Acteoside promotes autophagy and apoptosis of hepatoma cells by regulating JNK signaling pathway / 中国中药杂志

This study explored the molecular mechanism of acteoside against hepatoma 22(H22) tumor in mice through c-Jun N-terminal kinase(JNK) signaling pathway. H22 cells were subcutaneously inoculated in 50 male BALB/c mice, and then the model mice were classified into model group, low-dose, medium-dose, and high-dose acteoside groups, and cisplatin group. The administration lasted 2 weeks for each group(5 consecutive days/week). The general conditions of mice in each group, such as mental status, diet intake, water intake, activity, and fur were observed. The body weight, tumor volume, tumor weight, and tumor-inhibiting rate were compared before and after administration. Morphological changes of liver cancer tissues were observed based on hematoxylin and eosin(HE) staining, and the expression of phosphorylated(p)-JNK, JNK, B-cell lymphoma-2(Bcl-2), Beclin-1, and light chain 3(LC3) in each tissue was detected by immunohistochemistry and Western blot. qRT-PCR was performed to detect the mRNA expression of JNK, Bcl-2, Beclin-1, and LC3. The general conditions of mice in model and low-dose acteoside groups were poor, while the general conditions of mice in the remaining three groups were improved. The body weight of mice in medium-dose acteoside group, high-dose acteoside group, and cisplatin group was smaller than that in model group(P<0.01). The tumor volume in model group was insignificantly different from that in low-dose acteoside group, and the volume in cisplatin group showed no significant difference from that in high-dose acteoside group. Tumor volume and weight in medium-dose and high-dose acteoside groups and cisplatin group were lower than those in the model group(P<0.001). The tumor-inhibiting rates were 10.72%, 40.32%, 53.79%, and 56.44% in the low-dose, medium-dose, and high-dose acteoside groups and cisplatin group, respectively. HE staining showed gradual decrease in the count of hepatoma cells and increasing sign of cell necrosis in the acteoside and cisplatin groups, and the necrosis was particularly obvious in the high-dose acteoside group and cisplatin group. Immunohistochemical results suggested that the expression of Beclin-1, LC3, p-JNK, and JNK was up-regulated in acteoside and cisplatin groups(P<0.05). The results of immunohistochemistry, Western blot, and qRT-PCR indicated that the expression of Bcl-2 was down-regulated in the medium-dose and high-dose acteoside groups and cisplatin group(P<0.01). Western blot showed that the expression of Beclin-1, LC3, and p-JNK was up-regulated in acteoside and cisplatin groups(P<0.01), and there was no difference in the expression of JNK among groups. qRT-PCR results showed that the levels of Beclin-1 and LC3 mRNA were up-regulated in the acteoside and cisplatin groups(P<0.05), and the level of JNK mRNA was up-regulated in medium-dose and high-dose acteoside groups and cisplatin group(P<0.001). Acteoside promotes apoptosis and autophagy of H22 cells in mice hepatoma cells by up-regulating the JNK signaling pathway, thus inhibiting tumor growth.

Acteoside Inhibits Epithelial Mesenchymal Transformation of Hepatoma Cells Through Regulation of ERK1/2 Signaling Pathway / 肿瘤防治研究

Objective To investigate the effect and mechanism of acteoside (ACT) in inhibiting epithelial-mesenchymal transition (EMT) in human hepatoma HCCLM3 cells by regulating the ERK1/2 pathway. Methods CCK-8 assay was used to detect the effect of hepatocellular carcinoma cell proliferation. The invasion and migration of HCC cells were detected by scratch and Transwell tests. The mRNA and protein expression levels of the ERK1/2 signaling pathway and EMT-related genes (E-cadherin and N-cadherin) were detected by real-time PCR and Western blot analyses. Results ACT reduced the activity of HCCLM3 cells and inhibited the proliferation of HCC cells, and the effects had certain correlation with drug concentration and time. ACT inhibited the migration and invasion process of HCCLM3 cells in a concentration-dependent manner. ACT downregulated the mRNA and protein expression of genes related to the ERK1/2 signaling pathway. It increased the mRNA and protein expression levels of the EMT-related gene E-cadherin but decreased those of N-cadherin. Conclusion ACT could inhibit EMT and the invasion and migration of HCCLM3 cells in human hepatoma, and the underlying mechanism is closely related to the downregulation of the ERK1/2 signaling pathway.

In vitro inhibitory effects of acteoside on 6 kinds of CYP enzymes in liver microsomes of rats / 中国药房

OBJECTIVE To inv estigate the in vitro inhibitory effects of acteoside on cytochrome P 450（CYP）enzymes in liver microsomes of rats. METHODS Using probe substrates method ，acteoside（0.1，0.3，1，3，10，30 μmol/L）was incubated with probe substrates phenacetin ，mephentoin，diclofenac，coumarin，dextromethorphan and testosterone （substrates of CYP 1A2， CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）in liver microsomes of rats. Another blank control group and positive inhibitor group [ α-naphthoflavone，ticlopidine，sulfabendazole，pilocarpine，quinidine and ketoconazole （inhibitors of CYP 1A2，CYP2C19，CYP2C9，CYP2A6，CYP2D6 and CYP 3A4 enzymes，respectively）] were set up. Using indapamide as the internal standard ， the contents of corresponding metabolites （acetaminophen， 4-hydroxymephenytoin， 4-hydroxydiclofenac，7-hydroxycoumarin，dextran，6 β-hydroxytestosterone） were detected by ultra high performance liquid chromatography-tandem mass spectrometry . The IC 50 values were calculated by GraphPad v 8.0 software. By computer molecular docking technology ，acteoside and positive inhibitors were molecularly docked with the CYP enzyme ，and the binding mode and strength of the two molecules were analyzed. RESULTS The IC 50 values of acteoside to CYP 1A2 and CYP 2A6 enzymes were more than 30 μmol/L，and those of acteoside to CYP 2D6，CYP2C19，CYP2C9 and CYP 3A4 enzymes were 24.87，21.52，12.56 and 7.55 μmol/L，respectively. The hydrogen bond and hydrophobic force could form between acteoside and CYP 3A4 enzyme，and the hydrogen bond and electrostatic interaction could form between ketoconazole and CYP 3A4 enzyme. The binding free energy of acteoside and ketoconazole to CYP 3A4 enzyme were － 10.2 and － 12.4 kcal/mol （1 kcal/mol＝4.19 kJ），respectively. CONCLUSIONS Acteoside shows moderate inhibitory effect on CYP 3A4 enzyme in liver microsomes of rats ，and its affinity is equivalent to that of positive inhibitor ；the compound shows weak inhibitory effect on other 5 CYP enzymes.

Full-length transcriptome sequence and identification of genes involved in phenylethanol glycoside biosynthesis in Rehmannia glutinosa / 药学学报

italic>Rehmannia glutinosa belongs to the Scrophulariaceae family with important medicinal value. In order to effectively explore the transcriptome information of R. glutinosa and identify the genes encoding enzymes involved in phenylethanol glycoside (PhGs) biosynthesis, the leaves, stems and tuberous roots of R. glutinosa were used for transcriptome sequencing using Pacific Biosiences RS II platform. A total of 27 773 transcripts were generated with an average length of 2 380 bp, and 27 236 coding sequences (CDS) were predicted. Using BLAST software, non-redundant transcript sequences were annotated with NR, NT, GO, COG, KEGG, SwissProt and Interpro databases and a total of 27 399 annotated genes were obtained. Among them, the number of genes related to Sesamum indicum in the NR database was the highest (81.44%), which is consistent with their evolutionary relationship. Enzymes likely involved in the biosynthesis of isoacteoside, echinacoside, cistanosides A, cistanosides F, 2′-acetylacteoside and leonoside F were identified, and 143 genes were identified in R. glutinosa full-length transcriptome. The expression levels of 19 genes correlated with acteoside content in twelve tissues of R. glutinosa, and most showed higher expression levels in leaf tissues and floral organs. This study provides more reliable transcriptome data for screening R. glutinosa for functional genes and provides a foundation for the study of the molecular mechanisms of PhGs biosynthesis.

Effects of acteoside on hypoxia/reoxygenation-induced cardiomyocyte damage by regulating the Rnd 3/NF-κB pathway / 中国药房

OBJECTIVE To explore the effects of acteoside on hypoxia/reoxygena tion（H/R）-induced cardiomyocyte damage by regulating Rho family GTPase 3（Rnd3）/nuclear factor κB（NF-κB）pathway. METHODS The H 9c2 cardiomyocyte were divided into control group （no administration ，no modeling ），H/R group （only modeling ），H/R+AS-L group ，H/R+AS-M group ， H/R+AS-H group （10，30，90 μmol/L acteoside for above 3 groups firstly ，and then modeling ），H/R+pcDNA group [transfecting pcDNA （empty vector ） firstly，and then modeling] ，H/R + pcDNA-Rnd 3 group [overexpression of Rnd 3 by transfecting pcDNA-Rnd3（Rnd3 overexpression vector ）firstly，and then modeling] ，H/R+AS-H+si-NC group [transfecting si-NC （negative control）firstly，and then giving 90 μmol/L acteoside and modeling]，H/R+AS-H+si-Rnd3 group [inhibiting overexpression of Rnd 3 by transfecting si-Rnd 3 （Rnd3 small interfering RNA ） firstly，and then giving 90 μ mol/L acteoside and modeling]. After corresponding treatment ，the apoptotic rate ，release of lactate dehydrogenase （LDH），malondialdehyde（MDA）level，the activity of superoxide dismutase （SOD），the level of tumor necrosis factor α（TNF-α），interleukin 1β（IL-1β）and interleukin- 6（IL-6）， mRNA and protein expression of Rnd 3 and NF-κB subunit p65（NF-κB p65），the expression of aspartate proteolytic enzyme 3 （Cleaved Caspase- 3）protein and Cleaved Caspase- 9 protein were detected. RESULTS Different concentrations of acteoside could reduce the apoptotic rate of H/R-induced H 9c2 cardiomyocyte，the protein expressions of Cleaved Caspase- 3 and Cleaved Caspase-9，mRNA and protein expressions of NF-κB p65，the levels of LDH release and MDA ，TNF-α，IL-1β and IL-6，while increase the activity of SOD and mRNA and protein expressions of Rnd 3（P＜0.05），in a dose-dependent manner. Overexpression of Rnd 3 could decrease the apoptotic rate of H 9c2 cardiomyocyte，protein expressions of NF-κB p65，Cleaved Caspase- 3 and Cleaved Caspase- 9， the levels of LDH release ， MDA， TNF-α，IL-1β and IL-6，while increase the protein expression of Rnd 3 and the activity of SOD （P＜0.05）. The inhibition overexpression of Rnd 3 could weaken the inhibitory effects of acteoside on H/R-induced apoptosis of H 9c2 cardiomyocyte， oxidative stress and inflammatory reaction （P＜0.05）. CONCLUSIONS Acteoside could regulate Rnd 3/NF-κ B pathway by promoting the expression of Rnd 3 and inhibiting the expression of NF-κB p65，inhibit cardiomyocyte apoptosis ，oxidative stress and inflammation reaction so as to relieve the H/R-induced cardiomyocyte damage.

Effect of acteoside on delaying senescence and improving immune function in SAMP8 mice / 中国药理学通报

Aim To investigate the effect of acteoside on the changes of immune function and aging process in SAMP8 mice via detecting the proportion of immune cells and the changes of immune factors. Methods Sixty male healthy SAMP8 mice and twelve SAMR1 mice were randomly divided into six groups. They were administered by gavage once a day for 90 days. Aging scoring test was conducted regularly. The immune cells and immune factors in plasma and liver were detected. Results Compared with model group, acteoside could increase CD4+lymphocytes in SAMP8 mice and effectively inhibit the increase of CD8+lymphocytes; it played a greater role in spleen than in peripheral blood. At the same time, Thl/Th2 was significantly reduced in model group, and the ratio of Thl/Th2 factor increased in low and medium dose group. Conclusions Acteoside can increase the number of helper lymphocytes, effectively inhibit the increase of killer lymphocytes, and regulate the dynamic balance of Th1 and Th2 type immune inflammatory factors in SAMP8 mice, so as to improve the immune function of the body and delay the aging process of the body.

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.

Distribution and dynamic changes of chemical constituents in Callicarpa nudiflora / 中草药

Objective: To provide the theoretical basis for determining the best harvesting plant organ and harvesting period, and investigate the content of chemical constituents of Callicarpa nudiflora in different plant organs and different growth periods. Methods: The contents of total flavonoids, total phenolic acid and total saponins were determined by ultraviolet spectrophotometry, and the seven components were determined by HPLC. The ANOVA and PCA methods were used to analyze the content of each constituent. Results: The dry extract rate, the contents of total flavonoids, total phenolic acid, total saponins, forsythiaside B, acteoside, isoacteoside, and apigenin-7-O-β-D-glucopyranoside in functional leaves were the highest, while the contents of caffeic acid, galuteolin and luteolin in tender leaves were the highest, and all of them were significantly different from the young shoots (P 0.05). The contents of total phenolic acid, total saponins, forsythiaside B, and acteoside were the highest in the FB period, and there was no significant difference with the EFS period (P > 0.05). The contents of galuteolin and apigenin-7-O-β-D-glucopyranoside were the highest in the earlier fruit maturation (EFM) period and the later fruit maturation (LFM) period, respectively, and there was no significant difference with the EFS period (P > 0.05). The contents of each chemical component were reduced to the minimum at the fruit-drop (FD) period, and it was significantly different from that at the EFS period and the FB period (P < 0.05). According to the comprehensive evaluation model constructed by PCA, the comprehensive score of the EFS period was the highest (F = 3.252), followed by the FB period (F = 3.011). Conclusion: Main chemical constituents of C. nudiflora were significantly different in harvesting parts and growth periods. The contents of main chemical constituents were higher in functional leaves and tender leaves, and the contents of main chemical constituents were higher from FB period to EFS period.

Cloning, subcellular location and expression analysis of an acteoside synthase gene from Rehmannia glutinosa / 中草药

Objective: To clone the acteoside synthase gene (RgAcS1) from Rehmannia glutinosa, and analyze its subcellular localization and expression pattern. Methods: The cDNA sequence of RgAcS1 was identified based on the annotation of the transcriptome data of R. glutinosa, and the RgAcS1 gene was cloned by polymerase chain reaction (PCR). Constructing the GFP fusion expression vector and observing the subcellular localization of RgAcS1 mediated by Agrobacterium tumefaciens. The expression pattern of RgAcS1 in different parts of tuberous root of R. glutinosa was detected by real-time fluorescence quantitative PCR (qRT-PCR). Results: A full-length coding sequence of a shikimate-O-hydroxy cinnamoyl transferase from R. glutinosa was obtained and named RgAcS1. The length of the RgAcS1 cDNA was 1659 bp, including an open reading frame (ORF) of 1 296 bp, encoding 431 amino acid residues, the molecular weight of the protein was 475 900, and it has a typical domain of shikimic acid-O-hydroxy cinnamoyl transferase. The result of subcellular localization showed that RgAcS1 was mainly distributed in cytoplasm and also in nucleus. The qRT-PCR analysis showed that the expression levels of RgAcS1 were higher in the periderm and root hair of R. glutinosa tuberous root, but lower in the xylem and phloem. The expression levels of RgAcS1 were higher in non-radial striation than that in radial striation of BJ1, QH1 and 85-5. Conclusion: In this study, we obtained the cDNA sequence of RgAcS1, and analyzed the subcellular location and expression patterns of RgAcS1, which will lay foundations for further study on roles of RgAcS1 gene in the synthesis of acteoside in R. glutinosa.

Dissolution rule of Siwu Decoction by different decocting methods based on chemistry holography / 中草药

Objective: To study the content variation and chemical composition of Siwu Decoction between mixed decoction and single decoction comprehensively, and then explore variation rule of Siwu Decoction by different decocting methods based on material basis. Methods: Components of Siwu Decoction were identified by LC-MS/MS and an UPLC wavelength switching method for simultaneously determining the contents of multiple compounds in Siwu Decoction was established based on the idea of TCM chemistry holography. The mixed and single decoction samples were prepared and tested. Experimental data was compared to analyze material basis differences and variation rule of Siwu Decoction by different decocting methods. Results: A total of 72 compounds were identified and assigned, 18 compounds were quantitative detected and all of 18 analytes showed good linearity (R2 ≥ 0.999) within the test range. The relative standard deviations of the precision, repeatability and stability were not exceeding 2.0%, and the recoveries were in the range of 97%-105%. Analysis of Siwu Decoction samples showed dissolution of ligustilide, 3-n- butylphthalide, catechin, gallic acid and paeoniflorin was affected by the change of solvent volume and dissolution of aucubin, catechin, oxypaeoniflorin, paeoniflorin and acteoside were higher in mixed decoction than single decoction obviously. Compared to single decoction, the kinds of compounds in mixed decoction did not change significantly but the content showed notable variety. Conclusion: Through the study of chemistry holography, the composition and content of compounds in TCM mixed decoction and herbs single decoction can be compared and analyzed comprehensively to provide a new perspective for the study on the rule of TCM decoction and dissolution. TCM chemistry holography study may become a useful exploration of the TCM quality study.

Establishment of HPLC fingerprints of Qiwei Tangmaishu tablet and simultaneous determination of six constituents / 国际药学研究杂志

Objective: To establish HPLC fingerprints of Qiwei Tangmaishu tablet and simultaneously determine the contents of the six constituents, acteoside, martynoside, calycosin 7-O-β- D-glucopyranoside, schisandrin, formononetin and deoxyschizan- drin. Methods: The analysis of 50% methanol extract of this drug was performed on a 30℃ thermostatic Waters Symmetry C18 column, with the mobile phase comprising of acetonitrile(A)-0.2% formic acid solution(B)flowing at 1.0 ml/min in a gradient elution manner. The detection wavelength was set at 330 nm for acteoside and martynoside, 254 nm for calycosin 7-O-β-D-glucopyranoside, schisandrin, formononetin and deoxyschizandrin. Results: There were thirteen common peaks in the fingerprints of ten batches of samples with the similarities more than 0.9. Acteoside, martynoside, calycosin 7-O-β-D-glucopyranoside, schisandrin, formononetin and deoxyschizandrin showed good linear relationship within the ranges of 1.47-36.75, 0.66-16.50, 0.89-22.25, 2.58-64.50, 1.91-47.75 and 0.77-19.25 μg/ml(r≥0.9993), and their average recoveries were 97.57%, 99.22%, 96.69%, 100.01%, 98.79% and 96.77%, with the RSD of 0.79%, 1.54%, 0.61%, 0.64%, 0.83% and 0.36%, respectively. Conclusion: The established method is easily operable and repeatable, which could be used for quality control of Qiwei Tangmaishu tablet.

Effects of acteoside on learning, memory and neurotransmitters level in brain tissue of SAMP8 mice / 中华行为医学与脑科学杂志

Objective@#To evaluate the effect of acteoside on learning, memory and neurotransmitter in SAMP8 mice.@*Methods@#The 6-month-old rapidly aging SAMP8 mice were randomly divided into model group, namenda group, low-dose acteoside group(30 mg·kg-1 ·d-1), medium-dose acteoside group(60 mg·kg-1 ·d-1) and high-dose acteoside group(120 mg·kg-1 ·d-1) according to the digital table method, with 12 in each group.And 12 SAMR mice with the same age resistance were used as the control group.After 75 days of continuous intragastric administration, Morris water maze method and spontaneous activity experiment were used to investigate the effects of acteoside on learning, memory and anxiety of mice.The levels of neurotransmitters acetylcholine(ACh), serotonin(5-HT), norepinephrine(NE) and dopamine(DA) in mouse brain tissue(cortex and hippocampus) were detected by ELISA.@*Results@#(1)In the Morris water maze test, compared with the model group, the acteoside significantly reduced the escape latency of SAMP8 mice in training period.(2)In the experiment of autonomic activity, compared with the model group, the average speed and total distance of the low-dose acteoside group were significantly increased(t=15.0, 20.8, both P<0.05); the number of vertical movements and the average speed of the medium-dose acteoside group were significantly increased(t=15.8, 13.6, both P<0.05). The average speed and total distance of the high-dose acteoside group were remarkarbly increased(t=30.9, 29.7, both P<0.05), and the number of defecations in the mice of the lav-dose acteoside group, medium-dose acteoside group, high-dose acteoside group were((2.83±1.19)particles, (3.25±1.29)particles, (2.58±1.16)particles), they were obviously lower than that of the model group ((5.25±1.48) particles)(t=15.7, 20.1, 13.5, all P<0.01). (3)Simultaneously, the contents of ACh, NE and DA in the hippocampus and cortex of the model group were significantly lower than those in the normal group (hippocampus: t=10.3, 12.7, 13.2, all P<0.05; cortex: t=11.7, 10.5, 12.4, all P<0.05). Compared with the model group, the contents of ACh, NE, DA and 5-HT in the hippocampus and cortex of the low, medium and high doses of acteoside were significantly increased(hippocampus: t=31.4, 20.3, 10.7, 12.9, all P<0.05; cortex: t=33.7, 29.4, 14.5, 12.7, all P<0.05).@*Conclusion@#The acteoside can enhance the ability of spatial learning and memory of SAMP8 mice, and can regulate the depression and anxiety of animals.The mechanism may be related to the ACh content and the monoamine neurotransmitters increase in the brain.

Effects of acteoside on learning,memory and neurotransmitters level in brain tissue of SAMP8 mice / 中华行为医学与脑科学杂志

Objective To evaluate the effect of acteoside on learning,memory and neurotransmitter in SAMP8 mice. Methods The 6-month-old rapidly aging SAMP8 mice were randomly divided into model group,namenda group,low-dose acteoside group(30 mg·kg-1 ·d-1),medium-dose acteoside group(60 mg ·kg-1 ·d-1) and high-dose acteoside group(120 mg·kg-1 ·d-1 ) according to the digital table method, with 12 in each group. And 12 SAMR mice with the same age resistance were used as the control group. After 75 days of continuous intragastric administration,Morris water maze method and spontaneous activity experi-ment were used to investigate the effects of acteoside on learning,memory and anxiety of mice. The levels of neurotransmitters acetylcholine ( ACh), serotonin ( 5-HT ), norepinephrine ( NE ) and dopamine ( DA ) in mouse brain tissue(cortex and hippocampus) were detected by ELISA. Results (1) In the Morris water maze test,compared with the model group,the acteoside significantly reduced the escape latency of SAMP8 mice in training period. (2)In the experiment of autonomic activity,compared with the model group,the aver-age speed and total distance of the low-dose acteoside group were significantly increased(t=15. 0,20. 8,both P<0. 05);the number of vertical movements and the average speed of the medium-dose acteoside group were significantly increased(t=15. 8,13. 6,both P<0. 05). The average speed and total distance of the high-dose acteoside group were remarkarbly increased(t=30. 9,29. 7,both P<0. 05),and the number of defecations in the mice of the lav-dose acteoside group, medium-dose acteoside group, high-dose acteoside group were ((2. 83±1. 19)particles,(3. 25± 1. 29) particles,(2. 58± 1. 16) particles),they were obviously lower than that of the model group ((5. 25±1. 48) particles)(t=15. 7,20. 1,13. 5,all P<0. 01). (3) Simultaneously, the contents of ACh,NE and DA in the hippocampus and cortex of the model group were significantly lower than those in the normal group (hippocampus:t=10. 3,12. 7,13. 2,all P<0. 05;cortex:t=11. 7,10. 5,12. 4, all P<0. 05). Compared with the model group,the contents of ACh,NE,DA and 5-HT in the hippocampus and cortex of the low,medium and high doses of acteoside were significantly increased ( hippocampus: t=31. 4, 20. 3,10. 7,12. 9,all P<0. 05;cortex:t=33. 7,29. 4,14. 5,12. 7,all P<0. 05). Conclusion The ac-teoside can enhance the ability of spatial learning and memory of SAMP8 mice,and can regulate the depres-sion and anxiety of animals. The mechanism may be related to the ACh content and the monoamine neuro-transmitters increase in the brain.

Effects of different drying methods on multiple chemical components in Leonurus japonicus based on UPLC-QTRAP®/MS2 / 中草药

Objective To evaluate the effects of 15 kinds of drying methods including sun-drying, shade drying, infrared drying (50, 60, 70, 80 ℃), microwave drying (50, 60, 70, 80, 100 ℃), and hot-air drying (50, 60, 70, 80 ℃) on the quality of Leonurus japonicus through the content of multiple chemical components, and then optimize suitable drying methods for L. japonicus. Methods UPLC-QTRAP®/MS2 method was developed to determine the content of three alkaloids (stachydrine hydrochloride, leonurine hydrochloride, trigonelline), four phenolic acids (benzoic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid), five phenylpropanoids (salidroside, acteoside, chlorogenic acid, caffeic acid, ferulic acid), 11 flavonoids (rutin, isoquercitrin, hyperoside, wogonin, kaempferol-3-O-rutinoside, genkwanin, apigenin, kaempferol, isorhamnetin, hesperetin, quercetin), and one iridoid glycoside (ajugol) in L. japonicus. The principal component analysis (PCA) and TOPSIS analysis were performed to evaluate the quality of the L. japonicus samples obtained by different drying methods. Results Different drying methods exerted significant effects on the content of 24 chemical ingredients in L. japonicus. The PCA analysis divided 15 drying methods into three types based on the content of 24 compounds. Moreover, the comprehensive evaluation of TOPSIS was carried out, and the top three drying methods were 70 ℃ hot-air drying, 60 ℃ hot-air drying, and 100 ℃ microwave drying, which largely retained the active ingredients of L. japonicus. Conclusion Combined with practice, we found that 70 ℃ hot-air drying was the optimized drying process of L. japonicus, which provides guarantee for the quality of L. japonicus and provides scientific basis for the production and processing of L. japonicus.

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.

Optimization of steaming technology for decoction pieces of Scrophularia ningpoensis by D-optimal response surface methodology based on AHP- entropy weight method / 中草药

Objective :To determine the weight coefficient and optimize the steaming technology of decoction pieces of Scrophularia ningpoensis (SN). Methods The contents of harpagide, harpagoside, aucubin, acteoside, angoroside C, and cinnamic acid were simultaneously determined by HPLC. The weight coefficient of each component was evaluated by AHP-entropy (analyitc hierarchy process) method. With composite score as index, D-optimal response surface methodology was adopted to investigate the effects of soaking time, steaming time, and drying temperature on the quality of processed products and optimize the processing technology of decoction pieces of SN. Results :Optimal processing parameters were as follows: soaking time was 15.63 min, steaming time was 85 min, drying temperature was 60 ℃, and the synthetical mark was 97.20. Considering the actual situation, the optimum processing technology of SN was obtained by fine-turning the soaking time. The soaking time was 15 min, the steaming time was 85 min, and the drying temperature was 60 ℃. Also, the synthetical mark were 98.53, 99.39, 98.86, and its RSD was 0.47% through the obtained conditions in parallel with three batches of samples. Conclusion: The optimized steaming technology is simple and feasible, which can provide a reference for the steaming of decoction pieces of SN. The method established to simultaneously determine the contents of six components in decoction pieces of SN is rapid and reliable for controlling the quality of decoction pieces of SN.

Preparation and process quality control of substance benchmarks of classical famous prescription of Baihe Dihuang Decoction / 中草药

Objective: To prepare substance benchmarks of Baihe Dihuang Decoction (BDD), and evaluate the scientificity and rationality of preparation process by analyzing the process quality. Methods: Fifteen batches of substance benchmarks were prepared according to the ancient method, the content of catalpol and acteoside in the preparation process was determined, and the transfer rate and extractum rate were calculated. Fingerprints of 15 batches of decoction pieces, decoction, concentrate and substance benchmarks were detected by HPLC, and the common peaks of fingerprints were attributed and identified; In addition, the similarity of fingerprints were evaluated. Results: In 15 batches of substance benchmarks, the transfer rates of catalpol and acteoside were 81.40%—92.88% and 28.90%—41.41% respectively, the extractum rate was 36.06%—41.71%, and without discrete data. During the process of decoction, concentration and freeze-drying, the transfer rates of effective components were stable. In the fingerprints of substance benchmarks, 16 common peaks were determined, of which six peaks belong to Lilii Bulbus, nine peaks belong to Rehmanniae Radix. Three peaks were identified. The similarity of fingerprints of decoction pieces, decoction, concentrate, and substance benchmarks were all over 0.9. The similarity of reference fingerprints of decoction, concentrate, and substance benchmarks were over 0.99. Conclusion: The fingerprint method is reasonable and feasible, which can be used for simultaneously determining the fingerprint of decoction pieces, intermediates and substance benchmarks. The preparation process is scientific and reasonable, and will not changes substance basis significantly. The paper establishes a foundation for the development of preparation of BDD, and provides a new idea for the evaluation of process and quality of the substance benchmarks of classical famous prescriptions.

Multi-index optimization of water extraction process of Siwu Decoction by BP neural network combined with entropy weight method / 中草药

Objective: To optimize the water extraction process of Siwu Decoction by BP neural network combined with orthogonal experiment. Methods: The water amount, the extraction time, and the extraction times were taken as factors. Entropy weight method was used to calculate the comprehensive scores of the multi-indicators of eight active components of 5-hydroxymethylfurfural, chlorogenic acid, caffeic acid, paeoniflorin, ferulic acid, verbascoside, senkyunolide A, and ligustilide in R language environment. Using comprehensive score as an evaluation indicator, the BP neural network model was established by orthogonal experiment design, and the optimal water extraction process of Siwu Decoction was predicted through network training. Results: The optimized extraction process of Siwu Decoction was carried out by adding 8 times of water and extracting 3 times for 1 h. The relative error between the network predicted value and the actual measured value of the test sample was less than 1%. Conclusion: The established mathematical model can analyze and predict the water extraction process of Siwu Decoction. The obtained process is stable and feasible, and can effectively extract the active ingredients in Siwu Decoction.

Simultaneous Determination of the Contents of 8 Components in Pingzang Tiaoshen Granule by HPLC / 中国药房

OBJECTIVE:To establish a method for simultaneous determination of berberine hydrochloride,icariin,acteoside,isoflavone glucoside,paeoniflorin,mangiferin,salvianolic acid B and puerarin in Pingzang tiaoshen granule.METHODS:HPLC method was adopted.The determination was performed on InertSustain C18 column with mobile phase consisted of acetonitrile-0.05mol/L potassium dihydrogen phosphate solution (pH to 3,gradient elution) with the flow rate of 1.0 mL/min.The detection wavelength was set at 250 nm (0-23 min,puerarin,mangiferin),230 nm (＞23-30 min,paeoniflorin),220 nm (＞30-50 min,isoflavone glucoside,acteoside),286 nm (＞50-60 min,salvianolic acid B),265 nm (＞60-75 min,berberine hydrochloride),220 nm (＞75-90 min,icariin).The column temperature was set at 30 ℃,and sample size was 10 μL.RESLUTS:The linear range of berberine hydrochloride,icariin,acteoside,isoflavone glucoside,paeoniflorin,mangiferin,salvianolic acid B and puerarin were 4.000-400.0,4.843-484.3,0.498-49.8,2.366-236.6,23.26-2 326.0,3.067-306.7,3.629-362.9 μg/mL,48.23-4 823.2 μg/mL(r≥0.999 4),respectively.The limits of detection were 0.02,0.02,0.02,0.02,0.01,0.02,0.01,0.01 μg/mL;the limits of quantitation were 0.07,0.05,0.06,0.05,0.03,0.07,0.02,0.03 μg/mL,respectively.RSDs of precision,stability (24 h)and repetition tests were a11＜2.0％ (n=6).The average recoveries were 95.77％-103.50％ (RSD=0.77％-2.22％,n=6).CONCLUSIONS:Established method can be used for simultaneous determination of 8 components such as berberine hydrochloride in Pingzang tiaoshen granule.

Effect of acteoside on dysfunction of learning and memory and oxidative stress in rats with vascular dementia / 中华行为医学与脑科学杂志

Objective To explore the effects of acteoside on dysfunction of learning and memory and the protective effect of oxidative stress in rats with vascular dementia.Methods 30 SD rats were randomly divided into sham group,model group,nicergoline group,low-dose acteoside group and high-dose acteoside group,with 6 rats in each group.Preparation of vascular dementia model by 2-vessed occlusion.The ability of exploring and learning and memory in rats were detected by step down test and avoid dark test.Determination of malondialdehyde (MDA),reactive oxygen species (ROS) and glutathione peroxidase (GSH-PX) activity in serum and brain tissue was conducted by Elisa.Results Autonomic activity test showed that the frequency and activity of autonomous activity in the model group were significantly lower than those in the sham group(P＜0.01),the frequency of autonomous activity in each administration group was significantly higher than that in the model group (P＜0.01),and the central activity time in the high-dose group was significantly higher than that in the model group (P＜0.01).Step down test and avoid dark test showed that the latency of the model group was significantly lower than that of the sham group.(Model group of step down test:(25.33 ± 3.01) s,Sham group of step down test:(56.83 ± 15.90)) (P＜ 0.01).(Model group of avoid dark test:(15.67 ± 3.61) s,Sham group of avoid dark test:(135.82±44.00) s) (P＜0.01).The latency of low dose group was significantly higher than that of model group.(Low dose group of step down test:(46.40±14.32) s) (P＜0.01).(Low dose group of avoid dark test (44.20± 8.26)s) (P＜0.05).Step down test and avoid dark test showed that the number of mistakes in the model group was significantly higher than that in the sham operation group(P＜0.01).The number of errors in nicergoline group and the low dose group was significantly lower than that in the model group (P＜0.01,P＜0.05).In serum,the content of MDA and ROS in model group was significantly higher than that in sham group (P＜0.01) while the activity of GSH-PX in model group was significantly lower than that of sham group.The content of MDA in the other groups was significantly lower than that in the model group (P＜0.01).The content of ROS in the nicergoline group and low dose group was significantly lower than that in the model group (P＜0.05,P＜0.0l).The activity of GSH-PX in high dose group was significantly higher than that in model group (P＜0.01).In brain tissue,the content of MDA and Ros in model group was significantly higher than that in sham group (P＜0.01).The content of MDA and ROS in low dose group and high dose group were significantly lower than that in model group (P＜0.05,P＜0.01).Conclusion Acteoside can improve the dysfunction of learning and memory and depressive mood disorder caused by vascular dementia and reduce oxidative stress injury by decreasing the content of MDA-ROS and increasing the activity of GSH-PX enzyme.