Method for preparing amphoteric radix pseudostellariae polysaccharide-curcumin nanoparticles through Pickering emulsion polymerization
By grafting hydrophobic chain segments on the polysaccharide molecular chain, the amphoteric genus polysaccharide-curcumin nanocomplex was constructed, and the problem of poor stability of curcumin was solved, the effective load and stable delivery of curcumin were achieved, and its biological activity was improved. Application potential.
Patent Information
- Application Number
- CN202411051530.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-01
- Publication Date
- 2024-12-06
AI Technical Summary
The poor stability of curcumin leads to its limited application in medical chemistry, especially in light, heat and alkaline environments, which affects the delivery and stability of its physiologically active substances.
Using Pickering emulsion technology, the synthesis of amphoteric ginseng polysaccharides by grafting hydrophobic chains onto the polysaccharide molecular chains to construct curcumin-Prigorous ginseng polysaccharide nanocomplex, realize the effective load, transport and controlled release of curcumin, and improve its biological ability. and sex and bioavailability.
The stability and biological activity of curcumin have been maintained, and its retention and antioxidant activity under different pH values and light conditions have been improved, which has significantly improved the bioavailability of curcumin.
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Figure CN119081162A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by Pickering emulsion polymerization and the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles. Background Art
[0002] Curcumin (3-methoxy-4-hydroxy-phenyl-1,6-heptadiene-3,5-dione) is a natural low molecular weight polyphenol compound extracted from the rhizomes of plants such as Zingiberaceae and Araceae. 21 H 20 O6, with a relative molecular mass of 368.39, is the main active ingredient in curcumin and a polyphenol compound belonging to the β-diketone functional group. A large number of studies have shown that it has good physiological activities, such as anti-inflammatory, antiviral, anti-infection and antioxidant activities. It is widely used in the pharmaceutical, food and other industries with few adverse reactions.
[0003] Curcumin molecules have high hydrophobicity and low water solubility (11ng / ml). They are easily soluble in solutions such as ethanol, propylene glycol, alkali and glacial acetic acid, but insoluble in water and ether. Curcumin has a melting point of up to 183°C and has a high lattice energy. Compared with the amorphous state, the solubility of curcumin in the crystalline state in water is lower. In acidic and neutral aqueous solutions, curcumin mainly exists in the keto configuration and has a low solubility (11ng / mL; pH=5.0). In an alkaline environment (pH>12), the configuration of curcumin is mainly enol. At present, people have carried out a lot of research on the separation and purification, physical and chemical properties, and physiological effects of curcumin, but its poor stability has greatly limited its further application. As a highly photosensitivity substance, the degradation rate of curcumin can reach 68.9% after 5 days of outdoor light exposure. Its thermal stability is also poor. When the temperature is above 70°C, the structure of curcumin begins to be destroyed, and in boiling water for 20 minutes, the loss rate of curcumin can reach 30%. Curcumin molecules are also difficult to exist stably under strong acid and strong base conditions, especially under alkaline conditions, they are more likely to decompose and produce substances such as vanillic acid, vanillin, and ferulic acid. Therefore, how to achieve the steady-state delivery of curcumin and achieve the desired biochemical effect is an urgent problem to break through its application limitations in pharmaceutical chemistry.
[0004] In recent years, Pickering emulsions have become a hot topic in the study of drug stable delivery due to their excellent interfacial stability. This emulsion is composed of solid particles adsorbed on the oil-water interface. Compared with traditional small molecule emulsifiers, it can better prevent the aggregation of droplets, thereby improving the stability of the emulsion. Among them, protein-based Pickering emulsions have been widely studied and developed, but the higher-order structure of proteins is easily affected by environmental conditions. For example, high temperature, salt, and pH value can change the secondary structure of proteins and thus affect their emulsification stability, limiting their application in medicine, food, etc. In comparison, polysaccharides are less affected by environmental factors, but since most polysaccharides are water-soluble and difficult to encapsulate fat-soluble drugs, they cannot be directly used as solid particles to stabilize Pickering emulsions. Compared with natural polysaccharides, amphiphilic polysaccharides can significantly reduce interfacial tension and effectively improve emulsion stability. Therefore, constructing amphiphilic polysaccharide-based colloidal particles stabilized Pickering emulsions as delivery carriers is an important way to solve this problem.
[0005] The invention uses Radix Pseudostellariae polysaccharide as a raw material, grafts hydrophobic segments onto polysaccharide molecular chains, and synthesizes amphoteric Radix Pseudostellariae polysaccharide, thereby constructing a Pickering emulsion delivery system that stabilizes curcumin-Pseudostellariae polysaccharide nanocomposite, achieving effective loading, delivery and controlled release of curcumin, maintaining the stability of curcumin, and improving the bioaccessibility and bioavailability of curcumin, providing a new idea for solving the delivery and stability of bioactive substances. Summary of the invention
[0006] The purpose of the present invention is to provide a method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by Pickering emulsion polymerization with a simple process and the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles. The amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles prepared by the present invention can effectively load, transport and control the release of curcumin, maintain the stability of curcumin, improve the bioaccessibility and bioavailability of curcumin, and provide a new idea for solving the transportation and stability of bioactive substances.
[0007] The purpose of the present invention is achieved through the following technical solutions:
[0008] A method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by Pickering emulsion polymerization, comprising the following process steps:
[0009] (1) Preparation of amphoteric Pseudostellariae polysaccharide: Dissolving the purified Pseudostellariae polysaccharide in water and reacting it with glutaric anhydride ethanol solution to prepare amphoteric Pseudostellariae polysaccharide;
[0010] (2) Preparation of amphoteric Pseudostellariae Polysaccharide-Curcumin Nanoparticles by Anti-Solvent Method:
[0011] The amphoteric Pseudostellariae polysaccharide is dissolved in ethanol to obtain an amphoteric Pseudostellariae polysaccharide ethanol solution, a certain amount of curcumin is added to the amphoteric Pseudostellariae polysaccharide ethanol solution, and the mixture is stirred to obtain a mixed solution; the mixed solution is slowly dripped into a container containing a dispersion, and then centrifuged, the supernatant is removed, and freeze-dried to obtain the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles.
[0012] Wherein, the method for preparing amphoteric Pseudostellariae polysaccharide in step (1) is:
[0013] Take Radix Pseudostellariae polysaccharide and dissolve it in water, dissolve glutaric anhydride in anhydrous ethanol, add the glutaric anhydride ethanol solution dropwise into the Radix Pseudostellariae polysaccharide solution, control the reaction temperature at 35°C, use 3% sodium hydroxide to maintain the pH of the system between 8.0 and 9.0 during the reaction, after reacting for 2 hours, use a 3% hydrochloric acid solution to adjust the pH of the system to about 6.5, terminate the reaction, and filter. Wash the product with distilled water for 3 times, dry it in an oven at about 40°C for 10-12 hours, and obtain amphoteric Radix Pseudostellariae polysaccharide.
[0014] The synthetic route of amphoteric Pseudostellariae polysaccharide is as follows:
[0015]
[0016] The preparation of the amphoteric Pseudostellariae polysaccharide in step (1) also includes the steps of extracting and purifying the Pseudostellariae polysaccharide, specifically:
[0017] 1.1 Preparation of Radix Pseudostellariae Concentrate:
[0018] Take the fine powder of Radix Pseudostellariae Polysaccharides and add ethanol, extract in a water bath at 90°C for 2-3h, filter while hot, then dry the ethanol in a 50°C oven, put it in a round-bottom flask, add water, extract in a boiling water bath at reflux for 2-3h, discard the precipitate, take the supernatant, and concentrate the supernatant in a boiling water bath to 1 / 3 of the original volume;
[0019] 1.2 Extraction of crude polysaccharides from Radix Pseudostellariae:
[0020] Slowly pour 5 times the volume of anhydrous ethanol into the concentrated solution obtained in step 1.1, gently shake, seal with plastic wrap, and place in a 4°C refrigerator for 3-5 hours, then discard the supernatant, wait for the ethanol in the lower precipitate to evaporate, add 5-10 ml of water, stir to dissolve the lower precipitate, and freeze the polysaccharide sample in a -18°C refrigerator. After the polysaccharide sample is completely frozen, place it in a freeze dryer for freeze drying;
[0021] 1.3 Purification of Radix Pseudostellariae Polysaccharide:
[0022] DEAE Cellulose-52 was used as the column filler, and the crude polysaccharide was dissolved in Tris-HCl buffer and loaded, the crude polysaccharide concentration was 1 mg / mL, and 0.1-0.3 mol / L NaCl salt solution was used for elution, the flow rate was 2.0 mL / min, and the first main peak was collected, concentrated by rotary evaporation, dialyzed for desalination, and freeze-dried to obtain Pseudostellaria heterophylla polysaccharide;
[0023] The specific method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by anti-solvent method in step (2) is as follows:
[0024] 2.1 Preparation of amphoteric Pseudostellariae polysaccharide solution:
[0025] Using 70% ethanol solution as the solvent of the amphoteric Pseudostellariae polysaccharide, adding the amphoteric Pseudostellariae polysaccharide, and stirring with a magnetic force in a 50° C. water bath until the amphoteric Pseudostellariae polysaccharide is completely dissolved, thereby obtaining the amphoteric Pseudostellariae polysaccharide solution;
[0026] 2.2 Construction of amphoteric Pseudostellariae polysaccharide-curcumin pickering emulsion delivery system
[0027] Curcumin is added to the obtained amphoteric Pseudostellariae polysaccharide solution to form a system with a mass ratio of amphoteric Pseudostellariae polysaccharide to curcumin of 1 to 4:1, and the mixture is stirred at a constant temperature of 50°C for 2 to 3 hours to fully dissolve the mixture to obtain a mixed solution; the mixed solution is transferred to a constant pressure dropping funnel with a bottom valve aperture of 0.45 mm, and the mixture is slowly dripped into a container containing a dispersion liquid at a rate of 30 drops / min, wherein the dispersion liquid is in a state of rotating flow stirred by a magnetic stirrer, and the magnetic stirring rate is 1000 r / min. After the mixed solution is dripped, the mixture is stirred for another 10 to 15 minutes, and then centrifuged, the supernatant is removed, and the mixture is freeze-dried to obtain the powdered amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles; the entire preparation process is protected from light to prevent curcumin from decomposing under light.
[0028] The dispersion liquid is pure water at room temperature.
[0029] The invention also provides amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles prepared by the method.
[0030] Compared with the prior art, the advantages of the present invention are:
[0031] 1. The present invention uses Pseudostellariae Radix Polysaccharide as a raw material, grafts hydrophobic segments onto the polysaccharide molecular chain, and synthesizes amphoteric Pseudostellariae Radix Polysaccharide, thereby constructing a Pickering emulsion delivery system that is stable for curcumin-Pseudostellariae Radix Polysaccharide nanocomplex, achieving effective loading, delivery and controlled release of curcumin, maintaining the stability of curcumin, and improving the bioaccessibility and bioavailability of curcumin, providing a new idea for solving the delivery and stability of bioactive substances.
[0032] 2. Because Pseudostellariae polysaccharide is water-soluble, while curcumin is fat-soluble, the overall composite effect is relatively poor. The present invention modifies Pseudostellariae polysaccharide into an amphoteric polysaccharide by grafting glutaric anhydride, thereby achieving efficient loading of curcumin.
[0033] 3. The amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles prepared by the present invention have a photoprotective effect on curcumin, and the retention rate of curcumin in the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles is higher than that of free curcumin; the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles have excellent pH stability, and when the pH values are 5, 7, and 9, the retention rate of curcumin in the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles is higher than that of free curcumin; the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles have good in vitro antioxidant activity, which is higher than the in vitro antioxidant activity of free curcumin. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is a scanning electron micrograph of the amphoteric Pseudostellariae Polysaccharide (PHP)-Curcumin (cur) particles obtained in Example.
[0035] Figure 2 This is the retention rate of curcumin in PHP-Cur nanoparticles under Xe light irradiation.
[0036] Figure 3 is the retention rate of curcumin in PHP–Cur nanocomposite at pH 5.0.
[0037] Figure 4 is the retention rate of curcumin in PHP–Cur nanocomposite at pH 7.0.
[0038] Figure 5 is the retention rate of curcumin in PHP–Cur nanocomposite at pH 9.0.
[0039] Figure 6 is the scavenging activity of PHP–Cur nanocomplex on DPPH free radicals.
[0040] Figure 7 is the scavenging activity of PHP–Cur nanocomplex on ABTS free radicals. DETAILED DESCRIPTION
[0041] The present invention is described in detail below with reference to the accompanying drawings and embodiments:
[0042] Example 1: Preparation of amphoteric Pseudostellariae Polysaccharide
[0043] 1.1 Preparation of Radix Pseudostellariae Concentrate
[0044] Take 5g of Pseudostellariae polysaccharide powder, add 200ml of 80% (volume ratio) ethanol, reflux extract in a water bath at 90℃ for 2h, filter while hot, dry the ethanol in a 50℃ oven, put it in a round-bottom flask, add 200ml of water, reflux extract in a boiling water bath for 2h, discard the precipitate, take the supernatant, and concentrate it to 1 / 3 of the original volume in a boiling water bath.
[0045] 1.2 Extraction of crude polysaccharides from Pseudostellaria heterophylla
[0046] Slowly pour 5 times the volume of anhydrous ethanol into the above concentrate, shake gently, seal with plastic wrap, and place in a 4°C refrigerator for 3 hours. After 3 hours, discard the supernatant, and after the ethanol in the lower precipitate evaporates, add 5 ml of water, stir to dissolve the lower precipitate, and freeze the polysaccharide sample in a -18°C refrigerator. After the polysaccharide sample is completely frozen, put it into a freeze dryer.
[0047] 1.3 Purification of Radix Pseudostellariae Polysaccharides
[0048] DEAE Cellulose-52 from Beijing Biodi Biotechnology Co., Ltd. was used as the column filler, and crude polysaccharide was dissolved in tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer (pH 7.6) and loaded. The crude polysaccharide concentration was 1 mg / mL, and 0.1-0.3 mol / L NaCl salt solution was used for elution at a flow rate of 2.0 mL / min. The first main peak was collected, concentrated by rotary evaporation, desalted by dialysis, and freeze-dried to obtain Panax pseudoginseng polysaccharide.
[0049] 1.4. Preparation of amphoteric Pseudostellariae polysaccharides
[0050] Take 1g of Radix Pseudostellariae polysaccharide and dissolve it in 50mL of water, 0.5g of glutaric anhydride and dissolve it in 50mL of anhydrous ethanol, add the glutaric anhydride ethanol solution dropwise to the Radix Pseudostellariae polysaccharide solution, control the reaction temperature at 35°C, use 3% sodium hydroxide to maintain the pH of the system between 8.0 and 9.0 during the reaction, after reacting for 2h, use 3% (mass fraction) hydrochloric acid solution to adjust the pH of the system to about 6.5, terminate the reaction, and filter. Wash the product with distilled water 3 times, dry it in an oven at about 40°C for 12h, and obtain amphoteric Radix Pseudostellariae polysaccharide.
[0051] Example 2: Preparation of amphoteric Pseudostellariae Polysaccharide-Curcumin Nanoparticles (PHP-Cur Nanoparticles) by Antisolvent Method
[0052] 2.1 Preparation of amphoteric Pseudostellariae polysaccharide solvent
[0053] 100 ml of 70% (volume ratio) ethanol solution was used as the solvent of Pseudostellariae Radix Polysaccharide, 0.03 g of amphoteric Pseudostellariae Radix Polysaccharide was added, and magnetic stirring was performed in a 50° C. water bath until it was completely dissolved.
[0054] 2.2 Construction of amphoteric Pseudostellariae polysaccharide-curcumin pickering emulsion delivery system
[0055] 0.03 g of curcumin is added to the above solution to form a system with a mass ratio of 1:1 of amphoteric Pseudostellariae polysaccharide / curcumin, and the mixture is stirred at a constant temperature of 50°C for 2-3 hours to fully dissolve the mixture to obtain a mixed solution; the mixed solution is transferred to a constant pressure dropping funnel with a bottom valve aperture of 0.45 mm, and the mixture is slowly dripped into a 500 mL dispersion liquid (normal temperature pure water) container at a rate of 30 drops / min, wherein the dispersion liquid is in a state of rotating flow stirred by a magnetic stirrer, and the magnetic stirring rate is 1000 r / min. After the mixed solution is dripped, the mixture is stirred for another 10-15 minutes, and then centrifuged, the supernatant is removed, and the mixture is freeze-dried to obtain the powdered amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles PHP 1-Cur1; the preparation process should be kept away from light to prevent curcumin from decomposing under light.
[0056] Example 3 is a method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by an anti-solvent method. The method is similar to that of Example 2, except that the system is formed with a mass ratio of amphoteric Pseudostellariae polysaccharide / curcumin of 2:1. The specific operation method of Example 3 is as follows:
[0057] 3.1 Preparation of amphoteric Pseudostellariae polysaccharide solvent
[0058] 100 ml of 70% (volume ratio) ethanol solution was used as the solvent of Pseudostellariae Radix Polysaccharide, 0.06 g of amphoteric Pseudostellariae Radix Polysaccharide was added, and magnetic stirring was performed in a 50° C. water bath until it was completely dissolved.
[0059] 3.2 Construction of amphoteric Pseudostellariae polysaccharide-curcumin pickering emulsion delivery system
[0060] Add 0.03 g of curcumin to the above solution to form a system with a mass ratio of 2:1 of amphoteric Pseudostellariae polysaccharide / curcumin, and stir at a constant temperature of 50°C for 2-3 hours to fully dissolve it to obtain a mixed solution; transfer the mixed solution to a constant pressure dropping funnel with a bottom valve aperture of 0.45 mm, adjust the rate of 30 drops / min and slowly drip it into a 500 mL dispersion liquid (normal temperature pure water) container, wherein the dispersion liquid is in a state of stirring and rotating flow using a magnetic stirrer, and the magnetic stirring rate is 1000 r / min. After the mixed solution is dripped, stir for another 10-15 minutes, then centrifuge, remove the supernatant, and freeze-dry to obtain the powdered amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles PHP2-Cur1; the preparation process should be kept away from light to prevent curcumin from decomposing under light.
[0061] Example 4 is a method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by an anti-solvent method. The method is similar to that of Example 2, except that the system is formed with a mass ratio of amphoteric Pseudostellariae polysaccharide / curcumin of 3:1. The specific operation method of Example 4 is as follows:
[0062] 4.1 Preparation of amphoteric Pseudostellariae polysaccharide solvent
[0063] 100 ml of 70% (volume ratio) ethanol solution was used as the solvent of Pseudostellariae Radix Polysaccharide, 0.09 g of amphoteric Pseudostellariae Radix Polysaccharide was added, and magnetic stirring was performed in a 50° C. water bath until it was completely dissolved.
[0064] 4.2 Construction of amphoteric Pseudostellariae polysaccharide-curcumin pickering emulsion delivery system
[0065] Add 0.03 g of curcumin to the above solution to form a system with a mass ratio of 3:1 of amphoteric Pseudostellariae polysaccharide / curcumin, and stir at a constant temperature of 50°C for 2-3 hours to fully dissolve it to obtain a mixed solution; transfer the mixed solution to a constant pressure dropping funnel with a bottom valve aperture of 0.45 mm, adjust the rate of 30 drops / min and slowly add it dropwise to a 500 mL dispersion liquid (normal temperature pure water) container, wherein the dispersion liquid is in a state of stirring and rotating flow using a magnetic stirrer, and the magnetic stirring rate is 1000 r / min. After the mixed solution is dripped, stir for another 10-15 minutes, then centrifuge, remove the supernatant, and freeze-dry to obtain the powdered amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles PHP3-Cur1; the preparation process should be kept away from light to prevent curcumin from decomposing under light.
[0066] Example 5 is a method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by an anti-solvent method. The method is similar to that of Example 2, except that the system is formed with a mass ratio of amphoteric Pseudostellariae polysaccharide / curcumin of 4:1. The specific operation method of Example 4 is as follows:
[0067] 5.1 Preparation of amphoteric Pseudostellariae polysaccharide solvent
[0068] 100 ml of 70% (volume ratio) ethanol solution was used as the solvent of Pseudostellariae Radix Polysaccharide, 0.12 g of amphoteric Pseudostellariae Radix Polysaccharide was added, and magnetic stirring was performed in a 50° C. water bath until it was completely dissolved.
[0069] 5.2 Construction of amphoteric Pseudostellariae polysaccharide-curcumin pickering emulsion delivery system
[0070] Add 0.03 g of curcumin to the above solution to form a system with a mass ratio of 4:1 of amphoteric Pseudostellariae polysaccharide / curcumin, and stir at a constant temperature of 50°C for 2-3 hours to fully dissolve it to obtain a mixed solution; transfer the mixed solution to a constant pressure dropping funnel with a bottom valve aperture of 0.45 mm, adjust the rate of 30 drops / min and slowly add it dropwise to a 500 mL dispersion liquid (normal temperature pure water) container, wherein the dispersion liquid is in a state of stirring and rotating flow using a magnetic stirrer, and the magnetic stirring rate is 1000 r / min. After the mixed solution is dripped, stir for another 10-15 minutes, then centrifuge, remove the supernatant, and freeze-dry to obtain the powdered amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles PHP4-Cur1; the preparation process should be kept away from light to prevent curcumin from decomposing under light.
[0071] In order to verify the special loading effect of the amphoteric Pseudostellariae polysaccharide on curcumin, the inventors also synthesized some Pseudostellariae polysaccharide-curcumin nanoparticles for comparison. The specific synthesis method is shown in Example 6.
[0072] Example 6
[0073] 6.1 Preparation of Radix Pseudostellariae Polysaccharide Solvent
[0074] 100 ml of 70% (volume ratio) ethanol solution was used as the solvent of Pseudostellariae Radix Polysaccharide, 0.09 g of purified Pseudostellariae Radix Polysaccharide was added, and magnetic stirring was performed in a 50° C. water bath until it was completely dissolved.
[0075] 6.2 Synthesis of Pseudostellariae Polysaccharide-Curcumin Nanoparticles Control Group
[0076] 0.03g of curcumin was added to the above solution to form a system with a mass ratio of 3:1 of Radix Pseudostellariae Polysaccharides / Curcumin, and the mixture was stirred at a constant temperature of 50°C for 2-3h to fully dissolve the mixture to obtain a mixed solution; the mixed solution was transferred to a constant pressure dropping funnel with a bottom valve aperture of 0.45mm, and the mixture was slowly dripped into a 500mL dispersion liquid (normal temperature pure water) container at a rate of 30 drops / min, wherein the dispersion liquid was in a state of rotating flow with a magnetic stirrer, and the magnetic stirring rate was 1000r / min. After the mixed solution was dripped, it was stirred for another 10-15min, and then centrifuged, the supernatant was removed, and the powdered Radix Pseudostellariae Polysaccharides-Curcumin Nanoparticles were obtained by freeze drying; the preparation process should be kept away from light to prevent curcumin from decomposing under light. The prepared Radix Pseudostellariae Polysaccharides-Curcumin Nanoparticles were used as a control group in subsequent performance test experiments.
[0077] Example 7 Performance Test
[0078] 7.1 Scan the electron microscope images of the amphoteric Pseudostellariae polysaccharide (PHP)-curcumin (cur) nanoparticles obtained in Examples 2-5, respectively. Figure 1 shown.
[0079] 7.2 Test of the photoprotective effect of amphoteric Pseudostellariae polysaccharide on curcumin:
[0080] The PHP-Cur nanoparticles obtained in Examples 2-5, the free curcumin solution, and the Pseudostellariae polysaccharide-curcumin nanoparticles obtained in Example 6 were dissolved in pure water to prepare corresponding solutions.
[0081] The PHP-Cur solution obtained in Examples 2-5 and the free curcumin solution and the solution corresponding to the Pseudostellariae polysaccharide-curcumin nanoparticles obtained in Example 6 were placed under a 300W Xe lamp for continuous irradiation for 12 hours, 1 mL of the solution was drawn every 1 hour, and the curcumin content in the solution was determined using an ultraviolet spectrophotometer. Figure 2 As shown, under Xe light irradiation, the retention rate of curcumin in PHP-Cur is always higher than that of free curcumin. After 12 hours of light irradiation, the retention rate of free curcumin is 26.8%, the retention rate of PHP 1-Cur1 is 75.3%, the retention rate of PHP2–Cur1 is 85.4%, the retention rate of PHP3–Cur1 is 91.8%, and the retention rate of PHP4–Cur1 is 86.9%. The retention rate of Pseudostellariae polysaccharide-curcumin nanoparticles obtained in Example 6 is 53.2%.
[0082] 7.3 pH Stability of Amphoteric Pseudostellariae Polysaccharide-Curcumin Nanoparticles
[0083] The PHP-Cur nanoparticles obtained in Examples 2-5, the free curcumin solution, and the Pseudostellariae polysaccharide-curcumin nanoparticles obtained in Example 6 were respectively prepared into 2 mg / mL solutions, the pH value of the solution was adjusted with 0.1 M hydrochloric acid or sodium hydroxide solution, and the initial absorbance A0 at 426 nm was measured. At 25° C. and in the dark, the ratio A / A0 of the absorbance A measured at different time points to the initial absorbance A0 was used to represent the curcumin retention rate of each product under different pH environments.
[0084] like Figure 3-5 As shown in the figure, at pH 5, 7, and 9, the retention rate of curcumin in PHP-Cur nanoparticles was higher than that of free curcumin and the control group of Pseudo-Pseudo-curcumin nanoparticles.
[0085] Under the environment of pH = 5, after 6 hours, the retention rate of free curcumin was only 36.2%, while the retention rate of curcumin in the control group of Pseudo-Pseudo-curcumin nanoparticles was 68.1%, and the retention rates of curcumin in PHP1-cur1, PHP2-cur1, PHP3-cur1 and PHP4-cur1 were 85.8%, 91.2%, 95.0% and 87.9%, respectively.
[0086] Under the environment of pH = 7, after 6 hours, the retention rate of free curcumin was 28.1%, the retention rate of curcumin in the control group of Pseudo-curcumin nanoparticles was 40.9%, and the retention rates of curcumin in PHP1-cur1, PHP2-cur1, PHP3-cur1 and PHP4-cur1 were 85.3%, 88.7%, 91.8% and 86.5%, respectively.
[0087] Under the environment of pH = 9, after 6 hours, the retention rate of free curcumin was 29.6%, the retention rate of curcumin in the control group of Pseudo-Pseudo-curcumin nanoparticles was 47.6%, and the retention rates of curcumin in PHP1-cur1, PHP2-cur1, PHP3-cur1 and PHP4-cur1 were 78.6%, 76.3%, 85.5% and 69.3%, respectively.
[0088] 7.4 Effect of amphoteric Pseudostellariae polysaccharide-curcumin nanocomplex on in vitro antioxidant activity.
[0089] The experiment compared the scavenging abilities of free curcumin, PHP–Cur nanoparticles, and Pseudostellaria polysaccharide-curcumin nanoparticle control group on DPPH and ABTS free radicals.
[0090] The PHP-Cur nanoparticles obtained in Examples 2-5, the free curcumin solution, and the Pseudostellariae polysaccharide-curcumin nanoparticle control group obtained in Example 6 were dissolved in pure water to prepare solutions of different concentrations of 1-10 μg / mL. Then, according to the instructions for use of the kit, the DPPH free radical scavenging ability detection kit and the ABTS free radical scavenging ability detection kit of Beijing Solebaugh Technology Co., Ltd. were used to detect free radicals using UV-visible absorption spectrophotometry, and the detection wavelengths were 515 nm (DPPH free radical) and 405 nm (ABTS free radical), respectively.
[0091] Distilled water was used as a blank group. The free radical scavenging activity of Pseudostigma protein-polysaccharide nanoparticles was calculated according to the following formula:
[0092] Free radical scavenging rate (%) = (1-A 空白 / A 样品 )×100
[0093] like Figure 6 As shown, when each substance was at the maximum concentration, the scavenging rate of free curcumin on DPPH free radicals was 18.6%, the Pseudostellaria polysaccharide-curcumin nanoparticle control group was 33.4%, and the scavenging rates of PHP1-cur1, PHP2-cur1, PHP3-cur1 and PHP4-cur1 on DPPH free radicals were 58.4%, 67.2%, 73.3% and 66.0%, respectively.
[0094] like Figure 7 As shown in the figure, when each substance was at the maximum concentration, the scavenging rate of free curcumin on ABTS free radicals was 23.3%, the control group of Pseudo-curcumin nanoparticles was 67.1%, and the scavenging rates of PHP1-cur1, PHP2-cur1, PHP3-cur1 and PHP4-cur1 on ABTS free radicals were 91.2%, 95.3%, 99.6% and 93.4%, respectively. This indicates that the embedding of Pseudo-curcumin polysaccharide can improve the retention rate of curcumin, thereby improving the in vitro antioxidant activity of curcumin in PHP-Cur nanocomplexes.
[0095] The above are only preferred specific implementation modes of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and novel inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by Pickering emulsion polymerization, characterized in that: It includes the following process steps: (1) Preparation of amphoteric Pseudostellariae polysaccharide: Dissolving the purified Pseudostellariae polysaccharide in water and reacting it with glutaric anhydride ethanol solution to prepare amphoteric Pseudostellariae polysaccharide; (2) Preparation of amphoteric Pseudostellariae Polysaccharide-Curcumin Nanoparticles by Anti-Solvent Method: The amphoteric Pseudostellariae polysaccharide is dissolved in ethanol to obtain an amphoteric Pseudostellariae polysaccharide ethanol solution, a certain amount of curcumin is added to the amphoteric Pseudostellariae polysaccharide ethanol solution, and the mixture is stirred to obtain a mixed solution; the mixed solution is slowly dripped into a container containing a dispersion, and then centrifuged, the supernatant is removed, and freeze-dried to obtain the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles.
2. The method according to claim 1, characterized in that: The method for preparing amphoteric Pseudostellariae polysaccharide in step (1) is: Take Radix Pseudostellariae polysaccharide and dissolve it in water, dissolve glutaric anhydride in anhydrous ethanol, add the glutaric anhydride ethanol solution dropwise into the Radix Pseudostellariae polysaccharide solution, control the reaction temperature at 35°C, use 3% sodium hydroxide to maintain the pH of the system between 8.0 and 9.0 during the reaction, after reacting for 2 hours, use a 3% hydrochloric acid solution to adjust the pH of the system to about 6.5, terminate the reaction, and filter. Wash the product with distilled water for 3 times, dry it in an oven at about 40°C for 10-12 hours, and obtain amphoteric Radix Pseudostellariae polysaccharide.
3. The method according to claim 1, characterized in that: The preparation of the amphoteric Pseudostellariae polysaccharide in step (1) also includes the steps of extracting and purifying the Pseudostellariae polysaccharide, specifically: 1.1 Preparation of Radix Pseudostellariae Concentrate: Take the fine powder of Radix Pseudostellariae Polysaccharides and add ethanol, extract in a water bath at 90°C for 2-3h, filter while hot, then dry the ethanol in a 50°C oven, put it in a round-bottom flask, add water, extract in a boiling water bath at reflux for 2-3h, discard the precipitate, take the supernatant, and concentrate the supernatant in a boiling water bath to 1 / 3 of the original volume; 1.2 Extraction of crude polysaccharides from Radix Pseudostellariae Radix: Slowly pour 5 times the volume of anhydrous ethanol into the concentrated solution obtained in step 1.1, gently shake, seal with plastic wrap, and place in a 4°C refrigerator for 3-5 hours, then discard the supernatant, wait for the ethanol in the lower precipitate to evaporate, add 5-10 ml of water, stir to dissolve the lower precipitate, and freeze the polysaccharide sample in a -18°C refrigerator. After the polysaccharide sample is completely frozen, place it in a freeze dryer for freeze drying; 1.3 Purification of Radix Pseudostellariae Polysaccharide: DEAE Cellulose-52 was used as the column filler, and the crude polysaccharide was dissolved in Tris-HCl buffer and loaded. The loading concentration of the crude polysaccharide was 1 mg / mL. 0.1-0.3 mol / L NaCl salt solution was used for elution at a flow rate of 2.0 mL / min. The first main peak was collected, concentrated by rotary evaporation, desalted by dialyzation, and freeze-dried to obtain Pseudomonas heterophylla polysaccharide.
4. The method according to claim 1, characterized in that: The specific method for preparing amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles by anti-solvent method in step (2) is as follows: 2.1 Preparation of amphoteric Pseudostellariae polysaccharide solution: Using 70% ethanol solution as the solvent of the amphoteric Pseudostellariae polysaccharide, adding the amphoteric Pseudostellariae polysaccharide, and stirring with a magnetic force in a 50° C. water bath until the amphoteric Pseudostellariae polysaccharide is completely dissolved, thereby obtaining the amphoteric Pseudostellariae polysaccharide solution; 2.2 Construction of amphoteric Pseudostellariae polysaccharide-curcumin pickering emulsion delivery system Add curcumin to the obtained amphoteric Pseudostellariae polysaccharide solution to form a system with a mass ratio of amphoteric Pseudostellariae polysaccharide to curcumin of 1 to 4:1, and continue to stir at a constant temperature of 50°C for 2-3 hours to fully dissolve the amphoteric Pseudostellariae polysaccharide to obtain a mixed solution; transfer the mixed solution to a constant pressure dropping funnel with a bottom valve aperture of 0.45 mm, adjust the rate to 30 drops / min and slowly drip it into a container containing a dispersion liquid, wherein the dispersion liquid is in a state of stirring and rotating flow using a magnetic stirrer, and the magnetic stirring rate is 1000r / min. After the mixed solution is dripped, stir for another 10-15 minutes, then centrifuge, remove the supernatant, and freeze-dry to obtain the amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles in powder form; The entire preparation process was kept away from light to prevent curcumin from decomposing due to light.
5. The method according to claim 1, characterized in that: The dispersion liquid is pure water at room temperature.
6. A kind of amphoteric Pseudostellariae polysaccharide-curcumin nanoparticles prepared by the method according to any one of claims 1 to 5.