Effects of taxifolin on aspirin-induced gastric damage in rats: macroscopic and biochemical evaluation

Abstract

Taxifolin (dihydroquercetin) is a flavanonol isolated from various plants and has antioxidant effects. The aim of our study was to macroscopically and biochemically investigate the effects of taxifolin on aspirin-induced oxidative gastric damage in rats and to evaluate them by comparison with those of famotidine. Rats were divided into four drug administration groups: a healthy control group, an aspirin-only group (ASG), a taxifolin + aspirin group (TASG), and a famotidine + aspirin group (FASG). The results revealed that in light of the results that we obtained, 50 mg/kg taxifolin had anti-ulcer effects. At this dose, taxifolin was able to bring COX-1 activities to a level close to those seen in healthy rats with appropriate macroscopic, oxidant/antioxidant, and biochemical parameters. Based on these results, it can be said that taxifolin may be successfully used as a more potent alternative to famotidine, which is the currently accepted treatment for aspirin-induced ulcers.

Introduction

Aspirin, an O-acetyl derivative of salicylic acid, is a nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic, and antipyretic effects [1]. Today, aspirin is successfully used in the chronic treatment of acute coronary heart diseases, cerebral sinovenous thrombosis, and inflammatory diseases such as arthritis, carditis, and rheumatoid fever [2]. Like other NSAIDs, aspirin inhibits cyclooxygenase (COX) activity, which is responsible for the synthesis of prostaglandins (PGs) that cause inflammation, edema, pain, and fever [3]. The known, the COX enzyme has structural COX-1 and inducible COX-2 isoforms [4]. COX-1 is responsible for the protection of the stomach and many other organs and tissues, while COX-2 plays a role in inducing pathological processes such as inflammation [5]. The inhibitory effect of aspirin on COX-1 is greater than its effect on COX-2 [6]. The gastrointestinal toxic effects of NSAIDs are known to result from the inhibition of COX-1 production [7]. Aspirin is a drug that has gained worldwide acceptance in the treatment of various cardiovascular and inflammatory diseases. However, aspirin therapy causes toxic effects, especially on the stomach and liver [8]. Even low-dose aspirin administration leads to an increased risk of gastroduodenal ulcers and upper gastrointestinal bleeding [9]. It has been reported that aspirin causes damage by increasing the oxidant status level in the stomach tissue while decreasing the antioxidant and COX-1 levels [10]. In addition, it has been documented that aspirin increases the production of malondialdehyde (MDA), the toxic end product of lipid peroxidation in gastric tissues, and decreases the level of glutathione (GSH), which is known as an endogenous antioxidant parameter [11]. All this information suggests that drugs that inhibit the increase in oxidant levels and the decrease in antioxidant and COX-1 levels may be beneficial in the treatment of the gastrotoxic effects of aspirin.

Taxifolin (dihydroquercetin) is a flavanonol isolated from various plants, such as the thistle, onion, and tamarind, and bark of the French maritime pine [12]. Recent studies have documented the antioxidant effects of taxifolin [13]. Taxifolin has been shown to create an antioxidant effect by inhibiting the production of reactive oxygen species (ROS) [14]. It has also been reported that taxifolin protects stomach tissues from oxidative stress with this antioxidant effect [15]. However, no studies exploring whether the gastroprotective effect of taxifolin is associated with the COX-1 enzyme were found in the literature. The aim of our study was to macroscopically and biochemically investigate the effects of taxifolin on aspirin-induced oxidative gastric damage in rats and to evaluate them by comparison with those of famotidine.

Materials and Methods

Animals

This research involved a total of 24 three-month-old male albino Wistar rats with weights ranging from 275 to 287 g. All of the animals were obtained from the Erzincan Binali Yildirim University Medical Experimental Application and Research Center (Erzincan, Türkiye). Before the experiment, the animals were housed in groups in an animal room with an antimicrobial fan system for one week at normal room temperature (22 ± 2°C) and 55 ± 5% relative humidity with 12 h of light and 12 h of darkness. They were fed ad libitum with standard rat chow (Bayramoglu feed, Bayramoğlu Trade Co., Erzurum, Türkiye) and tap water. The compliance of the research with ethical principles was confirmed by the Erzincan Binali Yildirim University Animal Experiments Local Ethics Committee (dated 07.02.2022 and No. 22/01).

Chemicals

Thiopental sodium (CAS no. 76-75-5; 0.5 g/20 ml; lot no. (01) 08699508270385) was obtained from Ulagay (Istanbul, Türkiye), taxifolin (CAS no. 480-18-2; 25 mg/tablet; lot no. 74034) was obtained from Evalar (Moscow, Russia), aspirin (CAS no. 50-78-2; 100 mg/tablet; lot no. (01) 08699514040019) was obtained from Abdi İbrahim Pharmaceuticals (Istanbul, Türkiye), and famotidine (CAS No. 76824-35-6; 20mg/tablet; lot no. (21) A0000004347152) was obtained from Sandoz Drugs, Istanbul, Türkiye.

Experimental groups

The rats were divided into four drug administration groups: a healthy control (HC) group, an aspirin-only (AS) group, a taxifolin + aspirin (T+AS) group, and a famotidine + aspirin (F+AS) group .

Experimental procedure

Experimental applications were carried out in the laboratories of the Erzincan Binali Yildirim University Medical Experimental Application and Research Center. The animal rooms in the laboratory were closed to the outside and ventilated with a HEPA-filtered ventilation system. In addition, animal rooms and cages were periodically washed with high-pressure hot water. In this way, the experimental environment was protected from microbial pathogens. The surgical materials, gloves, and clothing used by personnel during the experiment were cleaned of pathogens with the appropriate disinfection and sterilization methods. Sterilization processes were carried out using an autoclave.

Pharmaceutical tablets, each containing 25 mg of taxifolin, were ground into powder with a mortar and mixed in distilled water. The drugs to be administered to the rats were suspended in distilled water in the laboratory environment and made suitable for oral use. A solution containing taxifolin at a concentration of 10 mg/ml (w/v) was prepared. For famotidine, 100 mg pharmaceutical tablets containing 20 mg of famotidine were ground into powder in a mortar and mixed with distilled water. A solution containing famotidine at a concentration of 30 mg/ml (w/v) was prepared. To conduct the planned experiment, the animals were fasted for 12 h (rats were allowed to drink water). Taxifolin was given at 50 mg/kg by oral gavage to the rats in the T+AS group. This dose was chosen because, in a previous study, administration of 50 mg/kg of taxifolin prevented liver damage by reducing pazopanib-induced oxidative stress [16]. Famotidine 20 mg/kg were administered through oral gavage to the F+AS group. This dose was chosen because famotidine showed anti-ulcer activity at a dose of 20 mg/kg in a previous study [17]. As a solvent, the same volume of normal saline (0.9% NaCl) was administered orally to the HC and AS groups. For aspirin, 130 mg pharmaceutical tablets containing 100 mg aspirin were ground into powder in a mortar and mixed with distilled water. A solution containing aspirin at a concentration of 30 mg/ml (w/v) was prepared. One hour after the administration of taxifolin, famotidine, and 0.9% NaCl solution, aspirin was administered at a dose of 100 mg/kg by oral gavage directly into the stomachs of rats in the T+AS, F4AS, and AS groups. This dose of aspirin is the dose that causes damage to animal stomachs [18]. Six hours after aspirin administration, all animals were euthanized with high-dose thiopental anesthesia (50 mg/kg). After macroscopic evaluation of the inner surfaces of the removed stomachs, the gastric tissues were biochemically examined.

Macroscopic analyses

Stomach tissue was evaluated according to the ulcerative areas (black-colored bleeding areas) and the degree of erosion. The ratio of ulcerative areas on the mucosa to the entire stomach surface was calculated by using squared paper and measured one by one. The protective effect of taxifolin on gastric ulcer was compared with the results obtained for the AS group. The mean total ulcer area (mm²) of each rat was determined, and the percent anti-ulcerative effect (%) was calculated according to the following formula:

Anti-ulcerative effect (%) = (control group mean total ulcer area − treatment group mean total ulcer area) / control group mean total ulcer area × 100.

Biochemical analyses

Determination of tissue MDA and total glutathion (tGSH): The MDA measurements were based on the technique used by Ohkawa et al., which involved the spectrophotometric measurement of the absorbance of the pink-colored complex formed by thiobarbituric acid (TBA) and MDA [19]. The tGSH measurements, on the other hand, were based on the method presented by Sedlak and Lindsay [20].

Determination of tissue total oxidant status and total antioxidant status: Total oxidant status (TOS) and total antioxidant status (TAS) levels of tissue homogenates were determined using a novel automated measurement method and commercially available kits (Rel Assay Diagnostics, Gaziantep, Türkiye), both of which were developed by Erel [21, 22].

Measurement of COX activity: We measured the COX activity in rat stomachs in this series of experiments using a COX Activity Assay Kit (Item No. 760151, Cayman Chemical, Ann Arbor, MI, USA). Stomach tissue was removed from stomach membranes and washed thoroughly with ice-cold Tris buffer (pH 7.4) containing 0.16 mg/ml of heparin to remove any red blood cells and clots, and then stored at −80°C until assayed. For each rat, a sample of stomach tissue was homogenized in 5 ml of cold buffer (0.1 M Tris-HCl, pH 7.8, containing 1 mM EDTA) per gram of tissue and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was removed for the assay and stored on ice. We then measured the protein concentration in the supernatant using the Bradford method [23]. The COX Activity Assay Kit measures the peroxidase activity of COX. This is assayed colorimetrically by monitoring the appearance of oxidized N,N,N’,N’-tetramethyl-p-phenylenediamine at 590 nm. We measured COX-2 activity using the COX-1-specific inhibitor [24]. Results for COX-1 and COX-2 activity are given as units per milligram of protein. The activity of COX in the tissue was expressed as nmol/min/mg protein (U/mg protein).

Statistical analysis: For statistical analysis, IBM SPSS Statistics for Windows 22 (IBM Corp., Armonk, NY, USA) was used. The results are presented as the mean ± SEM. For comparisons of groups, one-way ANOVA was used. After ANOVA, according to the homogeneity of variances, the post hoc LSD (least significant difference) test was used. The statistical level of significance for all tests was considered to be 0.05.

Results

Macroscopic findings for gastric tissue

As seen in Fig. 1, no hyperemia, edema, or damage to the gastric mucosa of healthy animals was observed. However, the gastric mucosa tissue of the animals who were given aspirin only had different numbers and diameters of foci of damage (ulcers). The ulcers consisted of round to oval and irregular mucosal defects with different depths, and the edges of the ulcers were edematous. The boundaries of the ulcers were clear. However, the gastric tissues had mild hyperemia and edema, while the ulcer foci were less numerous and smaller in the T4AS and F+AS groups samples. The mean total ulcer area in the gastric tissue of the AS group rats was 83.33 ± 1.45 mm²(Table 1). However, the mean total ulcer area was 1.33 ± 0.42 mm²(Table 1) in the T+AS group rats. In the F+AS group rats, the mean total ulcer area was 5.00 ± 0.57 mm²(Table 1).

Fig. 1.

Effects of taxifolin on aspirin-induced ulcers in rats. HC, healthy control; AS, 100 mg/kg aspirin; T+AS, 50 mg/kg taxifolin + 100 mg/kg aspirin; F+AS: 20 mg/kg famotidine + 100 mg/kg aspirin. Arrows indicate ulcer foci, Stars indicate mucosal edema.

Table 1. Effects of taxifolin on aspirin-induced ulcers in rats.

Group	Number of animals	Total ulcer area (mm2)	Anti-ulcer effect (%)
HC	6	0	–
AS	6	83.33 ± 1.45*	0
T+AS	6	1.33 ± 0.42**	98.4
F+AS	6	5 ± 0.57**	93.9

HC, healthy control; AS, 100 mg/kg aspirin; T+AS, 50 mg/kg taxifolin + 100 mg/kg aspirin; F+AS: 20 mg/kg famotidine + 100 mg/kg aspirin. The results are presented as the mean ± SEM. For comparisons of groups, one-way ANOVA was used. After ANOVA, according to the homogeneity of variances, the post hoc LSD (least significant difference) test was used. *P<0.001 compared with the HC group. **P<0.001 compared with the AS group.

Biochemical findings

The mean ± SE values of biochemical findings are shown in Table 2. The tGSH levels were found to be significantly higher in the HC, T+AS, and F4AS groups than in the ASG group (P<0.001). MDA levels were significantly lower in the HC, T+AS, and F+AS groups than in the AS group (P<0.001). The MDA and tGSH levels of the TASG group were similar to those of the HC group.

Table 2. Glutathione (GSH), malondialdehyde (MDA), total oxidant capacity (TOS), and total antioxidant status (TAS) measurement results for aspirin-induced ulcers in rats.

Group	tGSH (nmol/g protein)	MDA (µmol/g protein)	TOS (nmol H2O2/mg protein)	TAS (µmol Trolox equiv./mg protein)
HC	6.25 ± 0.06	3.20 ± 0.04	2.25 ± 0.05	5.18 ± 0.05
AS	3.48 ± 0.11*	5.47 ± 0.09*	4.09 ± 0.16*	2.70 ± 0.23*
T+AS	5.60 ± 0.09**	3.37 ± 0.06**	2.47 ± 0.16**	4.61 ± 0.13**
F+AS	5.77 ± 0.04**	3.27 ± 0.03**	2.36 ± 0.08**	4.86 ± 0.08**

HC, healthy control; AS, 100 mg/kg aspirin; T+AS, 50 mg/kg taxifolin + 100 mg/kg aspirin; F+AS: 20 mg/kg famotidine + 100 mg/kg aspirin.The results are presented as mean ± SEM. For comparisons of groups, one-way ANOVA was used. After ANOVA, according to the homogeneity of variances, the post hoc LSD (least significant difference) test was used. * P<0.001 compared with the HC group. ** P<0.001 compared with the AS group.

The measured TOS levels were quite close to the MDA levels, while the TAS levels were quite close to the tGSH levels. The highest TOS value was found to be 4.09 nmol H₂O₂/mg protein in the AS group, while the lowest TOS value was 2.25 nmol H₂O₂/mg protein in the HC group. The TOS values in the TASG and FASG groups were significantly lower compared with those in the AS group (P<0.001). The highest TAS value was 5.18 µmol Trolox equiv./mg protein in the HC group, while the lowest TAS value was 2.70 µmol Trolox equiv./mg protein in the AS group. The TAS values in the T+AS and F+AS groups were significantly higher compared with those in the AS group (P<0.001).

COX enzyme findings

COX enzyme findings are shown in Fig. 2. Compared with the AS group, COX-1 showed significantly higher levels of activity in the HC, T+AS, and F+AS groups (P<0.001). Considering the COX-2 activity results, it is seen that the HC and F+AS groups showed significantly higher levels of activity (P<0.001) compared with the AS group, while the T+AS group was not different in this regard.

Fig. 2.

Cyclooxygenase enzyme activity results for aspirin-induced ulcers in rats. HC, healthy control; AS, 100 mg/kg aspirin; T+AS, 50 mg/kg taxifolin + 100 mg/kg aspirin; F+AS: 20 mg/kg famotidine + 100 mg/kg aspirin. *P<0.001 compared with the AS group

Discussion

This study investigated the effects of taxifolin (dihydroquercetin), a flavonol, on aspirin-induced gastric ulcer damage in rats. The potential effect of taxifolin on aspirin-induced oxidative gastric injury in rats was macroscopically and biochemically investigated, and it was evaluated by comparing taxifolin with famotidine.

Aspirin is a very widely used NSAID. Gastric ulcer formation, a well-known side effect of aspirin, is used experimentally in animals to create ulcer models in pharmacology. In our study, significant total ulcer areas were formed in the group given aspirin at a dose of 100 mg/kg. The total ulcer area was smaller in the famotidine + aspirin group than in the aspirin group. Taxifolin, the substance that we tested, showed a stronger anti-ulcer effect than famotidine. The anti-ulcer effect of taxifolin against aspirin was calculated to be 98.4%, while the effect of famotidine was calculated to be 93.9%. When we reviewed the literature for studies related to the effects of taxifolin on the gastrointestinal tract, we found many studies related to the protective effects of taxifolin. For example, a fairly recent study conducted by Xie et al. showed that taxifolin suppresses malignancy in gastric cancers [25]. In another recent study, taxifolin supported gastric recovery in vivo and was able to inhibit Helicobacter pylori infection in vitro [26]. In a celiac artery ligation model in rats, taxifolin prevented oxidative gastric damage [15]. Similarly, the protective effects of taxifolin have been shown in other studies in the literature [16, 27].

Flavonoids constitute one of the main groups of plant phenolic antioxidants, and they have high chelating properties. Taxifolin (3,5,7,3,4-pentahydroxy flavanone or dihydroquercetin) is a flavonoid commonly found in the onion, milk thistle, and bark of the French maritime pine and Douglas fir [28]. Taxifolin has exhibited promising pharmacological activities in the management of inflammation, tumors, microbial infections, oxidative stress, and cardiovascular and liver disorders [28]. Among these activities, the antioxidant effects of taxifolin have particularly drawn attention [29]. Taxifolin has been shown to exert antioxidant effects by inhibiting ROS production [14]. It has also been reported that taxifolin protects stomach tissues from oxidative stress with those antioxidant effects [15].

In our study, it was observed that taxifolin inhibited the decrease in COX-1 levelinduced by aspirin in gastric tissue. The COX-1 enzyme plays a critical role in maintaining the integrity of the GI mucosa [2]. In the literature, it has been reported that aspirin causes damage by lowering the COX-1 level [30]. This indicates that the gastroprotective effect of taxifolin is due to its protective effect on COX-1. COX-2 inhibition is responsible for the therapeutic effects of aspirin and other NSAIDs, and COX-1 enzyme inhibition is responsible for its side effects [3]. As can be understood from our experimental results, aspirin, an anti-inflammatory drug, significantly reduced the activity of the COX-2 enzyme. Taxifolin did not significantly alter the inhibitory effect of aspirin on COX-2. This shows that the anti-inflammatory effect of aspirin is not suppressed by taxifolin. Furthermore, taxifolin is known to inhibit COX-2 and create an anti-inflammatory effect [31].

A study conducted by Naito et al. showed that ROS have a role in the etiopathogenesis of gastric damages induced by NSAIDs [32]. In tissues, enzymatic and nonenzymatic defense mechanisms (or antioxidant mechanisms) develop against these damaging free oxygen radicals (FORs) [33, 34]. Tissue damage begins with the formation of lipid radicals on the cell membrane. These radicals then cause damage by first turning into lipid hydroperoxides then ultimately turning and finally into toxic products such as aldehydes, alkanes, and MDA [32, 35].

GSH is a tripeptide that can be synthesized from the amino acids glycine, cysteine, and glutamate. It is a very important antioxidant that protects cells from oxidative damage by reacting with free radicals and peroxides. GSH and other antioxidant substances (e.g., melatonin or vitamins) prevent tissue damage by ensuring that the amounts of FORs in cells remain at certain concentrations and low levels [36]. MDA, another biomarker of oxidative damage, appears in the blood and urine, and it is the end product of lipid peroxidation. Therefore, the amount of MDA in biological material is an indicator of lipid peroxidation. Nonenzymatic lipid peroxidation is a very harmful chain reaction. It directly damages the membrane structure and indirectly damages other cell components through the reactive aldehydes that it produces. In this way, it causes tissue damage and many diseases [37]. Toxic oxygen radicals formed in excess amounts in tissues exposed to oxidative stress are known to stimulate lipid peroxidation, which leads to the formation of MDA [38]. High MDA levels in damaged gastric tissues have also been shown in a previous study [39]. In our study, measurements showed low GSH and high MDA levels in the AS group, while taxifolin increased the GSH levels and reduced the MDA levels. Measurements of TOS and TAS showed results similar to those for MDA and GSH levels. Taxifolin showed an anti-ulcer effect by protecting the stomach from aspirin-induced oxidative stress.

In conclusion, in light of the results obtained, 50 mg/kg taxifolin was revealed to have anti-ulcer effects. At this dose, taxifolin was able to bring COX-1 activities to a level close to those seen in healthy rats with appropriate macroscopic, oxidant/antioxidant, and biochemical parameters. Based on these results, it can be said that taxifolin may be successfully used as a more potent alternative to famotidine, which is the currently accepted treatment for aspirin-induced ulcers.