Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2013 , Vol 17 , Num 1
The effect of betulinic acid on TNBS-induced experimental colitis
Tarık Emre Şener1, Rıza Can Kardaş1, Ahmet Özer Şehirli2, Emel Ekşioğlu-Demiral3, Meral Yüksel4, Şule Çetinel5, Berrak C. Yeğen6, Göksel Şener2
1Marmara University Medical Student, Istanbul, Türkiye
2Marmara University School of Pharmacy, Department of Pharmacology, Istanbul, Türkiye
3Marmara University School of Medicine, Department of Hematology & Immunology, Istanbul, Türkiye
4Marmara University, Vocational School of Health Related Professions, Istanbul, Türkiye
5Marmara University School of Medicine, Department of Histology & Embryology, Istanbul, Türkiye
6Marmara University School of Medicine, Department of Physiology, Istanbul,Türkiye
DOI : 10.12991/201317393


Bu çalışmada betulinik asitin sıçanlarda kolondaki inflamasyon üzerinde muhtemel koruyucu etkileri araştırıldı. Her iki türden Sprague-Dawley sıçanlarda 1 ml trinitrobenzen sülfonik asit (TNBS)'in intrakolonik uygulaması ile kolit oluşturuldu. Kolit oluşturulan sıçanlara oral gavaj ile taşıyıcı (0.05% DMSO) veya betulinik asit (50 mg/kg/gün) 3 gün süreyle uygulandı. 72 saat sonra dekapite edilen hayvanlardan kan örnekleri alınarak TNF-α, IL-1Β, laktat dehidrojenaz (LDH) düzeyleri ve antioksidan kapasite (AOK) tayinleri yapıldı. Kolon dokusunun 8 cmlik distal kısmı makroskopik olarak skorlandı ve dokuda oksidatif hasar malondialdehit (MDA) ve glutatyon (GSH) düzeyleri, myeloperoksidaz (MPO) aktivitesi, kollagen içereği incelemeleri ve histolojik analizler yapıldı. Oksidan türevlerin oluşumu luminol ve lusigenin kemiluminesans (KL) ile değerlendirildi. Kolit oluşumu kolon dokusunda KL değerlerini, makroskopik skoru, MDA, MPO ve kollagen düzeylerini artırırken GSH düzeyleri anlamlı derecede azaldı. Benzer şekilde serum TNF-α, IL-1Β, ve LDH artarken AOK ise azaldı. Buna karşılık betulinik asit uygulaması TNBS uygulamasının neden olduğu tüm biyokimyasal ve histopatolojik değişimleri geri çevirdi. Bu bulgular betulinik asitin kolonda radikal süpürücü ve antioksidan etkileri ile koruyucu olduğunu düşündürmektedir.


Inflammatory bowel diseases (IBD) are idiopathic chronic inflammation in gut with diffuse inflammation of the colon and rectum[1]. Although the precise etiology of IBD is not known, many factors have been implicated; including neutrophil infiltration and overproduction of proinflammatory mediators such as cytokines, arachidonate metabolites and reactive oxygen mediators[2]. Medication of IBD includes glucocorticoids, 5-aminosalicylic acid and immunosuppressive agents[3]. However due to adverse effects of steroids, reduced effectiveness of 5-aminosalicylic acid in severe IBD or serious complications of immunosuppressive agents, their usage in IBD is limited and novel agents useful for the treatment of IBD are being developed.

Different parts of the plants are used in the traditional system of medicine for the treatment of various human ailments[4]. Plant-derived biological compounds with antioxidant properties may contribute to the protection of cells and tissues against deleterious effects of reactive oxygen species (ROS)[6,7]. Betulinic acid is a naturally occurring pentacyclic triterpene. It has several botanical sources, but can also be chemically derived from betulin, a substance found in abundance in the outer bark of white birch trees (Betula alba)[7]. Betulinic acid has been found to selectively kill human melanoma cells while leaving healthy cells alive and also several betulinic acid derivatives are potent and highly selective inhibitors of HIV-1[8]. Since triterpenoids have a similarity to steroidal compounds, their effects have often been attributed to a mechanism related to antiinflammatory action. In our previous study we have demonstrated that betulinic acid attenuates ischemia/reperfusion-induced oxidant responses and improved renal function by regulating apoptotic function of leukocytes and inhibiting neutrophil infiltration[9].

On the basis of this background, using biochemical and histological examination, we aimed to study the putative protective effects of betulinic acid on the colonic tissue in a rat model of colitis.


Adult Sprague-Dawley rats (250–300, both sexes) were kept in a light- and temperature-controlled room with 12:12-h light– dark cycles, where the temperature (22±0.5 °C) and relative humidity (65–70 %) were kept constant. The animals were fed a standard pellet and food was withdrawn overnight before colitis induction. Access to water was allowed ad libitum. Experiments were approved by the Marmara University Animal Care and Use Committee.

Induction of colitis and drug administration
Animals were fasted for 18 h before the induction of colitis. Under light ether anesthesia, a polyethylene catheter (PE-60) was inserted into the colon with its tip positioned 8 cm from the anus. To induce colitis, a single solution of 1 ml of a 30 mg/ ml trinitrobenzene sulphonic acid (TNBS) solution, dissolved in 40 % ethanol in saline was instilled. The rats in the control group were subjected to the same procedure with the exception that an equal volume of isotonic saline was substituted for TNBS. Betulinic acid [(3beta)-3-hydroxylup-20(29)-en-28-oic acid; Sigma-Aldrich, St. Louis, MO, USA,] was dissolved in 0.05% DMSO as a vehicle. Betulinic acid (50 mg/kg; i.p.; colitis+BA group) or vehicle were given orally 5 min after induction of colitis and the treatment was continued for the following 3 days. Similarly, control rats were also treated with either betulinic acid or vehicle. Each group consists of 8 rats. At the 72nd hour of experiment, rats were decapitated and trunk blood was collected for the assessment of lactate dehydrogenase (LDH) activity, as a marker of tissue injury, and the levels of the pro-inflammatory cytokines, TNF- α and IL-1Β and antioxidant capacity (AOC). Distal 8 cm of the colon obtained from each animal were initially examined for recording macroscopic damage scores and tissue wet weight index (WWI), and then stored at -80 °C until the determination of malondialdehyde (MDA), glutathione (GSH) levels, myeloperoxidase activity (MPO), collagen content and luminol and lucigenin chemiluminescence (CL). For the histological analysis, extra 1-square cm samples were obtained from each animal at 8 cm from anus to be fixed in formaldehyde.

Assessment of colitis severity
The distal 8 cm of the colons were opened longitudinally down their mesenteric borders, cleansed of luminal contents, gently rinsed in saline and dried on filter paper. The severity of colitis was assessed using macroscopic and microscopic damage scoring, WWI and tissue collagen content.

Three days after the induction of colitis, all rats were decapitated. The last 8 cm of the colon was excised, opened longitudinally, and rinsed with saline solution. The mucosal lesions were scored macroscopically using the criteria outlined in Table 1[10]. The scoring of colonic damage was performed by an observer who was unaware of the treatments received by the rats. After scoring, tissue weights were recorded, corrected for body weight and expressed as tissue WWI (g/100 g body weight).

TABLE 1: Criteria for macroscopic scoring of colonic lesions

Biochemical analysis
Measurement of serum lactate dehydrogenase (LDH) activity, and cytokine levels
Lactate dehydrogenase (LDH) activity, an indicator of tissue damage, was determined spectrophotometrically using an automated analyzer (Bayer Opera biochemical analyzer, Germany)[11]. Plasma levels of TNF-α, and IL-1β were quantified using enzyme-linked immunosorbent assay (ELISA) kits specific for the rat cytokines according to the manufacturer’s instructions and guidelines (Biosource Europe S. A., Nivelles, Belgium). The total AOC in plasma was measured by using colorimetric test system (ImAnOx, cataloge no.KC5200, Immunodiagnostic AG, D-64625 Bensheim, Germany), according to the instructions provided by the manufacturer. These particular assay kits were selected because of their high degree of sensitivity, specificity, inter- and intraassay precision and small amount of plasma sample required conducting the assay.

Chemiluminescence (CL) assay
To assess the contribution of reactive oxygen species in ethanol- induced tissue damage, luminol and lucigenin chemiluminescences were measured as indicators of radical formation. Measurements were made at room temperature using Junior LB 9509 luminometer (EG&G Berthold, Germany). Specimens were put into vials containing PBS-HEPES buffer (0.5 M PBS containing 20 mM HEPES, pH 7.2). ROS were quantitated after the addition of enhancers, lucigenin or luminal, for a final concentration of 0.2 mM. Counts were obtained at 1 min intervals and the results were given as the area under curve (AUC) for a counting period of 5 min. Counts was corrected for wet tissue weight and expressed as relative light units (rlu/mg tissue)[12].

Tissue malondialdehyde (MDA) and glutathione (GSH) assays
Colonic samples were homogenized in ice-cold 150 mM KCl for the determination of MDA and GSH levels. The MDA levels were assayed for products of lipid peroxidation[13]. Results were expressed as nmol MDA g-1 tissue. GSH was determined by the spectrephotometric method using Ellman’s reagent[14] and the results were expressed as µmol GSH g-1 tissue.

Tissue myeloperoxidase (MPO) activity
The activity of tissue-associated myeloperoxidase (MPO), a natural constituent of primary granules of neutrophils, was determined in the colonic samples according to the method of Hillegass et al.[15]. Since a direct relationship between the tissue MPO activity and the number of neutrophils was previously shown[16], MPO activity was regarded as an indication of neutrophil accumulation. All reagents for MPO assay were obtained from Sigma. The tissue samples (0.2–0.3 g) were homogenized in 10 volumes of ice-cold potassium phosphate buffer (50 mM K2HPO4, pH 6.0) containing hexadecyltrimethylammonium bromide (HETAB; 0.5%, w/v). Tissue samples were homogenized in 50 mM potassium phosphate buffer (PB, pH 6.0), and centrifuged at 41,400 g (10 minutes); pellets were suspended in 50mMPB containing 0.5 % hexadecyltrimethylammonium bromide. After three freeze and thaw cycles, with sonication between cycles, the samples were centrifuged at 41,400 g for 10 minutes. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue.

Tissue collagen was measured as a free radical-induced fibrosis marker. Tissue samples were cut with a razor blade, immediately fixed in 10% formalin then samples were embedded in paraffin, and sections, approximately 15 μm thick were obtained. The evaluation of collagen content was based on the method published by Lopez De Leon and Rojkind[17], which is based on selective binding of the dyes Sirius Red and Fast Green FCF to collagen and noncollagenous components, respectively. Both dyes were eluted readily and simultaneously by using 0.1 N NaOH–methanol (1:1, v/v). Finally, the absorbances at 540 and 605 nm were used to determine the amount of collagen and protein, respectively.

Histological evaluation
Full-thickness colon samples were collected for histological examination and fixed in 10% neutral buffered formalin solution. After fixation, tissue samples were dehydrated in graded ethanol series, cleared in toluene and embedded in paraffin. Tissue sections (5 microns thick) were stained by routine hematoxylin and eosin (H&E) stain for general morphological evaluation and Alcian Blue stain for mucin demonstration. Stained sections were examined and photographed under an Olympus BX51 photomicroscope (Tokyo, Japan).

Statistical analysis
All data are expressed as mean ± SEM. Statistical analysis was carried out using Instat statistical package (GraphPad Software, San Diego, CA, USA). Following the assurance of normal distribution of data, groups of data were compared with one-way analysis of variance (ANOVA) followed by Tukey– Kramer post hoc test for multiple comparisons. Values of p<0.05 were regarded as significant.


Severity of colonic injury
When compared with the colonic tissue of the control group, TNBS administered rats showed increased tissue WWI and the macroscopic damage score (p<0.001). On the other hand, betulinic acid treatment reduced both parameters significantly (p<0.05- 0.01), however they were still higher than control (p<0.001; Figure 1).

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FIGURE 1: a) Macroscopic scores, b) wet weight indices (WWI) in the colonic tissues of vehicle- or betulinic acid (BA) treated control and colitis groups. (n=8 per group). ***p<0.001 compared to control group. +p<0.05, ++p<0.01, +++p<0.001 compared to vehicle-treated colitis groups.

Biochemical parameters in the plasma
As shown in Figure 2, plasma cytokines, TNF-α and IL-1β, and lactate dehydrogenase (LDH) activity were significantly elevated in the vehicle-treated colitis group (p<0.001), while AOC was decreased. On the other hand, betulinic acid treatment decreased the plasma TNF-α, IL-1β levels and LDH activity and increased AOC levels (p<0.05-0.01).

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FIGURE 2: Plasma a) Tumor necrosis factor-alpha (TNF-α), b) interleukin 1-beta (IL-1 Β), c) lactate dehydrogenase (LDH) levels and d) antioxidant capacity (AOC) in the vehicle- or betulinic acid (BA) treated control and colitis groups. (n=8 per group). **p<0.01, ***p<0.001 compared to control group. +p<0.05, ++p<0.01, compared to vehicle-treated colitis groups.

Luminol and lucigenin chemiluminescence (CL) levels
Luminol and lucigenin CL levels in the vehicle-treated colitis group were increased dramatically (p<0.01-0.001) as compared to those in the control group, while erdosteine treatment following colitis induction prevented radical formation (p<0.01-0.001; Figure 3).

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FIGURE 3: a) Luminol, b) lucigenin chemiluminescence (CL) levels in the colonic tissues of vehicle- or betulinic acid (BA) treated control and colitis groups. (n=8 per group). ***p<0.001 compared to control group. +++p<0.001 compared to vehicle-treated colitis groups.

Colonic malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content
Colitis induction with TNBS followed by vehicle treatment significantly increased MDA level and decreased GSH level in the colonic tissue (p<0.001; Figure 4), as compared to control group; while betulinic acid treatment abolished colitis-induced elevation in MDA level and decrease in GSH level (p<0.01).

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FIGURE 4: a) Malondialdehyde (MDA) and b) glutathione (GSH) levels in the colonic tissues of vehicle- or betulinic acid (BA) treated control and colitis groups. (n=8 per group). **p<0.01, ***p<0.001 compared to control group. +p<0.05, compared to vehicle-treated colitis groups.

Intracolonic instillation of TNBS, as assessed by elevated MPO activity in the colonic tissues of the vehicle-treated group, caused a significant increase in neutrophil infiltration when compared to the control groups (p<0.001; Figure 5a). On the other hand, betulinic acid treated colitis group the colonic MPO activity back to the control level (p<0.001).

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FIGURE 5: a) Myeloperoxidase (MPO) activity and b) collagen contents in the colonic tissues of vehicle- or betulinic acid (BA) treated control and colitis groups. (n=8 per group). *p<0.05, ***p<0.001 compared to control group. +p<0.05, compared to vehicle-treated colitis groups.

As an indicator of enhanced tissue fibrotic activity caused by oxidative stress, the collagen content in the colonic tissue of vehicle-treated colitis group was markedly increased (p <0.001) with respect to control groups, while betulinic acid treatment prevented the increase fibrotic activity significantly (p<0.001; Figure 5b).

Light microscopic evaluation of stained sections both in the vehicle- or BA-treated control groups revealed regular colon mucosa with surface epithelium (Figure 6a, b). In the vehicle-treated colitis group, severe damage of mucosa with epithelial degeneration, necrosis, submucosal edema and inflammatory cell infiltration were observed (Figure 6c). However, the BA-treated colitis group showed healed colonic epithelium with mild inflammatory cell infiltration (Figure 6d).

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FIGURE 6: Micrographs illustrating the histological appearences of colonic tissues in different experimental groups. Control group (a), regular colon morphology (arrows); inset, normal colonic epithelium (arrow), Alcian blue staining; BA group (b), normal colon morphology (arrows); inset, normal colonic epithelium (arrow), Alcian blue staining; Colitis DMSO group (c), severe damage of mucosa with epithelial degeneration (arrows), necrosis, severe submucosal edema (double-headed arrow) and inflammatory cell infiltration (*); inset, necrotic colonic epithelium (arrow), Alcian blue staining; Colitis Betulinic acid group (d), mucosa with no localized epithelial degeneration (arrows), submucosal edema healing with mild inflammatory cell infiltration (*); inset, healed colonic epithelium (arrow), Alcian blue staining. H&E staining, original magnifications, ×200, insets Alcian blue staining, original magnifications, x200.


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