Anthocyanin content, lipid peroxidation and cyclooxygenase enzyme inhibitory activities of sweet and sour cherries.

Cherries contain bioactive anthocyanins that are reported to possess antioxidant, anti-inflammatory, anticancer, antidiabetic and antiobese properties. The present study revealed that red sweet cherries contained cyanidin-3-O-rutinoside as major anthocyanin (>95%). The sweet cherry cultivar "Kordia" (aka "Attika") showed the highest cyanidin-3-O-rutinoside content, 185 mg/100 g fresh weight. The red sweet cherries "Regina" and "Skeena" were similar to "Kordia", yielding cyanidin-3-O-rutinoside at 159 and 134 mg/100 g fresh weight, respectively. The yields of cyanidin-3-O-glucosylrutinoside and cyanidin-3-O-rutinoside were 57 and 19 mg/100 g fresh weight in "Balaton" and 21 and 6.2 mg/100 g fresh weight in "Montmorency", respectively, in addition to minor quantities of cyanidin-3-O-glucoside. The water extracts of "Kordia", "Regina", "Glacier" and "Skeena" sweet cherries gave 89, 80, 80 and 70% of lipid peroxidation (LPO) inhibition, whereas extracts of "Balaton" and "Montmorency" were in the range of 38 to 58% at 250 microg/mL. Methanol and ethyl acetate extracts of the yellow sweet cherry "Rainier" containing beta-carotene, ursolic, coumaric, ferulic and cafeic acids inhibited LPO by 78 and 79%, respectively, at 250 microg/mL. In the cyclooxygenase (COX) enzyme inhibitory assay, the red sweet cherry water extracts inhibited the enzymes by 80 to 95% at 250 microg/mL. However, the methanol and ethyl acetate extracts of "Rainier" and "Gold" were the most active against COX-1 and -2 enzymes. Water extracts of "Balaton" and "Montmorency" inhibited COX-1 and -2 enzymes by 84, and 91 and 77, and 87%, respectively, at 250 microg/mL.

Withanolide sulfoxide from Aswagandha roots inhibits nuclear transcription factor-kappa-B, cyclooxygenase and tumor cell proliferation.

Investigation of the methanol extract of Aswagandha (Withania somnifera) roots for bioactive constituents yielded a novel withanolide sulfoxide compound (1) along with a known withanolide dimer ashwagandhanolide (2) with an S-linkage. The structure of compound 1 was established by extensive NMR and MS experiments. Compound 1 was highly selective in inhibiting cyclooxygenase-2 (COX-2) enzyme by 60% at 100 microm with no activity against COX-1 enzyme. The IC(50) values of compound 1 against human gastric (AGS), breast (MCF-7), central nervous system (SF-268) and colon (HCT-116) cancer cell lines were in the range 0.74-3.63 microm. Both S-containing dimeric withanolides, 1 and 2, completely suppressed TNF-induced NF-kappaB activation when tested at 100 microm. The isolation of a withanolide sulfoxide from W. somnifera roots and its ability to inhibit COX-2 enzyme and to suppress human tumor cell proliferation are reported here for the first time. In addition, this is the first report on the abrogation of TNF-induced NF-kappaB activation for compounds 1 and 2.

Non-nutritive functional agents in rattan-shoots, a food consumed by native people in the Philippines.

The tender shoots of Calamus ornatus, one of the food items consumed by the native people, Kanawan Aytas, in the Bataan region of the Philippines, have not been studied before. A bioassay-guided investigation of its methanolic extract afforded non-nutritive functional agents (NFAs), steroidal saponins 1-3, along with its aglycone (4). The NFAs 1-4 inhibited cyclooxygenase enzymes, COX-1 and -2, by 47%, 43%, 33%, and 53% and 71%, 75%, 78%, and 73%, respectively, at 28.2, 24.2, 21.2 and 60.4µM. Treatment of breast (MCF-7), CNS (SF-268), lung (NCI-H460), colon (HCT-116) and gastric (AGS) cancer cell lines with the extract at 100µg/ml reduced cell proliferation. Similarly, the pure NFAs 2 and 3 reduced the cell viability of breast, CNS, lung, colon and gastric cancer cell lines by 37.5%, 22.4%, 53.3%, 58.2%, 40.3% and 29.8%, 21.3%, 45.6%, 37.1%, 25.0%, respectively, at 24.2 and 21.2µM. The 50% reduction in cell viability (IC50) concentrations of 2 and 3 against these cancer cell lines were 8.8, 6.1, 7.5, 23.8, 12.1 and 3.8, 7.1, 3.3, 14.3, 12.1µM, respectively. This is the first report on the isolation of steroidal saponins from C. ornatus shoots and their antiinflammatory and tumor cell proliferation inhibitory activities. Therefore, our results suggest that the Kanawan Aytas may yield health benefits from rattan-shoots in their diet.

Lipid peroxidation and cyclooxygenase enzyme inhibitory activities of acidic aqueous extracts of some dietary supplements.

The botanical supplement market is growing at a fast pace with more and more people resorting to them for maintaining good health. Echinacea, garlic, ginkgo, ginseng, Siberian ginseng, grape seed extract, kava kava, saw palmetto and St John's wort are some of the popular supplements used for a variety of health benefits. These supplements are associated with various product claims, which suggest that they possess cyclooxygenase (COX) enzyme and lipid s inhibitory activities. COX enzymes are found to be at elevated levels in inflamed and cancerous cells. To test some of the product claims, selected supplements were analysed for their ability to inhibit COX-1 and -2 enzymes and lipid peroxidation in vitro. The supplements were extracted with acidified water (pH 2) at 37 degrees C to simulate the gastric environment. The supplements tested demonstrated varying degrees of COX enzyme inhibition (5-85% for COX-1 and 13-28% for COX-2). Interestingly, extracts of garlic (Meijer), ginkgo (Solaray), ginseng (Nature's Way), Siberian ginseng (GNC, Nutrilite, Solaray, Natrol), kava kava (GNC, Sundown, Solaray) and St John's wort (Nutrilite) selectively inhibited COX-2 enzyme. These supplements also inhibited lipid peroxidation in vitro (5-99%). The results indicated that the consumption of these botanical supplements studied possess health benefits.

Impact of alkyl esters of caffeic and ferulic acids on tumor cell proliferation, cyclooxygenase enzyme, and lipid peroxidation.

The antioxidant ferulic and caffeic acid phenolics are ubiquitous in plants and abundant in fruits and vegetables. We have synthesized a series of ferulic and caffeic acid esters and tested for tumor cell proliferation, cyclooxygenase enzymes (COX-1 and -2) and lipid peroxidation inhibitory activities in vitro. In the tumor cell proliferation assay, some of these esters showed excellent growth inhibition of colon cancer cells. Among the phenolics esters assayed, compounds 10 (C12-caffeate), 11 (C16-caffeate), 21 (C8-ferulate), and 23 (C12-ferulate) showed strong growth inhibition with IC50 values of 16.55, 13.46, 18.67, and 7.57 microg/mL in a breast cancer cell line; 9.65, 7.45, 17.05, and 4.35 microg/ mL in a lung cancer cell line; 5.78, 3.5, 4.29, and 2.46 microg/mL in a colon cancer cell line; 12.04, 12.21, 14.63, and 8.09 microg/ mL in a central nervous system cancer cell line; and 8.62, 7.76, 11.0, and 5.37 in a gastric cancer cell line. In COX enzyme inhibitory assays, ferulic and caffeic acid esters significantly inhibited both COX-1 and COX-2 enzymes. Caffeates 5-10 (C4-C12), inhibited COX-1 enzyme between 50% and 90% and COX-2 enzyme by about 70%, whereas ferulates 15-21 (C3-C8) inhibited COX-1 and COX-2 enzymes by 85-95% 25 microg/mL. Long-chain caffeates 11-14 (C16-C22) and short-chain ferulates 15-20 (C3-C5) were the most active in lipid peroxidation inhibition and showed 60-70% activity at 5 microg/mL concentration.

Topography of the prostaglandin endoperoxide H2 synthase-2 in membranes.

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.

Synthetic curcuminoids modulate the arachidonic acid metabolism of human platelet 12-lipoxygenase and reduce sprout formation of human endothelial cells.

Platelet 12-lipoxygenase (P-12-LOX) is overexpressed in different types of cancers, including prostate cancer, and the level of expression is correlated with the grade of this cancer. Arachidonic acid is metabolized by 12-LOX to 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], and this biologically active metabolite is involved in prostate cancer progression by modulating cell proliferation in multiple cancer-related pathways inducing angiogenesis and metastasis. Thus, inhibition of P-12-LOX can reduce these two processes. Several lipoxygenase inhibitors are known, including plant and mammalian lipoxygenases, but only a few of them are known inhibitors of P-12-LOX. Curcumin is one of these lipoxygenase inhibitors. Using a homology model of the three-dimensional structure of human P-12-LOX, we did computational docking of synthetic curcuminoids (curcumin derivatives) to identify inhibitors superior to curcumin. Docking of the known inhibitors curcumin and NDGA to P-12-LOX was used to optimize the docking protocol for the system in study. Over 75% of the compounds of interest were successfully docked into the active site of P-12-LOX, many of them sharing similar binding modes. Curcuminoids that did not dock into the active site did not inhibit P-12-LOX. From a set of the curcuminoids that were successfully docked and selected for testing, two were found to inhibit human lipoxygenase better than curcumin. False-positive curcuminoids showed high LogP (theoretical) values, indicating poor water solubility, a possible reason for lack of inhibitory activity or/and nonrealistic binding. Additionally, the curcuminoids inhibiting P-12-LOX were tested for their ability to reduce sprout formation of endothelial cells (in vitro model of angiogenesis). We found that only curcuminoids inhibiting human P-12-LOX and the known inhibitor NDGA reduced sprout formation. Only limited inhibition of sprout formation at approximately IC(50) concentrations has been seen. At IC(50), a substantial amount of 12-HETE can be produced by lipoxygenase, providing a stimulus for angiogenic sprouting of endothelial cells. Increasing the concentration of lipoxygenase inhibitors above IC(50), thus decreasing the concentration of 12(S)-HETE produced, greatly reduced sprout formation for all inhibitors tested. This universal event for all tested lipoxygenase inhibitors suggests that the inhibition of sprout formation was most likely due to the inhibition of human P-12-LOX but not other cancer-related pathways.

Antioxidant and cyclooxygenase activities of fatty acids found in food.

Several commercially available C-8 to C-24 saturated and unsaturated fatty acids (1-29) were assayed for cyclooxygenase-I (COX-I) and cyclooxygenase-II (COX-II) inhibitory and antioxidant activities. Among the saturated fatty acids tested at 60 microg mL(-1), there was an increase in antioxidant activity with increasing chain length from octanoic acid to myristic acid (C-8-C-14) and a decrease thereafter. All unsaturated fatty acids tested at 60 microg mL(-1) showed good antioxidant activity except for undecylenic acid (12), cis-5-dodecenoic acid (13), and nervonic acid (29). The highest inhibitory activities among the saturated fatty acids tested on cyclooxygenase enzymes COX-I and COX-II were observed for decanoic acid to lauric acid (3-5) at 100 microg mL(-1). Similarly, among the unsaturated fatty acids tested, the highest activities were observed for cis-8,11,14-eicosatrienoic acid (25) and cis-13,16-docosadienoic acid (27) at 100 microg mL(-1).