• Title/Summary/Keyword: LDH activity assay

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Homeopathic mother tincture of Conium initiates reactive oxygen species mediated DNA damage and makes HeLa cells prone to apoptosis

  • Bishayee, Kausik;Mukherjee, Avinaba;Paul, Avijit;Khuda-Bukhsh, Anisur Rahman
    • CELLMED
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    • v.2 no.3
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    • pp.26.1-26.5
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    • 2012
  • Adverse side-effects and lack of scientific validation of some chemotherapeutic agents prevent the use of many traditional medicines claimed to have anti-cancer effects. Ethanolic extract of Conium maculatum has long been used in traditional and alternative systems of medicine including homeopathy for the treatment of glandular enlargements, cancerous tumours or hard lumps of testicles, prostate, ovaries, breasts and/ or uterus, particularly in the breast. However, if and how it acts still remains scientifically unknown. This study aims to test if Conium extract (CE), used as mother tincture of Conium in homeopathy, has demonstrable anti-cancer potentials without having much cytotoxicity in normal cells. Cytotoxicity of the drug was tested by conducting MTT assay on both normal (peripheral blood mononuclear cells) and HeLa cells. We also evaluated DNA fragmentation and DNA damage by DAPI and diphenylamine assay. The LDH activity assay was done to evaluate the percentages of apoptosis and necrosis. ROS accumulation also was evaluated to pin-point the actual events of apoptosis. Administration of drug clearly demonstrated its anti-cancer potentials as evidenced by the DNA damage analysis. The ROS activity also increased in case of the CE treated cells. LDH data revealed that the mode of cell death was mainly apoptotic and not necrotic. CE appears to induce apoptosis of cancer cells through ROS mediated pathway, and has negligible cytotoxicity against normal cells.

Evaluation of Safety with Gamgung-tang Using Rec assay and Enzymatic Methods (Rec assay 및 효소학적 방법을 이용한 감궁탕의 안전성평가)

  • Shon, Yun-Hee;Baek, Tae-Seon;Moon, Ji-Sun;Kim, Mee-Kyung;Kim, Cheorl-Ho;Jeon, Byung-Hun;Nam, Kyung-Soo
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.19 no.1
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    • pp.98-101
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    • 2005
  • Gamgung-tang(GGT) that is included in Gamdu-tang(consists of Glycyrrhizae Radix, black beans) and Gunggui-tang(consists of Angelicae Radix and Cnidii Rhizoma), showed therapeutic effects of autoimmume thyroiditis in the previous reports. GGT was tested for the safety using Rec assay and enzymatic methods. In the Rec assay, Bacillus subtilis H-17$(Rec^+)$ and M-45$(Rec^-)$ strains were used to test DNA damage activity. From the results, there was no DNA damage of GGT. Hepatotoxicity of GGT to female ICR mice was also monitored by the measurements of serum(s)-GOT, s-GPT and LDH activities after oral feeding for 15 days. GGT was not shown any significant changes of s-GOT, s-GPT and LDH activities in mice sera.

Antioxidant and Neuronal Cell Protective Effects of Methanol Extract from Schizandra chinensis using an in vitro System (In vitro system에서 오미자 메탄올 추출물의 항산화 및 신경세포 보호효과)

  • Kim, Ji-Hye;Jeong, Chang-Ho;Choi, Gwi-Nam;Kwak, Ji-Hyun;Choi, Sung-Gil;Heo, Ho-Jin
    • Korean Journal of Food Science and Technology
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    • v.41 no.6
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    • pp.712-716
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    • 2009
  • In this study, the antioxidant and neuronal cell protective effects of methanol extract from Schizandra chinensis were evaluated. The proximate composition and total phenolics content of the extract were as follows: 64.88% nitrogen free extract, 10.56% crude fiber, 10.22% moisture, 8.33% crude protein, 5.05% ash, 0.96% crude fat, and 83.04 mg/g of total phenolics. In assays the methanol extract of Schizandra chinensis presented ferric reducing/antioxidant power (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity in a dose-dependent manner. In a cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT), the methanol extract showed protective effect against $H_2O_2$-induced neurotoxicity, and lactate dehydrogenase (LDH) release into medium was also inhibited by various concentrations of extracts (68-80%). Cell viability after treatment of the methanol extract was higher than that shown for vitamin C ($100\;{\mu}M$) using a neutral red uptake (NRU) assay. Therefore, these data suggest that the methanol extract of Schizandra chinensis may be useful for neurodegenerative diseases including Alzheimer's disease.

Effects of Sophorae Radix Extract in Rat Cardiac Endothelial Cells (고삼 추출물이 배양 심장내피세포에 미치는 영향)

  • Kwon Kang Beom;Park Cheon Su;Kim In Gyu;Kim Hyun Gyu;Choi Ki Bang;Kim Yong Bok;Ryu Do Gon
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.17 no.1
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    • pp.220-224
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    • 2003
  • To test the protective effect of Sophorae Radix (SR) on the damage of cardiac endothelial cells by xanthine oxidase (XO)/hypoxanthine (HX)-induced oxygen tree radical, Neutral Red (NR), lactate dyhydrogenase (LDH), and c-fos immunopositive cells assay were used in the presence of SR extract. The results of these experiments were obtained as follows ; Cardiac endothelial cells treated with XO/HX showed the cytotoxicity such as a decrease in viability, and increases in LDH activity and c-fos immunopositive cells. Cardiac endothelial cells pretreated with SR extract protected the increase of LDH activity. Alos, cardiac endothelial cells pretreated with SR extract inhibited the increase of c-fos immunopositive cells. These results show that XO/HX induces toxic effects in cultured cardiac endothelial cells derived from neonatal rat, and suggest that SR extract is very effective in the prevention of XO/HX-induced toxicity.

Protective Effect of Methanolic Extracts from Dendrobium nobile Lindl. on $H_{2}O_{2}$-induced Neurotoxicity in PC12 cells (석곡 MeOH 추출물이 $H_{2}O_{2}$에 의한 신경세포 보호효과에 미치는 영향)

  • Yoon, Mi-Young;Kim, Ju-Young;Hwang, Ji-Hwan;Cha, Mi-Ran;Lee, Mi-Ra;Jo, Kyung-Jin;Park, Hae-Ryong
    • Applied Biological Chemistry
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    • v.50 no.1
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    • pp.63-67
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    • 2007
  • The neuroprotective effect of methanolic extracts from Dendrobium nobile Lindl. (DME) against $H_{2}O_{2}$-induced neurotoxicity in PC12 cells was investigated. The treatment of PC12 cells with various DME concentrations under $H_{2}O_{2}$ resulted in the induction of protective effect in a dose-dependent manner, as determined by the results of an MTT reduction assay, an LDH release assays, and a morphological assay. Interestingly, we also detected reduction of apoptotic bodies and inhibition of caspase-3 activity by DME in $H_{2}O_{2}$-indeced PC12 cells. These data show that the neuroprotective effect of DME against PC12 cells might be related to the suppression of caspase-3 activation. Therefore, these results suggest that DME could be a new potential candidate as chemotherapeutic agents against neuronal diseases.

Protective Effects of Jisilhaebaekgyeji-tang and Constituents Extract on Cultured Rat Myocardial Cell treated by XO/HX (XO/HX에 의해 손상된 배양 심근세포에 대한 지실해백계지탕과 구성약물 추출물의 방어효과)

  • Jang Seung Ho;Kwon Kang Beom;Kim In Su;Kang Gil Seong;Kim In Gyu;Kim In Seob;Ryu Do Gon
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.17 no.4
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    • pp.952-957
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    • 2003
  • To certify the protective effect of herbal medicine against oxygen free radical-induced myocardiotoxicity, cytotoxicity was measured using LDH activity and TBARS assay in the presence of Jisilhaebaekgyejitang(JHGT) extracts or single constituents of this prescription, In the present study, xanthine oxidase/hypoxanthine (XO/HX) resulted in a cell damage such as increases in LDH activity in culture medium and lipid peroxidation in cultured myocardial cells. In the effect of JHGT extract and its single constituents, which are Fructus Ponciri Seu Aurantii Immaturus (FPSAI), Cortex Magnoliae Officinalis (CMO), Bulbus Allii Macrostemi (BAM), Ramulus Cinnamomi (RC) and Fructus Trichosanthis (FT), they showed the prevention from the XO/HX-induced cardiotoxicity by the decrease of LDH activity and lipid peroxidation. From these results, they show that XO/HX is cardiotoxic in cultured myocardial cells derived from neonatal rat, and it suggests that JHGT, FPSAI, PT, CMO, BAM, RC and FT extracts are positively effective in the blocking in XO/HX-induced cardiotoxicity.

Purification and characterization of biochemical properties of hemolysin from Vibrio fluvialis (Vibrio fluvialis 유래의 hemolysin 정제와 생화학적 특성)

  • 이종희;한정현;안선희;김선회;이은미;공인수
    • Journal of Life Science
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    • v.12 no.4
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    • pp.490-495
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    • 2002
  • Hemolysin (VFH) of V. fluvialis, which is a pathogenic bacteria, causing watery diarrhea with vomiting, abdominal croup, was purified. V. fluvialis was cultivated in BHI medium and the culture supematant was precipitated by ammonium sulfate. The protein was purified by chromatographies on columns of DEAE-cellulose and Mono-Q. Molecular weight of the purified VFH was estimated as 79kDa by SDS-PAGE. The optimal temperature for a maximum hemolytic activity was at around 35$^{\circ}C$ and the activity was decreased at 4$0^{\circ}C$ Cytotoxicity of VFH was also investigated using RTG-2 cell line. LDH assay study showed that 50$\mu\textrm{g}$/m1 of VFH release 80% of total cellular LDH (lactate dehydrogenase) from RTG-2 cell and microscopic observation also showed the morphological change of cell.

Cirsium japonicum Extracts Show Antioxidant Activity and PC12 Cell Protection against Oxidative Stress (좁은잎 엉겅퀴 추출물의 산화방지 활성 및 산화적 스트레스에 대한 PC12 세포 보호효과)

  • Jang, Miran;Kim, Gun-Hee
    • Korean Journal of Food Science and Technology
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    • v.48 no.2
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    • pp.172-177
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    • 2016
  • The phenolic compounds, antioxidant activity and neuronal cell protective effect of Cirsium japonicum extract were evaluated in this study. High performance liquid chromatography mass analysis showed that C. japonicum was composed of chlorogenic acid, linarin, and pectolinarin. C. japonicum extract showed its antioxidant activity with half-maximal inhibitory concentrations of 567 and $130{\mu}g/mL$ by DPPH and ABTS radical scavenging activity, respectively. The total antioxidant capacities of C. japonicum via DPPH, ABTS, and FRAP assays were 11.32, 100.15, and $12.76{\mu}g/mL$ trolox equivalents, respectively. In addition, the neuroprotective effect of C. japonicum extract was investigated by measuring cell viability via MTT, LDH and DCF-DA assay using $H_2O_2-damaged$ PC12 cells. C. japonicum extract showed neuronal cell protective effects in a dose-dependent manner. These results indicated that C. japonicum extract has potent antioxidant and neuronal protective effects. Therefore, C. japonicum can be regarded as an effective and safe functional food resource as natural antioxidants, and may decrease the risk of neurodegenerative disorders.

Effect on Alzheimer's Disease by Sesim-tang in CT105-overexpressed SK-N-SH Cell Lines (CT105로 유도된 신경모세포종 세포주에서 세심탕의 항치매 효과)

  • 권형수;박치상;박창국
    • The Journal of Korean Medicine
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    • v.25 no.2
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    • pp.138-150
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    • 2004
  • Objectives : Alzheimer's disease (AD) is a geriatric dementia that is widespread in old age. In the near future AD will be the biggest problem in public health service. Although a variety of oriental prescriptions, including Sesim-tang, have been traditionally utilized for the treatment of AD, their pharmacological effects and action mechanisms have not yet been fully elucidated. The present study investigated the effects of Sesim-tang on apoptotic cell death induced by CT105 (carboxy terminal 105 amino acid peptide fragment of APP) overexpression in SK-N-SH neuroblastoma cell lines. Methods: We studied the regenerative and inhibitory effects on Alzheimer's disease in CT105-induced SK-N-SH cell lines by Sesim-tang water extract. We examined for cell morphological pattern, DNA fragmentation, LDH activity assay, zymography assay, and immunohistochemistric analysis. Additionally, we investigated the association between the CT105 and neurite degeneration caused by CT105-induced apoptotic response in neurone cells. Results: Findings from our experiments have shown that Sesim-tang inhibits the synthesis or activities of CT105, which has neurotoxicities and apoptotic activities in the cell line. In addition, pretreatment with Sesim-tang ($>50\mu\textrm{g}/ml$ for 12 hours) partially prevented CT105-induced cytotoxicity in SK-N-SH cell lines. SK-N-SH cell lines overexpressed with CT105 exhibited remarkable apoptotic cell damage. Based on morphological observations by phase-contrast microscope and LDH activity measurements in the culture media, the CT105-induced cell death was significantly inhibited by Sesim-tang water extract. Sesim-tang was found to reduce the expression of APP and caspase-3 induced by CT105 in SK-N-SH cell lines and in rat hippocampus. Conclusions: As the result of this study, in the Sesim-tang group, apoptosis in the nervous system is inhibited, the repair against the degeneration of SK-N-SH cell lines by CT105 expression is promoted. Hence, Sesim-tang may be beneficial for the treatment of AD.

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Neuroprotective effects of L-carnitine against oxygen-glucose deprivation in rat primary cortical neurons

  • Kim, Yu-Jin;Kim, Soo-Yoon;Sung, Dong-Kyung;Chang, Yun-Sil;Park, Won-Soon
    • Clinical and Experimental Pediatrics
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    • v.55 no.7
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    • pp.238-248
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    • 2012
  • Purpose: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). Methods: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 ${\mu}M$, 10 ${\mu}M$, and 100 ${\mu}M$) on OGD-induced neurotoxicity. Results: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 ${\mu}M$ and 100 ${\mu}M$ of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 ${\mu}M$ significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). Conclusion: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.