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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1978-1983
Laboratory of Nutrition Chemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Intravenous injection of stearoyl vanillylamide (C18-VA), a nonpungent capsaicin (CAP) analog, enhances adrenaline secretion significantly and as effectively as CAP in rats. Because swimming capacity was enhanced by CAP in mice due to CAP-induced adrenal catecholamine secretion, we investigated the effects of oral administration of C18-VA on swimming capacity using an adjustable-current water pool. Male Std ddY 6-wk-old mice were fed a commercial diet for this study and one group was orally administered C18-VA via a stomach tube. Treated mice were able to swim longer before exhaustion than the control mice (62.9 ± 5.6 vs. 49.6 ± 7.0 min, P < 0.05). The swimming capacity of two groups administered C18-VA (0.02 and 0.033 mmol/kg) was significantly greater than that of those administered vehicle alone, (P < 0.05). Substance P concentration in cerebrospinal fluid, which is involved in pain transmission and is the first direct measure of pungency, was not affected by C18-VA administration. In an experiment examining the effects of C18-VA on serum adrenaline concentration, adrenaline was significantly greater in C18-VA treated mice than in controls at 2-h post-dose (C18-VA group, 26.09 ± 2.82; control group 13.29 ± 0.96 µg/L, P < 0.01). In a separate study free fatty acids in serum were elevated in treated mice at 2-h post-dose (P < 0.01). While serum glucose concentration was not affected. These results suggest that C18-VA increased swimming capacity of mice via adrenaline release, independent of pungency. In addition, the present study suggests the usefulness of its application to humans.
KEY WORDS: capsaicin analog · swimming capacity · adrenaline · mice stearoyl vanillylamide
Capsaicin (CAP3, N-vanillyl-8-methyl-6-nonenamide), the pungent component of hot pepper, has the combined structure of a fatty acid and vanillylamide (Crombie et al. 1955 Many CAP analogs exist among the natural capsacinoids and several more analogs composed of a long alkyl chain were found to be minor components of capsaicinoids in Capsicum fruit (Suzuki and Iwai 1984
In the present study, we investigated the effect of C18-VA (stearoyl vanillylamide), a nonpungent CAP analog, on the swimming capacity of mice.
Materials.
3,4-Dihydroxybenzylamide (DHBA) and adrenaline were from Aldrich Chemical Co. (Milwaukee, WI) and Sigma Chemical Co. (St. Louis, MO), respectively. The substance P (SP) enzyme immunoassay kit was purchased from Cayman Chemical Co. (Ann Arbor, MI). The CAP analog (C18-VA) was synthesized by the condensation of corresponding carboxyl chlorides (Sigma Chemical) with vanillylamine hydrochloride (VA, Aldrich Chemical Co.) in pyridine (Watanabe et al. 1994 Animal treatment.
Male Std ddY 6-wk-old mice (Japan SLC, Hamamatsu, Japan) were used and housed in standard cages (33 × 23 × 12 cm, 6 mice/cage) under controlled conditions of temperature (22 ± 0.5°C), humidity (50%) and lighting (light from 0700 to 1900 h). During the study period, the mice were given free access to water and a commercial diet (MF; Oriental Yeast Co., Ltd., Tokyo, Japan), containing the following (g/kg diet): water, 80; protein, 246; fat, 56; fiber, 31; carbohydrate 523. The care and treatment of experimental animals conformed to the Kyoto University (Kyoto, Japan) guidelines for the ethical treatment of laboratory animals.
Current swimming pool.
An adjustable-current water pool was used to determine swimming capacity. The details were described previously (Matsumoto et al. 1996 Evaluation of the swimming capacity of mice.
To avoid circadian variations in physical activity, experiments were carried out from 1100 to 1700, a period in which minimal variation of swimming capacity was confirmed in mice (Matsumoto et al. 1996 Experimental design.
Experiment 1: Effects of CAP and C18-VA administration on swimming capacity of mice. The commercial diet was fed to 11 mice (6-wk-old). On d-4, they were forced to swim for 30 min at a flow rate of 6 L/min to accustom them to swimming and on d-0 randomly divided into two groups (Group 1, n = 5; Group 2, n = 6). The first measurement of swimming time was evaluated 2 h after the single oral administration of 400 µL C18-VA (0.033 mmol/kg body weight, Group 1) or vehicle (Group 2) via a stomach tube. On d-2, the swimming time of Group 1 (n = 5) administered C18-VA at the first swimming capacity measurement was evaluated 2 h after vehicle administration and also that of Group 2 (n = 6) administered vehicle at the first measurement was evaluated 2 h after C18-VA administration. In this study, we defined this measurement method as cross-experimentation. The swimming time data for C18-VA and vehicle of total 11 mice were compared by the paired Student's t test. In the experiment the effect of CAP on swimming capacity was also measured by cross-experimentation as the previously described method using 10 mice (Group 1, n = 5; Group 2, n = 5). C18-VA or CAP was suspended in 9 g/L of NaCl solution containing 30 g/L of ethanol and 100 g/L of Tween 80, and placebo (control)
Statistical analysis.
Data were expressed as means ± sem. Comparisons between the means of vehicle and C18-VA determined by cross-experimentation were performed by the paired Student's t test (Expts. 1, 2). Differences in means between placebo and four groups (30, 60, 120, 180 min) were assessed using one-way analysis of variance (ANOVA) and the Tukey-Kramer Multiple Comparison Test (Expt. 3). Differences in the adrenaline, serum glucose and FFA concentration between treated and placebo groups for each time point were tested by the unpaired nonparametric (Mann-Whitney) test (Expts. 4, 5). Differences in SP concentration among three groups (placebo, CAP, C18-VA) for each time point were analyzed by one-way ANOVA with Tukey-Kramer Multiple Comparison Test (Expt. 6). An INSTAT software package (Macintosh Version 2.00, GraphPad Software Inc., San Diego, CA) was used for calculation and a level of P < 0.05 as the criterion for statistical significance.
The effect of C18-VA on swimming endurance capacity.
Swimming capacity was greater in mice administered C18-VA (Fig. 2, Expt. 1). Mice given C18-VA (0.033/mmol/kg) 2 or 3 h before swimming swam longer than those administered vehicle (Fig. 3, Exp. 3) (P < 0.05), and the swimming time did not differ from that in mice given CAP (data not shown). The swimming capacity determined 2 h after administration was significantly greater in mice administered C18-VA dosages of 0.02 and 0.033 mmol/kg (Fig. 4, Expt. 2).
The effects of CAP and C18-VA on the concentration of SP in CSF (Exp. 6).
The SP concentration in CSF of mice administered CAP but not in those administered C18-VA was significantly different from that of the mice administered vehicle. (Fig. 5.). Therefore, C18-VA is a nonpungent CAP analog that is not nociceptive in mice.
Serum Adrenaline Concentration in Mice Administered CAP or C18-VA (Expt. 5).
The adrenaline concentration in serum of CAP- or C18-VA-treated mice was significantly greater than that of the vehicle-administered mice at 120 min post-dose. (Fig. 6).
The effect of C18-VA on serum glucose and FFA concentrations (Expt. 4).
The serum glucose concentration was unaffected by C18-VA but was elevated immediately after CAP administration compared to that of vehicle-administered mice (Fig. 7). Serum FFA concentrations were greater at 1, 2 and 3 h post-dose in mice administered CAP than in those treated with vehicle and at 2 h post-dose in those administered C18-VA (Fig. 8).
We reported that the swimming capacity of mice was increased by ingestion of CAP, which is the major pungent component of hot red pepper, using an adjustable-current swimming pool (Kim et al. 1997
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Nelson and Dawson 1923, Suzuki and Iwai 1984). We showed that swimming capacity was increased by oral ingestion of CAP (Kim et al. 1997), observed only when CAP was ingested 2 h before swimming. The oral administration of CAP produced a marked biphasic increase in serum adrenaline concentration of 30 min and 2 h after ingestion, and the endurance-enhancing effect of CAP was not observed in adrenalectomized mice. These results suggest that the effect of CAP was due to the enhancement of fatty acid metabolism via adrenaline secretion from the adrenal gland. However, the amount of CAP required to increase swimming endurance capacity was 0.033 mmol/kg, which is equivalent to about 100 g fresh red pepper in humans. It is impossible for humans to ingest such an amount and mass ingestion of hot red pepper has also been reported to cause gastric disorders (Monsereenusorn et al. 1982, Surh and Lee 1996, Suzuki and Iwai 1984).
, Todd et al. 1977). CAP analogs with a long alkyl chain were reported to have little or no pungency (Todd et al. 1977 and Watanabe et al. 1994). Furthermore, Watanabe et al. (1994) showed that the CAP analogs with a C14 to C20 side chain had no pungency (Fig. 1) and that like the pungent CAP, such nonpungent CAP analogs promoted adrenaline secretion. This suggests that the secretion of adrenaline is independent of pungency and that nonpungent CAP analogs may increase swimming capacity in mice as does CAP.
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Fig 1.
Structures of capsaicin (CAP) analogs.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Compounds with more than 95% purity, as verified by gas-liquid chromatography and high-performance chromatography (HPLC) were used in the experiment (Shimomura et al. 1989). Other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries (Osaka, Japan).
): acrylic plastic pool (90 × 45 × 45 cm) filled with water to a depth of 38 cm; a smooth and clear tank surface to prevent the animal from supporting itself while swimming; the pool current generated with a pump (type C-P60H; Hitachi, Tokyo, Japan). Water was returned to the pump through a narrow slit in a plastic pipe set on the bottom of the pool. The strength of the current was adjusted by changing the water flow by opening and closing the valve and was monitored with a water flowmeter (Ashida Co., Kyoto, Japan). The distribution of surface current speed was measured with a digital current meter (type SPC-5; Sanko Industry Co., Tokyo, Japan) at 12 points at equal intervals on the water surface. The water temperature was strictly maintained at 34°C by a water heater and thermostat. The high reproducibility and sensitivity of this apparatus were previously reported (Matsumoto et al. 1996).
). Mice were assessed as fatigued when they failed to rise to the surface of the water to breathe within a 7-s period. A period of longer than 7 s frequently resulted in drowning while less than 5 s reduced the reproducibility of the test (Matsumoto et al. 1996). The total swimming time until exhaustion was used as the index of swimming capacity.
20°C until measurement of free fatty acids (FFA) and glucose. Serum FFA were determined by the acyl CoA-synthetase and acyl CoA oxidase enzyme method with a commercial kit (NEFA C-Test; Wako, Wako Pure Chemical Industries). Glucose was assayed by a combination of mutase and glucose oxidase with a commercial kit (Glucose CII; Test Wako, Wako Pure Chemical Industries).
20°C until use. The serum sample was pretreated with aluminum oxide by the method of Anton and Sayre (1962). To serum samples (100 µL) were added 100 g/L Na2S2O5 (50 mL/L) and DHBA (40 µg/L) as the internal standard in 100 µL of 2 mol/L Tris-HCl buffer at pH 8.6 containing aluminum oxide (100 g/L). The mixture was shaken in a microtube mixer for 10 min, the supernatant was removed and the aluminum oxide washed twice with methanol and distilled water. Adrenaline was eluted with 60 µL of 0.5 mol/L HCl. The eluate was assayed by HPLC-EC as described previously (Watanabe et al. 1988).
70°C until assayed for SP by enzyme immunoassay (EI) kit.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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Fig 2.
The effect of capsaicin (CAP) and stearoyl vanillylamide (C18-VA) on swimming capacity of mice (Expt. 1). The swimming capacity of mice was measured at a flow rate of 7 L/min and analyzed by paired Student's t test. Values are means ± SEM, n = 10 or 11. * Significantly different from placebo, P < 0.05.
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Fig 3.
The effect of administration time of stearoyl vanillylamide (C18-VA) on swimming capacity of mice (Expt. 3). The swimming capacity of mice was measured at a flow rate of 7 L/min and analyzed by Tukey's comparison test. Values are means ± SEM, n = 18-26. Different letters indicate significant difference from placebo (P < 0.05).
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Fig 4.
The effect of stearoyl vanillylamide (C18-VA) concentration on swimming capacity of mice (Expt. 2). The swimming capacity was analyzed by a paired Student's t test. Values are means ± SEM, n = 9,10. * Significantly different from placebo, P < 0.05.
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Fig 5.
The effect of capsaicin (CAP) and stearoyl vanillylamide (C18-VA) on substance P (SP) concentration of cerebrospinal fluid (CSF) of mice (Expt. 6). Values are means ± SEM, n = 3 or 4. Different letters at each time point indicate significant differences among groups by Tukey's comparison test (P < 0.05).
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Fig 6.
Serum adrenaline concentration in mice administered stearoyl vanillylamide (C18-VA), capsaicin (CAP) or placebo (Expt. 5). The data were analyzed by the unpaired nonparametric test. Values are means ± SEM, n = 3-8. * Significantly different from placebo, P < 0.01.
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Fig 7.
Serum glucose in mice administered stearoyl vanillylamide (C18-VA), CAP or placebo (Expt. 4). The data were analyzed by the unpaired nonparametric test. Values are means ± SEM, n = 3-6. Significant difference from placebo, * P < 0.05, ** P < 0.01.
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Fig 8.
Serum fatty acid concentration in mice administered capsaicin (CAP), stearoyl vanillylamide (C18-VA) or placebo (Expt. 4). The data were analyzed by the unpaired nonparametric test. Values are means ± SEM, n = 5-10. Significant difference from placebo, * P < 0.05, ** P < 0.01.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). In that experiment the mean swimming time was significantly prolonged by CAP administered 2 h before swimming. Here we showed that this effect of CAP is due to CAP-induced adrenaline secretion that results in the sparing of muscle glycogen due to enhancement of fatty acid utilization (Kim et al. 1997). We also showed that oral administration of CAP (0.033 mmol/kg) caused a marked increase in serum adrenaline concentration in resting mice. These findings suggest that the increase in serum adrenaline secretion 2 h after CAP ingestion was advantageous to the mobilization and utilization of fat for the enhancement of swimming capacity in mice.
). The total amount of catecholamine secreted from adrenal medulla induced by the CAP analog with a C18 side chain was almost equal to that secreted in response to CAP administration. From these findings, we speculated that the mechanism of sensing the pungency of CAP and that of adrenal catecholamine secretion by CAP may not be the same, and we examined the swimming capacity-enhancing effect of the nonpungent compound, C18-VA.
, Nutt et al. 1980). C18-VA has no pungency in humans (Watanabe et al. 1994), but the pungency in mice has not been tested. Therefore, we measured the concentration of SP in CSF, which mediates the transmission of pungency induced by CAP (Gamse et al. 1979). As shown in Fig. 5, the SP concentration in CSF was significantly greater than in control 30 min after ingestion of CAP, but was not changed by ingestion of nonpungent C18-VA. This means that C18-VA has no pungency in mice.
). The second, slower stimulation of adrenal sympathetic efferent nerve activity was common to the pungent CAP and the nonpungent, CAP analog, C18-VA. Further investigations are required to elucidate the mechanisms underlying the adrenaline secretion induced by CAP and its analogs.
, Edrickson et al. 1987, Ivy et al. 1979). Costill et al. (1978) proposed that caffeine elevated the plasma catecholamine concentration that stimulated fat metabolism, either by the increase of adipose tissue and/or muscle triacylglyceride lipolysis and consequently FFA oxidation. Plasma FFA were elevated 1 h after caffeine ingestion in some studies (Essig et al. 1980, Powers et al. 1983, Sasaki et al. 1987, Tarnapolsky et al. 1989). The failure to increase FFA may be the result of an increased uptake by the muscle during exercise. The enhanced fat oxidation may subsequently reduce muscle glycogenolysis during exercise and may delay exhaustion in prolonged endurance exercise (Essig et al. 1980). Therefore, we examined whether the stimulation of adrenal secretion by C18-VA ingestion enhances the swimming capacity as does CAP. A significant increase in the swimming capacity was observed 2 h after C18-VA ingestion, when the serum adrenaline concentration was significantly increased. We also suggest that the C18-VA-induced increase in serum FFA, accompanied by the elevation of the serum adrenaline, may increase the utilization and/or uptake of serum FFA by the muscle. Furthermore, serum FFA after C18-VA administration were significantly greater than in placebo-treated mice, but the serum glucose concentration was not affected (Figs. 7, 8). Under such conditions, a sufficient amount of FFA was recruited without an increase of serum glucose, and this may be advantageous to increase endurance in mice. Increased fat oxidation prolongs exercise performance in mice.
). These facts and the present findings suggest that nonpungent C18-VA may be used as a nutritional aid to enhance the exercise capacity in humans. Detailed studies on C18-VA in hot pepper are underway.
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FOOTNOTES |
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Manuscript received 11 March 1998. Initial reviews completed 5 May 1998. Revision accepted 10 August 1998.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-adrenergic action on energy metabolism in rats: influence of capsaicin on oxygen consumption, the respiratory quotient, and substrate utilization.
Proc. Soc. Exp. Biol. Med.
1986;
183:250-256[Abstract]
a literature survey.
CRC Crit. Rev. Toxicol.
1982;
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