Lemon Balm Extract ALS-L1023 Regulates Obesity and Improves Insulin Sensitivity via Activation of Hepatic PPARα in High-Fat Diet-Fed Obese C57BL/6J Mice.

Our previous studies demonstrated that peroxisome proliferator-activated receptor α (PPARα) activation reduces weight gain and improves insulin sensitivity in obese mice. Since excess lipid accumulation in non-adipose tissues is suggested to be responsible for the development of insulin resistance, this study was undertaken to examine whether the lemon balm extract ALS-L1023 regulates hepatic lipid accumulation, obesity, and insulin resistance and to determine whether its mechanism of action involves PPARα. Administration of ALS-L1023 to high-fat-diet-induced obese mice caused reductions in body weight gain, visceral fat mass, and visceral adipocyte size without changes of food consumption profiles. ALS-L1023 improved hyperglycemia, hyperinsulinemia, glucose and insulin tolerance, and normalized insulin-positive ß-cell area in obese mice. ALS-L1023 decreased hepatic lipid accumulation and concomitantly increased the expression of PPARα target genes responsible for fatty acid ß-oxidation in livers. In accordance with the in vivo data, ALS-L1023 reduced lipid accumulation and stimulated PPARα reporter gene expression in HepG2 cells. These effects of ALS-L1023 were comparable to those of the PPARα ligand fenofibrate, while the PPARα antagonist GW6471 inhibited the actions of ALS-L1023 on lipid accumulation and PPARα luciferase activity in HepG2 cells. Higher phosphorylated protein kinase B (pAkt)/Akt ratios and lower expression of gluconeogenesis genes were observed in the livers of ALS-L1023-treated mice. These results indicate that ALS-L1023 may inhibit obesity and improve insulin sensitivity in part through inhibition of hepatic lipid accumulation via hepatic PPARα activation.

Determination of Five Chemical Markers in DF Formula, the Herbal Composition of Ephedra intermedia, Rheum palmatum, and Lithospermum erythrorhizon, Using High-performance Liquid Chromatography-ultraviolet Detection.

BACKGROUND: DF formula is a herbal preparation comprised three medicinal herbs, namely, Ephedra intermedia, Rheum palmatum, and Lithospermum erythrorhizon, which is being used for the treatment of obesity and liver fibrosis in Korean local clinics. OBJECTIVE: Since the abovementioned three herbs exist with different proportions in DF formula and their chemical markers have different physiochemical properties; it is quite challenging to develop an analytical methodology for the determination of these chemical markers. MATERIALS AND METHODS: For the analysis of the three herbs, five chemicals, (+)-pseudoephedrine (1) and (-)-ephedrine (2) for E. intermedia, aloe-emodin (3), and chrysophanol (4) for R. palmatum, and shikonin (5) for L. erythrorhizon, were selected for method validation of DF formula, and the analytical conditions were optimized and validated using high-performance liquid chromatography coupled with an ultraviolet detector (HPLC-UV). RESULTS: The specificities for the five compounds 1-5 were determined by their UV absorption spectra (1-4: 215 nm and 5: 520 nm). Their calibration curves showed good linear regressions with high correlation coefficient values (R2 > 0.9997). The limits of detection of these five markers were in the range 0.4-2.1 ng/mL, with the exception of 5 (12.7 ng/mL). The intraday variability for all the chemical markers was less than a Relative standard deviation (RSD) of 3%, except for 5 (RSD = 12.6%). In the case of interday analysis, 1 (1.0%), 2 (3.1%), and 4 (3.7%) showed much lower variabilities (RSD < 5%) than 3 (7.6%) and 5 (8.2%). Moreover, the five chemical markers showed good recoveries with good accuracies in the range of 90%-110%. CONCLUSIONS: The developed HPLC-UV method for the determination of the five chemical markers of the components of DF formula was validated. SUMMARY: DF formula, the herbal composition of Ephedra intermedia, Rheum palmatum and Lithospermum erythrorhizonFive chemical markers in DF formula were (+)-pseudoephedrine (1) and (-)-ephedrine (2) for E. intermedia, aloe-emodin (3) and chrysopanol (4) for R. palmatum, and shikonin (5) for L. erythrorhizon, with quite different physico-chemical propertiesFive chemical markers in DF formula were determined by HPLC-UV Abbreviations used: EP: (-)-ephedrine; PSEP: (+)-pseudoephedrine; HPLC: High-performance liquid chromatography; UV: Ultraviolet; LOD: Limit of detection; LOQ: Limit of quantification; RSD: Relative standard deviation.

Effect of Gangjihwan on hepatic steatosis and inflammation in high fat diet-fed mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gangjihwan (DF), a polyherbal drug composed of Ephedra intermedia Schrenk et C. A. Mayer (Ephedraceae), Lithospermum erythrorhizon Siebold et Zuccarini (Borraginaceae), and Rheum palmatum L. (Polygonaceae), is used to treat obesity in local Korean clinics. The constituents of DF have traditionally been reported to exert anti-obesity and anti-nonalcoholic fatty liver disease (NAFLD) effects. Thus, we investigated the effects of DF on obesity and NAFLD and the underlying mechanisms. MATERIALS AND METHODS: DF was extracted with water (DF-FW), 30% ethyl alcohol (DF-GA30), or 70% ethyl alcohol (DF-GA70). The chemical profile of DF was monitored using high performance liquid chromatography (HPLC)-ultraviolet analysis. The effects of DF on indices of obesity and NAFLD in high fat diet (HFD)-fed C57BL/6J mice and HepG2 cells were examined using quantitative real-time polymerase chain reaction, Oil red O staining, hematoxylin-eosin staining, toluidine blue staining, and immunohistochemistry. RESULTS: The presence of ephedrine, pseudoephedrine, aloe-emodin, and emodin in DF was determined by 3D chromatography using HPLC. Administration of DF-GA70 to HFD-fed obese mice decreased body weight, epididymal adipose tissue mass, and epididymal adipocyte size. DF-GA70 reduced serum levels of free fatty acids and triglycerides. All three DF extracts lowered serum alanine transaminase levels, hepatic lipid accumulation, and infiltration of macrophages, with the largest effects observed for DF-GA70. DF-GA70 increased mRNA levels of fatty acid oxidation genes and decreased mRNA levels of genes for lipogenesis and inflammation in the liver of obese mice. Treatment of HepG2 cells with a mixture of oleic acid and palmitoleic acid induced significant lipid accumulation, whereas all three DF extracts inhibited lipid accumulation. DF-GA70 also altered the expression of lipolytic and lipogenic genes in HepG2 cells. CONCLUSIONS: These results indicate that DF inhibits obesity and obesity-induced severe hepatic steatosis and inflammation without any adverse effects and that these effects may be mediated by regulation of the hepatic expression of lipid metabolism and inflammatory genes. These findings suggest that DF is a safe and efficient anti-obesity and anti-nonalcoholic steatohepatosis drug.