Abstract
Background/Aim: Ulcerative colitis (UC) is colonic inflammation associated with increased production of pro-inflammatory cytokines, oxidative stress, and disturbances of immune responses. Mitragynine is the most abundant active alkaloid in Mitragyna speciosa (kratom) and may have anti-inflammatory, antioxidant, and antispasmodic properties. In this study, we investigated the palliative effects of mitragynine in kratom leaf extract on the symptoms of UC.
Materials and Methods: Mice were divided into six groups (n=9): control; colitis; colitis plus syrup with kratom extract containing 5, 10, or 20 mg/kg mitragynine; and a positive control group treated with 4 mg/kg loperamide. The treatments were orally administered for 5 days after colitis was induced by transrectal administration of 5% acetic acid.
Results: The results showed that syrup with 10 and 20 mg/kg mitragynine significantly alleviated colonic tissue damage caused by acetic acid-induced colitis. Furthermore, the disease activity index, colonic weight, colonic lesions, and levels of malondialdehyde and inflammatory cytokines (tumor necrosis factor-α and interleukin-1β) decreased in these groups in comparison with the colitis-only group. With regard to antispasmodic activity, kratom extract significantly increased colonic smooth muscle relaxation by acting on μ-opioid receptor signaling and inhibited induced muscular contraction in mice with colitis. Moreover, kratom extract attenuated nitric oxide levels and enhanced the phagocytic activity of mouse peritoneal macrophages.
Conclusion: Kratom leaf extract, which contains mitragynine, alleviated acetic acid-induced colitis in mice by modulating immune responses and by its anti-inflammatory, antioxidative, and antispasmodic effects. Therefore, kratom leaves may be an effective therapeutic candidate for subsequent development as a multitarget drug for UC.
- Mitragyna speciosa
- inflammatory bowel disease
- colonic inflammation
- colonic function
- smooth muscle contraction
Introduction
Ulcerative colitis (UC) is a category of inflammatory bowel disease characterized by chronic inflammation of the large intestine (1). The etiology and pathogenesis of the condition are still poorly understood, but it has been associated with the overproduction of pro-inflammatory cytokines, reactive oxygen species (ROS), and inappropriate immune responses (2, 3). The typical symptoms of UC include abdominal pain, diarrhea with blood/mucus, and peritonitis. UC is associated with diffuse inflammation of the intestinal mucosa due to the production of ROS such as nitric oxide (NO) and malondialdehyde (MDA), and of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and prostaglandin E2 (4-6). Furthermore, it was associated with the release of these mediators from macrophage cells, resulting in colonic inflammation leading to increased colonic motility and contractility (7, 8).
Currently, there are no therapeutic drugs for UC. Instead, the treatment aims to reduce inflammation by suppressing pro-inflammatory cytokines with sulfasalazine, immune modifiers, and corticosteroids. These drugs have known adverse effects, such as gastric ulcer formation, bloating, muscle weakness, increased rates of infection, and malignancy because of a dysregulated immune system (7, 9, 10). Diarrhea due to inflammatory bowel disease is treated with loperamide to reduce intestinal motility and transit, and increase the absorption of fluids and nutrients via μ-opioid effects (11). Many studies have reported that the anti-nociceptive and anti-inflammatory effects of loperamide are due to the inhibition of TNF-α that activates a peripheral-selective μ-opioid receptor (MOR) (12, 13). However, several adverse effects have also been reported. Therefore, pharmacological therapy may offer a more benign novel treatment for patients with UC.
Mitragyna speciosa (Korth.) Havil. (kratom) is a plant native to tropical and subtropical Southeast Asia (14). Kratom leaves have been used in traditional medicine to treat diarrhea and intestinal infections (15). It has been investigated for its antidiarrheal, anti-inflammatory, and antioxidative properties, and for its ability to modulate the immune system (16, 17). Mitragynine is the major indole alkaloid and the most abundant active alkaloid in the plant, and is responsible for the opioid agonist effects of kratom and for its selective MOR agonism (18-20). A previous investigation demonstrated that mitragynine inhibited the contraction of smooth muscle in the guinea pig ileum via activation of MOR through the blockage of neuronal Ca2+ channels (21). Mitragynine was also shown to inhibit cyclo-oxygenase-2 and prostaglandin E2 expression in RAW 264.7 macrophage cells treated with lipopolysaccharide, but did not inhibit cyclo-oxygenase-1 (22).
Currently, there are few studies on the anti-inflammatory effects of kratom extract in the literature. Here, we explored the inhibition of the expression of inflammatory cytokines and the antioxidative and antispasmodic effects of kratom leaf extract on colonic tissue. The mechanisms of action and improvements in the symptoms of UC in mice with acetic acid-induced colitis are investigated, and the potential of kratom leaves for UC treatment is discussed.
Materials and Methods
Materials and reagents. Kratom leaf extract was prepared as a syrup containing the equivalent of 2 mg of mitragynine/ml. Glacial acetic acid was purchased from J.T. Baker (Avantor, Leicester, UK). Carbachol and potassium chloride (KCl) were purchased from Merck (Darmstadt, Germany). Serotonin, loperamide, and naloxone hydrochloride were from Sigma-Aldrich (St. Louis, MO, USA). ColoScreen ES for fecal occult blood testing was from Helena Laboratories (Beaumont, TX, USA). Rabbit anti-β-actin (#4976), rabbit anti-TNF-α (#11948), and rabbit anti-IL-1β (#31202) were purchased from Cell Signaling Technology (Danvers, MA, USA). Other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) except where specified.
Preparation of kratom extract and syrup. Kratom extract and kratom syrup were prepared following the method of Nukitram et al. (23). Fresh kratom leaves were collected, identified, and deposited as a voucher specimen (no. Wungsintaweekul, J. N5/001) at Prince of Songkla University, Thailand. Ethanolic extract was obtained by macerating the leaves in ethanol and drying the filtrate. Mitragynine content (12% w/w) was quantified by high-performance liquid chromatography according to Limsuwanchote et al. (24). Kratom extract was utilized in isometric measurement of isolated colonic tissue, whereas in other studies, a kratom syrup was used and formulated using solubilizing agents, artificial sweeteners, and flavoring agents to yield a final concentration equivalent to 2 mg mitragynine/mL.
Animals. Male ICR mice, 6 to 8 weeks old (30-35 g), were obtained from the National Laboratory Animal Center, Mahidol University, Thailand, and housed at the Southern Laboratory Animal Facility, Prince of Songkla University, Thailand. They were housed in appropriate cages at 22±2°C under a 12-h photoperiod, at room humidity and temperature, with free access to standard diet and water. All animals were allowed to adapt to the environment for 1 week before the experiments began. The experiments were approved by the Animal Ethics Committee of Prince of Songkla University (MOE 0521.11/1071).
Experimental design. After the adaptation period, 54 mice were randomly divided into six groups (nine mice per group). Group I was the control group; in this group, mice were treated only with a syrup solution (vehicle). Group II was the colitis control group; in this group, colitis was induced with acetic acid, and the mice were treated with the same syrup as for group I. For groups III-V, colitis was induced with acetic acid, and the mice received kratom syrup daily at doses equivalent to 5, 10, and 20 mg/kg mitragynine, respectively. Group VI was a positive control group that was treated with 4 mg/kg loperamide. All interventions were orally administered in a single dose for 5 days, starting 4 h after the induction of colitis. Only tissue from groups I and II were included in isometric measurements.
Colitis induction. Colitis induction was performed on lightly anesthetized mice on day 1. First, colonic lavage was administered for 1 h, using a polyethylene tube to introduce 100 μL of 0.9% saline solution intrarectally. Then, a 5% acetic acid solution was prepared in 0.9% saline solution, and 100 μL of the prepared solution was injected through the tube approximately 3 cm into the colon. The control group was treated with the same volume of 0.9% saline solution without acetic acid. The animals were then held in a supine position for 30 s to prevent leakage of the solution (25). The body weight and food and water intakes of all animals were recorded daily before and after treatment.
Evaluation of disease activity. The disease activity index (DAI), which is an indication of the severity of colitis, was measured on day 5 and calculated by combining scores from several parameters shown in Table I. Defecation and water content in feces were measured. On day 6, mice were injected with a single dose of thiopental sodium (70 mg/kg, intraperitoneally). Blood was immediately collected by cardiac puncture to record complete blood counts. Peritoneal macrophages were quickly collected for the assessment of NO production and macrophage phagocytic activity. The colon and spleen were removed to measure weight and length. Whole colonic motility and colonic smooth muscle contraction were recorded, and the remaining colonic tissues were collected for western blot analysis and assessment of macroscopic and microscopic colonic changes.
Criteria used in evaluation of disease activity index (DAI).
Macroscopic assessments. Colonic tissues were quickly removed, the luminal content was flushed with Krebs solution, and the colon was opened longitudinally. Following the method of a previous study, the severity of colonic lesions was blindly assessed using the following scoring system: 0 points=macroscopic changes absent; 1 point=mucosal erythema only; 2 points=mild mucosal edema, slight bleeding, or small erosions; 3 points=moderate edema, ulcers or erosions bleeding; and 4 points=severe ulceration, edema, erosions, and tissue necrosis (26).
Histopathology of colonic tissues. To evaluate histopathological changes in the colons of mice with colitis, colonic segments (1 cm long) were collected and fixed in 10% formalin for 24 h at room temperature. The formalin solution was changed, and the segments were fixed in 50% ethanol for 48 h. The tissue samples were then dehydrated, embedded in paraffin, and sectioned at 5 μm thick. The paraffin sections were mounted on slides, deparaffinized, hydrated, stained with hematoxylin and eosin, dehydrated, and cleared in xylene. For histopathological observations, photographs were taken with an Olympus DP-71 microscope (Olympus Corporation, Tokyo, Japan). The histological grading of tissue inflammation followed previously described criteria shown in Table II (27).
Criteria for histological scoring of the severity of tissue inflammation.
Estimation of oxidative stress. Colonic samples were collected and homogenized in phosphate-buffered saline (PBS) (pH 7.4; 1 g tissue/10 mL). The homogenate was centrifuged at 2,935×g for 30 min at 4°C. The supernatant was separated to measure MDA, which is a product of lipid peroxidation. MDA was measured by the thiobarbituric acid reactive substances assay. The supernatant (100 μL) was mixed with 900 μL of a 1:1 solution of 20% trichloroacetic acid and 0.5% thiobarbituric acid. The mixture was incubated at 95°C for 30 min. The incubated samples were immediately cooled on ice and centrifuged at 10,000×g for 10 min. The absorbance of the supernatant was measured at 532 nm using a spectrophotometer. The concentration of MDA was calculated using the molar extinction coefficient of 1.56×105 M−1cm−1, measured simultaneously with each test and expressed as nmol MDA/g wet tissue weight (28).
Hematological analysis. Blood samples were immediately collected and analyzed. Hematocrit, hemoglobin, and red blood cell (RBC), white blood cell (WBC), platelet, and WBC differential counts were measured with a Mindray BC-2800Vet® auto hematology analyzer (Shenzhen, P.R. China) (29).
Measurement of whole colonic tissue movement. The whole colon (without flushing natural fecal pellets) was placed in a gastrointestinal motility monitor (GIMM) organ bath (Catamount Research and Development, Inc., St. Albans, VT, USA) and continuously perfused at 50 mL/min with fresh oxygenated Krebs working solution (119 mM NaCl, 4.5 mM KCl, 2.5 mM MgSO4, 25 mM NaHCO3, 2.5 mM CaCl2, 1.2 mM KH2PO4, and 11.1 mM glucose) at 37°C. The tissues were equilibrated for 30 min. The movement of the pellets was recorded using a video camera connected to a computer running GIMM software (Catamount Research and Development, Inc.). The results of motility patterns are shown for peristalsis and segmentation contractions.
Isometric measurement of isolated colonic tissue. Colonic segments from the control and colitis groups (groups I and II) were used in this experiment. Colonic luminal content was cleared by carefully flushing out with the Krebs working solution, and the colon was transversely cut into 1 cm segments. The longitudinal segments were mounted in a 10 mL organ bath containing oxygenated Krebs working solution at 37°C. The segments were stretched passively to an initial tension of 0.5 g and equilibrated for 30 min. The equilibrated segments were exposed to 50 mM KCl, 1 μM carbachol, or 1 μM serotonin. The segments were exposed to 50 mM KCl for 5 min to stimulate contraction and then to kratom leaf extract (equivalent to 10−5 M mitragynine) or 10−5 M loperamide (a MOR agonist). Longitudinal colonic smooth muscle contractions were recorded and analyzed by a PowerLab® system and LabChart 7 software (AD Instruments Ltd., Bella Vista, New South Wales, Australia) (30), and tension, amplitude, and frequency of contraction are reported. In a second experiment, 10−5 M naloxone (a MOR antagonist) was added to the bath for 10 min before stimulation with KCl, and then 50 mM KCl and 10−5 M mitragynine or 10−5 M loperamide were continuously added to the bath for 5 min, and longitudinal colonic smooth muscle contractions were recorded and analyzed.
Western blot analysis. Distal colonic tissues were cut and snap-frozen in liquid nitrogen and stored at −80°C until expression of pro-inflammatory cytokines was measured. The frozen samples were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer containing a protease inhibitor cocktail (1:1,000) (Sigma-Aldrich). The homogenate was centrifuged at 17,949×g at 4°C for 30 min. The protein concentration in the supernatant was determined using a bicinchoninic acid assay protein kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (20 μg) were diluted with six×Laemmli sample buffer and incubated in boiling water for 10 min. The proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (GE Healthcare, Chicago, IL, USA) using a semi-dry transfer system. The transferred proteins were blocked for 1 h at room temperature in 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST). The blocked proteins were then incubated overnight at 4°C with anti-TNF-α (1:500), anti-IL-1β (1:500), and anti-β-actin (1:500), diluted in blocking buffer. After washing three times in TBST for 10 min on a shaker, proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) for 1 h at room temperature and washed again three times in TBST. Immunodetection was performed using an ECL Western blot method with hypersensitive film exposure. Band intensity was evaluated using ImageJ software (National Institutes of Health, Bethesda, MA, USA).
Culture of peritoneal macrophages. Peritoneal macrophages were immediately collected. The mouse was injected intraperitoneally with 3 mL of 2% fetal bovine serum in PBS. The resulting suspensions were harvested, and red blood cells were lysed in lysis buffer (4.145 g NH4Cl, 0.5 g KHCO3, 18.6 mg EDTA/2Na, and 500 mL deionized water) for 10 min. The lysates were centrifuged twice at 2,935×g for 10 min. The precipitated cells were adjusted to 1×106 cells/mL in RPMI-1640 medium supplemented with 15% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine. The viability of cells was evaluated using the trypan blue method, in which more than 95% of cells had to survive. Subsequently, 100 μL of suspension was incubated in a 96-well flat-bottom microplate for 30 min at 37°C in 5% CO2. Cells adhering to the bottom of 96-well plates were counted.
Assay of NO production by mouse peritoneal macrophages. Macrophages were seeded in 100 μL of RPMI-1640 medium and incubated in a 96-well plate at 37°C with 5% CO2 for 6 h to allow adherence. Following incubation, 100 μL of the culture supernatant was collected and mixed with an equal volume of Griess reagent. The mixture was then incubated in the dark for 10 min to assess NO production. Absorbance was then measured at 570 nm at room temperature using a spectrophotometer and NO was calculated from a standard curve of NaNO2, measured simultaneously with each test. NO production was expressed in terms of NO concentration (μM).
Assay of phagocytosis by mouse peritoneal macrophages. Adherent macrophage cells were cultured in 100 μL of RPMI-1640 medium and incubated in a 96-well plate at 37°C with 5% CO2 for 24 h. Following incubation, the culture supernatant was carefully removed, and 200 μL of 0.075% neutral red solution in PBS was added to each well. The cells were then incubated for an additional 30 min, after which the dye was removed, and the wells were washed three times with PBS. Finally, 200 μL of lysis solution [1:1 of 1 M acetic acid: ethanol (v/v)] was added, and the cells were incubated overnight at 4°C. The absorbance was then read at 540 nm, and phagocytosis was calculated in triplicate and reported from three experiments.
Statistical analysis. Results are expressed as the mean ± standard error of the mean. Statistical analysis was performed using one-way or two-way analysis of variance followed by Bonferroni’s test. Significance was considered at a value of p<0.05. Data were analyzed using GraphPad Prism (version five) software (GraphPad Software, San Diego, CA, USA).
Results
Kratom leaf extract alleviated UC symptoms. Mice with acetic acid-induced colitis developed signs and symptoms of severe colitis, including rectal bleeding, diarrhea, bloody stool adhesion on the anus, and abdominal pain from bending over. Oral gavage for 5 days with syrup of kratom leaf extract containing 10 or 20 mg/kg mitragynine ameliorated signs of acetic acid-induced colitis and bloody stool adhesion, as well as significantly reducing defecation frequency and water content in feces (Figure 1A-C, respectively). Mice with acetic acid-induced colitis showed a high mortality rate, but survival rates were higher in those treated with syrup containing 10 or 20 mg/kg mitragynine (Figure 2A). Mice with acetic acid-induced colitis also exhibited reduced food intake and body weight loss, but mice in the groups treated with syrup containing 10 and 20 mg/kg mitragynine exhibited increased body weight and food intake (Figure 1D and Figure 2B). Likewise, water intake showed a downward trend in the untreated colitis group, but the change was not significant (Figure 2C). Moreover, the untreated colitis group showed a significant increase in DAI score, wet colonic weight, and spleen weight index (Figure 3B and C, and Figure 2D) along with a reduction in colonic length (Figure 3A and D). In contrast, treatment with kratom syrup at doses of 10 and 20 mg/kg mitragynine effectively ameliorated all symptom severity-related parameters (Figure 2D and Figure 3A-D).
Kratom leaf extract alleviated signs and symptoms of colitis induced in mice by intrarectal injection with 5% acetic acid in saline solution. (A) Bloody stool adhesion on the anus and stool characteristics in normal control mice treated with a syrup vehicle, mice with colitis treated or not with kratom syrup containing 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. (B) Frequency of defecation, (C) fecal water content, and (D) body weight of mice in each group. The results are expressed as the mean ± standard error of the mean. Significantly different at: *p<0.05, **p<0.01 and ***p<0.001 from the control group; #p<0.05, ##p<0.01 and ###p<0.001 from the colitis group. Scale bar = 1 cm.
Kratom leaf extract affected the survival rate, food and water intake, and spleen weight in mice with colitis. (A) Survival of normal control mice treated with a syrup vehicle, mice with colitis treated or not with kratom syrup containing 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. (B) Food intake, (C) water intake, and (D) spleen weight index of mice in each group. The results are expressed as the mean ± standard error of the mean. Significantly different at: *p<0.05 and ***p<0.001 from the control group; #p<0.05, ##p<0.01 and ###p<0.001 from the colitis group.
Kratom leaf extract reduced the development of severe colitis. (A) Gross appearance of the colon of normal control mice treated with a syrup vehicle, mice with colitis treated or not with kratom syrup containing 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. (B) Disease activity index (DAI) score, (C) colonic weight, and (D) colonic length of mice in each group. The results are expressed as the mean ± standard error of the mean. Significantly different at: ***p<0.001 from the control group; #p<0.05, ##p<0.01 and ###p<0.001 from the colitis group.
Kratom leaf extract attenuated colonic lesions and histopathology in acetic acid-induced UC. In mice with acetic acid-induced colitis, colonic tissues developed multiple lesions (Figure 4A), leading to significantly higher macroscopy scores (Figure 4C). The control group showed no inflammation. However, treatment with syrup containing 10 and 20 mg/kg mitragynine reduced the severity of lesions, resulting in significantly lower macroscopic scores in these groups of mice (Figure 4A and C). In addition, the histopathological features in the untreated colitis group included necrosis and hyperplasia of crypt architecture, epithelial loss and ulceration, mucosal, submucosal, and transmural inflammation, and goblet cell loss (Figure 4B). These features led to significantly higher microscopy scores (Figure 4D). After oral gavage for 5 days with kratom syrup containing 10 or 20 mg/kg mitragynine, these histopathological changes were not observed. These results suggested that these doses of kratom leaf extract healed colonic lesions in acetic acid-induced UC.
Kratom leaf extract appeared to heal pathophysiological lesions of the colon in mice with acetic acid-induced colitis. (A) Representative examples of morphological lesions of the distal colon of normal control mice treated with a syrup vehicle, mice with colitis treated or not with kratom syrup containing 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. Colonic tissues in the colitis group developed multiple lesions, including mucosal edema (black arrows), bleeding ulcers (green hash), erosions (red arrowheads), and necrosis (red asterisks). (B) Histopathological features of the distal colon. Yellow and blue asterisks indicate inflammatory cell infiltration and smooth muscle layer, respectively. (C) Macroscopy and (D) microscopy scores of mice in each group. Significantly different at: ***p<0.001 from the control group; ###p<0.001 from the colitis group. Scale bar=20 μm.
Kratom leaf extract inhibited colonic motility in acetic acid-induced UC. Segmentation and peristaltic contractions of the colon in the untreated colitis group were significantly increased in comparison to the control group (Figure 5A and B). The mice with colitis treated with kratom leaf extract containing 10 or 20 mg/kg mitragynine in reduced these parameters. Contractions were also inhibited in the positive control group (4 mg/kg loperamide). The results suggest that 10 and 20 mg/kg mitragynine in kratom leaf extract reduced colonic motility in acetic acid-induced acute colitis.
Kratom leaf extract inhibited colonic motility. (A) Number of non-propagated (segmentation) contractions and (B) number of propagated (peristaltic) contractions of normal control mice treated with a syrup vehicle, mice with colitis treated or not with 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. The results are expressed as the mean ± standard error of the mean. Significantly different at: **p<0.001 from the control group; ##p<0.01 from the colitis group.
Kratom leaf extract inhibited colonic contraction induced by KCl, carbachol, and serotonin. The administration of kratom extract containing 10 or 20 mg/kg mitragynine and 4 mg/kg loperamide inhibited the tension, amplitude, and frequency of contractions that were previously stimulated with the spasmogens KCl and carbachol (Figure 6A and B). The frequency of serotonin-induced contractions was inhibited, and this effect was observed only in the groups treated with kratom leaf extract with 10 mg/kg mitragynine or 4 mg/kg loperamide (Figure 6C). Therefore, the results indicate that kratom leaf extract and loperamide have potential antispasmodic effects in acetic acid-induced acute colitis.
Kratom leaf extract promoted the recovery of colonic smooth muscle contraction in mice with acetic acid-induced colitis. Tension, amplitude, and frequency of distal colonic smooth muscle contraction induced by (A) KCl, (B) carbachol (Cch), and (C) serotonin in normal control mice treated with a syrup vehicle, mice with colitis treated or not with kratom syrup containing 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. The results are expressed as the mean ± standard error of the mean. Significantly different at: *p<0.05 and **p<0.01 from the control group; #p<0.05 and ##p<0.01 from the colitis group.
Kratom leaf extract promoted the relaxation of KCl-stimulated colonic contraction. We explored the mechanism of action of kratom extract on colonic smooth muscle contraction in acetic acid-induced UC by directly treating colonic tissues. Contraction stimulated by 50 mM KCl was significantly higher in the untreated colitis group than in the control group. However, the continuous addition of kratom extract (with 10−5 M mitragynine) to the organ bath produced no significant relaxation in the untreated colitis group compared with the control group, whereas the addition of 10−5 M naloxone significantly inhibited colonic relaxation compared to the control group (Figure 7A). In the same protocol, 10−5 M loperamide was added. Results were similar, as shown in Figure 7B. The results suggest that, similar to the effect of loperamide, colonic relaxation due to mitragynine in kratom leaf extract was related to the MOR-agonistic activity of mitragynine.
Kratom leaf extract inhibited colonic smooth muscle contraction by μopioid receptor agonist activity. (A) Relaxation response to mitragynine (MG) in kratom leaf extract and (B) relaxation response to loperamide (LP) and with/without naloxone in normal control and colitis mice. Data are presented as mean ± standard error of the mean. *Significantly different at p<0.05 versus the respective control group.
Kratom leaf extract regulated oxidative stress in acetic acid-induced UC. MDA production was measured by the thiobarbituric acid reactive substances assay. Acetic acid-induced UC effectively upregulates MDA production. However, mice in the groups treated with kratom syrup containing 10 and 20 mg/kg mitragynine showed downregulation of MDA (Figure 8A). The results indicate that kratom leaf extract has a potential antioxidative effect against acetic acid-induced UC.
Kratom leaf extract modulated oxidative stress and immune responses in mice with acetic acid-induced colitis. (A) Malondialdehyde (MDA) production, (B) nitric oxide (NO) production, and (C) phagocytic activity in normal control mice treated with a syrup vehicle, and mice with colitis treated or not with 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. The results are expressed as the mean±standard error of the mean. Significantly different at: *p<0.05 and ***p<0.001 from the control group; #p<0.05, ##p<0.01 and ###p<0.001 from the colitis group.
Kratom leaf extract affected hematological parameters in acetic acid-induced UC. Levels of WBC, granulocytes, and monocytes were significantly higher in the untreated colitis group when compared with the control group. However, levels of lymphocytes and RBC were significantly lower in comparison to the control group. The mice treated with kratom syrup containing 10 and 20 mg/kg mitragynine exhibited higher lymphocyte and RBC counts and lower counts of WBC, granulocytes, and monocytes when compared with the untreated colitis group (Table III). The results demonstrated that oral administration of kratom syrup with doses of 10 and 20 mg/kg mitragynine for 5 days significantly increased RBC counts compared to the untreated colitis group. In addition, these mitragynine concentrations potentially modulated the immune system by reducing WBC, granulocyte, and monocyte counts.
Effect of kratom leaf extract on blood parameters in mice with acetic acid-induced acute colitis. Hematological parameters were assessed in normal control mice treated with a syrup vehicle, and mice with colitis treated or not with 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control.
Kratom leaf extract inhibited protein expression of inflammatory cytokines in acetic acid-induced UC. Upregulation of TNF-α and IL-1β causes inflammation and malignant conditions in UC. We found that these cytokines were highly expressed in the colitis group. However, their expression levels were reduced in both the syrup- and loperamide-treated groups compared to the untreated colitis group (Figure 9). Therefore, the results suggest that kratom leaf extract and loperamide attenuated inflammation in acetic acid-induced UC.
Kratom leaf extract attenuated inflammation in mice with acetic acid-induced acute colitis. Western blot and quantification of tumor necrosis factor-alpha (TNF-α) (A) and interleukin-1-beta (IL-1β) (B) expression in distal colonic tissue from normal control mice treated with a syrup vehicle, and mice with colitis treated or not with kratom syrup containing 5, 10, or 20 mg/kg of mitragynine (MG), and mice treated with 4 mg/kg loperamide (LP) as a positive control. Lanes in the western blot follow the series shown in the charts. The results are expressed as the mean ± standard error of the mean. Significantly different at: *p<0.05 and **p<0.01 from the control group; #p<0.05, ##p<0.01 and ###p<0.001 from the colitis group.
Kratom leaf extract inhibited NO production by peritoneal macrophages in acetic acid-induced UC. To further investigate and confirm the potential anti-inflammatory effects of kratom extract in peritoneal macrophages from mice with acetic acid-induced colitis, the NO production of peritoneal macrophages was determined using the Griess reagent assay. NO levels were higher in the untreated colitis group than in the control group. The treatments with kratom leaf extract at 10 and 20 mg/kg inhibited NO expression (Figure 8B). This indicates the potential anti-inflammatory effects of kratom leaf extract on acetic acid-induced acute colitis.
Kratom leaf extract regulated phagocytosis activity of peritoneal macrophages in acetic acid-induced UC. To further assess and confirm the modulation of the immune system by kratom extract, we evaluated the phagocytic ability of peritoneal macrophages in acetic acid-induced colitis mice by using the neutral red assay. Macrophage phagocytosis activity was significantly lower in the colitis group than in the control group. However, this activity was significantly higher macrophages from mice treated with syrup containing 10 and 20 mg/kg mitragynine than in the colitis group (Figure 8C). This result showed that kratom leaf extract increased phagocytic ability to regulate the immune response of peritoneal macrophages in acetic acid-induced acute colitis.
Discussion
We investigated for the first time whether oral administration of kratom leaf extract would be able to alleviate colonic inflammation in mice with acetic acid-induced colitis. After the oral administration of single doses of kratom leaf syrup containing 10 and 20 mg/kg mitragynine for 5 days to mice with induced colitis, the survival rate, body weight, food intake, and colonic length were significantly higher, while DAI and colonic and spleen weights were significantly lower. Moreover, the signs and symptoms of colitis, including diarrhea, oxidative stress, and inflammation, were attenuated in mice that were treated with kratom leaf syrup containing 10 and 20 mg/kg mitragynine. The immune system was also modulated. These results indicate for the first time that kratom may be a candidate as an effective multitarget drug for UC.
UC is a well-known colonic disorder that is characterized by chronic and relapsing inflammation of the gastrointestinal tract. Acetic acid is component of a standardized method widely used to induce the pathophysiological process of UC in animal models, and it has a characterized severity that closely resembles UC severity in humans (31, 32). Previous studies examined colitis induced in mice intrarectally with 5% acetic acid to observe inflammation and ulceration. The results showed that oxidative destruction was the pathogenetic factor, and damage was also observed in the gross morphology of the colon (25, 33). These findings occurred in the same way as in humans (34). The common signs and symptoms of UC are abdominal pain, diarrhea with blood/mucus, and peritonitis. In addition, in the chronic state of the disease, the intestine develops epithelial regeneration, resulting in pseudopolyps, as well as mucosal atrophy and featurelessness, leading to narrowing and shortening of the colon (35). Currently, there are no therapeutic drugs for UC. We showed here that treatment with syrup with kratom leaf extract equivalent to 10 and 20 mg/kg mitragynine attenuated the signs and symptoms of acetic acid-induced colitis in mice.
Inflammation is a major response to the pathogenesis and development of UC, leading to colonic tissue injury and motor dysfunction. The mediators are products caused by an inflammatory response. Several studies showed that UC is associated with diffuse inflammatory mediators in the intestinal mucosa, including ROS such as NO and lipid peroxide, and inflammatory cytokines such as TNF-α, IL-1β, IL-6, and prostaglandin E2 (4-7). Ulceration, erosion, and necrosis in the colon result, and they lead to colonic motility disturbances. TNF-α is a pleiotropic cytokine. It is mainly released by activated macrophages, monocytes, neutrophils, and smooth muscle cells, and it is active in acute inflammation and infection through nuclear factor kappa B (NF-κB) signaling (36, 37). NF-κB has been associated with the pathophysiology in patients with UC and animal models of colitis (38-42). NF-κB activation promoted the expression of several pro-inflammatory mediators such as TNF-α, IL-1β, and inducible nitric oxide synthase (40, 43). In the present study, we observed that inflammatory cytokine levels, including TNF-α and IL-1β, were elevated in the colonic tissue of mice with acetic acid-induced UC. However, the oral administration of kratom leaf extract and loperamide reduced TNF-α and IL-1β levels in the colon. These results indicate that kratom leaf extract and loperamide alleviated acetic acid-induced UC by suppressing the expression of TNF-α and IL-1β. In addition, previous studies have reported that MOR agonists can inhibit NF-κB activation (44). Thus, our study suggests that kratom leaf extract and loperamide may act by inhibiting the NF-κB signaling pathway. We have shown for the first time that kratom leaf extract containing mitragynine alleviates acetic acid-induced UC in mice by suppressing inflammatory mediators, including TNF-α and IL-1β.
The local immune response in UC, which is characterized by the recruitment of macrophages, leads to the release of pro-inflammatory mediators and ROS. ROS are a product of oxidative stress produced during pathological reactions, including inflammation and infection. MDA is an end-product of lipid peroxidation. It is a common indicator of oxidative stress alongside NO. Several studies have indicated that MDA and NO were significantly increased in the colonic tissues of mice with acetic acid-induced colitis (32, 45, 46). Similarly, in the present study, we observed that the MDA level in the colon was increased in mice with acetic acid-induced colitis. Kratom leaf extract significantly reduced levels of MDA in mice with acetic acid-induced colitis. When colitis is induced, macrophages, a type of WBC of the innate immune system, infiltrate intestinal tissues and then release or generate NO and pro-inflammatory mediators, leading to tissue inflammation (7). In the present study, we found that peritoneal macrophage cells in mice with acetic acid-induced colitis significantly increased their production of NO. However, treatment with kratom leaf extract inhibited the expression of NO. The results indicate that kratom has the potential to reduce oxidative stress in mice with acetic acid-induced colitis by downregulating MDA and NO levels.
We also evaluated hematological indicators in mice with acetic acid-induced colitis. Previous studies showed that acetic acid-induced colitis had a significant effect on hematological composition because the inflammatory response causes the infiltration of WBC, monocytes, and macrophages into serum and tissues (28, 29). In our study, WBC, granulocytes, and monocytes were significantly increased in the serum of mice with acetic acid-induced UC. Treatment with kratom leaf extract reduced WBC, granulocytes, and monocytes levels. This demonstrates that oral administration of kratom extract has the potential to modulate the immune response. However, levels of lymphocytes and RBC were significantly reduced in the serum of mice with acetic acid-induced colitis. Previous studies showed that dextran sulfate sodium-induced colitis caused anemia due to reduced RBC counts (47, 48). In the present study, RBC counts increased in mice treated with kratom leaf extract. This demonstrates that the extract may enhance erythropoiesis or support RBC production, potentially improving hematological parameters in colitis-induced mice.
Abnormal intestinal motility and smooth muscle contraction are other effects of UC. Several studies in both human and animal models have demonstrated that UC is associated with disturbances in colonic motility. Patients with UC exhibit abdominal pain, diarrhea, and increased colonic motility (49-51). In addition, in-vitro studies have shown that circular and longitudinal colonic smooth muscle contraction increases within 1 week and after 4 weeks in 2,4,6-trinitrobenzenesulfonic acid-induced colitis (52, 53). In patients with UC compared to healthy controls, muscarinic and 5-hydroxytryptamine receptors in the intestine were significantly increased, leading to hypercontraction involving the stimulation of acetylcholine, which regulates the release of intracellular Ca2+ and Ca2+/calmodulin-dependent activation (51). Several studies have demonstrated that inflammation of intestinal smooth muscle cells leads to the expression of inflammatory mediators, such as TNF-α and IL-1β, which are activated by transcription factors including NF-κB (8, 54, 55). Furthermore, colonic longitudinal smooth muscle and colonic longitudinal muscle strips cultured with TNF-α or IL-1β exhibited hypercontractility in mice with colitis (56). The NF-κB signaling pathway was involved in muscle contraction in response to acetylcholine by attenuating AMP-activated kinase activity and augmenting myosin light-chain kinase activity (8). We observed that the segmentation and peristaltic contractions of the colon in mice with acetic acid-induced colitis significantly increased in comparison to the control group. In addition, colonic smooth muscle contraction stimulated by KCl, carbachol, and serotonin was significantly increased in mice with acetic acid-induced colitis compared to the control group. Oral administration of kratom syrup reduced the tension, amplitude, and frequency of contractions and restored colonic motility patterns in both segmentation and peristaltic contractions. These results indicate that kratom extract inhibited contractility and motility effects by the downregulation of TNF-α and IL-1β, which may be associated with the NF-κB signaling pathway in the colonic tissue of mice with acetic acid-induced UC.
The MOR is a member of the G-protein-coupled receptor family. These receptors are abundantly expressed in the central nervous system and also in the peripheral nervous system. In the peripheral nervous system, MORs are highly expressed in the enteric nervous system in the gastrointestinal tract. In the colon, MORs have been shown to inhibit smooth muscle contraction and impair peristalsis. This effect is caused by the activation of μ-opioid agonist binding MOR in presynaptic neurons; the release of acetylcholine is then inhibited. Several studies have reported that MORs are highly expressed in the inflamed intestinal tract of UC animal models and patients with UC (57-60). Moreover, the protection against intestinal inflammation provided by selective MOR agonists in mice with colitis was associated with the downregulation of TNF-α and IL-1β in colonic tissues, and with their significant upregulation in MOR-knockout mice (58, 61). In our study, after stimulating contraction of colonic tissues with 50 mM KCl, we showed that in vitro treatment with 10−5 M mitragynine from kratom extract (a MOR agonist) produced insignificant inhibition of relaxation compared to the control group. However, co-treatment with 10−5 M naloxone significantly inhibited kratom extract-induced relaxation in the colitis group compared with the control. Treatment with 10−5 M loperamide led to similar results. The findings indicate that kratom extract alleviates colon muscle spasms, thereby leading to reduced abdominal pain, due to the antispasmodic effect related to MOR agonistic activity.
Conclusion
In conclusion, our results showed for the first time that kratom leaf extract reduced oxidative stress, inflammatory response, and colonic smooth muscle contraction by inhibiting pro-inflammatory mediators in mice with acetic acid-induced colitis. This activity may be associated with the NF-κB signaling pathway. Moreover, colonic smooth muscle relaxation involves the MOR-agonistic property of kratom leaf extract to selectively activate MOR in the peripheral nervous system. Therefore, we propose that kratom may be an effective therapeutic candidate for development as a multitarget drug for the treatment of ulcerative colitis.
Acknowledgements
The Authors are grateful to Mr. Thomas Coyne, Faculty of Science, Prince of Songkla University, for assisting in proofreading and providing feedback on the manuscript.
Footnotes
Authors’ Contributions
SP, CTY, JW, and PK contributed to the experimental design. SP, CTY, CT, FH, NS, JW, and PK contributed to the conducted research. SP, CTY, CT, FH, and PK contributed to analyzing data. SP, CTY, CT, FH, JW, and PK wrote the manuscript. All Authors reviewed and approved the final version of the manuscript.
Conflicts of Interest
The Authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding
This work was supported by grants from the Agricultural Research Development Agency (ARDA; Public Organization) (Contract number: CRP6105022660) and a Research Grant for Thesis in the Fiscal Year 2020, Prince of Songkla University.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools were used in the preparation, writing, or editing of this manuscript. All research, analysis, and written content were completed solely by the author(s), in accordance with institutional guidelines for academic integrity and originality.
- Received September 3, 2025.
- Revision received October 17, 2025.
- Accepted November 4, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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