Effect of alfalfa saponins on intestinal cholesterol absorption in rats” 2 M. R. Malinow, Carolyn Stafford,3 Peter R. Cheeke,6
.
M.D. B.S., Ph.D.
Phyllis George
ABSTRACT
Five
intragastrically were by
estimation
of
hydrolysis tion.
.
also
fecal
Alfalfa
of alfalfa Am.
Kohler,5
mg
partial
sterols top Nut,’.
hydrolysis.
by
ChemicaLs We obtained [la, 2a(n)-3HJ-cholesterol (s.a. 60 Ci/mmole) and [4-’4CJ-cholesterol (s.a. 56 mCi/ mmole) from Amersham/Searle, Arlington Heights, Ill.; [22,23-3HF-sitosterol (s.a. 60 Ci/mmole) from New England Nuclear, Boston, Mass.; and -sitosterol (99%) from Applied Science Laboratories, Philadelphia , Pa . Saponins from alfalfa tops were prepared by the method of Walter et al. (10). Saponins from alfalfa roots were extracted into 50% ethanol and purified by decolorization with charcoal and precipitation from
ofClinical
Nutrinon
30:
intravenous dual
DECEMBER
Downloaded from https://academic.oup.com/ajcn/article-abstract/30/12/2061/4650853 by University of Glasgow user on 19 July 2018
th3 Livingston,5
alfalfa
tops
isotope their
of cholesterol
were
to
cholesterol. cholesterol
saponins
determined
assay of
inhibit
introduced The
was
involving absorption
ability
and
cholesterol.
technique reduced
M.S., B.A.,
or roots
ring-labeled Absorption
enhanced
30: 2061-2067,
Methods
Journal
a
saponins
,
American
from
acid
Hypercholesterolemia induced by cholesterol feeding is prevented in rabbits (1 2) and monkeys (3) by inclusion of alfalfaalfalfa tops, sun-cured, ground alfalfa hay (Medicago sativa)in th diet. The hypocholesterolemic effect of alfalfa may be associated with its content of saponins (4) since other saponins suppress diet-induced hypercholesterolemia in chickens (5-7) and a nitrogen analog of saponins inhibits intestinal absorption of cholesterol in rats (8). Moreover, alfalfa root saponins prevent the expected rise in cholestemolemia associated with a diet rich in butter and cholesterol in monkeys (9) and decrease intestinal absomption of cholesterol in rats (9). In the present paper, we demonstrate that intestinal absorption of cholesterol is inhibited in varying degrees by different alfalfa saponins and that this effect is modified by alteration of the saponins.
The
obtained and
.,
(nonhydrolyzed)
top or root
J. Clin.
Lynne Lyle
oral and
saponins
A.
Ph.D.,
of saponins
receiving
and after
before
radioactivity
0.
to 20
in rats
tested
B.A
,
of
plasma Acid absorp-
1977.
95% ethanol (1 1). Both types of saponins were partially hydrolyzed in 1 .5 N alcoholic HCI which was gently refluxed for 30 mm ; the saponins were purified by decolorization with charcoal and precipitated from 95% ethanol (1 1). For the sake of brevity, the altered saponins will be designated “hydrolyzed saponins.” Animals Male adult Sprague Dawley rats were used. The animals were fed Purina rat chow except from 7:30 AM to 4 PM on the day of the experiment; water was offered ad libitum throughout. The rats were kept in wire mesh cages and housed in a room lighted 1 2 hr per day. The room’s temperature was maintained at 24 C. Experiments that required changes in feeding patterns and in caging are noted below. The animals were lightly anesthetized with ether at about 10:30 AM. A tail vein was cannulated with PE-lO tubing according to the method described by Bollman and Van Hook (12) and injected with 5 .tCi of [3HJcholesterol freshly dissolved in 95% ethanol and suspended in 1 ml of saline. A cannula (feeding tube SF, Pharmaseal, Toa Alta , Puerto Rico) was then introduced into the stomach and the following were given
‘ Publication 941 of the Oregon Regional Primate Research Center. Supported with Grants RR00163 and HL 16587 of the National Institutes of Health. 2 Address reprint requests to: M. R. Malinow, M.D., Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97005. 3 Department of Cardiovascular Diseases, Oregon Regional Primate Research Center, Beaverton, Oregon 97005 . Department of Medicine, University of Oregon Health Sciences Center, Portland, Oregon 97201 . ‘ Western Regional Research Laboratory, Agricultural Research Service, USDA, Berkeley, California 94716. ‘ Department of Animal Science, Oregon State University, Corvallis, Oregon 97331.
1977,
pp.
2061-2067.
Printed
in U.S.A.
2061
2062
MALINOW
consecutively: 0.5 ml of safflower oil, alfalfa saponins in 0.7 to 1 .4 ml of 50 or 95% ethanol, about 2 Ci of [‘4Cjcholesterol dissolved with 2 mg of cholesterol in 0.5 ml ethanol and 1 .0 ml water. Control rats were studied similarly but received 0.7 to 1 .4 ml of 50 or 95% ethanol instead of a saponin solution. Certain experiments departed from this protocol. One series of animals received [3H]-f3-sitosterol dissolved with 0.2 mg of f3-sitosterol mixed with the oral I ‘4CJ-cholesterol; these animals were not given radioactive cholesterol intravenously . Another group of rats was kept in Bollman restraining cages to prevent coprophagy. A third group was fed a semipunfied diet containing 10% safflower oil, 25% casein, 48% sugar, 10% cellulose, and vitamins and salts starting 2 days before the experiment . The animals were fasted for 18 hr; they then received orally 7 mg of [‘4CJ-cholesterol dissolved in safflower oil and suspended via sonication in 0.6 ml of 6.8% skim milk powder as described by Zilversmit (1 3) . They also received [3H]-cholesterol as indicated above. Animals were stratified and assign.ed to the experimental or control groups according to a strictly random procedure. Four preparations were tested: nonhydrolyzed and hydrolyzed alfalfa top saponins and nonhydrolyzed and hydrolyzed alfalfa root saponins. The experimental design, including amounts of saponins and radioactive tracers used, is shown in Table 1. Analysis
Blood from the
with heparin as anticoagulant was obtained tail under light ether anesthesia 48 and 72 hr after injection of the isotopes. A plasma aliquot was saponified with 33% K OH in a boiling water bath for 1 hour and extracted into petroleum ether (bp 30 to 60 C). The solvent was washed with water. Feces were collected daily for 3 days after injection, pooled for each rat, and frozen until analyzed. The feces were then thawed, mixed with 50% methanol, and homogenized in a blender (Osterizer, Oster Corp., Milwaukee, Wis.); an aliquot by weight (approximately 1 ml) was transferred to a tared 50-ml, screw-cap tube . The feces were saponified under reflux with 2 ml of 33% K OH for 90 mm at 90 C: small condensers7 were adapted to the screw-cap tubes. The saponified feces were cooled; 2 ml of ethanol were added; and the mixture was extracted into 20 ml of petroleum ether. The solvent was washed with 5 ml of water and an aliquot was dried under N2. To decrease quenching, the residue was dissolved in chloroform and ozone was dispersed until no distinguishable color remained (14). Radioactivity
measurements
ET
AL.
receiving L’4C/3H1..cholesterol, the ‘4C/3H ratio was 1:2 to 1 :5 in plasma extracts and it was approximately 3:1 in fecal extracts. In extracts containing [3H]-f3-sitosterol , the ‘4C/3H ratio was about 1 : 5 . The error of counting was less than 1 % . An automatic external standard was used to calculate DPM. Calculation absorption
of intestinal
The radioactivity culated by weight
received by each animal was calof the syringe before and after injection. The excreted labeled neutral sterols were considered to represent the nonabsorbed cholesterol; absorbed cholesterol is thus shown in the tables as “ 1 00-feces .“ This estimation is based on the assumption that loss of radioactive cholesterol due to degradation to a chemical form not recovered by the method of analysis is equal to the fecal excretion of “endogenous” cholesterol and of an opposite sign . In certain experiments, 100-feces was compared to “corrected absorption” (see below,Table 3). Losses caused by incomplete collection and by degradation of cholesterol to substances not recovered by the method of analysis were estimated from losses in [3H]-6-sitosterol used as internal standard; absorption of [3H1-fJ-sitosterol was not measured, but it was assumed to be 5% of the injected dose (13, 14). Figures for ‘4C-nonsaponifiable material excreted after absorption were calculated as the product of the nonsaponifiable 3H excreted in the feces times the average ‘4C/3H plasma ratio determined at 48 and 72 hr; these figures were corrected for losses and degradation (see below, Table 3).
Results Validation
ofpresent
methods
Recovery of ‘4C-cholesterol added to an aliquot of feces was 97.9 ± 0.4% (mean ± SD, N = 4); it was 96.0 ± 6.8% (N = 4) after the addition of hydrolyzed alfalfa top saponins and 93.0 ± 3.8% (N = 4) after the addition of hydrolyzed alfalfa root saponins. The saponins were added in a S :1 (w/w) ratio with respect to cholesterol. Recovery of 3H-3-sitosterol was 1 00 .5 ± 4.0% (mean ± SD, N = 7). The fecal excretion of neutral ‘4C-stemols and of 3H-f3-sitosterol in three groups of rats is shown on Table 2 Prevention of coprophagy did not influence neutral sterol excretion; cholesterol absorption was 51.4 ± 1 .4% (mean ± SE) in rats kept in wire mesh cages and 52 .0 ± 2 .9% in rats kept in Bollman restraining cages. Rats that received saponins excreted 78.0 ± 2.3% of ‘4C-neutmal sterols and 86.8 ± 0.6% of /3sitosterol compared to the control rats, which excreted 48.6 ± 1 .4% and 82.8 ± 0.8% respectively. Consequently, choles.
Aliquots of the plasma extract were dried under N2 and were dissolved in 1 0 ml of toluene scintillator containing 5 .0 g PPO/liter and 0. 1 g dimethyl POPOP/ liter. The ozonated fecal extract was dried under N2, and the radioactivity was assayed in 0.5 ml of water and 12 ml of 10% Bio-Solv Solubilizer (Beckman Instruments, Fullerton, Calif.). Radioactivity was assayed in a liquid scintillation spectrometer. In rats
7
Manufactured
ate Center, Oregon
19600 97005.
by Mr. NW.
Alan Ryall, Oregon GraduWalker Road, Beaverton,
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,
ALFALFA TABLE Experimental Series and group
SAPONINS
AND
CHOLESTEROL
1 design No. of rats
Table
Body
Alfalfa
sapomns
Dosage
4,5 4, 5 4,5
4,5
a ‘4C-C,
254 ± 262 ± 259 ± 385 ± 394 ± 304±2 301 ± 339 ± 336 ± 322±5 322 ± 322 ± 322 ± 321 ± 321 ±
6 6 6 6 6 6 6 6 6 6 6 6 6
3
Comments Oral
5 5
2
isotopc
Injected
wtSE(mean
[4-’4C]-cholesterol;
IV’
mg
g
I, a I, b I, c II, a II , b III,a III, b IV, a IV, b V,a V, b V, c V, d VI,a VI, b
2063
ABSORPTION
2 2 4 6 6
Roots,
hydrolyzed
2 6
Roots,
5
Tops,
nonhydrolyzed
4 4 4 16 3
Roots, Roots, Roots,
hydrolyzed hydrolyzed hydrolyzed
Tops, 3H-C,
[icr,
‘4C-C,
0 0 20 0 0 0 10 0 20 0 5 10 20 0
nonhydrolyzed
‘4C-C, 14C-C, ‘4C-C ‘4C-C 14C-C ‘4C-C ‘4C-C ‘4C-C ‘4C-C ‘4C-C ‘4C-C 14C-C ‘4C-C
2a(n)-3H]-cholesterol;
3H-S,
Wire mesh Restraining Wire mesh Semipurified Chow diet
3H-C 3H-C 3H-C 3H-C 3H-C 3H-C 3H-C 3H-C 3H-C 3H-C 3H-C 3H-C
‘4C-C
5
hydrolyzed
3H-S 3H-S 3H-S
[22,23-3HJ--sitosterol.
b
cage cage cage diet
Intravenous.
TABLE 2 Effect of coprophagy and alfalfa root saponin (hydrolyzed) on neutral sterol fecal excretion and cholesterol absorption in rats (% ID/72 hr;5 mean ± SE)
.
Feral
Senes and group
C-neutral
I, a I, b I, c
Control, Control, Saponins,
P (Student’s aversusb aversusc a
0.
Injected b
Not
t
wire mesh cage restrained wire mesh cage
48.6 48.0 78.0
excretion
± ± ±
Cholesterol absorption (100-feces)
.
sterols
H-$-sitosterol
1 .4 2.9 2.3
82.8 78.9 86.8
0.8 1.5 0.6
± ± ±
51 .4 52.0 22.0
± ± ±
1.4 2.9 2.3
test) NSb