A microcapsule structure, its preparation method and application

Abstract

The application provides a microcapsule structure and a preparation method and application thereof, and the microcapsule structure comprises Chinese Litsea cubeba essential oil, beta-cyclodextrin, chitosan and inulin; the Chinese Litsea cubeba essential oil is located in the porous structure of the beta-cyclodextrin to form a complex; the complex is located in a three-dimensional structure formed by graft polymerization of the chitosan and the inulin to form the microcapsule structure. The Chinese Litsea cubeba essential oil is coated in the beta-cyclodextrin-chitosan-inulin to form the microcapsule structure, so that the irritability and volatility of the Chinese Litsea cubeba essential oil are greatly reduced, and the water solubility, stability and antibacterial activity are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a microcapsule structure and a preparation method and application thereof. BACKGROUND

[0002] Litsea cubeba essential oil (LCEO) is a natural essential oil extracted from the bark, fruit and leaves of Litsea cubeba in the Lauraceae family, which contains various components and has a wide range of applications in food, medicine and agriculture. Studies have shown that LCEO has inhibitory effects on various gram-negative and gram-positive bacteria, fungi, and has antioxidant, antiasthmatic, antiallergic and anthelmintic effects. However, due to its strong volatility, poor water solubility and strong irritation, its application is limited.

[0003] Beta-cyclodextrin (β-CD) has a special cavity structure and can complex various water-insoluble drugs, providing a microenvironment to enhance stability, delay volatilization, and easily form stable hydrates. However, this technology cannot achieve complete embedding of active substances, i.e. some active substances are lost during microencapsulation.

[0004] Chitosan has antibacterial activity against bacteria, algae, fungi and other microorganisms, but generally requires a relatively high dose of chitosan to achieve optimal antibacterial activity, especially against bacteria in biofilms. Chitosan can adsorb anions, making its surface gather numerous chlorine aggregates and become rougher; in addition, the low water solubility and high viscosity of chitosan limit its application.

[0005] Therefore, there is an urgent need to develop a new substance to coat LCEO to achieve sustained release and protection of LCEO. SUMMARY

[0006] In view of the above constraints, the present application provides a microcapsule structure; LCEO is coated in beta-cyclodextrin-chitosan-inulin, which can greatly reduce the irritation and volatility of LCEO, while enhancing water solubility, improving stability and antibacterial activity, overcoming the deficiencies and defects mentioned in the background art.

[0007] To achieve the above purpose, the present application adopts the following technical solutions:

[0008] The present application provides a microcapsule structure, which comprises LCEO, beta-cyclodextrin, chitosan and inulin; LCEO is located in the porous structure of beta-cyclodextrin to form a complex; the complex is located in the three-dimensional structure formed by grafting polymerization of chitosan and inulin to form a microcapsule structure.

[0009] Optionally, the ratio of the mass of β-cyclodextrin to the sum of the mass of chitosan and inulin is (1-5):1; the wall-to-core ratio of β-cyclodextrin, chitosan, inulin and Litsea cubeba essential oil is (1-10):1.

[0010] Optionally, the ratio of the mass of β-cyclodextrin to the sum of the mass of chitosan and inulin is 2:1; the wall-to-core ratio of β-cyclodextrin, chitosan, inulin and Litsea cubeba essential oil is 3:1.

[0011] Optionally, the particle size of the complex is 90-190 nm; the particle size of the microcapsule structure is 450-850 nm. Optionally, the mass ratio of chitosan to inulin is 1:(1-10).

[0012] Another application point of the present application is to provide a preparation method of the microcapsule structure as described in any of the above. Optionally, the preparation method comprises: (1) adding Litsea cubeba essential oil into a solution containing β-cyclodextrin to perform inclusion to obtain a complex; (2) mixing inulin and chitosan in a solution, adjusting the pH to 3.5-5.5, performing steam pressure and cooling to obtain a solution containing chitosan-inulin graft polymer; (3) adding the complex into the solution containing the chitosan-inulin graft polymer in the presence of a crosslinking agent to perform crosslinking to obtain the microcapsule structure.

[0013] Optionally, step (1) comprises: adding Litsea cubeba essential oil dropwise into a saturated aqueous solution of β-cyclodextrin and stirring at a temperature of 20-60°C to obtain the complex.

[0014] Optionally, in step (2), the inulin and chitosan are dissolved in a solution respectively to form an inulin solution and a chitosan solution, and then the solution mixing is performed; in the inulin solution, the mass-to-volume ratio of inulin is 0.1-5.0%; in the chitosan solution, the mass-to-volume ratio of chitosan is 0.1-1.0%.

[0015] Optionally, step (3) comprises: adding the complex into the graft polymer solution, continuously stirring at a speed of 1000-1400 r / min under heating conditions of 30-60°C for 0.5-1 h; then adding a crosslinking agent and adjusting the pH to 5.5-6.5 to obtain the microcapsule structure; the crosslinking agent comprises at least one of sodium tripolyphosphate, glutaraldehyde and formaldehyde.

[0016] Another application point of the present application is to provide a use of the microcapsule structure as described in any of the above in the preparation of a bacterial infection drug.

[0017] Compared with the prior art, the technical solution of the present application has the following beneficial effects:

[0018] (1) The application utilizes the anion adsorption capacity of chitosan and the cavity structure of β-cyclodextrin to prepare chitosan / β-cyclodextrin complexes, which have increased specific surface area, increased adsorption sites and increased adsorption rate. After in-grafting inositol with chitosan, the physical and chemical properties of chitosan can be changed, the surface roughness can be avoided, the solubility and physical stability of chitosan can be increased, and the biological activity can be improved. The prepared Litsea cubeba essential oil-β-cyclodextrin-chitosan-inositol microcapsule structure (LCEO / β-CD / CS-IL NPs) not only improves the stability and water solubility of Litsea cubeba essential oil, but also masks the irritating odor of the essential oil. With the participation of inositol, the copolymer microcapsule can not be destroyed by gastric juice and small intestinal juice, so that it can smoothly enter the colon and be degraded and released in the colon, and has obvious colon targeting effect.

[0019] Compared with Litsea cubeba essential oil-β-cyclodextrin and Litsea cubeba essential oil-hydroxypropyl-β-cyclodextrin inclusion complex, the LCEO / β-CD / CS-IL NPs microcapsule structure of the application has obvious sustained release effect and colon targeting effect, greatly reduces the irritation of Litsea cubeba essential oil to the stomach, has more prominent bacteriostatic effect, and has wider application space in the fields of veterinary drugs, feed additives and the like. The application of the microcapsule structure in animal pathogenic bacterial diseases has good effect on preventing and treating animal bacterial infections, and has important significance for the prevention and control of bacterial diseases in animal husbandry.

[0020] (2) The application prepares LCEO / β-CD / CS-IL NPs using LCEO as raw material, solves the stability and volatility problems of LCEO, and provides experimental basis for the development and application of LCEO in the field of veterinary medicine. Moreover, the preparation method of the application is simple and reliable, and is convenient for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a schematic diagram of the assembly process and release process of the microcapsule structure of the application.

[0022] Figure 2 It is an infrared spectrum diagram of β-cyclodextrin, Litsea cubeba essential oil, complex, chitosan-inositol graft copolymer and microcapsule structure in an embodiment of the application; (A) is LCEO, β-CD, LCEO / β-CD ICs, the abscissa is wavelength, unit: cm -1 ; ordinate is transmittance, unit: %; (B) is LCEO / β-CD ICs, CS-IL, LCEO / β-CD / CS-IL NPs, the abscissa is wavelength, unit: cm -1 ; ordinate is transmittance, unit: %.

[0023] Figure 3Particle size distribution diagram of the complex and microcapsule structure in an embodiment of the present application; (A) is LCEO / β-CD ICs, the abscissa is size, unit: nm; the ordinate is percentage, unit: %; (B) is LCEO / β-CD / CS-IL NPs, the abscissa is size, unit: nm; the ordinate is percentage, unit: %.

[0024] Figure 4 Scanning electron microscope diagram of the complex and microcapsule structure in an embodiment of the present application; (A) is the complex; (B) is the microcapsule structure.

[0025] Figure 5 In-vitro release curve diagram of the complex (LCEO / β-CD ICs) and microcapsule structure (LCEO / β-CD / CS-IL NPs) in an embodiment of the present application; the abscissa is time, unit: min; the ordinate is cumulative release amount, unit: %.

[0026] Figure 6 Scanning electron microscope diagram of the copolymer in Comparative Example 3 of the present application.

[0027] Figure 7 In-vitro release curve diagram of the copolymer (LCEO / β-CD / CS-IL in situ) in Comparative Example 3 of the present application and the microcapsule structure (LCEO / β-CD / CS-IL) in Test Example 1; the abscissa is time, unit: min; the ordinate is cumulative release amount, unit: %.

[0028] Figure 8 Scanning electron microscope of the microcapsule structure in Test Example 1 of the present application after acting on E. coli and Staphylococcus aureus; (A) is E. coli; (B) is Staphylococcus aureus; (C) is E. coli after adding microcapsules containing essential oil; (D) is Staphylococcus aureus after adding microcapsules containing essential oil.

[0029] Figure 9 Particle size distribution diagram of the microcapsule structure prepared at 20℃ and 30℃ in Embodiment 6 of the present application; (A) is 20℃; (B) is 30℃. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application, and is not used to limit the scope of the present application.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0032] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments. Figure 1 This is a schematic diagram of the preparation method of this application, as shown below. Figure 1 As shown, β-cyclodextrin has a unique cavity structure. Litsea cubeba essential oil is encapsulated within the cavity of β-cyclodextrin, forming a stable hydrate, i.e., a complex. Through graft copolymerization of chitosan and inulin, specifically by redox reactions of glucose residues at the inulin terminal with amino and hydroxyl functional groups of chitosan, a chitosan-inulin graft copolymer is formed. Under the cross-linking action of a cross-linking agent, the complex cross-links with the chitosan-inulin graft copolymer, causing the complex to be located in the voids of the chitosan-inulin graft copolymer, forming an intertwined and multi-layered microcapsule structure. After entering the target body, corresponding enzymes can decompose the wall material, thereby releasing Litsea cubeba essential oil.

[0033] Example 1

[0034] This embodiment provides a microcapsule structure comprising Litsea cubeba essential oil, β-cyclodextrin, chitosan, and inulin; the Litsea cubeba essential oil is located within the porous structure of β-cyclodextrin, forming a complex; the complex is located within a three-dimensional structure formed by the graft polymerization of chitosan and inulin, forming a microcapsule structure.

[0035] β-Cyclodextrins (β-CD) possess a unique cavity structure, allowing them to encapsulate guest molecules within the cavity without altering their chemical structure, forming stable hydrates. As a novel pharmaceutical excipient, β-CD can not only mask the irritating odor of drugs and increase their stability, but also improve their solubility and bioavailability.

[0036] Inulin is a low-toxicity, highly biocompatible, water-soluble polysaccharide with some antibacterial properties. It often ends with a glucose residue, allowing it to form polymers with other macromolecules through redox reactions. Chitosan (CS), a cationic polysaccharide derived from the deacetylation of chitin, is one of the most promising polymers currently available, possessing many significant properties. These properties include biofunctionality, biocompatibility, biodegradability, chemical resistance, relative thermal stability, and non-toxicity. Furthermore, due to the presence of amino and hydroxyl functional groups, this polymer can form complexes with inulin and other macromolecules through certain chemical reactions.

[0037] Inulin is a water-soluble polydisperse oligomer containing 10-65 fructose units (usually 12-15) and can be used as dietary fiber, prebiotic sugar, fat / sugar substitute, thickening agent and gelling agent in low-calorie food. Small intestinal enzymes (such as maltase, sucrase, a-amylase) in the digestive tract of mammals cannot degrade inulin, but can be selectively fermented by probiotics in the colon to form low molecular weight compounds such as organic acids. The presence of fructose, glucose and levan produced by natural enzymatic or hydrolysis reaction indicates that inulin contains about 4.1% reducing sugar, which can be combined with chitosan through Maillard reaction to prepare a chitosan-inulin conjugate with antibacterial properties as a new coating material, further improving the physical properties of microspheres and better protecting the drug from degradation during simulated gastrointestinal digestion.

[0038] Neither gastric nor small intestinal fluid contains enzymes and other substances that can degrade inulin, and only some bacteria living in the colon can ferment it, so grafting inulin with chitosan to form a new polymer can effectively protect the β-cyclodextrin inclusion compound from being destroyed by gastrointestinal fluid and also reduce the irritation of the essential oil of Litsea cubeba to the gastrointestinal tract.

[0039] The inclusion technology can be used to package the essential oil of Litsea cubeba, which can reduce its irritability, enhance its water solubility, and significantly improve its stability and preservation rate, thereby further broadening its industrial application.

[0040] The ratio of the mass of β-cyclodextrin to the sum of the mass of chitosan and inulin is (1-5): 1.

[0041] When the ratio of β-cyclodextrin is > 5:1, it is difficult to form complete microcapsules, and the protective effect of β-cyclodextrin on the essential oil of Litsea cubeba is significantly reduced; when the ratio is < 1:1, the yield of microcapsules decreases, the formed microcapsules are sticky, the particle size is uneven, and clumping occurs, which causes certain difficulties for drug delivery; and the sustained-release process also cannot effectively release the drug due to the adhesion.

[0042] Preferably, the ratio of the mass of β-cyclodextrin to the mass of chitosan-inulin graft copolymer is 2:1.

[0043] The hydrophilic groups (amide and carboxylic acid groups) on the surface of β-cyclodextrin can form intermolecular hydrogen bonds with the free hydroxyl groups on chitosan, thereby generating a cyclodextrin-chitosan inclusion compound. The chitosan-inulin graft copolymer formed after the Maillard reaction retains the original hydroxyl groups of chitosan and also forms a Schiff base (C=double bond), further improving the binding capacity of the copolymer with β-cyclodextrin, i.e., relative to chitosan, the chitosan-inulin graft copolymer also has other sites that can bind to β-cyclodextrin.

[0044] Therefore, β-cyclodextrin can be combined with both the free hydroxyl groups on chitosan and the Schiff base on the chitosan-inulin graft copolymer, and the combination is most stable when the ratio is 2:1.

[0045] Although more (or less) β-cyclodextrin can be encapsulated by the chitosan-inulin graft copolymer, the stability, encapsulation rate and yield are all lower than the ratio of 2:1.

[0046] When the ratio of β-cyclodextrin: chitosan-inulin graft copolymer is 2:1, the microcapsule has the highest encapsulation rate (yield 79.33%, encapsulation rate 84.40%) and the best sustained-release effect, and when the ratio is 1:1, the yield is only 64.34% and the encapsulation rate is only 71.22%; when the ratio is 3:1, the yield is 61.70% and the encapsulation rate is 67.71%; when the ratio is 4:1, the yield is 53.64% and the encapsulation rate is 66.42%; and when the ratio is 5:1, the yield is 39.54% and the encapsulation rate is 51.73%.

[0047] The ratio of β-cyclodextrin, chitosan, inulin and Litsea cubeba essential oil is (1-10):1.

[0048] Preferably, the ratio of β-cyclodextrin, chitosan, inulin and Litsea cubeba essential oil is 3:1.

[0049] The mass ratio of chitosan and inulin is 1:(1-10), and preferably 1:5.

[0050] The particle size of the complex is 90-190 nm, and the particle size of the microcapsule structure is 450-850 nm.

[0051] Example 2

[0052] The present example provides a preparation method of a microcapsule structure, which has the same description as the microcapsule structure of Example 1 and will not be repeated here.

[0053] The preparation method comprises: (1) adding Litsea cubeba essential oil into a solution containing β-cyclodextrin to perform inclusion to obtain a complex; (2) mixing inulin and chitosan in a solution, adjusting the pH to 3.5-5.5, performing steam pressure and cooling to obtain a solution containing chitosan-inulin graft polymer; and (3) adding the complex into the solution containing the chitosan-inulin graft polymer in the presence of a crosslinking agent to crosslink, thereby obtaining a microcapsule structure. Step (1) comprises: adding Litsea cubeba essential oil dropwise into a saturated aqueous solution of β-cyclodextrin, and stirring at a temperature of 20-60°C to obtain the complex.

[0054] The saturated aqueous solution of β-cyclodextrin is continuously stirred at a speed of 400-600 r / min for 0.5-2 h at a temperature of 30-60°C; preferably, the saturated aqueous solution of β-cyclodextrin is continuously stirred at a speed of 400 r / min for 1 h at a temperature of 40°C.

[0055] The complex is obtained by adding the oil of Litsea cubeba dropwise to a saturated aqueous solution of β-cyclodextrin, and continuously stirring at a speed of 600-1200 r / min at a temperature of 20-60°C for 2-6 h; preferably, the complex is obtained by continuously stirring at a speed of 1000 r / min at a temperature of 40°C for 4 h.

[0056] After step (1), the solution of the complex is first cooled to room temperature, and then placed in a 4°C refrigerator to cool overnight, and the precipitate is obtained by reduced pressure filtration, and the precipitate is washed with anhydrous ethanol and distilled water for three times, respectively, to obtain the complex in powder form.

[0057] In step (2), the inulin and chitosan are dissolved in solutions respectively to form an inulin solution and a chitosan solution, and then the solutions are mixed; in the inulin solution, the mass-volume ratio (w / v) of inulin is 0.1-5.0%; in the chitosan solution, the mass-volume ratio of chitosan is 0.1-1.0%.

[0058] The chitosan or inulin is dissolved in a 1% v / v glacial acetic acid solution, and stirred at a speed of 400-800 r / min at 40°C for 1-5 h to prepare a 1% w / v chitosan solution or inulin solution, and the solution is stored at 4°C for further hydration for 12-24 h.

[0059] The chitosan or inulin is dissolved in a 1% v / v glacial acetic acid solution, and stirred at a speed of 600 r / min at 40°C for 2 h to prepare a 1% w / v chitosan solution or inulin solution, and the solution is stored at 4°C for further hydration for 24 h. The inulin solution and the chitosan solution are mixed at a ratio of (1-10):1, and the pH of the solution is maintained at 3.5-5.5, and the polymerization is performed at 80-110°C.

[0060] Preferably, the pH is adjusted to 3.5-5.5 by using 0.1M NaOH.

[0061] Preferably, the inulin solution and the chitosan solution are mixed at a ratio of 5:1, and stirred for 1 h, the pH is adjusted to 5.0 by using 0.1M NaOH, and the heating is performed at 80-110°C to obtain the chitosan-inulin graft polymer.

[0062] The chitosan-inulin graft polymer needs to be heated at 80-110°C for 5-15 min for polymerization, and the pH of the solution is maintained at 3.5-5.5.

[0063] Too low temperature (<80°C) cannot form the polymer, too high temperature (>110°C) can cause the polymer to discolor and paste, and the pH of the solution >5.5 can cause the chitosan to precipitate and cannot be polymerized, and the pH of the solution <3.5 can cause the polymerization rate to be too low, and the polymer formed has too low degree of polymerization and is easy to decompose.

[0064] The solution of the chitosan-inulin polymer is then rapidly cooled in an ice water bath.

[0065] Step (3) comprises: adding the complex to the graft polymer solution, stirring at a speed of 1000-1400 r / min under heating conditions at 30-60℃ for 0.5-1 h; then adding a crosslinking agent, adjusting the pH to 5.5-6.5, to obtain a microcapsule structure.

[0066] The crosslinking agent comprises at least one of sodium tripolyphosphate, glutaraldehyde (2.5% v / v), and formaldehyde (2.5% v / v). The amount of the crosslinking agent added is 2.5%-10% of the mass of the chitosan-inulin graft polymer.

[0067] 6.0% sodium tripolyphosphate (TPP) is added for crosslinking for 1 h, and the pH of the microcapsule solution is adjusted to 5.5-6.5 using a 0.1 mol sodium hydroxide (NaOH) solution.

[0068] 6.0% sodium tripolyphosphate (TPP) is added for crosslinking for 1 h, and the pH of the microcapsule solution is adjusted to 6.2 using a 0.1 mol sodium hydroxide (NaOH) solution.

[0069] Step (3) is followed by step (4); step (4) comprises: rapidly cooling the solution containing the microcapsule structure to 4℃ in an ice water bath, continuing to stir for 1 h, cooling in a 4℃ refrigerator overnight, and filtering the precipitate under reduced pressure.

[0070] Step (4) is followed by step (5); step (5) comprises: washing the precipitate twice with petroleum ether and anhydrous ethanol, respectively, and freeze-drying to obtain the finished microcapsule structure.

[0071] Example 3

[0072] The present example provides a use of the microcapsule structure as described in any of the above in the preparation of a bacterial infection drug. The bacteria is Escherichia coli or Staphylococcus aureus.

[0073] Example 4

[0074] According to the content of the present application, the microcapsule structure of Example 1 and the preparation method of Example 2 are specifically described as follows:

[0075] The encapsulation efficiency calculation formula is:

[0076]

[0077] The yield calculation formula of the microcapsule structure is:

[0078]

[0079] The formula for calculating the overall score is: Overall score = 30 × microcapsule yield + 70 × encapsulation rate.

[0080] Experimental Example 1

[0081] (1) 1.0 g of Litsea cubeba essential oil was slowly added dropwise to 2.0 g of saturated aqueous solution of β-cyclodextrin; the mixture was heated at 40 °C and stirred continuously at 1000 r / min for 4 h, cooled to room temperature, and then cooled overnight in a refrigerator at 4 °C to obtain a Litsea cubeba essential oil-β-cyclodextrin inclusion complex solution. The solution was filtered under reduced pressure, and the precipitate was washed three times with anhydrous ethanol and double-distilled water, respectively. The washed precipitate was collected and freeze-dried to obtain β-CD / LCEO ICs powder (complex).

[0082] (2) Inulin and chitosan were dissolved separately in acetic acid solution to obtain a solution with a content of 1% w / v. The inulin and chitosan solutions were mixed and stirred for 1.0 h in a ratio of 5:1. The pH was adjusted to 5.0 with 0.1M NaOH solution to obtain a chitosan-inulin mixed solution. According to the Maillard reaction principle, the chitosan-inulin solution was heated at 100℃ for 15 minutes and then rapidly cooled in an ice water bath to form a solution containing chitosan-inulin grafted polymer.

[0083] (3) The β-CD / LCEO ICs powder (complex) was slowly added to a solution containing inulin-chitosan grafted polymer (1.0 g), and stirred continuously at 1200 r / min for 0.5 h under heating conditions at 40 °C. Then, 6.0% sodium tripolyphosphate (TPP) was added to the solution for crosslinking for 1 h, and the pH was adjusted to 6.2 with 0.1 mol sodium hydroxide (NaOH) solution to obtain a Litsea cubeba essential oil-β-cyclodextrin-chitosan-inulin microcapsules (LCEO / β-CD / CS-IL NPs) solution (a solution containing microcapsule structure).

[0084] (4) Place the solution containing the microcapsule structure in an ice-water bath and cool it rapidly to 4°C. Continue stirring for 1 hour and then place it in a 4°C refrigerator overnight. Filter under reduced pressure to obtain the precipitate. Wash the precipitate twice with petroleum ether and anhydrous ethanol, respectively, and freeze-dry to obtain the finished microcapsule structure.

[0085] Figure 2 Figures (A) and (B) contain the infrared spectra of β-cyclodextrin (β-CD), Litsea cubeba oil (LCEO), complex (LCEO / β-CD-ICs), chitosan-inulin graft copolymer (CS-IL), and microcapsule structure (LCEO / β-CD / CS-IL NPs).

[0086] Among them, by Figure 2 As shown in Figure (A), the infrared spectrum of β-CD has three characteristic peaks, namely at 2922 cm⁻¹. -1nearby -CH2, -CH3 stretching vibration peak, 1 630 cm -1 nearby C=O stretching vibration peak and at 1157 cm -1 nearby C-O-C stretching vibration peak. In the infrared spectrum of the complex, the three characteristic peaks can be observed, the position of each characteristic absorption peak is basically similar, the peak intensity difference is not big, indicating that the basic skeleton of β-CD before and after inclusion still exists and does not change greatly. In the infrared spectrum of Litsea cubeba essential oil, 1620~1680 cm -1 are two characteristic absorption peaks of its main component citral, the two absorption peaks do not appear on the spectrum of the complex, which may be due to the fact that after the essential oil enters the cavity of β-CD, the conjugation effect occurs between the C=O of β-CD at 1630 cm -1 , resulting in the decrease of the peak intensity of the microcapsule at this point, the peak shape is broadened and red shifted, and the two characteristic peaks of Litsea cubeba essential oil are completely covered by the host molecule β-CD. It can be judged that the Litsea cubeba essential oil and β-CD form a complex.

[0087] From the (B) figure of Figure 2 , the -CH2, -CH3 stretching vibration peak existing near 2920 cm -1 is covered by CS-IL, and the phenomenon of smaller absorption peak appears, a new amide peak appears at 1560 cm -1 of the spectrum of the microcapsule structure, which may be due to the fact that the electrostatic interaction between the chitosan-inulin graft copolymer and the phosphate group of TPP causes the blue shift of the absorption wave at 1674 cm -1 of the complex. The C-C bond existing near 937 cm -1 is covered by the CS-IL polymer; the above infrared spectrum shows that the microcapsule structure is formed.

[0088] The structure and particle size distribution of the complex and the microcapsule structure are also detected, and the results are shown in Figure 3 and Figure 4 . The particle size of the complex ranges from 91 to 190 nm, and its morphology is plate-shaped crystalline state, and the crystals are not connected with each other, which is the same as the structure of β-cyclodextrin, indicating that Litsea cubeba essential oil is located inside β-cyclodextrin and does not affect the overall structure of β-cyclodextrin; after being coated with chitosan-inulin graft copolymer, each crystalline state becomes elliptical state and is connected with each other, and the particle size also increases to 458~825 nm, and the corresponding electron microscope image also shows that the particle size of the microcapsule structure formed after being coated with chitosan-inulin graft copolymer is larger than that of the complex.

[0089] To verify the sustained-release effect of the microcapsule structure, experiments were conducted on the complex and the microcapsule structure. The procedure was as follows: 0.500 g of the microcapsule structure or complex was added to a flask containing SGF solution (artificial gastric fluid, 30 mL), the flask was sealed, and the mixture was shaken at 70 rpm for 2 hours in a 37°C water bath. Then, 30 mL of SIF solution (artificial intestinal fluid) was added, mixed thoroughly, and sealed. Digestion was simulated for 2 hours under the same conditions. For each digestion solution, 1 mL of the dissolution was collected every 10 minutes for the first 30 minutes, and then every 15 minutes thereafter, continuing for 2 hours. An equal volume of blank solution was added after each collection to ensure a constant volume. All collected liquids were stored on ice. After all collections were completed, the collected dissolutions were centrifuged at 6000 rpm for 5 minutes, filtered through a 0.22 μm filter, and the content was analyzed. Results are shown below. Figure 5 .

[0090] like Figure 5 As shown, the Litsea cubeba essential oil in the complex (LCEO / β-CD-ICs) was completely dissolved within 120 min, while the microcapsule structure (LCEO / β-CD / CS-IL NPs) had a cumulative release of 71.36% within 240 min, indicating that the microcapsule structure has a certain sustained-release effect compared to the complex.

[0091] Experimental Examples 2-5

[0092] The specific process is the same as in Experiment 1, except that the parameters are changed, as shown in Table 1.

[0093] Table 1

[0094]

[0095]

[0096] Experimental Example 6

[0097] The specific process is the same as in Experimental Example 1, except that the temperature in step (1) is changed. Specifically, 1.0g of Litsea cubeba essential oil is slowly added dropwise to 2.0g of β-cyclodextrin saturated aqueous solution, and the mixture is stirred continuously for 4 hours at 20℃, 30℃, 50℃, and 60℃ respectively. Everything else is the same as in Experimental Example 1.

[0098] At 20℃ and 30℃, the microcapsules formed have slightly poorer shape retention and the particle size is relatively less uniform. Figure 9As shown in Table 2, at 50°C and 60°C, the microcapsules formed had good formability, uniform particle size, and similar size without significant difference, similar to the results of Test Example 1. However, at 50°C and 60°C, the microcapsules formed had a certain degree of decrease in encapsulation efficiency and yield (at 50°C, the encapsulation efficiency was 73.54% and the yield was 68.82%; at 60°C, the encapsulation efficiency was 66.38% and the yield was 60.97%; compared to 40°C, the encapsulation efficiency decreased by 12.86% and 21.35%, respectively, and the yield decreased by 13.24% and 23.14%, respectively).

[0099] Comparative Example 1

[0100] As in Test Example 1, except that step (2) was omitted, i.e., the chitosan-inulin graft copolymer in step (3) was replaced by chitosan, and the microcapsule structure formed was: Litsea cubeba essential oil-β-cyclodextrin-chitosan.

[0101] Comparative Example 2

[0102] As in Test Example 1, except that step (2) was omitted, i.e., the chitosan-inulin graft copolymer in step (3) was replaced by inulin, and the microcapsule structure formed was: Litsea cubeba essential oil-β-cyclodextrin-inulin.

[0103] Comparative Example 3

[0104] As in Test Example 1, except that step (2) was omitted, i.e., the chitosan-inulin graft copolymer in step (3) was replaced by chitosan and inulin, and the copolymer was generated in situ in step (3).

[0105] Figure 6 The scanning electron microscope image of the copolymer generated in situ is shown in Figure 2. As can be seen from the image, the Litsea cubeba essential oil-β-cyclodextrin-chitosan / inulin inclusion complex generated in situ could not form microcapsules, and the surface of the inclusion complex was rough and had no fixed shape, which was significantly different from the microcapsules formed by graft copolymerization and then inclusion.

[0106] The dissolution time of the copolymer generated in situ was also detected, as shown in Table 3. The in vitro dissolution experiment also showed that the inclusion complex generated in situ was completely dissolved within 150 min, although it had a certain sustained-release property, but it was much smaller than the microcapsules formed by graft copolymerization and then inclusion, especially the stability in simulated gastric fluid (SGF) was significantly different from the microcapsules formed by graft copolymerization and then inclusion. Figure 7

[0107] The microcapsule structures of the above test examples and comparative examples were subjected to antibacterial experiments to determine the antibacterial activity against two common pathogenic bacteria (Escherichia coli and Staphylococcus aureus) of animals.

[0108] The experimental process was as follows:

[0109] ​The blank control was used as the negative control group, the Litsea cubeba essential oil was used as the positive control group, and the microcapsule structure of the test examples and comparative examples was used as the experimental group. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the LCEO / β-CD / CS-IL NPs microcapsule to E. coli, S. aureus, Salmonella and Streptococcus were determined by the double dilution method on a 96-well plate.

[0110] After the bacteria solution was cultured at 37°C in a constant temperature shaker at a speed of 120 r / min for 5 h, the bacteria solution was diluted to 1.0×10 6 CFU was added to the 96-well plate, and 100 μL of the bacteria solution was added to each well. A certain amount of Litsea cubeba essential oil was added to the broth, and after being fully mixed by vortexing at a speed of 12000 r / min for 5 min, the double dilution method was used to make the concentration of the essential oil in each well be 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.19, 0.095, 0.048, 0.024 μL / mL, and 100 μL of the essential oil solution was added to each well. The amount of the microcapsule structure containing the same essential oil concentration was calculated according to the encapsulation rate, and the same method was used for mixing and dilution and then added to the 96-well plate. The 96-well plate was cultured overnight in a constant temperature incubator at 37°C, and the last well with sterile growth was defined as the MIC, and the second last well with sterile growth on the 96-well plate was defined as the MBC. The results are shown in Table 2.

[0111] Table 2

[0112]

[0113]

[0114] As shown in Table 2, the Litsea cubeba essential oil has good antibacterial effect on E. coli and S. aureus, and the microcapsule structure further improves the antibacterial effect of the Litsea cubeba essential oil on E. coli and S. aureus, which may be due to the fact that chitosan itself has certain antibacterial activity, and the polymer formed after grafting with inulin still retains the original antibacterial activity, thereby increasing the antibacterial effect of the microcapsule. In addition, the microcapsule significantly reduces the volatilization of the antibacterial components in the essential oil, thereby improving the bactericidal effect and the action duration of the essential oil. Figure 8 The figures respectively show the bacterial damage after the action of the essential oil and the essential oil microcapsule on E. coli and S. aureus. Among them, Figures A and B are the action of the essential oil on E. coli and S. aureus, respectively, and it can be seen from the figures that the bacterial bodies of the two kinds of bacteria appear obvious shrinkage phenomenon after the action of the essential oil. Figures C and D are the action of the microcapsule on E. coli and S. aureus, respectively, and compared with Figures A and B, the bacterial body shrinkage phenomenon of the two kinds of bacteria is more obvious, and many rupture phenomena also appear, indicating that the action of the microcapsule on the two kinds of bacteria is more obvious.

[0115] The above only is the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement or improvement etc. made in the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A microcapsule structure, characterized in that, It includes Litsea cubeba essential oil, β-cyclodextrin, chitosan and inulin; Litsea cubeba essential oil is located in the porous structure of β-cyclodextrin to form a complex; the complex is located in the three-dimensional structure formed by the graft polymerization of chitosan and inulin to form a microcapsule structure; The mass ratio of β-cyclodextrin to the sum of the masses of chitosan and inulin was 2:1; the wall-to-core ratio of β-cyclodextrin, chitosan, inulin to Litsea cubeba essential oil was 3:

1.

2. The microcapsule structure according to claim 1, characterized in that, The particle size of the composite is 90–190 nm; the particle size of the microcapsule structure is 450–850 nm.

3. The microcapsule structure according to claim 1, characterized in that, The mass ratio of chitosan to inulin is 1:(1-10).

4. A method for preparing a microcapsule structure, characterized in that, include: (1) Add Litsea cubeba essential oil to a solution containing β-cyclodextrin for inclusion to obtain a complex; (2) Inulin and chitosan are mixed in a solution at pH 3.5 to 5.5 and grafted copolymerized to obtain a solution containing chitosan-inulin grafted polymer; (3) In the presence of a crosslinking agent, the composite is added to a solution containing chitosan-inulin grafted polymer and crosslinked to obtain a microcapsule structure.

5. The preparation method according to claim 4, characterized in that, Step (1) includes: adding Litsea cubeba essential oil dropwise to a saturated aqueous solution of β-cyclodextrin and stirring at a temperature of 20℃~60℃ to obtain a complex.

6. The preparation method according to claim 4, characterized in that, In step (2), inulin and chitosan are dissolved in a solution to form an inulin solution and a chitosan solution, and then the solutions are mixed. In the inulin solution, the mass-to-volume ratio of inulin is 0.1–5.0%; in the chitosan solution, the mass-to-volume ratio of chitosan is 0.1–1.0%. The conditions for graft copolymerization are: temperature 80–110℃, time 5–15 min.

7. The preparation method according to claim 4, characterized in that, Step (3) includes: adding the composite to the grafted polymer solution and stirring continuously at a speed of 1000-1400 r / min for 0.5-1 h under heating conditions of 30-60°C; then adding a crosslinking agent and adjusting the pH to 5.5-6.5 to obtain a microcapsule structure; The crosslinking agent includes at least one of sodium tripolyphosphate, glutaraldehyde, and formaldehyde.

8. The use of the microcapsule structure according to any one of claims 1 to 4 in the preparation of bacterial infection drugs.

Images