fecal soluble substances toward Caco-2 cells, a model of colonocyte (36). All these results 5
suggest that KGM and inulin may reduce the oxidative challenge to the colonocytes due to 6
the antioxidant capacity of their fermentation products.
7 8
The effects of dietary fibers on hepatic antioxidant enzymes have been rarely examined.
9
As the oxidative stress induced by a cystine-rich fiber-free diet, pectin effectively retained the 10
total superoxide dismutase and Cu, Zn-superoxide dismutase activities in the liver (37). A 11
recent study indicated that fructo-oligosaccharide, similar to vitamin E, effectively reduced 12
the alterations in the hepatic superoxide dismutase and glutathione peroxidase activities in 13
mice subcutaneously administered with D-galactose (38). Our study demonstrates that 14
hepatic MDA level (nmole/mg protein) was effectively reduced by more than 30% with either 15
KGM or inulin, which was in agreement with the elevated gene expressions of the hepatic 16
superoxide dismutase and catalase. Therefore, previous and current studies support that the 17
utilization of dietary fiber in the large intestine exerts antioxidative effects in the liver.
18 19
The effect of fiber consumption on the blood antioxidative status also has not been well 20
studied. Oligofructoses have been shown to reduce the plasma thiobarbituric acid-reactive 21
substances (TBARS) and promote the plasma vitamin E/triglycerol levels in rats fed a 22
high-fructose diet (39). A placebo-controlled and diet-controlled trial indicated that 23
fructo-oligosaccharide supplement beneficially reduced the plasma TBARS in constipated 24
nursing-home residents (40). In agreement with these previous studies, the current study 25
indicates that both KGM and inulin diets reduced the index of plasma oxidative stress, the 26
DNA breakage of lymphocytes, and increased the plasma -tocopherol/total lipid level, as 27
compared with the fiber-free counterpart. Although the mechanism is unclear, the prebiotic 28
effect of KGM and inulin may partially contribute to the reduced blood oxidative status, as a 29
previous study showing decreased oxidative stress markers in human blood and urine with 30
probiotic-fermented milk consumption (41).
31 32
In conclusion, addition of 5% (w/w) KGM or inulin into a high-fat fiber-free diet 33
effectively up-regulated the gene expressions of antioxidant enzymes throughout the colon, 34
and in the liver. The in vivo antioxidative effects of dietary fibers may reach the whole body 35
by reducing the oxidative damage towards the blood lymphocytes and increasing plasma 36
antioxidant levels. The local and systemic antioxidative effects of KGM and inulin are likely 37
to be related to their fermentation in the cecum and colon, and their prebiotic effects.
38 39
ABBREVIATIONS USED 40
KGM, konjac glucomannan; MDA, malondialdehyde; ROS, reactive oxidative 41
substances; FPG, formamidopyrimidine glycosylase; PCR, polymerase chain reaction.
42 43
ACKNOWLEGEMENT 44
The research was supported by grants from the National Science Council 45
(NSC-99-2320-B-040-014-MY2), Republic of China.
46 47
Literature Cited 48
(1) Kehrer, J. P. Free radicals as mediators of tissue injury and disease. Crit. Rev.
49
Toxicol. 1993, 23, 21-48.
50
(2) Halliwell, B. Free radicals, antioxidants, and human disease: curiosity, cause, or 1
consequence? Lancet 1994, 344, 721-724.
2
(3) Noda, Y.; Kaneyuki, T.; Mori, A.; Packer, L. Antioxidant activities of pomegranate fruit 3
extract and its anthocyanidins: delphinidin, cyanidin, and pelargonidin. J. Agric. Food 4
Chem. 2002, 50, 166-171.
5
(4) Henning, S. M.; Niu, Y.; Lee, N. H.; Thames, G. D.; Minutti, R. R.; Wang, H.; Go, V. L.;
6
Heber, D. Bioavailability and antioxidant activity of tea flavanols after consumption of 7
green tea, black tea, or a green tea extract supplement. Am. J. Clin. Nutr. 2004, 80, 8
1558-1564.
9
(5) Jaramillo, K.; Dawid, C.; Hofmann, T.; Fujimoto, Y.; Osorio, C. Identification of 10
antioxidative flavonols and anthocyanins in Sicana odorifera fruit peel. J. Agric. Food 11
Chem. 2011, 59, 975-983.
12
(6) Cummings, J. H. The effect of dietary fiber on fecal weight and composition. In Dietary 13
fiber in human nutrition; Spiller, G., Ed.; CRC Press, New York, 2001; pp 183-252.
14
(7) Beylot, M. Effects of inulin-type fructans on lipid metabolism in man and in animal 15
models. Br. J. Nutr. 2005, 93, s163-s168.
16
(8) Qi, L.; van Dam, R. M.; Liu, S.; Franz, M.; Mantzoros, S.; Hu, F. B. Whole-grain, bran, 17
and cereal fiber intakes and markers of systemic inflammation in diabetic women.
18
Diabetes Care 2006, 29, 207-211.
19
(9) Babbs, C. F. Free radicals and the etiology of colon cancer. Free Radic. Biol. Med. 1990, 20
8, 191-200.
21
(10) Seril, D. N.; Liao, J.; Yang, G.Y.; Yang, C. S. Oxidative stress and ulcerative 22
colitis-associated carcinogenesis: studies in humans and animal models. Carcinogenesis 23
2003, 24, 353-362.
24
(11) Kong, F.; Zhang, M.; Liao, S.; Yu, S.; Chi, J.; Wei, Z. Antioxidant activity of 25
polysaccharide-enriched fractions extracted from pulp tissue of Litchi Chinensis Sonn.
26
Molecules 2010, 15, 2152-2165.
27
(12) Shimada, K.; Fujikawa, K.; Yahara, K.; Nakamura, T. Antioxidative properties of 28
xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J. Agric. Food Chem.
29
1992, 40, 945-948.
30
(13) Doi, K. Effect of konjac fibre (glucomannan) on glucose and lipids. Eur. J. Clin. Nutr.
31
1995, 49, s190-s197.
32
(14) Wang, C.-H.; Lai, P.; Chen, M.-E.; Chen, H.-L. Antioxidative capacity produced by 33
Bifidobacterium- and Lactobacillus acidophilus-mediated fermentations of konjac 34
glucomannan and glucomannan oligosaccharides. J. Sci. Food Agric. 2008, 88, 35
1294-1300.
36
(15) Chen, H.-L.; Lin, Y.-M.; Wang, Y.-C. Comparative effects of cellulose and soluble fibers 37
(pectin, konjac glucomannan, inulin) on fecal water toxicity toward Caco-2 cells, fecal 38
bacteria enzymes, bile acid, and short-chain fatty acids. J. Agric. Food Chem. 2010, 58, 39
10277-10281.
40
(16) Wu, W.-T.; Chen, H.-L. Effects of konjac glucomannan on putative risk factors for colon 41
carcinogenesis in rats fed a high-fat diet. J. Agric. Food Chem. 2011, 59, 989-994.
42
(17) Guide for the Care and Use of Laboratory Animals;Publication 85-23, rev.; National 43
Research Council, National Institute of Health: Bethesda, MD, 1985.
44
(18) Report of the American Institute of Nutrition ad hoc committee on standards for 45
nutritional studies. J. Nutr. 1977, 107, 1340-1348.
46
(19) Ching, S. Y.; Prins, A. W.; Beilby, J. P. Stability of ascorbic acid in serum and plasma 47
prior to analysis. Ann. Clin. Biochem. 2002, 39, 518-520.
48
(20) Lee, H. S.; Csallany, A. S. Measurement of free and bound malondialdehyde in vitamin 49
E-deficient and -supplemented rat liver tissues. Lipids 1987, 22, 104-107.
50
(21) Catignani, G. L.; Bieri, J. G. Simultaneous determination of retinol and alpha-tocopherol 1
in serum or plasma by liquid chromatography. Clin. Chem. 1983, 29, 708-712.
2
(22) Frings, C. S; Dunn, R. T. A colorimetric method for determination of total serum lipids 3
based on the sulfo-phospho-vanillin reaction. Am. J. Clin. Pathol. 1970, 53, 89-91.
4
(23) Chuang, C.-H.; Hu, M.-L. Use of whole blood directly for single-cell gel electrophoresis 5
(comet) assay in vivo and white blood cells for in vitro assay. Mutat. Res. 2004, 564, 6
75-82.
7
(24) Singh, N. P.; McCoy, M. T.; Tice, R. R.; Schneider, E. L. A simple technique for 8
quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 1988, 175, 9
184-191.
10
(25) Wu, W.-T.; Cheng, H.-C.; Chen, H.-L. Ameliorative effects of konjac glucomannan on 11
human faecal -glucuronidase activity, secondary bile acid levels and faecal water 12
toxicity towards Caco-2 cells. Br. J. Nutr. 2010, 105, 593-600.
13
(26) Phillips, H. J. Dye exclusion tests for cell viability. In Tissue Culture. Method and 14
Applications; Kruse, P. F.; Patterson, M. K., Eds.; Academic Press, New York, 1973; pp 15
2973.
16
(27) Collins, A. R.; Ma, A. G.; Duthie, S. J. The kinetics of repair of oxidative DNA damage 17
(strand breaks and oxidised pyrimidines) in human cells. Mutat. Res. 1995, 336, 69-77.
18
(28) Lopez-Oliva, M. E.; Agis-Torres, A.; Goñi, I.; Muñoz-Martinez, E. Grape antioxidant 19
dietary fibre reduced apoptosis and induced a pro-reducing shift in the glutathione redox 20
state of the rat proximal colonic mucosa. Br. J. Nutr. 2010, 103, 1110-1117.
21
(29) Stein, K.; Borowicki, A.; Scharlau, D.; Glei, M. Fermented wheat aleurone induces 22
enzymes involved in detoxification of carcinogens and in antioxidative defence in human 23
colon cells. Br. J. Nutr. 2010, 104, 1101-1111.
24
(30) Roediger, W. E., Utilization of nutrients by isolated epithelial cells of the rat colon.
25
Gastroenterology 1982, 83, 424-429.
26
(31) Scharlau, D.; Borowicki, A.; Habermann, N.; Hofmann, T.; Klenow, S.; Miene, C.;
27
Munjal, U.; Stein, K.; Glei, M. Mechanisms of primary cancer prevention by butyrate and 28
other products formed during gut flora-mediated fermentation of dietary fibre. Mutat. Res.
29
2009, 682, 39-53.
30
(32) Rosignoli, P.; Fabiani, R.; De Bartolomeo, A.; Spinozzi, F.; Agea, E.; Pelli, M. A.;
31
Morozzi, G. Protective activity of butyrate on hydrogen peroxide-induced DNA damage 32
in isolated human colonocytes and HT29 tumour cells. Carcinogenesis 2001, 22, 33
1675-1680.
34
(33) Vanhoutvin, S. A.; Troost, F. J.; Kilkens, T. O.; Lindsey, P. J.; Hamer, H. M.; Jonkers, D.
35
M.; Venema, K.; Brummer, R. J. The effects of butyrate enemas on visceral perception in 36
healthy volunteers. Neurogastroenterol Motil. 2009, 21, 952-e76.
37
(34) Kullisaar, T.; Zilmer, M.; Mikelsaar, M.; Vihalemm, T.; Annuk, H.; Kairane, C.; Kilk, A.
38
Two antioxidative lactobacilli strains as promising probiotics. Int. J. Food Microbiol.
39
2002, 72, 215-224.
40
(35) Sun, J.; Hu, X. L.; Le, G. W.; Shi, Y. H. Lactobacilli prevent hydroxy radical production 41
and inhibit Escherichia coli and Enterococcus growth in system mimicking colon 42
fermentation. Lett. Appl. Microbiol. 2010, 50, 264-269.
43
(36) Yeh, S.-L.; Lin, M.-S.; Chen, H.-L. Inhibitory effects of a soluble dietary fiber from 44
Amorphophallus konjac on cytotoxicity and DNA damage induced by fecal water in 45
Caco-2 cells. Planta Med. 2007, 73, 1384-1388.
46
(37) He, G.; Aoyama, Y. Effects of adding some dietary fibers to a cystine diet on the 47
activities of liver antioxidant enzymes and serum enzymes in rats. Biosci. Biotechnol.
48
Biochem. 2003, 67, 617-621.
49
(38) Chen, H.-L.; Wang, C.-H.; Kuo, Y.-W.; Tsai, C.-H. Antioxidative and hepatoprotective 50
effects of fructo-oligosaccharide in D-galactose-treated Balb/cJ mice. Br. J. Nutr. 2011, 1
105, 805-809.
2
(39) Busserolles, J.; Gueux, E.; Rock, E.; Demigné, C.; Mazur, A.; Rayssiguier, Y.
3
Oligofructose protects against the hypertriglyceridemic and pro-oxidative effects of a 4
high fructose diet in rats. J. Nutr. 2003, 133, 1903-1908.
5
(40) Yen, C.-H.; Kuo, Y.-W.; Tseng, Y.-H.; Lee, M.-C.; Chen, H.-L. Beneficial effects of 6
fructo-oligosaccharides supplementation on fecal bifidobacteria and index of peroxidation 7
status in constipated nursing-home residents—A placebo-controlled, diet-controlled trial.
8
Nutrition 2011, 27, 323-328.
9
(41) Kullisaar, T.; Songisepp, E.; Mikelsaar, M.; Zilmer, K.; Vihalemm, T.; Zilmer, M.
10
Antioxidative probiotic fermented goats' milk decreases oxidative stress-mediated 11
atherogenicity in human subjects. Br. J. Nutr. 2003, 90, 449-456.
12 13
Figure Legend 14
15
Figure 1. DNA damage of lymphocytes with basal condition or in combination with 1 g/mL 16
FPG (basal + FPG). Lymphocytes were isolated from Sprague-Dawley rats fed a high corn oil 17
diets containing no fiber (white bar), 5% (w/w) KGM (gray bar) or inulin (black bar). The 18
DNA damage was determined by comet assay and expressed as tail moment. Bars are 19
presented as mean ± SE (n = 8 animals per group). Different letters denote significant 20
differences across groups as analyzed by one-way ANOVA followed by Duncan test (p 21
0.05). KGM, konjac glucomannan; FPG, formamidopyrimidine DNA-glycosylase.
22 23
Figure 2. The gene expression of glutathione peroxidase (GPx), superoxide dismutase (SOD), 24
and catalase (CAT) in the (A) proximal, (B) distal colonic mucosa, and (C) liver in 25
Sprague-Dawley rats fed a high corn oil diet containing no fiber, 5% (w/w) KGM or inulin.
26
Data (mean ± SE, colonic mucosa, n = 4 animals per group; liver, n = 8 animals per group) 27
are reported as fold difference with respect to fiber-free diet after normalization for GAPDH.
28
Different superscript letters denote significant differences across groups as analyzed by one- 29
way ANOVA followed by Duncan test (p ≤ 0.05). KGM, konjac glucomannan.
30
Table 1. Composition of Experimental Dietsa 1
Fiber-free KGM Inulin
Ingredients — g/kg diet —
Corn starch 500 437.5 441.5 Casein 200 200 200 Corn oil 250 250 250
Inulinb - - 58.5
KGMc - 62.5 -
Methioine 3 3 3
Choline 2 2 2 AIN Mineral Mix 76-A 35 35 35 AIN Vitamin Mix 76-A 10 10 10 Total energy (MJ/kg diet) 21.1 20.1 20.1
aThe diets were modified from AIN-76 (18).
2
bThe purity of inulin was 85.5%.
3
cThe purity of KGM was 94.8%.
4 5 6
Table 2. MDA Levels in the Proximal Colon, Distal Colon, and Liver of 7
Sprague-Dawley Rats Fed a High Corn Oil Diet Containing no Fiber, 5% (w/w) 8
KGM or Inulina 9
Fiber-free KGM Inulin
— nmole/mg Protein — Colon
Proximal 2.72 ± 0.19b 2.31 ± 0.15b 1.56 ± 0.16a Distal 3.79 ± 0.33b 2.65 ± 0.24a 3.03 ± 0.28ab Liver 1.03 ± 0.12b 0.68 ± 0.04a 0.59 ± 0.06a
aData are presented asmeans ± SE (n = 8 animals per group). Different 10
superscript letters denote significant differences across groups as analyzed by one 11
way ANOVA followed by Duncan test (p＜0.05) 12
13 14
Table 3. Plasma -Tocopherol, Ascorbic Acid and MDA Levels in Sprague-Dawley 15
Rats Fed a High Corn Oil Diet Containing no Fiber, 5% (w/w) Konjac Glucomannan 16
or Inulina 17
Fiber-free KGM Inulin
-tocopherol (mole/g Plasma Lipid) 1.87 ± 0.23a 3.10 ± 0.24b 2.63 ± 0.28b Ascorbic acid (mM) 0.20 ± 0.04 0.21 ± 0.03 0.19 ± 0.03
MDA (M) 8.2 ± 0.6 7.9 ± 0.5 7.4 ± 0.5
aData are expressed as mean ± SE (n = 8 animals per group). Different superscript 18
letters denote for significant differences across groups as analyzed by one-way 1
ANOVA followed by Duncan test (p＜0.05).
2 3
Table 4. Concentration, Daily Excretion and Relative Proportion of Fecal 4
Short-Chain Fatty Acids in Sprague-Dawley Rats Fed a High Corn Oil Diet 5
Containing no Fiber, 5% (w/w) KGM or Inulina 6
Fiber-free KGM Inulin
Concentration (mole/g of Wet Feces)
Acetate 64.3 ± 7.9a 104.4 ± 6.8b 90.9 ± 11.0ab Propionate 10.9 ± 2.2 22.4 ± 4.9 12.2 ± 3.3 Butyrate 5.1 ± 1.1a 9.9 ± 0.8b 13.2 ± 0.9c Total SCFAb 80.3 ± 10.5a 136.7 ± 11.7b 116.2 ± 13.9ab
Daily Fecal Excretion (mole/Day)
Acetate 155.2 ± 18.6a 389.9 ± 16.8c 244.7 ± 30.0b Propionate 26.4 ± 6.0a 84.4 ± 25.1b 29.6 ± 5.6a Butyrate 12.3 ± 2.9a 38.8 ± 4.9b 35.5 ± 2.0b Total SCFA 193.8 ± 26.0a 513.2 ± 40.7c 309.8 ± 30.6b
Relative Ratio (% total)
Acetate 80.9 ± 2.8 77.3 ± 2.0 78.0 ± 2.1 Propionate 13.1 ± 1.9 15.5 ± 2.1 9.5 ± 1.7 Butyrate 6.1 ± 0.9a 7.3 ± 0.3a 12.5 ± 2.0b
aData are presented asmean ± SE (n = 8 animals per group). Different superscript 7
letters denote significant differences between treatments as analyzed by one-way 8
ANOVA followed by Duncan test (p＜0.05).
9
bSum of acetate, propionate, and butyrate 10
11 12 13
0 2 4 6 8 10
Basal Basal + FPG FPG lesion Fiber-free KGM Inulin
1
DNA damage (Tail moment)
a b a
a
a b
a b c
Figure 1
0 1 2 3 4 5 6
GPx SOD CAT
Fiber-free KGM Inulin
0 1 2 3 4
GPx SOD CAT
Fiber-free KGM Inulin
0.0 0.5 1.0 1.5 2.0 2.5
GPx SOD CAT
Fiber-free KGM Inulin
A
B
C
a b
b
a
b b a
b b
a
b b
Relative expressionRelative expression
b b
Relative expression
a
Figure 2