The present study evaluates the protective effects of an antioxidant-rich extract of
The metabolic syndrome (MetS), also known as syndrome X, insulin resistance syndrome, or dysmetabolic syndrome, is a cluster of metabolic risk factors that come together in a single individual. The risk factors include atherogenic dyslipidemia, hypertension, hyperglycemia, abdominal obesity, insulin resistance, a proinflammatory state, and a prothrombic state [
Dyslipidemia is an integral part of MetS which includes hypertriglyceridemia, hypercholesterolemia, low HDL, high LDL, and VLDL levels. Individuals with MetS, particularly those with abdominal obesity, exhibit a highly atherogenic lipid profile, which may account for their high risk of CVD. Central fat accumulation and the presence of insulin resistance have both been associated with dyslipidemia [
The MetS is known to be caused by insulin resistance, a condition whereby the body’s cells are incapable of taking up glucose from the blood [
Homocysteine is known to be a strong and independent marker of risk for the development of cardiovascular disease [
Fructose has been implicated as a contributor to nearly all of the classic manifestations of MetS. Increased fructose consumption can lead to hyperlipidemia, development of insulin resistance, inflammation, oxidative stress, obesity, and comorbidities such as hypertension and T2DM, all risk factors for cardiac dysfunction [
Recently, much attention has been focused on plant foods that may be beneficial in preventing the metabolic syndrome and possibly reducing the risk of diabetes and cardiovascular disease. Dietary patterns high in green leafy vegetables are generally found to be associated with lower prevalence of the metabolic syndrome [
Flavonoids found ubiquitously in most edible vegetables and fruits and constituting a major portion of micronutrients in diet have been known to possess good antidiabetic and antihyperlipidemic activities. A study on spinach has shown its flavonoids to alleviate hyperlipidemia in rats by decreasing oxidative stress [
Spinach was purchased from a farm on the outskirts of Mumbai, India, and authenticated at the Blatter Herbarium, St. Xavier’s College, Mumbai, after matching with the existing specimen (specimen no.TK-1). The leaves were washed, shade dried, and ground to obtain a dry powder. This powder was extracted using a mixture of methanol : water (70 : 30)
Epinephrine, 5,5′-dithiobis (2-nitrobenzoic acid)—(DTNB) and trichloro acetic acid (TCA) were purchased from Sigma Chemical Co., St Louis, MO, USA. Thiobarbituric acid (TBA), reduced glutathione, oxidized glutathione, and nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from Himedia Laboratories, Mumbai, India. All other chemicals were obtained from local sources and were of analytical grade.
High performance thin layer chromatography (HPTLC) of NAOE was carried out on the CAMAG HPTLC system for the determination of natural antioxidants, flavonoids, and phenolic compounds. Total flavonoids were determined by HPTLC in our earlier studies [
Sprague Dawley female rats (150–200 g) were acquired from Glenmark Pharmaceuticals Ltd., India. They were housed in clean polypropylene cages under standard conditions of humidity (50 ± 5%), temperature (25 ± 2°C), and light (12 h light/12 h dark cycle) and fed with a standard diet (Amrut laboratory animal feed, Pranav Agro Industries, India) and drinking water ad libitum. All animals were handled with humane care. Experimental protocols were reviewed and approved by the Institutional Animal Ethics Committee (Animal House Registration no. 25/1999/CPCSEA) and conform to the Indian National Science Academy Guidelines for the Use and Care of Experimental Animals in Research.
Fructose solution (20%
After acclimatization for 7 days in the animal quarters, rats were randomly divided into 7 groups of 6 animals each (groups I to VII) and treated in the following way.
Group I (normal control): rats received drinking water (1 mL/kg, p.o) daily for 45 days. Group II (toxicant control): rats received fructose (20% Group III (NAOE200): rats received fructose (20% Group IV (NAOE400): rats received fructose (20% Group V (standard): rats received fructose (20% Group VI (AE): rats received fructose (20% Group VII (NAOEAE): rats received fructose (20%
All the animals were maintained on standard rat chow diet and drinking water ad libitum
Insulin resistance was determined from the formula:
LDL and VLDL were calculated as per Friedevald’s equation as follows:
The atherogenic index (AI) was calculated using the following formula:
Animals received glucose solution (1.5 g/kg) orally 30 minutes after their respective treatments. Blood glucose levels were determined at 0, 30, 90, and 120 min after glucose administration.
The insulin assay was carried out in serum using an ELISA kit supplied by Genxbio Health Sciences Ltd., India.
TC, TG, and HDL levels were determined by the CHOD-PAP method, GPO-Trinder method, and the phosphotungstic acid method, respectively, using kits supplied by Erba (Mumbai, India).
The marker enzymes AST, LDH, and CK-MB were assayed in serum using standard kits supplied by Erba (Mumbai, India). The results were expressed as IU/L.
Uric acid levels were determined by the modified Trinder’s end point method using kits supplied by Erba (Mumbai, India).
CRP was measured in serum using a standard agglutination test kit supplied by Spectrum Diagnostics, Egypt, according to the method of Pepys et al.
Hcy levels were determined in the serum of experimental animals by using an ELISA kit supplied by Cell Biolabs, Inc., USA.
The quantification of LPO was done by determining the concentration of thiobarbituric acid reactive substances (TBARS) in the heart using the method of Ohkawa et al. [
GSH level was estimated in the heart homogenate using DTNB by the method of Ellman [
SOD was assayed by the method of Sun and Zigman [
Hearts stored in 10% buffered formalin were embedded in paraffin, sections cut at 5
The results are expressed as mean ± SEM from 6 animals in each group. Results were statistically analyzed using one-way ANOVA followed by the Bonferroni’s multiple comparison test;
HPTLC analysis at 366 nm showed clear separation of 13 antioxidants and 10 phenolic acids from NAOE (Figures
(a) HPTLC fingerprinting of NAOE for total antioxidants. (b) HPTLC chromatogram of total antioxidants of NAOE. (c) HPTLC fingerprinting of NAOE for phenolic acids. (d) HPTLC chromatogram of phenolic acids of NAOE.
The effect of various treatments on body weight of rats recorded on the 1st, 15th, 30th, and 45th days is shown in Table
Effect of NAOE, AE, NAOEAE, and gemfibrozil on body weight in fructose-fed rats.
Treatment groups | Body weight (g) at different days | |||
---|---|---|---|---|
Day 1 | Day 15 | Day 30 | Day 45 | |
Normal control | 240.33 ± 4.91 | 250.16 ± 4.71 | 259.33 ± 4.60 | 269.16 ± 4.09 |
Toxicant control | 221.66 ± 5.58 | 251.33 ± 6.51 | 279.16 ± 6.88 |
325.83 ± 10.12 |
Aerobic exercise | 237.50 ± 6.29 | 218.33 ± 6.41 | 203.33 ± 5.72 |
195.00 ± 3.65 |
NAOE200 | 214.16 ± 5.23 | 200.83 ± 2.71 | 189.16 ± 3.96 |
183.33 ± 4.01 |
NAOE400 | 235.00 ± 4.08 | 225.83 ± 3.51 | 208.33 ± 2.47 |
199.16 ± 1.54 |
NAOEAE | 237.50 ± 6.29 | 218.33 ± 6.41 | 203.33 ± 5.72 |
193.33 ± 3.65 |
Gemfibrozil (60 mg/kg) | 216.16 ± 5.23 | 200.00 ± 2.71 | 189.16 ± 3.96 |
183.33 ± 4.01 |
All values are mean ± SEM;
The effect of various treatments on blood glucose levels is shown in Figure
Effect of NAOE, AE, NAOEAE, and gemfibrozil on blood glucose levels in fructose-fed rats. All values are mean ± SEM;
The effect of various treatments on OGTT is depicted in Table
Effect of NAOE, AE, NAOEAE, and gemfibrozil on oral glucose tolerance test (OGTT) in fructose-fed rats.
Treatment groups | Blood glucose levels at different intervals (mg/dL) | |||
---|---|---|---|---|
0 min | 30 min | 60 min | 120 min | |
Normal control | 87.33 ± 2.37 | 108.51 ± 2.71 | 99.11 ± 2.32 | 86.51 ± 1.91 |
Toxicant control | 197.21 ± 13.91a | 227.12 ± 12.97a | 203.71 ± 11.28a | 189.31 ± 12.34a |
Aerobic exercise | 133.32 ± 2.56 |
157.31 ± 2.43 |
146.82 ± 2.21 |
133.22 ± 2.27 |
NAOE200 | 146.71 ± 3.75 |
177.71 ± 2.74 |
155.51 ± 2.95 |
142.31 ± 3.12 |
NAOE400 | 138.71 ± 2.86 |
165.51 ± 4.42 |
148.31 ± 3.85 |
130.51 ± 3.24 |
NAOEAE | 99.67 ± 5.90 |
150.04 ± 7.47 |
129.22 ± 3.02 |
103.72 ± 4.44 |
Gemfibrozil | 168.52 ± 2.29 |
157.51 ± 2.46 |
152.31 ± 2.07 |
155.81 ± 2.33 |
All values are mean ± SEM;
Figures
(a) Effect of NAOE, AE, NAOEAE, and gemfibrozil on insulin levels in fructose-fed rats. (b) Effect of NAOE, AE, NAOEAE, and gemfibrozil on insulin resistance (HOMA-IR) in fructose-fed rats. All values are mean ± SEM;
The effect of gemfibrozil, NAOE, AE, and NAOEAE on lipid profile and the atherogenic index in fructose-fed rats is summarized in Figures
(a) Effect of NAOE, AE, NAOEAE, and gemfibrozil on lipid profile in fructose-fed rats. (b) Effect of NAOE, AE, NAOEAE, and gemfibrozil on the atherogenic index in fructose-fed rats. All values are mean ± SEM;
The effect of gemfibrozil, NAOE, AE, and NAOEAE on the activities of marker enzymes is demonstrated in Figure
Effect of NAOE, AE, NAOEAE, and gemfibrozil on AST, LDH, and CK-MB in fructose-fed rats. All values are mean ± SEM;
The effect of gemfibrozil, NAOE, AE, and NAOEAE on serum uric acid, CRP, and Hcy in fructose-fed rats is shown in Table
Effect on NAOE, AE, NAOEAE, and gemfibrozil on uric acid, CRP, and Hcy in fructose-fed rats.
Treatment groups | CRP (mg/dL) | Uric acid (mg/dL) | Homocysteine ( |
---|---|---|---|
Normal control | 0.80 ± 0.12 | 0.52 ± 0.04 | 1.71 ± 0.04 |
Toxicant control | 3.00 ± 0.21a | 3.19 ± 0.05a | 4.26 ± 0.14a |
Aerobic exercise | 1.80 ± 0.12 |
1.95 ± 0.05 |
2.32 ± 0.03 |
NAOE200 | 2.20 ± 0.12 |
5.30 ± 0.07 |
2.82 ± 0.07 |
NAOE400 | 1.60 ± 0.12 |
1.48 ± 0.12 |
1.88 ± 0.04 |
NAOEAE | 0.90 ± 0.13 |
0.78 ± 0.09 |
2.36 ± 0.02 |
Gemfibrozil | 1.20 ± 0.21 |
3.32 ± 0.05 | 2.64 ± 0.13 |
All values are mean ± SEM;
The effect of gemfibrozil, NAOE, AE, and NAOEAE on LPO, GSH, and some antioxidant enzymes is described in Table
Effect on NAOE, AE, NAOEAE, and gemfibrozil on LPO, GSH, SOD, CAT, GPx, and GR in fructose-fed rats.
Treatment groups | LPO (nmol MDA/min/mg protein) | GSH ( |
SOD (U/mg protein) | CAT (U/mg protein) | GPx (U/mg protein) | GR (U/mg protein) |
---|---|---|---|---|---|---|
Normal control | 18.15 ± 0.95 | 3.00 ± 0.11 | 24.13 ± 0.26 | 10.69 ± 0.30 | 9.39 ± 0.21 | 242.91 ± 9.85 |
Toxicant control | 36.18 ± 0.51a | 0.34 ± 0.02a | 14.81 ± 0.49a | 4.58 ± 0.09a | 4.96 ± 0.17a | 87.71 ± 3.50a |
Aerobic exercise | 27.41 ± 0.85 |
1.54 ± 0.03 |
18.35 ± 0.19 |
6.74 ± 0.11 |
7.11 ± 0.14 |
103.61 ± 2.58 |
NAOE200 | 29.36 ± 0.75 |
1.97 ± 0.04 |
19.51 ± 0.19 |
7.34 ± 0.17 |
7.14 ± 0.13 |
129.12 ± 2.21 |
NAOE400 | 26.27 ± 0.65 |
2.32 ± 0.04 |
19.54 ± 0.47 |
8.81 ± 0.26 |
7.66 ± 0.12 |
150.51 ± 7.67 |
NAOEAE | 19.15 ± 0.74 |
3.19 ± 0.03 |
22.13 ± 0.29 |
11.37 ± 0.23 |
9.34 ± 0.18 |
206.11 ± 8.75 |
Gemfibrozil (60 mg/kg) | 29.36 ± 0.71 |
2.38 ± 0.86 |
21.18 ± 0.63 |
8.07 ± 0.12 |
8.33 ± 0.14 |
192.41 ± 5.70 |
All values are mean ± SEM;
Significant (
Histopathological studies on heart tissue of the normal control group rats (Figure
(a) Hematoxylin and eosin staining of the heart of rat of normal control group (hematoxylin-eosin, original magnification ×100). Hollow arrow indicates normal histoarchitecture of the heart. (b) Hematoxylin and eosin staining of the heart of rat of toxicant (fructose) control group (hematoxylin-eosin, original magnification ×100). Hollow arrow indicates lymphocytic infiltrate, and solid arrow indicates myocardial necrosis and severe myofibrillar loss. (c) Hematoxylin and eosin staining of the heart of rat of NAOE200 group (hematoxylin-eosin, original magnification ×100). Hollow arrow indicates splitting and swaying of myocardial fibers with moderate lymphocytic infiltrate around blood vessels. (d) Hematoxylin and eosin staining of the heart of rat of NAOE400 group (hematoxylin-eosin, original magnification ×100). The big hollow arrow indicates focal hyalinized area in myocardium, and the small arrow indicates lymphocytic infiltration. The rest of the myocardial fibers show no histomorphological abnormality. (e) Hematoxylin and eosin staining of the heart of rat of AE group (hematoxylin-eosin, original magnification ×100). Hollow arrow indicates the splitting of myocardial fibers, and solid arrow indicates minimal vacuolar degeneration. (f) Hematoxylin and eosin staining of the heart of rat of NAOEAE group (hematoxylin-eosin, original magnification ×100). Arrows indicate minimum separation of fibers. (g) Hematoxylin and eosin staining of the heart of rat with standard (gemfibrozil) group (hematoxylin-eosin, original magnification ×100). Arrows indicate minimal separation of fibers.
The major contributing factor for progression of cardiovascular disease by fructose overload in drinking water is known to be oxidative stress [
Fructose intake may induce hypertriglyceridemia and lipogenesis [
Metabolism of fructose.
In the present study, fructose given in drinking water for 6 weeks could induce metabolic alterations such as hyperglycemia, dyslipidemia, and insulin resistance. This result indicates that a low carbohydrate diet would play a major role in ameliorating pathological conditions such as cardiovascular diseases and diabetes.
Aerobic exercise such as swimming increases oxygen consumption, number, size, and density of mitochondria and oxidative enzymes, thus increasing the rate of aerobic fat catabolism leading to energy expenditure and fat burn. Exercise also contributes to increase in the metabolic rate by improving cardiovascular capacity and suppressing proinflammatory cytokine production, in addition to increasing the release of nitric oxide and the consumption of free fatty acids (FFA), thus enhancing insulin sensitivity [
Gemfibrozil is a popular fibrate from the PPAR-alpha agonists clinically used as antihyperlipidemics, more recently reported to have beneficial effects on cardiovascular function. Treatment with fibrates has reduced oxidative stress in rat hearts, reduced LDH and CK-MB in coronary effluent, decreased C-reactive protein levels and TBARS and superoxide anion generation, and consequently increased reduced glutathione levels [
A major contributor to the development of insulin resistance is an overabundance of circulating FFA released from an expanded adipose tissue mass [
Roden et al. demonstrated that FFA could compete with glucose for substrate oxidation, thereby increasing fat oxidation and causing insulin resistance associated with diabetes and obesity. The possible mechanism is that increased free fatty acid oxidation causes an elevation of intramitochondrial acetyl CoA and NASH/NAD+ ratios with inactivation of pyruvate dehydrogenase [
Insulin resistance is closely linked to lipid metabolism as insulin resistance patients have high ectopic lipid deposition generating the toxic derivatives diacylglycerol and fatty acyl CoA. The presence of these metabolites leads to high serine phosphorylation of IRS-I (insulin receptor substrate-1) thus reducing insulin signaling and causing hyperinsulinemia [
Evaluation of insulin resistance (or sensitivity) and
The increased serum insulin levels and fasting glucose levels resulted in rise in HOMA-IR score in fructose-fed rats. Treatment groups showed significant reduction in HOMA-IR score indicating their ability to alleviate insulin resistance.
Fructose increases uric acid levels by the depletion of ATP and inorganic phosphate. Uric acid could be a prognostic marker for CV events in MetS. It can induce proinflammatory changes in the adipocyte that are similar to those observed in the prediabetic subjects. Uric acid inhibits endothelial function by impairing NO-induced vasodilation which is necessary for insulin to stimulate glucose uptake into tissues [
MetS is associated with inflammation as evidenced by an increase in levels of the proinflammatory cytokines IL-6, resistin, TNF, and C-reactive protein (CRP). CRP is an acute phase reactant made by the liver and released into blood stream within few hours after tissue injury, the start of an infection, or other cause of inflammation. The released CRP binds to lipoproteins (LDL and VLDL), upregulating adhesion molecule expression and progression of atherosclerosis and MetS [
Homocysteine is a breakdown product of the amino acid methionine in the body. Hyperhomocysteinemia is an independent and graded risk factor for the development of cardiovascular diseases. Elevated plasma homocysteine may cause or result from insulin resistance and may indicate vascular risk or be actively involved in atherogenesis [
Conversion of homocysteine to methionine and cysteine.
The diagnostic marker enzymes AST, LDH, and CK-MB are present plentifully in the heart. When injury to the heart occurs, these enzymes spill into blood stream. Thus, elevated levels of these enzymes released from the myocardium into blood indicate myocardial necrosis. Hyperglycemia produced by fructose overload in this model causes excessive formation of ROS which causes oxidation of the myocardial membrane leading to its damage [
High fructose administration induces oxidative stress and leads to insulin resistance and type II diabetes in rats [
Reduced glutathione is a nonenzymatic defense antioxidant present abundantly in the body. Together with GPx, GR, CAT, and SOD, it efficiently scavenges free radical species such as superoxide anions, alkoxy radicals, and H2O2. It is a substrate for the antioxidant enzymes GPx and GST and functions to protect cellular constituents from ROS and peroxides formed during metabolism. Depletion of GSH levels in fructose-fed rats may be due to its exhaustive utilization for augmenting the activities of GPx and glutathione S-transferase. Glutathione levels depleted by fructose were significantly elevated by NAOE, AE, NAOEAE, and gemfibrozil treatments. It may be understood that the increased levels of GSH could be due to its enhanced synthesis in the presence of these treatments.
SOD, CAT, and GPx constitute a mutually supportive enzyme system of the first line cellular defense against oxidative injury, decomposing O2− and H2O2 before they combine to form the more harmful hydroxyl (OH·) radical. In the present study, SOD activity decreased significantly in the fructose-fed group of animals, may be due to an excessive formation of O2− due to fructose-induced oxidative stress. The excessive O2− formed lead to exhaustion of the available SOD. The activities of CAT and GPx also decreased significantly in the fructose-fed rats. SOD dismutates O2− into H2O2which is the substrate for CAT and GPx. Due to unavailability of H2O2 as a result of SOD depletion, these H2O2 scavenging enzymes are left with no substrate to act and hence a depletion in their activity was observed. Administration of NAOE, AE, NAOEAE, and gemfibrozil to fructose-fed rats effectively prevented the decrease in SOD, CAT, and GPx activities, which may be due to scavenging of free radicals by NAOE, AE, NAOEAE, and gemfibrozil, resulting in prevention of their depletion.
GR is an antioxidant enzyme involved in reduction of GSSG (an end product formed by GPx) to GSH. In fructose-fed rats due to reduction in GPx activity, lesser GSSG was available to GR to act upon, which resulted in decreased activity of GR. Pretreatment of NAOE, AE, NAOEAE, and gemfibrozil to fructose-fed rats restored the activity of GPx, thus accelerating the conversion of GSSG to GSH by GR.
To summarize, NAOE (400 mg/kg) treatment combined with aerobic exercise for 45 days to fructose-fed rats was more effective than NAOE (200 mg/kg or 400 mg/kg) administration and AE alone. The protective activity of NAOE in MetS may be attributed to the potent antioxidant activity of its abundant NAO, especially the flavonoids. Flavonoids of NAOE stabilize the ROS by reacting with them and getting oxidized in turn to more stable less reactive radicals. Presumably, the high reactivity of OH group of flavonoids is responsible for this free radical scavenging activity [
Flavonoid (OH) + R
This study thus shows the way towards controlling MetS, which is a regular aerobic exercise along with adequate consumption of antioxidant-rich foods such as spinach in diet.
The authors declare that there is no conflict of interest regarding the publication of this paper.
The authors are extremely thankful to Glenmark Pharmaceuticals Ltd., India, for providing animals for this study. The authors also thank Mr. Mustansir Bhori, Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y. Patil University, Mumbai, India, for his valuable assistance in ELISA.