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Exploration research on hepatotoxic constituents from Polygonum multiflorum root

何首乌肝毒性物质基础探索研究



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Explorationresearchonhepatotoxicconstituentsfrom
Polygonummultiflorumroot
YANGMin1,2,LIUTing1,FENGWeihong1,3,HUILianqiang1,LIRaorao1,3,LIUXiaoqian1,3,
CHENAnjia2,LIChun1,3,WANGZhimin1,3
(1.InstituteofChineseMateriaMedica,ChinaAcademyofChineseMedicalSciences,Beijing100700,China;
2.ShanxiMedicalUniversity,Taiyuan030001,China;
3.NationalEngineeringLaboratoryforQualityControlTechnologyofChineseHerbalMedicines,Beijing100700,China)
[Abstract] ByobservingthecytotoxicefectsofanthraquinonesonHepG2celandusingtheprecisioncutliverslicestechniqueto
authenticatethecytotoxicconstituents,thepaperaimstoexplorethematerialbasisofPolygonummultiflorumroottocauselivertoxici
tyFirstly,MTTmethodwasusedtodetecttheefectof11anthraquinonederivativesonHepG2celThen,theclearcytotoxicingredi
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Vol41,No.7 April,2016
entswerecoculturedwithratliverslicesfor6hrespectively,andthelivertissuehomogenatewaspreparedBCAmethodwasusedto
determinethecontentofproteininthehomogenateandcontinuousmonitoringmethodwasusedtomonitortheleakageofalanineamin
otransferase(ALT),aspartateaminotransferase(AST),gammaglutamineaminotranspeptidase(GGT)andlactatedehydrogenase
(LDH)ThetoxicefectoftheseingredientsonlivertissuewastestedbycalculatingtheleakagerateofthemonitoredenzymesAsa
result,rhein,emodin,physcion8OβDglucopyranosideandphyscion8O(6′Oacetyl)βDglucopyranosideshowedcytotoxic
efectsonHepG2celandtheirIC50valueswere7107,12562,24227,40232μmol·L
-1respectively,buttheother7compounds
arelesstoxicandtheirIC50valuescannotbecalculatedTheprecisioncutliverslicestestsshowedthatrheingroupof400μmol·L
-1
concentrationsignificantlyincreasedtheleakagerateofALT,ASTandLDH(P<001),andtherheingroupof100μmol·L-1con
centrationonlyincreasedtheleakagerateofLDH(P<005)Withtheincreaseofrheinconcentration,theproteincontentinliver
slicesdecreasedsignificantly(P<005)withacertainrangeofdoesEmodingroupof400μmol·L-1concentrationsignificantlyin
creasedtheleakagerateofALT,GGTandLDH(P<001)Physcion8OβDglucopyranosidegroupof800μmol·L-1concentra
tionalsosignificantlyincreasedtheleakagerateofALT,ASTandLDH(P<001orP<005),butthegroupof200μmol·L-1con
centrationonlysignificantlyincreasedtheLDHleakage(P<005)Alongwiththeincreaseoftheconcentrationofphyscion8OβD
glucopyranoside,theleakagerateofALT,ASTandLDHshowedatrendofincrease,buttheproteincontentinliversliceswasinde
clineFurthermore,MTTreductionabilityofliverslicessignificantlydecreased(P<001)inthephyscion8OβDglucopyranoside
groupof800μmol·L-1concentrationTheresultssuggestedthatrhein,emodinandphyscion8OβDglucopyranosideathighcon
centrations(≥400μmol·L-1)canproducesomedamagetothelivertissueHowever,theexposurelevelsoftheseconstituentsare
verylow,sotoreachthetoxicconcentration(400μmol·L-1or800μmol·L-1)anadultof65kgbodyweightwilneedatleasta
singleoral4898g,339gand5581gofPmultiflorumrootrespectively,whichisfarfromthestatutorydoseofcrudePmultiflorum
root(36g)oritsprocessedproduct(612g)Therefore,theconclusionthatanthraquinonesaretheprimeconstituentsofthehepato
toxicityofPmultiflorumrootarestilnotbeproved
[Keywords] Polygonummultiflorumroot;anthraquinones;hepatoxicity;precisioncutliverslicestechnique;hepatoxicconstituents
doi:10.4268/cjcmm20160721
  
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Zahµƒ
fHg$
11
U°–—ì
HepG2
Za±Za
h烜æ]*
1,2。
Ͼhi

‹B…À¼

…À
Û

…ÀۇC
8OβD·¸£ê、…ÀۇC8
O(6′O
m 1
)βD·¸£êìHepG2Za-B
[â±Zah0ž

§Óâ_V˜±Zah0ž{
„:

rx-d
IC50;§åB±_V˜›…ÀÛ1
OβD·¸£ê{ì HepG2Za±ø™áÖ0
ž

ïð

…À¼

…ÀÛ

…À¢

ñû…ÀÛ

…À
Û
8OβD·¸£ê¬…À¢8OβD·¸£ê
ÝžOì
HepG2
Za*±•9Ë0ž

§tž
O{ìZaÂËBÖ0ž

’[ëÑ^”kڜ
æ€V
[1718,27]。
32 
~c‚d-ƒ
…À¼
25,100,400μmol·L-1тd-”4½
6h
Y
,400μmol·L-1{Íá‚d- ALT,AST,
LDH
îd‡±h·ÜÝ
(P<001),100μmol·L-1
{Íá‚d-
LDH
îd‡±h·ÜÝ
(P<005);
Ð,˜Å¾ø…

|}ëßh·@ä
(P<005),
å
hi[â±ßhÅ

…ÀÛ
25,100,400μmol·
L-1
тd-”4½
6h
Y
,400μmol·L-1MÍ
á‚d-
ALT,GGT,LDH
îd‡±h·ÜÝ
(P<
001)。
…À¼¬…ÀÛì‚d-
MTT
Ár{ñu
rúhòó

…ÀۇC
8OβD·¸£ê 50,
200,800μmol·L-1тd-”4½ 6hY,800
μmol·L-1{Íá‚d- ALT,AST,LDHîd‡±
h·ÜÝ
(P<001
p
P<005);200μmol·L-1{
Íá‚d-
LDH
îd‡±h·ÜÝ
(P<005),
å
Ð,˜Å¾±ø…‚d-
ALT,AST,LDH
îd‡Û
ÜÝÞß

|}ëßÛ@äÞß

ž•
800
μmol·L-1±…ÀۇC8OβD·¸£êтd
-” 4 ½
6h
Y
MTT
Á r { ñ h · @ ä
(P<001),
]*
3。
·2921·
P 


fHg‚hµ˜Þ1i4j«¬
*
1 
¥¦~,fHg$
11
U°–—ì
HepG2
Za±0ž
Table1 Thecytotoxicefectof11compoundsonHepG2cel
`
Ãß
/μmol·L-1
…ÀÛ
…ÀۇC
8
OβD·¸£ê
…ÀۇC
8O(6′O
m 1
)βD·¸£ê
…ÀۇC …À¢ ñû…ÀÛ

9ˇ
/%

9ˇ
/%

9ˇ
/%

9ˇ
/%

9ˇ
/%

9ˇ
/%
Ž}
- 106±005 0 087±005 0 087±005 0 092±004 0 117±005 0 093±005 0
ìH
- 104±005 0 088±003 0 088±003 0 090±003 0 117±005 0 092±005 0
~,
400 021±0032) 8037 038±0052) 5635 043±0032) 5077 051±0052) 4384 066±0042) 4365 073±0052) 2019
100 060±0032) 4215 054±0032) 391 062±0042) 2996 066±0022) 2646 084±0052) 282 078±005 1485
25 091±0052) 1285 070±0042) 1991 072±0042) 1825 076±0042) 1534 092±0052) 2113 095±003 -391
625 136±0042)-3041 080±0032) 931 081±0041) 767 084±0031) 686 117±004 -024 101±0032)-1046
156
039
`
Ãß
/μmol·L-1
…À¼ …ÀÛ
8OβD·¸£ê …À¢8OβD·¸£ê 8úÈ8OβD·¸£ê …ÀÛ1OβD·¸£ê

9ˇ
/% A
9ˇ
/% A
9ˇ
/% A
9ˇ
/% A
9ˇ
/%
Ž}
- 109±004 0 097±004 0 092±003 0 093±005 0 089±005 0
ìH
- 105±005 0 096±004 0 092±003 0 092±005 0 088±004 0
~,
400 007±0042) 9345 065±0032) 3195 073±0032) 2101 086±005 63 105±0052) -1857
100 042±0042) 6007 072±0042) 2489 080±0032) 1359 086±005 631 107±0012) -2139
25 085±0042) 1933 080±0012) 1647 080±0032) 1284 078±0022) 1492 110±0032) -2512
625 131±0022) -2436 082±0032) 1441 085±0042) 788 073±0042) 2002 115±0052) -2999
156 138±0032) -3066 091±005 5 091±002 148 076±0022) 1715
039 154±0032) -4651 118±0032) -2313 106±0042) -1542 092±005 013
  
å

ÑìHñò
1)P<005,2)P<001(
*

¦
)。
*
2 
fHg$ìíĖ—ì
HepG2
Za±
IC50
Table2 TheIC50valuesof11compoundsonHepG2cel
μmol·L-1
_V˜
IC50
…À¼
7107
…ÀÛ
12562
…ÀۇC
8OβD·¸£ê 24227
…ÀۇC
8O(6′O
m 1
)βD·¸£ê 40232
…ÀۇC

…À¢

…ÀÛ
8OβD·¸£ê -
…À¢
8OβD·¸£ê -
ñû…ÀÛ

8úÈ
8OβD·¸£ê -
…ÀÛ
1OβD·¸£ê -
  
å
:-
x-¥d
IC50。
4 
Ús
ÎyÏ

fHg±‚hµ’“îÍáD’“x
%ÓÔ±å

§4¬Óhµ˜Þ1i–D)“
å±õë

9«¬Ã’[õ뒓ÖnD4Ú

fHg$L/ëB€ƒmBêĬìí`˜
œ…Ä–—

9çƒb«¬±
11
_V˜†B¦f
Hg$—ž<ä±^”kÚ
[14],
¬åÓ$±
10
ì
íU°Gô•ÉfHg$±L/ìíĖ—
[28]。
HepG2
ZaÏ>Ÿ$‚¢Za/

Óbë±Â
˜o_P›qÑ$?w‚çÞZa-B¦>µ

Ó
—_¹¾òÝ

¬ådÒDòدåðµKâ±Â
˜o_P›q

É«¬,˜‚hµwž±ZaPQ
ý[
[2930]。
ÑìíÄ_V˜ñò

8úÈ
8OβD
·¸£êì
HepG2
Za19rh

>ï[á-Bì
íÄ»±_V˜ŽtãÂZahµ

‘žìí–êY‚Zah0žä¿Éø…ÁÉ
&ê¥{[¯§s

Á:/-°_V˜-°—à

хÀۀñ

…ÀÛ
8OβD·¸£ê¬…ÀÛ
1OβD·¸£ê±‚Zahµúhät,§å–
êíL¥¦

ÓìZa±òó祦

…ÀÛ
R4í
OH
–êY

‚ZahµBbät


R1í–ê
O

Óì
HepG2
Za¥OƒB9Ë0ž

Ö§Ma
ÖZa±ø™

хÀ¢€ñ

¦èž@…À
¢
8OβD·¸£ê±‚ZahµçBbät。}
É

хÀۇC€ñ

…ÀۇC
8OβD·¸£
ꬅÀۇC
8O(6′O
m 1
)βD·¸£ê
ì
HepG2
Za±hµ{uBbøÅ

·3921·
2016
y

z ³
41
ų

}
Vol41,No.7 April,2016
*
3 
…À¼

…ÀÛ

…ÀۇC
8OβD·¸£êì…R~c‚d-ALT,AST,GGT,LDHîd‡、|}ëßÊ MTTÁr
{ñ±òó
(珋x±s,n=3)
Table3 Theinfluenceofrhein,emodinandphyscion8OβDglucopyranosideonALT,AST,GGTandLDHleakagerate,protein
content,MTTreductionabilityofratliverslices(珋x±s,n=3)
`
Ãß
/μmol·L-1
ALT AST GGT LDH
|}
/mg·L-1
MTTA/g
‚d-Þß
ìH
  - 145±039 008±003 181±027 415±055 206163±48004 8186±245
…ÀÛ
25 161±045 008±003 219±034 415±035 213848±22407 7884±788
100 166±026 007±003 186±011 391±029 245744±18203 8627±457
400 234±0102) 011±002 255±0242) 561±0712) 166717±22637 8009±649
…ÀۇC
8OβD·¸£ê 50 130±042 009±003 154±035 389±062 228618±45628 8581±373
200 184±026 011±004 205±063 501±0341) 186451±21438 7632±559
800 226±0022) 012±0021) 168±033 604±0802) 141866±27940 5830±7772)
  
å
:ALT,AST,GGT,LDH
îd‡•|
1μg|}$±îd‡。
  11
_V˜$

…À¼±hµ€ìQe

§’
11
_V˜$‹B…À¼
R2í÷B#1,àï[á
#1±¿M……øeìí»±‚Zahµ

…ÀÛ

…ÀۇC¬…À¢Lü±œ÷‹B
R3ÖP1BÔ`,§‰Šì HepG2Za±hµ•…
ÀÛ

…ÀۇC≈…À¢,ÊïéúÃì‚Za
hµ§ê

ÖP1ììíÄ_V˜±ZahµÕ”
ʅä:·º•
OH>H≈OCH3。ïð,…À¼、…
À¢Êñû…Àۜ÷÷‹B
R2ÖP1¥¦,Ó
ì
HepG2
Za±hµÊ…ä:·º•…À¼

…
À¢

ñû…ÀÛ

Êï[áìí_V˜$ÖP1
ì
HepG2
Za±Zah0žòóʅä:•
COOH>CH3>CH2OH。^”@¸ñò…À¼¬ñ
û…ÀÛ±ðµ

R2íÖP1©Uï{ñâ
e

ì$‚ˆZa9Ë0žâe±œs
[31]。
9çƒ
œæ*ú©Uïx{ÇììíÄ_V˜±‚hµB
[âòó

}¬¥ØwùV|‹š

ïð

Zah烜æhi

…À¼¬…ÀÛì
HepG2
Za±hµQe

Óº•…ÀۇC
8Oβ
D
·¸£ê¬…ÀۇC
8O(6′O
m 1
)βD
·¸£ê

’

_V˜ì
HepG2
Za±9Ë0ž
uО±øݧø…

å¿Ýž0ž@Búh
±Zah0ž

§…ÀÛ

…À¼

…ÀۇC
8
OβD·¸£ê±hµ€ìòe,àï,0üpž~
c‚d-()ì’

–—±‚hµÖnDÇO
«¬

~c‚d-()ÉןwxÑZa¢%ýþ±
°ð烠¨

ÓdD[â±Fœ÷

dÒDb
BÄQ±Za

ZaþUÅÊx‚,q±ðµ

€ìŸZa¢%

Ž-µÑ¯°µ

{ò÷FP
a°8±‚M²

MÖ,˜¿°8±çpP›¸
¹

-BÁU



Ý@ßè½ë
[32]。ALT
¬
AST
ÉÀíQwž±Ö‚¥{ƒÈQ*汉
Â
,ALT
L/¿Ÿ‚Zao8

§
AST
L/¿
Ÿ‚Za›(°$

”Za#@´µø…O
,ALT
îd‡øÅ

§”‚ZaÔ.O k.O

›(°8
AST
&Ï;dÏ
。GGT
¿‚8ʂZa›(°ã
Â

ÓL/—‹¿‚Za#Ê[Z†3÷A
,LDH
É°8{ßP›¸¹$±[./±q

”‚Za
ƒ„pk.O

Za#ã䃄
,GGT
¬
LDH
±î
d‡Ã+ÜÝ
[3334]。
…†÷¤fHg]c‚¥
{ƒÈ±ûüӂÂ_‰Â–ë•
ALT
¬
AST
u
h·ÜÝ

¦OçuB
GGT
Ê
LDH
±ÜÝ
[3537]。
ïð

¶˜°8烜æ“*ú…RÆù¥¦Ãß
±fHgY


ALT
¬
AST
uB¥¦¹¾±Ü
Ý

Ãßâ…

~,OþâËÓÜݹ¾âú
h
[8,3839]。
~c‚d-烜æhi

¥¦Å¾±
…À¼Ñ‚d-”4½
6h
Y
,400μmol·L-1M
I
ALT,AST,LDH
îd‡øÅ
,100μmol·L-1MI
LDH
îd‡ÜÝ

åÐ,˜Å¾±ø…|}ëßh
·@ä

åhi[â±hɁÅ

ž•
400
μmol·L-1±…ÀÛMÍá ALT,GGT,LDH±îd
‡øÅ

Êïéú…Ãß±…À¼

…ÀÛ¿°ð
ì‚FuM*±d[â±hµ

…ÀۇC
8
OβD·¸£êž•200μmol·L-1O{Íá‚
d-
LDH
îd‡±h·ÜÝ

žø…Ÿ
800
μmol·L-1O‚d- ALT,AST,LDH±îd‡h·
ÜÝ

¦O

Ð,˜Å¾±ø…’

q±îd‡Û
·4921·
P 


fHg‚hµ˜Þ1i4j«¬
ÜÝÞßå|}ëßÛ@äÞß

@¸‚d-
MTT
çƒÁm±

‚d-Ñž•
800μmol·L-1
±…ÀۇC
8OβD·¸£ê”4½ 6hYÓ
MTT
Ár{ñh·@ä

ZahµçƒÊ~c‚d-çƒuŠú…À
¼

…ÀÛʅÀۇC
8OβD·¸£ê¿…Ã
ßO¿[ⱂhµ

|œæ*ú1—ìíì°
ð‚ZaʂFîçMãÂ[ⱃÈ0ž

}
É

@¸ìfHg$…À¼

…ÀÛʅÀۇC

8OβD·¸£ê±ëßÖnuœ<:fHg$
…À¼±Þߗ=ŒC•
0~00232%;
…ÀÛÞ
ߗ=ŒC•
00007% ~0319%;
…ÀۇC
8
OβD·¸£ê±Þߗ=ŒC• 0014% ~
0064%[29,4046]。
§åB^”kÚ.Iü©
50mg·
kg-1(
€”Ÿ…ÀÂ,
035g·kg-1)
UºŒá…À
¢#˜
,HPLC
áâ|µ¢#˜$…À¼Þߗ=
•
12308%,
‚áYm±…À¼¿$°8±Q…
¢,ž•
1428μmol·L-1[47]。ÊïM65kg
±.IüUºŒá…À¼±ß•
141μmol,Ö§[
-d$°±*—‹ê2ȕ
10L。
ûüážf
Hg±Ã߅1—•w‹Ãß
[6,48],
,˜‹âÂH
g±žß•
3~6g,
ËHg±žß•
6~12g[2],
P
©Q…Ãß
12g
ážfHg

…À¼©Q…Þߗ
=
00232%
x-

…ÀÛ©Q…Þߗ=
0319%
x-

…ÀۇC
8OβD·¸£ê©Q…Þߗ
=
0064%
x-
),
{…À¼

…ÀÛ¬…ÀۇC

8OβD·¸£êqrŸ°8±Q…ž‚—`•
098,1416,172μmol·L-1,P/sä°ðçƒ$
ã‚hµ±Å¾

…À¼¬…Àە
400
μmol·L-1,…ÀۇC8OβD·¸£ê• 800
μmol·L-1),{:/—`Uºáž4898,339,5581

fHg,Š

§’hÌÉ¥VS¬¥M{ç±±

àï
,“
ìíĖ—ÉfHg‚hµ–—

±éÉ
éˆ&Š>?+±


„q=

[1] 
IÕ:

ìfHg1r¬¤±Wôõ
[J].
$%$,-.

2013,38(22):3988.
[2] 
$%,˜

[1
[S].2015:175.
[3] KyoungAJ,HyunJM,SeungSY,etal.Druginducedliverin
jury:twentyfivecasesofacutehepatitisfolowingingestionofPo
lygonummultiflorumThunb[J].GutAndLiver,2011,5(4):
493.
[4] LeiX,ChenJ,RenJT,etal.LiverdamageassociatedwithPo
lygonummultiflorumThunb.:asystematicreviewofcasereports
andcaseseries[J].EvidBasedComplAlt,2015,doi:101155/
2015/459749.
[5] 
N¿f



fHgÊÓËÀ‚ƒÈ%8^”–—Ñ
—à
[J].
,˜¥Õւ-.
,2010,12(1):26.
[6] 
!|



b$

fHgc,˜µ‚ƒ„
36
¨…†—à
[J].
H9A
,2013,24(2):235.
[7] 
†:

s%s

fË|

è

ŒáfHgc‚ƒÈ
40¨
…†—
à
[J].
59%2A,
,2009,11(3):209.
[8] 
ÀO

!ð&

Nz

fHg¥¦—Uº~,ì:R‚hµ

ß

O

h

Å«¬
[J].
$%,˜<=
,2011,8(4):193.
[9] ChoiYJ,YoonY,ChoiHS,etal.Efectsofmedicinalherbex
tractsandtheircomponentsonsteatogenichepatotoxicityinSk
hep1cels[J].ToxicolRes,2011,27(4):211
[10] 


Fø}

Dùá

è

ËfHg¥¦ÃßхR‚ƒ
„¹¾±çƒ«¬
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