Protective effect of Ecklonia cava enzymatic extracts on hydrogen peroxide-induced cell damage

In this study, Ecklonia cava was enzymatically hydrolyzed to prepare water-soluble extracts, using five carbohydrases (Viscozyme, Celluclast, AMG, Termamyl, and Ultaraflo) and five proteases (Protamex, Kojizyme, Neutase, Flavourzyme, and Alcalase), and the potential antioxidant activity of each was assessed. The Celluclast and Viscozyme extracts of E. cava evidenced good hydrogen peroxide (H₂O₂) scavenging activities (73.25% and 72.92%, respectively) as compared to those of other enzymatic extracts. Therefore, the Celluclast enzymatic extract was selected for use in further experiments, and separated into four different molecular weight fractions (<1, 1–10, 10–30 and >30 kDa). Among these fractions, the >30 kDa fraction manifested the most profound H₂O₂ scavenging activity, with a measured IC₅₀ of 13 μg/ml. The >30 kDa fraction also strongly enhanced cell viability against H₂O₂-induced oxidative damage, and evidenced relatively good lipid peroxidation inhibitory activity in a Chinese hamster lung fibroblast (V79-4) cell line. This fraction also effected a reduction in the proportion of cells undergoing H₂O₂-induced apoptosis, as was demonstrated by a decreased quantity of sub-G₁ hypodiploid cells and decreased apoptotic body formation on the flow cytometry assay. These results clearly indicate that the >30 kDa fraction of E. cava possesses good antioxidant activity against H₂O₂ mediated cell damage in vitro.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

In vitro antiplasmodial activity of extract and constituents from Esenbeckia febrifuga, a plant traditionally used to treat malaria in the Brazilian Amazon

Esenbeckia febrifuga (Rutaceae) is a plant traditionally used to treat malaria in the Brazilian Amazon region. Ethanol extract of stems displayed a good antiplasmodial activity against Plasmodium falciparum strains W-2 (IC₅₀ 15.5±0.71 μg/ml) and 3 D7 (IC₅₀ 21.0±1.4 μg/ml). Two coumarins (bergaptene 1 and isopimpinellin 2), five alkaloids (flindersiamine 3, kokusaginine 4, skimmiamine 5, γ-fagarine 6 and 1-hydroxy-3-methoxy-N-methylacridone, 7), besides a limonoid (rutaevine 8), have been isolated for the first time from this species. Antiplasmodial activity of compounds 3, 5–8 has been evaluated in vitro against P. falciparum strains (W-2 and 3D7) and the furoquinolines 5 and 6 were the most potent displaying IC₅₀ values <50 μg/ml; flindersiamine (3) showed a weak activity while alkaloid 7 and rutaevine (8) were inactive (IC₅₀>100 μg/ml).     

Purification and characterization of angiotensin I converting enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin

Proteolytic digestion of gelatin extracts from Alaska Pollack (Theragra chalcogramma) skin brings about a high angiotensin I converting enzyme (ACE) inhibitory activity. Gelatin extracts were hydrolyzed by serial protease-treatments in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. Fragments arising from the third step were composed of peptides ranging from 0.9 to 1.9 kDa and responsible for ACE inhibitory activity. Catalytically active two peptides were separated by the consecutive chromatographic methods including gel filtration, ion-exchange chromatography, and reverse-phase high performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Met and showed IC₅₀ values of 2.6 and 17.13 μM, respectively. These results suggested that Gly-Pro-Leu would be useful as a new antihypertensive agent.     

Inhibitory activity of phosphorylated chitooligosaccharides on the formation of calcium phosphate

Chitooligosaccharides (COSs) were successfully prepared using an ultrafiltration membrane bioreactor system, and named as COS-I (10–5 kDa), COS-II (5–3 kDa), COS-III (3–1 kDa), and COS-IV (below 1 kDa), respectively. In addition, their phosphorylated derivatives were prepared by a P₂O₅ in methanesulfonic acid solution, and inhibitory activity on the formation of insoluble calcium phosphate of phosphorylated chitooligosaccharides. Phosphorylated COS-IV exhibited the highest inhibitory activity of calcium phosphate precipitation among tested chitooligosaccharides. Its inhibitory activity, especially at the concentration more than 4 mg/ml, was similar to that of CPP, which is widely used as a calcium fortifying agent increasing calcium absorbability. Therefore, phosphorylated chitooligosaccharides may be potential inhibitors of calcium phosphate precipitation.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Anti-allergic substances from the rhizomes of Dioscorea membranacea

Extracts of five species of Thai medicinal plants, locally known as Hua-Khao-Yen, were screened for anti-allergic activities using RBL-2H3 cells. Of the five species studied, the ethanolic extract of Dioscorea membranacea exhibited potent inhibitory activity against β-hexosaminidase release as a marker of degranulation in RBL-2H3 cells, with an IC₅₀ value of 37.5 μg/mL. Eight compounds were isolated from this crude ethanolic extract, [two naphthofuranoxepins (1, 2), one phenanthraquinone (3), three steroids (4–6), and two steroidal saponins (7, 8)], and tested for their anti-allergic activities. The results showed that dioscorealide B (2) possessed the highest activity with an IC₅₀ value of 5.7 μM, followed by dioscoreanone (3, IC₅₀ = 7.7 μM), dioscorealide A (1, IC₅₀ = 27.9 μM), and diosgenin (9, IC₅₀ = 29.9 μM). Structure–activity relationship studies of naphthofuranoxepins on anti-allergic activity revealed that the hydroxylation at position 8 conferred higher activity than methoxylation. For diosgenin derivatives, the aglycone was found to possess higher activity than the diglucosylated molecule; whereas substitution with rhamnoglucosides apparently results in loss of activity. Furthermore, effects of dioscorealide A, dioscorealide B, and dioscoreanone on antigen-induced release of TNF- and IL-4 in the late phase reaction were also examined.     

Synthesis, spectral studies and in vitro assessment for antiamoebic activity of new cyclooctadiene ruthenium(II) complexes with 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones

We report here the synthesis, characterization and in vitro antiamoebic activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones (TSC), 1–5, and their bidentate complexes [Ru(η⁴-C₈H₁₂)(TSC)Cl₂] 1a–5a. The biological studies of these compounds were investigated against HK-9 strain of Entamoeba histolytica and the concentration causing 50% cell growth inhibition (IC₅₀) was calculated in the micromolar range. The ligands exhibited antiamoebic activity in the range (2.05–5.29 μM). Screening results indicated that the potencies of the compounds increased by the incorporation of ruthenium(II) in the thiosemicarbazones. The complexes 1a–5a showed antiamoebic activity with an IC₅₀ of 0.61–1.43 μM and were better inhibitors of growth of E. histolytica, based on IC₅₀ values. The most promising among them is Ru(II) complex 2a having 1,2,3,4-tetrahydroquinoline as N⁴ substitution.     

Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines

Chalcones xanthohumol (X) and desmethylxanthohumol (DMX), present in hops (Humulus lupulus L.), and the corresponding flavanones isoxanthohumol (IX, from X), 8-prenylnaringenin (8-PN, from DMX), and 6-prenylnaringenin (6-PN, from DMX), have been examined in vitro for their anti-proliferative activity on human prostate cancer cells PC-3 and DU145. X proved to be the most active compound in inhibiting the growth of the cell lines with IC₅₀ values of 12.3±1.1 μM for DU145 and 13.2±1.1 μM for PC-3. 6-PN was the second most active growth inhibitor, particularly in PC-3 cells (IC₅₀ of 18.4±1.2 μM). 8-PN, a highly potent phytoestrogen, exhibited pronounced anti-proliferative effects on PC-3 and DU145 (IC₅₀ of 33.5±1.0 and 43.1±1.2 μM, respectively), and IX gave comparable activities (IC₅₀ of 45.2±1.1 μM for PC-3 and 47.4±1.1 μM for DU145). DMX was the least active compound. It was evidenced for the first time that this family of prenylated flavonoids from hops effectively inhibits proliferation of prostate cancer cells in vitro.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

Aromatic analogs of arcaine inhibit MK-801 binding to the NMDA receptor

Aromatic analogs of arcaine were shown to have inhibitory effects on the binding of the channel blocking drug [³H]MK-801 to the NMDA receptor complex. The most potent compound of the series was an N,N′-bis(propyl)guanidinium which inhibited [³H]MK-801 binding with an IC₅₀ of 0.58 μM and an IC₅₀ of 12.17 μM upon addition of 100 μM spermidine. The increase in IC₅₀ upon addition of spermidine suggests competitive antagonism between the inhibitor and spermidine at the arcaine-sensitive polyamine site of the NMDA receptor complex.     

Crystallographic and NMR studies of antiinfective tricyclic guanidine alkaloids from the sponge Monanchora unguifera

Three tricyclic guanidine alkaloids, including 1,8a;8b,3a-didehydro-8β-hydroxyptilocaulin (1), 1,8a;8b,3a-didehydro-8-hydroxyptilocaulin (2) and mirabilin B (3), were identified from the marine sponge Monanchora unguifera. 1,8a;8b,3a-Didehydro-8-hydroxyptilocaulin (2) is a new stereoisomer of 1, the structure of which was elucidated by spectroscopic analysis, comparison of its spectral data with those of 1, and confirmed by X-ray analysis. Compounds 1 and 2 co-crystallized in an unusual perfect order and packed around an approximate inversion center. A mixture of 1 and 2 is active against the malaria parasite Plasmodium falciparum with an IC₅₀ value of 3.8 μg/mL while mirabilin B (3) exhibited antifungal activity against Cryptococcus neoformans with an IC₅₀ value of 7.0 μg/mL and antiprotozoal activity against Leishmania donovani with an IC₅₀ value of 17 μg/mL.     

Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors

A myeloperoxidase inhibitory kaempferol derivative, namely pterogynoside (1), was isolated from fruits of Pterogyne nitens, along with six known flavonols, kaempferol, afzelin, kaempferitrin, quercetin, isoquercetrin and rutin. The structures of all compounds were elucidated primarily from 1D and 2D NMR spectroscopic analyses, as well as by high resolution mass spectrometry. All flavonols were screened to identify secondary metabolites as potential myeloperoxidase (MPO) inhibitors, and at concentrations of 0.50–50 nM, quercetin (5), isoquercitrin (6) and rutin (7) exhibited strong inhibitory effects with IC₅₀ values of 1.22 ± 0.01, 3.75 ± 0.02 and 3.60 ± 0.02, respectively. The MPO activity detected for the new derivative 1 was markedly decreased (IC₅₀ 10.3 ± 0.03) when compared with known flavonols 5–7, and interestingly increased when tested against ABTS scavenging activity.     

Substituted phenanthrene imidazoles as potent, selective, and orally active mPGES-1 inhibitors

Phenanthrene imidazole 3 (MF63) has been identified as a novel potent, selective, and orally active mPGES-1 inhibitor. This new series was developed by lead optimization of a hit from an internal HTS campaign. Compound 3 is significantly more potent than the previously reported indole carboxylic acid 1 with an A549 whole cell IC₅₀ of 0.42 μM (50% FBS) and a human whole blood IC₅₀ of 1.3 μM. It exhibited a significant analgesic effect in a guinea pig hyperalgesia model when orally dosed at 30 and 100 mg/kg.     

Purification and characterization of xylanase from Aspergillus ficuum AF-98

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH₄)₂SO₄, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7 U/ mg protein) was a monomeric protein with a molecular mass of 35.0 kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45 °C and 5.0, respectively. The xylanase was activated by Cu²⁺ up to 115.8% of activity, and was strongly inhibited by Hg²⁺, Pb²⁺ up to 52.8% and 89%, respectively. The xylanase exhibited K_m and V_max values of 3.267 mg/mL, 18.38 M/min/mg for beechwood xylan and 3.747 mg/mL, 11.1 M/min/mg for birchwood xylan, respectively.     

Extraction and purification of phycocyanin from Calothrix sp.

The extraction and purification of phycocyanin from Calothrix sp., cyanobacteria isolated from rice fields in Cuernavaca, Morelos, Mexico is described. Phycocyanin was extracted with 2 mg of lysozyme/g wet biomass, and purified by anion chromatography using Q-Sepharose fast-flow (Pharmacia^®, 1.5 cm×10 cm) column and hydrophobic interaction chromatography with methyl macro-prep (Bio-Rad^®, 1.5 cm×20 cm) column. The purified protein showed a pI of 5.2 and has two subunits with apparent molecular mass of 21–17 kDa each. The estimated molecular mass of native purified phycocyanin was 114 kDa, suggesting a stereochemistry of (β)₃.     

Design, synthesis, and biological evaluation of glycine-based molecular tongs as inhibitors of Abeta1-40 aggregation in vitro

A series of N-terminus benzamides of glycine-based symmetric peptides, linked to m-xylylenediamine and 3,4′-oxydianiline spacers, were prepared and tested as inhibitors of β-amyloid peptide Aβ_1–40 aggregation in vitro. Compounds with good anti-aggregating activity were detected. Polyphenolic amides showed the highest anti-aggregating activity, with IC₅₀ values in the micromolar range. Structure–activity relationships suggested that π–π stacking and hydrogen-bonding interactions play a key role in the inhibition of Aβ_1–40 self-assembly leading to amyloid fibrils.     

Inhibition of Candida rugosa lipase by saponins, flavonoids and alkaloids

Lipase inhibitors have generated a great interest because they could help in the prevention or the therapy of lipase-related diseases. Therefore, the aim of the work was to evaluate by HPLC, and using Candida rugosa lipase as model, the inhibitory effect of several saponins: β-aescin, digitonin, glycyrrhizic acid (GA) and Quillaja saponin (QS); flavonoids: 3-hydroxyflavone, 5-hydroxyflavone, (±)-catechin and kaempferol; and alkaloids: aspidospermine, papaverine, physostigmine, pilocarpine, raubasine, rescinnamine, reserpine and trigonelline.

The inhibition produced by most of these compounds is described here for the first time. Saponins appeared very active, being β-aescin and digitonin the most active compounds (IC₅₀ = 0.8–2.4 × 10⁻⁵ M). The inhibitory activity of flavonoids was lower than that of saponins (except GA), and (±)-catechin and kaempferol were the most active. Alkaloids was the most heterogeneous group assayed, varying from rescinnamine, with an IC₁₆ similar to that of digitonin, to papaverine and others which showed almost no inhibition.

In conclusion, β-aescin, digitonin, kaempferol or (±)-catechin, strong lipase inhibitors with a low toxicity and present herbal drugs used for lipase-related diseases such as acne or ulcer, are promising candidates for the prevention or the treatment of these diseases.