Abstract

BACKGROUND: The present study was designed to investigate the hypolipidaemic and hypoglycaemic effects of total flavonoids from seed residues of Hippophae rhamnoides L. (FSH) in a high-fat diet fed mouse model. Consumption of a high-fat diet (HFD) for 4 weeks caused a significant rise of serum total cholesterol in mice. These hypercholesterolaemic mice then were orally administrated with different doses of FSH (50, 100 and 150 mg kg⁻¹ body weight) and simvastatin (20 mg kg⁻¹ body weight) for another 12 weeks under continuous HFD feeding.

RESULTS: FSH administration markedly reduced total mouse body, liver, and epididymal fat pad weights. Serum total cholesterol and low density of lipoprotein-cholesterol levels were also significantly decreased by FSH treatment. Additionally, FSH significantly lowered total cholesterol and triglyceride concentrations in liver, and the results were corroborated by transmission electron microscope findings. The rise in serum glucose was significantly suppressed by FSH treatment while improving impaired glucose tolerance.

CONCLUSION: These results suggest that FSH possesses hypolipidaemic and hypoglycaemic properties in mice fed a high-fat diet and could be developed as a supplement in healthcare foods and drugs. Copyright © 2011 Society of Chemical Industry

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INTRODUCTION

Hippophae rhamnoides L. (sea buckthorn), of the Elaeagnaceae family, is a plant naturally distributed over Asia and Europe. It can be found growing widely in northern and western regions of China; locally known as ‘Shaji’. Containing many chemical compounds, such as flavonoids, carotenoids, tocopherols, sterols, lipids, ascorbic acid, tannins, etc.,1 all parts of this plant have been considered rich sources of bioactive substances with high medicinal and nutritional properties. Fruit, leaf and seed extracts, juice, pulp oil and seed oil have been reported to possess antioxidative, antibacterial, anti-inflammatory, immunomodulatory and radioprotective activities.2–5 Therapeutic efficacy has been demonstrated in atopic dermatitis, gastric ulcers, liver injury, wound healing, and cardiovascular diseases.6–13

Used in folk medicine, the oil of Hippophae rhamnoides L. occupies 12–13% of the seed.14 However, after oil extraction, a large amount of seed residue remains with bioactive substances, which may have potential therapeutic use in food supplements. In our previous study, the total flavonoids from seed residues of Hippophae rhamnoides L. (FSH) exhibited positive effects on blood lipid and glucose levels in different animal models, such as normal mice,15 rats with streptozotocin-induced diabetes,16 climacteric rats,17 and rats with hypertension induced by sucrose feeding.18 However, its efficacy in modulating blood lipid and glucose levels has not been investigated in depth, specifically the correlation between dyslipidaemia and hyperglycaemia. Although abnormal glucose metabolism defines type 2 diabetes mellitus (T2DM), the pathogenesis of T2DM are increasingly focused on disordered lipid metabolism.19 The presence of metabolic syndrome, characterised by impaired insulin sensitivity, hyperglycaemia, dyslipidaemia, abdominal obesity and hypertension, increases individual risk of T2DM and cardiovascular disease.20 In rodents, a high-fat diet has been shown to induce obesity and metabolic disorders that resemble the human metabolic syndrome.21 We used mice fed a high-fat diet to evaluate the anti-hyperlipidaemic and anti-hyperglycaemic effects of FSH.

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MATERIALS AND METHODS

Plant material and preparation of FSH

The seed residues of Hippophae rhamnoides L. were obtained from Inner Mongolia Yuhangren Hi-Tech Industrial Co., Ltd. (Hohhot, Inner Mongolia, China). The corresponding seeds were collected in Chifeng, Inner Mongolia, in November 2006, and authenticated by Associate Professor Hongqing Li, School of Life Science, East China Normal University, Shanghai. A voucher specimen (No.: Wang S.Y. 2 006 001) was deposited in the herbarium of East China Normal University (Shanghai, China). The preparation of FSH was as described by Pang et al.18 In brief, 100 g crushed powder of seed residues was boiled twice (6 h each) in 300 mL petroleum ether at 70 °C. The powder was air dried in a ventilated cabinet and then incubated with 700 mL L⁻¹ ethanol (600 mL) at 80 °C four times (2 h each) to extract crude total flavones. The crude total flavonoids were then applied to a D101 macroporous resin column (2 002 031; Cangzhou Bonchem Co., Ltd, Hebei, China) and eluted with ethanol from 300 mL L⁻¹ to 500 mL L⁻¹ at the speed of 2 mL min⁻¹. The fractions containing FSH were collected and dried in vacuum cabinet (model ZK-82A; Shanghai Laboratory Instrument Works Co., Ltd, Shanghai, China) at 4 °C. The purity of the resulting FSH was (699.2 ± 18.4) g kg⁻¹ determined by spectrophotometry as described by the Chinese Pharmacopoeia Committee.22

Animals

Male ICR mice weighing 18–20 g, were purchased from SIPPR/BK Lab Animal Co., Ltd. (Shanghai, China), and were housed in an environmentally controlled room with a 12-h light/12-h dark cycle at 22 ± 2 °C. All the mice were fed a standard chow diet (92 mL water, 221 g crude protein, 52.8 g crude fat, 52 g crude ash, 41.2 g crude fibre, 12.4 g calcium, 9.2 g phosphorus, 7.2 g mixture of dl-methionine and dl-leucine, 13.4 g lysine and 520 g nitrogen-free extract kg⁻¹) obtained from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and water ad libitum for a 1-week acclimatisation.

The licence number for experimental animals use was SYXK (SH) 2004-0001. All protocols for animal maintenance and handling were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.23

Experimental design

Hyperlipidaemia in mice was induced by the procedure of Huang et al.24 After acclimatisation, the mice were randomly divided into two groups: fed either the standard chow diet (normal control group, n = 10) or a high-fat diet (HFD, 150 g lard, 20 g cholesterol, 5 g bile salt and 825 g standard chow diet kg⁻¹). After 4 weeks of treatment, blood samples were obtained from the tail vein for assay of fasting serum total cholesterol (TC). Then the HFD treated mice were divided into five groups (10 mice each) according to the TC level. One served as high-fat diet control group, the others served as treatment groups. The two control group mice continued to receive their original diet of standard chow or a HFD, with the addition of equal-volume distilled water (vehicle). The treatment groups were fed a HFD with either simvastatin (Hangzhou MSD Pharmaceutical Co., Ltd, Hangzhou, China) at a dose of 20 mg kg⁻¹ body weight (BW) or with FSH at three different dose levels (50, 100 and 150 mg kg⁻¹ BW) by intragastric administration once a day for a subsequent 12 weeks. Body weights were recorded weekly; serum TC levels were analysed every other week. Upon completion of the experiment, all the mice were sacrificed after fasting for 12 h. Blood samples were collected and centrifuged for biochemical analysis. Liver tissue samples were fixed in 25 g L⁻¹ glutaraldehyde for transmission electron microscopy. Serum samples and isolated liver tissues were stored at − 80 °C until analysis.

Oral glucose tolerance test

After 11 weeks of administration of simvastatin or FSH, an oral glucose tolerance test was performed after an overnight fast of 12 h. An oral dose of distilled water, simvastain (20 mg kg⁻¹ BW) or FSH (50, 100 and 150 mg kg⁻¹ BW) was given to the respective groups, 30 min prior to oral glucose administration (2 g kg⁻¹ BW). Blood samples were obtained from the tail vein to monitor blood glucose (BG) levels at zero, 30, 60, and 120 min after oral glucose administration using MediSense Optium Xceed sensor and Optium blood glucose electrodes (Abbott Diabetes Care Inc., Alameda, CA, USA). The formula for determining the area under the curve (AUC in mmol L⁻¹ h) is:25

Biochemical analysis

Fasting serum TC, triglycerides (TG), glucose, and high density lipoprotein-cholesterol (HDL-C) levels were determined by using commercial kits purchased from Shanghai Kexin Biotech Institute (Shanghai, China). Low density lipoprotein-cholesterol (LDL-C, in mmol L⁻¹) was calculated with the Friedewald formula:26

Fasting serum insulin was measured by a double-antibody radioimmunoassay (RIA) method. Insulin sensitivity index (ISI) was calculated according to the formula given by Li and Pan:27

where FPG represents fasting plasma glucose and FINS represents fasting plasma insulin.

Liver tissue was homogenised with 9 g L⁻¹ cold saline on ice. After the 100 g L⁻¹ homogenates was centrifuged, the clear supernatant was collected for biochemical analysis.

Morphological examination

Small pieces of liver were prefixed with 25 g L⁻¹ glutaraldehyde and post-fixed with 10 g L⁻¹ osmium tetroxide and then embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined under a JEM-2100 transmission electron microscope (JEOL, Tokyo, Japan).

Statistical analysis

All data are expressed as mean ± SD. Statistical analysis was performed by one-way analysis of variance followed by Duncan’s multiple range test or Games–Howell using the SPSS program. Values with P < 0.05 were considered to be significant.

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RESULTS

Body, liver and fat weights

There was no obvious difference between the body weight of mice in the NC group and those in the HFD group, but the weights of the liver and epididymal fat pad were significantly increased by HFD feeding (P < 0.01) (Table 1). FSH showed a significant effect to control body weight, at the doses of 100 and 150 mg kg⁻¹ BW (P < 0.05). The high dose of FSH (150 mg kg⁻¹ BW) could decrease liver weight by 8.74% relative to HFD control group (P < 0.05). The epididymal fat pad weight also had a considerably reduction in the 50, 100 and 150 mg kg⁻¹ BW FSH treated groups with reductions of 23.87%, 20.90% and 24.11%, respectively (P < 0.05).

Table 1. Effects of FSH on body, liver and fat weights in high-fat diet fed mice

	Body weight (g)
Group	Initial	Final	Liver weight (g kg−1 BW)	Epididymal fat pad weight (g kg−1 BW)
Values are represented as mean ± SD (n = 10). * P < 0.05, and ** P < 0.01 compared with NC group; † P < 0.05 compared with HFD group.  NC, normal control; HFD, high-fat diet; Sim, simvastatin; FSH, total flavonoids from seed residues of Hippophae rhamnoides L.
NC	38.33 ± 2.38	40.94 ± 2.76	33.14 ± 1.75	20.89 ± 7.42
HFD	38.44 ± 2.15	41.58 ± 3.12	37.96 ± 2.23**	40.72 ± 9.60**
HFD + Sim20	38.78 ± 2.33	40.94 ± 3.06	37.13 ± 4.25*	39.87 ± 10.22**
HFD + FSH50	37.97 ± 3.13	38.63 ± 2.64	36.62 ± 3.26*	31.00 ± 7.62*,†
HFD + FSH100	37.27 ± 2.02	38.47 ± 3.96†	35.06 ± 3.53	32.21 ± 8.63**,†
HFD + FSH150	37.96 ± 2.05	38.02 ± 2.89†	34.64 ± 3.58†	30.90 ± 6.72*,†

Serum lipid profiles

The oral administration of FSH for 12 weeks showed a significant hypolipidaemic effect in the mice fed a high-fat diet (Table 2). In comparison to the NC group, the high-fat diet induced a significant elevation in serum TC, HDL-C and LDL-C to 2.14-fold, 1.33-fold and 15.07-fold, respectively. HFD also decreased the level of TG and HDL-C/TC by 0.48-fold and 0.62-fold (P < 0.01). Administration of FSH at 50, 100 and 150 mg kg⁻¹ BW produced a significant reduction in serum TC in comparison with the HFD control group (P < 0.05) by 13.56%, 12.47% and 12.99%, respectively. The 50 mg kg⁻¹ BW FSH treatment could significantly increase the HDL-C/TC level by 12.99% (P < 0.05), decrease the LDL-C level by 26.01% (P < 0.05). Simvastatin had only a slight effect on these parameters.

Table 2. Effects of FSH on serum lipid profiles in mice fed a high-fat diet

Group	TC (mmol L−1)	TG (mmol L−1)	HDL-C (mmol L−1)	HDL-C/TC	LDL-C (mmol L−1)
Values are represented as mean ± SD (n = 10). ** P < 0.01 compared with NC group; † P < 0.05 compared with HFD group.  NC, normal control; HFD, high-fat diet; Sim, simvastatin; FSH, total flavonoids from seed residues of Hippophae rhamnoides L.
NC	2.80 ± 0.34	1.87 ± 0.47	1.73 ± 0.21	0.62 ± 0.04	0.22 ± 0.17
HFD	6.02 ± 0.81**	0.90 ± 0.11**	2.31 ± 0.32**	0.38 ± 0.03**	3.30 ± 0.55**
HFD + Sim20	5.68 ± 0.85**	1.03 ± 0.20**	2.28 ± 0.24**	0.40 ± 0.06**	2.94 ± 0.78**
HFD + FSH50	5.20 ± 0.81**,†	1.14 ± 0.26**	2.25 ± 0.36**	0.43 ± 0.05**,†	2.44 ± 0.61**,†
HFD + FSH100	5.27 ± 0.86**,†	1.07 ± 0.12**,†	2.17 ± 0.42**	0.41 ± 0.05**	2.61 ± 0.62**
HFD + FSH150	5.24 ± 0.69**,†	1.05 ± 0.32**	2.22 ± 0.28**	0.43 ± 0.06**	2.54 ± 0.62**

Serum glucose, insulin and insulin sensitivity index

Serum glucose and insulin levels increased after the induction of a high-fat diet, with a simultaneous decrease of the ISI (P < 0.05 or P < 0.01) (Table 3). Lower serum glucose levels were seen in all treatment groups: 17.15% for Sim20 (P < 0.01), 22.46% for FSH50 (P < 0.01), 13.22% for FSH100 (P < 0.05), and 13.32% for FSH150 (P < 0.05), without a significant effect on insulin level and ISI compared to the HFD control group.

Table 3. Effects of FSH on serum glucose, insulin and insulin sensitivity index (ISI) in mice fed a high-fat diet

Group	Glucose (mmol L−1)	Insulin (µIU mL−1)	ISI
Values are represented as mean ± SD (n = 10). * P < 0.05, and ** P < 0.01 compared with NC group; † P < 0.05, and †† P < 0.01 compared with HFD group.  NC, normal control; HFD, high-fat diet; Sim, simvastatin; FSH, total flavonoids from seed residues of Hippophae rhamnoides L.
NC	5.72 ± 1.16	6.76 ± 1.27	− 3.62 ± 0.27
HFD	8.21 ± 0.90**	12.54 ± 3.93*	− 4.59 ± 0.26**
HFD + Sim20	6.80 ± 0.72*,††	13.14 ± 4.25**	− 4.44 ± 0.26**
HFD + FSH50	6.37 ± 0.81††	16.54 ± 5.77**	− 4.60 ± 0.41**
HFD + FSH100	7.12 ± 1.00**,†	13.50 ± 5.26*	− 4.50 ± 0.37**
HFD + FSH150	7.12 ± 1.00**,†	16.06 ± 10.31	− 4.59 ± 0.59**

Oral glucose tolerance test

After 11 weeks of treatment, an oral glucose tolerance test was performed (Fig. 1). Blood glucose levels at 0 min in HFD fed mice were higher than those in NC group (P < 0.01). A glucose challenge dramatically increased the blood glucose levels in HFD fed mice compared to those in NC group at 60 and 120 min time points (P < 0.01) suggesting a impaired glucose tolerance (IGT) state. FSH treated groups significantly prevented the blood glucose levels from rising, especially at 30 and 60 min time points (P < 0.05 or P < 0.01) (Fig. 1A). The Sim20 group showed a decreased blood glucose level at 30 min, but the rise at 120 min (P < 0.05) was not suppressed. The FSH50, FSH100 and FSH150 groups showed a reduction in AUC by 8.37%, 7.37% and 8.57%, respectively (P < 0.05) (Fig. 1B).

Figure 1. Blood glucose (A) and area under the curve (B) in oral glucose tolerance test. Values are represented as mean ± SD (n = 10). *P < 0.05, and **P < 0.01 compared with NC group; ^†P < 0.05, and ^††P < 0.01 compared with HFD group. NC, normal control; HFD, high-fat diet; Sim, simvastatin; FSH, total flavonoids from seed residues of Hippophae rhamnoides L.

Liver lipid levels

Liver TC and TG levels determined upon completion of the experiment (Table 4) showed a dramatic increase of 3.78-fold and 1.76-fold, respectively, in the HFD control group compared to the NC group (P < 0.01). FSH100 mice had a 36.18% decrease in hepatic TC compared to the HFD control mice (P < 0.01). All treatment groups significantly decreased the hepatic TG concentration: 20.14% for Sim20, 36.35% for FSH50, 40.23% for FSH100, and 34.58% for FSH150 (P < 0.01).

Table 4. Effects of FSH on liver lipid levels

Group	TC (mg mg−1 protein)	TG (mg mg−1 protein)
Values are represented as mean ± SD (n = 10). ** P < 0.01 compared with NC group; †† P < 0.01 compared with HFD group.  NC, normal control; HFD, high-fat diet; Sim, simvastatin; FSH, total flavonoids from seed residues of Hippophae rhamnoides L.
NC	0.019 ± 0.004	0.077 ± 0.020
HFD	0.072 ± 0.015**	0.135 ± 0.022**
HFD + Sim20	0.056 ± 0.015**	0.108 ± 0.022**,††
HFD + FSH50	0.052 ± 0.020**	0.086 ± 0.028††
HFD + FSH100	0.046 ± 0.014**,††	0.081 ± 0.020††
HFD + FSH150	0.054 ± 0.010**	0.088 ± 0.014††

Morphological results

We also observed the morphological effect of FSH on liver tissue by transmission electron microscopy. The nucleus (N), mitochondria (M), and rough endoplasmic reticulum (RER) were clearly observed in normal hepatocytes (Fig. 2A). In the hepatocytes of HFD control mice, the gathering and excess lipids droplets (L) were observed (Fig. 2B); however, all treatment groups showed improvement in the altered structure with a specific decrease in the lipid deposition (Fig. 2C–F).

Figure 2. Transmission electron micrographs of hepatocytes from (A) NC group, (B) HFD control group, (C) HFD + Sim20 group, (D) HFD + FSH50 group, (E) HFD + FSH100 group and (F) HFD + FSH150 group. N, nucleus; M, mitochondria; RER, rough endoplasmic reticulum; L, lipid droplet; NC, normal control; HFD, high-fat diet; Sim, simvastatin; FSH, total flavonoids from seed residues of Hippophae rhamnoides L.

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DISCUSSION

The current rise in prevalence of T2DM and the metabolic syndrome is believed to be a result of increasingly sedentary lifestyle combined with ingestion of energy-rich food in genetically susceptible individuals.19 In the present study, ICR mice fed a high-fat diet (HFD) developed a hyperlipidaemic state associated with visceral obesity, hyperglycaemia and hyperinsulinaemia. Treatment with FSH, the total flavonoids from seed residues of Hippophae rhamnoides L., had the ability to suppress the development of hyperlipidaemia and hyperglycaemia in spite of continued access to the HFD.

High serum cholesterol, particularly low density lipoprotein cholesterol is a major risk factor for cardiovascular disease.28 Excess LDL in the blood can deposit in the walls of blood vessels and become a major component of atherosclerotic plaque lesions. Another risk factor for developing atherosclerosis is the reduced serum high density lipoprotein cholesterol level. HDL carries cholesterol and cholesterol esters from the peripheral tissues and cells to the liver for metabolism, and plays a very important role in inhibiting atherosclerotic plaque formation.29 Thus, regulating the serum cholesterol level is important in atherosclerosis prevention. In this animal model, HFD feeding induced the remarkably increase in serum TC and LDL-C level, with a decrease in HDL-C/TC radio (shown in Table 2). We showed FSH administration lowers the elevated serum TC and LDL-C levels while elevating HDL-C/TC suggesting FSH may be helpful to decrease the incidence of cardiovascular disease.

Impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) represent intermediate states of abnormal glucose regulation that exist between normal glucose homeostasis and diabetes; most individuals with these pre-diabetic states eventually develop diabetes.30 As shown in Table 3 and Fig. 1, a long-term HFD induced an elevated serum glucose level and impaired glucose tolerance, abnormalities which could be reversed by FSH supplement, suggesting that FSH may have an effect in preventing the development of diabetes. In a previous report from our laboratory, FSH exerted antihypertensive effects at least in part by improving insulin sensitivity;18 however, no significant alterations were observed in serum insulin level and insulin sensitivity index (ISI) after 12 weeks administration of FSH. Further investigation is needed to illustrate the possibility.

In this experiment, HFD feeding did not lead to obvious weight gain, though increases in epididymal fat pad and liver weights, as well as liver TC and TG levels (shown in Tables 1 and 4, and Fig. 2) were demonstrated. Individuals with central obesity have an increased risk of metabolic and cardiovascular complications, whereas those with peripheral obesity are at low risk. The release of free fatty acids (FFAs) from stored triglycerides occurs at a higher rate in visceral adipocytes compared to those of the subcutaneous fat resulting in high FFA concentrations delivered to the liver via the portal vein.31 Elevated FFA might cause the accumulation of fat depots in liver, skeletal muscle and pancreas, and could interfere with metabolic signalling in these tissues. In muscle, high FFA concentrations appear to decrease insulin-mediated glucose transport by modifying upstream signalling events. In liver, raised FFAs contribute to resistance to the action of insulin by stimulate endogenous glucose production. Chronically raised FFAs also have a lipotoxic effect on the pancreas, and lipid accumulation in beta cells might lead to reductions in insulin secretion.19, 31 These are collectively associated with the development of hyperglycaemia. Besides, the accumulation of TG in liver by high FFA concentrations also lead to non-alcoholic fatty liver disease (NAFLD).32 In this investigation, we found FSH treatment could reduce the weight of the epididymal fat pad, liver and decrease liver lipid levels implying FSH may prevent excess lipid accumulation in tissues and favourable glucose homeostasis. Therefore, we suppose that the hypoglycaemic effect of FSH, of suppressed serum glucose rise and improve IGT, may partially be explained through prevention of visceral obesity.

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CONCLUSION

The study demonstrated FSH exerted beneficial effects on glucose and lipid homeostasis. FSH not only reduced lipid levels in HFD fed mice, but also corrected associated hyperglycaemia, potentially by preventing visceral obesity. Further studies are required to reveal the precise mechanisms of its hypolipidaemic and hypoglycaemic activities.

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Acknowledgements

This work was supported by a science foundation grant to Weijing Qu from the Science and Technology Commission of Shanghai Municipality (07DZ12043).

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