Role of ascorbic acid in counteracting ferric nitrilotriacetate-induced nephrotoxicity in rats

Abstract

Context: Ascorbic acid (AA) is a naturally occurring organic compound with antioxidant properties. It is necessary for normal growth and development, and has been shown to protect against tissue toxicity and oxidative stress.

Objective: The protective effect of AA against nephrotoxicity induced in albino rats by ferric nitrilotriacetate (Fe-NTA) was evaluated.

Materials and methods: Male albino rats of Wistar strain (4–6 weeks old) weighing 125–150?g were used in this study. Animals were given a single dose of Fe-NTA (9?mg/kg body weight, intraperitoneal) after a week of treatment with AA (1 and 2?mg/animal/day).

Results: Fe-NTA treatment enhanced microsomal lipid peroxidation (LPO) and hydrogen peroxide (H₂O₂) generation to 1.7- to 2.2-fold, glutathione (GSH) levels were decreased by two-fold and the activities of GSH metabolizing enzymes decreased to a range of 2.2- to 2.5-fold in renal tissue. These changes were reversed significantly in animals receiving pretreatment of AA. Treatment of rats with AA prior to the treatment with Fe-NTA decreased microsomal LPO and H₂O₂ generation to 124 and 172%, and also resulted in the recovery of reduced levels of GSH, GSH-metabolizing enzymes to almost 92% at the higher dose level of AA.

Discussion and conclusion: AA protects against Fe-NTA-induced nephrotoxicity and renal damage. AA has a beneficial impact on Fe-NTA-induced toxicity due to its scavenging and antioxidant effect in albino rats.     

Phytochemical investigation and radical scavenging activities of Melia azedarach and its DNA protective effect in cultured lymphocytes

Abstract

Context: Melia azedarach Linn (Meliaceae) is an Ayurvedic medicinal plant which is native to India. It is traditionally used for the treatment of leprosy, inflammation, scrofula, anthelmintic, antilithic, diuretic, deobstruent and cardiac disorders.

Objective: To evaluate the phytochemical constituents and antioxidant activities of the ethanol leaf extract of Melia azedarach (MA) and its protective effect against H₂O₂-induced cellular damage in cultured lymphocytes.

Materials and methods: The dose-dependent study of MA (20, 40, 60, 80, 100?µg/ml) was used to study in vitro radical scavenging assays. The effective dose of MA (60?µg/ml) was further used to study the H₂O₂-induced DNA damage (comet assay and DNA fragmentation assay) in cultured lymphocytes.

Results: The ethanol extract of MA (20, 40, 60, 80, 100?µg/ml) exhibited a significant dose-dependent inhibition of in vitro radical scavenging assays and their corresponding IC₅₀ values as follows: hydroxyl radical (26.50?±?0.26?µg/ml), superoxide anion (30.00?±?0.32?µg/ml), nitric oxide radical (48.00?±?0.48?µg/ml), DPPH radical (30.55?±?0.32?µg/ml) and reducing power (22.00?±?0.22?µg/ml). The increase in the severity of DNA damage and TBARS was increased significantly (p?<?0.05) at 500?µM H₂O₂-treated cultured lymphocytes and RBC cellular membranes. The phytochemical screening studies identified 13 chemical constituents present in the leaf extract of MA.

Discussion and conclusion: The results of this study demonstrate that MA offers protection against H₂O₂-induced cellular damage and it can be developed as an effective antioxidant during oxidative stress.     

Lutein protects retinal pigment epithelium from cytotoxic oxidative stress

Context: Lutein (LUT) and zeaxanthin (ZEA) are currently under investigation in clinical trials as prophylactic nutritional agents for age-related macular degeneration (AMD). However, dose used in these trials is empirical and not been investigated in in vitro studies.

Objective: In this study, we investigated the dose–response effect of LUT and ZEA in protecting retinal pigment epithelium (RPE) from oxidative stress, a common underlying pathology in AMD.

Methods: Three thousand cultured human retinal pigment epithelial cells (ARPE-19) were plated in 72-well plate and after 24?h were exposed to increasing concentrations of hydrogen peroxide (H₂O₂). ARPE-19 cells were exposed to four different concentrations of LUT (0.5, 1, 2 and 4?µg/mL) and ZEA (0.1, 0.2, 0.4 and 0.8?µg/mL). After 24?h incubation, cells were subjected to oxidative stress induced with H₂O₂. Cultures containing saline solution and dichloromethane served as controls. Cell viability was assessed using the WST-1 assay. Pathophysiological pathways were evaluated by measuring caspase-3 levels as an indicator of apoptosis induction. Reactive oxygen species (ROS) levels were measured using dihydrorhodamine-123.

Results: Cell viability as a percentage of control was 81.3%, 81.1%, and 88.8% at 0.5, 1, and 2?µg/ml, respectively of LUT (p?p?=?0.02).

Discussion and conclusions: Results from our study provide in vitro data to support the epidemiologic studies, which are currently underway to provide evidence that lutein may act as cofactor that modulates processes implicated in AMD pathogenesis.     

Digitally enhanced thin layer chromatography: further development and some applications in isotopic chemistry

Improvements to thin layer chromatography (TLC) analysis can be made easily and cheaply by the application of digital colour photography and image analysis. The combined technique, digitally enhanced TLC (DE‐TLC), is applicable to the accurate quantification of analytes in mixtures, to reaction monitoring and to other typical uses of TLC. Examples are given of the application of digitally enhanced TLC to: the deuteromethylations of theophylline to [methyl?²H₃]caffeine and of umbelliferone to [²H₃]7‐methoxycoumarin; the selection of tertiary amine bases in deuterodechlorination reactions; stoichiometry optimisation in the borodeuteride reduction of quinizarin (1,4‐dihydroxyanthraquinone) and to the assessment of xanthophyll yields in Lepidium sativum seedlings grown in deuterated media.     

Effect of Hedyotis diffusa water extract on protecting human hepatocyte cells (LO2) from H2O2-induced cytotoxicity

Context: Natural products are good sources of natural dietary antioxidants that are believed to protect the body against hepatotoxic effect induced by oxidative stress. Hedyotis diffusa Willd (Rubiaceae) (HDW) is a traditional Chinese medicinal herb that has been shown to possess a variety of antioxidant properties.

Objective: The present study examines and explains the cell protective property of HDW water extract (WEHDW).

Materials and methods: 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay was used to measure the free radical scavenging property of WEHDW (0.001–10?mg/mL). The protective effect of WEHDW (0.3–10?mg/mL 2?h pretreatment) against hydrogen peroxide (H₂O_2, 200?μM for 6?h) induced cytotoxicity in human hepatic cells, LO₂, was evaluated using cell viability assay and nuclear staining. The molecular pathway of WEHDW's effect was investigated by using Western blot assay.

Results: WEHDW had a 50% scavenging concentration (SC₅₀) at 0.153?mg/mL in the DPPH assay. Exposure of LO₂ cells to H₂O₂ resulted in apoptosis which could be markedly attenuated by pre-treating WEHDW in a concentration-dependent manner (0.5, 1, 3, 5, or 10?mg/mL) (all with p?<?0.001, versus control). Moreover, Hoechst (nuclear) staining showed that 1?mg/mL WEHDW could protect LO₂ cells by attenuating apoptotic cell death mediated by H₂O₂. It was found that WEHDW reversed H₂O₂-induced activation of MEK/ERK pathway and H₂O₂-induced inhibition of P13-K/AKT/GSK3β pathway in LO₂ cells.

Discussion and conclusion: WEHDW may help to improve the antioxidant defense system, resulting in prevention of oxidative stress-related fatty liver diseases.     

Kaurane diterpenes as mitochondrial alterations preventive agents under experimental oxidative stress conditions

Context: Foliol, linearol, and sidol are the most common diterpenes found in Sideritis L. spp. (Lamiaceae) with a wide range of demonstrated properties including anti-inflammatory, antioxidant, and anti-apoptotic effects.

Objective: For the first time, the present work was studied for the potential protective role of these kaurane-type diterpenes on mitochondrial oxidative stress induced by H₂O₂ in the human astrocytoma U373-MG cell line and in the rat adrenal pheochromocytoma PC12 cell line.

Materials and methods: Mitochondrial protection was assayed at 5 and 10?µM concentrations for 24?h (for kaurane diterpenes) and H₂O₂ as oxidative stress inducer (0.1?mM for PC12 cells and 1?mM for U373-MG, for 30?min). ATP concentration was determined by high-performance liquid chromatography (HPLC), and changes in mitochondrial membrane potential, caspase-3 activity as well as in cytosolic and mitochondrial calcium levels were assessed by fluorometric techniques, by using specific fluorescent probes.

Results: Pretreatments for 24?h with linearol and sidol, prior to H₂O₂ exposure, acted as mitochondrial alterations preventive agents by increasing membrane potential (over 40–60% in PC12 cells and over 10–20% in U373-MG), restoring both cytosolic and mitochondrial calcium homeostasis (linearol at 10?µM caused a 3.5-fold decrease in cytosolic calcium concentration in PC12 cells), decreasing caspase-3 activity (over 1.25–1.5-fold for linearol and sidol) and avoiding ATP depletion (linearol increased over 20% ATP level in both cell types).

Conclusion: Our results suggest that linearol and sidol could provide protective activity by targeting mitochondria in response to the deleterious changes induced by H₂O₂.     

Cytotoxic effects of Eryngium kotschyi and Eryngium maritimum on Hep2, HepG2, Vero and U138 MG cell lines

Abstract

Context: Eryngium maritimum L. and the endemic Eryngium kotschyi Boiss. of the Apiaceae family are used for antiinflammatory, antivenom, antinociceptive and diuretic purposes in folk medicine in Turkey.

Objective: This study investigated the cytotoxic effects of the plant extracts belonging to Eryngium L. genus on various cell lines.

Materials and methods: Cytotoxic activites of the lyophilized aqueous aereal and root parts of the plant extracts on human hepatocellular carcinoma (HepG2), human laryngeal epidermoid carcinoma (Hep2), human glioma (U138-MG) and African green monkey kidney epithelial (Vero) cell lines at 8.33–266.62?µg/ml concentrations were analyzed by lactate dehydrogenase (LDH) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) cell viability assays.

Results: Inhibitory concentration 50 (IC₅₀) values were found <100?µg/ml in most cases varying around 16.33–125.66?µg/ml. IC₅₀ values for E. kostchyi and E. maritimum root parts on Hep2 cells (32.86 and 30.25?µg/ml, respectively), E. kotschyi aereal, E. maritimum aereal and root parts on HepG2 cells (31.75, 32.42 and 35.01?µg/ml, respectively) by MTT assay were found to be close to the US National Cancer Institute (NCI) recommendations (IC₅₀?<?30?µg/ml) to define the antivity aganist cancer cells. The lowest IC₅₀ values according to the LDH method were observed in Hep2 cells and the highest in U138-MG cells. Root parts were found to be more toxic than aereal parts for both plants in both methods in general.

Discussion and conclusion: Both plant extracts exerted cytotoxic activity aganist Hep2 and HepG2 cells, with low IC₅₀ values defining their promising anticancer property according to NCI; however, further analysis are needed to confirm their activity.     

Protective effects of raw and cooked blackcurrant extract on DNA damage induced by hydrogen peroxide in human lymphoblastoid cells

Context: Blackcurrant (Ribes nigrum L.) is a classical fruit that has long been used to make juice, liqueur and sometimes medicines in Europe. The beneficial effects of blackcurrant, which are inhibition of lipopolysaccharide-stimulated inflammatory, anticarcinogenesis and other health effects, have been reported.

Objective: Previously, we reported the antimutagenic activities of blackcurrant using a yeast gene mutation assay. In this study, we investigated whether this antimutagenicity of blackcurrant was confirmed in human cells.

Materials and methods: We prepared four types of aqueous blackcurrant extracts (BCE) from mature and premature with or without heat treatment by microwave. Antioxidant activities of BCE were measured by the DPPH radical scavenger assay. In the DPPH radical scavenger assay, the maximum concentration of BCE was 1.6?mg/reaction. We investigated the antigenotoxic activities of BCE by the comet assay and micronucleus test using the human lymphoblastoid cell line TK6. In the comet assay, TK6 was treated with 300?μM?H₂O₂ without or with BCE at concentrations of 0.5,?1.0,?2.0 and 3.0?mg/mL. In the micronucleus test, TK6 was treated with 1?mg/mL BCE without or with H₂O₂.

Results: All BCEs exhibited more than 90% of inhibition rates of DPPH radicals at the maximum concentration of BCE. DNA damage and micronuclei induced by H₂O₂ significantly decreased in the each BCE-treated condition.

Conclusion: The results suggest that BCE treatment can reduce the genomic instability induced by H₂O₂ in human cells. We consider that these antigenotoxic effects are related to polyphenols, l-ascorbic acid and other antioxidant compounds.     

Protection by genistein on cortical neurons against oxidative stress injury via inhibition of NF-kappaB,JNK and ERK signaling pathway

Abstract

Context: Genistein, one of the isoflavones derived from soybean seeds, has been reported to exert multiple bioactivities. However, the mechanism of its action on the central nervous system is not fully understood.

Objective: To investigate the cytoprotection of genistein and its molecular mechanism against H₂O₂-induced cell death in primary rat cortical neurons.

Materials and methods: Genistein (0.01, 0.1, and 1?μM) were added into the primary rat neurons 24?h before and co-cultured with 500?μM H₂O₂ for 1?h. Neuronal injury was assessed by MTT, lactate dehydrogenase (LDH) assay, and Hoechst33258 staining. Intracellular reactive oxygen species (ROS) generation induced by H₂O₂ was determined. Neuronal apoptosis was evaluated by Bcl-2/Bax ratio as well as by caspase-9 and caspase-3 activities. The protein levels and phosphorylation of NF-κB/p65, IκB, JNK, and ERK were detected by western blots.

Results: Genistein pretreatment attenuated H₂O₂-mediated neuronal viability loss, nuclear condensation, and ROS generation in a concentration-dependent manner. Genistein exerted anti-apoptotic effects by reversing the apoptotic factors Bcl-2 and Bax ratio, along with the suppression of caspase-9 and caspase-3 activities. In addition, genistein down-regulated the expression of NF-κB/p65, and suppressed the phosphorylation of p65 and IκB. Genistein also inhibited H₂O₂-induced activation of the MAPK-signaling pathway including JNK and ERK.

Discussion and conclusion: The results indicated that genistein effectively protects cortical neurons against oxidative stress at least partly via inactivation of NF-κB as well as MAPK-signaling pathways, and suggested the possibility of this antioxidant for the prevention and treatment of stroke.     

Agkistrodon acutus-purified protein C activator protects human umbilical vein endothelial cells against H2O2-induced apoptosis

Context: Recent studies show that the Agkistrodon acutus (Viperidae) (syn. Deinagkistrodon acutus) protein C activator (PCA) treats acute myocardial infarction and ischaemia-reperfusion animal models effectively, while the underlying mechanism remains unknown.

Objective: To study the effect of PCA on the injury of human umbilical vein endothelial cells (HUVECs) induced by H₂O₂ and the underlying mechanism.

Materials and methods: Primary cultured HUVECs were pretreated with PCA (20, 40 and 80?μg/mL) for 1?h, then HUVEC apoptosis was induced by 300?μmol/mL H₂O₂. Apoptosis was analyzed by AnnexinV-FITC/PI, and reactive oxygen species (ROS) level was tested by flow cytometry. Colorimetric methods were used to detect the levels of NO and IL-1. In addition, real-time PCR and western blot analyses were used to detect the expression of eNOS and phospho-p38/MAPK.

Results: Morphological changes were induced by H₂O₂ in HUVECs. The cell survival rate was increased by 43.9, 64.0 and 80.6% in each PCA pretreated group (20, 40 and 80?μg/mL) compared to the model group. In each PCA pretreated group, oxidative stress level was also decreased to 54.7, 42.7 and 25.1%. Moreover, the level of IL-1 was decreased to 83.3, 62.2 and 30.7%. The level of NO was increased by 155.9, 232.4 and 317.6%. Apoptosis rate was decreased to 59.0, 47.7 and 32.7%. Phospho-p38 expression was downregulated, but eNOS expression was upregulated.

Discussion and conclusion: The results suggest that PCA can effectively protect the endothelial cells from injury induced by H₂O₂, which may be associated with antioxidation, upregulation of eNOS and downregulation of p38-MAPK.     

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

Context: Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture.

Objectives: This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells.

Materials and methods: Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0?µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot.

Results: Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123?µmol/ml at 24, 48 and 72?h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5?μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins.

Conclusion: Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.     

Angiotensin II type 1 receptor blockade suppresses H2O2-induced retinal degeneration in photoreceptor cells

Exposure to reactive oxygen species (ROS) leads to the development and progression of retinal degenerative diseases. However, the exact mechanisms are not fully understood. In this article, the role of angiotensin II type 1 receptor (AT1R) signaling in H₂O₂-induced retinal damage was examined. Mouse photoreceptor-derived 661?W cells were treated with the AT1R blockers valsartan, losartan and candesartan before exposure to H₂O₂. Cell viability, intracellular ROS level, mitochondrial membrane potential (MMP), cytochrome-c level, DNA fragmentation, caspase activity and gene expression were detected. Pre-treatment of 661?W cells with AT1R blockers significantly decreased H₂O₂-mediated toxicity and reduced the ROS level. In addition, apoptosis-related biochemical indicators showed that pre-incubation of AT1R blockers would elevate the MMP, decrease the release of cytochrome-c and formation of DNA fragmentation, and inhibit activities of caspase-3 and caspase-9 in exogenous H₂O₂-treated 661?W cells. Moreover, treatment with AT1R blockers suppressed the expression of Egr1, Fosl1 and Lox12. These results suggest that AT1R signaling mediates H₂O₂-induced apoptosis, at least partially through generating the ROS and increasing the levels of proapoptotic molecules in 661?W cells. AT1R blockade may provide a new therapeutic approach for preventing oxidative stress-induced retinal neural damage.     

Use of in vitro assays to assess the potential cytotoxic,genotoxic and antigenotoxic effects of vanillic and cinnamic acid

Vanillic acid (VA) found in vanilla and cinnamic acid (CA) the precursor of flavonoids and found in cinnamon oil, are natural plant phenolic acids which are secondary aromatic plant products suggested to possess many physiological and pharmacological functions. In vitro and in vivo experiments have shown that phenolic acids exhibit powerful effects on biological responses by scavenging free radicals and eliciting antioxidant capacity. In the present study, we investigated the antioxidant capacity of VA and CA by the trolox equivalent antioxidant capacity (TEAC) assay, cytotoxicity by neutral red uptake (NRU) assay in Chinese Hamster Ovary (CHO) cells and also the genotoxic and antigenotoxic effects of these phenolic acids using the cytokinesis-blocked micronucleus (CBMN) and the alkaline comet assays in human peripheral blood lymphocytes. At all tested concentrations, VA (0.17–67.2?μg/ml) showed antioxidant activity but CA (0.15–59.2?μg/ml) did not show antioxidant activity against 2,2-azino-bis (3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS). VA (0.84, 4.2, 8.4, 16.8, 84 and 168?μg/ml) and CA (0.74, 3.7, 7.4, 14.8, 74, 148?μg/ml) did not have cytotoxic and genotoxic effects alone at the studied concentrations as compared with the controls. Both VA and CA seem to decrease DNA damage induced by H₂O₂ in human lymphocytes.     

Horseradish peroxidase-mediated encapsulation of mammalian cells in hydrogel particles by dropping

Abstract

We report a method for preparation of mammalian cell-enclosing hydrogel particles through horseradish peroxidase (HRP)-catalysed hydrogelation by dropping cell-suspending aqueous solution into an aqueous coagulation solution. An aqueous solution of 10% (w/v) gelatin derivative possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph), HepG2 cells and 10?U/mL HRP was dropped into an aqueous coagulation solution containing 1?mM H₂O₂. The resultant hydrogel formed through the HRP-catalysed reaction consuming H₂O₂ had a spherical shape. The sphericity decreased with decreasing concentrations of Gelatin-Ph, HRP and H₂O₂. The thickness of the hydrogel membrane layer of the hydrogel particles could be controlled by altering incubation time in the H₂O₂ solution. The cells encapsulated in the particles with a thinner hydrogel membrane grew faster. These results demonstrate that we successfully established the method of cell-encapsulation in hydrogel particles based on dropping aqueous polymer solution into aqueous coagulation solution through HRP-catalysed reaction.     

The effect of intracellular antioxidant delivery (catalase) on hydrogen peroxide and proinflammatory cytokine synthesis: a new therapeutic horizon

Reactive oxygen species synthesized by endothelial cells may be responsible for cell damage and altered physiologic function. After endotoxin stimulation, free radicals including H₂O₂ are produced. We have developed a method of intracellular drug delivery using albumin microcapsules. Catalase would be an excellent compound to alter H₂O₂ production. However, the large molecular size of catalase limits cellular penetration. Endothelial cells have been previously shown to readily phagocytoze albumin microcapsules.

Methods: Catalase was added to an albumin solution to form a 10% solution of catalase. Microspheres from 2 to 7 μm in size were formed using a Bucchi spray dryer. Human endothelial cells were incubated with varying concentrations of microencapsulated catalase. The cells were then exposed to Escherichia coli endotoxin to determine if increased intracellular penetration of catalase would inhibit H₂O₂, nitrate, and cytokine synthesis.

Results: There was a 7.2-fold increase in endothelial intracellular catalase after 48?h incubation. H₂O₂ was inhibited by 72%, nitrate 96%, TNF 90%, IL1 21%, IL6 42%.

Conclusions: These results demonstrate that inhibition of H₂O₂ as a result of increased intracellular delivery of catalase inhibits proinflammatory cytokine synthesis after endotoxin exposure.     

Lutein improves cell viability and reduces Alu RNA accumulation in hydrogen peroxide challenged retinal pigment epithelial cells

Purpose: Dysfunction of the microRNA (miRNA)-processing enzyme DICER1 and Alu RNA accumulation are linked to the pathogenesis of age-related macular degeneration (AMD). This study determined the optimal dose of lutein (LUT) and zeaxanthin (ZEA) to protect human retinal pigment epithelium (RPE) cells against hydrogen peroxide (H₂O₂). The effect of the optimal dose of LUT and ZEA as DICER1 and Alu RNA modulators in cultured human RPE cells challenged with H₂O₂ was investigated.

Materials and methods: ARPE-19 cells were pre-treated with LUT, ZEA, or both for 24?h before 200?μM H₂O₂ challenge. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. DICER1 and Alu RNA were quantified by western blotting and real-time polymerase chain reaction, respectively.

Results: H₂O₂ increased cell Alu RNA expression and decreased cell viability of ARPE-19, but had no significant impact on the DICER1 protein level. LUT, alone and in combination with ZEA pre-treatment, prior to H₂O₂ challenge significantly improved cell viability of ARPE-19 and reduced the level of Alu RNA compared to the negative control.

Conclusions: These results support the use of LUT alone, and in combination with ZEA, in AMD prevention and treatment. This study is also the first to report LUT modulating effects on Alu RNA.     

Flavonoid constituents of Dobera glabra leaves: amelioration impact against CCl4-induced changes in the genetic materials in male rats

Context: Dobera glabra (Forssk.) Poir (Salvadoraceae) is a highly valued tree with diverse importance as special mineral sourced feed and a folkloric tool for forecasting droughts. However, there are no reports on its phytochemical and biological investigations.

Objective: Phytochemical investigation of D. glabra leaves and its protective potential against CCl₄ inducing changes in the genetic materials.

Materials and methods: D. glabra extract, DGE (70% MeOH/H₂O), was applied to polyamide column chromatography, eluting with MeOH/H₂O of decreasing polarities, followed by preparative chromatographic tools, yielded seven compounds. Three DGE doses (50, 100 and 200?mg/kg bw/d) were administrated for 8 weeks intragastrically to male albino rats prior treated with CCl₄ (0.5?mL/kg/bw). The reactive oxygen species (ROS) levels, expression changes of glutamate transporters (GLAST, GLT-1 and SNAT3) mRNA, DNA fragmentation and glutathione peroxidase (GPx) activity were investigated in the liver tissues of these rats.

Results: Isorhamnetin-3-O-β-glucopyranoside-7-O-α-rhamnopyranoside, isorhamnetin-3-O-α-rhamnopyranoside-7-O-β-glucopyranoside, kaempferol-3,7-di-O-α-rhamnopyranoside, isorhamnetin-3-O-β-glucopyranoside, kaempferol-3-O-β-glucopyranoside, isorhamnetin and kaempferol were identified. DGE (200?mg/kg bw)?+?CCl₄ exhibited the most significant reduction in ROS levels and DNA fragmentation with 251.3% and141% compared to 523.1% and 273.2% for CCl₄, respectively. Additionally, it increased significantly the mRNA expression of GLAST, GLT-1 and SNAT3 to 2.16-, 1.72- and 2.09-fold, respectively. Also, GPx activity was increased to 4.8?U/mg protein/min compared to CCl₄ (1.8?U/mg protein/min).

Discussion and conclusion: Flavonoid constituents, antioxidant effect and genotoxic protection activity of D. glabra were first reported. DGE may be valuable in the treatment and hindrance of hepatic oxidative stress and genotoxicity.     

Hepatoprotective effect of ganoderma triterpenoids against oxidative damage induced by tert-butyl hydroperoxide in human hepatic HepG2 cells

Context: Ganoderma triterpenoids (GTs) have been recognised as an important bioactive ingredient in Ganoderma Lucidum (Leyss. ex Fr.) Karst. (Polyporaceae), widely used for treating and preventing chronic hepatopathy of various etiologies.

Objective: The objective of this study is to better understand the hepatoprotective effect of GTs and to enhance their use in food supplement pharmaceutical and medical industries.

Materials and methods: HepG2 cells were pretreated in the presence or absence of GTs (50, 100 and 200?μg/ml) for 4?h, then exposed with 60?μmol/L of t-BHP for an additional 4?h. The cell viability was evaluated by MTT method. ALT, AST and LDH production in culture medium and intracellular MDA, GSH and SOD levels were determined. Moreover, the total triterpenoid content and chemical constituents in GTs were detected by ultraviolet spectrophotometry and HPLC/Q-TOF-MS, respectively.

Results: GTs (50, 100 and 200?μg/ml) significantly increased the relative cell viability by 4.66, 7.78 and 13.46%, respectively, and reduced the level of ALT by 11.44%, 33.41% and 51.24%, AST by 10.05%, 15.63% and 33.64%, and LDH by 16.03%, 23.4% and 24.07% in culture medium, respectively. GTs could also remarkably decrease the level of MDA and increase the content of GSH and SOD in HepG2 cells. Furthermore, the total triterpenoid content in GTs was 438?mg GAAEs/g GTs. And 16 triterpenoids in GTs were identified or tentatively characterised.

Discussion and conclusion: Our results showed that GTs had potent cytoprotective effect against oxidative damage induced by t-BHP in HepG2 cells, thus suggesting their potential use as liver protectant.     

Cardioprotective effects of fucoidan against hypoxia-induced apoptosis in H9c2 cardiomyoblast cells

Abstract

Context: Cardiomyocyte apoptosis plays a critical role in the progress of heart diseases. Fucoidan, a complex-sulfated polysaccharide, has been reported to possess potential cardioprotective efficacy in vivo.

Objective: The present study determines whether fucoidan could provide cardioprotection on hypoxia-induced cardiomyocyte apoptosis.

Materials and methods: H9c2 cardiomyoblast cells were incubated with various concentrations (15, 30, and 60?μg/ml) of fucoidan in a humidified incubator at 37?°C with 95% O₂ and 5% CO₂. After 6?h, hypoxia was processed and the cardioprotective effects of fucoidan were evaluated by applying MTT, ELISA, Hoechst 33258 nucleus staining, and western blot.

Results: Following a 6?h exposure of H9c2 to hypoxic condition, significant reduction was found in cell survival (0.57-fold) and superoxide dismutase (SOD) activity (0.56-fold), which were associated with the increase of malondialdehyde (MDA) level (2.58-fold), creatine phosphokinase (CK, 3.57-fold), and lactate dehydrogenase (LDH) activities (2.39-fold). Moreover, hypoxia-induced apoptosis was confirmed by Hoechst 33258 nuclear staining, and these changes were accompanied by the increase of Bcl-2 (1.27-fold) and Bax expression (2.6-fold). However, preincubation of the cells with fucoidan prior to hypoxia exposure elevated the cell viability (30?μg/ml, 1.18-fold; 60?μg/ml, 1.32-fold) and SOD activity (30?μg/ml, 1.12-fold; 60?μg/ml, 1.25-fold), but decreased the MDA level (30?μg/ml, 0.70-fold; 60?μg/ml, 0.80-fold), CK (30?μg/ml, 0.69-fold; 60?μg/ml, 0.76-fold), and LDH (30?μg/ml, 0.67-fold; 60?μg/ml, 0.86-fold) leakages. Hoechst 33258 nuclear staining observations demonstrated the same protective effect of fucoidan on hypoxia-induced myocardial injury. Also, cardioprotective effects of fucoidan were reflected by increasing Bcl-2 (60?μg/ml, 1.84-fold), as well as decreasing Bax (60?μg/ml, 0.6-fold).

Conclusion: Fucoidan had protective effect against hypoxia-induced cardiomyocytes apoptosis, and the mechanism might involve protections of the cell from oxidative injury.