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Optimization of reaction conditions and methodological investigation on microtox-based fast testing system for traditional Chinese medicine injection

基于Microtox技术的中药注射剂微毒快速测试体系反应条件的优化与方法学的考察



全 文 :2016
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Vol41,No.9 May,2016
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ò9mm_^

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<5%,
©nÕ]^
<10%,
ì\^¤
858% ~1032%。
kJD‘`Î>¤
386~7727mg·
L-1,
ëìüúú“Ó
y=2178lnx-1514,R2=0998。
ÚúûS
ZnSO4·7H2OÏ7iiΜIC50Ó1990mg·L
-1;
ÏkJ
D‘`…X>‚…†‡ˆ…a¡^JKüý
120,8h,
‚
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íµ÷øSÓ`!/
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CS234
Optimizationofreactionconditionsandmethodologicalinvestigationon
microtoxbasedfasttestingsystemfortraditionalChinesemedicineinjection
XIONGJingyue1,2,LIXiaorong2,YANLiangchun2,ZHAOJunning2
(1.ChengduUniversityofTraditionalChineseMedicine,Chengdu610072,China;
2.SichuanAcademyofChineseMedicineSciences,BiologicalEvaluationofTraditionalChineseMedicineQualityofthe
StateAdministrationofTraditionalChineseMedicine,Chengdu610041,China)
[Abstract] VibriofischeriCS234wasusedtoestablishandoptimizemicrotoxassaysystem,layingafoundationfortheapplicationof
thismethodincomprehensiveacutetoxicitytestoftraditionalChinesemedicine(TCM)injectionsFirstly,thePlacketBurmanmeth
odwascariedouttooptimizethefactorswhichwouldafectVibriofischeriCS234luminescenceSecondly,ZnSO4·7H2Owaschosen
asreferencesubstancetoestablishitsreactionsystemwithqualitycontrolsamplesTheoptimalluminescenceconditionswereachieved
asfolows:①Atatemperatureof(15±1)℃,VibriofischeriCS234lyophilizedpowderswerebalancedfor15min,then,1mLresus
·2261·
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()œ$,ëìíîïðñºòóôõö÷øœù_Öúûœüý
citationfluidwasaddedandblendedfor10min100μLbacteriasuspensionwastakentomeasuretheinitialluminescenceintensity,
andthen1mLresuscitationfluidortestsamplewasimmediatelyadded;afterreactionfor10min,corespondingluminescenceintensity
wasmeasuredagainResuscitationdiluent,osmoticpressureregulatorandZnSO4·7H2Ostocksolutionshowednointerferencetothe
determinationofVibriofischeriCS234luminescenceintensity,sothismethodwasofgoodspecificityThewithinandbetweenbatch
precisionsofqualitycontrolsandthelowerlimitofquantification(LLOQ)sampleswere<5% and<10% respectively,whiletheac
curacyrangedbetween858% and1032%ThestandardcurveequationofZnSO4·7H2Orangedfrom386mg·L
-1to7722mg
·L-1(finalconcentrations)wasy=2178lnx-1514,R2=0998;meanwhile,IC50ofZnSO4·7H2OtoVibriofischeriCS234was
1990mg·L-1ZnSO4·7H2Ostocksolutionanditsqualitycontrolswerecontinuouslyinvestigatedfor120hand8hrespectively,
andtheirRSDwaslowerthan2%,indicatingstabilityatroomtemperatureand4℃ storageconditionsBetweenpH4580,lumi
nescenceintensityofVibriofischeriCS234wascontroledwithin±10%,andsuchpHvaluerangecouldmeetthetestingneedsofthe
vastmajorityoftraditionalChinesemedicineinjectionsTheVibriofischeristrainCS234assaysystemwasspecific,stable,sensitive,
accurateandadaptableafteroptimization,soitwassuitableforthecomprehensiveacutetoxicityassessmentofTCMinjections
[Keywords] traditionalChinesemedicineinjection;microtoxassay;acutetoxicity;VibriofischeriCS234
doi:10.4268/cjcmm20160909
  
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à’Üò
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23*#_¶()ô´BCD
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m_ΜºB͇Ƥ
(15±1)℃
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1 PlacketBurman
ïs›¶ÖJþ
Table1 PlacketBurmanexperimentalfactorsandlevels


þEOn
/min

øä


f+>óE
/mL

øä


ÜîOn
/min

øä


Î>óE
/μL

øä


…aóE
/mL

øä


õöOn
/min
-1 10 - 1 - 10 - 50 - 1 - 10
1 15 - 2 - 15 - 100 - 2 - 15
·3261·
2016
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Vol41,No.9 May,2016
22 
ºòúûœyzüý
221 
ÈE!
 
¡^3f+)½>

Y̒»Á
>
、ZnSO4·7H2O…X>#7iiÎ CS234Î>x
01mL
ê
485nm
‘ºòm_a^

222 
ú!Lò

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X3
ZnSO4·7H2O500mgÞê500mL†Á$,ž
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、`
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1000mg·L-1
…X>

3…X>¯ž¹J)½

<…†™^¡^Ó
50,
200,400,800mg·L-1
ôOÎ>

_,x™^ë
ìï¨ÖY̒»Á>Â
17∶3
ܪ

‡Óëìüú
œxç

é–
21
ºù_œ÷ø‹nºò
,ZnSO4·
7H2Oëìüúxçœõöʙ^Ó386,1545,
3091,6182,7727mg·L-1。
ÂD(
(1)
stMˆ
ª
Ht#Ǝ¹ D,q HtZnSO4·7H2O™^‡
q

R2;
-Ò^¯`†S
C~
386mg·L-1(
ʙ^

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ì\^ŸÄ

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ZnSO4·7H2O…X>¯ž¹J
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150,500,900mg·L-1
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¡ŸŸÄ

fkt=Ikt/I0 (1)
D=(fkt±fkt)/fkt×100% (2)
Ict=Ic×fkt (3)
Ht=(Ict-It)/Ict×100% (4)
I0ÓϖKm_Î9mm_a^,IktÓϖK
m_ÎÖf+)½>ãq[`Onÿœm_a^

fktÓm_ô=。DÓm_ô=œÆŽ¹,fktÓ 2
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,IcӅaKm_Î9mm_a^。It
ӅaKm_Îօaãq[`Onÿœm_a
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223 
Ó`!
 
3
ZnSO4·7H2O…X>150,
500,900mg·L-1
…†‡ˆ…a‹n
Zn2+
Ó`!
üý

wê^¶”²¡__^sº†
Zn2+
Ϝ!
Lò¤
02~09mg·L-1,
£,…X>)½W
0538mg·L-1(
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Zn2+
s

ÿüý
0,2,4,6,8,24,
48,72,120h
œÓ`!

…†‡ˆ…a¡^)½W
0137,0457,0822mg·L-1
üý
0,2,4,6,8h
œ
Ó`!

qÂâö™^œY̒»Á>‡Óϖ

8
è…a]µÿÞ¤
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ìZ8íµ

224 pH
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ºò¥¦
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(40,45,50,60,70,80,90)
Ïm_
a^œØÙ

¯
1mol·L-1HCl
Î>î
NaOH
Î>
»Á
pH,
2ù…a$œóE¥žuóEœ
5%,
|C
pH
ï

CþnK

f

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wD(
(3),(4)
stpH
Ï7iiÎ
CS234
m_a^œM
ˆª‚þƎ

23 
=ò¡¢
DesignExpert805b
rø‹n
PlacketBurman
noïs=ò¡¢
。PEMS31
rø‹nëìü
úœ%ª
IC50œst。
3 
á 
31 
õö÷øù_
wêxºòÒ=©sE

Øé–
ISO
113483:2007
œëì÷ø
D<3%
•ÓªJÒª
[3],
„Ÿ
2。
À
3,4

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<3%,
KÂå•
»ù

£÷`m_÷øÓ7iiÎ
CS234
à’Üê
©Z$þE
15min,
2ùf+>
1mL
ÿÜî
10
min,
3
100μLÎ>ºò9mm_a^ÿz~2ù
1mL
f+>

ϖ

…a

õö
10min
ÿ_
}ºòϖK…aKm_a^

32 
ÈE!
f+)½>

Y̒»Á>
、ZnSO4·7H2O…
X>œm_a^Ƥ
001~002,
Í7iiÎ
CS234
Î>œm_a^¤
1152~1177,
H¡åL
•¥’M7iiÎm_a^œº`

õúûÈE!
a

„Ÿ
3。
33 
ú!Lò

-Ò^

Õ]^ì\^
`†SC…a…†‡ˆ…aÕ]^Æ

10%,
ì\^¤
858%~1032%,
„Ÿ
4。ZnSO4·
7H2OÎ>¤386~7727mg·L
-1,
ëìüúú“
Ó
y=2178lnx-1514,R2 =0998,
õúûS
ZnSO4·7H2OÏ7iiÎ CS234œ IC50Ó 1990
mg·L-1。
34 
Ó`!
ZnSO4·7H2O…X>‚…†‡ˆ…a¡^
JKüý
120,8h,
‚
RSD
Æ
<2%,
2Ĥð


íµ÷øSÓ`!/

„Ÿ
5。
35 pH
Ï7iiÎ
CS234
œØÙ
pH45~80
O

7iiÎ
CS234
m_œÃ
ƒ
<10%,
„Ÿ
6。
·4261·
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Microtox
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Ÿ
2 PlacketBurman
ïsá 
(n=2)
Table2 PlacketBurmanexperimentaldesignsandresults(n=2)
No A B C D E F G H J K L D1 D2 D3
1 -1 -1 1 -1 1 1 -1 1 1 1 -1 -048 575 -880
2 -1 1 1 -1 1 1 1 -1 -1 -1 1 113 530 289
3 1 -1 -1 -1 1 -1 1 1 -1 1 1 -067 034 -131
4 -1 1 1 1 -1 -1 -1 1 -1 1 1 122 118 -093
5 1 1 -1 -1 -1 1 -1 1 1 -1 1 -842 220 -338
6 -1 -1 -1 1 -1 1 1 -1 1 1 1 -544 066 -391
7 1 1 -1 1 1 1 -1 -1 -1 1 -1 -030 207 531
8 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 050 067 -527
9 -1 1 -1 1 1 -1 1 1 1 -1 -1 315 343 236
10 1 -1 1 1 1 -1 -1 -1 1 -1 1 101 494 -391
11 1 1 1 -1 -1 -1 1 -1 1 1 -1 -297 486 157
12 1 -1 1 1 -1 1 1 1 -1 -1 -1 -025 411 -448
Ÿ
3 
ÈE!üý
(n=2)
Table3 Experimentaldesignsofspecificityvalidationandcor
respondingresults(n=2)
No
m_a^
f+)½> Y̒»Á>
ZnSO4·7H2O…X> 7iiÎ
1 001 001 001 1152
2 001 002 001 1177
3 001 001 001 1155
4 
s
7iiÎä>ê γNôÎé Gammaproteobac
teria
iΧ
Vibrio,
û[±©Õ¤H[$œr‚ð!
Î

©}ÞÃS—mìÖÃÕÓ
420~670nm、
µŽ
m
490nm
œsÛ?„ÞðíÅ_

ÊËö¯ê°%
Microtox Model500# DeltaTox Analyzer,Â%
ToxAlert#LUMIStoxê‚BioToxTMÉ©¨ï
!ºòô©
[4]。
‚m_Pא“„q
1。
Ÿ
4 ZnSO4·7H2O…†‡ˆ…aÕ]^、ì\^`†SC-Ò^üý(n=2)
Table4 Experimentaldesignsofstandardcurve,precision,accuracyvalidationandcorespondingresultsinZnSO4·7H2OQCsam
ples(n=2)
ZnSO4·7H2O
ʙ^
/mg·L-1
H10
/%
©8Õ]^
/%
1 2 3
©nÕ]^
/%
ì\^
/%
1 2 3
`†SC…a
386 140 423 338 217 273 9272 9926 10060
…†‡ˆ…a
1 1159 354 107 151 278 547 10315 8782 8581
…†‡ˆ…a
2 3864 615 192 038 178 167 9934 9800 9197
…†‡ˆ…a
3 6955 730 137 080 001 416 9332 8828 9604
Ÿ
5 ZnSO4·7H2O…X>…†‡ˆ…aÓ`!üýá (n=3)
Table5 ExperimentaldesignsofstabilityvalidationandcorespondingresultsinZnSO4·7H2OstocksolutionandQCsamples(n=3)
Ò^
…†™^
/mg·L-1
”_^
0h 1h 2h 4h 6h 8h 24h 48h 72h 120h
RSD
/%
ZnSO4·7H2O…X> 0.538 0.037 0.036 0.036 0.037 0.037 0.036 0.038 0.036 0.037 0.037 19
ZnSO4·7H2O…†‡ˆ…a 0.137 0.024 0.023 0.023 0.023 0.023 0.024 - - - - 15
0.457 0.080 0.081 0.081 0.080 0.081 0.081 - - - - 057
0.822 0.143 0.145 0.146 0.145 0.144 0.146 - - - - 071
Ÿ
6 pH
Ï7iiÎ
CS234
m_œØÙ
(n=2)
Table6 ExperimentaldesignsofpHefectsonVibriofischeriCS234andcorespondingresults(n=2)
äå
pH40 pH45 pH50 pH60 pH70 pH80 pH90
1 2 1 2 1 2 1 2 1 2 1 2 1 2
H10 131 134 -07 70 -69 06 -84 28 -88 71 -88 -07 -103 117
D 68 09 08 -05 -23 05 -13 -01 -14 -03 12 -09 05 -73
·5261·
2016
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Vol41,No.9 May,2016
q
1 
7iiΜm_Pא“
Fig1 LuminescentmetabolismprocessofVibriofischeri
å僯œm_Î*!ï!ºòúûÑ_Bï
¯7iiÎ
NRRLB11177
’œ%pëì_Ò¸
ISO113483:2007《WaterqualityDeterminationof
theinhibitoryefectofwatersamplesonthelightemis
sionofVibriofischeri(Luminescentbacteriatest)Part
3:Methodusingfreezedriedbacteria》
#ﯡôm
_ñÎ
T3:±œ$%%ë GB/T154411995《J…
*!ï!œº`m_lÎû
》[5],
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iiΜ*!ï!ºòúû

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。GB/T154411995
ÂâÏm_a^œþƎ
(%),
„D(
(5),(6)
…a…†™^
(mg·L-1)
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