Cimicifugae Rhizoma Research Articles

Role and mechanism of Actein on condylar bone metabolism in APOE deletion-induced osteoporotic mice

AimsTo investigate the effects of Actein from Cimicifugae Rhizoma on condylar bone and cartilage in APOE deletion-induced osteoporotic mice, and to preliminarily explore the underlying mechanism. MethodsSixty 8-week-old female mice were used, which underwent APOE−/− and ovariectomy procedures, followed by oral administration of Actein (15 mg/kg) and Atorvastatin Calcium (AC, 3 mg/kg) for eight weeks. Body weight, uterine weight, and systemic indexes related to bone metabolism and lipid metabolism were assessed in each group. Changes in condylar bone histomorphometric parameters were evaluated using Micro-CT. Morphological changes in the condyle were observed with Hematoxylin-Eosin (H&E), Safranin O/Fast Green, and Alcian Blue Hematoxylin/Orange G (ABH/OG) staining, with OARSI pathology scoring performed. Sirius red staining and immunofluorescence were used to determine the expression levels of Collagen I (Col I) and Collagen III (Col III) in bone matrix, and Col II in cartilage matrix. Immunohistochemistry assessed the relative expression levels of ALP and proteins associated with the Wnt/β-catenin/RUNX2 signaling pathway. ResultsAPOE−/− exacerbates ovariectomy -induced osteoporosis (OP) in condylar of mice. Actein and AC significantly reversed OP, improved bone mineral density (BMD), increased bone microarchitecture, and restored abnormal calcium and phosphorus metabolism in the blood and urine. Morphologically, APOE−/− and ovariectomy reduced condylar cartilage thickness, disrupted chondrocyte arrangement, chondrocyte cleavage, and clustered aggregation, resembling osteoarthritis (OA)-like changes. Actein and AC partially restored the disrupted chondrocyte arrangement, smoothed chondrocyte cleavage, and up-regulated the levels of chondrocyte matrix (Col II, aggrecan) and bone matrix (Col III, ALP). Actein reversed the OA process, potentially through the Wnt/β-catenin/RUNX2 signaling pathway. ConclusionAPOE−/− and ovariectomy induced OP, leading to OA-like lesions in condylar of mice. Actein promoted cartilage repair and trabecular bone recovery by increasing extracellular matrix synthesis (Col II, Col III, aggrecan), reversing the OA process, possibly through the Wnt/β-catenin/RUNX2 signaling pathway.

Phytochemical Identification and Anti-Oxidative Stress Effects Study of Cimicifugae Rhizoma Extract and Its Major Component Isoferulic Acid

Background and Objectives: Cimicifugae Rhizoma, also known as ‘Sheng ma’ in Madeiran, is a widely used Chinese herbal medicine that has several pharmacological qualities, one of which is its antioxidant activity. Isoferulic acid, a prominent phenolic compound found in Cimicifugae Rhizoma, has potent antioxidant properties. This study was aimed to comprehensively analyze the components in Cimicifugae Rhizoma and rat plasma to evaluate the in vitro antioxidant and anti-inflammatory properties of Cimicifugae Rhizoma extract and Isoferulic acid as potential candidates for developing herbal formulations targeting podocyte injury in diabetic nephropathy for further clinical utilization. Materials and Methods: UPLC/Q-TOF-MS and HPLC were utilized as analytical tools to identify components of Cimicifugae Rhizoma extract or rat plasma after administrating it. MPC5 cells were cultured with H2O2 and high glucose and subjected to oxidative stress injury. The CXCL12/CXCR4 system plays a crucial role at certain stages of multiple kidney diseases’ injury. Apoptosis-related and target CXCL12/CXCR4/mTOR/Caspase-3 and Cask protein levels were assessed, and the levels of inflammatory-related factors, motility, morphology, ROS level, and apoptosis in podocytes were tested. Results: A total of 82 and 39 components were identified in the Cimicifugae Rhizoma extract and plasma, and Isoferulic acid content was determined as 6.52 mg/g in the Cimicifugae Rhizoma extract. The Cimicifugae Rhizoma extract (1 μg/mL) and Isoferulic acid (10, 25, 50 μM) considerably decreased high glucose and oxidative-stress-mediated toxicity, impaired mobility and adhesion and apoptotic changes in MPC5 cells, and reversed inflammation response. Moreover, the Cimicifugae Rhizoma extract and Isoferulic acid down-regulated Cask, mTOR, and Caspase-3, while significantly blocking the overactivation of CXCL12/CXCR4 in podocytes stimulated by oxidative stress and high glucose. Conclusions: These results indicate that the renal protective mechanism of the Cimicifugae Rhizoma extract and Isoferulic acid on simulating H2O2-induced podocyte injury involves mainly the of CXCL12/CXCR4 pathways and the inactivation of oxidative-stress-mediated apoptotic pathways after comprehensive qualitative and quantitative research by UPLC/Q-TOF-MS and HPLC. These findings provide an important efficacy and ingredient basis for further study on the clinical utilities of Cimicifugae Rhizoma and Isoferulic acid on podocyte and kidney impairment.

Study on the immune-enhancing and inhabiting transmissible gastroenteritis virus effects of polysaccharides from Cimicifuga rhizoma

Cimicifugae rhizoma is a traditional Chinese herbal medicine in China, and modern pharmacological research showed that it has obvious antiviral activity. Many polysaccharides have been proved to have immune enhancement and antiviral activity, but there are few studies on the biological activity of Cimicifuga rhizoma polysaccharide (CRP). The aim was to explore the character of CRP and its effects on improving immune activity and inhibiting transmissible gastroenteritis virus (TGEV). The monosaccharide composition, molecular weight, fourier transform infrared spectra and electron microscopy analysis of CRP was measured. The effect of CRP on immune activity in lymphocytes and RAW264.7 cells were studied by colorimetry, FITC-OVA fluorescent staining and ELISA. The effect of CRP on TGEV-infected PK-15 cells was determined using Real-time PCR, Hoechst fluorescence staining, trypan blue staining, acridine orange staining, Annexin V-FITC/PI fluorescent staining, DCFH-DA loading probe, and JC-1 staining. Network pharmacology was used to predict the targets of CRP in enhancing immunity and anti-TGEV, and molecular docking was used to further analyze the binding mode between CPR and core targets. The results showed that CRP was mainly composed of glucose and galactose, and its molecular weight was 64.28 kDa. The content of iNOS and NO in CRP group were significantly higher than the control group. CRP (125 and 62.5 μg/mL) could significantly enhance the phagocytic capacity of RAW264.7 cells, and imprive the content of IL-1β content compared with control group. 250 μg/mL of CRP possessed the significant inhibitory effect on TGEV, which could significantly reduce the apoptosis compared to TGVE group and inhibit the decrease in mitochondrial membrane potential compared to TGVE group. The mRNA expression of TGEV N gene in CRP groups was significantly lower than TGEV group. PPI showed that the core targets of immune-enhancing were AKT1, MMP9, HSP90AA1, etc., and the core targets of TGE were CASP3, MMP9, EGFR, etc. Molecular docking show that CRP has binding potential with target. These results indicated that CRP possessed the better immune enhancement effect and anti-TGEV activity.

A comprehensive strategy integrating metabolomics with DNA barcoding for discovery of combinatorial discriminatory quality markers: A case of Cimicifuga foetida and Cimicifuga dahurica

The multiple species characteristics of traditional Chinese medicines (TCMs) are crucial for expanding TCMs sources, meeting the needs of the pharmaceutical industry and ensuring clinical requirements. It’s also one of the significant factors affecting the quality control of TCMs. Systematic differential analysis of original species in TCMs is an important link in achieving comprehensive quality control, ensuring the effectiveness and safety of clinical medication. The study aims to establish a reliable and efficient approach to screen combinatorial discriminatory quality markers for rapid differentiation of original species by metabolomics coupled with DNA barcoding as a case of Cimicifugae Rhizoma. DNA barcoding is used to identify the origin of Cimicifugae Rhizoma. The data-dependent acquisition mode integrated with the computerized intelligent filtering system was established for in-depth characterization of metabolites from Cimicifugae Rhizoma using ultra-high performance liquid chromatography to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). The untargeted metabolomics combined with multivariate statistical analysis was performed to screen and identify the potential combinatorial discriminatory quality markers. Finally, quantitative analysis and predictive model of these markers were employed to validate the feasibility of this strategy to distinguish the original species. Based on the scores of variable importance in projection greater than 1.0 and t-test (p < 0.05) in chemometric analysis, caffeic acid, cimifugin, ferulic acid and isoferulic acid were authenticated as combinatorial discriminatory quality markers for the two original species of Cimicifugae Rhizoma. In addition, the Fisher discriminant model successfully classified 56 batches of Cimicifugae Rhizoma with an accuracy of 94.4 %, showcased the practicality and scientific validity of this method. This study has provided a comprehensive strategy for efficient discrimination of multiple species of medicinal materials.

A comprehensive quality evaluation of Cimicifugae Rhizoma using UPLC-Q-Orbitrap-MS/MS coupled with multivariate chemometric methods.

Cimicifugae Rhizoma, known in Chinese as Shengma, is a common medicinal material in traditional Chinese medicine (TCM), mainly used for treating wind-heat headaches, sore throat, uterine prolapse, and other diseases. An approach using a combination of ultra-performance liquid chromatography (UPLC), mass spectrometry (MS), and multivariate chemometric methods was designed to assess the quality of Cimicifugae Rhizoma. All materials were crushed into powder and the powdered sample was dissolved in 70% aqueous methanol for sonicating. Chemometric methods, including hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA), were adopted to classify and perform a comprehensive visualization study of Cimicifugae Rhizoma. The unsupervised recognition models of HCA and PCA obtained a preliminary classification and provided a basis for classification. In addition, we constructed a supervised OPLS-DA model and established a prediction set to further validate the explanatory power of the model for the variables and unknown samples. Exploratory work research found that the samples were divided into two groups, and the differences were related to appearance traits. The correct classification of the prediction set also demonstrates a strong predictive ability of the models for new samples. Subsequently, six chemical makers were characterized by UPLC-Q-Orbitrap-MS/MS, and the content of four components was determined. The results of the content determination revealed the distribution of representative chemical markers caffeic acid, ferulic acid, isoferulic acid, and cimifugin in two classes of samples. This strategy can provide a reference for assessing the quality of Cimicifugae Rhizoma, which is significant for the clinical practice and quality control of Cimicifugae Rhizoma.

Combining network pharmacology, RNA-seq, and metabolomics strategies to reveal the mechanism of Cimicifugae Rhizoma - Smilax glabra Roxb herb pair for the treatment of psoriasis

BackgroundPsoriasis is a prevalent chronic inflammatory skin condition marked by immune cell infiltration and keratinocyte abnormal proliferation. Cimicifugae Rhizoma - Smilax glabra Roxb (CS) herb pair, the main component of Shengma Detoxification Decoction, has been proven effective for the treatment of psoriasis. However, the mechanism is yet to be deciphered. PurposeTo explore the mechanism of CS for the treatment of psoriasis. MethodsThe imiquimod-induced psoriasis-like lesion mouse model was used to identify the targets and the molecular mechanisms of CS. Network pharmacology combined with RNA-seq strategy was employed to predict the targets and mechanisms of CS for psoriasis. Metabolomics approaches were used to demonstrate the complexity of CS for the treatment of psoriasis. Finally, a compound-response-enzyme-gene network was constructed based on the multi-omics results to elucidate potential connections. ResultsThe CS herb pair could significantly improve psoriatic lesions and reduce the inflammatory cell infiltration and proliferation of keratinocytes in skin lesions. Network pharmacology predicted that TNF, JNK, IL-6, and IL-1β could be potential targets. RNA-seq data revealed that CS could significantly regulate genes and signaling pathways associated with Th17 responses, such as IL-36, IL-1β, CCl2, CXCL16, keratin 14, keratin 5, and antimicrobial peptides S100A8 and S100A9 well as MAPK, mTOR, and other signaling pathways. Further experimental data validated that CS treatment remarkably reduced the expression of inflammatory cytokines and factors, such as CCL2, CCL7, IL1F6, IL-17, IL-23, IL-1β, TNF-α, and IL-6, and inhibited the phosphorylation of p38 and ERK1/2. This indicated that CS exerts its therapeutic effect by inhibiting the MAPK signaling pathways. In addition, metabolomic analyses demonstrated that CS treatment improved seven metabolic pathways, these included phenylalanine, tyrosine, pyruvate metabolism, carnitine metabolism, etc. Four key metabolites (L-Arginine, L-Phenylalanine, L-Carnitine, O-Acetylcarnitine) and nine differential genes (CMA1, PCBD2, TPSAB1, TPSB2, etc.) were identified that affected amino acid metabolism, carnitine metabolism, and other pathways contributing to the infiltration of Th17 cells in psoriatic lesions. ConclusionCS could alleviate IMQ-induced psoriasis-like dermatitis by reducing the expression of cytokines and chemokines mediated by the MAPK pathway, and improved amino acid and carnitine metabolism in vivo. Our study is the first to demonstrate the complex mechanism of CS for the treatment of psoriasis and provides a new paradigm to elucidate the pharmacological effects of Traditional Chinese Medicine (TCM) drugs for psoriasis from multiple perspectives.

Secondary Metabolites with Anti-Inflammatory from the Roots of Cimicifuga taiwanensis.

The genus Cimicifuga is one of the smallest genera in the family Ranunculaceae. Cimicifugae Rhizoma originated from rhizomes of Cimicifuga simplex, and C. dahurica, C. racemosa, C. foetida, and C. heracleifolia have been used as anti-inflammatory, analgesic and antipyretic remedies in Chinese traditional medicine. Inflammation is related to many diseases. Cimicifuga taiwanensis was often used in folk therapy in Taiwan for inflammation. Phytochemical investigation and chromatographic separation of extracts from the roots of Cimicifuga taiwanensis has led to the isolation of six new compounds: cimicitaiwanins A–F (1–6, respectively). The structures of the new compounds were unambiguously elucidated on the basis of extensive spectroscopic data analysis (1D- and 2D-NMR, MS, and UV) and comparison with the literature data. The effect of some isolates on the inhibition of NO production in lipopolysaccharide-activated RAW 264.7 murine macrophages was evaluated. Of the isolates, 3–6 exhibited potent anti-NO production activity, with IC50 values ranging from 6.54 to 24.58 μM, respectively, compared with that of quercetin, an iNOS inhibitor with an IC50 value of 34.58 μM. This is the first report on metabolite from the endemic Taiwanese plant-C. taiwanensis.

Evaluation of the effect of Shengxian Decoction on doxorubicin-induced chronic heart failure model rats and a multicomponent comparative pharmacokinetic study after oral administration in normal and model rats

Shengxian Decotion (SXT), a well-known Traditional Chinese Medicine (TCM) formula composed of Astragali Radix, Bupleuri Radix, Cimicifugae Rhizoma, Anemarrhenae Rhizoma and Platycodonis Radix, is clinically considered as an effective formula against cardiovascular diseases. However, the exact effective substance of SXT in treating chronic heart failure (CHF) still remains unclear. In the current study, we investigated the benefit of SXT in doxorubicin (DOX)-induced CHF rats and established a UHPLC-MS/MS method to simultaneously determine 18 key compounds in a subsequent comparative pharmacokinetic study in normal and CHF rats. Histopathological studies, transmission electron microscopy, and echocardiography were applied to assess the therapeutic effect of SXT on DOX-induced CHF rats, which indicated that SXT significantly ameliorated DOX-induced CHF, similar to enalapril. In addition, we successfully established a UHPLC-MS/MS method to determine the pharmacokinetics of the components in rat plasma, which was validated with good linearity, inter-day and intra-day precisions and accuracies, matrix effects, extraction recovery, and stability values. Our results showed that only astragaloside IV showed increased plasma exposure in the CHF rats, while saikosaponin A, quercetin, timosaponin B-II, ferulic acid, isoferulic acid and formononetin decreased compared to their pharmacokinetic characteristics in the normal and CHF rats. This study demonstrates that SXT enjoys obvious therapeutic effect on DOX-induced CHF rats, and the altered metabolism of some compounds in SXT is affected by the pathological state of CHF rats. Our findings provide a better understanding of the in vivo exposure to complex compounds of SXT, supporting effective substance screening and further investigation of the therapeutic mechanism.

Cimicifugae Rhizoma Extract Attenuates Oxidative Stress and Airway Inflammation via the Upregulation of Nrf2/HO-1/NQO1 and Downregulation of NF-κB Phosphorylation in Ovalbumin-Induced Asthma

Cimicifugae Rhizoma has been used as a medicinal herb for fever, pain, and inflammation in East Asia. We conducted this study because the effect of Cimicifugae Rhizoma extract (CRE) on allergic asthma has not yet been evaluated. To induce allergic airway inflammation, we intraperitoneally injected ovalbumin (OVA) mixed with aluminum hydroxide into mice twice at intervals of 2 weeks (Days 0 and 14) and then inhaled them thrice with 1% OVA solution using a nebulizer (Days 21 to 23). CRE (30 and 100 mg/kg) was administered orally daily for 6 days (Days 18 to 23). The mice showed remarkable reduction in allergic inflammation at 100 mg/kg of CRE, as evidenced by decreased inflammatory cell counts, pro-inflammatory cytokine levels, OVA-specific immunoglobulin E level, airway hyperresponsiveness, and production of mucus. Additionally, these effects were involved with the enhancement of heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase (NQO1), and nuclear factor erythroid 2-related factor 2 (Nrf2) expression and reduction of nuclear factor-κB (NF-κB) phosphorylation and matrix metalloproteinase-9 expression. Our findings indicated that CRE effectively protected against OVA-induced inflammation and oxidative stress via upregulation of the Nrf2/HO-1/NQO1 signaling and downregulation of NF-κB phosphorylation in asthma caused by OVA.

Use of an electrophysiological technique for stepwise detection of trace agonist constituents of Hochuekkito in Xenopus oocytes injected with serotonin 2C receptor mRNA.

An electrophysiological bioassay was used to isolate the active compound from Hochuekkito (HET), which the current authors previously described as having potent agonist action against serotonin 2C receptors (5-HT2CR). Synthetic 5-HT2CR mRNA was injected into Xenopus oocytes to specifically express these receptors. Crude extracts and purified products were subjected to an electrophysiological bioassay using the voltage clamp method. HET stimulated a 5-HT2CR-induced current response, whereas Juzentaohoto (JTT), which has anti-depressive action similar to that of HET, did not. Current responses were not observed with an extract mixed with five types of herbal medicines common to HET and JTT but were detected with an extract with the five types of herbal medicines found in HET alone (Hoc5). When the responses to each of the five types of Hoc5 were examined, current responses were noted with Cimicifugae rhizoma (CR) and Citrus unshiu Markovich extracts. Since efficacy and the EC50 value were higher for CR, its constituents were separated using three-dimensional high-performance liquid chromatography and the current response at each of the isolated peaks was examined. One constituent displayed a strong response and was identified as a single substance with a molecular weight of 283.1393 based on liquid chromatography/mass spectrometry. These results will contribute to the isolation of 5-HT2CR-stimulating constituents in HET and the identification of trace constituents with agonist action.

Effective components of Shengxian Decoction and its mechanism of action in treating chronic heart failure based on UHPLC-Q-TOF-MS integrated with network pharmacology

This study aimed to elucidate the effective components of Shengxian Decoction and its mechanism of action in treating chronic heart failure. Firstly, UHPLC-Q-TOF-MS was established to identify the main chemical constituents in the rat serum after intragastric administration with Shengxian Decoction. Secondly, the absorbed components in serum were then used for the network pharmacology analysis to infer the mechanism and effective components. Targets for constituents in serum were predicted at TCMSP and Swiss-TargetPrediction database. An association network map was drawn by network visualization software Cytoscape 3.6.1. Finally, GO enrichment analysis and KEGG pathway enrichment analysis were carried out for the core target genes. By UHPLC-Q-TOF-MS, 18 prototype compounds were definitely identified, including five compounds from Astragali Radix, four compounds from Anemarrhenae Rhizoma, four compounds from Bupleuri Radix, four compounds from Cimicifugae Rhizoma, and one compound from Platycodonis Radix. Those components of Shengxian Decoction were closely associated with 13 key protein targets, including inflammatory factors, like IL6, IL1 B, TNF, PTGS2, IL10; redox enzymes CAT, HMOX1, and MPO; cardiovascular targets, like VEGFA, NOS3, and NOS2; and transmememial proteins CAV1 and INS. Network pharmacology analysis showed that the 18 compounds could be responsible for the treatment of chronic heart failure by regulating HIF-1 signaling pathways, PI3 K-Akt signaling pathways, cGMP-PKG signaling pathways, cAMP signaling pathways and TNF signaling pathways. This study provided a scientific basis for mechanism and effective ingredients of Shengxian Decoction.

Profiling of 120 types of herbal extracts and their effects on morphology in cultured neuronal or glial cell lines, followed by RNA extraction for a cDNA library: Consideration for use in studies based on Kampo theories

ABSTRACTAimKampo medicine is composed of a variety of crude drugs. Each crude drug has its own effects, and the interaction of all drugs produces the therapeutic effect of Kampo medicine. In order to examine the characteristics of Kampo medicine and its crude drugs, 120 types of herbal medicines were characterized based on their three‐dimensional high‐performance liquid chromatography (3D‐HPLC) profiles as well as their effects on cell morphology/proliferation when added to cultured cells and gene expression changes such as on BNIP‐3 mRNA levels as an example.MethodsThe cultured neural cell line N18‐TG‐2 and glial cell line C6Bu‐1 were used in experiments. After the acquisition of 3D‐HPLC data, each crude drug was added to the cell culture medium at concentrations of 10 and 100 μg/mL, images were taken of morphological changes, and RNA was extracted to prepare a complementary DNA (cDNA) library.ResultsAll the results obtained from the treatments with 120 crude drugs are listed in the Supporting Information. Based on these data, as an example the expression of antidepressant‐related factor BNIP‐3, which we had previously found to increase in expression after treatment with Hochuekkito, was studied and the results obtained revealed that it was increased by Astragali Radix, Cimicifugae Rhizoma, Glycyrrhizae Radix and others.ConclusionThe present results, which were collected from observations under unified conditions, include information that will promote herbal medicine research. Future studies to examine the effects of crude drug combinations are also discussed, suggesting that the information files and cDNA library presented here can be the basis for many future integrated studies such as new Wakan‐yaku prescription developments.

Analysis on quality value transfer of substance benchmarks of Qingwei San

By preparing 10 batches of substance benchmarks freeze-drying powder( lyophilized powder),the methodology of the characteristic spectrum and the content of index component for substance benchmarks of Qingwei San was established. The characteristic peaks and the similarity range of the characteristic spectrum,the contents and the transfer rate range of isoferulic acid,palmatine and paeonol,and the paste-forming rate range were determined to define key quality attributes of substance benchmarks of Qingwei San. In the10 batches of substance benchmarks of Qingwei San,the similarity of characteristic spectrum was higher than 0. 90. In further comparison of the characteristic peak information,a total of 16 characteristic peaks were identified,including 5 characteristic peaks from Cimicifugae Rhizoma,5 characteristic peaks from Coptidis Rhizoma,2 characteristic peaks from Angelicae Sinensis Radix and 4 characteristic peaks from Moutan Cortex. The content of isoferulic acid was 0. 10%-0. 18%,with the average transfer rate of 49. 82%±4. 02%. The content of palmatine was 0. 17%-0. 31%,with the average transfer rate of 15. 84% ±2. 39%. The content of paeonol was 0. 41%-0. 75%,with the average transfer rate of 23. 41%±3. 23%. The paste-forming rate of the 10 batches of substance benchmarks were controlled at 27%-33%,with the transfer rate between the theoretical paste-forming rate and the actual paste-forming rate was 86. 59%±3. 39%. In this study,the quality value transfer of substance benchmarks of Qingwei San was analyzed by the combination of characteristic spectrum,the content of index component and the paste-forming rate. A scientific and stable evaluation method was preliminarily established,so as to provide the basis for subsequent development and quality control of relevant preparations of Qingwei San.

Regularity of Wind-dispelling Medication Prescribed by LI Dong-Yuan: A Data Mining Technology-based Study

ObjectiveTo extract and analyze information on the regularity with which wind-dispelling medication was prescribed by LI Dong-Yuan and many doctors of his school of thought and to provide theoretical basis and ideas for modern clinical application to facilitate the use, research, and development of these medications. MethodsOriginal data on wind-dispelling medication described in LI Dong-Yuan’s works were collected, sorted and organized. Data mining and comprehensive analysis were performed by using a series of data processing softwares, such as SPSS 22.0, Modeler 18.0 and xMiner. By reviewing LI Dong-Yuan's writings on wind-dispelling medication, the number of prescriptions for wind-dispelling medication were counted. Frequency of use of wind-dispelling medication was determined, and association rules analysis, factor analysis, and core drug network analysis were used to reveal associations of the symptoms of syndromes and treatment with wind-dispelling medication and to reveal the regularity with which these medications were prescribed by LI Dong-Yuan. ResultsA total of 356 prescriptions and 121 flavors of wind-dispelling medication were observed. Among them, five kinds of medicinal properties, seven kinds of medicinal tastes, and 12 kinds of meridians were identified. Furthermore, herbs were divided into 18 categories according to their efficacy. Statistical analysis showed that there were 23 wind-dispelling medications that were prescribed with a frequency of ≥ 60. The mainly used drugs were Saposhnikoviae Radix (Fang Feng, 防风), Bupleuri Radix (Chai Hu, 柴胡), Cimicifugae Rhizoma (Sheng Ma, 升麻), Notopterygii Rhizoma Et Radix (Qiang Huo, 羌活), Puerariae Lobatae Radix (Ge Gen, 葛根), Chuanxiong Rhizoma (Chuan Xiong, 川芎), and Ephedrae Herba (Ma Huang, 麻黄). In addition, we analyzed the association rules wind-dispelling medication and obtained 23 one-to-one drug association rules and 11 multiple-to-one drug association rules. The core drug network analysis data visualization showed core drugs used in the treatment of external wind assailing the exterior (wind-cold, wind-heat, wind-dampness, etc.), wind-stroke, and tonifying middle and replenishing Qi. Factor analysis was performed on the drugs used by LI Dong-Yuan, and finally 4 groups of wind medicine combinations were obtained. ConclusionsThis data mining-based study on the regularity of wind-dispelling medication described in LI Dong-Yuan's works is of great significance, because it reveals the clinical application of its theoretical formulae and medications.

Comparative Analysis of Actaea Chloroplast Genomes and Molecular Marker Development for the Identification of Authentic Cimicifugae Rhizoma.

Actaea (Ranunculaceae; syn. Cimicifuga) is a controversial and complex genus. Dried rhizomes of Actaea species are used as Korean traditional herbal medicine. Although Actaea species are valuable, given their taxonomic classification and medicinal properties, sequence information of Actaea species is limited. In this study, we determined the complete chloroplast (cp) genome sequences of three Actaea species, including A. simplex, A. dahurica, and A. biternata. The cp genomes of these species varied in length from 159,523 to 159,789 bp and contained 112 unique functional genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Gene order, orientation, and content were well conserved in the three cp genomes. Comparative sequence analysis revealed the presence of hotspots, including ndhC-trnV-UAC, in Actaea cp genomes. High-resolution phylogenetic relationships were established among Actaea species based on cp genome sequences. Actaea species were clustered into each Actaea section, consistent with the Angiosperm Phylogeny Group (APG) IV system of classification. We also developed a novel indel marker, based on copy number variation of tandem repeats, to facilitate the authentication of the herbal medicine Cimicifugae Rhizoma. The availability Actaea cp genomes will provide abundant information for the taxonomic and phylogenetic analyses of Actaea species, and the Actaea (ACT) indel marker will be useful for the authentication of the herbal medicine.

Natural Compound Mixture, Containing Emodin, Genipin, Chlorogenic Acid, Cimigenoside, and Ginsenoside Rb1, Ameliorates Psoriasis-Like Skin Lesions by Suppressing Inflammation and Proliferation in Keratinocytes.

Herbal combinations of Rhei Radix et Rhizoma, Gardeniae Fructus, Cimicifugae Rhizoma, and Ginseng Radix have been used in traditional formulas to treat the symptoms of heat and dryness. This study investigated the therapeutic effects of a natural compound mixture (PSM) of these herbal combinations, containing emodin, genipin, chlorogenic acid, cimigenoside, and ginsenoside Rb1, for the treatment of psoriasis and its underlying molecular mechanisms. PSM was applied topically to the dorsal skin lesions of imiquimod- (IMQ-) induced C57BL/6 mice, and the expression of the proinflammatory mediators was investigated. The topical application of 1% PSM reduced psoriasis-like symptoms in IMQ-induced C57BL/6 mice significantly. PSM also attenuated the production of IFN-γ, IL-1β, and IL-6 in skin lesions. Histological analysis showed that PSM had antipsoriatic effects by reducing the lesional epidermal thickness. Either M5 (IL-1α, IL-17A, IL-22, oncostatin M, and TNF-α, 10 ng/ml each) or IL-22- (100 ng/ml) stimulated HaCaT cells were used to examine the efficacy and underlying mechanism of PSM. In M5-stimulated HaCaT cells, PSM inhibited the production of C-X-C motif chemokine ligand (CXCL) 10 and C-C motif chemokine ligand (CCL) 20 effectively. Moreover, compared to the use of a single compound, it had synergistic inhibitory effects in CXCL8 production. PSM suppressed the phosphorylation of ERK1/2, p38, and STAT3 signaling pathways in M5-stimulated HaCaT cells. Furthermore, PSM reduced the proliferation rate and K16 and K17 expressions in IL-22-stimulated HaCaT cells by inhibiting the Akt/mTOR signaling pathway. These results suggest that PSM may have a therapeutic potential in the treatment of psoriasis lesions.

Simultaneous determination of 19 constituents in Cimicifugae Rhizoma by HPLC–DAD and screening for antioxidants through DPPH free radical scavenging assay

Cimicifugae Rhizoma (sheng ma) is a well-known traditional Chinese medicine, which has been demonstrated to possess anti-inflammatory, antipyretic and analgesic activities. In the present study, a simple and efficient HPLC-DAD (high-performance liquid chromatography coupled with diode array detection) method was developed and validated for simultaneous quantification of 19 chemical components (including 16 phenolic acids, one coumarin and two alkaloids) in Cimicifugae Rhizoma. The result indicated that this method could effectively evaluate the quality of Cimicifugae Rhizoma and provide a valuable reference for further study. Additionally, the antioxidant activity of Cimicifugae Rhizoma was evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay. The results showed that the content of phenolic acids and antioxidant activity exhibited significant correlation coefficients.

Abstract 4280: The cycloartane triterpenoid ADCX impairs autophagic degradation through Akt overactivation and promotes apoptotic cell death in multidrug-resistant HepG2/ADM cells

Abstract Multidrug resistance is the main obstacle in cancer chemotherapy. Emerging evidence has demonstrated the important role of autophagy in cancer cell resistance to chemotherapy. Therefore, autophagy inhibition may be a promising strategy for conquering drug resistance in liver cancer cells. Methods: HepG2/ADM cells were treated with ADCX in the presence or absence of relevant inhibitors, apoptosis was measured by flow-cytometry and expression of autophagy and apoptosis related proteins was detected by western blotting. Confocal microscope was used for GFP-LC3 assay, tandem mRFP-GFP-LC3 assay, DQ-BSA method and colocalization of GFP-LC3 and LAMP1. Celluar ultrastructure was observed by transmission electronic microscopy. Results: ADCX, a natural cycloartane triterpenoid extracted from the traditional Chinese medicine (TCM) source Cimicifugae Rhizoma (Shengma), inhibited autophagic degradation by impairing the maturation of lysosomal cathepsins in multidrug resistant liver cancer HepG2/ADM cells, thereby leading to autophagic flux inhibition. Interestingly, Akt was over-activated by ADCX treatment, which was associated with impairment of cathepsin maturation. Moreover, impairing autophagic degradation promoted ADCX-induced apoptotic cell death in HepG2/ADM cells. Conclusions: Our study indicates that impairing autophagic degradation may be an effective approach for overcoming resistance and also suggests a novel role for Akt in autophagic degradation. Citation Format: NAN YAO, Haiyan Sun, Maohua Huang, Wencai Ye, SiBao Chen, Dong-Mei Zhang. The cycloartane triterpenoid ADCX impairs autophagic degradation through Akt overactivation and promotes apoptotic cell death in multidrug-resistant HepG2/ADM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4280.

In vivo metabolic profiles of Bu-Zhong-Yi-Qi-Tang, a famous traditional Chinese medicine prescription, in rats by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been extensively used for conditioning sub-health status and diseases caused by spleen-qi deficiency in China for over 700 years. BZYQT is prevalent not only in China, but also in Japan and South Korea for the clinical treatment of chronic diseases, such as fatigue, tuberculosis and loss of appetite after surgery. However, due to a lack of research on the holistic metabolism of BZYQT, the in vivo bioactive components of BZYQT remain unclear, hindering further study of its in vivo mechanism of action and quality control. In the present study, a four-step integrated strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) was established to systematically screen the in vivo xenobiotics of BZYQT. Ultimately, a total of 162 xenobiotics (59 prototypes and 103 metabolites) were identified or tentatively characterized, including 48 in plasma, 147 in urine and 58 in feces, while the in vivo metabolic profile of atractylenolide III (a major component of BZYQT) was elucidated for the first time. The xenobiotics of BZYQT mainly included flavonoids from Astragali Radix, Glycyrrhizae Radix et Rhizoma and Citrus reticulatae Pericarpium; lactones from Angelicae Sinensis Radix and Atractylodis Macrocephalae Rhizoma; and triterpenoid saponins from Cimicifugae Rhizoma. After oral administration, BZYQT-related components underwent diverse metabolic pathways. Among them, flavonoids mainly underwent glucuronidation, sulfation and demethylation, while lactones mainly underwent hydroxylation and acetylcysteine conjugation, and deglycosylation was the major metabolic reaction of saponins. Our investigation gives a comprehensive analysis of the metabolic characteristics of BZYQT and will provide an important basis for further studying the pharmacokinetics of BZYQT to explore its in vivo disposal features and discover its in vivo bioactive components.

Mucosal immunomodulatory evaluation and chemical profile elucidation of a classical traditional Chinese formula, Bu-Zhong-Yi-Qi-Tang

Ethnopharmacological evidenceWith fast development and high pace life in modern society, autoimmune diseases like inflammatory bowel disease had become increasingly common. Bu-Zhong-Yi-Qi-Tang (BZYQT), a famous traditional Chinese medicine prescription (TCMP), has been used for 700 years mainly in Eastern Asia countries for the treatment of gastrointestinal and respiratory disorder, and weakness after serious diseases. These diseases were proved to be closely related to human immune system, among which, mucosal immune system is the largest immune system. So it is necessary to discover the mucosal immune related bioactive components of BZYQT. Aim of the studyTo evaluate the mucosal immunomodulatory bioactivity of BZYQT and ingredients. Materials and methodsPeyer's patches were collected from mice administrated orally with BZYQT, its related Octadecylsilane (ODS) fractions and polysaccharide part. Productions of several cytokines including IL-2, IL-4, IL-5, and IFN-γ from T lymphocytes were tested with enzyme linked immunosorbent assay (ELISA) by Peyer's patch cells ex vivo experiments. Chemical profile including low molecular part and polysaccharide part were investigated. Low molecular part of BZYQT and related ODS fractions were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on LC-MS information from self-established compound library. exclusion chromatography, and chemical property has been analyzed. ResultThree-days’ administration of BZYQT enhanced productions of IL-4 and IFN-γ in T lymphocytes of Peyer's patches in addition to IL-2. Some hydrophobic low molecular weight fractions (30%, 70% and 100% MeOH ODS fraction), which were fractionated from BZYQT by ODS column chromatography, showed enhancing or suppressive effects on productions of IL-2, IL-4 or IL-5 in T lymphocytes of Peyer's patches after oral administration. Besides, 161 components from hydrophobic low molecular weight fractions of BZYQT were unequivocally identified or tentatively characterized by UPLC-Q/TOF-MS according to retention time behaviors and fragments, and most of them were flavonoids and saponins from Glycyrrhizae Radix, Citri Reticulatae Pericarpium, and Cimicifugae Rhizoma. Polysaccharide part was separated and purified both by anion-exchange and size. BZYQT also contained at least one neutral and three weakly or strongly acidic polysaccharides, and analysis of their chemical properties indicated that a neutral polysaccharide was glucan, and acidic polysaccharides possessed heteroglycan and pectic arabinogalactan features. Murine administration of polysaccharide fractions of BZYQT induced different changes on functions of T lymphocytes in Peyer's patches from hydrophobic low molecular weight fractions. By experiment using intranasally-immunized mice, BZYQT negatively regulated antibody response in lung as combinatorial actions of its low molecular weight ingredients and polysaccharides. ConclusionBZYQT contains several low and macromolecular weight ingredients, which affect to immune-function of T lymphocytes in Peyer's patches, and the formula expresses its regulative activity on lower respiratory immune system through combinatorial actions of these ingredients on immunocompetent cells in Peyer's patches.