WO2020220863A1 - 大麻油的制备方法及其制备的大麻油 - Google Patents

大麻油的制备方法及其制备的大麻油 Download PDF

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WO2020220863A1
WO2020220863A1 PCT/CN2020/080807 CN2020080807W WO2020220863A1 WO 2020220863 A1 WO2020220863 A1 WO 2020220863A1 CN 2020080807 W CN2020080807 W CN 2020080807W WO 2020220863 A1 WO2020220863 A1 WO 2020220863A1
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preparation
oil
volume
ethanol
hemp
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PCT/CN2020/080807
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English (en)
French (fr)
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于朝晖
常坦然
高伟博
柳旭
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云南汉盟制药有限公司
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • 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
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • 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
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/84—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by crystallisation
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C11B1/108—Production of fats or fatty oils from raw materials by extracting after-treatment, e.g. of miscellae
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/10—Refining fats or fatty oils by adsorption
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/12—Refining fats or fatty oils by distillation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/55—Liquid-liquid separation; Phase separation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Definitions

  • the invention relates to the technical field of plant extraction, in particular to a preparation method of hemp oil and hemp oil prepared therefrom.
  • Cannabis sativa L. also known as hemp and hemp
  • hemp and hemp belong to the moraceae and hemp genus. Because the tetrahydrocannabinol (THC) content in the mosaic during the growth period is less than three thousandths, this Different from medical marijuana and recreational marijuana, large-scale cultivation and industrial development and utilization can be legally carried out, and it is widely used in the fields of daily chemicals, materials, energy and medicine.
  • THC tetrahydrocannabinol
  • the extraction of industrial hemp is mainly concentrated on the fiber or individual monomer components of the hemp itself, such as the extraction of cannabidiol (CBD) and the extraction of cannabinoids.
  • CBD cannabidiol
  • other legal, safe and effective cannabis active substances also have great development and application value, such as cannabis extracts obtained by leaching and concentrating with different solvents. Or the water extract dry powder that has lost most of the active ingredients.
  • the extraction of hemp oil is mostly prepared by supercritical or subcritical carbon dioxide fluid.
  • THC tetrahydrocannabinol
  • the technical problem to be solved by the present invention is to provide a preparation method of cannabis oil and cannabis oil prepared therefrom.
  • the cannabis oil prepared by the preparation method of the invention has a wide variety of cannabis oils, a clear composition, and basically does not contain THC.
  • the preparation method of hemp oil provided by the present invention includes:
  • Step 1 Extract raw materials with 4-10 times the volume of 30vol%-100vol% ethanol to obtain an extract; the raw materials are hemp leaves and/or hemp flowers;
  • Step 2 Recover the ethanol in the extract to a density of 1.05-1.25 (50°C) to prepare a liquid paste;
  • Step 3 Mix the liquid paste with an equal volume of water, boil at normal pressure for 1 to 3 hours, add hot water at 50°C to 100°C and stir to obtain a dispersion; the volume-mass ratio of the dispersion to the raw material is ( 3 ⁇ 8):1;
  • Step 4 Cool the dispersion to 40°C-50°C, separate and discard the insoluble matter to obtain a filtrate;
  • Step 5 Adsorb the filtrate on a macroporous resin column, and elute with 50vol% ⁇ 80vol% ethanol for 4 ⁇ 5BV, collect the eluate as target segment-I; elution with 95vol% ethanol for 3 ⁇ 5BV, collect the eluate as Target segment-II;
  • Step 6 After the target section I is concentrated under reduced pressure to remove water, it is dissolved in organic solvent A at 30°C to 50°C, and the solution is adsorbed by an adsorbent to prepare raw solution I; the adsorbent is clay and/or silicon Algae earth
  • Step 7 After concentrating the target section II under reduced pressure to remove water, dissolving it in organic solvent B at 35°C ⁇ 50°C, the solution is adsorbed on a silica gel solid phase extraction column, and eluting 1-3 times column with eluent A Volume, discard the eluent; then eluate 2-5 times the column volume with eluent B, and collect the eluent as stock solution II;
  • the eluent A is composed of heptane: ethyl acetate (100:1 ⁇ 2 ) Composition;
  • the eluent B is composed of heptane: ethyl acetate (100: 15-20);
  • Step 8 Combine the original liquid I and the original liquid II, concentrate under reduced pressure to remove water, and then wash with water to obtain hemp oil.
  • the hemp flower and hemp leaf are crushed before being extracted; the crushed to 20-60 mesh. In some embodiments, it is crushed to 20 mesh; in other embodiments, it is crushed to 60 mesh. Changing the decarboxylation process of flower leaf high temperature baking to boiling decarboxylation in the tank after extraction makes the decarboxylation process get rid of the dependence on equipment, decarboxylation is more complete and the high-temperature volatile terpenoid components are also better retained.
  • the volume fraction of ethanol is 60% to 90%; in some embodiments, the volume fraction of ethanol is 60%; in other embodiments, the volume fraction of ethanol is 90%.
  • the mass-volume ratio of the raw material to ethanol is 1:(4-8); in some embodiments, the mass-volume ratio of raw material to ethanol is 1:8; in other embodiments, the mass-volume ratio of raw material to ethanol The ratio is 1:4.
  • the mass-volume ratio of the raw material to ethanol refers to the ratio of the mass of the raw material to the volume of ethanol, for example, 4-8L of ethanol is used for extraction per kg of raw material.
  • the conditions of the extraction are normal temperature and normal pressure, and the number of extractions is 2 to 3 times. In some embodiments, leaching is 2 times; in other embodiments, leaching is 3 times.
  • step 2 the concentration is concentrated to a density of 1.10 or 1.20.
  • step 3 the temperature of the normal pressure boiling is 94°C, and the time is 1.2-2h. In some embodiments, the boiling time is 1.2h, and in other embodiments, the boiling time is 2h.
  • the temperature of the hot water is 50° C. to 70° C.; the stirring time is 15 min; and the volume-mass ratio of the dispersion to the raw material is (4-8):1.
  • the supplementary heating water is added while it is hot.
  • the temperature of the hot water is 50°C; in other embodiments, the temperature of the hot water is 70°C.
  • the volume-mass ratio of the dispersion to the raw materials is 4:1; in other embodiments, the volume-mass ratio of the dispersion to the raw materials is 8:1.
  • the mass-volume ratio of the raw material to the dispersion liquid refers to the ratio of the mass of the raw material to the volume of the dispersion liquid.
  • the volume of the dispersion liquid prepared per kg of the raw material is 4-8L.
  • the separation method is filtration or centrifugation; the pore size of the filtration is 200 mesh; and the rotation speed of the centrifugation is 800 rpm.
  • the cooling is to 50°C, and in other embodiments, the cooling is to 40°C.
  • the macroporous resin is selected from ADS-17, AB-8, D-101, XDA-8, LSA-7, D-941, DM-130, SP-825 or HPD-600, HPD-007 At least one of
  • the adsorption time is 0.5h to 1h; in some embodiments, the adsorption time is 0.5h; in other embodiments, the adsorption time is 1h.
  • the target segment-I is eluted with 65 vol% to 75 vol% ethanol, and the flow rate is 1 to 2 BV/h.
  • 65 vol% ethanol is used for the elution of the target segment-I
  • the flow rate is 1 BV/h
  • the elution is 5 BV.
  • 75 vol% ethanol is used for the elution of the target segment-I
  • the flow rate is 2 BV/h
  • the elution is 4 BV.
  • the flow rate of the target section-II is 0.25 to 1 BV/h.
  • the target segment-II is eluted with 95 vol% ethanol, the flow rate is 0.25 BV/h, and the elution is 3 BV; in other embodiments, the target segment-II is eluted with 95 vol% ethanol, and the flow rate is 1 BV. /h, 5BV is eluted.
  • the unit BV refers to a multiple of the column volume, for example, 5 BV refers to 5 times the column volume.
  • the organic solvent A is heptane or a mixture of hexane and ethyl acetate; the volume ratio of the hexane to ethyl acetate is (6-10):1; the dissolution temperature is 35°C ⁇ 50°C or 40 ⁇ 50°C.
  • the adsorbent is a mixture of diatomaceous earth and calcium phosphate, wherein the mass ratio of diatomaceous earth to calcium phosphate is 1: (2 ⁇ 3); or a mixture of white earth and diatomaceous earth, wherein the mass ratio of white earth and diatomaceous earth The mass ratio is 3:1;
  • the adsorption is to allow the solution to pass through the adsorbent, and the passing speed is 3 to 5 BV/h.
  • the passing rate is 3 BV/h; in other embodiments, the passing rate is 5 BV/h.
  • the organic solvent B is heptane or hexane; in some embodiments, the organic solvent B is heptane, and in other embodiments, the organic solvent B is hexane.
  • the temperature of the dissolution is 35°C or 50°C.
  • the filler of the silica gel solid phase extraction column is 100-500 mesh silica gel.
  • step 7 the volume ratio of the solution to the silica gel column packing is 1 ⁇ 4:1 (V/V).
  • the elution flow rate of the eluent A is 0.5-2.5 BV/h; the elution flow rate of the eluent B is 0.5-1.5 BV/h.
  • step 8 the washing with water is specifically: adding an equal volume of water, then concentrating to an anhydrous thick paste, and repeating 1 to 3 times.
  • the cannabis oil prepared by the preparation method of the present invention.
  • the raw material used for extracting hemp oil in the present invention is industrial hemp, and the extracted parts are the flowers and leaves of industrial hemp.
  • the full-spectrum hemp oil mentioned in this field refers to a refined oil that mainly contains cannabinoids and also includes active ingredients such as flavonoids and terpenes.
  • the cannabis oil prepared by the method of the present invention contains cannabinoids, flavonoids, and terpenes, but does not contain THC. Therefore, the hemp oil prepared by the method of the present invention is a full-spectrum hemp oil.
  • the hemp oil includes the following parts by mass: total cannabinoids 79.23 parts to 81.57 parts; total flavonoids 2.44 parts to 2.53 parts; total terpenes 0.46 parts to 0.58 parts.
  • the present invention proves through experiments that after the orphan mice are injected with oil, the frequency of opening arms of the elevated plus maze that they explore and induce anxiety is significantly increased, and the frequency of closing arms that they prefer is reduced.
  • the products are medicines, health care products, special functional foods and/or daily chemicals.
  • the invention provides the application of the cannabis oil in the preparation of medicines, health products, special functional foods and/or daily chemicals.
  • the medicines, health products, special functional foods and/or daily chemicals have an anti-anxiety effect.
  • the invention also provides medicines, health products, special functional foods and/or daily chemicals containing the hemp oil.
  • the present invention also provides an anti-anxiety method, which is administering a product containing the cannabis oil.
  • the mode of administration is injection, oral, sublingual, rectal or spray inhalation.
  • the beneficial effects of the present invention in the conventional technology in the field, medicinal materials are usually processed first and then extracted, and many active ingredients will be lost during the processing. Especially when processed at high temperature, volatile components such as terpenes are seriously lost.
  • the present invention proposes a post-processing method.
  • the conventional mosaic high-temperature baking and decarboxylation process is changed to the boiling decarboxylation in the tank after extraction, so that the decarboxylation step is eliminated Relying on equipment, decarboxylation is more complete and terpenoids that are volatile at high temperature are also better retained.
  • the use of hot water dispersion technology can remove a large amount of oil and pigments, complete the primary purification purpose and make the insoluble cannabis extract easier to load evenly.
  • Chromatography step and silica gel solid phase extraction can retain almost all the effective ingredients in hemp flowers and leaves, and make THC at a very low level (content ⁇ 0.1%).
  • the product prepared by the method of the present invention is a yellow-brown to red-brown oily fluid with a resin-specific fragrance.
  • the total content of known components reaches 80% to 90%, covering almost all the main active ingredients in hemp mosaic (including cannabinoids and flavonoids). And terpenes, etc.), the ingredients are clear, the quality is controllable, and the THC content of toxic ingredients is ⁇ 0.1%.
  • the hemp oil prepared by the method of the invention can be directly applied to the fields of medicines, health care products, special functional foods, daily chemicals and the like.
  • Figure 1 shows the chromatogram of the mixed standard product detected by high pressure liquid chromatography/DAD detector
  • Figure 2 shows the chromatogram of the whole spectrum of oil product-1 detected by high pressure liquid chromatography/DAD detector
  • Figure 3 shows the chromatogram of the full-spectrum oil product-2 detected by high pressure liquid chromatography/DAD detector
  • Figure 4 shows the chromatogram of the full-spectrum oil sample-1 detected by high pressure liquid chromatography/DAD detector
  • Figure 5 shows the chromatogram of the full-spectrum oil sample-2 detected by high pressure liquid chromatography/DAD detector
  • Figure 6 shows the chromatogram of the reference substance detected by gas chromatograph headspace sampling/FID detector
  • Figure 7 shows the chromatogram of the full-spectrum oil product-1 detected by gas chromatograph headspace sampling/FID detector
  • Figure 8 shows the chromatogram of the full-spectrum oil product-2 detected by gas chromatograph headspace sampling/FID detector
  • Figure 9 shows the chromatogram of the full-spectrum oil sample-1 detected by gas chromatograph headspace sampling/FID detector
  • Figure 10 shows the chromatogram of the full-spectrum oil sample-2 detected by gas chromatograph headspace sampling/FID detector
  • Figure 11(a) shows the chromatogram of silica gel solid phase extraction impurity removal liquid/DAD detector
  • Figure 11(b) shows the chromatogram of silica gel solid phase extraction eluent/DAD detector detection.
  • the present invention provides a preparation method of hemp oil and hemp oil prepared therefrom.
  • Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve.
  • all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • Invent technology is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • the instruments used in the present invention are all common commercially available products, and all are available in the market.
  • step 6 Concentrate the target segment-I of the full-spectrum oil obtained in step 6), concentrate under reduced pressure to anhydrous thick paste, dissolve it in heptane at 35-50°C, quickly pass through the adsorbent short column at a flow rate of 3BV/h ( White clay 3:1 diatomaceous earth), and wash the column with a small amount of solvent to recover the remaining part, and combine to obtain the decolorized full-spectrum cannabis oil stock solution-I;
  • the target segment-II of the full-spectrum oil obtained in the concentration step 6) was concentrated under reduced pressure to an anhydrous thick paste, dissolved in heptane at 35°C, and applied to a 200-300 mesh silica gel solid-phase extraction column, with heptane: Ethyl acetate (100:1-2) to wash away the oil and THC part, eluting with heptane: ethyl acetate (100:15), until HPLC determination or TLC thin layer shows no related cannabinoid components, the decolorized whole Pedigree cannabis oil stock-II;
  • step 6 Concentrate the target segment-I of the full-spectrum cannabis oil obtained in step 6), concentrate under reduced pressure to an anhydrous thick paste, dissolve it at 40-50°C with hexane: ethyl acetate (6-10:1), 5BV /h Pressurized and quickly pass through the mixed adsorbent of diatomaceous earth: calcium phosphate (1:2-3) to obtain the decolorized full-spectrum cannabis oil stock-I;
  • the target segment-II of the full-spectrum oil obtained in the concentration step 6) was concentrated under reduced pressure to an anhydrous thick paste, dissolved in hexane at 50°C, and applied to a 200-300 mesh silica gel solid phase extraction column, with heptane: Ethyl acetate (100:1-2) to wash away the oil and THC part, eluting with heptane: ethyl acetate (100:20), until HPLC determination or TLC thin layer shows no related cannabinoid components, a decolorized whole Pedigree cannabis oil stock-II;
  • the cannabis flower and leaf extract is processed as follows:
  • step 6) Concentrate the full-spectrum oil stock solution of step 5) under reduced pressure to an anhydrous thick paste, add an equal volume of purified water, and continue to concentrate to an anhydrous thick paste, repeat 1-3 times to obtain a full-spectrum hemp oil with low organic solubility Sample 1.
  • step (2) Extract the medicinal powder obtained in step (1) 3 times with 6 times n-hexane, combine and concentrate at 60°C
  • step (3) Add sodium hydroxide to the thick paste of step (2) to adjust the pH to 11, fully stir for 1 hour, and pass
  • step (3) Dissolve the insoluble precipitate obtained in step (3) with petroleum ether, pass it through silica gel column chromatography, eluate 5 times the column volume with acetone: petroleum ether (1:1, V/V) to remove impurities, and recover the solvent; :Petroleum ether (7:3, V/V) is eluted with 6 times the column volume, the second part of the eluate is collected, and after concentration, it is the full spectrum oil sample 2.
  • Preparation of the test solution Take about 20mg of the finished cannabis oil of the whole spectrum, accurately weigh it, place it in a 100ml measuring flask, add 100ml methanol, sonicate for 10 minutes, dilute with methanol to the mark, shake it, and use a microporous filter membrane (0.45 pm) Filter, take the additional filtrate, and get it.
  • Total cannabinoid content CBD%+CBDV%+CBG%+THCV%+CBN%+CBC%+CBL%;
  • CBD cannabidiol cannabidiol
  • CBDV cannabidiol or hypocannabidiol
  • CBG cannabidiol cannabidiol
  • CBC cannabinene CBN cannabidiol
  • THCV tetrahydrocannabinol or hypotetrahydrocannabinol
  • THC tetrahydrocannabinol CBL cannabidiol
  • test product Take 25mg of hemp oil of the whole spectrum and dissolve it in 25ml of methanol:chloroform (1:1) mixed solvent to prepare a test solution of 1mg/ml for test product.
  • Determination method accurately draw 0.8ml each of the reference solution and the test solution, inject the sample in the headspace according to the above chromatographic conditions, measure the peak area, and obtain the content of each component according to the following correction factors.
  • the number of compounds refers to the types of cannabinoids + flavonoids + terpenoids that can be measured by various measurement methods, and is counted with peaks above signal: noise (100:1).
  • the preparation method of the patent has enriched almost all the effective ingredients of cannabis, obtained better traits, and greatly improved the quality controllability.
  • the finished product 1 prepared in Example 1 has the most types of compounds, and besides total cannabinoids, it also contains flavonoids and terpenoids, but no THC is detected.
  • the total cannabinoid content obtained by extraction in Example 1 is higher.
  • Ethanol extraction concentration must be limited to 60-80% (V/V), less than 60% ethanol cannot completely extract cannabinoid components, and more than 80% ethanol will bring too much oil and pigment to the subsequent process Purification brings more trouble, especially this part of oil pigments will also entrap a large amount of cannabinoids and terpenes, causing loss.
  • Heptane-ethyl acetate impurity removal and elution ratio 200-300 mesh silica gel solid phase extraction column on the full-spectrum oil target segment-II, must be limited to heptane: ethyl acetate (100: 1-2)
  • heptane: ethyl acetate 100: 1-2
  • more than 100: 2 a large amount of CBC and CBL will be eluted; and must be limited to heptane: ethyl acetate (100: 10-20) elution (see Figure 11b) , Less than 100:10, the remaining CBD and CBDV components cannot be eluted.
  • Table 4 records part of the screening scheme of the process parameters of the present invention. Except for the parameters described in Table 4, other parameters are consistent with Example 1. Judging from the test results, none of these parameters can achieve the effects of Examples 1 to 2. The total cannabinoid content in the extracted cannabis oil is significantly different from that in Example 1, p ⁇ 0.05.
  • mice 1 Orphan mouse model: After weaning, the mice are raised in a single cage in a quiet environment, and the litter is changed once a week. Room temperature (24 ⁇ 2) °C, free food and water, feeding time is 6 weeks. After long-term isolation and rearing, the appearance of the mice is characterized by anxiety, fear, irritability, and aggressiveness. 2Group-raising mice: 5 mice in each cage, kept in the same environment, and mice of the same age were taken during the experiment. All mice weighed 18-22g.
  • the elevated cross labyrinth of mice consists of two 30cm ⁇ 5cm open arms and two 30cm ⁇ 5cm ⁇ 15cm closed arms.
  • the upper part of the closed arms is open, and there is a 5cm ⁇ 5cm open part in the center.
  • the arms of each type are in opposite positions. , Get lost 45cm from the ground.
  • the maze is made of wood, and the bottom and the walls of the closed arms are black. To prevent the mouse from falling, there is a border 0.25 cm higher on the periphery of the open arm.
  • mice were randomly divided into 6 groups, each with 20 animals: group-raising blank control group (equal saline), orphan model blank control group (equal saline), positive control group (Diamen 2.5mg/kg), injection
  • group-raising blank control group equal saline
  • phan model blank control group equal saline
  • positive control group Diamen 2.5mg/kg
  • injection 25mg/kg
  • all experiments were carried out in a quiet environment from 9:00 to 14:00 every day.
  • the mice were placed in the center of the elevated cross labyrinth with their heads open arms, and the mice's activities were observed within 5 minutes.
  • the full-spectrum oil product provided by this patent has a safer and more obvious sedative effect than other cannabis oil samples. The reason may be that the cannabinoids, flavonoids, terpenoids and other components in cannabis have certain Synergistic or auxiliary effect.

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Abstract

大麻油的制备方法，以大麻叶和/或大麻花为原料，经包括乙醇浸提、制备流膏、热水分散、冷却分离、层析、硅胶固相萃取的步骤制成全谱系大麻油，其中含有大麻素、黄酮、萜类且不含有四氢大麻酚。还公开了由该方法制得的大麻油及其在药品、保健品、特殊功能食品或日化品制备中的应用。

Description

大麻油的制备方法及其制备的大麻油
本申请要求于2019年04月30日提交中国专利局、申请号为201910360887.8、发明名称为“大麻油的制备方法及其制备的大麻油”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及植物提取技术领域，尤其涉及大麻油的制备方法及其制备的大麻油。
背景技术
工业大麻(学名：Cannabis sativa L.)，又名汉麻、火麻，为桑科、大麻属植物，因生长期花叶中的四氢大麻酚(THC)含量小于千分之三，以此区别于医用大麻和娱乐大麻，可以合法进行规模化种植与工业化开发利用，在日化、材料、能源和医药等领域都有广泛的应用。
医学古籍对大麻花，叶，果实(火麻仁)均有记载。其中，大麻叶药性药理为：辛，有毒，归肺、膀胱、大肠经，主治气喘，跌扑疼痛，虫病等；大麻花药性药理为：苦，辛，性温，有毒，用于祛风，活血，生发，主风病肢体麻木，遍身瘙痒，闭经等。现代的检测技术表明，工业大麻化学成分非常复杂，已鉴别出至少483种单体化合物，其中60多种大麻素成分是大麻植株特有的。其他还包括类脂、黄酮、萜烯、碳氢化合物、非环形大苯酚、生物碱，木质素等，而这些成分中，一些已经被报道具有较好的药理活性，如香叶基黄酮和萜烯等就发挥着抗氧化、抗炎、杀菌等功效。
目前对工业大麻的提取主要集中在大麻本身纤维或个别单体成分，如对大麻二酚(CBD)的提取、对大麻素的提取等。除了大麻二酚(CBD)等已知的有效成分之外，合法安全有效的其他大麻活性物质也具备极大的开发和应用价值，例如采用不同溶媒浸提浓缩而得的大麻提取物浸膏，或是损耗了大部分有效成分的水提物干粉。目前对大麻油的提取，多采用超临界或亚临界二氧化碳流体的方法制备。为了剔除提取物中的四氢大麻酚(THC)，目前采用的工艺尚不成熟，有些需要较长的周期，而有些则需 要较高的成本。如何在去除THC的同时，保留更多的活性物质，提高大麻油的收率是本领域仍然在不断研究的课题。
发明内容
有鉴于此，本发明要解决的技术问题在于提供大麻油的制备方法及其制备的大麻油，本发明所述的制备方法制得的大麻油种类丰富，成分组成明确，而且基本不含THC。
本发明提供的大麻油的制备方法，包括：
步骤1：将原料以4～10倍体积的30vol％～100vol％乙醇浸提获得提取液；所述原料为大麻叶和/或大麻花；
步骤2：回收所述提取液中的乙醇至密度为1.05～1.25(50℃)，制得流膏；
步骤3：将所述流膏与等体积水混合，常压煮沸1～3h后，补加50℃～100℃热水经搅拌获得分散液；所述分散液与原料的体积-质量比为(3～8)∶1；
步骤4：所述分散液冷却至40℃～50℃，分离弃除不溶物，获得滤液；
步骤5：将滤液上大孔树脂柱吸附，以50vol％～80vol％乙醇洗脱4～5BV，收集洗脱液为目标段-I；以95vol％乙醇洗脱3～5BV，收集洗脱液为目标段-II；
步骤6：将所述目标段I减压浓缩去除水后，于30℃～50℃以有机溶剂A溶解后，溶液以吸附剂吸附后制得原液I；所述吸附剂为白土和/或硅藻土；
步骤7：将所述目标段II减压浓缩去除水后，于35℃～50℃以有机溶剂B溶解后，溶液上硅胶固相萃取柱吸附，以洗脱液A洗脱1-3倍柱体积，弃除洗脱液；再以洗脱液B洗脱2-5倍柱体积，收集洗脱液为原液II；所述洗脱液A由庚烷∶乙酸乙酯(100∶1～2)组成；所述洗脱液B由庚烷∶乙酸乙酯(100∶15～20)组成；
步骤8：合并原液I与原液II，减压浓缩去除水分后，再以水清洗，制得大麻油。
所述大麻花、大麻叶在提取前，经过粉碎；所述粉碎至20～60目。一些实施例中，粉碎至20目；另一些实施例中，粉碎至60目。将花叶高温烘烤脱羧工艺改为提取后罐内煮沸脱羧，使得脱羧环节摆脱对设备的依赖，脱羧更完全并且使高温易挥发的萜类组分也得到较好的保留。
步骤1中，所述提取中，乙醇的体积分数为60％～90％；一些实施例中，乙醇的体积分数为60％；另一些实施例中，乙醇的体积分数为90％。
所述原料与乙醇的质量-体积比为1∶(4～8)；一些实施例中，原料与乙醇的质量-体积比为1∶8；另一些实施例中，原料与乙醇的质量-体积比为1∶4。所述原料与乙醇的质量-体积比，是指原料的质量与乙醇的体积之比，例如，每kg原料采用4～8L的乙醇进行提取。
所述浸提的条件为常温、常压，浸提次数为2～3次。一些实施例中，浸提2次；另一些实施例中，浸提3次。
步骤2中，所述浓缩至密度为1.10或1.20。
步骤3中，所述常压煮沸的温度为94℃，时间为1.2～2h。一些实施例中，所述煮沸的时间为1.2h，另一些实施例中，所述煮沸的时间为2h。
步骤3中，所述热水的温度为50℃～70℃；搅拌的时长为15min；所述分散液与原料的体积-质量比为(4～8)∶1。所述补加热水为趁热加入。一些实施例中，热水的温度为50℃；另一些实施例中，热水的温度为70℃。一些实施例中，所述分散液与原料的体积-质量比为4∶1；另一些实施例中，所述分散液与原料的体积-质量比为8∶1。所述原料与分散液的质量-体积比，是指原料的质量与分散液的体积之比，例如，每kg原料制得的分散液的体积为4～8L。
步骤4中，所述分离的方式为过滤或离心；所述过滤的孔径为200目；所述离心的转速为800rpm。一些实施例中，所述冷却至50℃，另一些实施例中，所述冷却至40℃。经过步骤3的热水分散工艺，可以剔除大量的油脂和色素。
步骤5中，所述大孔树脂选自ADS-17、AB-8、D-101、XDA-8、LSA-7、D-941、DM-130、SP-825或HPD-600、HPD-007中至少一种；
所述吸附的时间为0.5h～1h；一些实施例中，所述吸附的时间为0.5h； 另一些实施例中，所述吸附的时间为1h。
所述目标段-I的洗脱采用65vol％～75vol％乙醇，流速为1～2BV/h。一些实施例中，目标段-I的洗脱采用65vol％乙醇，流速为1BV/h，洗脱5BV。另一些实施例中，目标段-I的洗脱采用75vol％乙醇，流速为2BV/h，洗脱4BV。
所述目标段-II的流速为0.25～1BV/h。另一些实施例中，目标段-II的洗脱采用95vol％乙醇，流速为0.25BV/h，洗脱3BV；另一些实施例中，目标段-II的洗脱采用95vol％乙醇，流速为1BV/h，洗脱5BV。
本发明中，所述单位BV是指柱体积的倍数，例如5BV是指5倍柱体积。
步骤6中，所述有机溶剂A为庚烷，或为己烷与乙酸乙酯的混合物；所述己烷与乙酸乙酯的体积比为(6～10)∶1；所述溶解的温度为35℃～50℃或40～50℃。
所述吸附剂为硅藻土与磷酸钙的混合物，其中硅藻土与磷酸钙的质量比为1∶(2～3)；或为白土与硅藻土的混合物，其中白土与硅藻土的质量比为3∶1；
所述吸附为使溶液通过吸附剂，所述通过的速度为3～5BV/h。一些实施例中，所述通过的速率为3BV/h；另一些实施例中，所述通过的速率为5BV/h。
步骤7中，所述有机溶液B为庚烷或己烷；一些实施例中，有机溶剂B为庚烷，另一些实施例中，有机溶剂B为己烷。所述溶解的温度为35℃或50℃。
所述硅胶固相萃取柱，其填料为100～500目硅胶。
步骤7中，所述溶液与硅胶柱填料的体积比为1～4∶1(V/V)。所述洗脱液A洗脱的流速为0.5-2.5BV/h；所述洗脱液B洗脱的流速为0.5-1.5BV/h。
步骤8中，所述以水清洗具体为：加入等体积的水，再浓缩至无水稠膏，重复1～3次。
本发明所述制备方法制得的大麻油。
本发明提取大麻油采用的原料为工业大麻，提取的部位是工业大麻的花和叶片。本领域所述全谱系大麻油(即国际称谓“full spectrum hemp oil”)，是指以大麻素为主，同时涵盖黄酮、萜类等活性成分的成品油。本发明所述方法制得的大麻油中含有大麻素、黄酮、萜类，而不含有THC，因此，本发明方法制得的大麻油为全谱系大麻油。
本发明实施例中，所述大麻油中包括如下质量份的：总大麻素79.23份～81.57份；总黄酮2.44份～2.53份；总萜0.46份～0.58份。
本发明所述的大麻油在制备抗焦虑的产品中的应用。本发明通过实验证明，孤养小鼠在注射油之后，明显提升其探索诱发焦虑的高架十字迷宫开臂频次，降低了其更喜欢的闭臂频次。所述产品为药品、保健品、特殊功能食品和/或日化品。本发明提供了所述大麻油在制备药品、保健品、特殊功能食品和/或日化品中的应用。所述药品、保健品、特殊功能食品和/或日化品的作用为抗焦虑。
本发明还提供了含有所述大麻油的药品、保健品、特殊功能食品和/或日化品。
本发明还提供了一种抗焦虑的方法，其为给予含有所述大麻油的产品。
所述给予的方式为注射、口服、舌下、直肠或喷雾吸入。
本发明有益效果：在本领域常规技术里，药材通常是先炮制后再提取，在炮制的过程中很多有效成分会损失，特别高温烘烤炮制的时候，萜烯等易挥发成分损失严重。为了更好的保留大麻里的有效成分，制备全谱系大麻油，本发明提出了后炮制的方法，将常规的花叶高温烘烤炮制脱羧工艺改为提取后罐内煮沸脱羧，使得脱羧环节摆脱对设备的依赖，脱羧更完全并且使高温易挥发的萜类组分也得到较好的保留。采用热水分散工艺，可以剔除大量的油脂和色素，完成初级纯化目的并使得难溶性的大麻提取物更容易上样均匀。采用层析步骤再加硅胶固相萃取，可以保留几乎所有大麻花叶中的有效成分，并使得THC处在一个极低的水平(含量≤0.1％)。本发明方法制备所得产品为黄棕至红棕色油性流体，有树脂特有芳香，其中已知总成分含量达80％～90％，涵盖大麻花叶中几乎所有主要的有效成 分(包括大麻素、黄酮和萜烯等)、成分清晰、质量可控，且有毒成分THC含量≤0.1％。
本发明方法制备的大麻油，可以直接应用于药品、保健品、特殊功能食品、日化品等领域。
附图说明
图1示混合标品经高压液相色谱/DAD检测器检测色谱图；
图2示全谱系油成品-1经高压液相色谱/DAD检测器检测色谱图；
图3示全谱系油成品-2经高压液相色谱/DAD检测器检测色谱图；
图4示全谱系油样品-1经高压液相色谱/DAD检测器检测色谱图；
图5示全谱系油样品-2经高压液相色谱/DAD检测器检测色谱图；
图6示对照品经气相色谱顶空进样/FID检测器检测色谱图；
图7示全谱系油成品-1经气相色谱顶空进样/FID检测器检测色谱图；
图8示全谱系油成品-2经气相色谱顶空进样/FID检测器检测色谱图；
图9示全谱系油样品-1经气相色谱顶空进样/FID检测器检测色谱图；
图10示全谱系油样品-2经气相色谱顶空进样/FID检测器检测色谱图；
图11(a)示硅胶固相萃取除杂液/DAD检测器检测色谱图；
图11(b)示硅胶固相萃取洗脱液/DAD检测器检测色谱图。
具体实施方式
本发明提供了大麻油的制备方法及其制备的大麻油，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明采用的仪器皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
1)将大麻花叶粉碎20目，得到药材粉末；
2)将药材粉末采用8倍量(V/W)60％(V/V)的乙醇常温常压浸提3次，得到提取液；
3)将提取液浓缩至密度1.20(50℃)，回收乙醇，得到流膏；
4)将此流膏，加入等体积纯化水常压煮沸(94℃)1.2h，趁热补充70℃热水至药材4倍量(V/W)体积，搅拌15min，得分散液；
5)将分散液静置，直至温度测定为50℃，板框过滤(200目)，除去底部不溶物，得乳浊态分散液清液；
6)将清液上HPD007层析柱吸附30min后，直接以65％乙醇，1BV/h流速洗脱5BV，得富含CBD的全谱系大麻油目标段-I，以95％乙醇，0.25BV/h冲柱3BV，得富含THC的全谱系大麻油目标段-II；
7)浓缩步骤6)得到的全谱系油目标段-I，减压浓缩至无水稠膏，以庚烷，在35-50℃下溶解，快速以3BV/h流速，通过吸附剂短柱(白土3∶1硅藻土)，并以少量溶剂冲柱回收残留的部分，合并得脱色的全谱系大麻油原液-I；
8)将浓缩步骤6)得到的全谱系油目标段-II，减压浓缩至无水稠膏，以庚烷在35℃下溶解，上200-300目硅胶固相萃取柱，以庚烷∶乙酸乙酯(100∶1-2)洗除油脂和THC部分，以庚烷∶乙酸乙酯(100∶15)洗脱，直至HPLC测定或TLC薄层显示无相关大麻素成分，得脱色的全谱系大麻油原液-II；
9)将步骤7)的全谱系油原液-I和步骤8)的全谱系油原液-II合并，减压浓缩至无水稠膏，加入等体积纯化水，继续浓缩至无水稠膏，重复1-3次，得低有机溶残的全谱系大麻油成品1。
实施例2
1)将大麻花叶粉碎60目，得到药材粉末；
2)将药材粉末采用4倍量(V/W)90％(V/V)的乙醇提取2次， 得到提取液；
3)将提取液浓缩至密度1.10(50℃)，回收乙醇，得到流膏；
4)将此流膏，加入等体积纯化水常压煮沸(94℃)2h，趁热补充50℃热水至药材8倍量(V/W)体积，搅拌15min，得分散液；
5)将分散液静置，至温度测定40℃，800rpm离心，弃去底部不溶物，得乳浊态分散液清液；
6)将清液上SP825层析柱吸附1h后，直接以75％乙醇，2BV/h洗脱4BV，得富含CBD的全谱系大麻油目标段-I，以95％乙醇1BV/h冲柱5BV，得富含THC的全谱系大麻油目标段-II；
7)浓缩步骤6)得到的全谱系大麻油目标段-I，减压浓缩至无水稠膏，以己烷∶乙酸乙酯(6-10∶1)，在40-50℃下溶解，5BV/h加压快速通过硅藻土∶磷酸钙(1∶2-3)的混合吸附剂，得脱色的全谱系大麻油原液-I；
8)将浓缩步骤6)得到的全谱系油目标段-II，减压浓缩至无水稠膏，以己烷在50℃下溶解，上200-300目硅胶固相萃取柱，以庚烷∶乙酸乙酯(100∶1-2)洗除油脂和THC部分，以庚烷∶乙酸乙酯(100∶20)洗脱，直至HPLC测定或TLC薄层显示无相关大麻素成分，得脱色的全谱系大麻油原液-II；
9)将步骤7)的全谱系油原液-I和步骤8)的全谱系油原液-II合并，减压浓缩至无水稠膏，加入等体积纯化水，继续浓缩至无水稠膏，重复1-3次，得低有机溶残的全谱系大麻油成品2。
对比例1：
综合专利文献中提取和脱色的方法，对大麻花叶提取物进行以下处理：
1)将大麻花叶粉碎至40-60目、150℃烘烤2h，得到药材粉末；
2)将药材粉末采用甲醇∶氯仿(9∶1)提取，得到提取液；
3)将提取液减压浓缩至稠膏，回收有机相，得到富含大麻素的浓缩膏；
4)将此浓缩膏趁热，以65-80％乙醇，在60℃转溶两次，合并上清液，得转溶液；
5)将转溶液继续在60℃下，按固含物总量计加入0.5％活性炭，搅拌脱色0.5-1h，过滤，得全谱系大麻油原液；
6)将步骤5)的全谱系油原液减压浓缩至无水稠膏，加入等体积纯化水，继续浓缩至无水稠膏，重复1-3次，得低有机溶残的全谱系大麻油样品1。
对比例2：
1)、将大麻花、叶粉碎至60目，在150℃下，烘烤80min，得到药材粉末；
2)、将步骤(1)所得药材粉末用6倍正己烷萃取3次，合并，60℃浓缩
至稠膏(50℃，相对密度1.08)；
3)、向步骤(2)的稠膏加入氢氧化钠调节pH至11，充分搅拌1h，过
滤，滤液再加入乙酸调节pH至4.6，过滤，得不溶沉淀物；
4)、将步骤(3)得到的不溶沉淀物用石油醚溶解，过硅胶柱层析，丙酮∶石油醚(1∶1，V/V)洗脱5倍柱体积除杂，回收溶剂；丙酮∶石油醚(7∶3，V/V)洗脱6倍柱体积，收集第二部分洗脱液，浓缩后即为全谱系油样品2。
实验例1：
全谱系大麻油成品1-2和全谱系大麻油样品1和2各组分含量情况
1)大麻素与黄酮含量测定
色谱条件与系统适用性试验以十八烷基硅烷键合硅胶为填充剂；以乙腈为流动相A，以0.5％甲酸-水为流动相B，按A(％)∶B(％)＝70∶30进行等度洗脱；检测波长为210nm。理论板数按大麻二酚(CBD)峰计算应不低于2500。
混合对照品溶液的制备精密称取大麻二酚(CBD)、四氢大麻酚(THC)、香叶基黄酮A(CFLA)对照品各10mg，置100ml量瓶中，加甲醇100ml溶解制成每1ml各含0.1mg的对照品溶液，即得。
供试品溶液的制备取全谱系大麻油成品约20mg，精密称定，置100ml量瓶中，加甲醇100ml，超声处理10分钟，加甲醇稀释至刻度，摇勾， 用微孔滤膜(0.45pm)滤过，取续滤液，即得。
测定法分别精密吸取对照品溶液与供试品溶液各10μl，注入液相色谱仪，测定，按照以下矫正因子计算即得。结果如表1：
表1 大麻素和黄酮校正因子和保留时间
Figure PCTCN2020080807-appb-000001
总大麻素含量＝CBD％+CBDV％+CBG％+THCV％+CBN％+CBC％+CBL％；
总黄酮＝CFLA％+CFLB％+CFLC％
表中英文简写说明：
CBD大麻二酚；CBDV大麻素(或次大麻二酚)；CBG大麻萜酚；CBC大麻色烯；CBN大麻酚；THCV四氢大麻素(或次四氢大麻酚)；THC四氢大麻酚；CBL大麻环酚；CFLA/B/C香叶基黄酮A/B/C。
2)萜类测定：
气相色谱条件：30ml顶空瓶，加热温度90℃，平衡时间30min；HP-5MS石英毛细柱：30m×0.25mm×0.25μm；FID检测器(7820A)：250℃；进样口：200℃；接口180℃；1∶20分流进样；进样量0.8ml；氮气：2ml/min；尾吹：25ml/min；空气300ml/min；氢气：30ml/min；柱温50℃(2min)--20℃/min--200℃(10min)。
混合标准品制备与标准曲线：准确称取石竹烯25mg，溶解于25ml甲醇∶氯仿(1∶1)的混合溶剂，制成1mg/ml的母液。而后，倍比稀释(25、50、100、200、400)5个浓度梯度，分别顶空进样0.8ml，得到浓度-峰面积的标准曲线：y＝32.9446x+0.5867r＝0.9999
供试品制备：取全谱系大麻油25mg，溶解于25ml甲醇∶氯仿(1∶1)的混合溶剂，制成1mg/ml的供试品试液。
测定方法：分别精密吸取对照品溶液与供试品溶液各0.8ml，按照以上色谱条件顶空进样，测定峰面积，按照以下矫正因子即得各组分含量。
总萜＝叶绿醇％+丁香酚％+石竹烯％+芳樟醇％
表2 萜类校正因子和保留时间
名称 叶绿醇 芳樟醇 丁香酚 石竹烯
相对于石竹烯的f值 1.21 0.65 0.58 1
保留时间 4.78 8.52 9.22 9.68
3)测定结果汇总：
表3 大麻油检测结果
Figure PCTCN2020080807-appb-000002
注：化合物种数为各种测定方法所能测得的大麻素+黄酮+萜类的种类，以信号∶噪声(100∶1)以上的峰统计。
综上，本专利制备方法在富集了几乎所有大麻有效成分之外，获得了较好的性状特征，并使得质量可控性大幅提升。其中，实施例1制得的成品1所得化合物种类最多，除总大麻素外还含有黄酮和萜类物质，但未检测到THC存在。实施例中，实施例1提取获得的总大麻素含量更高。
实验例2：
实施例1～2记载的工艺参数为当前条件下甄选的最优模型，超出规 定范围是无法达到类似效果甚至是无法实现的。例如
1)乙醇提取浓度：必须限定在60-80％(V/V)，低于60％乙醇是无法完全浸出大麻素组分的，而高于80％乙醇则提出太多油脂和色素给后续工艺纯化带来较大麻烦，尤其这部分油脂色素还会裹挟大量的大麻素和萜类，造成损耗。
2)热水分散：在对清膏煮沸脱羧后，必须限定补充55-100℃热水，低于55℃是无法将此具有一定油脂性的物料完全分散的，仍会裹挟大麻素和萜烯组分被弃去。
3)庚烷-乙酸乙酯除杂和洗脱比例：全谱系油目标段-II上200-300目硅胶固相萃取柱，必须限定以庚烷∶乙酸乙酯(100∶1-2)洗除油脂和THC部分(见图11a)，超过100∶2，则大量CBC和CBL被洗脱下来；且必须限定以庚烷∶乙酸乙酯(100∶10-20)洗脱(见图11b)，达不到100∶10，则无法洗脱残留的CBD和CBDV组分。
表4 不同工艺参数制备方法所得组分含量
  THC含量/％ 总大麻素/％ 总黄酮/％ 总萜/％
55％醇提液浓缩膏 未检出 0.42 未检出 来检出
75％醇提液浓缩膏 5.72 38.75 0.18 0.05
85％醇提液浓缩膏 4.21 20.69 0.11 0.03
50℃水沉液过滤渣样 5.85 39.98 1.76 未检出
70℃水沉液过滤渣样 0.08 0.67 0.01 未检出
庚烷100∶3乙酸乙酯除杂液浓膏 67.86 0.57 未检出 未检出
庚烷100∶8乙酸乙酯洗脱液浓膏 未检出 56.19 1.77 0.21
表4记载了本发明工艺参数的部分筛选方案，除表4记载的参数外，其他参数与实施例1一致。从检测结果来看，这些参数都无法获得实施例1～2的效果，提取所得大麻油中的总大麻素含量与实施例1相比，存在显著性的差异，p＜0.05。
实验例3：孤养小鼠的镇静模型测试
1)实验小鼠的准备：①孤养小鼠模型：小鼠断奶后单只单笼饲养于 安静环境中，每周更换一次垫料。室温(24±2)℃，自由摄食饮水，饲养时间为6周。经长期隔离饲养后，小鼠在外观上的特征性表现为焦虑不安、恐惧、易激惹、攻击性强等特点。②群养组小鼠：每笼5只，饲养于相同环境中，实验时，取同龄小鼠。所有小鼠，体重均在18～22g。
2)注射剂的准备：①空白对照组：使用灭菌的生理盐水，2ml；②阳性对照组：使用2mg/kg的安定，2ml；③实验组：分别取上述四种提取物样品，以注射级大豆油配制25mg/ml的全谱系油的注射剂，2ml。以上均通过0.22μm有机膜过滤除菌。
3)镇静模型测试：
小鼠高架十字迷路包括2个30cm×5cm的开臂和2个30cm×5cm×15cm的闭臂，闭臂上部敞开，中央有一个5cm×5cm的开阔部位，每种类型的两臂呈相对位置，迷路距地面45cm。迷路由木板制成，底部和闭臂的壁均为黑色。为防止小鼠跌落，开臂的周边有一高出0.25cm的边界。
小鼠随机分成6组，每组20只：群养空白对照组(等量生理盐水)、孤养模型空白对照组(等量生理盐水)、阳性对照组(安定2.5mg/kg)、注射给药实验组(25mg/kg)，所有实验均在每天9：00～14：00于安静环境下进行。腹腔给药30min后，将小鼠置于高架十字迷路中央，头朝开臂，观察5min内小鼠的活动情况。观察并记录以下各项指标：进入开臂次数、进入闭臂次数及在两臂滞留的时间；分别计算小鼠进入开臂的时间和次数占总时间(开臂和闭臂滞留的时间之和)和总次数(开臂与闭臂之和)的百分比。观察时以小鼠四爪均进入开臂或闭臂为准。每只动物受试后用湿海绵将十字迷路彻底清洗并擦干排除残留气味对小鼠行为的影响。
表5 动物实验结果
Figure PCTCN2020080807-appb-000003
Figure PCTCN2020080807-appb-000004
其中，与孤养空白组相比，*P＜0.05，**P＜0.01，.
4)实验数据：
综上，孤养小鼠在注射全谱系油之后，明显提升其探索诱发焦虑的高架十字迷宫开臂频次，降低了其更喜欢的闭臂频次。然而，注射全谱系油样品3(含12.78％的THC)的所有小鼠均呈现一种不同程度的无序状态，有8只完全不具备统计意义，可能是由于样品中THC含量较高所致。所以，本专利提供的全谱系油成品相比于其他组分的大麻油样品，具有更安全、更明显的镇静作用，其原因可能是大麻中大麻素、黄酮、萜类等多成分具有一定的协同或辅助作用。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 大麻油的制备方法，包括：
    步骤1：将原料以4～10倍体积的30vol％～100vol％乙醇浸提获得提取液；所述原料为大麻叶和/或大麻花；
    步骤2：回收所述提取液中的乙醇至密度为1.05～1.25(50℃)，制得流膏；
    步骤3：将所述流膏与等体积水混合，常压煮沸1～3h后，补加50℃～100℃热水经搅拌获得分散液；所述分散液与原料的体积-质量比为(3～8)∶1；
    步骤4：所述分散液冷却至40℃～50℃，分离弃除不溶物，获得滤液；
    步骤5：将滤液上大孔树脂柱吸附，以50vol％～80vol％乙醇洗脱4～5BV，收集洗脱液为目标段-I；以95vol％乙醇洗脱3～5BV，收集洗脱液为目标段-II；
    步骤6：将所述目标段I减压浓缩去除水后，于30℃～50℃以有机溶剂A溶解后，溶液以吸附剂吸附后制得原液I；所述吸附剂为白土和/或硅藻土；
    步骤7：将所述目标段II减压浓缩去除水后，于35℃～50℃以有机溶剂B溶解后，溶液上硅胶柱吸附，以洗脱液A洗脱1～3倍柱体积，弃除洗脱液；再以洗脱液B洗脱2～5倍柱体积，收集洗脱液为原液II；所述洗脱液A由庚烷∶乙酸乙酯(100∶1～2)组成；所述洗脱液B由庚烷∶乙酸乙酯(100∶15～20)组成；
    步骤8：合并原液I与原液II，减压浓缩去除水分后，再以水清洗，制得大麻油。
  2. 根据权利要求1所述的制备方法，其特征在于，步骤1中所述提取中，乙醇的体积分数为60％～90％；所述原料与乙醇的质量-体积比为1∶(4～8)；所述浸提的条件为常温、常压；浸提次数为2～3次。
  3. 根据权利要求1或2所述的制备方法，其特征在于，步骤3中，所述常压煮沸的温度为94℃，时间为1.2～2h；所述热水的温度为50℃ ～70℃；搅拌的时长为15min；所述分散液与原料的体积-质量比为(4～8)∶1。
  4. 根据权利要求1～3任一项所述的制备方法，其特征在于，步骤4中，所述分离的方式为过滤或离心；所述过滤的孔径为200目；所述离心的转速为800rpm。
  5. 根据权利要求1～4任一项所述的制备方法，其特征在于，步骤5中，所述大孔树脂为HPD007层析柱或SP825层析柱；所述吸附的时间为0.5h～1h；所述目标段-I的洗脱采用65vol％～75vol％乙醇，流速为1～2BV/h；所述目标段-II的流速为0.25～1BV/h。
  6. 根据权利要求1～5任一项所述的制备方法，其特征在于，步骤6中所述有机溶剂A为庚烷，或为己烷与乙酸乙酯的混合物；所述己烷与乙酸乙酯的体积比为(6～10)∶1；所述吸附剂为硅藻土与磷酸钙的混合物，其中硅藻土与磷酸钙的质量比为1∶(2～3)；或为白土与硅藻土的混合物，其中白土与硅藻土的质量比为3∶1；所述吸附为使溶液通过吸附剂，所述通过的速度为3～5BV/h。
  7. 根据权利要求1～6任一项所述的制备方法，其特征在于，步骤7中所述有机溶液B为庚烷或己烷；所述硅胶固相萃取柱填料为100～500目填料。
  8. 根据权利要求1～7任一项所述的制备方法，其特征在于，步骤8中所述以水清洗具体为，加入等体积的水，再浓缩至无水稠膏，重复1～3次。
  9. 权利要求1～8任一项所述制备方法制得的大麻油。
  10. 权利要求1～8任一项所述制备方法制得的大麻油在药品、保健品、特殊功能食品或日化品领域的应用。
PCT/CN2020/080807 2019-04-30 2020-03-24 大麻油的制备方法及其制备的大麻油 WO2020220863A1 (zh)

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