Effect of roasting process on the antigenotoxic properties of
Cassia tora L.
Chi-Hao Wu and Gow-Chin Yen
Department of Food Science, National Chung Hsing University, 250
Kuokuang Road, Taichung 40227, Taiwan
Abstract
Antigenotoxic properties of water extracts from Cassia tora L.
(WECT) treated with different degrees of roasting (unroasted and roasted
at 150℃ and 250℃) were evaluated by Ames test and the Comet assay,
and the possible mechanisms were discussed. The reference mutagens
used were 2-amino-6-methyldipyrido(1,2-a:3':2'-d)imidazole (Glu-P-1)
and 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1).
Results
indicated that WECT, especially unroasted C. tora (WEUCT), markedly
suppressed the mutagenicity of Glu-P-1 and Trp-P-1.
The IC50 of
WEUCT toward Glu-P-1 and Trp-P-1 were 0.15, 0.57 and 0.20, 0.17
mg/mL for strains TA98 and TA100, respectively.
In the Comet assay
performed on human lymphocytes, WECT exhibited no cytotoxic effect
at a concentration of 0.1-0.5 mg/mL, the cell viability were greater than
95 %.
All three types of WECT (unroasted and roasted at 150, and 250
℃) exhibited protective effects on DNA damage in human lymphocytes
induced by Trp-P-1. At a concentration of 0.5 mg/mL, the inhibitory
effects were observed in the order of unroasted (55%)＞roasted at 150℃
53
(42%)＞roasted at 250℃ (29%). It revealed that increasing roasting
degrees of the Cassia seeds might decrease their antigenotoxic potency
both in prokaryotic (Ames test) and eukaryotic (Comet assay)
genotoxicity assays.
Mutagen-inhibitor interaction (molecular complex
formation) was identified in spectrophotometry studied, suggesting that
WEUCT may produce complexes with Trp-P-1.
Using a modified
Comet assay procedure, WEUCT exhibited 38.7% scavenging effect on
reactive intermediates of Trp-P-1 generated from metabolism system.
Pre-treatment of the human lymphocytes with WEUCT for 0.5 h resulted
in a modest repression of DNA damage (30 %). However, no significant
effect on excision-repair system was found during DNA damage
expression time in post-treatment scheme.
Further, the active
anthraquinone (AQ) substances: chrysophanol, emodin and rhein were
determined by HPLC.
The AQ contents decreased with increased
roasting.
these
Each
of
AQ
also
antigenotoxicity in the Comet assay.
demonstrated
significant
The inhibitory effects of
chrysophanol, emodin and rhein in Trp-P-1-mediated DNA damage in
human lymphocytes were 70, 58 and 21%, respectively, at 50 μM.
These findings suggested that the decrease of antigenotoxicity of the
roasted samples might be, at least partly, due to the reduction of their
anthraquinones.
Keyword ︰ Cassia tora L., roasting, Ames test, Comet assay,
antigenotoxicity,
DNA damage,
heterocyclic
mechanism, molecular complex, anthraquinones
54
amines,
Introduction
Concern about the role of diet is generally accepted as one of the
important factors in human cancer development.
A series of highly
mutagenic heterocyclic amines (HCAs) have been identified in foods
such as meat and fish prepared under typical cooking practices (Sugimura
et al., 1977).
HCAs, examples such as MeIQx, PhIP, Trp-P-1 and
Trp-P-2 are shown to be excreted in human urine after intake of cooked
foods (Ushiyama et al., 1991).
In addition, HCAs were demonstrated to
be carcinogenic for rodent gastrointestinal tract, liver, blood vessel,
mammary glands and urinary glands (Wakabayashi et al., 1992;
Hasegawa et al., 1993). Most people are exposed to extremely low
dosages of HCAs in the environment. Research indicates that contacting
even a low dosage of mutagen could result in a genotoxic effect for a
bioorganism.
However, it is increasingly becoming evidence that
naturally occurring plant extracts, which consist of antimutagens, may
afford protection against carcinogenic effects.
The Chinese herb “Jue-ming-zi”, which is the seed of the plant
Cassia tora L. (Leguminosae), has been used as a laxative and a tonic for
several centuries. Anthraquinones (AQ) were reported to be the main
active substances in C. tora, including chrysophanol, emodin, rhein, etc
(Duke, 1992; Zhang et al., 1996a; Choi et al., 1997). Choi et al. (1997)
indicated that AQ aglycons from C. tora exhibited inhibitory effect
against aflatoxin B1 in Ames test. Several reports shown that AQ can
act as an antimutagens, which suppress the mutagenicity of mycotoxins,
polycyclic aromatic hydrocarbons and HCAs (Hao et al., 1995). C. tora
55
extracts were found to promote hepatic enzymes in rats with
ethanol-induced hepatotoxicity, including catalase, superoxide dismutase
and glutathione peroxidase (Choi et al., 2002). In our previous work,
water extracts of C. tora (WECT), particularly unroasted sample
(WEUCT), markedly inhibited the mutagenicity of IQ and B[a]P, and the
possible mechanism was through suppressing the CYP-450 activity in rat
liver microsomal activation system (Wu and Yen, 1999). WECT also
moderated the oxidative DNA damage in human lymphocytes, induced by
hydrogen peroxide as evaluated by the Comet assay (Yen et al., 2002).
Yen et al. (1998) revealed that the major antioxidant compound of C. tora
was isolated and identified as emodin.
The commercial products of C. tora include both unroasted and
roasted samples, and the laxative effect was found to be higher in
unroasted C. tora than in the roasted products. Roasted C. tora has a
special flavor and color, and it is popularly used as a health tea drink.
Zhang et al. (1996b) revealed that some components, for example,
chrysophanol, in C. tora decreased after the roasting process.
Moreover,
the anti-hepatotoxic effect of C. tora decreased with an increase roasting
temperature (Wong et al., 1989). In view of these, this paper describes
efforts to clarify the effects of roasting conditions on the antigenotoxic
properties, the changes in active AQ compounds of WECT, and the
possible mechanisms of C. tora against food mutagens.
56
Materials and methods
Materials
The seeds of Cassia tora L. were obtained from a local market at
Taichung,
Taiwan.
2-Amino-6-methyldipyrido(1,2-a:3':2'-d)imidazole
(Glu-P-1), 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1) and
ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) were obtained
from Wako Pure Chemical Co. (Osaka, Japan). Chrysophanol, emodin,
rhein, N-lauroyl sarcosinate, ethidium bromidine, Triton X-100,
glucose-6-phosphate, β-NADP+, 7,8-benzoflavone (7,8-BF), histidine,
biotin, and trypan blue were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Dimethyl sulfoxide (DMSO), sodium dihydrogen
phosphate, disodium hydrogen phosphate, sodium chloride were
purchased from the E. Merck Co. (Darmstadt, Germany).
Normal
melting point agarose (NMA), low melting point agarose (LMA) and
RPMI 1640 medium were purchased from Gibco BRL Co (Grand Island,
NY). Ficoll-Paque separation media were purchased from Pharmacia
Biotech Co. (Sweden). Tris and protein assay kit were purchased from
Bio-Rad laboratories. (CA, USA).
2-Methylanthraquinone was
purchased from Tokyo Kasei Organic Chemicals Co. (Tokyo, Japan).
Sample Preparation
To obtained C. tora L. seeds with different degrees of roasting,
samples were washed and sun-dried and then left unroasted or roasted at
150 and 250 ℃ (internal temperature) for 5 min using a roasting
57
machine (rate 20 rotation/min, Nankung Machine Co., Taiwan). Each
unroasted and roasted sample (50 g) was extracted with boiling water
(500 mL) for 5 min, and the filtrate was freeze-dried. The yields of
extracts from unroasted, roasted at 150 °C, and roasted at 250 °C of C.
tora were 5.92, 6.00, and 3.90%, respectively.
Cell preparation
Human lymphocytes were isolated from fresh whole blood by
adding blood to RPMI 1640, then underlaying it with Ficoll-Paque before
centrifuging at 800g for 15 min. The lymphocytes were separated as a
pink layer at the top of the Ficoll-Paque.
Cells were washed with
phosphate buffered saline (PBS, pH 7.4) and suspended in buffer at
approximately 1106/mL.
In certain experiments, the viability of
lymphocytes was over 90 %, as determined by trypan blue exclusion
(Pool-Zobel et al., 1993).
Cell viabilities (%) = [ nonstained cells /
(nonstained cells + stained cells)] × 100.
Antimutagenicity assay
The antimutagenic effect of each WECT was assessed using the
Ames test (Maron and Ames, 1983). The histidine-requiring strains of
Salmonella typhimurium TA98 and TA100 were kindly supplied by Dr. B.
N. Ames (University of California, Berkeley). An aliquot of WECT (0.1
mL) was added to a mixture containing an overnight culture of S.
typhimurium TA98 or TA100 (0.1 mL), a mutagen dissolved in phosphate
buffer (0.2 g/mL Glu-P-1; 0.5 g/mL Trp-P-1), and 0.5 mL S9 mix.
58
After a 20 min preincubation at 37℃, 2 mL molten top agar was added to
the mixture and the entire liquid was poured onto an agar plate.
Histidine-revertant colonies were counted after a 48 h incubation at 37℃.
Each assay was performed in triplicate, and the data presented are the
means of at least three experiments. It was found that the doses (0.1-0.5
mg/plate) of the WECT tested in the present study were not toxic or
mutagenic to the bacteria with or without S9 mix. The mutagenicity of
each mutagen in the absence of WECT (control) was defined as 100 %.
The dose of WECT required to inhibit the mutagenicity of Glu-P-1 or
Trp-P-1 was interpolated from the multiple dose-response curve.
Comet assay
Genotoxicity of WECT, Chrysophanol, Emodin, and Rhein toward
Human Lymphocytes.
The comet assay was performed according to the methods of Singh
et al. (1988) and Anderson et al. (1997) with slight modifications. For
the genotoxicity studies, cells were treated with different concentrations
of
WECT
(0.1-0.5
mg/mL)
or
anthraquinones
dissolved
in
DMSO/chrysophanol, emodin, and rhein (1-50 μM) (the DMSO
concentration in the incubation medium never exceeded 1%).
incubations contained the same concentration of DMSO or PBS.
were incubated as mentioned above.
Cells
After incubation, cells were
centrifuged at 800g for 5 min at 4 °C.
59
Control
Cell number and viability
(Trypan blue exclusion) were determined by using a Neubauer improved
haemocytometer, before and after samples were treated. After
centrifugation, cells were resuspended in 75 μL of LMA (1% in PBS
without calcium and magnesium) and plated on fully frosted slides, which
had been covered with 75 μL of NMA (1% in PBS without calcium and
magnesium).
The slides were kept on ice for 5 min. After solidification,
a top layer of 75 μL of LMA was added, then allowed to solidify for 5
min. Slides were then immersed in freshly prepared lysing solution (2.5
M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, 1% N-lauroyl
sarsosinate, 1% (v/v) Triton X-100, and 10% DMSO) for 1 h at 4 °C.
The slides were then left in electrophoresis buffer (0.3 M NaOH and 1
mM Na2EDTA) for 20 min at 4 °C and placed in a gel electrophoresis
tank. Electrophoresis was conducted at 4 °C for 20 min with 25 V and
300 mA current. After electrophoresis, the excess alkali was neutralized
twice in Tris buffer (0.4 M Tris, pH 7.5) for 5-10 min. Finally, the
slides were stained with 50 μL of ethidium bromide (20 μL/mL) and
examined at a Nikon EFD-3 fluorescence microscope (Japan) with
excitation filter BP at 543/10 nm and a 590 nm emission barrier filter.
Objective measurements of the distribution of DNA were performed for a
sample of cells by using a Komet 3.1 (Kinetic Imaging Ltd., Liverpool,
UK).
One hundred cells on each slide (scored at random) were
classified according to the relative intensity of fluorescence in the tail.
The degree of DNA damage was scored by tail moment (TM; tail
moment = tail length x tail DNA% / 100).
Effects of WECT, Chrysophanol, Emodin, and Rhein on Trp-p-1-mediated
60
DNA Damage toward Human Lymphocytes.
As described above, human lymphocytes were treated with a series
of concentrations of WECT (final concentrations 0.1-2 mg/mL) or
chrysophanol, emodin, rhein (final concentrations 1-50 μM), or 7,8-BF
(100 μM) and Trp-P-1 (400 μM). The plates were incubated at 37 °C for
0.5h in 5% CO2. After incubation, the cells were washed with PBS
twice. Medium (1 mL) was added, cells were centrifuged at 800g for 5
min at 4°C, the supernatant was discarded, and the cells were
resuspended in 75 μL of LMA for comet analysis.
All test AQ
(chrysophanol, emodin, and rhein) and 7,8-BF were dissolved in DMSO;
DMSO concentration in the incubation medium never exceeded 1%.
Control incubations contained the same concentration of DMSO or PBS.
Studies of molecular complex formation
To investigate the possibility of a direct interaction (molecular
complex formation) between Trp-P-1 and WEUCT, spectrophotometric
titrations were performed as described by Connors (1987). Experiments
were conducted using matched quartz cuvettes in a double-beam U-3000
UV/VIS spectrophotometer.
Absorption spectra were measured from
220-340 nm for 1 mL solutions containing 17 M Trp-P-1 in 0.1 M
Tris-HCl buffer, pH 7.4.
Both sample and reference cuvettes
subsequently were treated with small volumes (2 L) of WEUCT (10-200
μg/mL) and scanned after each addition.
61
Scavenging effect of WECT on reactive intermediates of Trp-P-1
In order to assess whether the WECT neutralise the electrophilic
intermediates of the food carcinogen Trp-P-1, the Comet procedure was
modified.
Initially the human blood lymphocytes, Trp-P-1 and
activation system (S9 mix) were pre-incubated for 20 min in a shaking
water bath at 37℃ to allow the generation of the genotoxic products.
Microsomal metabolism was terminated by the addition of 50 L of
7,8-BF (500 M) and a second 20 min pre-incubation was carried out in
the presence of WECT. 7,8-BF is a potent inhibitor of the cytochrome
1A-dependent bioactivation of chemical carcinogens (Yamazoe et al.,
1983). After incubation the lymphocytes were harvested by centrifuged
at 800g for 5 min at 4℃ and the cells were resuspended in LMA for
Comet analysis.
Effects of pre- and post-treatment protocols of WECT on
Trp-P-1-mediated-DNA damage toward human lymphocytes
In the pre-treatment protocol, cells were first treated with different
concentrations of WECT (the final concentration was 0.1-0.5 mg/mL) for
0.5 h at 37℃. After the cells were washed three times with PBS, 400
M Trp-P-1 and 10 % (v/v) S9 mix were added for a second 0.5 h
incubation at 37℃. In the post-treatment protocol, lymphocytes were
treated with 400 M Trp-P-1 and 10 % (v/v) S9 mix for 0.5 h at 37℃ to
caused DNA damage. After the cells were washed three times with PBS,
different concentrations of WECT (the final concentration was 0.1-0.5
62
mg/mL) were added for 0.5 h at 37℃.
After treatment cells were
washed, harvested by centrifuged at 800g for 5 min at 4 ℃ and
resuspended in RPMI 1640 for Comet analysis.
HPLC analysis of anthraquinones in WECT
Standard
solution.
Chrysophanol,
emodin,
rhein
and
2-methylanthraquinone (internal standard) were dissolved in a small
amount of methanol and diluted with acetonitrile to prepare various stock
solutions of proper concentration.
Sample preparation for HPLC. The extraction scheme used was
modified as described by van der Berg and Labadie (1985).
WECT (1.0
g) and 2-methylanthraquinone (10 mg) were refluxed in a boiling water
bath for 2 h with 50 mL of 2.5 M HCl.
After cooling the hydrolysate
was extracted with 50 mL chloroform for 1 h, then re-extractd as above
three times.
The sample solution were combined and evaporated to
dryness. Dried extracts were dissolved in a few methanol and diluted
with acetonitrile to 50 mL for RP-HPLC analysis.
HPLC.
HPLC analysis was performed with a Hitachi liquid
chromatograph (Hitachi Ltd., Tokyo, Japan), consisting of a model
L-6200 pump, a Rheodyne model 7125 syringe-loading sample injector, a
model D-2000 integrator, and a model L-4200 UV-VIS detector set at 280
nm. A LiChrospher 100 RP-18 reverse phase column (5.0 m, 4×150
mm) was used for analysis. The volume injected was 10 L. The
elution solvents were acetonitrile/2 % acetic acid (55:45, v/v). The flow
rate was set at 1.5 mL/min.
63
Statistical analysis
All analyses were run in triplicate and averaged. Statistical
analyses were performed according to the SAS Institute User’s Guide.
Analyses of variance were performed using the ANOVA procedure.
Significant differences (P < 0.05) between the means were determined
using Duncan’s multiple range test.
64
Results and disscussion
Antimutagenic activity of WECT toward Trp-P-1 and Glu-P-1
In preliminary study (Wu and Yen, 1999), no cytotoxicity or
mutagenic effect was observed for any WECT at a dose of 0.25-5 mg per
plate.
As shown in Table 1, WECT, especially unroasted sample
(WEUCT), efficiently inhibited the mutagenicity of Trp-P-1 and Glu-P-1.
For strain TA98, the IC50 of WEUCT toward Trp-P-1 and Glu-P-1 were
0.15 and 0.57 mg/mL; whereas for strain TA100, the IC50 were 0.20 and
0.17 mg/ml, respectively.
The antimutagenicity of WECT decreased
with an increasing roasting temperature in the following order: unroasted
＞150℃＞250℃.
Effects of WECT on human lymphocytes DNA damage induced by
Trp-P-1
As Figure 2 shows, unroasted C. tora, roasted with 150 °C, and
roasted with 250°C at 0.5 mg/mL had suppressing effects of 55, 42, and
29%, respectively, on DNA damage of human lymphocytes (TM=38.9)
induced by Trp-P-1. Unroasted samples exhibited the best inhibitory
effect. The higher degree of roasting resulted in less protecting effects.
Results of this experiment have proved that WECT possessed protective
effect toward DNA damage induced by Trp-P-1 in the presence of S9 mix,
and that WECT had the same antigenotoxic effect shown previously by
the Ames test in a bacterial system (Table 1).
65
Analysis of molecular complex formation
To study the effects of WEUCT on the molecular complex
formation with Trp-P-1, changes in the absorption spectra of Trp-p-1
were monitored.
As Fig. 2 shows, with increasing concentration of
WEUCT, the peak at 263 nm was quenched in a manner indicative of
complex formation.
Hydrolysis and separation of AQ
AQ have been reported to be the main active components in C. tora.
The individual AQ content in extracts of Cassia tora were also measured
according to the method of van den Berg and Labadie (1985) that was
through acid hydrolysis, chloroform extraction, and determination by
HPLC. Three AQ, chrysophenol, emodin, and rhein, were detected in
WECT under the experimental conditions described above.
The
unroasted sample contains the AQ contents, the contents of rhein,
chrysophanol, and emodin was 10.42, 0.61, and 0.28 mg/g extracts,
respectively. The AQ content decreased with increased roasting. The
content of those three AQ for the sample roasted at 150 ℃ was 4.8, 0.14
and 0.10 mg/g extracts, respectively.
However, the extracts of Cassia
tora prepared by roasting at 250 ℃ did not show any detectable AQ.
Zhang et al. (1996) indicated that AQ in C. tora were degraded to a free
form (aglycon) by roasting treatment.
The content of these three AQ has
only one-eighth of the total content of AQ compared with the results
66
reported by the Yen and Chung (1999).
Most individual AQ have
shown antigenotoxicity activity in Ames test (Hao et al., 1995). Thus,
the decrease in antigenotoxicity activity of roasted Cassia tora was
related to the decrease in AQ.
Cytotoxicity, genotoxicity, and antigenotoxicity of chrysophanol,
emodin, and rhein on human lymphocytes.
Natural herbs contain a large number of potential anticancer
materials. For example, within Rhei rhizoma, Scutellariae radix, and
Rehmanniae radix, there is antimutagenicity that suppresses B[a]P (Sakai
et al., 1988). The major active component is probably a derivative of AQ;
and its mechanism was considered as suppressing exogenic activation
enzymes (Hao et al., 1995). To infer the previous results of this study
(Trp-P-1/Comet assay), strength of the antigenotoxicity of WECT
depended on the relative quantities of AQ. Therefore, this experiment
used these three AQ compounds to explore antigenotoxicity in cells. As
Figure 4 shows, the TM of chrysophanol was not statistically different (P
> 0.05) on DNA damage from that of the control group, suggesting that it
had no genotoxicity. Adding Trp-P-1 or not did not affect the toxicity of
chrysophanol on cells.
The cell viability was all over 90% and would
not pose interference on genotoxicity experiments. As for DNA damage
induced by Trp-P-1 (TM=28), chrysophanol that suppressed Trp-P-1 the
most possessed 31% protective effect even at low concentration (1 μM).
At a concentration of 50 μM, the highest suppressing effect was reached
70 %. Emodin (1-50 μM) showed neither cell toxicity nor genotoxicity
67
on human lymphocytes. There were suppressing effects of 29-58% in
selected concentrations (1-50 μM) of emodin.
The suppressing effect of rhein in low concentration was obviously
low (Figure 6). It possessed antigenotoxicity when its concentration was
reached 50 μM and could reduce, at most, 21% of DNA damage.
Moreover, it did not affect the viability of human lymphocytes, which
always came to more than 90%, whether the inducer Trp-P-1 was added
or not. Deserving of note is that, after electrophoresis, lymphocytes
came to 5.2 TM after being simply treated with 50 μM rhein. It had
significant difference (P < 0.05) compared to, and had slight DNA
damage on, human lymphocytes.
Conclusion
WECT showed a marked antigenotoxic potential against dietary
mutagens Glu-P-1 and Trp-P-1 in both the Ames test and the Comet assay,
and in the order of unroasted > roasted at 150℃ > roasted at 250℃. WECT
might produce molecular complexes with mutagens, and exhibited
scavenging effect on the reactive intermediates of Trp-P-1. The reduction
in the AQ content during roasting process was associated with the
decrease in antigenotoxic effects.
Chrysophanol, emodin and rhein were
found to have protective effects on DNA damage induced by Trp-P-1.
Apart from the traditional pharmacological effects of C. tora., the water
extracts of unroasted C. tora may have a potential health activity on the
cancer chemoprevention.
68
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Yao Tsa Chih. 21(11): 663-665.
71
Table 1. Inhibitory effect of water extracts from Cassia tora L. prepared
under different degrees of roasting on the mutagenicity of
Trp-P-1 and Glu-P-1 toward S. typhimurium TA98 and TA100
in the presence of S9 mix
Trp-P-1
Sample
Glu-P-1
IC50 (mg/mL) * % Inhibition ** IC50 (mg/mL) * % Inhibition **
TA98
0.15
96.8 ± 0.4
0.57
94.7 ± 0.7
150 ℃
0.22
94.9 ± 0.6
1.28
83.3 ± 1.4
250 ℃
0.59
88.4 ± 0.7
1.81
63.1 ± 1.8
Unroasted
Roasted
TA100
0.20
98.2 ± 1.4
0.17
86.2 ± 5.8
150 ℃
0.35
94.0 ± 0.6
0.64
79.6 ± 2.1
250 ℃
0.46
75.6 ± 1.6
2.47
58.4 ± 0.4
Unroasted
Roasted
*
**
The IC50 was defined as the concentration of the 50% inhibition.
The % Inhibition was evaluated as the inhibition percentage at the
sample concentration of 2 mg/ml per plate. Results are mean ±
SEM for n=3.
72
60
Unroasted
150¢J
250¢J
Tail moment
50
40
30
20
10
0
100
200
300
400
500
Concentration (g/mL)
Figure 1.
Inhibitory effect of water extracts from Cassia tora L.
prepared under different degrees of roasting on the
genotoxicity of Trp-P-1 toward human blood lymphocytes in
the presence of S9 mix.
73
Results are mean ± SEM for n=3.
1.0
a
Absorbance
0.8
e
b
c
d
0.6
0.4
0.2
0.0
200
220
240
260
280
300
320
340
Wavelength (nm)
Figure 2.
Changes in the UV spectrum illustrates the interaction
of Trp-P-1 with water extracts of unroasted Cassia tora L.
(WEUCT). Trp-P-1 was added to the sample cuvette at a
concentration of 17 μM in 0.1 M Tris-HCl buffer, pH 7.4. Titrations
with ligand were carried out by the addition of 0 (a), 10 (b), 50 (c),
100 (d) and 200 (e) μg/mL WEUCT to both sample and reference
cuvettes.
74
Table 2. Scavenging effects of the reactive intermediates of Trp-P-1 by water extracts of Cassia tora L. (WECT)
prepared under different degrees of roasting *
Unroasted
3.0±1.1
150℃
250℃
3.0±1.1
3.0±1.1
－
44.5±6.0
44.5±6.0
44.5±6.0
Trp-P-1 + BF
－
5.8±1.0
5.8±1.0
5.8±1.0
Trp-P-1
BF
34.1±2.8
34.1±2.8
34.1±2.8
Trp-P-1
BF + WEUT (0.25 mg/mL)
26.9±2.5
31.4±2.9
35.4±3.2
Trp-P-1
BF + WEUT (0.5 mg/mL)
20.9±3.4
26.7±1.3
32.9±3.6
1st preincubation
－
2nd preincubation
－
Trp-P-1
* The carcinogen Trp-P-1 was mixed with hepatic microsomals derived from Aroclor 1254-induced rats (10 %, v/v) and
human blood lymphocytes, and incubated for 30 min at 37℃ in a shaking waterbath (1st incubation). Following
termination of the microsomal metabolism by the addition of 25 μM BF (7,8-benzoflavone), WECT were added and a
further preincubation was carried out (2nd preincubation). Results are mean ± SEM for n=3.
.
75
(A)
80
*
60
Tail moment
40
20
0
(B)
60
40
20
0
0
0.1
0.25
0.5
Concentration (mg/mL)
Figure 3. Effect of water extracts of unroasted Cassia tora L. (WEUCT)
on Trp-P-1-induced DNA damage via different reaction steps
in human blood lymphocytes. Human lymphocytes were
pre-incubated for 30 min with WEUCT before exposure to
Trp-P-1 (A). Following the DNA damage induced by
Trp-P-1, the cells were washed twice in PBS to remove food
mutagen. WEUCT was added and a further post-incubation
was carried out for 30 min (B). Results are mean ± SEM for
n=2. *p＜0.05 refers to difference between Trp-P-1 treated
lymphocytes pre-incubation with or without WEUCT.
76
Table 4. Contents of anthraquinones in water extracts from Cassia tora
L. (WECT) prepared under different degrees of roasting
Content (mg/g extract) *
Sample
Chrysophanol
Emodin
Rhein
0.61 ± 0.06
0.28 ± 0.04
10.42 ± 1.18
150 ℃
0.10 ± 0.02
0.14 ± 0.03
4.77 ± 1.99
250 ℃
ND **
ND
ND
Unroasted
Roasted
*
Contents of anthraquinones in WECT were expressed by
anthraquinone (mg)/(g) WECT. Results are mean ± SEM for
n=3.
**
ND is not detection.
77
Genotoxicity
Chrysophanol (C)
C+S9
C+S9+Trp-P-1
Viability (%)
C
C+S9
70
100
80
50
31%
40
*
60
56%
57%
*
*
30
70%
40
*
20
20
10
0
0
0
1
10
25
50
Concentration (M)
Figure 4.
Inhibitory effect of chrysophanol on human lymphocytes
DNA damage induced by Trp-P-1. Chrysophanol-mediated
inhibition of DNA damage is shown as percentage.
Results are mean ± SEM for n=3. * p ＜ 0.05 is
significantly different by comparison with the control .
78
Viability (%)
Tail moment
60
Genotoxicity
Emodin (C)
C+S9
C+S9+Trp-P-1
Viability (%)
C
C+S9
70
100
80
50
29%
40
*
35%
58%
*
*
30
60
40
20
20
10
0
0
0
1
10
25
50
Concentration (M)
Figure 5.
Inhibitory effect of emodin on human lymphocytes DNA
damage
induced
by
Trp-P-1.
Emodin-mediated
inhibition of DNA damage is shown as percentage. Results
are mean ± SEM for n=3. * p＜0.05 is significantly
different by comparison with the control value.
79
Viability (%)
Tail moment
60
Genotoxicity
Rhein (R)
R+S9
R+S9+Trp-P-1
Viability (%)
C
C+S9
70
100
80
50
21%
*
40
30
60
40
20
20
10
*
0
0
0
1
10
25
50
Concentration (M)
Figure 6.
Inhibitory effect of rhein on human lymphocytes DNA
damage induced by Trp-P-1. Rhein-mediated inhibition
of DNA damage is shown as percentage. Results are mean
± SEM for n=3. * p＜0.05 is significantly different by
comparison with the control.
80
Viability (%)
Tail moment
60