Improvement effect and mechanism of sanguinarine on inflammatory pain in rats with lumbar disc herniation / 中国药房

OBJECTIVE To study the improvement effect and mechanism of sanguinarine （SG） on inflammatory pain in rats with lumbar disc herniation （LDH） and its mechanism. METHODS LDH model rats were established and divided into model group， SG low-dose， medium-dose and high-dose groups （1.00， 2.50， 6.25 mg/kg）， high-dose of SG+Anisomycin [mitogen-activated protein kinase （MAPK） activator] group （6.25 mg/kg SG+5 mg/kg Anisomycin）， with 10 rats in each group. Another 10 rats were included as the control group. Each group was given corresponding drugs intraperitoneally， while the control group and model group were given an equal volume of normal saline intraperitoneally， once a day， for 7 consecutive days. The general situation and neurological changes of rats in each group were observed， and the pain threshold [including paw withdrawal mechanical threshold （PWMT） and paw withdrawal thermal latency （PWTL）] of rats was determined； the histopathological changes of dorsal root ganglion （DRG） were observed in rats. The serum levels of inflammatory factors [tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）] and pain factors [neuropeptide Y （NPY）， 5-hydroxytryptamine （5-HT）] in rats were detected.The positive expressions of ionized-calcium binding adaptor molecule-1 （Iba-1） in spinal cord microglia and glial fibrillary acidic protein （GFAP） in astrocytes were observed. The expressions of proteins related to MAPK/extracellular signal-regulated kinase （ERK）/nuclear factor κB （NF-κB） signaling pathway， TNF-α and IL-1β proteins were detected in DRG tissue of rats. RESULTS Compared with the control group， the rats in the model group showed decreased appetite， hindlimb movement disorders， and disordered neuronal cell arrangement， the neurological score， the levels of TNF-α， IL-1β， NPY， the positive expressions of Iba-1 and GFAP， the phosphorylations of p38 MAPK， ERK1/2 and NF-κB p65， the protein expressions of TNF-α and IL-1β were significantly increased （P＜0.05）； PWMT， PWTL and the levels of 5-HT were significantly reduced （P＜0.05）. Compared with the model group， the rats of SG groups showed some relief in their mental appetite and hindlimb motor disorders， the intervertebral disc structure of DRG was restored， and the levels of the above quantitative indicators had significantly reversed （P＜0.05）. Anisomycin reversed the improvement effect of SG on inflammatory pain in LDH rats. CONCLUSIONS SG can improve inflammatory pain by inhibiting the activation of microglia in DRG tissue of LDH rats， reducing the release of inflammatory factors， and increasing pain threshold， and its mechanism of action may be related to the inhibition of MAPK/ERK/NF- κB signaling pathway.

Preventive and therapeutic effects of sanguinarine chloride on sodium arsenite-induced liver damage in mice / 环境与职业医学

Background Natural product sanguinarine chloride (SC) can significantly alleviate liver fibrosis and acute liver injury in mice, but whether it has a protective effect on mouse liver injury caused by sodium arsenite (SA) has not been studied. Objective To verify if SC may present preventive and therapeutic effects on SA-induced liver injury in mice. Methods A total of 140 SPF male Kunming mice were randomly divided into two sub-studies, which included a prevention sub-study and a treatment sub-study. In each sub-study, a blank group (normal saline), a model group (5 mg·kg−1 SA), and a positive control group (11.375 mg·kg−1 bicyclol and 182 mg·kg−1 glutathione), as well as SC low, medium, and high dose groups (25, 50, and 100 mg·kg−1) were arranged with 10 mice in each group. In the prevention sub-study, the blank group was given normal saline, the model group was given SA, and the other groups (the SC low, medium, and high dose groups and the positive control group) were given the corresponding treatment 30 min before gavage of SA, once a day, for 28 d. In the treatment sub-study, except for the blank group which was given normal saline, the other groups were given SA for 28 d, then the model group was given normal saline, and the other groups were given the corresponding treatment every day for 28 d. After the experiment, the mice were sacrificed to evaluate selected physiological and biochemical indicators in serum and liver tissue and to observe histopathological changes after HE staining. Results In either sub-study of preventive effect or treatment effect: compared with the blank group, body weight, liver weight, liver coefficient, as well as serum alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), malondialdehyde (MDA), glutathione peroxidase (GSH), and superoxide dismutase (SOD) among all SC groups were not significantly different (P>0.05); but compared with the model group, the SC groups showed increased body weight (P<0.01), decreased liver weight and liver coefficient (P<0.01), reduced ALT, AST, TBIL, and MDA (P<0.05 or P<0.01), and increased GSH and SOD with (P<0.05 or P<0.01) or without significance; compared with the positive control group, no differences were found in the above indicators (P＞0.05). The result of histopathological evaluation showed that the SC groups had a clear liver lobule structure, neatly arranged hepatic cords, and less infiltration of inflammatory cells. Conclusion SC has both preventive and therapeutic effects on SA-induced liver injury in mice.

Effect of sanguinarine combined with cisplatin on the apoptosis of bladder cancer EJ cells / 中国药科大学学报

@#To investigate the effects of sanguinarine (Sang) combined with cisplatin (Cis) in accelerating the apoptosis of bladder cancer EJ cells, CCK-8 method was used to detect the proliferation of bladder cancer EJ cells treated with different concentrations of Sang with the IC50 values calculated. Annexin V FITC/PI method was used to detect cell apoptosis in the control group, Sang group, Cis group and the combination group. Flow cytometer was used to detect cell cycle arrested. Western blot was used to detect the influence of Bcl-2 expression in the control group, Sang group, Cis group and the combination group. Nude mouse subcutaneous tumor model was constructed to verify that the combination group could accelerate the apoptosis of bladder cancer EJ cells and reduce the side-effects on mice. The safety of the Sang was evaluated by HE staining of vital organs in mice. In vitro, Sang could significantly inhibit the proliferation of EJ cells. Compared with the control group, the number of apoptosis EJ cells in the combination group was significantly increased (P < 0.05), and more cells were arrested in G2/M phase. The expression of Bcl-2 was significantly down-regulated in the combination group (P <0.001). In vivo, compared with the control group, the tumor growth was significantly slower, and a large number of apoptotic cells were inspected (P < 0.05) of the combination group. The side effects of cisplatin were reduced in the combination group. Sang has high biosafety and little side effect. Combined Sang and Cis can increase cell cycle G2/M block, down-regulate Bcl-2 expression, promote cell apoptosis and inhibit tumor growth.

Optimizations of an ELISA-like high-throughput screening assay for the discovery of β-catenin/TCF4 interaction antagonists / 生物工程学报

In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.

Development of a fluorescence polarization-based high-throughput screening assay to identify antagonists targeting β-catenin/TCF4 interaction / 药学学报

To develop a fluorescence polarization (FP)-based high-throughput screening (HTS) assay to identify novel small-molecule antagonists targeting β-catenin/TCF4 (T-cell factor 4) interaction, recombinant human β-catenin was expressed in Escherichia coli Rosetta (DE3) cells and purified by HisTrapTM column. The bioactivity of purified β-catenin was further analyzed by enzyme-linked immunosorbent assay (ELISA). According to FP principle, the β-catenin/TCF4 binding model was performed, and fluorescence isothiocyanate (FITC) labeled TCF4 peptide (FITC-TCF4) served as the molecular probe of adaptor for binding to β-catenin. The FITC-TCF4 and β-catenin working concentration were optimized, and the binding conditions (complex stability and dimethylsulfoxide (DMSO) tolerance) have been investigated yet for further hits screening. The results showed that recombinant human β-catenin was successfully expressed and purified β-catenin exhibited favorable bioactivity in ELISA binding assay. Subsequently, the FP-based HTS assay was performed using 20 nmol·L-1 FITC-TCF4 and 100 nmol·L-1 β-catenin. Under these optimized conditions, a high Z´factor of 0.88 was achieved in a 384-well format and this FP-based HTS assay was very stable with regard to DMSO. Through screening of a natural-based product library (NBPL) using the established FP-based HTS assay, three hits (sanguinarine, chelerythrine, and compound S720) were identified as potential β-catenin/TCF4 interaction antagonists. Taken together, we have successfully developed a simple, robust and reliable FP-based HTS assay for screening of novel antagonists targeting β-catenin/TCF4 interaction.

Síntese, avaliação biológica e modelagem molecular de potenciais antitumorais análogos da sanguinarina planejados por simplificação molecular / Synthesis, biological evaluation and molecular modeling of potential antitumors analogues from sanguinarine designed by molecular simplification

A sanguinarina é um alcaloide capaz de inibir Bcl-xL, uma proteína antiapoptótica que se encontra superexpressa em linhagens tumorais e que está frequentemente relacionada à resistência destas frente a quimioterápicos antineoplásicos. No intuito de identificar potenciais agentes antitumorais, o objetivo deste trabalho foi sintetizar três séries de análogos da sanguinarina planejados por simplificação molecular e avaliar sua atividade biológica. Dez N-benzil-naftil-aminas (3a-e; 4a-e) e dez arilisoquinolinas (6a-e; 7a-e) foram sintetizadas em duas a três etapas reacionais, utilizando-se métodos de aminação redutiva e acoplamento de Suzuki. Insucesso na etapa de reação de Heck impossibilitou a síntese da terceira série, benzofenantridínica, apesar de testadas diversas condições reacionais. Avaliação da citotoxicidade em linhagens de glioblastoma U87MG revelou que a série N-benzilnaftil-amina apresenta melhor atividade quando comparada às aril-isoquinolinas, sendo para ambas, observada atividade superior à temozolamida, principal fármaco para o tratamento de glioblastoma. Estudos em linhagem não tumorigênica MRC-5 demonstraram que os análogos foram significativamente superiores à sanguinarina em relação à seletividade. Os compostos mais mais promissores, 4a e 6e, induziram morte celular por apoptose e causaram despolarização da membrana mitocondrial, indicando morte apoptótica pela via extrínseca. Ademais, 4a interrompeu o ciclo interrompeu o ciclo celular na fase G2/M, indicando que o mesmo seria um agente ciclo celular específico. Simulações de dinâmica molecular sugerem que os compostos interagem com a proteína Bcl-xL principalmente por interações hidrofóbicas, e que o composto 4a apresentaria afinidade com o alvo semelhante à sanguinarina, embora esta tenha apresentado atividade superior em células U87. Perspectivas incluem estudos das vias de indução de morte celular, além da expansão do painel de células. Conclui-se, portanto, que os análogos da sanguinarina representam um arcabouço a ser explorado pelos químicos medicinais no desenvolvimento de potenciais antineoplásico

Sanguinarine is an alkaloid able to inhibit Bcl-xL, an antiapoptotic protein which is overexpressed in tumor cells and related to their resistance against antineoplastic chemotherapy. Regarding to develop potential antitumor agents, the aim of this work was the synthesis of three series of sanguinarine analogues designed by molecular simplification and their biological evaluation. Ten N-benzyl-naphtyl-amines (3a-e; 4ae) and ten aryl-isoquinolines (6a-e; 7a-e) were synthesized in two or three reaction steps through reductive amination and Suzuki coupling. Failure about Heck-type reaction had impaired the synthesis of the thirth series, benzophenanthridine, although several conditions were tested. Cytotoxicity evaluation against U87MG glioblastoma cell line showed that N-benzyl-naphtyl-amines are more active than aryl-isoquinolines and both series were superior to temozolamide, the main drug for glioblastoma treatment. Tests against non-tumorigenic cell MRC-5 indicated that the analogues were significantly superior to sanguinarine regarding selectivity. The most promising compounds, 4a e 6e, induced cell death by apoptosis and mitochondrial membrane depolarization, indicating apoptotic death by extrinsic pathway. 4a provide cell cycle arrest at G2/M phase, suggesting that it is a specific cell cycle agent. Molecular dynamics suggested that compounds interact with Bcl-xL mainly by hydrophobic interactions and 4a has affinity to the protein like sanguinarine, although the last showed superior activity against U87 cells. Perspectives include mechanistics studies about cell death pathway and expanding cell panel. In conclusion, sanguinarine anlogues represent a scaffold to be explored by medicinal chemists to the development of potential antitumor agent

Sanguinarine inhibits proliferation, migration and cell cycle of human gastric cancer SGC-7901 cells / 中国药理学通报

Aim: To explore the mechanism of the effects of sanguinarine on the proliferation, invasion and cell cycle of gastric cancer cells. Methods: MTT assay was used to detect the inhibitory effect of sangui-narine(3, 6, 9 μmol · L-1) on the cell viability of gastric cancer SGC-7901 cells. Wound healing and flow cytometry were applied to detect cell migration and cell cycle respectively. Western blot was employed to determine the protein expression levels of migration and cell cycle related proteins MMP-2, MMP-9, cyclin Dl, CDK4, and pi 6. Results: Compared with control group, the cell viability decreased significantly with the increase of drug concentration after 24 h treatment (P < 0. 05). Flow cytometry results showed that the proportion of cells in Gl phase after treatment with 3, 6, 9 μmol · L-1 of sanguinarine significantly increased (P < 0. 05). Wound healing results indicated that the migration ability of gastric cancer cells was significantly inhibited after sanguinarine treatment. After sanguinarine treatment, the protein expression levels of MMP-2, MMP-9, cyclin Dl and CDK4 were significantly down-regulated (P < 0. 05), whereas the protein expression level of pi 6 was significandy up-regulated (P < 0. 05). Conclusions: Sanguinarine significantly inhibits the proliferation, reduces the migration ability of cells, induces cell arrest in Gl phase, and its anticancer effect is related to its regulation of MMP-2, MMP-9, cyclin Dl, CDK4 and p16.

Mechanism study of sanguinarine inhibiting tumor growth of ovarian cancer by apoptosis induction / 实用肿瘤学杂志

Objective The aim of this study was to investigate the mechanism of sanguinarine(SANG)on the inhibitory pro-liferation in ovarian cancer SKOV3 cells. Methods CCK-8 assay was used to detect proliferation of SKOV3 cells. Flow cytometry was used to detect the effect of SANG on apoptosis in SKOV3 cells. The spectrophotometer was used to detect the production of reac-tive oxygen species(ROS)by SANG. The mouse ovarian cancer xenograft model was used to detect the inhibitory effect of SANG on tumor growth. Results SANG promoted apoptosis in SKOV3 cells in a dose-and time-dependent manner. The SANG-induced ap-optosis was associated with the production of ROS,Activated the c-Jun-N-terminal kinase( JNK) and nuclear factor-κB( NF-κB)signaling pathways. In mouse model of ovarian cancer xenografts,after intravenous injection of mice with SANG,SANG was signifi-cantly inhibited the growth of ovarian cancer xenografts when compared to the control group. SANG also significantly induced apoptosis in ovarian cancer xenografts. Conclusion SANG can significantly inhibit the proliferation of ovarian cancer SKOV3 cells,induce ap-optosis,increase the production of ROS,and inhibit the growth of ovarian cancer.

Study on Separation and Purification Technology of Sanguinarine from the Extract of Macleaya cordata by Ion Exchange Resin / 中国药房

OBJECTIVE： To establish the separation and purification technology of sanguinarine from the extract of Macleaya cordata with ion exchang resin. METHODS： The content of sanguinarine from the extract of M. cordata was determined by HPLC， with  Cosmosil C18-R-Ⅱ column （250 mm×4.6 mm，5 μm）， mobile phase of acetonitrile-0.2％ acetic acid solution （25 ∶ 75，V/V）， the flow rate of 1 mL/min， detection wavelength of 270 nm， column temperature of 30 ℃， and sample size of 20 μL. Static adsorption and desorption tests were carried out to compare the adsorption and desorption properties of 8 ion exchange resins for sanguinarine. The optimum concentration of sample solution， pH value and volume of sample were investigated by optimum ion exchange resin. APPS 10D liquid phase preparation system was used to investigate the dynamic elution conditions and obtain M. cordata refined extract solution. The refined purified product of M. cordata was obtained by desalination， elution on a reversed-phase （RP） C18 column and drying.  The purity of the purified product was analyzed by HPLC. The structure of the purified product was confirmed by HPLC， UV spectrophotometry， MS and NMR. RESULTS： CM-FF resin was screened for the separation and purification of sanguinarine from M. cordata extract. It was eluted with 20 mmol/L ammonium acetate solution 100 mL containing 20％ methanol and 0.25 mol/L sodium chloride. The optimal dynamic absorption condition included that the concentration of sample was 6.0 mg/mL at pH 5.0，and the loading amount was 25 mL； after desalination and refinement， for the eluted refined extract， the purified product with 97％ purity （purified yield  of 71％） was obtained， and its structure was confirmed to be sanguinarine. CONCLUSIONS： The optimal separation and purification technology by ion exchange resin is green， safe， efficient and easy to operate， which can be used for the separation and purification of sanguinarine from M. cordata extract and is suitable for industrial production.

Fingerprint profiling of Macleaya cordata fruits from different regions / 中草药

Objective To establish the fingerprint profiling of fruits of Macleaya cordata and study the method for its quality evaluation. Methods The fingerprint profiling of M. cordata fruits from different regions was established using high performance liquid chromatography (HPLC) based on the local standard which was the determination method of the content of protopine, allocryptopine, sanguinarine, and chelerythrine from Changsha of Hunan Province in 2009. The principal component analysis (PCA) and cluster analysis (CA) were performed to explore the correlation among the common fingerprint peaks, origins, and quality of M. cordata fruits. Results Eleven common fingerprint peaks were identified in the fingerprint profiling of chemical constituents of M. cordata fruits from different regions. M. cordata fruits produced from eight areas were classified into two classes by PCA and CA method, and there were five common peaks, including peak 5, 7, 8, 9, and 11 with significant contribution on the regional difference of the fingerprint. Also, common peak 6 was the right peak as the reference peak because of its less variation, appropriate retention time and intensity. Conclusion The fingerprint profiling of chemical constituents of M. cordata fruits established in this study has good precision, repeatability, and stability, which can be used to evaluate the quality of fruits of M. cordata from different producing areas.

Sanguinarine promotes apoptosis of HepG2 cells by inducing ROS / 中国药理学通报

Aim To investigate the effect of sanguina-rine on regulating the pathway of cell apoptosis by in-ducing reactive oxygen species (ROS) in HepG2 cells. Methods MTT method was used to detect the cell viability of HepG2 cell after the treatment of san-guinarine. The changes of ROS were observed by indi-cator DCFH-DA and DHE staining. The apoptosis was detected by Hoechst 33342 and Annexin V/PI stai-ning;Rhodamine 123 staining was used to detect mito-chondrial membrane potential. Western blot was used to detect expressions of key cell-apoptotic protein. Re-sults The cell viability of HepG2 cells showed a de-creasing trend with the increasing concentration of san-guinarine. Sanguinarine could significantly increase cellular ROS,decrease mitochondrial membrane poten-tial in HepG2 cell, and promote apoptosis of HepG2 cells. The expression of Bax, cleaved-caspase-3 and cytoplasmic Cyt-C significantly increased after the treatment of sanguinarine, however, the expression of Bcl-2 was inhibited. Conclusion Sanguinarine could activate mitochondrial pathway of apoptosis mediated by cellular uncontrolled ROS and promote apoptosis of HepG2 cells.

Sanguinarine Attenuates Lipopolysaccharide-induced Inflammation and Apoptosis by Inhibiting the TLR4/NF-κB Pathway in H9c2 Cardiomyocytes / 华中科技大学学报（医学）（英德文版）

The inflammatory response is involved in the pathogenesis of the most common types of heart disease.Sanguinarine (SAN) has various pharmacological properties such as anti-inflammatory,antioxidant,antibacterial,antitumor,and immune-enhancing properties.However,few studies have investigated the effects of SAN on lipopolysaccharide (LPS)-induced inflammatory and apoptotic responses in H9c2 cardiomyocytes.Therefore,in this study,H9c2 cells were co-treated with SAN and LPS,and the mRNA levels of pro-inflammation markers and the apoptosis rate were measured to clarify the effect of SAN on cardiac inflammation.The underlying mechanism was further investigated by detecting the activation of Toll-like receptor (TLR)4/nuclear factor-κB (NF-κB) signaling pathways.As a result,increased mRNA expression of interleukin (IL)-1 β,IL-6,and TNFα induced by LPS was attenuated after SAN treatment;LPS-induced apoptosis of H9c2 cardiomyocytes and cleaved-caspase 8,9,3 were all significantly reduced by SAN.Further experiments showed that the beneficial effect of SAN on blocking the inflammation and apoptosis of H9c2 cardiomyocytes induced by LPS was associated with suppression of the TLR4/NF-κB signaling pathway.It was suggested that SAN suppressed the LPS-induced inflammation and apoptosis of H9c2 cardiomyocytes,which may be mediated by inhibition of the TLR4/NF-κB signaling pathway.Thus,SAN may be a feasible therapy to treat sepsis patients with cardiac dysfunction.

Sanguinarine Attenuates Lipopolysaccharide-induced Inflammation and Apoptosis by Inhibiting the TLR4/NF-κB Pathway in H9c2 Cardiomyocytes / 华中科技大学学报（医学）（英德文版）

The inflammatory response is involved in the pathogenesis of the most common types of heart disease.Sanguinarine (SAN) has various pharmacological properties such as anti-inflammatory,antioxidant,antibacterial,antitumor,and immune-enhancing properties.However,few studies have investigated the effects of SAN on lipopolysaccharide (LPS)-induced inflammatory and apoptotic responses in H9c2 cardiomyocytes.Therefore,in this study,H9c2 cells were co-treated with SAN and LPS,and the mRNA levels of pro-inflammation markers and the apoptosis rate were measured to clarify the effect of SAN on cardiac inflammation.The underlying mechanism was further investigated by detecting the activation of Toll-like receptor (TLR)4/nuclear factor-κB (NF-κB) signaling pathways.As a result,increased mRNA expression of interleukin (IL)-1 β,IL-6,and TNFα induced by LPS was attenuated after SAN treatment;LPS-induced apoptosis of H9c2 cardiomyocytes and cleaved-caspase 8,9,3 were all significantly reduced by SAN.Further experiments showed that the beneficial effect of SAN on blocking the inflammation and apoptosis of H9c2 cardiomyocytes induced by LPS was associated with suppression of the TLR4/NF-κB signaling pathway.It was suggested that SAN suppressed the LPS-induced inflammation and apoptosis of H9c2 cardiomyocytes,which may be mediated by inhibition of the TLR4/NF-κB signaling pathway.Thus,SAN may be a feasible therapy to treat sepsis patients with cardiac dysfunction.

Syntheses of alkyl-de-sanguinarine-N5-methyl derivatives and evaluation of in vitro growth inhibitory activities against cancer cell lines / 药学学报

2,3:7,8-Bis(methylenedioxy)benzo[c]phenanthridine was synthesized in a strategy of converging synthesis with 6-bromo-2,3-dihydroxybenzaldehyde, 5-nitronaphthalene-2,3-diol, and dibromomethane, respectively, as starting materials. The reaction process included dioxy-de-dibromo nucleophilic substitution under alkaline condition, reduction reaction, Schiff base-forming reaction, and an arene radical cyclization step under the presence of Bu3SnH and AIBN as radical initiator, among others. The 2,3:7,8-bis(methylenedioxy)benzo[c] phenanthridine as intermediate was reacted with NaBH4 and different aliphatic acids as alkylation agent to afford 2,3:7,8-bis(methylenedioxy)-5,6-dihydro-N5-alkylbenzo[c]phenanthridines. These dihydro-type products were aromatized using DDQ as oxidant under alkaline condition, and then, salinized using HCl as source of equilibrium anion to yield the series of target alkyl-de-sanguinarine-N5-methyl derivatives. All the synthesized alkyl-de-sanguinarine-N5-methyl derivatives exhibited significantly improved in vitro growth inhibitory activities against cancer cell lines as compared with sanguinarine and the positive control. In pharmacological experiments targeting five cancer cell lines, the target compounds showed activities five-fold active than that of sanguinarine. The findings of this study indicated that the structure modification strategy of substituting n-alkyls for the N5-methyl of natural sanguinarine can be used to improve the growth inhibitory activities against cancer cell lines through increasing liposolubility and steric hindrance to protect the active 5,6-imine structure.

Effect of sanguinarine on biomechanics of rat airway smooth muscle cells / 生物医学工程学杂志

This study aimed to evaluate the effect of sanguinarine on biomechanical properties of rat airway smooth muscle cells (rASMCs) including stiffness, traction force and cytoskeletal stress fiber organization. To do so, rASMCs cultured were treated with sanguinarine solution at different concentrations (0.005~5 μmol/L) for 12 h, 24 h, 36 h, and 48 h, respectively. Subsequently, the cells were tested for their viability, stiffness, traction force, migration and microfilament distribution by using methylthiazolyldiphenyl-tetrazolium bromide assay, optical magnetic twisting cytometry, Fourier transform traction microscopy, scratch wound healing method, and immunofluorescence microscopy, respectively. The results showed that at concentration below 0.5 μmol/L sanguinarine had no effect on cell viability, but caused dose and time dependent effect on cell biomechanics. Specifically, rASMCs treated with sanguinarine at 0.05 μmol/L and 0.5 μmol/L for 12 and 24 h exhibited significant reduction in stiffness, traction force and migration speed, together with disorganization of the cytoskeletal stress fibers. Considering the essential role of airway smooth muscle cells (ASMCs) biomechanics in the airway hyperresponsiveness (AHR) of asthma, these findings suggest that sanguinarine may ameliorate AHR via alteration of ASMCs biomechanical properties, thus providing a novel approach for asthma drug development.

Technology Optimization of Ultrasonic-enzyme-assisted Extraction of Sanguinarine from Zanthoxylum nitidum / 医药导报

Objective To optimize the technology conditions of ultrasonic-enzyme-assisted extraction for sanguinarine from Zanthoxylum nitidum.Methods Extraction rate of sanguinarine determined by HPLC was served as an index.The applicability of the extraction solvent added with acid and enzymatic hydrolysis pretreatment to the ultrasonic-enzyme-assisted extraction of Zanthoxylum nitidum was investigated.Ultrasonic power,extraction frequency and solvent volume were optimized by orthogonal experiment.Finally,ultrasonic extraction time was optimized in dynamic process.Results The optimal process was as follows:Zanthoxylum nitidum powder was extracted 3 times by ultrasonic-wave (250 W) with 40％ ethanol (0.2％hydrochloride) as solvent (extracted for 15 rmin with 6-fold solvent at the first time,then extracted for 12 min with 3-fold solvent at the second and the third time,respectively).The extraction rate of sanguinarine was 88.6％.Conclusion The process is economic,efficient,energy-and time-saving,and provides experimental basis for industrial production of sanguinarine.

Study on effect mechanism of sanguinarine on proliferation, apoptosis, invasion and migration of cervical cancer cells / 重庆医学

Objective To investigate the effect mechanism of sanguinarine on the proliferation,apoptosis,invasion and migration abilities of cervical cancer cells.Methods MTT,flow cytometer,cell scratch test and Transwell chamber assay were respectively used to detect the cellular proliferation,apoptosis,migration and invasion abilities after sanguinarine action.The expression levels of E-Cadherin,PTEN,β-catenin and MMP2 protein of cervical cancer cells after sanguinarine action were detected by Western blot.Results 0.6,0.8 μmol/L sanguinarine had the inhibitory effect on the proliferation of cervical cancer cells.After 0.8μmol/L sanguinarine action for 48 h,cervical cancer HeLa and Siha cells apoptosis rate were up to (45.68± 2.26)％ and(31.89 ± 3.80)％ respectively.0.8 μmol/L sanguinarine action for 3 h,cervical cancer cells HeLa and Siha adhesion rates were only (67.45 ± 2.13)％and(73.59± 2.61)％.0.8 mol/L sanguinarine action for 16 h,the invasion numbers of cervical cancer Hela and Siha cell were (39.64 ±1.98) and (43.87 ± 2.83) respectively.The expression amount of E-Cadherin and PTEN in cervical cancer cells after sanguinarine action was increased,while the expression amount of E-Cadherin and PTEN was weakened.Conclusion Sanguinarine has the proliferation inhibiting and apoptosis promoting effect on cervical cancer cells,its mechanism may be related to adhesion protein E-Cadherin,β-catenin and PTEN,MMP2.

Determination of antidiabetic compounds from Helicteres isora fruits by oral glucose tolerance test.

The aim of this study was to analyze compounds from the methanolic extract of fruits of Helicteres isora for antidiabetic activity. Sanguinarine, berberine chloride (BC) and muscimol were found to be as major compounds in the methanolic fruits extract of H. isora. BC was isolated from the methanolic fruit extract of H. isora by using HPLC and other compounds were commercially acquired. Diabetes was induced in rats by a single dose of intraperitonial injection of streptozotocin (STZ). Sanguinarine (50 mg/kg b.w), BC (50 mg/kg b.w) and muscimol (50 mg/kg b.w) were evaluated by Oral Glucose Tolerance Test (OGTT) in normal and STZ induced diabetic rats. Among the three compounds, BC significantly reduced the blood glucose levels when compared to sanguinarine and muscimol. The OGTT study clearly indicates, BC possess promising antidiabetic activity against STZ induced diabetic rats.

Inhibitory effect of sanguinarine on growth of xenograft tumors of gastric cancer GTL-16 cells in nude mice and its related mechanism / 肿瘤

Objective: To investigate the antitumor effect of sanguinarine (SAN) in vivo and its possible mechanism. Methods: The tumor-bearing BALB/c male nude mouse models were established by subcutaneous inoculation of gastric cancer GTL-1 6 cells. The mouse models were divided into three groups [14 mice in each group were intragastrically administered with 0.9% NaCl solution (as the control group), 2.5 mg/kg SAN and 5.0 mg/kg SAN every other day for 7 times, respectively]. The general status of the mice was observed, and the tumor volume was measured. After 28 days, the mice were sacrificed, then the tumor volume and weight were recorded. The histopathologic examination of tumor tissues was performed using hematoxylin-eosin (HE) staining. Then the protein expression of epidermal growth factor receptor (EGFR) in tumor tissues was detected by Western blotting. Results: As compared with the control group, the tumors grew slowly in SAN treatment groups. At autopsy, the volume and weight of tumors in SAN treatment groups were all smaller than those in the control group (P < 0.01), and the tumor volume and weight were smallest in 5.0 mg/kg SAN group (P < 0.01). The result of histopathologic examination revealed that SAN treatment could reduce the accumulation of tumor cells and epidermal invasion of tumor cells. The expression levels of EGFR were significantly lower in SAN treatment groups than that in the control group (P < 0.05). Conclusion: SAN inhibits the growth of tumor in nude mice inoculated with gastric cancer GTL-16 cells, and which may be related to the downregulation of EGFR expression.

Study on the antibacterial effect of active ingredient of se-enriched Macleya cordata / 中国生化药物杂志

Objective To study on antibacterial effect of active ingredient of se-enriched Macleya cordata.Methods Penicillin sodium and saline as control, detected the average inhibition zone diameter of se-enriched Macleya cordata group ( containing 0.96 mg/mL sanguinarine) on 9 common bacteria with disc agar diffusion method;20% methanol and saline as control, se-enriched Macleya cordata group which the average inhibition zone diameter was minimum, without se-enriched Macleya cordata group and penicillin sodium were diluted with 20% methanol, made concentration of 0.48, 0.24,0.12,0.06, 0.03 mg/mL.minimal inhibitory concentration ( MIC) of each treatment group was determined by agar dilution method, evaluated antibacterial effect of se-enriched Macleaya cordata group.Results se-enriched Macleya cordata group could improve antibacterial effect significantly, the average inhibition zone of 100 μg/mL sodium selenite treatment group was minimum(Φ15.33 mm);MIC of se-enriched Macleya cordata group on E.coli O1 , E.coli P22 , E.Dublin reduced from 0.96 to 0.24 mg/mL;MIC of S.typhimurium and S.suipare were 0.12 mg/mL;MIC of S.aureus and S.suis were 0.06 mg/mL; MIC of E.Dublin and L.murrayi were less than 0.03 mg/mL.Conclusion Se-enriched Macleya cordata improves the antibacterial effect signififcantly, and it lays the theoretical foundation for development and utilization of se-enriched Macleya cordata for future.