Title
Protective activity from hydrophilic and lipophilic f
ree radical generators of Wen-Pi-Tang and its crud
e drug extracts in LLC-PK_1 cells
Author(s)
CHO, Eun Ju, Yokozawa, Takako, RHYU, Dong You
ng, Mitsuma, Tadamichi, Terasawa, Katsutoshi, PA
RK, Jong Cheol
Citation
和漢医薬学雑誌 = Journal of traditional medicines,
17(6): 245-252
Issue Date
2000-12
Type
Article
Text version
URL
Rights
publisher
http://hdl.handle.net/10110/1950
rights: 本文データは和漢医薬学会の許諾に基づき複製
したものである
http://utomir.lib.u-toyama.ac.jp/dspace/
Joumal of Traditional Medicines17，245−252，2000
245
Protective activity from hydrophilic and lipophilic
free radical generators ofWen−Pi−Tang and
its crude d：rug extracts in LLC−PKI cells
Eun Ju CHo夢）Takako YoKozAwA銃a）Dong Young RHYu夢）Tadamichi
MITSUMAl）
Katsutoshi TERAsAwAc）and Jong Cheol PARKd）
a）ノ勿s！伽陀げハ観％瓶1〃16づづ6i彫，T9ツα窺召〃6‘！io召1αnづPhα”n召66厩乞6αl Un♂∂6鴬勿
b）、0吻7加翻げ加αn6s607i6吻1（働柳o）〃6砒乞n6，万z漁∬o幼吻l
c）Pの）απ窺6n！げノ砂αn6s60万6n云π1〃6漉6in6，7bッα窺α〃6漉6α1α％‘！P：1短7窺召66％♂i6αl UnJ∂6短砂
d）Pのαz加6n！げ0吻nゑ81〃6漉6初61∼6so％π6，Coll昭6‘ゾハ履％瓶l S6乞6％66，S％n6ho盟翫渉io襯l Un歪∂6鴬勿
（ノ∼606♂z／6‘！／1㎎z‘s渉1．2000．／16α4）！6ゴSの！6ηz加725，2000．）
Abstract
WeinvestigatedWen−Pi−Tanganditscmdedmgextractstodeterminetheirprotectiveeffectfrom
oxidative stress caused by the hydrophilic and lipophilic free radical generators，2，2ヲーazobis（2−amidino−
propane）dihydrochloride（AAPH）and2，2なazobis（2，4−dimethylvaleronitrile）（AMVN）in LLGPKl
renal tubular epithelial cells．In response to AAPH and AMVN treatment，cell viability decreased
significantly and significantly enhanced thiobarbituric acid−reactive substances（TBARS）formation
was observed．However，Wen−Pi−Tang and its cmde drug extracts showed scavenging of peroxyl
radicals，which were generated by AAPH and AMVN，resulting in greater cell viability and lower
TBARS fomationthancontrolstreatedonlywithfreeradicalgenerators．Inparticular，Wen−Pi−Tang，
Rhei Rhizoma and Ginseng Radix demonstrated high protective activity，whereas Aconiti Tuber，
Zingiberis Rhizoma and Glycyrrhizae Radix showed relatively low activity．This result suggests that the
antioxidant activity of Wen−Pi−Tang was attributable to the cmde extracts，and that both act as
hydrophilic and lipophilic antioxidants．
Key words Wen−Pi−Tang，Rhei Rhizoma，Ginseng Radix， LLC−PK1，2，2’一azobis（2−amidino一
propane）dihydrochloride，2，2’一azobis（2，4−dimethylvaleronitrile）．
∂吻o，while carefully considering what differences if
Introduction
any exist between theガn∂i！70and in∂i∂o systems．
To generate free radicals at a controlled andwe11−
It is well accepted that free radica1−mediated
defined rate，azo compounds are widely employed，
oxidative stress results in a variety of pathologica1
since they produce free radicals without biotransfor−
conditions． Although several possible sources and
reactions of free radicals are known，it remains un−
mation in response to spontaneous thermal decompo一
sltlon．Noguchiαα1． have used azo compounds
clear how free radicals are initially generated in∂i∂o，
successfully in zノ〃70 and in z／iz／o not only on the
and the time，site and damage of oxygen radical
actions of free radicals upon biological molecules but
formation have not been definitively established．
also on the protective effects of antioxidants．As free
Therefore，in∂i！zo model experiments should be
radicals can be generated in either the aqueous or lipid
performed to clarify our understanding of the mecha−
phase， the efficiency of free radical scavenglng
nisms and dynamics of oxidations taking place in
should be determined in these two phases for hydro一
＊〒930−0194富山市杉谷2630
和漢医薬学雑誌17，245−252，2000
富山医科薬科大学和漢薬研究所 横澤 隆子
2630Sugitani，Toyama930−0194，Japan
246
Free radical protective activity of Wen−Pi−Tang
philic and lipophilic antioxidants．
In recent years，a great deal of attention has been
1，000mlwaterfor5∼65min，accordingtotheWen−Pi一
Tang preparation procedure described previously，
focused on traditional Chinese medicines which have
and each extract was concentrated under reduced
been recognized as antioxidant agents for radica1−
pressure to leave a residue．The yields of Rhei
scavenging activity，as well as on the various physio−
Rhizoma，Ginseng Radix，Aconiti Tuber，Zingiberis
10gical functions of crude drugs frequently prescribed
Rhizoma and Glycyrrhizae Radix were21％，32％，
in clinical practice．Chinese herbal medicines have
37％，11％and20％，respectively，by weight，of the
been regarded as promising new antioxidants and
original preparation．
have thus generated a great deal of research interest
1∼6㎎6n云sαn4耀4i卿n：AAPH and AMVN were
due to the fact that antioxidant therapy apparently
obtained from Wako Pure Chemical Industries，Ltd．
offers protection against a wide range of free radica1一
（Osaka，Japan）and3一（4，5−dimethy1−2−thiazoly1）一2，5−
induceddiseases．Ofthese，Wen−Pi−Tang，theChinese
dipheny1−2H tetrazolium bromide（MTT）was pur−
traditional prescription composed of Rhei Rhizoma as
chased from Sigma Chemical Co．（St．Louis，MO，
the main ingredient，together with Ginseng Radix，
USA）．Dulbeccoりs modified Eagle medium／nutrient
Aconiti Tuber，Zingiberis Rhizoma and Glycyrrhizae
mixture F−12（D−MEM／F−12）and fetal calf serum
Radix，is reported to have a radical scavenging
（FCS）were purchased from Life Technologies，Inc．
． 5−7）
actlon．
（Grand Island，NY，USA）and Cell Culture Labora−
The present study uses azo compounds，2，2’一
tories（Cleveland，OH，USA），respectively．
azobis（2−amidinopropane）dihydrochloride（AAPH）
C麗1魏7646611s6ゆ6吻n6n1：Commercially avai1−
and2，2’一azobis（2，4−dimethylvaleronitrile）（AMVN）
able LLC−PKI cells were maintained at37。C in a
as hydrophilic and lipophilic free radical generators，
humidified atmosphere of5％CO2in air（routine
respectively，to investigate the protective activity of
conditions） in culture plates with 5％ FCS−sup−
Wen−Pi−Tang and its crude dmg extracts on the
plemented D−MEM／F−12medium．After confluence
LLC−PKl renal tubular epithelial ce111ine，which is
was reached，the cells were seeded into96−well cu1−
susceptible to oxidative stress．
ture plates at lO4cells per welL Two hours later，1mM
of AAPH or AMVN treatment was performed，and
Materials and Methods
test samples were then added。The plates were in−
cubated under routine conditions for24h．Fifty mi．
晩n一一Pガー7初g：The composition of Wen−Pi一
croliters of MTT（l mg／m1）solution was added to
Tang used in this study was l5g Rhei Rhizoma
each we11．After incubation for4h at37QC，the MTT
（ノ∼h6雄n 罐6inα16 BAILLON），3g Ginseng Radix
solution was removed from the medium．The resultant
（P厩砿gins6ng C．A．MEYER），9g Aconiti Tuber
formazan crystals in the renal cells were solubilized
（、460ni渉卿n メ勿on才6雄n THUNBERG），3g Zingiberis
with100μl of dimethylsulfoxide．Absorbance at540
Rhizoma （Z乞ngi加7 瞬6i％α／6 ROSCOE） and 59
nm of each well was then read using a Microplate
Glycyrrhizae Radix （G凌yのノ77h乞詔glα加召LINN。var。
Reader（Mode13550−UV，BIO−RAD，Tokyo，Japan）．
glαn4κ1加π∼REGEL et HERDER）．Ginseng Radix was
The level of lipid peroxidant released from the cul．
produced in Korea，Aconiti Tuber was from Japan
tured cells was estimated as thiobarbituric acid−
and all other ingredients were from China．As de一
scribed previously，an extract was obtained by boi1−
reactive substances （TBARS） according to the
の
methods of Yagi and Yokodeαα乙 with a slight
ing the above cmde dmgs gently in1，000ml water for
modification．One aliquot of medium was mixed with
65min，which yielded approximately500ml of decoc−
l．5ml of O．67％TBA aqueous solution and L5ml of
tion，which was then concentrated under reduced
20％trichloroaceti（｝acid，and boiled at95−100℃ for
pressure to leave a brown residue with a yield of
45min．The mixture was cooled with water and
about30％，by weight，of the original preparation。
shaken vigorously with3．O ml of n−butanoL After
Cn64647㎎s：One hundred grams of each crude
centrifugation at4，000g for lO min，the n−butanol
dmgcomponent ofWen−Pi−Tangwasboiledgentlyin
layer was removed，and the fluorescence was mea一
て｝いユ
247
Joumal of Traditional Medicines（Vol．17No．62000）
sured on a fluorescence spectrophotometer（Model
Results
RF−550，SHIMADZU，Kyoto，Japan）．
S観ゑ語os：Results were presented as the mean±
S．E．of5determinations．The data were analyzed for
／L4、PH
statistical significance using Dunnett’s test．Signifi−
Table I shows the effect ofWen−Pi−Tang and its
cance was accepted at1）＜0．05．
crude drug extracts on the viability of LLC−PKI renal
epithelial cells treated with AAPH，which is a hydro−
philic free radical generator．The viability of LLC−
Table I Effect of Wen−Pi−Tang and its crude drug
extracts on viability of cells treated with AAPH．
Material
Concentration（μg／m1）Cell viability（％）
Normal
100．0±2．1
Contro1
Wen−Pi−Tang
75．1 ± 3．4c
0．5
1
Rhei Rhizoma
Ginseng Radix
with the controL In particular，Ginseng Radix
produced greater cell viability when compared with
5
92．7 ± 3．2a・f
non AAPH−treated cells．On the other hand，Aconiti
10
94．1 ± 3．9f
Tuber，Zingiberis Rhizoma and Glycyrrhizae Radix
25
95．6 ± 5．5f
produced relatively low cell viability in comparison
0．5
88．0±3．gc・f
with the other extracts，although they did maintain
greater viability than the contro1．
1
94．0 ± 2．7a・f
2．5
95．3 ± 4．2f
5
95．8 ± 3．Of
As shown in Table II，AAPH enhanced lipid
10
96．1 ± 1．6f
peroxidation in LLC−PKI renal tubular epithelial
25
98．4 ± 1．9f
cells，while Wen−Pi−Tang，Rhei Rhizoma andGinseng
99．9 ± 5．4f
Radix significantly decreased the formation of
0．5
5
GIycyrrhizae Radix
Pi−Tang，Rhei Rhizoma and Ginseng Radix while
significantly preserving cell survival in comparison
84．0 ± 3．3c・e
89．0 ± 3．5b・f
2．5
Zingiberis Rhizoma
AAPH could be attenuated by incubation with Wen−
84．6 ± 3．6c・e
2．5
1
Aconiti Tuber
PKI cells declined to75．1％following AAPH treat−
ment．However，we found that the cytotoxic effect of
102．9 ± 6．7f
106．4±6．1f
109．9 ± 5．7f
TBARS by AAPH．As the treatment concentration
was increased，the peroxidation by AAPH d．eclined
10
112．8 ± 7．6a・f
exponentially．Aconiti Tuber，Zingiberis Rhizoma and
25
1！2．8±9．4a・f
Glycyrrhizae Radix showed only relatively low inhibi．
0．5
76．0 ± 2．3c
tory effects on TBARS formation．
1
81．4 ± 0．8c
、4〃’1々V
2．5
84．6 ± 5．1c・d
5
85．7 ± 6．4c・d
TableIIIshowstheprotectiveactivityofWen−Pi−
10
86．2 ± 6．5c・e
Tang and its crude drug extracts against cellular
25
86．2 ± 4．gc・e
damage mediated by AMVN．AMVN induced a loss
0．5
81．9 ± 1．5c・f
of LLC−PKI renal tubular epithelial cells，and a
1
83．7 ± 0．8c・f
decrease in cell viability to59．9％．However，the
2．5
84．3 ± 1．6c・f
5
84．4 ± 2．oc・f
presence ofWen−Pi−Tang and its crude drugextracts
produced a dose−dependent recovery of viability of
10
87．3 ± 1．3c・f
25
88．1 ± 0．8c・f
LLC−PKI cells．Of these，Wen−Pi−Tang（25μg／ml）
0．5
80．5 ± 1．7c・f
showed the highest protective activity，producing
nearly lOO％cell viability．Moreover，Rhei Rhizoma
1
82．1 ± 0．8c・f
2．5
82．3 ± 0．5c・f
5
82。3 ± 0．4c・f
10
83．7 ± 1．oc・f
25
87．3 ± 0．7c・f
and Ginseng Radix also have strong antioxidant activ−
ity against peroxyl radicals generated by AMVN．On
the other hand，Aconiti Tuber，Zingiberis Rhizoma
Statistical significance： a／）＜0．05， b1）＜0．0！， c／）＜0．001 vs．
and Glycyrrhizae Radix showed relatively weak
normal values，dρ＜0．05，eヵ＜0．01，fヵ＜0．001vs．AAPH−treat−
effects．
ed control values．
248
Free radical protective activity of Wen−pi−Tang
Table II Effect of Wen−Pi−Tang and its cmde dmg
extracts on the formation of TBARS by AAPH．
Material
Concentration（μ9／ml）
TBARS
（nmo1／wel1）
Norma1
0．072 ± 0．004
Control
0．140 ± 0．006b
Wen−Pi−Tang
Rhei Rhizoma
Ginseng Radix
Aconiti Tuber
Zingiberis Rhizoma
Glycyrrhizae Radix
0．5
0．142 ± 0．010b
Table III Effect of Wen−Pi−Tang and its crude dmg
extracts on viability of cells treated with AMVN．
Material
Concentration（μg／ml）Cell viability（％）
Normal
100．0±0．7
59．9 ± 1．3b
Control
Wen−Pi−Tang
0．5
82．4 ± 3．4b・d
1
90．2 ± 0．8b・d
2．5
91．4 ± 2．7b・d
5
93．0 ± 2．4b・d
1
O．134 ± 0．006b
2．5
O．130 ± 0．006b
5
O．118±0．008b・d
10
94．1 ± 3．4a・d
10
O．108 ± 0．010b・e
25
98．1 ± 1．6d
25
O．098 ± 0．008b・e
0．5
0．136 ± 0．004b
Rhei Rhizoma
0．5
75．0 ± 0．1b・d
1
79．4 ± 1．1b・d
2．5
85．6 ± 1．1b・d
5
85．8±1．3b・d
1
O．130 ± 0．004b・c
2。5
O．124 ± 0．006b・e
5
O．112 ± 0．004b・e
10
88．9 ± 1．7b・d
10
O．100 ± 0．006b・e
25
92．6±2．8b・d
25
O．092 ± 0．004b・e
0．5
0．134 ± 0．008b
1
O．132 ± 0．006b
2．5
O．126 ± 0．006b
5
O．112 ± 0．006b・e
10
O．098 ± 0．008b・e
25
O．090 ± 0．004a・e
Ginseng Radix
Aconiti Tuber
0．5
83．5 ± 1．5b・d
1
83．9 ± 3．7b・d
2．5
85．7 ± 3．6b・d
5
90．3±3．3b・d
10
94．0 ± 3．oa・d
25
95．7±3．4d
0．5
72．1±1．5b・d
0．5
0．142±0．004b
1
1
73．9 ± 1．8b・d
O．140 ± 0．008b
2．5
77．3±1．5b・d
2．5
O．140 ± 0．008b
78．4 ± 1．6b・d
5
O．134 ± 0．004b
5
10
79．7±1．7b・d
10
O．130 ± 0．006b
25
82．6 ± 1．7b・d
25
O．122 ± 0．006b・d
0．5
0．138 ± 0．006b
Zingiberis Rhizoma
0．5
61．7 ± 0．7b
1
63．7 ± 0．6b・c
2．5
68．2 ± 0．7b・d
5
70．7 ± 0．6b・d
1
O．140 ± 0．004b
2．5
O．134 ± 0．006b
5
O．128 ± 0．004b・c
10
72．2 ± 1．3b・d
10
O．122 ± 0．006b・e
25
73．4 ± 2．7b・d
25
O．118 ± 0．006b・e
0．5
0．136 ± 0．004b
Glycyrrhizae Radix
0．5
69．9 ± 2．2b・d
1
71．1 ± 1．9b・d
2．5
79．9 ± 3．Ob・d
5
80．5 ± 2．4b・d
1
O．138 ± 0．006b
2．5
O．132 ± 0．006b
5
O．126 ± 0．008b・d
10
81．8 ± 1．1b・d
10
O．120 ± 0．006b・e
25
83．8 ± 5．6b・d
25
O．114 ± 0．004b・e
Statistical significance：aρ＜0．01，bρ＜0．001vs。normal val−
Statistical significance：aヵ＜0。05，bρ＜0．001vs。normal val−
ues，cρ＜0．01，dρ＜0．001vs．AMVN−treated control values．
ues，c1）＜0．05，dρ＜0．01，eヵ＜0．001vs．AAPH−treated control
values．
AMVN−mediated lipid peroxidation．In a similar
The protective activity from lipid peroxidation
mamer，Wen−Pi−Tang，Rhei Rhizoma and Ginseng
against AMVN of the various extracts is shown in
Radix inhibited lipid peroxidation more significantly
Table IV．AMVN enhancedlipidperoxidationinLLC−
than the other extracts．In particular，at a concentra−
PKI cells，and significantly increased TBARS forma−
tion of25μ9／m1，Ginseng Radix extract combined
tion．Wen−Pi−Tang and its cmde dmg extracts
with AMVN treatment resulted in the formation of
demonstrated significant protective activity against
O．136nmo1／well of TBARS，whereas O．204nmo1／we11
Joumal of Traditional Medicines（Vol．17No．62000）
Table IV Effect of Wen−Pi−Tang and its crude dmg
extracts on the formation of TBARS by AMVN．
Materia1
Concentration（μ9／m1）
TBARS
（nmo1／we11）
NormaI
0．076 ± 0．004
Contro1
0．204 ± 0．012a
Wen−Pi−Tang
Rhei Rhizoma
Ginseng Radix
Aconiti Tuber
Zingiberis Rhizoma
0．5
0．198 ± 0。006a
1
O．192 ± 0．008a
の
cals，Play an important role in tissue injury． ∫n∂z！zo
and in∂i∂o studies have implicated reactive oxygen
metabolites in various forms of toxic nephropa一
ユ thy． The putative site of injury in most forms of
toxic nephropathy is the epithelial cells lining the
の
tubules． Although various kinds of free radical initi−
ators are known，azo compounds have been used
easily and successfully as radical initiators，since they
2．5
O．184 ± 0．012a・b
5
O．174 ± 0．004a・d
generate free radicals at a measurable and constant
10
O．160 ± 0．008a・d
rate吻thermal decomposition without biotransfor−
25
O．152 ± 0．008a・d
0．5
0．184 ± 0．008a・c
1
O．180 ± 0．006a・d
2．5
O．170 ± 0．006a・d
5
O．164 ± 0．008a・d
10
O．156 ± 0．006a・d
25
O．146 ± 0．008a・d
0．5
0．188±0．008a・b
1
O．178 ± 0．006a・d
2．5
O．166 ± 0．008a・d
5
mation．The free radicals generated from azo com−
pounds react with oxygen radicals，attack other lipid
τnolecules to form lipid hydroperoxide and new lipid
radicals．This reaction，which induces physiochemical
alterations and cellular damage takes place repeated．
1y with a resultant attack upon various biologica1
つ molecules． The free radicals ultimately cause a
diverse array of pathological changes．
O．158 ± 0．008a・d
Under hydrophilic and lipophilic conditions，gen−
10
O．144 ± 0．006a・d
erators of free radicals may be located in two differ−
25
O．136 ± 0．004a・d
0．5
0．208 ± 0．004a
1
O．202 ± 0．008a
2．5
O．198 ± 0．008a
5
O．194 ± 0．004a
10
O．184 ± 0．008a・c
25
O．176 ± 0．004a・d
0．5
0．206 ± 0．004a
1
O．200 ± 0．008a
2．5
O．196 ± 0．006a
ent cellular compartments，namely the cytosol and
membrane．These areas represent aqueous and hydro一
の
phobic phases，respectively． Therefore，we chose
AAPH and AMVN as the source of hydrophilic and
lipophilic free radical initiators，since unlike most
experimental systems suitable for the induction of
oxidative stress，these compounds do not require the
addition of potentially interferring cofactors and tran−
O．184 ± 0．004a・c
sition metals．For these reasons，they are useful tools
10
O．180 ± 0．006a・d
for studying the damage induced by free radicals on
25
O．172 ± 0．008a・d
5
Glycyrrhizae Radix
249
biological systems．Hydrophilic AAPH added to the
0．5
0．206 ± 0．004a
1
O．200 ± 0．008a
2．5
O．198 ± 0．008a
5
O．186 ± 0．006a・c
region of micelles or membrane initially generates
10
O．180 ± 0．004a・d
18．21−26）
radicals within the lipid region．
25
O．170 ± 0．004a・d
Statistical significance：aρ＜0．001vs．normal values，
bρ＜0．05，cρ＜0．01，dρ＜0。001vs．AMVN−treated controI
values．
aqueous phase generates radicals in the aqueous
region，whereas lipophilic AMVN located in the lipid
We confirmed that both AAPH and AMVN
caused oxidative damage in LLC−PKI renal tubular
epithelial cells，resulting in the loss of cell viability．
One mM AAPH and AMVN caused declines in cell
viability to 75．1％ and 59．9％，respectively．In the
ofTBARS wasproducedinwellstreatedwithAMVN
present experiment，further evidence was obtained
alone．
that cells damaged by oxidative stress from free
radicals peroxidize more rapidly，resulting in the
1）iscussion
formation of lipid peroxidation end products，such as
TBARS，that provide a good index of cell destmction．
Reactive oxygen metabolites，including free radi一
Lipid peroxidation in LLC−PKI cells was increased by
250
Free radical protective activity of Wen−Pi−Tang
AMVN，a lipophilic free radical generator，more
significantly than by AAPH，a hydrophilic generator．
idative activity． Consequently，the localization of
antioxidants and the site of radicals generated should
The TBARS formation was enhanced from O．072to
be taken into account when assessing their antiox−
O．140nmo1／well and from O．076to O．204nmo1／well in
idant activities．In other words，hydrophilic antiox−
response to AAPH and AMVN treatment，respective−
idants scavenge the aqueous radicals efficiently，but
1y．These results demonstrate that the oxidative dam−
cannot scavenge lipophilic radicals within the lipid
age caused by AMVN is relatively more severe than
region．On the other hand， 1ipophilic antioxidants
that by AAPH．
scavenge lipophilic radicals within the liposomal
Antioxidants play a major role in protecting tis−
membranes，not hydrophilic radicals within the aque−
sue from cellular loss and lipid peroxidation causedby
ousregion．Withthisinmind，thefreeradicalscaveng−
reactive intermediates formed and released during
ing activity of Wen−Pi−Tang and each of its compo−
oxidative stress．Oriental medicines，which include a
nent cmde drugs were investigated using LLC−PKl
variety of antioxidant compounds，are still much in
cells，which are susceptible to oxidative stress．Wen−
demand despite the widespread use of conventional
Pi−Tang scavenged both AAPH and AMVN−generat−
medicines．Research is now being conducted in the
ed free radicals，suggesting that it plays a role both as
treatment of chronic diseases that respond poorly to
a hydrophilic and lipophilic antioxidant． Rhei
conventional dmg therapy，to determine the actual
Rhizoma and Ginseng Radix，two of the component
role of the antioxidants in oriental medicines and
cmde dmgs，had strong activity，while Aconiti Tuber，
medical prescriptions comprised of a combination of
Zingiberis Rhizoma and Glycyrrhizae Radix produced
several oriental medicines．This research also
a relatively weak effect．Thus，Wen−Pi−Tang was
attempts to clarify how active oxygen and its effects，
shown to inhibit the scavenging activity of peroxyl
such as lipid peroxidation，are involved in various
radicals generated by AAPH and AMVN，resulting in
diseases．Of the numerous prescription Chinese medi−
higher cell viability and lower MDA formation．Two
cines，the authors previously demonstrated the usefu1−
of its component crude drugs，Rhei Rhizoma and
ness ofWen−Pi−Tang as a conservativetreatmentfor
Ginseng Radix，were shown to play important contrib−
renal failure under enhanced oxidative conditions in
utory roles in such activity of Wen−Pi−Tang．
ク
り both experimental and clinical settlngs． To
experimentally establish the scientific basis for the
和文抄録
action of Wen−Pi−Tang，we further examined the
antioxidant capacity of Wen−Pi−Tang and its compo−
水溶性アゾ化合物のAAPH（2，2ヲーazobis（2−amidino−
nent cmde drugs both乞n∂i∂o and才n加ケo．The results
propane）dihydrochloride）と脂溶性アゾ化合物の
of our previous studies suggested that Wen−Pi−Tang
AMVN（2，2’一azobis（2，4−dimethylvaleronitrile））で腎
and some of its component crude drugs exert an
上皮細胞のLLC−PK1に酸化的ストレスを惹起させ，温
antioxidant action on the impaired kidney under
脾湯と5種類の構成和漢薬の効果を検討した。AAPH
57）
OxidatiVe StreSS．
とAMVNで処理した場合，細胞生存率が著しく低下し，
Antioxidants exert their actions by preventing
チオバルビツール酸反応物質の生成が著しく上昇した
the production of active oxygen radicals or by cap−
が，温脾湯と各構成和漢薬エキスをそれぞれ添加した場
ture／removal of the produced radicals．We examined
合，温脾湯と大黄，薬用人参では高い抗酸化活性を示し
the effects of Wen−Pi−Tang and its five component
た。しかし附子，乾姜，甘草では相対的に低い活性であっ
crude drugs on the latter anti−radical process．Recent−
た。このことから，温脾湯の抗酸化活性は構成和漢薬に
1y，the importance of different types of antioxidants，
起因し，また温脾湯は水溶性抗酸化物と脂溶性抗酸化物
water−soluble or lipid−soluble，has been the focus of
の両方の特徴を有していることが示唆された。
considerable research attention．Several reports sug−
gest that scavenging of aqueous and lipid peroxyl
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40）Terao，」．，Piskula，M．and Yao，Q．：Protective effect of epicate−
and protection by vitamin E．Fz66ぬ4ガ6．研ol．〃ε4．9，199−204，
chin，epicatechin gallate，and quercetin on lipid peroxidation in
1990．
phospholipid bilayers．／176h．B∫06h6”z．」Bぎ01）hツs．308，278−284，1994．
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2014 – 2015 PS184 課後班報名表

Valentines 2015 Newsletter