Nitric Oxide Inhibitory Activity Research Articles

Beneficial effects of methanolic extract of Indigofera linnaei Ali. on the inflammatory and nociceptive responses in rodent models

ABSTRACT Indigofera linnaei Ali. (Tamil Name: Cheppu Nerinjil) belongs to the family Fabaceae, used for the treatment of various ailments in the traditional system of medicine. In the present study, the beneficial effects of methanol extract of whole plant of I. linnaei (MEIL) were evaluated on inflammation and nociception responses in rodent models. In vitro nitric oxide (NO), lipoxygenase (LOX) and cyclooxygense (COX) inhibitory activities were also performed to understand the mode of action. MEIL at the dose of 200 & 400 mg/kg, p.o. significantly inhibited carrageenan induced rat paw volume and reduced the weight of granuloma in cotton pellet granuloma model. The results obtained were comparable with the standard drug aceclofenac. The anti-nociceptive effect of MEIL in mice was evaluated in hot plate and acetic acid induced writhing model. The plant extract significantly reduced the number of writhes and the analgesic effect was higher than that of the standard drug aspirin. However, the extract fails to increase the latency period in hot plate method suggesting that the extract produce nociception by peripheral activity. The extract produced inhibitory effect on NO, LOX and COX in concentration dependent manner. The extract exhibited pronounced and selective COX-2 inhibition. Altogether, these results suggested that the methanol extract of Indigofera linnaei could be considered as a potential anti-inflammatory and analgesic agent.

Anti-inflammatory action of high molecular weight Mytilus edulis hydrolysates fraction in LPS-induced RAW264.7 macrophage via NF-κB and MAPK pathways

Anti-inflammatory Mytilus edulis hydrolysates (MEHs) were prepared by peptic hydrolysis and MEH was further fractionated into three fractions based on molecular weight, namely >5kDa, 1–5kDa, and <1kDa. The >5kDa peptide fraction exerted the highest nitric oxide (NO) inhibitory activity and inhibited prostaglandin E2 (PGE2) secretion in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Pretreatment with the >5kDa peptide fraction markedly inhibited LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and gene expressions. Stimulation by LPS induced the production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and -1β (IL-1β), whereas co-treatment with the >5kDa peptide fraction suppressed pro-inflammatory cytokine production. The >5kDa peptide fraction inhibited the translocation of NF-κB (nuclear factor-kappa B) through the prevention of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation and also inhibited the MAPK signaling pathway in LPS-stimulated RAW264.7 macrophages.

In vitro Kinetic of Inhibition of Lipase, Antioxidant Activity, Glucose Entrapment and Polyphenolic Content of Solanum linnaeanum

Decoction as traditionally used and methanol extract of a commonly consumed vegetable Solanum linnaeanum (SL) was evaluated for any antioxidant potential (Ferric reducing power (FRAP) assay, nitric oxide, DPPH, and xanthine oxidase inhibitory activity). The inhibitory effect of SL against α-amylase, α-glucosidase and pancreatic lipase activity was studied and the type of inhibition was established by the Lineweaver-Burk plot in vitro. The entrapment ability in decreasing glucose movement in an in vitro model was also investigated. The total phenolic, flavonoid, anthocyanin and tannin content were determined. The aqueous extract (IC50=0.20 µg/ mL) was more active against pancreatic lipase than the methanol extract (IC50=0.40 µg/ mL) and the positive control Orlistat® (IC50=0.35 µg/ mL). An uncompetitive type of inhibition as indicated by a reduction in Vmax and Km was recorded. Methanol extract demonstrated a more efficient DPPH scavenging potential. The FRAP of the methanolic extract was 102.708±1.36 mM trolox equivalent (TE)/mg extract. Decoction extracts showed no significant difference (p>0.05) on glucose entrapment capacities (33.3 %) when compared to the positive control (Arabic gum). Phytochemical screening of SL indicated the presence of phenols (152.80±1.40 (GAE) [g/mL]) and flavonoids (4.34±0.16 Rutin equivalent[μg/mL]) in the methanol extract only and absence of tannin and anthocyanin in both methanol and aqueous extracts. This study is the first attempt to establish valuable baseline data on SL bioactivity. The results can be further exploited for the development of new therapeutic dietary approach for the management of diabetes and obesity.

WITHDRAWN: Determination of N-trans-feruloyltyramine content and nitric oxide inhibitory and antioxidant activities of Tinospora crispa

WITHDRAWN: Determination of N-trans-feruloyltyramine content and nitric oxide inhibitory and antioxidant activities of Tinospora crispa

Cytotoxicity, nitric oxide and acetylcholinesterase inhibitory activity of three limonoids isolated from Trichilia welwitschii (Meliaceae).

Background Limonoids are highly oxygenated compounds with a prototypical structure. Their occurrence in the plant kingdom is mainly confined to plant families of Meliaceae and Rutaceae. Owing to their wide range of pharmacological and therapeutic properties, this study was aimed at investigating the potential nitric oxide (NO) and acetylcholinesterase (AChE) inhibitory activity and the cytotoxicity of three limonoids: trichilia lactone D5 (1), rohituka 3 (2) and dregeanin DM4 (3), isolated from Trichilia welwitschii C.DC.ResultsResults indicated that the three limonoids had low cytotoxicity towards Vero cells with LC50 values ranging from 89.17 to 75.82 μg/mL. Compounds (2) and (3) had lower cytotoxicity compared to puromycin and doxorubicin used as reference cytotoxic compounds. Compound (1) (LC50 of 23.55 μg/mL) had good antiproliferative activity against RAW 264.7 cancer cells. At the lowest concentration tested (0.5 µg/mL), compound (2) and (3) released the lowest amount of nitric oxide (2.97 and 2.93 µM, respectively). The three limonoids had anti-AChE activity with IC50 values ranged of 19.13 μg/mL for (1), 34.15 μg/mL for (2) and 45.66 μg/mL for (3), compared to galantamine (IC50 of 8.22 μg/mL) used as positive control.ConclusionThe limonoid compounds studied in this work inhibited nitric oxide production in LPS-stimulated macrophages and had anti-AChE activity. Trichilia lactone D5 had potential antiproliferative activity against RAW 264.7 cancer cells. The limonoids had low cytotoxicity towards Vero cells lines. This study provided further examples of the importance of limonoids compounds as potential AChE inhibitors and anti-inflammatory agents targeting the inhibition of NO production.

Influence of Different Drying Treatments and Extraction Solvents on the Metabolite Profile and Nitric Oxide Inhibitory Activity of Ajwa Dates.

This study aimed to examine the variation in the metabolite profiles and nitric oxide (NO) inhibitory activity of Ajwa dates that were subjected to 2 drying treatments and different extraction solvents. (1)H NMR coupled with multivariate data analysis was employed. A Griess assay was used to determine the inhibition of the production of NO in RAW 264.7 cells treated with LPS and interferon-γ. The oven dried (OD) samples demonstrated the absence of asparagine and ascorbic acid as compared to the freeze dried (FD) dates. The principal component analysis showed distinct clusters between the OD and FD dates by the second principal component. In respect of extraction solvents, chloroform extracts can be distinguished by the absence of arginine, glycine and asparagine compared to the methanol and 50% methanol extracts. The chloroform extracts can be clearly distinguished from the methanol and 50% methanol extracts by first principal component. Meanwhile, the loading score plot of partial least squares analysis suggested that beta glucose, alpha glucose, choline, ascorbic acid and glycine were among the metabolites that were contributing to higher biological activity displayed by FD and methanol extracts of Ajwa. The results highlight an alternative method of metabolomics approach for determination of the metabolites that contribute to NO inhibitory activity. The association between metabolite profiles and nitric oxide (NO) inhibitory activity of the various extracts of Ajwa dates was evaluated by utilizing partial least squares (PLS) model. The validated PLS model can be employed to predict the NO inhibitory activity of new samples of date fruits based on their NMR spectra which was important for assessing fruit quality. The information gained might be used as guidance for quality control, nutritional values and as a basis for the preparation of any food supplements for human health that employs date palm fruit as the raw material.

Iridoid glycosides from the flower buds of Lonicera japonica and their nitric oxide production and α-glucosidase inhibitory activities

The flower buds of Lonicera japonica are commonly used as popular healthy beverages that are also effective in the prevention and cure of a variety of chronic diseases, such as diabetes, and also as an anti-inflammatory treatment for thousands of years. In this study, three new iridoid glycosides along with twenty-five known analogues were isolated from the aqueous extract of L. japonica. Their structures were determined by extensive 1D NMR, 2D NMR and HR-ESIMS spectroscopic data analyses, and all the isolates were investigated with regard to lipopolysaccharide (LPS)-induced nitric oxide (NO) production and α-glucosidase inhibitory effects. Among these compounds, 12 and 13 exhibited strong NO inhibitory activities and no cytotoxic effects. Moreover, most compounds were potent α-glucosidase inhibitors compared with the positive control acarbose. These findings will enhance knowledge about the bioactive components of L. japonica and help promote its potential application in functional foods.

Isoflavonoids from Caragana changduensis and their nitric oxideinhibitory activities

Ten isoflavonoids were isolated from the heartwoods of Caragana changduensis Lion f. by means of various column chromatographic techniques. Based on the detailed spectral data analysis (MS and NMR), as well as comparison with the literatures, their chemical structures were determined as 7,2'-dihydroxy-8,4'-dimethoxyisoflavone (1), 4'-hydroxy-7,3'-dimethoxyisoflavone (2), 5, 7, 4'-trihydroxy-2',5'-dimethoxyisoflavone (3), prunetin (4), afrormosin (5), odoratin (6), genistein (7), texasin (8), pratensein (9), and 6,7,3'-trihydroxy-4'-methoxyisoflavone (10). Among them, compounds 1-3 and 9-10 were isolated from the Caragana genus for the first time. All the compounds were obtained from this species for the first time. In the preliminary assays, compounds 1, 2, 6, and 7 possessed significant inhibitory effects on NO production, with IC50 values of 48.12, 25.32, 62.71, 43.59 μmol x L(-1), respectively.

Six new metabolites produced by Colletotrichum aotearoa 09F0161, an endophytic fungus isolated from Bredia oldhamii

Six new compounds, colletobredins A–D (1–4) and colletomelleins A and B (5 and 6), along with 12 previously identified compounds, were isolated from the culture broth of Colletotrichum aotearoa BCRC 09F0161, a fungal endophyte residing in the leaves of an endemic Formosan plant Bredia oldhamii Hook. f. (Melastomataceae). The structures of the new compounds were established by spectroscopic methods, including UV, IR, HR-ESIMS and extensive 1D and 2D NMR techniques. The effects of some isolates on the inhibition of nitric oxide (NO) production in lipopolysaccharide-activated murine macrophage RAW264.7 cells were evaluated. All these compounds inhibited NO production in activated macrophages without any cytotoxicity at a concentration of 100 μM. Of these isolates, 1 showed weak NO inhibitory activity with IC50 value of 182.2 μM. To the best of our knowledge, this is the first report on isochroman glycoside metabolites (1–4) from the genus Colletotrichum.

Echinochlorins A˜C from the grains of Echinochloa utilis (Barnyard Millet) and their anti-inflammatory activity

Three new compounds (echinochlorins A-C; 1, 8, and 9) together with eight known compounds, isolated form the methylene chloride and ethyl acetate fractions of grains of Echinochloa utilis, were examined with regard to their effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. The results indicated that compounds 3, 5, and 9 possessed potent NO inhibitory activity against lipopolysaccharide (LPS)-induced NO release with IC50 values of 36.04, 4.80, and 20.78µM, respectively. In summary, the isolates compounds showed novel biological activity of inhibition in NO production, suggesting that E. utilis may be applied as supplemental functional foods having a beneficial effect against inflammation.

Boerhavia diffusa L. ethanol extract suppresses inflammatory responses via inhibition of Src/Syk/TRAF6

Boerhavia diffusa L. is a green vegetable used as an herbal medicine in several Asian countries for the treatment of hepatitis, kidney stones, liver disorders, nephritis, urinary disorders, and urinary retention. In this study, the anti-inflammatory activity and the molecular inhibitory mechanisms of B. diffusa L. ethanol extract (Bd-EE) in vitro and in vivo were evaluated. Lipopolysaccharide (LPS)-activated peritoneal macrophage RAW264.7 cells and a mouse gastritis model induced by HCl/EtOH treatment were chosen to determine the in vitro and in vivo anti-inflammatory activities of Bd-EE. This extract (100 and 200 µg/ml) significantly (P < 0.01) inhibited nitric oxide (NO) and tumour necrosis factor (TNF)-α production in peritoneal macrophages and in RAW264.7 cells during LPS exposure at both. The inducible nitric oxide synthase (iNOS) and TNF-α mRNA expression levels were decreased. In addition, nuclear levels of p65, p50, c-Fos, and c-Jun transcription factors were reduced by Bd-EE treatment; moreover, it inhibited NF-κB upstream signalling via IκBα, PI3K, Syk, and Src. Furthermore, analysis of AP-1 upstream signalling revealed that the AP-1 activation pathway consisting of TRAF6, TAK1, MKK4/7, and MKK3/6 was also predominantly inhibited by Bd-EE. By HPLC analysis, luteolin was identified as a major ingredient displaying NO inhibitory activity. The data provided here strongly suggest that the anti-inflammatory property of Bd-EE is linked to the suppression of Syk, Src, and TRAF6.

Norlignans from Pouzolzia zeylanica var. microphylla and their nitric oxide inhibitory activity

Five new compounds, pouzolignan F [4-hydroxy-3-(3,5-dihydroxyphenyl)-2-[bis(4-hydroxy-3-methoxyphenyl)methyl]butyl acetate] (1), pouzolignan G [4-hydroxy-3-(3,5-dihydroxyphenyl)-2-[(4-hydroxy-3,5-dimethoxyphenyl)(4-hydroxy-3-methoxyphenyl)methyl]butyl acetate] (2), pouzolignan H [1,4-dihydroxy-3-(3,5-dihydroxyphenyl)-2-[bis(4-hydroxy-3-methoxyphenyl)methyl]butane] (3), pouzolignan I [1,4-dihydroxy-3-(3,5-dihydroxyphenyl)-2-[(4-hydroxy-3,5-dime thoxyphenyl)-(4-hydroxy-3-methoxyphenyl)methyl]butane] (4), and pouzolignan J [1,4-dihydroxy-3-(3,5-dihydroxyphenyl) -2-[(3,4,5-trimethoxyphenyl)(4-hydroxy-3-methoxyphenyl)methyl]butane] (5), along with two known compounds, indolyl-3-carboxylic acid (6) and uracil (7), were isolated from the aerial parts of Pouzolzia zeylanica (L.) Benn. var. microphylla (Wedd.) W.T.Wang. The structures of these compounds were characterized based on spectroscopic methods, including IR, NMR (1H–1H COSY, HSQC, HMBC, and NOESY), and HR-ESI/TOF-MS experiments. All the new norlignans were assayed for inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal macrophages.

Anti-inflammatory sesquiterpene pyridine alkaloids from Tripterygium wilfordii

During a screening procedure involving higher plants to find novel candidates for use as anti-inflammatory agents, Tripterygium wilfordii Hook. f. was shown to exhibit considerable inhibitory activity. Five new sesquiterpene pyridine alkaloids, tripterygiumines S–W (1–4,15), along with 14 known dihydroagarofuran derivatives, were isolated from the roots of the T. wilfordii Hook. f. Their structures were established by extensive use of spectroscopic techniques, including 1D and 2D NMR spectroscopy and high-resolution mass spectrometry. All compounds were evaluated for their anti-inflammatory activity by measuring the nitric oxide production by the LPS-induced murine macrophage cell line RAW264.7. It was found that 1, 5, and 19 possessed potent nitric oxide inhibitory activity with IC50 values ranging from 2.99 to 28.80μM, without any effect on the cell viability of RAW264.7 cells. Accordingly, compounds 1, 5, and 19, especially 5, were identified as promising candidates for further scientific investigation of their potential use as anti-inflammatory agents.

Nitric oxide inhibitory activity and antioxidant evaluations of 2-benzoyl-6-benzylidenecyclohexanone analogs, a novel series of curcuminoid and diarylpentanoid derivatives.

A series of twenty-four 2-benzoyl-6-benzylidenecyclohexanone analogs were synthesized and evaluated for their nitric oxide inhibition and antioxidant activity. Six compounds (3, 8, 10, 17, 18 and 19) were found to exhibit significant NO inhibitory activity in LPS/IFN-induced RAW 264.7 macrophages, of which compound 10 demonstrated the highest activity with the IC50 value of 4.2 ± 0.2 μM. Furthermore, two compounds (10 and 17) displayed antioxidant activity upon both the DPPH scavenging and FRAP analyses. However, none of the 2-benzoyl-6-benzylidenecyclohexanone analogs significantly scavenged NO radical. Structure-activity comparison suggested that 3,4-dihydroxylphenyl ring is crucial for bioactivities of the 2-benzoyl-6-benzylidenecyclohexanone analogs. The results from this study and the reports from previous studies indicated that compound 10 could be a candidate for further investigation on its potential as a new anti-inflammatory agent.

New ambuic acid derivatives from the solid culture of Pestalotiopsis neglecta and their nitric oxide inhibitory activity.

Four new ambuic acid derivatives (1–4), and four known derivatives (5–8), were isolated from the solid culture of a plant pathogenic fungus Pestalotiopsis neglecta. Their structures were elucidated by extensive NMR experiments. The absolute configuration of the C-16 secondary alcohol in 1 was deduced via the CD data of the in situ formed [Rh2(OCOCF3)4] complex with the acetonide derivative of 1. The absolute configuration in 3 was assigned by comparison of the experimental and simulated electronic circular dichroism (ECD) spectrum. The NMR data of compound 5 was reported for the first time. In the nitric oxide (NO) inhibition assay, compounds 4, 6 and 7 showed inhibitory activity against the NO production in the lipopolysaccharide (LPS)-induced macrophage with IC50 values of 88.66, 11.20, and 20.80 µM, respectively.

Coumarins from Angelica decursiva Inhibit Lipopolysaccharide‐induced Nitric Oxide Production in RAW 264.7 Cells

Angelica decursiva has been used in Korean traditional medicine as an antitussive, analgesic, antipyretic, and cough remedy. In a recent study, the 90% methanol fraction of the dichloromethane fraction showed the most promising anti‐inflammatory activity; however, compounds responsible for the anti‐inflammatory activity were not identified. In this study, bioactivity‐guided isolation yielded 8 coumarins along with a new coumarin, 3′‐angeloyloxy‐4′‐hydroxy‐3′,4′‐dihydroxanthyletin (4). The anti‐inflammatory activity of compounds was evaluated via inhibition of nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α), as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) expression in lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells. Among the tested compounds, edulisin II (1) exhibited the highest NO inhibitory activity, followed by decursidin (2), Pd‐C‐III (3), 4, Pd‐C‐I (5), and Pd‐C‐II (6). Structure‐activity relationships revealed that esterification of the hydroxyl at C‐3′/C‐4′ with an angeloyl/senecioyl/acetyl is essential for NO inhibitory activity; the number and the position of angeloyl/or senecioyl groups greatly affect the potency of coumarins. Coumarins 1~6 inhibited TNF‐α production and iNOS expression while 1~4 inhibited COX‐2 expression. Thus, coumarins isolated from A. decursiva might be used as potential leads for the development of therapeutic agents for inflammation.

Elucidation of the Chemical Structure and Determination of the Production Conditions for a Bioactive Maillard Reaction Product, [5-(5,6-Dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol, Isolated from a Glucose–Lysine Heated Mixture

We previously isolated a bioactive molecule, named F3-A, from an aqueous glucose (Glc) and lysine (Lys) Maillard reaction (MR) model system. Herein, F3-A was verified as [5-(5,6-dihydro-4H-pyridin-3-ylidenemethyl)furan-2-yl]methanol (5) and was subsequently synthesized for confirmation of bioactivity. Using Taguchi and factorial designs, we determined that the conditions which best increased the yield of F3-A were at pH 6 with a sugar:amino acid ratio of 2:1 and heating time of 12 h at 100 °C. The MR mixtures containing glucose produced highest yield, compared to fructose, lactose, and sucrose. Both the F3-A recovered from Glc-Lys MR mixture and the synthesized product exhibited significant (P < 0.05), dose dependent, nitric oxide (NO) inhibitory activity in Caco-2 cells that was comparable to aminoguanidine (AG) and pyrrolidine dithiocarbamate (PDTC), respectively. Finally, an additional inhibitory effect of F3-A was determined when coincubated with AG in cytokine-induced Caco-2 cells. This bioactivity points to a potential role in preventing intestinal inflammation.

Compounds from Geijera parviflora with prostaglandin E2 inhibitory activity may explain its traditional use for pain relief

Ethnopharmacological relevanceAustralian Aboriginal people used crushed leaves of Geijera parviflora Lindl. both internally and externally for pain relief, including for toothache (Cribb and Cribb, 1981). This study tested the hypothesis that this traditional use might be at least in part explained by the presence of compounds with anti-inflammatory activity. Materials and methodsA crude extract (95% EtOH) was prepared from powdered dried leaves. From the CH3Cl fraction of this extract compounds were isolated by bioassay-guided fractionation and tested for: (1) cytotoxicity in RAW 264.7 murine leukemic monocyte–macrophages, (2) prostaglandin E2 (PGE2) inhibitory activity in 3T3 Swiss albino mouse embryonic fibroblast cells, as well as (3) nitric oxide (NO) and (4) tumour necrosis factor alpha (TNFα) inhibitory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Isolated compounds were also tested for (5) antibacterial activity against a panel of Gram-positive (Staphylococcus aureus ATCC 29213 and ATCC 25923, Staphylococcus epidermidis ATCC 35984, biofilm-forming) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) strains by broth microdilution. ResultsEleven compounds were isolated, including one new flavone and one new natural product, with a further four compounds reported from this species for the first time. Some of the compounds showed good anti-inflammatory activity in vitro. In particular, flindersine (1) and N-(acetoxymethyl) flindersine (3) inhibited PGE2 release with IC50 values of 5.0μM and 4.9μM, respectively, without any significant cytotoxicity. Several other compounds showed moderate inhibition of NO (5, 6, 7) and TNF-α (6), with IC50 in the low micromolar range; however much of this apparent activity could be accounted for by the cytotoxicity of these compounds. None of the compounds showed anti-bacterial activity. ConclusionsThe inhibition of PGE2, an important mediator of inflammation and pain, by flindersine and a derivative thereof, along with the moderate anti-inflammatory activity shown by several other compounds isolated from Geijera parviflora leaf extract, support the traditional use of this plant for pain relief by Australian Aboriginal people.

Anti-inflammatory activity of a sulfated polysaccharide from the brown alga Sargassum cristaefolium

We screened the anti-inflammatory activities of polysaccharides from three algae species, such as Porphyra tenera (red algae), Monostroma nitidum (green algae), and Sargassum cristaefolium (brown algae) by measuring inhibition of LPS-induced nitric oxide (NO) production in murine RAW264.7 macrophages. In addition, we investigated the effects of sulfate content and molecular size of the S. cristaefolium polysaccharide on NO inhibitory activity. Among the three tested alga polysaccharide extracts, the S. cristaefolium extract had the highest NO inhibitory effect. The three polysaccharides with 1193.2, 864.4, 386.1 kDa in S. cristaefolium extract, in combination with their 4 acid-degraded polysaccharides with 106.3, 55.9, 15.4, and 1.9 kDa were prepared by Sephacryl S-400HR gel permeation chromatography, and the 386.1 kDa sample had the highest NO inhibitory effect. Three polysaccharide samples with sulfate contents of 0.84%, 9.42%, and 50.83% were obtained by NaBH4 desulfating or SO3–DMF sulfurization of the original S. cristaefolium polysaccharide with 386.1 kDa and 31.1% sulfate content. The highest NO inhibitory activity was observed in the polysaccharide with 9.42% sulfate content. The polysaccharide with 386.10 kDa significantly inhibited inducible nitric oxide synthase expression mediated by inhibiting the phosphorylation of the signal proteins of p-38, ERK and JNK, and nuclear factor-κB activation in lipopolysaccharide-stimulated RAW264.7 cells.

Effect of Three-spot Seahorse Petroleum Ether Extract on Lipopolysaccharide Induced Macrophage RAW264.7 Inflammatory Cytokine Nitric Oxide and Composition Analysis.

Three-Spot seahorse is a traditional medicine in Asian countries. However, the alcohol extract is largely unknown for its anti-inflammatory activity. This study aimed at elucidating fraction of potent anti-inflammatory activity of seahorse. A systematic solvent extraction method of liquid-liquid fractionation of ethanol crude extract gave four fractions petroleum ether (PE), and ethyl acetate (EtOAc), water saturated butanol (n-BuOH), water (H2O). In this study, PE extract was selected for further study after preliminary screening test, and was connected to silica column chromatography and eluted with different polarity of mobile phases, and obtained four active fractions (Fr I, Fr II, Fr III, Fr IV). Effect of separated fractions on inflammation was investigated in lipopolysaccharide (LPS) stimulated murine RAW264.7 cells in vitro. The result shows that seahorse extract was capable of inhibiting the production of nitric oxide (NO) significantly in a dose dependent manner and exhibited no notable cytotoxicity on cell viability. IC50 of fraction IV was 36.31 μg/mL, indicating that separated fraction possessed potent NO inhibitory activity against LPS-induced inflammatory response, thus, demonstrated its in vitro anti-inflammatory potentiality, it may be at least partially explained by the presence of anti-inflammation active substances, phenolic compounds, phospholipids and polyunsaturated fatty acids, especially phospholipids and polyunsaturated fatty acids. It could be suggested that seahorse lipid-soluble components could be used in functional food and anti-inflammatory drug preparations.