Pergamon 0306-3623(94)E0052-N

Gen. Pharmac. Vol. 25, No. 6, pp. 1237-1243, 1994 Copyright ~" 1994 Elsevier Science Ltd

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Oral Administration of Quercitrin Modifies Intestinal Oxidative Status in Rats

J U L I O G A L V E Z , I JOSE P E D R O DE LA C R U Z , z* A N T O N I O Z A R Z U E L O , I F E R M I N S A N C H E Z DE M E D I N A Jr, I JOSE J I M E N E Z ~ and

F E L I P E S A N C H E Z DE LA C U E S T A 2 1Department of Pharmacology', School of Pharmacy, Universidad de Granada, 18071 Granada, Spain

-'Department of Pharmacology and Therapeutics, School of Medicine, Universidad de Malaga, 29071 Malaga, Spain [Fax 34-5-213-1568]

(Receiced 14 January 1994)

Abstract--l. Oral administration of quercitrin to rats for 3 days increases the mucosal glutathione contents in ileum and colon as well as inhibits non-enzymatic lipid peroxidation induced in membrane fractions from jejunal and colonic mucosa.

2. After 7 days of treatment with quercitrin, rat intestinal oxidative status trends to normalize to control rats.

Key Words: Quercitrin, lipid peroxidation, glutathione, intestine

INTRODUCTION

Flavonoids comprise one of the large groups of secondary metabolites occurring widely throughout the plant kingdom, including food plants and crude drugs. In fact, they are ingested in daily quantities of 1-2g by humans who eat diets typical of those found in the Western world (Kuhnan, 1976). These compounds have been reported to have a wide range of possible uses in medicine (Pathak et al.,

1991). Quercetin is one of the most common flavonoids

in plants (Havsteen, 1983), but it occurs most frequently as its glycoside form, such as rutin (quercetin 3-rhamno-glucoside) and quercitrin (quercetin 3-rhamnoside). Several pharmacological activities have been demonstrated both in vivo and in vitro for these flavonoids, including anti- inflammatory (Mascolo et al., 1988), and anti- thrombotic (Landolfi et al., 1984; Gryglewski et al.,

1987) activities. The mechanisms of such pharmaco- logical activities have been related to inhibitory actions on several enzymes (Landolfi et al., 1984; Gryglewski et al., 1987) or to their actions as free radical scavengers and inhibitors of peroxidation (Mora et al., 1990).

*To whom all correspondence should be addressed.

However, little attention has been paid to their effects on the gastrointestinal system, even more if we take into account that quercetin and its glycosides are usually ingested in a normal diet. In previous studies we demonstrated that quercitrin showed antidiarrhoeic activity in mice and rats (Galvez et al.,

1993a; Galvez et al., 1993b), from doses of 25 mg/kg, in several models of experimentally-induced diarrhoea.

The aim of the present study was to evaluate the effects exerted by daily oral administration of quercitrin to rats at doses of 25 mg/kg for 3 and 7 days on intestinal physiology. First, we studied several enzymatic activities related to digestive and absorptive processes in the intestine, namely disac- charidase (maltase, sucrase and lactase) activities in jejunum and ileum, and alkaline phosphatase activity in colon; second, we evaluated the oxidative status in the intestinal mucosa, as quercitrin has been described to possess antioxidant properties (Galvez et al., 1993b), either by determination of glutathione content in the mucosa of jejunum, ileum and colon, or by inducing non-enzymatic lipid peroxidation in membrane fractions obtained from the above mentioned mucosa. The effects on the oxidative status could be interesting on the basis of its possible application in some intestinal diseases which have been related to the production of free radicals, such

1237

1238 Juuo GALVEZ et al.

as inflammatory bowel disease (Harris et al., 1992). Finally, the oxidative status in liver was also studied, in order to know the possible systemic effects that quercitrin might exert.

MATERIAL AND METHODS

Chemicals

All chemicals were obtained from Sigma Chemical Co. (St Louis, MO), except glutathione reductase (Boehringer Manheim, Barcelona, Spain) and malondialdehyde bis-diethylacetal (Aldrich-Chemic, Steinheim/Albuch, Germany).

EX VIVO EXPERIMENTS

Animals

Female Wistar rats of approx. 170 g wt were maintained in our laboratory and provided with free access to water and food. Animal quarters were air-conditioned (22'~C) and had a 12-hr l ight, lark cycle. On arrival at our laboratory, each rat was housed separately in a wire-bottomed polythene cage. The animals were separated into two groups of 20 rats each. Every day, quercitrin was administered orally to one of the groups at the dose of 25 mg/kg dissolved in l ml of isotonic saline solution. Rats from the control group received 1 ml of the vehicle. After 3 days of treatment, 10 animals of each group were sacrificed. The rest of the animals were sacrificed after seven days from the beginning of the experience.

Preparation o f tissues

The animals were sacrificed by decapitation between 9 and 11 a.m. The small intestine from the Treinz angle to the ileocecal valve was removed and divided into two halves. The proximal segment was defined as jejunum and the distal segment as ileum. The entire colon, from the caecum to the rectum, was also removed. All segments were trimmed of fat and mesentery, rinsed cautiously with cold saline solution (0.9% NaC1), and blotted on filter paper. The length (under a constant weight of 2 g) and weight of each segment was measured. Each segment was opened longitudinally and the mucosa was scraped using a glass slide, weighed separately on parafilm paper, and immediately frozen under liquid nitrogen. Mucosa samples were stored at -30°C until analysis. Rat liver samples were also obtained, weighed, frozen and stored at -30°C until analysis.

Analytical methods

Enzymatic assays. Mucosa from each rat was homogenized in a glass Potter-Evelhjeim with ice- cold Tris-HC1 buffer (50 mM Tris, 140mM NaCI

and 5 mM MgCI2, pH 7.4), using a ratio of I : 250 w/v for jejunum and ileum and 1:25 w/v for colon. Lactase, sucrase and maltase activities were assayed in jejunum and ileum by the Messer and Dalhquist method (Dalhquist, 1968). Alkaline phosphatase activity was assayed in colon by the method described by Bessey et al. (1946).

Rat liver homogenates were used to evaluate the enzymatic activities related to the glutathione system. All liver samples were diluted 1 : 6 (w/v) in potassium phosphate buffer (0.1 M, pH 7.0) containing 1 mmol/l EDTA and 0.01% digitonin. The samples were minced, homogenized, and centrifuged at 3000g for 15 rain at 4°C. The supernatant was immediately used for the enzyme assays. Glutathione peroxidase (GSH-px) activity was measured by using the con- secutive glutathione reductase reaction, according to the method of Floh6 and Gunzler (1984). The final concentration of tertbutyl hydroperoxide (TBHP) was 95#M. The assay solution contained 0.1M potassium phosphate buffer (pH7.0), l mmol/1 EDTA, 1.05mmol/l reduced glutathione (GSH), 0.118 mmol/l NADPH, 4/tl of glutathione reductase (600 U/ml) and a suitable sample (20/~g of protein) of enzyme solution. The reaction was started with the addition of the peroxide. The value for a blank reaction with the enzyme source replaced by water was substracted for each assay. The rate of reaction was recorded as 25'~C by following the decrease in absorbance at 340 nm. Activity was expressed as /~mol of NADPH oxidized, rain ~. mg ~ protein. Glutathione transferase (GSH-t) activity was measured by the method described by Warholm et al.

(1985). The assay mixture contained 0.1 M potassium phosphate buffer (pH 7.0), 1 mmol/l EDTA, 0.1 mmol/l GSH, 1 mmol/l 1-chloro-2,4-dinitroben- zene (CDNB) and a suitable amount (20/lg of pro- tein) of enzyme solution. The reference cuvette contained the complete assay mixture with the en- zyme solution replaced by water, The enzymatic assay was carried out at 25'C. Specific activity was expressed as/~mol of GSH conjugated, rain ~. m g ' protein. Glutathione reductase (GSSG-rx) activity was determined by the method described by Aceto et al. (1990). The assay mixture contained 0.1 M potassium phosphate buffer (pH7.0), l mmol/I EDTA, 3.4mmol/l oxidized glutathione (GSSG), 0.01 mmol/l NADPH and a appropriate amount (20/tg of protein) of enzyme source. The blank did not contain GSSG. Enzyme activity was determined at 25°C by measuring the disappearance of NADPH at 340 nm and it was expressed as l~mol of NADPH oxidized - min- ~ •mg t protein.

All measurements, in a final volume of 1 ml, were performed in duplicate.

Quercitrin and intestine 1239

The method of Bradford (1976) was used for the

determination of protein content, using bovine serum albumin as protein standard.

Totalglutathione content. Intestinal mucosa or liver samples from each rat were diluted 1:5 w/v in 5-sulfosalicyclic solution (5%), and homogenized in a glass Potter-Evelhjeim. The homogenates were centrifuged at 2000g for 5 rain at 4°C. The super- natants were removed and centrifuged in a Beckman microfuge (10,000g for 5min). The supernatants obtained were used for determination of total glutathione content utilizing the DTNB-GSSG Re- ductase Recycling assay described by Anderson (1985). The assay mixture contained 143 mM sodium phosphate buffer (pH 7.5), 6.3 mmol/l EDTA, NADPH (0.248mg/ml), 6mmol/1 5,5'-dithiobis(2- nitrobenzoic acid (DTNB), 2.5#I glutathione re- ductase (266 U/ml) and 10 ttl of sample supernatant. All measurements, in a final volume of l ml, were performed in duplicate. The amount of GSH was determined from a standard curve in which the GSH equivalents present (in nmol) were plotted against the rate of change of absorbance at 412 nm. Values are expressed as/~mol of glutathione per gram of tissue.

Lipid peroxidation experiments. Lipid peroxidation was measured both in intestinal and liver membrane fractions. Intestinal membranes were prepared following the method described by Van der Vliet et al. (1990). Liver membrane fractions were prepared according to the method described by De la Cruz et

al. (1992). The products resulting from the reaction with thiobarbituric acid (Thio-barbituric Acid Reactive Substances--TBARS), of which the most significant is malondialdehyde (MDA), were con- sidered as indicators of lipid peroxidation (Halliwell, 1990). Ferrous sulfate and ascorbic acid (FeAs) were used to induce lipid peroxidation via the formation of hydroxyl anions, following the method described by De la Cruz et al. (1991) with some modifications. Briefly, the reaction mixture contained 900/~1 of membrane fraction, 50/~1 ferrous sulfate and 50/~1 ascorbic acid in equimolecular concentrations (FeAs). The concentrations used were those which, in preliminary experiments, produced 100% of the maximum TBARS value in the different membrane fractions; 50/~mol/1 in colonic membranes, 100/~mol/1 in liver and ileal membranes and 500/~mol/1 in jejunal membranes. Samples were incubated at 37°C for 45 rain, and, subsequently, 500/11 of 0.5% thiobarbituric acid in 20% trichloroacetic acid were added. Samples were incubated at 100°C for 15 min and then centrifuged at 2750g for 15rain at 4°C. The amount of MDA produced was measured through spectrophotometric analysis of the supernatant at 532 nm. The ab-

sorbances obtained were compared to those of a standard curve using malondialdehyde-bis-diethyl- acetal. Results are expressed as nmol of MDA per mg of protein.

IN VITRO LIPID PEROXIDATION EXPERIMENTS

Rat liver or intestinal (jejunum, ileum and colon) mucosa samples were obtained from seven female Wistar rats of approx. 170 g wt. Non-enzymic lipid peroxidation assays were performed in membrane fractions obtained from liver or intestinal segments as described above in the ex vivo experiments. In this set of experiments, different concentrations of quercitrin were added before the first incubation at 37C, in order to test the antiperoxidative effect of this flavonoid. The results are expressed as percentage of inhibition of the MDA production in the control assays (without flavonoid). The concentrations which inhibited MDA baseline production by 50% (IC50) were calculated.

STATISTICS

All results are expressed as mean + SEM statisti- cal analyses were carried out using the Statgraphics program (STSC Inc. Rockville, Md). The non-paired Student's t-test was applied. Statistical significance was set at P < 0.05.

RESULTS

Effects o f quercitrin on jejunum, ileum and colon

Table 1 shows the length, total weight and mucosal weight, as well as the protein content per gram of mucosa, corresponding to the different intestinal segments from both experimental groups. There were no significant differences in these parameters between the quercitrin and control groups.

Specific dissaccharidase activities in jejunal and ileal mucosa are shown in Table 2. None of them was significantly modified by oral administration of quercitrin. Specific alkaline phophatase activity in colonic mucosa was also unaffected by either 3 (8 .9+0.9U/mg protein-control group vs 7.1 ___ 1.0U/mg protein-treated group) or 7 days of treatment (8.5 + 1.1 U/mg protein-control group vs 8.9 __+ 0.7 U/mg protein-treated group.

Figure 1 represents the total glutathione content in the different intestinal mucosa. Quercitrin treatment did not significantly modify this parameter in the jejunal mucosa, whereas the ileal glutathione content was increased by 34% in the treated animals (I.69 + 0.04/~mol GSH/g mucosa) compared to the

1240 JUL10 GALVEZ el al.

Table h Total weight, length, mucosal weight and protein content of the different intestinal segments obtained from rats treated with quercitrin (25 mg/kg, p.o.) for 3 and 7 days, and compared to control rats

Jejunum Ileum Colon

Control Treated Control Treated Control Treated

Total weight (g) 3 days 2 .28±0.18 2.25_+0.26 1.77±0.22 1.60+_0.18 0.704-0.10 0.68 ± 0.17 7 days 2.34_+ 0.14 2.15+_0.12 1.74_+0.19 1.46+_0.13 0.76 ± 0.14 0.61 4-0.08

Length (cm) 3 days 47.1 ±2 .5 46.7+_2.2 44.7+_3.4 44.7 ± 3.5 12.1 _ + 2 . 1 11.9_+2.3 7 d a y s 45.54-1,9 44 .4+2 .9 44 .2±3 .0 41.1+_3.9 13.1 ± hi 11.5_+1.2

Mucosal weight (g) 3 days 1.784-0.15 1.81 ±0.15 1.32_+0.18 1.22+_0.16 0 .39±0 .06 0 .39±0 .08 7 days 1.78 ± 0.14 1.64+_0.11 1.22+_0.12 1.07+_0.11 0.424-0.07 0.38+_0.06

Protein content (mg/g mucosa)

3 days 124.0 ± 13.8 134.3 4- 10.6 109.0+_ 13.8 118.4+_ 12.4 90.8_+ 17.1 101.7±9.5 7 d a y s 117.5+_18.1 114.4 +- 13.1 100.5 ± 9.3 108.5+_5.2 96 .2+17 .4 97.9+_8.6

Results are expressed as mean +_ SEM from 10 animals.

Table 2. Effects of quercitrin (25 mg/kg, p.o.), administered for 3 and 7 days, on maltase, sucrase and lactase activities of small intestinal segments in rats

Jejunum Ileum

Control Treated Control Treated

Maltase 3 days 301.9 ± 72.3 278.6 ± 40.7 265.9 ± 75.4 308.0 ± 79.6 7 days 382.3 ± 78.0 303.9±41.1 328.4_+60.1 341.04-84.3

Sucrase 3 d a y s 62 .2±8 .9 66.0+_9.2 30.0±12.8 30 .3±12.5 7 days 71.1 ±7 .9 65 .4± 12.1 29.04-7.6 34.6_+6.4

Lactase 3 days 18.9±6.5 14.8_+5.2 16.2_+6.8 17.0 ± 4.9 7 d a y s 23.3+_6.3 17.5_+3.7 21.8+_7.1 21.8+_7.4

Results are expressed as mean ± SEM from 10 animals. Specific activities are in #mol of substrate hydrolysed per min and gram of mucosal protein.

corresponding controls (1.26+_0.09pmol GSH/g mucosa, P < 0.001) after 3 days. The colonic mucosa showed a similar pattern, increasing its glutathione content by 42% after 3 days of flavonoid oral admin- istration (0.97 + 0.09 #mol GSH/g mucosa-treated

2-

o E

1- ¢¢ @

o

i

o3

O-

I

/ i / t / 1

3d 7d

jejunum

/ / / /

IS, / / / / / / / / / / / / / / / / / /

3d 7d ileum

control

I~1 treated

.ll-

lm 3d 7d colon

Fig. 1. Mucosal glutathione contents in the different intesti- nal segments of rats treated with quercitrin (25 mg/kg, p.o.) for 3 (3d) and 7 (7d) days compared to the corresponding

controls. (*) P < 0.05; (**) P < 0.01.

group vs 0.68 +0.05/~mol GSH/g mucosa-control group, P < 0.01).

Table 3 shows the maximum TBARS concentrations obtained after inducing lipid peroxi- dation with the corresponding concentrations of FeAs in different intestinal membrane fractions. In the jejunum, the peroxidation registered was lower in those animals treated with quercitrin, either for 3 or 7 days, this effect being most patent after 3 days of treatment. Lipid peroxidation was also lower in

Table 3. MDA contents (nmol/mg protein) after inducing non-enzy- matic lipid peroxidation with ferrous sulphate in membrane fractions obtained from different intestinal segments of rats treated with quercitrin (25 mg/kg, p,o.), for 3 and 7 days. Ferrous sulphate concentrations used were 501~mol/I in colonic membranes, 100/amol/l in ileal membranes and 500 pmol/I in jejunal membranes

Control Treated

Jejunum 3 days 9.59 4- 0.89 1.13 4- 0.21"** 7 days 7.52 4- 0.80 3.51 + 0.92**

Ileum 3 days 4.43 + 0.94 4.54 _+ 0.84 7 days 3.22 _+ 0.84 3.86 + 1.12

Colon 3 days 5.20 -+ 0.48 1.25 ± 0.60*** 7 days 6.20 + 1.04 6.01 4- 0.92

(**) P <0.01; (***) P < 0.001. Results are expressed as mean + SEM from 10 animals.

Quercitrin and intestine 1241

colonic mucosal membranes obtained from animals treated for 3 days with the flavonoid. However, no significant differences in the amount of MDA pro- duced in ileal membrane fractions from both groups of animals were observed.

Effects o f quercitrin on the liver

Table 4 shows the hepatic total glutathione content in the different groups of animals studied, as well as the enzymatic activities related to the glutathione system. None of these parameters was modified after treatment with quercitrin for 3 or 7 days.

When non-enzymatic lipid peroxidation was induced in liver membrane fractions, the production of TBARS was lower in those corresponding to 3 days of quercitrin treatment (5.24___ 0.87 nmol MDA/mg protein) compared to the controls (19.50 + 0.81 nmol MDA/mg protein, P < 0.001). This effect was also observed after 7 days of treatment, although a trend to normalization is evi- dent at this time point (17.19 _+ 0.11 nmol MDA/mg protein-treated group vs 19.94 + 0.41 nmol MDA/mg protein-control group, P < 0.01).

Effects o f quercitrin on in vitro lipid peroxidation

Quercitrin inhibited non-enzymatic lipid peroxidation induced both in intestinal and liver membranes. The ICm values were 8.19_+0.85pM, 7.49_0.84/zM and 7.14+0.78/zM in jejunum, ileum and colon membranes, respectively. There were no significant differences among them. The ICs0 value for liver membranes was 35.24_ 1.28/~M, which is significantly higher than the corresponding intestinal values (P < 0.001).

DISCUSSION

In a previous study it was demonstrated that quercitrin had antidiarrhoeic activity in several models of experimental diarrhoea in mice (Galvez et al., 1993b). In that study, the effects of quercitrin on small intestine transit as well as on fluid transport across the colonic mucosa in rats were also evaluated. Those assays pointed out that quercitrin delayed small intestine transit and increased electrolyte and fluid absorption when these functions were altered by agents like castor oil, sodium picosulphate or prostaglandin Ez, but not in normal conditions.

The results obtained in the present study show that quercitrin does not modify any of the macroscopic parameters studied, such as weight, length and mucosal weight, in the different intestinal segments: jejunum, ileum and colon. On the other hand, both dissaccharidases (maltase, sucrase and lactase) (Goda and Koldovsky, 1988) and alkaline phosphatase

Table 4. Glutathione contents and glutathione related enzymic activities [glutathione peroxidase (GSH-px), glutathione transferase (GSH-t) and glutathione reductase (GSSG-rd)], in rat liver after 3

and 7 days of treatment with quercitrin (25 mg/kg, p.o.)

Control Treated

GSH content (/a mol/g tissue)

3 days 4.04 + 0.35 3.43 ± 0.32 7 days 3.77 + 0.96 3.87 ± 0.71

GSH-px (U/mg protein)

3 days 3.62 ± 0.57 3.12 + 0.30 7 days 3.69 + 0.42 3.98 + 0.37

GSH-t (U/mg protein)

3 days 1.47 _+ 0,12 1.40 ± 0.13 7 days 1.49 ± 0.11 1.56 ± 0.11

GSSG-rd (U/mg protein)

3 days 0.57 ± 0.07 0.48 ± 0.11 7 days 0.54 ± 0.10 0.57_+ 0.10

Results are expressed as mean ± SEM from 10 animals. GSH-px and GSSG-rd activities are in #mol of NADPH oxidized per rain and mg of tissue protein; GSH-t activity is in #tool of glutathione conjugated per min and mg of tissue protein.

(Benjawatanapon et al., 1982) are commonly accepted as markers of absorption or active transport due to their membrane localization in intestinal epithelial cells. Oral administration of quercitrin for 3 or 7 days, produced no significant changes in these enzymatic nor in mucosal protein content, compared to control animals. We can therefore conclude that quercitrin does not alter the normal functionality of the intestine. This is in accordance with the previous study which revealed that quercitrin did not affect intestinal motility or absorptive processes in normal animals (Galvez et al., 1993b).

However, quercitrin is able to affect the intestinal oxidative status. Thus, the jejunal and colonic, but not the ileal membranes obtained from rats undergoing quercitrin treatment for 3 or 7 days were less susceptible to induced lypoperoxidation than the corresponding controls. This effect is more patent after 3 days of treatment and trends to disappear by the 7th day. Furthermore, the glutathione in ileal and colonic mucosa was increased after 3 days of oral administration of quercitrin, but had returned to normal after 7 days. In the jejunum, there is no significant modification in glutathione content either after 3 or 7 days in both groups of animals.

The fact that the oxidative status of the intestinal mucosa tends to normalize after 7 days of quercitrin treatment could be explained by the development of compensatory mechanisms at the intestinal level. However, the different behaviour showed in the three intestinal segments studied in this regard after 3 days of treatment may be explained by the existence of metabolizing products from quercitrin in the gut. The ability of mammals to hydrolyze fiavonoid glycosides to the corresponding aglycone has been reported

1242 JULIO GALVEZ et al.

(Hackett, 1986). Thus, when quercitrin gets to the intestine, it can be metabolized by microbial glycosidases present in the rat intestine to release its aglycone, quercetin (Bokkenheuser and Winter, 1988), Once quercetin is released, it can suffer ring cleavage by C-ring-cleaving bacteria in the gut, as proposed by Winter et al. (1989), to give phloro- glucinol and 3,4-dihydroxy-phenylacetic acid. However, a certain fraction of the quercetin released is not metabolized and can be absorbed from the digestive tract or excreted in the faeces (Ueno et al.,

1983). Although it is not clear in which part of the gut the glycoside hydrolysis and C-ring cleavage takes place, the results relative to the intestinal oxidative status in the present study could be explained by the different metabolic processes that quercitrin undergoes when administered orally.

Quercitrin is able to inhibit in vitro non-enzymatic lipid peroxidation induced on membrane fractions from all of the different segments of the gut studied. Therefore, the lower MDA production induced with FeAs in jejunum can be explained by the antiperoxi- dative effect exerted by this glycoside itself. As it was described above, quercitrin can be hydrolysed by bacterial glycosidases present in the gut to release quercetin. Part of this aglycone can suffer a C-ring fission, but other part remains unchanged. It has been described that autooxidation of quercetin can produce superoxide anions (Rueff et al., 1992). This superoxide anion production can be responsible for the increased levels of glutathione determined in ileum and colon, as a compensatory mechanism to avoid the possible effect of oxygen-derived species on intestinal mucosa. The lower induced peroxidation registered in colon can be attributed to the anti- peroxidative effect of quercetin, as this aglycone has been described to possess the ability of penetrate in the membranes (Price and Middleton, 1986) where it can exert its well known antioxidant properties (Mora et al., 1990), being also active when non- enzymatic lipid peroxidation was induced on intestinal membrane fractions (data not shown), in the same range of concentrations tht quercitrin did.

In the liver, oral administration of quercitrin to rats for 3 or 7 days does not induce significant changes either in total glutathione content or in the enzymatic activities related to this peptide. However, the liver membrane fractions obtained from quercitrin treated rats were less susceptible to non- enzymatic lipid peroxidation induced with FeAs. This effect can be attributed to the absorbed quercetin, released from quercitrin in the gut. Previous in vitro

experiments showed that quereitrin inhibits both non-enzymatic lipid peroxidation induced in liver membrane fractions (ICs0 = 8.50 + 0.41 #M, lower

than that of quercitrin) and glutathione related enzymes (unpublished data), although much higher concentrations were necessary for this latter effect.

In conclusion, the administration of quercitrin could be useful in those intestinal diseases which are mediated by free radicals and other reactive oxygen metabolites, such as in inflammatory bowel disease (Harris et al., 1992), since this ftavonoid is able to both increase the glutathione content and to inhibit lipid peroxidation in the intestinal mucosa. In fact, several authors have pointed out that the therapeutic activity of drugs used in inflammatory bowel disease in humans, such as sulfasalazine and its therapeuti- cally active metabolite 5-aminosalicylic acid, may be related to suppression of reactive oxygen production at intestinal mucosal level (Craven et al., 1987; Ahnfelt-Ronne et al., 1990).

SUMMARY

Quercitrin is a flavonoid (quercetin 3-rhamnoside) that shows antidiarrhoeic activity from doses of 25 mg • kg 1 in mice and rats. The effects exerted by daily oral administration of quercitrin (25 mg" kg- i) to rats for 3 and 7 days on intestinal physiology have been evaluated. Quercitrin is able to affect the intestinal oxidative status, and this effect is more patent after 3 days of treatment than after 7 days. In jejunum, the non-enzymatic lipid peroxidation induced in membrane fractions was lower in those animals treated with quercitrin either for 3 (88.2% of reduction in MDA concentration) or 7 days (53.3% of reduction). However, quercitrin treatment did not significantly modified glutathione content in the jejunal mucosa. In ileal membranes, no significant differences in the amount of MDA produced were observed between treated and control animals, whereas the ileal glutathione content was increased by 34% in the treated group compared to the corre- sponding control after 3 days. The colonic mucosa increased its glutathione content by 42% after 3 days of glavonoid oral administration, and the induced lipid peroxidation was lower (75.9% of reduction in MDA concentration) in colonic mucosal membranes obtained from animals treated for three days with the flavonoid. The different behaviour showed in the three intestinal segments studied in this regard after 3 days of treatment may be explained by the existence of metabolizing products from quercitrin in the gut. It is concluded that the administration of quercitrin could be useful in those intestinal diseases which are mediated by free radicals and other reactive oxygen metabolites, such as in inflammatory bowel disease, since this flavonoid is able both to increase the

Quercitrin and intestine 1243

glutathione content and to inhibit lipid peroxidat ion

in the intestinal mucosa.

Acknowledgement--This study was supported in part with DGICYT funds (PM90-0189) of the Spanish Ministry of Education and Science.

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GP 25/~-M