The effects of quercetin on oxidative stress resistance and physiological responses in olive flounder, Paralichthys olivaceus

어류는 독성 및 염분등과 같은 외부 인자에 의한 스트레스는 체내에서 유해산소인 reactive oxygen species (ROS)를 생성&#8228증가시켜 산화 스트레스를 유발시킨다. 따라서 생물체는 ROS에 의한 항산화 스트레스로부터 자신을 보호하고, 항상성을 유지하기 위하여 항산화 방어기작을 가지고 있는데 항산화 방어기작에 관여하는 대표적인 항산화 효소로는 superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) 및 glutathione S-transferase (GST)등이 존재하며, 항산화제로는 Metallothionein (MT), vitamin C 및 E 등이 항산화 작용을 하는 것으로 알려져 있다. 본 연구에서는 ROS 제거능이 우수한 천연 항산화제임과 동시에 면역 및 호르몬 증진 등 생리학적으로 뛰어나다고 알려져 있는 퀘르세틴을 넙치에게 공급한 후 독성물질인 Cd 및 저염분에 노출시켜 항산화 효소인 SOD와 CAT의 발현과 활성 변화를 조사하였으며, ROS의 일종인 H2O2농도, 지질 과산화(lipid peroxidation, LPO) 정도를 측정하였다. 퀘르세틴을 공급한 실험구와 일반 사료를 공급한 실험구를 Cd과 저염분에 노출시킨 후 항산화 효소인 SOD와 CAT 발현량을 quantitative real-time PCR (QPCR)을 이용하여 분석한 결과, 퀘르세틴을 공급한 실험구에서 일반 사료를 공급한 실험구보다 발현량이 낮았으며, SOD와 CAT 활성 또한 낮은 값을 나타내었다. 이를 뒷받침하는 결과로 혈장 H2O2 농도 또한 퀘르세틴을 공급한 실험구에서 더 낮은 수치값을 보였다. 이와 같이 생성된 ROS는 지질 과산화를 일으켜 세포를 손상시킨다고 알려져 있어, 본 연구에서도 퀘르세틴을 공급한 실험구를 Cd과 저염분에 노출시킨 후 측정한 결과 LPO 정도가 일반 사료를 공급한 실험구에 비하여 더 낮은 값을 보여, 퀘르세틴에 의해 제거된 ROS로 인하여 지질 과산화 정도가 낮음을 보여주고 있다. 덧붙여 퀘르세틴의 항산화 능력뿐만 아니라 면역능을 파악하기 위해 퀘르세틴을 공급한 실험구의 체내 lysozyme 활성을 측정한 결과 일반 사료를 공급한 실험구보다 더 높은 값을 보여 퀘르세틴이 체내 면역능도 향상시킨다는 사실을 알 수 있었으며, 호르몬 증진 효과를 알아보기 위하여 T3 농도를 측정한 결과 일반 사료를 공급한 실험구보다 높은 값을 보여 퀘르세틴이 체내 대사 작용 촉진을 위한 호르몬 증진에도 영향을 미친다는 것을 알 수 있었다.Contents i List of Table iii List of Figures iv Abstract (in Korean) v I. General Introduction 1 II. Experiment 1 3 Abstract 3 1. Introduction 4 2. Materials and methods 7 2.1. Experimental fish and conditions 7 2.2. Experimental diets 7 2.3. Plasma lysozyme activity and T3 analysis 10 2.4. Cd exposure 10 2.5. Quantitative PCR (QPCR) 11 2.6. SOD and CAT activity analysis 12 2.7. H2O2 assay 13 2.8. LPO assay 13 2.9. Statistical analysis 14 3. Results 15 3.1. Plasma lysozyme activity and T3 analysis 15 3.2. QPCR for SOD and CAT mRNA expression 18 3.3. SOD and CAT activity 22 3.4. H2O2 assay 26 3.5. LPO assay 29 4. Discussion 32 III. Experiment 2 36 Abstract 36 1. Introduction 38 2. Materials and methods 41 2.1. Experimental fish and conditions 41 2.2. Experimental diets 41 2.3. Plasma total cholesterol analysis 42 2.4. Osmotic stress produced by changes of salinity 42 2.5. Quantitative PCR (QPCR) 43 2.6. SOD and CAT activity analysis 44 2.7. H2O2 assay 45 2.8. Plasma lysozyme activity analysis 45 2.9. Plasma osmolality and cortisol analysis 46 2.10. Statistical analysis 46 3. Results 47 3.1. Plasma total cholesterol 47 3.2. SOD and CAT mRNA expression 50 3.3. SOD and CAT activities 54 3.4. H2O2 concentration 58 3.5. Plasma lysozyme activity 61 3.6. Plasma cortisol 64 3.7. Plasma osmolality 67 4. Discussion 70 IV. Conclusion 75 V. Acknowledgement 76 VI. References 7

Glycyrrhizin inhibits the invasion and metastasis of breast cancer cells via upregulation of expressions of miR-200c and e-cadherin

Purpose: To determine the inhibitory effect of glycyrrhizin (GLA) on cell invasion and metastasis in mammary carcinoma cells, and the mechanisms of actions involved.Methods: The effect of GLA at different concentrations on proliferation of breast cancer MDA-MB-231 and BT549 cells was assayed by MTT method. Transwell assay was used to determine the effect of GLA at different concentrations on invasiveness and metastasis of breast cancer MDA-MB-231 and BT549 cells. The influence of LGA on expressions of microRNA-200c and miR-200c was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR).Results: There was no statistically significant difference in cell proliferation amongst cells treated with 5 and 20 μM GLA and untreated breast cancer cells. However, the proliferation of cells treated with 40 μM GLA was significantly reduced (p < 0.05). In the cell invasion and migration experiments, cell population transferred to the base of Transwell chamber in the two cell lines treated with GLA was markedly decreased, relative to cells without GLA treatment, while the number of cells decreased with increase in GLA concentration (p < 0.05). Results from image-pro-plus analysis revealed that the population of cells quantitatively crossing the Transwell compartment membrane decreased with increase in GLA concentration (p < 0.05). The expression of e-cadherin was increased by GLA treatment in a concentration-dependent manner. Moreover, GLA treatment led to significant changes in amounts of miR-200s a, b and c, with changes in miR-200c being the most significant (p < 0.05).Conclusion: GLA suppresses the invasiveness and metastasis of breast cancer MDA-MB-231 and BT549 cells via upregulation of the expressions of miR-200c and e-cadherin. These findings provide a theoretical basis for the development of new breast cancer drugs. Keywords: Glycyrrhiza, GLA, miR-200c, E-cadherin, Inhibition, Breast cancer cells, Invasion, Metastasi

Formulation and characterization of an apigenin-phospholipid phytosome (APLC) for improved solubility, in vivo bioavailability, and antioxidant potential

The apigenin-phospholipid phytosome (APLC) was developed to improve the aqueous solubility, dissolution, in vivo bioavailability, and antioxidant activity of apigenin. The APLC synthesis was guided by a full factorial design strategy, incorporating specific formulation and process variables to deliver an optimized product. The design-optimized formulation was assayed for aqueous solubility, in vitro dissolution, pharmacokinetics, and antioxidant activity. The pharmacological evaluation was carried out by assessing its effects on carbon tetrachloride-induced elevation of liver function marker enzymes in a rat model. The antioxidant activity was assessed by studying its effects on the liver antioxidant marker enzymes. The developed model was validated using the design-optimized levels of formulation and process variables. The physical-chemical characterization confirmed the formation of phytosomes. The optimized formulation demonstrated over 36-fold higher aqueous solubility of apigenin, compared to that of pure apigenin. The formulation also exhibited a significantly higher rate and extent of apigenin release in dissolution studies. The pharmacokinetic analysis revealed a significant enhancement in the oral bioavailability of apigenin from the prepared formulation, compared to pure apigenin. The liver function tests indicated that the prepared phytosome showed a significantly improved restoration of all carbon tetrachloride-elevated rat liver function marker enzymes. The prepared formulation also exhibited antioxidant potential by significantly increasing the levels of glutathione, superoxide dismutase, catalase, and decreasing the levels of lipid peroxidase. The study shows that phospholipid-based phytosome is a promising and viable strategy for improving the delivery of apigenin and similar phytoconstituents with low aqueous solubility

Effects of enzymatically modified isoquercitrin in supplementary protein powder on athlete body composition: a randomized, placebo-controlled, double-blind trial

BackgroundEnzymatically modified isoquercitrin (EMIQ), a water-soluble quercetin, has been shown to intensify muscle hypertrophy in mice. We investigated the effect of EMIQ in supplementary protein powder on athlete body composition.MethodsForty Japanese males who played American football (age: 19.8 ± 1.4 years; body height: 174.1 ± 6.0 cm; body mass: 75.5 ± 10.7 kg) were assigned to a randomized, placebo-controlled, double-blind trial of parallel group. Participants received either EMIQ in whey protein (EW, n = 19) or contrast whey protein (W, n = 20) 6 days per week over 4 months. Body composition was assessed using dual-energy X-ray absorptiometry. Markers of oxidative stress, derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP), were assessed using a free radical analytical system. Data were analyzed using a univariate and repeated measures general model statistics.ResultsAfter 4 months, changes in lower limb fat-free mass and muscle mass were significantly greater in the EW group than in the W group (mean change ±95% CI; W: 324.1 ± 284.3, EW: 950.3 ± 473.2, p = 0.031, W: 255.7 ± 288.6, EW: 930.9 ± 471.5, p = 0.021, respectively). Moreover, the EW group exhibited a significantly higher BAP/d-ROMs ratio, antioxidation index, than the W group after 4 months (mean change ± SD; W: 8.8 ± 1.1, EW: 10.3 ± 2.8; p = 0.028). No significant differences in body mass, lean body mass, fat mass, or lower limb fat mass were observed between the groups.ConclusionIngestion of EMIQ in supplementary protein powder for 4 months exerts antioxidant effects and increases muscle mass among American football players

Analysis of phenolic and flavonoids of wild ephedra alata plant extractsby lc/pda and lc/ms and their antioxidant activity

Background: Ephedra is among Palestinian medicinal plants that are traditionally used in folkloric medicine for treating many diseases. Ephedra is known to have antibacterial and antioxidant effects. The goal of this study is to evaluate the antioxidant activity of different extracts from the Ephedra alata plant growing wild in Palestine, and to analyze their phenolic and flavonoid constituents by HPLC/PDA and HPLC/MS. Materials and Methods: Samples of the Ephedra alata plant grown wild in Palestine were extracted with three different solvents namely, 100% water, 80% ethanol, and 100% ethanol. The extracts were analyzed for their total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (AA), as well as phenolic and flavonoids content by HPLC/PDA/MS. Results: The results revealed that the polarity of the extraction solvent affects the TPC, TFC, and AA of extracts. It was found that both TPC and AA are highest for plant extracted with 80% ethanol, followed by 100% ethanol, and finally with 100% water. TFC however was highest in the following order: 100% ethanol > 80% ethanol > water. Pearson correlation indicated that there is a significant correlation between AA and TPC, but there is no correlation between AA and TFC. Simultaneous HPLC-PDA and UHPLC-MS analysis of the ethanolic plant extracts revealed the presence of Luteolin-7-O-glucuronide flavone, Myricetin 3-rhamnoside and some other major polyphenolic compounds that share myricetin skeleton. Conclusion Ephedra alata extract is rich in potent falvonoid glycosidic compounds as revealed by their similar overlaid UV-Vis spectra and UHPLC-MS results. On the basis of these findings, it is concluded that Ephedra alata constitutes a natural source of potent antioxidants that may prevent many diseases and could be potentially used in food, cosmetics, and pharmaceutical products

Anti-Senescence Therapy

The development of therapeutic strategies aimed at the aging process of cells has attracted increasing attention in recent decades due to the involvement of this process in the development of many chronic and age-related diseases. Interestingly, preclinical studies have shown the success of a number of anti-aging approaches in the treatment of a range of chronic diseases. These approaches are directed against aging processes such as oxidative stress, telomerase shortening, inflammation, and deficient autophagy. Many strategies has been shown to be effective in delaying aging, including antiaging strategies based on establishing healthy lifestyle habits and pharmacological interventions aimed at disrupting senescent cells and senescent-associated secretory phenotype. Caloric restriction and intermittent fasting were reported to activate autophagy and reduce inflammation. In turn, immune-based strategies, senolytic agents, and senomorphics mediate their effects either by eliminating senescent cells through inducing apoptosis or by disrupting pathways by which senescent cells mediate their detrimental effects. In addition, given the association of the decline in the regenerative potential of stem cells with aging, many experimental and clinical studies indicate the effectiveness of stem cell transplantation in preventing or slowing the progress of age-related diseases by enhancing the repairing mechanisms and the secretion of many growth factors and cytokines

Application of nano-curcumin as a natural antimicrobial agent against Gram-positive pathogens

Gram-positive bacteria cause various diseases from the superficial skin to deep tissue infections. The capability of causing numerous diseases is due to the production of virulence factors which are tightly regulated by the virulence genes. Various Gram-positive pathogenic bacteria e.g. Staphylococcus, Mycobacterium, and Listeria are capable of causing lethal infections in humans and animals. Conventional antibiotics, targeted antibiotics, and combinatorial drugs are used as therapeutic agents against Gram-positive pathogens. Due to intricate virulence pathway bacteria readily adopt resistance to available drugs. Therefore, there is need to develop some alternative approaches to combat these infections. Various natural extracts are effective against pathogenic bacteria with or without the available drugs. Curcumin is a natural extract of Curcuma longas rhizome, known as turmeric. Curcumin shows various biological activities such as antimicrobial, antioxidant and anti-inflammatory. It also shows strong antibacterial activity against Gram-positive and few Gram-negative bacteria. Besides all these beneficial applications, major drawbacks of curcumin are poor aqueous solubility and less bioavailability. However, drug delivery approaches including nanoformulation are developed to increase its stability in vitro and in vivo settings. The present review article focused on the translation of potential applications of curcumin in various diseases specifically caused by Gram-positive pathogens. Various methods used for the formulations of curcumin nanoparticles, combinatorial strategies with curcumin nanoparticles and their application in the prevention of infections have been discussed. The present article also discusses the future aspects of curcumin-nanoparticles and its use as an alternative therapeutic approach against pathogens