Protective effect of anthocyanins from black soybean seed coats on UVB-induced apoptotic cell death in vitro and in vivo

UVB radiation proves to be one of the most relevant environmental risks because of its hazardous effects, such as premature skin aging and especially skin photocarcinogenesis. Anthocyanins, water-soluble pigments present in plants, are known to be powerful antioxidants that help protect plants from UV damage. In this study, we aimed at investigating the protective effect of anthocyanins from black soybean [Glycine max (L.) Merr] seed coats on UVB-induced apoptosis, and furthermore, we investigated the molecular mechanism responsible for regulation of apoptosis in vitro and in vivo. Pretreatment with anthocyanins reduced UVB-induced reactive oxygen species levels and inhibited UVB-induced apoptotic cell death through the prevention of caspase-3 pathway activation and reduction of proapoptotic Bax protein levels. UVB irradiation induced apoptotic cell death, which was inhibited by topical application of anthocyanins in hairless mice. It is concluded that anthocyanins from the seed coat of black soybeans may be useful compounds to modulate UVB-induced photoaging.     

Effective separation of potent antiproliferation and antiadhesion components from wild blueberry (Vaccinium angustifolium Ait.) fruits

Extracts from wild blueberry (Vaccinium angustifolium Ait.) were separated into proanthocyanidin-rich fractions using liquid vacuum and open column chromatography on Toyopearl and Sephadex LH-20, respectively. Fractions were characterized using analytical tools including mass spectrometry and NMR spectroscopy; fraction composition was correlated with bioactivity using antiproliferation and antiadhesion in vitro assays. There was a significant positive correlation between proanthocyanidin content of different fractions and biological activity in both the antiproliferation and antiadhesion assays. Two fractions containing primarily 4-->8-linked oligomeric proanthocyanidins with average degrees of polymerization (DPn) of 3.25 and 5.65 inhibited adhesion of Escherichia coli responsible for urinary tract infections. Only the fraction with a DPn of 5.65 had significant antiproliferation activity against human prostate and mouse liver cancer cell lines. These findings suggest both antiadhesion and antiproliferation activity are associated with high molecular weight proanthocyanidin oligomers found in wild blueberry fruits.     

Photoprotective potential of strawberry (Fragaria × ananassa) extract against UV-A irradiation damage on human fibroblasts

Exposure to UV-A radiation is known to induce discrete lesions in DNA and the generation of free radicals that lead to a wide array of skin diseases. Strawberry (Fragaria × ananassa) contains several polyphenols with strong antioxidant and anti-inflammatory activities. Because the major representative components of strawberry are anthocyanins, these may significantly contribute to its properties. To test this hypothesis, methanolic extracts from the Sveva cultivar were analyzed for anthocyanin content and for their ability to protect human dermal fibroblasts against UV-A radiation, as assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide and Comet assays. Five anthocyanin pigments were identified using high-performance liquid chromatography-diode array detection-electrospray ionization/mass spectrometry. Moreover, the strawberry extract showed a photoprotective activity in fibroblasts exposed to UV-A radiation, increasing cellular viability, and diminishing DNA damage, as compared to control cells. Overall, our data show that strawberry contains compounds that confer photoprotective activity in human cell lines and may protect skin against the adverse effects of UV-A radiation.     

The UVA and aqueous stability of flavonoids is dependent on B-ring substitution

Flavonols such as kaempferol and quercetin are believed to provide protection against ultraviolet (UV)-induced damage to plants. Recent in vitro studies have examined the ability of flavonols to protect against UV-induced damage to mammalian cells. Stability of flavonols in cell culture media, however, has been problematic, especially for quercetin, one of the most widely studied flavonols. As part of our investigations into the potential for flavonols to protect skin against UV-induced damage, we have determined the stability of a series of flavonols that differ only in the number of substituents on the B-ring. We measured the stability of these flavonols over time to UVA radiation, Dulbecco's modified Eagle's medium (DMEM), and Dulbecco's phosphate-buffered saline (DPBS) using high performance liquid chromatography with UV detection (HPLC-UV). The identification of the breakdown products of flavonols was accomplished by using a hybrid quadrupole linear ion trap mass spectrometer coupled with liquid chromatography. Tandem mass spectrometric analysis (MS/MS) of flavonol photoproducts was confirmed by comparing with the known standard samples. We have determined that flavonol stability decreases with increasing B-ring substitution, suggesting that future investigation of potential photoprotective flavonols will need to be cognizant of this trend.     

Anthocyanins from black soybean seed coats inhibit UVB-induced inflammatory cylooxygenase-2 gene expression and PGE2 production through regulation of the nuclear factor-kappaB and phosphatidylinositol 3-kinase/Akt pathway

Ultraviolet (UV) radiation can cause inflammatory changes and may further contribute to skin carcinogenesis. Anthocyanins are known to be powerful antioxidants that help protect plants from UV damage. Recently, we isolated anthocyanins from black soybean [Glycine max (L.) Merr] seed coats. Thus, we investigated the protective effect of anthocyanins from black soybean seed coats on UVB radiation-induced inflammatory responses and the molecular mechanism responsible for regulation of apoptosis and inflammatory responses. Anthocyanins inhibited UVB-induced cylooxygenase-2 (COX-2) and PGE 2 production through a nuclear factor-kappaB-dependent pathway and regulation of the PI3 kinase/Akt pathway activated by UVB in a human keratinocyte cell line, HaCaT. Topical application of anthocyanins prior to UVB irradiation of hairless mice also inhibited induction of COX-2 and PGE 2. In conclusion, it is suggested that anthocyanins from the seed coat of black soybeans can be used as a useful drug to modulate oxidative disorders including UVB-induced inflammation.     

Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells

This study examined the growth inhibitory effects of theasinensin A (from oolong tea) and black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate (TF-2a) and theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3) in human cancer cells. Theasinensin A, TF-1, and TF-2 displayed strong growth inhibitory effects against human histolytic lymphoma U937, with estimated IC50 values of 12 microM, but were less effective against human acute T cell leukemia Jurkat, whereas TF-3 and (-)-epigallocatechin-3-gallate (EGCG) had lower activities. The molecular mechanisms of tea polyphenol-induced apoptosis as determined by annexin V apoptosis assay, DNA fragmentation, and caspase activation were further investigated. Loss of membrane potential and reactive oxygen species (ROS) generation were also detected by flow cytometry. Treatment with tea polyphenols caused rapid induction of caspase-3, but not caspase-1, activity and stimulated proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). Pretreatment with a potent caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluoromethyl ketone, inhibited theasinensin A induced DNA fragmentation. Furthermore, it was found that theasinensin A induced loss of mitochondrial transmembrane potential, elevation of ROS production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of caspase-9 activity. These results indicate that theasinensin A allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 (DNA fragmentation factor) degradation. The results suggest that induction of apoptosis by theasinensin A may provide a pivotal mechanism for their cancer chemopreventive function.     

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.     

Antioxidant activity of Glossogyne tenuifolia

Glossogyne tenuifolia is a native traditional anti-inflammatory herb in Taiwan. It has previously been shown that the ethanol extract of G. tenuifolia (GT) inhibited the LPS-induced inflammatory mediator release from murine macrophage cell line and human whole blood. In the present work, the ethanol extracts of G. tenuifolia and its major constituent, luteolin-7-glucoside, were shown to be scavengers of 1,1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl radicals. Moreover, copper-induced low-density lipoprotein oxidation was suppressed by GT and luteolin-7-glucoside as measured by decreased formation of malondialdehyde and conjugated diene as well as reduced electrophoretic mobility. GT and luteolin-7-glucoside were also against N-formyl-methionyl-leucyl-phenylalanine-induced reactive oxygen species (ROS) production in human polymorphonuclear neutrophils and peripheral blood mononuclear cells. In summary, these data indicated that GT is a potential ROS scavenger and may prevent atherosclerosis via inhibiting LDL oxidation or ROS production in human leukocytes. Moreover, luteolin-7-glucoside may serve as the active principal of GT.     

Alkyl hydroxytyrosyl ethers show protective effects against oxidative stress in HepG2 cells

Alkyl hydroxytyrosyl ethers (methyl, ethyl, propyl, and butyl ethers) have been synthesized from hydroxytyrosol (HTy) in response to the increasing food industry demand of new lipophilic antioxidants. Having confirmed that these compounds reach portal blood partially unconjugated and thus are effectively absorbed, their potential antioxidant activity was evaluated in the human hepatocarcinoma cell line (HepG2). The effects of 0.5-10 μM alkyl hydroxytyrosyl ethers on HepG2 cell integrity and redox status were assessed as well as the protective effect against oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Cell viability (Crystal violet) and cell proliferation (BrdU assay) were measured as markers of cell integrity, concentration of reduced glutathione (GSH), generation of reactive oxygen species (ROS), and activity of antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR) as markers of redox status and determination of malondialdehyde (MDA) as a marker of lipid peroxidation. Direct treatment of HepG2 with alkyl hydroxytyrosyl ethers induced slight changes in cellular intrinsic antioxidants status, reducing ROS generation and inducing changes in GPx and GR activities. Pretreatment of HepG2 cells with alkyl hydroxytyrosyl ethers counteracted cell damage induced by t-BOOH, partially after 2 h and completely after 20 h, by increasing GSH and decreasing ROS generation, MDA levels, and antioxidant enzyme (GPx and GR) activity. According to these results the alkyl hydroxytyrosyl ethers show clear protective effects against oxidative stress, related to their lipophilic nature, that are similar to or even higher than those of their precursor, HTy.     

High energy ultraviolet photo-oxidation: a novel technique for studying physically protected organic matter in clay- and silt-sized aggregates

A novel approach to the study of organic-matter distribution in soil microaggregates (<20 μm) using high-energy ultraviolet (UV) radiation in the presence of oxygen (photo-oxidation) is reported. The method quantitatively destroyed complex organic materials through oxidation, even in the presence of clay, provided the organic materials were directly exposed to the UV radiation. Photo-oxidation of clay and silt fractions for periods up to 8 h demonstrated that a considerable proportion of the organic matter was physically protected within clay- and silt-sized aggregates. In some clay fractions, up to 23% of the organic carbon could be considered as physically protected whereas in silt fractions this was as high as 36%. Infrared spectroscopy demonstrated that the materials external to both clay- and silt-sized aggregates were largely proteinaecous in nature, while the materials in the interior of the aggregates resembled humic acids. These humic materials appeared to be physically shielded against photo-oxidation, rather than being chemically recalcitrant. Using the clay- and silt-sized fractions from one soil, ¹⁴C accelerator mass spectrometry demonstrated that, although both clay and silt fractions contained essentially modern carbon, after 4 h of photo-oxidation much older organic carbon with a mean resonance time (MRT) of between 200±80 and 320±80 years before the present (BP) remained. This protection from photo-oxidation, therefore, appears to mirror the process which physically protects organic substances in soils against microbial degradation. Photo-oxidation of the clay-plus-silt fractions also resulted in a considerable reduction in particle size as the organic-cementing agents, consisting of proteinaecous and humic materials, were oxidized. Using data from the photo-oxidation method along with infrared spectroscopy, radiocarbon dating and scanning electron microscopy, a simple model is proposed that spatially relates the various organic structures present to their positions in the mineral aggregates.     

Lipid fractions with aggregatory and antiaggregatory activity toward platelets in fresh and fried cod (Gadus morhua): correlation with platelet-activating factor and atherogenesis

Cod (Gadus morhua) is a popular part of the diet in many countries on both sides of the North Atlantic; in most cases it is consumed fried. In this study, total lipids of cod muscle were separated into neutral and polar lipids, which were further fractionated by HPLC. The lipid fractions were tested in vitro, against washed rabbit platelets, for the probable existence of lipid compounds that either exhibit an action similar to that of platelet-activating factor (PAF) or inhibit the action of PAF. The platelet bioassay was used to evaluate total lipids, total polar lipids, and total neutral lipids, before any further separation. Detection of these compounds in fresh and fried cod could be used to evaluate the nutritional value of this important fish. The in vitro biological study of lipids showed that in fresh cod lipid fractions, ranges of PAF-like and anti-PAF-like activities were present, whereas in fried cod lipid fractions, both neutral and polar, anti-PAF activities were mainly observed. Because it has already been reported that PAF is involved in atheromatosis generation, the existence of PAF inhibitors in cod may contribute to the possible protective role of fish, in this case cod, against atherosclerosis.     

Wheat bran oil and its fractions inhibit human colon cancer cell growth and intestinal tumorigenesis in Apc(min/+) mice

This study was designed to investigate the cancer preventive activities of wheat bran (WB) oil. We studied the colon cancer preventive effects of WB oil and its subfractions in the Apc(min/+) mouse model, a recognized mouse model for human colorectal cancer, and used human colon cancer cell lines (HCT-116 and HT-29) to identify possible active fractions in WB oil. Our results showed that the oil fraction of WB was more active than the water fraction against the growth of human colon cancer cell lines and that 2% WB oil significantly inhibited the overall tumorigenesis by 35.7% (p < 0.0001) in the Apc(min/+) mouse model. The WB oil was further fractioned into nonpolar lipids and phytochemicals and the phytochemical fraction was fractionated into phytosterols and phytosterol ferulates, 5-alk(en)ylresorcinols, and unidentified constituents by normal phase silica gel column chromatography. Results on cell culture showed that the phytochemical fraction had a higher inhibitory effect on HCT-116 human colon cancer cells than that of WB oil, whereas the nonpolar lipid fraction had less growth inhibitory effectiveness. However, neither fractions showed a stronger inhibition than WB oil in the Apc(min/+) mouse model. The current results demonstrate, for the first time, the intestinal cancer preventive activity of WB oil. The active ingredients, however, remain to be identified.     

Influence of extraction parameters on the phytochemical characteristics of extracts from buckwheat (Fagopyrum esculentum) herb

In recent years, the interest in herbal medicinal products, especially in the field of dermatology and cosmetics, has risen enormously. Many plant-derived substances show photoprotective properties in terms of absorption of UV radiation and preventing photodamage to molecular structures of human skin. Modern phytopharmaceutics as well as phytocosmetics require standardized, defined extracts from the herbal matrix. Buckwheat herb is rich in flavonoids, which have been identified as potent antioxidants. Up to now, there have been no systematic investigations available concerning the extraction conditions for phenolic substances from buckwheat herb. In this paper, we report the influence of three extraction parameters, ethanol concentration, temperature, and extraction time, on the response variables extractable matter, antioxidant activity, and content of fagopyrin, rutin, and chlorogenic acid. Our results suggest that an extract with good antioxidant activity, a high content of phenolics, and a low content of the phototoxic fagopyrin can be yielded by agitated maceration with 30% ethanol at 60 degrees C for 2 h. Furthermore, there is good correlation between the antioxidant activity and the rutin content, whereas the extractable matter is not an appropriate parameter for extract quality. Huge differences in the content of rutin and chlorogenic acid when using herbal drugs from different suppliers confirm the demand of standardized procedures for the production of herbal drugs.     

Potential chemopreventive properties of extract from baked sweet potato (Ipomoea batatas Lam. Cv. Koganesengan)

The extract from baked sweet potato (Ipomoea batatas Lam. cv. Koganesengan) showed potential cancer-preventing effects. The extract was partially fractionated to four fractions (I, II-a II-b, and III) by Sephadex G-25 gel chromatography. The cytotoxicity against human myelocytic leukemia HL-60 cells, the suppression of TPA-induced transformation in mouse skin JB6 C141 cells, the apoptosis inducing activity in HL-60 cells, and the scavenging capacity against DPPH radical were tested on the four fractions. Fractions II-a and III showed markedly strong radical scavenging effects on the DPPH radical, coinciding with the high content of total phenolic compounds in the fractions. Both of these fractions suppressed strongly the proliferation of HL-60 cells with apoptosis induction in a dose-dependent manner. Moreover, the two fractions markedly blocked TPA-induced cell transformation in the JB6 cell line. Taken together, these data suggest that the water extract from baked sweet potato had potential chemopreventive properties.     

Antitumor activity of capsaicin on human colon cancer cells in vitro and colo 205 tumor xenografts in vivo

Capsaicin was reported to inhibit cancer cell growth. The aim of this study was to evaluate the antitumor potential of capsaicin by studying antitumor activity in vitro as well as in vivo. The in vitro studies are to examine the effects of capsaicin on human colon cancer colo 205 cells after exposure to capsaicin. The results showed that capsaicin induced cytotoxic effects in a time- and dose-dependent manner and increased reactive oxygen species (ROS) and Ca(2+) but decreased the level of mitochondrial membrane potential (ΔΨ(m)) in colo 205 cells. Data from Western blotting analysis indicated that the levels of Fas, cytochrome c, and caspases were increased, leading to cell apoptosis. Capsaicin decreased the levels of anti-apoptotic proteins such as Bcl-2 and increased the levels of pro-apoptotic proteins such as Bax. Capsaicin-induced apoptosis in colo 205 cells was also done through the activations of caspase-8, -9 and -3. In vivo studies in immunodeficient nu/nu mice bearing colo 205 tumor xenografts showed that capsaicin effectively inhibited tumor growth. The potent in vitro and in vivo antitumor activities of capsaicin suggest that capsaicin might be developed for the treatment of human colon cancer.     

Antioxidant capacity and other bioactivities of the freeze-dried Amazonian palm berry, Euterpe oleraceae mart. (acai)

The fruit of Euterpe oleraceae, commonly known as acai, has been demonstrated to exhibit significantly high antioxidant capacity in vitro, especially for superoxide and peroxyl scavenging, and, therefore, may have possible health benefits. In this study, the antioxidant capacities of freeze-dried acai fruit pulp/skin powder (OptiAcai) were evaluated by different assays with various free radical sources. It was found to have exceptional activity against superoxide in the superoxide scavenging (SOD) assay, the highest of any food reported to date against the peroxyl radical as measured by the oxygen radical absorbance capacity assay with fluorescein as the fluorescent probe (ORACFL), and mild activity against both the peroxynitrite and hydroxyl radical by the peroxynitrite averting capacity (NORAC) and hydroxyl radical averting capacity (HORAC) assays, respectively. The SOD of acai was 1614 units/g, an extremely high scavenging capacity for O2*-, by far the highest of any fruit or vegetable tested to date. Total phenolics were also tested as comparison. In the total antioxidant (TAO) assay, antioxidants in acai were differentiated into "slow-acting" and "fast-acting" components. An assay measuring inhibition of reactive oxygen species (ROS) formation in freshly purified human neutrophils showed that antioxidants in acai are able to enter human cells in a fully functional form and to perform an oxygen quenching function at very low doses. Furthermore, other bioactivities related to anti-inflammation and immune functions were also investigated. Acai was found to be a potential cyclooxygenase (COX)-1 and COX-2 inhibitor. It also showed a weak effect on lipopolysaccharide (LPS)-induced nitric oxide but no effect on either lymphocyte proliferation and phagocytic capacity.     

Glycosylated compounds from okra inhibit adhesion of Helicobacter pylori to human gastric mucosa

In Asian medicine the fruit of the okra plant, Abelmoschus esculentus (L.) Moench., is used as a mucilaginous food additive against gastric irritative and inflammative diseases. To find a rational basis for its use against these diseases, several crude and purified carbohydrate-containing fractions from immature okra fruits were isolated and analyzed, and their effects against Helicobacter pylori in an in situ adhesion model on sections of human gastric mucosa were determined. Pretreatment of the bacteria with a fresh juice preparation inhibited the bacterial adhesion almost completely. Lyophilization and reconstitution of an extract solution led to a reduction of this effect. A crude polysaccharide (RPS) isolated from the fresh juice by ethanolic precipitation showed strong inhibitory effects. Further fractionation of RPS revealed a purified, highly acidic subfraction (AF III) with high antiadhesive qualities. Carbohydrate analysis revealed the presence of rhamnogalacturonans with a considerable amount of glucuronic acid, whereas other inactive subfractions contained little glucuronic acid or were glucuronic acid-free. After heat denaturation of the fresh juice or protein precipitation with 5% TCA the antiadhesive activity of the fresh extract was reduced, indicating that besides polysaccharides, protein fractions also exhibited antiadhesive properties. SDS-PAGE analysis of the precipitate revealed several bands of glycosylated proteins between 25 and 37 kDa that were almost diminished in the nonactive supernatant. Preincubations of gastric tissue with any of the active fractions did not lead to reduced bacterial binding. The antiadhesive activity is therefore due to the blocking capacity of specific Helicobacter surface receptors that coordinate the interaction between host and bacterium. Neither of the active fractions showed inhibitory effects on bacterial growth in vitro. The antiadhesive qualities of okra were assumed to be due to a combination of glycoproteins and highly acidic sugar compounds making up a complex three-dimensional structure that is fully developed only in the fresh juice of the fruit.     

Chrysin protects epidermal keratinocytes from UVA- and UVB-induced damage

Chrysin (5,7-dihydroxyflavone), a natural flavonoid occurring in various plants and foods such as propolis and honey, reportedly opposes inflammation and carcinogenesis, but has rarely been applied in skin care. This study, therefore, aimed to explore the roles of chrysin in protection against UV-induced damage in HaCaT keratinocytes. Results showed that chrysin can attenuate apoptosis, reactive oxygen species (ROS) production, and cyclooxygenase 2 (COX-2) expression induced by UVB and UVA. Chrysin predominantly reversed the down-regulation of aquaporin 3 (AQP-3) by UVB. It predominantly reversed JNK activation and also mildly inhibited p38 activation triggered by UVA and UVB. Animal studies revealed that chrysin's topical application demonstrated efficient percutaneous absorption and no skin irritation. Overall, results demonstrated significant benefits of chrysin on the protection of keratinocytes against UVA- and UVB-induced injuries and suggested its potential use in skin photoprotection.