CN100467030C - Trachyostracous mussel extract and its prepn and use - Google Patents
Trachyostracous mussel extract and its prepn and use Download PDFInfo
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- CN100467030C CN100467030C CNB2005101100968A CN200510110096A CN100467030C CN 100467030 C CN100467030 C CN 100467030C CN B2005101100968 A CNB2005101100968 A CN B2005101100968A CN 200510110096 A CN200510110096 A CN 200510110096A CN 100467030 C CN100467030 C CN 100467030C
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Abstract
The present invention relates to medicine preparation with animal material, and is especially trachyostracous mussel extract and its preparation and use. The trachyostracous mussel extract features its total nitrogen content of 12-16 mg/ml, protein concentration of 15-20 mg/ml, amino nitrogen content of 8.5-15 mg/ml and pH 5.0-6.0. The preparation process includes washing trachyostracous mussel, eliminating shell, homogenizing meat to form homogeneous slurry, hydrolysis, eliminating acid and impurity with weak alkaline resin, concentration and other steps. The trachyostracous mussel extract of the present invention may be used in preparing medicines for resisting influenza, resisting SARS and regulating immunity.
Description
Technical field
The invention belongs to the medicinal preparation field of containing from the extract of the product of the raw material of the animal outside mammal or the birds or itself and not clear structure, specifically belong to extract, manufacture method and the application in pharmacy thereof of Mytilus crassitesta Lischke.
Background technology
The ocean is a huge drug resources.So far, the new compound that obtains from marine animal, plant kind more than 1000 wherein has pharmacologically actives such as antitumor, antibiotic, antiviral more than 1/2.The research of marine drug has become the modern biomedical research tendency at present.Mussel is compared with other shellfish, and output height and price are low.Mussel, different name: mussel, Fructus mail micromali, red clam etc., latin name: Mytilus edulis; English name: mussel.Principal item has: 1. Mytilus edulis (Mytilus galloprovincilis Linaeus) moves on the cay in offshore, inner bay shallow sea sexual maturity in a year, Shandong, the littoral output maximum in Liaoning; 2. Mytilus crassitesta Lischke (Mytiluscorusis Lischke), shell is longer and thick than Mytilus edulis, lives on the openr marine rock, and Zhejiang one band is many in 8-10 rice depths, and area, Zhoushan, Zhejiang output is the highest; 3. Perna uiridis (Linnaeus) (Perna viridis Linnaeus) moves in and does damp line to the rock at the unobstructed place of depth of water 5-6 rice current, is distributed in China East Sea and the South Sea, and is the highest with Hainan Island output.
Mussel nature and flavor temperature is salty, and is nontoxic, goes into liver, kidney channel.Have invigorating the liver and kidney, benefiting essence-blood, the goiter that disappears, the function of dysentery relieving just has medicinal record from ancient times.As supplement to the Herbal record mussel " main void is won strain, and is thin and weak because of producing, and the vim and vigour knot is long-pending, and abdomen is cold, borborygmus, dysentery pain in the lumbar region, diseases such as leukorrhagia "." Japan hanako materia medica " record " boil food, can tonifying five ZANG-organs, Yiyang thing, reason waist beriberi, the dyspepsia that disappears is except that cold air in the abdomen, mass beside the umbilicus etc. ".Mussel also can be treated rheumatism, dizziness and night sweat, and the effect of moistening the lung and resolving phlegm is arranged.
Influenza is called for short " influenza ", is the acute respiratory infectious disease with hyperinfection that is caused by influenza virus, also is the human worldwide public health problem that still can not effectively control so far.Influenza virus is a RNA viruses, and infectiousness is strong, propagates rapidly.Influenza virus also there is not specific drug at present.Mainly be symptomatic treatment, comprise antipyretic analgesic and prevent and treat the secondary bacterial infection.Tackle that influenza is most economical, effective method is to take positive preventive measure.But the influenza virus variation is fast, and the protection domain of vaccine is little, and the time is short, and all need inoculate every year.Therefore, exploitation can prevent and treat the influenza virus new drug becomes the present task of top priority.PCT patent documentation report (WO81/03124, the open date: on November 12nd, 1981), the polypeptides matter that extraction separation obtains from mussel (blue mussel, Perna uiridis (Linnaeus)) has biological activitys such as broad-spectrum antiviral, bacterial-infection resisting, promotion wound healing.Russ P (RU2043109, the open date: nineteen ninety-five JIUYUE 10 days and RU2165264, the open date: April 20 calendar year 2001) disclose Mytilidae (Mytilidae family) animal extracts and had the antiviral function, used as food additive.
China is to the development and use of mussel, and added value is also lower, has only dried food and nuts that mussel makes, canned food, flavouring agent, health food etc. in the market.Domestic relevant bibliographical information, a kind of saccharide of producing from mussel production and processing residue and protein complex are called Mi Jilan-novel immunomodulator (health food).Do not have toxicity and nonirritant and anaphylaxis effect, have significant antiinflammatory and necrosis effect.At present, still there is not the report that mussel class material is used for new drug both at home and abroad.
Summary of the invention
The inventor has contrasted four kinds of different mussel raw materials through broad research, has studied tens kinds of hydrolysis and process for extracting, extract of the present invention has been carried out widely the test of pesticide effectiveness in the active determination in vitro and animal body, thereby finished the present invention.
Therefore, one of technical problem to be solved by this invention provides the extract of Mytilus crassitesta Lischke, can be used to prepare medicine.
Two of technical problem to be solved by this invention provides the manufacture method of Trachyostracous mussel extract.
The technical problem that the present invention also will solve provides the medical usage of Trachyostracous mussel extract.
The invention discloses the extract of Mytilus crassitesta Lischke, it is characterized in that: total nitrogen 12-16mg/mL, protein concentration 15-20mg/mL, amino nitrogen content 8.5-15mg/mL, its pH5.0-6.0.
The extract of Mytilus crassitesta Lischke preferably, total nitrogen 12.5-14mg/mL, protein concentration 15-18mg/mL, amino nitrogen content 10-13mg/mL, its pH5.3-5.8.
The extract of Mytilus crassitesta Lischke more preferably, comprise aspartic acid, serine, threonine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, tyrosine, phenylalanine, lysine, histidine, arginine, total amino acid content is the 11-14g/100mL extract.
Total nitrogen is measured with Kjeldahl commonly used, protein concentration is measured with Coomassie brilliant blue method commonly used, amino nitrogen adopts commercial automatic amino acid analyzer and operates or use OPA method or PTC-AA method or ninhydrin method always according to this area and measure according to the method that producer provides with titration measuring commonly used, the mensuration of amino acid content.
The preparation method of extract of Mytilus crassitesta Lischke comprises the steps:
(a) Mytilus crassitesta Lischke cleans, and shells;
(b) Mytilus crassitesta Lischke meat homogenate;
(c) hydrolysis;
(d) with weakly base resin deacidification, roguing;
(e) solution after the roguing is concentrated.
Preferably, also comprise the step that concentrated solution is sterilized.
The mussel raw material that preparation method of extract adopted of Mytilus crassitesta Lischke of the present invention is the Mytilus crassitesta Lischke or the refrigerated Mytilus crassitesta Lischke of fresh collection.
The preparation method of extract of Mytilus crassitesta Lischke of the present invention, wherein the condition of hydrolysing step is: the homogenate pH of Mytilus crassitesta Lischke transfers to 6.0-7.5, adds the Flavourzyme enzyme of 0.1%-1.0% (percentage by weight), 45-55 ℃, stirring reaction 3h-20h obtains hydrolyzed solution.
Preferably, before the Flavourzyme enzyme hydrolysis, homogenate pH is transferred to 6.0-7.0, add the Neutrase enzyme of 0.1%-1.0% (percentage by weight), 40-50 ℃, stirring reaction 3h-4h; Then homogenate pH is transferred to 6.0-7.5, add the Flavourzyme enzyme of 0.1%-1.0% (percentage by weight), 50 ℃, stirring reaction 3h-20h obtains hydrolyzed solution.
The hydrolysing step of the preparation method of extract of Mytilus crassitesta Lischke of the present invention also can followingly carry out: before the Flavourzyme enzyme hydrolysis homogenate pH is transferred to 7.0-8.0, the Protamax enzyme that adds 0.1%-1.0% (percentage by weight), 50-60 ℃, stirring reaction 3h-5h; Then homogenate pH is transferred to 6.0-7.5, add the Flavourzyme enzyme of 0.5%-1.0% (percentage by weight), 50 ℃, stirring reaction 5h-10h obtains hydrolyzed solution.
More preferably, hydrolysing step also comprises the hydrochloric acid hydrolysis step, and for example the homogenate of Mytilus crassitesta Lischke preferably with 1.2N～3N or with 3N～6N hydrochloric acid hydrolysis 6-15h, carries out aforesaid enzyme hydrolysis operation then with 1.2N～6N hydrochloric acid hydrolysis 6-15h.
The Flavourzyme enzyme of mentioning in explanation of the present invention, Protamax enzyme, Neutrase enzyme all have commercial prod, the Novozyme product.The implication of enzyme dosage percentage by weight is represented the percentage by weight of enzyme and Mytilus crassitesta Lischke meat.
Wherein purification step dilutes 3-20 respectively doubly for the hydrolyzed solution that will obtain, with weak-base ion-exchange resin deacidification, roguing, and resin: hydrolyzed solution=1:2.8-1:5.6, the extract of acquisition Mytilus crassitesta Lischke.Said weak-base ion-exchange resin is known in this area, for example weakly base resin D303 or weakly base resin 701, deacidification operation or batch operation or adopt this area chromatographic column commonly used to carry out.
Spissated method is known in this area, as vacuum concentration etc.The method of sterilization is autoclaving or filtration sterilization.
Total nitrogen 12-16mg/mL, protein concentration 15-20mg/mL, amino nitrogen content 8.5-15mg/mL in the Trachyostracous mussel extract of the present invention, its pH5.0-6.0, content of beary metal meet the Pharmacopoeia of People's Republic of China requirement.External activity detects: maximal non-toxic concentration (TC0) 20%, minimum effectively inhibition concentration (IC0) 5%, TI value 4.And have 17 seed amino acids in the mensuration extract at least, comprise aspartic acid, serine, threonine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, tyrosine, phenylalanine, lysine, histidine, arginine, total amino acid content is 11-14g/100mL.
The preparation method of Trachyostracous mussel extract of the present invention is utilized China's output height, and the marine organisms Mytilus crassitesta Lischke that price is low is a raw material, and it is simple to develop technology, with low cost, and preparation technology is easy to the big technology path of producing.
The test of pesticide effectiveness of resisiting influenza virus proves: Trachyostracous mussel extract of the present invention has shown stronger inside and outside activity than Russ P product.The external model test shows, the tangible anti-SARS virus of Trachyostracous mussel extract tool of the present invention, the infected cell activity effect of protection.
Zoopery shows the big or middle dosage of extract of Mytilus crassitesta Lischke of the present invention to the certain potentiation of being formed with of mouse macrophage immunne response, and the humoral immune function of enhancing body recovers the antibody forming capacity of immunocompromised Mus to a certain extent.The extract of Mytilus crassitesta Lischke of the present invention has the effect of the low Mus bone-marrow-derived lymphocyte of enhance immunity conversion capability.Immunocompromised Mus T lymphocyte transformation ability there is trend of rising.
The extract of Mytilus crassitesta Lischke of the present invention is developed to the simple extract of dosage form, can be used for treating clinically or preventing influenza, instructions of taking: oral every day 3 times, dosage is 20mL/ time.
The extract of Mytilus crassitesta Lischke of the present invention is developed to the simple extract of dosage form, can be used for immunomodulating.
Describe the present invention in detail below in conjunction with concrete embodiment.After describing these concrete embodiments in detail, this area those skilled in the art can fully know how to design other reliable methods in conjunction with above-mentioned explanation, thereby obtain same information by using achievement of the present invention.Therefore, no matter these concrete embodiments records is how concrete in detail, all must not be interpreted as any restriction to scope of the present invention.Scope of the present invention is only limited by the structure of the law of claim.
The specific embodiment
The preparation of the extract of embodiment 1 Mytilus crassitesta Lischke
From China Zhejiang Zhoushan Islands gather fresh and alive, shell is vivid, the Mytilus crassitesta Lischke that individual size and weight equate substantially, flush away silt is shelled in cleaning.Deposit-20 ℃ of refrigerator preservations in.Take out the freezing product of preserving of Mytilus crassitesta Lischke, thaw, boil 1-2h, leach dry, homogenate.PH transfers to 6.0 with raw slurry, adds the Neutrase enzyme that 0.1% (with raw material ratio) Denmark Novozymes produces, and 45 ℃, stirring reaction 3h; Boil then, pH transfers to 6.8, adds 1% (with raw material ratio) Flavourzyme enzyme, and 50 ℃, stirring reaction 3h boils.Obtain hydrolyzed solution.The hydrolyzed solution that obtains is diluted 10 times, and with the ion exchange column roguing that weakly base resin D303 is housed, acquisition pH is about 5.0 thick product.The thick product that obtains is concentrated into the extract that the product nitrogen content is 10-15mg/ml in 50 ℃ rotation vacuum concentration instrument.
The raw-material selection of embodiment 2 mussels
Bought raw material back in the different places of production respectively.Be prepared into lyophilized powder, or the freezing preservation in back of shelling.Be prepared into sample with fixed production technology, measure its amino N, total N and activity in vivo.Compare each raw material quality, as table 1.
The raw-material selection of table 1 mussel
Annotate: dressing percentage=fresh scallop meat weight/band shell fresh scallop weight.Dry matter content=dry matter weight/fresh scallop meat weight.60 ℃ of constant temperature drying fresh scallop meat, constant weight three times is weighed.
It is high that total nitrogen content in dry and the acid hydrolysis solution, Mytilus crassitesta Lischke are obviously wanted.Dried mussel, Perna uiridis (Linnaeus) and Mytilus crassitesta Lischke are prepared sample carried out active testing in the mouse influenza virus model body, the average life natural law of statistics Mus, Mytilus crassitesta Lischke and positive control SHUANGHUANLIAN (9.61 days) be close to 9.33 days, higher 3.16 days than blank virus control (6.17 days).So make raw material with Mytilus crassitesta Lischke.
Amino acid content in embodiment 3 working samples
Get the extract of embodiment 1, measure amino acid content (Xia Qichang chief editor, " protein chemistry investigative technique and progress ", Beijing: Science Press, 1999: 30-32 according to this area RP-HPLC commonly used, OPA method.), the result is an amino acid/11 2.5g/100mL extract.
The every detection index of the extract of embodiment 4 Mytilus crassitesta Lischkes of the present invention and Russian commodity relatively
Product of the present invention and Russian commodity are detected every quality control index respectively, data as shown in Table 2: every index of the present invention such as total nitrogen, protein concentration, amino nitrogen content etc. are all approaching with Russian commodity, but hydrolysis conversion is higher.External lower to Madin-Darby canine kidney(cell line) (MDCK) toxicity, the external anti-influenza activity of product is very high.
The every detection index of table 2 the present invention and Russian commodity relatively
Annotate: 1) percent hydrolysis=amino nitrogen/total nitrogen
2) cytotoxicity: with the Madin-Darby canine kidney(cell line) (MDCK) is model, and sample is to Madin-Darby canine kidney(cell line) (MDCK) maximal non-toxic concentration.
3) external activity: with the Madin-Darby canine kidney(cell line) (MDCK) is model, and sample suppresses Madin-Darby canine kidney(cell line) (MDCK) pathological changes rate.
The external test of pesticide effectiveness of extract of embodiment 5 Mytilus crassitesta Lischkes of the present invention
Illustrate: 1
#Sample is a product of the present invention, 2
#Sample be Russ P health product rice its-the K test objective:
Observation sample is external to influenza virus (A
3/ capital section/30/95) drug action.
Test material:
1
#, 2
#Sample is provided by the present patent application people; Positive control medicine-ribavirin crude drug is mainly used in viral pneumonia and bronchitis that respiratory syncytial virus causes, and consumption is three times on the one, each 75mg/kg; Be Zhaoqing, Guangdong Xing Hu pharmaceutical factory product, lot number is 10638.Ribavirin test liquid: 1mg/ml ribavirin solution: take by weighing ribavirin raw material 10mg, add the cell culture fluid dissolving, be made into 1mg/ml solution, membrane filtration.Strain: influenza virus (A
3/ capital section/30/95), health and epidemic prevention station provides by Shanghai City, the preservation of going down to posterity.Cell model: Madin-Darby canine kidney(cell line) (MDCK) mdck cell.
The drug cell toxicity test:
Mdck cell is at 37 ℃ of 96 well culture plate monolayer culture, 5% CO
2Cultivated 24 hours, and added variable concentrations medicinal liquid (40,20,10,5,2.5%) respectively, observed toxicity through 72 hours and measure the OD value, calculate the cytotoxicity caused TC of medicine with mtt assay
0
The cytopathy inhibition test:
Mdck cell is at 37 ℃ of 96 well culture plate monolayer culture, 5% CO
2Cultivated 24 hours, and added 30TCID
50Virus liquid, 37 ℃, 5% CO
2Adsorbed 2 hours, and inhaled virus removal, add medicinal liquid under the maximal non-toxic concentration, through 37 ℃, 5% CO
2Cultivated 72 hours, and observed CPE (pathological changes), establish the cell matched group, the virus control group, positive controls (ribavirin) and medicine group are observed CPE simultaneously with experimental group, measure the OD value, determine that sample effectively suppresses pathological changes concentration (IC
0) and therapeutic index TI value.
Conclusion:
Show 1 through in vitro tests result (table 3)
#Sample is to mdck cell maximal non-toxic concentration (TC
0) be 20%, under 5% concentration, can suppress (IC
0) influenza A3 virus, the TI value is 4, suppresses effect and 250 μ g/ml ribavirins and is close.And Russian sample 2
#To mdck cell maximal non-toxic concentration (TC
0) be 2.5%, do not have and obviously suppress influenza A3 virus function.
Table 3 sample is to the cytopathogenic inhibitory action of influenza virus
Annotate :+expression suppresses pathological changes (〉=50%), and inhibition is not seen in-expression, and inhibition (≤25%) is slightly seen in ± expression.Do not see that toxicity represents cell survival 〉=90%, see that slightly toxicity represents cell survival 〉=80%, overt toxicity is represented cell survival≤50%
Test of pesticide effectiveness contrast in embodiment 6 the present invention and the Russ P body of commodity
Illustrate: 1
#Sample is a product of the present invention, 2
#Sample be Russ P health product rice its-K
Test objective:
The drug action of prevention and treatment A1 type (H1N1) influenza virus in the observation sample body.
Test material:
Be subjected to the reagent thing: the people provides by the present patent application; The positive control medicine: the ribavirin crude drug, be mainly used in viral pneumonia and bronchitis that respiratory syncytial virus causes, consumption is three times on the one, each 75mg/kg; Be Zhaoqing, Guangdong Xing Hu pharmaceutical factory product, lot number is 10638.75mg/kg ribavirin extract: take by weighing ribavirin raw material 300mg, add 0.5% CMC solution 100ml, be made into 0.3% suspension, each administration 0.5ml/20g mice, once-a-day.Strain: influenza virus A 1 type (H1N1) Mus lung adapted strain FM1 infected mice lung suspension, Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences provides by Beijing, through the preservation of going down to posterity.Animal: kunming mice, body weight 17～21 grams, male and female dual-purpose.The department of the Chinese Academy of Sciences of laboratory animal section of Fudan University provides credit number: scxk Shanghai 2002-0002.
The resisiting influenza virus test of pesticide effectiveness in the sample body:
1
#Sample is to the influence on dead mouse date
Method: get mice, random packet, 16 every group, male and female half and half, grouping and administration number of times and the same test of infective virus amount.Begin to observe the dead mouse situation after the infection, the record death toll.
Result of the test sees Table 4:
Sample adopts the dead median method of calculating to carry out date processing to the influence on dead mouse date.
Sample to the mice influenza after, life prolongs test:
Method: get mice (17-21g), random packet, 16 every group, male and female half and half, experiment is established:
(1), virus control group:
(2), positive controls (ribavirin): 75mg/kg
(3), 2
#Medicine, oral, the 0.5ml/20g Mus:
(4), 1
#Medicine, oral, the 1ml/20g Mus:
(5), 1
#Medicine, oral, the 0.5ml/20g Mus:
(6), 1
#Medicine, oral, the 0.25ml/20g Mus:
(7), normal control group: normal saline, 0.2ml/10g/d * 6
Oral administration, once a day, administration is 7 days altogether.Viral infection 10
-2, 55-60 μ L/ only.Continued administration three days, and observed a week altogether, the record death toll takes statistics to learn and handles, calculate dead median and date of death scope and average life day and life prolong %, and calculating P value.
Result of the test sees Table 5,6:
Sample adopts Stat View Ver 5.01 SAS Institute Inc softwares to carry out Mann-Whitney check and statistical procedures to the influence of mice survival natural law.Through test, the result shows and the virus control group compares administration 50ml/kg, 25ml/kg, 12.5ml/kg, 1
#Medicine life prolongs 60.1%, 52.8%, 47.2%, mice oral administration 25ml/kg, 2
#Medicine life prolongs 46.0%.
Table 4 sample is to the influence on dead mouse date
Conclusion:
According to above test as seen: sample is tested through mouse model, it is to the influence of mice survival natural law, adopt StatView Ver 5.01 SAS Institute Inc softwares to carry out Mann-Whitney check and statistical procedures, the result shows with the virus control group and compares, administration 50ml/kg, 25ml/kg, 12.5ml/kg, 1
#Medicine life prolongs 60.1%, 52.8%, 47.2%, mice oral administration 25ml/kg, 2
#Medicine life prolongs 46.0%.Prevention and treatment influenza virus A 1 type (H1N1) show under the same concentrations 1 through the mouse model test
#Medicine is than 2
#Medicine is effective.
Table 5 sample is to mice influenza inhibition test (the average life-time of X ± SD) (my god)
Annotate:
*P〉0.05
*P＜0.05
* *P＜0.01
Table 6 sample prolongs test to the mice life span
Embodiment 7 the present invention and influenza Chinese medicine SHUANGHUANLIAN drug effect relatively reach the dosage test
Test objective:
The drug action of prevention A1 type (H1N1) influenza virus in the observation sample body.
Test:
The reagent thing: the people provides by the present patent application, is the 1# medicine.
Positive control medicine: SHUANGHUANLIAN extract
Strain:
Influenza virus (A
3/ capital section/30/95), health and epidemic prevention station provides by Shanghai City, the preservation of going down to posterity.
Influenza virus A 1 type (H1N1) Mus lung adapted strain FM1 infected mice lung suspension, Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences provides by Beijing, through the preservation of going down to posterity.
Animal: kunming mice, body weight 13～15 grams, male and female dual-purpose.Credit number: scxk Shanghai 2002-0002
Experiment: get mice, random packet, 18 every group, male and female half and half, (1) virus control group; (2) positive controls (SHUANGHUANLIAN): 0.24ml/ Mus.Oral administration 7 days, viral infection 10
-2, 30 μ L/ only continue administration 6 days, observe altogether 14 days.Write down death toll every day, take statistics and learn to handle, calculate dead median and date of death scope and average life day and life prolong %, and calculating P value.Result of the test sees Table 7, table 8.Medicine 1# adopts SPSS For Windows11.5 software to carry out T check and statistical procedures to the influence of mice survival natural law.Through test, the result shows with the virus control group and compares, 1# medicine oral administration 0.3ml/ Mus, 0.55ml/ Mus, 0.8ml/ Mus, and life prolongs 51.22%, 22.37%, 16.21%.
Table 7 sample is to the influence on dead mouse date
Conclusion: this experimental result 1
#Pharmaceutical quantities 0.3ml/ Mus was with the interior drug effect result of the test of mouse body was identical in the past.1
#The effect of pharmaceutical quantities 0.3ml/ Mus and SHUANGHUANLIAN is approaching.
Table 8 sample is to mice influenza inhibition test (X ± SD)
Embodiment 8 extract anti-SARS virus test results of the present invention
Test philosophy:
As virus host cell (permissive cell), specimen is resisted the effect of virus infected cell with the Vero-E6 cell, and detecting index is cytopathy reaction (CPE) and infection cell protective rate.
Test material and method:
Strain: BJ-01 derives from Beijing Jun Keyuan microorganism epidemic research institute
Experiment place: the P3 of disease prevention and control center, Shanghai laboratory
Sample treatment: sample is dissolved in culture fluid or DMSO, is made into suitable initial concentration, 5 times of dilutions, 3 dilution factors.
Method of testing: the Vero-E6 cell at 96 well culture plates in 37 ℃, 5% CO
2Monolayer culture behind the SARS virus infection cell, adds the variable concentrations medicinal liquid respectively, establishes the cell matched group, observed result under the virus control group, and medicine group, every day mirror, record CPE, and, carries out the calculating and the evaluation of sample anti-SARS virus active function with reference to contrast with dimethyl diaminophenazine chloride dyeing mensuration OD value.
Table 9 extract anti-SARS virus test of the present invention
Cytopathy political reform (CPE): with "-" expression cell attachment complete form, do not see obvious disengaging, but cell quantity what do not compare, with "+" expression cytopathy degree,＜25%+, 25%～50%++, 50%～75%+++, 75%++++.
Infected cytoprotective rate: by comparing the OD value of virus control, cell contrast and sample contrast, calculation sample is to the protection activity of virus infected cell, protective rate〉EC
20Tentatively be considered to sample to being had the certain protection effect, antiviral activity is arranged by the cell of viral infection.
Sample cell toxicity: if the cytotoxicity of sample and cell contrast ratio are greater than 50 (〉 CC
50) time, do not estimate and protective rate calculating to CEP.
Conclusion: result of the test sees Table 9.Institute's test sample has obvious anti-SARS virus, protects infected cytosis in in-vitro screening model when 6.25% concentration.
Embodiment 9 specificity cellular immunity responses: delayed allergy
The animal Kunming mouse, male and female half and half, 19-23g.
Reagent and medicine
2.4-dinitrofluorobenzene (DNFB): in the fresh DNFB 50mg that takes by weighing on the experiment same day, put in the blue or green bottle of cleaning, (acetone: the 5ml mixing of Oleum Sesami=1:1), the adhesive plaster of jumping a queue sealing is made into 1%DNFB to add the solution for preparing in advance.
Levamisole hydrochloride [(-)-Tetramisole hydrochloride minimum]: sigma company, lot number: L9756-5G.Getting a certain amount of 40mg levamisole hydrochloride adds 0.5% CMC and grinds mixing (0.1ml/10g, 2mg/ml) 4 ℃ of storages are standby to deciding volume 20ml.Dosage 20mg/kg.
Experimental technique
Get mice (19-23g), random packet, 10 every group, male and female half and half, experiment is established:
(1), normal control group: normal saline, 0.5ml/20g;
(2), model group: 0.5% CMC, 0.5ml/20g;
(3), positive controls (levamisole): 20mg/kg;
(4), the extract heavy dose of embodiment 1 Mytilus crassitesta Lischke, 1ml/20g;
(5), dosage in the extract of embodiment 1 Mytilus crassitesta Lischke, 0.5ml/20g;
(6), the extract low dose of embodiment 1 Mytilus crassitesta Lischke, 0.25ml/20g;
Oral administration, beginning administration in 0 day, once a day (the heavy dose of group of the extract of embodiment 1 Mytilus crassitesta Lischke dosage every day divides secondary to give), administration is 6 days altogether.D1 except that the normal control group, every Mus abdominal part unhairing 3cm * 3cm, 50ul evenly is applied to the unhairing place with 1%DNFB solution.D6 evenly is applied in mouse right ear (two sides) with 1%DNFB solution 10ul and attacks, and normal group is coated with ear but abdominal part sensitization not equally.Attack back 24h, mice is put to death in the cervical vertebra dislocation, cuts left and right sides auricle, takes off the auricle of diameter 7mm with card punch, weighs.Difference with left and right sides auricle weight is the swelling degree, gets mouse thymus simultaneously and spleen is weighed.Spleen heavy (mg) and thymus with every 10g mice weighs (mg) as spleen index and thymus index respectively.Relatively each group difference is done check ANOVA and Fisher ' s PLSD check with the statview software of SAS company.
The result:
Compare with normal group, the model group mouse right ear is 24h after sensitinogen 2.4-dinitrofluorobenzene is attacked once more, auricle edema, obviously congested. spleen and thymus index do not have significant change, and levamisole 20mg/kg strengthens auris dextra swelling degree, and spleen and thymus index do not have significant change (seeing Table 10).
Compare with the model group mice, the extract heavy dose (1ml/20g) of oral embodiment 1 Mytilus crassitesta Lischke, auris dextra swelling degree has certain increase, but does not have statistical significance; Dosage (0.5ml/20g) auris dextra swelling degree significantly increases in the extract of oral embodiment 1 Mytilus crassitesta Lischke, and statistical significance is arranged.The no effect of extract low dose (0.25ml/20g) of embodiment 1 Mytilus crassitesta Lischke.
Dosage and levamisole 20mg/kg have obviously strengthened the mice delayed allergy in the extract of presentation of results embodiment 1 Mytilus crassitesta Lischke.The extract of embodiment 1 Mytilus crassitesta Lischke is heavy dose of may be because of twice administration, and the mice irriate is more, is formed with certain influence to what mouse immune was replied, and strengthening the swollen effect of ear only has certain trend and do not have upward significant difference of statistics.
Conclusion
Dosage (0.5ml/20g) and levamisole 20mg/kg can strengthen the delayed hypersensitivity that the 2.4-dinitrofluorobenzene brings out in the extract of embodiment 1 Mytilus crassitesta Lischke; The extract heavy dose (1ml/20g) of embodiment 1 Mytilus crassitesta Lischke has certain potentiation.
The extract of table 10. embodiment 1 Mytilus crassitesta Lischke is to the influence of mice delayed allergy
Annotate: medicine po qd * 6, n=10, x ± s. and normal control are relatively
aP＜0.05,
bP＜0.01,
cP＜0.001; Compare with model group
dP＜0.05,
eP＜0.01,
fP＜0.001.
The extract non-specific cell immunity of embodiment 10 Mytilus crassitesta Lischkes: mouse macrophage is engulfed neutral red test
The animal Kunming mouse, male, 22-24g.
Reagent and medicine
1. cyclophosphamide: 200mg/ bottle, Hualian Pharmaceutical Co., Ltd., Shanghai, lot number: 050115.Face with before, with physiological saline solution 5ml dissolving, prepare 0.2ml/10g to 50ml (4mg/ml), use for lumbar injection.Mice dosage 80mg/kg
2. levamisole hydrochloride [(-)-Tetramisole hydrochloride minimum]: sigma company, lot number: L9756-5G.(0.1ml/10g, 2mg/ml) 4 ℃ of storages are standby to deciding volume 20ml to get a certain amount of 40mg levamisole hydrochloride adding 0.5%CMC grinding mixing.Dosage 20mg/kg.
3.0.1% dimethyl diaminophenazine chloride: 0.05g dimethyl diaminophenazine chloride+50mlNS is heated to dissolving, filters, and uses preceding autoclaving.
Experimental technique
Get mice, random packet, 10 every group, experiment is established:
(1), normal control group: normal saline, 0.5ml/20g
(2), cyclophosphamide-a control: 0.5% CMC, 80mg/kg
(3), positive controls (levamisole)+cyclophosphamide pretreatment: 20mg/kg
(4), the extract heavy dose+cyclophosphamide pretreatment of embodiment 1 Mytilus crassitesta Lischke, 1ml/20g
(5), dosage+cyclophosphamide pretreatment in the extract of embodiment 1 Mytilus crassitesta Lischke, 0.5ml/20g
(6), the extract low dose+cyclophosphamide pretreatment of embodiment 1 Mytilus crassitesta Lischke, 0.25ml/20g
The mice random packet, 10 every group, comprise normal group, the pretreated negative control group of cyclophosphamide, cyclophosphamide pretreatment+levamisole group, each dosage group of cyclophosphamide pretreatment+reagent, levamisole and reagent po qd * 6, except that normal group, each organizes d
2Ip 5% mercaptoethanol acid sodium 0.5ml/ only; Each group is at d
3Ip cyclophosphamide 80mg/kg.D
66 groups of mice Mus are taken off neck put to death,, gently rub abdominal part 50 times immediately to intraperitoneal injection 1640 5ml, slowly extract peritoneal fluid 3ml then in centrifuge tube, (ice cube) 4 ℃ of 1000rpm, centrifugal 10 minutes, wash cell 1 time with 1640,1640-10% Fcs re-suspended cell is in culture fluid.Counting is regulated cell number.The mensuration of phagocytic function: get 0.2ml 2 * 106/ml peritoneal macrophage and add in 24 orifice plates, put under 37 ℃, 5% CO2 condition adherent 2 hours, supernatant is abandoned in taking-up, and the NS rinsing adds 0.1% dimethyl diaminophenazine chloride physiological salt liquid 0.1ml again, put under 37 ℃, 5% CO2 condition and cultivated 20 minutes, take out, abandon supernatant, NS dashes surplus liquid rinsing to the greatest extent, (ethanol: acetic acid=1: 1), fully the dissolving back is in 540nm wavelength place colorimetric to add the 0.1ml cytolysate.
The result:
Compare with normal group, ip cyclophosphamide 80mg/kg has suppressed the macrophage phagocytic dimethyl diaminophenazine chloride; Levamisole 20mg/kg can improve the effect of immunosuppressed mice macrophage phagocytic.(seeing Table 11)
Compare with cyclophosphamide group mice, the extract heavy dose (1ml/20g) of oral embodiment 1 Mytilus crassitesta Lischke, middle dosage (0.5ml/20g) has the effect of certain enhancing macrophage phagocytic, has statistical significance; The no effect of extract low dose (0.25ml/20g) of oral embodiment 1 Mytilus crassitesta Lischke.
The big or middle dosage of extract and the levamisole 20mg/kg of presentation of results embodiment 1 Mytilus crassitesta Lischke are to the certain potentiation of being formed with of mouse macrophage immunne response.
Conclusion:
The big or middle dosage of extract and the levamisole 20mg/kg of presentation of results embodiment 1 Mytilus crassitesta Lischke are to the certain potentiation of being formed with of mouse macrophage immunne response.
The extract of table 11. embodiment 1 Mytilus crassitesta Lischke is to the influence of macrophage phagocytic dimethyl diaminophenazine chloride
Annotate: medicine po qd * 6, n=10, x ± s. and normal control are relatively
aP＜0.05,
bP＜0.01,
cP＜0.001; Cyclophosphamide-a control relatively
dP＜0.05,
eP＜0.01,
fP＜0.001.
The extract specific humoral immunity of embodiment 11 Mytilus crassitesta Lischkes: serum hemolysin test
Animal: Kunming mouse, male, 22-24g.
Reagent and medicine
1. cyclophosphamide: 200mg/ bottle, Hualian Pharmaceutical Co., Ltd., Shanghai, lot number: 050115.Face with before, with physiological saline solution 5ml dissolving, prepare 0.2ml/10g to 50ml (4mg/ml), use for lumbar injection.Mice dosage 80mg/kg
2. levamisole hydrochloride [(-)-Tetramisole hydrochloride minimum]: sigma company, lot number: L9756-5G.(0.1ml/10g, 2mg/ml) 4 ℃ of storages are standby to deciding volume 20ml to get a certain amount of 40mg levamisole hydrochloride adding 0.5%CMC grinding mixing.Dosage 20mg/kg.
3. animal Sanguis caprae seu ovis (SRBC) preparation: go bail for and deposit Sanguis caprae seu ovis, 8-10 times of normal saline, light mixing, 2000rpm*10min*2 gets the SRBC hematocrit in certain volume normal saline preparation suspension, and ip in mice 2% suspension, 0.2ml/ are only.
4. preparation complement: arterial blood letting, at least 5 guinea pig serum are mixed, the centrifuging and taking supernatant, packing ,-70 degree are preserved.
5. test serum: eyeball is got blood, and room temperature was separated in 1 hour, packing, and-20 ℃ are frozen down.
Experimental technique
Get mice, random packet, 10 every group, experiment is established:
(1), normal control group: normal saline, 0.5ml/20g
(2), cyclophosphamide-a control: ip 80mg/kg, oral 0.5%CMC
(3), positive controls (levamisole)+cyclophosphamide pretreatment: 20mg/kg
(4), the extract heavy dose+cyclophosphamide pretreatment of embodiment 1 Mytilus crassitesta Lischke, 1ml/20g
(5), dosage+cyclophosphamide pretreatment in the extract of embodiment 1 Mytilus crassitesta Lischke, 0.5ml/20g
(6), the extract low dose+cyclophosphamide pretreatment of embodiment 1 Mytilus crassitesta Lischke, 0.25ml/20g
The mice random packet, 10 every group, comprise normal group, the pretreated negative control group of cyclophosphamide, cyclophosphamide pretreatment+levamisole group, each dosage group of cyclophosphamide pretreatment+reagent, d is respectively organized in levamisole and reagent po qd * 6 except that normal group
2Ip 2%SRBC 0.5ml/, d
3, d
5Ip cyclophosphamide 80mg/kg, d
6Extract eyeball and get blood, close at the plastics tubule.Get 2000r/min * 10min behind the blood 1h.Collect serum, press the 1:100 dilute serum, get dilute serum 0.2ml, 10%0.1ml SRBC, complement 0.2ml (1:10 dilution); Control tube is increase serum not, 37 ℃ of water-bath 30min, and ice bath is ended, and 5000r/min * 10min gets supernatant 0.2ml, 405nm wavelength colorimetric.50% haemolysis standard pipe: 10%SRBC 0.5ml is dissolved in the 1.5ml distilled water, is 100% haemolysis, with being 50% haemolysis behind the NS two-fold dilution (2ml), and mixing.Get supernatant 0.2ml, 405nm wavelength colorimetric.
CH50 (u/ml)=(measuring pipe A/50% haemolysis pipe A) * dilute serum multiple U/ml
The result: see Table 12., cyclophosphamide 80mg/kg significantly suppresses the generation of mice serum hemolysin, and levamisole has shown its promoting immunity restitution.The extract of embodiment 1 Mytilus crassitesta Lischke can partly resist the inhibitory action of cyclophosphamide, makes the serum hemolysin generation than the cyclophosphamide Mus certain rising (p=0.018) be arranged; But the effect of extract (1ml/20g) the rising serum hemolysin of only heavy dose of embodiment 1 Mytilus crassitesta Lischke has statistical significance (p=0.045)
The extract of table 12. embodiment 1 Mytilus crassitesta Lischke is to the influence of serum hemolysin
Annotate: medicine po qd * 6, n=10, x ± s. and normal control are relatively
aP＜0.05,
bP＜0.01,
cP＜0.001; Compare with the cyclophosphamide-a control group
dP＜0.05,
eP＜0.01,
fP＜0.001.
Conclusion: the extract of heavy dose of embodiment 1 Mytilus crassitesta Lischke is the humoral immune function of enhancing body to a certain extent, recovers the antibody forming capacity of immunocompromised Mus.
Embodiment 12 specific cellular immunities: lymphocyte transformation test
The animal Kunming mouse, male, 22-23g.
Reagent and medicine
Cyclophosphamide: 200mg/ bottle, Hualian Pharmaceutical Co., Ltd., Shanghai, lot number: 050115.Face with before, with physiological saline solution 5ml dissolving, prepare 0.2ml/10g to 50ml (4mg/ml), use for lumbar injection.Mice dosage 80mg/kg.
Levamisole hydrochloride [(-)-Tetramisole hydrochloride minimum]: sigma company, lot number: L9756-5G.Getting a certain amount of 40mg levamisole hydrochloride adds 0.5% CMC and grinds mixing (0.1ml/10g, 2mg/ml) 4 ℃ of storages are standby to deciding volume 20ml.Dosage 20mg/kg.
Experimental technique:
(1), normal control group: normal saline, 0.5ml/20g
(2), cyclophosphamide-a control: 0.5% CMC, 0.5ml/20g
(3), positive controls (levamisole)+cyclophosphamide pretreatment: 20mg/kg
(4), the extract heavy dose+cyclophosphamide pretreatment of embodiment 1 Mytilus crassitesta Lischke, 1ml/20g
(5), dosage+cyclophosphamide pretreatment in the extract of embodiment 1 Mytilus crassitesta Lischke, 0.5ml/20g
(6), the extract low dose+cyclophosphamide pretreatment of embodiment 1 Mytilus crassitesta Lischke, 0.25ml/20g
The mice random packet, every group 10, comprise normal group, cyclophosphamide pretreatment matched group, levamisole group, each dosage group of cyclophosphamide pretreatment+reagent, levamisole and po reagent qd * 6, except that normal group, respectively organize d3, d5 ip cyclophosphamide 80mg/kg, the d6 sacrificed by exsanguination is after every group of mouse spleen mixes, discharge splenocyte, destroy erythrocyte, wash secondary, transfer each splenocyte concentration to 1 * 10
7/ ml.Get the cell suspension 100 μ l of this concentration and 100 μ l ConA or LPS and add to 96 well culture plates, (splenocyte 1 * 10
7/ ml 0.1ml, ConA 6 μ g/ml 0.1ml/ holes, or LPS 20ug/ml 0.1ml/ hole), each sample adds the 4 multiple holes of ConA or LPS, does not add 8 multiple holes of stimulant, cultivates 48h, after adding 5mg/mlMTT 25ul (final concentration 2mg/ml) continuation cultivation 4h, take out culture plate (but 2000rpmX10min) centrifuging and taking supernatant, finish preceding 4 hours, every hole adds MTT 2mg/ml.After finishing to cultivate, add the DMSO lysate, purple crystal is dissolved fully.(λ=570nm) goes up colorimetric at enzyme-linked immunosorbent assay instrument.Represent the lymphopoiesis ability with optical density value.
The result
1. the results are shown in Table 13., cyclophosphamide makes the conversion capability of mouse lymphocyte significantly be subjected to press down, no matter the not existence of polyclone stimulant is arranged, the big or middle dosage of extract of levamisole and embodiment 1 Mytilus crassitesta Lischke all has recovery to a certain extent to the lymphopoiesis ability of the reduction of cyclophosphamide induction of immunity inhibition mice.Compare with cyclophosphamide transaction module Mus, it raises and all has statistical significance.
The influence of table 13. pair lymphocyte transformation test
Annotate: medicine po qd * 6, n=10, x ± s. and normal control are relatively
aP＜0.05,
bP＜0.01,
cP＜0.001; Cyclophosphamide-a control relatively
dP＜0.05,
eP＜0.01,
fP＜0.001.
2. when LPS exists, the big or middle dosage of extract of levamisole and embodiment 1 Mytilus crassitesta Lischke manifests obviously collaborative stimulation, and the cyclophosphamide induction of immunity is suppressed the lymphopoiesis ability of the reduction of mice, and recovery is to a certain extent all arranged.When ConA exists, the big or middle dosage of extract of levamisole and embodiment 1 Mytilus crassitesta Lischke suppresses the lymphopoiesis ability of the reduction of mice to the cyclophosphamide induction of immunity, recovery is to a certain extent all arranged, but rarely seen levamisole there is meaning on the statistics (P=0.0502)
Conclusion:
The big or middle dosage of extract of levamisole and embodiment 1 Mytilus crassitesta Lischke has the effect of the low Mus bone-marrow-derived lymphocyte of enhance immunity conversion capability.Immunocompromised Mus T lymphocyte transformation ability there is trend of rising.
Claims (7)
1. the extract of Mytilus crassitesta Lischke, comprise aspartic acid, serine, threonine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, tyrosine, phenylalanine, lysine, histidine, arginine, total nitrogen 12.5-14mg/mL, protein concentration 15-18mg/mL, total amino acids content are 11-14g/100mL extract, amino nitrogen content 10-13mg/mL, pH5.3-5.8, it is characterized in that it prepares by the following method:
(a) Mytilus crassitesta Lischke cleans, and shells;
(b) Mytilus crassitesta Lischke meat homogenate;
(c) hydrolysis;
(d) with weakly base resin deacidification, roguing; With
(e) solution after the roguing is concentrated,
Wherein the condition of hydrolysing step is: the homogenate pH of Mytilus crassitesta Lischke transfers to 6.0-7.5, adds the Flavourzyme enzyme of 0.1%-1.0% (percentage by weight), and 45-55 ℃, stirring reaction 3h-20h obtains hydrolyzed solution.
2. according to the extract of the Mytilus crassitesta Lischke of claim 1, it is characterized in that, before the Flavourzyme enzyme hydrolysis, homogenate pH is transferred to 6.0-7.0, add the Neutrase enzyme of 0.1%-1.0% (percentage by weight), 40-50 ℃, stirring reaction 3h-4h; Then homogenate pH is transferred to 6.0-7.5, add the Flavourzyme enzyme of 0.1%-1.0% (percentage by weight), 50 ℃, stirring reaction 3h-20h obtains hydrolyzed solution; The hydrolyzed solution that obtains is diluted 3-20 respectively doubly, use the weak-base ion-exchange resin roguing, resin: hydrolyzed solution=1:2.8-1:5.6, the extract of acquisition Mytilus crassitesta Lischke.
3. according to the extract of the Mytilus crassitesta Lischke of claim 1, it is characterized in that, before the Flavourzyme enzyme hydrolysis, homogenate pH is transferred to 7.0-8.0, add the Protamax enzyme of 0.1%-1.0% (percentage by weight), 50-60 ℃, stirring reaction 3h-5h; Then homogenate pH is transferred to 6.0-7.5, add the Flavourzyme enzyme of 0.5%-1.0% (percentage by weight), 50 ℃, stirring reaction 5h-10h obtains hydrolyzed solution; The hydrolyzed solution that obtains is diluted 3-20 doubly, use the weak-base ion-exchange resin roguing, resin: hydrolyzed solution=1:2.8-1:5.6, the extract of acquisition Mytilus crassitesta Lischke.
4. according to the extract of the Mytilus crassitesta Lischke of claim 1, it is characterized in that the homogenate of Mytilus crassitesta Lischke carries out enzymolysis then with 1.2N～6N hydrochloric acid hydrolysis 6-15h.
5. according to the application in the preparation Tamiflu of the extract of the arbitrary described Mytilus crassitesta Lischke of claim 1-4.
6 the application of extract in the anti-SARS medicine of preparation according to the arbitrary described Mytilus crassitesta Lischke of claim 1-4.
7 the application of extract in the immunoregulatory medicine of preparation according to the arbitrary described Mytilus crassitesta Lischke of claim 1-4.
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| CN102558296B (en) * | 2010-12-16 | 2014-01-15 | 浙江海洋学院 | Mytilus edulis enzymolysis polypeptide and preparation method and application thereof |
| CN102952838B (en) * | 2011-08-23 | 2015-05-06 | 浙江海洋学院 | Preparation method of immune-enhancing mussel enzymatic hydrolytic polypeptide and preparation method of corresponding tablet thereof |
| GB2523481B (en) * | 2012-10-24 | 2016-03-16 | Nikolay Vladimirovich Solovyev | Composition for parental administration, method for producing and use thereof |
| RU2526826C2 (en) | 2012-10-24 | 2014-08-27 | Николай Владимирович Соловьёв | Parenteral composition, method for preparing and using composition |
| WO2016153363A1 (en) * | 2015-03-24 | 2016-09-29 | The New Zealand Institute For Plant And Food Research Limited | Water-soluble mussel extract |
| CN104825494A (en) * | 2015-04-19 | 2015-08-12 | 青岛大学 | Mussel extract and application thereof in drugs for promoting post-natal uterine contraction |
| CN106913587A (en) * | 2015-12-25 | 2017-07-04 | 无限极（中国）有限公司 | Application of the Jadeite Mussel water extract in the health food with anti-oxidant and neuroprotection |
| WO2019011286A1 (en) * | 2017-07-14 | 2019-01-17 | Jiangyin Usun Pharmaceutical Co., Ltd. | Antiviral use of mussel adhesive proteins |
| JP7177438B2 (en) * | 2018-12-20 | 2022-11-24 | 株式会社渡辺オイスター研究所 | Production method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol |
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