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ANTICANCER AND ANTIOXIDANT EFFECTS OF FRUCTOOLIGOSACCHARIDE (FOS) ON CHEMICALLY-INDUCED COLON CANCER IN RATS
Sorial Adly Moharib
Biochemistry Department, National Research Center, Dokki, Cairo, Egypt
Onion (Cynara scolymus) and globe artichoke (Allium cepa) fruitswere
used as natural sources for isolation of fructooligosaccharides (FOS). Chemical
analyses of onion and globe artichoke dry matter revealed the major component
was carbohydrates (91.40 and 85.82% respectively). FOS from Onion (FOS1) andglobe
artichoke(FOS2) were isolated (45.70 and 42.91% of total carbohydrates respectively).
Quantitative and qualitative analysis revealed the presence of kestose, nystose,
and fructofuranosyl nystose as major components of FOS1 or FOS2. Cytotoxic activities
of FOS1 and FOS2 were examined in vitro using colon (HCT 116), liver (HEPG2),
cervical (HELA) and breast (MCF7) carcinoma cell lines. FOS1 or FOS2 showed a
higher percentage cell death of HCT-116 carcinoma cell lines than the other cell
lines, indicating promising anti-tumorigenic properties, and demonstrating their
direct effect on colon cancer cell proliferation. A marked reduction was observed
in the levels of ALP (62 and 61%), ALT (70 and 64%) and γ-GT (73 and 65%)
in sera of rat groups that first received FOS1 or FOS2 respectively, compared
to 1,2 dimethyl hydrazine (DMH) control rats, indicating protective effect of
both FOSs. Insignificant change was observed in AST level. Significant decreases
in the levels of lipid peroxidation (LP) in sera of rats administered with FOS1
or FOS2 compared to those DMH control rats. Highest significant decrease in the
level of LP was observed in sera of rats FOS2 administered more than those of
FOS1.The anticancer activity was evaluated through determination of CEA and C19.9
in DMH-induced colon cancer rat groups treated with either FOS1or FOS2 compared
with the carcinogenic control rat group (DMH). Therefore, FOS1or FOS2 is more
effective for inhibiting DMH-induced colon cancer. The present results showed
the activity of GSH-R was increased in liver (56 and 51%) and kidney (54 and
34%) of rat groups treated with FOS1or FOS2, respectively, compared to those
of DMH control rats. GSH-P activity was also increased in liver (61 and 58%)
and kidney (52 and 51%). FOS1or FOS2 did not modify GSH-T activity in rat groups.
FOS1or FOS2 exhibited a higher increase of SOD activity in liver (67 and 61%)
and kidney (59 and 54%), respectively, as compared to those of DMH control rats.
The most significant findings of the present study is that the FOS1 or FOS2 (200
mg/kg body weight) have shown a beneficial effect not only on colon cancer but
also on antioxidant enzymes activity in DMH - induced colon
cancer in rats, as well as protected the cell against DMH oxidative stress
by antagonizing DMH toxicity. According to these observations, the use of FOS
(FOS1or FOS2) can be recommended as anticancer and antioxidant agents.
Onion (Cynara scolymus) and globe artichoke (Allium cepa) fruitswere used as natural sources for isolation of fructooligosaccharides (FOS). Chemical analyses of onion and globe artichoke dry matter revealed the major component was carbohydrates (91.40 and 85.82% respectively). FOS from Onion (FOS1) andglobe artichoke(FOS2) were isolated (45.70 and 42.91% of total carbohydrates respectively). Quantitative and qualitative analysis revealed the presence of kestose, nystose, and fructofuranosyl nystose as major components of FOS1 or FOS2. Cytotoxic activities of FOS1 and FOS2 were examined in vitro using colon (HCT 116), liver (HEPG2), cervical (HELA) and breast (MCF7) carcinoma cell lines. FOS1 or FOS2 showed a higher percentage cell death of HCT-116 carcinoma cell lines than the other cell lines, indicating promising anti-tumorigenic properties, and demonstrating their direct effect on colon cancer cell proliferation. A marked reduction was observed in the levels of ALP (62 and 61%), ALT (70 and 64%) and γ-GT (73 and 65%) in sera of rat groups that first received FOS1 or FOS2 respectively, compared to 1,2 dimethyl hydrazine (DMH) control rats, indicating protective effect of both FOSs. Insignificant change was observed in AST level. Significant decreases in the levels of lipid peroxidation (LP) in sera of rats administered with FOS1 or FOS2 compared to those DMH control rats. Highest significant decrease in the level of LP was observed in sera of rats FOS2 administered more than those of FOS1.The anticancer activity was evaluated through determination of CEA and C19.9 in DMH-induced colon cancer rat groups treated with either FOS1or FOS2 compared with the carcinogenic control rat group (DMH). Therefore, FOS1or FOS2 is more effective for inhibiting DMH-induced colon cancer. The present results showed the activity of GSH-R was increased in liver (56 and 51%) and kidney (54 and 34%) of rat groups treated with FOS1or FOS2, respectively, compared to those of DMH control rats. GSH-P activity was also increased in liver (61 and 58%) and kidney (52 and 51%). FOS1or FOS2 did not modify GSH-T activity in rat groups. FOS1or FOS2 exhibited a higher increase of SOD activity in liver (67 and 61%) and kidney (59 and 54%), respectively, as compared to those of DMH control rats. The most significant findings of the present study is that the FOS1 or FOS2 (200 mg/kg body weight) have shown a beneficial effect not only on colon cancer but also on antioxidant enzymes activity in DMH - induced colon cancer in rats, as well as protected the cell against DMH oxidative stress by antagonizing DMH toxicity. According to these observations, the use of FOS (FOS1or FOS2) can be recommended as anticancer and antioxidant agents.
Key words: FOS, Onion, Artichoke, Anticancer, Antioxidant, Rat.
- (ALP) Alkaline phosphatase
- (ALT) alanine aminotransferase
- (AST) Aspartate aminotransferase
- (CEA and CA 19.9) Tumor markers
- (DMH) 1, 2 dimethylhydrazine dihydrochloride
- (DP) Degree of polymerization
- (DP3) Kestose
- (DP4) Nystose
- (DP5) fructosylnystose
- (FOS) Fructooligosaccharides
- (FOS1) Fructooligosaccharide from onion (Allium cepa)
- (FOS2) Fructooligosaccharide from globe artichoke (Cynara scolymus)
- (γ-GT) Gamma glutamyl transferase
- (GSH-P) glutathione peroxidase
- (GSH-R) glutathione reductase
- (GSH-T) glutathione transferase
- (LP) Lipid peroxidation
- (SCFA) Short chain fatty acids
- (SOD) superoxide dismutase
Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. Malignant cancers are the second leading cause of death, among them colorectal cancer  is the most malignant tumor. Numerous studies suggest that certain plant materials might be useful as anticancer and chemopreventive agents , amid reports that the colon cancer is considered a preventable disease. Plant polysaccharides can be considered as bioactive molecules in medicine, they have been demonstrated to have antihyperlipidemic effect . Fructans are produced naturally in different plant species and follow the starch in carbohydrates occurring in nature . The most common sources of fructans are chicory, artichoke, asparagus, and onion which are used by ancient peoples as food, feed or medicine .
Fructooligosaccharides (FOS) as dietary fibers have physiological actions on gastrointestinal tract . Considering the continuing need for effective anticancer agents, natural product and medicinal plants might be an inexhaustible source of anticancer drugs . Several studies have been shown the role of FOS in food and some functions defence [9, 11]. The higher sources of fructans are onion and artichoke . FOS is none digestible carbohydrates and act as prebiotics to selectively promote the growth of colonic bifidobacteria, thereby improving human gut health . Other investigators provided evidence that plant polysaccharides consumption results in treated and protection against chemically induced colon cancer [8, 14]. Cancer is the leading cause of mortality worldwide and exhibit severe toxicity to normal tissues when applied as the surgical adjuvant treatments of cancer . Plant polysaccharides have been demonstrated to have chemopreventive effects . Recent study has shown that some polysaccharides intake in rats cause improve some biochemical parameters of oxidative stress and exhibited reduce the risk of some diseases [17,18].
Natural products are rich resources of cancer chemotherapy drugs and anticancer potentials . Fructans are naturally occurring with different percentages in some plant species, particularly onion and artichoke . Colorectal cancer is the most malignant tumor with very high morbidity and mortality rates . Prebiotics being associated with improved bowel functions and reduced risk of colon cancer . Other studies revealed antioxidative properties of different constituents of some seeds on lipid metabolism in rats . Some non-digestible fructans such as inulin and inulin-like oligosaccharides confer potentially interesting prebiotic properties in human health . The nutritional effect of FOS in promotion of fecal nitrogen excretion was estimated by other investigators . FOS improves the gut ecosystem by increasing bifidobacteria, shortens gut transit time in DMH treated rats [15, 33]. Other investigator reported that plant polysaccharides, with different molecular weight, have different effects in rats . Some studies have shown the role of FOS like polysaccharides in control of diabetes . Recent study showed that the most chemotherapeutic and radiotherapeutic agents exhibit different side eﬀects in the cancer treatments . Epidemiological investigations indicated that the diets with high fruits and vegetables provide a means of cancer chemoprevention due to their phytochemical constituents . Natural products are rich resource of anticancer potential in a variety of bioassay systems and animal models . Other investigators provided evidence that the polysaccharides consumption results beneficial affects cecal fermentation and serum parameters in rats [7, 38]. Current research concentrates heavily on novel anticancer drug development from natural products particularly the biopolymer compounds with potential antitumor activity and protection against chemically induced colon cancer . Recent study has shown that some plants intake in rats cause an increase in the antioxidant enzymes activity and exhibited decrease in lipid peroxidation, which may reduce the risk of some diseases [34, 40]. However, the main and most abundant polysaccharides from different plants, include starch, cellulose and hemicelluloses, pectins, inulin and inulin related oligofructoses, are used as functional food ingredients . Other workers studied the effective of medicinal plants extract as anticancer agents . Plant-derived diets containing phytochemicals and /or polysaccharides could be used in preventive strategies to reduce the risk and inhibit or retard the development of colon cancer [42, 43]. The short chain fatty acids referred as prebiotics may be partially responsible for their some physiological effects .
FOS including inulin, are widespread carbohydrates belonging to a group of compounds known as fructans  which are polysaccharides made up mainly of a chain of fructose molecules (fructooligosaccharide). These FOSs exhibited different effects on the levels of antioxidant enzymes activity and decrease lipid peroxidation . FOSs consider as responsible agents for protective effects against some diseases may be their effects as free radical scavengers . Moreover, several studies have been shown the role of FOSs in carbohydrate and lipid metabolism in rats . However, the principle components of carbohydrates are glucose, fructose, sucrose and a series of FOSs with different degrees of polymerization . Recent study on anticancer drug development from plant biopolymer compounds with potential antitumor activity .
Indigestible carbohydrates, such as FOSs, dietary fiber, indigestible oligosaccharides or resistant starch, reach the large intestine intact and are fermented by bacteria in the intestinal lumen resulting in the production of organic acids such as acetate, propionate and butyrate [48, 50]. These polysaccharides are non-toxic, biodegradable and water soluble that consequently suitable for different pharmaceutical and biomedical uses and play important roles in several physiological and pathological conditions[50, 52]. However, the nutritional effects of FOS are known to include stimulation of mineral absorption [41, 52]. Different researchers have been conducted on the role of some phytochemical compounds present in onion and artichoke. Therefore, the aim of the present study was designed to focusing on the isolated FOS1 from onion and FOS2 from artichoke for its antioxidant and anticancer activities in DMH- induced colon cancer in vivo using rats. The cytotoxicity and anticancer effect were also done in vitro using four different carcinoma cell lines. The results will may helpful to develop a novel anticancer and antioxidant drugs as well as some functional foods.
MATERIALS AND METHODS
Carcinogenic material used in this study was 1, 2 dimethylhydrazine dihydrochloride 99+% (DMH) was obtained from Sigma-Aldrich® chemie, Gmbh, Riedstr. 2, D-89555 Steinheim, Germany. Onion (Allium cepa) and globe artichoke (Cynara scolymus) fruits were obtained from the local market.
Preparation and chemical analysis of samples
Dried samples of onion and artichoke according to the association of official agricultural chemists  were ground to fine powders, sifted through a 16-mesh sieve, packed in well sealed polyethylene bags and stored at room temperature until uses. Total carbohydrate was determined using phenol-sulfuric acid method . The protein content was also determined . Total lipid was removed and estimated according to the method previously described . Ashes were quantified gravimetrically after incineration in a muffle oven at 550°C. Extraction of FOSs using hot water (80°C) for 18 hours and cooled at room temperature as previously described . The isolated FOS1 from the dried Onion and FOS2 from artichoke used in the present study were analysis using HPLC in the Central Laboratory of National Research center (NRC). Carbohydrate analysis column, Aminex HPX-42C (Biorad) was used with Refractive Index detector. The column temperature was 80°C and the mobile phase was distilled H2O with flow rate of 0.6 ml/min. The standards of Kestose and Nystose were injected individually and separately with a known concentration.
In vitro studies
Cytotoxicity tests of the obtained FOS1 and FOS2 were done in vitro using different human cancer cell lines, particularly those of colon (HCT 116), liver (HEPG2), cervical (HELA) and brest (MCF7) carcinoma cell lines. Measurements of potential cytotoxicity of the samples were assayed [39, 68].
In vivo studies
Induction of colon cancer in rats using 1,2 dimethyl hydrazine (DMH) was done experimentally according to method previously described .
Thirty five male albino rats, 10 weeks of age, weighing about 190±1.2 g were purchased from the National Research Center for biological products. The rats were divided into five groups (7 rats/group) and housed in a wire screen cage. The rats had free access to fed commercial diet and tap water. The animal room was controlled (25±1ºC) and had a 12-hour light-dark cycle and humidity at 60±5%. The rats were acclimatized for a period of one week. Three groups of rats administrated for 5 weeks (twice / week) subcutaneous injections of DMH at a dose of 40 mg/kg body weight [39, 68]. The first group was maintained without any treatment over experimental period (16 weeks) and used as carcinogenic control group (C). The other 2 groups of rat administrated DMH for 5 weeks (twice /week) were then treated with oral dose (200 mg/kg body weight) of FOS1 and FOS2(C/FOS1 group and C/FOS2 group respectively) by special syringe from week 6 till the end of experimental period (16 weeks). The other two groups of rat were administrated with oral dose (200 mg/kg body wt) for 5 weeks, from the first week, of FOS1 and FOS2 (FOS1/C group and FOS2/C group respectively) and then they were administrated for 5 weeks (twice/week) subcutaneous injections of DMH at a dose of 40 mg/kg body weight and treated with oral dose (200 mg/kg body weight) of FOS1 and FOS2(C/FOS1 group and C/FOS2 group respectively) from week 6 till the end of experimental period (16 weeks). The experimental protocol was done according to Moharib et al. .
At the end of experimental period (16 weeks), blood samples were drawn from 7 rats per each group separately using capillary tubes, centrifuged at 4000xg for 10 min. Separated sera or plasma were stored at -60ºC till used. Liver, kidney and Heart tissues were removed immediately, weighed, washed (using saline 0.9%), minced and homogenized (10% w/v) separately with cold sodium potassium phosphate buffer (0.01 M, pH 7.4) using homogenizer (Precision model Warsaw MPW-309, Poland). The homogenates were centrifuged at 10,000 g for 20 min at 4ºC and the resultant supernatants were stored at -70ºC till used. Stored sera or plasma and tissues homogenates were used for estimation of the activities of glutathione transferase (GSH-T), glutathione peroxidase (GSH-P), glutathione (GSH-R) and superoxide dismutase (SOD) and other biochemical parameters. Liver and colon were also used for pathological examinations.
Total protein was estimated using Biodignostic kits, Egypt according to the method of Bradford . Alkaline phosphatase (ALP) level was carried out referring the DGKC indications, Germany . Serum albumin level was measured according to the method of Doumas et al. . SOD (EC 126.96.36.199) activity was measured by the NADH oxidation procedure, as described by Elstner et al. . GSH-R (EC188.8.131.52) activity was assayed using the method of Goldberg and Spooner 1992 . GSH-P (EC184.108.40.206) activities in plasma and homogenates of liver, kidney, and heart tissues were assessed by the method of Habig et al. . Lipid peroxidation (LP) was estimated according to the method of Ohkawa et al. . The GSH-T (EC 220.127.116.11) activities in plasma and homogenates of liver, kidney, and heart tissues were assessed by the method of Paglia and Valentine . CA 19.9 was performed  with commercially available Enzyme Immunoassay Kit. Globulin was calculated by subtracting albumin form the total protein . Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured according to the method of Reitman and Frankel , using kits of QCA, Spain. Gamma glutamyl transferase (γ-GT) was carried out according to the kinetic colorimetric method of Szasz  using Biodignostic kits, Egypt. Determination of CEA was performed with commercially available Enzyme Immunoassay Kit (Bio Check, Inc. catalog number: BC-1011) according to the method of Uotila et al. .
Histological assessments of colon tissues were carried out according to Scheuer and Chalk  using Hematoxyline and Eosin (H&E) staining technique.
Data from the biochemical analysis was statistically analyzed according to the method of Fisher  using student T-test.
Chemical analyses of onion and artichoke dry matter revealed the presence of different components of carbohydrate, protein, lipid and ashes. The results showed the amount of carbohydrate, as major portion in onion, was higher (91.4%) than that (85.82%) of artichoke dry matter. The protein content was lower (4.1%) in onion than that (7.4%) of artichoke dry matter. Less constituent was observed in the levels of total lipid (1.1 and 1.58%) and ashes (3.4 and 5.2%) in onion and artichoke respectively.
The present results indicated the amounts of FOS1 and FOS2 were about 45.70 and 42.91% of the total carbohydrate of onion and artichoke respectively. The FOS1 and FOS2 composition (mg/g) yields by HPLC analysis as shown in Table 1. The concentrations of Kestose (DP3), (DP6) and (DP8) in FOS1 were higher than those of FOS2. The concentrations of Nystose (DP4) in FOS2 were higher than those of FOS1. Fructosyl-nystose (DP5), (DP6) and (DP8) in FOS1 were higher than those of FOS2. On contrast the concentrations of fructosyl-nystose (DP7) and (DP10) in FOS2 were higher than those of FOS1. The concentration of (DP9) was nearly the same in both FOS1 and FOS2 obtained.
Table 1. FOS component in onion (Allium cepa) and globe artichoke (Synara scolymus)
Mean of three samples
In vitro studies
The present study was carried out to evaluate the potential efficacy of FOS1and FOS2 against four different carcinoma cell lines in vitro (Fig. 1, 2). The effect of FOS1 and FOS2 used in vitro cytotoxicity test was done to identify activity of the FOS1 and FOS2 in growth inhibition of four different carcinoma cell lines, colon (HCT-116), liver (HEPG2), cervical (HELA) and Brest (MCF7). Results showed that FOS1and FOS2 were more effective in inhibition of HCT-116 but lower effective against HEPG2, HELA and MCF7 cancer cell lines. FOS1 exhibited more effectiveness on HCT 116 and HEPG2 but less on MCF7 and HELA cancer cell lines (Fig. 1). Results also showed that FOS2 was more effective in inhibition of HCT-116 and HepG2 but not effective against MCF7 and HELA cancer cells (Fig. 2). The effect of FOS1 and FOS2 on HCT 116 cancer cell line in vitro revealed that FOS1 and FOS2 inhibit cell proliferation of human HCT-116. Results in Figure 3, illustrate the dose response (IC50) of FOS1 and FOS2 on HCT116. The present results showed the growth inhibitory effect of FOS1 and FOS2 on HCT-116. The data show that FOS1 and FOS2 have a higher cytotoxic activity against HCT116 than the other all cell lines (Fig. 1, 2). These results indicated that FOS1 and FOS2 have more anticancer effect against HCT116. The FOS1 and FOS2 reduced the survival fraction to 50% (kills 50% of the cancer cells) where less than 5 μg of FOS1 and FOS2 killed 50% of cancer cells, particularly HCT116 (Fig. 3). The most commonly used prebiotics are beta-fructans oligosaccharides. Inulin and oligofructose consider as natural food ingredients or dietary fibers present in certain plants as storage carbohydrates.
Fig. 1, 2. In vitro cytotoxic effect of FOS1 (above) and FOS2 (below) on different human cancer cell line particularly colon (HCT-116), liver (HEPG2), breast (MCF7) and cervical (HELA) carcinoma cell line
Fig. 3. IC50 of FOS1 and FOS2 on HCT-116
DMH was used as a potent and complete carcinogen for the colon, since it has been reliably used to induce the initiation and promotion steps of colon carcinogenesis after five doses over 5 successive weeks. The colon cancer was induced by intraperitoneal injection of DMH at a dose of 40 mg/kg body weight (twice a week for 5 weeks).
The present study establishes that FOS1 isolated from onion has appreciable anti-cancer activity greater than that of FOS2 isolated from artichoke. Oral administration of FOS1and FOS2 at a dose of 200 mg/kg did not produce any signs of toxicity and no any animal was died. It showed that FOS1and FOS2 were nontoxic in rat up to an oral dose of 200 mg/kg. Therefore, the investigation of anticancer activity was carried out using dose at level of 200 mg/kg. However, based on the published studies, administration of onion and artichoke to man is simple, since they are used as common dietary constituents in many parts of the world.
Effect of FOS1, FOS2 on total protein, ALP,
ALT, AST, γ-GT
The present results in Table 2 illustrate the potential effects of FOS1 and FOS2 on the levels of total protein, albumin, globulin and liver marker enzymes (ALP, ALT, AST, γ-GT) in sera of carcinogenic control and treated rat groups. These biochemical parameters were altered in DMH induced colon cancer rats (group C). Significant reductions in serum total protein, albumin and globulin levels were observed in DMH-induced colon cancer rats (group C). Higher significant increases were observed in the levels of total protein, albumin and globulin in sera of rat groups (Tab. 2). administered FOS1 (FOS1/C and C/FOS1) and FOS2 (FOS2/C and C/FOS2) compared to control rat group (C). Thus the administrated rats with FOS1 and FOS2 at doses of 200 mg/kg, showed higher increases in total protein, albumin and globulin levels compared to C control rats. The present results indicated higher significant increases in the level of ALP, ALT, γ-GT and AST in sera of rats administered DMH (group C). Higher significant decreases were observed in the levels of ALP, ALT, γ-GT and AST in sera of rats administered FOS1 and FOS2 compared to those administered DMH (control group C). Results also showed highly significant decreases in the levels of ALP (46 and 39%) and ALT (55 and 44%) in sera of treated rat groups (C/FOS1 and C/FOS2) respectively, compared to those of control rat group (C). Insignificant changes were observed in AST. A marked reductions were observed in the levels of ALP (62 and 61%) and ALT (70 and 64%) in sera of rat groups (FOS1/C and FOS2/C) respectively, compared to rat group C. AST level was deceased significantly in sera of rat groups administered FOS (FOS1/C and FOS2/C). The present results showed higher reduction in the level of γ-GT (70 and 46%) in sera of rat groups (C/ FOS1 and C/ FOS2) respectively, compared to rat group C. Higher reduction in the levels of γ-GT (73 and 65%) was observed in sera of rat groups given FOS1 (FOS1/C) and FOS2 (FOS2/C) respectively, compared to rat group C (Tab. 2). On contrast, significant decrease in the level of lipid peroxidation (LP) was observed in sera of rats administered FOS1 (FOS1/C and C/ FOS1) and FOS2 (FOS2/C and C/ FOS2), compared to those administered DMH (control group C). The highest significant decrease in the levels of LP was observed (Fig. 4) in sera of rats administered FOS2 (FOS2/C and C/ FOS2) more than those of FOS1 (FOS1/C and C/ FOS1). The present data showed higher significant decrease in the level of CEA in treated rats with FOS1 (C/ FOS1) and FOS2(C/ FOS2), compared to group C. A marked reduction in the level of CEA was observed in rat group received FOS1 (FOS1/C) and FOS2 (FOS2/C). Insignificant difference was observed in the levels of CA 19.9 (Fig. 5). Regarding, CEA and CA-19.9, showed a marked decrease in rat groups given FOS1 and FOS2 before DMH induction more than that decrease shown in rat groups received FOS1 and FOS2 after DMH induction.
Table 2. Biochemical parameters in sera of experimental rat groups
Data was presented as mean value ± SE of 7 rats / group (P<0.05 significant and P<0.01 higher significant)
Fig. 4. Lipid peroxide levels in sera of experimental rat groups
Fig. 5. CEA and C 19.9 levels in sera of experimental rat groups
Antioxidant enzymes activities
Generally, DMH showed significantly decrease in the activities of GSH-T, GSH-P, GSH-R and SOD in plasma and tissue homogenates of liver, kidney and heart of rat group C (Fig. 6).The present results showed the activity of GSH-P was significant increase (Fig. 6B) in plasma (47 and 54%), liver (40 and 63%), kidney (57 and 30%) and heart (36 and 28%) of rat treated with FOS1(C/FOS1). The activity of GSH-R was significant increase (Fig. 6C) in plasma (45 and 43%), liver (58 and 53%), kidney (54 and 34%) and heart (35 and 21%) of rat treated with FOS2 (C/FOS2). Rats received FOS1 and FOS2 (FOS1/C and FOS2/C) showed increase in the activities of GSH-P (40 and 44%) and GSH-R (64 and 61%) in plasma (Fig. 6B, C). Rats received FOS1 and FOS2 (FOS1/C and FOS2/C) showed increase in the activities of GSH-P (26 and 29%) and GSH-R (11 and 13%) in heart (Fig. 6B, C). GSH-R activity was increase in liver (56 and 51%) and kidney (25 and 30%) in rat groups (FOS1/C and FOS2/C respectively), compared to those of control rats C (Fig. 6C). GSH-P activity was also increase (Fig. 6B) in liver (61 and 58%) and kidney (52 and 51%). GSH-P is responsible for most of the decomposition of lipid peroxidation in cells and thus may protect the cell from the deleterious effects of peroxidation. In the present investigation, higher GSH-P and GSH-R activities were observed in liver and kidney of rat groups given FOS (C/FOS1and C/FOS2), compared to those of rat group C. However, the enhanced lipid peroxidation in heart tissue of rat may be due to lower activities of GSH-P and GSH-R (Fig. 6BC) of control rat group (C). The FOS1 and FOS2 did not modify GSH-T activity in plasma or liver of rats. The rat groups received FOS1 and FOS2 (C/FOS1and C/FOS2) exhibited higher SOD activity in liver (67 and 61%) and kidney (59 and 54%) respectively, compared to those of rat group C (Fig. 6D). Rats received FOS1 and FOS2 (FOS1/C and FOS2/C) showed increase in the activities of SOD in liver (58 and 55%) and kidney (27 and 21%) respectively as compared to those of rat group C (Fig. 6D). In the present study, combined treatment of DMH with FOS1 or FOS2 resulted in maintained the activities of SOD and GSH-dependent antioxidant enzymes, when compared to control rat group C, indicating the protective role of FOS1 or FOS2 against DMH-induced oxidative stress. The chief characteristics of these natural products are that they are rich in kestose, nystose and fructosyle-nystose exhibits relatively high antioxidant activity. The most significant findings of the present study is that the FOS1 or FOS2 at the dose of 200 mg/kg body weight for 16 weeks have shown beneficial effect not only on colon cancer but also on antioxidant activity in DMH-induced colon cancer in rats. Moreover, it ameliorated DMH-induced decrease the antioxidant activity of some enzymes. Therefore, the present results revealed that the FOS1 or FOS2 may be used as a protective effect by antagonizing DMH toxicity. The present study examined possible usefulness of FOS1 or FOS2 as natural sources of antioxidant agents to treat and protect the rat against cancer effect of DMH and improve antioxidant enzymes which can protect cell against oxidative stress in DMH-induced cancer. Antioxidant capacity depends on redox and free radical scavenging characteristics of the medium along with the content of several compounds (polyphenols, phytochemicals and other). Globe artichoke is rich in polyphenols, phytochemicals, and these substances are likely significant factors in the antioxidant status of large intestine health. Thus, intake of globe artichoke in rats modified microbial enzymatic activities and enhanced antioxidant free radical scavenging capacity.
Fig. 6. Antioxidant enzymes activities in plasma and homogenates of liver, kidney, and heart of experimental rats. GST-T (6A), GSH-P (6B), GSH-R (6C) and SOD (6D). Mean value ± SE of 7 rats/group
Examined sections of rat colons from carcinogenic group (C) revealed necrosis and showing lymphoid follicle hyperplasia with infiltration of the covering mucosa as recorded in Figure 7a. Colons from rat groups (C/FOS1 and C/FOS2), showed normal mucosal lining with mucosa secreting cells and the lamina propria showed minimal inflammatory cells (Fig. 7b,c). Colons from rat groups (FOS1/C and FOS2/C), showed submucosa, musclosa and serosa are within normal with no pathological changes (Fig. 7d,e). However, the histopathological studies indicated that the use of FOS1 and FOS2 (200 mg/kg) showed improved the histology in rats received the carcinogen DMH.
DMH-induced colon cancer rats group C (a)
Treated rats group C / FOS1 (b)
Treated rats group C / FOS1 (c)
Treated rats group FOS1/C (d)
Treated rats group FOS1/ C (e)
Fig. 7. Sections of DMH-induced colon cancer rats group C (a), treated rats groups (b,c) and (d,e)
The present study will focus on a more detailed review of new findings on dietary fibre inulin-type fructans (FOS) and their potential role in cancer prevention . FOS effect on carbohydrate and lipid metabolism is also well established . The β-2,1 glycoside bond inulin-type fructans have been shown to resist hydrolysis by enzymes in the human small intestine. They are fermented extensively by large bowel microflora to SCFA [32, 50], which can be absorbed, metabolised and inhibited cell growth . One dietary fibre that could be of relevance in colon cancer prevention is the group of the inulin-type fructans . Onion and artichoke have been primarily described as an overall stimulatory effect on specific as well as non-specific immune functions [2, 57]. A number of foods, such as garlic, onion, artichoke and asparagus, contains high levels of inulin-type fructans, has reduction of colon cancer risk . FOS improves antioxidant enzyme activity and liver function enzymes [59, 60]. FOS is a common name for fructose oligomers, and these are usually understood as inulin-type oligosaccharides. FOS constitute a series of homologous oligosaccharides derived from sucrose which are mainly composed of 1-kestose (DP2), nystose (DP3), and fructofuranosyl nystose (DP4), in which two, three, and four fructosyl units are bound at the β-2,1 position of glucose  reported that the main chemical constituents of carbohydrate of onion are polysaccharides and saponins as major constituents of dietary fibres . FOS are fermentable fibers that provide protection against earlier stages of colon carcinogenesis and reduce the number of DMH-induced aberrant crypt foci in rats [61, 64], attributed to the pH-lowering effect of bifidobacteria in the colon, which subsequently inhibited the growth .
The present results showed the carbohydrate in onion and artichoke accounts the major portions of dry matter contributing as much as 91.4 and 85.82% respectively. The principle component of these carbohydrates is a series of fructosylpolymer of degree polymerization (DP) ranged from 3:10. These results are similar to those reported by other investigators , stated that the FOS classified as oligosaccharides with a DP between 3 and 10. The degree of polymerization (DP) of these fructans can vary to a large extent amongst Allium species [20, 50]. Other investigators suggested chain length of FOS with a degree of polymerization (DP) between 2 and 8 [6, 66].
In the present results, FOS are group of glycosyl-fructosyl polymer with DP (3-5) occur in onion and artichoke. These results are in accordance with those reported by other investigators [12, 49]. However, the health benefits of these carbohydrates have been reported in the last decade and their prebiotic effect demonstrated the content of FOS that depends on the DP. The main constituents of FOS in onion and artichoke are kestose (274.6 and 122.26 mg/g DM), nystose (52.40 and 270.28 mg/g DM) and fructosyl-nystose (46.20 and 24.40 mg/g DM), respectively (Tab. 1). The present results are in the range with those reported by other investigators [48, 63]. Kestose and fructosyl-nystose are the main components in FOS1 while nystose and fructosyl-nystose is the main contents in FOS2. The percentage of kestose and nystose accounts for FOS1 and FOS2 are 70 and 78% respectively. In plants, two or more different types of fructosyltransferases are thought to be involved in fructan biosynthesis [6, 48, 56]. However, onion and artichoke are generally consumed for its nutritive values and medicinal power has been appreciated .
The present study was carried out to screen the isolated compounds (FOS1 and FOS2) using in vitro cytotoxicity test to identify their activity in growth inhibition of different carcinoma cell lines (HEPG2, HCT 116, MCF7 and HELA) in vitro [34, 36, 67]. The cytotoxic activity of FOS1 and FOS2 was observed against HCT116. Similar results were found when examined Raphanus sativus extract on colon carcinoma cell line in vitro . FOS1or FOS2 reduce the survival fraction to 50%, it means that FOS1 and FOS2 kill 50% of the colon cancer cells lines. Similar results were found by other workers [42, 49]. It can be observed that FOS1 and FOS2 inhibits cell proliferation of HCT-116 (human colon cancer cell line) that could arrest the cell cycle and generate apoptosis, which explain the in vitro antioxidant and anti-proliferative effect of fructooligosaccharides (FOS) and or polysaccharides [36, 69, 70], they reported similarity effect between them. In the present work in order to investigate the anticancer activity of FOS1 and FOS2 on chemically induced colon cancer in vivo, The carcinogenic compound commonly used is DMH that specifically targets the colon of rats, where it induces DNA damage [15, 56], preneoplastic lesions and is detected as aberrant crypts and tumors [7, 63, 71].
The colon cancer in the present study was induced by intraperitoneal injection of a dose of DMH at a dose of 40 mg/kg body weight twice a week for 5 weeks. DMH was used as a potent and complete carcinogen for the colon, since it has been reliably used to induce the initiation and promotion steps of colon carcinogenesis after five doses over 5 successive weeks . Previous studies indicated that longer periods (twice a week for 8 or 12 weeks) of DMH treatment led to the development of colon carcinoma . However, in the present study the dose of DMH was sufficient to induce colon cancer in rats. Moreover, DMH-induced tumors share many histopathologic characteristics with human tumors .
Serum transaminases are considered to be sensitive indicators of liver injury. In DMH-induced cancer rats, the liver was necrotized. The hepatic damage was indicated by increases in ALP, ALT and AST levels. ALP, ALT, AST are reliable markers of liver function [19, 59, 60]. The increased serum ALP, ALT, AST levels were reported in cancer and it may be due to liver dysfunction . An increase in the ALT, AST levels in plasma might be mainly due to the leakage of these enzymes from the liver into the blood stream which gives an indication of the hepatotoxic effect [35, 67]. However, the ALP, ALT, AST levels were significantly elevated in DMH carcinogenic rats and consistent with other previous reports [4, 38]. Treatment of DMH carcinogenic rats with FOS1or FOS2 reduced the activity of ALP and ALT in plasma and consequently alleviated liver damage caused by DMH-induced colon cancer. Moreover, the values of ALT and AST of rats administered DMH reflected abnormal liver function. The value of ALT and AST activities in sera of rats received FOS1and FOS2 reflected their improvements of liver function. Liver damage induced by chronic treatment that leads to liver cell necrosis and consequently elevated levels of serum transaminases. Other investigators studied the hepatoprotective effects induced liver damage in rats [19, 34, 67]. The degree of protection was evaluated by determining the marker enzymes (AST, ALT and ALP) and total proteins. In the present study, higher decrease was observed in the levels of AST, ALT and ALP in sera of rats received FOS1or FOS2 (FOS1/C and FOS2/C) than those of rats treated with FOS1 and FOS2 (C/FOS1 and C/FOS2). These results are consistent to other studies [4, 49]. Administration of FOS1or FOS2 in DMH-induced rats showed significant reductions in serum protein, albumin and globulin due to inhibition of protein degradation .
Regarding, CEA and CA-19.9, they showed a marked decrease in rat groups treated with FOS1 or FOS2 before DMH-induced colon cancer more than the decrease shown in rat groups treated with these FOS1or FOS2 after DMH-induced tumor. Moreover, both rat groups are markedly decreased CEA and CA-19.9 levels, compared to carcinogenic control group (C). The results in the present study indicating the similar role of treatment with FOS as dietary fibre or polysaccharides should be considered [1, 47, 49].
DMH-induced colon cancer showed significantly decreased in the activities of GSH-T, GSH-P, GSH-R and SOD in plasma and tissue homogenates of liver, kidney and heart of control rat (group C). Oxidation phenomena have been implicated in many illnesses, such as diabetes mellitus, arteriosclerosis and cancer. Oxidation of DNA, proteins and lipids plays an important role in aging and in a wide range of common diseases, including cardiovascular, inflammatory and cancer [8, 19, 36, 69]. In the present study, the results showed that, GSH-R concentration in the liver tissue were significantly higher in rats treated with FOS1or FOS2 than that in rats received the carcinogenic material DMH (group C). The results of the present study indicated that, FOS1or FOS2 tend to improve GSH-R concentration in rat tissue homogenates. These results are consistent to studies reported by other investigators [33, 47, 59, 69].
The present results showed the activities of GSH-P and GSH-R were significant increases in liver and kidney of rat treated with FOS1 or FOS2 compared to DMH-induced colon cancer rat (C). Several studies reported that the natural products induced a significant increase in GSH-P and GSH-R activities and exerted a protective and antioxidant effects [40, 46, 47]. Moreover, the primary radicals, by donating hydrogen radicals, are reduced to non-radical chemical compounds and this action helps in protecting the body from degenerative diseases [34, 59, 69]. Recent studies on the antioxidant properties of some plant materials revealed their stimulatory action on antioxidative enzymes [47, 69]. Other studies demonstrate decrease in GSH-P activity in liver of rats [17, 29], they demonstrate alterations in the liver antioxidants in rats. Results obtained from the present study are very much promising and similar to the observation reported in streptozotocin induced diabetic rats [35, 40].
In the present experiment the activity of SOD in liver and kidney was investigated. SOD, one of the major antioxidant enzymes, decomposes superoxide peroxide, blocks lipid peroxidation and protect the tissue against oxidative damage [17, 33, 35]. Free radicals are produced in the body as the result of metabolic processes. The imbalance between radical-generating and radical scavenging systems produce oxidative stress [18, 34, 57]. Free radicals are the source of lipid peroxidation derived from oxygen, and the first line of defense against them is SOD . A study shows that antioxidant substances which scavenge free radicals play an important role in the prevention of free radical-induced diseases . The principal agents responsible for the protective effects could be the presence of antioxidant substances that exhibit their effects as free radical scavengers [18, 57, 75]. FOS1or FOS2 increases the activity of SOD and it scavenges superoxide radicals and reduces myocardial damage caused by free radicals . Hence, the increased SOD activity in liver (61–67%) and kidney (54–59%) suggests that the absence of accumulation of superoxide anion radical might be responsible for decreased lipid peroxidation in these tissues [34, 40]. This is also evident from the fact that relatively higher decrease in lipid peroxidation in liver and kidney of rats given FOS being accompanied by the relatively higher increase in SOD activity in these tissues , they reported that FOS had antioxidant activity and protected the organs from free radicals and might be retard the progress of diseases. However FOS1 or FOS2 had antioxidant activity and protected some organs from free radicals in the present study. These results are consistent with other investigators demonstrated alteration in liver antioxidants in rats [47, 49]. From these results, it appeared that there was a positive correlation with the FOS1 or FOS2 contents and SOD scavenging activity [9, 18].
The superoxide scavenging ability of the FOS1 or FOS2 may be due to the presence of kestose, nystose and frutosyl-nystose as reported by other investigators [8, 46, 47, 71]. Antioxidant dietary fibre can be defined as a fibre containing significant amounts of natural antioxidants associated to the fibre [29, 33]. The antioxidant activity of kestose,nystose and frutosyl- nystose compounds is mainly due to their properties like dietary fibre which play an important role as free radical scavengers [9, 18, 76]. kestose, nystose and frutosyl-nystose are very important FOS constituents because of their antioxidant activity [9, 69]. However, the chemical properties of FOS (FOS1or FOS2) in terms of the availability of FOS1 and FOS2 as radical scavengers predict their antioxidant activity. Treatment of rats orally with Nigella sativa extract (50 and 100 mg/kg body weight) resulted in significant decrease in lipid peroxidation, incidence of tumours, oxidative stress and renal carcinogenesis in rats . Several investigators concluded that, the hepatoprotective effects of Nigella sativa extract against oxidative damage may be due to its antioxidant and free radical-scavenging activity [40, 69]. The present study establishes that FOS1or FOS2 have appreciable anticancer and antioxidant activities. However, based on the published studies, administration of Onion (Allium cepa) and artichoke (Cynara scolymus) to man is simple, since, they are used as common dietary constituents in many parts of the world.
It is known that most drugs isolated for cancer therapy are not cancer specific and, therefore, may be higher toxic to normal tissues, leading to serious adverse effects. FOS1or FOS2 might be considered alternative sources for adjuvant cancer therapy, as they have no adverse effects, activate the cells of the immune system, and reduce free radicals. Further studies, however, including the isolation and chemical characterization of the major compounds that contribute to inhibition of carcinogenesis, are needed and may generate new targets for therapy. Moreover, the present study establishes that FOS of onion (FOS1) and artichoke (FOS2) have appreciable anti-cancer and antioxidant activities and may improve health.
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Accepted for print: 23.02.2016
Sorial Adly Moharib
Biochemistry Department, National Research Center, Dokki, Cairo, Egypt
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