Method for extracting apigenin and apigenin from selaginella tamariscina

Abstract

The invention relates to a method for extracting amentoflavone and apigenin from selaginella tamariscina, which comprises the following specific steps: alcohol extraction, concentration, ethyl acetate extraction, alkali liquor extraction, column loading, crystallization of amentoflavone and apigenin and the like to prepare high-purity amentoflavone and apigenin products. According to the method disclosed by the invention, two flavonoid components of amentotaxus biflavone and apigenin are efficiently separated through alcohol extraction and combined extraction of ethyl acetate and alkali liquor, and are respectively crystallized to obtain high-content amentotaxus biflavone and apigenin products, so that the oxidation speed of the amentotaxus biflavone can be effectively avoided, the extraction rate of the amentotaxus biflavone is greatly increased, and the production cost of the amentotaxus biflavone is reduced.

Description

Technical Field

[0001] The invention specifically relates to a method for extracting aconitum biflavonoids and apigenin from Selaginella, and belongs to the technical field of natural product utilization. Background Art

[0002] Selaginella is a native or rock-dwelling fern of the genus Selaginella in the family Selaginaceae. Selaginella is native to China and is now distributed in provinces and regions such as Hebei, Henan, Hubei, Guangxi and the southwest of China. It likes warm, humid and semi-shady environments, and is mostly grown on sunny hillside rocks or in dry rock cracks, and reproduces by spores. According to the "Compendium of Materia Medica", the whole herb of Selaginella is used as medicine, which is rich in flavonoids and has the effects of promoting blood circulation and menstruation, removing blood stasis and stopping bleeding. It can be used to treat amenorrhea, dysmenorrhea, lumps and masses, hematemesis, metrorrhagia, bloody stools, and prolapse of the anus. It is rich in linoleic acid, β-sitosterol, 7-carbonyl β-sitosterol, sinomenine, arctigenin, emodin, chrysophanol, flavin methyl ether, apigenin, dihydroapigenin, polydatin, osthole, chlorogenic acid, podophyllotoxin, vanillic acid, scutellaria scabrachoa biflavonoids, baicalin, aloe-emodin, and arbutin.

[0003] The patent with the publication number CN1562994 discloses an extraction process of the biflavonoid extract of Selaginella tataricola. The extraction process is as follows: crush and dry the whole herb of Selaginella, reflux and extract the extract twice, filter the extract, and combine the extract; vacuum concentrate the extract, recover the extraction solvent, filter the concentrate after cooling and settling, and remove impurities; pour the sedimentation filtrate onto a macroporous resin column, control the flow rate during adsorption, then wash with pure water, and then analyze with an analytical solvent, vacuum concentrate the analytical solution, adjust the pH value to acidic, filter after cooling and settling, obtain a filter cake, and freeze-dry to obtain the product.

[0004] The patent with publication number CN101461829 discloses a Selaginella extract and its extraction method and application. The extract is extracted by the following method: the whole plant of Selaginella is repeatedly cold-soaked with a solvent for 2-4 times, each time for 6-8 days, filtered, the filtrate is combined, and extracted with an extractant for 2-4 times to obtain an effective part; the effective part is chromatographed on a silica gel atmospheric pressure column (200-300 mesh), and after elution, the part containing sedum sedum is combined by TLC detection, and eluted with acetone on a Sephadex LH-20 column chromatography to obtain a monomer compound sedum sedum.

[0005] The patent with publication number CN101606955 discloses a method for extracting scutellaria aviculare. The whole plant of Selaginella is extracted with a mixture of acetone and water for 2-4 times at room temperature, each time for 6-9 days, and then filtered. The filtrate is combined and the solvent is recovered under reduced pressure to obtain a concentrated extract. The extract is dispersed in water, extracted with ethyl acetate for 2-4 times to obtain the effective lipid-lowering part of Selaginella, namely the ethyl acetate part, and subjected to silica gel atmospheric pressure column chromatography, and gradient eluted with petroleum ether-acetone at a volume ratio of 10:1-7:3. The parts containing scutellaria aviculare are combined by TLC detection, and eluted with acetone on Sephadex LH-20 column chromatography to obtain scutellaria aviculare.

[0006] The patent with the publication number CN101606954 discloses a method for extracting and purifying flavonoids from Selaginella. After the dried Selaginella is crushed and sieved, it is extracted with ethanol aqueous solution by reflux, and the extract is filtered while hot, and the ethanol is recovered by vacuum distillation, and vacuum dried to constant weight to obtain the Selaginella extract; the extract obtained above is transferred to a polyamide resin column, and the impurities are first eluted with ethanol aqueous solution with a volume concentration of 10-60%, and then eluted with ethanol aqueous solution with a volume concentration of 70-90%, and the eluent flow rate is 0.5-2mL / min, and the eluent is collected, and the ethanol is recovered by vacuum distillation, and vacuum dried to constant weight to obtain the Selaginella flavonoids.

[0007] The patent with publication number CN102178703A discloses a method for extracting Selaginella biflavonoids, which belongs to the technical field of chemical extraction process of medicinal plants. The method uses Selaginella as raw material, extracts with petroleum ether and acetone in sequence, adds inorganic salt solution and polyethylene glycol to the acetone extract, stirs to make it fully mixed and extracted, and forms two phases after standing. Selaginella biflavonoids enters the polyethylene glycol upper phase, and the upper phase liquid uses ultrafiltration membrane method to separate Selaginella biflavonoids and polyethylene glycol, and the Selaginella biflavonoids are concentrated by nanofiltration membrane, and then concentrated and dried to obtain Selaginella biflavonoids product.

[0008] The patent with publication number CN104248648A discloses a method for extracting total flavonoids from Selaginella, which is to use surfactant-assisted microwave extraction of total flavonoids from Selaginella. The whole plant of Selaginella is dried and crushed, immersed in a dilute ethanol aqueous solution containing a surfactant of appropriate concentration, and extracted by microwave irradiation for multiple times. After extraction, it is quickly adsorbed on a macroporous resin column, eluted with ethanol and dried to obtain the total flavonoids of Selaginella.

[0009] The patent with publication number CN105954377A discloses a method for extracting spicate biflavonoids from Selaginella, comprising the following steps: step 1: crush the Selaginella and sieve it; step 2: weigh 0.2 parts by weight of Selaginella powder and mix it evenly with 1 part by weight of diatomaceous earth for standby use; step 3: put the mixture obtained in step 2 into an extraction pool with a filter membrane placed in advance, and add an appropriate amount of diatomaceous earth until it reaches the pool mouth of the extraction pool; cover the extraction pool with a lid for static extraction; obtain an extract after the extraction is completed; the extraction conditions are: the extraction solvent is methanol; the extraction temperature is 110°C; the static extraction time is 5 minutes; the flushing volume is 80%; the number of static cycles is 3 times.

[0010] The patent with publication number CN107382939A discloses a method for extracting flavonoid compounds from Selaginella glabra with supercritical carbon dioxide. The Selaginella glabra is dried, crushed and then soaked, and 60-95% ethanol is used as an entrainer. The extraction is carried out for 2-7 hours at a pressure of 20-35Mpa and a temperature of 40-60°C; a first-level decompression separation pressure of 5-10Mpa and a temperature of 40-60°C is carried out in a separation kettle; a second-level decompression separation pressure of 4-7Mpa and a temperature of 35-50°C is carried out, and the product is obtained from the separation kettle of the supercritical carbon dioxide extraction device.

[0011] The patent with publication number CN109320486A discloses a method for infrared extraction of selaginella biflavonoids from Selaginella. The method of the present invention uses an infrared lamp to irradiate a solution of a Selaginella sample, and after a certain period of extraction, the extract is processed and separated and analyzed using a high performance liquid chromatograph. Qualitative and quantitative analysis is performed based on the chromatographic information of the sample and the reference substance.

[0012] The patent with publication number CN109400566A discloses a method for extracting and separating high-purity sedum biflavonoids from Selaginella plants. The invention adopts a non-polar macroporous adsorption resin as a separation material and an aqueous ethanol-pH gradient elution process to separate crude sedum biflavonoids from the alcohol extract of the Selaginella plants. The crude sedum biflavonoids are further separated by alkaline methanol dissolution, acid precipitation and ethanol recrystallization to obtain high-purity sedum biflavonoids with a content of more than 98% and a process transfer rate of more than 50%.

[0013] A search of journal literature revealed that many studies on the chemical composition of Selaginella mainly focused on extraction and purification, with the aim of obtaining high levels of selaginella flavonoids. Some literature also reported on the content of other components in Selaginella, but there is currently no report on a method for simultaneously extracting selaginella flavonoids and apigenin. Summary of the invention

[0014] Aiming at the phenomenon that other flavonoid components cannot be fully utilized when extracting tadalafil biflavonoids, the present invention provides a method for extracting and purifying tadalafil biflavonoids and apigenin. The method simultaneously extracts various flavonoid components, separates and purifies tadalafil biflavonoids and apigenin by chromatography and crystallization technology, fully utilizes the extracted flavonoid components, and effectively reduces its production cost.

[0015] The present invention aims to provide a method for extracting aconitum biflavonoids and apigenin from Selaginella, and the specific steps are as follows:

[0016] (1) Alcohol extraction: crush the Selaginella, add an ethanol solution and perform high temperature reflux extraction for 60 to 150 minutes, filter to obtain a filtrate, wherein the ratio of the amount of the ethanol solution added L to the weight of the Selaginella is 5 to 8:1;

[0017] (2) Concentration: The filtrate is concentrated under reduced pressure at 60-70° C. to 1 / 20-1 / 40 of the original volume to obtain a concentrated solution;

[0018] (3) Ethyl acetate extraction: ethyl acetate is added to the concentrated solution and extracted at room temperature to obtain an extract phase, wherein the volume ratio of the added ethyl acetate L to the concentrated solution L is 3 to 5:1;

[0019] (4) Alkaline solution extraction: The extract phase is concentrated under reduced pressure at 60-70°C to 1 / 10-1 / 30 of its original volume to obtain a concentrated solution, which is then cooled to room temperature, and then an equal volume of potassium hydroxide solution is added and stirred for 30-50 minutes. The solution is allowed to stand and separate into ethyl acetate phase and aqueous phase;

[0020] (5) Loading on the column: adsorbing the macroporous resin on the ethyl acetate phase, and eluting with 3 to 5 times the column volume of ethanol solution to obtain eluent I;

[0021] (6) Crystallization of sedum biflavonoids: The eluate I was concentrated under reduced pressure at 60-70°C to 1 / 10-1 / 30 of its original volume to obtain a concentrated solution, which was placed in a refrigerator at 4°C overnight and filtered to obtain pure sedum biflavonoids;

[0022] (7) Crystallization of apigenin: The aqueous phase was adjusted to a pH of 6-6.5 with 0.1 mol / L hydrochloric acid, and concentrated under reduced pressure at 60-70°C to 1 / 4-1 / 8 of the original volume to obtain a concentrated solution, which was placed in a refrigerator at 4°C overnight and filtered to obtain pure apigenin.

[0023] The above filtration is performed once using a ceramic membrane, and the high temperature reflux extraction is performed at 70-85°C.

[0024] In a specific embodiment, the ethanol solution in steps (1) and (5) is a 70-80% ethanol aqueous solution.

[0025] In a specific embodiment, the potassium hydroxide solution in step (4) is a potassium hydroxide aqueous solution with a pH of 8-9.

[0026] Technical Effects

[0027] 1. The present invention uses alcohol extraction, ethyl acetate and alkali solution combined extraction to efficiently separate two flavonoid components, sedum biflavone and apigenin, and crystallizes them separately to obtain high-content sedum biflavone and apigenin products.

[0028] 2. The oxidation rate of the flavonoids of Metasequoia gmelinii can be effectively avoided by treating them with ethyl acetate and alkali solution at room temperature, which greatly improves their extraction rate and reduces their production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Chromatogram of the reference substance of aconitum biflavonoids;

[0030] Figure 2 : Chromatogram of pure product of aconitum biflavonoids;

[0031] Figure 3 :Chromatogram of apigenin reference substance;

[0032] Figure 4 : Chromatogram of pure apigenin. DETAILED DESCRIPTION

[0033] Hereinafter, the present invention will be further described using embodiments, but it is not limited to any one of these embodiments or similar examples.

[0034] Example 1

[0035] Take 10kg of crushed Selaginella, add 70L of 75% ethanol solution to carry out high-temperature reflux extraction for 120min, and filter to obtain 60L of filtrate; concentrate the filtrate under reduced pressure at 70°C to obtain 2L of concentrated solution; add 8L of ethyl acetate to the concentrated solution and extract at room temperature to obtain an extract phase; concentrate the extract phase under reduced pressure at 60°C to obtain 0.4L of concentrated solution, cool to room temperature, add 0.4L of potassium hydroxide solution and stir for 30min, stand and separate to obtain ethyl acetate phase and aqueous phase; adsorb the ethyl acetate phase on macroporous resin, and elute with 5 times column volume of 80% ethanol solution to obtain 2.5L of eluent I; concentrate the eluent I under reduced pressure at 70°C to obtain 0.15L of concentrated solution, place it in a refrigerator overnight, and filter to obtain 58.5g of pure apigenin; adjust the pH of the aqueous phase to 6 with 0.1mol / L hydrochloric acid, concentrate it under reduced pressure at 70°C to obtain 0.1L of concentrated solution, place it in a refrigerator overnight, and filter to obtain 142.8g of pure apigenin.

[0036] HPLC detection equipment: Waters1525, chromatographic column is Boston-C 18(4.6*250mm) column. The HPLC detection conditions of flavonoids of tachyphylla: the ratio of acetonitrile to 0.1% phosphoric acid aqueous solution is 38:62 (V / V), the detection wavelength is 330nm, the column temperature is 35℃, the flow rate is 1ml / min, and the injection volume is 10μl; the HPLC detection conditions of apigenin: the ratio of methanol to water is 70:30 (V / V), the column temperature is 35℃, the detection wavelength is 270nm, the flow rate is 1ml / min, and the injection volume is 10μl. The HPLC detection results show that the content of apigenin in the sample is 98.56%, and the content of flavonoids of tachyphylla is 97.28%.

[0037] Comparative Example 1

[0038] Take 10kg of crushed Selaginella, add 70L of ethanol solution and perform high-temperature reflux extraction for 120min, filter to obtain 60L of filtrate; concentrate the filtrate under reduced pressure at 70°C to obtain 2L of concentrated solution; add 8L of ethyl acetate to the concentrated solution, extract at room temperature to obtain an extract phase; concentrate the extract phase under reduced pressure at 60°C to obtain 0.4L of concentrated solution; adsorb the ethyl acetate phase on a macroporous resin, and elute with 5 times the column volume of 40% ethanol solution to obtain 2.5L of eluent I; elute with 5 times the column volume of 70% ethanol solution to obtain 2.5L of eluent II; concentrate the eluent I under reduced pressure at 70°C to obtain 0.15L of concentrated solution, place it in a refrigerator overnight, filter to obtain 95.4g of pure apigenin, and concentrate the eluent II under reduced pressure at 70°C to obtain 0.15L of concentrated solution, place it in a refrigerator overnight, filter to obtain 41.1g of pure apigenin. The HPLC test showed that the content of apigenin in the sample was 92.15%, and the content of aconitum biflavonoids was 93.47%.

[0039] Example 2

[0040] Take 100kg of crushed Selaginella, add 600L of ethanol solution to carry out high-temperature reflux extraction for 150min, and filter to obtain 500L of filtrate; concentrate the filtrate under reduced pressure at 70°C to obtain 20L of concentrated solution; add 70L of ethyl acetate to the concentrated solution and extract at room temperature to obtain an extract phase; concentrate the extract phase under reduced pressure at 60°C to obtain 3L of concentrated solution, cool to room temperature, add 3L of potassium hydroxide solution and stir for 50min, and stand to separate to obtain ethyl acetate phase and aqueous phase; adsorb the ethyl acetate phase on macroporous resin, and elute with 5 times column volume of ethanol solution to obtain 18L of eluent I; concentrate the eluent I under reduced pressure at 70°C to obtain 1L of concentrated solution, place it in a refrigerator overnight, and filter to obtain 627.8g of pure apigenin; adjust the pH of the aqueous phase to 6 with 0.1mol / L hydrochloric acid, concentrate it under reduced pressure at 70°C to obtain 0.8L of concentrated solution, place it in a refrigerator overnight, and filter to obtain 1685.3g of pure apigenin. The HPLC test showed that the content of apigenin in the sample was 98.64%, and the content of aconitum biflavonoids was 97.41%.

Claims

1. A method for extracting aconitum biflavonoids and apigenin from Selaginella, the specific steps of which are as follows: (1) Alcohol extraction: crush the Selaginella, add an ethanol solution and perform high temperature reflux extraction for 60 to 150 minutes, filter to obtain a filtrate, wherein the ratio of the amount of the ethanol solution added L to the weight of the Selaginella is 5 to 8:1; (2) Concentration: The filtrate is concentrated under reduced pressure at 60-70° C. to 1 / 20-1 / 40 of its original volume to obtain a concentrated solution; (3) Ethyl acetate extraction: ethyl acetate is added to the concentrated solution and extracted at room temperature to obtain an extract phase, wherein the volume ratio of the added ethyl acetate L to the concentrated solution L is 3 to 5:1; (4) Alkaline solution extraction: The extract phase is concentrated under reduced pressure at 60-70°C to 1 / 10-1 / 30 of its original volume to obtain a concentrated solution, which is then cooled to room temperature, and then an equal volume of potassium hydroxide solution is added and stirred for 30-50 minutes. The solution is allowed to stand and separate into ethyl acetate phase and aqueous phase; (5) Loading on the column: adsorbing the macroporous resin on the ethyl acetate phase, and eluting with 3 to 5 times the column volume of ethanol solution to obtain eluent I; (6) Crystallization of sedum biflavonoids: The eluate I was concentrated under reduced pressure at 60-70°C to 1 / 10-1 / 30 of its original volume to obtain a concentrated solution, which was placed in a refrigerator at 4°C overnight and filtered to obtain pure sedum biflavonoids; (7) Crystallization of apigenin: The aqueous phase was adjusted to a pH of 6-6.5 with 0.1 mol / L hydrochloric acid, and concentrated under reduced pressure at 60-70°C to 1 / 4-1 / 8 of the original volume to obtain a concentrated solution, which was placed in a refrigerator at 4°C overnight and filtered to obtain pure apigenin.

2. According to the method of claim 1, the ethanol solution in steps (1) and (5) is a 70-80% ethanol aqueous solution.

3. According to the method of claim 1, the potassium hydroxide solution in step (4) is a potassium hydroxide aqueous solution with a pH of 8 to 9.

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