CN106511401A - Preparation method and application of cordyceps taii mycelia polysaccharides - Google Patents
Preparation method and application of cordyceps taii mycelia polysaccharides Download PDFInfo
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Abstract
The invention discloses a preparation method and an application of cordyceps taii mycelia polysaccharides. According to the invention, a cordyceps taii GYYA0601 strain is screened, and it is discovered that the mycelia polysaccharides, which are prepared in accordance with the strain, have a good effect when used as a medicine for treating diabetics; the mycelia polysaccharides can inhibit the activity of [alpha]-glucosidase, significantly reduce the blood glucose level of a patient with the diabetics, improve the injury degree of pancreatic [beta] cells and increase the secretion of insulin; in addition, the mycelia polysaccharides also can boost body immunity; and the mycelia polysaccharides, on preventing and treating the diabetics, have the advantages of being good in curative effect, low in side effects and the like.
Description
Technical field
The present invention relates to biomedicine technical field, particularly a kind of DAISHICHONGCAO mycelium polysaccharides
Preparation method and application.
Background technology
Glucose -lipid metabolism disorder disease of the diabetes with hyperglycemia as principal character, is to be only second to the heart
Cerebrovascular disease and cancer, endanger the third-largest disease (Association of human health most serious
AD.Diagnosis and classification of diabetes mellitus.Diabetes Care,
2012,35,Suppl(1):S42–7；http://www.idf.org/diabetesatlas).Medicine is controlled
Treatment is the primary treatment strategy of diabetes, but, sulfonylurea, biguanideses, glucosides enzyme level
The most frequently used clinical line hypoglycemic drug such as agent class, insulin sensitivity enhancing class, has the secondary work of more poison
With, and easily cause secondary failure, invalid to diabetes and complication (Turner RC, Cull CA,
Frighi V,et al.Glycemic control with diet,sulfonylurea,metformin,or
insulin in patients with type 2diabetes mellitus:progressive requirement
for multiple therapies(UKPDS 49).Journal of the American Medical
Association,1999,281(6):2005-12).So, develop the drop of new and effective, low toxicity
Hypoglycemic medicament has become the study hotspot of biomedicine field.
Polysaccharide is main active component in natural drug, and biological activity is wide, and has efficiently low
Malicious characteristic, is more satisfactory medicine source.Found that fungus polysaccharide had good blood sugar lowering in recent years
Effect.Cordycepses are the fungus resources that a class has important application prospect in biomedicine field.Research
Show, Cordyceps, the mycelium polysaccharides of Cordyceps militaris (L.) Link. have obvious blood sugar lowering, anti-diabetic
Effect (Kiho T, Ookubo K, Usui S, et al.Structural features and
hypoglycemic activity of a polysaccharide(CS-F10)from the cultured
mycelium of Cordyceps sinensis.Biological&Pharmaceutical Bulletin,
1999,22(9):966-70；Li SP,Zhang GH,Zeng Q,et al.Hypoglycemic
activity of polysaccharide,with antioxidation,isolated from cultured
Cordyceps mycelia.Phytomedicine 2006；13(6):428-33；Zhang G,Huang
Y,Bian Y,et al.Hypoglycemic activity of the fungi Cordyceps militaris,
Cordyceps sinensis,Tricholoma mongolicum,and Omphalia lapidescens
in streptozotocin-induced diabetic rats.Applied Microbiology and
Biotechnology 2006；72(6):1152-6), antidiabetic medicine and health product are prepared
Good source.DAISHICHONGCAO (Metarhizium taii) Cordyceps taii are China's fungus scholar's beams
Zong Qi teaches new Cordyceps in Guizhou Province's reported first, and Guizhou is among the people to be mistakenly considered the winter always
Worm summer grass is used for nourishing and body-building.
The content of the invention
The technical problem to be solved is：A kind of DAISHICHONGCAO mycelium polysaccharides are provided
Preparation method.
Present invention also offers a kind of DAISHICHONGCAO mycelium polysaccharides are preparing antidiabetic medicine
In application.
What the present invention was realized in：The preparation method of DAISHICHONGCAO mycelium polysaccharides, including such as
Lower step：
1) DAISHICHONGCAO mycelium is prepared using DAISHICHONGCAO GYYA0601 bacterial strain；
2), after by the drying of DAISHICHONGCAO mycelium, 60-100 mesh molecular sieves were crushed to, were obtained
DAISHICHONGCAO erinaceus mycelium powder；
3) 1 will be pressed in DAISHICHONGCAO erinaceus mycelium powder:The solid-liquid ratio of 3-5 adds petroleum ether degreasing,
The unit of solid-liquid ratio is g/ml；Mycelium powder after defat is pressed into 1：5-15 solid-liquid ratios add distilled water,
The unit of solid-liquid ratio is g/ml, extracts crude polysaccharides using the method for water extract-alcohol precipitation；By crude polysaccharides Jing
After Deproteinization, decolouring and dialysis, precipitate with ethanol is concentrated, DAISHICHONGCAO mycelium polysaccharides are obtained.
Application of the DAISHICHONGCAO mycelium polysaccharides in antidiabetic medicine is prepared.
Modern pharmacology research result is pointed out, and DAISHICHONGCAO mycelium crude polysaccharides have immunity tune
The various actives such as section, antitumor, mutation (Liang Zongqi. Chinese funguses will. volume 32, worm
Grass category. Beijing：Science Press, 2007).Applicant is gathered and is divided on one, Kweiyang tea plantation
From to DAISHICHONGCAO GYYA0601 bacterial strain (Metarhizium taii Metarhizium taii
GYYA0601 bacterial strains), deposit number is CGMCC No.2880.By further grinding
Study carefully discovery, its mycelium polysaccharides carrys out slow down aging via anti-oxidation stress and immunomodulating, and this is more
Sugar is a kind of gala glucomannan (Xiao JH, Xiao DM, Chen DX, et al.
Polysaccharides from the medicinal mushroom Cordyceps taii show
antioxidant and immunoenhancing activities in a D-galactose-induced
aging mouse model.Evidence-Based Complementary and Alternative
Medicine Volume,2012(2012),Article ID 273435,15pages).It is worth proposing
, oxidative stress is one of pathomechanism of diabetes.Body anti-oxidative defense system is difficult to
The response to oxidative stress that free radicals cause in a large number caused by high sugar state is stabilized, causes islet cellss to receive
Damage, while also reduce peripheral tissues produces insulin resistant to the sensitivity of insulin, these
Factor promote diabetes and complication generation development (Forbes JM, Coughlan MT,
Cooper ME(2008)Oxidative stress as a major culprit in kidney disease in
diabetes.Diabetes 57:1446–54；Stadler K.Oxidative stress in diabetes.
Advances in Experimental Medicine and Biology.2012；771:272-87).And
And, oxidative stress and insulin resistant are to complement each other, and insulin resistant promotes oxidative stress again
(Dong F,Fang CX,Yang X,et al.Cardiac overexpression of catalase
rescues cardiac contractile dysfunction induced by insulin resistance:
Role of oxidative stress,protein carbonyl formation and insulin
sensitivity.Diabetologia.2006；49(6):1421-33).Therefore, reduce the oxygen of body
Change stress possibly prevent and treat the effective ways of diabetes and its complication,
Novel antidiabetic and its complication medicine are developed using antioxidation natural product
New direction.Therefore, Dai Shi worm of the applicant to have obvious anti-oxidation stress ability
Careless mycelium polysaccharides be object of study, systematically have studied first its preparation method and
To alpha-glucosidase, the effect of diabetes animal model.The DAISHICHONGCAO bacterium that the present invention is provided
Mycelium polysaccharide preparation method and the application in antidiabetic medicine or assistant hypoglycemic healthcare product, extremely
The present has no report.
The present invention is more to prepare DAISHICHONGCAO mycelium by DAISHICHONGCAO GYYA0601 bacterial strain
Sugar, it is found by the applicant that the mycelium polysaccharides have good effect on the medicine as treatment diabetes
With, it can Inhibiting α-glucosidase activity, can significantly reduce diabetes patient blood glucose value,
Improve the beta Cell of islet extent of damage, increase insulin secretion, can also enhancing human body immunity power,
To preventing and treating diabetes, there are good effect, Small side effects.
Description of the drawings
Accompanying drawing 1 is DAISHICHONGCAO mycelium polysaccharides to diabetic mouse model islet tissue cell
Affect (200 ×)；
A- normal group NG；B- model group MCG；C- positive controls PG；D- high dose groups HG；
E- middle dose groups MG；F- low dose groups LG.
Specific embodiment
Embodiments of the invention 1：The production of DAISHICHONGCAO fermentation product
1st, polysaccharide is from Cordyceps fungus DAISHICHONGCAO (Metarhizium taii) GYYA0601 bacterial strains
It is isolated in fermentation mycelium.DAISHICHONGCAO culture presevation numbering is CGMCC NO.2880,
The granted patent (ZL 200910301440) of applicant's earlier application is disclosed.
2nd, DAISHICHONGCAO inclined-plane seed culture based formulas include sucrose 25g, Semen Glyciness 3g, ferment
Female cream 5g, peptone 2g, Testa Tritici 3g, KH2PO41g、MgSO4·7H2O 0.5g、
Agar 18g, distilled water 1L, initial pH 7.0；DAISHICHONGCAO liquid seeds or/fermentation training
Foster based formulas include sucrose 30g, peptone 5g, yeast extract 3g, KH2PO41g, MgSO4·7H2O
0.5g, distilled water 1L, initial pH 7.0.
3rd, 23-28 DEG C of DAISHICHONGCAO inclined-plane seed is cultivated 7-15 days, is placed in 4 DEG C of preservations standby.
Transplanting slant strains in the shaking flask of 500mL specifications for filling 120mL liquid seed culture mediums,
Rotary shaker is subsequently placed in, 120-180r/min, 25-30 DEG C of culture 4d prepare fermentation liquid
Body seed.The canned 33L fermentation medium of 50L fermentations, by the ratio of volume factor 4% (v/v)
Example inoculation fermentation liquid seeds, 150-250r/min, 28 DEG C of condition bottom fermentation 60-80h.
After fermentation terminates, reduce pressure sucking filtration tunning, is cleaned with distilled water 3 times, is placed on baking oven,
45-60 DEG C of drying weighs fermentation product dry weight to constant weight.Dried fermentation product are pounded powder,
60-100 mesh molecular sieves are crossed, -20 DEG C of cold preservations of sealing are standby.
Embodiments of the invention 2：The preparation of DAISHICHONGCAO mycelium polysaccharides
1 will be pressed in DAISHICHONGCAO erinaceus mycelium powder:The solid-liquid ratio of 3-5 adds petroleum ether degreasing, material
The unit of liquor ratio is g/ml；Mycelium powder after defat is pressed into 1：5-15 solid-liquid ratios add distilled water,
The unit of solid-liquid ratio is g/ml, and extracts 1-3 hours in boiling water bath, repeats 2-4 time, takes out
Then filter, merging filtrate are evaporated to 1/3-1/4 volumes, and concentration filtrate is placed in beaker
In, add the ethanol that mass percent is 80-90%, 4 DEG C of refrigerators to stand overnight, centrifuging and taking
Obtain precipitate；1 is pressed again：The solid-liquid ratio (unit g/ml) of 1-3 adds distilled water dissolution precipitation
Thing, is subsequently adding the ethanol that mass percent is 80-90%, and 4 DEG C of refrigerators stand overnight, and repeat
This operation is until ethanol is without obvious color；By isolated total drying precipitate, that is, obtain
Crude polysaccharides；Crude polysaccharides are extracted using the method for water extract-alcohol precipitation；Sevag is taken to remove floating preteinses,
Then activated carbon or hydrogen peroxide for decoloration is adopted, then is dialysed with the convection current water law of 3500kD molecular weight
24 hours, after concentration precipitate with ethanol, obtain DAISHICHONGCAO mycelium polysaccharides.
Embodiments of the invention 3：Suppression of the DAISHICHONGCAO mycelium polysaccharides to alpha-glucosidase
Activity
1st, activity of the positive drug Acarbose to alpha-glucosidase：Enzyme reaction system is built,
Microscreen standard reaction system totally 140 μ L, first phosphorate respectively 20 μ L of phthalate buffer per hole,
Take 0.001 again respectively, 0.01,0.1, each 20 μ L are anti-in standard for 1mg/mL acarbose solutions
In answering system, the alpha-glucosaccharase enzymatic solution and 10 μ L of 60 μ L 20U/ml are finally sequentially added
The PNPG solution of 10mmol/L, after mix homogeneously, is incubated 30min in 37 DEG C, then
30 μ L 1mol/L Na are added per hole2CO3Terminating reaction, is now presented yellow, and microplate reader is surveyed
Determine the OD values at 405nm, while setting blank and ground control, each sample does 3
Individual multiple holes, average.Acarbose is calculated to alpha-glucosidase according to following equation
Inhibitory activity (is shown in Table 1)：
2nd, activity of the DAISHICHONGCAO mycelium polysaccharides to alpha-glucosidase：Build with more than
Screening methods for alpha-glucosidase inhibitors from Chinese medicinal herbs to DAISHICHONGCAO mycelium polysaccharides carry out screening and most
Good dose of quantifier elimination, and obtain IC50.Add 20 μ L phosphate buffers respectively per hole, then divide
Do not take 5,10,20,40,80,200,20 μ L of 300mg/ml concentration polysaccharide solution add
Alpha-glucosaccharase enzymatic solution and 10 μ L 10 in enzyme standard reaction system, with 60 μ L 20U/mL
After the PNPG solution mix homogeneously of mmol/L, in 37 DEG C of common incubation 30min, terminate liquid
Terminating reaction, microplate reader determine the OD values at 405nm, while setting blank and background
Control, each sample are done 3 multiple holes, are averaged.Equally, calculated according to above-mentioned formula
Inhibitory activity of the DAISHICHONGCAO mycelium polysaccharides to alpha-glucosidase.1 is the results are shown in Table, is calculated
Its IC50It is worth for 105.95mg/mL.
Inhibitory action of the 1 DAISHICHONGCAO mycelium polysaccharides of table to alpha-glucosidase
Embodiments of the invention 4：The antidiabetic effect of DAISHICHONGCAO mycelium polysaccharides
1st, the structure of diabetes model and administration：Male mouse of kunming (20-30g), in cleaning
Level standard laboratory (temperature：25 ± 2 DEG C of humidity：60 ± 5%, illumination：12h/d, ventilation
After well) raising 7d adaptation environment, mice is randomly divided into 2 groups, wherein Normal group
(normal control group, NC) 8, remaining mice is used to modeling, modeling group mice
After fasting 12h (free water) overnight, 100mg/kg disposable celiacs injection STZ (lemons
Lemon phthalate buffer, pH=4.44), after STZ injection 4h, feed the glucose of mice 5%
Solution, it is to avoid mice is dead because of hypoglycemia, and after 5d, tail vein is detected with blood glucose meter after taking blood
Fasting glucose, continuous 3 times, blood glucose value>11.1mmol/L is modeling success.Normal group is little
1 property of Mus injects citrate buffer, physiology salt of the drug control group using 0.5ml 0.9%
Water.Modeling success mice is randomly divided into into 5 groups, respectively normal group (NG, 0.9% physiology
Saline), model control group (MCG, 0.9% normal saline), positive controls (PG, 300
The metformin of mg/kg/d), low dosage polysaccharide group (LG, 100mg/kg/d), middle dosage it is many
Sugared group (MG, 200mg/kg/d), high dose polysaccharide group (HG, 400mg/kg/d), per group
12.
2nd, impact of the DAISHICHONGCAO mycelium polysaccharides to blood glucose in diabetic mice:As shown in table 2,
After modeling group mice 100mg/kg STZ single intraperitoneal injections 5d, compared with normal group,
Mice fasting glucose significantly raises (P ＜ 0.01), illustrates that diabetes model is successfully constructed；With mould
Type group is compared, and CTP gavages treatment group's blood glucose value is substantially reduced in dosage period, and has system
Meter learns meaning.Therefore, DAISHICHONGCAO mycelium polysaccharides can substantially reduce the blood glucose of diabetic mice
Level.
The shadow of the blood glucose in diabetic mice that 2 DAISHICHONGCAO mycelium polysaccharides of table are induced to STZ
Ring (N=12)
Note:Compare with MCG,aP<0.05,bP<0.01；Compare with NG：cP<0.01
3rd, impact of the DAISHICHONGCAO mycelium polysaccharides to diabetic mice serum insulin：Such as table
Shown in 3, normal group is compared, model group serum insulin content be decreased obviously (aP ＜ are 0.05)；
Compared with model group, all administration group serum insulin contents are significantly raised, andbP<0.01
It is statistically significant in level.Therefore, DAISHICHONGCAO mycelium polysaccharides can promote diabetes little
The secretion of Mus insulin.In addition, pathological analysis result is pointed out (see Fig. 1), NG group mice pancreases
Island cell is agglomerating to be distributed in pancreatic exocrine gland, islets of langerhans edge shape rule, and divides with outer
Secrete circumference between gland to understand, island inner cell number is more, rounded blue the dye of islet cellss core,
The abundant lightpink of kytoplasm dye；MCG group mouse islets edge shape is irregular, islets of langerhans atrophy,
Central part vacuolar degeneration, beta Cell of islet number are reduced, and islet cellss distribution is mixed and disorderly, part hypertrophy
Glandular tube spline structure is arranged in, part compensatory cellular is loose；Including four administration groups including PG
Mice pancreatic is more complete, and beta Cell of islet quantity is more compared with model group and is evenly distributed, and illustrates Dai Shi
Cordyceps mycelium polysaccharide have protection islet cellss function, and during high dose group effect is better than, it is low
Dosage group.
Impact of the 3 DAISHICHONGCAO mycelium polysaccharides of table to diabetic mice serum insulin ( N=12)
Note:Compare with NG：aP<0.01；Compare with MCGbP<0.01；
Model group MCG；C- positive controls PG；D- high dose groups HG；E- middle dose groups MG；F- low dose groups LG.
4th, impact of the DAISHICHONGCAO mycelium polysaccharides to diabetic mice peripheral blood leucocyte：By table 4
Understand, diabetic model group murine interleukin quantity is less than normal group, but no significant difference；
Compared with model group, DAISHICHONGCAO mycelium polysaccharides gavage treatment group quantity of leucocyte significantly rises
High (P ＜ 0.01)；Quantity of leucocyte increases the agent certain with the presentation of DAISHICHONGCAO mycelium polysaccharides
Amount dependence；When given low is 400mg/kg/d, effect is double better than positive drug diformazan
The curative effect of guanidine, illustrates that DAISHICHONGCAO mycelium polysaccharides can lift the immanoprotection action of body.
Impact of the 4 DAISHICHONGCAO mycelium polysaccharides of table to diabetic mice peripheral white blood cell amount (N=12)
Note:Compare with MCGaP<0.01；
5th, impact of the DAISHICHONGCAO mycelium polysaccharides to mouse immune organ organ index：With just
Often group is compared, and model group mouse thymus index significantly reduces (P<0.05)；Compared with model group,
High dose administration group can significantly improve mouse thymus index (P<0.05), therefore, DAISHICHONGCAO
Mycelium polysaccharides can improve the cellular immune function of body.
Impact of the 8 DAISHICHONGCAO mycelium polysaccharides of table to diabetic mice immune organ organ index
(N=8-12)
Note:Compare with NG,aP<0.05；Compare with MCG,bP<0.05
Embodiments of the invention 5：Medicine (includes DAISHICHONGCAO mycelium polysaccharides alone or in combination
Pharmaceutical carrier or excipient or antidiabetic drug) add appropriate amount of auxiliary materials to mix by recipe quantity to be pressed into
The tablet (including plain piece, coated tablet, Film coated tablets etc.) of disc-shaped or different in nature lamellar；Or press
Recipe quantity adds proper auxiliary materials to be filled in Capsuleses or the capsule system being sealed in soft capsule material
Agent (including hard capsule, soft capsule, enteric coated capsule etc.)；Or add by recipe quantity appropriate auxiliary
Material formulates the granular preparation of certain particle size；Or add appropriate adhesive by recipe quantity or other are auxiliary
Material makes the pill of ball shape or class ball shape；Or add appropriate dense aqueous sucrose solution to make by recipe quantity
Syrup；Or after mixing with suitable substrate heating melting by recipe quantity, instill not miscible
Condensed fluid in, shrink condensation and made by spherical or spherical drop pill；Or press recipe quantity
Other water preparations for oral administration for adding tea or other adjuvants to be mixed.
Dosage of the invention depends on many factors, including the property for preventing or treating disease and sternly
Weight degree, the sex of animal or patient, age, body weight, individual reaction, route of administration, gives
Medicine number of times etc..Therefore, dosage has wide variation.Can be produced according to complex of the present invention
Preparation in contained actual drug quantity, suitably adjusted, to reach its therapeutic purposes.
Claims (2)
1. a kind of preparation method of DAISHICHONGCAO mycelium polysaccharides, it is characterised in that：Comprise the steps：
1）DAISHICHONGCAO mycelium is prepared using DAISHICHONGCAO GYYA0601 bacterial strain；
2）After DAISHICHONGCAO mycelium is dried, 60-100 mesh molecular sieves were crushed to, obtained DAISHICHONGCAO erinaceus mycelium powder；
3）1 will be pressed in DAISHICHONGCAO erinaceus mycelium powder:The solid-liquid ratio of 3-5 adds petroleum ether degreasing, and the unit of solid-liquid ratio is g/ml；Mycelium powder after defat is pressed into 1：5-15 solid-liquid ratios add distilled water, and the unit of solid-liquid ratio is g/ml, extracts crude polysaccharides using the method for water extract-alcohol precipitation；By crude polysaccharides Jing after Deproteinization, decolouring and dialysis, precipitate with ethanol is concentrated, DAISHICHONGCAO mycelium polysaccharides are obtained.
2. application of a kind of DAISHICHONGCAO mycelium polysaccharides as claimed in claim 2 in antidiabetic medicine is prepared.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108611282A (en) * | 2018-05-16 | 2018-10-02 | 江苏师范大学 | The preparation method and application of one plant of Hypocreales fungi, its exocellular polysaccharide |
| CN114292345A (en) * | 2022-01-25 | 2022-04-08 | 遵义医科大学附属医院 | Decolorization method of cordyceps sinensis polysaccharide |
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| CN103467618A (en) * | 2013-09-23 | 2013-12-25 | 江苏大学 | Method for separating and purifying cordyceps sinensis mycelia polysaccharides |
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| CN101857842A (en) * | 2009-04-09 | 2010-10-13 | 遵义医学院附属医院 | Metarhizium GYYA0601 strain capable of producing broad-spectrum antibiotics and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108611282A (en) * | 2018-05-16 | 2018-10-02 | 江苏师范大学 | The preparation method and application of one plant of Hypocreales fungi, its exocellular polysaccharide |
| CN114292345A (en) * | 2022-01-25 | 2022-04-08 | 遵义医科大学附属医院 | Decolorization method of cordyceps sinensis polysaccharide |
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Application publication date: 20170322 |