CN115772234B - Sulfated cannabis polysaccharide, preparation method, application and product thereof - Google Patents
Sulfated cannabis polysaccharide, preparation method, application and product thereof Download PDFInfo
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- CN115772234B CN115772234B CN202111043230.2A CN202111043230A CN115772234B CN 115772234 B CN115772234 B CN 115772234B CN 202111043230 A CN202111043230 A CN 202111043230A CN 115772234 B CN115772234 B CN 115772234B
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- cannabis
- polysaccharide
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- sulfated
- cannabis polysaccharide
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Abstract
The invention provides a sulfated cannabis polysaccharide, the molecular weight of the sulfated cannabis polysaccharide is between 1000Da and 10000Da, and the substitution degree is 0.13-0.27. The invention also provides a preparation method of the sulfated cannabis polysaccharide, which is to react cannabis polysaccharide with chlorosulfonic acid to obtain the sulfated cannabis polysaccharide. The obtained sulfated cannabis sativa polysaccharide has strong antioxidant activity and antitumor activity.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to sulfated cannabis polysaccharide, a preparation method, application and a product thereof.
Background
Polysaccharide (Polysaccharide) is also called as Polysaccharide, is distributed in cell membranes of higher animals and plants or microbial cell walls, is a natural polymer formed by connecting aldose or ketose through glycosidic bonds, is the most abundant biopolymer in nature, is one of four basic substances forming life activities, not only provides skeleton structures and energy sources for life, but also is widely involved in the regulation of various life phenomena and physiological processes of cells. With the research and development of molecular biology and chemistry of immune substances and the search and development of new drug resources, the research of polysaccharide is more and more widely paid attention. The polysaccharide can be structurally modified by chemical, physical, biological and other methods, so that the biological activity of the polysaccharide is obviously increased. The plant polysaccharide has wide biological activities of improving the immune function of organisms, resisting viruses and oxidation, inhibiting tumor growth, resisting aging and the like, has the characteristics of green and natural property, small toxic and side effects, difficult generation of drug resistance and the like, and is widely studied by scholars at home and abroad.
Cancer, also known as malignant tumor, is a disease caused by a disorder in the control of the cell proliferation mechanism. Cancer has become one of serious diseases that endanger human health along with environmental deterioration and the like, and the attack of cancer has been an important research topic in the world. The unbalanced match of cell proliferation and apoptosis and the metastasis and diffusion thereof are the basis of malignant tumor occurrence. Intervention of apoptosis, i.e. regulation of cell proliferation and apoptosis, is an important approach and means for the treatment of neoplastic diseases. Under normal physiological conditions, timely and moderate apoptosis regulation is very important for organisms to remove cells with aging and potential danger, maintain the stability of genome and normal individual development, and induce apoptosis of tumor cells in the presence of tumor so as to achieve the purpose of tumor treatment.
Oxidative stress refers to the fact that when the body is subjected to various harmful stimuli, high-activity molecular free radicals in the body are generated excessively, the oxidation degree exceeds the oxide scavenging capacity, and an oxidation system and an antioxidation system are dynamically unbalanced, so that tissue damage is caused. In the production chain of animal husbandry, the development of the livestock industry is affected by oxidative stress caused by the factors of intensive cultivation, malnutrition, feed fermentation, long-distance transportation, extreme environment and the like, so that huge economic loss is caused. In order to solve the production problems caused by oxidative stress, research on exogenous antioxidants has been widely conducted.
The active molecule has one or several atomic groups or atoms with unpaired electrons, and has certain stability, high oxidability, active chemical property and other features. Under normal conditions, free radicals have a regulatory effect on cell proliferation, differentiation, apoptosis and necrosis; the free radicals and the oxidation process are relied on to enable a plurality of organism tissues to generate energy; the oxidation and antioxidation of the body are in dynamic balance. However, various exogenous and endogenous oxidative stress reactions can influence the dynamic balance of free radicals in organisms, and particularly, excessive active oxygen can be generated when the organisms are in a pathological state, so that the organisms are in an oxidative stress state. Excessive free radicals and metabolites thereof in the body cause cellular metabolic dysfunction and damage, and excessive free radicals attack biological macromolecules such as DNA, protein, carbohydrate and the like to generate various different consequences, so that the free radicals are closely related to the generation of various diseases, such as cancers, cardiovascular diseases, rheumatoid arthritis, atherosclerosis and the like. In addition, aging of the body with age is also closely related to this. Free radicals in the body are generally scavenged by antioxidants. Antioxidants commonly used today are t-butyl-p-methoxyphenol formulation (BHA) and Butyl Hydroxy Toluene (BHT). Although BHA and BHT have excellent antioxidant effects, with the increasing awareness of environmental protection, it is suspected that such chemically synthesized antioxidants may induce liver damage and cause cancer. Therefore, it is an important direction to find new antioxidants from natural products.
A large number of researches show that many plants and active ingredients thereof have a certain antioxidation effect, but the antioxidation effect of the plants is weak because the ingredients are complex and the quality is difficult to control, and the effect is not ideal. Thus, the existing researches indicate that the polysaccharide with an active function can be separated for use, and on the basis, the activity of the separated polysaccharide can be improved in various ways so as to fully exert the application and value of the polysaccharide. The cannabis polysaccharide has wide biological activities of improving the immune function of organisms, resisting viruses and oxidation, inhibiting tumor growth, resisting aging and the like, has the characteristics of green and natural property, small toxic and side effects, difficult generation of drug resistance and the like, and is widely studied by scholars at home and abroad. Further studies on structural modification of polysaccharides are required to increase the activity of polysaccharides.
Disclosure of Invention
The hemp is industrial hemp which can be legally planted in large scale and industrially developed and utilized.
The invention aims to provide sulfated cannabis polysaccharide, and a preparation method, application and a product thereof.
It is an object of the present invention to provide a sulfated cannabis polysaccharide having a molecular weight of between 1000Da and 10000Da, preferably between 2000Da and 5000Da, more preferably between 3000Da and 5000Da, with a degree of substitution of 0.13-0.27, preferably 0.15-0.27, more preferably 0.27.
The second object of the present invention is to provide a process for preparing sulfated cannabis polysaccharides comprising the steps of:
(1) Dispersing cannabis polysaccharide into amide or pyridine solvents, and uniformly stirring to obtain a solution A;
(2) Slowly adding chlorosulfonic acid into DMF in ice water bath, stirring and mixing uniformly to obtain solution B;
(3) Mixing the solution A and the solution B for reaction to obtain a solution C;
(4) After the reaction is finished, the solution C is cooled to room temperature, absolute ethyl alcohol is slowly added for precipitation, the supernatant is removed by centrifugation, the precipitate is dissolved in water, the pH is adjusted to be neutral, dialysis and the trapped fluid is subjected to alcohol precipitation, centrifugation and precipitation drying, and the sulfated cannabis polysaccharide is obtained.
According to the preparation method of the sulfated cannabis polysaccharide, in the step (1), the mass-volume ratio of the cannabis polysaccharide to the amide or pyridine solvent is 1:20-50 (g/mL), wherein the mass-volume ratio of the cannabis polysaccharide to the amide solvent can be 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 and 1:50, and the mass-volume ratio of the cannabis polysaccharide to the pyridine solvent can be 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 and 1:50, but the invention is not limited to the listed ratio, and other non-listed ratios in the ratio range are equally applicable.
In the step (1), the amide solvent is one or more of formamide, acetamide and propionamide, namely, the amide solvent comprises but is not limited to formamide, acetamide or propionamide which are respectively used independently, or can be mixed solution of formamide and acetamide in unlimited proportion, or can be mixed solution of formamide and propionamide in unlimited proportion, or can be mixed solution of acetamide and propionamide in unlimited proportion, or can be mixed solution of formamide, acetamide and propionamide in unlimited proportion.
The preparation method of the sulfated cannabis polysaccharide according to the present invention, wherein in the step (1), the pyridine solvent is one or more of pyridine, picoline and ethylpyridine, that is, the pyridine solvent includes but is not limited to pyridine, picoline or ethylpyridine, respectively, and may be a mixed solution of pyridine and picoline in an unlimited proportion, a mixed solution of pyridine and ethylpyridine in an unlimited proportion, a mixed solution of picoline and ethylpyridine in an unlimited proportion, or a mixed solution of pyridine, picoline and ethylpyridine in an unlimited proportion.
The method for producing a sulfated cannabis polysaccharide according to the present invention, wherein in step (1), the stirring temperature is 30 to 80 ℃, wherein the stirring temperature may be 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, preferably 45 ℃, but is not limited to the values listed, and other non-listed values within the range of values are equally applicable.
The method for preparing the sulfated cannabis polysaccharide according to the present invention, wherein in step (1), the stirring time is 20-50 min, wherein the stirring time may be 20min, 25min, 30min, 35min, 40min, 45min, 50min, preferably 50min, but is not limited to the recited values, and other non-recited values within the range of values are equally applicable.
The preparation method of the sulfated cannabis polysaccharide according to the present invention, wherein in step (2), the volume ratio of chlorosulfonic acid to DMF is 1:3-8 (mL/mL), wherein the volume ratio of chlorosulfonic acid to DMF may be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, preferably 1:5, but is not limited to the listed ratios, and other non-listed ratios within the range of the ratio are equally applicable.
The preparation method of the sulfated cannabis polysaccharide according to the present invention, wherein in the step (2), the stirring time is 30-40 min, wherein the stirring time may be 30min, 31min, 32min, 33min, 34min, 35min, 36min, 37min, 38min, 39min, 40min, but is not limited to the recited values, and other non-recited values within the range of values are equally applicable.
According to the preparation method of the sulfated cannabis polysaccharide, in the step (3), the mass-to-volume ratio of cannabis polysaccharide in solution A to chlorosulfonic acid in solution B is 1:1-10 (g/mL), wherein the mass ratio of cannabis polysaccharide in solution A to chlorosulfonic acid in solution B can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, preferably 1:4, but not limited to the listed ratio, and other non-listed ratios within the ratio range are equally applicable.
The method for producing a sulfated cannabis polysaccharide according to the present invention, wherein in step (3), the reaction temperature is 30 to 80 ℃, wherein the reaction temperature may be 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, preferably 50 ℃, but is not limited to the values listed, and other non-listed values within the range of values are equally applicable.
The process for preparing a sulfated cannabis polysaccharide according to the present invention, wherein in step (3), the reaction time is 2 to 8 hours, wherein the reaction time may be 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, preferably 5 hours, but is not limited to the recited values, and other non-recited values within the range of values are equally applicable.
The method for preparing sulfated cannabis polysaccharide according to the present invention, wherein in step (3), the reaction process is enhanced by ultrasonic to achieve the purpose of promoting sulfation synthesis, the ultrasonic probe may be in the form of a focused or dispersed type, the ultrasonic power of ultrasonic wave applied to the solution system is 1-10W/L, the ultrasonic power of ultrasonic wave applied to the solution system may be enhanced by receiving ultrasonic wave of different powers of 1W, 2W, 3W, 4W, 5W, 6W, 7W, 8W, 9W, 10W per liter of solution, wherein 5W/L solution system is preferable, but not limited to the recited values, and other non-recited values within the range of values are equally applicable.
The method for preparing sulfated cannabis polysaccharides according to the present invention, wherein in step (4), the amount of absolute ethanol is 4 to 6 times the volume of the C solution, wherein the amount of absolute ethanol may be 4 times, 4.5 times, 5 times, 5.5 times, 6 times the volume of the C solution, preferably 4 times the volume of the C solution, but is not limited to the recited values, and other non-recited values within the range of values are equally applicable.
The process for preparing a sulfated cannabis polysaccharide according to the present invention, wherein, in step (4),
the absolute ethyl alcohol is precooled to 0-4 ℃;
Adjusting the pH to 7 by adopting a saturated NaOH aqueous solution;
the molecular weight of a dialysis membrane used for dialysis is 300Da-500Da, and the trapped fluid is mainly sulfated cannabis polysaccharide with the molecular weight of more than 1000 Da;
precipitating the trapped fluid with ethanol until the concentration of ethanol is more than 80%;
the precipitation drying mode is freeze drying or vacuum pulsation drying.
The invention also provides a preparation method of the cannabis polysaccharide, which comprises the following steps:
(i) Extraction of cannabis sativa polysaccharide: extracting Cannabis sativa with water, filtering, concentrating the clear solution to obtain crude polysaccharide solution;
(ii) Purification of cannabis polysaccharide: precipitating crude hemp polysaccharide solution with ethanol, separating solid from liquid, collecting precipitate, dissolving precipitate with water, removing impurities, purifying eluate with DEAE cellulose column, eluting with salt solution, desalting eluate, and drying to obtain refined hemp polysaccharide.
The method for preparing cannabis polysaccharide according to the present invention, wherein, in step (i), the cannabis is the cannabis leaf material (fresh or dry) or the residue after CBD extraction from cannabis leaves.
The preparation method of cannabis polysaccharide according to the present invention, wherein in step (i), the mass-to-volume ratio of cannabis to water is 1:5-1:30 (g/mL), wherein the mass-to-volume ratio of cannabis to water may be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, preferably 1:15, but are not limited to, the recited ratios, and other ratios not recited in this range of ratios are equally applicable.
The preparation method of the cannabis polysaccharide according to the invention, wherein in step (i), enzymes are added in the extraction process to enhance the extraction process, wherein the enzymes are one or more of cellulases, pectinases, amylases and proteases.
The method for preparing cannabis polysaccharide according to the present invention, wherein in step (i), the concentration process is vacuum reduced pressure concentration or membrane concentration, preferably to a solids content of 10-30%, wherein concentration to a solids content of 10%, 15%, 20%, 25%, 30%, preferably to a solids content of 30%, is employed, but not limited to the values listed, other non-listed values within the range of values are equally applicable.
According to the preparation method of cannabis polysaccharide of the present invention, in the step (ii), the volume ratio of the crude cannabis polysaccharide solution to the ethanol is 1:4-6 (mL/mL), wherein the volume ratio of the crude cannabis polysaccharide solution to the ethanol can be 1:4, 1:4.5, 1:5, 1:5.5 and 1:6, but not limited to the listed ratio, and other ratios not listed in the ratio range are equally applicable.
The method for preparing cannabis polysaccharide according to the present invention, wherein in step (ii), the concentration of ethanol is 80% -100%, wherein the concentration of ethanol may be 80%, 85%, 90%, 95%, 100%, but is not limited to the recited values, and other non-recited values within the range of values are equally applicable.
The method for preparing cannabis polysaccharide according to the present invention, wherein in step (ii), the impurity removing means is macroporous resin, wherein the macroporous resin is any one of D201, D900, 201×7, 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, HZ-818, HZ-816, HZ-835, AB-8, DM130, D312, HZ-20SS, XDA-8, XDA-6, LSA-21 resin, and other decolorized, deproteinized resins, etc., are not limited to the above-listed resins, and other unrecited resins are equally applicable.
The preparation method of cannabis polysaccharide according to the present invention, wherein in step (ii), the impurity removal means may be membrane filtration, wherein the membrane used for membrane filtration is a microfiltration membrane.
The preparation method of cannabis polysaccharide according to the present invention, wherein in step (ii), the salt solution is 0.1-0.5M NaCl solution, wherein the concentration of NaCl solution may be 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, but not limited to the recited values, other non-recited values within the range of values are equally applicable.
The method for preparing cannabis polysaccharide according to the present invention, wherein in step (ii), the drying is freeze-drying or vacuum pulse drying.
It is a further object of the present invention to provide the use of sulfated cannabis polysaccharides in the preparation of cosmetics.
The use of the sulfated cannabis polysaccharides according to the invention for the preparation of cosmetics, in particular for anti-oxidative, whitening, spot-lightening cosmetics, in which the sulfated cannabis polysaccharides are used in an amount of 0.1% to 99% by mass, wherein the amount used may be 0.1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% by mass, but is not limited to the values listed, as are other non-listed values within the range of values.
The sulfated cannabis polysaccharides according to the present invention are used in the preparation of cosmetics which further comprise optional cosmetically acceptable carriers such as glycerin, vitamin B5, vitamin E, lactic acid, petrolatum, seaweed extract, propylene glycol, polyethylene glycol, hyaluronic acid (hyaluronic acid), jojoba oil, amino acids, hydrolyzed collagen, lecithin, ceramides, cholesterol, stearates, myristates, palmitates, glycerides, sorbitol, urea, hydroxy acids and saccharides, unsaturated linoleic acid, linolenic acid, arachidonic acid, various vitamins, various vegetable oils, chitin, aloe, seaweed, avocado oil, fruit acids, kojic acid, lactic acid, arbutin, licorice, mulberry extract, amino acid polypeptides, salicylic acid, thrombin acid, citric acid, various vegetable extracts, various preservatives, etc., but are not limited to the listed carriers.
The fourth object of the invention is to provide the application of sulfated cannabis polysaccharide in preparing antitumor drugs.
The use of the sulfated cannabis polysaccharide according to the present invention in the preparation of an antitumor drug, wherein the amount of the sulfated cannabis polysaccharide used is 0.1% -99% by mass, wherein the amount may be 0.1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% by mass, but is not limited to the recited values, and other non-recited values within the range of values are equally applicable.
The sulfated cannabis polysaccharides according to the present invention are useful in the preparation of antitumor agents, which may further comprise optional pharmaceutically acceptable carriers, excipients or diluents, and the like, including but not limited to lubricants, colorants, wetting agents, fillers, disintegrants and fragrances, the carriers may be povidone, stearic acid, magnesium stearate, polyethylene glycol, titanium dioxide, butylated hydroxyanisole, talc, micro-silica gel, dextrin, starch, lactose, and the like, but are not limited to the listed carriers.
The fifth object of the present invention is to provide an antioxidant comprising sulfated cannabis polysaccharides.
The antioxidant containing sulfated cannabis polysaccharide according to the present invention may be used in an amount of 0.1 to 99% by mass, wherein the amount may be 0.1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% by mass, but is not limited to the recited values, and other non-recited values within the range are equally applicable.
The antioxidant containing sulfated cannabis polysaccharides according to the present invention further comprises optional pharmaceutically acceptable carriers, excipients or diluents, and the like, including but not limited to lubricants, colorants, wetting agents, fillers, disintegrants and fragrances, the carriers may be povidone, stearic acid, magnesium stearate, polyethylene glycol, titanium dioxide, butylated hydroxyanisole, talc, gum arabic, dextrin, starch, lactose and the like, but are not limited to the listed carriers.
In the present invention, the term "pharmaceutically acceptable carrier" refers to an adjuvant for administration of a therapeutic agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and that is not potentially toxic after administration or after administration of a health care product.
Advantageous effects
The invention prepares cannabis polysaccharide from cannabis leaves or residues after CBD extraction from cannabis leaves, and derivatizes and modifies the cannabis polysaccharide to obtain sulfated cannabis polysaccharide. The obtained sulfated cannabis sativa polysaccharide has strong antioxidant activity and antitumor activity. The cannabis polysaccharide and the sulfated cannabis polysaccharide derived from the cannabis polysaccharide have application prospects for improving the antioxidant, whitening and spot-lightening effects of cosmetics and preparing antitumor drugs when used as cosmetic additives.
Drawings
FIG. 1 is an infrared spectrum of cannabis polysaccharides and sulfated cannabis polysaccharides prepared in accordance with the present invention.
FIG. 2 shows a sulfate solution standard curve in the substitution degree determination.
FIG. 3 shows the degree of substitution of the sulfated cannabis polysaccharides prepared in accordance with the present invention.
FIG. 4 is a DPPH radical scavenging profile of the sulfated cannabis polysaccharides prepared in accordance with the present invention.
FIG. 5 shows that the sulfated cannabis polysaccharides of the present invention inhibit the cellular activity of hepatoma cells (HepG 2).
Detailed Description
The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention in any way. It is apparent to those skilled in the art that the present invention is not limited to the above-described embodiments or modifications.
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent.
The instrument information and test conditions for infrared test of the invention are as follows:
ThermoFisher Nicolet iS50 FT-IR spectrophotometer, 4000cm -1 ～400cm -1 .
The main material information used in the following examples is as follows:
TABLE 1
EXAMPLE 1 preparation of sulfated Cannabis polysaccharide
Extracting 1000g of hemp leaf raw material with water at a mass ratio of hemp leaf raw material to water of 1:5 for 2h, filtering, vacuum concentrating the clear liquid until the solid content is 10% to obtain coarse hemp polysaccharide solution.
Adding 90% ethanol into the crude hemp polysaccharide solution for precipitation, wherein the volume ratio of the crude hemp polysaccharide solution to the ethanol is 1:4, carrying out solid-liquid separation on the precipitation overnight, collecting polysaccharide precipitate, dissolving the polysaccharide precipitate in water, carrying out decolorization and deproteinization on LSA-21 resin, loading the eluent on a DEAE cellulose column, eluting with 0.15M NaCl solution, desalting the eluent, and freeze-drying to obtain refined hemp polysaccharide.
Dispersing cannabis polysaccharide into formamide solvent, wherein the mass-volume ratio of cannabis polysaccharide to formamide is 1:20, and stirring at 60 ℃ for 40min to obtain solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:3.
Mixing the solution A and the solution B for reaction, wherein the mass volume ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:2, the reaction process adopts ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic power of ultrasonic waves acting on a solution system is 5W/L, the ultrasonic waves are dispersed, the reaction temperature is 50 ℃, and the reaction time is 3 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 4 times of pre-cooled absolute ethyl alcohol with the temperature of 4 ℃ is slowly added for precipitation, the precipitation is carried out overnight, the supernatant is removed by centrifugation, the precipitation is dissolved in water, the pH of the solution is adjusted to 7 by saturated NaOH aqueous solution, the dialysis is carried out, the molecular weight of a dialysis membrane is 500Da, the trapped liquid is subjected to alcohol precipitation until the concentration of the alcohol is 80%, the solution is centrifuged, and the precipitation is freeze-dried, thus 65g of sulfated cannabis polysaccharide is obtained.
EXAMPLE 2 preparation of sulfated Cannabis polysaccharide
Extracting the residue of hemp leaves after CBD extraction by using 95% ethanol, drying, taking 1000g of the residue, adding water for extraction, adding cellulase and pectinase mixed enzyme for extraction according to the mass ratio of the residue to the water of 1:30, filtering, and concentrating the clear liquid until the solid content is 20%, thus obtaining a coarse hemp polysaccharide solution.
Precipitating with 85% ethanol, separating solid and liquid at a volume ratio of 1:6, collecting precipitate, dissolving the precipitate with water, purifying with HPD-600 macroporous resin, purifying the eluate with DEAE cellulose column, eluting with 0.45M NaCl solution, desalting the eluate, and vacuum pulse drying to obtain refined hemp polysaccharide.
Dispersing cannabis polysaccharide into pyridine solvent, stirring for 50min at 30 ℃ with the mass-volume ratio of cannabis polysaccharide to pyridine of 1:50, and obtaining solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:8.
Mixing the solution A and the solution B for reaction, wherein the mass volume ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:2, the reaction process adopts ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic power of ultrasonic waves acting on a solution system is 3W/L, the ultrasonic waves are of a energy-accumulating type, the reaction temperature is 40 ℃, and the reaction time is 6.5 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 6 times of precooled absolute ethyl alcohol with the temperature of 4 ℃ is slowly added for precipitation, the supernatant is removed by centrifugation, the precipitate is dissolved in water, the pH value is adjusted to be neutral, dialysis is carried out, the molecular weight of a dialysis membrane is 300Da, the trapped liquid is subjected to alcohol precipitation until the concentration of the alcohol is 80%, the precipitate is centrifugated, and 88g of sulfated cannabis polysaccharide is obtained by vacuum pulsation drying of the precipitate.
EXAMPLE 3 preparation of sulfated Cannabis polysaccharide
Extracting 1000g of defatted (i.e. defatted) cannabis sativa leaf raw material with water, wherein the mass ratio of the cannabis sativa leaf raw material to the water is 1:12, adding cellulase, pectase, amylase and protease mixed enzyme for extraction, filtering, concentrating the clear liquid film until the solid content is 30%, and obtaining a crude cannabis sativa polysaccharide solution.
Adding 80% ethanol into the crude hemp polysaccharide solution for precipitation, wherein the volume ratio of the crude hemp polysaccharide solution to the ethanol is 1:5.5, carrying out solid-liquid separation, collecting the precipitate, adding water into the precipitate for dissolving, passing through a microfiltration membrane for impurity removal, passing through a DEAE cellulose column, eluting with 0.5M NaCl solution, desalting the eluent, and carrying out freeze drying or vacuum pulse drying to obtain refined hemp polysaccharide.
Dispersing cannabis polysaccharide into a propionamide solvent, wherein the mass-volume ratio of the cannabis polysaccharide to the propionamide is 1:40, stirring uniformly at 70 ℃ for 35min to obtain a solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:8.
Mixing the solution A and the solution B for reaction, wherein the mass volume ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:6, the reaction process adopts dispersed ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, and the ultrasonic power of ultrasonic waves acting on a solution system is that 10W power ultrasonic wave stimulation reinforcement is received by each liter of solution, the reaction temperature is 80 ℃, and the reaction time is 2 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 5 times of pre-cooled absolute ethyl alcohol with the temperature of 4 ℃ is slowly added for precipitation, the supernatant is removed by centrifugation, the precipitate is dissolved in water, the pH value of the precipitate is adjusted to 7 by saturated NaOH aqueous solution, the dialysis membrane molecular weight is 500Da, the trapped liquid is subjected to alcohol precipitation until the concentration of the alcohol is 90%, the precipitate is centrifugated, and the precipitate is frozen and dried, so that 81g of sulfated cannabis polysaccharide is obtained.
EXAMPLE 4 preparation of sulfated Cannabis polysaccharide
Extracting dried residue 1000g of hemp leaves after CBD extraction with water at a mass ratio of residue to water of 1:20, ultrasonically extracting for 2h, filtering, concentrating the clear liquid until the solid content is 15%, and obtaining coarse hemp polysaccharide solution.
Precipitating with 80% ethanol, collecting precipitate by solid-liquid separation, dissolving precipitate in water, removing impurities with D113 macroporous resin, purifying eluate with DEAE cellulose column, eluting with 0.1M NaCl, filtering eluate film for desalting, and lyophilizing or vacuum pulse drying to obtain refined hemp polysaccharide.
Dispersing cannabis polysaccharide into acetamide solvent, wherein the mass volume ratio of cannabis polysaccharide to acetamide is 1:45, and stirring for 30min at 70 ℃ to obtain solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:5.
Mixing the solution A and the solution B for reaction, wherein the mass ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:1, the reaction process adopts ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic power of ultrasonic waves acting on a solution system is 6W/L, the ultrasonic waves are dispersed, the reaction temperature is 70 ℃, and the reaction time is 6 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 4 times of absolute ethyl alcohol with pre-cooling temperature of 4 ℃ is slowly added, precipitation, centrifugation is carried out to remove clear liquid, the precipitation is dissolved in water, saturated NaOH aqueous solution is used for adjusting pH to 7, dialysis is carried out, the molecular weight of a dialysis membrane is 400Da, the trapped liquid is subjected to alcohol precipitation until the concentration of the ethanol is 90%, centrifugation is carried out, and the precipitation is freeze-dried, thus obtaining 76g of sulfated cannabis polysaccharide.
EXAMPLE 5 preparation of sulfated Cannabis polysaccharide
Extracting the residue of hemp leaves after CBD extraction with 95% ethanol, drying, adding water with a mass of 15 times of 1000g, adding pectase, extracting at normal temperature for 1h, heating to 85deg.C, extracting for 1h, filtering, concentrating the clear liquid film until the solid content is 25%, and obtaining coarse hemp polysaccharide solution.
Precipitating with 5 times of anhydrous ethanol, separating solid and liquid, collecting precipitate, dissolving precipitate with water, removing impurities with HPD-600 macroporous resin, purifying eluate with DEAE cellulose column, eluting with 0.25M NaCl salt solution, desalting eluate, and vacuum pulse drying to obtain refined cannabis sativa polysaccharide.
Dispersing cannabis polysaccharide into picoline solvent, wherein the mass-volume ratio of cannabis polysaccharide to picoline is 1:20, stirring uniformly at 60 ℃ for 40min to obtain solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:7.
Mixing the solution A and the solution B for reaction, wherein the mass volume ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:4, the reaction process adopts energy-accumulating type ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, and the ultrasonic power of ultrasonic waves acting on a solution system can be that each liter of solution is subjected to 2W power ultrasonic wave stimulation reinforcement, the reaction temperature is 60 ℃, and the reaction time is 5 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 6 times of absolute ethyl alcohol precooled to 0 ℃ is slowly added, precipitation, centrifugal supernatant removal is carried out, the precipitation is dissolved in water, saturated NaOH aqueous solution is used for adjusting the pH value to 7, dialysis is carried out, the molecular weight of a dialysis membrane is 500Da, the trapped liquid is subjected to alcohol precipitation until the concentration of the alcohol is 85%, centrifugation and precipitation freeze drying are carried out, and 88g of sulfated cannabis polysaccharide is obtained.
EXAMPLE 6 preparation of sulfated Cannabis polysaccharide
Drying and degreasing hemp leaves, extracting 1000g with 20 times of water, adding cellulase and amylase for extraction, filtering, concentrating the clear liquid until the solid content is 15%, and obtaining a coarse hemp polysaccharide solution.
Precipitating with 5 times of anhydrous ethanol, separating solid and liquid, collecting precipitate, dissolving precipitate with water, removing impurities with AB-8 macroporous resin, purifying eluate with DEAE cellulose column, eluting with 0.25M NaCl salt solution, desalting eluate, and lyophilizing to obtain refined cannabis sativa polysaccharide.
Dispersing cannabis polysaccharide into ethylpyridine solvent, wherein the mass-volume ratio of cannabis polysaccharide to ethylpyridine is 1:40, stirring uniformly at 65 ℃ for 50min to obtain solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B.
Mixing the solution A and the solution B for reaction, wherein the mass ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:5, the reaction process adopts dispersed ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic power of ultrasonic waves acting on a solution system receives 8W power ultrasonic wave stimulation reinforcement per liter of solution, the reaction temperature is 50 ℃, and the reaction time is 4.5 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 6 times of precooled absolute ethyl alcohol at 4 ℃ is slowly added, precipitation is carried out, the clear liquid is removed by centrifugation, the precipitation is dissolved in water, the pH value is adjusted to be neutral, dialysis is carried out, the molecular weight of a dialysis membrane is 500Da, the trapped liquid is subjected to alcohol precipitation until the concentration of the ethanol is 90%, centrifugation is carried out, and the precipitation is frozen and dried, thus 79g of sulfated cannabis polysaccharide is obtained.
EXAMPLE 7 preparation of sulfated Cannabis polysaccharide
Extracting residue after CBD extraction of hemp leaves with water of 25 times of the dry powder of 1000g, filtering, concentrating the clear liquid until the solid content is 10%, and obtaining coarse hemp polysaccharide solution.
Precipitating crude hemp polysaccharide solution with 4 times volume of absolute ethanol, separating solid and liquid, collecting precipitate, dissolving precipitate with water, removing impurities with microfiltration membrane, eluting with DEAE cellulose column, eluting with 0.45M NaCl salt solution, desalting the eluate, and vacuum pulse drying to obtain refined hemp polysaccharide.
The cannabis polysaccharide is dispersed into a propylpyridine solvent, the mass-volume ratio of the cannabis polysaccharide to the propylpyridine is 1:35, and the cannabis polysaccharide and the propylpyridine are uniformly stirred at the temperature of 40 ℃ for 35min to obtain a solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:6.
Mixing the solution A and the solution B for reaction, wherein the mass ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:3.5, the reaction process adopts ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic power of ultrasonic waves acting on a solution system is 5W/L, the ultrasonic waves are energy-accumulating type, the reaction temperature is 65 ℃, and the reaction time is 4 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 6 times of absolute ethanol which is precooled at 4 ℃ is slowly added for precipitation, the supernatant is removed by centrifugation, the precipitate is dissolved in water, the pH of the precipitate is adjusted to 7 by saturated NaOH aqueous solution, dialysis is carried out, the molecular weight of a dialysis membrane is 300Da, the trapped liquid is subjected to ethanol precipitation until the ethanol concentration is 85%, and 86g of sulfated cannabis polysaccharide is obtained by centrifuging and freeze-drying the precipitate.
EXAMPLE 8 preparation of sulfated Cannabis polysaccharide
1000g of residue after CBD extraction of cannabis sativa leaves is added with water with the mass of 18 times for ultrasonic extraction, and is filtered, and clear liquid is concentrated to the solid content of 25% by membrane, so as to obtain crude cannabis sativa polysaccharide solution.
Precipitating with 80% ethanol of 6 times volume, separating solid and liquid, collecting precipitate, dissolving precipitate with water, purifying with HZ-835 macroporous resin, purifying eluate with DEAE cellulose column, eluting with 0.4M NaCl salt solution, desalting eluate, and vacuum pulse drying to obtain refined hemp polysaccharide.
Dispersing cannabis polysaccharide into formamide solvent, wherein the mass-volume ratio of cannabis polysaccharide to formamide is 1:20, stirring uniformly at 50 ℃ for 40min to obtain solution A.
Slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 40min at volume ratio of chlorosulfonic acid to DMF of 1:6.5, and mixing to obtain solution B.
Mixing the solution A and the solution B for reaction, wherein the mass ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:6, the reaction process adopts ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic power of ultrasonic waves acting on a solution system is 6W/L, the ultrasonic waves are dispersed, the reaction temperature is 80 ℃, and the reaction time is 2 hours, so that the solution C is obtained.
After the reaction is finished, the solution C is cooled to room temperature, 4 times of absolute ethyl alcohol with pre-cooling at 4 ℃ is slowly added for precipitation, the supernatant is removed by centrifugation, the precipitate is dissolved in water, the pH of the precipitate is adjusted to be neutral by saturated NaOH aqueous solution, dialysis is carried out, the molecular weight of a dialysis membrane is 500Da, the trapped liquid is subjected to alcohol precipitation until the concentration of the alcohol is 80%, and 86g of sulfated cannabis polysaccharide is obtained by centrifuging and freeze-drying the precipitate.
EXAMPLE 9 preparation of sulfated Cannabis polysaccharide
Drying residues after CBD extraction of cannabis sativa leaves, taking 1000g, adding 15 times of water, heating to 90 ℃ for extraction for 2 hours, filtering, concentrating clear liquid until the solid content is 30%, and obtaining crude cannabis sativa polysaccharide solution.
Adding 90% ethanol into the crude hemp polysaccharide solution for precipitation, wherein the volume ratio of the crude hemp polysaccharide solution to the ethanol is 1:5.5, carrying out solid-liquid separation, collecting precipitate, dissolving the precipitate in water, purifying with D301 macroporous resin, purifying the eluent with DEAE cellulose column, eluting with 0.35M NaCl salt solution, desalting the eluent, and freeze-drying or vacuum pulse drying to obtain refined hemp polysaccharide.
Dispersing cannabis polysaccharide into pyridine solvent, wherein the mass-volume ratio of cannabis polysaccharide to pyridine is 1:35, stirring uniformly, and stirring at 45 ℃ for 50min to obtain solution A.
And slowly adding chlorosulfonic acid into DMF in ice water bath, stirring for 30min, and uniformly mixing to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:5.
Mixing the solution A and the solution B for reaction, wherein the mass ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:4, the reaction process adopts ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic power of ultrasonic waves acting on a solution system is 5W/L, the ultrasonic waves are dispersed, the reaction temperature is 50 ℃, and the reaction is carried out for 5 hours to obtain the solution C.
After the reaction is finished, the solution C is cooled to room temperature, 4 times of pre-cooled absolute ethyl alcohol with the temperature of 4 ℃ is slowly added for precipitation, the supernatant is removed by centrifugation, the precipitate is dissolved in water, saturated NaOH aqueous solution is used for regulating to be neutral, dialysis is carried out, the molecular weight of a dialysis membrane is 500Da, the trapped liquid is added with ethanol for precipitation until the ethanol concentration is 90%, and the precipitate is centrifugated and freeze-dried, thus obtaining 91g of sulfated cannabis polysaccharide.
EXAMPLE 10 preparation of a concentrate containing sulfated Cannabis sativa polysaccharide
The sulfated cannabis polysaccharide prepared in the embodiment 1 of the invention is uniformly mixed to prepare essence with the functions of whitening, freckle removing, acne removing and antioxidation, and the essence comprises the following steps:
the raw materials with the following mass percentages are prepared:
(1) Fully mixing the raw materials of the group B, carrying out water bath at 50 ℃ for 3min until the raw materials are fully swelled, and uniformly stirring the raw materials without obvious particles;
(2) Mixing glycerol and xanthan gum in the raw materials of the group A, uniformly dispersing, adding deionized water in the group A, uniformly stirring, sequentially adding sulfated cannabis polysaccharide and other raw materials in the group A, uniformly stirring and mixing in a water bath at 80 ℃ and a rotating speed of 30R/S until all the raw materials are fully dissolved;
(3) Adding the mixture obtained in the step (1) into the mixture obtained in the step (2), and stirring at the water bath of 80 ℃ and the rotating speed of 50R/S until the mixture is uniformly mixed, so that no obvious particulate matters exist;
(4) The mixture obtained in the step (3) is stirred and cooled to 40 ℃ at the rotating speed of 50R/S in a water bath at 25 ℃, and the raw materials of group C are added and uniformly stirred;
(5) Stirring at a rotating speed of 50R/S, and cooling to room temperature to obtain the essence containing the sulfated cannabis polysaccharide.
The sulfated cannabis polysaccharide prepared in other examples an essence containing sulfated cannabis polysaccharide was prepared in the same manner as in example 10.
EXAMPLE 11 preparation of skin cream containing sulfated Cannabis sativa polysaccharide
The embodiment is to prepare the skin cream with whitening, freckle removing and acne removing antioxidant effects by utilizing the sulfated cannabis sativa polysaccharide prepared in the embodiment 2, and the skin cream is prepared by the following steps:
the raw materials with the following mass percentages are prepared:
(1) Fully mixing the raw materials of the group C, standing at room temperature for 1 hour until the raw materials are fully swelled, and uniformly stirring the raw materials without obvious particles;
(2) Mixing glycerol and xanthan gum in the raw materials of the group A, uniformly dispersing, adding deionized water in the group A, uniformly stirring, sequentially adding sulfated cannabis polysaccharide and other raw materials in the group A, uniformly stirring and mixing in a water bath at 80 ℃ and a rotating speed of 30R/S until all the raw materials are fully dissolved;
(3) Uniformly dispersing hydrogenated lecithin and caprylic/capric triglyceride in the raw materials of the group B, heating in a water bath at 80 ℃, stirring until the mixture is fully dissolved, sequentially adding other raw materials in the group B, heating in the water bath at 80 ℃, and stirring until the mixture is fully dissolved;
(4) Adding a small amount of the mixed material of the group B in the step (3) into the mixed material of the group A in the step (2) for many times, keeping a water bath at 80 ℃ and homogenizing at a high speed of 3000R/S for 3min;
(5) Adding the swollen C group raw material component in the step (1) into the mixture in the step (4) for 3 times, keeping the water bath at 80 ℃, and homogenizing at a high speed of 3000R/S for 3min until no obvious particulate matters exist;
(6) Adding the mixed D group material into the mixture obtained in the step (5), keeping a water bath at 80 ℃, and homogenizing at a high speed of 3000R/S for 3min until no obvious particulate matters exist;
(7) The mixture obtained in the step (6) is stirred and cooled to 40 ℃ at the rotating speed of 50R/S in a water bath at 25 ℃, and the raw materials of the group E are added and uniformly stirred;
(8) Stirring at 50R/S speed, and cooling to room temperature to obtain skin cream containing sulfated cannabis polysaccharide.
The sulfated cannabis polysaccharide prepared in other examples a skin cream containing sulfated cannabis polysaccharide was prepared in the same manner as in example 11.
EXAMPLE 12 preparation of emulsion containing sulfated Cannabis polysaccharide
The present example is an emulsion with whitening, freckle-removing and acne-removing antioxidant effects prepared by using the sulfated cannabis sativa polysaccharide prepared in example 9 of the present invention, and the emulsion comprises the following steps:
The raw materials with the following mass percentages are prepared:
(1) Fully mixing the raw materials of the group C, standing at room temperature for 1 hour until the raw materials are fully swelled, and uniformly stirring the raw materials without obvious particles;
(2) Mixing butanediol and xanthan gum in the raw materials of the group A, adding deionized water in the group A after uniform dispersion, stirring uniformly, sequentially adding sulfated cannabis polysaccharide and other raw materials in the group A, stirring uniformly in water bath at 80 ℃ and rotating speed of 30R/S until all the raw materials are fully dissolved;
(3) Uniformly dispersing hydrogenated lecithin and caprylic/capric triglyceride in the raw materials of the group B, heating in a water bath at 80 ℃, stirring until the mixture is fully dissolved, sequentially adding other raw materials in the group B, heating in the water bath at 80 ℃, and stirring until the mixture is fully dissolved;
(4) Adding a small amount of the mixed material of the group B in the step (3) into the mixed material of the group A in the step (2) for many times, keeping a water bath at 80 ℃ and homogenizing at a high speed of 3000R/S for 3min;
(5) Adding the swollen C group raw material component in the step (1) into the mixture in the step (4) for 3 times, keeping the water bath at 80 ℃, and homogenizing at a high speed of 3000R/S for 3min until no obvious particulate matters exist;
(6) Adding the mixed D group material into the mixture obtained in the step (5), keeping a water bath at 80 ℃, and homogenizing at a high speed of 3000R/S for 3min until no obvious particulate matters exist;
(7) The mixture obtained in the step (6) is stirred and cooled to 40 ℃ at the rotating speed of 50R/S in a water bath at 25 ℃, and the raw materials of the group E are added and uniformly stirred;
(8) Stirring at a rotating speed of 50R/S, and cooling to room temperature to obtain emulsion containing sulfated cannabis polysaccharide.
The sulfated cannabis polysaccharide prepared in other examples an emulsion containing sulfated cannabis polysaccharide was prepared in the same manner as in example 12.
Test examples structural characterization and Activity determination of sulfated Cannabis sativa polysaccharide
(1) Structure identification (Infrared analysis)
When the sample is irradiated by infrared light with continuously changing frequency, the molecules absorb radiation with some frequencies, so that the transition from the ground state to the excited state is caused, the projected light intensity corresponding to the areas is weakened, and different groups or atoms are excited by infrared light and are different, so that the movement of characteristic chemical bonds in the sample or the disappearance of the characteristic chemical bonds are observed. The samples used in this test were sample a, sample B, sample C and sample D, wherein sample a was the sulfated cannabis polysaccharide prepared in example 1 (product with a reaction time of 3 hours), sample B was the sulfated cannabis polysaccharide prepared in example 1 (product with a reaction time of 2.5 hours), sample C was the sulfated cannabis polysaccharide prepared in example 1 (product with a reaction time of 2 hours), and sample D was the cannabis polysaccharide prepared in example 1 (purified cannabis polysaccharide), and the test procedures were as follows:
Drying the sample and potassium bromide for 24 hours, taking out, and cooling by a dryer to prevent water absorption; weighing 1-2mg of sample, adding 100-200 times of potassium bromide, and grinding with agate mortar until no granular sensation exists; placing the ground sample into a tablet press, and paving a thin layer to obtain a transparent tablet; and (3) detection: air is tested as a blank, and then infrared detection of the sample is performed. The infrared detection result is shown in fig. 1. As can be seen from FIG. 1, the wavelength is 3500cm -1 About typical hydroxyl peak, waveLength 1250cm -1 About the peak of typical sulfate groups, confirming successful sulfation of cannabis polysaccharide.
(2) Degree of substitution measurement
By BaCl 2 -determination of the degree of substitution of the sulfated cannabis polysaccharide by gelatin turbidity.
Principle of: by BaCl 2 -gelatin turbidity, liberating sulfate groups from the sugar complex by acid hydrolysis, producing barium sulfate, measured spectrophotometrically.
The steps are as follows: a series of sulfate radical standard solutions (0.5, 0.625, 0.75, 0.875, 1, 1.125, 1.25 and 1.375 mL) with the concentration of 1mg/mL are sucked into a test tube, pure water is added to 4mL, hydrochloric acid with the concentration of 0.18mol/L is added to 4mL, gelatin solution is added to 0.4mL, shaking is carried out for 10s, barium chloride-gelatin solution is added rapidly for 1.6mL, shaking is carried out for 2min, and the mixture is placed for 20min, and the absorbance is measured with the wavelength of 360 nm. At the same time, a blank test (1.6 mL of gelatin solution was used instead of the barium chloride-gelatin solution) was performed. The sulfate standard curve is shown in figure 2. The degree of substitution of the sulfated cannabis polysaccharides prepared in examples 1-9 was calculated as shown in FIG. 3.
(3) Scavenging DPPH free radical
The DPPH method is widely used for quantitatively determining antioxidant capacity of biological samples, phenols and foods. The dibenzopicryl radical (DPPH) is a very stable nitrogen-centered radical whose stability is mainly due to steric hindrance of the 3 benzene rings of resonance stabilization, so that unpaired electrons on the intervening nitrogen atom cannot exert their due electron pairing. The test sample scavenges such radicals, indicating that the sample has an effective concentration of reducing hydroxyl, alkyl or peroxy radicals and the like and interrupting lipid peroxidation chain reactions. DPPH has single electron, spectrum scanning shows that the reaction system has strong absorption at 517nm, and after the sample is added, the condition of decreasing absorbance at 517nm can reflect the antioxidant activity of the sample, and the capability of scavenging free radicals is calculated. Five samples used in this test were Vc, the cannabis polysaccharide prepared in example 1 (refined cannabis polysaccharide, HP), and the sulfated cannabis polysaccharides prepared in examples 1, 8 and 9 (HPS-1, HPS-8 and HPS-9), respectively, and were tested as follows:
1. a methanol solution of DPPH (wherein the concentration of the methanol solution of DPPH is 7.9mg/100 mL) was prepared and used.
2. Preparing a sample solution: 2mL of DPPH in methanol, 2mL of methanol, 1mL of samples (Vc, HP, HPS-1, HPS-8 and HPS-9, each at a concentration of 0.5, 1, 1.5, 2, 2.5, 3mg/mL, respectively) were added.
3. Preparing a blank solution: 2mL of DPPH in methanol, 2mL of methanol and 1mL of distilled water were added.
4. Sample background value (Ab): 2mL of methanol, 2mL of distilled water, and 1mL of samples (Vc, HP, HPS-1, HPS-8, and HPS-9, each at a concentration of 0.5, 1, 1.5, 2, 2.5, 3mg/mL, respectively) were added.
5. 2, 3 and 4, and standing for 30min in the dark after shaking.
6. OD was measured at 517 nm.
The clearance was calculated as follows:
wherein: a is that 0 Absorbance values measured when water was used instead of the sample; a is that i Absorbance values measured for different concentrations of the sample; a is that j Is the background absorbance.
The DPPH free radical scavenging activity results of FIG. 4 show that the sulfated cannabis polysaccharides of the present invention are all higher in biological activity than the non-sulfated cannabis polysaccharides.
(4) Research on cell activity of sulfated cannabis sativa polysaccharide for inhibiting hepatoma cell (HepG 2)
The samples of the sample group in this test were respectively example 1 (HPS-1), example 8 (HPS-8), example 9 (HPS-9), and cannabis polysaccharide (HP), and liver cancer cells (HepG 2) were treated with 10. Mu.L of aqueous sample solutions having different mass concentrations of 20, 50, 100, 200, 500, 1000. Mu.g/mL, and the antitumor activity of the sulfated cannabis polysaccharide was examined. The control group was prepared by replacing 10. Mu.L of the above-mentioned aqueous sample solution of different mass concentration with 10. Mu.L of water.
mu.L of the cell fluid was taken in a 96-well plate, and the cell density was about 1X 10 5 Individual/mL, placed at 37℃in 5% CO 2 After 4h incubation in an incubator, 10. Mu.L of samples of different mass concentrations were added and incubation was continued for 24h, 20. Mu.L of MTT reagent 5mg/mL was added and incubated for 4h, the supernatant was removed, 200. Mu.L of DMSO was added, and after 10min shaking, the absorbance was measured at 570nm using a microplate reader. The calculation formula of the inhibition rate of polysaccharide to liver cancer cells is as follows:
inhibition ratio = [ (Abs 0-Abs 1)/Abs 0] ×100%,
wherein Abs1 and Abs0 are absorbance values of the sample group and the control group, respectively.
The results in FIG. 5 show that sulfated cannabis polysaccharides inhibited liver cancer cell (HepG 2) activity higher than that of unsulfated cannabis polysaccharides.
Claims (14)
1. A sulfated cannabis polysaccharide having a molecular weight of between 1000Da and 10000Da and a degree of substitution of 0.13-0.27;
wherein the sulfated cannabis polysaccharide is prepared by the steps of:
(1) Dispersing cannabis polysaccharide into amide or pyridine solvents, and uniformly stirring to obtain a solution A;
(2) Slowly adding chlorosulfonic acid into DMF in ice water bath, stirring and mixing uniformly to obtain solution B, wherein the volume ratio of chlorosulfonic acid to DMF is 1:3-8;
(3) Mixing the solution A and the solution B for reaction to obtain a solution C;
(4) After the reaction is finished, the solution C is cooled to room temperature, absolute ethyl alcohol is slowly added for precipitation, the supernatant is removed by centrifugation, the precipitate is dissolved in water, the pH is adjusted to be neutral, dialysis and the trapped fluid is subjected to alcohol precipitation, centrifugation and precipitation drying, and the sulfated cannabis polysaccharide is obtained.
2. The sulfated cannabis polysaccharide of claim 1, having a molecular weight between 2000Da and 5000Da and a degree of substitution of 0.15-0.27.
3. The sulfated cannabis polysaccharide of claim 1, having a molecular weight between 3000Da and 5000Da and a degree of substitution of 0.27.
4. A sulfated cannabis polysaccharide as claimed in any of claims 1-3, wherein in step (1),
the mass volume ratio of the cannabis polysaccharide to the amide or pyridine solvent is 1:20-50;
the amide solvent is one or more of formamide, acetamide and propionamide;
the pyridine solvent is one or more of pyridine, picoline and ethylpyridine;
stirring temperature is 30-80 ℃;
the stirring time is 20-50 min.
5. A sulfated cannabis polysaccharide as claimed in any of claims 1-3, wherein in step (2),
the stirring time is 30-40 min.
6. A sulfated cannabis polysaccharide as claimed in any of claims 1-3, wherein in step (3),
The mass volume ratio of the cannabis polysaccharide in the solution A to the chlorosulfonic acid in the solution B is 1:1-10;
the reaction temperature is 30-80 ℃;
the reaction time is 2-8 h;
the reaction process adopts ultrasonic reinforcement to achieve the purpose of promoting sulfation synthesis, the ultrasonic probe is in a form of energy-accumulating type or dispersing type, and the ultrasonic power of ultrasonic waves acting on a solution system is 1-10W/L.
7. A sulfated cannabis polysaccharide as claimed in any of claims 1-3, wherein in step (4),
the dosage of the absolute ethyl alcohol is 4-6 times of the volume of the solution C;
the absolute ethyl alcohol is precooled to 0-4 ℃;
adjusting the pH to 7 by adopting a saturated NaOH aqueous solution;
the molecular weight of a dialysis membrane used for dialysis is 300Da-500Da;
precipitating the trapped fluid with ethanol until the concentration of ethanol is more than 80%;
the precipitation drying mode is freeze drying or vacuum pulsation drying.
8. A sulfated cannabis polysaccharide according to any of claims 1-3, wherein the cannabis polysaccharide is prepared by the following process:
(i) Extraction of cannabis sativa polysaccharide: extracting Cannabis sativa with water, filtering, concentrating the clear solution to obtain crude polysaccharide solution;
(ii) Purification of cannabis polysaccharide: precipitating crude hemp polysaccharide solution with ethanol, separating solid from liquid, collecting precipitate, dissolving precipitate with water, removing impurities, purifying eluate with DEAE cellulose column, eluting with salt solution, desalting eluate, and drying to obtain refined hemp polysaccharide.
9. The sulfated cannabis polysaccharide according to claim 8, wherein in step (i), the cannabis is cannabis sativa leaf material or the residue after CBD extraction from cannabis sativa leaves;
the mass volume ratio of the hemp to the water is 1:5-1:30;
adding enzyme in the extraction process to strengthen the extraction process, wherein the enzyme is one or more of cellulase, pectase, amylase and protease;
the concentration process adopts vacuum reduced pressure concentration or membrane concentration.
10. The sulfated cannabis polysaccharide according to claim 9, wherein in step (i) the concentration process is vacuum reduced pressure concentration or membrane concentration to a solids content of 10-30%.
11. The sulfated cannabis polysaccharide according to claim 8, wherein in step (ii) the volume ratio of crude cannabis polysaccharide solution to ethanol is 1:4-6;
the concentration of the ethanol is 80% -100%;
the impurity removing mode is macroporous resin impurity removing, wherein the macroporous resin is 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, HZ-818, HZ-816, HZ-835, AB-8, DM130, D312, HZ-20SS, XDA-8, XDA-6 and LSA-21, or the impurity removing mode is membrane filtering, wherein the membrane adopted by the membrane filtering is a micro-filtration membrane;
The salt solution is 0.1-0.5M NaCl solution;
the drying is freeze drying or vacuum pulse drying.
12. Use of the sulfated cannabis polysaccharide of any of claims 1-11 in the manufacture of a cosmetic.
13. Use of the sulfated cannabis polysaccharide of any of claims 1-11 in the manufacture of an anti-tumor medicament.
14. An antioxidant comprising the sulfated cannabis polysaccharide of any of claims 1-11, wherein the amount of sulfated cannabis polysaccharide used in the antioxidant is 0.1% to 99% by mass, the antioxidant further comprising a pharmaceutically acceptable carrier, excipient or diluent.
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| CN101684163A (en) * | 2008-09-24 | 2010-03-31 | 中国科学院上海药物研究所 | Polysaccharide of gastrodia elata sulphated derivative and preparation method and antitumor application thereof |
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| CN101684163A (en) * | 2008-09-24 | 2010-03-31 | 中国科学院上海药物研究所 | Polysaccharide of gastrodia elata sulphated derivative and preparation method and antitumor application thereof |
| CN110003351A (en) * | 2019-04-08 | 2019-07-12 | 南昌大学 | A kind of sulphation bean jelly grass polysaccharide and preparation method thereof |
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