Effects of echinacoside on renal injury in uremia rats and its mechanism / 中国药房

OBJECTIVE To investigate the effects of echinacoside （ECH） on renal injury in uremia （URE） rats and its mechanism. METHODS URE model of the rat was established by 5/6 nephrectomy. Successfully modeled rats were grouped into uremia group （URE group）， ECH low-dose [10 mg/（kg·d）] group， ECH medium-dose [20 mg/（kg·d）] group， ECH high-dose [40 mg/（kg·d）] group， ECH high-dose+anisomycin [p38 mitogen-activated protein kinase （p38 MAPK） pathway activator] group [ECH-H+Ani group， 40 mg/（kg·d） ECH +2 mg/（kg·d） anisomycin]， with a sham operation group， 12 mice in each group. Each drug group was given corresponding ECH intragastrically， while ECH-H+Ani group was further injected with anisomycin via the tail vein， once a day， for 8 consecutive weeks. The serum levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， IL-6， blood urea nitrogen （BUN）， β2-microglobulin （β2-MG）， serum creatinine （Scr）， neutrophil gelatinase-associated lipocalin （NGAL）， kidney injury molecule-1 （KIM-1）， cystatin C （Cys-C） and 24 h urine protein （24 h UP） as well as the levels of malondialdehyde （MDA） and superoxide dismutase （SOD） activity in renal tissue were all detected； pathological changes of renal tissue were observed； the rate of positive expression of α-smooth muscle protein （α-SMA） and E-cadherin， and the phosphorylation of p38 MAPK and nuclear factor-κB （NF-κB） p65 were determined in renal tissue of rats. RESULTS Compared with URE group， glomerular swelling， damage and necrosis of renal tubular epithelial cells and inflammatory cell infiltration were relieved significantly in ECH groups. The renal injury score， levels of TNF-α， IL-1β， IL-6， BUN， Scr， β2-MG， 24 h UP， NGAL， KIM- 1， Cys-C and MDA， the positive expression rate of α-SMA in renal tissue， the phosphorylation of p38 MAPK and NF-κB p65 were decreased in dose-dependent manner， while SOD activity and the positive expression rate of E-cadherin were obviously increased in dose-dependent manner （P＜0.05）. Anisomycin significantly attenuated the improvement effect of high-dose ECH on renal injury in URE rats （P＜0.05）. CONCLUSIONS ECH may inhibit inflammation and oxidative stress， enhance renal function， and improve renal injury in uremic rats by inhibiting the activation of p38 MAPK/NF-κB signaling pathway.

Echinacoside hampers malignant progression of breast cancer MCF-7 cells by modulating AKR1B10/ERK signal transduction / 中国中药杂志

This study analyzes the impact of echinacoside(ECH) in the proliferation, metastasis and adriamycin(ADR) resistance of breast cancer(BC) MCF-7 cells via the modulation of aldo-keto reductase family 1 member 10(AKR1B10)/extracellular signal-regulated kinase(ERK) pathway. The chemical structure of ECH was firstly confirmed. MCF-7 cells were treated with different concentration(0, 10, 20, 40 μg·mL~(-1)) of ECH for 48 h. Western blot was used to analyze expression of AKR1B10/ERK pathway-associated proteins and cell counting kit-8(CCK-8) assay to determine cell viability. MCF-7 cells were collected and classified into control group, ECH group, ECH + Ov-NC group, and ECH + Ov-AKR1B10 group. Then Western blot was employed to analyze the expression of AKR1B10/ERK pathway-associated proteins. CCK-8 and 5-ethynyl-2'-deoxyuridine(EdU) assay were used to examine cell proliferation. Cell migration was appraised with scratch assay, Transwell assay, and Western blot. Eventually, MCF-7 cells were treated with ADR for 48 h to induce ADR resistance. Cell viability was tested by CCK-8 assay and cell apoptosis was estimated based on terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and Western blot. Based on Protein Data Bank(PDB) and molecular docking, the binding affinity of ECH to AKR1B10 was assessed. Various doses of ECH decreased the expression of AKR1B10/ERK pathway-associated proteins in a dose-dependent manner and declined cell viability compared with the control group. Compared with the control group, 40 μg·mL~(-1) ECH blocked the AKR1B10/ERK pathway in MCF-7 cells and inhibited the proliferation, metastasis and ADR resistance of the cells. Compared with the ECH + Ov-NC group, ECH + Ov-AKR1B10 group showed the recovery of some biological behaviors of MCF-7 cells. ECH also targeted AKR1B10. ECH can inhibit the proliferation, metastasis, and ADR resistance of BC cells by blocking AKR1B10/ERK pathway.

Echinacoside Alleviates Cognitive Impairment in Cerebral Ischemia Rats through α 7nAChR-Induced Autophagy / 中国结合医学杂志

OBJECTIVES@#To evaluate the effect of echinacoside (ECH) on cognitive dysfunction in post cerebral stroke model rats.@*METHODS@#The post stroke cognitive impairment rat model was created by occlusion of the transient middle cerebral artery (MCAO). The rats were randomly divided into 3 groups by a random number table: the sham group (sham operation), the MCAO group (received operation for focal cerebral ischemia), and the ECH group (received operation for focal cerebral ischemia and ECH 50 mg/kg per day), with 6 rats in each group. The infarct volume and spatial learning were evaluated by triphenyl tetrazolium chloride staining and Morris water maze. The expression of α7nAChR in the hippocampus was detected by immunohistochemistry. The contents of acetylcholine (ACh), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), activities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and catalase (CAT) were evaluated by enzyme linked immunosorbent assay. The neural apoptosis and autophagy were determined by TUNEL staining and LC3 staining, respectively.@*RESULTS@#ECH significantly lessened the brain infarct volume and ameliorated neurological deficit in infarct volume and water content (both P<0.01). Compared with MCAO rats, administration of ECH revealed shorter escape latency and long retention time at 7, 14 and 28 days (all P<0.01), increased the α7nAChR protein expression, ACh content, and ChAT activity, and decreased AChE activity in MCAO rats (all P<0.01). ECH significantly decreased MDA content and increased the GSH content, SOD, and CAT activities compared with MCAO rats (all P<0.05). ECH suppressed neuronal apoptosis by reducing TUNEL-positive cells and also enhanced autophagy in MCAO rats (all P<0.01).@*CONCLUSION@#ECH treatment helped improve cognitive impairment by attenuating neurological damage and enhancing autophagy in MCAO rats.

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

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

Echinacoside regulates SIRT1/STAT3 signaling pathway to improve liver injury and glucose metabolism disorder in CLP rats / 中国医师杂志

Objective:To observe the therapeutic effect of echinacoside (ECH) on liver injury and glucose metabolism disorder in sepsis rats induced by cecal ligation and puncture (CLP), and to explore its possible mechanism.Methods:Forty eight male Sprague Dawley (SD) rats were randomly divided into four groups: sham group (sham), model group (CLP), treatment group (CLP+ ECH) and inhibitor group (CLP+ ECH+ EX527). The sham group only received laparotomy, and the model group underwent CLP. The treatment group was intragastric administration of echinacea (30 mg/kg) every day after CLP modeling. The inhibitor group was injected with silence information regulator 1 (SIRT1) inhibitor EX527 (5 mg/kg) one hour before CLP, and then treated the same as the treatment group. Fasting blood glucose, insulin and serum biochemical indexes were detected in virous groups. The serum levels of interleukin (IL)-1 β, IL-6 and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). 2′, 7′- dichlorofluorescein diacetate (DCFH-DA) staining was used to observe the production of reactive oxygen species (ROS) in liver tissue of rats in each group; hematoxylin-eosin (HE) staining was used to observe the pathological changes of liver tissue in each group; The expressions of SIRT1, glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), phosphorylated signal transducers and activators of transcription 3 (p-STAT3) and phosphorylated protein ki-nase B(p-AKT) were detected by Western blot.Results:Compared with sham group, the levels of serum glucose, serum insulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), ROS, IL-1β, IL-6 and TNF-α in model group increased, while the liver glycogen and survival rate decreased (all P<0.05). After echinacoside treatment, the serum glucose, serum insulin, ALT, AST, ROS , IL-1β, IL-6 and TNF-α levels decreased, and the liver glycogen and survival rate increased (all P<0.05); After SIRT1 inhibitor intervention, the levels of serum insulin, ALT, AST, IL-6 and ROS in the inhibitor group increased ( P<0.05). HE staining showed that there were infiltration and necrosis of inflammatory cells in the liver tissue of model group, and echinacoside could significantly reduce the focal and massive necrosis; Western blot showed that compared with the sham group, the expression levels of SIRT1, p-STAT3 and p-AKT protein in the model group decreased, while the expression levels of G6Pase and PEPCK protein increased ( P<0.05); After echinacoside treatment, the expression levels of SIRT1, p-STAT3 and p-AKT increased, while the expression levels of G6Pase and PEPCK decreased ( P<0.05). After SIRT1 inhibitor intervention, the expression of SIRT1, p-STAT3 and p-AKT protein decreased, and the expression of G6Pase and PEPCK protein increased in the inhibitor group ( P<0.05). Conclusions:Echinacoside is a potential therapeutic agent for sepsis associated liver injury and glucose metabolism disorders, which may play a role by targeting SIRT1 to activate STAT3 and AKT in the liver.

Study on HPLC characteristic fingerprint of substance benchmark of classical famous prescription of Jichuan Decoction / 中草药

Objective: To establish an HPLC characteristic fingerprint of substance benchmark (standard decoction) of classical famous prescription of Jichuan Decoction (JD), and provide reference for the quality study of substance benchmark of JD. Methods: JD standard decoction was prepared according to the ancient method, 15 batches JD standard decoction were determined by HPLC. The similarity analysis and characteristic peak analysis of 15 batches JD were carried out by the "Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica 2012 version". Results: A total of 18 common characteristic peaks were screened by automatic matching method, peaks 1 and 3 were from Angelicae Sinensis Radix and Cimicifugae Rhizoma, peaks 2, 5, 6, 7, 9, 11 and 13 were from Cistanches Herba, peaks 4, 12, 14, 15 and 17 from were Cimicifugae Rhizoma, peaks 8, 10 and 18 from Aurantii Fructus, and peak 16 was from Angelicae Sinensis Radix. Seven characteristic components were identified by the reference substance, including caffeic acid (peak 1), echinacoside (peak 2), ferulic acid (peak 3), isoferulic acid (peak 4), mullein glycoside (peak 6), naringin (peak 8) and neohesperidin (peak 10). The similarities of 15 batches substance benchmark of JD were greater than 0.9. Conclusion: The HPLC method established for substance benchmark of JD is simple, accurate, stable and sensitive. It can be used for the quality study for JD substance benchmark, and provides a reference for the transformation and development of JD for modern preparations.

Simultaneous Determination of 4 Components in Huanshao Capsules by HPLC / 中国药房

OBJECTIVE：To estab lish a me thod for simultaneous determination of morroniside ，loganin，echinacoside and acteoside in Huanshao capsules. METHODS ：HPLC method was adopted. The determination was performed on Zhongpuhong RD-C18 column with mobile phase consisted of acetonitrile- 0.1％ formic acid solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelength was set at 240 nm （morroniside，loganin） and 330 nm （echinacoside，acteoside）. The column temperature was set at 35 ℃，and sample size was 10 μL. RESULTS：The linear range were 5.29-105.80 μg/mL for morroniside， 4.49-89.88 mg/L for loganin ，16.26-325.25 mg/L for echinacoside and 16.31-326.25 mg/L for acteoside ，r values were 0.999 9. RSDs of precision ，stability （24 h），reproducibility and durability tests were all lower than 2.0％ . The recoveries were 94.34％ -96.23％（RSD＝0.81％ ，n＝6），97.04％ -98.89％（RSD＝0.73％ ，n＝6），96.23％ -98.08％（RSD＝0.82％ ，n＝6）， 95.40％-98.47％（RSD＝1.23％，n＝6），respectively. The contents of above 4 components in 11 batches of Huanshao Capsules were 0.190-0.704，0.439-0.857，2.723-4.475 and 0.589-1.035 mg/g，respectively. CONCLUSIONS ：Established method is specific ， precise and can be used for content determination of 4 components in Huanshao capsules.

Effect of drought stress on accumulation of two respective phenylethanoid glycosides in tissue culture of Cistanche tubulosa / 中草药

Objective: To establish the plant tissue culture system of Cistanche tubulosa, and determine the effect of drought stress on accumulation of two respective phenylethanoid glycosides in it. Methods The major chemical constituents of C. tubulosa by tissue culture were analyzed by HPLC-UV and HR-MS. The cell growth curves were also determined. In addition, the effects of drought stress on the phenylethanoid glycosides (echinacoside and acteoside) content in the tissue culture system of C. tubulosa were also studied by using NaCl, mannitol and PEG6000 as osmotic regulators, respectively. Results:Chemical constituents analyses revealed that callus and suspension cultures of C. tubulosa could produce the respective phenylethanoid glycosides of echinacoside and acteoside as in wild plant; Cell growth curves indicated that 30 d were the optimum culture period of callus culture; The cell growth rate and the accumulation of echinacoside and acteoside were mostly inhibited when the callus cells were under drought stress induced by NaCl or mannitol. Meanwhile, the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa could be effectively enhanced by treatment with PEG6000. The maximum biomass of echinacoside and acteoside could reach to (1.07 ± 0.10) g/L and (0.12 ± 0.01) g/L 15 d after induction, respectively. And their contents were 20.94% and 2.27% separately based on the cell dry weight (DW) after 15 d of treatment with 6% PEG6000, which were 1.29 and 1.19 fold higher than the control group. Conclusion:Drought stress induced by PEG6000 could effectively enhance the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa.

Extraction parameters and drying methods of Cistanche deserticola / 中草药

Objective: The optimum extraction process parameters of Cistanche deserticola were selected to study the effects of different drying methods on five phenylethanoid glycosides. Methods: Single factor screening combined with Box-Behnken response surface method was used to optimize the extraction process. After optimal conditions were extracted, HPLC method was used to detect the content of echinacoside, cistanche A, verbascoside, isoacteoside, and 2’-acetylacteoside in different drying methods, and one-way ANOVA, cluster analysis, principal component analysis and close value analysis were used to analyze the content of five phenylethanoid glycosides to choose the best drying method. Results: Optimal extraction process was as following: methanol volume fraction was 55.14%, liquid to material ratio was 46.39, extraction time was 38.50 min. Cluster analysis, principal component analysis, and close value analysis showed that the quality of C. deserticola obtained by freeze-drying method was the best, followed by drying at 80 ℃ and the lowest at 40 ℃. Conclusion: Using this process to extract C. deserticola, the five phenylethanoid glycosides are completely and fully extracted. Although the freeze-drying method of C. deserticola has the highest active ingredient retention, from the production point of view, the 80 ℃ drying method can achieve a balance of cost and efficiency.

Advances in biosynthesis and chemical synthesis of phenylethanoid glycosides / 中草药

Phenylethanoid glycosides (PeGs) are natural active ingredients of many plants at home and abroad. They possess a spectrum of beneficial activities, such as anti-oxidant, hepatoprotective, whitening, and neuroprotective. However, the content of PeGs in food or medicinal materials is low and unstable. During the past decade, studies on biosynthesis and metabolic regulation of PeGs have been extensively carried out. Here, the recent achievements in biosynthesis and metabolic regulation of PeGs in plants and microorganisms are reviewed, as well as in total synthesis and semi-synthesis of PeGs. Hopefully, this work done so far will provide reference for elucidating the biosynthesis mechanism of PeGs, stabilizing and improving the content of PeGs in herbal materials, improving the quality, and synthesizing PeGs by microbial metabolic engineering or chemical synthesis.

Improving Effects of Echinacoside on Spatial Cognitive Function in Mice under Hypobaric Hypoxia Environment and Its Mechanism Study / 中国药房

OBJECTIVE： To study the improving effects of echinacoside （ECH） on spatial cognitive function in mice under hypobaric hypoxia environment and its mechanism. METHODS： Totally 60 mice were randomly divided into blank group （normal saline）， model group （normal saline）， positive group （Ginkgo leaf extract tablet，100 mg/kg） and ECH low-dose， medium-dose and high-dose groups （50， 75， 100 mg/kg）， with 10 mice in each group. Except for blank group， other groups were cultured in hypobaric oxygen chamber to simulate hypobaric hypoxia； they were given relevant medicine intragastrically once a day， for consecutive 7 d （Placing into hypobaric oxygen chamber immediately after medication）. Using the times of horizontal and vertical activities of mice in 2 min as index， negative emotions and spatial cognitive function were evaluated. Histopathological changes of hippocampus in mice were observed by microscopy after HE staining. The levels of SOD， CAT， GSH-Px and MDA in hippocampal tissue of mice were detected. RESULTS： Compared with blank group， the times of horizontal activities， MDA level were increased significantly in model group （P＜0.05）， while the times of vertical activities， the levels of SOD， CAT and GSH-Px were decreased significantly （P＜0.05）； the pyramidal cells in the CA1 area of the hippocampal tissue were arranged loosely， and many pyramidal cells were compressed and stained deeply. Compared with model group， the times of horizontal activities and MDA level were decreased significantly in positive group and ECH high-dose group （P＜0.05）， while the times of vertical activities， the levels of SOD， CAT and GSH-Px were increased significantly （P＜0.05）； the pyramidal cells in the CA1 region of the hippocampal tissue were abundant and closely arranged， and a few of them are constricted and deeply stained. CONCLUSIONS： ECH can improve spatial cognitive impairment of mice under hypobaric hypoxia environment， the mechanism of which may be associated with up-regulation of SOD， CAT and GSH-Px， down-regulation of MDA in the hippocampal tissue.

Method for active ingredients' in vivo target identification of traditional Chinese medicine using magnetic nanoparticles / 中国中药杂志

Target identification is an important prerequisite for the study of medicine action mechanism. Currently,drug target identification is mostly based on various cell models in vitro. However,the growth microenvironment,nutrition metabolism,biological properties as well as functions are quite different between in vitro cell culture and physiological environment in vivo; wherefore,it is a challenging scientific issue to establish an effective method for identifying drug targets in vivo condition. In this study,we successfully prepared a kind of magnetic nanoparticles( MNPs) which can be chemically modified by the hydroxyl structure of natural bioactive compound echinacoside( ECH) via the epoxy group label on the surface of MNPs. Therefore,organ-selective and recoverable nanoscale target-recognizing particles were prepared. We then intravenously injected the ECH-binding MNPs into rats and distributed them to specific organs in vivo. After cell endocytosis,ECH-binding MNPs captured target proteins in situ for further analysis. Based on this method,we discovered several potential target proteins in the spleen lysates for ECH,and preliminarily clarified the immuno-regulation mechanism of ECH. Collectively,our strategy developed a proof-of-concept technology using nanoparticles for in vivo target identification,and also provided a feasible approach for drug target prediction and pharmacological mechanism exploration.

Preliminary optimization of a Chinese herbal medicine formula based on the neuroprotective effects in a rat model of rotenone-induced Parkinson's disease / 中西医结合学报

<p><b>OBJECTIVE</b>The main objective of this study was to preliminarily determine the optimum formulation of a Chinese herbal formula that may have neuroprotective effects against rotenone-induced Parkinson's disease (PD).</p><p><b>METHODS</b>Seven recipes were made from Dihuang (DH, Rehmannia glutinosa Libosch), Roucongrong (RCR, Cistanche deserticola Y.C.Ma), Niuxi (NX, Achyranthes bidentata Bl.) and Shanzhuyu (SZY, Cornus officinalis Sieb. et Zucc) in different proportions, according to the principles of uniform design (4 factors 7 levels). Tyrosine hydroxylase (TH)-positive neurons in substantia nigra pars compacta (SNpc) were detected by immunohistochemistry and rotenone-exposure days necessary to induce PD symptoms were recorded. To probe one likely mechanism of the formulas, echinacoside (ECH) concentrations of all seven recipes were determined by high-performance liquid chromatography and related to number of TH-positive neurons.</p><p><b>RESULTS</b>The data showed that recipe 4 (DH:RCR:SZY:NX = 1:1:1:1) and recipe 7 (DH:RCR:SZY:NX = 7:5:3:1) partially reversed rotenone-induced death of TH-positive neurons in the SNpc and significantly increased rotenone-exposed days compared with model group. Pharmacologically, there was not a strong correlation between ECH concentration and TH-positive neurons.</p><p><b>CONCLUSION</b>The investigated formulations of Chinese herbs had neuroprotective effects against PD models, and the neuroprotective effects were weakly related to the proportion of key herbs. However the neuroprotective effects of the formula may not result from a single active constituent.</p>

Transformation pathways in methanol of echinacoside, a principal effective ingredient of Cistanches Herba / 中国中药杂志

Echinacoside (ECH) is one of the active ingredients in Cistanche Herba and the principal effective component of Memoregain© as well. Moreover, a new agent namely Naoqing Zhiming tablet, derived from ECH has been licensed for clinical trials. However, the knowledge regarding the stability of is limited, till now, initiating a significant barrier for its further development along with the clinical trials. Herein, we aim to in depth characterize the transformation pattern of ECH in methanol. When ECH was stored in methanol, two primary products (P1 and P2) could be observed in HPLC chromatogram. A home-made automated fraction collector was configured via employing two 2-phase/6-port electronic valves to prepare P1 and P2. Following ¹H-NMR and LC-MS/MS assays, P1 and P2 were unambiguously identified as acteoside and cistanoside A, respectively. Moreover, the existences of cis-ECH, cis-acteoside, and cis-cistanoside A were claimed after careful analysis of the ¹H-NMR spectra of ECH, P1 and P2. Above all, the primary transformation pathways of ECH in methanol included methylation as well as hydrolysis, and mild transformation could also be initiated by cis/trans- configuration transferring for the caffeoyl group. The findings obtained in current study are envisioned to provide useful insight for the further development of ECH and the impurity detection of Naoqing Zhiming tablet. Moreover, the automated fraction collector configured in current study is able to serve as a versatile tool for the collection of signals-of-interest within phytochemical evaluations and impurity isolation.

Study of anti-oxidants of Rehmanniae Radix and Rehmannia Radix Praeparata by HPLC-UV-DPPH method / 中草药

Objective To develop an HPLC-UV-DPPH method to compare anti-oxidants of Rehmanniae Radix and Rehmanniae Radix Praeparata sample from different manufactories and to provide an effective method for the processing and quality control of traditional Chinese medicine. Methods HPLC in HPLC-UV-DPPH system was performed on Aglient Extend C18 (250 mm × 4.6 mm, 5 μm) column with gradient elution of acetonitrile-0.1% acetic acid at the flow rate of 1.0 mL/min, and the detection wavelength was at 334 nm. The column temperature was 30 ℃. Flow rate of DPPH solution is 0.5 mL/min, and detection wavelength was set at 517 nm. The total activities of the samples and the contribution rate of each component to the total activity were evaluated by the “quantity-effect” research idea, and verbascoside was regarded as a positive reference. The main anti-oxidants in original and processed herbs were identified by HPLC-FT-MS and were compared. Results The detection of HPLC-UV-DPPH method showed that there were nine anti-oxidants in Rehmanniae Radix extract, while 13 anti-oxidants were found in Rehmanniae Radix Praeparata. There were eight common anti-oxidants in the two herbs. The anti-oxidants were obviously different before and after Rehmanniae Radix processed. The activities of the antioxidants in different samples were markedly different. Anti-oxidants with higher contributions were mussaenosidic acid, echinacoside, jionoside A1/A2, verbascoside, and isoverbascoside, respectively. Conclusion The HPLC-UV-DPPH method is stable, sensitive, reproducible, and suitable for rapid screening of anti-oxidants and quality evaluation of Rehmanniae Radix and Rehmannia Radix Praeparata.

Simultaneous determination of 10 components in Paidu Yangyan Capsules by HPLC wavelength change method / 中草药

Objective The contents of sennoside A, sennoside B, echinacoside, verbascoside, hesperidin, aloe-emodin, rhein, emodin, chrysophanol, and rheochrysidin in Paidu Yangyan Capsules (PYC) were determined by HPLC. The quality of different batches of psoralen were evaluated by chemical pattern recognition. Methods HPLC was established for the simultaneous determination of 10 components in 15 batches of PYC with variable wavelength detector, and the results were analyzed by principal component analysis and cluster analysis to comprehensively evaluate the difference in quality of PYC which is available in the market. Results The contents of sennoside A, sennoside B, echinacoside, verbascoside, hesperidin, aloe-emodin, rhein, emodin, chrysophanol, and rheochrysidin in PYC had good linear relationship in the ranges of 0.167 4—2.093 0, 0.093 5—1.177 0, 0.075 9—0.948 6, 0.049 9—0.623 7, 0.109 0—1.363 0, 0.033 5—0.418 4, 0.079 7—0.996 1, 0.013 9—0.174 3, 0.025 2—0.315 6, and 0.012 3—0.061 4 μg/mL, the average sample recovery rate range were 99.84%, 99.15%, 99.34%, 99.81%, 100.6%, 99.36%, 99.86%, 100.1%, 100.1%, and 99.76% (RSD < 2.0%), the detection limits were 0.64—2.18 ng/mL, quantitative limits were 3.19—9.55 ng/mL, the content of 10 active ingredients in 15 batches of samples respectively were 0.712 7—1.118 0, 0.403 9—0.522 0, 0.236 4—0.320 3, 0.671 1—1.183 0, 0.0580—0.1467, 0.108 7—0.2592, 0.0147—0.0501, 0.5173—0.5828, 0.2754—0.3051, and 0.1621—0.1853 mg/grain and there were some differences among the samples in different batches, mainly due to different rhubarb dosages. Conclusion The established method is simple, accurate and reproducible, and can be used to control the quality of PYC. It is suggested that the quality control of Rhei Radix et Rhizoma should be strengthened in the process of preparation production for providing a reference of PYC follow-up quality improvement.

Echinacoside and Cistanche tubulosa(Schenk)R.wight have male reproductiveprotection effect by targeting hypothalamic and rogen receptor / 中国药理学与毒理学杂志

OBJECTIVE Cistanche deserticola Y. C. Ma is an official plant that grows in arid or semi-arid areas.Our early work demonstrated that Cistanche extracts protect against sperm damage in mice under bisphenol A induced reproductive damage. Echinacoside (ECH)is one of the major active components. The present study investigated the mechanisms behind the possible protective effects of ECH against oligoasthenospermia in rat and identified the interaction between ECH and AR. METH-ODS The distribution of ECH was assayed by HPLC,the quantity and quality of sperm was evaluated and hormone levels were determined by radioimmunosorbent assay. The levels of androgen receptor (AR)and key steroidogenic-related genes were reduced as determined by Western blotting and qPCR analysis.The interaction between ECH and AR were evaluated by fluorescence localization assay,indi-rect ELISA and molecular docking. RESULTS ECH significantly increased the quantity of sperm and secretions of luteinizing hormone and testosterone.ECH was distributed to the hypothalamus but not in the testes.ECH combined with hypothalamic AR in the pocket of Met-894 and Val-713 to inhibit transfer of AR from the cytoplasm to nuclei in the hypothalamus.While negative feedback of sex hormone regula-tion was inhibited, positive feedback was stimulated to increase the secretion of luteinizing hormone and testosterone subsequently enhancing the quantity of sperm. C. militaris significantly alleviated the BPA-induced reproductive damage by increasing testicular superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and glutathione (GSH); as well as by reducing serum malondialdehyde (MDA). C. militaris not only obviously enhanced the levels of serum LH and T, but it also improved the sperm count and motility compared to the BPA-treated group.CONCLUSION C.militaris and ECH protect the BPA induced reproductive damage.ECH blocks AR activity in the hypothalamus to increase the quantity of sperm and protect against oligoasthenospermia in rats.

Echinacoside, an active constituent of Herba Cistanche, suppresses epileptiform activity in hippocampal CA3 pyramidal neurons

Echinacoside, an active compound in the herb Herba Cistanche, has been reported to inhibit glutamate release. In this study, we investigated the effects of echinacoside on spontaneous excitatory synaptic transmission changes induced by 4-aminopyridine (4-AP), by using the in vitro rat hippocampal slice technique and whole-cell patch clamp recordings from CA3 pyramidal neurons. Perfusion with echinacoside significantly suppressed the 4-AP-induced epileptiform activity in a concentration-dependent manner. Echinacoside reduced 4-AP-induced increase in frequency of spontaneous excitatory postsynaptic currents (sEPSCs) but it did not affect the amplitude of sEPSCs or glutamate-activated currents, implicating a presynaptic mechanism of action. Echinacoside also potently blocked sustained repetitive firing, which is a basic mechanism of antiepileptic drugs. These results suggest that echinacoside exerts an antiepileptic effect on hippocampal CA3 pyramidal neurons by simultaneously decreasing glutamate release and blocking abnormal firing synchronization. Accordingly, our study provides experimental evidence that echinacoside may represent an effective pharmacological agent for treating epilepsy.

Antinociceptive activity of Cistanche salsa stolons, growing in the Republic of Kazakhstan

Abstract Herba Cistanche (Cistanche species) in Traditional Chinese Medicine is used for the treatment of several diseases and symptoms, to include pain. The objective of this study was to evaluate the antinociceptive effect of the hydroethanol extract of Cistanche salsa (C.A.Mey.) Beck, Orobanchaceae, stolons in animal models of pain. Chemical composition of Herba Cistanche was analyzed by HPLC-UV. Mice Swiss Webster (25-30 g, n = 6) were orally pre-treated with Herba Cistanche (10, 30 or 100 mg/kg) and evaluated in the formalin test and in the capsaicin- or glutamate-induced licking response. Kazakh Herba Cistanche is composed mainly by phenylpropanoid glycosides, from which echinacoside, acteoside and tubuloside B are the main constituents. When Herba Cistanche was administered to mice it had an effect in both phases of the formalin test (77% activity at 30 mg/kg for phase 1 and 62% activity at 100 mg/kg for phase 2) suggesting analgesic and anti-inflammatory properties. Kazakh Herba Cistanche was able to reduce the animals licking time after injection of glutamate (81% reduction at 30 mg/kg) and capsaicin (81% reduction at 100 mg/kg). We conclude that phenolics present in the hydroethanol extract of C. salsa could be responsible for its pharmacological profile. In order to source a good quality raw material for Traditional Chinese Medicine we recommended this Kazakh species to be standardized using echinacoside and acteoside as markers.

Research Progress on the Pharmacological Effects of Echinacoside / 医药导报

Echinacoside has various pharmacological effects, such as antioxidative, antisenescence, neuroprotection, antiinflammation, promotion of cicatrization, hepatoprotection, promotion of bone formation, and antitumor activity. There are some progress in its pharmacokinetics study. Echinacea has therapeutic effect on diseases in various systems. It has great significance to further research and develop echinacoside.