Natural molecule-based active agent, preparation method and application thereof
An amphiphilic natural molecular-based surfactant was prepared by coupling reaction of cinnamic acid and deacetylated chitosan, which solved the problem of single function of existing natural molecular-derived surfactants and achieved multifunctional emulsification, antibacterial and adsorption effects, which can be applied to food, pharmaceuticals and biomaterials.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NORTHWEST A & F UNIV
- Filing Date
- 2023-09-06
- Publication Date
- 2026-05-19
AI Technical Summary
Existing natural molecular-derived surfactants have limited functionality and cannot simultaneously possess multiple functions such as emulsification, wetting, and adsorption.
Using cinnamic acid and deacetylated chitosan as raw materials, a natural molecular-based surfactant with amphiphilic properties was prepared by reacting deacetylated chitosan with a mixture of coupling reagents 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.
The prepared natural molecular-based surfactant has good emulsifying ability and antibacterial activity, can form a gel network, and can be applied to food, pharmaceuticals, biomaterials and adsorbent materials. It achieves stable emulsification and adsorption of the oil phase, has the property of self-assembling into a colloid, and is safe and non-toxic.
Smart Images
Figure HDA0004434319250000011 
Figure HDA0004434319250000021 
Figure HDA0004434319250000032
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of pharmaceuticals, food, pesticides, coatings, oil extraction, and environmental additives, and specifically relates to a natural molecular-based surfactant, its preparation method, and its application as a surfactant, antibacterial agent, biological matrix, or coating material. Background Technology
[0002] Natural molecules include a wide variety of surfactants derived from proteins, polysaccharides, lignin, and lipids, but they have limited functions and cannot simultaneously possess functions such as emulsification, wetting, and adsorption. Summary of the Invention
[0003] In view of the defects or deficiencies of the prior art, the present invention provides a natural molecular-based surfactant.
[0004] Therefore, the raw materials for preparing the natural molecular-based surfactant provided by the present invention include cinnamic acid, deacetylated chitosan, and coupling reagent; the coupling reagent is selected from one or a mixture of two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.
[0005] Alternatively, the degree of deacetylation of the deacetylated chitosan can be selected to a suitable value, such as a degree of deacetylation ≥ 95%.
[0006] An alternative is that the molar ratio of cinnamic acid, deacetylated chitosan (with a relative molecular weight of 161.16) and the coupling agent is in the range of 1:[1,2]:1; wherein the relative molecular weight of deacetylated chitosan is 161.16.
[0007] This invention also provides a method for preparing the above-mentioned natural molecular-based surfactant, the method comprising the following steps:
[0008] Step 1: Mix the coupling reagent and the cinnamic acid organic solution thoroughly;
[0009] Step 2: The active agent is prepared by mixing the mixed solution obtained in Step 1 with the deacetylated chitosan solution and reacting at room temperature; the deacetylated chitosan solution is prepared by deacetylated chitosan and an aqueous solution of acetic acid.
[0010] An optional approach is that the above preparation method further includes step 3, where the precipitate is collected after the reaction in step 2 is completed, the precipitate is washed and dried to obtain the natural molecular-based surfactant.
[0011] The natural molecular-based surfactants of the present invention can be used as surfactants, antibacterial agents, and as intestinal-targeted delivery matrices for nutrients or drugs.
[0012] The present invention also provides a quercetin emulsion, which is made from quercetin, edible oil and an aqueous solution of the active agent of claim 1.
[0013] The present invention also provides a culture medium that serves as a cell scaffold material, wherein the raw materials for preparing the culture medium include the active agent described in claim 1. In a further embodiment, the culture medium is prepared by mixing an aqueous solution of the natural molecular-based active agent described in claim 1 with n-hexane and then drying the mixture.
[0014] The present invention also provides an aerogel material, which is prepared by drying an aqueous solution containing the active agent of claim 1. The drying is preferably freeze-drying.
[0015] Compared to existing technologies:
[0016] (1) The natural molecular-based surfactant of the present invention has hydrophilic amino and hydroxyl groups and hydrophobic hydrocarbon phenyl groups, exhibiting amphiphilic properties, and is both hydrophilic and lipophilic; it has good emulsifying ability and antibacterial activity; at a certain concentration, it can self-assemble to form a gel network, showing great potential in food, pharmaceuticals, biomaterials and adsorbent materials.
[0017] (2) The natural molecular-based surfactant of the present invention can stabilize 30%-50% of the oil phase after ultrasonic crushing. After emulsification and dehydration, a high internal phase emulsion with an oil phase of ≥75% can be formed. These emulsions remain stable after 90 days of storage.
[0018] (3) The natural molecular-based active agent, preparation method and application of the present invention, a high internal phase emulsion prepared by dissolving the fat-soluble active substance in the oil phase in advance, can achieve intestinal delivery of nutrients or drugs by maintaining stable emulsion droplets after digestion in gastric juice for 2 hours in vitro, and breaking down the emulsion droplets to release the active substance after digestion in intestinal juice for 2 hours.
[0019] (4) The natural molecular-based activator, preparation method and application of the present invention, by replacing the oil phase with 10%-30% n-hexane for emulsification and then freeze-drying, the resulting cell scaffold is safe and non-cytotoxic, and the cells can colonize on the scaffold network structure and grow and reproduce, which is expected to be used in cell meat and tissue engineering.
[0020] (5) The natural molecular-based surfactant, preparation method and application of the present invention, the aerogel obtained by self-assembly and freeze-drying of high concentration natural molecular-based surfactant has excellent wettability and can adsorb methanol, ethanol, water, corn oil and machine oil by tens of times their mass; it can adsorb dyes and be recycled.
[0021] In summary, the active agent of this invention possesses both hydrophilic and hydrophobic groups, giving it amphiphilic properties; it exhibits excellent bactericidal effects against Escherichia coli and Staphylococcus aureus; the naturally derived active agent demonstrates excellent emulsifying properties and storage stability in the preparation of high internal phase emulsions; the high internal phase emulsion containing active substances prepared based on the naturally derived active agent exhibits high stability in gastric juice and has the potential to achieve intestinal-targeted delivery of active substances and drugs in practical applications; the cell scaffold synthesized based on the naturally derived active agent has good biocompatibility, allowing cells to colonize, grow, and reproduce on it; the aerogel formed by the self-assembly of the naturally derived active agent has good wettability to various solvents, enabling the adsorption and recycling of dyes; this naturally derived active agent as a whole possesses characteristics such as safety and non-toxicity, antibacterial properties, emulsification, self-assembly into a colloid, and adsorption. Attached Figure Description
[0022] Figure 1 The 1H NMR spectrum of chitosan, cinnamic acid, and the natural molecular-based surfactant of this invention, as well as the water contact angle, antibacterial activity, and cytotoxicity of the natural molecular-based surfactant; wherein, Figure 1 A is the 1H NMR spectrum of the natural molecular-based surfactant in Example 1. Figure 1 B represents the water contact angle between the natural molecular-based surfactant and chitosan in Example 1. Figure 1 C represents the antibacterial activity of the natural molecular-based surfactant prepared in Example 1. Figure 1 D represents the antibacterial activity of chitosan. Figure 1 E represents the cytotoxicity of the natural molecular-based active agent in Example 1.
[0023] Figure 2 The images shown are: a photograph of the corn oil emulsion prepared in Example 2, a TEM image of the natural molecular-based surfactant nanoparticles and the emulsion; wherein, Figure 2 Image A is a photograph of the corn oil emulsion prepared by emulsification with natural molecular-based surfactants in Example 2, captured by a camera. Figure 2 B is a photograph of the chitosan emulsion and cinnamic acid emulsion prepared in Example 2. Figure 2 C is a TEM image of the natural molecular-based surfactant nanoparticles prepared in Example 2. Figure 2 D is a TEM image of the 50% corn oil emulsion dilution prepared in Example 2.
[0024] Figure 3 The images show the physical composition, microstructure, and digestion process of the high internal phase emulsion prepared in Example 3, as well as the release of active substances during digestion. Figure 3 A is a photograph of a high internal phase emulsion. Figure 3 B is a microscopic image of the high internal phase emulsion stained with Nile Red and FITC, observed under a fluorescence microscope. Figure 3 C is a diagram of the microstructure of emulsion droplets during the digestion of high internal phase emulsions. Figure 3 D is a diagram showing the release of active substances during digestion.
[0025] Figure 4 The images show SEM images of the cell scaffold prepared in Example 4 and images of stained cell nuclei observed under a fluorescence microscope; wherein, Figure 4 A is a SEM image of the cell scaffold. Figure 4 B represents the cell scaffold used for cytotoxicity assessment. Figure 4 C is a SEM image of cells and cell scaffolds after 3 days of continuous culture. Figure 4 D shows the cell nuclei stained on the first and third days of incubation with the cell scaffold prepared in Example 4.
[0026] Figure 5 The image shows the adsorption diagram of the aerogel prepared in Example 5; wherein, Figure 5 A is a bar chart showing the adsorption capacity of Example 5 and chitosan aerogel for different solvents. Figure 5 B is a physical image of aerogel adsorbing dyes and recycling them. Detailed Implementation
[0027] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.
[0028] Deacetylated chitosan itself has a large number of hydrophilic groups and few hydrophobic groups. Based on this, the present invention introduces the phenyl group of cinnamic acid, which makes the deacetylated chitosan amphiphilic and a surfactant. While retaining the original properties and functions of chitosan, it also has emulsifying properties and stronger antibacterial properties.
[0029] The deacetylated chitosan and N-hydroxysuccinimide used in the following examples were purchased from Shanghai Aladdin Co., Ltd., and cinnamic acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were purchased from Shanghai Maclean Co., Ltd. The deacetylated chitosan solution was prepared by dissolving deacetylated chitosan (degree of deacetylation greater than 95%) in a 1% (v / v) aqueous acetic acid solution (the mass-volume ratio of deacetylated chitosan to 1% aqueous acetic acid solution was 1 g: 100 ml).
[0030] Example 1:
[0031] This embodiment describes a method for preparing a natural molecular-based surfactant, comprising the following steps:
[0032] (1) Disperse the cinnamic acid powder of the prescribed amount in an ethanol solution (444.5 mg cinnamic acid / 20 mL ethanol) and stir continuously; then add the 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide of the prescribed amount to the cinnamic acid ethanol solution, stir and react in the dark for 24 hours, wherein the molar ratio of cinnamic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 2:2:1;
[0033] (2) Add the solution obtained in step (1) to the deacetylated chitosan solution (1 g chitosan corresponds to 444.5 mg cinnamic acid), stir continuously at 30°C, and react in the dark for 24 hours.
[0034] (3) The reaction solution obtained in step (2) is precipitated with acetone, centrifuged and the precipitate is collected and washed with ethanol three times to remove impurities. After washing with water once, it is freeze-dried to obtain the natural molecular-based surfactant.
[0035] The natural molecular-based surfactant prepared in this embodiment was identified by 1H NMR spectroscopy, and the results are as follows: Figure 1 As shown in Figure A.
[0036] The water contact angles of the natural molecular-based surfactant and deacetylated chitosan prepared in this example were measured using a contact angle meter. Figure 1 As shown in B.
[0037] The antibacterial activity of the natural molecular-based active agent and deacetylated chitosan prepared in this embodiment was tested (the *Escherichia coli* used was ATCC 25922, and the *Staphylococcus aureus* used was ATCC 6538). The test method is described in: Xinyu Sun, Lihua Li, Hui Zhang, et al. Near-Infrared Light-Regulated Drug-Food Homologous Bioactive Molecules and Photothermal Collaborative Precise Antibacterial Therapy Nanoplatform with Controlled Release Property. Advanced Healthcare Materials. 2021, 2100546.; the results are as follows. Figure 1 As shown in C and 1D.
[0038] The cytotoxicity of the natural molecular-based bioactive agent prepared in this embodiment was evaluated using Caco-2 cells. The evaluation method was the MTT assay (see: Lihua Li, Xinyu Sun, Hui Zhang, et al. Amphiphilic nano-delivery system based on modified-chitosan and ovalbumin: Delivery and stability insimulated digestion. Carbohydrate Polymers, 2022, 294:119779). The results are as follows: Figure 1 As shown in E.
[0039] The natural molecular-based surfactant prepared in this embodiment successfully grafted cinnamic acid onto the chitosan molecular chain, with a water contact angle located at the boundary between hydrophilic and hydrophobic properties, exhibiting amphiphilic characteristics and excellent antibacterial activity.
[0040] Example 2:
[0041] This embodiment illustrates the application of natural molecular-based surfactants in emulsion preparation, including the following steps:
[0042] Step 1: The natural molecular-based surfactant obtained in Example 1 is used as an emulsifier and specifically dispersed in water at a concentration of 1 mg / mL. The mixture is then sonicated for 3 min to obtain an aqueous solution of nanoparticles.
[0043] Step 2: Add corn oil to the aqueous solution of nanoparticles obtained in Step 1, and continue to sonicate to prepare an emulsion containing 30%-50% (v / v) oil phase.
[0044] Cinnamic acid emulsion and deacetylated chitosan emulsion containing 50% corn oil were prepared as controls using the same method as in this embodiment (the activator in step 1 was replaced with cinnamic acid and deacetylated chitosan, respectively).
[0045] The physical images of the emulsion prepared in this embodiment were recorded using a camera, such as... Figure 2 As shown in Figure A. Cinnamic acid emulsion and deacetylated chitosan emulsion, as... Figure 2 As shown in B.
[0046] The microstructures of the natural molecular-based surfactant nanoparticles and the 50% oil-containing emulsion prepared in this example were observed using TEM, as shown below. Figure 2 As shown in C and 2D.
[0047] In this embodiment, a natural molecular-based surfactant is used as an emulsifier. The natural molecular-based surfactant can self-assemble into nanoparticles under ultrasound. As a Pickering emulsion, the nanoparticles surround the oil droplets and the emulsion with an oil content of 30%-50% is successfully prepared by ultrasound. These emulsions are stable and homogeneous, while cinnamic acid emulsion and deacetylated chitosan emulsion are unstable and show stratification. The natural molecular-based surfactant has better emulsifying ability.
[0048] Example 3:
[0049] This embodiment illustrates the application of natural molecular-based surfactants in the preparation of high internal phase emulsions, including the following steps:
[0050] Step 1: Add quercetin to corn oil (edible oil) at a concentration of 500 μg / mL, and sonicate until completely dissolved in corn oil to obtain quercetin oil solution;
[0051] Step 2: Following the method in Example 2 (i.e., replacing the corn oil in Step 2 of Example 2 with a quercetin oil solution), use the quercetin oil solution in Step 1 of this Example to prepare a quercetin oil emulsion (quercetin content of 50% by mass). Then place the emulsion in a graduated open container, such as a beaker, and incubate at 37°C to remove water until the oil content reaches 75%, thus obtaining a quercetin high internal phase emulsion.
[0052] The quercetin high internal phase emulsion prepared in this embodiment was photographed, observed under a fluorescence microscope after staining, and observed under an optical microscope during digestion (the digestion method is described in: Lihua Li, Zhanli Zhao, Xin Wang, et al. Self-assembled emulsion gel based on modified chitosan and gelatin: Anti-inflammatory and improving cellular uptake of lipid-soluble actives. International Journal of Biological Macromolecules. 2023, 231:123300.). The amount of active substance released was calculated (the method for testing and calculating the amount of active substance released is described in: Lihua Li, Xinyu Sun, Hui Zhang, et al. Amphiphilic nano-delivery system based on modified-chitosan and ovalbumin: Delivery and stability in simulated digestion. Carbohydrate Polymers, 2022, 294:119779.). Free pure quercetin was used as a control. The results are as follows: Figure 3 As shown in A-3D.
[0053] The high internal phase emulsion prepared in this embodiment is semi-solid, plastic, and storage stable. During digestion, it can maintain stable droplets in gastric juice without being broken. After digestion in intestinal juice, the droplets rupture and release a large amount of encapsulated active substances, which is expected to achieve intestinal-targeted delivery of nutrients and drugs. In contrast, free quercetin is 60% released after 2 hours of digestion in gastric juice and has no gastric juice stability or intestinal-targeting function.
[0054] Example 4
[0055] This embodiment illustrates the application of natural molecular-based surfactants in cell scaffold preparation, including the following steps:
[0056] Step 1: Disperse the natural molecular-based surfactant prepared in Example 1 in water at a concentration of 3 mg / mL, sonicate for 3 min, then add 10%-30% (the percentage of n-hexane volume to the total emulsion volume) of n-hexane, sonicate for 6 min, and emulsify to obtain n-hexane emulsions with volume percentages of 10%, 20%, and 30%, respectively.
[0057] Step 2: Pre-freeze the n-hexane emulsion from Step 1 at -80°C for 4 hours, then add liquid nitrogen to make the n-hexane evaporate instantly, and then freeze-dry it in a freeze dryer at -52°C to obtain three types of cell scaffolds.
[0058] Step 3: Seed NIH-3T3 cells onto the cell scaffold prepared in Step 2, which has been sterilized by overnight soaking in 75% ethanol. Add sufficient NIH-3T3 cell culture medium and culture continuously in a cell culture incubator for 1 day and 3 days. Discard the cell culture medium and critically dry or stain with DAPI to obtain the cell scaffold containing cells.
[0059] The cell scaffold prepared in this embodiment was observed by SEM, and the results are as follows: Figure 4 As shown in Figure A.
[0060] The cell scaffold prepared in this embodiment was evaluated for cytotoxicity, and the results are as follows: Figure 4 As shown in B.
[0061] The cell scaffold containing cells prepared in this embodiment was observed by SEM and by fluorescence microscopy after DAPI staining of cell nuclei. The results are as follows: Figure 4 As shown in C and 4D.
[0062] The cell scaffold network prepared in this embodiment is dense, has uniform pores, and is non-cytotoxic, allowing cells to successfully colonize the cell scaffold and continuously proliferate.
[0063] Example 5:
[0064] This embodiment illustrates the application of natural molecular-based surfactants in aerogel preparation, including the following steps:
[0065] After the natural molecular-based surfactant is dissolved in water, it is stirred continuously until it is evenly distributed in the water. Then, it is left to stand at room temperature for 4 hours to allow it to self-assemble into a gel network. It is then placed in a -80℃ freezer and finally freeze-dried directly under vacuum at -52℃ to obtain a sponge-like aerogel.
[0066] Meanwhile, chitosan aerogel was prepared by freeze-drying a deacetylated chitosan solution.
[0067] The adsorption capacity of the aerogels and chitosan aerogels prepared in this embodiment for different solvents was measured (the adsorption capacity measurement method is described in: Jung Hwal Shina, Jun-Ho Heo, Seunggyu Jeon, et al. Bio-inspired hollow PDMS sponge for enhanced oil–water separation. Journal of Hazardous Materials. 2019, 365:494-501.), and the results are as follows. Figure 5 As shown in Figure A.
[0068] The aerogel prepared in this embodiment shows physical images of the adsorption of dyes and their recycling (for the test methods of adsorption dyes and recycling, please refer to: Jung Hwal Shina, Jun-Ho Heo, Seunggyu Jeon, et al. Bio-inspired hollow PDMS sponge for enhanced oil–water separation. Journal of Hazardous Materials. 2019, 365:494-501.). The results are as follows. Figure 5 As shown in Figure B, the aerogel exhibits the ability to adsorb dyes in various solvents (corn oil, ethanol, and water). After adsorbing dyes, it can be further extracted and collected by soaking it in an ethanol solution, thus enabling the recycling and reuse of the aerogel. Taking ethanol as an example, when the dye concentration is low, the aerogel can be completely recycled, and the recycled aerogel is identical to the original aerogel.
[0069] The aerogel prepared using natural molecular-based surfactants in this embodiment has better wettability to methanol, ethanol, water, corn oil, and engine oil compared to pure chitosan aerogel. It can adsorb dyes in water, ethanol, and oil and recycle them.
Claims
1. The application of natural molecular-based surfactants as intestinal-targeted delivery matrices for nutrients or drugs; the raw materials for preparing the natural molecular-based surfactants include cinnamic acid, deacetylated chitosan, and a coupling agent; the coupling agent is selected from one or a mixture of two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the degree of deacetylation of the deacetylated chitosan is ≥95%; the molar ratio of cinnamic acid, deacetylated chitosan, and the coupling agent is in the range of 1:[1,2]:1, wherein the relative molecular weight of the deacetylated chitosan is 161.
16.
2. The application according to claim 1, characterized in that, The preparation method of the aforementioned natural molecular-based surfactant includes: Step 1: Mix the coupling reagent and the cinnamic acid organic solution thoroughly; Step 2: The active agent is prepared by mixing the mixed solution obtained in Step 1 with the deacetylated chitosan solution and reacting at room temperature; the deacetylated chitosan solution is prepared by deacetylated chitosan and an aqueous solution of acetic acid.
3. The application according to claim 2, characterized in that, The preparation method further includes step 3, where the precipitate is collected after the reaction in step 2 is completed, and the precipitate is washed and dried to obtain the natural molecular-based surfactant.