WO2020034600A1 - 一种制备马钱子提取物的方法、制得的提取物及其用途 - Google Patents

一种制备马钱子提取物的方法、制得的提取物及其用途 Download PDF

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WO2020034600A1
WO2020034600A1 PCT/CN2019/073450 CN2019073450W WO2020034600A1 WO 2020034600 A1 WO2020034600 A1 WO 2020034600A1 CN 2019073450 W CN2019073450 W CN 2019073450W WO 2020034600 A1 WO2020034600 A1 WO 2020034600A1
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Prior art keywords
solution
extract
mesh
strychnine
solid
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PCT/CN2019/073450
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English (en)
French (fr)
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张保献
吴珍珍
胡杰
薛春美
鞠志赫
曹瑛
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北京盈科瑞创新医药股份有限公司
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Publication of WO2020034600A1 publication Critical patent/WO2020034600A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of medicine, and particularly relates to a method for preparing a horsetail extract, and also relates to the prepared horsetail extract and its medical use.
  • Ma Qianzi also known as Panmuchi
  • Panmuchi was first published in the Compendium of Materia Medica. It has bitter, cold, toxic, liver, and spleen meridians. Its medicinal effects are Tongluo analgesic, loosening and swelling, and it is often used clinically. In the treatment of bruises, fractures, swelling and pain, rheumatism, paralysis, numbness, sore throat and sore throat. However, it is highly toxic. This characteristic severely affects its clinical application. The general treatment amount of horsetail is 0.3-0.6g, and poisoning is more likely to occur than this amount. Therefore, in recent years, the safety of using Ma Qianzi has become a research hotspot.
  • strychnine is total alkaloids, which include strychnine, strychnine, papaverine, strychnine, geniposide, and icaginin.
  • Strychnine has anti-inflammatory and analgesic effects
  • Shi Ning basically has no anti-inflammatory and analgesic effects
  • Shi Ning is the main toxic component of strychnine;
  • mice administer Shi Ning The LD50s of strychnine and strychnine are 3.27mg / kg, 233mg / kg, and 234.5mg / kg, respectively; the LD50s of mice injected with the aforementioned drugs are 1.53mg / kg, 69.77mg / kg, and 76mg / kg, respectively.
  • the oral poisoning dose of Shi Ning for adults is 5-10 mg, and the oral lethal dose for adults is 30 mg. Therefore, the method that takes into account the efficacy and safety of strychnine is to increase the strychnine content while reducing the content of strychnine.
  • Shi Ning and strychnine are both alkaloids with similar structures, as shown in the figure below.
  • the structural difference between Shi Ning and strychnine is that methoxy is connected to the 2, 3 position of the benzene ring of strychnine, and hydrogen is connected to the 2 and 3 position of the ring of the ring
  • the polarity is similar. Therefore, no effective method has been found to separate strenin and strychnine.
  • Strychnine X is CH 3 O-; Shi Ning: X is H-.
  • One of the purposes of the present invention is to provide a method for preparing a troche extract.
  • the troche extract has a higher trochine content, while the strychnine content is lower, and the trochine extract is extracted. The rate is higher.
  • the present invention also provides a pomegranate extract.
  • Yet another object of the present invention is to provide medicinal use of the extract of pomegranate.
  • the first aspect of the present invention relates to a method for preparing a pomegranate seed extract, comprising the following steps:
  • step (2) using ethyl acetate to extract the solid phase obtained in step (1), and separating the solid and liquid to obtain an extraction liquid;
  • the second alkaline solution is selected from at least one of an aqueous ammonia solution, a potassium carbonate solution, a sodium carbonate solution, a sodium bicarbonate solution, and a potassium bicarbonate solution;
  • step (4) dissolving the dried product obtained in step (4) in an acid solution, and separating the solid and liquid to obtain a liquid phase;
  • step (5) The pH value of the liquid phase obtained in step (5) is adjusted to 8 to 13 (for example, 9, 10, 11, 12), and solid-liquid separation is performed to obtain a solid phase;
  • step (6) The solid phase obtained in step (6) is recrystallized to obtain an extract.
  • the second alkaline solution is a saturated solution.
  • the second alkaline solution is selected from a saturated aqueous ammonia solution, a saturated potassium carbonate solution, a saturated sodium carbonate solution, a saturated sodium bicarbonate solution, and a saturated potassium bicarbonate. At least one of the solutions.
  • the second alkaline solution is selected from a saturated aqueous ammonia solution, a saturated potassium carbonate solution, a saturated sodium carbonate solution, and a saturated sodium bicarbonate solution at 10 ° C to 30 ° C. At least one of a solution and a saturated potassium bicarbonate solution.
  • the concentration of the second alkaline solution is 0.1% (V / V) to 2% (V / V), and preferably 0.5% (V / V) to 1% (V / V). V).
  • the volume of the second alkaline solution in the eluent is 0.05% to 5% of the volume of the absolute ethanol or ethanol solution, such as 0.07%, 0.1%, 0.2%, 0.3%, 0.5%, 0.7%, 0.9%, 1%, 1.5%, 2%, 2.3%, 2.6%, 3%, 3.2%, 3.4%, 3.6%, 3.5%, 3.8%, 4% , 4.3%, 4.5%, 4.7%, 4.9%.
  • the silica gel column is a 70-300 mesh silica gel column, such as 90 mesh, 100 mesh, 120 mesh, 140 mesh, 160 mesh, 180 mesh, 200 mesh. , 220 mesh, 240 mesh, 260 mesh, 280 mesh or 290 mesh silica gel column.
  • step (3) the eluate after 3 to 8 times (for example, 4, 5, 6, 7 times) the column volume is collected.
  • the method includes one or more of the following 1) to 22):
  • the first alkaline solution is selected from at least one of a potassium carbonate solution, a sodium bicarbonate solution, a sodium carbonate solution, a potassium bicarbonate solution, and an amine solution;
  • the first alkaline solution is selected from at least one of a potassium carbonate solution, a sodium bicarbonate solution, a sodium carbonate solution, a methylamine solution, a potassium bicarbonate solution, and an ethylamine solution;
  • the first alkaline solution is selected from at least one of a saturated potassium carbonate solution, a saturated sodium bicarbonate solution, a saturated sodium carbonate solution, a saturated methylamine solution, a saturated potassium bicarbonate solution, and a saturated ethylamine solution;
  • the first alkaline solution is selected from a saturated potassium carbonate solution, a saturated sodium bicarbonate solution, a saturated sodium carbonate solution, a saturated methylamine solution, a saturated potassium bicarbonate solution, and a saturated ethylamine solution at a temperature of 10 ° C to 30 ° C. At least one of
  • the ratio of the first alkaline solution and the pomegranate crude drug is (0.5 to 10): 1 (mL / g), preferably (0.5 to 3): 1 (mL / g), for example 0.8: 1 (mL / g), 1: 1 (mL / g), 2: 1 (mL / g), 3: 1 (mL / g), 4: 1 (mL / g), 5: 1 (mL / g), 6: 1 (mL / g), 7: 1 (mL / g), 8: 1 (mL / g), 9: 1 (mL / g);
  • the infiltration time is 1 to 10 hours, preferably 2 to 4 hours, and more preferably 3 hours, such as 2, 3, 4, 5, 6, 7, 8, 9 hours;
  • step (1) solid-liquid separation is performed by filtration
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • filter cloth is used for filtering
  • step (1) before the infiltration, crush the crude oil of the horsetail seed;
  • step (2) the ratio of ethyl acetate to the solid phase obtained in step (1) is (1 to 10): 1 (mL / g), preferably (4 to 6): 1 (mL / g ), Such as 2: 1 (mL / g), 3: 1 (mL / g), 4: 1 (mL / g), 5: 1 (mL / g), 6: 1 (mL / g), 7: 1 (mL / g), 8: 1 (mL / g), 9: 1 (mL / g);
  • the extraction time is 2 to 16 hours, preferably 6 to 12 hours, and more preferably 8 to 10 hours, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 hours;
  • step (2) leaching under reflux conditions
  • step (2) the number of extractions is one or more times, preferably one, two, three or four times;
  • step (2) solid-liquid separation is performed by filtration
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • filter cloth is used for filtering
  • steps (2) and (3) further including step (2 '): concentrating the extract obtained in step (2) to obtain a concentrated liquid; filtering the concentrated liquid, and using the obtained filtrate to perform step ( 3) silica gel column chromatography;
  • the volume of the concentrated liquid is 1/4 to 1/2, for example, 1/3, of the volume of the extraction liquid obtained in the step (2);
  • a filter cloth of 100 to 500 mesh (for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh) is used for filtering;
  • the concentration of the ethanol solution is 70% (W / W) to 99% (W / W), such as 75% (W / W), 80% (W / W), 85% (W / W), 90% (W / W), 95% (W / W), 98% (W / W);
  • steps (4) and (5) further including step (4 '): crushing the dried product obtained in step (4);
  • the acid solution is selected from at least one of a sulfuric acid solution, a hydrochloric acid solution, and a phosphoric acid solution;
  • the concentration of the acid solution is 0.6% (W / W) to 9% (W / W), such as 1% (W / W), 2% (W / W), 3% (W / W), 4% (W / W), 5% (W / W), 6% (W / W), 7% (W / W), 8% (W / W);
  • step (5) it is dissolved under heating conditions
  • the ratio of the acid solution to the dried product obtained in step (4) is (12-47): 1 (mL / g), such as 13: 1 (mL / g), 15: 1 (mL / g), 17: 1 (mL / g), 19: 1 (mL / g), 21: 1 (mL / g), 22: 1 (mL / g), 25: 1 (mL / g), 27 : 1 (mL / g), 29: 1 (mL / g), 31: 1 (mL / g), 33: 1 (mL / g), 35: 1 (mL / g), 37: 1 (mL / g), 39: 1 (mL / g), 40: 1 (mL / g), 42: 1 (mL / g), 44: 1 (mL / g), 46: 1 (mL / g);
  • step (5) solid-liquid separation is performed by filtration
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • filter cloth is used for filtering
  • step (6) adjust the pH value by adding an lye
  • the lye is a potassium hydroxide solution and / or a sodium hydroxide solution;
  • step (6) solid-liquid separation is performed by filtration
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • 100-500 mesh for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh
  • filter cloth is used for filtering
  • steps (6) and (7) further including step (6 '): drying and crushing the solid phase obtained in step (6);
  • step (7) the solid phase obtained in step (6) is recrystallized using an ethanol solution
  • the concentration of the ethanol solution is 10% (W / W) to 60% (W / W), such as 15% (W / W), 20% (W / W), 30% (W / W), 40 % (W / W), 50% (W / W), 55% (W / W);
  • the recrystallization is performed by the following steps:
  • step (6) dissolving the solid phase obtained in step (6) in an ethanol solution, filtering, and collecting the filtrate;
  • step (ii) leaving the filtrate collected in step (i) to crystallize, filter, and collect the filter residue as the extract;
  • the ratio of the ethanol solution to the solid phase is (10-50): 1 (mL / g), such as 12: 1 (mL / g), 14: 1 (mL / g) , 16: 1 (mL / g), 20: 1 (mL / g), 22: 1 (mL / g), 24: 1 (mL / g), 26: 1 (mL / g), 30: 1 ( mL / g), 32: 1 (mL / g), 35: 1 (mL / g), 37: 1 (mL / g), 40: 1 (mL / g), 43: 1 (mL / g), 46: 1 (mL / g), 47: 1 (mL / g), 49: 1 (mL / g);
  • filtering is performed by using a filter cloth of 100-500 mesh (for example, 200-300 mesh, 250 mesh, 350 mesh, 400 mesh, 450 mesh);
  • step (i) it is dissolved under heating conditions
  • step (ii) the crystals are left to stand for crystallization at room temperature
  • the room temperature is 10 ° C to 30 ° C;
  • the number of recrystallizations is one or more times, preferably one or two times.
  • step (5) the dried matter is partially dissolved in the acid solution.
  • step (i) the solid phase is partially dissolved in an ethanol solution.
  • the "drying" operation in steps (4) and (6 ') is performed at a temperature that does not destroy or affect the biological activity of the extracted components. It is well known to those skilled in the art that the temperature range does not destroy or affect the biological activity of the extracted components.
  • the "heating" operation in step (5) and step (i) is performed at a temperature that does not destroy or affect the biological activity of the extracted component. It is well known to those skilled in the art that the temperature range does not destroy or affect the biological activity of the extracted components.
  • the ethanol solution, ammonia solution, potassium carbonate solution, sodium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, and acid solution are all aqueous solutions.
  • the second aspect of the present invention relates to an extract of strychnine, wherein the weight percentage of strychnine is ⁇ 94.5% (preferably ⁇ 95%, ⁇ 96%, or ⁇ 97%, for example, 94.5% to 99.9% , 95% to 99.9%, 95% to 99.8%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%), and the weight percentage content of Shi Ning ⁇ 3% (preferably ⁇ 2.9%, ⁇ 2.8%, ⁇ 2.7%, ⁇ 2.3%, for example, 0.01% to 2.9%, 0.05% to 2.9%, 0.05% to 2.8%, 0.1%, 0.08%, 2.1%, 2.2%, 2.3% , 2.4%, 2.6%, 2.7%).
  • the weight percentage of strychnine is ⁇ 94.5% (preferably ⁇ 95%, ⁇ 96%, or ⁇ 97%, for example, 94.5% to 99.9% , 95% to 99.9%, 95% to 99.8%, 96%, 97%
  • the pomegranate extract is prepared by the method of the first aspect of the present invention.
  • a third aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the pomegranate extract according to the second aspect of the present invention and a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient is selected from the group consisting of a filler, a disintegrant, an excipient, a binder, a lubricant, a surfactant, a diluent, a preservative, and a stabilizer. At least one of an agent, a flavoring agent, a thickener, a glidant, a humectant, a pH adjuster, and a transdermal agent.
  • different pharmaceutically acceptable excipients can be selected for different pharmaceutical dosage forms; for example, fillers, disintegrating agents, and dispersing agents commonly used in solid preparations such as tablets, capsules, and granules.
  • Forming agents, adhesives, lubricants, surfactants, fillers, etc .; surfactants, diluents, preservatives, stabilizers, flavoring agents, thickeners, etc. are commonly used in liquid preparations such as syrups and oral liquids.
  • Glidants, etc . surfactants, thickeners, humectants, pH adjusters, transdermal agents, preservatives, etc. are commonly used in the form of external preparations.
  • Fillers include, but are not limited to, starch, lactose, dextrin, powdered sugar, microcrystalline cellulose, mannitol, xylitol, and the like.
  • Disintegrating agents include, but are not limited to, dry starch, sodium carboxymethyl starch, crospovidone, and the like.
  • Excipients include, but are not limited to, talc, magnesium stearate, and the like.
  • Binders include, but are not limited to, starch syrup, gelatin, carmellose.
  • Lubricants include, but are not limited to, magnesium stearate, micronized silica gel, corn starch, and the like.
  • Solubilizers include, but are not limited to, polysorbates, polyoxyethylene fatty acid esters, and the like.
  • Co-solvents include, but are not limited to, potassium iodide, sodium acetate, sodium benzoate, and the like.
  • Preservatives include, but are not limited to, parabens, benzoic acid, sorbic acid, potassium sorbate, hydroxyphenethyl, hydroxyphenylpropyl, phenol, and the like.
  • Flavoring agents include, but are not limited to, sucrose, xylitol, lemon, peppermint oil, gum arabic, and the like.
  • Suspensions include, but are not limited to, glycerol, sodium alginate, agar, methyl cellulose, and the like.
  • Wetting agents include, but are not limited to, polysorbates, poloxamers, and the like.
  • Surfactants include, but are not limited to, sodium stearate, monoglyceride fatty acid esters, polyoxyethylene (40) fatty acid esters, Tweens and the like.
  • Thickeners include, but are not limited to, cross-linked polypropylene resins, hydroxypropyl methyl cellulose, xanthan gum, and the like.
  • Humectants include, but are not limited to, glycerin, propylene glycol, isopropanol, hyaluronic acid, and the like.
  • pH adjusting agents include, but are not limited to, organic amines such as triethanolamine, triethylamine, diethylamine, sodium hydroxide, sodium bicarbonate, sodium carbonate, and the like.
  • Transdermal agents include, but are not limited to, Azone, menthol, essential oils, dimethyl sulfoxide, propylene glycol, myristyl isopropyl, N-methylpyrrolidone, lauric polyethylene glycol glyceride, polyglycerin fatty acid Ester, oleic acid polyethylene glycol glyceride, caprylic acid capric acid polyethylene glycol glyceride.
  • the pharmaceutical composition is prepared manually (for example, artificially mixing a pomegranate seed extract and a pharmaceutically acceptable excipient).
  • the fourth aspect of the present invention relates to a method for relieving pain or loosening swelling of Tongluo or a method for treating bruises, fractures, swelling and pain, rheumatism, numbness, paralysis, sore throat or sore throat.
  • the subject in need thereof is an effective amount of the pomegranate extract according to the second aspect of the present invention or the pharmaceutical composition according to the third aspect of the present invention.
  • the fifth aspect of the present invention relates to the horsetail extract according to the second aspect of the present invention or the pharmaceutical composition according to the third aspect of the present invention in the preparation of a medicament for relieving pain, dispersing and swelling of the collaterals, or preparing a treatment for bruises , Fracture swelling and pain, rheumatism, paralysis, numbness, or throat soreness.
  • the sixth aspect of the present invention relates to the pomegranate extract according to the second aspect of the present invention or the pharmaceutical composition according to the third aspect of the present invention, which is used for Tongluo analgesia, loosening and swelling, or treating bruises and fractures. Pain, rheumatism, paralysis, numbness, sore throat or sore throat.
  • the invention also relates to the following aspects:
  • a method for extracting, isolating and purifying strychnine comprising the following steps:
  • Step 1 Use the pomegranate medicinal material as a raw material, infiltrate with an alkaline solution, and extract with ethyl acetate as a solvent to obtain extract A;
  • Step 2 Using ethanol with alkaline water as the eluent (ie, the eluent), the extraction liquid A is separated through a silica gel chromatography column to obtain a dry isolate B;
  • Step 3 The dried isolate B is filtrated by acid to obtain filtrate C, and then subjected to alkali precipitation to obtain dry powder D;
  • Step 4 The dried powder D is recrystallized from ethanol to obtain strychnine.
  • the base is an inorganic base, preferably, the base is sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide , Potassium hydroxide, one or two or more combinations of ammonia water.
  • the base is an organic base, preferably, the base is an amine, an alkaloid, potassium tert-butoxide, sodium tert-butoxide, One, two or more combinations of n-butyllithium.
  • the volume of the alkali solution is 0.5-3 times the weight of the medicinal material, volume / weight: ml / g, and the infiltration time is 2-4h.
  • the volume of the alkali solution is 0.5-1.5 times the weight of the medicinal material, volume / weight: ml / g, and the infiltration time is 3 hours.
  • the ethyl acetate in the step 1 is subjected to reflux extraction for 1-4 times, each for 6-12h, preferably, reflux extraction is performed for 2 times, each for 8 -10h.
  • alkaline water in step 2 is one or two of sodium carbonate solution, sodium bicarbonate solution, potassium carbonate solution, potassium bicarbonate, and ammonia water. Or a combination of two or more.
  • the extraction liquid A in step 2 is separated by a silica gel chromatography column
  • the elution solution ie, the eluent
  • the column volume is collected.
  • alkaline solution refers to a solution having a pH value greater than 7 or a hydroxide ion concentration greater than a hydrogen ion concentration at normal temperature.
  • raw medicine refers to pure natural or unprocessed or simply processed Chinese herbal medicines of plants, animals and minerals.
  • leaching refers to the process of dissolving the active ingredient in a medicinal material in a suitable solvent to extract the active ingredient.
  • eluent refers to a solvent used during elution or elution during silica gel column chromatography.
  • saturated solution refers to a solution in which the maximum amount (solubility) of the solute dissolved in the solvent is reached at a certain temperature.
  • the content of strychnine in the pomegranate extract obtained by the method of the present invention is higher, and the content of toxic substance shining is lower, which improves the effectiveness and safety of the drug.
  • the extraction rate of daughter alkali is higher.
  • the horsetail seed extract of the present invention has the effects of Tongluo analgesic, loosening and swelling, and also has the medicinal effects for treating bruises, fractures, swelling and pain, rheumatism, paralysis, paralysis, sore throat or sore throat.
  • Step 1 Take 50 g of medicinal herbs, crush and add 150 mL of saturated potassium carbonate solution to soak for 4 h, and filter to obtain a solid. Then, use 250 mL of ethyl acetate to extract the solid three times under reflux for 10 h, and use 200-300 mesh filter. The cloth was filtered, and the three filtrates were combined, concentrated under reduced pressure to 1/3 of the original volume, and filtered again with a 200-300 mesh filter cloth to obtain an extract solution;
  • Step 2 Load the extract obtained in step 1 on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) ethanol with a 0.1% (V / V) saturated ammonia solution.
  • the solution that is, the volume of the saturated ammonia solution is 0.1% of the volume of the ethanol solution
  • the eluate after 5 times the column volume is collected, and the eluate is concentrated and dried, and crushed to obtain a dry isolate;
  • Step 3 Add 2% (W / W) sulfuric acid solution to the dried isolate obtained in step 2 to heat and dissolve.
  • the ratio of the volume of sulfuric acid solution to the weight of the dry isolate is 40: 1 (mL / g). Filter the cloth to collect the filtrate; add sodium hydroxide solution to the collected filtrate to adjust the pH to 11, and then leave it at room temperature, filter with 200-300 mesh filter cloth, take the filter cake and pulverize to obtain a dry powder;
  • Step 4 Add 35% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 20: 1 (mL / g).
  • High-performance liquid chromatography was used to detect the extract and the standard solution, and the contents of strychnine and strychnine in the extract were calculated.
  • the operating conditions of liquid chromatography are as follows: The chromatographic column is C18 (specification: 4.6mm ⁇ 250mm, 5 ⁇ m), and a mixed solution of acetonitrile-0.01mol / L sodium heptanesulfonate and 0.02mol / L potassium dihydrogen phosphate in an equal amount (using 10% phosphoric acid adjusted the pH to 2.8) (21:79) as the mobile phase, the detection wavelength was 260 nm, the column temperature was 40 ° C, and the flow rate was 1.0 mL / min.
  • Extraction rate of strychnine 100% ⁇ (content of strychnine in the extract ⁇ weight of the extract) / (content of strychnine in the crude drug ⁇ weight of the bitch)
  • Step 1 Take 50 g of medicinal herbs, crush, add 75 mL of saturated sodium bicarbonate solution to soak for 3 h, and filter to obtain a solid; then use 300 mL of ethyl acetate to extract the solid twice under reflux for 9 h each, using 200-300 Filter with a mesh filter cloth, combine the two filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 Load the extract obtained in step 1 on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) added with 0.25% (V / V) saturated potassium carbonate solution.
  • Ethanol solution that is, the volume of the saturated potassium carbonate solution is 0.25% of the volume of the ethanol solution
  • the eluate after 5 times the column volume is collected, the eluate is concentrated to dryness, and pulverized to obtain a dry isolate;
  • Step 3 Add 1% (W / W) phosphoric acid solution to the dry isolate obtained in step 2 to heat and dissolve.
  • the ratio of the volume of the phosphoric acid solution to the weight of the dry isolate is 30: 1 (mL / g).
  • Step 4 Add 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 20: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated according to the method in Example 1. As a result, the weight percentage of strychnine in the extract was about 96.8%. The weight percentage of Shi Ning is about 1.9%, and the extraction rate of strychnine is about 51.3%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 50 mL of saturated sodium carbonate solution for 2 h, filter, and obtain a solid; then use 250 mL of ethyl acetate to extract the solid twice under reflux for 10 h, use 200-300 mesh Filter the cloth, combine the filtrates twice, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 Load the extract obtained in step 1 on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) added with 0.5% (V / V) saturated sodium carbonate solution.
  • Ethanol solution that is, the volume of saturated sodium carbonate solution is 0.5% of the volume of ethanol solution
  • the eluate after 5 times the column volume is collected, and the eluate is concentrated to dryness and crushed to obtain a dry isolate;
  • Step 3 Add 1.5% (W / W) sulfuric acid solution to the dry isolate obtained in step 2 to heat and dissolve.
  • the ratio of the volume of sulfuric acid solution to the weight of the dry isolate is 30: 1 (mL / g).
  • Step 4 Add 25% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 20: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated.
  • the weight percentage of strychnine in the extract was about 97.2%.
  • the weight percentage of Shi Ning is about 1.9%, and the extraction rate of strychnine is about 52.9%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 75 mL of saturated sodium carbonate solution to soak for 3 h, and filter to obtain a solid; then use 250 mL of ethyl acetate to extract the solid twice under reflux for 8 h each, using 200-300 Filter with a mesh filter cloth, combine the two filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 Load the extract obtained in step 1 on a 100-200 mesh silica gel chromatography column for chromatography.
  • the eluent is a 95% (W / W) ethanol solution with 0.5% (V / V) saturated ammonia solution. (That is, the volume of the saturated ammonia solution is 0.5% of the volume of the ethanol solution), and the eluate after 5 times the column volume is collected, and the eluate is concentrated and dried, and crushed to obtain a dry isolate;
  • Step 3 Add 1% (W / W) hydrochloric acid solution to the dry isolate obtained in step 2 to heat and dissolve.
  • the ratio of the volume of the hydrochloric acid solution to the weight of the dry isolate is 30: 1 (mL / g).
  • Step 4 Add the 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 30: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated.
  • the weight percentage of strychnine in the extract was about 97.2%.
  • the weight percentage of Shi Ning is about 1.9%, and the extraction rate of strychnine is about 52.8%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 50 mL of saturated methylamine solution for 3h, filter, and obtain a solid; then use 250mL of ethyl acetate to extract the solid twice under reflux for 9h each time, use 200-300 mesh Filter the cloth, combine the two filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 The extract obtained in step 1 is loaded on a 100-200 mesh silica gel chromatography column for chromatography.
  • the eluent is 95% (W / W) of 0.75% (V / V) saturated sodium bicarbonate solution.
  • Ethanol solution that is, the volume of the saturated sodium bicarbonate solution is 0.75% of the volume of the ethanol solution
  • the eluate after 5 times the column volume is collected, the eluate is concentrated and dried, and pulverized to obtain a dry isolate;
  • Step 3 After adding the 1% (W / W) phosphoric acid solution to the dried isolate obtained in step 2 to heat and dissolve, the ratio of the volume of the phosphoric acid solution to the weight of the dry isolate is 35: 1 (mL / g). Filter with a mesh filter cloth and collect the filtrate; add potassium hydroxide solution to the collected filtrate to adjust the pH to 10, then leave it at room temperature, filter, take the filter cake and dry, crush to obtain a dry powder;
  • Step 4 Add 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 20: 1 (mL / g).
  • Example 1 The method of Example 1 was used to detect and calculate the content of strychnine in the extract, the content of strychnine, and the extraction rate of strychnine. As a result, the weight percentage of strychnine in the extract was about 97.4%. The weight percentage of Shi Ning is about 1.9%, and the extraction rate of strychnine is about 52.7%.
  • Step 1 Take 50g of pomegranate medicinal material, crush, add 50mL saturated sodium bicarbonate solution to soak for 4h, and filter to obtain a solid; then use 250mL ethyl acetate to extract the solid twice under reflux for 9h each time, using 200 ⁇ 300 Filter with a mesh filter cloth, combine the two filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 The extract obtained in step 1 is loaded on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) ethanol added with a 0.75% (V / V) saturated ammonia solution.
  • the solution that is, the volume of the saturated ammonia solution is 0.75% of the volume of the ethanol solution
  • the eluate after 5 times the column volume is collected, and the eluate is concentrated and dried, and crushed to obtain a dry isolate;
  • Step 3 Add 1% (W / W) hydrochloric acid solution to the dry isolate obtained in step 2 to heat and dissolve.
  • the ratio of the volume of the hydrochloric acid solution to the weight of the dry isolate is 35: 1 (mL / g).
  • Step 4 Add 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 20: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated.
  • the weight percentage of strychnine in the extract was about 97.5%.
  • the weight percentage of Shi Ning is about 1.9%, and the extraction rate of strychnine is about 52.8%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 100 mL of saturated potassium bicarbonate solution to soak for 4 h, and filter to obtain a solid; then use 200 mL of ethyl acetate to extract the solid three times under reflux for 9 h, using 200-300 mesh Filter the cloth, combine the three filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 Load the extract obtained in step 1 on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) ethanol with a 1% (V / V) saturated ammonia solution.
  • the solution that is, the volume of the saturated ammonia solution is 1% of the volume of the ethanol solution
  • the eluate after 5 times the column volume is collected, and the eluate is concentrated to dryness and crushed to obtain a dry isolate;
  • Step 3 After adding 2% (W / W) sulfuric acid solution to the dried isolate obtained in step 2 to heat and dissolve, the ratio of the volume of the sulfuric acid solution to the weight of the dried isolate is 20: 1 (mL / g). Filter with a mesh filter cloth and collect the filtrate; add sodium hydroxide solution to the collected filtrate to adjust the pH to 12, and then leave it at room temperature, filter, take the filter cake, and pulverize to obtain a dry powder;
  • Step 4 Add 20% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 30: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated according to the method in Example 1. As a result, the weight percentage of strychnine in the extract was about 97.0%. The weight percentage of Shi Ning is about 2.0%, and the extraction rate of strychnine is about 51.7%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 150 mL of saturated ethylamine solution to soak for 3 h, and filter to obtain a solid; then use 250 mL of ethyl acetate to extract the solid twice under reflux for 10 h each, using 200-300 mesh Filter the cloth and combine the two filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 Load the extract obtained in step 1 on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) added with 1.5% (V / V) saturated sodium carbonate solution.
  • Ethanol solution that is, the volume of the saturated sodium carbonate solution is 1.5% of the volume of the ethanol solution
  • the eluate after 5 times the column volume is collected, and the eluate is concentrated to dryness and crushed to obtain a dry isolate;
  • Step 3 After adding 1.5% (W / W) hydrochloric acid solution to the dry isolate obtained in step 2 to heat and dissolve, the ratio of the volume of the hydrochloric acid solution to the weight of the dry isolate is 30: 1 (mL / g), using 200-300 Filter with a mesh filter cloth and collect the filtrate; add potassium hydroxide solution to the collected filtrate to adjust the pH to 11, and then stand at room temperature, filter, take the filter cake and dry, crush to obtain a dry powder;
  • Step 4 Add 30 times the weight of a 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 30: 1 (mL / g). Filter through a 300-mesh filter cloth and collect the filtrate; crystallize the filtrate, filter with a 200-300-mesh filter cloth, dry the filter cake, and pulverize to obtain 0.2103 g of extract.
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated.
  • the weight percentage of strychnine in the extract was about 96.4%.
  • the weight percentage of Shi Ning is about 2.1%, and the extraction rate of strychnine is about 50.7%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 100 mL of saturated potassium carbonate solution to soak for 2 h, and filter to obtain a solid; then use 200 mL of ethyl acetate to extract the solid twice under reflux for 9 h each, using 200-300 mesh Filter the cloth and combine the two filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 The extract obtained in step 1 is loaded on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) ethanol added with a 1.75% (V / V) saturated ammonia solution.
  • Solution that is, the volume of the saturated ammonia solution is 1.75% of the volume of the ethanol solution
  • the eluate after 5 times the column volume is collected, and the eluate is concentrated to dryness and crushed to obtain a dry isolate
  • Step 3 Add 1% (W / W) hydrochloric acid solution to the dry isolate obtained in step 2 and heat to dissolve.
  • the ratio of the volume of the hydrochloric acid solution to the weight of the dry isolate is 20: 1 (mL / g).
  • Step 4 Add 25% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 25: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated.
  • the weight percentage of strychnine in the extract was about 95.3%.
  • the weight percentage of Shi Ning is about 2.1%, and the extraction rate of strychnine is about 50.7%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 50 mL of saturated sodium carbonate solution to soak for 3 h, and filter to obtain a solid; then use 250 mL of ethyl acetate to extract the solid twice under reflux for 9 h each, using 200-300 mesh Filter the cloth and combine the two filtrates, concentrate under reduced pressure to 1/3 of the original volume, and filter again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 Load the extract obtained in step 1 on a 100-200 mesh silica gel chromatography column for chromatography.
  • the eluent is 95% (W / W) added with 2% (V / V) saturated potassium carbonate solution.
  • Ethanol solution that is, the volume of the saturated potassium carbonate solution is 2% of the volume of the ethanol solution
  • the eluent solution after 5 times the column volume is collected, and the eluate is concentrated to dryness and crushed to obtain a dry isolate;
  • Step 3 After adding the 1% (W / W) hydrochloric acid solution to the dry isolate obtained in step 2 to heat and dissolve, the ratio of the volume of the hydrochloric acid solution to the weight of the dry isolate is 30: 1 (mL / g), using 200-300 Filter with a mesh filter cloth and collect the filtrate; add sodium hydroxide solution to the collected filtrate to adjust the pH to 13, and then stand at room temperature, filter with a 200-300 mesh filter cloth, take the filter cake and pulverize to obtain a dry powder ;
  • Step 4 Add the 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 30: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated.
  • the weight percentage of strychnine in the extract was about 95.3%.
  • the weight percentage of Shi Ning is about 2.1%, and the extraction rate of strychnine is about 50.4%.
  • Step 1 Take 50 g of medicinal herbs, crush, add 50 mL of saturated sodium carbonate solution to soak for 3 h, and filter to obtain a solid substance; then use 250 mL of ethyl acetate to reflux extract the solid three times, 10 hours each time, and use a 200-300 mesh filter Cloth filtration, the three filtrates were combined, concentrated under reduced pressure to 1/3 of the original volume, and filtered again with a 200-300 mesh filter cloth to obtain an extract;
  • Step 2 The extract obtained in step 1 is loaded on a 100-200 mesh silica gel column for chromatography.
  • the eluent is 95% (W / W) ethanol added with a 0.75% (V / V) saturated ammonia solution.
  • Solution that is, the volume of the saturated ammonia solution is 0.75% of the volume of the ethanol solution
  • collect the eluate after 4 times the column volume concentrate the eluate to dryness, and pulverize to obtain a dry isolate;
  • Step 3 After adding 30 times the weight of the 1% (W / W) hydrochloric acid solution to the dry isolate obtained in step 2 to heat and dissolve, the ratio of the volume of the hydrochloric acid solution to the weight of the dry isolate is 30: 1 (mL / g). Filter through a 200-300 mesh filter cloth and collect the filtrate; add sodium hydroxide solution to the collected filtrate to adjust the pH to 12, and then stand at room temperature, filter with a 200-300 mesh filter cloth, take the filter cake, and crush it To get dry powder;
  • Step 4 Add the 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 30: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine in the extract were measured and calculated.
  • the weight percentage of strychnine in the extract was about 99.1%.
  • the weight percentage of Shi Ning is about 0.1%, and the extraction rate of strychnine is about 51.1%.
  • Step 1 Take 50g of pomegranate medicinal material, crush, add 50mL saturated sodium bicarbonate solution to soak for 4h, and filter to obtain a solid; then use 250mL ethyl acetate to extract the solid twice under reflux for 9h each time, using 200 ⁇ 300 Filtered with a mesh filter cloth, combined the filtrates twice, concentrated under reduced pressure to 1/3 of the original volume, and filtered again with a 200-300 mesh filter cloth to obtain an extract solution;
  • Step 2 The extract obtained in step 1 is loaded on a 100-200 mesh silica gel chromatography column for chromatography.
  • the eluent is a 95% (W / W) ethanol solution with a saturated ammonia solution.
  • the volume ratio of the saturated ammonia solution to the ethanol solution in the eluent is shown in Table 1.
  • the eluate after 5 times the column volume was collected, and the eluate was concentrated to dryness and pulverized to obtain a dry isolate;
  • Step 3 Add 1% (W / W) hydrochloric acid solution to the dry isolate obtained in step 2 to heat and dissolve.
  • the ratio of the volume of the hydrochloric acid solution to the weight of the dry isolate is 35: 1 (mL / g).
  • Step 4 Add the 30% (W / W) ethanol aqueous solution to the dry powder obtained in step 3 to heat and dissolve.
  • the ratio of the volume of the ethanol solution to the weight of the dry powder is 30: 1 (mL / g).
  • the content of strychnine, strychnine, and strychnine extraction rate in the extract were measured and calculated. The results are shown in Table 1.
  • step 2 the aqueous ammonia solution was replaced with an ammonium acetate solution of the same concentration; the rest of the operations were the same as in Example 6; 0.1466 g of an extract was obtained.
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated according to the method in Example 1. As a result, the weight percentage of strychnine in the extract was about 85.4%. The weight percentage of Shi Ning is about 10.7%, and the extraction rate of strychnine is about 31.3%.
  • the content of strychnine in the extract prepared by the method of the present invention is significantly higher and the content of strychnine is significantly lower.
  • the separation effect of alkali and strenin is better, and the extraction rate of pomegranate is significantly higher.
  • step 2 the extract obtained in step 1 is loaded on an AB-8 macroporous adsorption resin for chromatography.
  • the eluent used is the same as in Example 11, and the rest of the operations are the same as in Example 11; the extract is obtained. 1.447g.
  • the content of strychnine, strychnine, and strychnine in the extract was measured and calculated according to the method in Example 1. As a result, the weight percentage of strychnine in the extract was about 25.9%. The weight percentage of Shi Ning is about 61.3%, and the extraction rate of strychnine is about 93.4%.
  • step 2 the extract obtained in step 1 is loaded on an alumina column for chromatography.
  • the eluent used is the same as in Example 11, and the remaining operations are the same as in Example 11; 1.5549 g of the extract is obtained.
  • the content of strychnine, strychnine, and strychnine in the extract were measured and calculated.
  • the weight percentage of strychnine in the extract was about 24.6%.
  • the weight percentage of Shi Ning is about 62.4%, and the extraction rate of strychnine is about 93.8%.
  • the saturated solution refers to a saturated solution at 10 ° C to 30 ° C.

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Abstract

本发明属于医药领域，涉及一种制备马钱子提取物方法，包括：(1)第一碱性溶液浸润马钱子，固液分离，得固相物；(2)乙酸乙酯浸提步骤(1)固相物，固液分离，得提取液；(3)对步骤(2)提取液硅胶柱层析，洗脱剂为添加选自氨水溶液、碳酸钾溶液、碳酸钠溶液、碳酸氢钠溶液和碳酸氢钾溶液中至少一种的无水乙醇或乙醇溶液，得洗脱液；(4)将洗脱液干燥，得干燥物；(5)将干燥物溶解在酸溶液，固液分离，得液相物；(6)将步骤(5)液相物pH值调至8～13，固液分离，得固相物；(7)步骤(6)固相物重结晶。本发明涉及马钱子提取物及医药用途。本发明马钱子提取物中马钱子碱含量较高，士的宁含量较低，使用安全、有效。

Description

一种制备马钱子提取物的方法、制得的提取物及其用途
本申请要求申请号为201810938283.2、发明名称为“一种马钱子碱的提取分离纯化方法”的中国发明申请的优先权。
技术领域
本发明属于医药领域，具体涉及一种制备马钱子提取物的方法，还涉及制得的马钱子提取物及其医药用途。
背景技术
马钱子又名番木鳖，始载于《本草纲目》，味苦，性寒，有大毒，归肝、脾经，其药用功效为通络止痛、散结消肿，临床上多用于治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒、咽喉肿痛等疾病。但是，马钱子有剧毒，这一特性严重影响了马钱子的临床应用，马钱子的一般治疗量为0.3-0.6g，超过该治疗量易发生中毒。因此，近年来，马钱子的使用安全性已成为一个研究热点。
研究表明，马钱子的主要有效成分是总生物碱，其包括马钱子碱、士的宁、番木鳖次碱、马钱子新碱、番木鳖甙、依卡精等；其中，马钱子碱具有抗炎、镇痛作用；士的宁基本无抗炎、镇痛作用，并且，士的宁是马钱子的主要毒性成分；有文献报道：小鼠灌服士的宁、马钱子碱、马钱子仁的LD50分别为3.27mg/kg、233mg/kg和234.5mg/kg；小鼠腹腔注射前述药物的LD50分别为1.53mg/kg、69.77mg/kg和76mg/kg；士的宁的成人口服中毒剂量为5～10mg，成人口服致死剂量为30mg。因此，兼顾马钱子的药效和用药安全性的做法是在降低士的宁含量的同时，提高马钱子碱含量。
士的宁和马钱子碱同属于生物碱，二者结构相似，如下图所示。士的宁和马钱子碱的结构差异在于：马钱子碱苯环的2,3位上连接有甲氧基，而士的宁苯环的2，3位上连接氢，这使得二者的极性较相近。因此，还未发现能将士的宁和马钱子碱分离的有效方法。
Figure PCTCN2019073450-appb-000001
马钱子碱：X为CH 3O-；士的宁：X为H-。
目前亟需一种制备马钱子提取物的方法，以降低士的宁含量同时提高马钱子碱的含量。
发明内容
本发明目的之一在于提供一种制备马钱子提取物的方法，制得的马钱子提取物中马钱子碱含量较高，同时士的宁含量较低，并且马钱子碱的提取率较高。在此基础上，本发明还提供了一种马钱子提取物。本发明又一目的在于提供马钱子提取物的医药用途。
本发明第一方面涉及一种制备马钱子提取物的方法，包括如下步骤：
(1)采用第一碱性溶液浸润马钱子生药，固液分离，得到固相物；
(2)采用乙酸乙酯浸提步骤(1)得到的固相物，固液分离，得到提取液；
(3)对步骤(2)得到的提取液进行硅胶柱层析，洗脱剂为添加有第二碱性溶液的无水乙醇或者添加有第二碱性溶液的乙醇溶液，收集洗脱液；其中，第二碱性溶液选自氨水溶液、碳酸钾溶液、碳酸钠溶液、碳酸氢钠溶液和碳酸氢钾溶液中的至少一种；
(4)将步骤(3)收集到的洗脱液进行干燥，得到干燥物；
(5)将步骤(4)得到的干燥物溶解在酸溶液中，固液分离，得到液相物；
(6)将步骤(5)得到液相物的pH值调节至8～13(例如9、10、11、12)，固液分离，得到固相物；
(7)将步骤(6)得到的固相物进行重结晶，得到提取物。
本发明第一方面的一些实施方式中，步骤(3)中，所述第二碱性溶液为饱和溶液。
本发明第一方面的一些实施方式中，步骤(3)中，所述第二碱性溶液选自饱和氨水溶液、饱和碳酸钾溶液、饱和碳酸钠溶液、饱和碳酸氢钠溶液和饱和碳酸氢钾溶液中的至少一种。
本发明第一方面的一些实施方式中，步骤(3)中，所述第二碱性溶液选自10℃～30℃的饱和氨水溶液、饱和碳酸钾溶液、饱和碳酸钠溶液、饱和碳酸氢钠溶液和饱和碳酸 氢钾溶液中的至少一种。
本发明第一方面的一些实施方式中，第二碱性溶液的浓度为0.1％(V/V)～2％(V/V)，优选为0.5％(V/V)～1％(V/V)。
本发明第一方面的一些实施方式中，步骤(3)中，洗脱剂中，第二碱性溶液体积为无水乙醇或乙醇溶液体积的0.05％～5％，例如0.07％、0.1％、0.2％、0.3％、0.5％、0.7％、0.9％、1％、1.5％、2％、2.3％、2.6％、3％、3.2％、3.4％、3.6％、3.5％、3.8％、4％、4.3％、4.5％、4.7％、4.9％。
本发明第一方面的一些实施方式中，步骤(3)中，所述硅胶柱为70～300目硅胶柱，例如90目、100目、120目、140目、160目、180目、200目、220目、240目、260目、280目或290目的硅胶柱。
本发明第一方面的一些实施方式中，步骤(3)中，收集3至8倍(例如4、5、6、7倍)柱体积之后的洗脱液。
本发明第一方面的一些实施方式中，所述方法包括如下1)至22)中的一项或多项：
1)步骤(1)中，所述第一碱性溶液选自碳酸钾溶液、碳酸氢钠溶液、碳酸钠溶液、碳酸氢钾溶液和胺类溶液中的至少一种；
优选地，所述第一碱性溶液选自碳酸钾溶液、碳酸氢钠溶液、碳酸钠溶液、甲胺溶液、碳酸氢钾溶液和乙胺溶液中的至少一种；
更优选地，所述第一碱性溶液选自饱和碳酸钾溶液、饱和碳酸氢钠溶液、饱和碳酸钠溶液、饱和甲胺溶液、饱和碳酸氢钾溶液和饱和乙胺溶液中的至少一种；
进一步优选地，所述第一碱性溶液选自10℃～30℃的饱和碳酸钾溶液、饱和碳酸氢钠溶液、饱和碳酸钠溶液、饱和甲胺溶液、饱和碳酸氢钾溶液和饱和乙胺溶液中的至少一种；
2)步骤(1)中，第一碱性溶液和马钱子生药的比例为(0.5～10):1(mL/g)，优选为(0.5～3):1(mL/g)，例如0.8:1(mL/g)、1:1(mL/g)、2:1(mL/g)、3:1(mL/g)、4:1(mL/g)、5:1(mL/g)、6:1(mL/g)、7:1(mL/g)、8:1(mL/g)、9:1(mL/g)；
3)步骤(1)中，浸润时间为1～10小时，优选为2～4小时，更优选为3小时，例如2、3、4、5、6、7、8、9小时；
4)步骤(1)中，通过过滤进行固液分离；
优选地，采用100～500目(例如200-300目、250目、350目、400目、450目)的滤布过滤；
5)步骤(1)中，浸润前，对马钱子生药进行破碎处理；
6)步骤(2)中，乙酸乙酯和步骤(1)得到的固相物的比例为(1～10):1(mL/g)，优选为(4～6):1(mL/g)，例如2:1(mL/g)、3:1(mL/g)、4:1(mL/g)、5:1(mL/g)、6:1(mL/g)、7:1(mL/g)、8:1(mL/g)、9:1(mL/g)；
7)步骤(2)中，浸提时间为2～16小时，优选为6～12小时，更优选为8～10小时，例如3、4、5、6、7、8、9、10、11、12、13、14、15小时；
8)步骤(2)中，在回流条件下浸提；
9)步骤(2)中，浸提次数为一次或多次，优选为一次、二次、三次或四次；
10)步骤(2)中，通过过滤进行固液分离；
优选地，采用100～500目(例如200-300目、250目、350目、400目、450目)的滤布过滤；
11)在步骤(2)和(3)之间，还包括步骤(2’)：将步骤(2)得到的提取液浓缩，得到浓缩液；将浓缩液过滤，得到的滤液用于进行步骤(3)中的硅胶柱层析；
优选地，步骤(2’)中，浓缩液体积为步骤(2)得到提取液体积的1/4～1/2，例如1/3；
优选地，步骤(2’)中，采用100～500目(例如200-300目、250目、350目、400目、450目)的滤布过滤；
12)步骤(3)中，所述乙醇溶液的浓度为70％(W/W)～99％(W/W)，例如75％(W/W)、80％(W/W)、85％(W/W)、90％(W/W)、95％(W/W)、98％(W/W)；
13)在步骤(4)和(5)之间，还包括步骤(4’)：对步骤(4)得到的干燥物进行破碎处理；
14)步骤(5)中，所述酸溶液选自硫酸溶液、盐酸溶液和磷酸溶液中的至少一种；
15)步骤(5)中，所述酸溶液的浓度为0.6％(W/W)～9％(W/W)，例如1％(W/W)、2％(W/W)、3％(W/W)、4％(W/W)、5％(W/W)、6％(W/W)、7％(W/W)、8％(W/W)；
优选地，步骤(5)中，加热条件下溶解；
16)步骤(5)中，酸溶液和步骤(4)得到的干燥物的比例为(12～47):1(mL/g)，例如13:1(mL/g)、15:1(mL/g)、17:1(mL/g)、19:1(mL/g)、21:1(mL/g)、22:1(mL/g)、25:1(mL/g)、27:1(mL/g)、29:1(mL/g)、31:1(mL/g)、33:1(mL/g)、35:1(mL/g)、37:1(mL/g)、39:1(mL/g)、40:1(mL/g)、42:1(mL/g)、44:1(mL/g)、46:1(mL/g)；
17)步骤(5)中，通过过滤进行固液分离；
优选地，采用100～500目(例如200-300目、250目、350目、400目、450目)的滤布过滤；
18)步骤(6)中，通过添加碱液调节pH值；
优选地，所述碱液为氢氧化钾溶液和/或氢氧化钠溶液；
19)步骤(6)中，通过过滤进行固液分离；
优选地，采用100～500目(例如200-300目、250目、350目、400目、450目)的滤布过滤；
20)在步骤(6)和(7)之间，还包括步骤(6’)：将步骤(6)得到的固相物进行干燥，破碎；
21)步骤(7)中，采用乙醇溶液对步骤(6)得到的固相物进行重结晶；
优选地，乙醇溶液的浓度为10％(W/W)～60％(W/W)，例如15％(W/W)、20％(W/W)、30％(W/W)、40％(W/W)、50％(W/W)、55％(W/W)；
优选地，通过如下的步骤进行重结晶：
(i)将步骤(6)得到的固相物溶解在乙醇溶液中，过滤，收集滤液；
(ii)将步骤(i)收集的滤液静置析晶，过滤，收集滤渣即为提取物；
更优选地，步骤(i)中，乙醇溶液和固相物的比例为(10～50):1(mL/g)，例如12:1(mL/g)、14:1(mL/g)、16:1(mL/g)、20:1(mL/g)、22:1(mL/g)、24:1(mL/g)、26:1(mL/g)、30:1(mL/g)、32:1(mL/g)、35:1(mL/g)、37:1(mL/g)、40:1(mL/g)、43:1(mL/g)、46:1(mL/g)、47:1(mL/g)、49:1(mL/g)；
更优选地，步骤(i)和(ii)中，采用100～500目(例如200-300目、250目、350目、400目、450目)的滤布过滤；
更优选地，步骤(i)中，加热条件下溶解；
更优选地，步骤(ii)中，室温下静置析晶；
进一步优选地，所述室温为10℃～30℃；
22)步骤(7)中，重结晶次数为一次或多次，优选为一次或两次。
本发明第一方面的一些实施方式中，步骤(5)中，干燥物部分溶解在酸溶液中。
本发明第一方面的一些实施方式中，步骤(i)中，固相物部分溶解在乙醇溶液中。
本发明第一方面的一些实施方式中，步骤(4)、步骤(6’)中的“干燥”操作在不破坏或不影响被提取成分生物活性的温度下进行。本领域技术人员公知不破坏或不影响被提取成分生物活性的温度范围。
本发明第一方面的一些实施方式中，步骤(5)、步骤(i)中的“加热”操作在不破坏或不影响被提取成分生物活性的温度下进行。本领域技术人员公知不破坏或不影响被提取成分生物活性的温度范围。
本发明中，所述乙醇溶液、氨水溶液、碳酸钾溶液、碳酸钠溶液、碳酸氢钠溶液、碳酸氢钾溶液、酸溶液均为水溶液。
本发明第二方面涉及一种马钱子提取物，其中，马钱子碱的重量百分含量≥94.5％(优选为≥95％、≥96％或≥97％，例如为94.5％～99.9％、95％～99.9％、95％～99.8％、96％、97％、98％、99％、99.2％、99.4％、99.6％)，并且，士的宁的重量百分含量≤3％(优选为≤2.9％、≤2.8％、≤2.7％、≤2.3％，例如0.01％～2.9％、0.05％～2.9％、0.05％～2.8％、0.1％、0.08％、2.1％、2.2％、2.3％、2.4％、2.6％、2.7％)。
本发明第二方面的一些实施方式中，所述马钱子提取物由本发明第一方面所述的方法制得。
本发明第三方面涉及一种药物组合物，其包本发明第二方面所述的马钱子提取物以及药学上可接受的辅料。
本发明第三方面的一些实施方式中，所述药学上可接受的辅料选自填充剂、崩解剂、赋形剂、粘合剂、润滑剂、表面活性剂、稀释剂、防腐剂、稳定剂、矫味剂、增稠剂、助流剂、保湿剂、pH调节剂和透皮剂中的至少一种。
本发明第三方面的一些实施方式中，针对不同药物剂型可选择不同的药学上可接受的辅料；例如，在片剂、胶囊剂、颗粒剂等固体制剂中常用填充剂、崩解剂、赋形剂、粘合剂、润滑剂、表面活性剂、填充剂等；在糖浆、口服液等液体制剂形式中常用表面活性剂、稀释剂、防腐剂、稳定剂、矫味剂、增稠剂、助流剂等；在外用制剂形式中常用表面活性剂、增稠剂、保湿剂、pH调节剂、透皮剂、防腐剂等。
填充剂包括但不限于淀粉、乳糖、糊精、糖粉、微晶纤维素、甘露醇、木糖醇等。崩解剂包括但不限于干淀粉、羧甲基淀粉钠、交联聚维酮等。赋形剂包括但不限于滑石粉、硬脂酸镁等。粘合剂包括但不限于淀粉浆、明胶、羧丙甲纤维素。润滑剂包括但不限于硬脂酸镁、微粉硅胶、玉米淀粉等。增溶剂包括但不限于聚山梨酯类、聚氧乙烯脂肪酸酯类等。助溶剂包括但不限于碘化钾、醋酸钠、苯甲酸钠等。防腐剂包括但不限于对羟基苯甲酸酯类、苯甲酸、山梨酸、山梨酸钾、羟苯乙酯、羟苯丙酯、苯酚等。矫味剂包括但不限于蔗糖、木糖醇、柠檬、薄荷油、阿拉伯胶等。助悬剂包括但不限于甘油、海藻酸钠、琼脂、甲基纤维素等。湿润剂包括但不限于聚山梨酯类、泊洛沙姆等。表面活性剂包括但不限于硬脂酸钠、单甘油脂肪酸酯、聚氧乙烯(40)脂肪酸酯、吐温类等。增稠剂包括但不限于交联聚丙烯树脂类、羟丙甲基纤维素、黄原胶等。保湿剂包括但不限于甘油、丙二醇、异丙醇、透明质酸等。pH调节剂包括但不限于有机胺类如三乙醇胺、三乙胺、二乙胺、氢氧化钠、碳酸氢钠、碳酸钠等。透皮剂包括但不限于氮酮、薄荷醇、精油、二甲基亚砜、丙二醇、肉豆蔻异丙基酸酯、N-甲基吡咯烷酮、月桂酸聚乙二醇甘油酯、聚甘油脂肪酸酯、油酸聚乙二醇甘油酯、辛酸癸酸聚乙二醇甘油酯。
本发明第三方面的一些实施方式中，所述药物组合物通过人工配制(例如，将马钱子提取物及药学上可接受的辅料人工混合)得到。
本发明第四方面涉及一种通络止痛或散结消肿的方法或者一种治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒或咽喉肿痛疾病的方法，包括给予有需求的受试者以有效量的本发明第二方面所述的马钱子提取物或者本发明第三方面所述的药物组合物。
本发明第五方面涉及本发明第二方面所述的马钱子提取物或者本发明第三方面所述的药物组合物在制备通络止痛、散结消肿的药物或者在制备治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒或咽喉肿痛疾病的药物中的用途。
本发明第六方面涉及本发明第二方面所述的马钱子提取物或者本发明第三方面所述的药物组合物，用于通络止痛、散结消肿或者治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒或咽喉肿痛疾病。
本发明还涉及如下几方面内容：
1、一种马钱子碱的提取分离纯化方法，包括如下步骤：
步骤1：以马钱子药材为原料，经碱溶液浸润后，以乙酸乙酯为溶剂提取，得提取液A；
步骤2：以加碱水的乙醇为洗脱液(即洗脱剂)，提取液A经硅胶层析柱分离，得干燥分离物B；
步骤3：干燥分离物B经酸溶得滤液C，再经碱沉，得干燥粉末D；
步骤4：干燥粉末D经乙醇重结晶，即得马钱子碱。
2、根据第1方面所述的提取分离方法，所述步骤1中，所述碱为无机碱，优选的，所述碱为碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、氢氧化钠、氢氧化钾、氨水中的一种、两种或两种以上的组合。
3、根据第1方面所述的提取分离方法，所述步骤1中，所述碱为有机碱，优选的，所述碱为胺类、生物碱类、叔丁醇钾、叔丁醇钠、正丁基锂中的一种、两种或两种以上的组合。
4、根据第1-3方面所述的任一种提取分离方法，所述步骤1中，碱溶液体积用量为0.5-3倍药材重量，体积/重量：ml/g，浸润时间为2-4h，优选的，碱溶液体积用量为0.5-1.5倍药材重量，体积/重量：ml/g，浸润时间为3h。
5、根据第1-4方面所述的任一种提取分离方法，所述步骤1中乙酸乙酯体积用量为4-6倍药材重量，体积/重量：ml/g。
6、根据第1-5方面所述的任一种提取分离方法，所述步骤1中乙酸乙酯回流提取1-4次，每次6-12h，优选的，回流提取2次，每次8-10h。
7、根据第1-6方面所述的任一种提取分离方法，所述步骤1提取液减压浓缩至原提取液体积的1/3-1。
8、根据第1-7方面所述的任一种提取分离方法，所述步骤2中碱水为碳酸钠溶液、碳酸氢钠溶液、碳酸钾溶液、碳酸氢钾、氨水的一种、两种或两种以上的组合。
9、根据第1-8方面所述的任一种提取分离方法，所述步骤2中碱水浓度为0.1％-2％，优选的，碱水浓度为0.5-1％。
10、根据第1-9方面所述的任一种提取分离方法，所述步骤2中提取液A经硅胶层析柱分离后，收集5倍柱体积后的洗脱溶液(即洗脱液)。
本发明中，如无特别说明，其中：
术语“碱性溶液”是指常温下pH值大于7或者其氢氧根离子浓度大于氢离子浓度的溶液。
术语“生药”是指纯天然未经过加工或者简单加工后的植物类、动物类和矿物类中药材。
术语“浸提”是指将药材中的有效成分溶解在适宜溶剂中，以提取出有效成分的过程。
术语“洗脱剂”是指在硅胶柱层析过程中淋洗或洗脱时使用的溶剂。
术语“洗脱液”是指在硅胶柱层析过程中流出柱子的那部分液体。
术语“饱和溶液”是指在一定温度下，溶剂中溶解的溶质达到最大量(溶解度)的溶液。
本发明取得了如下的至少一项有益效果：
1、本发明方法得到的马钱子提取物中马钱子碱含量较高，同时有毒物质士的宁的含量较低，提高了药物的有效性和安全性，并且，本发明方法的马钱子碱提取率较高。
2、本发明马钱子提取物具有通络止痛、散结消肿的功效，还具有治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒或咽喉肿痛的药效。
具体实施方式
下面将结合实施例对本发明的实施方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，决不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
步骤1：取马钱子药材50g，破碎，加入150mL饱和碳酸钾溶液浸润4h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取三次，每次10h，采用 200～300目滤布过滤，合并三次的滤液，减压浓缩至原体积的1/3，再次用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有0.1％(V/V)饱和氨水溶液的95％(W/W)乙醇溶液(即饱和氨水溶液体积为乙醇溶液体积的0.1％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入2％(W/W)硫酸溶液加热溶解，硫酸溶液体积与干燥分离物重量的比例为40:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至11，然后，室温下静置，用200～300目滤布过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入35％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为20:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2096g。
取士的宁对照品6mg，马钱子碱对照品5mg，精密称量，分别置于10mL量瓶中，加三氯甲烷适量使溶解并稀释至刻度，摇匀。分别精密量取2mL，置于同一10mL量瓶中，用甲醇稀释至刻度，摇匀，得到标准溶液，其中，士的宁含量为0.12mg/mL，马钱子碱含量为0.1mg/mL。
采用高效液相色谱法检测提取物及标准溶液，经计算得到提取物中马钱子碱和士的宁的含量。液相色谱的操作条件如下：色谱柱为C18(规格为4.6mm×250mm，5μm)，以乙腈-0.01mol/L庚烷磺酸钠与0.02mol/L磷酸二氢钾等量混合溶液(用10％磷酸调节pH值至2.8)(21:79)为流动相，检测波长为260nm，柱温为40℃，流速为1.0mL/min。
取马钱子药材破碎，取约0.6，精密称定，置具塞锥形瓶中，加氢氧化钠试液3mL混匀，放置30分钟，精密加入三氯甲烷20mL，密塞，称定重量，置水浴中回流提取2小时，放冷，再称定重量，用三氯甲烷补足减失重量，摇匀，分取三氯甲烷液，用铺有少量无水硫酸钠的滤纸滤过，弃去初滤液，精密量取续滤液3mL，置10mL量瓶中，加甲醇至刻度，摇匀，得到测试溶液。将测试溶液及前述标准溶液采用前述的高效液相色谱法检测，经计算得到马钱子生药中的马钱子碱含量。
然后，按照如下公式计算出马钱子碱的提取率。
马钱子碱的提取率＝100％×(提取物中马钱子碱的含量×提取物重量)/(生药中马钱子碱含量×马钱子生药重量)
结果得到，提取物中马钱子碱重量百分含量约为95.8％、士的宁重量百分含量约为2.1％，马钱子碱的提取率约为50.2％。
实施例2
步骤1：取马钱子药材50g，破碎，加入75mL饱和碳酸氢钠溶液浸润3h，过滤，得到固体物；然后采用300mL乙酸乙酯对固体物回流提取两次，每次9h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有0.25％(V/V)饱和碳酸钾溶液的95％(W/W)乙醇溶液(即饱和碳酸钾溶液体积为乙醇溶液体积的0.25％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1％(W/W)磷酸溶液加热溶解，磷酸溶液体积与干燥分离物重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钾溶液调节pH值至9，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为20:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2119g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为96.8％、士的宁重量百分含量约为1.9％，马钱子碱的提取率约为51.3％。
实施例3
步骤1：取马钱子药材50g，破碎，加入50mL饱和碳酸钠溶液浸润2h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取两次，每次10h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次用200～300 目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有0.5％(V/V)饱和碳酸钠溶液的95％(W/W)乙醇溶液(即饱和碳酸钠溶液体积为乙醇溶液体积的0.5％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1.5％(W/W)硫酸溶液加热溶解，硫酸溶液体积与干燥分离物重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至10，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入25％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为20:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2177g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为97.2％，士的宁重量百分含量约为1.9％，马钱子碱的提取率约为52.9％。
实施例4
步骤1：取马钱子药材50g，破碎，加入75mL的饱和碳酸钠溶液浸润3h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取两次，每次8h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有0.5％(V/V)饱和氨水溶液95％(W/W)乙醇溶液(即饱和氨水溶液体积为乙醇溶液体积的0.5％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1％(W/W)盐酸溶液加热溶解，盐酸溶液体积与干燥分离物重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至12，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2173g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为97.2％、士的宁重量百分含量约为1.9％，马钱子碱的提取率约为52.8％。
实施例5
步骤1：取马钱子药材50g，破碎，加入50mL饱和甲胺溶液浸润3h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取两次，每次9h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有0.75％(V/V)饱和碳酸氢钠溶液的95％(W/W)乙醇溶液(即饱和碳酸氢钠溶液体积为乙醇溶液体积的0.75％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1％(W/W)磷酸溶液加热溶解后，磷酸溶液体积与干燥分离物重量的比例为35:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钾溶液调节pH值至10，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为20:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2164g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为97.4％、士的宁重量百分含量约为1.9％，马钱子碱的提取率约为52.7％。
实施例6
步骤1：取马钱子药材50g，破碎，加入50mL饱和碳酸氢钠溶液浸润4h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取两次，每次9h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有0.75％(V/V)饱和氨水溶液的95％(W/W)乙醇溶液(即饱和氨水溶液体积为乙醇溶液体积的0.75％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1％(W/W)盐酸溶液加热溶解，盐酸溶液体积与干燥分离物重量的比例为35:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至10，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为20:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2166g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为97.5％、士的宁重量百分含量约为1.9％，马钱子碱的提取率约为52.8％。
实施例7
步骤1：取马钱子药材50g，破碎，加入100mL饱和碳酸氢钾溶液浸润4h，过滤，得到固体物；然后采用200mL乙酸乙酯对固体物回流提取三次，每次9h，采用200～300目滤布过滤，合并三次的滤液，减压浓缩至原体积的1/3，再次采用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有1％(V/V)饱和氨水溶液的95％(W/W)乙醇溶液(即饱和氨水溶液体积为乙醇溶液体积的1％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入2％(W/W)硫酸溶液加热溶解后， 硫酸溶液体积与干燥分离物重量的比例为20:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至12，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入20％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2132g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为97.0％、士的宁重量百分含量约为2.0％，马钱子碱的提取率约为51.7％。
实施例8
步骤1：取马钱子药材50g，破碎，加入150mL饱和乙胺溶液浸润3h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取两次，每次10h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次采用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有1.5％(V/V)饱和碳酸钠溶液的95％(W/W)乙醇溶液(即饱和碳酸钠溶液体积为乙醇溶液体积的1.5％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1.5％(W/W)盐酸溶液加热溶解后，盐酸溶液体积与干燥分离物重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钾溶液调节pH值至11，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30倍重量的30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2103g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为96.4％、士的宁 重量百分含量约为2.1％，马钱子碱的提取率约为50.7％。
实施例9
步骤1：取马钱子药材50g，破碎，加入100mL饱和碳酸钾溶液浸润2h，过滤，得到固体物；然后采用200mL乙酸乙酯对固体物回流提取两次，每次9h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次采用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有1.75％(V/V)饱和氨水溶液的95％(W/W)乙醇溶液(即饱和氨水溶液体积为乙醇溶液体积的1.75％)，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1％(W/W)盐酸溶液，加热溶解，盐酸溶液体积与干燥分离物重量的比例为20:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至11，然后，室温下静置，过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入25％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为25:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2128g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为95.3％、士的宁重量百分含量约为2.1％，马钱子碱的提取率约为50.7％。
实施例10
步骤1：取马钱子药材50g，破碎，加入50mL饱和碳酸钠溶液浸润3h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取两次，每次9h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次采用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有2％(V/V)饱和碳酸钾溶液的95％(W/W)乙醇溶液(即饱和碳酸钾溶 液体积为乙醇溶液体积的2％)，收集5倍柱体积之后的洗脱溶液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1％(W/W)盐酸溶液加热溶解后，盐酸溶液体积与干燥分离物重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至13，然后，室温下静置，用200～300目滤布过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物0.2115g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为95.3％、士的宁重量百分含量约为2.1％，马钱子碱的提取率约为50.4％。
实施例11
步骤1：取马钱子药材50g，破碎，加入50mL饱和碳酸钠溶液浸润3h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取三次，每次10h，采用200～300目滤布过滤，合并三次的滤液，减压浓缩至原体积的1/3，再次采用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有0.75％(V/V)饱和氨水溶液的95％(W/W)乙醇溶液(即饱和氨水溶液体积为乙醇溶液体积的0.75％)，收集4倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入30倍重量的1％(W/W)盐酸溶液加热溶解后，盐酸溶液体积与干燥分离物重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至12，然后，室温下静置，用200～300目滤布过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液，将滤液析晶，过滤，取滤饼干燥，粉碎，得到固体物；重复一次上述重 结晶的操作；即得到提取物0.2063g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为99.1％、士的宁重量百分含量约为0.1％，马钱子碱的提取率约为51.1％。
实施例12-18
步骤1：取马钱子药材50g，破碎，加入50mL饱和碳酸氢钠溶液浸润4h，过滤，得到固体物；然后采用250mL乙酸乙酯对固体物回流提取两次，每次9h，采用200～300目滤布过滤，合并两次的滤液，减压浓缩至原体积的1/3，再次采用200～300目滤布过滤，得到提取液；
步骤2：将步骤1得到的提取液加载至100-200目硅胶层析柱上进行层析，洗脱剂为添加有饱和氨水溶液的95％(W/W)乙醇溶液，其中，各实施例洗脱剂中饱和氨水溶液与乙醇溶液的体积比见表1，收集5倍柱体积之后的洗脱液，将洗脱液浓缩干燥，粉碎，得到干燥分离物；
步骤3：向步骤2得到的干燥分离物中加入1％(W/W)盐酸溶液加热溶解，盐酸溶液体积与干燥分离物重量的比例为35:1(mL/g)，采用200～300目滤布过滤，收集滤液；向收集的滤液中加入氢氧化钠溶液调节pH值至12，然后，室温下静置，用200～300目滤布过滤，取滤饼干燥，粉碎，得干燥粉末；
步骤4：向步骤3得到的干燥粉末中加入30％(W/W)乙醇水溶液加热溶解，乙醇溶液体积与干燥粉末重量的比例为30:1(mL/g)，采用200～300目滤布过滤，收集滤液；将滤液析晶，用200～300目滤布过滤，取滤饼干燥，粉碎，即得提取物。按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果见表1。
表1
Figure PCTCN2019073450-appb-000002
Figure PCTCN2019073450-appb-000003
对比例1
步骤2中，将氨水溶液替换为等浓度的醋酸铵溶液；其余操作均与实施例6相同；得到提取物0.1466g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为85.4％、士的宁重量百分含量约为10.7％，马钱子碱的提取率约为31.3％。
可见，与采用添加有醋酸铵的乙醇作洗脱液相比，本发明方法制得的提取物中马钱子碱含量明显更高、士的宁含量明显更低，本发明方法对马钱子碱和士的宁的分离效果更佳，对马钱子的提取率明显更高。
对比例2
步骤2中，将步骤1得到的提取液加载至AB-8大孔吸附树脂上进行层析，采用的洗脱剂与实施例11中相同，其余操作也均与实施例11相同；得到提取物1.447g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为25.9％、士的宁重量百分含量约为61.3％，马钱子碱的提取率约为93.4％。
可见，与采用AB-8大孔吸附树脂进行层析相比，本发明方法制得的提取物中马钱子碱含量显著提高，同时士的宁含量显著降低，对马钱子碱和士的宁的分离效果明显更佳。
对比例3
步骤2中，将步骤1得到的提取液加载至氧化铝柱上进行层析，采用的洗脱剂与实施例11中相同，其余操作也均与实施例11相同；得到提取物1.5549g。
按照实施例1中的方法检测并计算出提取物中马钱子碱含量、士的宁含量以及马钱子碱提取率，结果得到，提取物中马钱子碱重量百分含量约为24.6％、士的宁重量百分含量约为62.4％，马钱子碱的提取率约为93.8％。
可见，与采用氧化铝柱进行层析相比，本发明方法制得的提取物中马钱子碱含量显著提高，同时士的宁含量显著降低，对马钱子碱和士的宁的分离效果明显更佳。
上述实施例和对比例中，饱和溶液均指10℃～30℃的饱和溶液。
最后应当说明的是：以上实施例仅用以说明本发明的技术方案而非对其限制；尽管参照较佳实施例对本发明进行了详细的说明，所属领域的普通技术人员应当理解：依然可以对本发明的具体实施方式进行修改或者对部分技术特征进行等同替换；而不脱离本发明技术方案的精神，其均应涵盖在本发明请求保护的技术方案范围当中。

Claims (12)

  1. 一种制备马钱子提取物的方法，包括如下步骤：
    (1)采用第一碱性溶液浸润马钱子生药，固液分离，得到固相物；
    (2)采用乙酸乙酯浸提步骤(1)得到的固相物，固液分离，得到提取液；
    (3)对步骤(2)得到的提取液进行硅胶柱层析，洗脱剂为添加有第二碱性溶液的无水乙醇或者添加有第二碱性溶液的乙醇溶液，收集洗脱液；其中，第二碱性溶液选自氨水溶液、碳酸钾溶液、碳酸钠溶液、碳酸氢钠溶液和碳酸氢钾溶液中的至少一种；
    (4)将步骤(3)收集到的洗脱液进行干燥，得到干燥物；
    (5)将步骤(4)得到的干燥物溶解在酸溶液中，固液分离，得到液相物；
    (6)将步骤(5)得到液相物的pH值调节至8～13，固液分离，得到固相物；
    (7)将步骤(6)得到的固相物进行重结晶，得到提取物。
  2. 根据权利要求1所述的方法，其中，步骤(3)中，所述第二碱性溶液为饱和溶液；
    优选地，所述第二碱性溶液选自饱和氨水溶液、饱和碳酸钾溶液、饱和碳酸钠溶液、饱和碳酸氢钠溶液和饱和碳酸氢钾溶液中的至少一种；
    优选地，所述第二碱性溶液的浓度为0.1％(V/V)～2％(V/V)，更优选为0.5％(V/V)～1％(V/V)。
  3. 根据权利要求1所述的方法，其中，步骤(3)中，洗脱剂中，第二碱性溶液体积为无水乙醇或乙醇溶液体积的0.05％～5％。
  4. 根据权利要求1所述的方法，其中，步骤(3)中，所述硅胶柱为70～300目硅胶柱。
  5. 根据权利要求1所述的方法，其中，步骤(3)中，收集3至8倍柱体积之后的洗脱液，优选为收集5倍柱体积之后的洗脱液。
  6. 根据权利要求1所述的方法，其特征在于如下1)至22)中的一项或多项：
    1)步骤(1)中，所述第一碱性溶液选自碳酸钾溶液、碳酸氢钠溶液、碳酸钠溶液、碳酸氢钾溶液和胺类溶液中的至少一种；
    优选地，所述第一碱性溶液选自碳酸钾溶液、碳酸氢钠溶液、碳酸钠溶液、甲胺溶液、碳酸氢钾溶液和乙胺溶液中的至少一种；
    更优选地，所述第一碱性溶液选自饱和碳酸钾溶液、饱和碳酸氢钠溶液、饱和碳酸钠溶液、饱和甲胺溶液、饱和碳酸氢钾溶液和饱和乙胺溶液中的至少一种；
    2)步骤(1)中，第一碱性溶液和马钱子生药的比例为(0.5～10):1(mL/g)，优选为(0.5～3):1(mL/g)；
    3)步骤(1)中，浸润时间为1～10小时，优选为2～4小时，更优选为3小时；
    4)步骤(1)中，通过过滤进行固液分离；
    优选地，采用100～500目的滤布过滤；
    5)步骤(1)中，浸润前，对马钱子生药进行破碎处理；
    6)步骤(2)中，乙酸乙酯和步骤(1)得到的固相物的比例为(1～10):1(mL/g)，优选为(4～6):1(mL/g)；
    7)步骤(2)中，浸提时间为2～16小时，优选为6～12小时，更优选为8～10小时；
    8)步骤(2)中，在回流条件下浸提；
    9)步骤(2)中，浸提次数为一次或多次，优选为一次、二次、三次或四次；
    10)步骤(2)中，通过过滤进行固液分离；
    优选地，采用100～500目的滤布过滤；
    11)在步骤(2)和(3)之间，还包括步骤(2’)：将步骤(2)得到的提取液浓缩，得到浓缩液；将浓缩液过滤，得到的滤液用于进行步骤(3)中的硅胶柱层析；
    优选地，步骤(2’)中，浓缩液体积为步骤(2)得到提取液体积的1/4～1/2；
    优选地，步骤(2’)中，采用100～500目的滤布过滤；
    12)步骤(3)中，所述乙醇溶液的浓度为70％(W/W)～99％(W/W)；
    13)在步骤(4)和(5)之间，还包括步骤(4’)：对步骤(4)得到的干燥物进行破碎处理；
    14)步骤(5)中，所述酸溶液选自硫酸溶液、盐酸溶液和磷酸溶液中的至少一种；
    15)步骤(5)中，所述酸溶液的浓度为0.6％(W/W)～9％(W/W)；
    优选地，步骤(5)中，在加热条件下溶解；
    16)步骤(5)中，酸溶液和步骤(4)得到的干燥物的比例为(12～47):1(mL/g)；
    17)步骤(5)中，通过过滤进行固液分离；
    优选地，采用100～500目的滤布过滤；
    18)步骤(6)中，通过添加碱液调节pH值；
    优选地，所述碱液为氢氧化钾溶液和/或氢氧化钠溶液；
    19)步骤(6)中，通过过滤进行固液分离；
    优选地，采用100～500目的滤布过滤；
    20)在步骤(6)和(7)之间，还包括步骤(6’)：将步骤(6)得到的固相物进行干燥，破碎；
    21)步骤(7)中，采用乙醇溶液对步骤(6)得到的固相物进行重结晶；
    优选地，乙醇溶液的浓度为10％(W/W)～60％(W/W)；
    优选地，通过如下的步骤进行重结晶：
    (i)将步骤(6)得到的固相物溶解在乙醇溶液中，过滤，收集滤液；
    (ii)将步骤(i)收集的滤液静置析晶，过滤，收集滤渣即为提取物；
    更优选地，步骤(i)中，乙醇溶液和固相物的比例为(10～50):1(mL/g)；
    更优选地，步骤(i)和(ii)中，采用100～500目的滤布过滤；
    更优选地，步骤(i)中，在加热条件下溶解；
    更优选地，步骤(ii)中，室温下静置析晶；
    22)步骤(7)中，重结晶次数为一次或多次，优选为一次或两次。
  7. 一种马钱子提取物，其中，马钱子碱的重量百分含量≥94.5％，并且，士的宁的重量百分含量≤3％。
  8. 根据权利要求7所述的马钱子提取物，其由权利要求1至6中任一项所述的方法制得。
  9. 一种药物组合物，其包权利要求7或8所述的马钱子提取物以及药学上可接受的辅料。
  10. 一种通络止痛或散结消肿的方法或者一种治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒或咽喉肿痛疾病的方法，包括给予有需求的受试者以有效量的权利要求7或8所述的马钱子提取物或者权利要求9所述的药物组合物。
  11. 权利要求7或8所述的马钱子提取物或者权利要求9所述的药物组合物在制备通络止痛、散结消肿的药物或者在制备治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒或咽喉肿痛疾病的药物中的用途。
  12. 权利要求7或8所述的马钱子提取物或者权利要求9所述的药物组合物，用于通络止痛、散结消肿或者治疗跌打损伤、骨折肿痛、风湿顽痹、麻木瘫痪、痈疽疮毒或咽喉肿痛疾病。
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