COX-2 Assays Research Articles

Synthesis, molecular docking evaluation for LOX and COX-2 inhibition and determination of in-vivo analgesic potentials of aurone derivatives

In the current study, seven (7) aurone derivatives (ADs) were synthesized and employed to in-vitro LOX and COX-2 assays, in-vivo models of acetic acid-induced mice writhing, formalin-induced mice paw licking and tail immersion test to evaluate their analgesic potential at the doses of 10 mg and 20 mg/kg body weight. Molecular docking was performed to know the active binding site at both LOX and COX-2 as compared to standard drugs. Among the ADs, 2-(3,4-dimethoxybenzylidene)benzofuran-3(2H)-one (WE-4)possessed optimal LOX and COX-2 inhibitory strength (IC50=0.30 μM and 0.22 μM) as compared to standard (ZileutonIC50 = 0.08 μM, CelecoxibIC50 = 0.05 μM). Similarly in various pain models compound WE-4 showed significantly (p < 0.05) highest percent analgesic potency as compared to control at a dose of 20 mg/kg i.e. 77.60 % analgesic effect in acetic acid model, 49.97 % (in Phase-1) and 70.93 % (inPhase-2) analgesic effect in formalin pain model and 74.71 % analgesic response in tail immersion model. By the administration of Naloxone, the tail flicking latencies were reversed (antagonized) in all treatments. The WE-4 (at 10 mg/kg and 20 mg/kg) was antagonized after 90 min from 11.23 ± 0.93 and 13.41 ± 1.21 to 5.30 ± 0.48 and 4.80 ± 0.61 respectively as compared to standard Tramadol (from 17.74 ± 1.33 to 3.70 ± 0.48), showing the opiodergic receptor involvement. The molecular docking study of ADs revealed that WE-4 had a higher affinity for LOX and COX-2 with docking scores of -4.324 and -5.843 respectively. As a whole, among the tested ADs, compound WE-4 demonstrated excellent analgesic effects that may have been caused by inhibiting the LOX and COX-2 pathways.

Modification of 4-(4-chlorothiophen-2-yl)thiazol-2-amine derivatives for the treatment of analgesia and inflammation: synthesis and in vitro, in vivo, and in silico studies.

Inflammation is a protective response to a variety of infectious agents. To develop a new anti-inflammatory drug, we explored a pharmacologically important thiazole scaffold in this study. In a multi-step synthetic approach, we synthesized seven new thiazole derivatives (5a-5g). Initially, we examined the in vitro anti-inflammatory potentials of our compounds using COX-1, COX-2, and 5-LOX enzyme assays. After in vitro confirmation, the potential compounds were subjected to in vivo analgesic and anti-inflammatory studies. The hot plate method was used for analgesia, and carrageenan-induced inflammation was also assayed. Overall, all our compounds proved to be potent inhibitors of COX-2 compared to celecoxib (IC50 0.05μM), exhibiting IC50 values in the range of 0.76-9.01μM .Compounds 5b, 5d, and 5e were dominant and selective COX-2 inhibitors with the lowest IC50 values and selectivity index (SI) values of 42, 112, and 124, respectively. Similarly, in the COX-1 assay, our compounds were relatively less potent but still encouraging. Standard aspirin exhibited an IC50 value of 15.32μM. In the 5-LOX results, once again, compounds 5d and 5e were dominant with IC50 values of 23.08 and 38.46μM, respectively. Standard zileuton exhibited an IC50 value of 11.00μM. Based on the COX/LOX and SI potencies, the compounds 5d and 5e were subjected to in vivo analgesic and anti-inflammatory studies. Compounds 5d and 5e at concentrations of 5, 10, and 20mg/kg body weight were significant in animal models. Furthermore, we explored the potential role of compounds 5d and 5e in various phlogistic agents. Similarly, both compounds 5d and 5e were also significantly potent in the anti-nociceptive assay. The molecular docking interactions of these two compounds with the target proteins of COX and LOX further strengthened their potential for use in COX/LOX pathway inhibitions.

Combinative effects of akarkara root-derived metabolites on anti-inflammatory and anti-alzheimer key enzymes: integrating bioassay-guided fractionation, GC-MS analysis, and in silico studies

BackgroundAnacyclus pyrethrum L. (Akarkara root), a valuable Ayurvedic remedy, is reported to exhibit various pharmacological activities. Akarkara root was subjected to bioassay-guided fractionation, to isolate its active constituents and discover their potential bioactivities, followed by computational analysis.MethodsThe methanol extract and its fractions, methylene chloride, and butanol, were assessed for their antioxidant, anti-inflammatory, and anticholinergic potentials. The antioxidant activity was determined using DPPH, ABTS, FRAP, and ORAC assays. The in vitro anticholinergic effect was evaluated via acetyl- and butyryl-cholinesterase inhibition, while anti-inflammatory effect weas determined using COX-2 and 5-LOX inhibitory assays. The methylene chloride fraction was subjected to GC/MS analysis and chromatographic fractionation to isolate its major compounds. The inhibitory effect on iNOS and various inflammatory mediators in LPS-activated RAW 264.7 macrophages was investigated. In silico computational analyses (molecular docking, ADME, BBB permeability prediction, and molecular dynamics) were performed.ResultsForty-one compounds were identified and quantified and the major compounds, namely, oleamide (A1), stigmasterol (A2), 2E,4E-deca-2,4-dienoic acid 2-phenylethyl amide (A3), and pellitorine (A4) were isolated from the methylene chloride fraction, the most active in all assays. All compounds showed significant in vitro antioxidant, anticholinergic and anti-inflammatory effects. They inhibited the secretion of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in activated RAW macrophages. The isolated compounds showed good fitting in the active sites of acetylcholinesterase and COX-2 with high docking scores. The ADME study revealed proper pharmacokinetics and drug likeness properties for the isolated compounds. The isolated compounds demonstrated high ability to cross the BBB and penetrate the CNS with values ranging from 1.596 to -1.651 in comparison with Donepezil (-1.464). Molecular dynamics simulation revealed stable conformations and binding patterns of the isolated compounds with the active sites of COX-2 and acetyl cholinesterase.ConclusionsUltimately, our results specify Akarkara compounds as promising candidates for the treatment of inflammatory and neurodegenerative diseases.

In Vitro Anti-Inflammatory and Anticancer Evaluation of Mentha spicata L. and Matricaria chamomilla L. Essential Oils.

Spearmint, Mentha spicata L., and the German chamomile, Matricaria chamomilla L., preparations are used against inflammatory conditions traditionally and in modern medicinal applications. This present study aimed to evaluate pharma-grade essential oils for their in vitro anti-inflammatory and anticancer effects using COX-1, COX-2, and 5-LOX enzyme assays, as well as their apoptosis potential through the caspase pathway. In addition, the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay was applied to evaluate the in vitro cytotoxic effects using HEK293/A549, MCF7, and PC3 cell lines. Major components of M. spicata essential oil were confirmed both by gas chromatography (GC)-flame ionization detector (FID) and GC/mass spectrometry (MS) as 72.8% carvone, 12.6% limonene, 2.2% 1,8-cineole, 1.3% myrcene, and 1% trans-dihydrocarvone. The major components of M. chamomilla essential oil were also confirmed as 47.9% α-bisabolol oxide A, 16.8% α-bisabolol, 13.8%, (Z)-β-farnesene, 5.8% α-bisabolol oxide, and 4.7% α-bisabolene oxide A. The IC50 values for M. chamomilla essential oil on A549, MCF7, PC3, and HEK293 cells were calculated as 208.54 ± 1.39, 315.44 ± 1.17, 197.52 ± 0.98, and 638.79 ± 1.15 μg/mL, respectively, whereas the IC50 values for M. spicata essential oil on A549, MCF7, and PC3 cells were 672.13 ± 2.57, 708.27 ± 2.05, and 206.49 ± 1.48 μg/mL, respectively. For M. spicata essential oil, no cytotoxic effects on healthy HEK293 cells were observed at the tested concentrations. The essential oils increased the apoptotic activity, where all results were statistically significant. According to the anti-inflammatory evaluation, both M. chamomilla and M. spicata oils showed selective COX-2 inhibitions, where the SI values were calculated as 0.30 and 0.67, respectively. Overall, both M. spicata and M. chamomilla essential oils showed selective inhibition on the COX-2 enzyme and apoptosis against the selected cancer cell lines for the first time, to the best of our knowledge, with this specific dual mode of action. The initial results encourage further detailed in vivo experimental evaluations.

Anti-Inflammatory, Analgesic and Antioxidant Potential of New (2S,3S)-2-(4-isopropylbenzyl)-2-methyl-4-nitro-3-phenylbutanals and Their Corresponding Carboxylic Acids through In Vitro, In Silico and In Vivo Studies.

In the current study, a series of new (2S,3S)-2-(4-isopropylbenzyl)-2-methyl-4-nitro-3-phenylbutanals (FM1-6) with their corresponding carboxylic acid analogues (FM7-12) has been synthesized. Initially, the aldehydic derivatives were isolated in the diastereomeric form, and the structures were confirmed with NMR, MS and elemental analysis. Based on the encouraging results in in vitro COX 1/2, 5-LOX and antioxidant assays, we oxidized the compounds and obtained the pure single (major) diastereomer for activities. Among all the compounds, FM4, FM10 and FM12 were the leading compounds based on their potent IC50 values. The IC50 values of compounds FM4, FM10 and FM12 were 0.74, 0.69 and 0.18 µM, respectively, in COX-2 assay. Similarly, the IC50 values of these three compounds were also dominant in COX-1 assay. In 5-LOX assay, the majority of our compounds were potent inhibitors of the enzyme. Based on the potency and safety profiles, FM10 and FM12 were subjected to the in vivo experiments. The compounds FM10 and FM12 were observed with encouraging results in in vivo analgesic and anti-inflammatory models. The molecular docking studies of the selected compounds show binding interactions in the minimized pocked of the target proteins. It is obvious from the overall results that FM10 and FM12 are potent analgesic and anti-inflammatory agents.

One pot domino synthesis of new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazine-2-thiones (THTTs) as anti-inflammatory and antinociceptive candidates: A proof from in-vivo to in-vitro and in-silico mechanistic studies

A series of alkyl/aryl/aralkylamines or amino acids appended tetrahydro-2H-1,3,5-thiadiazine-2-thiones (4a-i, 5a-g, 6 and 7) were synthesized via one pot domino synthesis. The synthesis involved reacting alkyl/aryl/aralkylamines or amino acids with carbon disulfide employing basic aqueous medium and further cyclization with formaldehyde and alkyl/aryl/aralkylamines or amino acids. In addition, the carboxy-functionalized 1,3,5-thiadiazine-2-thione 6 was further subjected to esterification. All the structures were confirmed through spectral techniques i.e IR, 1H NMR, 13C NMR, and MS analysis. Furthermore, the newly synthesized compounds were biologically assessed via in vitro COX-2 and 5-LOX assays, in vivo anti-nociceptive and anti-inflammatory activities. Among the screened compounds, 6, 5f, and 7 exhibited highest inhibitory potency against COX-2 with IC50 values of 11.96, 13.54, and 13.93 μM, respectively. Moreover, compounds 6 and 7 exhibited excellent inhibitory potential against 5-LOX with IC50 values of 14.01 and 14.13 μM. The in-vivo anti-inflammatory bioassay studies showed that compounds 6, 7 and 5f dramatically reduced the paw edema size at 1 h and 3 h time intervals. In the anti-nociceptive activity, compound 6 showed pain protection comparative to Tramadol in all tested time intervals. In addition, studies of molecular docking revealed the compounds binding modes in the allosteric site of COX-2 and active site of 5-LOX, where these compounds exhibited higher binding scores and good binding interactions.

Trimethoxyphenyl containing compounds: Synthesis, biological evaluation, nitric oxide release, modeling, histochemical and histopathological studies

Novel series of trimethoxy phenyl containing chalcone 3, 5, 6, 7, pyrazoline 4a&b, 9a-h and pyrazole 10a&b scaffolds were designed and synthesized. They were characterized by spectral data and elemental analyses. All newly synthesized compounds were screened for their in vitro COX-1/COX-2 inhibitory activities and in vivo anti-inflammatory activity. All the target compounds showed COX-2 inhibitory activity over COX-1. Compound 5c was the most active derivative with higher COX-2 inhibitory activity (IC50 = 0.039 µM) than celecoxib (IC50 = 0.045 µM), and selectivity index value of 321.28 nearly equal to that of celecoxib (S.I. = 326.66). Four additional derivatives 5a, 6, 8b and 9f exhibited excellent COX-2 inhibitory activity (IC50 = 0.041 – 0.049 µM) if compared to the reference drug, celecoxib, with selectivity index values (S.I. = 230.61 – 278.05). Additionally, prolonged in vivo A.I activity was observed in compounds 9e, 9 g, 10a and 10b with % inhibition ranged from 33.21 to 44.52%, after 7 h from carrageenan injection. Compound 9e appeared normal without degeneration similar to celecoxib as resulted from histolopathogical study. Compounds containing NO releasing moieties, 7, 10a and 10b were assesses to overcome the gastrointestinal side effects. Molecular modeling study was operated and achieved a parallel correlation with in vitro COX-2 assay results. Pharmacokinetic study for all the prepared compounds was developed.

Potential multiple bioactive components from Sinopodophyllum hexandrum explored by affinity ultrafiltration with four drug targets

Background: Sinopodophyllum hexandrum (S. hexandrum) is a typical Chinese herbal medicine with multiple components and pharmacological activities. However, the specific phytochemicals responsible for its anti-proliferative, anti-inflammatory and anti-viral effects remain unexplored. Purpose: This study aimed to explore the specific bioactive phytochemicals from S. hexandrum responsible for its multiple remarkable pharmacological in an effort to construct and decipher its empirical therapeutic roles. Study design: Bio-affinity ultrafiltration (AUF) with multiple drug targets corresponding to its empirical anti-proliferative, anti-inflammatory and anti-viral activities was developed to screen for potential bioactive ligands from S. hexandrum, and then in vitro bioactivity assays or molecular docking methods were used to verify those bioactive ligands. Methods: The integrative analytical strategy was developed to rapidly screen and identify bioactive ligands from S. hexandrum. The in vitro anti-proliferative, COX-2 and ACE2 inhibitory assays of ligands were further verified by SRB cell proliferation and cytotoxicity detection, COX-2 and ACE2 inhibitor screening kits, respectively. Molecular docking analysis was also implemented by the AutoDockTools 1.5.6 software. Results: Ten, seven, nine and nine phytochemicals were screened out and identified as the potential Topo I, Topo II, COX-2 and ACE2 ligands, respectively. Hereinto, podophyllotoxin and quercetin with higher EF values displayed strong inhibitory effects on A549 and HT-29 cells comparable with etoposide and 5-FU as the positive controls. Furthermore, compared with indomethacin at 0.73 ± 0.07 μM, kaempferol and podophyllotoxin with higher EF values exerted stronger inhibitory effects with IC50 values at 0.36 ± 0.02 μM and 10.49 ± 0.61 μM, respectively. Isorhamnetin with a higher EF value exerted stronger inhibitory effect against ACE2 with IC50 value at 118.46 ± 14.78 μM in contrast to podophyllotoxin with relatively lower EF value at > 970.00 μM. In addition, the optimal binding sites and mode of action between multiple drug targets and bioactive ligands were determined by molecular docking. Conclusion: This study showcased a quick and reliable experimental strategy for unveiling the underlying mechanism of S. hexandrum, which could also provide valuable information for better understanding the therapeutic targets and ligands of other herbal medicines.

Penghambatan ganda COX/LOX: Pemeriksaan dan Penilaian Kesan Asid Pyroligneous Fraksi Shell Kernel Sawit sebagai Ejen Anti-radang

Inflammation is treated using Steroidal (SAIDs) and Non-steroidal (NSAIDs) anti-inflammatory drugs. These synthetic drugs act by inhibiting the activity of inflammatory mediators (Prostaglandins and Leukotrienes) and are known to have various side effects. The use of natural products as alternatives is gaining prominence due to effective therapeutic function with reduced side effects. Palm kernel shell biomass can be thermo-chemically converted into Pyroligneous Acid (PA), which have been found to contain phenols that have antioxidant activities and potential anti-inflammatory effects. In this study, the PA is derived from slow pyrolysis of palm kernel shell by fractionation using column chromatography. The fractions are then screened for Total Phenolic content using Folin ciocalteau assay and free radical scavenging activity using FRAP and DPPH procedures. Anti-inflammatory activity of COX and LOX pathways with the screened fractions, is determined using the LOX-5 and COX-2 assay by ELISA method. The fractions were found to have phenolic and free radical scavenging activity with high percentage yield. The fractions were also found to have dual inhibition for COX and LOX enzymes with IC50 values ranging for COX enzymes IC50 (17.04% - 48.42%) and LOX with IC50 (5.23% -53.34%). The findings of the present study indicate the potential for use of various fractions of pyroligneous palm kernel shell as a viable source of anti-inflammatory agents.

Anti-inflammatory and antiproliferative compounds from Sphaeranthus africanus

BackgroundSphaeranthus africanus has been used in traditional Vietnamese medicine to treat sore throat, and to relieve pain and swelling. However, the anti-inflammatory activity of this plant had not yet been investigated. Previously, we isolated five carvotacetones (1–5) from this plant that displayed cytotoxicity against several cancer cell lines. PurposeThe objective of this study was to isolate further constituents from S. africanus and to investigate the anti-inflammatory activity of all constituents. Furthermore, the anti-proliferative activity of the newly isolated compounds was evaluated. Study design and methodsCompounds were isolated from the upper parts of S. africanus by chromatographic methods. Structures were determined using spectroscopic techniques, like NMR and MS. All nine compounds isolated from S. africanus were evaluated for inhibitory activity against COX-1 and COX-2 isoenzymes in-vitro, COX-2 mRNA expression and influence on NO production. The anti-proliferative activities of newly isolated compounds (6–9) were evaluated by XTT viability assay with four cancer cell lines, namely CCRF-CEM, MDA-MB-231, HCT-116, and U-251 cells. ResultsTwo diastereomeric carvotacetones (3-angeloyloxy-5-[2″S,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (6) and 3-angeloyloxy-5-[2″R,3″R-dihydroxy-2″-methyl-butanoyloxy]-7-hydroxycarvotacetone (7), asperglaucide (8) and chrysoplenol D (9) were isolated from S. africanus. COX-1 and COX-2 assays of compounds 1–9 revealed that compounds 1 and 2 possess potent and selective COX-2 inhibitory activity with IC50 values of 3.6 and 0.5 μM, respectively. COX-2 gene expression assay showed that some carvotacetones exhibited inhibitory effects on COX-2 gene expression in THP-1 macrophages. Compound 4 is the most active compound inhibiting the synthesis of COX-2 by 55% at 2.06 μM. In the iNOS assay, all seven carvotacetones inhibited NO production in BV2 and RAW cell lines with IC50 values ranging from 0.2 to 2.9 μM. Compound 4 showed potent inhibitory activity with IC50 values of 0.2 μM in both BV2 and RAW cell lines. Molecular docking studies revealed the binding orientations of 1 and 2 in the active sites of COX-2. XTT assay of the newly isolated compounds revealed that the two isomeric carvotacetones (6–7) exhibited considerable anti-proliferative activity against four cancer cell lines (CCRF-CEM, MDA-MB-231, HCT-116, U-251) with IC50 values ranging from 1.23 to 8 μM. ConclusionFor the first-time, the diastereomeric carvotacetones (6–7) were isolated as separate compounds, and their anti-proliferative activity was determined. Selective COX-2 inhibitory, COX-2 mRNA expression and NO production inhibitory activities by some of the major constituents of S. africanus supports the traditional medical application of this plant for the treatment of inflammation-related disorders.

Focused library design and synthesis of 2-mercapto benzothiazole linked 1,2,4-oxadiazoles as COX-2/5-LOX inhibitors

Mercapto benzothiazole linked 1,2,4-oxadiazole derivatives were designed (4a-u) as new anti-inflammatory agents using bioisosteric approach and docking studies. The docking results clearly indicated that the compounds 4a-u shown good docking interaction towards COX-2 enzyme. In silico drug-like properties were also calculated for compounds (4a-u) and exhibited significant H-bond acceptor ratio. All compounds were synthesized and biologically evaluated using in vitro COX-1, COX-2 and 5-LOX assays. Compound 4k and 4q (IC50 = 6.8 μM and IC50 = 5.0 μM) found to be potent, selective COX-2 inhibitors and display better anti-inflammatory activity than standard Ibuprofen. Compound 4l and 4e found to be potent inhibitors against 5-LOX (IC50 = 5.1 μM and IC50 = 5.5 μM). The in vivo anti-inflammatory activity studies shown that the compounds 4q and 4k effectively reducing the paw edema volume at 3h and 5h than standard drug Ibuprofen. The DPPH radical scavenging activity provided anti-oxidant activity of compound 4e (IC50 = 25.6 μM) than reference standard Ascorbic acid.

Anti-inflammatory Activity of Natural Geranylated Flavonoids: Cyclooxygenase and Lipoxygenase Inhibitory Properties and Proteomic Analysis.

Geranyl flavones have been studied as compounds that potentially can be developed as anti-inflammatory agents. A series of natural geranylated flavanones was isolated from Paulownia tomentosa fruits, and these compounds were studied for their anti-inflammatory activity and possible mechanism of action. Two new compounds were characterized [paulownione C (17) and tomentodiplacone O (20)], and all of the isolated derivatives were assayed for their ability to inhibit cyclooxygenases (COX-1 and COX-2) and 5-lipoxygenase (5-LOX). The compounds tested showed variable degrees of activity, with several of them showing activity comparable to or greater than the standards used in COX-1, COX-2, and 5-LOX assays. However, only the compound tomentodiplacone O (20) showed more selectivity against COX-2 versus COX-1 when compared with ibuprofen. The ability of the test compounds to interact with the above-mentioned enzymes was supported by docking studies, which revealed the possible incorporation of selected test substances into the active sites of these enzymes. Furthermore, one of the COX/LOX dual inhibitors, diplacone (14) (a major geranylated flavanone of P.tomentosa), was studied in vitro to obtain a proteomic overview of its effect on inflammation in LPS-treated THP-1 macrophages, supporting its previously observed anti-inflammatory activity and revealing the mechanism of its anti-inflammatory effect.

Characterization of 3,3-dimethyl substituted N-aryl piperidines as potent microsomal prostaglandin E synthase-1 inhibitors

Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine inhibitors of microsomal prostaglandin E synthases-1(mPGES-1). Example 14 potently inhibited PGE2 synthesis in an ex vivo human whole blood (HWB) assay with an IC50 of 7nM. In addition, 14 had no activity in human COX-1 or COX-2 assays at 30μM, and failed to inhibit human mPGES-2 at 62.5μM in a microsomal prep assay. These data are consistent with selective mPGES-1-mediated reduction of PGE2. In dog, 14 had oral bioavailability (74%), clearance (3.62mL/(min*kg)) and volume of distribution (Vd,ss=1.6L/kg) values within our target ranges. For these reasons, 14 was selected for further study.

COX-1 inhibitory effect of medicinal plants of Ghana

Plants used to treat inflammatory ailments, pain, fever and infections in Ghana, were tested for COX-1 inhibitory activity. Ethanolic extracts of 17 species were tested in a COX-1 assay. The extracts of Gardenia ternifolia, Thonningia sanguinea, Triumfetta rhomboidea, and the root of Zanthoxylum zanthoxyloides showed an inhibitory effect over 90% in the final concentration 0.1μg/μL. The HPLC profiles indicated that the extracts of the four active species did not contain tannins. The observed in vitro activities support the use of some of the plant species in the traditional medicine system in Ghana.

2-Acetoxyverecynarmin C, a New Briarane COX Inhibitory Diterpenoid from Pennatula aculeata

A new briarane-type diterpenoid, named 2-acetoxyverecynarmin C, was isolated from the methanolic extract of an octocoral, Pennatula aculeata, that exhibited cyclooxygenase (COX) inhibitory activity. The structure of the compound was elucidated by ESI-HRMS, 1D and 2D NMR spectroscopy and comparison of the measured spectral data with those reported in the literature. The relative stereochemistry at chiral carbons was established from 2D NOESY experiments. 2-Acetoxyverecynarmin C is a tricyclic compound containing a furan ring at C-7,8 of a briarane skeleton. 2-Acetoxyverecynarmin C showed moderate inhibitory activity in in vitro COX-1 and COX-2 assays.

Anti-inflammatory, antioxidant, anti-cholinesterase activity and mutagenicity of South African medicinal orchids

While the role of various processes in inflammatory-related degenerative disorders is still being researched, many avenues of research have concentrated on the treatment and/or prevention of these disorders. Inflammatory-responses, the cholinergic system and oxidative stress have often been linked to the symptoms prevalent in aged persons and Alzheimer's patients. The current research explored the selective inhibition of cyclooxygenase (COX) enzymes, antioxidant and anti-cholinesterase activities of selected South African orchid extracts, currently traded in herbal markets along the east coast of South Africa. Out of a total of 53 evaluated extracts, significant anti-inflammatory activity was observed in nearly 40% of extracts in the COX-1 assay and 25% of extracts in the COX-2 assay. Overall, the DCM root extract of Ansellia africana was the most potent, the DCM tuber extract of Eulophia hereroensis was the only extract to significantly inhibit both COX enzymes, while all Bulbophyllum scaberulum organic root extracts exhibited COX-2 selective inhibitory activity. Bulbophyllum scaberulum DCM root extract was also the most effective anti-cholinesterase extract, performing better than galanthamine. In the single electron transfer (SET) (2,2′-diphenylpicrylhydrazyl (DPPH) free radical scavenging assay and ferric reducing antioxidant power (FRAP) reaction based assays, E. petersii pseudobulb and A. africana root extracts performed better than other extracts. In the hydrogen atom transfer (HAT) (β-carotene/linoleic acid assay) reaction-based assay, the leaf extract of Tridactyle tridentata and root extracts of Cyrtorchis arcuata and E. hereroensis exhibited the best antioxidant effects. The mutagenicity (Salmonella/microsome assay) was also determined. Organic leaf and root extracts of C. arcuata produced no genotoxic effects in comparison to the other tested species. None of the crude extracts tested demonstrated mutagenic effects using S. typhimurium strain TA98 with metabolic activation. The results obtained in this study validate the use of certain orchid species in South African traditional medicine for inflammation-related degenerative disorders.

Myo-Inositol esters of indomethacin as COX-2 inhibitors

Ester prodrugs have the potential to eliminate the gastrotoxicity associated with the carboxylic acid group of indomethacin. 4,6-Bis-O-2′-[1′-(4″-chlorobenzoyl)-5′-methoxy-2′-methyl-1′H-indol-3′-acetyl]-myo-inositol-1,3,5-orthoacetate (2) was synthesised and evaluated as a COX-2 inhibitor. It adopts a conformationally restricted chair with two indomethacin groups in the sterically hindered 1,3-diaxial positions. Acid-induced cleavage of the orthoacetate lock of the prodrug leads to a ring flip of the myo-inositol ring with the two indomethacin groups now in 1,3-diequatorial positions. This increases the susceptibility of hydrolysis of the ester groups to release indomethacin under acidic conditions. The long half-life (152min) of decomposition of (2) at ∼pH 1–2 suggests that it may bypass the stomach with minimal hydrolysis upon oral administration. Indomethacin ester (2) was completely stable at pH 4.0–8.5 over 24h at 37°C and showed comparable activity to indomethacin in a COX-2 assay (pH 8.0).

Semisynthesis and pharmacological investigation of lipo-alkaloids prepared from aconitine

Processed aconite drugs are widely used in Eastern medicine as painkillers and antirheumatic agents. It is known that the traditional processing of aconite drugs increases the amount of lipo-alkaloids. In order to obtain information about the pharmacological potential of these compounds, semisynthesis of 9 aconitine-derived lipo-alkaloids was carried out and their COX-1, COX-2 and LTB 4 formation inhibitory activities were investigated. It was found that compounds esterified with unsaturated fatty acids demonstrated significant COX-2 inhibitory effects, while in the COX-1 assay only 14-benzoylaconine-8- O-eicosapentaenoate exerted remarkable activity. The inhibition of LTB 4 formation was pronounced in cases of long chain fatty acid derivatives.

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens was done. Dried samples were ground to fine powders and their total iridoid (colorimetric method), phenolic [Folin–Ciocalteu (Folin C) method] and gallotannin (Rhodanine assay) contents as well as anti-inflammatory activity [cyclooxygenase assays (COX-1 and COX-2)] were determined. The tissue culture-derived tubers had the highest total iridoid content which was significantly higher than that of the tubers collected from the wild and other tissue cultured materials evaluated. This suggests that cultivated plants can be a viable alternative source of the active principle(s). The total phenolic and gallotannin contents of the wild tubers were significantly higher than the tissue culture-derived tubers and other in vitro-derived plant materials. The presence of phenolic compounds including gallotannins in the tissue cultured materials is of interest from a pharmacological point of view given the pharmacological role of phenolics. In general, extracts from wild tubers demonstrated better inhibitory activities in both COX-1 and COX-2 assays when compared to the tissue culture-derived tubers. All the petroleum ether (PE) and dichloromethane (DCM) extracts showed moderate (50–70%) to good (> 70%) inhibitory activities whereas the ethanol (EtOH) extracts showed poor or no inhibition in both assays. Based on previous reports indicating weak inhibition of COX-2 enzyme by harpagoside, the inhibitory activities of both COX enzymes exhibited by PE and DCM extracts in the current study could be due to the presence of other constituents in the extracts. This points towards the need to identify other active constituents and evaluate their role and mode of action in relation to harpagoside — the main active principle.

In vitro biological activities of niloticane, a new bioactive cassane diterpene from the bark of Acacia nilotica subsp. kraussiana

Ethnopharmacological relevance Acacia nilotica subsp. kraussiana was reported in African traditional medicine for the treatment of various ailments. Isolation of an active compound in this study from the bark extract may lead to the validation of its efficiency as a traditional crude drug. Aims of the study This study aimed to isolate active compound(s) from an ethyl acetate bark extract of Acacia nilotica subsp. kraussiana and to investigate some of its biological activity. Materials and methods The isolation process was carried out using bioassay-guided fractionation. The isolated compound was tested for antibacterial activity using the micro-dilution assay; anti-inflammatory activity using the COX-1 and COX-2 assays and investigated for inhibitory effect against acetylcholinesterase using the microplate assay. Results A new bioactive compound was isolated and identified as a cassane diterpene, niloticane. Niloticane showed antibacterial activity against Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus with MIC values of 4 and 8 μg/mL, respectively. With Gram-negative bacteria, niloticane showed weak activity. MIC values obtained were 16 and 33 μg/mL against Klebsiella pneumonia and Escherichia coli, respectively. In the cyclooxygenase test, niloticane possessed activity with IC 50 values of 28 and 210 μM against COX-1 and COX-2, respectively. IC 50 values observed with indomethacin (positive control) were 3.6 μM for COX-1 and 189 μM for COX-2. In the acetylcholinesterase test, niloticane showed anti-cholinesterase activity with an IC 50 value of 4 μM. IC 50 values obtained by the galanthamine (positive control) was 2.0 μM. Conclusion The results obtained support the traditional uses of the bark of Acacia nilotica subsp. kraussiana in African traditional medicine for the treatment of some ailments that relate to microbial diseases, inflammation and central nervous system disorders.