Preparation method and application of nano-active euphorbia helioscopia extract

Abstract

The invention relates to the technical field of nano-carriers, and particularly discloses a preparation method and application of a nano-active millettia extract, the preparation method comprises the following steps: constructing a nano-carrier to wrap the millettia extract, and providing dual drug protection through a bimolecular layer lipid core and a chitosan shell, so that the nano-active millettia extract is obtained. The stability of the millettia bicolor extract in the nano-carrier is improved, and the requirements of the market for high efficiency and stability of a millettia bicolor extract nano-delivery system are met.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanocarriers, and particularly relates to a preparation method and application of a nano active spilanthes acuta extract. BACKGROUND

[0002] Spilanthes acuta is a plant widely used in traditional medicine, and its extract is rich in various bioactive components. Among them, the most core and representative is a class of compounds called N-alkyl amides, and spilanthal is the key functional molecule in N-alkyl amides. Under the optimized extraction process, the proportion of spilanthal in total N-alkyl amides can be as high as more than 85%, and it dominates in the extract. Spilanthal can produce a unique "tingling sensation" by acting on nerve endings, and it can act as an agonist of the TRPV1 (Transient Receptor Potential Vanilloid Type 1) channel, regulating the activity of sensory neurons, thereby producing analgesic and nerve regulation effects. Spilanthal also has significant anti-inflammatory activity, and its mechanism of action involves inhibiting inflammatory signaling pathways such as MAPK (Mitogen-Activated Protein Kinase) and NF-κB (Nuclear Factor kappa B) pathways, thereby reducing the production of various pro-inflammatory factors such as IL-6 and TNF-α. At the same time, because it can inhibit the microcontraction of subcutaneous muscles, spilanthal is also known as "botulinum toxin of plant origin" in the cosmetic field, and is used for anti-wrinkle and skin tightening. However, spilanthal itself is a lipophilic molecule with poor water solubility, and it is unstable under light and heat conditions. These physical and chemical properties greatly limit its direct application in medical and cosmetic formulations.

[0003] In order to overcome the application limitations of spilanthes acuta extract, relevant researchers turned to nanodelivery systems and considered improving its performance by coating it in nanocarriers. There are related nanotechnology of plant extracts in existing patents, such as the nanolipid carrier and its application disclosed in patent application CN117883295A. However, patents specifically targeting "nanotechnology coated spilanthes acuta extract" are not common.

[0004] Although nanotechnology has broad prospects, the liposome nanocarriers disclosed in the above-mentioned patent still face many challenges in actual application, such as insufficient stability, difficulty in ensuring encapsulation efficiency and drug loading, and difficulty in large-scale production. Therefore, developing a more stable, efficient and easy-to-prepare nanodelivery system for spilanthes acuta extract is still a problem to be solved in current technology. SUMMARY

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present application improves the stability of the spilanthol extract in the nanocarrier by constructing a nanocarrier to wrap the spilanthol extract and optimizing the structure of the nanocarrier, thereby meeting the high efficiency and stability requirements of the spilanthol extract nanodelivery system in the market.

[0006] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows: The present application provides a preparation method of nanometer active spilanthol extract, which specifically comprises the following steps: S1, stirring the lipids, spilanthol extract and anhydrous ethanol at 45-55 DEG C for 20-50 min to obtain a drug core; S2, dissolving chitosan in 0.5-3 v / v% of an ice acetic acid aqueous solution, stirring at room temperature for 1-2 h, and then removing the undissolved impurities by a filter membrane to obtain a chitosan solution; S3, under the ultrasonic power of 100-200 W, injecting the drug core into the chitosan solution at 5-10 DEG C through a multi-hole syringe at the rate of 1-5 mL / min per hole, and continuously ultrasonic for 5-10 min, and then rotary evaporation to obtain a nanoemulsion; S4, adding trehalose to the nanoemulsion, stirring for 20-30 min, and then freezing to obtain the nanometer active spilanthol extract.

[0007] In some embodiments, in step S1, the lipids comprise functionalized lipids and cholesterol.

[0008] The present application wraps the hydrophobic spilanthol extract with a lipid bilayer, improves the encapsulation efficiency of the lipid-soluble drug, effectively protects the active substance spilanthol amide in it from the damage of the external environment such as light, oxygen and pH change, and thereby improves the stability.

[0009] In some embodiments, the preparation steps of the functionalized lipids are as follows: Dissolve PEG and phospholipids in an aqueous ethanol solution, add a photoinitiator to ensure complete dissolution, and obtain a reaction solution; irradiate the reaction solution under a light source for 5-15 min under the protection of inert gas, and then purify after the reaction is completed to obtain the functionalized lipids.

[0010] In some embodiments, the PEG end contains a double bond, and the phospholipid end contains a thiol group.

[0011] Preferably, the type of the phospholipid end containing a thiol group is SH-PEG-DPPE.

[0012] In some embodiments, the molar ratio of the phospholipid and PEG is 1:(1.0-1.2).

[0013] Different from traditional amino reaction introduction, the application uses phospholipids with terminal thiol and functional PEG with terminal unsaturated double bond, and utilizes light-induced "thiol-ene" click chemistry reaction, thereby reducing solvent use, and having the advantages of high efficiency, rapidness, mild condition, and relative insensitivity to water and oxygen.

[0014] When the double PEG in the functionalized lipid is embedded on the surface of the nanoparticles, when two nanoparticles are close to each other, the PEG chains are compressed, the freedom is reduced, and a steric repulsion force is generated, which can effectively overcome the attraction caused by van der Waals force, preventing the aggregation and fusion of the nanoparticles during storage. During the freeze-drying process, the PEG chains of the PEGylated phospholipid can also play a protective role, and the steric hindrance effect can prevent the close contact of the nanoparticles during dehydration, thereby avoiding irreversible aggregation during freeze-drying and reconstitution, and ensuring that the particle size after reconstitution is basically consistent with that before freeze-drying.

[0015] In some embodiments, the mass ratio of the functionalized lipid, cholesterol, and spilanthol is (80-120):(20-30):(20-40).

[0016] In some embodiments, in step S2, the ratio of chitosan to glacial acetic acid aqueous solution is (1-4) mg / mL. The present application selects green solvent anhydrous ethanol to reduce potential toxicity, ensures that the two lipids of functionalized lipid and cholesterol and plant extract molecules are fully dispersed, and promotes the ordered arrangement of molecules by controlling the temperature to be above the lipid phase transition temperature. In the synthesis process, the chitosan solution with the positive charge characteristic is added to the drug core, and a stable complex structure is formed through electrostatic interaction, further improving the encapsulation efficiency and stability of the active ingredients, and preventing lipid oxidation and drug degradation by controlling the temperature under the overall ultrasonic.

[0017] In some embodiments, in step S4, the mass ratio of trehalose to nanoemulsion is 1:(15-25).

[0018] In some embodiments, in step S4, the freezing is: first freezing at-100 to-60℃ for 2-5h for pre-freezing, and then vacuum freezing at-60 to-40℃ for 20-30h for freeze-drying.

[0019] The present application uses trehalose to fill the gap of the lipid bilayer, prevents the structure from collapsing during freeze-drying, and reduces the ice crystal size through deep-freezing pre-freezing, thereby maintaining the integrity of the nanoparticle morphology during freeze-drying.

[0020] Another aspect of the present application provides the application of the nano active spilanthes extract prepared by the above preparation method in the field of adult lubricants.

[0021] The lipid nanocarriers themselves have physical stability problems, which can cause particle size increase, aggregation, fusion, and even active substance leakage during storage. Considering that the application of nanometer active spilanthes extract in the field of adult lubricants, in order to improve the stability of nanocarriers, the application uses chitosan shell to enhance the stability of nanoparticles, and the accompanying mucosal adhesion can also improve the onset speed of active substances.

[0022] Compared with the prior art, the application has the following beneficial effects: 1. The double-molecular layer lipid core and the chitosan shell provide double drug protection, wherein the inner layer of lipids encapsulates spilanthes extract through hydrophobic interaction to solve the problem of poor water solubility; the outer layer of chitosan stabilizes the lipid layer through electrostatic interaction to strengthen the stability; in addition, trehalose is used to fill the gap of the lipid bilayer to prevent the structure from collapsing during freeze-drying.

[0023] 2. By grafting PEG chains onto phospholipids, the steric hindrance repulsion is used to prevent nanoparticles from aggregating and fusing during storage, and at the same time, irreversible aggregation of nanocarriers during freeze-drying and reconstitution is avoided, so as to ensure that the particle size after reconstitution is basically the same as that before freeze-drying. In addition, in the grafting process, the application uses phospholipids with terminal thiol groups and functional PEG with terminal unsaturated double bonds, and uses the photo-initiated "thiol-ene" click chemistry reaction to replace the traditional solvent amino reaction, so that the grafting reaction has the advantages of high efficiency, rapidness, mild conditions, and relative insensitivity to water and oxygen.

[0024] 3. In terms of process, the green solvent anhydrous ethanol is selected to reduce potential toxicity, low-temperature ultrasonic and reduced pressure volatilization are used to avoid isomerization inactivation of active ingredients in spilanthes extract caused by high temperature; and deep cold pre-freezing is used to reduce the size of ice crystals, so as to maintain the integrity of the morphology of nanoparticles during freeze-drying. DETAILED DESCRIPTION

[0025] The application will be described below in conjunction with specific embodiments. It should be noted that the following examples are examples of the application and are only used to illustrate the application, but not to limit the application. Other combinations and various modifications within the concept of the application can be made without departing from the spirit or scope of the application. It should be noted that the raw materials used in the following preparation examples and examples are from any manufacturer on the market unless otherwise specified; The type of terminal double bond PEG is PEG-2 ALLy, Mw=600-1500; The type of terminal thiol phospholipid is SH-PEG-DPPE, Mw=1000-3000.

[0026] Preparation Example 1 The preparation steps of the functionalized lipids are as follows: 1 mol of terminal double bond PEG (Mw=1000), 1.1 mol of terminal thiol phospholipid (Mw=2000) were dissolved in 2 L of 60 wt% ethanol aqueous solution, 6.3 mmol of photoinitiator 2959 was added to ensure complete dissolution, to obtain a reaction solution; the reaction solution was irradiated under light source for 10 min under nitrogen protection, and the functionalized lipid was obtained by purification after the reaction was completed.

[0027] Example 1 A preparation method of a nano-active feverfew extract, specifically comprising the following steps: S1, 100 g of functionalized lipid, 25 g of cholesterol, 30 g of feverfew extract and 500 mL of anhydrous ethanol were stirred at 50°C for 30 min to obtain a drug core; S2, 100 mg of chitosan was dissolved in 200 mL of 1 v / v% concentrated acetic acid aqueous solution, stirred at room temperature for 1.5 h, and then passed through a 0.45 μm filter membrane to remove undissolved impurities to obtain a chitosan solution; S3, under the ultrasonic power of 150 W, 200 mL of the drug core was injected into 100 mL of 7°C chitosan solution through a multi-hole syringe at the rate of 2 mL / min per hole, and the ultrasonic was continued for 8 min, and then rotary evaporation was performed to obtain a nanoemulsion; S4, trehalose was added to the nanoemulsion at the mass ratio of trehalose to nanoemulsion of 1:20, stirred for 25 min, pre-frozen at -80°C for 3 h, and then freeze-dried under vacuum at -50°C for 24 h to obtain a nano-active feverfew extract.

[0028] Example 2 A preparation method of a nano-active feverfew extract, specifically comprising the following steps: S1, 80 g of functionalized lipid, 20 g of cholesterol, 20 g of feverfew extract and 500 mL of anhydrous ethanol were stirred at 55°C for 20 min to obtain a drug core; S2, 100 mg of chitosan was dissolved in 400 mL of 0.5 v / v% concentrated acetic acid aqueous solution, stirred at room temperature for 1 h, and then passed through a 0.45 μm filter membrane to remove undissolved impurities to obtain a chitosan solution; S3, under the ultrasonic power of 150 W, 200 mL of the drug core was injected into 100 mL of 5°C chitosan solution through a multi-hole syringe at the rate of 1 mL / min per hole, and the ultrasonic was continued for 5 min, and then rotary evaporation was performed to obtain a nanoemulsion; S4, trehalose was added to the nanoemulsion at the mass ratio of trehalose to nanoemulsion of 1:15, stirred for 20 min, pre-frozen at -60°C for 5 h, and then freeze-dried under vacuum at -40°C for 30 h to obtain a nano-active feverfew extract.

[0029] Example 3 A preparation method of a nano active spilanthes acuta extract, specifically comprising the following steps: S1, 120 g of functionalized lipids, 30 g of cholesterol, 40 g of spilanthes acuta extract and 500 mL of anhydrous ethanol are stirred at 45°C for 50 min to obtain a drug core; S2, 100 mg of chitosan is dissolved in 100 mL of 2v / v% concentrated acetic acid aqueous solution, stirred at room temperature for 2 h, and then passed through a 0.45 μm filter membrane to remove undissolved impurities, to obtain a chitosan solution; S3, under the ultrasonic power of 150 W, the drug core is injected into 100 mL of 10°C chitosan solution through a multi-hole syringe at a rate of 5 mL / min per hole, and the ultrasonic is continued for 10 min. Rotary evaporation is performed to obtain a nano emulsion; S4, add trehalose to the nano emulsion at a mass ratio of trehalose to nano emulsion of 1:25, stir for 30 min, pre-freeze at -100°C for 2 h, and then freeze dry at -60°C for 20 h, to obtain a nano active spilanthes acuta extract.

[0030] Example 4 The embodiment provides a preparation method of a nano active spilanthes acuta extract, and the specific implementation manner is the same as that in embodiment 1, and the difference lies in that the functionalized lipids in step S1 are replaced by the same amount of soybean phospholipids.

[0031] Example 5 The embodiment provides a preparation method of a nano active spilanthes acuta extract, and the specific implementation manner is the same as that in embodiment 1, and the difference lies in that the cholesterol in step S1 is replaced by the same amount of functionalized lipids.

[0032] Example 6 The embodiment provides a preparation method of a nano active spilanthes acuta extract, and the specific implementation manner is the same as that in embodiment 1, and the difference lies in that step S4 is replaced by: S4, add trehalose to the nano emulsion at a mass ratio of trehalose to nano emulsion of 1:20, stir for 25 min, and freeze dry at -50°C for 24 h, to obtain a nano active spilanthes acuta extract.

[0033] Comparative Example 1 The comparative example provides a preparation method of a nano active spilanthes acuta extract, and the specific implementation manner is the same as that in embodiment 1, and the difference lies in that step S4 is replaced by: S4, the nanoemulsion was first pre-frozen at -80℃ for 3h, and then freeze-dried at -50℃ for 24h under vacuum, to obtain the nanoactive feverfew extract.

[0034] Comparative Example 2 The comparative example provides a preparation method of a nanoactive feverfew extract, specifically comprising the following steps: S1, 100g of functionalized lipids, 25g of cholesterol, 30g of feverfew extract and 500mL of anhydrous ethanol were stirred at 50℃ for 30min to obtain a drug inner core; S2, 200mL of the drug inner core was injected into 100mL of purified water at 7℃ through a multi-hole syringe at a rate of 2mL / min per hole under 150W ultrasonic power for 8min, and rotary evaporation was performed to obtain a nanoemulsion; S3, trehalose was added to the nanoemulsion at a mass ratio of trehalose to nanoemulsion of 1:20, and stirred for 25min, and then pre-frozen at -80℃ for 3h, and then freeze-dried at -50℃ for 24h under vacuum, to obtain the nanoactive feverfew extract.

[0035] Performance test 1. Redispersibility test: 10mg of the freeze-dried nanoactive feverfew extract obtained in Examples 1-6 and Comparative Examples 1-2 was added to 10mL of purified water, and shaken at room temperature for 1min, and whether there were insoluble particles or lumps was observed.

[0036] 2. Polydispersity index PDI and particle size: 10mg of the freeze-dried nanoactive feverfew extract obtained in Examples 1-6 and Comparative Examples 1-2 was added to 10mL of purified water, and dynamic light scattering was used for measurement.

[0037] 3. Stability test: 10mg of the freeze-dried nanoactive feverfew extract obtained in Examples 1-6 and Comparative Examples 1-2 was taken in a vial, and the vial was placed in a condition of 40℃±2℃ / 75% RH±5% RH, and 1mg was taken out after 0, 20, 40 and 60 days, and the characteristic peak area of parthenolide in the freeze-dried nanoactive feverfew extract was analyzed by high performance liquid chromatography, and the percentage of the characteristic peak area of parthenolide in the freeze-dried nanoactive feverfew extract was calculated.

[0038] 4. Encapsulation efficiency test (the percentage of the amount of drug successfully wrapped into the nanoparticles to the total amount of drug): 1mL of the nanoemulsion obtained in Examples 1, 4-5 and Comparative Example 2 was centrifuged to completely settle the nanoparticles. All the supernatant was taken and recorded as S; the precipitate was subjected to demulsification and recorded as P; the parthenolide drug concentration in S and P was measured by HPLC, and the encapsulation efficiency was calculated. The encapsulation efficiency≥90% was recorded as excellent, 90%> encapsulation efficiency≥80% was recorded as good, and the encapsulation efficiency<80% was recorded as unqualified.

[0039] 5. Drug loading test (weight percentage of drug contained in nanoparticles per unit weight): Take 10 mg of nanoemulsion obtained from Example 1, Example 4-5 and Comparative Example 2, and determine the content of spirostanolamides therein using HPLC.

[0040] 6. In vitro sustained release effect test: adopt dialysis bag method, take 10 mL of nanoemulsion obtained from Example 1, Example 4-5 and Comparative Example 2, and load into a dialysis bag with a molecular weight cut-off of 10 kDa, immerse the dialysis bag in 10 mL of release medium; in order to simulate the lubricating environment of vaginal or skin contact, the release medium uses PBS (pH=5) and adds 0.5% w / v of Tween 80, and the temperature is maintained at 37°C to simulate human body temperature; at 0.5, 1, 2, 4 h, take 1 mL from the release medium, and determine the drug concentration using HPLC to calculate the cumulative release percentage at each time point.

[0041] The results of performance tests 1, 2 and 3 are shown in Table 1; the results of performance tests 4, 5 and 6 are shown in Table 2.

[0042] Table 1 Performance test results From the data in Table 1, it can be seen that the particle size of the nanoactive spirostanol extract of Examples 1-3 is in the range of 100-200 nm, and it can be completely reconstituted in one minute, dispersed into a uniform emulsion without obvious particles or lumps, indicating that the freeze-drying process is successful and the process is stable, and the combination of polydispersity index PDI further indicates that the nanoparticles do not undergo irreversible aggregation. In the stability test, the characteristic peak area of spirostanolamide at 20 days, 40 days and 60 days is 98.7%, 95.3% and 92.1% of the initial value, respectively, indicating that the nano-coating method provides good stability support for the active substance.

[0043] Compared with Example 1, Example 4 replaces the same amount of soybean phospholipid with functionalized lipids, although it still maintains the protection of the lipid bilayer, but the absence of PEG chains makes the nanoparticles partially aggregate and fuse during storage; although no insoluble particles or lumps appear during reconstitution, the polydispersity index PDI increases, the particle size range expands, and the stability decreases.

[0044] Compared with Example 1, Example 5 replaces cholesterol with an equal amount of functionalized lipids, which becomes a lipid monolayer wrapped around the spilanthes extract, which has little effect on the redispersibility, polydispersity index PDI, and particle size, but is not conducive to stability. In combination with the results of Comparative Example 1, it is found that if there is no trehalose to fill the lipid bilayer, the structure may also collapse during the freeze-drying process, which is also reflected in the decrease in stability. Example 6 does not use a pre-freezing process, which is not conducive to reducing ice crystal size, which in turn affects the integrity of the nanoparticles, resulting in a part of the stability decrease. Comparative Example 2 does not have a chitosan shell, which reduces the protective effect of the nanoparticle shell, mainly causing the stability to decrease.

[0045] Table 2 Performance test results In combination with Example 1 and Example 4, it can be known that the functionalized lipids, although there is a PEG chain compared with soybean phospholipids, do not have a great impact on the encapsulation efficiency and drug loading, but instead significantly improve the sustained-release effect. Compared with Example 1, Example 5 uses a functionalized lipid monolayer to wrap the spilanthes extract, which has some decrease in encapsulation efficiency, drug loading, and sustained-release effect. In combination with Table 1, it can be known that the stability of the nanoparticles in Comparative Example 2 decreases, which in turn causes the sustained-release process to be faster.

[0046] The above-described examples and comparative examples do not limit the present application in any form, although the present application has been disclosed as above with the preferred embodiments, however, it is not intended to limit the present application, any person skilled in the art can make some changes or modifications to the above-mentioned disclosed technical content as equivalent embodiments without departing from the scope of the technical solutions of the present application, as long as it does not deviate from the technical solution content of the present application, any simple modification, equivalent change and modification of the above examples according to the technical essence of the present application, all still belong to the scope of the technical solutions of the present application.

Claims

1. A method for preparing a nano-active chrysanthemum extract, characterized in that, Specifically, it includes the following steps: S1. Stir the lipids, chrysanthemum extract and anhydrous ethanol at 45-55℃ for 20-50 min to obtain the drug core; S2. Dissolve chitosan in a 0.5-3 v / v% aqueous solution of glacial acetic acid, stir at room temperature for 1-2 hours, and then filter through a membrane to remove undissolved impurities to obtain a chitosan solution. S3. Under ultrasonic power of 100-200 W, the drug core is injected into a chitosan solution at 5-10℃ through a multi-hole syringe at a rate of 1-5 mL / min per hole, and ultrasonication is continued for 5-10 min. Then, the solution is rotary evaporated to obtain nanoemulsion. S4. Add trehalose to the nanoemulsion, stir for 20-30 minutes, and freeze to obtain nano-active chrysanthemum extract.

2. The method for preparing nano-active chrysanthemum extract according to claim 1, characterized in that, In step S1, the lipids comprise functionalized lipids and cholesterol.

3. The method for preparing nano-active chrysanthemum extract according to claim 2, characterized in that, The preparation steps of the functionalized lipids are as follows: PEG and phospholipids were dissolved in an aqueous ethanol solution, and a photoinitiator was added to ensure complete dissolution, resulting in a reaction solution. The reaction solution was then irradiated under an inert gas atmosphere for 5–15 minutes. After the reaction was completed, the solution was purified to obtain the functionalized lipids.

4. The method for preparing nano-active chrysanthemum extract according to claim 3, characterized in that, The PEG ends contain double bonds, and the phospholipid ends contain thiol groups.

5. The method for preparing nano-active chrysanthemum extract according to claim 3, characterized in that, The molar ratio of the phospholipid to PEG is 1:(1.0 to 1.2).

6. The method for preparing nano-active chrysanthemum extract according to claim 2, characterized in that, The mass ratio of the functionalized lipids, cholesterol, and chamomile amide is (80-120):(20-30):(20-40).

7. The method for preparing nano-active chrysanthemum extract according to claim 1, characterized in that, In step S2, the ratio of chitosan to glacial acetic acid aqueous solution is (1-4) mg / mL.

8. The method for preparing nano-active chrysanthemum extract according to claim 1, characterized in that, In step S4, the mass ratio of trehalose to nanoemulsion is 1:(15-25).

9. The method for preparing nano-active chrysanthemum extract according to claim 1, characterized in that, In step S4, the freezing process involves: first pre-freezing at -100 to -60°C for 2 to 5 hours, and then freeze-drying under vacuum at -60 to -40°C for 20 to 30 hours.

10. The use of nano-active chrysanthemum extract obtained by the preparation method according to any one of claims 1 to 9 in an adult lubricant.

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