Preparation method and application of gamma-aminobutyric acid embedded chitosan-cellulose nanocrystalline nanoparticles

Abstract

The invention discloses a gamma-aminobutyric acid embedding system and a preparation method and application thereof, and the preparation method comprises the following steps: 1) adding gamma-aminobutyric acid into a chitosan solution, and then adding cellulose nanocrystals; and (2) adding a sodium tripolyphosphate solution into the chitosan solution containing gamma-aminobutyric acid in the step (1), and stirring to prepare the chitosan-cellulose nanocrystalline nanoparticles embedded with gamma-aminobutyric acid. The gamma-aminobutyric acid embedded chitosan-cellulose nanocrystalline nanoparticles prepared by the method can improve the embedding rate and stability of gamma-aminobutyric acid, can improve the sleep improvement effect of gamma-aminobutyric acid, and have practical application value in medicine production.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a γ-aminobutyric acid embedding system, a preparation method thereof, and an application thereof. Background Art

[0002] γ-aminobutyric acid is a neurotransmitter and an important inhibitory neuromodulator, which is widely present in animals and microorganisms. By binding to receptors, it inhibits the excitability of neurons, thereby regulating neurotransmission and neural electrical activities. It is crucial for maintaining the balance and normal functions of the nervous system and has various biological functions such as regulating neurotransmission, sleep, mood, and neural development.

[0003] However, the effect of using γ-aminobutyric acid alone is difficult to guarantee. Existing technologies have studied different usage methods, such as appropriate dosage forms. Through research, the present invention finds that preparing γ-aminobutyric acid into chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid can improve its stability and medicinal effect. Summary of the Invention

[0004] Aiming at the problem that the effect of using γ-aminobutyric acid alone in the prior art is difficult to guarantee, the present invention prepares γ-aminobutyric acid into chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid, which can improve its stability and medicinal effect. Through research, it is found that the chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid of the present invention can improve the stability of γ-aminobutyric acid and improve the effect of γ-aminobutyric acid in the process of promoting sleep.

[0005] In the first aspect of the present invention, a preparation method of a γ-aminobutyric acid embedding system is provided, including the following steps:

[0006] Step 1): Add γ-aminobutyric acid to a chitosan solution, and then add cellulose nanocrystals;

[0007] Step 2): Add a sodium tripolyphosphate solution to the chitosan solution containing γ-aminobutyric acid in Step 1) and stir to prepare chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid.

[0008] The present invention also provides an application of the chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid prepared by the preparation method of the present invention in the preparation of drugs for protecting the nervous system.

[0009] The present invention also provides an application of the chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid prepared by the preparation method of the present invention in the preparation of drugs for improving sleep.

[0010] The present invention also provides a pharmaceutical composition, which contains chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid prepared by the preparation method of the present invention.

[0011] The present invention has the following beneficial effects:

[0012] (1) The γ-aminobutyric acid encapsulation system of the present invention can improve the stability of γ-aminobutyric acid, relieve the balance regulation of the nervous system and poor sleep, and can be used for preparing nerve cell protection drugs and drugs for improving sleep, and has practical application value in pharmaceutical production.

[0013] (2) The preparation method of the γ-aminobutyric acid encapsulation system of the present invention is simple in operation, low in raw material cost, mild in reaction, low in energy consumption and high in encapsulation rate. Specific Embodiments

[0014] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise clearly stated, in the whole specification and claims, the term "comprising" or its variations such as "comprises" or "including" etc. will be understood to include the stated components or steps, without excluding other material components or steps.

[0015] In addition, for better illustration of the present invention, numerous specific details are given in the following specific embodiments.

[0016] Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some embodiments, the raw materials, methods, means, etc. well-known to those skilled in the art are not described in detail, so as to highlight the gist of the present invention.

[0017] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0018] In the present invention, the structural formulas and Chinese names of the compounds used are as follows:

[0019] In the present invention, the term "CS" refers to chitosan;

[0020] The term "CNC" refers to cellulose nanocrystals;

[0021] The term "TPP" refers to sodium tripolyphosphate;

[0022] The term "GABA" refers to γ-aminobutyric acid.

[0023] In the present invention, the term "room temperature" means that the temperature of an article is close to or the same as the temperature of the space (e.g., the location of the fume hood in which the article is located). Generally, room temperature is about 20 °C to about 30 °C, or about 22 °C to 27 °C, or about 25 °C.

[0024] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified in the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0025] The present invention first provides a method for preparing chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid, which comprises the following steps:

[0026] Step 1): Add γ-aminobutyric acid to the chitosan solution, and then add cellulose nanocrystals.

[0027] Step 2): Add the sodium tripolyphosphate solution to the chitosan solution containing γ-aminobutyric acid in Step 1) and stir to prepare chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid.

[0028] In the above preparation method, the chitosan solution in Step 1) is an aqueous acetic acid solution of chitosan, and the volume concentration of the aqueous acetic acid solution is 0.5 - 3%, preferably 1%; the mass concentration of chitosan in the aqueous acetic acid solution of chitosan is 0.05 - 5 mg / L. Under the above conditions, the encapsulation rate of the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid is relatively excellent.

[0029] In the above preparation method, the mass ratio of γ-aminobutyric acid to chitosan in Step 1) is 1:10 - 1:1, preferably 1:2, 1:3 and 1:4; the mass concentration of the cellulose nanocrystals is 0.005 - 0.2%. It is found that cellulose nanocrystals can well improve the encapsulation rate of the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid.

[0030] In the above preparation method, the sodium tripolyphosphate solution in Step 2) is preferably an aqueous solution of sodium tripolyphosphate, and particularly preferably an aqueous deionized solution of sodium tripolyphosphate; in Step 2), it is preferred to add a pH regulator to adjust the pH value to 7 - 8, preferably 7.4. Under the preferred conditions, the product performance of the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid is excellent.

[0031] In the above preparation method, the pH regulator described in step (ii) is 0.1 M sodium dihydrogen phosphate buffer solution.

[0032] In the above preparation method, the sodium tripolyphosphate solution in step (ii) is added dropwise to step (i) at a dropping rate of 3 mL per minute. During the dropping process, the stirring rate is 400 - 1000 rpm, preferably 800 rpm; the stirring time is 2 - 5 h, preferably 3 h.

[0033] Under the above preferred conditions, the product performance of the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid obtained by the preparation method of the present invention is excellent and the encapsulation rate is high.

[0034] The present invention also provides the application of the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid obtained by the preparation method of the present invention in the preparation of drugs for protecting the nervous system.

[0035] The present invention also provides the application of the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid obtained by the preparation method of the present invention in the preparation of drugs for improving sleep.

[0036] Research shows that the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid obtained by the preparation method of the present invention can significantly improve the application effect of γ-aminobutyric acid.

[0037] The present invention also provides a pharmaceutical composition, which comprises the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid obtained by the preparation method of the present invention.

[0038] The present invention will be further described below in conjunction with examples. Unless otherwise specified, the raw materials in the examples can be prepared by conventional methods or obtained by purchase.

[0039] Example 1: Preparation of chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid.

[0040] Dissolve 120 mg of chitosan in 100 mL of 1% aqueous acetic acid solution. Add 40 mg of γ-aminobutyric acid to this solution and stir at a speed of 500 rpm for 10 minutes to ensure complete dissolution. Dissolve 40 mg of cellulose nanocrystals in 100 mL of deionized water. After stirring evenly, add this solution to the chitosan-γ-aminobutyric acid solution. Meanwhile, dissolve 40 mg of sodium tripolyphosphate in 80 mL of deionized water, adjust the pH of the solution to 7.4 with 0.1 M sodium dihydrogen phosphate buffer solution and make up the volume to 100 mL. Slowly add 100 mL of sodium tripolyphosphate solution to the chitosan-γ-aminobutyric acid-cellulose nanocrystal mixed solution at a speed of 3 mL / min at room temperature and stir at a speed of 800 rpm for 180 minutes at room temperature. Finally, freeze-dry to obtain chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid (GABA-CS-CNC-NPs).

[0041] Example 2: Preparation of chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid

[0042] Dissolve 80 mg of chitosan in 100 mL of 1% aqueous acetic acid solution. Add 40 mg of γ-aminobutyric acid to this solution and stir at a speed of 500 rpm for 10 minutes to ensure complete dissolution. Dissolve 40 mg of cellulose nanocrystals in 100 mL of deionized water. After stirring evenly, add this solution to the chitosan-γ-aminobutyric acid solution. Meanwhile, dissolve 40 mg of sodium tripolyphosphate in 80 mL of deionized water, adjust the pH of the solution to 7.4 with 0.1 M sodium dihydrogen phosphate buffer solution and make up the volume to 100 mL. Slowly add 100 mL of sodium tripolyphosphate solution to the chitosan-γ-aminobutyric acid-cellulose nanocrystal mixed solution at a speed of 3 mL / min at room temperature and stir at a speed of 800 rpm for 180 minutes at room temperature. Finally, freeze-dry to obtain chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid (GABA-CS-CNC-NPs).

[0043] Example 3: Preparation of chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid

[0044] Dissolve 160 mg of chitosan in 100 mL of 1% aqueous acetic acid solution. Add 40 mg of γ-aminobutyric acid to this solution and stir at a speed of 500 rpm for 10 minutes to ensure complete dissolution. Dissolve 40 mg of cellulose nanocrystals in 100 mL of deionized water. After stirring evenly, add this solution to the chitosan-γ-aminobutyric acid solution. At the same time, dissolve 40 mg of sodium tripolyphosphate in 80 mL of deionized water, adjust the pH of the solution to 7.4 with 0.1 M sodium dihydrogen phosphate buffer solution, and make up the volume to 100 mL. Slowly add 100 mL of sodium tripolyphosphate solution to the chitosan-γ-aminobutyric acid-cellulose nanocrystal mixed solution at a speed of 3 mL / min at room temperature, and stir at a speed of 800 rpm for 180 minutes at room temperature. Finally, freeze-dry to obtain chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid (GABA-CS-CNC-NPs).

[0045] Example 4: Preparation of chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid.

[0046] Dissolve 120 mg of chitosan in 100 mL of 1% aqueous acetic acid solution. Add 40 mg of γ-aminobutyric acid to this solution and stir at a speed of 500 rpm for 10 minutes to ensure complete dissolution. Dissolve 20 mg of cellulose nanocrystals in 100 mL of deionized water. After stirring evenly, add this solution to the chitosan-γ-aminobutyric acid solution. At the same time, dissolve 40 mg of sodium tripolyphosphate in 80 mL of deionized water, adjust the pH of the solution to 7.4 with 0.1 M sodium dihydrogen phosphate buffer solution, and make up the volume to 100 mL. Slowly add 100 mL of sodium tripolyphosphate solution to the chitosan-γ-aminobutyric acid-cellulose nanocrystal mixed solution at a speed of 3 mL / min at room temperature, and stir at a speed of 800 rpm for 180 minutes at room temperature. Finally, freeze-dry to obtain chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid (GABA-CS-CNC-NPs).

[0047] Example 5: Preparation of chitosan nanoparticles embedding γ-aminobutyric acid.

[0048] Dissolve 120 mg of chitosan in 100 mL of 1% aqueous acetic acid solution. Add 40 mg of γ-aminobutyric acid to this solution and stir it at a speed of 500 rpm for 10 minutes to ensure complete dissolution. Dissolve 40 mg of sodium tripolyphosphate in 80 mL of deionized water, adjust the pH of the solution to 7.4 using 0.1 M sodium dihydrogen phosphate buffer, and make up the volume to 100 mL. Then, slowly add 100 mL of the sodium tripolyphosphate solution to the chitosan-γ-aminobutyric acid solution at a rate of 3 mL / min at room temperature and stir at a speed of 800 rpm for 180 minutes at room temperature. Finally, freeze-dry to obtain chitosan nanoparticles encapsulating γ-aminobutyric acid (GABA-CS-NPs).

[0049] Example 6 Determination of Encapsulation Efficiency of γ-aminobutyric acid

[0050] The free GABA content in the supernatant of GABA-CS-CNC-NPs and GABA-CS-NPs was detected by HPLC. Prepare 1 mL of suspensions of GABA-CS-CNC-NPs and GABA-CS-NPs nanoparticles, centrifuge them with a high-speed refrigerated centrifuge, wash the precipitate three times with an equal volume of ultrapure water, collect all the supernatants, filter them through a 0.22 μm filter membrane and add them to a liner tube, and then place them in a liquid-phase vial for on-machine detection. The calculation method of the encapsulation efficiency is as follows:

[0051]

[0052] Table 1 Test Results of Encapsulation Efficiency of γ-aminobutyric acid in GABA-CS-CNC-NPs and GABA-CS-NPs

[0053]

[0054] Example 7 In Vitro Simulated Digestion Experiment

[0055] 1. Preparation of Artificial Gastric Juice and Artificial Intestinal Juice

[0056] Artificial gastric juice: Add pepsin to an appropriate amount of 0.1 mol / L HCl solution to control the final concentration at 4 mg / mL. Artificial intestinal juice: Add trypsin to an appropriate amount of 0.1 mol / L sodium bicarbonate solution to control the final concentration at 2 mg / mL, and then mix it with an equal volume of 12 mg / mL sodium cholate salt.

[0057] 2. In Vitro Simulated Digestion Experiment

[0058] Prepare four 100 mL stoppered conical flasks, add 25 mL of normal saline and 4 mL of artificial gastric juice respectively, mix well, and adjust the pH value to 2.0 - 2.5 using hydrochloric acid standard solution. Add 15 mg of GABA standard to two of them, and add an equal amount of GABA-CS-CNC-NPs prepared according to Example 1 to the other two. After mixing, extract the solution once, then add NaHCO3 and place it in a constant temperature water bath at 37 °C and shake (100 r / min) for 2 h. Take two of them, digest with artificial gastric juice for 1 h, and then add artificial intestinal juice for digestion.

[0059] Add 4 ml of normal saline to the above solution digested with artificial gastric juice for 1 h, adjust the pH of the solution to 6.5 - 7.0 using 0.5 mol / L NaHCO3 solution, shake in a constant temperature water bath (37 °C, 100 r / min), then add 18 mL of artificial intestinal juice, adjust the pH to 7.0 - 7.5, and place it in a constant temperature water bath and shake for 3 h (37 °C, 100 r / min).

[0060] After ice-bathing all the above sample solutions, filter them through a 0.22 μm filter membrane, ultrasonically treat for 20 min, and analyze by HPLC method.

[0061] According to the contents measured by HPLC method after in vitro simulated digestion of GABA and CNF-GABA complex, after 2 h of simulated gastric juice digestion, the degradation rate of GABA is 25.3%, and the degradation rate of GABA-CS-CNC-NPs is 14.7%; after 3 h of simulated intestinal juice digestion, the degradation rate of GABA is 37.9%, and the degradation rate of GABA-CS-CNC-NPs is 22.5%. GABA-CS-CNC-NPs can effectively reduce the degradation rate of GABA in the gastric environment and be targeted and delivered to the intestine to play its role, improving the utilization rate of GABA to a certain extent.

[0062] Example 8 Evaluation of the sleep effect of GABA-CS-CNC-NPs

[0063] Select 80 male ICR mice (SPF level) with qualified quarantine, weighing 16.4 - 21.1 g, randomly divide them into 8 groups according to body weight (n = 10), namely normal control group, diazepam tablet group (1.3 mg / kg), low, medium, and high dose groups of GABA (50, 100, 150 mg / kg), and low, medium, and high dose groups of GABA-CS-CNC-NPs (50, 100, 150 mg / kg). After 1 week of adaptive feeding of the mice, continuously administer the drugs by gavage for 7 days, once a day. All mice received sample management and behavioral tests between 9:00 and 11:00 AM.

[0064] Pentobarbital sodium prolonged sleep time experiment

[0065] Thirty minutes after the last gavage, all mice were intraperitoneally injected with a suprathreshold dose of sodium pentobarbital (50 mg / kg), and the mice were placed on a warm pad (37 °C) with their abdomens up. The sleep latency (the time from the start of sodium pentobarbital injection to the disappearance of the righting reflex) and the sleep duration (the time from the disappearance of the righting reflex to the recovery of the righting reflex) were recorded. Mice that did not fall asleep within 15 minutes were excluded from the experiment.

[0066] Hypnotic Experiment with Subthreshold Dose of Sodium Pentobarbital

[0067] Thirty minutes after the last gavage, all mice were intraperitoneally injected with a subthreshold dose of sodium pentobarbital (40 mg / kg), and the mice were placed on a warm pad (37 °C) with their abdomens up. The criterion for falling asleep was the loss of the righting reflex for more than 1 minute, and the number of mice falling asleep in each group was recorded.

[0068] Table 2 Experiment on Prolonging Sleep Time with Sodium Pentobarbital (n = 10)

[0069]

[0070] Note: GABA-CS-CNC-NPs in Table 2 were prepared according to the method of Example 1. *P < 0.05 compared with the control group, **P < 0.001 compared with the control group; #P < 0.05 compared with the high-dose GABA group.

[0071] Table 3 Hypnotic Experiment with Subthreshold Dose of Sodium Pentobarbital (n = 10)

[0072]

[0073] Note: GABA-CS-CNC-NPs in Table 3 were prepared according to the method of Example 1.

[0074] As can be seen from Tables 2 and 3, the results of the experiment on prolonging sleep time with sodium pentobarbital and the hypnotic experiment with subthreshold dose of sodium pentobarbital were positive, proving that GABA-CS-CNC-NPs have the effect of improving sleep, and the high-dose group of GABA-CS-CNC-NPs significantly increased the sleep prolongation time of mice compared with the high-dose GABA group.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of chitosan-cellulose nanocrystal nanoparticles embedding γ-aminobutyric acid, characterized in that, It includes the following steps: Step 1): Add γ-aminobutyric acid to the chitosan solution, and then add the cellulose nanocrystal solution; the chitosan solution is an aqueous chitosan acetate solution, and the volume concentration of the acetic acid aqueous solution is 0.5-3%; the mass concentration of chitosan in the aqueous chitosan acetate solution is 0.05-5 mg / L; the mass ratio of γ-aminobutyric acid to chitosan is 1:10-1:

1. Step 2): Add the sodium tripolyphosphate solution to the chitosan solution containing γ-aminobutyric acid in Step 1) and stir to prepare chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid; the cellulose nanocrystal solution is an aqueous solution, and the mass percentage content of cellulose nanocrystals is 0.005-0.2%.

2. The preparation method according to claim 1, characterized in that, The volume concentration of the acetic acid aqueous solution is 1%; the mass concentration of chitosan in the aqueous chitosan acetate solution is 0.05-2 mg / L.

3. The preparation method according to claim 1, characterized in that, In Step 1), the mass ratio of γ-aminobutyric acid to chitosan is 1:2, 1:3 or 1:

4.

4. The preparation method according to claim 1, characterized in that, In Step 2), the sodium tripolyphosphate solution is an aqueous sodium tripolyphosphate solution; Step 2) also includes the step of adding a pH regulator to adjust the pH value to 7-8.

5. The preparation method according to claim 4, wherein, The pH regulator in Step 2) is 0.1 M sodium dihydrogen phosphate buffer solution.

6. The preparation method according to claim 4, characterized in that, In Step 2), the sodium tripolyphosphate solution is added dropwise to Step 1), and the addition is dropwise addition, the dropping rate is 3 mL per minute, and the stirring rate during the dropping process is 400-1000 rpm; the stirring time is 2-5 h.

7. Use of the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid prepared by the preparation method according to any one of claims 1-6 in the preparation of sleep-improving drugs.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the chitosan-cellulose nanocrystal nanoparticles encapsulating γ-aminobutyric acid prepared by the preparation method according to any one of claims 1-6.

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