1H-NMR-Based Metabolomics for the Classification of the Roots of Paeonia lactiflora, a Constituent of Kampo Medicines.

The root of Paeonia lactiflora (PAEONIAE RADIX) is a constituent of the traditional Japanese medicines (Kampo) and is known to have various effects. Peony roots cultivated in Japan and China are available in the Japanese market for medicinal use. In this study, the chemical diversity of ten available peony roots in the market that differed in their cultivation area was investigated using 1H-NMR metabolomics techniques. Principal component analysis and hierarchical cluster analysis of the 1H-NMR spectra of the peony roots methanolic extracts revealed a clear difference between the metabolic profiles of Japanese and Chinese peony roots. By preparative procedures using chromatography based on 1H-NMR spectra measurements, oxypaeoniflorin and (+)-catechin were found to be specific compounds for Japanese peony root. All peony roots used in this study were listed in the Japanese Pharmacopoeia. Therefore, the differences in the constituents of these peony roots might be attributed to growing conditions than differences in species. Cultivation conditions also influence the quality of natural medicines.

Pharmacokinetic study of Erlong Zuoci Pill in rats / 中国药科大学学报

@#To establish a quantitative LC-MS/MS method for the simultaneous detection of components of Erlong Zuoci Pill in rat plasma: verbascoside, oxypaeoniflorin, echinacoside and benzoylpaeoniflorin, and to evaluate the pharmacokinetic characteristics of Erlong Zuoci Pill in rats, plasma samples were purified by protein precipitation using methanol as a protein precipitant.Methanol was used as the organic phase and aqueous solution containing 0.1% formic acid was used as the water phase.The quantitative analysis method of verbascoside, oxypaeoniflorin, echinacoside and benzoylpaeoniflorin was established in negative ion mode, and the validation of bioanalytical method was carried out.Healthy SD rats were selected, and 20 mL/kg (equivalent to the original drug 10 g/kg dose) of Erlong Zuoci Pill extract was administered by intragastric administration.The plasma concentration of the target compounds at different time intervals after administration was determined using the established method, and the pharmacokinetic parameters was calculated by the Phoenix WinNonlin8.3 software using the non-compartmental model.The method validation results showed that verbascoside (r = 0.993 7) and oxypaeoniflorin (r = 0.994 6) had good linear relationship in the concentration range of 0.5-50 ng/mL, echinacoside (r = 0.993 6) and benzoylpaeoniflorin (r = 0.992 6) had good linear relationship in the concentration range of 1-100 ng/mL.The relative standard deviations of the inter- and intra- batch precision of the four compounds were all less than 15%, and the inter- batch and intra- accuracies were between 85% and 115%.Extraction recovery, matrix effect and stability met the relevant requirements.After a single gavage of Erlong Zuoci Pill extract in rats, all the four compounds were rapidly absorbed and eliminated.Oxypaeoniflorin, echinacoside, and benzoylpaeoniflorin showed two peaks in their drug concentration-time curves.Compared with the other three compounds, oxypaeoniflorin has the highest concentration in rat plasma with cmax1 of (24.40 ± 4.78) ng/mL and cmax2 of (22.50 ± 2.70) ng/mL. The results show that the validation results of this method are in line with the guiding principles of biological sample analysis methods, and it can be used to evaluate the pharmacokinetic characteristics of Erlong Zuoci Pill extract in rats.

Comparative analysis of monoterpene glycosides in Paeoniae Rubra Radix Formula Granule from different manufacturers / 中草药

Objective: To establish an HPLC method for simultaneous determination of 15 monoterpene glycosides pyrindyl- paeoniflorin, mudanpioside F, oxyalbiflorin, oxypaeoniflorin, 10-hydroxypaeoniflorin, albiflorin, paeoniflorin, oxypaeonidanin, 4-O- methyl-oxypaeoniflorin, galloylpaeoniflorin, 4-O-methyl-paeoniflorin, albiflorin R1, paeonidanin, benzoyloxypaeoniflorin, and benzoylpaeoniflorin in Paeoniae Rubra Radix Formula Granule, in order to compare the quality of Paeoniae Rubia Radix Formula Granule from different manufacturers and provide the basis for the establishment of a unified quality control method. Methods The separation was performed on an Agilent Zorbax SB-Aq C18 column (250 mm × 4.6 mm, 5 µm), using acetonitrile and potassium dihydrogen phosphate solution (pH 2.8) as the mobile phase at the flow rate of 1.0 mL/min for a gradient elution. The detection wavelength was set at 260 nm. Results: Paeoniflorin, albiflorin, and oxypaeoniflorin were the three main monoterpene glycosides with the highest content in the Paeoniae Rubra Radix Formula Granule. There were extremely significant difference among the contents of 15 monoterpene glycosides in Paeoniae Rubra Radix Formula Granule produced by different manufacturers. The sample of CSPFKL-KRT was remarkable for the highest content of paeoniflorin and oxypaeoniflorin (73.214 mg and 16.935 mg per gram sample, respectively) and the lowest content of albiflorin among all samples (2.343 mg per gram sample), while sample CSPFKL-XLS was remarkable for the lowest content of paeoniflorin and oxypaeoniflorin (26.327 mg and 4.165 mg per gram sample, respectively) and the highest content of albiflorin among all samples (18.893 mg per gram sample). Conclusion: There were extremely significant difference among the contents of main monoterpene glycosides in Paeoniae Rubra Radix Formula Granule produced by different manufacturers, which may affect the clinical use. The establishment of a unified quality standard plays an important role in the quality control of Paeoniae Rubia Radix Formula Granule.

Phytochemical variation among the traditional Chinese medicine Mu Dan Pi from Paeonia suffruticosa (tree peony).

Mu Dan Pi is a traditional Chinese medicine used to treat inflammation, cancer, allergies, diabetes, angiocardiopathy, and neurodegenerative diseases. In this study, the metabolome variation within Mu Dan Pi collected from 372 tree peony cultivars was systematically investigated. In total, 42 metabolites were identified, comprising of 14 monoterpene glucosides, 11 tannins, 8 paeonols, 6 flavonoids, and 3 phenols. All cultivars revealed similar metabolite profiles, however, they were further classified into seven groups on the basis of their varying metabolite contents by hierarchical cluster analysis. Traditional cultivars for Mu Dan Pi were found to have very low metabolite contents, falling into clusters I and II. Cultivars with the highest amounts of metabolites were grouped in clusters VI and VII. Five potential cultivars, namely, 'Bai Yuan Qi Guan', 'Cao Zhou Hong', 'Da Zong Zi', 'Sheng Dan Lu', and 'Cheng Xin', with high contents of monoterpene glycosides, tannins, and paeonols, were further screened. Interestingly, the majority of investigated cultivars had relatively higher metabolite contents compared to the traditional medicinal tree peony cultivars.

Effects of sulfur-fumigation on the pharmacokinetics, metabolites and analgesic activity of Radix Paeoniae Alba.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Paeoniae Alba (Baishao, BS), one of the most commonly used traditional Chinese medicinal herbs, has many pharmacological effects including analgesic activity. Previous studies found that sulfur-fumigation, a post-harvest handling process developed to prevent mold contamination of medicinal herbs, altered the quality of BS. However, whether sulfur-fumigation affects the pharmacokinetics, safety and efficacy of BS warrants further investigation. AIM OF THE STUDY: To evaluate the feasibility of sulfur-fumigation as a post-harvest handling process of BS from the viewpoints of pharmacokinetics, safety and efficacy. MATERIALS AND METHODS: The pharmacokinetic behaviors of four active components of BS and one characteristic component of sulfur-fumigated BS (S-BS) were evaluated by high performance liquid chromatography triple quadrupole mass spectrometry (HPLC-TQ-MS/MS). The safety was investigated using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS/MS) based metabolomics approach after intragastric (i.g.) administration of non-fumigated BS (N-BS) and S-BS in rats. The analgesic efficacy was compared using hot-plate test in mice, after i.g. administration of N-BS and S-BS, at both high and low dosages. RESULTS: Systemic exposures of paeoniflorin and oxypaeoniflorin, two analgesic components of BS, were significantly decreased in the S-BS treated group compared to the N-BS treated group, while paeoniflorin sulfonate, one of the sulfur-containing derivatives of S-BS, was detected in all time-points of S-BS treated group with the area under the plasma concentration-time curve (AUC0-t) and the maximum plasma concentration (Cmax) as high as 7077.06 ± 2232.97ng/mL*h and 1641.42 ± 634.79ng/mL respectively, which indicated that sulfur-fumigation altered the pharmacokinetic behaviors of BS. Besides, paeoniflorin sulfonate and its four metabolites with ambiguous toxicities, as well as one endogenous metabolite p-cresol glucuronide, the biomarker of disordered homeostasis of intestinal bacteria and bile acid, were identified as the characteristic metabolites in S-BS administered rats, suggesting that sulfur-fumigation reduced the safety of BS. Furthermore, the analgesic effects at both low and high dosages were decreased in different extent when compared to N-BS administered groups, indicating that sulfur-fumigation weakened the efficacy of BS. CONCLUSION: Sulfur-fumigation altered the pharmacokinetics, as well as reduced the safety and efficacy of BS, suggesting that sulfur-fumigation is not recommended for post-harvest handling of BS.

Quantitative Study on Simultaneous Determination of Nine Components in Renshen Nüjin Pills Based on QAMS / 中国药学杂志

OBJECTIVE: To establish a quantitative analysis of multi-components by single-marker (QAMS) method for simultaneous determination of senkyunolide H, senkyunolide I, senkyunolide A, ligustilide, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin and α-cyperone in Renshen Nüjin pills, in order to provide basis for studying its quality standards.METHODS: The analysis of the methanol extract of Renshen Nüjin pills was performed on Agilent Zorbax SB C18 column (4.6 mm×250 mm,5 μm), with mobile phase composed of methanol-acetonitrile (2∶1)-0.1% phosphoric acid solution at a flow rate of 0.9 mL•min-1 in gradient elution mode. The column temperature was maintained at 30 ℃, and the detection wavelengths were set at 280, 230 and 242 nm. Paeoniflorin was selected as the internal standard, and the relative correlation factors (RCF) of senkyunolide H, senkyunolide I, senkyunolide A, ligustilide, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin and α-cyperone were determined by HPLC. The accuracy and feasibility of the method were validated by comparing the results of QAMS method and external standard method.RESULTS: The standard curves of senkyunolide H, senkyunolide I, senkyunolide A, ligustilide, oxypaeoniflorin, albiflorin, paeoniflorin, benzoylpaeoniflorin and α-cyperone had good linear relationship in the ranges of the tested concentrations. The precision, stability and repeatability complied with the requirements of methodology. The recoveries were 97.38%, 98.16%, 98.84%, 99.63%, 97.04%, 99.14%, 100.04%, 96.93% and 98.48%, RSDs were 1.38%, 1.18%, 0.97%, 0.86%, 1.68%, 1.30%, 0.57%, 1.32% and 1.19%, respectively. No significant differences were observed in the determination results by QAMS method and external standard method.CONCLUSION: The QAMS method can be used for the content determination and quality control of the nine components in Renshen Nüjin pills.

Terpene glycoside component from Moutan Cortex ameliorates diabetic nephropathy by regulating endoplasmic reticulum stress-related inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Multiple lines of evidences have suggested that endoplasmic reticulum (ER) stress-related inflammatory responses play a critical role in the pathogenesis of diabetic nephropathy (DN). Moutan Cortex (MC), the root bark of Paeonia suffruticosa Andr., is a well-known traditional Chinese medicine (TCM), which has been used clinically for treating inflammatory diseases in China. The findings from our previous research suggested that terpene glycoside (TG) component of MC possessed favorable anti-inflammatory properties in curing DN. However, the underlying mechanisms of MC-TG for treating DN are still unknown. AIM OF THE STUDY: To explore the role of ER stress-related inflammatory responses in the progression of DN, and to investigate the underlying protective mechanisms of MC-TG in kidney damage. MATERIALS AND METHODS: DN rats and advanced glycation end-products (AGEs) induced HBZY-1 cell dysfunction were established to evaluate the protective effect of MC-TG on ameliorating renal injury. Evaluation of pathological lesions was performed by Masson staining and transmission electron microscopy (TEM). Interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), glucose regulated protein 78 (GRP78/Bip), as well as spliced X box binding protein 1(XBP-1(s)) levels in rat serum were detected by an enzyme-linked immunosorbent assay (ELISA). Furthermore, western blotting (WB) was applied to detect the protein expressions including IL-6, MCP-1, intercellular cell adhesion molecule-1 (ICAM-1), GRP78/Bip, XBP-1 (s), phosphorylated inositol-requiring enzyme-1α (p-IRE1α), cleaved activating transcription factor 6 (ATF6), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), and phosphorylated nuclear factor κB p65 (p-NF-κB p65) in vivo and in vitro. Immunohistochemistry (IHC) was carried out to determine the phosphorylation of IRE1α and NF-κB p65 in kidney tissues. RESULTS: Pretreatment with MC-TG could markedly improve renal insufficiency and pathologic changes. It could down-regulate ER stress-related factors GRP78/Bip, XBP-1(s) levels, and also reduce the pro-inflammatory molecules IL-6, MCP-1, and ICAM-1 expressions. Furthermore, a significant decrease in phosphorylation of IRE1α and NF-κB p65 by the treatment of MC-TG. CONCLUSIONS: These findings indicated that MC-TG ameliorated ER stress-related inflammation in the pathogenesis of DN, wherein the protective mechanism might be associated with the inhibition of IRE1/NF-κB activation. Thus, MC-TG might be a potential therapeutic candidate for the prevention and treatment of DN.

Combination of Ligusticum chuanxiong and Radix Paeoniae ameliorate focal cerebral ischemic in MCAO rats via endoplasmic reticulum stress-dependent apoptotic signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Combination of Ligusticum chuanxiong and Radix Paeoniae (XS) is highly effective in the treatment for focal cerebral ischemic, but the underlying mechanism is not clear. This study was conducted to evaluate the combinative effects of XS on MCAO rats and explore the underlying mechanisms. MATERIALS AND METHODS: MCAO rats were used to evaluate the protective effect of Ligusticum chuanxiong (CX), Radix Paeoniae Rubra (CS) and their combination (XS) on ameliorating focal cerebral ischemic. Cerebral ischemia deficits and infarct size were performed by 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (H-E) staining. Activities of SOD, CAT and GSH-Px, as well as levels of LPO and MDA were detected by commercial kits while ELISA kits for the content of plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA). Immunohistochemistry (IHC) and western blot analysis (WB) were carried out to examine the protein expressions including PKR-like endoplasmic reticulum kinase (PERK), cytoplasmic of glucose regulated protein 78 (GRP78), X box-binding protein-1 (XBP-1), activating transcription factor-6 (ATF-6), C/EBP-homologous protein (CHOP), metalloprotease-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), Bcl-2 associated X protein (Bax), and porcineB-cellleukemia/lymphoma-2 (Bcl-2) in brain tissues. Reverse transcription polymerase chain reaction (RT-PCR) and Quantitative PCR (Q-PCR) were applied to examine vascular endothelial growth factor (VEGF) and N-methyl-d-aspartate receptors (NMDAR1) mRNA levels. RESULTS: CX, CS and their combination (XS) could reduce cerebral ischemia deficits and infarct size of MCAO rats. They increased SOD, CAT and GSH-Px activities, and reduced MDA and LPO levels in serum, markedly. A significant decrease of endoplasmic reticulum stress-related factors PERK, XBP-1, ATF-6 and CHOP protein expression levels while an increase of GRP78 and MVD expression by the treatment of CX, CS and XS. It could also be observed that their treatment could reduce apoptotic damage of brain tissues by up-regulating Bax level and down-regulating Bcl-2 level. Furthermore, the levels of MMP-9 and PAI-1 in serum and tissues of rats were down-regulated remarkably while TIMP-1 and PA levels were up-regulated. VEGF mRNA level was up-regulated dramatically whereas NMDAR1 was reduced. Importantly, the combination of CX and CS, namely XS, has a more meaningful improvement on focal cerebral ischemic than CX or CS alone. CONCLUSION: All these revealed that the combined XS exerted more remarkable protective effects than alone. XS could inhibit neuronal apoptosis by attenuating ER-stress-dependent apoptotic signaling and protected the blood-brain barrier. These findings might supply beneficial hints for the synergy of CX and CS, and provide the basis for rationality of XS preparation and deserve further clinical investigations.

Fingerprint and multi-components quantitative determination for Huoxuetongluo Injection by UPLC / 中草药

Objective: To establish a UPLC fingerprint of the compounds in Huoxuetongluo Injection, and to make a quantitative analysis. Methods: The Thermo C18 (100 mm × 3 mm, 1.7 μm) column was used with a mobile phase of methyl alcohol-0.05% formic acid gradient elution, the flow rate was 0.4 mL/min, the column temperature was 20℃, and the detection wavelength was 260 nm. Results: The fingerprint chromatography included 21 mutual peaks, of which 11 mutual peaks from Paeoniae Radix Rubra, 7 mutual peaks from Chuanxiong Rhizoma, and 3 mutual peaks from Persicae semen. The similarity among the batches was more than 0.98. Based on the retention time, and UV absorption spectra of reference compounds, five components, amygdalin, oxypaeoniflorin, albiflorin, paeoniflorin, and ferulic acid, were identified and quantified. Conclusion: The method is rapid, simple, and accurate, and can be used for the quality control of Huoxuetongluo Injection.

Evaluation on processing technology for bran-fried Paeoniae Alba Radix by multi-index comprehensive weighted mark method / 中草药

Objective: To optimize the best processing technology for bran-fried Paeoniae Alba Radix (PAR). Methods: Taking the contents and extract yields of paeoniflorin, oxypaeoniflorin, albiflorin, and gallic acid in PAR as the indexes, the affecting factors on the processing technology of bran-fried PAR was investigated by orthogonal design test and multi-index comprehensive weighted mark method. Results: The best processing technology was to add 10% bran and fry for 8 min at 200°C. Conclusion: The optimal processing technology is stable, and could provide the references for the further study of PAR.

Absorption and transport characteristic of paeoniflorin and its derivatives in model of Caco-2 cell monolayers / 中草药

Objective: To study the absorption and transport characteristic of paeoniflorin (PF), oxypaeoniflorin (OP), benzoylpaeoniflorin (BP), tetraacetylpaeoniflorin (TP), pentaacetylpaeoniflorin (PP), and pentacacetylalbiflorin (PA) in human colon adenocarcinoma cell line Caco-2 cell monolayer model. Methods: The Caco-2 cell monolayers were used as an intestinal epithelial cell model. The permeability of the tested compounds from apical (AP) side to basolateral (BL) side or from BL side to AP side was evaluated. The concentration of the tested compounds was measured by HPLC coupled with UV detector. The transport parameters and apparent permeability coefficients (Papp) were calculated, and the Papp values were compared with the reported values for model compounds, Propranolol and Atenolol. Results: The Papp values of PF in the bi-directional transport and atenolol were at the quantitative degree of 10-7 cm/s. Whereas those of OP, BP, TP, PP, and PA were between atenolol and propranolol used as a control substance for low and high permeability, respectively. The absorption and transport of the tested compounds were concentration-dependent at the concentration range of 10-200 μmol/L for PF, OP, and BP, 10-150 μmol/L for TP and PA, and 10-100 μmol/L for PP. Conclusion: The six tested compounds could be absorbed across the intestinal epithelial cells by passive diffusion mechanism. PF is poorly absorbed compound and OP, BP, TP, PP, and PA are moderately absorbed compounds. BP has a role to promote atenolol uptake transporters in Caco-2 cell monolayer model.

Compositional differences in different processing technology of radix paeoniae alba and different origin of red peony / 中国基层医药

Objective To determine and compare the contents of oxypaeoniflorin and paeoniflorin of Paeoniae Radix,and to improve the quality standard of Radix Paeonia.Methods The contents of oxypaeoniflorin and paeoniflorin in different kind of White and Red Peony Root were successfully analyzed by HPLC.Results The content of oxypaeoniflorin in Red Peony Root is more than White Peony Root at 0.8361％ ;The content of paeoniflorin in White Peony Root is more than Red Peony Root at 0.2157％.The proportion of paeoniflorin and oxypaeoniflorin in White Peony Root is more than Red Peony Root.Conclusion With HPLC technology,quantitative analysis of active constituents in Paeoniae Radix was performed accurately.