An enhanced cardio-protective effect of nanoparticles loaded with active components from Polygonum orientale L. against isoproterenol-induced myocardial ischemia in rats.

In this study, nanoparticles loaded with active components from Polygonum orientale L. (PO), a traditional Chinese herb known for its anti-myocardial ischemic properties, were investigated for cardio-protective properties. Specifically, OVQ-Nanoparticles (OVQ-NPs) with Orientin (Ori), Vitexin (Vit), and Quercetin (Que) was obtained by double emulsion-solvent evaporation method. The OVQ-NPs exhibited a spherical shape, with a uniform size distribution of 136.77 ± 3.88 nm and a stable ζ-potential of -13.40 ± 2.24 mV. Notably, these nanoparticles exhibited a favorable sustained-release characteristic, resulting in an extended circulation time within the living organism. Consequently, the administration of these nanoparticles resulted in significant improvements in electrocardiograms and heart mass index of myocardial ischemic rats induced by isoproterenol, as well as decreased serum levels of CK, LDH, and AST. Furthermore, the results of histopathological examination, such as H&E staining and TUNEL staining, confirmed a reduced level of cardiac tissue pathology and apoptosis. Moreover, the quantification of biochemical indicators (SOD, MDA, GSH, NO, TNF-α, and IL-6) demonstrated that OVQ-NPs effectively mitigated myocardial ischemia by regulating oxidative stress and inflammatory pathways. In conclusion, OVQ-NPs demonstrate promising therapeutic potential as an intervention for myocardial ischemia, providing a new perspective on traditional Chinese medicine treatment in this area.

[Potential pharmacodynamic substances of Laportea bulbifera in treatment of rheumatoid arthritis based on serum pharmacochemistry and pharmacology].

The present study investigated the pharmacodynamic material basis of Laportea bulbifera in the treatment of rheumatoid arthritis. Firstly, human rheumatoid arthritis fibroblast-like synoviocyte line MH7A was cultured in vitro and treated with tumor necrosis factor alpha(TNF-α, 50 ng·mL~(-1)). The proliferation and the levels of inflammatory cytokines such as prostaglandin E2(PGE2), interleukin-1ß(IL-1ß), and interleukin-6(IL-6) of the MH7A cells exposed to the serum containing L. bulbifera were determined to evaluate the anti-rheumatoid arthritis effects of the serum. Furthermore, the ultra-performance liquid chromatography tandem mass spectrometry fingerprints of the L. bulbifera crude extract, the drug-containing serum, and the drug-free serum were compared to identify the compounds newly generated in the serum after oral administration of the extract. According to the peak areas of common peaks and the results of anti-rheumatoid arthritis effect test, the active components were identified. The serum containing L. bulbifera significantly inhibited the proliferation of the MH7A cells activated by TNF-α and the expression of PGE2, IL-6, and IL-1ß. Thirty newly generated compounds were detected in the drug-containing serum. Among them, neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, rutin, isoquercitrin, luteoloside, kaempferol-3-O-rutinoside, and quercitrin were also present in the crude extract. Twelve characteristic peaks(3, 7, 8, 14, 18, 19, 21, 23, 24, m6, m7, and m15) were significantly correlated with the pharmaceutical effect. According to the correlations, neochlorogenic acid, cryptochlorogenic acid, and chlorogenic acid had great contributions to the anti-rheumatoid arthritis activity. This study preliminarily clarified the potential pharmacodynamic substances of L. bulbifera in the treatment of rheumatoid arthritis, which laid a theoretical and experimental foundation for further development and application of the medicinal plant.

Comparative analysis of excretion of six major compounds of Polygonum orientale L. extract in urine, feces and bile under physiological and myocardial ischemia conditions in rats using UPLC-MS/MS.

Polygonum orientale L. is a traditional Chinese medicine having extensive pharmacological activities including antimyocardial ischemia (MI) injury properties. Isoorientin, orientin, vitexin, quercitrin, astragalin and protocatechuic acid are the main compounds in P. orientale extract. The aim of this study was to establish an ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of the content of these compounds in urine, feces and bile samples simultaneously and application of the method in a comparative excretion study in normal and MI model rats after oral administration of P. orientale extract. Chromatographic seperation was conducted on an Agilent Eclipse Plus C18 column with the mobile phase consisting of 0.1% formic acid-acetonitrile and 0.1% formic acid-water. Negative ion multiple reaction monitoring mode was used for quantification. The six compounds had good linearity (r ≥ 0.9921) and acceptable accuracy ranging from 10.10 to -5.82% The relative standard deviations of within-day precision and inter-day precision were <10.45 and 13.44%, respectively. The extraction recovery of the six analytes ranged from 80.31 to 101.47% and the matrix effect was 82.56-102.88%, indicating that the preparations of sample collected form urine, feces and bile were stable throughout analysis. The excretion amount of the six analytes increased in both normal and MI model rats' urine, feces and bile in a 24 h period and became stable between 36 and 48 h after administration. The total excretion rate of six compounds was <5% in urine, feces and bile of normal and MI model rats. The excretion peak period for all compounds in MI rats was slower than that in normal rats. This excretion study provides insights for further application and research on P. orientale.

Efficacy of water fraction from Dioscorea cirrhosa on oxidative stress and apoptosis in H9c2 cardiomyocytes induced by H2O2.

OBJECTIVE: To investigate the efficacy of water fraction from Dioscorea cirrhosa (WF) on oxidative damage and apoptosis of cardiomyocytes induced by H2O2, and to study its mechanism. METHODS: Cell viability was measured by the MST assay kit. The content of malondialdehyde (MDA), release of lactate dehydrogenase (LDH) and activity of catalase (CAT) and superoxide dismutase (SOD) were detected by biochemical kit. The content of reactive oxygen species (ROS) was assessed by nonfluorescent probe 2' ,7'-dichlorofluorescin diacetate (DCFH-DA). JC-1 was used to analyze the mitochondrial membrane potential (mtΔΨ) and Annexin-V-FITC/PI staining was applied to assess apoptosis of H9c2 by flow cytometry. Moreover, the expression of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), caspase-3, caspase-9, cleaved-caspase-3 and cleaved-caspase -9 proteins was determined by western blot analysis. RESULTS: WF increased cell viability and decreased LDH leakage in H9c2 cells exposed to H2O2. WF treatment decreased ROS and MDA level, enhanced SOD and CAT activities, improved mtΔΨ and inhibited apoptosis. Western blot analysis demonstrated that the ratio of Bcl-2/Bax was increased and the expression cleaved-caspase-3, caspase-3, cleaved-caspase-9 and caspase-9 were decreased in group treated with WF. CONCLUSION: WF protects H9c2 myocardial cells on H2O2-induced oxidative stress and apoptosis by scavenging ROS, improving antioxidant capacity, protecting mitochondrial and regulating the proteins expression related to apoptosis.

Gukang Capsule Promotes Fracture Healing by Activating BMP/SMAD and Wnt/ß-Catenin Signaling Pathways.

BACKGROUND: Gukang capsule (GKC) is a traditional Chinese medicine formulation which has been used extensively in the clinical treatment of bone fractures. However, the mechanisms underlying its effects on fracture healing remain unclear. METHODS: In this study we used a rabbit radius fracture model, and we measured the serum content of bone alkaline phosphatase (ALP), calcium, and phosphorus and examined pathology of the fracture site as indicators of the fracture healing effects of GKC. SaOS-2 human osteosarcoma cells were used to measure (i) ALP activity, (ii) ornithine transcarbamylase (OTC), calcium, and mineralization levels, (iii) the expression of osteogenic-related genes, that is, runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen I (COL-I), osteopontin (OPN), OTC, and osterix (Osx), and (iv) the expression of key proteins in the Wnt/ß-catenin and BMP/SMAD signaling pathways to study the mechanisms by which GKC promotes fracture healing. RESULTS: We found that GKC effectively promotes radius fracture healing in rabbits and enhances ALP activity, increases OTC and calcium levels, and stimulates the formation of mineralized nodules in SaOS-2 cells. Moreover, COL-I, OTC, Osx, BMP2, and OPN expression levels were higher in SaOS-2 cells treated with GKC than control cells. GKC upregulates glycogen synthase kinase 3ß (GSK3ß) phosphorylation and Smad1/5 and ß-catenin protein levels, thereby activating Wnt/ß-catenin and BMP/Smad signaling pathways. Inhibitors of the Wnt/ß-catenin and BMP/Smad signaling pathways (DKK1 and Noggin, respectively) suppress the osteogenic effects of GKC. CONCLUSIONS: GKC promotes fracture healing by activating the Wnt/ß-catenin and BMP/Smad signaling pathways and increasing osteoprotegerin (OPG) secretion by osteoblasts (OBs), which prevents receptor activator of nuclear factor kappa B ligand (RANKL) binding to RANK.

[Study on metabolites of Laportea bulbifera extract in rat feces based on UHPLC-Q-TOF-MS~E technique].

This project is to study the metabolites of Laportea bulbifera extract in rat feces. After the SD rats were gavaged with the extract(136 g·kg~(-1), according to the crude drug dose), the metabolites in their feces were detected by UHPLC-Q-TOF-MS~E technique, and the obtained mass spectrometry data was combined with UNIFI software for prediction. The prototype components and metabolites in rat feces were identified with reference materials and related literature. A total of 43 metabolites were identified(including 8 prototype components and 35 metabolites). The metabolic pathways mainly include monocaffeoylquinic acid(hydrogenation reduction, ring-opening cracking, sulfation, hydroxylation, glucuronidation), quercetin(O-C2 bond ring-opening cleavage, C2-C3 double bond reduction, rutin carbonylation) and so on. The metabolites and metabolic process of L. bulbifera extract in rat feces were clarified, which provided a basis for the study of the active substances and its mechanism of action.

[Differences in intestinal absorption characteristics of Laportea bulbifera extract in normal and rheumatoid arthritis model rats by isolated everted intestine model].

This work aimed to investigate the intestinal absorption characteristics of Laportea bulbifera extract in normal and rheumatoid arthritis model rats. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, kaempferol-3-O-rutinoside, galuteolin, quercetin and isoquercetin in intestinal absorption solution samples were detected by UPLC-MS/MS with 5.0 g·L~(-1) as the absorption concentration. The cumulative absorption(Q) and absorption rate constant(K_a) were calculated, and the absorption characteristics of different components of L. bulbifera in intestinal absorption solution of normal rats and rheumatoid arthritis rats were compared. The results showed that all the eight index components in the extract of L. bulbifera could be absorbed into the intestinal capsule, the cumulative absorption-time curve of each component showed an upward trend without saturation, and the correlation regression coefficient(R~2) was greater than 0.92, which is consistent with the zero-order absorption rate process. It was speculated that the possible absorption mode of each component was passive diffusion. In normal condition, the absorption of ileum was the best(except chlorogenic acid), and in pathological condition, duodenum was the best. The total absorption of 8 components in each intestinal segment of RA rats was better than that of normal rats, which speculated that rheumatoid arthritis may change the specific site of drug absorption. The experimental results showed that rheumatoid arthritis could change the intestinal absorption of the extract of L. bulbifera, and its mechanism needs further study.

Comparative pharmacokinetics of quercitrin, astragalin, afzelin and taxifolin in plasma after oral administration of Polygonum orientale inflorescence in sham-operated and myocardial ischemia-reperfusion injury rats.

The study aimed to compare the pharmacokinetic properties of quercitrin, astragalin, afzelin and taxifolin, four major bioactive components of Polygonum orientale inflorescence extracts, between sham-operated and myocardial ischemia-reperfusion injury (MIRI) rats.Rats were divided into two groups: MIRI model and sham-operated. The blood samples were collected according to the time schedule. The levels of quercitrin, astragalin, afzelin and taxifolin in the plasma at designated time points were determined using an HPLC-MS/MS method. Various pharmacokinetic parameters were estimated from the plasma concentration versus time data using non-compartmental methods. After the administration of the Chinese herb Polygonum orientale inflorescence extracts, the Cmax, AUC, as well as MRT, increased, while CL decreased, in MIRI model compared to the sham-operated animals.These results suggest that the pathological damage of ischemia-reperfusion had a significant impact on the pharmacological effects of Polygonum orientale inflorescence extracts on ischemic heart disease.The method had been successfully applied to evaluate the pharmacokinetics of quercitrin, astragalin, afzelin and taxifolin in rat plasma after the oral administration of Chinese herb Polygonum orientale inflorescence extracts in rats.

[Establishment of Flp-InTM CHO cell lines with double expression of CYP2A13 and MRP2].

Objective To construct a double transfected Flp-InTM CHO cell line stably expressing both cytochrome P450 family 2 subfamily A member 13(CYP2A13) and multidrug resistance-associated protein 2(MRP2). Methods We constructed the recombinant plasmids of pCMV6-NEO-CYP2A13 and pcDNA5-MRP2. The pCMV6-NEO-CYP2A13 recombinant plasmid was first transfected into Flp-InTM CHO cells, and CYP2A13-Flp-InTM CHO cells with higher CYP2A13 activity were screened using limiting dilution method and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) cytotoxicity assay. Thereafter, pcDNA5-MRP2 was transfected into CYP2A13-Flp-InTM CHO cells. The expression levels and activities of CYP2A13 and MRP2 in the double transfected cells and normal cells were detected by real-time quantitative PCR, Western blot analysis and NNK cytotoxicity assay in order to screen Flp-InTM CHO cells with stable expression of CYP2A13 and MRP2. Results Compared with non-transfected cells, the expression of CYP2A13 and the sensitivity of NNK toxicity in CYP2A13-Flp-InTM CHO cells increased. The expression of CYP2A13 and MRP2 in CYP2A13/MRP2-Flp-InTM CHO cells also increased significantly. Compared with CYP2A13-Flp-InTM CHO cells, CYP2A13/MRP2-Flp-InTM CHO cells showed no significant difference in CYP2A13 expression; the expression of MRP2 increased while the sensitivity of NNK toxicity decreased significantly. Conclusion The double transfected cell model of CYP2A13 and MRP2 has been successfully established, which lays the foundation for the study of in situ activation of respiratory carcinogens.

Simultaneous Determination of Three Bioactive Constituents from Bletilla striata by UPLC-MS/MS and Application of the Technique to Pharmacokinetic Analyses.

Bletilla striata has been widely used as a valuable hemostatic agent for thousands of years due to the high levels of bioactive constituents it contains. Here, we used a sensitive ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) method for the simultaneous determination of three major active ingredients of the B. striata extract, namely, α-isobutylmalic acid, gymnoside I, and militarine in rat plasma. The three major active ingredients were determined using the multiple reaction monitoring (MRM) mode at m/z 189 ⟶ 129 for α-isobutylmalic acid, m/z 457.2 ⟶ 285.1 for gymnoside I, m/z 725.3 ⟶ 457.2 for militarine, and m/z 417.0 ⟶ 267.0 for the IS puerarin. All calibration curves showed good linearity (R 2 ≥ 0.999) over the concentration range with the lower limit of quantification between 0.015 and 0.029 µg/mL. The relative standard deviations of intraday and interday measurements were less than 15%, and the method was accurate within 93.3-100.4%. The extraction recovery was 92.65-100.98%, and no matrix effect was observed. The results indicated that after oral administration of B. striata in rats, the T max of α-isobutylmalic acid was significantly longer than that of gymnoside I and militarine and the mean residence time and area under the curve of α-isobutylmalic acid and gymnoside I in rats were significantly higher than those of militarine. Moreover, the blood concentration-time curve of α-isobutylmalic acid showed double peaks, suggesting that α-isobutylmalic acid could exhibit the phenomenon of enterohepatic circulation or metabolic conversion. We also explored some of the pharmacokinetic characteristics of three ingredients from B. striata extract in vivo, and the data obtained may provide a basis for the further investigation of B. striata.

Comparative Pharmacokinetics of Gallic Acid, Protocatechuic Acid, and Quercitrin in Normal and Pyelonephritis Rats after Oral Administration of a Polygonum capitatum Extract.

Polygonum capitatum Buch.-Ham. ex D. Don is traditionally used by Hmong for the treatment of urinary tract infections and pyelonephritis. Information regarding the pharmacokinetic behavior of the extract in the condition of pyelonephritis is lacking. In the present study, we aimed to compare the pharmacokinetic properties of gallic acid (GA), protocatechuic acid (PCA), and quercitrin (QR)-the main bioactive constituents in the herb-in normal and pyelonephritis rats. The plasma samples were collected at various time points after administration of a single dose of Polygonum capitatum extract. The plasma level of GA, PCA, and QR at the designed time points was determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and drug concentration versus time plots were constructed to estimate the pharmacokinetic parameters. The AUC(0-t), AUC(0-∞), MRT(0-t), and CL of GA, PCA, and QR in pyelonephritis rats was significantly different from those of the normal rats. The results indicated that the three constituents have higher rate of uptake and slower rate of elimination in the rats with pyelonephritis, suggesting altered rate and extent of drug metabolism.

[Determination of plasma protein binding rates of nine compounds of Inula cappa extraction based on method of equilibrium dialysis].

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

(E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides as tubulin polymerization inhibitors: Structure-based bioisosterism design, synthesis, biological evaluation, molecular docking and in silico ADME prediction.

A series of (E)-N-Aryl-2-oxo-2-(3,4,5-trimethoxyphenyl)acetohydrazonoyl cyanides have been synthesized and evaluated for their anticancer activity in human hepatocellular liver carcinoma HepG2 and breast adenocarcinoma MCF-7 cell lines. Among all the tested compounds, compound 3a, 3e and 3n displayed more activity than lead compound with IC50 value of 0.26-0.61 µM. Meanwhile, these compounds (3a, 3e and 3n) showed potent antiproliferative activity against a panel of cancer cells and the HCT-8/T multidrug resistant cell line with IC50 values in the range of 0.077- 7.44 µM. Flow cytometric analyses revealed that compound 3n induced cell cycle arrest in G2/M phases in a dose dependent manner. The compound 3n also displayed potent tubulin polymerization inhibition with an IC50 value of 0.9 µM, with ten folds more active than colchicine (IC50 = 9 µM). Molecular docking studies revealed that compound 3n efficiently interacted with the colchicine binding site of tubulin through hydrophobic, cation-π and hydrogen bond interaction. Furthermore, in silico pharmacokinetic prediction shown that these compounds have a good ADME-related physicochemical parameters. These results demonstrate that 3n exhibits potent cytotoxicity in cancer cells by targeting the colchicine binding site of tubulin and potentially acts as a therapeutic lead compound for the development of anticancer drugs.

In Vivo and In Vitro Anti-Arthritic Effects of Cardenolide-Rich and Caffeoylquinic Acid-Rich Fractions of Periploca forrestii.

Periploca forrestii Schltr. (P. forrestii) is a species used in Traditional Chinese Medicine (TCM) known as "Miao medicine", and has a long history of use in the treatment of rheumatism, rheumatoid arthritis (RA), and joint pain. The present study aimed to evaluate the anti-arthritis effects of the cardenolide-rich and caffeoylquinic acid-rich fractions (CDLFs and CQAFs) of P. forrestii in collagen-induced arthritic (CIA) rats, and defined the mechanisms of therapeutic action in MH7A cells treated with TNF-α. Serum rheumatoid factor (RF), TNF-α, IL-6, IL-1ß, PGE2, NO, SOD, and MDA were determined by ELISA or other commercially assay kits. Histopathological changes in ankle joint tissues were examined. The mRNA expressions of IL-1ß, IL-6, COX-2, and iNOS in MH7A cells were measured by qRT-PCR assays. In addition, the expressions of iNOS, COX-2, and p65 proteins, and the phosphorylation of IκBα, p38, ERK1/2, and JNK proteins in MH7A cells were analyzed by Western blot. The results showed that CDLF and CQAF could suppress the paw swelling in CIA rats at different doses (125 mg/kg, 250 mg/kg, and 500 mg/kg). Histopathological examination suggests that the CDLF and CQAF significantly relieved the damage of the structure of the ankle joint in CIA rats. In addition, serum RF, TNF-α, IL-6, IL-1ß, PGE2, NO, and MDA were decreased, along with increased activity of serum SOD. Furthermore, CDLF and CQAF downregulated the expressions of IL-1ß, IL-6, COX-2, iNOS, and p65, and inhibited the phosphorylation of IκBα, p38, ERK1/2, and JNK in MH7A cells treated with TNF-α. These findings demonstrated that both CDLF and CQAF exhibited anti-arthritic activity, which might be associated with their inhibitory effects on the NF-κB and MAPK signaling pathways.

A UPLC-ESI-MS/MS Method for Simultaneous Quantitation of Chlorogenic Acid, Scutellarin, and Scutellarein in Rat Plasma: Application to a Comparative Pharmacokinetic Study in Sham-Operated and MCAO Rats after Oral Administration of Erigeron breviscapus Extract.

Erigeron breviscapus, a traditional Chinese medicine, is clinically used for the treatment of occlusive cerebral vascular diseases. We developed a sensitive and reliable ultra-performance liquid chromatography-electrospray-tandem mass spectrometry (UPLC-ESI-MS/MS) method for simultaneous quantitation of chlorogenic acid, scutellarin, and scutellarein, the main active constituents in Erigeron breviscapus, and compared the pharmacokinetics of these active ingredients in sham-operated and middle cerebral artery occlusion (MCAO) rats orally administrated with Erigeron breviscapus extract. Plasma samples were collected at 15 time points after oral administration of the Erigeron breviscapus extract. The levels of chlorogenic acid, scutellarin, and scutellarein in rat plasma at various time points were determined by a UPLC-ESI-MS/MS method, and the drug concentration versus time plots were constructed to estimate pharmacokinetic parameters. The concentration of chlorogenic acid in the plasma reached the maximum plasma drug concentration in about 15 min and was below the limit of detection after 4 h. Scutellarin and scutellarein showed the phenomenon of multiple absorption peaks in sham-operated and MCAO rats, respectively. Compared with the sham-operated rats, the terminal elimination half-life of scutellarein in the MCAO rats was prolonged by more than two times and the area under the curve of each component in the MCAO rats was significantly increased. The results showed chlorogenic acid, scutellarin, and scutellarein in MCAO rats had higher drug exposure than that in sham-operated rats, which provided a reference for the development of innovative drugs, optimal dosing regimens, and clinical rational drug use.

[Protective effect of Polygonum orientale flower extract on H2O2-induced oxidative damage of HUVEC cells].

To investigate the protective effects and mechanism of Polygonum orientale flower extract on H2O2-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H2O2 was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 µmol·L⁻¹ H2O2 for 0.5 h. Treatment of H2O2 also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H2O2 model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H2O2-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.

[Absorption of Inula cappa extract based on everted intestinal sac method].

To investigate the absorptive characteristics of Inula cappa extract based on the rat everted intestinal sac method in vitro. Nine representative ingredients in I. cappa extract were selected as the study objects. An UPLC-MS/MS method was established to determine and detect their cumulative absorption amount for expounding the absorptive characteristics of ingredients in different intestinal sections. According to the results， the transport mechanism of 8 compounds showed passive diffusion by the reverted gut sac method. And scopolin was actively transported in the intestine. The best absorption site of chlorogenic acid was duodenum. The best absorption site of cryptochlorogenic acid， 1，3-O-dicaffeoylquinic acid， luteolin-7-glucoside and 3，4-O-dicaffeoylquinic acid were jejunum. The best absorption site of neochlorogenic acid， scopolin， 4，5-O-dicaffeoylquinic acid and 3，5-O-dicaffeoylquinic acid was ileum. The absorption of all the compounds was affected by pH and bile. All of the nine ingredients in I. cappa extract could be absorbed in intestines， but with differences in the absorption rate， the best absorptive site and mechanism， indicating that the intestinal absorption of I. cappa extract was selective.

[Metabolism of Inula cappa extract by rat intestinal bacteria in vitro].

To investigate the metabolism of major components in Inula cappa by rat intestinal bacteria in vitro. I. cappa extract was incubated for 24 h with rat intestinal bacteria under anaerobic environment. After the samples were precipitated by n-butanol, UPLC-Q-TOF-MS/MS was applied for the qualitative analysis of the metabolites, combined with data software such as Metabolite Tools, Data Analysis and so on. The potential metabolites in rat intestinal bacteria were analyzed by comparing the total ion current of the test samples and blank samples and analyzing the quasi-molecular ion and fragment ion of all chromatograms. The results injected that fourteen metabolites were detected in rat intestinal flora. Various types of metabolic reactions happen to caffeoylquinic acid in intestinal flora, including isomerization, hydrolyzation, there were also methylation, hydrogenation and acetylation of caffeic acid. At the same time, a methylate of dicaffeoylquinic acid was also detected. Presumably, caffeoylquinic acids were gradually transformed into more hydrophobic metabolites with smaller molecular mass, which were better absorbed by the intestinal tract.

A UPLC-MS/MS Method for Simultaneous Determination of Free and Total Forms of a Phenolic Acid and Two Flavonoids in Rat Plasma and Its Application to Comparative Pharmacokinetic Studies of Polygonum capitatum Extract in Rats.

The principal active constituents of Polygonum capitatum are phenolic acids and flavonoids, such as gallic acid, quercitrin, and quercetin. The aim of this study was to develop and validate a method to determine the three constituents and the corresponding conjugated metabolites of Polygonum capitatum in vivo and to conduct pharmacokinetic studies on the herb, a well-known Miao medicinal plant in China. Gallic acid, quercitrin, and quercetin were analysed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). Protein precipitation in plasma samples was performed using methanol. For the determination of total forms of analytes, an additional process of hydrolysis was conducted using ß-glucuronidase and sulphatase. The analytes were separated on a BEH C18 column (50 mm × 2.1 mm; i.d., 1.7 µm) and quantified by multiple reaction monitoring (MRM) mode. The linear regression showed high linearity over a 729-fold dynamic range for the three analytes. The relative standard deviations of intra- and inter-day measurements were less than 9.5%, and the method was accurate to within -11.1% to 12.5%. The extraction recoveries for gallic acid, quercitrin, and quercetin were 94.3%-98.8%, 88.9%-98.8%, and 95.7%-98.5%, respectively. All samples were stable under short- and long-term storage conditions. The validated method was successfully applied to a comparative pharmacokinetic study of gallic acid, quercitrin, and quercetin in their free and total forms in rat plasma. The study revealed significantly higher exposure of the constituents in total forms for gallic acid and quercetin, while quercitrin was detected mainly in its corresponding free form in vivo. The established method was rapid and sensitive for the simultaneous quantification of free and total forms of multiple constituents of Polygonum capitatum extract in plasma.

Pharmacokinetic Comparison of Scutellarin and Paeoniflorin in Sham-Operated and Middle Cerebral Artery Occlusion Ischemia and Reperfusion Injury Rats after Intravenous Administration of Xin-Shao Formula.

Xin-Shao formula is a folk remedy widely used in China to prevent and cure stroke. Cerebral ischemic reperfusion (I/R) injury often takes place during the treatment of stroke. Information about the pharmacokinetic behavior of the remedy under cerebral I/R injury conditions is lacking. The present study aimed to compare the pharmacokinetic properties of scutellarin and paeoniflorin, two major bioactive components of Xin-Shao formula, under physiological state in cerebral I/R injury rats. Neurobehavioral dysfunction was evaluated and cerebral infarcted volume was measured in middle cerebral artery occlusion I/R injury (MCAO) rats. Plasma samples were collected at various time points after a single dose (intravenous, i.v.) of Xin-Shao formula. The levels of plasma scutellarin and paeoniflorin at the designed time points were determined by a UPLC-MS/MS method, and drug concentration versus time plots were constructed to estimate pharmacokinetic parameters. Increase in terminal elimination half-life (t1/2z) and mean residence time (MRT(0-t)) of scutellarin as well as elevation in area under the plasma drug concentration-time curve from 0 h to the terminal time point (AUC(0-t)) and maximum plasma drug concentration (Cmax) of paeoniflorin, along with decreased clearance of paeoniflorin and scutellarin as well as reduced apparent volume of distribution (Vz) of paeoniflorin, were observed in MCAO rats, compared with those in sham-operated animals. The elimination of scutellarin and paeoniflorin were reduced in cerebral I/R injury reduced rats.