Beneficial Effects of Fennel ( Foeniculum Vulgare ) in Treating Obesity in Rats

Obesity is associated with a number of serious medical complications, which are often referred to as the “insulin resistance syndrome”. The aim of the present study was performed to investigate the possible interaction between a conventional drug used for management of cholesterol and traditional herbal remedies on the obesity. This was carried through out: through estimation of blood test; Estimation of serum tests; Determination of oxidative stress biomarkers and the antioxidant enzymes activities in the liver were assayed; Histopathological examination of the liver and kidney of adult male albino rats were done .In the present study, the serum levels of the total protein and albumin in the obesity group (7.1± 0.2) and (4.78 ± 0.19); respectively were significantly (p ≤ 0.05) more than those of the control group (6.5±0.1) and (3.95± 0.1).The administration of (fennel group) revealed significant (P<0.05) decrease in the serum levels of the albumin and total protein (4.38± 0.1) and (6.65± 0.2); respectively as compared to the obesity group (4.78 ± 0.19) and (7.1± 0.2). The total cholesterol of the group(5) (fennel and ator) after two weeks from a high fat diet than treatment with fennel and Ator through six weeks equal 142.86±5.9, 100.4±8.68, 93.29±5.99, 87.1±11.28, 80.4±21.55, 78.1±6.7 and 77.1±6.87; respectively. The present study showed a significant (P<0.05) increase in the activities of ALT, AST and ALP in the obesity group which recorded as (60.5±11.45), (57.25±6.3) and (845.0±49.47); respectively as compared to the control group (28.25±1.7), (38.5±3.87) and (537.0±41.5); respectively. The fennel group caused significant decrease in the activities of these enzymes (41.0± 2.9), (42.25+3.2) and (717.75+48.6); respectively compared to the obesity group. Ator group showed a significant decrease in the activities of these enzymes (40.0±2.16), (42.5±3.1) and (679.25±41.16); respectively compared as obesity group. The activity of AlT, AST and ALP in the fennel and ator group (32.75±2.5), (40.5±2.38) and (601.25±17.5); respectively were near to the control group. DOI: 10.14302/issn.2574-450X.jom-18-2484 Corresponding author: Eman H. Radwan, Department of Zoology, Faculty of Science, Damanhour University, Egypt, Email: dr_eman_hashem@yahoo.com


Introduction
The most common complications involve alterations in metabolic function that are risk factors for cardiovascular disease, namely hyperglycemia, hyper-insulinemia, and glucose intolerance, liver fat accumulation, dyslipidemia (increased serum triglyceride and decreased serum HDL-cholesterol concentrations; a predominance of smaller LDL particles and an increased number of large VLDL particles), and endothelial dysfunction [1].Fat accumulation in non-adipose tissues such as in the liver, skeletal muscles, heart appears to be a key feature distinguishing metabolically healthy from metabolically abnormal subjects.Obesity has deleterious effects on metabolic homeostasis, and affects numerous body organs [2,3].Lipids are essential for membrane synthesis, maintenance of membrane integrity, as an energy source, as hormone precursors, and as signaling molecules.Hyperlipidemia is usually characterized by elevated serum total cholesterol and decrease in High density lipoprotein (HDL) cholesterol and increase in Low density lipoprotein (LDL) cholesterols.It is observed that there is also an association between hyperlipidemia, and serum triglycerides [4].In the presence of severe hypertriglyceridemia or mixed hyperlipidemia, the combination of statins and fibrates is a possible treatment [5].Fennel is an aromatic herb belonging to the Parsley family.It is used as a spice and possesses a sweet taste that is similar to anise.Fennel seed shape is oval and has a strong scent, while the fruit have a slightly sweet and slightly spicy.Sweet fennel oil is made up predominantly of anethole (50 to 80%), fenchone, and estragole.The seeds also contain fiber and complex carbohydrates [6].

Material and Methods
Experimental animals and treatment: Adult male Albino rats weighing 150-160gm were obtained from the animal house of the Faculty agriculture, Alexandria University, Egypt.They were housed in a clean and well ventilated animal house with a constant 12 hr light and 12 hr dark schedule.They lived in five different groups (seven animals per group) at 25-27 o C.The animals were provided with standard diet and tap water was supplied.
They were acclimatized under laboratory conditions for one week before the experiment.Diets; Composition of the experimental diet (g/kg diet) was according to the formula of [7].It is including the normal diet for the control rats (fat 5%, carbohydrates 65%, proteins 20.3%, fiber 5%, salt mixture 3.7% and vitamin mixture 1%).The high fat diet contained fat 46%, carbohydrates 24%, proteins 20.3%, fiber5%, salt mixture 3.7% and vitamin mixture 1%.Normal and HFD; high fat diet constituents were purchased from El-Gomhoria Company, Cairo, Egypt.HFD was preserved at 4 o C until being used.Atorvastatin was obtained from local pharmacies, Cairo, Egypt and ground using a mortar.The drugs were administered orally once a day .
Fennel (Foeniculum vulgare) seeds were obtained and dried fennel seeds (Shamar) were washed with tap water to remove possible potential dust.Afterwards, it was dried by cotton cloth to remove the excess liquid prior to drying.Drying was achieved at room temperature for 48 hr.Then a grinder mill and sieves were used to obtain a powder particle size of less than 0.2mm [8].
Experimental design :Thirty five Male Albino rats were randomly divided into five groups; (7  Determination of serum total protein was done according to Gornall et al. [10].Determination of serum albumin was done according to Doumas et al. [11]. Determination of serum total cholesterol was done according to Allain et al. [12].Determination of serum triglycerides was done according to Fassati and Prencipe [13].Determination of serum high density lipoproteins (HDL) Cholesterol levels and Determination of serum low density lipoproteins (LDL) Cholesterol levels according to Fruchart [14].Determination of serum urea was done according to Fawcett and Soctt [15].Determination of serum creatinine was done according to Bartles et al. [16].Determination of alanine aminotransferase(ALT) and Determination of aspartate aminotransferase activity (AST) according to Tietz [17].
Determination of Glutathione peroxide (GPx) was done according to Paglia and Valentine [23].and (2.68±0.28)as compared to the obesity group.In the opposite direction, the uric acid level of group (5) showed significant decrease comparing to group (3) and group (4).(Table 7)

Effect of Ator, Fennel and Their Combination on the Histology of the kidney of the Obese Rats
The histological examination of the kidney of the control rats fed on a standard diet showing normal rounded capsules with normal Bowman's glomeruli, round proximal tubules and elongated distal tubules with high cuboidal cells (fig.1).The rats fed (HFD) showed fatty degeneration of the tubules with eosinophilic material deposition, glomerular atrophy with wide urinary space and distal tubules with extrusion of nuclei into lumen (fig.2).By comparison kidneys of rats which were treated by fennel after being obese and the control rats observed partial improvement in both Bowman ' s capsules and proximal tubules.Note the distal tubules show less focal fatty infiltration (fig.3).While examination of rats kidney that were treated by ator after obesity showed improvement in Bowman ' s capsules with normal glomerular and partial improvement in proximal tubules and distal tubules, (fig.4).Kidney of rats that were treated by fennel and ator after obesity and the control rats showed high improvement in the tissues with normal glomerular and that Most of Bowman's capsules and renal tubules, restoring their normal appearance (fig.5).

Effect of Ator, Fennel and Their Combination on the Liver of Obese Rats
The histological examination of the livers of control rats feeding standard diet showed normal architecture hepatocytes, blood sinusoid and central vein, (fig.6).The rats which were fed on a (HFD), showed swollen hepatocytes with vacuolated cytoplasm

Discussion
Obesity is caused by various environmental and genetic factors: one of the main environmental factors causing obesity is the intake of a HFD.In this study, high fat diet (HFD) caused significant increase in the body weight of the obesity animal, this result was in agreement with Galisteo et al. [25] observed that food intake in obese rats was significantly increased after 12 or 24 weeks.This means that the total energy intake was higher in obese rats than that in the lean control one.Consumption of high fat diet led to obesity and overweight because it facilitates the development of a positive energy balance leading to an increase in visceral fat deposition, this lead to abdominal obesity in particular [26].Schrauwen-Hinderling et al. [27] found that high fat diet feeding is accompanied by molecular adaptation that favors fat storage in muscle rather than oxidation.
In this study the body weight of the group which treated with fennel herb significantly decreases, this results agree with Schone et al. [28] reported that food intake in rats treated with either one of the fennel extracts.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.
Weight loss Fennel increases the metabolism of fats and sugars in liver and pancreas.It dissolves fat deposits in bloodstream also and allows it to be used as an energy source.These aspects combined with its natural diuretic effect and a reputation as an appetite suppressant makes it an excellent remedy for weight loss [29].In the present study, fennel reduced the body weight gain.
This may be due to several isoflavans constituents of fennel as phytoestrogens, which like estradiol, affect the serotonergic system, inhibiting serotonin re-uptake and thereby increasing the levels of serotonin in synaptic clefts, this enhances satiety and promote weight gain [30].
In the current study the rats which treated with atorvastatin significantly decrease the body weight, this result was agreement with Erdmann et al. [31] observed that the treatment of over-weight and obesity has been shown to reduce plasma cholesterol.In this study it was observed that combination of fennel and atorvastatin showed significant decrease in the body weight compared to other group and reach to the normal.In this study, high fat diet caused significant increase in the protein of the obese animal.In this study the protein of the group which treated with fennel herb significantly decreases .In current study, the rats which treated with atorvastatin significantly decrease the protein.In this study it was observed that combination of fennel and atorvastatin showed significant decrease in the protein compared to other group and reach to the normal .In the present study the high fat diet caused significant increase in the albumin of the obese animal.In this study the albumin of the group which treated with fennel herb significantly decreases.In current study, the rats which treated with atorvastatin significantly decrease the albumin.
In the present study, HFD caused significant decrease in platelet, this result was agree with Chen et al. [32] reported that the effects of naturally-gained obesity on hematological and biochemical parameters are scarce.Chronic inflammation affects many hematological parameters, including erythrocytes, leukocytes and platelets.It is now well-established that obesity results in a state of chronic low-grade inflammation [33] .In the current study, the rats which treated with fennel herb showed increase in the platelet count, this result was in disagreement with Kaur [34] who reported that Fennel seeds are also known to have     The increased serum LDL level in obese rats has been also recorded in high fat diet supplemented rats [35].This 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 [36].Griffin et al. [39] found that patients with obesity-related glomerulopathy (ORG), defined as glomerular disease in obese patients in the absence of other renal disease-associated conditions, develop proteinuria prior to the onset of azotemia [40].In the current study indicates that fennel significant decrease creatinine and uric acid.The present results were in agreement with Fasset et al. [41] who observed that atorvastatin 10 mg would significantly slow the rate of decline of kidney function with chronic kidney disease.
In the present study , the obesity rats indicates the increase in the liver function this results was agree with Choi [42] who reported that elevated serum hepatic enzyme activities may be associated with a high prevalence of fatty liver, which is frequently observed in obese humans.In the present study, the rats which treated with fennel herb indicates significant decrease in the liver function this results was in agreement with Ozbek et al. [43] who observed that herbal drugs and essential oils of fennel have hepato-protective effects.In the present study, the rats which treated with atorvastatin indicates significant decrease in the liver function this results was agree with Ali et al. [44] who showed that oral atorvastatin is capable of improving liver function enzymes (ALT, AST, and ALP) and cause a significant decrement in their serum levels after induction of hypercholesterolemia in rats [45][46][47][48][49].
Previous studies illustrated that LPO and oxidative stress can severely elevated in patients with diabetes and hyperlipidemia [50] and that atorvastatin can improve serum levels of liver enzymes by its antioxidant effect against the liver damage.
The current study indicates that the HFD increase in oxidative stress, these results are in agreement with Diniz et al. [50] who reported that 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.The present data revealed that the lipid peroxidation product (MDA) recorded significant elevation in obese rats when compared with the lean control one.In the present study, the rats which treated with fennel herb indicate significant decrease in the oxidative stress, this result agreed with Choi and Hwang [6] who reported that 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 per oxidative capacity of fennel constituents in its methanolic extract.
Augmentation of the antioxidant defense system and the anti-lipid per oxidative activity of aqueous fennel extract has been reported [51].
The anti-inflammatory and antioxidant activities of fennel have been reported by Choi and Hwang [6].
Anti-oxidant Fennel contains flavonoid anti-oxidants compounds like kaempferol and quercetin which helps eliminate harmful free radicals from the body, thus protecting from infection and aging.Fennel seeds are generally eaten for the taste but also very healthy owing to the nutrition value attached to it.Fennel is used for various health benefits that are derived from its anti-oxidants.In the present study, the rats which treated with atorvastatin indicates significant decrease in the oxidative stress, this results was agree with Koter et al. [52] who reported that atorvastatin showed a significant reduction in the MDA level.Medicinal plants can be used in the treatment of various diseases [53], Fennel seeds contain antioxidants as kaemoferol and quercetin that prevent degenerative reactions [54].
Many researchers reported that micro vesicularsteatosis occurred due to changes in enzyme levels of hepatocytes.They emphasized that it was caused by oxidative stress resulting in mitochondrial degenerations [55].The present results were confirmed with histological changes of feeding rats with high fat diet only, which showed vaculation of tunica media and narrowing in the lumen of aorta sections as well as congestion of cardiac blood vessel and hyalinosis of its wall.This result was confirmed by Szilvassy et al. [56] who indicated that although hyperlipidemia increases oxidative stress in the cardiovascular system, it renders the heart and the vasculature more susceptible to stress.
Ouwens et al. [57] identified that development of hyper-cholestremia which is one of the risk factors for cardio vascular diseases.

Conclusion
The high fat diet administration was associated with a highly significant increase in urea level as compared to the control group.Treatment with fennel, ator and fennel with ator following high fat diet administration exhibited a high drop in this elevation as compared to the obesity group and this indicated also that treatment with fennel with ator was near to the control value.On the other hand, fennel group and ator group supplement successes partially in improving the creatinine level in comparing with the obesity group.
And although fennel and ator group indicated significant more decrease in creatinine level as compared to group Conventional techniques of Paraffin wax sectioning and Haematoxylin and Eosin staining were used for histological studies [24].Immediately pieces of each tissue were fixed in formalin 10% solution for 12 hours, dehydrated through ascending grades of ethyl alcohol until they reached the absolute alcohol (1 hour), they were then transferred to xylol.Tissues were placed in a mixture of melted wax and xylol (1:1) for about 10 minutes and transferred to paraffin wax 56ċ (3 changes for about 2 hours).For sectioning, the paraffin blocks were mounted in a microtome where successive sections adhere to form a straight ribbon.A drop of distilled water was spread on clean slides on which a small section of the paraffin ribbon was float.The slide was hold high up over a Bunsen burner flame till the sections flatten out and firmly adhere to the slide.Slides were immersed in xylol for three minutes to dissolve the paraffin and then transferred to absolute alcohol for one minute to remove the xylol, sections were dehydrated by Effect of Fennel and Ator on Low Density Lipoprotein (LDL) and Ratio (holesterol/HDL) of Obese rats A significant increase was obCserved in serum low density lipoproteins LDL and Ratio cholesterol/HDL levels in obesity rats reaching a value of (151.57±29.88)and (7.3±1.59)respectively in compare with the control group (62.86±12.1)and (2.69± 0.81); respectively.Fennel treatment in combination with obesity group improved these elevations to be (44.7±12.96)and (2.29±0.5).Also treatment with ator drug gave a significant decrease these parameters (36.4±8.4) and (1.98±0.2) as compared to the obesity group.The treatment with fennel and ator showed a significant decrease in these parameters(33.1±5.15)    and (1.85±0.2) compared to group (3) and group (4).(Table6)Obesity administration was associated with a highly significant increase in urea level reaching (43.25±4.65)as compared to the control group (27.75±2.6).Treatment with fennel, ator, fennel with ator following obesity administration exhibited a high drop in this elevation to (30.5±3.1),(31.0±3.16) and (28.0±2.16);respectively as compared to the obesity group and this indicated that treatment with fennel with ator was near to the control value (27.75±2.6).Fennel group and ator group supplement successes partially in improving the creatinine level to be (1.0±0.09),(0.9±0.06); respectively in comparing with the obesity group (0.58+0.07).Although fennel and ator group indicated significant more decrease in creatinine level (0.78±0.0) as compared to group(3) and group(4) but increase than the control ( 0.58±0.07).The Uric acid level showed a significant increase, reaching (3.8±0.25) in obesity group as compared as to the control value (2.18+0.28).The administration of fennel, ator and fennel with ator for six weeks resulted in a partial improvement in the uric acid level (2.8±0.2),(2.98±0.2)

Figure 1 .
Figure 1.Photomicrogragh of kidney section of control rat showing normal rounded capsules with normal Bowman , s glomeruli, round proximal tubules and elongated distal tubules with high cuboidal cells , (H&E) (40X).

Figure 2 .
Figure 2. Photomicrogragh of kidney section of obese rat showing fatty degeneration of the tubules with eosinophilic matrial deposition, glomrerular atrophy with wide urinary space and distal tubules with extrusion of nuclei into lumen , (H&E) (40X).

Figure 3 .Figure 4 .
Figure 3. Photomicrogragh of kidney section of treated rat with fennel herb showing partial improvement in both Bowman ' s capsules and proximal tubules.Note the distal tubules show less focal fatty infiltration, (H&E) (40X)

Figure 5 .
Figure 5. Photomicrograph of kidney section of treated rat with both fennel herb and Ator drug showing highly improved tissue with normal glomerular.Note, most Bowman ' s capsules and renal tubules, restoring their normal appearance (star).(H&E) (40X).

Freely
Available Online www.openaccesspub.org| JOM CC-license DOI : 10.14302/issn.2574-450X.jom-18-2484Vol-1 Issue 2Pg.no.-27 filled with fatty infiltration, congested central vein and disappearance of blood sinusoids, (fig 7).By comparison, the liver of rats that were treated by fennel after obesity and control mice observed nearly normal of the hepatocytes with eosinophilic cytoplasm, central vein and clear blood sinusoids and more bi-nucleated cells, (fig.8).While examination of rats' liver that were treated by Ator and after the obesity appears, they showed mild fatty change in hepatocytes, few hepatocytes retain is eosinophilic cytoplasm and central vein (fig.9).Liver of rats that were treated by fennel and Ator after obesity and control rats are showing nearly normal hepatocytes (fig.10).

Figure 7 .
Figure 7. Photomicrograph of liver section of obese rat showing swollen hepatocytes with vacuolated cytoplasm filled with fatty infiltration, congested central vein and disappearance of blood sinusoids, (H&E) (40X).

Figure 8 .
Figure 8. Photomicrograph of liver section of treated rat with fennel herb showing nearly normal of the hepatocytes with eosinophilic cytoplasm, central vein (CV) and clear blood sinusoids and more bi-nucleated cells , (H&E) (40X).

Figure 9 .
Figure 9. Photomicrogragh of liver section of treated rat with Ator drug showing mild fatty change in hepatocytes, few hepatocytes retain is eosinophilic cytoplasm and central vein , (H&E) (40X).

Figure 10 .
Figure 10.Photomicrograph of liver section of treated rat both with both fennel herb and Ator drug showing nearly normal hepatocytes (H&E) (40X).

( 3 )
and group(4) but increase than the control.Uric acid level showed a significant increase in obesity group as compared as to the control value.The administration of fennel, ator and fennel with ator resulted in a partial improvement in the uric acid level as compared to the diabetic group .In the opposite direction the uric acid level of group(5) showed significant decrease comparing to group (3) and group(4).The data reported in the present work indicates that administration of high fat diet provoked a significant increase in serum ALT, AST and ALP.Treatment with (fennel) and (ator) induced a partial recovery but treatment by fennel with ator more improving the ALT, AST and ALP.The finding of this study indicates that the concentration of Malondialdehyde (MDA) and Myeloperoxidase (MPO) in liver homogenates of the fennel and ator group significant decrease than group (3) and group (4) and obesity group.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.

Table 1 .
Effect of Fennel and Ator on the body weight of obese male albino rats

Table 2 .
Effect of Fennel and Ator on serum total protein level and albumin level of obese male albino rats:Means with Common letters are not significant (Means with Different letters are significant(F,p: F and p values for ANOVA test, Significance between groups was done using Post Hoc Test (LSD).*: Statistically significant at p ≤

Freely Available Online www
.openaccesspub.org| JOM CC-license DOI : 10.14302/issn.2574-450X.jom-18-2484Vol-1 Issue 2Pg.no.-20 after four weeks from treatment of fennel and ator which indicated that the body weight of obese rats (279.57±5.5)had increased more than the control group (212.57±17.2).On the other direction, group (3), group (4) and group (5) were able to recover this weight than treatment with fennel only group (3) or ator only group(4).The rats which were treated with fennel and ator after six weeks were recorded in table (1) as (184.29±8.38)significant decrease as compared to the group (3) and group (4) (193.14±6.67)and (188.43±25.15);respectively.Group (3) and group (4) significantly decreased more than group (2) which was recorded as (289.57±7.46)and group (2) significantly increased compared to the control group (230.1±25.97).Effect of Fennel and Ator on Serum Total Protein Level and Albumin level of obese Male Albino Rats

Table 2 )
Effect of Fennel and Ator on the Cholesterol Level of the Obese Rats (5)le(3)showed cholesterol of the five experimental groups of animals after two weeks form recieving a high fat diet then treatment with fennel and Ator through six weeks.Total cholesterol of the group(5)(fennel and ator) after two weeks form a high fat diet

Table 3 .
Effect of Fennel and Ator on cholesterol of obese rats Means with Common letters are not significant (Means with Different letters are significant (F,p: F and p values for ANOVA test, Significance between groups was done using Post Hoc Test (LSD).*: Statistically significant at p ≤ 0.05.www.openaccesspub.org| JOM CC-license DOI : 10.14302/issn.2574-450X.jom-18-2484Vol-1 Issue 2Pg.no.-21 than treatment with fennel and Ator through six weeks equal 142.86±5.9,100.4±8.68,93.29±5.99,87.1±11.28,80.4±21.55,78.1±6.7 and 77.1±6.87;respectively.It is indicating a decrease in the cholesterol and reaching to the normal control level which was Effect of Fennel and Ator on Triglyceride of Obese Rats

Table 4 .
Effect of Fennel and Ator on triglyceride of obese ratsMeans with Different letters are significant (F,p: F and p values for ANOVA test, Significance between groups was done using Post Hoc Test (LSD).

Table 5 .
Effect of Fennel and Ator on high density lipoprotein (HDL) of obese rats:Means with Different letters are significant (F, p: F and p values for ANOVA test, Significant between groups was done using Post Hoc Test (LSD)*: Statistically significant at p ≤ 0.05.

Table 7 .
Effect of fennel and ator on serum kidney function tests in obese rats

Table 8 .
Effect of fennel and ator on serum liver function tests in obese rats