阅读说明：本技术 一种负载百里香酚的环糊精络合物及其制备方法 (Thymol-loaded cyclodextrin complex and preparation method thereof ) 是由 黄强 潘晓丹 张斌 扶雄 于 2021-08-20 设计创作，主要内容包括：本发明公开了一种负载百里香酚的环糊精络合物及其制备方法,包括如下步骤：(1)将环糊精、含钾化合物与百里香酚充分溶解于水,得到母液；(2)在密闭容器中,将母液静置于易挥发醇类气氛中反应,直至络合物晶体从母液中析出；(3)将晶体从母液中分离,洗涤除去游离的百里香酚,离心,干燥后得到负载百里香酚的环糊精络合物。该方法采用一步共结晶法制备得到络合物,明显提高百里香酚的负载量与稳定性,与传统的γ-环糊精相比,负载量由124mg/g提升至250mg/g以上,50天室温条件下,百里香酚保留率由30％提高至60％以上；又因采用食品级原料与绿色、简便的合成工艺,该方法制备的络合物具有良好的生物安全性。(The invention discloses a thymol-loaded cyclodextrin complex and a preparation method thereof, wherein the preparation method comprises the following steps: (1) fully dissolving cyclodextrin, a potassium-containing compound and thymol in water to obtain a mother solution; (2) standing the mother solution in a closed container in an atmosphere of volatile alcohols to react until complex crystals are separated out from the mother solution; (3) separating the crystal from the mother liquor, washing to remove free thymol, centrifuging, and drying to obtain the thymol-loaded cyclodextrin complex. The method adopts a one-step co-crystallization method to prepare a complex, the loading capacity and the stability of the thymol are obviously improved, compared with the traditional gamma-cyclodextrin, the loading capacity is improved to more than 250mg/g from 124mg/g, and the thymol retention rate is improved to more than 60% from 30% under the room temperature condition of 50 days; and because of adopting food-grade raw materials and a green and simple synthesis process, the complex prepared by the method has good biological safety.)

1. A preparation method of a thymol-loaded cyclodextrin complex is characterized by comprising the following steps:

(1) fully dissolving cyclodextrin, a potassium-containing compound and thymol in water to obtain a mother solution;

(2) standing the mother solution in a closed container in an atmosphere of volatile alcohols to react until complex crystals are separated out from the mother solution;

(3) separating the crystal from the mother liquor, washing to remove free thymol, centrifuging, and drying to obtain the thymol-loaded cyclodextrin complex.

2. The method of claim 1, wherein the step ofThe cyclodextrin in the step (1) is one or more than two of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin; the potassium-containing compound is KOH or CH₃COOK and KCl.

3. The method according to claim 2, wherein the concentration of cyclodextrin in the mother liquor in step (1) is 0.01 to 0.25mol/L, the concentration of potassium-containing compound is 0.08 to 2mol/L, and the concentration of thymol is 0.1 to 1.25 mol/L.

4. The method according to claim 3, wherein the concentration of cyclodextrin in the mother liquor of step (1) is 0.05 to 0.1mol/L, the concentration of potassium-containing compound is 0.4 to 0.8mol/L, and the concentration of thymol is 0.25 to 1 mol/L.

5. The method according to claim 2, wherein the molar ratio of the cyclodextrin, the potassium-containing compound and the thymol in the step (1) is 1 (6-12) to (1-10).

6. The method according to claim 3, wherein the molar ratio of the cyclodextrin, the potassium-containing compound and the thymol in the step (1) is 1 (8-10) to (4-8).

7. The method according to any one of claims 1 to 6, wherein the reaction conditions in step (2) are as follows: the temperature is 25-50 ℃, and the reaction time is 5-20 days.

8. The preparation method according to claim 7, wherein the volatile alcohol in the step (2) is methanol or ethanol, and the volume of the alcohol is 2 to 5 times of the volume of the mother liquor.

9. The method according to any one of claims 1 to 6, wherein the free thymol in step (3) is removed by washing with ethanol 3 to 5 times, and after each washing, the mixture is centrifuged at 3000r/min to 4500r/min for 3 to 5 min; the drying is vacuum drying, the temperature is 30-50 ℃, and the treatment time is 4-6 h.

10. A thymol-loaded cyclodextrin complex made according to the method of any one of claims 1-9.

Technical Field

The invention relates to a preparation method of a thymol complex with efficient loading and sustained release effects, belonging to the fields of food and chemical industry.

Background

Thymol is a natural phenolic monoterpene mainly present in plants of the genus thymus and has a strong odor. Because of having excellent antibacterial, antiviral, anti-inflammatory, sedative and other functions, thymol is often used for a long time as an expectorant, an anti-inflammatory agent, an antiviral agent, an antibacterial agent and an antiseptic in the traditional medicine, and plays a good role in treating diseases such as upper respiratory infection, bronchitis, parasitic infection, dermatitis and the like.

Thymol has strong volatility and low water solubility, and the industrial application of thymol is severely restricted. Encapsulating it in a suitable wall material to slow down degradation and loss of thymol has become one of the main research contents at present. At present, polysaccharide, cyclodextrin, lipid, glycerol, protein and the like are mainly used as encapsulation wall materials and are complexed with thymol by means of spray drying, freeze drying, electrostatic spinning, coprecipitation and the like. However, in the compound constructed by using polysaccharide, cyclodextrin and protein as wall materials, the loading capacity of thymol is generally low, for example, in the thymol-chitosan oligosaccharide compound prepared by chinese patent CN111771968A, the highest loading capacity of thymol is 52.3 mg/g; in the fiber membrane synthesized by taking thymol, beta-cyclodextrin and cellulose acetate as raw Materials, the content of the thymol is 48.6mg/g (Materials Science & Engineering C,2020,115,111155); the polymer particles obtained by complexing soy protein isolate with thymol had a thymol content of 103.6mg/g (Food Chemistry,2021,334,127594)

The cyclodextrin-metal organic framework (CD-MOF) is a porous crystalline material which is formed by self-assembling cyclodextrin and alkali metal salt in a solution, has high specific surface area and adjustable pore diameter, and has a periodic network structure. Experiments prove that the CD-MOF prepared from the food-grade cyclodextrin and the food-grade alkali metal salt has the characteristics of no toxicity and high safety. In view of the specificity of applications in the fields of food, medicine and the like, raw materials for constructing complex systems need to have good biosafety, and green synthesis means is adopted, so as to be an excellent and green safe carrier, CD-MOF has been used for encapsulating bioactive substances such as glycyrrhizic acid, folic acid, curcumin, anise leaf polyphenol and the like. The complex of the thymol and the thymol can effectively overcome the application defect of the thymol.

The combination of the CD-MOF and the guest substance is usually realized by a mixed heating method or a dipping adsorption method, for example, the Chinese patent CN111513356A, beta-CD-MOF is used as an encapsulation wall material, and the efficient loading of the L-menthol is realized by a direct mixed heating method. For another example, guest substance (curcumin) is dissolved in methanol in advance, and γ -CD-MOF crystals are added and stirred for several hours for adsorption (Food Chemistry,2021,347,128978). Both methods are carried out by complexing and adsorbing on the basis of preparing CD-MOF crystals, and have long preparation period and complex procedures.

At present, the content of thymol is generally low by taking polysaccharide or cyclodextrin as a carrier, and the loading capacity of thymol is only 124mg/g by taking a compound of gamma-cyclodextrin and thymol as an example.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide the preparation method of the thymol-cyclodextrin complex which is high in thymol loading efficiency, high in biological safety, green and simple in process and beneficial to improvement of the dispersing ability and stability of thymol.

The purpose of the invention is realized by the following technical scheme:

a preparation method of a thymol-loaded cyclodextrin complex comprises the following steps:

(1) fully dissolving cyclodextrin, a potassium-containing compound and thymol in water to obtain a mother solution;

(2) standing the mother solution in a closed container in an atmosphere of volatile alcohols to react until complex crystals are separated out from the mother solution;

(3) separating the crystal from the mother liquor, washing to remove free thymol, centrifuging, and drying to obtain the thymol-loaded cyclodextrin complex.

Preferably, the cyclodextrin in the step (1) is one or more than two of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.

Preferably, the potassium-containing compound in the step (1) is KOH or CH₃COOK and KCl.

Preferably, the concentration of cyclodextrin in the mother liquor in the step (1) is 0.01-0.25 mol/L, the concentration of potassium-containing compound is 0.08-2 mol/L, and the concentration of thymol is 0.1-1.25 mol/L.

Preferably, the molar ratio of the cyclodextrin, the potassium-containing compound and the thymol in the step (1) is 1 (6-12) to 1-10; more preferably 1 (8-10) to (4-8).

Preferably, the reaction conditions in step (2) are: the temperature is 25-50 ℃, and the reaction time is 5-20 days.

Preferably, the volatile alcohol in the step (2) is methanol or ethanol, and the volume of the alcohol is 2-5 times of the volume of the mother liquor.

Preferably, the removal of free thymol in the step (3) is realized by washing with ethanol for 3-5 times, and after washing each time, the mixed solution is centrifuged for 3-5 min under the condition of 3000 r/min-4500 r/min; the drying is vacuum drying, the temperature is 30-50 ℃, and the treatment time is 4-6 h.

The invention uses cyclodextrin crystal prepared by recrystallization after cyclodextrin and potassium-containing compound (potassium salt) are dissolved together as the packaging wall material to realize the encapsulation and stabilization of thymol. The cyclodextrin coexisting in the mother liquor is spontaneously coordinated with potassium ions, and the cyclodextrin molecules are connected and stacked with each other through metal ion bridging to form a loose and porous cyclodextrin metal organic framework (CD-MOF). The CD-MOF has good thermal stability, high specific surface area and adjustable pores, and can form a stable inclusion compound with guest molecules. According to the invention, the cyclodextrin crystal (CD-MOF) is used as the packaging wall material to load thymol, so that the thymol loading material has the characteristics of high loading efficiency, high stability, greenness, no toxicity and the like, and provides more choices for the application fields of thymol in food preservation, oral care, medical antibacterial preparation development and the like.

Compared with the prior art, the invention has the advantages that:

(1) the complex is prepared by adopting a one-step co-crystallization method, the loading capacity and the stability of the thymol are obviously improved, compared with the traditional gamma-cyclodextrin, the loading capacity is improved to more than 250mg/g from 124mg/g, and the retention rate of the thymol is improved to more than 60% from 30% under the room temperature condition of 50 days; after being stored for 50 days in a low-temperature environment (4 ℃), the retention rate of thymol is more than 72 percent.

(2) According to the invention, the cyclodextrin complex loaded with thymol is prepared by adopting a one-step co-crystallization method, and the complexation of the thymol is synchronously carried out in the process of forming the CD-MOF crystal, so that the preparation process can be simplified, the preparation period is shortened, the energy consumption is reduced, the preparation cost is reduced, the preparation method is green and environment-friendly, and food-grade raw materials are selected in the preparation process, so that the complex can be endowed with good biological safety, and can be applied in the fields of food preservation, oral care, medical antibacterial agent development and the like.

Drawings

FIG. 1 is a standard graph of peak area versus thymol concentration.

FIG. 2 is a topographical view of a thymol-loaded cyclodextrin complex; a: gamma-CD-THY (comparative example 1); gamma-CD-MOF-THY (comparative example 2); c: gamma-CD-MOF-THY (example 1); d: beta-CD-MOF-THY (example 2); e: α -CD-MOF-THY (example 3); f: gamma-CD-MOF-THY (example 4); g: gamma-CD-MOF-THY (example 5).

Detailed Description

The invention will be further described with reference to the following examples for a better understanding of the invention, but the scope of the invention as claimed is not limited to the examples.

In the examples, the method for measuring thymol in the complex is as follows: accurately weighing 5mg of complex into a 5mL centrifuge tube, adding 1mL of distilled water, tightly covering a centrifuge tube cap, magnetically stirring at 1000r/min for 0.5h, adding 2mL of ethyl acetate for extraction, collecting supernatant, filtering with a 0.22 μm organic filter membrane, transferring into a gas bottle, and measuring the content of thymol by gas chromatography. The peak area of thymol was converted into the content of thymol and calculated from a thymol standard curve.

Comparative example 1

Complexes of gamma-cyclodextrin with thymol were used as a comparison: taking 2g of gamma-cyclodextrin and 1g of thymol, placing in a mortar, manually grinding for 3min, uniformly mixing, transferring to a closed reaction kettle, and compounding for 4h at 75 ℃. After compounding, washing the sample with ethanol for 3-5 times, centrifuging at the rotating speed of 4000r/min for 3min, and drying in vacuum at 40 ℃ for 6h to obtain a white powdery complex.

Tests show that the loading amount of thymol in the complex is 124mg/g, the thymol retention rate is about 30% after the complex is stored for 50 days in an open environment at normal temperature, and the thymol retention rate is about 58% after the complex is stored for 50 days in a low-temperature environment (4 ℃).

Comparative example 2

Mixing gamma-cyclodextrin and CH₃COOK is dissolved in distilled water according to the molar ratio of 1:8, and is stirred for 3 hours to be fully dissolved, so that mother liquor with the cyclodextrin concentration of 0.05mol/L is obtained; standing 20mL of mother liquor in an ethanol atmosphere at 25 ℃ for 10 days, wherein the dosage of ethanol in a closed container is 50 mL; separating the CD-MOF crystals from the mother liquor, washing with ethanol for 3 times, centrifuging at the rotating speed of 4000r/min for 3min, and vacuum drying at 40 ℃ for 5h to obtain crystal particles. In order to obtain the complex carrying thymol, the CD-MOF crystal and the thymol are manually ground for 3min according to the mass ratio of 1:1, uniformly mixed, transferred into a closed reaction kettle and compounded for 6h at the temperature of 75 ℃. And after compounding, washing the crystals for 3-5 times by using ethanol, centrifuging for 3min at the rotating speed of 4000r/min, and drying for 6h in vacuum at 40 ℃ to obtain the thymol complex crystals.

Tests show that the loading amount of thymol in the complex is 243mg/g, the thymol retention rate is about 54% after the complex is stored for 50 days in an open environment at normal temperature, and the thymol retention rate is about 66% after the complex is stored for 50 days in a low-temperature environment (4 ℃).

Example 1

Dissolving gamma-cyclodextrin, KOH and thymol in distilled water according to the molar ratio of 1:8:5, and stirring for 3 hours to fully dissolve the gamma-cyclodextrin, the KOH and the thymol to obtain a mother solution with the cyclodextrin concentration of 0.1 mol/L; standing 20mL of mother liquor in an ethanol atmosphere at 25 ℃ for 7 days, wherein the dosage of ethanol in a closed container is 50 mL; separating the crystal from the mother liquor, washing with ethanol for 3 times, centrifuging at 4000r/min for 3min, and vacuum drying at 40 deg.C for 5h to obtain crystal granule.

Tests show that the thymol loading amount in the crystal is 327mg/g, the crystal is stored for 50 days under a normal-temperature open environment, the thymol retention rate is about 65%, and the thymol retention rate can be nearly 80% under a low-temperature environment (4 ℃).

Example 2

Dissolving beta-cyclodextrin, KOH and thymol in distilled water according to the molar ratio of 1:8:8, stirring for 3 hours to fully dissolve the beta-cyclodextrin, the KOH and the thymol to obtain mother liquor with the cyclodextrin concentration of 0.1 mol/L; standing 20mL of mother liquor in an ethanol atmosphere at 50 ℃ for 10 days, wherein the dosage of ethanol in a closed container is 50 mL; separating the crystal from the mother liquor, washing with ethanol for 3 times, centrifuging at 4000r/min for 3min, and vacuum drying at 50 deg.C for 4h to obtain crystal granule.

Tests show that the thymol loading amount in the crystal is 269.2mg/g, the crystal is stored for 50 days in an open environment at normal temperature, the thymol retention rate is about 68%, and the thymol retention rate can be nearly 75% in a low-temperature environment (4 ℃).

Example 3

Mixing alpha-cyclodextrin and CH₃Dissolving COOK and thymol in distilled water at a molar ratio of 1:10:5, stirring for 3h to fully dissolve, and obtaining a mother solution with cyclodextrin concentration of 0.1 mol/L; standing 30mL of mother liquor in an ethanol atmosphere at 25 ℃ for 20 days, wherein the dosage of ethanol in a closed container is 75 mL; separating the crystal from the mother liquor, washing with ethanol for 3 times, centrifuging at 4000r/min for 3min, and vacuum drying at 30 deg.C for 6h to obtain crystal particles.

Tests show that the thymol loading capacity of the crystal is 284.6mg/g, the crystal can be stored for 50 days in an open environment at normal temperature, the thymol retention rate is about 63%, and the thymol retention rate can be more than 70% in a low-temperature environment (4 ℃).

Example 4

Mixing gamma-cyclodextrin and CH₃Dissolving COOK and thymol in distilled water at a molar ratio of 1:10:5, stirring for 3h to fully dissolve, and obtaining a mother solution with cyclodextrin concentration of 0.1 mol/L; standing 30mL of the mother liquor in an ethanol atmosphere at 40 ℃ for 10 days, wherein the dosage of ethanol in a closed container is 75 mL; separating crystal from mother liquor, washing with ethanol for 3 times, centrifuging at 4000r/min for 3min at 40 deg.CAfter drying in vacuo for 6h, crystalline particles were obtained.

Tests show that the thymol loading amount in the crystal is 313.5mg/g, the crystal can be stored for 50 days under a normal-temperature open environment, the thymol retention rate is about 78%, and the thymol retention rate can be about 82% under a low-temperature environment (4 ℃).

Example 5

Dissolving gamma-cyclodextrin, KCl and thymol in distilled water at a molar ratio of 1:8:4, stirring for 3h to fully dissolve the gamma-cyclodextrin, KCl and thymol to obtain a mother solution with cyclodextrin concentration of 0.15 mol/L; standing 25mL of mother liquor in an ethanol atmosphere at 25 ℃ for 15 days, wherein the dosage of ethanol in a closed container is 75 mL; separating the crystal from the mother liquor, washing with ethanol for 4 times, centrifuging at 4000r/min for 3min, and vacuum drying at 45 deg.C for 5.5h to obtain crystal granule.

Tests prove that the thymol loading amount in the crystal is 255.3mg/g, the crystal can be stored for 50 days under a normal-temperature open environment, the thymol retention rate is about 60%, and the thymol retention rate can be more than 72% under a low-temperature environment (4 ℃).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.