2009 03 28 content 2nd
TRANSCRIPT
Extracts from fermented black soybean by
immobilized Rhizopus oligosporous exhibit both
antioxidative and cytotoxic activities
Kuan-Chen Cheng1,*, †, Jiun-Tsai Lin1, and Wen-Hsiung Liu1
1Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan
*Corresponding author: Tel: (814)865-5616; Fax: (814)863-1031;
E-mail: [email protected]
†Present address: Agricultural and Biological Eng Bldg, Penn State University, University
Park, PA, 16802, USA
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Abstracts
Black soybeans are abundant in dietary fiber and provide 8 human essential amino
acids, which can improve gastrointestinal function as well as reduce uncomfortableness
induced by flatus. Other reports also proposed black soybeans exhibit some biological
functions, such as free radical scavenging activity, inhibit low density lipoprotein
oxidation, antitumor, and antiviral activity. In this study, ethanol extracts from 2-day
fermented back soybean broth by immobilized generally recognized as safe (GRAS)
filamentous-fungi R. oligosporous (NTU-5) were evaluated both its antioxidative and
tumoricidal activity. The results indicated that the fermented broth reached its maximum
2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging effect (76%) after 2-day cultivation,
which was not consistent with total amount of polyphenol and isoflavone. The extracts
were further fractionated and an unknown compound, FBE5-A, was obtained exhibiting
strong antioxidative activity. IC50 of DPPH scavenging effect of FBE5-A was 7.5
(g/mL), which is stronger than commonly used antioxidant, Vitamin E (-tocopherol;
17.4 g/mL) and similar to Vitamin D (ascorbic acid; 7.6 g/mL). The cytotoxic test
demonstrated that extracts of 2-day fermented broth significantly exhibited selective
cytotoxic activity toward human carcinoma cells Hep 3B (IC50 = 150.2 g/mL) and did
not affect normal human lung fibroblast MRC-5 (P < 0.05). The results implied the
potential applications of fermented black soybean as functional food, pharmaceutical
and/or cancer therapy formula.
KEYWORDS: Black soybean; Rhizopus oligosporous; Antioxidant; Cytotoxic activity;
Hep 3B; MRC 5; Isoflavone; Total polyphenol content
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INTRODUCTION
Black soybean [Glycine max (L.) Merrill], a variety type of soybean with a black
seed coat, has been widely utilized as food supplement and key ingredients in Chinese
herbal medicine for hundreds of years (Li, 1990). The seed coats of black soybeans
contain anthocyanins, which lead to their several therapeutic effects, and hence they
are darker than the seed coats of other strains of soybean (Choung et al., 2001). Black
soybeans are rich in dietary fiber and provide 8 human essential amino acids, which
can enhance gastrointestinal function and reduce uncomfortableness caused by flatus
(Choung et al., 2001).
Isoflavones, which are abundant in soybean, demonstrate several health-
enhancing properties such as ease of symptoms of postmenopausal women (Nagata,
Takatsuka, Kawakami & Shimizu, 2001), reducing the risk of osteoporosis (Cotter &
Cashman, 2003; Weaver & Cheong, 2005), prevention of cardiovascular disease
(Anthony, Clarkson, Hughes, Morgan & Burke, 1996; Goodman-Gruen & Kritz-
Silverstein, 2001) and antimutagenic effects (Hung, Huang & Chou, 2007). Hendrich
(2002) reported that biological activity of isoflavones is mainly from the aglycone
forms. It has also been demonstrated that in human, isoflavone aglycones are
absorbed faster and in greater amount than their glycosides (Setchell et al., 2003).
Besides isoflavones, black soybeans consist of other bioactive components like
flavonoids and saponins (Kao, Wu, Hong, Wu & Chen, 2007), which exhibit
biological functions, such as free radical scavenging activity and inhibit low density
lipoprotein (LDL) oxidation, antitumor and antiviral, and reducing the incidence of
DNA damage by cyclo-phosphamide (Liao, Chou, Wu, Khoo, Chen & Wang, 2001;
Yamai, Tsumura, Kimura, Fukuda, Murakami & Kimura, 2003; Ribeiro & Saloadori,
2003). Our previous study also supported these findings since the fermented black
soybean exhibited its maximum antioxidative activity, which was not consistent with
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the maximum of isoflavones obtained during cultivation implying the existence of
other antioxidants (Cheng, Lin, Wu & Liu, 2009).
Fermented black soybean also exhibited more bioactivity such as antimutagenic
and antioxidant activity (Hung et al., 2007; Lee, Hung & Chou, 2007). Compounds
isolated from fermented soybean by filamentous fungi demonstrated several bioactive
functions. Esaki, Hiromichi, Shunro & Toshihiko (1996) reported that 3–
hydroxyanthranilic acid (HAA) had higher antioxidation ability than Vitamin E at
same concentration. HAA also demonstrated its ability to induce apoptosis of liver
cancer cells (Matsuo, Nakamura, Shidoji, Muto, Esaki & Osawa, 1997). Several
antioxidants such as 6-hydroxydaidzein (6-OHD), 2,3-dihydroxybenzoic acid (2,3-
DHBA), 8-hydroxydaidzein (8-OHD), and 8-hydroxygenistein (8-OHG) were later
reported (Esaki, Hiromichi, Shunro & Toshihiko, 1997; Esaki, Hiromichi, Yasujiro,
Shunro & Toshihiko, 1998; Esaki, Kawakishi, Morimitsu & Osawa, 1999). Those
antioxidants are derivatives of daidzein and genistein from fermented soybean, which
is correlated to Aspergillus oryzae by secreting extracellular β-glucosidase during
fermentation process.
The evidences shown above clearly demonstrate the potential applications of
isoflavone and other bioactive components from soybeans in functional food,
pharmaceutical and cancer therapy area. The data, however, regarding the
antioxidation and cytotoxic activity by a mixture of isoflavones and extracts from
fermented soybean broth are rare. As a result, it would be of great advantage of
functional food industry if the fermented broth of black soybean can be proven with
antioxidative and cytotoxic activities without further isolation or purification.
Moreover, although many compounds with cytotoxic effect on cancer cells have been
reported, the side effect is that they often exhibit cytotoxicity toward normal cells at
the same time (Hadjur, Richard, Parat & Jardon, 1995).
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To promote the utilization of domestic black soybean as well as to evaluate the
antioxidative and cytotoxic activities of fermented black soybeans, a generally
recognized as safe (GRAS) filamentous-fungus, Rhizopus oligosporous (NTU5), and
an optimal cultivation condition reported in our previous study (Cheng et al., 2009)
was used for black soybean fermentation within a solid support bioreactor. The
extracts of fermented black soybean were further fractionated and purified in order to
evaluate its antioxidative activity and compared with commonly used antioxidants,
Vitamin D and Vitamin E. The cytotoxicity of fermented black soybean extracts was
also evaluated between human carcinoma cell lines and normal lung fibroblast cells.
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MATERIALS AND METHODS
Chemicals
Genistein, daidzein, genistin, and daidzin and other chemicals were purchased
from Sigma (St. Louis, MO). Liquid chromatography grade acetonitrile and methanol
were purchased from Merck (Darmstadt, Germany). Potato dextrose broth and Bacto-
agar were purchased from Difco (Detroit, MI). Dulbecco’s modified eagle media
(DMEM), fetal bovine serum (FBS) and trypsin-EDTA were obtained from Gibco
(Grand Island, NY). Ethyl acetate, n-hexane and thin-layer chromatography (TLC)
plates were obtained from Merck (Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium
iodide and all other chemicals were purchased from Sigma (St. Louis, MO). The black
soybean [Glycine max (L.) Merr.] (Tainan NO.3) was purchased from Shiuejia
farmers' cooperative (Tainan, Taiwan). The black soybeans were milled with a grinder
(Model: CP-75 S, Ladyship, Kaohsiung, Taiwan) and the powders after passing
through a 20-mesh sieve were used for medium preparation.
Microorganism
Filamentous-fungus, Rhizopus oligosporous (NTU-5) was previously isolated in
our lab. The working cultures were maintained on potato dextrose agar (PDA) slants
after growing at 30oC and subcultured biweekly.
Suspension spores preparation
The cultures of R. oligosporous were grown on PDA plates for 5 to 7 days at
30oC. Mycelia were then rinsed with 10 mL of 0.05% Tween 80 and scraped with
glass hockey stick to detach spores. The solution was then filtered through sterile
gauze to remove mycelium. The filtrate containing spores was adjusted to around 106
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spores /mL and stored at 4oC until used. For long term storage, the spore suspensions
in a 0.05% Tween 80 solution were stored at -80oC.
Black soybean fermentation by R. oligosporous
Black soybean fermentations were conducted in a 5-L bioreactor (Mituwa,
Japan) at 30oC. Seed culture was prepared as follow: 1 mL of prepared spore
suspension was added in 100 mL medium consisting of 2 g of glucose, 1 g of yeast
extract, 0.2 g of KH2PO4, and 0.1 g of MgSO4●7H2O per liter of distilled water (pH
6.0) in 500 mL Hinton flasks. Cultivation was carried out on a reciprocal shaker at
125 rpm and 30oC for 72 h. The seed culture was subsequently inoculated into
bioreactor (3 L working volume) for 6-day cultivation with the optimal conditions
from our previous study (Cheng et al., 2009). A luffa cylindrical fiber (10-cm O.D.
and 15-cm length) was implemented as biofilm support. Fermentation broth was
sampled daily and analyzed of its polyphenol and isoflavone content, 2,2-diphenyl-1-
picrylhydrazyl (DPPH) radical scavenging rate, and pH.
Isoflavone Quantification
Extraction of fermented broth was carried out as follow: 10 mL of 80% EtOH
was added to the lyophilized powder of 10 mL fermentation broth and then incubated
in shaker at 65oC, 125 rpm for 2 h. After incubation, the mixture was centrifuged at
12,000 x g for 15 min to remove insoluble debris. The supernatant was then filtered
through a 0.45 m nylon filter (Millipore, Bedford, MA) for the quantification of
isoflavones using high performance liquid chromatography (HPLC) modified from
the work of Wang and Murphy (1994). The HPLC system was consisted of a
Shimadzu SCL-10Avp system controller, two Shimadzu LC-10ATvp liquid
chromatograph pumps, and a Shimadzu SPD-M10Avp photodiode array detector
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(Shimadzu, Nakagyo-ku, Kyoto, Japan). A Chemcosorb 7-ODS column (250 X 4.6
mm) was used. A linear HPLC gradient was composed of 0.1% (v/v) glacial acetic
acid water solution (solvent A) and 80% (v/v) acetonitrile in 0.1% (v/v) glacial acetic
acid water solution (solvent B). After injection of 20 L sample, solvent B was
increased from 15% to 70% in 25 min, and then returned to 15% in 5 min. The solvent
flow rate was 1.6 mL min-1. Elution was monitored by UV absorbance at 262 nm.
Antioxidation activity
The antioxidation activity of fermented samples was determined using DPPH
scavenging method as an indicator to express the anti-oxidation activity. One mL of
extraction sample from fermented broth was added to 2 mL 400 mM DPPH methanol
solution and placed in dark for 30 min. The mixture was then monitored by
absorbance at 517 nm. The DPPH radical scavenging rate (%) was defined as follow:
[1-(absorbance of sample at 517 nm)/ (absorbance of control at 517 nm)] x 100.
Polyphenol quantification
Total amount of polyphenol was identified by the modified method of Shetty,
Korus & Crawford (1989). After 2-day cultivation, 2 mL of the culture broth was
centrifuged at 12,000 × g for 15 min at 15oC. One mL of supernatant was added with
1 mL of 95% EtOH, 5 mL of distilled H2O, and 0.5 mL of 50% Folin-Ciocalteau
reagent. After 5 min reaction, 1 mL 5% Sodium Carbonate was added for another 1 hr
reaction in dark room at room temperature. The mixture was then measured
absorbance at 750 nm. Gallic acid was used as standard for quantification of total
polyphenol of fermentation broth.
Thin layer chromatography for antioxidant separation
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Fermented black soybean was sampled daily and each sample was firstly
centrifuged at 12,000 × g for 15 min at 15oC and the supernatant was recovered and
lyophilized. One milligram of lyophilized sample was dissolved in 80% ethanol and
used for TLC analysis. The samples were applied with the help of capillary tubing.
Daidzein and genistein were used as standard. The solvent system used was: n-
hexane:ethyl acetate:methanol (2:7:1). The chromatograms were developed for about
10 min on a TLC plate for a 10 cm run (Silica gel 60 F254, Merck, Darmstadt,
Germany). After development, the silica gel plate was treated with 10% H2SO4 and
the spots were revealed by heating at 110oC for 5 min. The TLC plate was
subsequently sprayed with 400 mM DPPH methanol solution and placed in dark for
another 30 min. TLC image was taken using an UV lamp and marked (365 nm;
VinPro, Palm desert, CA). Sample exhibiting strongest antioxidative activity was
chosen for further separation.
Preparation and Fractionation of Ethanol Extracts
After 2-day cultivation, 1 L of the culture broth was centrifuged at 12,000 × g for
15 min at 15oC to remove insolubles. The lyophilized supernatant was extracted using
80% ethanol and the ethanol extract was then concentrated using a Rotavapor-R
rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) under reduced
pressure at 30oC. The residue was designated as the fermented black soybean extract
(FBE). The FBE (7.53 g) was then resuspended in n-hexane. After initial analysis, the
FBE in n-hexane was subjected to separation on a silica gel column (40 5 cm I.D.;
Merck, Darmstadt, Germany) with 50 g silica gel by loading in n-hexane and eluted
using a combination of n-hexane–ethyl acetate-methanol with different ratio (Figure
1). After silica gel column chromatography, each fraction was determined by its
polyphenol content and DPPH scavenging effect. Eight main fractions were obtained
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and the solvent was removed under reduced pressure at 40oC. The most antioxidative
fraction was further separated by HPLC system (Shimadzu LC-10A) using a
MetaChem Polaris C18-A column (5 μm particles, 150, 250 10 mm I.D., Varian
Inc., Palo Alto, CA) and eluted with 80% methanol in H2O. TLC development
described earlier was conducted to isolate potential antioxidant. We obtained a pure
compound termed FBE5-A which was evaluated by its antioxidative activities.
Cell Line and Culture Conditions
MRC-5, a human lung fibroblast cell line was obtained from the Bioresources
Collection and Research Center (Hsinchu, Taiwan). Hep 3B, a human hepatocellular
carcinoma cell line and Hep G2, a human hepatoblastoma cell line were a gift from
Dr. B.H. Chiang (Institute of Food Science and Technology, National Taiwan
University, Taipei, Taiwan). HeLa, a human cervical epithelioid carcinoma cell line
and CL1-1, a human lung adenocarcinoma cell line were a gift from Dr. F.H. Chang
(Institute of Biochemistry and Molecular Biology, National Taiwan University,
Taipei, Taiwan). All cells were cultured in 10% FBS in DMEM with 100 U/mL
penicillin and 100 μg/mL streptomycin at 37oC in a humidified atmosphere containing
5% CO2.
Cytotoxicity of Black Soybean Extracts from Submerged culture of R.
oligosporous NTU5
Cytotoxicity toward human cell lines was measured using the MTT assay, which
is based on the ability of cells to convert tetrazolium salt into purple formazan
(Mosmann, 1983). Briefly, cells were subcultured into a 96 well plate with 4,000 cells
per well in 100 μL medium. After 48 h incubation, the medium in each well was
discarded and replaced with 200 μL of fresh medium containing the test agents
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dissolved in 0.4% DMSO. DMSO alone was used as control. After 2 h incubation, the
medium was discarded and 55 μL of MTT solution (0.5mg/mL) were added to each
well. The plates were then incubated for another 4 h at 37oC. The supernatant was
removed and 100 μL of DMSO were added to each well to dissolve the formazan. The
absorbance at 570 nm was measured using a MRX II microplate reader (DYNEX,
Chantilly, VA). The inhibition of cell viability was expressed as a percentage of the
proliferation of the control cells.
Data analysis
All experiments were performed in triplicates and expressed as mean ± standard
deviation. The significant difference of the results was evaluated using the Tukey’s
honesty significant differences (HSD) multiple comparison module of the MINITAB
Statistical Software package (Release 13.30; State College, PA).
Results and Discussion
Time Course of Submerged Culture of R. oligosporous NTU5
The cultivation process was carried out in a 5-L reactor with luffa cylindrical
fiber as solid support. After 7-day cultivation, total amount of polyphenol, content of
four major isoflavones, DPPH scavenging activity, were summarized in Table 1. The
results demonstrated that fermented broth reached its maximum antioxidant activity
on second day (75%), however, which was not consistent with either amount of
polyphenol or isoflavone aglycones. Amount of polyphenol and isoflavone increased
and reached maximum on fifth and sixth day, respectively.
To further investigate this finding, samples obtained from each day were
developed on TLC plate and the results were shown in Figure 2. From TLC pattern,
fermented samples exhibited higher DPPH scavenging effect on both second and third
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day which agree with our previous result. Moreover, there was one spot exhibited
strong antioxidative activity around Retention factor (Rf) 7.0, which was neither
daidzein nor genistein. As a result, fermented broth of black soybean on second day
was chosen for further study.
Fractionation of fermented black soybean broth
After concentrating of fermented broth of black soybean extracts, 7.53 g FBE was
fractioned by eight different combinations of n-hexane, ethyl acetate, and methanol
solvents. Elutes were harvested, condensed and measure for their dry weight. Total
polyphenol and DPPH scavenging effect were determined and summarized in Table
2. From the results, fraction 5 (FBE5) yielded highest polyphenol content as well as
highest DPPH scavenging effect. Therefore, FBE5 was chosen for further separated
using HPLC method.
The HPLC results demonstrated that four compounds were separated and named
FBE5-A, FBE5-B, FBE5-C, and FBE5-D (Figure 3). One milligram of each major
compound was harvested and developed on TLC and imaged under UV light. The
spots which exhibited antioxidative activity were circled. From Figure 4, FBE5-A
exhibited strongest antioxidative activity. FBE-C and FBE-D were later identified as
daidzein and genistein using LC/MS (data not shown). DPPH scavenging effect was
conducted for comparison among FBE-A and other commercial antioxidants and the
results were summarized in Table 3. The results demonstrated that IC50 of FBE5-A
was 7.5 (g/mL) which is stronger than commonly used antioxidant, Vitamin E (-
tocopherol; 17.4 g/mL) and similar to Vitamin D (ascorbic acid; 7.6 g/mL). Pure
FBE5-A was obtained (Figure 5) and an attempt to identify the structure of FBE5-A
was performed. However, due to the limitation of instruments in our lab and the low
yield of product, only UV spectrometric data was obtained. UV λmax were 208 and 262
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nm.
Cytotoxicity of ethanol extract from the submerged culture of R. oligosporous
NTU5
The ethanol extract from supernatant (FBE) from 2-day culture broth was
evaluated for its cytotoxicity using cell viability assy. Four humane carcinoma cell
lines (HeLa, Hep G2, Hep 3B, and CL 1-1) were tested and normal human lung
fibroblast cell line, MRC 5, was used as control. The results demonstrated that FBE
exhibited selective cytotoxic activity toward human carcinoma cells Hep 3B (IC50 =
150.2 g/mL) and no significant effect on other three carcinoma cell lines (Figure 6
B). On the other hand, FBE did not affect the viability of normal human lung
fibroblast MRC-5 cells (Figure 6 A).
Discussion
Fermented black soybeans have long been used not only as a food or dessert but
also daily supplement in China and Japan for hundred years (Su, 1980). There are
many reports mainly focus on the conversion efficiency of deglycosylation of
isoflavone glycosides by different microorganisms since isoflavone aglycones exhibit
higher gastrointestinal absorption (Choi, Kim & Rhee, 2002). Moreover, other
bioactive functions, such as antioxidative activity, cytotoxicity toward carcinoma cells
of fermented soybeans are investigated (Cheng et al., 2009; Esaki et al., 1997; Hung
et al., 2007). Moreover, black soybeans exhibit high potential applications as food,
dessert, and condiments due to its physical nature, such as higher polyphenol content
and lower content of flatulence-causing raffinose and stachyose compared to common
soybean (Su, 1980; Li, 1990). Therefore, further studies on the fermented black
soybean are needed.
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This study may provide insight into the effects of fermented black soybean
toward its antioxidative and cytotoxic activities. From the time course of black
soybean fermented by R. oligosporous, DPPH scavenging effect was not consistent
with either total polyphenol or isoflavone aglycones. Extract of fermented broth
reached its highest antioxidativity on second day, however, the amount of polyphenol
and isoflavone aglycones were still accumulating through the cultivation process. This
inconsistency phenomenon also reported by Lee et al. (2007) when performing solid
state fermentation by Aspergillus awamori. The reason probably due to the
accumulation of specific phenolic compound or new isoflavone derivatives were
generated during fermentation since R. oligosporous has been reported possessing -
glucosidase and other enzymes which can convert two major native, daidzin and
genistin, into their either aglycones or derivatives (Toda, Uesugi, Hirai, Nukaya,
Tauji, & Ishida, 1999).
From the results of fractionation of fermented black soybean broth, an unknown
compound FBE5-A was isolated and exhibited strong antioxidative activity using
DPPH scavenging test (Figure 4), which is higher than Vitamin E and similar to
Vitamin C. FBE5-A is not one of the twelve major isoflavones after HPLC analysis
(data not shown) and could be a new derivative of polyphenol during cultivation.
Since oxygen free radicals and other reactive oxygen molecules can cause
deterioration on cell components, such as membrane proteins, enzymes, nucleic acid,
and lipids (Halliwell, Murcia, Chirco, and Aruoma, 1995), it is worthwhile for further
identification of FBE5-A and to evaluate its dosage of antioxidative activity.
Hung et al. (2007) reported that fermented black soybean exhibited antimutagenic
effect toward 4-NQO and B[a]P, which are carcinogenic compounds produced by
Salmonella Typhimurium. Black soybean also reported to effectively reduce the
incidence of DNA damage caused by cyclophosphamide (Ribeiro & Saloadori, 2003).
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In stead of prevention, an efficient formula for cancer therapy is also crucial.
Fermented black soybean by R. oligosporous exhibited selective cytotoxicity toward
human hepatocellular carcinoma cell, Hep 3B and no effect on normal human lung
fibroblast MRC-5 cells. The relatively high cytotoxic activities noted in the fermented
black soybean extracts is generally associated with the higher contents of total
phenolic compounds, anthocyanin and isoflavone aglycones, in fermented black
soybean (Lee et al., 2007). However, it is also possible resulted from the formation of
other antimutagenic metabolites during cultivation (Park, Jung, Rhee & Choi, 2003)
and varies with different fermentation conditions.
In summary, this study found that fermented black soybean extract proved itself
antioxidative activity which is not consistent with either polyphenol or isoflavone
content. Further fractionation of the extract demonstrated that an unknown compound,
FBE5-A, was isolated exhibiting strong DPPH scavenging effect and served as an
antioxidant candidate. The cell viability test also suggested that fermented black
soybean extracts possessed selective cytotoxicity toward carcinoma cells but not
healthy ones. Further study on the identification of FBE5-A is needed. The
mechanism of cell death triggered by FBE is another issue for cancer therapy study.
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Acknowledgement
This research was financially supported by The National Science Council, ROC
(Taiwan). (NSC 92-2321-B-002-012). Authors would like to thank Dr. Ali Demirci at
Agricultural and Biological Engineering, Pennsylvania State University for technical
review of the manuscript. Thanks also to Dr. Yi-Ling Chiang at the Institute of
Biochemistry and Molecular Biology of National Taiwan University for useful discussion
of cell culture experiments.
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Table 1. Time course of black soybean fermentation by R. oligosporous in a 5-L bioreactora.
DayGlycoside (mg/ml) Aglycone (mg/ml) Total isoflavone
(mg/ml)
Scavenging effect
(%)
Polyphenol
Daidzin Genistin Daidzein Genistein (mg/mL)
0 32.2±0.6 32±1.3 4.9±0.3 5.1±0.2 74.2±2.4 52±0.5 235.5±12.6
1 6.9±0.3 3.7±0.4 5.3±0.5 5.0±0.5 20.9±1. 60±0.3 260.8±8.3
2 NDb 1.8±0.2 8.2±0.3 5.2±0.3 15.2±0.8 75±0.3 317.4±10.9
3 NDb NDb 7.9±0.4 5.2±0.3 13.1±0.7 72±0.4 325.9±11.2
4 NDb NDb 8.0±0.3 5.8±0.5 13.8±0.8 68±0.6 348.4±9.6
5 NDb NDb 9.6±0.6 6.3±0.6 15.9±1.2 66±0.5 350.1±12.5
6 NDb NDb 14.7±0.5 7.5±0.4 22.2±0.9 62±0.3 322.3±16.7
aCultivation conditions were as follows: 30oC, 6% (w/v) black soybean powder as medium, aeration at 0.1 vvm,
agitation at 100 rpm with 10% inoculum.
bNot detected.
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Table 2. Results of silica gel chromatography of fermented black soybean extracts.
FractionWeight Yield
Polyphenol
content
DPPH Scavenging
Effect
(mg) (%) (g/mg)a (%)
1 2.9 0.03 2.7 1.4
2 98.1 1.29 18.4 1.7
3 269.4 3.56 21.8 3.4
4 65.7 0.87 14.9 30.1
5 431.3 5.71 36.1 74.7
6 1596.9 21.14 17.5 37.4
7 2441.4 32.31 15.4 37.2
8 1884.2 24.94 17.3 52.3
Total 6790.2 89.98 144.1
aThe total phenolic content in FBE was determined as g/mg of gallic acid equivalent.
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Table 3. Comparison of IC50 of DPPH scavenging
effect among different samples.
Sample IC50 (μg/mL)a
Ascorbic acid 7.6
α-Tocopherol 17.4
FBE 1323.6
FBE5 17.8
FBE5-A 7.5
aThe IC50 value was defined as the concentration of
each sample causing 50% reduction in the DPPH free
radical.
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LEGENDS TO FIGURES
Figure 1. Diagram of isolation and purification of antioxidants from fermented
black soybean by R. oligosporousa.
aSolvent composition: Fraction 1: n-hexane; Fraction 2: n-hexane: ethyl acetate (2:1);
Fraction 3: n-hexane: ethyl acetate (1:2); Fraction 4: ethyl acetate; Fraction 5: ethyl
acetate: methanol (2:1); Fraction 6: ethyl acetate: methanol (1:2); Fraction7:
methanol; Fraction 8: 80% methanol.
Figure 2. Antioxidative activity analysis of fermented black soybean extractsa,b. (A)
TLC results of fermented black soybean extracts, and (B) DPPH scavenging effect of
fermented black soybean extracts. .
aLane 1~Lane 6: Crude extracts of fermented black soybean extracts (0~5 day). Lane
7: Daidzein (Standard) ,Lane 8: Genistein (Standard).
bTLC image was taken under an UV lamp (365 nm) and spots which exhibited
antioxidative activity were circled.
cRetention factor (Rf) is defined as follow: (distance traveled by the compound/
distance traveled by the solvent front).
Figure 3. HPLC results of fraction 5 (FBE5).
Figure 4. TLC results from fraction 5 (FBE 5)a.
aLane 1: FBE5; Lane 2: FBE5-A: Lane 3: FBE5-B; Lane 4: Daidzein; Lane 5:
Genistein.
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bTLC image was taken under an UV lamp (365 nm) and spots which exhibited
antioxidative activity were circled.
Figure 5. Chromatograms of FBE5-A. UV spectrum of FBE5-A (A); FBE5-A was
detected at 262 nm (B).
Figure 6. Effect of fermented black soybean extracts (FBE) on cell viability of
MRC-5 (A), and carcinoma cell lines, HeLA, Hep G2, Hep 3B and CL 1-1 (B)
(n=5).
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7
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9
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Figure 1. Diagram of isolation and purification of antioxidants from fermented black
soybean by R. oligosporousa.
aSolvent composition: Fraction 1: n-hexane; Fraction 2: n-hexane: ethyl acetate
(2:1); Fraction 3: n-hexane: ethyl acetate (1:2); Fraction 4: ethyl acetate; Fraction 5:
Ethyl acetate: methanol (2:1); Fraction 6: ethyl acetate: methanol (1:2); Fraction7:
methanol; Fraction 8: 80% methanol.
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Figure 2. Antioxidative activity analysis of fermented black soybean extractsa,b,c. (A)
TLC results of fermented black soybean extracts, and (B) DPPH scavenging effect of
fermented black soybean extracts. .
aLane 1~Lane 6: Crude extracts of fermented black soybean extracts (0~5 day). Lane
7: Daidzein (Standard) ,Lane 8: Genistein (Standard).
bTLC image was taken under an UV lamp (365 nm) and spots which exhibited
antioxidative activity were circled.
cRetention factor (Rf) is defined as follow: (distance traveled by the compound/
distance traveled by the solvent front).
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Figure 3. HPLC results of fraction 5 (FBE5).
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1 2 3 4 5
Figure 4. TLC results from fraction 5 (FBE 5)a,b.
aLane 1: FBE5; Lane 2: FBE5-A: Lane 3: FBE5-B; Lane 4: Daidzein; Lane
5: Genistein.
bTLC image was taken under an UV lamp (365 nm) and spots which
exhibited antioxidative activity were circled.
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Figure 5. Chromatograms of FBE5-A. UV spectrum of FBE5-A (A); FBE5-A was
detected at 262 nm (B).
.
(A)
(B)
Time (min.)
nm
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1
0
20
40
60
80
100
120
0 50 100 150 200
FBE concentration (mg/ mL)
Ce
ll V
iab
ilit
y (
%)
MRC 5
0
20
40
60
80
100
120
0 50 100 150 200
FBE concentration (mg/ mL)
Ce
ll V
iab
ilit
y (
%)
HeLa
Hep G2
Hep 3B
CL 1-1
Figure 6. Effect of fermented black soybean extracts (FBE) on cell viability of
MRC-5 (A), and carcinoma cell lines, HeLA, Hep G2, Hep 3B and CL 1-1 (B)
(n=5).
(A)
(B)
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