A nanocomposite emulsion of dihydromyricetin and its preparation method and application

Abstract

The present invention belongs to the field of biological preparations, and specifically relates to a nanocomposite emulsion of dihydromyricetin, a preparation method thereof, and an application thereof. The nanocomposite emulsion of dihydromyricetin proposed by the present invention comprises the following components, calculated by weight percentage: 0.5-3.0 wt.% dihydromyricetin, 2.0-8.0 wt.% hydrophilic core solvent, 12.5-25.5 wt.% lipid carrier with a bidirectional hydrophilic structure, and 63.5-85.0 wt.% external aqueous phase. The present invention first uses a hydrophilic core solvent to completely dissolve DHM. The invention overcomes the defects of the prior art, greatly improves the solubility of DHM, and significantly increases its room temperature stability. The DHM lipid particles can reach the nanometer level and are not easily segregated, which significantly improves the permeability of DHM and enhances its bioavailability.

Description

Technical Field

[0001] The invention belongs to the field of biological preparations, and particularly relates to a dihydromyricetin nanocomposite emulsion and a preparation method and application thereof. Background Art

[0002] Dihydromyricetin (DHM) is a natural dihydroflavonol compound with high content in the pseudofruit of large-leaf vine and sour jujube. Its molecular formula is C 15 H 12 O8, molecular weight is 320.25g / mol, its molecular structure is shown in Figure 1 DHM has poor chemical stability, mainly because it contains 5 phenolic hydroxyl groups, which are easily oxidized by trace amounts of Fe 3+ 、Cu 2+ It can be chemically degraded by metal ions such as ions and photocatalytic oxidation. It can exist relatively stably in a low-temperature weakly acidic environment, but its oxidation degree will increase with the increase of pH value and temperature. DHM has poor water solubility and fat solubility. Its solubility in water at 25°C is 0.2 mg / mL, and its oil-water (n-octanol-water) partition coefficient is only 0.45. Low water solubility and fat solubility lead to low bioavailability of DHM. Literature reports that the intestinal mucosal permeability of DHM in rats is 9.3×10 -6 cm / s, but there is no report on the bioavailability of DHM in humans. The intestinal mucosal permeability in the human gastrointestinal tract is estimated to be less than 10%. The pharmacokinetic results of DHM showed that after oral administration of 100 mg / kg body weight to rats, the peak time of DHM was 32.2 minutes, and the highest concentration in rat plasma was C max It is only about 80 ng / mL, the absorption phase half-life is 13.2 min, and the elimination phase half-life is 17.9 min, indicating that DHM has a short half-life in animals, rapid absorption and metabolism, and very low oral bioavailability.

[0003] Pharmacological studies have shown that DHM possesses multiple pharmacological activities, including antioxidant, anticancer, anti-inflammatory, antimicrobial, hepatoprotective, and lipid and glucose metabolism regulation, along with a good safety profile, making it highly promising for development. The solubility and stability of flavonoids are key factors in determining their in vivo bioavailability. However, due to its low solubility and low permeability, DHM is classified as a Class IV2 compound according to biopharmaceutical classification standards. Furthermore, DHM's poor molecular stability and short half-life result in low bioavailability and poor drugability, limiting its clinical application.

[0004] For Class IV compounds, generally speaking, improving solubility and permeability helps improve their clinical performance. In order to improve the above-mentioned problems of DHM, in recent years, research on improving the stability, solubility and bioavailability of DHM by adopting various new formulation technologies and new dosage forms has become a hot topic. Studies on DHM liposomes, microemulsions, microcapsules, nanomicelles, etc. have emerged, but none of them have completely solved the problem of significantly improving the solubility and in vitro stability of DHM. According to literature reports, some scholars have made DHM into liposomes, which improved the stability of DHM, but only increased its solubility from the original 0.2 mg / ml to 1.02 mg / ml, and the solubility increase level was only 5 times. Other scholars have made DHM into microemulsions or microcapsules. Although the solubility was effectively improved, the particle size of the droplets in the system was large, all above 1000 nm, which made the droplets easy to aggregate, thereby affecting the stability of DHM in the system. Developing a new emulsion tailored to the characteristics of DHM to solve its problems such as low solubility, low permeability, poor stability, and short half-life, thereby increasing its bioavailability, improving its drugability, and expanding the clinical application prospects of DHM has significant scientific research significance and market value. Summary of the Invention

[0005] In view of the above-mentioned characteristics of DHM, the technical problem solved by the present invention is to propose a new nanocomposite emulsion of dihydromyricetin and a preparation method thereof. DHM is first completely dissolved in a hydrophilic core solvent and then encapsulated in a natural or synthetic lipid carrier with a bidirectional hydrophilic structure to form a water-in-oil nanoemulsion. Utilizing the bidirectional hydrophilicity of the lipid carrier, the water-in-oil nanoemulsion is then dispersed in an external aqueous phase to form a new composite emulsion with a particle size of 10 to 500 nanometers. The present invention overcomes the defects of the existing technology, can greatly improve the solubility of DHM, and significantly increase its room temperature stability. The DHM lipid particles can reach the nanometer level and are not easily segregated, which significantly improves the permeability of DHM and increases its bioavailability.

[0006] The present invention provides a nanocomposite emulsion of dihydromyricetin, which comprises the following components in weight percentage: 0.5-3.0 wt.% of dihydromyricetin, 2.0-8.0 wt.% of a hydrophilic core solvent, 12.5-25.5 wt.% of a lipid carrier with a bidirectional hydrophilic structure, 63.5-85.0 wt.% of an external aqueous phase,

[0007] Preferably, the hydrophilic core solvent includes glycerol, propylene glycol and water;

[0008] Preferably, in terms of weight percentage, the ratio of glycerol to propylene glycol in the hydrophilic core solvent is 0.2 to 1:1;

[0009] Preferably, the glycerol in the hydrophilic core solvent can be replaced with PEG300 or PEG400;

[0010] DHM has poor water solubility and fat solubility. Its solubility in water at 25°C is only 0.2 mg / mL, and its oil-water (n-octanol-water) partition coefficient is only 0.45. According to the principle of like dissolves like, a non-aqueous solvent with a strong polarity can effectively increase the concentration of DHM in solution. The present invention has selected four solvents, glycerol, propylene glycol, PEG300, and PEG400, and obtained the optimal ratio between the solvents. However, DHM still has the problem of poor stability in polar non-aqueous solvents. In order to increase the stability of the product, DHM needs to be prepared into a composite nanoemulsion.

[0011] Preferably, the bidirectionally hydrophilic lipid carrier comprises a water-in-oil emulsifier, an emulsifier co-emulsifier and oil;

[0012] Preferably, the water-in-oil emulsifier is any one of triglycerol dioleate, sorbitan monostearate, and sorbitan monooleate;

[0013] Preferably, the co-emulsifier is any one of natural or synthetic soybean lecithin, natural or synthetic egg yolk lecithin;

[0014] Preferably, the oil comprises any one of natural or purified coconut oil, natural or synthetic medium-chain triglyceride fatty acid ester, olive oil, avocado oil, or soybean oil;

[0015] Preferably, in terms of weight percentage, the content of the water-in-oil emulsifier in the bi-directionally hydrophilic lipid carrier is 2.5 wt.% to 4 wt.%, the content of the co-emulsifier is 1.5 wt.% to 2.5 wt.%, and the content of the oil is 3.4 wt.% to 7.6 wt.%;

[0016] This bi-directionally hydrophilic lipid carrier protects the stability of DHM within the hydrophilic core, preventing oxidation and precipitation of DHM from the solution due to environmental changes. During this phase, the bi-directionally hydrophilic lipid carrier serves as the external phase, while the hydrophilic core serves as the internal aqueous phase. The lipid carrier contains an oil phase, stabilized by a water-in-oil emulsifier, and lecithin serves as a co-emulsifier to maintain affinity for the internal aqueous phase.

[0017] Preferably, the external aqueous phase comprises an oil-in-water emulsifier, calcium disodium EDTA, vitamin C, an antibacterial agent, and water;

[0018] Preferably, the oil-in-water emulsifier is any one of Tween 80, vitamin E derivatives, lauroyl macrogol glyceride or polyoxyethylene hydrogenated castor oil derivatives;

[0019] Preferably, the vitamin E derivative is vitamin E polyethylene glycol succinate;

[0020] Preferably, the antibacterial agent is any one of sodium benzoate or potassium sorbate;

[0021] Preferably, in terms of weight percentage, the content of the oil-in-water emulsifier in the external aqueous phase is 4-6 wt.%, the content of calcium disodium ethylenediaminetetraacetic acid is not more than 0.0025 wt.%, the content of vitamin C is 0.1 wt.%-0.5 wt.%, and the content of the antibacterial agent is not more than 0.1 wt.%;

[0022] The external aqueous phase primarily increases the compatibility of this product with other hydrophilic solutions, accommodating consumer taste adjustments. An oil-in-water emulsifier is added to this external aqueous phase to enhance affinity with lipid carriers. The chelating agent calcium disodium EDTA prevents metal ions from oxidizing the product. Vitamin C, an antioxidant, consumes oxygen in the product, reducing DHM oxidation. The antibacterial agent inhibits bacterial growth, ensuring product safety.

[0023] The present invention provides a method for preparing a nanocomposite emulsion of dihydromyricetin, comprising the following steps:

[0024] S1: Accurately weigh the material components of dihydromyricetin and the hydrophilic core solvent according to weight percentage, first place the components of the hydrophilic core solvent in an appropriate container, stir and mix evenly, and heat the solvent to 50-60° C. in a water bath to prepare a hydrophilic core solvent, then pass the solid powder of dihydromyricetin through a 40-mesh sieve and slowly sieve it into the hydrophilic core solvent being stirred to fully dissolve it, thereby preparing a dihydromyricetin-hydrophilic core solution;

[0025] S2: Accurately weigh the water-in-oil emulsifier, co-emulsifier, and oil according to weight percentage, mix the three materials in a suitable container, and heat to 50-60°C in a water bath. After fully melted, continue to stir the solution to mix evenly, thereby preparing a lipid carrier with a bidirectional hydrophilic structure;

[0026] S3: adding the dihydromyricetin-hydrophilic core solution prepared in step S1 to the bidirectional hydrophilic lipid carrier solution prepared in step S2 at a rate of 1 drop / s, magnetically stirring the mixed solution while adding, and continuing magnetic stirring for 30 minutes after the addition is completed, and then ultrasonically treating the mixed solution to obtain a water-in-oil nanoemulsion.

[0027] S4: Accurately weigh the materials of the external aqueous phase according to weight percentage, add the oil-in-water emulsifier, calcium disodium EDTA, vitamin C and antibacterial agent to water, heat to 50-60° C., and stir thoroughly to dissolve to obtain the external aqueous phase;

[0028] S5: stirring the water-in-oil nanoemulsion prepared in step S3, and slowly adding the external aqueous phase prepared in step S4 while stirring, and performing ultrasonic treatment again to finally prepare the dihydromyricetin nanocomposite emulsion.

[0029] Preferably, the ultrasonic treatment in step S3 and step S5 is as follows: in an ice bath, ultrasonic treatment is performed for 5 minutes each time, with an interval of 2 minutes between each treatment, and repeated 3 times, with an ultrasonic power of 500 W and an ultrasonic amplitude of 80%;

[0030] Preferably, the ultrasonic treatment in step S3 and step S5 can also be replaced by a homogenization treatment, wherein the mixed solution is placed in a homogenizer for homogenization, the power of the homogenizer is 200W, the temperature is controlled at 50-60°C, 2 minutes / time, each interval is 3 minutes, and repeated 6 times.

[0031] The present invention provides a use of a nanocomposite emulsion of dihydromyricetin in medicines, health products or health drinks. The medicines include one or more of the medicines for anti-oxidation, anti-cancer, anti-inflammatory, anti-microbial, liver protection and hangover relief.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The solubility of DHM was significantly improved, increasing the solubility of DHM by at least 100 times, from 0.2 mg / ml to at least 20 mg / ml;

[0034] (2) Significantly improved the stability of DHM: the stability of DHM at room temperature can reach 12 months, ensuring

[0035] The medicines and health care products prepared with it have a good shelf life, making the clinical administration of DHM possible;

[0036] (3) Significantly improved the permeability of DHM: the lipid particles of DHM reached the nanometer level of 10-500 nanometers,

[0037] It is easy to aggregate and more stable; when the lipid particles decrease from 200 microns to 200 nanometers, the ratio of the particles

[0038] The surface area increases by more than 1000 times, which improves the affinity and penetration of DHM on the surface of gastrointestinal mucosa.

[0039] permeability, thereby improving the oral bioavailability of DHM, and promoting the development of DHM in antioxidant,

[0040] It has clinical applications in a variety of pharmacological activities such as cancer, anti-inflammatory, antimicrobial, and liver protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 This is the molecular structure diagram of dihydromyricetin;

[0043] Figure 2 Schematic diagram of the preparation process of the dihydromyricetin nanocomposite emulsion disclosed in the present invention;

[0044] Figure 3 This is the particle size analysis graph of the sample prepared in Example 1;

[0045] Figure 4 This is the particle size analysis graph of the sample prepared in Example 2;

[0046] Figure 5 This is the particle size analysis graph of the sample prepared in Example 3;

[0047] Figure 6 This is the chromatogram of the sample prepared in Example 1;

[0048] Figure 7 The macromorphology of the samples prepared in Examples 1 to 3; DETAILED DESCRIPTION

[0049] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the chemical reagents used in the examples are conventional commercial reagents, and the technical means used in the examples are those of ordinary skill in the art.

[0050] The specific implementation of the present invention is as follows:

[0051] A method for preparing a nanocomposite emulsion of dihydromyricetin comprises the following steps:

[0052] S1: Accurately weigh the material components of dihydromyricetin and the hydrophilic core solvent according to weight percentage, first place the components of the hydrophilic core solvent in an appropriate container, stir and mix evenly, and heat the solvent to 50-60° C. in a water bath to prepare a hydrophilic core solvent, then pass the solid powder of dihydromyricetin through a 40-mesh sieve and slowly sieve it into the hydrophilic core solvent being stirred to fully dissolve it, thereby preparing a dihydromyricetin-hydrophilic core solution;

[0053] S2: Accurately weigh the water-in-oil emulsifier, co-emulsifier, and oil according to weight percentage, mix the three materials in a suitable container, and heat to 50-60°C in a water bath. After fully melted, continue to stir the solution to mix evenly, thereby preparing a lipid carrier with a bidirectional hydrophilic structure;

[0054] S3: adding the dihydromyricetin-hydrophilic core solution prepared in step S1 to the bidirectionally hydrophilic lipid carrier solution prepared in step S2 at a rate of 1 drop / s, magnetically stirring the mixed solution while adding, and continuing magnetic stirring for 30 minutes after the addition is completed, and then ultrasonically treating or homogenizing the mixed solution to obtain a water-in-oil nanoemulsion.

[0055] S4: Accurately weigh the materials of the external aqueous phase according to weight percentage, add the oil-in-water emulsifier, calcium disodium EDTA, vitamin C and antibacterial agent to water, heat to 50-60° C., and stir thoroughly to dissolve to obtain the external aqueous phase;

[0056] S5: stirring the water-in-oil nanoemulsion prepared in step S3, and slowly adding the external aqueous phase prepared in step S4 while stirring, and performing ultrasonic treatment or homogenization treatment again to finally prepare the dihydromyricetin nanocomposite emulsion.

[0057] The ultrasonic treatment in step S3 and step S5 is as follows: in an ice bath, ultrasonic treatment is performed for 5 minutes each time, with an interval of 2 minutes between each treatment, and repeated 3 times, with an ultrasonic power of 500 W and an ultrasonic amplitude of 80%;

[0058] The ultrasonic treatment in step S3 and step S5 can also be replaced by a homogenization treatment, wherein the mixed solution is placed in a homogenizer for homogenization, the power of the homogenizer is 200W, the temperature is controlled at 50-60°C, 2 minutes / time, each interval is 3 minutes, and repeated 6 times.

[0059] Example 1

[0060] Example 1 was prepared according to the above specific implementation method. Table 1 shows the material components and preparation methods involved in Example 1.

[0061] Table 1 Material components and preparation methods involved in Example 1

[0062]

[0063]

[0064] Example 2

[0065] Example 2 was prepared according to the above specific implementation method. Table 2 shows the material components and preparation methods involved in Example 2.

[0066] Table 2 Material components and preparation methods involved in Example 2

[0067]

[0068] Example 3

[0069] Example 3 was prepared according to the above specific implementation method. Table 3 shows the material components and preparation methods involved in Example 2.

[0070] Table 3 Material components and preparation methods involved in Example 3

[0071]

[0072]

[0073] Comparative Example 1

[0074] Comparative Example 1 was prepared according to the above specific implementation method. Table 4 shows the material components and preparation methods involved in Comparative Example 1.

[0075] Table 4 Material components and preparation methods involved in Comparative Example 1

[0076]

[0077] Comparative Example 2

[0078] Comparative Example 2 was prepared according to the above specific implementation method. Table 5 shows the material components and preparation methods involved in Comparative Example 1.

[0079] Table 5 Material components and preparation methods involved in Comparative Example 2

[0080]

[0081]

[0082] Comparative Example 3

[0083] Comparative Example 3 was prepared according to the above specific implementation method. Table 6 shows the material components and preparation methods involved in Comparative Example 3.

[0084] Table 6 Material components and preparation methods involved in Comparative Example 3

[0085]

[0086] Comparative Example 4

[0087] Comparative Example 4 was prepared according to the above specific implementation method. Table 7 shows the material components and preparation methods involved in Comparative Example 4.

[0088] Table 7 Material components and preparation methods involved in Comparative Example 4

[0089]

[0090] Comparative Example 5

[0091] Comparative Example 5 was prepared according to the above specific implementation method. Table 8 shows the material components and preparation methods involved in Comparative Example 5.

[0092] Table 8 Material components and preparation methods involved in Comparative Example 5

[0093]

[0094] According to the commonly used quality evaluation method for nanoemulsions, relevant performance tests were performed on the dihydromyricetin nanocomposite emulsions prepared in Examples 1 to 3 and Comparative Examples 1 to 5. The test results are shown in Table 9.

[0095] Table 9 Performance test results of nanocomposite emulsions prepared in Examples and Comparative Examples

[0096]

[0097] As shown in Table 9, the DHM content of the DHM nanocomposite emulsions prepared in the examples of the present invention can reach up to 3 wt.%, and the stability of DHM at both room temperature and low temperature can exceed 4 months. At the same time, the DHM content is not lost, and the average size of the lipid particles can reach a minimum of 80 nm, showing good overall performance. However, the nanocomposite emulsions prepared in Comparative Examples 1 to 3 have lower DHM contents, and the DHM content is lost during storage at room temperature and low temperature, and their stability is lacking. The average size of the lipid particles is also relatively large. The nanocomposite emulsions prepared in Comparative Examples 4 and 5 have poor stability, and a significant white precipitate occurs after one week at room temperature and three days at low temperature.

[0098] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A nanocomposite emulsion of dihydromyricetin, characterized in that: The invention comprises the following components in weight percentage: 0.5-3.0 wt.% dihydromyricetin, 2.0-8.0 wt.% hydrophilic core solvent, 12.5-25.5 wt.% lipid carrier with a bidirectional hydrophilic structure, and 63.5-85.0 wt.% external aqueous phase; The hydrophilic core solvent comprises glycerol, propylene glycol and water, and the ratio of glycerol to propylene glycol in the hydrophilic core solvent is 0.2 to 1:1 by weight. Optionally, the glycerol in the hydrophilic core solvent is replaced by PEG300 or PEG400. The lipid carrier with a bidirectional hydrophilic structure includes a water-in-oil emulsifier, an emulsifier and oil; The water-in-oil emulsifier is any one of triglycerol dioleate, sorbitan monostearate, and sorbitan monooleate; the co-emulsifier is any one of natural or synthetic soybean lecithin and natural or synthetic egg yolk lecithin; the oil includes any one of natural or purified coconut oil, natural or synthetic medium-chain triglyceride, olive oil, avocado oil, or soybean oil; The external aqueous phase includes an oil-in-water emulsifier, calcium disodium EDTA, vitamin C, an antibacterial agent, and water; The oil-in-water emulsifier is any one of Tween 80, vitamin E derivatives, and lauroyl macrogol glyceride; the vitamin E derivative is vitamin E macrogol succinate; and the antibacterial agent is any one of sodium benzoate and potassium sorbate.

2. The nanocomposite emulsion of dihydromyricetin according to claim 1, characterized in that In terms of weight percentage, the content of the water-in-oil emulsifier in the bidirectionally hydrophilic lipid carrier is 2.5 wt.%~4 wt.%, the content of the co-emulsifier is 1.5 wt.%~2.5 wt.%, and the content of oil is 3.4 wt.%~7.6wt.%.

3. The nanocomposite emulsion of dihydromyricetin according to claim 1, characterized in that In terms of weight percentage, the content of water-in-oil emulsifier in the external aqueous phase is 4-6 wt.%, the content of calcium disodium ethylenediaminetetraacetic acid is not more than 0.0025 wt.%, the content of vitamin C is 0.1 wt.%-0.5 wt.%, and the content of antibacterial agent is not more than 0.1 wt.%.

4. A method for preparing the nanocomposite emulsion of dihydromyricetin according to claim 1, characterized in that: The preparation method comprises the following steps: S1: Accurately weigh the material components of dihydromyricetin and the hydrophilic core solvent according to weight percentage, first place the components of the hydrophilic core solvent in an appropriate container, stir and mix evenly, and heat the solvent to 50-60°C in a water bath to prepare the hydrophilic core solvent, then pass the solid powder of dihydromyricetin through a 40-mesh sieve and slowly sieve it into the stirring hydrophilic core solvent to fully dissolve it, thereby preparing a dihydromyricetin-hydrophilic core solution; S2: Accurately weigh the water-in-oil emulsifier, co-emulsifier, and oil according to weight percentage, mix the three materials in a suitable container, and heat to 50-60°C in a water bath. After fully melting, continue to stir the solution to mix it evenly, thereby preparing a lipid carrier with a bidirectional hydrophilic structure; S3: adding the dihydromyricetin-hydrophilic core solution prepared in step S1 to the bidirectionally hydrophilic lipid carrier solution prepared in step S2 at a rate of 1 drop / s, magnetically stirring the mixed solution while adding, and continuing magnetic stirring for 30 minutes after the addition is completed, and then ultrasonically treating the mixed solution to obtain a water-in-oil nanoemulsion; S4: Accurately weigh the materials of the external aqueous phase according to weight percentage, add the oil-in-water emulsifier, calcium disodium EDTA, vitamin C and antibacterial agent to water, heat to 50-60°C, and stir thoroughly to dissolve to prepare the external aqueous phase; S5: stirring the water-in-oil nanoemulsion prepared in step S3, and slowly adding the external aqueous phase prepared in step S4 while stirring, and performing ultrasonic treatment again to finally prepare the dihydromyricetin nanocomposite emulsion.

5. The method for preparing the nanocomposite emulsion of dihydromyricetin according to claim 4, wherein: The ultrasonic treatment in step S3 and step S5 is as follows: in an ice bath, ultrasonic treatment is performed for 5 minutes each time with an interval of 2 minutes each time, repeated 3 times, with the ultrasonic power of 500 W and the ultrasonic amplitude of 80%.

6. The method for preparing the nanocomposite emulsion of dihydromyricetin according to claim 4, wherein: The ultrasonic treatment in step S3 and step S5 is replaced by a homogenization treatment, wherein the mixed solution is placed in a homogenizer for homogenization. The power of the homogenizer is 200 W, the temperature is controlled at 50-60 ° C, 2 min / time, 3 min interval each time, and repeated 6 times.

7. A nanocomposite emulsion of dihydromyricetin according to any one of claims 1 to 3, characterized in that: It is used in medicines, health products or health drinks.

Images