CN105176844A - Method for bi-directional solid-state fermentation of inonotus obliquus on Chinese herbal material or Chinese medicine residues - Google Patents
Method for bi-directional solid-state fermentation of inonotus obliquus on Chinese herbal material or Chinese medicine residues Download PDFInfo
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Abstract
The invention discloses a method for bi-directional solid-state fermentation of inonotus obliquus on a Chinese herbal material or Chinese medicine residues. The method comprises the steps that a fermentation substrate is prepared, namely a medicinal substrate is processed; a fermentation strain, namely the inonotus obliquus, is processed; the inonotus obliquus is inoculated to the medicinal substrate; bi-directional solid-state fermentation is performed under a certain condition; medicinal mycoplasm is collected, the medicinal substrate is smashed in processing, an appropriate amount of water is added, and high-pressure sterilization is performed after bottling, wherein a fermentation strain processing method comprises the steps of activating the inonotus obliquus and performing multiplication culture at the 28 DEG C temperature for 14-16 d, and a multiplication culture method is performed by adopting the liquid culture medium and shake flasks for culture. The inonotus obliquus is inoculated to the medicinal substrate of the Chinese herbal material or the Chinese medicine residues for bi-directional solid-state fermentation, the produced medicinal mycoplasm is non-toxic and has good anti-oxidation effect, hypoglycemic effect, immune adjustment effect and the like, the problem of lack of a precious and rare medicinal fungi-inonotus obliquus resource is effectively solved, and a new way is opened for effective reutilization of the Chinese medicine residues.
Description
Technical field
The present invention relates to Chinese traditional medicine biology technical field, specifically a kind of Chinese medicinal materials of Phaeopoms obliquus or the two-way solid fermentation method of Chinese medicine slag.
Background technology
Phaeopoms obliquus Inonotusobliquus (Ach.exPers.) Pil á t is a kind of rare medicinal fungi, since 16-17 century, Eastern Europe, Russia, Poland, Finland etc. are among the people extensively utilizes it to prevent and treat various difficult and complicated cases, as various cancer, heart trouble, diabetes etc.Experimentation on animals and clinically to show: Phaeopoms obliquus has no side effect, has anticancer, treatment diabetes, prevents AIDS, the function such as anti-ageing, strengthening immunity.For tumour, diabetes, cardiovascular system diseases, antidotal prevention and therapy, therefore Phaeopoms obliquus has broad application prospects and Development volue in fields such as medicine, food, healthcare products, and market demand continues to increase.But current Phaeopoms obliquus wild resource is exhausted, artificial domesticating cultivation is not yet successful, and the fermentation condition of liquid fermenting requires strict, the easy microbiological contamination of fermenting process, facility investment is excessive and dry matter content is low, production cost is high, causes Phaeopoms obliquus scarcity of resources.
Chinese medicine slag derives from the manufacturing enterprise of processing Chinese medicine material medicine, the processing of the prepared slices of Chinese crude drugs and the process of preparing Chinese medicine and the light industry product enterprise etc. containing traditional Chinese medicine ingredients, and the annual emissions of national Chinese medicine slag reaches more than 3,000 ten thousand tons.Chinese medicine slag is generally regarded as refuse and abandons, and as adopted mode process such as stacking landfill burning, causes the significant wastage of resource and serious environmental pollution.At present, Chinese medicine slag field of comprehensive utilization has obvious limitation, substantially be around agricultural (plant husbandry, aquaculture) direction in the majority, next is paper-making industry, but successful example is few, the exploratory preliminary study of many genus, particularly Chinese medicine slag recycling research phoenix feathers and unicorn horns especially medically.
First professors Zhuan Yi etc. create " two-way solid fermentation " and try out, medicinal fungi is seeded in the property of medicine matrix of Chinese medicinal materials or Chinese medicine slag, carry out solid fermentation under given conditions, produce the medicinal fungal substance with new constituent, new effect, because " property of medicine matrix " not only can provide fungi desired nutritional, the simultaneously structural constituent of the enzyme of fungi also decomposable asymmetric choice net Chinese medicinal materials or Chinese medicine slag, thus produce new composition and effect, therefore " two-way fermentation " is claimed, Here it is medicinal fungi novel (amphicheirality) solid fermentation, is called for short two-way fermentation.Two-way solid fermentation resource requirement is abundant, method is easy, with low cost, Be very effective, medicinal fungi novel amphicheirality's solid fermentation engineering is that study of tcm new drug opens a frontier, i.e. fungus medicine, medicinal fungal substance has an important sources of independent intellectual property right new drug (see Zhuan Yi, a frontier of Chinese medicine by becoming China.New Chinese medicine and clinical pharmacology.1992，3(2)：49-51)。
The content such as theoretical basis, the design of fementative composition, the pharmacological screening of mycoplasma of medicinal fungi novel amphicheirality's solid fermentation engineering can see document (Zhuan Yi, medicinal fungi novel (two-way type) solid fermentation engineering.Edible fungi of china, 2002,21 (4): 3-6).Show that the medicinal fungal substance using this technology to produce has good drug effect, be mainly manifested in the aspects such as synergy, broadened application, removing toxic substances (see 1, the development of medicinal fungi novel solid fermentation engineering and Chinese scholartree stilbene mycoplasma. Chinese Pharmaceutical Journal, 2004,39 (3): 175-178; 2, the foundation of the two-way zymotechnique of trypterygine removing toxic substances holding effect. fungus journal, 2010, (02): 294-299; 3, the removing toxic substances lasting effect of the two-way fermentation trypterygine of glossy ganoderma, herbal medicine, 2009, (12): 1925-1929; 4, medicinal fungi novel (amphicheirality) solid fermentation engineering is to the preliminary study of trypterygine removing toxic substances holding effect, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2009, (16): 2083-2087.)
There is no the Chinese medicinal materials of bibliographical information Phaeopoms obliquus or the pharmacotoxicological effect of the two-way solid-fermented technique of Chinese medicine slag and medicinal fungal substance thereof at present, based on current Phaeopoms obliquus scarcity of resources, a large amount of generations of Chinese medicine slag, need badly and solve the shortage of resources problem of Phaeopoms obliquus, and open up new way for effective recycling of Chinese medicine slag.
Summary of the invention
The object of the present invention is to provide the two-way solid fermentation method of a kind of Chinese medicinal materials of Phaeopoms obliquus or Chinese medicine slag, obtain the medicinal fungal substance with effects such as good antioxygenation, blood sugar reducing function and immunomodulatorys, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
The Chinese medicinal materials of Phaeopoms obliquus or a two-way solid fermentation method for Chinese medicine slag, concrete steps comprise: the preparation of fermented substrate, namely to the process of property of medicine matrix; The process of fermented bacterium Phaeopoms obliquus; Phaeopoms obliquus is inoculated in property of medicine matrix; Carry out two-way solid fermentation; Collect medicinal fungal substance.
As the further scheme of the present invention: described property of medicine matrix, when processing, is pulverized property of medicine matrix, added water and make its water content be 50-65%, bottling is 121 DEG C of autoclaving 1h also.
As the further scheme of the present invention: the treatment process of described fermented bacterium is after being activated by Phaeopoms obliquus, and under 28 DEG C of conditions amplification cultivation 14-16d, the method for amplification cultivation adopts liquid nutrient medium shake-flask culture.
As the further scheme of the present invention: the fermentation condition of described two-way solid fermentation is temperature range 26 DEG C-28 DEG C, and fermentation number of days is 40-60d.
As the further scheme of the present invention: described property of medicine matrix refers to Chinese medicinal materials or Chinese medicine slag.
As the further scheme of the present invention: described Chinese medicinal materials or Chinese medicine slag are the root of kudzu vine, GEGEN QINLIAN TANG prescription medicinal material, LIUWEI DIHUANG WAN prescription medicinal material, the root of kudzu vine dregs of a decoction, the GEGEN QINLIAN TANG dregs of a decoction or the LIUWEI DIHUANG WAN dregs of a decoction.
As the present invention's further scheme: described medicinal fungal substance is Phaeopoms obliquus solid fermentation and mycoplasma produced in the property of medicine matrix containing Chinese medicinal materials or Chinese medicine slag.
Compared with prior art, the invention has the beneficial effects as follows:
Phaeopoms obliquus is seeded in Chinese medicinal materials or Chinese medicine slag property of medicine matrix and carries out two-way solid fermentation by the present invention, the medicinal fungal substance nontoxicity of producing, there is the effects such as good antioxygenation, blood sugar reducing function and immunomodulatory, efficiently solve the shortage of resources problem of rare medicinal fungi-Phaeopoms obliquus, and open new way for effective recycling of Chinese medicine slag.
Accompanying drawing explanation
Fig. 1 is mycoplasma DPPH free radical scavenging activity schematic diagram.
Fig. 2 is mycoplasma OH free radical scavenging activity schematic diagram.
Fig. 3 is the change schematic diagram of FRAP value with concentration of different solvents extraction components.
Fig. 4 is the α-amylase inhibiting rate schematic diagram of different solvents extract part.
Fig. 5 is the alpha-glucosaccharase enzyme inhibition rate schematic diagram of different solvents extract part.
Embodiment
Be described in more detail below in conjunction with the technical scheme of embodiment to this patent.
Embodiment 1
(1) slant medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the agar of 7.5g, the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(2) liquid nutrient medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(3) preparation of fermented bacterium: Phaeopoms obliquus is inoculated on slant medium, cultivate 5-7d for 28 DEG C, now mycelial growth is intensive, with inoculating needle picking 1cm
2bacterium block be inoculated into and be equipped with in the culturing bottle of liquid nutrient medium, put in shaking table and cultivate, shaking speed 130r/min, cultivate 15d for 28 DEG C, make it to produce a large amount of size evenly, as the bacterium ball of " pearl shape ", obtain bacterium ball liquid.
(4) preparation of fermented substrate: after root of kudzu vine drying treatment, is crushed to particle as mung bean, and every bottled 200g, in solid fermentation bottle, adds water and makes the fermented substrate of water content 50%-65%, lid bottle stopper, and also 121 DEG C of autoclaving 1h are for subsequent use in bottling;
(5) two-way solid fermentation: aseptic technique, in each solid fermentation bottle, inoculation bacterium ball liquid about 10mL, is placed in 26 DEG C of-28 DEG C of proving rooms and cultivates, cover with after bottle until mycelia, continue fermentation 30d, stop fermentation, draw bottle, collect medicinal fungal substance by fermentation flask.
(6) medicinal fungal substance aftertreatment: the medicinal fungal substance of collection is dry at 50 DEG C of temperature in time.
Embodiment 2
(1) slant medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the agar of 7.5g, the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(2) liquid nutrient medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(3) preparation of fermented bacterium: Phaeopoms obliquus is inoculated on slant medium, cultivate 5-7d for 28 DEG C, now mycelial growth is intensive, with inoculating needle picking 1cm
2bacterium block be inoculated into and be equipped with in the culturing bottle of liquid nutrient medium, put in shaking table and cultivate, shaking speed 130r/min, cultivate 15d for 28 DEG C, make it to produce a large amount of size evenly, as the bacterium ball of " pearl shape ", obtain bacterium ball liquid.
(4) preparation of fermented substrate: after the process of GEGEN QINLIAN TANG prescription medicinal material drying, be crushed to particle as mung bean, every bottled 200g, in solid fermentation bottle, adds water and makes the fermented substrate of water content 50%-65%, lid bottle stopper, also 121 DEG C of autoclaving 1h are for subsequent use in bottling;
(5) two-way solid fermentation: aseptic technique, in each solid fermentation bottle, inoculation bacterium ball liquid about 10mL, is placed in 26 DEG C of-28 DEG C of proving rooms and cultivates, cover with after bottle until mycelia, continue fermentation 30d, stop fermentation, draw bottle, collect medicinal fungal substance by fermentation flask.
(6) medicinal fungal substance aftertreatment: the medicinal fungal substance of collection is dry at 50 DEG C of temperature in time.
Embodiment 3
(1) slant medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the agar of 7.5g, the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(2) liquid nutrient medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(3) preparation of fermented bacterium: Phaeopoms obliquus is inoculated on slant medium, cultivate 5-7d for 28 DEG C, now mycelial growth is intensive, with inoculating needle picking 1cm
2bacterium block be inoculated into and be equipped with in the culturing bottle of liquid nutrient medium, put in shaking table and cultivate, shaking speed 130r/min, cultivate 15d for 28 DEG C, make it to produce a large amount of size evenly, as the bacterium ball of " pearl shape ", obtain bacterium ball liquid.
(4) preparation of fermented substrate: after the process of LIUWEI DIHUANG WAN prescription medicinal material drying, be crushed to particle as mung bean, every bottled 200g, in solid fermentation bottle, adds water and makes the fermented substrate of water content 50%-65%, lid bottle stopper, also 121 DEG C of autoclaving 1h are for subsequent use in bottling;
(5) two-way solid fermentation: aseptic technique, in each solid fermentation bottle, inoculation bacterium ball liquid about 10mL, is placed in 26 DEG C of-28 DEG C of proving rooms and cultivates, cover with after bottle until mycelia, continue fermentation 30d, stop fermentation, draw bottle, collect medicinal fungal substance by fermentation flask.
(6) medicinal fungal substance aftertreatment: the medicinal fungal substance of collection is dry at 50 DEG C of temperature in time.
Embodiment 4
(1) slant medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the agar of 7.5g, the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(2) liquid nutrient medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(3) preparation of fermented bacterium: Phaeopoms obliquus is inoculated on slant medium, cultivate 5-7d for 28 DEG C, now mycelial growth is intensive, with inoculating needle picking 1cm
2bacterium block be inoculated into and be equipped with in the culturing bottle of liquid nutrient medium, put in shaking table and cultivate, shaking speed 130r/min, cultivate 15d for 28 DEG C, make it to produce a large amount of size evenly, as the bacterium ball of " pearl shape ", obtain bacterium ball liquid.
(4) preparation of fermented substrate: after root of kudzu vine dregs of a decoction drying treatment, be crushed to particle as mung bean, every bottled 200g, in solid fermentation bottle, adds water and makes the fermented substrate of water content 50%-65%, lid bottle stopper, and also 121 DEG C of autoclaving 1h are for subsequent use in bottling;
(5) two-way solid fermentation: aseptic technique, in each solid fermentation bottle, inoculation bacterium ball liquid about 10mL, is placed in 26 DEG C of-28 DEG C of proving rooms and cultivates, cover with after bottle until mycelia, continue fermentation 30d, stop fermentation, draw bottle, collect medicinal fungal substance by fermentation flask.
(6) medicinal fungal substance aftertreatment: the medicinal fungal substance of collection is dry at 50 DEG C of temperature in time.
Embodiment 5
(1) slant medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the agar of 7.5g, the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(2) liquid nutrient medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(3) preparation of fermented bacterium: Phaeopoms obliquus is inoculated on slant medium, cultivate 5-7d for 28 DEG C, now mycelial growth is intensive, with inoculating needle picking 1cm
2bacterium block be inoculated into and be equipped with in the culturing bottle of liquid nutrient medium, put in shaking table and cultivate, shaking speed 130r/min, cultivate 15d for 28 DEG C, make it to produce a large amount of size evenly, as the bacterium ball of " pearl shape ", obtain bacterium ball liquid.
(4) preparation of fermented substrate: after GEGEN QINLIAN TANG dregs of a decoction drying treatment, be crushed to particle as mung bean, every bottled 200g, in solid fermentation bottle, adds water and makes the fermented substrate of water content 50%-65%, lid bottle stopper, also 121 DEG C of autoclaving 1h are for subsequent use in bottling;
(5) two-way solid fermentation: aseptic technique, in each solid fermentation bottle, inoculation bacterium ball liquid about 10mL, is placed in 26 DEG C of-28 DEG C of proving rooms and cultivates, cover with after bottle until mycelia, continue fermentation 30d, stop fermentation, draw bottle, collect medicinal fungal substance by fermentation flask.
(6) medicinal fungal substance aftertreatment: the medicinal fungal substance of collection is dry at 50 DEG C of temperature in time.
Embodiment 6
(1) slant medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the agar of 7.5g, the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(2) liquid nutrient medium preparation: potato 100g peels, cuts into slices, wheat bran 25g adds appropriate tap water and decocts 30min, and gauze filters, and filtrate adds the glucose of 7.5g, the MgSO of 0.75g
4, the KH of 1.5g
2pO
4, add tap water to 500mL, 121 DEG C of autoclaving 30min;
(3) preparation of fermented bacterium: Phaeopoms obliquus is inoculated on slant medium, cultivate 5-7d for 28 DEG C, now mycelial growth is intensive, with inoculating needle picking 1cm
2bacterium block be inoculated into and be equipped with in the culturing bottle of liquid nutrient medium, put in shaking table and cultivate, shaking speed 130r/min, cultivate 15d for 28 DEG C, make it to produce a large amount of size evenly, as the bacterium ball of " pearl shape ", obtain bacterium ball liquid.
(4) preparation of fermented substrate: after LIUWEI DIHUANG WAN dregs of a decoction drying treatment, be crushed to particle as mung bean, every bottled 200g, in solid fermentation bottle, adds water and makes the fermented substrate of water content 50%-65%, lid bottle stopper, also 121 DEG C of autoclaving 1h are for subsequent use in bottling;
(5) two-way solid fermentation: aseptic technique, in each solid fermentation bottle, inoculation bacterium ball liquid about 10mL, is placed in 26 DEG C of-28 DEG C of proving rooms and cultivates, cover with after bottle until mycelia, continue fermentation 30d, stop fermentation, draw bottle, collect medicinal fungal substance by fermentation flask.
(6) medicinal fungal substance aftertreatment: the medicinal fungal substance of collection is dry at 50 DEG C of temperature in time.
Pharmacological experiment
One, the extraction of medicinal fungal substance reactive site and assay:
(1) extraction of medicinal fungal substance reactive site:
The extracting and separating of alcohol extract: get medicinal fungal substance meal 1kg, in the ratio of 1:12 (m/v), adds 80% alcohol reflux (1h/ time, extract 3 times).Dried by the dregs of a decoction after extraction and continue to employ, decompression and solvent recovery after extracting solution merges, obtains ethanol extract 132.1g.Be suspended in by ethanol extract in water, use sherwood oil, ethyl acetate, n-butanol extraction successively, respectively decompression and solvent recovery, vacuum-drying obtains ligroin extraction 1.2g, ethyl acetate extract 21.6g, n-butanol extract 39.3g, water layer extract 53.7g.
Water extract-alcohol precipitation extracts Crude polysaccharides: the dregs of a decoction after alcohol extracting are in the ratio adding distil water refluxing extraction (1h/ time of 1:10 (m/v), extract 3 times), united extraction liquid decompression and solvent recovery is to appropriate, add ethanol to ultimate density 75%, 4 DEG C of standing 24h, abandon supernatant, gained are precipitated vacuum-drying, obtain Crude polysaccharides, phend-sulphuric acid measures polysaccharide content.
(2) Determination of Polyphenols of medicinal fungal substance different solvents extract part measures:
1, standard curve making:
Precision takes gallic acid standard substance and is about 1mg, dissolves and be settled to 10mL volumetric flask to obtain standard solution.The standard solution of accurate absorption 0,0.25,0.5,0.75,1.00,1.25,1.50mL, in 25mL test tube, adds the forint phenol of 3mL respectively, shakes up, and leaves standstill 30s, respectively adds the Na of 12%
2cO
3solution 6.0mL, distilled water is settled to 25mL, and 25 DEG C of lucifuges leave standstill 2h, makes blank 760nm survey OD value with the sample of 0mL.With gallic acid standard substance content (μ g) for X-coordinate, absorbancy is ordinate zou, and drawing standard curve also obtains its regression equation: y=0.0045x+0.0202 (r=0.9995).
2, assay:
Sample thief 1.0mL, takes the method for above production standard curve, measures the content of total polyphenols in each component, in gallic acid.78.84mg/g, 53.96mg/g is respectively by calculating polyphenol content contained by ethyl acetate and propyl carbinol component; Water layer extract and Crude polysaccharides polyphenol content are respectively 5.51mg/g, 15.29mg/g, are starkly lower than ethyl acetate and n-butanol portion.
(3) total triterpene contents of medicinal fungal substance different solvents extract part measures:
1, standard curve making:
Precision takes trochol reference substance, with anhydrous alcohol solution, be made into the standardized solution that mass concentration is 160 μ g/mL, accurate absorption 0.1, 0.2, 0.3, 0.4 and 0.5mL be placed in 5mL volumetric flask respectively, evaporate to dryness in 100 DEG C of water-baths, 5% Vanillin-the glacial acetic acid solution and the 0.6mL perchloric acid that add 0.3mL brand-new shake up, 70 DEG C of water bath heat preservation 15min, cool with frozen water again and put to room temperature, add glacial acetic acid to hold to 5mL, shake up, blank is made with dehydrated alcohol, light absorption value is surveyed with trochol quality (μ g) for X-coordinate at 550nm place, light absorption value is ordinate zou, drawing standard curve also obtains its regression equation: y=0.103x+0.0248 (r=0.9985).
2, assay:
The sample 0.5mL of sample thief 1mg/mL, measure by the method income samples contg of production standard curve, by calculating ethyl acetate, propyl carbinol, Crude polysaccharides, the total triterpene contents of water layer extract is respectively: 34.40mg/g, 26.44mg/g, 3.69mg/g, 4.08mg/g, can find out: the triterpenes content of ethyl acetate and propyl carbinol is far above Crude polysaccharides and water layer extract.
Two, the antioxygenation of medicinal fungal substance different solvents extract part:
1, DPPH free radical scavenging activity measures:
In test tube, add 140 μm of ol/LDPPH ethanolic soln 2mL, then add 1mL test sample, mixing is also fully vibrated.Dark at room temperature place leaves standstill 30min, measures light absorption value in 517nm place.Control group replaces sample liquid with distilled water, and sample controls group replaces DPPH with ethanol, and all the other all add all identical, measures light absorption value with under condition, take Vc as positive control, parallel three experiments.Refer to Fig. 1, clearance rate is by following Equation for Calculating:
(formula 1)
In formula: A
0absorbancy is tested in-contrast (distilled water)
A
xthe absorbancy of-polysaccharide sample liquid
A
x0the experiment of-background absorbs (without DPPH in reaction system)
Can obviously be found out by Fig. 1, VC> ethyl acetate extract > n-butanol extract > water layer extract > Crude polysaccharides > ligroin extraction is followed successively by DPPH free radical scavenging activity is descending, ethyl acetate and n-butanol portion have larger scavenging(action) to DPPH free radical, water layer extract, Crude polysaccharides scavenging(action) are more weak, and the clearance rate of petroleum ether layer is 0 substantially.
2, hydroxyl radical free radical scavenging capacity measures:
Reaction system (4mL) comprising: 1mL sample, the FeSO of 7mmol/L
4solution 1mL, 1mL Whitfield's ointment ethanolic soln (7mmol/L), 0.3mLH
2o
2(0.012%), H
2o
2finally add to start whole reaction.Reaction system is incubation 30min under 37 DEG C of conditions, then centrifugal 10min under 3000r/s condition.Finally detect absorbancy at 510nm place, using Vc as positive control.Refer to Fig. 2, clearance rate is by lower Equation for Calculating:
(formula 2)
In formula: A
0absorbancy is tested in-contrast (distilled water)
A
xthe absorbancy of-polysaccharide sample liquid
A
x0the experiment of-background absorbs (without H in reaction system
20
2)
Can obviously be found out by Fig. 2, VC> ethyl acetate extract > n-butanol extract > water layer extract ≈ Crude polysaccharides > ligroin extraction is followed successively by OH free radical scavenging activity is descending, ethyl acetate and n-butanol portion have larger scavenging(action) to OH free radical, water layer extract, Crude polysaccharides scavenging(action) are more weak, and the clearance rate of petroleum ether layer is 0 substantially.
3, the resistance of oxidation of FRAP method working sample:
FRAP standard curve making: add 1.5mmol/LFeSO4 solution from 0 ~ 400 μ L by 50 μ L amount of spaces, supply less than the distilled water of 400 μ L, add the acetate buffer 4 μ L of 300mmol/L, PH3.6 again, the TPTZ solution 400 μ L of 10mmol/L, shake up rear 37 DEG C of reaction 10min, replace the FeSO of 1.5mmol/L with 400 μ L distilled water
4solution, as blank, surveys absorbancy in 593nm.Absorbance (A) is X-coordinate, Fe
2+concentration (μm ol/L) is ordinate zou, obtains FRAP typical curve and obtains its regression equation:
FRAP value=C (Fe
2+) (μm ol/L)=aA+b (formula 3),
Y=1446.2x-49.419 (r=0.9982) (formula 4).
Adopt the resistance of oxidation of FRAP method working sample, get 0.4mL concentration respectively and be respectively 500,400, the sample solution of 300,200,100 μ g/mL and the FRAP working fluid (sodium acetate buffer of 0.3mol/L, PH3.6 of 3.0mL, the iron trichloride of TPTZ and 20mmol/L of 10mmol/L is with volume ratio 10:1:1 mixing) mixing, leave standstill 10min, measure OD value in 593nm place, replace FeCl with distilled water
3solution is done blank, operates with method.Make positive control with Vc, test parallel 3 times.Refer to Fig. 3, light absorption value substitutes into FRAP graticule and obtains corresponding FRAP value.
The overall trend of the resistance of oxidation (FRAP value) of ethyl acetate and propyl carbinol component is apparently higher than water layer extract and Crude polysaccharides as shown in Figure 3, by calculating, the total antioxidant capacity of ethyl acetate component, namely FRAP value is (1.82 ± 0.03) mmol/L (FeSO
47H
2o
2a great deal of); Propyl carbinol is (0.8375 ± 0.04) mmol/L (FeSO
47H
2o
2a great deal of), Crude polysaccharides and water layer extract are respectively (0.176 ± 0.02) mmol/L (FeSO
47H
2o
2a great deal of), (0.199 ± 0.04) mmol/L (FeSO
47H
2o
2a great deal of), sherwood oil be 0.
Experimental result shows that the medicinal fungal substance that the present invention obtains has good antioxygenation, all show in 3 anti-oxidant experiments ethyl acetate extract part and n-butanol extraction position anti-oxidant activity apparently higher than Crude polysaccharides and water layer extract, may with the polyphenol of ethyl acetate, n-butanol portion and triterpenes content higher relevant.
Three, the blood sugar reducing function of medicinal fungal substance different solvents extract part:
1, the α-amylase inhibit activities of medicinal fungal substance different solvents extract part measures:
Refer to Fig. 4, application of sample system according to the form below carries out, and acarbose does positive control;
| Enzyme liquid | Inhibitor | Starch solution | DNS | |
| Blank tube | 0.3mL | _ | 0.4mL | 0.5mL |
| Blank pipe | _ | _ | 0.4mL | 0.5mL |
| Killer tube | 0.3mL | 1mL | 0.4mL | 0.5mL |
| Ground control pipe | _ | 1mL | 0.4mL | 0.5mL |
As shown in Figure 4, Crude polysaccharides to α-amylase inhibiting rate compared with positive control acarbose, significant difference, but higher than the inhibiting rate at ethyl acetate extract part, n-butanol extraction position, water layer extract, Petroleum ether extraction position, and difference has statistical significance (P<0.01).
2, the alpha-glucosaccharase enzyme inhibition activity of medicinal fungal substance different solvents extract part measures:
Sample tube get 0.2mL mass concentration be respectively 2.0,1.0,0.5,0.25,0.125 and the sample solution (with pH6.8,0.2mol/L phosphate buffered saline) of 0.0625mg/mL add in 25mL test tube, add 0.2mL alpha-glucosaccharase enzyme solution for 3.75U/mL more alive than enzyme respectively to mix in time, then add P-oil of mirbane-α-glucopyranoside solution (phosphate buffered saline) mixing that 0.2mL concentration is 6mmol/L.Control tube 0.2mL phosphate buffered saline buffer replaces sample solution, and the effective 0.2mL phosphate buffered saline buffer of background replaces P-oil of mirbane-α-glucopyranoside solution.37 DEG C of water bath with thermostatic control 30min, then add 6mLNa
2cO
3termination reaction, 400nm place surveys light absorption value, makes positive control, refer to Fig. 5 with acarbose:
Alpha-glucosaccharase enzyme inhibition rate=[1-(A
sample-A background)/A
contrast] × 100% (formula 5).
As shown in Figure 5, Crude polysaccharides to alpha-glucosaccharase enzyme inhibition rate compared with positive control acarbose, significant difference, but higher than the inhibiting rate of ethyl acetate extract, n-butanol portion, water layer extract, petroleum ether part, and difference has statistical significance (P<0.01).
Experimental result shows that the medicinal fungal substance that the present invention obtains has good blood sugar reducing function.The blood sugar reducing function of medicinal fungal substance different solvents extract part compares, and Crude polysaccharides is better than n-butanol portion, ethyl acetate extract, water layer extract and petroleum ether part.
Four, the immunoregulation effect of medicinal fungal substance:
1, experiment material and method:
By one, the medicinal fungal substance Crude polysaccharides (JZPC) that obtains of (extraction of medicinal fungal substance reactive site and assay) (one) (extracting method of medicinal fungal substance reactive site).
The preparation of medicinal fungal substance Crude polysaccharides purified components:
The polysaccharide soln of Sevag method deproteination matter 5% mixes with Sevag reagent equal-volume, and the centrifugal 5min of jolting 30min, 4000r/min, discards metaprotein layer, and upper water liquid operates with method, until metaprotein layer disappears.Coomassie Brilliant Blue measures protein content.
Dialysis is removed the first tap water flowing water of the Deproteinated polysaccharide soln of small-molecule substance 5% and is dialysed 2 days, then deionized water is dialysed 1 day, within every 3 hours, changes time water.Be concentrated into proper volume after having dialysed, vacuum-drying obtains refined polysaccharide, called after JZPJ.
DEAE-SepharoseCL-6B column chromatography grading purification (0.5%, W/V) mycoplasma refined polysaccharide upper prop is separated, use deionized water, 0.1mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.9mol/L, 1.5mol/LNaCl eluant solution successively, volumetric flow rate 1.0ml/min, every bottle of 5ml, phend-sulphuric acid tracing detection.With bottle number for X-coordinate, light absorption value is that ordinate zou draws elution curve, merges same composition, reconcentration according to elution curve, and dialysis final vacuum is dry, obtains polysaccharide purification component JZP1, JZP2, JZP3.
The preparation of mouse spleen lymphocyte suspension: aseptic technique after sacrifice, gets spleen and prepares cell suspension, and Trypan Blue also counts, and viable count is greater than 95%, adjustment cell concn to 4 × 10
6/ ml.
Mice spleen lymphocytes proliferation experiment (mtt assay): in 96 orifice plates, every hole adds above-mentioned cell suspension 100 μ L; The every hole of sample group adds tested material makes its final concentration be 20,100,500,1000 μ g/mL; Blank group adds RPMI1640 nutrient solution; The every hole of ConA group adds canavaline, and final concentration is 5 μ g/mL; The every hole of LPS group adds LPS, final concentration 10 μ g/mL; Each group complements to 200 μ L with RPMI1640 nutrient solution, and 6 multiple holes, put 5%C0
2, 37 DEG C of incubators cultivate 48 hours.Stop the every hole of 4h before cultivating and add the MTT20 μ L of 5mg/mL, continue after mixing to cultivate.After cultivation terminates, inhale gently and abandon supernatant liquor, every hole adds the DMSO of 150 μ L, and vibration 10min makes purple crystal dissolve completely, and microplate reader measures the absorbancy at 570nm wavelength place.Proliferation rate calculates:
Proliferation rate (%)=(by prospect hole OD value-control wells OD value)/control wells OD value × 100% (formula 6).
2, experimental result
Different concns medicinal fungal substance polysaccharide on the impact of spleen lymphocyte proliferation in table 1
Table-1 different concns medicinal fungal substance polysaccharide to lymphocytic proliferation function (OD value,
)
Note: compare with blank group, * P<0.01.
Mtt assay detects medicinal fungal substance polysaccharide affects result as table 1 to Proliferation of lymphocytes, compare with blank group, the Proliferation of lymphocytes of ConA and LPS process is extremely remarkable, 4 concentration of JZPC, JZPJ, JZP3 test all can promote lymphopoiesis (p<0.01), JZP1 and JZP2 increases with concentration, spleen lymphocyte proliferation rate is reduced gradually, when 1000 μ g/mL concentration, play restraining effect (p<0.01) to lymphopoiesis, inhibiting rate is respectively 15.66%, 13.25%.
Experimental result shows that the medicinal fungal substance that the present invention obtains has good immunoregulation effect.
Five, the acute toxicity of medicinal fungal substance:
1, experiment material:
Medicinal fungal substance Crude polysaccharides: by one, the medicinal fungal substance Crude polysaccharides that obtains of (one) (extracting method of medicinal fungal substance reactive site).
Laboratory animal: kunming mice, male and female half and half, body weight 18-22g, University Of Nanchang's medical board Experimental Animal Center provides.Buy rear normal diet and drinking-water to conform 3-5d, feeding environment room temperature 20-25 DEG C, and observe the defective animal of removing.
2, experimental technique:
Carry out preliminary experiment according to Horn method, kunming mice is divided into 3 groups at random, often organizes 2, test adopts given low to be respectively 0.1,1.0,10.0g/kgBW.Adopt corresponding dosage all only to carry out a gavage to each group of mouse, the symptom that after gavage, in 24h, close observation mouse occurs and death toll, estimate minimum 100% lethal quantity (Dm) roughly and maximum 0 lethal quantity (Dn).
If preliminary experiment can obtain Dm and Dn, then carry out LD by karber's method
50mensuration.Dm and Dn is scaled conventional logarithm, then by the highest difference with the logarithm of lowest dose level, is equidistantly divided into 5 logarithms, if 5 dosage groups, often organize 10 animals, male and female half and half, weigh successively, record body weight also dye number.Before experiment, mouse fasting can't help water 12h, each 0.2mLl/10g of mouse stomach capacity.Continuous Observation 1h after administration, then in 2,4,6,8,12,24h observes 1 time respectively, the later morning and afternoon every day same time observes 1 time, and Continuous Observation 14d also carries out observed and recorded, and weighs in 7d and 14d same time, and records body weight.Observed and recorded index comprises active situation, the build outward appearance of mouse, the mental status, search for food, by hair, external reaction, abnormal secretion thing, stool and urine color and shape, the toxic reactions such as breathing and death condition, if there is death, just dissect immediately, the main organs quality color and luster changing conditions such as the visual inspection heart, liver, spleen, lung, kidney, stomach, intestine and small intestine, brain.Find that there is the organ Ying Zuo histopathologic examination of pathology.By following formulae discovery LD50 and standard error, 95% fiducial limit.
LogLD50=XK-d (∑ p-0.5) (formula 7),
The standard error (S) of LD50:
95% fiducial limit (X) of LD50:
X=log
-1(logLD50 ± 1.96 × S
logLD50) (formula 9)
If preliminary experiment cannot measure Dm, then carry out mtd test.
Animal divides into groups: each 10 of male female mice, weighs successively, record body weight, and dye number.
Before on-test, mouse fasting can't help water 16 hours, gives mouse stomach according to tested material maxima solubility and maximum gavage volume (20mL/kgBW).In all mouse 24h, gavage number of times can not more than 3 times, interval time 4-6h.After administration, 2h provides food, and Continuous Observation 1h, in 2,4,6,8,12,24h observes 1 time respectively, at least each 1 time of later morning and afternoon every day same time, Continuous Observation 14d carries out observed and recorded, and weighs in 7d and 14d same time, and makes a record.Observation index comprises activity, the build outward appearance of mouse, the mental status, search for food, by hair, external reaction, abnormal secretion thing, stool and urine color and shape, the toxic reactions such as breathing and death condition, if there is death, dissect immediately, the appearance change of the main organs such as the visual inspection heart, liver, spleen, lung, kidney, stomach, intestine and small intestine, brain.Continuous Observation 14d also records animal poisoning manifestations and feature, toxic reaction appearance, toxic reaction extinction time and death time of animal, after observation period terminates, the sacrifice often organizing survival is dissected, check the color and luster of the organs such as mouse heart, lung, liver, thymus gland, kidney, cerebral tissue, stomach and enteron aisle, position and quality etc., the each internal organs of visual inspection have without exception, if any macroscopic pathology, carry out histopathological examination.
3, experimental result:
Phaeopoms obliquus mycoplasma polysaccharide gives to observe after mouse through gavage, mouse behavior act and control group indifference.Within after administration the 1st day, observe, mouse behavioral activity and control group are not significantly distinguished, and rapid to irritant reaction, eyes, nostril have no secretory product, but stool consistency is comparatively large, and color is dark, may produce containing polyose just.Within after administration the 2nd day, observe, other observation items such as the behavioral activity of mouse and control group are as good as, and mouse diet is normal.Administration is observed on the 3rd day, and mouse behavioral activity and control mice are as good as, and eyes nose also has no secretory product, observe its stool colour all identical with control group with viscosity.Within the 4th day after administration, until the 14th day, namely at the end of experimental observation, every observation index of mouse is all as good as with Normal group, and none example of mouse is dead.At the end of experimental observation, de-white the putting to death of mouse carries out gross anatomy, contrasts contents such as examining each internal organs color quality, all no abnormal situation with control group.Accumulative mouse stomach dosage, experiment mice is greater than 21g/kg to medicinal fungal substance polysaccharide maximum tolerated dose.Its body weight change and death condition are in table 2.
Table 2-acute toxicity test Mouse Weight change and death condition (
)
| Group | Dosage (g/kg) | Number of animals (only) | Initial weight (g) | Heavy (g) on the 7th | Heavy (g) on the 14th | Animal dead number (only) |
| Control group | - | 10 | 20.01±1.47 | 26.24±2.96 | 28.69±4.07 | 0 |
| Test group | 21 | 20 | 20.50±2.49 | 26.50±4.00 | 29.05±4.88 | 0 |
Experimental result shows the minimum or nontoxicity of medicinal fungal substance toxicity that the present invention obtains.
Phaeopoms obliquus is seeded in Chinese medicinal materials or Chinese medicine slag property of medicine matrix and carries out two-way solid fermentation by the present invention, the medicinal fungal substance nontoxicity of producing, there is the effects such as good antioxygenation, blood sugar reducing function and immunomodulatory, efficiently solve the shortage of resources problem of rare medicinal fungi-Phaeopoms obliquus, and open new way for effective recycling of Chinese medicine slag.
Above the better embodiment of this patent is explained in detail, but this patent is not limited to above-mentioned embodiment, in the ken that one skilled in the relevant art possesses, various change can also be made under the prerequisite not departing from this patent aim.
Claims (7)
1. the Chinese medicinal materials of Phaeopoms obliquus or a two-way solid fermentation method for Chinese medicine slag, it is characterized in that, concrete steps comprise: the preparation of fermented substrate, namely to the process of property of medicine matrix; The process of fermented bacterium Phaeopoms obliquus; Phaeopoms obliquus is inoculated in property of medicine matrix; Carry out two-way solid fermentation; Collect medicinal fungal substance.
2. the Chinese medicinal materials of Phaeopoms obliquus according to claim 1 or the two-way solid fermentation method of Chinese medicine slag, it is characterized in that, described property of medicine matrix, when processing, is pulverized property of medicine matrix, adding water makes its water content be 50-65%, and bottling is 121 DEG C of autoclaving 1h also.
3. the Chinese medicinal materials of Phaeopoms obliquus according to claim 1 or the two-way solid fermentation method of Chinese medicine slag, it is characterized in that, the treatment process of described fermented bacterium is after being activated by Phaeopoms obliquus, and under 28 DEG C of conditions amplification cultivation 14-16d, the method for amplification cultivation adopts liquid nutrient medium shake-flask culture.
4. the Chinese medicinal materials of Phaeopoms obliquus according to claim 1 or the two-way solid fermentation method of Chinese medicine slag, is characterized in that, the fermentation condition of described two-way solid fermentation is temperature range 26 DEG C-28 DEG C, and fermentation number of days is 40-60d.
5. the Chinese medicinal materials of Phaeopoms obliquus according to claim 1 or the two-way solid fermentation method of Chinese medicine slag, is characterized in that, described property of medicine matrix refers to Chinese medicinal materials or Chinese medicine slag.
6. the Chinese medicinal materials of Phaeopoms obliquus according to claim 1 or the two-way solid fermentation method of Chinese medicine slag, it is characterized in that, described Chinese medicinal materials or Chinese medicine slag are the root of kudzu vine, GEGEN QINLIAN TANG prescription medicinal material, LIUWEI DIHUANG WAN prescription medicinal material, the root of kudzu vine dregs of a decoction, the GEGEN QINLIAN TANG dregs of a decoction or the LIUWEI DIHUANG WAN dregs of a decoction.
7. the Chinese medicinal materials of Phaeopoms obliquus according to claim 1 or the two-way solid fermentation method of Chinese medicine slag, is characterized in that, described medicinal fungal substance is the mycoplasma that Phaeopoms obliquus produces through solid fermentation in the property of medicine matrix containing Chinese medicinal materials or Chinese medicine slag.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108450656A (en) * | 2016-12-13 | 2018-08-28 | 湖南民康生物技术研究所 | A kind of method that Chinese herb residues orientation fermentation prepares functional feedstuff additive |
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| CN108795770A (en) * | 2017-04-27 | 2018-11-13 | 天明制药股份有限公司 | To improve the process for solid culture of polysaccharides content in inonotus obliquus |
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| CN113768042A (en) * | 2021-07-30 | 2021-12-10 | 吉林省农业科学院 | Traditional Chinese medicine residue fermentation compound and preparation method and application thereof |
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