CN100486595C - Brazil mushroom soluble small molecular extract, its preparation technology and use - Google Patents
Brazil mushroom soluble small molecular extract, its preparation technology and use Download PDFInfo
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Abstract
A water-soluble micro-molecular extract extracted from the sporophores and mycelia of Brazi edible mushroom and able to supress the growth of tumor cells and regulate immunity of preparing the medicines to prevent and treat tumor and the diseases associated with immune has the molecular weight less than 4000. It contains 9 active components including nannitol, mycose, Phe, Glu, etc. Its preparing process is also disclosed.
Description
Technical field
The invention belongs to health food and medical technical field, be specifically related to a kind of edible fungus Brazil mushroom soluble small molecular extract and preparation technology thereof and the purposes aspect health food and Medicines.
Technical background
Agaricus blazei Murrill claims Brazilian mushroom again, is a kind of edible fungus of preciousness, on classification of fungi, belongs to Basidiomycotina, Hymenomycetes, and the Zoopagales that looses, Agaricus edibilis, Agaricus, Latin formal name used at school are Agaricus blazei Murill..This bacterium originates in Latin Americans such as Brazil, the U.S., Peru.Japan passes to south east asia such as Vietnam, Thailand, Indonesia and Taiwan subsequently in succession, and imports China into the beginning of the nineties in last century in the promptly successful introducing and planting seventies in last century.Fujian, zhejiang and other places have a large amount of cultivations now.Simultaneously, the relevant mycelial liquid culture technology of Agaricus blazei Murrill been has also has been researched and developed success and has been carried out large-scale production.On the basis of successfully having carried out artificial large-scale planting and mycelium large-scale production; the Japan scholar takes the lead in again the chemical constituent and the medical health care function thereof of Agaricus blazei Murrill are furtherd investigate, and finds that its hot water extract and water soluble polysaccharide have physiologically actives such as significant antitumor and adjusting immunity.Chinese scholar has also been carried out the research of its active component and pharmacologically active thereof in succession in the later stage nineties, confirm multiple biological activitys such as its hot water extract and water soluble polysaccharide have antitumor, regulate immunity, protect the liver, blood sugar lowering.Up to now, relevant research document and patent only find and confirm that the water soluble polysaccharide among this fungus hot water extract partly is main active that its molecular weight belongs to the macromole active component all more than 10,000.Molecular weight does not see that less than the composition of 4,000 soluble small molecular composition and biological activity thereof patent and document deliver in the relevant Agaricus blazei Murrill.
Summary of the invention
The object of the present invention is to provide a kind of Brazil mushroom soluble small molecular extract and preparation technology thereof and the purposes aspect health food and Medicines.
Brazil mushroom soluble small molecular extract provided by the present invention, be from the mycelium of edible fungi Agaricus blazei Murrill sporophore or artificial culture, to extract, molecular weight is lower than 4, the compositions of 000 micromolecular compound, the wherein clear and definite macromolecular components such as the polysaccharide of molecular weight more than 10,000, protein that do not contain.
The inventor finds under study for action, and the hot water extract of Agaricus blazei Murrill is 10,000 and 4 with molecular cut off respectively, after 000 molecular sieve carries out ultra-filtration and separation, the hot water extract is divided into molecular weight greater than 10,000,4,000～10,000 and molecular weight less than 4,000 three parts.Subsequently pharmaceutical research confirms that these three parts all have significant antitumor activity to the S180 tumor-bearing mice, and with molecular weight less than 4,000 soluble small molecular part activity the strongest (seeing embodiment 1).
By to this molecular weight less than 4,000 active soluble small molecular position is carried out the chemical constituent of system and is separated and evaluation, be divided into from having identified 9 chemical compounds, its title is: mannitol (mannitol), trehalose (trehalose), phenylalanine (phenylalanine), glutamic acid (glutamic acid), the fast morpholine nucleoside of gland (adenosine), uracil (uracil), uridine (uridine), deoxyuridine (dexoxyuridine) and 2,6-deoxy fructosazine 2,6-deoxyfructosazine, promptly 2-(1 ', 2 ', 3 ', 4 '-tetrahydroxy butyl)-5-(2 ", 3 ", 4 "-the trihydroxy butyl)-pyrazine [2-(arabo-tetrahydroxybutyl)-5-(erythro-2; 3,4-trihydroxylbutyl) pyrazinetrehalose].These 9 chemical compounds are the inventor and find (seeing embodiment 2) first from this funguses.
Adopt HPLC-ELSD that main component mannitol (Mannitol) in this active site and trehalose (Trehalose) are carried out the assay analysis, the result shows that mannitol content is greater than 35% in this position, content of trehalose is greater than 4%, and both are the main component (seeing embodiment 3) of Agaricus blazei Murrill small molecular extract.
The present invention also provides a kind of optimum extraction and preparation technology of Brazil mushroom soluble small molecular extract, abbreviates alcohol extraction process as.
The concrete grammar of this technology and step are: to edible fungi Agaricus blazei Murrill sporophore or artificial culture mycelium, doubly measuring (volume (liter)/weight (kilogram)) concentration with 6-20 is that the alcoholic solution of 15-95% extracts, each extraction time is 1-3 hour, extracts 1-3 time merge extractive liquid, continuously.Through the orthogonal experiment design, determined that optimum extraction process is: used concentration of alcohol is 75%, and its consumption (volume) is 8 times (liter/kilograms) of Agaricus blazei Murrill raw material consumption (weight), extracts 1.5 hours at every turn, extracts continuously 2 times.
After said extracted liquid merges, can pass through to obtain clarifying extracting solution after sucking filtration, ultrafiltration or the centrifugal filtration.If before sucking filtration, ultrafiltration or centrifugal filtration, use a certain amount of clarifier, as 101 fruit juice clarifiers, ZTC1+1 natural clarifying agent etc., then filtration and clarifying effect are even more ideal.Extracting solution after the clarification is carried out ethanol to be reclaimed and concentration; Extracting solution after concentrating again by vacuum drying, spray drying or lyophilization, is obtained Powdered extract, for use in various preparations.Also the extracting solution dilute with water after concentrating can be made into finite concentration and be directly used in liquid preparations (seeing embodiment 4) such as preparing oral liquid etc.
The present invention also provides the another kind of effectively extraction and preparation technique of Brazil mushroom soluble small molecular extract, abbreviates aqueous extraction-alcohol precipitation technology as.
The method of this technology with usually used from plant, extract prepare polysaccharide component method roughly the same.The different just target extracts of this technology is the clear liquor part (containing micromolecule) behind the water extract-alcohol precipitation, rather than precipitation part (containing macromole such as polysaccharide, protein).This preparation technology's concrete steps are: Agaricus blazei Murrill sporophore or mycelium material, doubly measure the water heating extraction of (weight ratio) with 6-20, and heated 1-3 hour at every turn, extract 1-3 time continuously.Merge extractive liquid, is clarified with suction method, ultrafiltration or centrifugal filtration process.After clear liquor was condensed into thick extractum, slowly adding concentration under stirring condition was the ethanol liquid of 50-95%, left standstill more than 8 hours after fully stirring, and obtained clear liquor.Divide and get supernatant, concentrate, be dried to powder, or direct rare water soluble preparation (seeing embodiment 5) that is made into.
The present invention also provides another effective preparation technology of Brazil mushroom soluble small molecular extract, is called for short water and carries ultrafiltration technology.
The water of this technology is carried step and the water of aforementioned decoction and alcohol sedimentation technique, and to put forward step identical, be Agaricus blazei Murrill sporophore or mycelium material, doubly measure the water heating extraction of (weight ratio) with 6-20, each heating 1-3 hour, extract 1-3 times continuously, merging filtrate carries out preliminary defecate and purify with suction method or centrifuging.Being that with above-mentioned alcohol precipitation process difference this law is not to adopt alcohol precipitation process to separate micromolecule and macromole, is that 4,000 ultrafilter membrane carries out ultra-filtration and separation but adopt molecular cut off.Extracting solution behind the above-mentioned defecate and purify, after ultrafilter membrane by molecular weight 4,000 carries out ultrafiltration repeatedly, be not macromolecular polysaccharide on the film by part, clear liquor by film is molecular weight and is lower than 4,000 soluble small molecular extracting solution (extracting flow process sees shown in Figure 1).To directly be mixed with liquid preparation such as oral liquid after this partial concentration, or be prepared into powder, for preparation tablet, capsule, the usefulness of solid preparations such as mixture (seeing embodiment 6) by spraying, vacuum drying or lyophilization.
The present invention has carried out zoopery to above-mentioned Brazil mushroom soluble small molecular extract, shows that this extract is nontoxic, has no side effect, and the growth of tumor cell is had the obvious suppression effect, has tangible immunoloregulation function simultaneously.Concrete result of the test is as follows:
Test example 1: acute toxicity testing
Acute toxicity test in mice is the result show, with Cmax, maximum administration volume is irritated stomach and given mice, and its maximum dosage-feeding is 80.0g/kg, is about 160 times of clinical recommended dose.
Test example 2: Brazil mushroom soluble small molecular extract is to the inhibitory action of mice S180 sarcoma
(1) test objective
The test Brazil mushroom soluble small molecular extract is to the inhibitory action of mice S180 sarcoma growth.
(2) be subjected to the reagent thing
Title: Brazil mushroom soluble small molecular extract (A), the inventor prepares voluntarily.
Preparation: use the distilled water wiring solution-forming
(3) control sample
Polysaccharide-peptide capsule, Shanghai Xinkang Pharmaceutical Factory produces, lot number: 990801, specification: every 0.34g.
(4) animal
Kunming mouse, male, body weight: 18-22g, is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center by 10 every group.
(5) transplanted tumor: mice S180 sarcoma, available from Shanghai cell institute of the Chinese Academy of Sciences.
(6) test method
Get well-grown mice S180 sarcoma ascites, diluted with 1: 4, every the right axil subcutaneous vaccination of mice 0.2ml, random packet with normal saline.Brazil mushroom soluble small molecular extract (A) establishes 0.5,0.25, three dosage groups of 0.125g/kg, polysaccharide-peptide matched group 0.5g/Kg, and administration is played next day in the inoculation back, and the administration volume is the 0.5m1/20g body weight, continuous irrigation stomach 7 days.Inoculate back 10 days and take off neck execution animal, weigh, dissect and get the tumor piece, claim tumor heavy.The result judges according to following formula:
(7) result of the test
Brazil mushroom soluble small molecular extract (A) is respectively 76.11,57.52,59.73% to the suppression ratio of mice S180 sarcoma when dosage 0.5,0.25,0.125g/kg, with matched group tool significant difference relatively.Result of the test sees Table 1 and picture 1.
(8) conclusion (of pressure testing)
Brazil mushroom soluble small molecular extract (A) has the obvious suppression effect to the growth of mice S180 sarcoma.
Test example 3. Brazil mushroom soluble small molecular extracts are to Immune Effects
1. to the influence of tumor-bearing mice spleen lymphocyte proliferation
Table 1. Brazil mushroom soluble small molecular extract (A) is to the tumor-inhibiting action of mice S180 sarcoma
Compare with matched group: * * P＜0.01.
(1) test objective
Observe the effect of Brazil mushroom soluble small molecular extract to the tumor-bearing mice spleen lymphocyte proliferation.
(2) be subjected to the reagent thing
Title and compound method are with test 2.
(3) control sample
With test 2.
(4) animal
Female C57BL/6 mice, body weight: 18-20g is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center.
(5) other material
Culture medium: RPMI-1610, the Difco product includes 15%NBS, mercaptoethanol, Hepes etc.
3H-TdR: Shanghai nuclear research institute, radioactive concentration 1mci/ml.
Con A (ConA): Sigma product, 50ug/ml.
(6) test method
30 of C57BL/6 mices, every Mus axil subcutaneous vaccination Lewis lung cancer cell about 2 * 10
6Individual, be divided into 5 groups at random, 6 every group, i.e. Brazil mushroom soluble small molecular extract (A) 0.125,0.25,0.5g/kg, Polysaccharide-peptide capsule 0.5g/kg, normal saline matched group, all po * 7.Administration finishes the back and puts to death animal, gets spleen under the aseptic condition, the counting splenocyte, and the adjustment cell concentration is 1 * 10
7Individual/ml, every hole adds cell suspension 100ul on 96 well culture plates, ConA 50ul and culture fluid, and each group is all established three multiple holes, 37 ℃, 5%CO
2Cultivated 18 hours under the condition, add
3H-TdR 0.5uci/ hole continues to cultivate 18 hours.With bull cell harvestor collecting cell, on liquid scintillation instrument, survey the CPM value, and compare with matched group, the results are shown in Table 2.
(7) result of the test
Brazil mushroom soluble small molecular extract has the effect of tangible promotion tumor-bearing mice spleen lymphocyte proliferation when 0.125g/kg dosage, along with the increase of dosage, promote the spleen lymphocyte proliferation effect also to strengthen thereupon.
Table 2. Brazil mushroom soluble small molecular extract is to the influence of tumor-bearing mice spleen lymphocyte proliferation
Compare with matched group: * P＜0.05; * P＜0.01
2. Brazil mushroom soluble small molecular extract is to the influence of tumor-bearing mice NK cytoactive
(1) test objective
Observe the oral influence of Brazil mushroom soluble small molecular extract to tumor-bearing mice NK cytoactive
(2) be subjected to the reagent thing
With test 2.
(3) control sample
Polysaccharide-peptide capsule, the source, lot number, specification is the same.
(4) experimental animal
Strain, body weight, sex and originate the same.
(5) other material
Culture medium:
3H-TdR is the same.The YAC-1 cell is provided by Chinese Academy of Sciences's Shanghai cell.
(6) experimental technique
The labelling of target cell: get the back 24 hours well-grown YAC-1 cells that go down to posterity, by 1 * 10
6Individual/ml cell suspension adds
3H-TdR 10uci, in 37 ℃, 5%CO
2Cultivated 2 hours in the incubator, vibrated once in per 30 minutes, the cell behind the labelling is resuspended in the culture fluid with culture fluid washing 3 times, and making cell concentration is 1 * 10
5/ ml.
The NK cytoactive is measured: 30 of C57BL/6 mices, every Mus axil subcutaneous vaccination Lewis lung cancer cell about 2 * 10
6Individual, be divided into 5 groups at random, 6 every group, promptly Brazil mushroom soluble small molecular extract 0.125,0.25,0.5g/kg, Polysaccharide-peptide capsule 0.5g/kg, normal saline matched group, all po * 7.The last administration finishes the back and puts to death animal, gets spleen under the aseptic condition, the preparation splenocyte, and adjusting cell concentration is 1 * 10
6Individual/ml does the effector lymphocyte, and other gets and cultivates 24 hours YAC-1 cells, and adjusting cell concentration is 1 * 10
4Individual/ml is as target cell, is that the cell of 1:100 is added on the 96 porocyte culture plates with target cell and effector lymphocyte's ratio, adds
3Three multiple holes, 37 ℃, 5%CO are all established for every group in the H-TdR0.5uci/ hole
2Cultivate under the condition after 24 hours, collecting cell is surveyed the CPM value, calculates specificity and suppresses percentage rate (Pi) expression NK cytoactive.
(7) experimental result
Successive administration can obviously activate the NK cell activity after 7 days, and dosage and facilitation have certain dependency, activated the polysaccharide-peptide that the NK cell activity is better than Isodose during 0.5g/kg.The results are shown in Table 3.
3. Brazil mushroom soluble small molecular extract is to the influence of tumor-bearing mice IL-2 generation
(1) test objective: observe the influence that Brazil mushroom soluble small molecular extract produces tumor-bearing mice IL-2
(2) be subjected to the reagent thing: the same.
Table 3. Brazil mushroom soluble small molecular extract is to the active influence of tumor-bearing mice NK
Compare with the normal saline group: * * P＜0.01.
(3) control sample
Polysaccharide-peptide capsule, the source, lot number, specification is the same.
(4) experimental animal
Strain, body weight, sex, it is the same to originate.
(5) other material
Culture medium,
3H-TdR is the same.
(6) test method
30 of C57BL/6 mices, every Mus axil subcutaneous vaccination Lewis lung cancer cell about 2 * 10
6Individual, the random packet administration, promptly Brazil mushroom soluble small molecular extract 0.125,0.25,0.5g/kg, polysaccharide-peptide 0.5g/kg, normal saline matched group, all po * 7.The last administration finishes the back and puts to death animal, gets spleen under the aseptic condition, the preparation splenocyte suspension, and adjusting cell concentration is 1 * 10
7Individual/ml, every hole adds 2ml cell and ConA 5ug/ml on 24 orifice plates, 37 ℃ of 5%CO
2Cultivate under the condition and collected supernatant in 24 hours, with IL-2 dependent cell strain CTLL, with
3H-TdR mixes method and measures the IL-2 activity, and compares with matched group.The results are shown in Table 4.
(7) experimental result
The Brazil mushroom soluble small molecular extract successive administration can promote the generation of IL-2 after 7 days, its 0.5g/kg group effect is the most obvious.
Table 4. Brazil mushroom soluble small molecular extract produces the influence of IL-2 to tumor-bearing mice
Compare with the normal saline group: * P＜0.05; * P＜0.01.
4. to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic function
(1) test objective
The test Brazil mushroom soluble small molecular extract is to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic function
(2) be subjected to the reagent thing
The same.
(3) control sample
Polysaccharide-peptide capsule, the source, lot number, specification is the same.
(4) experimental animal
Kunming mouse, body weight: 20-22g, male, provide by Chinese Academy of Sciences's Shanghai animal center.Every treated animal number: 8
(5) test method
Brazil mushroom soluble small molecular extract is established three dosage groups (0.125,0.25,0.5g/kg), other establishes normal saline group, polysaccharide-peptide group (0.5g/kg), continuous oral administration 6 days, the last administration finishes the every Mus lumbar injection 20% chicken erythrocyte suspension 1ml in back, and animal is put to death in the cervical vertebra dislocation after 30 minutes, and the abdominal cavity injects normal saline 2ml, after rotating mice, sucking-off abdominal cavity washing liquid is dripped on microscope slide, after rinsing, fixing, dyeing, carries out the macrophage counting under the oily mirror.
The result judges according to following formula:
(6) result of the test
Normal saline group mice phagocytic percentage and phagocytic index are respectively 22.10% and 0.24; Behind the Brazil mushroom soluble small molecular extract successive administration 6 days, high, medium and low dosage group all can obviously activate the phagocytic function of Turnover of Mouse Peritoneal Macrophages, phagocytic percentage is respectively 58.63%, 46.96% and 36.88%, phagocytic index is respectively 0.64,0.56 and 0.40, the action intensity of high, middle dosage group be better than Polysaccharide-peptide capsule.Result of the test sees Table 5.
(7) conclusion (of pressure testing)
The administration of Brazil mushroom soluble small molecular extract continuous oral is after 6 days, and high, medium and low dosage group all can obviously activate the phagocytic function of Turnover of Mouse Peritoneal Macrophages.
Table 5. Brazil mushroom soluble small molecular extract is to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic function
Compare with the normal saline group: * * P＜0.01.
The present invention also proposes the purposes of described Brazil mushroom soluble small molecular extract, it can be used to prepare medicine and functional health-care food separately, also can with other any Chinese and western drugs or food, especially with some have antitumor and (or) immunoregulatory medicine or food compatibility, be used to prepare medicine and functional health-care food.
Independent medicine and functional health-care food by extract preparation of the present invention, or compound medicine and the functional health-care food formed by this extract and other Chinese and western drugs or food, all have antitumor, regulate body immunity, pharmacologically active such as health invigorating and energy can be used for: all kinds of tumors are prevented and treated in (1); (2) strengthen that tumor is put, the curative effect of chemotherapy; (3) reduce that tumor is put, the toxicity, side effect of chemotherapy; (4) quality of life of raising tumour patient, life-saving; (5) prevent and treat other Ia diseases, as rheumatism, rheumatoid, lupus erythematosus, all kinds of hepatitis, diabetes, asthma, chronic tracheitis etc.; (6) immunity of raising human body, this kind sub-health state is prevented and cured diseases, looks improving and the skin nourishing, life lengthening.
When this extract, or comprise the medicine and the combinations of foods of this extract, when being used for above-mentioned medical treatment and health care purpose, can adopt known method of those skilled in the art and technology, directly, make several formulations finished products such as capsule, tablet, injection, granule, oral liquid, syrup, soft extract, medicated wine, beverage, fruit juice, instant tea, electuary with the adjuvant that adds necessity.When extract of the present invention was made into tablet, the excipient that it contains had: diluent, as starch, dextrin, lactose etc.; Wetting agent or binding agent, as: water, ethanol, starch slurry, dextrin, gelatine size, low-substituted hydroxypropyl cellulose, polyvinylpyrrolidone, Polyethylene Glycol etc.; Disintegrating agent, as: dry starch, gas-producing disintegrant, surfactant etc.; Lubricant is as Pulvis Talci, magnesium stearate, liquid paraffin, polyethylene glycol 6000 or 4000 etc.When extract of the present invention was made into capsule, the excipient that it contains had: diluent, as: starch, dextrin, lactose, magnesium oxide, magnesium carbonate etc.; Wetting agent or binding agent, as: water, ethanol, starch slurry, dextrin slurry, gelatine size, low-substituted hydroxypropyl cellulose, polyvinylpyrrolidone, Polyethylene Glycol etc.; Disintegrating agent, as: dry starch, gas-producing disintegrant, surfactant etc.; And select gelatin hard softgel shell or soft capsule shell for use.When pharmaceutical composition of the present invention was made into injection, the excipient that it contains had: solubilizing agent, as: tween 80, glycerol etc.; Suspensoid, as: hydroxy methocel, polyvinylpyrrolidone, methylcellulose etc.; Antioxidant, as: sodium sulfite, sodium pyrosulfite, sodium thiosulfate etc.; Osmotic pressure regulator is as sodium chloride or glucose etc.; The additives that ease the pain, as: benzyl alcohol, procaine hydrochloride etc.When pharmaceutical composition of the present invention was made into oral liquid or beverage, the excipient that it contains had: aqueous sucrose solution, correctives; Suspending agent is as hydroxy methocel, polyvinylpyrrolidone, methylcellulose etc.; Antiseptic is as Ethyl Hydroxybenzoate or Buddhist nun uncle tortoise beetle ester, propylene glycol, benzoic acid, sorbitol etc.
Description of drawings
Fig. 1 is the ultra-filtration and separation flow chart of Agaricus blazei Murrill hot water extraction liquid.
Fig. 2 is the inhibitory action result of the test of Brazil mushroom soluble small molecular extract to mice S180 sarcoma.
Fig. 3 is the inhibitory action result of the test of each position of Agaricus blazei Murrill hot water extract to mice S180 sarcoma.
The specific embodiment
Embodiment 1 Agaricus blazei Murrill hot water extract's ultra-filtration and separation and antitumor cytolytic activity thereof
1, experiment purpose:
Press the solubility property and the molecular weight size separation Agaricus blazei Murrill hot water extract of chemical compound, determine the anti-tumor activity at each position.
2, ultra-filtration and separation:
Agaricus blazei Murrill material powder 1.0kg, with 1:10 hot water extraction 2 times, each 2 hours.Merge extractive liquid,, the D101 macroporous adsorptive resins (include 1.0kg resin) of clear liquor by handling well in advance got in centrifugal filtration after the standing over night.Allow all purified rinse water post beds by back reuse 3 times of bed volumes of extracting solution.Merga pass liquid and water elution liquid.Get wherein 1/3 eluent earlier, be concentrated into thick extractum (proportion〉1.10), carry out precipitate with ethanol with 75% ethanol, the limit adds the ethanol limit stirs, divide after the standing over night and get precipitation, vacuum drying, water extract-alcohol precipitation part (E).2/3 eluent in addition is that 10,000 film carries out ultrafiltration with molecular cut off earlier, and film top concentrates, vacuum drying, must molecular weight greater than 10,000 part (A).Changing molecular cut off again by liquid is that 4,000 film carries out ultrafiltration, and film top concentrates, vacuum drying, the part (B) of molecular weight 4,000～10,000.Concentrate by liquid, vacuum drying, molecular weight less than 4,000 part (C).Resin column 95% ethanol elution gets 95% ethanol elution part (being the water-insoluble micromolecule) (D).Its extraction separation flow process as shown in Figure 1.
3, each position (A, B, C, D, E) is to the inhibitory action of mice S180 sarcoma growth
Animal: Kunming mouse, male, body weight: 18-22g, is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center by 10 every group.
Transplanted tumor: mice S180 sarcoma, available from Shanghai cell institute of the Chinese Academy of Sciences.
The contrast medicine: Polysaccharide-peptide capsule, Shanghai Xinkang Pharmaceutical Factory produces, lot number: 990801, specification: every 0.34g.
Coriolous Dersicolor (Fr.) Quel elite capsule, BAICAO hall company limited, lot number: 3YZ11057, specification: every 0.4g.
Test method: get well-grown mice S180 sarcoma ascites, diluted with 1: 4, every the right axil subcutaneous vaccination of mice 0.2ml, random packet with normal saline.A, B, C, D respectively establish a dosage group, 0.5g/Kg; E establishes 0.5g/Kg and two dosage groups of 1.5g/Kg; Polysaccharide-peptide and Coriolous Dersicolor (Fr.) Quel elite matched group are respectively established 0.5g/Kg.Administration is played next day in the inoculation back, and the administration volume is the 0.5ml/20g body weight, continuous irrigation stomach 7 days.Inoculate back 10 days and take off neck execution animal, weigh, dissect and get the tumor piece, claim tumor heavy.The result judges according to following formula;
Result of the test: A, B, C, D, each position of E are respectively 27.45,30.39 to the suppression ratio of mice S180 sarcoma when dosage 0.5g/kg, and 50.33,41.50 and 48.37%, with matched group tool significant difference relatively.It is active best wherein to be lower than 4,000 soluble small molecular part (C) with molecular weight.Result of the test sees Table 6 and picture 2.
The separation and the evaluation of embodiment 2. Brazil mushroom soluble small molecular chemical compounds
1. extract and separate
The dry sporophore 1Kg of Agaricus blazei Murrill, be ground into powder just, with 8 liters of reflux, extract, of 75% ethanol 2 times, each 2 hours, thin up filtered to 3000mL after 50 ℃ of extracting solution were evaporated to about 500mL, and filtrate is earlier with D101 macroporous adsorptive resins absorption weeding of grease solubility impurity, by also being concentrated into 500mL, low temperature
Each position of table 6. Agaricus blazei Murrill water extraction is to the tumor-inhibiting action of mice S180 sarcoma
Compare with matched group: * P＜0.05; * P＜0.01.
Separate out white crystals after the placement.Crystallization is by getting Compound I behind Sephadex LH-20 purification; Mother solution carries out column chromatography repeatedly through SephadexLH-20, with first-pure water gradient elution, obtains Compound I I～IX successively.
2. compound structure is identified
Compound I: colourless needle, soluble in water, be insoluble in methanol.EI/MS m/z：183[M+H]
+，165[M+H-1H
2O]
+，147[M+H-2H
2O]
+，129[M+H-3H
2O]
+；
1H NMR(500MHz，DMSO-d
6 δ：Hz)：4.39(2H，d，J＝5.5Hz，OH-2，5)，4.30(2H，t，J＝5.5Hz，OH-1，6)，4.11(2H，d，J＝7.0Hz，OH-3，4)，3.60(2H，m，H-1
a，6
a)，3.54(2H，t，J＝7.OHz，H-3，4)，3.46(2H，m，H-2，5)，3.37(2H，m，H-1
b，6
b)；
13C NMR(500MHz，DMSO-d
6，δ：Hz)：71.2(C-2，5)，69.6(C-3，4)，63.8(C-1，6)。Above data are consistent with reported in literature, are mannitol (mannitol).
Compound I I: colourless needle, soluble in water, methanol, ethanol.
1H NMR (500MHz, D
2O δ: Hz): 7.42,7.40,7.38,7.36,7.35,7.31,7.30 (5H, single-substituted rings), 3.93 (1H, t, J=8.3Hz, H-2), 3.26 (1H, dd, J
1=15.0Hz, J
2=8.3Hz, H-3
a), 3.08 (1H, dd, J
1=15.OHz, J
2=8.3Hz, H-3
b);
13C NMR (500MHz, D
2O, δ: Hz): 175.5 (C-1), 137.2 (C-1 '), 131.4 (C-3 ', 5 '), 131.1 (C-2 ', 6 '), 129.7 (C-4 '), 58.0 (C-2), 38.4 (C-3).Above data are consistent with reported in literature, are phenylalanine (phenylalanine).
Compound III: colourless needle, soluble in water, methanol, ethanol.
1H NMR (500MHz DMSO-d
6, δ: Hz): 8.35 (s, H-8), 8.14 (s, H-2), 7.35 (2H, s add D
2O disappears, NH
2), 5.88 (J=6.2Hz), 5.43 (1H, OH-2 ' add D for 1H, d
2O disappears), 5.42 (1H, OH-3 ' add D
2O disappears), 5.18 (1H, 0H-5 ' add D
2O disappears), 4.60 (1H, dd, J
1=6.2Hz, J
2=5.7Hz, H-2 '), 4.14 (1H, dd, J
1=5.7Hz, J
2=4.2Hz, H-3 '), 3.96 (1H, dt, J
1=4.2Hz, J
2=3.1Hz, H-4 '), 3.67 (1H, dd, J
1=12.0Hz, J
2=3.1Hz, H-5 '
a), 3.55 (1H, dd, J
1=12.OHz, J
2=3.1Hz, H-5 '
b);
13C NMR (500Hz, D
20, δ: Hz): 156.6 (C-6), 153.1 (C-2), 149.7 (C-4), 140.7 (C-8), 119.9 (C-5).Above data are consistent with reported in literature, are ribosidoadenine (adenosine).
Compound IV: white indefiniteness powder, soluble in water.UV
(nm)：210；274.8；IR
(cm
-1)：3284、2935、1537、1047、1018、876；ESIMS(m/z)：327.6[M+Na]
+；
1H NMR(500MHz DMSO-d
6，δ：Hz)：8.54(1H，s，H-3)，8.33(1H，s，H-5)，5.33(1H，d，J＝4.2Hz，OH-1’)，4.94(1H，bs，H-1’)，4.72(1H，bs，OH-4″)，4.67(1H，bs，OH-3’)，4.62(1H，d，J＝5.7，OH-2″)，4.43(1H，bs，OH-2’)，4.41(1H，bs，OH-3″)，4.39(1H，bs，OH-6’)，3.74(1H，m，H-3’)，3.64(1H，m，H-4’
a)，3.62(1H，m，H-2’)，3.60(1H，m，H-2″)，3.56(1H，m，H-4″
a)，3.45(1H，m，H-4’
b)，3.41(1H，m，H-4″
b)，3.38(1H，m，H-3″)，3.06(1H，dd，J
1＝14.0Hz，J
2＝2.1Hz，H-1’’
b)，2.71(1H，dd，J
1＝14.0HZ，J
2＝9.7Hz，H-1”
a)；
13C NMR(500MHZ，DMSO-d
6，δ：ppm)：158.22(C-2)、141.02(C-3)、143.35(C-5)、154.36(C-5)、72.0(C-1’)、74.3(C-2’)、71.8(C-3’)、64.0(C-4’)、39.2(C-1”)、71.7(C-2”)、75.5(C-3”)、63.7(C-4”)。HSQC[DMSO-d
6, 500MHZ δ: ppm, charcoal spectrum (hydrogen related spectrum)]: 143.4 (8.54), 141.0 (8.33), 75.5 (3.38), 74.3 (3.62), 72.0 (4.94), 71.8 (3.74), 71.7 (3.60), 64.0 (3.64,3.45), 63.7 (3.56,3.41), 39.2 (2.71; 3.06); HMBC is shown as 2,5 and replaces pyrazine.Above data core bibliographical information unanimity, for deoxyfructosazine[2-(arabo-tetrahydroxybutyl)-5-(erythro-2,3,4-trihydroxylbutyl) pyrazinetrehalose].
Chemical compound V: white needle, soluble in water.
1H NMR(500MHz DMSO-d
6，δ：Hz)：4.87(2H，d，J＝3.6Hz，H-1，1’)，4.77(2H，t，J＝5.7Hz，OH-4，4’)，4.76(2H，t，J＝5.7Hz，OH-2，2’)，4.60(2H，d，J＝6.2Hz，OH-3，3’)，4.35(2H，t，J＝5.7Hz，OH-6，6’)，3.64(2H，m，H-5，5’)，3.56(2H，m，H-4，4’)，3.54(2H，m，H-6a，6’
a)，3.47(2H，m，H-3，3’)，3.23(2H，m，H-6b，6’
b)，3.12(2H，dd，J
1＝5.7HZ，J
2＝3.6Hz，H-2，2’)；
13C NMR(500MHZ，DMSO-d
6，δ：ppm)：93.6(C-1)，73.4(C-3)，72.9(C-2)，72.1(C-4)，70.6(C-5)，61.3(C-6)。Above data core bibliographical information unanimity is trehalose (trehalose).
Compound VI: white crystallization on chip, soluble in water.
1H NMR(500MHz，DMSO-d
6，δ：Hz)：4.09(1H，dd，J
1＝9.0Hz，J
2＝4.4Hz，H-2)，2.32(1H，m，H-4
a)，2.13(2H，m，H-3)，1.96(1H，m，H-4
b)，
13C NMR(500MHz，DMSO-d
6，δ ppm)：178.9(C-5)，176.2(C-1)，56.6(C-2)，30.8(C-4)，26.4(C-3)。Above data core bibliographical information unanimity is glutamic acid (glutamic acid).
Compound VI I: white needle, soluble in water, methanol.
1H NMR(500MHz，DMSO-d
6，δ：Hz)：7.39(1H，d，J＝7.6Hz，H-5)，5.44(1H，d，J＝7.6Hz，H-6)；
13C NMR(500MHZ，DMSO-d
6，δ：ppm)：166.1(C-4)，153.3(C-2)，144.0(C-6)，102.0(C-5)。Above data core bibliographical information unanimity is uracil (Uracil).
Compound VIII: white needle, soluble in water, methanol.
1H NMR(500MHz，D
2O，δ：Hz)：7.75(1H，d，J＝8.0Hz，H-5)，5.78(1H，d，J＝5.7Hz，H-1’)，5.76(1H，d，J＝8.0Hz，H-6)，4.33(1H，dd，J
1＝5.7Hz，J
2＝5.2Hz，H-2’)，4.21(1H，dd，J
1＝5.7Hz，J
2＝5.2Hz，H-3’)，4.12(1H，m，H-4’)，3.90(1H，dd，J
1＝12.8Hz，J
2＝2.4Hz，H-5’
a)，3.79(1H，dd，J
1＝12.8Hz，J
2＝4.2Hz，H-5’
b)；
13C NMR(500MHZ，D
2O，δ：ppm)：168.1(C-4)，153.6(C-2)，143.7(C-6)，104.2(C-5)，91.3(C-1’)，86.2(C-4’)，75.6(C-3’)，71.4(C-2’)，62.7(C-5’)。Above data core bibliographical information unanimity is uridine (uridine).
Compound I X: white needle, soluble in water, methanol.
1H NMR(500MHz，D
2O，δ：Hz)：7.75(1H，d，J＝8.0Hz，H-5)，5.76(1H，d，J＝8.0Hz，H-6)，4.71(1H，t，J＝4.6Hz，H-1’)，4.53(1H，m，H-3’)，4.25(1H，m，H-4’)，3.89(1H，dd，J
1＝12.8Hz，J
2＝3.0Hz，H-5’
a)，3.78(1H，dd，J
1＝12.8Hz，J
2＝3.0Hz，H-5’
b)，2.84(1H，dd，J
1＝18.3Hz，J
2＝5.9Hz，H-2’
a)，1.82(1H，dd，J
1＝18.3Hz，J
2＝5.9Hz，H-2’
b)；
13C NMR(500MHZ，D
2O，δ：ppm)：168.1(C-4)，153.6(C-2)，143.7(C-6)，104.2(C-5)，82.1(C-4’)，79.0(C-1’)，69.2(C-3’)，66.7(C-5’)，39.6(C-2’)。Above data core bibliographical information unanimity is deoxyuridine (deoxyuridine).
The content of mannitol and trehalose in the embodiment 3.HPLC-ELSD method mensuration Brazil mushroom soluble small molecular extract
1, instrument and reagent;
Angilent 1100 chromatographs comprise the G1311A quaternary pump, the online degasser of G1322A, PL-ELS1000 ELSD detector, HP ChemStation (Rev.A.07.01) liquid chromatograph work station.
Reference substance mannitol, trehalose are this chamber preparation, and purity is greater than 98%;
Acetonitrile, water are chromatographically pure.
2, assay method:
(1) chromatographic condition: chromatographic column Waters Spherisorb NH
2(4.6 * 250nm, 5 μ m); Mobile phase 75% acetonitrile: 25%water; Flow velocity: 1.2mL/min.
(2) standard curve: accurately take by weighing mannitol and each 200.0mg of trehalose of being dried to constant weight, be dissolved in after the mixing in an amount of distilled water, add water and be settled to 50ml as operating level liquid.Get 1.0,2.0,3.0,4.0,6.0 and 8.0mL operating level liquid, add water and be settled to 10mL; Other gets 2.5mL and 5.0mL operating level liquid, adds water and is settled to 100mL, is made into the series standard product solution of content 100-4000 μ g/mL.Analyze sample size 20 μ L with above-mentioned chromatographic condition.With the peak area is vertical coordinate, and (μ g/mL) is abscissa with sample concentration, and the drawing standard curve calculates regression equation and correlation coefficient r.The regression equation that records mannitol is Y=0.00082876X
2+ 2.81355X-195.19, r=0.9999; The regression equation of trehalose is Y=0.00036772X
2+ 1.84019X-52。39314，r＝0.9999。
(3) precision experiment: take by weighing Brazil mushroom soluble small molecular extract sample 0.5g and be dissolved in the 100mL distilled water, fixed molten to scale.Therefrom essence is got 8.0mL, is settled to 10ml with distilled water, with 5 parts of method preparations.Sample introduction 20uL analyzes by above-mentioned condition respectively, and measurement result is as follows:
(4) response rate experiment: get above-mentioned sample solution 8mL, add 14.0mg mannitol and 13.0mg trehalose, distilled water is settled to 10ml, is equipped with 5 parts with legal system.Measure with method, get the response rate and be respectively 102.8% and 105.8%.
(5) stability experiment; Get the sample of preparation in above-mentioned (3), measure content respectively at 0min, 30min, 60min, 2h, 4h and 8h, measurement result is as follows:
Precision experimental result response rate experimental result
The stability experiment result
3. measurement result
Take by weighing Brazil mushroom soluble small molecular extract sample 500.0mg, with dissolved in distilled water and fixed molten to 100mL, sample introduction 20 μ L, the method for using is measured.Measure 5 times with method, recording its meansigma methods is mannitol 37.8%; Trehalose 4.62%,
The alcohol extraction process of embodiment 4. Brazil mushroom soluble small molecular extracts
1. the orthogonal experiment optimization of extraction process:
(1) Orthogonal Experiment and Design:
A: concentration of alcohol
A
1 0％；A
2 15％；A
3 30％；A
4 45％；A
5 60％；A
6 75％；A
7 85％；A
8 95％.
B: sample (kilogram)/quantity of solvent (liter)
B
1 1:6；B
2 1:8
C: extraction time (h)
C
1 1.5；C
2 3.0
D: extraction time
D
1 2；D
2 3.
According to above factor and level design L
16(8 * 2
8) orthogonal table sees Table 7.
Table 7 L
16(8 * 2
8) the gauge outfit design
| 1 2 3 4 5 6 7 8 9 A B C D | |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 | 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 1 1 3 1 1 2 2 1 1 2 2 3 2 2 1 1 2 2 1 1 4 1 1 2 2 2 2 1 1 4 2 2 1 1 1 1 2 2 5 1 2 1 2 1 2 1 2 5 2 1 2 1 2 1 2 1 6 1 2 1 2 2 1 2 1 6 2 1 2 1 1 2 1 2 7 1 2 2 1 1 2 2 1 7 2 1 1 2 2 1 1 2 8 1 2 2 1 2 1 1 2 8 2 1 1 2 1 2 2 1 |
(2) judge index and measurement result:
Evaluation of this orthogonal experiment and judge index are the total amount of the content of trehalose in the extractum and trehalose, glucose, mannitol.Its content all adopts HPLC-ELSD method to measure, and concrete assay method is seen embodiment 3.Experimental result sees Table 8:
(3) variance analysis:
According to above index component content measurement result, carry out variance analysis with the total amount of trehalose and trehalose, glucose and mannitol respectively, the results are shown in Table 9 and table 10.
(4) experimental result: the The results of analysis of variance of table 9 and table 10 shows, no matter be to be separately evaluation index with the trehalose, still the total amount with trehalose, glucose and mannitol is an evaluation index, and Orthogonal experiment results all shows A6, B2, C1/C2, D2 is an optimum process condition, promptly uses 75% ethanol, and consumption is 1:8, extraction time is 1.5～3 hours, extracts continuously 2 times.
2. according to the definite alcohol extraction preparation technology of orthogonal experiment
Agaricus blazei Murrill sporophore coarse powder 1.0Kg extracts with 8 liter 75% alcoholic solution, and each extraction time is 1.5 hours, extracts merge extractive liquid, continuously 2 times.In extracting solution, add 5% ZTC1+1 natural clarifying agent, leave standstill after fully stirring and put coldly, filter with centrifugal filtration process and to obtain clarifying extracting solution.Extracting solution after the clarification reclaims ethanol and is concentrated to thick extractum (proportion～1.10) with Rotary Evaporators, with this extractum vacuum drying oven drying, pulverizes, and promptly gets Brazil mushroom soluble small molecular extract 153g.
Table 8 is respectively organized experimental index composition measurement result
| No. | Extractum content of trehalose (0.01g) trehalose, glucose, mannitol total content (0.01g) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 | 3.6622 255.8718 257.2306 256.5512 4.1247 152.0225 152.2165 152.1195 291.1428 288.2532 289.698 3.7349 160.0411 156.2394 158.1403 277.9825 273.5839 275.7832 3.7762 157.1902 158.475 157.8326 271.2742 276.9974 274.1358 3.833 160.4048 160.6231 160.5139 276.2048 276.3011 276.253 3.9064 161.7486 158.6477 160.1982 277.3642 281.3509 279.3576 4.3116 171.9223 172.7599 172.3411 311.8061 317.3923 314.5992 3.8596 155.4669 151.1828 153.3249 277.1263 272.8327 274.9795 4.0872 168.5316 171.4922 170.0119 295.5863 296.4133 295.9998 4.0354 168.3896 163.7394 166.0645 300.0441 295.1978 297.6209 3.7791 171.5623 173.5924 172.5773 290.6255 295.6512 293.1384 4.2435 184.3077 193.2801 188.7939 312.2091 326.4581 319.3336 3.7931 194.7373 194.3611 194.5492 318.8485 318.6545 318.7515 2.8245 120.467 123.2558 121.8614 226.8674 232.3605 229.6139 2.4524 95.74547 98.79878 97.27213 192.6088 195.9428 194.2758 3.2339 146.2279 151.2973 148.7626 259.1611 268.2342 263.6976 |
Table 9. is the variance analysis of index with the trehalose
Table 10. is the variance analysis of index with trehalose, glucose and mannitol summation
The aqueous extraction-alcohol precipitation technology of embodiment 5. Brazil mushroom soluble small molecular extracts
Agaricus blazei Murrill sporophore coarse powder 1.0Kg extracts with 10 liters water, extracts 1.5 hours at every turn, extracts merge extractive liquid, continuously 2 times.In extracting solution, add 5% ZTC1+1 natural clarifying agent, leave standstill after fully stirring and put coldly, filter with centrifugal filtration process and to obtain clarifying extracting solution.This extracting solution adds 75% ethanol liquid 5000mL, standing over night after being concentrated to thick extractum (proportion～1.10) with Rotary Evaporators while stirring.Inclining supernatant, after reclaiming ethanol and be concentrated to thick extractum (proportion～1.10) with Rotary Evaporators, uses the vacuum drying oven drying, pulverizes, and promptly gets Brazil mushroom soluble small molecular extract 112g.
The water of embodiment 6. Brazil mushroom soluble small molecular extracts is carried ultrafiltration technology
Agaricus blazei Murrill sporophore coarse powder 1.0Kg extracts with 10 liters water, extracts 1.5 hours at every turn, extracts merge extractive liquid, continuously 2 times.In extracting solution, add 5% ZTC1+1 natural clarifying agent, leave standstill after fully stirring and put coldly, filter with centrifugal filtration process and to obtain clarifying extracting solution.Extracting solution is pumped in the ultrafilter, with molecular cut off is 4,000 ultrafilter membrane carries out ultrafiltration, after several is carried out in circulation, collect clear liquor, be concentrated to thick extractum (proportion～1.10), reuse vacuum drying oven drying with Rotary Evaporators by film, pulverize, promptly get Brazil mushroom soluble small molecular extract 128g.
Embodiment 7. contains the preparation of Brazil mushroom soluble small molecular extract oral liquid:
Agaricus blazei Murrill sporophore coarse powder 1.0Kg extracts with 8 liter 75% alcoholic solution, and each extraction time is more than 1.5 hours, extracts merge extractive liquid, continuously 2 times.In extracting solution, add 5% ZTC1+1 natural clarifying agent, leave standstill after fully stirring and put coldly, filter with centrifugal filtration process and to obtain clarifying extracting solution.Extracting solution after the clarification reclaims ethanol and is concentrated to thick extractum (proportion～1.10) with Rotary Evaporators, and this extractum is diluted to 1000mL with water for injection, adds saccharin sodium 0.8g, makes dissolving, left standstill 24 hours, and filtered, add water and adjust total amount, stir evenly to 1000mL, fill, sterilization, promptly.
The compound capsule that embodiment 8. is made up of Brazil mushroom soluble small molecular extract and ganoderan preparation:
Brazil mushroom soluble small molecular extract 300g
Ganoderan extract (content 56%) 200g
The said components mix homogeneously, in the hard gelatin capsule of packing into, totally 1000 capsules.
Claims (8)
1, a kind of soluble small molecular extract of Agaricus blazei Murrill obtains from the mycelium of the sporophore of edible fungi Agaricus blazei Murrill or its artificial culture, it is characterized in that being lower than 4,000 micromolecular compound by water miscible, molecular weight forms, and is extracting solution or dried powder.
2, extract according to claim 1 is characterized in that, contains following 9 kinds of compositions in this extract: mannitol, trehalose, phenylalanine, glutamic acid, gland fast morpholine nucleoside, uracil, uridine, deoxyuridine and 2,6-deoxy fructosazine.
3, a kind of preparation technology of Brazil mushroom soluble small molecular extract as claimed in claim 1, it is characterized in that, the mycelium of the sporophore of edible fungi Agaricus blazei Murrill or artificial culture adopted 75% ethanol extract, the ethanol consumption is a per kilogram Agaricus blazei Murrill material with 6-20 liter 75% ethanol, extraction time is 1-3 hour, extraction time is 1-3 time, obtains extracting solution.
4, preparation technology according to claim 3 is characterized in that, after this extracting solution process filtration, ethanol recovery and the concentration, adopts vacuum drying, spray drying or freeze-drying to make dried powder.
5, a kind of preparation technology of Brazil mushroom soluble small molecular extract as claimed in claim 1, it is characterized in that, adopt hot water to extract to edible fungi Agaricus blazei Murrill sporophore or artificial culture mycelia, the consumption of water is 6-20 a times of Agaricus blazei Murrill weight, be 1-3 hour heat time heating time, and extraction time is 1-3 time; Crude extract filtered with sucking filtration, ultrafiltration or centrifuging clarify, clear liquor is condensed into thick extractum, thick extractum is carried out precipitate with ethanol with the ethanol of 50-95%, fully leave standstill more than 8 hours after the stirring, obtain the clarification extracting solution; Be that 4,000 ultrafilter membrane carries out ultra-filtration and separation perhaps, obtain the clarification extracting solution the extracting solution molecular cut off.
6, preparation technology according to claim 5 is characterized in that, this clarification extracting solution is made dried powder with vacuum drying, spray drying or freeze-drying after reclaiming, concentrating.
7, a kind of application of extract as claimed in claim 1 is characterized in that, the extracting solution of this extract or dried powder, separately or with other Chinese and western drugses or food compatibility, be used to prepare the medicine or the functional health-care food of prevention and treatment tumor disease.
8, the application of extract according to claim 7, it is characterized in that, the extracting solution of this extract or dried powder, or comprise the medicine and the combinations of foods of this extracting solution or dried powder, make capsule, tablet, injection, granule, oral agents, syrup, soft extract, medicated wine, electuary and beverage.
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