CN111747958A - Method for separating multiple active ingredients from African Voacanga chalotiana - Google Patents
Method for separating multiple active ingredients from African Voacanga chalotiana Download PDFInfo
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Abstract
The invention provides a method for separating various active ingredients from African strobilus cuscutae, which comprises the following steps: (1) extracting and separating the Voacanga seed volatile oil: crushing Voacanga chalotiana Linn raw material, adding lipophilic organic solvent, heating and reflux-extracting, passing the extractive solution through silica gel chromatographic column, and concentrating the eluate to obtain Voacanga chalotiana Linn volatile oil; (2) extraction and separation of water-soluble polysaccharide: adding water into the organic solvent extraction residues to obtain a water extraction solution, sequentially passing through an anion exchange resin column and a cation exchange resin column, concentrating and drying to obtain the water-soluble polysaccharide of the Voacanga seed; (3) extraction and separation of tabersonine: adding acid water containing betaine into the water extraction residue to obtain acid water extractive solution; the tabersonine finished product is obtained through the subsequent steps of centrifugation, ceramic membrane filtration, alkali neutralization, chromatographic alumina resin column adsorption, acetic acid aqueous solution elution, extraction and the like. Can comprehensively utilize the strobilus cuscutae resources, has coherent and simple process, low production cost, high content of various active ingredients and high yield.
Description
Technical Field
The invention belongs to the field of natural extracts, and particularly relates to a method for separating active ingredients of African strobilus cusia, in particular to a method for separating various active ingredients from African strobilus cusia.
Background
African muguet (Voacanga Africana), also known as African Voacanga, is a perennial arbor of the genus Marguet of the family Apocynaceae, and is mainly distributed between African Sagnac and Sudan, Angora, and West African, Congo, and tanzania. African lilac fruit has a long medicinal history in traditional medicine in African regions, and the whole strain can be used as a medicine and is often used for treating diseases such as leprosy, dysentery, malaria, systemic edema, epilepsy and infantile convulsion. Pharmacological activity research shows that the African voaca has the activities of resisting tumor, bacteria, inflammation and malaria, inhibiting angiogenesis, improving memory, calming, hypnotizing, giving up drugs, removing addiction and the like, and has extremely high medicinal value. At present, African Voacanga chalotiana has been introduced and domesticated by domestic scientific research units.
African strobilus custard is rich in indole alkaloids with cardiotonic, antibacterial, antiulcer, and antioxidant effects, such as: tabersonine, laojiu amine, laojiu alkali, laojiu Ling, voacangine, etc. In particular, tabersonine (tabersonine), also known as tabersonine, has the highest attention because it is a precursor of vincristine, a cancer chemotherapy drug, and has been industrially produced at present in China.
Besides tabersonine active substances with high economic value, the Voacanga chalotiana Franch also contains abundant volatile oil, and the Voacanga chalotiana Franch volatile oil has effects of manufacturing and repairing cell membrane, enhancing cell resistance, and preventing skin inflammation, and can be applied to cosmetics and health products. The literature reports that the Voacanga seed volatile oil mainly comprises fatty acids, esters, hydrocarbons and a small amount of phenols.
CN201010609832.5 discloses a method for extracting tabersonine from Voacanga chalotiana, which comprises the steps of crushing Voacanga chalotiana as a raw material, extracting with acid water, adjusting alkali, filtering, extracting, crystallizing and the like to obtain a tabersonine product. The yield of the alkali adjusting and precipitating process is difficult to control, and limited tabersonine is suspended or dissolved in a large amount of alkali liquor after alkali adjustment and is difficult to completely separate out and precipitate, so the yield of tabersonine prepared by the method is low.
CN201210165108.7 discloses a new process for extracting tabersonine from Voacanga chalotiana Pierre ex Stapf based on enzymatic hydrolysis, which takes dry Voacanga chalotiana Pierre ex Stapf seeds as raw materials, and obtains tabersonine products through the steps of crushing, petroleum ether degreasing, enzymolysis, lower alcohol extraction, reduced pressure distillation concentration, drying crystallization and the like. In the enzymolysis process of the method, tabersonine is unstable in property and has the risk of being degraded; if the degreasing is insufficient, a large amount of fat-soluble impurities are leached in the subsequent alcohol extraction process, so that the yield and the content of tabersonine crystals are influenced.
CN201810284666.2 discloses a method for extracting tabersonine from African voacanga fruit, which takes seeds of African voacanga fruit as raw materials, and obtains tabersonine products through the steps of crushing, sulfuric acid solution soaking and percolating, filtering, macroporous resin adsorption and desorption, concentration and centrifugation and the like. The content of tabersonine product obtained by the method is low.
CN201410053307.8 discloses a novel extraction and separation technology of tabersonine hydrochloride in African plant Voacanga chalotiana, which comprises the steps of taking African Voacanga chalotiana as a raw material, extracting by alkaline methanol or ethanol, dissolving by acid water, degreasing, alkalifying, extracting by an organic solvent, concentrating, back extracting by acid water, concentrating, crystallizing and the like to obtain tabersonine hydrochloride (i.e. tabersonine hydrochloride). The method has complicated steps, and the dosage of acid, alkali and organic solvent is large, so the method is not suitable for industrial production.
Therefore, a new method which can comprehensively utilize African Voacanga chalotiana Linn resources, is suitable for industrial production and can continuously extract various Voacanga chalotiana Linn active ingredients is needed at present.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art, provide a brand-new method for continuously extracting various active ingredients of African strobilus custard, comprehensively utilize strobilus custard resources, have coherent and simple process and strong operability, can recycle used solvents and various resins, has low production cost and high content and yield of various active ingredients, and is suitable for industrial production.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for separating multiple active ingredients from African muguet comprises the following steps:
(1) extracting and separating the Voacanga seed volatile oil: crushing the acorn raw material, adding a lipophilic organic solvent, heating, refluxing, extracting and filtering to obtain an organic solvent extracting solution, wherein organic solvent extracting residues are used for standby; passing the organic solvent extractive solution through silica gel chromatographic column, and concentrating the eluate to obtain Voacanga seed volatile oil;
(2) extraction and separation of water-soluble polysaccharide: evaporating residual organic solvent in the organic solvent extraction residue in the step (1), adding water, stirring, filtering to obtain water extraction solution, and extracting residue with water for later use; sequentially passing the water extract through an anion exchange resin column and a cation exchange resin column, collecting the effluent of the cation exchange resin column, concentrating and drying to obtain the water-soluble polysaccharide of the Voacanga seed;
(3) extraction and separation of tabersonine: adding acid water containing betaine into the water extraction residue in the step (2), stirring and extracting at room temperature, and filtering to obtain an acid water extracting solution; centrifuging the acid water extract, filtering with ceramic membrane, neutralizing with alkali, adsorbing with chromatographic alumina resin column, eluting with acetic acid water solution, extracting the eluate with alkyl halide, drying, filtering, concentrating under reduced pressure, cooling, crystallizing, vacuum filtering, and drying to obtain tabersonine product.
Preferably, in the step (1), the lipophilic organic solvent is one or more of diethyl ether, petroleum ether, n-hexane, cyclohexane, 6# solvent oil and 120# solvent oil, the dosage of the lipophilic organic solvent is 8-10 times (L/kg) of the weight of the African strobilus Linteus raw material in sequence, and the reflux extraction time is 2-3 hours. The purpose of extracting by using a lipophilic organic solvent is to fully leach the volatile oil in the Voacanga seed raw material by utilizing the principle that the Voacanga seed volatile oil is easily dissolved in the lipophilic organic solvent; secondly, after the volatile oil and other fat-soluble components in the Voacanga chalotiana Linn raw material are fully leached, water or acid in the subsequent steps can be favorably permeated into the cell tissues of the raw material, and the leaching of polysaccharide and tabersonine can be favorably realized.
Preferably, in the step (1), the dosage of the silica gel for column chromatography is 0.05-0.1 time (L/kg) of the weight of the African voaca fruit raw material, the height-diameter ratio of the silica gel chromatographic column is 5-8: 1, and the flow rate of the silica gel chromatographic column is 0.5-1.0 BV/h. The organic solvent extract is passed through silica gel chromatographic column to remove impurities such as liposoluble pigment, etc., and improve the purity of Voacanga Argus volatile oil.
Preferably, in the step (2), the solvent is distilled off by using water vapor to distill off the residual organic solvent. Steam distillation is the most easily realized industrialized means, and the distilled gas can be completely recovered through a condenser, the steam is condensed and converted into water, and the organic solvent is volatilized at high temperature and then condensed and converted into organic solvent liquid. The water and the organic solvent liquid are layered, and the organic solvent can be recycled and reused through liquid separation.
Preferably, in the step (2), the water is selected from distilled water, mineral water, deionized water and pure water. The amount of the water is 8-10 times (L/kg) of the weight of the African voaca fruit raw material, and the stirring and extraction time at room temperature is 6-8 hours. Adding water, stirring and extracting to extract water-soluble components in African muguet fruit raw material, such as polysaccharide, water-soluble flavone, water-soluble pigment, inorganic salt, etc.
Preferably, in the step (2), the type of the anion exchange resin is macroporous strongly basic anion exchange resin with the models of D941, D945, LX-T5, LSD-762, LSA-700 and LX-22, the dosage of the anion exchange resin is 0.1-0.2 times (L/kg) of the weight of the African fructus Voacanthi Tricuspidatae raw material, the height-diameter ratio of an anion exchange resin column is 3-8: 1, and the flow rate of the material passing through the anion exchange resin column is 1-2 BV/hour. The purpose of using anion exchange resin column is to adsorb water-soluble flavone, water-soluble pigment and other impurities, so as to raise the purity of Voacanga seed polysaccharide in water extract. If the amount of the anion exchange resin used is too small, the aspect ratio of the anion exchange resin column is too small, or the flow rate of the material passing through the anion exchange resin column is too high, the above-mentioned objects cannot be sufficiently achieved. If the dosage of the anion exchange resin is too much, the height-diameter ratio of the anion exchange resin column is too large or the flow rate of the material passing through the anion exchange resin column is too slow, the waste of energy or material is caused.
Preferably, in the step (2), the cation exchange resin is a strong-acid styrene cation exchange resin with the type of 001 × 7, 001 × 8, 001 × 12 and 001 × 16, the dosage of the cation exchange resin is 0.1-0.2 times (L/kg) of the weight of the African strobilus Linnaeus raw material, the height-diameter ratio of the cation exchange resin column is 3-8: 1, and the flow rate of the material passing through the cation exchange resin column is 1-2 BV/h. The purpose of using a cation exchange resin column is desalting. If the amount of the cation exchange resin used is too small, the aspect ratio of the cation exchange resin column is too small, or the flow rate of the material passing through the cation exchange resin column is too high, the above-mentioned object cannot be sufficiently achieved. If the dosage of the cation exchange resin is too much, the height-diameter ratio of the cation exchange resin column is too large or the flow rate of the material passing through the cation exchange resin column is too slow, the waste of energy or material is caused.
Preferably, in the step (3), the acid water is an aqueous solution of sulfuric acid and hydrochloric acid, the mass percentage concentration of the acid in the acid water is 1 to 20 per mill, the amount of the acid water is 10 to 20 times (L/kg) of the weight of the African voacanga fruit raw material, and the stirring and extraction time at room temperature is 10 to 12 hours. Adding acid water, stirring and extracting at room temperature to leach the tabersonine.
Preferably, in the step (3), the dosage of the betaine is 3-5 per mill of the weight of the African voacanthus fruit raw material. The purpose of adding betaine into acid water is to generate hydrochloride and/or sulfate of the betaine, and firstly, the betaine hydrochloride or sulfate is a good surfactant, so that the surface tension of acid water can be reduced, and the leaching rate of the acid water on tabersonine is increased; secondly, the molecular structure of tabersonine (salt) in the acid water extracting solution can be protected from being oxidized and decomposed.
In the step (3), the centrifugation is not particularly limited, and for example, a horizontal decanter centrifuge is used for centrifugation.
Preferably, in the step (3), the pore diameter of the ceramic membrane is 20-50 nm. The purpose of filtration using a ceramic membrane is to remove fine raw material particles and macromolecular substances such as proteins leached with acid water from the filtrate.
Preferably, in the step (3), the pH value after the neutralization with the base is 7-8, the base is not particularly limited, and the base may be an aqueous solution of inorganic base commonly used in the art, such as at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and saturated aqueous solution of potassium bicarbonate.
Preferably, in the step (3), the mesh number of the chromatographic alumina is 80-300 meshes, and the dosage of the chromatographic alumina is 10-20% of the weight of the African voacanthus fruit raw material. The purpose of using chromatographic alumina is to absorb and enrich tabersonine.
Preferably, in the step (3), the aspect ratio of the chromatographic alumina resin column is 3-8: 1, the flow rate of the material on the column is 0.5-1.0 BV/h.
Preferably, in the step (3), the mass percentage concentration of the acetic acid aqueous solution is 0.5-5%, the usage amount of the acetic acid aqueous solution is 1-2 BV, and the flow rate of elution is 0.5-2.0 BV/h. The purpose of eluting the chromatographic alumina resin column with an aqueous acetic acid solution is to desorb the tabersonine adsorbed on the chromatographic alumina resin.
Preferably, in the step (3), the alkyl halide is chloroform and/or dichloromethane, and the amount of the alkyl halide is 0.5-1 times (L/L) of that of the acetic acid eluent. The purpose of extracting the eluent by the alkyl halide is to transfer the tabersonine in the acetic acid eluent from a water phase to an organic phase, which is beneficial to low-temperature concentration and crystallization.
Preferably, in the step (3), the drying agent is one or more of anhydrous sodium sulfate, anhydrous magnesium sulfate, anhydrous calcium sulfate or anhydrous copper sulfate, and the amount of the drying agent is 5-10% (Kg/L) of the volume of chloroform or dichloromethane. The drying agent is added into the chloroform or dichloromethane layer, and the stirring is carried out at room temperature, so that a small amount of water remained in the chloroform or dichloromethane layer is removed, and the existence of the water can negatively influence the crystallization of the tabersonine.
Preferably, in the step (3), the concentration of the solid in the concentrated solution is 20-25%, the temperature for cooling is 5-10 ℃, the stirring speed is 30-90 r/min, and the time for crystallization is 12-24 hours.
In the present invention, 1BV is 1 packed column volume.
The principle of the method of the invention is as follows:
after crushing the African voaca pomacea raw material, firstly using a lipophilic organic solvent to fully leach fat-soluble voaca pomacea volatile oil, and decoloring the lipophilic organic solvent extract by a silica gel chromatographic column to obtain the high-purity voaca pomacea volatile oil; in the degreased raw material slag, the components of the Voacanga seed polysaccharide, the water-soluble flavone, the water-soluble pigment, the inorganic salt and the like can be leached out by cold water, and the water-soluble flavone, the water-soluble pigment, the inorganic salt and the like can be removed by treating with anion and cation exchange resin, so that the Voacanga seed water-soluble polysaccharide with higher purity can be obtained; the tabersonine is insoluble in lipophilic organic solvent and cold water, exists in cold water extraction residue, is subjected to acid water room temperature extraction, centrifugation, ceramic membrane filtration and neutralization, is adsorbed and desorbed by alumina resin, is extracted by chloroform or dichloromethane, is concentrated and crystallized, and a tabersonine finished product with high content can be obtained.
The method has the following beneficial effects:
(1) the method provided by the invention provides a brand-new method for continuously extracting various active ingredients of African strobilus Linteus, can comprehensively utilize strobilus Linteus resources, has the advantages of coherent and simple process, strong operability, recyclable solvent and various resins, low production cost, high content of various active ingredients and high yield, and is suitable for industrial production.
(2) In the production process, the extraction conditions of tabersonine are mild, and the molecular structure of tabersonine (or salt) is not oxidized and decomposed due to the addition of the protective agent betaine, so that the yield of tabersonine is guaranteed.
Detailed Description
The present invention will be further described with reference to the following examples.
The voacanga seed raw material used in the embodiment of the invention is purchased from African gardner, wherein the content of water-soluble polysaccharide is 2.89%, and the content of tabersonine is 2.35%; the anion and cation exchange resins used in the embodiment of the invention are all purchased from Xian lan Xiao science and technology New materials GmbH; the ceramic membrane used in the embodiment of the invention is purchased from Nanjing Fulinde environmental protection science and technology Limited; the adjuvants or chemicals used in the examples of the present invention are commercially available in the usual manner unless otherwise specified.
In the embodiment of the invention, the content of the water-soluble polysaccharide of the Voacanga chalotiana Linn is detected by adopting a phenol-sulfuric acid colorimetric method, and the content of the tabersonine is detected by adopting a High Performance Liquid Chromatography (HPLC) external standard method.
Example 1
(1) Extracting and separating the Voacanga seed volatile oil: 200kg of African voaca seed raw material is taken, crushed to the particle size of 1-2 mm, put into an extraction tank, added with 2000L of No. 6 solvent oil, heated and refluxed for extraction for 2 hours, filtered to obtain No. 6 solvent oil extraction liquid, and No. 6 solvent oil extraction residues for later use. And (3) enabling the 6# solvent naphtha extract to pass through a silica gel chromatographic column at the flow rate of 0.5 BV/h, enabling the using amount of column chromatography silica gel to be 10L, enabling the height-diameter ratio of the silica gel chromatographic column to be 8:1, collecting effluent of the silica gel chromatographic column, and concentrating until no solvent exists to obtain 10.15kg of brown yellow oily matter, namely the Voacanga chalotiana volatile oil.
(2) Extraction and separation of water-soluble polysaccharide: and (2) distilling the residual organic solvent in the 6# solvent oil extraction residue in the step (1) by using water vapor, adding 2000L of pure water, stirring and extracting at room temperature for 6 hours, filtering to obtain a water extraction solution, and extracting the residue with water for later use. Sequentially passing the water extract through an anion exchange resin column and a cation exchange resin column (wherein the type of the anion exchange resin is D941, the using amount of the anion exchange resin is 20L, the height-diameter ratio of the anion exchange resin column is 8:1, the type of the cation exchange resin is 001 x 7, the using amount of the anion exchange resin is 20L, and the height-diameter ratio of the anion exchange resin column is 8:1) at the flow rate of 1 BV/h, collecting effluent of the ion exchange resin column, concentrating under reduced pressure, and drying by microwave to obtain 6.17kg of the Voacanga chalotiana water-soluble polysaccharide.
(3) Extraction and separation of tabersonine: adding 3500L hydrochloric acid aqueous solution (containing 0.6kg of betaine) with the mass percent concentration of 8 per mill into the water extraction residue in the step (2), stirring and extracting at room temperature for 12 hours, and filtering to obtain acid aqueous extract. Centrifuging the acid water extractive solution with horizontal screw centrifuge, and filtering with ceramic membrane with aperture of 20nm to obtain ceramic membrane filtrate. Neutralizing the ceramic membrane filtrate with a sodium hydroxide saturated aqueous solution to a pH value of 7, and passing through a chromatographic alumina resin column (wherein the type of the chromatographic alumina is neutral alumina, the mesh number of the chromatographic alumina is 100-200 meshes, the using amount of the chromatographic alumina is 40L, and the height-diameter ratio of the chromatographic alumina resin column is 4: 1) at a flow rate of 0.5 BV/h. After the column loading is finished, eluting the chromatographic alumina resin column by using 60L of acetic acid aqueous solution with the mass percentage concentration of 3 percent, wherein the elution flow rate is 0.5 BV/h, collecting acetic acid eluent, and extracting the eluent by using 60L of dichloromethane. Separating, collecting dichloromethane layer, adding 3kg anhydrous sodium sulfate, stirring at room temperature for 2 hr, filtering, concentrating under reduced pressure until the concentration of solid in the concentrated solution is 22%, cooling to 5 deg.C, stirring at 60r/min for crystallization for 16 hr, vacuum filtering, and drying to obtain 4.53kg tabersonine product.
The content of the water-soluble polysaccharide of the African muguet fruit obtained in the embodiment is 85.19% and the yield of the water-soluble polysaccharide of the African muguet fruit is 91.09% by the phenol-sulfuric acid colorimetric method; the content of tabersonine obtained in this example was 98.73% and the yield of tabersonine was 94.32% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
Example 2
(1) Extracting and separating the Voacanga seed volatile oil: taking 200kg of African voaca seed raw material, crushing the raw material until the particle size is 1-2 mm, putting the crushed raw material into an extraction tank, adding 1600L of petroleum ether, heating and refluxing for extraction for 3 hours, filtering to obtain petroleum ether extracting solution, and using the petroleum ether extracting slag for later use. Passing the petroleum ether extractive solution through silica gel column chromatography at flow rate of 0.8 BV/hr (the amount of silica gel for column chromatography is 18L, and the height/diameter ratio of silica gel column chromatography is 6:1), collecting the effluent of silica gel column chromatography, and concentrating until no solvent is present to obtain brown yellow oily substance 9.80kg, i.e. Voacanga chalotiana Linn volatile oil.
(2) Extraction and separation of water-soluble polysaccharide: and (2) distilling the residual organic solvent in the petroleum ether extraction residue obtained in the step (1) by using water vapor, adding 2000L of pure water, stirring and extracting at room temperature for 7 hours, filtering to obtain a water extraction solution, and extracting the residue with water for later use. And (2) sequentially passing the water extract through an anion exchange resin column and a cation exchange resin column (wherein the model of the anion exchange resin is D945, the dosage of the anion exchange resin is 40L, the height-diameter ratio of the anion exchange resin column is 5:1, the model of the cation exchange resin is 001 multiplied by 8, the dosage of the anion exchange resin is 40L, and the height-diameter ratio of the anion exchange resin column is 5:1) at the flow rate of 1.5 BV/h, collecting effluent of the ion exchange resin column, concentrating under reduced pressure, and drying by microwave to obtain 6.18kg of the Voacanga chalotiana water-soluble polysaccharide.
(3) Extraction and separation of tabersonine: and (3) adding 4000L of sulfuric acid aqueous solution (containing 0.8kg of betaine) with the mass percentage concentration of 6 per mill into the water extraction residue in the step (2), stirring and extracting for 10 hours at room temperature, and filtering to obtain acid water extracting solution. Centrifuging the acid water extractive solution with horizontal screw centrifuge, and filtering with ceramic membrane with pore diameter of 50nm to obtain ceramic membrane filtrate. Neutralizing the pH value of the ceramic membrane filtrate with a potassium hydroxide saturated aqueous solution to be 8, and passing through a chromatographic alumina resin column (wherein the type of the chromatographic alumina is alkaline alumina, the mesh number of the chromatographic alumina is 100-200 meshes, the using amount of the chromatographic alumina is 25L, and the height-diameter ratio of the chromatographic alumina resin column is 8:1) at the flow rate of 1.0 BV/h. After the column loading, the chromatographic alumina resin column was eluted with 50L of 2% by mass aqueous acetic acid solution at a flow rate of 1 BV/hr, and the acetic acid eluate was collected and extracted with 50L of chloroform. Separating, collecting chloroform layer, adding 2.5kg anhydrous magnesium sulfate, stirring at room temperature for 2 hr, filtering, concentrating under reduced pressure until the concentration of solid in the concentrated solution is 25%, cooling to 8 deg.C, stirring at 40r/min for crystallization for 20 hr, vacuum filtering, and drying to obtain 4.49kg tabersonine product.
The content of the water-soluble polysaccharide of the African muguet fruit obtained in the embodiment is 86.33% and the yield of the water-soluble polysaccharide of the African muguet fruit is 92.15% through the determination of a phenol-sulfuric acid colorimetric method; the content of tabersonine obtained in this example was 99.14% and the yield of tabersonine was 95.55% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
Example 3
(1) Extracting and separating the Voacanga seed volatile oil: taking 200kg of African voaca seed raw material, crushing the raw material until the particle size is 1-2 mm, putting the crushed raw material into an extraction tank, adding 1800L of cyclohexane, heating, refluxing and extracting for 3 hours, filtering to obtain cyclohexane extract, and obtaining cyclohexane extraction residues for later use. Passing the cyclohexane extractive solution through silica gel column chromatography at flow rate of 0.5 BV/hr (the amount of silica gel for column chromatography is 12L, and the height/diameter ratio of silica gel column chromatography is 7:1), collecting the effluent of silica gel column chromatography, and concentrating until no solvent is present to obtain 10.33kg of brown yellow oily substance, i.e. Voacanga chalotiana Linn volatile oil.
(2) Extraction and separation of water-soluble polysaccharide: and (2) distilling the residual organic solvent in the cyclohexane extraction residue obtained in the step (1) by using water vapor, adding 1800L of pure water, stirring and extracting at room temperature for 8 hours, and filtering to obtain a water extraction solution, wherein the water extraction residue is used for later use. Sequentially passing the water extract through anion-cation exchange resin columns (wherein the type of anion exchange resin is LX-T5, the dosage of the anion exchange resin is 25L, the height-diameter ratio of the anion exchange resin column is 7:1, the type of the cation exchange resin is 001 × 16, the dosage of the anion exchange resin is 25L, and the height-diameter ratio of the anion exchange resin column is 7:1) at the flow rate of 1 BV/h, collecting effluent of the ion exchange resin columns, concentrating under reduced pressure, and drying by microwave to obtain 6.13kg of the Voacanga chalotiana water-soluble polysaccharide.
(3) Extraction and separation of tabersonine: adding 3000L of 10 per mill hydrochloric acid aqueous solution (containing 1kg of betaine) into the water extraction residue in the step (2), stirring and extracting at room temperature for 11 hours, and filtering to obtain an acid aqueous extract. Centrifuging the acid water extractive solution with horizontal screw centrifuge, and filtering with ceramic membrane with aperture of 20nm to obtain ceramic membrane filtrate. Neutralizing the ceramic membrane filtrate with a sodium hydroxide saturated aqueous solution to a pH value of 7, and passing through a chromatographic alumina resin column (wherein the type of the chromatographic alumina is neutral alumina, the mesh number of the chromatographic alumina is 200-300 meshes, the using amount of the chromatographic alumina is 30L, and the height-diameter ratio of the chromatographic alumina resin column is 6:1) at a flow rate of 0.6 BV/h. After the column loading, the chromatographic alumina resin column was eluted with 60L of 2.5% by mass aqueous acetic acid at a flow rate of 0.8 BV/hr, and the acetic acid eluate was collected and extracted with 60L of dichloromethane. Separating, collecting a dichloromethane layer, adding 3.6kg of anhydrous calcium sulfate, stirring at room temperature for 2 hours, filtering, concentrating under reduced pressure until the concentration of solid matters in the concentrated solution is 20%, cooling to 10 ℃, stirring at the rotating speed of 30r/min for crystallization for 24 hours, performing suction filtration, and drying to obtain 4.45kg of tabersonine finished product.
The content of the water-soluble polysaccharide of the African muguet fruit obtained in the embodiment is 85.16% and the yield of the water-soluble polysaccharide of the African muguet fruit is 90.32% by the phenol-sulfuric acid colorimetric method; the content of tabersonine obtained in this example was 99.16% and the yield of tabersonine was 93.89% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
Example 4
The other steps and conditions are the same as those in the example 2, except that in the step (3) of the extraction and separation of tabersonine, 3500L of hydrochloric acid aqueous solution with the mass percent concentration of 8 per mill is added into water extraction residues, the addition amount of betaine is 0.4kg, the content of tabersonine obtained in the example is 98.72 percent and the yield of tabersonine is 91.48 percent through the determination of a High Performance Liquid Chromatography (HPLC) external standard method.
Example 5
The other steps and conditions are the same as those in the example 2, except that in the step (3) of the extraction and separation of the tabersonine, 3500L of hydrochloric acid aqueous solution with the mass percent concentration of 8 per mill is added into water extraction residues, the addition amount of the betaine is 1.2kg, the content of the tabersonine obtained in the example is 99.17 percent, and the yield of the tabersonine is 92.85 percent through the determination of a High Performance Liquid Chromatography (HPLC) external standard method.
Example 6
The other steps and conditions are the same as those in example 2, except that in the step (3) of extraction and separation of tabersonine, the ceramic membrane filtrate is neutralized by a sodium carbonate saturated aqueous solution to have a pH value of 6.5, and the content of tabersonine obtained in the example is 99.03% and the yield of tabersonine is 92.72% as determined by a High Performance Liquid Chromatography (HPLC) external standard method.
Example 7
The other steps and conditions are the same as those in example 2, except that in the step (3) of extraction and separation of tabersonine, the ceramic membrane filtrate is neutralized to pH value of 8.5 by using potassium hydroxide saturated aqueous solution, and the content of tabersonine obtained in the example is 99.12% and the yield of tabersonine is 93.60% by using a High Performance Liquid Chromatography (HPLC) external standard method.
Comparative example 1
The other steps and conditions are the same as those in example 1, except that in the step (3) of extraction and separation of tabersonine, 3500L of hydrochloric acid aqueous solution with the mass percent concentration of 8 per mill is added into the water extraction residues, and betaine is not contained. The content of tabersonine obtained in this example was 97.48% and the yield of tabersonine was 87.47% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
Comparative example 2
The other steps and conditions were the same as in example 2 except that in the step (2) of extracting and separating the water-soluble polysaccharide, the aqueous extract was passed through cation exchange resin and then anion exchange resin column, i.e., the order of anion and cation exchange resin was changed. The content of the water-soluble polysaccharide of the African muguet fruit obtained in the embodiment is 81.64% and the yield of the water-soluble polysaccharide of the African muguet fruit is 85.72% as determined by a phenol-sulfuric acid colorimetric method; the content of tabersonine obtained in this example was 99.13% and the yield of tabersonine was 93.82% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A method for separating multiple active ingredients from African muguet comprises the following steps:
(1) extracting and separating the Voacanga seed volatile oil: crushing the acorn raw material, adding a lipophilic organic solvent, heating, refluxing, extracting and filtering to obtain an organic solvent extracting solution, wherein organic solvent extracting residues are used for standby; passing the organic solvent extractive solution through silica gel chromatographic column, and concentrating the eluate to obtain Voacanga seed volatile oil;
(2) extraction and separation of water-soluble polysaccharide: evaporating residual organic solvent in the organic solvent extraction residue in the step (1), adding water, stirring, filtering to obtain water extraction solution, and extracting residue with water for later use; sequentially passing the water extract through an anion exchange resin column and a cation exchange resin column, collecting the effluent of the cation exchange resin column, concentrating and drying to obtain the water-soluble polysaccharide of the Voacanga seed;
(3) extraction and separation of tabersonine: adding acid water containing betaine into the water extraction residue in the step (2), stirring and extracting at room temperature, and filtering to obtain an acid water extracting solution; centrifuging the acid water extract, filtering with ceramic membrane, neutralizing with alkali, adsorbing with chromatographic alumina resin column, eluting with acetic acid water solution, extracting the eluate with alkyl halide, drying, filtering, concentrating under reduced pressure, cooling, crystallizing, vacuum filtering, and drying to obtain tabersonine product.
2. The method according to claim 1, wherein in the step (1), the lipophilic organic solvent is one or more selected from diethyl ether, petroleum ether, n-hexane, cyclohexane, 6# solvent oil and 120# solvent oil, the amount of the lipophilic organic solvent is 8-10 times (L/kg) of the weight of the African strobilus Linteus raw material, and the reflux extraction time is 2-3 hours.
3. The method according to claim 1, wherein in the step (1), the amount of the column chromatography silica gel is 0.05-0.1 times (L/kg) of the weight of the African fringed fruit raw material, the height-diameter ratio of the silica gel chromatography column is 5-8: 1, and the flow rate of the silica gel chromatography column is 0.5-1.0 BV/h.
4. The method according to claim 1, wherein in the step (2), the water is selected from distilled water, mineral water, deionized water, pure water; the amount of the water is 8-10 times (L/kg) of the weight of the African voaca fruit raw material, and the stirring and extraction time at room temperature is 6-8 hours.
5. The method according to claim 1, wherein in the step (2), the anion exchange resin is macroporous strongly basic anion exchange resin with the types of D941, D945, LX-T5, LSD-762, LSA-700 and LX-22, the dosage of the anion exchange resin is 0.1-0.2 times of the weight of the African voacanthus fruit raw material (L/kg), the height-diameter ratio of an anion exchange resin column is 3-8: 1, and the flow rate of the material passing through the anion exchange resin column is 1-2 BV/h;
the cation exchange resin is strong-acid styrene cation exchange resin with the types of 001 multiplied by 7, 001 multiplied by 8, 001 multiplied by 12 and 001 multiplied by 16, the dosage of the cation exchange resin is 0.1-0.2 times (L/kg) of the weight of the African strobilus cusia raw material, the height-diameter ratio of a cation exchange resin column is 3-8: 1, and the flow rate of the material passing through the cation exchange resin column is 1-2 BV/h.
6. The method according to claim 1, wherein in the step (3), the acid water is an aqueous solution of sulfuric acid and/or hydrochloric acid, the mass percentage concentration of the acid in the acid water is 1 to 20 per mill, the amount of the acid water is 10 to 20 times (L/kg) of the weight of the African horse berry raw material, and the stirring extraction time at room temperature is 10 to 12 hours.
7. The method of claim 1, wherein in step (3), the betaine is present in an amount of 3% to 5% by weight of the African horse berry source.
8. The method of claim 1, wherein in step (3), the pH value after neutralization with the base is 7 to 8.
9. The method according to claim 1, wherein in the step (3), the mesh number of the chromatographic alumina is 80-300 meshes, and the dosage of the chromatographic alumina is 10-20% of the weight of the African strobilus Linteus raw material; the height-diameter ratio of the chromatographic alumina resin column is 3-8: 1, the flow rate of the material on the column is 0.5-1.0 BV/h; and/or
The mass percentage concentration of the acetic acid aqueous solution is 0.5% -5%, the dosage of the acetic acid aqueous solution is 1-2 BV, and the flow rate of elution is 0.5-2.0 BV/h.
10. The method according to claim 1, wherein in the step (3), the alkyl halide is chloroform and/or dichloromethane, and the amount of the alkyl halide is 0.5-1 times (L/L) that of the acetic acid eluent; and/or
The concentration of solid matters in the concentrated solution is 20-25%, the temperature for cooling is 5-10 ℃, the stirring speed is 30-90 r/min, and the crystallization time is 12-24 hours.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477035A (en) * | 2010-11-23 | 2012-05-30 | 成都合盛生物技术有限公司 | Cleaning process for extracting and purifying tabersonine from voacango Africana stapf seeds |
| CN104829618A (en) * | 2014-02-12 | 2015-08-12 | 李玉山 | New extraction and separation technology of tabersonine hydrochloride from African plant voacanga chalotiana |
| CN106243106A (en) * | 2016-09-23 | 2016-12-21 | 成都合盛生物技术有限公司 | A kind of method extracting tabersonine from the volt health seeds of trees |
| CN106397438A (en) * | 2016-09-23 | 2017-02-15 | 成都合盛生物技术有限公司 | Method for extracting tabersonine from voacanga seeds |
| CN108484603A (en) * | 2018-04-02 | 2018-09-04 | 太阳树（厦门）生物工程有限公司 | The method that tabersonine is extracted from African Voacanga |
-
2020
- 2020-07-29 CN CN202010745864.1A patent/CN111747958B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477035A (en) * | 2010-11-23 | 2012-05-30 | 成都合盛生物技术有限公司 | Cleaning process for extracting and purifying tabersonine from voacango Africana stapf seeds |
| CN104829618A (en) * | 2014-02-12 | 2015-08-12 | 李玉山 | New extraction and separation technology of tabersonine hydrochloride from African plant voacanga chalotiana |
| CN106243106A (en) * | 2016-09-23 | 2016-12-21 | 成都合盛生物技术有限公司 | A kind of method extracting tabersonine from the volt health seeds of trees |
| CN106397438A (en) * | 2016-09-23 | 2017-02-15 | 成都合盛生物技术有限公司 | Method for extracting tabersonine from voacanga seeds |
| CN108484603A (en) * | 2018-04-02 | 2018-09-04 | 太阳树（厦门）生物工程有限公司 | The method that tabersonine is extracted from African Voacanga |
Non-Patent Citations (1)
| Title |
|---|
| 王荣: "非洲马铃果中柳叶水甘草碱的定性定量分析及提取分离工艺研究", 《西北大学硕士学位论文》 * |
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