Background. Ulcerative colitis is a worldwide chronic gastrointestinal disease characterized by variable extensions of colon mucosal inflammation. The available drugs have an incomplete response with various side effects and socioeconomic impacts. Aloe barbadensis Miller (Aloe vera) is a well-known medicinal plant with diverse pharmacological and therapeutic activities. As a result, in the current study, Aloe vera was selected to evaluate its therapeutic effects on experimental colitis in rats. Methods. This study is intended to evaluate the possible beneficial effect of Aloe vera for the treatment of experimental colitis. Trinitrobenzenesulfonic acid (TNBS) was used to induce experimental colitis in 60 of 70 Wistar rats. The rats were grouped in 7 clusters including healthy control, negative, positive control (received sulfasalazine), and test groups treated with Aloe vera extracts via oral or rectal routes. Macroscopic and histologic factors as well as the biochemical parameters were evaluated on day 7. Results. In the present study, it was found that serum levels of tumor necrosis factor-α (75 vs. 44 pg./ml), interleukin-6 (41 vs. 21 pg/ml), and nitric oxide (24 vs. 6 μm/ml) in TNBS-induced untreated colitis treatment were significantly increased as compared to healthy control. Similar patterns were also observed in malondialdehyde (76.41 vs. 236.35 μg/mg) and myeloperoxidase (4.24 vs. 29.38 U/mg) in colonic tissue. Among different treatments, rectal administration of Aloe vera extract (400 mg/kg) exhibited the best result in which serum concentration of tumor necrosis factor-α (55 pg/ml), interleukin-6 (24 pg/ml), and nitric oxide (10 μm/ml) and the levels of malondialdehyde (102.67 μg/mg), as well as myeloperoxidase (12.29 U/mg) in colon tissue, were reduced as compared to the untreated group. Also, the body weight and colon weight/length ratios were more improved in the treated group with 400 mg/kg Aloe vera extract, rectally. Conclusion. Aloe vera extract exhibited a therapeutic effect in TNBS-induced colitis, and local, rectal administration of Aloe vera extract was more effective than oral administration.
Ulcerative colitis (UC) as an annoying chronic problem is one of the two major subtypes of inflammatory bowel diseases (IBDs) with different geographic prevalences and worldwide distribution [1, 2]. Although UC may present insidiously, its hallmark is subacute bloody diarrhea, accompanied by anemia and fatigue. It also may change to acute severe colitis, presenting temperature above 37.5°C, heart rate above 90/min, and hemoglobin concentration below 10.5 g/dL with more than 6 bloody stools daily [3, 4].
Its manifestation is due to continuous inflammation of the rectum with the variable extension but usually with decreasing severity up to the cecum . Its etiology and exact underlying pathophysiologic aspects are unclear, but most probably is due to aberrant deregulated mucosal immune responses (humoral and cellular immunity) to environmental factors in a genetically susceptible population. Following epithelial barrier dysfunction and immune cell activation, inflammatory cytokines and mediators (interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-13, IL-17, IL-23, and tumor necrosis factor α (TNF-α)) are released which may be used as disease activity indicators [6, 7]. The diagnosis of UC is based on clinical presentation and chronic colon inflammation confirmed by histology . Uncertain definitive pathogenesis, variable presentation, natural course, and lack of standard disease activity index are obstacles for definite therapeutic effect assessment. In any case, the accepted therapeutic goals are (a) accentuating induction of remission and maintenance period, (b) improving the nutritional status, (c) decreasing disease complications, and (d) considering side effects and cost effectiveness. In current medicine, the main treatments are focused on 5-ASA and steroids. Biologic therapies such as antitumor necrosis factor antibodies are prescribed for resistant patients. Antiadhesion molecules and kinase inhibitors are under research for UC treatment [9–12]. The inadequate response, frequent relapse, steroid dependency, and side effects result in developing a new candidate as the second line of treatment. Considering therapeutic effects of some herbal medicine such as heartleaf houttuynia [13, 14], boswellic acid [15, 16], diamonnium glycyrrizhinate , slippery elm , fenugreek , devil’s claw , tormentil , and wei tong ning  in various diseases, especially in China, Middle-East, and Africa, new research in this field is rational.
Aloe vera (Aloe barbadensis Mill.) belongs to the Aloeaceae family with thick, tapered, green lance-shaped, juicy, sharp, and edged leaves . Aloe vera grows in dry regions of Africa, Europe, Asia, and America. Aloe vera is probably the most applied medicinal plant commercially and the most popular plant worldwide . Various parts of the plant contain amino acids, sugars, enzymes, vitamins, minerals, saponins, anthraquinones, lignin, and salicylic acid. Also, the leaves are the source of various organic acids, phenolic compounds, minerals, and vitamins . Therapeutic effects of Aloe vera in wound healing , inflammation, intestinal absorption, and reducing oxidative status were assessed in recent research . It also has been used empirically to increase high-density lipoprotein, reduce low-density lipoprotein, and decrease glycemia in diabetics . Furthermore, the anti-inflammatory effects of Aloe vera in the human colon were confirmed in vitro by Langmead et al. . In 2017, the healing effect of the aqueous leaf extract of Aloe vera in an animal model of experimentally induced colitis was investigated. The favorable effects confirmed through the significant reduction in Bax mRNA expression and elevation in Bcl-2 mRNA expression when compared with the colitis group without treatment . In another study, 50 and 300 mg/kg of Aloe vera gel extract were used to evaluate the improvement in the symptoms of UC in rats. According to microscopy and macroscopic observations, the symptoms of UC were improved significantly . Park et al. showed that 0.1% and 0.5% aloesin supplement (one of the compounds of Aloe vera) decreased the myeloperoxidase (MPO) activities as well as TNF-α and interleukin-1β (IL-1β) mRNA expressions on the UC rat colitis model . In another study, glucomannan extracted from Aloe vera balanced pro- and anti-inflammatory cytokines regulated the expressions of TLR-2 and improved the health state of colitis in mice . Similarly, assessments on polysaccharides extracted from Aloe vera on UC-animal models depicted an improvement in colitis, via JAK2, p-JAK2, STAT-3, and p-STAT3 protein expression . In a randomized, double-blind, placebo-controlled trial, oral Aloe vera gel (100 mL twice daily for 4 weeks, in a 2 : 1 ratio) was administered for active UC patients. The supplement reduced the clinical colitis activity index and histological scores significantly during treatment with Aloe vera .
However, it seems that further evaluation about the therapeutic potential of Aloe vera extract on UC as well as its effect on new biochemical factors related to UC is needed to get more insight into signaling pathways. Furthermore, in the current study, for the first time, the different routes and doses of Aloe vera administrations (intragastrically and rectally) were studied. Regarding the therapeutic dose of Aloe vera used in the previous studies with no report of toxicity in the tested range, 200 and 400 mg/kg Aloe vera extract were chosen for further study [31–35]. This study was designed to evaluate and compare the dose and route treatment of Aloe vera extract on colitis in rats and its impacts on proinflammatory cytokines.
2. Materials and Methods
2.1. Ethical Statement
The animal experiments were performed in accordance with the guidelines of the Laboratory Animal Center of Shiraz Medical University (No. 91-01-36-4560). All the experimental procedures were strictly conducted according to the international standards and national legislation on animal care and the Animal Research Reporting In Vivo Experiments (ARRIVE) guidelines. Experimental research on the plant was under international legislation and guidelines of the Pharmacognosy Department of Shiraz University of Medical Sciences, Shiraz, Iran. At the end of the study, rats were euthanized with the rapid and humane method using a 70% volume displacement rate of CO2 increased to around 100% in the induction chamber.
2.2. Study Design and Induction of Colitis
The Laboratory Animal Center of Shiraz University of Medical Sciences with a pathogen-free environment, constant temperature (23 ± 2), and acceptable humidity (55 ± 5%) provided us with 70 male Wistar rats (175–215 grams) supplied with a balanced diet along with free access to water. The rats were fasted with free access to water for 24 h before induction of colitis. After rats were anesthetized with ketamine (50 mg/kg i.p), the rubber-tipped gavage needle was inserted into the anus of rats (7 cm) and 1 ml solution of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 150 mg/kg dissolved in ethanol) was slowly injected into the colon while the control group received only ethanol. Animals were held in the head-down position for 30 seconds and then returned to their cages [36–38]. Later, water and food were available. 12 hours after colitis induction, the treatments were started and continued one a day for six consecutive days. The effectiveness of treatment was assessed by clinical, macroscopic, biochemical, and histopathological assessments. The rats’ general conditions were assessed daily.
2.3. Experimental Animals
A total of 70 Sprague Dawley male rats (aged 10-12 weeks, weighing initially 220 ± 20 gram) were obtained from the Laboratory Animal Center of Shiraz University of Medical Sciences. Animals were divided into seven groups (10 rats per group, n = 10). Group (1): healthy control group Group (2): TNBS-induced colitis untreated rats Groups (3): TNBS-induced colitis treated rats who received 200 mg/kg Aloe vera extract once a day, intragastrically Groups (4): TNBS-induced colitis treated rats who received 400 mg/kg Aloe vera extract once a day, intragastrically Groups (5): TNBS-induced colitis treated rats who received 200 mg/kg Aloe vera extract once a day, rectally Groups (6): TNBS-induced colitis treated rats who received 400 mg/kg Aloe vera extract once a day, rectally Group (7): TNBS-induced colitis treated rats who received 500 mg/kg sulfasalazine once a day, intragastrically as a positive control group
The dose of Aloe vera extract for treatments was selected according to previously reported research [33–35]. According to the published articles, evaluation on the acute and subacute toxicity of Aloe vera in rats indicated that the methanol extract at the doses of 1, 2,4, 8, and 16 g/kg B.wt did not produce significant toxic effects . In the other study, assessments on the subacute toxicity test showed that Aloe vera did not produce marked subacute toxic effects up to a maximum concentration of 3330 mg/kg body weight on rats with no mutagenic activity in ICR mice exposed to 10000 mg Aloe vera/kg body weight . As a result, at the tested dose of Aloe vera extract, the toxic effect in rats without colitis was not assessed.
Sulfasalazine also was purchased from Merck chemical company. The appropriate amount of extract or sulfasalazine based on the treated group was dissolved in sterile water. Intragastric administration was used in conscious rats with biomedical needles (length 76.2 mm, diameter 3 mm, straight). To prepare extract for rectal administration, 5% glycerol was mixed with 2% sodium carboxymethyl cellulose (NaCMC) as an inert preservative substance . Next, 200 and 400 mg/kg body weight of the dried extract was dissolved in deionized water, and the mixtures were gradually added to the glycerol-NaCMC solution. The gel was homogenized for 30 minutes, and the gel was collected in an aluminum tube in the refrigerator. For rectal administration, the gavage needle was inserted into the anus of rats (7 cm) and 1 ml of the prepared gel was injected .
2.4. Plant Extract
Aloe vera leaves were obtained in Shiraz, Fars Province, Iran, and its species was endorsed by SUMS taxonomists at a pharmacy school. 100 g dried Aloe vera was powdered and percolated with 70% ethanol (3 times), at room temperature, and the extracts were filtered and evaporated under reduced pressure to acquire 9.8 g of dried extracts (9.8% yield). This procedure was repeated several times to get enough amounts of extract for in vitro and in vivo studies.
2.5. Macroscopic Scoring
The dosage and period of treatments were accompanied by daily body weights, gross stool evaluation for visible and/or occult bleeding. On the last day of the experiment (7th), the degree of colonic inflammation and damage was scored (Table 1) as described by Morris et al. with slight modifications [41, 42].
Localized hyperemia, but no ulcers or erosions
Ulcers or erosions with no significant inflammation
Ulcers or erosions with inflammation at one site
Two or more sites of ulceration and/or inflammation
Two or more major sites of inflammation and ulceration or one major site of inflammation and ulceration extending >1 cm along the length of the colon