CN106309502B - Application of the eupolyphoge sinensis enzymatic hydrolyzed extract in preparation prevention and/or treatment carbon tetrachloride hepatic injury drug - Google Patents
Application of the eupolyphoge sinensis enzymatic hydrolyzed extract in preparation prevention and/or treatment carbon tetrachloride hepatic injury drug Download PDFInfo
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- CN106309502B CN106309502B CN201610916770.XA CN201610916770A CN106309502B CN 106309502 B CN106309502 B CN 106309502B CN 201610916770 A CN201610916770 A CN 201610916770A CN 106309502 B CN106309502 B CN 106309502B
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- eupolyphoge sinensis
- hepatic injury
- carbon tetrachloride
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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Abstract
The invention belongs to pharmaceutical technology fields, and in particular to application of the eupolyphoge sinensis enzymatic hydrolyzed extract in preparation prevention and/or treatment carbon tetrachloride hepatic injury drug.The eupolyphoge sinensis enzymatic hydrolyzed extract is prepared by the method included the following steps: eupolyphoge sinensis powder is soluble in water, papain is added, for the pH value of regulation system to 6.0, supernatant is collected in enzyme digestion reaction, centrifugation.Eupolyphoge sinensis enzymatic hydrolyzed extract can significantly lower CCl4Damage to mouse liver cell enhances the oxidation resistance of acute hepatic injury mice, is likely to become a kind of very outstanding prevention and/or treats the drug candidate of carbon tetrachloride hepatic injury.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to eupolyphoge sinensis enzymatic hydrolyzed extract is in preparation prevention and/or treatment tetrachloro
Change the application in carbon liver injury medicament.
Background technique
Liver is one of most important internal organs of body, the function of metabolism and bioconversion is carry, vulnerable to internal
The invasion of outer various pathogenic factors and stimulating factor, cause the damage and inflammatory reaction of liver.Hepatic injury is various adverse factors
Such as virus, bacterium, drug, alcohol, ischemic, hepatic portion excision, direct or indirect liver injury cause the inflammation of liver cell
A series of lesions such as disease, necrosis, even apoptosis and fibrosis are the common pathologic basis of various hepatopathys.Therefore, research has shield
The health food or drug of liver liver protection function are very important to protection human health.
Carbon tetrachloride has light anesthesia effect, has serious detrimental effect to liver, kidney, can be changed into free radical in vivo,
The metabolism of lipoids on liver plasma membrane is upset, necrosis of liver cells is caused, is common cause hepatic injury compound, utilizes its building
Liver damage animal model is the preferred experimental animal model for the hepatic injury that current research chemical damage and oxidative stress mediate
One of, and one of the common model of exploitation liver protecting class drug.
Eupolyphoge sinensis is China's tradition blood-activating stasis-removing kind Chinese medicine, and modern medicine study and clinical test prove, eupolyphoge sinensis, which has, to be inhibited
Tumour adjusts blood lipid, anti-aging, anti-mutation, resist oxygen lack and other effects, containing alkaloid, protein, amino acid, phenols, carbohydrate,
The multiple nutritional components such as steroidal, oil have high medicinal and health value.The current chemistry in relation to eupolyphoge sinensis biological function
(component) basis and Biological Mechanism not yet illustrate completely.
Summary of the invention
The object of the present invention is to provide a kind of new medicine uses of eupolyphoge sinensis enzymatic hydrolyzed extract.
The new application of eupolyphoge sinensis enzymatic hydrolyzed extract provided by the present invention is it in preparation prevention and/or treatment carbon tetrachloride liver
Application in damage medicine.
The eupolyphoge sinensis enzymatic hydrolyzed extract is prepared by the method included the following steps:
Eupolyphoge sinensis powder is soluble in water, it is added papain, the pH value of regulation system to 6.0-7.0, enzyme digestion reaction, from
The heart collects supernatant.
In the above method, the material-water ratio of eupolyphoge sinensis powder and water is 1g:15mL-1g:25mL, concretely 1g:15mL.
The water concretely distilled water.
The proportion of eupolyphoge sinensis powder and papain is 1g:500U-1g:700U, concretely 1g:500U.
The reaction temperature of the enzyme digestion reaction is 55 DEG C, time 3-5h, specifically can be with 4h.
The condition of the centrifugation are as follows: be centrifuged 15min under 10000r/min.
The above method further includes being placed in the enzymatic hydrolysis system in 100 DEG C of boiling water before being centrifuged to enzymatic hydrolysis system
10min is with the operation of enzymolysis reaction.
Pharmaceutical preparation comprising above-mentioned eupolyphoge sinensis enzymatic hydrolyzed extract also belongs to protection scope of the present invention.
Said medicine preparation can be made into various forms of oral preparations, comprising: capsule, tablet, granule, powder, ball
Agent, pill, sustained-release preparation, oral solution, mixture and syrup.The drug of above-mentioned various dosage forms can be according to pharmaceutical field
Conventional method preparation.
The present invention uses mouse CCl4It causes liver injury model to carry out zoopery, is damaged by spectrophotometry Acute Hepatic
Hurt AST, ALT vigor in mice serum, compared with model group, eupolyphoge sinensis enzymatic hydrolyzed extract group serum alt, AST vigor are extremely aobvious
Writing reduces, the extremely significant raising of CAT vigor in eupolyphoge sinensis enzymatic hydrolyzed extract group murine liver tissue, and middle high dose group SOD, GSH-Px is living
Power significantly increases, and the content of low high dose group MDA is substantially reduced, in addition, in eupolyphoge sinensis enzymatic hydrolyzed extract group murine liver tissue
TNF-α, IL-6 and iNOS content and gene relative expression quantity significantly reduce.To sum up illustrate, eupolyphoge sinensis enzymatic hydrolyzed extract can be significant
Lower CCl4Damage to mouse liver cell enhances the oxidation resistance of acute hepatic injury mice, is likely to become a kind of very excellent
The drug candidate of elegant prevention and/or treatment carbon tetrachloride hepatic injury.
Detailed description of the invention
Fig. 1 shows influence of the eupolyphoge sinensis enzymatic hydrolyzed extract to acute hepatic injury mice level of fibrosis.
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, biomaterial etc., are commercially available unless otherwise specified.
1 materials and methods
1.1 test material
Carbon tetrachloride (CCl4) it is purchased from Beijing Chemical Plant, Silymarin Capsule is purchased from Madaus AG, alanine
Aminopherase (ALT), aspartate amino transferase (AST), superoxide dismutase (SOD), glutathione peroxidating
Object enzyme (GSH-Px), catalase (CAT), malonaldehyde (MDA), total protein quantification kit build up biotechnology purchased from Nanjing
Co., Ltd, TUREscript 1st Strand cDNA Synthesis Kit are purchased from the limited public affairs of Beijing Ai Delai biotechnology
Department,Premix Ex TaqTM (Tli RNaseH Plus) (RR420A) is purchased from TaKaRa company, Japan, used to draw
Object is synthesized by Hua Da scientific & technical corporation, and eupolyphoge sinensis enzymatic hydrolyzed extract is prepared by this laboratory.
Healthy kunming mice, male, weight 20-22g are purchased from Chinese military medicine academy of sciences Experimental Animal Center [SCXK-
(army) 2012-0004].
1.2 method
1.2.1 the extraction of eupolyphoge sinensis zymolyte
It takes eupolyphoge sinensis powder to be dissolved in distilled water in the ratio that material-water ratio is 1g:15mL, is added according to the material enzyme ratio (W/U) of 1g:500U
Enter papain, adjusting pH is 6.0, and above-mentioned solution is placed in 55 DEG C of water-baths after water-bath 4h and is placed in 100 DEG C of boiling water again
10min is with enzymolysis reaction, and then 10000r/min is centrifuged 15min, draws supernatant and obtains eupolyphoge sinensis enzymolysis liquid.
1.2.2 test grouping
60 healthy mices are taken to be randomly divided into I group (solvent group), II group (model group), III group of (silymarin 100mg/
Kg), IV~VI group (eupolyphoge sinensis enzymatic hydrolyzed extract 50,100,200mg/kg), every group 10.Solvent group and model group gavage life daily
Salt water is managed, positive controls gavage silymarin, and eupolyphoge sinensis enzymatic hydrolyzed extract group gavages various dose zymolyte, once a day, even
After continuous 7d, last dose 2h, in addition to solvent group (olive oil), 0.1%CCl is injected intraperitoneally in model group and each administration group4Olive
Oil solution 10ml/kg, is deprived of food but not water, and 18h posterior orbit blood sampling, centrifugation prepares serum, separates hepatic tissue, and mouse left lobe of liver is taken to make
HE dyeing carries out histological examination, and it is spare that the homogenate of 10% tissue is made in rest part.
1.2.3 liver function index detects
Illustrated according to ALT, AST kit, detects serum alt, AST content.
1.2.4 the measurement of oxidation resistance
Illustrated according to SOD, CAT, GSH-Px and MDA kit, detects SOD, CAT, GSH-Px vigor and MDA in hepatic tissue
Content.
1.2.5 TNF-α, IL-6 and iNOS content and the measurement of mRNA relative expression quantity in liver
Illustrated according to TNF-α, IL-6 and iNOS kit, detects TNF-α in liver, IL-6 content and iNOS activity.
Trizol method extracts total serum IgE in hepatic tissue, RNA Oligo (dT) 18Primer reverse transcription is synthesized the first chain of cDNA, with cDNA
PCR amplification is carried out for template.Finally PCR number is analyzed using with the mating Step OneSoftware v2.1 of fluorescence quantitative PCR instrument
According to.
1.2.6 hepatic tissue section is observed
After 4 DEG C of normal saline flushings of murine liver tissue, filter paper is wiped dry, and it is molten to be soaked in 4% paraformaldehyde of volume fraction
Fixed in liquid, the pathology of paraffin embedding, conventional section, hematoxylin eosin staining, optical microphotograph microscopic observation hepatic tissue section becomes
Change.
1.3 statistical analysis
Test data is indicated with mean+SD, carries out single factor test variance using Graphpad Prism6.0 software
It analyzes (one-way ANOVA), Multiple range test between LSD each group is carried out when significant difference, P < 0.05 is significant difference.2 results
Influence of the 2.1 eupolyphoge sinensis enzymatic hydrolyzed extracts to acute hepatic injury mice liver function
Transaminase is to reflect an important indicator of liver function, passes through spectrophotometry acute hepatic injury mice serum
Middle AST, ALT vigor, the results are shown in Table 1, and compared with solvent group, ALT, AST content in model group mice serum are significantly risen
High (P < 0.01), illustrates CCl4Cause mouse liver injury modeling success.Compared with model group, positive controls and eupolyphoge sinensis enzymolysis and extraction
The extremely significant reduction (P < 0.01) of object group serum alt, AST vigor.
Influence of the 1 eupolyphoge sinensis enzymatic hydrolyzed extract of table to acute hepatic injury mice liver function
Note: compared with solvent group, P < 0.01 * *；Compared with model group,++P<0.01。
Influence of the 2.2 eupolyphoge sinensis enzymatic hydrolyzed extracts to acute hepatic injury mice oxidation resistance
As shown in Table 2, it is compared with solvent group, SOD, CAT, GSH-Px vigor in liver injury model group murine liver tissue are equal
It significantly reduces (P < 0.01 or P < 0.05), and MDA content significantly increases (P < 0.01).Compared with model group, eupolyphoge sinensis enzymolysis and extraction
The extremely significant raising (P < 0.01) of CAT vigor in object group murine liver tissue, middle high dose group SOD, GSH-Px vigor significantly increase
(P < 0.01 or P < 0.05), and the content of low high dose group MDA is substantially reduced (P < 0.01 or P < 0.05).
Influence of the 2 eupolyphoge sinensis enzymatic hydrolyzed extract of table to acute hepatic injury mice liver oxidation resistance
Note: compared with solvent group, P < 0.05 * * P < 0.01, *；Compared with model group,++P < 0.01,+P<0.05。
2.3 eupolyphoge sinensis enzymatic hydrolyzed extracts are to TNF-α, IL-6 and iNOS content and mRNA phase in acute hepatic injury mice hepatic tissue
Influence to expression quantity
As shown in Table 3, it is compared with solvent group, TNF-α, IL-6 and iNOS content are bright in liver injury model group murine liver tissue
It is aobvious to increase (P < 0.05 or P < 0.01).It is compared with model group, in positive controls and eupolyphoge sinensis enzymatic hydrolyzed extract group murine liver tissue
The extremely significant reduction (P < 0.01) of TNF-α, IL-6 and iNOS content.
By fluorescence quantitative PCR detection murine liver tissue TNF-α, IL-6 and iNOS mRNA relative expression quantity, as a result such as table
It shown in 4, is compared with solvent group, TNF-α, IL-6 and iNOS mRNA relative expression quantity are bright in liver injury model group murine liver tissue
It is aobvious to increase (P < 0.01).It is compared with model group, TNF-α, IL- in positive controls and eupolyphoge sinensis enzymatic hydrolyzed extract group hepatic tissue
6mRNA relative expression quantity significantly reduces (P < 0.01), iNOS mRNA relative expression quantity decrease to some degree, but difference is not
Significantly (P > 0.05).
Influence of the 3 eupolyphoge sinensis enzymatic hydrolyzed extract of table to TNF-α, IL-6 and iNOS content in acute hepatic injury mice hepatic tissue
Note: compared with solvent group, P < 0.05 * * P < 0.01, *；Compared with model group,++P<0.01。
4 eupolyphoge sinensis enzymatic hydrolyzed extract of table is to TNF-α, IL-6 and iNOS mRNA in acute hepatic injury mice hepatic tissue with respect to table
Up to the influence of amount
Note: compared with solvent group, P < 0.01 * *；Compared with model group,++P<0.01。
Influence of the 2.4 eupolyphoge sinensis enzymatic hydrolyzed extracts to acute hepatic injury mice blood lipid
Fig. 1 shows influence of the eupolyphoge sinensis enzymatic hydrolyzed extract to acute hepatic injury mice level of fibrosis.
As shown in Figure 1, solvent group mouse lobuli hepatis structure is normal, liver Cable Structure is clear and legible, and it is bright to have no that liver cell occurs
The pathological changes such as aobvious denaturation, necrosis.Model group mouse lobuli hepatis loses normal configuration, and liver cell volume increases, and balloon sample is presented
Lesion.Compared with model group, positive controls and eupolyphoge sinensis enzymatic hydrolyzed extract group mouse lobuli hepatis structure are obviously improved, CCl4Induction
Phenomena such as liver cell oedema for generating in course of liver damage, necrosis has weakens to some extent, and normal liver cell number increases.
Claims (5)
1. application of the eupolyphoge sinensis enzymatic hydrolyzed extract in preparation prevention carbon tetrachloride hepatic injury drug；Wherein, the eupolyphoge sinensis enzymatic hydrolysis mentions
Taking object is prepared by the method included the following steps:
Eupolyphoge sinensis powder is soluble in water, papain is added, to 6.0-7.0, enzyme digestion reaction is centrifuged the pH value of regulation system, is received
Collect supernatant.
2. application according to claim 1, it is characterised in that: in the method, the material-water ratio of eupolyphoge sinensis powder and water is 1g:
15mL-1g:25mL；
The proportion of eupolyphoge sinensis powder and papain is 1g:500U-1g:700U；
The reaction temperature of the enzyme digestion reaction is 55 DEG C, time 3-5h；
The condition of the centrifugation are as follows: be centrifuged 15min under 10000r/min；
The method further includes that the enzymatic hydrolysis system is placed in 10min in 100 DEG C of boiling water before being centrifuged to enzymatic hydrolysis system
With the operation of enzymolysis reaction.
3. application of the eupolyphoge sinensis enzymatic hydrolyzed extract in preparation treatment carbon tetrachloride hepatic injury drug；Wherein, the eupolyphoge sinensis enzymatic hydrolysis mentions
Taking object is prepared by the method included the following steps:
Eupolyphoge sinensis powder is soluble in water, papain is added, to 6.0-7.0, enzyme digestion reaction is centrifuged the pH value of regulation system, is received
Collect supernatant.
4. a kind of drug for preventing carbon tetrachloride hepatic injury, active constituent is eupolyphoge sinensis enzymatic hydrolyzed extract；Wherein, the eupolyphoge sinensis enzyme
Solving extract is prepared by the method included the following steps:
Eupolyphoge sinensis powder is soluble in water, papain is added, to 6.0-7.0, enzyme digestion reaction is centrifuged the pH value of regulation system, is received
Collect supernatant.
5. a kind of drug for treating carbon tetrachloride hepatic injury, active constituent is eupolyphoge sinensis enzymatic hydrolyzed extract；Wherein, the eupolyphoge sinensis enzyme
Solving extract is prepared by the method included the following steps:
Eupolyphoge sinensis powder is soluble in water, papain is added, to 6.0-7.0, enzyme digestion reaction is centrifuged the pH value of regulation system, is received
Collect supernatant.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101596230A (en) * | 2009-07-14 | 2009-12-09 | 广西中医学院附属瑞康医院 | A kind of pharmaceutical composition for the treatment of hepatopathy |
| CN104398971A (en) * | 2014-11-04 | 2015-03-11 | 王松华 | Traditional Chinese medicine composition for treating liver cirrhosis, and preparation method and application thereof |
-
2016
- 2016-10-20 CN CN201610916770.XA patent/CN106309502B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101596230A (en) * | 2009-07-14 | 2009-12-09 | 广西中医学院附属瑞康医院 | A kind of pharmaceutical composition for the treatment of hepatopathy |
| CN104398971A (en) * | 2014-11-04 | 2015-03-11 | 王松华 | Traditional Chinese medicine composition for treating liver cirrhosis, and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 土鳖四逆散对肝纤维化大鼠TGF-β_1和α-SMA表达及肝损伤的影响;方承康等;《辽宁中医药大学学报》;20071130;第9卷(第06期);176-178 |
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