Unveiling potential inhibitors targeting the nucleocapsid protein of SARS-CoV-2: Structural insights into their binding sites.

The Nucleocapsid (N) protein of SARS-CoV-2 plays a crucial role in viral replication and pathogenesis, making it an attractive target for developing antiviral therapeutics. In this study, we used differential scanning fluorimetry to establish a high-throughput screening method for identifying high-affinity ligands of N-terminal domain of the N protein (N-NTD). We screened an FDA-approved drug library of 1813 compounds and identified 102 compounds interacting with N-NTD. The screened compounds were further investigated for their ability to inhibit the nucleic-acid binding activity of the N protein using electrophoretic mobility-shift assays. We have identified three inhibitors, Ceftazidime, Sennoside A, and Tannic acid, that disrupt the N protein's interaction with RNA probe. Ceftazidime and Sennoside A exhibited nano-molar range binding affinities with N protein, determined through surface plasmon resonance. The binding sites of Ceftazidime and Sennoside A were investigated using [1H, 15N]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. Ceftazidime and Sennoside A bind to the putative RNA binding site of the N protein, thus providing insights into the inhibitory mechanism of these compounds. These findings will contribute to the development of novel antiviral agents targeting the N protein of SARS-CoV-2.

Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

INTRODUCTION: Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. OBJECTIVE: To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. METHODOLOGY: This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. RESULTS: When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 µg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. CONCLUSION: The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

Potential inhibitors of RPS6KB2 and NRF2 in head and neck squamous cell carcinoma.

Among the major altered pathways in head and neck squamous cell carcinoma, AKT/mTORC1/S6K and NRF2/KEAP1 pathway are quite significant. The overexpression and overstimulation of proteins from both these pathways makes them the promising candidates in cancer therapeutics. Inhibiting mTOR has been in research from past several decades but the tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms, encourages to explore other downstream targets for inhibiting the pathway. One such downstream effectors of mTOR is S6K2. It is reported to be overexpressed in cancers such as head and neck cancer, breast cancer and prostate cancer. In case of NRF2/KEAP1 pathway, nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2) is overexpressed in ∼90% of head and neck squamous cell carcinoma (HNSCC) cases. It associates with poor survival rate and therapeutic resistance in HNSCC treatment. NRF2 pathway is the primary antioxidant pathway in the cell which also serves pro-tumorigenic functions, such as repression of apoptosis, cell proliferation support and chemoresistance. The aim of this work was to explore S6K2 and NRF2 and identify novel and potential inhibitors against them for treating head and neck squamous cell carcinoma. Since the crystal structure of S6K2 was not available at the time of this study, we modelled its structure using homology modelling and performed high throughput screening, molecular dynamics simulations, free energy calculations and protein-ligand interaction studies to identify the inhibitors. We identified natural compounds Crocin and Gypenoside XVII against S6K2 and Chebulinic acid and Sennoside A against NRF2. This study provides a significant in-depth understanding of the two studied pathways and therefore can be used in the development of potential therapeutics against HNSCC.Communicated by Ramaswamy H. Sarma.

Dysregulated expression of slingshot protein phosphatase 1 (SSH1) disrupts circadian rhythm and WNT signaling associated to hepatocellular carcinoma pathogenesis.

Growing evidence underscores the circadian rhythm's essential function in liver stability and disease. Its disruption is progressively linked with metabolic issues, oncogene triggers, and heightened cancer susceptibility. Research points to slingshot protein phosphatase 1 (SSH1), a modulator of cofilin-1 (CFL-1), as instrumental in the reformation of the actin cytoskeleton, thereby impacting the invasiveness of various cancer types. Yet, the dynamics of SSH1's influence on liver cell stemness and circadian activity remain unclear. Through in-silico, tissue analysis, and functional assays, the study reveals a significant SSH1 expression in HCC samples, compared to non-cancerous counterparts, across six HCC platforms (AUC between 0.62 and 0.77, p < 0.01). The aberrant expression of SSH1 was correlated with poor patients' survival (HR = 1.70, p = 0.0063) and progression-free (HR = 1.477, p = 0.0187) survival rates. Targeting SSH1, either via Sennoside A or CRISPR SSH1 in Huh7 cells (Huh7-SSH1-/-) significantly suppressed cell viability, migration, invasion, colony and tumorsphere formation of the Huh7-SSH1-/- cells. Mechanistically, we showed that downregulated SSH1 expression suppressed CLOCK, BMAL1, WNT3, ß-catenin, LRP5/6, BCL2, VIM and Snail, with concomitant upregulated CFL-1/2, and CRY1 expression, indicating dysregulated circadian rhythm and WNT/ß-catenin oncogenic pathway deactivation. Treatments in reflected notable tumor size reductions in the mice treated with SenAlight (1.76-fold, p < 0.01) and SenAdark (3.79-fold, p < 0.01). The expression of SSH1, CLOCK, BMAL1 and ß-catenin proteins were significantly downregulated in the SenAlight and SenAdark mice; this was more so in the SenAdark mice. This reveals a potential treatment approach for HCC patients.

Sennoside A induces autophagic death of prostate cancer via inactivation of PI3K/AKT/mTOR axis.

Prostate cancer (PC) is the most common malignancy in male reproductive system. Sennoside A (SA) is an anthraquinone active ingredient extracted from Rheum officinale Baill., which exerts anti-tumor activity on different tumors. In the present study, the toxicity of SA on PC3 and DU 145 cells was detected via CCK-8. The effects of SA on growth, apoptosis, and autophagy were determined through CCK-8, Hoechst stain, flow cytometry, western blot, and immunofluorescence examinations. An in vivo experiment was performed in xenografted mice with intraperitoneal introduction of 10 mg/kg SA and validated via TUNEL, immunohistochemistry and western blot. The results showed that SA inhibited the cell viability with a IC50 value of 52.36 and 67.48 µM in DU 145 and PC3 cells respectively, and enhanced the apoptosis of PC3 and DU 145 cells. Additionally, SA elevated the relative LC3B expression, and the relative protein expression of LC3II/LC3I and Beclin-1, but diminished the P62 protein expression. The relative protein level of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR was reduced with SA treatment, which was verified by the 740 Y-P application. The 740 Y-P treatments also restored the SA-induced the cell viability, apoptosis rate and relative LC3B expression. Meanwhile, SA inhibited the growth of PC cell and the relative protein level of PI3K/AKT/mTOR axis in vivo. Taken together, SA regulated the proliferation, apoptosis and autophagy via inactivating the PI3K/AKT/mTOR axis in PC.

Comparison of the conventional and immersing powdered crude drugs (IPCD) methods for color and extraction of quantitative indicator ingredients in the Kampo formula decoction of daiokanzoto.

AIM: The immersing powdered crude drugs (IPCD) method is a quick and simple method for preparing decoctions. Here, the conventional and IPCD methods were compared for the color and extraction of quantitative indicator ingredients in the daiokanzoto decoction solution, and the suitability of the IPCD method was assessed. METHODS: The color of decoction solutions was visually observed, and the Commission Internationale de L'éclairage (CIE) L*a*b*color parameters were measured using conventional and IPCD methods. The extracted amounts of sennoside A and glycyrrhizic acid, which are quantitative indicator ingredients of rhubarb and glycyrrhiza, respectively, were quantified. RESULTS: Using both methods, the decoction solution colors were strong for rhubarb alone and daiokanzoto but weak for glycyrrhiza alone. The color change of daiokanzoto was thought to be primarily caused by rhubarb alone. The L*a*b* values of the decoction solution determined by the IPCD method were comparable to those determined by the conventional method (60 min). Using the conventional method, sennoside A and glycyrrhizic acid were mostly extracted in 10 and 30 min, respectively. Using the IPCD method, both sennoside A and glycyrrhizic acid were fully extracted in 2 min. The IPCD method yielded significantly more sennoside A and glycyrrhizic acid (2 times and 1.5 times, respectively) than the conventional method (60 min). CONCLUSION: The IPCD method was found to be comparable to the conventional method in terms of the color, and using IPCD method, the same or greater amounts of quantitative indicator ingredients of crude drugs in the decoction of daiokanzoto compared to the conventional method. It was suggested that there are limitations to assessing the equivalence of decoctions from decoction color. The IPCD method may be a useful method although it is prudent to use the IPCD method for Kampo formula decoction in clinical practice with a certain degree of caution.

Sennoside A restrains TRAF6 level to modulate ferroptosis, inflammation and cognitive impairment in aging mice with Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is a common neurodegenerative disease and a momentous cause of dementia in the elderly. Sennoside A (SA) is an anthraquinone compound and possesses decisive protective functions in various human diseases. The purpose of this research was to elucidate the protective effect of SA against AD and investigate its mechanism. METHODS: Male APPswe/PS1dE9 (APP/PS1) transgenic mice with a C57BL/6J background were chosen as AD model. Age-matched nontransgenic littermates (C57BL/6 mice) were negative controls. SA's functions in AD in vivo were estimated by cognitive function analysis, Western blot, hematoxylin-eosin staining, TUNEL staining, Nissl staining, detection of Fe2+ levels, glutathione and malondialdehyde contents, and quantitative real-time PCR. Also, SA's functions in AD in LPS-induced BV2 cells were examined using Cell Counting Kit-8 assay, flow cytometry, quantitative real-time PCR, Western blot, enzyme-linked immunosorbent assay, and analysis of reactive oxygen species levels. Meanwhile, SA's mechanisms in AD were assessed by several molecular experiments. RESULTS: Functionally, SA mitigated cognitive function, hippocampal neuronal apoptosis, ferroptosis, oxidative stress, and inflammation in AD mice. Furthermore, SA reduced BV2 cell apoptosis, ferroptosis, oxidative stress, and inflammation induced by LPS. Rescue assay revealed that SA abolished the high expressions of TRAF6 and p-P65 (NF-κB pathway-related proteins) induced by AD, and this impact was reversed after TRAF6 overexpression. Conversely, this impact was further enhanced after TRAF6 knockdown. CONCLUSIONS: SA relieved ferroptosis, inflammation and cognitive impairment in aging mice with AD through decreasing TRAF6.

Inhibitory effect of Bofutsushosan (Fangfengtongshengsan) extract on the absorption of fructose in rats and mice.

Bofutsushosan (BTS; fangfengtongshengsan in Chinese) is a formula in traditional Japanese Kampo and Chinese medicine comprising 18 crude drugs and used to treat obesity and metabolic syndrome. In our previous study, BTS boiling water extract inhibited the uptake of fructose absorbed via glucose transporter 5 into cultured cells. In this study, the inhibitory effect of BTS extract on the absorption of fructose from the intestine was investigated in vivo. The extract of BTS was orally administered to rats at doses equivalent to 25-fold of the daily dose for humans. One minute after sample administration, fructose was orally administered and blood samples were collected from the jugular vein 0.5, 1, 1.5, 2, and 4 h after the administration of fructose. The absorption of fructose from the intestine was significantly reduced by treatment with BTS extract, and this in vivo study reproduced previous in vitro results. Subsequently, the blood samples were collected from the portal vein 30 min after the oral administration of fructose in mice. BTS extract significantly reduced fructose absorption in mice, and compared the effect of modified BTS samples by removing one to several crude drugs from BTS. We found that the dried rhizome of Rheum palmatum (RR) significantly contributed to the inhibitory effect of BTS on fructose absorption. We found sennoside A to be the active ingredient of RR for the inhibition of fructose absorption, and that its effect almost saturated at a dose of 3 mg/kg. These results support the action mechanisms of BTS when used for the treatment of obesity in clinics and drug stores.

Time-dependent laxative effect of sennoside A, the core functional component of rhubarb, is attributed to gut microbiota and aquaporins.

ETHNOPHARMACOLOGICAL RELEVANCE: Sennoside A is a natural anthraquinone component mainly derived from rhubarb and has been routinely used as a clinical stimulant laxative. However, long-term application of sennoside A may lead to drug resistance and even adverse reactions, thus limiting its clinical use. Therefore, to reveal the time-dependent laxative effect and potential mechanism of sennoside A is of critical importance. AIM OF THE STUDY: This study was conducted to investigate the time-dependent laxative effect of sennoside A and unveil its underlying mechanism from the perspective of gut microbiota and aquaporins (AQPs). MATERIALS AND METHODS: Based on a mouse constipation model, 2.6 mg/kg sennoside A was administered orally for 1, 3, 7, 14 and 21 days, respectively. The laxative effect was assessed by the fecal index and fecal water content, the histopathology of the small intestine and colon was evaluated by hematoxylin-eosin staining. Gut microbiota changes was observed by 16S rDNA sequencing, and colonic AQPs expression was analyzed by quantitative real-time polymerase chain reaction and western blotting. Partial least-squares regression (PLSR) was used to screen out the effective indicators contributing to the laxative effect of sennoside A. The effective indicators were then fitted to time by a drug-time curve model to analyze the trend of efficacy of sennoside A, and the optimal time of administration was derived by comprehensive analysis with a three-dimensional (3D) time-effect image. RESULTS: Sennoside A had a significant laxative effect at 7 days of administration with no pathological changes in the small intestine or colon; however, at 14 or 21 days of administration, the laxative effect diminished and slight damage to the colon was observed. Sennoside A affects the structure and function of gut microbes. The alpha diversity showed that the abundance and diversity of gut microorganisms reached the highest value after 7 days of administration. Partial least squares discriminant analysis showed that the composition of the flora was close to normal when administered for less than 7 days, but was closest to the composition of constipation over 7 days. The expression of aquaporin 3 (AQP3) and aquaporin 7 (AQP7) decreased gradually after the administration of sennoside A, with the lowest expression at 7 days, and then increased gradually afterwards, while the expression of aquaporin 1 (AQP1) was the opposite. The PLSR results showed that AQP1, AQP3, Lactobacillus, Romboutsia, Akkermansia and UCG_005 contributed more to the laxative effect of the fecal index, and after fitting with the drug-time curve model, each index showed a trend of increasing and then decreasing. The comprehensive evaluation of the 3D time-effect image concluded that the laxative effect of sennoside A reached its best after 7 days of administration. CONCLUSION: Sennoside A should be used in regular dosages for less than one week, as it provides significant relief of constipation and exhibits no colonic damage within 7 days of administration. In addition, Sennoside A exerts its laxative effect by regulating gut microbiota of Lactobacillus Romboutsia, Akkermansia and UCG_005 and water channels of AQP1 and AQP3.

Sennoside A inhibits quorum sensing system to attenuate its regulated virulence and pathogenicity via targeting LasR in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen, and the emergence of drug resistance greatly increased the difficulty of treating its infection. Cell density-dependent quorum sensing (QS) system not only regulates the virulence but also associates with the drug resistance of P. aeruginosa. Screening for agents targeting QS to inhibit bacterial virulence and pathogenicity is considered a promising strategy to combat P. aeruginosa infection. In the present study, sennoside A was found to be able to inhibit the QS expression of P. aeruginosa at subinhibitory concentrations. The QS-regulated virulence factors, including protease, elastase, rhamnolipid, and pyocyanin, were also inhibited by sennoside A at both transcriptional and translational levels. Moreover, sennoside A could suppress the motility of twitching, swimming, and swarming as well as the biofilm formation, which is associated with the acute and chronic infections of P. aeruginosa in a dose-dependent manner. The attenuated pathogenicity of P. aeruginosa by sennoside A was further verified by Chinese cabbage, Drosophila melanogaster, and Caenorhabditis elegans infection analysis. Further study found that sennoside A might target the las system, mainly LasR, to interfere with QS. All the results indicate that sennoside A could inhibit the QS system to attenuate its regulated virulence and pathogenicity via mainly targeting LasR in P. aeruginosa and further research to identify its anti-QS activity for other Gram-negative bacteria is warranted.

Response Surface Methodology (RSM)-Based Optimization of Ultrasound-Assisted Extraction of Sennoside A, Sennoside B, Aloe-Emodin, Emodin, and Chrysophanol from Senna alexandrina (Aerial Parts): HPLC-UV and Antioxidant Analysis.

In this study, ultrasound-assisted extraction conditions were optimized to maximize the yields of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol from S. alexandrina (aerial parts). The three UAE factors, extraction temperature (S1), extraction time (S2), and liquid to solid ratio (S3), were optimized using response surface methodology (RSM). A Box-Behnken design was used for experimental design and phytoconstituent analysis was performed using high-performance liquid chromatography-UV. The optimal extraction conditions were found to be a 64.2 °C extraction temperature, 52.1 min extraction time, and 25.2 mL/g liquid to solid ratio. The experimental values of sennoside A, sennoside B, aloe-emodin, emodin, and chrysophanol (2.237, 12.792, 2.457, 0.261, and 1.529%, respectively) agreed with those predicted (2.152, 12.031, 2.331, 0.214, and 1.411%, respectively) by RSM models, thus demonstrating the appropriateness of the model used and the accomplishment of RSM in optimizing the extraction conditions. Excellent antioxidant properties were exhibited by S. alexandrina methanol extract obtained using the optimized extraction conditions with a DPPH assay (IC50 = 59.7 ± 1.93, µg/mL) and ABTS method (47.2 ± 1.40, µg/mL) compared to standard ascorbic acid.

Pharmacology, Toxicology, and Metabolism of Sennoside A, A Medicinal Plant-Derived Natural Compound.

Sennoside A (SA) is a natural dianthrone glycoside mainly from medicinal plants of Senna and Rhubarb, and used as a folk traditional irritant laxative and slimming health food. Accumulating evidences suggest that SA possesses numerous pharmacological properties, such as laxative, anti-obesity, hypoglycemic, hepatoprotective, anti-fibrotic, anti-inflammatory, anti-tumor, anti-bacterial, anti-fungal, anti-viral, and anti-neurodegenerative activities. These pharmacological effects lay the foundation for its potential application in treating a variety of diseases. However, numerous published studies suggest that a long-term use of SA in large doses may have some adverse effects, including the occurrence of melanosis coli and carcinogenesis of colon cancer, thereby limiting its clinical use. It remains to be established whether SA or its metabolites are responsible for the pharmacological and toxicity effects. In this review, the latest advances in the pharmacology, toxicology, and metabolism of SA were summarizedbased on its biological characteristics and mechanism.

Sennoside A alleviates inflammatory responses by inhibiting the hypermethylation of SOCS1 in CCl4-induced liver fibrosis.

Liver fibrosis is the consequence of chronic liver injury and is a major challenge to global health. However, successful therapy for liver fibrosis is still lacking. Sennoside A (SA), a commonly used clinical stimulant laxative, is reported to improve hepatic disease, but the underlying mechanisms remain largely elusive. Here, we show for the first time that SA enhanced suppressor of cytokine signaling 1 (SOCS1) expression in a DNA methyltransferase 1 (DNMT1)-dependent manner and thereby attenuated liver fibrosis. Consistently, SA inhibited the expression of the liver fibrogenesis markers α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) and suppressed inflammatory responses in vivo and in vitro. Coculture experiments with macrophages/hepatic stellate cells (HSCs) revealed that SA suppressed HSC proliferation by downregulating proinflammatory cytokines in macrophages. Mechanically, SA promoted the aberrant expression of SOCS1 in liver fibrosis. However, blocking SOCS1 expression weakened the inhibitory effect of SA on HSC proliferation, indicating that SOCS1 may play an important role in mediating the antifibrotic effect of SA. Furthermore, SA inhibited DNMT1-mediated SOCS1 and reduced HSC proliferation by inhibiting inflammatory responses in carbon tetrachloride (CCl4) -induced liver fibrosis.

Clubbed Digits Presumably Caused by Lubiprostone.

Digital clubbing has been regarded as an important sign in medicine. A 33-year-old woman with no history of hepatic, pulmonary, or malignant disease was referred to our hospital. She had been taking lubiprostone every day for three years for constipation. Clubbing in her upper and lower limb digits began gradually about two years ago. The results of laboratory investigations were almost normal. We suspected the clubbed digits were a side effect of lubiprostone and confirmed that the levels of urinary prostaglandin E2 (PGE2), which can cause clubbed digits, were elevated. Thus, we instructed the woman to stop taking lubiprostone and monitored this lab value. However, the value continued to rise over 2 months to 41.9 µg/g Cr. During that time, she had been taking sennoside A B calcium instead of lubiprostone for constipation. Since sennoside A B calcium also has the effect of increasing PGE2, we ordered the discontinuation. Her urinary PGE2 to creatinine level normalized, and the clubbing improved after the discontinuation of these two medications.

Organ Specific Differences in Alteration of Aquaporin Expression in Rats Treated with Sennoside A, Senna Anthraquinones and Rhubarb Anthraquinones.

Senna and rhubarb are often used as routine laxatives, but there are differences in mechanism of action and potential side effects. Here, we studied metabolites of senna anthraquinones (SAQ), rhubarb anthraquinones (RAQ) and their chemical marker, sennoside A (SA), in a rat diarrhea model. In in vitro biotransformation experiments, SAQ, RAQ and SA were incubated with rat fecal flora solution and the metabolites produced were analyzed using HPLC. In in vivo studies, the same compounds were investigated for purgation induction, with measurement of histopathology and Aqps gene expression in six organs. The results indicated that SAQ and RAQ had similar principal constituents but could be degraded into different metabolites. A similar profile of Aqps down-regulation for all compounds was seen in the colon, suggesting a similar mechanism of action for purgation. However, in the kidneys and livers of the diarrhea-rats, down-regulation of Aqps was found in the RAQ-rats whereas up-regulation of Aqps was seen in the SAQ-rats. Furthermore, the RAQ-rats showed lower Aqp2 protein expression in the kidneys, whilst the SA-rats and SAQ-rats had higher Aqp2 protein expression in the kidneys. This may have implications for side effects of SAQ or RAQ in patients with chronic kidney or liver diseases.

Inhibition behavior of Sennoside A and Sennoside C on amyloid fibrillation of human lysozyme and its possible mechanism.

Amyloid proteins were recognized as the crucial cause of many senile diseases. In this study, the inhibitory effects of Sennoside A (SA) and Sennoside C (SC) on amyloid fibrillation were evaluated by the combination of biophysical approaches and molecular docking tool using human lysozyme (HL) as amyloid-forming model. The results of thioflavin-T (ThT), 8-anilino-1-naphthalenesulfonic acid (ANS) and congo red (CR) assays indicated that both SA and SC could inhibit the amyloid fibrillation of HL in a dose-dependent manner. The IC50 value of SA and SC on HL fibrillation was 200.09 µM and 186.20 µM, respectively. These findings were further verified by transmission electron microscopy (TEM) and atomic force microscopy (AFM), which showed that the addition of SA or SC could sharply reduce the amyloid fibrillation of HL. Additionally, the interactions of HL with SA and SC were investigated by steady-state fluorescence spectra and molecular docking studies. The results suggested that both SA and SC could bind to the binding pocket of HL and form a stable complex mainly via hydrogen bonds, van-der-Waals forces and hydrophobic interactions. In conclusion, our experiments revealed that both SA and SC can significantly inhibit amyloid fibrillation of HL.

[Determination of sennoside B, sennoside A and physcion in slimming health foods].

OBJECTIVE: To establish a quantitative analysis method for sennoside A, sennoside B and physcion by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). METHODS: The sample was extracted by methanol-2 mmol/L ammonium formate(9∶1) at 40 ℃ for 1 h. The separation was performed using Agilent Eclipse Plus C_(18 )(2. 1 mm × 50 mm, 1. 8 µm) column with gradient elution. The mobile phase was consisted of 0. 1% formic acid and methanol. Qualitative and quantitative analysis was conducted with an electrospray ionization source operated in the negative ionization(ESI~-) mode and multiple reaction monitoring(MRM) mode. RESULTS: The linear range of three compounds were from 0. 1 to 10 µg/mL with the correlation coefficients(r) above 0. 995. The spiked recoveries were in the range of 81. 9% to 114. 5% at the concentrations of 0. 02, 0. 15 and 1. 60 mg/g with relative standard devisions(RSDs) ranged from 0. 30% to 3. 43%(n=6). The detection limits of sennoside A and sennoside B were 1. 2 µg/g. The detection limit of physcion was 2. 4 µg/g. Sennoside A, sennoside B or physcion were detected in 19 out of 40 batches of samples. The content of sennoside A ranged from 0. 184 to 6. 33 mg/g and the content of sennoside B ranged from 0. 202 to 7. 23 mg/g. The content of physcion ranged from 0. 042 to 0. 79 mg/g. CONCLUSION: The method is simple, accurate and suitable for the determination of sennoside A, sennoside B and physcion.

Sennoside A prevents liver fibrosis by binding DNMT1 and suppressing DNMT1-mediated PTEN hypermethylation in HSC activation and proliferation.

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.

Sennoside A drug capped biogenic fabrication of silver nanoparticles and their antibacterial and antifungal activities.

Sennoside A (dianthrone glycoside) shows laxative properties and used as a folk traditional medicine. Sennoside A capped silver nanoparticles (Ag/sennoside A) were synthesized at room temperature for the first time by using sennoside A as reducing and capping agent. UV-visible spectroscopic data reveals that the absorption peaks of pure sennoside A was appeared at 266, and 340 nm, which red shifted to 304, and 354 nm at higher sennoside A concentration. Upon addition of the Ag+ ions, an additional peak also observed at 398 nm, indicating the formation of spherical sennoside A capped silver nanoparticles (Ag/sennoside A). Cetyltrimethylammonium bromide (CTAB) was used a stabilizing agent to determine the role of cationic micelles on the nucleation and growth processes of Ag/sennoside A NPs formation. The 2,2-diphenyl-1-picrylhydrazyl nitrogen radical (DPPH · ), two bacteria strains (Staphylococcus aureus and Escherichia coli) and two yeast strains (Candida albicans ATCC 10231 and Candida parapsilosis ATCC 22019) were used to determine the antioxidant and antimicrobial properties of Ag/sennoside A NPs. In addition, Rhein-9-anthrone (4,5-dihydroxy-10-oxo-9H-anthracene-2-carboxylate) was isolated from the acidic hydrolysis of glycoside linkage of sennoside A and characterized. The antioxidant and antimicrobial activities of rhein-9-anthrone were also determined against DPPH radical, antibacterial and antifungal strains. The minimum inhibitory concentration was determined and discussed.

Sennoside A Induces GLP-1 Secretion Through Activation of the ERK1/2 Pathway in L-Cells.

PURPOSE: Glucagon-like peptide-1 (GLP-1) is secreted from the intestinal L-cells to stimulate insulin secretion in the blood glucose control. Our previous study indicates that Sennoside A (SA) can increase the plasma GLP-1 level in a mouse model of type 2 diabetes. However, the mechanism of SA activity remains largely unknown. This issue was explored in this study. MATERIALS AND METHODS: C57BL/6 mice were randomly divided into four groups: a control group without drug treatment, and the other groups with different SA dosages, respectively. Blood glucose was assayed by oral glucose tolerance test (OGTT). Plasma GLP-1 and insulin were investigated. Colon tissues were collected for mRNA or Western blot analysis. Immunofluorescence staining assays were performed to evaluate the number of ß-cells and L-cells. In NCI-H716 cells, extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors were employed to investigate the SA-induced GLP-1 secretion mechanism. RESULTS: In this work, the SA was found to improve OGTT in mice. Plasma GLP-1 and insulin were markedly elevated by SA at the dosage of 45 mg/kg/day. Meanwhile, the increased phosphorylation status of EKR1/2 and prohormone convertase 1/3 (PC1/3) proteins were observed in the colon of SA-treated mice. The number of L-cells exhibited no change in each group. In the NCI-H716 cells, GLP-1 secretion induced by SA was blocked by the ERK1/2 inhibitor. CONCLUSION: The present study provides a direct evidence for the interaction between SA and L cells for induction of GLP-1 secretion. These data suggest that GLP-1 secretion induced by SA is dependent on the ERK1/2 signaling pathway. Therefore, the SA is a new drug candidate for the type 2 diabetes treatment by induction of GLP-1 secretion.