Industrial Crops and Products 12 2000 183 – 192
The detrimental effect of simmondsin on food intake and body weight of rats
David A. York
a,
, Lori Singer
a
, Julian Oliver
b
, Thomas P. Abbott
b
, George A. Bray
a
a
Pennington Biomedical Research Center,
6400
Perkins Road, Baton Rouge, LA
70808 4124
, USA
b
New Crops Research, USDA-ARS-NCAUR,
1815
N. Uni6ersity Street, Peoria, IL
61604
, USA Accepted 12 May 2000
Abstract
Background
:
Simmondsin is a cyanomethylene glycoside, derived from the desert shrub, Simmondsia chinensis. Simmondsin has been reported to produce weight loss and to decrease food intake, an effect that can be blocked by
treatment with an inhibitor of cholecystokinin. Study design
:
Six experiments were conducted on male Sprague – Daw- ley rats. Results
:
In two acute experiments where simmondsin was either added to the diet or injected, there was a dose-related reduction in food intake. The CCK
A
antagonist, lorglumide, did not block the acute inhibitory effects of simmondsin on food intake and simmondsin did not produce conditioned taste aversion. In two chronic feeding
studies, the high dose of simmondsin 0.5 in the diet produced profound weight loss and death in rats. At autopsy, the kidney, heart and liver of the treated animals were larger than the pair-fed animals and there was a marked
suppression of the bone marrow elements with severe anemia. Conclusion
:
Simmondsin is toxic with profound effects on the hematopoietic system. © 2000 Elsevier Science B.V. All rights reserved.
Keywords
:
Energy expenditure; Cholecystokinin; Lorglumide; Conditioned taste aversion; Capsaicin vagotomy www.elsevier.comlocateindcrop
1. Introduction
Simmondsia chinensis is a desert shrub that grows in the Sonoran desert of the Southwestern
US. The jojoba meal extract from this plant is toxic to animals Booth et al., 1974; Verbiscar et
al., 1980. Elliger et al. 1974a,b have identified a compound called simmondsin [2-cyano-methyl-
ene-3-hydroxy-4,5-dimethoxycyclohexyl b-
D
-glu- coside] from jojoba meal. Cokelaere et al. 1992b
have taken up the question of whether the toxic effects of jojoba meal and the reduction of food
intake have been separated with the purified sim- mondsin. This compound appears to be well ab-
sorbed whether given intragastrically or mixed with food Flo et al., 1997. Cokelaere et al.
1995a have reported that this effect is blocked by devazepide, an antagonist of the cholecys-
tokinin A receptor. The possibility that CCK
A
receptors were involved in the mechanism of ac-
Corresponding author. Tel.: + 1-225-7632548; fax: + 1- 225-7632525.
E-mail address
:
yorkdapbrc.edu D.A. York. 0926-669000 - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 6 - 6 6 9 0 0 0 0 0 0 7 0 - 4
tion of this compound prompted the present studies.
It has recently become evident that the majority of orexigenic and anorectic compounds also have
effects on energy expenditure through their recip- rocal effects on the autonomic responses to feed-
ing Bray and York, 1998. Thus, orexigenic agents tend to inhibit the sympathetic drive to
brown adipose tissue BAT to reduce BAT ther- mogenesis, whereas anorectic agents promote
BAT thermogenesis and weight loss. We have investigated the possibility that simmondsin in-
creases energy expenditure in addition to its anorectic properties. We have also undertaken
studies to show that the anorexia induced by simmondsin does not result from the induction of
a conditioned-taste aversion.
2. Methods and materials
2
.
1
. Animals Male Sprague – Dawley rats were purchased
from Harlan Sprague – Dawley Indianapolis, IN. They were maintained on a 12:12 h light:dark
cycle 08:00 – 20:00 h with tap water available through an automatic system.
2
.
2
. Diets The animals were maintained on a chow diet
Ralston-Purina Co., St. Louis, MO, Lot 5001. In some experiments, powdered chow was mixed
with simmondsin at one of several concentrations. Food intake was measured at intervals of 0.5, 1,
2, 4, 6, and 24 h in the acute experiments and daily during the chronic experiments.
2
.
3
. Procedures
2
.
3
.
1
. Experiment
1
Male Sprague – Dawley rats, weighing 220 – 260 g, were housed singly and allowed to eat
powdered chow that was provided in a food jar within the cage. On the experimental day at the
beginning of the dark cycle 20:00 h all rats were given fresh diet with either no simmondsin N =
4, or simmondsin added at 0.1, 0.25, 0.5, 0.75, 1.0 or 2.0 in the powdered chow with four rats
at each dose. Food intake was measured at 1, 2, 4, 6 and 24 h. After 24 h, the diet was replaced with
normal powdered chow. Each rat received only one treatment.
2
.
3
.
2
. Experiment
2
After completion of Experiment 1 7 days, the male Sprague – Dawley rats were fasted overnight
to enhance their hunger and then given intraperi- toneal injections of 2 mlkg containing either sa-
line 0.9 wv vehicle N = 6 or 200, 400, 800 or 1600 mg simmondsin with N = 6 rats at each
dose. Food was returned within 5 min and food intake was measured at 0.5, 1, 2, 4, 6 and 24 h.
2
.
3
.
3
. Experiment
3
This experiment investigated the ability to block anorectic response to simmondsin with the
CCK
A
receptor antagonist lorglumide. Thirty male Sprague – Dawley rats 200 – 240 g were
housed singly and adapted to a powdered chow diet. All rats were deprived of food for 2 h
09:00 – 11:00 h before they received either a sa- line vehicle injection N = 10, simmondsin, 600
mgkg i.p. plus saline N = 10 or lorglumide 600 mgkg and simmondsin 600 mgkg N = − 10.
The dose of lorglumide was injected 10 min before simmondsin. This dose of lorglumide has previ-
ously been shown to block the anorectic response to CCK, but lorglumide in this dose had no effect
on food intake by itself data not shown. Food intake was measured at 0.5, 1, 2, 4, 6 and 8 h.
2
.
3
.
4
. Experiment
4
Conditioned taste
aversion was
tested in
Sprague – Dawley rats by comparing the intake of a saccharine-sweetened solution 0.15 in a two-
bottle test against water when no food was avail- able. Four male rats were injected with saline, six
rats were injected with simmondsin 600 mgkg i.p. and five rats were injected with lithium chlo-
ride in the presence of the saccharine solution on two occasions, 2 days apart. After an interval of 2
more days following the injection of lithium, rats were provided with a choice of saccharine solu-
tion or water to drink and the consumption of
each was measured over a 30-min period. The intake of saccharine solution is expressed as a
percent of total fluid intake.
2
.
3
.
5
. Experiment
5
The chronic effect of simmondsin 0.5 in the chow diet on food intake and body weight was
compared in groups of ten animals fed ad-lib and ten animals that were pair-fed to the simmondsin
group. Pair feeding was achieved by providing each rat with the intake of a simmondsin-fed rat
on the previous day after correcting for spillage from the simmondsin-fed rats.
Energy expenditure of the rats was measured between the third and fifth weeks. Animals were
placed in individual metabolic cages and the con- sumption of oxygen and production of carbon
dioxide over 24 h was measured with a Columbus Instruments apparatus Columbus, OH.
2
.
3
.
6
. Experiment
6
A chronic dose-ranging study compared doses of 0.015, 0.05, 0.15 and 0.5 to a control group
and a capsaicin-treated control group to a capsa- icin-treated group receiving 0.15 simmondsin.
Rats were fed ground chow with or without the simmondsin for 4 weeks. Capsaicin treatment was
performed in two groups of rats 2 weeks prior to the beginning of the study as previously described
Lin et al., 2000.
2
.
4
. Pathology At autopsy, dissected tissues from multiple or-
gans from 16 rats in Experiment 5 four survivors on the 0.5 simmondsin, four pair-fed rats and
eight controls were placed in 10 formalin for fixation and transferred to the veterinary patholo-
gist. Tissues were embedded in paraffin, cut in 4-mm sections and stained with hematoxylin and
eosin. Lesions, when present, were graded as nor- mal, mildly affected, moderately affected or
severely affected.
2
.
5
. Reagents The simmondsin used in these experiments was
kindly prepared by Thomas Abbott, Ph.D. of the USDA Laboratories in Peoria, IL.
Simmondsin was purified by the procedure de- scribed by Abbott et al. 1999. Analysis by five
different laboratories determined the purity to be 94.3 – 100 and averaging 99.48 purity Abbott
et al., 2000. Analysis of this simmondsin sample C was performed by HPLC by an independent
laboratory, comparing it to the same material recrystallized twice more TC and the residue
from the recrystallization R. A Sphersorb Nov- Pak C-18, 4-mm, 4.6 × 150 mm column Waters
was eluted isocratically with acetonitrilewater acetic acid 250 ml acetonitrile in 4 l water and
with pH adjusted with acetic acid to pH 3.5, eluting at 0.5 mlmin. Detection was with UV at
220 nm, refractive index RI and scanning fluorescence, 320 nm excitation, 450 nm emission.
Only simmondsin S, RT 13.3 min and demethyl- simmondsin DMS peaks two isomers gave two
peaks, RT 7.1 and 10.0 min, were observed. C gave a purity of 98.58 S and 0.50 DMS one
peak, RT 10.1 compared to 98.28 S, 0.91 DMS one peak, RT 10.1 for TC, based on UV
peak area of the observed components. R tested to be 83.67 S, 15.06 DMS by UV. Peak areas
in RI gave 99.32 S, 0.35 DMS for TC, 99.33 S, 0.23 DMS for C and 88.27 S, 9.05 DMS
for R. LC-MS was also performed on the three materials. The only three materials found in C
and TC were identified as S and two DMS iso- mers. Very minor peaks were observed in R,
which were not identifiable at these very low levels. Thin layer chromatography TLC on silica
gel in 9010 acetonitrilewater, 8020 chloroform methanol and 8020 methanolwater and detec-
tion by UV, exposure to iodine or charring with sulfuric acidphenol gave at most two spots for C
and TC and three spots for R, none of which were at the origin. The mother liquor from the prepara-
tion of C gave five spots, some with the same R
f
values as in C, TC and R. Preparative TLC was used to separate the individual spots for identifi-
cation by NMR. Based on these analyses, the C and TC samples appear to be very similar in
composition and of high purity with DMS as the principal minority impurity and no other detected
impurities.
The capsaicin was purchased from Sigma St. Louis, MO. It was dissolved in 0.9 saline vehi-
cle containing 10 ethanol, 10 Tween-80 before being administered to rats on three occasions at
doses of 25, 25 and 50 mgkg with 18 and 6 h between successive treatments. The effectiveness
of the capsaicin treatment in destroying non- myelinated afferent fibers was confirmed by the
failure to exhibit corneal reflex in response to 1 NH
4
OH. The lorglumide was purchased from Sigma and was dissolved in 0.9 saline before
being administered to the rats.
2
.
6
. Statistical analysis Data are expressed as mean 9 S.E.M. An a of
0.05 was accepted as the level of statistical signifi- cance. ANOVA was used with Bonferroni’s cor-
rection for multiple comparisons to compare repeat measure studies.
3. Results
