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OBJECTIVE@#To investigate the effects of stachydrine (STA) on apoptosis of Aβ-induced PC12 cells mimicking Alzheimer's disease and explore the mechanisms.@*METHODS@#The differential genes of STA were analyzed based on GSE85871 data, and the target genes of STA were identified using STITCH database. PC12 cells were treated with Aβ to establish a cell model of Alzheimer's disease, and the changes in cell viability and cell cycle in response to STA treatment were assessed using MTT assay and flow cytometry, respectively. RT-PCR and Western blotting were used to detect the relevant gene or protein expressions in the treated cells.@*RESULTS@#GSE85871 data showed 37 up-regulated genes and 48 down-regulated genes in cells following treatment with STA. Analysis of the data from the STITCH database indicated that RPS8 and EED were the target genes of STA. Treatment of PC12 cells with Aβ significantly lowered the cell viability ( < 0.05) and the expressions of RPS8 and EED at both the mRNA and protein levels ( < 0.05), and obviously inhibited the expression of apoptosis-related proteins Bcl-2 and p53 ( < 0.05). STA treatment of the cells significantly reversed the effect of Aβ and induced cell cycle arrest in G2/M phase, causing also significantly increases in the expression levels of RPS8, EED, Bcl-2 and p53 ( < 0.05).@*CONCLUSIONS@#STA plays an important role in inhibiting the apoptosis of PC12 cells induced by Aβ possibly by regulating RPS8 and EED expression to promote the expressions of Bcl-2 and p53.
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Animales , Ratas , Enfermedad de Alzheimer , Péptidos beta-Amiloides , Apoptosis , Supervivencia Celular , Células PC12 , Fragmentos de PéptidosRESUMEN
Based on the charge repulsion and solution-diffusion effect in nanofiltration separation, the correlation among mass transfer behavior, solution environment and molecular structure of three typical alkaloids from medicine was analyzed by nanofiltration mass mathematical model. The experiment revealed a linear relationship between ln[(1-Ro)·Jv/Ro] and Jv, and the regression coefficients were all greater than 0.9. Compared with the ultrafiltration separation behavior conforming to molecular sieve, the mass transfer coefficient of three alkaloids under different pH was pH 3.00 < pH 7.00 < pH 10.00. As the pH changed, the state of alkaloid transit from ionic state to a free state, the alkaloid could easily approach the membrane surface and pass through the nanofiltration membrane with charge repulsion and solution-diffusion effects, and the results were verified by the membrane adsorption tendency. The nanofiltration mass transfer of alkaloids is related to the state and molecular weight. In the ionic state, the charge effect produces separation behavior, and the molecular state is related to the molecular weight. The separation mechanism of nanofiltration for alkaloids was clarified further by analyzing the correlation of nanofiltration mass transfer behavior and molecular structure. The results of nanofiltration technology provide references for separation of alkaloids at room temperature with fast separation and low energy consumption.
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OBJECTIVE: To investigate the effect of stachydrine on tumor growth of colon cancer and its mechanisms. METHODS: MTS was used to test the effect of stachydrine on proliferation of HCT116 colon cancer cell. HCT116 colon cancer xenograft model was used to detect the effect of stachydrine on tumor growth in vivo. Immunohistochemical was used to determine the effect of stachydrine on expression of smooth muscle actin 2 (ACTG2) in subcutaneous transplantation tumor. The transfection of ACTG2 siRNA into HCT116 cells was conducted to study the effect of ACTG2 intervene on tumor angiogenesis by tube formation experiment in colon cancer. The protein expression of VEGF, bFGF, TNF-α and PDGF after ACTG2 intervene were examined by Western blotting to explore the potential mechanisms of stachydrine on regulation of tumor angiogenesis. RESULTS: Compared with the control group, the proliferation and growth of HCT116 colon cancer cells were significant inhibited by stachydrine in vitro and in vivo. Immunohistochemical showed that the expression of ACTG2 was significantly decreased by treatment with stachydrine. ACTG2 siRNA transfection could significantly decreased the inhibition effect of stachydrine on tumor angiogenesis. Mechanistic study found that stachydrine could inhibit the expression of VEGF, bFGF, TNF-α and PDGF, however, ACTG2 siRNA transfection could significantly reverse the expression of above mentioned angiogenesis-related factors. CONCLUSION: Stachydrine could inhibit the growth of colon cancer by suppressing tumor angiogenesis, and such effect is dependent on ACTG2 expression.
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To build an evaluation standard for quality of Leonuri Herba standard decoction. 13 batches of Leonuri Herba standard decoction with different quality were prepared. The contents of leonurine hydrochloride and stachydrine hydrochloride were determined; then the transfer rate and the extract rate were calculated and pH value was measured; and HPLC fingerprint method was established for analysis. The results of 13 batches of samples revealed that the transfer rate of leonurine hydrochloride and stachydrine hydrochloride was 30.0%-53.4% and 67.0%-82.6%, respectively; the extract rate was 12.1%-18.3% and the pH value was 5.87-6.22. Moreover, 12 common chromatographic peaks were determined based on fingerprint by using Similarity Evaluation System for Chromatographic fingerprint of Traditional Chinese Medicine (2012A). The similarity of 13 batches of samples was analyzed and compared, and the results showed that the similarity was higher than 0.9. In this study, the preparation method for Leonuri Herba decoction was standard, with high similarity in fingerprint, showing high precision, stability and repeatability in fingerprint analysis. Thus, this study can provide a reference for the quality control of Leonuri Herba dispensing granules.
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AIM:To observe the therapeutic effect of stachydrine hydrochloride on experimental acute cerebral infarction in rats and to explore the underlying mechanisms .METHODS:SD rats ( n=75) were randomly divided into 5 groups:sham group, cerebral infarction model group , and stachydrine hydrochloride (10 mg/kg, 20 mg/kg and 40 mg/kg) treatment groups .After the establishment of cerebral infarction model , the rats were given stachydrine hydrochloride at dose of 10 mg/kg, 20 mg/kg or 40 mg/kg by gavage daily for 14 d.The impairment of neurological function in each group was scored .The cerebral infarction volume and brain water content were measured .Moreover , the protein levels of β-cate-nin, cyclin D1, glycogen synthase kinase 3β(GSK-3β) and p-GSK-3βin the brain tissues were detected by Western blot . RESULTS:Compared with cerebral infarction group , the score of neurological function impairment , cerebral infarction volume and brain water content were significantly decreased in stachydrine hydrochloride treatment groups .In addition , the protein levels of β-catenin, cyclin D1 and p-GSK-3βwere markedly increased after stachydrine hydrochloride treatment . CONCLUSION:Stachydrine hydrochloride protects against experimental acute cerebral infarction through activation of Wnt/β-catenin signaling pathway .
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AIM:To observe the therapeutic effect of stachydrine hydrochloride on experimental acute cerebral infarction in rats and to explore the underlying mechanisms .METHODS:SD rats ( n=75) were randomly divided into 5 groups:sham group, cerebral infarction model group , and stachydrine hydrochloride (10 mg/kg, 20 mg/kg and 40 mg/kg) treatment groups .After the establishment of cerebral infarction model , the rats were given stachydrine hydrochloride at dose of 10 mg/kg, 20 mg/kg or 40 mg/kg by gavage daily for 14 d.The impairment of neurological function in each group was scored .The cerebral infarction volume and brain water content were measured .Moreover , the protein levels of β-cate-nin, cyclin D1, glycogen synthase kinase 3β(GSK-3β) and p-GSK-3βin the brain tissues were detected by Western blot . RESULTS:Compared with cerebral infarction group , the score of neurological function impairment , cerebral infarction volume and brain water content were significantly decreased in stachydrine hydrochloride treatment groups .In addition , the protein levels of β-catenin, cyclin D1 and p-GSK-3βwere markedly increased after stachydrine hydrochloride treatment . CONCLUSION:Stachydrine hydrochloride protects against experimental acute cerebral infarction through activation of Wnt/β-catenin signaling pathway .
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To optimize the concentrate process of alkaloid from Leonurus japonicus by nanofiltration-ultrafiltration coupling technology with response surface methodology. The experiment showed that after ultrafiltration pre-treatment, the total protein removal rate was 94.38% in aqueous extract from L. japonicus, and the nanofiltration technology had obvious advantages over the conventional concentrate process. The optimal concentrate conditions were as follows：molecular weight cut-off 450, pH 3.07, concentration of stachydrine hydrochloride 80.15 mg•L⁻¹, and concentration of the total alkaloid 285.73 mg•L⁻¹. The cut-off rate was 93.37% and 95.85% respectively for stachydrine hydrochloride and the total alkaloid under the optimum conditions, with a relative error of 0.79% and 1.16% respectively. The combination of Box-Behnken design and response surface analysis can well optimize the concentrate process of L. japonicus by nanofiltration, and the results provide the basis for nanofiltration concentrate for heat-sensitive traditional Chinese medicine.
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Objective To study the difference ofLeonurus japonicus germplasm resources and provide good materials for breeding.Methods Totally 20 wildLeonurus japonicus germplasm resources from different places of China were collected. Experiment was conducted in homogeneous garden in Quzhou City of Zhejiang Province. Phenological phase, cold endurance and habitat adaptability were observed; the plant height, fresh weight and dry weight in early flowering were measured; the contents of stachydrine hydrochloride and leonurine hydrochloride were determined in early flowering and out-of-season cultivation.Results Considering the habitat adaptability, dry plant weight in early flowering, the contents of stachydrine hydrochloride and leonurine hydrochloride separately in early flowering and out-of-season cultivation, it was believed that the germplasms from Linbao County, Sheqi County in Henan Province and Guidong County in Hunan Province were better, in which Linbao germplasm was the best: the dry plant weight was 30.6 g, the content of stachydrine hydrochloride and leonurine hydrochloride were 1.31% and 0.19% respectively in early flowering, and were 3.44% and 0.37% respectively under anti-season cultivation, and it can be well adapted in Zhejiang Province.Conclusion The germplasm ofLeonurus japonicas from Lingbao can be the best materials for breeding.
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To establish the quantitative method of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba. The contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba were determined by HPLC-MS. The chromatographic column was Waters XBridge Amide(4.6 mm×250 mm,5 μm). The mobile phase was acetonitrile-0.1% formic acid in gradient mode,at the flow rate of 1.0 mL• min⁻¹,with the split ratio of 1∶4. MS conditions for the ESI ion source,positive ion mode,selective ion scan(SIM) of stachydrine hydrochloride(m/z 144.0) and leonurine hydrochloride(m/z 312.0) was measured. The linear ranges of stachydrine hydrochloride was 0.562 8-281.4 μg•L-1（r=0.999 8）. The linear ranges of leonurine hydrochloride was 0.521 2-260.6 μg•L-1（r=0.999 8）. The method is accurate,simple,and reliable,and can be used to determine the contents of stachydrine hydrochloride and leonurine hydrochloride in the preparations of Leonuri Herba.
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Objective To optimize the preparative procedure for stachydrine in Fructus Leonuri. Methods The preparation was screened by orthogonal experiment, and a mathematical model of relationship of extraction time, methanol concentration, and solid-liquid ratio with the content of stachydrine hydrochloride was established by using back-propagation (BP) neural network. And the process parameters were optimized with genetic algorithm (GA) . Results The optimum process parameters were as follows: extraction with 69% of methanol concentration and with solid-liquid ratio being 11 times for 62 min. The content of stachydrine obtained by BP neural network modeling and GA was higher than that achieved by orthogonal experiment. Conclusion The optimum preparative procedure could be achieved by combining BP modeling with GA. The model developed in this study was proved to be predictable and feasible for the optimization of process parameters of multi-dimension nonlinear system.
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Objective: To investigate the effect of stachydrine on the expression of protein kinase R-like endoplasmic reticulum kinase (PERK) in renal tissue of rats with unilateral ureteral obstruction (UUO). Methods: To establish the animal model of renal interstitial fibrosis induced by UUO. The rats were randomly divided into the Sham, model Enalapril, high-, mid-, and low-dose stachydrine groups. Rats were sacrificed to collect serum for determining the serum creatinine (Scr) and blood urea nitrogen (BUN) after day 14 of surgery. HE staining was used for observation of kidney pathological changes and the assessment of renal tubular damage indexes. Masson staining was applied to the semi-quantitative analysis of relative area of renal interstitial fibrosis (RIF) and degree of RIF. The expression of PERK, activiting transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) was detected by immunohistochemical methods. Results: Compared with the model group, the levels of Bun, Scr, the tubular injury, the relative area of renal interstitial collagen, and the expression of PERK, ATF4, and CHOP was significantly reduced in the treatment groups (P < 0.05, 0.01). The high stachydrine was more effective than Enalapril in inhibiting RIF (P < 0.05). Conclusion: The stachydrine could inhibit the increasing expressions of ATF4 induced by PERK signaling pathway and CHOP activated by ATF4. The apoptosis are blocked to slow down the occurrence and development of RIF.
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Objective To control the quality of Herba Leonuri by studying the content of stachydrine hydrochloride in different parts of it. Method A HPLC method was developed for the content detetmination of stachydrine hydrochloride in flower, stem, leafage from Herba Leonuri. Spherisorb SCX colum was used with mobile phase of 20 mmol/L sodium dihydrogen phosphate (containing 0.04% triethylamine and 0.15% phosphoric acid). The colum temperature was 25 ℃, the detective wavelength was 192 nm. Results Stachydrine hydrochloride content was the highest in leafage and the lowest in stem. Conclusion To ensure the quality of Herba Leonuri, it is significant to choose medical material with more leafage.
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OBJECTIVE:To establish TLC scanner method for the determination of stachydrine hydrochloride in xue?niaokang granule.METHODS:Silica gel G thin layer plate was adopted in the determination with n-butanol-hydrochloric acid-ethyl acetate(8∶3∶1)used as developer and thin potassium heptaiodobismuthate test solution used as color-developing agent,the detection wavelength was515nm,the reference wavelength was700nm and the slit size was6.00mm?0.45mm.RESULTS:Good linear correlation with the peak area score of spots achieved when the concentration range of stachydrine hydrochloride was within5.06?g～25.32?g(r=0.9978),the average recovery was96.67%(RSD=2.01%).CONCLUSION:The method was accurate,simple,reliable,sensitive and reproducible,and it can be used for the determination of xueniaokang granule and its quality control.
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OBJECTIVE:To determine the contents of hydrochloric stachydrine in the shenning granule by TLC-scanning Method.METHODS:The acetone-dehydrated alcohol-HCl(10∶6∶1)were used as developing agents,the potassium hep-taiodobismuthate test solution-1%trichloride ferric dehydrated alchhol solution(2∶1)were used as the color-developing a-gents,the detection wavelength was530nm,the reference wavelength was670nm,the slit size was0.4mm?0.4mm,the lin-earization parameter Sx=3.RESULTS:The spot peak area score assumed a satisfactory linear relationship when the electric sample size for the hydrochloric stachydrine at a range of2.412?g～6.432?g(r=0.9972),the recovery was98.93%(RSD=1.15%).CONCLUSION:This method is simple,accurate,and this determination method has a good reproducibility,which can be used for the quality control of the shenning granule.
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Objective To establish the fingerprints method of capillary electrophoresis for Leonurus heterophyllus Injection.Methods Method of capillary zone electrophoresis was developed to establish the fingerprints of Leonurus heterophyllus Injection with the peak of stachydrine as reference.Results Fingerprints of Leonurus heterophyllus Injection obtained contained 12 major common peaks.Conclusion The method has good reproducibility and resolution which can be used for the quality control of Leonurus heterophyllus Injection.
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Objective To establish a HPLC method for the separation and determination of stachydrine hydrochloride in fresh medicinal material of Herba Leonuri.Methods The NH2 column(250 mm? 4.6 mm,5 ? m) was adopted with acetonitrile-water(80 ∶ 20) as mobile phase at a flow rate of 1.0 mL/min.The detection wavelength was at 192 nm.The column temperature was at 22 ℃.The solution for content determination was obtained after extracted with ethanol and then filtered and purified through an activated charcoal-aluminium oxide column with ethanol as eluant.Results Stachydrine hydrochloride in fresh Herba Leonuri can be separated and determined on NH2 column.A good linear range was within 2.29～ 11.46 ? g.The average recovery for stachydrine hydrochloride was 98.26 % and RSD was 1.52 %(n=5).Conclusion The method is steady and with good repeatability,and can be used to determine the content of stachydrine hydrochloride in fresh Herba Leonuri.
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Objective To establish the quality standard for Chanhou Zhuyu Capsules.Methods Leonurus Japonicus and Ligusticum chuanxiong in Chanhou Zhuyu Capsules were identified by TLC,and the content of stachydrine hydrochloride was determined by TLC Scanning.Results The quality identification by TLC was specific.Stachydrine hydrochloride had a good linearity in the range of 4.0～12.0 ?g,r=0.999 7,and the average recovery was 99.87 %,RSD=1.67 %.Conclusion The methods of identification and content determination are simple,reliable and reproducible.They can be used effectively for the quality control of Chanhou Zhuyu Capsules.
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Objective: To establish the quality standard for Guangxiao Liniao Capsules (Herba Verbenae, Herba Leonuri, Rhizoma Alismatis, Radix Linderae, etc.). Methods: Herba Verbenae, Herba Leonuri, Rhizoma Alismatis, Radix Linderae were identified by TLC, and the content of stachydrine hydrochloride was determined by TLC scanning. Results: Herba Verbenae, Herba Leonuri, Rhizoma Alismatis, Radix Linderae could be identified by TLC. Stachydrine hydrochloride showed a good linear relationship at a range of 4?g～20?g, r = 0.9975. The average recovery was 97.9%, and RSD was 1.20%. Conclusion: The methods are accurate and can be used for the quality control of Guangxiao Liniao Capsules
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Objective:To investigate the quality of Herba Leonuri Preparations sold in market. Methods: The alkaloid content in preparations was determined according to the determination method in China Pharmacopoeia (1995). Results: There were significant differences among Herba Leonuri Preparations from different pharmaceutical factories, place of origin and batches. Conclusions: It is suggested that the determination item of stachydrine hydrochloride be added.