Seabuckthorn Leaves Extract and Flavonoid Glycosides Extract from Seabuckthorn Leaves Ameliorates Adiposity, Hepatic Steatosis, Insulin Resistance, and Inflammation in Diet-Induced Obesity

The aim of the current study was to elucidate the effect of seabuckthorn leaves (SL) extract and flavonoid glycosides extract from seabuckthorn leaves (SLG) on diet-induced obesity and related metabolic disturbances, and additionally, to identify whether flavonoid glycosides and other components in SL can exert a possible interaction for the prevention of metabolic diseases by comparing the effect of SL and SLG. C57BL/6J mice were fed a normal diet (ND, AIN-93G purified diet), high-fat diet (HFD, 60 kcal% fat), HFD + 1.8% (w/w) SL (SL), and HFD + 0.04% (w/w) SLG (SLG) for 12 weeks. In high fat-fed mice, SL and SLG decreased the adiposity by suppressing lipogenesis in adipose tissue, while increasing the energy expenditure. SL and SLG also improved hepatic steatosis by suppressing hepatic lipogenesis and lipid absorption, whilst also enhancing hepatic fatty acid oxidation, which may be linked to the improvement in dyslipidemia. Moreover, SL and SLG improved insulin sensitivity by suppressing the levels of plasma GIP that were modulated by secreted resistin and pro-inflammatory cytokine, and hepatic glucogenic enzyme activities. SL, especially its flavonoid glycosides (SLG), can protect against the deleterious effects of diet-induced obesity (DIO) and its metabolic complications such as adiposity, dyslipidemia, inflammation, hepatic steatosis, and insulin resistance.


Resistance
The blood glucose concentration was measured by the glucose oxidase method using a glucose analyzer (Glucocard, Arkray, Japan) in whole blood obtained from the tail vein after food withholding for 12 h. The intraperitoneal glucose tolerance test (IPGTT) was performed at week 11. After 12 h of fasting, the mice were injected intraperitoneally with glucose (0.5 g/kg of body weight). The blood glucose level was determined from the tail vein at 0, 30, 60, and 120 min after the glucose injection.

Hepatic and Fecal Lipid Contents
Hepatic and fecal lipids were extracted as previously described [1], and then dried lipid residues were dissolved in 1 mL of ethanol for triglyceride, cholesterol, and fatty acid (FA) assays. Triton X-100 and a sodium cholate solution in distilled water were added to 200 μL of a dissolved lipid solution for emulsification. Hepatic and fecal triglyceride, cholesterol, and FA contents were analyzed with the same enzymatic kits that were used for the plasma analysis.
The protein concentrations were determined using the Bradford method.
Phosphoenolpyruvate carboxykinase (PEPCK) activity was monitored in the direction of oxaloacetate synthesis using a spectrophotometric assay developed by Bentle and Lardy [8]. Fatty acid β-oxidation was measured spectrophotometrically by monitoring the reduction of NAD to NADH in the presence of palmitoyl-CoA as described by Lazarow [4], with a slight modification.

Analysis of Gene Expression
The liver were homogenized in the TRIzol reagent (Invitrogen, Grand Island, NY, USA), and total RNA was isolated according to the manufacturer's instructions. The total RNA was converted to cDNA using the QuantiTect Reverse Transcription kit (Qiagen Gmbh, Hilden, Germany). mRNA expression was quantified by quantitative real-time polymerase chain reaction (PCR) using the QuantiTect SYBR Green PCR kit (Qiagen) and SDS7000 sequence detection system (Applied Biosystems, CA, USA).
Each cDNA sample was amplified using primers for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene) labeled with SYBR green dye.
The amplification was performed as follows: 10 min at 90 °C, 15 s at 95 °C, and 60 s at 60 °C for a total of 40 cycles. The cycle threshold (Ct) was defined as the cycle at which a statistically significant increase in the SYBR green emission intensity occurred. The Ct data were normalized relative to those for the housekeeping gene, GAPDH, which is stably expressed in mice. Relative gene expression was calculated with the 2 ∆∆Ct method [9].

Primer
The primer were designed using a Primer 5.0 software (Primer-E Ltd., Plymouth, UK), SREBP1c

Statistical Analysis
The parameter values were expressed as the mean (standard error of the mean (SEM)). Significant differences between the ND and HFD groups were determined by student's t-test and significant differences among the HFD, SL and SLG groups were determined by one-way ANOVA using the SPSS program (SPSS Inc., Chicago, IL). Results were considered statistically significant at p < 0.05.