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    Article

    Protective Effects of Methanolic Leaf Extract of Citrullus colocynthis Combined with Kefir against 1,2-Dimethylhydrazine and High-Fat Diet-Induced Colorectal Cancer in Wistar Rats

    Sanjay Samanth Manthena1, Eswar Kumar Kilari2, Satya Obbalareddy3, Santosh Kumar Ranajit4★★ Corresponding author

    1. 1Department of Pharmacy practice, AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, INDIA.
    2. 2Department of Pharmacology, AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, INDIA.
    3. 3Department of Health System Management Studies, JSS Academy of Higher Education and Research, Mysuru, Karnataka, INDIA.
    4. 4Department of Pharmacology, School of Pharmacy and Life Sciences, Centurion University of Technology and Management, Bolangir, Odisha, INDIA.

    CORRESPONDENCE

    Santosh Kumar Ranajit

    Department of Pharmacology, School of Pharmacy and Life Sciences, Centurion University of Technology and Management, Bolangir, Odisha, INDIA.

    sanrancol@gmail.com

    Received: 02-04-2026; Revised: 29-05-2026; Accepted: 13-07-2026.

    Volume 18, Issue 4 · pp. 1243–1251 · PUBLISHED Oct-Dec 2026 · DOI: 10.5530/pres.20260208

    ABSTRACT

    Background Colorectal Cancer (CRC) is a major global malignancy strongly influenced by diet and lifestyle. Natural products and probiotics are being explored for their chemopreventive potential. Objectives To evaluate the combined protective effects of Methanolic Leaf Extract of Citrullus colocynthis (MLECC) and kefir against CRC induced by 1,2-Dimethylhydrazine (DMH) and a High-Fat Diet (HFD) in Wistar rats. Materials and Methods Forty-two male Wistar rats were divided into four groups: control, disease (DMH + HFD), probiotic (DMH + HFD + kefir), and treatment (DMH + HFD + kefir + MLECC). Hematological indices, liver enzymes (AST, ALT, ALP), C-reactive protein, microbial enzyme activity, oxidative-stress biomarkers, Aberrant Crypt Foci (ACF), Ki-67 expression, and histopathology were evaluated. Data were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. Results DMH + HFD exposure caused anemia, hepatic injury, inflammation, oxidative imbalance, and high ACF incidence. Kefir partly reversed these effects, while the MLECC + kefir combination normalized biochemical and histological parameters, reduced ACF by ≈60%, and suppressed Ki-67 expression (p<0.05 vs disease). Conclusion Co-administration of MLECC and kefir exerts synergistic antioxidants, anti-inflammatory, and antiproliferative actions, supporting their potential as adjuncts in colorectal cancer prevention.

    KEYWORDS

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    INTRODUCTION

    Colorectal Cancer (CRC) remains a major global health concern, ranking among the top three most diagnosed malignancies and the second leading cause of cancer mortality worldwide. According to GLOBOCAN 2020, CRC accounted for ~1.9 million new cases and 0.94 million deaths. Although incidence in India is comparatively lower, it has shown a steady rise in urban populations, largely due to dietary changes, obesity, and reduced physical activity.

    CRC develops through a multistep process that includes transformation of benign adenomatous polyps into invasive adenocarcinomas. This progression is driven by mutations in genes such as KRAS and TP53, and dysregulation of signaling cascades including Wnt/β-catenin, NF-κB, and PI3K/AKT. Aberrant Crypt Foci (ACF) represent early microscopic lesions predictive of tumorigenesis.

    1,2-Dimethylhydrazine (DMH) is widely used to induce experimental CRC, as its metabolites closely mimic human oncogenic mutations. When combined with a High-Fat Diet (HFD), it promotes oxidative stress, inflammation, and gut dysbiosis, intensifying carcinogenic potential.

    Natural products and probiotics have gained attention as complementary strategies for chemoprevention. Citrullus colocynthis (L.) Schrad. (bitter apple) is a traditional medicinal plant containing cucurbitacins, flavonoids, and glycosides with reported anti-inflammatory and antiproliferative activities. Kefir, a fermented milk drink rich in lactic acid bacteria and yeasts, modulates gut microbiota and suppresses inflammatory responses.

    The rationale of this work was to evaluate whether combining the Methanolic Leaf Extract of Citrullus colocynthis (MLECC) with kefir could yield synergistic protection against DMH + HFD-induced CRC. The study examined biochemical, hematological, oxidative, microbial, and histological parameters to elucidate their potential mechanisms of chemoprevention.

    MATERIALS AND METHODS

    Chemicals and reagents

    1,2-Dimethylhydrazine (DMH) and analytical-grade reagents were obtained from Sri GhaMa Enterprises (India). The high-fat diet was purchased from VRK Nutritional Solutions (India). Methanolic leaf extract of C. colocynthis was procured from Kshipra Biotech Pvt. Ltd., Indore, India. Phytochemical profiling by HPLC confirmed the presence of cucurbitacins and flavonoids.

    Preparation of kefir

    Kefir grains were cultured in sterilized whole milk at room temperature for 24 hr. The fermented beverage was filtered to remove grains and stored at 4ºC until use. Microbial composition was confirmed by plating on selective media for Lactobacillus, Lactococcus, and Saccharomyces species.

    Experimental animals and ethics

    42 healthy male Wistar rats (130-140 g) were housed under controlled temperature (25±2ºC), humidity (45-55%), and a 12 hr light/dark cycle, with free access to food and water. All procedures followed CPCSEA guidelines and were approved by the Institutional Animal Ethics Committee (CPCSEA Registration No: 516/01/A/CPCSEA).

    Experimental design

    Rats were randomized into four groups (n=10 each).

    • Control: vehicle (0.5% CMC in saline) + normal diet.
    • Disease: DMH (40 mg/kg twice weekly) + HFD.
    • Probiotic: DMH + HFD + kefir (3.87 mg/mL orally).
    • Treatment: DMH + HFD + kefir + MLECC (200 mg/kg orally).

    The study lasted 13 weeks. On day 91, animals were euthanized under CO₂ asphyxiation. Blood, serum, and colon tissues were collected for analysis.

    Hematological and biochemical assays

    Blood was analyzed for RBC, WBC, hemoglobin, and platelet counts using an automated hematology analyzer. Serum AST, ALT, ALP, and C-reactive protein were measured using commercial colorimetric and immunoturbidimetric kits.

    Microbial enzyme activities

    Colonic and fecal homogenates were assayed for β-glucuronidase, β-glucosidase, and mucinase activities following standard protocols, with results normalized to protein content (Bradford method).

    Oxidative stress parameters

    Colon tissue homogenates were evaluated for reduced Glutathione (GSH), Glutathione Peroxidase (GPx), Catalase (CAT), and Lipid Peroxidation (LPO) as Thiobarbituric-acid reactive substances (TBARS).

    Aberrant Crypt Foci (ACF) assessment

    Fixed colons were stained with methylene blue, and ACF were counted microscopically at ×10 magnification.

    Molecular and histological analysis

    Gene expression of Ki-67 and BCL-2 was quantified by qRT-PCR relative to GAPDH. Histopathology was performed on H&E-stained sections, and β-catenin localization assessed by immunohistochemistry.

    Statistical analysis

    Results are expressed as mean±SEM. Data was analyzed by one-way ANOVA followed by Dunnett’s post hoc test using GraphPad Prism 8.0. Differences were considered significant at p<0.05.

    RESULTS AND DISCUSSION

    Body weight and survival

    DMH + HFD exposure caused a marked reduction in weight gain and growth rate, with 40% mortality compared to the control group. Animals treated with kefir or the MLECC + kefir combination exhibited improved body weight and survival, with mortality decreasing to 30% and 20%, respectively.

    Changes in body weight, growth rate, survival, and mortality across experimental groups are summarized in Table 1.

    Table 1: Impact of treatment on body weight and survival in DMH + HFD-induced CRC rats.
    Group Name (n=6)Initial body weight (g)Final body weight (g)Weight gain (g)Growth rateNo. of Initial animalsNo. of deaths% Mortality
    Control135±6.732273±16.17138±11.511.741000%
    Disease133±5.971210.6±5.56#77.6±12.51#1.0210440%
    Kefir134.33±8.787239.6±2.36*107.3±3.09*1.2710330%
    CC+Kefir134.0±5.856251.63±3.9*117.6±4.52*1.410220%

    These improvements suggest that probiotic supplementation, particularly when combined with C. colocynthis extract, enhances resilience against carcinogen-induced metabolic stress.

    Hematological parameters

    The disease group showed anemia (decreased RBC and hemoglobin) and leukocytosis with elevated neutrophils, indicative of systemic inflammation. Kefir treatment partially corrected these alterations, while the MLECC + kefir combination restored hematological parameters close to normal levels (p<0.05).

    The effects of MLECC and kefir on hematological parameters including RBC, WBC, hemoglobin, platelet count, neutrophils, and lymphocytes are presented in Table 2.

    Table 2: Effects of MLECC and Kefir on Hematological Parameters.
    RBC (×106/μL)WBC (×103/μL)HGB (g/dL)Platelets (103/μL)Neutrophil countLymphocyte count
    Control6.633±0.1878157±132.813.63±0.3284826.67±77.6021.19±1.15066.00±3.510
    Disease4.580±0.5003#10890±315.1#10.18±0.5829#1493.33±52.11#47.33±3.502#34.67±2.160#
    Kefir5.893±0.1598*8987±279.5 *12.10±0.1256*1369.52±48.25*41.87±2.482 *45.67±2.160*
    CC+kefir6.387±0.1846*8436±253.6*12.52±0.4983*1221.39±50.13*32.49±1.105*49.89±2.585*

    The effect of MLECC and kefir on hematological parameters in DMH + HFD-induced colorectal cancer rats is illustrated in Figure 1.

    Figure 1: Effect of Methanolic Leaf Extract of Citrullus colocynthis (MLECC) and kefir on hematological parameters (WBC, RBC, hemoglobin, platelets, neutrophils, and lymphocytes) in DMH + HFD-induced colorectal cancer in Wistar rats. Bar graphs showing alterations in RBC, WBC, hemoglobin, platelet count, neutrophils, and lymphocytes among experimental groups. The disease group exhibited anemia and leukocytosis, whereas kefir and MLECC+kefir treatments significantly restored hematological indices toward normal values. Data are presented as mean±SEM (n=10). p<0.05 versus disease control.

    This recovery reflects the combined antioxidant and immunomodulatory effects of MLECC and kefir, which are likely to protect erythrocytes from oxidative damage and normalize leukocyte function.

    Liver enzymes and inflammatory markers

    DMH + HFD caused significant elevation of serum AST, ALT, ALP, and CRP, reflecting hepatic stress and systemic inflammation. Both kefir and MLECC reduced these elevations, with the combination producing near-normal values (p<0.05).

    Serum liver enzymes (AST, ALT, ALP) and C-reactive protein levels across experimental groups are shown in Table 3.

    Table 3: Effects of MLECC and kefir on liver enzymes and C-reactive protein.
    Group Name (n=6)SGPT (U/L)SGOT (U/L)ALP (U/L)Serum C-reactive protein (mg/dL)
    Control56.83±4.26260.5±3.507138.5±3.61914.73±3.46
    Disease125.3±4.457#120.7±4.010#284.8±6.942#36.12±2.97#
    Kefir103.7±4.546*110.6±4.819*248.1±6.128*28.17±3.11*
    CC+kefir87.69±4.429*86.54±3.509*191.7±5.167*19.01±2.53*

    The observed hepatoprotection may arise from flavonoids and cucurbitacins in MLECC stabilizing hepatocyte membranes, while probiotic peptides from kefir suppress inflammatory mediators such as NF-κB and IL-6.

    Microbial enzyme activity

    DMH + HFD increased β-glucuronidase, β-glucosidase, and mucinase activity, which promote carcinogen reactivation and mucus barrier degradation. Kefir reduced these enzyme levels, and the combination with MLECC achieved the most pronounced reduction (p<0.05).

    Alterations in fecal pH and fecal microbial enzyme activities, including β-glucuronidase, β-glucosidase, and mucinase, across experimental groups are presented in Table 4.

    Table 4: Effects on microbial enzyme levels in fecal homogenates.
    Group Name (n=6)Fecal Matter pHß-Glucuronidaseß- GlucosidaseMucinase
    Control5.567±0.166.033±0.2311.56±0.173.365±0.14
    Disease8.163±0.15#10.42±0.45#15.5±0.71#7.73±0.11#
    Kefir7.833±0.25*9.82±0.24*14.51±0.35*5.158±0.11*
    CC+kefir6.524±0.35*9.11±0.54*12.80±0.36*4.686±0.15*

    The changes in fecal pH and fecal microbial enzyme activities following MLECC and kefir treatment are depicted in Figure 2.

    Figure 2: Effect of MLECC and kefir on fecal microbial enzyme activities (β-glucosidase, β-glucuronidase, and mucinase) and fecal pH in DMH + HFD-induced colorectal cancer in Wistar rats. Changes in β-glucuronidase, β-glucosidase, mucinase activity, and fecal pH in DMH + HFD-induced colorectal cancer rats. Disease induction reminds increased enzyme activities associated with carcinogen activation, whereas kefir and MLECC+kefir significantly reduced these microbial markers, indicating modulation of gut microbiota.

    The effects of MLECC and kefir on colonic mucosal microbial enzyme activities are summarized separately in Table 5.

    Table 5: Effects on microbial enzyme levels in colon homogenates.
    Group name (n=6)ß-Glucuronidaseß- GlucosidaseMucinase
    Control1.482±0.07191.35±0.08741.435±0.0977
    Disease7.510±0.1657#3.905±0.1945#4.325±0.1734#
    Kefir6.332±0.1286*3.435±0.3986*3.932±0.0920*
    CC+kefir4.227±0.195*2.749±0.128*3.167±0.125*

    The modulation of colonic mucosal microbial enzyme activities by MLECC and kefir is shown in Figure 3.

    Figure 3: Effect of MLECC and kefir on colonic mucosal microbial enzymes (β-glucuronidase, β-glucosidase, and mucinase) in DMH + HFD-induced colorectal cancer rats. Enzyme activities of β-glucuronidase, β-glucosidase, and mucinase measured in colon tissue homogenates. Combined treatment markedly suppressed microbial enzyme levels compared with disease control, suggesting protection against mucosal degradation and carcinogenic metabolite production.

    These findings support a synergistic microbiota-modulating effect, where kefir alters bacterial composition and MLECC polyphenols exhibit prebiotic-like properties.

    Oxidative stress markers

    The disease group showed depleted GSH, GPx, and CAT, along with elevated lipid peroxidation (TBARS). Treatment with kefir and MLECC significantly enhanced antioxidant enzyme activities and reduced lipid peroxidation (p<0.05).

    The effects of treatments on antioxidant status and lipid peroxidation in colon tissue are summarized in Table 6.

    Table 6: Effects on antioxidant markers in colon tissue.
    Group Name (n=6)GSH (μM/g tissue)GPx (µgm GSH utilized/min/mg protein)LPO (nmol MDA/mL plasma)CAT (units/mg protein
    Control5.671±0.307434.56±1.2450.1036±0.0600.0991±0.0076
    Disease1.711±0.4296#17.14±0.534#0.3100±0.054#0.0446±0.0092#
    Kefir3.910±0.5142*27.50±0.529*0.2048±0.045*0.0741±0.0086*
    CC+kefir4.621±0.3360*31.16±1.098*0.1790±0.043*0.0845±0.0065*

    The effects of MLECC and kefir on antioxidant status and lipid peroxidation in colon tissue are presented in Figure 4.

    Figure 4: Effect of MLECC and kefir on oxidative stress biomarkers (GSH, GPx, LPO, and catalase) in colon tissue homogenates of DMH + HFD-induced colorectal cancer rats. Levels of reduced Glutathione (GSH), Glutathione Peroxidase (GPx), Catalase (CAT), and lipid peroxidation (LPO/TBARS) in experimental groups. DMH + HFD reduced antioxidant defenses and elevated oxidative damage, while combined treatment restored antioxidant enzyme activity and reduced lipid peroxidation. Data expressed as mean±SEM (n=10).

    The results indicate potent free-radical-scavenging activity and reinforcement of endogenous defense systems. Cucurbitacins and flavonoids in C. colocynthis, combined with probiotic metabolites, may act synergistically to counteract DMH-induced oxidative injury.

    Aberrant crypt foci and proliferation markers

    Aberrant Crypt Foci (ACF) were abundant in the disease group, confirming successful CRC induction. Kefir reduced ACF incidence to ≈ 57%, while MLECC + kefir reduced it further to ≈ 40%. Ki-67 mRNA expression, elevated in the disease group, was significantly suppressed in the combination treatment (p<0.05).

    The distribution and incidence of aberrant crypt foci among treatment groups are presented in Table 7.

    Table 7: Effects on ACF count.
    Group Name (n=6)Number of aberrant crypts per ACFTotal no. ACF% of incidence
    1 crypt2 crypts3 crypts4 crypts
    ControlNILNILNILNILNILNIL
    Disease22.80±5.217.33±3.4414.0±1.1822±1.5876.13±6.53#100
    Kefir10.98±2.89.21±1.639±0.9814±2.043.19±3.94*56.73
    CC+Kefir7.76±1.366.09±1.0926.02±0.92111.16±1.17830.47±2.38*40.02

    Relative Ki-67 gene expression levels across experimental groups are summarized in Table 8.

    Table 8: Effects on Ki-67 expression.
    Group Name (n=6)Ki67
    Control0.76±0.066
    Disease1.59±0.057#
    Kefir1.28±0.072*
    CC+kefir1.04±0.052*

    The impact of MLECC and kefir on aberrant crypt foci formation and Ki-67 expression is illustrated in Figure 5.

    Figure 5: Effect of MLECC and kefir on Aberrant Crypt Foci (ACF) formation and Ki-67 expression in colon tissues of DMH + HFD-induced colorectal cancer rats. Quantitative analysis of ACF incidence and relative Ki-67 mRNA expression in colon tissue. Combination therapy significantly reduced ACF number and suppressed proliferation marker expression compared with disease control, indicating inhibition of early tumorigenesis.

    Reduced ACF formation and proliferation marker expression confirm that both interventions hinder early tumor promotion. Comparable effects have been observed with other antioxidant plant compounds such as curcumin and quercetin.

    Histopathology and β-catenin immunohistochemistry

    Histological analysis revealed dysplastic lesions, loss of goblet cells, and inflammatory infiltrates in the disease group. Kefir treatment improved mucosal integrity, while the combination group exhibited near-normal colonic architecture with intact mucous-secreting cells. β-catenin immunostaining showed abnormal nuclear localization in disease samples, which was restored to membranous localization in the treatment group, indicating normalization of Wnt signaling.

    Representative peripheral blood smear morphology across experimental groups is shown in Figure 6.

    Figure 6: Representative blood smear morphology: (A) Normal control showing uniform erythrocytes; (B) DMH + HFD group showing abnormal cell morphology; (C) Kefir-treated group showing partial improvement; (D) MLECC + kefir group showing near-normal cell structure.

    Histopathological changes and treatment-induced restoration of colonic architecture are illustrated in Figure 7.

    Figure 7: Representative histopathological sections of colon tissue (H&E, 40×): (A) Normal architecture in control group; (B) Distorted crypts and dysplasia in DMH + HFD group; (C) Partial restoration in kefir-treated group; (D) Improved mucosal and glandular structure in MLECC + kefir co-treated group.

    Immunohistochemical localization of β-catenin in colon tissue and its modulation by MLECC and kefir treatment are shown in Figure 8.

    Figure 8: Immunohistochemical staining of β-catenin in colon tissue sections (40×). The disease control group (DMH + HFD) shows prominent nuclear and cytoplasmic accumulation of β-catenin, indicating enhanced proliferative signaling. Treatment with kefir and Methanolic Leaf Extract of Citrullus colocynthis (MLECC) restored predominant membranous localization, reflecting suppression of Wnt/β-catenin activation. Black arrows indicate β-catenin localization, and red boxes highlight preservation of cell junction integrity.

    These observations demonstrate normalization of Wnt/β-catenin signaling, consistent with cucurbitacin-mediated inhibition of oncogenic transcription and probiotic restoration of epithelial barrier function.

    Overall interpretation

    The combined administration of MLECC and kefir provided comprehensive protection against DMH + HFD-induced CRC. Improvements spanned hematological, hepatic, microbial, and oxidative parameters, along with reduced ACF and normalized histology. The synergy between plant-derived phytochemicals and probiotic constituents appears to target multiple hallmarks of cancer—sustained proliferation, inflammation, and oxidative stress.

    CONCLUSION

    The combined administration of Methanolic Leaf Extract of Citrullus colocynthis (MLECC) and kefir demonstrated marked chemo preventive effects against 1,2-dimethylhydrazine (DMH) and High-Fat Diet (HFD)-induced colorectal cancer in Wistar rats. The treatment corrected hematological disturbances, reduced hepatic enzyme elevation and systemic inflammation, suppressed microbial enzyme activity, restored antioxidant balance, and reduced aberrant crypt formation.

    Histopathological and molecular findings, including normalization of β-catenin localization and downregulation of Ki-67, confirmed the reversal of carcinogenic alterations. The results underscore the synergistic antioxidant, anti-inflammatory, and antiproliferative mechanisms of MLECC and kefir. These findings provide a scientific basis for further exploration of plant-probiotic combinations as potential adjuvants in colorectal cancer prevention and therapy.

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    Manthena, S. S., Kilari, E. K., Obbalareddy, S., & Ranajit, S. K. (2026). Protective Effects of Methanolic Leaf Extract of Citrullus colocynthis Combined with Kefir against 1,2-Dimethylhydrazine and High-Fat Diet-Induced Colorectal Cancer in Wistar Rats. Pharmacognosy Research, 18(4), 1243–1251. https://doi.org/10.5530/pres.20260208