CN115154498A - Astragalus membranaceus fermented extract rich in astragaloside IV and preparation method and application thereof - Google Patents
Astragalus membranaceus fermented extract rich in astragaloside IV and preparation method and application thereof Download PDFInfo
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- CN115154498A CN115154498A CN202210710955.0A CN202210710955A CN115154498A CN 115154498 A CN115154498 A CN 115154498A CN 202210710955 A CN202210710955 A CN 202210710955A CN 115154498 A CN115154498 A CN 115154498A
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- astragaloside
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- 235000006533 astragalus Nutrition 0.000 title claims abstract description 56
- 239000000284 extract Substances 0.000 title claims abstract description 37
- QMNWISYXSJWHRY-YLNUDOOFSA-N astragaloside IV Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)[C@H]4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)C[C@H]3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-YLNUDOOFSA-N 0.000 title claims abstract description 22
- QMNWISYXSJWHRY-BCBPIKMJSA-N astragaloside IV Natural products CC(C)(O)[C@@H]1CC[C@@](C)(O1)[C@H]2[C@@H](O)C[C@@]3(C)[C@@H]4C[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@@H](O)[C@H]5O)[C@H]6C(C)(C)[C@H](CC[C@@]67C[C@@]47CC[C@]23C)O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O QMNWISYXSJWHRY-BCBPIKMJSA-N 0.000 title claims abstract description 22
- PFKIBRPYVNVMRU-UHFFFAOYSA-N cyclosieversioside F Natural products CC(C)(O)C1COC(C)(C1)C2C(O)CC3(C)C4CC(OC5OC(CO)C(O)C(O)C5O)C6C(C)(C)C(CCC67CC47CCC23C)OC8OCC(O)C(O)C8O PFKIBRPYVNVMRU-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 241000045403 Astragalus propinquus Species 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 43
- 230000004151 fermentation Effects 0.000 claims abstract description 43
- 241001061264 Astragalus Species 0.000 claims abstract description 35
- 210000004233 talus Anatomy 0.000 claims abstract description 35
- SMDOOINVMJSDPS-UHFFFAOYSA-N Astragaloside Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)=C1 SMDOOINVMJSDPS-UHFFFAOYSA-N 0.000 claims abstract description 19
- QMNWISYXSJWHRY-XWJCTJPOSA-N astragaloside Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)C4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)CC3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-XWJCTJPOSA-N 0.000 claims abstract description 19
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 10
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 10
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- 238000009630 liquid culture Methods 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 235000015278 beef Nutrition 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 8
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 7
- 102000002322 Egg Proteins Human genes 0.000 claims description 7
- 108010000912 Egg Proteins Proteins 0.000 claims description 7
- 235000014103 egg white Nutrition 0.000 claims description 7
- 210000000969 egg white Anatomy 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 6
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 6
- 235000019206 astragalus extract Nutrition 0.000 claims description 5
- 208000024172 Cardiovascular disease Diseases 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 4
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 1
- 239000004480 active ingredient Substances 0.000 abstract description 3
- 239000000419 plant extract Substances 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 13
- 239000009636 Huang Qi Substances 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 9
- 229930182490 saponin Natural products 0.000 description 6
- 235000017709 saponins Nutrition 0.000 description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 5
- 241000612118 Samolus valerandi Species 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 150000007949 saponins Chemical class 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 241001669263 Bacillus amyloliquefaciens DSM 7 Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 206010019668 Hepatic fibrosis Diseases 0.000 description 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 229940107666 astragalus root Drugs 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 208000029028 brain injury Diseases 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- -1 saponin compound Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/481—Astragalus (milkvetch)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Medical Informatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Heart & Thoracic Surgery (AREA)
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Abstract
The invention belongs to the technical field of research on plant extract medicaments, and particularly relates to an astragalus mongholicus fermented extract rich in astragaloside IV, and further discloses a preparation method and application thereof. On the basis of the traditional astragalus fermentation process by using bacillus subtilis, the astragalus fermentation extract is fermented together by adding bacillus amyloliquefaciens, so that the content of active ingredients in the astragalus fermentation extract is effectively improved, particularly the content of astragaloside is obviously improved, and the astragalus fermentation extract rich in astragaloside can be obtained.
Description
Technical Field
The invention belongs to the technical field of research on plant extract medicaments, and particularly relates to an astragalus mongholicus fermented extract rich in astragaloside IV, and further discloses a preparation method and application thereof.
Background
Astragalus (Radix Astragali) is one of the commonly used Chinese medicines, and is dried root of Astragalus membranaceus Bunge (Fischer) Bunge and Astragalus membranaceus Mongolica Bunge (Fisch.) Bunge var. Mongholicus (Bunge) P.K.Hsiao which are leguminous plants. The astragalus root has been used for more than 2000 years, is sweet and slightly warm in nature, enters lung, spleen, liver and kidney meridians, and has the functions of enhancing the immunologic function of an organism, protecting the liver, promoting urination, resisting aging, resisting stress, reducing blood pressure and having wider antibacterial action.
Astragaloside IV (ASI) is a saponin compound with multiple pharmacological activities separated from radix astragali, and is an index component for quality identification of radix astragali preparation. Studies show that the astragaloside IV has wide pharmacological action in cardiovascular system, immune system, nervous system, digestive system, urinary system, circulatory system, endocrine system and the like. Astragaloside is reported to alleviate the increase in blood brain barrier permeability caused by cerebral ischemia reperfusion; has protective effect on ischemic brain injury; exerting anticancer activity by regulating the expression of oncogenes; has blood sugar lowering effect on diabetic mice; has strong anti-hepatitis B virus activity; can effectively inhibit or reverse the hepatic fibrosis of a mouse model induced by PS; the medicinal preparation is developed into a myocardial protectant for treating coronary heart disease and angina pectoris. However, in the industrial extraction of astragalus, the content of astragaloside in astragalus is extremely low, and the effect of the astragalus extract is greatly influenced by the production place and season.
For example, chinese patent CN102559828A discloses a method for preparing astragaloside iv by microbial transformation of total saponins of astragalus, which takes total saponins of astragalus as a substrate, and hydrolyzes acetyl and glycosyl in total saponins of astragalus by using microorganisms containing deacetylase and glycosidase under the action of bacteria, mold or yeast as microorganisms, thereby increasing the content of astragaloside iv. The method can effectively increase the content of astragaloside IV in the fermentation broth. However, the method needs microbial transformation treatment by taking the total saponins of astragalus as a substrate, and also needs steps of extracting and purifying to obtain the total saponins of astragalus, and the process steps are relatively complicated. Therefore, the method for fermenting by directly taking astragalus as a substrate and obtaining the fermentation product with high astragalus glucoside content is expected to be developed by the technical personnel in the field, and has positive significance for the application and development of astragalus.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a preparation method of astragalus mongholicus fermented extract rich in astragaloside, wherein the astragalus mongholicus is used as a substrate for fermentation, so that the fermented extract with high astragaloside content can be obtained;
the second technical problem to be solved by the invention is to provide the application of the astragalus mongholicus fermented extract rich in astragaloside IV in preparing the medicines for preventing and treating cardiovascular diseases.
In order to solve the technical problems, the preparation method of the astragalus mongholicus fermentation extract rich in astragaloside comprises the following steps:
(1) Preparing a fermentation culture medium from the crushed astragalus powder for later use;
(2) Inoculating the preserved bacillus subtilis into a first seed liquid culture medium for culture to obtain a first seed liquid for later use;
(3) Inoculating the preserved bacillus amyloliquefaciens into a second seed liquid culture medium for culture to obtain a second seed liquid for later use;
(4) Respectively inoculating the first seed liquid and the second seed liquid into the fermentation culture medium for fermentation culture, and collecting fermentation products.
Specifically, in the step (1), the fermentation medium comprises the following components: 150-250g/L of astragalus powder, 30-80g/L of glucose, 30-80g/L of peptone, 5-15g/L of egg white, 0.4-0.6g/L of dipotassium phosphate, 0.8-1.2g/L of sodium acetate and pH7.0-7.2.
Preferably, the fermentation medium comprises the following components: 200g/L of astragalus powder, 50g/L of glucose, 50g/L of peptone, 10g/L of egg white, 0.5g/L of dipotassium hydrogen phosphate, 1g/L of sodium acetate and 7.0-7.2 of pHs.
Specifically, in the step (2), the first seed liquid culture medium comprises the following components: 20-40g/L of glucose, 10-20g/L of beef extract, 5-15g/L of peptone, 0.5-1.0g/L of sodium chloride, 0.5-1.0g/L of sodium dihydrogen phosphate and pH value of 7.0-7.2.
Preferably, the first seed liquid culture medium comprises the following components: 30g/L glucose, 15g/L beef extract, 10g/L peptone, 0.8g/L sodium chloride, 0.8g/L sodium dihydrogen phosphate, and pH7.0-7.2.
Specifically, in the step (2), the temperature of the first seed liquid culture step is 32-38 ℃, and the culture is carried out for 12-24h at 100-150 rpm.
Specifically, in the step (3), the second seed liquid culture medium comprises the following components: 20-40g/L of glucose, 10-20g/L of beef extract, 5-15g/L of urea, 0.5-1.0g/L of dipotassium phosphate, 0.5-1.0g/L of monopotassium phosphate and 7.0-7.2 of pH value.
Preferably, the second seed liquid culture medium comprises the following components: 30g/L glucose, 15g/L beef extract, 10g/L urea, 0.8g/L dipotassium phosphate, 0.8g/L potassium dihydrogen phosphate and pH7.0-7.2.
Specifically, in the step (3), the temperature of the second seed liquid culture step is 22-28 ℃, and the culture is carried out for 24-36h at 50-100 rpm.
Specifically, in the step (4), the first seed liquid and the second seed liquid are 5-15v/v% independently based on the inoculation amount of the fermentation liquid;
preferably, the ratio of the inoculation amount of the first seed liquid to the inoculation amount of the second seed liquid is 2:1.
specifically, in the step (4), the temperature of the fermentation culture step is 30-32 ℃, and the culture time is 48-72h.
The invention also discloses the astragalus mongholicus fermented extract rich in astragaloside prepared by the method.
The invention also discloses application of the astragalus mongholicus fermented extract rich in astragaloside IV in preparing a medicament for preventing and treating cardiovascular diseases.
On the basis of the traditional process for fermenting astragalus by using bacillus subtilis, the astragalus fermented extract is fermented together by adding the bacillus amyloliquefaciens, so that the content of active ingredients in the astragalus fermented extract is effectively improved, particularly the content of astragaloside is obviously improved, and the bacillus amyloliquefaciens has stronger enzyme production characteristics, so that the bacillus amyloliquefaciens is presumed to utilize an enzyme production system to assist the bacillus subtilis to ferment the metabolism of the astragalus by fermenting the astragalus, and the astragalus fermented extract rich in the astragaloside can be obtained; the efficiency of producing astragaloside by fermenting and metabolizing astragalus by bacillus amyloliquefaciens is not ideal, and the performance and the effect of assisting bacillus subtilis to ferment astragalus are proved laterally.
The astragalus mongholicus fermented extract has high content of astragaloside IV and can be used for preparing pharmaceutical preparations for treating cardiovascular diseases.
Detailed Description
Preparation example 1
The preparation example is used for obtaining a first seed liquid, and the first seed liquid comprises the following components: 30g/L glucose, 15g/L beef extract, 10g/L peptone, 0.8g/L sodium chloride, 0.8g/L sodium dihydrogen phosphate, pH7.0-7.2, and sterilizing at 121 deg.C.
Taking a ring of the bacillus subtilis ATCC6633 preserved on the slant, inoculating the bacillus subtilis ATCC6633 into the first seed solution, shaking the flask to contain 40ml/250ml of liquid, and culturing overnight at 35 ℃ and 150rpm to obtain the first seed solution.
Preparation example 2
The preparation example is used for obtaining a second seed solution, and the second seed solution comprises the following components: 30g/L glucose, 15g/L beef extract, 10g/L urea, 0.8g/L dipotassium phosphate, 0.8g/L potassium dihydrogen phosphate, pH7.0-7.2, and sterilizing at 121 deg.C.
Inoculating a loop of the slant-preserved Bacillus amyloliquefaciens ATCC23350 into the second seed solution, shaking the flask to contain 40ml/250ml, and culturing at 25 ℃ and 80rpm for 24h.
Example 1
Pulverizing cleaned and dried radix astragali to obtain radix astragali powder, and preparing the required fermentation culture medium according to the following components: 200g/L of astragalus powder, 50g/L of glucose, 50g/L of peptone, 10g/L of egg white, 0.5g/L of dipotassium hydrogen phosphate and 1g/L of sodium acetate, wherein the pH value is 7.0-7.2, and the sterilization is carried out at 121 ℃.
Inoculating the first seed solution and the second seed solution obtained in the preparation examples 1-2 to the fermentation medium according to the inoculation amount of 10v/v% and 5v/v%, culturing at 30 deg.C and 100rpm for 72h, and collecting the fermentation product.
Example 2
Pulverizing cleaned and dried radix astragali to obtain radix astragali powder, and preparing the required fermentation culture medium according to the following components: 200g/L of astragalus powder, 50g/L of glucose, 50g/L of peptone, 10g/L of egg white, 0.5g/L of dipotassium hydrogen phosphate and 1g/L of sodium acetate, wherein the pH value is 7.0-7.2, and the sterilization is carried out at 121 ℃.
Inoculating the first seed solution and the second seed solution obtained in the preparation examples 1-2 to the fermentation medium according to the inoculation amount of 10v/v% and 10v/v%, culturing at 30 deg.C and 100rpm for 72h, and collecting the fermentation product.
Example 3
Pulverizing cleaned and dried radix astragali to obtain radix astragali powder, and preparing the required fermentation culture medium according to the following components: 200g/L of astragalus powder, 50g/L of glucose, 50g/L of peptone, 10g/L of egg white, 0.5g/L of dipotassium hydrogen phosphate and 1g/L of sodium acetate, wherein the pH value is 7.0-7.2, and the sterilization is carried out at 121 ℃.
Inoculating the first seed solution and the second seed solution obtained in the preparation examples 1-2 to the fermentation medium according to the inoculation amount of 10v/v% and 15v/v%, culturing at 30 deg.C and 100rpm for 72h, and collecting the fermentation product.
Comparative example 1
The fermented extract of astragalus membranaceus described in this comparative example was prepared in the same manner as in example 1, except that only the bacillus subtilis was inoculated and the fermentation was performed under the same conditions.
Comparative example 2
The fermented extract of astragalus membranaceus described in this comparative example was prepared in the same manner as in example 1, except that only the strain of bacillus amyloliquefaciens was inoculated for fermentation under the same conditions.
Comparative example 3
The fermented extract of astragalus membranaceus according to this comparative example was prepared in the same manner as in example 1, except that the same amount of beef extract was added to the fermentation medium without the addition of egg white.
Examples of the experiments
In this experimental example, the content of astragaloside in the fermentation products collected in examples 1 to 3 and comparative examples 1 to 3 was measured, and the content of astragaloside in the fermentation medium before fermentation was used as a control, and the measurement results are shown in table 1 below.
The determination method of astragaloside IV comprises the following steps: high performance liquid chromatography. Taking 10g of fermentation product, evaporating to dryness in a water bath at 60 ℃, putting into a Soxhlet extractor, adding 40mL of methanol, and carrying out cold soaking overnight; adding appropriate amount of methanol, heating in water bath at 80 deg.C, refluxing for 4 hr, and concentrating the extractive solution to dryness; dissolving the residue in 10mL of water, slightly heating to dissolve, extracting with water saturated n-butanol under shaking for 4 times (40 mL each time), mixing n-butanol solutions, washing with ammonia solution for 2 times (40 mL each time), discarding ammonia solution, evaporating n-butanol layer, and diluting to 5mL with methanol for testing. And taking astragaloside to prepare a control sample with the concentration of 0.5 mg/mL. Chromatographic conditions and system applicability: a C18 column (150 mm. Times.4.6 mm,5 μm) was selected by pre-run, mobile phase acetonitrile: water =32:68 isocratic elution is carried out, the detection wavelength is 202nm, the flow rate is 0.8mL/min, the column temperature is 30 ℃, the sample injection amount is 10 mu L, and the quantification is carried out by adopting an external standard method.
TABLE 1 content of astragaloside in fermentation products
| Number of | Content of Astragaloside IV (mg/g) |
| Example 1 | 0.140 |
| Example 2 | 0.142 |
| Example 3 | 0.145 |
| Comparison ofExample 1 | 0.078 |
| Comparative example 2 | 0.054 |
| Comparative example 3 | 0.122 |
| Control of | 0.043 |
Therefore, on the basis of the traditional astragalus fermentation process by using the bacillus subtilis, the astragalus fermentation extract is fermented together by adding the bacillus amyloliquefaciens, so that the content of active ingredients, particularly astragaloside IV, in the astragalus fermentation extract is effectively improved.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. A preparation method of astragalus mongholicus fermented extract rich in astragaloside is characterized by comprising the following steps:
(1) Preparing a fermentation culture medium from the crushed astragalus powder for later use;
(2) Inoculating the preserved bacillus subtilis into a first seed liquid culture medium for culture to obtain a first seed liquid for later use;
(3) Inoculating the preserved bacillus amyloliquefaciens into a second seed liquid culture medium for culture to obtain a second seed liquid for later use;
(4) And respectively inoculating the first seed solution and the second seed solution into the fermentation culture medium for fermentation culture, and collecting fermentation products.
2. The method for preparing Astragaloside IV-enriched Astragalus fermented extract as claimed in claim 1, wherein in the step (1), the fermentation medium comprises the following components: 150-250g/L of astragalus powder, 30-80g/L of glucose, 30-80g/L of peptone, 5-15g/L of egg white, 0.4-0.6g/L of dipotassium phosphate, 0.8-1.2g/L of sodium acetate and pH7.0-7.2.
3. The method for preparing Astragalus membranaceus fermented extract rich in Astragaloside IV according to claim 1 or 2, wherein in the step (2), the first seed liquid culture medium comprises the following components: 20-40g/L of glucose, 10-20g/L of beef extract, 5-15g/L of peptone, 0.5-1.0g/L of sodium chloride, 0.5-1.0g/L of sodium dihydrogen phosphate and pH value of 7.0-7.2.
4. The method for preparing fermented Astragalus extract rich in astragaloside IV according to claim 3, wherein in the step (2), the first seed liquid culture step is carried out at 32-38 deg.C and 100-150rpm for 12-24h.
5. The method for preparing fermented astragalus extract enriched in astragaloside according to any one of claims 1-4, wherein in the step (3), the second seed liquid culture medium comprises the following components: 20-40g/L of glucose, 10-20g/L of beef extract, 5-15g/L of urea, 0.5-1.0g/L of dipotassium phosphate, 0.5-1.0g/L of monopotassium phosphate and 7.0-7.2 of pH value.
6. The method for preparing fermented Astragalus extract rich in astragaloside IV according to claim 5, wherein in the step (3), the temperature of the second seed liquid culture step is 22-28 deg.C, and the culture is carried out at 50-100rpm for 24-36h.
7. The method for preparing fermented Astragalus membranaceus extract rich in astragaloside IV according to any one of claims 1-6, wherein in the step (4), the first seed solution and the second seed solution are 5-15v/v% independent of each other based on the amount of inoculation of the fermentation broth.
8. The method for preparing fermented astragalus extract enriched in astragaloside according to any one of claims 1-7, wherein in the step (4), the temperature of the fermentation culture step is 30-32 ℃ and the culture time is 48-72h.
9. Fermented extract of astragalus membranaceus, enriched in astragaloside IV, obtained by the process according to any one of claims 1-8.
10. Use of the astragaloside-enriched fermented extract of astragalus membranaceus according to claim 9 for the preparation of a medicament for the prevention and treatment of cardiovascular diseases.
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| CN101002787A (en) * | 2006-01-20 | 2007-07-25 | 天津药物研究院 | Inhalant containing active component of methyl astragaloside |
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| CN101002787A (en) * | 2006-01-20 | 2007-07-25 | 天津药物研究院 | Inhalant containing active component of methyl astragaloside |
| CN105725008A (en) * | 2016-02-20 | 2016-07-06 | 三株福尔制药有限公司 | Natto bacillus subtilis fermented milkvetch root composition and preparation method and application thereof |
| CN109331641A (en) * | 2018-12-03 | 2019-02-15 | 武汉轻工大学 | A kind of preparation method of garbage deodorant |
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