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Effects of pure capsaicinoids (capsaicin and dihydrocapsaicin) on plasma lipid and lipoprotein concentrations of turkey poults
Atherosclerosis,
Elsevier
64 (1987)
Scientific
85
85-90
Publishers
Ireland,
Ltd.
ATH 03920
Effects of pure capsaicinoids (capsaicin and dihydrocapsaicin) on plasma lipid and lipoprotein concentrations of turkey poults J.A. Negulesco Departments
‘, S.A. Noel 2, H.A.I. Newman 2, E.C. Naber and D.T. Witiak 4
of ’ Anatom.v,
’ Pathology,
’ Pot&v
Science and 4 College of Pharmacv,
Columhtu,
3, H.B. Bhat 4
The Ohio State iJnioersrt_v.
OH 43210 (U.S.A.)
(Received 31 July. 1985) (Revised, received 15 August, 1986) (Accepted 27 August, 1986)
The effects of capsaicin and dihydrocapsaicin on blood lipid and lipoprotein concentrations were determined in two groups of turkeys. The first group was maintained on a cholesterol-free diet, while the second received a diet supplemented with 0.2% cholesterol. Daily administration of capsaicinoids occurred at a dose of 4 mg per animal. Neither drug had an effect on serum triglyceride concentrations in the total cholesterol, LDL-cholesterol and HDLanimals receiving the cholesterol-free diet. However, cholesterol concentrations were increased significantly, while VLDL cholesterol concentrations were decreased significantly by both drugs relative to controls. In the cholesterol-fed group triglycerides, total cholesterol and LDL-cholesterol decreased significantly with dihydrocapsaicin treatment. Both compounds reduced VLDL-cholesterol and increased HDL-cholesterol in the cholesterol-fed animals. Dihydrocapsaitin had a greater efficacy in producing benificial anti-hyperlipidemic effects in the cholesterol-fed animals.
Key words:
Capsaicin;
Cholesterol;
Dihydrocapsaicin;
Introduction Capsaicin, the acylamide of vanillylamine 8-methyl-6-trans-nonenoic acid [l] is one
and of 5
This investigation was supported, in part, by the Departments of Anatomy, Pathology and Poultry Science of the Ohio State University (OSU), the OSU Small Research Grant Program (Project 221543). and by the Central Ohio Heart Association (Project 714555). Correspondence address: Dr. J.A. Negulesco, Department of Anatomy, The Ohio State University, Columbus. OH 43210, U.S.A.
OO21-9150/R7/$03.50
‘9 1987 Elsevier
Scientific
Publishers
Ireland.
Lipoproteins;
Triglycerides;
Turkeys
analogs of the group of compounds called capsaicinoids (capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin) [2]. These compounds are widely distributed in nature and are part of the diet of various ethnic groups as spicy fruit of the genus Capsicum (red cluster pepper, cone pepper, short pepper, cayenne, chili and long yellow peppers) [3]. Consumption of red peppers by hyperlipidemic rats has been shown to decrease liver and serum cholesterol concentrations [4], and liver triglyceride concentrations [S]. Recently, we have Ltd
86 reported the effects of the parent compound, capsaicin (80% pure), on serum lipid concentrations of hypercholesterolemic turkeys [6] and rabbits [7]. In both models, capsaicin effectively decreased serum cholesterol concentrations. Serum triglyceride concentrations were decreased , while high density lipoprotein cholesterol (HDL-C) concentrations were significantly increased in capsaitin-treated rabbits. Because the previous work [4,6,7] involved a mixture of capsaicins (80% capsaicin and 20% dihydrocapsaicin) our present investigation was designed to ascertain, the effects of pure capsaicin and dihydrocapsaicin on serum lipid concentrations. Turkey poults were selected for this study since it was suggested that, although this animal model develops aortic atherosclerotic lesions over a relative short experimental period [8], there are some similarities to the aortic insults of man [9]. Materials and methods
Experimental design Forty-eight turkey poults (Melaegris gallopouo) received one-day post hatch were allowed to acclimatize under poultry house conditions: 36-24OC *, 45% relative humidity, a 12-h light, 12-h dark cycle, ad libitum food (turkey starter diet) and water consumption over a 7-day period. On day eight, the animals were divided randomly into two equal groups of 24 birds each and treated as follows for a 6 weeks experimental period: Group 1. Cholesterol-free poultry diet: A, = control subgroup: drug-free placebo (n = 8); B, = capsaicin (98% pure, Sigma Co.)-thated subgroup: 4 mg p animal p day (PAPD) (n = 8); C, = dihydrocapsaicin (98% pure) treated subgroup: 4 mg PAPD (n = 8). Group 2: 0.2 5%cholesterol-supplemented poultry diet: A, = control subgroup: drug-free placebo (n = 8); Bz = capsaicin-treated subgroup: 4 mg PAPD (n = 8); C, = dihydrocapsaicin-treated subgroup: 4 mg PAPD (n = 8). Divided in groups of 4 animals per cage, the turkeys were allowed unlimited access to water
* Temperature was 36°C the first week and dropped 2°C per week until it reached 24OC.
(trough). A sufficient amount of feed (200 g) was supplied daily, and feed consumption and/or spillage was determined. Animal body weight and fecal output were measured and recorded on a weekly basis. Diets The 0.2% cholesterol diet was prepared by mixing the cholesterol suspended in cholesterol-free vegetable oil, heating (56°C) and mixing the solution with the turkey starter diet. The control cholesterol-free diet included equivalent amounts of vegetable oil mixed into the turkey starter diet. Drug administration Capsaicin (Sigma Co.; 98% pure) was converted to dihydrocapsaicin in our laboratory. The dihydro-analog was determined to be 98% pure using NMR and mass spectrometry. Both compounds were suspended in 0.2 ml cholesterol-free vegetable oil, and were administered daily via buccal route (5 ml syringe adapted with a 5 cm long x 2 mm diameter blunt metal needle terminally fitted with a polyethylene cuff). Control animals received the drug-free suspension medium (0.2 ml cholesterol-free vegetable oil). Blood collection After anesthetizing the animals, blood samples taken by cardiac puncture (21-gauge needle into lo-ml syringes) were collected into 10 ml EDTAcoated tubes. Plasma was obtained by immediately transferring a portion of whole blood into a tube containing 200 ~1 of 0.2 M EDTA and centrifuging at 2000’ rpm (800 X g) for 10 min. Serum was obtained by allowing whole blood to clot in a separate tube and centrifuging in the same manner. Quantitative methodr Plasma cholesterol concentration was determined by the enzymatic method of Allain et al. [lo] and the serum triglyceride concentration was determined enzymatically by the method of Bucolo and David [ll]. High density lipoproteins (HDL) were separated from other lipoprotein fractions by the precipitation technique of War-nick et al. [12]. Dextran sulfate and Mg*+ were used to precipi-
87 Results
tate the low density (LDL) and very low density (VLDL) lipoproteins. The precipitate was separated by centrifugation at 4000 rpm (1600 X g) for 30 min at 4°C and high density lipoprotein cholesterol (HDL-C) was determined in the supernatant as previously described. Lipoproteins were also separated using isopychnic ultracentrifugation at the following densities: VLDL, 0.950-1.006; LDL, 1.006-1.063; HDL, 1.063-1.210. The specimens were centrifuged at 36000 rpm (104400 X g) for 24 h for the VLDL and LDL separation and 48 h for the HDL separation. Following dialysis with 0.004 M EDTA buffer (pH 7.5), the cholesterol concentration was determined in each fraction as previously described. Statistical analyses involved two-way ANOVA with post-hoc Dunnett’s test [13].
Mean body weight and weight gain in the final week of the study did not differ significantly among the subgroups of animals fed the cholesterol-free diet (Table 1). However, capsaitin-treated turkeys had significantly lower final body weight and weight gain than that of controls and dihydrocapsaicin-treated animals. Capsaicin had no effect on body weight in previous studies [4,6,7]. In the final week mean body weight gain of cholesterol-fed animals was greater with dihydrocapsaicin treatment compared to controls, although the final mean body weight was unchanged. Results of serum or plasma lipid analyses are presented in Table 2. Serum triglyceride concentration of capsaicin- or dihydrocapsaicin-
TABLE 1 COMPARISON OF CAPSAICIN (C) AND DIHYDROCAPSAICIN (D) ADMINISTRATION ON THE MEAN BODY WEIGHT, FOOD CONSUMPTION AND FECAL ELIMINATION OF TURKEY POULTS MAINTAINED ON A CHOLESTEROL-FREE OR A 0.2% CHOLESTEROL DIET FOR 6 WEEKS Mean f SD; all values in grams. Cholesterol-free Control Initial body wt Mean body wt at end of exp. period (week 6)
diet Ca
Da
91*12
86k
1307+ 12
1454F205
I
85+
16
0.2% Cholesterol
diet
Control
Ca
92k
8
1535& 117
1423+137
D” 89+
1397+87
6
92+
*
1524+ 128 ***
Mean body wt gain between week 5-6
301 f 88
344* 173
350+
Food consumption per animal/day (week 6)
111
113
109
106
109
113
Wet fecal mass per animal/day (week 6)
176
158
181
158
184
169
Dry fecal mass per animal/day (week 6)
34
33
39
33
29
36
a 4 mg/animal/day. * P -c0.05 between ** P i 0.01 between *** P c 0.05 between t P < 0.01 between
drug treatments and their control. drug treatments and their control. the two drug treatments. the two drug treatments.
86
361+
42
298+40
**
8
406k
40 *.+
88 TABLE
2
COMPARISON OF CAPSAICIN (C) AND DIHYDROCAPSAICIN (D) ADMINISTRATION ON THE MEAN PLASMA LIPID AND LIPOPROTEIN CONCENTRATIONS OF TURKEY POULTS MAINTAINED ON A CHOLESTEROL-FREE OR A 0.2% CHOLESTEROL DIET FOR 6 WEEKS Mean
*SD;
mg/dl. Cholesterol-free
diet
No drugs n=8
C” n=8
Da n=8
No drugs n=8
C” n=8
D” n=8
205 f 36
193 If: 16
191* 31
195k26
170*40
159*12
112_+ 9
131+10
143 _+29
148 + 30
123+
58k 8 63,lO
76& 71*
8** 8*
80* 84k
36+ 8+
56+ 5*
6** 1**
89 + 12 **.+ 2* 0.2 **.+
0.2% Cholesterol
diet
Plasma triglycerides Plasma
total
cholesterol Plasma HDL-Cc HDL-C b LDL-Cb VLDL-C a b ’ * ** ’
*
b
8 2
**
173*15
**.+ 5 ** 2 **,+
59k 65k 67k lo?
8 3 4 3
72k 7** 73*17 63*15 5* 4**
81+ 79* 39+ 5k
4 3** 5* 6 **.+ 2**
4 mg/animal/day. Ultracentrifugation technique; Precipitation technique. P < 0.05 between drug treatments and their control. P < 0.01 between drug treatments and their control. P < 0.01 between the two drug treatments.
treated animals maintained on a cholesterol-free diet did not differ significantly from that of controls. Total plasma cholesterol concentration of animals maintained on the cholesterol-free diet increased significantly when the animals received either capsaicin (17%) or dihydrocapsaicin (55%). Notably, both drugs significantly increased the plasma HDL-C concentration of animals maintained on a cholesterol-free diet; a 31% increase in capsaicin-treated turkeys and a 38% increase in dihydrocapsaicin-treated animals wa> observed. Both compounds significantly increased the LDLC and decreased the VLDL-C concentration from plasma samples of animals maintained on a cholesterol-free diet. Serum triglyceride concentration of cholesterolfed animals was not changed significantly by capsaicin treatment. Administration of dihydrocapsaicin to animals fed the 0.2% cholesterol diet resulted in an 19% decrease in serum triglyceride concentration. Interestingly, both drug when administered to the cholesterol-fed animals maintained total plasma cholesterol concentrations similar to those of cholesterol-fed controls. This drug action was significant because animals fed a
cholesterol-free diet with a concomitant drug treatment had elevated cholesterol concentrations. Plasma HDL-C concentration (separated by precipitation technique) of cholesterol-fed animals was increased significantly by capsaicin (22%) and dihydrocapsaicin (37%). Separated by ultracentrifugation, plasma HDL-C increased in concentration by 22% when cholesterol-fed animals received only dihydrocapsaicin. Plasma LDL-C concentration of dihydrocapsaicin-treated (Group 2) animals was decreased 42% compared to controls. Both compounds significantly decreased the plasma VLDL-C concentration of hypercholesterolemic animals. Discussion The significant weight loss (9%) observed in capsaicin-treated turkeys maintained on the 0.2% cholesterol diet may be attributed to intensified gastric and intestinal peristalsis [14]. Clearly, the wet fecal weight of this subgroup (&) was increased above the respective controls although the dry weight was lower. Since in this animal the cloaca is also the conduit for urine it suggests that
89 there was increased urine excretion perhaps secondary to increased water intake. These results involving turkeys agree with our previous work in rabbits [7]. Anorexia was omitted as the cause of weight loss because food consumption did not differ among the treatment groups. Administered as pure (98%) compounds, capsaicin and dihydrocapsaicin did not alter the serum triglyceride concentration of young turkeys fed a cholesterol-free diet. Pure dihydrocapsaicin was more efficacious than capsaicin in decreasing the mean serum triglyceride concentration when the animals were maintained on a 0.2% cholesterol diet. Serum triglyceride concentration was reduced by mixtures of capsaicinoids (80% capsaicin, 20% dihydrocapsaicin, Sigma, Co.) in our previous experiments with rabbits rendered hyperlipidemic due to ingestion of exogenous cholesterol (0.5%) over a 5-week experimental period [7]. Administration of pure capsaicin or dihydrocapsaicin to turkey poults maintained on a cholesterol-free diet increased plasma total cholesterol concentrations. This hypercholesterolemic effect was not observed in our previous work with mammals in which mixtures of capsaicinoids produced no mean differences between total plasma cholesterol concentration of capsaicin treated or capsaicin-free rabbits maintained on a cholesterolfree diet [7]. Throughout the experimental period, total plasma cholesterol concentration of control animals maintained on the 0.2% cholesterol diet was slightly higher (30 mg/dl) than that of cholesterol-free controls. It was significant to note that capsaicinoid treatment of the 0.2% cholesterol-fed animals decreased total plasma cholesterol concentration, whereas the reverse was observed with animals fed a cholesterol-free diet. Mean differences for total plasma cholesterol concentration of dihydrocapsaicin-free or dihydrocapsaicin-treated hyperlipidemic animals were not significant because of significant decreases in LDL-C with offsetting increases in HDL-C. Mixtures of the drug (capsaicin and dihydrocapsaicin) may have a potentiating hypocholesterolemic effect when administered to hypercholesterolemic animals as observed in our previous work with turkeys [6] and rabbits [7]. The increased plasma LDL-C concentration of
control animals maintained on the 0.2% cholesterol diet was important in the utility of this model, since it resembles the hypercholesterolemia found in man and in some cholesterol-fed animals [15]. Under hyperlipidemic conditions capsaicin did not augment the already elevated LDL-C concentration of plasma. In contrast with this effect the drug increased LDL-C concentration of plasma when administered to animals maintained on the regular diet. Dihydrocapsaicin appears to have a significant anti-LDL-C effect as indicated by the significantly decreased mean plasma LDL-C concentration of cholesterol-fed turkeys. Binding of the drug to bile acids in the intestinal lumen may produce the anti-LDL-C effect, while decreasing cholesterol absorption. Capsaicin treatment has been shown to increase fecal bile acid [4] and cholesterol excretion [5]. Excessive elimination of bile acids may also decrease plasma LDL-C concentration by induced expression of hepatic LDL-receptors [16,17]. This mechanism may be similar to that suggested for bile acid sequesterants [18] but appears to occur only in the presence of a cholesterol diet. Regardless of diet, plasma HDL-C concentration was significantly increased in capsaicinoidtreated turkeys relative to untreated controls. Pure dihydrocapsaicin had a greater efficacy for increasing plasma HDL-C concentration than that of pure capsaicin when both drugs were compared to their control groups. HDL-C concentration was similarly elevated in our previous work [7] when mixtures of capsaicinoids were administered to hypercholesterolemic animals. Plasma HDL-C concentrations were increased in studies employing hypercholesterolemic diets [19-211. This effect, however, was not observed in the present study. The increased plasma HDL-C concentration may result from a capsaicinoid enhanced lipoprotein lipase (LPL) activity. In animal models as well as in humans, ‘surface remnants’ are produced by LPL catalyzed lipolysis of triglyceriderich lipoproteins (VLDL), and these ‘surface remnants’ are believed to be a major source of HDL [22]. One may infer that an increased LPL activity may account for the increased HDL-C concentration. Another mechanism by which capsaicinoids may elevate HDL-C concentration is an increase in the lecithin : cholesterol acyl transferase (LCAT)
90 activity. LCAT transforms the discoidal nascentHDL particles to spherical HDL by conversion of free-cholesterol to cholesterol esters. The spherical HDL is capable of acting as a scavenger of tissue cholesterol thus, maintaining cholesterol homeostasis (231. Dihydrocapsaicin appears to have beneficial effects in studies of atherosclerosis greater than those of capsaicin. Dihydrocapsaicin significantly increased HDL-C concentration of plasma concomitant to a decreased VLDL-C concentration in turkey poults, regardless of dietary conditions (cholesterol-free or cholesterol-rich diet). Similarly, the drug appears to work best under hyperlipidemic conditions since it decreases plasma LDL-C concentrations of cholesterol-fed animals. Acknowledgements
The writers are most indebted to Mr. Richard Jandry and Ms. Kory Ward for assistance with the animals, Ms. Kreter for the typing, and Dr. Larry Sachs for the statistical analysis. References Szolcsanyi, J., Capsaicin: hot new pharmacological tool, Trends Phar. Sci., 4 (1983) 495 (Letter). Suzuki, T., Kawada, T. and Iwai, K., Effective separation of capsaicin and its analogues by reversed-phase high-performance thin-layer chromatography, J. Chromatogr., 198 (1980) 217. Paech, K. and Tracy, M.V., Modem Methods of Plant Analysis, Springer-Verlag, Berlin, 1955, pp. 475-550. Sambaiah, K. and Satyanarayana, M.N., Hypocholesterolemic effect of red pepper and capsaicin, Indian J. Exp. Biol., 18 (1980) 898. Sambaiah, K. and Satyanarayana, M.N., Influence of red pepper and capsaicin on body composition and lipogenesis in rats, J. Biosci., 4 (1982) 425. Ki, P., Negulesco, J.A. and Mumane, M., Decreased total serum, myocardial and aortic cholesterol levels following capsaicin treatment, IRCS Med. Sci., 10 (1982) 446. Negulesco, J.A., White, R. and Ki, P., Capsaicin lowers plasma cholesterol and triglycerides of logomorphs, Artery, 12 (1985) 301. Middleton, C.D., Naturally occurring atherosclerosis in broad-breasted bronze turkeys. In: J.C. Roberts (Ed.), Comparative Atherosclerosis, Harper and Row, New York, 1965, ‘pp. 59-61.
G.A. and Howard, A.N., Studies on aortic 9 Gresham, atherosclerosis in the turkey. In: J.C. Roberts (Ed.), Comparative Atherosclerosis, Harper and Row, New York, 1965, pp. 62-65. 10 Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. determination of 11 Bucolo, G. and David, H., Quantitative serum triglycerides by the use of enzymes, Clin. Chem., 19 (1973) 476. 12 Wamick, G.R., Benderson J.M. and Albers, J.J., Dextran sulfate-Mg*+ precipitation procedure for quantitation of high-density lipoprotein cholesterol, Clin. Chem., 28 (1982) 1379. procedure for compar13 Dunnett, C., A multiple comparison ing several treatments with a control, J. Amer. Stat. Ass., 50 (1955) 1096. 14 Molnar, T., Die pharmakologischen Wirkungen des Capsaicins, des scharf schmeckenden Wirkstoffes in Paprika, Arzneim.-Forsch. (Drug Res.), 15 (1965) 718. 15 Mahley, R.W., Alterations in plasma lipoproteins induced by cholesterol feeding in animals including man. In: J.M. Dietschy, A.M. Gotto, Jr. and J.A. Ontoko (Eds.), Disturbances in Lipid and Lipoprotein Metabolism, American Physiological Society, Bethesda, MD, 1978, pp. 181-197. 16 Brown, M.S., Kovanen, P.T. and Goldstein, J.L., Regulation of plasma cholesterol by lipoprotein receptors, Science, 212 (1981) 628. 17 Angelin, B., Raviola, C.A., Innerarity, T.L. and Mahley, R.W., Regulation of hepatic lipoprotein receptors in dog, J. Clin. Invest., 71 (1983) 816. C.J., Bicker, S. and Shepherd, J., 18 Slater, H.R., Packard, Effects of cholestyramine on receptor mediated plasma clearance and tissue uptake of human low density lipoproteins in the rabbit, J. Biol. Chem., 255 (1980) 10210. R.W., Weisgraber, K.H. and Innerarity, T.L., 19 Mahley, Atherogenic hyperlipoproteinemia induced by cholesterol feeding the Patas monkey, Biochemistry, 15 (1976) 2979. 20 Mahley, R.W., Innerarity, T.L., Bersotry, T.P., Lipson, A. and Margolis, S., Alterations in human high-density lipoproteins with or without increased plasma-cholesterol, induced by diets high in cholesterol, Lancet, ii (1978) 807. K.H., Innerarity, T.L., Brewer, 21 Mahley, R.W., Weisgraber, H.B. and Assmann, G., Swine lipoproteins and atherosclerosis. Changes in plasma lipoproteins and apoproteins induced by cholesterol feeding, Biochemistry, 14 (1975) 2817. 22 Eisenberg, S., High density lipoprotein metabolism, J. Lipid Res., 25 (1984) 1017. 23 Mahley, R.W., Atherogenic hyperlipoproteinemia: the cellular and molecular biology of plasma lipoproteins altered by dietary fat and cholesterol, Med. Clin. N. Amer., 66 (1982) 375.
Elsevier
64 (1987)
Scientific
85
85-90
Publishers
Ireland,
Ltd.
ATH 03920
Effects of pure capsaicinoids (capsaicin and dihydrocapsaicin) on plasma lipid and lipoprotein concentrations of turkey poults J.A. Negulesco Departments
‘, S.A. Noel 2, H.A.I. Newman 2, E.C. Naber and D.T. Witiak 4
of ’ Anatom.v,
’ Pathology,
’ Pot&v
Science and 4 College of Pharmacv,
Columhtu,
3, H.B. Bhat 4
The Ohio State iJnioersrt_v.
OH 43210 (U.S.A.)
(Received 31 July. 1985) (Revised, received 15 August, 1986) (Accepted 27 August, 1986)
The effects of capsaicin and dihydrocapsaicin on blood lipid and lipoprotein concentrations were determined in two groups of turkeys. The first group was maintained on a cholesterol-free diet, while the second received a diet supplemented with 0.2% cholesterol. Daily administration of capsaicinoids occurred at a dose of 4 mg per animal. Neither drug had an effect on serum triglyceride concentrations in the total cholesterol, LDL-cholesterol and HDLanimals receiving the cholesterol-free diet. However, cholesterol concentrations were increased significantly, while VLDL cholesterol concentrations were decreased significantly by both drugs relative to controls. In the cholesterol-fed group triglycerides, total cholesterol and LDL-cholesterol decreased significantly with dihydrocapsaicin treatment. Both compounds reduced VLDL-cholesterol and increased HDL-cholesterol in the cholesterol-fed animals. Dihydrocapsaitin had a greater efficacy in producing benificial anti-hyperlipidemic effects in the cholesterol-fed animals.
Key words:
Capsaicin;
Cholesterol;
Dihydrocapsaicin;
Introduction Capsaicin, the acylamide of vanillylamine 8-methyl-6-trans-nonenoic acid [l] is one
and of 5
This investigation was supported, in part, by the Departments of Anatomy, Pathology and Poultry Science of the Ohio State University (OSU), the OSU Small Research Grant Program (Project 221543). and by the Central Ohio Heart Association (Project 714555). Correspondence address: Dr. J.A. Negulesco, Department of Anatomy, The Ohio State University, Columbus. OH 43210, U.S.A.
OO21-9150/R7/$03.50
‘9 1987 Elsevier
Scientific
Publishers
Ireland.
Lipoproteins;
Triglycerides;
Turkeys
analogs of the group of compounds called capsaicinoids (capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin) [2]. These compounds are widely distributed in nature and are part of the diet of various ethnic groups as spicy fruit of the genus Capsicum (red cluster pepper, cone pepper, short pepper, cayenne, chili and long yellow peppers) [3]. Consumption of red peppers by hyperlipidemic rats has been shown to decrease liver and serum cholesterol concentrations [4], and liver triglyceride concentrations [S]. Recently, we have Ltd
86 reported the effects of the parent compound, capsaicin (80% pure), on serum lipid concentrations of hypercholesterolemic turkeys [6] and rabbits [7]. In both models, capsaicin effectively decreased serum cholesterol concentrations. Serum triglyceride concentrations were decreased , while high density lipoprotein cholesterol (HDL-C) concentrations were significantly increased in capsaitin-treated rabbits. Because the previous work [4,6,7] involved a mixture of capsaicins (80% capsaicin and 20% dihydrocapsaicin) our present investigation was designed to ascertain, the effects of pure capsaicin and dihydrocapsaicin on serum lipid concentrations. Turkey poults were selected for this study since it was suggested that, although this animal model develops aortic atherosclerotic lesions over a relative short experimental period [8], there are some similarities to the aortic insults of man [9]. Materials and methods
Experimental design Forty-eight turkey poults (Melaegris gallopouo) received one-day post hatch were allowed to acclimatize under poultry house conditions: 36-24OC *, 45% relative humidity, a 12-h light, 12-h dark cycle, ad libitum food (turkey starter diet) and water consumption over a 7-day period. On day eight, the animals were divided randomly into two equal groups of 24 birds each and treated as follows for a 6 weeks experimental period: Group 1. Cholesterol-free poultry diet: A, = control subgroup: drug-free placebo (n = 8); B, = capsaicin (98% pure, Sigma Co.)-thated subgroup: 4 mg p animal p day (PAPD) (n = 8); C, = dihydrocapsaicin (98% pure) treated subgroup: 4 mg PAPD (n = 8). Group 2: 0.2 5%cholesterol-supplemented poultry diet: A, = control subgroup: drug-free placebo (n = 8); Bz = capsaicin-treated subgroup: 4 mg PAPD (n = 8); C, = dihydrocapsaicin-treated subgroup: 4 mg PAPD (n = 8). Divided in groups of 4 animals per cage, the turkeys were allowed unlimited access to water
* Temperature was 36°C the first week and dropped 2°C per week until it reached 24OC.
(trough). A sufficient amount of feed (200 g) was supplied daily, and feed consumption and/or spillage was determined. Animal body weight and fecal output were measured and recorded on a weekly basis. Diets The 0.2% cholesterol diet was prepared by mixing the cholesterol suspended in cholesterol-free vegetable oil, heating (56°C) and mixing the solution with the turkey starter diet. The control cholesterol-free diet included equivalent amounts of vegetable oil mixed into the turkey starter diet. Drug administration Capsaicin (Sigma Co.; 98% pure) was converted to dihydrocapsaicin in our laboratory. The dihydro-analog was determined to be 98% pure using NMR and mass spectrometry. Both compounds were suspended in 0.2 ml cholesterol-free vegetable oil, and were administered daily via buccal route (5 ml syringe adapted with a 5 cm long x 2 mm diameter blunt metal needle terminally fitted with a polyethylene cuff). Control animals received the drug-free suspension medium (0.2 ml cholesterol-free vegetable oil). Blood collection After anesthetizing the animals, blood samples taken by cardiac puncture (21-gauge needle into lo-ml syringes) were collected into 10 ml EDTAcoated tubes. Plasma was obtained by immediately transferring a portion of whole blood into a tube containing 200 ~1 of 0.2 M EDTA and centrifuging at 2000’ rpm (800 X g) for 10 min. Serum was obtained by allowing whole blood to clot in a separate tube and centrifuging in the same manner. Quantitative methodr Plasma cholesterol concentration was determined by the enzymatic method of Allain et al. [lo] and the serum triglyceride concentration was determined enzymatically by the method of Bucolo and David [ll]. High density lipoproteins (HDL) were separated from other lipoprotein fractions by the precipitation technique of War-nick et al. [12]. Dextran sulfate and Mg*+ were used to precipi-
87 Results
tate the low density (LDL) and very low density (VLDL) lipoproteins. The precipitate was separated by centrifugation at 4000 rpm (1600 X g) for 30 min at 4°C and high density lipoprotein cholesterol (HDL-C) was determined in the supernatant as previously described. Lipoproteins were also separated using isopychnic ultracentrifugation at the following densities: VLDL, 0.950-1.006; LDL, 1.006-1.063; HDL, 1.063-1.210. The specimens were centrifuged at 36000 rpm (104400 X g) for 24 h for the VLDL and LDL separation and 48 h for the HDL separation. Following dialysis with 0.004 M EDTA buffer (pH 7.5), the cholesterol concentration was determined in each fraction as previously described. Statistical analyses involved two-way ANOVA with post-hoc Dunnett’s test [13].
Mean body weight and weight gain in the final week of the study did not differ significantly among the subgroups of animals fed the cholesterol-free diet (Table 1). However, capsaitin-treated turkeys had significantly lower final body weight and weight gain than that of controls and dihydrocapsaicin-treated animals. Capsaicin had no effect on body weight in previous studies [4,6,7]. In the final week mean body weight gain of cholesterol-fed animals was greater with dihydrocapsaicin treatment compared to controls, although the final mean body weight was unchanged. Results of serum or plasma lipid analyses are presented in Table 2. Serum triglyceride concentration of capsaicin- or dihydrocapsaicin-
TABLE 1 COMPARISON OF CAPSAICIN (C) AND DIHYDROCAPSAICIN (D) ADMINISTRATION ON THE MEAN BODY WEIGHT, FOOD CONSUMPTION AND FECAL ELIMINATION OF TURKEY POULTS MAINTAINED ON A CHOLESTEROL-FREE OR A 0.2% CHOLESTEROL DIET FOR 6 WEEKS Mean f SD; all values in grams. Cholesterol-free Control Initial body wt Mean body wt at end of exp. period (week 6)
diet Ca
Da
91*12
86k
1307+ 12
1454F205
I
85+
16
0.2% Cholesterol
diet
Control
Ca
92k
8
1535& 117
1423+137
D” 89+
1397+87
6
92+
*
1524+ 128 ***
Mean body wt gain between week 5-6
301 f 88
344* 173
350+
Food consumption per animal/day (week 6)
111
113
109
106
109
113
Wet fecal mass per animal/day (week 6)
176
158
181
158
184
169
Dry fecal mass per animal/day (week 6)
34
33
39
33
29
36
a 4 mg/animal/day. * P -c0.05 between ** P i 0.01 between *** P c 0.05 between t P < 0.01 between
drug treatments and their control. drug treatments and their control. the two drug treatments. the two drug treatments.
86
361+
42
298+40
**
8
406k
40 *.+
88 TABLE
2
COMPARISON OF CAPSAICIN (C) AND DIHYDROCAPSAICIN (D) ADMINISTRATION ON THE MEAN PLASMA LIPID AND LIPOPROTEIN CONCENTRATIONS OF TURKEY POULTS MAINTAINED ON A CHOLESTEROL-FREE OR A 0.2% CHOLESTEROL DIET FOR 6 WEEKS Mean
*SD;
mg/dl. Cholesterol-free
diet
No drugs n=8
C” n=8
Da n=8
No drugs n=8
C” n=8
D” n=8
205 f 36
193 If: 16
191* 31
195k26
170*40
159*12
112_+ 9
131+10
143 _+29
148 + 30
123+
58k 8 63,lO
76& 71*
8** 8*
80* 84k
36+ 8+
56+ 5*
6** 1**
89 + 12 **.+ 2* 0.2 **.+
0.2% Cholesterol
diet
Plasma triglycerides Plasma
total
cholesterol Plasma HDL-Cc HDL-C b LDL-Cb VLDL-C a b ’ * ** ’
*
b
8 2
**
173*15
**.+ 5 ** 2 **,+
59k 65k 67k lo?
8 3 4 3
72k 7** 73*17 63*15 5* 4**
81+ 79* 39+ 5k
4 3** 5* 6 **.+ 2**
4 mg/animal/day. Ultracentrifugation technique; Precipitation technique. P < 0.05 between drug treatments and their control. P < 0.01 between drug treatments and their control. P < 0.01 between the two drug treatments.
treated animals maintained on a cholesterol-free diet did not differ significantly from that of controls. Total plasma cholesterol concentration of animals maintained on the cholesterol-free diet increased significantly when the animals received either capsaicin (17%) or dihydrocapsaicin (55%). Notably, both drugs significantly increased the plasma HDL-C concentration of animals maintained on a cholesterol-free diet; a 31% increase in capsaicin-treated turkeys and a 38% increase in dihydrocapsaicin-treated animals wa> observed. Both compounds significantly increased the LDLC and decreased the VLDL-C concentration from plasma samples of animals maintained on a cholesterol-free diet. Serum triglyceride concentration of cholesterolfed animals was not changed significantly by capsaicin treatment. Administration of dihydrocapsaicin to animals fed the 0.2% cholesterol diet resulted in an 19% decrease in serum triglyceride concentration. Interestingly, both drug when administered to the cholesterol-fed animals maintained total plasma cholesterol concentrations similar to those of cholesterol-fed controls. This drug action was significant because animals fed a
cholesterol-free diet with a concomitant drug treatment had elevated cholesterol concentrations. Plasma HDL-C concentration (separated by precipitation technique) of cholesterol-fed animals was increased significantly by capsaicin (22%) and dihydrocapsaicin (37%). Separated by ultracentrifugation, plasma HDL-C increased in concentration by 22% when cholesterol-fed animals received only dihydrocapsaicin. Plasma LDL-C concentration of dihydrocapsaicin-treated (Group 2) animals was decreased 42% compared to controls. Both compounds significantly decreased the plasma VLDL-C concentration of hypercholesterolemic animals. Discussion The significant weight loss (9%) observed in capsaicin-treated turkeys maintained on the 0.2% cholesterol diet may be attributed to intensified gastric and intestinal peristalsis [14]. Clearly, the wet fecal weight of this subgroup (&) was increased above the respective controls although the dry weight was lower. Since in this animal the cloaca is also the conduit for urine it suggests that
89 there was increased urine excretion perhaps secondary to increased water intake. These results involving turkeys agree with our previous work in rabbits [7]. Anorexia was omitted as the cause of weight loss because food consumption did not differ among the treatment groups. Administered as pure (98%) compounds, capsaicin and dihydrocapsaicin did not alter the serum triglyceride concentration of young turkeys fed a cholesterol-free diet. Pure dihydrocapsaicin was more efficacious than capsaicin in decreasing the mean serum triglyceride concentration when the animals were maintained on a 0.2% cholesterol diet. Serum triglyceride concentration was reduced by mixtures of capsaicinoids (80% capsaicin, 20% dihydrocapsaicin, Sigma, Co.) in our previous experiments with rabbits rendered hyperlipidemic due to ingestion of exogenous cholesterol (0.5%) over a 5-week experimental period [7]. Administration of pure capsaicin or dihydrocapsaicin to turkey poults maintained on a cholesterol-free diet increased plasma total cholesterol concentrations. This hypercholesterolemic effect was not observed in our previous work with mammals in which mixtures of capsaicinoids produced no mean differences between total plasma cholesterol concentration of capsaicin treated or capsaicin-free rabbits maintained on a cholesterolfree diet [7]. Throughout the experimental period, total plasma cholesterol concentration of control animals maintained on the 0.2% cholesterol diet was slightly higher (30 mg/dl) than that of cholesterol-free controls. It was significant to note that capsaicinoid treatment of the 0.2% cholesterol-fed animals decreased total plasma cholesterol concentration, whereas the reverse was observed with animals fed a cholesterol-free diet. Mean differences for total plasma cholesterol concentration of dihydrocapsaicin-free or dihydrocapsaicin-treated hyperlipidemic animals were not significant because of significant decreases in LDL-C with offsetting increases in HDL-C. Mixtures of the drug (capsaicin and dihydrocapsaicin) may have a potentiating hypocholesterolemic effect when administered to hypercholesterolemic animals as observed in our previous work with turkeys [6] and rabbits [7]. The increased plasma LDL-C concentration of
control animals maintained on the 0.2% cholesterol diet was important in the utility of this model, since it resembles the hypercholesterolemia found in man and in some cholesterol-fed animals [15]. Under hyperlipidemic conditions capsaicin did not augment the already elevated LDL-C concentration of plasma. In contrast with this effect the drug increased LDL-C concentration of plasma when administered to animals maintained on the regular diet. Dihydrocapsaicin appears to have a significant anti-LDL-C effect as indicated by the significantly decreased mean plasma LDL-C concentration of cholesterol-fed turkeys. Binding of the drug to bile acids in the intestinal lumen may produce the anti-LDL-C effect, while decreasing cholesterol absorption. Capsaicin treatment has been shown to increase fecal bile acid [4] and cholesterol excretion [5]. Excessive elimination of bile acids may also decrease plasma LDL-C concentration by induced expression of hepatic LDL-receptors [16,17]. This mechanism may be similar to that suggested for bile acid sequesterants [18] but appears to occur only in the presence of a cholesterol diet. Regardless of diet, plasma HDL-C concentration was significantly increased in capsaicinoidtreated turkeys relative to untreated controls. Pure dihydrocapsaicin had a greater efficacy for increasing plasma HDL-C concentration than that of pure capsaicin when both drugs were compared to their control groups. HDL-C concentration was similarly elevated in our previous work [7] when mixtures of capsaicinoids were administered to hypercholesterolemic animals. Plasma HDL-C concentrations were increased in studies employing hypercholesterolemic diets [19-211. This effect, however, was not observed in the present study. The increased plasma HDL-C concentration may result from a capsaicinoid enhanced lipoprotein lipase (LPL) activity. In animal models as well as in humans, ‘surface remnants’ are produced by LPL catalyzed lipolysis of triglyceriderich lipoproteins (VLDL), and these ‘surface remnants’ are believed to be a major source of HDL [22]. One may infer that an increased LPL activity may account for the increased HDL-C concentration. Another mechanism by which capsaicinoids may elevate HDL-C concentration is an increase in the lecithin : cholesterol acyl transferase (LCAT)
90 activity. LCAT transforms the discoidal nascentHDL particles to spherical HDL by conversion of free-cholesterol to cholesterol esters. The spherical HDL is capable of acting as a scavenger of tissue cholesterol thus, maintaining cholesterol homeostasis (231. Dihydrocapsaicin appears to have beneficial effects in studies of atherosclerosis greater than those of capsaicin. Dihydrocapsaicin significantly increased HDL-C concentration of plasma concomitant to a decreased VLDL-C concentration in turkey poults, regardless of dietary conditions (cholesterol-free or cholesterol-rich diet). Similarly, the drug appears to work best under hyperlipidemic conditions since it decreases plasma LDL-C concentrations of cholesterol-fed animals. Acknowledgements
The writers are most indebted to Mr. Richard Jandry and Ms. Kory Ward for assistance with the animals, Ms. Kreter for the typing, and Dr. Larry Sachs for the statistical analysis. References Szolcsanyi, J., Capsaicin: hot new pharmacological tool, Trends Phar. Sci., 4 (1983) 495 (Letter). Suzuki, T., Kawada, T. and Iwai, K., Effective separation of capsaicin and its analogues by reversed-phase high-performance thin-layer chromatography, J. Chromatogr., 198 (1980) 217. Paech, K. and Tracy, M.V., Modem Methods of Plant Analysis, Springer-Verlag, Berlin, 1955, pp. 475-550. Sambaiah, K. and Satyanarayana, M.N., Hypocholesterolemic effect of red pepper and capsaicin, Indian J. Exp. Biol., 18 (1980) 898. Sambaiah, K. and Satyanarayana, M.N., Influence of red pepper and capsaicin on body composition and lipogenesis in rats, J. Biosci., 4 (1982) 425. Ki, P., Negulesco, J.A. and Mumane, M., Decreased total serum, myocardial and aortic cholesterol levels following capsaicin treatment, IRCS Med. Sci., 10 (1982) 446. Negulesco, J.A., White, R. and Ki, P., Capsaicin lowers plasma cholesterol and triglycerides of logomorphs, Artery, 12 (1985) 301. Middleton, C.D., Naturally occurring atherosclerosis in broad-breasted bronze turkeys. In: J.C. Roberts (Ed.), Comparative Atherosclerosis, Harper and Row, New York, 1965, ‘pp. 59-61.
G.A. and Howard, A.N., Studies on aortic 9 Gresham, atherosclerosis in the turkey. In: J.C. Roberts (Ed.), Comparative Atherosclerosis, Harper and Row, New York, 1965, pp. 62-65. 10 Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. determination of 11 Bucolo, G. and David, H., Quantitative serum triglycerides by the use of enzymes, Clin. Chem., 19 (1973) 476. 12 Wamick, G.R., Benderson J.M. and Albers, J.J., Dextran sulfate-Mg*+ precipitation procedure for quantitation of high-density lipoprotein cholesterol, Clin. Chem., 28 (1982) 1379. procedure for compar13 Dunnett, C., A multiple comparison ing several treatments with a control, J. Amer. Stat. Ass., 50 (1955) 1096. 14 Molnar, T., Die pharmakologischen Wirkungen des Capsaicins, des scharf schmeckenden Wirkstoffes in Paprika, Arzneim.-Forsch. (Drug Res.), 15 (1965) 718. 15 Mahley, R.W., Alterations in plasma lipoproteins induced by cholesterol feeding in animals including man. In: J.M. Dietschy, A.M. Gotto, Jr. and J.A. Ontoko (Eds.), Disturbances in Lipid and Lipoprotein Metabolism, American Physiological Society, Bethesda, MD, 1978, pp. 181-197. 16 Brown, M.S., Kovanen, P.T. and Goldstein, J.L., Regulation of plasma cholesterol by lipoprotein receptors, Science, 212 (1981) 628. 17 Angelin, B., Raviola, C.A., Innerarity, T.L. and Mahley, R.W., Regulation of hepatic lipoprotein receptors in dog, J. Clin. Invest., 71 (1983) 816. C.J., Bicker, S. and Shepherd, J., 18 Slater, H.R., Packard, Effects of cholestyramine on receptor mediated plasma clearance and tissue uptake of human low density lipoproteins in the rabbit, J. Biol. Chem., 255 (1980) 10210. R.W., Weisgraber, K.H. and Innerarity, T.L., 19 Mahley, Atherogenic hyperlipoproteinemia induced by cholesterol feeding the Patas monkey, Biochemistry, 15 (1976) 2979. 20 Mahley, R.W., Innerarity, T.L., Bersotry, T.P., Lipson, A. and Margolis, S., Alterations in human high-density lipoproteins with or without increased plasma-cholesterol, induced by diets high in cholesterol, Lancet, ii (1978) 807. K.H., Innerarity, T.L., Brewer, 21 Mahley, R.W., Weisgraber, H.B. and Assmann, G., Swine lipoproteins and atherosclerosis. Changes in plasma lipoproteins and apoproteins induced by cholesterol feeding, Biochemistry, 14 (1975) 2817. 22 Eisenberg, S., High density lipoprotein metabolism, J. Lipid Res., 25 (1984) 1017. 23 Mahley, R.W., Atherogenic hyperlipoproteinemia: the cellular and molecular biology of plasma lipoproteins altered by dietary fat and cholesterol, Med. Clin. N. Amer., 66 (1982) 375.