CN107303263B - Tripterygium glycosides nanoemulsion gel and preparation method thereof - Google Patents
Tripterygium glycosides nanoemulsion gel and preparation method thereof Download PDFInfo
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- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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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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- 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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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
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- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0014—Skin, i.e. galenical aspects of topical compositions
Abstract
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to tripterygium glycosides nanoemulsion gel and a preparation method and application thereof. The invention discloses tripterygium glycosides nanoemulsion gel, which mainly comprises the raw material tripterygium glycosides, an oil phase, a surfactant, a cosurfactant, a gel matrix and deionized water. Mixing tripterygium glycosides with oil phase, surfactant and cosurfactant, adding deionized water dropwise into the mixed solution, magnetically stirring to obtain tripterygium glycosides nanoemulsion, and adding gel matrix to obtain tripterygium glycosides nanoemulsion gel. The invention selects O/W type nanoemulsion as a carrier for transdermal drug delivery to prepare tripterygium glycosides nanoemulsion gel transdermal preparation, and provides a novel effective pharmaceutical preparation for clinical application of tripterygium glycosides.
Description
Technical Field
The invention relates to the technical field of medicines, and particularly relates to tripterygium glycosides nanoemulsion gel and a preparation method and application thereof.
Background
Tripterygium wilfordii hook.f. series Celastraceae Erycibe woody vine, which is bitter in nature and toxic, and has the effects of promoting blood circulation, removing blood stasis, clearing heat, detoxicating, relieving swelling, resolving hard mass, killing parasites, stopping bleeding, etc. Tripterygium glycosides are the main active site of Tripterygium wilfordii, have antiinflammatory, analgesic, antitumor and immunoregulatory effects, and are clinically used for treating dermatitis, immunosuppression, malignant tumor, etc. Tripterygium glycosides have remarkable clinical treatment effect, but have obvious peak-valley phenomenon due to oral absorption, easily cause large toxic reaction, and the adverse reaction incidence rate of digestive system and genitourinary system is 35.8% and 22.8%, respectively. Meanwhile, because tripterygium glycosides are insoluble in water and poorly absorbed in vivo, the improvement of the patent medicine property and the bioavailability of the tripterygium glycosides is influenced, and the clinical application of the tripterygium glycosides is severely limited. Therefore, it is very necessary to develop a tripterygium glycosides preparation with non-oral administration route, so as to reduce the toxic and side effects of tripterygium glycosides, improve the bioavailability of the medicine and the compliance of patients with the medicine, and provide a novel effective medicine preparation for the treatment of immune diseases such as clinical rheumatoid arthritis, psoriasis and the like.
The nano-emulsion is a transparent or semitransparent system which is composed of a water phase, an oil phase, a surfactant and a cosurfactant and has stable thermodynamics and kinetics with the particle size of 10-100 nm. Structurally, nanoemulsions can be classified into oil-in-water (O/W), water-in-oil (W/O), and bicontinuous types. The nanoemulsion is used as a novel drug carrier, has simple preparation and high safety, can increase the solubility of insoluble drugs and improve the stability of easily hydrolyzed drugs, has slow release and targeting property, can improve the bioavailability of the drugs, and is suitable for oral administration, injection and transdermal administration. When used as a carrier of a transdermal drug delivery system, the nanoemulsion is easy to moisten the skin and change the physicochemical property of the stratum corneum structure due to the reduction of the specific surface tension, and therefore, has attracted great attention in the research aspect of transdermal absorption preparations.
The transdermal drug delivery system can avoid the first pass effect of liver and the inactivation of the drug in gastrointestinal tract, and the absorption of the drug is not influenced by the factors of the gastrointestinal tract. The sustained-release tablet maintains constant effective blood concentration or physiological effect, has long action time, avoids peak valley phenomenon of blood concentration caused by oral administration, reduces toxic and side effects, is convenient to use, and can be independently taken by patients or withdrawn at any time.
The gel has the advantages of smooth appearance, transparency, fineness, proper consistency and viscosity, easy coating and the like, so that the preparation of the tripterygium glycosides into the nano-emulsion gel has good research and development values.
Disclosure of Invention
The invention aims to provide a tripterygium glycosides nanoemulsion gel preparation, the other purpose of the invention is to provide a preparation method of the tripterygium glycosides nanoemulsion gel, and the third purpose of the invention is to provide application of the tripterygium glycosides nanoemulsion gel in medicines for treating immune diseases such as rheumatoid arthritis, dermatitis, eczema, psoriasis and the like.
The most critical problem of transdermal drug delivery is the transdermal absorption of the drug, so the penetration promoting technology becomes one of the key technologies of the preparation process. In the invention, in the preliminary experiment of the gel preparation, the influence of the oil phase proportion, the ratio of the surfactant to the cosurfactant and the proportion of the water phase in the total amount of the prescription on the in vitro transdermal accumulated permeation quantity is also found to be great.
Then, the prescription of tripterygium glycosides nanoemulsion gel is screened by adopting a star point Design-effect surface method, data is processed by adopting Design-Expert software, and the deviation between the optimal prescription and the accumulated permeation quantity obtained by an effect surface diagram and a binomial regression model and an actual measurement value is small, so that the established model is good in predictability.
In a first aspect of the invention, a tripterygium glycosides nanoemulsion gel is provided.
A tripterygium glycosides nanoemulsion gel comprises the following main drugs and auxiliary materials in percentage by weight:
0.33 to 3.30 percent of tripterygium glycosides,
10 to 20 percent of oil phase,
20 to 40 percent of surfactant,
10 to 20 percent of cosurfactant,
1 to 5 percent of gel matrix,
the balance being water.
The gel matrix is carbomer 940.
Further, the tripterygium glycosides nanoemulsion gel is preferably prepared by mixing tripterygium glycosides, tripterygium glycosides and tripterygium glycosides,
0.5 to 3.3 percent of tripterygium glycosides,
15 to 20 percent of oil phase,
25 to 35 percent of surfactant,
12 to 18 percent of cosurfactant,
9401 to 2 percent of gel matrix carbomer,
the balance being water.
The oil phase is propylene glycol monocaprylate, caprylic capric polyethylene glycol glyceride, polyoxyethylene castor oil and glyceryl triacetate, and is preferably propylene glycol monocaprylate.
The surfactant is OP-10 emulsifier, Tween-80, propylene glycol monocaprylate, caprylic capric polyethylene glycol glyceride, preferably OP-10 emulsifier.
The cosurfactant is 1, 2-propylene glycol, tween-80, caprylic capric acid polyethylene glycol glyceride, preferably 1, 2-propylene glycol.
The water is purified water or water for injection.
Further, the tripterygium glycosides nanoemulsion gel is preferably prepared by mixing tripterygium glycosides, tripterygium glycosides and tripterygium glycosides,
1 to 3.3 percent of tripterygium glycosides, optimally 3 percent,
17 to 19 percent of propylene glycol monocaprylate, optimally 18 percent,
29 to 31 percent of OP-10 emulsifier, optimally 30 percent,
14 to 16 percent of 1, 2-propylene glycol, optimally 15 percent,
9401 to 2 percent of carbomer, optimally 1.5 percent,
the balance being water.
In a second aspect of the invention, a preparation method of the tripterygium glycosides nanoemulsion gel is provided.
The preparation method of the tripterygium glycosides comprises the following steps:
A. dissolving tripterygium glycosides in a mixed solution of propylene glycol monocaprylate, OP-10 emulsifier and 1, 2-propylene glycol for later use;
B. adding purified water dropwise into the solution, and stirring to obtain tripterygium glycosides nanoemulsion;
C. adding appropriate amount of deionized water into carbomer 940, and swelling at 4 deg.C overnight to obtain transparent gel matrix;
D. adding tripterygium glycosides nanoemulsion into the gel matrix, and stirring well to obtain the final product.
The stirring speed is 300rpm/min, and the stirring time is 10-30 min.
The tripterygium glycosides nanoemulsion gel prepared by the invention has the characteristics of transparency, fineness, proper viscosity, easiness in coating, no stimulation to skin and the like. In the hydrogel preparation, the permeation promoting mechanism of OP-10 and propylene glycol monocaprylate is probably related to disturbing the ordered structure of the stratum corneum and influencing the fluidity of cell membranes, and 1, 2-propylene glycol is probably used for promoting the percutaneous absorption of the drug by improving the solubility of the drug in the stratum corneum, so that the OP-10 and the propylene glycol monocaprylate have the combined and synergistic permeation promoting effect.
The tripterygium glycosides nanoemulsion gel prepared by the invention can be used for treating immune diseases such as rheumatoid arthritis, dermatitis eczema, psoriasis and the like, and can replace tripterygium glycosides oral preparations, so that the administration is convenient, the first pass effect of the liver is avoided, the toxic and side effects of the tripterygium glycosides are reduced, a medicine storage library is formed on the skin by the gel, the sustained release effect is realized, and the bioavailability of the medicine and the medication compliance of patients are improved.
Drawings
FIG. 1 is a transmission electron micrograph (× 20000) of tripterygium glycosides nanoemulsion of the present invention;
FIG. 2 is a distribution diagram of the particle size of tripterygium glycosides nanoemulsion of the present invention;
FIG. 3 chromatogram of triptolide standard HP L C;
FIG. 4 three-dimensional effect surface plot (X) of influence factors and response values1: the percentage of oil in the oil phase (w/w); x2: the ratio of surfactant to co-surfactant (w/w); x3: the percentage of water in the nanoemulsion (w/w)); wherein: a X1(%) and X2(Km)；b X1(%) and X3(％)；c X2(Km) And X3(％)；
FIG. 5 influence factors andtwo-dimensional contour map (X) of response values1: the percentage of oil in the oil phase (w/w); x2: the ratio of surfactant to co-surfactant (w/w); x3: the percentage of water in the nanoemulsion (w/w)); wherein: a X1(%) and X2(Km)；b X1(%) and X3(％)；c X2(Km) And X3(％)。
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Materials:
tripterygium glycosides (Hibiscus makinoi Biotech Co., Ltd.);
triptolide standards (batch No. 120416, Shanghai Po pharmaceutical science and technology Co., Ltd.);
1, 2-propanediol (batch No. 20140503, Yonghua chemical science and technology Co., Ltd.);
azone (batch No. 20131115, chemical reagents of national drug group, Ltd.);
tween80 (batch No. 20150121, Shanghai Lingfeng Chemicals Co., Ltd.);
propylene glycol monocaprylate (batch No. 150857, Jiafa lion);
OP-10 (batch No. H1504057, Aladdin);
glyceryl triacetate (batch number: 0050613, Beijing chemical Co., Ltd.)
Polyoxyethylene castor oil (batch number: A1506033, alatin);
methanol (batch No. 14090703, TEDIA);
SD rats, male, body weight 20 + -2 g, provided by animal testing center of second department of military medical university. The license number of the experimental animal: SCXK (Shanghai) 2015-10-20.
Example 1
Weighing 0.6g tripterygium glycosides, 3.63g propylene glycol monocaprylate, 5.94g OP-10, 2.97g1, 2-propylene glycol, stirring, mixing, slowly adding 7.46g purified water into the solution, and stirring to obtain tripterygium glycosides nanoemulsion.
Weighing 0.3g of carbomer 940, adding a proper amount of purified water to ensure that the poloxamer is totally submerged below the water surface, and placing the mixture in a refrigerator at 4 ℃ to ensure that the mixture is fully swelled.
And after 24h, taking out the swelled carbomer, adding the tripterygium glycosides nanoemulsion while stirring, and uniformly stirring to obtain a yellow and transparent tripterygium glycosides nanoemulsion gel preparation.
Example 2
Weighing 0.6g tripterygium glycosides, 3.63g propylene glycol monocaprylate, 5.94gOP-10, 2.97g tween80, stirring, mixing, slowly adding 7.46g purified water into the solution, and stirring to obtain tripterygium glycosides nanoemulsion.
Weighing 0.3g of carbomer 940, adding a proper amount of purified water to ensure that the poloxamer is totally submerged below the water surface, and placing the mixture in a refrigerator at 4 ℃ to ensure that the mixture is fully swelled.
And after 24h, taking out the swelled carbomer, adding the tripterygium glycosides nanoemulsion while stirring, and uniformly stirring to obtain a yellow and transparent tripterygium glycosides nanoemulsion gel preparation.
Example 3
Weighing 0.6g tripterygium glycosides, 3.63g propylene glycol monocaprylate, 5.94g tween80 and 2.97g1, 2-propylene glycol, stirring, mixing, slowly adding 7.46g purified water into the solution, and stirring to obtain tripterygium glycosides nanoemulsion.
Weighing 0.3g of carbomer 940, adding a proper amount of purified water to ensure that the poloxamer is totally submerged below the water surface, and placing the mixture in a refrigerator at 4 ℃ to ensure that the mixture is fully swelled.
And after 24h, taking out the swelled carbomer, adding the tripterygium glycosides nanoemulsion while stirring, and uniformly stirring to obtain a yellow and transparent tripterygium glycosides nanoemulsion gel preparation.
Example 4
Weighing 0.6g tripterygium glycosides, 3.63g propylene glycol monocaprylate, 5.94g polyoxyethylene castor oil and 2.97g1, 2-propylene glycol, stirring, mixing, slowly adding 7.46g purified water into the solution, and stirring to obtain tripterygium glycosides nanoemulsion.
Weighing 0.3g of carbomer 940, adding a proper amount of purified water to ensure that the poloxamer is totally submerged below the water surface, and placing the mixture in a refrigerator at 4 ℃ to ensure that the mixture is fully swelled.
And after 24h, taking out the swelled carbomer, adding the tripterygium glycosides nanoemulsion while stirring, and uniformly stirring to obtain a yellow and transparent tripterygium glycosides nanoemulsion gel preparation.
Example 5
Weighing 0.6g tripterygium glycosides, 3.63g triacetin, 5.94g propylene glycol monocaprylate and 2.97g1, 2-propylene glycol, stirring, mixing, slowly adding 7.46g purified water into the solution, and stirring to obtain tripterygium glycosides nanoemulsion.
Weighing 0.3g of carbomer 940, adding a proper amount of purified water to ensure that the poloxamer is totally submerged below the water surface, and placing the mixture in a refrigerator at 4 ℃ to ensure that the mixture is fully swelled.
And after 24h, taking out the swelled carbomer, adding the tripterygium glycosides nanoemulsion while stirring, and uniformly stirring to obtain a yellow and transparent tripterygium glycosides nanoemulsion gel preparation.
Example 6
Weighing 0.6g tripterygium glycosides, 3.63g propylene glycol monocaprylate, 5.94g caprylic/capric polyethylene glycol glyceride and 2.97g1, 2-propylene glycol, stirring, mixing, slowly adding 7.46g purified water, and stirring to obtain tripterygium glycosides nanoemulsion.
Weighing 0.3g of carbomer 940, adding a proper amount of purified water to ensure that the poloxamer is totally submerged below the water surface, and placing the mixture in a refrigerator at 4 ℃ to ensure that the mixture is fully swelled.
And after 24h, taking out the swelled carbomer, adding the tripterygium glycosides nanoemulsion while stirring, and uniformly stirring to obtain a yellow and transparent tripterygium glycosides nanoemulsion gel preparation.
Example 7
Weighing 0.6g tripterygium glycosides, 3.63g triacetin, 5.94g OP-10 emulsifier, 2.97g1, 2-propylene glycol, stirring, mixing, slowly adding 7.46g purified water, and stirring to obtain tripterygium glycosides nanoemulsion.
Weighing 0.3g of carbomer 940, adding a proper amount of purified water to ensure that the poloxamer is totally submerged below the water surface, and placing the mixture in a refrigerator at 4 ℃ to ensure that the mixture is fully swelled. And after 24h, taking out the swelled carbomer, adding the tripterygium glycosides nanoemulsion while stirring, and uniformly stirring to obtain a yellow and transparent tripterygium glycosides nanoemulsion gel preparation.
Example 8: in vitro transdermal test
Preparation of in vitro skin: unhairing SD rat abdomen, standing for 1d to ensure no damage to abdominal skin, taking the complete abdominal unhaired skin immediately after sacrifice, scraping redundant fat and tissue on the skin, cleaning with normal saline, and soaking in fresh normal saline at 4 deg.C.
The method is carried out by adopting a Franz diffusion cell method: PBS (containing 20% absolute ethyl alcohol) which is in constant temperature water bath at 32 ℃ and stirred at a constant speed is taken as receiving liquid. The treated in vitro skin was fixed on Franz diffusion cell with stratum corneum facing upwards, and 0.1g of prepared Tripterygium glycosides nanoemulsion gel (prepared in example 1) was uniformly applied and sealed with sealing film. Taking 1ml of receiving solution for content detection at 1,2, 3, 4, 6, 8 and 12 hours respectively, and adding 1ml of PBS at the same temperature into the receiving pool.
Calculation of in vitro transdermal cumulative permeation:
from Q ═ CnV + Σ (C)i×1)]The cumulative permeation of the drug per unit area was calculated as/S. Wherein Q is the cumulative transdermal flux of the drug per unit area, Cn is the concentration of the drug in the receiving solution at the sampling point, V is the volume of the receiving solution, CiThe cumulative concentration of the drug in the receiving solution from the 1 st sampling point to the last sampling point, and S is the effective transdermal area of the drug.
Example 9: pharmacodynamic experiment
Rats were randomly anesthetized by intraperitoneal injection. After anesthesia, the rat test site was depilated. After 3 days of adaptive feeding, the rats are anesthetized again, and the line marks are drawn on the toe parts to be measured of the rats. The toe volume measuring instrument detects the normal toe volume of the rat before model building. After detection, the tripterygium glycosides nanoemulsion gel prepared in example 1, blank nanoemulsion gel (no drug), dexamethasone acetate and normal saline each 0.5g are smeared on the toes of rats, 10 per group.
And starting molding the next day. 300mg/kg of chloral hydrate is injected into the abdominal cavity of the rat for anesthesia. A1 ml syringe sucks Freund's complete adjuvant and injects the Freund's complete adjuvant into the skin of a rat footpad until the inflammatory volume is 0.1 ml/mouse, and presses a needle hole for 20s to prevent the adjuvant from overflowing. After 10min, the toe of each rat in each test group was smeared with the corresponding test drug. After 24h of administration, the rats were anesthetized again and the toe volume meter measured the volume of the toes after the rats had reached inflammation.
The results of the adjuvant arthritis model experiments in rats are shown in table 1. Before inflammation, the average level is about 1.80ml which is close to the normal toe volume, after Freund's complete adjuvant is received to inflammation, the toes of the rats are obviously swelled, and the swelling degree of the substrate control group reaches 41.4%. The positive control group had 18.4% swelling, which was significantly different (p < 0.001) compared to the vehicle control group. The swelling degree of the tripterygium gel administration group reaches 29.7 percent, and the difference is obvious compared with that of a matrix control group (p is less than 0.05). From the experimental results, the tripterygium glycosides nanoemulsion gel has the effect of treating the adjuvant arthritis of rats.
TABLE 1 Effect of Tripterygium wilfordii gel on adjuvant arthritis in rats (n ═ 10)
Model control group with P < 0.05X P < 0.001VS
Example 10: optimization of prescription of tripterygium glycosides nanoemulsion gel by star point design-effect surface method
1 establishment of content determination method
1.1 chromatographic/Mass Spectrometry conditions
The chromatographic column is ZORBAX extended-C18 column (2.1mm × 100mm, 3.5um), the fluidity is methanol, 0.1% (v/v) formic acid water solution is 60: 40, the flow rate is 0.3ml/min, the ultraviolet detection wavelength is 280nm, the sample introduction amount is 3ul, and the column temperature is 40 ℃.
The mass spectrum conditions comprise that an ion source is an ESI source, cation mode detection, a scanning mode is multi-reaction monitoring (MRM), ions for quantitative analysis are m/z 361.3/105.2, internal standard removal m/z 361.3/147, capillary voltage 4000V, drying gas flow rate 8L/min, drying gas temperature 350 ℃, atomizer pressure 33psi, cracker voltage 168eV and collision energy 41.
1.2 preparation of the solution
Accurately weighing 1mg of triptolide standard substance in a 100ml volumetric flask, dissolving with methanol to a constant volume, mixing well, transferring 1ml of the solution to a 50ml volumetric flask to a constant volume, and shaking well to obtain the triptolide standard substance solution.
Accurately weighing 0.1g of tripterygium glycosides nanoemulsion gel, adding into a 10ml volumetric flask, adding a proper amount of methanol, performing ultrasonic emulsification for 20min, adding methanol to a constant volume, and shaking up to obtain the tripterygium glycosides test solution.
The triptolide standard solution and tripterygium glycosides sample solution are filtered through 0.45 μm microporous membrane, and the obtained chromatogram is shown in FIG. 3 under 1.1 chromatographic/mass spectrometric conditions. From the spectrogram, the triptolide in the standard substance and the gel has good peak shape, and other components in the gel have no interference to absorption of tripterygium glycosides.
1.3 establishment of standard curve precisely absorbs 0.1ml, 0.25ml, 0.5ml, 1.0ml, 2.0ml, 4.0ml, 5.0ml to 10ml of standard solution in 1.2 items, uses methanol to dilute and fix the volume to obtain a series of concentrations, shakes uniformly, samples according to 1.1 chromatographic conditions, determines peak area (A), and uses A to make linear regression on concentration (C) to obtain regression equation: a is 22.487C + 10.586, (R)20.9993). The linear range of the concentration of tripterygium glycosides is 2-100 ng/ml.
1.4 precision test sample introduction of the standard solution in item 1.2 is repeated for 6 times according to 1.1 chromatographic conditions, peak areas are recorded, and the measurement results are shown in Table 2, which shows that the precision of the instrument test is good.
TABLE 2 results of daily and intraday precision measurements
1.5 stability test 1.2 standard solution is taken, sample injection is carried out for 0h, 1h, 2h, 4h, 6h and 8h respectively according to 1.1 chromatographic conditions, peak area values are recorded, the measurement result is shown in table 3, and the result shows that the test sample is basically stable within 8h after treatment.
Table 3 stability test results
1.6 sample application recovery test 9 parts of tripterygium glycosides gel (sample prepared in example 1) with known content are added with a proper amount of precisely measured reference solution, 3 parts are 1 group, are dissolved and filtered with a proper amount of methanol, sample injection is carried out according to 1.1 chromatographic conditions, peak areas are recorded, and the measurement results are shown in Table 4. The result shows that the method has good recovery rate, and other components in the preparation have no interference on the content determination of the triptolide.
TABLE 4 sample recovery test results
1.7 content determination 3 batches of 3% tripterygium glycosides nanoemulsion gel are taken, sample solution is prepared according to the method of 1.2 items, sample injection is carried out according to the chromatographic condition of 1.1 items, peak area is recorded, and the content of the tripterygium glycosides in the gel is calculated according to linear regression.
2 tripterygium glycosides nanoemulsion gel preparation process
2.1 Tripterygium glycosides nanoemulsion gel comprises Tripterygium glycosides 0.6g, propylene glycol monocaprylate 3.63g, OP-10 emulsifier 5.94g, 1, 2-propylene glycol 2.97g, and purified water 20 g.
2.2 preparation of tripterygium glycosides nanoemulsion gel firstly, mixing tripterygium glycosides, propylene glycol monocaprylate, OP-10 emulsifier and 1, 2-propylene glycol uniformly, stirring and dispersing, then adding purified water drop by drop, and stirring uniformly; obtaining tripterygium glycosides nanoemulsion; adding appropriate amount of purified water into carbomer 940, and swelling at 4 deg.C overnight to obtain transparent gel matrix; adding tripterygium glycosides nanoemulsion into the gel matrix, and stirring uniformly to obtain a yellow transparent gel preparation. The pH value of the prepared gel preparation is 7.0-7.4.
3 in vitro transdermal test
3.1 preparation of in vitro skin SD rats were dehaired on the abdomen to ensure no damage to the abdominal skin. And (3) killing the mice after 1d, immediately taking the complete abdomen of the mice to remove the skin, scraping redundant fat and tissues on the skin, cleaning and soaking the skin by using normal saline, and storing the skin for a short time at 4 ℃ for later use.
3.2 in vitro transdermal experiments were performed using the Franz diffusion cell method. The devices used were Franz diffusion cell and self-made transdermal diffusion apparatus, the effective diffusion inner diameter was 0.9cm, the receiving chamber volume was 5ml, PBS (containing 20% absolute ethanol) was used as the receiving solution, and stirred at constant speed in a constant temperature water bath at 32 ℃. Collecting the in vitro skin treated under 3.1 items, drying with filter paper, fixing the stratum corneum upwards on Franz diffusion cell, uniformly coating 0.1g Tripterygium glycosides nanoemulsion gel on rat skin, and sealing with sealing membrane. 1ml of receiving solution is taken at 1,2, 3, 4, 6, 8 and 12 hours respectively, and 1ml of PBS with the same temperature is added into a receiving pool. The received solution was filtered through a 0.45 μm microporous membrane, and sample injection was carried out under 1.1 chromatographic conditions, and the peak area was recorded.
3.3 calculation of in vitro cumulative permeation volume the regression equation was used to calculate the tripterygium glycosides content in the sink at different time points from Q ═ CnV + Σ (C)i×1)]The cumulative permeation of the drug per unit area was calculated as/S. Wherein Q is the cumulative transdermal flux of the drug per unit area, Cn is the concentration of the drug in the receiving solution at the sampling point, V is the volume of the receiving solution, CiThe cumulative concentration of the drug in the receiving solution from the 1 st sampling point to the last sampling point, and S is the effective transdermal area of the drug.
Optimization prescription of 4-star point design-effect surface method
4.1 Star point design
The prescription is optimized by adopting a Box-Behnken design-effect surface method. On the basis of the previous experiments, 3 factors which have more remarkable influence on the properties of the nanoemulsion are selected, namely X1: percentage of oil in total mass of oil and emulsifier (w/w), X2: ratio of surfactant to co-surfactant (w/w), X3: the percentage (w/w) of water in the nanoemulsion is optimized and researched on 3 levels, the low level, the middle level and the high level of each factor are respectively marked as-1, 0 and +1, the accumulated transmittance (K) is used as an evaluation index to arrange a test, and the setting of the factor levels and the design of the experimental star points are shown in tables 5 and 6.
TABLE 5 Box-Behnken test factor levels
TABLE 6 Star design experiment table and results
4.2 optimization of the surface of Effect
Regression analysis was performed using Design-Expert software (8.0.5b master) to create a quadratic polynomial regression model with confidence (P) as the criterion for model decision:
Y＝A0+A1X1+A2X2+A3X3+A4X1X2+A5X2X3+A6X1X3+A7X1 2+A8X2 2+A9X3 2
and drawing an effect surface graph of X1, X2 and X3 to each evaluation index by the model obtained by fitting, reflecting the influence trend of each dependent variable on the response value from the effect surface graph of each index, and determining a better optimization parameter from the relationship between the effect value and the investigation factor.
The quadratic polynomial regression equation obtained was based on the cumulative permeation rate at each factor level (see table 5): y-14.08331 +0.98805X1+1.49515X2+0.60686X3-0.098629X1X2+8.73102E-003X1X3-0.013432X2X3-0.023179X12-2.29345X22-8.42670E-003X 32. Three-dimensional effect surfaces and two-dimensional contour maps are drawn by polynomial regression equations, see fig. 4 and 5.
The optimized actual prescription proportion is as follows: the oil phase accounts for 18.15% of the total amount of the prescription, the S/CO-S accounts for 44.54% of the total amount of the prescription, and the distilled water accounts for 37.31% of the total amount of the prescription.
4.3 optimal prescription verification
Preparing 3 batches of tripterygium glycosides nanoemulsion gel according to the optimal prescription, carrying out transdermal experiment according to an in vitro transdermal experiment method, and calculating in vitro accumulated transdermal permeation quantity by a formula. Deviation as an indicator of whether the model created is predictive, deviation (%) The mean cumulative transdermal penetration rate (15.612 μ g.cm) of the 3 batches of formulation was determined at × 100% where measured-predicted)/predicted ×% values were 100%-2.h-1) And predicted value (16.486. mu.g.cm)-2.h-1) Substituting into the deviation formula to obtain 5.3% deviation. This indicates that the predictability of this experiment is good. See table 7 for details.
TABLE 7 prescription verification results
The experimental results show that: when the optimal mixture ratio of tripterygium glycosides nanoemulsion is 18.15 percent of propylene glycol monocaprylate, 29.69 percent of OP-10 emulsifier and 14.85 percent of 1, 2-propylene glycol, the cumulative transdermal permeability for 12 hours is 15.612 mu g.cm-2.h-1And the method has good conformity with the predicted value.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (6)
1. The tripterygium glycosides nanoemulsion gel is characterized by comprising the following components in percentage by weight:
0.5 to 3.0 percent of tripterygium glycosides,
15 to 20 percent of oil phase,
25 to 35 percent of surfactant,
12 to 18 percent of cosurfactant,
9401 to 2 percent of carbomer,
the balance of water;
the oil phase is propylene glycol monocaprylate;
the surfactant is OP-10 emulsifier;
the cosurfactant is 1, 2-propylene glycol.
2. The tripterygium glycosides nanoemulsion gel of claim 1, wherein the tripterygium glycosides nanoemulsion gel comprises the following components in percentage by weight:
3. a method for preparing tripterygium glycosides nanoemulsion gel of claim 1 or 2, wherein the method for preparing the tripterygium glycosides nanoemulsion gel comprises the following steps:
A. mixing Tripterygium glycosides with oil phase, surfactant and cosurfactant at a certain proportion, and stirring;
B. dropwise adding deionized water into the solution, and stirring uniformly to obtain tripterygium glycosides nanoemulsion;
C. adding appropriate amount of deionized water into carbomer 940, and swelling at 4 deg.C overnight to obtain transparent gel matrix;
D. adding tripterygium glycosides nanoemulsion into the gel matrix, and stirring well to obtain the final product.
4. The method for preparing tripterygium glycosides nanoemulsion gel of claim 3, wherein the stirring speed in steps B and D is 100-500 rpm/min, and the stirring time is 10-30 min.
5. The use of the tripterygium glycosides nanoemulsion gel of claim 1 or 2 in the preparation of a medicament for treating immune diseases.
6. The use of the tripterygium glycosides nanoemulsion gel of claim 5 in the preparation of a medicament for treating an immunological disorder, wherein the immunological disorder is rheumatoid arthritis, eczema dermatitis or psoriasis.
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