The chemical and hydroxyl radical scavenging activity changes of ginsenoside-Rb1 by heat processing

Article abstract of DOI:10.1016/j.bmcl.2008.07.056

The chemical and hydroxyl radical ({radical dot}OH) scavenging activity changes of ginsenoside Rb₁ (Rb₁) by heat processing were investigated in this study. Rb₁ was changed into 20(S)-Rg₃, 20(R)-Rg₃,

Full text of DOI:10.1016/j.bmcl.2008.07.056

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4515–4520
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The chemical and hydroxyl radical scavenging activity changes
of ginsenoside-Rb by heat processing
1
a
b
a,c
a
c
a,*
Yong Jae Lee , Hyun Young Kim , Ki Sung Kang , Jin Gyun Lee , Takako Yokozawa , Jeong Hill Park
a
Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Shillim-Dong San 56-1, Seoul 151-742, Republic of Korea
Department of Food Science, JinJu National University, Jinju 660-758, Republic of Korea
Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 February 2008
Revised 3 July 2008
Accepted 12 July 2008
Available online 17 July 2008
The chemical and hydroxyl radical (ÅOH) scavenging activity changes of ginsenoside Rb1 (Rb ) by heat
1
processing were investigated in this study. Rb
heat processing through glucosyl elimination and epimerization of carbon-20 by SN1 reaction. The glu-
cosyl moiety, separated from Rb , made Maillard reaction product (MRPs) with glycine. The generations
of 20(S)-Rg and MRPs were related to the increased ÅOH scavenging activity of Rb by heat processing.
Ó 2008 Elsevier Ltd. All rights reserved.
1 3 3 1 5
was changed into 20(S)-Rg , 20(R)-Rg , Rk , and Rg by
1
3
1
Keywords:
Ginsenoside Rb
1
Hydroxyl radical
Maillard reaction
Glycine
The root of ginseng, Panax ginseng C. A. Meyer (Araliaceae), has
been heat processed to improve its medicinal efficacies in Korea.
Steaming process is known to induce a structural change of ginse-
als such as amino reductants during heat treatment.12,13 Rb
has
1
glucosyl moiety at carbon-20, which can be easily separated by
heat processing, and glycine is a frequently used amino acid in
the Maillard reaction model system, and is also contained in Panax
1
–4
noside and to enhance the biological activities of ginseng.
1
4,15
Although there are several reports about the heat processing or
intestinal bacteria-induced chemical and biological activity
changes of ginsenosides, the interaction of ginsenoside with amino
ginseng.
generation of Maillard reaction products (MRPs) from the heat pro-
cessing model experiment using Rb and glycine. However, argi-
nine, major amino acid of ginseng, inhibited Maillard reaction of
Rb
with unknown reason from our previous report.6 Therefore,
by heat
In addition, we already demonstrated the significant
1
5
,6
acids during heat processing was not fully elucidated yet. There-
fore, we have been investigating the chemical and antioxidant
activity changes of ginsenosides by Maillard reaction.
1
the chemical and ÅOH scavenging activity changes of Rb
1
Ginsenoside-Rb
1
(Rb
1
) is a well-known diol-type triterpene gly-
processing with glycine were investigated in this study before
the further study on the other amino acids.
coside that exists most abundantly in Panax ginseng, and is known
to have anti-inflammatory and antioxidant effects.7 Rb
–9
rescued
Rb
1
1
consists of four hydrophobic ring steroid-like structures
2,16
hippocampal neurons from lethal damage caused by the hydroxyl
radical (ÅOH)-promoting agent FeSO in vitro, and the Fenton reac-
tion containing p-nitrosodimethylaniline confirmed the ÅOH inhib-
with hydrophilic sugar moieties at carbon-3 and -20 (Fig. 1).
4
To elucidate the chemical and antioxidant activity changes of Rb
1
1
0
iting activity of Rb
1
.
In addition, the ÅOH scavenging activity test
using electron spin resonance spectrometer (ESR) was suggested to
be the most appropriate to test the antioxidant activities of
ginsenosides, and this effect was closely related to the ferrous
metal ion-chelating activities of ginsenosides from our previous
Glc(1 - 6)Glc-O
OH
20
12
5
,6,11
researches.
On the other hand, most major Maillard reactions are repre-
sented by sugar-amino acid groups, and the reaction between
reducing sugars and amino acid produces strong reducing materi-
3
Glc(1 - 2)Glc-O
*
Corresponding author. Tel.: +82 2 8807857; fax: +82 2 8748928.
E-mail address: hillpark@snu.ac.kr (J.H. Park).
1
Figure 1. The chemical structure of ginsenoside Rb . –Glc, D-glucopyranosyl.
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.056

4
516
Y. J. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4515–4520
by Maillard reaction, we investigated the Maillard reaction model
experiment using Rb
and glycine,17 and the ÅOH scavenging
activity changes were tested with ESR.
(D)). On the other hand, the change in content of glycine was not
confirmed because its peak was overlapped with glucose peak pro-
duced by steaming as shown in Fig. 3(D). 20(S)-ginsenosides and
20(R)-ginsenosides are epimers of each other depending on the
geometrical position of the hydroxyl group (OH) on carbon-20.
Especially, this epimerization is known to occur by the selective at-
tack of the OH group after the elimination of the glycosyl residue at
1
18
Figure 2 shows the
, heat processed
-glycine mixture.
strongly inhibited ÅOH production to about 24% at the concen-
comparisons in ÅOH scavenging activities of Rb
1
1
Rb
Rb
1 1
, Rb -glycine mixture, and heat processed Rb
1
tration of 0.5%, but the effect after heat processing was lowered to
2% (Fig. 2(A) and (B)). On the other hand, Rb -glycine mixture
inhibited ÅOH production to about 50%, which effect was about
the half of Rb , but the effect of heat processed Rb -glycine mixture
1
4,16
4
1
carbon-20 during the steaming process.
polar ginsenosides such as Rk and Rg are known to be easily pro-
duced by the elimination of H O at carbon-20 of Rg under high
pressure and temperature conditions like in autoclaving. There-
fore, Rb was gradually changed into 20(S)-Rg , 20(R)-Rg , Rk , and
Rg by heat processing, but the generated content of 20(S)-Rg was
higher than that of 20(R)-Rg when Rb was heat processed with
In addition, more less-
1
5
1
1
2
3
3,5
had a stronger value of 37% at the concentration of 0.5%. In addi-
tion, there showed nearly no ÅOH scavenging activity in glycine
1
3
3
1
(
Rb
data not shown). Consequently, the ÅOH inhibiting activity of
1
5
3
was decreased by heat processing, but it was increased when
3
1
6
heat processed with glycine (Fig. 2(B) and (C)). Therefore, compar-
glycine (Fig. 3(B) and (D)) as determined previously, and there
was a need to examine the ÅOH scavenging activities of these
less-polar ginsenosides produced by heat processing for the identi-
fication of active components.
isons of the constituent analysis19 and ÅOH scavenging activities of
components produced by the heat processing of Rb
1
-glycine mix-
ture were carried out.
As shown in the HPLC chromatograms of Rb
cessed Rb , Rb (1 mg) was detected at about 17 min when not
steamed (Fig. 3(A)) and it disappeared, and the contents of 20(S)-
Rg (146 g), 20(R)-Rg (201 g), Rk (102 g), and Rg
110 g) were increased by heat processing (Fig. 3(B)). In addi-
tion, the increased peak area at about 2.3 min shown in Figure
(B) was determined as glucose from the GC-MS analysis (data
not shown). In the case of steaming model experiment using
Rb -glycine mixture, the glycine and Rb were detected at about
.3 and 17.0 min, respectively, when not steamed (Fig. 3(C)). Then,
all of the Rb disappeared, and the contents of 20(S)-Rg (196 g),
0(R)-Rg (167 g), Rk (102 g), and Rg (108 g) were increased
as shown by steaming of Rb , but the generated content of 20(S)-
Rg was higher than that of heat processed Rb (Fig. 3(B) and
1
and heat pro-
When the ÅOH scavenging activities of Rb
20(R)-Rg , Rk , and Rg were compared, Rb
1
duction to about 47% at the concentration of 0.05%, but glycine
showed slight or no ÅOH scavenging activity (Fig. 4(A)). In the
comparison of ginsenosides produced by steaming, 20(S)-Rg
3
1
, glycine, 20(S)-Rg
inhibited ÅOH pro-
3
,
1
1
3
1
5
3
l
3
l
1
l
5
2
0
(
l
and Rg
respectively, at the concentration of 0.05% (Fig. 4(A)), but the ef-
fects of 20(R)-Rg and Rk were comparably lower than that of
Rb (Fig. 4(A) and (B)). The stronger ÅOH inhibiting activities of
20(S)-Rg and Rg than their epimers were suggested to be med-
iated by geometrically closer double bond and OH group at car-
5
strongly inhibited ÅOH generation to about 16 and 22%,
3
3
1
1
1
1
2
3
5
1
3
l
1
1
2
3
l
1
l
1
5
l
bon-20 to OH group at carbon-12. Although the strongly ÅOH
inhibiting ginsenosides such as 20(S)-Rg and Rg were generated
by heat processing, it accompanied the increases in 20(R)-Rg and
3
5
3
1
3
1 1 1 1
Figure 2. The comparison in the ÅOH scavenging activities of Rb , heat processed Rb , Rb -glycine mixture, and heat processed Rb -glycine mixture.

Y. J. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4515–4520
4517
1 1 1 1
Figure 3. HPLC chromatograms of (A) Rb , (B) heat processed Rb , (C) Rb -glycine mixture, and (C) heat processed Rb -glycine mixture.
1 3 3 1 5
Figure 4. The comparison in the ÅOH scavenging activities of Rb , glycine, 20(S)-Rg , 20(R)-Rg , Rk , and Rg .
Rk
1
weak ÅOH inhibitors. Therefore, the effect of these mixed gin-
was heat processed with glycine (Fig. 3(B) and (D)), and the in-
creased ÅOH scavenging activity of Rb -glycine mixture by heat
processing was thought to be partially mediated by effect of
20(S)-Rg . These results were similar with our previous report
using Rb
senosides was thought to be neutralized or lowered, and there
1
showed no significant increase in the ÅOH inhibiting activity when
the Rb
0(S)-Rg
1
was heat processed. However, the generated content of
3
2
1
2
3
was more higher than that of 20(R)-Rg
3
when Rb
1
2
, another major ginsenoside, and glycine.

4
518
Y. J. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4515–4520
We have confirmed that the MRPs may have little or no effects
on the increased ÅOH scavenging activity of Rb -glycine mixture by
heat processing in our previous report.21 The novel point of this
study is that the MRPs, which generated from Rb -glycine mixture
added glycine, but we could not identify the molecular structures
2
of MRPs in present study because their structures are extremely
21
complex and largely unknown. Then, the browning compound le-
1
vel and ÅOH scavenging activity changes of the MRPs produced
from glucose–glycine and maltose–glycine mixtures were
examined.
by heat processing, have ÅOH scavenging activity. The development
of color is known as an important and obvious feature of the
Maillard reaction, and heat-induced antioxidants including
melanoidins and reductones are known to be formed by this
Figure 6 shows the changes in ÅOH scavenging activities and
browning compound levels of MRPs generated from glucose–
glycine and maltose–glycine mixtures. MRPs generated from glu-
cose–glycine and maltose–glycine mixtures inhibited ÅOH genera-
tion to about 37% and 77%, respectively, at the concentration of
0.05% (Fig. 6 (A)). In addition, the browning compound levels at
the concentration of 0.05% of MRPs generated from glucose–gly-
cine and maltose–glycine mixtures were 1.019 and 0.117 A.U.,
respectively. These values were dose-dependently increased as
shown in Fig. 6(B) and (C), and the brown color of MRPs generated
from glucose–glycine mixture was stronger than that of maltose–
glycine mixture. In addition, the ÅOH scavenging activity of glu-
1
3,22
reaction.
of un-treated Rb
out glycine. The absorbance values at 420 nm of un-treated Rb
Figure 5 shows the comparison in browning levels
1
and its steamed products at 120 °C with or with-
1
and glycine were 0.0053 and 0.0002 A.U., respectively, and they
were increased to 0.0073 and 0.0042 A.U. when heat processed
at 120 °C for 3 h, respectively (Fig. 5). No significant changes in
the browning compound levels were noted when Rb
were heat processed separately, but the browning level of heat
processed Rb -glycine mixture was significantly high value of
.3182 A.U. than the others (Fig. 5). Therefore, heat-induced anti-
1
and glycine
1
0
oxidants including melanoidins and reductones were thought to
be generated by the Maillard reaction as mentioned above. Consid-
cose–glycine mixture was stronger than that of Rb
concentration (Figs. 4 (B) and 6(A)). Therefore, it is clear that the
MRPs generated from the separated glucosyl moiety of Rb and
1
at the same
ering the HPLC data of un-treated and heat processed Rb
mixture, Rb was gradually changed into 20(S)-Rg , 20(R)-Rg
Rk , and Rg by heat processing, and its sugar moieties at car-
1
-glycine
1
1
3
3
,
glycine have ÅOH scavenging activity, and the changes in browning
levels of ginsengs or ginsenosides by heat processing were thought
to be related to the increase in ÅOH scavenging activity. On
1
5
bon-20 were separated (Fig. 3(B)). The separated sugar moieties
such as glucose or maltose were thought to form MRPs with the
2
the other hand, the little or no effects of MRPs from Rb -glycine
Figure 5. The graph compares the browning compound levels in Rb
1 1
, glycine, heat processed Rb , heat processed glycine, and heat processed Rb
1
-glycine mixture at the
concentration of 0.05%.
*
Figure 6. The graphs compare the ÅOH scavenging activities and browning compound levels of MRPs generated from glucose–glycine and maltose–glycine mixtures. p < 0.01
1
vs. Rb .

Y. J. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4515–4520
4519
Figure 7. The graphs compare (A) DPPH radical scavenging and (B) ferrous metal ion-chelating activities of MRPs generated from glucose–glycine and maltose–glycine
mixtures.
mixture from our previous report were thought to be resulted from
Acknowledgments
22
the difference in sugar types between Rb
1
and Rb
2
.
1
Rb generates
glucose, but Rb generates arabinose during heat processing.
Although we could not expend our conclusion to the other ginse-
nosides and amino acids in present study, the identification of indi-
2
We thank Hyea-Sook Shin, a research support specialist at Na-
tional Center for Inter-University Research Facilities, SNU, for tech-
nical assistance. This work was supported by a grant of Health and
Medical Technology R&D Program (No. 040021) funded by the
Ministry for Health, Welfare and Family Affairs, Republic of Korea.
rect antioxidant effect of Rb
1
by generating glucose to make
antioxidant MRPs with glycine is novel idea in ginsenoside
research.
Subsequently, we examined 1,1-diphenyl-2-picrylhydrazyl
DPPH) radical scavenging and ferrous metal ion (Fe )-chelating
References and notes
2+
(
5
1. Kitagawa, I.; Yoshikawa, M.; Yoshihara, M.; Hayashi, T.; Taniyama, T. Yakugaku
Zasshi 1983, 103, 612.
activities of MRPs from Rb -glycine mixture to identify its ÅOH
1
scavenging mechanism. The ÅOH scavenging in present study can
be accomplished by direct scavenging of free radical or via preven-
tion of ÅOH formation through the chelation of Fe , and the ÅOH
inhibiting effect of ginsenoside is known to be mediated by Fe
chelating activity. DPPH is a stable free radical, and has been
widely used to test the ability of compounds to act as free radical
2.
Matsuura, H.; Hirao, Y.; Yoshida, S.; Kunihiro, K.; Fuwa, T.; Kasai, R.; Tanaka, O.
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2
3
scavengers. In the DPPH radical scavenging activity test of MRPs,
MRPs generated from glucose–glycine mixture showed stronger
activity than maltose–glycine mixture (Fig. 7(A)). However, both
of the MRPs generated from glucose–glycine and maltose–glycine
mixtures showed no Fe2 -chelating activity changes (Fig. 7(B)).
The correlation between DPPH radical and ÅOH scavenging activi-
ties of MRPs was observed (Figs. 6(A) and 7(A)). Therefore, the
ÅOH scavenging of MRPs was thought to be mediated by direct free
6
7
.
.
Kang, K. S.; Lee, Y. J.; Park, J. H.; Yokozawa, T. Biol. Pharm. Bull. 2007, 30, 1975.
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13. Samaras, T. S.; Camburn, P. A.; Chandra, S. X.; Gordon, M. H.; Ames, J. M. J. Agric.
As shown from the above results, we confirmed that the Rb
was gradually changed into 20(S)-Rg , 20(R)-Rg , Rk , and Rg by
heat processing, and its sugar moieties at carbon-20 were sepa-
rated (Fig. 3). The ÅOH inhibiting activities of 20(S)-Rg and Rg
were stronger than that of Rb , but 20(R)-Rg and Rk showed
1
Food Chem. 2005, 53, 8068.
14. Park, J. D. Korean J. Ginseng Sci. 1996, 20, 389.
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3
1
5
1
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3
5
16. Shoji, J. In Recent Advances in Ginseng Studies; Shibata, S., Ohtsuka, Y., Sato, S.,
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3
1
Eds.; Hirokawa Publishing: Tokyo, 1990; p 11.
weak or no ÅOH inhibiting activities. The neutralized ÅOH scaveng-
ing activity of these mixtures of strong and weak ÅOH inhibiting
17. Rb
975, 77, 1057. The molecular formula of Rb
high-resolution mass spectrum (m/z 1109.6115 [M+H] , calculated for
23 1109.6115). The same amounts (w/w) of Rb and glycine were
steamed together at 120 °C. After drying at 50 °C, un-treated and heat
processed Rb -glycine mixtures at 120 °C were prepared. The samples were
dissolved in distilled water (D.W.)-acetonitrile (1:1, v/v), and the absorbance at
20 nm was measured in a 1 cm glass cuvette using a UV-1200 UV–Vis
1
was isolated and identified from Panax ginseng as described in J. Biochem.
1
1
was given as C54 23 from the
92
H O
+
ginsenosides of heat processed Rb
un-treated Rb . However, the generated content of 20(S)-Rg
higher than that of 20(R)-Rg when the Rb was heat processed
with glycine, and its browning compound level and ÅOH scavenging
activity were significantly increased than that of un-treated Rb
Therefore, the increase in ÅOH scavenging activity of Rb by heat
processing with glycine was medicated by the generations of
1
was not stronger than that of
C
54
H
93
O
1
1
3
was
1
3
1
4
1
.
spectrophotometer (Shimadzu, Kyoto, Japan) to measure the extent of
browning. The heat processing and measurement were repeated three times
for each sample. The results for each group are expressed as mean ± SE values.
Individual differences between groups were evaluated using Student’s t-test,
and those at p < 0.05 were considered significant.
1
2
4
0(S)-Rg
3
and MRPs, which have ÅOH scavenging activities (Figs.
1
8. The ESR spectra were recorded on a JES-TE100 ESR spectrometer (JEOL, Tokyo,
Japan). The experimental parameters were as follows: temperature, ambient;
microwave power, 1.02 mW; modulation frequency, 100 kHz; modulation
width, 0.16 mT; sweep width, 5.0 mT; sweep time, 0.5 min; center field,
and 6). The biological and chemical roles of ginsenosides in terms
of self-mediated and indirectly mediated actions such as via the
Maillard reaction are thought to be valuable in order to understand
the complex efficacy changes of ginseng by heat processing.
339.550 mT; time constant, 0.03 s; and receiver gain, 1. 5,5-dimethyl-1-

4
520
Y. J. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4515–4520
2
+
pyrroline N-oxide (DMPO) was used as a spin-trapping reagent for ÅOH. Mn
acetonitrile (solvent B), and the flow rate was 1 mL/min. The detector was a
SEDEX 55 evaporative light scattering detector (Sedere, France). The gradient
elution was used as follows: 0 min, 15% B; 10 min, 34.5% B; 25 min, 47.5% B;
40 min, 80% B; and 50 min, 100% B.
was used as an external standard to calculate the relative amounts from the
ESR signal intensity. Twenty microliters of DMPO (1/10 diluted with D.W., v/v)
were mixed with 38
diethylenetriaminepentaacetic acid. The mixture was stirred with 30
sample solution and 75 L of 1 mM hydrogen peroxide. The solutions were
l
L
of 0.2 mM ferrous sulfate and 37
lL
of 1 mM
l
L of
20. The BuOH fraction of heat processed Panax ginseng, as described in J. Nat. Prod.
l
3
2000, 63, 1702, was applied to a silica gel column eluting with CHCl -MeOH
transferred to a capillary tube and placed in the cavity of the ESR spectrometer
for measurement. After 5 min, the ESR signal was taken to measure the yield of
the inhibition of ÅOH by samples. Measurement was repeated three times for
each sample. The inhibition of ÅOH was determined by the ratio of peak height
(30:1 ? 20:1 ? 10:1 ? 1:1) to afford subfractions, and it was further purified
with preparative HPLC with a detection wavelength of 203 nm. The structures
of 20(S)-Rg , 20(R)-Rg , Rk , and Rg were confirmed by comparing the
3 3 1 5
13 1
chemical shifts in C NMR and H NMR with standard samples.
21. Borrelli, R. C.; Visconti, A.; Mennella, C.; Anese, M.; Fogliano, V. J. Agric. Food
Chem. 2002, 50, 6527.
2
+
of the DMPO-OH spin adduct to the signal of Mn and compared to the ratio of
the control.
1
9. The changes in constituents by heat processing were analyzed with a Hitachi
22. Kang, K. S.; Kim, H. Y.; Baek, S. H.; Yoo, H. H.; Park, J. H.; Yokozawa, T. Biol.
Pharm. Bull. 2007, 30, 724.
23. Hatano, T.; Edamatsu, R.; Hiramatsu, M.; Mori, A.; Fujita, Y.; Yasuhara, T.;
Yoshida, T.; Okuda, T. Chem. Pharm. Bull. 1989, 37, 2016.
(
Tokyo, Japan) L-7100 liquid chromatograph fitted with a C-18, reversed-phase
column (5
m, 25 cm  4.6 mm I.D.; Phenomenex Luna) utilizing the solvent
gradient system. The mobile phase consisted of water (solvent A) and
l