CN111978417A - Extraction method of hemp polysaccharide, product and application thereof - Google Patents
Extraction method of hemp polysaccharide, product and application thereof Download PDFInfo
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Abstract
The invention relates to a method for extracting hemp polysaccharide, a product and application thereof. The extraction method comprises the steps of soaking a hemp raw material, extracting, carrying out solid-liquid separation, concentrating filtrate, and precipitating concentrated solution in alcohol to obtain the hemp polysaccharide. The extraction, separation and purification method provided by the invention is simple and easy to operate, and the prepared hemp polysaccharide has high purity, has obvious effects of removing DPPH free radicals, resisting oxidation, whitening, resisting aging, improving immunity, regulating blood fat, resisting tumors and the like, and can be widely applied to cosmetics, health products, foods or medicines. Meanwhile, the raw materials of the extraction method comprise residues after the cannabinoids active ingredients such as CBD and the like are extracted from the hemp flowers and leaves, so that the extraction method also provides a new strategy for recycling the residues after the cannabinoids active ingredients such as CBD and the like are extracted from the hemp flowers and leaves, changes waste into valuable and realizes high-value recycling of resources.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for extracting hemp polysaccharide, a product and application thereof.
Background
Industrial hemp (Cannabis sativa L.) is a plant of Cannabis of Moraceae, has Tetrahydrocannabinol (THC) content of less than three thousandths in the flower and leaf in the growth period, has no value of extracting tetrahydrocannabinol or can be directly sucked as drug, can be legally planted in large scale and industrially exploited, and has extremely high economic and medicinal values. China has records of planting marijuana as early as 3500 years ago. Hemp is widely used, hemp skin can be peeled to be made into fiber woven cloth, paper and rope, seeds can be eaten and used for pressing oil, flowers and leaves can be used as medicine, and stalks can be used for manufacturing novel composite materials such as density boards, so the hemp and hemp products thereof are once and once applied to life of people and industrial and agricultural production. Cannabis is a very promising medicinal plant, and its effectiveness in treating various diseases has been reported in many studies. At present, research on active substances of cannabis is mainly focused on cannabinoids, which are important active substances contained in cannabis plants, and mainly include Tetrahydrocannabinol (THC), Tetrahydrocannabinoids (THCV), Cannabidiol (CBD), Cannabigerol (CBG), sub-Cannabidiol (CBDV), and the like, and the five substances account for more than 90% of the phenolic compounds of cannabis. Cannabidiol (CBD), one of the most important non-addictive components in plants, has pharmacological activities such as anti-spasmodic, anti-rheumatic arthritis, and anti-anxiety, and can hinder the adverse effects of Tetrahydrocannabinol (THC) on the human nervous system, and has become a hot spot in drug development.
Plant polysaccharides, also known as plant polysaccharides, are polysaccharides with a degree of polymerization of more than 10 produced by plant cell metabolism. Generally, plant polysaccharides consist of more than 100 monosaccharide groups and even thousands of monosaccharide groups, and the properties of the plant polysaccharides are greatly different from those of monosaccharides, such as sweetness and strong reducibility disappear. Plant polysaccharides are divided into two categories according to their function in the plant body: one is the supporting tissue that forms the plant, such as cellulose; one is the plant's stored food, which is soluble in hot water to form a colloidal solution, and can be hydrolyzed by enzymes to release monosaccharides to supply energy, such as starch, inulin, etc. Classifying according to the existing parts: extracellular polysaccharides, cell wall polysaccharides and intracellular polysaccharides. Common plant polysaccharides in life include starch, cellulose, polysaccharides, pectin, and the like.
The industrial hemp leaves contain a large amount of plant polysaccharide, are rarely developed and utilized at present, and after the hemp leaves are used for extracting the cannabinoids active ingredients such as CBD (CBD), residues are usually discarded and are not recycled, so that the environmental pollution and the resource waste are caused. Therefore, it is very significant to develop a method for recycling hemp flower and leaf residues with high value.
Disclosure of Invention
The hemp is industrial hemp which can be legally planted in a large scale and industrially developed and utilized.
Aiming at the defects of the prior art, the invention aims to provide a hemp polysaccharide extraction method, a product and an application thereof, the extraction method is simple and easy to operate, the extracted polysaccharide has high purity, has good effects of scavenging free radicals, resisting oxidation, whitening, resisting aging, improving immunity, regulating blood fat, resisting tumors and the like, and can be widely applied to cosmetics, foods, health products or medicines; meanwhile, the extraction method also provides a new strategy for recycling residues after the cannabinoids active ingredients such as CBD and the like are extracted from the hemp flowers and leaves. In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a method for extracting cannabis polysaccharide, which comprises the steps of soaking a cannabis raw material, extracting, carrying out solid-liquid separation, concentrating filtrate, and precipitating the concentrated solution in alcohol to obtain the cannabis polysaccharide.
The extraction method provided by the invention is simple and easy to operate, the extracted hemp polysaccharide has high purity, and has the remarkable effects of eliminating DPPH free radicals, resisting oxidation, whitening, resisting aging, improving immunity, regulating blood fat, resisting tumors and the like, and can be widely applied to cosmetics, foods or medicines; meanwhile, the raw materials of the extraction method comprise residues after the cannabinoids active ingredients such as CBD and the like are extracted from the hemp flowers and leaves, so that the extraction method also provides a new strategy for recycling the residues after the cannabinoids active ingredients such as CBD and the like are extracted from the hemp flowers and leaves, changes waste into valuable and realizes high-value recycling of resources.
Preferably, the cannabis raw material comprises cannabis flowers and/or cannabis leaves. Specifically, the extract comprises fresh flos Cannabis, dried flos Cannabis, fresh folium Cannabis, dried folium Cannabis, residue obtained by extracting cannabinoids such as CBD from flos Cannabis, and residue obtained by extracting cannabinoids such as CBD from folium Cannabis.
Preferably, the extraction method comprises the following steps:
(1) soaking hemp in water;
(2) extracting the raw materials soaked in the step (1);
(3) after the extraction in the step (2) is finished, carrying out solid-liquid separation, and concentrating the filtrate;
(4) and (4) mixing the concentrated solution obtained in the step (3) with alcohol for precipitation to obtain the hemp polysaccharide.
Preferably, the mass ratio of the hemp raw material to the water in the step (1) is 1 (5-30), such as 1:5, 1:6, 1:8, 1:10, 1:12, 1:15, 1:17, 1:18, 1:20, 1:22, 1:25, 1:28 or 1:30, and the like. Preferably 1: 12.
The quality ratio of the hemp raw material to the water has an important influence on the extraction rate of the hemp polysaccharide and the purity of the hemp polysaccharide, the specific selection is in a range of 1 (5-30) because the extraction rate of the hemp polysaccharide and the purity of the hemp polysaccharide are kept at higher levels in the range, the industrial production water and the subsequent cost for solution and sewage treatment are considered, the limit of the volume of an extraction container influences the efficiency of industrial extraction, the ratio of 1 (5-30) is a better ratio, and the comprehensive balance is that the ratio of 1:12 is a ratio with an optimal effect.
Preferably, the extraction manner of step (2) comprises ultrasonic extraction and/or heating extraction in an aqueous solution, preferably ultrasonic extraction. The ultrasonic extraction may be performed in a circulating ultrasonic extractor, and the heated extraction may be performed in a heated extraction tank.
Preferably, the power of the ultrasonic extraction is 500-. The duty ratio of the ultrasonic extraction is 1 (1-5), such as 1:1, 1:2, 1:3, 1:4 or 1: 5. The ultrasonic extraction time is 1-3h, such as 1h, 1.5h, 2h, 2.5h or 3 h.
Preferably, the temperature of the ultrasonic extraction is 10-45 ℃, such as 10 ℃, 12 ℃, 15 ℃, 17 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ or 45 ℃, preferably 30 ℃.
The temperature during extraction has important influence on the extraction rate and purity of the hemp polysaccharide, the extraction rate and purity of the hemp polysaccharide are higher within the range of 10-45 ℃, and the DPPH free radical removing effect of the extracted hemp polysaccharide is stronger, wherein the temperature of 30 ℃ is the temperature with the optimal effect.
Preferably, the ultrasonic extraction is performed in stirring at a rotation speed of 300-1200rpm, such as 300rpm, 400rpm, 500rpm, 600rpm, 700rpm, 800rpm, 900rpm, 1000rpm, 1100rpm, 1200rpm, or the like.
Preferably, the pH of the aqueous solution at the time of extraction in step (2) is 5 to 8, for example, pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, pH 8, or the like.
The pH value during extraction has important influence on the extraction rate and purity of the hemp polysaccharide, the extraction rate and purity of the hemp polysaccharide are higher within the pH range of 5-8, and the DPPH free radical removing effect of the extracted hemp polysaccharide is stronger.
Preferably, an enzyme is added during the extraction in step (2), the enzyme includes any one or a combination of at least two of cellulase, pectinase, beta-glucanase, xylanase, amylase, protease or lipase, the combination of at least two of cellulase and pectinase, xylanase and amylase, beta-glucanase and protease, and the like, and any other possible combination modes are not repeated herein.
The enzyme is added in the extraction process to strengthen the extraction process, promote the decomposition of cellulose, starch, xylan and the like except the target polysaccharide and be beneficial to separating and purifying the target polysaccharide.
Preferably, the concentration of the enzyme in the aqueous solution is 0.1% to 0.5%, such as 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, etc.
And (3) performing solid-liquid separation by using a mode of coarse filtration and fine filtration, wherein the coarse filtration can adopt a drum-type filter device with a filter screen, and the fine filtration can adopt a disc centrifuge or a tubular centrifuge or a plate-and-frame filtration.
Preferably, the solid-liquid separation in step (3) is followed by membrane purification, and the membrane is a microfiltration membrane with molecular weight cut-off of 300-600KDa, such as 300KDa, 350KDa, 400KDa, 450KDa, 500KDa or 600 KDa.
Preferably, the concentration in the step (3) is performed by vacuum decompression and/or ultrafiltration membrane.
Preferably, the ultrafiltration membrane has a molecular weight cut-off of 300-1000Da, such as 300Da, 400Da, 500Da, 600Da, 700Da, 800Da, 900Da or 1000Da, etc.
Preferably, the concentration of step (3) is 1/8-1/4, such as 1/8, 1/7, 1/6, 1/5 or 1/4 of the filtrate volume.
Preferably, the alcohol of step (4) is ethanol.
Preferably, the concentration of ethanol is 75% to 100%, e.g., 75%, 78%, 80%, 85%, 88%, 90%, 95%, 98%, or 100%, etc.
Preferably, the final concentration of ethanol in the mixed system is 75% to 95%, such as 75%, 80%, 82%, 85%, 90%, or 95%, etc., preferably 80%.
The final concentration of the ethanol in the mixed system has important influence on the extraction rate and the purity of the hemp polysaccharide, the extraction rate and the purity of the hemp polysaccharide are higher within the range of 75-95%, the DPPH free radical removing effect of the extracted hemp polysaccharide is stronger, and the effect is optimal when the concentration is 80%.
Preferably, the temperature for precipitation in step (4) is-15 to 10 ℃, for example-15 ℃, -10 ℃, -5 ℃, 0 ℃, 2 ℃, 5 ℃ or 10 ℃, preferably-5 ℃.
The temperature during precipitation is also a key factor influencing the extraction efficiency and purity of the cannabis polysaccharide, and when the temperature is too high, part of polysaccharide with lower molecular weight can not be precipitated, so that the extraction efficiency is reduced; too low a level will precipitate many impurities, which will affect the purity of the cannabis polysaccharide. The optimum range is-15 to 10 ℃, wherein-5 ℃ is the optimum value.
Preferably, the precipitation in step (4) is carried out while stirring, and the stirring time is 2-10h, such as 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h or 10h, etc., preferably 5 h.
Preferably, the precipitate is separated after the cannabis polysaccharide is obtained in step (4), and the precipitate is dissolved in water and dried.
Preferably, the drying manner includes any one or a combination of at least two of ultrasonic drying, spray drying, boiling drying or freeze drying, for example, a combination of ultrasonic drying and spray drying, a combination of spray drying and boiling drying, a combination of boiling drying and freeze drying, and the like, and any other feasible combination manner is not repeated here.
The conditions for the ultrasonic drying are preferably: the ultrasonic power is 0.1-100W/cm2Preferably 10W/cm2(ii) a The conditions of the spray drying are preferably: the air inlet temperature is 120-220 ℃, the preferred temperature is 175 ℃, and the air outlet temperature is 70-110 ℃; the feeding density is 1.01-1.05g/cm3Preferably 1.03g/cm3。
Preferably, the hemp polysaccharide obtained in step (4) is further separated and purified, the separation and purification manner includes any one or a combination of at least two of membrane filtration, DEAE cellulose resin chromatography or macroporous resin chromatography, the combination of at least two of the membrane filtration and DEAE cellulose resin chromatography, the combination of membrane filtration and macroporous resin chromatography, and the like, and any other feasible combination manner is not repeated herein.
The membrane filtration method can adopt a membrane with the molecular weight cutoff of 50000Da for impurity removal.
The DEAE cellulose resin chromatography process comprises the following steps: preparing polysaccharide to 1%, loading on column, eluting with water and salt.
The filler for macroporous resin chromatography is as follows: any one of D201, D900, 201 multiplied by 7, D113, D72, D301, D-101, D318, D208, HPD-100, HPD300, HPD-600, HPD-100A, HPD-400A, HPD-722, DM301, ADS-17, NHK, ADS-8, ADS-1 or AB-8 resin. The required control parameters during the macroporous resin chromatography are as follows: the diameter-height ratio of the chromatographic column is 1:3-1:15, preferably 1:7-1:10, more preferably 1: 8; the hemp polysaccharide loading concentration is 3-60%, preferably 20-40%, more preferably 35%; the loading volume of the hemp polysaccharide is 1-3 column volumes, preferably 2 column volumes; the volume of eluent is 1-5 column volumes, preferably 4 column volumes; the regeneration of the resin adopts ethanol gradient elution.
As a preferred technical scheme of the invention, the extraction method specifically comprises the following steps:
(1) soaking hemp raw material in water, wherein the ratio of the raw material to the liquid is 1 (5-30);
(2) ultrasonically stirring and extracting the raw materials soaked in the step (1) in an aqueous solution, wherein the power is 500-2000W, the duty ratio is 1 (1-5), the time is 1-3h, the temperature is 10-45 ℃, and the pH value of the aqueous solution is 5-8;
(3) after the extraction in the step (2) is finished, carrying out solid-liquid separation, carrying out membrane impurity removal, carrying out vacuum decompression and/or ultrafiltration membrane concentration on the filtrate, and concentrating to 1/8-1/4 of the volume of the filtrate;
(4) and (3) mixing the concentrated solution obtained in the step (3) with 75% -100% of ethanol, precipitating at-15-10 ℃ until the final concentration of the ethanol in a mixed system is 75% -95%, simultaneously stirring, separating the precipitate, dissolving the precipitate in water, and drying to obtain the hemp polysaccharide.
In another aspect, the present invention provides a cannabis polysaccharide extracted by the extraction method as described above.
In a further aspect, the present invention provides the use of a cannabis polysaccharide as described above in the preparation of a cosmetic, food, health product or pharmaceutical product. Specifically, cosmetics with functions of scavenging free radicals, resisting oxidation, whitening skin and resisting aging, foods or health products for improving immunity and anti-tumor medicines are preferably selected. The content of Cannabis polysaccharide in the above product is 0.01-99%, preferably 20-50%, and more preferably 30-40%.
Compared with the prior art, the invention has the following beneficial effects:
the extraction method provided by the invention is simple and easy to operate, the extracted hemp polysaccharide has high purity, has obvious effects of removing DPPH free radicals, resisting oxidation, whitening, resisting aging, improving immunity, resisting tumors and the like, and can be widely applied to cosmetics, foods, health-care products or medicines; meanwhile, the raw materials of the extraction method comprise the residues after the CBD and other cannabinoids active ingredients are extracted from the hemp flowers and leaves, so that the extraction method also provides a new strategy for recycling the residues after the CBD and other cannabinoids active ingredients are extracted from the hemp flowers and leaves, changes waste into valuable and realizes high-value recycling of resources.
Drawings
FIG. 1 is a graph of a glucose standard curve for polysaccharide purity testing by phenol-sulfuric acid method;
FIG. 2 is an HPLC plot of the first component of example 6;
FIG. 3 is an HPLC plot of the second component of example 6;
FIG. 4 is a graph of the ultraviolet absorption spectrum of the first component in example 6;
FIG. 5 is a graph of the ultraviolet absorption spectrum of the second component in example 6;
FIG. 6 is a standard graph of HPLC determination of polysaccharide molecular weight;
FIG. 7 is a chart of the infrared absorption spectrum of cannabis polysaccharide.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
This example provides a method for extracting cannabis sativa polysaccharide, comprising:
(1) soaking 1kg of residue raw material obtained after CBD extraction of cannabis sativa leaves in water, wherein the mass ratio of the raw material to the water solution is 1: 12;
(2) placing the raw materials soaked in the step (1) in a circulating ultrasonic extractor to perform ultrasonic stirring extraction in an aqueous solution, wherein the power is 1200W, the duty ratio is 1:1, the time is 2h, the temperature is 30 ℃, and the pH value of the aqueous solution is 6;
(3) after the extraction in the step (2), performing coarse filtration and fine filtration, wherein the coarse filtration adopts a drum-type filter device with a filter screen, the fine filtration adopts a disc centrifuge for filtration, then a microfiltration membrane with the molecular weight cutoff of 300KDa is adopted for impurity removal, and the filtrate is subjected to vacuum reduced pressure concentration and concentrated to 1/8 of the volume of the filtrate;
(4) mixing the concentrated solution obtained in the step (3) with 95% ethanol, precipitating at-5 deg.C to make the final concentration of ethanol in the mixed system be 80%, stirring for 5h, standing for 6h after stirring, separating precipitate, dissolving the precipitate in water, and spray drying under the conditions of: the air inlet temperature is 175 ℃, the air outlet temperature is 100 ℃, and the feeding density is 1.03g/cm 3To obtain 150g of the hemp polysaccharide.
And (3) detecting the purity of the extracted cannabis sativa polysaccharide by using a phenol-sulfuric acid method (the glucose standard curve for detecting the purity of the cannabis sativa polysaccharide by using the phenol-sulfuric acid method is shown in figure 1), so as to obtain the cannabis sativa polysaccharide with the purity of 40%.
Example 2
This example provides a method for extracting cannabis sativa polysaccharide, comprising:
(1) soaking 1kg of dried cannabis sativa raw material in water, wherein the mass ratio of the raw material to the aqueous solution is 1: 30;
(2) putting the raw material soaked in the step (1) into a heating extraction tank to extract in an aqueous solution, wherein the temperature is 80 ℃, the pH value of the aqueous solution is 7, and additionally adding 0.1% of cellulase;
(3) after the extraction in the step (2), performing coarse filtration and fine filtration, wherein the coarse filtration adopts a drum-type filter device with a filter screen, the fine filtration adopts a disc centrifuge for filtration, then a microfiltration membrane with the molecular weight cutoff of 300KDa is adopted for impurity removal, and the ultrafiltration membrane concentration is performed on the filtrate until the volume of the filtrate is 1/4;
(4) mixing the concentrated solution obtained in step (3) with 90% ethanol, precipitating at 10 deg.C to obtain ethanol with final concentration of 95% in the mixed system, stirring for 5 hr, standing for 6 hr, separating precipitate, dissolving the precipitate with water, and adding into water Ultrasonic drying is carried out, and the ultrasonic power is 10W/cm2To obtain 200g of the hemp polysaccharide.
And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 35%.
Example 3
This example provides a method for extracting cannabis sativa polysaccharide, comprising:
(1) soaking 1kg of fresh hemp leaf raw material in water, wherein the mass ratio of the raw material to the water solution is 1: 5;
(2) placing the raw materials soaked in the step (1) in a circulating ultrasonic extractor to perform ultrasonic stirring extraction in an aqueous solution, wherein the power is 1000W, the duty ratio is 1:3, the time is 2h, the temperature is 45 ℃, and the pH value of the aqueous solution is 5; in addition, a mixture of cellulase, pectinase, beta-glucanase and xylanase in equal proportion is added, and the enzyme concentration is 0.5%;
(3) after the extraction in the step (2), performing coarse filtration and fine filtration, wherein the coarse filtration adopts a drum-type filter device with a filter screen, the fine filtration adopts a disc centrifuge for filtration, then a microfiltration membrane with the molecular weight cutoff of 300KDa is adopted for impurity removal, and the filtrate is subjected to vacuum reduced pressure concentration and concentrated to 1/5 of the volume of the filtrate;
(4) and (3) mixing the concentrated solution obtained in the step (3) with 75% ethanol, precipitating at-15 ℃ until the final concentration of the ethanol in a mixed system is 75%, stirring for 2 hours, standing for 8 hours after stirring, separating precipitates, dissolving the precipitates in water, and freeze-drying to obtain 180g of the hemp polysaccharide.
And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 43%.
Example 4
This example provides a method for extracting cannabis sativa polysaccharide, comprising:
(1) soaking 1kg of dry hemp leaf raw material in water, wherein the mass ratio of the raw material to the water solution is 1: 20;
(2) placing the raw materials soaked in the step (1) in a circulating ultrasonic extractor to perform ultrasonic stirring extraction in an aqueous solution, wherein the power is 500W, the duty ratio is 1:3, the time is 2h, the temperature is 35 ℃, and the pH value of the aqueous solution is 7; in addition, a mixture of cellulase and pectinase is added according to the proportion of 2:1, and the enzyme concentration is 0.5%;
(3) after the extraction in the step (2), performing coarse filtration and fine filtration, wherein the coarse filtration adopts a drum-type filter device with a filter screen, the fine filtration adopts a disc centrifuge for filtration, then a microfiltration membrane with the molecular weight cutoff of 500KDa is adopted for impurity removal, and the filtrate is subjected to vacuum reduced pressure concentration and concentrated to 1/5 of the volume of the filtrate;
(4) and (3) mixing the concentrated solution obtained in the step (3) with 95% ethanol, precipitating at-10 ℃ until the final concentration of the ethanol in a mixed system is 80%, stirring for 3 hours at the same time, standing for 6 hours after stirring, separating the precipitate, dissolving the precipitate in water, and performing boiling drying to obtain 160g of the hemp polysaccharide.
And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 45%.
Example 5
This example further purifies the cannabis polysaccharide obtained in example 1 as follows:
the cannabis polysaccharide prepared in example 1 was subjected to membrane filtration using a 50000Da cut-off membrane to remove impurities followed by macroporous resin chromatography. The filler for macroporous resin chromatography is as follows: d201, the diameter-height ratio of the chromatographic column is 1: 8; the hemp polysaccharide loading concentration is 35%; the sample loading volume of the hemp polysaccharide is 2 column volumes; eluent volume was 4 column volumes; the eluent is 10% ethanol. Concentrating and drying the eluent to obtain further purified hemp polysaccharide, and detecting the purity of the polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 80%.
Example 6
This example further purifies the cannabis polysaccharide obtained in example 2 by the following method:
the hemp polysaccharide prepared in example 2 was chromatographed on DEAE cellulose resin, the process of DEAE cellulose resin chromatography was: preparing the polysaccharide into 1% concentration, loading onto column, eluting with water, eluting with salt, desalting, concentrating, drying to obtain further purified Cannabis sativa polysaccharide, and detecting the purity of the polysaccharide by phenol-sulfuric acid method to obtain Cannabis sativa polysaccharide with purity of 95%.
After the hemp polysaccharide is subjected to gradient elution by solutions with different salt concentrations, according to the enrichment of different polysaccharide molecular weights in an analysis gradient eluent, the polysaccharide can be considered to be separated into two components, and the two components are respectively subjected to HPLC (high performance liquid chromatography) and ultraviolet analysis, wherein the HPLC chart of the first component is shown in FIG. 2, and the chart shows that: the highest peak appears at 12.983min, the molecular weight is 1000Da, and the content is highest; the second component HPLC chart is shown in FIG. 3, which shows that: the highest peak appears at 13.374min, the molecular weight is 300Da, and the content is the highest. The ultraviolet absorption pattern of the first component is shown in fig. 4, from which it can be seen that: the highest absorption peak is at 210nm, and absorption exists at 280nm, which is considered as the absorption peak of the benzene ring; the ultraviolet absorption pattern of the second component is shown in fig. 5, and it can be seen from the figure that: the highest absorption peak is also at 210nm, but unlike the first component, there is no UV absorption at 280nm, and it can be considered that the two components are structurally distinct and belong to two different species.
A standard curve of the molecular weight of the polysaccharide determined by HPLC is plotted, as shown in FIG. 6 (the ordinate lgM in the figure is the logarithm of the molecular weight of the polysaccharide). The molecular weight of the first component is about 1000Da and the molecular weight of the second component is about 300Da according to the standard curve.
Example 7
This example further purified the cannabis polysaccharide obtained in example 3 by the following method:
the cannabis polysaccharide prepared in example 3 was subjected to macroporous resin chromatography with the following packing: HPD-600, the diameter-height ratio of the chromatographic column is 1: 10; the hemp polysaccharide loading concentration is 20%; the sample loading volume of the hemp polysaccharide is 1 column volume; eluent volume was 3 column volumes; the eluent is distilled water. Concentrating and drying the eluent to obtain further purified hemp polysaccharide, and detecting the purity of the polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 76%.
Example 8
This example further purified the cannabis polysaccharide obtained in example 4 by the following method:
the cannabis polysaccharide prepared in example 4 was subjected to membrane filtration using a 50000Da cut-off membrane to remove impurities followed by macroporous resin chromatography. The filler for macroporous resin chromatography is as follows: AB-8 resin, the diameter-height ratio of the chromatographic column is 1: 7; the hemp polysaccharide loading concentration is 10%; the sample loading volume of the hemp polysaccharide is 3 column volumes; eluent volume was 3 column volumes; the eluent is distilled water. Concentrating and drying the eluent to obtain further purified hemp polysaccharide, and detecting the purity of the polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 89%. FTIR infrared spectroscopy of the obtained cannabis polysaccharides is shown in FIG. 7. As can be seen from the figure: at 3500- -1The broad peak of (A) is an O-H stretching vibration absorption peak, which indicates that the separated component possibly contains an O-H bond; at 2930cm-1The characteristic peak is the stretching vibration absorption peak of the C-H bond, which indicates that the C-H bond is possibly present; at 1590cm-1The absorption peak of (a) is a C ═ O symmetric stretching vibration absorption peak, which indicates that the compound contains carbonyl; at 1420cm-1The absorption peak at (A) may be due to C-O stretching vibrations, thus taking into account the possible presence of-COOH in the polysaccharide structure; at 1240cm-1An absorption peak exists among the groups, which is probably caused by O-H variable angle vibration and is presumed to exist hydroxyl; at 1080cm-1The absorption peak may be caused by C-O stretching vibration, and may have an aliphatic ether or cyclic ether structure, and thus it is considered that the separated component contains a polysaccharide.
Example 9
This example provides a process for extracting cannabis polysaccharide, which differs from example 1 only in that the mass ratio of the raw material to the aqueous solution in step (1) is 1:5, and the other steps are kept the same. The procedure of example 5 was then repeated. And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 62%.
Example 10
This example provides a process for extracting cannabis polysaccharide, which differs from example 1 only in that the mass ratio of the raw material to the aqueous solution in step (1) is 1:30, and the other steps are kept the same. The procedure of example 5 was then repeated. And detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 75%.
Example 11
This example provides a process for extracting cannabis polysaccharide which differs from example 2 only in that the pH of the aqueous solution in step (2) is 8, the other steps remaining the same. The procedure of example 6 was then repeated. And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 82%.
Example 12
This example provides a process for extracting cannabis polysaccharide which differs from example 2 only in that the pH of the aqueous solution in step (2) is 4, the other steps remaining the same. The procedure of example 6 was then repeated. And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 86%.
Example 13
This example provides a process for extracting cannabis polysaccharide which differs from example 1 only in that the temperature in step (2) is 45 ℃ and the other steps remain the same. The procedure of example 5 was then repeated. And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 78%.
Example 14
This example provides a process for extracting cannabis polysaccharide which differs from example 1 only in that the temperature in step (2) is 10 ℃, and the other steps remain the same. The procedure of example 5 was then repeated. And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 71%.
Example 15
This example provides a process for extracting cannabis polysaccharide which differs from example 4 only in that the final concentration of ethanol in the mixed system in step (4) is 95% and the other steps are kept the same. The procedure of example 8 was then repeated. And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 83%.
Example 16
This example provides a process for extracting cannabis polysaccharide which differs from example 4 only in that the final concentration of ethanol in the mixed system in step (4) is 75%, and the other steps are kept the same. The procedure of example 8 was then repeated. And (3) detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 77%.
Example 17
This example provides a process for extracting cannabis polysaccharide which differs from example 1 only in that the precipitation is carried out at-15 ℃ in step (4), the other steps remaining the same. The procedure of example 5 was then repeated. And detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 75%.
Example 18
This example provides a process for extracting cannabis polysaccharide which differs from example 1 only in that the precipitation is carried out at 10 ℃ in step (4), the other steps remaining the same. The procedure of example 5 was then repeated. And detecting the purity of the extracted hemp polysaccharide by using a phenol-sulfuric acid method to obtain the hemp polysaccharide with the purity of 72%.
Example 19
The results of the removal of DPPH free radicals by the cannabis polysaccharides obtained in examples 5-18 (vitamin C control) were evaluated in this example, by: respectively taking 5mg of samples in examples 5-18 to prepare 0.5mg/mL aqueous solution samples, accurately measuring 1mL, adding 1.0mL of 0.6mmol/L DPPH ethanol solution, supplementing 1mL of water, uniformly mixing, and reacting for 15min at room temperature in a dark place. The absorbance A of the sample at a wavelength of 517nm was then determined1(ii) a The control group uses equal volume of deionized water to replace the extracting solution, and the corresponding absorbance A is measured0(ii) a Absorbance of sample background A2The DPPH ethanol solution was replaced with an equal volume of absolute ethanol. DPPH radical clearance was determined as described above using Vc as a positive control. The formula for DPPH clearance is:
the results are shown in Table 1, and it is clear from Table 1 that:
TABLE 1
The applicant states that the present invention is illustrated by the above examples to describe the extraction method of cannabis polysaccharide, its products and applications, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A method for extracting hemp polysaccharide is characterized in that the extraction method comprises the steps of soaking hemp raw materials, extracting, carrying out solid-liquid separation, concentrating filtrate, and precipitating concentrated solution in alcohol to obtain the hemp polysaccharide.
2. The extraction process of claim 1, wherein the cannabis material comprises cannabis flowers and/or cannabis leaves.
3. The extraction method according to claim 1 or 2, characterized in that it comprises the steps of:
(1) soaking hemp in water;
(2) extracting the raw materials soaked in the step (1);
(3) After the extraction in the step (2) is finished, carrying out solid-liquid separation, and concentrating the filtrate;
(4) and (4) mixing the concentrated solution obtained in the step (3) with alcohol for precipitation to obtain the hemp polysaccharide.
4. The extraction method according to claim 3, wherein the mass ratio of the hemp raw material to the water in the step (1) is 1 (5-30), preferably 1: 12;
preferably, the extraction manner of step (2) comprises ultrasonic extraction and/or heating extraction in an aqueous solution, preferably ultrasonic extraction;
preferably, the power of the ultrasonic extraction is 500-; the duty ratio of ultrasonic extraction is 1 (1-5); the ultrasonic extraction time is 1-3 h;
preferably, the ultrasonic extraction is carried out in stirring, and the stirring speed is 300-1200 rpm;
preferably, the temperature of the ultrasonic extraction is 10-45 ℃, preferably 30 ℃.
5. The extraction process according to claim 3 or 4, wherein the pH of the aqueous solution at the time of the extraction in the step (2) is 5 to 8;
preferably, an enzyme is added during the extraction in the step (2), and the enzyme comprises any one or a combination of at least two of cellulase, pectinase, beta-glucanase, xylanase, amylase, protease or lipase;
Preferably, the concentration of the enzyme in the aqueous solution is between 0.1% and 0.5%.
6. The extraction process according to any one of claims 3 to 5, wherein the solid-liquid separation in step (3) is followed by membrane purification, wherein the membrane is a 600KDa microfiltration membrane with molecular weight cut-off of 300-;
preferably, the concentration in the step (3) is performed by vacuum decompression and/or ultrafiltration membrane;
preferably, the ultrafiltration membrane has a molecular weight cut-off of 300-1000 Da;
preferably, the concentration of step (3) is 1/8-1/4 of the volume of the filtrate.
7. The extraction process according to any one of claims 3 to 6, wherein the alcohol of step (4) is ethanol;
preferably, the concentration of the ethanol is 75% -100%;
preferably, the final concentration of ethanol in the mixed system is 75% -95%, preferably 80%;
preferably, the temperature for precipitation in the step (4) is-15 to 10 ℃, preferably-5 ℃;
preferably, the precipitation in the step (4) is carried out while stirring, and the stirring time is 2-10h, preferably 5 h;
preferably, the precipitate is separated after the cannabis polysaccharide is obtained in the step (4), and the precipitate is dissolved in water and dried;
preferably, the drying mode comprises any one or combination of at least two of ultrasonic drying, spray drying, boiling drying or freeze drying;
Preferably, the hemp polysaccharide obtained in step (4) is further separated and purified, and the separation and purification mode comprises any one or a combination of at least two of membrane filtration, DEAE cellulose resin chromatography or macroporous resin chromatography.
8. The extraction method according to any one of claims 3 to 7, characterized in that it comprises in particular the steps of:
(1) soaking hemp raw material in water, wherein the ratio of the raw material to the liquid is 1 (5-30);
(2) ultrasonically stirring and extracting the raw materials soaked in the step (1) in an aqueous solution, wherein the power is 500-2000W, the duty ratio is 1 (1-5), the time is 1-3h, the temperature is 10-45 ℃, and the pH value of the aqueous solution is 5-8;
(3) after the extraction in the step (2) is finished, performing solid-liquid separation, performing membrane impurity removal, performing vacuum decompression and/or ultrafiltration membrane concentration on the filtrate, and concentrating to 1/8-1/4 of the volume of the filtrate;
(4) and (3) mixing the concentrated solution obtained in the step (3) with 75% -100% of ethanol, precipitating at-15-10 ℃ until the final concentration of the ethanol in a mixed system is 75% -95%, simultaneously stirring, separating the precipitate, dissolving the precipitate in water, and drying to obtain the hemp polysaccharide.
9. A cannabis polysaccharide extracted by the extraction method of any one of claims 1 to 8.
10. Use of cannabis polysaccharide according to claim 9 for the preparation of a cosmetic, food, health product or pharmaceutical product;
preferably, the cosmetic is an antioxidant, whitening or anti-aging cosmetic;
preferably, the food is a food for improving immunity;
preferably, the health care product is a health care product for improving immunity;
preferably, the drug is an anti-tumor drug.
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