Swertiamarin attenuates paraquat-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Abstract The aim of the present study was to investigate the effect of swertiamarin (STM) in attenuating paraquat (PQ)-induced human lung alveolar epithelial-like cell (A549) apoptosis and the underlying mechanisms. A549 cells were pretreated with different concentrations of STM for 2 hr and then cultured with or without PQ (700 µM) for 24 hr. Cell survival was determined using the CCK8 assay. Morphological changes, MDA content, inflammatory factors, fibrogenesis parameters, apoptosis rates, redox status and mitochondrial membrane potential (MMP) were evaluated. The expression of several genes involved in the modulation of redox status was measured by Western blotting. Cell viability and MMP were decreased, but the apoptosis rate and DCFH oxidation were elevated by PQ exposure. STM pretreatment notably increased cell viability and MMP and reduced the apoptosis rate and DCFH oxidation. Furthermore, TLR4- NOX4 signaling was significantly inhibited by STM. The downregulation of NOX4 by siRNA exerted the same protective effects as STM. This study provides the first evidence that STM attenuates PQ-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Effect of swertiamarin, gentiopicrin and sweroside on cell apoptosis and expression of Bcl-2 in rheumatoid arthritis fibroblast-like synoviocytes / 中国中药杂志

The aim of this paper was to discuss the effect of swertiamarin, gentiopicrin and sweroside on rheumatoid arthritis fibroblast-like synoviocytes(RA-FLSs) and B-cell lymphoma-2(Bcl-2) and their mechanisms. ZINC database and RCSB PDB database were retrieved for 3 D chemical structures of swertiamarin, gentiopicrin and sweroside and 3 D target protein structures. AutoDock Mgltools 1.5.6, AutoDockVina 1.1.2 and pyMOL 2.2.0 were applied for molecular docking to analyze the relationship between Bcl-2(1 GJH) target protein and important ingredients. The cell apoptosis of RA-FLSs was tested by Annexin V-FITC. The Bcl-2 protein expression of RA-FLSs treated with different ingredients was tested by Western blot. The Bcl-2 mRNA expression of RA-FLSs treated with different ingredients was tested by RT-PCR. Swertiamarin, gentiopicrin and sweroside were docked well with Bcl-2(1 GJH). The binding energy of swertiamarin was-6.9 kcal·mol~(-1), the binding energy of gentiopicrin was-6.7 kcal·mol~(-1) and the binding energy of sweroside was-6.4 kcal·mol~(-1). Compared with the blank group, the Bcl-2 protein expression of each group were reduced, while that of the gentiopicrin group was the highest(P<0.01). Compared with the blank group, the Bcl-2 mRNA expression of each groups were reduced. Gentiopicrin can reduce the Bcl-2 protein expression and the Bcl-2 mRNA expression, so as to promote the RA-FLSs apoptosis.

Inhibitory Effect of Swertiamarin on MGC803 Cells in vitro and in vivo / 胃肠病学

Background: Gastric cancer is one of the most commonly seen malignant tumors. Inhibiting the proliferation of gastric cancer cells is the key for the development of anticancer drugs. Aims: To investigate the effect of swertiamarin on proliferation of MGC803 cells in vitro and in vivo. Methods: MGC803 cells were cultured with different concentrations of swertiamarin (0, 30, 60 and 90 μg/mL) for 48 hours. The cell activity were detected by MTT. The apoptosis rate was measured by Annexin V-FITC/PI assay. The contents of Bax and Bcl-2 were detected by ELISA. MGC803 cell xenograft tumor in BALB/c nude mice was established, and different concentrations of swertiamarin (0, 40, 60 and 80 mg/kg) were injected through caudal vein. Nude mice were killed when the volume of tumor was 1 000 mm3. The initial and final body weights of nude mice were recorded. The protein expression of Ki-67 was detected by immunohistochemistry. Results: Compared with control group, different concentrations of swertiamarin effectively inhibited the proliferation of MGC803 cells, induced cell apoptosis, increased the content of Bax, while the content of Bcl-2 was significantly decreased (P<0.05). In BALB/c nude mice with MGC803 cell xenograft tumor, the volume of tumor, protein expression of Ki-67 decreased significantly in groups injected with different concentrations of swertiamarin than in control group (P<0.05). Conclusions: Swertiamarin is a safe and effective anti-gastric tumor plant agent.

HPLC fingerprint of Lomatogonium rotatum / 中草药

Objective To study the fingerprints of 15 batches of Mongolian medicinal materials of Lomatogonium rotatum collected from different places by HPLC, and to evaluate the quality of the medicinal materials of L. rotatum by similarity calculation. Methods A total of 15 batches of ribbed flowers were collected by HPLC. Chromatographic column: YMC C18 column (250 mm × 4.6 mm, 5 μm), mobile phase: acetonitrile-0.4% phosphoric acid-methanol, gradient elution, flow rate was 0.8 mL/min, detection wavelength was 254 nm, column temperature was 30 ℃. The similarity of fingerprints was evaluated by using the “Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine 2004A Edition”. Results The fingerprints of L. rotatum of Mongolian medicine were established, 15 common peaks were identified, five common peaks were identified, and the similarity among 15 batches of L. rotatum and the fingerprints of control was in the range of 0.881—0.997. The fingerprints of erect column flowers have good precision, stability and reproducibility. Conclusion The characteristic fingerprint of L. rotatum was established for the first time, which not only provides a new scientific basis for the identification and quality control of L. rotatum, but also has important significance for the quality evaluation of L. rotatum.

A new secoiridoid glycoside from Comastoma traillianum / 中草药

Objective To research the chemical constituents in Comastoma traillianum. Methods The chemical constituents from C. traillianum were isolated and purified by column chromatographic methods of silica gel, RP-HPLC, Sephadex LH-20, and RP-C18. Their structures were elucidated by modern spectroscopy methods, including 1D and 2D NMR, HRESIMS, IR, and UV techniques. The anti-inflammatory activity of compounds against LPS and IFN-γ-induced mouse RAW 264.7 macrophages was determined by Griess, and the IC50 value was calculated to evaluate their anti-inflammatory activity. Results Four secoiridoid glycosides (1-4) were isolated from C. traillianum, and identified as comtraide A (1), deacetylcentapicrin (2), swertiamarin (3), and gentiopieroside (4). Compound 1 is a new compound named comtraide A. All compounds were tested for their in vitro anti-inflammatory activity and no activity was found. Conclusion Compounds 1-4 are isolated for the first time from C. traillianum, and compound 1 is a new compound. However, no significant anti-inflammatory activities were detected in all these compounds at concentrations up to 100 μmol/L.

Swertiamarin ameliorates carbon tetrachloride-induced hepatic apoptosis via blocking the PI3K/Akt pathway in rats

Swertiamarin (STM) is an iridoid compound that is present in the Gentianaceae swertia genus. Here we investigated antiapoptotic effects of STM on carbon tetrachloride (CCl₄)-induced liver injury and its possible mechanisms. Adult male Sprague Dawley rats were randomly divided into a control group, an STM 200 mg/kg group, a CCl₄ group, a CCl₄+STM 100 mg/kg group, and a CCl₄+STM 200 mg/kg group. Rats in experimental groups were subcutaneously injected with 40% CCl₄ twice weekly for 8 weeks. STM (100 and 200 mg/kg per day) was orally given to experimental rats by gavage for 8 consecutive weeks. Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins were evaluated by western blot analysis. The expression of TGF-β1, collagen I, collagen III, CTGF and fibronectin mRNA were estimated by qRT-PCR. The results showed that STM significantly reduced the number of TUNEL-positive cells compared with the CCl₄ group. The levels of Bax and cleaved caspase-3 proteins, and TGF-β1, collagen I, collagen III, CTGF, and fibronectin mRNA were significantly reduced by STM compared with the CCl₄ group. In addition, STM markedly abrogated the repression of Bcl-2 by CCl₄. STM also attenuated the activation of the PI3K/Akt pathway in the liver. These results suggested that STM ameliorated CCl₄-induced hepatocyte apoptosis in rats.

Swertiamarin attenuates carbon tetrachloride (CCl4)-induced liver injury and inflammation in rats by regulating the TLR4 signaling pathway

The aim of the present study is to illustrate the effects of swertiamarin (STM), a natural iridoid from herbal medicines, on hepatic inflammation induced by carbon tetrachloride (CCl4) in rats. Male Sprague Dawley rats were exposed to CCl4 with or without STM co-administration for 8 weeks. Our results revealed that STM administration (100 and 200 mg/kg b.w.) significantly attenuated inflammation in livers of CCl4-treated rats. STM remarkably reduced the production of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-1a (MIP-1α), and monocyte chemotactic protein-1 (MCP-1) in liver tissue of CCl4-treated rats. In addition, STM treatment downregulated connective tissue growth factor (CTGF) and ser307pIRS-1 expression, which was induced by CCl4 exposure. In the process of exploring the anti-inflammatory mechanisms of STM action, we demonstrated that STM significantly inhibited Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NF-κB) p65 expression in the liver. In conclusion, these results suggested that the inhibition of CCl4-induced inflammation by STM was, at least in part, due to its regulation of the TLR4 /NF-κB signaling pathway

UPLC fingerprint for three species of folk medicine in Swertia L / 中草药

Objective: To accelerate the development of nation medicine, ultra-high performance liquid chromatography (UPLC) combined with content analysis of index compound, cluster analysis (HCA), and principal component analysis (PCA) was used to evaluate and discriminate three kinds of national folk medicinal plants, Swertia davidi, S. punicea, and S. angustifolia. Methods: The chromatograms of 27 samples from three kinds of Swertia L. were collected. The chromatograms were imported in Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica 2004A to obtain data retention time and peak area of samples. Three sets of samples of similarity were analyzed. The peak area data were imported in SPSS and SIMCA-P+ software for cluster analysis and principal component analysis, dendrogram and principal component scores were obtained, and the clustering effect was observed. Results: The line relationship of this way was good (R2 > 0.9993), with high precision instrument (RSD of 0.19%-2.45%), the method had good reproducibility (RSD of 0-1.77%), recovery was between 95.8% and 102.3% (RSD of 0.45%-2.81%). Three kinds of Swertia L. medicinal plants were different in the contents of index compounds, and the contents of index compounds in the same species were different. Swertiamarin: S. davidi (0.54-10.05 mg/g), S. angustifolia (undetectable), S. punicea (1.76-15.62 mg/g); gentiopicroside: S. davidi (4.27-11.18 mg/g), S. angustifolia (0.26-3.58 mg/g), S. punicea (1.13-16.11 mg/g); sweroside: S. davidi (0.02-0.28 mg/g), S. angustifolia (0.02-0.14 mg/g), S. punicea (0.11-44.52 mg/g); mangiferin: S. davidi (0.14-0.55 mg/g), S. angustifolia (0.03-0.04 mg/g), S. punicea (0.01-13.49 mg/g). The accuracy of dendrogram classification was 85.2%. The contents of index compounds of three false anomalies were less than the remaining nine samples of S. punicea. The effect of discrimination by PCA scores plot of three Swertia L. plants was good, and S. punicea with dispersive distribution indicated that the individuals were significant difference. Conclusion: The method to evaluate and discriminate three Swertia L. medicinal plants is feasible by UPLC combined with analysis of index compound HCA and PCA. In this article, S. punicea is more valuable compared S. davidi and S. angustifolia.

Anti-mutagenicity of Swertiamarin and Its Metabolite in Incubated System of Human Intestinal Flora / 中草药·英文版

Objective To study the biotransformation regulation and pharmacological effect of swertiamarin and its metabolite in incubated system of human intestinal flora. Methods Incubated system of human intestinal flora was utilized to research the intestinal metabolism of swertiamarin. Furthermore, mutagenic test and anti-mutagenic test were carried out to research the activity relationship of swertiamarin and its metabolite. Results Gentianine was found in the metabolites of swertiamarin. The pharmacological experiment indicated that swertiamarin and its metabolite both had good anti-mutagenic effect. Conclusion Swertiamarin is partly metabolized to gentianine after oral administration. They show similar anti-mutagenicity effects.

Contents of seven indicative constituents in seven kinds of Gentiana macrophylla by UPLC / 中草药

Objective: An ultra performance liquid chromatographic (UPLC) method was developed for simultaneously determining seven constituents, such as loganic acid, swertiamarin, 6'-O-β-D-glucosyl gentiopicroside, gentiopicroside, sweroside, isoorientin, and isovitexin, from the medicinal plants of Gentiana macrophylla. Methods: The separation was performed on an Acquity UPLC® BEH C18 column (50 mm ×2.1 mm, 1.7 μm) through a gradient elution of methanol-0.04% aqueous phosphorite at a flow rate of 0.3 mL/min. The detection wavelength was 242 nm, and the column temperature was set at 30℃. Results: For the seven analytes, loganic acid, swertiamarin, 6'-O-β-D-glucosyl gentiopicroside, gentiopicroside, sweroside, isoorientin, and sovitexin, a good linearity (r≥0.9995) was obtained in the range of 2.100-537.100, 1.050-270.000, 0.920-236.000, 11.100-2830.000, 0.750-192.000, 0.167-102.000, and 0.216-52.800 μg, respectively. Their average recoveries (n=6) were 97.83%-100.08%, respevtively, with RSD values less than or equal to 3.76%. Conclusion: The UPLC method is simpler and more effective than HPLC, and can be used for the simultaneous determination of seven indicative constituents in medicinal plants of G. macrophylla.

Improved RP-HPLC Method for Quantitation of Swertiamarin from Swertia Densifolia Leaves and Marketed Herbal Formulations.

Objective: Present study aims at developing and validating an improved RP-HPLC method for determination of Swertiamarin from extracts of Swertia densifolia (Griseb.) Kashyap leaves and marketed herbal formulations. Methods: Developed RP-HPLC method is validated and is used for determination of Swertiamarin from hydroalcoholic extracts of test plant material and marketed herbal formulations. The separation is carried out on C18 column. The mobile phase used is 10 mM Ammonium acetate: Acetonitrile: Formic acid in ratio 90:10:0.2% (v/v/v/). The wavelength selected is 237 nm using UV detector. Results and Discussion: A seven-point calibration curve over the concentration range of 50.00 –20000.00 ng/mL for Swertiamarin provides an optimum linear detector response (with r2>0.9970). The % R.S.D. (n = 6) for the low, middle and high quality control samples are within <6.44 and < 9.04 for inter-day and intra-day precision and accuracy respectively. Conclusion: A simple, sensitive and precise method is developed for quantitation of SWM. Method is validated as per International Conference on Harmonization (ICH) guidelines and its applicability is demonstrated by successful measurement of Swertiamarin from hydroalcoholic extracts of Swertia densifolia leaves and selected marketed herbal formulations.

Chemical constituents from swertia cincta burk. and their antagonistic effects on benign prostatic hyperplasia / 中国药学杂志

OBJECTIVE: To study the chemical constituents extracted from the whole plants of Swertia cincta Burk. and their antagonistic effects on benign prostatic hyperplasia (BPH). METHODS: The compounds were isolated by various chromatographic methods including silica gel, reverse silica gel, RP-HPLC, Sephadex LH-20, preparative TLC, and so on. Their structures were i-dentified by extensive analysis of the spectroscopic data. Experimental prostatic hyperplasia was produced in male Sprague-Dawley (SD) rats by testosterone injection for 28d, and then the prostate index(PI) was measured. RESULTS: Eleven compounds were isolated from the whole plants of Swertia cincta Burk. and identified as swertiamarin(1), (R)-(-)-gentiopicroside(2), sweroside(3), 6'-O-β-D-glucopyranosylsweroside(4), 6'-O-β-Z)-glucopyranosylgentiopicroside(5), loganic acid(6), oleanolic acid(7), 2α, 3β-dihydroxyolean-12-en-28-oicacid-28-O-β-D-glucopyranoside (8), β-daucosterol (9), 4-hydroxyl-3, 5-dimethoxyl-6-O-β-D-glu-cosebenzene(10), and isoorientin (11). Swertiamarin significantly decreased PI in SD rats. CONCLUSION: Compounds 5 and 8 were isolated irom the plants of Swertia cincta for the first time. Compound 1 has favorable effect for inhibiting prostatic hyperplasia in SD rats.

Determination of seven components in flowers of Gentiana tibetica and Gentiana dendrologi by HPLC / 中草药

Objective: To establish an HPLC method for simultaneously determining seven components, such as loganic acid, 6′-O-β-D-glucopyranosyl gentiopicroside, swertiamarine, gentiopicroside, swertiamarin, isoorientin, and isovitexin, from the flowers of Gentiana tibetica and G. dendrology. Methods: Chromatographic analysis was achieved on an Agilent Zorbax ODS column (150 mm × 4.6 mm, 5 μm) by gradient elution of acetonitrile-0.4% acetic acid in water at 30℃. The flow rate was 0.4 mL/min and the detection wavelength was 254 nm. Results: The calibration curves of all the seven constituents showed good linearity in a relatively wide concentration range. The linear ranges of loganic acid, 6′-O-β-D-glucopyranosyl gentiopicroside, swertiamarine, gentiopicroside, gentiopicroside, isoorientin, and isovitexin were 0.228-2.280, 0.680-6.800, 0.220-2.20, 1.476-14.760, 0.200-2.000, 0.436-4.360, and 0.276-2.760 μg (R2 ≥ 0.999 2), respectively. The recoveries (n = 9) of loganic acid, 6′-O-β-D-glucopyranosyl gentiopicroside, swertiamarine, gentiopicroside, gentiopicroside, isoorientin, and isovitexin were 100.9%, 99.4%, 101.0%, 105.7%, 103.1%, 98.4%, 104.2%. Conclusion: This method is simple, accurate, and specific, and can be used for the determination of seven constituents in the flowers of G. tibetica and G. dendrologi.

HPLC Determination of Swertiamarin in Gantaishu Capsules / 中国中医药信息杂志

Objective To establish an HPLC method for determining the content of swertiamarin in Gantaishu capsules. Methods Using a Waters Symmetry C18 column (4.6 mm×250 mm, 5μm), the mobile phase was acetonitrile-0.02%phosphoric acid, with flow rate of 1.0 mL/min. The detection wavelength was at 250 nm, the column temperature was room temperature. Results The content of swertiamarin showed a good linear relationship in the range of 0.373 556-2.988 448 μg, r 2=1.000 0. The average recovery was 106.81%, and RSD was 3.7%. Conclusion This method is fast, accurate and simple with good reproducibility, which can be applied to the quality control of Gantaishu capsules.

Chemical constituents of Swertia macrosperma / 中草药

Objective: To study the chemical constituents from Swertia macrosperma. Methods: HPLC, column chromatography, and recrystallization techniques were used for the separation and purification of the compounds. Their structures were elucidated by physiochemical properties and spectral analyses. The inhibitory activity against tobacco mosaic virus (TMV) was screened by half-leaf blight spot assay. Results: Eight compounds were isolated from S. macrosperma and their structures were identified as 9, 10-dihydroxyl-sweroside (1), 3'-O-(3-hydroxybenzoyl)-swertiamarin (2), mangiferin (3), 1-O-β-glucopyranosyl-2, 6, 8-trihydroxyl-xanthone (4), campestroside (5), bellidifolin (6), 4, 4'-dihydroxy-Z-stilbene (7), and 4, 4'-dihydroxy-E-stilbene (8). Conclusion: Compound 1 is a new compound named macrospermaoside A, and exhibits weak anti-TMV activity. Compounds 2, 7, and 8 are isolated from the plants of this genus for the first time.

Biotransformation optimization of swertiamarin by Aspergillus niger / 中草药

Objective: With swertiamarin as the substrate, biotransformation conditions of swertiamarin were optimized by Aspergillus niger. Methods: With productivity ratio of EHPO {(2)-5-ethylidene-8-hydroxy-3,4,5,6,7,8-hexahydro-1H-pyrano [3,4-c] pyridine-1-one} and transformation ratio of swertiamarin as indexes, training time, carbon resource, nitrogen resource, metal ions, phosphate, growth factor (yeast extract paste), inoculum size, substrate concentration, initial pH value, and tempuraure were evaluated to optimize the biotransformation of swertiamarin. Results: It confirmed the optimization cultivating condition that glucose concentration was 10 g/L, peptone concentration was 5 g/L, yeast extract paste was 5 g/L, KH2PO4 concentration was 5 g/L, MgSO4 and CaCl2 concentration was 1 g/L, initial pH value was 6.0, inoculum size was 0.5%, substrate concentration was 1 mg/mL, cultivation temperature was 28 °C, and cultivating time was 5 d. Conclusion: The biotransformation ratio for EHPO into swertiamarin biotransformed by A. niger could steadily be about 8%.

Evaluation of effect of aqueous extract of Enicostemma littorale Blume in streptozotocin-induced type 1 diabetic rats.

The present investigation was undertaken to standardize and study the dose-dependent effect of three weeks treatment with hot and cold aqueous extract of E. littorale (0.5, 1 and 2 g/kg, po) on streptozotocin (STZ) induced type I diabetic (confirmed by histopathology) rats (45 mg/kg, iv single dose). Treatment of rats with STZ produced cardinal signs of diabetes-mellitus like a significant loss of body weight, polyuria and polydipsia. There was also a significant increase in fasting blood glucose levels and AUCglucose associated with decrease in insulin levels and AUCinsulin in STZ-diabetic rats. Treatment with E. littorale hot extract (1 and 2 g/kg) significantly reduced the elevated food intake and water intake, glucose and AUCglucose levels of diabetic rats. There was also a significant increase in serum cholesterol, serum triglyceride in the STZ diabetic rats. Treatment with E. littorale hot extract (1 and 2 g/kg) significantly decreased all these elevated levels in diabetic rats. Hot aqueous extract of E. littorale at 0.5 g/kg produced a significant decrease in serum glucose and triglycerides. At this doses serum cholesterol and AUCglucose were not found to be altered significantly.TLC finger-print profiles were established for the aqueous extract using HPTLC. Swertiamarin, which was used as a chemical marker, was found to be one of the major components in the hot extract while it was absent in cold extract. The results suggest that E. littorale possesses potential antidiabetic activity and improves lipid profile at a small dose of 0.5 g/kg.

Determination of Swertiamarin in Lomatogoniopsis alpina by HPLC / 中国中医药信息杂志

Objective To establish a method for content determination of swertiamarin in Lomatogoniopsis alpina. Methods HPLC method for determination was used. Chromatographic column: Alltimal C18 (250 mm?4.6 mm, 5 ?m). Mobile phase: methanol-water (including 0.05% H3PO4), and gradient elution. Flow rate:1 mL/min. Wavelength:234 nm. Column temperture:30 ℃. Results The calibration curve of swertiamarin was in good linearity over the range of 3.10～30.98 (r =0.999 1). The average recoveries were 98.0%, with RSD=2.66% (n =6). Conclusion It is a simple and sensitive method in controlling the quality of Lomatogoniopsis alpina.

Swertiamarin of related overground and underground organ of Gentiana / 中成药

flower, changed with the blossoming period. CONCLUSION: It may be a relationship between the content and blossoming period.