全 文 :2016
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Vol41,No.9 May,2016
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ì\^¤
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L-1,
ëìüúúÓ
y=2178lnx-1514,R2=0998。
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ZnSO4·7H2OÏ7iiÎIC50Ó1990mg·L
-1;
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D`
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CS234
Optimizationofreactionconditionsandmethodologicalinvestigationon
microtoxbasedfasttestingsystemfortraditionalChinesemedicineinjection
XIONGJingyue1,2,LIXiaorong2,YANLiangchun2,ZHAOJunning2
(1.ChengduUniversityofTraditionalChineseMedicine,Chengdu610072,China;
2.SichuanAcademyofChineseMedicineSciences,BiologicalEvaluationofTraditionalChineseMedicineQualityofthe
StateAdministrationofTraditionalChineseMedicine,Chengdu610041,China)
[Abstract] VibriofischeriCS234wasusedtoestablishandoptimizemicrotoxassaysystem,layingafoundationfortheapplicationof
thismethodincomprehensiveacutetoxicitytestoftraditionalChinesemedicine(TCM)injectionsFirstly,thePlacketBurmanmeth
odwascariedouttooptimizethefactorswhichwouldafectVibriofischeriCS234luminescenceSecondly,ZnSO4·7H2Owaschosen
asreferencesubstancetoestablishitsreactionsystemwithqualitycontrolsamplesTheoptimalluminescenceconditionswereachieved
asfolows:①Atatemperatureof(15±1)℃,VibriofischeriCS234lyophilizedpowderswerebalancedfor15min,then,1mLresus
·2261·
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citationfluidwasaddedandblendedfor10min100μLbacteriasuspensionwastakentomeasuretheinitialluminescenceintensity,
andthen1mLresuscitationfluidortestsamplewasimmediatelyadded;afterreactionfor10min,corespondingluminescenceintensity
wasmeasuredagainResuscitationdiluent,osmoticpressureregulatorandZnSO4·7H2Ostocksolutionshowednointerferencetothe
determinationofVibriofischeriCS234luminescenceintensity,sothismethodwasofgoodspecificityThewithinandbetweenbatch
precisionsofqualitycontrolsandthelowerlimitofquantification(LLOQ)sampleswere<5% and<10% respectively,whiletheac
curacyrangedbetween858% and1032%ThestandardcurveequationofZnSO4·7H2Orangedfrom386mg·L
-1to7722mg
·L-1(finalconcentrations)wasy=2178lnx-1514,R2=0998;meanwhile,IC50ofZnSO4·7H2OtoVibriofischeriCS234was
1990mg·L-1ZnSO4·7H2Ostocksolutionanditsqualitycontrolswerecontinuouslyinvestigatedfor120hand8hrespectively,
andtheirRSDwaslowerthan2%,indicatingstabilityatroomtemperatureand4℃ storageconditionsBetweenpH4580,lumi
nescenceintensityofVibriofischeriCS234wascontroledwithin±10%,andsuchpHvaluerangecouldmeetthetestingneedsofthe
vastmajorityoftraditionalChinesemedicineinjectionsTheVibriofischeristrainCS234assaysystemwasspecific,stable,sensitive,
accurateandadaptableafteroptimization,soitwassuitableforthecomprehensiveacutetoxicityassessmentofTCMinjections
[Keywords] traditionalChinesemedicineinjection;microtoxassay;acutetoxicity;VibriofischeriCS234
doi:10.4268/cjcmm20160909
îïºò()û[±Ê¯m_lÎn©¨
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(15±1)℃
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1 PlacketBurman
ïs¶ÖJþ
Table1 PlacketBurmanexperimentalfactorsandlevels
Jþ
A
þEOn
/min
B
øä
1
C
f+>óE
/mL
D
øä
2
E
ÜîOn
/min
F
øä
3
G
Î>óE
/μL
H
øä
4
J
aóE
/mL
K
øä
5
L
õöOn
/min
-1 10 - 1 - 10 - 50 - 1 - 10
1 15 - 2 - 15 - 100 - 2 - 15
·3261·
2016
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Vol41,No.9 May,2016
22
ºòúûyzüý
221
ÈE!
¡^3f+)½>
、
YÌ»Á
>
、ZnSO4·7H2O
X>#7iiÎ CS234Î>x
01mL
ê
485nm
ºòm_a^
。
222
ú!Lò
、
-Ò^
、
Õ]^ì\^
Õ]
X3
ZnSO4·7H2O500mgÞê500mLÁ$,
¹J~¡ÎÝ
、`
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~<
1000mg·L-1
X>
。
3
X>¯¹J)½
,
<
^¡^Ó
50,
200,400,800mg·L-1
ôOÎ>
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ìï¨ÖYÌ»Á>Â
17∶3
ܪ
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Óëìüú
xç
,
é
21
ºù_÷ønºò
,ZnSO4·
7H2OëìüúxçõöÊ^Ó386,1545,
3091,6182,7727mg·L-1。
ÂD(
(1)
stM
ª
Ht#ƹ D,q HtZnSO4·7H2O^
q
,
%ª
-Ò^¯`S
C~
386mg·L-1(
Ê^
)
ëìï¨Õ]^#
ì\^Ä
。
_3
ZnSO4·7H2O
X>¯¹J
Ö
150,500,900mg·L-1
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17∶3
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1159,3864,6955mg·L-1,
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RSD
Ä
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¡Ä
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D=(fkt±fkt)/fkt×100% (2)
Ict=Ic×fkt (3)
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m_ÎÖf+)½>ãq[`Onÿm_a^
,
fktÓm_ô=。DÓm_ô=ƹ,fktÓ 2
CþnK
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223
Ó`!
3
ZnSO4·7H2O
X>150,
500,900mg·L-1
an
Zn2+
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üý
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Zn2+
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0137,0457,0822mg·L-1
üý
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Ó`!
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qÂâö^YÌ»Á>ÓÏ
,
8
è
a]µÿÞ¤
4℃
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224 pH
Ï7iiÎ
CS234
ØÙ
ºò¥¦
pH
f+>
(40,45,50,60,70,80,90)
Ïm_
a^ØÙ
。
¯
1mol·L-1HCl
Î>î
NaOH
Î>
»Á
pH,
2ù
a$óE¥uóE
5%,
|C
pH
ï
2
CþnK
,
f
2
}
。
wD(
(3),(4)
st
Ï7iiÎ
CS234
m_a^M
ªþÆ
。
23
=ò¡¢
DesignExpert805b
røn
PlacketBurman
noïs=ò¡¢
。PEMS31
rønëìü
ú%ª
IC50st。
3
á
31
õö÷øù_
wêxºòÒ=©sE
,
Øé
ISO
113483:2007
ëì÷ø
D<3%
ÓªJÒª
[3],
2。
À
3,4
}
D
Æ
<3%,
KÂå
»ù
,
£÷`m_÷øÓ7iiÎ
CS234
àÜê
©Z$þE
15min,
2ùf+>
1mL
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10
min,
3
100μLÎ>ºò9mm_a^ÿz~2ù
1mL
f+>
(
Ï
)
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a
,
õö
10min
ÿ_
}ºòÏK
aKm_a^
。
32
ÈE!
f+)½>
、
YÌ»Á>
、ZnSO4·7H2O
X>m_a^Ƥ
001~002,
Í7iiÎ
CS234
Î>m_a^¤
1152~1177,
H¡åL
¥M7iiÎm_a^º`
,
õúûÈE!
a
,
3。
33
ú!Lò
、
-Ò^
、
Õ]^ì\^
`SC
a
aÕ]^Æ
<
10%,
ì\^¤
858%~1032%,
4。ZnSO4·
7H2OÎ>¤386~7727mg·L
-1,
ëìüúú
Ó
y=2178lnx-1514,R2 =0998,
õúûS
ZnSO4·7H2OÏ7iiÎ CS234 IC50Ó 1990
mg·L-1。
34
Ó`!
ZnSO4·7H2O
X>
a¡^
JKüý
120,8h,
RSD
Æ
<2%,
2Ĥð
4
℃
íµ÷øSÓ`!/
,
5。
35 pH
Ï7iiÎ
CS234
ØÙ
pH45~80
O
,
7iiÎ
CS234
m_Ã
<10%,
6。
·4261·
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2 PlacketBurman
ïsá
(n=2)
Table2 PlacketBurmanexperimentaldesignsandresults(n=2)
No A B C D E F G H J K L D1 D2 D3
1 -1 -1 1 -1 1 1 -1 1 1 1 -1 -048 575 -880
2 -1 1 1 -1 1 1 1 -1 -1 -1 1 113 530 289
3 1 -1 -1 -1 1 -1 1 1 -1 1 1 -067 034 -131
4 -1 1 1 1 -1 -1 -1 1 -1 1 1 122 118 -093
5 1 1 -1 -1 -1 1 -1 1 1 -1 1 -842 220 -338
6 -1 -1 -1 1 -1 1 1 -1 1 1 1 -544 066 -391
7 1 1 -1 1 1 1 -1 -1 -1 1 -1 -030 207 531
8 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 050 067 -527
9 -1 1 -1 1 1 -1 1 1 1 -1 -1 315 343 236
10 1 -1 1 1 1 -1 -1 -1 1 -1 1 101 494 -391
11 1 1 1 -1 -1 -1 1 -1 1 1 -1 -297 486 157
12 1 -1 1 1 -1 1 1 1 -1 -1 -1 -025 411 -448
3
ÈE!üý
(n=2)
Table3 Experimentaldesignsofspecificityvalidationandcor
respondingresults(n=2)
No
m_a^
f+)½> YÌ»Á>
ZnSO4·7H2O
X> 7iiÎ
1 001 001 001 1152
2 001 002 001 1177
3 001 001 001 1155
4
s
7iiÎä>ê γNôÎé Gammaproteobac
teria
iΧ
Vibrio,
û[±©Õ¤H[$rð!
Î
,
©}ÞÃSmìÖÃÕÓ
420~670nm、
µ
m
490nm
sÛ?ÞðíÅ_
,
ÊËö¯ê°%
Microtox Model500# DeltaTox Analyzer,Â%
ToxAlert#LUMIStoxêBioToxTMÉ©¨ï
!ºòô©
[4]。
m_P×q
1。
4 ZnSO4·7H2O
aÕ]^、ì\^`SC-Ò^üý(n=2)
Table4 Experimentaldesignsofstandardcurve,precision,accuracyvalidationandcorespondingresultsinZnSO4·7H2OQCsam
ples(n=2)
ZnSO4·7H2O
Ê^
/mg·L-1
H10
/%
©8Õ]^
/%
1 2 3
©nÕ]^
/%
ì\^
/%
1 2 3
`SC
a
386 140 423 338 217 273 9272 9926 10060
a
1 1159 354 107 151 278 547 10315 8782 8581
a
2 3864 615 192 038 178 167 9934 9800 9197
a
3 6955 730 137 080 001 416 9332 8828 9604
5 ZnSO4·7H2O
X>
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 19
ZnSO4·7H2O
a 0.137 0.024 0.023 0.023 0.023 0.023 0.024 - - - - 15
0.457 0.080 0.081 0.081 0.080 0.081 0.081 - - - - 057
0.822 0.143 0.145 0.146 0.145 0.144 0.146 - - - - 071
6 pH
Ï7iiÎ
CS234
m_ØÙ
(n=2)
Table6 ExperimentaldesignsofpHefectsonVibriofischeriCS234andcorespondingresults(n=2)
äå
pH40 pH45 pH50 pH60 pH70 pH80 pH90
1 2 1 2 1 2 1 2 1 2 1 2 1 2
H10 131 134 -07 70 -69 06 -84 28 -88 71 -88 -07 -103 117
D 68 09 08 -05 -23 05 -13 -01 -14 -03 12 -09 05 -73
·5261·
2016
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5
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}
Vol41,No.9 May,2016
q
1
7iiÎm_P×
Fig1 LuminescentmetabolismprocessofVibriofischeri
åå¯m_Î*!ï!ºòúûÑ_Bï
¯7iiÎ
NRRLB11177
%pëì_Ò¸
ISO113483:2007《WaterqualityDeterminationof
theinhibitoryefectofwatersamplesonthelightemis
sionofVibriofischeri(Luminescentbacteriatest)Part
3:Methodusingfreezedriedbacteria》
#ﯡôm
_ñÎ
T3:±$%%ë GB/T154411995《J
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》[5],
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。GB/T154411995
ÂâÏm_a^þÆ
(%),
D(
(5),(6)
a
^
(mg·L-1)
%ª~[}ú!úäÄ
a*!ï!
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[6],
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fkt,D((1),[âÏ©`m
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、
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PlacketBurman
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qù_Ö.Ó`
m_õöóô
。
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aKm_a^
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yzâ
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ZnSO4·7H2OÓëìï¨
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bñ7¡É
Ýnëìï¨
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Zn2+
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Ù+
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æüýâ
pH
Ï7iiÎm_ØÙ
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ëìíö¼BÖt>âÉîâã
pH,
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pH
4~9,
tu
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¡\ÚLòû`ªm_lΩ
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,6ãª`î難ºòóôö¯Lò
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á VÄ
,pH45~80
ÏlÎm_Mª
¤
±10%
8
,
?>?®wZ=$,ëìí¶º
8_
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bf
,
õnoð@Ï7iiÎõö÷øù
_úûüý
,
yzâ`¯ê
CS234
[Î
ºòóô
,
õúû¼B/ÈE!
、
Ó`!
、
-
Ò^
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ú^T
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,2013,9(2):137.
[3] ISO113483:WaterqualityDeterminationoftheinhibitoryefect
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