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.

Polygonum orientale L. Alleviates Myocardial Ischemia-Induced Injury via Activation of MAPK/ERK Signaling Pathway.

Although Polygonum orientale L. (PO) has a beneficial effect on treatment of myocardial ischemia (MI), its mechanism remains unclear. This study aimed to explore the pharmacological mechanism of PO against MI through MAPK signaling pathways. Firstly, the therapeutic effect of PO was evaluated for treatment of MI mice. Using Western blot and immunohistochemistry, the influence of PO on MAPK signaling pathways and cell apoptosis was investigated. Subsequently, one key pathway (ERK) of MAPK signaling pathways was screened out, on which PO posed the most obvious impact. Finally, an inhibitor of ERK1/2 was utilized to further verify the regulatory effect of PO on the MAPK/ERK signaling pathway. It was found that PO could reduce the elevation of the ST segment; injury of heart tissue; the activity of LDH, CK, NOS, cNOS and iNOS and the levels of NO, BNP, TNF-α and IL-6. It is notable that PO could significantly modulate the protein content of p-ERK/ERK in mice suffering from MI but hardly had an effect on p-JNK/JNK and p-p38/p38. Additionally, the expressions of bax, caspase3 and caspase9 were inhibited in heart tissue in the PO-treated group. To evaluate whether ERK1/2 inhibitor (PD98059) could block the effect of PO on treatment of MI, both PO and PD98059 were given to mice with MI. It was discovered that the inhibitor indeed could significantly reverse the regulatory effects of PO on the above indicators, indicating that PO could regulate p-ERK/ERK. This study provides experimental evidence that PO extenuates MI injury, cardiomyocyte apoptosis and inflammation by activating the MAPK/ERK signaling pathway.

Antibacterial Activity and Mechanism of Polygonum orientale L. Essential Oil against Pectobacterium carotovorum subsp. carotovorum.

Infected by Pectobacterium carotovorum subsp. carotovorum (Pcc), the quality of Chinese cabbage could severely decline. Using chemical bactericides to control Pcc could cause food safety problems. Thus, we investigated the optimum extraction conditions, antibacterial activity, chemical compounds and antibacterial mechanism of Polygonum orientale L. essential oil (POEO) against Pcc in order to search a new way to control Pcc. The optimum extraction conditions of POEO (soaking time 2.6 h, extraction time 7.7 h and ratio of liquid to solid 10.3 mL/g) were optimized by response surface methodology. The minimum inhibitory concentration (MIC) of POEO against Pcc was 0.625 mg/mL. The control efficiency of protective activity of POEO against Pcc was 74.67~92.67%, and its curative activity was 76.00~93.00%. Then, 29 compounds were obtained by GC-MS; the prime compounds of POEO were phytol, phytone, n-pentacosane, 1-octen-3-ol and ß-ionone. It was verified that, compared with control samples, POEO destroyed cell morphology. It increased surface potential, increased hydrophobicity, damaged cell walls, destroyed the integrity and permeability of cell membrane, reduced membrane potential (MP), and changed membrane protein conformation. It inhibited the activities of pyruvate kinase (PK), succinate dehydrogenase (SDH) and adenosine triphosphatase (ATPase). Briefly, the results of this study demonstrate that POEO showed effective inhibitory activity against Pcc, thus POEO could have potential application in controlling Pcc.

[Pharmacokinetic study of Polygonum orientale extract in H9c2 cells by UPLC-MS/MS].

A detection method of ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was established to detect concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-ß-D-glucoside in H9 c2 cells and applied to the pharmacokinetic study of Polygonum orientale extract in the cells. H9 c2 cells were treated with 100 µg·mL~(-1) P. orientale extract and then they and the corresponding nuclei, mitochondria and Golgi bodies were collected at the set time. After protein precipitation, UPLC-MS/MS was used to determine concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-ß-D-glucoside in the whole cells and subcellular structures. Also, related pharmacokinetic parameters were calculated. The results showed that the peak time was 8 h for all these components. Orientin, vitexin, quercetin and isoorientin have high affinities to nuclei and mitochondria, while the affinity of kaempferol-3-O-ß-D-glucoside is higher with mitochondria compared to nuclei. It is suggested that these chemical components of P. orientale may mainly act on nuclei or mitochondria to exert pharmacological effects of protecting cardiomyocytes.

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.

[Intestinal absorption characteristics of Polygonum orientale extract in normal and isoproterenol-induced myocardial ischemia model rats via everted intestinal sac models].

The present study is to investigate the absorption characteristics of the main components in Polygonum orientale extract in normal and isoproterenol-induced myocardial ischemia model rats with everted intestinal sac models. Intestinal sac fluid samples were collected in different part of intestine(duodenum, jejunum, ileum, colon) at different time after administration of different concentration of P. orientale extract(5.0,10.0, 20.0 mg·mL~(-1)). An UPLC-TQD method was employed for the determination of six components including orientin, isoorientin, vitexin, protocatechuic acid, kaempferol-3-O-ß-D-glucoside and quercitrin in the intestinal sac samples. The absorption rate and cumulative absorption were calculated to analyze the intestinal absorption characteristics of six components in normal and myocardial ischemia model rats. The P-glycoprotein(P-gp) inhibitor was applied to investigate influence of intestinal absorption of six components in P. orientale extract. The results showed that the main absorption sites were concentrated on the duodenum at low concentration, while they were the colon at the medium concentration and the ileum at high concentration in control groups. In the condition of myocardial ischemia model, the main absorption sites focus on the ileum and jejunum at low concentration; the main absorption sites were in the ileum at the medium concentration and main absorption sites were the duodenum and ileum at high concentration. Compared with the normal group, the absorption rate and cumulative absorption of the six components significantly decreased in the model group. P-gp inhibitor markedly increased the absorption rate and cumulative absorption of six components in the model group, inferring that the 6 components may be the substrates of P-gp, and the mechanism needs further study. In this study, it is revealed that the six components of P. orientale extract can be absorbed into the intestinal sac, and it is an effective method to assess the intestinal absorption characteristics of P. orientale extract through everted intestinal sac model, providing data support for the clinical application and further development of P. orientale.

A strategy for component-based Chinese medicines design approach of Polygonum orientale L. against hypoxia/reoxygenation based on uniform design-stepwise regression-simulated annealing.

Presently, optimal proportions and synergistic mechanisms of component-based Chinese medicines are critical for developing novel strategies to treat cardiovascular diseases (CVDs). A new multi-objective optimization algorithm based on uniform design (UD) and stepwise regression (SR) modeling is proposed to find the synergistic effect of orientin (Ori), quercitrin (Que) and vitexin (Vit), the three effective components from Polygonum orientale L., using the H9c2 cells injury induced by hypoxia/reoxygenation (H/R). The optimal proportion of these three components was calculated by simulated annealing (SA). In this research, the excellent combination named OQV-e (Ori: Que: Vit =12.55 µM: 39.99 µM: 19.99 µM) could exert significant cardioprotection against the H9c2 cells injury induced by H/R through increasing cell viability, decreasing leakage rate of lactate dehydrogenase (LDH) and the level of nitric oxide (NO). Moreover, western blot analysis revealed that OQV-e could activate autophagy by inhibiting the p-JNK/JNK signaling pathway, which showed that the method (UD-SR-SA) was a feasible strategy. Mathematical system modeling may be a considerable approach for the powerful mathematical analysis of the complex pharmacological effects of component-based Chinese medicines from herbal medicines, which might greatly enhance the efficiency to find new modern Chinese drugs for CVDs based on Chinese herbal medicine (CHM) with affirmative therapeutic effect.

Intestinal absorption characteristics of Polygonum orientale extract in normal and isoproterenol-induced myocardial ischemia model rats via everted intestinal sac models / 中国中药杂志

The present study is to investigate the absorption characteristics of the main components in Polygonum orientale extract in normal and isoproterenol-induced myocardial ischemia model rats with everted intestinal sac models. Intestinal sac fluid samples were collected in different part of intestine(duodenum, jejunum, ileum, colon) at different time after administration of different concentration of P. orientale extract(5.0,10.0, 20.0 mg·mL~(-1)). An UPLC-TQD method was employed for the determination of six components including orientin, isoorientin, vitexin, protocatechuic acid, kaempferol-3-O-β-D-glucoside and quercitrin in the intestinal sac samples. The absorption rate and cumulative absorption were calculated to analyze the intestinal absorption characteristics of six components in normal and myocardial ischemia model rats. The P-glycoprotein(P-gp) inhibitor was applied to investigate influence of intestinal absorption of six components in P. orientale extract. The results showed that the main absorption sites were concentrated on the duodenum at low concentration, while they were the colon at the medium concentration and the ileum at high concentration in control groups. In the condition of myocardial ischemia model, the main absorption sites focus on the ileum and jejunum at low concentration; the main absorption sites were in the ileum at the medium concentration and main absorption sites were the duodenum and ileum at high concentration. Compared with the normal group, the absorption rate and cumulative absorption of the six components significantly decreased in the model group. P-gp inhibitor markedly increased the absorption rate and cumulative absorption of six components in the model group, inferring that the 6 components may be the substrates of P-gp, and the mechanism needs further study. In this study, it is revealed that the six components of P. orientale extract can be absorbed into the intestinal sac, and it is an effective method to assess the intestinal absorption characteristics of P. orientale extract through everted intestinal sac model, providing data support for the clinical application and further development of P. orientale.

Pharmacokinetic study of Polygonum orientale extract in H9c2 cells by UPLC-MS/MS / 中国中药杂志

A detection method of ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was established to detect concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-β-D-glucoside in H9 c2 cells and applied to the pharmacokinetic study of Polygonum orientale extract in the cells. H9 c2 cells were treated with 100 μg·mL~(-1) P. orientale extract and then they and the corresponding nuclei, mitochondria and Golgi bodies were collected at the set time. After protein precipitation, UPLC-MS/MS was used to determine concentrations of isoorientin, orientin, quercetin, vitexin and kaempferol-3-O-β-D-glucoside in the whole cells and subcellular structures. Also, related pharmacokinetic parameters were calculated. The results showed that the peak time was 8 h for all these components. Orientin, vitexin, quercetin and isoorientin have high affinities to nuclei and mitochondria, while the affinity of kaempferol-3-O-β-D-glucoside is higher with mitochondria compared to nuclei. It is suggested that these chemical components of P. orientale may mainly act on nuclei or mitochondria to exert pharmacological effects of protecting cardiomyocytes.

Study on Improvement Effects of Polygonum orientale Flower Extract on Hypoxia-reoxygenation Injury of H 9c2 Cardiomyocytes / 中国药房

OBJECTIVE：To study the improvement effects and mechanism of Polygonum orientale flower extract on hypoxia- reoxygenation injury of H 9c2 cardiomyocytes. METHODS ：H9c2 cardiomyocytes were divided into normal control group ，model group and low- ，medium- and high-concentrations groups of P. orientale flower extract （20，40，80 μg/mL）. Except for normal control group ，other groups were given 800 μmol/L CoCl2 to induce hypoxia-reoxygenation injury model. Cell apoptosis was observed. The levels of Ca 2+（in cytoplasm ），mitochondrial membrane potential （MMP），ATP enzyme （Na+-K+-ATP enzyme ，Ca2+-Mg2+-ATP enzyme） activities， the ratio of cytochrome c （Cyto c ）， protein in cytosol to mitochondria ，phosphorylation levels of reperfusion injury salvage kinase （RISK） signaling pathwayrelated protein [protein kinase B （Akt）and extracellular signal regulated kinase 1/2（ERK1/2）] as well as protein expression of HIF- 1 α were detected respectively. In addition，the cells were divided into normal control group ，model group and P. orientale flower extract group （80 μ g/mL），PI3K inhibitor LY294002+CoCl2 group（15 μmol/L LY294002+80 μmol/L ，LY294002+P. orientale flower extract group （15 μmol/L LY294002+80 μg/mL P. orientale flower extract ），MEK inhibitor PD98059+CoCl2 group（25 μmol/L PD98059+800 μmol/L CoCl2），PD98059+P. orientale flower extract group （25 μmol/L PD98059+80 μg/mL P. orientale flower extract ）. After cultured by the same method ，the phosphorylation levels of Akt protein and ERK1/2 protein in the cells were measured to verify the activation of P. orientale flower extract to RISK signaling pathway. RESULTS：Compared with model group ，nuclear pyknosis and the number of apoptotic bodies were reduced in different concentrations groups of P. orientale flower extract. ROS level ，Ca2+ level（except for low-concentration group ），MMP，ratio of Cyto c in cytoplasm to Cyto c in mitochondria ，protein expression of HIF- 1α were decreased significantly（P＜0.05 or P＜0.01）； the activity of ATP enzyme （except for the low-concentration group ），Akt protein and ERK 1/2 protein phosphorylation level were significantly increased （P＜0.01）. After treated with PI 3K inhibitor LY 294002 and MEK inhibitor PD 98059，Akt protein and ERK 1/2 protein phosphorylation level in cadiomyocyte were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：P. orientale flower extract can improve hypoxia-reoxygenation injury of H 9c2 cardiomyocytes，the mechanism of which may be associated with inhibiting cardiomyocyte apoptosis ，improving ATPase activity ，protecting mitochondria ，regulating RISK signaling pathway related proteins and HIF- 1α protein expression.

Preventive effects of Polygonum orientale L. on ovariectomy-induced osteoporosis in rats.

Objective: The aim of the present study was to evaluate the effects of ethyl acetate extract of Polygonum orientale L. (POE) on ameliorating postmenopausal osteoporosis in ovariectomized (OVX) rats.Methods: Six-month-old female rats were randomly divided into seven groups: sham-operated; OVX; OVX with estradiol valerate; OVX with alendronate; and OVX with POE in graded doses (3.75, 5.0, or 7.5 g/kg/day). Administration began at week 6 after ovariectomy for 12 weeks. A comprehensive assessment of bone quality was performed, including serum biochemical markers, serum inflammatory factors, bone oxidative stress markers, bone mechanics, and bone histomorphometry.Results: POE treatment significantly decelerated OVX-induced body weight gain without affecting the uterus index and produced a significant decrease in the levels of serum bone turnover markers (p < 0.05 or p < 0.01). Biomechanical testing demonstrated that POE (5.0 and 7.5 g/kg/day) treatments significantly prevented the reduction in maximum stress and Young's modulus in OVX rats (p < 0.05). Compared with the OVX group, POE (3.75, 5.0, or 7.5 g/kg/day) treatments significantly increased trabecular bone mineral density by 35.03, 38.42, and 42.02%, respectively.Conclusion: Our findings suggest that POE has potential effects in regulation of bone metabolism and prevention of bone loss in postmenopausal osteoporosis.

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.

Preliminary study on Q-markers of anti-tumor activity extracts of Polygonum orientale / 中草药

Objective: Based on the concept of quality marker (Q-marker), the components and the quality of the ethyl acetate extract of Polygonum orientale (POEa) was analyzed and studied. Methods: Firstly, the components of POEa were identified using the UPLC-ESI-HRMS method and standard compounds. Secondly, the main active compounds were determined by HPLC. Antitumor activities of these compounds were reviewed and its Q-marker was predicted. Finally, we evaluated the effects of POEa and the compound of gallic acid, isoquercetin, valerin, vitexin, luteolin, and quercetin on proliferation, apoptosis and migration of A549 cells. Results: A new quality method for simultaneous determining these six compounds of POEa was established. The six chemical ingredients were detected in each sample and the total content was more than 10%. The number of apoptotic cells in A549 cells treated with POEa and six chemical mixtures were all substantial increased, and the migration amount were significantly decreased. Tow groups showed no significantly differeances. Conclusion: The six components are scientific and reasonable to be considered as potential Q-marker represented the anti-tumor activity of POEa. The HPLC method can be used as accurate and stable quality control strategy of POEa.

Study on Intestinal Absorption Characteristic Differences of 6 Active Constituents of Polygonum orientale in Normal and Myocardial Ischemia Model Rats / 中国药房

OBJECTIVE：To s tudy the intestinal absorption differences of 6 kinds of active constituents of Polygonum orientale （kaempferol，isokaempferol，vitexin，protocatechuic acid ，kaempferol-3-O-β-D-glucoside and quercetin ）in normal and myocardial ischemia（MI）model rats. METHODS ：UPLC-MS/MS method was adopted to determine the contents of 6 active components in the intestinal circulatory perfusion fluid. Totally male SD 80 rats were divided into normal group and model group ，with 40 rats in each group. Model group was given isoproterenol hydrochloride （50 mg/kg） subcutaneously to induce MI model；normal group was given constant volume of normalsaline， once a day ， for consecutive 2 days. 24 h after successful molding ，normal group and model group received in-situ intestinal circulatory perfusion experiment. The effects of different concentration s of P. orientale extract（5.0，10.0， 20.0 mg/mL），different intestinal segments （duodenum，jejunum，ileum，colon），P-glycoprotein（P-gp）inhibitors（verapamil） and bile on the intestinal absorption of each constituent were explored. RESULTS ：The linear ranges of concentrations of kaempferol， isokaempferol， vitexin， protocatechuic acid ， kaempferol-3-O-β-D-glucoside and quercetin were 3.15-50.40， 3.21-51.31，1.63-52.43，1.60-50.94，1.31-20.97，8.07-129.25 µg/mL（r＞0.999）. The lower limits of quantification were 7.86， 8.45，6.52，4.00，3.28，16.14 ng/mL，respectively. RSDs of precision ，matrix effect and stability tests were all lower than 11％； the accuracy were 85.64％-107.65％，which were in line with the requirements of biological sample quantification analysis. Except for there was no statistical significance in the absorption of kaempferol absorption in duodenum of model group at different concentrations，absorption of other five constituents in duodenum of normal and model rats increased with the increase of the concentration of active constituents ，and absorption of medium- and/or high- concentration active constituents （except quercetin ）in model group was significantly lower than normal group （P＜0.05）. In normal group ，the absorption of kaempferol was more in jejunum，ileum and colon ，isokaempferol was more in ileum ，vitexin and protocatechuic acid were more in jejunum and ileum ， kaempferin-3-O- β-D-glucoside was more in duodenum ，jejunum and colon ，quercetin was more in colon ；in the model group ，the absorption of Polygonum orientale in jejunum and colon was more ，the absorption of isokaempferol in 4 intestinal segments was little different ，vitexin was mainly absorbed in ileum ，protocatechuic acid and kaempferol- 3-O-β-D-glucoside was mainly absorbed in jejunum ，quercetin was mainly absorbed in duodenum and ileum ；in the same intestine ，the absorption of constituents in the model group was less than normal group. After adding verapamil ，absorption of all constituents in the normal group increased ，but the difference was not statistically significant （P＞0.05）；absorption of kaempferol ，isokaempferol，vitexin，protocatechuic acid and kaempferol- 3-O-β-D-glucoside were all increased significantly in model group （P＜0.05），while there was no statistical significance in the increase of quercetin （P＞0.05）. After the bile flowed into the duodenum ，absorption of protocatechuic acid was increased significantly in normal group （P＜0.05）；absorption of other active constituents were increased significantly in model group，except for isokaempferol and quercetin （P＜0.05）. CONCLUSIONS ：Six active constituents of P. orientale were absorbed in the whole intestine of normal and MI model rats ，and the absorption of above constituents may be enhanced more significantly by P-gp inhibitor and bile under pathological condition.

[UPLC-Q-TOF-MS/MS analysis on prototypes components and metabolites of effective fractions of Polygonum orientale flower in rat serum and urine].

Ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS) method was applied to analyze the prototypes and metabolites of the effective components of Polygonum orientale in SD rat serum and urine. The separation was performed on Agilent Eclipse Plus C_(18) column( 2. 1 mm×100 mm,1. 8 µm),with 0. 1% formic acid solution( A)-acetonitrile( B) as the mobile phase for gradient elution. Mass spectrometry data of biological samples were obtained under positive and negative electrospray ion mode. By comparing chromatogram differences between blank samples and drug treatment samples,prototype components and metabolites of the effective components of P. orientale extract were identified. The results showed that 12 metabolites were detected in serum and 26 metabolites in urine( including cross-components) of rats. The main metabolic pathways included hydrogenation,hydroxylation,glucuronidation,sulfation reaction,and methylation-glucuronidation,etc. The method established in this study was reliable and effective for studying the metabolic characteristics of the effective components of P. orientale in rats,and it can provide a reference for further studies on therapeutic material basis of this herb.

Study on Protective Effects of Polygonum orientale Extract on Myocardial Ischemia-reperfusion Injury Model Rats / 中国药房

OBJECTIVE： To study the protective effects of Polygonum orientale extract on myocardial ischemia-reperfusion injury （MIRI） model rats， and to provide reference for it’s deeply development of medicinal source. METHODS： Totally 24 rats were randomly divided into sham operation group （normal saline）， model group （normal saline）， Compound danshen tablet group （positive group， 0.17 g/kg） and P. orientale extract group （86 g/kg， calculated by crude drug）， with 6 rats in each group. All groups were given drugs 2 mL/100 g intragastrically once a day. After 4 d of consecutive administration， MIRI model was induced by the left anterior descending branch of arteria coronaria in all groups except for sham operation group. 24 h after reperfusion， they were given related medicine again. After medication， the changes of electrocardiogram ST segment were monitored in each group. The plasma levels of LDH， CK-MB， CK， cTn-I， SOD and NO were detected in each group. The myocardial infarction rate in each group was calculated and the pathomorphological changes in the myocardium were observed. RESULTS： Compared with sham operation group， ST segment of myocardial electrocardiogram was increased in model group （P＜0.01）. The plasma levels of LDH， CK， CK-MB and cTn-I were increased significantly （P＜0.01）， while the plasma levels of SOD and NO were decreased significantly （P＜0.01）. The rate of myocardial infarction was increased significantly （P＜0.01）， and pathomorphological changes were observed in myocardial tissue such as infiltration of inflammatory cells and loose cytoplasm of cardiac myocytes. Compared with model group， ST segment of myocardial electrocardiogram was decreased significantly in Compound danshen tablet group and P. orientale extract group （P＜0.05）； the plasma levels of LDH， CK， CK-MB and cTn-I were decreased significantly （P＜0.05）， while the plasma levels of SOD and NO were increased significantly （P＜0.05）； the rate of myocardial infarction was decreased significantly （P＜0.05）， and inflammatory cell infiltration and tissue edema in myocardium were relieved to varying degrees. CONCLUSIONS： The protective effect of P. orientale extract protect on MIRI may be exerted by anti-oxidative damage.

UPLC-Q-TOF-MS/MS analysis on prototypes components and metabolites of effective fractions of Polygonum orientale flower in rat serum and urine / 中国中药杂志

Ultra performance liquid chromatography coupled with time-of-flight mass spectrometry( UPLC-Q-TOF-MS/MS) method was applied to analyze the prototypes and metabolites of the effective components of Polygonum orientale in SD rat serum and urine. The separation was performed on Agilent Eclipse Plus C_(18) column( 2. 1 mm×100 mm,1. 8 μm),with 0. 1% formic acid solution( A)-acetonitrile( B) as the mobile phase for gradient elution. Mass spectrometry data of biological samples were obtained under positive and negative electrospray ion mode. By comparing chromatogram differences between blank samples and drug treatment samples,prototype components and metabolites of the effective components of P. orientale extract were identified. The results showed that 12 metabolites were detected in serum and 26 metabolites in urine( including cross-components) of rats. The main metabolic pathways included hydrogenation,hydroxylation,glucuronidation,sulfation reaction,and methylation-glucuronidation,etc. The method established in this study was reliable and effective for studying the metabolic characteristics of the effective components of P. orientale in rats,and it can provide a reference for further studies on therapeutic material basis of this herb.

Effect of Intestinal Flora in Rats on Metabolism and Transformation of Effective Components in Polygonum orientale Inflorescences / 中国实验方剂学杂志

Objective:To observe the metabolic characteristics of effective components from Polygonum orientale inflorescences in intestinal flora of rats. Method:The incubating samples of effective components from P. orientale inflorescences in rat intestinal flora in vitro were detected by UPLC-ESI-Q-TOF-MS/MS, the mobile phase was consisted of 0.1%formic acid solution-0.1%formic acid acetonitrile solution and eluted in gradient mode at a flow rate of 0.3 mL·min-1.The mass spectral analysis was detected with electrospray ionization under positive ion mode and negative ion mode.The metabolites and possible biotransformation pathways of effective components form P. orientale inflorescences in rat intestinal flora in vitro was analyzed by Metabolite ToolsTM, mass defect filtration(MDF) and other metabolite analysis techniques and combined with the accurate relative molecular weight of the compounds, the fragment ion information and the literature data. Result:Eighteen metabolites were detected after incubation of effective components from P. orientale inflorescences in rat intestinal flora.The main biotransformation pathways were reduction, oxidation, hydrolysis in Ⅰ phase reaction and methylation in Ⅱ phase reaction. Conclusion:The effective components of P. orientale inflorescences can be transformed into a variety of metabolites under the action of intestinal flora in rats.It is suggested that whether the metabolites are bioactive components should be considered when P. orientale inflorescences is used as medicine.

Anti-rheumatoid arthritis effects in adjuvant-induced arthritis in rats and molecular docking studies of Polygonum orientale L. extracts.

BACKGROUND AND PURPOSE: Polygonum orientale L. (family: Polygonaceae), named Hongcao in China, has effects of dispelling wind and dampness, promoting blood circulation, and relieving pain. Our group has already studied and confirmed that POEa and POEe (ethyl acetate and ethyl ether extract of P. orientale, respectively) had anti-inflammatory and analgesic effects in early research, which was mainly relevant to the existence of flavonoids. According to the clinical application of P. orientale in traditional Chinese medicine, it has long been used for rheumatic arthralgia and rheumatoid arthritis. Therefore, our group further explored whether flavonoids of P. orientale have anti-rheumatoid arthritis effect and how does they play this role. METHODS: Dried small pieces of the stems and leaves of P. orientale were decocted with water and partitioned successively to obtain POEa and POEe, respectively. The anti-rheumatoid arthritis effect of P. orientale was studied by using a Freund's complete adjuvant (FCA)-induced arthritis (AIA) in a rat model. The levels of PGE2, TNF-α, and IL-1ß in serum of AIA rats were detected by enzyme linked immunosorbent assay (ELISA) to explore its mechanisms. In addition, we computationally studied the relationships between the 15 chemical components of POEa and POEe, and the currently focused 9 target proteins of rheumatoid arthritis by molecular docking. RESULTS: Pharmacological experiments showed that POEa and POEe significantly ameliorate symptoms of rheumatoid arthritis via reducing paw swelling volume, arthritis score, and thymus and spleen indices, as well as increasing body weight in AIA rats. Simultaneously, the concentrations of PGE2, TNF-α, and IL-1ß were significantly decreased by POEa and POEe. Histopathology revealed noticeable reduction in bone and cartilage, synovial hyperplasia, inflammatory cell infiltration, cartilage surface erosion, and joint degeneration by POEa and POEe treatment. In addition, the molecular docking studies showed that docking scores of 14 chemical compositions (including 12 flavonoids and 2 phenolic acids) of POEa and POEe with anti-rheumatoid arthritis protein targets were better than the complexed ligands of the anti-rheumatoid arthritis protein targets. Among them, six flavonoids in POEa and POEe had more docking protein targets (n ≥ 3). Five anti-rheumatoid arthritis targets including high-temperature requirement A1 protease (HtrA1), janus kinase 1 (JAK1), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (i-NOS), and prostaglandin E2 (PGE2) had better docking score compared with the complexed ligands. Moreover, most of the chemical components in POEa and POEe showed strong interaction with HtrA1. CONCLUSIONS: The flavonoids of P. orientale have anti-rheumatoid arthritis effect. In addition, the molecular docking results indicate that quercetin, catechol, orientin, and other six flavonoids may be closely related to HtrA1, JAK1, COX-2, i-NOS, and PGE2 protein target receptors. It suggests that these chemical compositions form strong protein-ligand complexes with these protein targets, especially HtrA1 to exert anti-rheumatoid arthritis. Further experimental studies show that mechanisms of anti-rheumatoid arthritis effects may also be relevant to inhibit the levels of PGE2, TNF-α, and IL-1ß in serum. Therefore, our group can further explore the possible active ingredients and mechanisms of the anti-rheumatoid arthritis effects of flavonoids, and focus on the inhibition of the expression of inflammatory factors and the TGF-ß1/Smad signaling pathway associated with HtrA1 protein target receptors, which can provide a direction and powerful reference for the action mechanism and drug research of anti-rheumatoid arthritis of flavonoids in P. orientale.

[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.