NO Production In LPS-stimulated RAW264 Research Articles

Four new tirucallane-type triterpenoids from Aphanamixis polystachya

Four new tirucallane-type triterpenoids, polystanins H–K (1–4), were obtained from the stems and leaves of Aphanamixis polystachya. Their structures were elucidated by analysis of the spectroscopic data and comparison with literature data. Compounds 1 and 2 showed week inhibitory effects against NO production in LPS-stimulated RAW264.7 cells. All the isolates were investigated for their antifungal activities against drug-resistant Candida albicans.

The Antioxidant and Anti-Inflammatory Properties of Wild Bilberry Fruit Extracts Embedded in Mesoporous Silica-Type Supports: A Stability Study.

Polyphenolic extracts from wild bilberries (Vaccinium myrtillus L.) have shown antioxidant and anti-inflammatory effects, but they are prone to degradation when exposed to environmental factors, limiting their use in biomedical applications. To overcome this issue, this study proposed the embedding of wild bilberry fruit ethanolic extracts in pristine mesoporous silica functionalized with organic groups (mercaptopropyl and propionic acid), as well as coated with fucoidan, a biopolymer. Herein, we report a stability study of free and incorporated extracts in mesoporous silica-type supports in high-humidity atmospheres at 40 °C up to 28 days, using HPLC analysis, thermal analysis, and radical scavenging activity determination. Better chemical and thermal stability over time was observed when the extracts were incorporated in mesoporous silica-type supports. After 12 months of storage, higher values of antioxidant activity were determined for the extract embedded in the supports, silica modified with mercaptopropyl groups (MCM-SH), and fucoidan-coated silica (MCM-SH-Fuc) than that of the free extract due to a synergistic activity between the support and extract. All encapsulated extracts demonstrated remarkable effects in reducing NO production in LPS-stimulated RAW 264.7 cells. The treatment with extract embedded in MCM-SH-Fuc in a dose of 10 μg/mL surpassed the effect of free extract in the same concentration. For the extract encapsulated in an MCM-SH support, a lower IC50 value (0.69 μg/mL) towards COX-2 was obtained, comparable with that of Indomethacin (0.6 μg/mL). Also, this sample showed a higher selectivity index (2.71) for COX-2 than the reference anti-inflammatory drug (0.98). The developed formulations with antioxidant and anti-inflammatory properties could be further used in nutraceuticals.

Phytochemical Constituents and Potential of Different Extracts from Eleutherine bulbosa on Skin Infectious Treatment : Anti-fungal, Anti-bacterial, Anti-inflammatory and Cytotoxic Activities

Eleutherine bulbosa is a plant commonly employed in traditional medicine across various tropical regions, including Thailand. The aim of this study was to identify the phytochemical constituents of E. bulbosa, evaluate the inhibitory effects on skin fungal and bacterial pathogens, and examine the anti-inflammatory and cytotoxic properties of extracts obtained from E. bulbosa bulbs through n-Hexane, EtOAc, and EtOH extraction methods. Agar diffusion, MIC, and MBC assays were employed to determine the fungistatic, bacteriostatic, and bactericidal activities. The EtOAc extract of E. bulbosa bulbs demonstrated the highest inhibition against fungal and bacterial strains when compared to other solvents. The results revealed that the EtOAc extract exhibited a potent anti-fungal effect on T. rubrum (the zones of inhibition = 35.00 ± 0.00 mm) comparable to Ketoconazole (the zones of inhibition = 34.33 ± 1.16 mm). The highest antibacterial activity against S. pyogenes was observed, with a zone diameter of 23.00 ± 1.00 mm, while the lowest MIC and MBC values were recorded at 19.53 and 78.12 μg/ml, respectively. Additionally, we demonstrated significant inhibitory properties of E. bulbosa bulbs extracted with n-Hexane on NO production in LPS-stimulated RAW 264.7 cells compared to other solvent extracts. This occurred without affecting cell viability across a concentration range of 6.25 to 25 μg/ml. Moreover, the less affected viability of fibroblasts suggests that the extract may be useful in preventing inflammatory diseases mediated by excessive production of NO with low toxicity to normal cells. Phytochemical screening indicated the presence of various compounds with biological activities including alkaloids, flavonoids, phenols, and tannins in all different solvent extracts. These findings suggest that E. bulbosa bulb extracts possess beneficial effects and hold potential for further development as a new natural source of multifunctional therapeutic agents, which are safe and effective for a broad spectrum of skin infectious microorganisms and also potentially reduce inflammation that is associated with an infection caused by microorganisms.

Identification of diverse sesquiterpenoids with anti-fibrotic potential from Inula japonica Thunb.

In the chemical investigation of Inula japonica, a total of 29 sesquiterpenoids (1–29) were obtained, including pseudoguaine-, xanthane-, eudesmane-, and 1,10-secoeudesmane-type compounds, as well as their dimers. Among them, six new dimeric sesquiterpenoids, bisinulains A–F (1–5, 7), characterized by a [4 + 2] biogenetic pathway between different sesquiterpenoid monomers were identified. Additionally, three new monomers named inulaterins A-C (13, 18 and 21) were discovered. The structures of these compounds were determined through analysis of spectroscopic data, X-ray crystallographic data, and ECD experiments. To assess their potential anti-inflammatory activities, the sesquiterpenoid dimers were tested for their ability to inhibit NO production in LPS-stimulated RAW 264.7 cells. Furthermore, the compounds that exhibited anti-inflammatory effects underwent evaluation for their anti-fibrotic potential using a TGF-β-induced epithelial-mesenchymal transition model in A549 cells. As a result, bisinulain B (2) was screened out to significantly inhibit the production of cytokines involved in pulmonary fibrosis such as NO, α-SMA, collagen I and fibronectin.

Three new anti-inflammatory stilbenoids and a diphenyl ether derivative from Cajanus cajan

Three new stilbenoids, namely two rare plant-derived phenanthrenes denominated Cajananthrenes A and B (1, 2) and one bibenzyl named Cajanbenzyl (3), together with a diphenyl ether derivative designated Cajanether (4), as well as five other known compounds (5-9) were isolated from the ethanolic extract of the leaves of Cajanus cajan. Their structures were determined through extensive spectroscopic analysis including UV, IR, NMR (1D and 2D) and HRESIMS as well. A plausible biogenesis pathway was proposed for the biosynthesis of compounds 1-3. Compounds 1 and 2 displayed moderate anti-inflammatory activity as evident from the inhibitory effect on NO production in LPS-stimulated RAW 264.7 macrophages with IC50 values of 73.6 and 44.6 μM respectively.

Isolation, Identification, Anti-Inflammatory, and In Silico Analysis of New Lignans from the Resin of Ferula sinkiangensis.

Ferula sinkiangensis K. M. Shen (Apiaceae) is distributed in arid desert areas of Xinjiang, and its resin is a traditional Chinese medicine to treat gastrointestinal digestive diseases. To explore bioactive components from F. sinkiangensis, three new lignans and thirteen known components were isolated. The structural elucidation of the components was established utilizing spectroscopic analyses together with ECD calculations. Griess reaction results indicated new compounds 1 and 2 significantly decreased NO production in LPS-stimulated RAW 264.7 macrophages, and ELISA results indicated that they effectively attenuated LPS-induced inflammation by inhibiting TNF-α, IL-1β, and IL-6 expressions. The in silico approach confirmed that compound 1 docked into the receptors with strong binding energies of -5.84~-10.79 kcal/mol. In addition, compound 6 inhibited the proliferation of AGS gastric cancer cells with IC50 values of 15.2 μM by suppressing the cell migration and invasion. This study disclosed that F. sinkiangensis might be a promising potential resource for bioactive components.

Design, Synthesis, and Evaluation of the COX-2 Inhibitory Activities of New 1,3-Dihydro-2H-indolin-2-one Derivatives.

Thirty-three 1,3-dihydro-2H-indolin-2-one derivatives bearing α, β-unsaturated ketones were designed and synthesized via the Knoevenagel condensation reaction. The cytotoxicity, in vitro anti-inflammatory ability, and in vitro COX-2 inhibitory activity of all the compounds were evaluated. Compounds 4a, 4e, 4i-4j, and 9d exhibited weak cytotoxicity and different degrees of inhibition against NO production in LPS-stimulated RAW 264.7 cells. The IC50 values of compounds 4a, 4i, and 4j were 17.81 ± 1.86 μM, 20.41 ± 1.61 μM, and 16.31 ± 0.35 μM, respectively. Compounds 4e and 9d showed better anti-inflammatory activity with IC50 values of 13.51 ± 0.48 μM and 10.03 ± 0.27 μM, respectively, which were lower than those of the positive control ammonium pyrrolidinedithiocarbamate (PDTC). Compounds 4e, 9h, and 9i showed good COX-2 inhibitory activities with IC50 values of 2.35 ± 0.04 µM, 2.422 ± 0.10 µM and 3.34 ± 0.05 µM, respectively. Moreover, the possible mechanism by which COX-2 recognized 4e, 9h, and 9i was predicted by molecular docking. The results of this research suggested that compounds 4e, 9h, and 9i might be new anti-inflammatory lead compounds for further optimization and evaluation.

Identification of volatile compounds and their bioactivities from unpolar fraction of Alpinia oxyphylla Miq. and mining key genes of nootkatone biosynthesis

In this study, analysis of the chemical constituents and bioactivities of the unpolar fractions [petroleum ether (PE) and chloroform (C)] of fruits and leaves of Alpinia oxyphylla Miq. were carried out, as well as the bioactivities of the main compounds nootkatone and valencene. From PE and C fractions of the fruits, and PE fraction of the leaves, 95.80%, 59.30%, and 82.11% of the chemical constituents respectively were identified by GC-MS. Among these identified compounds, nootkatone was the main compound in all of three fractions, while valencene was the second main compound in the PE fractions of the fruits and leaves. The bioactivities results showed that all of the fractions and the major compound nootkatone showed tyrosinase inhibitory, as well as inhibitory effect on NO production in LPS-stimulated RAW264.7 cells. While valencene only presented inhibitory activity on NO production in RAW264.7 cells. The critical genes involved in nootkatone biosynthesis in A. oxyphylla were identified from the public transcriptome datasets, and protein sequences were preliminarily analyzed. Our studies develop the usage of the unpolar fractions of A. oxyphylla, especially its leaves as the waste during its production, and meanwhile provide the gene resources for nootkatone biosynthesis.

Anti-inflammatory ent-cleistanthane-type diterpenoids from Phyllanthus rheophyticus

A bioactivity-guided isolation from the aerial parts of Phyllanthus rheophyticus obtained 17 undescribed ent-cleistanthane-type diterpenoids, namely phyllarheophols A−Q, as well as 12 known analogs. Their structures were characterized by a combination of spectroscopic data interpretation, single-crystal X-ray diffraction and ECD analysis. The anti-inflammatory activities of these compounds were evaluated by measuring their inhibitory effects on NO production in LPS-stimulated RAW264.7 macrophages, and their preliminary structure-activity relationships were also discussed. Further study showed that promising compounds phyllarheophol D and phyacioid B significantly suppressed the expressions of cytokines and nitric oxide synthase through the NF-κB signaling pathway.

Chemical constituents from the aerial parts of Rubia cordifolia L. with their NO inhibitory activity

A new naphthoquinone derivative (1) together with twenty-three known compounds (2–24), were isolated from the aerial parts of Rubia cordifolia L. Their structures were elucidated on the basis of NMR and HR-ESIMS data. Compounds 1–13 were assessed for their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophage cells. Compounds 2-6 exhibited significant inhibitory activities with IC50 values of 21.37, 13.81, 24.56, 20.32, and 30.08 μmol·L−1, respectively.

Moutan Cortex Extract Modulates Macrophage Activation via Lipopolysaccharide-Induced Calcium Signaling and ER Stress-CHOP Pathway.

Moutan Cortex, Paeonia suffruticosa root, has long been used as a medicine for the treatment of inflammatory diseases. The aim of this study was to evaluate the modulative properties of Moutan Cortex water extract (CP) on endoplasmic reticulum (ER) stress-related macrophage activation via the calcium-CHOP pathway. RAW 264.7 mouse macrophages were activated by lipopolysaccharide (LPS), and the levels of various inflammatory mediators from RAW 264.7 were evaluated. The multiplex cytokine assay was used to investigate both cytokines and growth factors, and RT-PCR was used to investigate the expressions of inflammation-related genes, such as CHOP. Data represent the levels of NO and cytosolic calcium in LPS-stimulated RAW 264.7 were significantly inhibited by CP as well as hydrogen peroxide (p < 0.05). Minutely, NO production in LPS-stimulated RAW 264.7 incubated with CP at concentrations of 25, 50, 100, and 200 µg/mL for 24 h was 97.32 ± 1.55%, 95.86 ± 2.26%, 94.64 ± 1.83%, and 92.69 ± 2.31% of the control value (LPS only), respectively (p < 0.05). Calcium release in LPS-stimulated RAW 264.7 incubated with CP at concentrations of 25, 50, 100, and 200 µg/mL for 18 h was 95.78 ± 1.64%, 95.41 ± 1.14%, 94.54 ± 2.76%, and 90.89 ± 3.34% of the control value, respectively (p < 0.05). Hydrogen peroxide production in LPS-stimulated RAW 264.7 incubated with CP at concentrations of 25, 50, 100, and 200 µg/mL for 24 h was 79.15 ± 7.16%, 63.83 ± 4.03%, 46.27 ± 4.38%, and 40.66 ± 4.03% of the control value, respectively (p < 0.05). It is interesting that the production of IL-6, TNF-α, G-CSF, MIP-1α, MIP-2, and M-CSF in LPS-stimulated RAW 264.7 were significantly inhibited by CP (p < 0.05), while the production of LIX, LIF, RANTES, and MIP-1β showed a meaningful decrease. CP at concentrations of 25, 50, 100, and 200 µg/mL significantly reduced the transcription of Chop, Camk2α, NOS, STAT1, STAT3, Ptgs2, Jak2, c-Jun, Fas, c-Fos, TLR3, and TLR9 in LPS-stimulated RAW 264.7 (p < 0.05). CP at concentrations of 25, 50, and 100 µg/mL significantly reduced the phosphorylation of STAT3, p38 MAPK, and IκB-α in LPS-stimulated RAW 264.7 (p < 0.05). These results suggest that CP might modulate macrophage activation via LPS-induced calcium signaling and the ER stress-CHOP pathway.

Development of fermented rice cake containing strawberry showing anti-inflammatory effect on LPS-stimulated macrophages and paw edema induced mice.

Strawberry (Fragaria ananassa) is one of the richest sources containing a wide variety of nutritive compounds. Anti-inflammatory activities of fermented rice cake made of strawberry powder as well as rice powder were evaluated. The fermented rice cake containing strawberry powder (SRC) significantly and dose-dependently inhibited NO production in LPS-stimulated RAW264.7 cells without cytotoxicity. Also, SRC effectively suppressed inflammatory gene expression, including iNOS, COX-2, IL-1β, IL-6, and TNF-α. In addition, the production of PGE2, IL-1β, IL-6, and TNF-α was significantly reduced. Furthermore, the anti-inflammatory effect of SRC was investigated using carrageenan-induced paw edema of ICR mice. It was demonstrated that pre-orally administration of SRC at a dose of 50 and 100 mg/kg BW significantly inhibited paw edema induced by carrageenan. This study suggested that the anti-inflammation activities of strawberry rice cake give the potential for increasing the commercialization of rice cake and rice products.

Anti-inflammatory Activity Guided Fractionation of Aerial Parts of Clematis gouriana Roxb. ex DC

Clematis gouriana Roxb. ex DC. also known as Indian Traveler’s Joy is used in different regions of India for curing skin infections, cuts and wounds. The study was performed on 70% methanol extract of C. gouriana aerial parts and its n-hexane, ethyl acetate and n-butanol fractions. The in vitro and in vivo anti-inflammatory potential was evaluated using inhibition of NO production in LPS-stimulated RAW 264.7 macrophages cell line and carrageenan-induced rat paw edema model in vivo, respectively. All the extracts and fractions exhibited concentration-dependent inhibitory effects. It was found that ethyl acetate fraction was the most potent fraction showing 77.24±0.71% inhibition of NO release at 100 μg/mL concentration. The ethyl acetate fraction of C. gouriana also showed significant anti-inflammatory activity in the carrageenan-induced rat paw edema model at a dose of 400 mg/Kg. Further, the phytochemical investigation of the bioactive ethyl acetate fraction led to isolation of five compounds belonging to phenolics and flavonoids type of natural products.

Two new chlorinated sesquiterpenes from Sigesbeckia orientalis

Two new chlorinated guaianane-type sesquiterpenes (named chlosigesolides A and B) together with eight known compounds were isolated from the leaves and twigs of Sigesbeckia orientalis. Their structures were determined by analysis of HR-ESI-MS, 1D- and 2D-NMR spectral data as well as comparison with the literature. Absolute configurations of new compounds were elucidated by NOESY and ECD methods. Chlosigesolide A inhibited NO production in LPS-stimulated RAW264.7 macrophages with IC50 value of 10.9 ± 0.8 μM. Other compounds exhibited inhibitory activity at IC50 in range of 26.5 to 49.7 μM.

Anti-Inflammatory and α-Glucosidase Inhibitory Activities of Chemical Constituents from Bruguiera parviflora Leaves

Bruguiera parviflora (Rhizophoraceae) is one of the Bruguiera genus-based mangrove plants which has not been investigated for the chemical compositions as well as biological activities so far. The present study was aimed at investigating the phytochemicals as well as anti-inflammatory and α-glucosidase inhibitory activities of B. parviflora leaves. The results showed that the crude extract and its fractions significantly increased the percentage inhibitory activity against α-glucosidase and decreased NO production in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. The most effective fraction BP5 was further chromatographed and purified. As a result, eight compounds were isolated and elucidated, including five flavonoids (1–5) and three triterpenoids (6–8). All isolated compounds were evaluated for the anti-inflammatory and α-glucosidase inhibitory effects. The results indicated that flavonoids namely taxifolin (1), quercetin (2), myricetin (3), rutin (4), and kaempferol (5) exhibited potent anti-inflammatory as well as α-glucosidase inhibitory activities. Among them, compound 2 showed the most potent inhibitory effect against an α-glucosidase activity with an I C 50 value of 3.4 ± 0.5   μ g / mL and the LPS-induced NO production of 11.8 μM at the concentration of 100 μg/mL. These findings suggest that flavonoids (1–5) from B. parviflora leaves may be useful as the potential α-glucosidase inhibitor as well as anti-inflammatory agent.

Anthraquinones from the Aerial Parts of Rubia cordifolia with Their NO Inhibitory and Antibacterial Activities.

The present study aimed to identify the composition of the aerial parts of Rubia cordifolia L. A chemical investigation on the EtOAc extracts from the aerial parts of Rubia cordifolia resulted in the isolation of four new anthraquinones, namely Cordifoquinone A–D (1–4), along with 16 known anthraquinones. Their structures were elucidated on the basis of NMR and HR-ESIMS data. All isolates were assessed for their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophage cells. Compounds 1, 3 and 10 exhibited significant inhibitory activities with IC50 values of 14.05, 23.48 and 29.23 μmol·L−1, respectively. Their antibacterial activities of four bacteria, Escherichia coli (ATCC 25922), Staphylococcus aureus subsp. aureus (ATCC 29213), Salmonella enterica subsp. enterica (ATCC 14028) and Pseudomonas aeruginosa (ATCC 27853), were also evaluated. Our results indicated that the antibacterial activity of these compounds is inactive.

Analysis of Antioxidant Phytochemicals and Anti-Inflammatory Effect from Vitex rotundifolia L.f.

An extraction method using 80% EtOH was selected and applied to obtain the total extracts from leaves, flowers, fruits, twigs, and roots of Vitex rotundifolia L.f. based on the antioxidant activity-guided experiments. Subsequently, total extract from each part of V. rotundifolia was successfully partitioned into fractions, which were evaluated for their antioxidant and anti-inflammatory properties via DPPH, ABTS, and NO assays, respectively. Among them, EtOAc (E) and n-butanol (B) fractions showed the potent antioxidant activity and the methylene chloride (MC) fractions of roots, leaves, and fruits that exhibited strong scavenging activity on DPPH and ABTS radicals. In the anti-inflammatory assay, n-hexane (H) and MC fractions of leaves potently inhibited NO production in LPS-stimulated RAW264.7 cells, followed by E fractions derived from fruits, flowers, twigs, and roots, along with B fractions from flowers and twigs. Additionally, a comprehensive HPLC-decoupled MS profiling was established and validated using seven isolated marker compounds (1–7), which were identified by analysis of their UV, NMR, and MS data. The established method was also applied for quantification of these marker compounds in each organ collected from different locations, and to assess their antioxidant capacity by a screening DPPH-HPLC method. Principal component analysis suggested the botanical organs from this plant correlated with the marker compound contents in association with bioactivity. The study results are a prelude to further studies involving the active fractions and provide a comprehensive insight into the functional products of this plant against oxidative diseases.

Recurvataside, a new saponin from aerial parts of Mussaenda recurvata

A new glycoside, recurvataside (1) and six known compounds, quinovic acid (2), quinovic acid 28-O-β-D-glucopyranosyl ester (3), 3-O-β-D-glucopyranosylquinovic acid (4), 3-O-β-D-glucopyranosylquinovic acid 28-O-β-D-glucopyranosyl ester (5), pomolic acid (6), and ursolic acid (7) were isolated from aerial parts of Mussaenda recurvata. The structure of compound 1 was identified from its spectroscopic data and by comparison with the literature. Recurvataside represents the first occurrence of δ-oleanolic acid saponin bearing two D-glucose units at C-3 and C-28 in nature. This is the first time δ-oleanane-type saponin reported in the genus Mussaenda. Compounds 1-7 were evaluated the cytotoxicity against two cancer cell lines MCF-7 and HepG2. Among them, only compound 7 exhibited moderate activity against MCF-7 and HepG2 cell lines with IC50 value of 16.97 ± 1.55 and 20.28 ± 1.00 μM, respectively. Compounds 1–7 were also tested for their inhibitory NO production in LPS-stimulated RAW264.7 cells. Compounds 3, 5, and 7 showed significant reduction of nitrite accumulation in LPS-stimulated RAW 264.7 cells with the IC50 values of 8.81 ± 0.48, 13.42 ± 0.84, and 18.37 ± 0.67 μM, respectively.

Zizaane-Type Sesquiterpenoids and Their Rearranged Derivatives from Agarwood of an Aquilaria Plant

Nine new sesquiterpenoids (1–9) were isolated from ethyl ether extract of agarwood originated from Aquilaria sp., including three novel sesquiterpenoids (1–3) derived from zizaane, together with six zizaane-type sesquiterpenoids (4–9). All structures were unambiguously elucidated based on 1D and 2D NMR spectra as well as by HRESIMS data. The absolute configuration of sesquiterpenoids was determined by comparison of the experimental and computed ECD spectra. In vitro anti-inflammatory assessment showed that compound 9 exhibited inhibition of NO production in LPS-stimulated RAW264.7 cells with an IC50 value of 62.22 ± 1.27 μM.

Anti‐Inflammatory Properties of Longifuran A, a New Benzofuran from the Stems of Amomum longiligulare

In this study, the following compounds were isolated from the dichloromethane fraction of the stems of Amomum longiligulare and then characterized: a new benzofuran, namely, longifuran A (1); five other phenolic compounds, namely, 4-methoxycinnamic acid (2), 2,5-dimethoxyphenol (3), eudesmic acid (4), 1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (5), and 4,4'-dihydroxychalcone (6); and two triterpenoids, namely, 24-methylcycloartan-3β-ol (7) and 24-methylencycloartan-3β-ol (8). They were evaluated in terms of their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophages. Results indicated that 1 and 5 exhibited promising inhibitory activities against NO generation with IC50 of 10.47±1.02 μM and 8.51±1.14 μM, respectively. Enzymatic assays demonstrated that they remarkably suppressed the secretion of two pro-inflammatory cytokines (i. e., IL-6 and TNF-α). They also dose-dependently inhibited the expression of inducible nitric oxide synthase and cyclooxygenase-2, two important enzymes modulating inflammation. Therefore, 1 and 5 could be targets for the development of new anti-inflammatory therapeutics.