CN110279724A - Cicada fungus active material and its purposes for preventing, delaying or treating cataract - Google Patents
Cicada fungus active material and its purposes for preventing, delaying or treating cataract Download PDFInfo
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Abstract
The present invention provides a kind of cicada fungus active material and its purposes for preventing, delaying or treating cataract, belong to active material technical field, the preparation method of cicada fungus active material, including, the rear Cordyceps cicadae strain activated in PDA slant medium is inoculated in seed liquid culture medium and is cultivated, seed liquor is obtained；Seed liquor is inoculated in the PDA liquid culture medium containing Chinese medical extract and Cajanonic acid A and is cultivated, then dry, pulverize mycelium and fermentation liquid, obtains the fermentation material comprising active material；Fermentation material is extracted in distilled water, obtains extracting solution, is concentrated into medicinal extract, it is dry to get cicada fungus active material.Each active constituent in cicada fungus active material that preparation method of the present invention obtains can play gain effect; there is protective effect for crystalline body function; delay the pathogenesis of posterior capsule opacification; the development for delaying cataract is expected in the prevention and treatment for being applied to age-related cataract and After Cataract.
Description
Technical field
The invention belongs to active material technical fields, and in particular to cicada fungus active material and its for preventing, delaying or control
Treat the purposes of cataract.
Background technique
Cataract refers to due to various reasons such as aging, local dystrophia, immune and metabolic disorder, wound, poisoning, spoke
It penetrates etc. and to cause crystalline lens metabolic disorder, make denaturation of lens proteins and muddiness occurs, eventually lead to what visual impairment was even lost
Disease seriously affects daily life and the life quality of patient.With the aging of China's population, cataract has become most
One of main diseases causing blindness.Currently, the main mode for the treatment of cataract is to carry out lens replacement surgery, that is, take out muddiness
Crystalline lens, implantable artificial crystal.Although cataract surgery has graduallyd mature, operative treatment not only gives patient house
Front yard and society bring huge economic problems, especially for many impoverished patients of developing country, mostly because of financial difficulties
And operative treatment cannot be mostly carried out in time.Furthermore there is also certain complication, such as inverse position method are white interior for cataract operation itself
Barrier, forfeiture of adjusting force of intraocular lens etc..There is investigation to show, if the age of onset of cataract delays 10 years, operation
Rate will decline 45%, this not only has great importance to the life quality for improving China human mortality；Moreover, a large amount of to saving
Human and financial resources, the overall cost for reducing social development all have important practical significance.Therefore, cataract is actively inquired into
Pathogenic factor and pathogenesis, delay and prevent the occurrence and development of cataract, it has also become one of ophthalmology worker is important
Project.Seek medical treatment cataract with important economic benefit and social benefit.
Crystalline lens is made of lens capsule, lens fibers, is had on single layer crystalline lens under anterior lens capsule and ambitus capsule
Chrotoplast, lenticular muddiness are known as cataract.The pathogenesis of cataract is there is no unified final conclusion, according in recent years
It studies, oxidative damage caused by free radical is the common pathway for causing various cataract, and the apoptosis of lens epithelial cells is institute
There is the Cytological Basis of type Cataractogenesis.Therefore, inhibiting lens epithelial cell apoptosis is the pass that prevention occurs
Key.Cicada fungus (Isaria cicadae Miquel), is a kind of rare traditional Chinese medicine, is a kind of Cordyceps Militaris colonized on cicada larva.
Belong to Ascomycota (Ascomycota), the cup fungi subphylum (Pezizomycotina), excrement shell Gammaproteobacteria of mycota (Fungi)
(Sordariomycetes), Hypocreales (Hypocreales), cordyceps sinensis section (Cordycipitaceae), Isaria category
(Isaria).Traditional application of set prescription such as existing literature report " the cicada fungus five tastes dissipate " and " ten thousand answer cicada fungus to dissipate " is in eye related disease
Research.Peng Guanghua of Hospital No.1 Attached to Henan Medical Univ.'s ophthalmology etc. was once dissipated with the raw liquid of Chinese medicine and " the cicada fungus five tastes dissipate " cooperation west
Medicine treats traumatic hypotony.Traumatic hypotony is a kind of common complication of eye traumas, can seriously affect visual function.This grinds
Study carefully 14 people of western medicines in treatment group as the result is shown and reach normal intraocular tension (> 1.33kPa), effective percentage accounts for 46.67%, averagely raising intraocular pressure
0.76kPa, 8 people reach normal intraocular tension in 30 people of western medicine group, and effective percentage accounts for 26.67%, averagely raising intraocular pressure 0.41kPa；In
Washout treatment spring conjunctivitis 100 for oral administration of the observation such as institute of traditional Chinese medicine, Xinxiang City, state Xu great Mei " ten thousand answer cicada fungus to dissipate " plus-minus is clinical to be seen
It examines, and does paired observation with western medicine.As a result treatment group's cure rate is 78%, control group 26%.After 1 year, treatment group is multiple
Hair rate is 22%, control group 88%.
Summary of the invention
Sufficient nutrition is provided for Cordyceps cicadae strain the purpose of the present invention is to provide a kind of, promotes its growth and breeding, effectively
Content, the content of cordycepic acid and cordycepin of polysaccharide in cicada fungus active material are improved, the activity of gained cicada fungus active material is improved
Cicada fungus active material preparation method.
The technical solution that the present invention is taken to achieve the above object are as follows:
The preparation method of cicada fungus active material, including,
1) the rear Cordyceps cicadae strain activated in PDA slant medium is inoculated in seed liquid culture medium and is cultivated, planted
Sub- liquid；
2) seed liquor of step 1) is inoculated in liquid fermentation medium and is cultivated, then do mycelium and fermentation liquid
It is dry, it crushes, obtains the fermentation material comprising active material；
3) fermentation material of step 2) is extracted in distilled water, obtains extracting solution, be concentrated into medicinal extract, it is dry to get cicada fungus
Active material；
Aforesaid liquid fermentation medium is the PDA liquid culture medium containing Chinese medical extract and Cajanonic acid A.
On the one hand the presence of liquid fermentation medium Chinese medical extract and Cajanonic acid A can change Cordyceps cicadae strain cell wall
Structure, increase wall permeability, accelerate the rate that Cordyceps cicadae strain absorbs nourishment from culture medium, significantly improve cicada fungus bacterium
Kind is conducive to generate new secondary to the consumption of carbohydrate in liquid fermentation medium to influence intracellular metabolism
The secretion of metabolite and metabolite improves polysaccharide and cordyceps sinensis in cicada fungus active material while promoting Cordyceps cicadae strain growth
The content of acid, to change the activity of cicada fungus active material；On the other hand coding adenylosuccinate synthetase base can be raised
Because of the transcriptional level of-purA gene, the content that AMP key enzyme is synthesized by IMP is improved, to promote cordycepin during the cultivation process
Synthesis, improve cicada fungus active material in cordycepin content.It follows that present invention liquid fermentation medium can not only be
Cordyceps cicadae strain provides sufficient nutrition, promotes its growth and breeding, and can effectively improve the content of polysaccharide in cicada fungus active material,
The content of cordycepic acid and cordycepin improves the activity of gained cicada fungus active material.
Preferably, Chinese medical extract is selected from wolfberry fruit extract, Honegsukle flower P.E, Astragalus Root P.E, tree beans extract, purple
At least one of wingceltis stilbene extract.
Preferably, Chinese medical extract is water extract of TCM.Water extract of TCM is prepared by the following procedure method and is made: by 50-100
Mesh traditional Chinese medicine powder adds 5-8 times of water logging alveolation 5-15min, then extracts at 90-100 DEG C and boils 30-60min, extraction is collected by filtration
Liquid, repetition are extracted 1-2 times, and filtrate is gone out after merging presses concentration, and Chinese medicine water extract is obtained after freeze-drying.
It is furthermore preferred that Chinese medical extract and 0.1-10mg/L Cajanonic acid A in PDA liquid culture medium containing 2-8g/L.
The activity for the cicada fungus active material that PDA liquid culture medium in the concentration range makes reaches best.
Preferably, step 2) content >=145.36mg/g of polysaccharide in mycelium, the content of cordycepic acid >=
202.47mg/g, content >=324.37 μ g/g of cordycepin.
Preferably, the solid-to-liquid ratio of fermentation material and distilled water is 1:8-15g/mL in step 3), and Extracting temperature is 85-95 DEG C,
Time is 5-7h.
It is another object of the present invention to provide one kind to have protective effect for crystalline body function, delays Lens capsular muddy
Turbid pathogenesis delays the development of cataract, is expected to be applied to the prevention of age-related cataract and After Cataract
With the cicada fungus active material in treatment.
The technical solution that the present invention is taken to achieve the above object are as follows:
Cicada fungus active material is obtained by the preparation method of above-mentioned cicada fungus active material.
Each active constituent in cicada fungus active material of the present invention can play gain effect, by enhance it is anti-oxidant it is related because
The expression of son plays cytoprotection, makes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxide
The expression quantity of compound enzyme (GSH-Px) increases, and improves lens epithelial cells (LECs) oxidation resistance under oxidative stress status,
Effective protection lens epithelial cells and crystalline body function；Meanwhile cicada fungus active material of the present invention can obviously inhibit to be given birth to by conversion
Lens epithelial cells abnormality proliferation, migration, the conversion of epithelium mesenchyma, collagen deposition and the fiber of the long factor (TGF-β 2) induction
It organizes the formation of, delays the pathogenesis of posterior capsule opacification.Therefore, cicada fungus active material of the present invention has crystalline body function and protects
Shield effect, can reduce lenticular turbidity, delay the development of cataract, be expected to be applied to age-related cataract and
In the prevention and treatment of After Cataract.
Preferably, it is 2.6 × 10 that active material, which contains molecular weight,4The active polysaccharide of D.
Preferably, active polysaccharide is made of mannose, glucose and rhamnose.The active polysaccharide is one mainly by sweet dew
The gala glucomannan of sugar composition, wherein the molar ratio of mannose, glucose and galactolipin is 6.6:1.3:1.
Invention additionally discloses cicada fungus active materials in preparing the medical composition for preventing, delaying or treat cataract
Purposes.
Compared with prior art, the invention has the benefit that preparation method of the present invention with liquid fermentation medium not only
Sufficient nutrition can be provided for Cordyceps cicadae strain, promote its growth and breeding, and polysaccharide in cicada fungus active material can be effectively improved
The content of content, cordycepic acid and cordycepin improves the activity of gained cicada fungus active material；It is each in cicada fungus active material of the present invention
Active constituent can play gain effect, improve lens epithelial cells oxidation resistance under oxidative stress status, hence it is evident that inhibit
By the lens epithelial cells abnormality proliferation of transforming growth factor induction, migration, the conversion of epithelium mesenchyma, collagen deposition and fiber
It organizes the formation of, there is protective effect for crystalline body function, delay the pathogenesis of posterior capsule opacification, delay the hair of cataract
Exhibition is expected in the prevention and treatment for being applied to age-related cataract and After Cataract.
Cicada fungus active material is provided present invention employs above-mentioned technical proposal and its for preventing, delaying or treating cataract
Purposes, compensate for the deficiencies in the prior art, reasonable design, easy operation.
Detailed description of the invention
Fig. 1 is the standard curve of 2 middle-molecular-weihydroxyethyl of test example of the present invention measurement；
Fig. 2 is the HPLC map of active polysaccharide in test example 2 of the present invention；
Fig. 3 is the HPLC chromatogram of 10 kinds of monosaccharide standard derivatives in test example 2 of the present invention；
Fig. 4 is the HPLC chromatogram of active polysaccharide derivative in test example 2 of the present invention；
Fig. 5 is cicada fungus active material in test example 4 of the present invention to TGF-β2Induce the shadow of lens epithelial cells survival rate
It rings；
Fig. 6 is cicada fungus active material in test example 4 of the present invention to TGF-β2The migration distance for inducing lens epithelium thin
It influences；
Fig. 7 is cicada fungus active material in test example 4 of the present invention to TGF-β2Induce lens epithelial cells epithelium mesenchyma
The influence of conversion.
Specific embodiment
Described below is to enable to have in field usually intellectual and understand and use of the invention and of the invention
Preferred embodiment.However, because general scope principle has been proposed in the present invention, so any scope that do not depart from
Obvious modification should all belong to the range of the present invention.
The present invention relates to a kind of preparation methods of cicada fungus active material, including,
1) the rear Cordyceps cicadae strain activated in PDA slant medium is inoculated in seed liquid culture medium, at 20-30 DEG C
(such as 22 DEG C, 22.5 DEG C, 23 DEG C, 24 DEG C, 27 DEG C, 28 DEG C etc.), 100-150r/min (such as 105r/min, 110r/min,
124r/min, 130r/min, 140r/min, 145r/min etc.) under shaking table culture 2-5d (such as 2.5d, 3d, 3.5d, 4d, 4.5d
Deng), obtain seed liquor；
2) by the seed liquor of step 1) with 5-15%'s (such as 5.4%, 7%, 8%, 10.8%, 13%, 14.5% etc.)
Inoculum concentration is inoculated in liquid fermentation medium, at 20-30 DEG C (such as 22 DEG C, 22.5 DEG C, 23 DEG C, 24 DEG C, 27 DEG C, 28 DEG C
Deng), shaking table under 50-100r/min (such as 55r/min, 60r/min, 74r/min, 80r/min, 90r/min, 95r/min etc.)
It cultivates 2-5d (such as 2.5d, 3d, 3.5d, 4d, 4.5d etc.), mycelium and fermentation liquid is freeze-dried, 20-100 is smashed it through
Mesh obtains the fermentation material comprising active material；Replace heating, drying that extract is dried using Freeze Drying Technique, energy
Enough avoid damage to the nutritional ingredient of extract；
3) fermentation material of step 2) is extracted in distilled water, obtains extracting solution, medicinal extract is concentrated at 50 DEG C, freezing is dry
It is dry, 20-100 mesh is smashed it through to get cicada fungus active material；
Aforesaid liquid fermentation medium is the PDA liquid culture medium containing Chinese medical extract and Cajanonic acid A.
On the one hand the presence of liquid fermentation medium Chinese medical extract and Cajanonic acid A can change Cordyceps cicadae strain cell wall
Structure, increase wall permeability, accelerate the rate that Cordyceps cicadae strain absorbs nourishment from culture medium, significantly improve cicada fungus bacterium
Kind is conducive to generate new secondary to the consumption of carbohydrate in liquid fermentation medium to influence intracellular metabolism
The secretion of metabolite and metabolite improves polysaccharide and cordyceps sinensis in cicada fungus active material while promoting Cordyceps cicadae strain growth
The content of acid, to change the activity of cicada fungus active material；On the other hand coding adenylosuccinate synthetase base can be raised
Because of the transcriptional level of-purA gene, the content that AMP key enzyme is synthesized by IMP is improved, to promote cordycepin during the cultivation process
Synthesis, improve cicada fungus active material in cordycepin content.It follows that present embodiment with liquid fermentation medium not only
Sufficient nutrition can be provided for Cordyceps cicadae strain, promote its growth and breeding, and polysaccharide in cicada fungus active material can be effectively improved
The content of content, cordycepic acid and cordycepin improves the activity of gained cicada fungus active material.
In the embodiment of the present invention, Chinese medical extract is selected from wolfberry fruit extract, Honegsukle flower P.E, Astragalus Root P.E, tree
At least one of beans extract, pterostilbene extract.
In the embodiment of the present invention, Chinese medical extract is water extract of TCM.Method system is prepared by the following procedure in water extract of TCM
: 50-100 mesh traditional Chinese medicine powder is added into 5-8 times of water logging alveolation 5-15min, is then extracted at 90-100 DEG C and boils 30-60min, mistake
Extracting solution is collected in filter, and repetition is extracted 1-2 times, and pressure of going out after filtrate merging concentration obtains Chinese medicine water extract after freeze-drying.
In the embodiment of the present invention, in PDA liquid culture medium containing 2-8g/L (such as 2.2g/L, 3.5g/L, 4g/L,
5.1g/L, 7g/L, 7.5g/L etc.) Chinese medical extract and 0.1-10mg/L Cajanonic acid A (such as 0.5mg/L, 1.0mg/L,
2.3mg/L, 4.5mg/L, 6.8mg/L, 8.0mg/L etc.).The cicada fungus that PDA liquid culture medium in the concentration range makes is living
The activity of property substance reaches best.
In the embodiment of the present invention, step 2) obtains content >=145.36mg/g of polysaccharide in mycelium, and cordycepic acid contains
Amount >=202.47mg/g, content >=324.37 μ g/g of cordycepin.
In the embodiment of the present invention, in step 3) solid-to-liquid ratio of fermentation material and distilled water be 1:8-15g/mL (such as 1:
8.2g/mL, 1:9.4g/mL, 1:10g/mL, 1:11g/mL, 1:12g/mL, 1:14.5g/mL etc.), Extracting temperature is 85-95 DEG C
(such as 86 DEG C, 87 DEG C, 88 DEG C, 90.5 DEG C, 92 DEG C, 93 DEG C etc.), the time be 5-7h (such as 5.2h, 5.5h, 5.8h, 6.5h,
6.7h, 6.8h etc.).
The invention further relates to a kind of cicada fungus active materials, are obtained by the preparation method of above-mentioned cicada fungus active material.
Each active constituent in present embodiment cicada fungus active material can play gain effect, by enhancing anti-oxidant phase
The expression for closing the factor plays cytoprotection, makes catalase (CAT), superoxide dismutase (SOD) and glutathione
The expression quantity of peroxidase (GSH-Px) increases, and improves lens epithelial cells (LECs) anti-oxidant energy under oxidative stress status
Power, effective protection lens epithelial cells and crystalline body function；Meanwhile present embodiment cicada fungus active material can obviously inhibit by
The lens epithelial cells abnormality proliferation of transforming growth factor (TGF-β 2) induction, migration, the conversion of epithelium mesenchyma, collagen deposition
And fibr tissue is formed, and the pathogenesis of posterior capsule opacification is delayed.Therefore, present embodiment cicada fungus active material is for crystalline lens
Function has protective effect, can reduce lenticular turbidity, delays the development of cataract, is expected to be applied to age correlation
In the prevention and treatment of cataract and After Cataract.
In the embodiment of the present invention, it is 2.6 × 10 that active material, which contains molecular weight,4The active polysaccharide of D.
In the embodiment of the present invention, active polysaccharide is made of mannose, glucose and rhamnose.The active polysaccharide is one
The gala glucomannan being mainly made of mannose, wherein the molar ratio of mannose, glucose and galactolipin is 6.6:
1.3:1 is mainly made of mannose, and structure feature is main chain by 1,6- and 1, and the Portugal of the mannose residue composition of 3- connection is sweet
Glycan.
The invention further relates to cicada fungus active materials in preparing the medical composition for preventing, delaying or treat cataract
Purposes.
In the following, being described further in conjunction with specific embodiments to embodiment of the present invention.
Embodiment 1:
The preparation method of cicada fungus active material, including,
1) the rear Cordyceps cicadae strain activated in PDA slant medium is inoculated in seed liquid culture medium, 20 DEG C,
Shaking table culture 2d under 100r/min obtains seed liquor；
2) seed liquor of step 1) is inoculated in liquid fermentation medium with 5% inoculum concentration, in 20 DEG C, 50r/min
Mycelium and fermentation liquid are freeze-dried by lower shaking table culture 2d, smash it through 20 meshes, obtain the hair comprising the active material
Ferment object；
3) it is 1:8g/mL by the fermentation material addition distilled water of step 2) by solid-to-liquid ratio, extracts 5h at 85 DEG C, mentioned
Liquid is taken, medicinal extract is concentrated into, is freeze-dried, smashes it through 20 meshes to get cicada fungus active material；
Aforesaid liquid fermentation medium is the PDA liquid of the fructus lycii water extract containing 2g/L and 0.1mg/L Cajanonic acid A
Culture medium.
Embodiment 2:
The preparation method of cicada fungus active material, including,
1) the rear Cordyceps cicadae strain activated in PDA slant medium is inoculated in seed liquid culture medium, 25 DEG C,
Shaking table culture 3d under 120r/min obtains seed liquor；
2) seed liquor of step 1) is inoculated in liquid fermentation medium with 10% inoculum concentration, in 25 DEG C, 80r/min
Mycelium and fermentation liquid are freeze-dried by lower shaking table culture 3d, smash it through 60 meshes, obtain the hair comprising the active material
Ferment object；
3) it is 1:12g/mL by the fermentation material addition distilled water of step 2) by solid-to-liquid ratio, 6h is extracted at 90 DEG C, is obtained
Extracting solution is concentrated into medicinal extract, and freeze-drying smashes it through 60 meshes to get cicada fungus active material；
Aforesaid liquid fermentation medium is the PDA liquid of the fructus lycii water extract containing 5g/L and 2.0mg/L Cajanonic acid A
Culture medium.
Embodiment 3:
The preparation method of cicada fungus active material, including,
1) the rear Cordyceps cicadae strain activated in PDA slant medium is inoculated in seed liquid culture medium, 30 DEG C,
Shaking table culture 5d under 150r/min obtains seed liquor；
2) seed liquor of step 1) is inoculated in liquid fermentation medium with 15% inoculum concentration, in 30 DEG C, 100r/
Mycelium and fermentation liquid are freeze-dried by shaking table culture 5d under min, smash it through 100 meshes, and obtaining includes the active material
Fermentation material；
3) it is 1:15g/mL by the fermentation material addition distilled water of step 2) by solid-to-liquid ratio, 7h is extracted at 95 DEG C, is obtained
Extracting solution is concentrated into medicinal extract, and freeze-drying smashes it through 100 meshes to get cicada fungus active material；
Aforesaid liquid fermentation medium is the PDA liquid of the fructus lycii water extract containing 8g/L and 10mg/L Cajanonic acid A
Culture medium.
Embodiment 4:
Cicada fungus active material
With embodiment 2 the difference is that: liquid fermentation medium be the honeysuckle flower water extract containing 5g/L and
The PDA liquid culture medium of 2.0mg/L Cajanonic acid A.
Embodiment 5:
Cicada fungus active material
With embodiment 2 the difference is that: liquid fermentation medium be the Radix Astragali water extract containing 5g/L and
The PDA liquid culture medium of 2.0mg/L Cajanonic acid A.
Embodiment 6:
Cicada fungus active material
With embodiment 2 the difference is that: liquid fermentation medium be the tree beans water extract containing 5g/L and
The PDA liquid culture medium of 2.0mg/L Cajanonic acid A.
Embodiment 7:
Cicada fungus active material
With embodiment 2 the difference is that: liquid fermentation medium be the pterostilbene water extract containing 5g/L and
The PDA liquid culture medium of 2.0mg/L Cajanonic acid A.
Comparative example 1:
Compared with Example 2, the difference of this comparative example are as follows: liquid fermentation medium is that the fructus lycii water containing 5g/L mentions
Take the PDA liquid culture medium of object.
Comparative example 2:
Compared with Example 2, the difference of this comparative example are as follows: liquid fermentation medium is to contain 2.0mg/L tree beans ketone
The PDA liquid culture medium of sour A.
Comparative example 3:
Compared with Example 2, the difference of this comparative example are as follows: liquid fermentation medium is PDA liquid culture medium.
Test example 1:
1. fermentation liquid biomass estimation
After the completion of liquid state fermentation culture culture, after mycelium drying, weigh to calculate mycelial weight.
2. the measurement of polysaccharide in mycelium
The dry erinaceus mycelium powder of 0.05g is accurately weighed, is placed in 150mL triangular flask, is added 20mL water, used in micro-wave oven
Middle high fire extracts 1.5min, is then centrifuged 15min at 5000rpm, 95% ethyl alcohol (matter of 3 times of volumes is added in supernatant
Measure concentration), stand 12h at 4 DEG C, take out centrifugation, abandon supernatant, it is to be precipitated dry after water is added, obtain Thick many candies solution；Thick
The H of 1mL mass concentration 30% is added in polysaccharide solution2O2, it is made into the mixed liquor that whole mass concentration is 3%, is decolourized in 60 DEG C of heat preservations
After 2h, H is driven away in heating2O2, and examine decoloration whether complete with potassium permanganate solution；2.4 times of Sevage liquid are added into solution
(chloroform: n-butanol=5:1) extracts 25min, and centrifugation takes top layer's clear liquid, and 95% ethyl alcohol of 3 times of volumes is added, and (quality is dense
Degree), 12h is stood at 4 DEG C, takes out centrifugation, abandons supernatant, and precipitating is dried as mycelium polysaccharides.
The measurement of polysaccharide uses sulfuric acid-phynol method, its absorbance is measured at 490nm.Blank is done with water, with the micro- of polysaccharide
Grams is abscissa, using absorbance value as ordinate, draws standard curve.
The absorbance of sample liquid is measured, polyoses content (C) is calculated with standard curve.The results are shown in Table 1.
Calculation formula: polyoses content (mg/g)=C × 10 in mycelium-3×V×W-1,
In formula, C- polyoses content (μ g/mL)；
V- sample volume (mL)；
W- dry erinaceus mycelium powder quality (g).
3. the measurement of cordycepic acid in mycelium
The erinaceus mycelium powder for taking 0.05g dry, is put into 50mL colorimetric cylinder, and 10mL distilled water is added.It is placed in micro-wave oven
Fire processing 2min, is then centrifuged 10min at 5000rpm, takes supernatant constant volume in 100mL volumetric flask.
Cordycepic acid measuring method uses sodium metaperiodate colorimetric method, prepares PEARLITOL 25C standard liquid, is existed with ultraviolet specrophotometer
Light absorption value is measured at 420nm, blank is done with water, is vertical sit with absorbance value using the micrograms of PEARLITOL 25C content as abscissa
Mark draws standard curve.
The OD value for measuring sample liquid, carries it into standard curve to calculate cordycepic acid content in sample liquid (C).As a result such as
Shown in table 1.
Calculation formula: cordycepic acid content (mg/g)=C × 10 in mycelium-3×100×V×m-1,
In formula, C- cordycepic acid content (μ g/g)；
Constant volume (mL) after the extraction of 100- sample；
M- dry erinaceus mycelium powder quality (g).
4. the measurement of cordycepin in mycelium
Cordycepin content is measured using high performance liquid chromatography (HPLC), the erinaceus mycelium powder for taking 0.1g dry is put into
In 10mL tool plug test tube, 8mL distilled water is added.It is placed in microwave ingle processing 2min, is then centrifuged 10min at 5000rpm,
Supernatant is spare.
Dry cordycepin standard items 1.733mg is weighed, is dissolved in distilled water, 10mL is settled to and is configured to 173.3 μ g/
The standard solution of mL concentration；Take standard solution 0.5mL, add distilled water two-fold dilution, and so on to the 5th dilution, cordyceps sinensis
Rhzomorph concentration is respectively 173.3,86.65,43.33,21.66,10.83 and 5.42 μ g/mL, obtains cordycepin series of concentrations standard
Solution.
Supernatant is detected on HPLC.Chromatographic condition: chromatographic column Kromasil C18 (250mm × 4.6mm, 5 μ
M), 525 types it is ultraviolet/visible detection device, Detection wavelength 259nm；Flow: 1.0mL/min；Sensitivity 1.0AUFS；20 μ of sample volume
l；30 DEG C of column temperature；Mobile phase: methanol: 0.01M potassium dihydrogen phosphate aqueous solution=15:85.It is control with cordycepin standard items, efficiently
Liquid chromatography for measuring cordycepin content.The results are shown in Table 1.
The content of polysaccharide, cordycepic acid, cordycepin in 1 fermentation liquid biomass of table and mycelium
From table 1 it follows that polysaccharide in the fermentation liquid biomass and mycelium of the embodiment of the present invention 2, embodiment 4-7,
Cordycepic acid, cordycepin content obviously higher than comparative example 1-3, this illustrates that liquid fermentation medium of the invention can not only promote
The growth and breeding of Cordyceps cicadae strain, and containing for the content of polysaccharide in cicada fungus active material, cordycepic acid and cordycepin can be effectively improved
Amount.
Test example 2:
Embodiment 2, embodiment 4-7 are obtained into mycelium polysaccharides according to the measurement of polysaccharide in mycelium in test example 1, added double
Double distilled water stirring and dissolving, centrifugation.Supernatant is separated with DEAE- cellulose column (1.6cm × 60cm).Eluent is followed successively by
The Na Cl of water, 0.05,0.1,0.2,0.3,0.5mol/L, automatic portion collection instrument Fraction collection flow point, according to sulfuric acid-phynol
Faxian colour response result merges identical flow point, is successively collected into saccharic composition W1, W2, W3, W4, W5 and W6.Saccharic composition W1 is successively used
Sephacryl S-200 and Sephadex G-100 carry out column chromatography, obtain neutral polysaccharide A (account for applied sample amount 15%) and B (is accounted for
The 85% of applied sample amount) and C.Wherein B is the active polysaccharide referred in summary of the invention.
Comparative example 1-3 obtains mycelium polysaccharides according to the measurement of polysaccharide in mycelium in test example 1, and dual distilled water is added to stir
Dissolution is mixed, is centrifuged.Supernatant is separated with DEAE- cellulose column (1.6cm × 60cm).Eluent be followed successively by water, 0.05,
0.1, the Na Cl of 0.2,0.3,0.5mol/L, automatic portion collection instrument Fraction collection flow point develop the color anti-according to sulfuric acid-phynol method
Should result merge identical flow point, be successively collected into saccharic composition W1, W2, W3, W4, W5 and W6.Saccharic composition W1 successively uses Sephacryl
S-200 and Sephadex G-100 carries out column chromatography, does not obtain neutral polysaccharide B, that is, do not obtain referring in summary of the invention
Active polysaccharide.
1. the molecular weight determination of active polysaccharide
Active polysaccharide is made into 5mg/mL solution, sample introduction after 0.45 μm of membrane filtration with 0.003mol/L sodium acetate respectively.
Chromatographic condition: TSK-GEL G-3000PWXL column (7.8mm × 300mm), mobile phase are 0.003mol/L sodium acetate, and flow velocity is
0.5m L/min, column temperature are 30 DEG C, and sample and standard items sample volume are 20 μ L.It is determined according to sample HPLC chromatogram peak peak shape pure
Degree, with the molecular weight logarithm of each standard polysaccharide (2000KDa, 500KDa, 70KDa, 40KDa, 10KDa) for ordinate, when reservation
Between for abscissa formulate standard polysaccharide molecular weight standard curve.Further according to the retention time of active polysaccharide, its molecular weight is calculated.
Standard curve is as shown in Figure 1, being computed standard curve equation of linear regression is y=21.013-1.572*x, R2=0.9934,
Middle x is the logarithm (lgMw) of molecular weight, and y is retention time.The HPLC map of active polysaccharide is as shown in Fig. 2, be shown as single right
Claim peak, shows that sample is homogeneous components, the retention time of active polysaccharide is 12.5min.It is calculated to obtain according to equation of linear regression
Molecular mass is 2.6 × 104D。
2. the monosaccharide composition analysis of active polysaccharide
It weighs 10mg active polysaccharide to cut open in bottle in peace, adds the sulfuric acid solution of 5mL 2mol/L, alcolhol burner sealing, 100 DEG C of water
8h is solved, barium carbonate neutralizes, and centrifugation takes supernatant, is concentrated to dryness, obtains polysaccharide hydrolysis object.It is separately added into 10mg hydrochloric acid hydroxyl
Amine and 1m L pyridine, 90 DEG C of water-bath 30min are cooled to room temperature, and 1mL acetic anhydride is added, and 90 DEG C of water-bath 30min are cold
But to room temperature, saccharogenesis nitrile acetyl ester derivant is prepared, carries out GC analysis.Chromatographic condition: HP-1 21000mm × 0.2mm elasticity stone
English capillary chromatographic column；Column temperature: 130 DEG C of initial temperature, retain 5min, 4 DEG C/min of temperature programming, 240 DEG C of final temperature, keep
10min；250 DEG C of temperature of vaporization chamber；250 DEG C of detector temperature.The derivatization method of mixing monosaccharide standard is same as above, according to reservation
Time determines the type of simple monosaccharide composition, according to the molar ratio between calculated by peak area simple monosaccharide.10 kinds of monosaccharide standards are spread out
The HPLC chromatogram of biology (1- mannose, 2- ribose, 3- rhamnose, 4- glucuronic acid, 5- galacturonic in figure as shown in Figure 3
Acid, 6- glucose, 7- galactolipin, 8- xylose, 9- arabinose, 10- fucose).The HPLC chromatogram of active polysaccharide derivative
As shown in figure 4, as shown in Figure 4, active polysaccharide is made of mannose, glucose and rhamnose, wherein mannose, glucose and half
The molar ratio of lactose is 6.6:1.3:1.
Test example 3:
Cicada fungus active material is to H2O2The protective effect of the lens epithelial cells of induced oxidation damage
1. cell culture
Lens epithelial cells are placed in green containing 15% fetal calf serum of volume fraction, 100 μ g/mL streptomysins and 100U/mL
In the DMEM culture medium of mycin, 37 DEG C, volume fraction 5%CO2Cell incubator in culture.After cell fusion to 80%,
It digested, passed on trypsase, subsequent experimental is used for after sub-bottle culture.
2. experimental group
Blank control group: with normal culture solution culture；
H2O2Damage group: 100 μm of ol/L of the every hole addition of lens epithelial cells that culture Fusion Strain reaches 80% are given
H2O2, act on 12h；
Test group: it is incubated for 100 μm of ol/L embodiments 2, embodiment 4-7 cicada fungus active material (being dissolved in sterile PBS) thin
Born of the same parents for 24 hours after, 100 μm of ol/L H are added2O2, act on 12h；
Control group: (nothing is dissolved in 100 μm of ol/L comparative example 1-3 cicada fungus active materials, active polysaccharide, cordycepic acid, cordycepins
In bacterium PBS) incubated cell for 24 hours after, 100 μm of ol/L H are added2O2, act on 12h.
3. oxidative stress Indexs measure
Cell is collected after trypsin digestion, 1200r/min is centrifuged 10min, and phosphate buffer is added 30 after rinsing 2 times
μ L cell pyrolysis liquid is placed in low temperature on ice and is incubated for 20min, and 3000r/min is centrifuged 10min and uses 721D by kit specification
Intracellular CAT, SOD and GSHPx content of spectrophotometric determination.The results are shown in Table 2, from Table 2, it can be seen that with blank pair
It is compared according to group, H2O2CAT, SOD and GSH-Px content reduce in damage group lens epithelial cells, test group and control group
CAT, SOD and GSH-Px content are above H2O2Damage group, and CAT, SOD and GSH-Px content of test group are above control group,
This illustrates that cicada fungus active material of the present invention can be such that the expression quantity of CAT, SOD and GSH-Px increases, and improves brilliant under oxidative stress status
The oxidation resistance of shape body epithelial cell, effective protection lens epithelial cells and crystalline body function.
The variation of 2 lens epithelial cells CAT, SOD and GSH-Px content of table
Test example 4:
Cicada fungus active material is to TGF-β2The protective effect of the lens epithelial cells of induction
1. cell culture
Lens epithelial cells are placed in green containing 15% fetal calf serum of volume fraction, 100 μ g/mL streptomysins and 100U/mL
In the DMEM culture medium of mycin, 37 DEG C, volume fraction 5%CO2Cell incubator in culture.After cell fusion to 80%,
It digested, passed on trypsase, subsequent experimental is used for after sub-bottle culture.
2. experimental group
Blank control group: with normal culture solution culture；
TGF-β2Group: 10 μm of ol/L of the every hole addition of lens epithelial cells that culture Fusion Strain reaches 80% are given
TGF-β2, act on 12h；
Test group: it is incubated for 100 μm of ol/L embodiments 2, embodiment 4-7 cicada fungus active material (being dissolved in sterile PBS) thin
Born of the same parents for 24 hours after, 10 μm of ol/L TGF-β are added2, act on 12h；
Control group: (nothing is dissolved in 100 μm of ol/L comparative example 1-3 cicada fungus active materials, active polysaccharide, cordycepic acid, cordycepins
In bacterium PBS) incubated cell for 24 hours after, 10 μm of ol/L TGF-β are added2, act on 12h.
3. cicada fungus active material is to TGF-β2Induce the influence of lens epithelial cells abnormality proliferation
Group of cells vigor is detected using CCK-8 method, by above-mentioned group technology culture cell, culture is abandoned after culture
Liquid, phosphate buffer rinse cell, and CCK-8 liquid is added in 1:10 ratio in each culture hole, and the light that microplate reader measures 492nm is close
Angle value, every hole are surveyed 3 times, are averaged.The survival rate (%) of lens epithelial cells=(group of cells OD value/blank pair
According to a group cell OD value) × 100%.As a result (1- blank control group, 2-TGF- β in figure as shown in Figure 52Group, 3- embodiment 2
Test group, 4 test group of 4- embodiment, 5 test group of 5- embodiment, 6 test group of 6- embodiment, 7 test group of 7- embodiment, 8- comparison
1 control group of example, 2 control group of 9- comparative example, 3 control group of 10- comparative example, 11- active polysaccharide control group, 12- cordycepic acid control group,
13- cordycepin control group), from figure 5 it can be seen that compared with blank control group, after the processing of TGF-β 2, TGF-β2Group cell is living
Power significantly increases, and the cell viability of test group and control group is below TGF-β2Group, and the cell viability of test group is below
Control group, this illustrates that cicada fungus active material of the present invention can obviously inhibit on the crystalline lens induced by transforming growth factor (TGF-β 2)
Chrotoplast abnormality proliferation.
4. cicada fungus active material is to TGF-β2Induce the influence of lens epithelial cells migration
Lens epithelial cells make scratch in six orifice plate centers with 10 μ L pipette tips, by above-mentioned group technology culture cell,
Image Pro Plus software detects the cell migration distance after the processing of TGF-β 2 12h is added respectively.As a result (scheme as shown in Figure 6
Middle 1- blank control group, 2-TGF- β2Group, 2 test group of 3- embodiment, 4 test group of 4- embodiment, 5 test group of 5- embodiment, 6-
6 test group of embodiment, 7 test group of 7- embodiment, 1 control group of 8- comparative example, 2 control group of 9- comparative example, 10- comparative example 3 compare
Group, 11- active polysaccharide control group, 12- cordycepic acid control group, 13- cordycepin control group), from fig. 6 it can be seen that and blank
Control group is compared, after the processing of TGF-β 2, TGF-β2Group group cell migration distance is significant to be increased, the group cell of test group and control group
Migration distance is below TGF-β2Group, and the group cell migration distance of test group is below control group, this illustrates cicada of the present invention
Flower active material can obviously inhibit by transforming growth factor (TGF-β 2) induce lens epithelial cells group cell migration away from
From.
5. cicada fungus active material is to TGF-β2Induce the influence of lens epithelial cells epithelium mesenchyma conversion
By above-mentioned group technology culture cell, RNA, RewerTra Ace qPCR RT are extracted using Trizol kit
Kit kit is to RNA reverse transcription.Real-Time PCR reaction is carried out using SYBR Green PCR Master kit.Inspection
Survey EMT marker type i collagen (collagen I alpha 1chain, COL1A1 in each group sample；collagen I alpha
2chain, COL1A2), type III collagen (collagen III, COL3), IV Collagen Type VI (collagen IV, COL4), fine even egg
The expression of white (fibronectin, Fn), α-smooth muscle actin (α-smoothmuscle actin, α-SMA).Draw
Object sequence is shown in Table 3, as a result using GADPH as internal reference, counts with 2-ΔΔCtValue compares mRNA expression.As a result (scheme as shown in Figure 7
Middle 1- blank control group, 2-TGF- β2Group, 2 test group of 3- embodiment, 4 test group of 4- embodiment, 5 test group of 5- embodiment, 6-
6 test group of embodiment, 7 test group of 7- embodiment, 1 control group of 8- comparative example, 2 control group of 9- comparative example, 10- comparative example 3 compare
Group, 11- active polysaccharide control group, 12- cordycepic acid control group, 13- cordycepin control group), it can be seen from figure 7 that and blank
Control group is compared, after the processing of TGF-β 2, TGF-β2In group epithelium mesenchyma conversion marker COL1A1, COL1A2, COL3,
The middle epithelium mesenchyma of the mRNA expression significant raising respectively of COL4, α-SMA, Fn, test group and control group, which converts, to be indicated
Object COL1A1, COL1A2, COL3, COL4, α-SMA, Fn mRNA expression be below TGF-β2Group, and in test group on
Skin mesenchyma converts marker COL1A1, COL1A2, COL3, COL4, α-SMA, the mRNA expression of Fn is below control group,
This illustrates that cicada fungus active material of the present invention can obviously inhibit the lens epithelial cells induced by transforming growth factor (TGF-β 2)
Epithelium mesenchyma conversion.
3 RT-PCR primer sequence of table
The prior art of routine techniques dawn known to those skilled in the art in above-described embodiment, therefore herein no longer in detail
It repeats.
The above embodiments are only used to illustrate the present invention, and not limitation of the present invention, the ordinary skill people of this field
Member can also make a variety of changes and modification without departing from the spirit and scope of the present invention.Therefore, all equivalent
Technical solution also belong to scope of the invention, scope of patent protection of the invention should be defined by the claims.
Claims (10)
1. the preparation method of cicada fungus active material, it is characterised in that: including,
1) the rear Cordyceps cicadae strain activated in PDA slant medium is inoculated in seed liquid culture medium and is cultivated, obtain seed liquor；
2) seed liquor of step 1) is inoculated in liquid fermentation medium and is cultivated, then that mycelium and fermentation liquid is dry, powder
It is broken, obtain the fermentation material comprising the active material；
3) fermentation material of step 2) is extracted in distilled water, obtains extracting solution, be concentrated into medicinal extract, it is dry to get cicada fungus activity
Substance；
The liquid fermentation medium is the PDA liquid culture medium containing Chinese medical extract and Cajanonic acid A.
2. the preparation method of cicada fungus active material according to claim 1, it is characterised in that: the Chinese medical extract is selected from
At least one of wolfberry fruit extract, Honegsukle flower P.E, Astragalus Root P.E, tree beans extract, pterostilbene extract.
3. the preparation method of cicada fungus active material according to claim 1 or 2, it is characterised in that: the Chinese medical extract
For water extract of TCM.
4. the preparation method of cicada fungus active material according to claim 3, it is characterised in that: the PDA liquid culture medium
In the Chinese medical extract containing 2-8g/L and 0.1-10mg/L Cajanonic acid A.
5. the preparation method of cicada fungus active material according to claim 1 or 2, it is characterised in that: the step 2) obtains bacterium
Content >=145.36mg/g of polysaccharide in filament, content >=202.47mg/g of cordycepic acid, content >=324.37 μ of cordycepin
g/g。
6. the preparation method of cicada fungus active material according to claim 1, it is characterised in that: fermentation material in the step 3)
Solid-to-liquid ratio with distilled water is 1:8-15g/mL, and Extracting temperature is 85-95 DEG C, time 5-7h.
7. cicada fungus active material, it is characterised in that: pass through the preparation side of cicada fungus active material described in any one of claims 1-6
Method obtains.
8. cicada fungus active material according to claim 7, it is characterised in that: it is 2.6 that the active material, which contains molecular weight,
×104The active polysaccharide of D.
9. cicada fungus active material according to claim 7 or 8, it is characterised in that: the active polysaccharide is by mannose, grape
Sugar and rhamnose composition.The active polysaccharide is the gala glucomannan being mainly made of mannose, wherein mannose,
The molar ratio of glucose and galactolipin is 6.6:1.3:1.
10. cicada fungus active material described in claim 7 or 8 or 9 is preparing the medicine for preventing, delaying or treating cataract
Purposes in composition.
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