CN114452316A - Walnut mountain ash flavone component nanoemulsion preparation and preparation method and application thereof - Google Patents
Walnut mountain ash flavone component nanoemulsion preparation and preparation method and application thereof Download PDFInfo
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- CN114452316A CN114452316A CN202210091420.XA CN202210091420A CN114452316A CN 114452316 A CN114452316 A CN 114452316A CN 202210091420 A CN202210091420 A CN 202210091420A CN 114452316 A CN114452316 A CN 114452316A
- Authority
- CN
- China
- Prior art keywords
- nanoemulsion
- flavone
- flavone component
- juglans mandshurica
- component
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 150000002212 flavone derivatives Chemical class 0.000 title claims abstract description 73
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
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Abstract
The invention provides a nano-emulsion preparation of a flavone component of Juglans mandshurica, and a preparation method and application thereof, wherein a surfactant and a cosurfactant are mixed to prepare a mixed surfactant, then an oil phase is added into the mixed surfactant and uniformly mixed, a constant-temperature magnetic stirrer is used for stirring for 1h, distilled water is dropwise added under the magnetic stirring state until a nano-emulsion with clear and transparent appearance is formed, then a flavone component of the Juglans mandshurica is added, and the nano-emulsion preparation of the flavone component of the Juglans mandshurica is obtained after magnetic stirring and dissolution.
Description
Technical Field
The invention belongs to the field of medicines, and discloses a walnut tree flavone component nanoemulsion preparation as well as a preparation method and application thereof.
Background
Liver cancer is one of the most common malignant tumors worldwide. In malignant tumors in China, the morbidity and mortality of liver cancer are the fourth and the second place respectively. However, since the pathogenesis of liver cancer is complex and the onset of liver cancer is hidden, most patients have already entered the middle and late stages of the diagnosis, and the treatment methods are very limited. In recent years, traditional Chinese medicinal materials are gradually concerned by researchers due to the advantages of multiple specific targets, multiple levels, less adverse reactions and the like. A large number of research reports suggest that natural, effective and low-toxicity chemical components of traditional Chinese medicines have great potential in the aspect of resisting liver cancer.
Juglans mandshurica (Juglans mandshurica) is a species of noble trees of the genus Juglans, the family Juglandaceae, and is a heterogynic deciduous broad-leaf arbor. As an important medicinal source plant in China, the juglans mandshurica maxim has remarkable antibacterial, anti-inflammatory and antioxidant effects. The juglans mandshurica branches contain active ingredients such as flavonoids, anthraquinones, alkaloids, terpenoids and the like, so that the juglans mandshurica branches have good medicinal prospect and development value. The flavonoid compounds are also called flavonoid compounds, are important secondary metabolites produced by the juglans mandshurica maxim, have wide pharmacological activities such as tumor resistance, bacteriostasis, inflammation resistance, oxidation resistance and the like, and have become research hotspots at home and abroad. Among them, flavonoids have been shown to be effective against various types of tumors. However, the characteristics of the flavonoid compound such as small solubility, fast metabolism, poor oral absorption and the like limit the exertion of the anti-tumor activity.
In recent years, the development of nanotechnology greatly promotes the application development of insoluble drugs in the field of medicine. The nano emulsion is a novel oral drug delivery system, and two mutually insoluble solutions form a dispersion system with transparent or semitransparent appearance and 1-100nm particle size under the action of an emulsifier/co-emulsifier. The drug-loading system can improve the stability of the embedded drug, the solubility of the insoluble drug, the bioavailability of the macromolecular water-soluble drug, the sustained-release effect of the drug and the like. Although researchers have carried out corresponding formulation improvement research on partial flavonoids compounds, no report is found about the research on the nano preparation of the total flavonoids of the juglans mandshurica maxim.
Disclosure of Invention
The invention provides a walnut tree flavone component nanoemulsion preparation and a preparation method thereof. In the invention, the preparation process of the flavonoid component nanoemulsion of the juglans mandshurica maxim is simple, the average particle size of the obtained nano preparation is 20-35nm, and the defects of low water solubility, poor bioavailability and the like of the flavonoid compound of the juglans mandshurica maxim are solved. In addition, the nano-emulsion also has good anti-oxidation and anti-liver cancer activities, and has good application prospects in the fields of tumor treatment, food health care, cosmetics and the like.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a walnut tree flavone component nanoemulsion is composed of the following raw materials in percentage by mass: the surfactant is a mixture of Tween 80 and span 80, and the mass ratio of the Tween 80 to the span 80 is 1:1, the content of the two in the system is 10.0-30.0%, the content of PEG400 is 10.0-30.0%, the content of isopropyl myristate is 1.0-6.0%, the content of flavones of juglans mandshurica maxim is 0.5-6.0%, and the balance is water phase, namely distilled water.
2. A preparation method of walnut tree flavone component nanoemulsion comprises the following steps: mixing a surfactant and a cosurfactant according to a volume ratio of 1:1, adding proper isopropyl myristate (IPM), stirring for 1h by magnetic force to completely mix, and adding distilled water dropwise under the condition of magnetic stirring until the system becomes clear and transparent to obtain blank nano emulsion. Adding 228mg of Juglans mandshurica Maxim flavone component into 10mL of blank nano emulsion, and magnetically stirring until the Juglans mandshurica Maxim flavone component is completely dissolved to obtain the Juglans mandshurica Maxim flavone component nano emulsion.
(2) In the preparation method of the juglans mandshurica maxim flavone component nanoemulsion, the preferable surfactant is compounded of tween 80 and span 80, the cosurfactant is PEG400, and the contents of the tween 80 and the span 80 in a nanoemulsion system are 10.0-30.0%; the preferable oil phase is isopropyl myristate (IPM) 1.0-6.0%, the flavone component of Juglans mandshurica Maxim 0.5-6.0%, and the rest is water phase which is distilled water. The prepared walnut tree flavone component nanoemulsion is clear and transparent and has good stability.
3. Application of walnut tree flavone component nanoemulsion in cancer treatment and antioxidant product development
The invention has the beneficial effects that: the nanoemulsion preparation containing the flavone component of the juglans mandshurica maxim provided by the invention has stronger anti-tumor and anti-oxidation capabilities, and can be applied to a plurality of industries such as medical treatment and health, food health care, cosmetics and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Wherein:
FIG. 1 shows the screening results of the walnut tree flavone component nanoemulsion oil phase.
FIG. 2 shows the screening results of the Juglans mandshurica Maxim flavone component nanoemulsion surfactant.
FIG. 3 shows the screening results of Juglans mandshurica Maxim flavone component nanoemulsion cosurfactant.
FIG. 4 shows the dyeing results of blank nanoemulsion of Juglans mandshurica Maxim flavone component.
FIG. 5 is an appearance of a nanoemulsion containing Juglans mandshurica flavone.
FIG. 6 shows the effect of different concentrations of Juglans mandshurica flavone component nanoemulsion on HepG2 cell morphology.
FIG. 7 shows the apoptotic effect of nanoemulsion of different concentrations of Juglans mandshurica flavone components on HepG 2.
FIG. 8 shows the DPPH scavenging ability measurement results of different concentrations of Juglans mandshurica Maxim flavone component nanoemulsion.
Detailed Description
To facilitate an understanding of the present invention, the following examples are specifically exemplified. Its purpose is to be construed as illustrative of the invention and not as limiting it in any way.
Example 1: optimization of preparation process of walnut mountain ash bark flavone component nanoemulsion
(1) Oil phase screening
6g of Tween 80 and span 80 compound surfactant (4.6: 1) and 2g of absolute ethyl alcohol are weighed, namely the total amount of the mixed surfactant is 8 g. Under a constant-temperature magnetic stirrer at 25 ℃, 1g of various oil phases (castor oil, olive oil, isopropyl myristate, isopropyl palmitate and sweet almond oil) are added, and after stirring for 1h, distilled water is slowly dropped, if the system can become clear and transparent or can be considered to form a nano-emulsion. If a plurality of systems form the nano-emulsion, the optimal oil phase is further screened by constructing a pseudo-ternary phase diagram. The result shows that isopropyl myristate (IPM) and isopropyl palmitate (IPP) can form clear and transparent nano emulsion, and isopropyl myristate (IPM) is finally selected as the oil phase prepared by the nano emulsion through a pseudo-ternary phase diagram.
(2) Surfactant screening
The influence of single or compound surfactants on the formation of the nanoemulsion is examined in a plurality of ways by adopting single-factor tests. The surfactants to be selected in this test were as follows: tween 80 and span 80 (4.6: 1), Tween 20 and span 80 (1.73: 1), castor oil polyoxyethylene ether (EL40) and polyoxyethylene hydrogenated castor oil (RH 40). The result shows that the Tween 80 and the span 80 can be compounded, RH40 and EL40 can form nano-emulsion, and the Tween 80 and the span 80 are finally selected to be compounded as the surfactant through a pseudo-ternary phase diagram.
(3) Cosurfactant screening
The influence of various cosurfactants on the formation of the nanoemulsion is tested by adopting a single factor. The cosurfactants to be selected in the test are ethanol, propylene glycol, glycerol and PEG 400. The results show that ethanol, propylene glycol and PEG can form a nano-emulsion, and finally PEG400 is used as a cosurfactant through a pseudo-ternary phase diagram.
(4) Km value screening
The Km value is the ratio of the mass of the surfactant to the mass of the cosurfactant, and the size of the forming area of the nanoemulsion when the Km value is 3:1, 2:1 and 1:1 is respectively inspected according to the pre-test result. The results show that the formed nanoemulsion region is the smallest when Km is 3:1, the difference in the area of the nanoemulsion region is small when Km is 2:1 and 1:1, but gel is easily formed when Km is 2:1 in the experimental process, so the ratio of the mass of the finally selected surfactant to the mass of the cosurfactant is 1: 1.
(5) Determination of optimal prescription
According to the pseudo ternary phase diagram of (4) when Km is 1:1, firstly, determining that S/O (mass ratio of the mixed surfactant to the oil phase) of 4 alternative prescriptions is 8.5/1.5, 8/2, 7.5/2.5 and 7/3 respectively, and then measuring the relation between water content and electric conductivity by using a conductivity meter to determine the type of the nanoemulsion and determine the water content in the nanoemulsion simultaneously. The results show that the conductivity of the four alternative formulas changes basically in a consistent way with the increase of the water content, the conductivity begins to decrease after the water content reaches 70%, and at the moment, the nano emulsion can be considered to be O/W type, so that the water phase content is selected to be 70% -80%, and S/O (mass ratio of the mixed surfactant to the oil phase) is selected to be 8/2 by measuring the particle size and PDI of each alternative formula when the water content is 80%. The blank nanoemulsion prescription obtained by final optimization is as follows: the ratio of the surfactant (compounded by tween 80 and span 80 in a ratio of 4.6: 1) to the cosurfactant (PEG400) is 1:1, the content of the two in the system is 15.7-23.5%; the oil phase is isopropyl myristate (IPM) with a content of 3.9-5.0%; the rest is water phase;
example 2: characterization of walnut mountain ash flavone component nanoemulsion
(1) Preparation of walnut tree flavone component nanoemulsion
After blank nano-emulsion is prepared according to the optimized prescription, 220mg of the juglans mandshurica maxim flavone purified product is slowly added into 10g of the blank nano-emulsion, and is stirred to be completely dissolved, so that the juglans mandshurica maxim flavone component nano-emulsion with the content of 22.0mg/g is prepared.
(2) Particle size analysis of walnut mountain ash flavone component nanoemulsion preparation
The particle size of the emulsion droplets is an important characteristic parameter of the nanoemulsion and is one of the most important properties for evaluating the stability of the nanoemulsion, and a laser particle size analyzer is used for measuring the particle size of a sample. The result shows that the average grain diameter of the walnut tree flavone component nanoemulsion is 22.5 nm.
(3) Dyeing analysis of walnut mountain ash flavone component nanoemulsion preparation
The type of the nanoemulsion is identified by a dyeing method, two parts of nanoemulsion are taken, the water-based dye methylene blue and the oil-based dye Sudan red with the same volume are respectively dripped, the diffusion condition of the two dyes in the nanoemulsion is observed, and the result shows that the diffusion speed of the methylene blue is obviously high. In sudan red dye, the prepared nanoemulsion was described as being of the oil-in-water type.
(4) Stability analysis of walnut mountain ash flavone component nanoemulsion preparation
Taking Juglans mandshurica Maxim flavone nanoemulsion, standing at 4 deg.C in dark place for 30 days at room temperature, taking out at 0, 5, 10, 15, 20, 30 days, observing appearance and determining nanoemulsion particle size. The result shows that the juglans mandshurica flavone nanoemulsion has good stability at normal temperature and in a storage environment of 4 ℃.
Example 3: research on anti-liver cancer effect of walnut tree flavone component nanoemulsion
(1) Cell line source and culture method
HepG2 and HL02 cells were cultured according to a conventional method, specifically, in a DMEM medium containing penicillin/Streptomyces (1%) and fetal bovine serum (10%), in an incubator at 37 ℃ with 5% CO 2.
(2) Influence of walnut tree flavone component nanoemulsion on HepG2 cell morphology
HepG2 cells were selected for log phase growth at 3X 10 per well5The individual cells were seeded in 6-well plates, incubated in an incubator for 24h, and the solution was discarded. By nuclei of different concentrationsAfter the aronia melanocarpa flavone component nanoemulsion (40, 80 and 120 mu g/mL) is treated for 48 hours, the cell morphology is observed under an inverted microscope, when the flavone concentration is 0 mu g/mL, the cells are in a polygon with close and regular arrangement, the cell arrangement becomes loose and the cells are elongated with the increase of the medicine concentration, and are in an irregular polygon or triangle, so that a large number of vacuoles are generated in the cells.
(3) Influence of walnut tree flavone component nanoemulsion on HepG2 cell proliferation
The effect of different concentrations of nanoemulsion (10, 20, 40, 60, 80, 100, 120, 140 mug/mL) on the proliferation of HepG2 cells is detected by using CCK8, and the result shows that the inhibitory effect of the nanoemulsion on the proliferation of HepG2 is dose-dependent, and the IC50 of the nanoemulsion at 24h and 48h is 119.0 mug/mL and 49.21 mug/mL respectively.
(4) Influence of walnut tree flavone component nanoemulsion on HepG2 cell migration
The effect of the nanoemulsion on HepG2 cell migration was examined using the scratch test. The result shows that the walnut tree flavone component nanoemulsion can inhibit the migration of HepG2 cells, and the higher the treatment concentration is, the higher the inhibition rate is.
(5) Influence of walnut tree flavone component nanoemulsion on HepG2 cell apoptosis
The flow cytometry is adopted to detect the influence of the nanoemulsion on the HepG2 cell apoptosis, and the result shows that the cell apoptosis rate is increased along with the increase of the concentration of the Juglans mandshurica flavone component nanoemulsion, which indicates that the Juglans mandshurica flavone component nanoemulsion can induce the apoptosis of HepG2 cells.
(6) Influence of walnut tree flavone component nanoemulsion on HL02 cells and proliferation
The toxicity of the nanoemulsion on normal liver cells HL02 is detected by using CCK 8. The result shows that the walnut tree flavone component nanoemulsion has no obvious toxicity to HL02 cells, and is relatively safe at the cell level.
Example 4: research on antioxidant effect of walnut tree flavone component nanoemulsion
The effect of the walnut tree flavone component nanoemulsion on eliminating DPPH free radicals is evaluated. Adding a certain amount of nanoemulsion or vitamin C standard solution into ethanol. Mix well with 2.4mL of DPPH reagent. The mixture was left to react in the dark for 40 minutes, the absorbance at 520nm was measured, and then the DPPH radical scavenging ability of the sample was calculated. The result shows that the walnut tree flavone component nanoemulsion has good capability of clearing DPPH free radicals.
The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not substantially depart from the spirit and scope of the embodiments of the present invention, and are intended to be included within the scope of the present invention.
Claims (9)
1. A walnut tree flavone component nanoemulsion preparation is characterized by comprising a walnut tree flavone component serving as an effective active ingredient and auxiliary additive ingredients matched with medicines, wherein the effective active ingredient is obtained from a macroporous resin purified walnut tree.
2. The nanoemulsion formulation for flavonoid component of juglans mandshurica maxim as claimed in claim 1, wherein: the nanometer emulsion is prepared by mixing flavone component of Juglans mandshurica Maxim as effective active component with oil phase, surfactant and cosurfactant.
3. The nanoemulsion formulation for the flavone component of juglans mandshurica maxim as claimed in claim 2, wherein: finally selecting the surfactant as Tween 80 and span 80 according to the mass ratio of 1: 1-8:1, wherein the content of the two in the system is 10.0-30.0%; the cosurfactant is PEG400, and the content of the cosurfactant is 10.0-30.0%; the oil phase is isopropyl myristate, the content of isopropyl myristate is 1.0-6.0%, the flavone component of the juglans mandshurica maxim is 0.5-6.0%, and the balance is water phase, and the water phase is distilled water.
4. The nanoemulsion formulation for the flavone component of juglans mandshurica maxim as claimed in any one of claims 1-3, characterized in that: the nanoemulsion of the juglans mandshurica maxim flavone component has the granularity of 20-35nm and the polymer dispersity index of 0.15-0.17.
5. The nanoemulsion formulation for the flavone component of juglans mandshurica maxim as claimed in any one of claims 2-3, wherein the mass ratio of the surfactant to the cosurfactant is 1:1, and the mass ratio of the mixed surfactant to the oil phase is 8: 2.
6. The method for preparing the nanoemulsion formulation for the flavone component of juglans mandshurica maxim as claimed in any one of claims 1 to 5, comprising the steps of:
mixing a surfactant and a cosurfactant to prepare a mixed surfactant, adding an oil phase into the mixed surfactant, uniformly mixing, stirring for 1 hour by using a constant-temperature magnetic stirrer, and dropwise adding distilled water under the magnetic stirring state until a clear and transparent nanoemulsion is formed;
slowly adding 220mg of walnut tree flavone purified product into 10g of the nanoemulsion, and stirring to completely dissolve the mixture to prepare the walnut tree flavone component nanoemulsion with the content of 22.0 mg/g;
performing stability analysis on the walnut tree flavone component nanoemulsion; taking Juglans mandshurica Maxim flavone nanoemulsion, standing at 4 deg.C in dark for 30 days, taking out at 0, 5, 10, 15, 20, 30 days, observing appearance and measuring nanoemulsion particle diameter to determine stability.
7. The preparation method of the juglans mandshurica maxim flavone component nanoemulsion preparation as claimed in any one of claim 6, wherein the particle size of the juglans mandshurica maxim flavone component nanoemulsion is analyzed by a laser particle size analyzer, and the average particle size of the selected juglans mandshurica maxim flavone component nanoemulsion is 22.5 nm.
8. The method for preparing a nanoemulsion formulation containing Juglans mandshurica Maxim flavone components as claimed in claim 7, wherein the type of the nanoemulsion formulation containing Juglans mandshurica Maxim flavone components is identified by dyeing; and (3) taking two parts of the walnut tree flavone component nanoemulsion, respectively dropwise adding the same volume of water-based dye methylene blue and oil-based dye Sudan red, and observing the diffusion conditions of the two dyes in the walnut tree flavone component nanoemulsion so as to determine the type of the walnut tree flavone component nanoemulsion.
9. Use of the walnut tree flavone component nanoemulsion preparation according to any one of claims 1 to 5 in preparation of foods, medicines, health products and cosmetics with antioxidation and anticancer effects.
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