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WO2020119001A1 - 一种利用高速逆流色谱分离纯化制备大麻二酚的方法 - Google Patents

一种利用高速逆流色谱分离纯化制备大麻二酚的方法 Download PDF

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WO2020119001A1
WO2020119001A1 PCT/CN2019/083401 CN2019083401W WO2020119001A1 WO 2020119001 A1 WO2020119001 A1 WO 2020119001A1 CN 2019083401 W CN2019083401 W CN 2019083401W WO 2020119001 A1 WO2020119001 A1 WO 2020119001A1
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cannabidiol
speed countercurrent
countercurrent chromatography
purification
speed
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French (fr)
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邓秋云
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Techson Industry Co Ltd
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Techson Industry Co Ltd
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Priority to US17/311,991 priority Critical patent/US11267775B2/en
Priority to EP19896174.0A priority patent/EP3896050B1/en
Priority to ES19896174T priority patent/ES2991161T3/es
Priority to CA3121924A priority patent/CA3121924A1/en
Priority to JP2021528864A priority patent/JP2022510832A/ja
Priority to AU2019395470A priority patent/AU2019395470B2/en
Publication of WO2020119001A1 publication Critical patent/WO2020119001A1/zh
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    • 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
    • C07C37/72—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by liquid-liquid treatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0215—Solid material in other stationary receptacles
    • B01D11/0223—Moving bed of solid material
    • B01D11/0242—Moving bed of solid material in towers, e.g. comprising contacting elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/265—Adsorption chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424—Elution mode
    • B01D15/426—Specific type of solvent
    • 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
    • C07C37/004—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by obtaining phenols from plant material or from animal material
    • 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
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated

Definitions

  • the invention belongs to the field of cannabidiol extraction, and particularly relates to a method for separating and purifying cannabidiol by high-speed countercurrent chromatography.
  • Marijuana (scientific name: Cannabis sativa L.), cannabis, cannabis is an annual herb. Also known as hemp, hemp, hemp, mountain silk seedlings, jute, has important agricultural and medicinal value. At present, more than 500 substances have been isolated from cannabis plants, of which at least 86 cannabis phenolic compounds. It mainly includes tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN) and cannabichromene (CBC), etc., the first three of which account for 90% of cannabidiol compounds. Above 4%, tetrahydrocannabinol can cause psychedelic addiction, can be used as a drug, and has been banned for a long time.
  • THC tetrahydrocannabinol
  • CBD cannabidiol
  • CBN cannabinol
  • CBC cannabichromene
  • the raw material cannabis for industrial use is simply referred to as "industrial cannabis".
  • the content of THC in cannabis flowers and leaves is less than 0.3%, and it is not available.
  • the value of extracting the toxic component tetrahydrocannabinol or directly as a drug can be legally cultivated on a large scale and industrialized development and utilization.
  • cannabidiol is not neurotoxic and is a non-addictive active ingredient with obvious medicinal value.
  • Pharmacological research shows that it can antagonize the effect of tetrahydrocannabinol on the human nervous system, and has anti-spasmodic, sedative and hypnotic, anti-rheumatic arthritis, anti-anxiety and other pharmacological activities, is a very promising application in the field of medicine and food Natural active ingredients.
  • composition of cannabinoids in industrial cannabis plants is complex. There are more than 80 known compounds with many similar polar components. After being extracted and refined by traditional methods, the purity of CBD in the final product is not high.
  • the technical problem to be solved by the present invention is to provide a method for separating and purifying cannabidiol using high-speed countercurrent chromatography.
  • This method uses a combination of a macroporous resin chromatography column and a high-speed countercurrent chromatography to remove impurities and tetrahydrocannabinol. it is good.
  • the invention provides a method for separating and purifying cannabidiol by high-speed countercurrent chromatography, which includes:
  • n-hexane-ethyl acetate-methanol-water also a three-solvent system composed of n-hexane-methanol-water
  • the separation solvent system for high-speed countercurrent chromatography separation and purification, collect cannabidiol distillate fractions, and recover the solvent , Post-processing cannabidiol.
  • the alcohol extraction and concentration in the step (1) uses an ethanol solution with a volume percentage concentration of 50-95%, and the mass-volume ratio of industrial cannabis flowers or leaves to the ethanol solution is 1 g: 5-10 mL.
  • the water sink temperature in the step (1) is 5-8°C, and the water sink time is 24 hours.
  • the macroporous resin in the step (2) is D101, AB-8 or HPD-100.
  • an ethanol solution with a volume percentage concentration of 5-85% is used for gradient elution; the elution section of the ethanol solution with a volume percentage concentration of 70-85% is collected.
  • the volume ratio of n-hexane-ethyl acetate-methanol-water in the step (3) is 5:0 ⁇ 1:5:1 ⁇ 3.
  • the high-speed countercurrent chromatography separation and purification in the step (3) specifically includes: taking one phase in the separation solvent system as the stationary phase and the other phase as the mobile phase, and pumping the stationary phase into the system at a flow rate of 30-50 mL/min
  • the mobile phase is pumped into the mobile phase at a flow rate of 5 to 10 mL/min under the conditions of 25 to 35° C. and the host speed of 700 to 1000 r/min.
  • the cannabidiol The crude extract is dissolved in the mobile phase and injected, and after detection by the detector, the cannabidiol fraction is collected.
  • the upper phase in the solvent system is the stationary phase and the lower phase is the mobile phase; or the lower phase in the solvent system is the stationary phase and the upper phase is the mobile phase.
  • the high-speed counter-current chromatograph can adopt forward-forward mode and forward-backward mode.
  • the positive connection is the connection mode from the head end to the tail end.
  • the concentration of the cannabidiol crude extract dissolved in the mobile phase is 50-100 mg/mL, the injection volume is 20 mL; the detection wavelength is 220 nm.
  • the post-processing in the step (3) includes concentration under reduced pressure, crystallization and vacuum freeze-drying.
  • the sample of the present invention is roughly separated by the macroporous adsorption resin, most of the impurities in the sample are removed, and cannabidiol is enriched; and then the impurities are further refined by high-speed countercurrent chromatography to remove impurities, especially to remove tetrahydrocannabinol, reduce The wastage makes the preparation of cannabidiol large-scale.
  • the high-speed countercurrent chromatography adopted by the present invention does not use solid carriers, so there is no irreversible adsorption and loss of samples caused by the solid carriers, the separation effect is high, the raw materials can be used to the maximum, and the production is reduced cost. Moreover, the entire separation process is in a closed device, the preparation process is simple, safe, environmentally friendly, and can be carried out continuously. It is an efficient and fast method for separating high-purity CBD from industrial cannabis.
  • the invention adopts a combination of a macroporous resin chromatography column and a high-speed countercurrent chromatograph, and optimizes the process parameters to separate high-purity cannabidiol from industrial cannabis flowers and leaves, while removing the psychotoxic component tetrahydrocannabinol and the solvent used Environmental protection, no residue, low cost, recyclable, suitable for industrial production.
  • Example 1 is a high performance liquid chromatogram of crude extract of cannabidiol in Example 1;
  • Example 2 is a high performance liquid chromatogram of the final product cannabidiol in Example 1;
  • FIG. 3 is a graph of the crude extract of cannabidiol separated and purified by high-speed countercurrent chromatography in Example 1.
  • the mobile phase is pumped into the mobile phase at a flow rate of 5mL/min.
  • 1000mg of crude cannabidiol extract is dissolved in the 20mL mobile phase, injected and detected under a UV detector.
  • the target peak component was collected and concentrated under reduced pressure to remove the organic phase.
  • the precipitate precipitated during the depressurization was filtered with suction and freeze-dried to obtain cannabidiol monomer with a purity of 99.12%. THC was not detected, as shown in Figures 2 and 3. Show.
  • D101 macroporous resin is soaked in ethanol for 24 hours, then loaded into the chromatography column, washed with ethanol until the eluate plus an equal volume of deionized water is a transparent solution. Then wash with deionized water until the effluent is neutral; dissolve the crude cannabis extract with ethanol and inject it into D101 macroporous resin until the adsorption volume reaches 2/3 of the total volume of the resin. First rinse the resin with deionized water at a flow rate of 2.5BV/h, then rinse with 10%, 30%, 70%, 80% ethanol aqueous solution at a flow rate of 2.5BV/h, and collect the 70-80% eluted fraction . The ethanol was removed by vacuum rotary evaporation at 45°C to obtain a crude extract of cannabidiol.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Botany (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Pyrane Compounds (AREA)

Abstract

涉及一种利用高速逆流色谱分离纯化制备大麻二酚的方法，包括：醇提水沉、大孔树脂吸附、高速逆流色谱分离。采用大孔树脂层析柱和高速逆流色谱仪相结合，通过优化工艺参数，从工业大麻花叶中分离得到高纯度的大麻二酚，同时去除了精神毒性成分四氢大麻酚，所用溶剂环保、无残留、低成本，可循环利用，适合工业化生产。

Description

一种利用高速逆流色谱分离纯化制备大麻二酚的方法 技术领域
本发明属于大麻二酚提取领域，特别涉及一种利用高速逆流色谱分离纯化制备大麻二酚的方法。
背景技术
大麻(学名：Cannabis sativa L.)，大麻科、大麻属一年生草本植物。又名麻、汉麻、火麻、山丝苗、黄麻，具有重要的农用及药用价值。目前，人们已从大麻植株中分离出了500余种物质，其中大麻酚类化合物至少有86种。主要包括四氢大麻酚(tetrahydrocannabinol，THC)、大麻二酚(cannabidiol，CBD)、大麻酚(cannabinol，CBN)及大麻萜酚(cannabichromene，CBC)等，其中前三者占大麻酚类化合物的90％以上，四氢大麻酚可使人致幻成瘾，可作毒品，曾在相当长时期内禁种。
由于大麻的经济、药用价值极高，专供工业用途的原料大麻简称为“工业大麻”，其生长期内大麻花、叶中的四氢大麻酚(THC)含量低于0.3％，不具备提取毒性成分四氢大麻酚的价值或直接作为毒品吸食，可以合法进行规模化种植与工业化开发利用。
近年来，通过对大麻活性成分的研究发现，大麻二酚(cannabidiol)不具有神经毒性，为非成瘾性活性成分，药用价值明显。药理研究表明，其能拮抗四氢大麻酚对人体神经系统的影响，并具有抗痉挛、镇静催眠、抗风湿性关节炎、抗焦虑等药理活性，是一种在医药、食品领域极具应用前景的天然活性成分。
肖培云等在《中国医药工业杂志》(2008年39卷4期)发表了对在不同生长期工业大麻中THC和CBD含量的测定方法比较的研究。其中提到在速长期、始花期和盛花期THC的含量小于千分之三，达到工业大麻的标准，同时CBD的含量也小于千分之三，说明CBD在工业大麻中的含量也很低，如何尽量去除THC等具有致幻及成瘾作用成分并保证高纯度CBD的产率，是CBD开发应用的前提。
目前，公开信息中有一些关于从工业大麻中提取大麻二酚的方法的报道，多采用各种柱层析技术，如使用大孔吸附树脂，MCI树脂或十八烷基键合硅胶等。通过对比研究，现有技术中所提及的从工业大麻中提取大麻二酚的方法，主要存在以下问题：
1)工业大麻植物中大麻素类成分复杂，已知的有80多种，极性相似成分较多，采用传统方法提取、精制后，最终产品中CBD纯度不高。
2)提取纯化后，仍可检出精神毒性成分THC，产品安全性不能得到保证，产品流通受限，影响工业化生产和应用。
3)传统技术中反复柱层析分离纯化，势必会造成CBD的损耗，产率下降，使产能受到限 制。
发明内容
本发明所要解决的技术问题是提供一种利用高速逆流色谱分离纯化制备大麻二酚的方法，该方法采用大孔树脂层析柱和高速逆流色谱仪相结合，去除杂质和四氢大麻酚效果更好。
本发明提供了一种利用高速逆流色谱分离纯化制备大麻二酚的方法，包括：
(1)以工业大麻花或叶为原料，经醇提浓缩、水沉、真空旋转蒸发得到大麻粗提物；
(2)将得到的大麻粗提物使用乙醇(稀)溶解后注入大孔树脂，经梯度洗脱，收集富含大麻二酚的洗脱段，真空旋转蒸发得到大麻二酚粗提物；
(3)以正己烷-乙酸乙酯-甲醇-水(也可以是正己烷-甲醇-水组成的三溶剂体系)为分离溶剂体系进行高速逆流色谱分离纯化，收集大麻二酚馏分段，回收溶剂，后处理得到大麻二酚。
所述步骤(1)中的醇提浓缩采用体积百分浓度为50～95％的乙醇溶液，工业大麻花或叶与乙醇溶液的质量体积比为1g:5～10mL。
所述步骤(1)中的水沉温度为5～8℃，水沉时间为24h。
所述步骤(2)中的大孔树脂为D101、AB-8或HPD-100。
所述步骤(2)中的梯度洗脱采用体积百分浓度为5～85％的乙醇溶液进行梯度洗脱；收集体积百分浓度为70～85％的乙醇溶液洗脱段。
所述步骤(3)中的正己烷-乙酸乙酯-甲醇-水的体积比为5:0～1:5:1～3。
所述步骤(3)中的高速逆流色谱分离纯化具体为：以分离溶剂体系中的一相为固定相，另一相为流动相，以30～50mL/min的流速将所述固定相泵入高速逆流色谱仪中，在25～35℃、主机转速为700～1000r/min的条件下，以5～10mL/min的流速泵入所述流动相，待两相达到平衡后，将大麻二酚粗提物用流动相溶解后进样，经检测器检测后，收集大麻二酚馏分段。
其中，以溶剂体系中的上相为固定相，下相为流动相；或者以溶剂系统的下相为固定相，上相为流动相。高速逆流色谱仪可以采用正接正转模式，也可以采用正接反转模式。所述正接即从首端和尾端的连接模式。
所述大麻二酚粗提物用流动相溶解后的浓度为50～100mg/mL，进样体积为20mL；检测的波长为220nm。
所述步骤(3)中的后处理包括减压浓缩、结晶和真空冷冻干燥。
本发明的样品经过大孔吸附树脂粗分离后，样品中的大部分杂质被除去，大麻二酚得到富集；再经过高速逆流色谱仪进一步精分离去除杂质，尤其是去除四氢大麻酚，降低损耗，使大麻二酚的制备实现规模化。
本发明所采用的高速逆流色谱法与柱层析等方法相比，不使用固态的载体，因此不存在 固态载体造成的样品不可逆吸附和损耗，分离效果高，能最大限度的利用原料，降低生产成本。而且整个分离过程在封闭设备中，制备过程简便、安全、环保，能够连续进行，是一种高效快捷的从工业大麻中分离高纯度CBD的方法。
有益效果
本发明采用大孔树脂层析柱和高速逆流色谱仪相结合，通过优化工艺参数，从工业大麻花叶中分离得到高纯度的大麻二酚，同时去除了精神毒性成分四氢大麻酚，所用溶剂环保、无残留、低成本，可循环利用，适合工业化生产。
附图说明
图1为实施例1中大麻二酚粗提物的高效液相色谱图；
图2为实施例1中最终产物大麻二酚的高效液相色谱图；
图3为实施例1中用高速逆流色谱仪分离纯化大麻二酚粗提物的图谱。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
(1)将10kg工业大麻粉碎烘干，按照料液比1:5(w/v，g/mL)的比例加入到70％乙醇水溶液充分混合，超声提取120min，(控制温度在45℃以下，避光)，超声结束后真空抽滤，得到的滤渣按上述条件重复提取2次，合并滤液，在45℃下真空旋转蒸发除去乙醇，浓缩至相对密度为1.2，加入5-7倍纯化水，在5-8℃条件下，水沉24h，过滤，沉淀物减压干燥得到大麻粗提物。
(2)将AB-8大孔树脂用乙醇浸泡24h后装入层析柱中，用乙醇洗至洗出液加等体积去离子水为透明溶液为止。然后用去离子水洗至流出液为中性；将大麻粗提物用乙醇溶解后注入AB-8大孔树脂中，直至吸附体积达到树脂总体积的2/3。先用去离子水以2BV/h的流速冲洗树脂，然后分别用10％，30％，50％，70％的乙醇水溶液以2BV/h的流速冲洗，并收集70％洗脱部分。在45℃下真空旋转蒸发除去乙醇，得到大麻二酚粗提物，高效液相色谱图如图1所示。
(3)将正己烷:乙酸乙酯:甲醇:水按5:0.5:5:1的体积比置于分液漏斗中，充分摇匀，静置20min后，将上下相分开，超声脱气20min。将上相作为固定相，下相作为流动相。将高速逆流色谱仪启动预热30min后，循环水浴设置为25℃，将固定相以30mL/min的流速泵入仪器，然后正接正转，启动仪器，使主机转速至800r/min。转速稳定后，以5mL/min的流速 泵入流动相，两相在管路中达到平衡后，将1000mg大麻二酚粗提物溶于20mL流动相中，进样并在紫外检测器下检测，收集目标峰组分并减压浓缩除去有机相，减压过程中析出的沉淀物抽滤并冷冻干燥即得大麻二酚单体，纯度为99.12％，未检出THC，如图2和3所示。
实施例2
(1)将10kg工业大麻粉碎烘干，按照料液比1:10(w/v，g/mL)的比例加入到80％乙醇水溶液充分混合，超声提取100min，(控制温度在45℃以下，避光)，超声结束后真空抽滤，得到的滤渣按上述条件重复提取2次，合并滤液，在45℃下真空旋转蒸发除去乙醇，浓缩至相对密度为1.2，加入5-7倍纯化水，在5-8℃条件下，水沉24h，过滤，沉淀物减压干燥得到大麻粗提物。
(2)将D101大孔树脂用乙醇浸泡24h后装入层析柱中，用乙醇洗至洗出液加等体积去离子水为透明溶液为止。然后用去离子水洗至流出液为中性；将大麻粗提物用乙醇溶解后注入D101大孔树脂中，直至吸附体积达到树脂总体积的2/3。先用去离子水以2.5BV/h的流速冲洗树脂，然后分别用10％，30％，70％，80％的乙醇水溶液以2.5BV/h的流速冲洗，并收集70-80％洗脱部分。在45℃下真空旋转蒸发除去乙醇，得到大麻二酚粗提物。
(3)将正己烷:甲醇:水按5:5:2.5的体积比置于分液漏斗中，充分摇匀，静置20min后，将上下相分开，超声脱气20min。将下相作为固定相，上相作为流动相。将高速逆流色谱仪启动预热30min后，循环水浴设置为25℃，将固定相以30mL/min的流速泵入仪器，然后正接反转，启动仪器，使主机转速至850r/min。转速稳定后，以10mL/min的流速泵入流动相，两相在管路中达到平衡后，将1000mg大麻二酚粗提物溶于20mL固定相中，进样并在紫外检测器下检测，收集目标峰组分并减压浓缩除去有机相，得到的粉末用80％乙醇结晶处理，过滤得到晶体，减压真空干燥即得大麻二酚单体，纯度为99.75％，未检出THC。
实施例3
(1)将10kg工业大麻粉碎烘干，按照料液比1:8(w/v，g/mL)的比例加入到80％乙醇水溶液充分混合，超声提取120min，(控制温度在45℃以下，避光)，超声结束后真空抽滤，得到的滤渣按上述条件重复提取2次，合并滤液，在45℃下真空旋转蒸发除去乙醇，浓缩至相对密度为1.2，加入5-7倍纯化水，在5-8℃条件下，水沉24h，过滤，沉淀物减压干燥得到大麻粗提物。
(2)将AB-8大孔树脂用乙醇浸泡24h后装入层析柱中，用乙醇洗至洗出液加等体积去离子水为透明溶液为止。然后用去离子水洗至流出液为中性；将大麻粗提物用乙醇溶解后注入AB-8大孔树脂中，直至吸附体积达到树脂总体积的2/3。先用去离子水以2BV/h的流速冲洗树脂，然后分别用10％，30％，50％，80％的乙醇水溶液以2BV/h的流速冲洗，并收集 80％洗脱部分。在45℃下真空旋转蒸发除去乙醇，得到大麻二酚粗提物。
(3)将正己烷:甲醇:水按5:5:1的体积比置于分液漏斗中，充分摇匀，静置20min后，将上下相分开，超声脱气20min。将上相作为固定相，下相作为流动相。将高速逆流色谱仪启动预热30min后，循环水浴设置为25℃，将固定相以30mL/min的流速泵入仪器，然后正接正转，启动仪器，使主机转速至800r/min。转速稳定后，以5mL/min的流速泵入流动相，两相在管路中达到平衡后，将2000mg大麻二酚粗提物溶于20mL流动相中，进样并在紫外检测器下检测，收集目标峰组分并减压浓缩除去有机相，减压过程中析出的沉淀物抽滤并冷冻干燥即得大麻二酚单体，纯度为99.50％，未检出THC。

Claims (9)

  1. 一种利用高速逆流色谱分离纯化制备大麻二酚的方法，包括：
    (1)以工业大麻花或叶为原料，经醇提浓缩、水沉、真空旋转蒸发得到大麻粗提物；
    (2)将得到的大麻粗提物使用乙醇溶解后注入大孔树脂，经梯度洗脱，收集富含大麻二酚的洗脱段，真空旋转蒸发得到大麻二酚粗提物；
    (3)以正己烷-乙酸乙酯-甲醇-水为分离溶剂体系进行高速逆流色谱分离纯化，收集大麻二酚馏分段，回收溶剂，后处理得到大麻二酚。
  2. 根据权利要求1所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述步骤(1)中的醇提浓缩采用体积百分浓度为50～95％的乙醇溶液，工业大麻花或叶与乙醇溶液的质量体积比为1g:5～10mL。
  3. 根据权利要求1所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述步骤(1)中的水沉温度为5～8℃，水沉时间为24h。
  4. 根据权利要求1所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述步骤(2)中的大孔树脂为D101、AB-8或HPD-100。
  5. 根据权利要求1所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述步骤(2)中的梯度洗脱采用体积百分浓度为5～85％的乙醇溶液进行梯度洗脱；收集体积百分浓度为70～85％的乙醇溶液洗脱段。
  6. 根据权利要求1所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述步骤(3)中的正己烷-乙酸乙酯-甲醇-水的体积比为5:0～1:5:1～3。
  7. 根据权利要求1所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述步骤(3)中的高速逆流色谱分离纯化具体为：以分离溶剂体系中的一相为固定相，另一相为流动相，以30～50mL/min的流速将所述固定相泵入高速逆流色谱仪中，在25～35℃、主机转速为700～1000r/min的条件下，以5～10mL/min的流速泵入所述流动相，待两相达到平衡后，将大麻二酚粗提物用流动相溶解后进样，经检测器检测后，收集大麻二酚馏分段。
  8. 根据权利要求7所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述大麻二酚粗提物用流动相溶解后的浓度为50～100mg/mL，进样体积为20mL；检测的波长为220nm。
  9. 根据权利要求1所述的一种利用高速逆流色谱分离纯化制备大麻二酚的方法，其特征在于：所述步骤(3)中的后处理包括减压浓缩、结晶和真空冷冻干燥。
PCT/CN2019/083401 2018-12-14 2019-04-19 一种利用高速逆流色谱分离纯化制备大麻二酚的方法 Ceased WO2020119001A1 (zh)

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