Rapid Model to Evaluate the Anti-Obesity Potential of a Combination of Syzygium aromaticum (Clove) and Cuminun cyminum (Cumin) on C57BL6/j Mice Fed High-Fat Diet

Summary

This work presents a protocol to explore the anti-obesity effect of two plants used together, for a 5-week duration. There was a combined administration of the extract and high-fat-diet (HFD) in the obese mice. The method can promote the benefits of plants in the treatment of obesity.

Abstract

Several studies have demonstrated that the phytochemical contents of plants are potential anti-obesity agents. In this study we examine the effect of using a combination of dry buttons from Syzygium aromaticum and seeds from Cuminum cyminum (CC) on C57BL6/J mice induced with obesity via high-fat-diet (HFD). The aim of this study is to demonstrate that the method proposed in the study reduced obesity significantly after several weeks of experimentation. The extract from both plants was extracted using ultrasound to enhance the extraction of phytochemicals. Optimum extraction conditions were obtained with ethanol as follows: 50:50 v/v water with an ultrasound power of 300 W, and ultrasonication time of 30 minutes. The simultaneous administration of the CC extract in HFD for 5 weeks led to the regulation of lipid profiles (cholesterol and triglycerides), reduction of food intake, weight gain, adipose tissue and liver weight. Findings obtained by this obese model indicate that CC extract can prevent obesity. Compared with the traditional 16 weeks method (8 weeks to get fat, and 8 weeks to lose weight), similar results were obtained in the present study obese model in less time of experimentation.

Introduction

Excess body fat accumulation is a characteristic of obesity. The disequilibrium between energy intake and consumption leads to the storage of excess energy in adipocytes, which is related to metabolic risk factors for hyperglycemia and insulin resistance in type 2 diabetes, hypertension, hypercholesterolemia, and cardiovascular disease^1,2.

Natural products with minimum side effects and low cost have received increased attention since previous studies have reported bioactive phytochemicals and potential anti-obesity agents with mechanisms that reverse or delay metabolic syndrome and associated pathologies⁸.

Several medicinal plants have been studied to prevent obesity and related diseases. Among them, Syzygium aromaticum has been investigated for its anti-obesity potential in in vitro treatment on 3T3-L1 cells and in vivo treatment on mice fed with a high-fat diet¹¹. In addition, significant anti-overweight effects were observed in a multi-center open trial of Cuminum cyminum on extremely obese subjects¹². In this study, C57BL6/J mice was used to investigate the experimental fast model to evaluate a potential anti-obesity agent using a combination of both edible plants.

Protocol

All animal experiments were approved by the Animal Experiment Committee of Escuela Nacional de Ciencias Biologicas (IPN; Protocol No. 2732).

1. Preparation of the extract of the combination of cloves and cumin

NOTE: The edible plants, Syzygium aromaticum (clove) and Cuminum cyminum (cumin) were purchased from Central de Abastos CDMX, Mexico.

1. Grind 500 g of seeds from the plants and perform ultrasonic-assisted extraction. Sonicate them (320 Watts; 24 kHz frequency, 30 min) with ethanol:water (60:40 v/v) at a temperature range of 30 ± 4 °C.¹³
2. Filter the solids using Whatman number 2 filter paper under vacuum and concentrate the extract with a rotary evaporator.
3. Measure the UV-vis spectrum of the extract of both plants (CC).

2. Animals

1. Use male C57BL6/J mice of six weeks old (48 in total, body weight 24-29 g) for the study.
2. House the animals in groups of eight in standard laboratory conditions: temperature (20-22 ± 1 °C), relative humidity (45-54 ± 2%), lighting (08:00-20:00 h) with food and water ad libitum. Acclimate the mice in these conditions for a week before the experiment.

3. Experimental Procedure

1. Prepare the high fat diet¹⁴ as in Table 1.
2. At the beginning of the test, subject each of the groups formed (n = 8) to different treatments: Feed Group 1 on a normal diet; Group 2, on high-fat diet; Group 3, on high-fat diet + 100 mg/kg of CC extract; Group 4, on high-fat diet + 250 mg/kg of CC extract; Group 5, on high-fat diet + 450 mg/kg of CC extract; Group 6, on high-fat diet + phentermine (an antiobesity drug used as positive control)¹⁵.
3. Feed all the groups of mice for 5 weeks and record the amount of food consumed daily.
   1. In a test tube with stopper, homogenize the extract or the drug with the dose of water corresponding to the group to which the treatment is being administered. This is done with vortex shaker.
   2. Once homogenized, add all the corresponding treatment into the drinker of each group.

4. Sample collection

1. Measure both food intake and body weight once per week. Fast the mice overnight for a 5-week period.
2. Sacrifice them by cervical dislocation and necropsy.
3. After the mice have been sacrificed, immobilize them in a supine position.
4. Using clean surgical forceps and scissors, locate and lift the central skin near the genitals and make a small 0.2 mm incision.
5. Insert the flat scissors horizontally into the incision and carefully separate the abdominal skin from the abdominal wall.
6. With the forceps, lift the central skin and with the surgical scissors cut it at the level of the rib cage. Then cut the skin above both of the hind limbs to facilitate the collection of adipose tissue.
7. Ensure the collection of all adipose tissue from the body by blunt dissection with scissors and forceps. Once collected, place the adipose tissue in aluminum foil. Remember to collect the fat around the reproductive organs. Avoid the accumulation of hair and skin to not alter the results.
8. To access the visceral adipose tissue, cut the abdominal muscle with surgical scissors from the genitals to the rib cage, making an incision in the abdominal muscles from the genitals towards the mouse's back.
9. Perform a hepatectomy to remove 100% of the liver. To externalize the liver, remove unnecessary organs and cut the hepatic veins and arteries to separate the liver from the rest of the body. As the last step, precisely extract the gallbladder from the liver¹⁶.
10. Measure hypercholesterolemia (cholesterol) and hypertriglyceridemia (triglycerides) using commercial assay kits according to the manufacturer's indications.

5. Statistical analysis

NOTE: All experimental results should be representative from three independent assays, expressed as mean ± standard deviation.

1. Calculate standard error with a one-way ANOVA analysis of variance followed by Tukey's range test. Consider a P< 0.05 as statistically significant.

Representative Results

Ultrasonic-assisted extraction
The optimum extraction condition for the extraction was ethanol:water (50:50, v/v) with an ultrasound power of 300 W and an ultrasonication time of 30 min. This way, the extraction was faster than conventional extraction methods (Table 2). The extract showed an intense absorption peak at 320 nm in UV-visible spectra (Figure 1).

Food intake and body weight
The changes in the body weight of the mice given different concentrations of the extract (100, 200 and 450 mg/kg) for 5 weeks are shown in Figure 2A. The percentages of weight reduction in groups 3, 4 and 5 were 12.2%, 30%, and 41%, respectively. This shows the lowest body weight gain was observed in all the groups; however, the HFD group had an increase of 2.1x compared to the normal fat diet group. These data indicate that a 450 mg/kg dose of CC is more effective than 30 mg/kg phentermine (39% reduction) in reducing body weight gain.

The mean food intake at the end of the experiment in the HFD + CC (450 mg/mL) was 31% lower than that in the obese group (HFD), with 1.3 g reduction in food intake compared to the obese group (Figure 2B). Food intake in the HFD-fed mice was higher by 1.5-fold than in the normal control, thus it significantly (P<0.05) decreased in all CC groups in a range of 7.14% to 31%. The diet efficiency rate in HFD group was 16%, which was significantly different (P< 0.05) from that of the control group (ND) at 11%. These results demonstrate that CC can reduce HFD-induced body weight gain.

Adipose tissue and liver weight
The weight of epididymal WAT, subcutaneous fat and perirenal WAT adipose tissues was higher in the HFD- group, but not in the HFD-CC groups (Table 3). The liver weight was significantly (P<0.05) decreased in the CC group (100 to 450 mg/kg) with a range of 15%-28.4% compared to those of the HFD group (Table 4).

Liver lipids and serum
The levels of serum triacylglycerol (TG) and total cholesterol (TC) after 5 weeks of treatment are shown in Figures 3A and 3B, respectively. The triglycerides and total cholesterol level were significantly elevated by 1.32-fold and 1.31-fold, respectively, in HFD groups compared to those in the ND groups. The increased levels of plasma TG and TC in the HFD diet were significantly attenuated by the administration of 200 and 450 mg/kg/day CC extract by 18.18% and 22.7% for TG and 16.41% and 20.61% for TC, respectively compared to the control (ND).

Figure 1. UV-vis spectrum of clove and cumin extract (CC) Please click here to view a larger version of this figure.

Figure 2. Effects of CC on: (A) body weight (g), (B) food intake in High-Fat Diet C57BL6/J mice at 4 weeks of treatment. Data are represented as mean ± SD. For each group, n=8. Different letter showed significant difference (p<0.05). Please click here to view a larger version of this figure.

Figure 3. Effects of CC on: (A) Triglycerides, (B) Total cholesterol. All experimental data were Mean ± SD mice (n = 8). Mice were treated with 100, 200 and 450 mg/kg of CC for daily oral administration with simultaneous feeding of HFD for 5 weeks. Different letters indicate significant differences among all the groups (P < 05). Please click here to view a larger version of this figure.

Ingredient	Quantity (g/kg)
Casein	140
L-Cysteine	1.8
lard	120
Soybean oil	40
Maltodextrin 10	150
Sucrose	450
Cellulose	50
Vitamins and Minerals	Tablet equivalent
Choline Bitartrate	2.5

Table 1. High fat diet for C57BL6/J mice.

Power	370 Watts
Water temperature	30 °C (86 °F)
Extraction time	30 min
Solvent concentration	50% ethanol/50% distilled water
Number of extractions	2 extractions

Table 2. Optimal ultrasound conditions for the preparation of the clove and cumin extract

Group	Epididymal WAT (g)	Perirenal WAT (g)	Subcutaneous fat (g)	Fat/Body Weight (g/100)
ND	0.32 ± 0.04a	0.05 ± 0.06a	0.08 ± 0.01b	1.22 ± 0.09a
HFD	2.30 ± 0.09b	0.93 ± 0.07b	0.15 ± 0.04a	6.38 ± 1.02b
HFD + CC 100 mg/kg	1.26 ± 0.08b	0.50 ± 0.05b	0.12 ± 0.02a,b	5.12 ± 1.93b
HFD + CC 200 mg/kg	0.90 ± 0.05b	0.35 ± 0.04b	0.11 ± 0.04a,b	3.21 ± 0.32b
HFD + CC 400 mg/kg	0.42 ± 0.07a	0.16 ± 0.02c	0.09 ± 0.03b	2.30 ± 0.45c
HFD + PHE 30 mg/kg	0.49 ± 0.05a	0.19 ± 0.08c	0.10 ± 0.01b	2.41 ± 0.18c

Table 3. Effect of CC in diet-induced obese mice on white adipose tissue weight. Each value is expressed as the mean ± SD (n = 8). Different letter showed significant difference (P<0.05) vs white adipose tissue (WAT)

Group	Liver weight (g)
ND	1.01 ± 0.05a
HFD	1.48 ± 0.08b
HFD + CC 100 mg/kg	1.26 ± 0.09b
HFD + CC 200 mg/kg	1.12 ± 0.03b
HFD + CC 400 mg/kg	1.06 ± 0.06a
HFD + PHE 30 mg/kg	1.08 ± 0.07a

Table 4. Effect of CC in diet-induced obese mice on liver tissue weight. Each value is expressed as the mean ± SD (n = 8). Different letter showed significant difference (P<0.05) vs white adipose tissue (WAT).

Discussion

In this study we evaluated, for the first time, the effect of oral administration of a combination of cloves and cumin (CC) extract on lipid profiles and obesity in mice fed with a high-fat diet for 5 weeks. Findings indicated that the HFD groups showed a significantly higher body weight gain compared to the ND group, which showed that the induction of obesity in the obese model was successful. The administered dose of CC (100, 200 and 450 mg/kg/day) produced a reduction in food intake and body weight. Consequently, the reduction in food intake must be due to a decline in the animals' appetite.

The reduction of dyslipidemia is very important to prevent obesity-related disorders. In our study, the total cholesterol levels and triglycerides were lower in the CC group compared to those in HFD and phentermine groups used as positive control. Findings suggested that CC may inhibit hyperlipidemia and its complications.

Our study showed that the HFD group had increased body fat, including abdominal and liver fat that were significantly reduced by the CC extract supplementation. This shows that the CC extract treatment on the HFD mice reduces lipid absorption due to its anti-adipogenic activity.

In this study, we evaluated a fast model to determine the anti-obesity effect of an extract from a combination of edible plants, Syzygium aromaticum (clove) and Cuminum cyminum (cumin) on C57BL6/J mice, which rapidly developed obesity with a high-fat diet. The CC treatment significantly prevented the development of obesity and ameliorated hyperlipidemia induced by HFD given to the mice. Altogether, findings in this research provide solid evidence that this fast method of 5 weeks duration developed in our laboratory could be used to assess whether a plant has the capacity to be a potential agent for treating obesity.

Materials

Name	Company	Catalog Number	Comments
Distilled Water	Any vendor	n/a	Available for other vendors as well
Ethanol	Fermont	6063	99.8% purity
Diet Ingredients
Casein	Any vendor	n/a
Cellulose	Any vendor	n/a
Centrum balance multivitamin	Pfizer	n/a
Choline Bitartrate	Any vendor	n/a
L- cystein	Sigma Aldrich	168149	Available for other vendors as well
Lard	Any vendor	n/a
Maltodextrin 10	Any vendor	n/a
Pellets Nutricubos	Purina	n/a	Available for other vendors as well
Soybean Oil	Any vendor	n/a
Sucrose	Any vendor	n/a
Extraction Equipment
Rotavapor	Buchi	R-300
Shimadzu UV-1800 UV/Visible Scanning Spectrophotometer	Cole Parmer	T-83400-20	Available for other vendors as well
Ultrasonic Unit	Elma	TI-H-20	Available for other vendors as well
Vacuum pump	Buchi	V-100