CN108338987A - The purposes of sea urchin yellow polysaccharide anti-hepatitis B virus - Google Patents
The purposes of sea urchin yellow polysaccharide anti-hepatitis B virus Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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Abstract
The invention discloses the application in sea urchin yellow polysaccharide on a cellular level anti-hepatitis B virus, the concrete mode of application is：By sea urchin yellow polysaccharide for the inhibition to HBV DNA replication dnas outside the expression of HepG2.2.15 cells HBsAg, HBeAg, the transcription of pgRNA and total RNA, the expression of core albumen and intracellular.The present invention provides a kind of new applications of sea urchin yellow polysaccharide, and sea urchin yellow polysaccharide is used for anti-hepatitis B virus, and a kind of new mode is provided for treatment hepatitis type B virus.
Description
Technical field
The present invention relates to a kind of new applications of sea urchin yellow polysaccharide, and in particular to sea urchin yellow polysaccharide resists B-mode on a cellular level
The effect of hepatitis virus belongs to the application field of natural active matter.
Background technology
Hepatitis type B virus (HBV) infection is the main of oxyhepatitis, chronic hepatitis, liver fibrosis, hepatic sclerosis and liver cancer
Inducement, the life and health of serious prestige victimization class.The whole world about 2,500,000,000 people's hepatitis b virus infections at present, there is about 400,000,000
People gradually develops into chronic hepatitis B.Hepatitis type B virus can be divided into 8 different genotype, i.e. A, B, C, D, E, F, G, H.
The variability of HBV height determines the presence of numerous gene hypotypes with diversity, between gene hypotype sequence difference at least more than
4%.Hepatitis type B virus can cause very serious complication and death, prevent it and control expends a large amount of society
Resource.
China is the district occurred frequently of hepatitis B virus infection, according to Ministry of Public Health's data announced in 2013, national population in 2006
Seroepidemiological survey shows that the hepatitis B infected prevalence rate in China is that 34.28%, HBsAg prevalence rates are 7.18%, calculates China
The people for infecting hepatitis B at present has nearly 500,000,000, HBsAg carrier, 93,000,000 people.From 1992, since China implements newly
The policy of raw youngster's free vaccination vaccine, 5 years old or less children's carrying rate have dropped down to the more original 10- of 0.96%, HBsAg prevalence rates
15% decreased significantly and become Medium endemic area.For chronic HBV infection, still lack the curative medicine for thoroughly removing virus
Object, it is necessary to reduce serum HBV copy number dependent on nucleoside analog and interferons drug for a long time.Such as due to nucleoside analog
There is after drug withdrawal the problems such as rebound, medicament-resistant mutation, crossing drug resistant for Lamivudine, lamivudine etc.；And the recombination of high dose is dry
Disturbing element also has the limitations such as persistently response rate is low, side effect is big；Therefore, relevant disease still lacks caused by treating HBV infection
Highly effective drug and means.
Scientists from all over the world are studied extensively by the polysaccharide to separate sources, it is found that it is antitumor, cardiovascular polysaccharide has
The effect of disease treatment, immunological regulation, antiviral etc..Polysaccharide is widely studied as drug the 1950s, but
So far it has been found that there is hundreds of polysaccharide bioactivity, research to find that a variety of polysaccharide have the activity of anti-hepatitis virus, packet
Include animal, plant and microbial polysaccharide.Sea urchin yellow polysaccharide (SEP) is the polysaccharide isolated and purified from Strongylocentrotus nudus,
Its structure is identified using physicochemical analysis and GC analyses, it was demonstrated that for a kind of glucan being separated to from sea urchin Huang.
Pharmacological experiment study shows that sea urchin yellow polysaccharide not only has immunoregulatory activity, also has natural antitumor activity.It grinds before
Study carefully and find that sea urchin yellow polysaccharide plays antitumor activity by immune regulation mechanism in vivo and in vitro, but for sea urchin yellow polysaccharide Anti-HBV activity
Activity have not been reported, so we have studied the effects of sea urchin yellow polysaccharide Anti-HBV activity once.
Invention content
For the technical deficiencies of existing treatment HBV, the present invention provides the new applications of sea urchin yellow polysaccharide, utilize sea urchin Huang
The effect of polysaccharide Anti-HBV activity on a cellular level, to provide a kind of substance of new Anti-HBV activity.
In view of the existing deficiency in HBV treatment technologies, the present invention provides a kind of new technical solutions, by sea urchin Huang
Polysaccharide is on a cellular level as the drug of Anti-HBV activity.
Specific implementation mode is to utilize the secretion of sea urchin yellow polysaccharide inhibition HBsAg, HBeAg and the transcription to HBV RNA
Inhibit the inhibition of DNA replication with HBV DNA.Its main contents detects sea urchin Huang to HepG2.2.15's including the use of mitochondria mtt assay
Cytotoxicity finds that it is smaller to the cytotoxicity of HepG2.2.15 under the concentration of 800 μ g/ml.Use enzyme linked immunosorbent assay
Influence of the sea urchin yellow polysaccharide to HBsAg and HBeAg is detected, it is found that it in 400 μ g/ml couple, two kinds of antigens there is apparent inhibition to make
With there are the dependency relationships of dosage and time.With fluorescence quantitative PCR detection sea urchin yellow polysaccharide to the shadow of HBV RNA and DNA
Ring, find its 400 μ g/ml to HBV RNA and DNA expression there are the inhibiting effect of conspicuousness, determine it in virus replication
The transcription and replication of HBV is inhibited in level.
Inventor has detected cytotoxicity of the sea urchin yellow polysaccharide to HepG2.2.15 cells under various concentration, finds its tool
There is smaller cytotoxicity.Pharmacological tests show, sea urchin yellow polysaccharide by induce the expression of antiviral protein IFN-β to
Play its suppression to the secretion of HBsAg and HBeAg in the transcription of pgRNA and total RNA, supernatant and the duplication of HBV DNA
It makes and uses, to influence the synthesis of HBV.The nucleic acid drug clinically used is due to existing drug resistance risk, but polysaccharide
Class drug not will produce drug resistance generally.Therefore, Anti-HBV drugs of the sea urchin yellow polysaccharide as effective low toxicity, it is contemplated that clinically
Treatment for resistance of hepatitis B.
Description of the drawings
The inhibiting effect that Fig. 1 sea urchin yellow polysaccharide secretes HBeAg
The inhibiting effect that Fig. 2 sea urchin yellow polysaccharide secretes HBsAg
Inhibiting effect of Fig. 3 sea urchin yellow polysaccharide to HBV pgRNA
Inhibiting effect of Fig. 4 sea urchin yellow polysaccharide to HBV total RNA
Inhibiting effect of Fig. 5 sea urchin yellow polysaccharide to born of the same parents Inner HBV DNA
Inhibiting effect of Fig. 6 sea urchin yellow polysaccharide to the HBV DNA secreted in HepG2.2.15 cell conditioned medium culture mediums
Inhibiting effect of Fig. 7 sea urchin yellow polysaccharide to born of the same parents' Inner HBV core albumen
Fig. 8 sea urchin yellow polysaccharide influences the up-regulation of IFN-β expression quantity in supernatant
Here is the specific implementation case of sea urchin yellow polysaccharide Anti-HBV activity of the present invention：
Embodiment 1
HepG2.2.15 cell culture
10% fetal calf serum of HepG2.2.15 cell lines, 100 μ g/ml, G 418 of penicillin 100IU/ml and streptomysin
The DMEM medium cultures of 250 μ g/ml, 37 DEG C, 5%CO2Under conditions of cultivate.
Embodiment 2
Sea urchin yellow polysaccharide is to HepG2.215 cytotoxicities
HepG2.2.15 cells press 1 × 103A/hole concentration is inoculated into 96 orifice plates, sets 37 DEG C, 5%CO2It is trained in incubator
It supports overnight.After cell is adherent, cell is washed 2 times with PBS, is separately added into containing various concentration (800,400,200,100 μ g/ml)
4 multiple holes are arranged in 100 μ L cell maintenance mediums of sea urchin yellow polysaccharide, experimental setup normal cell controls group, each concentration.
HepG2.215 cells are cultivated 9 days in 37 DEG C, are renewed fresh administration culture medium every three days, are observed cell state daily.Finally use
Mtt assay measures cell viability, and MTT 15 μ L, avoid light place 4h are added per hole and sucks supernatant, and DMSO150 μ l cracking is added per hole
Cell detects its absorbance at 490nm.Experiment is repeated 3 times altogether, calculates cytotoxicity.Cell survival rate %=(ODDosing group-
ODNormal cell controls group)/ODNormal cell controls group× 100%
Table one the experimental results showed that：Sea urchin yellow polysaccharide is under 1600 μ g/ml concentration to HepG2.2.15 cell acellular poisons
Property, but its CC50More than 3200 μ g/ml.
Table one：Sea urchin yellow polysaccharide is to HepG2.215 cytotoxicities
Embodiment 3
The inhibiting effect that sea urchin yellow polysaccharide secretes HBV antigens
HepG2.2.15 cells press 5 × 104A/hole concentration is inoculated into 24 orifice plates, sets 37 DEG C, 5%CO2It is trained in incubator
It supports overnight.DMEM culture medium maintaining liquids containing various concentration sea urchin yellow polysaccharide are added in HepG2.2.15 cells so that hole
Middle final concentration of 0,100,200,400, the 800 μ g/ml of drug.Experiment is additionally provided with the normal cell controls group and the positive of not dosing
Medicine lamivudine group.Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.After 72h, collects supernatant and managed in 1.5ml EP
It is interior, it is placed in -20 DEG C of refrigerators and preserves.The cell maintenance medium of the new concentration containing different pharmaceutical is added per hole, co-cultures 9 days.Use ELISA
Kit detects influence of the sea urchin yellow polysaccharide to processing 6 days, the secretion of 9 days HBV antigens.
Table 2 and table 3 to HBsAg and HBeAg the experimental results showed that all there is inhibiting effect in sea urchin yellow polysaccharide, and with agent
The increase inhibiting rate of amount also increases；With the extension of drug treating time, inhibiting rate also increases.Illustrate sea urchin yellow polysaccharide pair
There are dosage and Time Dependent sexual intercourse for the inhibiting effect of the secretion of HBsAg and HBeAg.
Table two：Inhibiting effect of the sea urchin yellow polysaccharide to HBsAg
Table three：Inhibiting effect of the sea urchin yellow polysaccharide to HBeAg
With cell controls group ratio, P ＜ 0.05 and P ＞ 0.01 are expressed as " * ", P ＜ 0.01 are expressed as " * * "
Example IV
Inhibiting effect of the sea urchin yellow polysaccharide to HBV RNA
HepG2.2.15 cells press 5 × 104A/hole concentration is inoculated into 24 orifice plates, sets 37 DEG C, 5%CO2It is trained in incubator
It supports overnight.The DMEM culture medium maintaining liquids of the sea urchin yellow polysaccharide containing various concentration are added in HepG2.215 cells so that hole Chinese medicine
Final concentration of 0,100,200,400,800 μM of object.Experiment is additionally provided with the normal cell controls group and positive drug rummy of not dosing
Husband determines group.Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Primary fresh pastille culture medium is changed within every 3 days, it is sharp after 6 days
With Trizol methods extraction born of the same parents Inner RNA.Detecting sea urchin yellow polysaccharide influences the transcription of born of the same parents' Inner HBV correlations RNA.
Fig. 3 is the experimental results showed that sea urchin yellow polysaccharide can significantly affect the transcription of HBV, and sea urchin yellow polysaccharide is to HBV's
The inhibition of total serum IgE is preferable, is respectively 35.51%, 50.64%, Fig. 4 experimental results to the inhibiting rate of HBV at 400,800 μM
Show influence of the sea urchin yellow polysaccharide to HBV pgRNA, it is found that it is to the inhibiting rate of HBV pgRNA at 400,800 μM
22.74%, 52.27%.To sum up the result shows that sea urchin yellow polysaccharide is all up to 50% in 800 μ g/ml to the inhibiting rate of HBVRNA, say
Bright its can inhibit the transcription of HBV correlations RNA with conspicuousness, and there are dose-dependent relationships.
Embodiment 4
Inhibiting effect of the sea urchin yellow polysaccharide to HBV correlations DNA
HepG2.2.15 cells press 1 × 105A/hole concentration is inoculated into 6 orifice plates, sets 37 DEG C, 5%CO2It is cultivated in incubator
Overnight.Various concentration sea urchin yellow polysaccharide administration culture medium maintaining liquid will be contained to be added in HepG2.2.15 cells so that hole Chinese medicine
Final concentration of 0,100,200,400, the 800 μ g/ml of object.Experiment sets normal cell controls group and the positive drug rummy husband of not dosing
Determine group (2.45 μ g/ml).Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Change within every 3 days primary fresh drug containing culture
Base collects cell conditioned medium and collects born of the same parents Inner DNA using cell pyrolysis liquid after 6 days.Sea urchin yellow polysaccharide is detected to born of the same parents Inner HBV DNA's
It influences.DNA in supernatant illustrates the copy number of extraction and application qPCR instrument detection HBV DNA according to DAN extracts kits.
There are inhibiting effect the experimental results showed that HBV is to born of the same parents Inner HBV DNA by Fig. 5.It is in 100,200,400,800 μ g/ml
Inhibiting rate to born of the same parents Inner HBV DNA is respectively 40.5%, 36.17%, 54%, 82.87%.Show sea urchin yellow polysaccharide to born of the same parents Inner
There are dose-dependent relationships for the inhibiting rate of HBV DNA.And Fig. 6 the experimental results showed that sea urchin yellow polysaccharide to supernatant HBV DNA's
There are inhibition relationships for secretion.In HepG2.2.15 cells, sea urchin yellow polysaccharide is in 100,200,400,800 μ g/ml to HBV DNA
Inhibiting rate be 33.08%, 61.28%, 67.33%, 74.21%.The above result shows that sea urchin yellow polysaccharide is to HBV in supernatant
There are dose-dependent relationships for the inhibition of DNA.To sum up the result shows that inhibition of the sea urchin yellow polysaccharide to the HBV DNA outside HBV born of the same parents Inner
All there is dose-dependent relationship in effect.
Embodiment 5
Inhibiting effect of the sea urchin yellow polysaccharide to born of the same parents' Inner HBV core albumen
HepG2.2.15 cells press 1 × 105A/hole concentration is inoculated into 6 orifice plates, sets 37 DEG C, 5%CO2It is cultivated in incubator
Overnight.Various concentration sea urchin yellow polysaccharide administration culture medium maintaining liquid will be contained to be added in HepG2.2.15 cells so that hole Chinese medicine
Final concentration of 0,100,200,400, the 800 μ g/ml of object.Experiment is additionally provided with the normal cell controls group of not dosing and positive drug is drawn
Meter Fu Ding groups (2.45 μ g/ml).Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Change within every 3 days primary fresh drug containing training
Base is supported, cell is collected after 6 days, born of the same parents' Inner albumen is collected using cell pyrolysis liquid, albumen concentration is detected using BCA methods.Utilize SDS-
The influence of gel electrophoresis and Westonblot detection sea urchin yellow polysaccharide to HBV core albumen.
Finding expression of the sea urchin yellow polysaccharide to HBV core albumen by gray analysis, there are inhibition relationships.Fig. 5 is indicated
400,800 μ g/ml are to the expression quantity of its albumen there are the inhibition relationship of conspicuousness, inhibiting rate is respectively 28.85%,
41.26%.
Embodiment 6
In 24 orifice plate of influence of the sea urchin yellow polysaccharide to intracellular IFN-β, 37 DEG C, 5%CO are set2Overnight incubation in incubator.It will
It is added in HepG2.215 cells containing various concentration sea urchin yellow polysaccharide administration culture medium maintaining liquid so that drug final concentration in hole
For 0,100,200,400,800 μ g/ml.Cell is placed in 37 DEG C, 5%CO2It is cultivated in incubator.Cell conditioned medium is collected after 16h,
The expression of IFN-β in supernatant is detected using ELISA.
The experimental results showed that the expression of broad-spectrum antiviral protein I FN- β can be obviously raised after administration 16h, and there are dosage
Dependency relationships, in 800 μ g/ml to the expression quantity of IFN-β from 3.5ng/ml it is notable on be transferred to 16.4ng/ml, to play
The effect of its Anti-HBV activity.
Claims (3)
1. sea urchin yellow polysaccharide is preparing the application in treating hepatitis b virus infected disease product.
2. the application described in claim 1, it is characterised in that：Sea urchin yellow polysaccharide (200~800 μ g/ml) is in virus replication water
The flat upper synthesis for inhibiting HBV DNA；Sea urchin yellow polysaccharide (400~800 μ g/ml) inhibits HBsAg and HBeAg secretions；Sea urchin is yellow more
The inhibition of sugared (400~800 μ g/ml) to HBV pgRNA and HBV total RNA；Sea urchin yellow polysaccharide (400~800 μ g/ml) presses down
HBV core protein expressions processed.
3. new application of the sea urchin yellow polysaccharide described in claim 1 in preparing anti-hepatitis B virus infective medicament.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507430A (en) * | 2018-09-06 | 2019-03-22 | 重庆医科大学 | The function and purposes of SIRT7 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103479663A (en) * | 2012-06-11 | 2014-01-01 | 上海市公共卫生临床中心 | Application of yeast-origin glucan on preparation of drugs for curing hepatitis B virus infection |
| CN104497162A (en) * | 2015-01-05 | 2015-04-08 | 中国药科大学 | Urchin yellow polysaccharide with liver protecting function and application thereof |
-
2018
- 2018-02-06 CN CN201810131663.5A patent/CN108338987A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103479663A (en) * | 2012-06-11 | 2014-01-01 | 上海市公共卫生临床中心 | Application of yeast-origin glucan on preparation of drugs for curing hepatitis B virus infection |
| CN104497162A (en) * | 2015-01-05 | 2015-04-08 | 中国药科大学 | Urchin yellow polysaccharide with liver protecting function and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 郑建仙主编: "《功能性食品（第二卷）》", 31 January 2002 * |
| 高松主编: "《辽宁中药志》", 31 July 2015 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109507430A (en) * | 2018-09-06 | 2019-03-22 | 重庆医科大学 | The function and purposes of SIRT7 |
| CN109507430B (en) * | 2018-09-06 | 2022-03-11 | 重庆医科大学 | Function and application of SIRT7 |
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