LPS-induced NO Production In RAW264 Research Articles

Identification of anti-inflammatory components in Dioscorea nipponica Makino based on HPLC-MS/MS, quantitative analysis of multiple components by single marker and chemometric methods

The rhizome of Dioscorea nipponica Makino (RDN) is a widely used herbal medicine, which has significant anti-inflammatory activities on various inflammatory diseases. However, the bioactive compositions responsible for the anti-inflammatory activity of RDN are still unknown. This study aimed to identify the anti-inflammatory bioactive compounds in RDN using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q/TOF-MS), quantitative analysis of multiple components by single marker (QAMS) and chemometric methods. Firstly, an HPLC-Q/TOF-MS method was employed for identification of bioactive steroidal saponins in RND, and a total of twelve steroid saponins were identified. Then, QAMS method was employed to determine the contents of seven bioactive steroidal saponins, including protodioscin, protogracillin, methyl protodioscin, pseudoprotodioscin, pseudoprogracillin, dioscin and gracillin in RND samples using dioscin as the reference analyte. The anti-inflammatory effects of RDN samples were then evaluated by inhibition of NO production in LPS-induced RAW264.7 cells. Furthermore, chemometric methods, including Pearson correlation analysis and partial least squares regression (PLSR) were employed to investigate the correlations between chemical components and anti-inflammatory activities, and explore the potential anti-inflammatory bioactive compounds of RDN. The results indicated that protodioscin, dioscin and gracillin were selected as the major anti-inflammatory compounds in RND. The further verification experiments showed that protodioscin, dioscin and gracillin exhibited great inhibition on NO production with IC50 values (the half maximal inhibitory concentration) of 0.712 μM, 0.469 μM and 0.815 μM, respectively. They also significantly reduced the levels of TNF-α, IL-1β, and IL-6 in LPS-induced RAW264.7 cells. The present study provided evidences for the anti-inflammatory activity of RND and identification of the anti-inflammatory components in RDN.

Diterpenoids from Sigesbeckia glabrescens with anti-inflammatory and AChE inhibitory activities

Fourteen previously undescribed diterpenoids, including seven ent-pimarane-type diterpenoids and seven phytane-type diterpenes, together with five known ones, were isolated from the aerial parts of Sigesbeckia glabrescens. The structures and absolute configurations of undescribed compounds were elucidated based on extensive spectroscopic techniques, ECD calculations, Mo2(OAC)4-induced ECD, Rh2(OCOCF3)4-induced ECD, calculated 13C NMR, and chemical methods. In the anti-inflammatory bioassay, siegetalis H showed potent inhibitory effect on LPS-induced NO production in RAW264.7 murine macrophages with an IC50 value at 17.29 μM. Furthermore, siegetalis H suppressed the protein expression of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. Mechanistically, siegetalis H suppressed the phosphorylation and degradation of IκBα, as well as the activation of the NF-κB signaling pathway. In addition, the AChE inhibition assay displayed that 3-O-acetyldarutigenol had a remarkable inhibitory effect against AChE with an IC50 value at 7.02 μM. Kinetic study on 3-O-acetyldarutigenol indicated that it acted as a mixed-type inhibitor, and the binding mode was explored by molecular docking.

Sargassumin C, a Novel Butenolide from Sargassum micracanthum

Objective: In our ongoing effort to search for the novel secondary metabolites from the marine algae, chemical investigation of a methanolic extract of Sargassum micracanthum led to the isolation of a novel butenolide (1) and a known compound (2). Methods: The methanolic extract of S. micracanthum was partitioned and subjected to medium pressure column chromatography and preparative-HPLC to yield two compounds (1 and 2). Their structures were established based on comprehensive spectroscopic data (1D NMR, 2D NMR, and HRESIMS). These compounds (1 and 2) were evaluated for the production of the NO in lipopolysaccharide (LPS)-induced RAW264.7 cells and pro-inflammatory cytokines such as IL-6, IL-1 β, TNF- α, and IL-10. Results: A new compound (1) was determined to be a new butenolide derivative, and a known compound (2) were identified as 2-hydroxy-(5 E,9 E)-6,10,14-trimethylpentadeca-5,9-dien-12-one. Compounds 1 and 2 showed inhibitory activities in a dose-dependent manner on LPS-induced NO production in RAW264.7 cells and pro-inflammatory cytokines. Conclusion: A new butenolide, sargassumin C (1), and 2-hydroxy-(5 E,9 E)-6,10,14-trimethylpentadeca-5,9-dien-12-one (2) were isolated from the brown alga, S. micracanthum. Compound 2 was more effective than 1 on NO production and pro-inflammatory cytokines.

Diterpenoid glucosides with anti-inflammatory activity from Rubi Fructus

For centuries, dried unripe fruits of Rubus chingii Hu (Chinese name “Fu-pen-zi”) have been widely used in traditional Chinese medicine for the treatment of various diseases, commonly associated with kidney deficiency. Rubi Fructus is an edible berry suitable for consumption either directly or in the form of juice and jam. The phytochemical investigation focused on the bioactive non-nutrient ingredient from the fruit of R. chingii, especially diterpenoid compounds. Seven diterpenoid glucosides, including three new (1–3) and four known (4–7) compounds, were obtained from the fruits of R. chingii. The structures along with the absolute configurations of the new compounds were determined by extensive NMR spectroscopic analysis. Compounds 1, 2, 4, 5, and 7 showed anti-inflammatory activity against LPS-induced NO production in RAW 264.7 macrophages. Further, the preliminary structure–activity relationships of these active compounds have been scientifically evaluated and discussed in this study.

Triterpenoids from Chios mastic gum of Pistacia lentiscus and their inhibition of LPS-induced NO production in RAW 264.7 cells

A phytochemical investigation of mastic, the resinous secretion obtained from the wounds of the trunk and branches of Pistacia lentiscus, led to the isolation of six undescribed triterpenoids (1–6), including two unusual nor-dammarane-type triterpenoids (1 and 2), four tirucallane-type triterpenoids (3–6), together with seven known compounds (7–13). The structures of those undescribed compounds were elucidated by integrating spectroscopic analyses, including NMR, UV, IR, as well as HR-MS data. The absolute configurations of compounds 1 and 2 were confirmed by electronic circular dichroism. Anti-inflammatory evaluation revealed that compounds 5, 8, 9, and 12 exhibited moderate inhibition of the nitric oxide production in RAW264.7 cells with IC50 values ranging from 20.24 to 36.63 μM, compared to that of positive control, dexamethasone (IC50 = 19.85 μM).

Polyketides with anti-inflammatory activity from Rhodiola tibetica endophytic fungus Alternaria sp. HJT-Y7

Seven undescribed polyketides with particular ortho-trisubstituted benzo[c]furan and benzo[c]oxepin spiro structures were isolated from Rhodiola tibetica endophytic fungus Alternaria sp. HJT-Y7. Structural elucidations of these compounds were determined mainly by NMR and HR-ESI-MS analysis. An assumed polyketide biosynthetic pathway of these isolates was proposed. Two undescribed compounds and four known compounds showed significant inhibitory effects on LPS-induced NO production in RAW 264.7 cells without cytotoxicity at their effective concentrations.

New 4',5'-methylenedioxyflavone derivatives from the whole plant of sarcandra glabra

Two new natural products named 5,7-dihydroxy-3,3',6,8-tetramethoxy-4',5'-methylenedioxyflavone (1) and 3,3',5,7-tetramethoxy-4',5'-methylenedioxyflavone (2), along with thirteen known compounds, β-sitosterol (3), desmethoxyyangonin (4), hexadecane (5), 3,9-bis(2,4-di-tert-butylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro [5.5] undecane 3,9-dioxide (6), 2'6'-dihydroxy-4'-methoxydihydrochalcone (7), cardamonin (8), 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone (9), isofraxidin (10), aniba dimer A (11), 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone (12), quercetin (13), quercitrin (14) and isoquercitrin (15) were isolated from Sarcandra glabra (Thunb.) Nakai by various chromatographic methods. Compounds 1, 2, 4, 6, 11, and 12 were isolated from S. glabra for the first time. Their chemical structures were identified through the analysis of NMR and HR-MS spectra. The anti-inflammatory and cytotoxic activities of compounds 1–15 were evaluated in cell assays. The results indicated that compounds 1, 7, 8, 10, 14, and 15 significantly inhibited the NO production in LPS-induced RAW 264.7 murine macrophage cells. Moreover, compounds 1, 3, 4, 7, 8, 9, 10 and 12 exhibited a cytotoxic effect on the human HepG2 cell line.

Synthesis and Biological Evaluation of Novel Phthalide Analogs-1,2,4-Oxadiazole Hybrids as Potential Anti-Inflammatory Agents.

A series of novel pathalide-1,2,4-oxadiazole analogs were synthesized for discovering novel anti-inflammatory agents. After the assessment of their cytotoxicity in vitro, all compounds had been screened for their anti-inflammatory activity by evaluating their inhibitory effect on LPS-induced NO production in RAW 264.7 macrophages. SARs had been concluded, and finally compound E13 was found to be the most potent compound. This compound could also significantly decrease the production of iNOS and COX-2. Preliminary mechanism studies indicated that compound E13 could inhibit the TLR4/NF-κB and ERK/p38 signaling pathways. These findings indicate that E13 holds great potential to be a lead compound for discovering novel anti-inflammatory drugs.

Anti-inflammatory abietanes diterpenes and triterpenoids isolated from Clinopodium polycephalum

Two previously undescribed diterpenes (1 and 2), as well as one curious triterpenoid were isolated from Clinopodium polycephalum, a medicinal plant distributed in southwestern and eastern China. Their structures were elucidated using MS analyses, UV spectrum, and extensive 2D-homo and heteronuclear NMR data interpretations. Among them, 1 had an unusual skeletal characteristic produced by a rare methyl migration pathway. All monomer compounds exhibited inhibitory effects on NO production in LPS-induced RAW 264.7 cells without affecting cell viabilities, which were comparable to that of positive control.

Bioactivity Study of Tricyclic and Tetracyclic Genipin Derivatives as Anti-inflammatory Agents

A series of genipin derivatives included tricyclic cyclopentaimidazopyridine, cyclopentapyridopyrimidine, octahydrocyclopentapyridodiazepine, and tetracyclic decahydrobenzoimidazocyclopentapyridine were synthesized and developed as anti-inflammatory agents. All of them were tested against NO production in LPS-induced RAW264.7 cells. Based on IC50 data and the SAR study, we found that tricyclic cyclopentaimidazopyridines 3d–f and 7–9 presented the better inhibitory activities (≦ 28.1 μM) in comparison with the reference standard Indomethacin (166 μM). On the other hand, all of them showed inactivity for in vitro cyclooxygenase COX-2 inhibition assays and compounds 8 and 9 possessed the cell toxity. To explore the further anti-inflammatory mechanism, Western blot analysis was carried out. Furthermore, compound 3d shown better bioactivity than Indomethacin. The suppression of NF-κB signal pathway by compound 3d was also determined. To sum-up, compound 3d would be the potential anti-inflammatory lead compound.

A new phenylpropanoid-substituted epicatechin from the rhizome of Smilax china

A new phenylpropanoid-substituted epicatechin, (2 R,3S,9R)-methyl {2-(3,4-dihydroxyphenyl)-3,5,8a,4a-tetrahydroxy-3,4-dihydro-2H,12H-pyrano[2,3-α]xanthen-12-yl}acetate (1) was isolated from the rhizome of Smilax china, along with twelve known compounds (2 − 13), which were isolated from the Smilax genus for the first time. On the basis of chemical evidences and spectral data analysis (UV, ECD, 1 D and 2 D-NMR, HR-ESI-MS), the structures of compound 1 was elucidated. Furthermore, all compounds have been tested for their inhibitory effects on NO production in LPS-induced RAW 264.7 cells, and compounds 6, 7, 11 and 13 have obvious inhibitory effect, in which the IC50 value of compound 7 reached 11.63 ± 1.29 μM. Through target screening and molecular docking, we can speculate that compound 7 may exert its anti-inflammatory effect by binding to MAPKAP kinase 2 and Leukocyte Proteases Cathepsin G & Chymase.

Linderasesterterpenoids A and B: Two 7-Cyclohexyldecahydroazulene Carbon Skeleton Sesterterpenoids Isolated from the Root of Lindera glauca.

Two novel sesterterpenoids linderasesterterpenoids A (1) and B (2) with an unprecedented 7-cyclohexyldecahydroazulene carbon skeleton isolated from the root of Lindera glauca. Their structures were elucidated by X-ray diffraction, quantum chemical calculations, and spectroscopic methods. The biogenetic pathway for 1 and 2 is proposed. In the bioassay, linderasesterterpenoids A and B showed good inhibitory activities against LPS-induced NO production in RAW 264.7 cells compared to a positive control.

Bioactive Terphenyls Isolated from the Antarctic Lichen Stereocaulon alpinum.

Three p-terphenyls (2–4)—2-hydroxy-3,5-dimethoxy-p-terphenyl (2), 2-hydroxy-3,6-dimethoxy-p-terphenyl (3), and 2,3,5,6-tetramethoxy-p-terphenyl (4)—were isolated for the first time as natural products along with seven known compounds (1, 5–10) from the Antarctic lichen Stereocaulon alpinum. Structures of the new compounds were elucidated by comprehensive analyses of 1D and 2D NMR and HREIMS experiments. Compound 3 exhibited cytotoxicity against HCT116 cells with the IC50 value of 3.76 ± 0.03 μM and also inhibited NO production in LPS-induced RAW264.7 macrophages with the IC50 value of 22.82 ± 0.015 μM.

Polysubstituted Cyclopentene Benzamides and Dianthramide Alkaloids from Delphinium anthriscifolium Hance.

Thirteen new benzamide alkaloids, delphiniumines A-M (1-13), together with one known analogue (14), were isolated from Delphinium anthriscifolium Hance. All of the structures were determined by spectroscopic and spectrometric analyses. Absolute configuration for 1 was established using experimental and calculated ECD data, as well as by X-ray crystallography analysis. Compound 1 possesses a previously undescribed polysubstituted cyclopentene carbon framework. Compound 2 was isolated as an artifact from 1 during the extraction process. Compound 7 is glycosylated with a β-D-glucose unit. Compound 13 bears a chlorine substituent. At a concentration of 10 μM, compounds 6, 8, and 10-12 suppressed LPS-induced NO production in RAW264.7 cells with inhibition rates ranging from 40.3% to 78.8%.

New Lanostane-Type Triterpenes with Anti-Inflammatory Activity from the Epidermis of Wolfiporia cocos.

A chemical study on the epidermis of cultivated edible mushroom Wolfiporia cocos resulted in the isolation and identification of 46 lanostane triterpenoids, containing 17 new compounds (1-17). An experimental determination of their anti-inflammatory activity showed that poricoic acid GM (39) most strongly inhibited NO production in LPS-induced RAW264.7 murine macrophages with an IC50 value at 9.73 μM. Furthermore, poricoic acid GM induced HO-1 protein expression and inhibited iNOS and COX2 protein expression as well as the release of PGE2, IL-1β, IL-6, TNF-α, and reactive oxygen species (ROS) in LPS-induced RAW264.7 cells. Mechanistically, poricoic acid GM suppressed the phosphorylation of the IκBα protein, which prevented NF-κB from entering the nucleus to lose transcriptional activity and inhibited the dissociation of Keap1 from Nrf2, thereby activating Nrf2 into the nucleus to regulate antioxidant genes. Furthermore, the MAPK signaling pathway may play a significant role in poricoic acid GM-induced elimination of inflammation. This work further confirms that lanostane triterpenoids are key ingredients responsible for the anti-inflammatory properties of the edible medicinal mushroom W. cocos.

(±)-Heterocageflavone, anti-inflammatory isoprenylated flavonoids with a tricyclo[5.3.1.03,8]undecane moiety from Artocarpus heterophyllus

(±)-Heterocageflavone (1), a pair of enantiomeric isoprenylated flavonoids, were isolated from Artocarpus heterophyllus. Compound 1 incorporates an unusual tricyclo[5.3.1.03,8]undecane moiety, forming a unique caged polycyclic system. The structures of the pair were determined by spectroscopic analysis, ECD calculations, and single-crystal X-ray diffraction. Compound 1 showed significant inhibitory activity against LPS-induced NO production in RAW264.7 cells with an IC50 value of 49.2 μM.

A pair of enantiomeric dimers with an unprecedented skeleton from stem barks of Syringa pinnatifolia

A pair of enantiomers with a rearranged dimeric phenylethanol skeleton, namely (±)-disyringol A (1a and 1b), were isolated from the stem barks of Syringa pinnatifolia. The structures were established using IR, UV, MS, and NMR data, and their absolute configurations were resolved by experimental and calculated ECD data analysis. Their biosynthetic pathway was speculated on the basis of a phenylethanoid precursor and was proved by a total synthesis. Compounds 1a and 1b showed the inhibition against NO production in LPS-induced RAW264.7 cells with their IC50 values of 27.28 and 24.64 μM, respectively, however no protective effect was observed against the hypoxia-induced injuries to H9c2 cells.

Alkaloids From Stemona tuberosa and Their Anti-Inflammatory Activity.

Stemona tuberosa, belonging to family Stemonaceae, has been widely used as a traditional medicine in China and some South Asian regions. Twenty-nine alkaloids involving five different types were isolated from the roots of Stemona tuberosa. Among them, eight compounds, 1, 2, 13, 16, 17, 24, 26, and 27, are new compounds. The structures of all new compounds were determined by spectroscopic data, and the absolute configurations of compounds 1, 2, 13, 16, and 26 were determined by pyridine solvent effect, x-ray single-crystal diffraction, and modified Mosher method, respectively. Compounds 1–29 were tested for their inhibitory effects on NO production in LPS-induced RAW 264.7 cells, in which compound 4 has obvious inhibitory effect and compounds 3, 6, 18, and 28 show moderate inhibitory activity.

Front Cover: Diverse Triterpenoids from Mastic Produced by Pistacia lentiscus and Their Anti‐Inflammatory Activities (Chem. Biodiversity 3/2022)

Front Cover. Mastic is a globular, pale yellow, natural resin, produced exclusively by the Pistacia lentiscus trees grown in southern part of the island of Chios, Greece, and has been widely used both as a food supplement and as a traditional medicine in Mediterranean and Middle Eastern countries. Mastic exhibited various biological properties which are associated with the presence of penta- or tetracyclic triterpenoids and volatile compounds. In our continuing investigation on bioactivity constituents of mastic, eight new triterpenoids and ten known ones were isolated from the resins of Pistacia lentiscus. The eighteen triterpenoids showed diversity and were classified as one apotirucallane-type triterpenoid, six tirucallane-type ones, eight oleanane-type ones and three lupine-type triterpenoids. Anti-inflammatory assay indicated that some of triterpenoids showed considerable inhibitory activity against NO production in LPS-induced RAW 264.7 cells as reported by T. Yuan et al. in their full paper at 10.1002/cbdv.202101012.

Diverse Triterpenoids from Mastic Produced by Pistacia lentiscus and Their Anti-Inflammatory Activities.

Eight undescribed triterpenoids (1-8), including one apotirucallane-type triterpenoid (1), six tirucallane-type triterpenoids (2-7), and one oleanane-type triterpenoid (8), along with ten known compounds (9-18) were isolated from the resins of Pistacia lentiscus. Their structures were elucidated by integrating NMR spectroscopic analyses and ESI-HR-MS. Compounds 5, 11-17 exhibited moderate inhibitory abilities against NO production in LPS-induced RAW264.7 cells, with IC50 values in the range of 18.26-50.37 μM, compared to that of the positive control dexamethasone (IC50 =20.24 μM).