Effect of diet treatments on body weight food consumption
Obesity is considered to be a disorder of energy balance, occurring when energy expenditure is no longer in equilibrium with daily energy intake, so as to ensure body weight homeostasis [
44]. Although the etiology of obesity is complex, dietary factors, particularly the consumption of a HFD, is considered a risk factor for its development [
45].
The current results showed that body weight increased significantly in the HFD group compared with the normal group (Figure
1), a result in accordance with that of
Xu et al [
46]; this is associated with increased food intake.
Consumption of the HFD led to obesity because it facilitates the development of a positive energy balance leading to an increase in visceral fat deposition; this led to abdominal obesity in particular. Moreover,
Schrauwen-Hinderling et al [
47] found that HFD feeding is accompanied by molecular adaptations that favor fat storage in muscle rather than oxidation.
In the current study, rats fed HFD consumed considerably more food than the control rats throughout the experiment (Table
2 and Figure
3). So their caloric intake was increased and they showed a large increase in perirenal visceral adipose tissue mass (Table
3), suggesting that the excess energy led to the build up of adiposity. This is the source of the increase in body weight. Rat consuming the high fat ration actually received about 27% more kilocalories, more weight, and had larger fat pads than rats fed only chow. Rats consuming a palatable dietary fat in addition to a standard chow diet take in about 10% more calories a day than rats fed only the chow diet and over time, become obese and exhibit a number of complications of obesity.
Our results showed a significant decrease in food intake, whole body weight, and adipose tissue accumulation from oral administration of L-carnitine (Tables
1,
2). Interest in the role of L-carnitine as a feed additive to improve whole body composition arose from the desire to partition nutrients away from lipid accretion, causing improvement of nitrogen balance; L-carnitine also attenuated visceral fat accumulation and accelerated the normalization of food intake [
48].
The current results due to the effect of HMF, showed a significant decrease in body weight and food consumption (Table
1,
2) in accordance with the findings of
York et al [
49]. Several isoflavans constituents of licorice are unique 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 resembles the action of sibutramine, but naturally [
50].
Dietary phytoestrogens may also activate AMPK leading to a reduction in food intake [
51]. Our study is a novel trial for using a phytoestrogen in fennel, licorice and anise as a natural serotonin reuptake inhibitor instead of sibutramine.
Anetholein fennel as a carminative herb improves the digestion by stabilization of the gastrointestinal mucous membrane and causing the pancreas to increase its secretions [
11]. Hydroxyl-anthracene glucosides, especially sennosides A B in senna and T. chebula of HMF improve gastrointestinal motility and influence colonic motility thereby reducing fluid absorption and facilitate weight loss.
Effect of diet treatments on serum lipid profile in rats
In the present study, a high fat diet resulted in dyslipidemic changes as illustrated by increasing triglycerides, VLDL, total cholesterol and low density lipoprotein LDL and a decrease in serum level of high density lipoprotein HDL (Table
3) a finding in accordance with that of
Woo et al. [
52].
L-carnitine supplementation produced significant decreases in serum TG, VLDL, and T-cholesterol, LDL-C while there were significant increases in HDL cholesterol in obese rats. These results are in agreement with those of
González-Ortiz et al. [
53] and
El-Metwally et al., [
54] who reported that oral L-carnitine increases plasma free carnitine levels, improves dyslipidemia and decreases oxidative stress, with reduction of cardiac parameters.
L-carnitine administration to obese rats reduces significantly serum hypertriglyceridemia (Table
3) via decreased synthesis of triglycerides by the liver or by inhibition of triglyceride release from the liver. Also L-carnitine induced marked reductions in total serum cholesterol in skeletal muscles of obese rats [
55].
L-carnitine is necessary for mitochondrial transport metabolism of long-chain fatty acids, thus for myocardial energetic metabolism. Fatty acids cross mitochondrial membranes as acyl-carnitine derivatives to enter pathways for oxidation, acylation, chain shortening or chain elongation-desaturation. Therefore, L-carnitine-dependent fatty acid transfer is central to lipid metabolism; dietary supplementation of L-carnitine improves the utilization of fat providing marked reduction in plasma levels of TG [
56].
In our experimental rat model of obesity, treatment with HMF led to significant decrease in TG, VLDL, T-cholesterol and LDL-C but a significant increase in HDL cholesterol (Table
3), a result in agreement with that of
Murali et al [
57].
T. chebula components of HMF have hypocholesterolemic activity that might be mediated through increased cholesterol excretion in the feces. In addition anthraquinone glycosides from rhubarb in the HMF have lipid-lowering effects, resulting in depression of lipid accumulation. It consequently has anti-atherosclerotic properties [
58].
Glycyrrhizin, the active constituent in licorice root, which is another plant in the mixture, exerts hypocholesterolemic action by stimulating the conversion of cholesterol into bile acids without effect on cholesterol synthesis [
59]. Another herbal supplement in the HMF, Nigella sativa, contains active constituents, phytosterols (mostly b-sitosterol, stigmasterol campesterol) with the ability to reduce the intestinal absorption of diet biliary cholesterol [
60].
Effect of diet treatments on cardiac biomarkers in rats
The obese rats showed a significant increase in the activity of serum LDH, CK-NAC and CK-MB when compared to the normal rats (Table
4). Similar results were reported by
Diniz et al. [
61]. Reduced muscle mitochondrial content function with increasing obesity would lower the total cellular ATP yield, which would result most notably in increased mitochondrial volume, and increased glycolytic enzymes necessitating increased activity of creatine kinase, as this enzyme is responsible for rapidly transferring high-energy phosphate groups from the site of production to the site of use [
62].
The increased blood levels of total cholesterol, LDL, VLDL as well as lowered levels of HDL in HFD rat have been identified in the development of hypercholestremia, which is one of the risk factors for CAD [
63]. Administration of L-carnitine produces a significant decrease in the activity of CK-NAC, CK-MB and LDH (Table
4). This is in agreement with
Ferrari et al. [
64], who reported a reduction in cardiac markers, with L-carnitine having a good protective effect on myocardium.
In conclusion, L-carnitine stimulates the activity of pyruvate dehydrogenase (PDH) which is an important enzyme catalyzing the rate-limiting step in lactate utilization. In type 2 diabetes this led to significant decrease in the activity of LDH [
65]. Moreover, L-carnitine in the β-oxidation of fatty acid as parallel source for energy acts synergistically with the creatine/phosphocreatine/creatine kinase system to produce significantly decreased activity of creatine kinase.
This effect is mainly attributable to the vasodilatation property of L-carnitine, which both improves energetic metabolism of the hypoxic/damaged muscle and enhances wash-out of algogenic metabolites.
Our findings showed that obese rats treated with the HMF exhibited significant decreases in LDH, CK-NAC and CK-MB activity (Table
4), in with the findings of
Suchalatha et al. [
66], who reported that T. chebula extract treatment ameliorates the effect of lipid peroxide formation that is related to the activities of diagnostic myocardial marker enzymes.
The rhubarb content in our mixture could prevent the development of atherosclerosis through regulating vascular inflammatory processes in rats fed with an atherogenic diet [
67]; and the thymoquinone content in
N. sativa normalized cardiac histopathology, as it decreased lipid peroxidation [
20,
21].
Effect of treatments on renal function tests of HFD in rat
The obese rats showed a highly significant increase in the concentration of serum urea, uric acid creatinine, compared with the normal group (Table
4) which is in agreement with the results of
Cindik et al. [
68].
Abnormal renal function is mainly associated with diabetic nephropathy. The pathophysiology involves glucose that binds irreversibly to proteins in the kidney circulation to form advanced glycosylation end products that can form complexes that contribute to renal damage by stimulation of fibrotic growth factors [
69].
HFD induces alteration of renal lipid metabolism by an imbalance between lipogenesis and lipolysis in the kidney, as well as systemic metabolic abnormalities and subsequent renal lipid accumulation leading to renal injury [
70]. There is an association between CKD and insulin resistance that contributes to increased VLDL production and decreased HDL levels, both of which are considered risk factors for the development of kidney dysfunction and markers for progression of CKD [
71].
In addition HFD resulted in hyperinsulinemia, activation of the renin-angiotensin system, glomerular hyperfiltration and structural changes in the kidney that may be the precursors of more severe glomerular injury associated with prolonged obesity [
72].
The oral administration of L-carnitine (Table
4) shows that serum concentration of urea, uric acid and creatinine were highly significantly decreased. The effect of L-carnitine on renal lipid metabolism could serve as a new therapeutic approach, as it counters the renal changes associated with metabolic syndrome [
73]. Hence, L-carnitine has beneficial effects on renal function.
Although there is still debate on the significance of uric acid as a risk factor for cardiovascular disease, many physicians do consider elevated uric acid to be a component of the metabolic syndrome. There is little support for an independent causal role for serum uric acid in the development of CHD. However, uric acid may provide useful prognostic information in subjects with hypertensive vascular disease, suggesting that the influence of uric acid on CHD is explained by the secondary association of it with other risk factors such as dyslipidemia, hyperinsulinaemia and obesity [
74].
The oral administration of HMF extract (Table
4) clarified that serum concentration of urea; uric acid and creatinine were significantly decreased. Rhubarb results in lowering the serum creatinine level causing decrease in urinary protein excretion, attenuation of lipid derangements, decreased oxygen consumption and the hypertrophy of the remnant kidney [
75]. Rhein, another ingredient in rhubarb of HMF, improves cell metabolism through glucose transporter-1: it decreases cell hypertrophy, indicating that there are multiple active ingredients even in a single herbal medicine involved in the multiple therapeutic effects of rhubarb in CKD, and suggesting that HMF might delay the progression of renal failure.
The herbal drugs containing tannins have a uremic-toxin-decreasing action, whereas rhubarb's tannins significantly improved BUN creatinine, glomerular filtration rate and renal blood flow [
76]. In this respect rhubarb has proven effective as a diuretic in rabbit models, apparently by blocking sodium-potassium ATPase in the renal medulla [
77].
The HMF therapy retains the balance between lipogenesis and lipolysis in the kidney to counteract the obesity-associated renal damage, in addition to maintaining cellular hydration due to laxative effect of senna, which is associated with diuretic effect of rhubarb, collectively leading to improvement in renal blood flow and consequent improvement in kidney function.
Effect of treatments on glucose and insulin of HFD rats
Our results showed that a high fat diet results in significant increase in serum glucose, insulin level and insulin resistance (Table
5), which parallels the results obtained from
Zhang et al. [
78]. Diminished hepatic and muscular uptake of glucose produced hyperlipidemia due to increased fat mobilization from adipose tissue and resistance to the antilipolytic actions of insulin. Impaired insulin action is associated with an oversupply of lipids. This increased availability led to either elevated lipid stored in insulin target tissues (e.g. muscle, liver adipose) or increased plasma FFA or triglyceride [
79].
The weight loss in this study due to oral administration of L-carnitine is associated with hypoglycemia, as it promotes insulin sensitivity, thus lowering insulin resistance in obese rats, possibly by regulating the cell energy metabolism or reducing free fatty acids as shown also by
González-Ortiz et al. [
53] and [
55].
Obesity is associated with endothelial dysfunction through direct mechanisms, as insulin resistance in association with diabetes mellitus and dyslipidemia, indirectly, by the production of adipokines and pro-inflammatory cytokines, induces oxidative stress that affect the role of endothelium in modulating vascular function structure [
80].
Our findings (Table
5) indicated a significant decrease of serum glucose, insulin and its resistance in obese rats treated with extracts of HMF. Some prenylflavonoids, such as glycycoumarin, glycyrin, dehydroglyasperin C and dehydroglyasperin D, in licorice ethanolic extract, are effective in preventing and ameliorating diabetes, abdominal obesity and preventing hypertension and metabolic syndrome [
81].
In addition, hypoglycemia induced by T. chebula is probably mediated through enhanced secretion of insulin from the beta-cells of the pancreatic islets or through an extra pancreatic mechanism. Moreover, T. chebula may reduce the effect of inflammatory cytokine release during diabetes which may be one of the causative agents for the insulin resistance [
69].
The hypoglycemic action of thymoquinone due to Nigella sativa in HMF could be partly due to preservation of β-cell integrity of the pancreatic islets causing a significant increase in insulin secretion. It also sensitized hepatocytes to the action of insulin. Furthermore, NS treatment exerts a therapeutic protective effect on diabetes by decreasing oxidative stress, preserving pancreatic beta-cell integrity [
21].
Effect of treatments on hepatic enzymes and lipid peroxidation in HFD-rats
The current data showed a significant increase in the activity of enzymes AST and ALT in the obese compared with normal rats (Table
4). Liver is bombarded by the free fatty acids (FFA) that pour out of the adipose tissue into the portal blood. This can directly cause inflammation within the liver cells, which then release further pro-inflammatory cytokines, leading to more hepatocyte injury and affecting the integrity of liver cells [
82].
Administration of L-carnitine produces a significant lowering effect in the activity of AST and ALT in obese rats, a result in agreement with
Yapar et al. [
83]. Improvement of hepatocyte integrity does not seem to be limited to its obligatory role in the transmembrane import of fatty acids for mitochondrial β-oxidation, but L-carnitine prevents lipotrope methyl group wastage and increased production of polyamines with known immunomodulatory properties. Additionally L-carnitine can directly modify cytokine responses and reduce TNF-production. Moreover, lipid peroxidation is significantly blunted by oral administration of L-carnitine [
84].
The present results demonstrate that the mixture of HMF showed a significant decrease in the activity of both AST and ALT (Table
4) agreeing with the resulted obtained by
Celik and
Isik [
19].
D-limonene and β-myrcene in fennel
(Foeniculum vulgare) has a potent hepatoprotective action; D-limonene increases the concentration of liver GSH which is required by several enzymes that participate in the formation of the correct disulfide bonds of many proteins. Polypeptide hormones participate in the metabolism of xenobiotics. β-myrcene elevates the levels of apoproteins CYP2B1 CYP2B2, which are subtypes of P450 enzyme system that catalyse the oxidative metabolism of a wide variety of exogenous chemicals including drugs, toxins, and endogenous compounds such as fatty acids [
85]. Nigella sativa and licorice root extract of HMF possesses hepatoprotective effects in some models of liver toxicity [
86].
HFD generates oxidative stress in obese rats as shown by a marked increase in the levels of MDA and a distinct diminution in hepatic GSH, as well as activities of the antioxidant enzyme catalase. All showed reduced activity in hyperlipidemic rats (Table
5).
Hyperglycemia in the HFD group activates different pathways leading to increased oxidative stress. Increased activity of the polylol pathway inhibition of the pentose phosphate pathway as a result of hyperglycemia resulted in decreased intracellular levels of NADPH, which is required for regeneration of GSH from its oxidized form GSSG [
87]. The net result was non-enzymatic disruption of H
2O
2 and increased levels of cellular superoxides, hydroperoxides, hydroxyl radicals as well as other radicals.
In addition oxidative stress may be increased in metabolic syndrome due to dyslipidemia resulting from increased levels of FFA and TGs that led to increased formation of foam cells, rendering LDL less dense and more vulnerable to oxidation and uptake by macrophages [
88].
Our results indicated that L-carnitine produced a significant inhibition of MDA production and a significant increase in GSH and activity of catalase (Table
5). L-carnitine reduces significantly the content of thiobarbituric acid reactive substances (TBARS), and causes marked increase in activity of catalase in skeletal muscles of obese rats [
55]. Moreover L-carnitine favorably modulates oxidative stress causing a reduction in oxidized LDL cholesterol levels [
89].
L-carnitine effectively protects and improves mitochondrial function in vivo: it acts as an antioxidant, so by inhibiting ROS it protects the vascular endothelial tissues against oxidative damage in hypertension [
90]. Thus, L-carnitine treatment effectively protected the liver tissue against oxidative damage and showed marked improvement in its antioxidant status.
The current results demonstrated an antioxidative effect of HMF, as indicated by increased GSH level and hepatic catalase activity in comparison with obese rats (Table
5). Administration of aqueous extract of T. chebula effectively modulated oxidative stress and enhanced antioxidant status in the liver [
91], which may be due to the total polyphenol content, expressed as gallic acid. T. chebula extract has the potential to play a role in the prevention of oxidative damage in living systems which can be attributed to its membrane stabilizing activities [
92]. It has stronger antioxidant activity than α-tocopherol, due to the presence of hydroxybenzoic acid derivatives, flavonol aglycones and their glycosides.
Glabridin, a natural polyphenolic isoflavone antioxidant from licorice root extract in HMF possesses potent free radical scavenging activity that could promote a decrease in lipid peroxidation and could protect LDL from oxidation [
93]. This would occur via a direct interaction with the lipoprotein and/or an indirect effect through accumulation in arterial macrophages. Therefore licorice represents a potent nutrient, which can attenuate the development of atherosclerosis, secondary to its antioxidant properties against lipids peroxidation in arterial cells [
94].
Polyphenols, the main compound of N. sativa oil in HMF, have many biological properties. They possess powerful antioxidative components, which can inhibit membrane lipid peroxidation [
28], and their administration exerts a therapeutic protective effect by decreasing oxidative stress.
Foeniculum vulgare extract, another constituent of the HMF, has potent antioxidant activity, as has been documented with various antioxidant assays. These activities include total antioxidant, free radical scavenging, superoxide anion radical scavenging, and hydrogen peroxide scavenging [
95]. Antioxidant activity of the anthraquinones of rhubarb in the HMF may help protect against lipid peroxidation and free radical damage; its extracts will probably be useful for the development of safe food additives [
12].
This study showed the antioxidant effect of dietary supplement represented by L-carnitine and free radical scavenger activities of herbal supplements represented by T. chebula, licorice, fennel, aniseed and Nigella sativa extract in the HMF.
Based on these broad observations, we suggest that high fat diet-induced obesity resulted in deleterious effects in kidney and liver tissues. HMF extract or carnitine supplementation counteracted the injuries, and ameliorated or normalized most the biochemical parameters.
Collectively obesity is associated with cardiometabolic complications, including insulin resistance, dyslipidemia, cardiovascular disease (CVD). When comparing therapies for obesity, both L-carnitine and HMF have shown improvements in body weight, lipid profile, glucose levels, liver, and kidney and cardiac marker of function, as well as insulin resistance and oxidative stress markers.
It can be concluded that in this study HFD induced a model of obesity associated with high body weight, hyperlipaemia, hyperinsulinemia, insulin resistance, hepatic oxidative stress, associated hepatic, cardiac renal function marker disturbance in rats. The obesity and its associated problems in this study could be ameliorated in different degrees by using L-carnitine or HMF extract.
The study also showed that herbal extract mixture significantly improved body weight, cholesterol, LDL triglyceride concentrations, creatinine, urea, ALT, oxidative stress, resulting from the high-fat diet. So it also had hypolipidemic, renoprotective, hapatoprotective, antioxidant and antiobesity effect.
It could be helpful to use HMF in conjunction with drugs; herbal extract might be a safer combination to surmount the overweight state. Positive results from such trials would confirm the medicinal usefulness of empirical combinations of traditional Egyptian medicines.