A Comparison of Hypocholesterolemic Activity of ß-Sitosterol and ß-Sitostanol in Rats1 MICHIHIRO SUGANO,2 HIDEKAZU MORIOKA ANDIKUO IKEDA Laboratory of Nutrition Chemistry, Kyushu University School of Agriculture, Fukuoka 812, Japan ABSTRACT The hypocholesterolemic activity of ¿8-sitosteroland its hydrogenated product, /3-sitostanol ( dihydrositosterol or stigmastanol ) has been compared in young male rats. When cholesterol was included in the diet, sitostanol consistently exhibited significantly greater hypocholesterol emic activity than sitosterol. There were no apparent differences in the effects of the sterol and the stanol on the concentration of liver cholesterol and triglycéride.Increases in plasma triglycéridedue to feeding sitosterol were not observed with sitostanol. Incorporation of dietary sitostanol into plasma, liver and other tissues was always negligible, and thus this stanol was almost completely recovered in feces, while there was considerable deposition of sitosterol (mean fecal recovery being 85% to 92%). The in crease in fecal output of dietary cholesterol was significantly greater with the stanol than with the sterol. There was no demonstrable negative effect on growth and weight of major visceral tissues in rats fed the sterol as well as the stanol. These observations together with those reported previously indicate that hydrogénationof phytosterols is a novel approach to enhance their hypocholesterolemic activities without influencing the relative safety of the initial sterols. J. Nutr. 107: 2011-2019, 1977. INDEXING KEY WORDS sitosterol • sitostanol • plasma cholesterol The hypocholesterolemic action of phytosterols in experimental animals and man has been known for many years. /8-Sitosterol is a cholesterol-lowering compound of modest to moderate efficacy, and seems remarkably free of subjective side-effects for the patient with hypercholesterolemia (1). Use of a phytosterol mixture containing considerable amount of campesterol is not recommended because of an appreciable incorporation of this sterol into plasma, although there is no evidence that campesterol is more atherogenic than cholesterol (2). Thus, it appears likely that the preference of phytosterols for use in the treatment of hypercholesterolemia depends on

low levels of absorption are desirable. On the other hand, few data are available indieating postabsorptive effects of phytosterols (4). Hydrogénation of phytosterols to the corresponding phytostanols results in à significant reduction in absorption (5) similar to the observation with cholesterol and cholestanol (6). Only limited information is available as to the occurrence of stanols in plant sterol fractions ( 7 ). Preliminary experiments showed that commercial unhydrogenated vegetable oils contained demonstrable amounts; up to 25% of the total sterol

their chemical evidpnrp eviaence that mat

*Preliminary part of this study was presented at the Iv japan,August International Symposium on Atherosclerosis, Tokvo, me (Abstract pp.236).

from the „„„tl._

Structures. Since there is nlanr plant sfornir sterols nrimnatÃ-nrr originating

diet may initiate 4. • j i

development ¿L

of /n\

xantnomatosis and perhaps atheroma(3),

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Receivedfor publicationNovember19, me. 2Thls study was supported grant from the Ministry of

in part by the research Education, Science and

culture(Scientific ResearchNO.147107). 2011

2012

SUGANO, MORIOKA AND IKEDA TABLE

1

Effects of ß-sitosterol and ß-sitostanol on the

dietExp

Sterols added to the basal

No.'123456Groups (No. rats)1 of

fats%Safflower,

sterol%00.50000.500000.501.000.500.5000.5000.50Sitostanol%000.50000.500000.501.000.500.5000.5000.5PlasmaCholes terolmg/dl2120 terol%00000.50.50.500.50.5*0.5«0.540.540.540.50.50.50.50.50.50.50.50.50.5Sitoéridesmg/dl2214

5Safflower, 5Safflower, 5Safflower, 5Safflower, 5Safflower, 5Safflower, 5Safflower, 5Safflower, 5Safflower, 5Safflower, (6)12 5Safflower, (6)13(7)14(7)155Safflower, 5Safflower, 5Corn, 5Corn, (7)16 (8)17 5Lard, (7)18 5Lard, (8)19 5Safflower, (6)20(7)21 20Safflower, 20Safflower, (7)22 20Lard, (6)23(7)24 20Lard, 20Lard, (7)Dietary 20Choles (6)2 (6)3 (6)4 (6)5 (6)6 (6)7 (6)8 (6)9 (6)10(6)11

fraction was stanols. The corresponding value for ß-sitosterolpreparations was 10% to 15%. Since our previous experiments demonstrated that in rats, the hypocholesterolemic action of phytostanol mixtures was demonstrably greater than that of phytosterol mixtures (5), a comparison be tween ß-sitosterol and ß-sitostanol was made in the present study.

±30257 ±11126 6115 ± ±33179 598.5± ± ±13138 ±18126 5.0141 4117 ± ±16274 4C95.7± ± ±30"138 5.0'''107 ±23°^121 9166 ± ±13133 ±12426 ±21214 ±18152 ±35190 ±19«166 ±14'138 6C120± ±15°122 ±1097.3± 796.2± ± 3.5«97.6± 5.2158 4.180.7± ±17111 4.3698.3± 8»160 ± 5.686.5± 9112 ± 3.4'83.9± 9°67.3± ± 3.677. 5.4102 5C78.2 ± 3.659.0± 1± 6.8«81. ± 1.2°.'87.3± 5.0133 1± 3.078.3± 8C90.8± ± 1.7°66.7± 6.4« 3.0".'Triglyc

chromatography (TLC) (8) and by GLC on a glass capillary column (5). Judging from GLC and TLC analyses, the hydrogenated compound still contained approxi mately 3% of the unhydrogenated sterols, and the apparent composition of the re duction products was 93% ß-sitostanoland 7% campestanol. Animals and diets. Male Wistar rats ob tained from a commercial breeder5 and MATERIALS AND METHODS weighing 85 to 115 g were used throughout Materials. ß-Sitosterol3 was repeatedly the experiment. The composition or the recrystallized from ethyl acetate until over basal diet was (in %) (5): vitamin free 98% purity as the sterols was achieved as casein,6 20; mineral mixture, 4; vitamin measured by gas-liquid chromatography mixture, 1; choline chloride, 0.15; cellulose powder,7 4 and sucrose to 100. The vitamin ( GLC ). The composition of the sterol frac tion of this preparation was 93% ß-sito (water soluble) and mineral mixtures were according to Harper (9).8 Fats, cholesterol, sterol and 7% campesterol. Capillary col umn GLC revealed the occurrence of stanols in this preparation (0.5% campe3 E. Merck, Darmstadt. «Japan Electron Optic Laboratory JNM-MH-100 stanol and 12.5% sitostanol). Hydrogéna NMR spectrometer, Tokyo. tion of ß-sitosterolwas performed as re 5 Kyudo Co., Kumamoto. « ICN Pharmaceuticals Inc., Cleveland, Ohio. ported previously (5). The purity of the 7 Toyo Kagaku Sangyo Co., cellulose powder Type E, Tokyo. product was checked by nuclear magnetic 8 The Tanabe Amino Acid Research Foundation, resonance spectrometry,4 by Ag+-thin-layer Osaka.

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SITOSTANOL, HYPOCHOLESTEROLEMIC ACTIVITY

2013

concentration of plasma and liver lipids

Plasma Choles sterol% terolSito terolCampes-

Liver

terolmg/g22.52±0.082.25±0.062.45±0.103.15±0.2022.5 éridemg/g217.6±1.119.6±1.516.8±0.417.7±1.541.4±2.222.6± terolCampessterol%terolSito

sterols298.0±0.389.4±1.097.5±0.3«98.5±0.299.6±0.193.8±0.6C98.6±0.1«.'99.2±0.110010097.8±0.2'99.4±0.1«96.0±0.699.3±0 of total sterols298.4±0.189.7±0.697.7±0.2°98.7±0.110096.5±0.5 of total wt.24.48±0.114.57±0.224.13±0.124.40±0.264.90±0.124.62±0.18¿4.26

±2.04.00±0.24'4.09±0.21'2.94±0.0823.1 0.4±0.0°.