Plant glycogen sulfate and its preparation method and application

Abstract

The present invention belongs to the technical field of organic synthesis of natural products and discloses a phytoglycogen sulfate, its preparation method, and application. The preparation method comprises: mixing phytoglycogen-containing plants with water and crushing the mixture to obtain a phytoglycogen preformed liquid; using sodium trisulfonamide as an esterifying agent, mixing the mixture with the phytoglycogen preformed liquid, conducting an esterification reaction under ultrasonic conditions, performing solid-liquid separation, and collecting the liquid component; concentrating the liquid component and precipitating it in a precipitant, performing solid-liquid separation, and obtaining a precipitate; dialyzing the precipitate and drying it to obtain the phytoglycogen sulfate. The preparation method of the present invention is simple to operate, has a mild reaction, is relatively easy to control, and is environmentally friendly. Furthermore, high-purity, high-yield, and high-degree-of-substitution phytoglycogen sulfate can be obtained without the addition of an additional catalyst.

Description

Technical Field

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

[0002] Phytoglycogen is a soluble, nanostructured branched polysaccharide linked by α-1,4 and α-1,6 glycosidic bonds. This nanopolysaccharide is widely found in the endosperm of plants such as maize, rice, and Kale. Structurally, phytoglycogen is similar to amylopectin, but it has more numerous and shorter branches, resulting in a globular structure that is loose on the outside and tight on the inside. Its surface possesses numerous hydroxyl groups, giving it excellent water-binding capacity. However, due to its lack of other active groups, phytoglycogen exhibits poor performance in other biological activities, such as antioxidant activity. Therefore, introducing additional active groups into the phytoglycogen structure is an effective approach to enhancing its bioactivity.

[0003] Polysaccharide sulfate refers to a polysaccharide derivative with a relatively complex chemical structure, formed by replacing the hydroxyl groups on the macromolecular chain with sulfate groups. As important molecules in organisms, polysaccharides have various physiological activities, including but not limited to antioxidant, anti-aging, blood pressure lowering, and lipid-lowering functions. However, not all polysaccharides found in nature possess these physiological activities, which has prompted scientists to actively explore. Extensive research has shown that by changing the chemical structure of polysaccharides through molecular modification, their physical and chemical properties can be effectively controlled, and even new activities that are contrary to their natural activities can be induced. For example, after some polysaccharides are modified by sulfation, the resulting polysaccharide sulfates can exhibit high anti-HIV activity, providing potential possibilities for their application in antiviral drug research.

[0004] Sulfation modification, an important molecular modification method for polysaccharides, encompasses both homogeneous and heterogeneous phases. Traditional chemical agents used for sulfated polysaccharide modification include sulfur trioxide and concentrated sulfuric acid. While the sulfur trioxide method offers mild reaction, simple operation, and high yield, sulfur trioxide, as an anhydride of sulfuric acid, has a low degree of substitution, weak nucleophilic reactivity, and a low degree of sulfated modification. Concentrated sulfuric acid, while offering advantages such as ease of operation, few side reactions, stable reaction conditions, and low chemical reagent toxicity, suffers from low product substitution and recovery rates, and can lead to polysaccharide degradation and carbonization. In recent years, scientists have made some progress in improving sulfated synthesis methods. For example, the chlorosulfonic acid-pyridine method offers high yields and a high degree of substitution, but its operation is complex, the reaction is violent, and it is difficult to control. Summary of the Invention

[0005] To address the deficiencies in the prior art, the present invention provides a plant glycogen sulfate, a preparation method thereof, and applications thereof. The present invention aims to provide a method for preparing a plant glycogen sulfate having antioxidant and tyrosinase inhibitory activities, thereby obtaining a glycogen sulfate with high product yield and recovery rate and a high degree of substitution.

[0006] The plant glycogen sulfate and its preparation method and application are achieved through the following technical solutions:

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

[0008] Step 1: mixing plants containing phytoglycogen with water and crushing the mixture 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, 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 its esterification to obtain phytoglycogen sulfate, in some preferred embodiments of the present invention, a water-adding crushing method is adopted to fully release the phytoglycogen in the phytoglycogen-containing plants into the water.

[0011] In order to ensure that phytoglycogen is fully released into 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 crusher is used for crushing, and the rotation speed of the crusher 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 phytoglycogen pre-liquid, performing an esterification reaction, performing solid-liquid separation, collecting the liquid component; and concentrating the liquid component to obtain a concentrated solution.

[0014] It should be noted that, in order to increase the degree of substitution and yield of phytoglycogen sulfate, the present invention preferably uses sodium triammonium sulfonate as an esterifying agent. This allows direct sulfation of phytoglycogen without the addition of a catalyst to obtain phytoglycogen sulfate. Furthermore, to ensure that sodium triammonium sulfonate reacts with the phytoglycogen in the phytoglycogen preformed solution to produce phytoglycogen sulfate, in some preferred embodiments of the present invention, the mass ratio of sodium triammonium sulfonate to the phytoglycogen-containing plant in the phytoglycogen preformed solution is 4 to 6:1. Furthermore, to ensure that sodium triammonium sulfonate is thoroughly mixed with the components of the phytoglycogen preformed solution in the subsequent esterification reaction, in some preferred embodiments of the present invention, when mixing sodium triammonium sulfonate with the phytoglycogen preformed solution, the total solid-to-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 is mixed with the phytoglycogen preformed liquid, a water bath heating method under ultrasonic conditions is adopted to allow sodium ammonium trisulfonate and the phytoglycogen to undergo sulfate 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 a more complete esterification reaction; it can also further promote the release of more phytoglycogen in the plant residue into the water under heating conditions, thereby increasing the phytoglycogen content in the solution; that is, the present invention achieves the combination of extraction and esterification by heating the plant 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] To ensure that the above-mentioned technical effects can be achieved by water bath heating, in some preferred embodiments of the present invention, the temperature of the esterification reaction is 45° C. to 55° C. Moreover, at this reaction temperature, to further ensure that sodium trisulfonamide can fully contact and react with the phytoglycogen in the phytoglycogen preformed solution, in some preferred embodiments of the present invention, the esterification reaction time is 3 h to 5 h.

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

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

[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 and mixed thoroughly to facilitate the subsequent reaction during the stirring reaction to obtain sodium trisulfonate. In some preferred embodiments of the present invention, the 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 to obtain sodium trisulfonate by stirring in water, 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-mentioned stirring reaction is carried out in a solution environment, so that the obtained sodium trisulfonate exists in the form of an aqueous solution. In order to facilitate the subsequent esterification reaction of sodium trisulfonate in a solid form, in some preferred embodiments of the present invention, the aqueous solution containing sodium trisulfonate 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 placed in a vacuum freeze dryer for freeze drying to obtain a solid sodium trisulfonate 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 operational convenience and separation effect, in some preferred embodiments of the present invention, centrifugation is preferably used to achieve solid-liquid separation. Furthermore, to ensure that the plant residue can be precipitated by centrifugation, the supernatant obtained is the liquid component containing phytoglycogen sulfate. In some preferred embodiments of the present invention, the centrifugation speed is 8000 rpm to 12000 rpm, and the centrifugation time is 15 min to 25 min.

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

[0027] Step 3: subjecting the concentrated liquid to precipitation treatment in a precipitant, performing solid-liquid separation, and obtaining a precipitate.

[0028] It should be noted that the present invention takes into account the fact that the concentrated liquid contains salts and other small molecular impurities, so the concentrated liquid 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 liquid 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 precipitation treatment, the product obtained still contains a precipitated component and a liquid component. At this time, the component containing phytoglycogen sulfate is transferred from the concentrated liquid to the precipitated component. Therefore, after removing the supernatant after precipitation, 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. Therefore, 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 the acidic aqueous solution, so as to induce protein precipitation through the acidic solvent, thereby achieving protein removal. In addition, the present invention takes into account the factors of molecular thermal motion and allows the dispersed solution system to be allowed to stand at 3 to 5 ° C to achieve the purpose of rapid impurity removal. After standing, the phytoglycogen sulfate will be transferred to the acidic aqueous solution. Therefore, after the product after standing is solid-liquid separation, the supernatant containing the phytoglycogen 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 also dialyzes the supernatant to remove residual protein and inorganic salts, taking into account the possibility that the supernatant may contain residual protein and inorganic salts. In some preferred embodiments of the present invention, the dialysis procedure involves placing the supernatant into a dialysis bag with a molecular weight cutoff of 8 to 14 kDa, sealing both ends, and dialysis using deionized water for 30 to 42 hours, with the deionized water replaced every 5 to 7 hours.

[0033] The present invention takes into account the presence of solvent in the dialysate obtained after dialysis. Therefore, in some preferred embodiments of the present invention, the dialysate is dried to remove excess solvent to obtain a solid powder, which 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. 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.

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

[0035] The present invention first mixes phytoglycogen-containing plants with water and then crushes them to achieve preliminary extraction of phytoglycogen from the phytoglycogen-containing plants. The crushed products are then mixed with an esterifying agent, sodium trisulfonamide, and then subjected to an esterification reaction under ultrasonic conditions. This allows esterification to be directly performed after the phytoglycogen is extracted under ultrasonic conditions, thereby combining extraction and esterification, reducing the number of operating steps, improving the preparation efficiency of phytoglycogen sulfate, and thereby significantly improving the yield of phytoglycogen sulfate. The ultrasonic product is then subjected to solid-liquid separation, after removing plant residues, followed by concentration, precipitation, and solid-liquid separation. The precipitate is then dialyzed and dried to purify the phytoglycogen sulfate, thereby obtaining high-purity phytoglycogen sulfate.

[0036] The present invention uses sodium trisulfonamide as an esterifying agent, and can directly achieve sulfation of phytoglycogen without adding a catalyst. The obtained phytoglycogen sulfate has both antioxidant and tyrosinase activity inhibition capabilities, making it possible to use it 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, easy to control, and environmentally friendly. Furthermore, high-purity, high-yield, and high-degree-of-substitution phytoglycogen sulfate can be obtained without the need for adding an additional catalyst. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The following are Fourier infrared spectra of Comparative Example 1 and Example 1.

[0039] Figure 2 The hydrated particle size distribution of Comparative Example 1 and Example 1.

[0040] Figure 3 These are the test results of the surface potential charge and its distribution in Comparative Example 1.

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

[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 following are the test results of the hygroscopic properties of Comparative Example 1, Example 1, glycerol and hyaluronic acid.

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

[0046] Figure 9The figures are the test results of DPPH radical scavenging rate of Comparative Example 1, Example 1 and VC.

[0047] Figure 10 The figures are the test results of hydroxyl radical scavenging rates of Comparative Example 1, Example 1 and VC.

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

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

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

[0051] The technical solutions in the embodiments of the present invention are described below in a clear and complete manner. 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 commercially.

[0052] Example 1

[0053] This embodiment provides a phytoglycogen 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 5 mol:1 mol:50 mL, weigh the corresponding mass of NaHSO3, NaNO2 and the corresponding volume of deionized water respectively and set aside.

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

[0057] 3) The obtained aqueous solution containing sodium trisulfonate was directly 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 to obtain a solid crude product of sodium trisulfonate.

[0058] Step 2, prepare 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 sodium trisulfonate amine product obtained in step 1 and the plant glycogen pre-solution 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 h 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 10,000 rpm 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.

[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) Weigh the corresponding mass of precipitate and the corresponding volume of 1% acetic acid solution at a ratio of 5 mL of acetic acid to precipitate. Disperse the precipitate in the 1% acetic acid solution. After standing at 4°C for 6 h, centrifuge at 10,000 rpm for 20 min and collect the supernatant.

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

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

[0068] Example 2

[0069] This embodiment provides a phytoglycogen 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 4.5 mol:1 mol:50 mL, weigh the corresponding mass of NaHSO3, NaNO2 and the corresponding volume of deionized water respectively and set aside.

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

[0073] 3) The obtained aqueous solution containing sodium trisulfonate was directly 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 to obtain a solid crude product of sodium trisulfonate.

[0074] Step 2, prepare 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 sodium trisulfonate amine product obtained in step 1 and the plant glycogen prefabricated solution obtained in step 2 were mixed in 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 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) Weigh the corresponding mass of precipitate and the corresponding volume of 0.5% acetic acid solution at a ratio of 4 mL of acetic acid to precipitate. Disperse the precipitate in the 0.5% acetic acid solution. After standing at 3°C ​​for 4 h, centrifuge at 10,000 rpm for 15 min and collect the supernatant.

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

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

[0084] Example 3

[0085] This embodiment provides a phytoglycogen 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 5.5 mol:1 mol:50 mL, weigh the corresponding mass of NaHSO3, NaNO2 and the corresponding volume of deionized water respectively and set aside.

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

[0089] 3) The obtained aqueous solution containing sodium trisulfonate was directly 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 to obtain a solid crude product of sodium trisulfonate.

[0090] Step 2, prepare 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 sodium trisulfonate amine product obtained in step 1 and the plant glycogen pre-solution 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 12000 r / min at 5° C. 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) Weigh the corresponding mass of precipitate and the corresponding volume of 1.5% acetic acid solution at a ratio of 6 mL of acetic acid to precipitate. Disperse the precipitate in the 1.5% acetic acid solution. After standing at 5°C for 8 h, centrifuge at 15,000 rpm for 25 min and collect the supernatant.

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

[0099] 5) The dialysate obtained was frozen at -30°C for 8 hours, then placed in a vacuum freeze dryer and vacuum freeze-dried at -80°C for 36 hours. The resulting 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, prepare 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-solution 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 10,000 rpm 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) Weigh the corresponding amount of precipitate in a 1% acetic acid solution at a ratio of 5:1 acetic acid aqueous solution to precipitate. Disperse the solution in 1% acetic acid aqueous solution. Allow the solution to stand at 4°C for 12 h, then centrifuge at 10,000 rpm for 20 min, and collect the supernatant.

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

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

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

[0113] In this comparative example, no esterification reaction was performed.

[0114] Experimental part

[0115] (1) 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 test on them respectively, and the test results are as follows: Figure 1 shown.

[0117] According to Figure 1 From the test results, we can see that PG is at 3320cm -1 and 2926cm -1 The typical -OH stretching vibration absorption peak and the CH asymmetric stretching vibration absorption of the C6 site on the sugar ring appear. In addition, 1640 cm -1 The peak at 1250cm is attributed to the C=O stretching vibration absorption of glucose C1 site. 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 the S=O and COS bond absorption peaks, respectively. These characteristics are the characteristic absorption of sulfonic acid groups. Therefore, the above results confirmed the successful synthesis of phytoglycogen sulfate.

[0118] (2) 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 test method is as follows: PG and PG-S are dissolved in deionized water respectively to obtain a 1% PG dilute solution and a 1% PG-S dilute solution, and the particle size and distribution of PG and PG-S are determined by dynamic light scattering.

[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 dispersion; 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] (3) Determination of average surface charge and its distribution

[0123] The present invention uses a Zeta potential meter to test the surface potential charge and its 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 distribution of PG-S in Example 1 are shown in FIG. Figure 3 and Figure 4 The surface charge of pure PG is -6.41±0.7 mV, primarily due to the presence of numerous hydroxyl groups on the PG surface. However, after modification with sulfonic acid groups, the surface charge of PG-S decreases to -17.93±1.01 mV, due to the presence of two negative charges on the sulfonic acid groups. This result further confirms the successful preparation of PG sulfate.

[0125] (4) 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. Figure 5 and Figure 6 As can be seen, PG and PG-S exhibit an overall spherical structure, slightly smaller than their hydrated size. This is due to the removal of the hydration layer during sample drying. Further observation revealed the presence of some larger particles in both sample groups. This is because PG is synthesized non-uniformly in vivo, resulting in plant polysaccharides with a range of molecular weights.

[0128] (5) Determination of moisture absorption and moisture retention properties

[0129] 1) Determination of hygroscopicity

[0130] The present invention takes the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and takes two common skin care additives glycerin and hyaluronic acid 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 create an experimental environment with a relative humidity of RH = 43%. Weigh appropriate amounts of dry PG, PG-S, glycerin and hyaluronic acid into 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 The test results show 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 the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and takes two common skin care additives glycerin and hyaluronic acid as control groups, and tests 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, and place them in a desiccator. Measure and record the sample mass at regular intervals. Perform the experiment three times in parallel, and calculate the moisturizing rate of each sample. The moisturizing rate is used 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 the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and takes VC as the control group, and tests the DPPH free radical scavenging rates thereof respectively, and the test results are as follows: Figure 9 shown.

[0142] The test method is:

[0143] A series of test solutions with gradient concentrations 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 added with different concentrations of the test solutions. The solution was shaken and reacted in the dark for 30 minutes at room temperature. The solution was then 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. A series of VC solutions with gradient concentrations of 0.5 mg / mL, 1.0 mg / mL and 5.0 mg / mL were prepared with deionized water to replace the test solution as a positive control group. Distilled water was used instead of the test solution as a blank control group. The absorbance was measured at 517 nm. The experiment was repeated three times to calculate the DPPH free radical scavenging rate of each sample.

[0144] according to Figure 9 The test results show 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 the PG of comparative example 1 and the PG-S of embodiment 1 as examples, and takes VC as the control group, and tests the hydroxyl radical scavenging rates thereof respectively, and the test results are as follows: Figure 10 shown.

[0147] The test method is:

[0148] A series of sample solutions with different concentration gradients were diluted according to the same operation as above. 1.0 mL of the sample solution to be tested 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. Then, 2.0 mL of 8.8 mmol / L H2O2 was added to start the reaction. After incubation in a water bath at 37°C for 60 min, the plates were transferred to 96-well cell culture plates and placed in a spectrophotometer. The absorbance was measured at 510 nm. The measurements were repeated three times and post-processed in the same manner to calculate the hydroxyl radical scavenging rate of each sample.

[0149] according to Figure 10The test results show that the scavenging efficiency of PG and PG-S on -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 Figure 9 and Figure 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 evaluated the reducing power of PG and PG-S, and the evaluation results are summarized as follows: Figure 11 As shown in the figure, 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 tyrosinase activity in anti-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. Figure 12 shown.

[0154] The test method is:

[0155] B16 cells in the logarithmic growth phase were taken and cultured at a cell density of 5×10 4 The cells were seeded into 96-well plates and incubated for 24 hours. Appropriate amounts of PG and PG-S were added, followed by an equal amount of arbutin solution. After incubation for 48 hours, the supernatant was removed and the cells were rinsed twice with PBS solution. 50 μL of 1% Triton X-100 was added and the cells were frozen at -80°C for 1 hour. 100 μL of 0.1% L-DOPA solution was added and the cells were incubated at 37°C for 2 hours. The absorbance was measured at a wavelength of 490 nm to calculate the tyrosinase activity of the cells.

[0156] according to Figure 12 The test results show that PG and PG-S can inhibit tyrosinase activity in a dose-dependent manner; further comparison found 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: Figure 13 shown.

[0159] The test method is:

[0160] PG-S was 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 cells to adhere and grow. Prepare a concentration gradient of 31.25μg / mL, 62.5μg / mL, 125μg / mL, 250μg / mL, 500μg / mL, and 1000μg / mL PG-S solution. Aspirate the old culture medium and replace with new culture medium, 180μL per well, and then add 20μL of PG-S solution of different concentrations. Set up blank 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 Figure 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 1000 μg / 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing plant glycogen 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, mixing it with the plant glycogen pre-prepared 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 liquid is subjected to precipitation treatment in a precipitant to separate the solid and the liquid to obtain a precipitate; dialyzing and drying the precipitate to obtain the phytoglycogen sulfate; The mass ratio of the sodium trisulfonate ammonium to the plant containing phytoglycogen in the phytoglycogen pre-solution is 4-6:1; When mixing the sodium trisulfonate ammonium chloride and the phytoglycogen pre-solution, the total solid-liquid mass ratio of the mixture is controlled to be 1:11-13; The power of ultrasound is 100W~140W, the temperature of the esterification reaction is 45℃~55℃, and the time of the esterification reaction is 3h~5h.

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

3. The preparation method according to claim 2, wherein The usage ratio of the sodium bisulfite, sodium nitrite and water is 4.5 mol~5.5 mol:1 mol:50 mL.

4. The preparation method according to claim 2, wherein The temperature of the stirring reaction is 85°C to 95°C.

5. The preparation method according to claim 1, wherein The volume of the concentrated solution is 1 / 11 to 1 / 9 of the volume of the liquid component.

6. A phytoglycogen sulfate prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The phytoglycogen sulfate is water-soluble nano-scale phytoglycogen sulfate.

7. Use of the phytoglycogen sulfate according to claim 6 in the preparation of antiviral drugs and skin care products.

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