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Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan;
*
Department of Biopharmaceutics, Tokyo University of Pharmacy and Life Science, Tokyo 192-0355, Japan; and
Central Research Laboratories, Wakunaga Pharmaceutical Company, Hiroshima 739-1105, Japan
2To whom correspondence should be addressed. E-mail: horieto{at}p.chiba-u.ac.jp.
ABSTRACT
Antitumour drugs such as methotrexate (MTX) and 5-fluorouracil (5-FU) induce intestinal damage. This is a serious side effect of cancer chemotherapy. The present studies examined whether or not aged garlic extract (AGE) protects against damage from these antitumor drugs. Both drugs were administered orally for 4 or 5 d to rats fed a standard laboratory diet with and without 2% AGE. The small intestinal absorption of the poorly absorbable compound, fluorescein isothiocyanate–labeled dextran (FD-4; average molecular weight, 4400) was used to evaluate the damage to the intestine using the in vitro everted intestine technique and the in situ intestinal loop technique. FD-4 absorption increased in the antitumour drug–treated rats fed the diet without garlic. Interestingly, FD-4 absorption was depressed in rats fed the diet containing AGE. These results suggest that AGE may protect the small intestine of rats from antitumour drug–induced damage.
KEY WORDS: • aged garlic extract • methotrexate • 5-fluorouracil • intestinal damage • protection
Antitumor drugs such as methotrexate
(MTX)3
and 5-fluorouracil (5-FU) are known to cause damage to the small
intestine, leading to its dysfunction (Altmann 1974
,
Capel et al. 1979
). Nausea, vomiting, diarrhea,
stomatitis and gastrointestinal ulceration are reported to occur after
the use of these chemotherapeutic agents (Frei et al. 1975
, Rosen et al. 1979
). These and other side
effects can interfere with cancer chemotherapy.
Antitumour drug–induced damage to the intestine can be evaluated by
various methods including morphological, biochemical and
physicochemical changes (Tsurui et al. 1990
). Both
transcellular and paracellular transport routes are observed in the
intestinal epithelium (Powell 1981
). Fluorescein
isothiocyanate (FITC)-labeled dextran (FD-4; average molecular weight,
4400) absorption has been used to examine paracellular absorption. FD-4
is poorly permeable in the normal intestine. Recently, we discovered
that intestinal damage increased the permeation of FD-4
(Nakamaru et al. 1998
).
Garlic is thought to have various pharmacologic properties. For
example, it has been found to lower serum and liver cholesterol
(Kamanna and Chandrasekhara 1984
, Qureshi et al. 1983
), inhibit platelet aggregation (Apitz-Castro et al. 1983
, Ariga et al. 1981
), inhibit bacterial
growth (Cavallito and Bailey 1944
) and reduce oxidative
stress (Horie et al. 1989
, 1992
). This
paper reports that a commercially available form of garlic, aged garlic
extract (AGE), alleviates the small intestinal damage in rats induced
by MTX and 5-FU.
MATERIALS AND METHODS
Chemicals.
MTX was purchased from Wako Pure Chemical(Osaka, Japan). FITC-dextran and 5-FU were from Sigma Chemical (St. Louis, MO). All other reagents were of analytical grade.
Animals.
Male Wistar rats (8 wk old) (Japan SLC, Shizuoka, Japan) used in those studies had free access to food and water. They were housed in a room with a 12-h light:dark cycle and an ambient temperature of 25°C. Rats were acclimated for at least 1 wk before experimental use. Rats were first fed a standard laboratory diet (Oriental Yeast, Tokyo, Japan) and then a CE-2 diet (standard laboratory diet commercially available, Clea Japan, Tokyo, Japan) or the CE-2 diet containing 2% AGE for 8–11 d, depending on the study. MTX (15 mg/kg body) or 5-FU (30 or 60 mg/kg body) was administered orally once each day for 4 or 5 d. Treated rats were deprived of food overnight before experimental use. AGE was obtained by extracting cracked garlic bulbs (Allium sativum Linn) with ethanol at 4°C for 15 h, followed by evaporation under reduced pressure below 40°C and lyophilization.
In vitro permeation of FD-4.
The intestinal permeation was studied in vitro using everted segments
of small intestine as described previously (Yamamoto et al. 1997
). Rats were anesthetized with ethyl ether and the
intestines were excised. Intestinal segments (12 cm) were cut off 3 cm
from the end of the duodenum. The segments were everted in ice-cold
saline solution. An L-shaped glass cannula was inserted
into each end (1 cm) of the everted segments and a 10-cm plastic
syringe was attached to the exposed end of each cannula, a modification
of a technique of Doluisio et al. (1969)
. The segments
were then placed in 40 mL of 0.05 mol/L phosphate buffer/0.9% saline
solution (pH 6.5) containing FD-4. The buffer solution (5 mL) was put
into the serosal side of the segments. The plungers were gently moved
up and down and the permeation experiments were started after the
intestinal segments were incubated for 7 min at 37°C. Gas (95%
O2/5% CO2) was gently bubbled into the mucosal
side solution during the permeation experiments. At designated times,
0.3 mL was taken from the serosal side for determination of FD-4 and
replaced with an equal volume of buffer. Additionally, 0.1 mL of the
mucosal solution was taken for FD-4 determination.
In situ absorption of FD-4.
FD-4 absorption was studied by the in situ intestinal loop technique. Treated rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body). A cannulation with a polyethylene tube was made in the femoral artery of rats. A jejunal loop 10 cm in length was prepared, and then FD-4 solution (1 mmol/L, 1.3 mL) was put into the loop. Blood was withdrawn from the femoral artery at designated times to determine the plasma concentration of FD-4. The blood samples were centrifuged for 2 min at 14,000 g in a Beckmann Microfuge E (Palo Alto, CA) and the plasma concentration of FD-4 was determined.
Determination of FD-4.
FD-4 was determined according to Nakamaru et al. (1998)
.
The sample solutions taken from the serosal and mucosal sides were
diluted with 0.05 mol/L phosphate buffer/0.9% saline solution (pH
6.5). The fluorescence intensity of FD-4 in the sample solutions
prepared as above and in the plasma separated from the blood samples as
above was determined at an excitation wavelength of 495 nm and an
emission wavelength of 515 nm using a Hitachi fluorescence spectrometer
F-2000 (Tokyo, Japan).
Permeation clearance.
Permeation clearance of FD-4 was calculated using the following
equation (Horie et al. 1998
):
 |
where Ct1 and
Ct2 are the concentration of FD-4 in
the serosal side at times t1 and
t2, respectively; V is the
volume of the buffer solution in the serosal side after the completion
of permeation experiment after the time interval 
t
(t2 -
t1);
Cout is the concentration of FD-4 on
the mucosal side; and L is the length of the small
intestine.
Statistical analysis.
Statistical analysis was performed using Student’s t test. Results were considered significant at P < 0.05.
RESULTS
FD-4 permeability
The time course of FD-4 permeability of the small intestine is
shown in Figure 1
. It increased linearly from 10 to 30 min. The permeated amounts of FD-4
through the small intestine of the MTX-administered rats at 20 and
30 min were significantly greater than those of the controls. The
permeation clearance of FD-4 in the MTX-administered rats was
significantly higher than that in the controls (Fig. 2
).
|
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. Body weight was reduced by 5-FU treatment. Treatment with 5-FU (30
mg/kg body) for 4 d resulted in a similar permeation clearance of
FD-4 to that observed after treatment with 5-FU for 5 d (data not
shown). The FD-4 permeation through the small intestine increased,
depending on the administered dose of 5-FU (Fig. 4
). The FD-4 permeation in rats treated with 5-FU (60 mg/kg body) was
greater than that in rats treated with 5-FU (30 mg/kg body). The
permeation clearance of FD-4 in rats treated with 5-FU was greater than
that of the untreated rats and increased depending on the dose of 5-FU
(Fig. 5
).
|
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Two groups of rats were fed the experimental diets for 15 d.
One was fed a standard laboratory diet and the other the standard
laboratory diet plus AGE. The oral administration of MTX (15 mg/kg
body) to the two groups of rats was started on d 11 after the start of
consumption of the above diets and carried out once each day for 4 d. As shown in Figure 6
, rats fed the standard laboratory diet lost considerable weight after d
13, i.e., d 3 after MTX administration. Rats fed AGE were less markedly
influenced. The enhanced permeation of FD-4 through the small intestine
in MTX-treated rats was muted by the provision of AGE (Fig. 7
). The permeation clearance was 0.346 ± 0.061
µL/(min · cm) for rats fed the standard diet and 0.225
± 0.039 µL/(min · cm) for rats supplemented with
AGE.
|
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) (Table 1
). The AUC0–90 min for the 5-FU–treated rats fed
the standard diet was 6.60 ± 2.72
nmol · min · mL-1;
for the 5-FU–treated rats supplemental with AGE, it was reduced to
5.43 ± 2.24
nmol · min · mL-1.
|
DISCUSSION
Oral administration of MTX to rats results in an increase in FD-4
permeation through the small intestine as shown by the in vitro everted
intestine experiment, compared with those not treated with the drug.
These results are consistent with those obtained in mice (Horie et al. 1998
, Nakamaru et al. 1998
). The
increased permeability of the poorly absorbable compound, FD-4, is
attributed to damage to the intestine. Oral administration of 5-FU to
rats also enhanced the FD-4 permeation through the small intestine.
Again, this change in permeability likely reflects damage to the
intestine (Horie et al. 1998
, Nakamaru et al. 1998
).
The effect of AGE on the intestinal damage to the MTX- or 5-FU–treated rats was consistent. Weight loss in rats treated with MTX was reduced by the provision of AGE. Accompanying this was a lower permeability of FD-4.This suggests that AGE protected the small intestine from MTX-induced damage.
Intestinal damage induced by the administration of antitumour drugs
such as MTX and 5-FU is attributed to damage to the crypt cells
(Altmann 1974
, Kosakai et al. 1991
,
Taminiau et al. 1980
). Vitamin A coadministration with
MTX has been demonstrated to protect the small intestine from damage
(Tsurui et al. 1990
). Vitamin A may influence the crypt
cells by activating protein synthesis (Kosakai et al. 1991
). The present studies suggest that damage to the small
intestine induced by the antitumour drugs such as MTX and 5-FU is
reduced by AGE. The mechanism accounting for the protection is unknown.
Similarly, it is unclear whether other garlic preparations would also
have this effect on the small intestine.
FOOTNOTES
1 Presented at the conference "Recent Advances
on the Nutritional Benefits Accompanying the Use of Garlic as a
Supplement" held November 15–17, 1998 in Newport Beach, CA. The
conference was supported by educational grants from Pennsylvania State
University, Wakunaga of America, Ltd. and the National Cancer
Institute. The proceedings of this conference are published as a
supplement to The Journal of Nutrition. Guest editors:
John Milner, The Pennsylvania State University, University Park, PA and
Richard Rivlin, Weill Medical College of Cornell University and
Memorial Sloan-Kettering Cancer Center, New York, NY. 
3 Abbreviations used: AGE, aged garlic extract;
AUC, area under the curve; FD-4, fluorescein isothiocyanate–labeled
dextran; FITC, fluorescein isothiocyanate; 5-FU, 5-fluorouracil; MTX,
methotrexate.
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) Without MTX treatment;
(•) with MTX treatment. Each value represents the mean ± SEM, n = 3–6. *P
< 0.05, **P < 0.01, significantly different
from rats without MTX treatment.
) with 5-FU (60 mg/kg body) treatment. The data
represent the mean ± SEM, n = 3.
When absent, the bars were within the symbols. *P
< 0.05, significantly different from rats without 5-FU
treatment.
