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      • Molecular Mechanism of Crocin Induced Caspase Mediated MCF-7 Cell Death: In Vivo Toxicity Profiling and Ex Vivo Macrophage Activation

        Bakshi, Hamid A,Hakkim, Faruck Lukmanul,Sam, Smitha Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.3

        Background: Crocus sativus and its major constituent crocin are well established to have anti-cancer properties in breast cancer cells (MCF-7). However the role of C. sativus extract (CSE) and crocin on caspase signaling mediated MCF-7 cell death at molecular level is remains unclear. In this study, we tried to unravel role of CSE and crocin on caspase mediated MCF-7 cells death and their in vivo preclinical toxicity profiling and immune stimulatory effect. Materials and Methods: CSE extract was fractionated by HPLC and crocin was isolated and characterized by NMR, IR, and MS. MCF-7 cells were treated with both CSE and crocin and expression of Bcl-2 and Bax was assessed after 24 and 36 hours. Furthermore, caspase 3, caspase 8 and caspase 9 expression was determined by Western blotting after 24 hours of treatment. DNA fragmentation analysis was performed for genotoxicity of CSE and crocin in MCF-7 cells. The in vivo toxicity profile of CSE (300 mg/kg of b.wt) was investigated in normal Swiss albino mice. In addition, peritoneal macrophages were collected from crocin (1, 1.5 and 2 mg/kg body weight) treated mice and analyzed for ex vivo yeast phagocytosis. Results: Immunoblot analysis revealed that there was time dependent decline in anti-apoptotic Bcl-2 with simultaneous upregulation of Bax in CSE and crocin treated MCF-7 cells. Further CSE and crocin treatment downregulated caspase 8 and 9 and cleaved the caspase 3 after 24 hours. Both CSE and crocin elicited considerable DNA damage in MCF-7 cells at each concentration tested. In vivo toxicity profile by histological studies revealed no observable histopathologic differences in the liver, kidney, spleen, lungs and heart in CSE treated and untreated groups. Crocin treatment elicited significant dose and time dependent ex vivo yeast phagocytosis by peritoneal macrophages. Conclusions: Our study delineated involvement of pro-apoptotic and caspase mediated MCF-7 cell death by CSE and crocin at the molecular level accompanied with extensive DNA damage. Further we found that normal swiss albino mice can tolerate the maximum dose of CSE. Crocin enhanced ex vivo macrophage yeast phagocytic ability.

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        Antioxidant property of leaves and calluses extracts of in-vitro grown 5 different Ocimum species

        송혁,Prem Kumar,Girija Arivazhagan,Sang-Il Lee,Hyung Moon Yoon,김익희,권혁중,김종문,Faruck Lukmanul Hakkim 한국식물생명공학회 2012 식물생명공학회지 Vol.39 No.3

        In this study, the antioxidant property of leaf and callus extracts of five selected in vitro grown Ocimum species (Ocimum sanctum, Ocimum kilimandscharicum, Ocimum gratissimum, Ocimum basilicum, and Ocimum americanum) and their respective callus extracts was investigated. The callus cultures were successfully initiated on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) (1 mg・L) combined with different concentrations (0.1-0.4 mg・L) of kinetin as plant growth regulators. Total phenolic contents were estimated using the Folin-Ciocalteu reagent. 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power, Fe2+ chelating activity, and β-carotenelinoleic acid bleaching assays were used to determine the biological effects of the extracts. Interestingly, all the callus extracts exhibited significant (p<0.05) increase in phenolic contents and antioxidant activity. Furthermore, a liner correlation was obtained between the total phenolic contents and free radical scavenging activity (R2 = 0.783). The extracts of leaves and calluses of Ocimum species exhibited activity in all the in vitro antioxidant assays, but its extent was less potent that the positive controls butylated hydroxyl anisole (BHA) and ascorbic acid. A higher accumulation of phenolics in the callus extracts suggests that isolation of high-concentration materials with antioxidant activivity is possible from in vitro callus cultures rather than field-grown plant organs. Furthermore, these extracts may be used as an effective preservative in the food industry.

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        Antioxidant property of leaves and calluses extracts of in-vitro grown 5 different Ocimum species

        Song, Hyuk,Kumar, Prem,Arivazhagan, Girija,Lee, Sang-Il,Yoon, Hyung-Moon,Kim, Ick-Hee,Kwon, Hyuk-Jung,Kim, Jong-Moon,Hakkim, Faruck Lukmanul The Korean Society of Plant Biotechnology 2012 식물생명공학회지 Vol.39 No.3

        In this study, the antioxidant property of leaf and callus extracts of five selected in vitro grown Ocimum species (Ocimum sanctum, Ocimum kilimandscharicum, Ocimum gratissimum, Ocimum basilicum, and Ocimum americanum) and their respective callus extracts was investigated. The callus cultures were successfully initiated on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) (1mg L) combined with different concentrations (0.1-0.4 mg L) of kinetin as plant growth regulators. Total phenolic contents were estimated using the Folin-Ciocalteu reagent. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power, $Fe^{2+}$ chelating activity, and ${\beta}$-carotenelinoleic acid bleaching assays were used to determine the biological effects of the extracts. Interestingly, all the callus extracts exhibited significant (p<0.05) increase in phenolic contents and antioxidant activity. Furthermore, a liner correlation was obtained between the total phenolic contents and free radical scavenging activity ($R^2$ = 0.783). The extracts of leaves and calluses of Ocimum species exhibited activity in all the in vitro antioxidant assays, but its extent was less potent that the positive controls butylated hydroxyl anisole (BHA) and ascorbic acid. A higher accumulation of phenolics in the callus extracts suggests that isolation of high-concentration materials with antioxidant activivity is possible from in vitro callus cultures rather than field-grown plant organs. Furthermore, these extracts may be used as an effective preservative in the food industry.

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