Plant glycogen sulfate as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of organic synthesis of natural products, and discloses plant glycogen sulfate as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing a plant containing plant glycogen with water, and crushing to obtain a plant glycogen prefabricated solution; taking sodium amine trisulfonate as an esterifying agent, uniformly mixing the esterifying agent with the plant glycogen prefabricated liquid, performing esterification reaction and solid-liquid separation under an ultrasonic condition, and collecting a liquid component; concentrating the liquid component, precipitating in a precipitating agent, and carrying out solid-liquid separation to obtain a precipitate; and dialyzing the precipitate, and drying to obtain the plant glycogen sulfate. The preparation method disclosed by the invention is simple to operate, mild in reaction, relatively easy to control, green and environment-friendly, and the plant glycogen sulfate with high purity, high yield and relatively high substitution degree can be obtained without additionally adding a catalyst.

Description

Technical Field

[0001] The invention relates to the technical field of natural product organic synthesis, and in particular to a plant glycogen sulfate and a preparation method and application thereof. Background Art

[0002] Phytoglycogen is a soluble nano-branched polysaccharide connected by α-1,4 and α-1,6 glycosidic bonds. This nano-polysaccharide is widely present in the endosperm of sul mutants, such as corn, rice, and Kale. Phytoglycogen is similar to amylopectin in structure, but it has more and shorter branches, presents a spherical structure that is loose on the outside and tight on the inside, and has a large number of hydroxyl groups on the surface, so it has good water binding ability. However, due to its lack of other active groups, phytoglycogen performs poorly in other biological activities, such as antioxidant. Therefore, introducing other active groups into the structure of phytoglycogen is an effective way to improve its biological activity.

[0003] Polysaccharide sulfate refers to a polysaccharide derivative with a relatively complex chemical structure formed by replacing the hydroxyl group on the macromolecular chain with a sulfate group. As an important molecule in the body, polysaccharides have a variety of physiological activities, including but not limited to anti-oxidation, anti-aging, lowering blood pressure and lowering blood lipids. However, not all polysaccharides existing in nature have these physiological activities, which has prompted scientists to actively explore. Extensive studies have shown that changing the chemical structure of polysaccharides by molecular modification can effectively regulate their physical and chemical properties, and even induce new activities that are contrary to their natural activities. For example, after some polysaccharides are modified by sulfation, the resulting polysaccharide sulfates can show higher anti-HIV activity, providing potential possibilities for their application in antiviral drug research.

[0004] Sulfate modification is an important molecular modification method for modifying polysaccharides, including homogeneous and heterogeneous phases. The chemical agents used for traditional sulfated polysaccharide modification include sulfur trioxide, concentrated sulfuric acid, etc. Among them, although the sulfur trioxide method has mild reaction, simple operation and high yield, sulfur trioxide, as an anhydride of sulfuric acid, has a low degree of substitution, weak nucleophilic reaction performance and low degree of sulfate modification. Although the concentrated sulfuric acid method has the advantages of easy operation, few side reactions, stable reaction conditions, and low toxicity of chemical reagents, the product substitution degree and recovery rate are low, and the polysaccharide will be degraded and carbonized. In recent years, scientists have made some progress in improving the sulfate synthesis method. For example, the chlorosulfonic acid-pyridine method, although this method has the advantages of high yield and high degree of substitution, its operation is complicated and the reaction is violent, which is not easy to control. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the present invention provides a plant glycogen sulfate and a preparation method and application thereof. The present invention aims to provide a preparation method of a plant glycogen sulfate having antioxidant and tyrosinase inhibitory activities, so as to obtain a glycogen sulfate with high product yield and recovery rate and high degree of substitution.

[0006] The plant glycogen sulfate and its preparation method and application of the present invention are achieved through the following technical scheme:

[0007] The first object of the present invention is to provide a method for preparing phytoglycogen sulfate, comprising the following steps:

[0008] Step 1: Mix the plant containing phytoglycogen with water, and then crush it to obtain a phytoglycogen pre-liquid.

[0009] It should be noted that the preparation method of the present invention is applicable to a variety of different phytoglycogens. For example, the corresponding phytoglycogen-containing plants can be selected according to actual needs. For example, in some preferred embodiments of the present invention, the phytoglycogen-containing plants used are any one or more of corn, sorghum, rice and barley.

[0010] In order to ensure that the phytoglycogen in the phytoglycogen-containing plants is fully released so as to facilitate esterification thereof to obtain phytoglycogen sulfate, in some preferred embodiments of the present invention, a water-adding crushing method is adopted so that the phytoglycogen in the phytoglycogen-containing plants is fully released into the water.

[0011] In order to ensure that the phytoglycogen is fully released into the water, in some preferred embodiments of the present invention, the solid-to-liquid mass ratio of the phytoglycogen-containing plant to water is 1:5-7.

[0012] In order to further ensure that the phytoglycogen is fully released into the water, in some preferred embodiments of the present invention, a pulverizer is used for crushing, and the rotation speed of the pulverizer is 800 r / min to 1200 r / min, and the crushing time is 1 min to 3 min.

[0013] Step 2, using sodium trisulfonamide as an esterifying agent, mixing it with the plant glycogen pre-liquid, performing an esterification reaction, separating the solid and the liquid, collecting the liquid component; and concentrating the liquid component to obtain a concentrated solution.

[0014] It should be noted that, in order to improve the degree of substitution and yield of phytoglycogen sulfate, the present invention preferably uses sodium trisulfonate as an esterifying agent, and can directly achieve sulfation of phytoglycogen without adding a catalyst to obtain phytoglycogen sulfate. And in order to ensure that sodium trisulfonate can react with phytoglycogen in the phytoglycogen preformed liquid to obtain phytoglycogen sulfate, in some preferred embodiments of the present invention, the mass ratio of the sodium trisulfonate to the plant containing phytoglycogen in the phytoglycogen preformed liquid is 4 to 6:1. And in order to ensure that in the subsequent esterification reaction, sodium trisulfonate can be fully mixed with the components in the phytoglycogen preformed liquid in a solution environment, in some preferred embodiments of the present invention, when mixing the sodium trisulfonate with the phytoglycogen preformed liquid, the total solid-liquid mass ratio of the mixed solution is controlled to be 1:11 to 13.

[0015] In order to ensure that sodium ammonium trisulfonate can fully contact and react with the phytoglycogen in the phytoglycogen preformed liquid, in some preferred embodiments of the present invention, after sodium ammonium trisulfonate and the phytoglycogen preformed liquid are mixed, a water bath heating method is used under ultrasonic conditions to make sodium ammonium trisulfonate and phytoglycogen undergo sulfuric acid esterification. It should be emphasized that since the present invention directly uses the phytoglycogen preformed liquid obtained by crushing the plant containing phytoglycogen with water as the phytoglycogen source, during the reaction process, ultrasound can not only promote the efficient molecular collision between the phytoglycogen originally released by crushing and sodium ammonium trisulfonate in the solution, thereby promoting the esterification reaction to occur more fully; moreover, it can further promote more phytoglycogen in the plant residue to be released into the water under heating conditions, thereby increasing the phytoglycogen content in the solution; that is, the present invention realizes the combination of extraction and esterification by heating in a water bath under ultrasonic conditions, thereby reducing the operation steps and improving the preparation efficiency of phytoglycogen sulfate.

[0016] In order to ensure that the above technical effects can be achieved through ultrasound, in some preferred embodiments of the present invention, the frequency of the ultrasound used is 100W to 140W.

[0017] In order to ensure that the water bath heating can achieve the above technical effects, in some preferred embodiments of the present invention, the temperature of the esterification reaction is 45° C. to 55° C. And at this reaction temperature, in order to further ensure that sodium trisulfonamide can fully contact and react with the phytoglycogen in the phytoglycogen prefabricated solution, in some preferred embodiments of the present invention, the esterification reaction time is 3h to 5h.

[0018] It should also be noted that, in some preferred embodiments of the present invention, the sodium amine trisulfonate is prepared by the following steps:

[0019] Sodium bisulfite and sodium nitrite are used as raw materials, dissolved in water, and stirred for reaction to obtain sodium trisulfonate amine.

[0020] Among them, the present invention takes into account the factors such as the violent reaction and danger of traditional sulfate reagents such as sulfuric acid, and preferably uses sodium bisulfite and sodium nitrite as raw materials to achieve the purpose of mild reaction, economy and easy operation.

[0021] In order to ensure that sodium bisulfite and sodium nitrite can fully react to obtain sodium trisulfonate, in some preferred embodiments of the present invention, the two are first fully dissolved in water, so that the two are fully mixed, so as to react in the subsequent stirring reaction process to obtain sodium trisulfonate. In some preferred embodiments of the present invention, the amount ratio of sodium bisulfite, sodium nitrite and water is 4.5 mol to 5.5 mol: 1 mol: 50 mL.

[0022] In order to further ensure that sodium bisulfite and sodium nitrite can fully react in water to obtain sodium trisulfonate, in some preferred embodiments of the present invention, the stirring reaction temperature is 85° C. to 95° C. In order to further ensure that sodium bisulfite and sodium nitrite can fully react to obtain sodium trisulfonate at this temperature, in some preferred embodiments of the present invention, the stirring reaction time is 1.5 h to 2.5 h.

[0023] The present invention takes into account that the above stirring reaction is carried out in a solution environment, so that the obtained sodium trisulfonate amide exists in the form of an aqueous solution. In order to facilitate the subsequent esterification reaction of sodium trisulfonate amide in a solid form, in some preferred embodiments of the present invention, the aqueous solution containing sodium trisulfonate amide obtained by the stirring reaction is first frozen at -30°C to -10°C for 8h to 16h to freeze it into ice cubes, and then put it into a vacuum freeze dryer for freeze drying to obtain a solid sodium trisulfonate amide crude product. In some preferred embodiments of the present invention, the vacuum freeze drying temperature is -80°C to -40°C, and the time is 36h to 60h.

[0024] The present invention takes into account that since the present invention directly uses the phytoglycogen pre-liquid obtained by crushing the plant containing phytoglycogen with water as the phytoglycogen source, the product obtained after the esterification reaction contains plant residues in addition to the phytoglycogen sulfate. In order to prevent the plant residues from affecting the subsequent further purification of the phytoglycogen sulfate, in some preferred embodiments of the present invention, the product obtained after the esterification reaction is subjected to solid-liquid separation to remove the plant residues.

[0025] Considering the convenience of operation and the separation effect, in some preferred embodiments of the present invention, the present invention preferably uses centrifugation to achieve solid-liquid separation. In order to ensure that the plant residue can be precipitated by centrifugation, the obtained supernatant is the liquid component containing plant glycogen sulfate. In some preferred embodiments of the present invention, the centrifugation rate is 8000r / min to 12000r / min, and the centrifugation time is 15min to 25min.

[0026] The present invention takes into account that the obtained supernatant contains a large amount of solvent in addition to phytoglycogen sulfate. In order to facilitate the subsequent further purification treatment, in some preferred embodiments of the present invention, the obtained supernatant is first concentrated to remove most of the excess water solvent. And in order to ensure that the above technical effects can be achieved through concentration treatment, in some preferred embodiments of the present invention, a rotary evaporator is used to perform rotary evaporation to remove most of the excess water solvent in the supernatant, and the rotary evaporation is performed until the obtained concentrated solution is 1 / 11 to 1 / 9 of the volume of the original supernatant.

[0027] Step 3, subjecting the concentrated solution to precipitation treatment in a precipitant, separating the solid from the liquid, and obtaining a precipitate.

[0028] It should be noted that the present invention takes into account the fact that the concentrated solution contains salt and other small molecular impurities, so the concentrated solution obtained is first precipitated in a precipitant to achieve the purpose of impurity removal and purification. And in order to ensure that the precipitant used can achieve the above-mentioned technical effects, in some preferred embodiments of the present invention, ethanol is used as a precipitant to achieve the purpose of removing fat-soluble impurities, pigments, etc. through ethanol. In order to ensure that ethanol can achieve the above-mentioned technical effects, in some preferred embodiments of the present invention, the volume ratio of ethanol to the concentrated solution is 2 to 4:1, and the standing temperature of the precipitation treatment is 3°C to 5°C, and the standing time is 3h to 5h. In some more preferred embodiments of the present invention, the standing temperature of the precipitation treatment is 4°C.

[0029] The present invention takes into account that after the precipitation treatment, the product obtained still contains a precipitation component and a liquid component. At this time, the component containing phytoglycogen sulfate is transferred from the concentrated liquid to the precipitation component. Therefore, after the supernatant after precipitation is removed, the precipitate obtained is the component containing phytoglycogen sulfate.

[0030] Step 4, dialyzing and drying the precipitate to obtain the phytoglycogen sulfate.

[0031] It should be noted that the present invention takes into account the presence of macromolecular impurities such as proteins in the precipitate, so in some preferred embodiments of the present invention, the present invention first uses an acidic aqueous solution as a dispersant to disperse the precipitate in an acidic aqueous solution, so as to induce protein precipitation through an acidic solvent, thereby achieving protein removal. And the present invention takes into account the factors of molecular thermal motion, and the dispersed solution system is allowed to stand at 3 to 5 ° C to achieve the purpose of rapid impurity removal, and after standing, the plant glycogen sulfate will be transferred to the acidic aqueous solution, so after the product after standing is solid-liquid separation, the supernatant containing the plant glycogen sulfate is collected for subsequent further purification. In some preferred embodiments of the present invention, the acidic aqueous solution used is an acetic acid aqueous solution, and the ratio of the acetic acid aqueous solution to the precipitate is 4 to 6 mL: 1 mg, wherein the mass concentration of acetic acid in the acetic acid aqueous solution is 0.5% to 1.5%.

[0032] The present invention takes into account that the obtained supernatant may contain residual protein and inorganic salts, so the present invention also dialyzes the obtained supernatant to remove the residual protein and inorganic salts. In some preferred embodiments of the present invention, the specific dialysis operation is to put the supernatant into a dialysis bag with a molecular weight cutoff of 8 to 14 KDa, seal both ends, and dialyze with deionized water for 30 hours to 42 hours, during which the deionized water is replaced every 5 to 7 hours.

[0033] The present invention takes into account that there is still solvent in the dialysate obtained after dialysis, so in some preferred embodiments of the present invention, the dialysate is dried to remove excess solvent, and the solid powder obtained is plant glycogen sulfate. In order to ensure that the excess solvent can be removed by drying, in some preferred embodiments of the present invention, the dialysate is first frozen into ice cubes at -30°C to -10°C, and then placed in a vacuum freeze dryer for drying to achieve solid-liquid separation and obtain the product. And in some preferred embodiments of the present invention, the temperature of vacuum freeze drying is -80°C to -40°C, and the time is 36h to 60h.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention first mixes the plant containing phytoglycogen with water and then crushes it to achieve the preliminary extraction of phytoglycogen in the plant containing phytoglycogen, then mixes the crushed product with the esterifying agent sodium trisulfonamide and then performs an esterification reaction under ultrasonic conditions, so that the esterification can be directly performed after the phytoglycogen is extracted under ultrasonic conditions, and the extraction and esterification are combined, the operation steps are reduced, the preparation efficiency of phytoglycogen sulfate is improved, and the yield of phytoglycogen sulfate is greatly improved. Then, the ultrasonic product is subjected to solid-liquid separation, and after removing the plant residue, it is sequentially concentrated and precipitated, and then solid-liquid separation is performed, and then the precipitate is dialyzed and dried to achieve the purification of phytoglycogen sulfate and obtain high-purity phytoglycogen sulfate.

[0036] The present invention uses sodium trisulfonamide as an esterifying agent, and can directly realize the sulfation esterification of phytoglycogen without adding a catalyst, and the obtained phytoglycogen sulfate has both antioxidant and tyrosinase activity inhibition capabilities, so that it can be used in the preparation of antiviral drugs and skin care products.

[0037] The preparation method of the present invention is simple to operate, mild in reaction, relatively easy to control and environmentally friendly, and can obtain plant glycogen sulfate with high purity, high yield and high degree of substitution without the need to add an additional catalyst. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the Fourier infrared spectra of comparative example 1 and embodiment 1.

[0039] Figure 2 It is the hydrated particle size distribution of Comparative Example 1 and Example 1.

[0040] Figure 3 The test results of the surface potential charge and its distribution of comparative example 1 are shown in FIG.

[0041] Figure 4 The test results of the surface potential charge and its distribution in Example 1 are shown in FIG.

[0042] Figure 5 This is the transmission electron microscope image of Comparative Example 1.

[0043] Figure 6 This is a transmission electron microscope image of Example 1.

[0044] Figure 7 The hygroscopicity test results of Comparative Example 1, Example 1, glycerol and hyaluronic acid are shown in FIG.

[0045] Figure 8 The following are the test results of the moisturizing properties of Comparative Example 1, Example 1, glycerin and hyaluronic acid.

[0046] Fig. 9The DPPH free radical scavenging rate test results of Comparative Example 1, Example 1 and VC are shown in FIG.

[0047] Fig.10 The hydroxyl radical scavenging rate test results of Comparative Example 1, Example 1 and VC are shown in FIG.

[0048] Fig.11 The reducing power evaluation results of Comparative Example 1 and Example 1 are shown.

[0049] Fig.12 These are the test results of the ability of Comparative Example 1 and Example 1 to resist tyrosinase activity in melanoma cells.

[0050] Fig.13 The figure shows the test result of the cytotoxicity of Example 1 in 3T3 fibroblasts. DETAILED DESCRIPTION

[0051] The technical solutions in the embodiments of the present invention are described clearly and completely below. For ease of description, in the following embodiments, sodium bisulfite is represented by its chemical formula NaHSO3, and sodium nitrite is represented by its chemical formula NaNO2. It should be noted that the reagents and cells used in the following embodiments of the present invention were purchased from the market.

[0052] Example 1

[0053] This embodiment provides a plant glycogen sulfate, which is prepared by the following steps:

[0054] Step 1, preparation of esterifying agent sodium trisulfonate amine:

[0055] 1) According to the usage ratio of NaHSO3, NaNO2 and deionized water of 5mol:1mol:50mL, weigh the corresponding mass of NaHSO3, NaNO2 and the corresponding volume of deionized water respectively and set aside.

[0056] 2) The weighed NaHSO3 and NaNO2 are placed in deionized water, and then placed in a constant temperature magnetic stirring oil bath pot, and reacted at 90°C for 2 hours. After the reaction is completed, an aqueous solution containing sodium trisulfonate amine is obtained.

[0057] 3) The obtained aqueous solution containing sodium amide trisulfonate was directly frozen at -20°C for 12 hours, and then placed in a vacuum freeze dryer for vacuum freeze drying at -60°C for 48 hours to obtain a solid crude sodium amide trisulfonate product.

[0058] Step 2, preparing phytoglycogen pre-solution:

[0059] Sweet corn kernels were used as the plant containing phytoglycogen. The sweet corn kernels were mixed with deionized water at a solid-liquid mass ratio of 1:6, placed in a grinder, and crushed at a speed of 1000 r / min for 2 minutes to obtain a phytoglycogen pre-liquid.

[0060] Step 3, esterification reaction:

[0061] The crude product of sodium amine trisulfonate obtained in step 1 and the pre-made solution of phytoglycogen obtained in step 2 were mixed at a mass ratio of 5:1, and then reacted at a constant temperature of 50° C. for 4 hours under ultrasonic conditions with an ultrasonic power of 120 W.

[0062] Step 4, subsequent purification:

[0063] 1) The reaction solution of the esterification reaction was centrifuged at 4° C. and a centrifugal speed of 10,000 r / min for 20 min, the supernatant was collected, and the supernatant was concentrated to 1 / 10 of the original volume using a rotary evaporator to obtain a concentrated solution.

[0064] 2) Using ethanol as a precipitant, the concentrated solution was added to a corresponding volume of ethanol at a volume ratio of ethanol to concentrated solution of 3:1, and the mixture was allowed to stand at 4° C. for 4 h to achieve precipitation, and the precipitate was collected.

[0065] 3) According to the ratio of acetic acid aqueous solution to precipitate of 5 mL:1 mg, weigh the corresponding mass of precipitate and the corresponding volume of 1% acetic acid aqueous solution, and disperse the precipitate in the 1% acetic acid aqueous solution. Then, after standing at 4°C for 6 hours, centrifuge at a centrifugal speed of 10,000 r / min for 20 minutes, and collect the supernatant.

[0066] 4) The supernatant is dialyzed using a dialysis bag to remove residual protein and inorganic salts to obtain a dialysate.

[0067] 5) The dialysate obtained was frozen at -20°C for 12 hours, and then placed in a vacuum freeze dryer and vacuum freeze-dried at -60°C for 48 hours. The obtained white powder was phytoglycogen sulfate, which was recorded as PG-S.

[0068] Example 2

[0069] This embodiment provides a plant glycogen sulfate, which is prepared by the following steps:

[0070] Step 1, preparation of esterifying agent sodium trisulfonate amine:

[0071] 1) According to the usage ratio of NaHSO3, NaNO2 and deionized water of 4.5mol:1mol:50mL, weigh the corresponding mass of NaHSO3, NaNO2 and the corresponding volume of deionized water respectively and set aside.

[0072] 2) The weighed NaHSO3 and NaNO2 were placed in deionized water, and then placed in a constant temperature magnetic stirring oil bath pot, and reacted at 85°C for 3 hours. After the reaction was completed, an aqueous solution containing sodium trisulfonate was obtained.

[0073] 3) The obtained aqueous solution containing sodium amide trisulfonate was directly frozen at -10°C for 16 hours, and then placed in a vacuum freeze dryer for vacuum freeze drying at -40°C for 60 hours to obtain a solid crude sodium amide trisulfonate product.

[0074] Step 2, preparing phytoglycogen pre-solution:

[0075] Sweet corn kernels were used as the plant containing phytoglycogen. The sweet corn kernels were mixed with deionized water at a solid-liquid mass ratio of 1:6, placed in a grinder, and crushed at a speed of 800 r / min for 3 minutes to obtain a phytoglycogen pre-liquid.

[0076] Step 3, esterification reaction:

[0077] The crude product of sodium amine trisulfonate obtained in step 1 and the pre-made phytoglycogen solution obtained in step 2 were mixed at a mass ratio of 4:1, and then reacted at a constant temperature of 45° C. for 5 h under ultrasonic conditions with an ultrasonic power of 100 W.

[0078] Step 4, subsequent purification:

[0079] 1) The reaction solution of the esterification reaction was centrifuged at 8000 r / min at 3° C. for 25 min, the supernatant was collected, and the supernatant was concentrated to 1 / 9 of the original volume using a rotary evaporator to obtain a concentrated solution.

[0080] 2) Using ethanol as a precipitant, the concentrated solution was added to a corresponding volume of ethanol at a volume ratio of ethanol to the concentrated solution of 2:1, and the mixture was allowed to stand at 3° C. for 5 h to achieve precipitation, and the precipitate was collected.

[0081] 3) According to the ratio of acetic acid aqueous solution to precipitate of 4 mL:1 mg, weigh the corresponding mass of precipitate and the corresponding volume of 0.5% acetic acid aqueous solution, and disperse the precipitate in the 0.5% acetic acid aqueous solution, then let it stand at 3°C ​​for 4 hours, centrifuge at a centrifugal speed of 10000 r / min for 15 minutes, and collect the supernatant.

[0082] 4) The supernatant is dialyzed using a dialysis bag to remove residual protein and inorganic salts to obtain a dialysate.

[0083] 5) The dialysate obtained was frozen at -10°C for 16 hours, and then placed in a vacuum freeze dryer and vacuum freeze-dried at -40°C for 60 hours. The obtained white powder was phytoglycogen sulfate.

[0084] Example 3

[0085] This embodiment provides a plant glycogen sulfate, which is prepared by the following steps:

[0086] Step 1, preparation of esterifying agent sodium trisulfonate amine:

[0087] 1) According to the usage ratio of NaHSO3, NaNO2 and deionized water of 5.5mol:1mol:50mL, weigh the corresponding mass of NaHSO3, NaNO2 and the corresponding volume of deionized water respectively and set aside.

[0088] 2) The weighed NaHSO3 and NaNO2 were placed in deionized water, and then placed in a constant temperature magnetic stirring oil bath pot, and reacted at 95°C for 1 hour. After the reaction was completed, an aqueous solution containing sodium trisulfonate was obtained.

[0089] 3) The obtained aqueous solution containing sodium amide trisulfonate was directly frozen at -30°C for 8 hours, and then placed in a vacuum freeze dryer for vacuum freeze drying at -80°C for 36 hours to obtain a solid crude sodium amide trisulfonate product.

[0090] Step 2, preparing phytoglycogen pre-solution:

[0091] Sweet corn kernels were used as the plant containing phytoglycogen. The sweet corn kernels were mixed with deionized water at a solid-liquid mass ratio of 1:6, placed in a grinder, and crushed at a speed of 1200 r / min for 1 min to obtain a phytoglycogen pre-liquid.

[0092] Step 3, esterification reaction:

[0093] The crude product of sodium amine trisulfonate obtained in step 1 and the pre-made solution of phytoglycogen obtained in step 2 were mixed at a mass ratio of 6:1, and then reacted at a constant temperature of 55° C. for 3 h under ultrasonic conditions with an ultrasonic power of 140 W.

[0094] Step 4, subsequent purification:

[0095] 1) The reaction solution of the esterification reaction was centrifuged at 5° C. and a centrifugal speed of 12000 r / min for 25 min, the supernatant was collected, and the supernatant was concentrated to 1 / 11 of the original volume using a rotary evaporator to obtain a concentrated solution.

[0096] 2) Using ethanol as a precipitant, the concentrated solution was added to a corresponding volume of ethanol at a volume ratio of ethanol to concentrated solution of 4:1, and the mixture was allowed to stand at 8° C. for 3 h to achieve precipitation, and the precipitate was collected.

[0097] 3) According to the ratio of acetic acid aqueous solution to precipitate of 6 mL:1 mg, weigh the corresponding mass of precipitate and the corresponding volume of 1.5% acetic acid aqueous solution, and disperse the precipitate in the 1.5% acetic acid aqueous solution, then let it stand at 5°C for 8 hours, centrifuge at a centrifugal speed of 15000 r / min for 25 minutes, and collect the supernatant.

[0098] 4) The supernatant is dialyzed using a dialysis bag to remove residual protein and inorganic salts to obtain a dialysate.

[0099] 5) The dialysate obtained was frozen at -30°C for 8 hours, and then placed in a vacuum freeze dryer and vacuum freeze-dried at -80°C for 36 hours. The obtained white powder was phytoglycogen sulfate.

[0100] Comparative Example 1

[0101] This comparative example provides a phytoglycogen, which is prepared by the following steps:

[0102] Step 1, preparing phytoglycogen pre-solution:

[0103] Sweet corn kernels were used as the plant containing phytoglycogen. The sweet corn kernels were mixed with deionized water at a solid-liquid mass ratio of 1:6, placed in a grinder, and crushed at a speed of 1000 r / min for 2 minutes to obtain a phytoglycogen pre-liquid.

[0104] Step 2, ultrasonic treatment:

[0105] The phytoglycogen pre-liquid obtained in step 1 was ultrasonically treated at 50° C. for 4 h under ultrasonic conditions with an ultrasonic power of 120 W.

[0106] Step 3, subsequent purification:

[0107] 1) The ultrasonically treated reaction solution was centrifuged at 10000 r / min for 20 min at 4° C., the supernatant was collected, and the supernatant was concentrated to 1 / 10 of the original volume using a rotary evaporator to obtain a concentrated solution.

[0108] 2) Using ethanol as a precipitant, the concentrated solution was added to a corresponding volume of ethanol at a volume ratio of ethanol to concentrated solution of 3:1, and the mixture was allowed to stand at 4° C. for 4 h to achieve precipitation, and the precipitate was collected.

[0109] 3) According to the ratio of acetic acid aqueous solution to precipitate of 5:1, weigh the corresponding mass of precipitate, disperse it in 1% acetic acid aqueous solution, then let it stand at 4°C for 12 hours, centrifuge it at a centrifugal speed of 10000 r / min for 20 minutes, and collect the supernatant.

[0110] 4) The supernatant is dialyzed using a dialysis bag to remove residual protein and inorganic salts to obtain a dialysate.

[0111] 5) The dialysate obtained was freeze-dried at -20°C for 12 h, and then placed in a vacuum freeze dryer and vacuum freeze-dried at -60°C for 48 h. The obtained white powder was phytoglycogen, which was recorded as PG.

[0112] That is, the difference between this comparative example and Example 1 is only that:

[0113] This comparative example did not undergo esterification reaction.

[0114] Experimental Section

[0115] (I) Fourier transform infrared spectroscopy

[0116] The present invention takes the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and performs Fourier transform infrared spectroscopy tests on them respectively, and the test results are as follows: Figure 1 shown.

[0117] According to Figure 1 From the test results, it can be seen that PG at 3320cm -1 and 2926cm -1 The typical -OH stretching vibration absorption peak and the CH asymmetric stretching vibration absorption of the C6 position on the sugar ring appear. In addition, 1640cm -1 The peak at 1250cm is attributed to the C=O stretching vibration absorption of the C1 site of glucose. After PG is modified by sulfate esterification, the -OH absorption becomes narrower and other sites also shift significantly. -1 and 856cm -1 New absorption peaks appeared at , which were attributed to S=O and COS bond absorption peaks, and these characteristics were the characteristic absorption of sulfonic acid group. Therefore, the above results confirmed the successful synthesis of phytoglycogen sulfate.

[0118] (II) Determination of hydrated particle size distribution

[0119] The present invention takes the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and respectively measures the hydrated particle size distribution thereof, and the test results are as follows: Figure 2 shown.

[0120] The testing method is: dissolve PG and PG-S respectively in deionized water to obtain a 1% PG dilute solution and a 1% PG-S dilute solution, and use dynamic light scattering to determine the particle size and distribution of PG and PG-S.

[0121] According to Figure 1From the test results, it can be seen that PG has a smaller particle size, with an average size of about 68nm, and good dispersibility; while the average particle size of PG-S is slightly larger, about 85nm. This is because the sulfonic acid group can bind more water molecules than the hydroxyl group, resulting in a thicker hydration layer and larger particle size.

[0122] (III) Determination of average surface charge and its distribution

[0123] The present invention uses a Zeta potential instrument to test the surface potential charge and distribution of the PG of Comparative Example 1 and the PG-S of Example 1, and the test results are as follows: Figure 3 and Figure 4 shown.

[0124] in, Figure 3 The test results of the surface potential charge and its distribution of PG in comparative example 1 are as follows: Figure 4 The test results of the surface potential charge and its distribution of PG-S in Example 1 are shown in Table 1. Figure 3 and Figure 4 It can be seen that the surface charge of pure PG is -6.41±0.7mV, which is mainly due to the presence of a large number of hydroxyl groups on the surface of PG; after modification with sulfonic acid groups, the surface charge of PG-S is reduced to -17.93±1.01mV, which is because the sulfonic acid groups carry two molecules of negative charge. This result further confirms that PG sulfate was successfully prepared.

[0125] (IV) Transmission electron microscopy measurement

[0126] The present invention takes the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and performs transmission electron microscopy test on them respectively, and the test results are as follows: Figure 5 and Figure 6 shown.

[0127] in, Figure 5 is the transmission electron microscope image of PG of Comparative Example 1, Figure 6 This is a transmission electron microscope image of PG-S of Example 1. By comparison Figure 5 and Figure 6 It can be seen that PG and PG-S have a spherical structure as a whole, and the size is slightly smaller than the hydrated size. This is because the samples were dried to remove the hydration layer. Further observation revealed that there were some larger particles in both groups of samples. This is because PG is not uniformly synthesized in vivo, and plant polysaccharides with a certain molecular weight range are obtained.

[0128] (V) Determination of moisture absorption and moisture retention properties

[0129] 1) Determination of hygroscopicity

[0130] The present invention takes PG of comparative example 1 and PG-S of embodiment 1 as examples, and takes glycerin and hyaluronic acid, two common skin care additives, as control groups, and tests their hygroscopic properties respectively, and the test results are as follows: Figure 7 shown.

[0131] Test method:

[0132] Place a beaker filled with saturated sodium carbonate solution in a desiccator to form an experimental environment with a relative humidity of RH = 43%. Weigh appropriate amounts of dry PG, PG-S, glycerin and hyaluronic acid in weighing bottles and place them in the desiccator. Measure and record the sample mass at regular intervals. Repeat the experiment three times, take the average value of the data, and determine the moisture absorption rate of each sample. The moisture absorption rate is used as an indicator to evaluate the moisture absorption performance.

[0133] according to Figure 7 From the test results, it can be seen that glycerol has strong hygroscopicity, with a moisture absorption rate of more than 100% in 48 hours, while hyaluronic acid has relatively weak hygroscopicity, with a moisture absorption rate of only 45.56% in 48 hours; the moisture absorption rates of the two PG samples gradually increase with the extension of treatment time, among which PG-S is significantly higher than PG, which is because the sulfonic acid group can bind to more water molecules.

[0134] 2) Determination of moisturizing properties

[0135] The present invention takes PG of comparative example 1 and PG-S of embodiment 1 as examples, and takes glycerin and hyaluronic acid, two common skin care additives, as control groups, to test their moisturizing properties respectively, and the test results are as follows: Figure 8 shown.

[0136] Test method:

[0137] Add appropriate amount of pure water to weighing bottles containing equal amounts of PG, PG-S, glycerin and hyaluronic acid, respectively, place them in a desiccator, measure and record the sample mass at regular intervals, conduct three parallel experiments, calculate the moisturizing rate of each sample, and use the moisturizing rate as an indicator to evaluate the moisturizing performance.

[0138] according to Figure 8 The test results show that several samples have good moisturizing properties after 48 hours of treatment, suggesting that PG and its derivatives have application potential for commercial transformation.

[0139] (VI) Determination of DPPH free radical scavenging rate and hydroxyl free radical scavenging rate

[0140] 1) Determination of DPPH free radical scavenging rate

[0141] The present invention takes PG of comparative example 1 and PG-S of embodiment 1 as examples, and takes VC as a control group, and tests the DPPH free radical scavenging rate thereof respectively, and the test results are as follows: Fig. 9 shown.

[0142] The test method is:

[0143] A series of sample solutions with concentration gradients of PG and PG-S were prepared with deionized water, and the concentrations of the sample solutions were 0.5 mg / mL, 1.0 mg / mL and 5.0 mg / mL, respectively. 1.0 mL of 0.2 mmol / L DPPH ethanol solution was taken into a test tube, and different concentrations of the sample solutions were added, respectively, and the solution was shaken and reacted at room temperature in the dark for 30 minutes. Then, the solution was transferred to a 96-well cell culture plate and placed in a spectrophotometer. The absorbance was measured at 517 nm. The experiment was repeated three times in parallel. A series of VC solutions with concentration gradients of 0.5 mg / mL, 1.0 mg / mL and 5.0 mg / mL were prepared with deionized water to replace the sample solution as a positive control group, and distilled water was used to replace the sample solution as a blank control group to measure the absorbance at 517 nm. The experiment was repeated three times in parallel to calculate the DPPH free radical scavenging rate of each sample.

[0144] according to Fig. 9 From the test results, it can be seen that the scavenging efficiency of PG and PG-S on DPPH free radicals gradually increases with the increase of sample concentration, showing a typical dose-dependence; further comparison shows that the ability of PG-S to scavenge DPPH free radicals is better than that of pure PG.

[0145] 2) Determination of hydroxyl radical scavenging rate

[0146] The present invention takes PG of comparative example 1 and PG-S of embodiment 1 as examples, and takes VC as a control group, and tests the hydroxyl radical scavenging rates thereof respectively, and the test results are as follows: Fig.10 shown.

[0147] The test method is:

[0148] A series of sample solutions with different concentration gradients were diluted according to the above operation. 1.0 mL of the sample solution was added with 2.0 mL of 9.0 mmol / L FeSO4 solution and 2.0 mL of 9.0 mmol / L salicylic acid solution in sequence, and then 2.0 mL of 8.8 mmol / L H2O2 was added to start the reaction. After 60 min in a water bath at 37°C, the plates were transferred to a 96-well cell culture plate and placed in a spectrophotometer to measure the absorbance at 510 nm. The plates were measured three times in parallel and post-processed in the same way to calculate the hydroxyl radical scavenging rate of each sample.

[0149] according to Fig.10From the test results, it can be seen that the scavenging efficiency of PG and PG-S for -OH free radicals gradually increases with the increase of sample concentration, showing a typical dose-dependence; further comparison shows that the ability of PG-S to scavenge -OH free radicals is better than that of pure PG.

[0150] And by comparison Fig. 9 and Fig.10 It can be found that the effects of PG and PG-S in scavenging DPPH free radicals are higher than those in scavenging ·OH free radicals, indicating that PG hydroxyl derivatives are more sensitive to organic free radicals.

[0151] The present invention also evaluates the reducing power of PG and PG-S, and the evaluation results are summarized as follows: Fig.11 As shown, it can be seen that the reducing power of pure PG is weak, while the reducing power of PG after sulfate esterification is significantly enhanced.

[0152] (VII) Determination of anti-tyrosinase activity in melanoma cells

[0153] The present invention takes the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and tests the tyrosinase activity of the PG and PG-S against melanoma cells respectively. The test results are as follows: Fig.12 shown.

[0154] The test method is:

[0155] B16 cells in the logarithmic growth phase were taken at a cell density of 5×10 4 Inoculate into a 96-well plate, incubate for 24 hours, add appropriate amounts of PG and PG-S, and then add an equal amount of arbutin solution. After incubation for 48 hours, remove the supernatant, rinse twice with PBS solution, add 50 μL of 1% Triton X-100, freeze at -80°C for 1 hour, add 100 μL of 0.1% L-DOPA solution, incubate at 37°C for 2 hours, measure the absorbance at a wavelength of 490 nm, and calculate the tyrosinase activity of the cells.

[0156] according to Fig.12 The test results show that PG and PG-S can inhibit tyrosinase activity in a dose-dependent manner; further comparison revealed that PG-S has a stronger ability to inhibit tyrosinase than pure PG. This may be because the sulfonic acid group is more easily bound to the enzyme active site, interfering with enzyme activity.

[0157] (VIII) Determination of PG-S cytotoxicity in 3T3 fibroblasts

[0158] The present invention takes the PG-S of Example 1 as an example, and tests its cytotoxicity in 3T3 fibroblasts, and the test results are as follows: Fig.13 shown.

[0159] The test method is:

[0160] PG-S were sterilized by ultraviolet irradiation, and 3T3 cells in the logarithmic growth phase were taken at a cell density of 5×10 4 Inoculate into 96-well plates and culture in a cell culture incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere to the wall and grow. Prepare PG-S solutions with concentration gradients of 31.25μg / mL, 62.5μg / mL, 125μg / mL, 250μg / mL, 500μg / mL and 1000μg / mL. Aspirate the old culture medium and replace with new culture medium, 180μL per well, and then add 20μL of PG-S solutions of different concentrations. Set up blank controls and negative controls and culture in an incubator for 24 hours. After 24 hours of culture, use the MTT method to measure cell viability.

[0161] according to Fig.13 The test results show that PG-S is non-toxic to cells at lower concentrations and can promote cell proliferation; when the sample concentration is increased to 1000μg / mL, PG-S has slight cytotoxicity, suggesting that attention should be paid to the dosage when using PG-S.

[0162] Obviously, the above embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing phytoglycogen sulfate, characterized in that: The following steps are involved: The plant containing phytoglycogen is mixed with water and crushed to obtain a phytoglycogen pre-liquid; wherein the plant containing phytoglycogen is any one or more of corn, sorghum, rice and barley; Using sodium trisulfonamide as an esterifying agent, and mixing it with the plant glycogen pre-made solution to obtain a mixed solution; Under ultrasonic conditions, the mixed solution is subjected to an esterification reaction, solid-liquid separation is performed, and a liquid component is collected; and the liquid component is concentrated to obtain a concentrated solution; The concentrated solution is subjected to precipitation treatment in a precipitant to separate the solid from the liquid to obtain a precipitate; The precipitate is dialyzed and dried to obtain the phytoglycogen sulfate.

2. The preparation method according to claim 1, characterized in that The mass ratio of the sodium trisulfonate amine to the plant containing phytoglycogen in the phytoglycogen prefabricated liquid is 4 to 6:

1.

3. The preparation method according to claim 1, characterized in that: When the sodium ammonium trisulfonate and the phytoglycogen prefabricated solution are mixed, the total solid-liquid mass ratio of the mixed solution is controlled to be 1:11-13.

4. The preparation method according to claim 1, characterized in that: During the esterification reaction, the ultrasonic power is 100W-140W, and the temperature of the esterification reaction is 45°C-55°C.

5. The preparation method according to claim 1, characterized in that: The sodium amine trisulfonate is prepared by the following steps: Sodium bisulfite and sodium nitrite are used as raw materials, dissolved in water, and stirred for reaction to obtain sodium trisulfonate amine.

6. The preparation method according to claim 5, characterized in that: The dosage ratio of the sodium bisulfite, sodium nitrite and water is 4.5 mol to 5.5 mol: 1 mol: 50 mL.

7. The preparation method according to claim 5, characterized in that: The temperature of the stirring reaction is 85°C to 95°C.

8. The preparation method according to claim 1, characterized in that: The volume of the concentrated solution is 1 / 11 to 1 / 9 of the volume of the liquid component.

9. A phytoglycogen sulfate prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The phytoglycogen sulfate is water-soluble nano-scale phytoglycogen sulfate.

10. Use of the phytoglycogen sulfate according to claim 9 in the preparation of antiviral drugs and skin care products.

Images