Ethanol extracts of Hizikia fusiforme induce apoptosis in human prostate cancer PC3 cells via modulating a ROS-dependent pathway

Objective: To investigate whether ethanol extracts of Hizikia fusiforme could induce apoptosis in human prostate cancer PC3 cells.Methods: Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis and mitochondrial membrane potential (MMP) were measured using flow cytometry in PC3 cells. DNA damage was assessed by nuclear staining and DNA fragmentation assay. Expressions of apoptosis-associated proteins were determined by Western blotting assays. Activities of caspase-3, -8, and -9 were determined by colorimetric assay. Moreover, intracellular reactive oxygen species (ROS) generation was detected using a flow cytometer and fluorescence microscope. Results: Treatment of PC3 cells with ethanol extracts of Hizikia fusiforme inhibited proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL), Bax and tBid, and decreased expression of Bcl-2. In addition, ethanol extracts of Hizikia fusiforme reduced c-Flip expression and activated caspase-8, -9 and -3, resulting in an increase in poly (ADP-ribose) polymerase (PARP)cleavage. However, in the presence of a pan-caspase inhibitor, ethanol extracts of Hizikia fusiforme-mediated growth inhibition and apoptosis were significantly attenuated. Ethanol extracts of Hizikia fusiforme also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by treatment with ethanol extracts of Hizikia fusiforme, which was significantly suppressed by the ROS scavenger N-acetyl-L-cysteine. Further investigation of whether ethanol extracts of Hizikia fusiforme-induced apoptosis was related to the generation of ROS was conducted and the results showed that N-acetyl-L-cysteine fully blocked ethanol extracts of Hizikia fusiforme-induced apoptotic events including loss of MMP, activation of caspase-3, the cytosolic release of cytochrome c and cytotoxicity.Conclusions: Ethanol extracts of Hizikia fusiforme have chemopreventive potential via induction of ROS-dependent apoptosis. Therefore, ethanol extracts of Hizikia fusiforme may be useful for developing effective and selective natural sources to inhibit cancer cell proliferation.

Ethanol extracts of Hizikia fusiforme induce apoptosis in human prostate cancer PC3 cells via modulating a ROS-dependent pathway

Objective: To investigate whether ethanol extracts of Hizikia fusiforme could induce apoptosis in human prostate cancer PC3 cells. Methods: Cell viability was evaluated using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis and mitochondrial membrane potential (MMP) were measured using flow cytometry in PC3 cells. DNA damage was assessed by nuclear staining and DNA fragmentation assay. Expressions of apoptosis-associated proteins were determined by Western blotting assays. Activities of caspase-3, -8, and -9 were determined by colorimetric assay. Moreover, intracellular reactive oxygen species (ROS) generation was detected using a flow cytometer and fluorescence microscope. Results: Treatment of PC3 cells with ethanol extracts of Hizikia fusiforme inhibited proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL), Bax and tBid, and decreased expression of Bcl-2. In addition, ethanol extracts of Hizikia fusiforme reduced c-Flip expression and activated caspase-8, -9 and -3, resulting in an increase in poly (ADP-ribose) polymerase (PARP)cleavage. However, in the presence of a pan-caspase inhibitor, ethanol extracts of Hizikia fusiforme-mediated growth inhibition and apoptosis were significantly attenuated. Ethanol extracts of Hizikia fusiforme also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by treatment with ethanol extracts of Hizikia fusiforme, which was significantly suppressed by the ROS scavenger N-acetyl-L-cysteine. Further investigation of whether ethanol extracts of Hizikia fusiforme-induced apoptosis was related to the generation of ROS was conducted and the results showed that N-acetyl-L-cysteine fully blocked ethanol extracts of Hizikia fusiforme-induced apoptotic events including loss of MMP, activation of caspase-3, the cytosolic release of cytochrome c and cytotoxicity. Conclusions: Ethanol extracts of Hizikia fusiforme have chemopreventive potential via induction of ROS-dependent apoptosis. Therefore, ethanol extracts of Hizikia fusiforme may be useful for developing effective and selective natural sources to inhibit cancer cell proliferation.

Anti-inflammatory activity of a sulfated polysaccharide isolated from an enzymatic digest of brown seaweed Sargassum horneri in RAW 264.7 cells

BACKGROUND/OBJECTIVES: Sargassum horneri is an edible brown alga that grows in the subtidal zone as an annual species along the coasts of South Korea, China, and Japan. Recently, an extreme amount of S. horneri moved into the coasts of Jeju Island from the east coast of China, which made huge economic and environmental loss to the Jeju Island. Thus, utilization of this biomass becomes a big issue with the local authorities. Therefore, the present study was performed to evaluate the anti-inflammatory potential of crude polysaccharides (CPs) extracted from S. horneri China strain in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. MATERIALS/METHODS: CPs were precipitated from S. horneri digests prepared by enzyme assistant extraction using four food-grade enzymes (AMG, Celluclast, Viscozyme, and Alcalase). The production levels of nitric oxide (NO) and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β were measured by Griess assay and enzyme-linked immunosorbent assay, respectively. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPKs) were measured by using western blot. The IR spectrums of the CPs were recorded using a fourier transform infrared spectroscopy (FT-IR) spectrometer. RESULTS: The polysaccharides from the Celluclast enzyme digest (CCP) showed the highest inhibition of NO production in LPS-stimulated RAW 264.7 cells (IC₅₀ value: 95.7 µg/mL). Also, CCP dose-dependently down-regulated the protein expression levels of iNOS and COX-2 as well as the production of inflammatory cytokines, including TNF-α and IL-1β, compared to the only LPS-treated cells. In addition, CCP inhibited the activation of NF-κB p50 and p65 and the phosphorylation of MAPKs, including p38 and extracellular signal-regulated kinase, in LPS-stimulated RAW 264.7 cells. Furthermore, FT-IR analysis showed that the FT-IR spectrum of CCP is similar to that of commercial fucoidan. CONCLUSIONS: Our results suggest that CCP has anti-inflammatory activities and is a potential candidate for the formulation of a functional food ingredient or/and drug to treat inflammatory diseases.

Antihypertensive effect of an enzymatic hydrolysate from Styela clava flesh tissue in type 2 diabetic patients with hypertension

BACKGROUND/OBJECTIVES: In this randomized, placebo-controlled, double-blind study, we evaluated the antihypertensive effects of enzymatic hydrolysate from Styela clava flesh tissue in patients with type 2 diabetes mellitus (T2DM) and hypertension. SUBJECTS/METHODS: S. clava flesh tissue hydrolysate (SFTH) (n = 34) and placebo (n = 22) were randomly allocated to the study subjects. Each subject ingested two test capsules (500 mg) containing powdered SFTH (SFTH group) or placebo capsules (placebo group) during four weeks. RESULTS: In the SFTH group, systolic and diastolic blood pressure decreased significantly 4 weeks after ingestion by 9.9 mmHg (P < 0.01) and 7.8 mmHg (P < 0.01), respectively. In addition, the SFTH group exhibited a significant decrease in hemoglobin A1c with a tendency toward improvement in homeostasis model assessment of insulin resistance, triglyceride, apolipoprotein B and plasma insulin levels after 4 weeks. No adverse effects were observed in other indexes, including biochemical and hematological parameters in both groups. CONCLUSION: The results of our study suggested that SFTH exerts a regulatory, antihypertensive effect in patients with T2DM and hypertension.

Protective effect of phloroglucinol against gamma radiation-induced oxidative stress in hair follicles

When exposed to gamma-rays, hair follicular cells immediately go through apoptosis, which hampers their rapid differentiation essential for the regeneration of hair. Phloroglucinol (PG) is a phenolic compound of Ecklonia cava, brown algae abundant in Jeju island, Korea. Containing plentiful polyphenols, PG is known for its instructive effects by inhibiting apoptosis, scavenging oxygen radicals, and protecting cells against oxidative stress. In this study, we demonstrate that PG rescues radiosensitive hair follicular cells from gamma radiation-induced apoptosis and DNA damage. To identify protective capacity of PG on hair follicles, we irradiated with 8.5 Gy (1.5 Gy/min) of gamma-rays to the whole body of C57BL/6 mice at day 6 after depilation with or without PG. In mice exposed to radiation, the expression of proapoptotic molecule p53 was downregulated in the skin of PG treated group. On immunohistochemical observation of the skin, PG inhibited the immunoreactivity of p53 and cleaved caspase-3. PG treatment protected hair follicular cells from cell death due to gamma-radiation. Our results suggest that PG presents radioprotective effects by inhibiting apoptosis of radiosensitive hair follicular cells and can protect hair follicular cells from gamma-ray induced damage.

Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model

BACKGROUND/OBJECTIVES: In this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model. MATERIALS/METHODS: We investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model. RESULTS: Among the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate. CONCLUSION: The results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.

Protective effect of Hizikia fusiforme on radiation-induced damage in splenocytes / 대한수의학회지

The immune system is specifically sensitive to oxidative stress induced by ionizing radiation because of its rapid proliferative activity. For this reason, an instructive immune system is one of the best ways to minimize side effects, such immunodeficiency, of gamma radiation. Over the past few decades, several natural plants with antioxidant and immunomodulatory properties have been identified as adjuncts for nontoxic and successful radiotherapy. Hizikia fusiforme extract (HFE) containing plentiful dietary fiber and fucoidan is known for its instructive antioxidant capacity, immunomodulation abilities, and immune activation. In this study, we determined whether HFE protects radiosensitive immune cells from gamma radiation-induced damage. C57BL/6 mice were irradiated with gamma-ray. The effect of HFE on the ionizing radiation damage of immune cells was then evaluated with an MTT assay, 3H-thymidine incorporation assay, and PI staining. We found that HFE stimulated the proliferation of gamma-ray irradiated immune cells without cytotoxic effects. We also observed that HFE not only decreased DNA damage but also reduced gamma radiation-induced apoptosis of the immune cells. Our results suggest that HFE can protect immune cells from gamma-ray damage and may serve as an effective, non-toxic radioprotective agent.

The Effect of Metformin Treatment on CRBP-I Level and Cancer Development in the Liver of HBx Transgenic Mice

Retinoids regulate not only various cell functions including proliferation and differentiation but also glucose and lipid metabolism. After we observed a marked up-regulation of cellular retinol-binding protein-I (CRBP-I) in the liver of hepatitis B virus x antigen (HBx)-transgenic (HBx Tg) mice which are prone to hepatocellular carcinoma (HCC) and fatty liver, we aimed to evaluate retinoid pathway, including genes for the retinoid physiology, CRBP-I protein expression, and retinoid levels, in the liver of HBx Tg mice. We also assessed the effect of chronic metformin treatment on HCC development in the mice. Many genes involved in hepatic retinoid physiology, including CRBP-I, were altered and the tissue levels of retinol and all-trans retinoic acid (ATRA) were elevated in the liver of HBx Tg mice compared to those of wild type (WT) control mice. CRBP-I protein expression in liver, but not in white adipose tissue, of HBx Tg mice was significantly elevated compared to WT control mice while CRBP-I protein expressions in the liver and WAT of high-fat fed obese and db/db mice were comparable to WT control mice. Chronic treatment of HBx Tg mice with metformin did not affect the incidence of HCC, but slightly increased hepatic CRBP-I level. In conclusion, hepatic CRBP-I level was markedly up-regulated in HCC-prone HBx Tg mice and neither hepatic CRBP-I nor the development of HCC was suppressed by metformin treatment.

Antibacterial effect of citrus press-cakes dried by high speed and far-infrared radiation drying methods

In this study, the antibacterial effect was evaluated to determine the benefits of high speed drying (HSD) and far-infrared radiation drying (FIR) compared to the freeze drying (FD) method. Citrus press-cakes (CPCs) are released as a by-product in the citrus processing industry. Previous studies have shown that the HSD and FIR drying methods are much more economical for drying time and mass drying than those of FD, even though FD is the most qualified drying method. The disk diffusion assay was conducted, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with methanol extracts of the dried CPCs against 11 fish and five food-related pathogenic bacteria. The disk diffusion results indicated that the CPCs dried by HSD, FIR, and FD prevented growth of all tested bacteria almost identically. The MIC and MBC results showed a range from 0.5-8.0 mg/mL and 1.0-16.0 mg/mL respectively. Scanning electron microscopy indicated that the extracts changed the morphology of the bacteria cell wall, leading to destruction. These results suggest that CPCs dried by HSD and FIR showed strong antibacterial activity against pathogenic bacteria and are more useful drying methods than that of the classic FD method in CPCs utilization.

Evaluation of antioxidant properties of a new compound, pyrogallol-phloroglucinol-6,6'-bieckol isolated from brown algae, Ecklonia cava

In this study, antioxidant and free radical scavenging activities of the natural antioxidative compound, pyrogallol-phloroglucinol-6,6'-bieckol (PPB) isolated from brown algae, Ecklonia cava was assessed in vitro by measuring the radical scavenging activities (DPPH, alkyl, hydroxyl, and superoxide) using electron spin resonance (ESR) spectrometry, intracellular reactive oxygen species (ROS) scavenging activity, and DNA damage assay. According to the results of these experiments, the scavenging activity PPB against difference radicals was in the following order: DPPH, alkyl, hydroxyl, and superoxide radicals (IC50; 0.90, 2.54, 62.93 and 109.05 microM). The antioxidant activities of PPB were higher than that of the commercial antioxidant, ascorbic acid. Furthermore, PPB effectively inhibited DNA damage induced by H2O2. These results suggest that the natural antioxidative compound, PPB, can be used by the natural food industry.

Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line

Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H2O2-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H2O2-induced cell damage in vitro.

Composition of Flavonoids and Antioxidative Activity from Juice of Jeju Native Citrus Fruits during Maturation

This study aims to evaluate the changes of flavonoid contents and antioxidants activity of Jeju native citrus fruits juice according to the harvest date. Flavonoids such as quercatagetin, narirutin, hesperidin and neohesperidin were contained most plentifully in the juice of Jigak (Citrus aur- antium) by 573.73 mg/100 mL, Sadoogam (C. pseudogulgul) by 393.99 mg /100 mL, Soyooja by 29.63 mg/100 mL and Jigak (C. aurantium) by 201.23 mg/100 mL in the late August, respectively. The highest contents of nob-iletin, sinensetin and tangeretin among polymethoxyflavones were found in the juice of Hongkyool (C. tachibana) by 7.39 mg/100 mL, 2.24 mg/100 mL, 0.63 mg/100 mL in the late August, respectively. 3,5,6,7,8,3',4'- Heptamet- hoxyflavone recorded the highest amount in Punkyool (C. tangerina) by 0.27 mg/100 mL in the late August, but the other polymethoxyflavones including 3',4',7,8-tetramethoxyflavone, 3',4'-dimethoxyflavone, 4'-methoxyflavone, 5,6,7,3',4',5'-hexamethoxyflavone, scutellarein tetramethylether were observed only trace amount in all the citrus fruits. Flavonoid contents in the citrus fruit juices were the highest during early maturation and decreased rapidly while ripening. Total polyphenol contents were the highest in the late August and decreased with ripening. However from the late December, the contents were increased again. Antioxidant activities of the fruits were evaluated as electron donating ability and were the lowest in the late September and increased with the fruit ripening. These results suggest that quercetagetin among all the flavonoids was most plentiful in Jigak and Dangyooja (C. grandis), so that the fruits could be used for industrial material of flavonoids and antioxidant agents.