Chitosan modified dihydromyricetin liposome as well as preparation method and application thereof

Abstract

The invention discloses a chitosan modified dihydromyricetin liposome as well as a preparation method and application thereof, and belongs to the technical field of liposome preparation. The chitosan modified dihydromyricetin liposome is prepared from the following raw materials: dihydromyricetin, phospholipid and chitosan according to the mass ratio of (0.25 to 0.5) to (1 to 1.5) to (0.25 to 1); the chitosan modified dihydromyricetin liposome takes dihydromyricetin as a core material, and a coating structure comprises an inner layer and an outer layer; the inner layer is a phospholipid bilayer spherical vesicle formed by self-assembly of phospholipid in an aqueous solution state, namely lipidosome; and the outer layer is a chitosan coating. The chitosan modified dihydromyricetin lipidosome disclosed by the invention has excellent properties of oxidation resistance, alpha-glucosidase inhibitory activity and the like, and is small in particle, uniform in particle size distribution, good in stability, good in slow-release effect and high in bioavailability.

Description

Technical Field

[0001] The invention relates to the technical field of liposome preparation, in particular to a chitosan-modified dihydromyricetin liposome and a preparation method and application thereof. Background Art

[0002] Ampelopsis grossedentata Hand.-Mazz. is a wild vine plant of the genus Ampelopsis in the family Vitaceae. It is widely distributed. The stems and leaves of Ampelopsis grossedentata contain a large number of flavonoid compounds, among which the main active ingredient is dihydromyricetin (DMY), also known as dihydromyricetin, ampelopsis, and ampelopsis. The total flavonoid content in Ampelopsis grossedentata tea leaves is 45%, and DMY is 16.32-33.03%, which is the flavonoid compound with the highest content. DMY has multiple pharmacological effects such as antioxidant, antibacterial, antiviral, anti-tumor, hypoglycemic, alcohol detoxification and liver protection. However, DMY (flavonoid substance) has poor fat solubility and is easily oxidized, so its oral bioavailability is often less than 10%, and its absolute bioavailability is only 4.02%, which has caused great difficulties in the research and development of nutritional preparations with practical utility value, which greatly limits its application in clinical and food industries. Therefore, in order to overcome the above shortcomings, it is urgent to seek new methods and technologies to improve the stability and bioavailability of dihydromyricetin.

[0003] Studies have shown that liposomes have advantages over traditional formulations in improving the biostability, absorption and bioavailability of active substances. Liposomes are an artificial phospholipid bilayer membrane with a structure similar to that of a cell membrane. They can carry hydrophobic and hydrophilic compounds separately / simultaneously as a physical and chemical barrier to increase drug solubility. Encapsulating bioactive substances such as peptides and polyphenols in liposomes can play a role in sustained release and improve their stability and bioavailability. However, conventional liposomes are easily oxidized during storage and have poor structural stability. After oral administration, they are easily damaged by enzymes and pH values ​​in the gastrointestinal tract, which in turn affects their drug absorption. Surface modification can reduce environmental degradation and drug leakage from the phospholipid bilayer, and can protect the load in the liposome system. Therefore, surface modification of traditional liposomes may provide a method to effectively improve the stability and bioavailability of dihydromyricetin and avoid its damage by enzymes and pH values ​​in the gastrointestinal tract after oral administration. Summary of the invention

[0004] The purpose of the present invention is to provide a chitosan-modified dihydromyricetin liposome and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The chitosan-modified dihydromyricetin liposome of the present invention uses natural soybean lecithin (PC for short) as the first coating layer and chitosan (CHI for short) as the second coating layer, which can effectively improve the stability and bioavailability of dihydromyricetin.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: a chitosan-modified dihydromyricetin liposome, the raw materials comprising dihydromyricetin, phospholipid and chitosan in a mass ratio of (0.25-0.5):(1-1.5):(0.25-1);

[0007] The chitosan modified dihydromyricetin liposome comprises an inner layer and an outer layer; the inner layer is a dihydromyricetin liposome formed by phospholipid-coated dihydromyricetin; and the outer layer is chitosan.

[0008] Furthermore, the phospholipid comprises soybean lecithin.

[0009] Furthermore, the raw materials include dihydromyricetin, phospholipids and chitosan in a mass ratio of 0.375:1:0.25.

[0010] The second technical solution of the present invention: A method for preparing the above-mentioned chitosan-modified dihydromyricetin liposomes, comprising the following steps:

[0011] Dissolve dihydromyricetin and phospholipids in organic solvents respectively and mix them evenly, then remove the solvent to obtain a uniform film, then add a buffer solution for hydration reaction, and filter through a microporous filter membrane (pore size of 0.22 μm) to obtain a dihydromyricetin liposome (DMY-Lips for short);

[0012] The dihydromyricetin liposome solution is added to the acidic solution of chitosan, and an outer layer encapsulation treatment (ie, magnetic stirring treatment) is performed, and the mixture is filtered through a microporous filter membrane (pore size of 0.45 μm) to obtain the chitosan-modified dihydromyricetin liposome (CHI-DMY-Lips for short).

[0013] Furthermore, the concentration of dihydromyricetin in the dihydromyricetin liposome solution is 0.5-1 mg / mL, and the concentration of phospholipid is 2-3 mg / mL.

[0014] Furthermore, the concentration of chitosan in the acidic chitosan solution is 0.5-2 mg / mL; and the volume ratio of the acidic chitosan solution and the dihydromyricetin liposome solution when mixed is 1:1.

[0015] Furthermore, the concentration of dihydromyricetin in the dihydromyricetin liposome solution is 0.75 mg / mL, and the concentration of phospholipids is 2 mg / mL; the concentration of chitosan in the acidic chitosan solution is 0.5 mg / mL. The average particle size of DMY-Lips prepared under this condition is 171.04 nm, the Zeta potential is -19.8 mV, and the DMY encapsulation efficiency is 41.01%. The average particle size of CHI-DMY-Lips is 345.21 nm, the Zeta potential is 23.5 mV, and the DMY encapsulation efficiency is 54.00%. The PDI of DMY-Lips and CHI-DMY-Lips is around 0.1 to 0.3, proving that they are evenly distributed and stable in the aqueous phase.

[0016] Furthermore, the method for removing the solvent includes rotary evaporation under reduced pressure, and the evaporation temperature is 30°C.

[0017] Furthermore, the buffer solution includes PBS phosphate buffer with a concentration of 0.01 mol / L and a pH of 7.2.

[0018] Furthermore, the hydration reaction specifically includes: conducting a hydration reaction under ultrasonic conditions, the temperature is 30° C., the ultrasonic power is 200-300W, the frequency is 40KHz, and the ultrasonic method is to ultrasonicate for 30s and stand for 30s as one cycle, and a total of 4 cycles.

[0019] Furthermore, the encapsulation treatment time is 1 to 4 hours.

[0020] The third technical solution of the present invention: an application of the chitosan modified dihydromyricetin liposome in the preparation of antioxidant, anticancer, liver-protecting, antibacterial or hypoglycemic drugs.

[0021] The chitosan-modified dihydromyricetin liposome of the invention has anti-oxidation and α-glucosidase activity inhibition effects; and using the chitosan-modified liposome as a drug-carrying system can effectively improve the stability and bioavailability of dihydromyricetin.

[0022] The chitosan-modified dihydromyricetin liposome prepared by the invention is beneficial to improving the mechanical strength of the entire drug-carrying system, ensuring that the chitosan-modified dihydromyricetin liposome has a good DMY sustained-release effect under a certain pH environment, and effectively improving the absorption and utilization rate of DMY.

[0023] The present invention discloses the following technical effects:

[0024] (1) The chitosan-modified dihydromyricetin liposomes of the present invention have antioxidant and α-glucosidase activity inhibitory effects.

[0025] (2) The CHI-DMY-Lips of the present invention does not involve modification and activation through chemical reactions during the entire preparation process, and there is no organic solvent residue. The prepared nanoliposome particles have good biosafety.

[0026] (3) The CHI-DMY-Lips obtained by modifying and encapsulating DMY-Lips in the present invention can not only retain the antioxidant and α-glucosidase activity inhibition properties of the raw material DMY itself, but also significantly improve the water solubility, stability (under different storage environments) and bioavailability of DMY, delay the release of DMY, have significant sustained-release characteristics, effectively improve the burst release behavior of DMY, and increase the absorption and utilization of active substances in the gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Schematic diagram of the preparation process of DMY-Lips and CHI-DMY-Lips;

[0029] Figure 2 The particle size distribution diagram of DMY-Lips prepared in Example 1 and CHI-DMY-Lips prepared in Example 1;

[0030] Figure 3 The UV absorption spectra of DMY, blank liposomes (CHI-Blank-Lips for short), DMY-Lips prepared in Example 1, and CHI-DMY-Lips prepared in Example 1;

[0031] Figure 4 TEM images of DMY-Lips prepared in Example 1 and CHI-DMY-Lips prepared in Example 1, wherein A is DMY-Lips and B is CHI-DMY-Lips;

[0032] Figure 5The encapsulation efficiency, particle size, PDI and potential changes of DMY-Lips prepared in Example 1 and CHI-DMY-Lips prepared in Example 1 stored at different temperatures for different days, wherein A is the encapsulation efficiency of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days; B is the particle size of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days; C is the potential of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days; D is the PDI of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days;

[0033] Figure 6 The in vitro release of DMY, DMY-Lips prepared in Example 1 (reconstituted after freeze-drying), and CHI-DMY-Lips prepared in Example 1 (reconstituted after freeze-drying) at pH 6.8;

[0034] Figure 7 DPPH radical scavenging activity of DMY (concentration of 0.75 mg / mL), DMY-Lips prepared in Example 1, CHI-DMY-Lips prepared in Example 1, and samples of DMY and DMY-Lips prepared in Example 1 diluted in half (DMY-HD and DMY-Lips-HD, DMY content of 0.375 mg / mL);

[0035] Figure 8 The inhibitory effect of DMY (concentration of 0.75 mg / mL), DMY-Lips prepared in Example 1, CHI-DMY-Lips prepared in Example 1, and samples of DMY and DMY-Lips prepared in Example 1 diluted in half (DMY-HD and DMY-Lips-HD, DMY content of 0.375 mg / mL) on α-glucosidase activity. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. The instruments and equipment used in the preparation experiments and product analysis and detection are all conventional instruments and equipment commonly used. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0042] Materials and methods used in the present invention:

[0043] 1. Main Materials and Reagents

[0044] The main chemical reagents involved in the experiment are shown in Table 1. Unless otherwise specified, all water used in the experiment was ultrapure water.

[0045] Table 1 Main chemical reagents

[0046] Reagent name Specification Manufacturer Dihydromyricetin AR Aladdin Reagents (Shanghai) Co., Ltd. Anhydrous ethanol AR Guangzhou Guanghua Technology Co., Ltd. Soy lecithin AR Sigma-Aldrich (USA) Chitosan MV=50000 Shanghai MacLean Biochemical Technology Co., Ltd. Chloroform AR Guangzhou Chemical Reagent Factory Phosphate buffered saline 0.01mol / L, pH7.2 Shanghai Yuanye Biotechnology Co., Ltd. 1% acetic acid aqueous solution / Shanghai Yuanye Biotechnology Co., Ltd.

[0047] 2. Main instruments and equipment

[0048] The main instruments and equipment involved in the experiment are shown in Table 2.

[0049] Table 2 Main instruments and equipment

[0050] Instrument Name model Manufacturer Rotary evaporator EV400H Labtech Ltd. Ultrasonic Cleaner LC-MUC-100S Shanghai Lichen Bangxi Instrument Co., Ltd. UV-Vis Spectrophotometer Carly60Uv-Vis Agilent Technologies ELISA reader VarioskanLUX Thermo Fisher Scientific Analytical Balance BSA224S Odoris Scientific Instruments (Beijing) Co., Ltd. Magnetic stirrer -- Nanjing Huaduo Pharmaceutical Technology Co., Ltd. Constant temperature vibration shaker THZ-82 Guohua Instrument Manufacturing Co., Ltd. Dialysis Bags 8000～14000 Shanghai Jizhi Biochemical Technology Co., Ltd. Transmission electron microscopy JEM1400PIus Japan Electronics Co., Ltd. (JEOL) Nanoparticle size analyzer ZETASUZERNANOZSE Malvern Panalytical Instruments Ltd.

[0051] 3. Data Analysis

[0052] Origin 2024 software was used to analyze the differences among the groups and draw relevant graphs.

[0053] Example 1

[0054] A preparation method of chitosan modified dihydromyricetin liposomes:

[0055] (1) The liposomes were prepared by thin film hydration method: an appropriate amount of PC was dissolved in chloroform to obtain a PC solution; an appropriate amount of DMY was dissolved in anhydrous ethanol to obtain a DMY solution; the PC solution and the DMY solution were mixed in a volume ratio of 2:1 to obtain a mixed solution; the mixed solution was transferred to a 250 mL round-bottom flask, and the organic solvent was removed by rotary evaporation at 30°C for 10 min under reduced pressure to obtain a uniform thin film; the round-bottom flask with the phospholipid film was further purged with nitrogen for 5 min at room temperature to completely remove the residual organic solvent, and then 20 mL of PBS solution (0.01 mol / L, pH 7.2) was vortexed and hydrated at room temperature for 2 min to pre-dissolve the phospholipid film; finally, an ultrasonic cleaner was used in a water bath for 2 min (power 240 W, frequency 40 kHz; ultrasonic 30 s, standing 30 s as one cycle, a total of 4 cycles) to obtain a liposome suspension containing 0.75 mg / mL The dihydromyricetin liposome (DMY-Lips) solution of DMY and 2 mg / mL PC was stored at 4°C until use.

[0056] (2) Weigh an appropriate amount of CHI and mix it with 1% acetic acid aqueous solution to obtain a chitosan mixed solution with a concentration of 0.5 mg / mL. DMY-Lips is added to the chitosan mixed solution by dripping (the volume ratio of DMY-Lips solution to chitosan mixed solution is 1:1), and magnetic stirring (200 r / min) is performed for 1 h. The mixture is filtered through a 0.45 μm microporous membrane to obtain chitosan-modified dihydromyricetin liposomes (CHI-DMY-Lips) containing 0.375 mg / mL DMY. The schematic diagram of the preparation process of DMY-Lips and CHI-DMY-Lips is shown in Figure 1 .

[0057] Comparative Example 1

[0058] How to prepare CHI-Blank-Lips:

[0059] The preparation method of CHI-Blank-Lips is the same as that of Example 1, except that the dihydromyricetin ethanol solution is replaced by an equal volume of ethanol solution.

[0060] Effect Example 1

[0061] Characterization of dihydromyricetin liposomes (DMY-Lips) and chitosan-modified dihydromyricetin liposomes (CHI-DMY-Lips):

[0062] (1) Determination of average particle size, zeta potential and polydispersity index

[0063] 1 mL of DMY-Lips (prepared in Example 1) and CHI-DMY-Lips (prepared in Example 1) were diluted 5 times respectively, and the average particle size, Zeta potential and polydispersity index of DMY-Lips and CHI-DMY-Lips were tested by a nanoparticle size analyzer. The results are shown in Table 3.

[0064] The size of liposomes is one of the factors to evaluate their performance. The size of liposomes is usually measured in nanometers (nm), and its size ranges from a few nanometers to a few micrometers. The size of nanoliposomes used for medical applications is usually between 50nm and 450nm. The average particle size of DMY-Lips and CHI-DMY-Lips is 171.04±2.91nm and 345.21±9.32nm, respectively (Table 3). The particle size distribution of the two is shown in Figure 3. Figure 2 The results showed that the average size of nanoliposomes increased after chitosan modification, which may be attributed to the interaction between chitosan and the phospholipid bilayer - chitosan was modified by electrostatic interaction and attached to the surface of liposomes to form a chitosan coating, which in turn led to an increase in the particle size of liposomes.

[0065] The absolute value of the Zeta potential of liposomes will affect the dispersion between vesicles. Zeta potential is an indicator that assists in evaluating the stability of liposomes and is a parameter that represents the surface charge of particles. Studies have found that a Zeta potential between ±(0-10) mV indicates that the liposomes are extremely unstable, and a Zeta potential between ±(20-30) mV indicates that the liposomes are relatively stable. The larger the absolute value, the more charge the liposome surface carries, the stronger the repulsive force between the vesicles, thereby avoiding aggregation and adhesion between liposome particles, and the better the stability of the liposomes. The Zeta potentials of DMY-Lips and CHI-DMY-Lips were -19.8±0.90 mV and 23.5±1.02 mV, respectively (Table 3). After the liposomes were modified with chitosan, the Zeta potential changed from the original negative potential to a positive potential. This potential change was due to the ionic attraction between the positively charged chitosan amino groups and the negatively charged phospholipid group surface. The positive chains of chitosan were adsorbed on the membrane surface through electrostatic interactions and gradually saturated. Therefore, the negative charge was neutralized and the particles became more stable. The reason was related to the interaction between the chitosan amino groups and phospholipids through electrostatic adsorption. The results showed that chitosan was successfully coated on the surface of dihydromyricetin liposomes.

[0066] In addition, the polydispersity index (PDI) is a dimensionless value of particle size distribution, which is also measured by dynamic light scattering. It is usually used to evaluate the quality and stability of materials and is also an important indicator for measuring the uniformity of particle size distribution. The PDI of DMY-Lips and CHI-DMY-Lips are 0.239±0.01 and 0.178±0.022, respectively (Table 3 and Figure 2 ). The results showed that both DMY-Lips and CHI-DMY-Lips had good PDI, indicating that the particle size distribution range was narrow. In summary, the basic physicochemical properties of DMY-Lips and CHI-DMY-Lips showed that chitosan-coated liposomes changed their particle size and surface charge, but had little effect on the particle size distribution (PDI). In general, chitosan coating can effectively improve the stability of dihydromyricetin liposomes.

[0067] (2) Determination of encapsulation efficiency

[0068] 2 mL of DMY-Lips (prepared in Example 1) or CHI-DMY-Lips (prepared in Example 1) was taken into a centrifuge tube and centrifuged at 10000 r / min for 20 min. 1 mL of the supernatant was taken and diluted to a certain multiple, and its absorbance was measured at 290 nm. The encapsulation efficiency was calculated according to the following formula:

[0069] EE(%)=(m 投药总量 -m 游离药量 ) / m 投药总量 ×100%.

[0070] The encapsulation efficiency (or drug loading) is often used to evaluate whether nanoparticles can load enough drugs to achieve their pharmacological effects. The encapsulation efficiency is an important indicator for evaluating the intrinsic quality of liposomes. It reflects the retention capacity of the embedded active substances in the liposomes. The size of the encapsulation efficiency directly reflects the quality of the liposomes. The encapsulation efficiency of DMY-Lips and CHI-DMY-Lips was 41.01±2.09% and 54.00±0.29%, respectively (Table 3). The results showed that after DMY-Lips was coated with CHI, the encapsulation efficiency of DMY increased by 12.99%, indicating that the coating effect of chitosan on the pores on the surface of the liposome bilayer membrane can prevent the leakage of the embedded substances, improve its stability, and make the nanoparticles have better quality.

[0071] Table 3 Physicochemical properties of liposomes

[0072] sample Particle size (nm) Zeta potential (mV) PDI EE(%) DMY-Lips 171.04±2.91 -19.8±0.90 0.239±0.01 41.01±2.09 CHI-DMY-Lips 345.21±9.32 23.5±1.02 0.178±0.022 54.00±0.29

[0073] (3) UV Characterization of DMY-Lips and CHI-DMY-Lips

[0074] The absorption wavelengths of CHI-Blank-Lips (prepared in Comparative Example 1), DMY-Lips (prepared in Example 1), CHI-DMY-Lips (prepared in Example 1) and DMY solution in the range of 200 to 800 nm were recorded using an ultraviolet spectrophotometer (UV-vis). The concentrations were diluted to appropriate values. The results are shown in Table 1. Figure 3 .

[0075] UV spectroscopy can determine the skeleton and hydroxyl substitution position of flavonoids, and plays a very important role in the determination of flavonoid structures. DMY belongs to the dihydroflavonol class of substances. In order to determine the successful coating of DMY, UV-vis analysis was performed on DMY, CHI-Blank-Lips, DMY-Lips and CHI-DMY-Lips. Figure 3As shown in the figure, DMY exhibits two main absorption bands in the UV-Vis region, and CHI-Bank-Lips has no obvious ultraviolet absorption peak in the range of 200-450nm. DMY has an obvious absorption peak at 290nm, which corresponds to the B ring part and is the result of the energy level transition of its cinnamoyl system. After being coated with PC, the absorption peak of the cinnamoyl system blue-shifts to 325nm due to the change of the conjugated system of DMY. When chitosan is coated on the surface of liposomes (CHI-DMY-Lips), it interacts with the phospholipid molecules on the surface of liposomes to form a protective chitosan film. This process may change the physicochemical properties of liposomes, including their ultraviolet absorption characteristics. Therefore, the position and intensity of the ultraviolet absorption peak of the chitosan-coated liposomes (CHI-DMY-Lips) will change. This is because the addition of chitosan changes the local environment of dihydromyricetin and affects the mode of its electronic transition, which leads to the movement of the ultraviolet absorption peak, and the absorption peak of its cinnamoyl system red-shifts to 290nm. Therefore, this UV analysis can preliminarily confirm the successful coating of DMY.

[0076] (4) Morphological observation

[0077] The microscopic morphology of DMY-Lips (prepared in Example 1) and CHI-DMY-Lips (prepared in Example 1) was observed by transmission electron microscopy, and the specific method was as follows:

[0078] DMY-Lips and CHI-DMY-Lips were diluted 10 times with ultrapure water and dropped onto a copper grid. Then 2% uranyl acetate was added to induce staining. After drying, the samples were carefully observed under a transmission microscope (TEM) and photographed. The results are shown in Figure 4 , where A is DMY-Lips and B is CHI-DMY-Lips.

[0079] TEM is often used to examine the microscopic morphology of nanoparticles. Figure 4 It can be seen that although there are still a few traces of residual dye, most of the particles show effective dispersion effect without obvious agglomeration. DMY-Lips and CHI-DMY-Lips appear as relatively evenly distributed round particles, indicating that in the solution state, DMY-Lips and CHI-DMY-Lips are spherical or quasi-spherical vesicle structures (showing a full granular vesicle structure), with particle sizes of about 170nm and 345nm, respectively. The system is stable, without rupture, uniform in particle size, uniform in distribution, and round and regular in appearance. In addition, the TEM of CHI-DMY-Lips shows that there is a thin film on the outermost layer, indicating that CHI can be used to modify liposomes without changing their microstructure or morphology.

[0080] (5) Solubility determination

[0081] Appropriate amounts of DMY API, DMY-Lips (prepared in Example 1) lyophilized powder, and CHI-DMY-Lips (prepared in Example 1) lyophilized powder were weighed, and ultrapure water was added for 20 minutes of ultrasound. The upper suspension was taken under the condition of centrifugation at 12000 r / min for 20 minutes, and the DMY concentration was measured using an ultraviolet spectrophotometer. The solubility results are shown in Table 4.

[0082] The results showed that the solubility of DMY-Lips (prepared in Example 1) and CHI-DMY-Lips (prepared in Example 1) lyophilized powders was significantly different from that of the raw materials (p < 0.001), and the solubility was increased by 2.01 times and 3 times, respectively.

[0083] Table 4 Solubility test results (n=3)

[0084] sample Solubility (μg / mL) DMY API 904.23±1.54 DMY-Lips freeze-dried powder 1815.4±1.28*** CHI-DMY-Lips Lyophilized Powder 2714.4±1.57***

[0085] Note: Compared with DMY API, ***p<0.001.

[0086] (6) Storage stability of liposomes

[0087] DMY-Lips (prepared in Example 1) and CHI-DMY-Lips (prepared in Example 1) were packaged in glass sample bottles and stored at room temperature and in a refrigerator at 4°C for 0, 5, 10, 15, 20, 25, and 30 days, respectively. Three vials were taken each time to test their encapsulation efficiency, particle size, PDI, and potential, and the average values ​​were taken to investigate the stability of the liposomes. The results are shown in Figure 5 .

[0088] Storage stability refers to storing the liposome suspension at different temperatures (4°C, room temperature) for several days, observing the changes in liposome morphology during storage, and evaluating the stability of liposomes by measuring indicators such as liposome encapsulation efficiency, average particle size, polymer dispersibility index, and potential.

[0089] Figure 5 Figure A shows the encapsulation efficiency of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days; Figure B shows the particle size of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days; Figure C shows the potential of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days; Figure D shows the PDI of DMY-Lips and CHI-DMY-Lips stored at 4°C and room temperature (RT) for different days.

[0090] Although the DMY-Lips encapsulation rate at both ambient temperatures was significantly different from the initial encapsulation rate (p < 0.05), the change at 4°C was relatively smaller than that at room temperature, indicating that DMY-Lips is more conducive to preservation at 4°C. Liposomes still have the phenomenon of leakage of embedded materials, aggregation and fusion, lipid oxidation and hydrolysis caused by environmental changes during storage and transportation. Therefore, it is necessary to modify liposomes with biomacromolecules such as chitosan to improve their dynamics, maintain structure and activity unchanged, and increase mechanical stability. After storage at the same 4°C for 30 days, the retention rate of DMY in CHI-DMY-Lips obtained after chitosan modification was higher than that of the initial single-layer liposomes (DMY-Lips), but its encapsulation rate at room temperature was lower than that at 4°C. This may be because the surface modification of chitosan can improve the stability of liposomes and the chitosan coating can improve the stability of active ingredients encapsulated in liposomes, reduce the leakage of contents to a certain extent, and is more conducive to the preservation of liposomes at 4°C.

[0091] The particle size of DMY-Lips increased with the extension of storage time from 0 to 25 days under different temperature environments. This may be due to the aggregation and fusion of phospholipid molecules during storage. However, under 4°C storage conditions, the changes were smaller than those under room temperature conditions. As the storage time increased, small phospholipid vesicles gradually aggregated into larger vesicles, resulting in an increase in the average particle size of the liposomes. This phenomenon is particularly evident at higher temperatures, because high temperatures increase the kinetic energy of phospholipid molecules and promote their aggregation and fusion. Between 25 and 30 days, the particle size of DMY-Lips decreased under different temperature environments, which may be because the phospholipid components of the liposomes may degrade during storage. This degradation may change the composition and properties of the phospholipid membrane, thereby affecting the particle size of the liposomes. The degradation of phospholipids may cause the membrane to become looser or more unstable, thereby reducing the particle size of the liposomes. Within 0 to 30 days, PDI was relatively stable, with little change and no significant difference.

[0092] Under different temperature conditions, the particle size of CHI-DMY-Lips increased with the extension of storage time from 0 to 10 days. This may be because chitosan has adhesive properties and will adhere together in small amounts during storage, causing the particle size to increase, but the change in storage under 4°C conditions is relatively small. From 15 to 30 days, the particle size of CHI-DMY-Lips stored under different temperature conditions decreased, and the particle size reduction was greater at room temperature than that under 4°C storage conditions. After chitosan modifies liposomes, a protective layer will be formed on the surface of the liposomes. With the extension of storage time, this protective layer may change, resulting in the release of part of the liposome contents or the contraction of the liposome membrane, which in turn manifests as a decrease in particle size. Chitosan molecules may be more likely to undergo conformational changes at higher temperatures, resulting in a decrease in the particle size of the liposomes. Within 0 to 30 days, PDI was relatively stable, with little change and no significant difference.

[0093] DMY-Lips was stored at 4℃ for 30 days, and its potential was relatively stable with no significant difference. At room temperature, it remained relatively stable from 0 to 10 days, but its potential began to become unstable and began to rise from 15 to 30 days. CHI-DMY-Lips was stored at 4℃ for 25 days, and its potential was around +21 to 23 mV, with little change, showing relatively stable performance. After 30 days of storage, it was significantly different from the initial state (p < 0.05); but after 10 days of storage at room temperature, its potential began to decrease, which was significantly different from the initial state (p < 0.05). The potential change results show that 4℃ is more conducive to the preservation of liposomes, and chitosan coating can improve its stability.

[0094] (7) In vitro release

[0095] The in vitro release of DMY-Lips (prepared in Example 1) and CHI-DMY-Lips (prepared in Example 1) was measured by equilibrium dialysis, and DMY was used as a control.

[0096] The specific operation is as follows: freeze-dried DMY-Lips and CHI-DMY-Lips (DMY concentration is consistent) are re-dissolved with 2 mL 7.2 PBS buffer, and then placed in a dialysis bag (retained molecular weight 8000-14000 Da), and placed in 100 mL of release medium (0.1 mol / L pH 6.8 PBS buffer, containing 0.5% Tween 80), respectively, and shaken at 37°C and 100 r / min. 1 mL of release medium is sampled at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, 72, 96, and 120 h, and replaced with 1 mL of new release medium at the same time, and then the amount of DMY in the sampled release medium is determined by ultraviolet spectrophotometry, and the cumulative release rate (Q) is calculated. The control sample DMY solution (with the same DMY concentration in the liposome sample) is measured under the same conditions.

[0097] Q i =(C 1 +C 2 +C 3 +……+C i )V / m

[0098] Where m is the mass of DMY added to the dialysis bag, V is the volume of the dialysis medium, and Ci is the DMY mass concentration measured at each time point.

[0099] The results are as follows Figure 6 As shown in the figure, compared with the free drug DMY, DMY-Lips and CHI-DMY-Lips showed obvious sustained release effect at pH 6.8. At 120h, the cumulative release of DMY free drug was 68.98%, the cumulative release of DMY-Lips was 58.23%, and the cumulative release of CHI-DMY-Lips was 54.50%; during the medium release process, DMY-Lips and CHI-DMY-Lips did not show a sudden release phenomenon, and the cumulative release of CHI-DMY-Lips was lower than that of DMY-Lips, because chitosan increased the thickness of the liposome membrane, thereby delaying the diffusion and release rate of the drug, reducing the exposure of the liposome, and thus reducing the leakage of the drug.

[0100] Zero-order kinetics, first-order kinetics, Higuchi equation fitting, and Weibull equation fitting were used to fit the in vitro cumulative release results of free DMY, DMY-Lips (prepared in Example 1), and CHI-DMY-Lips (prepared in Example 1). The fitting results are shown in Table 5. The results show that the first-order kinetic model has the highest fitting degree for DMY, DMY-Lips (prepared in Example 1), and CHI-DMY-Lips (prepared in Example 1) in pH 6.8 PBS solution, and the correlation coefficient R 2They are 0.91372, 0.89838 and 0.89636 respectively.

[0101] Table 5 Drug release kinetic equations and correlation coefficients of DMY, DMY-Lips and CHI-DMY-Lips

[0102]

[0103] (8) In vitro antioxidant activity

[0104] Using a DMY solution with a concentration of 0.75 mg / mL, a DMY solution with a concentration of 0.375 mg / mL (DMY-HD), and a liposome solution containing 0.375 mg / mL DMY (DMY-Lips prepared in Example 1 was obtained by diluting the DMY-Lips in half, recorded as DMY-Lips-HD) as controls, the antioxidant capacity of DMY-Lips (prepared in Example 1) and CHI-DMY-Lips (prepared in Example 1) was determined, and the specific method is as follows:

[0105] Determination of DPPH free radical scavenging activity: The scavenging ability of the free radical 2,2-diphenyl-1-trinitrophenylhydrazine (DPPH) was measured by the determination method. 2.958 mg of DPPH powder was weighed and diluted to 50 mL with anhydrous ethanol to obtain a DPPH solution with a concentration of 0.15 mmol / L. 2 mL of the test sample solution was added to 2 mL of DPPH ethanol solution, mixed evenly and reacted in the dark for 30 minutes. Then, the absorbance was measured at 517 nm using an ultraviolet spectrophotometer, and the calculation formula is as follows:

[0106] DPPH free radical scavenging rate (%) = 1-(A 2 -A 1 ) / A 0 ×100%;

[0107] Where: A 0 A is the absorbance value of 2 mL DPPH solution plus 2 mL ethanol solution; 1 A is the absorbance value of 2 mL of the test sample solution plus 2 mL of ethanol solution; 2 It is the absorbance value of 2 mL of test sample solution plus 2 mL of DPPH solution.

[0108] The lone pair of electrons of DPPH has a maximum absorption peak at 517nm, and DPPH can exist stably in pure ethanol solution. When the sample to be tested has antioxidant activity, it converts the lone pair of electrons into paired electrons by providing electrons, thereby reducing the DPPH absorbance. The antioxidant activity of the sample can be reflected by comparing the absorbance values ​​before and after the reaction. DMY is a flavonol with high antioxidant capacity. The DPPH free radical scavenging method was used to evaluate the ability of DMY to reduce the organic compound 2,2-diphenyl-1-trinitrochloride (DPPH) in the presence of hydrogenated antioxidants. The scavenging percentages of DMY, DMY-HD, DMY-Lips, DMY-Lips-HD, and CHI-DMY-Lips for DPPH were 94.59±0.0002%, 93.62±0.0006%, 95.05±0.0025%, 92.65±0.0121%, 98.29±0.0078% ( Figure 7 ). From the DPPH antioxidant results, it can be seen that the DMY-coated liposomes still retain the ability of DMY to scavenge DPPH free radicals. This phenomenon may be because DMY is encapsulated in liposomes, which reduces the possibility of DMY self-oxidation in the solution and protects its antioxidant ability. And the antioxidant capacity of CHI-DMY-Lips obtained by CHI modification is higher than that of DMY, DMY-HD, DMY-Lips, and DMY-Lips-HD (p<0.05). The antioxidant results show that CHI-DMY-Lips has the strongest antioxidant activity among all samples, which may be because the outermost CHI layer of CHI-DMY-Lips also has antioxidant activity. The synergistic effect of DMY and CHI improves the antioxidant effect of the drug delivery system.

[0109] (9) Inhibitory activity of DMY-Lips and CHI-DMY-Lips on α-glucosidase

[0110] The inhibitory activity of DMY-Lips (prepared in Example 1) and CHI-DMY-Lips (prepared in Example 1) on α-glucosidase was determined, and a DMY solution with a concentration of 0.75 mg / mL, a DMY solution with a concentration of 0.375 mg / mL (DMY-HD) and a liposome solution containing 0.375 mg / mL DMY (DMY-Lips prepared in Example 1 was obtained by diluting the DMY-Lips in half, recorded as DMY-Lips-HD) were used as controls. In the inhibition test, α-glucosidase and PNP-G were dissolved in phosphate buffer (0.1M, pH 6.8) to obtain an α-glucosidase solution with a concentration of 2U / mL and a PNP-G solution with a concentration of 10mM; acarbose, a test sample (1mL each) and an α-glucosidase solution (1mL, 2U / mL) were mixed and cultured at 37°C for 10min; then 1mL of 10mM PNP-G solution was added. The resulting mixture was kept at 37°C for 20 min, 1 mL of ethanol was added to terminate the reaction, and the absorbance of the treated sample was measured at 405 nm (A Sample ). For the blank control sample, 1 mL of PBS was used instead of the sample solution, and the operation was the same as above.

[0111] The calculation formula of α-glucosidase inhibitory activity is as follows:

[0112] α-glucosidase inhibitory activity = (A control -A sample ) / A control ×100%

[0113] Where: A control is the absorbance value of the blank control group, A sample is the absorbance value of the tested sample group.

[0114] Type 2 diabetes has developed into one of the chronic diseases that seriously endangers human health in the world. Controlling postprandial blood sugar is an effective means to prevent diabetes and its complications. Inhibiting the activity of carbohydrate digestive enzymes in the intestine, such as α-glucosidase, is a very effective way to lower postprandial blood sugar. At present, there are three drugs on the market that can be used as α-glucosidase inhibitors to control postprandial blood sugar, namely acarbose, voglibose and miglitazone, but the complex production process and large side effects of these drugs limit their widespread use. A large number of studies have shown that DMY can lower postprandial blood sugar levels and prevent diabetes. Therefore, using acarbose as a positive control, a series of test samples were tested for their inhibitory activity on α-glucosidase.

[0115] Depend on Figure 8It can be seen that the inhibition rate of acarbose on α-glucosidase activity is 98.36±0.0003%; the inhibition rates of DMY, DMY-HD, DMY-Lips, DMY-Lips-HD, and CHI-DMY-Lips on α-glucosidase activity are 97.03±0.11%, 96.17±0.02%, 53.52±4.35%, 44.54±1.20%, and 92.14±0.24%, respectively. The inhibition rate of DMY-Lips on α-glucosidase activity is lower than that of free DMY, which may be because DMY is encapsulated in liposome nanoparticles and its release rate is too slow, which may affect the effective concentration and action time of α-glucosidase inhibitors in the body, resulting in a decrease in the effective concentration that actually reaches the target. After CHI modified liposomes, the enzyme activity inhibition rate was close to that of DMY. This may be because the hypoglycemic activity of the outermost CHI layer of CHI-DMY-Lips, chitosan can directly bind to the active site of α-glucosidase, thereby blocking the binding of the enzyme to the substrate and reducing the production of glucose; and after chitosan is absorbed in the human body, it forms a protective film in the intestine, covering the surface of the small intestinal mucosa, improving the intestinal microenvironment, and delaying the absorption rate of glucose. This improvement may indirectly affect the activity of α-glucosidase and further reduce blood sugar levels. Therefore, it can be explained that the synergistic effect of DMY and CHI improves the hypoglycemic effect of the drug delivery system.

[0116] Comparative Example 2

[0117] Referring to the preparation method of the patent "A tea saponin-chitosan coated dihydromyricetin liposome and its preparation method and application", the raw materials and formula of this patent were used to prepare comparative example 2:

[0118] (1) DMY-Lip solution was prepared by the oil-in-water method as follows:

[0119] Appropriate amounts of sodium dihydrogen phosphate and disodium hydrogen phosphate were weighed and dissolved in ultrapure water respectively, and mixed evenly according to a certain ratio to obtain 20 mL of phosphate buffer solution (pH 6.5), i.e., the aqueous phase solution; 0.04 g of soybean lecithin and 0.015 g of DMY were dissolved in 10 mL of chloroform and 5 mL of anhydrous ethanol respectively, and stirred to fully dissolve them to obtain a DMY phase solution; under the condition of 300 rpm magnetic stirring, the DMY phase solution was added dropwise to the aqueous phase solution, and after the addition was completed, an emulsion was obtained; the emulsion was stirred at a speed of 100 rpm at 43 ° C for 3 h until the chloroform and anhydrous ethanol in the emulsion were removed, and after the solution maintained a constant volume, a DMY-Lips solution (the content of DMY in DMY-Lips was 0.75 mg / mL) was obtained.

[0120] The encapsulation efficiency of DMY in the DMY-Lips solution was determined to be 22.65±3.53%, the particle size was 1138.33±17.21 nm, the PDI was 0.87±0.216, and the potential was -59.9±2.49 mV.

[0121] (2) preparing chitosan mixed solution, specifically as follows:

[0122] An appropriate amount of CHI was weighed and mixed with 1% acetic acid aqueous solution to obtain a chitosan mixed solution with a concentration of 0.5 mg / mL.

[0123] (3) Preparation of CHI-DMY-Lips, specifically as follows:

[0124] At room temperature and 200 rpm magnetic stirring, 20 mL of the DMY-Lips solution in step (1) was added dropwise to 20 mL of the chitosan solution in step (2), and stirring was continued for 4 h to obtain a CHI-DMY-Lips solution.

[0125] The encapsulation efficiency of DMY in CHI-DMY-Lips solution was determined to be 44.66±0.77%, the particle size was 2255±29.21 nm, the PDI was 0.93±0.117, and the potential was 34.7±1.36 mV.

[0126] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A chitosan-modified dihydromyricetin liposome, characterized in that: The raw materials include dihydromyricetin, phospholipids and chitosan in a mass ratio of (0.25-0.5):(1-1.5):(0.25-1); The chitosan modified dihydromyricetin liposome comprises an inner layer and an outer layer; the inner layer is a dihydromyricetin liposome formed by phospholipid-coated dihydromyricetin; and the outer layer is chitosan.

2. The chitosan-modified dihydromyricetin liposome according to claim 1, characterized in that: The phospholipids include soybean lecithin.

3. A method for preparing chitosan-modified dihydromyricetin liposomes according to any one of claims 1 to 2, characterized in that: The following steps are involved: Dissolving dihydromyricetin and phospholipids in organic solvents respectively and mixing them evenly, then removing the solvent to obtain a uniform film, and then adding a buffer solution for hydration reaction to obtain a dihydromyricetin liposome solution; The dihydromyricetin liposome solution is added to the acidic solution of chitosan, and an outer layer encapsulation treatment is performed to obtain the chitosan-modified dihydromyricetin liposome.

4. The preparation method according to claim 3, characterized in that: The hydration reaction specifically includes: conducting a hydration reaction under ultrasonic conditions, the temperature is 30° C., the ultrasonic power is 200-300W, the frequency is 40KHz, and the ultrasonic method is to ultrasonicate for 30s and stand for 30s as one cycle, and a total of 4 cycles.

5. The preparation method according to claim 3, characterized in that: The concentration of dihydromyricetin in the dihydromyricetin liposome solution is 0.5-1 mg / mL, and the concentration of phospholipid is 2-3 mg / mL.

6. The preparation method according to claim 3, characterized in that: The concentration of chitosan in the acidic chitosan solution is 0.5-2 mg / mL; the volume ratio of the acidic chitosan solution to the dihydromyricetin liposome solution is 1:

1.

7. The preparation method according to claim 3, characterized in that: The encapsulation treatment time is 1 to 4 hours.

8. Use of the chitosan-modified dihydromyricetin liposome according to any one of claims 1 to 2 in the preparation of antioxidant, anticancer, liver-protecting, antibacterial or hypoglycemic drugs.

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