Apigenin Isolated from Carduus crispus Protects against H2O2-Induced Oxidative Damage and Spermatogenic Expression Changes in GC-2spd Sperm Cells.

Testicular oxidative stress is one of the most common factors underlying male infertility. Welted thistle, Carduus crispus Linn., and its bioactive principles are attracting scientific interest in treating male reproductive dysfunctions. Here, the protective effects of apigenin isolated from C. crispus against oxidative damage induced by hydrogen peroxide (H2O2) and dysregulation in spermatogenesis associated parameters in testicular sperm cells was investigated. Cell viabilities, ROS scavenging effects, and spermatogenic associated molecular expressions were measured by MTT, DCF-DA, Western blotting and real-time RT-PCR, respectively. A single peak with 100% purity of apigenin was obtained in HPLC conditions. Apigenin treated alone (2.5, 5, 10 and 20 µM) did not exhibit cytotoxicity, but inhibited the H2O2-induced cellular damage and elevated ROS levels significantly (p < 0.05 at 5, 10 and 20 µM) and dose-dependently. Further, H2O2-induced down-regulation of antioxidant (glutathione S-transferases m5, glutathione peroxidase 4, and peroxiredoxin 3) and spermatogenesis-associated (nectin-2 and phosphorylated-cAMP response element-binding protein) molecular expression in GC-2spd cells were attenuated by apigenin at both protein and mRNA levels (p < 0.05). In conclusion, our study showed that apigenin isolated from C. crispus might be an effective agent that can protect ROS-induced testicular dysfunctions.

Pectinase-treated Panax ginseng extract (GINST) rescues testicular dysfunction in aged rats via redox-modulating proteins.

The root of Panax ginseng improves testicular function both in humans and animals. However, the molecular mechanism by which ginseng exerts this effect has not been elucidated. Changes in protein expression in the rat testis in response to a pectinase-treated P. ginseng extract (GINST) were identified using 2-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF MS. Number of sperm, Sertoli cells and germ cells, and the Sertoli Cell Index decrease in the testis of aged rats (AR) relative to young control rats (YCR). However, those parameters were completely restored in GINST-treated AR (GINST-AR). A proteomic analysis identified 14 proteins that were differentially expressed between vehicle-treated AR (V-AR) and GINST-AR. Out of these, the expression of glutathione-S-transferase (GST) mu5 and phospholipid hydroperoxide (PH) glutathione peroxidase (GPx) was significantly up-regulated in GINST-AR compared to V-AR. The activity of GPx and GST, as well as the expression of glutathione, in the testis of GINST-AR was higher than that in V-AR. The levels of lipid peroxidation (LPO) increased in AR compared with YCR, but this change was reversed by GINST-AR. These results suggest that the administration of GINST enhances testicular function by elevating GPx and GST activity, thus resulting in increased glutathione, which prevents LPO in the testis.

Panax ginseng reduces oxidative stress and restores antioxidant capacity in aged rats.

Nutritional antioxidants interact with cells in an active mode, including retrieving and sparing one another, to diminish oxidative stress. However, the intracellular balance of prooxidants and antioxidants becomes unbalanced, favoring prooxidants during the aging process. One hypothesis is that an aging-associated increase in oxidative stress is the primary cause of aging. Hence, the research hypothesis for this study is that Korean red ginseng reduces oxidative stress in vivo. Therefore, we investigated the efficacy of Korean red ginseng water extract (GWE) in reducing aging-associated oxidative stress by measuring lipid peroxidation and antioxidant levels in older rats compared with young rats. We observed a significant increase in the markers for oxidative damage (eg, lipid peroxidation) and markers for vital organ damage (eg, aspartate aminotransferase, alanine aminotransferase, urea, and creatinine levels) in aged rats. The oxidative damage was accompanied by a significant decrease in enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase, and nonenzymatic antioxidants such as reduced glutathione, vitamin E, and vitamin C. Aged rats fed a diet supplemented with Korean red ginseng water extract had significantly less oxidative damage, possibly by enhancing the enzymatic and nonenzymatic antioxidants status. Our data suggest that consumption of Korean red ginseng reduces lipid peroxidation and restores antioxidant capacity by suppressing oxidative stress in rats.

Protection of apigenin against kainate-induced excitotoxicity by anti-oxidative effects.

Apigenin (5,7,4'-trihydroxyflavone) is a principal ingredient of Cirsium japonicum. These experiments were performed to determine whether apigenin has neuroprotective effects against kainic acid (KA)-induced excitotoxicity in vitro and in vivo. Intraperitoneal (i.p.) administration of apigenin (25, 50 mg/kg) decreased the seizure scores induced by KA injection (40 mg/kg, i.p.) in mice. In addition, the convulsion onset time was significantly delayed by apigenin administration. Moreover, we found that apigenin blocked KA-induced seizure-form electroencephalogram (EEG) discharge activity in the brain cortex. In hippocampal cells, apigenin inhibited KA-induced excitotoxicity in a dose-dependent manner as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To study the possible mechanisms underlying the in vitro neuroprotective effects of apigenin against KA-induced cytotoxicity, we also examined the effect of apigenin on intracellular reactive oxygen species (ROS) elevations in cultured hippocampal neurons and found that apigenin treatment dose-dependently inhibited intracellular ROS elevation. The remarkable reduction of glutathione (GSH) levels induced by KA in hippocampal tissues was reversed by apigenin in a dose-dependent manner. In addition, similar results were obtained after pretreatment with free radical scavengers such as trolox and dimethylthiourea (DMTU). Finally, after confirming the protective effect of apigenin in hippocampal CA3 region, we found apigenin is an active compound in KA-induced neuroprotection. These results collectively indicate that apigenin alleviates KA-induced excitotoxicity by quenching ROS as well as inhibiting GSH depletion in hippocampal neurons.

Cordycepin (3'-deoxyadenosine) attenuates age-related oxidative stress and ameliorates antioxidant capacity in rats.

Free radical-induced oxidative damage is considered to be the most important consequence of the aging process. The activities and capacities of antioxidant systems of cells decline with increased age, leading to the gradual loss of pro-oxidant/antioxidant balance and resulting in increased oxidative stress. Our investigation was focused on the effects of cordycepin (3'-deoxyadenosine) on lipid peroxidation and antioxidation in aged rats. Age-associated decline in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), reduced glutathione (GSH), vitamin C and vitamin E, and elevated levels of malondialdehyde (MDA) were observed in the liver, kidneys, heart and lungs of aged rats, when compared to young rats. Furthermore, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine were found to be significantly elevated in aged rats compared to young rats. Aged rats receiving cordycepin treatment show increased activity of SOD, CAT, GPx, GR and GST, and elevated levels of GSH, and vitamins C and E such that the values of most of these parameters did not differ significantly from those found in young rats. In addition, the levels of MDA, AST, ALT, urea and creatinine became reduced upon administration of cordycepin to aged rats. These results suggest that cordycepin is effective for restoring antioxidant status and decreasing lipid peroxidation in aged rats.

Enhancement of pentobarbital-induced sleep by apigenin through chloride ion channel activation.

This experiment was performed to investigate whether apigenin has hypnotic effects and/or enhances pentobarbital-induced sleep behaviors through the GABAergic systems. Apigenin prolonged sleep time induced by pentobarbital similar to muscimol, a GABA(A) receptors agonist. Apigenin also increased sleep rate and sleep time in the combined administration with pentobarbital at the sub-hypnotic dosage, and showed synergic effects with muscimol in potentiating sleep onset and enhancing sleep time induced by pentobarbital. In addition, both of apigeinin and pentobarbital increased chloride influx in primary cultured cerebellar granule cells. Apigenin increased glutamate decarboxylase (GAD) and had no effect on the expression of GABA(A) receptor α-, ß-, γ-subunits in n hippocampus of mouse brain, showing different expression of subunits from pentobarbital treatment group. In conclusion, it is suggested that apigenin augments pentobarbital-induced sleep behaviors through chloride ion channel activation.

Effect of fermented Panax ginseng extract (GINST) on oxidative stress and antioxidant activities in major organs of aged rats.

The intracellular levels of oxidant and antioxidant balances are gradually distorted during the aging process. An age associated elevation of oxidative stress occurring throughout the lifetime is hypothesized to be the major cause of aging. The present study was undertaken to evaluate the putative antioxidant activity of the fermented Panax ginseng extract (GINST) on lipid peroxidation and antioxidant status of major organs of aged rats compared to young rats. Increased levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine were observed in the serum of aged rats. Increased levels of malondialdehyde (MDA) and significantly lowered activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) were observed in the liver, kidneys, heart and lungs of aged rats, when compared with those in young rats. Quantitative analysis of the non-enzymatic antioxidants such as reduced glutathione (GSH), ascorbic acid and α-tocopherol levels showed significantly lower values in the liver, kidneys, heart and lungs of aged rats. On the other hand, administration of the fermented Panax ginseng extract (GINST) to aged rats resulted in increased activities of SOD, CAT, GPx, GR and GST as well as elevation in GSH, ascorbic acid and α-tocopherol levels. Besides, the level of MDA, AST, ALT, urea and creatinine were reduced on administration of GINST to aged rats. These results suggested that treatment of GINST can improve the antioxidant status during aging, thereby minimizing the oxidative stress and occurrence of age-related disorders associated with free radicals.