Regulation of Obesity and Lipid Disorders by Foeniculum vulgare Extracts and Plantago ovata in High-fat Diet-induced Obese Rats

INTRODUCTION

Obesity is the result of an energy imbalance caused by an increased ratio of caloric intake to energy expenditure. In spite of the number of studies to prevent or treat obesity, its prevalence continues to rise and it has become one of the major threats to health throughout the world (Lopez-Jimenez and Cortes-Bergoderi, 2011). In fact, the dysregulated energy homeostasis stems from a societal reduction in physical activity, energy-dense foods, combined with a myriad of genetic, social and economic complicating factors (Mitchell et al., 2005). High fat diet is certainly of importance to obesity incidence and to its negative consequences such as type II diabetes (Samaras and Campbell, 2000), dyslipidemia (Novelli et al., 2007), cardiovascular disease (Diniz et al., 2005), aging (Hart et al., 1999) and cancer (Bianchini et al., 2002). Adipose tissue has been considered as a highly specialized, endocrine and paracrine organ producing an array of mediators called adipokines. One of the most important adipokines is leptin which significantly correlates with the amount of fat tissue in humans and animals (Maffei et al., 1995). Ordinary, leptin functions to reduce food intake and maintain energy homeostasis but in obesity, the development of a state of leptin resistance results in a dysfunctional energetic state (Sahu, 2004).

Oxidative stress was found to be associated with obesity. There is a growing awareness that obesity is a prime risk factor for the development of dyslipidemic profile and that oxidative stress may play a role in various adverse effects of obesity (Diniz et al., 2005). Pharmacologic therapy used for the treatment of obesity is formulated to reduce energy intake or increase energy output (Fujioka, 2002). However, the effectiveness and tolerableness of these medications are limited because they have well recognized potential side effects (Sindler, 2001). American Association of Clinical Endocrinologists reported that natural therapies for obesity may be beneficial but studies were needed to prove their safety and efficacy (AACE/ACE obesity Task Force, 1998).

Foeniculum vulgare (Fennel) fruits and their essential oil are used as flavoring agents in food products such as liqueurs, bread, pickles, pastries and cheese. They are also used as a constituent of cosmetic and pharmaceutical products (Piccaglia and Marotti, 2001). The anti inflammatory and antioxidant activities of fennel have been reported (Choi and Hwang, 2004). There are three species of Plantago, Plantago ovata, Plantago psyllium and Plantago indica. The seeds of this plant are used for medical purposes from many thousand years (Kritchevsky et al., 1995). It has been reported that obese patients treated with a rational diet supplemented with 15 g Plantago psyllium for two months lost their weight significantly compared with the untreated obese ones (Moran et al., 1997). It has been demonstrated that Plantago ovata husk-supplemented diet prevents endothelial dysfunction, hypertension and ameliorates dyslipidemia and hyperinsulinemia in obese Zucker rats (Galisteo et al., 2005).

The current study was designed to evaluate the therapeutic potential and the possible mode of action of Foeniculum vulgare (fennel) extracts as well as Plantago ovata (Plantago) in the treatment of obesity in adult female rats. This goal could be achieved through using anthropometrical parameters and testing the hypothesis that the anthropometrical index may predict obesity adverse effects on lipid profile and oxidative stress and may assess the effectiveness of the herbal therapy in the treatment of obesity in rats.

MATERIALS AND METHODS

A: Phytochemical study
Plant materials: Foeniculum vulgare fruits and Plantago ovata seeds were obtained from local market at Harraz Herbal Drugstore (2 Cairo, Egypt) in September 2010 and identified by Prof. Dr Ibrahem Elgarf, Department of Botany, Faculty of Science, Cairo University, Cairo, Egypt. A voucher specimen has been deposited at the Herbarium of the National Research Centre, Cairo, Egypt.

Preparation of fennel extracts and Plantago seeds:

•	Methanol extract, 3 kg powder of fennel fruit was mixed five times with 5 L methanol. Extraction continued until the extraction solvents became colorless. The obtained extracts were filtered over Whatman No. 1 paper and the filtrate was collected, then methanol was removed by a rotary evaporator at 50°C to give 150 g
•	Water extract, 3 kg of fennel fruit was grounded into a fine powder in a mill and mixed with 5 L boiling water obtained using an ultrasonic bath for 1/2 h to ensure an exhaustive extraction. Then the extract was filtered over Whatman No. 1 paper. The filtrate was frozen and lyophilized to give 250 g
•	Essential oil was obtained by hydrodistillation of the powdered dry fennel fruits (1 kg) according to Shahat et al. (2011). The oil phase was separated, dried over anhydrous sodium sulfate and kept in a dark glass bottle at 4°C until the analyses and biological evaluation
•	Plantago ovata seeds are grounded using the electric blender and added on the pulverized standard diet of rats in a dose of 35 g kg^-1 diet

B: Biological study: This study was conducted in accordance with the principles and guidelines of the Ethical Committee for animal care and protection of the National Research Centre, Egypt.

Animals and experimental protocol: The present study was conducted on sixty adult female sprague Dawley rats weighing 130±10 g at 90 days of age obtained from the animal house colony of the National Research Centre, Cairo, Egypt in October 2010. The animals were housed 5/cage in polypropylene cages in an environmentally controlled clean air room with a temperature of 24±1°C, a 12 h light/12 h dark cycle, a relative humidity of 60±5% and free access to tap water and food. Rats were allowed to adapt to these conditions for two weeks before beginning the experimental protocol.

After the acclimatization period, ten rats were given water ad libitum and were fed a standard rodent chow with 26.5% protein, 3.8% fat, 40% carbohydrate, 4.5% crude fibre in 100 g of chow during 24 weeks of the experimental period and served as lean control group. The other fifty rats were received water ad libitum and were fed a High-fat Diet (HFD) with 19.93% protein, 15.39% fat, 57.50% carbohydrate, 2.81% dietary fibre in 100 g of chow. The dietary ingredients were homogenized in distilled water at 60°C and the homogenate was used to prepare the pellets. Diets were given fresh each day as dry pellets; therefore there was no spillage (Novelli et al., 2007). These rats were randomly assigned to five groups (10 rats group^-1) as follows; group one in which the rats were fed a HFD diet, for induction of obesity (Soliman et al., 2012), for 24 weeks and served as obese control group, the groups two and three in which the rats were fed a HFD diet for 24 weeks but at the 12th week, they were orally administered with fennel methanolic extract in a dose of 200 mg kg^-1 b.wt., fennel aqueous extract in a dose of 300 mg kg^-1 b.wt. by Choi and Hwang (2004) and Birdane et al. (2007), respectively daily for 12 weeks. Group four in which the rats were fed a HFD diet for 24 weeks but at the 12th week the HFD was supplemented with 100 mg fennel oil kg^-1 diet (Schone et al., 2006) and the rats were fed this fennel oil supplemented diet for the other 12 weeks and group five in which the rats were fed a HFD diet for 24 weeks but at the 12th week, the HFD diet was supplemented with grounded Plantago ovata seeds in a dose of 35 g kg^-1 diet (Galisteo et al., 2005) and the rats were fed this Plantago ovata-supplemented diet for the other 12 weeks. Food intake was measured after 12 weeks and also after 24 weeks i.e., at the end of the experiment at the same time (09:00-10:00 h).

Anthropometrical determinations: The Abdominal Circumference (AC) (immediately anterior to the forefoot), Thoracic Circumference (TC) (immediately behind the foreleg), body length (nose-to-anus or nose-anus length) were determined in all rats at the end of the experimental period (24 weeks). The measurements were made in anaesthetized rats (0.1 mL intraperitoneally of 1% sodium barbiturate). The body weight and body length were used to determine the body mass index (Novelli et al., 2007):

Biochemical determinations: After obtaining anthropometrical measurements, rats were fasted overnight (12-14 h) and the blood samples were withdrawn, under diethyl ether anaesthesia, from the retro-orbital pleux in a clean dry centrifuge tubes and allowed to clot to obtain the sera. Serum samples were separated by centrifugation at 800 xg for 10 min at 4°C. Aliquots of serum were frozen and stored at -20°C for further determinations of biochemical markers.

Serum Total Cholesterol (TC) and Triglycerides (TG), High Density Lipoprotein (HDL) and Low Density Lipoprotein (LDL) were assayed by enzymatic method using StanBio Laboratory kits (Boerne, Texas, USA) according to Allain et al. (1974) Fossati and Prencipe (1982), Lopes-Virella et al. (1977) and Assmann et al. (1984) methods respectively. Serum malondialdehyde (MDA) was determined by colorimetric methods using Biodiagnostic kit (Egypt) according to the method described by Satoh (1978). Serum insulin was estimated by enzyme linked immunosorbent assay (ELIZA) procedure using DRG kit (Germany) according to the method of Temple et al. (1992). Serum glucose was measured by colorimetric method using Biodiagnostic kit (Egypt) according to the method described by Trinder (1969). Serum leptin was assayed by ELIZA technique using DRG kit (Germany) according to Considine et al. (1996) method.

Statistical analysis: The statistical analysis was performed using SPSS/PC statistical program (version 11.0 for Windows; SPSS, Inc). Descriptive data were expressed as Mean±SE. Differences between each two groups were analyzed by the student unpaired t-test. A probability of 0.05 was chosen as the significant level (Steel and Torrie, 1984).

RESULTS

This study was constructed to evaluate the anti-obesity influence of fennel methanolic, aqueous and oil extracts as well as Plantago seeds in high fat diet-fed rats. Table 1 illustrates the results of average food intake in the different studied groups after 12 and 24 weeks from starting the experiment. It was observed that there was significant increase (p<0.01) in average food intake of obese rats after 12 or 24 weeks when compared with that in lean control rats.

Table 1:	Effect of fennel extracts and Plantago seeds on the average food intake in female rats after 12 and 24 weeks

Values are Mean±SE for 8 animals group^-1, ^,*Significant at p<0.05 and p<0.01, respectively

In contrast, the average food intake of obese rats treated with either one of fennel extracts or those treated with Plantago seeds showed significant decrease (p<0.01) after 12 or 24 weeks when compared with that of obese rats at each time interval.

The results of the current study revealed that there was significant increase in Thoracic Circumference (TC) (p<0.01) and Abdominal Circumferences (AC) (p<0.05) of obese group that supplemented with HFD when compared with lean control one kept on standard rodent chow. However, there was significant decrease (p<0.01) in TC of obese rats treated with either fennel methanolic extract or fennel oil extract when compared with obese control rats. Also, Plantago seeds-treated group showed significant decrease (p<0.05) in TC when compared with obese control group. Significant decrease (p<0.05) in AC was detected only in obese rats treated with fennel oil extract. Meanwhile, obese rats treated with Plantago seeds showed insignificant decrease (p>0.05) in AC when compared with AC of obese control rats. Body Mass Index (BMI) of obese group showed significant increase (p<0.01) when compared with that of lean control group. However, significant decrease (p<0.01) in BMI was recorded in obese groups treated with each one of the three fennel extracts and also in a group of rats treated with Plantago seeds when compared with BMI of obese control group (Table 2).

The results depicted in Table 3 showed the effect of treatment with either one of fennel extracts or Plantago seeds on lipid profile of obese female rats. The data revealed that there was significant increase (p<0.01) in serum cholesterol and LDL levels in obese rats compared with lean control rats. Triglycerides showed insignificant increase (p>0.05) in obese rats as compared with that in lean control rats. While, serum HDL level recorded significant decrease (p<0.01) in obese rats when compared with lean control rats (Table 3). However, treatment of obese rats with either one of fennel extract or with Plantago seeds resulted in significant decrease (p<0.01) in serum cholesterol level when compared with that in obese control rats. Serum LDL level revealed significant decrease (p<0.05) only in rats treated with either fennel methanolic extract or Plantago seeds when compared with that in obese control rats. Triglycerides level in serum of the all treated groups showed insignificant decrease (p>0.05) when compared with that in untreated obese control rats. Serum HDL level recorded significant increase only in rats treated with fennel methanolic extract (p<0.01) and in those treated with Plantago seeds (p<0.05) as compared with that recorded in obese control rats (Table 3).

The data illustrated in Table 3 also revealed that serum lipid peroxide level represented by malondialdehyde (MDA) recorded significant increase (p<0.01) in obese rats compared with lean control rats. On the other hand, treatment of obese rats with fennel methanolic extract or fennel aqueous extract or fennel oil extract or even Plantago seeds produced significant decrease (p<0.01) in serum MDA level as compared with that in untreated obese control rats (Table 3).

Table 2:	Effect of fennel extracts and Plantago seeds on anthropometric parameters of female rats

Values are Mean±SE for 8 animals group^-1, ^,*Significant at p<0.05 and p<0.01, respectively

Table 3:	Effect of fennel extracts and Plantago seeds on lipid profile of female rats

Values are Mean±SE for 8 animals group^-1, ^,*Significant at p<0.05 and p<0.01, respectively

Table 4:	Effect of fennel extracts and Plantago seeds on biochemical parameters of female rats

Values are Mean±SE for 8 animals group^-1, ^,*Significant at p<0.05 and p<0.01, respectively

Serum insulin level showed significant increase (p<0.01) in obese rats compared with lean control rats. Significant decrease (p<0.05) in serum insulin level was detected only in obese rats treated with fennel methanolic extract. Similarly, obese rats treated with Plantago seeds showed significant decrease (p<0.05) in serum insulin level compared with that in obese control rats (Table 4).

Serum glucose level showed significant increase (p<0.01) in obese rats as compared with that in lean control rats. In contrast, significant decrease (p<0.01) in serum glucose level was recorded only in obese rats treated with fennel methanolic extract. Similarly, significant decrease (p<0.01) in serum glucose level was demonstrated in obese rats treated with Plantago seeds as compared with that in obese control rats (Table 4).

Concerning serum leptin level, obese rats showed significant increase (p<0.01) compared with serum leptin level in lean control rats. However, serum leptin level showed significant depletion in obese rats treated with fennel methanolic extract (p<0.01) or with fennel aqueous extract (p<0.05) or with fennel oil extract (p<0.05) or even with Plantago seeds (p<0.05) when compared with that in obese control rats (Table 4).

DISCUSSION

In the present study, the potential effects of fennel methanolic, fennel aqueous and fennel oil extracts as well as Plantago seeds on food intake, anthropometric measures, lipid profile, oxidative stress marker, insulin, glucose and leptin circulating levels in obese rats were investigated.

The present data revealed that food intake in obese rats was significantly increased after 12 or 24 weeks, this finding is in agreement with that of Galisteo et al. (2005). This means that the total energy intake was higher in obese rats than that in the lean control one. This indicates that diet is certainly of importance to obesity incidence. The diminished food intake in rats treated with either one of the fennel extracts agreed with the previous report of Schone et al. (2006). In this respect, it can be proposed that trypsin inhibitors in fennel reduce food intake and stimulate cholecystokinin release, increasing satiety and that is the reason for fennel's association with weight control. Regarding the effect of Plantago seeds on reducing food intake in obese rats, Galisteo et al. (2005) attributed this effect to the reduction in ghrelin production and also to the low energy intake of Plantago seeds due in part to dilution of nutrients by fiber incorporation.

The present data revealed that both TC and AC were significantly increased in obese rats. In addition, BMI showed significant increase in obese rats when compared with lean control rats. These results indicate that there was fat accumulation in the thoracic and abdominal regions due to high fat diet (Novelli et al., 2007). This means that the increased body weight due to excessive energy intake was the adipose tissue. There were positive correlations between daily lipid intake and BMI as well as fat deposition (Rodrigues et al., 2012) demonstrating that BMI is a simple reliable estimate of body fat and obesity in rats (Novelli et al., 2007). Treatment of obese rats with the different fennel extracts, particularly fennel oil extract, resulted in a reduction in both TC and AC. Methanolic extract of fennel and Plantago seeds exerted their reducing effects on TC only. In general, treatment with the different fennel extracts as well as Plantago seeds exhibited significant reduction in BMI of obese rats. These findings indicate that fennel extracts could reduce body fat percentage in obese rats. This might be explained by the capacity of fennel active ingredients to reduce the expression of sterol regulatory element binding protein 1 which inhibits lipid accumulation and expression of lipogenic genes, resulting in the reduction in body fat. In addition, promoting satiation and altering secretion of gut hormones might be the possible mechanisms involved in weight loss and BMI regulation associated with the treatment with fennel extracts in obese rats (Schone et al., 2006). The observed reduction in TC and BMI of obese rats treated with Plantago seeds was due, at least in part, to a reduced food intake. The weight reducing effect of Plantago was previously asserted by Galisteo et al. (2005).

The current results revealed significant increase in serum cholesterol and LDL levels accompanied with insignificant increase in serum triglycerides level in obese rats. While, significant depletion in serum HDL level was recorded in obese rats as compared to lean control rats. Hypercholesterolemia and hypertriglyceridemia have been previously detected in obese rats (Son et al., 2012). It has been demonstrated that lipids in adipose tissue are largely derived from circulating triglycerides especially during high-fat diet feeding (Fruchart et al., 1998) and the reduction in serum triglycerides leads to decreased adipose tissue mass (Jeong et al., 2004). The increased serum LDL level in obese rats has been also recorded in high fat diet supplemented rats (Novelli et al., 2007). This fact was explained by the decreased HDL level, as recorded in our study, thus decreasing the reverse cholesterol transport from the blood stream to the liver (Raveh et al., 2001). Different fennel extracts and Plantago seeds used in the present study could reduce serum cholesterol level in obese rats. Nonappreciable reduction in serum triglycerides level was recorded in obese rats treated with either one of fennel extracts or with Plantago seeds. Fennel methanolic extract and Plantago seeds treatment could significantly decrease serum LDL level and increase serum HDL level in obese rats. The other fennel extracts could insignificantly reduce LDL level and rise HDL level in obese rats. These results indicated that the constitution of fennel and Plantago seeds plays an important role in improving blood lipid profile. This could be explained as fennel methanolic extract (Choi and Hwang, 2004) and Plantago seeds (Ziai et al., 2005) could significantly increase HDL level. This type of lipoprotein could stimulate the reverse cholesterol transport from the blood stream to the liver (Raveh et al., 2001). Furthermore, it has been shown that fennel, involved in herbal formulation, could delay upper gastrointestinal transit which promotes a decrease in fat and sugar absorption (Capasso et al., 2007). Regarding the hypolipidemic effect of Plantago seeds, our results are in agreement with those of Galisteo et al. (2005). The possible hypocholesterolemic mechanisms of Plantago were related to decreased cholesterol absorption or inhibition of the enterohepatic circulation of bile acids, due to the physicochemical properties of Plantago and also to the increasing meal viscosity (MacMahon and Carless, 1998). The hypotriglyceridemic effect of Plantago is consistent with a possible delay in the absorption of triglycerides and sugars from the small intestine (Ebihara and Schneeman, 1989).

The present data revealed that the lipid peroxidation product (MDA) recorded significant elevation in obese rats when compared with the lean control one. This result agrees with that of Prasanna and Purnima (2011). The increased MDA level in obese rats could be explained as the increased caloric intake represents an important factor in decreasing the mitochondrial membrane fluidity and increasing the generation of ROS (Esposito et al., 1999). The study of Novelli et al. (2007) revealed that there was a positive correlation between BMI and lipid peroxidation products concentration. The significant reduction in serum MDA level observed in obese rats treated with either one of the fennel extracts could be attributed to the anti-lipid peroxidative capacity of fennel constituents in its methanolic extract (Choi and Hwang, 2004). Augmentation of the antioxidant defense system and the anti-lipid peroxidative activity of aqueous fennel extract has been also reported (Birdane et al., 2007). The efficacy of fennel oil extract to decrease the percentual feed intake in pigs (Schone et al., 2006) and in rats as shown in our study indicated that fennel oil could reduce the production of ROS and in turn oxidative stress indirectly via decreasing the caloric intake. Treatment of obese rats with Plantago seeds resulted in significant reduction in serum MDA level. The effect of Plantago in lowering food intake and energy intake in obese rats has been previously reported (Galisteo et al., 2005). This effect of Plantago could be responsible for the depletion of oxidative stress and lipid peroxidation product (MDA) observed in the current study.

Our results revealed that there was significant increase in serum insulin level in obese rats compared with that in lean control one. This finding is in agreement with that of Galisteo et al. (2005). This could be explained as obesity is associated with low-grade chronic systemic inflammation which potentially leads to insulin resistance (Mori et al., 2011). Treatment of obese rats with fennel methanolic extract significantly decreased serum insulin level compared with that in obese control rats. This result could be explained via the powerful antiinflammatory action of fennel methanolic extract (Choi and Hwang, 2004). This property contributed in reducing the proinflammatory cytokine production and promoting the antiinflammatory mediators. In this way, fennel methanolic extract could reduce hyperinsulinemia in obese rats. Plantago seeds supplementation has been found to reduce serum insulin level significantly in obese rats. This result agreed well with that of Galisteo et al. (2005) who explained this phenomenon by the ability of Plantago seeds to reduce food intake and ghrelin concentration in obese rats.

The present results showed that obese rats recorded significant elevation in serum glucose level. This finding is in agreement with that in the study of Galisteo et al. (2005). This result could be explained by the presence of high lipolytic activity in fat accumulation that results in high free fatty acids mobilization to the liver. The elevated fatty acids flux to liver accelerates gluconeogenesis and decreases the effect of insulin on peripheral glucose disposal (Ginsberg and Stalenhoef, 2003). The ability of fennel methanolic extract to reduce serum glucose level in obese rats could be attributed to the effect of this extract in lowering fat accumulation and hence improving the glycemic status of obese rats via reducing the gluconeogenesis process and elevating the efficacy of insulin on glucose disposal. Obese rats supplemented with Plantago seeds recorded significant reduction in serum glucose level in comparison with that in obese control one. Plantago has been found to have glucose lowering effect in serum via its ability to inhibit glucose absorption from the small intestine (Hannan et al., 2006). Additionally Plantago could reduce the concentration free fatty acids which promote gluconeogenesis and inhibit the effect of insulin on peripheral glucose disposal (Galisteo et al., 2005).

Serum leptin level showed significant increase in obese rats as compared with lean control one. Leptin is a cytokine like polypeptide produced by the adipocytes and it is overproduced during obesity (Assal et al., 2007). The observed significant lowering effect of either fennel methanolic, aqueous or oil extract or even Plantago seeds on serum leptin level in obese rats indicates that both fennel and Plantago seeds have an ability to reduce fat mass in the treated groups. Thus, the reduction in fat mass, due to fennel administration (Choi and Hwang, 2004) or Plantago seeds supplementation (Ziai et al., 2005), attenuates the proinflammatory environment associated with obesity (Cottam et al., 2002) leading to depletion of leptin production and hence serum leptin level.

CONCLUSION

In conclusion, the present study showed that the prolonged intake of fennel extracts or Plantago seeds-supplemented diet retards obesity. The effect that was accompanied by improvement of BMI, amelioration of the dyslipidemia, hyperinsulinemia and hyperleptinemia, modulation of glycemic status and reduction of the oxidative stress in high-fat diet-induced obesity in rats. The observed effects of fennel extracts or Plantago seeds could be attributed to their properties such as hypolipidemic, antioxidant and antiinflammatory. Thus, fennel extracts, particularly, methanolic extract and Plantago seeds may be useful for treating obese patients with hypercholesterolemia and hypertriglyceridemia. Further studies will be necessary to identify and characterize active compounds responsible for these effects.