CN117323350A - Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus - Google Patents
Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus Download PDFInfo
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- CN117323350A CN117323350A CN202311443467.9A CN202311443467A CN117323350A CN 117323350 A CN117323350 A CN 117323350A CN 202311443467 A CN202311443467 A CN 202311443467A CN 117323350 A CN117323350 A CN 117323350A
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- polyphenol
- botrytis cinerea
- ethanol
- macroporous resin
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- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 89
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 89
- 208000007536 Thrombosis Diseases 0.000 title claims abstract description 17
- 239000003814 drug Substances 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 229940079593 drug Drugs 0.000 title abstract description 10
- 241000123650 Botrytis cinerea Species 0.000 claims abstract description 55
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 59
- 239000011347 resin Substances 0.000 claims description 38
- 229920005989 resin Polymers 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 29
- 238000011068 loading method Methods 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 19
- 238000001179 sorption measurement Methods 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 11
- 238000004108 freeze drying Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 238000002390 rotary evaporation Methods 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 239000000287 crude extract Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 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 4
- 238000005406 washing Methods 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- 238000007873 sieving Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 239000000284 extract Substances 0.000 abstract description 13
- 238000000605 extraction Methods 0.000 description 27
- 239000000243 solution Substances 0.000 description 18
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 14
- 239000012153 distilled water Substances 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 10
- 238000011835 investigation Methods 0.000 description 10
- 238000003795 desorption Methods 0.000 description 9
- 238000010828 elution Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 229940074391 gallic acid Drugs 0.000 description 7
- 235000004515 gallic acid Nutrition 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 238000007781 pre-processing Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- MYKOKMFESWKQRX-UHFFFAOYSA-N 10h-anthracen-9-one;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 MYKOKMFESWKQRX-UHFFFAOYSA-N 0.000 description 4
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 4
- 229960001138 acetylsalicylic acid Drugs 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 230000002785 anti-thrombosis Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000001186 cumulative effect Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000000465 moulding Methods 0.000 description 3
- 241000737252 Botrychium Species 0.000 description 2
- 241001465180 Botrytis Species 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 241000195628 Chlorophyta Species 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 2
- 241000581650 Ivesia Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 206010037437 Pulmonary thrombosis Diseases 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000002526 effect on cardiovascular system Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 241001290342 Caulerpa Species 0.000 description 1
- 241001339217 Caulerpa lentillifera Species 0.000 description 1
- 241001493811 Caulerpaceae Species 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 229940127218 antiplatelet drug Drugs 0.000 description 1
- 229940127217 antithrombotic drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000003073 embolic effect Effects 0.000 description 1
- 229960003072 epinephrine hydrochloride Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
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- 239000011521 glass Substances 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000000106 platelet aggregation inhibitor Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
Classifications
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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
- A61K36/02—Algae
- A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- 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
-
- 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
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
The invention relates to the technical field of marine organisms, in particular to application of Botrytis cinerea polyphenol in preparation of a medicine for treating thrombus. Therefore, provides the application of the extract in preparing medicines for treating thrombus and further provides a preparation method of the Botrytis cinerea polyphenol.
Description
Technical Field
The invention relates to the technical field of marine organisms, in particular to application of Botrytis cinerea polyphenol.
Background
Botrytis cinerea (Caulerpa lentillifera) belonging to genus Botrytis (Caulerpa) of family Caulerpaceae of order Botrytis (Brilloplaceae) belonging to the order Chlorophyta (Chlorophyta), is a plant which can be eaten and can be used for medicines.
Thrombotic diseases are a group of diseases which can involve whole body tissue organs and seriously damage human life health, and can cause ischemia and necrosis of tissue organs due to blood flow interruption caused by thrombus formation or downstream blood flow interruption caused by embolic shedding. Statistics show that about 1700 ten thousand people die from various cardiovascular and cerebrovascular diseases worldwide each year, and thrombosis is an important factor for initiating various severe cardiovascular and cerebrovascular diseases, so that thrombotic diseases have become one of the main threats to life health of the public today. At present, the antiplatelet drugs and anticoagulants for treating thrombus still have the defects of bleeding event, insufficient drug effect, poor selectivity and the like, and development of novel antithrombotic drugs has been paid attention. Marine plants have characteristics which are not possessed by some terrestrial plants because of unique living environments, the marine plants are widely distributed and huge in quantity, and the acquisition of the effective components with the thrombus treatment activity from the marine plants is a novel and development prospect thought.
Disclosure of Invention
The invention aims to solve the technical problem of providing a novel marine plant extract which can be used for treating and preventing thrombus.
In order to achieve the purpose of the invention, the following technical means are specifically adopted:
the application of the Botrytis cinerea polyphenol in preparing the medicine for treating thrombus is characterized in that the preparation method of the Botrytis cinerea polyphenol comprises the following steps:
(1) Freeze-drying fresh Botrytis cinerea for 48 hours, shearing the Botrytis cinerea, and then putting the Botrytis cinerea into a pulverizer and sieving the Botrytis cinerea with a 60-mesh sieve to obtain Botrytis cinerea powder;
(2) Accurately weighing Botrytis cinerea powder, adding 80% ethanol water solution according to a feed-liquid ratio of 1:15, fully mixing and soaking;
(3) Extracting polyphenol with a numerical control ultrasonic cleaner for 40min at 50deg.C, and vacuum filtering to obtain crude extract of Botrytis cinerea polyphenol;
(4) Performing primary purification on the crude extract of the Botrytis cinerea polyphenol by using macroporous resin BS-45, loading the sample at a flow rate of 1BV/h, standing for adsorption for 1h, washing with deionized water of 0.78BV to remove impurities, eluting with 80% ethanol of 2.5BV, collecting the eluent, concentrating by vacuum rotary evaporation at 40-50 ℃ and freeze-drying to obtain Botrytis cinerea polyphenol powder;
(5) Taking the Botrytis cinerea polyphenol purified by macroporous resin, precisely weighing, ultrasonically dissolving with a small amount of water, centrifuging, loading the supernatant on a sephadex LH-20 chromatographic column, eluting with water, 80% ethanol and absolute ethanol respectively, and performing rotary evaporation and freeze-drying on the obtained 80% ethanol eluent to obtain the Botrytis cinerea polyphenol.
Advantageous effects
The invention has the beneficial effects that:
(1) The invention provides a preparation method of high-purity Botrytis cinerea polyphenol.
(2) The Botrytis cinerea polyphenol prepared by the method shows remarkable antithrombotic activity on a mouse animal model.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 method for determining extraction yield and purity of Polyphenol
The polyphenol content was measured by Folin-Ciocalteu (Folin-Ciocalteu) method, and gallic acid was used as a standard. The method comprises the following specific steps: precisely removing 0.1mg/mL gallic acid standard solution 0.0, 0.2, 0.4, 0.6, 0.8, 1.0mL, respectively adding2.5mL of 10% Fu Lin Fen reagent is added into a 10mL volumetric flask respectively, and the reaction is carried out for 5min in a dark place. Then 7.5% Na was added separately 2 CO 3 2mL of solution, mixing uniformly, and then respectively using distilled water to fix the volume to 10mL, and carrying out light-shielding reaction for 2h. The absorbance of each tube was measured at 765nm wavelength by zeroing with distilled water using an ultraviolet spectrophotometer. And drawing a Gallic Acid (GA) standard curve by taking the concentration of the gallic acid standard solution as an abscissa and the absorbance measured by each tube as an ordinate.
Determination of the extraction rate of the grape fern algae polyphenol: after the extract was diluted, 1mL of the diluted extract was added to a 10mL volumetric flask, and 2.5mL of 10% Fu Lin Fen reagent was added thereto to react for 5 minutes in the dark. Then 7.5% Na was added 2 CO 3 2mL of solution, mixing uniformly, then using distilled water to fix the volume to 10mL, and reacting for 2h in a dark place. Absorbance was measured at 765nm wavelength by zeroing with distilled water using a spectrophotometer. The polyphenol concentration was determined using the gallic acid standard curve equation, and the extraction rate was calculated according to the following formula.
Extraction yield (mg GAE/g) =c×nV/M
Wherein: GAE represents gallic acid equivalent; c represents polyphenol concentration (mg/mL); n represents dilution factor; v represents the volume (mL) of the extract; m represents the mass (g) of the seaweed dry powder.
The extract is subjected to vacuum freeze drying to obtain polyphenol solid extract. The dried product was weighed and dissolved in distilled water to prepare a sample solution. Polyphenol concentration, polyphenol purity (%) =polyphenol concentration×sample solution volume/polyphenol solid extract mass×100 was determined using the foliosol method.
Example 2 extraction Process of Polyphenol from Botrytis cinerea
The part adopts a single factor method to screen the extraction process of polyphenol.
(1) Investigation of ultrasound temperature
Weighing 2g of Botrytis cinerea powder, adding 60% ethanol according to the feed liquid ratio of 1:20 (g/mL), respectively extracting at 30 ℃,40 ℃,50 ℃, 60 ℃ and 70 ℃ by ultrasonic for 40min, filtering, taking supernatant, evaporating and concentrating at 40-50 ℃. And measuring the polyphenol concentration of the Botrytis cinerea in the concentrated solution, calculating the extraction rate, and examining the influence of temperature on the polyphenol extraction rate.
| Extraction temperature (. Degree. C.) | 30 | 40 | 50 | 60 | 70 |
| Extraction yield (mg GAE/g) | 0.3318 | 0.4342 | 0.5486 | 0.4858 | 0.4552 |
According to the above results, the extraction temperature was set to 50 ℃.
(2) Investigation of ethanol concentration
Weighing 2g of Botrytis cinerea powder, adding ethanol with different concentrations (60%, 70%, 80%, 90%, 95%) according to the feed liquid ratio of 1:20 (g/mL), carrying out ultrasonic extraction at 50 ℃ for 40min, carrying out suction filtration, taking supernatant, and evaporating and concentrating at 40-50 ℃. And measuring the polyphenol concentration of the Botrytis cinerea in the concentrated solution, calculating the extraction rate, and examining the influence of the ethanol concentration on the polyphenol extraction rate.
| Ethanol concentration (%) | 60 | 70 | 80 | 90 | 95 |
| Extraction yield (mg GAE/g) | 0.4858 | 0.5195 | 0.8460 | 0.6689 | 0.6667 |
From the above results, the ethanol concentration was determined to be 80%.
(3) Investigation of feed-to-liquid ratio
Weighing 2g of Botrytis cinerea powder, respectively adding 80% ethanol according to the feed liquid ratio of 1:10, 1:15, 1:20, 1:25 and 1:30 (g/mL), ultrasonically extracting at 50 ℃ for 40min, suction filtering, taking supernatant, and evaporating and concentrating at 40-50 ℃. And measuring the polyphenol concentration of the Botrytis cinerea in the concentrated solution, calculating the extraction rate, and examining the influence of the feed-liquid ratio on the polyphenol extraction rate.
| Feed liquid ratio (g/mL) | 1∶10 | 1∶15 | 1∶20 | 1∶25 | 1∶30 |
| Extraction yield (mg GAE/g) | 0.6876 | 1.122 | 0.8460 | 0.7750 | 0.6271 |
According to the result, the feed-liquid ratio is determined to be 1:15.
(4) Investigation of extraction time
Weighing 2g of Botrytis cinerea powder, adding 80% ethanol according to the feed liquid ratio of 1:15 (g/mL), respectively carrying out ultrasonic extraction for 20, 30, 40, 50 and 60min at 50 ℃, carrying out suction filtration, taking supernatant, and evaporating and concentrating at 40-50 ℃. And measuring the polyphenol concentration of the Botrytis cinerea in the concentrated solution, calculating the extraction rate, and examining the influence of the extraction time on the polyphenol extraction rate.
| Extraction time (min) | 20 | 30 | 40 | 50 | 60 |
| Extraction yield (mg GAE/g) | 0.5695 | 1.071 | 1.122 | 1.079 | 1.058 |
According to the result, the extraction time is determined to be 40min.
In conclusion, according to the single-factor experimental result, the optimal extraction process of the Botrytis cinerea polyphenol comprises the following steps: the extraction temperature is 50 ℃, the ethanol concentration is 80%, the feed-liquid ratio is 1:15, and the extraction time is 40min.
EXAMPLE 3 macroporous resin purification Process of Botrytis cinerea Polyphenol
Purifying the ultrasonic extracting solution by using macroporous resin.
(1) Macroporous resin pretreatment
The macroporous resin is soaked in twice amount of 95% ethanol for 24 hours to fully swell, the macroporous resin is filled with 95% ethanol and then is washed with 95% ethanol until effluent liquid is water without white turbidity, and distilled water is washed until no ethanol smell exists.
(2) Macroporous resin static experimental screening
Accurately sucking 0.066g crude drug/mL Botrytis cinerea polyphenol extract with 25mL volume, adding into conical flask with 2g pretreated dry resin plug, and shaking once every half hour to make resin fully adsorb components. After shaking 8 times, the mixture was allowed to stand for 3 hours. The mixture was filtered through 4 layers of gauze into a 100mL Erlenmeyer flask to obtain a filtrate. The polyphenol concentration of the filtrate was measured.
To the filtered resin, 25mL of 80% ethanol was added, and the mixture was shaken once every half an hour for 8 times and allowed to stand for 3 hours. The solution was filtered through a 4-layer gauze into a 100mL Erlenmeyer flask to obtain a desorption solution. The concentration of polyphenols in the desorption solution was determined.
The adsorption amount (mg), adsorption rate (%), desorption amount (mg) and desorption rate (%) of the macroporous resin are calculated according to the following formulas:
adsorption amount (mg) = (C) 0 -C 1 )V 1
Adsorption rate (%) = [ (C) 0 -C 1 )/C 0 ]×100％
Desorption amount (mg) =c 2 V 2
Desorption rate (%) = [ C 2 V 2 /(C 0 -C 1 )V 1 ]×100％
Wherein: c (C) 0 : initial total polyphenol concentration; c (C) 1 : the concentration of total polyphenol in the filtrate at the end of adsorption; c (C) 2 : total polyphenol concentration of desorption liquid; v (V) 1 : the volume of the extract; v (V) 2 : volume of desorption solution
The experimental results are shown in the following table:
| resin model | Adsorption Rate (%) | Desorption rate (%) |
| AB-8 | 56.91 | 28.67 |
| XDA-7 | 56.71 | 28.90 |
| BS-45 | 63.86 | 38.58 |
| NKA-9 | 55.49 | 26.23 |
| ADS-17 | 52.69 | 31.23 |
| ADS-21 | 53.10 | 23.54 |
| HPD100 | 55.54 | 28.35 |
| LS-305A | 57.68 | 34.01 |
| DA201 | 53.39 | 29.81 |
| XAD16N | 49.84 | 33.97 |
The results of the table show that the adsorption rate and the resolution rate of the BS-45 macroporous resin are the highest, and the BS-45 macroporous resin is selected for subsequent investigation.
(3) Dynamic adsorption diameter-height ratio investigation of macroporous resin
Four parts of BS-45 macroporous resin (17.376 g, 34.752g, 52.128g and 69.504 g) (equivalent to 5g, 10g, 15g and 20g of dry weight of macroporous resin) are weighed respectively, and pretreatment is carried out according to macroporous resin pretreatment. Is placed in four glass columns of the same inner diameter to have diameter to height ratios of about 1:1.4, 1:2.8, 1:4.2 and 1:5.6, respectively. The extract was loaded (BV, column volume) at a flow rate of 1BV/h with 0.066g crude drug/mL Botrytis cinerea polyphenol extract of 25mL, 50mL, 75mL, 100mL, respectively. And standing and adsorbing for 1h after the sample loading is finished. The resin column was eluted with 2BV distilled water at a rate of 2BV/h, and each was eluted with 2BV by 80% ethanol by volume at a flow rate of 2BV/h. And measuring the polyphenol content of the Botrytis cinerea in the eluent and calculating the transfer rate of the Botrytis cinerea.
The experimental results are shown in the following table
| Aspect ratio | Transfer Rate (%) |
| 1∶1.4 | 23.59 |
| 1∶2.8 | 28.09 |
| 1∶4.2 | 35.63 |
| 1∶5.6 | 30.13 |
As can be seen from the table results, the transfer rate of the polyphenol of the Botrytis cinerea is the highest when the diameter-to-height ratio is 1:4.2 by taking the transfer rate of the polyphenol of the Botrytis cinerea as an index, so that the next factor investigation is carried out by selecting the ratio of the diameter-to-height ratio to be 1:4.2.
(4) Dynamic adsorption loading quantity investigation of macroporous resin
Weighing up Kong Shula 52.128g of BS-45, and preprocessing according to macroporous resin preprocessing. 100mL of crude drug/mL of Botrytis cinerea polyphenol extracting solution with the concentration of 0.066g is measured, then the pretreated macroporous resin is loaded, the loading flow rate is 1BV/h, and one loading effluent is collected every 5 mL. And (5) measuring the polyphenol content of the effluent Botrytis cinerea and calculating the polyphenol leakage rate. The effluent is considered to be a leak when the mass of polyphenols in the effluent is about 10% of the mass of polyphenols in the loading solution, at which point the loading is the optimal loading.
Leakage rate (%) =effluent polyphenol mass/polyphenol mass in loading solution×100%;
cumulative leakage rate = Σleakagerate
The experimental results are shown in the following table:
| leakage liquid pipe number | Leakage Rate (%) | Cumulative leakage Rate (%) |
| 1～8 | 0 | 0 |
| 9 | 0.598 | 0.598 |
| 10 | 0.952 | 1.550 |
| 11 | 2.083 | 3.633 |
| 12 | 2.674 | 6.307 |
| 13 | 3.261 | 9.568 |
| 14 | 3.833 | 13.401 |
The accumulated leakage rate of polyphenols reached 9.568% when the loading liquid volume reached 65mL, and the mass of polyphenols in the effluent was considered to be about 1/10 of the mass of polyphenols in the loading liquid to be the leakage point, determining the optimal loading amount to be 65mL (0.72 BV).
(5) Dynamic adsorption water-washing volume investigation of macroporous resin
And eluting with distilled water to remove water-soluble impurities such as saccharides in the sample liquid, and detecting anthrone sulfuric acid reaction in the water eluting liquid to determine the volume of water required when the reducing sugar is completely eluted.
Weighing 52.128g of BS-45 macroporous resin, and preprocessing according to macroporous resin preprocessing. Measuring 65mL of crude drug/mL of Botrytis cinerea polyphenol extract with the concentration of 0.066g, loading the pretreated macroporous resin with the loading flow rate of 1BV/h, and standing for adsorption for 1h after loading. And then eluting the resin column by using 150mL of distilled water at the eluting speed of 2BV/h, collecting water eluent by using 10mL of distilled water as one part, and simultaneously detecting each part of water eluent by using an anthrone sulfuric acid reagent respectively until the reaction is negative (reducing sugar is completely eluted), and stopping eluting by using water to remove impurities. The experimental results are shown in the following table:
| sample name | Anthrone sulfuric acid reaction results |
| Aqueous eluent 1 | Positive and negative |
| …… | …… |
| …… | …… |
| Aqueous eluent 6 | Positive and negative |
| Aqueous eluent 7 | Negative of |
When the water elution amount was 70mL, the anthrone sulfuric acid reaction of the water elution solution showed a negative result, which proves that the impurity of the reducing sugar had been eluted, so the water elution amount was determined to be 70mL (0.78 BV) in the experiment.
(6) Macroporous resin dynamic adsorption 80% ethanol elution volume investigation
Weighing 52.128g of BS-45 macroporous resin, and preprocessing according to macroporous resin preprocessing. Measuring 65mL (0.72 BV) of crude drug/mL of Botrytis cinerea polyphenol extract with the concentration of 0.066g, loading the pretreated macroporous resin with the flow rate of 1BV/h, standing and adsorbing for 1h after loading, flushing the resin column with 70mL (0.78 BV) of distilled water with the flow rate of 2BV/h, eluting with 4BV of 80% ethanol, collecting the eluent with the flow rate of 1 part per 0.5BV, and collecting 8 parts. And measuring the polyphenol content in each eluent, and calculating the polyphenol transfer rate.
Transfer rate (%) =mass of polyphenol in eluent/mass of polyphenol in loading solution×100%
The experimental results are shown in the following table:
| elution volume (BV) | Cumulative transfer Rate (%) |
| 0.5 | 17.07 |
| 1 | 29.77 |
| 1.5 | 37.17 |
| 2 | 41.29 |
| 2.5 | 44.30 |
| 3 | 44.69 |
| 3.5 | 43.88 |
| 4 | 44.57 |
As a result, the polyphenol elution capacity was maximized when using an 80% ethanol elution volume of 2.5BV, and the subsequent basic equilibrium was determined, i.e., the optimal elution volume was 2.5BV.
Summarizing macroporous resin purification process: BS-45 resin with the diameter-to-height ratio of 1:4.2, the eluting solvent of 80% ethanol, the loading flow rate of 1BV/h, the loading concentration of 0.066g crude drug/mL, the loading volume of 0.72BV, the standing adsorption of 1h, the water washing volume of 0.78BV, the eluting flow rate of 2BV/h and the eluting solvent volume of 2.5BV (BV represents column volume).
(7) Sample preparation
By adopting the process, the polyphenol sample liquid is prepared repeatedly for many times by amplifying for 20 times, and is concentrated by rotary evaporation, freeze-dried and freeze-dried powder is used for subsequent further purification.
EXAMPLE 4 Sephadex LH-20 was further purified
Taking 100mg of the freeze-dried powder of the grape fern algae polyphenol purified by the macroporous resin, dissolving in 5mL of water, centrifuging at 12000 Xg low temperature (4 ℃) for 5 minutes, and putting the supernatant on a Sephadex LH-20 gel column drop by drop.
After the sample permeates the gel bed, sequentially eluting with 100mL of distilled water, 80% ethanol and absolute ethanol at a flow rate of 0.7mL/min, collecting 1 tube per 10mL, measuring polyphenol concentration by using a Fu Lin Fen method, drawing a curve by taking the number of the tubes as an abscissa and the polyphenol concentration as an ordinate, and observing the eluting condition of the polyphenol. Mixing 1-10 tubes, 11-20 tubes and 21-30 tubes respectively, concentrating by rotary evaporation, freeze-drying, and measuring the weight of the freeze-dried powder and the purity of polyphenol. Purity (%) = polyphenol mass/lyophilized powder mass x 100%
Experimental results show that the purity of the Botrytis cinerea polyphenol in the 80% ethanol phase is highest and is 65.98%, so that the 80% ethanol phase component is selected for the following study.
EXAMPLE 5 antithrombotic Activity Studies
(1) Pulmonary thrombosis model
Mouse pulmonary thrombosis modeling agent: 0.5mg/mL type I collagen and 0.05mg/mL epinephrine hydrochloride.
Male ICR mice were taken in 50 groups of 10 randomly. The compositions were divided into a blank group, a model group, an aspirin positive control group (administration dose: 20 mg/kg), a polyphenol low dose group (administration dose: 20 mg/kg), and a polyphenol high dose group (administration dose: 40 mg/kg). Mice were dosed at a volume of 20mL/kg, 1 time per day for 5 consecutive days, and the blank and model groups were dosed with equal volumes of saline. After the last 2h of administration, the volume of the mouse tail intravenous injection molding agent was 10mL/kg except for the blank group. And observing death condition within 15min after the mice are molded, and calculating the survival rate of the mice after 15min after the mice are molded.
The survival rate of mice within 15min after molding is shown in the following table:
| group of | Number of deaths (only) | Survival number (only) | Survival (%) |
| Blank group | 0 | 10 | 100％ |
| Model group | 10 | 0 | 0 |
| Aspirin positive control group | 5 | 5 | 50 |
| Low dosage set of polyphenols | 6 | 4 | 40 |
| High dosage set of polyphenols | 5 | 5 | 50 |
(2) Mouse tail thrombus model
Male ICR mice were taken in 50 groups of 10 randomly. The compositions were divided into a blank group, a model group, an aspirin positive control group (administration dose: 20 mg/kg), a polyphenol low dose group (administration dose: 20 mg/kg), and a polyphenol high dose group (administration dose: 40 mg/kg). Mice were dosed 1 time a day for 7 consecutive days at a volume of 20mL/kg by gavage, and the blank and model groups were perfused with an equal volume of physiological saline. After 1 hour of dosing on the last day, the back of each group of mice except the blank group was subcutaneously injected with 0.1% carrageenan-producing agent. The tail length and tail thrombus formation length of the mice are measured after 24 hours of molding, and the ratio of the thrombus formation length to the total tail length is the relative average thrombus formation length, namely the black tail rate (%).
| Group of | Black tail ratio (%) |
| Blank group | 0 |
| Model group | 84.8±3.1 |
| Aspirin positive control group | 44.9±2.6*** |
| Low dosage set of polyphenols | 67.4±3.1*** |
| High dosage set of polyphenols | 55.3±2.9*** |
Note that: ratio to model group, P < 0.001
From the above results, it is clear that the Botrytis cinerea polyphenol prepared by the present invention has a remarkable antithrombotic effect.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (1)
1. The application of the Botrytis cinerea polyphenol in preparing the medicine for treating thrombus is characterized in that the preparation method of the Botrytis cinerea polyphenol comprises the following steps:
(1) Freeze-drying fresh Botrytis cinerea for 48 hours, shearing the Botrytis cinerea, and then putting the Botrytis cinerea into a pulverizer and sieving the Botrytis cinerea with a 60-mesh sieve to obtain Botrytis cinerea powder;
(2) Accurately weighing Botrytis cinerea powder, adding 80% ethanol water solution according to a feed-liquid ratio of 1:15, fully mixing and soaking;
(3) Extracting polyphenol with a numerical control ultrasonic cleaner for 40min at 50deg.C, and vacuum filtering to obtain crude extract of Botrytis cinerea polyphenol;
(4) Performing primary purification on the crude extract of the Botrytis cinerea polyphenol by using macroporous resin BS-45, loading the sample at a flow rate of 1BV/h, standing for adsorption for 1h, washing with deionized water of 0.78BV to remove impurities, eluting with 80% ethanol of 2.5BV, collecting the eluent, concentrating by vacuum rotary evaporation at 40-50 ℃ and freeze-drying to obtain Botrytis cinerea polyphenol powder;
(5) Taking the Botrytis cinerea polyphenol purified by macroporous resin, precisely weighing, ultrasonically dissolving with a small amount of water, centrifuging, loading the supernatant on a sephadex LH-20 chromatographic column, eluting with water, 80% ethanol and absolute ethanol respectively, and performing rotary evaporation and freeze-drying on the obtained 80% ethanol eluent to obtain the Botrytis cinerea polyphenol.
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