In vitro anti-inflammatory and xanthine oxidase inhibitory activity of Tephrosia purpurea shoot extract

The methanolic extract of Tephrosia purpurea (Leguminosae) shoots was evaluated in-vitro for its anti-inflammatory and xanthine oxidase inhibitory activity. Anti-inflammatory activity was measured by the Diene-conjugate, HET-CAM and beta-glucuronidase methods. The enzyme inhibitory activity was tested against isolated cow milk xanthine oxidase. The average anti-inflammatory activity of T. purpurea shoot extract in the concentration range of 1-2 microg/mL in the reacting system revealed significant anti-inflammatory activities, which, as recorded by the Diene-conjugate, HET-CAM and beta-glucuronidase assay methods, were 45.4, 10.5, and 70.5%, respectively. Screening of the xanthine oxidase inhibitory activity of the extract in terms of kinetic parameters revealed a mixed type of inhibition, wherein the Km and Vmax values in the presence of 25 to 100 microg/mL shoot extract was 0.20 mM/mL and 0.035, 0.026, 0.023 and 0.020 microg/min, while, for the positive control, the Km and Vmax values were 0.21 mM/mL and 0.043 microg/min, respectively. These findings suggest that T. purpurea shoot extract may possess constituents with good medicinal properties that could be exploited to treat the diseases associated with oxidative stress, xanthine oxidase enzyme activity and inflammation.     

A highly sensitive RP-HPLC-fluorescence method to study aldehyde oxidase activity

Aldehyde oxidase is a widely distributed enzyme that is involved in the metabolism of an extensive range of aldehydes and N-heterocyclic compounds with physiological, pharmacological, and toxicological relevance. In the present study, a highly sensitive RP-HPLC-fluorescence method based on the oxidation of phenanthridine to phenanthridinone has been developed and validated to assay aldehyde oxidase activity in biological samples. Determination of phenanthridinone was achieved on a C18 column using 10 mmol/L phosphate buffer (pH 5.0) containing 0.1 mmol/L EDTA-acetonitrile (40 + 60, v/v) as the mobile phase. The fluorescence intensity of phenanthridinone was measured at 364 nm with excitation at 236 nm. The proposed method was precise, accurate, specific and rapid (analysis time, approximately 8 min) with a mean RSD of 2.54%. Peak responses were linear from 0.5 to 100 nmol/L, with an LOD of 0.125 nmol/L. The applicability of the method was demonstrated by measurement of aldehyde oxidase activity in rat liver, kidney, ovary, and heart fractions.     

Phenolic constituents from the heartwood of Artocapus altilis and their tyrosinase inhibitory activity

From the MeOH extract of the heartwood of Artocapus altilis, thirteen phenolic compounds have been isolated, namely curcumin (1), desmethoxycurcumin (2), retrodihydrochalcone (3), apigenin (4), tangeretin (5), nobiletin (6), O-methyldehydrodieugenol (7), dehydrodieugenol (8), beta-hydroxypropiovanillone (9), p-coumaric acid (10), p-hydroxybenzaldehyde (11), vanillin (12), and vanillic acid (13). This is the first report on the presence of these compounds in the heartwood of A. altilis. Compounds 1, 2, and 10 showed more potent tyrosinase inhibitory activities, with IC50 values ranging from 2.3 to 42.0 microM, than the positive control kojic acid (IC50, 44.6 microM). The most active compound, p-coumaric acid (10) (IC50, 2.3 microM), was 22 times more active in tyrosinase inhibitory activity than kojic acid.     

Selective inhibition of bovine plasma amine oxidase by homopropargylamine, a new inactivator motif

Propargylic and activated allylic amines are known to inactivate the quinone-dependent plasma amine oxidases, possibly through active-site modification by the alpha,beta-unsaturated aldehyde turnover products. Although homopropargylamine (1-amino-3-butyne, 1) is a nonobvious candidate as a mechanism-based inhibitor, 1 was found to be an unusually potent time- and concentration-dependent irreversible inactivator of bovine plasma amine oxidase (BPAO), exhibiting a 30 min IC(50) of 2.9 microM at 30 degrees C ([BPAO] = 1.2 microM). Preserved cofactor redox activity of the denatured inactivated enzyme indicates that inactivation by 1 involves either a cofactor modification that reverses upon enzyme denaturation or a modification of an active-site residue. Because inactivation by 1 may involve enzyme alkylation by the reactive 2,3-butadienal (3) tautomer of the 3-butynal turnover product of 1, aldehyde 3 was prepared and was found to inactivate BPAO, but only at high concentration. In addition, whereas inhibition by 3 was blunted by the presence of mercaptoethanol, no such protection was observed against 1. The amine whose turnover should lead directly to 3 was prepared (1-amino-2,3-butadiene, 4) and was found to be an even more potent inactivator of BPAO than 1, exhibiting a 5 min IC(50) of 1.25 microM. Rat liver mitochondrial monoamine oxidase was also inactivated by 4, as expected, but only very weakly by 1. Potential mechanisms explaining the selective inhibition of BPAO by 1 are discussed.     

Multifunctional approaches to provide potential pharmacophores for the pharmacy shelf: Heracleum sphondylium L. subsp. ternatum (Velen.) Brummitt.

Heracleum sphondylium L. subsp. ternatum (Velen.) Brummitt. commonly known as “hogweed” is traditionally used to manage several human ailments. This investigation assessed, for the first time, the enzyme inhibitory properties, antioxidant activity, phytochemical profile, antimutagenic, and antimicrobial potential of the ethyl acetate, methanol, and water extracts of H. sphondylium. We also established the possible interactions of identified phenolic compounds with cholinesterases, amylase, glucosidase, and tyrosinase using in silico docking studies. Chlorogenic acid was found in high amounts in the methanol extract of H. sphondylium. The methanol extract was an effective inhibitor of acetylcholinesterase (1.70 mg galantamine equivalent (GALAE)/g extract) while the ethyl acetate extract showed pronounced inhibitory action against butyrylcholinesterase (1.77 mg GALAE/g extract). The extracts exhibited low inhibition against amylase (0.12-0.84 mmol acarbose equivalent (ACAE)/g extract) and a more pronounced inhibition against glucosidase (2.29–3.65 mmol ACAE/g extract). In silico results showed that rutin and quercetin (-70.4 and -72.2 Kcal/mol, for rutin and quercetin respectively) docked to the enzymatic cavity of acetylcholinesterase but these phenolic compounds showed less affinity with butyrylcholinesterase (-15.0 and -5.2 Kcal/mol, for rutin and quercetin respectively). The extracts did not induce any mutations on the bacterial strains, while they have excellent antimutagenic capacity against well-known mutagens (inhibition values 98%, 97% and 96%). The methanol extract (0.78 mg/ml) showed moderate antifungal activity while the ethyl acetate extract (0.78–3.12 mg/ml) showed weak to moderate antimicrobial activity. This study provides valuable baseline data which might serve for the development of future pharmacophores for the management of human ailments.     

Lignin biotransformations by an aromatic aldehyde oxidase produced byStreptomyces viridosporus T7A

Streptomyces viridosporus produces an intracellular aromatic aldehyde oxidase that oxidizes aromatic and α, β-unsaturated aromatic aldehydes to their corresponding acids. It also produces extracellular oxidase as shown by zones of clearing when grown on agar containing insoluble dehydrodivanillin (DHDV). This extracellular form may be responsible for oxidizing aldehyde groups in lignin. The extracellular oxidase was expressed maximally after 3 d growth in medium containing only yeast extract. However, higher levels were produced when lignocellulose was in the medium. The enzyme was partially purified and its molecular weight was approximated to be about 80,000 daltons. Mutant cultures that had lost the ability to produce zones of clearing on DHDV-containing agar solubilized smaller quantities of lignin as compared to the wild type, except for one strain. A partially purified oxidase preparation was shown to oxidize a natural lignocellulose substrate.     

Potent inhibitory effects of N-aryl S-alkylthiocarbamate derivatives on the dopa oxidase activity of mushroom tyrosinase

This study reports the potent inhibitory effect of N-aryl S-alkylthiocarbamate derivatives on mushroom tyrosinase (MT) activity. N-Aryl S-alkylthiocarbamate derivatives were found to exhibit a potent inhibitory effect on the dopa (3,4-dihydroxyphenylalanine) oxidase activity of mushroom tyrosinase. Most of the N-aryl S-alkylthiocarbamate derivatives (compounds from A to J) exhibited higher inhibitory effects than kojic acid (IC50=318 microM), a well known tyrosinase inhibitor. Tyrosinase was the most inhibited by S-phenetyl N-phenylthiocarbamate (compound E, IC50=7.25 microM), and this inhibition was 44 times stronger than that of kojic acid. Compound E exhibited 95.0% of inhibition at 100 microM. A kinetic study of MT inhibition by compound E using the Lineweaver-Burk plots analysis was performed. And the kinetics profiles observed suggest that compound E competitively inhibits MT.     

Propolis oil extract: quality analysis and evaluation of its antimicrobial activity

Designing propolis products for external use involves determining the optimal form of propolis for the introduction into dermatological pharmaceuticals and cosmetic preparations. As a potent ingredient, propolis oil extract from raw material harvested in Lithuania was analysed. The rheological characteristics, content of phenolic compounds, major compounds and antimicrobial activity of the propolis oil extract are investigated here for the first time. The propolis oil extract was produced by maceration using different solvents, raw material was collected in Lithuania. Solvent mixture with 96% ethanol increased the rheological stability and extracted amount of phenolic compound. High-performance liquid chromatography identified the potent quality markers for Lithuanian propolis, phenylpropanoid vanillin, coumaric acid and ferulic acid. Antimicrobial activity of propolis oil extract was evaluated in experimental studies in vitro, and the minimal concentration of phenolic compounds that inhibited respective microorganisms was determined. The results demonstrate that phenolic compounds have effective antimicrobial activity in propolis oil extract; thus, it can be compatible with the semisolid preparation.     

Antioxidant, diuretic activities and polyphenol content of Stereospermum kunthianum Cham. (Bignoniaceae)

Stereospermum kunthianum was used for biological and phytochemical investigations. In biological studies, antioxidant activities were investigated with water, methanol and aqueous acetone extracts. Furthermore, the xanthine oxidase inhibitory activity and the diuretic activity of an aqueous acetone extract were evaluated. In the phytochemical investigations, the flavonoids and polyphenols were quantified spectrophotometrically in all extracts followed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of an aqueous acetone extract. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP) and 2,2'-azinobis (3-ethylbenzoline-6-sulphonate (ABTS) methods have shown that the aqueous acetone extract presents the best antioxidant activities. This aqueous acetone extract was further proven to have interesting xanthine oxidase inhibitory activity, but only a weak diuretic activity. This aqueous acetone extract also possessed the highest phenolic and flavonoid contents. HPLC-MS analysis allowed identifying and quantifying, rutin, isoquercitrin, quercetin, hyperoside, quercitrin and luteolin and the glycosides of ferulic, sinapic p-coumaric acids and kaempferol, apigenin in aqueous-acetone extract.     

A pinacol rearrangement/oxidation synthetic route to hydroxyphenstatin

In an attempt to develop biologically active compounds from the inactive trans isomer (3a) of stilbene 1a, after asymmetric dihydroxylation to optically pure (R,R)-diol 8 the unexpected racemic diphenylacetaldehyde (9) was generated via a Pinacol rearrangement. Several derivatives of diphenylacetaldehyde 9 were synthesized (11-15) and reported. Further reaction of aldehyde 9 during desilylation through autoxidative decarbonylation afforded benzophenone 2b, designated hydroxyphenstatin, a potent antitumor and antimitotic agent. Hydroxyphenstatin showed potent inhibition of the tubulin assembly (IC(50) 0.82 microM) and exhibited an ED(50) of 2.5 microg/mL against the P388 lymphocytic leukemia cell line.     

DPPH radical scavenging and xanthine oxidase inhibitory activity of Terminalia macroptera leaves

From a methanol extract of the leaves of the Malian medicinal tree Terminalia macroptera, cis-polyisoprene (1), chebulic acid trimethyl ester (2), methyl gallate (3), shikimic acid (4), corilagin (5), rutin (6), narcissin (7), chebulagic acid (8) and chebulinic acid (9), were isolated. Cispolyisoprene (1) was the major non-polar constituent. The novel compound 2 showed high radical scavenging activity (IC50 4.7 microg/mL), but was inactive as xanthine oxidase inhibitor. The major substituent of the crude extract, substance 5, showed a high radical scavenger effect (IC50 2.7 microg/mL) and weak xanthine oxidase inhibition (IC50 ca 105 microg/mL). The antioxidant and radical scavenging effects of some of the substances identified in this study may to some extent explain the medical use of this tree in West Africa.     

Validation assay for total flavonoids, as rutin equivalents, from Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract

An ultraviolet spectrophotometric method was validated for total flavonoid quantitation, as rutin equivalents, present in the Trichilia catigua Adr. Juss (Meliaceae) and Ptychopetalum olacoides Bentham (Olacaceae) commercial extract. Parameters as linearity, interval (range), specificity, estimated limit of detection (LOD, microg/mL), estimated limit of quantitation (LOQ, microg/mL), recovery (R, %), precision or relative standard deviation (RSD, %), and accuracy (E, %) were established. The analytical method was validated according to the experimental results: correlation coefficient (r = 0.9997); interval (RSD = 0.15-0.47%; E = 98.98-101.24%); specificity to total flavonoids quantitation, as rutin equivalents, at wavelength 361.0 nm; LOD = 0.09 microg/mL and LOQ = 0.27 microg/mL; R = 99.36-102.14%; adequate intra- and interrun precision (0.30-0.49% and 0.31-0.81%), and intra- and interrun accuracy (100.60-102.38% and 98.58-100.38%).     

Xanthine oxidase inhibitors from the heartwood of Vietnamese Caesalpinia sappan

From the MeOH extract of Vietnamese Caesalpinia sappan, a novel biogenetically exclusive benzindenopyran, with a new carbon framework, neoprotosappanin (1), and a new compound, protosappanin A dimethyl acetal (3), were isolated together with protosappanin E-2 (2), neosappanone A (4), and 13 previously reported phenolic compounds (5-17). Their structures were elucidated on the basis of spectroscopic data. Compounds 1-4, 7, 13, and 15-17 showed significant xanthine oxidase inhibitory activity in a concentration-dependent manner, and sappanchalcone (17) showed the most potent activity with an IC50 value of 3.9 microM, comparable to that of positive control allopurinol (IC50, 2.5 microM). The kinetic study of these inhibitors indicated that they are competitive inhibitors, the same as allopurinol, except for 1 and 16 which are noncompetitive inhibitors.     

Chemical Profile,Antimicrobial and Antioxidant Activity Assessment of the Crude Extract and Its Main Flavonoids from Tartary Buckwheat Sprouts

The purpose of this study was to investigate the major flavonoids content and bioactivities of Tartary buckwheat sprouts. The crude methanol extract (ME) of Tartary buckwheat sprouts was abundant in flavonoids, and six major flavonoids, including isoorientin, vitexin, isovitexin, rutin, quercetin, and kaemferol were successfully determined from the sprouts by the high-performance liquid chromatography (HPLC) method. Generally, the flavonoid content of buckwheat sprouts was in the order of rutin > quercetin > isovitexin > vitexin> isoorientin > kaemferol. The highest rutin content of the ME and sprout cultures was 89.81 mg/g and 31.50 mg/g, respectively. Antibacterial activity results indicated the ME displayed notable inhibitory activity against the five tested bacteria, and its minimum inhibitory concentration (MIC) values ranged from 0.8 mg/mL to 3.2 mg/mL. Among the six flavonoids, quercetin was the most active compound, which exhibited strong activity against all tested bacteria except for E. coli and S. epidermidis, with its MIC values ranging from 0.2 mg/mL to 0.4 mg/mL. For the antifungal activity assay, the ME of Tartary buckwheat sprouts and four flavonoids could significantly inhibit the spore germination of two pathogenic fungi, and their inhibitory efficiency was concentration dependent. Quercetin was the most active one, which significantly inhibited the spore germination of F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cucumerinum, and its median effective inhibitory concentration (IC₅₀) value was 42.36 and 32.85 µg/mL, respectively. The antioxidant activity results showed that quercetin, kaemferol, and rutin displayed excellent antioxidant activity in the DPPH radical scavenging test, and their IC₅₀ value was calculated as 5.60, 16.23, and 27.95 µg/mL, respectively. This is the first report on the antimicrobial activity of the crude extract of Tartary buckwheat sprouts. These results indicated that the methanol extract of Tartary buckwheat sprouts could be used as a potential antimicrobial or antioxidant agent in the future.     

Anti-plasmodial and cholinesterase inhibiting activities of some constituents of Psorospermum glaberrimum

Glaberianthrone (1), a new bianthrone was isolated from the hexane extract of the stem bark of Psorospermum glaberrimum together with thirteen known compounds: 3-geranyloxyemodin anthrone (2), friedelan-3-one (3), 3-prenyloxyemodin anthrone (4), 3-geranyloxyemodin (5), 3-prenyloxyemodin (6), friedelan-3-ol (7), acetylvismione D (8), betulinic acid (9), 2-geranylemodin (10), bianthrone A2b (11), bianthrone 1a (12), emodin (13) and 2-prenylemodin (14). The structures of the isolated compounds were established by means of spectroscopic methods. The extracts and the isolated compounds were tested in vitro for their anti-plasmodial activity against Plasmodium falciparum (chloroquine resistant strain W2) and for their acetyl- and butyrylcholinesterase inhibitory properties. The n-hexane extract showed good anti-plasmodial activity against P. falciparum W2 strain, with IC(50) of 0.87 microg/ml. It also exhibited 65.5% and 98.2% of acetyl- and butyrylcholinesterase inhibition at 0.2 mg/ml, respectively. Compounds 2 and 8 showed the best potencies against P. falciparum W2 strain with IC(50) of 1.68 microM and 0.12 microM, (0.66 microg/ml and 0.054 microg/ml) respectively. All tested compounds showed good butyrylcholinesterase inhibition activities with compound 12 displaying the best potency (IC(50) 9.25+/-0.25 microM). All the tested compounds showed weak inhibitory activity against acetylcholinesterase.     

Piperine from the fruits of Piper longum with inhibitory effect on monoamine oxidase and antidepressant-like activity

A bioassay-guided isolation of the ethanol extract from the fruits of Piper longum yielded a known piperidine alkaloid, piperine, as a monoamine oxidase (MAO) inhibitor. Piperine showed an inhibitory effect against MAO-A (IC50 value: 20.9 microM) and MAO-B (IC50 value: 7.0 microM). Kinetic analyses by a Lineweaver-Burk plot clearly indicated that piperine competitively inhibited MAO-A and MAO-B with Ki values of 19.0+/-0.9 microM and 3.19+/-0.5 microM, respectively. The inhibition by piperine was found to be reversible by dialysis of the incubation mixture. In addition, the immobility times in the tail suspension test were significantly reduced by piperine, similar to that of the reference antidepressant fluoxetine, without accompanying changes in ambulation when assessed in an open-field. These results suggest that piperine possesses potent antidepressant-like properties that are mediated in part through the inhibition of MAO activity, and therefore represent a promising pharmacotherapeutic candidate as an antidepressant agent.     

Purification and Characterization of NAD+-Dependent Salicylaldehyde Dehydrogenase from Carbaryl-Degrading Pseudomonas sp. Strain C6

NAD⁺-dependent salicylaldehyde dehydrogenase (SALDH) which catalyzes the oxidation of salicylaldehyde to salicylate was purified form carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a functional homotrimer (150 kDa) with subunit molecular mass of 50 kDa and contained calcium (1.8 mol/mol of enzyme). These properties were found to be unique. External addition of metal ions showed no effect on the activity and addition of chelators showed moderate inhibition of the activity. Potassium ions were found to enhance the activity significantly. SALDH showed higher affinity for salicylaldehyde (K _m?=?4.5 μM) and accepts mono- as well as di-aromatic aldehydes; however it showed poor activity on aliphatic aldehydes. Chloro-/nitro-substituted benzaldehydes were potent substrate inhibitors as compared to benzaldehyde and 3-hydroxybenzaldehyde, while 2-naphthaldehyde and salicylaldehyde were moderate. The kinetic data revealed that SALDH, though having broad specificity, is more efficient for the oxidation of salicylaldehyde as compared to other aromatic aldehyde dehydrogenases which gives an advantage for Pseudomonas sp. strain C6 to bioremediate carbaryl and other aromatic aldehydes efficiently.     

Xanthine Oxidase Inhibitors from Filipendula ulmaria (L.) Maxim. and Their Efficient Detections by HPTLC and HPLC Analyses

Filipendula ulmaria is a plant commonly used for the treatment of several pathologies, such as diarrhoea, ulcers, pain, stomach aches, fevers, and gout. Our study focused on the use of F. ulmaria for the treatment of gout disease. We first studied the chemical composition of a methanolic extract of the aerial parts and demonstrated its xanthine oxidase (XO) inhibitory activity. Then, we performed a fractionation and evaluated the most XO inhibitory active fractions by UV measurement. Purification of some fractions allowed the determination of the inhibitory activity of pure compounds. We demonstrated that spiraeoside, a glycosylated flavonoid, possesses an activity around 25 times higher than allopurinol, used as a reference in the treatment of gout disease. In order to easily and quickly identify potent inhibitors in complex matrix, we developed a complementary strategy based on an HPLC method and an Effect Directed Assay (EDA) method combining HPTLC and biochemical assays. The HPLC method, capable of determining compounds exhibiting interactions with the enzyme, could be an efficient strategy for evaluating potent enzyme inhibitors in a complex mixture. This strategy could be applied for quantitative assays using LC/MS experiments.     

Purification and characterization of a microbial dehydrogenase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M _r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.     

Phytopathogenic fungal inhibitors from celery seeds

Extract of celery (Apium graveolens L.) seeds was investigated against phytopathogenic fungi. The light petroleum extract showed promising inhibition activities in the tests against Rhizoctonia solani and Fusarium oxysporum f. sp. vasinfecum. Chromatographic separation of the extract gave 19 fractions, one of which, QCZ-4, possessed significant inhibitory rates of 64.6%, 88.4% and 54.7% at a concentration of 100 ppm against R. solani, F. oxysporium f. sp. vasinfecum and Alternaria alternata, respectively. Major components in the active fraction were identified by GC-MS as p-(2-aminoethyl)phenol (39.7%), 3-(3,4-dimethybenzoyl) propionic acid (32.6%) and p-heptylphenol (26.9%).