• Journal of Nutrition and Health
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• v.34 no.8
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• pp.896-904
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• 2001

Conjugated linoleic acid(CLA) is a group of positional and geometric isomers of linoleic acid(LA) and exhibits anticarcinogenic activity in multiple experimental animal models. Cis-9,trns-11(c9t11) and trans-10,cis-12(t10c12) CLA are the principal isomers found in foods. The present study was performed to determine whether CLA and the two isomers inhibits HT-29 cell proliferation and to assess whether such an effect was related to changes in secretion of eicosanoids. Cells were incubated in serum-free medium with various concentrations(0 to 20$\mu$M) of CLA or LA. CLA inhibited cell proliferation in a dose-dependent manner, with maximal inhibition(70 $\pm$ 1%) observed at 20$\mu$M concentration after 96 hours. However, LA had no effect at the same concentration range. To compare the ability of c9f11 and t10c12 to inhibit cell proliferation, cells were incubated with increasing concentrations(0 to 4$\mu$M) of these isomers. T10c12 inhibited cell proliferation in a dose-dependent manner. A 66 $\pm$ 2% decrease in cell number was observed within 96 hours after addition of 4$\mu$M t10c12. By contrast, c9t11 had no effect. The concentrations of CLA and the two isomers in the plasma membrane were increased when they were added to the incubation medium. However, they did not alter the levels of arachidonic acid in plasma membrane. To assess whether the proliferation inhibiting effect of CLA was related to changes in eicosanoid production, prostaglandin E$_2$(PGE$_2$) and leukotriene B$_4$(LTB$_4$) concentrations in conditioned media were estimated by a competitive enzyme immunoassay. Both CLA and t10c12 increased the production of materials reactive to PGE$_2$ and LTB$_4$ antibodies in a dose-dependent manner. By contrast, c9t11 had no effect. These results indicate that inhibition of HT-29 cell proliferation by CLA is attributed to the effect of the t10v12 isomer. The materials reactive to PGE$_2$ and LTB$_4$ antibodies may inhibit growth stimulatory effect of arachidonic acid-derived eicosanoids on HT-29 cell proliferation.

• Journal of Nutrition and Health
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• v.37 no.7
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• pp.533-539
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• 2004

It has been reported that CLA decreases fat deposition in vivo and in vitro experiments. Among CLA isomers, c9t11 and t10c12 have been shown to exert active biological activities. For example, t10c12 reduces body weight and increases lean body mass, whereas, c9t11 has little effect on body fattness. However, the underlying molecular mechanism for the anti-obesity action of CLA isomers are not well understood. The purpose of this study was to examine the effects of t10c12 and c9t11 on lipid accumulation, cell proliferation, cell death and the expression levels of Ucp genes which are proposed as targets for anti-obesity in 3T3-L1 preadipocytes. Isomers of CLA at 50$\mu$M were added into preadipocyte differentiation medium for 3, 6 and 9days. Control cells received only the vehicle in the differentiation medium. Cytochemical analyses for lipid accumulation, cell proliferation and apotosis were carried out to compare lipidogenesis and cellular activity. RT-PCR analysis of GAPDH, Ucp 2,3 and 4 were also performed to find any modulatory effects of CLA isomers on the metabolic genes. Lipid accumulation indicated by Oil Red-O staining was inhibited in CLA isomers as compared to the control. T10c12 isomer showed less lipidogenesis than c9t11 did. A decrease occurred in CLA isomers as shown by BrdU incorporation. Apotosis has occured at higher level in t10c12 when compared to that of t9c11. Ucp 2, 3 and 4 genes were also upregulated in CLA isomers. T10c12 showed higher level of Ucp gene expressions than the c9t11 did. The biological activities of CLA isomers were also found to be different during differentiation of 3T3-L1 preadipocytes, suggesting that different isomers may be active in certain stage of lipidogenesis. The results indicate that both c9t11 and t10c12 CLA isomers decrease lipidogenesis, inhibit cell proliferation, increase cell death and upregulate in Ucp gene expressions during 3T3-L1 preadipocyte differentiation. T10c12 isomer was more effective than c9t11 in overall anti-obesity activity.

• Korean Journal of Crystallography
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• v.5 no.1
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• pp.1-6
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• 1994

Two possible space groups of the comfound, VP6N3018C24H:60, are: P 1, a=14.022(1), b=12.644(2), c= 12.640(1)A, a=8038(1), B=102.12(1), r=102.16(1), V=2124.1A3, Z=2, μ=0.47cm-1, d=1.46g/cm3, R=0.083 for 3350 independent reflections with Fo>4o IFI, and C2/c, a=19.32(2), b=16.32(2), c=14.02(1)A, B=105.98(5), β=105.98(5), V=4248.2A3, Z=4 R=0.083 for 1590 independent reflections with Fo>4c IFoI . In the space group P T, there are two monlecules in a unit cell. Vanadium atoms in the two monlecules occupy the two different special positions such that the complete monlecules are accomplished by the two independent center of symmetry. Therefore two different half molecules of bis(trimetaphosphate)vanadate and three molecules of tetraethylammonium are the asymmetric unit in a unit cell. In the space group C2/c, however, the vanadium atom is located at a special position with centrosymmetry, and a two-fold symmetry axis passes through C2/c, N2 and C25 atoms. Therefore the asymmrtic unit in a unit cell consists of a half molecule of bis(trimetaphosphate)vanadate and one and a half molecules of tetraethylammonium. All the molecular conformations in both space groups are very similar: six oxygen atoms coordinated to a vanadium atom in the bi s(trimetaphosphate)vanadate molecule form an octahedron and the four carbon atoms bonded to a nitrogen atom in the tetraethylammonium molecule are disordered so that the eight carbon atoms around nitrogen atom exhibit an irregular dodecahedral form.

• Journal of Korean Medicine Rehabilitation
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• v.34 no.2
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• pp.15-28
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• 2024

Objectives We used the D-galactose (D-gal) induced C2C12 myoblast senescence model to investigate whether ethanol extract of Perilla. fructescens leaves (EEPF) could delay cellular senescence and regulate related mechanisms. Methods C2C12 myogenic cells were cultured in an incubator under 37 ℃ and 5% CO₂ conditions. EEPF, dried perilla leaves were pulverized and extracted at 1:10 (v/v) at 50 ℃ for 4 hours. Cell counting kit-8 and western blot analysis was performed. Annexin V-FITC apoptosis detection kit and DAPI staining was applied. Catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde analysis kits were used. To measure the level of reactive oxygen species generation, staining and flow cytometry was used. To analyze the mitochondrial activity, membrane potential changes were measured using JC-1. 𝛽-gal activity was analyzed using SA-𝛽-gal staining solution, and DNA damage was analyzed by using 𝛾-H2AX. Quantikine ELISA kit was used to analyze inflammatory cytokine production. Results According to the results of this study, EEPF significantly alleviated the decrease in cell viability in C2C12 cells treated with D-gal and suppressed the decrease in the expression of proliferating cell nuclear antigen. EEPF also markedly blocked D-gal-induced C2C12 cell apoptosis and restored reduced activity of CAT, GSH-Px, T-AOC, SOD. In addition, EEPF suppressed the decrease in 𝛽-galactosidase activity, the induction of DNA damage and the increase in expression of senescence-associated secretory phenotype proteins such as p16, p53 and p21 in D-gal-treated C2C12 cells. Furthermore, EEPF significantly attenuated D-gal-induced production and expression of inflammatory cytokines such as interleukin (IL)-6 and IL-18. Conclusions The results of this study indicate that EEPF can be used as a potential candidate for the prevention and treatment of muscle aging.

• Journal of Life Science
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• v.33 no.1
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• pp.64-72
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• 2023

Despite several advances in identification of cardiac transcription factors, there are still needs to find new bioactive molecules that promote cardiomyogenesis from stem cells to highly efficient myocardial differentiation. We analyzed Illumina expression microarray data of mouse embryonic stem cells (mESCs)-derived cardiomyocytes. 276 genes were upregulated (≥ 4fold) in mESCs-derived cardiomyocytes compared undifferentiated ESCs. Secreted phosphoprotein 2 (Spp2) is one of candidates and is known to inhibit bone morphogenetic protein 2 (BMP2) signal transduction as a pseudoreceptor for BMP2. However, its function in cardiomyogenesis is unknown. We confirmed that Spp2 expression increased during the differentiation into functional cardiomyocytes using mESCs, TC-1/Kh2 and E14. Interestingly, Spp2 secretion transiently increased 3 days after formation of embryoid bodies (EBs), indicating that the extracellular secretion of Spp2 is involved in the differentiation of ESCs into cardiomyocytes. To characterize Spp2, we performed experiments using the C2C12 mouse myoblast cell line, which has the property of shifting the differentiation pathway from myoblastic to osteoblastic by treatment with BMP2. Similar to the differentiation of ESCs, transcription of Spp2 increased as C2C12 myoblasts differentiated into myotubes. In particular, Spp2 secretion increased dramatically in the early stage of differentiation. Furthermore, treatment with Spp2-Flag recombinant protein promoted the differentiation of C2C12 myoblasts into myotubes. Taken together, we suggest a novel bioactive protein Spp2 that differentiates ESCs into cardiomyocytes. This may be useful for understanding the molecular pathways of cardiomyogenesis and for experimental or clinical promotion of stem cell therapy for ischemic heart diseases.

• Journal of Plant Biology
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• v.12 no.2
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• pp.7-14
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• 1969

Dunaliella tertiolecta did not show any increase in respiration rate when supplied with glucose, glycerol, sucrose, L-alanine, acetate, pyruvate and succinate. This was in contrast to Chlorella pyrenoidosa, which, under identical conditions, showed significant increase when supplied with glucose or acetate but not with the other compounds. Production of 14CO2 from added 14C-glucose in D. tertiolecta was lower than the other 14C-labelled substrates: L-alinine, glycerol, succinate, but higher than 14C-sucrose addition. And it was also lower than C. pyrenoidosa experiments which was added 14C-glucose as a substrate. Light reduced amounts of labelled carbon dioxide from 14C-glucose or 14C-acetate and increased incorporation of 14C from the substrates to cell materials in either D. tertiolecta or C. pyrenoidosa. The contribution of 14C from 14C-glucose to 14CO2 in cell-free system of D. tertiolecta were much higher than in whole cell suspension. It was contrast to C. pyrenoidosa which were showed reduction of 14CO2 production in cell-free systems than whole cell suspensions. When cell-free systems of D. tertiolecta and C. pyrenoidosa were supplied with ATP, NAD, NADP or/and hexokinase, it was remarkably increased production of 14CO2 from the substrates than the control. It was concluded that the low ability of D. tertiolecta to metabolize glucose were caused by the impermeability of the cell membrane to glucose and were not due to deficiencies of enzyme systems concerning glucose metabolism. In the cell-free systems, it seemed to be more active pentose phosphate pathway than glycolytic pathway in D. tertiolecta.

• Journal of Nutrition and Health
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• v.37 no.9
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• pp.763-770
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• 2004

This study was designed to compare the anti-carcinogenic effect of conjugated linoleic acid isomers on tumor incidence, cell proliferation and the levels of thromboxane (TX) B$_2$, prostaglandin (PG) E$_2$ and 1,2-diacylglycerol (DAG), and the related enzyme expression of cyclooxygenase (COX)-2 and protein kinase C (PKC) in colonic mucosa of 1,2-dimethy- lhydrazine (DMH) -treated rats. One hundred eight male Sprague Dawley rats were randomly divided into 3 groups depending on the types of CLA isomers, i.e. control group (no CLA contained), c9t11 group (cis-9, trans-11 CLA contained), and t10c12 group (trans-10, cis-12 CLA contained). The experimental diet was composed of protein at 20%, carbohydrate at 56.2%, and fat at 14.5% including 1.0% CLA isomers by weight. The experimental diet was fed for 30 weeks with the initiation of intramuscular injection of DMH, which was injected twice a week for 6 weeks to give total dose of 180 mg per kg body weight. Two CLA isomers (c9, t11, t10, c12) significantly reduced tumor incidence and cell proliferation by reducing the protein expression of COX-2 and PKC, and the level of TXB$_2$, PGE$_2$, and DAG in colonic mucosa. However, there was no significant difference in anti-carcinogenic effect between c9t11-CLA and t10c12-CLA.

• Herbal Formula Science
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• v.29 no.4
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• pp.181-189
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• 2021

Objectives : The calyx of Diospyros kaki Thunb. has usually been used to treat obstinate hiccup. This study investigated the protective effects of Diospyros kaki using C2C12 myoblasts of H₂O₂-induced oxidative stress. Methods : Cell viability and cytotoxicity were determined by MTT assay. The level of reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH) were measured by using optical properties. Results : The calyx of Diospyros kaki Thunb. extract showed no toxicity to C2C12 myoblasts until 20 ㎍/mL concentration and increased cell viability compared to the H₂O₂ treated group. The calyx of Diospyros kaki Thunb. extract inhibited the production of ROS and MDA at all concentrations. In addition, the calyx of Diospyros kaki Thunb. extract increased the concentration of GSH. Conclusion : This study provides that the calyx of Diospyros kaki Thunb. can be used as a potential material that exhibit antioxidative and protective effects on H₂O₂-induced oxidative stress in C2C12 Myoblasts.

• Herbal Formula Science
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• v.25 no.2
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• pp.179-191
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• 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 Food Hygiene and Safety
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• v.38 no.2
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• pp.69-78
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• 2023

This study aimed to investigate the protective effect of enzymatically modified stevia (EMS) on C2C12 cell-based model of dexamethasone (DEX)-induced muscle atrophy to provide baseline data for utilizing EMS in functional health products. C2C12 cells with DEX-induced muscle atrophy were treated with EMS (10, 50, and 100 ㎍/mL) for 24 h. C2C12 cells were treated with EMS and DEX to test their effects on cell viability and myotube formation (myotube diameter and fusion index), and analyze the expression of muscle strengthening or degrading protein markers. Schisandra chinensis Extract, a common functional ingredient, was used as a positive control. EMS did not show any cytotoxic effect at all treatment concentrations. Moreover, it exerted protective effects on C2C12 cell-based model of DEX-induced muscle atrophy at all concentrations. In addition, the positive effect of EMS on myotube formation was confirmed based on the measurement and comparison of the fusion index and myotube diameter when compared with myotubes treated with DEX alone. EMS treatment reduced the expression of muscle cell degradation-related proteins Fbx32 and MuRF1, and increased the expression of muscle strengthening and synthesis related proteins SIRT1 and pAkt/Akt. Thus, EMS is a potential ingredient for developing functional health foods and should be further evaluated in preclinical models.