaturus 719412818@qq.com （P＜0.05）. CONCLUSIONS ：There are significant differences between Chebulae Fructus and Chebulae Fructus Immaturus gallic acid ，punicalagin A ，punicalagin B and chebulagic acid are the main difference components for identification.

Mechanism of detoxification of Chebulae Fructus against Aconiti kusnezoffii radix toxicity based on network pharmacology / 药学学报

The mechanism of detoxification of Chebulae Fructus against Aconiti kusnezoffii radix toxicity, which was known as Mongolian medical theory, was studied by establishing network of active components-targets-pathways of detoxification and enrichment analysis of targets and pathways based on network pharmacology. Firstly, the targets of active components collected from TCMSP and TCM Database@Taiwan were obtained through SwissTargetPrediction compared with disease targets from OMIM, TTD, DiGSeE. Then, the target enrichment analysis of GO functional annotations and KEGG pathways and protein function were analyzed by Metascape, furthermore, the action between main active ingredients and targets was assessed by SystemsDock Web Site. At last, the Cytoscape was used to construct the network of active components-targets-pathways. In conclusion, there were 15 components and 40 targets related to the cardiotoxicity caused by Aconiti kusnezoffii radix. Furthermore, Chebulae Fructus could regulate cardiac function to detoxify the toxicity by Aconiti kusnezoffii radix through the biological process of negative regulation of blood vessel diameter, regulation of ion transport circulatory system process, muscle contraction inorganic ion homeostasis and the pathways of neuroactive ligand-receptor interaction, calcium signaling pathway, adrenergic signaling in cardiomyocytes, etc.

Study on Improving Quality Standard of Qiwei Maqianzi Pills / 中国药房

OBJECTIVE:To improve the quality standard for Qiwei maqianzi pills.METHODS:TLC was used for the qualitative identification of Chebulae Fmctus and Aucklandiae Radix in the preparation.HPLC method was used for the content determination of hydroxy safflor yellow A,brucine and strychnine in preparation.The determination was performed on Phenomenex Prodigy C18 column with mobile phase consisted of methanol-acetonitrile-0.7％ phosphoric acid soulution(26 ∶ 2 ∶ 72,V/V/V,for hydroxy safflor yellow A),acetonitrile-0.01 mol/L sodium heptanesulfonate mixed with same quantity of 0.02 mol/L potassium dihydrogen phosphate (pH adjusted to 2.8 using 10％ phosphoric acid,21 ∶ 79,V/V,for brucine and strychnine) at the flow rate of 1.0 mL/min.The detection wavelengths were 403 nm (for hydroxy safflor yellow A) and 260 nm (for brucine and strychnine).The column temperature was 25 ℃C,and the injection volume was 10 μL.RESULTS:TLC spots of Chebulae Fructus and Aucklandiae Radix were clear and well-separated without interference from negative control.The linear range was 6.29-62.94 μg/mL for hydroxy safflor yellow A(r=0.999 3),1.83-18.30 μg/mL for brucine(r=0.999 4) and 2.11-21.11 μg/mL for strychnine (r=0.999 6).RSDs of precision,stability and reproducibility tests were lower than 2.0％.The recoveries were 101.66％-104.91％(RSD=1.14％,n=6),99.58％-104.55％ (RSD=1.75％,n=6) and 101.22％-104.04％ (RSD=0.99％,n=6),respectively.CONCLUSIONS:Improved standard can be better used for quality control of Qiwei maqianzi pills.

Simultaneous Determination of Contents of Seven Tannins Active Ingredients in Chebulae Fructus Retz from Different Areas by HPLC / 中国药学杂志

OBJECTIVE: To develop an HPLC method for simultaneous determination of seven tannins in Chebulae Fructus, including gallic acid, chebulic acid, corilagin, ethyl gallate, ellagic acid, chebulagic acid and 1, 2, 3, 4, 6-O-pentagalloylglucose and determine the contents of the seven tannins in Chebulae Fructus Retz from different areas. METHODS: The HPLC analysis was carried out on an Hypersil ODS2 C18 (4.6 mm×250 mm, 5 μm) column with acetonitrile (A) and 0.05% formic acid solution in water (B) as mobile phase in a linear gradient elution mode. The UV detection wavelength was set at 290 nm and the flow rate was 1.0 mL·min-1. RESULTS: The calibration curves of the seven tannins all showed good linearity (r>0.999 8). The recovery rates were in the range of 95.2% to 98.4%. All the seven tannins could be detected in the two kinds of Chebulae Fructus Retz from eight regions, but the amounts of these tannins varied significantly. The contents of the seven tannins active ingredients in Chebulae Fructus of Terminalia chebula Retz from Hainan, Guangxi, Guangdong and Xinjiang were much higher than those from other areas, while those in Chebulae Fructus of Terminalia chebula Retz. var. tomentella Kurt were higher in Guangdong and Guangxi than other areas. CONCLUSION: The method is proved to be accurate and valid, and can be used for the quality control of Terminalia chebula Retz.

Research on compatibility mechanism of Mongolian medicine Sendeng-4 based on metabonomic methods / 中草药

Objective: To explore the mechanism of the compatibility of Mongolian medicine Sendeng-4. Methods: Sendeng-4 was composed of Xanthoceratis Sorbifoliae Lignum seu Ramulus et Folium and its co-decoction with Gardeniae Fructus, Toosendan Fructus, and Chebulae Fructus with a mass ratio of 5:3:1:1. BEH C18 column (50 mm×2.1 mm, 1.7 μm) was used as the chromatographic column, water (0.1% formic acid)-methanol were used as mobile phase for the gradient elution, and the ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS), principle component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze the chemical constituent changes of Xanthoceratis Sorbifoliae Lignum seu Ramulus et Folium and its co-decoction. Results: The significant differences between the group of Xanthoceratis Sorbifoliae Lignum seu Ramulus et Folium and Sendeng-4 were observed in PCA and OPLS-DA model. There were significant differences among eight chemical compounds after compatibility (P<0.05). The relative contents of catechin, (-)-gallocatechin, dihydromyricetin, quercetin decreased, xanthocerasic acid, 3β-hydroxytirucalla-7,24-dien-21-oic acid, 3-oxotirucalla-7,24-dien-21-oic acid, and rutin were increased. Conclusion: Maybe the anti-inflammatory and anti-rheumatic activity of Sendeng-4 have the relationship with the content changes of some index component in Xanthoceratis Sorbifoliae Lignum seu Ramulus et Folium after compatibility.

Determination of gallic acid in chebulae fructus immaturus of different area by RP-HPLC / 国际中医中药杂志

Objective To establish an HPLC method for determining gallic acid in Chebulae fructus immaturus of different area.Methods The ZORBAX SB-C18 (250 mm×4.6 mm,5 μm) column was used,the mobile phase consisted of acetonitrile: 0.1％ H3PO4(29: 71),the flow rate was 1.0 ml/min,the column temperature was 30℃ the detecting wavelength was at 273 nm.Results Gallic acid was successfully separated within 20 min,the linear response range was 0.5611～5.611 μg.The average recovery was 99.9％,and RSD was 2.35％.Conclusion The method is simple,accurate and repeatable; it can provide evidence for further development and utilization of this crude drug.