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.
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.
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 disease1,2.
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.
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.
2. Animals
3. Experimental Procedure
4. Sample collection
5. Statistical analysis
NOTE: All experimental results should be representative from three independent assays, expressed as mean ± standard deviation.
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).
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.
The authors have nothing to disclose.
This research was supported by Instituto Politécnico Nacional México.
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 |