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Abstract
Colorectal cancer is one of the most common cancers occuring in men and women in the western world. Dietary factors are thought to play a predominant role in the causation of colorectal cancer. Diets high in fat and / or meat and low in fruits, cereals and / or vegetables have been found to be associated with a higher risk of colorectal cancer. The reduced risk of colorectal cancer in association with consumption of fruits, cereals and vegetables may be explained by certain micronutrients.
Folate (a water soluble vitamin) which is found primarily in organ meats, whole wheat products, soy bean, yeast, wheat germ and green leafy vegetable, might have a role in carcinogenesis. This has been best studied for colorectal cancer. Some conducted epidemiological studies that investigated the relationship between the folate status and the risk of colorectal cancer showed a significant relationship. Collectively, these retrospective studies suggest a 40% reduction in the risk of colorectal neoplasms in subjects with the highest dietary folate intake compared with those with the lowest intake.
Bakery products were found to be poor sources of folic acid, so our study aimed to estimate the level of folic acid content in soy bean flour and wheat flour (82% extraction rate), to use soy bean flour and pure folic acid in the production of bakery products rich in folic acid, to determine the effect of processing on the folic acid content in the final products, and to study the possible effect of folic acid in fortified products on the prevention of colon cancer development in rats. To achieve this aim, the following methods were carried out:
Methods
1. Wheat flour samples (82% extraction rate) and soy bean flour were analyzed to determine their folic acid contents using the High Performance Liquid Chromatography (HPLC).
2. Unfortified and folic acid fortified Baladi breads were prepared: 1- unfortified Baladi bread, prepared using wheat flour (82% extraction rate) only, 2- Baladi bread fortified with 5% soy bean flour (0.24 mg folic acid / kg wheat flour), 3- Baladi bread fortified with pure folic acid (8 mg folic acid / kg wheat flour), and 4- Baladi bread fortified with both 5% soy bean flour and 8 mg pure folic acid /kg wheat flour (8.24 mg folic acid / kg wheat flour).
3. During the preparation of each bread, samples of each dough of bread were taken after mixing and fermentation processes, to be analyzed for folic acid concentration. Moreover, other samples were taken from each bread after baking to be analyzed for folic acid content using HPLC.
4. Protein, fat, ash, fibers and carbohydrates percentages were also determined in each bread.
5. Finally, rat diets were prepared from the Baladi breads prepared. After an adaptation period of 2 weeks, rats were divided into 5 groups, four of them were injected subcutaneously with dimethylhydrazine (DMH) (colon cancer-induced substance) at a dosage of 400 mg/kg body weight as follow:
a. Group1 (-ve control group) received a diet containing Baladi bread (containing 0.30 mg folic acid / kg bread).
b. Group2 (+ve control group) received a diet containing Baladi bread (containing 0.30 mg folic acid / kg bread) and subcutaneously injected with DMH.
c. Group3: This group received the diet that had the bread fortified with 5% soy bean flour (containing 0.48 mg folic acid / kg bread) and subcutaneously injected with DMH.
d. Group4: This group received the diet that had the bread fortified with 0.8% pure folic acid (containing 7.04 mg folic acid / kg bread) and subcutaneously injected with DMH.
e. Group5: This group received the diet that had the bread fortified with a mixture of 5% soy bean flour and 0.8% pure folic acid (containing 6.56 mg folic acid / kg bread) and subcutaneously injected with DMH.
6. All rat diets were supplemented with nutrients (casein, corn oil, wheat bran, vitamin mixture and mineral mixture) at varying concentrations to obtain balanced diets.
7. During the period of rat housing (15 weeks), the amount of feed eaten by the rat was weighed and the rat weight and mortality % was recorded on weekly basis. In addition, they were examined visually for morphological signs.
8. After 15 weeks, the rats had been fasted overnight and then sacrificed under diethyl ether anesthesia for pathological examination.
This study revealed the following results:
1. The folic acid content in wheat flour (82% extraction rate) was highly significantly lower than the folic acid content in soybean flour.
2. After fermentation of dough, folic acid content increased, while after baking, folic acid content in all breads was found to decrease significantly.
3. After baking, the added folic acid was most stable in pure folic acid fortified Baladi bread (Retention percentage = 84.25 %). This was followed by soy bean flour and pure folic acid fortified Baladi bread (Retention percentage = 75.97 %), then by soy bean flour fortified Baladi bread (Retention percentage = 75.00 %).
4. The highest protein and fat contents were found in soybean flour fortified Baladi bread, followed by soybean flour and pure folic acid fortified Baladi bread, then by unfortified Baladi bread, and finally by pure folic acid fortified Baladi bread.
5. The highest carbohydrate percentage was in unfortified Baladi bread, followed by pure folic acid fortified Baladi bread, then by soybean flour + pure folic acid fortified Baladi bread, and finally by soy bean flour fortified Baladi bread. There was a significant difference at P < 0.01 in carbohydrate percentages among all breads.
6. Folic acid content increased in the following order: unfortified Baladi bread (0.30 ± 0.02 mg/kg bread), soy bean flour fortified Baladi bread (0.48 ± 0.02), soybean flour + pure folic acid fortified Baladi bread (6.56 ± 0.10), then the highest folic acid percentage was in pure folic acid fortified Baladi bread (7.04 ± 0.07 mg/kg bread).This difference in folic acid contents among all breads was highly significant at P > 0.01.
7. The mean body weight of rats in group 1 (-ve control group) increased significantly throughout the 15 weeks of experimental period. Similarly, the mean body weight of rats in group 4 (fed Baladi bread fortified with 8 mg folic acid /kg wheat flour), increased significantly. On the other hand, rats in groups 2, 3, and 5 showed a significant decrease in mean body weights beginning from weeks 10, 10, and 13, respectively. The overall mean body weight was highest in rats of –ve control group (group1) (97.39 ± 40.09 g), followed by rats fed pure folic acid fortified diets; group 4 (81.62 ± 26.78 g) and group 5 (fed soy flour + pure folic acid fortified diet) (76.67 ± 18.24 g), then by rats fed soy flour fortified diets; group 3 (73.49 ± 17.34 g) and finally by rats fed unfortified diets; group 2 (+ve control group) (62.73 ± 13.08 g).
8. The highest significant mean feed intake allover the 15 weeks of experimental period was for rats of group 4 (10.82 ± 3.21 g/rat), whereas the lowest significant mean feed intake was for rats of group 3 (5.27 ± 0.55 g/rat)
9. The highest mean of mortality percentages during the 15 weeks were found in group 4 (46.67 ± 8.80 %), followed by group 5 (45.00 ± 8.80 %), then by group 1 (36.11 ± 16.57 %), and by group 2 (15.56 ± 9.89 %), and finally by group 3 (10.56 ± 4.95 %).
10. Control group (-ve Control group, fed unfortified diet) (Group 1) showed normal structures of the colon and small intestine. The mucosa of the colon was formed of tubular glands, lined by tall columnar epithelium and goblet cells, separated by lamina propria, resting on muscle layers.The mucosa of the small intestine was formed of tubular glands thrown into villi, lined by columnar epithelium, resting on the muscle layer.
11. Group 2 (+ve Control group, fed unfortified diet & injected with DMH) showed chronic non specific inflammation in the colon, where inflammatory cells such as lymphocytes, plasma cells and histiocytes were seen infiltrating the lamina propria in between the mucosal glands. This severe inflammation may proceed to benign lymphoid hyperplasia (lymphoid follicles of the mucosa were hyper plastic with prominent germinal centers) where also seen in both large and small intestine in 50% of this group. Also, focally the colons in this group showed aberrant crypts (seen as focus of dilated crypts as compared to the surrounding normal sized glands).
12. Group 3 (Soy bean flour group, fed diet fortified with 5% soy bean flour & injected with DMH) showed chronic active colitis, enteritis, and benign lymphoid hyperplasia. The colons were formed of successive segments of stenosis or strictures alternating with areas of colonic dilatation, their lumens showed mucosal hypertrophy, where the mucosa was thrown into folds, or showing mucosal hyperplasia, in the form of marked thickening of the mucosa due to proliferating glands. These colonic changes caused formation of segments of luminal stenosis or constriction, causing the accumulation of digested food, pressing the wall of the colon causing its inflammation and atrophy alternating with areas of marked atrophy of the colonic mucosa and muscle layers. Moreover, colonic mucosa showed severe dysplasia (pre malignant) in the form of pleomorphic hyper chromatic nuclei, prominent nucleoli, and abnormal mitotic figures. All these changes were seen in 100% of the colons of this group.
13. Folic acid group (Group 4) (fed diet fortified with 8 mg pure folic acid/kg wheat flour & injected with DMH) showed normal small intestine structure with foci of enteritis, but in the colon, it showed a focus of aberrant crypts, foci of mild mucosal hyperplasia and mucin depletion. Focus of excessive mucin production, associated with small foci of mild dysplasia, mild chronic colitis, and benign lymphoid hyperplasia were also shown in the colon. Severe mucosal atrophy and colonic dilatation were also detected.These gross pathological changes were found in all specimens provided as seen in groups 2, 3. But neither malignancy nor dysplasia (premalignant changes) was detected.
14. Mild chronic colitis and enteritis were detected in group 5 (Soy bean flour and Folic acid group, fed diet fortified with a mixture of 5% soy flour + 8 mg folic acid / kg wheat flour & injected with DMH) . In this group, multiple tubular adenomas were seen as proliferated tightly packed tubular colonic glands lined by columnar cells and showing depletion of mucin secreting cells; these lesions are considered as pre cancerous. Also, this group showed areas of atrophy, others of hypertrophy, and / or hyperplasia as in group 3. But neither dysplasia, nor malignancy was detected in this group.