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Malignant melanoma (MM) is one of the malignant tumors with highly metastatic and aggressive biological actions. Schizandrin A (SchA) is a bioactive lignin compound with strong anti-oxidant and anti-aging properties, which is stable at room temperature and is often stored in a cool dry place. Hence, we investigated the effects of SchA on MM cell line A375 and its underlying mechanism. A375 cells were used to construct an in vitro MM cell model. Cell viability, proliferation, apoptosis, and migration were detected by Cell Counting Kit-8, BrdU assay, flow cytometry, and transwell two-chamber assay, respectively. The cell cycle-related protein cyclin D1 and cell apoptotic proteins (Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9) were analyzed by western blot. Alteration of H19 expression was achieved by transfecting with pEX-H19. PI3K/AKT pathway was measured by detecting phosphorylation of PI3K and AKT. SchA significantly decreased cell viability in a dose-dependent manner. Furthermore, SchA inhibited cell proliferation and cyclin D1 expression. SchA increased cell apoptosis along with the up-regulation of pro-apoptotic proteins (cleaved-caspase-3, cleaved-caspase-9, and Bax) and the down-regulation of anti-apoptotic protein (Bcl-2). Besides, SchA decreased migration and down-regulated matrix metalloproteinases (MMP)-2 and MMP-9. SchA down-regulated lncRNA H19. Overexpression of H19 blockaded the inhibitory effects of SchA on A375 cells. SchA decreased the phosphorylation of PI3K and AKT while H19 overexpression promoted the phosphorylation of PI3K and AKT. SchA inhibited A375 cell growth, migration, and the PI3K/AKT pathway through down-regulating H19.
Subject(s)
Humans , Polycyclic Compounds/pharmacology , Down-Regulation/drug effects , Cell Movement/drug effects , Apoptosis/drug effects , Lignans/pharmacology , Cyclooctanes/pharmacology , Cell Proliferation/drug effects , Melanoma/pathology , Signal Transduction/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Blotting, Western , MicroRNAs/metabolism , Cell Line, Tumor , Real-Time Polymerase Chain Reaction , RNA, Long NoncodingABSTRACT
Objective: To optimize the preparation process of Schisandrae Chinensis Fructus granules(SCFG) to obtain a stabilized and qualified SCFG-preparation. Methods: The Schisandrae Chinensis Fructus raw materials were extracted by decoction with water,and the extraction process was optimized by the orthogonal test. In the orthogonal test, the yield of total extract and the extraction rate of schizandrin were used as evaluation index,and the effect of solid-liquid ratio,decoction time and extraction times on the indexes were investigated to optimize the related parameters of these fac- tors. Meanwhile,the concentration process,drying process and granulation process were also investigated to finally opti- mize the preparation process of SCFG products. The content of schizandrin was determined by the HPLC method. Re- sults Under the HPLC conditions in the Pharmacopia of the People’s Republic of China(2015 edition),schizandrin showed a good linearity in the range of 0.03598-0.28784 μg(r=1.0000),with the average recovery rate 99.32% and RSD 2.17%. The conditions for the optimized extraction process were 3 times of extraction,with 0.5 h of each decoction time and with the 1:8,1:6 and 1:6 solid- liquid ratio in turn. The conditions for the optimized concentration process were concentrating at 60℃ and -0.08 MPa vacuum to a certain concentration. The conditions for the optimized drying pro- cess were drying at 60℃ and -0.09 MPa vacuum to a dryness. The conditions for the optimized granulation process were adding appropriate amount of dextrin,using 90% alcohol as moistening agent,and sifting with 14 mesh sieve and desic- cating at 60℃. Conclusion: The improved method is feasible,simple,stable and suitable for large-scale production of SCFG products.
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AIM To establish an HPLC-DAD method for the simultaneous content determination of six constituents in Maiwei Dihuang Pills (Ophiopogonis Radix,Schisandrae chinensis Fructus,Rehmanniae Radix Praeparata,etc.).METHODS The analysis of 50％ methanol extract of this drug was performed on a 35 ℃ thermostatic Agilent ZORBAX SB-C18column (4.6 mm × 150 mm,5 μm),with the mobile phase comprising of acetonitrile-water (containing 0.2％ phosphate acid) flowing at 0.8 mL/min in a gradient elution manner,and the detection wavelengths were set at 220,230,236 and 274 nm.RESULTS Deoxyschizandrin,schizandrin B,schisandrin,paeoniflorin,paeonol and loganin showed good linear relationships within the ranges of 10-70,6.5-45.5,33.5-234.5,17-119,31-217 and 34-238 μg/mL (r ＞0.990 0),whose average recoveries (RSDs) were 99.6％ (1.7％),100.4％ (1.8％),100.7％ (1.8％),102.9％ (1.7％),102.2％ (1.5％) and 99.7％ (1.2％),respectively.CONCLUSION This simple and reproducible method can be used for the rapid quality control of Maiwei Dihuang Pills.
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Objective: To study the relativity between color and chemical composition in fruits of Schisandra chinensis by fruits' surface color value (L*, a*, and b*) and contents of active ingredients of fructus core S. chinensis). Methods: The surface chromatic value (multivariate linear regression equation of L*, a*, b* values and deoxyschizandrin, schizandrin B, schizandrol A, schinsantherin A) was established by SPSS 20.0 with mathematical statistics method, then the influence degree of the active ingredients of. S. chinensis was detected by F test. Results: The value of L* were significant negative with deoxyschizandrin (r ≥ 0.5), and other chromaticity values were very low or low-grade correlated with lignans Conclusion: The effective ingredient content of fruit of S. chinensis can be estimated by its chromatic values, and also can be used in the hierarchies of the fruit of S. chinensis, in order to provide better scientific basis of the quality of S. chinensis.
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Objective To establish a quantitative analysis of multi-component with a single-marker (QAMS) method for the quality control of Wuzi Yanzong Pills (WYP). Methods Six main effective components (schisandrin, hyperin, quercitrin, kaempferol 3-O-rutinoside, deoxyschizandrin, and γ-schizandrin) of WYP were simultaneously separated on a reversed-phase column (Ultimate LP-C18) with high-resolution of each chromatographic peak by high performance liquid chromatography (HPLC). Schisandrin was selected as the internal reference, and the relative correlation factors (RCFs) of other five components were calculated to achieve QAMS. The ruggedness of RCFs was tested on different instruments and columns. Moreover, results of the QAMS were compared with the external standard method. Results Within a certain linear range, the RCFs of hyperin, quercitrin, kaempferol 3-rutinoside, deoxyschizandrin, and γ-schizandrin were 0.36, 4.86, 0.88, 7.34, and 6.35, respectively. The repeatability was good under different experimental conditions. There were no significant differences between the calculated value and estimated value on QAMS and external standard method. Conclusion The QAMS method can be used to assay the content of six components of WYP simultaneously and control the quality of WYP simplely, reliably, and accurately.
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This study was conducted to test the effects of schizandrin B (Sch B) on clozapine (CLZ) induced chronic liver injury in mice and the mechanism of action, and this may provide a new approach for clinical prevention of CLZ-induced side effects. The CLZ was given to mice for three weeks alone or co-administration with Sch B. The changes of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and antioxidation indexes superoxide dismutase (SOD), malonic dialdehyde (MDA), glutathione (GSH) and liver histological evaluation were determined. Expression of Nrf2 was assayed in hepatic cells by immunohistochemical staining and Western blotting. The changes of relative gene expression of NAD(P)H:quinone oxidoreductase l (NQO1) and heme oxygenase 1 (HO-1) were assayed by real-time Q-PCR. The results showed that pretreatment with a lower dosage of Sch B (25, 50 mg·kg-1) prevented CLZ-induced liver injury as indicated by the reduced levels of ALT, AST and ALP, and the preserved activities of SOD, GSH and inhibiting MDA. It was shown that Sch B could up-regulate Nrf2 expression leading to nuclear accumulation of Nrf2 to induce oxidative response genes such as NQO1 and HO-1. These results suggest that Sch B could protect against liver injury induced by CLZ via the activation of the Nrf2/ARE signal pathway in a dose-dependent manner.
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Objective:To investigate the effects of schizandrin B on P- glycoprotein by a classical in vitro cell model. Methods:Caco-2 cell model was used as the carrier,and rhodamine 123 and cyclosporin A were employed as the P-gp substrates, the transmembrane transportation of schizandrin B,rodamine 123 and cyclosporin A were detected by HPLC and a liquid scintillation counting assay,and the apparent permeability coefficient and permeability directional ratio were calculated. Results:The bidirectional transportation rates of schizandrin B(20 μg·ml -1 ,40 μg·ml -1 and 80μg·ml-1 )were similar,and showed non-selective difference. Schizandrin B(20-160 μg ·ml -1 )significantly inhibited the BL → AP directional transportation of rhodamine 123 and cyclosporine A in Caco-2 cell model(P < 0. 05) in a concentration-dependent manner. Conclusion:Schizandrin B is a P-gp inhibitor,while it isn’t a P-gp substrate.
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OBJECTIVE:To establish a method for simultaneous contents determination of schizandrin,schizandrin B and cin-namaldehyde in Xiaoqinglong granule. METHODS:HPLC was performed on the column of Wondasil-C18 with the mobile phase of acetonitrile-0.1% phosphoric acid solution(gradient elution)at the flow rate of 1.0 ml/min,the detection wavelength was 245 nm, temperature was 25 ℃ and volume was 20 μl. RESULTS:The linear range was 1.008-20.16 μg/ml(r=0.999 8) for schizandrin, 0.496-9.92μg/ml(r=0.999 7)for schizandrin B,and 1.012-20.24μg/ml(r=0.999 6)for cinnamaldehyde;RSDs of precision,sta-bility and reproducibility tests were no more than 1.33%;the average recoveries were respectively 98.9%(RSD=1.71%,n=6), 99.9%(RSD=1.50%,n=6) and 98.7%(RSD=2.10%,n=6). CONCLUSIONS:The method is simple,accurate and reproduc-ible,and can be used for the quality control of Xiaoqinglong granule.
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Aim To investigate the inhibitory effect of schizandrin B( SchB) on ultraviolet radiation b ( UVB) radiation-induced apoptosis of HaCaT cells. Methods Methyl thiazolyl tetrazolium ( MTT ) assay was used to examine the effect of SchB on cell viability recovery. Cell apoptosis and necrosis were measured by Ho-chest33342 staining. The p53, p21 and Caspase-3 mRNA expressions were examined by RT-PCR. Results In this study, we found that Sch B attenuated UVB-in-duced toxicity in HaCaT cells. Through Hoechst 33342 stain, we visualized that SchB could inhibit UVB-in-duced HaCaT cell death. The result demonstrated that p53 , p21 and Caspase-3 mRNA levels decreased com-pared with the control group. Conclusions Sch B at-tenuates the UVB-induced toxicity of HaCaT by inhibi-ting apoptotic gene expression. It plays a role in anti-photoaging.
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Objective To explore the protective effect of schisandrin B (SchB) on the permanent damage of human keratinocytes (HaCat) induced by long wave ultraviolet (UVA),and its possible mechanism thereof. Methods After HaCat was treated by 5 J/cm2 UVA, different concentrations of SchB (0.1, 0.01, 0.001 and 0.000 1μmol/L) were used to treat HaCat cells. The levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity, lactate dehydrogenase (LDH) and NO content were detected. Results The levels of SOD and GSH-Px activity were decreased, and he levels of LDH and NO content were increased in HaCat cells after being treated by UVA. The different concentrations of SchB showed significant ef-fects on the increased levels of SOD, GSH-Px activity and decreased levels of LDH and NO, and improved the survival rate of HaCat cells. The 0.001 μmol/L SchB showed the strongest protective effect. Conclusion The 0.001 μmol/L SchB showed the best effect on the damage of HaCat cells induce the UVA.
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Objective:To study the effect of artificial gastric juice on the dissolution of schizandrin A to provide the parameters for the best extraction method of Schisandra chinensis. Methods:Schisandra chinensis was respectively extracted by artificial gastric juice and water. Schizandrin A in the extracts was determined by HPLC, and the dissolution of schizandrin A in artificial gastric juice and water was studied and compared. Results:At 60 min, schizandrin A dissolution was 0. 483% in artificial gastric juice, and 0. 362%in water. Conclusion:The dissolution of schizandrin A in artificial gastric juice is 33. 4% higher than that in water, suggesting artifi-cial gastric juice can significantly improve the dissolution of schisandrin A.
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Objective To explore the possible mechanism of SchA ,which decreases MPP+induce SH-SY5Y cell damage .Meth-ods Cultured cells were divided into 5 groups ,one as control group ,cultured by free-blood serum media;the other 4 groups were treated with different concentrations of SchA(1 ,3 ,5 μmol/L) and MPP+ (1 mmol/L) for 48 h named model group ,1 ,3 ,5 μmol/L SchA group respetivly .The content of nitric oxide(NO) were measured by NO kit ;The expression levels of total Akt and p-Akt proteins were detected by Western blot .Results Compared with the control group ,the content of NO in group significantly in-creased after MPP+stimulating(P0 .05) .The expression levels of p-Akt in model group significantly lowered ,while SchA(1、3、5 μmol/L) significantly increased the expression levels of p-Akt in comparision with cells in model group .Conclusion Decreasing MPP+ induced SH-SY5Y cell damage of SchA may be related to the content of NO and p-Akt expression .
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OBJECTIVE: To study the application of Multi-objective Genetic Algorithm in optimization of micro-extraction technology of Schisandrae Chinensis Fructus. METHODS: Sub-target model of extraction rate, schizandrin and the total lignan was established based on micro-extraction Schisandrae Chinensis Fructus experimental data resulted from uniform design. Applying MOGA, the optimal extracting conditions were explored, and the results were compared with those of Single-objective genetic algorithm. Using SGALAB beta5008 of the Matlab2009a plug-in tool-box, the genetic algorithm optimization was achieved. SPSS13.0 software was used for statistical analysis. RESULTS: The optimal conditions were as follows; microwave power 270 W, ethanol 87%, extraction time 7 min, ethanol amount 1:4.5, grinding degree 70. Under these conditions, the extract rate was 21.40%, Schizandrin recovery was 4.72%, and the total lignan recovery was 10.84%. CONCLUSION: MOGA provides a reasonable pareto ensuring the optimal multi-objective, which provides a reasonable method for extraction of Schisandrae Chinensis Fructus. This method can be applied in the selection of extraction technology of other drugs. Copyright 2012 by the Chinese Pharmaceutical Association.
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Objective To optimize the extract technology of active lignins from the fruits of Schisandra chinensis.Methods The content of schizandrin,gomisin A,and deoxyschizandrin were selected as standards to evaluate the efficiency of smashing tissue extraction (STE).Solid-liquid ratio,extracting times,ethanol concentration,and extracting time were investigated through orthogonal test.Results The optimized conditions for STE were ten times amount of 80％ EtOH,extracting for three times,and 2 min for each time.Conclusion STE could obtain relatively higher yield,simplicity of operation,and benefit for environment protection.It could be better choice for the extraction ofS.chinensis.
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The effectiveness of traditional Chinese medicine (TCM) against various diseases urges more low cost,speed and sensitive analytical methods for investigating the phamacology of TCM and providing a theoretical basis for clinical use.The potential of second-order calibration method was validated for the quantification of two effective ingredients of Schisandra chinensis in human plasma using spectrofluorimetry.The results obtained in the present study demonstrate the advantages of this strategy for multi-target determination in complex matrices.Although the spectra of the analytes are similar and a large number of interferences also exist,second-order calibration method could predict the accurate concentrations together with reasonable resolution of spectral profiles for analytes of interest owing to its ‘second-order advantage'.Moreover,the method presented in this work allows one to simply experimental procedure as well as reduces the use of harmful chemical solvents.
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Objective: To establish an HPLC method for simultaneous determination of syringin, schizandrin, deoxyschizandrin and schisandrin B in Shuangwu capsules. Methods: The HPLC method was employed using a Diamonsil C18 column (200 mm X 4.6 mm, 5μm) with a mobile phase of methanol-acetonitrile (1 : 1, A) and water (B). The gradient elution program was as follows, 0-5 min, 35%-60%A; 5-10 min, 60%-70%A; 10-50 min, 70%-90%A; 50-90 min, 90%A. The flow rate was 1 ml/min. The detection wavelength was set at 220 nm and the temperature was at 35°C. Results: The linearity was obtained within the range of 1.28-20.40 μg/ml for syringin (r=0.999 7), 6.30-100.80μg/ml for schisantherin (r=0.999 6), 1.20-19.20μg/ml for deoxyschizandrin (r=0.999 8), and 3.75-60. 00 μg/ml for schisandrin B (r=0. 999 6). The RSD values of precision were less than 1% for all the four components. The results showed that the samples were stable in the room temperature for at least 24 h. The average recovery rates of syringin, schizandrin, deoxyschizandrin and schisandrin B were 99.47%, 102.50%, 99.21% and 101.86%, respectively. Conclusion: Our method is rapid, easy to perform and accurate; it can be used to control the quality of Shuangwu capsules.
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PURPOSE: This study investigated the effect of reducing cisplatin induced nephrotoxicity with DWP-04 that is the compound of Schizandrin C derivative biphenyldimethyl dicarboxylate (DDB), glutathione and selenium. For the purpose of observation is that how DWP-04 has influence on mechanism of reducing cisplatin induced nephrotoxicity with renal function test, free radical formation and detoxification enzyme system in renal tissue. METHODS: Five groups of rats were dosed with vehicle, cisplatin (2 mg/kg i.p.), cisplatin+DWP-04 (100, 200 mg/kg po), or cisplatin+sodium thiosulfate (200 mg/kg i.p.) daily for 4 weeks. RESULTS: Serum creatinine, lactate dehydrogenase and activity of hydroxy radical increased in the cisplatin group and suppressed in the cisplatin+DWP-04 group compared to the cisplatin group. The renal tissue concentration of lipid peroxidase and lipofuscin were increased in the cisplatin group compared to the other groups. The activity of aminopyrine N-demethylase, aniline hydroxylase, aldehyde oxidase and xanthine oxidase, of which free radical formation system in kidney was also decreased in the cisplatin+DWP-04 group compared to the cisplatin and cisplatin+sodium thiosulfate group. The activity of detoxification system of free radical, such as glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase were markedly increased in the cisplatin+DWP-04 group than the cisplatin and the cisplatin+sodium thiosulfate group (p<0.05). CONCLUSION: It can be concluded that the mechanism of decreasing cisplatin-induced nephrotoxicity by DWP-04 is that the decreasing of the amount of lipid peroxide and lipofuscin in the renal tissue by increasing activity of the antioxidant defense system and the decreasing of reactive oxygen species by increasing detoxification enzyme activity.
Subject(s)
Animals , Rats , Aldehyde Oxidase , Aminopyrine N-Demethylase , Aniline Compounds , Aniline Hydroxylase , Antioxidants , Catalase , Cisplatin , Creatinine , Cyclooctanes , Glutathione , Glutathione Peroxidase , Glutathione Transferase , Kidney , L-Lactate Dehydrogenase , Lignans , Lipofuscin , Peroxidase , Polycyclic Compounds , Reactive Oxygen Species , Renal Insufficiency , Selenium , Superoxide Dismutase , Xanthine OxidaseABSTRACT
Schizandrae Fructus has been used for controlling respiratory allergic or inflammatory diseases in folk medicine and their components, schizandrin, schizandrin-A and gomisin-A were reported to have diverse biological effects. In this study, we investigated whether schizandrin, schizandrin-A and gomisin-A affect adenosine triphosphate (ATP)-induced mucin secretion from cultured airway epithelial cells. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using 3H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on 3H-mucin secretion. The results were as follows: 1) schizandrin significantly inhibited ATP-induced mucin secretion; 2) However, schizandrin-A and gomisin-A did not affect ATP-induced mucin secretion, significantly. We conclude that schizandrin can inhibit ATP-induced mucin secretion by directly acting on airway mucin-secreting cells. Therefore, schizandrin should further be investigated for the possible use as mucoregulators in the treatment of inflammatory airway diseases.
Subject(s)
Animals , Cricetinae , Adenosine Triphosphate , Adenosine , Epithelial Cells , Medicine, Traditional , Mucins , SchisandraABSTRACT
OBJECTIVE:To determine the content of ?-schizandrin,one of the effective ingredients,in Shuangjia Wuling capsules METHODS:The RP-HPLC method was performed with YWG C18 column(4 6mm?250mm) The mobile phase consisted of methanol-water(72∶28) The detecting wavelength was 254nm RESULTS:The calibration curve for ?-schizandrin was linear in the range of 0 0 207～0 4 130mg/ml(r=0 9 999) The average recovery was 97 68% with RSD=1 85% CONCLUSION:The method is simple,rapid and reliable for quality control of the capsules
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Objective To establish a method for the determination of schizandrin, deoxyschizandrin and ?-schizandrin in Jiangtang soft capsule. Methods The sample was extracted by methanol. The chromatographic conditions were: a gradient mobile phase of methanol-water(65: 35)within 0～20 rain and methanol-water(70: 30)within 20～80 min, the wavelength at 250 nm. Results A linear range of schizandrin, deoxyschizandrin and ?-schizandrin was within 0.71 ?g～3.53 ?g(r=0.99998, n=5), 0.19?g～0.95 ?g(r=0.99993, n=5)and 0.36?g～1.80?g(r=0.99997, n=5) and the average recoveries were 100.44%, 98.06% and 101.14% respectively. Conclusion This method is easy, sensitive, specific and accurate for the determination of schizandrin, deoxyschizandrin and ?-schizandrin in Jiangtang soft capsule.