Preparation of Rubra (Paeonia lactiflora Pall.) extract and studies in activity and skin penetration.

The inhibition rate of tyrosinase activity was used to determine extraction solvent of Paeoniae Radix Rubra extract (PRRE), which was established quality control standards by HPLC and verified the antioxidant activity. Ternary phase diagram was used to screen the best formulation of PRRE nanoemulsion, the skin permeability of PRRE and nanoemulsion were compared. The results show that 70% ethanol as the extraction solvent were highest (88.89%) and the contents of catechin (CC) and paeoniflorin (PF) in PRRE were 0.145 ± 0.0006 µg/mg and 21.783 ± 0.0247 µg/mg, respectively. The inhibition rate of PRRE on pyrogallol autoxidation was 6.94% ± 0.53%. The optimal formulation is Isopropyl myristate (IPM) as oil phase, Ethoxylated hydrogenated castor oil (RH40) as emulsifier, glycerine as coemulsifier, Km 3:1. The skin penetration of CC in PRRE nanoemulsion (0.79 ± 0.04 µg·cm-2) was significantly higher than that PRRE (0.17 ± 0.09 µg·cm-2) after 12 h.

Paeoniae radix rubra:A review of ethnopharmacology, phytochemistry, pharmacological activities, therapeutic mechanism for blood stasis syndrome, and quality control.

Paeoniae Radix Rubra (PRR) known as Chishao, in China, is the dried root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, with a history of over 2000 years in traditional Chinese medicine, is employed to clear heat, cool the blood, dispel blood stasis, and alleviate pain. Phytochemical investigations identified 264 compounds that contained monoterpenes and their glycosides, sesquiterpenes, triterpenes, steroids, flavonoids, lignans, tannins, volatile oils, and other compounds. It has been reported to have different pharmacological activities, including cardiovascular-protective, antidepressive, neuroprotective, antitumor, hepatoprotective, and anti-inflammatory effects. This study offers a comprehensive review covering ethnopharmacology, phytochemistry, pharmacological activities, therapeutic mechanism for blood stasis syndrome, and quality control of PRR. The comprehensive analysis aims to achieve a thorough understanding of its effects and serves as a foundation for future research and development.

A systematic review and meta-analysis of the anti-tumor effects of Paeoniae Radix Rubra in animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniae Radix Rubra (PRR) is the dried root of Paeonia lactiflora Pall, which has been widely used to anti-thrombotic, lipid-lowering, anti-spasmodic, antioxidant, antibacterial, hepatoprotective, and anti-tumor in Chinese clinical practice. Recent research has demonstrated that PRR plays a significant anti-tumor role in animal models of tumor-bearing. AIM OF THE STUDY: There has not been the evaluation of the anti-tumor effects of PRR. This study conducts a meta-analysis to assess the anti-tumor efficacy of PRR on animal models, providing scientific evidence for clinical application of PRR in the adjuvant therapy of tumors. MATERIALS AND METHODS: English databases (PubMed, The Cochrane Library, Embase, and Web of Science) and Chinese databases (CNKI, WanFang, SinoMed, CTSJ-VIP) were used to search all pertinent animal studies investigating the anti-tumor effects of PRR and its extracts. The quality of the included studies was evaluated using the SYRCLE animal experiment risk assessment tool, and statistical analysis was carried out using Revman 5.3 software. Egger's test and funnel plots were used to assess potential publication bias in the studies. RESULTS: The initial search produced a total of 3905 potentially pertinent studies, and 24 studies met the inclusion criteria. These studies included animal tumor models of hepatocellular carcinoma, lung cancer, sarcoma, bladder cancer, leukemia, colon cancer, glioblastoma, and pancreatic cancer. The meta-analysis findings demonstrated that both PRR and its extracts significantly inhibited tumor growth in animals. Compared with the control group, PRR substantively inhibited tumor volume (SMD, -3.09; 95% CI, [-4.05, -2.13]; P < 0.0001), reduced tumor weight (SMD, -1.08; 95% CI, [-1.37, -0.78]; P < 0.0001), decreased tumor number (SMD, -2.16; 95% CI, [-3.45, -0.86]; P = 0.001), and prolonged the survival duration time (SMD, 0.97; 95% CI, [0.23, 1.71]; P = 0.01) on the experimental animals. CONCLUSIONS: PRR displayed a potential therapeutic efficacy on eight tumors in animal models including hepatocellular carcinoma, lung cancer, sarcoma, bladder cancer, leukemia, colon cancer, glioblastoma, and pancreatic cancer. However, the quality and quantity of included studies may affect the accuracy of positive results. In the future, more high-quality randomized controlled animal experiments are need for meta-analysis.

Chishao (Paeoniae Radix Rubra) alleviates intra-hepatic cholestasis by modulating NTCP in rats.

Background: Cholestasis is a common pathological manifestation dominated by accumulation of potentially toxic biliary compounds. Na+-taurocholate cotransporting polypeptide (NTCP) plays a critical role in protection from cholestasis and can be targeted therapeutically. Chishao (Paeoniae Radix Rubra) is a clinically efficacious agent for treating cholestasis, but the underlying mechanism has not been fully clarified. Objective: To evaluate the effects of Chishao on the expression of NTCP in rats with alpha-naphthylisothiocyanate (ANIT)-induced cholestasis. Methods: Chishao extracts were obtained by water decoction. Cholestasis model induced by ANIT in rats were established. Thirty rats were divided into five groups: control group (C), ANIT model group (M), 10 g/kg Chishao group (LD), 20 g/kg Chishao group (MD) and 40 g/kg Chishao group (HD). The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), direct bilirubin (DB), alkaline phosphatase (ALP) and total bile acid (TBA) were detected. The mRNA and protein expression of NTCP, multidrug resistance associated protein 2 (MRP2) and bile salt export pump (BSEP) were detected by reverse transcription qPCR and Western blotting respectively. To assess the effects of Chishao on NTCP, MRP2 and BSEP localized at the membrane of hepatocytes, an in vitro experiment involving primary hepatocytes was conducted via the utilization of laser scanning confocal microscopy. Results: The extracts of Chishao significantly improved serum ALT, AST, ALP, TB, DB and TBA (p < 0.05), especially ALP in the HD group (p < 0.01). The histological pathological findings were also reversed in LD, MD and HD groups. The mRNA level of MRP2 was significantly downregulated after treatment with ANIT, whereas it was reversed in MD and HD groups (p < 0.05). The mRNA expression of NTCP was significantly downregulated after ANIT treatment, but dramatically upregulated in the HD group. The expressions of BSEP and MRP2 were similar, but that of NTCP decreased after ANIT treatment, which was reversed significantly by Chishao extracts in a dose-dependent manner. The expression of NTCP in hepatocytes from rats increased dose-dependently after Chishao treatment in vitro. Conclusion: Chishao extracts can improve the serum and histological performances of intra-hepatic cholestasis caused by ANIT, probably by working on transport proteins in liver cell membranes.

Paeoniae Radix Rubra extract attenuates cerebral ischemia injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, has been widely used to promote blood circulation and eliminate blood stasis in Chinese clinical practice, but its effect on cerebral ischemia is still rarely reported. AIM OF THE STUDY: The present study aimed to assess the potential therapeutic possibilities of the extract of PRR (PRRE) on cerebral ischemia, further exploring the underlying mechanism, and preliminary screening of the corresponding active components. MATERIALS AND METHODS: The neuroprotective effects of PRRE in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO) injury and mouse hippocampal neuronal cells (HT22 cell line) following oxidative stress were confirmed. The mechanism was investigated using immunohistochemical staining, western blotting, transmission electron microscopy (TEM), and immunofluorescence. The active components of PRRE were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecular docking. RESULTS: The in vivo study showed that PRRE reduced infarct volume and improved neurological deficits in rats, and the expression of GPX4, FTH1, Beclin1, LC3 II, and p-Akt was upregulated in the rat hippocampi. In addition, the vitro research indicated that PRRE can also alleviate H2O2-induced HT22 cell damage by regulating cytokines such as malondialdehyde (MDA), reduced glutathione (GSH) and reactive oxygen species (ROS), and the expressions of GPX4 and Beclin1 were observed to be elevated. The PI3K/Akt signalling pathway was inhibited by LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K). Furthermore, the effective components of PRRE in regulating ferroptosis and autophagy are mainly defined as albiflorin, paeoniflorin, benzoyl paeoniflorin, oleanolic acid, and hederagenin. CONCLUSION: PRRE exerts neuroprotective effects against cerebral ischaemic injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway. This study provides an experimental basis for the potential application of PRRE as a novel therapeutic drug, and PI3K/Akt-associated ferroptosis and autophagy as therapeutic targets for cerebral ischemia.

[Monoterpenoids from Paeoniae Radix Rubra based on UPLC-Q-TOF-MS].

The ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to conduct the qualitative analysis of the monoterpene chemical components from Paeoniae Radix Rubra. Gradient elution was performed on C_(18) HD(2.1 mm×100 mm, 2.5 µm) column with a mobile phase of 0.1% formic acid(A) and acetonitrile(B). The flow rate was 0.4 mL·min~(-1) and the column temperature was 30 ℃. MS analysis was conducted in both positive and negative ionization modes using electrospray ionization(ESI) source. Qualitative Analysis 10.0 was used for data processing. The identification of chemical components was realized by the combination of standard compounds, fragmentation patterns, and mass spectra data reported in the literature. Forty-one monoterpenoids in Paeoniae Radix Rubra extract were identified. Among them, 8 compounds were reported in Paeoniae Radix Rubra for the first time and 1 was presumed to be the new compound 5″-O-methyl-galloylpaeoniflorin or its positional isomer. The method in this study realizes the rapid identification of monoterpenoids from Paeoniae Radix Rubra and provides a material and scientific basis for quality control and further study on the pharmaceutical effect of Paeoniae Radix Rubra.

The mechanism of action of paeoniae radix rubra-angelicae sinensis radix drug pair in the treatment of rheumatoid arthritis through PI3K/AKT/NF-κB signaling pathway.

Objective: To investigate the effects and mechanisms of Paeoniae radix rubra-Angelicae sinensis radix (P-A) drug pair in the treatment of rheumatoid arthritis (RA). Methods: Mass spectrometry was employed to accurately characterize the main components of the P-A drug pair. Network pharmacology was used to analyze the main components and pathways of the P-A drug pair in the treatment of RA, and Discovery Studio software was used to molecularly dock the key proteins on the pathway with their corresponding compounds. The levels of serum TNF-a, IL-1ß, and IL-6 were measured by enzyme linked immunosorbent assay (ELISA). The histopathology of the ankle joint was observed by hematoxylin-eosin (HE) staining, and the positive expression of p-PI3K, p-IKK, p-NF-κB, and p-AKT in the synovial tissue of the ankle joint was detected by immunohistochemical analysis. Finally, the expression of PI3K, IKK, and AKT and their phosphorylation levels were determined by western blot in each group of rats. Results: Network pharmacology combined with molecular docking analysis revealed that the pharmacodynamic mechanism of the P-A drug pair for the treatment of RA may be related to the contents of caffeic acid, quercetin, paeoniflorin, and baicalein in the regulation of the expression of the PI3K/AKT/NF-κB signaling pathway and the targets of PIK3CA, PIK3R1, AKT1, HSP90AA1 and IKBKB in the pathway. Compared with the model group, the P-A drug pair significantly improved the pathological changes of the synovial tissue and reduced feet swelling in RA model rats. Moreover, it regulated the levels of TNF-α, IL-1ß, and IL-6 in serum (p < 0.05). The results of the immunohistochemical analysis and western blot showed that the expression of PI3K, IKK, NF-κB, and AKT decreased after phosphorylation in the synovial tissue (p < 0.05). Conclusion: The P-A drug pair exhibited an inhibitory effect on the hyperactivation of the PI3K/AKT/NF-κB signaling pathway in the synovial membrane of RA rats. The mechanism may be related to the downregulation of the phosphorylation levels PI3K, IKK, NF-κB, and AKT, which in turn decreased inflammatory cell infiltration and synovial membrane proliferation.

First Report of Root Rot caused by Coniella fragariae on Paeoniae radix Rubra in China.

Paeoniae radix Rubra is a traditional Chinese herbal medicine, which has the effect of clearing heat and cooling blood, activating blood and removing stasis. It has become popular in the Chinese market in recent years due to its extremely high medicinal value and showy flower color. In May 2021, typical symptoms of root rot were observed in a field (35°7'12″ N, 103°58'48″ E) in Dingxi, Gansu province, China. Approximately 10% of the plants in the field had typical root rot symptoms, and the root of each affected plant is at least 5% severe. The roots of the naturally infected plants in the field discolored and decayed with black brown spots on the surface of the root bark, the root bark detached from the phloem，and some leaves were chlorosis, shrunken and smaller, and the branches were dead and underdeveloped. In the transverse section, the xylem was black diffusion and abnormal odor. Three diseased plants with typical symptoms were chosen at random and brought back to the lab. Small pieces cut from the margins of lesions were surface disinfested with 75% ethanol for 15 s, and 0.5% NaClO solution for 30 s, rinsed three times in sterile distilled water, dried on sterile filter paper, plaed onto potato dextrose agar (PDA), and incubated at 25 ± 1℃ for 7 days in the dark. The pure cultures were obtained by single-spore isolation. All isolates produced wavy on the surface, radial from the inside out, initially white or milky white to orange colonies with abundant black brown oily conidiomata pycnidia on PDA at 25 ± 1℃ after 15 days in the dark. The conidiomata pycnidia is spherical to irregularly spherical, 231.5 to 512.4 µm, initially transparent with age turning brown, with a dark brown internal conidial mass inside, and with a 13.1 to 45.4 µ m wide ostiole central. Young conidia (n=100) developed from conidiogenous cells, which were simple, tapering, hyaline, smooth, and 12.3 to 18.0 × 2.5 to 4.6 µm, 1.0 to 1.5 µm wide at apex. Mature conidia (n=100) were ellipsoid, apices tapering, subobtusely rounded, brown, and 6.5 to 11.0 × 4.1 to 7.5 µm. The morphological characteristics of the isolates were consistent with previous descriptions of the genus Coniella (Crous et al., 2014). A representation isolate CS-1 was deposited in the Institute of Plant Protection, Gansu Academy of Agricultural Sciences and used for further studies. To confirm the identity of the causal fungus, the internal transcribed spacer (ITS), 28S large subunit of nuclear ribosomal RNA (LSU) and partial translation elongation factor 1-alpha (TEF1-α) gene of the representative isolate CS-1 were amplified and sequenced using primers ITS1/ITS4 (White et al., 1990), LROR/LR7 (Chethan et al., 2017) and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively, and deposited on GenBank with accession numbers OP824764 (ITS), OP824767 (LSU)/span>and OP903926 (TEF1-α). Blastn analysis of all sequences resulted in E-value of 0.0 (ITS and LSU) and nearly 0.0 (TEF1-α), with Query cover values of 90% to 99% identity with C. fragariae, confirming the hypothesis based on morphological features examination. To conduct a pathogenicity test, three root segments of healthy plants were wounded using sterilized needles and inoculated by pipetting 10 µL of conidial suspension (1×107 conidia/mL) onto each wound, and controls were inoculated with 10 µL sterile distilled water. These root segments were kept in a moist chamber at 25°C in the dark. The experiment was repeated three times. After 14 days, root rot symptoms were observed on all of the inoculated root segments and identical to those observed in the field, whereas control root segments did not develop symptoms. The pathogen was re-isolated from the lesions of inoculated root segments, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of C. fragariae causing root rot on P. radix Rubra in China. This identification can aid in the selection of appropriate management measures for this disease.

Effect of Paeoniae Radix Rubra (Paeonia lactiflora Pall.) extract on mucin secretion, gene expression in human airway epithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall., is a traditional Chinese medicine which has the effects of regulating various inflammatory diseases, treating blood stasis, and enhancing blood circulation. AIM OF THE STUDY: This study examined whether Paeoniae Radix rubra extract (PRRE) and Paeoniflorin (PF) affect mucin production, gene expression including MUC5AC, and protein expression related to the ERK pathway induced by TNF-α from human airway epithelial cells. MATERIALS AND METHODS: NCI-H292 cells induced by TNF-α were treated with each agent. MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription polymerase chain reaction, staining, and enzyme-linked immunosorbent assay. Western blot was used to investigate the cell signaling pathways. RESULTS: PRRE and PF inhibited the production of MUC5AC mucin protein and gene expression in TNF-α-induced H292 cells. In Western blot, PRRE was involved in protein expression related to the ERK pathway. CONCLUSIONS: Overall, PRRE effectively inhibited the MUC5AC, and inflammatory cytokines expression caused by TNF-α, which was closely involved in the ERK pathway. PRRE may have the potential for treating mucus producing respiratory inflammation.

Monoterpenoids from Paeoniae Radix Rubra based on UPLC-Q-TOF-MS / 中国中药杂志

The ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to conduct the qualitative analysis of the monoterpene chemical components from Paeoniae Radix Rubra. Gradient elution was performed on C_(18) HD(2.1 mm×100 mm, 2.5 μm) column with a mobile phase of 0.1% formic acid(A) and acetonitrile(B). The flow rate was 0.4 mL·min~(-1) and the column temperature was 30 ℃. MS analysis was conducted in both positive and negative ionization modes using electrospray ionization(ESI) source. Qualitative Analysis 10.0 was used for data processing. The identification of chemical components was realized by the combination of standard compounds, fragmentation patterns, and mass spectra data reported in the literature. Forty-one monoterpenoids in Paeoniae Radix Rubra extract were identified. Among them, 8 compounds were reported in Paeoniae Radix Rubra for the first time and 1 was presumed to be the new compound 5″-O-methyl-galloylpaeoniflorin or its positional isomer. The method in this study realizes the rapid identification of monoterpenoids from Paeoniae Radix Rubra and provides a material and scientific basis for quality control and further study on the pharmaceutical effect of Paeoniae Radix Rubra.

Study on the mechanism of Danpi-Chishao in the treatment of sepsis based on network pharmacology / 中国医师杂志

Objective:To analyze the mechanism of Danpi-Chishao in treatment of sepsis based on network pharmacology.Methods:The corresponding targets of Danpi-Chishao and sepsis were carried out through TCMSP database, OMIM database and Genecards database. Cystoscope 3.8.2 software was used to construct the " Chinese medicine-active components-target-disease" network diagram. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were carried out by DAVID database. Weisheng cloud platform was used to draw bubble map.Results:A total of 36 effective components of Danpi-Chishao was obtained, mainly including quercetin, kaempferol, baicalin, β-sitosterol, stigmasterol, paeoniflorin and so on. There were 96 potential common key targets between Danpi-Chishao and sepsis, such as prostaglandin-endoperoxide synthase 2 (PTGS2), transcription factor p65 (RELA), phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG), B-cell lymphoma 2 (BCL-2)-associated X (BAX), BCL-2, Caspase-3 (CASP3) with a degree value>4.9. The result of protein-protein interaction (PPI) network analysis showed that there were 10 important target proteins, including alpha serine/threonine-protein kinase (AKT1), interleukin-6 (IL-6), tumor necrosis factor (TNF), interleukin-1β (IL-1β), vascular endothelial growth factor A (VEGFA), cellular tumor antigen p53 (TP53), matrix metalloproteinase-9 (MMP9), CASP3, PTGS2, C-C motif chemokine ligand 2 (CCL2). The pathways obtained by GO and KEGG enrichment analysis included atherosclerosis pathway, advanced glycation end products (AGE)-receptor for advanced glycation end products (RAGE) signal pathway, cancer pathway, tumor necrosis factor signal pathway, hypoxia-inducible factor (HIF) signal pathway, IL-17 signal pathway and other pathway.Conclusions:The mechanism of the intervention effect of Danpi-Chishao on sepsis may be that the active components such as quercetin, kaempferol, paeoniflorin act on target proteins such as PTGS2, RELA, PIK3CG, BAX, BCL2, CASP3, and through TNF-related signal pathway, HIF-1 signal pathway, IL-17 signal pathway, etc. Nonetheless, the conclusion needs further experimental verification.

Paeoniae Radix Rubra can enhance fatty acid ß-oxidation and alleviate gut microbiota disorder in α-naphthyl isothiocyanate induced cholestatic model rats.

Cholestasis is the most destructive pathological manifestation of liver disease and available treatments are very limited. Paeoniae Radix Rubra (PRR) is an important traditional Chinese drug used to treat cholestasis. This study combined targeted metabonomics, PCR array analysis, and 16S rRNA sequencing analysis to further clarify the mechanisms of PRR in the treatment of cholestasis. PRR conspicuously reversed the elevation of fatty acids (FFA 14:0 and other 14 fatty acids) and the decrease of organic acids (pyruvic acid and citric acid) in a cholestatic model induced by α-naphthyl isothiocyanate (ANIT). Eight elevated amino acids (L-proline, etc.) and five elevated secondary bile acids (taurohyodeoxycholic acid, etc.) in model rats were also reduced by PRR. Pathway analysis revealed that PRR significantly alleviated eight pathways (ß-alanine metabolism). Furthermore, we found that PRR significantly reversed the decrease of Cpt1a, Hadha, Ppara, and Slc25a20 (four genes relevant to fatty acid ß-oxidation) mRNAs caused by ANIT, and PRR conspicuously decreased nine acylcarnitines (the forms of fatty acids into mitochondria for ß-oxidation) that increased in model rats. These results indicate that PRR could enhance fatty acid ß-oxidation, which may be the way for PRR to reduce the levels of 15 fatty acids in the serum of model rats. 16S rRNA sequencing analysis revealed that PRR alleviated gut microbiota disorders in model rats, including upregulating four genera (Coprococcus, Lactobacillus, etc.) and downregulating four genera (Bacteroides, Escherichia, etc.). As the relative abundance of these eight genera was significantly correlated with the levels of the five secondary bile acids (deoxycholic acid, taurolithocholic acid, etc.) reduced by PRR, and Bacteroides and Escherichia were reported to promote the production of secondary bile acid, we inferred that the downregulation of PRR on five secondary bile acids in model rats was inseparable from gut microbiota. Thus, the gut microbiota also might be a potential pharmacological target for the anticholestatic activity of PRR. In conclusion, we consider that the mechanisms of PRR in treating cholestasis include enhancing fatty acid ß-oxidation and alleviating gut microbiota disorders.

Metabolism of Paeoniae Radix Rubra and its 14 constituents in mice.

Objective: Paeoniae Radix Rubra (PRR) is a commonly used traditional Chinese medicine with the effects of clearing away heat, cooling the blood, and relieving blood stasis. To 1) elucidate the metabolites and metabolic pathways of PRR and its 14 main constituents in mice and 2) reveal the possible origins of the known effective forms of PRR and their isomers, the metabolism of PRR in mice was systematically studied for the first time. Methods: PRR and its 14 constituents were administered to mice by gavage once a day for seven consecutive days, respectively. All urine and feces were collected during the 7 days of dosing, and blood was collected at 1 h after the last dose. Metabolites were detected and identified using high performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (HPLC-DAD-ESI-IT-TOF-MSn). Results: In total, 23, 16, 24, 17, 18, 30, 27, 17, 22, 17, 33, 3, 8, 24, and 31 metabolites of paeoniflorin, albiflorin, oxypaeoniflorin, benzoylpaeoniflorin, hydroxybenzoylpaeoniflorin, benzoyloxypaeoniflorin, galloylpaeoniflorin, lactiflorin, epicatechin gallate, catechin gallate, catechin, ellagic acid, 3,3'-di-O-methylellagic acid, methylgallate, and PRR were respectively identified in mice; after eliminating identical metabolites, a total of 195 metabolites remained, including 8, 11, 25, 17, 18, 30, 27, 17, 21, 17, 1, 2, 8, 20, and 20 newly identified metabolites, respectively. The metabolic reactions of PRR and its 14 main constituents in mice were primarily methylation, hydrogenation, hydrolysis, hydroxylation, glucuronidation, and sulfation. Conclusion: We elucidated the metabolites and metabolic pathways of PRR and its 14 constituents (e.g., paeoniflorin, catechin, ellagic acid, and gallic acid) in mice and revealed the possible origins of the 10 known effective forms of PRR and their isomers. The findings are of great significance to studying the mechanism of action and quality control of PRR.

Elucidation of the Mechanisms and Effective Substances of Paeoniae Radix Rubra Against Toxic Heat and Blood Stasis Syndrome With a Stage-Oriented Strategy.

In the clinical practice of traditional Chinese medicine, toxic heat and blood stasis syndrome (THBSS) is a common syndrome observed in various critical diseases. Paeoniae Radix Rubra (PRR) has known therapeutic effects on THBSS. However, its pharmacodynamic mechanisms and effective substances in the treatment of THBSS still need further elucidation. Our previous study indicated that THBSS had three stages of progression, and the abnormal biochemical indices of each stage were different. Therefore, this study aimed to elucidate the pharmacodynamic mechanisms and effective substances of PRR for the treatment of THBSS with a stage-oriented strategy. Specifically, research was performed separately in two stable stages of THBSS: the excessive heat and little blood stasis (EHLBS) and blood stasis (BS) stages. THBSS model rats, at different time periods after syndrome initiation (first 5 h for EHLBS and 24 h later for BS), were used to conduct the two-stage investigation. Targeted metabonomics analysis was performed to elucidate the pharmacodynamic mechanisms of PRR in the treatment of EHLBS or BS. Based on the relationship between the individual differences in blood drug concentrations and pharmacodynamic effects, partial least squares regression analysis was employed to screen for the effective substances from the original constituents and metabolites of PRR. We found that PRR could upregulate primary bile acid biosynthesis, glycerophospholipid metabolism, ether lipid metabolism, and five amino acid metabolic pathways (e.g., arginine and proline metabolism) to treat EHLBS. Meanwhile, PRR alleviated BS by upregulating primary bile acid biosynthesis and downregulating glycerophospholipid metabolism. But PRR had no obvious effects on ether lipid metabolism and amino acid metabolism in this stage. In total, 17 and 9 potential effective substances were found in the EHLBS and BS stages, respectively, among which there were only five common compounds between the two stages. To our knowledge, sixteen compounds were regarded as potential effective substances of PRR for the first time. Therefore, the pharmacodynamic mechanisms and effective substances of PRR in the treatment of EHLBS and BS were partly different. Overall, this stage-oriented strategy provides a new way to study the pharmacodynamic mechanisms and effective substances of traditional Chinese drugs.

Mechanism of Scutellariae Radix-Paeoniae Radix Rubra Combination of Different Proportions Against Hepatic Fibrosis in Rats / 中国实验方剂学杂志

ObjectiveTo observe the effects of Scutellariae Radix （SR）-Paeoniae Radix Rubra （PRR） combination of different proportions on the expression of the Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear transcription factor κB （NF-κB） and phosphatidylinositol kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathways in liver tissues of rats with hepatic fibrosis and explore the mechanism against hepatic fibrosis. MethodSixty male SD rats of SPF grade were randomly divided into a normal group， a model group， a positive control （silymarin） group， and SR-PRR 1∶1， SR-PRR 1∶2， and SR-PRR 1∶4 groups， with 10 rats in each group. The hepatic fibrosis model was induced in rats except for those in the normal group by intraperitoneal injection of 40% tetrachloromethane （CCl4）-olive oil solution at 3 mL·kg-1， 5 mL·kg-1 for the first time， for 8 weeks， twice per week. After 4 weeks， rats were treated correspondingly at 10 mL·kg-1 by intragastric administration， and the body weight of rats in each group was weighed for 8 weeks. After administration， histopathological changes in the liver were observed. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， hyaluronic acid （HA）， laminin （LN）， albumin （ALB）， alkaline phosphatase （AKP）， and superoxide dismutase （SOD） activities， malondialdehyde （MDA）， and hydroxyproline （HYP） content in liver tissues were detected. The mRNA expression levels of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats were detected by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the model group， SR-PRR combination of different proportions could recover the body weight and improve the pathological injury of the liver. As revealed by enzyme linked immunosorbent assay （ELISA） results， compared with the normal group， the model group showed increased ALT， AST， HA， LN， AKP， MDA， and HYP levels to different degrees （P<0.05）. Compared with the model group， the groups with drug intervention showed decreased levels of ALT， AST， HA， LN， AKP， MDA， and HYP， potentiated SOD activity， and increased level of ALB （P<0.05）. As revealed by Real-time PCR results， compared with the normal group， the model group showed increased mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR （P<0.05）. Compared with the model group， the groups with drug intervention showed reduced mRNA expression of TLR4， MyD88， NF-κB， PI3K， Akt， and mTOR in the liver of rats （P<0.05）. ConclusionSR-PRR combination of different proportions can improve the histopathological injury in liver tissues caused by CCl4， with the optimal effect observed in the SR-PRR 1∶4 group. SR-PRR may inhibit the development of liver fibrosis by inhibiting the expression of TLR4/MyD88/NF-κB and PI3K/Akt/mTOR signaling pathways， thereby alleviating chemical-induced liver injury.

Aqueous extract of Paeoniae Radix Rubra prevents deep vein thrombosis by ameliorating inflammation through inhibiting GSK3ß activity.

BACKGROUND: Deep vein thrombosis (DVT) is a kind of blood stasis syndrome. Paeoniae Radix Rubra (PRR) has long been widely used for eliminating blood stasis in China, but its effect on DVT has not yet been reported. PURPOSE: The present study aimed to assess the potential inhibitory effect of the aqueous extract of PRR (i.e.,PRR dispensing granule, PRRDG) on DVT and explore the underlying mechanism. STUDY DESIGN/METHODS: The chemical profile of PRRDG was analyzed by high-performance liquid chromatography. Sprague-Dawley rats were intragastrically treated with PRRDG (0.625, 1.25 and 1.875 g crude drug/kg/d) once daily for 7 consecutive days. On the sixth day, a model of inferior vena cava (IVC) stenosis-induced DVT was established. All rats were sacrificed on the seventh day. Serum was collected for enzyme-linked immunosorbent assay. Thrombus-containing IVC was weighed and further processed for histopathologic examination, immunohistochemical analysis and western blotting. LiCl and LY294002 were adopted to block and increase the activity of glycogen synthase kinase 3ß (GSK3ß), respectively. RESULTS: The chemical profile analysis showed that paeoniflorin, benzoylpaeoniflorin, albiflorin, gallic acid and catechin were the main constituents of PRRDG. LiCl decreased thrombus weight, reduced the number of inflammatory cells in thrombus and vein wall, down-regulated phosphorylated NF-κB p65 (p-p65) protein expression. Similarly, PRRDG decreased thrombus weight and tissue factor (TF) protein expression. PRRDG reduced the protein expression levels of P-selectin, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in venous endothelium, serum levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and the number of inflammatory cells in thrombus and vein wall. Moreover, PRRDG down-regulated p-p65 protein expression and up-regulated phosphorylated GSK3ß (p-GSK3ß) protein expression. LY294002 abrogated the inhibitory effects of PRRDG on thrombus weight, TF protein expression, TNF-α and IL-1ß serum levels, inflammatory cells influxes, and p-p65 protein expression. CONCLUSION: PRRDG prevents DVT by ameliorating inflammation through inhibiting GSK3ß activity.

[Mechanism of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on intervention of cerebral ischemia based on network pharmacology-molecular docking].

Cerebral ischemia is one of the most common diseases in China, and the drug pair of Chuanxiong Rhizoma and Paeoniae Radix Rubra can intervene in cerebral ischemia to reduce the inflammatory response of cerebral ischemia and apoptosis. To reveal the intervention mechanism of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on cerebral ischemia systematically, computer network pharmacology technology was used in this paper to predict the target and signaling pathway of the drug pair on the intervention of cerebral ischemia, and then the molecular docking technology was used to further analyze the mechanism of the intervention. The target results were then verified by the rat cerebral ischemia model. The target network results showed that the active compounds of Chuanxiong Rhizoma-Paeoniae Radix Rubra for cerebral ischemic disease contained 30 compounds, 38 targets and 9 pathways. The main compounds included phenolic acids in Chuanxiong Rhizoma and monoterpene glycosides in Paeoniae Radix Rubra. The key targets involved mitogen-activated protein kinase 1(MAPK1), steroid receptor coactivator(SRC), epidermal growth factor receptor(EGFR), mitogen-activated protein kinase 14(MAPK14), caspase-3(CASP3), caspase-7(CASP7), estrogen receptor 1(ESR1), and mitogen-activated protein kinase 8(MAPK8), etc. The target gene functions were biased towards protein kinase activity, protein autophosphorylation, peptidyl-serine phosphorylation and protein serine/threonine kinase activity, etc. The important KEGG pathways involved Ras signaling pathway, ErbB signaling pathway and VEGF signaling pathway. Molecular docking results showed that catechin, oxypaeoniflorin, albiflorin, paeoniflorin and benzoylpaeoniflorin had strong binding ability with MAPK1, SRC, EGFR, MAPK14 and CASP7. MCAO rat experimental results showed that Chuanxiong Rhizoma-Paeoniae Radix Rubra significantly improved the cerebral ischemia injury and interstitial edema, and significantly reduced the activation of caspase-7 and the phosphorylation of ERK1/2. The Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair alleviated cerebral ischemia injury through a network model of multi-phenotype intervention by promoting cell proliferation and differentiation, reducing inflammatory factor expression, protecting nerve cells from death and figh-ting against neuronal cell apoptosis, with its action signaling pathway most related to Ras signaling pathway, ErbB signaling pathway and VEGF signaling pathway. This study provides the basis for clinical intervention of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on cerebral ischemia, and also provides ideas for the modernization of drug pairs.

Review of pharmacological effects of Paeoniae Radix Rubra / 药学实践杂志

Paeoniae Radix Rubra has the effects of clearing heat, cooling blood, dissipating blood stasis and pain relieving (in terms of Chinese medicine). Paeoniae Radix Rubra and its active ingredients have significant pharmacological effects in anti-tumor，protecting liver, nerve and heart. By reviewing the relevant literatures published in recent years, we found that the studies on Paeoniae Radix Rubra are mainly focused in the mechanism of action, drug development and clinical application. In this review, we summarize the research results of the pharmacological effects of Paeoniae Radix Rubra and its active ingredients in order to provide the reference for the future research and clinical application of Paeoniae Radix Rubra.

Chemical Constituents and Pharmacological Effects of Paeoniae Radix Rubra： A Review / 中国实验方剂学杂志

Paeoniae Radix Rubra is a traditional Chinese medicine commonly used in clinical practice， it is mostly wild and widely distributed in different areas of China. In addition， the plant of Paeoniae Radix Rubra also has ornamental value. Modern phytochemical researches showed that the chemical constituents of Paeoniae Radix Rubra were complex. Up to now， more than 300 chemical constituents have been found， mainly including monoterpene glycosides， triterpenoids， flavonoids， tannins， phenolic acids， saccharides， steroids， volatile oils and so on. Among them， the content of monoterpene glycosides was the highest， and the types of volatile oil were the most. Paeoniae Radix Rubra has a wide range of pharmacological effects， exerting different curative effects in multiple systems such as blood， cardiovascular， nervous and digestive system. It can protect myocardial cells and nerve cells， stabilize microcirculation， anti-endotoxin， anti-atherosclerosis， reduce pulmonary hypertension， anti-depression， protect liver， anti-gastric ulcer， anti-tumor， slow down aging， treat Parkinson's syndrome and diabetes and its complications， anti-radiation， anti-inflammatory， anti-virus and so on. Through reviewing the literature on chemical constituents and pharmacological effects of Paeoniae Radix Rubra， it was found that total glycosides and monomers such as paeoniflorin， albiflorin， benzoylpaeoniflorin and gallic acid may be the main active components of Paeoniae Radix Rubra. At present， the research on Paeoniae Radix Rubra mainly focused on monoterpene glycosides， while the research on flavonoids and volatile oil in Paeoniae Radix Rubra was less. It is suggested that research on these two components should be strengthened in the future.

Mechanism of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on intervention of cerebral ischemia based on network pharmacology-molecular docking / 中国中药杂志

Cerebral ischemia is one of the most common diseases in China, and the drug pair of Chuanxiong Rhizoma and Paeoniae Radix Rubra can intervene in cerebral ischemia to reduce the inflammatory response of cerebral ischemia and apoptosis. To reveal the intervention mechanism of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on cerebral ischemia systematically, computer network pharmacology technology was used in this paper to predict the target and signaling pathway of the drug pair on the intervention of cerebral ischemia, and then the molecular docking technology was used to further analyze the mechanism of the intervention. The target results were then verified by the rat cerebral ischemia model. The target network results showed that the active compounds of Chuanxiong Rhizoma-Paeoniae Radix Rubra for cerebral ischemic disease contained 30 compounds, 38 targets and 9 pathways. The main compounds included phenolic acids in Chuanxiong Rhizoma and monoterpene glycosides in Paeoniae Radix Rubra. The key targets involved mitogen-activated protein kinase 1(MAPK1), steroid receptor coactivator(SRC), epidermal growth factor receptor(EGFR), mitogen-activated protein kinase 14(MAPK14), caspase-3(CASP3), caspase-7(CASP7), estrogen receptor 1(ESR1), and mitogen-activated protein kinase 8(MAPK8), etc. The target gene functions were biased towards protein kinase activity, protein autophosphorylation, peptidyl-serine phosphorylation and protein serine/threonine kinase activity, etc. The important KEGG pathways involved Ras signaling pathway, ErbB signaling pathway and VEGF signaling pathway. Molecular docking results showed that catechin, oxypaeoniflorin, albiflorin, paeoniflorin and benzoylpaeoniflorin had strong binding ability with MAPK1, SRC, EGFR, MAPK14 and CASP7. MCAO rat experimental results showed that Chuanxiong Rhizoma-Paeoniae Radix Rubra significantly improved the cerebral ischemia injury and interstitial edema, and significantly reduced the activation of caspase-7 and the phosphorylation of ERK1/2. The Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair alleviated cerebral ischemia injury through a network model of multi-phenotype intervention by promoting cell proliferation and differentiation, reducing inflammatory factor expression, protecting nerve cells from death and figh-ting against neuronal cell apoptosis, with its action signaling pathway most related to Ras signaling pathway, ErbB signaling pathway and VEGF signaling pathway. This study provides the basis for clinical intervention of Chuanxiong Rhizoma-Paeoniae Radix Rubra drug pair on cerebral ischemia, and also provides ideas for the modernization of drug pairs.