• Herbal Formula Science
• /
• v.25 no.2
• /
• pp.179-191
• /
• 2017

Objectives : Increased oxidative stress by reactive oxygen species (ROS) has been suggested as a major cause of muscle fatigue. Although several studies have demonstrated the various biological properties of Sophora flavescens Aiton, Glycyrrhiza uralensis Fischer and Dictamnus dasycarpus Turcz, but the antioxidative potentials have not been clearly demonstrated. The present study was designed to investigate the protective effects of their water and ethanol extract mixtures (medicinal herbal mixtures, MHMIXs) on hydrogen peroxide ($H_2O_2$)-induced cell damage and apoptosis in C2C12 myoblasts. Methods : Cytotoxicity was assessed by an MTT assay. Quantitative evaluation of apoptosis induction and ROS production was evaluated by flow cytometry analysis. Expression levels of apoptosis regulatory and DNA-damage proteins were detected by Western blotting. Result : The inhibition of $H_2O_2$-induced cell proliferation was effectively blocked in extracts of 3: 1: 1 (EMHMIXs-1) or 2: 2: 1 (EMHMIXs-2) of S. flavescens, G. uralensis and D. dasycarpus Turcz, ethanol extracts from various complex extracts in C2C12 myoblasts. EMHMIXs-1 and EMHMIXs-2 also effectively attenuated $H_2O_2$-induced C2C12 cell apoptosis, which was associated with the restoration of the upregulation of Bad and death receptor 4, and downregulation of XIAP and cIAP-1 induced by $H_2O_2$. In addition, these herbal mixtures significantly blocked the $H_2O_2$-induced ROS generation and phosphorylation of $p-{\gamma}H2A.X$, which suggests that they can prevent $H_2O_2$-induced cellular DNA damage. Conclusions : The results suggest that EMHMIXs-1 and EMHMIXs-2 could block the DAN damage and apoptosis of C2C12 myoblasts by oxidative stress through blocking ROS generation.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.41 no.1
• /
• pp.41-48
• /
• 2012

The purpose of this study was to assess the antioxidant activity of vegetable extracts (pumpkin, aloe, and artichoke) containing Protaetia brevitarsis (PB) and the clinical and pathological changes in ICR mice after a single oral administration. The total polyphenol (TP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability, total radical trapping antioxidant potential (TRAP), oxygen radical absorbance activity (ORAC), and single cell gel electrophoresis assay were done to measure their antioxidant activities. The effect of vegetable extracts containing PB in TP and the ORAC value was significantly higher than those without PB. In addition, all extracts had effective $DPPH{\cdot}$ scavenging and $ABTS{\cdot}+$ scavenging activities. The protective effect of vegetable extracts with/without PB on $H_2O_2$-induced DNA damage was found. In a single-dose toxicity study, mortality, body weight, physiological signs, and biochemical analysis were analyzed. Seventy mice were randomly assigned to 7 experimental groups and were administered three vegetable extracts with and without PB (2 g/kg). A full 14 days after administration, no mice mortality was observed in any group. Body weight, physiological signs, and biochemical analysis were never significantly different from those of the control group. Taken together, these findings indicate that vegetable extracts containing PB with antioxidant activities and safety could be applied as medicinal and edible resources in an industrial area.

• Journal of Oriental Neuropsychiatry
• /
• v.21 no.2
• /
• pp.125-140
• /
• 2010

Objectives : The purpose of this study is to examine from various angles the protective effect of Gastrodia elata Blume (GEB) against nerve cell death induced by $\beta$-amyloid by using the cell line SH-SY5Y, which is commonly utilized for toxicity testing in nerve cells, and to find out its mechanism of action. Methods : To begin with, as a result of assessing the rate of cell survival by employing MTT reduction assay, the treatment with $\beta$-amyloid at different concentrations caused cytotoxicity, which was inhibited by preprocessing GEB extract. In addition, after $\beta$-amyloid was processed with the cell SH-SY5Y, apoptosis progressed, which was reduced effectively by processing GEB extract. Results : When cytotoxicity was caused by using hydrogen peroxide, a representative ROS, in order to examine the antioxidant effect of GEB, its protective effect was also observed. Apart from ROS, reactive nitrogen species (RNS) are also known to play a crucial role in nerve cell death. The treatment with the NO donor SNAP increased the production of nitric oxide and the expression of iNOS, which was also inhibited by GEB extract. Meanwhile, as an attempt to find out the mechanism of action explaining the antioxidant effect, the intracellular antioxidant enzyme expressions were measured by RT-PCR, which showed that GEB extract increased the expressions of heme oxygenase-1, GAPDH and $\gamma$-glutamate cysteine ligase. Lastly, GEB extract had a protective effect against impaired memory induced by scopolamine in animal models (in vivo). Conclusions : These findings indicate that GEB has a protective effect against the death of cranial nerve cells, suggesting possibilities for the prevention and treatment of AD.

• Journal of Life Science
• /
• v.25 no.11
• /
• pp.1197-1203
• /
• 2015

Salmonella Typhimurium (ST) JOL389 and χ3339 are strong virulent strains against mouse. ST χ8554 is derived by deletion of the asd gene from ST χ3339. Plasmid pMMP184 carrying a ghost cassette was transformed into ST χ8554, and ST χ8554 ghost cells were prepared and administrated via the oral route to BALB/c mice. Change in the amount of total IgG was not elicited to boosting of single ST χ8554 ghost cells, but the content was increased from 6 weeks after the 3^rd administration. However, when the ST JOL389 ghost cells is administered together with ST χ8554 ghost cells, the content of total IgG was increased in 2 weeks post primary administration. It was found that the content of total IgG of the group mixed with ST JOL389 ghost cells showed an increased value of 8 times or more at 10 weeks when compared with the group of ST χ8554 ghost cells. The content of IgG1, IgG2a, and sIgA in both groups increased from 4 weeks postprimary administration. As a challenge test of virulent ST χ3339, χ8554 (pMMP184) and χ8554 (pMMP184)/JOL389 ghost cell groups showed protection of 50% or more when compared to the control group. These results suggest that the preparation of combined ghost cells from a strong virulent ST increases immunity more than a single strain.

• The Journal of Internal Korean Medicine
• /
• v.31 no.1
• /
• pp.54-65
• /
• 2010

Objective : The water extract of Chungsimyeonja-eum (CSYJE) has traditionally been used in treatments of heart diseases and brain diseases in Oriental medicine. However, little is known about the mechanism by which CSYJE protects neuronal cells from injury damages. Therefore, in this study we attempted to elucidate the mechanism of the cytoprotective effect of the CSYJE extract on glutamate-induced C6 glial cell death. Methods : Cultured cells were pretreated with CSYJE and exposed to glutamate, cell damage was assessed by using MTT assay and propidium iodide (PI), probe 2',7'-dichlorofluorescein diacetate (DCF-DA) staining. Western blotting was performed using anti-procaspase-3 and anti-PARP, respectively. Result : We determined the elevated cell viability by CSYJE extract on glutamate-induced C6 glial cell death. Glutamate induced DNA fragmentation on C6 glial cells but pre-treatment with CSYJE inhibited DNA fragmentation. One of the main mediators of glutamate-induced cytotoxicity was known to generation of reactive oxygen species (ROS). Pre-treatment with CSYJE inhibited this ROS generation from glutamate-stimulated C6 glial cells. Also, we identified that the ROS-induced DCF-DA green fluorescence was reduced by CSYJE pre-treatment. The critical markers of apoptotic cell death are the cleavages of procaspase-3 protease and PARP proteins, so we checked the expression level and cleavages of procaspase-3 protease and PARP proteins. Glutamate-treated C6 glial cells showed the cleavages of procaspase-3 protease and PARP proteins and followed the reduction of expression of these proteins. Conclusion : These findings indicate that CSYJE may prevent cell death from glutamate-induced C6 glial cell death by inhibiting the ROS generation and procaspase-3 and PARP expression.

• The Journal of Internal Korean Medicine
• /
• v.34 no.2
• /
• pp.178-191
• /
• 2013

Objectives : In this study, we made an effort to investigate the protective mechanism of Ukgan-san (UGS) extracts on hypoxia-induced C6 glial cell death. Methods : The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MMT) assay and cell morphological changes were analysed with microscope after staining with crystal violet (CV). Reactive oxygen species (ROS) formation was assessed by flow cytometer after staining with 2'7'-dichlorofluorescein diacetate (DCF-DA). We also analyzed expression of hypoxia-inducible factor-1 alpha (HIF-$1{\alpha}$) and p53, processing of procaspase-3 and procyclic acidic repetitive protein (PARP) by western blot method. Results : We estimated the elevated cell viability by UGS extract on $CoCl_2$-induced C6 glial cells. UGS attenuated $CoCl_2$-induced ROS formation in C6 glial cells and also showed a protective activity compared to antioxidants and exhibited abrogation of LDH-released by $CoCl_2$. UGS suppressed the typical apoptotic cell death markers, caspase-3 and PARP activation. UGS inhibited $CoCl_2$-induced HIF-1${\alpha}$ expression which is known as a major regulator for hypoxia-induced cell death, and suppressed p53 expression. Conclusions : These results suggest that UGS extract contains protective constituents for hypoxia-induced C6 glial cell death.

• The Korea Journal of Herbology
• /
• v.30 no.1
• /
• pp.67-75
• /
• 2015

Objectives : The aim of this study is to examine the neuroprotective effect of wheat bran extract (WBE) against ${\beta}$-amyloid ($A{\beta}$)-induced apoptotic cell death in SH-SY5Y human neuroblastoma cells and memory impairment in triple transgenic animal model's of Alzheimer's disease (3xTg AD mice). Methods : In SH-SY5Y cells, MTT assay and TUNEL staining were conducted to evaluate the protective effect of WBE against $A{\beta}_{25-35}$-induced neurotoxicity and apoptosis. Alterations in mitochondrial transmembrane potential (MMP), expression of proapoptotic Bax and antiapoptotic Bcl-2 proteins, cleavage of PARP, and brain-derived neurotrophic factor (BDNF) levels were analyzed to elucidate the neuroprotective mechanism of WBE. To further investigate the memory enhancing effect of WBE, Morris water maze test was performed in 3xTg AD mice. Results : In SH-SY5Y cells, WBE protected against $A{\beta}_{25-35}$-caused cytotoxicity and apoptosis as shown by the restoration of cell viability in MTT assay and inhibition of DNA fragmentation in TUNEL staining. $A{\beta}_{25-35}$-induced apoptotic signals such as dissipation of MMP, decreased Bcl-2/Bax ratio, and cleavage of PARP were suppressed by WBE. Moreover, WBE up-regulated the protein levels of BDNF, which seemed to be mediated by activation of cAMP response element-binding protein (CREB). In 3xTg AD mice, oral administration of WBE attenuated learning and memory deficit as verified by reduced mean escape latency in water maze test. Conclusions : WBE protects neuronal cells from $A{\beta}_{25-35}$-induced apoptotic cell death and restores learning and memory impairments in 3xTg AD mice. These findings suggest that WBE exhibit neuroprotective potential for the management of AD.

• Journal of Nutrition and Health
• /
• v.36 no.2
• /
• pp.125-132
• /
• 2003

The present study was attempted to investigate and compare the antioxidant potency of several well-know flavonoids, antioxidant vitamin and commercially available popular beverages. The antioxidant potency was assessed by the effect on reducing oxidative DNA damage of human lymphocytes. Cellular oxidative DNA damage was measured by SCGE (single-cell gel electrophoresis), also known as comet assay. Lymphocytes were pre-treated for 30 minutes with wide ranges of doses of apigenin, kaempferol, luteolin, myricetin, rutin, quercetin, $\alpha$-tocopherol (10,25,50,100,200,500,1000 $\mu$M) ,green tea extract or grape juice (10,50,100,250,500,1000 $\mu$g/mL) followed by a $H_2O$$_2$(100 $\mu$M) treatment for 5 min as an oxidative stimulus. The physiological function of each antioxidant substance on oxidative DNA damage was analyzed as tail moment (tail length $\times$ percentage migrated DNA in tail) and expressed as relative DNA damage score after adjusting by the level of control treatment. Cells treated with $H_2O$$_2$alone (positive control) had an extensive DNA damage compared with cells treated with phosphate buffered saline (PBS, negative control) or pre-treated with all the tested samples. Of all the six flavonoids, quercetin was the most potent antioxidant showing the lowest $ED_{50}$/ of 8.5 $\mu$g/mL (concentration to produce 50% protection of relative DNA damage). The antoxidant potency of individual flavonoids were ranked as follows in a decreasing order; luteolin (18.4 $\mu$g/mL), myricetin (19.0 $\mu$g/mL) , rutin (22.2 $\mu$g/mL) , apigenin (24,3 $\mu$g/mL) , kaempferol (25.5 $\mu$g/mL). The protective effect of $\alpha$-tocopherol was substantially lower (highest $ED_{50}$value of 55.0 $\mu$g/mL) than all the other flavonoids, while the protective effect was highest in green tea and grape juice with low ED5O value of 7.6 and 5.3, respectively. These results suggest that flavonoids, especially quercetin, and natural compounds from food product, green tea and grape juice, produced powerful anti-oxidative activities, even stronger than $\alpha$-tocopherol. Taken together, supplementation of antioxidants to lymphocytes followed by oxidative stimulus inhibited damage to cellular DNA, supporting a protective effect against oxidative damage induced by reactive oxygen species.

• The Korean Journal of Pharmacology
• /
• v.21 no.2
• /
• pp.79-89
• /
• 1985

Mechanism of calcium transport inhibition of cardiac sarcoplasmic reticulum (SR) by oxygen free radicals was examined. Effects of oxygen free radicals generated by xanthine/xanthine oxidase (X/XO) system on isolated porcine ventricle SR were studied with respect to its calcium binding, lipid peroxidation, SH-group content and alteration of membrane protein components. The results are as follows. 1) Calcium binding of isolated SR was markedly inhibited by X/XO. 2) During the incubation of sarcoplasmic reticulum with xanthine/xanthine oxidase, there were marked inclose in lipid peroxidation and reduction of SH-group content. 3) An antioxidant, p-phenylenediamine effectively prevented the lipid peroxidation but partially prevented the calcium binding inhibition of X/XO treated SR. 4) The reduction of SH-group content of SR treated with X/XO was partially prevented by p-phenylendiamine. 5) When modifying SH-group of SR by treatment with DTNB, the inhibition of calcium binding activity was partially prevented. 6) On gel-permeation chromatography of X/XO-treated sarcoplasmic reticulum, there was an increase of small molecular weight products, probably protein degradation products. 7) Semicarbazide, which prevents the cross-linking reaction of protein components, did not affect the calcium binding inhibition of X/XO-treated SR. From these results, it is suggested that the inhibition of calcium binding of SR by oxygen free radicals results from the consequence of multiple changes of SR components, which are lipid peroxidation, SH-group oxidation and degradation of protein components.

• The Journal of Internal Korean Medicine
• /
• v.31 no.2
• /
• pp.254-263
• /
• 2010

Objectives : The neuroprotective effects of bee venom (BV) have been demonstrated in many studies, but bee venom has many side effects. So we used sweet bee venom (SBV), which has high molecular elements removed to reduce the side effects. I examined the neuroprotective effect of sweet bee venom in 1-methyl-4-phenylpyridine ($MPP^+$)-induced human neuroblastoma SH-SY5Y cells. Methods : To observe the possible toxicity of SBV itself, SH-SY5Y cells were treated with SBV in various concentrations for 3 h and $MPP^+$ in concentrations (1 and 5mM) for 24h. To investigate the protective effect of SBV against $MPP^+$ toxicity, SH-SY5Y cells were pretreated with vehicle or nontoxic concentrations of SBV for 3h and the cells were not washed, followed by incubation with respective concentrations of SBV and 1 mM $MPP^+$ for 24h. To investigate the protective effect of SBV against $MPP^+$ toxicity, SH-SY5Y cells were pretreated with vehicle or nontoxic concentrations of SBV for 3h and the cells were not washed, followed by incubation with respective of SBV(0.5%), 1 mM $MPP^+$, 5uM AKT inhibitor(LY984002) and 10uM ERK inhibitor(PD98059) for 24 h. The protective effect was measured by cell viability assay. To investigate the degree of apoptosis, caspase-3 enzyme activity was measured in control, $MPP^+$, SBV+$MPP^+$. Results : SBV (0.5%) pretreatment protected the SH-SY5Y cells against $MPP^+$-induced apoptotic cell death. The cell viability was higher in the SH-SY5Y cells that were pretreated with vehicle or nontoxic concentrations of SBV than those not pretreated. The caspase-3 activity was lower in the pretreated groups than these not pretreated. ERK and AKT enzymes have a role in the neuroprotective effects of the sweet bee venom. Conclusions : The results demonstrate that SBV has a protective effect on dopaminergic neurons against $MPP^+$ toxicity. This data suggest that SBV could be a potential therapeutic tool for neurodegenerative diseases such as Parkinson's disease(PD).