CN111961021B - Separation and purification process of geranylflavone A - Google Patents
Separation and purification process of geranylflavone A Download PDFInfo
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- CN111961021B CN111961021B CN201911400597.8A CN201911400597A CN111961021B CN 111961021 B CN111961021 B CN 111961021B CN 201911400597 A CN201911400597 A CN 201911400597A CN 111961021 B CN111961021 B CN 111961021B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/40—Separation, e.g. from natural material; Purification
Abstract
The application discloses a method for preparing geranylflavone A, which at least comprises the following steps: a) pretreatment of hemp flower and leaf raw materials; b) extracting the pretreated hemp flower and leaf raw material by using a solvent I, concentrating an extracting solution, and separating to obtain a supernatant; c) adsorbing the supernatant through macroporous adsorption resin, washing out the supernatant through a solvent II, and concentrating to obtain a geranylflavone A crude product; d) the geranylflavone A crude product is subjected to trans-dissolution by a solvent III, and insoluble substances are obtained through separation; e) recrystallizing the insoluble substance to obtain geranylflavone A primary product. The separation and purification process has high product transfer rate, and the purity of geranylflavone A can reach more than 95%. In the operation, a liquid sealing film is selected to isolate air, and the finished product can be quickly dried in vacuum, so that the original color of the finished product can be kept. The process can realize production amplification, and the macroporous adsorption resin can be repeatedly used after proper regeneration treatment, so that the process has a good industrial preparation prospect.
Description
Technical Field
The application relates to a separation and purification process of geranylflavone A, belonging to the field of chemical industry.
Background
The whole strain of hemp has more than 500 compounds, wherein more than 20 flavonoid compounds are reported, and apigenin, luteolin, quercetin, orientin, rutin, vitexin and the like are common. In addition, geranylflavone A (Cannflavin A) and geranylflavone B (Cannflavin B) are two flavonoid compounds specific to hemp plants.
Cannabinoids have been shown to possess a variety of pharmacological activities, as Barrett et al reported that geranylflavone A/B in Cannabis can potentiate anti-inflammatory effects greatly, and that prostaglandin E-2 is inhibited 30-fold more than aspirin in rheumatoid synovial cells (Barrett, M.L., A.M.Scutt, et al, "Canunflavin A and B, pre-esterified flavones from Cannabis sativa". Experientia 42(4): 452 453). US20190083452a1 discloses the use of cannabinoids and derivatives thereof for the prevention and treatment of neurodegenerative diseases. US patent 20180353462a1 discloses the use of cannabixanthone derivatives for the treatment and prevention of cancer.
However, to date, this plant-derived compound has not been developed and used. The geranylflavone A content in industrial hemp flowers is about two ten-thousandth, and the content of geranylflavone B is as low as one hundred ten-thousandth, so that the mass preparation of samples is difficult, and no standard product is sold in the market at present, and the separation method found in the current literature is basically limited to the vegetative separation of mg grade, such as chloroform 9: 1, or sequentially extracting petroleum ether normal hexane, chloroform, ethyl acetate methanol water and purifying by means of silica gel column chromatography and the like, only a small amount of samples can be prepared, and the method is not suitable for industrial production.
Therefore, the comprehensive development and utilization of geranylflavone A firstly urgently needs to solve the preparation method, and at present, no industrial preparation scheme is reported at home and abroad.
Disclosure of Invention
The invention provides a process for industrially preparing geranylflavone A. Wherein the purity of geranylflavone A can reach more than 95%.
According to one aspect of the present application, there is provided a process for the preparation of geranylflavone a, characterized in that it comprises at least the following steps:
a) pretreatment of hemp flower and leaf raw materials;
b) extracting the pretreated hemp flower and leaf raw material by using a solvent I, concentrating an extracting solution, and separating to obtain a supernatant;
c) adsorbing the supernatant through macroporous adsorption resin, washing out the supernatant through a solvent II, and concentrating to obtain a geranylflavone A crude product;
d) the geranylflavone A crude product is subjected to trans-dissolution by a solvent III and is separated to obtain insoluble substances;
e) recrystallizing the insoluble substance to obtain geranylflavone A primary product.
Optionally, in step a), the pre-processing includes: pulverizing hemp flower and leaf to 10-60 mesh.
Optionally, the pre-treatment further comprises degreasing.
Optionally, the degreasing comprises: pulverizing Cannabis sativa flower and leaf to obtain Cannabis sativa flower and leaf raw material of 10-60 meshes, and extracting with petroleum ether, n-hexane or n-heptane.
Optionally, the degreasing comprises: pulverizing Cannabis sativa flower and leaf to obtain Cannabis sativa flower and leaf raw material of 10-60 meshes, and leaching with petroleum ether, n-hexane or n-heptane for 2-4 hr.
Optionally, step a) comprises: the hemp flower and leaf raw material with 10-60 meshes obtained by crushing hemp flower and leaf is leached for 2-4h by petroleum ether (or normal hexane, normal heptane) with the amount of 2-5 times (M/V), and then is dried by a residue drying machine under reduced pressure (45-55 ℃ and-0.06-0.08 MPa) to obtain the pretreated hemp flower and leaf raw material.
Optionally, step a) comprises: baking folium Cannabis until water content is below 15% (baking step is easy for storage), and pulverizing into 10-60 mesh to obtain non-defatted folium Cannabis raw material; and crushing the raw material to 10-60 meshes, leaching the raw material for 2-4h by petroleum ether (or normal hexane and normal heptane) with the amount of 2-5 times (M/V), and drying the raw material by a slag drying machine under reduced pressure (45-55 ℃ and-0.06-0.08 MPa) to obtain the pretreated hemp flower and leaf raw material.
Most of oil in the hemp flower and leaf raw material can be removed by degreasing, and the post-treatment pressure is relieved.
Optionally, in step a), the amount of the hemp flower leaf raw material is above 300 kg.
Optionally, in step a), the amount of hemp flower leaf material is between 300kg and 1200 kg.
Optionally, in step b), the solvent I is selected from at least one of methanol, ethanol and acetone.
Optionally, in step b), cold soaking or hot refluxing with 65-95 wt% solvent I with volume (W/V) 5-20 times of the weight of folium Cannabis raw material for 1-3h, extracting for 2-4 times, mixing, concentrating to solvent I concentration of 50-70 wt%, and separating to obtain supernatant.
Optionally, in step c), the macroporous adsorbent resin is selected from at least one of HPD series, ADS series, HZ series, XAD series, SP series macroporous adsorbent resins or polyamide resins.
Optionally, the macroporous adsorbent resin is selected from at least one of AB-8, D-101, HPD700, XAD2, XDA-8, LSA-7, D-941, DM-130, ADS600, ADS-17, SP-825, HPD-600, and SP825 macroporous adsorbent resins.
Optionally, the amount of the macroporous adsorbent resin is 10-75% of the feed mass of the cannabis sativa leaf raw material.
Optionally, the amount of the macroporous adsorbent resin is 15-60% of the feed mass of the cannabis sativa leaf raw material.
Optionally, the amount of the macroporous adsorbent resin is 18-53% of the feed mass of the cannabis sativa leaf raw material.
Optionally, the amount of the macroporous adsorbent resin is selected from 15%, 18%, 20%, 30%, 40%, 42.5%, 50%, 52.7%, 53%, 60%, 70% or 75% of the upper limit of the charged mass of the hemp flower leaf raw material; the lower limit is selected from 10%, 15%, 18%, 20%, 30%, 40%, 42.5%, 50%, 52.7%, 53%, 60% or 70%.
In the application, the macroporous adsorption resin has the following structural characteristics: 1) the resin has permanent micropores, the size, the number and the distribution of the pore passages can be controlled and adjusted through a pore-foaming agent and the crosslinking degree in the preparation process according to needs, and meanwhile, a user is provided with a selection space. For example, in practical experiments, it was found that the pore size was superior to other specifications at 80-100A °.
(2) Since the resin is porous and spongy, the surface area is extremely large (as large as 1000 m)2More than g), can be processed in large scale.
(3) After proper regeneration treatment, the filler can be repeatedly used, and the cost consumption is greatly reduced.
In the application, the macroporous adsorption resin is used, large-scale treatment can be carried out, and the filler can be repeatedly used after appropriate regeneration treatment, so that the cost consumption is greatly reduced. The regeneration can be completed by flushing the column with 2-5BV (BV refers to the volume of the column bed, namely the actual column filling volume of the resin layer) of 95 wt% ethanol, the regeneration can be repeatedly used, the laboratory repeated experiment investigation can be carried out, the regeneration can be repeated for more than 50 times, and the adsorption and desorption are not influenced.
The macroporous adsorption resin can be used for treating a large amount of supernatant containing geranylflavonoid A separated after alcohol extraction. The existing purification means such as silica gel column chromatography can only process a very small amount of samples, and the purification time is long. The macroporous adsorption resin is utilized, the treatment of industrial quantity can be realized, and the treatment time period is short.
Optionally, in step c), the solvent II is selected from at least one of methanol, ethanol, isopropanol, and acetone.
Optionally, in the step c), the supernatant is subjected to macroporous adsorption resin, and is washed out by 30-60 wt% of solvent II to obtain a geranylflavone A crude product.
Optionally, in the step c), the supernatant is subjected to macroporous adsorption resin, and 70-100 wt% of solvent II is used for partially washing out Cannabidiol (CBD) -based cannabinoid components.
Optionally, in step d), the solvent III is selected from at least one of non-polar alkanes, halogenated alkanes, esters, higher aliphatic alcohols.
Optionally, the non-polar alkane is selected from at least one of petroleum ether, diethyl ether, n-hexane and n-heptane.
Optionally, the alkyl halide is at least one selected from dichloromethane and chloroform.
Optionally, the ester is selected from at least one of ethyl acetate and butyl acetate.
Optionally, the higher aliphatic alcohol is selected from at least one of n-octanol, sec-octanol, and heptanol.
In the present application, the higher aliphatic alcohol means "aliphatic alcohol having 5 to 10 carbon atoms".
Optionally, the solvent III is selected from one of n-heptane, n-octanol, a mixed solution of n-heptane and ethyl acetate.
Optionally, in the step d), the number of times of the transferring and dissolving is 1-3, and no other ointment-like impurities are taken as a transferring and dissolving end point.
Alternatively, the brown-yellow insoluble residue can be uniformly dispersed in the transfer solution or other auxiliary supporting medium, and no other ointment-like impurities are taken as the transfer solution end point.
Optionally, the solvent for recrystallization is selected from at least one of halogenated alkanes, esters, ketones, a binary system of methanol/ethanol and water.
Optionally, the alkyl halide is at least one selected from dichloromethane and chloroform.
Optionally, the ester is selected from at least one of ethyl acetate and butyl acetate.
Optionally, the ketone is selected from at least one of acetone and butanone.
Alternatively, the recrystallization solvent is used in an amount corresponding to a saturation concentration at which the sample is dissolved at room temperature up to a limit concentration at which precipitation is possible.
Alternatively, the recrystallization solvent is used in an amount of 0.5 to 5 times (M/V) the amount of the solid crude product.
Optionally, the recrystallization condition is crystallization at-20 to 10 ℃ for 4 to 24 hours.
Optionally, filtering the crystallized sample, and washing with the same recrystallization solvent at-20-0 deg.C to obtain amorphous geranylflavonoid A.
The amount of washing solvent is only required to wet the entire sample layer.
Alternatively, the washing solvent is used in an amount of 0.05 to 0.2 times the amount of the sample (M/V).
Optionally, in step e), spraying a solvent IV on the surface of the recrystallized geranyl flavone a to form a liquid-sealed film, so as to obtain a primary geranyl flavone a product.
Optionally, the solvent IV is selected from at least one of ethyl acetate, water, n-heptane.
In practical application, only a layer of organic solvent or water mist is needed to be sprayed on the surface of the geranylflavone A, and the operation is simple and convenient, so that the air isolation can be realized, and the excellent effect of keeping the natural color of the finished product can be realized.
Optionally, drying the geranyl flavone A primary product obtained in the step e) under reduced pressure to obtain a finished geranyl flavone A product.
Optionally, the reduced pressure drying is carried out under the conditions of pressure of 0.06-0.10Mpa, temperature of 30-60 deg.C, and time of 6-36 h.
In the present application, "CFLA" represents geranylflavonoid a.
The beneficial effects that this application can produce include:
1) the geranylflavone A separation and purification process provided by the application adopts the preparation methods of alcohol extraction, macroporous resin purification and solvent crystallization, can realize production amplification, and has a good industrial preparation prospect.
2) The separation and purification process of geranyl flavone A provided by the application has high transfer rate, and the purity of geranyl flavone A can reach more than 95%.
3) The separation and purification process of geranylflavonoid A provided by the application isolates air by a liquid sealing membrane, and can keep the natural color of a finished product by rapid vacuum drying.
4) The process for separating and purifying geranylflavone A provided by the application uses macroporous adsorption resin, can be used for large-scale treatment, and can be used repeatedly after appropriate regeneration treatment, so that the cost consumption is greatly reduced.
Drawings
FIG. 1 is a liquid chromatogram of a flower and leaf extract stock solution of example 1;
FIG. 2 is a liquid chromatogram of geranylflavonoid A end product of example 1;
FIG. 3 shows the geranylflavonoid A end product of example 11H NMR (600MHz, DMSO-d6) chart;
FIG. 4 shows an embodiment1 geranylflavone A finished product13C NMR (600MHz, DMSO-d6) chart.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.
The flower and leaf raw material of the application is mature hemp flower and leaf of 7-9 months, and is industrial hemp with Tetrahydrocannabinol (THC) content below 0.3% after primary drying by farmers.
The product analysis method in the examples of the present application is as follows:
by means of hydrogen spectra1H NMR and carbon Spectroscopy13C NMR analysis was carried out using a Bruker AM-600 nuclear magnetic resonance spectrometer (Bruker, Germany) and deuterated dimethyl sulfoxide (DMSO-d6, sigma) as solvent.
Analyzing the raw solution of the flower and leaf extraction and the finished product of the geranylflavonoid A by using high performance liquid chromatography, wherein an analytical instrument is an Agilent 1260 liquid chromatograph (Agilent, USA), and octadecylsilane chemically bonded silica is used as a filler; taking acetonitrile as a mobile phase A, taking 0.5% formic acid-water as a mobile phase B, and carrying out isocratic elution according to the ratio of A (%): B (%): 70: 30; the detection wavelength was 210 nm.
In fig. 1, CBG represents cannabigerol, CBD represents cannabidiol, THCV represents tetrahydrocannabivarin, CBN represents cannabinol, and THC represents tetrahydrocannabinol.
The content of geranylflavone a in the floral leaf material used in the present application was determined by the following method:
and (3) standard product configuration: accurately weighing CFLA working reference substances (standard substances are prepared and standardized by laboratories), and dissolving with methanol to prepare a standard substance test solution with the concentration of 0.1000 mg/ml.
Preparation of a flower and leaf test sample: weighing 5.0000g of flower leaves, stirring and degreasing with n-hexane of which the mass volume is 5 times that of the flower leaves (W/V) at room temperature for 20min, volatilizing the n-hexane, adding 75% methanol-water solution of which the mass volume is 15 times that of the flower leaves (W/V), carrying out ultrasonic extraction for 15min, and taking a 0.45-micron needle type filter to continue filtrate to enter a liquid phase for detection. Octadecylsilane chemically bonded silica is used as a filling agent; taking acetonitrile as a mobile phase A, taking 0.5% formic acid-water as a mobile phase B, and carrying out isocratic elution according to the ratio of A (%): B (%): 70: 30; the detection wavelength was 210 nm.
The CFLA content of the raw material (CFLA sample liquid phase peak area, sample concentration, sample extracting solution volume), CFLA sample peak area, water retaining dry material mass of the flower leaf raw material) is 100 percent
The content of geranylflavone A in the raw material of the flower and leaf used in the application is measured to be 2.3 parts by weight.
The purity of the geranylflavone A finished product in the embodiment of the application is calculated through a liquid chromatogram, and the purity is consistent with the liquid phase peak percentage area.
The transfer rate of geranylflavonoid a (cfla) finished product was calculated as follows:
the transfer rate of CFLA is (CFLA finished product quality: purity)/(flower leaf raw material water retaining dry material quality: 0.00023): 100%
EXAMPLE 1 Geranylflavone A was prepared as follows
1) The hemp flowers and leaves are crushed to 10-20 meshes, uniformly mixed and sampled, the moisture content is measured to be 12.56%, and 300kg is weighed and put into an extraction tank.
2) Cold soaking the flower and leaf raw materials in a 95 wt% ethanol aqueous solution with the volume (W/V) 5 times of the mass of the flower and leaf raw materials for 2 hours, stirring at 100rpm, extracting for 2 times, combining, concentrating until the ethanol concentration is 50-70 wt%, and settling overnight or centrifuging to obtain a supernatant.
3) Washing the clear solution with macroporous adsorption resin HPD700 (158 kg), 30 wt% ethanol for 5BV (BV is column bed volume, namely actual column volume of the resin layer), mixing, and concentrating under reduced pressure (0.06-0.08 MP) at 60 ℃ by a scraper concentrator to obtain 4.45kg of a target component geranylflavone A crude product; eluting with 70 wt% ethanol, and concentrating under reduced pressure to obtain extract of cannabinoid component mainly containing CBD.
4) And transferring the geranylflavone A crude product of the target component to a reaction kettle, stirring and dissolving 4.5L of n-hexane with the same amount (M/V) at 40 ℃, passing through a three-effect filter, transferring the insoluble substance to the reaction kettle again, repeatedly transferring and dissolving for 3 times under the same condition, primarily drying by the three-effect filter, removing the surface organic solvent, and finally obtaining 52.28g of brown-yellow residual insoluble substance.
5) Dissolving the insoluble substances in 100mL of 2 times (M/V) acetone, incubating at-20 deg.C for crystallization for 4h, filtering with Buchner funnel, washing with 20mL of cold acetone at-10 deg.C for three times, and draining to obtain brown amorphous geranylgeranyl flavone A.
6) And (3) quickly transferring the geranyl flavone A primary product to a vacuum drying oven, and drying for 36 hours under reduced pressure at 30 ℃ and 0.10MPa to obtain 38.73g of a brown yellow geranyl flavone A finished product.
The resulting final product of the tan geranylflavonoid A was recorded as sample 1, with a transfer rate of 61.91%. FIG. 1 is a liquid chromatogram of a raw liquid of a flower and leaf extract; FIG. 2 is a liquid chromatogram of sample 1; the chromatographic purity was 96.45%.
FIG. 3 is a drawing of sample 11H NMR (600MHz, DMSO-d6) chart,1the H NMR values were assigned as follows:1H NMR(600MHz,DMSO-d6)δ13.21(s,1H),10.76(s,1H),9.93(s,1H),7.52(s,2H),6.93(d,J＝8.2Hz,1H),6.86(d,J＝2.4Hz,1H),6.54(s,1H),5.17(t,J＝6.8Hz,1H),5.01(s,1H),3.89(s,3H),3.22(d,J＝6.7Hz,2H),1.98(d,J＝5.9Hz,2H),1.91(s,2H),1.72(s,3H),1.56(s,3H),1.50(s,3H)。
FIG. 4 shows sample 113C NMR (600MHz, DMSO-d6) chart,13the C NMR values were as follows:13C NMR(600MHz,DMSO-d6)δ181.92,163.49,161.90,158.49,155.18,150.74,148.12,134.24,130.72,124.19,122.14,121.75,120.36,115.87,111.05,110.21,103.63,103.25,93.34,56.04,39.36,26.28,25.53,21.07,17.59,16.01。
the nuclear magnetic spectrum of sample 1 was compared well with the literature "Young Hae Choi, Arno Hazekamp, NMR Assignments of the Major and Cannabiflavonoids Isolated from Flowers of Cannabis sativa, phytochem, anal.15, 345-354 (2004)". It was confirmed that the obtained sample 1 was geranylflavone A.
Example 2 Geranylflavone A was prepared as follows
1) Crushing the hemp leaves to 20-40 meshes, uniformly mixing and sampling, measuring the moisture content to be 15.26%, weighing 600kg, putting into an extraction tank, adding 120L petroleum ether, stirring for 4h, and drying by a slag drying machine under reduced pressure to obtain the degreased hemp leaf raw material.
2) Extracting the above flower and leaf raw materials with 20 times volume (W/V) of 65 wt% ethanol under reflux for 2h, stirring at 100rpm for 1 time, and settling or centrifuging to obtain supernatant.
3) Passing the clear solution through XAD2 macroporous adsorbent resin (255kg) chromatographic column, washing with 60 wt% ethanol for 3.5BV, mixing, and concentrating under reduced pressure at 60 deg.C with scraper concentrator to obtain 7.19kg crude geranylflavone A; eluting with 90 wt% ethanol, and concentrating under reduced pressure to obtain extract of cannabinoid component mainly containing CBD.
4) Transferring the geranylflavone A crude product of the target component to a reaction kettle, adding 1/2 times (M/V) of heptane: 3.6L of ethyl acetate (100: 6, V/V) mixed solvent is stirred and dissolved at the temperature of 60 ℃, insoluble substances are leached on the surface of the mixed solvent by a small amount of the binary mixed solvent through a three-effect filter, the mixed solvent is primarily dried by the three-effect filter, the organic solvent on the surface is removed, and finally 125.42g of brown-yellow residual insoluble substances are obtained.
5) Dissolving the insoluble substances in 65mL of 1/2 times (M/V)60 wt% ethanol, incubating at 10 ℃ for 24h for crystallization, quickly filtering through a Buchner funnel, and spraying a small amount of purified water on the surface to form a liquid seal film, thus obtaining the primary product of the early geranylflavonoid A with the amorphous yellow color of the goose.
6) And (3) quickly transferring the geranyl flavone A primary product to a vacuum drying oven, and drying for 16 hours at 60 ℃ under 0.10MPa under reduced pressure to obtain 88.76g of the finished product of the gooseyellow geranyl flavone A.
The resulting final product of the tan geranylflavonoid A was recorded as sample 2 and the transfer rate was 74.57%. The liquid chromatogram of sample 2 showed a purity of 98.25%. Of sample 21H NMR (600MHz, DMSO-d6) chart and13the C NMR (600MHz, DMSO-d6) pattern is consistent with that of sample 1.
Example 3 Geranylflavone A was prepared as follows
1) The hemp flowers and leaves are crushed to 40-60 meshes, uniformly mixed and sampled, the moisture content is measured to be 10.14%, and 1200kg of hemp flowers and leaves are weighed and put into an extraction tank.
2) Extracting the above flower and leaf raw materials with 75 wt% ethanol with 8 times volume (W/V) under reflux for 1h, stirring at 100rpm for 2 times, mixing, concentrating to ethanol content of 50 wt%, and settling overnight or centrifuging to obtain supernatant.
3) Passing the clear solution through ADS600 macroporous adsorbent resin (216kg) chromatographic column, washing with 50 wt% ethanol for 4BV, mixing, and concentrating under reduced pressure at 60 deg.C with scraper concentrator to obtain crude geranylflavone A11.63 kg; eluting with 90% ethanol, and concentrating under reduced pressure to obtain extract containing CBD as main cannabinoid component.
4) Transferring the geranylflavone A crude product of the target component to a reaction kettle, stirring to form a fluid paste at 60 ℃ by 1/3 times (M/V) of 4L of n-hexane, adding 1/10 times (M/M) of diatomite: 1.2kg of silica gel (3: 1, M/M) are mixed well, passed through a triple-effect filter pre-coated with a layer of diatomaceous earth, and subsequently purified with an equivalent amount (M/V) of heptane: the solid mixture was preliminarily dried by a triple effect filter by washing 11.5L with a mixed solvent of ethyl acetate (100: 6, V/V).
5) And (3) rinsing the solid mixture by using 1.2L of equivalent (M/M) ethyl acetate, incubating and crystallizing at-10 ℃ for 6h, filtering by using a Buchner funnel, washing by using a small amount of cold ethyl acetate at-10 ℃ for 100ml for three times, and leaving a thin layer of ethyl acetate on the surface to form a liquid-sealed film so as to obtain the early product of the gooseyellow amorphous geranylflavonoid A.
6) And (3) quickly transferring the geranyl flavone A primary product to a vacuum drying oven, and drying for 24 hours under reduced pressure at 40 ℃ and 0.10MPa to obtain 152.50g of the finished product of the gooseyellow geranyl flavone A.
The resulting final product of the tan geranylflavonoid A was recorded as sample 3, with a transfer rate of 60.92%. The liquid chromatogram of sample 3 showed a purity of 99.08%. The 1H NMR (600MHz, DMSO-d6) and 13C NMR (600MHz, DMSO) profiles of sample 3 were consistent with those of sample 1.
The above examples are summarized in table 1 below.
TABLE 1
Example 4 macroporous adsorbent resin recycle experiment
The procedure of example 1 was repeated by flushing the macroporous adsorbent resin HPD700 of example 1 through 95 wt% ethanol at 3BV (BV means bed volume, i.e. the actual packed volume of the resin layer), and the experimental results were consistent with the use of fresh macroporous adsorbent resin HPD 700. The performance is not reduced after the compound is repeatedly used for 50 times.
In conclusion, the process for separating and purifying geranylflavone A provided by the application can treat the dosage of more than 300kg by using macroporous adsorption resin, the product transfer rate is kept above 60%, the purity of geranylflavone A can reach more than 95%, and the industrial preparation can be realized. The macroporous adsorption resin can be regenerated by flushing the column with 2-5BV 95 wt% ethanol, and the regeneration can be repeated more than 50 times by repeated experimental investigation in a laboratory, so that adsorption and desorption are not affected.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (10)
1. A method for preparing geranylflavone A, characterized by comprising at least the following steps:
a) pretreatment of hemp flower and leaf raw materials;
b) extracting the pretreated hemp flower and leaf raw material by using a solvent I, concentrating an extracting solution, and separating to obtain a supernatant;
c) adsorbing the supernatant through macroporous adsorption resin, washing out the supernatant through a solvent II, and concentrating to obtain a geranylflavone A crude product;
d) the geranylflavone A crude product is subjected to trans-dissolution by a solvent III and is separated to obtain insoluble substances;
e) recrystallizing the insoluble substance to obtain geranylflavone A primary product;
in the step c), the solvent II is 30-60 wt% of ethanol;
in the step d), the solvent III is at least one selected from petroleum ether, diethyl ether, n-hexane, n-heptane, dichloromethane, chloroform, ethyl acetate, butyl acetate, n-octanol, sec-octanol and heptanol;
in step e), the solvent for recrystallization is at least one selected from dichloromethane, chloroform, ethyl acetate, butyl acetate, methanol and water, ethanol and water, acetone and butanone;
the macroporous adsorption resin is at least one of AB-8, D-101, HPD700, XAD2, XDA-8, LSA-7, D-941, DM-130, ADS600, ADS-17, SP-825, HPD-600 and SP825 macroporous adsorption resin.
2. The method according to claim 1, wherein in step a), the pre-processing comprises: pulverizing hemp flower and leaf to 10-60 mesh.
3. The method of claim 2, wherein the pre-treating further comprises degreasing;
the degreasing comprises the following steps: pulverizing Cannabis sativa flower and leaf to obtain Cannabis sativa flower and leaf raw material of 10-60 meshes, and extracting with petroleum ether, n-hexane or n-heptane.
4. The method according to claim 1, wherein the solvent I in step b) is at least one selected from methanol, ethanol and acetone.
5. The method as claimed in claim 1, wherein in step b), the mixture is extracted by cold soaking or hot refluxing with 65-95 wt% ethanol 5-20 times the volume of the hemp leaves for 1-3h for 2-4 times, and then concentrated to ethanol concentration of 50-70 wt%, and the supernatant is separated.
6. The method as claimed in claim 1, wherein the amount of macroporous adsorbent resin used is 10-75% of the feed mass of the cannabis sativa leaf raw material.
7. The method as claimed in claim 1, wherein the amount of macroporous adsorbent resin used is 15-60% of the feed mass of the cannabis sativa leaf raw material.
8. The method according to claim 1, wherein in the step d), the number of times of the re-dissolution is 1-3, and no other ointment-like impurities are taken as a re-dissolution end point.
9. The method as claimed in claim 1, wherein in step e), the surface of the recrystallized geranylflavone A is sprayed with a solvent IV to form a liquid-tight film, so as to obtain a geranylflavone A primary product;
the solvent IV is at least one selected from ethyl acetate, water and n-heptane.
10. The method according to claim 1 or 9, further comprising drying the primary geranylflavonoid a obtained in step e) under reduced pressure to obtain a final geranylflavonoid a product.
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