Nanometer lipid transfer body capable of efficiently loading plant active ingredients as well as preparation method and application of nanometer lipid transfer body

Abstract

The invention discloses a preparation method of a nano lipid transfer body for efficiently loading active ingredients of medicinal plants, which comprises the following steps: adding egg yolk lecithin, cholesterol and a surfactant into an organic solvent to obtain a mixed solution, and carrying out ultrasonic treatment on the mixed solution in a water bath to obtain an oil phase solution; dissolving the active ingredients of the medicinal plants in a buffer solution, and performing ultrasonic treatment in a water bath to obtain a water-phase solution; and preparing the nano lipid transfer body loaded with the active ingredients of the medicinal plants through a film hydration method and sieve pore filtration. The invention also discloses a nano-liposome transfer body capable of efficiently loading the active ingredients of the medicinal plants, a bimolecular layer nano-liposome is constructed by phospholipid, cholesterol and a surfactant, the active ingredients of the medicinal plants are coated by a phospholipid bimolecular layer and an inner water phase, and the active ingredients of the medicinal plants are encapsulated in the nano-liposome, so that the nano-liposome transfer body is obtained. And in combination with mass transfer gel formed by carboxymethyl chitosan and calcium chloride, effective transmission and release of drugs are realized, and active ingredients of medicinal plants are efficiently loaded.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery systems, and in particular relates to a nano lipid mass transfer body capable of efficiently loading plant active ingredients, and a preparation method and application thereof. Background Art

[0002] With the development of modern medicine, people's requirements for drug delivery systems are increasing, especially in terms of improving drug efficacy, reducing side effects, and increasing patient compliance. Although the traditional oral administration method is convenient, the bioavailability is not high because the drug needs to go through the metabolic process of the digestive tract and liver. In addition, for some situations that require continuous or timed administration, oral administration methods often cannot meet the needs. Therefore, exploring more effective drug delivery routes has become the focus of research.

[0003] Transdermal drug delivery systems have attracted much attention due to their advantages of avoiding first-pass effect, reducing dosing frequency, and reducing systemic side effects. However, as the largest barrier organ in the human body, the existence of the stratum corneum of the skin greatly limits the effective penetration of most drugs.

[0004] Active ingredients from medicinal plants have important clinical application prospects due to their wide range of biological activities, such as antioxidant, anti-inflammatory, and anti-cancer effects. However, these active ingredients generally have problems such as low solubility and poor stability, which limit their clinical application.

[0005] In recent years, the development of nanotechnology has provided new ideas for solving the above problems. As a new type of drug carrier, nanoliposomes have shown great potential in increasing drug loading and sustained drug release. Nanoliposomes are tiny vesicles composed of phospholipid bilayers, which can be loaded with water-soluble drugs, while the lipid bilayer can be embedded with fat-soluble drugs. In addition, by regulating the size, surface charge and composition of liposomes, the transdermal performance of drugs can be effectively changed.

[0006] Despite this, existing nanoliposomes still have certain shortcomings in the actual application of encapsulating active ingredients of medicinal plants, such as limited drug loading capacity and low delivery efficiency. In addition, as the number of active medicinal ingredients loaded simultaneously by the existing drug delivery system increases, the drugs will affect the liposomes, or the drugs will affect each other, which will greatly reduce the drug loading capacity, making it impossible to load multiple liposomes at the same time. Summary of the invention

[0007] In order to overcome the deficiencies of the above prior art, the purpose of the present invention is to provide a method for preparing a nano-lipid mass transfer body that efficiently loads plant active ingredients, which not only improves the drug loading and skin penetration rate of plant active ingredients, but also loads multiple drugs without affecting each other's drug loading.

[0008] Another object of the present invention is to provide a nano lipid mass transfer body that can efficiently load plant active ingredients.

[0009] Another object of the present invention is to provide an application of a nano lipid mass transfer body for efficiently loading plant active ingredients.

[0010] The purpose of the present invention is achieved through the following technical solutions: A method for preparing a nano-lipid mass transfer body that efficiently loads active ingredients of medicinal plants comprises the following steps: (1) adding egg yolk lecithin, cholesterol and a surfactant to an organic solvent to obtain a mixed solution, and ultrasonicating the mixed solution in a water bath for 10-30 minutes to obtain an oil phase solution; dissolving the active ingredients of the medicinal plant in a buffer solution, and then ultrasonicating the mixed solution in a water bath for 5-30 minutes to obtain an aqueous phase solution; (2) subjecting the oil phase solution to rotary evaporation to remove the organic solvent to obtain a lipid film; mixing the lipid film with the aqueous phase solution, fully shaking and hydrating the mixture, and filtering the mixture to obtain a drug-loaded liposome solution; (3) mixing the drug-loaded liposome solution and the carboxymethyl chitosan solution and adding the mixture to a calcium chloride solution to form a gel, thereby obtaining a nano-lipid mass transfer body that efficiently loads the active ingredients of medicinal plants; The buffer solution in step (1) is a mixture of glycerol and a phosphate solution with a pH of 6-8.

[0011] Preferably, the concentration of the active ingredient of the medicinal plant in the buffer solution is 1-5 mg / ml.

[0012] Preferably, the volume ratio of the glycerol to the phosphate solution is 1:8-10.

[0013] Preferably, the surfactant is one or more of bile salt, Tween-80, polysorbate or glycyrrhizate.

[0014] Preferably, the mass ratio of the egg yolk lecithin, cholesterol and surfactant in step (1) is 4-12:1-2:1-3.

[0015] Preferably, in step (2), the volume ratio of the oil phase solution to the water phase solution is 1-5:1.

[0016] Preferably, the organic solvent in step (2) is chloroform or methanol.

[0017] Preferably, the concentration of the egg yolk lecithin in the organic solvent is 10-20 mg / ml.

[0018] Preferably, the concentration of the active ingredient of the medicinal plant in the organic solvent is 15-45 mg / mL.

[0019] Preferably, the specific step of filtering in step (2) is to filter through a 100 nm microporous sieve.

[0020] Preferably, the specific step of shaking hydration in step (2) is ultrasonic water bath for 1-5 minutes.

[0021] Preferably, the specific step of the rotary evaporation in step (2) is to remove the solvent by a reduced pressure rotary evaporation method using a rotary evaporator at 40-50°C.

[0022] Preferably, in step (3), the mass fraction of the carboxymethyl chitosan solution is 2-3wt%, and the mass fraction of the calcium chloride solution is 2-5wt%.

[0023] Preferably, in step (3), the volume ratio of the liposome solution to the carboxymethyl chitosan solution is 1:1-2.

[0024] Preferably, the solvent used for the carboxymethyl chitosan solution and the calcium chloride solution in step (3) is deionized water.

[0025] A nanolipid mass transfer body for efficiently loading active ingredients of medicinal plants comprises nanoliposomes and mass transfer body gel. The nanoliposomes comprise active ingredients of medicinal plants, egg yolk lecithin, cholesterol and a surfactant, and the mass transfer body comprises carboxymethyl chitosan and calcium chloride.

[0026] The invention discloses an application of a nano lipid mass transfer body for efficiently loading active ingredients of medicinal plants in a transdermal drug delivery system.

[0027] Preferably, the method is applied to a transdermal drug delivery system for simultaneously loading two or more active ingredients of medicinal plants.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention constructs bilayer nanoliposomes by using phospholipids, cholesterol and surfactants. The phospholipid bilayer and the inner aqueous phase encapsulate the active ingredients of medicinal plants, encapsulate the active ingredients of medicinal plants in the nanoliposomes, and combine with the mass transfer body gel formed by carboxymethyl chitosan and calcium chloride to achieve effective drug transmission and release, and efficiently load the active ingredients of medicinal plants. This structure not only improves the transdermal efficiency and bioavailability of the drug, but also the prepared lipid mass transfer body can achieve efficient loading of multiple active ingredients of medicinal plants at the same time. The drug loading amount is not significantly changed compared with two active ingredients of medicinal plants, which enriches the administration method of traditional Chinese medicine prescriptions.

[0029] (2) The present invention uses materials from natural sources and sets the mass ratio of egg yolk lecithin phospholipids, cholesterol, and surfactant to 4-12:1-2:1-3, thereby ensuring the biocompatibility and safety of the preparation; through thin film hydration method and sieve filtration, the preparation cost is greatly simplified, the drug encapsulation rate and the uniformity of liposomes are improved, and it is conducive to large-scale production and application; (3) The present invention uses a mixture of glycerol and phosphate with a pH of 6-8 as a buffer for dissolving the active ingredients of medicinal plants, which does not affect the basic parameters of the mass transfer body and can also increase the solubility of the active ingredients of medicinal plants in the mass transfer body, thereby achieving a higher drug loading amount, making it easier for the drug to enter the liposome and be stored, thereby increasing the encapsulation rate of the drug.

[0030] (4) The raw materials and their mass ratio, the preparation method and the setting of the buffer solution enable the lipid mass transfer body prepared by the present invention to not only improve the stability and bioavailability of the drug, but also enhance the transdermal absorption capacity of the drug and reduce systemic side effects.

[0031] (5) The nanolipid mass transfer body disclosed in the present invention improves drug absorption, enables drugs to better penetrate the skin barrier and reach the lesions directly, improves the therapeutic effect, has a good sustained release effect, and can also ensure the continuous release of drugs; secondly, through local administration, the distribution of drugs in other parts of the body is reduced, reducing potential side effects; thirdly, the administration method is simplified, avoiding the inconvenience that may be caused by traditional oral or injection administration, and improving the patient's medication experience and compliance. In addition, the application of this technology promotes technological innovation in the pharmaceutical field, provides new ideas and methods for the development of more efficient drug delivery systems, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The particle size, polydispersity coefficient and Zeta potential of the nanolipid mass transfer body of Example 1 and Comparative Examples 1 and 2 are shown.

[0033] Figure 2 The drug loading capacity of the nano lipid mass transfer bodies of Example 1 and Comparative Examples 1 and 2 for the active ingredients of medicinal plants.

[0034] Figure 3 The encapsulation efficiency of the nano lipid mass transfer bodies of Example 1 and Comparative Examples 1 and 2 for the active ingredients of medicinal plants.

[0035] Figure 4 The in vitro drug release diagram of the nanolipid mass transfer body of Example 1 and Comparative Examples 1 and 2.

[0036] Figure 5 The transdermal and intradermal drug release of the nanolipid mass transfer bodies of Example 1 and Comparative Examples 1 and 2.

[0037] Figure 6 The transdermal and intradermal drug release rates of the nanolipid mass transfer bodies of Example 1 and Comparative Examples 1 and 2 are shown.

[0038] Figure 7 The drug loading capacity of the nano lipid mass transfer bodies of Example 2 and Comparative Examples 3, 4, and 5 for the active ingredients of medicinal plants. DETAILED DESCRIPTION

[0039] The purpose of the present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples cannot be described one by one here, but the implementation methods of the present invention are not therefore limited to the following examples.

[0040] Example 1 Material preparation: Active ingredients of medicinal plants: one of gallic acid, quercetin or hyperoside; egg yolk lecithin; cholesterol; surfactant: Tween-80; carboxymethyl chitosan; calcium chloride; organic solvent: chloroform; buffer: glycerol and phosphate buffer with pH=7.4 mixed in a volume ratio of 1:9.

[0041] Preparation steps: (1) Preparation of oil phase solution: Weigh egg yolk lecithin (120 mg), cholesterol (30 mg) and Tween-80 (30 mg), dissolve in 10 mL of chloroform to obtain an oil phase solution. Ultrasonicate the mixture in a water bath for 20 minutes to mix it thoroughly.

[0042] (2) Preparation of aqueous solution: Prepare 5 mL of a buffer solution of glycerol and pH 7.4 phosphate in a volume ratio of 1:9. Dissolve 5 mg of the active pharmaceutical ingredient in the buffer solution and sonicate in a water bath for 15 minutes to ensure complete dissolution.

[0043] (3) Formation of nanolipid mass transfer bodies: The oil phase solution was subjected to reduced pressure rotary evaporation at 50°C using a rotary evaporator to remove chloroform in the oil phase solution to form a uniform lipid film. The lipid film was mixed with the above aqueous phase solution at a volume ratio of 2:1 and fully shaken for 3 minutes. The drug-loaded liposome solution was obtained by filtering through a 100 nm microporous membrane.

[0044] Prepare 2 wt% carboxymethyl chitosan solution and 2 wt% calcium chloride solution. Mix the drug-loaded liposome solution and carboxymethyl chitosan solution in a volume ratio of 1:2. Add the mixture to the calcium chloride solution and stir evenly to form a stable drug-loaded nanolipid mass transfer body.

[0045] Through the above preparation steps, gallic acid-loaded lipid mass transfer body, quercetin-loaded lipid mass transfer body and hyperoside-loaded lipid mass transfer body are prepared respectively.

[0046] Example 2 The difference between this embodiment and embodiment 1 is that the medicinal plant active ingredients include three components: gallic acid, quercetin and hyperoside, and the added amount of each component is 5 mg.

[0047] The remaining parameters and preparation methods are the same as those in Example 1.

[0048] Comparative Example 1 The difference between this comparative example and Example 1 is that the buffer solution is 5 mL of a phosphate solution with a pH of 7.4.

[0049] The remaining parameters and preparation methods are the same as those in Example 1.

[0050] Comparative Example 2 The difference between this comparative example and Example 1 is that the buffer solution is 5 mL of a mixture of sodium hyaluronate and a phosphate solution with a pH value of 7.4 at a mass ratio of 0.1:100.

[0051] The remaining parameters and preparation methods are the same as those in Example 1.

[0052] Comparative Example 3 The difference between this comparative example and Example 2 is that the medicinal plant active ingredients include two components, gallic acid and quercetin, and the added amount of each component is 5 mg.

[0053] The remaining parameters and preparation methods are the same as those in Example 2.

[0054] Comparative Example 4 The difference between this comparative example and Example 2 is that the medicinal plant active ingredients include two components, gallic acid and hyperoside, and the added amount of each component is 5 mg.

[0055] The remaining parameters and preparation methods are the same as those in Example 2.

[0056] Comparative Example 5 The difference between this comparative example and Example 2 is that the medicinal plant active ingredients include two components, quercetin and hyperoside, and the added amount of each component is 5 mg.

[0057] The remaining parameters and preparation methods are the same as those in Example 2.

[0058] The evaluation method for the encapsulation efficiency, in vitro drug release, and transdermal drug release of the lipid mass transfer body described in the embodiments of the present invention is as follows: The encapsulation efficiency test method is as follows: The instrument is Shimadzu LAD-20; the mobile phase is acetonitrile-0.4% phosphoric acid aqueous solution; the chromatographic column is a C18 reversed phase chromatographic column; the column temperature is 40°C; the flow rate is 1.0 ml / min; the detector wavelength is 254 nm; the elution gradient is 0-5 min, 5-15% acetonitrile, 5-10 min, 15-17% acetonitrile, 10-25 min, 17% acetonitrile, 25-35 min, 17-26% acetonitrile, 35-60 min, 26-56% acetonitrile.

[0059] Ultrafiltration centrifugation was used to separate liposomes and drugs, and the separated drugs were detected and analyzed by high performance liquid chromatography, and the encapsulation efficiency of various drugs was calculated.

[0060] The encapsulation efficiency calculation formula is: (W total - W travel) / W total × 100% Wherein Wtotal is the total drug content in the liposome solution, and Wfree is the free drug content in the separated liposome solution.

[0061] The in vitro drug release test method of nanolipid mass transfer body is: A mass transfer gel containing 30 mg of active ingredients of medicinal plants was placed in a 50 mL centrifuge tube, 15 mL of ultrapure water was added to the centrifuge tube to completely immerse the hydrogel, and the centrifuge tube was placed in a constant temperature shaker. 2 mL of the extract was taken at 1, 2, 4, 8, 12, 24, and 48 h and supplemented with 2 mL of ultrapure water. The extract was detected using a high performance liquid chromatograph using the method in Test Example 1. The obtained chromatogram was combined with the obtained standard curve to calculate the content of various drug components in the hydrogel extract at different times. The cumulative permeation amount was calculated according to the following formula, and the in vitro drug release curve was plotted: Wherein, Qt is the cumulative permeation amount of the drug (μg); Cn is the drug concentration at the nth time (μg / mL); V is the volume of the extract (mL); and Vn is the volume of each sampling (mL).

[0062] The test method of the nanolipid mass transfer body drug transdermal release experiment is as follows: The abdominal skin of C57 mice was taken, the hair on the outer surface of the skin was removed with a razor, the fat on the inner surface was removed with a cotton swab, and then washed with water. The prepared ex vivo mouse abdominal skin and the transdermal diffusion instrument were used to simulate the transdermal diffusion experiment to determine the drug scalp release performance of the hydrogel. The hydrogels of each group with the same amount of drug release as the in vitro drug release were used. The experimental conditions were temperature 37 ℃, rotation speed 350 rpm, and the experiment lasted for 48 h. After the experiment, 2 mL of the receiving pool solution was taken, and the surface of the ex vivo mouse skin was wiped clean with a cotton swab and cut into pieces. 4 mL of 50% methanol aqueous solution was used for ultrasonic extraction at 60 ℃ for 60 min, and then centrifuged at 1500r / min for 10 min, and the supernatant was taken to determine the drug content retained in the skin. The obtained solution was detected by high performance liquid chromatography using the method in the encapsulation rate test experiment, and the obtained chromatogram was combined with the standard curve obtained from the in vitro release experiment of the mass transfer body drug to calculate the content of various drug components in the solution.

[0063] Figure 1 The particle size, polydispersity coefficient, and Zeta potential of the nanolipid mass transfer body of Example 1 and Comparative Examples 1 and 2 are shown in FIG. Figure 1 It can be seen that the particle size, polydispersity coefficient, and Zeta potential of Example 1 (using glycerol-phosphate buffer) are basically not significantly different from those of Comparative Example 1 (adding only phosphate buffer), indicating that the glycerol in Example 1 does not affect the basic parameters of the mass transfer body. However, the particle size, polydispersity coefficient, and Zeta potential of Comparative Example 2 using sodium hyaluronate-phosphate buffer have changed significantly compared with Example 1, which has a greater impact on the basic parameters of the mass transfer body.

[0064] Figure 2 The drug loading of the nanoliposome mass transfer bodies of Example 1 and Comparative Examples 1 and 2 for the active ingredients of medicinal plants. Figure 2 It can be seen that the introduction of glycerol in Example 1 and the introduction of sodium hyaluronate in Comparative Example 2 can increase the solubility of the active ingredients of the medicinal plants in the mass transfer body, thereby achieving a higher drug loading. Comparing Example 1 with Comparative Example 2, it can be found that the loading of the active ingredients of the medicine in Example 1 is slightly higher than that in Comparative Examples 1 and 2.

[0065] Figure 3 The encapsulation efficiency of the nanoliposome mass transfer body for the active ingredients of medicinal plants in Example 1 and Comparative Examples 1 and 2. Figure 3 It can be seen that the encapsulation efficiency of each medicinal plant active ingredient in Example 1 is substantially better than that in Comparative Examples 1 and 2. This is due to the fact that the addition of glycerol increases the fluidity of the liposome vesicles, making it easier for the drug to enter the liposome and be stored, thereby increasing the encapsulation efficiency of the drug.

[0066] Figure 4 The in vitro drug release diagram of the nanoliposome mass transfer body of Example 1 and Comparative Examples 1 and 2. Figure 4It can be seen that Example 1 not only has a good sustained release effect, but also can ensure the continuous release of drugs. Compared with the performance of the explosive release in the early stage of Comparative Examples 1 and 2, and the inability to release excess drugs in the later stage, the sustained release effect of Example 1 can avoid excessive accumulation of drugs in the patient's body and excessive drug concentration leading to adverse consequences, and can also ensure that the drugs can be continuously delivered to the patient, prolonging the drug action time.

[0067] Figure 5 and Figure 6 The transdermal and intradermal drug release and release rate of the nanoliposome mass transfer bodies of Example 1 and Comparative Examples 1 and 2. It can be seen that although the drug transdermal rates of Example 1, Comparative Example 1, and Comparative Example 2 are basically the same, the drug transdermal amount of Example 1 is slightly better than that of Comparative Example 1 and Comparative Example 2. The intradermal drug release and release rate of Comparative Examples 1 and Comparative Example 2 are higher than those of Example 1, indicating that the drug loaded by Comparative Examples 1-2 has a high release in the skin layer, resulting in accumulation, and more drugs stay in the skin and cannot penetrate the skin to reach the desired part. In Example 1, more drugs penetrate the skin and reach the drug administration site, thereby achieving more efficient drug administration efficiency.

[0068] Figure 7 The drug loading capacity of the nano lipid mass transfer bodies for the active ingredients of medicinal plants in Example 2 and Comparative Examples 3, 4, and 5. Figure 7 It can be seen that when the nanolipid mass transfer body is loaded with three medicinal plant active ingredients at the same time, the drug loading amount is basically the same as the drug loading amount when two medicinal plant active ingredients are loaded, indicating that the nanolipid mass transfer body can efficiently load more than two medicinal plant active ingredients. And the content of the loaded medicinal plant active ingredients is within the required range. For drug loading, especially for traditional Chinese medicine prescriptions with multiple complex plant active ingredients, the nanoliposome mass transfer body enriches the administration method of traditional Chinese medicine prescriptions.

[0069] The above specific implementation modes are preferred embodiments of the present invention and cannot be used to limit the present invention. Any other changes or other equivalent replacement methods that do not deviate from the technical solution of the present invention are included in the protection scope of the present invention.

Claims

1. A method for preparing a nano-lipid mass transfer body that efficiently loads active ingredients of medicinal plants, characterized in that: The following steps are involved: (1) adding egg yolk lecithin, cholesterol and a surfactant to an organic solvent to obtain a mixed solution, and ultrasonicating the mixed solution in a water bath for 10-30 minutes to obtain an oil phase solution; The active ingredients of the medicinal plants are dissolved in a buffer solution, and then placed in a water bath for ultrasonication for 5-30 minutes to obtain an aqueous phase solution; (2) subjecting the oil phase solution to rotary evaporation to remove the organic solvent to obtain a lipid film; mixing the lipid film with the aqueous phase solution, fully shaking and hydrating the mixture, and filtering the mixture to obtain a drug-loaded liposome solution; (3) mixing the drug-loaded liposome solution and the carboxymethyl chitosan solution and adding the mixture to a calcium chloride solution to form a gel, thereby obtaining a nano-lipid mass transfer body that efficiently loads the active ingredients of medicinal plants; The buffer solution in step (1) is a mixture of glycerol and a phosphate solution with a pH of 6-8.

2. The method for preparing the nano lipid mass transfer body according to claim 1, characterized in that: The volume ratio of the glycerol to the phosphate solution is 1:8-10.

3. The method for preparing the nano lipid mass transfer body according to claim 1, characterized in that: The mass ratio of egg yolk lecithin, cholesterol and surfactant in step (1) is 4-12:1-2:1-3.

4. The method for preparing the nano lipid mass transfer body according to claim 1, characterized in that: The volume ratio of the oil phase solution to the water phase solution in step (2) is 1-5:

1.

5. The method for preparing the nano lipid mass transfer body according to claim 1, characterized in that: The organic solvent in step (2) is chloroform or methanol.

6. The method for preparing the nano lipid mass transfer body according to claim 1, characterized in that: The specific steps of the rotary evaporation in step (2) are to remove the solvent by reduced pressure rotary evaporation using a rotary evaporator at 40-50°C.

7. The method for preparing the nano lipid mass transfer body according to claim 1, characterized in that: In step (3), the mass fraction of the carboxymethyl chitosan solution is 2-3wt%, and the mass fraction of the calcium chloride solution is 2-5wt%.

8. The method for preparing the nano lipid mass transfer body according to claim 1, characterized in that: In step (3), the volume ratio of the liposome solution to the carboxymethyl chitosan solution is 1:1-2.

9. A nanolipid mass transfer body for efficiently loading active ingredients of medicinal plants, prepared by the preparation method of the nanolipid mass transfer body according to any one of claims 1 to 8, characterized in that: The invention comprises nano liposome and mass transfer body gel. The nano liposome comprises active ingredients of medicinal plants, egg yolk lecithin, cholesterol and surfactant, and the mass transfer body comprises carboxymethyl chitosan and calcium chloride.

10. The use of the nano lipid mass transfer body for efficiently loading active ingredients of medicinal plants as claimed in claim 9, characterized in that: Used in transdermal drug delivery systems.

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