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Ethnopharmacological relevance Radix Rehmanniae (RR) is the tuber root of Rehmannia glutionsa Libosch, which was firstly recorded in Shennong's Classic of Materia Medica (⟪神农本草经⟫). RR is a non-toxic and wide used traditional Chinese medicine. RR has the effect of clearing heat, generating essence, cooling blood, stopping bleeding, nourishing yin and blood, and filling marrow. It is used in clinic in the form of processed decoction pieces, including Dry Radix Rehmnniae (DRR) and Rehmanniae Radix Praeparata (RRP). The application of RR in traditional Chinese medicine (TCM) prescriptions can treat various diseases, such as anemia, irregular menstruation, deficiency of liver yin, renal failure and so on. Aim of review This paper aims to provide a comprehensive and productive review of RR, which mainly contains botanical characteristics, processing methods, traditional application, chemical composition, quality control and pharmacological action. Materials and methods Literature search was conducted through the Web of Science, Baidu Scholar, ScienceDirect, PubMed, CNKI, and WanFang DATA using the keywords “Radix Rehmnniae”, “Rehmanniae Radix Praeparata”, “processing”, “clinical application”, “chemical composition”, “quality control”, and “pharmacological action”. In addition, information was collected from relevant textbooks, reviews, and documents. Results RR is a traditional Chinese herbal medicine with clinical value and rich resources. More than 100 components have been isolated and identified from RR. It has multiple pharmacological actions, such as hemostasis, antioxidation, anti-osteoporosis, lowering blood sugar, improving renal function, anti-inflammation, protecting neuronal function, antidepression and anti-anxiety. DRR and RRP are two different processed products of RR. After processing, there are great changes in property, taste, efficacy, clinical application, chemical composition and pharmacological action. At present, identifying chemical constituents of RR and its medicinal value has been deeply studied. However, there is a lack of research on the reasons for the differences in pharmacological effects between DRR and RRP. The reasons for these differences need to be further verified. Catalpol, the active component of RR, has been studied extensively in the literature, but the pharmacological effects of catalpol cannot represent the pharmacological effects of the whole RR. In the future, effective components such as rehmannioside D, polysaccharide, total glycosides, and effective parts in RR need to be further studied and developed. The pharmacodynamic material basis and mechanism of RR need to be further discussed. The scientific connotation and processing methods of RRP need to be studied and standardized.

Ethnopharmacological relevance Curcumae Radix (Yujin) has a long medicinal use history in China, which is used to cure diseases like jaundice, cholelithiasis caused by dampness-heat of gallbladder and liver, and so on. It comes from the dried tuberous roots of C. kwangsiensis (Guiyujin), C. longa (Huangyujin), C. phaeocaulis (Lvyujin) and C. wenyujin (Wenyujin). Though there are differences in chemical compositions and pharmacological activities among the four species of Yujin, they have not been differentiated well in clinical application due to their similar morphological characterizations. Aim of the study In this study, the four species of Yujin were rapidly and accurately discriminated. The potential volatile markers for varietal recognition were identified. Materials and methods Attenuated total reflection fourier transformed infrared (ATR-FTIR) spectroscopy combined with chemometrics was used to rapidly discriminate the four species of Yujin. Headspace-gas chromatography-mass spectrometry (HS-GC-MS) technology coupled with chemometrics was employed to characterize volatile profiling, differentiate species and select potential markers for varietal recognition of Yujin. Results By applying PCA (principal components analysis) and HCA (hierarchical cluster analysis), HS-GC-MS realized complete differentiation of the four species of Yujin, while ATR-FTIR only recognized Guiyuijin. Back propagation neural network (BP-NN), KNN (K-nearest neighbor) and LDA (linear discriminant analysis) models based on spectral data achieved 100% discriminant accuracies. Support vector machines (SVM), KNN and PLS-DA (partial least square discriminant analysis) models based on volatile compounds also realized 100% discriminant accuracies. Additionally, the potential volatile markers for varietal recognition of Yujin were screened using PLS-DA, including 2 for Guiyujin, 6 for Lvyujin, 9 for Wenyujin and 13 for Huangyujin. Conclusions The present study developed reliable methods for the varietal discrimination and volatile compounds characterization of Yujin, which will provide references for its quality control and clinical efficacy.

Ethnopharmacological relevance Diabetic retinopathy (DR) is a kind of complex complication of late diabetes mellitus with high incidence and risk of blindness. Bushen Huoxue Prescription (BHP), which consists of Rehmanniae radix (RR), Salviae miltiorrhizae radix et rhizoma (SMRR), Ginseng radix et rhizome (GRR) and Puerariae lobatae radix (PLR), has an active effect on the treatment of DR. However, the quality markers (Q-markers) of BHP are not entirely clear. Purpose This study aimed to screen the Q-markers of BHP for DR treatment based on the establishment of spectrum-effect relationship and verified experiment. Materials and methods In this study, 12 BHP samples (S1–S12) for fingerprint analysis and pharmacological evaluation were prepared according to a four-factor and twelve-level uniform design. High performance liquid chromatography-ultraviolet detector-evaporative light scattering detector (HPLC-UV-ELSD) was employed to analyze the fingerprint on the basis of the characteristics of BHP components. The evaluation of sample similarity was carried out by similarity analysis (SA) and hierarchical cluster analysis (HCA). The pharmacological indicators, including expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) in the retina of Sprague Dawley (SD) rats induced by streptozotocin (STZ), were detected by enzyme-linked immunosorbent assay (ELISA). Besides, the spectrum-effect relationship between common peaks of fingerprints and the pharmacological results was investigated by partial least squares regression (PLSR) and canonical correlation analysis (CCA). The results of spectrum-effect relationship were verified by the expression of VEGF and HIF-1α on primary culture retinal Muller cells induced by hyperglycemia and hypoxia. Results In the HPLC-UV-ELSD fingerprint, 23 common peaks in UV and 14 common peaks in ELSD were identified. The pharmacological results indicated that the expression of VEGF and HIF-1α in the retina of SD rats were inhibited by 12 BHP samples to varying degrees compared with the model group. Based on SA and heatmap of HCA, S4 and S8 were clearly distinguished from other samples. The results of PLSR and CCA revealed that the contents of puerarin, daidzin, salvianolic acid B and ginsenoside Rb1 were inversely correlated with the expression of VEGF and HIF-1α. Hence, the four compounds may be the main active components to prevent and treat DR. The results of intervention on primary culture retinal Muller cells showed that puerarin, daidzin, salvianolic acid B, and ginsenoside Rb1 can significantly inhibit the expression of VEGF and HIF-1α. Conclusions The spectrum-effect relationship of BHP was successfully established, and the Q-markers of BHP for the prevention and treatment of DR were preliminarily confirmed. It provides a feasible method for the research of quality control.

Ethnopharmacological relevance San-Ye-Tang-Zhi-Qing formula (SYTZQ) is an effective prescription for the treatment of pre-diabetes disorders of glycolipid metabolism in type 2 diabetes mellitus (T2DM). It consists of five Chinese herbs including Mori Folium, Nelumbinis Folium, Crataegi Folium, Salviae Miltiorrhizae Radix et Rhizoma and Paeoniae Radix Rubra. Aim of the study This study was aimed to reveal the pharmacological mechanism of pharmacokinetic target components of SYTZQ for the treatment of T2DM. Materials and methods A rapid, precise and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify simultaneously nuciferin, vitexin-4″-O-glucoside, vitexin-2″-O-rhamnoside, paeoniflorin and rosmarinic acid in rat plasma after oral administration of SYTZQ. The network pharmacology was used to analyze the effect of the compounds absorbed into the blood of SYTZQ on T2DM. The effects of paeoniflorin, nuciferine and rosmarinic acid on adipogenic differentiation were validated in vitro experiments. Results The separation was performed on an ACQUITY UHPLC HSS T3 column (2.1 mm × 100 mm, 1.7 μm) using acetonitrile and 0.1% (v/v) formic acid in water as the mobile phase in gradient elution. The calibration curves of five analytes showed good linearity (r ≥ 0.9991) with the lower limits of quantification (LLOQ) between 0.3 and 5.0 ng/mL. The recoveries and matrix effects of five analytes ranged from 81.1% to 113%. The RSDs of inter-day and intra-day precision were all within 13.7%. The validated method was successfully applied to the pharmacokinetic study of five ingredients after oral administration of SYTZQ to rat. 39 major targets and 22 candidate pathways of five compounds absorbed into the blood of rats after administration of SYTZQ were identified and successfully constructed a compound-target-disease-pathway network. It was confirmed that paeniforin, nuciferine and rosmarinic acid could decrease the adipogenicity differentiation in vitro experiments. Conclusions The pharmacokinetic parameters indicated that the five components (nuciferin, vitexin-4″-O-glucoside, vitexin-2″-O-rhamnoside, paeoniflorin and rosmarinic acid) were absorbed and eliminated quickly in vivo. These five absorbed components were associated with 22 pathways, including insulin resistance, regulation of lipolysis in adipocytes, PI3k/AKT-, TNF-, cAMP- and cGMP-PKG-signaling pathway. Paeoniflorin, nuciferine and rosmarinic acid have the effect of inhibiting adipocyte differentiation. This study could provide more reference for quality control, and provide a firm basis for evaluating the clinical efficiency of SYTZQ.

Ethnopharmacological relevance Ophiopogonis Radix, the commonly used traditional Chinese medicine in clinic for treating cardiovascular diseases, is returned to the stomach, lung and heart meridian. It is reported to nourish yin, moisten lung and is used to treat heart yin deficiency syndromes and asthenia of heart and lung, which indicated that Ophiopogonis Radix may have a protective effect on heart disorders. Atherosclerosisis is an important process in the development of cardiovascular diseases and abnormal lipid deposition induced macrophage foam cells is its crucial foundation. Our previous study showed the extract of Ophiopogonis Radix (EOR) ameliorates atherosclerosis in vitro. However, it may protect against cardiovascular diseases through inhibiting macrophage foam cell formation and its potential effective components and mechanisms are still unclear. Aim of the study Our study aimed to investigate the effect of Ophiopogonis Radix on macrophage foam cell formation and its potential active constituents and mechanisms. Materials and methods Ox-LDL induced macrophage cells were employed to evaluate the effect of Ophiopogonis Radix on macrophage foam cell formation. Then the potential active constituents inhibited formation of macrophage foam cells were screened by biospecific cell extraction and its underlying mechanisms were also explored by Western blot. Results The extract of Ophiopogonis Radix was found to significantly inhibit macrophage foam cell formation, evidenced by the decrease of TG and TC and Oil Red O staining analysis in macrophage cells, which indicated that EOR reduced the formation of macrophage foam cells. At the same time, EOR was showed to increase antioxidant capacity in macrophage cells. After treatment with EOR, two potential active components interacted with macrophage foam cells specifically were identified to inhibit macrophage foam cell formation including methylophiopogonanone A and methylophiopogonanone B. Methylophiopogonanone A was then proved to decrease the expression of CD36, Lox-1 and SREBP2, increase the expression of ABCA1 obviously, while the expression of ABCG1 and SREBP1 had no changes. Conclusions In our study, Ophiopogonis Radix was found to protect against atherosclerosis through suppressing ox-LDL induced macrophage foam cell formation and two potential compounds were identified by biospecific cell extraction including methylophiopogonanone A and methylophiopogonanone B. Moreover, methylophiopogonanone A was proved to inhibit foam cells through reducing uptake, synthesis and increasing efflux, which may provide guidance and reference for application of Ophiopogonis Radix and investigation of the effective components of TCMs.

Ethnopharmacological relevance Aconiti Lateralis Radix Preparata (AR) is the most frequently used herb to generate heat and treat symptoms associated with coldness in Asia. Aims of the study The hypothalamus is one of the master regulators to maintain constant core body temperature. Chronic exposure to cold stress disturbs homeostatic regulation, gradually resulting in hypothalamic inflammation. This study investigate the effects of AR, on the chronic intermittent cold (CIC)-induced release of pro-inflammatory signaling molecules in the mouse hypothalamus. Materials and methods Aconiti Lateralis Radix Preparata extract (ARE) were solubilized in distilled water and diluted with saline before administration. Male ICR mice (7 weeks old, 30–32 g) were divided randomly into 6 groups: (1) control, (2) cold stress, (3) ARE 30, (4) ARE 100, (5) ARE 300, and (6) ARE 1000 mg/kg groups. Groups (2)-(6) were exposed to CIC stress once a day for 14 days. CIC stress was achieved by exposing the mice to 4 °C and 60 ± 10% humidity for 120 min once a day. Rectal temperature was measured after terminating cold stress. Cortisol levels were measured from serum. Hypothalamus tissue was used for western blot analysis, and IL-9, IL-13, PGE1, and PGE2 levels were assessed. Results ARE treatment prevented the CIC-induced decrease in rectal temperature and increase in serum cortisol level. ARE-treated CIC-exposed mice demonstrated decrease in nuclear c-Fos levels dose-dependently compared to CIC-exposed mice. Nuclear NF-kB expression showed significant increase in CIC-exposed mice. ARE treatment significantly blunted the increase in nuclear NF-kB expression. CIC-exposed mice had significantly increased levels of both IL-9 and IL-13. Treatment with ARE suppressed the elevated IL-9 and IL-13 levels. Between control and CIC-exposed mice PGE1 levels showed no difference. However ARE (1000 mg/kg)-treated CIC-exposed mice had a significant increase in PGE1 level compared to CIC-exposed mice. PGE2 levels were significantly higher in CIC-exposed mice compared to control mice. ARE treatment significantly attenuated the increase in PGE2 levels. Conclusions Our findings suggest CIC stress disturbs the anti-inflammatory effect of cortisol and maintenance of the body temperature. Thus AR contributes to suppress the activated proinflammatory factors, IL-9, IL-13, and PGE-2, and to increase the heat production.

Background Due to the complexity of traditional Chinese medicine (TCM), the current quality evaluation of TCM are difficult to associate with clinical efficacy. Shenqi Jiangtang Granule (SJG), a classical TCM formula, is proven as a therapy for treatment of type II diabetes mellitus (DM) and complications while the substantial basis of the therapeutic effects is not clear. Purpose The present study proposed an integrated approach to discriminate the quality markers (Q-markers) based on multi-dimensional characteristic network for quality control of TCM. Methods The multi-dimensional characteristic network was established by “Spider-web” mode, which was comprehensively integrating “compatibility-content-activity- efficiency-stability” of the candidate ingredients. The activity dimension was evaluated by the inhibitory activity of SJG on α-glucosidase and aldose reductase. The efficacy dimension was assessed through the association between the compounds and the target pathway of diabetic nephropathy (DN) based on integrated pharmacology platform. Each dimension for the feature network was quantified by multivariate statistical analysis, and regression area of the candidate compounds was constructed in the network. Finally, the candidate compounds were sorted comprehensively by the regression area. Results A total of 30 chemical compounds with effective hypoglycemic activity were identified as the potential Q-markers. From the data analysis, three dimensions of activity, efficacy and content performed a greater impact on the regression area of the characteristic network. Among these compounds, ginsenoside Re, ginsenoside Rd, ginsenoside Rg1, calycosin, ginsenoside Rb1, formononetin, astragaloside IV, ginsenoside Rf, ginsenoside Rc, notoginsenoside Fe, schisandrol A, gomisin D were screened out as the candidate Q-markers of SJG. Conclusion The multi-dimensional characteristic network integrating compatibility, content, activity, efficiency and stability is efficient to discriminate the potential Q-markers of TCM prescription. Our results demonstrated that 12 candidate compounds from Panax Ginseng, Radix Astragali and Schisandrae Chinensis might select as Q-markers for qualitative evaluation of SJG.

Ethnopharmacological relevance Duijinsan (DJS) is a famous Chinese medicine prescription composed of Radix scutellariae (RS) and Rhei Radix (RRR), which has been mainly used for treating migraine. Aim of the study This study aimed to uncover the anti-migraine active compounds from DJS and preliminary predicted the pharmacological mechanism by evaluating the spectrum-effect relationship between high-performance liquid chromatography (HPLC) fingerprints and anti-migraine effects of Duijinsan (DJS) extract combined with molecular docking. Materials and methods HPLC and LC-MS were applied for chemical analyses of DJS extracts in different proportions. Inhibition of DJS extracts on trigeminal nerve cell releasing calcitonin gene related peptide (CGRP) experiment was performed. The active compounds were screened by spectrum-effect relationship analysis and confirmed by molecular docking and the activities of major predicted compounds were validated in vitro. Results Twenty-six common peaks were assigned and identified from the fingerprints of different proportions DJS extracts. In vitro experimental results showed that DJS extracts inhibited inflammation and release of CGRP from trigeminal nerve cells. Five predicted active compounds, Chrysin 6-C-arabinoside 8-C-glucoside, Chrysin 6-C-glucoside 8-C-arabinoside, baicalin, Chrysin-7-O-Beta-D-glucoronide and Oroxylin A 7-O-glucuronide were sorted out according to spectrum-effect relationship analysis and molecular docking comprehensively. In vitro validation experiments showed that all the predicted compounds inhibited the CGRP releasing and the activation of TRPV1 channel. Baicalin, chrysin-7-O-β-D-glucuronide and Oroxylin A-7-glucoronide significantly inhibited the activation of TRPV1 channel. Conclusion Chrysin 6-C-arabinoside 8-C-glucoside, Chrysin 6-C-glucoside 8-C-arabinoside, baicalin, Chrysin-7-O-Beta-D-glucoronide and Oroxylin A 7-O-glucuronide which can inhibit the CGRP releasing and the activation of TRPV1 channel were screened as the anti-migraine active compounds by spectrum-effect relationship analysis and molecular docking.

Ethnopharmacological relevance Fangji is an ancient combinatorial formula. The compatibility mechanisms that how component herbs of Fangji work cooperatively to achieve the executive framework remain unexplored. Aim of the study Toexplore compatibility mechanism and systematical effects of Fangjis by taking Wenxin Keli decoction (WXKL), a classical Fangji constituted by Codonopsis Radix, PolygonatiRhizoma, Notoginseng Radix Et Rhizoma, Ambrum, and Nardostachyos Radix Et Rhizoma., as example. Main methods Here, we employed bioinformatics approach, including cluster analysis, cooperative module pair analysis, primary module identification, and proximity examination among target profile of herbs, to investigate compatibility characterization and anti-arrhythmia mechanism of WXKL. Finally, core mechanisms of WXKL were validatedby in vivo experiments. Results As a result, we identified 695 putative target proteins and 27 clusters (W-modules) inWXKL target network (W-network), in which W-module 1, 2, 4, 8, 10 were primary modules. The cooperative module pairs were W-module 2 and 4, W-module 2 and 8, and W-module 2 and 1, all of which existed in Codonopsis Radix- or Notoginseng Radix Et Rhizoma.-condition. And Nardostachyos Radix Et Rhizoma only yielded cooperation between W-module 1 and 2. The proximity of herbs’ target profiles of Codonopsis Radix and Notoginseng Radix Et Rhizoma were similar, and Nardostachyos Radix Et Rhizoma and Ambrum were similar. For the compatibility framework, Codonopsis Radix general regulated 70.67% targets and majority W-modules (81.48%) as sovereign herb, contributing to primary therapeutic effect, mainly involving neurohormonal regulation, vasomotor, inflammation and oxidative stress. Other herbs assisted Codonopsis Radix to enhance major outcomes through common modules, and acted as complementary roles through unique process including mitotic cell cycle, biosynthetic and catabolic process, etc. Furthermore, WXKL regulated 66.67% hub proteins of arrhythmia-network, 68.18% and 47.37% proteins in primary arrhythmia-module 1 and 2, mainly involving ion channel activity, neurohormonal regulation, and stress response processes, to constitute regulatory network focusing on cardiovascular, renal, nervous system, to reverse the pathological process of arrhythmia. In vivo experiments demonstrated WXKL can attenuate adrenergic activation induced sympathetic atrial fibrillation by inhibiting calmodulin expression (CaM) and ryanodine receptor 2 (RYR2) phosphorylation to regulate neurohormonal action. Conclusion This strategy provided an overarching view of anti-arrhythmia mechanism of WXKL and its internal compatibility, and may facilitate the understanding of compatibility in Fangjis from the perspectives of modern biology.

Ethnopharmacological relevance In traditional Chinese medicine (TCM) and modern pharmacodynamics, dried Rehmannia Radix (DRR) possesses prominent anti-thrombotic activity that decreases after processing by nine steaming and drying cycles to develop processed Rehmannia Radix (PRR). Due to the complexity of the DRR components, the chemical mechanism leading to efficacy changes of DRR caused by processing is still unclear. Aim of study This study aimed to trace the anti-thrombotic active compounds of DRR and different degrees of processed RR (PRR) and to evaluate the synergistic effects among different active components. Materials and methods The anti-thrombotic active chemical fraction of DRR extracts was evaluated. Targeted fractions of the processed products of RR were prepared at different processing stages. The changes in monosaccharides, oligosaccharides and secondary metabolites during processing were characterized by multidimensional high-performance liquid chromatography (HPLC). The anti-thrombotic effects of targeted fractions of different RR samples were evaluated by analyzing the length of tail thrombus (LT) and serum biochemical indicators in carrageenan-induced tail-thrombus mice. The spectrum-effect relationships were investigated by partial least squares regression (PLSR) analysis and gray correlation analysis (GRA). Finally, the active compounds were screened by spectrum-effect relationship analysis and validated in vivo, and their synergistic effects were determined by Webb's fraction multiplication method. Results Six ingredients highly associated with anti-thrombotic activities were screened out by the spectrum-effect relationship analysis, of which oligosaccharides (stachyose, sucrose and raffinose) and iridoid glycosides (catalpol, leonuride and melitoside) possessed a synergistic effect on tumor necrosis factors (TNF-α), interleukin 1β (IL-1β) and plasminogen activator inhibitor 1 (PAI-1)/tissue-type plasminogen activator (t-PA) ratio in vivo with synergistic coefficient (SC) > 1. Conclusion The main material basis of the anti-thrombotic activities of DRR is oligosaccharide components of stachyose, raffinose and sucrose, iridoid glycosides components of catalpol, leonuride and melittoside. The two kinds of components exert synergistic anti-thrombotic effects by inhibiting the expression of inflammatory factors and regulating the balance of the fibrinolysis system.

Polygoni Multiflori Radix is the dried root of Polygonum multiflorum Thunb officially recorded in the Chinese Pharmacopoeia as HeShouWu (HSW) in Chinese pinyin. The processed HSW are commonly used in TCM to treat liver disease and Chinese Pharmacopoeia has described the actions of it to tonify liver-kidney, replenish essence and blood, blacken beard and hair, strengthen sinew and bone, and resolve turbidity and lower lipid hence making it use not only as a herbal medicine in TCM but also as supplementary food in health care. Aim of the study Concerns about the hepatotoxicity in association with Polygoni Multiflori Radix and its processed products have been reported in some countries. In the present study, we aim to investigate the potential hepatotoxicity of HSW in rats with oral administration of 95% ethanol-extracts of Polygoni Multiflori Radix by using metabolomics method. Materials and methods Here, male rats with 150–180 g body weight were received vehicle control or Polygoni Multiflori Radix extracts (HSW-Ex) orally at 19.2 (low dose), 192 (medium dose), or 1920 mg/kg/day (high dose), respectively, for 28 consecutive days. Signs of HSW-induced toxicity were monitored by traditional toxicity assessments (e.g., clinical pathology and histopathology). Metabolomics investigation of serum was performed to identify potential endogenous metabolites which may be relevant to liver injury. Results Rats received High and Medium dose of HSW-Ex showed greater sign of liver injury with increased levels of ASP, ALT, and AST, as well as reduced SOD activity when compared to vehicle control. In contrast, there are no significant changes relevant to liver injury observed in rats by receiving the low dose of HSW-Ex. Metabolomics analyses have identified ten potential endogenous metabolites varied significantly among the treatment groups with varying doses of HSW-Ex, of which might be related to liver injury. Conclusion Our data has further suggested that liver damage resulting from HSW-Ex consumption is dosage dependent in rats. It is possible that disruption in amino acid and energy metabolism might lead to subsequent oxidative damage in the liver of rats. Because the clinic practice often use low dose in a short time, therefore HSW usage in TCM still keep safe currently, but we present a warning to the clinical doctors and make them has some concern about high dose of HSW usage in a long term that has potential danger to damage liver.

Wine-processed Radix scutellariae (RS) is the processed product of RS, which is the dried root of Scutellaria baicalensis Georgi. It is recorded in Chinese traditional formula that wine-processed RS has the effect of anti-migraine, while the effect has not been confirmed and the possible mechanism remains unclear.To verify the anti-migraine effect of wine-processed RS in nitroglycerin (NTG)-induced rats and explore the correlation between compounds dissolution and the pore structure based on fractal theory.In the validation of pharmacodynamics, the effects of wine-processed RS on migraines were firstly evaluated by observing the number of head-scratching of rats, then investigated by determining the levels of nitric oxide (NO), calcitonin gene-related peptide (CGRP) and the expression of c-Fos in the brain of NTG-induced rat models using ELISA and immunohistochemical assessments. In the correlation study, the stir-frying time of RS was set to 5 min, 10 min and 15 min. The scanning electron microscope (SEM) and mercury intrusion method were used to explore the pore structure and main parameters of the pore structure including pore size distribution, pore volume, porosity, surface area and fractal dimension. The compounds dissolution of total flavonoids and five major components containing baicalein, baicalin, scutellarin, wogonin and wogonoside was determined by UV-Vis spectrophotometry and HPLC separately.The animal experiments had shown that wine-processed RS could significantly reduce the head-scratching times of NTG-induced rat models (p 0.01) and markedly decrease the levels of NO (p 0.01), CGRP (p 0.05) and the expression of c-Fos (p 0.01) compared with model group. The data indicated that wine-processing would affect the dissolution of compounds by changing the pore structure of RS. The order of positive correlation between pore structure parameters and compounds' dissolution was total surface area fractal dimension (r 0) and the order of negative correlation was average pore size total porosity total volume (r 0). Compared with the other sample groups (p 0.05), the wine-processed RS stir-fried for 10 min had a pore structure which was more favorable for compounds dissolution.Wine-processing could strengthen the anti-migraine effect of RS by changing the pore structure of RS, which is linked to the dissolution of compounds. The RS stir-fried for 10 min may be more effective in treating migraine.

Ethnopharmacological relevance The influence of sulfur fumigation processing on chemical profile, pharmacological activity and safety of Chinese herbs has attracted great attention. Panacis Quinquefolii Radix (PQR) was more widely used as edible and medicinal than Ginseng because of its tonifying effect and characteristic of not getting inflamed. The disadvantage of sulfur fumigated (SF) Ginseng has been reported, but the systematic study of SF-PQR is deficient and urgently needed. Aim of the study To systematically describe the influence of sulfur fumigation on chemical profile, characteristic products, immunoregulation and liver and kidney injury of PQR. Materials and methods ICP-MS and HPLC-DAD were used to detect 11 inorganic elements and 3 ginsenosides, respectively. Principal component analysis (PCA) was used to distinguish SF-PQR from non-sulfur fumigated (NSF)-PQR by combining the content changes of inorganic elements and ginsenosides. UPLC/Orbitrap-MS was applied to screen the characteristic products (m/z) after sulfur fumigation. For the effectiveness and safety, male KM mice were used to compare the immunomodulatory effects of NSF-PQR or SF-PQR under both healty and cyclophosphamide induced immunosuppressive conditions by net growth rate of body weight, thymus and spleen indices, serum IL-6, SOD, BUN, AST levels, and HE staining of liver and kidney. Results Sulfur fumigation processing significantly reduced the contents of ginsenosides Rb1, Re and Rg1 with the elevation of inorganic elements in 20 batches PQR. Based on the scatter distribution of PCA, SF-PQR and NSF-PQR can be distinguished. According to the Rt, Precursor ion (m/z) and Product ion (m/z) produced by UPLC/Orbit trap-MS, R1-SO3 (m/z, 1059.53), Re-SO3 (m/z, 1025.55), Rg1-SO3 (m/z, 878.47), Ro-SO3 (m/z, 1035.32), Rb1–SO3 (m/z, 1179.58), and Rk3-SO3 (m/z, 745.40) could be confirmed as important markers for identifying SF-PQR. The effect of SF-PQR on reversing immunosuppression induced by cyclophosphamide was significantly reduced (P Conclusion Sulfur fumigation processing not only reduced the immunomodulatory effect of PQR, but also brought the hidden danger in liver and kidney injury. The sulfonated products provided in this paper can be applied for the identification of SF-PQR accurately.

Ethnopharmacological relevance Asari Radix (Xixin, Asarum heterotropoides Fr. Schmidt var. mandshuricum Kitag., Asarum sieboldii Miq., or Asarum sieboldii Miq. var. seoulense Nakai, Asarum spp.) is the only herbal medicine containing aristolochic acid that can be used in medical practice. However, scientific evidence regarding its safe use in relation to hepatocellular carcinoma (HCC) is lacking. Aim of the study The aim of this study was to use post-marketing surveillance to provide a scientific understanding of the relationship between Asari Radix and the development of HCC and suggest the maximum allowable amount of Asari Radix. Materials and methods A retrospective, population-based cohort study was conducted, with patients randomly selected and divided into three cohorts: a non-hepatitis B virus (HBV)/hepatitis C virus (HCV) cohort, a HBV cohort, and a HCV cohort. Data were retrieved from the National Health Insurance Research Database of Taiwan from January 1, 1997 to December 31, 2013. The study period covered the initial 10 years of exposure to persistent HBV or HCV, followed by exposure to Asari Radix for an additional 8 years. Results After propensity score matching, 106,942, 3818, and 928 patients were included in the non-HBV/HCV, HBV, and HCV cohorts, respectively. These cohorts included 75, 50, and 42 HCCs and 1,564,943, 30,956, and 6938 person-years, respectively. All hazard ratios of exposure to 1–30 g, 31–60 g, 61–100 g, and 101–200 g of Asari Radix in these three cohorts showed negative associations between Asari Radix exposure and HCC development. Furthermore, the three cohorts demonstrated that exposure to under 200 g of Asari Radix was safe. Conclusions Post-marketing surveillance showed that Asari Radix has no relationship with HCC development at an intake of under 200 g. The study is persuasive in furthering our knowledge of the maximum allowable amount of Asari Radix.

Ethnopharmacological relevance Arnebiae Radix, a common herbal medicine in China, is often utilized to treat blood-heat syndrome and has been reported to exert an effect on the heart. Aim of the study The combination of acetylcholine (Ach) and CaCl2 has been widely used to induce atrial fibrillation (AF) in animals. However, whether Arnebiae Radix displays any preventive action on Ach-CaCl2 induced AF in rats remains uncertain. In our study, we attempted to investigate the protective effects of Arnebiae Radix on Ach-CaCl2 induced AF compared to amiodarone, which was employed as the positive control. Materials and methods To establish the AF model, SD rats were treated with a mixture of 0.1 mL/100 g Ach-CaCl2 (60 μg/mL Ach and 10 mg/mL CaCl2) by tail vein injection for 7 days. Rats were also given a gavage of Arnebiae Radix (0.18 g/mL) one week before or concurrently with the establishment of the AF model. At the end of the experimental period, the induction, duration and timing of AF were monitored using electrocardiogram recordings. Left atrial tissues were stained to observe the level of fibrosis. Electrophysiological measurements were used to examine atrial size and function. Results In Ach-CaCl2-induced AF rats, Arnebiae Radix decreased AF induction, duration and susceptibility to AF. In addition, Arnebiae Radix significantly reduced atrial fibrosis and inhibited atrial enlargement induced by Ach-CaCl2. Moreover, there was an apparent improvement in cardiac function in the Arnebiae Radix-treated group. Conclusions Our findings indicate that Arnebiae Radix treatment can attenuate Ach-CaCl2-induced atrial injury and serve as an effective therapeutic strategy for the treatment of AF in the future.

Ethnopharmacological relevance CaoWu ( Aconiti Kusnezoffii Radix ), well known for its high toxicity leading to fatal ventricular arrhythmias, is detoxified by HeZi ( Terminalia Chebula Retz ) decoction to prepare ZhiCaoWu ( Aconiti Kusnezoffii Radix Preparata ) as one part of ingredients of NaRu-3 pill which is used for the treatment of rheumatoid arthritis (RA). Aconitine (AC) is a highly toxic alkaloid of CaoWu and it is used as toxic target marker for the quality control (QC) of ZhiCaoWu. In the traditional processing method, the vanish of astringent or spicy feeling in tongue is the important detoxification indicator of ZhiCaoWu. However, how CaoWu is detoxified to ZhiCaoWu and whether the appropriate content of AC in ZhiCaoWu can be efficiently perceived after the empirical detoxification still lack factual basis. Aim of the study The present study aimed to optimize the traditional processing method for precision detoxification of CaoWu through biomimetic linking kinetics and human toxicokinetics (TK) of AC, with a view of providing insights into the changes of toxic target marker. Materials and methods CaoWu medicinal slices (Mes) and coarse powder (Cop) were processed by blank HeZi decoction through the soaking method for 7 days. High-performance liquid chromatography (HPLC) was used for the analysis of the samples. The acidity of blank HeZi decoction and HeZi processing decoction was directly determined by pH meter. The non-compartment analysis (NCA) was used to have an intuitive appreciation for AC and pH changes in HeZi processing decoction while the compartment model method was used to build the biomimetic linking kinetics model with the covariate. The inter-species scaling of animal TK parameters was conducted to predict human AC TK profiles. The possible uptake ways of AC (rapid-release or extended-release) for humans were attempted to assess the poisoning risk of AC in NaRu-3 pill. Based on the target content of AC in ZhiCaoWu, the biomimetic linking kinetics model was explored to optimize the traditional processing detoxification method of CaoWu. The assays of determining inflammatory cytokines in lipopolysaccharides (LPS)-induced RAW264.7 cells were performed to investigate the inflammatory modulation effects of AC in vitro. Results ZhiCaoWu was prepared by eliminating redundant AC in CaoWu through the repeatable replacement of HeZi processing decoction in which its acidity (pH) was affected. AC-pH changes in HeZi processing decoction were adequately depicted by a biomimetic linking kinetics model whose predictive power was determined by comparing the predictions of AC in ZhiCaoWu with the reported data. Rapid-release AC at the converted dose of 111.1 and 417.6 μg (0.011 and 0.042% of AC in NaRu-3 pill) reached maximum blood concentrations of 26.1 and 98.1 ng/mL at 0.3 h, in comparison with minimum human lethal concentration (100 ng/mL). Achieving the target content of AC (0.04%) in ZhiCaoWu or AC (0.011%) in NaRu-3 pill to precisely control the poisoning risk, the potential optimized protocols were that the processing time at 0.2–0.8% of AC in CaoWu was 2.0–4.4 days for Cop and 2.7–6.2 days for Mes. Correspondingly, pH values in HeZi processing decoction were 3.95 and 3.77 for Cop and Mes, respectively. Meanwhile, Lipopolysaccharides (LPS)-induced RAW264.7 cells were exposed to 0, 20, and 200 μM of AC for 12 h and AC at 20 μM enhanced the levels of IL-6, IL-10 and TNF-α. Conclusions Thus, for the first time, a biomimetic linking kinetics model was built to optimize the traditional detoxification method. Moreover, pH changes could be developed as surrogate endpoint for guiding the processing detoxification of CaoWu. Notably, setting the content limit of AC (0.011%) was very rational to control the poisoning risk of NaRu-3 pill. In addition, it was possible that there existed the more complex mechanisms of AC for inflammatory modulation in vitro.

Ethnopharmacological relevance Codonopsis Radix is a commonly used traditional Chinese medicine, and has the effect of strengthening spleen and tonifying lung, nourishing blood and engendering liquid. In addition, it is also used as important food materials. Aim of the study The aim of the study was to explain the underlying correlations between chemical constituents and pharmacological effects and explore the bioactive markers of Codonopsis Radix. Materials and methods Codonopsis Radix samples from Min county, Gansu province processed with different methods were taken as the materials, UPLC-ESI-Q-TOF-MS/MS analysis was conducted to identify the compounds and establish UPLC fingerprint. Meanwhile, hematopoietic and immunologic functions of Codonopsis Radix were investigated to obtain relevant pharmacological index. Then, the correlation analysis between chemical constituents in UPLC fingerprints and pharmacological effects was carried out. The plant name was confirmed to the database “The Plant List” ( www.theplantlist.org ). Results According to the results of canonical correlation analysis, tryptophan, syringin, tangshenoside I, codonopyrrolidium A, lobetyolin and two unknown compounds might be the potential bioactive markers related to the hematopoietic and immunologic functions of Codonopsis Radix, which could be recommended as the index compounds. Conclusion This study illustrated the underlying correlations between chemical constituents and pharmacological effects, explored the pharmacological material basis, and could lay a foundation for the improvement of quality standard of Codonopsis Radix.

Ethnopharmacological relevance Clematis chinensis Osbeck (C. chinensis), Clematis hexapetala Pall (C. hexapetala) and Clematis terniflora var. mandshurica Rupr (C. mandshurica) are collectively referred to as Clematidis Radix et Rhizome (CRR) in China. CRR is widely distributed in China, which is used as a traditional Chinese medicine to treat rheumatic arthralgia, limb numbness, tendon constriction and inconvenience in flexion and extension. Aims of this review This review systematically summarized the research progress on uses, chemical components, pharmacological activities and toxicology of CRR, listed the chemical structures of main compounds for clarifying the differences in chemical compositions. Meanwhile, the review will provide a theoretical and practical basis for the further research and development of CRR. Materials and methods The available information on CRR was collected using published materials and electronic databases, including ancient and modern books, Chinese Pharmacopoeia, Ph.D. and M. Sc. dissertations, CNKI, SciFinder, WanFang data, PubMed, ScienceDirect and Web of Science. The starting and ending years of references is 1965–2020, the search strategy was conducted by key words such as uses, chemical components, pharmacology and toxicology of CRR. Results Up to now, CRR has been used to treat various diseases/disorders, such as relieving rheumatism pain, treating cervical spondylopathy and scapulohumeral periarthritis, treating hepatic carcinoma and gastrointestinal, etc. In addition, more than 200 compounds have been isolated from the three plant species of Clematidis. Moreover, the crude extracts and isolated compounds of CRR have been reported to have a wide range of pharmacological activities, such as anti-inflammatory, anti-tumor, antimicrobial and antioxidant activities, etc. Toxicity studies have shown that CRR can cause oral burning, swelling, abdominal pain or severe diarrhea, difficulty breathing, dilated pupils, renal tissue structural changes, and severe death. Conclusions Researches in recent years mainly focused on C. chinensis and C. mandshurica, while there are a few reports on the pharmacological studies of C. hexapetala. Therefore, it is necessary to conduct further research on C. hexapetala. Meanwhile, it is important to pay attention to pursue research on the similarities and differences between the three plant species of Clematidis to find their respective advantages and make rational use of CRR. In addition, there is no report on the mechanism of toxicity research, which needs more attention.

Ethnopharmacological relevance Aconiti Lateralis Radix Praeparata (the Chinese name is Fuzi, FZ), the lateral or daughter root of Aconitum carmichaelii Debx. (Ranunculaceae), is a controversial traditional Chinese medicine (TCM) that is universally distributed and applied in many countries, such as China, Japan, Korea, and India. FZ can be used to treat various diseases, including rheumatic fever, rheumatism, painful joints, syncope, collapse, bronchial asthma, some endocrinal disorders, etc. However, quality control and assessment of FZ are challenging due to its obvious and high toxicological risks, and only its processed products are allowed to be used clinically according to the relative safety regulations. Consequently, it is necessary to analyze the whole chemical composition and the dynamic changes of FZ before and after processing. Addressing the changes in the chemical substance of raw and processed products is a way to reduce toxicity. Aim of the study In this article, the whole chemical composition of FZ is analyzed, the differences between raw and processed FZ are evaluated, and possible factors that influence the reduced toxicity of processed FZ are explained from the perspective of its chemical composition using qualitative and quantitative analysis methods. Materials and methods A novel strategy of multiple data collection and processing based on ultra-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) method in the positive ion mode, together with Global Natural Product Social Molecular Networking (GNPS) and multivariate statistical analysis, was established to systematically identify the chemical constituents of FZ and comprehensively investigate the chemical markers that can be used to differentiate FZ processed with vinegar and honey from its raw product. Combined with the qualitative analysis results, 12 components, including 8 chemical marker compounds and 4 toxicity components, were quantitatively analyzed by using high-performance liquid chromatography equipped with triple-quadrupole mass spectrometry (HPLC-MS/MS). Results Using the molecular networking (MN) analysis method, a total of 145 compounds were identified, of which 13 were identified using reference compounds. Seventy seven chemical markers were also detected between raw and processed FZ. The identification results of the chemical markers were also verified by orthogonal partial least squares discriminant analysis (OPLS-DA). The quantitative results indicated that the contents of 12 important components all decreased, especially diester-diterpenoid alkaloids (DDAs), after processing. Conclusion The decrease of toxicity of FZ after processing is closely related to the changes in its chemical composition. The method developed in this study is a comprehensive analysis technique for quality assessment of FZ, and this study provides a useful and quick strategy to characterize chemical compounds of TCM and explore the different chemical markers between raw and processed Chinese herbal medicine.

Ethnopharmacological relevance Ophiopogonis Radix (Maidong), derived from the dried root tuber of Ophiopogon japonicus (Thunb.) Ker Gawl., has been widely used in the treatment of chronic inflammatory and cardiovascular diseases. However, Ophiopogonis Radix is often adulterated with some species because of morphological similarities. Adulterants circulating in herbal markets are a latent threat to the clinical safety and consumers’ interest. Aim of the study We aimed to develop a nucleotide signature for identification of Ophiopogonis Radix and its Chinese patent medicines. Materials and methods A total of 255 ITS2 sequences representing 39 species and 4 varieties were used to develop a nucleotide signature of Ophiopogonis Radix. The nucleotide signature was used to investigate 17 commercial crude drugs and eight batches of Chinese patent medicines. Results A 69 bp nucleotide signature unique to Ophiopogonis Radix was found. The survey revealed that 2 of 17 crude drug samples were adulterants detected as Liriopes Radix (Shanmaidong). Fortunately, no adulterants were detected in the eight batches of Chinese patent medicines. Conclusions The newly developed nucleotide signature could be efficiently applied to identify Ophiopogonis Radix and its Chinese patent medicines, aiding in the authentication, quality control, and supervision of processed products in herbal markets.

Purpose This paper aimed to study the active compounds of Astragali Radix (AR) in the treatment of adriamycin nephropathy (AN) by a combination of network pharmacology and transcriptomics. Methods The chemical compounds of AR were screened out by text mining and database searching. Pharm Mapper was used to predict the targets of these chemical compounds. Potential targets of AN were screened by integrating the data from network pharmacology with known transcriptomics analysis results of kidney tissue. Compound-active target-potential target interactions networks were constructed so as to illustrate the relationship between compounds and targets, and obtain the chemical compounds directly related to potential targets of AN. The formula of compound contribution index (CI) based on algorithm was used to screen the active compounds of AR in the treatment of AN. In addition, we established an adriamycin-induced cell damage model with MPC5 cell, and used MTT assay, trypan blue dyeing and western blot analyses to validate the pharmacodynamic effect of the active compounds. Results 27 chemical compounds and 376 targets in AR were obtained by network pharmacology. Through Compound-active target-potential target interactions networks analysis, 22 compounds and 9 active targets as well as 130 potential targets were linked through 282 edges. The CI of every chemical compounds was further calculated by formula, the first four chemical compounds, including astragaloside IV, formononetin, quercetin and calycosin, whose cumulative contribution rate reached 87.28%, were considered to be active compounds. The results of MTT and trypan blue staining indicate that four active compounds had the significant protective effect on adriamycin-induced cell damage with MPC5 cell. Western blot result showed that four active compounds could significantly increase the expression of podocin protein in MPC5 cell. Conclusion The active compounds of AR in the treatment of AN were successfully identified by using a network pharmacology and transcriptomics approach. This approach is expected to be beneficial to the study of the pharmacodynamic material basis of traditional Chinese medicine (TCM) in treating specific diseases.

Ethnopharmacological relevance Astragali Radix (AR, Huangqi in Chinese), the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or A. membranaceus (Fisch.) Bge., possesses diverse therapeutic effects against fatigue, dyspepsia, diarrhea, heart diseases, hepatitis, and anemia. In recent years, increasing evidence has indicated the multiple immunomodulatory activities of AR in preclinical and clinical studies. Aim of the review This review attempts to elaborate the immunomodulatory effects of AR and its potential application in the treatment of immune related diseases. Materials and methods A comprehensive literature search AR was carried out using multiple internationally recognized databases (including Web of Science, Google Scholar, PubMed, ScienceDirect, Wiley, ACS, Springer, Taylor & Francis, and CNKI). Results The immunomodulatory effects of AR are closely attributed to its active constituents such as polysaccharides, saponins, and flavonoids. We also demonstrate that AR can be used as a potential therapeutic intervention for immune related diseases through regulating immune organs, mucosal immune, and immune system (innate immunity and acquired immunity). Conclusion AR promotes the development of immune organs, enhances mucosal immune function, increases the quantity and phagocytic capacity of innate immunity, promotes the maturation and differentiation of acquired immunity cells, and improves the expression of antibodies in acquired immunity. We believe that AR has a broad research space in the adjuvant treatment of immune related diseases, which could be a breakthrough point to improve the application value of AR.

Ethnopharmacological relevance Lithospermi radix has been prescribed in traditional folk medicine to treat diverse diseases like cancer. Aim of the study The present study assessed the sub-chronic oral toxicity of an aqueous extract of lithospermi radix (WLR) in Fischer 344 rats over a period of 13 weeks. Materials and methods The chemical compositions of WLR were analyzed using ultra-high performance liquid chromatography (UHPLC). WLR was daily administered to Fischer 344 rats at 0, 500, 1000, and 2000 mg/kg body weights (bw) for 13 weeks via oral gavage. Changes in mortalities, body weights, and intakes of food and water were monitored during the WLR treatment period. Urine was collected and analyzed 12 h before necropsy. Organ weights, hematological parameters, and plasma biochemical parameters were determined along with histopathological examination. Results When compared with the normal control group, no remarkable toxic signs or parameter variations related with WLR treatment were observed in mortality, body weights, organ weights, food and water consumptions, urinalysis, hematological and plasma biochemical analyses, and histopathological examination. Mortalities observed in one male at 2000 mg/kg bw and three females at 1000 mg/kg bw were not related with WLR treatment because no gross findings of toxicity were observed in both morphological and histological examination. Some significant changes in clinical parameters or histological lesions observed in WLR-treated animals were not related with WLR treatment because the differences were marginal and did not show dose-dependent or directional changes. Conclusions Based on these findings, the calculated no-observed-adverse-effect-level (NOAEL) in rats was higher than 2000 mg/kg bw.

Scutellariae radix (Scutellaria baicalensis Georgi) and Coptidis rhizoma (Coptis chinensis Franch), known as traditional Chinese medicine (TCM), have been widely used with the effects of suppressing fever, dispelling dampness, purging fire and removing toxicosis. Owing to their unimaginable complexity, it is difficult to understand their pharmacokinetic properties in detail. The aim of this study was to develop an optimal proteomics approach to analyze the protein profiling related with ADME/Tox in rat liver treated with S. radix and C. rhizoma as well as their compatibility.Male rats were respectively administered the extracts of S. radix, C. rhizoma and their mixture for 7 days, and their liver tissue samples were prepared for the comparative proteomic analysis. The significantly differentially expressed proteins between the experimental groups and the control group were found and identified by 2-DE and MALDI-TOF-MS analyses. To validate the proteomic analysis results, glutathion peroxidase, catalase and betaine homocysteine methyl transferase were selected and confirmed by western blotting.Seventy eight significantly differentially expressed proteins between the experimental groups and the control group were found and identified. By querying the relational databases, the identified differentially expressed proteins were summarized and classified into three groups, phase I drug metabolic enzymes, phase II drug metabolic enzymes and the rest proteins which mainly involve in energy metabolism, signal transduction and cytoskeleton. These proteins involved in ADME/Tox may be the targets for metabolic studies or markers for toxicity.Our findings indicated S. radix and C. rhizoma as well as their compatibility can assuredly influence the expression of the proteins in rat liver. After administration, the majority of these expressions presented a downward trend, which may be closely related to the pharmacological properties of the medicine. The method in this study may open up a new road for the complementary tests for ADME/Tox properties of S. radix and C. rhizoma as well as their compatibility.

Ethnopharmacological relevance Danggui Buxue Tang (DBT) is a classical Chinese herbal decoction containing two herbs, Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), which serves as dietary supplement for treating women menopausal syndromes. Pharmacological studies indicate that DBT has estrogenic, erythropoietic and osteogenic properties; however, the action mechanism for this complex herbal decoction is not known. Calycosin, a major flavonoid in AR, shares similar structure with β-estradiol, and thus which is hypothesized to be the critical compound of DBT. Here, we aim to investigate the role of calycosin in DBT in terms of its biological functions by using a calycosin-depleted DBT decoction (DBTΔcal). The biological functions of DBTΔcal and parental DBT were systematically compared. Materials and methods In order to standardize DBT decoction, four chemical markers were determined and quantified by HPLC. A semi-preparative HPLC method was utilized to prepare DBTΔcal. The authenticity of DBTΔcal was evaluated by LC-QQQ-MS/MS. To reveal the effect of calycosin on DBT functions, several cell assays related to the known properties of DBT were revealed, including estrogenic, erythropoietic and osteogenic functions. Results As compared to parental DBT, the estrogenic, erythropoietic and osteogenic abilities were markedly reduced in DBTΔcal. However, calycosin alone did not show significant responses. Conclusions Our results suggest that calycosin is a bioactive chemical in DBT decoction, and which could play a key linker in orchestrating multi-components of DBT as to achieve maximal functions. These discoveries should be invaluable in drug development and in investigating the modernization of traditional Chinese medicine from a new perspective.

Glycyrrhizae radix et rhizoma (Gancao) is often prescribed together with Sophorae flavescentis radix (Kushen) in traditional Chinese medicinal practice to increase the efficacy on the treatment of hepatitis and hepatic fibrosis. Meanwhile, long-term single used Gancao can cause adverse reactions, lead to pseudohypercorticosteroidism especially. But the side effects of Gancao are significantly reduced when combined with Kushen; the reasons are still unknown. The aim of this study was to elucidate potential pharmacokinetic interaction between Kushen and Gancao, and to provide guidance for clinical medicine safety.A specific and rapid HPLC-MS method was established to quantify the four main activity ingredients matrine (MT), oxymatrine (OMT), glycyrrhizin (GL) and glycyrrhetinic acid (GA) in rat plasma. In this study, the pharmacokinetic parameters and the pharmacokinetic differences of the four main activity ingredients MT, OMT, GL and GA in single herb and Kushen-Gancao couple were obtained.Compared with oral administration of Gancao extract, K10 and Tmax of GA significantly increased to 0.43 h(-1)and 30 h after giving Kushen-Gancao (p0.05), but T1/2 and Vd were reduced to 0.73 L kg(-1)and 4.98 h (p0.05). In addition, the AUC of GA was increased, and the other three activity ingredients all decreased.GA as the main factor leading to the sodium-water retention side effects of Gancao. The result found that the absorption of GA was significantly slowed down and the metabolism rate was accelerated in Kushen-Gancao than single herb. So the attenuated toxicity mechanism may be because the accumulation of GA reduced in vivo. The conclusion has important meaning to the compatibility of Chinese med.

Puerariae Lobatae Radix (PLR) and Puerariae Thomsonii Radix (PTR) are traditional Chinese medicines used for the treatment of diabetes and cardiovascular diseases. These two herbs are used interchangeably in clinical practice, even though they possess significantly different chemical profiles. In the case of Pueraria species, the misidentification is related to the multiple Chinese common names in clinical practice and variable pharmaceutical Latin names in different versions of the Pharmacopoeia of the People's Republic of China. In addition, there is lack of evidence demonstrating how the differences in the chemical profile would impact on the pharmacological activity of the two herbs. Therefore, the aim of this study was to compare the microscopic, phytochemical profiles and anti-diabetic activity of PLR and PTR so that the two species can be differentiated.The microscopic characteristics of the PLR and PTR were observed and measured by an optical microscope. The major compounds were quantified by ultra-performance liquid chromatography (UPLC) and total flavonoid content (TFC) colorimetric assay. The free radical scavenging capacity was assessed by 2,2-diphenyl-2-picrylhydrazyl (DPPH) antioxidant assays. Anti-diabetic activity was determined by the inhibition of porcine pancreatic α-amylase and rat intestinal α-glucosidase activities.Microscopic results illustrated that the size of xylem vessels (PLR: 0.1390 ± 0.0184 mm; PTR: 0.0471 ± 0.0109 mm), number of fibre per bundle (PLR: 32.6800 ± 2.8780; PTR: 16.5900 ± 0.9982) and the size of fibre (PLR: 0.0075 ± 0.0003 mm(2); PTR: 0.0025 ± 0.0002 mm(2)) in PLR were significantly greater than that in PTR (p0.01). PLR possessed a significantly lower total starch content (PLR: 0.5288 ± 1.2559 mg starch/g DM; PTR: 76.7954 ± 2.9905 mg starch/g DM) and total dietary fibre content (PLR: 4.2886 ± 0.3466 g/100g DM; PTR: 12.4148 ± 0.4541 g/100g DM) as compared to PTR. Isoflavonoids including puerarin, daidzin, genistin and daidzein were the major chemical constituents in both species. However, the average content of puerarin in PLR was found to be eleven times greater than that in PTR. Furthermore, the TFC, DPPH free radical scavenging capacity, anti-α-amylase and anti-α-glucosidase in the PLR extracts were 4.42, 4.91, 3.10 and 4.22 times greater than in the PTR extracts.This study provides a comprehensive investigation on the two medicinal valuable Pueraria species and allows differences to be ascertained. This information can be used to update monographs which will help practitioners and dispensers differentiate the herbs. Further study on the interchangeable use of PLR and PTR in clinical practice is urgently warranted.

Ethnopharmacological relevance D anggui B uxue T ang (DBT), a Chinese herbal decoction commonly used in treating women׳s ailments, contains two herbs: Angelica Sinensis Radix (ASR) and Astragali Radix (AR). Traditionally, ASR had to be pre-treated with yellow wine before the herbal preparation, which reduced the amount of volatile oil in water extract of ASR and DBT, and meanwhile the volatile oil-reduced DBT processed better bioactivities in cell cultures. The present study aimed to investigate the effect of volatile oil from ASR (Angelica oil) on the solubility of AR-derived ingredients and the biological properties of DBT. Materials and methods To standardize Angelica oil, four marker chemicals in ASR were determined by GC-QQQ-MS/MS. Subsequently, fifteen gram of AR was boiled with different amounts of Angelica oil. The amounts of astragaloside IV, calycosin, formononetin, total polysaccharides, total saponins and total flavonoids, all derived from AR, were extracted and determined by HPLC-UV/ELSD. To reveal the effect of Angelica oil on DBT functions, several cell assays related to the traditional functions of DBT were selected, including anti-platelet aggregation, induction of NO production, hematopoetic, estrogenic and osteogenic properties. Results The inclusion of Angelica oil in AR during preparation significantly decreased the amount of AR-derived astragaloside IV, calycosin, formononetin, total saponins and total flavonoids in the final water extract. In parallel, an inclusion of Angelica oil caused a decrease of DBT׳s estrogenic and hematopoetic activities in cultured cells. Moreover, the Angelica oil decreased DBT-induced cell proliferation of cultured MG-63 and endothelial cells. Conclusions The results indicated that Angelica oil was a negative regulator for DBT chemically and biologically, which supported the traditional practice of preparing DBT by using the wine-treated ASR.

Ethnopharmacological relevance Traditional Chinese medicine (TCM) has been used in clinical practice for several thousand years. TCM has played an indispensable role in the prevention and treatment of disease, especially the complicated and chronic ones. In TCMs, many ingredients which are known to have biological effects just pass through the gut, they do not get into the bloodstream. Study on interactions of these active ingredients with the intestinal bacteria is very helpful to unravel how TCM works. Radix scutellariae was widely used alone or in combination with other medicinal herbs to the treatment of type 2 diabetes mellitus for a long time in China even in Asia. Additionally, the incidence of type 2 diabetes is closely related to the changes of intestinal flora. In this paper, the metabolism of baicalin in Radix scutellariae extract by normal and type 2 diabetic mice intestinal bacteria were firstly investigated. Materials and methods Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/QTOF-MS) technique combined with MetabolynxTM software was used for analysis of the metabolic profile of baicalin in Radix scutellariae extracts by the intestinal bacteria from normal and type 2 diabetic mice. Results The amount of baicalin׳s aglycone (baicalein) in type 2 diabetic mice samples were remarkably more than that in normal mice samples and oroxylin A only existed in type 2 diabetic mice samples. Intestinal bacteria produced not only a small amount of baicalein, but also some conjugates such as hydrogenated baicalin and methylated baicalin. Conclusions We proposed that β- d -glucuronidases contributed to the deglycosylation prior to absorption. Intestinal bacteria from pathological state mice produced more baicalein, which was well absorbed contributing to the treatment of type 2 diabetes. Additionally, the pharmacological effects of oroxylin A were associated with type 2 diabetes. Hence, the production of metabolites of baicalin might influence the effects of traditional medicines. Thus the study on the metabolism of baicalin by intestinal bacteria from normal and type 2 diabetic mice was of great importance to understanding the effects of traditional medicines. Furthermore, this work demonstrated the potential of the ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry approach with MetaboLynx for quite rapid, simple, reliable and automated identification of metabolites of natural products.

Ethnopharmacological relevance To investigate the synergistic property of Platycodonis Radix (PG) in a classic Traditional Chinese Medicine (TCM) prescription Shengxian Decoction (SXT) by combining chemical profile with pharmacokinetic analysis strategy. The synergized prescription consisted of Astragali Radix, Anemarrhenae Rhizoma, Bupleuri Radix, and Cimicifuage Rhizoma. Materials and methods Ultra-performance liquid chromatography/quadruple time-of-flight mass spectrometry (UPLC–Q-TOF/MS) was employed to investigate the chemical fingerprints of SXT and decreased SXT (SXT that removed Platycodonis Radix, SXT-PG). A reliable LC–MS/MS method was developed to examine the pharmacokinetics of 9 marker compounds (including formononetin, calycosin-7-O-β- d -glucoside, ononin, caffeic acid, isoferulic acid, mangiferin, timosaponin E1, timosaponin B-II and timosaponin B) following oral administration of SXT and SXT-PG in rats. Both in vitro chemical profiles and in vivo pharmacokinetic parameters differences between SXT and SXT-PG were conducted. Results By using UPLC–Q-TOF/MS method, a total of 25 compounds identified from SXT, including 13 triterpenoids, 5 caffeinic derivatives, 4 isoflavonoids and 3 xanthone glycosides. Comparing the chemical fingerprints between SXT and decreased SXT did not reveal significant difference in the chemical profile of other four TCMs. The improved pharmacokinetic profiles of mangiferin, timosaponin E1, timosaponin B-II and timosaponin B were found in SXT group, suggesting the quicker distribution and more effective absorption, when compared with those in the SXT-PG group. Conclusions These results indicated that PG did not increase the dissolution of synergized prescription when co-decocting, but guided the synergized prescription to target location, reflecting the courier role of PG, which was in line with the clinical principle of TCM. It also established a useful method for TCM synergistic property research.

Ethnopharmacological relevance Osteoporosis is one of the most common bone diseases; it is characterized by bone loss and is a risk factor for hip fracture. Chinese herbal medicines (CHMs) and their related natural compounds have been used for treating many diseases, including bone diseases, since ancient times in China and are regarded as a cost-effective complementary therapy. Aim of the study The goal of this study was to investigate the osteoprotective mechanisms of these three Chinese herbs and their related natural compounds. The effects of CHMs and related natural compounds on RANKL-induced osteoclastogenesis in vitro were investigated. Materials and methods A network pharmacology method was applied to study CHM-related natural compounds and their osteoporosis targets. In addition, their effect on RANKL-induced osteoclastogenesis in RAW264.7 cells was also investigated in vitro. Results Radix dipsaci, Eucommiae cortex, and Rhizoma drynariae exhibited protective effects against mortality in hip fracture patients. Furthermore, these three herbs inhibited RANKL-induced TRAP activities and reduced the expression of bone resorption-related genes in RAW264.7 cells. Network analysis of natural compound (ingredient)-target interactions identified 11 natural compounds. Signal pathway analyses suggested that these compounds may target cytokine-cytokine receptor interactions, including RANKL-induced osteoclastogenesis. Five novel natural compounds exhibited reduced RANKL-induced TRAP activities and bone resorption-related gene expression. Conclusion The clinically used CHMs, Radix dipsaci, Eucommiae cortex, and Rhizoma drynariae, and natural compounds obtained from them may suppress RANKL-induced osteoclastogenesis in vitro.

Ethnopharmacological relevance Niuhuang Jiedu Tablet (NJT) is an effective prescription of traditional Chinese medicine (TCM) used in treating acute tonsillitis, pharyngitis, periodontitis and mouth ulcer. NJT is prepared from Xionghuang ( Realgar , As 2 S 2 ), Rengong Niuhuang ( Bovis Calculus Artificialis ), Bingpian ( Borneolum Synthcticum ), Shigao ( Gypsum Fibrosum ), Dahuang ( Rhei Radix et Rhizoma ), Huangqin ( Scutellariae Radix ), Jiegeng ( Platycodonis Radix ) and Gancao ( Glycyrrhizae Radix et Rhizoma ). In the prescription, significant level of realgar (As 2 S 2 ) as a potentially toxic element is contained. Aim of the study In this study, 1 H NMR-based metabonomics approach has been used to investigate the toxicity of realgar (As 2 S 2 ) after being counterbalanced by other TCMs in NJT. Materials and methods Male Wistar rats were divided into five groups: control, group I (treated with Realgar ), group II (treated with Realgar , Bovis Calculus Artificialis , Borneolum Synthcticum , Gypsum Fibrosum , Rhei Radix et Rhizoma , Scutellariae Radix , Platycodonis Radix and Glycyrrhizae Radix et Rhizoma ), group III (treated with Realgar , Bovis Calculus Artificialis , Borneolum Synthcticum and Gypsum Fibrosum ) and group IV (treated with Realgar , Rhei Radix et Rhizoma , Scutellariae Radix , Platycodonis Radix and Glycyrrhizae Radix et Rhizoma ). Based on 1 H–NMR spectra of urine and serum from rats, PCA and PLS-DA were performed to identify different metabolic profiles. Liver and kidney histopathology examinations and serum clinical chemistry analysis were also performed. Results PLS-DA scores plots demonstrated that the cluster of group I was separated from that of control rats, while group II was located close to control rats, indicating that metabolic profiles of group II were restored toward those of control rats. The metabolic profiles of group III were similar to those of group I, while the metabolic profiles of group II were almost in line with those of group II. Statistics results were confirmed by the histopathological examination and biochemical assay. Conclusion Our results indicated that it was more secure and much less toxic for counterbalanced realgar (As 2 S 2 ) in NJT. The effective material bases of toxicity alleviation to realgar (As 2 S 2 ) were Dahuang ( Rhei Radix et Rhizoma ), Huangqin ( Scutellariae Radix ), Jiegeng ( Platycodonis Radix ) and Gancao ( Glycyrrhizae Radix et Rhizoma ), which regulated energy metabolism, choline metabolism, amino acid metabolism and gut flora disorder affected by realgar (As 2 S 2 ) exposure.

Aconiti Radix (AC) and Aconiti Kusnezoffii Radix (AK) are two traditional Chinese medicines commonly used to treat joint pain and arthritis. In Southwestern China, Huangcaowu (AV), the root of Aconitum vilmorinianum Kom., has long been used as a local substitute for these herbs for analgesia and anti-inflammation. However, its anti-arthritic effects have not been investigated.To investigate the anti-arthritic effects of Huangcaowu (AV).Mono-arthritis in SD rats was induced by unilateral intra-articular injection of Freund's complete adjuvant. Physiological saline was injected in the contralateral knee. Seventy five percent ethanol extracts of AV (10 mg/kg/day and 100 mg/kg/day), AC (100 mg/kg/day) and AK (100 mg/kg/day) were administered to rats by oral gavage for 14 consecutive days (Day -6 to Day 7) while arthritis was induced at the seventh day (Day 0). The anti-arthritic effects of the herbs were assessed by measuring allodynia, swelling, hyperaemia and the vascular permeability of the knee joints.AV (10 mg/kg/day and 100 mg/kg/day), AC (100 mg/kg/day) and AK (100 mg/kg/day) suppressed joint allodynia. AV (10 mg/kg/day and 100 mg/kg/day) and AK (100 mg/kg/day) significantly reduced join swelling and hyperaemia while AC (100 mg/kg/day) did not. AV (100 mg/kg/day) attenuated vascular permeability while AC (100 mg/kg/day) and AK (100 mg/kg/day) showed no improvement.Huangcaowu (AV) significantly improved allodynia, swelling, hyperaemia and vascular permeability in arthritic knee joints. It showed the highest anti-arthritic effects among the three tested Aconitum herbs.

Ethnopharmacological relevance Angelicae Dahurica ( Hoffm. ) Benth.& Hook.f.ex Franch.&Sav combined with Pueraria labota ( Willd. ) Ohwi has been widely used as herb-pairs in traditional Chinese medicine (TCM) for utilization of antipyretic analgesic and anti-inflammatory drugs, and modern pharmacological studies have shown that application compatibility of the two drugs has the effects of cardiovascular disease treatment. The previous study has proved that Radix Angelicae Dahuricae extract could enhance the intestinal absorption of puerarin in Pueraria. But the underlying compatibility mechanism of the two herbs remains unknown. In this study we tried to further evaluate the improvement of Radix Angelicae Dahuricae extract on the puerarin using the Caco-2 cell model and explore the transport properties of puerarin through the above research to discuss the possible effect mechanism of Radix Angelicae Dahuricae extract on the transport of puerarin and the underlying compatibility mechanism of the two herbs. Aim of study The aim of this work was to study the transport properties of puerarin in Radix Pueraria across Caco-2 cell membrane and to explore how the Radix Angelicae Dahuricae extract affected the transport of puerarin using the well-characterized, human-based intestinal Caco-2 cell model as a platform. Materials and methods The bidirectional transport, and the effects of time, drug concentration, pH, P-gp inhibitors (Verapamil, Cyclosporin A), MRP inhibitor (MK-571) and EDTA-Na 2 (tight junction modulator) on the absorption of puerarin were observed. Then the influence of extract of Radix Angelicae Dahuricae on the transport of puerarin was studied. Drug concentration was measured by HPLC and the apparent permeability coefficients ( Papp ) and apparent permeability ratio ( PDR ) were calculated. Results and conclusions The results showed that the transport ( Papp ) of puerarin in Caco-2 cell monolayer model had time and concentration dependence, and the transport showed saturation characteristics with the time and concentration of puerarin to a certain degree. The Papp of puerarin transported on Caco-2 cell monolayer model was significantly changed when the specified inhibitors of P-gp were added to the model and the PDR decreased from 1.74 to 0.43. The absorption of puerarin was improved when combined with Radix Angelicae Dahuricae . The intestinal absorption of puerarin is by passive diffusion as the dominating process and active transportation was mediated by P-gp and MRP transporter in Caco-2 cell monolayer model, and Radix Angelicae Dahuricae could enhance the intestinal absorption of puerarin.

Ethnopharmacology relevance Post-traumatic stress disorder (PTSD) is a severe psychiatric disorder that is characterized by symptoms of re-experiencing, avoidance and hyperarousal, as well as social and professional dysfunction at least one month after the exposure to a traumatic event. Biosynthesis of allopregnanolone has been suggested as one of the important contributors to PTSD. Albiflorin (AF) extracted from Radix paeoniae Alba had been shown to be effective in the therapy of depression. However, few studies were concerned about the anti-PTSD-like effects of AF. Aim of the study The current study aimed to evaluate the anti-PTSD-like effects of AF in an animal model and its possible mechanism. Materials and methods To evaluate this, the single prolonged stress (SPS) model was used in the present study. The SPS rats were administered by AF (at doses of 3.5, 7 and 14.0 mg/kg, i.g.) after induction of SPS from days 2–13. After the exposure to SPS, behavioral assessments were conducted, including contextual fear paradigm (CFP), elevated plus-maze test (EPMT), open-field test (OFT). The rats were decapitated at the end of the behavioral tests and levels of allopregnanolone in prefrontal cortex, hippocampus and amygdala were measured by enzyme linked immunosorbent assay (ELISA). Results It had been shown that behavioral deficits of SPS rats were reversed by AF (7.0 and 14.0 mg/kg, i.g.), which attenuated the PTSD-like associated contextual freezing behavior in CFP and improved PTSD-like associated anxiogenic behavior in EPMT without affecting locomotor activity in OFT. Moreover, decreased levels of allopregnanolone in prefrontal cortex, hippocampus, and amygdala were reversed by AF (7.0 and 14.0 mg/kg, i.g.), respectively. Conclusion In summary, the present study indicated that AF exerted the anti-PTSD-like effects, which maybe associated with allopregnanolone biosynthesis in the brain.

Ethnopharmacological relevance Roots of Angelica sinensis (Oliv.) Diels (AS) a commonly used herbal, always act as an anti-inflammatory drug in Chinese traditional therapy. In clinical use, AS is always processed before being used for the reason that processing can increase its therapeutic effect. Recent studies have shown that volatile oil of AS (VOAS), an important component in AS, has evident anti-inflammatory activities. Aim of the study In this study, our aim is to evaluate the anti-inflammatory effects of volatile oils from processed products of AS. Materials and methods In this paper, volatile oils from stir-fried AS (C-VOAS), parched AS with alcohol (J-VOAS), parched AS with soil (T-VOAS), and parched AS with sesame oil (Y-VOAS) were applied to intervene the carrageenan-induced acute inflammation model rats. GC–MS based metabolomics was utilized to determine different metabolites in the inflammatory exudate and plasma samples. Results The results showed that VOASs could significantly inhibit the release of PGE2, HIS, 5-HT and TNF-α, among which C-VOAS and J-VOAS expressed better effect. Otherwise, 14 potential biomarkers were identified respectively in inflammatory exudate and plasma, which changed highly significantly (P Conclusions We inferred that the anti-inflammatory effect of C-VOAS and J-VOAS were superior to other VOASs.

Ethnopharmacological relevance Radix astragali (RA) was the most frequently used traditional Chinese medicine (TCM) according to the statistics on 52 anti-diabetic formulas recorded in New National Traditional Chinese Medicine; it was employed in 34 out of the 52 formulas. The aim of this study was to elucidate potential pharmacokinetic interaction between RA and pioglitazone, and to provide guidance for clinical medicine safety. Materials and methods A specific and rapid UPLC–MS/MS method was established to quantify pioglitazone in rat plasma. Then healthy and type 2 diabetes mellitus (T2DM) rats were each divided into control and RA decoction (RAD) administration groups—healthy, healthy-RAD, T2DM, T2DM–RAD; pharmacokinetics of pioglitazone was carried out after RAD was administrated to rats for 7 days. Results The established UPLC–MS/MS method was rapid, specific, and precise. Between healthy and healthy-RAD groups, half-life (T1/2), area under the curve (AUC 0–t), Vz/F, and Cl/F showed mild yet statistically significant differences; no significant difference for any above parameter was detected between T2DM and T2DM–RAD groups. Conclusion RAD co-administration did not affect the pharmacokinetics of pioglitazone especially in diabetic groups; RA and pioglitazone might be feasible herb–drug co-effectiveness.

Ethnopharmacological relevance Dipsaci radix, the dried root of Dipsacus asperoides C.Y. Cheng et al T.M.Ai is used as a medicinal plant in oriental clinics for the treatment of bone diseases and functions by strengthening bone and healing bone fractures. Aim of the study This study investigated the therapeutic efficacy of Dipsaci radix in treating rheumatoid arthritis using a type II collagen (CII)-induced arthritis (CIA) mouse model. Materials and methods Arthritis was induced in male DBA/1 mice by immunization with CII. Dipsaci radix water (DR-W) extract at 50 mg/kg and 100 mg/kg was orally administered from days to after the induction of arthritis. Arthritic score, serum levels of anti-CII IgG2a, the inflammatory mediator prostaglandin E 2 (PGE 2 ), and inflammatory cytokines (TNF-α, IL-1β and IL-6), and histological changes in the ankle joint were analyzed in CIA mice. Results Arthritic induction increased the arthritic score, as well as serum levels of anti-CII IgG2a antibody, PGE 2 , TNF-α, IL-1β and IL-6 in mice. However, administration of DR-W extract in CIA mice significantly reduced arthritic scores and serum levels of anti-CII IgG2a antibody, PGE 2 , TNF-α, IL-1β and IL-6 compared with those in vehicle-treated CIA mice. Furthermore, histopathological improvement in joint architecture was also observed in DR-W extract-treated CIA mice. Conclusions DR-W extract has anti-inflammatory and anti-arthritic effects in arthritic mice. This suggests that Dipsaci radix might be used as a therapeutic agent for the treatment of human arthritis.

Ethnopharmacological relevance Huang-Lian-Jie-Du decoction (HLJDD) (Oren-gedoku-to in Japanese) as a famous traditional Chinese recipe is composed of Rhizoma coptidis , Radix scutellariae , Cortex phellodendri and Fructus gardeniae . It has been used to treat inflammation for nearly two thousand years. Aim of the study To explore the material base for the anti-inflammatory activity of formula HLJDD, its extract was fractionated on D101 macroporous resin to afford two fractions, HLJDD-1 and HLJDD-2. The whole formula, HLJDD-1 and HLJDD-2, and four typical component compounds were then evaluated for their effects on inflammation-related parameters using lipopolysaccharide (LPS)-induced RAW264.7 cells as a model system. Materials and methods The effect of HLJDD on carrageenan-induced mice paw edema was first evaluated. A series of inflammation-related parameters including malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), prostaglandin E 2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) were then measured in LPS-induced RAW264.7 cells treated with HLJDD, its two fractions, and four typical component compounds (geniposide, baicalin, berberine and baicalein). Results With the help of principal component analysis (PCA) technique, the data obtained revealed that the two fractions and the major group of compounds in HLJDD (iridoids, flavonoids and alkaloids) complement each other with particular emphasis to synergistically exert anti-inflammatory effects. Conclusions This study demonstrated that HLJDD exhibited anti-inflammatory effect as a “whole”, which justified the combined use of the four component herbs forming the compound prescription and suggested quality control of HLJDD based on its three types of components.

An investigation was designed and conducted to compare the pharmacokinetics difference of paeoniflorin after oral administration of the extracts of Radix Paeoniae Rubra and Radix Paeoniae Alba to rats on separate occasions. Quantification of paeoniflorin in rat plasma was achieved using a simple and rapid HPLC method for pharmacokinetic study. After oral administration of decoctions of Radix Paeoniae Rubra and Radix Paeoniae Alba, paeoniflorin was absorbed and reached a maximum concentration of 3.69+/-1.46 and 1.46+/-0.29 (p

Huang-Lian-Jie-Du-Tang (HLJDT) is an important "heat-clearing" multiherb remedy of traditional Chinese medicine, and Radix scutellariae (Scutellaria baicalensis Georgi, Labiatae) is a key ingredient herb in it. Baicalin and wogonoside are two main effective ingredients enriched in Radix scutellariae. In the present study, pharmacokinetic differences of baicalin following oral administration of pure baicalin, Radix scutellariae extract, baicalin co-administrated with extract of the other three herbs of HLJDT and HLJDT were investigated in male S.D. rats with approximately the same dose of 200 mg/kg baicalin. The pharmacokinetic comparison of wogonoside was conducted only in Radix scutellariae extract and HLJDT. Plasma concentrations of baicalin and wogonoside were determined using HPLC method. Unpaired Student's t-test was used for statistical comparison. The results indicated that baicalin and wogonoside demonstrated bimodal phenomenon in the plasma profile. Some ingredients in the other three herbs of HLJDT, not in Radix scutellariae itself, had pharmacokinetic interaction with baicalin and wogonoside and hence decreased their systematic exposure level (p

Ethnopharmacological relevance Astragali radix is one of the well-known traditional Chinese herbal medicine, and possesses various biological functions, such as hepatoprotective and anticancer. In present study, to investigate the metabolism and pharmacokinetics of the major constituents of A. radix, a sensitive ultra-performance liquid chromatography–electrospray ionization–mass spectrometry (UPLC–MS/MS) method with shorter chromatographic running time was developed and validated for simultaneous quantification of formononetin, ononin, calycosin, calycosin-7-β-glucoside, astragaloside IV and their glucuronide metabolites in rat plasma after oral administration of water extract of A. radix at two different doses. Materials and methods The chromatographic separation was achieved on a C18 column with gradient elution by using a mixture of 0.1% formic acid aqueous solution and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. A tandem mass spectrometric detection was conducted using multiple-reaction monitoring (MRM) via electrospray ionization (ESI) source in positive ionization mode. Samples were pre-treated by a single-step protein precipitation with methanol, and erlotinib was used as internal standard (IS). Results The current UPLC–MS/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery, matrix effects and stability. The lowest limit of quantifications (LLOQ) were 1 ng/mL for all analytes. After oral administration, the plasma concentrations of the glucuronides, especially calycosin-3′-glucuronide, were much higher than the parent compounds. The mean half-life (t1/2) was between 1 and 5 h, and the metabolites were eliminated faster than the parent constituents. The median (range) time to reach maximum plasma concentration (Tmax) was between 0.5 and 1 h. Conclusions This is the first study of the pharmacokinetic study of bioactive compounds and their glucuronides in male rat plasma after oral administration of water extract of A. radix. The results demonstrated the biotransformation between the bioactive isoflavonoids and their glucuronides was extensive in rats and provided a significant basis for better understanding the absorption and metabolism mechanism of A. radix. Furthermore, this study could suggest that future studies should focus on the metabolites and biotransformation between the bioactive constituents when conducting a drug efficacy study.

Ethnopharmacological relevance Shenqi Fuzheng Injection (SFI) is an injectable traditional Chinese herbal formula comprised of two Chinese herbs, Radix codonopsis and Radix astragali, which were commonly used to improve immune functions against chronic diseases in an integrative and holistic way in China and other East Asian countries for thousands of years. Materials and methods This present study was designed to explore the bioactive components on immuno-enhancement effects in SFI using the relevance analysis between chemical fingerprints and biological effects in vivo. According to a four-factor, nine-level uniform design, SFI samples were prepared with different proportions of the four portions separated from SFI via high speed counter current chromatography (HSCCC). SFI samples were assessed with high performance liquid chromatography (HPLC) for 23 identified components. For the immunosuppressed murine experiments, biological effects in vivo were evaluated on spleen index (E1), peripheral white blood cell counts (E2), bone marrow cell counts (E3), splenic lymphocyte proliferation (E4), splenic natural killer cell activity (E5), peritoneal macrophage phagocytosis (E6) and the amount of interleukin-2 (E7). Based on the hypothesis that biological effects in vivo varied with differences in components, multivariate relevance analysis, including gray relational analysis (GRA), multi-linear regression analysis (MLRA) and principal component analysis (PCA), were performed to evaluate the contribution of each identified component. Results The results indicated that the bioactive components of SFI on immuno-enhancement activities were calycosin-7-O-β- d -glucopyranoside (P9), isomucronulatol-7,2′-di-O-glucoside (P11), biochanin-7-glucoside (P12), 9,10-dimethoxypterocarpan-3-O-xylosylglucoside (P15) and astragaloside IV (P20), which might have positive effects on spleen index (E1), splenic lymphocyte proliferation (E4), splenic natural killer cell activity (E5), peritoneal macrophage phagocytosis (E6) and the amount of interleukin-2 (E7), while 5-hydroxymethyl-furaldehyde (P5) and lobetyolin (P13) might have negative effects on E1, E4, E5, E6 and E7. Finally, the bioactive HPLC fingerprint of SFI based on its bioactive components on immuno-enhancement effects was established for quality control of SFI. Conclusions In summary, this study provided a perspective to explore the bioactive components in a traditional Chinese herbal formula with a series of HPLC and animal experiments, which would be helpful to improve quality control and inspire further clinical studies of traditional Chinese medicines.

Ethnopharmacological relevance Angelicae Dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav combined with Radix Scutellariae baicalensis Georgi has been widely used in traditional Chinese medicine (TCM) as an antipyretic analgesic and anti-inflammatory drug. Modern pharmacological studies have demonstrated that the compatible application of these two drugs is an effective treatment for hepatitis. A previous study indicated that a Radix Angelicae Dahuricae extract enhanced the intestinal absorption of the baicalin found in Radix Scutellariae ; however, the underlying compatibility mechanism of these two herbs remains unknown. In this study, we further examined the effect of a Radix Angelicae Dahuricae extract on the absorption and transport properties of baicalin in a Caco-2 cell model to determine the compatibility mechanism of these two herbs. Aim of the study The aim of this work was to study the transport properties of baicalin in Radix Scutellariae across cell membranes and the effects of a Radix Angelicae Dahuricae extract on baicalin absorption using the well-characterized, human-based intestinal Caco-2 cell model. Materials and methods We assessed the absorption, bidirectional transport and toxicity of baicalin using a range of parameters, including drug concentration, pH, a P-glycoprotein (P-gp) inhibitor (Verapamil), an MRP inhibitor (MK-571) and EDTA-Na 2 (tight junction modulator). Next, we studied the influence of a Radix Angelicae Dahuricae extract on the transport of baicalin under the same conditions. Drug concentration was measured by HPLC, and the apparent permeability coefficient (Papp) and apparent permeability ratio (PDR) were subsequently calculated. Results The results showed that baicalin is non-toxic within a concentration range of 800 µg/mL to 4800 µg/mL. The transport of baicalin showed time and concentration dependence. The absorption of baicalin was optimal at pH 7.4 in 37 °C; however, the absorption decreased at 4 °C. The Papp of baicalin transport through the Caco-2 cell monolayer model was altered when specific inhibitors of P-gp or MRP were added to the cells. However, there was no significant difference in the PDR value. The Papp of baicalin improved when it was combined with the Radix Angelicae Dahuricae extract. The influence of EDTA-Na 2 on the transport of baicalin showed that the permeability of baicalin significantly increased. The result further indicated that the mechanism of baicalin intestinal absorption in the Caco-2 cell monolayer involves passive transcellular diffusion. Conclusions Passive diffusion is the main mode of intestinal absorption of bacalin and it involved in the efflux of proteins. The enhanced intestinal absorption of baicalin by Radix Angelicae Dahuricae can be due to opening of the tight junctions between cells and inhibition of MRP efflux protein expression or function.

Ethnopharmacological relevance Radix Aucklandiae (RA), a well known traditional Chinese medicine, is widely used for treating various problems in digestive system. A selective and sensitive high-performance liquid chromatography coupled with mass spectrometry method was first developed and validated for simultaneous quantification of costunolide and dehydrocostuslactone in rat plasma with diazepam as internal standard after oral administration of RA extraction. Materials and methods Plasma samples were extracted via solid-phase extraction and detected by multiple-reaction monitoring mode under positive electrospray. Chromatographic separation was accomplished on an Agilent C18 column (2.1 mm×150 mm, 5 µm), with 0.1% formic acid and acetonitrile (1:1) as the mobile phase at a flow rate of 0.5 mL/min. Results The quantification was performed using the transitions of m/z 233/187 for costunolide, m/z 231/185 for dehydrocostuslactone and m/z 285/193 for diazepam, respectively. Calibration curves were linear over the concentration range of 0.7–769.7 ng/mL for costunolide and 0.9–956.0 ng/mL for dehydrocostuslactone. The intra-day and inter-day precisions (RSD%) for two compounds was less than 8.76% and 9.70% and the accuracy (RE%) range from 6.14% to 5.35%. The time to reach the maximum plasma concentration (Tmax) was 10.46 h for costunolide, 12.39 h dehydrocostuslactone. The elimination half-time (t1/2) of costunolide and dehydrocostuslactone was 5.54±0.81 and 4.32±0.71 (h). The AUC of costunolide and dehydrocostuslactone was 308.83 and 7884.51 respectively (ng h/mL). Conclusions It was the first report for the study of pharmacokinetic profile of costunolide and dehydrocostuslactone in rat plasma after oral administration of RA extract. These results provided a meaningful basis for better understanding the absorption of traditional medicine, RA, and provide useful scientific data for clinical application.

The processed radix polygoni multiflori (P-RPM) are produced from the raw radix polygoni multiflori (R-RPM) steamed with black bean juice, but the two traditional Chinese medicines are used to treat the different diseases in clinic. In order to clarify the influence of processing on pharmacological properties of radix polygoni multiflori, an investigation was carried out to compare the pharmacokinetics of typical constituents after oral administration of P-RPM and R-RPM extractsA simple, rapid and sensitive high performance liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the simultaneous determination of gallic acid, polydatin, 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside (PM-SG), resveratrol, and emodin in rat plasma. Male Sprague-Dawley rats were orally administered the two extracts with approximately the same dosage.It was found that gallic acid was distributed as opened one-compartment model while polydatin, PM-SG and emodin were fitted to an open two-compartment model after oral administration of raw and processed radix polygoni multiflori extract. Cmax and AUC of gallic acid were increased (P0.01), but Cmax and AUC of PM-SG were descreased (P0.05). AUC of polydatin and emodin were similar with that of PM-SG. However, resveratrol was not detected in plasma collected at certain intervals following oral administration of the two extracts.These results indicate that influence of the processing could improve the bioavailability of gallic acid and reduce the absorption of PM-SG, polydatin and emodin in rats. The LC-MS/MS method could be used to evaluate the effect of processing on pharmacokinetic of typical constituents in radix polygoni multiflori after oral administration.

Ethnopharmacological relevance Licorice (Glycyrrhiza uralensis Fisch., Leguminosae) has been used in herbal medicine and food supplement worldwide for centuries. Licorice is a common ingredient of several prescriptions of traditional Chinese medicine which have been proved to inhibit infection of human respiratory syncytial virus (HRSV). There are two preparations of licorice, Radix Glycyrrhizae and Radix Glycyrrhizae Preparata. However, it is unknown whether licorice or which preparation of licorice is effective against HRSV, nor is its active constituent. Aim of the study We tested the hypothesis that Radix Glycyrrhizae can effectively decrease HRSV-induced plaque formation in respiratory mucosal cell lines. We also tried to find out the active constituent. Materials and methods Anti-HRSV activities of hot water extracts of preparations of licorice, glycyrrhizin and 18β-glycyrrhetinic acid (18β-GA), the active constituents of licorice, were examined by plaque reduction assay in both human upper (HEp-2) and low (A549) respiratory tract cell lines. Abilities of crude licorice to inhibit viral replication and to stimulate IFN-β were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Results Radix Glycyrrhizae and Radix Glycyrrhizae Preparata dose-dependently inhibited HRSV-induced plaque formation in both HEp-2 and A549 cell lines (p, Graphical abstract

Shakuyakukanzoto (SKT) composed of Glycyrrhizae radix (G. radix) and Paeoniae radix (P. radix) has been traditionally used in Japan, Korea and China as an antispasmodic drug for the treatment of skeletal muscle cramps and intestinal cramps.To evaluate the antispasmodic activity of SKT and its two components, as well as to identify the key constituents of the components which mediate this effect in skeletal muscles in vivo.An experimental cramp model was constructed to evaluate the effects of peripherally-acting muscle relaxants on electrically-induced cramps under physiological conditions. This was accomplished by surgically isolating the motor supply to the gastrocnemius muscle in an anesthetized rat and delivering electrical stimuli to an isolated tibial nerve to induce tetanic contractions. We first tested dantrolene, a well-known peripherally-acting relaxant, to determine the sensitivity and reliability of our experimental model. We then evaluated the effects of SKT, P. radix, G. radix, and the eight active constituents of G. radix against tetanic contractions.We found that dantrolene (10 and 30 mg/kg, i.d.) rapidly and significantly inhibited tetanic contractions (P0.01) irrespective of dose. SKT (0.5, 1.0, and 2.0 g/kg, i.d.) and G. radix (0.5 and 1.0 g/kg, i.d.) also significantly inhibited tetanic contractions (P0.01) but in a dose-dependent manner owing to the actions of six of the eight active constituents in G. radix (liquiritin apioside, liquiritigenin, isoliquiritin apioside, isoliquiritigenin, glycycoumarin, and glycyrrhetinic acid, 20 μmol/kg, i.v.). These constituents, which include flavonoids, a triterpenoid, and a courmarin derivative, demonstrated temporal variations in their inhibitory activity. In contrast, P. radix (0.5 and 1.0 g/kg, i.d.) did not show a statistically significant antispasmodic effect in our study; however, we previously found that it had a significant antinociceptive effect.Our findings show that SKT inhibits tetanic contractions in vivo and that G. radix is the main antispasmodic component due to the actions of its active constituents, thus supporting the traditional use of SKT. We further propose that SKT containing the antispasmodic G. radix and antinociceptive P. radix is a pharmaceutically elegant option for muscle cramps as treatment requires a two-pronged approach, i.e., inhibition of hyperexcitable skeletal tissues and modulation of the pain accompanying cramps.