Mitigating Effect of Dietary Dioclea reflexa (Hook F) Seed Inclusion in Experimental Colon Carcinogenesis

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Introduction
Most colorectal cancers (CRCs) arise from benign, noncancerous tumors called polyps, which grow along the inner lining of the colon or rectum.Although the rate of CRC in Africa was relatively lower than in developed countries [1], the age-standardized incidence rate (ASR) for colon cancer in males and females in many countries in Africa has been increasing steadily in recent decades [2], presumably, as a result of increasing adoption of Western diets.Fortunately, this type of cancer is preventable and even curable, if identifed early.Recent estimates suggest that colorectal cancer was one of the leading cancer groups in Africa, being among the top-10 cancer groups in all 54 countries of Africa (Bray and Parkin [2]), and being the third, in terms of incidence in men [3].
Environmental chemical carcinogens are known to induce and promote carcinogenesis through oxidative assault, and antioxidants as contained in certain foods and medicinal plants [4,5] are reputed for their housekeeping roles by mopping up free radicals and reducing oxidative stress, with consequent protective capacity against cell-damaging oxidative processes as in the etiology of diseases such as cancer [6].In these medicinal and food plants, antioxidant polyphenols, an increasingly popular class of substances, are receiving increasing interest from consumers and food manufacturers for their probable roles in the prevention of various diseases associated with oxidative stress, including cancer [7,8].
Te plant, Dioclea refexa (Hook F), belongs to the Fabaceae family.Its seed is popular in eastern and central Nigeria as well as many central African countries as traditional soup thickeners and has been reported to be suitable as a rheology modifer in processed foods [9].Te seed is also traditionally prescribed in the management of pile and related anorectal disorders, including colon carcinogenesis [10], gastrointestinal tract infection [11], and asthmatic treatment [12], while constituents have been demonstrated to have biological activities.Previously, we reported the hypolipidemic, antioxidant, and organ-protective properties of the methanolic extract of this seed in acute and chronic oxidative injuries [9].Building on that report, Oladele Oladimeji et al. [13] have isolated two antioxidant favonoids, named dioclins A and B from the seed, while Balapangu et al. [14] have demonstrated that the acidic eluates of the seed metabolites exhibited profound in vitro antiproliferative efects on breast cancer, Michigan Cancer Foundation-7 (MCF-7) cells.Tus, the present study focused on evaluating the preventive efect of D. refexa seeds in experimental colon carcinogenesis using an animal model, which is considered superior to in vitro experiments, in view of the complex interactions that occur in biological systems.

Plant Collection and Authentication. Seeds of D. refexa
were collected from Achalla village, Anambra State, Nigeria, with geographical (GPS) coordinates of 6 * 20′54′N, 6 * 59′0′E′ in the month of June and identifed at the Botany Department's Herbarium Unit of Ahmadu Bello University, Zaria, Nigeria, where voucher specimen with number 1286 was deposited.Te seeds were dried under shade and pulverized using a laboratory mortar and pestle.Te sieved material was stored in airtight polyethylene bags and kept at room temperature until required.

Ethical Clearance and Care for Experimental Animals.
Four weeks old, apparently healthy male Albino rats of the Wistar strain, weighing between 80 and 100 g, were acquired from the Laboratory Animal Facility of the Department of Physiology, Ahmadu Bello University.While undergoing quarantine, the animals were fed with the basal diet and allowed access to drinking water ad libitum for a period of two weeks to condition.Prior to the commencement of the experiment, rats were fed a basal (normal) diet, maintained under a 12-hour light/dark cycle, weighed, and divided randomly into two replicates of six groups with 7 animals each.All animal experiments were approved by the Ethical Committee of Ahmadu Bello University, Zaria, and were undertaken in accordance and compliance with national and international guidelines (ARRIVE; U.K. Animals Scientifc Procedures and Associated Guidelines Act, 1986; EU Directive 2010/63/EU for animal experiments).

Diet Preparation and Feeding
Regimen.Growers' pellet (Grand Cereals, Bukuru-Jos, Plateau State, Nigeria) was used as the basal diet.Te powdered D. refexa seed was prepared in clean laboratory containers and homogeneously mixed with the basal diet using clean drinking water, and an electrical blender was used to create a uniform blend of nutrients at 0%, 2.5%, 5%, and 10% inclusion rates.Te feeds were then remolded into pellets and dried under the sun before being ofered to the rats in the respective groups.Te normal diet control and N-methyl-N-nitrosourea (MNU) reference group were fed with the basal diet only.Following two weeks of acclimatization, feeding of experimental diets lasted 11 weeks, but concomitant MNU intoxication stopped after the frst ten weeks.

Extraction of Total Polyphenol and Flavonoid Contents.
Te D. refexa seed-included diet samples were pulverized by a mechanical grinder, and 0.2 g portions were suspended in 10 ml methanol (100% and 70% for total polyphenol and favonoid determinations, respectively).Te mixtures were incubated at room temperature for 2 hours with shaking every 15 minutes using an autoshaker.Tey were then centrifuged (3000 rpm for 5 min), and the supernatants were collected into capped sample bottles and stored in the deep freezer at −4 °C until required for analysis [5,15,16].

Determination of Total Polyphenols.
Te total polyphenols content of extracts was determined with the Folin-Ciocalteu reagent according to the method described by Savitree et al. [17] using 0.25 ml of 500 mg/L methanolic extract, 2.5 ml of Folin-Ciocalteu reagent, 2 ml of 1.0 M sodium carbonate, and adjusted to 10 ml with distilled water.Utilizing a spectrophotometer (Jenway 20305 model, 50/ 60 Hz, U.K.), the absorbance was measured at 760 nm, and quantitation was achieved by means of extrapolation from a standard curve (0, 50, 100, 150, and 200 mg/L gallic acid).Te total polyphenols content was expressed as mg/g gallic acid equivalents (GAE).

Determination of Total Flavonoids Content.
Te total favonoids content of the extracts was estimated by the method described by Lachman et al. [18] using 10% aqueous aluminium chloride and 1 M potassium acetate.Te intensity of the pink color developed was measured at 415 nm, while quantitation was achieved with a quercetin (QU) standard curve (0 μg, 12.5 μg, 25 μg, 50 μg, and 100 μg).Te results were expressed as quercetin equivalents (μg quercetin/gm dried extract).

Crude Fiber Determination.
Te crude fber was determined gravimetrically using 2 g of the sample after chemical digestion and solubilization of other materials present.Te digestion and solubilization were achieved with 1.25% sodium hydroxide solution, 1.25% sulfuric acid solution, 3 × 50 ml portions of distilled water, and 25 ml of alcohol, respectively.Te organic matter present was then incinerated at 600 ± 15 °C for 30 minutes.Te percentage of crude fber was calculated using the method enumerated by Holst [19].

Instillation Protocol for Induction of Colon Carcinogenesis.
As described in our earlier reports [5,20], every seventy-two (72) hours for ten (10) weeks, rats in each test group and the MNU control group were intrarectally instilled with 0.2 ml of 1.2% MNU containing 1.9% citric acid, while the normal (basal) control and D. refexa control groups were administered normal saline for the same period using an 8 cm cannula mounted on a syringe (about 5.5 cm from the anal cavity).One week following the termination of the induction experiment, all the rats were sacrifced following mild chloroform anesthesia.Blood for serum separation, organs, 2 Journal of Food Biochemistry and tissues were collected for biochemical, histological, and immunohistochemical assays, respectively.
2.6.Lipid Peroxidation Assay.Lipid peroxidation was determined as a thiobarbituric acid reactive substance (TBARS) as described by Ohkawa et al. [21], using trichloroacetic acid and thiobarbituric acid.Tis assay is based on the principle that lipid peroxidation generates peroxide intermediates, which upon cleavage, releases malondialdehyde, which reacts with thiobarbituric acid to form a colored complex that was estimated at 535 nm.Concentrations of TBARS are expressed in nmol/mg protein.

Glutathione S-Transferase (GST) Assay
. Glutathione stransferases activity assay was estimated according to the procedure of Habig et al. [22] using the GST colorimetric activity assay kit (BioVision incorporated, catalog number #K263-100).Te procedure is based on the principle that GST catalyze the reaction between GSH and the GST substrate, CDNB (1-chloro-2,4-dinitrobenzene) to produce GS-DNB (a dinitrophenyl thioether), which can be monitored spectrophotometrically at 340 nm.

Catalase Activity Determination.
Catalase activity was measured using the method of Abei [23].In this procedure, the enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water.Te reaction was initiated by adding 0.1 cm 3 of fresh 30 mM hydrogen peroxide, and the decomposition rate of hydrogen peroxide was measured spectrophotometrically at 240 nm after 5 minutes.Te molar extinction coefcient (ε) of 0.041 mM −1 cm −1 was used to calculate the catalase activity.

Superoxide Dismutase (SOD) Activity Determination.
Superoxide dismutase activity assay was based on the SODmediated decrease in the rate of autoxidation of hematoxylin in aqueous alkaline solution to yield a chromophore with maximum absorbance at 560 nm [24].Te SOD concentration was calculated as percentage inhibition of the rate of autoxidation of hematoxylin.

Determination of Carcinoembryonic Antigen (CEA).
Carcinoembryonic antigen level was determined via the "Sandwich" enzyme-linked immunosorbent assay (ELISA) using a CEA kit containing a microtitre plate that was precoated with an antibody specifc to CEA (USCN Life Science Inc., Cloud-Clone Corp., USA).Te reactions were then terminated by the addition of sulfuric acid solution, and the color change was measured spectrophotometrically at 450 nm.Te concentrations of CEA in the samples were then extrapolated from a standard curve [25].

Immunohistochemical Analysis of MLH1 Protein Expression in Colon
Tissues.Formalin-fxed, parafnembedded tissues were studied for MLH1 expression using the method involving the avidin-biotin-peroxidase complex (ABC) in the immunoperoxidase technique described by Hsu et al. [26].In the analysis, embedded tissues (2 microns thick) were treated for antigen retrieval, peroxidase blocking, protein blocking using avidin, and further blocking with endogenous biotin before incubation with the respective primary antibody, MLH1.Ten, treatment with the biotylinated secondary antibody, streptavidin, and DAB/ substrate followed.Te postanalytical procedure involved stain interpretation by a pathologist in context with positive and negative tissue controls using bright-feld microscopy (magnifcation at ×40). Brown staining of the cytoplasm and nucleus of the cells was viewed, and the percentage area of expression was quantifed with an immunohistochemistry image analysis tool (ImageJ) as described by Tuominen et al. [27].
2.12.Histological Assessment of Colon.Histological examination was performed on the colon tissue fragments collected from the distal part of the large intestine that had been fxed in 10% formalin, dehydrated in ascending grades of alcohol, cleared in xylene, and embedded in parafn as described by Drury and Wallington [28].Parafn sections (5 µm thickness) using a cryostat were stained with hematoxylin and eosin and examined under a light microscope (Leica Bufalo, N.Y.14240 USA.Model CME Microscope 220-240 VAC 50/60 Hz).

Hematological Assay for Neutrophil-to-Lymphocyte
Counts Ratio (NLR) Calculation.Te determination of hematological indices was carried out using a hematology auto analyzer (Sysmex).Blood samples collected in K 3 EDTA tubes were passed through automated analysis following the manufacturer's operational guidelines.Te NLR was then calculated through dividing neutrophil counts by lymphocyte counts as described by Lee et al. [29].

Diets Total Polyphenols, Flavonoids, and Crude Fiber
Content.Te concentration of total polyphenols, total favonoids, and dietary fber in diferent percentages of D. refexa seed-included diets showed total polyphenols to range from 83.2 ± 0.08 to 287.8 ± 0.01 mg/L, favonoids to range from 21.8 ± 0.04 to 54.6 ± 0.002 μg/ml, and dietary fber to range from 3.46 to 4.34%.Generally, these parameters were increasing with the inclusion rates (Figure 1).
Journal of Food Biochemistry

Carcinoembryonic Antigen (CEA) Level.
A statistically signifcant diference (p < 0.05) in CEA level was observed between the group administered MNU only and that fed a basal diet only, and this was higher than all other groups (Table 1).However, there were no signifcant diferences (p > 0.05) among the MNU groups which fed higher percentages of D. refexa seed-included diets compared to the normal control (Table 1).

Liver Glutathione S-transferase Activity.
Te activity of glutathione s-transferase is represented in Table 1.Te inclusion of D. refexa seed at diferent levels in the diet signifcantly (p < 0.05) prevented MNU-induced depletion in the activity of glutathione s-transferase, and this was largely independent of the inclusion rates.

Level of Malondialdehyde (MDA) in the Colon.
Compared with the normal control, a signifcant (p < 0.05) elevation of the MDA level was observed in the colon of the MNU control group.However, D. refexa seed at all levels of inclusion signifcantly (p < 0.05) prevented the MNUinduced elevation in the levels of MDA (Table 1).

Activities of Colon Catalase and Superoxide Dismutase (SOD).
A signifcant (p < 0.05) reduction in the activities of catalase and superoxide dismutase enzymes in the MNU control group was observed when compared to the normal control group.However, feeding with D. refexa seedincluded diets was able to prevent the depletion of these enzymes, except in the colon of the rats fed the lowest percentage (2.5%) of the included diet (Table 1).

Immunohistochemical Observations.
Te results indicated reduced expression of the mismatch repair protein (MLH1) in the MNU reference group and the MNU group fed with 2.5% D. refexa-included diet.However, in normal, D. refexa seed control groups and experimental groups were fed with 5 and 10% included diets, and the percentage expression of mismatch repair protein (MLH1) was not signifcantly afected (Figure 3 and Table 1).

Discussion
Many plant-derived agents are being reported for their signifcant potency in the management and treatment of oxidative stress-related diseases such as cancer [7,20,30].Hence, in the continued attempt to unravel new therapeutic agents that lack the toxic side efects associated with orthodox chemotherapeutic agents, we extended our earlier study [5,20], by examining the efects of dietary inclusion of D. refexa seed in the prevention of colon carcinogenesis, since it is popular as a soup thickening agent that is widely used in the traditional management of colorectal disorders.Te relevance of this study lies in the fact that food with high content of phenolic compounds [13] and food materials that have demonstrated signifcant antioxidant properties such as D. refexa seed [9,31] are receiving increasing interest from consumers and food manufacturers for their roles in the prevention of various diseases associated with oxidative stress, including cancer [7,8].Chemoprevention of cancer requires long-term administration of a compound with little or no toxicity through diets or the oral route.Like in previous reports [9], in this study, D. refexa seed-included diets were well tolerated by rats as there were no observable changes in the gross behavior of the rats and biochemical and histological   carcinogenesis would be consistent with the basic requirements that chemopreventive natural products should be safe and well-tolerated over a long period of usage.Te colon carcinogenesis preventive efect of D. refexa seed constituents was refected in the levels of carcinoembryonic antigen, a parameter that has been reported to be a sensitive and reliable biomarker in cancer, especially, colon carcinogenesis [32], and malondialdehyde (MDA), which is also a reliable marker for lipid peroxidation and the presence of oxidative stress [33].Tus, D. refexa seed-included diets signifcantly mitigated carcinogen-induced elevation in the levels of CEA and MDA, suggesting that this seed is rich in constituents that play important roles in mitigating colon carcinogenesis.Tis is because oxidative stress and the resulting oxidative damage are important hallmarks of carcinogenesis.However, in this study, these efects were signifcantly prevented in the MNU-intoxicated rats through continuous feeding with diferent levels of D. refexa seedincluded diets.Tis appears to confrm the potency of D. refexa seed constituents in the prevention of chemically induced colon carcinogenesis.
Te observed MNU-induced signifcant depletion in GST activity (Table 1) is rather not surprising, since MNU elicits its toxicity by transferring its methyl group to nucleobases in nucleic acids.Tis can lead to AT : GC transition mutations [34] with consequent deactivation.Tis mutation caused by MNU may also afect other drugmetabolizing enzymes of the cytochrome P 450 group that normally have the capacity to counteract the toxic efects of MNU.It is noteworthy, however, that the signifcant increase in GST activity with increased levels of D. refexa seed inclusion inversely correlated with CEA levels (Table 2), suggesting that D. refexa seed has components that prevented or retarded the initiation or activation and progression of MNU-induced carcinogenesis.It is, therefore, not surprising that one of the proposed mechanisms for protection against cancer, especially colon carcinogenesis by dietary constituents, is the induction of glutathione stransferases [20,35].Tis detoxifying enzyme system participates in preventing the initiation of carcinogenesis through detoxifcation of oxidized metabolites of potentially carcinogenic xenobiotics into relatively inert and less toxic forms that can be easily excreted [35].
Dietary inclusion of D. refexa seed signifcantly retarded MNU-induced depletion in the activities of catalase and superoxide dismutase in the colon tissues (Table 1).It is known that superoxide dismutase is present in the mitochondria, while catalase is found principally in peroxisomes and tissues with high peroxisomal content [36] and to a lesser extent in the cytosol and microsomal fraction of the cell.Tus, the reduced activity following MNU treatment suggests an increased tendency towards oxidative stress consequent on the overwhelming rate of reactive oxygen species generation and the increasingly reduced capacity of the endogenous antioxidant system to neutralize them.Te ability of D. refexa seed to protect the colon against oxidative stress-related damage could be as a result of its polyphenols and favonoids content, since polyphenols, especially favonoids, have been reported to exert positive efects on human health through their antioxidant and antimutagenic properties [9,15,37].Tis observation is in agreement with the report that D. refexa root crude extract demonstrated signifcant activity against prostate cancer cell line, presumably as a result of its content of phytochemicals such as refevone together with mearnsetin, 7,4′-dihydroxyfavone, phytosterols, β-sitosterol, and stigmasterol [38].Tese authors [13] have indeed isolated two favonoids, dioclins A and B, from the seed of Dioclea refexa seed.Tese bioactive compounds also exhibited inhibitory efects against urease and lipoxygenases, as well as free radical scavenging activities [38], and exhibited in vitro antiproliferative efects in breast cancer cells [14].Te possible role of favonoids in the preventive capacity of Dioclea refexa seed is validated by the negative correlation between favonoids, polyphenols, and MDA levels (Table 2).Besides, diocleinae lectins (ConA-like lectins) isolated from Dioclea refexa seed have been demonstrated to possess antiinfammatory, immunomodulatory, antiproliferative, and antitumor activities via induction of cell death through the mitochondrial apoptotic pathway and G2/M cell cycle arrest [39][40][41].
Based on the boost in activities of antioxidant enzymes in animals fed with D. refexa seed-included diets (Figure 1), it may be suggested that the antioxidant compounds in D. refexa seed signifcantly contributed to the prevention of MNU-induced cellular damage.Accordingly, feeding with D. refexa seed-included diets signifcantly prevented MNUinduced deep mucosal ulcerations and moderate interglandular infammations (Figure 2).It has been reported that excessive damage to cells lead to several pathological conditions such as ulcerations and infammations, which are established preludes to carcinogenesis.Te specifc roles of antioxidants in the prevention of these MNU-induced pathologies have been previously reported [31].Moreover, earlier reports have suggested the antioxidant [9] and antiinfammatory [44] efects of D. refexa seed extracts in the paw edema model.In this experiment, this appears to be confrmed by the strong negative correlation between CEA and MDA and the antioxidant enzyme activities (Table 2).
Biomarkers that examine molecules at the cellular level are some of the most reliable tools for diagnosis of carcinogenesis, since oxidative damage can lead to DNA modifcations in several ways [42].One of these modifcations is mismatch repair (MMR) gene defciency, measured as MLH1 protein expression.Tis may be detected by immunohistochemical assays, whose reliability is said to be comparable to that of molecular techniques [43].Results of this assay revealed that D. refexa seed-included diets virtually protected against damage to the colon mismatch repair system (Figure 3), and this corresponded with the favourable histological observations (Figure 2) and the carcinoembryonic antigen profle (Table 1).Correlation analysis (Table 2) revealed that the preventive efect of D. refexa seed in colon carcinogenesis, may, to a large extent, be attributable to the antioxidant constituents of the seeds, which has been amply demonstrated [9], with the resultant mitigating roles in infammations [44], oxidative damage, and carcinogenesis [5].

8
Journal of Food Biochemistry Te carcinogenesis preventive capacity of a D. refexa seed-included diet could equally be partly attributed to its fber content (Figure 1), as may be extrapolated from the negative correlation between crude fber and carcinogenesis biomarkers such as MDA concentration and expression of the mismatch repair gene that were determined.It has been reported that dietary fber can reduce the risk of cancer development through various mechanisms, including improving serum lipid concentrations and reducing infammations [45].Moreover, dietary fber has been demonstrated to participate in the modulation of the gut microbiota landscape to a healthier composition by providing substrates for bacterial fermentations.Tis decreases the risk of colon cancer by improving immunity [46], since compromised immunity is a major risk factor in carcinogenesis.
Te neutrophil-to-lymphocyte ratio was found to be higher in the MNU reference group compared with D. refexa seed-included diets and normal control animals.It is reported that an NLR value greater than 2.88 may be clinically useful in predicting malignancy since elevated NLR could be indicative of relative neutrophilia or lymphopenia [47].Tis is because neutrophils are commonly present in the early stages of acute infammation, whereas lymphocytes are enrolled in chronic infections.Moreover, neutrophils have been associated with an increase in angiogenesis through the vascular endothelial growth factor, which in turn promote the development and spread of cancer cells.Hence, the reduced NLR in D. refexa-included diets, indicated reduced neutrophilia and lymphopenia, suggesting that constituents of D. refexa seed prevented malignancy in carcinogen-exposed animals.Recently, some favonoids of Dioclea refexa seed, namely, dioclins A and B, demonstrated to have an antiproliferative efect in breast cancer cells [14].However, this is the frst report of the cancer preventive efects of whole seed consumption in complex biological systems.Tus, the fnding that Dioclea refexa seed-included diets exhibited capacity to prevent colon carcinogenesis is particularly signifcant since animal experiments better mimic the human body.Te whole seed material that was used adequately refected the public health implication of the consumption of this seed and its constituents by the local population in colon cancer prevention.Besides, emerging evidence suggests that a specifc combination of phytochemicals as present in whole diets may be more efective in protecting against cancer than isolated compounds [48].However, in the future, it would be necessary to evaluate the mechanisms by which favonoids such as dioclins A and B, which are unique to Dioclea refexa, specifcally mitigate colon carcinogenesis.

Conclusion
Te results presented here suggest that Dioclea refexa seed is endowed with phytochemicals, including polyphenols and favonoids, which have the potent capacity to mitigate oxidative stress and other early indices of chemically induced colon carcinogenesis.Hence, Dioclea refexa seed could be exploited in diets to prevent colon cancer in populations that consume it regularly.
Colon.Some pathological features such as infammatory cell infltrations, erosion of the epithelial layer, necrotizing ulcer, and aggregation of infammatory cells, as well as some goblet cells, were observed in the MNU control group (Figure2(b)).However, only mild ulceration with submucosal infammations was observed in the test group fed with 2.5% D. refexa seedincluded diets (Figure2(c)), while normal mucosal epithelia with no dysplasia were observed in other test groups fed with higher percentages of D. refexa seed-included diets and the normal control groups (Figures2(a) and 2(d)-2(f )), ×40 magnifcation).

Table 1 :
Efect of dietary inclusion with diferent levels of D. refexa seeds on oxidative stress, some endogenous antioxidant enzymes, and indices of early carcinogenesis in rats following aValues are means ± SD.Values with diferent superscripts (a, b, c) down the column are signifcantly diferent (p < 0.05).

Table 2 :
Correlation analysis of biochemical parameters and dietary constituents following 10 weeks of concomitant MNU instillations and feeding with diferent levels of D. refexaseed-included diets.