Tetramethylpyrazine and paeoniflorin combination(TMP-PF)alleviates atherosclerosis progress by reducing hyperlipemia and inhibiting plaque angiogenesis via the NR4A1/VEGFR2 pathway

Atherosclerosis remains a great threat to human health worldwide.Previous studies found that tetramethylpyrazine(TMP)and paeonifl orin(PF)combination(TMP-PF)exerts anti-atherosclerotic effects in vitro.However,whether TMP-PF improves atherosclerosis in vivo needs further exploration.The present study aims to assess the anti-atherosclerotic properties of TMP-PF in ApoE^(-/-)mice and explore the related molecule mechanisms.Results showed that TMP and high-dose TMP-PF decreased serum triglyceride and low-density lipoprotein cholesterol levels,suppressed vascular endothelial growth factor receptor 2(VEGFR2)and nuclear receptor subfamily 4 group A member 1(NR4A1)expression in aortic tissues,inhibited plaque angiogenesis,reduced plaque areas,and alleviated atherosclerosis in ApoE^(-/-)mice.Also,TMP-PF exhibited a better modulation effect than TMP or PF alone.However,NR4A1 agonist abolished the anti-atherosclerotic effects of TMP-PF.In conclusion,TMP-PF was first found to alleviate atherosclerosis progression by reducing hyperlipemia and inhibiting plaque angiogenesis via the NR4A1/VEGFR2 pathway,indicating that TMP-PF had a positive effect on reducing hyperlipemia and attenuating atherosclerosis development.

Paeoniflorin ameliorates chronic colitis via the DR3 signaling pathway in group 3 innate lymphoid cells

Inhibiting the death receptor 3(DR3)signaling pathway in group 3 innate lymphoid cells(ILC3s)presents a promising approach for promoting mucosal repair in individuals with ulcerative colitis(UC).Paeoniflorin,a prominent component of Paeonia lactiflora Pall.,has demonstrated the ability to restore barrier function in UC mice,but the precise mechanism remains unclear.In this study,we aimed to delve into whether paeoniflorin may promote intestinal mucosal repair in chronic colitis by inhibiting DR3 signaling in ILC3s.C57BL/6 mice were subjected to random allocation into 7 distinct groups,namely the control group,the 2%dextran sodium sulfate(DSS)group,the paeoniflorin groups(25,50,and 100 mg/kg),the anti-tumor necrosis factor-like ligand 1A(anti-TL1A)antibody group,and the IgG group.We detected the expression of DR3 signaling pathway proteins and the proportion of ILC3s in the mouse colon using Western blot and flow cytometry,respectively.Meanwhile,DR3-overexpressing MNK-3 cells and 2%DSS-induced Rag1^(-/-)mice were used for verification.The results showed that paeoniflorin alleviated DSS-induced chronic colitis and repaired the intestinal mucosal barrier.Simultaneously,paeoniflorin inhibited the DR3 signaling pathway in ILC3s and regulated the content of cytokines(interleukin-17A,granulocyte-macrophage colony stimulating factor,and interleukin-22).Alternatively,paeoniflorin directly inhibited the DR3 signaling pathway in ILC3s to repair mucosal damage independently of the adaptive immune system.We additionally confirmed that paeoniflorin-conditioned medium(CM)restored the expression of tight junctions in Caco-2 cells via coculture.In conclusion,paeoniflorin ameliorates chronic colitis by enhancing the intestinal barrier in an ILC3-dependent manner,and its mechanism is associated with the inhibition of the DR3 signaling pathway.

Changes in the root system of the herbaceous peony and soil properties under different years of continuous planting and replanting

The herbaceous peony(Paeonia lactiflora Pall.)has high ornamental value.Replanting problems occur when seedlings are replanted into previous holes.We studied the root system and soil environment of the'Dongjingnvlang'variety under a continuous planting regime of one,four,and seven years,and a replanting regime of one and four years.Under the condition of continuous planting,with the increase of number of years,pH,ammonium nitrogen,and nitrate nitrogen decreased in the rhizosphere and non-rhizosphere soils,whereas organic matter,available phosphorus and potassium,enzyme activities,and the number of bacteria,fungi,and actinomycetes increased.Under the condition of replanting,with the increase of number of years,fungi and actinomycetes in both soils increased,while pH,organic matter,nutrients,enzyme activities,and bacterial number decreased.pH,organic matter,nutrient content,enzyme activity and the number of bacterial were lower in soil replanted for four years,whereas the abundance of fungi and actinomycetes was higher,altering the soil from“bacterial high-fertility”to“fungal low-fertility”with increasing years of replanting.The activity of antioxidant enzymes and MDA content in roots of peony in replanting were higher than those in continuous planting,while the content of osmotic regulatory substances in replanting was lower than that in continuous planting.The results showed that there were no obvious adverse factors in soil during seven years of continuous planting,and herbaceous peony could maintain normal growth and development.However,soils after four years of replanting were not suitable for herbaceous peony growth.Benzoic acid increased with years of replanting,which potentially caused replanting problems.This study provides a theoretical basis for understanding the mechanism of replanting problems in the herbaceous peony.

Population pharmacokinetics of paeoniflorin in guanxin Ⅱ prescription

To evaluate the effect of components in Guanxin Ⅱ prescription on the pharmacokinetic profiles of paeoniflorin. Plasma concentration of Paeoniflorin in rats after intravenous injection of Paronia Pall Extract （PPE） and oral administration of PPE and three types of decoctions in Guanxin Ⅱ prescription, respectively, were determined by HPLC analyses. NONMEM （nonlinear mixed-effect modeling） method was used to analyze full set of pharmacokinetic data directly. A two-compartment model with first-order degradation in absorption compartment was employed for the data analysis. The mean of population parameters, CL1, V1, CL2, V2, Ka0, and Kal, were measured to be 0.509 L/h, 0.104 L, 0.113 L/h, 0.123 L, 0.135/h, and 0.0135/h, respectively. Inter-individual variabilities were estimated and dose formulation （DF） was identified as a significant covariate of Ka 1, Ka0, and V1. It is concluded that the pharmacokinetic behaviors of paeoniflorin in rats can alter with different dose formulations.

Paeoniflorin inhibits human gastric carcinoma cell proliferation through up-regulation of microRNA-124 and suppression of PI3K/Akt and STAT3 signaling

AIM: To examine the potential anti-tumor activity of paeoniflorin in the human gastric carcinoma cell line MGC-803.METHODS: Cell viability and cytotoxic effects in MGC-803 cells were analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay, respectively. Cell apoptosis of MGC-803 cells was measured using flow cytometry,DAPI staining assay and caspase-3 activity assay.Quantitative reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the expression of microRNA-124(miR-124) in response to paeoniflorin.The expression of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt), phospho-Akt(p-Akt) and phospho-signal transducer and activator of transcription3(p-STAT3) were also measured by quantitative RTPCR and Western blot analysis in normal, miR-124 and anti-miR-124 over-expressing MGC-803 cells, treated with paeoniflorin.RESULTS: Paeoniflorin was found to inhibit MGC-803 cell viability in a dose-dependent manner. Paeoniflorin treatment was associated with the induction of apoptosis and caspase-3 activity in MGC-803 cells. Paeoniflorin treatment significantly increased miR-124 levels and inhibited the expression of PI3 K, Akt, p-Akt and p-STAT3 in MGC-803 cells. Interestingly, the over-expression of miR-124 inhibits PI3K/Akt and phospho-STAT3 expressions in MGC-803 cells. PI3 K agonist(IGF-1, 1μg/10 μL) or over-expression of STAT3 reversed the effect of paeoniflorin on the proliferation of MGC-803 cells. Over-expression of anti-miR-124 in MGC-803 cells reversed paeoniflorin-induced up-regulation.CONCLUSION: In summary, the in vitro data suggest that paeoniflorin is a potential novel therapeutic agent against gastric carcinoma, which inhibits cell viability and induces apoptosis through the up-regulation of miR-124 and suppression of PI3K/Akt and STAT3 signaling.

Intranasal delivery of paeoniflorin nanocrystals for brain targeting

Paeoniflorin(PA) is an anti-Parkinson Chinese medicine with inferior bioavailability and difficulty in delivery to the brain. This research is to develop an efficacious PA nanocrystal formulation(PA-NCs) that is suitable for intranasal administration to treat Parkinson’s disease(PD). PA-NCs were fabricated through an antisolvent precipitation method using TPGS as the stabilizer. The rod-shaped PA-NCs had particle size of 139.6 ± 1.3 nm and zeta potential of-23.2 ± 0.529 mV. A molecular dynamics simulation indicated that van der Waals forces are the primary drivers of interactions between PA and TPGS. In the ex vivo nasal mucosa permeation assay, the cumulative drug release at 24 h was 87.14% ± 5.34%,which was significantly higher than that of free PA. PA-NCs exhibited substantially improved cellular uptake as well as permeability on Calu-3 cells as compared to PA alone. FRET imaging analysis demonstrated that intact NCs could be internalized into Calu-3 cells.Moreover, PA-NCs conferred desirable protective effect against MPP+-induced SH-SY5Y cellular damage. Pharmacokinetic studies revealed a higher PA concentration in the brain following intranasal delivery of PA-NCs. In summary, the intranasal administration of PANCs is a promising treatment strategy for PD.

Influence of puerarin,paeoniflorin,and menthol on structure and barrier function of tight junctions in MDCK and MDCK-MDR1 Cells

Objective:In this study,the influence of puerarin,paeoniflorin,and menthol on the structure and barrier function of tight junctions(TJs)in MadineDarby canine kidney epithelial(MDCK)and MDCK-multi-drug resistance 1(MDR1)cells was evaluated to determine the mechanisms by which the drugs cross the bloodebrain barrier(BBB).Method:Cells were treated with puerarin,paeoniflorin,and menthol followed by immunohistochemical staining with occludin,claudin-1,and F-actin.The cells were then observed using laser-scanning confocal microscopy.Average optical density(AOD)of the immunofluorescence images of the proteins were analyzed using ImageJ software while Transepithelial electrical resistance(TEER)was measured using an epithelial voltohmmeter.Results:Confocal microscopy revealed that puerarin-and paeoniflorin-treated tight junction proteins were conspicuous while menthol suppressed their expression.Correspondingly,AOD values of cells treated with puerarin or paeoniflorin,or both showed no difference compared to the control group(P>.05)while the menthol group value was downregulated.In 3 h,TEER of cells not treated with menthol were similar to the control group,while treatment with menthol significantly decreased TEER value(P<.05).In addition,application of menthol decreased TEER in MDCK cells earlier than in MDCK-MDR1 cells.Conclusion:Menthol but not puerarin and paeoniflorin may enhance paracellular transport and improve drug penetration of the BBB by disrupting the structure and,thereby,weakening the barrier function of TJs.

Advance in Pre-Clinical Pharmacokinetics of Paeoniflorin, a Major Monoterpene Glucoside from the Root of <i>Paeonia lactiflora</i>

Paeoniflorin (PF) is one of the main bioactive components of total glucosides of paeony (TGP) extracted from the root of Paeonia lactiflora Pall. TGP exhibit various biological activities such as improvement in memory, hepatoprotection, antimutagenic properties and platelet aggregation inhibition. The aim of this paper is to review the pharmacokinetics (PK) of PF as a pure compound and in single or multiple herb(s) of traditional Chinese medicine (TCM) prescriptions. The distribution of PF or PF in TCM fitted one or two compartmental model after oral administration or intravenous injection, respectively. However, PF has a low bioavailability (BA) in rabbit (7.24%) and rat (3.24%) after oral administration. The PK profiles and BA of PF were remarkably improved when co-administered with sinomenine or glycyrrhizin acid. The PK profiles and BA of PF in Radix Paeonia Rubra (RP-R) and Jing-zhi guan-xin were improved, but in co-administration of RP-R with Radix Angelicae Sinensis, the BA was significantly reduced. PK profiles and BA of PF in Shan yao gan-cao tang or Danggui-Shaoyao-San was either remarkably improved or not. However, neither the PK profiles nor the BA of PF in Radix paeonia alba, Huangqin-tang Si ni san or Tang-Min-Ling-Wan was improved. Metabolism in the liver did not play any role in the low oral BA of PF. The low BA was thus attributed to poor permeation due to low lipophilicity, P-glycoprotein mediated efflux, intestinal bacteria and hydrolytic degradation in the intestine by the intestinal brush border lactase phlorizin hydrolase (LPH) and certain esterases. These findings show the in vivo course of PF and provide information on the maximum biological actions of PF that may help traditional Chinese herbal medicinal practitioners.

Protective Effect of Paeoniflorin against Optic Nerve Crush

In order to evaluate the efficacy of traditional paeonia extract paeoniflorin against optic nerve crush, 16 Brown Norway rats were divided into two groups at random, with 8 rats in each group. In paeoniaflorin-treated group, 2 mg paeoniaflorin （total volum： 1 mL） was injected into rat＇s peritoneum one time a day for a period of 8 days. Rats in untreated group were given a single dose of vehicle. The optic nerve was crushed by a special forceps for 30 s in the left eye and a sham procedure was performed in the right eye on the 2nd day after the first injection. The retrograde fluorogold labeling of ganglion cells was conducted 5 days after optic nerve crush. The whole retina was flat-mounted thereafter. The ganglion cells that survived the crush were counted under fluorescent microscope by using an automatic counting software. As compared with the contralateral eye, the survival rate of ganglion cells in the left eye increased from 40.22% to 64.53% with a significant difference found between them （t=2.55, P=0.023）. The results suggested that the paeonia extract paeoniflorin possessed a protective effect against optic nerve crush.

Simultaneous LC-MS/MS Determination of Danshensu and Paeoniflorin for Permeability Studies in Caco-2 Intestinal Absorption Model

A high sensitive method based on liquid chromatography tandem mass spectrometry（LC-MS/MS） was developed and validated for the study of permeability of danshensu（DS） and paeoniflorin（PF） in Caco-2 intestinal absorption model. The DS and PF were extracted from cell culture by vacuum-lyophilizing and then separated on a Zorbax Stable Bond C18 column with 0.1% acetic acid aqueous solution and methanol as mobile phase. Detection was carried out by negative electrospray ionization（ESI ） with selected reaction monitoring（SRM） mode. The apparent permeability coefficients（Papp） of DS and PF in Caco-2 cell medium were calculated and the effects of verapamil on the coefficients Papp of the two test compounds were also illustrated. The permeability of PF was much better than that of DS when the two compounds were administrated individually. Co-administration of DS and PF led to the decrease of the transport from apical side to basolateral side for both the compounds. However, the transport in the contrary direction were accelerated. It was also observed that verapamil could accelerate the transport of the test compounds from apical side to basolateral side. However, the absorption-enhanced effect of verapamil was attenuated when DS and PF were co-administrated. These observations suggest that both passive diffusion and active efflux involved in P-gp would effect the passage of DS and PF across Caco-2 cell monolayer. At the same time, the co-administration of DS and PF to an alteration of transport behavior, which suggests that the interaction must be taken into account when ‘n-in-one＇ samples were used in Caco-2 intestinal model.

Simultaneous Determination of Six Constituents in Cangbaiqutong Capsule by HPLC Wavelength Switching Technique

[Objectives] To establish a high performance liquid chromatography (HPLC) wavelength switching method for the simultaneous determination of content of six constituents (phellodendrine chloride, gentiopicrin, paeoniflorin, tetrandrine, berberine hydrochloride and paeonol) in Cangbaiqutong capsules, and provide a scientific basis for the comprehensive evaluation of the quality of Cangbaiqutong capsule.[Methods] The chromatographic column of Waters XSELECT CSH-C 18 (4.6 mm × 150 mm, 5 μm), the mobile phase of acetonitrile-0.1% phosphate solution, gradient elution (0-15 min,10%-18% A;15-30 min,18%-50% A;30-35 min, 50%-10% A);the flow rate of 1.0 mL/min, wavelength switching of 284 (0-7 min, phellodendrine), 274 (7-10 min, gentiopicrin), 230 (10-14 min, paeoniflorin) and 274 nm (14-35 min, tetrandrine, berberine hydrochloride, paeonol), the injection volume of 10 μL.[Results] There was a good linear relationship between the area of chromatographic peak and the injection volume of phellodendrine chloride, gentiopicrin, paeoniflorin, tetrandrine, berberine hydrochloride and paeonol in the range of 0.150-1.504, 0.768-7.680, 1.096-10.960, 0.220-2.200, 0.296-2.956, 0.0345-0.345 μg, respectively;the average recovery rates ( n =6) were 98.3%, 99.2%, 98.8%, 98.8%, 99.1% and 98.2%, respectively;the RSD value was 1.32%, 1.46%, 1.08%, 1.31%, 1.26% and 1.21%, respectively.[Conclusions] The method can be used to determine many kinds of constituents at the same time, and the operation is simple, accurate and reproducible, and can be used for the quality control of compound Cangbaiqutong capsules.

Study on the Multi-Component Quantitation of Cortex Moutan and Its Intestinal Absorption Characteristics

Cortex Moutan (Paeonia suffruticosa Andr.) is a common traditional Chinese medicine and has been widely used in clinic for 2000 years in China. As sources for this crude drug are always mixed with other species, many cultivars on herbal market may lead to quality instability. Multi-component quantitative analysis is an efficient method to reflect chemical profiles of herb medicine and is always taken as the main method for quality evaluation. So, the aim of this work is to develop analytical method to quantify paeonol, paeoniflorin, gallic acid, oxypaeoniflorin, benzoylpaeoniflorin and paeonolide in Cortex Moutan (CM) to evaluate the chemical qualities of CM from different species or cultivars. Besides, we also study the intestinal absorption characteristics of paeonol and paeoniflorin for further pharmacological evaluation. In the present study, all of the standard markers were performed on an Ecosil C18 (250 mm × 4.6 mm, 5 μM, Lubex Co., Guangzhou, China) with linear gradient elution of 0.2% formic acid water and acetonitrile. The proposed method was applied to analyze 50 batches of samples with acceptable linearity (R2, 0.9995 - 0.9999), precisions (RSD, 0.47% - 2.08%), repeatability (RSD, 039% - 2.63%), stability (RSD, 0.52% - 2.45%), and recovery (RSD, 0.72% - 3.03%) of the six compounds. Furthermore, the Hierarchical Cluster Analysis was applied to classify the 50 samples based on contents of the six compound markers. The results obtained from multi-component quantification of CM clearly indicated that CM originated from P. suffruticosa and P. ostii presented different chemical properties, and that samples from the two materials could be gathered into one branch, respectively, while CM sourced from cultivars of P. suffruticosa showed great variety on chemical quality. The results from Hierarchical Cluster Analysis implied that the established method could be used as a powerful tool for the quality evaluation of CM. The intestinal absorption study indicated that the intestinal absorption activities for paeoniflorin and paeonol showed an increasing absorption with time. Paeonol had lower absorption rate (6.69% - 15.93%) than that of paeoniflorin (19.0% - 30.70%). As a result, the established method is suitable for the quality evaluation of CM. The results of intestinal absorption characteristics of paeonol and paeoniflorin offer an insight for pharmacological evaluation and clinical efficacy research of CM.

Pharmacological Modulation by Shakuyakukanzoto (Shao-Yao-Gan-Cao-Tang) and the Ingredients in Rat Intestinal Smooth Muscle

Shakuyakukanzoto (Shao-Yao-Gan-Cao-Tang), a formulation of Japanese herbal (Kampo) medicines, is composed of Paeoniae Radix and Glycyrrhizae Radix. Effects of Shakuyakukanzoto and the ingredients on rat intestinal tract were examined. Shakuyakukanzoto (0.01 - 0.3 mg/ml) relaxed a carbachol (CCh, 0.3 μM) - induced contraction in a concentration-dependent manner. Both components (Paeoniae radix and Glycyrrhizae radix) also relaxed the CCh-induced contraction. At 0.1 to 1 mM, their constituents (paeoniflorin and glycyrrhetic acid) and the metabolic products (18-α- and 18-β-glycyrrhetinic acids) exerted almost the same actions. The relaxations induced by Shakuyakukanzoto were not modified by 1 μM nicardipine, 10 μM suramin (ATP receptor inhibitor) and several K+ channel inhibitors, but was attenuated by 20 μM IBMX (a phosphodiesterase inhibitor). Also, IBMX inhibited the relaxations induced by paeoniflorin and glycyrrhetic acid, but not by other ingredients. Nicardipine decreased the relaxation of just 18-α-glycyrrhetinic acid. Even in non-treatment with CCh, Shakuyakukanzoto relaxed the intestinal tract. CCh (0.3 μM) elicited spontaneous contractions in 23% specimens, depressed by application of Shakuyakukanzoto. These results indicate that Shakuyakukanzoto causes a remarkable relaxation by the anti-cholinergic and the PDE inhibitory actions, but by minor contribution of Ca2+ channel inhibition. Thus, Shakuyakukanzoto exerts an anti-spasmodic action due to the interaction with pharmacological effects of its ingredients.

Adsorption equilibria of paeoniflorin and albiflorin on cyano-silica column from supercritical carbon dioxide/ethanol

Adsorption equilibria of paeoniflorin and albiflorin on a cyano-silica column(CN column) from the solution of supercritical carbon dioxide(scCO2) modified with ethanol were studied. The adsorption capacity at 308.15 K,313.15 K, 318.15 K and 323.15 K under pressures corresponding to carbon dioxide/ethanol densities from0.347 g·cm^-3 to 0.662 g·cm^-3 were determined using the elution by characteristic point method(ECP). The effects of temperature and pressure on the solute loading were investigated. The results showed that the lower the temperature, the higher the adsorption capacity. With the decrease of density of scCO2, the adsorption capacity strengthens. The maximum adsorption capacity of paeoniflorin(albiflorin) on the CN column was15.24 mg·ml^-1(31.14 mg·ml^-1) in the range of 0–1.84 mg·ml^-1(0–1.67 mg·ml^-1) of paeoniflorin(albiflorin)standard solution. The adsorption capacity of albiflorin was twice as much as that of paeoniflorin under the same conditions. Adsorption data of paeoniflorin and albiflorin could be well described by the Langmuir model and Freundlich model. Compared with the two model fitting results, the adsorption of paeoniflorin and albiflorin belonged to the monolayer adsorption under conditions of 308.15–323.15 K and 10–17 MPa.

Paeoniflorin protects HUVECs against AOPP-induced oxidative injury by blocking ROS-HIF-1α/VEGF pathway

Advanced oxidation protein products （AOPPs）, as a novel indicator of oxidative stress, are thought to be involved in aging-related diseases. The excessive AOPPs were served as independent risk factor for coronary artery disease （CAD）, atherosclerosis and carotid intima media thickness, which mainly through hypoxia inducible factor- l OL （HIF-Iot） and vascular endothelial growth factor （VEGF） pathway. Paeoniflorin, a monoterpene glycoside, ex- erts well protective effect in vascular as its good antioxidant property. However, there is no research that has reported whether Paeoniflorin has the protective effect on oxidative damage induced by AOPPs in HUVECs, and also it is little known about this underlying mechanism. The protective effect of Paeoniflorin on oxidative damage induced by AOPPs was investigated in HUVECs. The cell viability was assessed by MTT colorimetric assay. The fluorescence intensity of 2＇, 7＇-dichlorofluorescein-diacetate （DCFH-DA） staining was detected for intracellular reactive oxygen species （ROS） production. And mitochondrial membrane potential （MMP） was measured byflow cytometry and confocal mi- croscopy staining with Mito Tracker Deep Red/ MitoTracker Green. The intracellular adenosine triphosphate （ATP） was measured by ATP Determination Kit. Nox2, Nox4, HIF-lα, VEGF and NF-KB p65 protein expressions were detected by western blot. The results showed that AOPPs significantly decreased MMP and ATP in a dose-de- pendent manner. Furthermore, AOPPs increased HW-1α, VEGF protein expression, and also partly increased NF-KB p65 expression may through increase of ROS production by up-regulations of Nox2, Nox4 and RAGE expression in HUVECs. These effects were remarkably reversed by pre-treatment of Paeoniflorin, which indicated that Paeoniflorin inhibited Nox2/Nox4 expression, restored ATP depletion and mitochondria dysfunction via ROS suppression, and down-regulated HIF-lα/VEGF possibly via ROS-NF-KB axis. In conclusion, these results suggesting that Paeoniorin had a protective effect against AOPP-induced oxidative damage in HUVECs and that HIF-lα/VEGF might be inter- vention site in this process.

Effect of 6＇-acetylpaeoniflorin on dinitrochlorobenzene induced allergic contact dermatitis in BALB/c mice

Aim The present study aimed to investigate anti-inflammatory activity of 6-AP on murine model of aller- gic contact dermatitis （ACD）. Methods 6＇-acetylpaeoniflorin （6-AP） was synthesized from paeoniflorin （Pae） via acetylation and the structure was characterized by 1H-NMR, 13C-NMR and EI-MS. ACD model was established by repeated application of dinitrochlorobenzene （DNCB） to induce skin immune inflammation. The mice were oral- ly administered 6-AP （35, 70, 140 mg. kg-1 ·d^-l）, Pae （70 rag. kg-1·d^-1） and prednisone （Pre, 5 rag. kg^- 1· d^-1 ） from day 1 to day 7 after Cutaneous inflammation was evaluated by ear swelling and histological exami- nation. Splenocyte proliferation was assayed by the 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyl-2H tetrazolium bromide assay. The cytokine production in the splenocytes was measured by enzyme-linked immunosorbent assay. Results Topical application of DNCB to the skin provoked obvious swelling and inflammatory cell infiltration. 6- AP significantly inhibited ear swelling, decreased inflammatory cell infiltration and epidermal keratinization. Ad- dtionally, 6-AP obviously alleviated the hyperplasia of red pulp and germinal center （GC） appearance, decreased spleen index, decreased spleen index, and inhibited splenocyte proliferation in ACD model, compared to that of Pae. Further, the study indicated that 6-AP treatment could increase IL-10 level, while reduce IL-17 level in splenocytes simultaneously. The correlation analysis displayed significantly positive correlations between IL-17 level and the severity of skin inflammation, while negative correlations between IL-10 level and skin inflammation. Con- clusion 6-AP has a significantly higher anti-inflammatory effect than Pae, and it may be a useful treatment for ACD.

A comparison of pharmacokinetic interaction of paeoniflorin, albiflorin and total peony glucosides

Aim Paeoniflorin （PF） and albiflorin （AF） are the main components of total peony glucosides （TPG）, which has been widely used as the valuable monoterpene glycosides in Paeouia lactiflora Pall and have many biological activities such as anti-inflammatory, anti-hypertension and antioxidation effects. In this study, the pharmacokinetic interaction and possible interaction mechanism among Pie and A1e and TGP were conducted and in- vestigated. A sensitive and specific ultra high-performance liquid chromatography - tandem mass spectrometry method （UPLC-MS） was successfully developed and validated for simultaneous quantification of Pie and AF in rat plasma after intragastric administration and the comparative study of pharmacokinetics for PF and A1e in different combinations. The combinations of Pie （ 80 mg· kg^- 1 ）, Ale （ 14 mg ·kg^- 1 ）, co-administration of Pie （ 80 mg · kg^-1） and Ale （14 mg· kg^-1）, TPG （Pie 80 rag. kg^-1） and TPG （Alel4 mg.·1kg^-1） were orally administrated to rats respectively. Chromatographic separation was performed on a ZORBAX Eclipse plus Cls column using 0.2% formic acid-methanol （79：21, V/V） as mobile phase. The calibration curves were linear in ranges of 0.5 - 1000 μg · L^-1. The results indicated that the main pharmacokinetic parameters including AUC, MRT and tl/2 between the single ingredient （ Pie and Ale） and TPG had significant difference （ P 〈 0.05 or P 〈 0.01 ）. The different phar- macokinetics data implied that the multiple active components of TPG resulting some potential drug-drug interac- tions, which may promote the absorption and decease the elimination of Pie and Ale. Our results could be helpful for further investigation of the interaction mechanism of Pie and Ale.

Absorption characteristic of Paeoniflorin-6＇O-benzene sulfonate in an situ single-pass intestinal perfusion model

Aim Paeoniflorin-6＇O-benzene sulfonate （CP-25） was synthesized to improve oral absorption. This study was performed to investigate absorptive behavior and mechanism of CP-25 in intestine. Methods The effects of drug concentration, intestinal segments, gender as well as ATP binding cassette （ABC） transporter inhibitors on absorption of CP-25 were studied in a situ single-pass intestinal perfusion rat model. Meanwhile, Paeoniflorin （Pae） was tested and served as control group. The concentration of tested drugs was measured by HPLC. Results The results showed intestinal absorption of CP-25 was neither segmental dependent changes nor gender difference. Transepithelial transportation would not change with increasing concentrations of CP-25, which suggest a passive transport was the main pattern of CP-25. Additionally, absorption of CP-25 was much better than that of Pae in small intestine. When compared with Pae, CP-25 gave a 1.82-fold permeability rate. Finally, the results indicated Pae was substrate of P-glycoprotein （P-gp） , but was not the substrate of breast cancer resistance protein and muhi- drug resistance associated protein 2. Among the used ABC inhibitors, the absorption rate of Pae could only be in- creased by？ P-gp inhibitor Verapamil and GF120918, while CP-25 had no remarkable alteration. Conclusion CP-25 has better absorptive features than that of Pae, which may be attributed to its lipophicity enhancement and be unaffected by P-gp efflux.

Production of paeoniflorin and albiflorin by callus tissue culture of Paeonia lactiflora Pall

In order to facilitate the preparation of paeoniflorin(PF)and albiflorin(AF),two chief bioactive constituents in Paeonia lactiflora Pall(PL),induction and culture of callus from PL were studied.With a modified woody plant medium supplemented with 0.5 mg·L-16-benzylaminopurine,1.0 mg·L-1naphthylacetic acid,0.1 mg·L-1thidiazuron and 30 g·L-1sucrose,callus was induced from four kinds of explants:leaf,stems,petiole,and root.The potency to form callus varies between different explants and leaf explants exhibits the highest capacity(100%).On the other hand,root-derived callus(R-callus)produces the highest level of total amount of PF and AF,31.8 mg·g-1dry mass,which is higher than the corresponding level in the root of field cultivated PL.Furthermore,the time needed is only 40 days,remarkably shorter than the cultivation time of PL,about 4–5 years.Higher accumulation levels of PF and AF with shorter production time indicate that callus culture of PL is a promising powerful tool for production of PF and AF in the future.

Application of an ultra performance liquid chromatography-tandem mass spectrometry method to the pharmacokinetics study of paeoniflorin-6'-O-benzene sulfonate and its active paeoniflorin metabolite in rat plasma

OBJECTIVE To establish a rapid,simple,precise and sensitive ultra performance liquid chromatography(UPLC) method for determination of paeoniflorin-6'-O-benzene sulfonate(acylated derivative of Pae,CP-25) and its primary metabolite(Pae,M1) in rat plasma,and to investigate the effects of gender,food and disease status on the pharmacokinetics after oral administration in rats.METHODS Plasma samples and calibrators were extracted with methanol after addition of the internal standard solution.After evaporation of the methanol layer,the residue was reconstituted in mobile phase,and a 2 μL of the sample was injected into a Waters ACQUITY UPLC BEH C18(2.1 mm × 50 mm,1.7 μm) column for separation at a flow rate of 0.5 mL·min^(-1) at 40℃.The mobile phase was composed of 0.1% formic acid in water and methanol(68∶32,V/V).RESULTS The developed UPLC-MS/MS method was linear in the concentration range of 2-800 mg·L^(-1).Validation of the method proved that the method′s precision,selectivity and stability were all within the acceptable limits.Pharmacokinetics study showed that CP-25 could have more extensive distribution in females than that in males but no differences in M1.Food intake could also increase the extent of absorption and decrease the rate of clearance of CP-25 and M1 after oral administration in rats.The disease status would decrease the absorption of CP-25 in rats.Comparing single-and multiple-dosing in adjuvant arthritis(AA) model,absorption of CP-25 and M1 was improved with increasing dosage.CONCLUSION The developed UPLC-MS/MS method is sensitive,specific and was successfully applied to the pharmacokinetics of CP-25 and M1 in rats.And a significant gender,food intake and disease status differences for CP-25 and M1 were observed in this study.