Yaobitong capsules reshape and rebalance the gut microbiota and metabolites of arthritic rats: An integrated study of microbiome and fecal metabolomics analysis.

Yaobitong capsule (YBTC), a Chinese medicine compound preparation, has been demonstrated to affect multiple pathways associated with inflammation and exhibit potential anti-arthritis effect. In this study, an integrated omic approach based on UHPLC-Q-TOF MS and 16S rRNA sequencing analyses was proposed to reveal the anti-arthritis effect and possible mechanism of YBTC. The AIA rat model showed that YBTC significantly alleviated the typical symptoms of AIA rats such as weight, spleen index and pro-inflammatory cytokines. Fecal metabolomics results identified 41 differential metabolites, which mainly referred to tryptophan, bile acid and fatty acid metabolism. The gut microbiota played a crucially important role in anti-inflammatory immunity, 16S rRNA results indicated that YBTC changed the community structure and alleviated the microecological imbalance caused by rheumatoid arthritis (RA). Further ROC curve analysis demonstrated that it was reliable to identify RA by using 5 metabolites and 3 microorganisms (AUC > 0.83). In summary, it was the first time that the preventive effect of YBTC in RA was confirmed. The secretion of the microbiota-mediated metabolites was significantly improved by YBTC, through its callback effect on the disturbed gut microbiota. Thus, we have indicated a potential novel strategy for the prevention of RA via evaluation of intervention effects of YBTC on AIA rat model.

Integrative serum metabolomic analysis for preventive effects of Yaobitong capsule in adjuvant-induced rheumatoid arthritis rat based on RP/HILIC-UHPLC-Q-TOF MS.

Yaobitong capsule (YBTC) has been used for the prevention and treatment of inflammation-related lumbago and leg pain. However, its intervention mechanism still remains unclear. This study was aimed to evaluate the control efficiency of YBTC on adjuvant-induced rheumatoid arthritis (RA) rats by metabonomic method and to explore its possible anti-arthritis mechanism. Taking into account the complexity of endogenous metabolites in serum samples, an integrated metabolomics method based on RP/HILIC-UHPLC-Q-TOF MS was developed, to overcome the limitations of a single chromatographic in this study. The results showed that 32 potential biomarkers of arthritis were identified, primarily related to amino acid metabolism, glucose metabolism, lipid metabolism and nucleotide metabolism. Further receiver operating characteristic analysis revealed that the area under the curve of two down-regulated metabolites (3-Hydroxy-hexadecanoic acid, 2-Oxoarginine) and one up-regulated metabolite (l-Glutamic acid) among 32 biomarkers were 0.906, 0.969 and 1.000, respectively, indicating that high predictive ability of this method for RA. In this study, an integrated serum metabolomics method based on high-resolution mass spectrometry was successfully established for the first time to study the intervention mechanism of YBTC in RA, providing evidence regarding the clinical application of YBTC and a new insight for the prevention of RA in the future.

Nondestructive qualitative and quantitative analysis of Yaobitong capsule using near-infrared spectroscopy in tandem with chemometrics.

The purpose of the study is to present a nondestructive qualitative and quantitative approach of hard-shell capsule using near-infrared (NIR) spectroscopy combined with chemometrics. The Yaobitong capsule (YBTC) was used for demonstration of the proposed approach and the NIR spectra were collected using a handheld fiber probe (FP) without the damage of capsule shell. By comparing the differences and similarities of the NIR spectra of capsule shells, contents and intact capsules, a preliminary conclusion can be drawn that the NIR spectra contained the information of the contents. Characteristic variables were selected by competitive adaptive weighted resampling (CARS) method, and least squares support vector machine (LSSVM) method based on particle swarm optimization (PSO) algorithm was applied to the construction of quantitative models. The relative standard error of prediction (RSEP) values of five saponins including notoginsenoside R1, ginsenoside Rg1, Re, Rb1, and Rd were 3.240%, 5.468%, 5.303%, 5.043%, and 3.745%, respectively. In addition, for qualitative model, three different types of adulterated capsules were designed. The model established by data driven version of soft independent modeling of class analogy (DD-SIMCA) demonstrated a satisfactory result that all adulterated capsules were identified accurately after an appropriate number of principal components (PCs) were chosen. The results indicated that although the NIR spectra collection was affected by capsule shell, sufficient content information can be obtained for quantitative and qualitative analysis after combining with chemometrics. It further proved that acquired NIR spectra do contain the effective component information of the capsule. This study provided a reference for the rapid nondestructive quality analysis of traditional Chinese medicine (TCM) capsule without damaging capsule shell.

Systematically characterized mechanism of treatment for lumbar disc herniation based on Yaobitong capsule ingredient analysis in rat plasma and its network pharmacology strategy by UPLC-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Yaobitong capsule (YBTC) was a traditional Chinese medicine (TCM) and it had clinically used to treat lumbar disc degeneration (LDH) for a long time. However, the active ingredients of YBTC absorption into the plasma and its pharmacological mechanism of treatment for LDH still remained unclear. AIM OF THE STUDY: In this study, our research committed to identify the absorbed active ingredients of YBTC in rat plasma, and it may be a potential mechanism of action on LDH by the biological targets regulating related pathways. MATERIALS AND METHODS: An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to identify the absorption components and metabolites of YBTC in rat plasma, and the network pharmacology was further investigated to illuminate its potential mechanism of treatment for LDH by the biological targets regulating related pathways. RESULTS: The network analysis found that 56 components were identified as its main active ingredients including ginsenoside Rg1, ginsenoside Rb1, senkyunolide H, and tetrahydropalmatine, etc. Combining with biological process, cellular component and molecular functions of GO, and kyotoencyclopedia of genes and genomes pathway enrichment analysis to perform network topology analysis on core targets. These active ingredients regulated 29 mainly pathways by 87 direct target genes including MAPK, Ras, PI3K-Akt, and NF-kappa B signaling pathway, etc. CONCLUSION: In this study, the absorption active ingredients of YBTC in rat plasma were firstly combined with the network pharmacology investigation to elucidate its biological mechanism of treatment for LDH in vivo. It inhibited excessive inflammatory reactions, thereby reducing the sensitivity of the nerves to reduce pain and relieve LDH, and potential medicine targets could be identified to clarify the molecular mechanism of YBTCs' regulation of LDH.

UPLC-MS/MS simultaneous determination of seven active ingredients of Yaobitong capsule in rat plasma and its integrated pharmacokinetic application.

A reliable and sensitive UPLC-MS/MS method was first established and validated for the simultaneous determination of seven active ingredients of Yaobitong capsule in rat plasma: ginsenoside Rg1, ginsenoside Rb1, osthole, tetrahydropalmatine, paeoniflorin, albiflorin, and ferulic acid. And this method was further applied for the integrated pharmacokinetic study of Yaobitong capsule in rats after oral administration. Plasma samples (100 µL) were precipitated with 300 µL of methanol using carbamazepine as internal standard. Chromatographic separation was achieved using an Aquity UPLC BEH C18 column (100 × 2.1 mm, 1.7 µm), with the mobile phase consisting of 0.1% formic acid and acetonitrile. The method was validated using a good linear relationship (r ≥ 0.991), and the lower limit of quantification of the analytes ranged from 0.5 to 40 ng/mL. In the integrated pharmacokinetic study, the weight coefficient was calculated by the ratio of AUC0-∞ of each component to the total AUC0-∞ of the seven active ingredients. The integrated pharmacokinetic parameters Cmax , Tmax , and t1/2 were 81.54 ± 9.62 ng/mL, 1.00 ± 0.21 h, and 3.26 ± 1.14 h, respectively. The integration of pharmacokinetic parameters showed a shorter t1/2 because of fully considering the contribution of the characteristics of each active ingredient to the overall pharmacokinetics.

Extraction optimization of accelerated solvent extraction for eight active compounds from Yaobitong capsule using response surface methodology: Comparison with ultrasonic and reflux extraction.

This work described the development of a novel method for simultaneous extraction of eight active compounds (including catechin, albiflorin, paeoniflorin, ferulic acid, ginsenoside Rg1, tetrahydropalmatine, ginsenoside Rb1 and osthole) from Yaobitong capsule by accelerated solvent extraction (ASE). Response surface methodology (RSM) with desirability functions was employed to optimize the extraction conditions yielding the optimal conditions of ASE (extraction time 8 min, extraction temperature 80 °C, extraction solvent 70% methanol and flushing volume 100%). A high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) method was developed and validated for simultaneous quantification of the eight compounds in Yaobitong capsule. The values of correlation coefficient (R) were satisfactory between 0.9992 and 0.9999 over the linear concentration range of 0.5-1000 µg mL-1. It was found that the limits of detection (LODs) and the limits of quantification (LOQs) for the eight active compounds were 0.10-2.90 µg•mL-1 and 0.30-9.40 µg•mL-1, respectively. The recoveries of the eight main active compounds in Yaobitong capsule were in the range of 93.31%-106.22%. And the contents of the analytes extracted by ASE under the optimal conditions were compared to traditional solvent extraction methods, such as ultrasonic and reflux extraction. The results indicated that the ASE method proved to be more suitable for the extract of active compounds in Yaobitong capsule, which could obtain higher extraction efficiency. At last, the proposed method was applied to analyze ten batches of actual samples, which provided high extraction efficiency and had wide potential application in the analysis of traditional Chinese medicines.

Simultaneous determination of five saponins in Yaobitong capsule by HPLC-CAD / 药学学报

A high performance liquid chromatography charged aerosol detector (HPLC-CAD) method was established for the simultaneous determination of five saponins (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rd) in Yaobitong capsule, providing a method for quality control. The sample was extracted with methanol and chromatographic separation was performed on a Waters Xbridge Phenol column (150 mm×4.6 mm, 3.5 μm) using acetonitrile-water as the mobile phase with gradient elution at a flow rate of 1.0 mL·min-1. The column temperature was 30 ℃ and the injection volume was 10 μL. The nebulizer temperature of CAD was 35 ℃ and the air pressure was 60.2 psi, the filtration was 3.6 s, and the collection frequency was 5 Hz. Notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd showed a good linear relationship in the range of 16.96-203.5 μg·mL-1 (R2 = 0.999 3), 54.46-653.5 μg·mL-1 (R2 = 0.999 3), 10.96-131.5 μg·mL-1 (R2 = 0.999 6), 51.50-618.0 μg·mL-1 (R2 = 0.999 0), 15.94-191.3 μg·mL-1 (R2 = 0.999 4), respectively. The average recoveries were 98.96%, 100.8%, 94.76%, 100.1%, 103.1%, and RSDs were 0.87%, 1.46%, 1.85%, 2.06%, 0.96% (n = 6), respectively. The proposed method is accurate, simple and reliable, and can be used for the determination of five saponins in Yaobitong capsule.

Study on chemical constituents in Yaobitong Capsule (I) / 中草药

Objective To investigate the chemical constituents from the ethanol extract of Yaobitong Capsule. Methods The chemical constituents were isolated by repeated silica gel column, dynamic axial compression chromatography, Sephadex LH-20 column, and prep-HPLC. Their structures were identified on the basis of NMR spectral data analysis. Results Sixteen compounds were isolated and identified to be nodakenetin (1), columbianetin (2), ferulic acid (3), umbelliferone (4), bergaptol (5), xanthotoxin (6), isoimperatorin (7), scoparone (8), (Z)-6-hydroxy-7-methoxydihydroligustilide (9), 4-hydroxy-3-butylphthalide (10), 3-(3′-hydroxy)- butylphthalide (11), senkyunolide C (12), senkyunolide H (13), senkyunolide I (14), fraxidin (15), and isofraxidin (16). Conclusion Compounds 1—16 are isolated from Yaobitong Capsule for the first time, and compound 11 is obtained by the means of separation for the first time.

Studies on chemical constituents in Yaobitong Capsule (II) / 中草药

Objective To investigate the anti-inflammatory constituents from the ethanol extract of Yaobitong Capsule. Methods The chemical constituents were isolated by repeated silica gel column, Sephadex LH-20, and preparative HPLC. The structures were identified by various spectral data analysis and physicochemical properties. The anti-inflammatory effects were evaluated by in vitro model of LPS-stimulated rat thoracic aortic smooth muscle cells (A7r5). Results Eleven compounds were isolated from the contents of the capsule, including 2 organic acids, 1 alkaloid, 2 senkyunolides, 1 sesquiterpene, and 5 coumarins, which identified as ethyl caffeate (1), ferulic acid (2), corydaline (3), senkyunolide H (4), senkyunolide I (5), ligustiphenol (6), columbianetin (7), ulopterol (8), meranzin hydrate (9), angelitriol (10), and 6-[1(R),2(R)-1,2,3-thihydroxy-3-methylbutyl]-7-methoxycoumarin (11). Compounds 1-6 showed the anti-inflammatory activity in different degrees. Conclusion Compounds 1, 3, 6, and 8-11 are isolated from Yaobitong Capsule for the first time, and organic acids and senkyunolide compounds may be the anti-inflammatory pharmacodynamic material bases of Yaobitong Capsule.

Identification of metabolites of Yaobitong Capsule in rat urine and bile after intragastric administration / 中草药

Objective The main metabolites of Yaobitong Capsule (YC) in rat urine and bile were investigated by UPLC-Q-TOF- MS/MS, and their major metabolic pathways were summarized. Methods To compare with the control samples, the possible metabolites were found. Their structures were speculated by exact mass and fragment ions. Results YC had 31 metabolites in urine and 35 metabolites in bile under positive and negative ion mode. They were mainly the metabolites of ligustilide, 3-butyl-phthalide, osthole, senkyunolide A, sankyunolide A, emodin, decursinol/marmesin/columbianetin, corypalmine/ isocorypalmine, corybulbine/isocorybulbine/coryphenanthrine, allocryptopine, ferulic acid, cryptochlorogenic acid, angelol and so on. Their major metabolic pathways included hydrolysis of the lactone ring, hydroxylation, demethylation, dehydration, dehydrogenation, dehydroxylation, hydrogenation, methylation, methyl oxidation, glucuronidation, sulphate-binding, N-acetylcysteine-binding, cysteine-binding, cysteinyl acid-glycine-binding, glutathione-binding and so on. In addition, three prototype components, namely paeoniflorin, albiflorin, and ginsenosides Rg1, were detected in rats’ bile. Conclusion The established UPLC-Q-TOF-MS/MS method can analyze the metabolites of YC capsule in rat urine and bile. The in vivo metabolic characteristics of YC have been initially clarified. The results provide a foundation for elucidating the in vivo efficacy ingredients of YC.

Effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38mark phosphorylation in autologous nucleus pulposus transplantation model of rats.

OBJECTIVE: To discuss the effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38MARK phosphorylation in autologous nucleus pulposus transplantation model of rats. METHODS: A total of 60 SD rats were randomly divided into the blank group, model group and Yaobitong capsule group, with 20 rats in each group. The animal model of autologous nucleus pulposus transplantation around the lumbar nerve root was built. Three days after the modeling, rats were given the drugs for the first time, while rats in the model group were given the equivalent normal saline. After 30 d of continuous administration, samples were collected from rats. HE staining was performed on the dorsal root ganglion of L4 and L5 spinal cord of rats in each group and the expression of p38MARK phosphorylation was measured. All data were treated with the statistical analysis. RESULTS: The histological examination showed that the histomorphology of dorsal root ganglion in the Yaobitong capsule group was more significantly improved than the one in the model group, while the results of western blot showed that Yaobitong capsule could significantly inhibit the level of p38MAPK phosphorylation of dorsal root ganglion cells. CONCLUSIONS: Yaobitong capsule can improve the symptoms and nerve radiculopathy of autologous nucleus pulposus transplantation of rats and its mechanism may be associated with its inhibiting effect on the level of p38MAPK phosphorylation.

Effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38mark phosphorylation in autologous nucleus pulposus transplantation model of rats

Objective: To discuss the effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38MARK phosphorylation in autologous nucleus pulposus transplantation model of rats. Methods: A total of 60 SD rats were randomly divided into the blank group, model group and Yaobitong capsule group, with 20 rats in each group. The animal model of autologous nucleus pulposus transplantation around the lumbar nerve root was built. Three days after the modeling, rats were given the drugs for the first time, while rats in the model group were given the equivalent normal saline. After 30 d of continuous administration, samples were collected from rats. HE staining was performed on the dorsal root ganglion of L4 and L5 spinal cord of rats in each group and the expression of p38MARK phosphorylation was measured. All data were treated with the statistical analysis. Results: The histological examination showed that the histomorphology of dorsal root ganglion in the Yaobitong capsule group was more significantly improved than the one in the model group, while the results of western blot showed that Yaobitong capsule could significantly inhibit the level of p38MAPK phosphorylation of dorsal root ganglion cells. Conclusions: Yaobitong capsule can improve the symptoms and nerve radiculopathy of autologous nucleus pulposus transplantation of rats and its mechanism may be associated with its inhibiting effect on the level of p38MAPK phosphorylation.

Effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38mark phosphorylation in autologous nucleus pulposus transplantation model of rats

OBJECTIVE@#To discuss the effect of Yaobitong capsule on histomorphology of dorsal root ganglion and on expression of p38MARK phosphorylation in autologous nucleus pulposus transplantation model of rats.@*METHODS@#A total of 60 SD rats were randomly divided into the blank group, model group and Yaobitong capsule group, with 20 rats in each group. The animal model of autologous nucleus pulposus transplantation around the lumbar nerve root was built. Three days after the modeling, rats were given the drugs for the first time, while rats in the model group were given the equivalent normal saline. After 30 d of continuous administration, samples were collected from rats. HE staining was performed on the dorsal root ganglion of L4 and L5 spinal cord of rats in each group and the expression of p38MARK phosphorylation was measured. All data were treated with the statistical analysis.@*RESULTS@#The histological examination showed that the histomorphology of dorsal root ganglion in the Yaobitong capsule group was more significantly improved than the one in the model group, while the results of western blot showed that Yaobitong capsule could significantly inhibit the level of p38MAPK phosphorylation of dorsal root ganglion cells.@*CONCLUSIONS@#Yaobitong capsule can improve the symptoms and nerve radiculopathy of autologous nucleus pulposus transplantation of rats and its mechanism may be associated with its inhibiting effect on the level of p38MAPK phosphorylation.