阅读说明：本技术 一种低水溶性多酚类药物载体及其制备方法和应用 (Low-water-solubility polyphenol drug carrier and preparation method and application thereof ) 是由 王仲妮 刘玮 于 2021-08-03 设计创作，主要内容包括：本发明涉及药剂制备技术领域,为了解决现有技术存在的药物载体缓释时间短,无法实现前期快速释放,后期缓慢释放的问题,本发明提出一种低水溶性多酚类药物载体及其制备方法和应用。利用甘油酸单油酯(GMO)、山梨醇酐油酸酯(Span80)、油酸乙酯、水制备了能够缓释低水溶性多酚类药物的药物载体,该药物载体是一种基于表面活性剂的聚集体,可以使低水溶性多酚类药物持续释放。就整体释放周期而言,药物释放时间至少可达38h,具有缓释效应,可以减少药物二氢杨梅素的服用次数。且载体对低水溶性多酚类药物的累积释放率可达92％以上。(The invention relates to the technical field of medicament preparation, and provides a low water-solubility polyphenol medicament carrier and a preparation method and application thereof in order to solve the problems that the medicament carrier in the prior art is short in slow release time, cannot realize early-stage quick release and later-stage slow release. The drug carrier capable of slowly releasing the polyphenol drugs with low water solubility is prepared by using monooleyl Glycerate (GMO), sorbitan oleate (Span80), ethyl oleate and water, and is an aggregate based on a surfactant, so that the polyphenol drugs with low water solubility can be continuously released. In terms of the whole release period, the release time of the medicine can reach at least 38h, the sustained release effect is realized, and the taking frequency of the medicine dihydromyricetin can be reduced. And the accumulative release rate of the carrier to the polyphenol drugs with low water solubility can reach more than 92 percent.)

1. The carrier is characterized in that the carrier takes a monoglyceride aggregate as a matrix, the aggregate comprises a surfactant, ethyl oleate and water, and the surfactant is monoglyceride and sorbitan oleate.

2. The low water-solubility polyphenol drug carrier of claim 1, wherein the dihydromyricetin carrier comprises 2.8-68 parts by mass of surfactant, 0-25.2 parts by mass of ethyl oleate, 32-78 parts by mass of water and nonzero part by mass of ethyl oleate.

3. The low water solubility polyphenol based drug carrier in claim 1, wherein the water is double distilled water;

preferably, the low water-soluble polyphenol drug carrier also comprises a low water-soluble polyphenol drug.

4. The method for preparing a low water-solubility polyphenol pharmaceutical carrier of any one of claims 1 to 3, which comprises: mixing monoglyceride, sorbitan oleate, ethyl oleate and water.

5. The method for preparing a polyphenol based drug carrier having low water solubility according to claim 4 wherein monoglyceride, sorbitan oleate and ethyl oleate are mixed at temperature I, water is added and mixing is carried out at temperature II;

preferably, the mass ratio of the monoglyceride to the sorbitan oleate is 1:0.5-1.5, preferably 1: 1;

preferably, the temperature I is 60-70 ℃;

preferably, the temperature II is 25 ℃.

6. The method for preparing the low water solubility polyphenol based drug carrier as claimed in claim 4, wherein the mixing is all performed under the condition of water bath;

preferably, water is added dropwise;

preferably, the moisture is added multiple times;

preferably, the water adding amount is 1-3 parts by mass each time;

preferably, the preparation method further comprises the step of adding the low water-solubility polyphenol drug, mixing the low water-solubility polyphenol drug with the sorbitan oleate, and then adding the monooleate glycerate, the ethyl oleate and the water for mixing.

7. The use of the low water-soluble polyphenol pharmaceutical carrier of any one of claims 1 to 3 in the preparation of anti-oxidant drugs, antibacterial drugs, anti-cancer drugs, liver-protecting drugs, and blood lipid-regulating drugs.

8. A carrier drug comprising the low water-soluble polyphenol drug carrier of any one of claims 1 to 3.

9. A carrier drug comprising the low water-soluble polyphenol drug carrier of any one of claims 1 to 3 and a low water-soluble polyphenol drug;

preferably, the low water-soluble polyphenol drug is dihydromyricetin;

preferably, the mass of the low water-solubility polyphenol medicine is 0.26mg/g based on the total mass of the carrier medicine.

10. An antioxidant drug comprising the low water-soluble polyphenol drug carrier of any one of claims 1 to 3.

Technical Field

The invention relates to the technical field of medicament preparation, in particular to a low water-solubility polyphenol medicament carrier and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Dihydromyricetin (DMY) is extracted from a woody vine of Ampelopsis of Vitaceae, and is also extracted from Hovenia dulcis Thunb, wherein the main active ingredient is flavonoid, and the material has effects of scavenging free radicals, resisting oxidation, resisting thrombi, resisting tumor, and relieving inflammation; the dihydromyricetin is a special flavonoid compound, and has the general characteristics of the flavonoid compound, and also has the effects of relieving alcoholism, preventing alcoholic liver and fatty liver, inhibiting hepatocyte deterioration, reducing incidence of liver cancer and the like. Is a good product for protecting liver, dispelling the effects of alcohol and sobering up.

Dihydromyricetin is a polyphenol drug with low water solubility, has various pharmacological effects and biological activities, such as the like, but has poor solubility in an aqueous solution, so that the dihydromyricetin is difficult to be absorbed and utilized by the small intestine when being orally taken, and the application of the dihydromyricetin in practice is severely limited. In order to overcome the defects of low water-solubility polyphenol drugs such as dihydromyricetin, a plurality of preparations and carriers are researched in recent years to improve the defects of the drugs, such as liposome, microemulsion, lyotropic liquid crystal, hydrogel and the like.

The surfactant is an amphiphilic compound having a hydrophilic head and a hydrophobic tail, and the hydrophobic tails of molecules associate with each other to form micelles, and when the concentration of the surfactant exceeds a certain value, i.e., exceeds a critical micelle concentration, surfactant-based aggregates are spontaneously formed in an aqueous solution. The aggregate has a lipophilic region for encapsulating the drug with low water solubility, and can realize the solubilization of the hydrophobic drug. And the aggregate structure has viscoelasticity, can protect the medicament and can realize the slow release of the medicament.

However, the inventor researches and discovers that the carrier drug containing the surfactant has short sustained release time and low accumulative release rate. In addition, the drug release rate of the existing low water-solubility polyphenol drug carrier is only 20-30% in the first 500min, but in actual use, some drugs need to have higher release rate in the early stage and need to be continuously and slowly released as consolidation reinforcement in the later stage, and the existing drug carriers cannot meet the use requirement.

Disclosure of Invention

The invention provides a low water-solubility polyphenol drug carrier and a preparation method and application thereof, aiming at solving the problems that the drug carrier in the prior art has short slow release time, can not realize quick release at the early stage and slow release at the later stage. The drug carrier capable of slowly releasing the polyphenol drugs with low water solubility is prepared by using monooleyl Glycerate (GMO), sorbitan oleate (Span80), ethyl oleate and water, and is an aggregate based on a surfactant, so that the polyphenol drugs with low water solubility can be continuously released. In addition, the release rate of the carrier within 500min reaches 50-70%, which is beneficial to quickly playing a role and slowly releasing in a later period, and is used for strengthening and enhancing the effect. In terms of the whole release period, the release time of the medicine can reach at least 38h, the sustained release effect is realized, and the taking frequency of the medicine dihydromyricetin can be reduced. And the accumulative release rate of the carrier to the polyphenol drugs with low water solubility can reach more than 92 percent.

Specifically, the invention is realized by the following technical scheme:

in the first aspect of the invention, the low water solubility polyphenol drug carrier is provided, wherein the carrier takes a monoglyceride aggregate as a matrix, the aggregate comprises a surfactant, ethyl oleate and water, and the surfactant is monoglyceride and sorbitan oleate.

In a second aspect of the present invention, a method for preparing a low water-solubility polyphenol pharmaceutical carrier is provided, which comprises: mixing monoglyceride, sorbitan oleate, ethyl oleate and water.

In a third aspect of the invention, an application of a low water-solubility polyphenol drug carrier in preparing an antioxidant drug, an antibacterial drug, an anticancer drug, a liver protection drug and a blood fat regulation drug is provided.

In a fourth aspect of the invention, a carrier drug is provided, which comprises a low water-solubility polyphenol drug carrier.

In a fifth aspect of the invention, a carrier drug is provided, which comprises a low water-soluble polyphenol drug carrier and a low water-soluble polyphenol drug.

In a sixth aspect of the invention, an antioxidant drug is provided, which comprises a low water-solubility polyphenol drug carrier.

One or more of the technical schemes have the following beneficial effects:

1) the drug carrier capable of slowly releasing the low water-soluble polyphenol drugs is prepared from GMO, Span80, ethyl oleate and water, and is an aggregate based on a surfactant, so that the low water-soluble polyphenol drugs, particularly dihydromyricetin, can be continuously released.

2) The prepared drug carrier has the release effect that the release time of the dihydromyricetin can reach at least 38h, and has the slow release effect. Can reduce the frequency of taking dihydromyricetin. And the accumulative release rate of the carrier to the dihydromyricetin can reach more than 92 percent.

3) The release rate of the drug carrier within 500min reaches 50-70%, which is beneficial to the rapid effect and the slow release in the later period, and is used for strengthening and enhancing the effect. In addition, the prepared carrier can change the in vitro release rate of the medicine by adjusting the temperature. Experimental data show that the release of the dihydromyricetin by the carrier can reach 53h at room temperature and 38h at simulated human body temperature.

4) The drug-loaded carrier prepared by the technical scheme of the invention has good antioxidant activity.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows GMO-Span80 (C.sub.251:1)/EtOL/H₂Quasi-ternary phase diagram of O system.

Fig. 2(a) storage modulus G' (solid) and loss modulus G "(open) of drug loaded samples a1, a2, A3 at 25 ℃ as a function of shear stress. (b) Frequency sweep curves for drug-loaded samples a1, a2, A3 at 25 ℃ (G "is open and G' is solid). (c) Shear viscosity of drug loaded samples a1, a2, A3 at 25 ℃ as a function of shear rate.

FIG. 3 is a standard curve of dihydromyricetin with the inset showing the absorption spectrum of dihydromyricetin.

FIG. 4 is an in vitro release profile of dihydromyricetin at 37 ℃ in samples (a) A1, A2, A3, (B) B1, B2, B3, (C) C1, C2, C3.

FIG. 5 is an in vitro release profile of dihydromyricetin at 25 ℃ in samples (a) A1, A2, A3, (B) B1, B2, B3, (C) C1, C2, C3, (d) A1, A5, C5, E1.

Fig. 6 is a graph of the scavenging activity of drug-loaded samples a1, a2, A3 on DPPH free radicals, and the inset is a graph of the scavenging activity of dihydromyricetin ethanol solution on DPPH free radicals.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The invention provides a low water-solubility polyphenol drug carrier and a preparation method and application thereof, aiming at solving the problems that the drug carrier in the prior art has short slow release time, can not realize quick release at the early stage and slow release at the later stage. The drug carrier capable of slowly releasing the polyphenol drugs with low water solubility is prepared by using monooleyl Glycerate (GMO), sorbitan oleate (Span80), ethyl oleate and water, and is an aggregate based on a surfactant, so that the polyphenol drugs with low water solubility can be continuously released. In addition, the release rate of the carrier within 500min reaches 50-60%, which is beneficial to quickly playing a role and slowly releasing in a later period, and is used for strengthening and enhancing the effect. In terms of the whole release period, the release time of the medicine can reach at least 38h, the sustained release effect is realized, and the taking frequency of the medicine dihydromyricetin can be reduced. And the accumulative release rate of the carrier to the polyphenol drugs with low water solubility can reach more than 92 percent.

Specifically, the invention is realized by the following technical scheme:

in a first aspect of the present invention, there is provided a low water solubility polyphenol type drug carrier, wherein the carrier uses a monoglyceride aggregate as a matrix, the aggregate comprises a surfactant, ethyl oleate (EtOL) and water, and the surfactant is monoglyceride and sorbitan oleate.

Some embodiments of the invention prepare GMO-Span80/EtOL/H₂And O system. When the aggregate based on the surfactant is used as a carrier, a green low-toxicity raw material harmless to organisms needs to be selected, and the monoglyceride is a nonionic surfactant and has good emulsifying property. Because of its good biocompatibility, it is widely used in the industries of medicine, cosmetics and textile industry. Moreover, the monoglyceride has the characteristic of biodegradability and is often used as an auxiliary material of a drug delivery system.

Span80 (Span80) is chemically called as sorbitan oleate, is a yellow viscous liquid, belongs to a nonionic surfactant, is a good emulsifier, and is widely applied to the production of food, cosmetics, medicines, textiles and the like. Ethyl oleate is a substance with good food safety, is widely used in food additives, and is often used as a raw material for preparing drug carriers.

In one or more embodiments of the invention, the dihydromyricetin carrier comprises, by mass, 2.8-68 parts of a surfactant, 0-25.2 parts of ethyl oleate, 32-78 parts of water, and the mass of the ethyl oleate is not zero.

In order to further improve the slow release effect of the carrier and realize the effects of quick release at the early stage and slow release at the later stage, the mass ratio of the surfactant to the ethyl oleate is 4-6:4-6, preferably 6:4, 5:5 and 4: 6.

The ratio of monoolein glycerate to sorbitan oleate affects the stability and sustained release of the carrier, and in some embodiments, the mass ratio of monoolein glycerate to sorbitan oleate is 1:0.5 to 1.5, preferably 1: 1.

In one or more embodiments of the invention, the water is double distilled water;

preferably, the low water-soluble polyphenol drug carrier also comprises a low water-soluble polyphenol drug.

In a second aspect of the present invention, a method for preparing a low water-solubility polyphenol pharmaceutical carrier is provided, which comprises: mixing monoglyceride, sorbitan oleate, ethyl oleate and water.

Because the reduction includes multiple esters, in order to improve the mixing effect and avoid the adverse effect of agglomeration or uneven mixing on the carrier, in some embodiments, the low water-solubility polyphenol drug is prepared by mixing monoolein glycerate, sorbitan oleate and ethyl oleate at a temperature I, adding water, and mixing at a temperature II;

preferably, the temperature I is 60-70 ℃;

preferably, the temperature II is 25 ℃.

The mixing is carried out under the condition of water bath for heat preservation;

preferably, water is added dropwise;

preferably, the moisture is added multiple times;

preferably, the water adding amount is 1-3 parts by weight each time, and the water adding interval is 5 min.

In the carrier system, if all water is added at one time, the mixing of the monoglyceride, the monoglyceride and the ethyl oleate is not easy to be facilitated. And because the two temperatures before and after the water is added, enough water is added at one time, the carrier system is influenced from the aspects of mixing, uniform temperature change and reaction uniformity, and the slow-release effect or stability of the carrier is further reduced.

Preferably, the preparation method further comprises the step of adding the low water-solubility polyphenol drug, mixing the low water-solubility polyphenol drug with the sorbitan oleate, and then adding the monooleate glycerate, the ethyl oleate and the water for mixing.

In a third aspect of the invention, an application of a low water-solubility polyphenol drug carrier in preparing an antioxidant drug, an antibacterial drug, an anticancer drug, a liver protection drug and a blood fat regulation drug is provided.

In a fourth aspect of the invention, a carrier drug is provided, which comprises a low water-solubility polyphenol drug carrier.

In a fifth aspect of the invention, a carrier drug is provided, which comprises a low water-soluble polyphenol drug carrier and a low water-soluble polyphenol drug.

Preferably, the low water-soluble polyphenol drug is dihydromyricetin;

preferably, the mass of the low water-solubility polyphenol medicine is 0.26mg/g based on the total mass of the carrier medicine.

In a sixth aspect of the invention, an antioxidant drug is provided, which comprises a low water-solubility polyphenol drug carrier.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Instruments and reagents

TABLE 1 reagents used in the examples section of the present invention

TABLE 2 instruments used in the section of the embodiments of the invention

Drawing of phase diagrams

Taking a mixed phase of monoglyceride glycerate and Span80 with the mass ratio of 1:1 as a surfactant phase and ethyl oleate as an oil phase, weighing the surfactant and the oil phase according to the mass ratio of 10:0 to 0:10, placing the weighed mixed phase into a colorimetric tube, placing the colorimetric tube into a water bath at 65 ℃, fully stirring and mixing, and then dropwise adding double distilled water by a gradient of water content increased by 2 wt% and fully mixing. And (3) placing the uniformly mixed mixture in a constant-temperature water bath at 25 ℃ for balancing, and observing and recording the experimental phenomenon after the balancing is finished. The equilibration time is suitably extended as the phase boundary is approached. The phase boundaries are preliminarily judged by observing the color, transparency, viscosity, etc. of the sample.

The present invention will be further described with reference to the following examples.

Example 1

The ratio of the surfactant to the oil was 6:4, the mass ratio of the monoglyceride and sorbitan oleate was 1:1, the water content was 80%, GMO and Span80 (as surfactants) were weighed into a colorimetric cylinder, ethyl oleate was added thereto, and the mixture was stirred and mixed in a water bath at 65 ℃. Finally, double distilled water was added dropwise to the colorimetric cylinder, the water content was increased at intervals of 2%, the mixture was stirred uniformly using a magnetic stirrer, and then placed in a water bath at 25 ℃ for equilibration, the phase state of the aggregates and the change in appearance were observed and recorded, and the equilibration time was appropriately prolonged when the phase boundary was approached. The phase boundaries are preliminarily judged by observing the color, transparency, viscosity, etc. of the aggregates. Denoted as a 1.

Example 2

The ratio of the surfactant to the oil was 6:4, the mass ratio of the monoglyceride and sorbitan oleate was 1:1, the water content was 68%, GMO and Span80 (as surfactants) were weighed into a colorimetric cylinder, ethyl oleate was added thereto, and the mixture was stirred and mixed in a water bath at 65 ℃. Finally, double distilled water was added dropwise to the colorimetric cylinder, the water content was increased at intervals of 2%, the mixture was stirred uniformly using a magnetic stirrer, and then placed in a water bath at 25 ℃ for equilibration, the phase state of the aggregates and the change in appearance were observed and recorded, and the equilibration time was appropriately prolonged when the phase boundary was approached. The phase boundaries are preliminarily judged by observing the color, transparency, viscosity, etc. of the aggregates. Denoted B1.

Example 3

The ratio of the surfactant to the oil was 6:4, the mass ratio of the monoglyceride and sorbitan oleate was 1:1, the water content was 56%, GMO and Span80 (as surfactants) were weighed into a colorimetric cylinder, ethyl oleate was added thereto, and the mixture was stirred and mixed in a water bath at 65 ℃. Finally, double distilled water was added dropwise to the colorimetric cylinder, the water content was increased at intervals of 2%, the mixture was stirred uniformly using a magnetic stirrer, and then placed in a water bath at 25 ℃ for equilibration, the phase state of the aggregates and the change in appearance were observed and recorded, and the equilibration time was appropriately prolonged when the phase boundary was approached. The phase boundaries are preliminarily judged by observing the color, transparency, viscosity, etc. of the aggregates. Denoted as C1.

Example 4

The weight ratio of the surfactant to the oil is 5:5, the mass ratio of the monoglyceride and the sorbitan oleate is 1:1, the water content is 80%, GMO and Span80 (serving as the surfactant) are weighed and placed in a colorimetric cylinder, ethyl oleate is added into the colorimetric cylinder, and the mixture is stirred and mixed uniformly in a water bath at 65 ℃. Finally, double distilled water was added dropwise to the colorimetric cylinder, the water content was increased at intervals of 2%, the mixture was stirred uniformly using a magnetic stirrer, and then placed in a water bath at 25 ℃ for equilibration, the phase state of the aggregates and the change in appearance were observed and recorded, and the equilibration time was appropriately prolonged when the phase boundary was approached. The phase boundaries are preliminarily judged by observing the color, transparency, viscosity, etc. of the aggregates. Denoted as a 2.

Example 5

The ratio of the surfactant to the oil was 4:6, the mass ratio of the monoglyceride and sorbitan oleate was 1:1, the water content was 80%, GMO and Span80 (as surfactants) were weighed into a colorimetric cylinder, ethyl oleate was added thereto, and the mixture was stirred and mixed in a water bath at 65 ℃. Finally, double distilled water was added dropwise to the colorimetric cylinder, the water content was increased at intervals of 2%, the mixture was stirred uniformly using a magnetic stirrer, and then placed in a water bath at 25 ℃ for equilibration, the phase state of the aggregates and the change in appearance were observed and recorded, and the equilibration time was appropriately prolonged when the phase boundary was approached. The phase boundaries are preliminarily judged by observing the color, transparency, viscosity, etc. of the aggregates. Denoted as a 3.

Example 6

The ratio of the surfactant to the oil was 6:4, the mass ratio of the monoglyceride and sorbitan oleate was 1:1, the water content was 45%, GMO and Span80 (as surfactants) were weighed into a colorimetric cylinder, ethyl oleate was added thereto, and the mixture was stirred and mixed in a water bath at 65 ℃. Finally, double distilled water was added dropwise to the colorimetric cylinder, the water content was increased at intervals of 2%, the mixture was stirred uniformly using a magnetic stirrer, and then placed in a water bath at 25 ℃ for equilibration, the phase state of the aggregates and the change in appearance were observed and recorded, and the equilibration time was appropriately prolonged when the phase boundary was approached. The phase boundaries are preliminarily judged by observing the color, transparency, viscosity, etc. of the aggregates. Denoted as C5.

Preparation of drug-loaded samples

Accurately weighing 0.3g dihydromyricetin, adding into a color comparison tube with a plug containing 3.0g Span80, and vortex mixing for 20 minutes. Sealing, and stirring in 60 deg.C water bath in dark for 12 hr to dissolve dihydromyricetin. Mixing Span80 containing dihydromyricetin with GMO, adding ethyl oleate at a certain proportion, mixing at 65 deg.C, adding required amount of double distilled water in batches, magnetically stirring, mixing, and balancing in water bath at 25 deg.C.

Rheology test

The instrument used for measuring the rheological properties of the sample is a Discovery HR-2 rheometer, the measuring jig is a measuring plate with a diameter of 2cm and a cone angle of 2 deg.. The temperature of the bath set for the measurement was 25 ℃. During measurement, the head of the rheometer is lifted to a specified position, and a sample to be measured is placed in the center of the sensor. And adjusting the instrument, lowering the machine head to a specified position, controlling the thickness of the central sample of the sensor to be 0.053mm, setting instrument parameters, balancing the sample for 5min, and starting to measure.

Fixing frequency, selecting the range of stress value to be 0.1-10000Pa, carrying out stress scanning on all sample points, and determining a linear viscoelastic region. Selecting proper stress value in the linear viscoelastic region, re-loading the sample, and performing frequency scanning on the sample, wherein the scanning frequency range is 0.03-500 rad/s. Then, the sample is scanned in a steady state, and the shear rate is set to be 0.1-100s^-1. The temperature deviation should be less than 0.1 ℃ when measured.

In vitro drug release study

The in vitro release experiment of DMY in the aggregate is carried out by dialysis at room temperature of 25 ℃ and physiological temperature of 37 ℃. The small intestine environment was simulated with phosphate buffered saline (pH 6.8). 1.0g of the drug-loaded sample was placed in a dialysis bag (1000Da) which was immersed in a beaker containing 60.0ml of PBS buffer and stirred at a constant speed at 100rpm using a magneton. At regular intervals, 3.0ml of release medium was removed and the same volume of fresh release medium was added to the beaker. The absorbance of dihydromyricetin is measured by an ultraviolet spectrophotometer (X-3, Shanghai Yuan analysis instruments Co., Ltd.) at the wavelength of 293nm, and the release amount of the drug is calculated to calculate the cumulative release rate of the drug.

The cumulative release rate of the drug is multiplied by 100 percent based on the total amount of the drug in the carrier

Research on Oxidation resistance

Diluting the sample with ethanol to 8 different concentrations by DPPH free radical scavenging activity method, and preparing the concentration of DPPH ethanol solution to 6 × 10^-5mol/L. Adding 2.0ml sample solution with different dilution concentrations into 1.0ml DPPH ethanol solution, mixing uniformly by a vortex mixer, and reacting for 30min in a dark place. Measuring the absorbance at a wavelength of 450-650nm by using an ultraviolet spectrophotometer, determining the measurement wavelength at which the absorbance of the sample is measured, and recording the absorbance as A₁The absorbance of the same volume of DPPH in ethanol was designated as A₀。

RSA％＝(1-A₁/A₀)×100％

Results and discussion

(1) Act of phase

GMO-Span80/EtOL/H at 25 ℃ was studied₂Quasi-ternary phase diagram of O system. As shown in figure 1, a total of six regions appear in the phase diagram of the system, including a golden yellow viscous flow region (I), a light creamy yellow semi-fluid viscous region (II), and a light creamy yellow non-fluid regionThe region (III) with higher kinematic viscosity and the region (IV) with higher light cream yellow non-flow viscosity and elasticity show a region (V) with lower light cream yellow non-flow elasticity and viscosity with increasing water content, and a region (VI) with light cream yellow flow also shows with increasing water content in samples with larger surfactant ratio.

(2) Rheology of drug loaded samples

The stress scanning is carried out on the drug-loaded sample at a fixed frequency, and a relation graph of the elastic modulus G ', the viscous modulus G' and the shear stress can be obtained. It can be seen from fig. 2(a) that the elastic modulus and the viscous modulus of the aggregate remain substantially unchanged by varying the magnitude of the external shear stress within a certain range, and this region becomes a linear viscoelastic region in which the elastic modulus of the sample is higher than the viscous modulus, indicating that the elastic properties are dominant. The elastic modulus of the sample decreased rapidly with increasing stress value when the stress increased to a certain value, and the stress value at which the elastic modulus began to decrease was called critical stress (σ)_c) Generally, at the critical stress value, the greater the elastic modulus, the greater the ability of the sample to resist external forces.

To further explore the effect of shaking on the internal structure of the samples, the samples were frequency scanned at 25 ℃ with a fixed shear stress. The variation curve of elastic modulus versus viscous modulus with frequency is shown in fig. 2(b), and the elastic modulus is greater than the viscous modulus for all three samples over the entire vibration frequency range, indicating that the elastic properties are dominant. Wherein the elastic modulus of 12/8/80 sample (representing 12/8/80% of surfactant/ethyl oleate/water) is substantially unchanged at vibration frequency ranging from 0.03 to 100rad/s, and the elastic modulus of the sample shows a tendency to increase when the frequency is more than 100 rad/s. 10/10/80 the modulus of elasticity of the sample is substantially constant at vibration frequencies in the range of 0.03 to 40rad/s, and the modulus of elasticity of the sample tends to decrease at frequencies greater than 44 rad/s. 8/12/80 the modulus of elasticity of the sample is substantially constant at vibration frequencies in the range of 0.03 to 100rad/s, and the modulus of elasticity of the sample tends to decrease at frequencies greater than 100 rad/s. The viscous moduli of the three samples were substantially constant at frequencies ranging from 0.03 to 40rad/s, and increased at frequencies greater than 44 rad/s. And converting the dynamic viscoelasticity result into a relaxation map to obtain the change trend of the relaxation modulus along with time. From the relaxation spectra, it can be seen that the relaxation modulus of the 12/8/80 sample is higher than that of the 10/10/80 sample at the early relaxation time, and that the relaxation modulus of the 10/10/80 sample is higher than that of the 12/8/80 sample at the late relaxation time.

The steady-state rheological property can indicate the capacity of the aggregate to resist external shearing force, and the higher the shear viscosity, the stronger the capacity of the sample to resist the external shearing force. The shear viscosity versus shear rate curve for all sample points is given in FIG. 2(c), with the shear viscosity of all three samples decreasing with increasing shear rate, showing the non-Newtonian fluid property of shear thinning. And the viscosity of the sample is higher as the proportion of the surfactant is higher, which is probably because the content of dihydromyricetin in the sample is higher as the content of the surfactant is higher, so that the viscosity of the sample is increased.

(3) In vitro Release assay

FIG. 3 is a standard curve of dihydromyricetin, with the inset showing the absorption spectrum of dihydromyricetin. As can be seen from the ultraviolet absorption spectrum of the dihydromyricetin, the maximum absorption wavelength of the dihydromyricetin is 293nm, the absorbance of the dihydromyricetin with different concentrations at the maximum absorption wavelength is measured, and a standard curve of the dihydromyricetin obtained by fitting is as follows: abs-0.01505 +35.99C (mg/mL) R²＝0.9961

Wherein Abs is the absorbance of dihydromyricetin at 293nm, and C is the concentration of dihydromyricetin

Table 3 shows the names and compositions of the respective samples

FIG. 5(a) is a graph of the release profile for samples containing 80% water at 25 ℃ with varying ratios of surfactant to oil in zone V. As can be seen from the release time of each sample in the figure, the four samples can realize the slow release of the dihydromyricetin (the release time can last for 58 h). In the early stage of release (before 8h), the release rate of dihydromyricetin is high, the cumulative release rate reaches 60%, and the reason for the phenomenon is probably that part of drug molecules adhered to the surface of a sample are dissolved out, so that the release rate is high; with the increase of the release time, the dihydromyricetin dissolved in the surfactant and the oil phase escapes through the surface membrane substance, the dissolution becomes relatively difficult, the release rate is slow, the rising rate of the cumulative release rate is slow, and finally the release platform is reached. From the release profile, it can be found that the more the surfactant content in the sample, the smaller the cumulative release rate of dihydromyricetin, which can be explained by the stability of the sample, and the more the surfactant content, the stronger the stability of the sample, the stronger the interaction between dihydromyricetin and the carrier, so the smaller the cumulative release rate.

FIG. 4(a) is a graph of the release profile for samples containing 80% water at 37 ℃ with varying ratios of surfactant to oil in zone V. As can be seen from the release curve, the four samples can still realize the slow release of the dihydromyricetin at the simulated physiological temperature (the release time can last for 38 h). The faster release rate at 37 ℃ compared to the release profile at 25 ℃ is probably due to the increased thermal movement of the molecules and the increased diffusion rate of the drug at higher temperatures, resulting in an increased release rate and an increased cumulative release rate. According to the application, different release kinetic models are adopted to fit a release curve, the fitting result is shown in table 2, the fitting result shows that the release curve of 8/12/80 samples conforms to a Korsmeyer-Peppas equation under the condition of 25 ℃, the release index of the samples in the early stage is larger than 0.89, the release type in the period belongs to super Case II delivery, and the release in the period is mainly controlled by the erosion effect of a polymer chain; and in the middle and later release period, the release index of the sample is less than 0.5, which indicates that the release of the dihydromyricetin at the period belongs to Fickian diffusion, and indicates that the release of the sample is controlled by erosion swelling action of the polymer. 6/14/80 the early and middle release phases of the sample are in accordance with zero order kinetics equation, which shows that the release process in this phase is almost not controlled by concentration diffusion and always releases at a constant rate; the middle and later release periods accord with a first-order kinetic equation, and the release is controlled by concentration diffusion. 10/10/80 the sample accords with zero order kinetic equation in the early stage of release, first order kinetic equation in the middle stage of release and Korsmeyer-Peppas equation in the later stage of release, showing that the release of the sample is controlled by the combination of concentration diffusion and polymer swelling erosion. 12/8/80 the early phase of release of the sample followed zero order kinetics equations, indicating that the early phase release was almost not controlled by concentration diffusion; the middle and later release stages conform to a first order kinetic equation, and the release of the sample at the stage belongs to concentration diffusion controlled release. The fitting results at 37 ℃ compare with those at 25 ℃, the release medium periods of 12/8/80, 10/10/80 and 8/12/80 samples at the former temperature all conform to the Korsmeyer-Peppas equation, wherein the release index of 12/8/80 sample is more than 0.45 and less than 0.89, which indicates that the release at this stage belongs to non-Fickian diffusion, namely the release at this stage is controlled by the diffusion and erosion together; whereas the release indices for both 10/10/80 and 8/12/80 samples were less than 0.45, indicating that it is a Fickian diffusion. At the latter temperature, only the mid-release phase of the 8/12/80 sample was in accordance with the Korsmeyer-Peppas equation and the release type was hypercase II transport, indicating that temperature increases change the release from erosion control of the polymer chains to diffusion and erosion co-control, which again explains the increase in cumulative release rate and release rate caused by temperature increases. FIG. 5(b) is a graph of the release profile for 68% water samples at 25 ℃ with different surfactant to oil ratios in zone IV. It can be seen from the release curve that the less the surfactant content in the sample, the greater the cumulative release rate of dihydromyricetin. The early stage of release of the 19/13/68 sample was found to follow the zero order kinetics equation by release kinetics analysis, indicating that the release at this stage is less controlled by concentration diffusion; the release medium period of the sample conforms to a Korsmeyer-Peppas equation, and the release index is less than 0.45, which indicates that the release medium period belongs to Fickian diffusion; the later release period conforms to a first order kinetic equation, which shows that the later release period is controlled by concentration diffusion. The release profiles of samples 16/16/68 and 13/19/68 fit the first order kinetics equation, indicating that their release is under concentration diffusion control. The early stage and the middle stage of the release of the 9.6/22.4/68 sample accord with Korsmeyer-Peppas equation, the early stage release index is more than 0.89, the release type in the period belongs to super Case II transport, and the release in the period is mainly controlled by the erosion action of a polymer chain; the release index in the middle release period is less than 0.45, which indicates that the release in the middle release period belongs to Fickian diffusion; and the later release period conforms to the first order kinetic equation, which shows that the release at the period is controlled by concentration diffusion.

FIG. 4(b) is a graph of the release profile for 68% water samples at 37 ℃ with different surfactant to oil ratios in zone IV. From the release profile, it can be found that the sample containing a larger amount of the surfactant has a smaller cumulative release rate, similarly to the 25 ℃ condition, which is probably because the larger amount of the dihydromyricetin has a stronger interaction with the carrier, resulting in a decreased cumulative release rate. The release kinetics analysis shows that the release of the four samples conforms to a first order kinetics equation, and the fitting indexes are all larger than 0.92, which indicates that the release of the phase region samples is controlled by concentration diffusion at the condition of 37 ℃.

In addition, fig. 4 shows that the release rate of the carrier reaches 50-70% within 500min, which is beneficial to quickly playing the effect and slowly releasing in the later period for strengthening and enhancing the effect.

FIG. 5(c) is a graph showing the release profiles of samples containing 56% water at 25 ℃ and different ratios of surfactant to oil in zone III, and it can be seen that the sustained release of dihydromyricetin can be achieved in all four samples. In the early stage of release (before 10 h), the release rate of dihydromyricetin is higher, the cumulative release rate reaches 70%, and the reason for the phenomenon is probably that part of drug molecules adhered to the surface of a sample are dissolved out, so that the release rate is higher; with the increase of the release time, the dihydromyricetin dissolved in the surfactant and the oil phase escapes through the surface membrane substance, the dissolution becomes relatively difficult, the release rate is slow, the rising rate of the cumulative release rate is slow, and finally the release platform is reached. The release profile of the 26.4/17.6/56 sample was found by release kinetics analysis to conform to the first order kinetics equation, indicating that it is primarily controlled by concentration diffusion. 22/22/56 the sample was in accordance with Korsmeyer-Peppas equation at the early stage of release, with a release index greater than 0.89, indicating that the release pattern at this stage is of the super Case II transport, indicating that the release at this stage is primarily controlled by polymer chain erosion; the middle release period conforms to a first-order kinetic equation and is controlled by concentration diffusion; the late release period conforms to the Korsmeyer-Peppas equation, and the release index is less than 0.45, which indicates that the release at the period belongs to Fickian diffusion. The early stage and the later stage of the release curve of the 17.6/26.4/56 sample accord with a first-order kinetic equation, which indicates that the release curve is controlled by concentration diffusion; while the middle period of the release curve conforms to the Korsmeyer-Peppas equation, and the release index is less than 0.45, which indicates the release type Fickian diffusion at the middle period. The release curve of the 13.2/30.8/56 sample conforms to a first order kinetic equation at the early stage, and the release at the stage is mainly controlled by concentration diffusion; the middle release period and the later release period both accord with Korsmeyer-Peppas equation, the release indexes are both less than 0.45, and the release type at the period is Fickian release.

FIG. 4(c) is a graph showing the release profiles of 56% water samples with different surfactant to oil ratios in zone III at 37 deg.C, from which it can be seen that the release of dihydromyricetin can be achieved in all four samples. The release curves of the three samples 26.4/17.6/56, 17.6/26.4/56 and 13.2/30.8/56 all conform to the first order kinetic equation, and the fitting indexes of the release curves are all larger than 0.96, which indicates that the release of the release curves is mainly controlled by concentration diffusion; the early stage of the release curve of the 22/22/56 sample conforms to a zero-order kinetic equation, which shows that the early stage is not controlled by concentration diffusion and releases at a constant rate all the time; the middle and later release phases conform to a first order kinetic equation, indicating that the middle and later release phases are controlled by concentration diffusion.

The components and compositions in the carrier have obvious influence on the release behavior of the drug. The present study explored the effect of samples with different water contents on the release behavior of dihydromyricetin, and fig. 5(d) shows the release curves of samples with different water contents at 25 ℃ on dihydromyricetin. It can be seen from the graph that the more water content in the remaining samples except 54/36/10 shows burst release, the faster the release rate and the greater the cumulative release rate, which is probably due to the increased water content, the enhanced swelling effect of the carrier, and the easier the dihydromyricetin to escape from the surfactant and oil phase to the water phase, so the release rate is increased and the cumulative release rate is increased.

Table 4 shows the release kinetics of samples coated with dihydromyricetin at 37 ℃ over different periods of time

Table 5 shows the release kinetics model and fitting parameters of samples encapsulating dihydromyricetin at 25 deg.C

The K-P equation is called Korsmeyer-Peppas equation

(4) Oxidation resistance test

The method adopted in the antioxidant experiment is DPPH free radical scavenging activity. Fig. 6 is an anti-oxidation curve of the drug-loaded sample, and the inset is the anti-oxidation curve of the dihydromyricetin ethanol solution. As can be seen from the figure, the scavenging efficiency of the free radicals is increased along with the increase of the content of the dihydromyricetin in the sample. IC of all samples compared to Dihydromyricetin EtOH solution₅₀The value is two orders of magnitude lower than that of the dihydromyricetin ethanol solution, which shows that the aggregate coated with the dihydromyricetin has better antioxidant activity.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.