Capsaicinoids but Not Their Analogue Capsinoids Lower Plasma

Jul 31, 2014 - ABSTRACT: Capsaicinoids exist in chili peppers, whereas capsinoids are ... compounds found in the fruits of chili pepper plant Capsicum...
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Capsaicinoids but Not Their Analogue Capsinoids Lower Plasma Cholesterol and Possess Beneficial Vascular Activity Weihuan Huang,†,§ Wai San Cheang,‡ Xiaobo Wang,† Lin Lei,† Yuwei Liu,† Ka Ying Ma,† Fangrui Zheng,# Yu Huang,‡ and Zhen-Yu Chen*,† †

Food and Nutritional Sciences Program, School of Life Sciences, ‡School of Biomedical Sciences, and #Department of Chemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China § Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, China S Supporting Information *

ABSTRACT: Capsaicinoids exist in chili peppers, whereas capsinoids are present in some sweet peppers. The present study investigated the effects of capsaicinoids and capsinoids on plasma lipids, relaxation of the aorta, atherosclerotic plaque development, and fecal sterol excretion in hamsters fed a high-cholesterol diet. Five groups of male hamsters were given the control diet or one of the four experimental diets containing 1.3 mmol of capsaicinoids (NL), 2.6 mmol of capsaicinoids (NH), 1.3 mmol of capsinoids (OL), or 2.6 mmol of capsinoids (OH), respectively. Results showed capsaicinoids but not capsinoids could decrease plasma total cholesterol (TC), reduce the formation of atherosclerotic plaque, and relax the aortic artery. This was accompanied by a 28−175% increase in fecal excretion of acidic sterols in hamsters fed the diets containing capsaicinoids. Similarly, capsaicinoids but not capsinoids could decrease the pad weights of epididymal and prerenal adipose tissues. It was concluded that capsaicinoids but not capsinoids could favorably modulate plasma lipids and possess beneficial vascular activity. KEYWORDS: aorta, capsaicinoids, capsinoids, cholesterol, atherosclerosis, pepper

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INTRODUCTION Capsaicinoids refer to a group of well-known pungent compounds found in the fruits of chili pepper plant Capsicum, whereas capsinoids are the nonpungent analogues of capsaicinoids isolated in the fruits of sweet pepper Capsicum CH-19.1,2 Capsaicinoids consists of mainly two isomers, namely, capsaicin and dihydrocapsaicin, whereas capsinoids also have two major isomers, capsiate and dihydrocapsiate (Figure 1). The structural difference is that capsaicinoids are fatty acid amides linked with vanillylamine, whereas capsinoids are fatty acid esters linked with vanillyl alcohol. Health benefits associated with the consumption of capsaicinoids have been well reported. It has been shown that capsaicinoids can promote weight reduction by enhancing fat oxidation and increasing adrenergic activity and energy expenditure.3 Research has also demonstrated that capsaicinoids have some beneficial effect on the human cardiovascular system, possess some antitumor activity, and also decrease plasma lipids.4−8 Like capsaicinoids, capsinoids are also able to reduce body fat by increasing thermogenesis9 and possess some antitumor activity.4 Capsaicinoids are beneficial for people who love spicy foods, whereas capsinoids may be an alternative for people who cannot tolerate spicy foods. In view of the structural similarity between capsaicinoids and capsinoids, the present study was the first to examine if capsaicinoids and capsinoids were both able to reduce plasma cholesterol concentration and formation of atherosclerosis plaque. We also sought to ascertain whether capsaicinoids and capsinoids could improve endothelial function and possess beneficial vascular activity. The major findings were that only capsaicinoids but not capsinoids could © 2014 American Chemical Society

reduce the plasma cholesterol concentration and possessed vasorelaxation activity.

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MATERIALS AND METHODS

Capsinoid Synthesis. Capsinoids are not commercially available in large quantities. We therefore had to use capsaicinoids as starting materials to synthesize their corresponding capsinoids. Capsaicinoids containing 51.3% capsaicin and 46.2% dihydrocapsaicin were purchased from Henan Bis-Biotech Co. Ltd. (Henan, China). Vanillyl alcohol was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Lipase enzyme (Lipozyme 435) was purchased from Guangzhou Minagyao Trading Co. Ltd. (Guangzhou, China). To obtain a capsinoid mixture of 51.3% capsiate and 46.2% dihydrocapsiate, the mixture of acyl donors was prepared with capsaicinoids according to the method described by Kobata et al.10 and Dong et al.11 In brief, 3 g of capsaicinoids was dissolved in 1 L of 4 N HCl in 67% methanol and was refluxed for 48 h. Then n-hexane was added to the solution to extract the acyl donors (Figure 1). Capsinoids were synthesized by adding 10 g of Lipozyme 435 into a solution of acetone (1 L) containing vanillyl alcohol (7.7 g) and acyl donors (14.2 g). After being incubated in a shaker at 25 °C for 48 h, the reaction mixture was filtered to remove the enzymes, and the solvent was evaporated in a rotary evaporator. The crude capsinoids were loaded onto a silica column and eluted using a solvent mixture of hexane and ethyl acetate (6:1, v/v), producing 4 g of capsinoids. Structures of synthesized capsinoids were confirmed using NMR (Supporting Information Supplementary Figures 1 and 2). HPLC analysis found that capsinoids consisted of 52.4% capsiate and 44.9% dihydrocapsiate Received: Revised: Accepted: Published: 8415

June 18, 2014 July 31, 2014 July 31, 2014 July 31, 2014 dx.doi.org/10.1021/jf502888h | J. Agric. Food Chem. 2014, 62, 8415−8420

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Figure 1. Synthesis of capsinoids (capsiate and dihydrocapsiate) from capsaicinoids (capsaicin and dihydrocapsaicin). Modified from reference 11. with such a ratio being similar to that in sweet pepper (Supporting Information Supplementary Figure 3). Diet. Five diets were prepared by modifying the formulation as we previously described.12 The control diet was prepared by mixing all powdered ingredients (g/kg): corn starch, 507; casein, 241; lard, 50; sucrose, 119; mineral mix, 40, vitamin mix, 20; geltatin, 20; DLmethionine, 1; cholesterol, 2 (Table 1). The four experimental diets

12/12 h light/dark cycle. The hamsters were allowed free access to food and water. Blood sample was taken from the retro-orbital sinus under light anesthesia using a mixture of ketamine, xylazine, and saline (4:1:5; v/v/v) after overnight fasting at weeks 0, 3, and 6. At the end of experiment, all hamsters were killed under carbon dioxide anesthesia; the abdomen was opened with blood being sampled from the aorta into a syringe. The fecal output from each hamster was collected and pooled at weeks 1 and 6, then freeze-dried, ground, and saved for neutral and acidic sterol analyses. Plasma Lipids. Plasma total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triacylglycerols (TG) were measured using their respective commercial enzymatic kits from Infinity (Waltham, MA, USA) and Stanbio Laboratories (Boerne, TX, USA), respectively. Non-HDL cholesterol (non-HDL-C) was calculated by deducting HDL-C from the TC. Atherosclerotic Plaque. The aorta was used to assess atherosclerosis as we previously described.12 In brief, the thoracic aorta was cut open vertically followed by staining in a 10 mL ethanol solution containing 0.5 g of Sudan III for 3 h. Afterward, Sudan III was washed with distilled water three times and scanned with a table scanner. The area of atherosclerotic plaque was measured with the aid of a computer image analysis program (Sigma Scan Pro 5.0, SPSS, Inc., Chicago, IL, USA).12 Hepatic Cholesterol. Total cholesterol in the liver was quantified as previously described.15 Total liver cholesterol was extracted using a mixture of chloroform/methanol (2:1, v/v). 5α-Cholestane as an internal standard was added during the extraction. Other lipids were saponified and removed. The nonsaponified fraction containing cholesterol was saved, and free cholesterol was converted to its TMS-ether derivative by a commercial TMS reagent (Sigma, Hong Kong). Analysis of the cholesterol TMS-ether derivative was performed in an SAC-5 column in a Shimadzu GC-14B GLC equipped with a flame ionization detector. Hepatic cholesterol was calculated according to the amount of internal standard 5α-cholestane added. Fecal Neutral and Acidic Sterols. The total fecal output was collected and saved for the measurement of total fecal neutral and acidic sterols as previously described.15 In brief, total fecal sterols were extracted with 5α-cholestane being added as an internal standard for quantification of fecal neutral sterols, whereas hyodeoxycholic acid was added as an internal standard for the quantification of total acidic sterols. First, the fecal samples were saponified, the total neutral sterols were extracted into cyclohexane, and the neutral sterols were converted into their TMS derivatives. Second, total fecal acidic sterols in the aqueous phase after cyclohexane extraction were also extracted

Table 1. Composition of the Control Diet and Four Experimental Diets Containing 1.3 mmol of Capsaicinoids (NL), 2.6 mmol of Capsaicinoids (NH), 1.3 mmol of Capsinoids (OL), or 2.6 mmol of Capsinoids (OH), Respectively ingredient (per kg diet)

control

NL

NH

OL

OH

corn starch (g) casein (g) sucrose (g) lard (g) mineral mixture AIN-76 (g) vitamin mixture AIN-76A (g) gelatin (g) DL-methionine (g) cholesterol (g) capsaicinoids (mmol) capsinoids (mmol)

507 241 119 50 40 20 20 1 2 0 0

507 241 119 50 40 20 20 1 2 1.3 0

507 241 119 50 40 20 20 1 2 2.6 0

507 241 119 50 40 20 20 1 2 0 1.3

507 241 119 50 40 20 20 1 2 0 2.6

were similarly prepared by mixing the control diet with 1.3 mmol of capsaicinoids (NL), 2.6 mmol of capsaicinoids (NH), 1.3 mmol of capsinoids (OL), or 2.6 mmol of capsinoids (OH), respectively. Such amounts of capsaicinoids and capsinoids added into diets were equivalent to a daily dose of 7−14 mg/kg body weight, which could be achieved by humans who love chili hot foods.13 All five diets contained 0.2% cholesterol by weight and had 68.2, 26.4, and 5.4% energy from carbohydrate, protein, and fat, respectively. Hamsters. We chose hamsters as model because cholesterol metabolism in hamsters is close to that in humans.14 The experiments were approved and conducted in accordance with the guidelines set by the Animal Experimental Ethics Committee, The Chinese University of Hong Kong. Golden hamsters (n = 50, body weight = 116 ± 6 g) were divided into five groups and fed the control or one of the four experimental diets as described above for 6 weeks. All hamsters were given fresh diet daily and housed in an animal room at 23 °C with a 8416

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Table 2. Change in Food Consumption, Body Weight, and Relative Organ Weights in Hamsters Fed the Control and the Four Experimental Diets Diets Containing 1.3 mmol of Capsaicinoids (NL), 2.6 mmol of Capsaicinoids (NH), 1.3 mmol of Capsinoids (OL), or 2.6 mmol of Capsinoids (OH), Respectively, at Week 6a control food intake (g/day) body weight (g) initial final relative weight (% body weight) liver heart kidney testis epididymal fat pad perirenal fat pad a

NL

NH

OL

OH

8.5 ± 0.3

7.9 ± 0.4

7.8 ± 0.3

8.5 ± 0.4

7.8 ± 0.3

116.7 ± 4.3 125.8 ± 4.7

115.1 ± 6.6 121.9 ± 5.0

118.9 ± 5.2 124.4 ± 1.0

116.4 ± 6.9 124.1 ± 8.6

116.5 ± 7.8 121.7 ± 8.5

5.0 ± 0.3 0.33 ± 0.02 0.9 ± 0.1 2.7 ± 0.3 1.9 ± 0.3a 1.2 ± 0.3a

4.9 ± 0.3 0.32 ± 0.01 0.9 ± 0.1 2.6 ± 0.4 1.6 ± 0.3b 1.0 ± 0.2ab

5.2 ± 0.5 0.32 ± 0.01 1.0 ± 0.1 2.3 ± 0.8 1.5 ± 0.2b 0.8 ± 0.2b

5.2 ± 0.2 0.32 ± 0.02 0.9 ± 0.1 2.2 ± 0.5 1.8 ± 0.3ab 1.1 ± 0.2ab

5.1 ± 0.3 0.33 ± 0.02 0.9 ± 0.1 2.5 ± 0.5 1.7 ± 0.2ab 1.1 ± 0.1ab

Data are expressed as the mean ± SD; n = 10. Means in the same row with different letters differ significantly at P < 0.05.

Table 3. Changes in Plasma Total Cholesterol (TC), Triacylglycerols (TG), HDL-Cholesterol (HDL-C), Non-HDL-Cholesterol (Non-HDL-C), Non-HDL-C/HDL-C, HDL/TC, Liver Total Cholesterol, and Aortic Atherosclerotic Plaque in Hamsters Fed the Control or One of the Four Experimental Diets Containing 1.3 mmol of Capsaicinoids (NL), 2.6 mmol of Capsaicinoids (NH), 1.3 mmol of Capsinoids (OL), or 2.6 mmol of Capsinoids (OH), Respectivelya control

NL

NH

OL

OH

week 0 TC (mg/dL) HDL-C (mg/dL) TG (mg/dL) non-HDL-C (mg/dL) non-HDL-C/HDL-C HDL/TC

127.9 ± 9.7 65.3 ± 13.8 79.0 ± 24.4 62.6 ± 14.1 1.0 ± 0.4 0.5 ± 0.1

127.9 ± 9.5 64.0 ± 14.3 80.7 ± 38.4 63.9 ± 14.4 1.0 ± 0.4 0.5 ± 0.1

125.1 ± 10.2 65.1 ± 20.7 77.8 ± 30.1 60.0 ± 26.6 1.1 ± 0.6 0.5 ± 0.2

127.3 ± 13.3 65.2 ± 23.6 80.4 ± 36.9 62.1 ± 19.9 1.2 ± 0.8 0.5 ± 0.2

127.8 ± 12.0 67.7 ± 17.7 79.1 ± 23.6 60.0 ± 17.0 1.0 ± 0.6 0.5 ± 0.1

TC (mg/dL) HDL-C (mg/dL) TG (mg/dL) non-HDL-C non-HDL-C/HDL-C HDL/TC

240.6 ± 27.6a 117.6 ± 13.6a 150.8 ± 63.4ab 123.1 ± 20.9a 1.05 ± 0.19ab 0.48 ± 0.05ab

217.1 ± 29.7ab 102.2 ± 12.2b 138.3 ± 31.5b 114.9 ± 18.6ab 1.12 ± 0.08a 0.47 ± 0.05b

202.8 ± 36.3b 100.5 ± 19.2b 121.0 ± 33.9b 102.4 ± 20.4b 1.04 ± 0.18ab 0.49 ± 0.05ab

237.8 ± 25.2a 120.8 ± 11.8a 181.7 ± 43.5a 117.2 ± 18.8ab 0.98 ± 0.16b 0.51 ± 0.04a

239.5 ± 29.7a 120.8 ± 10.0a 163.1 ± 41.7ab 118.6 ± 15.6ab 0.98 ± 0.11b 0.51 ± 0.04ab

TC (mg/dL) HDL-C (mg/dL) TG (mg/dL) non-HDL-C (mg/dL) non-HDL-C/HDL-C HDL-C/TC liver cholesterol (mg/g) aortic atherosclerotic plaque (% area)

216.2 ± 35.3a 114.7 ± 13.5a 171.2 ± 13.2 101.2 ± 25.6abc 0.88 ± 0.18b 0.53 ± 0.05b 53.5 ± 16.6a 25.7 ± 4.6a

203.9 ± 30.1ab 108.8 ± 21.5a 168.8 ± 42.3 95.2 ± 15.8bc 0.86 ± 0.19b 0.53 ± 0.05b 43.2 ± 12.0b 18.1 ± 6.3b

179.3 ± 56.7b 90.4 ± 28.4b 169.4 ± 56.2 88.9 ± 27.2c 0.98 ± 0.29ab 0.50 ± 0.15ab 37.9 ± 9.7b 17.8 ± 6.7b

220.9 ± 30.5a 103.8 ± 15.9ab 173.5 ± 43.5 117.0 ± 20.4a 1.18 ± 0.22a 0.47 ± 0.05a 50.6 ± 11.2ab 26.8 ± 5.5a

219.2 ± 33.0a 109.0 ± 16.9a 151.5 ± 39.2 110.2 ± 22.1ab 1.01 ± 0.19a 0.5 ± 0.05a 56.6 ± 10.4a 24.1 ± 3.0a

week 3

week 6

a

Data are expressed as the mean ± SD; n = 10. Means in the same row with different letters differ significantly at P < 0.05. three times. Phenylephrine at 1 μmol/L was used to induce steady contraction of the ring and the effect of capsaicinoids or capsinoids was then determined. Culture of Human Umbilical Cord Vein Endothelial Cells (HUVECs). HUVECs (CC-3317, Clonetics, Lonza, Walkersville, MD, USA) were grown in endothelial cell growth medium (Clonetics) supplemented with bovine brain extract and 1% penicillin and streptomycin. Cells were grown in 75 cm2 flasks and maintained at 37 °C in a 95% humidified air/5% CO2 atmosphere. Medium was changed every 2 days. Confluent cells were passaged by trypsinization (0.25% trypsin with 2.5 mmol/L EDTA in PBS). Experiments were performed on cells at passage 4−8 when 80−90% confluency was achieved. Measurement of Intracellular Ca2+ Concentration by Fluorescence Microscopy. HUVECs were treated at 37 °C for 60 min with 10 μmol/L calcium indicator fluo-4 AM (Molecular Probes,

and similarly converted into their TMS derivatives. Individual neutral and acidic sterol TMS derivatives were then subjected to GC analysis on an SAC-5 column in a Shimadzu GC-14B GLC equipped with a flame ionization detector. Endothelium-Dependent Relaxation in Rat Mesenteric Artery. To assess the effect of capsaicinoids and capsinoids on the functionality of the artery, endothelium-dependent relaxation was measured in rat mesenteric artery as previously described.16 In brief, rats were sacrificed by CO2 suffocation. Superior mesenteric arteries were dissected out and placed in ice-cold Krebs solution (mmol/L): 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 D-glucose. Arteries were cleaned of adhering tissue and cut into ring segments of 2 mm in length. Rings were suspended in a myograph (Danish Myo Technology, Aarhus, Denmark) for recording of changes in isometric tension. After a 60 min equilibration period, each ring was first contracted by 60 mmol/L KCl and then washed in Krebs solution 8417

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Table 4. Cholesterol Intake and Changes in Fecal Sterol Profile in Hamsters Fed the Control or One of the Four Experimental Diets Containing 1.3 mmol of Capsaicinoids (NL), 2.6 mmol of Capsaicinoids (NH), 1.3 mmol of Capsinoids (OL), or 2.6 mmol of Capsinoids (OH), Respectively, at Week 6a cholesterol intake (mg/hamster/day) fecal neutral sterols (mg/hamster/day) coprostanol coprostanone cholesterol dihydrocholesterol total sterols fecal acidic sterols (mg/hamster/day) lithocholic acid deoxycholic acid chenodeoxycholic acid total acid sterols a

control

NL

NH

OL

OH

16.98 ± 0.54

15.78 ± 0.72

15.58 ± 0.68

16.98 ± 0.85

15.98 ± 0.69

0.32 0.31 0.82 0.17 1.62

± ± ± ± ±

0.10 0.15 0.58 0.10 0.57

0.49 0.26 0.71 0.12 1.58

± ± ± ± ±

0.31 0.26 0.37 0.04 0.59

0.64 0.10 1.01 0.17 1.91

± ± ± ± ±

0.10 0.06 0.22 0.02 0.28

0.47 0.09 0.58 0.17 1.32

± ± ± ± ±

0.07 0.04 0.23 0.03 0.30

0.38 0.09 0.56 0.13 1.15

± ± ± ± ±

0.04 0.03 0.09 0.02 0.11

1.39 0.37 0.47 2.23

± ± ± ±

0.46b 0.16 0.21b 0.71b

1.66 0.44 0.74 2.85

± ± ± ±

0.62b 0.15 0.46b 1.23b

4.00 0.58 1.55 6.13

± ± ± ±

0.78a 0.07 0.26a 0.7a

1.45 0.41 0.61 2.48

± ± ± ±

0.53b 0.32 0.27b 0.58b

1.30 0.28 0.48 2.08

± ± ± ±

0.63b 0.17 0.20b 0.54b

Data are expressed as the mean ± SD; n = 10 each group. Means in the same row with different letters differ significantly at P < 0.05.

Eugene, OR, USA) in normal physiological saline solution containing (mmol/L) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES (pH ∼7.4). Fluorescent images were obtained under a fluorescence microscope (Olympus, Tokyo, Japan) (excitation, 495 nm; emission filter, 505−525 nm) every 5 s continuously. Changes in intracellular Ca2+ concentration triggered by the addition of capsaicinoids or capsinoids was expressed as F1/F0 ratio, where F1 is the fluorescence intensity at a specific time point and F0 is the signal measured at the starting point of image recording. Statistical Analysis. Data are expressed as the mean ± SD (n = 10 for the experiment on hamsters and n = 4 for the experiment on aortic relaxation). One-way analysis of variance followed by using Fisher’s LSD method was used to statistically evaluate differences in plasma lipids, aortic functions, and atherosclerotic plaque among the five groups.

Fecal Neutral and Acidic Sterols. In the colon, cholesterol is subjected to microbial bioconversion, producing series of cholesterol microbial metabolites with coprostanol and dihydrocholesterol being the major products. To simplify the presentation, only data on fecal samples collected from week 6 are reported. As shown in Table 4, capsaicinoids had a trend of increasing the excretion of fecal cholesterol and coprostanol; however, the effect was statistically insignificant. Capsinoids had no effect on total excretion of fecal neutral sterols. With regard to fecal acidic sterol excretion, it was evident that capsaicinoids but not capsinoids increased their excretion. Endothelium-Dependent Relaxation. In phenylephrinecontracted superior mesenteric arteries, capsaicinoids beneficially induced relaxation in a dose-dependent manner. By contrast, capsinoids were without any beneficial effect (Figure 2A). Ca2+ Influx. Acute exposure to capsaicinoids stimulated an increase of intracellular Ca 2+ level in HUVECs in a concentration-dependent manner (Figure 2B), whereas capsinoids led to little rise of intracellular Ca2+ (Figure 2C).

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RESULTS Food Intake and Body and Organ Weights. No differences in food intake, body weight, and relative weights of heart, kidney, and testis were seen among the five groups (Table 2). However, the relative weights of epididymal and perirenal fat pats were smaller in NL and NH groups fed the diets containing capsaicinoids compared with those in the control hamsters (P < 0.05). However, no significant difference was seen among the control, OL, and OH hamsters. Plasma TC, HDL-C, Non-HDL-C, and TG. The five groups of hamsters had similar concentrations of plasma TC, HDL-C, non-HDL-C, and TG at week 0 (Table 3). When the experiment reached the end of weeks 3 and 6, NH groups had plasma TC, non-HDL-C, and TG concentrations significantly lower than the control hamsters. It was clear that capsaicinoids decreased plasma cholesterol in a dose-dependent manner (P < 0.05). However, two capsinoids groups, OL and OL, had no effect on plasma TC, non-HDL-C, and HDL (Table 3). Liver Cholesterol and Atherosclerotic Plaque. Two capsaicinoids groups, NL and NH, significantly decreased hepatic cholesterol concentrations in a dose-dependent manner compared with the control hamsters, whereas two capsinoids groups, OL and OH, had no effect on hepatic cholesterol concentration (Table 3). A similar trend in atherosclerotic plaque was seen. Hamsters fed NL and NH diets had less atherosclerotic plaque, whereas those fed OL and OH groups had levels of atherosclerotic plaque similar to those of the control hamsters (Table 3).

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DISCUSSION Capsaicinoids are present in large quantity in most chili peppers and can induce strong pungent sensations, whereas capsinoids are not pungent and are present in only a small amount in sweet pepper CH-19.1 It has been estimated that the consumption of chili pepper by humans who like hot spicy foods could reach 2.5−20 g/person/day, equivalent to 0.5−4 mg capsaicinoids/kg body weight/day provided that capsaicinoids in chili pepper are 1% by weight.17 In contrast, no data are available for us to assess the intake of capsinoids in humans. Structurally, capsaicinoids and capsinoids are similar except that the former are the amides of vanillylamine with fatty acids, whereas the latter are the esters of vanillyl alcohol with fatty acids (Figure 1). For individuals who like spicy foods, capsaicinoids provide a pleasurable sensation upon ingestion. In addition to the use for releasing pain, capsaicinoids also act as an antioxidant and increase the oxidation of body fat, thus being used as a target for obesity management.18,19 However, the burning and pungent sensation may prevent their wide application, particularly for people who dislike or cannot tolerate spicy foods. In this regard, capsinoids could be an alternative if they had bioactivity similar to that of capsaicinoids because they are nonpungent. In fact, capsinoids have been 8418

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sterols. It was clear that both capsaicinoids and capsinoids had no significant effect on the excretion of fecal neutral sterols. However, only capsaicinoids but not capsinoids could increase the excretion of fecal bile acids (Table 4). To be specific, it was found that dietary capsaicinoids at 1.3 and 2.6 mmol in the diet could increase the excretion of fecal bile acids by 28 and 175%, respectively. The present result was in agreement with that of Zhang et al., who found that the gavage of capsaicinoids at 10 mg/kg body weight could increase the excretion of fecal bile acids by 13% in rats given a 2% cholesterol diet.21 In contrast, no significant effect of capsinoids on the excretion of fecal bile acids was seen. Because excess cholesterol in mammals is usually disposed of via bile fluid duct or by conversion to bile acids, the increase in bile acid excretion is likely the primary mechanism underlying the plasma TC-reducing activity of capsaicinoids. Capsaicinoids have been shown to decrease atherosclerotic plaque and possess some beneficial effect on aortic function.13,21 To ascertain if capsinoids also had similar activity, we studied the vasodilatory effect of both capsaicinoids and capsinoids in the mesenteric arteries from rats. It was clear that only capsaicinoids but not capsinoids beneficially induced the relaxation of mesenteric arteries. Moreover, acute treatment of capsaicinoids triggered a favorable increase of intracellular Ca2+ concentration in endothelial cells, which in turn promotes nitric oxide formation and causes endothelium-dependent relaxation. It has been shown that the favorable effect of capsaicinoids on the aorta is at least in part mediated by their inhibition on either activity or expression of cylco-oxygenase 2 (COX-2).13 Up-regulation of COX-2 is associated with endothelial dysfunction, atherosclerotic plaque, and inflammation.22 In summary, capsaicinoids and capsinoids are naturally present in chili or sweet peppers; however, their biological properties differ in many ways. Under the present physiological conditions, capsaicinoids were able to favorably modulate plasma lipoprotein profile and possess beneficial vascular activity, whereas analogue capsinoids had no such properties. It was therefore concluded that nonpungent capsinoids could not be used as an alternative of pungent capsaicinoids in relation to their favorable modification of plasma lipids and cardiovascular benefit.

Figure 2. Effect of capsaicinoids and capsinoids on the relaxation of rat mesenteri artery and intracellular Ca2+ concentration in human umbilical cord endothelial cells (HUVECs). (A) Capsaicinoids but not capsinoids caused vasodilatation on phenylephrine (1 μmol/L)contracted mesenteric arteries. (B) Representative tracing and (C) summarized data show that capsaicinoids stimulated Ca2+ entry in HUVECs in a concentration-dependent manner, whereas capsinoids had little effect. Data are expressed as the mean ± SEM; n = 4.

similarly proposed to be a dietary supplement in weight management, as they are equally effective in increasing the energy expenditure by increasing fatty acid oxidation in vivo.20 The present results demonstrated that capsaicinoids could reduce the size of both epididymal and prerenal fat pads, which were the two visible adipose tissues in rodents (Table 2). However, the effects of dietary capsinoids on two fat pads were found to be insignificant. This discrepancy with previous studies was probably because the duration of the present study was only 6 weeks, which might be not long enough for capsinoids to exert any antiobesity effect in hamsters. The present study was the first time to compare the effect of capsaisinoids on plasma TC with that of capsinoids. Results clearly demonstrated that capsaicinoids but not capsinoids were able to decrease plasma TC in hamsters fed a 0.2% cholesterol diet (Table 3). Reduction in plasma TC caused by dietary capsaicinoids was accompanied by a decrease in both liver cholesterol and atherosclerotic plaque. The result regarding the effect of capsaicinoids on plasma TC was in agreement with that of Liang et al.13 In another study, it was found that 0.015% capsaicinoids in the diet could reduce serum TC by 23% in Sprague−Dawley rats fed a 30% fat diet.7 In the present hamster model fed a diet containing 5% lard and 0.2% cholesterol, we found that reduction in plasma TC could reach 16%. To explain why capsaicinoids but not capsinoids could decrease plasma TC, we quantified the excretion of fecal total

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ASSOCIATED CONTENT

* Supporting Information S

Supplementary Figures 1−3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(Z.-Y.C.) Fax: (852) 2603-7246. E-mail: zhenyuchen@cuhk. edu.hk. Funding

This project was supported by a grant from the Hong Kong Research Grant Council (CUHK 462813). Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Yazawa, S.; Suetome, N.; Okamoto, K.; Namiki, T. Content of capsaicinoids and capsaicinoid-like substances in fruit of pepper (Capsicum annuum L.) hybrids made with ‘CH-19 sweet’ as a parent. J. Jpn. Soc. Hortic. Sci. 1989, 58, 601−607. 8419

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dx.doi.org/10.1021/jf502888h | J. Agric. Food Chem. 2014, 62, 8415−8420