CN105463061B - A method of for assessing fleece-flower root Drug-induced liver - Google Patents
A method of for assessing fleece-flower root Drug-induced liver Download PDFInfo
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Abstract
The application provides a kind of method for assessing fleece-flower root Drug-induced liver, and the method includes the following steps: the preparation of a. fleece-flower root solution；B. the preparation of endotoxin solution；C. zoopery；D. Testing index；E. statistical procedures.Method for assessing fleece-flower root Drug-induced liver provided by the present application based on LPS can provide reference with the Drug-induced liver of the Fast Evaluation fleece-flower root for fleece-flower root hepatotoxicity wind agitation evaluation model, the research of science mechanism and clinical application.
Description
Technical field
This application involves a kind of methods for assessing fleece-flower root Drug-induced liver
Background technique
The fleece-flower root is the root tuber of polygonum multiflorum thunb Polygonum multiflorum Thunb., is famous simply
Traditional help class Chinese medicine is widely used in nourishing liver and kidney, black hair, the Chinese materia medica preparation of reducing blood lipid, health food, public daily food and drink, food
Treat health care and Shampoo & Conditioner etc..However, recent domestic is increased significantly in relation to the report of fleece-flower root adverse reaction, packet
It includes and takes Radix Polygoni Multiflori, prepared fleece flower root, the compound containing the fleece-flower root, Chinese patent drug or health food etc..Domestic and foreign literature report is related
Fleece-flower root cases of liver damage about 150.2006, Britain and Australian drug administration department successively issue fleece-flower root hepatic lesion were alert
Accuse information；2013, China's drug administration department also issued the warning of the hepatic lesion of preparation containing the tuber of multiflower knotweed.Just so-called " fleece-flower root in thousand, today
Hepatotoxicity wind agitation ", the hepatotoxic problem of the fleece-flower root cause highest attention both domestic and external.The fleece-flower root is widely used, and China is containing head according to statistics
About 500 kinds of Chinese patent drug, about 200 kinds of health food of crow.If the hepatotoxic Resolving probiems of the fleece-flower root are bad, what will seriously affect
The tuber of multiflower knotweed and Related product clinical application safety, and may to Chinese medicine international fame and industry sound development bring it is huge unfavorable
It influences.It is clinical rational drug use, going after profits and advoiding disadvantages provides science it would therefore be highly desirable to illustrate the hepatotoxic related science mechanism of the fleece-flower root
Foundation.
However, currently based on common intact animal to the toxic dose of the fleece-flower root hepatotoxic evaluation result and clinical patients
Significant difference.Consulting the fleece-flower root hepatotoxicity wind agitation Research Literature that domestic more units independently carry out shows the fleece-flower root in clinic
Equivalent dose can not do the liver damage to experimental animal, need in over much dosage (40-60g crude drug/kg), long term administration (4-
12 weeks) in the case of can just cause rat hepatic lesion.But in fact, there is the trouble of hepatotoxicity in process of clinical application in the fleece-flower root
Person, many there is no overdoses to take, and taking dose is within States Pharmacopoeia specifications range (Radix Polygoni Multiflori 3-6g/ days).The liver of drug
Dysentery side reaction can be divided into intrinsic hepatotoxicity wind agitation (intrinsic hepatotoxicity) and Drug-induced liver
(idiosyncratic hepatotoxicity) two classes, wherein the former refers to that drug-induced Liver occurs and dosage, time positive
It closes, medication inter-individual difference is unobvious；And the latter refers to drug-induced Liver that there are significant individual differences, only part population
It can occur.Although the fleece-flower root causes the True incidences data of hepatic lesion unclear, according to the data of National Center for Adverse Reactions,
And 302 hospital's medicamentous liver lesion clinical database statistics, thus it is speculated that the overall incidence of fleece-flower root hepatic lesion is lower, therefore
Consider that fleece-flower root hepatic lesion may look like Drug-induced liver.Evaluation study in relation to fleece-flower root Drug-induced liver there is not yet
Report.
The generation of Drug-induced liver is often unrelated with dosage, administration time, it is difficult to predict.Drug-induced liver evaluates mould
Type is always the hot spot and advanced subject of domestic and international toxicologic study.Currently, the mechanism about Drug-induced liver generation is also not
It is fully apparent from, many studies have shown that, it is immune using joint endotoxin such as bacteria lipopolysaccharide (lipopolysaccharide, LPS)
The animal model intervened in advance and clinical medicine Drug-induced liver are more approximate.However, whether the related fleece-flower root can use LPS
There is not been reported for the method for progress Drug-induced liver evaluation.
Summary of the invention
The purpose of the application is to provide a kind of method for assessing fleece-flower root Drug-induced liver.
Specifically, the application provides a kind of method for assessing fleece-flower root Drug-induced liver, the method includes under
State step:
A. the preparation of fleece-flower root solution
Prepare the dried extract of the fleece-flower root, calculated yield；
When test, the amount of dried extract needed for calculating according to the dosage of the fleece-flower root is dissolved using sterile distilled water,
Storage, is preferably stored at 4 DEG C, spare；
B. the preparation of endotoxin solution
Endotoxic amount needed for calculating according to endotoxic dosage, is dissolved using sterile distilled water, is stored, preferably
It is stored at 4 DEG C, it is spare；
C. zoopery
Rat is grouped at random, weighs and records before testing, give fleece-flower root solution described in rat by stomach-filling, later,
Tail vein injection endotoxin solution, then, anesthetized rat, inferior caval vein take blood, and centrifugation acquires plasma specimen；
D. Testing index
With the liver function index Serum alanine amino transaminases (ALT) and aspartic acid of microplate reader method detection plasma specimen
Amino transaminases (AST)；
E. statistical procedures
For statistical analysis to ALT and AST using SPSS software, measurement data uses one-way analysis of variance, P < 0.05
It is statistically significant.
Certainly, it will be appreciated by those skilled in the art that can also be counted using other statistical procedures softwares
According to analysis.
In some embodiments, the dried extract can pass through following preparation:
Fleece-flower root medicine materical crude slice is weighed, cold soaking extracts at room temperature with solvent, and for extracting solution vacuum concentration to remove solvent, vacuum is dry
It is dry, obtain dried extract；
In some embodiments, the diameter of the fleece-flower root medicine materical crude slice can be about 1cm.
In some embodiments, the solvent can be ethyl alcohol, it is preferable that the solvent can be percentage by volume and be
50% ethyl alcohol.
In some embodiments, the volume in terms of milliliter of the solvent can be for the fleece-flower root medicine materical crude slice in gram
5-15 times of weight.
In some embodiments, the cold soaking time can be 12-72 hours.
In some embodiments, the dried extract can pass through following preparation:
Weigh the medicine materical crude slice of the fleece-flower root, the ethyl alcohol cold soaking that percentage by volume is 50% be added and extracts, wherein ethyl alcohol with milliliter
The volume of meter is 8 times of the weight in gram of the fleece-flower root medicine materical crude slice, and cold soaking extracts 2 times, and each 48h, combined extract subtracts
Pressure concentration and recovery uses up ethyl alcohol, is dried in vacuo, and obtains dry crude extract.
In some embodiments, the endotoxin can be bacteria lipopolysaccharide.
In some embodiments, yellow Jackets anesthetized rat can be used.
In some embodiments, in animal experiments, fleece-flower root dosage can be 0.54g/kg~75.6g/kg
Preferably, 1.08g/kg~50.4g/kg is to lead to hepatotoxicity wind agitation range.
Wherein, in this application, the unit g/kg of fleece-flower root dosage refers to that the weight of every kg rat is calculated to g is given
Fleece-flower root crude drug amount, for example 0.54g/kg refers to that the weight of every kg rat gives 0.54g fleece-flower root crude drug amount.Wherein, He Shou
Black crude drug amount can pass through following calculating: weight × yield of the medicine materical crude slice of the weighed fleece-flower root.
In some embodiments, in animal experiments, endotoxin administration dosage can be 1.4mg/kg~5.6mg/kg.
Preferably, endotoxin administration dosage can be 1.4mg/kg~2.8mg/kg.
It in some embodiments, in animal experiments, can be after giving fleece-flower root solution 2~5 hours, to big rat-tail
It is injected intravenously endotoxin solution.Preferably, endogenous toxic material can be injected to rat tail vein after giving fleece-flower root solution 2~4 hours
Plain solution.
In some embodiments, in animal experiments, it can be anaesthetized big after endotoxin injection solution 7~10 hours
Mouse；Preferably, can be after endotoxin injection solution 7~9 hours, anesthetized rat.
In some embodiments, testing rat used can be Wistar rat, SD rat or F344 rat, preferably
Ground can be SD rat.
In some embodiments, the weight of the rat can be 150g~300g.Preferably, the weight of the rat
It can be 200g~250g.
The application, based on endotoxic Drug-induced liver animal model, investigates the fleece-flower root using current wide use
Damaging action and its amount-poison relationship to rat liver, to the research for fleece-flower root hepatotoxicity wind agitation evaluation model and science mechanism
Reference is provided.
Compared with existing fleece-flower root hepatotoxicity wind agitation evaluation method, the application's is used to assess fleece-flower root Drug-induced liver
Method has the advantages that
1) the application can evaluate the special of the fleece-flower root based on the method for assessing fleece-flower root Drug-induced liver of LPS
Matter hepatotoxicity wind agitation；
2) the application based on LPS for assess fleece-flower root Drug-induced liver method can close to clinical dosage,
Shorter time inner evaluation fleece-flower root hepatotoxicity wind agitation, relatively with fleece-flower root clinic Drug-induced liver phenomenon；
3) in the method for assessing fleece-flower root Drug-induced liver of the application, the fleece-flower root combine LPS administration group with
Control group is compared, significant difference (P < 0.05), compared with the conventional hepatotoxic method of the assessment fleece-flower root, easy to operate, the used time
Shorter, economic cost is low.
Therefore, the method for assessing fleece-flower root Drug-induced liver provided by the present application based on LPS can be commented quickly
The Drug-induced liver of the valence fleece-flower root provides for fleece-flower root hepatotoxicity wind agitation evaluation model, the research of science mechanism and clinical application
With reference to.
Detailed description of the invention
Influence of Fig. 1 LPS different dosing initial time to rat ALT, AST vigor；
To ALT, the influence of AST vitality test after Fig. 2 various dose LPS joint fleece-flower root；
Fig. 3 LPS combines influence of the fleece-flower root administration to ALT, AST vigor of the rat of different lines；
Fig. 4 LPS combines influence of the fleece-flower root administration to ALT, AST vigor of the rat of different weight；
Fig. 5 anesthetized rat and when being detected under the different time points after LPS administration, studies this different time point
Influence to ALT, AST vigor of rat；
Fig. 6 LPS combines influence of the high dose fleece-flower root to ALT, AST vigor of rat；
Fig. 7 LPS combines influence of the fleece-flower root close to clinical equivalent dosage to ALT, AST vigor of rat；
The light microscopy checking result of Fig. 8 hepatic tissue.
Specific embodiment
Presently filed embodiment is described below by embodiment, it will be appreciated by the person skilled in the art that these
Specific embodiment only indicates to achieve the purpose that the application and the implementation technical solution that selects, is not to technical solution
Limitation.According to teachings of the present application, be to the improvement of technical scheme in conjunction with the prior art it is obvious, belong to the application
The range of protection.
The substance used in following embodiment, except where noted, remaining is commercially available.
Embodiment 1
The fleece-flower root (is purchased from Beijing green field pharmaceutcal corporation, Ltd, lot number 13101701, the place of production: Hubei).
Lipopolysaccharides (lipopolysaccharide, LPS) is purchased from Sigma company (lot number L2880,100mg/ branch).Penta bar
Than appropriate sodium (Sigma company).ALT, AST kit are purchased from Nanjing and build up Bioisystech Co., Ltd, lot number 20140420.Its
Its agents useful for same is that analysis is pure.
It wherein, in the microplate reader method being described below, is tested using kit.
Used SPSS software is SPSS13.0 software below.
Hereinafter, the fleece-flower root is indicated with PM.
Also, hereinafter the dried extract of the used fleece-flower root is by following preparation:
The fleece-flower root medicine materical crude slice 1000g that diameter is about 1cm is weighed, the ethyl alcohol 8000ml cold soaking that percentage by volume is 50% is added
It extracts, wherein the volume in terms of milliliter of ethyl alcohol is 8 times of the weight in gram of the fleece-flower root medicine materical crude slice, and cold soaking extracts 2 times,
Each 48h, combined extract are concentrated under reduced pressure and recycle ethyl alcohol to the greatest extent, and vacuum drying obtains dry crude extract 150g, calculated yield
15%.
When test, the amount of dried extract needed for being calculated according to the dosage of the fleece-flower root, and dissolved with sterile distilled water
At solution, stored at 4 DEG C, it is spare.
Hereinafter the dried extract of used Radix Glycyrrhizae is by following preparation:
Radix Glycyrrhizae 100g is weighed, addition percentage by volume is 800ml water refluxing extraction, and wherein the volume in terms of milliliter of water is
8 times of the weight in gram of the licorice piece, refluxing extraction 2 times, first time 1h, the 2nd time 0.5h, combined extract subtract
Pressure is concentrated and dried, and obtains dry crude extract 10g, calculated yield 10%.
When test, the amount of dried extract needed for being calculated according to the dosage of Radix Glycyrrhizae, and be dissolved into sterile distilled water
Solution is stored at 4 DEG C, spare.
Wherein, the endotoxin solution hereinafter used passes through following preparation:
Endotoxic amount needed for being calculated according to endotoxic dosage, and it is dissolved into solution with sterile distilled water, at 4 DEG C
Lower storage, it is spare.
It will be appreciated by those skilled in the art that the preparation of the above-mentioned fleece-flower root, Radix Glycyrrhizae and endotoxic solution can be only
A small amount of water, which is added, dissolves corresponding dried extract or drug just, then applies；Solution can certainly be carried out
It is being used after dilution, condition is the total dosage for guaranteeing to be administered every time.
The determination of a.LPS administration initial time
SD rat 80 is only randomly divided into 10 groups (n=10): Normal group；1h LPS is administered alone group, and 2h LPS is independent
Administration group, 3h LPS are administered alone group, and 4h LPS is administered alone group, and 5h LPS is administered alone group, wherein 1h, 2h, 3h, 4h or 5h
Time point with LPS joint PM administration group in PM administration after the time point of 1h, 2h, 3h, 4h and 5h it is identical and give medicament
Amount is 2.8mg/kg；PM is administered alone group, and wherein dosage is 18.9g/kg and the administration time of PM combines PM's with LPS
The PM administration time of administration group is identical；1h LPS combines the administration group of PM, and 2h LPS combines the administration group of PM, and 3h LPS combines PM
Administration group, 4h LPS combine PM administration group, 5h LPS combine PM administration group, wherein giving PM's by stomach-filling respectively
After 1h, 2h, 3h, 4h and 5h, tail vein injection LPS, wherein the dosage of PM is 18.9g crude drug/kg and LPS to medicament
Amount is 2.8mg/kg.
After giving LPS7 hours, using 1% yellow Jackets (dosage 50mg/kg) by all rat anesthesias,
Inferior caval vein takes blood, and centrifugation takes blood plasma, and acquire plasma sample and hepatic tissue sample.
Liver function index ALT and AST are detected with microplate reader method, for statistical analysis using SPSS software, measurement data is adopted
With one-way analysis of variance, P < 0.05 be it is statistically significant, as a result as shown in Figure 1.
It will be seen from figure 1 that compared with Normal group, 1h, 2h, 3h, 4h and 5h LPS be administered alone and PM individually to
ALT the and AST vigor of medicine group increases (P > 0.05) without conspicuousness；The ALT and AST that 1h LPS combines the administration group of PM are living
Power also increases (P > 0.05) without conspicuousness；ALT the and AST vigor of the administration group of 2h, 3h, 4h LPS joint PM has aobvious
It writes sex differernce (P < 0.01)；ALT the and AST vigor of the administration group of 5h LPS joint PM also has significant difference (P < 0.05).
It is therefore preferred that 2-5h is administered in PM, LPS is given after more preferably 2h~4h.
The b.LPS joint fleece-flower root leads to the determination of the LPS dosage of hepatic injury
Experimental animal SD rat 80 is only randomly divided into 8 groups (n=10): (dosage is for Normal group, PM group
(dosage is by 18.9g/kg, LPS (dosage 0.7mg/kg) group, LPS (dosage 1.4mg/kg) group, LPS
2.8mg/kg) (dosage is by group, LPS (dosage 5.6mg/kg) group, LPS (dosage 1.4mg/kg)+PM
18.9g/kg) (dosage is by group, LPS (dosage 2.8mg/kg)+PM (dosage 18.9g/kg) group, LPS
5.6mg/kg)+PM (dosage 18.9g/kg) group.By rat weight, record, wherein PM is given by stomach-filling, after 3h, tail
It is injected intravenously the LPS of above-mentioned dosage, it is using 1% yellow Jackets (dosage 50mg/kg) that all rats are numb after 7h
Liquor-saturated, inferior caval vein takes blood, and centrifugation takes blood, and acquire plasma sample and hepatic tissue sample.The specific time, which calculates, refers to the portion a
Point.
Liver function index ALT, AST are detected with microplate reader method, for statistical analysis using SPSS software, measurement data uses
One-way analysis of variance, P < 0.05 be it is statistically significant, as a result as shown in Figure 2.
Figure it is seen that compared with plasma A LT, AST of Normal group, the fleece-flower root is applied alone, and (dosage is
The no significant difference of 18.9g/kg group (P > 0.05)；It is applied alone the LPS group of 0.7mg/kg, 1.4mg/kg and 2.8mg/kg also not have
There is significant difference (P > 0.05)；It is applied alone 5.6mg/kg LPS group to have significant difference (P < 0.05)；0.7mg/kgLPS combination
The fleece-flower root group of 18.9g/kg does not have significant difference (P > 0.05)；1.4mg/kg, 2.8mg/kg and 5.6mg/kg LPS combination
The fleece-flower root group of 18.9g/kg has significant difference (P < 0.01), and what head of 5.6mg/kg LPS combination 37.8g/kg
There is death condition in crow group rat, therefore it is preferred that the dosage of LPS is 1.4mg/kg~5.6mg/kg, more preferably 1.4mg/
Kg~2.8mg/kg.
C. the determination of different lines rat
3 strain rats: Wistar rat, SD rat and F344 rat are selected, every group of rat, which is all weighed, to be recorded and be divided into
LPS is administered alone group, PM is administered alone group and LPS joint PM administration group (the rat number of each administration group be 10, i.e.,
N=10).Wherein, the dosage of the fleece-flower root is 18.9g/kg, and gives radix-polygoni multiflori extract by stomach-filling.The administration of LPS
Time is to be given by tail vein injection after radix-polygoni multiflori extract 3h is given in stomach-filling and dosage is 2.8mg/kg.LPS
After 7h is administered, using 1% yellow Jackets (dosage 50mg/kg) by all rat anesthesias, inferior caval vein takes blood, from
The heart takes blood plasma, and acquires plasma sample and hepatic tissue sample.The specific time, which calculates, refers to the part a.
Liver function index ALT, AST are detected with microplate reader method, for statistical analysis using SPSS software, measurement data uses
One-way analysis of variance, P < 0.05 be it is statistically significant, as a result as shown in Figure 3.
From figure 3, it can be seen that compared with being administered alone fleece-flower root group (18.9g/kg), be administered alone LPS Wistar,
ALT the and AST vigor of F344 and SD rat increases (P > 0.05) without conspicuousness；In LPS and the united administration group of the fleece-flower root
In, ALT the and AST vigor of Wistar rat has conspicuousness to increase (P < 0.05), ALT the and AST vigor of F344 and SD rat is equal
There is significant difference (P < 0.01), since F344 rat is more expensive, Wistar rat sensibility is strong without other two strains, therefore
It is preferred that SD rat is as research object.
D. the determination of rat body weight
The SD rat of following different weight: 150g, 200g, 250g and 300g is selected, every group of rat weight records and divide
Group is administered alone at LPS, PM is administered alone group and the administration group (n=10) of LPS joint PM.Fleece-flower root dosage is 18.9g/kg
And it is given by stomach-filling.After giving radix-polygoni multiflori extract 3h, 1% penta bars are used after the LPS of tail vein injection 2.8mg/kg, 7h
Than appropriate sodium (dosage 50mg/kg) by all rat anesthesias, inferior caval vein takes blood, and centrifugation takes blood plasma, and acquire blood plasma sample
Sheet and hepatic tissue sample.The specific time, which calculates, refers to the part a.
Liver function index ALT, AST are detected with microplate reader method, for statistical analysis using SPSS software, measurement data uses
One-way analysis of variance, P < 0.05 be it is statistically significant, as a result as shown in Figure 4.
From fig. 4, it can be seen that being administered alone LPS (2.8mg/kg) compared with being administered alone fleece-flower root group (18.9g/kg)
150g, 200g, 250g and 300g SD rat ALT and AST vigor also without conspicuousness increase (P > 0.05)；LPS and what
Tuber of multiflower knotweed drug combination, ALT the and AST vigor of 150g, 200g, 250g and 300g rat have significant difference (P < 0.01), therefore
It is preferred that the SD rat of 150g~300g is as research object.But 150g rat, since weight is too light, sensibility is stronger, it is dead compared with
Seriously, it will cause Testing index difficulty, and 300g rat wastes medicine, therefore it is preferred that the SD rat of 200g~250g is as research pair
As.
When e.LPS joint PM causes hepatotoxic-poison experiment
70 weighings of experimental animal SD rat are recorded and are randomly divided into 7 groups (n=10): Normal group, LPS group, PM
Group, 5h LPS+PM group, 7h LPS+PM group, 9h LPS+PM group and 10h LPS+PM group.Rat oral gavage gives PM, and dosage is
18.9g/kg.After giving PM3h, the LPS of tail vein injection 2.8mg/kg distinguishes 5h, 7h, 9h, 10h upon administration, uses
For 1% yellow Jackets (dosage 50mg/kg) by all rat anesthesias, inferior caval vein takes blood, and centrifugation takes blood plasma, and adopt
Collect plasma sample and hepatic tissue sample.The specific time, which calculates, refers to the part a.
Liver function index ALT and AST are detected with microplate reader method, for statistical analysis using SPSS software, measurement data is adopted
With one-way analysis of variance, P < 0.05 be it is statistically significant, as a result as shown in Figure 5.
Rat from fig. 5, it can be seen that compared with Normal group, in the LPS group that is administered alone, the PM group being administered alone
ALT and AST vigor without conspicuousness increase (P > 0.05)；In each group of the LPS joint fleece-flower root, the 5h after LPS is administered
There was no significant difference with AST vigor (P > 0.05) by the ALT that anesthetized rat is detected；7h, 9h and 10h anesthesia are big after LPS is administered
The ALT and AST vigor that mouse is detected have significant difference (P < 0.01), therefore 7h~10h is selected to carry out as anesthetized rat
The time of detection.But 10h rats death is more serious, may will affect subsequent index determining, therefore more select 7h~9h as
The time that anesthetized rat is detected.
F. high dose PM combines amount-poison experiment of LPS
80 weighings of SD rat are recorded and are randomly divided into 8 groups (n=10): Normal group, LPS group, PM is administered alone
3 dosage groups, LPS combine PM administration 3 dosage groups.Wherein, the dosage for 3 dosage groups that PM is administered alone is respectively as follows:
35.7g/kg, 50.4g/kg and 75.6g/kg.3 dosage groups of LPS joint PM administration are respectively as follows: LPS+PM2.8mg/kg+
35.7g/kg, 2.8mg/kg+50.4g/kg and 2.8mg/kg+75.6g/kg.PM is given by stomach-filling.After giving PM3h, tail
It is injected intravenously the LPS of 2.8mg/kg, using 1% yellow Jackets (dosage 50mg/kg) by all rat anesthesias after 7h,
Inferior caval vein takes blood, and centrifugation takes blood plasma, and acquire plasma sample and hepatic tissue sample.The specific time, which calculates, refers to the portion a
Point.
Liver function index ALT and AST are detected with microplate reader method, for statistical analysis using SPSS software, measurement data is adopted
With one-way analysis of variance, P < 0.05 be it is statistically significant, as a result as shown in Figure 6.
From fig. 6, it can be seen that compared with Normal group, the LPS group being administered alone, the PM group for being administered alone various dose
In rat ALT and AST vigor without conspicuousness increase (P > 0.05)；LPS (2.8mg/kg) combines 35.7,50.4 and
ALT the and AST vigor of rat in 75.6g/kg PM group has conspicuousness to increase (P < 0.01), and shows significantly amount-
Malicious relationship, it is therefore preferred to high doses to be less than or equal to 75.6g/kg.Therefore there are the phenomena of mortality in 75.6g/kg dosage group rat,
Therefore preferably, the fleece-flower root with dosage less than or equal to 50.4g/kg is tested.
G. low dosage PM combines amount-poison experiment of LPS
It is tested according to the same procedure of the part f, in addition to three dosages of PM are respectively 0.54g/kg, 1.08g/
3 dosage groups of kg, 2.16g/kg and LPS joint PM administration are respectively as follows: LPS+PM (2.8mg/kg+0.54g/kg, 2.8mg/kg
+ 1.08g/kg and 2.8mg/kg+2.16g/kg).In addition, also increasing Radix Glycyrrhizae (Gly) as negative medicine control, Gly is respectively set
It is administered alone group, dosage 2.16g/kg and Gly combine LPS administration group, and dosage is Gly+LPS (2.16g/kg
+2.8mg/kg).As a result as shown in Figure 7.Wherein, Radix Glycyrrhizae solution is also to be administered in the form of gastric infusion.
From figure 7 it can be seen that compared with Normal group, the LPS group, Radix Glycyrrhizae (Gly) group and the fleece-flower root that are administered alone
(PM) ALT, AST vigor of the rat in group there are no significant change (P > 0.05)；LPS combines 1.08 and 2.16g/kg PM's
ALT, AST vigor conspicuousness of rat in group increase (P < 0.05), and the rat in the group of LPS joint 0.54g/kg PM
ALT, AST are without significantly sexually revising (P > 0.05)；LPS combines ALT, AST of the rat in the group of 2.16g/kg Radix Glycyrrhizae also without significant
Sex differernce (P > 0.05), illustrating LPS not is to combine to will lead to hepatotoxicity wind agitation with all Chinese medicines, therefore, as diathesis liver poison
Journal of Sex Research model is reliable.
H. liver pathology inspection
Liver tissues of rats 1.5cm × 1cm × 0.5cm of the above-mentioned part a is taken, 10% formaldehyde is fixed, routine pathology slice, HE
Dyeing, light microscopic observation, as a result as shown in Figure 8.From figure 8, it is seen that Normal group and fleece-flower root group liver histopathology
There are slight inflammatory cell infiltration in variation that there are no significant, LPS group portal area, and the visible center of LPS use in conjunction fleece-flower root group is quiet
Arteries and veins expansion, inner membrance fall off, central vein week swelling of liver cell, necrosis, there are a large amount of inflammatory cell infiltrations, middle area part in portal area
Swelling of liver cell, necrosis.Illustrate that LPS can enhance fleece-flower root hepatotoxicity wind agitation, and what the Drug-induced liver model based on LPS can be used for
The research of tuber of multiflower knotweed Drug-induced liver.
The foregoing is merely preferred embodiment of the present application, not to the application make in any form with substantial limit
System.Those skilled in the art, in the range of not departing from technical scheme, in using disclosed above technology
Hold and the equivalent variations of a little change, modification and differentiation made is the equivalent embodiment of the application；Meanwhile it is all according to this Shen
Change, modification and differentiation of any equivalent variations to the above embodiments of substantial technological please etc. are in the application by weighing
Benefit requires in the range of defining.
Claims (13)
1. a kind of method for detecting fleece-flower root Drug-induced liver, the method includes the following steps:
A. the preparation of fleece-flower root solution
Prepare the dried extract of the fleece-flower root, calculated yield；
When test, the amount of dried extract needed for calculating according to the dosage of the fleece-flower root is dissolved using sterile distilled water, is store
It deposits, it is spare；
B. the preparation of endotoxin solution
Endotoxic amount needed for calculating according to endotoxic dosage, is dissolved using sterile distilled water, is stored, spare；
Wherein the endotoxin is bacteria lipopolysaccharide；
C. zoopery
Rat is grouped at random, weighs and records before testing, give fleece-flower root solution described in rat by stomach-filling, and giving
After stating fleece-flower root solution 2~5 hours, to rat tail vein endotoxin injection solution, then, anesthetized rat, inferior caval vein takes blood,
Centrifugation acquires plasma specimen；
In animal experiments, fleece-flower root dosage is 0.54g/kg~75.6g/kg, and endotoxic dosage is 1.4mg/
Kg~5.6mg/kg；
D. Testing index
Turned with the liver function index Serum alanine amino transaminases ALT and aspartic acid amino of microplate reader method detection plasma specimen
Adnosine deaminase AST；
E. statistical procedures
For statistical analysis to ALT and AST using SPSS software, measurement data uses one-way analysis of variance, and P < 0.05 is to have
Statistical significance.
2. according to the method described in claim 1, wherein, the dried extract passes through following preparation:
Weigh the medicine materical crude slice of the fleece-flower root, the ethyl alcohol cold soaking that percentage by volume is 50% be added and extracts, wherein ethyl alcohol in terms of milliliter
Volume is 8 times of the weight in gram of the fleece-flower root medicine materical crude slice, and cold soaking extracts 2 times, each 48h, and combined extract depressurizes dense
It retracts and receives ethyl alcohol to the greatest extent, vacuum drying obtains dry crude extract.
3. according to the method described in claim 1, wherein, the storage in step a and step b is stored at 4 DEG C.
4. according to the method described in claim 1, wherein, using yellow Jackets anesthetized rat.
5. according to the method described in claim 1, wherein, in animal experiments, fleece-flower root dosage be 1.08g/kg~
50.4g/kg。
6. according to the method described in claim 1, wherein, in animal experiments, endotoxic dosage be 1.4mg/kg~
2.8mg/kg。
7. according to the method described in claim 1, wherein, in animal experiments, after giving fleece-flower root solution 2~4 hours, to
Rat tail vein endotoxin injection solution.
8. according to the method described in claim 1, wherein, in animal experiments, after endotoxin injection solution 7~10 hours,
Anesthetized rat.
9. according to the method described in claim 8, wherein, in animal experiments, after endotoxin injection solution 7~9 hours, fiber crops
Liquor-saturated rat.
10. according to the method described in claim 1, wherein, testing rat used is that Wistar rat, SD rat or F344 are big
Mouse.
11. according to the method described in claim 10, wherein, testing rat used is SD rat.
12. according to the method for claim 11, wherein the weight of the SD rat is 150g~300g.
13. according to the method for claim 12, wherein the weight of the SD rat is 200g~250g.
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| 何首乌肝毒性客观性、临床标志物及损伤机制的初步研究;马致洁;《中国博士学位论文全文数据库 医药卫生科技辑》;20140715(第7期);第E057-10页 |
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