CN107163013B - Method for preparing osthole from plant and application of osthole in preparing cosmetics - Google Patents
Method for preparing osthole from plant and application of osthole in preparing cosmetics Download PDFInfo
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- CN107163013B CN107163013B CN201710343858.1A CN201710343858A CN107163013B CN 107163013 B CN107163013 B CN 107163013B CN 201710343858 A CN201710343858 A CN 201710343858A CN 107163013 B CN107163013 B CN 107163013B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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Abstract
The invention discloses a method for preparing osthole from plants and application of osthole in preparing cosmetics. The method comprises the steps of pretreating the fruit part of the plant containing the osthole, and then carrying out ethanol extraction, liquid-residue separation, crude osthole precipitation, ethanol crystallization and recrystallization with accurate concentration and the like to obtain the osthole monomer active ingredient. The method provided by the invention can effectively remove impurities and has high osthole transfer rate. The obtained components have antibacterial, antiinflammatory, parasite killing, itching relieving, antiallergic, spasmolytic, blood pressure lowering, arrhythmia resisting, and immunity enhancing effects, and can be prepared into emulsifying system and surfactant system for use as skin caring product with antiallergic, parasite killing, itching relieving, antibacterial, antiinflammatory, and acne removing effects.
Description
Technical Field
The invention belongs to the field of medicines and cosmetics, and particularly relates to a method for preparing osthole from plants and application of osthole in preparing cosmetics.
Background
Osthole, also known as methoxy-parsley phenol, or parsley methyl ether, or Woodward (Ost), is mainly present in Umbelliferae and Rutaceae plants, is the main active ingredient in dried mature fruit of Umbelliferae plant, Cnidium monnieri (L.) Cuss. or Cnidiumformisanum YABE, and is chemically named 7-methoxy-8-isopentenyl coumarin (7-methoxy-8- [3-methyl pent-2-eny 1)]columin) is the most abundant one of the effective chemical components of alkyl coumarins in fructus Cnidii, and is colorless columnar crystal with molecular formula C15H16O3The relative molecular mass is 244.29, and the melting point is 82-84 ℃. Pharmacological experimental research of osthole shows that: osthole has effects in resisting hypertension, arrhythmia, aging, tumor, osteoporosis, inflammation and allergy, and enhancing immunity. In the aspect of daily chemical external application, the osthole has the anti-allergic reaction effect, has stronger antihistamine effect, can obviously antagonize histamine and chronic reaction substances, and has stronger antiallergic effect; has good treatment and health care effects on various allergies, skin itch, psoriasis, herpes zoster and eczema; in addition, the osthole has strong pathogenic microorganism resisting effect, has inhibiting effect on Epidermophyton floccosum, Paecilomyces gypseus, Paecilomyces lanuginosus and broad-spectrum bacteria, has strong killing effect on trichomonas vaginalis, can inhibit the formation of various inflammatory factors, and has good antibacterial, anti-inflammatory, parasite killing, itching relieving and acne removing effects.
In order to meet the increasing demand of osthole, the preparation research of osthole has been developed rapidly, but all have obvious defects. For example, the invention patent CN201110151743.5 discloses a process method for preparing osthole and isoanetholide from fructus cnidii; the method comprises subjecting fructus Cnidii powder to supercritical CO2Extracting to obtain fructus Cnidii extract, and separating with polyamide resinPurifying to obtain crude product, and further purifying by high speed countercurrent chromatography to obtain pure osthole and isoanetholide; the process has the advantages of high equipment cost and high production cost, and is not suitable for large-scale industrial production. For another example, patent CN201010182729.7 discloses a "process for extracting osthole", in which microfiltration, ultrafiltration, nanofiltration of petroleum ether crystallization, and repeated recrystallization with absolute ethanol are employed, and the process is relatively complex and costly, and the petroleum ether is easy to produce potential safety hazard when used in large-scale industrial production. In addition, for example, patent CN 201010531931.6 discloses a method for preparing osthole, in the method, strong alkali such as NaOH and KOH is used for treatment, and the strong alkali is easy to change the structure of osthole to generate side reaction. Also, for example, the invention patent CN201010527673.4 discloses "a method for extracting osthole from traditional Chinese medicine fructus cnidii", which is simple and suitable for large-scale industrial production; however, the process method uses an ethanol solution with the concentration of more than 90% for crystallization and recrystallization, and because the 90% ethanol solution has strong solubility to osthole and impurities, the recrystallization is required for a plurality of times, and the influence on the yield of the product is large. The method for extracting and separating the osthole can overcome the defects, and the method for producing the osthole has the advantages of high yield, simple operation, low energy consumption and easy realization of industrial amplification, and has not been reported at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for preparing osthole from plants.
Another object of the present invention is to provide osthole prepared by the above method.
Still another object of the present invention is to provide the above method and use of osthole in the preparation of cosmetics.
The purpose of the invention is realized by the following technical scheme: a method for preparing osthole from plants comprises the following steps:
(1) extracting osthole: selecting fruits of Umbelliferae plants, cleaning, drying, and pulverizing to obtain coarse powder; heating and extracting or heating, warm dipping and ultrasonic extracting by using 65-95% ethanol solution by volume percentage; after extraction, carrying out solid-liquid separation to obtain an extract containing osthole;
(2) separating the crude cnidium lactone: taking an extract containing osthole, and recovering ethanol until the ethanol is completely recovered to obtain a concentrated solution; adding hot water with the temperature of 40-80 ℃ into the concentrated solution for dissolving, then cooling, standing at the temperature of 1-25 ℃ for more than 24 hours, carrying out solid-liquid separation, removing the liquid, and collecting the solid to obtain a crude extract containing osthole;
(3) refining and purifying osthole monomer component through step ① or steps ① and ②;
① crystallizing for the first time, adding 50-60 vol% ethanol water solution into the crude extract of osthole, heating to dissolve, cooling to room temperature, standing for the first time, separating solid and liquid, collecting liquid, discarding solid, standing the liquid obtained from solid-liquid separation at 0-10 deg.C for the second time until crystal is fully separated out, separating solid and liquid, discarding liquid, and collecting crystal;
② and recrystallizing the obtained crystal according to step ① to obtain refined and purified crystal of osthole.
The Umbelliferae plant of step (1) is preferably Cnidium monnieri (L.) Cusson or Cnidium formosanum YABE.
The coarse powder in the step (1) is preferably coarse powder with 15-20 meshes.
The ethanol solution in the step (1) is preferably an ethanol solution with the concentration of 85-90% by volume percentage.
The heating extraction times in the step (1) are preferably 2-3 times; the specific steps are preferably as follows: when the extraction times are 2 times: adding ethanol solution with volume (L) which is 10 times of the mass (kg) of the coarse powder for the first time, fully stirring and uniformly mixing, and performing reflux extraction at 70-100 ℃ for 1-3 hours; adding ethanol solution with volume equivalent to 8 times of the mass of the coarse powder for the second time, and performing reflux extraction at the temperature of 70-100 ℃ for 0.5-2 hours; when the extraction times are 3 times: adding ethanol solution with volume equivalent to 8 times of the mass of the coarse powder for the third time when the times of the first two times of extraction are 2 times, and carrying out reflux extraction at 70-100 ℃ for 0.5-2 hours; after extraction, carrying out solid-liquid separation, and combining the liquid obtained by 2-3 times of separation to obtain an osthole-containing extracting solution; the specific steps are more preferably as follows: when the extraction times are 2 times: adding ethanol solution with volume (L) equivalent to 10 times of the mass (kg) of the coarse powder for the first time, fully stirring and uniformly mixing, and performing reflux extraction at 80 ℃ for 2 hours; adding ethanol solution with volume amount of 8 times of the coarse powder for the second time, and performing reflux extraction at 80 ℃ for 1.5 hours; when the extraction times are 3 times: adding ethanol solution with volume equivalent to 8 times of the coarse powder mass for the third time when the times of the first two times of extraction are 2 times, and performing reflux extraction at 80 ℃ for 1.5 hours; and after extraction, performing solid-liquid separation, and combining the liquid obtained by 2-3 times of separation to obtain the osthole-containing extracting solution.
The ultrasonic condition in the heating warm immersion ultrasonic extraction in the step (1) is preferably 200-300 Hz; more preferably 250 Hz.
The frequency of the heating warm-dipping ultrasonic extraction in the step (1) is preferably 2-3 times; the specific steps are preferably as follows: when the extraction times are 2 times: adding ethanol solution with volume (L) which is 10 times of the mass (kg) of the coarse powder for the first time, fully stirring and uniformly mixing, and carrying out warm immersion ultrasonic extraction at the temperature of 60-80 ℃ for 0.5-2 hours; adding ethanol solution with volume amount being 8 times of the mass of the coarse powder for the second time, and carrying out warm immersion ultrasonic extraction at the temperature of 60-80 ℃ for 0.5-1.5 hours; when the extraction times are 3 times: adding ethanol solution with volume equivalent to 8 times of the mass of the coarse powder for the third time when the times of the previous two times of extraction are 2 times, and carrying out warm immersion ultrasonic extraction for 0.5-1.5 hours at the temperature of 60-80 ℃; after extraction, carrying out solid-liquid separation, and combining the liquid obtained by 2-3 times of separation to obtain an osthole-containing extracting solution; the specific steps are more preferably as follows: when the extraction times are 2 times: adding ethanol solution with volume (L) equivalent to 10 times of the mass (kg) of the coarse powder for the first time, fully stirring and uniformly mixing, and carrying out warm immersion ultrasonic extraction at 60 ℃ for 1.5 hours; adding ethanol solution with volume amount of 8 times of the coarse powder mass for the second time, and soaking at 60 deg.C and ultrasonic extracting for 1 hr; when the extraction times are 3 times: adding ethanol solution with volume equivalent to 8 times of the coarse powder mass for the third time when the times of the previous two times of extraction are 2 times, and carrying out warm immersion ultrasonic extraction for 1 hour at 60 ℃; and after extraction, performing solid-liquid separation, and combining the liquid obtained by 2-3 times of separation to obtain the osthole-containing extracting solution.
The hot water in the step (2) is preferably hot water with the temperature of 50-70 ℃; more preferably hot water at 60 c.
The addition amount of the hot water in the step (2) is preferably calculated according to the volume of 1-2L of the extract concentrated solution obtained by adding 1kg of raw materials (fruits of Umbelliferae plants) into the hot water; more preferably, the volume of the hot water added is 1.5-1.8L.
The cooling in the step (2) is preferably natural cooling.
The degree of cooling in step (2) is preferably to below 30 ℃; more preferably to 25 ℃.
The standing condition in the step (2) is preferably to be 2-8 ℃ for 24-30 h; more preferably still standing for 26-28 h at 3-6 ℃.
The concentration of the ethanol aqueous solution in the step (3) ① is preferably 53-57% by volume, and more preferably 55-56% by volume.
The addition amount of the ethanol aqueous solution in the step (3) ① is preferably calculated according to the proportion of 1-3L ethanol aqueous solution to the crude extract obtained from 1kg raw material (fruits of Umbelliferae plants), and more preferably calculated according to the proportion of 2L ethanol aqueous solution.
The room temperature in the step (3) ① is 5-35 ℃, preferably 10-30 ℃, more preferably 20-28 ℃, and most preferably 25 ℃.
The time for the first standing in the step (3) ① is preferably 7-8 hours.
The time for the second standing in step (3) ① is preferably 48 hours or more, more preferably 72 hours.
The temperature of the second standing in the step (3) ① is preferably 0-8 ℃, and more preferably 1-2 ℃.
The number of recrystallization in the step (3) ② is preferably 1, and the purity content of the obtained refined and purified osthole crystals is not less than 98% (w/w).
The solid-liquid separation in the present invention may be performed by centrifugation or by filtration.
The method is applied to the preparation of cosmetics.
A refined osthole is prepared by the above method, and has purity content of 98% (w/w) or more.
The refined osthole is applied to the preparation of cosmetics.
The cosmetic has antiallergic, antibacterial, antiinflammatory, and acne removing effects.
Antiallergic agent is prepared from osthole and can inhibit histamine release, strongly inhibit passive skin anaphylaxis, significantly inhibit delayed hypersensitivity, and has antiallergic and antipruritic effects; the antibacterial anti-inflammatory and acne removing means that cosmetics prepared from osthole have strong antibacterial and anti-inflammatory effects, and can inhibit staphylococcus aureus, staphylococcus aureus resistant bacteria, escherichia coli, shigella dysenteriae, salmonella typhimurium, pseudomonas aeruginosa and other bacteria; can obviously inhibit various acute and chronic inflammations, and has good antibacterial, anti-inflammatory and acne removing effects.
The application of the refined osthole in preparing cosmetics comprises the following steps: dissolving or heating to dissolve the osthole in pharmaceutically acceptable excipient or carrier, and mixing with other components.
The content of the refined osthole in the cosmetic is therapeutically effective amount.
The cosmetically acceptable pharmaceutical excipients or carriers for the preparation may be selected from: solvents, solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel modifiers, surfactants, emulsifiers, propellants/propellants, fragrances, pigments, and other performance additives.
The cosmetic is in the form of emulsifying system, surfactant system, topical liniment, and ointment.
In order to illustrate the beneficial effects of the osthole prepared from plants and the application of the osthole in cosmetics, staphylococcus aureus resistant bacteria, escherichia coli, shigella dysenteriae, salmonella typhimurium and pseudomonas aeruginosa are used as research objects, and the prepared osthole has remarkable antibacterial and anti-inflammatory effects through the experiment of a drug sensitive paper method and a two-fold dilution method; the osthole of the invention is proved to have obvious anti-inflammatory and analgesic effects by taking a male mouse as a research object and carrying out an experiment of a mouse otochitis inflammation-causing method; mice and rats are taken as research objects, and the osthole has obvious antiallergic effect by inhibiting Passive Cutaneous Anaphylaxis (PCA) reaction of the mice;
compared with the prior art, the invention has the following advantages and effects:
(1) the invention relates to a method for preparing osthole from plant cnidium fruit, which overcomes the defect of the prior CO prepared by osthole2The defects of the methods such as a supercritical fluid extraction method, an acid-base treatment method, a petroleum ether treatment crystallization method, a high-concentration alcohol treatment crystallization method and the like are that according to the properties and characteristics of the osthole, a new method for removing impurities and crystals by 50-60% of medium-concentration alcohol treatment is invented, and the content and activity of the osthole are retained to the maximum extent.
(2) In the method for preparing osthole from plant cnidium fruit, the osthole monomer component is refined and purified, and the method for adding ethanol aqueous solution with medium concentration for hot dissolution, removing impurities at room temperature and refrigerating crystallization is invented for the first time, so that not only can the impurities be effectively removed, but also the transfer rate of osthole is high (about 88%).
(3) Because osthole has poor water solubility, the osthole is not beneficial to being directly applied to cosmetics, and is often processed in an inclusion mode, otherwise, the prepared cosmetics are easy to generate mud-scooping feeling and active ingredients are difficult to be absorbed by skin. The invention skillfully utilizes the characteristics of the osthole and the multiphase composition of the cosmetic preparation, the osthole is firstly dissolved or heated and dissolved in fat-soluble excipient or carrier allowed by the preparation of the cosmetic, and the osthole does not need to be included, thus overcoming the defects and greatly saving the cost.
(4) The method provided by the invention has the advantages of simple process flow, convenient operation, convenient product quality control and operability, and is a safe and reliable preparation method suitable for industrial production.
Drawings
FIG. 1 is a photographic picture of Cnidium lactone prepared in example 1 from fruit of Cnidium monnieric (L.) cusson.
FIG. 2 is an HPLC chromatogram of osthole prepared from fruit of Cnidium monnieri (L.) Cusson of example 1; wherein panel A is a reference Cnidium lactone and panel B is a sample of Cnidium lactone prepared from fruit of Cnidium monnieric (L.) cusson.
FIG. 3 is a photograph showing the Cnidium fruit essence prepared from Cnidium formosanum YABE in example 2.
FIG. 4 is an HPLC chromatogram of osthole prepared from Cnidium formosanum YABE of example 2; wherein, the picture A is a osthole reference substance, and the picture B is a osthole sample prepared in the osthole Cnidium formosanum YABE.
FIG. 5 is a photographic picture of Cnidium lactone prepared in example 1 from fruit of Cnidium monnieric (L.) cusson.
FIG. 6 is an HPLC chromatogram of osthole prepared from fruit of Cnidium monnieri (L.) Cusson of example 1; wherein panel A is a reference Cnidium lactone and panel B is a sample of Cnidium lactone prepared from fruit of Cnidium monnieric (L.) cusson.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
Method for extracting and preparing osthole-I from fruit of Cnidium monnieri (L.) Cusson
(1) Extraction of osthole
Fruit of Cnidium monnieri (L.) cusson is used as raw material, cleaned, dried and crushed into 20-mesh coarse powder. Taking 1000g of fructus cnidii coarse powder, adding 10 times (10L) of 85 vol% ethanol aqueous solution, stirring thoroughly, heating to 80 deg.C, reflux-extracting for 2 hr, filtering, and collecting filtrate; adding 8 times (8L) of 85% (v/v) ethanol water solution into the filter residue, stirring, heating to 80 deg.C, reflux extracting for 1.5 hr, and filtering to obtain filtrate; adding 8 times (8L) of 85% (v/v) ethanol water solution into the residue, stirring, heating to 80 deg.C, reflux-extracting for 1.5 hr, filtering, and mixing the filtrate with the previous two filtrates to obtain osthole extractive solution; the filter residue is discarded.
(2) Separation of crude cnidium lactone
Collecting the above osthole extractive solution, recovering ethanol under reduced pressure to obtain concentrated solution (about 800mL), adding 60 deg.C hot water to adjust to 1500mL solution, stirring, cooling to room temperature, standing in about 5 deg.C cold storage chamber for 28 hr, centrifuging at 4000r/min for 20min, discarding supernatant, and collecting precipitate to obtain about 185g of osthole crude extract precipitate.
(3) Purification of osthole monomer component
Precipitating the crude extract of osthole, adding 55% (v/v) ethanol water solution 2000ml, heating at 60 deg.C, stirring for dissolving, filtering, discarding precipitate, cooling the filtrate to room temperature (about 25 deg.C), standing for 8 hr, filtering, discarding precipitate; placing the filtrate in a refrigerating chamber at 2 ℃, standing for 72 hours for crystallization, filtering and separating again, discarding the filtrate, and collecting crystals to obtain 22.8g of osthole crude crystals; the obtained crystal was further treated and recrystallized once as described above to obtain 20.3g of purified and purified osthole. The purity content of osthole is 98.9% determined by determination method of osthole content in pharmacopoeia fructus Cnidii, and its osthole photo graph and determination chromatogram are shown in figure 1 and figure 2 respectively. The osthole content in the raw material fructus cnidii (fructus cnidii) is 2.31%, and the osthole transfer rate is 87.9%.
Comparative example 1
According to the method of the invention patent CN201010527673.4 'A method for extracting osthole from fructus Cnidii' in example 3 of the invention, fruit of Cnidium monnieri (L.) Cusson is used as raw material, 1000g of fructus Cnidii coarse powder is taken to extract and prepare osthole, 10.2g of refined and purified crystal of osthole is obtained, the purity content of osthole is 99.1%, and the osthole transfer rate is 44.2%.
Example 2
Method for extracting and preparing osthole from fruit of Cnidium formosanum YABE
(1) Extraction of osthole
Fruits of Cnidium fortinum YABE are used as raw materials, cleaned, dried and crushed into coarse powder of about 18 meshes. Taking 1000g of fructus cnidii coarse powder, adding 10 times (10L) of 85% (v/v) ethanol aqueous solution, fully stirring and uniformly mixing, heating to 80 ℃, performing reflux extraction for 2 hours, and filtering to obtain filtrate for later use; adding 8 times (8L) of 85% (v/v) ethanol water solution into the filter residue, stirring, heating to 80 deg.C, reflux extracting for 1.5 hr, and filtering to obtain filtrate; adding 8 times (8L) of 85% (v/v) ethanol water solution into the residue, stirring, heating to 80 deg.C, reflux-extracting for 1.5 hr, filtering, and mixing the filtrate with the previous two filtrates to obtain osthole extractive solution; the filter residue is discarded.
(2) Separation of crude cnidium lactone
Collecting the above osthole extractive solution, recovering ethanol under reduced pressure to obtain concentrated solution (about 750mL), adding 60 deg.C hot water to adjust to 1500mL solution, stirring, cooling to room temperature, standing in about 5 deg.C cold storage chamber for 26 hr, centrifuging at 4000r/min for 20min, discarding supernatant, and collecting precipitate to obtain about 181g of osthole crude extract precipitate.
(3) Purification of osthole monomer component
Precipitating the crude extract of osthole, adding 55% (v/v) ethanol water solution 2000ml, heating at 60 deg.C, stirring for dissolving, filtering, discarding precipitate, cooling the filtrate to room temperature (about 25 deg.C), standing for 7 hr, filtering, discarding precipitate; placing the filtrate in a refrigerating chamber at 1 deg.C, standing for 72 hr for crystallizing, filtering for separating, discarding filtrate, and collecting crystals to obtain 21.3g of osthole crude crystals; the obtained crystal was further treated and recrystallized once as described above to obtain 19.7g of purified and purified crystals of osthole. The purity of osthole is 99.1% according to determination method of osthole in pharmacopeia fructus Cnidii, and its picture and chromatogram are shown in figure 3 and figure 4 respectively. The osthole content in the raw material fructus cnidii (fructus cnidii) is 2.27%, and the osthole transfer rate is 86.8%. The picture and the chromatogram of determination of osthole are shown in FIG. 3 and FIG. 4, respectively.
Comparative example 2
According to the method of the invention patent CN201010527673.4 'method for extracting osthole from traditional Chinese medicine fructus Cnidii' in example 3, fruit of plant Cnidium monnieri (Cnidium fruit) Yabe is used as raw material, 1000g of fructus Cnidii coarse powder is taken to extract and prepare osthole, 10.8g of refined and purified crystal of osthole is obtained, and the purity content of osthole is measured to be 98.8%, and the osthole transfer rate is measured to be 47.6%.
Example 3
Method for extracting osthole II from fruit of Cnidium monnieri (L.) Cusson
(1) Extraction of osthole
Fruit of Cnidium monnieri (L.) cusson is used as raw material, cleaned, dried and crushed into coarse powder of 15 meshes. Taking 1000g of fructus cnidii coarse powder, adding 10 times (10L) of 90% (v/v) ethanol aqueous solution, fully stirring and uniformly mixing, heating to 60 ℃, performing ultrasonic extraction (250Hz) for 1.5 hours, and filtering to obtain filtrate for later use; adding 8 times (8L) of 90% (v/v) ethanol water solution into the filter residue, stirring, heating to 60 deg.C, extracting with ultrasound (250Hz) for 1.0 hr, and filtering to obtain filtrate; adding 8 times (8L) of 90% (v/v) ethanol water solution into the residue, stirring, heating to 60 deg.C, ultrasonic extracting at 250Hz for 1.0 hr, filtering, and mixing the filtrate with the filtrate of the previous and second times to obtain osthole extractive solution; the filter residue is discarded.
(2) Separation of crude cnidium lactone
Collecting the above osthole extractive solution, recovering ethanol under reduced pressure to obtain concentrated solution (about 700mL), adding 60 deg.C hot water to adjust to 1800mL solution, stirring, cooling to room temperature, placing in 5 deg.C cold storage chamber, standing for 28 hr, centrifuging at 4000r/min for 20min, removing supernatant, and collecting precipitate to obtain about 173g of osthole crude extract precipitate.
(3) Purification of osthole monomer component
Precipitating the crude extract of osthole, adding 2000ml ethanol water solution with concentration of 56% (v/v), heating at 60 deg.C, stirring for dissolving, filtering, discarding precipitate, cooling the filtrate to room temperature (about 25 deg.C), standing for 8 hr, filtering, discarding precipitate; placing the filtrate in a refrigerating chamber at 1 deg.C, standing for 72 hr for crystallizing, filtering for separating, discarding filtrate, and collecting crystal to obtain osthole coarse crystal 22.4 g; the obtained crystal was further treated and recrystallized once as described above to obtain 20.1g of purified and purified crystals of osthole. The purity content of osthole is 99.4% according to determination method of osthole in pharmacopeia fructus Cnidii, and its osthole photo graph and determination chromatogram are shown in FIG. 5 and FIG. 6 respectively. The osthole content in the raw material fructus cnidii (fructus cnidii) is 2.31%, and the osthole transfer rate is 87.0%.
Example 4: the invention relates to a research on the external bacteriostatic action of osthole extracted and separated by the method
Cnidium fruit is the fruit of Cnidium (Cnidium monnieri (L.) Cusson.) which is a plant of Cnidium of Umbelliferae, and Cnidium fruit is often referred to as Cnidium fruit. Another is Cnidium formosanum YABE. The two kinds of common cnidium fruit have been grown in various places in China, but one kind of common cnidium fruit has been grown in the past. Jiangsu and Anhui provinces are the main origins of cnidium fruit. The cnidium fruit is used as an external medicine since ancient times, the ancient medical works of China have little record about the application of the cnidium fruit, and the cnidium fruit is mainly externally used for treating skin diseases, for example, the Shen nong Ben Cao Jing has the main treatment on tinea pedis and malignant boil; the formula is recorded in Ji Jian Fang, which is recorded as the recipe of woman pruritus vulvae, the powder of fructus cnidii and lard for application; ben Cao gang mu records that it can remove yin sweat and damp tinea, and is mainly indicated for trichomonas vaginalis and eczema of skin. The osthole has good antibacterial and anti-inflammatory effects, can inhibit staphylococcus aureus, staphylococcus aureus drug-resistant bacteria, escherichia coli, shigella dysenteriae, salmonella typhimurium, pseudomonas aeruginosa and other bacteria, and has wide antibacterial spectrum, and the experimental method and the results are as follows:
1 materials and methods
1.1 instruments
DY6000 II electric heating constant temperature incubator (Tianjin Tester instruments, Ltd.); HT-20-5MT double super clean bench (Suzhou Antai air technologies, Inc.); micropipettes (Shanghai thermoelectric instruments, Inc.); LDZX-50K autoclave (Shanghai Sanshen medical instruments, Inc.).
1.2 reagent
Osthole is extracted from fruit of plant Cnidium monnieri (L.) Cus-son according to the method of the present invention, and its purity is 98.9% as determined by HPLC control of osthole reference substance and determination method of osthole content in pharmacopeia Cnidium fruit, and its photograph and chromatogram are shown in fig. 1 and fig. 2 respectively.
1.3 bacterial species
Staphylococcus aureus (CMCC26003), Staphylococcus aureus resistant bacteria (S4050), Escherichia coli (JM110), Shigella dysenteriae (CMCC51252), Salmonella typhimurium (CMCC50115), and Pseudomonas aeruginosa (CMCC10104) were purchased from Guangdong province culture Collection of microorganisms.
2 methods and results
2.1 preparation of osthole fat emulsion solution and blank fat emulsion solution
2.1.1 preparation of an osthole fat emulsion solution
Adding 1.5g of the osthole prepared in the example 1 into 15ml of soybean oil, heating in a water bath at 60 ℃ until the osthole is completely dissolved, adding 1.5g of soybean phospholipid into the soybean oil under the condition of introducing nitrogen, mashing at high speed until the soybean phospholipid is completely dissolved, and keeping the temperature at 60 ℃ to obtain an oil phase; putting 50mL of purified water into a container, slowly adding 15g of glycerol while stirring, heating to 60 ℃, and uniformly stirring to obtain a water phase; slowly adding the oil phase into the water phase under the conditions of constant temperature of 60 deg.C and high-speed dispersion emulsification at 12000rpm, mixing, adding purified water to adjust to 100mL, homogenizing in a homogenizer at 60Mpa and 60 deg.C for 10 times, filtering with 350 mesh filter to obtain 15 mg/mL-1And (4) preparing the osthole fat emulsion solution for later use.
2.1.2 preparation of blank fat emulsion solution
Adding soybean phospholipid 1.5g into 15ml soybean oil under nitrogen gas, mashing at high speed of 12000rpm until soybean phospholipid is completely dissolved, and maintaining the temperature at 60 deg.C to obtain oil phase; putting 50mL of purified water into a container, slowly adding 15g of glycerol while stirring, heating to 60 ℃, and uniformly stirring to obtain a water phase; slowly adding the oil phase into the water phase under the conditions of constant temperature of 60 ℃ and high-speed dispersion emulsification at 12000rpm, mixing uniformly, adding purified water to adjust to 100mL, putting into a homogenizer, adjusting the homogenization pressure to 60Mpa, the homogenization temperature to 60 ℃, homogenizing for 10 times, and filtering by a 350-mesh filter to obtain an empty fat emulsion solution for later use.
2.2 preparation of bacterial liquid
And respectively inoculating single colonies of different strains into LB culture solution, and carrying out shake cultivation at a constant temperature of 37 ℃ for 18-24 h. Taking out a small amount of bacteria solution, and diluting with sterilized 0.9% sodium chloride solution to 10%7～108CFU·mL-1
2.3 bacteriostatic experiments
2.3.1 method of drug sensitive paper
Preparing the drug sensitive paper sheets containing osthole with different concentrations and with the diameter of 5 mm. Coating 100 mu L of different strains on an LB culture medium, after the surface of the culture medium is dried, sticking paper sheets on the surface of the culture medium, culturing for 24h at 37 ℃, observing and measuring the diameter of a bacteriostatic ring, repeating for 3 times, and using blank fat emulsion solution paper sheets for a negative control group.
2.3.2 two-fold dilution method
2.0mL of the culture medium required by each strain, 8 tubes for each strain, was added to a 5mL sterile test tube with a stopper. Numbering 1-8, respectively adding 2mL osthole fat emulsion solution into the 1 st tube, mixing, taking out 2.0mL, placing into the 2 nd tube, and sequentially absorbing the medicinal liquid into 1: 2, 1: 4, 1: 8, 1: 16, 1: 32, and 1: 64 series solutions with dosage of 7.50, 3.75, 1.88, 0.94, 0.47, and 0.24 mg/mL by double dilution method－1. Each tube was charged at a concentration of about 5X 105 CFU. mL－10.2mL of the bacterial liquid of each pathogenic indicator bacterium, and a negative control tube (containing the bacterial liquid without the bacterial liquid) and a positive control tube (containing the bacterial liquid without the medicine) are respectively arranged at the same time. And culturing in an incubator at 37 ℃ for 24 hours, and observing the growth condition of the strain. Pathogenic bacteria concentration calculation method-units of Colony Formation (CFU) observed on the plate. The concentration of CFU less than or equal to 5 is bacteriostatic concentration, the mark is "-", the concentration of CFU more than or equal to 5 is non-bacteriostatic concentration, the mark is "+", and the minimum concentration producing bacteriostatic action represents the dose of the tubeI.e., the Minimum Inhibitory Concentration (MIC) of the test agent.
2.4 results and discussion
2.4.1 results
The paper method for determining the bacteriostatic activity of osthole on 6 bacteria is shown in table 1; the MIC values of osthole against 6 bacteria determined by the double dilution method are shown in Table 2.
TABLE 1 mean zone diameter mm of osthole against 6 bacteria
TABLE 2 MIC values n of osthole for 6 bacteria 3, mg. mL-1
As can be seen from tables 1 and 2, osthole has obvious bacteriostatic action on 6 kinds of bacteria, and the bacteriostatic action is enhanced along with the increase of the concentration. Wherein the antibacterial effect on Staphylococcus aureus, Staphylococcus aureus resistant bacteria, Pseudomonas aeruginosa and Escherichia coli is strongest, and MIC is 0.94 mg/mL-1(ii) a Secondly, the bacteriostatic action on the salmonella typhimurium is that the MIC is 1.88 mg.mL-1(ii) a Has the weakest bacteriostatic action on Shigella dysenteriae, and the MIC is 3.75 mg/mL-1。
2.4.2 discussion
According to the invention, the anti-bacterial effects of osthole on staphylococcus aureus, staphylococcus aureus resistant bacteria, escherichia coli, shigella dysenteriae, salmonella typhimurium and pseudomonas aeruginosa are investigated by a drug sensitive paper sheet method and a test tube double dilution method. The research result shows that the osthole has the strongest bacteriostasis effect on staphylococcus aureus, staphylococcus aureus drug-resistant bacteria, pseudomonas aeruginosa and escherichia coli, and secondly has the weak effect on salmonella typhimurium and shigella dysenteriae. And (4) prompting by a result: the osthole and the cosmetic preparation thereof have strong antibacterial and anti-inflammatory effects clinically, and can inhibit staphylococcus aureus, staphylococcus aureus resistant bacteria, escherichia coli, shigella dysenteriae, salmonella typhimurium, pseudomonas aeruginosa and other bacteria; has remarkable antibacterial, anti-inflammatory and acne removing effects.
Example 5: the osthole extracted and separated by the method of the invention has the effect of diminishing inflammation and relieving pain
The osthole fat emulsion solution has good antibacterial effect, and can inhibit Staphylococcus aureus, Staphylococcus aureus resistant bacteria, Escherichia coli, Shigella dysenteriae, Salmonella typhimurium, Pseudomonas aeruginosa, etc.; in addition, the osthole has the functions of diminishing inflammation and relieving pain, and the experimental method and the result are as follows:
1 reagent
1.1 preparation of osthole fat emulsion solution and blank fat emulsion solution prepared as described in 2.1 of example 4 above for intraperitoneal injection.
1.2 Indomethacin, a product of Shanghai pharmaceutical company, which is prepared into 5mg/ml before use and is used for intraperitoneal injection.
2 Experimental animals and Environment
Kunming mouse, male, weight 20 + -2 g, provided by Guangdong province medical experiment animal center, animal quality qualification certification serial number: no. 44411600000901. License number for experimental animals: SYXK (Yue) 2013-.
The experimental environment conditions comprise that the temperature is 23 +/-2 ℃, the relative humidity is 60 +/-10%, the indoor natural light illumination is mainly used, the ventilation is good, the environment is kept quiet, and the environmental facilities of SPF-level experimental animals are provided.
120 mice were divided into 2 large groups of 60 mice each; each large group is divided into a blank fat emulsion solution control group, an indomethacin positive control group and three dosing groups of small, medium and large dose of osthole fat emulsion solution, and each group has 12 patients.
3 Experimental methods
Method II according to the guideline compilation of the medical and political administration of the Ministry of public health of the people's republic of China for the Pre-clinical research guidelines of New drugs
3.1 mouse writhing experiment
After the mice are grouped, a blank fat emulsion solution is used as a negative control, indomethacin is used as a positive control, the mice are administrated by intraperitoneal injection, 0.6 percent acetic acid 0.2m1 is intraperitoneally injected after 40-60 minutes, and the times of writhing of the mice are observed within 15 minutes. Compared with the group significant differences (the mouse writhing reaction refers to the phenomenon that the mouse is sunken in the abdomen, stretched in the trunk and the hind legs and raised in the hip due to the pain and the insignia).
3.2 methods of inflammation of mouse Eggshell
After the mice are grouped, blank fat emulsion solution is used as negative control, indomethacin is used as positive control, the medicine injection is injected into the abdominal cavity, and after 30 minutes of administration, 0.03-0.05ml of dimethylbenzene is smeared inside and outside the right ear shells of the mice. After 3 hours, the mice were sacrificed and the right ear shells were removed, while the left ear shells were removed as controls, and 8mm punches were used to punch off the round ear pieces from the left and right ears, respectively, and then weighed on an analytical balance to calculate the swelling degree. (degree of swelling-right ear concha weight-left ear concha weight). Significant differences between groups were compared.
4 results of the experiment
TABLE 3 inhibitory Effect of osthole on acetic acid-induced writhing of the Min: (
n＝12)
*P<0.05，**P<O1 compared to the blank fat emulsion solution group.
TABLE 4 inhibition of osthole on xylene-induced swelling of mouse concha: (
n＝12)
*P<0.05，**P<0.01 compared to the blank fat emulsion solution group.
The experimental result shows that the osthole has obvious inhibiting effect on the writhing of the mice induced by acetic acid, and the action intensity is related to the dosage. And 2 large and medium dose groups are superior to indomethacin. The results are shown in Table 3.
The experimental results show that: the osthole has obvious inhibition effect on mouse concha swelling, the inhibition effect is increased along with the increase of the dosage, and the action intensity of the large and medium 2 dosages is stronger than that of the indomethacin group. The results are shown in Table 4. 5 conclusion
The experiment result shows that the osthole has obvious anti-inflammatory and analgesic effects. Has stronger action on relieving inflammation and pain. Can be used for preparing natural cosmetics or anti-inflammatory and analgesic drugs.
Example 6: the anti-allergic reaction effect experiment of the osthole extracted and separated by the method of the invention
Another important role of osthole in cosmetics is its anti-allergic reaction, which is demonstrated by using mice and rats as research objects, inhibiting Passive Cutaneous Anaphylaxis (PCA) reaction of mice and degranulation of mast cells in abdominal cavity of rats, and the results are summarized as follows:
1 reagent
1.1 preparation of osthole fat emulsion solution and blank fat emulsion solution prepared as above 2.1 in example 4 for oral administration and intraperitoneal injection.
1.2 radix trichosanthis injection: shanghai biochemical pharmaceutical factory product, using 0.5% Evans blue, physiological saline to prepare 1.00mg/ml, 1.25mg/ml for injection; the aluminum hydroxide gel is commercially available.
1.3 antiserum: prepared according to the method of Xutuyun and the like, which is mainly compiled in pharmacological experimental methodology (1 st edition, Beijing: people's health publishing agency, 1982: 923).
1.4 sodium cromoglycate injection: the Shanghai biochemical pharmaceutical factory product is prepared into 5.8mg/ml by using normal saline for injection.
2 laboratory animals
Kunming mouse (weight 20 + -2 g), Wister rat (weight 200g + -20 g), provided by Guangdong province medical experiment animal center, animal quality qualification certification serial number: no. 44411600000901. License number for experimental animals: SYXK (Yue) 2013-.
3 Experimental methods
3.1 mouse Passive skin allergy (PCA) test
Taking a healthy mouse, unhairing the abdomen, injecting 0.03mL (serum is diluted by 10 times) of antiserum into the skin, injecting 0.2mL of trichosanthes injection (1.25mg/mL) into the tail vein after 48 hours, attacking for 30min, cutting off the head, killing the animal, turning over the abdomen skin, measuring the diameter of the blue spot, cutting off the skin sheet of the blue spot, respectively soaking in 5mL of acetone physiological saline (7:3), eluting after 24 hours, carrying out color comparison at the wavelength of 610nm by using a spectrophotometer, and reading the optical density.
3.2 rat peritoneal mast cell degranulation test
Taking healthy rats, injecting a trichosanthes root-aluminum hydroxide gel suspension into soles, injecting 0.4ml into 4 soles together, namely injecting 1mg of trichosanthes root into each rat, injecting 15 days after injection, injecting a tested medicament into the abdominal cavity, performing ether anesthesia after 0.5h, bleeding femoral artery, killing the animals, cutting off the abdominal skin, injecting 10ml of Hank's solution (containing no phenol red) with the pH of 6.9 into the abdominal cavity, containing 0.005% of heparin, massaging the abdominal cavity for 2min lightly, then opening the abdominal cavity, collecting the abdominal cavity solution into a test tube by a dropper, centrifuging for 15min at 1000r/min after cooling in an ice bath, discarding supernatant, suspending precipitate in 2ml of Hank's solution, and placing the test tube for standby.
Taking 0.5ml of the mast cell suspension, adding an equivalent amount of antigen solution (containing 1mg/ml of radix Trichosanthis), keeping the temperature at 37 ℃ for 15min, staining with 0.125% neutral red, observing 100 mast cells under a high power microscope, and calculating the inhibition percentage of the medicament on the degranulation of the mast cells according to a formula of (1-a/q) multiplied by 100%. Wherein a and q represent the numbers of degranulated cells in the administration group and the control group, respectively.
4 results of the experiment
4.1 inhibition of mouse PCA reaction by osthole
30 mice are taken, after the intradermal injection of antiserum on the abdomen, the mice are divided into three groups, the control group is infused with blank fat emulsion solution, and the other two groups are infused with the fat emulsion solution of osthole 100mg/kg and 200mg/kg respectively. The diameter of the blue spot and the optical density of the three groups of animals are shown in Table 5. The result shows that the osthole has stronger inhibition effect on mouse PCA reaction.
TABLE 5 osthole passivity in miceEffect of inhibition Rate of skin allergy (PCA) (PCA)
n＝10)
| Osthole mg/kg | Diameter of blue spot (mm) | Optical density |
| 0 | 1.21±0.23 | 0.167±0.07 |
| 100 | 0.51±0.25* | 0.074±0.04* |
| 200 | 0.43±0.28** | 0.051±0.02** |
*P<0.05，**P<0.01 compared to the blank fat emulsion solution group.
4.2 inhibition of mast cell degranulation by osthole
Taking 40 rats, dividing into four groups, injecting control group with normal saline 0.7 mL/rat and cromolyn sodium 0.7 mL/rat, and injecting administration group with osthole fat emulsion solution 0.7 mL/rat and 1.4 mL/rat. For rat abdominal cavity mast cell degranulation caused by trichosanthes root antigen, the inhibition rate of cromolyn sodium is 64.0%, and the inhibition rates of osthole are 57.7% and 79.1%, respectively, and compared with a control group, the significant difference shows that the osthole has significant inhibition effect on rat abdominal cavity mast cell degranulation. The experimental results are shown in Table 6.
TABLE 6 inhibition of mast cell degranulation by osthole: (
n＝10)
Discussion of 5
Experiments prove that: the osthole has strong inhibition effect on the PCA reaction of mice for 48 hours, which shows that the osthole can obviously inhibit the formation of IgE antibodies and has inhibition effect on mast cell degranulation in allergic reaction. Osthole has important effect in preparing antiallergic cosmetics.
Example 7: examples of preparation of cnidium lactone cosmetics
⑴ preparation method of facial cream
The formula is as follows:
the preparation process comprises the following steps:
mixing osthole, cetearyl glucoside, cetostearyl alcohol, caprylic/capric triglyceride, jojoba oil, isopropyl palmitate and glycerol, heating to 60 deg.C to obtain phase A, and keeping the temperature; taking dipotassium glycyrrhizinate, adding xanthan gum, carbomer 940, sodium hyaluronate and deionized water, mixing and heating to 60 ℃, uniformly dispersing to serve as phase B, and keeping the temperature for later use; slowly adding phase A into phase B which is dispersed uniformly in advance and heated to 80 deg.C under homogenizing condition, adding cyclodimethylsilicone 345, homogenizing for 3 min, adding sodium hydroxide to adjust pH to 6-7, stirring and cooling to 50 deg.C, adding essence, stirring, and cooling to 35 deg.C.
⑵ preparation of emulsion
The formula is as follows:
the preparation process comprises the following steps:
mixing osthole, cyclopentasiloxane 995, vaseline, tween-80, vitamin E acetate, and glycerol, heating to 60 deg.C to obtain phase A, and keeping the temperature for use; taking dipotassium glycyrrhizinate, adding xanthan gum, Carbopol Ultrez 10, 1, 3-butanediol and deionized water, mixing and heating to 60 ℃, uniformly dispersing to serve as a phase B, and keeping the temperature for later use; slowly adding phase A into phase B which is dispersed uniformly in advance and heated to 80 deg.C under homogenizing condition, homogenizing for 3 min, adding triethanolamine to adjust pH to 6-7, stirring and cooling to 50 deg.C, adding essence and antiseptic, stirring, and cooling to 35 deg.C.
⑶ preparation of eye cream
The formula is as follows:
the preparation process comprises the following steps:
taking dipotassium glycyrrhizinate, adding 1, 3-butanediol, green tea extract, disodium ethylene diamine tetraacetate and deionized water, uniformly dispersing, heating to 50 ℃, and stirring until the mixture is clear; slowly adding the organic silicon elastomer under stirring to serve as a phase A for later use; mixing osthole, DC 245, jojoba oil, vitamin E acetate, and vitamin A palmitate, heating to 60 deg.C to obtain phase B, and keeping the temperature; slowly adding phase A into phase B which is dispersed uniformly in advance and heated to 60 deg.C under homogenizing, homogenizing for 3 min, and cooling to 35 deg.C to obtain the final product.
Preparation of ⑷ jelly
The formula is as follows:
the preparation process comprises the following steps:
adding polyacrylic acid, glycerol, 1, 3-butanediol, fucoidan sulfate, and sodium hyaluronate into osthole and dipotassium glycyrrhizinate, stirring and dispersing uniformly to obtain phase A for use; adding deionized water into phase A, dispersing, heating to 60 deg.C, adding triethanolamine to adjust pH to 6-7, stirring, cooling to 50 deg.C, adding essence and methyl isothiazolinone, stirring, and cooling to 35 deg.C.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A method for preparing osthole from plants is characterized by comprising the following steps:
(1) extracting osthole: selecting fruit of cnidium monnieri as raw material, cleaning, drying, and pulverizing to obtain coarse powder; heating and extracting or heating, warm dipping and ultrasonic extracting by using 65-95% ethanol solution by volume percentage; after extraction, carrying out solid-liquid separation to obtain an extract containing osthole;
(2) separating the crude cnidium lactone: taking an extract containing osthole, and recovering ethanol until the ethanol is completely recovered to obtain a concentrated solution; adding hot water with the temperature of 40-80 ℃ into the concentrated solution for dissolving, then cooling, standing at the temperature of 1-25 ℃ for more than 24 hours, carrying out solid-liquid separation, removing the liquid, and collecting the solid to obtain a crude extract containing osthole;
(3) refining and purifying osthole monomer component through step ① or steps ① and ②;
① crystallizing for the first time, adding 50-60 vol% ethanol water solution into the crude extract of osthole, heating to dissolve, cooling to room temperature, standing for the first time, separating solid and liquid, collecting liquid, discarding solid, standing the liquid obtained from solid-liquid separation at 0-10 deg.C for the second time until crystal is fully separated out, separating solid and liquid, discarding liquid, and collecting crystal;
② and recrystallizing the obtained crystal according to step ① to obtain refined and purified crystal of osthole.
2. The method for producing osthole from plants according to claim 1, wherein:
the ethanol solution in the step (1) is 85-90% by volume;
the hot water in the step (2) is hot water with the temperature of 50-70 ℃;
the concentration of the ethanol water solution in the step (3) ① is 53-57% by volume.
3. The method for producing osthole from plants according to claim 1, wherein:
the heating extraction times in the step (1) are 2-3; the method comprises the following specific steps: when the extraction times are 2 times: adding ethanol solution with the volume equivalent to 10 times of the mass of the coarse powder for the first time, fully stirring and uniformly mixing, and performing reflux extraction at 70-100 ℃ for 1-3 hours; adding ethanol solution with volume equivalent to 8 times of the mass of the coarse powder for the second time, and performing reflux extraction at the temperature of 70-100 ℃ for 0.5-2 hours; when the extraction times are 3 times: adding ethanol solution with volume equivalent to 8 times of the mass of the coarse powder for the third time when the times of the first two times of extraction are 2 times, and carrying out reflux extraction at 70-100 ℃ for 0.5-2 hours; after extraction, carrying out solid-liquid separation, and combining the liquid obtained by 2-3 times of separation to obtain an osthole-containing extracting solution;
the times of the heating warm-dipping ultrasonic extraction in the step (1) are 2-3 times; the method comprises the following specific steps: when the extraction times are 2 times: adding ethanol solution with volume equivalent to 10 times of the mass of the coarse powder for the first time, fully stirring and uniformly mixing, and carrying out warm immersion ultrasonic extraction at the temperature of 60-80 ℃ for 0.5-2 hours; adding ethanol solution with volume amount being 8 times of the mass of the coarse powder for the second time, and carrying out warm immersion ultrasonic extraction at the temperature of 60-80 ℃ for 0.5-1.5 hours; when the extraction times are 3 times: adding ethanol solution with volume equivalent to 8 times of the mass of the coarse powder for the third time when the times of the previous two times of extraction are 2 times, and carrying out warm immersion ultrasonic extraction for 0.5-1.5 hours at the temperature of 60-80 ℃; and after extraction, performing solid-liquid separation, and combining the liquid obtained by 2-3 times of separation to obtain the osthole-containing extracting solution.
4. The method for producing osthole from plants according to claim 3, wherein:
the ultrasonic condition in the heating warm immersion ultrasonic extraction in the step (1) is 200-300 Hz.
5. The method for producing osthole from plants according to claim 1, wherein:
the standing condition in the step (2) is standing for 24-30 h at the temperature of 2-8 ℃;
the first standing time in the step (3) ① is 7-8 hours;
the time for the second standing in the step (3) ① is more than 48 hours;
and (3) ①, wherein the temperature of the second standing is 0-8 ℃.
6. The method for producing osthole from plants according to claim 1, wherein:
the adding amount of the hot water in the step (2) is calculated according to the volume of 1-2L of the extracted concentrated solution obtained by 1kg of raw materials after the hot water is added;
the addition amount of the ethanol aqueous solution in the step (3) ① is calculated according to the proportion of 1-3L ethanol aqueous solution to the crude extract obtained from 1kg raw materials.
7. The method for producing osthole from plants according to claim 1, wherein:
the snake bed in the step (1) is a snake bedCnidium monnieric(L.) Cusson. orCnidium formosanumYABE；
The coarse powder in the step (1) is 15-20 meshes of coarse powder;
the number of recrystallization in the step (3) ② was 1.
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