Food Sci. Technol. Res., ++ (.), -10ῌ-13, ,**/

Konjac Glucomannan Consumption May Enhance Production in Mice

῍ Motoi TAMURA , Tojiro TSUSHIDA and Kazuki SHINOHARA

National Food Research Institute, ,ῌ+ῌ+, Kannondai, Tsukuba, Ibaraki -*/ῌ20.,, Japan

Received September ,,, ,**/; Acepted November +., ,**/

The e#ects of konjac glucomannan on isoflavonoid levels in the plasma and cecum were assessed in adult mice. Male /-week-old mice were fed on a konjac glucomannan-isoflavone (KM) diet or a cellulose- isoflavone (control) diet for three weeks. After this period, plasma equol levels were significantly higher in the KM group, but there was no significant di#erence in the plasma concentration. The ratio of plasma equol to plasma daidzein in the KM group was significantly higher than in the control group. The total amount of equol present as aglycone in the cecum was significantly greater in the KM group, but there was no significant di#erence in the total daidzein present as cecal aglycone. The total amount of present as aglycone in the cecum was significantly greater in the KM group. We have demonstrated that the ingestion of konjac glucomannan may enhance equol production by a#ecting the metabolic activity of intestinal microflora.

Keywords: daidzein, equol, mouse

Introduction Ltd. (Osaka, Japan). It contained -+.+ῌ (w/w) , Much attention has been focused on the health benefits 3.0ῌ (w/w) and -2./ῌ (w/w) glycitin. Daidzein of soy-based foods, which have been largely attributed to and equol used as standards for HPLC analysis were isoflavones. Daidzin, glycitin, daidzein (the aglycone of purchased from LC laboratories (Woburn, MA, USA). daidzin) and glycitein (the aglycone of glycitin) are Glycitein and konjac glucomannan were purchased from isoflavones found in soy products. Equol is a bacterial Wako Pure Chemical Industries, Ltd. (Osaka, Japan). metabolite of the widespread isoflavone daidzein (Bowey Beta-glucuronidase type H-/ was obtained from Sigma et al., ,**-). These isoflavonoids are estrogenic com- (St. Louis, MO, USA). pounds (). In animals, phytoestrogens Treatment of animals Male Crj: CD-+ (ICR) mice (/ exert estrogenic e#ects on the central nervous system, weeks old) were purchased from Charles River Japan, Inc. induce estrus and stimulate the growth of the female (Kanagawa, Japan). All mice were specific pathogen-free genital tract (Lieberman, +330). Investigating the absorp- (SPF) and the animals were housed in conventional condi- tion and metabolism of isoflavonoids is essential for un- tions in our laboratory. The mice were randomly divid- derstanding their biological activity. ed into two groups of six animals each. The animals Akaza et al., (,**,) reported that either equol itself or were housed in suspended stainless-steel cages with wire some unknown factor regulating the metabolism of mesh bottoms, in a room kept at ,.῍*./῎ and a relative daidzein is deeply involved in prostate cancer, which is humidity of 0/ῌ, with +,-hr periods of light and dark. known to be responsive to estrogen therapy. Previous They were fed an AIN 10 diet for one week. After one studies have shown that only about -*ῌ.*ῌ of individuals week, the diet was replaced with a konjac glucomannan- excrete significant quantities of equol after isoflavone isoflavone (KM) diet or cellulose-isoflavone (control) diet consumption (Lampe et al., +332; Setchell, et al., +32.). which was continued for three weeks. The composition Human gastrointestinal bacteria seem to play an impor- of each diet is shown in Table +. The KM diet contained tant role in equol production and daidzein metabolism. /ῌ konjac glucomannan and *..ῌ isoflavone mixture. As there are few reports of the e#ects of konjac The control diet contained /ῌ cellulose and *..ῌ glucomannan on cecal and plasma isoflavonoids, the aim isoflavone mixture. Body weight and food consumption of the present study was to investigate these e#ects in were measured during the experiment. After being fed mice. on the experimental diets for the specified period, the mice were sacrificed and blood and cecal contents were Materials and methods collected. The blood samples were then centrifuged, and Materials A extract with a high isoflavone- the plasma was stored at ῌ2*῎ until HPLC analysis for glucoside content was kindly provided by Fuji Oil Co., isoflavonoids. Cecal contents were stored at ῌ2*῎ until HPLC analysis for isoflavonoids. ῍ To whom correspondence should be addressed. This study was carried out in accordance with the E-mail: motoita@a#rc.go.jp “Guidelines for Animal Care and Experimentation” of the Konjac Glucomannan May Enhance Equol Production 377

Table +. Composition of the experimental diet.

National Food Research Institute. For analysis of cecal isoflavone aglycones, cecal con- Analysis of plasma and cecal isoflavonoids The anal- tents were treated with , mL of methanol/acetic acid ysis of plasma isoflavonoids was performed as follows. (+**//, v/v), vortexed for -* s, sonicated for -* s, vortexed A total of ,** mL plasma was added to ,** mL of beta- again for -* s, and centrifuged at /***῎g for +* min. The glucuronidase type H-/ solution (-/ mg/mL, Sigma, MO, supernatant was then transferred to an eggplant-type USA) in *., M sodium acetate bu#er (pH /.*). Next, the flask. Methanol/acetic acid (+**//, v/v), at the same mixture was incubated at -1῏ in a water bath for , hr with volume used in the first extraction, was added to the shaking, followed by treatment with -0** mL of methanol/ sediment, and the procedure was repeated. The superna- acetic acid (+**//, v/v), vortexing for -* s, sonication for tants from both extractions were pooled and evaporated -* s, vortexing again for -* s, and centrifugation at /***῎ completely using a rotary evaporator. The sample was g for +* min at .῏. The supernatants were transferred to then dissolved in *./ mL of the mobile phase of the HPLC an eggplant-type flask and evaporated completely using a system and filtered through a *., mm filter. HPLC analy- rotary evaporator. The sample was then dissolved in .** sis was carried out using the same method as described mLof2*ῌ methanol and filtered through a *.,-micrometer above. filter. During HPLC analysis, ,* mL of each preparation Statistics The data were expressed as means῍SE. was injected into a ,/*῎..0 mm Capcell Pak C+2-/ m Data were analysed using SigmaStat for windows (Jandel column (Shiseido, Tokyo, Japan). Isoflavonoids were Corporation, San Rafael, CA, USA) and t-test analysis. detected using an ECD with a guard cell (model /*,*), and an analytical cell (model /*+*) (Coulochem II; ESA Inc., Results Bedford, MA, USA). The mobile phase consisted of meth- Body weight, food consumption, and cecal contents No anol/acetic acid/water (-/: /: 0*, v/v/v). The HPLC con- significant di#erence in final body weight (g) was ob- ditions were as follows: column temperature, .*῏;flow served between the KM (.*.3῍+.+) and control (.+.1῍*.1) rate, + mL/min; guard cell, ῌ2/* mV; analytical cell, ῌ groups. No significant di#erence in food consumption -** mV for electrode + and ῌ2** mV for electrode ,. (g/day) was observed between the KM and control Electrochemical data were collected from electrode ,. groups. Significant di#erences were found in the cecal 378 M. TAMURA et al.

Fig. ,. Ratio of equol/daidzein in plasma of KM and Fig. +. Plasma isoflavonoids (aglyconesῌmetabolites) of control-group mice. Values are means῍SE. ῌ Significant mice fed on KM and control diets. Values are means῍SE. di#erence (p῎*.*/) from control group. ῌῌ Significant di#erence (p῎*.*+) from control group.

contents of the KM (*..2῍*.*.) and control (*.,*῍*.*-) groups (p῎*.**+). Plasma isoflavones and cecal isoflavone aglycones The plasma equol concentration was significantly higher in the KM group compared to the control group (Fig. +). Conversely, there were no significant di#erences in plasma daidzein and glycitein concentrations between the KM and control groups (Fig. +). There was a significant di#erence between the KM and control groups in the ratio of plasma equol to plasma daidzein (Fig. ,). The plasma equol/plasma daidzein ratio in the KM group was significantly higher than that of the control group. The total amount of equol present as aglycone in the cecum was significantly higher in the KM group than in the control group (Fig. -). However, there was no significant - di#erence in the total amount of daidzein present as agly- Fig. . Amounts of isoflavonoids present as aglycone in the cecum of mice in the KM and control groups. Values cone in the cecum (Fig. -). The total amount of glycitein are means῍SE. ῌῌ Significant di#erence (p῎*.*+) from present as aglycone in the cecum was significantly higher control group. in the KM group than in the control group (Fig. -).

Discussion Daidzein is a major component of isoflavones, and is control group, which suggests that equol productivity metabolized to equol by intestinal bacterial flora (Bowey was higher in the KM group than in the control group. et al., ,**-). Both compounds have an estrogenic e#ect It has been reported that konjac glucomannan undergoes and may contribute to protection against cardiovascular fermentation by human intestinal anaerobic bacteria to disease. It has been reported that equol is more es- produce formic acid, acetic acid, propionic acid, and +- trogenic than daidzein (Shutt and Cox, +31,). In our study, butyric acid (Matsuura et al., +332), and that it may modify plasma equol concentration was found to be significantly microbial metabolism (Fujiwara et al., +33+). From these higher in the KM diet group than in the control diet reports it may be deduced that konjac glucomannan is group. The amounts of equol existing as aglycone in the utilized by intestinal flora, and a#ects their metabolic cecum were also significantly greater in the KM diet activity. The konjac glucomannan that was used in the group than in the control diet group. KM diet is a water-soluble fiber, while the cellulose used Our results suggest that konjac glucomannan con- in the control group diet is a water-insoluble fiber. sumption may enhance the estrogenic e#ects of dietary Tamura et al., (+333) demonstrated that di#erent types of isoflavone in mice. We found that the plasma equol/ fiber have di#erent e#ects on the enzyme activities of daidzein ratio was higher in the KM group than in the cecal flora in mice. The e#ects of konjac glucomannan Konjac Glucomannan May Enhance Equol Production 379 on intestinal flora may be di#erent from those of cellu- isoflavones and lignans by the gut microflora: a study in germ- lose. A previous study has already shown that intestinal free and human flora associated rats. Food Chem. Toxicol., .+, 0-+ῌ0-0 flora have a significant e#ect on isoflavone metabolism in . Duncan, A.M., Merz-Demlow, B.E., Xu, X., Phipps, W.R. and Kurzer, vivo (Xu et al., +33/). Di#erent methods of daidzein metab- M.S. (,***). Premenopausal equol excretors show plasma hor- # olism by intestinal flora may be caused by di erent kinds mone profiles associated with lowered risk of breast cancer. of dietary fiber. Cancer Epidemiol. Biomarkers. Prev., 3, /2+ῌ/20. The amounts of glycitein present as aglycone in the Fujiwara, S., Hirota, T., Nakazato, H., Muzutani, T. and Mitsuoka, cecum of KM group were significantly greater than those T. (+33+). E#ect of konjac mannan on intestinal microbial of control group. As glycitein has a di#erent chemical metabolism in mice bearing human flora and in conventional F -.. ,3 0*+ῌ0*0 structure from daidzein, the degradation of cecal glycitein rats. Food Chem. Toxicol., , . Lampe, J.W., Karr, S.C., Hutchins, A.M. and Slavin, J.L. (+332). by intestinal flora may be di#erent from that of daidzein, Urinary equol excretion with a soy challenge: influence of which means that the degree of degradation of cecal habitual diet. Proc. Soc. Exp. Biol. Med., ,+1, --/ῌ--3 (+332). # glycitein may be lower in the KM group. Di erences in Lieberman, S. (+330). Are the di#erences between estradiol and glycitein metabolism by intestinal flora may also be other estrogens, naturally occurring or synthetic, merely caused by di#erent kinds of dietary fiber. semantical? J. Clin. Endocrinol. Metab., 2+, 2/*ῌ2/+. There is much evidence to support the hypothesis that Matsuura, Y. (+332). Degradation of konjac glucomannan by en- an adequate intake of isoflavonoids reduces cancer risk zymes in human feces and formation of short-chain fatty acids by intestinal anaerobic bacteria. J. Nutr. Sci. Vitaminol., .., .,-ῌ (Duncan et al., ,***; Akaza et al., ,**,). For this e#ect, .-0. the maintenance of a constant level of equol may be Setchell, K.D., Borriello, S.P., Hulme, P., Kirk, D.N. and Axelson, M. important. We have demonstrated for the first time that (+32.). Nonsteroidal estrogens of dietary origin: possible roles the ingestion of konjac glucomannan may enhance equol in hormone-dependent disease. Am. J. Clin. Nutr., .*, /03ῌ/12. production by a#ecting the metabolic activity of intesti- Shutt, D.A. and Cox, R.I. (+31,). Steroid and phyto-oestrogen nal microflora. binding to sheep uterine receptors in vitro. J. Endocrinol., /,, ,33ῌ-+*. +333 # References Tamura, M., Hirayama, K. and Itoh, K. ( ). E ects of guar gum and cellulose on cecal enzyme activity and cecal short-chain Akaza, H., Miyanaga, N., Takashima, N., Naito, S., Hirao, Y., fatty acids in young and aged mice. Ann. Nutr. Metab., .-, 0*ῌ Tsukamoto, T. and Mori, M. (,**,). Is daidzein non-metabolizer 0/. a high risk for prostate cancer? A case-controlled study of Xu, X., Harris, K.S., Wang, H.J., Murphy, P.A. and Hendrich, S. serum soybean isoflavone concentration. Jpn. J. Clin. Oncol., (+33/). Bioavailability of soybean isoflavones depends upon gut -,, ,30ῌ-**. microflora in women. J. Nutr., +,/, ,-*1ῌ,-+/. Bowey, E., Adlercreutz, H. and Rowland, I. (,**-). Metabolism of