Anti-oxidative Herbs and Indomethacin-Induced Rat Gastric Mucosal Lesions: Protection by GamiHyangsa-Yukgunja

Anti-oxidative Herbs and Indomethacin-Induced Rat Gastric Mucosal Lesions: Protection by GamiHyangsa-Yukgunja

Heung Mook Shin

(Accepted for publication November 1, 2000)

Abstract: This study investigates the protective effects of GamiHyangsa-Yukgunja (GHY, a popular herbal medicine formula) on indomethacin-induced gastric mucosal lesions and morphological change in rats. Subcutaneous injection of indomethacin (25 mg/kg) produced the following gastric morphological alterations: mucosa hemorrhagic infarct, mucosa cell necrosis, leukocyte infiltration, mucosa hemorrhagic erosion, and gastric pit disappearance. Tissue damages were accompanied by increased oxidative stress, lipid peroxidation, and decreases in superoxide dismutase (SOD), and catalase (CAT) activities, and glutathione (GSH) concentrations. Our results show that pretreatment of the rats with orally administered GHY extract (3.3 ml/kg/day) significantly reduced gastric lesion formation and caused the amelioration of several pathological changes in the above-mentioned gastric mucosal lesions. Concomitantly, GHY-pretreatment increased gastric mucosal SOD and CAT activities and GSH concentrations. We therefore propose that GHY exerts a prophylactic effect on the indomethacin-induced gastric mucosal lesions by enhancing antioxidant defense systems.

Gastric diseases are widespread among the inhabitants of many countries. Reports suggest that reactive oxygen free radical species (ROS) play an important role in the pathophysiological processes of acute gastric lesions (Parks, 1989; Farber et al., 1990; Vaananen et al., 1991; Bulbena et al., 1993). Thus, recent studies to define the underlying mechanisms of gastric disease have focused on the investigation of ROS-induced damage. In this regard, studies on the means of gastric protection by attenuating ROS insult may provide the clues to the delineation of some of the responsible, underlying mechanisms which may lead to therapeutic applications.

One approach is to assess oxidative status in indomethacin-induced gastric lesions (Takeuchi et al., 1991), because some antioxidants are known to prevent gastric lesions through their cytoprotective properties (Bulbena et al., 1993; Cook et al., 1997; Robert et al., 1984; Szabo et al., 1992; Ueshima et al., 1992).

At present, no data are available on evaluating the gastroprotective prophylactic action of medicinal herbs based on their anti-oxidative actions. GamiHyangsa-Yukgunja (GHY) is a popular herbal medicine used in the Orient for the treatment of various symptoms of stomach disorders, including anorexia, nausea, belching, indigestion, gastritis, and gastric ulcer. However, the mechanisms underlying its efficacy are unknown.

In this study, we examined the putative action of GHY in its ability to protect against the oxidative stress underpinning indomethacin-induced gastric injury and morphologic changes in the rats.

Materials and Methods

Animals and Experimental Design

Male Spraque-Dawley rats, weighing approximately 300 g, were used for the experiments described in this study. The animals were deprived of food but allowed free access to water for 24 hr prior to the experiments.

Thirty rats were equally divided into the following five groups: A, Control (nontreated group); B, indomethacin-injected alone; C, GHY-pretreated for 1 day prior to indomethacin injection; D, GHY-pretreated for 2 days prior to indomethacin injection; E, GHY-pretreated for 3 days prior to indomethacin injection.

General Protocols

Gastric lesions were induced by subcutaneous (s.c.) injection of indomethacin at a dose of 25mg/kg body weight (Okada et al., 1988). As a pretreatment, the GHY extract was administered by gavage with 3.3 mi/kg/day for 1,2, and 3 days, respectively, then given a single dose of indomethacin, injected 1 hr after GHY administration. All rats were killed under deep diethyl ether anesthesia 4 hr after the indomethacin injection, and the stomachs were removed, immediately. The protocol for these experiments has been approved by the Institutional Animal Care Committee.

Herbal Composition and Extract Preparation

GamiHyangsa-Yukgunja (GHY) is a mixture of fourteen herbal drugs, which was obtained from the Oriental Herbal Center (OHC) of the Oriental Medical Hospital of Dongguk University College of Oriental Medicine (Kyoungju, Korea). The composition of the mixture is as follows: Panax ginseng C.A. Mey (8.16%), Atractylodis japonica Koidz (8.16%), Poria cocos (Schw.) Wolf (8.16%), Pinelliae ternata (Thunb.) Breit. (8.16%), Citrus unshiu Markovich (8.16%), Amomum xanthioides Wall. (4.08%), Agastache rugosa (Fisch. et Meyer) O. Kuntze (4.08%), Glycyrrhiza uralensis Fisch (4.08%), Crataegus pinnatifida Bge. var. major N.E. Br (8.16%), Massa medicata fermentata (8.16%), Hodeum vulgare var. hexastichon Aschers. (8.16%), Fossilia ossis mastodi (8.16%), Ostrea gigas Thunb. (8.16%), and Cyperus rotundus L. (6.12%).

To prepare GHY extract, GHY (98 g) was boiled with 1,000 ml of distilled water for 2 hr, then filtered through a fine mesh cloth. The extract was evaporated at 50 [degrees] C with a vacuum evaporator to make up a final volume of 100 mi, thus making the final product as a ten-fold concentrate.

Chemicals

Bovine serum albumin (BSA); 5,5′-dithio-bis-(2-nitrobenzoic acid); (DTNB); ethylenediamine tetraacetic acid, (EDTA); indomethacin, sodium lauryl sulfate; thiobarbituric acid (TBA), sulfosalicylic acid; ferricytochrome c; xanthine; and xanthine oxidase were purchased from Sigma Chemical Co. Hydrogen peroxide was obtained from Merck Co. and malondialdehyde tetrabutylammonium salt from Fluka Co. All other chemicals and reagents were of pure reagent grade purchased from commercial suppliers.

Tissue Preparation for Biochemical Assays

The stomachs were rapidly removed and rinsed in cold saline. Stomach tissues were homogenized in 4 volumes of 0.1 M potassium phosphate buffer (pH 7.5) and centrifuged at 600 x g at 4 [degrees] C for 5 min. The resultant supernatant fraction was used for the determination of lipid peroxidation and glutathione (GSH). The supernatant fraction was further centrifuged at 1,650 x g at 4 [degrees] C for 15 min to yield a second supernatant fraction that was used for the SOD and CAT assays.

Determination of Lipid Peroxidation

Lipid peroxidation accumulation products were evaluated as TBA reactive substances (TBARS), according to the modified method of Ohkawa et al. (1979). Briefly, the supernatant (0.2 ml) was supplemented with 0.1 M potassium phosphate buffer (0.6 ml), 8.1% sodium lauryl sulfate (0.2 ml), 20% acetic acid (1.5 ml) and 0.8% TBA (1.5 mi) to make up a total volume of 4 ml, and heated at 100 [degrees] C in water bath for 1 hr. After cooling, the reactants were mixed with 4 ml of the mixture of n-butanol and pyridine (15:1, v/v) with vigorous vortexing, followed by centrifugation for 20 min at 3,000 rpm. Absorbance of the supernatant was measured at 532 nm. The results were expressed as malondialdehyde (MDA) nmoles/g of tissue.

Determination of Glutathione

The amount of glutathione (GSH) in the gastric tissue was according to the modified Ellman method (1959). The supernatant (0.2 ml) was mixed with 50 mM potassium phosphate buffer (0.3 ml) and 4% sulfosalicylic acid (0.5 ml). The mixtures were vigorously vortexed and then centrifuged 3,000 rpm for 20 min. The supernatant (0.3 ml) was mixed with 2.7 ml of 1 mM DTNB in 0.1 M phophate buffer (pH 8.0) and incubated for 20 min at 25 [degrees] C. The absorbance was read at 412 nm. The results were expressed as reduced glutathione (GSH) nmoles/g of tissue.

The SOD activity was measured by monitoring the inhibition of ferricytochrome c reduction by xanthine/xanthine oxidase reaction, as described by McCord and Fridovich (1969). Briefly, the assay was performed in 3 ml of 300 mM potassium phosphate buffer at pH 7.8 containing 0.1 mM EDTA (0.5 ml) in a cuvette at 25 [degrees] C. The reaction mixture contained 300 mM potassium phosphate buffer (pH 7.8) containing 0.6 mM EDTA (0.5 ml), 60 M cytochrome c (0.5 ml), 0.3 mM xanthine (0.5 ml). The reaction was initiated by adding xanthine oxidase (0.2 ml). The rate of increase was at 550 nm of 0.025 absorbance unit per min. One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of reduction of cytochrome c by 50% at 25 [degrees] C (to a rate of 0.0125 absorbance unit per min).

The CAT activity assay was carried out by measuring the disappearance of peroxide followed spectrophotometrically at 240 nm according to the method of Decker (1977). The reaction mixture consisted of 59 mM hydrogen peroxide (1 ml), distilled water (1.9 ml) and enzyme sample (0.1 ml) in 3 ml total volume at 25 [degrees] C. One unit of enzyme activity is defined as decomposition 1 [Mu] mole of [H.sub.2][O.sub.2] per min.

The protein concentration was determined according to Lowry (1951) using a BSA as standard protein.

Histopathological Examination

The stomach was removed immediately and fixed in 10% neutral formalin for 24 hr and embedded in paraffin for 2 hr. Then stomach tissue was sliced into 5 [micro]m pieces with a microtome and stained with hematoxylin-eosin for photomicroscopic assessment.

Statistics

Data are presented as the mean [+ or -] SD from 6 rats per group. Statistical analysis was performed using unpaired t-test by the Sigma Plot program.

Results

Protective Effect of GHY on Lipid Peroxidation

As shown in Table 1, the indomethacin-injected rats in the control group showed a marked increase in stomach lipid peroxidation, as evidenced by elevated MDA. The GHY-pretreated rats showed significantly less lipid peroxidation than controls. The suppression was more evident in the 3-day pretreated group (Table 1).

Table 1. Effect of Indomethacin and GHY Pretreatment on LPO and GSH

LPO (MDA nmoles/

Groups g of tissue)

A: Control (non-treated) 10.77 [+ or -] 0.86

B: I.M. 18.90 [+ or -] 2.88(##)

C: GHY 1 + I.M. 15.95 [+ or -] 1.44(*)

D: GHY 2 + I.M. 15.29 [+ or -] 1.64(*)

E: GHY 3 + I.M. 10.54 [+ OR -] 0.80(**)

GSH (nmoles/

Groups g of tissue)

A: Control (non-treated) 2.30 [+ or -] 0.59

B: I.M. 1.59 [+ or -] 0.14(#)

C: GHY 1 + I.M. 2.16 [+ or -] 0.23(**)

D: GHY 2 + I.M. 2.17 [+ or -] 0.16(**)

E: GHY 3 + I.M. 2.13 [+ or -] 0.26(**)

I.M.: indomethacin-injected, GHY: 1, 2, 3: pretreatment (day). The

animals were killed 4 hr after indomethacin injection. Values are

mean [+ or -] SD (n = 6).

(#) p < 0.05,

(##) p < 0.01 compared with Control group;

(*) p < 0.05,

(***) p < 0.01, compared with indomethacin-injected group.

GHY Anti-oxidative Enzyme Activity

The antioxidant defense status of stomach was measured by assaying the activity of two major scavenger enzymes, SOD and CAT (Table 2). In the indomethacin-injected rats, SOD activity was significantly decreased (p [is less than] 0.05). In contrast, indomethacin did not affect a significant change in CAT activity. However, when rats were pretreated with GHY prior to indomethacin injection, the SOD activity did not show a significant difference in the 1-and-2-day pretreatment groups, but was significantly increased (p [is less than] 0.01) in the 3-day pretreatment group compared to control (Table 2). On the other hand, CAT activity showed no significant increases (p [is less than] 0.05, p [is less than] 0.01, p (0.05).

Table 2. Effect of Indomethacin and GHY Pretreatment on SOD and CAT

Activities

SOD (units/mg

Groups of protein)

A: Control (non-treated) 4.18 [+ or -] 0.45

B: I.M. 3.50 [+ or -] 0.36(#)

C: GHY 1 + I.M. 3.61 [+ or -] 0.71

D: GHY 2 + I.M. 3.71 [+ or -] 1.08

E: GHY 3 + I.M. 4.76 [+ or -] 0.26(***)

CAT (units/mg

Groups of protein)

A: Control (non-treated)

B: I.M. 9.92 [+ or -] 2.01

C: GHY 1 + I.M. 9.94 [+ or -] 1.67

D: GHY 2 + I.M. 15.18 [+ or -] 4.29(*)

E: GHY 3 + I.M. 13.87 [+ or -] 1.91(**)

12.63 [+ or -] 1.96(**)

I.M.: indomethacin, GHY: 1, 2, 3: pretreatment (day). The animals

were killed 4 hr after indomethacin injection. Values are mean

[+ or -] SD (n = 6).

(#) p < 0.05, compared with non-treated group;

(#) p < 0.05,

(**) p < 0.01,

(***) p < 0.001, compared with indomethacin-injected group.

GSH Concentrations and Gastric Protection

The non-enzymatic, antioxidant GSH concentrations in gastric tissues were significantly decreased (p [is less than] 0.05) by indomethacin injection. However, pretreatment with GHY prior to indomethacin injection prevented a decrease with similar results observed in all GHY-pretreated groups (Table 1).

Morphological Change in Gastric Lesion

Microscopic evidence shows morphologic alterations were readily detected in indomethacin-injected (25 mg/kg) rats. Visible, pronounced damage appeared in the glandular stomach along the mucosal folds. Many surface mucous cells showed signs of necrosis and a large portion of the gastric pits disappeared within hemorrhagic infarct (Figure B, C). However, in the GHY-pretreated rats, a clear protective effect was observed in the fewer changes in the hemorrhagic infarct of mucosa (Figure D).

[Figure 1 ILLUSTRATION OMITTED]

Discussion

Indomethacin, a nonsteroidal anti-inflammatory drug, is well known to produce lesions in the gastric mucosa of experimental animals and humans (Djahanguiri, 1969; Lanza, 1984). Injection of indomethacin at higher doses (25 mg/kg) is reported to cause nonhemorrhagic lesions within 1 hr, which later become hemorrhagic (Takeuchi et al, 1986; Okada et al., 1988). Some suggest the implication of free radicals as a possible culprit. For example, oxygen free radicals and lipid peroxidtion have been reported in the pathogenesis of gastric mucosal lesions induced by indomethacin in rats (Takeuchi et al., 1991).

Experimental evidence supporting the possibility comes from several studies on the protection from injury by some antioxidants, prostaglandins (PGs), and sulfhydryl-containing compounds (Soldato et al., 1985; Szabo et al., 1992; Bulbena et al., 1993; Pearson et al., 1996; Liebler et al., 1997).

Recent interest in herbal remedies as putative anti-oxidative stress are becoming increasingly popular (Liu et al., 1990; Liu et al., 1992; Singh et al., 1994). Therefore an urgent need for the systematic investigation of the anti-oxidant properties of herbal medicine and for defining their cellular mechanisms has developed.

Through long-term clinical use, GarniHyangsa-Yukgunja (GHY), a popular oriental herbal medicine, has been recognized to improve gastrointestinal function. However, as far as we know, no attempt has yet been made to establish its putative anti-oxidative effect at cellular levels. Thus, we have investigated the gastroprotective effects of GHY at multi-levels by documenting histological changes, measurement of oxidative status, and oxygen free radical scavenging enzyme levels in indomethacin-induced gastric mucosal lesions.

We found clear microscopic evidence that indomethacin given at higher doses (25 mg/kg) causes hemorrhagic erosion and necrosis in the superficial layer of mucus and a disappearance of the mucus cells from the upper portion of gastric glands pit (Figure B, C, D). These effects were similar to the histologic features commonly observed with indomethacin-induced mucosal lesions having deep mucosal erosion, necrotic cells at base, and the congestion and hemorrhages around it (Okada et al., 1989; Bulbena et al., 1993). However a pretreatment with GHY was shown to decrease the severity of the lesions and the mucosa showed only slight, superficial erosion. This amelioration was much pronounced in the 3-day pretreated group (Figure D).

The underlying gastroprotective effect of GHY was further delineated by measurements of anti-oxidant activity in isolated mucosal tissue. Oxygen free radicals are capable of damaging cell membranes and their components and can initiate lipid peroxidation reaction, which may lead to widespread tissue damage, a similar effect of the indomethacin injection. Thus, the inhibiting lipid peroxidation can be named as one of the best protections against oxidative cell injury in its ability to eliminate peroxide forming free radicals (Dormandy, 1983). Our findings on the significant inhibition of lipid peroxidation strongly suggest that GHY contains effective peroxide-removing components, which have yet to be identified.

Furthermore, our results show additional beneficial action by GHY in the enhancement of the anti-oxidative defense systems. Anti-oxidant enzymes are considered a primary defense against the prevention of the biological macromolecules that cause oxidative damage. SOD rapidly converts [O.sub.2]- to the less dangerous [H.sub.2][O.sub.2], which is further degraded by CAT. Our data showed that SOD activity in the stomach that was significantly decreased in the indomethacin-treated group was significantly increased by GHY pretreatment. To our knowledge, this is the first report that shows GHY inhibits lipid peroxidation and at the same time increases scavenging activity in the stomach.

GHY’s antioxidant effect was further exhibited by CAT activity and GSH levels. CAT catalyzes the decomposition of [H.sub.2][O.sub.2] to produce water and molecular oxygen, and plays a major role in protecting cells against tissue inflammation. Moreover, GSH is one of the principal protection mechanisms that neutralizes endogenous and exgenous toxic substance and free radical-mediated damage in gastric mucosa (Szabo et al., 1981; Hoppencamps et al., 1984; Szabo et al., 1992). In the present study, CAT activity in all GHY-pretreated groups was significantly increased, although no significant difference in stomach CAT activity was observed between the normal and the indomethacin-treated group. Similarly, GHY pretreatment increased GSH levels in all groups by counteracting significantly decreased GSH by indomethacin.

Taken together, the GHY pretreatment prior to the injection of indomethacin clearly provided overall protection by increased anti-oxidant activity of SOD, CAT, and GSH while inhibiting lipid peroxidation and oxidative damage. These anti-oxidant effects by GHY were most pronounced in the 3-day pretreated groups. These data provide us with a cellular explanation for the efficacy of GHY as a gastroprotective agent.

The biochemical and morphological evidence mentioned above clearly indicates that GHY has a protective effect on indomethacin-induced gastric lesions. Thus, based on this work, the use of GHY is beneficial to indomethacin- caused gastrointestinal disorder, but further studies are needed to define the active ingredients of GHY responsible for the antioxidant property.

Acknowledgment

The author thanks Dr. Byung Pal Yu, Department of Physiology, University of Texas Health Science Center at San Antonio for his encouragement and guidance for this study and critical evaluation for the preparation of this manuscript.

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Heung Mook Shin Department of Physiology, College of Oriental Medicine, Dongguk University, Kyoungju, Kyoungbuk, 780-714, Korea Present address: Boston Biomedical Research Institute, Inc., 64 Grove Street, Watertown MA 02472, U.S.A.

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