• Journal of Korean Medicine for Obesity Research
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• v.18 no.2
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• pp.106-114
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• 2018

Objectives: We investigated about the microbial properties and changes in the efficacy of the Codonopsis lanceolata (CL) by natural fermentation. Methods: CL was fermented for four weeks in a well-ventilated place with 2.5% salt. pH, total sugar, total polyphenol, and total flavonoid were measured to determine fermentation characteristics according to fermentation period and salt treatment. Polymerase chain reaction denaturing gradient gel electrophoresis and random amplification of polymorphic DNA-polymerase chain reaction were carried out for microbial analysis during fermentation. In addition, HepG2 cell was cultured to check the lipid accumulation through oil red O staining and the glucose uptake was analyzed by measuring the 2-NBDG at C2C12 cell. Results: The pH level and the total sugar decreased with the CL fermentation. Total polyphenol and flavonoid increased after CL fermentation. It was confirmed that Leuconostoc mesenteroides were maintained continuously during fermentation. In the salt treatment CL, there was a sharp increase in Rahnella aquatilis. Lactobacillus plantarum matrix was observed in fermented CL. In addition, Lactococcus lactis, Weissella koreensis, R. aquatilis, L. plantarum, Leu. mesenteroides have been added to the salt treatment. Glucose uptake were significantly increased after fermentation with salt for four weeks. Lipid accumulation in the HepG2 cells was observed that there was difference (P<0.01) between free fatty acid group (100%) and decreased 4 weeks after fermentation (90.38%) at $800{\mu}g/mL$. Conclusions: Total polyphenol and flavonoid were increased after CL fermentation. Especially, percentage of the glucose uptake and lipid accumulation inhibition increased in CL fermentation with salt. It is expected that fermentation of salt treated CL will be more effective in diabetes and fatty liver.

• Journal of Nutrition and Health
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• v.54 no.5
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• pp.448-458
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• 2021

Purpose: Mulberry (Morus alba L.) fruit is widely grown in Asia and consumed as fresh fruit, jam, and juices. The fruit has beneficial health effects, including anti-diabetic, anti-tumor, and anti-obesity properties. However, the mechanisms by which mulberry fruit juice powder (MJ) regulates inflammatory microRNAs (miRs) are not yet known. This study investigated the effect of mulberry fruit juice powder on the regulation of inflammation and miR-132/143 during 3T3-L1 adipocyte differentiation. Methods: The 3T3-L1 cells were induced to differentiate for 2 days and then treated with various concentrations of MJ for 7 days. Cytotoxicity was determined by evaluating cell viability using a water-soluble tetrazolium salt-8 assay kit. Intracellular lipid accumulation was evaluated by oil-red O staining. The levels of the expression of genes involved in adipogenesis and inflammation, and miR-132/143 were measured by quantitative real-time polymerase chain reactions. Results: MJ showed no cytotoxic effect on 3T3-L1 adipocytes at concentrations below 100 ng/mL. Intracellular lipid accumulation was reduced by MJ treatment at concentrations of 100 ng/mL. The messenger RNA (mRNA) levels of proliferator-activated receptor-γ, cytosine-cytosine-adenosine-adenosine-thymidine/enhancer-binding protein-α, and adipocyte protein 2, which are involved in adipogenesis, were suppressed by MJ. A reduction was also seen in mRNA levels of genes related to the inflammatory response, such as tumor necrosis factor-α, interleukin-6, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. The expression of the inflammatory miR-132 and miR-143 was also decreased by MJ. Conclusion: These results suggest that MJ may suppress adipogenesis and inflammation through the regulation of miR-132/143 expression in 3T3-L1 adipocytes. Thus, MJ may be useful as a food agent that prevents obesity-associated inflammation.

• Journal of Korean Medicine for Obesity Research
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• v.20 no.2
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• pp.78-87
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• 2020

Objectives: To expand and provide information on the efficacy of herbal medicines, anti-obesity effects were evaluated. In many studies, plant-derived components with anti-obesity efficacies have been investigated for their potential inhibitory effects on 3T3-L1 preadipocyte cells. The purpose of this study was to investigate the anti-obesity effects of 65 herbal medicine in 3T3-L1 preadipocyte cells. Methods: Preferentially, 3T3-L1 cells were treated with 65 herbal medicines (500 ㎍/mL) during differentiation for 8 days. Next, 3T3-L1 cells were treated with selected herbal medicines at concentrations ranging from 50 to 200 ㎍/mL during differentiation for 8 days. The accumulation of lipid droplets was determined by Oil Red O staining. The expressions of genes related to adipogenesis were measured by reverse transcription polymerase chain reaction and Western blot analyses. Results: Among the 65 kinds of herbal medicines, 13 herbal medicines that been shown to be effective against the accumulation of lipid droplets were selected. Finally, selected Banhasasim-tang and Samhwangsasim-tang showed inhibitory activity on adipocyte differentiation at 3T3-L1 preadipocytes without affecting cell toxicity. In addition, Banhasasim-tang and Samhwangsasim-tang significantly reduced the expression levels of several adipocyte marker genes including peroxisome proliferator activated receptor-γ and CCAAT/enhancer binding protein-α. Conclusions : These results suggest that the ability of Banhasasim-tang and Samhwangsasimtang has inhibited overall adipogenesis and lipid accumulation in the 3T3-L1 cells. Banhasasim-tang and Samhwangsasim-tang may be a promising medicine for the treatment of obesity and related metabolic disorders.

• Biomolecules & Therapeutics
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• v.30 no.3
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• pp.246-256
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• 2022

The present study focused on the potential mechanism of betulin (BT), a pentacyclic triterpenoid isolated from the bark of white birch (Betula pubescens), against chronic alcohol-induced lipid accumulation and metaflammation. AML-12 and RAW 264.7 cells were administered ethanol (EtOH), lipopolysaccharide (LPS) or BT. Male C57BL/6 mice were fed Lieber-DeCarli liquid diets containing 5% EtOH for 4 weeks, followed by single EtOH gavage on the last day and simultaneous treatment with BT (20 or 50 mg/kg) by oral gavage once per day. In vitro, MTT showed that 0-25 mM EtOH and 0-25 µM BT had no toxic effect on AML-12 cells. BT could regulate sterolregulatory-element-binding protein 1 (SREBP1), lipin1/2, P2X7 receptor (P2X7r) and NOD-like receptor family, pyrin domains-containing protein 3 (NLRP3) expressions again EtOH-stimulation. Oil Red O staining also indicated that BT significantly reduced lipid accumulation in EtOH-stimulated AML-12 cells. Lipin1/2 deficiency indicated that BT might mediate lipin1/2 to regulate SREBP1 and P2X7r expression and further alleviate lipid accumulation and inflammation. In vivo, BT significantly alleviated histopathological changes, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and triglyceride (TG) levels, and regulated lipin1/2, SREBP1, peroxisome proliferator activated receptor α/γ (PPARα/γ) and PGC-1α expression compared with the EtOH group. BT reduced the secretion of inflammatory factors and blocked the P2X7r-NLRP3 signaling pathway. Collectively, BT attenuated lipid accumulation and metaflammation by regulating the lipin1/2-mediated P2X7r signaling pathway.

• Animal Bioscience
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• v.35 no.5
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• pp.763-777
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• 2022

Objective: Excessive lipid accumulation in adipocytes results in prevalence of obesity and metabolic syndrome. Curcumin (CUR), a naturally phenolic active ingredient, has been shown to have lipid-lowering effects. However, its underlying mechanisms have remained largely unknown. Therefore, the study aims to determine the effect of CUR on cellular lipid accumulation in porcine subcutaneous preadipocytes (PSPA) and to clarify novel mechanisms. Methods: The PSPA were cultured and treated with or without CUR. Both cell counting Kit-8 and lactate dehydrogenase release assays were used to examine cytotoxicity. Intracellular lipid contents were measured by oil-red-o staining extraction and triglyceride quantification. Apoptosis was determined by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling assay. Adipogenic and apoptosis genes were analyzed by quantitative polymerase chain reaction and Western blot. Results: The CUR dose-dependently reduced the proliferation and lipid accumulation of PSPA. Noncytotoxic doses of CUR (10 to 20 μM) significantly inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and expression of adipogenic genes peroxisome proliferation-activity receptor-γ (PPAR-γ), CCAAT/enhancer binding protein-α, sterol regulatory element-binding protein-1c, adipocyte protein-2, glucose transporter-4 as well as key lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase, while ERK1/2 activation significantly reversed CUR-reduced lipid accumulation by increasing PPAR-γ. Furthermore, compared with differentiation induced media treated cells, higher dose of CUR (30 μM) significantly decreased the expression of AKT and B-cell lymphoma-2 (BCL-2), while increased the expression of BCL-2-associated X (BAX) and the BAX/BCL-2 expression ratio, suggesting triggered apoptosis by inactivating AKT and increasing BAX/BCL-2 ratio and Caspase-3 expression. Moreover, AKT activation significantly rescued CUR inhibiting lipid accumulation via repressing apoptosis. Conclusion: These results demonstrate that CUR is capable of suppressing differentiation by inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis via decreasing AKT and subsequently increasing BAX/BCL-2 ratio and Caspase-3, suggesting that CUR provides an important method for the reduction of porcine body fat, as well as the prevention and treatment of human obesity.

• The Korea Journal of Herbology
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• v.29 no.2
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• pp.15-21
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• 2014

Objectives : The purpose of this study was to investigate inhibitory effects of steamed Polygonatum odoratum extract (POE) on differentiation and adipogenesis in 3T3-L1 adipocytes. Methods : Polygonatum odoratum (P. odoratum) extract was extracted with ethyl acetate. Total phenolic and flavonoid contents in POE were measured for antioxidant activity. The spectrophotometric method was used to determine the DPPH and ABTS radical scavenging activity and ferric-reducing antioxidant potential (FRAP). MTT assay was examined for cell toxicity, oil red O staining was performed for intracelluar adipogenesis in differentiated 3T3-L1 adipocytes. Western blot analysis for measurement of CCAAT/enhancer-binding protein ${\alpha}$ ($C/EBP{\alpha}$), peroxisome proliferator-activated receptor${\gamma}$ ($PPAR{\gamma}$) and AMP-activated protein kinase (AMPK) expressions were performed. Results : The results revealed that POE has antioxidant activities. Contents of total polyphenolics and flavonoids were $50.83{\pm}1.52$ GAE mg/100g dry weight of POE and $17.05{\pm}2.47$ RE mg/100g dry weight of POE, respectively. DPPH radical scavenging activity, and FRAP in 10 mg/ml concentration were $92.1{\pm}0.6%$, $244.8{\pm}9.0{\mu}M$ Fe(II) and ABTS inhibition in 5 mg/ml concentration was $84.8{\pm}4.1%$. Treatment of POE in adipocytes inhibited the differentiation and adipogenesis of 3T3-L1 adipocytes compared to those of vehicle control. Additionally, protein expressions of $C/EBP{\alpha}$ and $PPAR{\gamma}$, major transcription factor for the adipogenic genes, were significantly decreased compared to those of vehicle control (p<0.05). Futhermore, phosphorylation of AMPK was increased in 3T3-L1 adipocytes treated with POE compared to that of vehicle control (p<0.05). Conclusions : we demonstrate that steamed P. odoratum extract (POE) has potentiating antioxidant activities, inhibits differentiation and lipid accumulation and also induces energy expenditure in adipocytes, which may contribute to antiobesity property.

• The Korea Journal of Herbology
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• v.29 no.1
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• pp.45-52
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• 2014

Objectives : The aim of this study was to investigate the effects water extract of fermented new korean medicinal mixture, combinations of Mori Folium, Adenophorae Radix, Phllostachyos Folium and Citri Pericarpium (F-MAPC), on adipocyte differentiation, adipogenesis and glucose uptake using undiffernentiated 3T3-L1 adipocytes and L6 myoblasts. Methods : Each herb and those mixture were respectively fermented and then extracted with water. We carried on MTT assay for check-up on cell toxicity, Oil Red O staining for determination of cell differentiation and intracelluar adipogenesis. Western blot analysis for measurement of pAMPK and pACC, $C/EBP{\alpha}$, $PPAR{\gamma}$ and Glut-4 protein expressions were performed. Results : F-MAPC showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 preadipocytes without affecting cell toxicity as assessed by measuring fat accumulation, and this effect was 2 fold higher in 0.2 mg/ml F-MAPC than that of the same dose of each fermented herbal extract alone. In addition, these effects were associated with modulation of adipogenic transcription factors, such as $C/EBP{\alpha}$, $PPAR{\gamma}$, as well as stimulated phosphorylations of AMPK and ACC. Translocation of Glut-4 was significantly increased by 10.2% in L6 cells treated with 0.2 mg/ml F-MAPC compared with that of control. Conclusions : These results demonstrate that F-MAPC may be an ideal candidate for therapy of obesity and diabetes by disturbing the differentiation into adipocytes, as well as the inducement of intramuscular glucose uptake from blood.

• The Korea Journal of Herbology
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• v.28 no.1
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• pp.23-31
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• 2013

Objectives : The purpose of this study was to determine the anti-obesity effect and molecular mechanism of YY312, a herbal extract composed of Imperatae Rhizoma, Citri Unshius Pericarpium Immaturus, and Evodiae Fructus, on a high-fat diet-induced animal model and on 3T3-L1 cells. Methods : C57BL/6 mice were fed for 6 weeks with a normal diet or a high-fat diet (HFD). Then they orally administered daily with 300 mg/kg YY312 for next 10 weeks. Body weight and food consumption were recorded weekly and daily, respectively. Tissue weights, serum lipid, and glucose levels were analyzed at the end of the study. Additionally, the effects of YY312 on adipocyte differentiation in 3T3-L1 cells were examined. After differentiating 3T3-L1 cells were treated with YY312, Oil-red O staining, RT-PCR, and Western blotting were performed for lipid accumulation, mRNA expression of adipogenesis gene, and AMP-activated protein kinase (AMPK) phosphorylation, respectively. Results : YY312-administered mice showed a significant reduction of body weights and abdominal adipose tissue weights. YY312 also reduced the serum levels of triglycerides and total cholesterol, compared with the HFD group. Treatment with YY312 inhibited lipid accumulation and blocked expression of adipogenic transcription factors and lipogenesis genes, including peroxisome proliferator-activated receptor ${\gamma}$, CCAT/enhancer binding protein ${\alpha}$ and fatty acid synthase. YY312 increased AMPK phosphorylation in 3T3-L1 adipocytes. Conclusions : This study showed that herbal extract YY312 has an anti-obesity effect in vitro and in vivo. Thus, YY312 could be developed as a supplement for reduction of body weight gain induced by an HFD.

• The Korea Journal of Herbology
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• v.33 no.1
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• pp.17-26
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• 2018

Objective : In this study, we investigated the effect of SAL5(mixing extracts of Schisandra chinensis Baillon, Artemisia capillaris Thunb., and Aloe vera Linne) on chronic ethanol-induced fatty liver model. Methods : Sprague-Dawley male rats were fed Liber-DeCarli (normal), ethanol liquid diet (control), SAL5 (200 mg/kg). We administrated the SAL5 on chronic ethanol-induced fatty liver model for 5 weeks. We measured alkaline phosphtase (ALP), alanine transminase (ALT), aspartate transminase (AST) and ${\gamma}-glutamyl$ transpeptase (${\gamma}-GTP$) in serum and triglyceride (TG), superoxide dismutase (SOD), catalase, glutathione (GSH) and malondialdehyde (MDA) level in liver. Liver histopathology was examined by Hematoxylin-eosin and Oil red O staining of the fixed liver tissues. Real-time PCR was performed to measure the mRNA expression of inflammatory cytokines and MMP-2, MMP-9. Results : SAL5 administration resulted in significantly decreased liver marker enzymes activities of alanine transminase (ALT), ${\gamma}-glutamyl$ transpeptase (${\gamma}-GTP$) in serum and triglyceride (TG) activities in liver. The control group decreased the activities of superoxide dismutase (SOD), catalase (CAT) with the reduced level of glutathione (GSH) in liver. On the other hand, SAL5 group increased the activities of SOD, CAT and the level of GSH. SAL5 delayed the development of an alcoholic fatty liver by reversing fat accumulation in the liver, as evidenced in histological observations. The gene expression of mRNA were significantly decreased at the $IL-1{\beta}$, $TNF-{\alpha}$, NOS-II and MMP-2 by SAL5. Conclusions : These results indicate that SAL5 might have protective effect chronic ethanol-induced fatty liver models.

• The Korea Journal of Herbology
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• v.33 no.2
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• pp.69-77
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• 2018

Objectives : Daesiho-tang (DSHT) has been widely used in the treatment of cerebral infarct in traditional medicine. However, there was not report on the anti-obesity-related diseases efficacy of DSHT. In this study, we investigated the effects for the new formulation of DSHT, on the adipocyte differentiation cycle in 3T3-L1 cells. Methods : 3T3-L1 cells were treated with DSHT (50, 100, $200{\mu}g/m{\ell}$) during differentiation for 6 days. Also, the inhibitory effect of DSHT against 3T3-L1 adipogenesis was evaluated in various stage of adipogenesis such as early (0-2day), intermediate (2-4day), and terminal stage (4-6day). The accumulation of lipid droplets was determined by Oil Red O staining. and, the expressions of genes related to adipogenesis were measured by RT-PCR and Western blot analyses. Results : DSHT showed inhibitory activity on adipocyte differentiation at 3T3-L1 preadipocytes without affect cell toxicity as assessed by measuring fat accumulation and adipogenesis. In addition, DSHT significantly reduced the expression levels of several adipocyte marker genes including proliferator activated $receptor-{\gamma}$ ($PPAR-{\gamma}$) and CCAAT/ enhancer-binding $protein-{\alpha}$ ($C/EBP-{\alpha}$). Also, the anti-adipogenic effect of DSHT was strongly limited in the intermediate (2-4 day), terminal stage (4-6 day) of 3T3-L1 adipogenesis. In addition, the DSHT treatment down- regulated mRNA expression levels of $PPAR-{\gamma}$,, $C/EBP-{\alpha}$ in mature 3T3-L1 adipocytes. Conclusions : These results suggest that, the ability of DSHT has inhibited overall adipogenesis and lipid accumulation in the 3T3-L1 cells. The new formulation of DSHT may be a promising medicine for the treatment of obesity and related metabolic disorders.