CN117159445A - Preparation method and application of curcumin nanoemulsion temperature-sensitive hydrogel - Google Patents
Preparation method and application of curcumin nanoemulsion temperature-sensitive hydrogel Download PDFInfo
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- CN117159445A CN117159445A CN202310076503.6A CN202310076503A CN117159445A CN 117159445 A CN117159445 A CN 117159445A CN 202310076503 A CN202310076503 A CN 202310076503A CN 117159445 A CN117159445 A CN 117159445A
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- curcumin
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention relates to the technical field of nano materials, in particular to a preparation method and application of curcumin nanoemulsion temperature-sensitive hydrogel. According to the invention, nanoemulsion is added into poloxamer to prepare nanoemulsion gel, so that the curcumin-loaded nanoemulsion temperature-sensitive hydrogel is obtained. The curcumin nanoemulsion temperature-sensitive hydrogel prepared by the invention can enable curcumin to be uniformly smeared on an affected part, and can prolong the residence time of the curcumin so as to continuously exert the drug effect.
Description
Technical Field
The invention relates to the technical field of nano materials, in particular to a preparation method and application of curcumin nanoemulsion temperature-sensitive hydrogel.
Background
Chronic eczema is a clinically common specific immune and inflammatory skin disease, and is mainly represented by skin erythema, pimple, pruritus, erosion, exudation and the like. The disease has the characteristics of intractable disease, repeated attack and lasting and can seriously influence the life quality and physical and mental health of patients. The cause of chronic eczema is various, and researches show that skin barrier defects, immune cell infiltration, pathogenic microorganism colonization, gene polymorphism and the like are all involved in the pathogenesis. However, the exact pathogenesis of chronic eczema is not completely defined, so that a eradication method is lacking, and long-term use of external medicines for control is a more practical solution. The traditional main medicine for clinically treating chronic eczema is glucocorticoid, and although the disease condition can be effectively controlled, adverse reactions such as easy infection of skin injury wound surface, skin atrophy, easy recurrence or aggravation of disease condition after stopping medicine and the like exist in long-term use, so that the compliance of patients is poor. Therefore, it is particularly necessary to find a safe and effective chronic eczema therapeutic drug which can be used for a long time.
Chinese traditional medicine resources in China are extremely rich, and are important sources for developing novel medicines. In recent years, the traditional Chinese medicine has a certain curative effect on chronic eczema, and has the advantages of good curative effect and small adverse reaction compared with western medicines. Curcumin is a chemical component containing phenolic group and quinone group extracted from rhizome of plants of Zingiberaceae and Araceae. At present, research at home and abroad finds that curcumin has various pharmacological activities, such as anti-inflammatory, anti-oxidative stress, anti-tumor, anti-fibrosis and the like, and can be used for treating tumors, autoimmune diseases, diabetic complications, cardiovascular diseases and the like. At present, the application of curcumin in the treatment of inflammatory immune diseases is an emerging hot spot and shows better effect in the treatment of various skin diseases, which indicates that curcumin has good prospect in the treatment of chronic eczema. However, curcumin belongs to class IV drugs of biopharmaceutical classification system, and has a solubility in water of only 11. Mu.g.l -1 And the skin permeability is poor, so that the clinical application is severely restricted. The nanoemulsion has the advantages of increasing the solubility of the medicament, improving the transdermal rate of the medicament, prolonging the acting time of the medicament and the like, provides an effective way for solving the percutaneous absorption of insoluble medicaments, and is increasingly acceptedThe researchers attach importance to each other. However, nanoemulsion has strong fluidity and has the problems of weak adhesion, poor skin spreadability, short retention time and the like when used as a transdermal drug delivery carrier. The nanoemulsion gel can effectively solve the problems as a novel transdermal drug delivery system. The nanoemulsion gel is a transparent and stable reticular colloid formed by adding water-soluble polymer materials such as carbomer, poloxamer and the like into nanoemulsion, and can lead the medicine to be uniformly smeared on an affected part, prolong the residence time of the medicine and further continuously exert the medicine effect. The curcumin nanoemulsion is prepared by the research, a single factor test and a Box-Behnken response surface method are adopted to optimize the prescription and the process, then the curcumin nanoemulsion with optimal conditions is added into poloxamer 188/407 gel matrix to construct transdermal thermosensitive hydrogel, and finally, the commercial compound dexamethasone acetate cream is used as a control, and pharmacodynamics evaluation is carried out by using the established mouse chronic eczema model, so that a new idea is provided for the treatment of chronic eczema.
Disclosure of Invention
The invention aims to provide a preparation method and application of curcumin nanoemulsion temperature-sensitive hydrogel, so as to provide a new idea for treating chronic eczema.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of curcumin nanoemulsion temperature-sensitive hydrogel, which comprises the following steps:
(1) Mixing the raw materials, ethanol and oil, and performing ultrasonic treatment to obtain an oil phase;
(2) Sequentially adding tween-80 and water into the oil phase obtained in the step (1), mixing by vortex, and then placing into an ultrasonic cell grinder for ultrasonic treatment to obtain curcumin nanoemulsion;
(3) Mixing the mixed solution of poloxamer 188 and poloxamer 407 with the curcumin nanoemulsion obtained in the step (2) to obtain a mixture;
(4) And (3) standing the mixture obtained in the step (3) to obtain the curcumin nanoemulsion temperature-sensitive hydrogel.
Preferably, the crude drug in the step (1) is curcumin; the ethanol is absolute ethanol; the oil is soybean oil.
Preferably, the mass-volume ratio of the bulk drug, the absolute ethyl alcohol and the soybean oil in the step (1) is 2.1-2.7 mg:0.1 to 0.3ml:0.11 to 0.17ml.
Preferably, the specific ultrasonic treatment method in the step (1) comprises the following steps: the temperature is 25-35 ℃, the power is 190-210 w, and the time is 10-20 min.
Preferably, the volume fraction of tween-80 in the step (2) is 15-25%; the water is ultrapure water.
Preferably, the volume ratio of the ultrapure water to the tween-80 in the step (2) is 0.1-0.7 ml:1.23 to 1.29ml.
Preferably, the mixing time in the step (2) is 10-20 min; the working parameters of the ultrasonic cell grinder are as follows: 290-310W, working for 1-3 s and intermittence for 1-3 s; the ultrasonic time is 1-7 min.
Preferably, the mass volume ratio of the poloxamer 188, the poloxamer 407 and the water in the step (3) is 0.10-0.70 g:1.20 to 1.26g: 6-12 ml; the curcumin nanoemulsion in the step (4) is used in an amount of 0.5-1.5 ml; the standing temperature is 1-7 ℃; the standing time is 21-27 h.
Preferably, the prepared curcumin nanoemulsion temperature-sensitive hydrogel.
Preferably, the curcumin nanoemulsion temperature-sensitive hydrogel is applied to preparation of chronic eczema pharmaceutical preparations.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, curcumin is firstly prepared into nanoemulsion to solve the problems of low solubility and poor skin permeability, and a good foundation is laid for the preparation of curcumin nanoemulsion temperature-sensitive hydrogel and the full play of drug effect.
2. The prepared hydrogel gel has good temperature sensitivity, which shows that the hydrogel gel can be quickly formed after being smeared on the surface of skin, and is beneficial to enhancing the adhesion to the skin so as to fully exert the drug effect; and the in vitro transdermal performance research result shows that the curcumin nanoemulsion temperature-sensitive hydrogel can promote the percutaneous penetration of curcumin, which is the result of the double functions of nanoemulsion and hydrogel, and the property is beneficial to the treatment of chronic eczema.
3. Curcumin is used as a natural component extracted from plants, has fewer toxic and side effects after long-term use, and can improve compliance of eczema patients.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of a specificity test: a: curcumin methanol solution, B: curcumin nanoemulsion solution, C: blank nanoemulsion solution;
fig. 2 is a single factor test result (n=3): a: dosage, B: emulsifier concentration, C: the amount of the auxiliary emulsifier, D: ultrasonic power, E: ultrasonic time;
fig. 3 is a 3D response surface plot of dependent variable Y: a: independent variable X 1 And X 2 Is, B: independent variable X 1 And X 3 Is, C: independent variable X 2 And X 3 Is a function of (1);
fig. 4 is an appearance of curcumin nanoemulsion temperature-sensitive gel at 4 ℃ and 37 ℃): a:4 ℃, B:37 ℃;
fig. 5 is SEM photograph (x 5000) of curcumin nanoemulsion temperature sensitive hydrogel;
fig. 6 is the transdermal properties of curcumin in curcumin solution and curcumin nanoemulsion temperature sensitive hydrogel (n=6);
fig. 7 is a photograph of the appearance of mouse ear tissue: a: normal group, B: a model group;
fig. 8 is a photograph of a pathological section of mouse ear tissue (x 400): a and B: normal group, C and D: a model group;
fig. 9 is the effect of different drugs on the appearance of mouse ear tissue: a: normal group, B: model group, C: positive drug group, D: curcumin solution group, E: example 3 group;
fig. 10 is the effect of different drugs on the difference in ear mass and swelling in mice (n=3): a: poor ear quality, B: ear swelling degree.
Detailed Description
The invention provides a preparation method of curcumin nanoemulsion temperature-sensitive hydrogel, which comprises the following steps:
(1) Mixing the raw materials, ethanol and oil, and performing ultrasonic treatment to obtain an oil phase;
(2) Sequentially adding tween-80 and water into the oil phase obtained in the step (1), mixing by vortex, and then placing into an ultrasonic cell grinder for ultrasonic treatment to obtain curcumin nanoemulsion;
(3) Mixing the mixed solution of poloxamer 188 and poloxamer 407 with the curcumin nanoemulsion obtained in the step (2) to obtain a mixture;
(4) And (3) standing the mixture obtained in the step (3) to obtain the curcumin nanoemulsion temperature-sensitive hydrogel.
In the invention, the bulk drug in the step (1) is curcumin; the ethanol is preferably absolute ethanol; the oil is preferably soybean oil.
In the invention, the mass-volume ratio of the bulk drug, the absolute ethyl alcohol and the soybean oil in the step (1) is 2.1-2.7 mg:0.1 to 0.3ml: 0.11-0.17 ml; preferably 2.2 to 2.6mg:0.15 to 0.25ml: 0.12-0.16 ml; further preferably 2.3 to 2.5mg:0.18 to 0.22ml: 0.13-0.15 ml; more preferably 2.4mg:0.2ml:0.14ml.
In the invention, the specific ultrasonic treatment method in the step (1) comprises the following steps: the temperature is 25-35 ℃, the power is 190-210 w, and the time is 10-20 min; preferably, the temperature is 27-33 ℃, the power is 193-207 w, and the time is 12-18 min; further preferably, the temperature is 29-31 ℃, the power is 197-203 w, and the time is 14-16 min; more preferably 30℃and 200w for 15min.
In the invention, the volume fraction of the tween-80 in the step (2) is 15-25%; preferably 17 to 23%; further preferably 19 to 21%; more preferably 20%.
In the present invention, the water of step (2) is preferably ultrapure water.
In the invention, the volume ratio of the ultrapure water and the tween-80 in the step (2) is 0.1-0.7 ml:1.23 to 1.29ml; preferably 0.2 to 0.6ml: 1.24-1.28 ml; more preferably 0.3 to 0.5ml:1.25 to 1.27ml; more preferably 0.4ml:1.26ml.
In the invention, the mixing time in the step (2) is 10-20 min; preferably 11 to 19 minutes; further preferably 13 to 17 minutes; more preferably 15min.
In the invention, the working parameters of the ultrasonic cell grinder in the step (2) are as follows: 290-310W, working for 1-3 s and intermittence for 1-3 s; preferably 293-307W, work for 1.5-2.5 s, and interval for 1.5-2.5 s; further preferably 297 to 303W, for 1.7 to 2.3 seconds, and intermittently for 1.7 to 2.3 seconds; more preferably 300W, 2s on duty, 2s off.
In the invention, the ultrasonic time in the step (2) is 1-7 min; preferably 2 to 6 minutes; further preferably 3 to 5 minutes; more preferably 4min.
In the invention, the mass volume ratio of the poloxamer 188, the poloxamer 407 and the water in the step (3) is 0.10-0.70 g:1.20 to 1.26g: 6-12 ml; preferably 0.20 to 0.60g:1.21 to 1.25g: 7-11 ml; more preferably 0.30 to 0.50g:1.22 to 1.24g: 8-10 ml; more preferably 0.40g:1.23g:9ml.
In the invention, the curcumin nanoemulsion in the step (4) is used in an amount of 0.5-1.5 ml; preferably 0.7 to 1.3ml; further preferably 0.9 to 1.1ml; more preferably 1.0ml.
In the invention, the standing temperature in the step (4) is 1-7 ℃; preferably 2 to 6 ℃; further preferably 3 to 5 ℃; more preferably at 4 ℃.
In the invention, the standing time in the step (4) is 21-27 h; preferably 22 to 26 hours; further preferably 23 to 25 hours; more preferably 24h.
In the invention, the prepared curcumin nanoemulsion temperature-sensitive hydrogel.
The invention discloses application of curcumin nanoemulsion temperature-sensitive hydrogel in preparation of a chronic eczema pharmaceutical preparation.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Instrument and reagent
LC-20AT type high performance liquid chromatograph (Shimadzu corporation); JY 92-II ultrasonic cell grinder (Ningbo Xinzhi biotechnology Co., ltd.); sorvall ST8R high-speed refrigerated centrifuge [ Simer Feichi technologies (China) Co., ltd ]; nanoZS90 particle size potentiometric analyzer (Malvern, uk); JEM-2100PLUS type transmission electron microscope (Japanese electronics Co., ltd.); sigma type 300 scanning electron microscope (ZEISS, germany); SHA-B type water bath constant temperature oscillator (Shanghai libosci instruments science and technology limited); PHS-25 pH meter (Shanghai Instrument electric science instruments Co., ltd.); KX-V/HDP horizontal transdermal diffusion instrument (large Lian Ke Xiang technology development Co., ltd.); SC600 type biological microscope (Optika, italy Co.).
Curcumin reference substance (content is more than or equal to 98%, lot number B20614) and curcumin bulk drug (content is more than or equal to 95%, lot number S19245) (Shanghai Yuan leaf Biotechnology Co., ltd.); poloxamer 188 (lot number M11GS 148056) and poloxamer 407 (lot number J24GS 1155964) (Shanghai source leaf biotechnology limited); soybean oil (Shandong Rui crude drug adjuvant Co., ltd., lot DD 20210710); polysorbate 80 (Tween-80, national pharmaceutical chemicals limited); methylene blue (lot RH 339484) and sudan red iii (lot RH 276492) (Shanghai Yi En technologies development limited); 2, 4-dinitrochlorobenzene [ DNCB, western Asia chemical technology (Shandong) Co., ltd., content of not less than 99%, lot number 20220111]; compound dexamethasone acetate cream (Huarun Sanjiu medicine Co., ltd., specification 20g:15mg, lot number 2104019S); absolute ethanol (lot 20220606, far eastern fine chemical company, smoke counter); methanol (content of chemical reagent of national medicine group, co., ltd., batch No. 20221113), acetonitrile (content of Beijing carboline scientific, co., ltd., batch No. L6C0V 46), and the like.
BALB/c mice (male, 18-22 g) and SD rats (male, 200-240 g) were supplied by Jinan Pengyue laboratory animal breeding Co., ltd, and the laboratory animal production license number was SCXK 2019-0003.
Example 1
A preparation method of curcumin nanoemulsion temperature-sensitive hydrogel comprises the following steps:
(1) Mixing curcumin, absolute ethyl alcohol and soybean oil, and performing ultrasonic treatment to obtain an oil phase; the mass volume ratio of curcumin, absolute ethyl alcohol and soybean oil is 2.1mg:0.1ml:0.11ml; the specific method of ultrasonic treatment comprises the following steps: the temperature is 25 ℃, the power is 190w, and the time is 10min;
(2) Sequentially adding 0.1ml of tween-80 with volume fraction of 15% and 1.23ml of ultrapure water into the oil phase obtained in the step (1), vortex mixing for 10min, and then placing into an ultrasonic cell grinder (290W, work 1s and intermittent 1 s) for ultrasonic treatment for 1min to obtain curcumin nanoemulsion;
(3) Mixing the mixed solution of poloxamer 188 and poloxamer 407 with the curcumin nanoemulsion obtained in the step (2) to obtain a mixture; wherein the mixed solution of poloxamer 188 and poloxamer 407 is prepared by mixing poloxamer 188, poloxamer 407 and ultrapure water, and the mass volume ratio is 0.10g:1.20g:6ml; the dosage of the curcumin nanoemulsion is 0.5ml;
(4) And (3) standing the mixture obtained in the step (3) in a refrigerator at the temperature of 1 ℃ for 21 hours to obtain the curcumin nanoemulsion temperature-sensitive hydrogel.
Example 2
Preparation method of curcumin nanoemulsion temperature-sensitive hydrogel
(1) Mixing curcumin, absolute ethyl alcohol and soybean oil, and performing ultrasonic treatment to obtain an oil phase; the mass volume ratio of curcumin, absolute ethyl alcohol and soybean oil is 2.7mg:0.3ml:0.17ml; the specific method of ultrasonic treatment comprises the following steps: the temperature is 35 ℃, the power is 210w, and the time is 20min;
(2) Sequentially adding 0.7ml of Tween-80 with volume fraction of 25% and 1.29ml of ultrapure water into the oil phase obtained in the step (1), vortex mixing for 10min, and then placing into an ultrasonic cell grinder (310W, working for 3s and intermittent for 3 s) for ultrasonic treatment for 7min to obtain curcumin nanoemulsion;
(3) Mixing the mixed solution of poloxamer 188 and poloxamer 407 with the curcumin nanoemulsion obtained in the step (2) to obtain a mixture; wherein the mixed solution of poloxamer 188 and poloxamer 407 is prepared by mixing poloxamer 188, poloxamer 407 and ultrapure water, and the mass volume ratio is 0.70g:1.26g:12ml; the dosage of the curcumin nanoemulsion is 1.5ml;
(4) And (3) standing the mixture obtained in the step (3) in a refrigerator at 7 ℃ for 27 hours to obtain the curcumin nanoemulsion temperature-sensitive hydrogel.
Example 3
Preparation method of curcumin nanoemulsion temperature-sensitive hydrogel
(1) Mixing curcumin, absolute ethyl alcohol and soybean oil, and performing ultrasonic treatment to obtain an oil phase; the mass volume ratio of curcumin, absolute ethyl alcohol and soybean oil is 2.4mg:0.2ml:0.14ml; the specific method of ultrasonic treatment comprises the following steps: the temperature is 30 ℃, the power is 200w, and the time is 15min;
(2) Sequentially adding 0.4ml of tween-80 with the volume fraction of 20% and 1.26ml of ultrapure water into the oil phase obtained in the step (1), carrying out vortex mixing for 10min, and then placing into an ultrasonic cell grinder (300W, working 2s and intermittent 2 s) for ultrasonic treatment for 4min to obtain curcumin nanoemulsion;
(3) Mixing the mixed solution of poloxamer 188 and poloxamer 407 with the curcumin nanoemulsion obtained in the step (2) to obtain a mixture; wherein the mixed solution of poloxamer 188 and poloxamer 407 is prepared by mixing poloxamer 188, poloxamer 407 and ultrapure water, and the mass volume ratio is 0.40g:1.23g:9ml; the dosage of the curcumin nanoemulsion is 1.0ml;
(4) And (3) standing the mixture obtained in the step (3) in a refrigerator at the temperature of 4 ℃ for 24 hours to obtain the curcumin nanoemulsion temperature-sensitive hydrogel.
Experimental example 1
Establishment of content analysis method
Chromatographic conditions
Chromatographic column nucifferac 18 Columns (4.6 mm. Times.250 mm,5 μm); mobile phase acetonitrile: 0.3% glacial acetic acid solution (60:40); the detection wavelength is 426nm; column temperature is 30 ℃; the flow rate is 1ml/min; the sample was introduced in an amount of 10. Mu.l.
Specificity test
And precisely transferring 20 μl of curcumin methanol solution, curcumin nanoemulsion solution and blank nanoemulsion solution, and performing sample injection measurement according to chromatographic conditions. The result is shown in figure 1, the curcumin appears peak around 5.6min, and the auxiliary materials have no interference to the measurement of the curcumin, so that the method has good specificity.
Linear relationship investigation
Precisely weighing 10mg of curcumin reference substance, placing in a 100ml measuring flask, adding 60ml of methanol, ultrasonically dissolving, and fixing volume to obtain curcumin stock solution. 0.125, 0.25, 0.5, 1.0, 1.5, 3.0ml of curcumin stock solution is precisely removed, and diluted with methanol to obtain curcumin standard solutions with concentrations of 0.5, 1, 2,4, 6 and 12 mug/ml. The peak area was recorded as measured by chromatographic conditions. And (3) drawing a standard curve by taking the concentration (c) of curcumin as an abscissa and the measured peak area (A) as an ordinate. The regression equation obtained is: a=119748c+6514.5 (r=0.9999), indicating that curcumin has a good linear relationship in 0.5 to 12 μg/ml.
Precision investigation
The curcumin standard solution with low concentration, medium concentration and high concentration is selected and continuously measured for 6 times in 1d respectively, and the RSD in the day and the daytime is examined by continuously measuring for 6 d. The results showed that the daily RSD (n=6) was 1.48%, 1.49% and 0.61% for the low, medium and high concentration samples, respectively, and the daily RSD (n=6) was 0.95%, 1.14% and 0.68% respectively, indicating that the instrument precision was good.
Stability investigation
Precisely transferring 1.0ml of curcumin stock solution prepared in linear relation investigation, placing the curcumin stock solution into a 25ml measuring flask to prepare 4 mug/ml curcumin standard solution (3 parts are prepared in parallel), sampling at 0, 1, 2,4, 6, 8 and 24 hours, measuring by sample injection according to chromatographic conditions, recording peak area, and calculating curcumin concentration according to the peak area. The results showed RSD (n=3) of 0.24%, 0.28%, 0.24%, respectively, indicating good stability of the test solution over 24h.
Recovery rate investigation
Precisely transferring 0.25ml, 1.0ml and 1.5ml of curcumin stock solution prepared in linear relation investigation, placing the stock solution into a 25ml measuring flask, adding 1ml of blank nanoemulsion to prepare curcumin sample solutions (3 parts of each concentration is prepared in parallel) of 1, 4 and 6 mug/ml respectively, measuring by sample injection according to chromatographic conditions, recording peak areas, and calculating curcumin concentration according to the peak areas. The results showed that the recovery rates of the low, medium and high concentration samples (n=3) were (99.46±1.21)%, (99.89±0.99)%, and (100.13 ±1.05)%, respectively.
Experimental example 2
Determination of nanoemulsion encapsulation efficiency
Examination of nanoemulsion permeation Capacity
Precisely transferring 1ml curcumin nanoemulsion, adding into an ultrafiltration centrifuge tube (with a molecular weight cut-off of 10 KD), centrifuging for 10min at 3000r/min, collecting filtrate, and detecting particle size by a particle size potential analyzer. The results show that the presence of curcumin nanoemulsion is not detected in the filtrate, indicating that the conditions can achieve the effect of completely entrapping curcumin nanoemulsion.
Ultrafiltration recovery test
0.25, 1.0 and 1.5ml of curcumin stock solution in experimental example 1 are precisely removed, placed in a 25ml measuring flask, and 1ml of blank nanoemulsion is added to prepare curcumin sample solutions of 1, 4 and 6 mug/ml respectively (3 parts of curcumin sample solutions are prepared in parallel for each concentration). And precisely transferring a proper amount of low, medium and high concentration sample solutions into an ultrafiltration centrifuge tube (with the molecular weight cut-off of 10 KD), centrifuging for 10min at 3000r/min, collecting filtrate, and carrying out sample injection measurement according to the chromatographic condition of experimental example 1 to calculate the ultrafiltration recovery rates (n=3) of the high, medium and low concentration sample solutions to be (98.99+/-0.18)%, (97.79+/-0.21)% and (99.64+/-0.19)%, respectively.
Determination of nanoemulsion encapsulation efficiency
Precisely transferring curcumin nanoemulsion 1ml, adding into ultrafiltration centrifuge tube (molecular weight cut-off of 10 KD), centrifuging at 3000r/min for 10min, collecting filtrate, diluting, and determining by sample injection under chromatographic condition of experimental example 1 to obtain the mass W of free drug in curcumin nanoemulsion f . Precisely transferring 1ml of curcumin nanoemulsion, adding into a centrifuge tube, performing ultrasonic demulsification with methanol, properly diluting, and performing sample injection measurement according to the chromatographic condition of experimental example 1 to calculate the mass W of the total medicine in the curcumin nanoemulsion t . Finally according to W f And W is equal to t Encapsulation efficiency was calculated.
Single factor test
In the single factor test, the investigation factors were changed in the following order: the dosage is 2.0 mg, 2.4mg, 2.8 mg and 3.2mg respectively; the concentration of the emulsifier Tween-80 is 10%, 15%, 20% and 25% respectively; the consumption of the auxiliary emulsifier absolute ethyl alcohol is 0.1ml, 0.2ml, 0.3ml and 0.4ml respectively; the ultrasonic power is 100W, 200W, 300W and 400W respectively; the ultrasonic time is respectively 1, 2, 3 and 4min. During the test, the curcumin nanoemulsion was prepared as in example 3 and its encapsulation efficiency was measured as in experimental example 1, except that the factors were not changed. The results are shown in FIG. 2, which shows that the encapsulation efficiency showed a tendency to increase with the increase in the amount of the administered and the amount of the co-emulsifier, and was not changed after reaching the peak at the administration amount of 2.4mg and the amount of the co-emulsifier of 0.2ml, respectively; the encapsulation efficiency showed a tendency to rise and then fall with increasing emulsifier concentration, ultrasonic power and ultrasonic time, and peaked at 20% emulsifier concentration, 300W ultrasonic power and 2min ultrasonic time, respectively.
Experimental example 3
Box-Behnken response surface method optimized prescription
Optimizing coding and level
Based on the single factor test results, ultrasonic power (X 1 ) Ultrasonic time (X) 2 ) And emulsifier concentration (X) 3 ) And taking the encapsulation efficiency (Y) as an evaluation index as a main influencing factor, and optimizing the curcumin nanoemulsion prescription and process by adopting a Box-Behnken response surface method. The factor levels are shown in Table 1, and the test arrangements and results are shown in Table 2.
TABLE 1Box-Behnken design factors and levels
TABLE 2 independent and dependent variables
Model fitting
The results were subjected to analysis of variance and fitting of multiple quadratic regression equations using Design experet 8.0 software. The regression equation model obtained by fitting is as follows:
Y＝82.76+2.22X 1 -0.66X 2 +12.31X 3 -2.82X 1 X 2 -3.48X 1 X 3 +5.81X 2 X 3 +1.32X 1 2 -0.078X 2 2 -6.05X 3 2 。
the model P is less than 0.0001, which shows that the statistical difference is extremely remarkable; the correlation coefficient r= 0.9876 indicates that the model predictive value has good correlation with the measured value. The mismatch term p= 0.3932 > 0.05, without significance, indicates that the equation fit and reliability are good.
Analysis of variance and significance test
Analysis of experimental data using ANOVA, as known from the significance test of each regression coefficient, X in the model equation 1 (p=0.0079) has a significant effect on the encapsulation efficiency, X 3 (P<0.0001 Has an extremely significant effect on the encapsulation efficiency, the others are not.
Response surface optimization and prediction
The results of the experiments were plotted as a contour plot and a three-dimensional response surface plot using Design expert8.0 software, and the results are shown in fig. 3, wherein the encapsulation efficiency increases with increasing ultrasonic time and decreases with increasing emulsifier concentration when the fixed ultrasonic power is 200W. When the fixed working time is 2min, the encapsulation efficiency increases with the ultrasonic power and the emulsifier concentration. When the concentration of the fixed emulsifier is 15%, the encapsulation efficiency increases with the increase of ultrasonic power and decreases with the increase of working time.
Fitting the obtained optimized prescription X 1 ＝208W，X 2 ＝3min，X 3 =19%, at which time y=95.78%. 3 batches of curcumin nanoemulsions were prepared according to the above-described optimization results, and the encapsulation efficiency was measured to be (95.69.+ -. 0.04)%, and the deviation from the expected result was 0.09%. The curcumin nanoemulsion prepared under the optimal conditions was used in the following experiments.
Experimental example 4
Quality evaluation of nanoemulsion temperature-sensitive hydrogel
Appearance characteristics
The appearance of the curcumin nanoemulsion temperature-sensitive hydrogel at 4 ℃ and 37 ℃ is shown in figure 4, and the curcumin nanoemulsion temperature-sensitive hydrogel is a pale yellow uniform solution at 4 ℃ and has good fluidity; is a pale yellow transparent semi-solid at 37 ℃ and has no fluidity, which indicates that the material has good temperature sensitivity.
Determination of gel temperature and time
Precisely transferring 1ml of curcumin nanoemulsion temperature-sensitive hydrogel into a 5ml penicillin bottle, placing a perforated rubber plug, and then inserting a thermometer (precision 0.1 ℃) to ensure that the sensing end of the curcumin nanoemulsion temperature-sensitive hydrogel is completely immersed into gel solution. The gel-containing portion of the vial was immersed in water, the water temperature was gradually increased at a rate of 0.5 c/min from room temperature, the test tube was taken out at 1 minute intervals to observe the gel state until the vial was inverted for 15s without flowing the gel, the temperature was recorded as the gelation temperature (6 parts measured in parallel), and the gelation temperature was (34.3±0.3) °c.
Precisely transferring 1ml of curcumin nanoemulsion temperature-sensitive hydrogel into a 5ml penicillin bottle, incubating in a constant-temperature water bath kettle at 37 ℃ (ensuring that the gel-containing part of the penicillin bottle is completely immersed in water), taking out a test tube at intervals of 25s to observe the gel state until the penicillin bottle is inverted for 15s and the gel does not flow any more, and recording the time to obtain the gel time (6 parts measured in parallel), wherein the gel time is (54.2+/-10.2) s.
Determination of the pH value
Precisely removing 5ml of curcumin nanoemulsion temperature-sensitive hydrogel, and measuring the pH value (6 parts in parallel) by using a pH meter, wherein the pH value is 6.65+/-0.10, and meets the requirement of transdermal administration preparation for pH 5-7.
Scanning electron microscope (scanning electron microscope, SEM) observations
And (3) placing 5ml of curcumin nanoemulsion temperature-sensitive hydrogel into a 15ml centrifuge tube, incubating for 30min at 37 ℃ under water bath condition until the curcumin nanoemulsion temperature-sensitive hydrogel is gel, pre-freezing for 12h at-20 ℃, and freeze-drying for 24h at-45 ℃ in a freeze dryer to obtain curcumin nanoemulsion temperature-sensitive hydrogel powder. And (3) carrying out metal spraying treatment on 10mg of powder, accelerating the voltage to 10.0kV, and observing the microstructure morphology of the curcumin nanoemulsion temperature-sensitive hydrogel after gelation by adopting SEM. As a result, as shown in fig. 5, the gelled sample showed a typical three-dimensional network structure, with irregular cells interconnected and having a certain cell wall thickness.
Experimental example 5
Transdermal performance study of nanoemulsion temperature-sensitive hydrogel
Preparation of ex vivo skin
SD male rats were anesthetized, then were shaved, the abdominal skin was rapidly separated and subcutaneous fat and mucous tissue were removed, and the rats were sealed in an aluminum foil bag after washing with physiological saline, and were stored frozen at-20 ℃.
In vitro transdermal experiments
The improved Franz diffusion cell is adopted, and the cell opening area is 1.767cm 2 The volume of the receiving tank is 10ml, the temperature of the circulating water bath is controlled to be (37.0+/-0.5) DEG C, and the rotating speed is set to be 200r/min. The isolated skin was placed between a diffusion cell and a receiving cell (the stratum corneum facing the diffusion cell), and 10ml of physiological saline containing 30% ethanol was injected into the receiving cell (ultrasound deaeration before use). Precisely transferring curcumin solution and 1ml of curcumin nanoemulsion temperature-sensitive hydrogel prepared in example 3, uniformly smearing on the skin surface (measuring 6 parts in parallel), precisely transferring 1ml of receiving solution (timely supplementing Wen Dengliang blank receiving solution) respectively for 2,4, 6, 8, 10 and 24 hours, centrifuging (7912 r/min) for 10min, taking supernatant, measuring according to the condition of experimental example 1, and calculating curcumin concentration and accumulated penetration (Q) n ). By Q n Plotting the time t and simultaneously carrying out linear regression on the straight line part in the curve, wherein the slope of the obtained straight line is the steady-state transdermal rate (J) s ). The results are shown in FIG. 6 and Table 3, and the cumulative permeation quantity Q of the curcumin nanoemulsion temperature-sensitive hydrogel for 24h 24h Is 9.927 +/-0.696 mu g cm -2 Steady state transdermal rate J s 0.707 mug.cm -2 ·h -1 The temperature-sensitive hydrogel is 2.45 times and 2.60 times of curcumin solution respectively, which shows that the prepared 1 nanometer emulsion temperature-sensitive hydrogel can promote the percutaneous permeation of curcumin.
Table 3 permeation parameters of curcumin solution and curcumin nanoemulsion temperature sensitive hydrogel (x±s, n=6)
Experimental example 6
Pharmacodynamics evaluation of nanoemulsion temperature-sensitive hydrogel
Establishment of chronic eczema model of mice
Precisely weighing 0.7g of 2, 4-Dinitrochlorobenzene (DNCB) powder, placing into a centrifuge tube, adding 2ml of olive oil and 8ml of acetone, and uniformly mixing to obtain a 7% DNCB solution. A1% DNCB solution was prepared in the same manner.
Except for the normal group, the mice in each group were subjected to abdominal dehairing treatment (dehairing area 2X 2 cm) 1 day before the experiment 2 ). 100 μl of 7% DNCB solution was precisely removed on day 1 of the experiment and applied to the dehairing area and sensitized by the same method on days 2 and 3. On day 7, except for the normal group, 100 μl of 1% DNCB solution was precisely removed and applied to the inner side of the right ear of each group of mice, 1 time every 3 days, and 3 times of continuous reinforcement. The appearance of the ear tissues of the normal mice and the model mice is shown in fig. 7, and the model mice are obviously red, swelling, curling and dander, and the normal mice do not have the phenomenon.
Normal group and model group mice were randomly selected, right ear was cut off along the outline of ear after cervical removal and sacrifice, and after washing with physiological saline, fixed with 4% paraformaldehyde solution, dehydrated step by step, paraffin-embedded, sectioned, hematoxylin-eosin (HE) stained, and ear histopathological changes were observed under 400 x-ray microscope. As a result, as shown in fig. 8, the epidermis was thickened and the stratum corneum was highly keratinized, intradermal edema and a large number of neutrophil infiltrates occurred in the model group compared to the normal group.
Group administration
50 BALB/c male mice were randomly divided into 5 groups (10 each) which were respectively a normal group, a model group, a positive drug group (compound dexamethasone acetate cream), a curcumin solution group and an example 3 group. Normal mice are normally raised, normal raising is carried out after the modeling of the mice in the model group is finished, 0.1g of compound dexamethasone acetate emulsifiable paste and 0.1ml of curcumin solution and curcumin nanoemulsion temperature-sensitive hydrogel are respectively weighed precisely after the modeling of the mice in the positive drug group, the curcumin solution group and the mice in the example 3 group is finished, and the mice are uniformly smeared on the right ear for 1 time per day and continuously dosed for 10 days.
Appearance evaluation
And during the drug treatment period after the establishment of the chronic eczema model, the red swelling and crusting degree of the right ear of the mouse are observed regularly, and the curative effect is evaluated. When the drug treatment is terminated, the appearance of the right ear of each group of mice is shown in fig. 9, and compared with the normal group, the right ear of the model group of mice shows curl, the skin is obviously thickened and has dandruff falling off; curcumin solution group mice had reduced swelling of the right ear but had some dander present; the positive drug group and the example 3 group mice had a reduced degree of swelling of the right ear and no dander was seen.
Poor ear quality and swelling degree
When the drug treatment is terminated, 3 mice are randomly selected from each group, left and right ears are cut off along the outline of the ears after rapid cervical removal and sacrifice, and ear tissue discs with the same aperture are cut off by a puncher with the diameter of 0.5 cm. The mass (M) of the left and right ears was weighed, and the difference (M) in ear mass was calculated Right ear -M Left ear ) The method comprises the steps of carrying out a first treatment on the surface of the The thickness (D) of the left and right ears is measured by a vernier caliper, and the ear swelling degree (D) is calculated Right ear -D Left ear ). The results are shown in FIG. 10, in which the model group mice had significantly higher ear mass differences (P < 0.01) than the normal group; the mice of example 3 group had significantly lower difference in both ears (P < 0.05) compared to the model group; the mice of example 3 group had a reduced difference in ear mass compared to the positive drug group, but no significance (P>0.05 A) is provided; the ear mass difference was significantly reduced (P < 0.05) in the mice of example 3 compared to the curcumin solution group. Model group mice had significantly increased ear swelling (P < 0.01) compared to normal group; the ear swelling was significantly reduced (P < 0.01) in the mice of example 3 compared to the model group; the mice of example 3 had reduced ear swelling compared to the positive drug group, but no significant differences (P>0.05 A) is provided; the ear swelling was significantly reduced (P < 0.05) in the mice of example 3 compared to the curcumin solution group.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the curcumin nanoemulsion temperature-sensitive hydrogel is characterized by comprising the following steps of:
(1) Mixing the raw materials, ethanol and oil, and performing ultrasonic treatment to obtain an oil phase;
(2) Sequentially adding tween-80 and water into the oil phase obtained in the step (1), mixing by vortex, and then placing into an ultrasonic cell grinder for ultrasonic treatment to obtain curcumin nanoemulsion;
(3) Mixing the mixed solution of poloxamer 188 and poloxamer 407 with the curcumin nanoemulsion obtained in the step (2) to obtain a mixture;
(4) And (3) standing the mixture obtained in the step (3) to obtain the curcumin nanoemulsion temperature-sensitive hydrogel.
2. The method of claim 1, wherein the drug substance of step (1) is curcumin; the ethanol is absolute ethanol; the oil is soybean oil.
3. The method of claim 1, wherein the mass-to-volume ratio of the bulk drug, the absolute ethyl alcohol and the soybean oil in the step (1) is 2.1-2.7 mg:0.1 to 0.3ml:0.11 to 0.17ml.
4. The method according to claim 1, wherein the specific ultrasonic treatment method in step (1) is as follows: the temperature is 25-35 ℃, the power is 190-210 w, and the time is 10-20 min.
5. The method of claim 1, wherein the tween-80 of step (2) is 15-25% by volume; the water is ultrapure water.
6. The method according to claim 1, wherein the volume ratio of ultrapure water to tween-80 in step (2) is 0.1 to 0.7ml:1.23 to 1.29ml.
7. The method according to claim 1, wherein the mixing time in step (2) is 10 to 20 minutes; the working parameters of the ultrasonic cell grinder are as follows: 290-310W, working for 1-3 s and intermittence for 1-3 s; the ultrasonic time is 1-7 min.
8. The method according to claim 1, wherein the mass-to-volume ratio of poloxamer 188, poloxamer 407 and water in step (3) is 0.10-0.70 g:1.20 to 1.26g: 6-12 ml; the curcumin nanoemulsion in the step (4) is used in an amount of 0.5-1.5 ml; the standing temperature is 1-7 ℃; the standing time is 21-27 h.
9. The curcumin nanoemulsion temperature-sensitive hydrogel prepared by the method of claims 1-8.
10. The curcumin nanoemulsion temperature-sensitive hydrogel prepared by the method of claims 1-8, and the application of the curcumin nanoemulsion temperature-sensitive hydrogel of claim 9 in preparation of chronic eczema pharmaceutical preparations.
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