CN105998222A - Probiotic fermented unprocessed common monkshood daughter root composition and preparation method and application thereof - Google Patents
Probiotic fermented unprocessed common monkshood daughter root composition and preparation method and application thereof Download PDFInfo
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- CN105998222A CN105998222A CN201610314984.XA CN201610314984A CN105998222A CN 105998222 A CN105998222 A CN 105998222A CN 201610314984 A CN201610314984 A CN 201610314984A CN 105998222 A CN105998222 A CN 105998222A
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- radix aconiti
- aconiti lateralis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
- A61K36/714—Aconitum (monkshood)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
Abstract
The invention discloses a probiotic fermented unprocessed common monkshood daughter root composition and a preparation method thereof. Unprocessed common monkshood daughter roots are smashed, water is added for soaking, pH is adjusted, enzymolysis and sterilization are carried out, the sterilized solution is inoculated with a precultured culture, fermentation is carried out, and then the probiotic fermented unprocessed common monkshood daughter root composition is obtained. The invention further discloses application of the probiotic fermented common monkshood daughter root composition to preparation of fermented type common monkshood daughter root Chinese medicinal granules and to extraction separation of polysaccharide and an alkaloid composition in fermented common monkshood daughter roots. The prepared fermented type common monkshood daughter root Chinese medicinal granules can remarkably reduce the content of toxic component diester-alkaloids, increase the content of medicinal component single ester type alkaloids, promote extraction of medicinal components to the greatest extent and increase the utilization ratio of drug, and is stable in component and convenient to use. Experiment results show that the content of high-toxicity diester-alkaloids in the fermented type common monkshood daughter root Chinese medicinal granules is very low, almost no diester-alkaloids is detected, and the content of the low-toxicity single ester type alkaloids is remarkably increased. Acute toxicity tests show that the fermented type common monkshood daughter root Chinese medicinal granules do not show toxicity.
Description
Technical field
The present invention relates to herb fermenting compositions and processing technique of Chinese herbs, particularly relate to Aconitum carmichaeli through fermentable
Reach attenuation and deposit effect purpose, prepare effective extract, particularly prepare fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule, belong to medical science
Field.
Background technology
Aconitum carmichaeli derives from ranunculaceae plant Aconitum carmichjaelii Debx. (Aconitum carmichaeli Debx.), its side root (daughter root)
It is Radix Aconiti Lateralis Preparata, acrid in the mouth, sweet, extremely hot in nature, poisonous, it is described as that " good general of troubled times, the first product of recuperating depleted YANG and rescuing the patient from collapse mend the gate of vitality kidney-yang first
Key medicine ", there is recuperating depleted YANG, by cold, the effect of wind-damp dispelling.Control loss of YANG due to profuse perspiration, four cold of the limbs are inverse, kidney yang is weak chills and pain of the waist and kness, cold physique are liked
Cold, lassitude and the disease such as wind-cold-dampness ache, tinea pedis.Later age is many has " malicious " to fear it because of it.Someone says that " Radix Aconiti Lateralis Preparata is most useful, also
It is most difficult to use ".Radix Aconiti Lateralis Preparata is the key medicine of clinical recuperating depleted YANG and rescuing the patient from collapse, has wide prospect in recent years, have in terms for the treatment of severe crisis
Analgesia, antiinflammatory, heart tonifying and the pharmacological action such as anticancer.
Radix Aconiti Lateralis Preparata is mainly composed of alkaloid, including diester-type alkaloids (aconitine, hypaconitine, mesaconitine etc.), list
Ester alkaloid (benzoyl aconine, benzoyl hypo-aconine, Benzoylmesaconine etc.), ammonia alcohol type alkaloid
(aconine, secondary aconine, new aconine etc.) and other Alkaloids are (such as neojiangyouaconitine, the nor-Radix Linderae of racemization
Alkali etc.), the Difference In Toxicity of these several alkaloids is very big, and wherein diester-type alkaloids toxicity is very big, aconitine, mesaconitine, secondary
The mice LD50 of aconitine is respectively 1.8 mg/kg, 1.9 mg/kg and 5.8 mg/kg.The di esters biology aqueous alkalis such as aconitine
Solution can generate the little monoesters Alkaloid of toxicity, and (benzoyl aconine, Benzoylmesaconine and benzoyl time Aconitum carmichjaelii Debx. is former
Alkali), its toxicity is only the 1/1000 of diester-type alkaloids.If monoester alkaloid further hydrolysis, become toxicity less or
Nontoxic alcamines alkaloid (aconine, new aconine, secondary aconine), its toxicity is only 1/2000 left side of aconitine
Right.Therefore, the toxicity index of Radix Aconiti Lateralis Preparata should be with diester-type alkaloids content as index.As concoct improper or dosage is excessive and decoct
Time is inadequate, all can cause toxic reaction.The toxicity of aconitine mainly acts on nervous system, digestive system, cardiovascular system
And respiratory system.For nervous system, aconitine can press down after various teleneurons and cental system are produced a first excitement
The process of system, and generation effect direct to cardiac muscle, in turn result in respiratory center paralysis, central blood pressure drops, even cause and stop up
Breath and dead.For cardiovascular system, aconitine is the most serious to the infringement of heart, and clinical manifestation is under cardiopalmus, uncomfortable in chest, blood pressure
Fall, arrhythmia, decreased heart rate or overrun, even there is cardiac arrest, acute heart source property cerebral ischemia syndrome etc. in severe patient.Right
In digestive system, aconitine excitation vagal to gastrointestinal can cause sialorrhea, dysphagia, vomiting nauseating, frequent, stomach
Portion is scorching hot, suffer from abdominal pain, suffer from diarrhoea, bowel sound hyperaction, and severe patient may result in vomiting coffee tinctorial pattern liquid and solves blood sample feces, after pain in the abdomen
Weight, exactly likes the performances such as dysentery.For respiratory system, the toxicity of aconitine may result in dyspnea, respiratory paralysis and even suffers a shock extremely
Die.
Owing to Radix Aconiti Lateralis toxicity is big, clinical practice Radix Aconiti Lateralis Preparata, the most all take much count of the Processing methods of Radix Aconiti Lateralis Preparata.
Depositing the purpose of effect for making Radix Aconiti Lateralis Preparata reach attenuation, Han dynasty has more than 70 processing and concocting methods planting Radix Aconiti Lateralis Preparata so far, such as,
Parch to black method, processed with honey method, cannon method, paper wrap up in stewing method, processed with Rhizoma Zingiberis Recens method, salt preparation method, glycyrrhiza decoction stir-fry method etc..Version " Chinese Pharmacopoeia " in 2015
Radix Aconiti Lateralis Preparata still uses gallbladder bar concocting method, by Radix Aconiti Lateralis edible gallbladder bar water soaking, well-done, water drift, section, again water drift, dye
Color, steam, be dried.Processing through repetitious water soaking, rinsing etc., processing procedure is loaded down with trivial details, and method is complicated, poor controllability,
Loss of effective components is serious.The research reports such as Tang little Long: in Radix Aconiti Lateralis Preparata concocting process, the minimizing of alkaloid essentially consists in gallbladder bar blister
(loss 31.6%), drift sheet (loss 33.6%), hydrolysis (loss 16.9%), alkaloid total amount decreases 81.3%, again because
The differences such as soak time, temperature, rinsing degree, different batches Radix Aconiti Lateralis Preparata decoction pieces component content difference may be up to 10 times.
There is Radix Aconiti Lateralis Preparata effective active composition and flow in a large number in the technological process with attenuation as target of the Aconitum carmichaeli traditional diamond-making technique
The drawback lost, reform traditional technique, science holds the hydrolysis of the dibasic acid esters alkaloids such as aconitine, it is to avoid waste of running off, effectively
Utilize natural resources of Chinese medicinal materials, be trend of the times.
Summary of the invention
For overcoming the deficiencies in the prior art, the invention provides compositions and the preparation thereof of a kind of probiotics fermention Radix Aconiti Lateralis
Method, is by preferred several strains, adds in pretreated Radix Aconiti Lateralis Preparata, utilizes growth of microorganism activity to produce active substance,
Realize the structural modification to Radix Aconiti Lateralis Preparata composition, to reach improve drug effect and drop hypotoxic purpose.
Microorganism species have oxidation, methylate, the multiple biotransformation capacity such as hydroxylating, deesterify, reducing.Pass through
Microbial Biotransformation concocts Chinese medicine, can strengthen larger than general process of preparing Chinese medicine means physically or chemically or adjust medicine
Property, thus improve Chinese Herbs, reduce toxic and side effects, expand Chinese medicine indication.
Antibacterial is fast with growth and breeding speed, with strong points, have stronger comprehensive conversion capability, in particular with human body intestinal canal
In profitable strain Chinese medicine is converted.
The compositions of probiotics fermention Radix Aconiti Lateralis of the present invention, is by after Radix Aconiti Lateralis pulverizing, immersion, enzymolysis, by benefit
The fermentation of raw bacterium prepares, and specifically comprises the following steps that
1, pulverize: take dry Radix Aconiti Lateralis and pulverize (10-100 mesh)；
2, soak: 5-20 times of water soaking of addition medical material amount 0.5-2.0 hour, obtain Radix Aconiti Lateralis water logging suspension；
3, adjust pH: Radix Aconiti Lateralis water logging suspension step 2 obtained, adjust pH6.0-7.5 with sodium hydroxide or calcium hydroxide；
4, enzymolysis: the Radix Aconiti Lateralis water logging suspension of modulated pH value step 3 obtained, is heated to 70-95 DEG C, doing by Radix Aconiti Lateralis
Heavily add the Thermostable α-Amylase of percentage by weight 0.1-1.0%, and keep 20-60 minute, sampling detection, make iodine test solution not
Become blue again, obtain the Radix Aconiti Lateralis Preparata mixed liquor of enzymolysis；
5, sterilizing: the Radix Aconiti Lateralis Preparata mixed liquor of the enzymolysis that step 4 is obtained, 105-121 DEG C of autoclave sterilization 20-120min；
6, inoculating: sterilizing Radix Aconiti Lateralis Preparata mixed liquor step 5 obtained, temperature is down to 30-37 DEG C, inoculating two kinds or two or more pre-training
The strain supported；
7, fermentation: vaccinated Radix Aconiti Lateralis Preparata mixed liquor step 6 obtained, under 25-37 DEG C of environment, fermentation condition is cultivated routinely
2-7 days, terminate fermentation, i.e. obtain the compositions of probiotics fermention Radix Aconiti Lateralis Preparata.
Described strain is selected from bacillus cereus, lactobacillus, bacillus bifidus, yeast, and inoculum concentration is respectively percent by volume
0.5%-2.0%。
Described bacillus cereus is selected from bacillus subtilis, Bacillus licheniformis；Described lactobacillus is selected from addicted to yogurt bar
Bacterium, Deshi Lactobacillus, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus paracasei, lactobacillus casei and lactobacillus rhamnosus；Institute
The bacillus bifidus stated is selected from bifidobacterium bifidum, bifidobacterium breve, bifidobacterium longum, animal bifidobacteria, bifidobacterium adolescentis
And bifidobacteria infantis；Described yeast is selected from Marx's Crewe yeast, saccharomyces cerevisiae.
Strain derives from China typical culture collection center (CCTCC) (China, Wuhan) or China General Microbiological bacterium
Kind of preservation administrative center (CGMCC) (China, Beijing) or Chinese industrial Microbiological Culture Collection administrative center (CICC) (in
State, Beijing).
The offer probiotics fermention Radix Aconiti Lateralis Preparata of the present invention application in field of medicaments is provided, according to
Purposes is different, relates to preparing fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule；Extract the polysaccharide component in separation and fermentation Radix Aconiti Lateralis Preparata and/or extraction
Alkaloid component in separation and fermentation Radix Aconiti Lateralis Preparata.
Described prepares fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule, is by the combination of the probiotics fermention Radix Aconiti Lateralis Preparata fermented
Thing carries out extracting, concentrates, is dried, detects, subpackage, obtains fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule.
Specifically include following steps:
A, extract, concentrate: the compositions of probiotics fermention Radix Aconiti Lateralis Preparata that will obtain, centrifugal or filter, separation and fermentation liquid, Chinese medicine residue
Add water (5-20 times), heating extraction 1-2 time, merge fermentation liquid and extracting solution, be evaporated to solid content more than 10%；
B, be dried: the concentrated solution that step a is obtained, be spray-dried；
C, detection, subpackage: dried powder step b obtained, detect composition, and specification subpackage on request.
Alkaloid in the described polysaccharide component extracted in separation and fermentation Radix Aconiti Lateralis Preparata and/or extraction separation and fermentation Radix Aconiti Lateralis Preparata becomes
Point, it is that the compositions of the probiotics fermention Radix Aconiti Lateralis fermented is carried out extracting and developing, purification.
Specifically include following steps:
1) extract: by centrifugal for the compositions of the probiotics fermention Radix Aconiti Lateralis obtained or filtration, separation and fermentation liquid, Chinese medicine residue adds again
Water (5-20 times), heating extraction 1-2 time, merge fermentation liquid and extracting solution, be evaporated to relative density 1.08-1.18；
2) separation and fermentation polysaccharide from aconite root composition: take the concentrated solution that step 1) obtains and add ethanol and make alcohol content be 75-90%, place
After 24 hours, separating supernatant and precipitation, take precipitation and be dissolved in water, filter, filtrate adds ethanol again makes alcohol content be 80-90%,
Placing 24 hours, again separate supernatant and precipitation, take precipitation washing with acetone 3 times, volatilize solvent, 50 DEG C of drying under reduced pressure must be sent out
Ferment Radix Aconiti Lateralis Preparata polysaccharide.
3) total alkaloids composition in separation and fermentation Radix Aconiti Lateralis Preparata: take the concentrated solution that step 1) obtains and add ethanol and make alcohol content be 80-
90%, after placing 24 hours, separate supernatant and precipitation, collect alcoholic supernatant, decompression recycling ethanol, and be concentrated into the closeest
Degree 1.01-1.03, uses D101 absorption with macroporous adsorbent resin, with 5 times of volume distilled water remove impurity, then is the second of 15% by volume fraction
Alcoholic solution 1.5～2 BV 5 times of volumes of h-1 flow velocity eluting, concentrate eluant, vacuum drying, obtain fermentation Radix Aconiti Lateralis Preparata total alkaloids.
Beneficial effect: the present invention is to be combined with modern biotechnology, biotechnology and enzyme engineering technology by Chinese medicine
A kind of new Chinese medicine processing method, be that Chinese medicine processing is organically combined with preparing Chinese medicinal granule.System of the present invention
Standby fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule, can significantly reduce the content of toxic component diester-type alkaloids, improves active ingredient
The content of monoester alkaloid, meanwhile, can promote the extraction of active ingredient, improve utilization ratio of drug, composition is steady to greatest extent
Fixed, easy to use.The fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule attenuation prepared for the detection present invention deposits effect, has carried out composition ratio respectively
Relatively experiment and acute toxicity comparative test.Comparison of ingredients experimental result shows: the fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule of the present invention
Middle high toxicity diester-type alkaloids content is the lowest, is nearly no detectable, and hypotoxicity monoester alkaloid content significantly improves.Anxious
Property toxicity test result shows: fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule does not show toxicity.
Accompanying drawing explanation
Fig. 1 is the thin of fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule and the Radix Aconiti Lateralis of the present invention and Radix Aconiti Lateralis Preparata decoction pieces (Radix Aconiti Lateralis Preparata) comparison of ingredients
Layer chromatography figure.
In figure: S, from top to bottom be respectively hypaconitine, aconitine, mesaconitine, benzoyl hypo-aconine, benzene first
Acyl aconine, Benzoylmesaconine；1, fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule A test sample；2, fermented type Radix Aconiti Lateralis Preparata Chinese medicine
Granule B test sample；3, fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule C test sample；0, Radix Aconiti Lateralis；A, Radix Aconiti Lateralis Preparata decoction pieces (Radix Aconiti Lateralis Preparata).
Detailed description of the invention
Below by way of the description of detailed description of the invention, the invention will be further described, but this is not the limit to the present invention
System, those skilled in the art are according to the basic thought of the present invention, and various modifications may be made or improves, but without departing from this
The basic thought of invention, the most within the scope of the present invention.
Embodiment 1: the preparation of fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule A
Taking Chinese medicine Radix Aconiti Lateralis coarse powder (20 mesh) 10kg, the 100L that adds water to soak 1.0 hours, adjusting pH with sodium hydroxide is 7.0, adds
Heat, to 90 DEG C, adds Thermostable α-Amylase 50g, and keeps 30 minutes, sampling detection, and iodine test solution no longer becomes blue.At 115 DEG C
Sterilizing 50 minutes, when temperature is down to 37 DEG C, inoculates the bifidobacterium breve (CGMCC of pre-incubated percent by volume 1.0% simultaneously
1.2213) bacterium solution and the bacillus acidophilus (CGMCC 1.1854) of percent by volume each 0.5%, Lactobacillus plantarum (CGMCC
1.19) and the beer yeast (CGMCC 2.1416) of percent by volume 1.0%, 37 DEG C ferment 24 hours, and then temperature is down to
Cultivating 48 hours for 30 DEG C, sampling detection, diester-type alkaloids speckle disappears, and terminates fermentation, obtains the group of probiotics fermention Radix Aconiti Lateralis
Compound.Centrifugation fermentation liquid, Chinese medicine residue adds water 150L, and heated and boiled is extracted 0.5 hour, centrifugal, merge fermentation liquid with
Extracting solution, is evaporated to solid content more than 10%, is spray-dried, obtains dry granule 3.62kg, detection, and subpackage to obtain final product.
Embodiment 2: the preparation of fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule B
Taking Chinese medicine Radix Aconiti Lateralis powder (60 mesh) 10kg, the 50L that adds water to soak 2.0 hours, adjusting pH with calcium hydroxide is 7.0, is heated to
70 DEG C, adding Thermostable α-Amylase 100g, and keep 20 minutes, sampling detection, iodine test solution no longer becomes blue.Go out at 121 DEG C
Bacterium 40 minutes, when temperature is down to 30 DEG C, inoculates the bacillus subtilis (CGMCC of pre-incubated percent by volume 2.0% simultaneously
1.2213) bacterium solution and Marx's Crewe yeast (CGMCC 2.3959) of percent by volume 2.0%, cultivate 7 days for 30 DEG C, sampling
Detection, diester-type alkaloids speckle disappears, and terminates fermentation, obtains the compositions of probiotics fermention Radix Aconiti Lateralis.Fermented product adds water 100L,
Heated and boiled is extracted 0.5 hour, and centrifugal, Chinese medicine residue adds water 100L, heating extraction 0.5 hour, united extraction liquid, reduces pressure dense
It is reduced to solid content more than 10%, is spray-dried, obtains dry granule 3.06kg, detection, subpackage, to obtain final product.
Embodiment 3: the preparation of fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule C
Taking Chinese medicine Radix Aconiti Lateralis powder (100 mesh) 10kg, the 200L that adds water to soak 2.0 hours, adjusting pH with sodium hydroxide is 7.0, heating
To 80 DEG C, adding Thermostable α-Amylase 10g, and keep 60 minutes, sampling detection, iodine test solution no longer becomes blue.Go out at 110 DEG C
Bacterium 90 minutes, when temperature is down to 37 DEG C, inoculates the bifidobacterium bifidum (CGMCC of pre-incubated percent by volume 1.0% simultaneously
1.5090) bacterium solution and the lactobacillus casei (CGMCC 1.2901) of percent by volume each 0.5%, Lactobacillus delbrueckii subsp. lactis
(CGMCC 1.2625), 37 DEG C ferment 48 hours, sampling detection, and diester-type alkaloids speckle disappears, and terminates fermentation, obtains probiotic bacteria
The compositions of fermentation Radix Aconiti Lateralis.Centrifugation fermentation liquid, Chinese medicine residue adds water 50L, and heated and boiled is extracted 0.5 hour, centrifugal,
Merge fermentation liquid and extracting solution, be evaporated to solid content more than 10%, be spray-dried, obtain dry granule 3.38kg, inspection
Survey, subpackage, to obtain final product.
Embodiment 4: extract the polysaccharide component in separation and fermentation Radix Aconiti Lateralis Preparata and alkaloid component
Taking Chinese medicine Radix Aconiti Lateralis coarse powder (80 mesh) 1kg, the 10L that adds water to soak 1.0 hours, adjusting pH with sodium hydroxide is 7.0, heating
To 90 DEG C, adding Thermostable α-Amylase 5g, and keep 30 minutes, sampling detection, iodine test solution no longer becomes blue.Go out at 115 DEG C
Bacterium 50 minutes, when temperature is down to 37 DEG C, inoculates the bifidobacterium breve (CGMCC of pre-incubated percent by volume 1.0% simultaneously
1.2213) bacterium solution and the bacillus acidophilus (CGMCC 1.1854) of percent by volume each 0.5%, Lactobacillus plantarum (CGMCC
1.19), 37 DEG C ferment 24 hours, and then temperature is down to 30 DEG C of cultivations 24 hours, terminates fermentation, obtains probiotics fermention Radix Aconiti Lateralis
Compositions.Centrifugation fermentation liquid, Chinese medicine residue adds water 15L, and heated and boiled is extracted 0.5 hour, centrifugal, merge fermentation liquid with
Extracting solution, is evaporated to relative density 1.10；
It is slowly added into ethanol and makes containing amount of alcohol to be 85%, after placing 24 hours, separate supernatant and precipitation, collect supernatant standby
With；Take precipitation add water 500ml dissolve, filter, filtrate adds ethanol makes alcohol content be 85%, places 24 hours, again separates supernatant
Liquid and precipitation, collect supernatant standby；Take precipitation washing with acetone 3 times, each 150ml, volatilize solvent, 50 DEG C of drying under reduced pressure,
Must be fermented Radix Aconiti Lateralis Preparata polysaccharide 62.6g.
Another 85% alcoholic supernatant merging collection, decompression recycling ethanol, and it is concentrated into relative density 1.02, big with D101
Macroporous adsorbent resin adsorbs, with the distilled water remove impurity of 5 times of volumes (BV), then with the ethanol solution 1.5～2 that volume fraction is 15%
BV h-1 flow velocity eluting 5BV, concentrate eluant, vacuum drying, must ferment Radix Aconiti Lateralis Preparata total alkaloids 10.8g.
Experimental example 1: fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule and Radix Aconiti Lateralis, Radix Aconiti Lateralis Preparata decoction pieces (Radix Aconiti Lateralis Preparata) comparison of ingredients
Taking Radix Aconiti Lateralis powder 5g and add ammonia solution 5ml moistening, the ultrasonic 30min of the 50ml that adds methylene chloride, filter, filtrate volatilizes, residue
Add dehydrated alcohol 1.0ml to dissolve, as Radix Aconiti Lateralis control sample；Separately take Radix Aconiti Lateralis Preparata powder 5g to prepare with method, as Radix Aconiti Lateralis Preparata decoction pieces
(Radix Aconiti Lateralis Preparata) control sample；Take fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule A, B, C more appropriate (being equivalent to Radix Aconiti Lateralis Preparata 5g), add ammonia solution
2.0ml moistening, the ultrasonic 30min of the 50ml that adds methylene chloride, to filter, filtrate volatilizes, and residue adds dehydrated alcohol 1.0ml and dissolves, as
Test sample 1,2,3；Take aconitine 3mg, hypaconitine 3mg, each 1.8mg of mesaconitine, benzoyl aconine 3.2mg, benzene again
Formyl new aconine 2.6mg, Benzoylmesaconine 1.9mg add dehydrated alcohol 2ml respectively and dissolve.Thin layer chromatography: according to thin
Layer chromatography (2015 editions general rules 0502 of Chinese Pharmacopoeia) is tested, draw reference substance solution, need testing solution 1,2,3, Radix Aconiti Lateralis pair
According to each 5ul of sample solution, put respectively on same silica gel g thin-layer plate, with n-hexane-ethyl acetate-methanol (6.4:3.6:1) be
Developing solvent, puts in the expansion cylinder of ammonia saturated with vapor 20min, launches, and takes out, dries, and spray is with dilute bismuth potassium iodide test solution.
Interpretation of result: fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule with diester-type alkaloids (hypaconitine, aconitine, newly crow
Head alkali) be hardly visible corresponding speckle on the corresponding position of reference substance, and with monoester alkaloid (benzoyl time Aconitum carmichjaelii Debx.
Alkali, benzoyl mesaconitine) on the corresponding position of reference substance, corresponding speckle is obvious, and fermented type Radix Aconiti Lateralis Preparata Chinese medicine formula is described
In Li, diester-type alkaloids content seldom, is nearly no detectable, and monoester alkaloid substantially increases；Radix Aconiti Lateralis control sample
Product diester-type alkaloids speckle clearly, illustrates that Radix Aconiti Lateralis diester-type alkaloids content is higher；In Radix Aconiti Lateralis Preparata, diester-type is biological
Alkaline spot point is more weak than Radix Aconiti Lateralis, but remains to detect, illustrates to still suffer from a certain amount of diester-type alkaloids in Radix Aconiti Lateralis Preparata.See accompanying drawing 1.
With thin-layer method, fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule is carried out comparison of ingredients, the most intuitively with Radix Aconiti Lateralis and Radix Aconiti Lateralis Preparata
Reflecting diester-type alkaloids content in Radix Aconiti Lateralis high, monoester alkaloid content is low, and Radix Aconiti Lateralis Preparata diester-type alkaloids content drops
Low, monoester alkaloid increases inconspicuous, illustrates that Radix Aconiti Lateralis Preparata component damages in concocting process is serious；And fermented type Radix Aconiti Lateralis Preparata Chinese medicine
Granule diester-type alkaloids content is the lowest, and monoester alkaloid content substantially increases, and illustrates severe toxicity in preparation process
Property diester-type alkaloids C position on acetyl group hydrolysis, lose a part acetic acid, obtain corresponding benzoyl monoester alkaloid,
Thus reduce toxicity.Additionally the acetyl group on aconite alkaloids C position is replaced by fatty acyl group under conditions of comparing mitigation, raw
Become the monoesters Alkaloid that toxicity is less.So fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule reduces the toxicity of Radix Aconiti Lateralis Preparata, save monoester type
The pharmacological effect of alkaloid, thus reach to be attenuated the purpose depositing effect, make clinical application safer, convenient.
Experimental example 2: fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule and Radix Aconiti Lateralis acute toxicity testing
Take mice 48, male and female half and half, be randomly divided into 6 groups according to sex and body weight: normal group, Radix Aconiti Lateralis group (Radix Aconiti Lateralis Preparata 1.0 g/
Ml), Radix Aconiti Lateralis Preparata group (Radix Aconiti Lateralis Preparata 1.0 g/ml), fermentation Radix Aconiti Lateralis Preparata granule A group (being equivalent to Radix Aconiti Lateralis Preparata 1.0 g/ml), fermentation Radix Aconiti Lateralis Preparata granule
B group (being equivalent to Radix Aconiti Lateralis Preparata 1.0 g/ml), fermentation Radix Aconiti Lateralis Preparata granule C group (being equivalent to Radix Aconiti Lateralis Preparata 1.0 g/ml).
Mice is removed food and is not removed after water 12 h by 40 mL/kg volume gastric infusions, every day 2 times, gives 3 days continuously.Connect after medicine
Continuous observation 7 d: after medicine in 30min, Continuous Observation；30min-2h after medicine, every 15 min observe once；2 h 4 h after medicine, often
30 min observe once；4 h 8 h after medicine, every 1 h observe once；8 h 24 h after medicine, every 4 h observe once；D2 after medicine
Rising, every day observes once, the record hair color of mice, spirit, dynamic spend, take food, ordinary circumstance, poisoning symptom and the death feelings such as drinking-water
Condition.Dead mice is dissected at once, observes the general pathology change of the important organs such as the heart, liver, spleen, lung, kidney.
Result
Hair color in 7 days after little raticide, two just etc. normal, dead 3 of Radix Aconiti Lateralis group, dead 0 of Radix Aconiti Lateralis Preparata group, fermentation Radix Aconiti Lateralis Preparata group are dead
Die 0.After mice gives Radix Aconiti Lateralis and Radix Aconiti Lateralis Preparata, Mouse Weight increasess slowly, and compares with normal group, and Radix Aconiti Lateralis group is in the most aobvious
Writing sex differernce, Radix Aconiti Lateralis Preparata group is significant difference, and each fermentation group there are no significant difference.And gavage Radix Aconiti Lateralis, Radix Aconiti Lateralis Preparata
Feeling sick in various degree, the prostrate symptom toxic reaction such as motionless it is presented with after decocting liquid, and mouse stomach fermented type Radix Aconiti Lateralis Preparata Chinese medicine
There is not corresponding toxicities symptom in granule, to mice postmortem no abnormality seen.
1. the impact on Mouse Weight
Mouse Weight change is shown in Table 1.
From table 1 result, after giving Radix Aconiti Lateralis and Radix Aconiti Lateralis Preparata, Mouse Weight increasess slowly, hence it is evident that less than normal group, with
Normal group compares, and Radix Aconiti Lateralis group is significant differences, and Radix Aconiti Lateralis Preparata group is significant difference, and each fermentation group there are no significant
Difference.
2. the impact on dead mouse situation
After continuous several times gives Radix Aconiti Lateralis Preparata, the death condition of mice is as shown in table 2.3 are caused after Radix Aconiti Lateralis group was administered at 2～4 days
Dead mouse, other are respectively organized without dead mouse.
3. mouse toxicity observation of symptoms
Radix Aconiti Lateralis Preparata difference sample is shown in Table 3 to mouse stomach signs of toxicity.
From table 3 result: the toxic reaction after mouse stomach Radix Aconiti Lateralis, Radix Aconiti Lateralis Preparata decocting liquid mainly has nauseating, prostrate
The symptom such as motionless；And there is not corresponding toxicities symptom in mouse stomach fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule, to mice postmortem no abnormality seen.
Claims (9)
1. the compositions of a probiotics fermention Radix Aconiti Lateralis, it is characterised in that after Radix Aconiti Lateralis pulverizing, immersion, enzymolysis, by prebiotic
Bacterium fermentation prepares, and specifically comprises the following steps that
1) pulverize: take dry Radix Aconiti Lateralis and pulverize (10-100 mesh)；
2) soak: 5-20 times of water soaking of addition medical material amount 0.5-2.0 hour, obtain Radix Aconiti Lateralis water logging suspension；
3) adjust pH: Radix Aconiti Lateralis water logging suspension step 2 obtained, adjust pH6.0-7.5 with sodium hydroxide or calcium hydroxide；
4) enzymolysis: the Radix Aconiti Lateralis water logging suspension of modulated pH value step 3 obtained, is heated to 70-95 DEG C, doing by Radix Aconiti Lateralis
Heavily add the Thermostable α-Amylase of percentage by weight 0.1-1.0%, and keep 20-60 minute, sampling detection, make iodine test solution not
Become blue again, obtain the Radix Aconiti Lateralis Preparata mixed liquor of enzymolysis；
5) sterilizing: the Radix Aconiti Lateralis Preparata mixed liquor of the enzymolysis that step 4 is obtained, 105-121 DEG C of autoclave sterilization 20-120min；
6) inoculating: sterilizing Radix Aconiti Lateralis Preparata mixed liquor step 5 obtained, temperature is down to 30-37 DEG C, inoculating two kinds or two or more pre-training
The strain supported；
7) fermentation: vaccinated Radix Aconiti Lateralis Preparata mixed liquor step 6 obtained, under 25-37 DEG C of environment, fermentation condition is cultivated routinely
2-7 days, terminate fermentation, i.e. obtain the compositions of probiotics fermention Radix Aconiti Lateralis；
Strain described in step 6) is selected from bacillus cereus, lactobacillus, bacillus bifidus or yeast, and inoculum concentration is respectively volume basis
Compare 0.5%-2.0%.
The compositions of probiotics fermention Radix Aconiti Lateralis the most according to claim 1, it is characterised in that described bacillus cereus choosing
From bacillus subtilis, Bacillus licheniformis；Described lactobacillus selected from bacillus acidophilus, Deshi Lactobacillus, Lactobacillus plantarum,
Lactobacillus salivarius, Lactobacillus paracasei, lactobacillus casei and lactobacillus rhamnosus；Described bacillus bifidus is selected from not tally bifid bar
Bacterium, bifidobacterium breve, bifidobacterium longum, animal bifidobacteria, bifidobacterium adolescentis and bifidobacteria infantis；Described yeast
Selected from Marx's Crewe yeast, saccharomyces cerevisiae.
3. the preparation method of the compositions of the probiotics fermention Radix Aconiti Lateralis described in claim 1 or 2, it is characterised in that step is such as
Under:
1) pulverize: take dry Radix Aconiti Lateralis and be crushed to 10-100 mesh；
2) soak: 5-20 times of water soaking of addition medical material amount 0.5-2.0 hour, obtain Radix Aconiti Lateralis water logging suspension；
3) adjust pH: Radix Aconiti Lateralis water logging suspension step 2 obtained, adjust pH6.0-7.5 with sodium hydroxide or calcium hydroxide；
4) enzymolysis: the Radix Aconiti Lateralis water logging suspension of modulated pH value step 3 obtained, is heated to 70-95 DEG C, doing by Radix Aconiti Lateralis
Heavily add the Thermostable α-Amylase of percentage by weight 0.1-1.0%, and keep 20-60 minute, sampling detection, make iodine test solution not
Become blue again, obtain the Radix Aconiti Lateralis Preparata mixed liquor of enzymolysis；
5) sterilizing: the Radix Aconiti Lateralis Preparata mixed liquor of the enzymolysis that step 4 is obtained, 105-121 DEG C of autoclave sterilization 20-120min；
6) inoculating: sterilizing Radix Aconiti Lateralis Preparata mixed liquor step 5 obtained, temperature is down to 30-37 DEG C, inoculating two kinds or two or more pre-training
The strain supported；
7) fermentation: vaccinated Radix Aconiti Lateralis Preparata mixed liquor step 6 obtained, under 25-37 DEG C of environment, fermentation condition is cultivated routinely
2-7 days, terminate fermentation, i.e. obtain the compositions of probiotics fermention Radix Aconiti Lateralis；
Strain described in step 6) is selected from bacillus cereus, lactobacillus, bacillus bifidus or yeast, and inoculum concentration is respectively volume basis
Compare 0.5%-2.0%.
The preparation method of the compositions of probiotics fermention Radix Aconiti Lateralis the most according to claim 3, it is characterised in that described
Bacillus cereus is selected from bacillus subtilis, Bacillus licheniformis；Described lactobacillus selected from bacillus acidophilus, Deshi Lactobacillus,
Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus paracasei, lactobacillus casei and lactobacillus rhamnosus；Described bacillus bifidus choosing
From bifidobacterium bifidum, bifidobacterium breve, bifidobacterium longum, animal bifidobacteria, bifidobacterium adolescentis and bifidobacteria infantis；
Described yeast is selected from Marx's Crewe yeast, saccharomyces cerevisiae.
5. the compositions of the probiotics fermention Radix Aconiti Lateralis described in claim 1 or 2 is preparing fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule
In application.
Application the most according to claim 5, it is characterised in that described prepares fermented type Radix Aconiti Lateralis Preparata Chinese medicinal granule, is
Carry out the compositions of probiotics fermention Radix Aconiti Lateralis extracting, concentrate, be dried, detect, subpackage, obtain fermented type Radix Aconiti Lateralis Preparata Chinese medicine formula
Granule, specifically comprises the following steps that
A, extract, concentrate: by centrifugal for the compositions of probiotics fermention Radix Aconiti Lateralis or filter, separation and fermentation liquid；Chinese medicine residue adds again
The water of 5-20 times of weight, heating extraction 1-2 time, obtain extracting solution；Merge fermentation liquid and extracting solution, be evaporated to solid content
More than 10%, obtain concentrated solution；
B, be dried: the concentrated solution that step a is obtained, be spray-dried, obtain dried powder；
C, detection, subpackage: dried powder step b obtained, detect composition, on request specification subpackage.
7. the compositions of the probiotics fermention Radix Aconiti Lateralis described in claim 1 or 2 polysaccharide in extracting separation and fermentation Radix Aconiti Lateralis Preparata becomes
Divide and/or extract the application in the alkaloid component in separation and fermentation Radix Aconiti Lateralis Preparata.
Application the most according to claim 7, it is characterised in that the described polysaccharide component extracted in separation and fermentation Radix Aconiti Lateralis Preparata
Step is as follows:
1) extract: by centrifugal for the compositions of probiotics fermention Radix Aconiti Lateralis or filtration, separation and fermentation liquid, Chinese medicine residue adds 5-20 times again
Water, heating extraction 1-2 time, merge fermentation liquid and extracting solution, be evaporated to relative density 1.08-1.18；
2) separation and fermentation polysaccharide from aconite root: take the concentrated solution that step 1) obtains and add ethanol and make alcohol content be 75-90%, place 24 little
Shi Hou, separates supernatant and precipitation, takes precipitation and be dissolved in water, and filters, and filtrate adds ethanol again makes alcohol content be 80-90%, places
24 hours, again separating supernatant and precipitation, take precipitation washing with acetone 3 times, volatilize solvent, 50 DEG C of drying under reduced pressure must ferment attached
Sub-polysaccharide.
Application the most according to claim 7, it is characterised in that the described alkaloid component extracted in separation and fermentation Radix Aconiti Lateralis Preparata
Step as follows:
1) extract: by centrifugal for the compositions of probiotics fermention Radix Aconiti Lateralis or filtration, separation and fermentation liquid, Chinese medicine residue adds 5-20 times again
Water, heating extraction 1-2 time, merge fermentation liquid and extracting solution, be evaporated to relative density 1.08-1.18；
2) total alkaloids in separation and fermentation Radix Aconiti Lateralis Preparata: take the concentrated solution that step 1) obtains and add ethanol and make alcohol content be 80-90%, place
After 24 hours, separate supernatant and precipitation, collect alcoholic supernatant, decompression recycling ethanol, and be concentrated into relative density 1.01-
1.03, use D101 absorption with macroporous adsorbent resin, with 5 times of volume distilled water remove impurity, then be the ethanol solution of 15% by volume fraction
1.5～2 BV 5 times of volumes of h-1 flow velocity eluting, concentrate eluant, vacuum drying, obtain fermentation Radix Aconiti Lateralis Preparata total alkaloids.
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