Grape Seed Extract Dose-Responsively Decreases Disease Severity in a Rat Model of Mucositis; Concomitantly Enhancing Chemotherapeutic Effectiveness in Colon Cancer Cells

Objective Mucositis is a serious disorder of the gastrointestinal tract that results from cancer chemotherapy. We investigated the effects of increasing grape seed extract doses on the severity of chemotherapy in a rat model and its coincident impact on chemotherapeutic effectiveness in colon cancer cells. Design Female Dark Agouti rats were gavaged with grape seed extract (400–1000 mg/kg) or water (day 3–11) and were injected intraperitoneally with 5-Fluorouracil (150 mg/kg) or saline (control) on day 9 to induce mucositis. Daily metabolic data were collected and rats were sacrificed on day 12. Intestinal tissues were collected for histological and myeloperoxidase analyses. Caco-2 cell viability was examined in response to grape seed extract in combination with 5-Fluorouracil by 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide) assay. Results Compared with 5-Fluorouracil controls, grape seed extract (400–1000 mg/kg) significantly decreased the histological damage score (P<0.05) in the jejunum. Grape seed extract (1000 mg/kg) increased jejunal crypt depth by 25% (P<0.05) in 5-Fluorouracil treated rats compared to 5-Fluorouracil controls, and attenuated the 5-Fluorouracil -induced reduction of mucosal thickness (25%, P<0.05). Grape seed extract (600 mg/kg) decreased myeloperoxidase activity by 55% (P<0.01) compared to 5-Fluorouracil controls. Grape seed extract was more effective at ameliorating 5-Fluorouracil induced intestinal injury, with effects most pronounced in the proximal jejunum. Grape seed extract (10–25 ug/mL) significantly enhanced the growth-inhibitory effects of 5-Fluorouracil by 26% (P<0.05) in Caco-2 cells and was more potent than 5-Fluorouracil at 50–100 µg/mL. Conclusion Grape seed extract may represent a new therapeutic option to decrease the symptoms of intestinal mucositis while concurrently impacting on the viability of colon cancer cells.


Introduction
Mucositis is a serious, debilitating consequence of cancer therapy, which significantly reduces quality of life in cancer patients [1]. Mucositis is a painful condition associated with inflammation and ulceration of the gastrointestinal tract; most commonly affecting the mucosa of the mouth (oral mucositis) and small intestine (intestinal mucositis). Intestinal mucositis is characterized by reduced enterocyte proliferation and increased apoptotic rate of crypt cells, resulting in malabsorption and disrupted barrier function [2,3]. Symptoms of mucositis include intense pain, diarrhoea, nausea, vomiting and anorexia. Often there is an increased risk of bacterial infection with associated mortality and morbidity [1]. Sometimes, gastrointestinal toxicity may lead to a reduction, or even termination, of the chemotherapy regimen [4]. Due to this, the chemotherapy dose administered to cancer patients often be sub-optimal; hence new regimens that reduce side-effects, maintaining efficacy are sought. Current mucositis treatments are largely ineffective as they target only the symptoms, but not the pathogenesis of the condition [5]. Thus, it is important to seek new alternative treatments which not only target mucositis but also enhance chemotherapeutic action without compromising the well-being of the patient. At present, the optimal combination of agents which could enhance both chemotherapeutic cytotoxicity against cancer cells and have minimal impact on normal cells, has not yet been determined.
Grape seed extract (GSE) is widely consumed as a dietary supplement on the basis of its potent anti-oxidant [6], antiinflammatory [7] and purported, anti-neoplastic [8] properties. Procyanidins (PCs) are a class of polyphenolic compounds composed of flavan-3-ol subunits (oligomers and polymers) [9]. PCs are widely found in other food sources such as tea, apples and red wine and are believed to be the key bioactive constituents in GSE [10,11]. Several studies have reported that the absorption and bioavailability of PCs in the gut is dependent upon their chemical structure and degree of polymerization [12]. PCs (degree of polymerization = 7 or higher) are retained in the intestinal tract, thereby increasing contact time with gut enterocytes to promote intestinal health [10]. Studies examining GSE in combination with 5-Fluorouracil (5-FU) in normal animals are limited. In addition, no studies have been published examining the combination of 5-FU and GSE in colon cancer models.
Previously, we demonstrated in a preliminary study that GSE (400 mg/kg) was able to reduce intestinal damage both in rat models of intestinal mucositis [13] and ulcerative colitis [14]. However, the dose required to achieve maximal therapeutic benefit, dose-responsiveness and safety of GSE remained undefined. Accordingly, we investigated GSE across a range of doses for its potential to optimally and safely reduce the severity of intestinal mucositis in a rat model. Increasing doses of GSE further prevented 5-FU-induced mucositis damage, and these treatments were well tolerated by the animals as no metabolic changes were observed compared to the healthy controls. In addition, we also investigated the effects of the combination of GSE with 5-FU chemotherapy on colonic neoplasia in an in vitro model compared to the effectiveness of 5-FU alone. The combination of GSE and 5-FU further enhanced toxicity in colon cancer cells.

Grape seed extract (GSE) preparation
Grape seed extract was kindly donated by Tarac Techologies (North Adelaide, South Australia, batch no: 02VIN03) and stored in an air-tight, light-resistant pack until being dissolved in distilled water prior to use. The GSE was derived from condensed tannin made from Australian white wine marc (residual skins and seeds from winemaking). The nutrition profile of GSE is listed in Table 1. The GSE utilized in the current study was obtained from the same source with the same batch number to that described by Cheah et al [13]. The polyphenolic content was measured previously by Folin-ciocalteau assay [13], and the chemical profile was quantified by phloroglucinolysis described below.

Folin-ciocalteau assay
The total phenolic content of GSE was previously quantified by Folin-ciocalteau assay [13]. Briefly, GSE samples were added in triplicate to non-sterile 96 well plates and incubated with Folinciocalteu reagent for 5 min. Sodium bicarbonate solution (7.5% w/v) was added and further incubated for 4 h in the dark. The plate was read at 740 nm by a spectrometer (MultiskanH Spectrum, Therma Electron Corporation, Vantaa, Finland) using Skanit software 2.2. Catechin standards were prepared from 1 mg/mL stock (1/2 serial dilutions) and used to generate a calibration curve. Data were analysed using GraphPad Prism version 4.0 for windowsH (GraphPad Software, San Diego, CA, USA) and expressed in mg/mL catechin equivalents.

Quantification of procyanidins (PCs) in GSE by phloroglucinolysis
The procyanidin profile of GSE was characterized by phloroglucinolysis which determines the subunit composition, mean degree of polymerization (mDP) and galloylation of PCs. Phloroglucinolysis was performed according to a previously described method [15]. GSE was dissolved in methanol (10 mg/ mL, v/v) and 25 mL of GSE was added to an equal volume of phloroglucinol solution (0.2 N HCL in methanol, 100 g/L of phloroglucinol and 20 g/L of ascorbic acid). The phloroglucinolysis reaction was carried out at 50uC for 25 min and analyzed by reverse phase-high pressure liquid chromatography (RP-HPLC) using (2)-epicatechin as quantitative standard [15].

Ethic Statements
This study followed the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and was approved by both the Animal Care and Ethics Committees of the Children, Youth and Women's Health Service and the University of Adelaide (AE:777-3-2011).

Animal studies
Female Dark Agouti rats (100-140 g, n = 64) were housed in individual metabolic cages (Tecniplast, Exton, PA, USA) in a temperature-controlled room (22uC) with a light-dark cycle of 12 h. Rats were given ad libitum access to water and food (18% casein-based diet) [16] in the Animal Care Facility of the Children, Youth and Women's Health Service, North Adelaide, South Australia. Polyphenol 6 (%) 43 1 The nutrition profile of GSE is represented as quantity/100 g. 2 The phenolic content was obtained from Folin-ciocalteau (FC) assays. doi:10.1371/journal.pone.0085184.t001 Rats were randomly allocated to 8 groups (n = 8): Water+Saline injection; GSE 400 mg/kg+Saline injection; GSE 600 mg/kg+Saline injection; GSE 1000 mg/kg+Saline injection; Water+5-FU injection (5-Fluorouracil: 150 mg/kg); GSE 400 mg/kg+5-FU injection; GSE 600 mg/kg+5-FU injection; and GSE 1000 mg/ kg+5-FU injection. Rats were acclimatized in metabolism cages from day 0-2 and then gavaged with 1 mL GSE dissolved in water (400 mg/kg, 600 mg/kg or 1000 mg/kg) or water from day 3-11. At day 9, all rats were intraperitoneally injected with either 5-FU or saline (controls). Daily measurements of body weight, food and water intake, and urine and faecal output were recorded. Rats were sacrificed by CO 2 asphyxiation followed by cervical dislocation on day 12. All visceral organs were weighed and discarded. The lengths and weights of the gastrointestinal organs (duodenum, small intestine and colon) were recorded. Representative samples (2 cm) of gastrointestinal organs were collected and fixed in 10% buffered formalin for histological analyses, while four cm samples were snap frozen in liquid nitrogen and stored at 280uC for biochemical analysis.

C-sucrose breath test (SBT)
The SBT is an indirect measure of intestinal sucrase activity and was performed according to the method described by Tooley et al. [17] In brief, rats were oro-gastrically gavaged with 1 mL of sucrose solution containing 13 C (250 mg/kg) and breath samples were collected at 15 min intervals for 120 min. Breath samples were analyzed for 13 CO 2 content by isotope ratio mass spectrometry (IRMS) equipped with a V410 data collection system (Europa Scientific, ABCA 20/20, Crewe, United Kingdom). The data were expressed as percentage cumulative dose at 90 min (%CD90) by calculating the change in breath 13 CO 2 levels from baseline for each time point of breath collection throughout the period of interval sampling. SBT determinations were performed at day 3 (before GSE treatment), day 9 (before 5-FU injection) and day 12 (before kill).

Myeloperoxidase (MPO) assay
Small intestinal tissue samples (4 cm) of jejunum, junction of jejunum and ileum (JI) and ileum were thawed on ice and homogenized with 1.5 mL of phosphate buffer (10 mM, pH 6.1) for 60 seconds until the solution was homogenous. The homogenates were kept frozen at 280uC until required.
MPO is an enzyme present in the intracellular granules of neutrophils, acting as an acute inflammation marker. The level of MPO in the small intestine was determined by a slight modification of the assay described by Krawisz et al. [18] Tissue homogenates were thawed on ice and centrifuged at 13000 g for 13 min. The supernatant was discarded and cell pellets were resuspended in hexadecyltrimethyl ammonium bromide (0.5%, pH 6.0). The samples were vortexed for 2 min and further centrifuged at 13000 g for 3 min. Supernatants were reacted with o-dianisidine and absorbance measured at 450 nm at 1 min intervals for a period of 15 min using a microplate reader (Sunrise Microplate Reader, Tecan Austria GmbH, Grodig, Austria). MPO activity was expressed as units MPO activity per gram of tissue.

Histological analyses
Gut tissue samples (2 cm) were embedded in paraffin wax and 4 mm sections were stained with haemotoxylin and eosin. The overall histological disease severity score (ODS) of intestinal sections was rated semi-quantitatively (0-3) based on 11 independent histological criteria according to a protocol described by Howarth et al. [19] Villus heights and crypt depths (40 villi and 40 crypts per section) were determined in the small intestinal sections including jejunum, junction of jejunum and ileum (JI) and ileum as described in Howarth et al. [19] The combined measurement of villus heights and crypt depths provided an approximation of total mucosal thickness in each small intestinal specimen. All microscope-based analyses were performed in a blinded fashion using a light microscope (Nikon, ProgResHCS, Tokyo, Japan) and image ProPlus software version 5.1 (Media Cybernetics, Silver Spring MD, USA).

Cell Culture
The human colon cancer cell line, Caco-2 was obtained from the American Type Culture Collection (ATCC, Manassas, USA). Caco-2 cells were maintained at 37uC in a humidified incubator with 5% CO 2 -95% air, and 90% relative humidity in Dulbecco's Mass conversion based on % recovery of procyanidin by phloroglucinolysis based on the gravimetric mass. 2 Mean degree of polymerization. 3 Estimated molecular mass based on subunit composition from phloroglucinolysis. 4 Percent composition of terminal and extension subunits (in moles) with the following subunit abbreviations: (-P), phloroglucinol adduct of extension subunit; C, (+)catechin; EC, (2)-epicatechin; ECG, (2)-epicatechin-3-O-gallate. doi:10.1371/journal.pone.0085184.t002 Table 3. Effects of increasing doses of grape seed extract (GSE; mg/kg) on cumulative body weight change, food and water intake, urine and faecal output in saline-injected rats over day 3-9.

Cell viability
The inhibition of Caco-2 cells viability was determined by 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide) (MTT) assay according to a previously described method by Huynh-Delerme et al. [20] Cells (5610 3 cells/well) were seeded on 96-well tissue culture plates for 48 hrs to allow attachment. GSE was prepared in dimethyl sulfoxide (DMSO) and diluted with DMEM and further filter-sterilized through a 0.22 mM filter (Millipore, South Australia, Australia). In all experiments, the concentration of DMSO in control and treated samples was less than 0.025%. After 48 h, culture medium was replaced with serum free media containing GSE at different concentrations (mg/ mL) and 5-FU (mM) and further incubated for either 24 or 48 h. MTT solution was prepared in Dulbecco's Phosphate Buffered Saline (1 mg/mL) and sterile-filtered to remove any biological contaminants. Next, 50 mL of MTT solution was added to each well and further incubated at 37uC for 4 h. Medium was replaced with 100 mL of DMSO to extract the formazan product. Plates were placed on a shaking incubator for 15 min and read by spectrometer at 570 nm. Data were expressed as number of viable cells as a percentage of control cells treated with serum free medium only.

Statistical analyses
Statistical analyses were conducted using PASW 18 (SPSS, Inc., Chicago, IL, USA) and XLSTAT version 2011.4.02 (Addinsoft SARL, France). All parametric data including bodyweight, daily metabolic data, SBT, MPO, villus height and crypt depth and cell viability were compared using one-way analysis of variance (ANOVA) with a Tukey's post-hoc test. The overall disease severity score (ODS) was compared by a Kruskal-Wallis test with a Mann Table 4. Effects of increasing doses of grape seed extract (GSE; mg/kg) on cumulative body weight change, food and water intake, urine and faecal output in saline-injected rats and in 5-Fluorouracil (5-FU) injected rats from day 10-12. Whitney U-test to identify significance between groups. Data were considered significant at P,0.05.

Daily metabolic parameters and bodyweight
Oral administration of GSE (400, 600 or 1000 mg/kg) between days 3 and 9 did not significantly affect body weight, food or water intake and urine or faecal output compared to rats receiving water (Table 3). 5-FU injection significantly increased water intake and urine output and reduced body weight, food intake and faecal output compared to rats receiving saline injection between days 10 and 12 (Table 4). GSE 600 and 1000 mg/kg in 5-FU treated rats significantly returned faecal output (P,0.01), back towards the values for normal saline-injected control rats.

Visceral organs
5-FU injection significantly reduced thymus weight by 51% (P,0.001) and spleen weight by 20% (P,0.01) compared to saline-injected rats. Whilst GSE did not prevent any of the 5-FUinduced changes in thymus and spleen weight, none of the GSE doses tested impacted negatively on visceral organ weights (Table 5) nor gastrointestinal organ weights and lengths (Table 6) in healthy animals. However, GSE 1000 mg/kg significantly increased stomach weight by 13% (P,0.05) compared to normal controls.

Sucrose breath test (SBT)
5-FU injection significantly (P,0.001) decreased the SBT (%CD90) by 70% compared to values in water+saline treated rats, indirectly indicating 5-FU injection had disrupted brush border sucrase activity (Figure 1). There were no significant differences in %CD90 among any of the 5-FU treated rats receiving GSE compared to 5-FU treated-control rats. In addition, no significant differences in %CD90 were observed between GSE and water treatment, implying none of the GSE doses had demonstrably impacted on brush border sucrase activity in healthy rats ( Figure 1).

Myeloperoxidase activity (MPO)
Following 5-FU injection, there was a significant (P,0.001) increase in MPO activity in the proximal jejunum, junction of jejunum and ileum (JI) and ileum of water+5-FU treated rats (1092%, 357% and 297% respectively) compared to water+saline treatment ( Figure 2). GSE 600 mg/kg significantly (P,0.01) reduced MPO activity by 55% in the JI compared to water+5-FU treated rats ( Figure 2B). There was no significant difference in MPO activity between GSE and water treatment in healthy rats (Figure 2), indicating that GSE administration did not affect MPO activity in healthy animals.

Effects of 5-FU on viability of Caco-2 cells
The dose responses of 5-FU (0-100,000 mM) on Caco-2 cells for 24 h and 48 h are illustrated in Figure 7. 5-FU doses were also tested at 72 h (Data not shown). The cell viability of Caco-2 cells was inhibited by 5-FU in a time-and dose-dependent manner. At 24 h, 5-FU at 100 mM significantly reduced viability in Caco-2 cells to 88% (P,0.05) of control values and a further reduction of cell viability was observed to 70% (P,0.05) of control values at 48 h. A 100 mM concentration of 5-FU was selected for the next experiment because this dose was able to reduce Caco-2 cell viability (70-85%) reflecting gastrointestinal toxicity commonly observed in cancer patients following chemotherapy.

Discussion
The present study represents the first report of GSE doseresponsively reducing severity of mucositis. Our findings suggest that higher doses of GSE are more effective at reducing the severity indicators of intestinal mucositis in rats and that these GSE-induced effects are largely dose-dependent and more evident in the proximal jejunum compared to the distal small intestine.
Injection of 5-FU impacts on the small intestine to a greater extent than the large intestine, presumably due to the greater cell turnover rate in the more proximal regions of the gut [3]. Our results are in agreement with previous studies [18,19,21], in which the 5-FU mucositis model resulted in severe intestinal injury 72 h after the induction of mucositis. This damage was characterized by a reduction of intestinal brush border enzyme activities [21], increased neutrophil infiltration [22], increased disease severity score [23] and decreased mucosal thickness [13]. Consistent with earlier studies [13], blunting of the villi and disorganization of crypts (location of stem cells) were the primary events associated with severe mucositis. The jejunum is the maximal site of injury induced by chemotherapy and the impact becomes less pronounced in the more distal regions of the small intestine [13]. This was demonstrated in the current study in which less injury was observed in terms of MPO activity (neutrophil infiltration), villus height and mucosal thickness at the distal end of the small intestine The bioavailability of PCs in the gut system has been well documented in other studies [24]. The unique polymerized structure of PCs inhibits absorption across the small intestine, as they adhere to the gut mucosa [25]. Tsang et al. [26] detected larger forms of PCs in the small intestine of rats up to 12 h after ingestion. Thus, an accumulation of relatively high PC concentrations can occur in the gut lumen to protect the intestinal barrier. In the current study, higher doses of GSE (1000 mg/kg) were effective at maintaining crypt depth and mucosal thickness in the jejunal region, with most values approaching the values of healthy  controls. Furthermore, the current study also showed improvement of fecal output (less severe diarrhoea) in chemotherapytreated rats receiving the higher dose of GSE (600 mg/kg and 1000 mg/kg), suggesting reduced disruption of the mucosal lining of the small intestine.
Although GSE in the current study was more effective in the jejunum, the site of major intestinal injury, bioactivity was reduced in the distal small intestine. This may have been due to degraded bioactive components reaching the distal region of the small intestine [27,28]. The cleavage, absorption and metabolism of GSE is important to identify the fate of bioactive compounds in GSE. In future studies it will be necessary to identify the form, size and bioactivity of procyanidins from GSE responsible for promoting intestinal health using in vitro and in vivo models of intestinal absorption. Additionally, future studies could examine protection of GSE, possibly by microencapsulation, or via suppository application, to better target GSE and improve its bioavailability in the more distal regions of the bowel. Due to the complexity of GSE content, it would be difficult to determine which factors are responsible for the observed bioactivity. For this reason, GSE, rather than alternative protein source such as bovine serum was used as its own control. Administration of GSE on normal animals allowed more precise comparison with GSEtreated rats receiving 5-FU chemotherapy.
Interest in GSE has been primarily due to its high antioxidant content. GSE is a more potent radical scavenger than other known anti-oxidants such as vitamin C and E [29]. In the present study, the partial reduction in acute inflammation by GSE, as indicated by the decrease of MPO activity, and reduction in lymphocyte infiltration recorded by the disease severity score analysis, could strengthen the potential role of GSE as a potent anti-oxidant and anti-inflammatory agent. A number of studies have described GSE as an anti-inflammatory agent. For example, GSE has been reported to reduce the expression of pro-inflammatory cytokines (TNF-a and IL-6) in mesenteric lymph nodes [30], rat plasma [31] and carrageenan-induced paw edema in rats [32]. The reduction of these activities may represent a consequence of GSE and its ability to prevent NF-kB activation and subsequently reduce the activation of nitric oxide and pro-inflammatory cytokines. Thus, inhibition of NF-kB activation may have been a possible mechanism by which GSE reduced mucosal injury and hence mucositis severity, in the current study. Other biomarkers such as inflammatory cytokines in tissue and blood could be measured in future studies to quantify GSE effects on the systemic and mucosal immune system.
PC rich food has been reported to be both beneficial and detrimental to human health due to its ability to interact with proteins (enzymes, toxins, hormones) [10,33]. The current study provides important information on the safety of GSE usage. Oral administration of GSE (400 mg/kg, 600 mg/kg and 1000 mg/kg) for nine days did not induce any deleterious side-effects in healthy animals. The increased of stomach weight in GSE treated rats might be due to indigestible of GSE PCs deposited in the stomach. GSE did not impact negatively on daily metabolic parameters, nor induce any side-effects in the small intestine. Moreover, the sucrose breath test indicated that GSE did not affect small intestinal brush border enzyme activity. These data concur with other studies [34] in which rats ingesting up to 2 g/kg of GSE showed no abnormal metabolic findings or toxicological effects. In the current study, GSE (1000 mg/kg) significantly increased stomach weight in healthy rats. This finding has not been reported previously [35], but could be a reflection of differing rat strains between studies. Thus, histological analyses on rat stomach should be conducted in future studies.
The promising effects of GSE in the mucositis rat model provided the impetus to further investigate its potential impact on the effectiveness of chemotherapy against transformed colonocytes. Recently, various strategies have been developed to counter the development of mucositis (reduced gastrointestinal toxicity) or to enhance the chemotherapeutic activity of 5-FU. To date, a number of strategies to enhance 5-FU efficacy on colon cancer have been investigated although none are clinically available. These include omega 3-fatty acid [36], chloroquine [37], violecin   [38] and ginseng [39] which have been reported to effectively improve 5-FU efficiency at killing cancer cells in vitro compared to the chemotherapy agent acting independently. In the current study, the effects of GSE and 5-FU on Caco-2 cell viability were examined at 24 h and 48 h. This was because 5-FU (100 uM) significantly reduced cell viability to 70-85%, reflecting gastrointestinal toxicity in cancer patients. Moreover, longer time exposure (24-48 h) of cancer cells to both GSE and 5-FU resulted in a further reduction in cell viability. However, the differences between treatments remained relatively unchanged even up to 72 h (data not shown). Importantly, GSE acted synergistically with 5-FU to inhibit Caco-2 cell proliferation.
GSE may act as a potent chemotherapeutic agent as it has been demonstrated to exert selective cytotoxicity against tumour cells compared to normal cells [40,41]. It has been suggested that GSE induces growth inhibition in cancer cells via induction of cell cycle arrest which eventually leads to the induction of caspasedependent apoptosis [8] and disruption of the mitochondrial membrane [42]. Moreover, the current study demonstrated that GSE alone at higher concentration, tended to induce greater growth inhibitory effects on Caco-2 cells compared to 5-FU alone. Thus, the current data support GSE as a promising anti-neoplastic adjunct to cancer treatment. Future in vitro studies including invasion, proliferation and growth analysis should be performed to identify the phenotypic changes of colon cancer cells after GSE treatment.
Although the current in vivo study revealed that GSE only minimally improved parameters of intestinal mucositis (disease severity score), future studies could examine the efficacy of higher doses of GSE or alternatively more highly purified PC compounds. Chemotherapy is likely to progress to a chronic condition. Future studies should investigate GSE effects in rat models over protracted periods of several weeks and months. The promising findings of GSE in the in vitro model also support further studies into the identification of bioactive components of GSE responsible for these effects. The present study was conducted using female Dark Agouti rats which can be manipulated to develop breast cancer [43]. Such studies would facilitate further investigations into GSE and its potential to modify tumour growth. The colon cancer cell line was selected on the basis that it could be translated later to an animal model of colon cancer. The current work represents the synergistic effect of GSE and 5-FU at partially preventing mucositis, whilst reducing Caco-2 cells viability. This will allow us to further investigate GSE for its potential to modify tumour growth with 5-FU by determining its effects on tumour growth such as that induced by azoxymethane in rat model [44].
In conclusion, the current investigation provides the first evidence for GSE to reduce the severity of intestinal mucositis in a dose-responsive manner while enhancing the impact of 5-FU chemotherapy on colon cancer cells. Dietary GSE could be a promising adjunctive approach for combating intestinal mucositis while concurrently potentiating the impact of conventional chemotherapy for colon cancer.