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.

Phenolic composition, antioxidant capacity and physical characterization of ten blackcurrant (Ribes nigrum) cultivars, their juices, and the inhibition of type 2 diabetes and inflammation biochemical markers.

The objective was to analyze the phenolic composition, antioxidant capacity, and physical characteristics of 10 blackcurrant cultivars, their juices, and the enzymatic inhibition of dipeptidyl peptidase-IV, α-amylase, α-glucosidase, nitric oxide synthase, and cyclooxygenase-2. Fruit masses ranged from 0.47 to 1.22 g and diameters from 7.42 to 14.42 mm. For the juices, pH ranged from 2.80 to 2.96, soluble solids from 11.33% to 17.5%, total acidity from 3.17 to 4.26 g/100 mL, and viscosity from 1.28 to 273.83 mPa·s. Total anthocyanins (TA) ranged from 1.81 to 5.48 mg eq cyanidin 3-O-glucoside/100 g, total polyphenols (TP) from 7.67 to 39.70 mg eq gallic acid/100 g, total condensed tannins from 3.24 to 7.76 g eq catechin/100 g, and antioxidant capacity from 219.24 to 499.26 µmol eq Trolox/100 g. Juices of the cultivars Coronet and Consort contained the highest levels of TA, TP, and antioxidants. Whistler cultivar contained high concentrations of major anthocyanins. Juices from all cultivars favorably inhibited the activities of enzymes used as surrogate biochemical markers for T2 diabetes and inflammation.

Screening and isolation of cyclooxygenase-2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry, and complex chromatography.

Nonsteroidal anti-inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti-inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase-2. The cyclooxygenase-2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase-2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi-preparative liquid chromatography was developed for the efficient scaled-up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase-2 inhibitors from complex samples. This could be used as an efficient method for the large-scale production of functional ingredients.

Screening for anti-proliferative and anti-inflammatory components from Rhamnus davurica Pall. using bio-affinity ultrafiltration with multiple drug targets.

Rhamnus davurica Pall. (R. davurica) has been used as a traditional medicine for many years in China and abroad and shown a wide spectrum of biological activities. Previously, we reported the phytochemical fingerprinting profile of R. davurica, its distinct anti-proliferative activities against HT-29 and SGC-7901 cell lines, and the topoisomerase I (Top I) ligands based on bio-affinity ultrafiltration and HPLC-MS (UF-HPLC-MS). Nevertheless, among the 32 peaks detected in the fingerprinting profile, the common bioactive constituents responsible for the anti-inflammatory and anti-proliferative activities in the extracts remain elusive. To further explore the specific responsible components for their diversified activities and their potential action targets/mechanisms, the method based on bio-affinity UF-HPLC-MS using therapeutic targets like Top I and cyclooxygenase 2 (COX-2) was established to rapidly screen and identify the ligands binding to these known target enzymes. As a result, 12 components were revealed as potential Top I ligands along with 11 components as potential COX-2 ligands, where several components were revealed to possess both activities. Further validations of these bioactive components have also been conducted and confirmed their highlighted activities. This integrated method of UF-HPLC-MS exhibits high efficiency in rapidly screening for multi-target bioactive components responsible for multiple pharmacological effects from the complex natural products and could be very useful to explain the complex action mechanisms of herb medicines in a complex multi-component and multi-target mode at the molecular level. Graphical abstract Schematic diagram of UF-HPLC-MS assay to screen for Top I and COX-2 ligands. The principle of the assay usually involves the following steps: incubation, ultrafiltration, and identification.

Flavonoid composition of orange peel and its association with antioxidant and anti-inflammatory activities.

Dried citrus peel derived from Citrus reticulata, also called "chenpi", possesses a complex mixture of flavonoids and has a history of traditional use to treat a variety of digestive disorders. We compared three sources of conventional chenpi from California (USA), Guangxi, Zhejiang, and two sources of "nchenpi", which contain greater nobiletin content, from Sichuan and Xinhui (China). Xinhui orange peel extract (OPE) had highest content of polymethoxylated flavones, along with greatest capacity to scavenge 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,2-diphenyl-1-pcrylhydrazyl (DPPH), and 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) radicals and nitric oxide (NO). OPE also had higher NO, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) inhibitory activity than an equivalent mixture of flavonoids (P<0.05). In conclusion, nobiletin is a good chemical marker for assessing the anti-inflammatory potential of OPE from different sources. Obtaining "nchenpi" from either Sichuan or Xinhui provided potentially superior health benefits compared to conventional chenpi sources.

Antioxidant and anti-inflammatory properties of an aqueous cyanophyta extract derived from Arthrospira platensis: contribution to bioactivities by the non-phycocyanin aqueous fraction.

The goal for this work was to characterize basic biological properties of a novel Arthrospira platensis-based aqueous cyanophyta extract (ACE), enriched in the known anti-inflammatory cyclooxygenase-2 (COX-2) inhibitor phycocyanin (PC), but also containing a high level of non-PC bioactive compounds. Antioxidant properties were tested in parallel in the Folin-Ciocalteu assay (chemical antioxidant capacity) and in the cellular antioxidant protection (CAP-e) bioassay, where both the PC and the non-PC fractions contributed to the antioxidant capacity and CAP of ACE. In contrast to the COX-2 inhibition seen in the presence of PC, the inhibition of enzymatic activity of the inflammatory mediator Lipoxygenase was associated specifically with the non-PC fraction of ACE. Inhibition of formation of reactive oxygen species (ROS) was evaluated using polymorphonuclear cells from healthy human donors. The inhibition of ROS formation was seen for both the PC and non-PC fractions, with ACE showing the most robust effect. The effects of PC, non-PC, and ACE on clotting and clot lysing was tested using a modified Euglobulin fibrinolytic assay in vitro. In the presence of PC, non-PC, and ACE, the time for clot formation and lysis was not affected; however, the clots were significantly more robust. This effect was statistically significant (p<.05) at doses between 125-500 µg/mL, and returned to baseline at lower doses. Both PC and the non-PC fraction contributed to the antioxidant properties and anti-inflammatory effects, without a negative impact on blood clotting in vitro. This suggests a potential benefit for the consumable ACE extract in assisting the reduction of inflammatory conditions.

Anti-inflammatory activity of grains of paradise (Aframomum melegueta Schum) extract.

The ethanolic extract of grains of paradise (Aframomum melegueta Schum, Zingiberaceae) has been evaluated for inhibitory activity on cyclooxygenase-2 (COX-2) enzyme, in vivo for the anti-inflammatory activity and expression of several pro-inflammatory genes. Bioactivity-guided fractionation showed that the most active COX-2 inhibitory compound in the extract was [6]-paradol. [6]-Shogaol, another compound from the extract, was the most active inhibitory compound in pro-inflammatory gene expression assays. In a rat paw edema model, the whole extract reduced inflammation by 49% at 1000 mg/kg. Major gingerols from the extract [6]-paradol, [6]-gingerol, and [6]-shogaol reduced inflammation by 20, 25 and 38%. respectively when administered individually at a dose of 150 mg/kg. [6]-Shogaol efficacy was at the level of aspirin, used as a positive control. Grains of paradise extract has demonstrated an anti-inflammatory activity, which is in part due to the inhibition of COX-2 enzyme activity and expression of pro-inflammatory genes.

Shuang-Huang-Lian exerts anti-inflammatory and anti-oxidative activities in lipopolysaccharide-stimulated murine alveolar macrophages.

Shuang-Huang-Lian (SHL) is a traditional Chinese compound formula prepared from Lonicerae Japonicae Flos, Scutellariae Radix and Fructus Forsythiae. In this study, we demonstrate the anti-inflammatory and anti-oxidative activities of SHL in lipopolysaccharide (LPS)-stimulated murine alveolar macrophages (MH-S). SHL significantly reduces the transcriptional and translational levels of iNOS and COX-2 as well as the production of NO and prostaglandin E2 (PGE2). It also suppresses the transcription and translation of inflammatory cytokines production, such as TNF-α, IL-1ß and IL-6. These inhibitory effects mainly act via ERK1/2- and p38-mediated AP-1 rather than the NF-κB pathway. In parallel with the anti-inflammatory activity, SHL suppresses LPS-induced intracellular total ROS levels by weakening NADPH oxidase activity, enhancing SOD activity and increasing GSH content. In addition, SHL directly scavenges OH and O2(-). Thus, our study elucidates the anti-inflammatory and anti-oxidative mechanisms of SHL in LPS-stimulated MH-S.

HPLC-PDA analysis and anti-inflammatory effects of Mori Cortex Radicis.

Mori Cortex Radicis (MCR, Moraceae) is used traditionally in the treatment ofjaundice, hematemesis, edema, and pollakisuria in Korea. In this study, the antiinflammatory effects of MCR extract were investigated using RAW 264.7 cells. The simultaneous analysis of five components present (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, and p-coumaric acid) in the MCR extract was performed using high-performance liquid chromatography (HPLC) coupled with photodiode array (PDA) detection. We determined the effects of MCR extract and its components on the production of nitric oxide (NO), prostaglandin E2 (PGE2), and mRNA expression of cyclooxygenase-2 (COX-2) in RAW 264.7 cells. MCR extract suppressed the production of NO and PGE2 in RAW 264.7 cells in a dose-dependent manner. None of the five components of the MCR extract had any influence on the production of NO. However, caffeic acid and p-coumaric acid inhibited the production of PGE2 and mRNA expression of COX-2 in RAW 264.7 cells. Our results suggest that MCR extract may offer potential as a therapeutic agent for the treatment of inflammation. The method we have established will help to improve the quality control of MCR extracts.

Inhibition of in vitro leukotriene B4 biosynthesis in human neutrophil granulocytes and docking studies of natural quinones.

Quinones are compounds frequently contained in medicinal plants used for the treatment of inflammatory diseases. Therefore, the impact of plant-derived quinones on the arachidonic acid metabolic pathway is worthy of investigation. In this study, twenty-three quinone compounds of plant origin were tested in vitro for their potential to inhibit leukotriene B4 (LTB4) biosynthesis in activated human neutrophil granulocytes with 5-lipoxygenase (5-LOX) activity. The benzoquinones primin (3) and thymohydroquinone (4) (IC50 = 4.0 and 4.1 microM, respectively) showed activity comparable with the reference inhibitor zileuton (1C50 = 4.1 microM). Moderate activity was observed for the benzoquinone thymoquinone (2) (1C50 = 18.2 microM) and the naphthoquinone shikonin (1) (IC50 = 24.3 microM). The anthraquinone emodin and the naphthoquinone plumbagin (5) displayed only weak activities (IC50 > 50 microM). The binding modes of the active compounds were further evaluated in silico by molecular docking to the human 5-LOX crystal structure. This process supports the biological data and suggested that, although the redox potential is responsible for the quinone's activity on multiple targets, in the case of 5-LOX the molecular structure plays a vital role in the inhibition. The obtained results suggest primin as a promising compound for the development of dual COX-2/5-LOX inhibitors.

Nutritional and nutraceutical comparison of Jamaican Psidium cattleianum (strawberry guava) and Psidium guajava (common guava) fruits.

Psidium cattleianum (strawberry guava) is one of many underutilised edible fruits that grow wild in Jamaica, and could potentially be commercially exploited to yield health and economic benefits. In this study, the total phenolics, proximate contents, and antioxidant, anti-inflammatory, and antimicrobial activities of P. cattleianum and P. guajava (common guava), a well-known species, were compared. Strawberry guavas were found to be superior to common guavas in antioxidant and antimicrobial activities, total phenolics and vitamin C content. They also possessed relatively high fibre content (24.9%). The hexane and ethyl acetate extracts of strawberry guavas showed cyclooxygenase-2 enzyme inhibitory activities of 18.3% and 26.5%, respectively (250 µg/mL), indicating anti-inflammatory activity. The EtOAc and MeOH extracts of P. guajava showed 56.4% (COX-2) and 44.1% (COX-1) inhibitory activity, respectively. Additionally, nine compounds were isolated from strawberry guava fruits, some of which demonstrated anti-inflammatory activity. These results indicate that strawberry guavas are beneficial for health.

Analgesic and anti-inflammatory activities of the methanol extract of Elaeagnus oldhamii Maxim. in mice.

We investigated possible mechanisms of analgesic and anti-inflammatory activities of the methanol extract from the leaf of Elaeagnus oldhamii Maxim. (EO(MeOH)). EO(MeOH) was evaluated for its analgesic activity in acetic acid-induced writhing response and formalin test, and anti-inflammatory effect was examined by λ-carrageenan-induced paw edema assay. We detected the activities of GPx, GRd and SOD in the liver, and the levels of inflammatory mediators including IL-1ß, IL-6, TNF-α, COX-2, MDA and NO in the edema paw to investigate the mechanism of action against inflammation. Total polyphenol, flavonoid and flavanol contents of EO(MeOH) were detected to explore its antioxidant activities. Results showed that, in the analgesic test, EO(MeOH) decreased acetic acid-induced writhing response and the licking time in the late phase of formalin test. In the anti-inflammatory test, EO(MeOH) decreased paw edema at the 2nd, 3rd, 4th and 5th h after λ-carrageenan had been injected. EO(MeOH) increased the activities of SOD and GPx in liver tissue and decreased MDA, NO, IL-1ß, IL-6, TNF-α and COX-2 levels in paw edema tissue at the 3rd h after λ-carrageenan-induced inflammatory reaction. EO(MeOH) exhibited abundant polyphenol, flavonoid and flavanol contents. In HPLC fingerprint test of EO(MeOH), two index ingredients, ursolic acid and pomolic acid, were isolated from EO(MeOH) and were exhibited in HPLC chromatographic analysis. The results demonstrated analgesic and anti-inflammatory effects of EO(MeOH). It was indicated that the anti-inflammatory mechanism of EO(MeOH) may be due to declined levels of NO and MDA in the edema paw through increasing the activities of SOD, GPx and GRd in the liver. Additionally, EO(MeOH) decreased IL-1ß, IL-6, TNF-α and COX-2 levels in the edema paw. The results suggested its value in future development of herbal medicine for the treatment of inflammatory diseases.

Flavonoids eupatorin and sinensetin present in Orthosiphon stamineus leaves inhibit inflammatory gene expression and STAT1 activation.

Cytokines and other inflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO) produced by cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively, activate and drive inflammation and therefore serve as targets for anti-inflammatory drug development. Orthosiphon stamineus is an indigenous medicinal plant of Southeast Asia that has been traditionally used in the treatment of rheumatoid arthritis, gout, and other inflammatory disorders. The present study investigated the anti-inflammatory properties of Orthosiphon stamineus leaf chloroform extract (CE), its flavonoid-containing CE fraction 2 (CF2), and the flavonoids eupatorin, eupatorin-5-methyl ether (TMF), and sinensetin, identified from the CF2. It was found that CE (20 and 50 µg/mL) and CF2 (20 and 50 µg/mL) inhibited iNOS expression and NO production, as well as PGE2 production. Eupatorin and sinensetin inhibited iNOS and COX-2 expression and the production of NO (IC50 5.2 µM and 9.2 µM for eupatorin and sinensetin, respectively) and PGE2 (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively) in a dose-dependent manner. The extracts and the compounds also inhibited tumor necrosis factor α (TNF-α) production (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively). Eupatorin and sinensetin inhibited lipopolysaccharide (LPS)-induced activation of transcription factor signal transducers and activators of transcription 1α (STAT1α). Furthermore, eupatorin (50 mg/kg i. p.) and sinensetin (50 mg/kg i. p.) inhibited carrageenan-induced paw inflammation in mice. The results suggest that CE and CF2, as well as the known constituents of CF2, i.e., eupatorin and sinensetin, have meaningful anti-inflammatory properties which may be utilized in the development of novel anti-inflammatory treatments.

Anti-inflammatory activities of aqueous extract of Mesona procumbens in experimental mice.

BACKGROUND: Mesona procumbens is consumed as a herbal drink and jelly-type dessert in Taiwan. The aim of this study was to determine the mechanism of anti-inflammatory activities of the aqueous extract of M. procumbens (AMP) using the λ-carrageenin (Carr)-induced mouse paw oedema model. The fingerprint chromatogram of AMP was obtained by high-performance liquid chromatography (HPLC) analysis. To investigate the anti-inflammatory mechanism of AMP, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) and the level of malondialdehyde (MDA) in paw oedema were monitored. Serum nitric oxide (NO), tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were also evaluated. RESULTS: The fingerprint chromatogram from HPLC indicated that AMP contained protocatechuic acid, chlorogenic acid, vanillic acid and caffeic acid. In the anti-inflammatory test, AMP decreased paw oedema after Carr administration and increased the CAT, SOD and GPx activities and decreased the MDA level in paw oedema at 5 h after Carr injection. AMP also affected the serum NO, TNF-α and IL-1ß levels at 5 h after Carr injection. Western blotting revealed that AMP decreased the expression of Carr-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). CONCLUSION: Mesona procumbens has the potential to provide a therapeutic approach to inflammation-associated disorders.

Analgesic and anti-inflammatory property of the methanol extract from Ligustrum morrisonense leaves in rodents.

Ligustrum morrisonense Kaneh and Sasaki (abbreviated as LM), an endemic Ligustrum plant in Taiwan, is similar to Ligustrum lucidum, which is usually used for curing hepatic and inflammatory disorders. The aim of this study was to evaluate the analgesic and anti-inflammatory properties of LM by chemical-induced algesia and carrageenan-induced inflammation in rodents. Its triterpenoid contents were measured by using high performance liquid chromatography-photodiode array detector. LM leaf extracts effectively inhibited writhing responses induced by 1% acetic acid and biphasic-licking responses caused by 1% formalin. LM leaf extract also reduced the edema induced by 1% carrageenan. Furthermore, LM leaf extract reduced the abdominal Evan's blue extravasations caused by lipopolysaccharide (LPS), serotonin, histamine and bradykinin. LM leaf extract has higher contents of amyrin and lupeol among six assayed triterpenoid compounds. In conclusion, LM is a potential analgesic and anti-inflammatory Ligustrum plant, and its anti-inflammatory effects are partially related to decreasing microvascular permeability via inflammatory mediators and inhibiting cyclooxygenase-2 activity.

Biochemical, biological and histological evaluation of some culinary and medicinal herbs grown under greenhouse and field conditions.

BACKGROUND: Increasing evidence supports the potential health benefits of herbal extracts displaying antioxidant, anti-inflammatory and antitumour activities. Environment can have a pronounced effect on phenolic content and antioxidant capacity. The objectives of this study were to evaluate the total phenolic contents and antioxidant capacities of five different herbs grown under greenhouse and field conditions and to assess their potential anti-inflammatory effects. RESULTS: High total polyphenolic (TPP) content (measured by the Folin-Ciocalteu reagent method) and high Trolox equivalent antioxidant capacity (TEAC) were observed in all herbs evaluated. Leaves from thyme, sage, spearmint and peppermint grown in the greenhouse showed significantly higher TPP content and TEAC than those grown under field conditions, with a threefold difference being observed in peppermint. Rosemary, spearmint and peppermint extracts showed stronger inhibition of cyclooxygenase COX-2 than of COX-1. CONCLUSION: The results show that producing herbs under greenhouse conditions can improve their biological activities by increasing TPP contents and antioxidant capacities. The selective inhibition of COX-2 activity by rosemary, spearmint and peppermint suggests that they may be useful as anti-inflammatory agents with fewer side effects than regular non-steroidal drugs.

Determination of two COX-2 inhibitors in serum and synovial fluid of patients with inflammatory arthritis by ultra performance liquid chromatography-inductively coupled plasma mass spectroscopy and quadrupole time-of-flight mass spectrometry.

The determination of two sulphur-containing drugs, the COX-2 inhibitors celecoxib and etoricoxib, in the serum and synovial fluid of inflammatory arthritis patients, is described using a sensitive ultra performance liquid chromatography-inductively coupled plasma mass spectroscopy (UPLC/ICPMS) method. Confirmation of the identity of the analytes in the samples was also performed by electrospray quadruple time-of-flight mass spectrometry in positive electrospray ionisation mode. The two COX-2 inhibitors were extracted from serum and synovial fluid following dilution with acetate buffer (pH 5) and liquid-liquid extraction (LLE) into ethyl acetate. Extracted samples were then analysed using UPLC/ICPMS with sulphur-specific detection. The limit of detection by UPLC/ICPMS was 0.45 ng/ml of sulphur in both serum and synovial fluid. The UPLC/ICPMS method was applied to the analysis of samples from patients receiving either 200 mg/day of celecoxib (2x 100 mg), 90 mg/day etoricoxib or placebo. The range of concentrations detected in the samples for the two drugs was from 0.3 to 3.3 microg/ml.

COX-1 and -2 activity of rose hip.

The objective of this study was to investigate whether the clinically observed efficacy of rose hip in the treatment of osteoarthritis is due to inhibition of cyclooxygenase-1 and -2. Water, methanol, dichloromethane and hexane extracts of rose hip were tested for in vitro COX-1 and 2 activity. The organic solvent extracts showed good inhibition of both COX-1 and 2. The methanol extract was most active in both assays with IC(50) values of 12 microg/mL for COX-1 and 19 microg/mL for COX-2. The clinically observed effect might be due to inhibition of cyclooxygenase.