CN111440186B - Coumarin norisoflavone compound extracted from small horse blebs and having liver protecting effect, and preparation method and application thereof - Google Patents
Coumarin norisoflavone compound extracted from small horse blebs and having liver protecting effect, and preparation method and application thereof Download PDFInfo
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- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 title claims abstract description 64
- 150000001875 compounds Chemical class 0.000 title claims abstract description 59
- 210000004185 liver Anatomy 0.000 title claims abstract description 58
- 229960000956 coumarin Drugs 0.000 title claims abstract description 32
- 235000001671 coumarin Nutrition 0.000 title claims abstract description 32
- 241000283073 Equus caballus Species 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 230000002633 protecting effect Effects 0.000 title claims abstract description 6
- 208000002352 blister Diseases 0.000 title description 3
- 230000000694 effects Effects 0.000 claims abstract description 42
- 239000003814 drug Substances 0.000 claims abstract description 10
- 239000000284 extract Substances 0.000 claims description 23
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- 206010067125 Liver injury Diseases 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 238000012216 screening Methods 0.000 claims description 15
- 231100000753 hepatic injury Toxicity 0.000 claims description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 210000005229 liver cell Anatomy 0.000 claims description 10
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 9
- 239000000741 silica gel Substances 0.000 claims description 9
- 229910002027 silica gel Inorganic materials 0.000 claims description 9
- 230000004071 biological effect Effects 0.000 claims description 7
- 230000006378 damage Effects 0.000 claims description 7
- 235000013399 edible fruits Nutrition 0.000 claims description 7
- 239000003208 petroleum Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 206010025421 Macule Diseases 0.000 claims description 6
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 238000000338 in vitro Methods 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000007791 liquid phase Substances 0.000 claims description 5
- 238000005191 phase separation Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 208000027418 Wounds and injury Diseases 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 208000014674 injury Diseases 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 238000002791 soaking Methods 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- NOTFZGFABLVTIG-UHFFFAOYSA-N Cyclohexylethyl acetate Chemical compound CC(=O)OCCC1CCCCC1 NOTFZGFABLVTIG-UHFFFAOYSA-N 0.000 claims description 3
- 240000000759 Lepidium meyenii Species 0.000 claims description 3
- 235000000421 Lepidium meyenii Nutrition 0.000 claims description 3
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 125000001033 ether group Chemical group 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 230000002443 hepatoprotective effect Effects 0.000 claims description 3
- 235000012902 lepidium meyenii Nutrition 0.000 claims description 3
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000012071 phase Substances 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- 238000010898 silica gel chromatography Methods 0.000 claims description 3
- 231100000234 hepatic damage Toxicity 0.000 claims description 2
- 239000013067 intermediate product Substances 0.000 claims description 2
- 230000008818 liver damage Effects 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 238000004440 column chromatography Methods 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 2
- 208000002991 Ring chromosome 4 syndrome Diseases 0.000 abstract 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000219112 Cucumis Species 0.000 description 2
- 235000010071 Cucumis prophetarum Nutrition 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 229930003427 Vitamin E Natural products 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229940046009 vitamin E Drugs 0.000 description 2
- 235000019165 vitamin E Nutrition 0.000 description 2
- 239000011709 vitamin E Substances 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 1
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 1
- 235000009831 Citrullus lanatus Nutrition 0.000 description 1
- 244000241257 Cucumis melo Species 0.000 description 1
- 244000022291 Cucumis melo subsp agrestis Species 0.000 description 1
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 1
- 241000219104 Cucurbitaceae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002481 ethanol extraction Methods 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/22—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains four or more hetero rings
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Engineering & Computer Science (AREA)
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- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
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- General Chemical & Material Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract
The invention belongs to the technical field of medicines, and particularly relates to a coumarin norisoflavone compound which is extracted from a small equine follicle and has a liver protection effect, and a preparation method and application thereof. The molecular formula of the coumarin norisoflavone compound is C27H22O10The chemical name is: 3- (4' -hydroxy-3 ',5' -dimethoxy-benzylidene) -5,6- (4' -methylene) -benzopyran-7, 8- (12, 13-dimethyl-pyran ring-4, 3' -diketone, the coumarin homoisoflavonoid compound has the effect of protecting the liver.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a coumarin norisoflavone compound which is extracted from a small equine follicle and has a liver protection effect, and a preparation method and application thereof.
Background
Komare (Cucumis bisexualis) is the fruit of an annual creeping herbaceous plant of the genus Cucumis of the family Cucurbitaceae, and is generally wild. The edible vinegar is widely distributed in the areas of the yellow river and the downstream of the Huaihe river in the southeast of Shandong, Anhui and the north of Jiangsu, has rich resources and large and rich yield, and can be eaten after being mature. Mostly growing beside roads and ridges, commonly called "horse sacks", which are called "horse treasures", wild melons, marmalade, Shuguo, Ma-soaked eggs and muskmelon eggs, and are also called "horse treasures eggs" in the southern river and rural areas. The review of the literature finds that the small equine vesicles have relatively little research on the medicine, and the potential medicinal value of the small equine vesicles needs to be further developed.
Disclosure of Invention
The invention provides a coumarin norisoflavone compound which is extracted from small equine vesicles and has a liver protection effect, and the compound can be extracted from the small equine vesicles and has the liver protection effect, so as to solve the technical problems.
The second objective of the invention is to provide a preparation method of the coumaroisoflavone compound.
The third purpose of the invention is to provide a product with the liver protection effect.
The invention also aims to provide application of the coumarin norisoflavonoid compound.
The coumarin norisoflavone compound extracted from the small horse blebs and having the liver protection effect adopts the following technical scheme: what is neededThe molecular formula of the coumarin norisoflavonoid compound is C27H22O10The structural formula is as follows:
the chemical name is:
3- (4' -hydroxy-3 ',5' -dimethoxy-benzylidene) -5,6- (4 "-methylene) -benzopyran-7, 8- (12, 13-dimethyl-pyran ring
-4,3 "-dione.
The preparation method of the coumarin norisoflavone compound adopts the following technical scheme: the coumarin norisoflavonoid compound is extracted from the small vesicles.
Preferably, the method comprises the following steps: (1) extracting the small horse macule fruits by adopting an ethanol solution, and further concentrating the extracting solution to obtain thick paste; (2) adding water into the thick paste obtained in the step (1), and uniformly stirring to obtain a suspension; (3) extracting the suspension by adopting different organic solvents to obtain a plurality of extracts, and collecting the liver protection extracts with the liver protection effect for later use; (4) purifying the liver protection extract to obtain the coumarin homoisoflavonoid compound.
Preferably, the purification method in step (4) includes, but is not limited to, passing the hepatoprotective extract into any one or a combination of a silica gel column, a gel column or a liquid chromatography column; the liver protection extract is obtained by screening according to the following method: respectively applying the plurality of extracts obtained in the step (3) to a liver injury model to obtain the liver protection extracts by screening by confirming the liver protection effect of the extracts based on a biological activity guidance separation method; the purification also comprises a step of applying the purified intermediate product to a liver injury model to confirm the liver protection effect of the liver injury model so as to realize the purification of the coumaroisoflavone compound based on a method for guiding separation by biological activity.
Preferably, the ethanol solution in the step (1) is 80-95% by volume, and before the extraction of the small horse macules, the method further comprises the steps of crushing the small horse macules and performing ethanol extraction on the small horse maculesSoaking in the solution; the volume ratio of the thick paste to the water in the step (2) is 1: (0.8-1.5); the liver injury model adopts H2O2And (3) treating the L-O2 human liver cell line to obtain a liver injury model.
Preferably, the liver protection extract is purified by adopting a mode of combining a silica gel column, a gel column and a liquid chromatography column.
Preferably, the preparation method of the coumarin norisoflavone compound comprises the following steps:
(1) pulverizing dried Lepidium meyenii Walp fruit to 60-100 mesh, soaking in 80-95% ethanol for 18-20 hr, heating and reflux extracting for 3 times (each time for 3-5 hr) to obtain extractive solution;
(2) mixing the extractive solutions obtained in step (1), concentrating and drying at 40-50 deg.C under vacuum condition until no alcohol smell exists to obtain total soft extract; (3) uniformly suspending the total thick paste obtained in the step (2) in water according to the volume ratio of 1:0.8-1.5, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain a petroleum ether part, an ethyl acetate part and an n-butanol part respectively;
(4) based on a biological activity guidance separation method, adopting an in vitro passage liver cell line L-O2 injury model to carry out liver protection activity screening on different extraction parts obtained in the step (3), and determining an ethyl acetate extraction part as an active part;
(5) performing 4-grade gradient elution on the 100-200-mesh silica gel column on the ethyl acetate extraction part with the liver protection activity determined in the step (4) by using a cyclohexane-ethyl acetate elution system with the volume ratio of 12:1-3:1 to obtain 4 fractions A, B, C, D; screening the fractions A, B, C and D for liver protection activity again to determine that D is liver protection activity fraction;
(6) subjecting the fraction D obtained in the step (5) to 200-300-mesh silica gel column, performing 3-stage gradient elution by using an elution system with the volume ratio of 6:1-2:1 to obtain sub-fractions D-1, D-2 and D-3, and determining that D-1 is the liver protection activity sub-fraction through liver protection activity screening;
(7) subjecting the liver protection activity sub-fraction D-1 obtained in the step (6) to Sephadex LH-20 gel column, eluting with methanol-dichloromethane with volume ratio of 1:1 to obtain sub-fractions D-1-1 and D-1-2 (since the homoisoflavonoid compound is a light yellow solid, in the actual separation process, appropriate fractions can be selected according to the colors of the fractions), and screening D-1-1 as liver protection activity sub-fractions through liver protection activity;
(8) performing liquid phase separation on the sub-fraction D-1-1 obtained in the step (7) to obtain the coumaroisoflavone compound; the liquid phase separation conditions are YMC-Pack ODS-A, 250X 20mm X5 μm chromatographic column, and the mobile phase is 20% -45% methanol by volume percentage.
The product with liver protection effect adopts the following technical scheme: a liver-protecting product, the raw material or the effective ingredient of which comprises the coumarinohomoisoflavone compound of claim 1, in the form of, but not limited to, food, pharmaceutical or beverage products.
The application of the coumarin norisoflavone compound adopts the following technical scheme: the application of the coumarin norisoflavone compound in preparing the medicine for treating liver injury.
The application of the coumarinohomoisoflavone compound in preparing the medicine for reducing ALT and/or AST of liver damaged cells; the liver injury cell includes but is not limited to H2O2The resulting liver damages cells.
The invention has the beneficial effects that: the coumarinohomoisoflavone compound has the effect of protecting the liver, and can be used for reducing the ALT (alanine aminotransferase) and AST (aspartate aminotransferase) contents of liver-damaged cells.
The coumarin norisoflavone compound can be extracted from the small vesicles, and the utilization rate of the small vesicles can be improved.
The invention adopts 80-95% ethanol to heat and reflux and extract folk wild fruit-small bulb for 3 times, ensures that the active ingredients for protecting liver are fully extracted and enriched, and can separate coumarin homoisoflavonoid compounds with liver protection function from active parts by comprehensively adopting silica gel column chromatography, gel chromatography and preparative liquid chromatography.
The preparation method of the coumarin norisoflavone compound is simple, rapid, efficient, thorough and easy to operate.
The coumarin norisoflavone compound can be used for preparing products with the liver protection effect, such as food, medicines or drinks, and has good medicinal value.
The invention adopts an in vitro passage liver cell strain L-O2 damage model to carry out liver protection activity screening on the extracted coumarin homoisoflavonoids, and discloses the application of the coumarin homoisoflavonoids in preparing liver protection medicaments for the first time.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of one of the processes for the preparation of coumarin norisoflavone compounds of the present invention;
FIG. 2 is a coupling correlation diagram of coumarin norisoflavone compounds of the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following examples are given to further illustrate the essence of the present invention, but should not be construed as limiting the scope of the present invention.
EXAMPLE 1 preparation of coumarin norisoflavone compounds extracted from Microequiburia for liver protection according to the present invention (see FIG. 1 of the drawings in the present application)
(1) Pulverizing dried Lepidium meyenii Walp fruit (15.5 kg) to 80 mesh, soaking in 90% ethanol for 19 hr, and reflux-extracting under heating for 3 times (4 hr/time) to obtain extractive solution;
(2) mixing the extractive solutions obtained in step (1), concentrating and drying at low temperature (40-50 deg.C) under vacuum condition until no alcohol smell exists to obtain total soft extract (1.6 kg);
(3) uniformly suspending the total thick paste obtained in the step (2) in water according to a certain proportion (volume ratio is 1:1, V/V), and sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain a petroleum ether part (98.6 g), an ethyl acetate part (235.5 g) and an n-butanol part (305.8 g), respectively;
(4) based on a biological activity guidance separation method, adopting an in vitro passage liver cell line L-O2 damage model (AST and ALT) to carry out liver protection activity screening on different extraction parts, and determining an ethyl acetate extraction part as an active part;
(5) the hepatoprotective active site (ethyl acetate extraction site) determined in step (4) was subjected to silica gel (100-200 mesh) column, and gradient elution was carried out using cyclohexane-ethyl acetate (12: 1 → 3:1, V/V) elution system to obtain 4 fractions A (29.6 g), B (59.5 g), C (72.8 g) and D (19.5 g). Screening the 4 fractions for liver protection activity again to determine D as the liver protection activity fraction;
(6) subjecting the fraction D obtained in the step (5) to silica gel (200-300 mesh) column, and performing gradient elution with petroleum ether-ethyl acetate (6: 1 → 2:1, V/V) elution system to obtain sub-fractions D-1(4.8 g), D-2(8.5 g) and D-3(3.2 g);
(7) putting the liver protection activity sub-fraction D-1 obtained in the step (6) on a Sephadex LH-20 gel column, and eluting with methanol-dichloromethane (1: 1, V/V) to obtain sub-fractions D-1-1(1.9 g) and D-1-2(2.1 g); since the homoisoflavonoid compound is a light yellow solid, a color band with color difference can be observed in the actual separation process, and proper fractions can be selected according to the color.
(8) The subfraction D-1-2 obtained in step (7) was subjected to preparative liquid phase separation (wavelength: 280nm, mobile phase: 35% methanol, YMC-Pack ODS-A column: 250X 20mm,5 μm) to give novel compound I (13.52 mg, purity 98.8%).
The new compound I is light yellow solid, HR-ESI-MS m/z 529.1118[M+Na]+Indicating that its molecular formula is C27H22O10(calcd.for C27H22O10Na, 529.1111). UV (MeOH) lambda of Compound Imax:205、238、280、360nm；IRνmax:3408、1736、1630、1364cm-1；1H NMR(CD3COCD3400MHz) and13C NMR(CD3COCD3100MHz) data are shown in Table 1. From the spectral data of compound I and HMBC and1H-1the related information of H COSY coupling (figure 2) is searched by SciFinder, and the compound I is identified as a novel coumarinohomoisoflavonoid compound, which is named as: 3- (4' -hydroxy-3 ',5' -dimethoxy-benzylidene) -5,6- (4 "-methylene) -benzopyran-7, 8- (12, 13-dimethyl-pyran ring) -4, 3" -dione.
TABLE 1 preparation of compound I1H NMR(400MHz,CD3COCD3)、13C NMR(100MHz,CD3COCD3) And HMBC related data
2. The liver protection activity of the coumarin homoisoflavonoid compound
(1) Experimental Material
RPMI-1640 medium, ALT, AST kit (Shanghai Yubo Biotech Co., Ltd.), vitamin E, 30% H2O2L-O2 human liver cell line (Shanghai YuBo Biotech Co., Ltd.), etc.
After dissolving the new compound I in DMSO, 2mg/mL of the new compound I was prepared in RPMI-1640 medium containing 10% calf serum and used.
(2) Experimental method
The coumarin norisoflavone compound is first diluted into 5 dosage groups of 100 microgram/mL, 20 microgram/mL, 4 microgram/mL, 0.8 microgram/mL and 0.16 microgram/mL. Then, the cells which grew vigorously in L-O2 were washed, digested with 0.25% trypsin and counted to adjust the number of cells to 2X 105Perml, seeded in 96-well plates at 0 per well1mL, placing the mixture in an incubator at 37 ℃ and 5% carbon dioxide for culturing, sucking out supernatant after culturing for 24 hours, respectively adding the 5 samples with different concentrations, wherein each concentration is provided with 3 multiple wells, and vitamin E group (50mmol/L) and H are provided2O2Model group (0.4mmol/L) and blank control group, each 0.1 mL/well. Finally, after culturing in a 5% carbon dioxide incubator at 37 ℃ for 6 hours, the supernatants were separately collected and the ALT and AST levels in hepatocytes were measured. Statistical analysis SPSS17.0 statistical software was used, experimental data are presented as X. + -. S, and a t-test was used for comparison between groups.
(3) Results of the experiment
TABLE 2 protective Action (ALT) of New Compound I against injury from in vitro passaged liver cell line L-O2
Note: n is 3; compared with the model group, P of the compound I is less than 0.05.
TABLE 3 protective action of Compound I against injury of in vitro passage liver cell line L-O2 (AST)
Note: n is 3; compared with the model group, the P of the compound I is less than 0.05.
As can be seen from tables 2 and 3, the ALT and AST of each concentration group of the new compound I are significantly different (P is less than 0.05) compared with the model group, and the ALT and AST contents are both significantly reduced, and the experimental result shows that the new compound I is used for H2O2The damage of acute liver cells caused by the traditional Chinese medicine has a certain protection effect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (10)
1. Small horse bubbleThe coumarin homoisoflavonoid compound extracted from the extract and having the effect of protecting the liver is characterized in that the molecular formula of the coumarin homoisoflavonoid compound is C27H22O10The structural formula is as follows:
2. the method of claim 1, wherein the coumarinohomoisoflavone compound is extracted from the vesicles.
3. The method of claim 2, comprising the steps of: (1) extracting the small horse macule fruits by adopting an ethanol solution, and further concentrating the extracting solution to obtain thick paste; (2) adding water into the thick paste obtained in the step (1), and uniformly stirring to obtain a suspension; (3) extracting the suspension by adopting different organic solvents to obtain a plurality of extracts, and collecting the liver protection extracts with the liver protection effect for later use; (4) purifying the liver protection extract to obtain the coumarin homoisoflavonoid compound.
4. The method of claim 3, wherein the purifying step (4) comprises subjecting the hepatoprotective extract to any one or a combination of silica gel column, or liquid chromatography column; the liver protection extract is obtained by screening according to the following method: respectively applying the plurality of extracts obtained in the step (3) to a liver injury model to obtain the liver protection extracts by screening by confirming the liver protection effect of the extracts based on a biological activity guidance separation method; the purification also comprises a step of applying the purified intermediate product to a liver injury model to confirm the liver protection effect of the liver injury model so as to realize the purification of the coumaroisoflavone compound based on a method for guiding separation by biological activity.
5. The method for preparing coumarinohomoisoflavone compound according to claim 3, wherein the ethanol solution in step (1) is 80-95% by volume, and the method further comprises the steps of pulverizing the fruit of the small horse macules and soaking in ethanol solution before extracting the small horse macules; the volume ratio of the thick paste to the water in the step (2) is 1: (0.8-1.5); the liver injury model adopts H2O2And (3) treating the L-O2 human liver cell line to obtain a liver injury model.
6. The method of claim 3, wherein the extract is purified by a combination of silica gel column, gel column and liquid chromatography column.
7. The method of any one of claims 2 to 6, comprising the steps of:
(1) pulverizing dried Lepidium meyenii Walp fruit to 60-100 mesh, soaking in 80-95% ethanol for 18-20 hr, heating and reflux extracting for 3 times (each time for 3-5 hr) to obtain extractive solution;
(2) mixing the extractive solutions obtained in step (1), concentrating and drying at 40-50 deg.C under vacuum condition until no alcohol smell exists to obtain total soft extract;
(3) uniformly suspending the total thick paste obtained in the step (2) in water according to the volume ratio of 1:0.8-1.5, and sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain a petroleum ether part, an ethyl acetate part and an n-butanol part respectively;
(4) based on a biological activity guidance separation method, adopting an in vitro passage liver cell line L-O2 injury model to carry out liver protection activity screening on different extraction parts obtained in the step (3), and determining an ethyl acetate extraction part as an active part;
(5) performing 4-grade gradient elution on the 100-200-mesh silica gel column on the ethyl acetate extraction part with the liver protection activity determined in the step (4) by using a cyclohexane-ethyl acetate elution system with the volume ratio of 12:1-3:1 to obtain 4 fractions A, B, C, D; screening the fractions A, B, C and D for liver protection activity again to determine that D is liver protection activity fraction;
(6) subjecting the fraction D obtained in the step (5) to 200-300-mesh silica gel column, performing 3-stage gradient elution by using an elution system with the volume ratio of 6:1-2:1 to obtain sub-fractions D-1, D-2 and D-3, and determining that D-1 is the liver protection activity sub-fraction through liver protection activity screening;
(7) putting the liver protection activity sub-fraction D-1 obtained in the step (6) on a Sephadex LH-20 gel column, eluting with methanol-dichloromethane with the volume ratio of 1:1 to obtain sub-fractions D-1-1 and D-1-2, and screening the sub-fraction D-1 to obtain liver protection activity sub-fractions through liver protection activity;
(8) performing liquid phase separation on the sub-fraction D-1-1 obtained in the step (7) to obtain the coumaroisoflavone compound; the liquid phase separation conditions are YMC-Pack ODS-A, 250X 20mm X5 μm chromatographic column, and the mobile phase is 20% -45% methanol by volume percentage.
8. The use of the coumarin norisoflavone compound of claim 1 in the preparation of a liver-protecting product.
9. The use of the coumarin norisoflavone compound of claim 1 in the preparation of a medicament for the treatment of liver injury.
10. The use of the coumarinohomoisoflavone compound of claim 1 in the preparation of a medicament for reducing ALT and/or AST in liver damaged cells; the liver injury cell is selected from H2O2The resulting liver damages cells.
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