Inhibition of cathepsin D by tripeptides containing statine analogs.

Various analogs of statine, a remarkable amino acid component of the protease inhibitor pepstatine, were synthesized and evaluated as tripeptide derivatives for their activity against cathepsin D and HIV-1 protease.     

Antiosteoarthritic effect of Punica granatum L. peel extract on collagenase induced osteoarthritis rat by modulation of COL‐2, MMP‐3, and COX‐2 expression

Osteoarthritis (OA) is a chronic degenerative and musculoskeletal disorder. The toxicity associated with nonsteroidal antiinflammatory drugs (NSAIDs) limits its use in the management of OA. To ameliorate these toxicities, natural antioxidants can be used as substitutes for the management of OA. Therefore, this study is aimed to investigate the prophylactic mechanisms of Punica granatum L. peel (PGP) in collagenase‐induced OA rat compared with indomethacin. OA was induced in female Sprague Dawley rats by intraarticular injection of collagenase type‐II and treated with PGP (250 and 500 mg/kg body wt) and a positive control (PC) indomethacin (3 mg/kg body wt). The results demonstrated that PGP reduced the collagenase induced OA as compared with indomethacin treated group through reducing blood ALP (P < .001) and significantly (P < .001) inhibited cartilage erosion as indicated in histological slides with retention of collagen and proteoglycan content. Quantitative real‐time PCR analysis revealed the considerable (P < .05) upregulation in the expression of COL‐2 gene and downregulation of MMP‐3 and COX‐2 genes in the PGP treated group. The high phenolic content (633 ± 1.16 mg/GAE) and flavonoid content (420.3 ± 2.14 mg/RE) contribute to the strong antioxidant activity with IC₅₀ value (320 ± 2.2 μg/mL) of DPPH free radical scavenging activity. These results need further validation in clinical studies and thus, PGP could be developed as a preventive drug treatment for OA.     

Antioxidant and antimutagenic activity of Carum copticum fruit extracts

The ajowain (Carum copticum (L.)) is a popular spice and traditionally used in Indian system of medicine. Considering the importance of natural products in modern phytomedicine, the antioxidant and antimutagenic activities of C. copticum fruits extract and its fractions were evaluated. The methanol fraction showed highest antioxidant activity by phosphomolybdenum (2087.7 μmol) and DPPH assay (90.2%) followed by other fractions comparable to ascorbic acid and BHT. Based on antioxidant activity, methanol fraction was evaluated for antimutagenic potential against direct acting mutagens sodium azide (NaN₃) and methyl methane sulphonate (MMS) and indirect acting mutagens 2-aminofluorene (2-AF) and benzo(a)pyrene (B(a)P), using Salmonella typhimurium (TA97a, TA98, TA100, and TA102) tester strains. The methanolic fraction showed no sign of mutagenicity at tested concentrations (25–100 μg/plate). Antimutagenic activity was recorded with inhibition of mutagenicity ranging from 10.8% to 83.1% in a concentration dependent manner. The phytochemical analysis by IR, HPLC, GC–MS, and total phenolic assay revealed a high content of phenolic terpenoids. Further, characterization of active principle is needed to understand the mechanism of action and therapeutic efficacy in vivo.     

Assessment of antioxidant,anti-inflammatory,anti-cholinesterase and cytotoxic activities of pomegranate (Punica granatum) leaves

This study evaluated antioxidant, anti-inflammatory, anti-cholinesterase and cytotoxic activities of extracts with different polarities (hexane, dichloromethane, ethyl acetate, ethanol and methanol) obtained from Punica granatum leaves. Total phenolics (8.8–127.3 mg gallic acid equivalent/g dry weight), flavonoids (1.2–76.9 mg quercetin equivalent/g dry weight), tannins (63.7–260.8 mg catechin equivalent/kg dry weight) and anthocyanins (0.41–3.73 mg cyanidin-3-glucoside equivalent/g dry weight) of different extracts were evaluated. The methanolic extract presented a good IC50 by DPPH and ABTS assays (5.62 and 1.31 mg/l respectively). The strongest 5-lipoxygenase (5-LOX), acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibition activities were obtained for the ethanol extract (IC50 values of 6.20, 14.83 and 2.65 mg/l, respectively) and the best cytotoxic activity against MCF-7 cells was obtained for the methanol extract (IC50 = 31 mg/l). These important biological activities showed that P. granatum leaves could be a potential source of the active molecules intended for applications in pharmaceutical industry, but only after additional in vivo experiments.     

Antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from Punica granatum L

The Punica granatum L. (pomegranate) by-product POMx was partitioned between water, EtOAc and n-BuOH, and the EtOAc and n-BuOH extracts were purified by XAD-16 and Sephadex LH-20 column chromatography to afford ellagic acid (1), gallagic acid (2), punicalins (3), and punicalagins (4). Compounds 1 - 4 and the mixture of tannin fractions (XAD-16 eluates) were evaluated for antioxidant, antiplasmodial, and antimicrobial activities in cell-based assays. The mixture of tannins (TPT), XAD-EtOAc, XAD-H2O, XAD-PJ and XAD-BuOH, exhibited IC50 values against reactive oxygen species (ROS) generation at 0.8 - 19 microg/mL. Compounds 1 - 4 showed IC50 values of 1.1, 3.2, 2.3 and 1.4 microM, respectively, against ROS generation and no toxicity up to 31.25 microg/mL against HL-60 cells. Gallagic acid (2) and punicalagins (4) exhibited antiplasmodial activity against Plasmodium falciparum D6 and W2 clones with IC50 values of 10.9, 10.6, 7.5 and 8.8 microM, respectively. Fractions XAD-EtOAc, XAD-BuOH, XAD-H2O and XAD-PJ compounds 1 - 4 revealed antimicrobial activity when assayed against Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Cryptococcus neoformans, methicillin-resistant Staphylococcus aureus (MRSA), Aspergillus fumigatus and Mycobacterium intracellulare. Compounds 2 and 4 showed activity against P. aeruginosa, C. neoformans, and MRSA. This is the first report on the antioxidant, antiplasmodial and antimicrobial activities of POMx isolates, including structure-activity relationships (SAR) of the free radical inhibition activity of compounds 1 - 4. Our results suggest a beneficial effect from the daily intake of POMx and pomegranate juice (PJ) as dietary supplements to augment the human immune system's antioxidant, antimalarial and antimicrobial capacities.     

Inhibition of CYP2D6 activity by bupropion

The purpose of this study was to assess the effect of bupropion on cytochrome P450 2D6 (CYP2D6) activity. Twenty-one subjects completed this repeated-measures study in which dextromethorphan (30-mg oral dose) was administered to smokers at baseline and after 17 days of treatment with either bupropion sustained-release (150 mg twice daily) or matching placebo. Subjects quit smoking 3 days before the second dextromethorphan administration. To assess CYP2D6 activity, urinary dextromethorphan/dextrorphan metabolic ratios were calculated after an 8-hour urine collection. Thirteen subjects received bupropion, and 8 received placebo. In those receiving active medication, the dextromethorphan/dextrorphan ratio increased significantly at the second assessment relative to the first (0.012 +/- 0.012 vs. 0.418 +/- 0.302; P < 0.0004). No such change was observed in those randomized to placebo (0.009 +/- 0.010 vs. 0.017 +/- 0.015; P = NS). At baseline, all subjects were phenotypically extensive CYP2D6 metabolizers (metabolic ratio <0.3); after treatment, 6 of 13 subjects receiving bupropion, but none of those receiving placebo, had metabolic ratios consistent with poor CYP2D6 metabolizers. Bupropion is therefore a potent inhibitor of CYP2D6 activity, and care should be exercised when initiating or discontinuing bupropion use in patients taking drugs metabolized by CYP2D6.     

Investigation of fruit peel extracts as sources for compounds with antioxidant and antiproliferative activities against human cell lines

The aim of this study was to evaluate antioxidant activity and cytotoxicity against human cell lines of fruit peel extracts from rambutan, mangosteen and coconut. The highest antioxidant activity was found from rambutan peel crude extract where the highest radical scavenging capacity via ABTS assay was from its ethyl acetate fraction with a TEAC value of 23.0 mM/mg and the highest ferric ion reduction activity via FRAP assay was from its methanol fraction with an EC value of 20.2 mM/mg. Importantly, using both assays, these fractions had a higher antioxidant activity than butylated hydroxyl toluene and vitamin E. It was shown that the ethyl acetate fraction of rambutan peel had the highest polyphenolic content with a gallic acid equivalent of 2.3 mg/mL. The results indicate that the polyphenolic compounds are responsible for the observed antioxidant activity of the extracts. Interestingly, the hexane fraction of coconut peel showed a potent cytotoxic effect on KB cell line by MTT assay (IC₅₀ = 7.7 μg/mL), and no detectable cytotoxicity toward normal cells. We concluded that the ethyl acetate fraction of rambutan peel is a promising resource for potential novel antioxidant agents whereas the hexane fraction of coconut peel may contain novel anticancer compounds.     

Inhibition of poly(ADP-ribose)polymerase activity by nucleoside analogs of thymidine.

The poly ADP-ribosylation of proteins catalyzed by poly(ADP-ribose)polymerase (PARP) is involved in a number of important cellular metabolic activities. We evaluated various analogs of deoxythymidine and deoxyuridine as inhibitors of PARP. Most of these compounds have antiviral and/or anticancer activities. The structural requirements for these nucleoside analogs to be inhibitors of PARP were determined. The compounds evaluated had various substitutions on the 2-, 4- and/or 5-position of the pyrimidine ring, as well as on the 2'-, 3'- and/or 5'-position of the pentose moiety. Inhibition of PARP was strongly dependent on the size of the alkyl or halogen substituent on the 5-position of the pyrimidine ring. Whereas the 5-position of the pyrimidine ring could be varied, alteration of the 2- or 4-position drastically decreased the inhibition of PARP. Kinetic analysis was performed with concentrations of 1-10 microM NAD+. The Ki values for many compounds were five to seven times lower than the Ki for 3-aminobenzamide, a previously described potent inhibitor of PARP. Compounds with combined substituents at both the 5-position of the pyrimidine ring and the 3'- or 5'-position of deoxyribose generally were potent inhibitors of PARP, as for example 3'-amino-2', 3'-dideoxy-(E)-5-(2-bromovinyl)uridine (Ki = 0.7 microM), or 5'-azido-2',5'-dideoxy-5-ethyluridine (Ki = 0.8 microM). The 5-halogenated analogs had Ki values of 18, 35, 110 and greater than 1000 microM for 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine, respectively, and the 5-alkyl analogs had Ki values of 45, 2.2, 7, 16 and 180 microM for 5-methyl-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 5-butyl-2'-deoxyuridine and 5-pentyl-2'-deoxyuridine, respectively. Two other compounds with substituents in the 5-position of the pyrimidine moiety also had potent activities: (E)-5-(2-bromovinyl)-2'-deoxyuridine (Ki = 6 microM) and 5-trifluoromethyl-2'-deoxyuridine (Ki = 1.6 microM). Compounds substituted in the 2'-, 3'- and/or 5'-position of the deoxyribose moiety were investigated and 5'-azido-5'-deoxythymidine, 5'-amino-5'-deoxythymidine, 3'-azido-3'-deoxythymidine and 3'-deoxythymidine (d2T) and Ki values of 12, 16, 18 and 30 microM, respectively.     

In vitro activity of a new semisynthetic echinocandin, micafungin, against clinical isolates of Candida species isolated in Tenri Hospital

We have examined the antifungal activities of the available antifungal agents including micafungin (MCFG), one of the echinocandin antifungal group, against 92 yeast-like fungi isolated at our hospital during a 3-month period from November 2002 to February 2003. Determination of the antifungal susceptibility was conducted in conformity with the Standards of the Japanese Society for Medical Mycology. The MIC 80% of the antifungal agents against 4 fungi species including C. albicans (55 strains), C. tropicalis (20 strains), C. glabrata (8 strains), C. krusei (5 strains) were as follows; MCFG: 0.03-0.125 microgram/ml, amphotericin-B: 0.125-0.25 microgram/ml, 5-fluorocytosine: 0.125-16 micrograms/ml, itraconazole: 0.25-2 micrograms/ml, fluconazole: 0.5-32 micrograms/ml. The isolation rate of the drug-resistant fungi was 20% for the fluconazole (FLCZ)-resistant C. tropicalis and 33% when including the susceptible dose dependent (S-DD) class. The rate was 5% for FLCZ-resistant strains of C. albicans and 11% when including the S-DD class. However, MCFG was shown to have an excellent antifungal activity against those azole-resistant strains of Candida species. An analysis of the randomly amplified polymorphic DNA pattern (RAPD) was carried out to assess the fingerprinting of the azole-resistant strains. The results demonstrated a common pattern in 3 of the 6 strains of C. tropicalis that showed MIC of > or = 16 micrograms/ml for fluconazole, while all of the 6 strains of C. albicans demonstrated their respective patterns.     

Beneficial Effects of Punica granatum L. Juice and Gallic Acid Against Kidney Oxidative Damage Caused by Sodium Fluoride

Pharmaceutical Chemistry Journal - The present work was undertaken to study the effect of sodium fluoride (NaF) exposure on renal oxidative stress status in rats and to evaluate the protective...     

Inhibition of TRPP3 channel by amiloride and analogs

TRPP3, a member of the transient receptor potential (TRP) superfamily of cation channels, is a Ca2+-activated channel permeable to Ca2+, Na+, and K+. TRPP3 has been implicated in sour tasting in bipolar cells of tongue and in regulation of pH-sensitive action potential in spinal cord neurons. TRPP3 is also present in excitable and nonexcitable cells of other tissues, including retina, brain, heart, testis, and kidney, with unknown functions. In this study, we examined the functional modulation of TRPP3 channel by amiloride and its analogs, known to inhibit several ion channels and transporters and respond to all taste stimuli, using Xenopus laevis oocyte expression, electrophysiology, and radiotracer measurements. We found that amiloride and its analogs inhibit TRPP3 channel activities with different affinities. Radiolabeled (45)Ca2+ uptake showed that TRPP3-mediated Ca2+ transport was inhibited by amiloride, phenamil, benzamil, and 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Two-microelectrode voltage clamp experiments revealed that TRPP3-mediated Ca2+-activated currents are substantially inhibited by amiloride analogs, in an order of potency of phenamil > benzamil > EIPA > amiloride, with IC50 values of 0.14, 1.1, 10.5, and 143 microM, respectively. The inhibition potency positively correlated with the size of inhibitors. Using cell-attached patch clamping, we showed that the amiloride analogs decrease the open probability and mean open time but have no effect on single-channel conductance. Study of inhibition by phenamil in the presence of previously reported inhibitor tetrapentylammonium indicates that amiloride and organic cation inhibitors compete for binding the same site on TRPP3. TRPP3 may contribute to previously reported in vivo amiloride-sensitive cation transport.     

Inhibition of chemiluminescence and chemotactic activity of phagocytes in vitro by the extracts of selected medicinal plants

The methanol extracts of 20 selected medicinal plants were investigated for their effects on the respiratory burst of human whole blood, isolated human polymorphonuclear leukocytes (PMNs) and isolated mice macrophages using a luminol/lucigenin-based chemiluminescence assay. We also tested the effect of the extracts on chemotactic migration of PMNs using the Boyden chamber technique. The extracts of Curcuma domestica L., Phyllanthus amarus Schum & Thonn and C. xanthorrhiza Roxb. were the samples producing the strongest oxidative burst of PMNs with luminol-based chemiluminescence, with IC₅₀ values ranging from 0.5 to 0.7 μg/ml. For macrophage cells, the extracts which showed strong suppressive activity for luminol-based chemiluminescence were C. xanthorrhiza and Garcinia mangostana L. Among the extracts studied, C. mangga Valton & Vazsjip, Piper nigrum L. and Labisia pumila var. alata showed strong inhibitory activity on lucigenin-amplified oxidative burst of PMNs, with IC₅₀ values ranging from 0.9 to 1.5 μg/ml. The extracts of Zingiber officinale Rosc., Alpinia galangal (L.) Willd and Averrhoa bilimbi Linn showed strong inhibition on the chemotaxic migration of cells, with IC₅₀ values comparable to that of ibuprofen (1.5 μg/ml). The results suggest that some of these plants were able to modulate the innate immune response of phagocytes at different steps, emphasizing their potential as a source of new immunomodulatory agents.     

Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits

We investigated the correlation between the flavonoid content and NO production inhibitory activity of fruit peel extracts using 20 citrus plants. The contents of seven flavonoids (naringin, naringenin, hesperidin, hesperetin, rutin, nobiletin, and tangeretin) were determined by HPLC analysis. Each citrus peel extract varied in flavonoid content, but the contents of nobiletin and tangeretin, which were contained in all 20 fruit peels, showed a positive and significant correlation with each other (r=0.879, p<0.0005 for immature fruit peels; r=0.858, p<0.0005 for mature fruit peels). All citrus peel extracts dose-dependently inhibited LPS-induced NO production in RAW 264.7 cells. This inhibitory effect was significantly and positively correlated with the content of nobiletin and tangeretin. Nobiletin showed a more potent NO production inhibitory activity (IC50=26.5 microM) compared to tangeretin (IC50=136.6 microM). This result supports the premise that nobiletin-rich citrus may provide protection against disease resulting from excessive NO production.     

Inhibition of MG-63 cell cycle progression by synthetic vitamin D3 analogs mediated by p27, Cdk2, cyclin E,and the retinoblastoma protein

Progression through eukaryotic cell division cycle is regulated by synergistic activities of both positive and negative regulatory factors. The active form of vitamin D₃ (1α,25(OH)₂D₃, 1,25D) and a number of its synthetic analogs have been shown to arrest cells in the G₁ phase of the cell cycle. In the present study, 1α,25(OH)₂D₃ and the analogs KH1060, EB1089, and CB1093 were used to study the mechanism of the cell cycle arrest and to compare the effectiveness of these compounds in human MG-63 osteosarcoma cells. The 20-epi analogs KH1060 and CB1093, as well as the 20-normal analog EB1089, were found to be more potent than 1α,25(OH)₂D₃ in inhibiting cell proliferation and arresting the MG-63 cells in the G₁ phase. These analogs were more active than 1α,25(OH)₂D₃ in increasing the cyclin dependent kinase inhibitor p27 protein levels (approximately 2.3-2.5-fold compared to 1α,25(OH)₂D₃) by both increasing its formation and decreasing its degradation rate. The increased p27 formation was accompanied by stabilization of binding of nuclear proteins to the Sp1+NF-Y responsive promoter region of the p27 gene. The increase in p27 protein levels and the simultaneous decrease in cyclin E protein levels was accompanied by decreased Cdk2 kinase activity, retinoblastoma (Rb) protein hypophosphorylation and, finally, cell cycle arrest in the G₁ phase. In summary, the analogs KH1060, EB1089, and CB1093 keep Rb protein in its growth-suppressing, hypophosphorylated form and prevent cell cycle progression through the restriction point. Therefore, these synthetic vitamin D₃ analogs may be potential candidates for treating diseases, where cell cycle regulation is needed.