CN107490645A - A kind of safe mass control method of prepared fleece flower root and application - Google Patents
A kind of safe mass control method of prepared fleece flower root and application Download PDFInfo
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
A kind of safe mass control method of prepared fleece flower root of disclosure, the cis-stilbene glycosides that methods described is included in measure prepared fleece flower root sample presses the weight percentage of prepared fleece flower root sample gross weight meter and controls the weight percentage within a limit value, to control the risk of liver injury of the prepared fleece flower root.Wherein, the risk of liver injury can be detected by the zoopery of correlation.
Description
Technical field
The application is related to medicinal material safety and quality control field, and in particular to a kind of safe mass controlling party of prepared fleece flower root
Method and application.
Background technology
The medicinal material fleece-flower root is polygonum multiflorum thunb Polygonum multiflorum Thunb. dried root.It is clinical
Using having, Radix Polygoni Multiflori and prepared fleece flower root are not, and health product can detoxify, the carbuncle that disappears, preventing malaria, relax bowel, and processed product prepared fleece flower root can
With filling liver kidney, benefiting essence-blood, black beard and hair etc..The fleece-flower root is curative for effect, is clinical conventional Chinese medicine and popular health food, traditionally
It is nontoxic.
However, the report for causing hepatic injury adverse reaction about the fleece-flower root and its preparation in recent years rolls up, cause state
Inside and outside extensive concern.Hepatic injury caused by the fleece-flower root is to take the disease of liver caused by the fleece-flower root, and cardinal symptom has jaundice, urine
Yellow, weak etc., severe patient causes hepatic failure even dead.The fleece-flower root is widely used in clinical and health care, and China is contained according to statistics
About 500 kinds of the Chinese patent drug of the fleece-flower root, about 200 kinds of health food.The liver toxicity issues of the fleece-flower root, will have a strong impact on the fleece-flower root and its
The drug safety of related preparations, cause worry of the public to Chinese native medicine security, also the modernization to traditional Chinese medicine, internationalization hair
Exhibition brings certain influence.
It would therefore be highly desirable to solve the liver toxicity issues of the fleece-flower root, especially liver toxicity issues of prepared fleece flower root, such as by from
Quality point reduces or controlled it that the risk of hepatic injury occurs.But so far, have no prepared fleece flower root safe mass controlling party
The report of method.
The content of the invention
This application provides a kind of safe mass control method of prepared fleece flower root.
Specifically, the application provides a kind of safe mass control method of prepared fleece flower root, and methods described includes determination sample
In cis-stilbene glycosides by prepared fleece flower root sample gross weight meter weight percentage and by the weight percentage control
System is within a limit value, to control the risk of liver injury of the prepared fleece flower root.
Wherein, the chemical full name of cis-stilbene glycosides is cis -2,3,5,4 '-tetrahydroxystilbene -2-O- β-D-
Glucoside.
More than or in other embodiment, the limit value can be 0.10%.
More than or in other embodiment, the cis-stilbene glycosides in determination sample presses prepared fleece flower root sample gross weight
The weight percentage of meter can include repeatedly using the cis-stilbene glycosides in solvent extraction prepared fleece flower root sample, with substantially
Cis-stilbene glycosides in prepared fleece flower root sample is extracted.
Wherein, substantially extracting the cis-stilbene glycosides in prepared fleece flower root sample can be by such as getting off really
It is fixed：The dregs of a decoction after multiple extraction are continued to use solvent extraction, and the cis hexichol in extract solution can not be detected by HPLC methods
Ethene glycosides, or the weight percentage of cis-stilbene glycosides are less than 0.005%.
It will be appreciated by those skilled in the art that the extraction of cis-stilbene glycosides can pass through methods known in the art
Come carry out.
More than or in other embodiment, the solvent can be ethanol.
More than or in other embodiment, the solvent can be the ethanol that volume weight fraction is 50%.
More than or in other embodiment, the cis-stilbene glycosides in determination sample presses prepared fleece flower root sample gross weight
The weight percentage of meter can include：
Prepared fleece flower root coarse powder is weighed, according to 8 times of amounts using the volume that milliliter mL is counted as the weight of the prepared fleece flower root in terms of gram g
The ethanol that percentage by volume is 50% is added, is extracted under the ultrasonic wave added that frequency is 40kHz and power is 500W, carried altogether
Take 2 times, each 30min, merge extract solution, the recovery ethanol that is concentrated under reduced pressure obtains concentrate.
Wherein, by it is above-mentioned under ultrasonic wave added with 50% ethanol extract 2 times after, the remaining dregs of a decoction continue to be carried with 50% ethanol
The weight content that the extract solution taken detects cis-stilbene glycosides by HPLC methods is less than 0.005%.
More than or in other embodiment, the weight percentage of the cis-stilbene glycosides in sample can pass through height
Effect liquid phase chromatogram method determines.It will be appreciated by those skilled in the art that the weight percent of the cis-stilbene glycosides in sample
Content can also be determined by other method known in the art, for example pass through LC-MS instrument detection etc..
More than or in other embodiment, the condition determination of high performance liquid chromatography can be：
ZORBAX Eclipse Plus C18 chromatographic columns, 250mm × 4.6mm, 5 μm；Mobile phase is water (A)-acetonitrile (B)；
Detection wavelength 280nm；30 DEG C of column temperature；Flow velocity 1mLmin-1；Linear gradient elution, 0~8min, 5%~35%A, 8~
10min, 35%~45%A, 10~15min, 45%~80%A, 15~18min, 80%~5%A；Theoretical cam curve is by cis
The calculated by peak area of Stibene-glucoside should be not less than 2000.
More than or in other embodiment, the step of passing through high effective liquid chromatography for measuring, can include：
Take appropriate concentrate and with methanol dilution, for example take 1ml concentrate and with methanol dilution to 100ml, use
0.22 μm of filtering with microporous membrane, produces need testing solution；
Cis-stilbene glycosides (cis-SG) reference substance and trans stilbene glycosides (trans-SG) appropriate reference substance are taken, essence
It is close weighed, add methanol that the solution of every 1mL cis-SG containing 0.25mg reference substances and 0.25mg trans-SG reference substances is made, use
0.22 μm of filtering with microporous membrane, produces mixed reference substance solution；
It is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, liquid chromatograph is injected, then passes through calculating
Peak area and by one point external standard method calculate need testing solution in cis-SG contents.
More than or in other embodiment, the risk of liver injury can be detected by following zoopery：
SD rats are grouped at random, weighs and records before experiment, give prepared fleece flower root sample described in rat respectively by gavage
(7.56g crude drug amounts/kg body weight, medicine liquid volume 1ml/100g body weight is administered) in product, after 3h, tail vein injection endotoxin solution
(2.8mg/kg body weight, administration medicine liquid volume 0.35ml/100g body weight), after 7h, yellow Jackets anesthetized rat is injected intraperitoneally, under
Vena cave takes blood, centrifugation collection plasma specimen, and gathers liver tissues of rats；
It is measured using at least one of group formed selected from following methods：
Alanine aminotransferase ALT and aspartic acid amino in plasma specimen is detected using automatic clinical chemistry analyzer
Transaminase AST is horizontal；IL-6, TNF-α and PPAR- γ contents using ELISA method by kit specification detection plasma specimen；
Liver tissues of rats Pathologic changes are observed using HE decoration methods；NF- κ B in rat liver tissue are determined using Immunohistochemical Method
P65 is expressed；The apoptosis of liver cell in rat liver tissue is detected using TUNEL methods；
Statistical analysis is carried out to data using SPSS softwares, measurement data uses one-way analysis of variance, wherein P ＜ 0.05
To be statistically significant, so as to judge risk of liver injury.
Present inventor's Comprehensive Experiment evaluation and the discovery of clinical analysis result, cis-stilbene glycosides (cis-SG)
A certain amount-malicious relation between content and hepatic injury be present.Therefore, start with from the content of cis-stilbene glycosides (cis-SG),
Its correlation with prepared fleece flower root hepatic injury is studied, and inquires into its possible margin of safety, is to establish that what head made from quality point
Black risk of liver injury control device and the horizontal offer reference of raising prepared fleece flower root data for clinical drug use.
A certain amount-malicious relation is had according to cis-SG contents and prepared fleece flower root diathesis hepatic injury, to reduce clinical use
Medicine risk, preliminary advice can produce the Quality Control limit of concocting process using cis-SG contents 0.10% as the fleece-flower root.
Other features and advantages of the present invention will be illustrated in the following description, also, partly becomes from specification
Obtain it is clear that or being understood by implementing the present invention.The purpose of the present invention and other advantages can be by specification, rights
Specifically noted structure is realized and obtained in claim and accompanying drawing.
Brief description of the drawings
Accompanying drawing is used for providing further understanding technical solution of the present invention, and a part for constitution instruction, with this
The embodiment of application is used to explain technical scheme together, does not form the limitation to technical solution of the present invention.
(wherein, I is reference substance to cis-SG and trans-SG HPLC figures, and II is RADIX POLYGONI MULTIFLORI PREPARATA in Fig. 1 display RADIX POLYGONI MULTIFLORI PREPARATA samples
Non- illumination sample, III is the sample of cis-SG contents 0.10% after RADIX POLYGONI MULTIFLORI PREPARATA illumination, and IV is cis-SG contents after RADIX POLYGONI MULTIFLORI PREPARATA illumination
0.35% sample, V is the sample of cis-SG contents 0.70% after RADIX POLYGONI MULTIFLORI PREPARATA illumination；1 is cis-SG, and 2 be trans-SG).
Fig. 2 shows and plasma A LT and AST is lived when the RADIX POLYGONI MULTIFLORI PREPARATA sample of different cis-SG contents is alone or joint LPS is used
Influence (wherein, the * P of power<0.05, * * P<0.01, * * * P<0.001vs control groups；▲P<0.05, ▲ ▲ P<0.01, ▲ ▲ ▲ P
<0.001vs model groups).
Fig. 3 is shown when the RADIX POLYGONI MULTIFLORI PREPARATA sample of different cis-SG contents is alone or joint LPS is used to liver histopathology
Change (HE dyeing 100 ×).
Fig. 4 show the RADIX POLYGONI MULTIFLORI PREPARATA sample of different cis-SG contents it is alone or joint LPS use when to Plasma TNF-α, IL-6,
The influence of PPAR- γ vigor is (wherein,*P<0.05,**P<0.01vs control groups；▲P<0.05vs model groups).
Fig. 5, Fig. 6 are shown when the RADIX POLYGONI MULTIFLORI PREPARATA sample of different cis-SG contents is alone or joint LPS is used to hepatic tissue NF- κ B
P65 influence (p65 SABCs 200 ×) is (wherein,*P<0.05,**P<0.01,***P<0.001vs control groups；▲P<0.05,▲▲
P<0.01vs model groups).
Fig. 7, Fig. 8 are shown when the RADIX POLYGONI MULTIFLORI PREPARATA sample of different cis-SG contents is alone or joint LPS is used to hepatocellular apoptosis
Influence (TUNEL apoptosis 200 ×) (wherein,***P<0.001vs control groups；▲P<0.05,▲▲P<0.01vs model groups).
Fig. 9 shows that cis-SG content situations in medicine materical crude slice are collected in the remaining medicine of clinical patients with liver deficiency and the place of production.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, below in conjunction with accompanying drawing to the present invention
Embodiment be described in detail.It should be noted that in the case where not conflicting, in the embodiment and embodiment in the application
Feature can mutually be combined.
Herein, LPS full name is lipopolysaccharides (lipopolysaccharide), is gram-negative bacterial cell wall
In a kind of composition, just discharged after bacterial death dissolves or destroys bacterium cell by artificial means, be a kind of important interior
Toxin.Micro LPS can cause gentle, undamaged inflammatory reaction, can't develop into obvious hepatotoxicity；But
The release of inflammatory factor is accompanied by, the stability of environment in tissue is destroyed, liver is greatly enhanced drug susceptibility, is showed
Go out medicine diathesis hepatic injury.The LPS and some medicine collective effects of non-toxic is used in combination, can produce similar clinical special
Heterogeneous hepatic injury reaction.
Gavage (intragastric administration, ig.)：Be in pharma-toxicology experiment it is a kind of it is conventional to
Prescription formula, animal is fixed, and using irrigation stomach device, decoction is directly injected into animal stomach by animal mouth.
It is injected intravenously (intravenous injection, iv.)：It is a kind of conventional administration in pharma-toxicology experiment
Mode, it is general to use tail vein injection more, animal is fixed, using syringe, decoction is injected directly into the lateral vein of rat-tail two
In blood vessel.
Glutamic-pyruvic transaminase (alanine aminotransferase, ALT) and glutamic-oxalacetic transaminease (aspartate
Transaminase, AST) be all liver function test on clinical medicine index, for judging whether liver suffers damage.
Tumor necrosis factor-alpha (tumour necrosis factor- α, TNF-α) and interleukin-6
(interleukin-6, IL-6) is all important proinflammatory factor, and its horizontal height is directly related with diseases associated with inflammation, in liver
Vital effect is played in the occurrence and development of damage.
Peroxisome proliferator-activated receptors-γ (peroxisome proliferator-activated
Receptor- γ, PPAR- γ) belong to nuclear receptor family, important adjustment effect is played in inflammation, can be from difference
Level modulation a plurality of Inflammatory Signal Transduction approach into the cell, wherein the Transcription inhibition to pro-inflammatory mediator gene such as NF- κ B is that it is anti-
The molecular basis of scorching effect.
The material used in following examples, except where noted, remaining is commercially available.
Material
1st, experimental animal
SPF level male SD rats, 180~200g of body weight, are purchased from Military Medical Science Institute's Experimental Animal Center, quality certification number
SCXK- (army) 2012-0004, in the hospital's Experimental Animal Center sub-cage rearing of PLA 302, free water and feed.Experiment is dynamic
Thing center room temperature (25 ± 2) DEG C, humidity 50~70%, indoor holding 12h illuminations replace with dark, and periodically carry out disinfection.
2nd, medicine and reagent
Prepared fleece flower root (abbreviation RADIX POLYGONI MULTIFLORI PREPARATA) (Radix Polygoni multiflori Preparata, RPMP) (lot number
15050401, place of production Hubei) Beijing green field pharmaceutcal corporation, Ltd is purchased from, its processing standard performs《Chinese Pharmacopoeia》With《Beijing
Prepared slices of Chinese crude drugs concocted specification》.Cis -2,3,5,4 '-tetrahydroxystilbene -2-O- β-D-Glucose glycosides (referred to as cis hexichol
Ethene glycosides, cis-SG, lot number 16091802, for assay, in terms of 98%), trans -2,3,5,4 '-tetrahydroxy hexichol second
Alkene -2-O- β-D-Glucose glycosides (abbreviation trans stilbene glycosides, trans-SG, lot number 16081506, for assay, with
98% meter) it is purchased from Chengdu Puffy moral Bioisystech Co., Ltd；Lipopolysaccharides (lipopolysaccharide, LPS) (lot number
046M4045V, 100mg/ branch) it is purchased from Sigma Co., USA；Yellow Jackets (lot number 57-33-0) are purchased from U.S. Sigma public affairs
Department；10% neutral formalin (fixer) (lot number 20160620) is purchased from Wan Bang bio tech ltd of BeiJing ZhongKe；AST is determined
Kit (MDH methods), ALT measure kits (lactic dehydrogenase enzyme process) are purchased from Beckman Coulter companies of the U.S.；Rat
IL-6 enzyme-linked immunosorbent assay kits (SEA079Ra 96T), rat TNF-α enzyme-linked immunosorbent assay kit
(SEA133Ra 96T) and P of Rats PAR- γ enzyme-linked immunosorbent assay kits (SEA886Ra 96T) are purchased from the U.S.
Cloud-Clone companies；Acetonitrile (chromatographic grade, lot number WXBB6406V) is purchased from Sigma Co., USA；Water is ultra-pure water, remaining examination
Agent is that analysis is pure.
3rd, instrument
Agilent1200 high performance liquid chromatographs (HP-12000DAD detectors) (the limited public affairs of U.S. Agilent science and technology
Department)；ZF-1 types ultraviolet analysis instrument for three purposed (Beijing Science and Technology Ltd. of departure BDCom)；XS205DU electronic balances (Switzerland
Mettler Toledo companies)；Rotary Evaporators R205B (SENCO) (Shensheng Science ＆ Tech. Co., Ltd., Shanghai)；KQ-500DE type numbers
Control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)；AU5400 automatic clinical chemistry analyzers (Japanese OLYMPUS optics
Co., Ltd.)；ELx808 absorbs light ELIASA (BioTek companies of the U.S.)；KD-P spreads out piece machine (section of Jinhua, Zhejiang Province city enlightening instrument
Equipment Co., Ltd)；Leica RM2235 paraffin slicing machines (Leica Microsystems GmbH, Shanghai)；JB-L7 paraffin bags
Bury machine (Wuhan person of outstanding talent Electronics Co., Ltd.)；The people of Nikon Ni-U tri- look at microscope (Japanese Nikon companies) etc. altogether.
Implement the determination of amount-malicious relation between 1 prepared fleece flower root and hepatotoxicity wind agitation
1. the preparation of RADIX POLYGONI MULTIFLORI PREPARATA sample
Prepare the sample of non-illumination：RADIX POLYGONI MULTIFLORI PREPARATA coarse powder 37.8g is taken, adds the body for 8 times of amounts that volume is RADIX POLYGONI MULTIFLORI PREPARATA coarse powder weight
The ethanol (i.e. plus 302.4mL 50% ethanol) that product percentage is 50%, ultrasonic (40KHz, 500W) extraction extract 2 times altogether, often
Secondary 30min, merge extract solution, the recovery ethanol that is concentrated under reduced pressure obtains concentrate (the preparation method bibliography of non-illumination sample：[1]
Lv's Yang, Wang Jiabai, Ji Yang, influence [J] Chinese experimental pharmacology of traditional Chinese medical formulae magazine of the Extraction solvents to fleece-flower root hepatotoxicity is waited,
2013,19 (20):268-272).
Wherein, by it is above-mentioned under ultrasonic wave added with 50% ethanol extract 2 times after, the remaining dregs of a decoction continue to be carried with 50% ethanol
The content that the extract solution taken detects cis-stilbene glycosides by HPLC methods is less than 0.005%.
The preparation of illumination sample：According to the non-illumination of above-mentioned preparation sample the step of be made extract solution, the step it is parallel enter
Row 3 times, so as to obtain 3 parts of extract solutions, 3 parts of extract solutions are placed in plate respectively, it is small to irradiate 3 respectively under 365nm uviol lamps
When, 15 hours and 80 hours so that trans-SG is converted into cis-SG, be concentrated under reduced pressure recovery ethanol obtain concentrate.Wherein, it is ultraviolet
Line irradiation is to simulate the condition that trans-SG changes into cis-SG.
2. cis-SG contents in high performance liquid chromatography (HPLC) detection sample
Chromatographic condition
Chromatographic column：ZORBAX Eclipse Plus C18 (4.6mm × 250mm, 5 μm)；Mobile phase：Water (A)-acetonitrile
(B)；Detection wavelength：280nm；Column temperature：30℃；Flow velocity：1ml/min；Linear gradient elution：0~8min, 5%~35%A, 8~
10min, 35%~45%A, 10~15min, 45%~80%A, 15~18min, 80%~5%A；Theoretical cam curve is by suitable
Formula -2,3,5,4 '-tetrahydroxystilbene -2-O- β-D-Glucose glycosides calculated by peak area should be not less than 2000.
The preparation of mixed reference substance solution：Take cis-SG reference substances, trans-SG reference substances appropriate, it is accurately weighed, add first
The solution of the trans-SG reference substances of cis-SG reference substance and 0.25mg of every 1mL containing 0.25mg is made in alcohol, with 0.22 μm of micropore
Membrane filtration, produce.
The preparation of need testing solution：The above-mentioned RADIX POLYGONI MULTIFLORI PREPARATA concentrate 1mL without after illumination and illumination is taken respectively, it is dilute with methanol
18 times are released, with 0.22 μm of filtering with microporous membrane, is produced.
Assay method：It is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, inject liquid chromatograph.Chromatogram
Figure is as shown in Figure 1.
Cis-SG contents in each sample are calculated according to one point external standard method, by calculating, respectively under 365nm uviol lamps respectively
Irradiation 3 hours, 15 hours and 80 hours after sample in cis-SG relative to the gross weight of prepared fleece flower root sample weight hundred
It is respectively about 0.10%, 0.35%, 0.70% to divide content.
Also, it was found from the II in Fig. 1, the sample cis-SG of non-illumination peak is very small and much smaller than in Fig. 1
The peak corresponding to cis-SG in the sample of III irradiation 3 hours, thus illustrate that the cis-SG contents of the sample of non-illumination are less than
0.10%.
Computational methods：Cis-SG concentration in need testing solution=(cis-SG peak areas/reference substance solution in need testing solution
Cis-SG concentration in middle cis-SG peak areas × reference substance solution), cis-SG contents (%)=test sample in prepared fleece flower root sample
The multiple of cis-SG concentration × dilution × concentrate cumulative volume/prepared fleece flower root sample sample weighting amount × 100% in solution.(above-mentioned meter
In calculation method, concentration unit mgmL-1, volume unit mL, sample weighting amount unit is g)
Determined again by LC-MS instrument and calculate the cis-SG contents in above-mentioned sample, acquired results are with passing through HPLC
The content of method measure is consistent.The liquid-phase condition of LC-MS instrument detection is identical with foregoing HPLC methods, Mass Spectrometer Method condition：ESI- sources
Desolvation N2Flow velocity 600L/min, spray pressure power 600kPa, 450 DEG C of desolvation temperature, capillary voltage 3.5kV；It is more
Reactive ion monitors (MRM) mode detection, parent ion 405 → daughter ions of m/z m/z 243, collision gas (He) 0.25mL/min.
3. zoopery
3.1 animal packet：80 SD rats are randomly divided into 10 groups (n=8)：Normal group, LPS model groups, system are first
The sample of cis-SG contents 0.10% is administered alone group, RADIX POLYGONI MULTIFLORI PREPARATA illumination after the non-illumination sample of crow is administered alone group, RADIX POLYGONI MULTIFLORI PREPARATA illumination
Afterwards the sample of cis-SG contents 0.35% be administered alone the sample of cis-SG contents 0.70% after group, RADIX POLYGONI MULTIFLORI PREPARATA illumination be administered alone group,
The sample administration group of cis-SG contents 0.10% after the non-illumination sample administration group of LPS joint RADIX POLYGONI MULTIFLORI PREPARATAs, LPS joint RADIX POLYGONI MULTIFLORI PREPARATA illumination,
Cis-SG contents after the sample administration group of cis-SG contents 0.35%, LPS joint RADIX POLYGONI MULTIFLORI PREPARATA illumination after LPS joint RADIX POLYGONI MULTIFLORI PREPARATA illumination
0.70% sample administration group.
3.2 administrations and collection of specimens：After Rat Fast be can't help into water 12h, weigh, be administered twice by body weight dose：First
In secondary administration, for needing the experimental group using RADIX POLYGONI MULTIFLORI PREPARATA, (7.56g crude drug amounts/kg body weight, decoction is administered in RADIX POLYGONI MULTIFLORI PREPARATA sample
Volume 1mL/100g body weight) (dosage bibliography：[2] Li Xiaofei, Li Na, Tu Can, is waited to be based on endotoxin diathesis mould
The Radix Polygoni Multiflori of type and the comparative studies of RADIX POLYGONI MULTIFLORI PREPARATA hepatotoxicity wind agitation [J] Chinese herbal medicines, 2015,46 (10):1481-1486.) given by gavage
Give；Second of administration after 3h, wherein for needing the experimental group using LPS to pass through tail vein injection after RADIX POLYGONI MULTIFLORI PREPARATA gives 3h
LPS (2.8mg/kg body weight, administration medicine liquid volume 0.35ml/100g body weight).After second of administration 7h, taken for all experimentss group
Sample is analyzed.Sampling is that inferior caval vein takes blood, and gathers liver by the way that yellow Jackets (50mg/kg) anesthetized rat is injected intraperitoneally
Sample (administration and methods of sample collection bibliography：[3] Yee SB, Hanumegowda UM, Copple BL, et
al.Endothelial cell injury and coagulation system activation during
synergistic hepatotoxicity from monocrotaline and bacterial
Lipopolysaccharide coexposure [J] .Toxicological Sciences, 2003,74 (1):203-214.
[4] Li Chunyu, Li Xiaofei, Tu Can, fleece-flower root diathesis hepatic injury evaluation [J] the Acta Pharmaceutica Sinicas of based on endogenous toxic material prime model are waited,
2015(1):28-33.)。
Group：+ second dosing instructions of administration are as follows for the first time：
Normal group (referred to as " control group ")：Physiological saline (ig.)+physiological saline (iv.)；
LPS model groups (referred to as " model group ")：Physiological saline (ig.)+LPS (iv.)；
The non-illumination sample of RADIX POLYGONI MULTIFLORI PREPARATA is administered alone group (referred to as " non-light group ")：The non-illumination sample of RADIX POLYGONI MULTIFLORI PREPARATA (ig.)+physiology
Salt solution (iv.)；
The sample of cis-SG contents 0.10% is administered alone group (referred to as " 0.10% group ") after RADIX POLYGONI MULTIFLORI PREPARATA illumination：RADIX POLYGONI MULTIFLORI PREPARATA illumination
The sample of cis-SG contents 0.10% (ig.)+physiological saline (iv.) afterwards；
The sample of cis-SG contents 0.35% is administered alone group (referred to as " 0.35% group ") after RADIX POLYGONI MULTIFLORI PREPARATA illumination：RADIX POLYGONI MULTIFLORI PREPARATA illumination
The sample of cis-SG contents 0.35% (ig.)+physiological saline (iv.) afterwards；
The sample of cis-SG contents 0.70% is administered alone group (referred to as " 0.70% group ") after RADIX POLYGONI MULTIFLORI PREPARATA illumination：RADIX POLYGONI MULTIFLORI PREPARATA illumination
The sample of cis-SG contents 0.70% (ig.)+physiological saline (iv.) afterwards；
The non-illumination sample administration group of LPS joint RADIX POLYGONI MULTIFLORI PREPARATAs (referred to as " the non-light groups of LPS+ ")：The non-illumination sample of RADIX POLYGONI MULTIFLORI PREPARATA
(ig.)+LPS(iv.)；
The sample administration group of cis-SG contents 0.10% (referred to as " LPS+0.10% groups ") after LPS joint RADIX POLYGONI MULTIFLORI PREPARATA illumination：System
The sample of cis-SG contents 0.10% (ig.)+LPS (iv.) after tuber of multiflower knotweed illumination；
The sample administration group of cis-SG contents 0.35% (referred to as " LPS+0.35% groups ") after LPS joint RADIX POLYGONI MULTIFLORI PREPARATA illumination：System
The sample of cis-SG contents 0.35% (ig.)+LPS (iv.) after tuber of multiflower knotweed illumination；
The sample administration group of cis-SG contents 0.70% (referred to as " LPS+0.70% groups ") after LPS joint RADIX POLYGONI MULTIFLORI PREPARATA illumination：System
The sample of cis-SG contents 0.70% (ig.)+LPS (iv.) after tuber of multiflower knotweed illumination.
4. Indexs measure
The detection of 4.1 blood plasma transaminases：Isolated rat blood is collected, centrifugation (3000r/min, 10min) takes blood plasma, uses
Automatic clinical chemistry analyzer detection glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST) are horizontal.
The detection of 4.2 liver histopathologies：Liver tissues of rats is gathered, is fixed with 10% neutral formalin, routine pathology
Section, hematoxylin eosin staining method (HE) dyeing, liver histopathology change is observed under an optical microscope.
4.3 Plasma Interleukins -6 (IL-6), tumor necrosis factor-alpha (TNF-α), peroxisome proliferator-activated receptor
Body-γ (PPAR- γ) detection：Using ELISA method by kit specification detection IL-6, TNF-α, PPAR- γ contents.
4.4 hepatic tissue nuclear transcription factor-kappa Bs (NF- κ B) p65 detection：Liver tissues of rats is gathered, with 10% neutral Fu Er
Malin is fixed, routine pathology section, and NF- κ B p65 are expressed in Immunohistochemical Method detection liver.
4.5 hepatocellular apoptosis detect：Liver tissues of rats is gathered, is fixed with 10% neutral formalin, routine pathology section,
Apoptosis cell is detected with TUNEL kits.
5. statistical analysis
Using SPSS19.0 softwares carry out statistical analysis, experimental data withRepresent, measurement data uses single factor test
Variance analysis (ANOVA), the horizontal P ＜ 0.05 of significance probability.
3rd, result
1. plasma A LT, AST measurement result compares
From Fig. 2 and table 1 as can be seen that compared with control group, non-light group, 0.10% group, 0.35% group, 0.70% group,
Rat plasma AST and the ALT vigor of the non-light group of LPS model groups, LPS+ and LPS+0.10% groups there are no significant difference (P>
0.05), and LPS+0.35% groups, LPS+0.70% groups are compared with control group and model group, and rat plasma AST and ALT vigor is equal
Significantly rise (P<0.05).
The RADIX POLYGONI MULTIFLORI PREPARATA sample of the different cis-SG contents of table 1 is alone or combines when LPS is used to plasma A LT and AST vigor
Influence
Note：*P<0.05, * * P<0.01, * * * P<0.001vs control groups；▲P<0.05, ▲ ▲ P<0.01, ▲ ▲ ▲ P<
0.001vs model groups
The result shows, the cis-SG (0.35% and 0.70%) of higher inversion quantity RADIX POLYGONI MULTIFLORI PREPARATA sample and LPS are combined,
Hepatic injury biochemical indicator ALT, AST notable rise can be caused.
2. pathology of hepar is analyzed
From figure 3, it can be seen that compared with control group, non-light group, 0.10% group, 0.35% group, 0.70% group without obvious
Change, liver tissues of rats pathological section visible cell marshalling, occasionally there is cell infiltration；The visible liver tissues of rats of model group is cut
The portal area inflammatory cell increase of piece, but without obvious pathological change；The non-light groups of LPS+, LPS+0.10% groups, it is seen that header
Area's inflammatory cell increase, but without obvious pathological change；And LPS+0.35% groups, LPS+0.70% groups, it is seen that central vein expands
, inner membrance come off, central vein week liver cell there is swelling, necrosis phenomena, portal area is by substantial amounts of cell infiltration.
The result shows, the cis-SG (0.35% and 0.70%) of higher inversion quantity RADIX POLYGONI MULTIFLORI PREPARATA sample and LPS are combined,
Significantly changing for pathology of hepar can be caused.
3. plasma IL -6, TNF-α, PPAR- γ changes of contents
It is and right from Fig. 4 and table 2 as can be seen that LPS can stimulate Monocytes/Macrophages to secrete substantial amounts of proinflammatory cytokine
Compared according to group, the significantly raised (P of TNF-α content of LPS model groups<0.05), elevated trend is presented in IL-6 but difference is without notable
Property (P>0.05), non-light group, 0.10% group, 0.35% group and 0.70% group there are no significant change (P>0.05)；With LPS
Model group is compared, and LPS+0.35% groups, the TNF-α of LPS+0.70% groups, IL-6 contents significantly raise (P<0.05), and LPS+ not
Light group, LPS+0.10% groups change (P without obvious>0.05).
Compared with control group, the PPAR- γ expression of LPS model groups is decreased obviously (P<0.05), non-light group, 0.10%
Group, 0.35% group, 0.70% group there was no significant difference (P>0.05)；Compared with LPS model groups, the non-light groups of LPS+, LPS+
0.10% group of PPAR- γ expression changes (P without obvious>0.05), LPS+0.35% groups, LPS+0.70% group PPAR- γ expression
Significantly reduce (P<0.05).
The RADIX POLYGONI MULTIFLORI PREPARATA sample of the different cis-SG contents of table 2 it is alone or joint LPS use when to Plasma TNF-α, IL-6,
The influence of PPAR- γ vigor
Note：*P<0.05,**P<0.01vs control groups；▲P<0.05vs model groups
The result shows, the cis-SG (0.35% and 0.70%) of higher inversion quantity RADIX POLYGONI MULTIFLORI PREPARATA sample and LPS are combined,
Inflammatory factor TNF-α, IL-6 notable rise can be caused, PPAR- γ's substantially reduces.4. in SABC measure rat liver
NF- κ B p65 are expressed
From Fig. 5, Fig. 6 and table 3 as can be seen that compared with control group, the p65 of model group expresses significantly raised (P<0.05),
Illustrate that LPS have activated the activity of NF- κ B paths, and non-light group, 0.10% group, 0.35% group and 0.70% group are without significant changes
(P>0.05)；Compared with model group, LPS+0.35% groups, LPS+0.70% groups, accumulation OD value (IOD) significantly rise (P<
0.05)；And the unobvious change (P of the IOD of the non-light groups of LPS+, LPS+0.10% groups>0.05).To occur palm fibre in nucleus
Yellow particle is positive criteria, and the height of positive expression amount is judged according to IOD sizes.
The release of inflammatory factor such as TNF-α can activate nuclear transcription factor-kappa B (nuclear factor kappa B, NF-
κ B) path, cause core to shift, NF- κ B can be combined with target target gene, start and regulate and control the expression of inflammatory mediator, further be added
Hyperphlogosis reacts, and mediates the damage of body tissue and cell.It is the common counter for detecting NF- κ B pathway activities that whether p65, which enters core,.
The RADIX POLYGONI MULTIFLORI PREPARATA sample of the different cis-SG contents of table 3 is alone or combines when LPS is used to hepatic tissue NF- κ B p65 shadow
Ring
Note：*P<0.05,**P<0.01,***P<0.001vs control groups；▲P<0.05,▲▲P<0.01vs model groups
The result shows, the cis-SG (0.35% and 0.70%) of higher inversion quantity RADIX POLYGONI MULTIFLORI PREPARATA sample and LPS are combined,
The notable rise of NF- κ B p65 expression quantity in liver can be caused.
5. the TUNEL analyses of liver organization Apoptosis
From Fig. 7, Fig. 8 and table 4 as can be seen that compared with control group, LPS model groups, LPS+0.35% groups, LPS+0.70%
The hepatocellular apoptosis number and apoptosis rate of group significantly raise (P<0.05)；And the unobvious change (P of the apoptosis rate of other groups
>0.05).Dyed with nucleus in sepia as positive criteria, apoptosis rate %=(apoptosis cell/TCS) *
100%.
Influence when the RADIX POLYGONI MULTIFLORI PREPARATA sample of the different cis-SG contents of table 4 is alone or combines LPS uses to hepatocellular apoptosis
Note：***P<0.001vs control groups；▲P<0.05,▲▲P<0.01vs model groups
The result shows, the cis-SG (0.35% and 0.70%) of higher inversion quantity RADIX POLYGONI MULTIFLORI PREPARATA sample and LPS are combined,
It can cause the notable rise of liver cell apoptosis rate, and the cis-SG (0.10%) of low inversion quantity RADIX POLYGONI MULTIFLORI PREPARATA sample and non-illumination
Without significant difference between the sample of conversion, it was demonstrated that cis-SG inversion quantities are too high to increase risk of liver injury, and controls cis-SG to turn
Change amount can reduce or control risk of liver injury.
Medicine materical crude slice content analysis is collected in the remaining medicine of 2 clinical patients with liver deficiency of embodiment and the place of production
By collecting from the RADIX POLYGONI MULTIFLORI PREPARATA medicine materical crude slice of different sources (Hunan, Guangdong, Hubei, Sichuan, Yunnan) and through liberation
Patients with liver deficiency caused by hospital of army 302 is diagnosed as RADIX POLYGONI MULTIFLORI PREPARATA takes remaining medicine (medicine materical crude slice, medicinal liquor and medicinal powder), and determines
Cis-SG contents, " cis-SG contents in high performance liquid chromatography (HPLC) detection sample " in assay method reference implementation example 1.
From fig. 9, it can be seen that the RADIX POLYGONI MULTIFLORI PREPARATA drink collected from different sources (Hunan, Guangdong, Hubei, Sichuan, Yunnan)
Cis-SG contents are universal relatively low (＜ 0.10%) in piece, and cis-SG contents are obvious higher in the medicine that patients with liver deficiency is taken
(＞ 0.40%), illustrate that the height of cis-SG contents and its diathesis hepatic injury are closely related in RADIX POLYGONI MULTIFLORI PREPARATA.
In conjunction with the embodiments 1 and embodiment 2 interpretation of result understand, all RADIX POLYGONI MULTIFLORI PREPARATA samples do not cause on normal rat
Hepatic injury；On endotoxin model, non-illumination, the RADIX POLYGONI MULTIFLORI PREPARATA sample of light conversion cis-SG contents 0.10% are showed no obvious liver
Damage, and the RADIX POLYGONI MULTIFLORI PREPARATA sample of light conversion cis-SG contents 0.35% and 0.70% causes obvious pathology of livers, shows
For swelling of liver cell is downright bad, a large amount of inflammatory cell infiltrations, hepatic tissue nuclear transcription factor-kappa B (NF- κ B) p65 expression quantity, cell wither
Die rate and dramatically increase (P<0.05), while liver function biochemical indicator ALT, AST and inflammatory factor TNF-α, IL-6 contents are aobvious
Write increase (P<0.05), peroxisome proliferator-activated receptors-γ (PPAR- γ) content significantly reduces (P<0.05)；Together
When, clinically RADIX POLYGONI MULTIFLORI PREPARATA causes the remaining analysis of drug content of patients with liver deficiency to find, its cis-SG content (>0.40%) it is high
In the place of production collect medicine materical crude slice (<0.10%).Comprehensive Experiment evaluation and the prompting of clinical analysis result, cis-SG contents and RADIX POLYGONI MULTIFLORI PREPARATA are special
A certain amount-malicious relation be present in matter hepatic injury；To reduce clinical application risk, preliminary advice can make cis-SG contents 0.10%
The Quality Control limit of concocting process is produced for the fleece-flower root.
Although disclosed herein embodiment as above, described content be only readily appreciate the present invention and use
Embodiment, it is not limited to the present invention.Technical staff in any art of the present invention, taken off not departing from the present invention
On the premise of the spirit and scope of dew, any modification and change, but the present invention can be carried out in the form and details of implementation
Scope of patent protection, still should be subject to the scope of the claims as defined in the appended claims.
Claims (10)
1. a kind of safe mass control method of prepared fleece flower root, methods described includes the cis hexichol in measure prepared fleece flower root sample
Ethene glycosides presses the weight percentage of the prepared fleece flower root sample gross weight meter and by weight percentage control in a limit
Within definite value, to control the risk of liver injury of the prepared fleece flower root.
2. according to the method for claim 1, wherein the limit value is 0.10%.
3. according to the method for claim 1, wherein, the cis-stilbene glycosides in the determination sample presses prepared fleece flower root sample
The weight percentage of product gross weight meter includes repeatedly using the cis-stilbene glycosides in prepared fleece flower root sample described in solvent extraction,
So that substantially the cis-stilbene glycosides in the prepared fleece flower root sample to be extracted.
4. according to the method for claim 3, wherein the solvent is ethanol.
5. according to the method for claim 4, wherein the solvent is the ethanol that volume weight fraction is 50%.
6. method of quality control according to claim 3, wherein the cis-stilbene glycosides in the determination sample is by system
The weight percentage of fleece-flower root sample gross weight meter includes：
Prepared fleece flower root coarse powder is weighed, according to 8 times of amount additions using the volume that milliliter mL is counted as the weight of the prepared fleece flower root in terms of gram g
Percentage by volume is 50% ethanol, is extracted under the ultrasonic wave added that frequency is 40kHz and power is 500W, extracts 2 altogether
It is secondary, each 30min, merge extract solution, the recovery ethanol that is concentrated under reduced pressure obtains concentrate.
7. according to the method for quality control any one of claim 1-6, wherein cis in the prepared fleece flower root sample
The weight percentage of Stibene-glucoside is by high effective liquid chromatography for measuring.
8. the condition determination of method of quality control according to claim 7, wherein high performance liquid chromatography is as follows：
ZORBAX Eclipse Plus C18 chromatographic columns, 250mm × 4.6mm, 5 μm；Mobile phase is water (A)-acetonitrile (B)；Detection
Wavelength 280nm；30 DEG C of column temperature；Flow velocity 1mLmin-1；Linear gradient elution, 0~8min, 5%~35%A, 8~10min,
35%~45%A, 10~15min, 45%~80%A, 15~18min, 80%~5%A；Theoretical cam curve presses cis hexichol second
The calculated by peak area of alkene glycosides should be not less than 2000.
9. quality and safety control method according to claim 8, wherein the step of passing through high effective liquid chromatography for measuring is wrapped
Include：
Take appropriate concentrate and with methanol dilution, with 0.22 μm of filtering with microporous membrane, produce need testing solution；
Cis-stilbene glycosides (cis-SG) reference substance and trans stilbene glycosides (trans-SG) appropriate reference substance are taken, precision claims
It is fixed, add methanol that the solution of the trans-SG reference substances of cis-SG reference substance and 0.25mg of every 1mL containing 0.25mg is made, with 0.22
μm filtering with microporous membrane, produces mixed reference substance solution；
It is accurate respectively to draw reference substance solution and each 10 μ L of need testing solution, liquid chromatograph is injected, then by calculating peak face
Accumulate and calculate cis-SG contents in need testing solution by one point external standard method.
10. according to the method for claim 1, wherein the risk of liver injury is detected by following zoopery
's：
SD rats are grouped at random, weighs and records before experiment, give prepared fleece flower root sample described in rat respectively by gavage
(7.56g crude drug amounts/kg body weight, administration medicine liquid volume 1mL/100g body weight), after 3h, tail vein injection endotoxin solution
(2.8mg/kg body weight, administration medicine liquid volume 0.35ml/100g body weight), after 7h, yellow Jackets anesthetized rat is injected intraperitoneally, under
Vena cave takes blood, centrifugation collection plasma specimen, and gathers liver tissues of rats；
It is measured using at least one of group formed selected from following methods：
Ammonia is turned using the alanine aminotransferase ALT in automatic clinical chemistry analyzer detection plasma specimen and aspartic acid amino
Enzyme AST is horizontal；IL-6, TNF-α and PPAR- γ contents using ELISA method by kit specification detection plasma specimen；Using
HE decoration methods observe liver tissues of rats Pathologic changes；NF- κ B p65 tables in rat liver tissue are determined using Immunohistochemical Method
Reach；With the apoptosis using liver cell in TUNEL methods detection rat liver tissue；
Statistical analysis is carried out to data using SPSS softwares, measurement data uses one-way analysis of variance, and wherein P ＜ 0.05 are to have
Statistical significance, so as to judge risk of liver injury.
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