CN110859842A - Tetrandrine composition, preparation method thereof, tetrandrine external preparation, preparation method and application thereof - Google Patents

Tetrandrine composition, preparation method thereof, tetrandrine external preparation, preparation method and application thereof Download PDF

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CN110859842A
CN110859842A CN201911285177.XA CN201911285177A CN110859842A CN 110859842 A CN110859842 A CN 110859842A CN 201911285177 A CN201911285177 A CN 201911285177A CN 110859842 A CN110859842 A CN 110859842A
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tetrandrine
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陈新
郑颖
陈少魁
林子贝
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University of Macau
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • 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
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/06—Antipsoriatics

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Abstract

The invention relates to the field of medicines, and in particular relates to a tetrandrine composition, a preparation method thereof, a tetrandrine external preparation, a preparation method thereof and application thereof. The tetrandrine composition comprises, by weight, 0.005-0.015 parts of tetrandrine, 1 part of an oil phase, 0.5 parts of an emulsifier, 7-8 parts of a water phase and 1 part of an auxiliary emulsion. The tetrandrine composition is made into the nano-emulsion by using the emulsifier, the auxiliary emulsion, the oil phase and the like, so that the solubility of the tetrandrine is improved, the retention amount of the tetrandrine on the skin is improved, the toxic and side effects of the tetrandrine are reduced, and the tetrandrine composition is low in economic cost. Meanwhile, the tetrandrine composition has good effects of inhibiting desquamation, erythema, epidermal thickening and the like of psoriasis vulgaris by inhibiting expression of related inflammatory factors.

Description

Tetrandrine composition, preparation method thereof, tetrandrine external preparation, preparation method and application thereof
Technical Field
The invention relates to the field of medicines, and in particular relates to a tetrandrine composition, a preparation method thereof, a tetrandrine external preparation, a preparation method thereof and application thereof.
Background
Psoriasis is a chronic inflammatory disease of the skin, commonly known as psoriasis. Clinically, it is divided into four types, including psoriasis vulgaris, psoriasis pustulosa, arthropathies and erythrodermic psoriasis. Psoriasis vulgaris is common and mainly manifests as thickening of skin scraps at a skin injury part to form plaques, and red rashes are often accompanied by white scales covering the plaques. So far, no method for completely curing psoriasis is available clinically, and patients with moderate and mild psoriasis usually adopt chemical medicine or Chinese herbal compound treatment, and can cure the psoriasis in a short period but easily relapse; patients with severe psoriasis are usually treated by antibody medicines, the curative effect is high but the cost is high, and no suitable traditional Chinese medicine can well treat psoriasis in the prior art.
Tetrandrine, also known as tetrandrine, is extracted from the root of Stephania tetrandra of Stephania of Menispermaceae, and is a bisbenzylisoquinoline compound, which is also contained in the tuber root of Stephania cepharantha, Royleaf Tetrastigmatis root, Menispermum dauricum stem and Shinyleaf-Johnston root. The previous experimental study shows that it has the functions of resisting hypertension, rheumatism, pain and the like. In recent years, researches show that tetrandrine has the effects of resisting inflammation, bacteria, tumors, multidrug resistance and the like, and can be used for preventing and treating cardiovascular system and digestive system diseases and treating scalds and ophthalmic diseases. The prior art does not describe that tetrandrine can well treat psoriasis.
At present, a large amount of tetrandrine in the market is tablets, injections and the like, and the medicines have the problems of large use amount, poor solubility, low bioavailability, poor targeting property, high renal toxicity and the like in clinic.
Disclosure of Invention
The invention provides a tetrandrine composition, a preparation method thereof, a tetrandrine external preparation, a preparation method thereof and application thereof.
The invention is realized by the following steps:
in the embodiment of the invention, the tetrandrine composition is provided and comprises 0.005-0.015 part of tetrandrine, 1 part of oil phase, 0.5 part of emulsifier, 7-8 parts of water phase and 1 part of auxiliary emulsion.
Further, in a preferred embodiment of the present invention, the active ingredient of the tetrandrine composition is only tetrandrine and its derivatives;
further, in a preferred embodiment of the present invention, the tetrandrine composition is a nano-emulsion having a nano-particle size;
preferably, the particle size of the nanoemulsion is 30-50 nm.
Further, in a preferred embodiment of the present invention, the oil phase includes at least one of an unsaturated oil, a fatty acid ester-based compound, and a grease polymer;
preferably, the unsaturated oil comprises any one of soybean oil, palm oil, linseed oil and castor oil;
preferably, the fatty acid ester compound comprises any one of isopropyl myristate, caprylic capric triglyceride, polyethylene glycol laurate, polyethylene glycol oleate, polyethylene glycol stearate, glyceryl triacetate, ethyl oleate, glyceryl monostearate and isopropyl palmitate;
preferably, the grease polymer is polyethylene glycol glyceryl oleate.
Further, in a preferred embodiment of the present invention, the emulsifier comprises a surfactant;
preferably, the surfactant includes any one of a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant and an anionic surfactant;
preferably, the nonionic surfactant includes any one of polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acid compounds, polyoxyethylene fatty alcohol ether compounds, fatty acid glyceride compounds, polysorbate compounds, sorbitan fatty acid compounds, polyoxyethylene-polyoxypropylene copolymer compounds and PEG; more preferably, the PEG is PEG 400;
preferably, the zwitterionic surfactant comprises any one of soybean lecithin, whey protein isolate and lactoferrin;
preferably, the cationic surfactant comprises any one of cetyltrimethylammonium bromide, lauroyl arginine ethyl ester and dodecyltrimethylammonium bromide;
preferably, the anionic surfactant includes any one of sodium dodecyl sulfonate and sodium dodecyl sulfate.
Further, in a preferred embodiment of the present invention, the co-emulsion comprises any one of alcohol and modified polyethylene glycol;
preferably, the modified polyethylene glycol compound is lithium polyethylene glycol dodecahydroxy stearate;
preferably, the alcohol species includes monohydric alcohols and polyhydric alcohols;
preferably, the monohydric alcohol comprises any one of ethanol and n-butanol;
preferably, the polyol includes any one of 1, 2-propanediol and glycerol.
Further, the embodiment of the present invention also provides a preparation method of the tetrandrine composition, which includes: mixing the oil phase, the emulsifier and the auxiliary emulsion, and then mixing with the tetrandrine and the water phase to form the tetrandrine composition. Further, the embodiment of the invention also provides a tetrandrine external preparation, which comprises the tetrandrine composition; preferably, the dosage form of the tetrandrine external preparation comprises any one of nano cream, gel, ointment, lotion, patch and film agent;
more preferably, when the tetrandrine external preparation is in the form of gel, the pH of the gel is 6-8.
Further, the embodiment of the present invention also provides a preparation method of the tetrandrine external preparation, and when the tetrandrine external preparation is a gel, the preparation method includes: mixing the tetrandrine composition with the swollen gel base material;
preferably, the preparation method further comprises: mixing the tetrandrine composition with the swollen gel matrix, adjusting the pH, and adjusting the pH to 6-8;
preferably, the preparation of the swollen gel matrix comprises: mixing a thickener with a solvent until the thickener is fully swollen;
preferably, the concentration of the swollen gel matrix is 0.8% -2%;
preferably, the volume ratio of the mixture to the swollen gel matrix is 3: 1.5-2.
Further, the embodiment of the invention also provides an application of the tetrandrine composition in preparing a medicine for treating psoriasis.
Wherein the psoriasis comprises: psoriasis vulgaris, psoriasis dropwise, psoriasis vulgaris, psoriasis pustulosa, psoriasis vulgaris, psoriasis erythrodermalis, and psoriasis arthropathica.
The invention has the beneficial effects that: the tetrandrine composition is prepared into the nano-emulsion by using the emulsifier, the co-emulsion, the oil phase and the like, so that the solubility of the tetrandrine is improved, the retention amount of the tetrandrine on the skin is improved, the toxic and side effects of the tetrandrine are reduced, and the tetrandrine composition is low in economic cost. Meanwhile, the tetrandrine composition forms a gel after being dispersed in the gel, can directly act on skin injury parts, improves the curative effect, and has good effects of inhibiting desquamation, erythema, epidermal thickening and the like of psoriasis vulgaris.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph showing the results of PASI scoring provided in Experimental example 1 of the present invention;
FIG. 2 is a comparative graph of the treatment of the skin on the back of psoriasis mice provided in Experimental example 1 of the present invention;
FIG. 3 is a comparison graph of HE staining provided in Experimental example 1 of the present invention;
FIG. 4 is a graph showing the detection of the expression level of mRNA provided in Experimental example 1 of the present invention;
FIG. 5 is a comparative graph of the treatment of the skin on the back of psoriasis mice provided in Experimental example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following embodiments of the present invention provide a tetrandrine composition, a method for preparing the tetrandrine composition, a tetrandrine external preparation, a method for preparing the tetrandrine external preparation, and applications of the tetrandrine external preparation.
Firstly, the invention provides a tetrandrine composition which comprises 0.005-0.015 part of tetrandrine, 1 part of oil phase, 0.5 part of emulsifier, 7-8 parts of water phase and 1 part of auxiliary emulsion in parts by weight
The tetrandrine composition is in the form of the nanoemulsion by utilizing the effective emulsifier, the co-emulsion and the like, so that the solubility of tetrandrine is improved, the bioavailability of tetrandrine is improved, and meanwhile, the toxic and side effects of the tetrandrine composition can be effectively reduced through the interaction of the substances, and the tetrandrine composition has a good treatment effect on psoriasis.
Furthermore, in the embodiment of the invention, the active ingredients of the tetrandrine composition are only tetrandrine and derivatives thereof,
the tetrandrine derivative comprises natural active compounds which naturally exist in tetrandrine plants and are close to the chemical structures of the tetrandrine, and also comprises new compounds which are structurally modified by taking the tetrandrine as a basic structure, such as the replacement of carboxyl hydrogen by functional groups such as halogen, alkyl and the like. These compounds have an effect on treating psoriasis, but have poor solubility and cannot be directly administered transdermally.
The active ingredients of the tetrandrine composition are only tetrandrine and the derivatives thereof, the active ingredients are single, the toxic and side effects are low, the treatment effect of tetrandrine on psoriasis can be further ensured, and the tetrandrine composition has less skin irritation compared with other psoriasis treatment medicines in the prior art.
Further, the tetrandrine composition is a nano-emulsion with nano-particle size; preferably, the particle size of the nanoemulsion is 30-50 nm; the tetrandrine composition is prepared into the nanoemulsion, so that the solubility of tetrandrine is increased, the skin retention amount of the tetrandrine composition is increased, the toxicity of the tetrandrine composition is reduced, and meanwhile, the amphiphilic property of the nanoemulsion, namely the hydrophily and lipophilicity, can be utilized to enable active ingredients to penetrate through a skin stratum corneum, so that the absorption and utilization of medicines are facilitated, the medicine concentration of a medicine part is sufficient, and the effect of the tetrandrine is fully exerted.
Further, the oil phase includes at least one of unsaturated oil, fatty acid ester-based compound, and grease polymer. Preferably, the unsaturated oil includes any one of soybean oil, palm oil, linseed oil and castor oil. Preferably, the fatty acid ester compound comprises any one of isopropyl myristate, caprylic/capric triglyceride, polyethylene glycol laurate, polyethylene glycol oleate, polyethylene glycol stearate, glyceryl triacetate, ethyl oleate, glyceryl monostearate and isopropyl palmitate. Preferably, the grease polymer is polyethylene glycol glyceryl oleate.
The oil phase is more beneficial to the dissolution of the tetrandrine and the formation of the nano-emulsion, thereby ensuring the drug effect of the tetrandrine composition.
Further, the emulsifier includes a surfactant. Preferably, the surfactant includes any one of a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant and an anionic surfactant. Preferably, the nonionic surfactant comprises any one of polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acid compounds, polyoxyethylene fatty alcohol ether compounds, fatty acid glyceride compounds, polysorbate compounds, sorbitan fatty acid compounds, polyoxyethylene-polyoxypropylene copolymer compounds and PEG; more preferably, the PEG is PEG 400.
Preferably, the zwitterionic surfactant comprises any one of soy lecithin, whey protein isolate and lactoferrin. Preferably, the cationic surfactant includes any one of cetyltrimethylammonium bromide, lauroyl arginine ethyl ester, and dodecyltrimethylammonium bromide. Preferably, the anionic surfactant includes any one of sodium dodecyl sulfonate and sodium dodecyl sulfate.
The auxiliary emulsion comprises any one of alcohol substances and modified polyethylene glycol compounds. Preferably, the modified polyethylene glycol compound is polyethylene glycol lithium lauryl stearate, and preferably, the alcohol substance comprises monohydric alcohol and polyhydric alcohol. Preferably, the monohydric alcohol includes any one of ethanol and n-butanol. Preferably, the polyol includes any one of 1, 2-propanediol and glycerol. The emulsifier and the co-emulsion are more favorable for forming hydrophilic and oleophilic nano-emulsion and are favorable for skin to absorb tetrandrine.
The water phase is water, which is beneficial to the formation of the tetrandrine composition and is more beneficial to carrying, the concentration of active ingredients of the medicinal part is increased, and the drug effect of the tetrandrine is favorably exerted.
The embodiment of the invention also provides a tetrandrine external preparation which comprises the tetrandrine composition; preferably, the dosage form of the tetrandrine external preparation comprises any one of a nano cream, a gel, an ointment, a lotion, a patch and a film agent.
The tetrandrine composition is prepared into external preparation, and can directly act on focus part, improve drug concentration of skin injury part, accelerate the cure of psoriasis, and reduce the influence of drug toxicity on other tissues due to entering blood circulation. Meanwhile, the medicine is more beneficial to patients to self-administer medicine, is more convenient to carry, can prolong the detention time of the medicine on the surface of the skin, and enhances the medicine effect.
More preferably, the tetrandrine external preparation is gel, and the prepared tetrandrine nanoemulsion is liquid with extremely strong fluidity and is inconvenient to apply. The gel matrix has effects of keeping moisture and isolating bacteria.
Furthermore, when the tetrandrine external preparation is in the form of gel, the pH of the gel is 6-8, the pH of the gel is controlled, the irritation of the gel to the skin is relieved, and meanwhile, the treatment effect of the tetrandrine external preparation is ensured
The embodiment of the invention also provides a preparation method of the tetrandrine composition, which comprises the following steps: mixing the oil phase, the emulsifier and the auxiliary emulsion, and then mixing with the tetrandrine and the water phase to form the tetrandrine composition.
Specifically, the oil phase, the emulsifier and the co-emulsion are uniformly mixed at the temperature of 40-60 ℃, and then the tetrandrine is added into the mixture and stirred until the tetrandrine is dissolved.
The aqueous phase is then heated to 40-60 ℃ and then added to the mixture of oil phase, emulsifier, co-emulsion and tetrandrine, the heating is stopped and stirring is continued to room temperature (typically 25 ℃).
The embodiment of the invention also provides a preparation method of the tetrandrine external preparation, and when the tetrandrine external preparation is gel, the preparation method comprises the following steps: mixing the tetrandrine composition with the swollen gel base material; preferably, the pH is adjusted after the tetrandrine composition is mixed with the swollen gel base material, and the pH is adjusted to 6-8; the substance selected for adjusting the pH is triethylamine, and the influence on active ingredients can be reduced by adopting triethylamine.
Preferably, the preparation method further comprises: mixing the tetrandrine composition with the swollen gel matrix and adjusting the pH; preferably, the preparation of the swollen gel matrix comprises: mixing a thickener with a solvent until the thickener is fully swollen; preferably, the thickener is present in the gel in an amount of 0.8 to 2% by mass.
The gel prepared by adopting the conditions is easier to apply, the retention time of the tetrandrine on the skin surface is prolonged, and the drug effect is enhanced.
The thickening agent is conventional thickening agent, such as carbomer, etc., and the solvent is water or other conventional solvent for swelling the thickening agent.
It should be noted that the present invention only provides a method for preparing the tetrandrine composition into gel, and other external application preparations can be prepared from the tetrandrine composition by using the existing conventional technology.
The invention also provides an application of the tetrandrine composition in preparation of a medicament for treating psoriasis. The tetrandrine composition has good treatment effect on psoriasis, wherein the psoriasis comprises the following components: psoriasis vulgaris, psoriasis dropwise, psoriasis vulgaris, psoriasis pustulosa, psoriasis vulgaris, psoriasis erythrodermalis, and psoriasis arthropathica.
The tetrandrine composition, the preparation method thereof, the tetrandrine external preparation, the preparation method thereof and the application thereof provided by the present invention are specifically described below with reference to specific examples.
Example 1
This example provides a tetrandrine composition, which includes 0.015 grams of tetrandrine composition, labrafil m1994CS1 grams, PEG4000.5 grams, solutol hs151 grams, and 7.5 grams of ultrapure water.
This example also provides a gel containing the tetrandrine composition, which contains Labrafilm1994CS1 g, PEG4000.5 g, SolutolHS151 g, ultrapure water 7.5 g and carbomer 974N, wherein the weight content of carbomer in the gel is 0.8%, and the pH of the gel is 7.
The present invention also provides a method for preparing a tetrandrine composition, which comprises:
labrafil M1994CS, SolutolHS15 and PEG400 were mixed in a beaker by magnetic stirring, and then in a thermostatic water bath at 50 deg.C, tetrandrine powder was added and stirred continuously until the powder was dissolved. Preheating the prepared ultrapure water to 50 ℃, dropwise adding the ultrapure water into the mixture, stopping heating in the water bath, and continuously stirring the mixture to room temperature.
And then detecting the concentration of the prepared tetrandrine to ensure that the tetrandrine in the gel obtained by subsequent preparation meets the requirement, demulsifying by using methanol, absorbing 10 mu l of nano-emulsion into 90 mu l of methanol, destroying the structure of the emulsion, ensuring complete demulsification by using a high-speed centrifugation method, releasing the tetrandrine wrapped in the emulsion, dissolving the released tetrandrine in the methanol, and detecting the concentration of the tetrandrine by using HPLC. The HPLC determination conditions are as follows: agilent 1200 Series HPLC (DAD detector); detection wavelength: 280nm, flow rate: 1 ml/min; the mobile phase is acetonitrile: methanol: water: acetic acid 40: 30: 30: 1.
the embodiment also provides a preparation method of the tetrandrine external preparation, which comprises the following steps:
mixing carbomer 974N with ultrapure water, fully stirring the mixture until the mixture is completely swelled, and mixing the prepared tetrandrine nanoemulsion according to the weight ratio of 3: 2, adding the mixture into the swelled gel, and adjusting the pH to be neutral by using triethylamine. Wherein the mass content of carbomer 974N in the gel is 0.8%.
Example 2 to example 3
A gel was prepared according to the preparation method of example 1, and the amounts of the respective substances in the tetrandrine composition were the same as in example 1 except that the mass content of carbomer 974N in the gel was different, the mass content of carbomer 974N in example 2 was 1.2%, and the mass content of carbomer 974N in example 3 was 1.6%.
Example 4-example 5
A gel was prepared according to the preparation method of example 1, and the amounts of ultrapure water, carbomer 974N, and tetrandrine in the composition were the same as in example 1 except for the amount of tetrandrine, which was 0.01 part in example 4 and 0.005 part in example 5.
Example 6 and example 7
The gel was prepared according to the preparation method of example 1, except that the amounts of ultrapure water, carbomer 974N, ultrapure water and triethylamine in the tetrandrine composition were the same as in example 1, except that the amounts of ultrapure water and triethylamine were different, i.e., the pH of the gel was different, example 6 was 7 parts ultrapure water, pH was 6, example 7: 8 parts of ultrapure water and 8 parts of pH.
Comparative example: a gel was prepared according to the method of example 1, using the same amount of ultrapure water, carbomer 974N, and tetrandrine as in example 1 except for the amount of tetrandrine except for the amount of 0 part in the comparative example, and 0.002 part of triptolide (wherein triptolide ointment is a drug for psoriasis treatment on the market, and adverse reactions thereof recorded in the specification of the drug show marked irritation symptoms including local flushing, burning sensation, small blisters, erosion, and exudation or pain of the skin 2 to 3 days after administration.
Experimental example 1
Experimental mice were randomly divided into 7 groups: a normal group, a model group, a blank preparation control group (0 part of tetrandrine, the other materials are the same as in example 1, and the preparation method is the same as in example 1), a high concentration group (the gel of example 1), a medium concentration group (the gel of example 4), a low concentration group (the gel of example 5), and a positive drug control group.
The operation is as follows: the whole animal experiment lasts for 8 days, and the skin on the back of all mice is preserved and depilated by a shaver and depilatory cream on the first day, so that the exposed area of the skin on the back is more than 6cm2. Every morning thereafter all mice were smeared with Imiquimod (IMQ) cream except for the normal group, and the blank formulation control group, high concentration group, medium concentration group, low concentration group, positive drug control group were smeared with the corresponding drugs every afternoon every day starting on the third day at the same time as the mice were dosed by body weight: the high concentration is 9mg/kg, the medium concentration is 6mg/kg, and the low concentration is 3 mg/kg. Mice were monitored daily for body weight to ensure compliance with animal ethical criteria and their skin PASI scores were recorded (day 1 IMQ cream application on first day) and sacrificed on the last dayMice, skin tissue samples were taken for subsequent analysis.
(1) PASI score for skin lesion status in different groups of mice
The scoring results are shown in FIG. 1.
As can be seen from fig. 1, the high concentration group, the medium concentration group, and the low concentration group were able to suppress erythema and desquamation caused by IMQ molding, and the high concentration group exhibited a better suppression effect, as compared with the model group. Meanwhile, the three high-concentration groups, the medium-concentration group and the low-concentration group can reduce the PASI score, and the PASI score of the high-concentration group is the lowest.
(2) The skin of the back was photographed before the mice were sacrificed on the last day. The test results are shown in FIG. 2.
As can be seen from fig. 2, the mice in the model group, i.e., IMQ group, had red skin on their backs and white clumpy scales, as compared with the mice in the normal group, i.e., Control group. Compared with the IMQ group, the positive drug control group, namely the tacrolimus group, the high-dose medium-concentration group and the low-concentration group can relieve the skin redness and the scale caused by IMQ modeling to different degrees. Wherein the tacrolimus and the tetrandrine in the high concentration group can obviously reduce the skin damage condition of the psoriasis mouse.
(3) After the mice were sacrificed, the skin on the back was removed and immediately fixed in 4% PFA overnight, and the next day the skin was removed and washed with PBS and dehydrated in graded alcohol. Dehydrating, and permeabilizing in xylene to make skin slightly transparent. The treated skin was embedded in paraffin, cut to a thickness of 5 microns, dewaxed and hydrated, stained with 10% hematoxylin, differentiated with 0.5% hydrochloric acid ethanol, stained with 1% eosin solution, and sealed with neutral gum for observation of skin morphology. The results of the test are shown in FIG. 3.
As can be seen from fig. 3, the skin epidermal layer of the IMQ group mice was significantly thickened compared to the Control group mice. Compared with the IMQ group, the tacrolimus group, the high-concentration group, the medium-concentration group and the low-concentration group can inhibit the thickening of the skin epidermal layer caused by IMQ molding to different degrees, wherein the tacrolimus and the tetrandrine in the high-concentration group can obviously relieve the thickening of the epidermal layer.
(4) The rest skin is cut open by scissors, and is respectively packaged in a 2mL sterile freezing tube, immediately put into liquid nitrogen for quick freezing, and then transferred into a low-temperature refrigerator at minus 80 ℃ for storage to be tested, and the expression levels of mRNA of TNF, IL-1 β, IL-6 and IL-17 are analyzed by Realtime PCR.
Specifically, the main reagents: RNAiosolplus (TotalRNA extraction reagent),
Figure BDA0002317784240000141
RTreagentkitwitthDNAera (cDNA Synthesis kit),
Figure BDA0002317784240000142
PremixExTaqTM (TliRNaseHPlus) (real-timePCR kit) was purchased from Bao bioengineering (Dalian) Ltd.
The main apparatus is as follows: 7500Real-timePCR instrument (applied biosystems), gradient temperature raising and lowering PCR instrument (TaKaRa), DYY-6C constant voltage constant current electrophoresis instrument, Nanodrop2000 ultramicro spectrophotometer (Thermo), refrigerated centrifuge (Sigma), Tanon-4100 gel imaging system (Shanghai Tianneng).
Tissue total RNA extraction: total RNA was extracted from each tissue using RNAiosolplus, and the extraction procedure was performed exactly as described in the kit instructions. The RNA concentration is detected by a Nanodrop2000 ultramicro spectrophotometer, the purity is expressed by a light absorption value 260/280 and ranges from 1.8 to 2.2, the integrity of the RNA is detected by agarose gel electrophoresis, and the RNA sample is stored at the temperature of minus 80 ℃.
Synthesis of cDNA ① removal of genomic DNA into a 200. mu.L Eppendorf tube, 1. mu.gRNA, 2. mu.L 5 XGdnaEraser buffer and 1. mu.L gDNAeraser in this order and RNaseFreeDH were added2O to 10 μ L, reaction at 42 deg.C for 2min, ② reverse transcription reaction, 4 μ L5
Figure BDA0002317784240000143
Buffer，
Figure BDA0002317784240000144
RTenzymemix, 1.0. mu.LRTprimix, 10. mu.L of ① reaction solution, plus RNaseFrededdH2O to 20. mu.L. Reverse transcription parameters: 15min at 37 ℃, 5s at 85 ℃, infinity at 4 ℃, and placing the reaction product at-20 ℃ for freezing and storing for later use after the reaction is finished.
Real-time fluorescent quantitative PCR:the 20. mu.L reaction system used was composed of 10. mu.L
Figure BDA0002317784240000145
PremixExTaqTM(2×)、0.4μLROXreferencedyeII(10×)、2μLcDNA、6.8μLRNasefreeddH2O, 0.4. mu.L of upstream primer (10. mu. mol/L), 0.4. mu.L of downstream primer (10. mu. mol/L). Amplification was performed using an ABI7500Real-timePCR instrument. Circulation parameters: firstly, 30s at 95 ℃; subsequently, 95 ℃ for 5s, 60 ℃ for 34s, 40 cycles were carried out. Each sample was replicated 3 times. Calculation of relative fluorescent quantitative PCR data Using Livak and Schmitgen (2001) 2-ΔΔCTGAPDH was used as an internal reference gene. The relative expression level of mRNA of each gene was expressed as an expression level relative to the control group by a normalization method.
The published gene sequences of TNF, IL-1 β, IL-6, IL-17 and GAPDH of mice are used to design Real-time PCR primers, synthesized cDNA is used as a template, 7 concentration standards are diluted according to 2-fold gradient, each gene is amplified on a Real-time PCR instrument, Ct values and template concentration log values of each gene are used to make a standard curve to detect the amplification efficiency of each gene, the amplification efficiency of each gene of the test is close to 100%, and the detection results are shown in FIG. 4.
From FIG. 4, it can be seen that the mRNA level of skin cytokines in mice of the IMQ group was significantly increased compared to the mice of the Control group, and the mRNA expression level of IL17 and IL-1 β in the medium concentration group was suppressed in the IMQ molding, while the mRNA expression level of cytokines in the low concentration group was not suppressed in the IMQ molding, and the mRNA expression level of these cytokines was significantly suppressed in tacrolimus and tetrandrine in the high concentration group.
Experimental example 2
Experimental mice were randomly divided into 3 groups: normal group, model group, comparative group.
The operation is as follows: the whole animal experiment lasts for 8 days, and the skin on the back of all mice is preserved and depilated by a shaver and depilatory cream on the first day, so that the exposed area of the skin on the back is more than 6cm2. Every day thereafter all mice were given imiquimod cream in the morning, except for the normal group, and the blank formulation was given to the control group, the high concentration group, the medium concentration group, the low concentration groupAnd in the degree group, the positive drug control group is coated with the corresponding drugs every afternoon at the same time starting on the third day. The body weight of the mice was monitored daily to ensure compliance with animal ethical standards, and the mice were sacrificed on the last day and their skin was recorded by photography.
The test results are shown in fig. 5, which shows that the mice in the model group, i.e., IMQ group, had red skin and white scales on the back, compared to the mice in the normal group, i.e., Control group. Compared with the IMQ group, the comparative example group was completely unable to alleviate redness and scaling of the skin caused by IMQ molding and even aggravate the skin lesions in psoriatic mice.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A tetrandrine composition is characterized by comprising 0.005-0.015 parts of tetrandrine, 1 part of oil phase, 0.5 part of emulsifier, 7-8 parts of water phase and 1 part of auxiliary emulsion in parts by weight.
2. The tetrandrine composition according to claim 1, wherein the active ingredient of the tetrandrine composition is only tetrandrine and its derivatives.
3. The tetrandrine composition according to claim 1, wherein the tetrandrine composition is a nanoemulsion with a nano particle size;
preferably, the particle size of the nanoemulsion is 30-50 nm.
4. The tetrandrine composition according to any one of claims 1 to 3, wherein the oil phase comprises at least one of unsaturated oil, fatty acid ester-based compound and oil-based polymer;
preferably, the unsaturated oil comprises any one of soybean oil, palm oil, linseed oil and castor oil;
preferably, the fatty acid ester compound comprises any one of isopropyl myristate, caprylic capric triglyceride, polyethylene glycol laurate, polyethylene glycol oleate, polyethylene glycol stearate, glyceryl triacetate, ethyl oleate, glyceryl monostearate and isopropyl palmitate;
preferably, the grease polymer is polyethylene glycol glyceryl oleate.
5. The tetrandrine composition according to claim 4, wherein the emulsifier comprises a surfactant;
preferably, the surfactant includes any one of a nonionic surfactant, a zwitterionic surfactant, a cationic surfactant and an anionic surfactant;
preferably, the nonionic surfactant includes any one of polyoxyethylene hydrogenated castor oil, polyoxyethylene fatty acid compounds, polyoxyethylene fatty alcohol ether compounds, fatty acid glyceride compounds, polysorbate compounds, sorbitan fatty acid compounds, polyoxyethylene-polyoxypropylene copolymer compounds and PEG; more preferably, the PEG is PEG 400;
preferably, the zwitterionic surfactant comprises any one of soybean lecithin, whey protein isolate and lactoferrin;
preferably, the cationic surfactant comprises any one of cetyltrimethylammonium bromide, lauroyl arginine ethyl ester and dodecyltrimethylammonium bromide;
preferably, the anionic surfactant includes any one of sodium dodecyl sulfonate and sodium dodecyl sulfate.
6. The tetrandrine composition according to claim 5, wherein the co-emulsion comprises any one of alcohol and modified polyethylene glycol;
preferably, the modified polyethylene glycol compound is lithium polyethylene glycol dodecahydroxy stearate;
preferably, the alcohol species includes monohydric alcohols and polyhydric alcohols;
preferably, the monohydric alcohol comprises any one of ethanol and n-butanol;
preferably, the polyol includes any one of 1, 2-propanediol and glycerol.
7. A process for the preparation of a tetrandrine composition according to any one of claims 1 to 6, comprising: mixing the oil phase, the emulsifier and the auxiliary emulsion, and then mixing with the tetrandrine and the water phase to form the tetrandrine composition.
8. A tetrandrine external preparation comprising the tetrandrine composition of any one of claims 1 to 6;
preferably, the dosage form of the tetrandrine external preparation comprises any one of nano cream, gel, ointment, lotion, patch and film agent;
more preferably, when the tetrandrine external preparation is in the form of gel, the pH of the gel is 6-8.
9. A method for preparing the tetrandrine external preparation of claim 8, which comprises the following steps when the tetrandrine external preparation is gel: mixing the tetrandrine composition of any one of claims 1-6 or the tetrandrine composition prepared by the method of claim 7 with the swollen gel matrix;
preferably, the preparation method further comprises: mixing the tetrandrine composition with the swollen gel matrix, adjusting the pH, and adjusting the pH to 6-8;
preferably, the preparation of the swollen gel matrix comprises: mixing a thickener with a solvent until the thickener is fully swollen;
preferably, the mass content of the thickening agent in the gel is 0.8% -2%.
10. Use of a tetrandrine composition according to any one of claims 1-6 in the manufacture of a medicament for the treatment of psoriasis.
CN201911285177.XA 2019-12-13 2019-12-13 Tetrandrine composition, preparation method thereof, tetrandrine external preparation, preparation method and application thereof Pending CN110859842A (en)

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