Comprehensive screening and separation of cyclooxygenase-2 inhibitors from Pterocephalus hookeri by affinity solid-phase extraction coupled with preparative high-performance liquid chromatography.

Pterocephalus hookeri, a classical Tibetan herb, is mainly used to treat rheumatoid arthritis (RA) and contains various constituents potentially with cyclooxygenase-2 (COX-2) selective inhibition. A novel strategy for screening and target separating COX-2 inhibitors from the extracts of P. hookeri based on affinity solid-phase extraction (ASPE) column combined with preparative high-performance liquid chromatography (pre-HPLC) was successfully developed. The potential COX-2 inhibitors of P. hookeri were screened and recognized by the ASPE-HPLC system, which strategy is to analyze the compounds isolated by the ASPE column. Then, the active compounds were targeted separated by pre-HPLC according to real-time chromatograms. The control drugs celecoxib and glipizide were analyzed to verify the specificity and accuracy of the developed method. As a result, two pure compounds with COX-2 binding affinities were successfully separated from P. hookeri. They were characterized as swertisin and scopoletin using 1H- and 13C NMR spectroscopy, and the in vitro COX-2 inhibitory activities were verified. Compounds with COX-2 inhibitory activities could be screened and targeted separated from crude extracts by this strategy, which indicated that the proposed method was feasible, robust and effective for rapid separation of COX-2 inhibitors from natural products.

Sonapatha (Oroxylum indicum) mediates cytotoxicity in cultured HeLa cells by inducing apoptosis and suppressing NF-κB, COX-2, RASSF7 and NRF2.

Oroxylum indicum (Sonapatha) is traditionally used to cure several human ailments. Therefore, the cell killing effect of chloroform, ethanol, and water extracts of Sonapatha was studied in cultured HeLa cells treated with 0-100 µg/mL of these extracts/doxorubicin by MTT assay. Since ethanol extract was most cytotoxic its effect was further investigated by clonogenic, apoptosis, necrosis, and lactate dehydrogenase assays. The mechanism of cytotoxicity of Sonapatha was determined at the molecular level by estimation of caspase 8 and 3 activities and Western blot analysis of NF-κB, COX-2, Nrf2, and RASSF7 which are overexpressed in neoplastic cells. HeLa cells treated with Sonapatha extract exhibited a concentration and time-dependent rise in the cytotoxicity as indicated by the MTT assay. Ethanol extract of Sonapatha (0, 20, 40, and 80 µg/mL) reduced clonogenicity, increased DNA fragmentation, apoptotic and necrotic indices, lactate dehydrogenase release, caspase 8 and 3 activities and inhibited the overexpression of NF-κB, COX-2, Nrf2, and RASSF7 in HeLa cells concentration-dependently.

Diverse anti-inflammation and anti-cancer polyketides isolated from the endophytic fungi Alternaria sp. MG1.

Six new polyketides, alternaritins A-D [(±)-1-4] and isoxanalteric acid I (8), and 25 known Alternaria toxins were isolated from the culture of an endophytic fungi Alternaria sp. MG1. 3 is a rare fungal metabolite. 6 is a new natural product, and 5, 7, and 9 are known previously but their absolute configurations have not been determined. Three enantiomers [(±)-1, (±)-7, and (±)-15] were separated via chiral HPLC resolution. The structures of those polyketides (1-9) were elucidated by spectrometric analysis using MS and NMR. The absolute configurations were established using X-ray diffraction analysis and statistical comparative analysis of the experimental ECD and OR data, in conjunction with quantum mechanical calculations. All of the compounds were evaluated for their bioactivities. Known compound 27 exerted the most potent cytotoxic activities against HT-1080 and NCI-H1299 cell lines. The new compounds, 2 and 3, showed moderate inhibition on COX-2, while a pair of isomers, 8 and 9, exhibited medium activity on COX-2 and uropathogenic Escherichia coli.

Zanthoxylum zanthoxyloides inhibits lipopolysaccharide- and synthetic hemozoin-induced neuroinflammation in BV-2 microglia: roles of NF-κB transcription factor and NLRP3 inflammasome activation.

OBJECTIVES: The effects of a root extract of Zanthoxylum zanthoxyloides on neuroinflammation in BV-2 microglia stimulated with LPS and hemozoin were investigated. METHODS: ELISA, enzyme immunoassay and Griess assay were used to evaluate levels of cytokines, PGE2 and NO in culture supernatants, respectively. Microglia-mediated neurotoxicity was evaluated using a BV-2 microglia-HT-22 neuron transwell co-culture. KEY FINDINGS: Treatment with Z. zanthoxyloides caused reduced elevated levels of TNFα, IL-6, IL-1ß, NO and PGE2, while increasing the levels of IL-10. In addition, there were reduced levels of iNOS and COX-2 proteins. This was accompanied by a prevention of microglia-mediated damage to HT-22 mouse hippocampal neurons. Z. zanthoxyloides reduced elevated levels of phospho-IκB and phospho-p65, while preventing degradation of IκB protein and DNA binding of p65. Further mechanistic studies revealed that Z. zanthoxyloides reduced the levels of pro-IL-1ß and IL-1ß in hemozoin-activated BV-2 microglia. This was accompanied by a reduction in caspase-1 activity and NLRP3 protein expression. Bioassay-guided fractionation resulted in the isolation of skimmianine as an anti-inflammatory compound in Z. zanthoxyloides. CONCLUSION: This is the first report showing the inhibition of neuroinflammation in LPS- and hemozoin-activated BV-2 microglia by the root extract of Z. zanthoxyloides by targeting the activation of both NF-κB and NLRP3 inflammasome.

A new prenylated benzoquinone from Cyathocalyx pruniferus abrogates LPS-induced inflammatory responses associated with PGE2, COX-2 and cytokines biosynthesis in human plasma.

In our previous laboratory findings, Cyathocalyx pruniferus extracts exhibited platelet-activating factor inhibition, suggesting their anti-inflammatory potential. Hence, this study was designed with the aim to isolate phyto-constituents from C. pruniferus with potent anti-inflammatory activities. Column and volume liquid chromatography were used for isolation of phyto-constituents. The structure elucidation was carried out using spectroscopic analysis (HRESI-MS, 1H and 13C-NMR) and compared with published literature. For cytotoxicity analysis, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide assay was performed on peripheral blood mononuclear cells. Anti-inflammatory activities were evaluated against the levels of inflammatory cytokines (IL-1ß and IL-6), prostaglandin-E2 (PGE2) and cyclooxegenase-2 (COX-2), in lipopolysaccharide (LPS)-induced human plasma using ELISA and radioimmunoassay (RIA). The chromatographic purification of methanol leaves extract afforded 13 (1-13) secondary metabolites. Additionally, cytotoxicity analysis suggested that isolates were non-cytotoxic at 100 µM. In anti-inflammatory evaluation, 2-octaprenyl-1, 4-benzoquinone (5) produced strong (≥ 70%) inhibition of PGE2, COX-2, IL-1ß and IL-6 at 50 µM. Moreover, 2-octaprenyl-1,4-benzoquinone (5) exhibited concentration-dependent inhibition with IC50 values (µM) of 11.21, 6.61, 2.20 and 3.56 as compared to controls; indomethacin for PGE2 (11.84) and dexamethasone in COX-2 (5.19), IL-1ß (1.83) and IL-6 (3.76) analysis, respectively. In conclusion, two new compounds including 2-octaprenyl-1, 4-benzoquinone (5) and 14-methyloctadec-1-ene (6) are reported for the first time from plant species. Additionally, 2-octaprenyl-1, 4-benzoquinone (5) dose-dependently suppressed the production of pro-inflammatory mediators involved in acute and chronic inflammation at non-cytotoxic concentrations.

Evaluation of Selective COX-2 Inhibition and In Silico Study of Kuwanon Derivatives Isolated from Morus alba.

Six kuwanon derivatives (A/B/C/E/H/J) extracted from the roots of Morus alba L. were evaluated to determine their cyclooxygenase (COX)-1 and 2 inhibitory effects. Cyclooxygenase (COX) is known as the target enzyme of nonsteroidal anti-inflammatory drugs (NSAIDs), which are the most widely used therapeutic agents for pain and inflammation. Among six kuwanon derivatives, kuwanon A showed selective COX-2 inhibitory activity, almost equivalent to that of celecoxib, a known COX inhibitor. Kuwanon A showed high COX-2 inhibitory activity (IC50 = 14 µM) and a selectivity index (SI) range of >7.1, comparable to celecoxib (SI > 6.3). To understand the mechanisms underlying this effect, we performed docking simulations, fragment molecular orbital (FMO) calculations, and pair interaction energy decomposition analysis (PIEDA) at the quantum-mechanical level. As a result, kuwanon A had the strongest interaction with Arg120 and Tyr355 at the gate of the COX active site (-7.044 kcal/mol) and with Val89 in the membrane-binding domain (-6.599 kcal/mol). In addition, kuwanon A closely bound to Val89, His90, and Ser119, which are residues at the entrance and exit routes of the COX active site (4.329 Å). FMO calculations and PIEDA well supported the COX-2 selective inhibitory action of kuwanon A. It showed that the simulation and modeling results and experimental evidence were consistent.

Discovery of cyclooxygenase-2 inhibitors from Kadsura coccinea by affinity ultrafiltration mass spectrometry and the anti-inflammatory activity.

The medicinal plant Kadsura coccinea distributing in South China, was widely used for reducing inflammation and relieving pain. Previous study in our laboratory had proved the significant therapeutic effects of K. coccinea extract on adjuvant arthritis rats. To explore the responsible components and possible mechanisms, an AUF-HPLC-Q-TOF/ MS method was employed for screening and characterizing COX-2 ligands from K. coccinea stems for the first time. Meanwhile, the molecular docking was performed to simulate the binding modes for ligands and COX-2, the cell-free enzyme activity assay was applied to verify the direct COX-2 inhibition of potential inhibitors, and the cell-based study on COX-2 expression was to evaluate the anti-inflammatory effect of (+)-Anwulignan. As a result, the potential COX-2 inhibitor (+)-Anwulignan significantly suppressing COX-2 expressions in LPS signaling pathways might be a good candidate for anti-inflammation and analgesia. In conclusion, AUF mass spectrometry combining the molecular docking and bioassays in vitro was an efficient approach for discovering enzyme inhibitors from traditional herbs.

Bioactive aromatic butenolides from a mangrove sediment originated fungal species, Aspergillus terreus SCAU011.

Seven new compounds including five aromatic butenolide analogues (1-5), one quinazolinone alkaloid (6) and one benzoic acid derivative (7), along with eleven known co-metabolites (8-18), were isolated from Aspergillus terreus SCAU011, a fungus from the rhizosphere sediment of a mangrove plant Rhizophora stylosa. The structures of these isolates were established by a combination of MS, NMR and ECD data analyses, as well as chemical method. Compound 3 is a rare ring-open aromatic butenolide, while 6 represents the first natural ring-open benzomalvin-type quinazolinone alkaloid. Also, the previously reported structures for asperlides A-C were proposed to be revised in the present work. The COX-2 inhibitory, α-glucosidase inhibitory, antioxidant and antibacterial activities of all the compounds were assessed. While compounds 4, 6, 11 and 18 exhibited better COX-2 inhibitory activity than the positive control celecoxib, compounds 9 and 10 showed significant α-glucosidase inhibitory activity with IC50 values of 56.1 and 12.9 µM, respectively. Meanwhile, half of the tested samples (1, 8-11 and 15-17) exerted similar or better antioxidant activity compared with the reference drug curcumin, and compounds 3, 9, 17 and 18 displayed moderate antibacterial effect against Staphylococcus aureus.

A hybrid platform featuring nanomagnetic ligand fishing for discovering COX-2 selective inhibitors from aerial part of Saussurea laniceps Hand.-Mazz.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea laniceps Hand.-Mazz. (Compositae) is a representative "snow lotus" herb well known in Chinese folk medicine to treat inflammation-related diseases such as arthritis. S. laniceps (SL) shows anti-inflammatory and analgesic potencies and contains various constituents potentially with cyclooxygenase-2 (COX-2) selective inhibition. The herb is a valuable source of natural alternatives to synthetic COX-2 selective nonsteroidal anti-inflammatory drugs, a common medication for rheumatoid arthritis (RA) and osteoarthritis (OA) reported with serious cardiovascular side effects. AIM OF THE STUDY: Based on an innovative drug screening platform, this study aimed to discover safe, effective COX-2 selective inhibitors from SL. MATERIALS AND METHODS: An enzyme-anchored nanomagnetic fishing assay was developed to separate COX-2 ligands from SL. Cell and animal models of cardiomyocytes, lipopolysaccharide-stimulated macrophages, rat adjuvant-induced arthritis, and anterior cruciate ligament transection-induced OA rats, were adopted to screen the single/combined ligands regarding toxicity and bioactivity levels. Molecular docking was employed to unravel binding mechanisms of the ligands towards COX-1 and COX-2. RESULTS: Four COX-2 selective compounds were separated from SL using optimized COX-2-functionalized magnetic nanoparticles. All the four ligands were proved with evidently lower cardiotoxicity both in vitro and in vivo than celecoxib, a known COX-2 selective inhibitor. Two ligands, scopoletin and syringin, exhibited potent anti-arthritic activities in rat models of RA and OA by alleviating clinical statuses, immune responses, and joint pathological features; their optimum combination ratio was discovered with stronger remedial effects on rat OA than single administrations. The COX-1/2 binding modes of the two phytochemicals contributed to explain their cardiac safety and therapeutic performances. CONCLUSIONS: The screened chemicals are promising to be developed as COX-2 selective inhibitors as part of treating RA and OA. The hybrid strategy for discovering therapeutic agents from SL is shown here to be efficient; it should be equally valuable for finding other active chemicals in other natural sources.

Dendropanax dentiger (Harms) Merr. root and its major constituents exert therapeutic effect on adjuvant-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Dendropanax dentiger (Harms) Merr. is a pivotal folk Chinese medicine against rheumatoid arthritis (RA) with no scientific validation. AIM OF THE STUDY: This study was conducted to explore the anti-RA effect of the D. dentiger extract on complete Freund's adjuvant-induced arthritis (AIA) in rats and identified its major bio-constituents. MATERIALS AND METHODS: Dendropanax dentiger roots extracts (127.5, 255.0 and 510.0 mg/kg, once daily) were orally at day 7 post-administration adjuvant and lasting for 22 days. The therapeutic effects of D. dentiger roots extract on AIA rats were investigated by body weight growth, arthritis score, thymus and spleen indices, and histopathological analysis. Moreover, the levels of rheumatoid factor (RF), C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), IL-4, IL-6, IL-10, IL-17, cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and matrix metalloproteinase-2 (MMP-2) were also evaluated. Finally, the major constituents were isolated and identified from D. dentiger roots extract with COX-2 inhibitory and antioxidant activities. RESULTS: Dendropanax dentiger roots extract remarkably alleviated the histological lesions of knee joint, increased body weight growth, decreased arthritis score, and reduced thymus and spleen indices in model rats. In parallel, the levels of RF, CRP, TNF-α, IL-1ß, IL-6, IL-17, COX-2, 5-LOX and MMP-2 were observably downregulated, while the levels of IL-4 and IL-10 were prominently upregulated in D. dentiger roots extract-treated rats. Meanwhile, 14 compounds were isolated and identified from D. dentiger roots extract, and four phenol derivatives (1, 4, 6 and 7) exhibited remarkable COX-2 inhibitory and antioxidant activities. CONCLUSIONS: Dendropanax dentiger roots extract possessed persuasive anti-RA effect may be partly responsible for phenol derivatives via modulation of inflammatory biomarkers, and supports the traditional folk use of D. dentiger in China.

Kirenol, darutoside and hesperidin contribute to the anti-inflammatory and analgesic activities of Siegesbeckia pubescens makino by inhibiting COX-2 expression and inflammatory cell infiltration.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese traditional medicine of Siegesbeckia pubescens Makino (SM), which has the effect of healing rheumatism and promoting joint health, is often used to treat rheumatoid arthritis and ischemic stroke. AIM OF THE STUDY: To clarify the mechanisms underlying the anti-inflammatory and analgesic influence of active components in the ethanol extract of Siegesbeckia pubescens Makino (ESM). MATERIALS AND METHODS: The active ingredients in the ESM were identified practicing high-performance liquid chromatography-diode array detection (HPLC-DAD). Four models including xylene-induced ear oedema, complete Freund's adjuvant (CFA)-induced hind paw oedema, acetic acid-induced pain writhing and lipopolysaccharide (LPS)-induced RAW264.7 cell migration, were used to clarify the anti-inflammatory and analgesic mechanisms of the active ingredients in the ESM. RESULTS: (1) Three active ingredients of kirenol, darutoside and hesperidin were identified in the ESM, with relative proportion of 0.6%, 0.2% and 0.01%, respectively; hesperidin was reported for the first time in the ESM. (2) Both the ESM and its active ingredients could effectively alleviate the degree of swelling of the auricle and toes, increase the threshold of heat pain, decrease the overexpression of inflammatory protein cyclooxygenase-2 (COX-2) in the skin tissue of the tested parts of the toes, and reduce the number of writhes induced by acetic acid in mice. (3) ESM and its active ingredients also dose-dependently inhibited the migration of RAW264.7 cells. CONCLUSIONS: ESM and its active ingredients can effectively attenuate the expression of inflammatory factors induced by chemical inflammation, prevent the infiltration of inflammatory cells, and exert good anti-inflammatory and antinociceptive activities.

Bioassay-guided isolation of cyclooxygenase-2 inhibitory and antioxidant phenylpropanoid derivatives from the roots of Dendropanax dentiger.

The root of Dendropanax dentiger (Harms) Merr. is a traditional Chinese medicine that has been used to treat inflammation-related diseases with little scientific validation. In this study, a bioassay-guided phytochemical investigation of D. dentiger led to the isolation of 19 phenylpropanoid derivatives including one new compound (1) and 18 known ones (2-19). Their structures were elucidated by NMR and HRMS as well as comparison with literature data. The ability of cyclooxygenase-2 (COX-2) inhibition and antioxidant of all isolated compounds were measured in vitro. Chlorogenic acid derivatives (14-19) exhibited outstanding COX-2 inhibitory (IC50 = 5.1-93.4 µM) and antioxidant (IC50 = 13.2-31.9 µM) activities. Moreover, the tight structure-activities relationships were proposed. This is the first report on the COX-2 inhibitory activity of phenylpropanoids and D. dentiger.

Periplanetols A-F, phenolic compounds from Periplaneta americana with potent COX-2 inhibitory activity.

Six new compounds, periplanetols A - F (1-4, 6 and 7), a compound isolated from natural origin for the first time (5), and nine known ones (8-16) were isolated from the 70% ethanol extract of the whole bodies of Periplaneta americana. Their structures including absolute configurations were unambiguously identified by comprehensive spectroscopic analyses and computational methods. Biological evaluation toward COX-2 inhibition revealed that compounds 1, 2, and 10 could inhibit COX-2 activity with the IC50 values of 768.0 nM, 617.7 nM, and 599.5 nM respectively, indicating their potential in developping novel agents against inflammation related disorders.

Racemic xanthine and dihydroxydopamine conjugates from Cyclopelta parva and their COX-2 inhibitory activity.

Seven new compounds including three pairs of enantiomeric xanthine analogues (1-3), a pair of enantiomeric hypoxanthine analogue (4), and three pairs of enantiomeric N-acetyldopamine dimers (6-8), together with a known one (5) were isolated from the insect Cyclopelta parva. Their structures including absolute configurations were assigned by using spectroscopic and computational methods. Chiral HPLC was used to separate racemic 1-8. Biological evaluation found that 6b and 7a are potent COX-2 inhibitory agents with IC50 values at 385.2 nM and 868.8 nM respectively.

pH-Zone-refining counter-current chromatography for two new lipo-alkaloids separated from refined alkaline extraction of Kusnezoff monkshood root.

An efficient and refined method for the separation of six aconitine-type alkaloids from the alkaline prepared "Kusnezoff monkshood root" was established. It is the first study that two new lipo-alkaloids were successfully isolated from refined sample by pH-zone-refining counter-current chromatography rather than synthetic method. It was of interest that a great deal of lipo-alkaloids was produced in crude extract from the alkalization of "Kusnezoff monkshood root." A refined sample method was proposed to enrich two types of alkaloids by liquid-liquid extraction, i.e. lipo-alkaloids and monoester-diterpenoid alkaloids. The pH-zone-refining counter-current chromatography was performed with an optimized two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:5:4:5, v/v), where upper organic phase was added to 3 mmol/L triethylamine as a retainer and lower aqueous mobile phase was added to 3 mmol/L hydrochloric acid as an eluter. As a result, six aconitum alkaloids, including two lipo-alkaloids (8-lino-14-benzoylaconine, 8-pal-14-benzoylaconine), three monoester-diterpenoid alkaloids (14-benzoylmesaconine, 14-benzoylaconine, beyzoyldeoxyaconine), and one aconine alkaloid (neoline) were acquired from the plant at the same time. The anti-inflammatory activities of the two new lipo-alkaloids were compared to the six alkaloids in vitro, in cyclo-oxygen-ase-2 inhibition assays. The separation mechanism of six alkaloids by pH-zone-refining counter-current chromatography was illustrated.

Anti-inflammatory effects of mulberry (Morus alba L.) root bark and its active compounds.

Mulberry (Morus alba L.) root bark (MRB) was extracted using methanol and the extracts were subjected to tests of anti-inflammatory effects. The ethyl acetate fraction demonstrated the best anti-inflammatory effects. Purified compounds, sanggenon B, albanol B and sanggenon D, showed inhibitory effects on NO production in LPS-stimulated RAW264.7 cells and albanol B demonstrated the best anti-inflammatory effects. Regarding the underlying molecular mechanisms, further investigations showed that treatments with Albanol B reduced production of pro-inflammatory cytokines and decreased expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). These results would contribute to development of novel anti-inflammatory drugs from MRB.

Prenylated benzenepropanoic acid analogues from the Citrus grandis (L.) Osbeck and their anti-neuroinflammatory activity.

Phytochemical studies of the air-dried pericarp of Citrus grandis led to the isolation of four new compounds including three prenylated benzenepropanoic acids (2, 3 and 5) and one alkamidic glycoside (6), together with ten known compounds (1, 4 and 7-14). The structures of these compounds were determined by the NMR spectroscopy, optical rotation data and modified Mosher's method. Meanwhile, the anti-neuroinflammatory activities of all isolated compounds were evaluated by detecting the production of nitric oxide (NO) in LPS-stimulated BV2 cells. The results showed that compounds 1, 2, 5 and 13 exhibited strong inhibition effects on NO production in LPS-stimulated BV2 cells. Mechanistically, compounds 1, 2 and 5 could suppress the expressions of iNOS. In addition, compounds 1, 2 and 5 also showed obvious inhibition effects on COX-2 expression, another vital enzyme in the inflammation process, in LPS-treated BV2 cells. These findings shed light on the potent anti-neuroinflammatory effects of Citrus grandis.

Sesquiterpenoids from Artemisia argyi and their COXs inhibitory activities.

Four new sesquiterpene lactones, named artemargyinolides A-D (1-4), and seven known sesquiterpenoids (5-11) were isolated from Artemisia argyi. Their structures were elucidated based on the extensive analysis of spectroscopic data. The absolute configuration of 1 was assigned by X-ray crystallographic analysis. Compound 1 is an unprecedented sesquiterpene dimer-monoterpene lactone. The cyclooxygenases (COX-1 and COX-2) inhibitory activities of all isolated compounds were evaluated. Compounds 1, 2, 4, and 6-11 showed inhibitory activity against COX-2 with IC50 values ranging from 35.78 ±â€¯0.39 to 256.07 ±â€¯0.38 µM. However, compounds 7, 9, and 10 exhibited weak inhibitory activity against COX-1 with IC50 values of 465.70 ±â€¯1.53, 281.43 ±â€¯3.56, and 490.45 ±â€¯6.07 µM, respectively. Other compounds are inactive against COX-1. Therefore, compounds 1, 2, 4, 6, 8, and 11 displayed selective COX-2 inhibitory activity.

Screening cyclooxygenase-2 inhibitors from Andrographis paniculata to treat inflammation based on bio-affinity ultrafiltration coupled with UPLC-Q-TOF-MS.

The cyclooxygenase-2 (COX-2) is a key enzyme in the synthesis of prostaglandins, its inhibitors are effective for the treatment of inflammation. In this study, an analytical method based on bio-affinity ultrafiltration coupled with ultra performance liquid chromatography and quadrupole-time-of-flight mass spectrometry (BAUF-UPLC-Q-TOF-MS) was established for rapidly screening and identifying COX-2 ligands. Meanwhile, the specificity of the method was verified by denatured COX-2 and inactive compound. Next, the biological activity of ligands screened was proved by enzyme inhibition assay. The preferred binding modes for these COX-2 inhibitors were then determined by molecular docking. Finally, network pharmacology was used to explore the pathways involved anti-inflammatory effects. As a result, five COX-2 inhibitors were selected in the extract of Andrographis Herba (AH), including andrographolide (1), 14-deoxy-11,12-didehydroandrographiside (2), andrographidine E (3), andrographidine D (4), and deoxyandrographolide (5). Among them, compounds 2, 3, 4 and 5 were reported to have COX-2 inhibitory activity for the first time. The result of COX-2 inhibition assay showed that compound 3 had an IC50 of 19 µM, compounds 2 and 5 had an IC50 of >200 µM. And each ligand could bind to multiple amino acid residues of COX-2 based on molecular docking. At last, combined with network pharmacology, these ligands could exert anti-inflammatory effects through three pathways related to COX-2, arachidonic acid metabolism, synthesis of prostaglandins, and prostanoid ligand receptors. The method established in the study could be used to rapidly screen other enzyme inhibitors in complex mixtures, and help to understand the mechanism of action.

New cyathane diterpenoids with neurotrophic and anti-neuroinflammatory activity from the bird's nest fungus Cyathus africanus.

Eleven new cyathane diterpenoids, designated cyafricanins A-K (1-11), were isolated from the culture broth of the baisidiomycete Cyathus africanus (Nidulariaceae, Bird's nest fungi). Their structures were elucidated by comprehensive analysis of their NMR and HRESIMS data. Cyafricanins A (1) was found to possess an unusual 3,4-seco­carbon skeleton. All compounds were evaluated for their neurotrophic activity in PC-12 cells and anti-neuroinflammatory activity in BV2 microglia cells. All of the diterpenoids showed nerve growth factor induced neurite outgrowth-promoting activity at concentration of 20 µM. Among them, cyafricanin B (2) and cyafricanin G (7) exhibited promising neurotrophic activity, and cyafricanin A (1) showed strong inhibitory effects on both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Furthermore, molecular docking studies revealed that cyafricanin A (1) showed strong interactions with the iNOs protein in the active cavity.