Preparation and application of a nanoemulsion of chrysanthemum luteolin

Abstract

The present invention relates to the field of medical technology, and specifically to the preparation and application of a chrysanthemin nanoemulsion. The specific technical solution comprises: a method for preparing a chrysanthemin nanoemulsion, comprising mixing chrysanthemin with an oil phase and a composite surfactant and stirring until the chrysanthemin dissolves; then slowly adding water dropwise and stirring until the emulsion becomes clear, transparent, and emits a bluish light, thereby obtaining the chrysanthemin nanoemulsion. The present invention solves the problems of poor water solubility and low oral bioavailability of chrysanthemin in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the preparation and application of a chrysanthemum luteolin nanoemulsion. Background Art

[0002] Isodon sculponeatus (Vaniot) Kudo, also known as stinking wormwood, square-stem perilla, and dysentery medicine, is the dried whole herb of Isodon sculponeatus (Vaniot) Kudo, a plant of the genus Isodon in the Lamiaceae family. It is primarily distributed in southwestern my country. Isodon sculponeatus is warm in nature and pungent in flavor, and has the effects of regulating qi and dampness, clearing heat and detoxifying. It is commonly used internally to treat dysentery, colds, and infantile malnutrition, and applied topically for itchy skin and athlete's foot, with good therapeutic effects.

[0003] Wang Xianrong et al. (Wang Xianrong, Wang Zhaoquan, Dong Jinguang. New antibacterial diterpenoids from S. chrysogenum [J]. Bulletin of Chinese Medicine, 1985(06):32-35.) isolated and obtained a variety of diterpenoids including sculponeatin A (STA), sculponeatin B and sculponeatin C from the ethanol extract of S. chrysogenum. Antibacterial experiments showed that these diterpenoids had strong antibacterial effects on Staphylococcus aureus, Shigella dysenteriae and Bacillus subtilis.

[0004] Sculponeatin A (sptA or STA) is a new diterpenoid compound isolated from the genus Sculponeatin of the Lamiaceae family. 20 H 24 O6, with the structural formula as follows and a molecular weight of 360.406, has a strong inhibitory effect on Staphylococcus aureus, Shigella flexneri, and Bacillus subtilis. Its minimum inhibitory concentration (MIC) was measured to be 125 μg / ml by the bacterial test tube method, and its antibacterial effect is relatively strong; there are no reports on the role and mechanism of chrysanthemin A in tumors. The research team of the present invention found in the study that chrysanthemin A has a strong anti-tumor effect and a broad anti-cancer spectrum, but it also has certain cardiovascular toxicity and liver toxicity. In addition, its solubility is poor and after injection into the tail vein of the injection solution prepared with PEG and ethanol, the activity of the experimental animals is significantly deteriorated.

[0005] Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides a preparation and application of a nanoemulsion of chrysanthemum luteolin A, which solves the problems of the existing technology such as poor water solubility and low oral bioavailability of chrysanthemum luteolin A.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] The invention discloses a preparation method of a chrysanthemin nanoemulsion. The method comprises the following steps: mixing chrysanthemin with an oil phase and a composite surfactant and stirring until the chrysanthemin is dissolved; slowly adding water dropwise and stirring until the emulsion is clear, transparent and blue-light-emitting, thereby obtaining the chrysanthemin nanoemulsion.

[0009] Preferably, the concentration of the chrysanthemum luteolin is 0.6-10 mg / mL.

[0010] Preferably, the mass ratio of the oil phase to the complex surfactant is 1:9 to 9:1.

[0011] Preferably, the oil phase is any one of medium chain triglycerides, soybean oil, oleic acid glycol glyceride, peanut oil, and sesame oil.

[0012] Preferably, the composite surfactant is any two of polyoxyethylene 40 hydrogenated castor oil, poloxamer 188, polyglycerol oleate, Tween 80 and Span 80, and the mass ratio of any two of the composite surfactants is 1:1 to 10:1.

[0013] Preferably, the composite surfactant is polyoxyethylene 40 hydrogenated castor oil and polyglycerol oleate, and the mass ratio of polyoxyethylene 40 hydrogenated castor oil to polyglycerol oleate is 4:1 to 1:1.

[0014] Preferably, based on mass fraction, the oil phase is 25%, polyoxyethylene 40 hydrogenated castor oil is 12.5-20%, polyglycerol oleate is 5-12.5%, and water is 50%.

[0015] Correspondingly, the above preparation method prepares the chrysanthemum luteolin nanoemulsion.

[0016] Correspondingly, the chrysanthemin nanoemulsion prepared by the above preparation method or the use of the chrysanthemin nanoemulsion in the preparation of a mitochondrial STAT3 protein degrader.

[0017] The present invention has the following beneficial effects:

[0018] The present invention prepares a nanoemulsion of chrysanthemin A by mixing chrysanthemin A with an oil phase and a complex surfactant. The preparation of chrysanthemin A into a nanoemulsion can improve the oral bioavailability of chrysanthemin A. Furthermore, experimental results show that chrysanthemin A can inhibit the expression of mitochondrial STAT3 protein, thereby inducing mitochondrial dysfunction. Furthermore, it can effectively inhibit tumor cell cloning. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1is the particle size and potential distribution diagram of the blank nanoemulsion;

[0020] Figure 2 This is the appearance of the nanoemulsion of chrysanthemum luteolin and the Tyndall phenomenon;

[0021] Figure 3 This is the structural type identification diagram of the chrysanthemum luteolin nanoemulsion;

[0022] Figure 4 This is the particle size and potential distribution diagram of the chrysanthemum luteolin nanoemulsion;

[0023] Figure 5 This is a diagram of a cell cloning experiment;

[0024] Figure 6 This is the dose-mortality curve of Sculponeatin A cardiovascular toxicity;

[0025] Figure 7 A typical diagram of cardiovascular toxicity in zebrafish after Sculponeatin A treatment (Note: the yellow dotted box represents the heart, and the red arrow points to bleeding);

[0026] Figure 8 Heart rate per minute of zebrafish 4 hours after Sculponeatin A treatment (compared with the solvent control group, ***p<0.001);

[0027] Figure 9 This is the dose-mortality curve of Sculponeatin A liver toxicity;

[0028] Figure 10 This is a typical diagram of zebrafish liver toxicity after Sculponeatin A treatment (Note: the yellow dotted box is the liver);

[0029] Figure 11 The zebrafish liver area and zebrafish liver fluorescence intensity after Sculponeatin A treatment;

[0030] Figure 12 The graph shows the blood drug concentration (NE). DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0033] The invention discloses a preparation method of a chrysanthemin nanoemulsion. The method comprises the following steps: mixing chrysanthemin with an oil phase and a composite surfactant and stirring until the chrysanthemin is dissolved; slowly adding water dropwise and stirring until the emulsion is clear, transparent and blue-light-emitting, thereby obtaining the chrysanthemin nanoemulsion.

[0034] The concentration of the chrysanthemin is 0.6 to 10 mg / mL. The mass ratio of the oil phase to the composite surfactant is 1:9 to 9:1. The oil phase is any one of medium-chain triglycerides, soybean oil, oleic acid glycol glyceride, peanut oil, and sesame oil. The composite surfactant is any two of polyoxyethylene 40 hydrogenated castor oil, poloxamer 188, polyglycerol oleate, Tween 80, and Span 80, and the mass ratio of any two of the composite surfactants is 1:1 to 10:1.

[0035] Furthermore, the composite surfactant is polyoxyethylene 40 hydrogenated castor oil and polyglycerol oleate, and the mass ratio of polyoxyethylene 40 hydrogenated castor oil and polyglycerol oleate is 4:1 to 1:1.

[0036] Furthermore, according to mass fraction, the oil phase is 25%, polyoxyethylene 40 hydrogenated castor oil is 12.5-20%, polyglycerol oleate is 5-12.5%, and water is 50%.

[0037] 2. The present invention discloses the use of the chrysanthemum luteolin nanoemulsion prepared by the above preparation method in the preparation of a mitochondrial STAT3 protein degrader.

[0038] The present invention will be further described below with reference to specific embodiments.

[0039] Example 1

[0040] 1. Screening of oil phase

[0041] Medium chain triglycerides (MCT), soybean oil, glycol oleate, peanut oil, sesame oil, etc. were used to conduct compatibility tests with three emulsifiers, namely Tween80, RH40, and ELP35.

[0042] At room temperature, the oil phase and the emulsifier are placed in a beaker at a weight ratio of 2:3, and are fully mixed under the action of a magnetic stirrer. Subsequently, 100 times the amount of deionized water is added, and stirring is continued to observe the degree of self-emulsification.

[0043] According to El-Badry et al., the self-emulsification grading system is divided into four levels: Grade A: a transparent emulsion with a bluish hue forms within 1 minute; Grade B: a translucent emulsion with a bluish hue forms within 2 minutes; Grade C: a white, opaque emulsion forms within 2 minutes; and Grade D: a grayish, opaque emulsion or one with oil droplets floating on the surface forms after more than 2 minutes. The solubility of surfactants in various oil phases was investigated to identify the most suitable oil phase. The results showed that medium-chain triglycerides (MCTs) were more effective as the oil phase.

[0044] 2. Screening of surfactants

[0045] Surfactants come in many varieties, including cationic, anionic, and nonionic types. Because ionic surfactants are highly toxic and unstable, a combination of polyoxyethylene 40 hydrogenated castor oil (RH40), poloxamer 188, polyglycerol oleate (CC497), Tween 80, and Span 80 was selected and mixed in a 1:1 mass ratio. The mixture was then added to the oil phase screened in the previous step at a ratio of 3:2:5. The formulation was evaluated based on the appearance and particle size of the nanoemulsions. Results showed that nanoemulsions formed when the composite surfactants consisted of RH40 and CC497, and RH40 and Span 80. RH40 and CC497 exhibited the best emulsification during nanoemulsion preparation. The addition ratio of RH40 and CC497 was further optimized.

[0046] 3. Optimization of the ratio of composite surfactants

[0047] Based on the oil phase and composite surfactants obtained above, MCT was selected as the oil phase, and composite surfactants RH40 and CC497 were used. The formulation was optimized based on the appearance, shape, and stability of the nanoemulsion. RH40 and CC497 were mixed and stirred at a mass ratio of 4:1 to 1:1 to form the composite surfactant. The composite surfactant was then mixed and stirred with the oil phase at a mass ratio of 1:9 to 9:1. Pure water was slowly added dropwise to the system with stirring. The water consumption at the phase transition point of the nanoemulsion was recorded to determine the optimal formulation. The optimal composite emulsifier composition was a 3:1 mass ratio of RH40 to CC497, and the nanoemulsion had the best appearance and flowability when the oil phase to composite surfactant ratio was 5:5. Therefore, an oil phase to surfactant ratio of 5:5 was selected as the optimal formulation.

[0048] According to the mass percentage, the final blank nanoemulsion formula was MCT 25%, RH4018.75%, CC4976.25%, and pure water 50%. The average particle size was 52.20 nm, the polydispersity coefficient was 0.069, and the Zeta potential was -23.91 mv. The particle size and potential distribution of the blank nanoemulsion are shown in Figure 1 .

[0049] 4. Preparation of Isocyanin-loaded Nanoemulsion

[0050] Accurately weigh 3 mg of chrysanthemum luteolin and place it in a 10 mL vial. Add 1.25 g of MCT and 1.25 g of complex surfactant, stir magnetically to dissolve the drug, slowly add 2.5 mL of pure water with a pipette, and stir magnetically for 20 minutes until the emulsion becomes clear and transparent with a blue light.

[0051] Example 2 Quality evaluation of blank nanoemulsion and chrysanthemin nanoemulsion

[0052] 1. Appearance inspection

[0053] The prepared chrysanthemum luteolin nanoemulsion was placed on the experimental table and its appearance was investigated. Figure 2 The appearance of the chrysanthemum luteolin nanoemulsion is clear and transparent with blue opalescence, good fluidity, and no unstable phenomena such as stratification, flocculation, and sedimentation. After laser irradiation, a bright pathway can be observed in the system, indicating that the system is a colloidal dispersion.

[0054] 2. Identification of the structural type of nanoemulsion

[0055] The type of nanoemulsion was determined by dyeing. Equal amounts of water-soluble dye methylene blue and fat-soluble dye Sudan red were added to the same nanoemulsion at the same time. The diffusion rates of the two were observed to determine the type of nanoemulsion. If the diffusion rate of methylene blue was fast, it was O / W type; otherwise, it was W / O type. Figure 3 Methylene blue quickly dissolved and diffused in the chrysanthemum luteolin nanoemulsion, while Sudan red did not dissolve and diffuse, indicating that the chrysanthemum luteolin nanoemulsion was an O / W nanoemulsion.

[0056] 3. Determination of Particle Size and Zeta Potential

[0057] At room temperature, the average particle size and Zeta potential of the nanoemulsion were measured using a Beckman nanoparticle size analyzer. Figure 4 As shown, the average particle size of the chrysanthemum luteolin nanoemulsion was 77.50 nm, the polydispersity coefficient was 0.168, and the Zeta potential was -13.15 mv (n=3) as determined by a laser particle size analyzer.

[0058] 4. Stability test of chrysanthemum luteolin nanoemulsion

[0059] The stability of the nanoemulsion of chrysanthemum luteolin was investigated by centrifugal acceleration test, dilution method and placement method.

[0060] An appropriate amount of chrysanthemin A nanoemulsion was placed in a centrifuge tube, placed in a centrifuge, and centrifuged at a speed of 9000 r / min for 30 minutes. The emulsion remained clear and transparent without phase separation or sedimentation.

[0061] Four portions of the chrysanthemum-derived luteolin nanoemulsion were diluted 50, 100, 500, and 1000 times with deionized water, respectively. No delamination or demulsification occurred, indicating that dilution did not affect the stability of the nanoemulsion, further demonstrating that the structure of the chrysanthemum-derived luteolin nanoemulsion was an O / W type.

[0062] In a sterile and alcohol-disinfected environment, the nanoemulsion of chrysanthemum luteolin was placed at room temperature. The solution was observed with the naked eye at 0, 10, and 30 days to see if there was flocculation or stratification. The results showed that after 30 days, it was still a clear and transparent liquid with a blue opalescence. No stratification, flocculation, or sedimentation of the nanoemulsion was observed, indicating that the system was very stable.

[0063] Example 3 Clone formation experiment

[0064] A cell cloning assay was performed to investigate the inhibitory effect of STA (0-50 nM) on the proliferation of human breast cancer cell lines MCF-7, MB-231, and BT-474. Tumor cells were seeded at a density of 500 cells per well in a 6-well plate. After cell attachment, 1 mL of sample solution was added to each well. After 24 hours of incubation, 2 mL of complete DMEM medium was added as a replacement. Culture was continued for 10 days, with fresh medium replaced every three days. Following the cloning assay, cells were washed two to three times with PBS, fixed with paraformaldehyde, and then washed with PBS. Crystal violet staining was applied, the stain was discarded, and the cells were washed two to three times with PBS. After drying, cells were photographed to visualize cloning patterns. The number of colonies and the rate of colony formation were calculated using ImageJ software.

[0065]

[0066] Crystal violet staining results and colony formation rate Figure 5 As shown, it shows that STA can effectively inhibit tumor cell clones.

[0067] Example 4 In vivo cardiovascular toxicity evaluation of STA in a zebrafish model

[0068] 2-dpf wild-type AB strain zebrafish were randomly selected and plated in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples (doses shown in Table 1) were administered intravenously. A normal control group and a solvent control group were also set up, with a volume of 3 mL per well. After 4 hours of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and placed under a dissecting microscope to measure their heart rate per minute. After 24 hours of treatment at 28°C, the heart, circulatory system, bleeding, and thrombosis of the zebrafish in each experimental group were observed under a dissecting microscope. The number of toxic events in each experimental group was counted, and typical toxic organs were photographed. Statistical analysis was used to evaluate the cardiovascular toxicity of the samples in zebrafish.

[0069] Under the experimental conditions, the MNLD and LD10 of Sculponeatin A for cardiovascular toxicity were simulated by OriginPro 8.0 software and were 45.0 ng / tail and 47.3 ng / tail, respectively. Figure 6 shown.

[0070] Table 1 Results of Sculponeatin A cardiovascular toxicity dose experiment (n=30)

[0071]

[0072] Under the experimental conditions, Sculponeatin A can induce cardiovascular toxicity, which is manifested as pericardial edema, abnormal heart rhythm (reduced heart rate), slow blood flow, circulation loss and bleeding. The lowest inducing dose is 45.0 ng / tail. See Table 2, Table 3, Figure 7 and Figure 8 shown.

[0073] Table 2 Results of Sculponeatin A cardiovascular toxicity evaluation experiment (n=10)

[0074]

[0075] Compared with the solvent control group, ***p<0.001.

[0076] Table 3 Statistics of the incidence of cardiovascular toxicity phenotypes of Sculponeatin A (n=30)

[0077]

[0078] Note: “-” indicates no obvious abnormality; the numbers in the table indicate: incidence.

[0079] Example 5 In vivo liver toxicity evaluation of STA in a zebrafish model

[0080] 3dpf transgenic zebrafish with green fluorescent livers were randomly selected and plated in 6-well plates, with 30 zebrafish treated in each well (experimental group). Samples were administered intravenously, and a normal control group was also set up. The volume per well was 3mL. After 48 hours of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. Liver area and liver fluorescence intensity were analyzed. Statistical analysis of the above indicators was used to evaluate sample hepatotoxicity. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. P < 0.05 indicated statistical significance.

[0081] Under the experimental conditions, the MNLD and LD10 of Sculponeatin A hepatotoxicity were simulated by OriginPro 8.0 software and were 6.82 ng / tail and 12.6 ng / tail, respectively. Figure 9 shown.

[0082] Table 4 Sculponeatin A hepatotoxicity dose experimental results (n=30)

[0083]

[0084] Under the experimental conditions, Sculponeatin A did not cause liver toxicity at doses of 6.82 ng / tail, 2.27 ng / tail, and 0.758 ng / tail. Figure 10 and Figure 11 shown.

[0085] Table 5 Results of Sculponeatin A liver toxicity evaluation experiment (n=10)

[0086]

[0087] Example 6 Pharmacokinetics Study of Isocarpin Nanoemulsion

[0088] 1. Pharmacokinetic Experimental Plan

[0089] Sprague-Dawley rats (220-250 g, female) were purchased from the Guangdong Medical Laboratory Animal Center and housed in an SPF-grade environment for one week. They were deprived of food but not water for 24 hours before the experiment. The rats were randomly divided into three groups: one group of four rats received an oral injection of a 6.0 mg / kg suspension of chrysanthemum luteolin (Citric acid), another group of four rats received an oral injection of a 6.0 mg / kg Citric acid nanoemulsion, and the remaining four rats received an intravenous injection of a 2.0 mg / kg Citric acid solution. Approximately 50 μL of blood was collected from the tail vein at designated time points, plasma was separated, and the blood samples were treated with organic solvent precipitation. The plasma concentration of Citric acid luteolin was determined by LC-MS / MS analysis. Pharmacokinetic parameters were obtained using the pharmacokinetic software das2.0 (Drug and Statistics Version 2.0).

[0090] 2. Processing of Plasma Samples

[0091] Plasma was thawed in a room temperature water bath, mixed, and 50 μL was precisely pipetted into a 2 ml centrifuge tube. Add 50 μL of a 1:1 methanol:2% formic acid solution and mix thoroughly. Add 150 μL of an acetonitrile solution containing arctiin (arcigenin concentration: 25 ng / mL) and vortex for 5 minutes. Centrifuge at 12,000 rpm and 4°C for 15 minutes. Filter the supernatant through a 0.22 μm filter membrane and sample the filtrate for analysis.

[0092] 3. Chromatographic and mass spectrometry conditions

[0093] The content of chrysanthemum luteolin in plasma samples was determined by LC-MS / MS using a Sepax HP-C18 (3*50 mm, 3 μm) column with a 1 μL injection volume and a 0.1% formic acid aqueous solution-methanol mobile phase for gradient elution. The elution gradient is shown in Table 6. An ESI ionization source was used in positive ion detection mode with multiple reaction monitoring (MRM). The capillary voltage was 0.5 kV, the ion source temperature was 150°C, the desolvation gas temperature was 500°C, and the desolvation gas flow rate was 1000 L / h.

[0094] The ion transition used for the quantitative analysis of chrysanthemum isatin was m / z 361.00→313.15, and the ion transition used for the analysis of the internal standard arctigenin was m / z 373.00→137.10.

[0095] Table 6 Elution gradient

[0096] Time (min) Flow rate (mL / min) A% (methanol) B% (0.1% formic acid water) 0.00～1.00 0.5 65 35 1.00～3.50 0.5 65～5 35～95 3.50～4.50 0.5 5 95 4.50～5.00 0.5 5～65 95～35 5.00～6.00 0.5 65 35

[0097] 4. Result calculation and analysis

[0098] After the plasma samples were treated according to method 2 and injected according to the conditions under item 3, no drug was detected in the rats that were orally administered with the 6.0 mg / kg suspension of chrysanthemum luteolin. The data obtained by orally administering the 6.0 mg / kg nanoemulsion of chrysanthemum luteolin and intravenously administering the 2.0 mg / kg solution of chrysanthemum luteolin were used to draw the blood drug concentration curve using Graphpad Prism 9.0 (see Figure 12 ), and the pharmacokinetic software Drug Analysis System 2.0 was used to process the plasma concentration-time curves, and the pharmacokinetic parameters were obtained by non-compartmental analysis (see Table 7). The final results showed that nanoemulsion can improve the oral bioavailability of chrysanthemin A.

[0099] Table 7 Calculation results of pharmacokinetic parameters of chrysanthemum luteolin (n=4)

[0100]

[0101] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a nanoemulsion of chrysanthemin A, characterized in that: Mixing chrysanthemin A with an oil phase and a composite surfactant and stirring until chrysanthemin A is dissolved, slowly adding water and stirring until the emulsion becomes clear and transparent with a blue light to obtain chrysanthemin A nanoemulsion; The mass ratio of the oil phase to the composite surfactant is 1:9 to 9:1, the oil phase is medium-chain triglycerides, the composite surfactant is polyoxyethylene 40 hydrogenated castor oil and polyglycerol oleate, and the mass ratio of the polyoxyethylene 40 hydrogenated castor oil and polyglycerol oleate is 4:1 to 1:

1.

2. The method for preparing a nanoemulsion of chrysanthemum luteolin according to claim 1, wherein: The concentration of the xanthophyllin A is 0.6-10 mg / mL.

3. The method for preparing a nanoemulsion of chrysanthemin A according to claim 1, wherein: Calculated by mass fraction, the oil phase is 25%, polyoxyethylene 40 hydrogenated castor oil is 12.5-20%, polyglycerol oleate is 5-12.5%, and water is 50%.

4. The chrysanthemum luteolin nanoemulsion prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the chrysanthemin nanoemulsion prepared by the preparation method according to any one of claims 1 to 3 or the chrysanthemin nanoemulsion according to claim 4 in the preparation of drugs for treating breast cancer.

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