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.

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 o...

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 ...

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