INTRODUCTION:

Uncontrolled and regular intake of diet containing high amount of fat can be the reason for a number of problems that include obesity, dyslipidemia, Diabetes (type 2), non alcoholic fatty liver and sometimes cancer because elevated amount of fat results in profound changes in physiological and metabolic functions of body^1,2. Obesity and hyperlipidemia are lifestyle lipid disorder problems often result due to long term imbalance in energy intake and its expenditure and are fast growing global health issue and reasons responsible are high fat, calorie rich diets, psycho social annoyance

and less active life style^3,4,5. Consumption of high fat diet give rise to dyslipidemia which is linked to oxidative leads to generation and accumulation of reactive oxygen species (ROS), a result of biochemical processes in body, pose

the increment in oxidative stress^6,7. It is assumed that oxidative stress give rise to wide range of problems that include pathologies caused by diabetes taking patient to premature morbidity and mortality^8,9.

High levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) in blood tend to increase risk of developing problems like narrowing of arteries leading to high blood pressure and further to coronary heart disease^10,11.

This systemic oxidation stress associated with High fat Diet (HFD) and obesity directly impacts insulin sensitivity of metabolic organs and contribute in the development of problems of dyslipidemia and type II diabetes¹. In order to prevent cardiovascular diseases and management of all other risk factors that includes diabetes and obesity; it will turn up to be a useful strategy for the discovery of new lead molecules with better potential and activity on regulating the different mechanism. It will help in maintaining the lipid metabolism and may be used in the treatment of hyperlipidemia that occurs due to various etiology¹².

Sweet lime (Citrus limetta) belongs to the family Rutaceae, locally called as “Mosambi”, in Indian peninsula and neighboring areas, a plant of Asian origin and best cultivated in India, China, Malaysia, Indonesia and Thailand. Its fruits used as rich source of vitamin C to replenish energy by squeezing the fruit’s glandular hair. D-limonene is the most abundant terpene found in the Citrus limetta, contains various clinically useful properties i.e gallstone dissolution^13,14, antibacterial and antifungal properties⁸, antihyperglycemic^15,16, antagonize the hypertensive effect of angiotensin II¹⁷, larvicidal activity¹⁸ and antiulcer activity¹⁹. Antidiabetic and antihyperlipidemic activity of Citrus limetta is reported under its traditional use²⁰.

Metformin, a biguanide, used for treatment of type 2 diabetes mellitus works by suppression of endogenous glucose production, primarily by the liver. It is also termed as insulin sensitizer due to the dropped levels of blood insulin during its use²¹. Role of Metformin for weight management in patients without type 2 is also reported. Both the drugs above show their effectiveness in weight loss fighting obesity and lowering the elevated amount of glucose in body. This study focuses on the comparative study evaluation of Metformin and citrus limetta’s main terepene constituent against HFD induced hyperlipidemia in STZ diabetic and non-diabetic rats.

MATERIALS AND METHODS:

Animals:

Present study was approved by the Institutional Animal Ethics Committee (IAEC) in Integral University, Lucknow. The approval number is IU/IAEC/16/01; Faculty of Pharmacy, Integral University, Dasauli, P.O. Bas-ha Kursi Road; Lucknow-226026 (U.P). Animals were acclimatized under standard laboratory conditions at 25 ± 2 ºC, relative humidity (50 ± 15%). Animals were kept in propylene cages (6 in each cage) under standard laboratory conditions (12h light and 12h light: dark::day: night cycle) and had a free access to commercial pellet diet and tap water ad libitum.

Drugs and chemicals:

D-limonene, purchased from Ecotech Technologies, private Ltd., Mumbai, India. Metformin was purchased locally as marketed tablets by Franco-Indian Pharmaceuticals under the name Glyciphage. Streptozocin was purchased by M.P Biomedicals Ltd. All chemicals used were of analytical grade. For biochemical assays, double distilled water was taken into use. The enzymatic kits were purchased from Kamineni life sciences private ltd.

Experimental Protocol:

After acclimatization, all the 42 animals were randomly divided into 9 groups of 6 animals each. All animals were fed with HFD except the ones in normal control group. High fat diet, prepared according to the formula and method used by Vijaya et al²². Water and food intake of the animals was monitored daily at the same time during the experimental period. Food efficiency ratio was calculated while the experiment was going on. Animals were pretreated as per the given plan under the treatment protocol. Diabetes was induced by subcutaneously administering Streptozocin; STZ (30 mg/kg)²³ on the 4^th day of study after fasting for 24 hours in groups other than normal control, high fat diet control, high fat diet control + limonene(400mg/kg)²⁴, high fat diet control + Metformin (200mg/kg)²⁵, high fat diet control + Citrus limetta extract (400mg/kg)²⁶. Diabetes was confirmed in all the administered groups by testing blood glucose level after 48 hours.

Measurements of Anthropometric parameters:

Fasting blood glucose was measured by withdrawing blood from the tail with the help of small pricking in the tail vein. Body weight of the rats, total food intake was noted during the study. Body weight gain, body mass index and food efficiency ratio was calculated at the last day of the study. Body mass index was calculated by the following formula^27,28.

Body weight (gm)

BM1 = -------------------------

Length² (cm)

While FER was calculated by formula below

Total body weight

FER = -----------------------------------------------------

Total food intake during the study period

Biochemical estimations in serum:

Retro-orbital plexus route was used to collect the blood of the overnight fasted rats with the help of micro-capillary on the 29^th day. Blood was centrifuged at 4000 rpm for 10 min to get the serum. Serum was taken into separated eppendorf tubes. The amount of Triglycerides, High Density Lipoproteins (HDL), Total Cholesterol (TC), low density lipoproteins (LDL) were calculated (Kamineni Life Sciences Pvt Ltd. Hyderabad. India) with the help of commercial kits.

Determination of antioxidants enzymes in liver tissue:

Homogenization of tissue was done with saline phosphate buffer at a pH of 7. Glutathione was measured by the process described by Sedlack²⁸ and Shanmukha²⁹. Superoxide dismutase (SOD), activity was determined by process of Kakkar et al³⁰, and Khatun et al³¹.Thiobarbituric acid relative substances (TBARS) by Ohkawa et al.³² and catalase activity by the process of Cohen et al³³ and Shokrzadh et al³⁴.

Determination of end organ weight and fat pad weight:

Overnight fasted rats on the final day of the study were sacrificed and then different organs (Heart, liver and kidney) and fat pads (epididymal, perirenal, mesentric) were removed, washed with normal saline and weighed.

Histomorphological studies:

The animal groups under study were sacrificed and fat (adipose) tissue samples were collected, fixed in 10% formalin buffered solution. After the fixation process cut sections of paraffin embedded tissues were stained. Haematoxylin and eosin were used for staining. The slides were examined under light microscope and photomicrographs were taken. Histopathological studies were done from RS Diagnostics center, Lucknow.

Statistical Analysis:

The results were expressed as Mean±SEM. Comparisons were done by applying analysis of variance (ANOVA) between the treatment groups and control, followed by Tuckey’s test. In all the tests, the criteria for statistical significance was p<0.05.

RESULTS:

Table1. Anthropometric parameters

Groups	Body mass index (gm/cm²)	Lee’s Index	Heart: Body (weight) ratio	Food efficiency Ratio Total B.W/Total food taken.
Normal control	0.555 ± 0.012	0.312 ± 0.002	3.5 ± 0.09	5.36 ± 0.042
HFD+Diabetic Control	0.676 ± 0.0217***	0.341 ± 0.012 ns	3.61 ± 0.03 ns	5.55 ± 0.15 ns
HFD control	0.491 ± 0.013 ns	0.316 ± 0.007 ns	5.60 ± 0.15***	6.50 ± 0.13**
HFD+Diabetes+Limonene (250mg/kg)	0.4183 ± 0.022***	0.296 ± 0.005***	7.18 ± 0.17***	7.31 ± 0.14***
HFD+Diabetes+ Metformin (200mg/kg)	0.616 ± 0.017 ns	0.322 ± 0.0056 ns	5.62 ± 0.66***	5.63 ± 0.031 ns
HFD+Limonene (250mg/kg)	0.505 ± 0.014 ns	0.313 ± 0.007 ns	7.46 ± 0.09***	7.30 ± 0.04 ns
HFD+Metformin (200mg/kg)	0.568 ± 0.021*	0.322 ± 0.004 ns	6.46 ± 0.25***	6.44 ± 0.24 ns
HFD+Diabetes+ Extract 400mg/kg	0.383 ± 0.005***	0.263 ± 0.002 ***	6.71 ± 0.06***	5.76 ± 0.38 ns
HFD+ Extract 400mg/kg	0.568 ± 0.007*	0.284 ± 0.003*	6.91 ± 0.05***	7.47 ± 0.11*

Table 1 Shows Body mass index, Lee’s Index, Heart: Body (weight) ratio, Food efficiency Ratio, where Values are expressed as mean ± SEM. where *P < 0.05, **P<0.01,***P<0.001 and ^nsP>0.05 when compared to HFD+Diabetic Control and HFD control.

Table2. Lipid Profile

Groups	Total Cholesterol (mg/dl)	Triglycerides (mg/dl)	High Density lipoprotein (mg/dl)	Low Density Lipoprotein (mg/dl)	Very low density lipoprotein (mg/dl)	Atherogenic Index
Groups	Total Cholesterol (mg/dl)	Triglycerides (mg/dl)	High Density lipoprotein (mg/dl)	Low Density Lipoprotein (mg/dl)	Very low density lipoprotein (mg/dl)	TC/HDL	LDL/HDL
Normal control	101.87 ± 0.806	68.17 ± 0.780	30.713 ± 0.419	58.39 ± 0.30	12.76 ± 1.082	3.318± 0.064	1.821± 0.0626
HFD+Diabetic Control	176.2 ± 1.186***	160.90 ± 1.172***	25.02 ± 0.466***	113.5 ± 0.615***	37.681 ± 1.084***	7.05 ± 0.1673***	4.505 ± 0.115***
HFD control	172.82 ± 1.821***	156.02 ± 1.105***	27.76 ± 0.4368***	109.12 ± 0.473***	35.93 ± 2.07***	6.22 ± 0.136***	3.93 ± 0.0625***
HFD+Diabetes+ Limonene (250mg/kg)	122.31 ± 0.739***	110.26 ± 1.11***	40.94 ± 0.3940***	57.07 ± 0.326***	24.295 ± 0.429***	2.983 ± 0.0215***	1.39 ± 0.012***
HFD+Diabetes+ Metformin (200mg/kg)	112.30 ± 1.188***	106.14 ± 0.926***	45.036 ± 0.401***	52.60 ± 0.557***	14.67 ± 1.195***	2.49± 0.004***	1.161 ± 0.020***
HFD+ Limonene (250mg/kg)	127.615 ± 1.180***	116.506 ± 0.97***	38.72 ± 0.504***	58.30 ± 0.306***	30.57 ± 1.71 ns	3.29 ± 0.0708***	1.501 ± 0.016***
HFD+ Metformin (200mg/kg)	119.15 ± 1.006***	111.79 ± 0.975***	46.90 ± 0.379***	54.24 ± 0.342***	18.10 ± 0.89***	2.538± 0.031***	1.151 ± 0.009***
HFD+Diabetes+ Extract 400mg/kg	118.64 ± 0.28***	104.75 ± 0.281***	43.89 ± 0.441***	53.83 ± 0.268***	20.91 ± 0.31***	2.69 ± 0.024***	1.224 ± 0.017***
HFD+ Extract 400mg/kg	124.61 ± 0.281*	111.83 ± 0.461***	40.48 ± 0.331***	55.83 ± 0.209***	28.39 ± 0.395**	3.07 ± 0.029***	1.375 ± 0.015***

Table 2: Lipid Profile, where values are expressed as mean ± SEM. where *P < 0.05, **P<0.01, ***P<0.001 and ^nsP>0.05 when compared to HFD+Diabetic Control and HFD control.

Table 3. End Organ weights

Groups	Liver (g)	Heart (g)	Kidney (Rt+ Lt) (g)	Visceral fat pad (g)
Normal control	6.88 ± 0.019	0.738 ± 0.0007	1.505 ± 0.007	1.354 ± 0.011
HFD+Diabetic Control	9.48 ± 0.135***	0.904 ± 0.014***	2.00 ± 0.021***	2.505 ± 0.007***
HFD control	8.40 ± 0.021***	0.849 ± 0.008***	1.635 ± 0.026***	2.65 ± 0.019***
HFD+Diabetes+Limonene (250mg/kg)	5.385 ± 0.059***	0.736 ± 0.007***	1.786 ± 0.006***	1.57 ± 0.009***
HFD+Diabetes+ Metformin (200mg/kg)	7.025 ± 0.06***	0.795 ± 0.0158***	1.685 ± 0.008***	1.48 ± 0.011***
HFD+Limonene (250mg/kg)	6.087 ± 0.03***	0.712 ± 0.009***	1.472 ± 0.018***	1.632 ± 0.014***
HFD+Metformin (200mg/kg)	6.172 ± 0.015***	0.758 ± 0.006***	1.535 ± 0.007***	1.538 ± 0.014***
HFD+Diabetes+ Extract 400mg/kg	5.79 ± 0.020***	0.734 ± 0.0008***	1.732 ± 0.007***	1.54 ± 0.0037***
HFD+ Extract 400mg/kg	6.23 ± 0.01***	0.714 ± 0.0008***	1.431 ± 0.006***	1.61 ± 0.0033***

Table 3: End Organ weights, where values are expressed as mean ± SEM. where *P < 0.05, **P<0.01, ***P<0.001 and ^nsP>0.05 when compared to HFD+Diabetic Control and HFD control

Fig1. Shows the Body weight gain (%) and fasting blood glucose (mg/dl) where Values are expressed as mean ± SEM. where *P < 0.05, **P<0.01,***P<0.001 and ^nsP>0.05 when compared to HFD+Diabetic Control and HFD control.

Table4. Biochemical Parameters

Groups	TBARS (MDA nmol/mg protein)	CAT (nmol H₂O₂-Consumed/min/mg protein)	SOD(IU/mg protein)	GSH (µmol of Phosphorus liberated/min/mg of Protein)
Normal control	0.317 ± 0.0004	37.65 ± 0.34	1.842 ± 0.01	27.79 ± 0.135
HFD+Diabetic Control	0.714 ± 0.001***	16.20 ± 0.317 ***	1.258 ± 0.008***	8.86 ± 0.107***
HFD control	0.699 ± 0.004***	19.16 ± 0.161***	1.238 ± 0.007***	7.67 ± 0.127***
HFD+Diabetes+Limonene (250mg/kg)	0.439 ± 0.005***	33.575 ± 0.327***	1.35 ± 0.011***	28.65 ± 0.348***
HFD+Diabetes+ Metformin (200mg/kg)	0.386 ± 0.003***	39.55 ± 0.196***	1.454 ± 0.011***	29.26 ± 0.303***
HFD+Limonene (250mg/kg)	0.409 ± 0.003***	34.04 ± 0.2904***	1.377 ± 0.004***	27.76 ± 0.28***
HFD+Metformin (200mg/kg)	0.375 ± 0.001***	37.70 ± 0.198***	1.450 ± 0.012***	30.31 ± 0.24***
HFD+Diabetes+ Extract 400mg/kg	0.413 ± 0.0009***	34.14 ± 0.077***	1.37 ± 0.0013***	28.80 ± 0.017***
HFD+ Extract 400mg/kg	0.392 ± 0.0009***	35.43 ± 0.068***	1.38 ± 0.0011***	28.185 ± 0.016***

Table 4: Biochemical Parameters, where values are expressed as mean ± SEM. where *P < 0.05, **P<0.01, ***P<0.001 and ^nsP>0.05 when compared to HFD+Diabetic Control and HFD control.

Fig. 2-Histological sections of fat tissues. Normal control, group with no treatment shows normal cell size and regular morphology. HFD-DC slide, high fat diet+diabetic group show the deteriorative changes in the fat tissue. HFD-C slide, high fat diet control group shows the swollen fat tissues. HFD-DC+Limonene slide, high fat diet fed diabetic group treated by limonene shows the changes with normal limit. HFD-DC+ Metformin slide, high fat diet fed diabetic group treated by Metformin shows the changes in normal limit with some congestions. HFD-C+Limonenesilde, high fat diet fed non-diabetic group shows the changes within the normal limit. HFD-C+Metforminslide, high fat diet fed non-diabetic group shows the changes within the normal limit. HFD-DC+Extractslide, high fat diet fed diabetic group treated by Citruslimetta extract shows the changes within the normal limit. HFD-C+Extract slide high fat diet fed non-diabetic group treated by Citruslimetta extract shows the changes within the normal limit.

DISCUSSION:

Results of human studies have concluded that exceeding fat consumption is associated with body weight gain that can lead to obesity and other related metabolic disorders. Animal rodent readily gain weight when fed with high fat diets making them useful tools for studying obesity³⁵. Nowadays plants are gaining their place in medicinal area as they have been used in traditional way of treating many diseases and currently available drugs used for hypolipidemia have a number of side effects associated with them^36,37.

The sweet lime (Citrus limetta), has been used in the Indian traditional medicine, in the Ayurvedic system of medicine. Fruit peel turn up to be the main source of flavonoids, pectin and essential oils³⁸. D-limonene which is the main constituent among citrus peel biochemical compounds possess the various properties like antimicrobial properties, primarily the exhibition of antibacterial activity against gram positive bacteria and also increases the effectiveness of sodium benzoate as a preservative^39,40. Because of its pleasant citrus fragrance it is used as additive in perfumes, soaps, foods, chewing gum and beverages. D-limonene, listed in the code of federal regulations and generally recognized as safe (GRAS) for a flavoring agent. It act as solvent of cholesterol, d-limonene has been clinically used to dissolve cholesterol-containing gallstones. Also used to relieve heartburn, because of its potential for neutralization of gastric acid and it promotes healthy peristalsis. D-limonene has well-established chemo preventive activity against many types of cancers. Phase-I clinical trials show a partial response in patient of breast cancer and stable disease for more than six months in three patients with colorectal cancer¹³.

The present study reported that oral administration of high fat diet (20 g/d/rat) to the Wistar albino rats for a period of 28 days produced significant obesity in rats as evidenced by increased in body mass index and body weight gain which is supported by Altunkaynak²⁶ who reported that BMI was significantly increased in rats with high fat diet fed for 8 weeks as compared to the control group i.e. (from 3.2 ± 0.3 kg/m^ to 5.6 ± 0.5 kg/m^), and Matsuo et al⁴¹ reported that body weight gain was greater in beef tallow diet group than in the other dietary groups.

D-limonene (200mg/kg), citrus peels extract (400mg/kg) and standard drug 200mg/kg (i.e Metformin) significantly reduced the BMI and body weight gain. This may be due to decrease in food intake and that leads to decrease in calorie intake⁴² and it is shown by studies that weight reduction is the means of reducing coronary risk incidence, high blood pressure and also to increase the level of HDL-Cholesterol in body⁴³.

Serum glucose levels were significantly increased in the HFD group (i.e. group II) as compared with those in the normal healthy control group (i.e. group I). Chang et a l,⁴⁴observed that diet-induced obesity dysregulated glucose homeostasis and causes hyperglycemia. Most abundant terpene D-limonene and extract significantly reduced the amount of elevated serum glucose in which extract remain a step ahead of limonene giving the indication that there may be more constituents that possess the power to reduce the serum glucose level in blood. Studies shown that glucose lowering effect of the drug may be due to increased peripheral glucose utilization and also may be due to inhibition of proximal tubular reabsorption mechanism of glucose in kidney⁴⁵.

There was significant increase in the lipids (TC, TGs, LDL-C, and VLDL-C) levels in the HFD group as compared to the normal healthy control group. Lavie and Milani⁴⁶ indicated that obesity adversely affects plasma lipids, especially by increasing TC, LDL- C, VLDL-C, TGs and decreasing the level o f HDL-cholesterol. The HFD might lead to an increase in the synthesis of phospholipids and cholesterol esters in rats⁴⁷. D-limonene and citrus peel extract significantly reduced the level of TC, LDL-C, VLDL-C, TGs and increased the level of HDL-C. Results show that citrus peel extract performed better in decreasing the level of bad cholesterols and increasing the levels of good cholesterol. The act of lipid lowering that was shown in the above study may be due to the presence of plant sterol as it works by reducing the absorption of cholesterol and thus fecal excretion of cholesterol is increased ultimately decreasing the level of systemic cholesterol⁴⁸.

Obesity along with hyperlipidemia is one of the conditions that decrease antioxidant capacity; obesity decreases the antioxidant defense by lowering the levels of antioxidant enzymes [catalase, glutathione peroxidase (GPx) and glutathione reductase (GRd)⁴⁹. It has been reported that high levels of fat increase fat-mediated oxidative stress and decrease antioxidative enzyme activity⁵⁰. Groups treated with D-limonene and Citrus peel extract were reported to be containing less level of oxidative stress markers in comparison with the other untreated control groups. This antioxidant activity shown by Citrus limetta and D-limonene might be due to their capacity of activating antioxidant enzymes⁵¹

CONCLUSION:

In accordance with the results obtained in the present study it may be confirmed that presence of active phyto-constituents in the citrus peel extract such as terpenes, flavonoids, alkaloids and glycosoids was the reason to lower the hyperlipidemia in the present study, however, a better result was shown by the peel extract when compared to the result obtained from a single terpene compound, D-limonene. While in the comparative evaluation of plant derived extract and drug to the synthetic drug, better readings of the result were seen leaning towards the synthetic drug, Metformin. To conclude all, the effect of Citrus limetta and metformin was studied in experimental rats, high fat diet were used to induce hyperlipidemia, while streptozosin was used to induce diabetes. The administration of Citrus limettainto the hyperlipedimic rats and hyperlipedemic plus diabetic rats significantly reduced total cholesterol, LDL, VLDL, Atherogenic index, blood glucose, body weight, Body mass index, TBARS while the level of HDL, CAT, SOD, GSH was increased. The administration of metformin in hyperlipedimic rats and hyperlipedemic plus diabetic rats revealed the significant reduction of total cholesterol, LDL, VLDL, Atherogenic index, while the level of HDL, CAT, SOD, GSH was increased as metformin is a well-established anti-diabetic drug. The result was supported by the average organ weight of liver, kidney, heart, visceral fat pad. This result suggests that Citrus limetta has a potent antihyperlipedimic property when compared to metformin, further clinical study can be performed to establish its effects in human.

AUTHOR’S CONTRIBUTION:

All the authors have contributed equally in the collection of data and to assemble information in writing of the manuscript.

ACKNOWLEDGMENTS:

Author thanks Prof. Syed Waseem Akhtar, Hon. Chancellor and Prof. Aqil Ahmad, Hon. Vice Chancellor for providing excellent research facility in the university. The university has provided a manuscript communication number for further communication (IU/R&D/2019-MCN000592).

CONFLICT OF INTEREST:

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Received on 15.03.2019 Modified on 27.03.2019

Research J. Pharm. and Tech. 2019; 12(3): 1244-1250.

DOI: 10.5958/0974-360X.2019.00207.5