Chemopreventive effect of a yerba mate extract on 1,2-Dimethylhydrazine-induced colon carcinogenesis in BALB/c mice

Chemopreventive effect of a yerba mate extract on 1,2-Dimethylhydrazine-induced colon carcinogenesis in BALB/c mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chemopreventive effect of a yerba mate extract on 1,2-Dimethylhydrazine-induced colon carcinogenesis in BALB/c mice Humberto Lamdan, Rocio S. Garcia-Lazaro, Norailys Lorenzo, Lorena G. Caligiuri, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7593777/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Yerba mate ( Ilex paraguariensis St. Hill. Aquifoliaceae ) is a plant native to South America with numerous medicinal properties, mainly attributed to its high content of phenolic compounds. Several studies have shown that these bioactive compounds can reduce the risk of developing chronic diseases, including cancer. In the present study, we evaluated the chemopreventive effect of yerba mate extract (YMe) against 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in BALB/c mice of both sexes. Colon tumors were induced by weekly intraperitoneal injections of DMH (20 mg/kg body weight) for 14 weeks. YMe (1.6 g/kg body weight/day) or maltodextrin (vehicle control) was administered to the animals via drinking water, starting four weeks before the first DMH injection, and continued until the end of the experiment. Presence of colonic lesions and incidence of animals with neoplastic tissue was confirmed by macroscopic examination and histopathological analysis. YMe treatment completely inhibited the development of tumor lesions in the colon of female mice, all of which exhibited a normal colonic mucosal architecture. In contrast, tubular and tubulo-villous adenomas were observed in male mice of both DMH-treated groups, regardless of YMe administration. These findings demonstrate a sex-specific chemopreventive effect of YMe against DMH-induced colon carcinogenesis, with significant protection observed in females but not in males. Further experiments are needed to elucidate the molecular mechanisms underlying these effects. Our results suggest that yerba mate could be used as a natural agent for colorectal cancer prevention and support its potential role in dietary-based chemopreventive strategies. Colon carcinogenesis Ilex paraguariensis Polyphenols Yerba mate Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Since ancient times, plants have played a very relevant role in the treatment of human diseases. The search for antitumor agents of plant origin began in the early 1940s and today approximately 50% of approved anticancer drugs are derived from this source [ 1 ]. Yerba mate ( Ilex paraguariensis St. Hill. Aquifoliaceae ) is a plant native to the subtropical region of South America. The infusion from Ilex paraguariensis , which is a very popular beverage in Argentina, southern Brazil, Paraguay and Uruguay, is called “mate”. This plant contains a large amount of bioactive compounds that contribute to their health benefits. It has been reported that yerba mate has a high antioxidant capacity, as well as anti-inflammatory and immunomodulatory effects, which correlates positively with the amount of polyphenols present in the plant [ 2 ]. The action of these secondary metabolites can reduce the incidence of certain chronic diseases, including cancer. Colorectal cancer (CRC) is a recurrent malignant neoplasm of the digestive system and the second leading cause of cancer deaths worldwide [ 3 ]. According to the clinical and histopathological data published to date, the majority of these neoplasms arise from adenomas, i.e ., pre-existing benign tumors. In consequence, CRC is currently considered a cancer that can be “prevented” by avoiding the adenoma-carcinoma sequence. During the last few decades, researchers have focused their studies on dietary factors due to the implication of diet in the carcinogenesis and progression of CRC [ 4 ]. Chemically induced carcinogenesis models have contributed significantly to the understanding of the mechanisms underlying the development of CRC. 1,2- Dimethylhydrazine (DMH) is a potent carcinogen widely used to induce tumors in the descending colon in experimental animals. DMH is metabolized in the liver to form azoxymethane (AOM) and methylazoxymethanol (MAM). The reactive metabolite, MAM, is transported to the colon via bile or blood to generate the alkylating methyldiazonium ion. The latter is capable of methylating macromolecules of colonic epithelial cells resulting in oxidative stress. In consequence, reactive oxygen species can induce DNA damage, which in turn can cause mutations that initiate malignant transformation [ 5 ]. The role of yerba mate in the development of cancer was under discussion for many years with controversial results. Many epidemiological studies from late 1980s to early 2000s indicated that yerba mate consumption was positively correlated with the risk of developing certain types of cancer, including oral cavity, oropharyngeal, bladder and lung cancer [ 6 , 7 ]. However, these studies did not take into account other risk variables such as the temperature of the infusion, the simultaneous consumption of tobacco and alcohol, age, sex, and other dietary or lifestyle factors that may affect carcinogenesis and tumor progression. More recently, based on evidence, the scientific community accepts that yerba mate has an antitumor effect. An inverse correlation has been reported between high consumption of mate and the risk of developing breast cancer [ 8 ] or CRC [ 9 ]. The protection observed in these studies could be associated with the high levels of antioxidants present in the plant, which can neutralize free radicals, reduce oxidative DNA damage, and prevent mutation-driven tumorigenesis [ 10 ]. For several years, our group has been working on the development and characterization of a yerba mate extract (YMe). We demonstrated that YMe has a high phenolic content and antioxidant activity. The identified bioactive compounds suggest that this extract is a biosource of health-promoting phenolic compounds and natural antioxidants. YMe showed a noticeable antiproliferative activity against colon and breast tumor cells. In addition, the extract suppressed cell adhesion, migration, and invasion. These results indicate that yerba mate could be able to modulate key cellular functions during metastatic development. Furthermore, YMe exerts in vivo antiangiogenic and antitumor effects [ 11 , 12 ]. Despite growing evidence of the antitumor activity of yerba mate, its potential chemopreventive role in CRC remains poorly investigated. Moreover, no studies to date have comparatively evaluated this effect in male and female animals using experimental models of colon carcinogenesis. Therefore, the main objective of this study was to evaluate the chemopreventive properties of YMe in a DMH-induced colon carcinogenesis model using both male and female BALB/c mice. To our knowledge, this is the first study to investigate the effects of long-term yerba mate supplementation on colon carcinogenesis in both sexes. Materials and Methods Preparation of YMe YMe was generated by aqueous extraction as described in our previous work [ 11 ]. Briefly, Ilex paraguariensis leaves were macerated at 95°C for 1 h and then, the mixture was concentrated until 25° Brix, using maltodextrin (MD, Mathiensen S.A., Argentina) as an encapsulating agent. The resulting solution was incorporated into a pilot scale spray dryer (Galaxie, model 1612). The powder was collected and stored in polyethylene bags at room temperature and protected from light. Prior to use, YMe solution was prepared by dissolving 10 g of powder in 1000 mL tap water and filtered using a sterile 0.22 µm membrane filter. The extract was standardized to main polyphenols previously identified by RP-HPLC method: chlorogenic acid (66.3 mg/g dry sample), rutin (6.783 mg/g dry sample), gallic acid (6.665 mg/g dry sample), caffeic acid (0.533 mg/g dry sample), and quercetin (0.229 mg/g dry sample) [ 11 ]. Animals Four-week-old pathogen-free male and female BALB/c mice weighing approximately 18 g were purchased from the Comparative Medicine Center of ICiVet-Litoral (UNL-CONICET, Santa Fe, Argentina). All animals were housed and maintained under standardized conditions (25°C, 60% relative humidity, 12 h light/12 h dark cycle) at the Animal Facility of National University of Quilmes. Food and water were provided ad libitum , and general health status of the animals was monitored daily. The experimental protocol was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL-UNQ) at the National University of Quilmes. Experimental Design All animals were acclimated for one week before starting the experiment. A total of 48 mice, 24 females and 24 males, were weighed and randomly assigned by sex into three treatment groups, with eight mice per group. Normal control group: animals received only drinking water and weekly intraperitoneal (i.p.) injections of saline solution. MD + DMH group: animals received MD (the vehicle of the extract), as their only source of drinking water, along with i.p. injections of DMH. YMe + DMH group: animals received YMe as their only source of drinking water, along with i.p. injections of DMH. Mice in the DMH-treated groups received i.p. injections of 1,2-dimethylhydrazine dihydrochloride (Sigma-Aldrich, D161802) in saline solution at a dosage of 20 mg/kg body weight, once per week for 14 weeks. YMe (1.6 g/kg body weight/day) or MD solution as a vehicle control were administered to the animals through the drinking water four weeks prior to the first DMH injection, and treatments were extended until the end of the experiment (Fig. 1 ). Body weight was recorded weekly throughout the protocol. Growth rate was calculated as the difference between final and initial body weight divided by the total number of days. Weight gain was calculated as the difference between final and initial body weight. The experiment concluded at week 32, corresponding to 28 weeks after the first DMH administration. Macroscopic Analysis of the Colon At the end of the experimental protocol, all mice were euthanized by cervical dislocation. A complete autopsy was performed and all visceral organs were examined for any gross pathological changes. The entire colon was removed, its length recorded (approximately 10 cm), then longitudinally opened, rinsed with PBS, and divided into three sections (ascending, transverse and descending). Each section was fixed flat between two sheets of filter paper in 10% buffered formalin overnight. The presence of macroscopic lesions in different regions of the colon was analyzed and recorded using a stereomicroscope. Histological Analysis Tissues from normal, as well as lesion regions of the colon, were collected, fixed in 10% paraformaldehyde in PBS, embedded in paraffin, sectioned at 5 µm and mounted on glass slides. Paraffin sections were stained with hematoxylin and eosin (H&E) for routine histological evaluation. Stained slides were examined under a light microscope at 40× magnification and assessed by a specialist in pathological anatomy. The colorectal mucosa was histopathologically classified into four main categories. Normal mucosa was defined as colonic tissue without significant alterations. Hyperplastic polyps were characterized by glandular hyperplasia within the colonic mucosa. Tubular adenomas exhibited tubular glandular epithelial proliferation, while tubulo-villous adenomas were defined by the presence of tubulo-villous glandular epithelial proliferation with moderate to high-grade dysplasia. For analytical purposes, tissues classified as normal included normal mucosa and glandular hyperplasia, whereas neoplastic tissues comprised tubular and tubulo-villous adenomas. The percentage of mice with neoplastic or normal tissue was calculated as the number of mice exhibiting each condition divided by the total number of mice in the corresponding treatment group. Statistical analysis All data analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software, San Diego, California, USA). The normality of the data was assessed using the Kolmogorov–Smirnov test. Results are presented as mean ± standard deviation (SD). Differences in final body weight and body weight gain were analyzed using one-way ANOVA followed by Tukey’s post hoc test. The incidence of neoplastic lesions was evaluated using the chi-square (χ²) test for proportions. A p -value of < 0.05 was considered statistically significant. Results Body Weight of Animals No general signs of toxicity were observed in mice treated with YMe throughout the 32-week experimental protocol, indicating that the extract was well tolerated. A total of six mice died during the study: in female groups, one from the control group and two from the YMe + DMH group; in male groups, two from the control group and one from the YMe + DMH group. Complete necropsies were not performed due to advanced postmortem changes. Body weight variation was recorded weekly over the course of the experiment. Table 1 summarizes the weight gain and growth rate for both female and male mice in each experimental group. Table 1 Effects of YMe and DMH on the body weight of female and male BALB/c mice Control MD + DMH YMe + DMH Female Male Female Male Female Male Initial Weight (g) 17.3 ± 1.02 21.15 ± 1.06 17.4 ± 1.23 19.85 ± 1.34 17.6 ± 1.17 19.25 ± 1.77 Final Weight (g) 26.24 ± 2.09 32.87 ± 3.45 a 26.85 ± 2.24 27.81 ± 1.67 b 25.42 ± 1.98 26.50 ± 3.15 b Weight Gain (g) 8.91 ± 2.09 11.71 ± 3.09 a 9.44 ± 2.24 7.96 ± 1.68 b 7.79 ± 1.98 7.25 ± 2.14 b Growth Rate 0.039 ± 0.009 0.053 ± 0.014 0.042 ± 0.010 0.036 ± 0.008 0.034 ± 0.009 0.032 ± 0.010 Data are presented as mean ± SD (n = 8 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Different superscript letters indicate statistically significant differences between groups ( p < 0.01) In females, neither DMH injections nor long-term YMe administration caused alterations in weight gain or growth rate compared to the healthy control group. Additionally, no significant differences in final body weight were observed among female treatment groups. In contrast, DMH-treated male mice, whether receiving YMe or MD (vehicle control), showed significantly lower final body weights compared to the normal control group. Macroscopic Assessment of Colonic Lesions After 32 weeks of treatment with YMe or MD, colons from all experimental groups were examined under a stereomicroscope. Lesions were identified and counted in different regions of the colon (ascending, transverse, and descending). In both female and male mice, lesions were observed exclusively in the descending colon, and they presented a polyp-like appearance (Fig. 2 ). In DMH-exposed female mice treated with YMe, no macroscopic lesions were detected, resembling the findings in healthy control animals. In contrast, two female mice from the MD + DMH group exhibited lesions measuring approximately 3–4 mm. In male mice, colonic lesions were observed in both DMH-exposed groups: two animals in MD + DMH group and three animals in YMe + DMH group. Histopathological Study The effect of YMe on DMH-induced histopathological alterations in the colon was evaluated in both female and male mice. The lesions generated by DMH were examined in longitudinal sections of the colon and classified as normal mucosa, hyperplasia, tubular adenoma, or tubulo-villous adenoma. In female mice, YMe treatment significantly inhibited adenoma formation compared to the MD + DMH group ( p < 0.001) (Fig. 3 a). Notably, all female mice treated with YMe preserved normal colonic mucosal architecture, similar to healthy control animals (Table 2 ; Fig. 4 a,d). In contrast, adenomas were observed in two female mice from the MD + DMH group. One lesion exhibited a tubular crypt arrangement, consistent with a tubular adenoma, while the other was classified as a tubulo-villous adenoma based on the presence of digitiform projections characteristic of the villous component (Table 2 ; Fig. 4 b,c). Table 2 Effects of YMe and DMH on the colonic architecture of female and male BALB/c mice Experimental Group N o of mice Histological classification Female Male Control 1 Normal colonic mucosa Normal colonic mucosa 2 Normal colonic mucosa Normal colonic mucosa 3 Normal colonic mucosa Normal colonic mucosa 4 Normal colonic mucosa Normal colonic mucosa 5 Normal colonic mucosa Normal colonic mucosa 6 Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp 7 Normal colonic mucosa. Mild glandular hyperplasia † 8 † † MD + DMH 1 Normal colonic mucosa Normal colonic mucosa 2 Normal colonic mucosa Normal colonic mucosa 3 Normal colonic mucosa Normal colonic mucosa 4 Normal colonic mucosa. Mild glandular hyperplasia Normal colonic mucosa 5 Normal colonic mucosa. Mild glandular hyperplasia Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp 6 Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp Tubular adenoma 7 Tubular adenoma Tubulo-villous adenoma with low-grade dysplasia 8 Tubulo-villous adenoma with low-grade dysplasia Tubulo-villous adenoma with moderate-grade dysplasia YMe + DMH 1 Normal colonic mucosa Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp 2 Normal colonic mucosa Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp 3 Normal colonic mucosa Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp 4 Normal colonic mucosa Normal colonic mucosa. Focal glandular hyperplasia. Hyperplastic polyp 5 Normal colonic mucosa Tubulo-villous adenoma with moderate-grade dysplasia 6 Normal colonic mucosa. Mild glandular hyperplasia Tubulo-villous adenoma with high-grade dysplasia 7 † Tubulo-villous adenoma with high-grade dysplasia 8 † † † Animal died before the end of the experiment In male mice, both tubular and tubulo-villous adenomas were observed in the MD + DMH and YMe + DMH groups (Table 2 ; Fig. 4 f–h), with incidences of 38% and 43%, respectively. No statistically significant difference was found between these groups ( p = 0.46) (Fig. 3 b). As expected, healthy control animals exhibited normal colonic mucosa (Fig. 4 e). These histopathological findings indicate that YMe effectively prevented colonic carcinogenesis in female mice but did not exert a significant protective effect in males. Discussion Despite the recent advances in cancer treatment, there is a growing opportunity and need for alternative strategies such as chemoprevention, the pharmacological approach to suppress, prevent or delay the initial phases of carcinogenesis or the progression of premalignant cells to invasive disease by using non-toxic agents [ 13 ]. Experimental models of carcinogenesis in rodents are useful tools for studying the chemopreventive effects of new therapeutic agents, including plant-derived extracts. In this work, we investigated the effect of YMe in cancer chemoprevention using a DMH-induced colon carcinogenesis model in both male and female BALB/c mice. It has been proposed that, in chemoprevention strategies, long-term treatments should be administered prior to cancer initiation and progression. This period is defined as a “window of opportunity” in the management of the disease. Translating this concept to the preclinical setting, the experimental protocol in the current study was designed to simulate the window period for chemoprevention by administering 1.6 g/kg body weight/day of YMe to the animals four weeks before chemical carcinogenesis induction with DMH. Treatment was then continued until the end of the experiment. Since the temperature of the yerba mate infusion has been associated with the development of certain types of cancer [ 14 ], the extract was provided at room temperature as the sole source of drinking fluid, thus excluding this variable considered as a confounding factor. We have previously demonstrated that this dose of YMe significantly inhibits angiogenesis and tumor growth in a murine colon cancer model [ 11 ], reduces tumor metastasis and increases survival in breast cancer models [ 12 ]. Similar doses (1.0 and 2.0 g/kg/day) of yerba mate administered during four weeks have been evaluated in previous studies by other authors, showing a reduction of systemic inflammation biomarkers in high-fat diet-fed Wistar rats, as well as protective effects against H 2 O 2 -induced DNA damage in mice, without significant differences between the two doses [ 15 , 16 ]. Only two studies to date have evaluated the chemopreventive properties of yerba mate in animal models of chemically induced colon carcinogenesis [ 17 , 18 ]. Zapaterini et al ., investigated the potential beneficial effects of mate tea-like infusions on the post-initiation stage of mammary and colon carcinogenesis induced by 7,12-dimethylbenz(a)anthracene and DMH in female Swiss mice [ 17 ]. In their study, the animals received mate tea-like infusions only after the initiation period with the carcinogens. Therefore, the experimental protocol did not include a window period for chemopreventive intervention. In the study by Puangpraphant et al ., although a chemopreventive intervention was simulated by administering yerba mate tea to the animals two weeks before chemical carcinogenesis induction, the main objective was to assess the ability of yerba mate to prevent colonic inflammation as an early step in colon carcinogenesis [ 18 ]. In the present study, YMe exhibited a preventive effect on the carcinogenesis process in female mice, as none of the animals in YMe + DMH group developed colonic lesions in comparison with MD + DMH group, in which most of the tumors found were classified as adenomas. However, in male animals, tubular and tubulo-villous adenomas were observed in both DMH-treated groups, regardless of YMe administration. The chemopreventive effects of YMe may be attributed to its content of phenolic compounds. It has been reported that polyphenols are able to interfere with cancer initiation, promotion, and progression, thereby acting as chemopreventive agents [ 19 ]. In a previous study, we identified five major polyphenolic compounds in YMe using RP-HPLC. Chlorogenic acid was the most abundant with 66.3 mg/g of dry sample, followed by rutin, gallic acid, caffeic acid, and quercetin [ 11 ]. The antioxidant and chemopreventive effects of chlorogenic acid in colorectal cancer have been the focus of numerous research studies. Its mechanisms of action encompass a range of cellular processes, including cell cycle arrest, apoptosis induction, the migration and invasion of cancer cells, and the modulation of key signaling pathways, alongside their roles in oxidative stress and inflammation management [ 20 ]. Chlorogenic acid has been reported to attenuate the early-stage of chemically DMH-induced mouse colon carcinogenesis by decreasing epithelial cell proliferation and increasing apoptosis in colonic crypts, and also by reducing the levels of proinflammatory cytokines in the colon [ 21 ]. Although chlorogenic acid is the main polyphenol in yerba mate, the health benefits attributed to this plant are not due solely to the content of this single compound, but also to the combined action of other phytochemicals (such as caffeic acid, rutin and quercetin), which may act additively or synergistically to exert its pharmacological effects. Quercetin and its derivatives interact with numerous molecular targets involved in the initiation and promotion/progression phases of CRC carcinogenesis. Among the anticarcinogenic activities of quercetin, the most notable described in CRC are inhibition of cellular proliferation, induction of apoptosis, reduction in tumor size, decrease in number of tumor nodule, suppression of metastasis, decrease in inflammation and antioxidant activity [ 22 ]. Rutin is also considered a promising natural product in colon cancer prevention. Many studies revealed that rutin exerts anti-inflammatory, anticarcinogenic, antiproliferative and antimetastatic effects through the regulation of several signaling pathways such as Wnt/β-catenin, PI3K/Akt, JAK/STAT, MAPK, p53, and NF-κB [ 23 ]. The DMH-induced colon carcinogenesis model used in this work is an experimental system that exhibits similar characteristics to sporadic forms of CRC in humans. The preneoplastic lesions and histopathological observations of DMH-induced colon tumors may provide typical understanding to accurately identify and interpret alterations that occur in the colonic mucosa when evaluating natural compounds with potential anticarcinogenic activities [ 24 ]. Susceptibility to DMH-induced colon carcinogenesis depends on several factors such as the strain of rodent, age, sex, dosage and route of DMH administration and the induction time. The genetic background of laboratory animals is a significant component of organ-specific carcinogenesis. The susceptibility to colon carcinogens differs greatly among the genetically defined inbred mouse strains [ 5 ]. It has been reported that BALB/c mouse strain is susceptible to DMH [ 25 ]. The success of carcinogenesis induction in this strain is described as moderate. Although repetitive exposure to DMH generates a large number of lesions in the crypts, few adenomas per animal colon are observed. This is consistent with our results, we found that only 25% of female mice and 38% of males in the MD + DMH group developed adenomas. Aberrant crypt foci (ACF) have been widely used as a surrogate biomarker of colon carcinogenesis [ 26 ]. However, some studies reported a disagreement between ACF and tumors, probably due to the heterogeneous nature of ACF, comprising both hyperplastic and dysplastic lesions [ 27 , 28 ]. Since ACF represent one of the first steps in the colon carcinogenesis process, these lesions could be found in the majority of animals exposed to the carcinogen. In the present study, the number of macroscopic tumors was used as an endpoint to evaluate the chemopreventive effect of YMe. Importantly, we confirmed these macroscopic lesions by histopathological analysis, and they were classified as tubular and tubulo-villous adenoma, i.e ., neoplastic tissue. The use of distinct endpoints could explain the differences in the incidence rate found in our study with respect to other published data. Susceptibility to DMH-induced colon carcinogenesis depending on the sex of the animals has been previously reported [ 29 ]. We found a higher incidence of colonic adenomas in male mice compared with female mice. These differences could be explained by the influence of sex hormones in the colon carcinogenesis process. It has been demonstrated in chemically induced carcinogenesis experimental models that castration reduces the number of adenomas in the colon of male rats and mice. Subsequent testosterone replacement reversed this number of colonic adenomas. However, ovariectomy and replacement of female hormones had no measurable effect on colonic adenomagenesis [ 30 ]. In contrast, Weyant et al . demonstrated that the ovariectomy in female C57BL/6J mice with a germ-line APC gene mutation increased the number intestinal adenomas compared to non-ovariectomized females. In addition, the supplementation of 17β-estradiol to ovariectomized female mice reduced the number of adenomas to the same level as non-ovariectomized mice [ 31 ]. In another animal study, the colon carcinogenesis incidence was higher in male mice than females in AOM/dextran sodium sulfate-induced colon cancer model, and the treatment with 17β-estradiol reduced the development of CRC [ 32 ]. These findings support the protective role of both endogenous and exogenous estrogens in the prevention of carcinogenesis. Our results demonstrated sex-specific differences in the chemopreventive properties of YMe, with significant protection observed in females but not in males. These differences may be attributed to variations in metabolism or interactions with sex hormones. However, further studies, including hormone profiling and analysis of estrogen receptor expression are necessary to confirm these observations. Some polyphenols such as flavonoids, lignans and stilbenes are considered phytoestrogens. These biologically active plant compounds are structurally similar to endogenous steroidal estrogens found in humans and animals, and act in a comparable manner by binding to estrogen receptors [ 33 ]. Estrogens may exert anti-tumor effects by selectively activating pro-apoptotic pathways mediated by estrogen receptor beta (ERβ), suppressing inflammatory signals and modulating the tumor microenvironment [ 34 ]. Considering the sex-related differences in chemoprevention observed in our study, it is plausible to hypothesize that flavonoids such as quercetin and rutin, present in YMe, could modulate key processes involved in the early stages of tumorigenesis. These flavonoids may exhibit selective estrogen receptor modulating activities, thus functioning as a primary chemopreventive agent. We have previously demonstrated that YMe inhibits colon cancer cell proliferation through the induction of apoptosis, both in vitro and in vivo [ 11 ]. Whether this mechanism also contributes to the sex-specific chemopreventive effects observed in the present study remains to be elucidated. Additionally, other biological processes, including the modulation of inflammatory responses and oxidative stress, should be investigated to better understand the molecular basis of these differential outcomes. Conclusions This study demonstrated the chemopreventive properties of YMe against chemically induced colon carcinogenesis in female mice, with no protective effect observed in males. These findings suggest that the effect of YMe may be influenced by sex-dependent biological factors, potentially involving the modulatory role of sex hormones in colorectal carcinogenesis. The observed sex-specific response underscores the importance of considering sex as a biological variable in chemoprevention studies. Further investigations are warranted to elucidate the molecular mechanisms underlying these effects. Overall, our results highlight the potential of yerba mate as a natural anticancer agent and support its relevance in the development of dietary-based chemopreventive strategies. Declarations Competing Interests The authors declare that they have no competing interests. Ethics Approval All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health. The experimental protocol was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL-UNQ) at the National University of Quilmes. Funding This research was supported by the National University of Quilmes (Grant No. 1287/19), the National Cancer Institute (Grant No. 827–1567/18), and the National Institute of Yerba Mate (Grant No. 827 − 0650/19). Author Contribution H.L, RS.GL, N.L, and LG.C performed the experiments and analyzed the data. A.S carried out the histopathological analysis. H.L drafted the manuscript. N.L and LG.C contributed to writing the manuscript and editing the figures. HG.F and DF.A conceived and designed the experiments. All authors participated in the revision of the manuscript and approved the submitted version. Humberto Lamdan and Rocio S. Garcia-Lazaro contributed equally to this work. Acknowledgement The authors wish to thank Victoria Romero for her valuable technical assistance in the care and maintenance of the experimental animals. Data Availability The datasets generated during the current study are available from the corresponding author upon reasonable request. References Naeem A, Hu P, Yang M, Zhang J, Liu Y, Zhu W, et al. Natural Products as Anticancer Agents: Current Status and Future Perspectives. Molecules. 2022;27:8367. https://doi.org/10.3390/molecules27238367 . José MFB, Machado RP, Araujo PAB, Speretta GF. Physiological effects of yerba maté (Ilex paraguariensis): a systematic review. Nutr Rev. 2023;81:1163–79. https://doi.org/10.1093/nutrit/nuac109 . Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74:229–63. https://doi.org/10.3322/caac.21834 . 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Toxicol Res (Camb). 2020;9:2–18. https://doi.org/10.1093/toxres/tfaa004 . Moen CJ, van der Valk M, Bird RP, Hart A, Demant P. Different genetic susceptibility to aberrant crypts and colon adenomas in mice. Cancer Res. 1996;56:2382–6. Femia AM, Caderni G. Rodent models of colon carcinogenesis for the study of chemopreventive activity of natural products. Planta Med. 2008;74:1602–7. https://doi.org/10.1055/s-2008-1074577 . Shih CK, Chiang W, Kuo ML. Effects of adlay on azoxymethane-induced colon carcinogenesis in rats. Food Chem Toxicol. 2004;42:1339–47. https://doi.org/10.1016/j.fct.2004.03.011 . Papanikolaou A, Wang QS, Papanikolaou D, Whiteley HE, Rosenberg DW. Sequential and morphological analyses of aberrant crypt foci formation in mice of differing susceptibility to azoxymethane-induced colon carcinogenesis. Carcinogenesis. 2000;21:1567–72. Moon RC, Fricks CM. Influence of gonadal hormones and age on 1,2-dimethylhydrazine-induced colon carcinogenesis. 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A review on phytoestrogens: Current status and future direction. Phyther Res. 2023;37:3097–120. https://doi.org/10.1002/ptr.7861 . Caiazza F, Ryan EJ, Doherty G, Winter DC, Sheahan K. Estrogen receptors and their implications in colorectal carcinogenesis. Front Oncol. 2015;5:19. https://doi.org/10.3389/fonc.2015.00019 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7593777","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":543436094,"identity":"6b0071ef-4374-4a42-ab34-be4e455cebf1","order_by":0,"name":"Humberto Lamdan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYBACAwYGNgiLHYh5GBjkQOwDDwhrAVLMEC3GYC0JpGhJbAAJ49NiLpGd9uDjnj9yDMzMTze8qbmTPj/s8EOgLXZyug3YtVjOyN1uOOOZgTEDM5vZzTnHnuVuvJ1mANSSbGx2AIfDbuRuk+Y5YJDYwMxgdpuH7XDuxtkJIC0HErfh0/IHrIX9222ef4fTDWenfyCshQGshcfsNm/b4QR56RwCtpx5u92w54CxMRszT9nNuX2HDTdI5xQcSDDA45fjudse/DggJ8fP3r7txptvh+XlZ6dv/vChwk4OlxY4YIMbAlZpQEA5CpBvIEX1KBgFo2AUjAQAAGJaZCJKBnnmAAAAAElFTkSuQmCC","orcid":"","institution":"National University of Quilmes","correspondingAuthor":true,"prefix":"","firstName":"Humberto","middleName":"","lastName":"Lamdan","suffix":""},{"id":543436095,"identity":"b5aa1fce-2848-4601-9c0c-7abe59cfef0c","order_by":1,"name":"Rocio S. Garcia-Lazaro","email":"","orcid":"","institution":"National University of Quilmes","correspondingAuthor":false,"prefix":"","firstName":"Rocio","middleName":"S.","lastName":"Garcia-Lazaro","suffix":""},{"id":543436096,"identity":"3bd81f4e-9312-4853-a491-e1185b22fb6a","order_by":2,"name":"Norailys Lorenzo","email":"","orcid":"","institution":"National University of Quilmes","correspondingAuthor":false,"prefix":"","firstName":"Norailys","middleName":"","lastName":"Lorenzo","suffix":""},{"id":543436097,"identity":"81ff431e-003f-4a24-85e9-effa48570e29","order_by":3,"name":"Lorena G. 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DMH (20 mg/kg body weight) was administered intraperitoneally once per week during 14 weeks. Normal control animals received only drinking water and weekly intraperitoneal injections of saline solution. Mice in the MD+DMH and YMe+DMH groups received MD (the vehicle of the extract) or YMe (1.6 g/kg body weight/day), respectively, instead of water, starting 4 weeks before the first DMH injection. Treatments continued until the end of the experiment. All mice were sacrificed 28 weeks after the initial DMH administration.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7593777/v1/c612c7530ff1d16e298fa806.jpeg"},{"id":96240138,"identity":"b33f68df-023d-4023-ae2e-a240d62a3fdc","added_by":"auto","created_at":"2025-11-19 07:08:28","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120589,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic analysis of colon sections in the different experimental groups. Representative images of descending colon from female and male mice. In female mice in the YMe+DMH group, no macroscopic lesions were observed, showing a preserved colonic mucosa similar to healthy controls. Polyps induced by DMH are indicated with black arrows\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7593777/v1/bc951e054201de2adc9d22cd.jpeg"},{"id":96240453,"identity":"6325f3a7-ff3b-44be-876e-5331d4330160","added_by":"auto","created_at":"2025-11-19 07:08:55","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":364945,"visible":true,"origin":"","legend":"\u003cp\u003eIncidence of neoplastic and normal colonic tissue in the different experimental groups. Bar graphs show the percentage of animals exhibiting either preserved normal colonic architecture or neoplastic lesions in each treatment group, for female \u003cem\u003e(a)\u003c/em\u003e and male \u003cem\u003e(b)\u003c/em\u003e mice. Statistical differences between groups were assessed using the chi-square (χ²) test for proportions. *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; \u003cem\u003ens\u003c/em\u003e: not statistically significant\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7593777/v1/5f4df8ffe0c51b71aea6bfab.jpeg"},{"id":95854811,"identity":"c4d762d1-d4a6-4289-8ecb-f09b5f95c1df","added_by":"auto","created_at":"2025-11-13 16:37:17","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":320359,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological analysis of colonic tissue in the different experimental groups. Hematoxylin and eosin staining of colonic sections from female \u003cem\u003e(a–d)\u003c/em\u003e and male \u003cem\u003e(e–h)\u003c/em\u003emice. Images were taken at 40× magnification. Scale bar: 500 µm. \u003cem\u003ea, d, e\u003c/em\u003e: normal colonic mucosa; \u003cem\u003eb, f\u003c/em\u003e: tubular adenoma; \u003cem\u003ec, g, h\u003c/em\u003e: tubulo-villous adenoma\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7593777/v1/d6bf35a59b83cbb1cfdf1db5.jpeg"},{"id":96452806,"identity":"cc916a73-5822-4b80-8e1f-6f423402fbb2","added_by":"auto","created_at":"2025-11-21 09:46:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1725804,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7593777/v1/c672e720-25b9-42b0-8876-9ea78c246cd3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chemopreventive effect of a yerba mate extract on 1,2-Dimethylhydrazine-induced colon carcinogenesis in BALB/c mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince ancient times, plants have played a very relevant role in the treatment of human diseases. The search for antitumor agents of plant origin began in the early 1940s and today approximately 50% of approved anticancer drugs are derived from this source [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Yerba mate (\u003cem\u003eIlex paraguariensis St. Hill. Aquifoliaceae\u003c/em\u003e) is a plant native to the subtropical region of South America. The infusion from \u003cem\u003eIlex paraguariensis\u003c/em\u003e, which is a very popular beverage in Argentina, southern Brazil, Paraguay and Uruguay, is called \u0026ldquo;mate\u0026rdquo;. This plant contains a large amount of bioactive compounds that contribute to their health benefits. It has been reported that yerba mate has a high antioxidant capacity, as well as anti-inflammatory and immunomodulatory effects, which correlates positively with the amount of polyphenols present in the plant [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The action of these secondary metabolites can reduce the incidence of certain chronic diseases, including cancer.\u003c/p\u003e\u003cp\u003eColorectal cancer (CRC) is a recurrent malignant neoplasm of the digestive system and the second leading cause of cancer deaths worldwide [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. According to the clinical and histopathological data published to date, the majority of these neoplasms arise from adenomas, \u003cem\u003ei.e\u003c/em\u003e., pre-existing benign tumors. In consequence, CRC is currently considered a cancer that can be \u0026ldquo;prevented\u0026rdquo; by avoiding the adenoma-carcinoma sequence. During the last few decades, researchers have focused their studies on dietary factors due to the implication of diet in the carcinogenesis and progression of CRC [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChemically induced carcinogenesis models have contributed significantly to the understanding of the mechanisms underlying the development of CRC. 1,2- Dimethylhydrazine (DMH) is a potent carcinogen widely used to induce tumors in the descending colon in experimental animals. DMH is metabolized in the liver to form azoxymethane (AOM) and methylazoxymethanol (MAM). The reactive metabolite, MAM, is transported to the colon via bile or blood to generate the alkylating methyldiazonium ion. The latter is capable of methylating macromolecules of colonic epithelial cells resulting in oxidative stress. In consequence, reactive oxygen species can induce DNA damage, which in turn can cause mutations that initiate malignant transformation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe role of yerba mate in the development of cancer was under discussion for many years with controversial results. Many epidemiological studies from late 1980s to early 2000s indicated that yerba mate consumption was positively correlated with the risk of developing certain types of cancer, including oral cavity, oropharyngeal, bladder and lung cancer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, these studies did not take into account other risk variables such as the temperature of the infusion, the simultaneous consumption of tobacco and alcohol, age, sex, and other dietary or lifestyle factors that may affect carcinogenesis and tumor progression. More recently, based on evidence, the scientific community accepts that yerba mate has an antitumor effect. An inverse correlation has been reported between high consumption of mate and the risk of developing breast cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] or CRC [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The protection observed in these studies could be associated with the high levels of antioxidants present in the plant, which can neutralize free radicals, reduce oxidative DNA damage, and prevent mutation-driven tumorigenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor several years, our group has been working on the development and characterization of a yerba mate extract (YMe). We demonstrated that YMe has a high phenolic content and antioxidant activity. The identified bioactive compounds suggest that this extract is a biosource of health-promoting phenolic compounds and natural antioxidants. YMe showed a noticeable antiproliferative activity against colon and breast tumor cells. In addition, the extract suppressed cell adhesion, migration, and invasion. These results indicate that yerba mate could be able to modulate key cellular functions during metastatic development. Furthermore, YMe exerts \u003cem\u003ein vivo\u003c/em\u003e antiangiogenic and antitumor effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite growing evidence of the antitumor activity of yerba mate, its potential chemopreventive role in CRC remains poorly investigated. Moreover, no studies to date have comparatively evaluated this effect in male and female animals using experimental models of colon carcinogenesis. Therefore, the main objective of this study was to evaluate the chemopreventive properties of YMe in a DMH-induced colon carcinogenesis model using both male and female BALB/c mice. To our knowledge, this is the first study to investigate the effects of long-term yerba mate supplementation on colon carcinogenesis in both sexes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of YMe\u003c/h2\u003e\u003cp\u003eYMe was generated by aqueous extraction as described in our previous work [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, \u003cem\u003eIlex paraguariensis\u003c/em\u003e leaves were macerated at 95\u0026deg;C for 1 h and then, the mixture was concentrated until 25\u0026deg; Brix, using maltodextrin (MD, Mathiensen S.A., Argentina) as an encapsulating agent. The resulting solution was incorporated into a pilot scale spray dryer (Galaxie, model 1612). The powder was collected and stored in polyethylene bags at room temperature and protected from light. Prior to use, YMe solution was prepared by dissolving 10 g of powder in 1000 mL tap water and filtered using a sterile 0.22 \u0026micro;m membrane filter. The extract was standardized to main polyphenols previously identified by RP-HPLC method: chlorogenic acid (66.3 mg/g dry sample), rutin (6.783 mg/g dry sample), gallic acid (6.665 mg/g dry sample), caffeic acid (0.533 mg/g dry sample), and quercetin (0.229 mg/g dry sample) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eFour-week-old pathogen-free male and female BALB/c mice weighing approximately 18 g were purchased from the Comparative Medicine Center of ICiVet-Litoral (UNL-CONICET, Santa Fe, Argentina). All animals were housed and maintained under standardized conditions (25\u0026deg;C, 60% relative humidity, 12 h light/12 h dark cycle) at the Animal Facility of National University of Quilmes. Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e, and general health status of the animals was monitored daily. The experimental protocol was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL-UNQ) at the National University of Quilmes.\u003c/p\u003e\n\u003ch3\u003eExperimental Design\u003c/h3\u003e\n\u003cp\u003eAll animals were acclimated for one week before starting the experiment. A total of 48 mice, 24 females and 24 males, were weighed and randomly assigned by sex into three treatment groups, with eight mice per group. Normal control group: animals received only drinking water and weekly intraperitoneal (i.p.) injections of saline solution. MD\u0026thinsp;+\u0026thinsp;DMH group: animals received MD (the vehicle of the extract), as their only source of drinking water, along with i.p. injections of DMH. YMe\u0026thinsp;+\u0026thinsp;DMH group: animals received YMe as their only source of drinking water, along with i.p. injections of DMH.\u003c/p\u003e\u003cp\u003eMice in the DMH-treated groups received i.p. injections of 1,2-dimethylhydrazine dihydrochloride (Sigma-Aldrich, D161802) in saline solution at a dosage of 20 mg/kg body weight, once per week for 14 weeks. YMe (1.6 g/kg body weight/day) or MD solution as a vehicle control were administered to the animals through the drinking water four weeks prior to the first DMH injection, and treatments were extended until the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBody weight was recorded weekly throughout the protocol. Growth rate was calculated as the difference between final and initial body weight divided by the total number of days. Weight gain was calculated as the difference between final and initial body weight. The experiment concluded at week 32, corresponding to 28 weeks after the first DMH administration.\u003c/p\u003e\n\u003ch3\u003eMacroscopic Analysis of the Colon\u003c/h3\u003e\n\u003cp\u003eAt the end of the experimental protocol, all mice were euthanized by cervical dislocation. A complete autopsy was performed and all visceral organs were examined for any gross pathological changes. The entire colon was removed, its length recorded (approximately 10 cm), then longitudinally opened, rinsed with PBS, and divided into three sections (ascending, transverse and descending). Each section was fixed flat between two sheets of filter paper in 10% buffered formalin overnight. The presence of macroscopic lesions in different regions of the colon was analyzed and recorded using a stereomicroscope.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eHistological Analysis\u003c/h3\u003e\n\u003cp\u003eTissues from normal, as well as lesion regions of the colon, were collected, fixed in 10% paraformaldehyde in PBS, embedded in paraffin, sectioned at 5 \u0026micro;m and mounted on glass slides. Paraffin sections were stained with hematoxylin and eosin (H\u0026amp;E) for routine histological evaluation. Stained slides were examined under a light microscope at 40\u0026times; magnification and assessed by a specialist in pathological anatomy. The colorectal mucosa was histopathologically classified into four main categories. Normal mucosa was defined as colonic tissue without significant alterations. Hyperplastic polyps were characterized by glandular hyperplasia within the colonic mucosa. Tubular adenomas exhibited tubular glandular epithelial proliferation, while tubulo-villous adenomas were defined by the presence of tubulo-villous glandular epithelial proliferation with moderate to high-grade dysplasia. For analytical purposes, tissues classified as normal included normal mucosa and glandular hyperplasia, whereas neoplastic tissues comprised tubular and tubulo-villous adenomas. The percentage of mice with neoplastic or normal tissue was calculated as the number of mice exhibiting each condition divided by the total number of mice in the corresponding treatment group.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software, San Diego, California, USA). The normality of the data was assessed using the Kolmogorov\u0026ndash;Smirnov test. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences in final body weight and body weight gain were analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. The incidence of neoplastic lesions was evaluated using the chi-square (χ\u0026sup2;) test for proportions. A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eBody Weight of Animals\u003c/h2\u003e\u003cp\u003eNo general signs of toxicity were observed in mice treated with YMe throughout the 32-week experimental protocol, indicating that the extract was well tolerated. A total of six mice died during the study: in female groups, one from the control group and two from the YMe\u0026thinsp;+\u0026thinsp;DMH group; in male groups, two from the control group and one from the YMe\u0026thinsp;+\u0026thinsp;DMH group. Complete necropsies were not performed due to advanced postmortem changes.\u003c/p\u003e\u003cp\u003eBody weight variation was recorded weekly over the course of the experiment. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the weight gain and growth rate for both female and male mice in each experimental group.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of YMe and DMH on the body weight of female and male BALB/c mice\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eMD\u0026thinsp;+\u0026thinsp;DMH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eYMe\u0026thinsp;+\u0026thinsp;DMH\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eInitial Weight (g)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e17.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.15\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e19.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e17.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e19.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFinal Weight (g)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e26.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32.87\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e26.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e27.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e25.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e26.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.15 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eWeight Gain (g)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.71\u0026thinsp;\u0026plusmn;\u0026thinsp;3.09 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e9.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e7.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGrowth Rate\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.053\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.042\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.036\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e0.034\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.032\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;8 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Different superscript letters indicate statistically significant differences between groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01)\u003c/p\u003e\u003cp\u003eIn females, neither DMH injections nor long-term YMe administration caused alterations in weight gain or growth rate compared to the healthy control group. Additionally, no significant differences in final body weight were observed among female treatment groups. In contrast, DMH-treated male mice, whether receiving YMe or MD (vehicle control), showed significantly lower final body weights compared to the normal control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMacroscopic Assessment of Colonic Lesions\u003c/h2\u003e\u003cp\u003eAfter 32 weeks of treatment with YMe or MD, colons from all experimental groups were examined under a stereomicroscope. Lesions were identified and counted in different regions of the colon (ascending, transverse, and descending). In both female and male mice, lesions were observed exclusively in the descending colon, and they presented a polyp-like appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In DMH-exposed female mice treated with YMe, no macroscopic lesions were detected, resembling the findings in healthy control animals. In contrast, two female mice from the MD\u0026thinsp;+\u0026thinsp;DMH group exhibited lesions measuring approximately 3\u0026ndash;4 mm. In male mice, colonic lesions were observed in both DMH-exposed groups: two animals in MD\u0026thinsp;+\u0026thinsp;DMH group and three animals in YMe\u0026thinsp;+\u0026thinsp;DMH group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHistopathological Study\u003c/h2\u003e\u003cp\u003eThe effect of YMe on DMH-induced histopathological alterations in the colon was evaluated in both female and male mice. The lesions generated by DMH were examined in longitudinal sections of the colon and classified as normal mucosa, hyperplasia, tubular adenoma, or tubulo-villous adenoma.\u003c/p\u003e\u003cp\u003eIn female mice, YMe treatment significantly inhibited adenoma formation compared to the MD\u0026thinsp;+\u0026thinsp;DMH group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotably, all female mice treated with YMe preserved normal colonic mucosal architecture, similar to healthy control animals (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,d). In contrast, adenomas were observed in two female mice from the MD\u0026thinsp;+\u0026thinsp;DMH group. One lesion exhibited a tubular crypt arrangement, consistent with a tubular adenoma, while the other was classified as a tubulo-villous adenoma based on the presence of digitiform projections characteristic of the villous component (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of YMe and DMH on the colonic architecture of female and male BALB/c mice\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExperimental Group\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN\u003csup\u003eo\u003c/sup\u003e of mice\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eHistological classification\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia. Hyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia.\u003c/p\u003e\u003cp\u003eHyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eMild glandular hyperplasia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eMD\u0026thinsp;+\u0026thinsp;DMH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eMild glandular hyperplasia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eMild glandular hyperplasia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia.\u003c/p\u003e\u003cp\u003eHyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia. Hyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTubular adenoma\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTubular adenoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTubulo-villous adenoma with low-grade dysplasia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTubulo-villous adenoma with low-grade dysplasia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTubulo-villous adenoma with moderate-grade dysplasia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eYMe\u0026thinsp;+\u0026thinsp;DMH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia. Hyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia. Hyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia. Hyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eFocal glandular hyperplasia. Hyperplastic polyp\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTubulo-villous adenoma with moderate-grade dysplasia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNormal colonic mucosa.\u003c/p\u003e\u003cp\u003eMild glandular hyperplasia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTubulo-villous adenoma with high-grade dysplasia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTubulo-villous adenoma with high-grade dysplasia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026dagger;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u0026dagger; Animal died before the end of the experiment\u003c/p\u003e\u003cp\u003eIn male mice, both tubular and tubulo-villous adenomas were observed in the MD\u0026thinsp;+\u0026thinsp;DMH and YMe\u0026thinsp;+\u0026thinsp;DMH groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef\u0026ndash;h), with incidences of 38% and 43%, respectively. No statistically significant difference was found between these groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.46) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). As expected, healthy control animals exhibited normal colonic mucosa (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). These histopathological findings indicate that YMe effectively prevented colonic carcinogenesis in female mice but did not exert a significant protective effect in males.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite the recent advances in cancer treatment, there is a growing opportunity and need for alternative strategies such as chemoprevention, the pharmacological approach to suppress, prevent or delay the initial phases of carcinogenesis or the progression of premalignant cells to invasive disease by using non-toxic agents [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eExperimental models of carcinogenesis in rodents are useful tools for studying the chemopreventive effects of new therapeutic agents, including plant-derived extracts. In this work, we investigated the effect of YMe in cancer chemoprevention using a DMH-induced colon carcinogenesis model in both male and female BALB/c mice.\u003c/p\u003e\u003cp\u003eIt has been proposed that, in chemoprevention strategies, long-term treatments should be administered prior to cancer initiation and progression. This period is defined as a \u0026ldquo;window of opportunity\u0026rdquo; in the management of the disease. Translating this concept to the preclinical setting, the experimental protocol in the current study was designed to simulate the window period for chemoprevention by administering 1.6 g/kg body weight/day of YMe to the animals four weeks before chemical carcinogenesis induction with DMH. Treatment was then continued until the end of the experiment. Since the temperature of the yerba mate infusion has been associated with the development of certain types of cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the extract was provided at room temperature as the sole source of drinking fluid, thus excluding this variable considered as a confounding factor.\u003c/p\u003e\u003cp\u003eWe have previously demonstrated that this dose of YMe significantly inhibits angiogenesis and tumor growth in a murine colon cancer model [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], reduces tumor metastasis and increases survival in breast cancer models [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similar doses (1.0 and 2.0 g/kg/day) of yerba mate administered during four weeks have been evaluated in previous studies by other authors, showing a reduction of systemic inflammation biomarkers in high-fat diet-fed Wistar rats, as well as protective effects against H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced DNA damage in mice, without significant differences between the two doses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOnly two studies to date have evaluated the chemopreventive properties of yerba mate in animal models of chemically induced colon carcinogenesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Zapaterini \u003cem\u003eet al\u003c/em\u003e., investigated the potential beneficial effects of mate tea-like infusions on the post-initiation stage of mammary and colon carcinogenesis induced by 7,12-dimethylbenz(a)anthracene and DMH in female Swiss mice [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In their study, the animals received mate tea-like infusions only after the initiation period with the carcinogens. Therefore, the experimental protocol did not include a window period for chemopreventive intervention.\u003c/p\u003e\u003cp\u003eIn the study by Puangpraphant \u003cem\u003eet al\u003c/em\u003e., although a chemopreventive intervention was simulated by administering yerba mate tea to the animals two weeks before chemical carcinogenesis induction, the main objective was to assess the ability of yerba mate to prevent colonic inflammation as an early step in colon carcinogenesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, YMe exhibited a preventive effect on the carcinogenesis process in female mice, as none of the animals in YMe\u0026thinsp;+\u0026thinsp;DMH group developed colonic lesions in comparison with MD\u0026thinsp;+\u0026thinsp;DMH group, in which most of the tumors found were classified as adenomas. However, in male animals, tubular and tubulo-villous adenomas were observed in both DMH-treated groups, regardless of YMe administration.\u003c/p\u003e\u003cp\u003eThe chemopreventive effects of YMe may be attributed to its content of phenolic compounds. It has been reported that polyphenols are able to interfere with cancer initiation, promotion, and progression, thereby acting as chemopreventive agents [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In a previous study, we identified five major polyphenolic compounds in YMe using RP-HPLC. Chlorogenic acid was the most abundant with 66.3 mg/g of dry sample, followed by rutin, gallic acid, caffeic acid, and quercetin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe antioxidant and chemopreventive effects of chlorogenic acid in colorectal cancer have been the focus of numerous research studies. Its mechanisms of action encompass a range of cellular processes, including cell cycle arrest, apoptosis induction, the migration and invasion of cancer cells, and the modulation of key signaling pathways, alongside their roles in oxidative stress and inflammation management [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Chlorogenic acid has been reported to attenuate the early-stage of chemically DMH-induced mouse colon carcinogenesis by decreasing epithelial cell proliferation and increasing apoptosis in colonic crypts, and also by reducing the levels of proinflammatory cytokines in the colon [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough chlorogenic acid is the main polyphenol in yerba mate, the health benefits attributed to this plant are not due solely to the content of this single compound, but also to the combined action of other phytochemicals (such as caffeic acid, rutin and quercetin), which may act additively or synergistically to exert its pharmacological effects. Quercetin and its derivatives interact with numerous molecular targets involved in the initiation and promotion/progression phases of CRC carcinogenesis. Among the anticarcinogenic activities of quercetin, the most notable described in CRC are inhibition of cellular proliferation, induction of apoptosis, reduction in tumor size, decrease in number of tumor nodule, suppression of metastasis, decrease in inflammation and antioxidant activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRutin is also considered a promising natural product in colon cancer prevention. Many studies revealed that rutin exerts anti-inflammatory, anticarcinogenic, antiproliferative and antimetastatic effects through the regulation of several signaling pathways such as Wnt/β-catenin, PI3K/Akt, JAK/STAT, MAPK, p53, and NF-κB [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe DMH-induced colon carcinogenesis model used in this work is an experimental system that exhibits similar characteristics to sporadic forms of CRC in humans. The preneoplastic lesions and histopathological observations of DMH-induced colon tumors may provide typical understanding to accurately identify and interpret alterations that occur in the colonic mucosa when evaluating natural compounds with potential anticarcinogenic activities [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Susceptibility to DMH-induced colon carcinogenesis depends on several factors such as the strain of rodent, age, sex, dosage and route of DMH administration and the induction time. The genetic background of laboratory animals is a significant component of organ-specific carcinogenesis. The susceptibility to colon carcinogens differs greatly among the genetically defined inbred mouse strains [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It has been reported that BALB/c mouse strain is susceptible to DMH [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The success of carcinogenesis induction in this strain is described as moderate. Although repetitive exposure to DMH generates a large number of lesions in the crypts, few adenomas per animal colon are observed. This is consistent with our results, we found that only 25% of female mice and 38% of males in the MD\u0026thinsp;+\u0026thinsp;DMH group developed adenomas. Aberrant crypt foci (ACF) have been widely used as a surrogate biomarker of colon carcinogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, some studies reported a disagreement between ACF and tumors, probably due to the heterogeneous nature of ACF, comprising both hyperplastic and dysplastic lesions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Since ACF represent one of the first steps in the colon carcinogenesis process, these lesions could be found in the majority of animals exposed to the carcinogen. In the present study, the number of macroscopic tumors was used as an endpoint to evaluate the chemopreventive effect of YMe. Importantly, we confirmed these macroscopic lesions by histopathological analysis, and they were classified as tubular and tubulo-villous adenoma, \u003cem\u003ei.e\u003c/em\u003e., neoplastic tissue. The use of distinct endpoints could explain the differences in the incidence rate found in our study with respect to other published data.\u003c/p\u003e\u003cp\u003eSusceptibility to DMH-induced colon carcinogenesis depending on the sex of the animals has been previously reported [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We found a higher incidence of colonic adenomas in male mice compared with female mice. These differences could be explained by the influence of sex hormones in the colon carcinogenesis process. It has been demonstrated in chemically induced carcinogenesis experimental models that castration reduces the number of adenomas in the colon of male rats and mice. Subsequent testosterone replacement reversed this number of colonic adenomas. However, ovariectomy and replacement of female hormones had no measurable effect on colonic adenomagenesis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In contrast, Weyant \u003cem\u003eet al\u003c/em\u003e. demonstrated that the ovariectomy in female C57BL/6J mice with a germ-line APC gene mutation increased the number intestinal adenomas compared to non-ovariectomized females. In addition, the supplementation of 17β-estradiol to ovariectomized female mice reduced the number of adenomas to the same level as non-ovariectomized mice [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In another animal study, the colon carcinogenesis incidence was higher in male mice than females in AOM/dextran sodium sulfate-induced colon cancer model, and the treatment with 17β-estradiol reduced the development of CRC [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These findings support the protective role of both endogenous and exogenous estrogens in the prevention of carcinogenesis.\u003c/p\u003e\u003cp\u003eOur results demonstrated sex-specific differences in the chemopreventive properties of YMe, with significant protection observed in females but not in males. These differences may be attributed to variations in metabolism or interactions with sex hormones. However, further studies, including hormone profiling and analysis of estrogen receptor expression are necessary to confirm these observations.\u003c/p\u003e\u003cp\u003eSome polyphenols such as flavonoids, lignans and stilbenes are considered phytoestrogens. These biologically active plant compounds are structurally similar to endogenous steroidal estrogens found in humans and animals, and act in a comparable manner by binding to estrogen receptors [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Estrogens may exert anti-tumor effects by selectively activating pro-apoptotic pathways mediated by estrogen receptor beta (ERβ), suppressing inflammatory signals and modulating the tumor microenvironment [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Considering the sex-related differences in chemoprevention observed in our study, it is plausible to hypothesize that flavonoids such as quercetin and rutin, present in YMe, could modulate key processes involved in the early stages of tumorigenesis. These flavonoids may exhibit selective estrogen receptor modulating activities, thus functioning as a primary chemopreventive agent.\u003c/p\u003e\u003cp\u003eWe have previously demonstrated that YMe inhibits colon cancer cell proliferation through the induction of apoptosis, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Whether this mechanism also contributes to the sex-specific chemopreventive effects observed in the present study remains to be elucidated. Additionally, other biological processes, including the modulation of inflammatory responses and oxidative stress, should be investigated to better understand the molecular basis of these differential outcomes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated the chemopreventive properties of YMe against chemically induced colon carcinogenesis in female mice, with no protective effect observed in males. These findings suggest that the effect of YMe may be influenced by sex-dependent biological factors, potentially involving the modulatory role of sex hormones in colorectal carcinogenesis. The observed sex-specific response underscores the importance of considering sex as a biological variable in chemoprevention studies. Further investigations are warranted to elucidate the molecular mechanisms underlying these effects. Overall, our results highlight the potential of yerba mate as a natural anticancer agent and support its relevance in the development of dietary-based chemopreventive strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003ch2\u003eEthics Approval\u003c/h2\u003e\u003cp\u003e All animal procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health. The experimental protocol was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL-UNQ) at the National University of Quilmes.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by the National University of Quilmes (Grant No. 1287/19), the National Cancer Institute (Grant No. 827\u0026ndash;1567/18), and the National Institute of Yerba Mate (Grant No. 827\u0026thinsp;\u0026minus;\u0026thinsp;0650/19).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.L, RS.GL, N.L, and LG.C performed the experiments and analyzed the data. A.S carried out the histopathological analysis. H.L drafted the manuscript. N.L and LG.C contributed to writing the manuscript and editing the figures. HG.F and DF.A conceived and designed the experiments. All authors participated in the revision of the manuscript and approved the submitted version.\u003c/p\u003e\u003cp\u003eHumberto Lamdan and Rocio S. Garcia-Lazaro contributed equally to this work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to thank Victoria Romero for her valuable technical assistance in the care and maintenance of the experimental animals.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNaeem A, Hu P, Yang M, Zhang J, Liu Y, Zhu W, et al. Natural Products as Anticancer Agents: Current Status and Future Perspectives. 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Front Oncol. 2015;5:19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fonc.2015.00019\u003c/span\u003e\u003cspan address=\"10.3389/fonc.2015.00019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Colon carcinogenesis, Ilex paraguariensis, Polyphenols, Yerba mate","lastPublishedDoi":"10.21203/rs.3.rs-7593777/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7593777/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eYerba mate (\u003cem\u003eIlex paraguariensis St. Hill. Aquifoliaceae\u003c/em\u003e) is a plant native to South America with numerous medicinal properties, mainly attributed to its high content of phenolic compounds. Several studies have shown that these bioactive compounds can reduce the risk of developing chronic diseases, including cancer. In the present study, we evaluated the chemopreventive effect of yerba mate extract (YMe) against 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in BALB/c mice of both sexes. Colon tumors were induced by weekly intraperitoneal injections of DMH (20 mg/kg body weight) for 14 weeks. YMe (1.6 g/kg body weight/day) or maltodextrin (vehicle control) was administered to the animals via drinking water, starting four weeks before the first DMH injection, and continued until the end of the experiment. Presence of colonic lesions and incidence of animals with neoplastic tissue was confirmed by macroscopic examination and histopathological analysis. YMe treatment completely inhibited the development of tumor lesions in the colon of female mice, all of which exhibited a normal colonic mucosal architecture. In contrast, tubular and tubulo-villous adenomas were observed in male mice of both DMH-treated groups, regardless of YMe administration. These findings demonstrate a sex-specific chemopreventive effect of YMe against DMH-induced colon carcinogenesis, with significant protection observed in females but not in males. Further experiments are needed to elucidate the molecular mechanisms underlying these effects. Our results suggest that yerba mate could be used as a natural agent for colorectal cancer prevention and support its potential role in dietary-based chemopreventive strategies.\u003c/p\u003e","manuscriptTitle":"Chemopreventive effect of a yerba mate extract on 1,2-Dimethylhydrazine-induced colon carcinogenesis in BALB/c mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 16:37:08","doi":"10.21203/rs.3.rs-7593777/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6e98e263-4e07-4012-b498-b560b465fd92","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T21:38:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-13 16:37:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7593777","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7593777","identity":"rs-7593777","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}