CN106214692A - The application of peoniflorin 6 O ' benzene sulfonate treatment hepatic fibrosis and confirmatory experiment method - Google Patents
The application of peoniflorin 6 O ' benzene sulfonate treatment hepatic fibrosis and confirmatory experiment method Download PDFInfo
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Abstract
The invention discloses application and the confirmatory experiment method of peoniflorin 6 O' benzene sulfonate treatment hepatic fibrosis, confirmatory experiment method comprises the following steps: the model construction of Liver Fibrosis Model mice and packet；Liver Fibrosis Model mouse stomach gives peoniflorin 6 O' benzene sulfonate, Radix Paeoniae Alba total glucosides, peoniflorin and colchicine；The each Indexs measure of Liver Fibrosis Model mice to after being administered: include transaminase, the detection of oxidation/Antioxidant Indexes, the mensuration of hepatic tissue hydroxyproline Hyp content, pathology of hepar section HE dyeing detection；Verify the effectiveness of peoniflorin 6 O' benzene sulfonate treatment hepatic fibrosis；Peoniflorin 6 O' benzene sulfonate be treatment hepatic fibrosis effect more preferably, the medicine material of less adverse effect, regulate antioxidant levels and immune level in the patient, improve the life quality of patient.
Description
Technical field
The present invention relates to field of medicaments, particularly relate to the application of peoniflorin-6-O'-benzene sulfonate treatment hepatic fibrosis and test
Card experimental technique.
Background technology
Hepatic fibrosis is that the reparation of chronic injury is reacted by hepatic tissue, is polytype cell, oxidative stress, cytokine
With the result of the series of complex effect such as somatomedin, with the hyperplasia of extracellular matrix components with abnormal deposition for main spy
Levy.Hepatic fibrosis is the pathological characters that chronic hepatopathy is important, be also liver cirrhosis occur prelude and must through intermediate link, be to face
The key link of bed treatment chronic hepatopathy.China is the district occurred frequently that viral hepatitis is popular, and chronic hepatitis patient is tens million of.There is money
Material display, the whole world there are about more than 3.5 hundred million hepatitis b virus carriers, and China's hepatitis B virus carriers just accounts for nearly 80%,
Drastically influence the healthy of our people.In HBV infection person, there is the patient of 10%～15% can develop into Chronic HBV
Infect, be that hepatic fibrosis the most only needed for 2～6 years from development of chronic hepatitis.It is reported, Chronic Hepatitis B generation hepatocarcinoma
Dangerous higher more than 200 times than ordinary people, the hepatocarcinoma patient of more than 95% is developed by chronic viral hepatitis B.Have been accepted as at present
Hepatic disease is typically all by the generation developmental pattern of acute-chronic-hepatic fibrosis-liver cirrhosis to hepatocarcinoma, therefore blocks liver
Fibrotic generation and development, significant to preventing and treating liver cirrhosis and hepatocarcinoma.Now generally believe hepatic fibrosis even liver
Hardening is all a dynamic process, belongs to reversibility pathological changes, if paathogenic factor obtains the most thoroughly treatment, hepatic injury stops sending out
Exhibition, then the many reversibles of hepatic fibrosis.Eliminating the cause of disease, blocking and suppressing the process of hepatic fibrosis is prevent and treat liver cirrhosis at present main
Means.
Although the medicine being currently used for treating hepatopathy is more, but always without important breakthrough, the treatment liver of exploitation treating both the principal and secondary aspects of a disease
The focus of scorching medicine research the most both at home and abroad and difficult point, have important real value.The herbal species of clinical practice at present
More, such as liver-protecting tablet, compound recipe 861, Fuzheng Huayu 319 Recipe, soft liver granule, FUFANG BIEJIA RUANGAN PIAN etc., there is certain curative effect.
But it is the highest that these pharmaceutical dosage forms mostly exist medical material utilization rate, medical material purifies excessively simple, and active constituent content is low, and impurity is many,
The problems such as quality control index and quality control method are unreasonable, and the uncertain therapeutic efficacy even having is cut, action character and machine-processed ten is hard to tell.
Hepatic fibrosis does not still have preferable medicine, chemical drugs treatment hepatic fibrosis often to have seriously the most clinically
Untoward reaction, and offer limited effectiveness, the medicine of invention better healing effect is problem in the urgent need to address.Peoniflorin be from
The effective ingredient extracted in the Chinese medicine Radix Paeoniae Alba, side effect is little, has a good antiinflammatory action, but to have hydrophilic readily soluble for peoniflorin
Yu Shui, is difficult to through cell membrane, and medicine is difficult to enter intracellular playing a role.
Summary of the invention
It is an object of the invention to provide a kind of medicine material peoniflorin-6-O'-benzene sulfonate treating hepatic fibrosis and
Confirmatory experiment method, and peoniflorin-6-O'-benzene sulfonate (Paeoniflorin-6-O '-benzene sulfonate, code name CP-
25), it is that Radix Paeoniae extracts peoniflorin (Paeoniflorin, Pae), and peoniflorin is carried out the lipotropy that structure of modification obtains
Compound, does not have the hydrophilic of peoniflorin and is insoluble in water, has lipotropy and easily passes through cell membrane, and medicine enters intracellular
Being more easy to play antiinflammatory immunity function, its action intensity is substantially better than Radix Paeoniae Alba total glucosides (TGP) and peoniflorin (Pae).
The present invention provide a kind of treat hepatic fibrosis effect more preferably, the medicine material of less adverse effect, regulate patient's body
Interior antioxidant levels and immune level, improve the life quality of patient.
In the present invention, utilize low (25mg/kg), in (50mg/kg), the CP-25 of high (100mg/kg) Three doses gradient
Liver is respectively organized with TGP, Pae, positive control colchicine (Col) give random packet the most in advance by the way of gavage respectively
Fibrosis mice, starts to be administered from modeling, is administered 4 weeks altogether on the 3rd week；After administration terminates, detect the transaminase level of mice, oxygen
Change/antioxidant levels, Liver Collagen level, and put to death animal detection hepatic pathology change；It is respectively compared before and after each group of medication each
Assessment of Changes CP-25, TGP and the Pae of index the therapeutic effect to hepatic fibrosis.
The experimental technique part of the present invention is as follows:
The model construction of Liver Fibrosis Model mice and packet:
By lumbar injection 10% carbon tetrachloride, 2 times a week, prepare hepatic fibrosis animal model, comprehensive according to mice
Situation is assigned randomly to model group and each medication group, ensures that the body weight of each group of mice and ordinary circumstance are close as far as possible.
The preparation of CP-25, TGP, Pae and Col and administration:
CP-25, white crystals sprills, purity > 95%, before internal medication, with micro ethanol dissolving, (every 50mg this product is molten
In 40 μ l ethanol), then add sodium carboxymethyl cellulose and make suspensoid, prepare low dosage (25mg/kg), middle dosage respectively
(50mg/kg), this medicine of high dose (100mg/kg) Three doses gradient, in 4 DEG C of refrigerators, save backup.Mouse weights, uses
Mouse stomach syringe needle and 1ml syringe, give mice CP-25 according to the capacity gavage of 0.1ml/10g body weight.
TGP, brownish-yellow powder, add sodium carboxymethyl cellulose before overall administration and make suspensoid, be prepared as 100mg/kg, give
Mouse weights before medicine, uses mouse stomach syringe needle and 1ml syringe, gives according to the capacity gavage of 0.1ml/10g body weight.
Pae, white powder, purity > 95%, add sodium carboxymethyl cellulose before overall administration and make suspensoid, be prepared as
100mg/kg, mouse weights before being administered, use mouse stomach syringe needle and 1ml syringe, fill according to the capacity of 0.1ml/10g body weight
Stomach gives.
Col: 1, commercially available colchicine tablet (0.5mg), after mortar is fully ground, adds sodium carboxymethyl cellulose, is prepared as
0.15mg/kg.Mouse weights before being administered, uses mouse stomach syringe needle and 1ml syringe, according to the capacity of 0.1ml/10g body weight
Gastric infusion.
The detection of transaminase:
Within 3rd week, starting to be administered from modeling, after being administered 4 weeks, mouse orbit rear vein beard is taken a blood sample, and centrifugal acquisition serum, by examination
Agent box description operation detection Serum ALT, the activity of AST.
Testing result shows, compared with normal group, model group Serum ALT, AST activity are increased significantly.Give CP-25
Can obviously reduce ALT and AST activity.CP-25 reduce be better than in terms of AST the peoniflorin Pae of same dosage, Radix Paeoniae Alba total glucosides TGP and
Col。
The detection of oxidation/Antioxidant Indexes:
After putting to death mice, take out liver immediately, wash away floating blood with cold saline, wipe dry, weigh 0.5g hepatic tissue, with cold
Normal saline makes the liver homogenate of 10%.By test kit description detection oxidation product malonaldehyde (MDA) level and antioxygen
Change enzyme superoxide dismutase (SOD) activity.
Result shows, compared with normal group, model group mouse liver even slurry oxidation product MDA content significantly raises, antioxygen
Change enzyme SOD activity substantially to reduce, give CP-25 and can obviously reduce MDA content, increased SOD activity.CP-25 is reducing antioxidation
Thing level, raising activities of antioxidant enzymes aspect are superior to the peoniflorin Pae of same dosage, Radix Paeoniae Alba total glucosides TGP and Col.
The mensuration of hepatic tissue hydroxyproline (Hyp) content:
After putting to death mice, take out liver immediately, weigh 0.5g hepatic tissue cold saline and wash away floating blood, with cold physiology salt
Water makes the liver homogenate of 10%.Content by test kit description detection collagen component Hyp.
Result shows, compared with normal group, model group mouse liver even slurry Hyp content significantly raises, and giving CP-25 can be bright
Aobvious reduction liver homogenate Hyp content.CP-25 is better than the peoniflorin Pae and Radix Paeoniae Alba total glucosides TGP of same dosage in terms of reducing Hyp.
Pathology of hepar detects: puts to death mice after being administered 4 weeks, takes mouse liver and do pathologic finding, liver lobus sinister, warp
10% formalin solution is fixed, 70%, 80%, 90%, 95%, 100% ethanol dehydration, and dimethylbenzene is transparent, paraffin embedding, section
Machine makees 5 μm sections, conventional dewaxing, and HE dyes, neutral gum mounting；Observing coloration result, CP-25 is to cell infiltration, collagen
The formation of the hypertrophy of fiber and the destruction of lobules of liver and pseudolobuli improves significantly.
Advantages of the present invention:
Peoniflorin-6-O'-benzene sulfonate in the present invention, is that peoniflorin carries out the lipotropy chemical combination that structure of modification obtains
Thing, does not have the hydrophilic of peoniflorin and is insoluble in water, has lipotropy and easily passes through cell membrane, and medicine enters intracellular being more easy to
Playing antiinflammatory immunity function, its action intensity is significantly better than that Radix Paeoniae Alba total glucosides TGP and peoniflorin Pae.Peoniflorin-6-O'-benzene sulphur
Acid esters be treatment hepatic fibrosis effect more preferably, the medicine material of less adverse effect, regulate antioxidant levels and exempting from the patient
Epidemic disease level, improves the life quality of patient.
Accompanying drawing explanation
Fig. 1 is to be administered pathology of hepar section HE after 4 weeks to dye testing result.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described:
Instrument reagent: analytical balance, eye scissors, ophthalmic tweezers, microplate reader, 1ml syringe；CP-25, Pae, TGP, Col, carboxylic
Sodium carboxymethylcellulose pyce (CMC-Na).
The structure of hepatic fibrosis mouse model and packet:
By lumbar injection 10% carbon tetrachloride, 2 times a week, prepare hepatic fibrosis animal model, comprehensive according to mice
Situation is assigned randomly to model group and each medication group, ensures that the body weight of each group of mice and ordinary circumstance are close as far as possible.
The preparation of CP-25 and administration:
CP-25, white crystals sprills, purity > 95%, before internal medication, with micro ethanol dissolving, (every 50mg this product is molten
In 40 μ l ethanol), then add sodium carboxymethyl cellulose and make suspensoid, prepare low dosage (25mg/kg), middle dosage respectively
(50mg/kg), this medicine of high dose (100mg/kg) Three doses gradient, in 4 DEG C of refrigerators, save backup.Mouse weights, uses
Mouse stomach syringe needle and 1ml syringe, give mice CP-25 according to the capacity gavage of 0.1ml/10g body weight.
TGP, brownish-yellow powder, add sodium carboxymethyl cellulose before overall administration and make suspensoid, be prepared as 100mg/kg, give
Mouse weights before medicine, uses mouse stomach syringe needle and 1ml syringe, gives according to the capacity gavage of 0.1ml/10g body weight.
Pae, white powder, purity > 95%, add sodium carboxymethyl cellulose before overall administration and make suspensoid, be prepared as
100mg/kg, mouse weights before being administered, use mouse stomach syringe needle and 1ml syringe, fill according to the capacity of 0.1ml/10g body weight
Stomach gives.
Col: 1, commercially available colchicine tablet (0.5mg), after mortar is fully ground, adds sodium carboxymethyl cellulose, is prepared as
0.15mg/kg.Mouse weights before being administered, uses mouse stomach syringe needle and 1ml syringe, according to the capacity of 0.1ml/10g body weight
Gastric infusion.
The detection of transaminase:
After experiment terminates, mouse orbit rear vein beard is taken a blood sample, centrifugal acquisition serum, by test kit description operation detection blood
The activity of clear ALT, AST.
Testing result shows, compared with normal group, model group Serum ALT, AST activity are increased significantly.Give CP-25
Can obviously reduce ALT and AST activity.CP-25 reduce be better than in terms of AST the peoniflorin Pae of same dosage, Radix Paeoniae Alba total glucosides TGP and
Col。
Table 1 CP-25 impact on the hepatic fibrosis mice serum transaminase of tetrachloro-methane induction
##P < 0.01vs normal group；**P < 0.01vs model group；△P < 0.05vs CP-25 (100mg/kg) group
The detection of oxidation/Antioxidant Indexes:
After putting to death mice, take out liver immediately, wash away floating blood with cold saline, wipe dry, weigh 0.5g hepatic tissue, with cold
Normal saline makes the liver homogenate of 10%.By test kit description detection oxidation product malonaldehyde (MDA) level and antioxidation
Enzyme superoxide dismutase (SOD) activity.
Result shows, compared with normal group, model group mouse liver even slurry oxidation product MDA content significantly raises, antioxygen
Change enzyme SOD activity substantially to reduce, give CP-25 and can obviously reduce MDA content, increased SOD activity.CP-25 is reducing antioxidation
Thing level, raising activities of antioxidant enzymes aspect are superior to the peoniflorin Pae of same dosage, Radix Paeoniae Alba total glucosides TGP and Col.
Table 2 CP-25 is on the hepatic fibrosis mouse liver even slurry MDA content of tetrachloro-methane induction and the impact of SOD activity
##P < 0.01vs normal group；*P<0.05,**P < 0.01vs model group；△P<0.05,△△P<0.01vs CP-25
(100mg/kg) group
The mensuration of hepatic tissue hydroxyproline (Hyp) content:
After putting to death mice, take out liver immediately, weigh 0.5g hepatic tissue cold saline and wash away floating blood, with cold physiology salt
Water makes the liver homogenate of 10%.Content by test kit description detection collagen component Hyp.
Result shows, compared with normal group, model group mouse liver even slurry Hyp content significantly raises, and giving CP-25 can be bright
Aobvious reduction liver homogenate Hyp content.CP-25 is better than the peoniflorin Pae and Radix Paeoniae Alba total glucosides TGP of same dosage in terms of reducing Hyp.
Table 3 CP-25 impact on the hepatic fibrosis mouse liver Hyp content of tetrachloro-methane induction
##P < 0.01vs normal group；**P < 0.01vs model group；△P < 0.05vs CP-25 (100mg/kg) group
Pathology of hepar detects:
Put to death mice after being administered 4 weeks, take mouse liver and do pathologic finding, liver lobus sinister, fix through 10% formalin solution,
70%, 80%, 90%, 95%, 100% ethanol dehydration, dimethylbenzene is transparent, paraffin embedding, and microtome makees 5 μm sections, conventional de-
Wax, HE dyes, neutral gum mounting.HE coloration result shows, model group portal area inflammatory cell, non-viable non-apoptotic cell increase, fiber
Interval in lobule stretch formed fiber package not of uniform size around, cell rope arrangement disorder, fibrous septum thickens, it is seen that pseudolobuli shape
Become.In addition to normal group, all visible significantly cell infiltration of the interstitial of each group.CP-25 group the most substantially alleviates carbon tetrachloride
Caused degree of hepatic fibrosis, to cell infiltration, the hypertrophy of collagen fiber and the destruction of lobules of liver and the formation of pseudolobuli
Deng all improving significantly.Section HE dyeing testing result is shown in Fig. 1.
Cut into slices testing result according to Fig. 1 and transaminase, the detection of oxidation/Antioxidant Indexes, hepatic tissue hydroxyproline Hyp contain
The measurement result analysis of amount, CP-25 treatment Liver Fibrosis Model mice can be obviously improved hepatic pathology change, reduce transaminase's water
Flat, improve liver oxidation resistance simultaneously.Result shows that CP-25 treats hepatic fibrosis definite effect, and drug effect is preferable.
Claims (2)
1. the application of peoniflorin-6-O'-benzene sulfonate treatment hepatic fibrosis.
2. the confirmatory experiment method of mice of peoniflorin-6-O'-benzene sulfonate treatment hepatic fibrosis, it is characterised in that include with
Lower step:
Step one, the model construction of Liver Fibrosis Model mice and packet；
Step 2, Liver Fibrosis Model mouse stomach gives peoniflorin-6-O'-benzene sulfonate, Radix Paeoniae Alba total glucosides, peoniflorin and autumn waters--limid eyes
Celestial alkali；
Step 3, each Indexs measure of Liver Fibrosis Model mice to after being administered:
The detection of transaminase: to the mouse orbit rear vein beard blood sampling of 4 weeks after being administered, the centrifugal serum that obtains, detection serum paddy third
Transaminase ALT, the activity of glutamic oxaloacetic transaminase, GOT AST；
Record testing result, compared with normal group, whether model group Serum ALT, AST activity are increased significantly, and administration group is
No can reduce ALT and AST activity；
The detection of oxidation/Antioxidant Indexes: put to death the mice of 4 weeks after being administered, take out liver immediately, wash away with cold saline floating
Blood, wipes dry, weighs 0.5g hepatic tissue, makes the liver homogenate of 10% with cold saline；Detection oxidation product malonaldehyde MDA water
Gentle antioxidant enzyme superoxide dismutase SOD activity；
Record testing result, compared with normal group, whether model group mouse liver even slurry oxidation product MDA content raises, antioxygen
Change whether enzyme SOD activity reduces；Whether administration group liver homogenate oxidation product MDA content reduces, and whether antioxidase SOD activity rises
High；
The mensuration of hepatic tissue hydroxyproline Hyp content: put to death the mice of 4 weeks after being administered, take out liver immediately, weigh 0.5g liver group
Knit and wash away floating blood with cold saline, make the liver homogenate of 10% with cold saline；Detection collagen component Hyp contains
Amount；
Record testing result, compared with normal group, whether model group mouse liver even slurry Hyp content raises, and whether administration group drops
Low liver homogenate Hyp content；
Pathology of hepar detects: putting to death mice after being administered 4 weeks, take mouse liver and do pathologic finding, liver lobus sinister, through 10%
Formalin solution is fixed, 70%, 80%, 90%, 95%, 100% ethanol dehydration, and dimethylbenzene is transparent, paraffin embedding, and microtome is made
5 μm sections, conventional dewaxing, HE dyes, neutral gum mounting；Observe coloration result, investigate peoniflorin-6-O'-benzene sulfonate pair
Whether the formation of cell infiltration, the hypertrophy of collagen fiber and the destruction of lobules of liver and pseudolobuli improves significantly.
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Cited By (2)
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| CN109481452A (en) * | 2019-01-07 | 2019-03-19 | 安徽医科大学 | A kind of purposes of Paeoniflorin -6-O '-benzene sulfonate |
| CN111543389A (en) * | 2020-05-06 | 2020-08-18 | 李立 | Method for constructing hepatic fibrosis rat model |
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| CN111543389A (en) * | 2020-05-06 | 2020-08-18 | 李立 | Method for constructing hepatic fibrosis rat model |
| CN111543389B (en) * | 2020-05-06 | 2022-03-08 | 李立 | Method for constructing hepatic fibrosis rat model |
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Application publication date: 20161214 |