The role of intestinal microflora on daidzein excretion in urine in humans treated with Chungpesagantang.

Abstract

This study examined the relationship between the metabolism of the constituents of herbal medicines by human intestinal microflora and the level of metabolites excreted in the urine. This was performed by administering Chungpesagantang (CST) to volunteers and measuring their fecal metabolic activity CST to and urine excretion of daidzein, one of the metabolite of CST The metabolic activity of of CST dadizein was 54.8 +/- 16.7 mmol/h/g wet feces. When CST was administered orally to the subjects, the amount of daidzein excreted in the urine over 24 h was 103.7 +/- 55.8 mg, which accounted for 20.2% of the puerarin, daidzin and daidzein contained in CST. However, neither puerarin nor daidzin were excreted in the urine. The profile of daidzein excreted in the urine was found to be in proportion to that of the metabolic activity of the CST components. This suggests that the daidzein level excreted in the urine of the subjects administered CST is associated with the daidzein glycoside-hydrolyzing activity of the fecal microflora.

Full text

Page 1

Arch Pharm Res Vol 30, No 4, 481-485, 2007
481
http://apr.psk.or.kr
The Role of Intestinal Microflora on Daidzein Excretion in Urine
in Humans Treated with Chungpesagantang
Young-Suk Kim, Hwa-Jun Lee, Ki-Ho Cho, Hyung-Sup Bae, Eun-Kyung Park1, and Dong-Hyun Kim1
College of Oriental Medicine, Kyung Hee University, Seoul 130-701, Korea and 1College of Pharmacy, Kyung Hee
University, Seoul 130-701, Korea
(Received October 12, 2006)
This study examined the relationship between the metabolism of the constituents of herbal
medicines by human intestinal microflora and the level of metabolites excreted in the urine.
This was performed by administering Chungpesagantang (CST) to volunteers and measuring
their fecal metabolic activity CST to and urine excretion of daidzein, one of the metabolite of
CST. The metabolic activity of of CST dadizein was 54.8 ± 16.7 mmol/h/g wet feces. When
CST was administered orally to the subjects, the amount of daidzein excreted in the urine over
24 h was 103.7 ± 55.8 mg, which accounted for 20.2% of the puerarin, daidzin and daidzein
contained in CST. However, neither puerarin nor daidzin were excreted in the urine. The pro-
file of daidzein excreted in the urine was found to be in proportion to that of the metabolic activ-
ity of the CST components. This suggests that the daidzein level excreted in the urine of the
subjects administered CST is associated with the daidzein glycoside-hydrolyzing activity of the
fecal microflora.
Key words: Chungpesagantang, Intestinal microflora, Metabolism, Daidzein, Excretion
INTRODUCTION
Functional foods and herbal medicines are commonly
consumed by humans. Therefore, their components are
inevitably brought into contact with the microflora of the
alimentary tract and are transformed before they are
absorbed (Kobashi and Akao, 1997; Kim, 2002).
All individuals have their own characteristic indigenous
strains of intestinal bacteria. Newly ingested bacteria cannot
necessarily colonize and proliferate in the intestine.
Therefore, an individual’s intestinal microflora in feces are
believed to be fairly stable over time in the absence of
disease and/or antimicrobial therapy (Cole et al., 1985;
Rumney and Rowland, 1992; Simon and Gorbach, 1986).
Ikeda et al. reported that the activity of some intestinal
bacterial enzymes does not appear to be associated with
specific populations (Ikeda et al., 1994). These fecal
bacterial enzyme activities are affected by diet (Ikeda et
al., 1994; Ling et al., 1994), but this effect is reversed if
the diet or supplements are stopped for a short time
(Goldin et al., 1980; Reddy et al., 1980). Kobashi et al
(1984). reported that there are significant individual
variations in some enzymes of intestinal bacteria. We
previously reported that some fecal bacterial enzymatic
activities related to the pharmacological actions of herbal
medicines vary between individuals (Yim et al., 2004).
The rhizome of Pueraria thunbergiana (family Le-
guminosae) is frequently used as a functional food or an
ingredient of herbal formulae (Lee, 1996; Jung et al.,
2003). Its main components are puerarin and daidzin,
which are daidzein glycosides. Kim et al. (1998) reported
that puerarin and daidzin are metabolized to daidzein by
human intestinal microflora. The in vitro anti-tumor, anti-
allergic and antioxidant activity of the metabolite, daidzein,
are higher than that of puerarin and daidzin. Therefore,
the components of arrowroot may need to be metabolized
by intestinal microflora in order to be pharmacologically
active. Nevertheless, the relationship between the meta-
bolism of the components of herbal medicines by human
intestinal microflora and the amount of absorbed or
excreted metabolites has not been studied.
Chungpesagantang (CST) is a formula containing the
rhizome of Pueraria thunbergiana that is frequently used
in Korea to prevent stroke (Jung et al., 2003; Park, 2003).
This study examined the metabolism of the isoflavone
Correspondence to: Dong-Hyun Kim, College of Pharmacy,
Kyung-Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701,
Korea
Fax: 82-2-957-5030
E-mail: dhkim@khu.ac.kr

Page 2

482 Y.-S. Kim et al.
glycosides of CST by human fecal microflora. In addition,
the amount of their metabolite, daidzein, excreted in the
urine of human subjects administered CST was measured,
and the relationship between the metabolic activity and
the amount of metabolite excreted in the urine was
determined.
MATERIALS AND METHODS
Materials
p-Nitrophenyl-β-D-glucopyranoside was purchased from
Sigma Co. (St. Louis, MO, U.S.A.). 52 g CST, a herbal
mixture containing 16 g arrowroot (the rhizome of Pueraria
thunbergiana), 8 g Scutellaria baicalensis rhizome, 8 g
Angelica tenussima rhizome, 4 g Cimicifuga heracleifolia
rhizome, 4 g Angelcai dahurica rhizome, 4 g Planticodon
gradiforum rhizome, 4 g Raphanus satius semen and 4g
Rheum palmatum rhizome, was extracted with 10 times
the total weight of water for 1 h and freeze-dried (yield,
21.2%: 3.93% puerarin, 0.42% daidzin and 0.31% ber-
berine).
Subjects
Fourteen male volunteers (age: 20-30 years; body
weight: 61-77 kg; height: 169-181 cm), who were found to
be healthy according to their medical history, physical
examination, electrocardiography, and laboratory testing,
were enrolled in this study. The exclusion criteria included
smoking and current medication, particularly the regular or
current use of antibiotics. All subjects were fully informed
of the risks and stresses associated with the project. This
study was approved by the Ethics Committee of the
Medical Center of Kyung Hee University.
Urine collection and analysis of metabolites
CST extract (11 g/person) was administered orally to
the volunteers and total urine was collected in a sterilized
urine collector at 0, 3, 6, 9, 12 and 24 h. 50 mL of each
urine sample was incubated for 20 h at 37oC with 3,000
units of β-glucuronidase (Sigma Co., St. Louis, MO, U.S.A.).
The level of the metabolite daidzein was measured using
HPLC (Hitachi system: column, Spherisorb ODS1 (3.0×
250 mm); elution solvent, 0.5M phosphate buffer:acetonitrile
(72:28); elution rate, 1.0 mL/min; detection wavelength, 280
nm).
Preparation of specimens
Fresh fecal specimens of volunteers (3 g) prepared
according to a previously described method (Lee et al.,
2003), were collected in plastic cups 9 h before oral
administration of CST, and then carefully mixed with a
spatula and suspended with 27 mL cold saline. The fecal
suspension was centrifuged at 100 g for 5 min. The
supernatant was then centrifuged at 10000 g for 20 min.
The resulting precipitates were used as a metabolic
enzyme source for the assay of enzyme activity. The
preparation and assay of the enzyme source were
performed within 24 h.
Assay of metabolic activities of herbal medicine
components by human fecal microflora
An assay of the metabolism of CST, puararin and
daidzin to daidzein by human fecal microflora was per-
formed according to the method reported in the literature
(Lee et al., 2003). Briefly, the above fecal precipitate (0.2
g) was suspended with 1.8 mL of 50 mM phosphate
buffer (pH 7.0) and used as a crude enzyme solution in
the present experiment.
The reaction mixture (2 mL) containing 0.4 mL of the
fecal suspension, 0.4 mL of 0.5 mM CST, puerarin, daidzin,
or p-nitrophenyl-β-D-glucopyranoside and 0.2 mL of 25
mM phosphate buffer (pH 7.0) was incubated at 37oC for
2 h, and the reaction mixture was extracted twice with 10
mL of ethyl acetate, and then evaporated under vacuum.
The ethyl acetate fraction was dissolved in methanol and
then analyzed by TLC.
Thin layer chromatography
TLC of daidzein was performed on silica gel plates
(silica gel 60F-254, Merck, Germany) with a developing
solvent system of CHCl3:MeOH = 6:1 (v/v) or CHCl3:
petroleum ether:acetic acid = 6:6:1 (v/v). The chromato-
grams were quantitatively assayed with a TLC scanner
(CS-9301PC, Shimadzu Co.).
Statistics
The data was analyzed using the SPSSwin 10.0
program. Correlations between the results were analyzed
using the Spearman test (p <0.1).
RESULTS AND DISCUSSION
The role of intestinal microflora in the absorption and
excretion of the components of CST was examined by
measuring the fecal metabolism of CST constituents to
daidzein. The metabolic activities of the fecal specimens
of the 14 subjects were between 32.3 and 79.9 µmol/h/g
wet feces (Table I). The average metabolic activity (mean
± S.D.) was 54.8 ± 16.7 µmol/h/g wet feces.
In order to understand the relationship between the
metabolism of the components of CST, puerarin and
daidzin, by human fecal microflora and the amount of their
metabolite(s) absorbed, the amount of daidzein excreted
in the urine of volunteers administered CST orally was
measured (Fig. 1). The accumulated amount of daidzein
excreted within 24 h of CST administration in all subjects

Page 3

Daidzein Excretion in Chungpesagantang-Treated Humans 483
was between 20.1 and 214.5 mg. The average amount
(mean ± S.D.) was 103.7 ± 55.8 mg, which corresponded
to 20.2% of the total amount of puerarin, daidzin and
daidzein contained in CST. However, no puerarin or
daidzin were detected in the urine.
All individuals possess their own characteristic indi-
genous strain of intestinal bacteria. This is due to the
affinity between the intestinal lumen of the individual and
the bacteria. Newly ingested bacteria cannot necessarily
colonize and proliferate in the intestine. Thus, intestinal
bacteria of each resident are not easily affected by
environmental factors. These results are supported by
previous reports that intestinal microflora in feces are
stable over time within individuals in the absence of
disease and antimicrobial therapy (Kim, 2002; Kobashi et
al., 1984; Rumney and Rowland, 1992). It has been
reported that these intestinal microflora may play an
important role in the pharmacological actions of herbal
medicines. Herbal medicines contain many glycosides
that cannot be absorbed from the human intestine due to
their polarity. These glycosides come into contact with the
microflora of the intestine, which metabolizes them to
metabolites that can be absorbed into the blood.
When herbal medicines and functional foods are
administered orally to humans, the absorption of their
components into blood is affected by intrinsic and extrinsic
factors, such as individual genetic properties, metabolic
potency of intestinal microflora, and polarity of their com-
ponents. Therefore, the fecal daidzein glycoside-hydrolyzing
activities of the volunteers as well as their urinary excretion
of daidzein after CST administration were investigated in
an attempt to further understand the pharmacological
actions of CST. The individual fecal microflora hydrolysis
profiles of both purararin and daidzin (but not purarin or
daidzein) in CST seemed to be in proportion to the amount
of daidzein excreted in the urine but this relationship did
not reach statistical significance (p=0.23). However, when
two specimens were excluded (circled in Fig. 2), the p
value of the correlation was 0.017. The urine excretion
profile was in proportion to the metabolism of the CST
components to daidzein. These results suggest that the
absorption of the metabolite in most volunteers may be
mainly dependent on the capacity of the intestinal micro-
flora to metabolize puerarin and daidzin, but a few of the
volunteers may be affected by other factors, such as
membrane absorption from the intestine and metabolism
in the liver.
The amount of daidzein excreted was 20.2% of the total
puerarin, daidzin and daidzein contained in CST. Most of
the daidzein excreted was detected in the urine between
6 to 12 h after administering CST. However, the amount of
daidzein absorbed was high compared to those of other
flavonoids, such as quercetin and hesperidin (Miyake et
al., 2006; Jaganath et al., 2006). Many researchers have
reported that flavonoids may be difficult to be absorbed
from the intestine, because they are hydrophilic and
metabolized to phenolic acids by intestinal microflora
(Park et al., 2005; Yamada et al., 2006). These findings
suggest that puerarin and daidzin are not absorbed in the
stomach due to their high polarity, but move to the small
and large intestines where they are metabolized by the
intestinal microflora to daidzein, which is hydrophobic, and
partially absorbed into the blood.
CST has been used to prevent stroke (Jung et al.,
2003) and its pharmacological activity may be due to the
metabolite daidzein, which inhibits platelet aggregation
and exhibit anti-inflammatory activity. Therefore, we believe
that the pharmacological activity of puerarin and daidzin
containing CST may be dependent on the metabolic activity
of the intestinal microflora.
Table I. Metabolic activity of CST (Puerariae Radix) and its con-
stituents to daidzein in 14 subjects
AgentRate metabolized (ìmol/h/g)
CST 54.8 ± 116.7
Puerarin15.9 ± 106.7
Daidzin98.0 ± 116.8
PNG93.9 ± 102.3
CST: Chungpesagantang; PNG: p-Nitrophenyl-β-D-glucopyranoside.
Results are expressed as the mean ± S.D. (n=14).
Fig. 1. Time course of daidzein accumulation in the urine of subjects
administered CST orally. The thick line represents the profile of the
mean of the daidzein excretion values.

Page 4

484Y.-S. Kim et al.
REFERENCES
Cole, C. B., Fuller, R., Mallet, A. K., and Rowland, I. R., The
influence of the host on expression of intestinal microbial
enzyme activities involved in metabolism of foreign com-
pounds. J. Appl. Bacteriol., 59, 549-553 (1985).
Goldin, B. R., Swenson, L., Dwyer, J., Sexon, M., and Gorbach,
G. L., Effect of diet and Lactobacillus acidophilus supplements
on human fecal bacterial enzymes. J. Natl. Cancer Inst., 64,
2555- 2261 (1980).
Ikeda, N., Saito, Y., Shimazu, J., Ochi, A., Mizutani, J., and
Watanabe, J., Variations in concentrations of bacterial
metabolites, enzyme activities, moisture, pH and bacterial
composition between and within individuals in faeces of
seven healthy adults. J. Appl. Bacteriol., 77, 185-194 (1994).
Jaganath, I. B., Mullen, W., Edwards, C. A., and Crozier, A., The
relative contribution of the small and large intestine to the
absorption and metabolism of rutin in man. Free Radic. Res.,
40, 1035-1046 (2006).
Jung, W. S., Choi, D. J., Cho, K. H., Lee, K. S., Moon, S. K.,
Kim, Y. S., and Bae, H. S., Safety and efficacy assessment of
Chungpyesagan-tang for acute ischemic stroke. Am. J. Chin.
Med., 31, 181-190 (2003).
Kim, D.-H., Yu, K. U., Bae, E. A., and Han, M. J., Metabolism of
puerarin and daidzin by human intestinal bacteria and their
relation to in vitro cytotoxicity. Biol. Pharm. Bull., 21, 628-630
(1998).
Kim, D.-H., Herbal medicines are activated by intestinal
microflora. Nat. Prod. Sci., 8, 35-43 (2002).
Kobashi, K., Nakata, H., Takebe, H., and Terasawa, K.,
Relationship between syo and intestinal microflora. Wakan-
iyaku-kaishi, 1, 166-167 (1984).
Kobashi, K. and Akao, T., Relation of intestinal bacteria to
pharmacological effects of glycosides. Biosci. Microflora, 16,
1-7 (1997).
Lee, J. M., Longevity and life Preservation in Oriental Medicine.
S.H. Choi (translated), Seoul: Kyung Hee Univ. Press, pp
153-175. 1996.
Lee, D. K., Kim, Y. S., Ko, C. N., Cho, K. H., Bae, H. S., Lee, K.
S., Kim, J. J., Park, E. K., and Kim, D. H., Fecal metabolic
activities of herbal components to bioactive compounds.
Arch. Pharm. Res., 25, 165-169 (2003).
Ling, W. H., Korpela, R., Mykkanen, H., Salminen, S., and
Hanniinen, O., Lactobacillus strain GG supplementation
decreases colonic hydrolytic and reductive enzyme activities
in healthy female adults. J. Nutr., 124, 18-23 (1994).
Miyake, Y., Sakurai, C., Usuda, M., Fukumoto, S., Hiramitsu,
M., Sakaida, K., Osawa, T., and Kondo, K., Difference in
plasma metabolite concentration after ingestion of lemon
flavonoids and their aglycones in humans. J. Nutr. Sci.
Fig. 2. Profile of the relationship between fecal metabolism of CST to daidzein and the amount of daidzein excreted in the urine of the subjects
administered CST orally. This relationship was examined for the metabolism of CST extract (A) and its constituents daidzin (B), puerarin (C) and p-
nitrophenyl-β-D-glucopyranoside (PNG; D).

Page 5

Daidzein Excretion in Chungpesagantang-Treated Humans 485
Vitaminol., 52, 54-60 (2006).
Mykkanen, H., Laiho, K., and Salminen, S., Variations in faecal
bacterial enzyme activities and associations with bowel
function and diet in elderly subjects. J. Appl. Microbiol., 85,
37- 41 (1998).
Park, J., Safety and efficacy needs to be tested in an
appropriate design. Am. J. Chin. Med., 31, 1001-1002 (2003).
Park, S. H., Park, E. K., and Kim, D. H., Passive cutaneous
anaphylaxis-inhibitory activity of flavanones from Citrus
unshiu and Poncirus trifoliata. Planta Med., 71, 24-27 (2005).
Reddy, B. S., Hanson, D., Manar, S., Mathews, L., Abaschnig,
M., Sharma, C., and Simi, B., Effect of high-fat, high-beef diet
and of mode of cooking of beef in the diet on fecal bacterial
enzymes and fecal bile acids and neutral sterols. J. Nutr.,
110, 1880-1887 (1980).
Rumney, C.J. and Rowland, I. R., In vivo and in vitro models of
the human colonic flora. Crit. Rev. Fd Sci. Nutr., 31, 299-331
(1992).
Simon, S. I. and Gorbach, S. I., The human intestinal microflora.
Digest. Dis. Sci., 31, S147-S162 (1986).
Yamada, M., Tanabe, F., Arai, N., Mitsuzumi, H., Miwa, Y.,
Kubota, M., Chaen, H., and Kibata, M., Bioavailability of
glucosyl hesperidin in rats. Biosci. Biotechnol. Biochem., 70,
1386-1394 (2006).
Yim, J. S., Kim, Y. S., Moon, S. K., Cho, K. H., Bae, H. S., Kim,
J. J., Park, E. K., and Kim, D. H., Metabolic activities of
ginsenoside Rb1, baicalin, glycyrrhizin and geniposide to
their bioactive compounds by human intestinal microflora.
Biol. Pharm. Bull., 27, 1580-1583 (2004).