Sinomenine inhibits the inflammatory responses of human fibroblast-like synoviocytes via the TLR4/MyD88/NF-κB signaling pathway in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder mainly characterized by inflammation of the synovial tissue that can lead to destruction of bone and cartilage. Sinomenine is an alkaloid extracted from the stem of the Chinese medicinal plant Sinomenium acutum. It has been reported that sinomenine has immunosuppressive and anti-inflammatory properties. However, the molecular mechanism underlying the effect of sinominine on IL-1ß-induced human RA fibroblast-like synoviocytes (RAFLS) is poorly understood. Therefore, in this study, we investigated the effect of sinomenine on the expression of inflammatory cytokines in IL-1ß-treated human RAFLS in vitro and the underlying mechanism. RAFLS viability was evaluated using the MTS assay after sinomenine treatment. The levels of inflammatory cytokines were measured with ELISA, RT-PCR and western blot, respectively. The levels of TLR4 and its downstream signaling targets were determined by western blot analysis. We found that sinomenine suppressed not only NO and PGE2 production but also iNOS and COX-2 expression in IL-1ß-induced RAFLS. It also inhibited the expression of TNF-α and IL-6 in IL-1ß-stimulated RAFLS. Furthermore, sinomenine prevented IL-1ß-induced TLR4, MyD88 and p-NF-κB p65 expression. Taken together, these results demonstrated that sinomenine prevented IL-1ß-induced inflammation in human RAFLS at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway, suggesting that sinomenine could be a potential agent in the treatment of RA.

A validated method for the simultaneous quantitation of bioactive alkaloid markers in the leaf ethanolic extract of Cissampelos sympodialis Eichl.: a phenological variation study.

INTRODUCTION: The leaf hydroalcoholic extract of Cissampelos sympodialis Eichl. (Menispermaceae) has shown promising activity in different animal models of asthma. Several alkaloids have been identified in the extract, including warifteine and methylwarifteine (bisbenzylisoquinoline), as well as milonine (morphinandienone). OBJECTIVE: To develop and validate an analytical method for the simultaneous quantitation of the bioactive markers of C. sympodialis hydroalcoholic leaf extract and to apply the method to a seasonal (phenological) study of the concentration of the alkaloid markers. METHODOLOGY: The method used reversed phase high performance liquid chromatography with UV detection and calibration by standard addition. Separation was achieved using a C18-column (250 × 4.6 mm, 5 µm) and a mobile phase consisting of a mixture of 0.05% aqueous (Et)3NH2 (A):MeOH(B) in gradient mode at a flow rate of 1.0 mL/min. RESULTS: The method proved to be linear in the concentration range tested (2-100 µg/mL, r² > 0,99), precise (RSD ≤ 15%), accurate (85-115%), selective and robust. Detection limits for warifteine, methyl-warifteine and milonine were 0.39, 1.10 and 1.77 µg/mL respectively. The highest concentration of total alkaloids (determined as the sum of the three alkaloids) in the hydroalcoholic extract of the leaves was 2.9 ± 0.2 mg/g extract (n = 3), prior to fruit development. Both warifteine and methylwarifteine were detected in the total alkaloid fraction of the ripened fruits. CONCLUSION: The results demonstrated that significant variations in the concentration of the biomarkers occurred throughout the vegetative cycle. The lowest concentration of the alkaloids in the leaves coincided with their appearance in the ripened fruits.

Two new morphinane alkaloids from Sinomenium acutum.

Two new morphinane alkaloids, 1-hydroxy-10-oxo-sinomenine (1) and 4,5-epoxy-14-hydroxy sinomenine N-oxide (2), have been isolated from the stems of Sinomenium acutum. Their structures were established by various spectral analyses, especially 2D NMR experiments. The structure of 2 was confirmed by single crystal X-ray diffraction. The absolute configurations of 1 and 2 were deduced by comparison of CD spectra with the known alkaloid sinomenine (3). Compound 1 was tested for DPPH inhibition and gave IC(50) of 27.9 µM. Compound 2 was tested for neuroprotective effect and showed significant activity against ß-amyloid(25-35)-induced oxidative injury (*P < 0.05) at 10 µM in PC-12 cells.

[Tracking the active component of Tebatan medicine Meconopsis quintuplinervia from Gansu].

OBJECTIVE: To study the active ingredients of analgesic and anti-inflammatory from Meconopsis quintuplinervia, Tracking the active ingredients of such medicines. METHODS: The compounds Meconopsis quintuplinervia,were separated with chromatography and its chemical structure was elucidated by means of MS, NMR spectroscopy methods respectively. RESULTS: Five compounds were obtaine as O-methylflavinantine (I), flavinantin (II), tricin (III), quercitrin (IV) and methyl linoleate (V). CONCLUSION: Compounds I is obtained from the plant for the first time.

Sinomenine Suppress the Vitamin D3 and High Fat Induced Atherosclerosis in Rats via Suppress of Oxidative Stress and Inflammation.

Atherosclerosis (AS) is a cardiovascular disease that arise due to dysfunction of lipid deposition and metabolism. AS is causes the mortality and morbidity worldwide. Sinomenine isolated from the Sinomenium acutum is used extensively against the various cardiac diseases in China. However, the anti-atherosclerosis effect of sinomenine still not explore. In this study, we explore the cardioprotective and anti-atherosclerosis effect of sinomenine against Vitamin D3 and High fat induced atherosclerosis in rats. Sprague Dawley (SD) rats were used in this study. The rats were received the vitamin D (60000) and High fat diet to induce the atherosclerosis and divided into groups and received the oral administration of sinomenine (2.5, 5 and 10 mg/kg) and simvastatin (5 mg/kg). Body weight, organ weight and biochemical parameters were estimated. The mRNA expression of MyD88, TLR4, NF-κB and IκB were estimated. Sinomenine treated rats significantly (p<0.001) suppressed the body weight and modulated the organ weight (hepatic, renal and heart). Sinomenine significantly (p<0.001) decreased the level of triacylglycerols (TG), low density lipoprotein cholesterol (LDL-c), total cholesterol (TC), very low-density lipoprotein cholesterol (VLDL-c) and augmented the level of high-density lipoprotein cholesterol (HDL-c). Sinomenine treatment also reduced the level of atherogenic index (TC/HDL-c and LDL-c/HDL-c). Sinomenine treatment decrease the ratio of HMG CoA/Mevalonate and level of collagen and total protein. Sinomenine significantly (p<0.001) altered the level of heart parameters, antioxidant parameters and inflammatory cytokines. Sinomenine significantly (p<0.001) reduced the expression of MyD88, TLR4, NF-κB and IκB. Taken together, sinomenine exhibited the protective effect against the atherosclerosis via alteration of TLR4/NF-κB signaling pathway.

[Study on the alkaloids in the stems and leaves of Stephania cepharantha (II)].

OBJECTIVE: To study the alkaloids in the stems and leaves of Stephania cepharantha Hayata. METHODS: The dried stems and leaves of Stephania cepharantha Hayata were percolated with 95% ethanol and the solvent was removed by rotary evaporation to give a concentrate, and the concentrate was extracted by petroleum ether and chloroform. Column chromatograghy on MCI CHP 20P, silica gel, Rp-18, Sephadex LH-20 and polyamide were applied for the isolation and purification of the chloroform fraction. The structures were elucidated by their physicochemical properties and spectral data. RESULTS: Five alkaloids were obtained and identified as, Stephasunoline (I) Aknadinine (II), Discretamine (III), Acutumine (IV), Sinomenine (V). CONCLUSION: Compounds I, III, IV are isolated from this plant for the first time, and compound IV is isolated from the genus for the first time.

Sample preparation and separation techniques for bioanalysis of morphine and related substances.

In present time the use or misuse of morphine and its derivatives are monitored by assaying the presence of the drug and its metabolites in biofluids. In the present review, focus is placed on the sample preparation and on the separation techniques used in the current best practices of bioanalysis of morphine and its major metabolites. However, as methods for testing the misuse of heroin, a morphine derivative, often involve bioanalytical methods that cover a number of other illicit drug substances, such methods are also included in the review. Furthermore, the review also includes bioanalysis in a broader perspective as analysis of plant materials, cell cultures and environmental samples. The review is not intended to cover all publications that include bioanalysis of morphine but is more to be considered a view into the current best practices of bioanalysis of morphine, its metabolites and other related substances.

A new morphinandienone alkaloid from the stems of Fissistigma tungfangense.

A new morphinandienone alkaloid, fissistigmine A (1), together with three known alkaloids (2-4), were isolated and identified from the stems of Fissistigma tungfangense. Among them, fissistigmine A (1) represents the first example of a novel naturally occurring morphinandienone alkaloid with a unique cleavage of the C-9-N-17 bond. All isolated compounds were evaluated for their anti-rheumatoid arthritis activities via examining their anti-proliferative effects on synoviocytes in vitro. Compound 1 exhibited inhibitory effect on the proliferation of synoviocytes with an IC50 value of 114.6 ± 2.2 µM.

Isolation and antimalarial activity of new morphinan alkaloids on Plasmodium yoelii liver stage.

Decoction of Strychnopsis thouarsii is used in the Malagasy traditional medicine to combat malaria. We have shown that this traditional remedy prevents malaria infection by targeting Plasmodium at its early liver stage. Bioassay-guided fractionation of S. thouarsii stem barks extracts, using a rodent Plasmodium yoelii liver stage parasites inhibition assay, led to isolate the new morphinan alkaloid tazopsine (1) together with sinococuline (2) and two other new related morphinan analogs, 10-epi-tazopsine (3) and 10-epi-tazoside (4). Structures were characterized by 2D NMR, MS, and CD spectral analysis. Compounds 1-3 were found to fully inhibit the rodent P. yoelii liver stage parasites in vitro.

Alkaloids from Papaver nudicaule L.

A new promorphinane alkaloid (-)-8,14-dihydroflavinantine was isolated from the aerial parts of Papaver nudicaule L. (Papaveraceae) growing in Mongolia. Six known isoquinoline alkaloids (+)-amuronine, pseudoprotopine, allocryptopine, (-)-dihydroamuronine, (-)-amurensinine N-oxide A and (-)-amurensinine N-oxide B were isolated, too. Pseudoprotopine has been found for the first time in a plant of the family Papaveraceae. (-)-dihydroamuronine, (-)-amurensinine N-oxide A and (-)-amurensinine N-oxide B are new for the genus Papaver. All structures were established using spectral and physical data.

A new 8,14-dihydropromorphinane alkaloid from Papaver nudicaule L.

A racem. 8,14-dihydroamurine is a new promorphinane alkaloid isolated from the aerial parts of Papaver nudicaule L. (Papaveraceae) of Mongolian origin. The known promorphinane and isopavine alkaloids (+)-amurine, (-)-amurensinine, (-)-O-methylthalisopavine, (-)-flavinantine and (-)-amurensine were also described. All structures were established by physical and spectral analysis. Flavinantine has been found for the first time in the species.

[Preparation of sinomenine microemulsion and its transdermal absorption].

OBJECTIVE: To screen the optimum formulation and prepare O/W sinomenine microemulsion and investigate its in vitro transdermal delivery ability. METHOD: The microemulsions were prepared with the formulation containing oleic acid-tween 80-dehydrated alcohol-water by the pseudo-ternary phase diagram. The permeation flux of sinomenine was determined in vitro by Franz diffusion cell fitted with rat skin. The sinomenine was determined by HPLC. The transdermal characteristics of sinomenine microemulsion were compared with that of sinomenine gels. RESULT: The steady state flux of sinomenine microemulsion was significantly higher than that of sinomenine gels. The average permeation rate of sinomenine microemulsion was 116. 44 microg x cm(-2) x h(-1) in vitro. CONCLUSION: These results indicated that the studied microemulsion system with high permeation rate may be a potential vehicle for the transdermal delivery of sinomenine.

Suppressing mPGES-1 expression by sinomenine ameliorates inflammation and arthritis.

Recently, microsomal prostaglandin E synthase 1 (mPGES-1) has attracted much attention from pharmacologists as a promising strategy and an attractive target for treating various types of diseases including rheumatoid arthritis (RA), which could preserve the anti-inflammatory effect while reducing the adverse effects often occur during administration of non-steroidal anti-inflammatory drugs (NSAIDs). Here, we report that sinomenine (SIN) decreased prostaglandin (PG)E2 levels without affecting prostacyclin (PG)I2 and thromboxane (TX)A2 synthesis via selective inhibiting mPGES-1 expression, a possible reason of low risk of cardiovascular event compared with NSAIDs. In addition, mPGES-1 protein expression was down-regulated by SIN treatment in the inflamed paw tissues both in carrageenan-induced edema model in rats and the collagen-II induced arthritis (CIA) model in DBA mice. More interestingly, SIN suppressed the last step of mPGES-1 gene expression by decreasing the DNA binding ability of NF-κB, paving a new way for drug discovery.

[Determination in vitro of rat plasma protein binding rate of sinomenine by using microdialysis method].

AIM: To determine in vitro the rat plasma protein binding rate by using microdialysis method. METHODS: The binding rate was determined by using microdialysis probe as sampling tools and zero-net flux method as calibrating method. The regression equation was made by the difference of concentrations between the dialysis sample and the perfusate. The x-intercept of regression equation was the free drug concentration (Cf). The plasma protein binding rate was calculated by using the following equation: f = ( C0 - Cf)/C0. RESULT: The binding rate was kept relatively stable in the studied concentration range. CONCLUSION: It is feasible that the plasma protein binding rate can be determined by using microdialysis method.

[Study on determination of entrapment efficiency of sinomenine liposomes].

OBJECTIVE: To establish an HPLC method for the determination of entrapment efficiency of sinomenine liposomes. METHOD: The liposomes and dissociated drugs were separated by sephadex filtration, mini-column centrifugation and dialysis. The methodology study and the optimization of determining condition were carried out at the same time. RESULT: Sephadex filtration could effectively separate the sinomenine liposomes from dissociated sinomenine. The column recovery was 98.8%, the average entrapment efficiency of three tests was64.9%, RSD 2.67%. CONCLUSION: The method was simple, exact, and had a good reappearance. It can be used to examine the entrapment efficiency of sinomenine liposomes.

Access to a Structurally Complex Compound Collection via Ring Distortion of the Alkaloid Sinomenine.

Many compound collections used in high-throughput screening are composed of members whose structural complexity is considerably lower than that of natural products. We previously reported a strategy for the synthesis of complex and diverse small molecules from natural products using ring-distortion reactions, called complexity-to-diversity (CtD), and herein, CtD is applied in the synthesis of 16 diverse scaffolds and 65 total compounds from the alkaloid natural product sinomenine. Chemoinformatic analysis shows that these compounds possess complex ring systems and marked three-dimensionality.

Supercritical fluid extraction of sinomenine from Sinomenium acutum (Thumb) Rehd et Wils.

Supercritical carbon dioxide, with and without a methanol modifier, was used to extract sinomenine from Sinomenium acutum (Thumb) Rehd et Wils. Sinomenine determinations were carried out using high-performance liquid chromatography (HPLC). The results show that the yield obtained after 2.5 h extraction with methanol-modified supercritical carbon dioxide was the highest (7.47 mg/g), while that obtained with only supercritical carbon dioxide was the lowest (0.17 mg/g). The recovery obtained with supercritical carbon dioxide, with and without a methanol modifier, could not be increased greatly by the method of the alkalinization of sample. Higher recoveries were obtained than extraction using methanol in Soxhlet extractor.

Automated multiple development thin-layer chromatography for separation of opiate alkaloids and derivatives.

There are three types of opiate alkaloids. First, the poppy alkaloids: morphine, codeine, thebaine, noscapine and papaverine; then, the semi-synthetic and synthetic derivatives used in therapy as antitussives and analgesics, such as pholcodine, ethylmorphine and dextromethorphan; at last narcotic compounds, diacetylmorphine (heroin) and opiates employed as substitutes in treatment of addiction: buprenorphine and methadone. For classical thin-layer chromatography (TLC) of opium alkaloids, it is necessary to use complex eluents with strong alkaline substances to obtain a clean separation between morphinan and isoquinoline compounds. This study purposes the planar chromatographic analysis of these substances by the automated multiple development (AMD) compared with results obtained by classical TLC method. The aim of this work was to achieve the best separation of these opiate alkaloids and derivatives by this modern technique of planar chromatography. The AMD system provided a clean separation for each of three opiates groups studied and the best results have been obtained with universal gradient: methanol 100, methanol-dichloromethane 50/50, dichloromethane 100, dichloromethane 100, hexane 100 for opium alkaloids and with gradient A: 5% of 28% ammonia in methanol 100, acetone 100, acetone 100, ethyl acetate-dichloromethane 50/50, dichloromethane 100 for antitussives and substitutes. Two reagents were used for the detection of alkaloids by spraying: Dragendorff and iodoplatinate reagents. The detection limits with these two reagents were 1 microg for ethylmorphine, thebaine, papaverine, codeine, and 2 microg for morphine and noscapine and other alkaloids.

[Immunopharmacological action of sinomenine, an alkaloid isolated from Sinomenium acutum, and its mechanism of action in treating rheumatoid arthritis].

AIM: To observe the effects of sinomenine on the immune functions and apoptosis of murine lymphocyte as well as on human synovial fibroblast proliferation. METHODS: Both in vivo and in vitro tests were adopted. The lymphocyte proliferation induced by mitogens was assayed by MTT method. Spleen T lymphocyte subtypes were tested with flow cytometry. Spleen lymphocyte apoptosis was analyzed by flow cytometry and DNA ladder methods. In vitro test was adopted to observe the effects of sinomenine on the proliferation of human fibroblast of rheumatoid arthritis. RESULTS: Sinomenine can inhibit the proliferation of mouse lymphocytes induced by ConA, LPS and anti-CD3 mAb but not PMA in vitro, and inhibit the proliferation induced by LPS and PMA in vivo. Sinomenine can reduce up-regulated CD4+/CD8+ ratio of T lymphocyte subtype in adjuvant arthritis rat. At the same concentration increased apoptosis ratio. As to human synovial fibroblast, sinomenine can significantly inhibit proliferation of human fibroblast. CONCLUSION: Sinomenine can inhibit the immunological function and correct imbalance of CD4+/CD8+ ratio of T lymphocyte subtype. It can also increase apoptosis ratio of spleen lymphocyte. This may be the mechanism of its immunological inhibitory effect.

[New study progress of sinomenine].

To further understand sinomenine, this paper has introduced the abstract technology, assaying, pharmaceutical chemistry, pharmacological action, pharmacotoxicology, pharmacokinetics and clinical application of sinomenine based on the important and significant contents of reference which have been consulted in the past ten years. Sinomenine is a kind of non-steroidal anti-inflammatory drugs with very effective and little side effect and expected it as a good new drug withdrawal medicine in the future.