The stem of E. aschenbornianum plants were harvested from Morelos, Mexico, which was made in 2015 during the spring season and were identified by Professor Carlos Francisco Cortés García, a Specialist in the Department of Taxonomy at the University of Guadalajara, Mexico. Leaves, branches and flowers of the plant were excluded, the remaining stem was dried at room temperature until the moisture content was ~7%, the stems were then immediately chopped and pulverized with a blender, the resultant powder obtained was filtered through a 30-mesh (0.595 mm). Finally, the E. aschenbornianum powder was vacuum-packed at room temperature for one week to avoid alteration of the sample (22).

A total of 23 male Wistar rats (age, 8 weeks; weight, 180–220 g) were purchased from the animal facility of the University of Guadalajara. All animal procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health from 1985. Animals were housed, handled and cared for in accordance with the official Mexican standards for the care and use of laboratory animals (no. NOM-062-ZOO-1999). An internal bioethical committee at the Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (Guadalajara, Mexico) reviewed and approved the animal study. Animals were housed with controlled conditions, light cycle (12-h light/dark cycle), relative humidity (46–50%) and temperature (22°C). The animals had free access to rat chow and water. All were maintained individually in polyethylene cages. The rats were fasted for 24 h prior to the start of the experiment but had free access to water (23).

The induction of gastric ulcers was performed according to the method described by Konturek et al (24), Lewis and Shaw (25) and Narayan et al (26). Rats were randomly divided into three groups containing six animals each. The first and second groups were induced by acetylsalicylic acid (ASA) by intragastric gavage at a dose of 150 mg/kg body weight to generate gastric ulcers, while the third group was administered saline solution. Immediately after induction, the second group was fed E. aschenbornianum powder at a dose of 400 mg/kg body weight (previous studies with few animals suggested anti-ulcerative effects at this dose) (22) mixed in standard rat chow, while the other groups were fed in the same manner without E. aschenbornianum powder. Animals were maintained individually in cages and were fed for a period of five days. The next day, all animals were euthanized by an intraperitoneal injection of sodium pentobarbital (120 mg/kg). Animals were dissected and the stomachs were carefully extracted. An incision was made on the stomach along the greater curvature, and the stomachs were then washed with PBS. Mucosal lesions were evaluated by macroscopic analysis.

Taking into account the severity of damage in the gastric mucosa, the UI was determined as follows: UI=[1× (number lesions of ≤1 mm) + 2× (number lesions of 1–2 mm) + 3× (number lesions >2 mm)]/10.

The overall score was divided among the Ulcer Index designated as 10 (27,28), while the percentage of ulcer protection was calculated as follows: Ulcer protection (%)=[(UI ulcer-induced group)-(UI treated group)]/(UI ulcer-induced group) ×100.

Gastric mucosal tissues (100 mg/ml) were placed into 20 mM Tris-HCl buffer (pH 7.4) at 4°C and homogenized using a Tissue-Tearor (BioSpec Products Inc.). The homogenates were centrifuged at 2,000 × g, at 4°C for 10 min; the supernatant was collected and stored at 4°C until use. The degree of lipid peroxidation was determined as the amount of malondialdehyde (MDA) and 4-hydroxynonenal (HNE) using the Lipid Hydroperoxide Assay kit (Bioquochem SL). In brief, 200 µl of the supernatant and 650 µl 10.3 nM N-methyl-2-phenylindole were added to a 1:3 mixture of acetonitrile and methanol. Subsequently, 150 µl methanesulfonic acid was added and the reaction mixture was incubated at 40°C for 40 min. Subsequently, the tubes were centrifuged at 5,000 × g at room temperature for 5 min. Finally, 200 µl of the supernatant was collected and the absorbance was measured at 586 nm. A standard calibration curve was generated simultaneously to establish the sample concentration.

According to the OECD 425 guidelines, it is possible to perform a limit test (a maximum fixed dose) with five animals using a dose of 2,000 mg/kg E. aschenbornianum as a suggestion (29); it was then decided to use a single administration of 400 mg/kg E. aschenbornianum in order to demonstrate that intake at this dose is safe. E. aschenbornianum powder was administered to five rats by intragastric gavage at a dose of 400 mg/kg body weight. Animals were observed for one week to detect any toxicity signs and an additional week for any delayed toxicity. Mortality, body weight and clinical signs were recorded. Blood samples were collected from the tail vein 14 days after E. aschenbornianum administration and separated to obtain serum, which was stored at −80°C until use for the analysis of biochemical parameters. Bilirubin was measured using the method of Jendsrassik and Grof (30), while the activities of alanine transaminase (ALT) and aspartate transaminase (AST) were measured according to the Reitman and Frankel method (31). Albumin was estimated by the method of Doumas et al (32), while alkaline phosphatase (ALP) activity was estimated by the method of Bessey et al (33).

Values are expressed as the mean ± standard error of the mean. Statistically significant differences between groups were determined by one-way analysis of variance, followed by a Tukey's post-hoc test. P<0.05 was considered to indicate statistical significance. Statistical analyses were performed using SigmaStat 8.0 software (Systat Software, Inc.).

In the ulcer-induced group, gastric mucosal lesions with a diameter of >2 mm were observed in the glandular regions of the stomach. These mucosal lesions appeared to be black and dark red with elongated bands. Of note, a gastroprotective effect was observed in rats administered E. aschenbornianum powder after the development of ASA-induced gastric ulcers. The severity of these mucosal gastric lesions was similar to that of the non-induced group, and the lesions were <1 mm in diameter (Fig. 1).

Administration of ASA caused severe gastric lesions to develop with a UI value of 9.65±0.89 in the ulcer-induced group. Rats of the ulcer-induced group administered E. aschenbornianum powder exhibited a statistically significant reduction in the severity and number of gastric lesions with a UI of 4.52±0.14. Unexpectedly, this group also exhibited a significant reduction in the UI value compared with the non-induced group (UI=5.04±0.30; Fig. 2).

Treatment with E. aschenbornianum powder in animals with gastric ulcers induced by ASA resulted in significantly reduced levels of MDA-HNE aldehydes (33.79±1.84 µM) in the gastric mucosa compared with those in the ASA-induced ulcer group (46.00±1.65 µM); of note, the MDA-HNE aldehydes in the treatment group were not significantly different compared with those in the non-induced group (39.16±2.15 µM; Fig. 3).

No mortality, clinical signs of toxicity or effects on behavior or appearance, including lethargy and immobility, were observed throughout the study. All animals survived during the whole study period. Similar food intake and consistent changes in body weight were observed in the non-induced and ulcer-induced groups (data not shown). The ulcer-induced group administered 400 mg/kg of E. aschenbornianum powder did not exhibit any alterations in the biochemical parameters analyzed compared with the non-induced group and the reference range (Table I) (34).