CN102260340B - Chinese magnoliavine protein, and preparation method and medicinal application thereof - Google Patents
Chinese magnoliavine protein, and preparation method and medicinal application thereof Download PDFInfo
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Abstract
The invention provides a Chinese magnoliavine protein, and a preparation method and medicinal application thereof, belonging to the field of traditional Chinese medicines. The extraction method comprises the following steps: pulverizing Chinese magnoliavine fruit or Chinese magnoliavine seed into fine powder, extracting by directly adding solvent, filtering, and treating the filtrate by a membrane filtration method or saturation precipitation method by adding solid ammonium sulfate; and directly carrying out freeze-drying on the product obtained by the membrane filtration method, or carrying out freeze-drying on the product obtained by the saturation precipitation method by adding solid ammonium sulfate after dialysis, thereby obtaining the Chinese magnoliavine protein. The protein content of the Chinese magnoliavine protein is higher than 50%. The corresponding preparation, which is prepared by using the Chinese magnoliavine protein as the main active ingredient and being assisted by a pharmaceutical acceptable carrier or auxiliary materials, can enhance the immunity of the organism, and has a certain anti-hypoxia action.
Description
Technical field
The invention belongs to the field of Chinese medicines, relate to a kind of albumen that obtains that extracts from schisandra fruit and shizandra berry seed, this protein extract has enhancing body immunizing power and has certain resisting oxygen lack human body.
Background technology
Shizandra berry is the dry mature fruit of magnoliaceae schisandra [Schisandra chinensis (Turcz.) Baill.] and schisandra chinensis [Schisandra sphenanthera Rehd.et Wils.].The former is recorded by Chinese Pharmacopoeia 2005 and one one of version in 2010 and is shizandra berry (being commonly called as Schisandra chinensis), and the latter records and is kadsura longepedunculata.Herbal textual, shizandra berry begin to be stated from Shennong's Herbal, classify as top grade.Shizandra berry is traditional medicinal fruit, and is warm in nature, flavor acid, sweet.Has convergence astringent or styptic treatment for spontaneous sweating, supplementing QI for promoting the production of body fluid, the effect of kidney calming.The clinical diseases such as chronic cough void is breathed heavily, emission, enuresis frequent micturition, chronic diarrhea, spontaneous perspiration, night sweat, neurasthenia, hepatitis that are used for the treatment of.
At present, research to shizandra berry mainly concentrates in the research of lignan component, modern chemistry and pharmacological research show, contain a large amount of lignanoids chemical compositions in the shizandra berry, 32 kinds of Lignanoids compounds monomers now from shizandra berry, have been isolated, found clear and definite pharmacological action for wherein 7 kinds, their chemical structure all belongs to cyclohexyl biphenyl octene class, pharmacological research and clinical proof: 1, have the effect that reduces the hepatitis serum GPT levels; 2, by oxyradical is removed, suppress the lipid peroxidation generation thereby reach, alleviate lipid peroxide to the damage of brain cell, brain cell is had provide protection, make brain have obvious antifatigue and oxygen lack resistant function, improve memory; 3, shizandra berry effective constituent can increase under the threshold sleep dosage pentobarbital sodium in mice sleep number of elements, prolongs the dosage pentobarbital sodium in mice length of one's sleep of above threshold sleeping, and has obvious improvement sleep, the effect of tranquilizing and allaying excitement.More for this bibliographical information to the extracting method of Lignans in Schisandra chinensis constituents and pharmacologically active (liver protecting, tranquilizing and allaying excitement), patent relates to extensively.
Summary of the invention
The invention provides a kind of shizandra berry albumen and preparation method thereof and pharmaceutical use.It is that shizandra berry or shizandra berry seed are pulverized, and through extracting, dialysis method after utilization membrane separation technique or the saturated precipitation of reinforcing body ammonium sulfate obtains highly purified water-soluble protein.Protein content reaches more than 50%, take this vegetable-protein as activeconstituents or auxiliary material can prepare oral drug and the protective foods of various formulations.The technical scheme that the present invention takes is:
(1) schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80～100 order fine powders;
(2) material after will pulverizing, the solvent that adds 5～15 times of amounts soaks in 1 ℃～10 ℃ and extracts 12～24h, or supersound extraction 0.5～2h, or microwave extraction 0.5～2h, adopts same procedure same solvent consumption to extract united extraction liquid 1～4 time;
(3) purifying of albumen:
After the extracting solution that merges is the tubular fibre membrane filtration of 0.22～0.65 μ m through membrane pore size, be the tubular fibre membrane ultrafiltration of 10～100kDa through molecular weight cut-off again, freeze-drying gets shizandra berry albumen;
Maybe with the extracting solution that merges, reinforcing body ammonium sulfate is to saturated, and 1～10 ℃ of static 12～24h gets precipitation after centrifugal, and is centrifugal with the dissolving of Tris-HCl damping fluid, filters, and gets subsequent filtrate, and through dialysis, freeze-drying gets shizandra berry albumen.
The present invention also provides the pharmaceutical preparation with shizandra berry albumen of the present invention and pharmaceutically acceptable carrier or vehicle preparation.These pharmaceutical preparations are selected from following formulation: soft capsule, hard capsule, microcapsule, microspheres agent, pill, tablet, oral liquid, powder, freeze-dried preparation, injection etc. can also be prepared into slowly-releasing or controlled release preparation as required.
Magnolia vine fruit kernel albumen of the present invention, when useful in preparing drug formulations, can add the medicine acceptable carrier, described medicine acceptable carrier is from oxidation inhibitor, the chelating agent, tensio-active agent, weighting agent, disintegrating agent, wetting agent, solvent, slow-release material, enteric material, pH adjusting agent, correctives, pigment etc., common carrier is such as mannitol, dextran, lactose, glucose, sorbyl alcohol, Xylitol, water for injection, injection ethanol, sodium-chlor, silicon derivative, Mierocrystalline cellulose, derivatived cellulose, gelatin, polyvinylpyrrolidone, glycerine, tween 80, agar, calcium carbonate, polyoxyethylene glycol, cyclodextrin, the phospholipid material, talcum powder, Magnesium Stearate, calcium stearate etc.
Extracting and preparing technique according to claim 3 is characterized in that: extract solvent and be 0.01mol/L Tris-HCl damping fluid, the pH=7.4 of distilled water, pH=7.4 the 0.01mol/L phosphoric acid buffer, contain a kind of solvent in the 0.01mol/L Tris-HCl damping fluid of pH=7.4 of 0.1～0.3mol/LNaCl.
Extracting and preparing technique according to claim 3 is characterized in that: extracting method is one or more technique combinations of the lixiviate of solvent room temperature, supersound extraction or microwave extraction.
Extracting and preparing technique according to claim 3 is characterized in that: extracting solution is with a kind of purifying that carries out albumen of step 3 or step 4.
The present invention is take schisandra fruit and shizandra berry seed as raw material, therefrom extract and isolated shizandra berry albumen, by studies show that this albumen has enhancing body immunizing power, radioprotective, antioxygenation, and have certain antifatigue, hypoxia tolerance, anti-inflammatory, analgesic activity.And preparation shizandra berry albumen and utilize the product of shizandra berry albumen so far there are no report and produce.This beneficial effect of the invention has been filled up the blank of preparation shizandra berry albumen and has been expanded its range of application at field of medicaments, thereby has improved the competitive power of such preparation in the world market.
Description of drawings
Fig. 1 is the comparison diagram of shizandra berry albumen Different Extraction Method, among the figure:
Mark: low molecular mass standard protein, I: embodiment 11 gained albumen, II: embodiment 12 gained albumen, III: embodiment 14 gained albumen;
Fig. 2 A is the HPLC figure of 17 seed amino acid standard substance mixtures, among the figure: 1-Asp; 2-Glu; 3-Ser; 4-Ser; 5-Arg; 6-Gly; 7-Th; 8-Pro; 9-Ala; 10-Val; 11-Cys; 12-Met; 13-Ile; 14-Leu; 15-Phe; 16-Lys; 17-Try,
Fig. 2 B is embodiment 11 gained shizandra berry albumen HPLC figure, among the figure: 1-Asp; 2-Glu; 3-Ser; 4-Ser; 5-Arg; 6-Gly; 7-Th; 8-Pro; 9-Ala; 10-Val; 11-Cys; 12-Met; 13-Ile; 14-Leu; 15-Phe; 16-Lys; 17-Try;
Fig. 2 C is embodiment 12 gained shizandra berry albumen HPLC figure, among the figure: 1-Asp; 2-Glu; 3-Ser; 4-Ser; 5-Arg; 6-Gly; 7-Th; 8-Pro; 9-Ala; 10-Val; 11-Cys; 12-Met; 13-Ile; 14-Leu; 15-Phe; 16-Lys; 17-Try;
Fig. 2 D is embodiment 14 gained shizandra berry albumen HPLC figure, among the figure: 1-Asp; 2-Glu; 3-Ser; 4-Ser; 5-Arg; 6-Gly; 7-Th; 8-Pro; 9-Ala; 10-Val; 11-Cys; 12-Met; 13-Ile; 14-Leu; 15-Phe; 16-Lys; 17-Try;
Fig. 3 is shizandra berry albumen is engulfed toluylene red on Turnover of Mouse Peritoneal Macrophages the figure that affects.
Embodiment
Embodiment 1:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80 order fine powders;
2, the material 100g after will pulverizing, the 0.01mol/L Tris-HCl damping fluid that adds the pH=7.4 of 5 times of amounts soaks in 1 ℃ of temperature and extracts 24h, adopts same procedure same solvent consumption to extract united extraction liquid 4 times;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.22 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of the 0.01mol/LTris-HCl damping fluid of pH=7.4, be the tubular fibre membrane ultrafiltration of 100kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen;
After working method described in the above-mentioned steps 2 is extracted for the first time, in the centrifugal 10～60min of 5000～10000r/min, get supernatant liquor, then add said extracted liquid in the residue after extract, extract so again united extraction liquid 3 times.
Embodiment 2:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 90 order fine powders;
2, the material 100g after will pulverizing, the 0.01mol/LTris-HCl damping fluid that adds the pH=7.4 that contains 0.1～0.3mol/L NaCl of 10 times of amounts soaks in 5 ℃ of temperature and extracts 18h, adopts same procedure same solvent consumption to extract united extraction liquid 3 times;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.45 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of the 0.01mol/LTris-HCl damping fluid of pH=7.4, be the tubular fibre membrane ultrafiltration of 50kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 3:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 100 order fine powders;
2, the material 100g after will pulverizing, the distilled water that adds 15 times of amounts soaks in 10 ℃ of temperature and extracts 24h, collects extracting solution;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.45 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of the 0.01mol/LTris-HCl damping fluid of pH=7.4, be the tubular fibre membrane ultrafiltration of 10kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 4:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 100 order fine powders;
2, the material 100g after will pulverizing adds the distilled water of 15 times of amounts in 1 ℃ of supersound extraction 2h of temperature, adopts same procedure same solvent consumption to extract united extraction liquid 1 time;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.65 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of distilled water, be the tubular fibre membrane ultrafiltration of 50kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 5:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 90 order fine powders;
2, the material 100g after will pulverizing adds the distilled water of 10 times of amounts in 4 ℃ of supersound extraction 1.2h of temperature, adopts same procedure same solvent consumption to extract united extraction liquid 2 times;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.22 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of distilled water, be the tubular fibre membrane ultrafiltration of 10kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 6:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80 order fine powders;
2, the material 100g after will pulverizing adds the distilled water of 5 times of amounts in 10 ℃ of supersound extraction 0.5h of temperature, adopts same procedure same solvent consumption to extract united extraction liquid 4 times;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.45 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of distilled water, be the tubular fibre membrane ultrafiltration of 100kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 7:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80 order fine powders;
2, the material 100g after will pulverizing adds the 0.01mol/L phosphoric acid buffer of pH=7.4 of 5 times of amounts in 4 ℃ of microwave extraction 24h of temperature, adopts same procedure same solvent consumption to extract united extraction liquid 4 times;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.22 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of the 0.01mol/L phosphoric acid buffer of pH=7.4, be the tubular fibre membrane ultrafiltration of 10kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 8:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 90 order fine powders;
2, the material 100g after will pulverizing adds the Tris-HCl damping fluid of 10 times of amounts in 1 ℃ of microwave extraction 18h of temperature, adopts same procedure same solvent consumption to extract united extraction liquid 3 times;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.45 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of the 0.01mol/L phosphoric acid buffer of pH=7.4, be the tubular fibre membrane ultrafiltration of 50kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 9:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 100 order fine powders;
2, the material 100g after will pulverizing adds the Tris-HCl damping fluid that contains 0.1mol/L NaCl of pH=7.4 of 15 times of amounts in 10 ℃ of microwave extraction 24h of temperature, collects extracting solution;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.65 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of the 0.01mol/L phosphoric acid buffer of pH=7.4, be the tubular fibre membrane ultrafiltration of 100kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Embodiment 10:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 100 order fine powders;
2, the material 100g after will pulverizing, the 0.01mol/LTris-HCl damping fluid that adds the pH=7.4 that contains 0.1～0.3mol/L NaCl of 10 times of amounts soaks in 4 ℃ of temperature and extracts 12h, adopts same procedure same solvent consumption to extract united extraction liquid 3 times;
3, with the vat liquor that merges, reinforcing body ammonium sulfate is to saturated, and 4 ℃ of static 12h get precipitation after centrifugal, and are centrifugal with the 0.01mol/L Tris-HCl damping fluid dissolving of pH=7.4, filter, and get subsequent filtrate, and through dialysis, freeze-drying gets shizandra berry albumen.
Embodiment 11:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80 order fine powders;
2, the material 100g after will pulverizing, the 0.01mol/L Tris-HCl damping fluid that adds the pH=7.4 of 15 times of amounts soaks in 4 ℃ of temperature and extracts 24h, adopts same procedure same solvent consumption to extract united extraction liquid 2 times;
3, with the vat liquor that merges, reinforcing body ammonium sulfate is to saturated, and 6 ℃ of static 24h get precipitation after centrifugal, and are centrifugal with the 0.01mol/L Tris-HCl damping fluid dissolving of pH=7.4, filter, and get subsequent filtrate, and through dialysis, freeze-drying gets shizandra berry albumen.
Embodiment 12:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 100 order fine powders;
2, the material 100g after will pulverizing adds the distilled water of 5 times of amounts in 4 ℃ of supersound extraction 1h of temperature, adopts same procedure same solvent consumption to extract united extraction liquid 2 times;
3, with the vat liquor that merges, reinforcing body ammonium sulfate is to saturated, and 1 ℃ of static 18h gets precipitation after centrifugal, and is centrifugal with dissolved in distilled water, filters, and gets subsequent filtrate, and through dialysis, freeze-drying gets shizandra berry albumen.
Embodiment 13:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80 order fine powders;
2, the material 100g after will pulverizing adds the 0.01mol/L phosphoric acid buffer of pH=7.4 of 10 times of amounts in 4 ℃ of microwave extraction 2h of temperature, adopts same procedure same solvent consumption to extract united extraction liquid 2 times;
3, with the vat liquor that merges, reinforcing body ammonium sulfate is to saturated, and 10 ℃ of static 24h get precipitation after centrifugal, and are centrifugal with dissolved in distilled water, filter, and get subsequent filtrate, and through dialysis, freeze-drying gets shizandra berry albumen.
Embodiment 14:
1, schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 100 order fine powders;
2, the material 100g after will pulverizing, the 0.01mol/LTris-HCl damping fluid that adds the pH=7.4 that contains 0.1～0.3mol/L NaCl of 10 times of amounts soaks in 4 ℃ of temperature and extracts 12h, adopts same procedure same solvent consumption to extract united extraction liquid 3 times;
3, the vat liquor that merges is the tubular fibre membrane filtration of 0.45 μ m through membrane pore size after, with a small amount of washing hollow-fiber film of the 0.01mol/LTris-HCl damping fluid of pH=7.4, be the tubular fibre membrane ultrafiltration of 10kDa again through molecular weight cut-off after the merging, with a small amount of washing hollow-fiber film of same solvent, filtrate freeze-drying in the gained gets shizandra berry albumen.
Experimental example 1: the qualitative and assay of shizandra berry total protein
Choose embodiment 11 gained albumen, embodiment 12 gained albumen, embodiment 14 gained albumen.
(1) SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Adopt the SDS-polyacrylamide gel electrophoresis.Low molecular mass standard protein (1.2 μ g μ L
-1), sample applied sample amount (2.4 μ g μ L
-1) be 20 μ L.Resolving gel concentration is 12%, and concentrated gum concentration is 5%.Concentrated glue voltage is 70mv, separation gel voltage 140mv.After electrophoresis finishes with coomassie brilliant blue R250 dyeing (methyl alcohol: water: glacial acetic acid=4.5: 4.5: 1), decolour with the aqueous solution of 7.5% methyl alcohol and 5% glacial acetic acid at last clear to background.
(2) assay of shizandra berry total protein
Precision takes by weighing the bovine serum albumin reference substance, is made into 1.37mgmL
-1Solution.Precision takes by weighing 3 kinds of protein extracts, is made into 1.5mg/mL solution.
Press the Lowry method, shizandra berry albumen is carried out assay.Accurate measuring albumin contrast solution 0,0.1,0.2,0.4 respectively, 0.6,0.8, put in the 10mL measuring bottle, reagent adding first 5mL, mixing, room temperature is placed 10min, add fast reagent second 0.5mL, room temperature is placed 1h behind the mixing, take the 1st pipe as blank, adopt spectrophotometry to measure its optical density in the 765nm place, measuring result is 0.095,0.170,0.322,0.462,0.602.(m, μ g) is X-coordinate with protein content, and optical density (A) is for ordinate zou carries out correlation analysis, and drawing regression equation is Y=0.7246X+0.0259, r=0.9998.
Measure respectively above-mentioned protein sample solution 1mL, calculate protein content with return law of the straight line.See the following form 1.
Table 1 shizandra berry total protein content measurement result
| The | Embodiment | 11 gained albumen | Embodiment 12 gained | Embodiment | 14 gained albumen |
| Protein content (%) | 51.2 | 57.3 | 55.6 |
(3) Contents of Amino Acids
The preparation of reference substance mixing solutions and need testing solution:
Precision takes by weighing the kilnitamin reference substance, and being made into content is 312.5 μ mol/L solution.
Precision takes by weighing three kinds of protein extracts, wiring solution-forming.Each adds 6mol/L hydrochloric acid 20mL, vacuumizes sealing, and 110 ℃ of hydrolysis of constant temperature 24h lets cool, and filters.Get the 2mL brown bottle, add respectively said hydrolyzed sample 30 μ L, NaHCO
3(pH=9.0) solution 30 μ L, 1%FDNB acetonitrile solution 10 μ L, 1h is placed in 60 ℃ of water-bath dark places, adds KH
2PO
4(pH=7.0) solution 1mL shakes up, and through 0.45 μ m filtering with microporous membrane, treats that HPLC analyzes.
Chromatographic condition:
Agilent ZORBAX Eclipse XDB-C
18Chromatographic column (5um, 4.6mm * 150mm).Moving phase: A, KH
2PO
4(pH=7.2,0.04molL
-1); B, acetonitrile-water (55: 45).Linear gradient elution program: 0～4min0%B, 4～10min 14%B, 10～20min 40%B, 20～21min 80%B, 21～25min 100%B.Detect wavelength: 360nm; Flow velocity: 1mL/min; Column temperature: 40 ℃.
Assay:
Get respectively standard solution, each 10 μ L of need testing solution, put in the sampler, qualitative with retention time, external standard method is quantitative, aminoacids content in the working sample.See Fig. 2 A～2D, table 2.
Table 2 sample Contents of Amino Acids result
Bottom further specifies the present invention by pharmacodynamic experiment.
Below choosing embodiment 11 gained shizandra berry albumen tests.
Experimental example 1: shizandra berry albumen is to the lymphocytic in-vitro multiplication effect of T (T lymphocyte transformation test)
Instrument: the Epoch microwell plate spectrophotometer of Biotex company
Shizandra berry albumen 0.03g adds 0.1M PBS to 0.6ml, and it is 50 μ g/ μ l. that the X medicine stores concentration
Positive control Con A saves as 200 μ g/ml
Experiment grouping: blank group, negative control group, shizandra berry protein groups (2.5,0.25,0.025,0.0025 μ g/ μ l), Con A group, 3 every group multiple holes.
Experimental technique:
1. get mouse spleen under the aseptic condition, smash to pieces with sterile gauze filtration cell suspension;
2. cell counting, the accent cell concn is 1 * 107/ml, the every hole of 96 orifice plates adds 100 μ l cell suspensions;
3. dosing: blank group only adds nutrient solution and does not add cell; Negative control group only adds nutrient solution and cell, not dosing; Shizandra berry protein groups medicine final concentration is respectively 2.5,0.25,0.025,0.0025 μ g/ μ l, and it is 5 μ g/ml that Con A organizes positive contrast final concentration, every hole final volume is 200 μ l;
4.37 ℃, cultivate 44h after, add MTT 10 μ l/ holes, 37 ℃ hatch 4h after, abandon supernatant, (100 μ l/ hole) are after the formazan dissolving, and microplate reader is surveyed the OD value at 570nm to add DMSO; The results are shown in following table 3
Table 3T lymphocyte transformation test result
SI=(the blank group of experimental group OD-OD)/(the blank group of negative control group OD-OD)
The result shows that the death of 2.5,0.25 μ g/ μ l group lymphocyte is more, illustrates that this concentration is larger to lymphocytotoxicity.0.0025 μ g/ μ l group is more obvious to the hormesis of spleen lymphocyte, but is lower than positive controls.
Experimental example 2: shizandra berry albumen is on the impact of mouse cell immunologic function
1, delayed allergy experiment
Get 48 of mouse, be divided at random negative control, low, in, 4 experimental group of high dosage, 3 experimental group give respectively 0.714g/ (kgbw), 1.428g/ (kgbw), 4.286g/ (kgbw), each group is pressed 0.2ml/10 (gbw) volume to mouse stomach, the control group gavage gives equal-volume distilled water, once a day, after continuous 30 days, mouse peritoneal injection 2% (V/V) SRBC, 4d measures left back sufficient sole of the foot thickness after the sensitization, and then at measuring point subcutaneous injection 20% (V/V) SRBC, every mouse is injected 20 μ l, inject and measured left back sufficient sole of the foot section thickness in rear 24 hours, represent the degree of DTH with sufficient sole of the foot thickness difference before and after attacking.The results are shown in following table 4.
Table 4 delayed allergy experimental result
The result shows, compares with negative control group, and swelling degree of the paw difference low, the high dose group mouse has statistical significance.
2, the impact of shizandra berry albumen mouse spleen lymphocyte conversion capability that ConA is induced
Get 48 of mouse, be divided at random negative control, low, in, 4 experimental group of high dosage, 3 experimental group give respectively 0.714g/ (kgbw), 1.428g/ (kgbw), 4.286g/ (kgbw), each group is pressed 0.2ml/10 (gbw) volume to mouse stomach, the control group gavage gives equal-volume distilled water, once a day, after continuous 30 days, the aseptic spleen of getting, the preparation splenocyte suspension is washed 2 times with Hanks liquid, each centrifugal 10min (1000r/min, centrifugal radius 9.2cm), in 1mL RPM I 1640 complete culture solutions, adjusting cell concn is 3 * 10 with cell suspension
6Individual/mL.Divide two holes to add in 24 well culture plates cell suspension, every hole 1mL, a hole adds 75 μ L ConA liquid, and another hole is put in 5%CO2,37 ℃ of incubators and is cultivated 72h in contrast.Cultivate and finish front 4h, supernatant liquor 0.7mL is drawn in every hole, adds the RPM I1640 complete culture solution that 0.7mL does not contain calf serum, adds simultaneously M TT50 μ L, continues to cultivate 4h.After cultivating end, every hole adds 1mL acid isopropyl alcohol, and piping and druming evenly makes purple crystal dissolve fully, measures optical density(OD) (OD) at wavelength 570nm place and is worth, and deducts the OD value representation lymphopoiesis ability that does not add the ConA hole with the OD value that adds the ConA hole.The results are shown in following table 5.
The mouse spleen lymphocyte transformation experiment result that table 5ConA induces
The result shows, the OD difference of each dosage group is all greater than negative control group, and wherein the difference of low dose group and control group has significance, shows the spleen lymphocyte proliferation that this albumen has stimulates mouse, the effect of conversion.
Experimental example 3: the Turnover of Mouse Peritoneal Macrophages toluylene red is engulfed in vitro tests
Instrument: the Epoch microwell plate spectrophotometer of Biotex company; Super clean bench
Experimental technique:
1. mouse peritoneal is injected 3% starch (3ml/ only), collects abdominal cavity cell afterwards with the about 10ml flushing abdominal cavity of the IMDM that contains 10% foetal calf serum in 1 day, centrifugal 1500r/min, and 5min abandons supernatant, adds 3ml nutrient solution re-suspended cell (cell counting approximately 106).Cell is inoculated in 96 orifice plates, and 100 μ l/ are empty, 37 ℃ hatch 4 hours after, abandon supernatant, remove not attached cell.
2. drug level: 1,0.25,0.05,0.01,0.002,0.0004,0.00008,0.000016ug/ul contrasts: 0ug/ul.Blank is established in every group 3 multiple holes simultaneously.
3.37 ℃ incubate after .24 hour and to discard supernatant, add 0.072% toluylene red dye liquor, 200 μ l/ holes, hatch after 4 hours for 37 ℃ and discard dye liquor, 0.1M PBS washes 3 times, adds lysate (0.1M Glacial acetic acid: dehydrated alcohol=1: 1), 2h is hatched for 4 ℃ in 200 μ l/ holes, and microplate reader is surveyed light absorption value at 540nm.
The results are shown in following table 6 and Fig. 3
Table 6 shizandra berry albumen is engulfed the impact of toluylene red on Turnover of Mouse Peritoneal Macrophages
Each administration group OD value all is higher than control group, and except 0.25 and 0.000016ug/ul group, all have statistical significance, the most obvious with 0.02ug/ul, prompting 0.02ug/ul group shizandra berry albumen can obviously promote Turnover of Mouse Peritoneal Macrophages to engulf toluylene red.Point out this medicine at external phagocytic function to peritoneal macrophage certain promoter action to be arranged.
Experimental example 4: shizandra berry albumen is on the impact of mouse monokaryon-macrophage phagocytic function
1, carbon is cleaned up ability
Get 40 of mouse, be divided at random negative control, 4 experimental group of basic, normal, high dosage, 3 experimental group give respectively 0.714g/ (kgbw), 1.428g/ (kgbw), 4.286g/ (kgbw), each group is pressed 0.2ml/10 (gbw) volume to mouse stomach, the control group gavage gives equal-volume distilled water, once a day, after continuous 30 days, the india ink of mouse tail vein injection dilution in 1: 3, treat that prepared Chinese ink injects immediately timing, injected behind the prepared Chinese ink 2,10 minutes, and got blood 20 μ L from the angular vein clump respectively, and it is added to 2mLNa
2CO
3In the solution, sentence Na with ultraviolet-visible pectrophotometer at the 600nm wavelength
2CO
3Solution is made blank and is surveyed the OD value.Mouse is put to death, get liver and spleen is weighed, the counting phagocytic index.Phagocytic index=end weight * K
1/3/ (liver weight+spleen is heavy); K=(logOD
1-logOD
2)/(t
2-t
1).The results are shown in following table 7.
Table 7 mouse carbon is cleaned up experimental result
The result shows, compares with negative control group, and the carbon of high dose group mouse is cleaned up capacity variance statistical significance.
2, macrophage phagocytic chicken red blood cell ability
Get 40 of mouse, be divided at random negative control, low, in, 4 experimental group of high dosage, 3 experimental group give respectively 0.714g/ (kgbw), 1.428g/ (kgbw), 4.286g/ (kgbw), each group is pressed 0.2ml/10 (gbw) volume to mouse stomach, and the control group gavage gives equal-volume distilled water, once a day, after continuous 30 days, mouse peritoneal is injected 20% chicken erythrocyte suspension 1mL, and interval 30min puts to death, be fixed on the mouse plate, cut off abdominal skin, injecting normal saline 2mL rotates mouse plate 1min, sucking-off abdominal cavity washing lotion 1mL, divide and drip on 2 slides, 37 ℃ of incubation 30min use the physiological saline rinsing, dry, fix with 1: 1 acetone methanol solution, the Giemsa 3min that dyes dries with the distilled water rinsing, survey with oily microscopy, calculate phagocytic rate and phagocytic index.
The scavenger cell number of the scavenger cell number of phagocytic rate (%)=engulf chicken red blood cell/counting * 100
The scavenger cell number of chicken red blood cell sum/counting that phagocytic index=quilt is engulfed the results are shown in following table 8.
Table 8 is engulfed the impact of chicken red blood cell phagocytic rate, phagocytic index on mouse macrophage
*P＜0.05,
*P＜0.01vs negative control group
The result shows, compares with negative control group, and peritoneal macrophage phagocytic percentage and the phagocytic index difference of 3 dosage group mouse all have statistical significance.
Experimental example 5: shizandra berry albumen improves the experiment of anoxia endurance
Experimental technique
1, normal pressure hypoxia tolerance experiment: get 40 of mouse, be divided at random negative control, low, in, 4 experimental group of high dosage, 3 experimental group give respectively 0.714g/ (kgbw), 1.428g/ (kgbw), 4.286g/ (kgbw), each group is pressed 0.2ml/10 (gbw) volume to mouse stomach, the control group gavage gives equal-volume distilled water, once a day, after continuous 30 days, 1h after last gives tested material, each group mouse is put into respectively the 250ml port grinding bottle (1 every bottle) that fills the 5g sodica calx, seal bottleneck with Vaseline, cover tightly, make it air tight, immediately manual time-keeping, take breath stopped as index, observe mouse because of the anoxic Post-dead duration.
2, Sodium Nitrite is poisoned to survive and is tested: get 40 of mouse, be divided at random negative control, low, in, 4 experimental group of high dosage, 3 experimental group give respectively 0.714g/ (kgbw), 1.428g/ (kgbw), 4.286g/ (kgbw), each group is pressed 0.2ml/10 (gbw) volume to mouse stomach, the control group gavage gives equal-volume distilled water, once a day, after continuous 30 days, 1h after last gives tested material, each treated animal is pressed 240mg/kg bw dosage abdominal injection Sodium Nitrite injection volume and is (0.1mg/10g), immediately manual time-keeping, the record animals survived time.
Experimental result
1, shizandra berry albumen is on the impact of Mouse Weight
The results are shown in Table following table 9, by as seen from Table 9, above each group and blank group be Mouse Weight no significant difference (P＞0.05) before and after each test group relatively.Show shizandra berry albumen to weight of mice without influence.
Table 9 shizandra berry albumen is on the impact of experiment mice body weight
2, normal pressure hypoxia tolerance experiment
The results are shown in following table 10, with the solvent control group relatively each dosage group can obviously prolong Hypoxia under normal pressure, each dosage group is all variant a significance (P＜0.05).Show that the shizandra berry egg has the effect of prolongation mouse because of the anoxic death time.
Table 10 shizandra berry albumen is on the impact of normal pressure hypoxia tolerance experiments experiment mouse survival time
3, Sodium Nitrite poisoning survival experiment
The results are shown in following table 11, each dosage group obviously prolongs Sodium Nitrite and poisons the rear mouse survival time, and wherein low, middle dosage group difference has significance (P＜0.05).Show that low, the middle dosage of shizandra berry albumen has the effect of prolongation mouse survival time after Sodium Nitrite is poisoned.
Table 11 shizandra berry albumen is on the impact of Sodium Nitrite poisoning survival experiments experiment mouse survival time
Claims (4)
1. shizandra berry albumen is characterized in that it is made by following method:
(1) schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80 order fine powders;
(2) material after will pulverizing, the 0.01 mol/L Tris-HCl damping fluid that adds the pH=7.4 of 15 times of amounts soaks in 4 ℃ of temperature and extracts 24h, adopts same procedure same solvent consumption to extract united extraction liquid 2 times;
(3) purifying of albumen:
With the vat liquor that merges, reinforcing body ammonium sulfate is to saturated, and 6 ℃ of static 24h get precipitation after centrifugal, and are centrifugal with the 0.01 mol/L Tris-HCl damping fluid dissolving of pH=7.4, filter, and get subsequent filtrate, and through dialysis, freeze-drying gets shizandra berry albumen.
2. the preparation method of shizandra berry albumen as claimed in claim 1 is characterized in that comprising the following steps:
(1) schisandra fruit or shizandra berry seed are cleaned, dry, be ground into 80 order fine powders;
(2) material after will pulverizing, the 0.01 mol/L Tris-HCl damping fluid that adds the pH=7.4 of 15 times of amounts soaks in 4 ℃ of temperature and extracts 24h, adopts same procedure same solvent consumption to extract united extraction liquid 2 times;
(3) purifying of albumen:
With the vat liquor that merges, reinforcing body ammonium sulfate is to saturated, and 6 ℃ of static 24h get precipitation after centrifugal, and are centrifugal with the 0.01 mol/L Tris-HCl damping fluid dissolving of pH=7.4, filter, and get subsequent filtrate, and through dialysis, freeze-drying gets shizandra berry albumen.
3. shizandra berry albumen as claimed in claim 1 is for the preparation of the application in the medicine of enhancing body immunizing power.
4. shizandra berry albumen as claimed in claim 1 is for the preparation of the application in the medicine with resisting oxygen lack.
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