The Effect of Capsaicin on the Metabolic Rate of Male Mus musculus

Linda Ko

Department of Biological Sciences

Saddleback College Mission Viejo, CA, 92692

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the chemical component in chili peppers that gives them heat. The purpose of this experiment was to examine the effect of capsaicin on the metabolic rate in male Mus musculus. To calculate the metabolic rate, the production of CO2 is used. The objective of the study was to see what the impact of capsaicin would have on the basal metabolic rates (BMR) of mice. Twelve mice were used at different times to examine the BMR of mice with no substances, a control and experimental solution. The control and experimental groups were stomach fed via a gavage tube. They were then put into a CO2 analyzer. The hypothesis being tested is that the consumption of capsaicin would have an effect on the metabolic rates of the mice. The average BMR of the mice without any substances was 0.419 mLCO2/g/min (±0.0310 SEM, N=12). The average BMR of mice with the control solution was 0.230mLCO2/g/min (±0.0284 SEM, N=12). The average BMR of mice with the experimental capsaicin solution was 0.341mLCO2/g/min (±0.0149 SEM, N=12). A One-Way Analysis of Variance test was used and the results indicate that there was a significant difference in the metabolic rate between the subject groups (p<0.0001. Tukey’s HSD Post Hoc test showed significant differences between all groups (p<0.05)

Introduction

Capsaicin (C18H27NO3), a chemical compound (8- methyl-N-vanillyl-6-nonenamide), is the key component in red chili peppers that gives your mouth a kick once it’s consumed (Hursel and Westerterp- Plantenga, 2009). This chemical compound is extremely pungent and irritates the skins of humans and animals. Capsaicin is notably found as a major pungent factor in the fruits of Capsicum and it has been stated that capsaicin increases the catecholamine secretion, energy outflow, and also reduces body fat in perspective of a long term treatment (Ohnuki et al., 2001). Capsaicin, a major component in red hot peppers, is a trigger for the body to undergo thermogenesis. Thermogenesis is a process in which the body temperature is increased due to many biochemical and metabolic events. (Kobayashi et al, 1998) With an increase in thermogenesis, the body’s metabolic rates are raised; therefore, stored fat cells in the body are utilized as energy to support the body’s increased metabolic rate, resulting in weight loss.

In a previously published study done by Ohnuki and his team (2001) at Kyoto University, researchers were able to see the result of M. musculus loss of

weight. Their study was successful; however, we differ in the intentions of our study. They were examining the long term effects of capsaicin on weight loss, and we were studying the effects on their metabolic rates. Ohnuki and his team also gave the mice CH-19 Sweet, the fruit of a non-pungent pepper, capsaicin that contains small amounts of capsaicinoids (Hursel and Westerterp-Plantenga, 2009; Kawabata et al., 2009; Ohnuki et al.,2001).

The objective was to determine if the basal metabolic rates (BMR) of the mice would be affected when given capsaicin. BMR is the metabolic rate of a fasting mammal resting at a normal temperature in a dark room, in a conscious state. The hypothesis being tested is that the mice given the capsaicin would have an effect on BMR compared to the two other groups.

Methods

Twelve male M. musculus were purchased and used as both the control and experimental group. They were kept in the guest bathroom at Linda Ko’s house where they remained for 8 days prior to the start of the experiment. This was done to give them ample time to adapt to their environment and stabilize. The cages were placed in an isolated enclosure to prevent them from being scared by outside environmental factors. The room was blocked off from natural light; instead lights were turned on for 12 hours a day. Due to the mice being of a circadian species, the tests were given during times between 11:00 to 16:00 to avoid disturbances. They were given fresh food and water daily. Three hours prior to the runs with the CO2 analyzer, their food was taken away in to prevent any factors that may have arisen. Each mouse was weighed, and then had various colored rings drawn on their tails for identification. The CO2 analyzer had air flowing through the first chamber, where the mouse was contained. It then went down through a tube containing Drierite (Xenia, Ohio), then into the last chamber where a Pasco GLX Carbon Dioxide Gas sensor was placed. The sensor was connected to a Pasco GLX unit that generated a graph. A Pasco GLX barometer and temperature sensors were also used. A FoxBox (Sable Systems, Las Vegas) was used to generate flow rate (Figure 1).

The first trials were the mice without any substances. A time of one minute was used to stabilize the machine. The mouse was put into the first tube and remained inside the tube for five minutes with a towel covering it. The mouse was then taken out and the investigators had to wait until the graph dropped down to the stabilization rate. It was then recorded for another minute. Calculations for the amount of solution given to the mice were done prior to stomach feeding them. Shown in a previous study, 10 mg of solution was distributed per kg body weight. The control solution consisted of 3mL ethanol, 10 mL Polysorbate 80 (Tween 80) and enough NaCl to make 100 mL (Ohnuki et al. 2001, Kawabata et al. 2009). The experimental solution consisted of 1.0 mg capsaicin, 3 mL ethanol, 10 mL Polysorbate 80 (Tween 80) and enough NaCl to make a total volume of 100 mL. The pure capsaicin was purchased from Sigma Aldrich. The mice were fed their control and experimental solution via a gavage tube and then tested one hour after all of them had been fed. Six stainless steel gavage tubes were purchased from Instech Laboratories, model FTSS- 18S-51. The Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco, states the standard procedure of oral gavage in mice is done by appropriately inserting the gavage tube into the mice. The length from the tip of the head to the last rib must be determined in order to prevent puncturing a hole in their stomach. A mouse must be scruffed by grasping its skin over the shoulders, utilizing the researcher’s thumb, middle finger, and pinky specifically for holding the tail. The fore legs must be extended out to the side in to prevent them kicking the tube away. The index finger will be placed on top of the head to bring it back. Doing so will create a straight line through the neck and esophagus. The tube was placed on either side of the mouse, but made sure it was over the tongue. The tube went through the pharynx, while pressing the gavage tube to the roof of the mouth, causing the mouth to elevate the head and the esophagus to straighten. Put the tube in. The tube was taken out the same way put in. The data found was used to calculate the rate of CO2 produced and amount of O2 consumed.

The rate of carbon dioxide production was calculated using the following equation:

VCO2= (Ve-V1)(Flow Rate mL/min)(1)

This volume was correct to STPD using Boyle’s law. Assuming a respiratory quotient of 0.8, we converted rate of carbon dioxide consumption t=into oxygen consumption. All rates were divided by animal weight to give weight specific data.

Figure 1. System for measuring CO2 production

55 Saddleback Journal of Biology Vol. 9, Spring 2011

Results

The end result turned out that there was a statistically significant difference between the three groups, which consist of the same group of male mice, without any substances, control solution, and experimental solution. The mice without any of the substances showed a mean of 7.74 mLCO2 /min (± 0.0310 SEM, N=12). The mice with the 10% Polysorbate 80 in 0.9% NaCl solution had a mean of 4.32 mLCO2 /min (± 0.0284 SEM, N=12). And the mice that was given the 0.1mg/mL Capsaicin mixed with 10% Polysorbate 80 in 0.9% NaCl had a mean of 6.50 mLCO2 /min (±0.0149 SEM, N=12). Therefore, the mice that were given the Capsaicin solution resulted in a significant difference (p=0.00372) with a PostHoc of Mean (M) 1 vs. M2 (p<0.01), M1 vs. M3 (nonsignificant), M2 vs. M3 (p<0.05).

When the BMR was calculated, the mean of the mice without any substances turned out to be 0.419 mLCO2/g/min (± 0.0310 SEM, N=12), the mice given the control solution turned out to have a mean of 0.230 mLCO2/g/min (± 0.0284 SEM, N=12), and the mice given the experimental solution had a mean of 0.341 mLCO2/g/min (± 0.0149 SEM, N=12). There was a statistically significant difference (p<0.0001, ANOVA. PostHoc: M1 vs. M2 (p<0.01), M1 vs. M3 (nonsignificant), M2 vs. M3 (p<0.01).

In order to show the relations of the different groups a line graph and a bar graph was constructed (Figure 2 and Figure 3).

Figure 2. BMR measurements of each mouse for every run (p<0.0001, ANOVA. PostHoc: M1 vs. M2 (p<0.01), M1 vs. M3 (nonsignificant), M2 vs. M3 (p<0.01)).

Figure 3. The average BMR of the mice of the three groups (none 0.419 mLCO2/g/min (± 0.0310 SEM, N=12), control solution 0.230 mLCO2/g/min (± 0.0284 SEM, N=12), experimental solution 0.341

Discussion

The study demonstrated that the hypothesis tested was proved wrong even though there was a significant difference. Capsaicin does increase the BMR of the mice, however, the group without any substances had an elevated BMR, overriding the capsaicin group. As seen in Figure 2, the group that had no substances had a higher BMR value than the group with capsaicin. The investigators suggest that the reason why this occurred is because the run, where nothing was given, was the mouse’s first time in the constricted chamber and the mice may have panicked. In Ohunki’s experiment, he and his team had an increase in oxygen consumption for the group that was given capsaicin (p<0.05). In the experiment conducted by the investigators, they too got a significant difference of O2 consumption (p=4.74 x 10-4), and also for CO2 production (p=3.72 x 10-3) and BMR (p<0.0001). P values were analyzed to determine if there is a significant difference. In order to run the values under ANOVA, each mouse did all three runs, but not all in the same day.

In future experiments, the investigators should make sure that the equipment’s being used for their project are working properly and let the mice be accustomed to the chamber beforehand.

Acknowledgements

Iwould like to thank Professor Steve Teh and Dr. Tony Huntley for all of their help during this project.

Literature Cited

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Kawabata F, Inoue N, Masamoto Y, Matsumura S, Kimura W, Kadowaki M, Higashi T, Tominaga M, Inoue K, Fushiki T. NonPungent Capsaicin Analogs (Capsinoids) Increase Metabolic Rate and Enhance Thermogensis via Gastrointestinal TRPV1 in Mice. Biosci Biotechnol Biochem. 2009 Dec;73(12):2690-2697. Epub 2009 Dec 7.

Kobayashi, Akiko, Osaka, Toshimasa, Namba, Yoshio, Inoue ,Shuji, Lee, Tai Hee, Kimura, Shuichi. Capsaicin activates heat loss and heat production simultaneously and independently in rats. American Journal of Physiology. (1998 July) 275:R92-R98.

Ohnuki, K., Haramizu, S., Oki K., Watanabe, T., Yazawa, S. Admisitration of Capsiate, a Non- Pungent Capsaicin Analog, Promotes Energy Metabolism and Suppresses Body Fat Accumulation in Mice. Biosci Biotechnol Biochem. 2001 Aug;65(12):2735-2740. Epub 2001 Aug 20.