CN117752604A - Compound micro-emulsion gel preparation for treating acne vulgaris and preparation method thereof - Google Patents

Abstract

The invention relates to a compound micro-emulsion gel preparation for treating acne vulgaris and a preparation method thereof, belonging to the technical field of medicines. The material consists of the following raw materials in percentage by mass: 98-99% of microemulsion and 1-2% of gel matrix, wherein the microemulsion is prepared from the following raw materials in percentage by mass: 5% -19.95% of oil phase, 0.7% -14% of emulsifying agent, 1.4% -28% of auxiliary emulsifying agent and 53% -77% of water phase, wherein the water phase is an aqueous solution prepared from ethanol extracts of rheum officinale, coptis chinensis, scutellaria baicalensis and liquorice. The invention has good biocompatibility, the matrix material in the microemulsion gel is usually natural or synthetic polymer or biological macromolecule with high biocompatibility, has good adsorption capacity, can be well absorbed by skin, has convenient use, definite curative effect and small toxic and side effect, has good drug delivery and controlled release characteristics, small droplet size of the microemulsion, large drug loading capacity and strong adsorptivity, and can lead the drug to achieve the slow release effect by adding the gel.

Description

Compound micro-emulsion gel preparation for treating acne vulgaris and preparation method thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a traditional Chinese medicine compound micro-emulsion gel preparation for treating acne vulgaris and a preparation method thereof.

Background

Acne vulgaris (acne vulgaris) is a chronic inflammatory disease of the skin with a complex pathogenesis. Since more acne is obtained in puberty, common acne is called as "whelk", the incidence rate is up to 40% -70%, but when acne vulgaris is obtained, not only in puberty, but also with the increase of life pressure, improvement of diet condition and other factors, the number of men and women in middle-aged men is increased. The chinese guidelines for acne treatment (revised 2014) considers that the pathogenesis of acne is mainly related to four factors, namely seborrheic hyperseborrhoea, follicular keratinization abnormality, propionibacterium acnes proliferation and inflammatory reaction, while propionibacterium acnes mass proliferation in the pilosebaceous glands is an important factor in the pathogenesis of acne.

Propionibacterium acnes (Propionibacterium acnes) is a common gram-positive baculous bacterium and is now known as keratobacterium cutanea (Cutibacterium acnes) according to the latest classification. The bacterium itself is less toxic, grows more rapidly in anaerobic environments at 30-37 ℃ and ph7.0, and is itself found in human hair follicles. At present, propionibacterium acnes infection is widely considered to be the main cause of acne formation.

Staphylococcus aureus (Staphylococcus aureus) is a gram-positive coccus, which causes folliculitis in skin diseases, and can also be found in lesions of acne, enter the human body to cause tissue necrosis to form cysts, and enter blood and lymph to cause septicemia.

In recent years, in order to better solve the chronic inflammatory skin diseases involving the sebaceous glands of hair follicles caused by acne, intensive studies have been conducted by the traditional Chinese and western medicine. Modern medical research shows that propionibacterium acnes is an important target for treating acne vulgaris, and medicines such as antibiotics, hormone and the like are generally used for clinically treating acne vulgaris. The chemical drug treatment mainly comprises tretinoin; antibiotics such as clindamycin, erythromycin, chloramphenicol and the like are used for local external medicine treatment, tetracycline, macrolide, antiandrogen oral medicine treatment and the like, and antibacterial therapy is used for treating acne vulgaris quite commonly, but the common antibiotics have the problems of poor absorption, easy bacteria generation, high drug resistance rate and the like, and have gastrointestinal tract reaction or certain side effects such as hepatorenal toxicity and the like. However, the traditional Chinese medicine external preparation has certain defects, such as ointment and cream can cause the obstruction of pilosebaceous glands and even aggravate the illness; the lotion is easily affected by sweat to reduce the adhesive force of the medicine and affect the medicine effect; tincture has a pungent smell and a large skin irritation, so that a new way for resisting bacteria is necessary.

Clinically, the traditional Chinese medicine has remarkable curative effect of treating the acne vulgaris, has less adverse reaction, and is helpful for searching the mechanism of the traditional Chinese medicine for treating the acne vulgaris from the modern medical angle, thereby providing a new basis and thought for treating the acne vulgaris by the traditional Chinese medicine.

Acne vulgaris is known as "acne" in traditional Chinese medicine, and is classified as "acne due to wind of lung". The traditional medicine considers that the common acne is caused by three factors of lung heat, blood heat and wind heat, damp heat is accumulated in the body due to wind heat affecting the lung and spleen and stomach movement and transformation disorder, the skin is affected by fumigation due to the overuse of the wind heat, and modern doctors put forward new views of blood stasis, damp heat and the like on the basis of the original theory. Sanhuang Xixin decoction has the functions of purging fire, removing toxin, eliminating dampness and relieving heat, is a common prescription for treating surgical sores and ulcers, and is a representative prescription in the fire purging agent. Rhubarb, coptis root and baikal skullcap root can release the fire of triple energizer, so excessive fire of triple energizer or invisible heat evil can be treated by Sanhuang Xiexin decoction in clinical application, so that a plurality of doctors use it to treat acne.

Sanhuang Xixin Tang comes from jin Kui Yao Lv Jing Yi Sheng Zhang Zhong Jing (the first medical science of the decoction of Sanhuang Xixin Tang for treating liver fire and surgical treatment of sores and ulcers. Has effects of purging pathogenic fire, removing toxic substances, eliminating dampness and clearing heat. The rheum officinale, the coptis chinensis and the scutellaria baicalensis in the soup are bitter and cold in nature, have the effects of diminishing inflammation, clearing heat, purging triple energizer, and the scutellaria baicalensis acts on upper energizer, and has the effects of purging lung and clearing intestine; coptis chinensis rhizoma acts on the middle energizer to relieve heart and clear stomach; da Huang acts on lower energizer to purge fire and purge fu organs, and can purge fire and detoxify, resolve dampness and dispel heat.

Coptis chinensis Franch (Coptis chinensis Franch.) is bitter in taste and cold in nature. It enters heart and spleen meridians. Clear heat and dry dampness, purge fire and detoxify. The coptis chinensis has the function of broad-spectrum antibiotics, and 4 coptis chinensis alkaloids including berberine, coptisine, palmatine and jateorhizine are all antibacterial active ingredients of the coptis chinensis, have basically the same antibacterial capacity on drug-resistant staphylococcus aureus and staphylococcus aureus, and have the function of eliminating drug resistance of the drug-resistant staphylococcus aureus.

Rhubarb (Rheum palmatum L.) is bitter and cold in nature, and the main antibacterial components are aloe-emodin and rhein, bitter in taste and cold in nature. Enter kidney and bladder meridians. Has the effects of clearing heat and drying dampness, purging fire and removing steam, and detoxifying and curing sores, and researches show that the coptis chinensis and the rheum officinale have strong antibacterial effect on propionibacterium acnes, and provide basis for developing new medicines for treating acnes.

The baical skullcap root (Scutellaria baicalensis Georgi) has slight smell and bitter taste, and flavonoid compounds baicalin and baicalein in the baical skullcap root have good antibacterial effect on common skin disease pathogenic bacteria, and can effectively treat skin itch, detumescence and inflammation removal after being added into soap and toothpaste; can be added into cosmetics for preventing and treating acne, etc.

Licorice (Glycyrrhiza uralensis Fisch.) is sweet in taste, mild in nature, good in spleen and qi, clear away heat and toxic materials, dispel phlegm and relieve cough, slow in urgency and stop pain, and harmonizes the medicines. The active ingredients of Glycyrrhrizae radix mainly comprise flavonoids and triterpene saponins, and have various biological activities such as antioxidant, antiviral, antibacterial, anticancer or antiinflammatory effects, liver protection, etc. The literature shows that licorice shows obvious antibacterial effect and almost no drug resistance when being used for treating acne. Research of Yang G and the like reports that liconone A extracted from licorice root can block P.acnes induced mouse macrophages and human SZ95 sebocytes from producing caspase-1 (p 10) and IL-1 beta, inhibit activation of NLRP3 inflammatory corpuscles and effectively inhibit occurrence of acne. Clinically, common fruit acid and licorzinc particles are used for treating acne vulgaris, and isotretinoin and licorzinc are used for treating acne with higher effective rate than isotretinoin alone.

The traditional decoction is applied to acne treatment, has obvious effect, but has long administration period and influences the treatment effect due to the injury to intestines and stomach and the first pass effect of liver, and according to the theory of traditional Chinese medicine: the traditional Chinese medicine can directly reach the affected part by external treatment, has the characteristic of rapid effect, combines the traditional Chinese medicine compound with the modern pharmaceutical technology, researches external preparations through proportion to treat acne, has convenient use, reduces toxic and side effects, improves the efficacy, is not easy to generate drug resistance, and has wide application prospect.

Microemulsion gels (MBGs) are new dosage forms that have been developed over the last decade, in which microemulsions are added to a gel matrix composed of natural polymers, cellulose derivatives, block polymers, etc., to form a transparent, homogeneous, stable gel network containing droplets of microemulsions.

Disclosure of Invention

The invention provides a compound microemulsion gel preparation for treating acne vulgaris and a preparation method thereof, which aim to improve the transdermal property of a medicament and have small toxic and side effects, microemulsion is adopted as a medicament carrier in a formula, and the characteristics of a gel agent are combined to prepare the flavored Sanhuangxiexin decoction microemulsion gel agent, so that the use of a large amount of grease can be avoided, the risk of pore blockage and bacterial growth is reduced, and the skin feel is fresh.

In the recipe, rhubarb has the characteristics of cold property and bitter taste, is a monarch drug, and can clear heat and purge fire, cool blood and detoxify, and lead fire to descend; coptis chinensis has cold property and bitter taste, and is used as ministerial drug for purging heart and clearing stomach, and eliminating dampness and detoxifying; baical skullcap root, radix Scutellariae is cold in nature and bitter in taste, and has the effects of purging lung-heat, eliminating dampness and removing toxin, and is used as an adjuvant drug; the liquorice with flat nature is used for detoxification, and the liquorice is used for assisting the monarch and minister in nourishing yin, clearing heat and detoxifying, and the medicines are combined to form a mild and non-irritating traditional Chinese medicine preparation, so that the effects of clearing heat, resolving masses, clearing heat and detoxifying can be achieved, and the effects of inhibiting Propionibacterium acnes and staphylococcus aureus are obvious; the traditional Chinese medicine composition has the advantages of high clinical curative effect, quick response, low recurrence rate, safety, reliability and no obvious adverse reaction through compatibility of medicines.

The invention adopts the technical scheme that the material consists of the following raw materials in percentage by mass: 98-99% of microemulsion and 1-2% of gel matrix, wherein the microemulsion is prepared from the following raw materials in percentage by mass: 5% -19.95% of oil phase, 0.7% -14% of emulsifying agent, 1.4% -28% of auxiliary emulsifying agent and 53% -77% of water phase, wherein the water phase is an aqueous solution prepared from ethanol extracts of rheum officinale, coptis chinensis, scutellaria baicalensis and liquorice.

The microemulsion is O/W type microemulsion.

The water phase is prepared from the following raw materials in parts by weight: 10-20 parts of rheum officinale, 10-20 parts of coptis chinensis, 5-10 parts of scutellaria baicalensis and 1-5 parts of liquorice, adding 75% ethanol for ultrasonic extraction, removing excessive ethanol by a rotary evaporator, and fixing the volume by distilled water until the crude drug content is 1g/mL.

The oil phase is ethyl oleate or isopropyl myristate.

The emulsifier is one or more of Tween-80, polyoxyethylene hydrogenated castor oil CO-40 and polyoxyethylene ether castor oil-40.

The auxiliary emulsifier is one or more of polyethylene glycol 400, 1,2 propylene glycol and isopropanol.

The gel matrix is carbomer 940.

The average grain diameter of the microemulsion gel is 56.32+/-3.55-97.19 +/-2.52 nm, and the polydispersity PDI is below 0.31+/-0.01.

A preparation method of a compound micro-emulsion gel preparation for treating acne vulgaris, which comprises the following steps:

(1) 10-20 parts of rheum officinale, 10-20 parts of coptis chinensis, 5-10 parts of scutellaria baicalensis and 1-5 parts of liquorice are taken according to parts by weight, 75% ethanol is added according to the weight-volume ratio of 1:10 feed liquid, ultrasonic extraction is carried out twice, each time for 1h, the two extraction solutions are combined, redundant ethanol is removed by a rotary evaporator, distilled water is used for constant volume until the content of crude drugs is 1g/mL, and the mixture is used as a water phase for standby;

(2) Taking 5% -19.95% of oil phase, 0.7% -14% of emulsifying agent and 1.4% -28% of auxiliary emulsifying agent according to mass percentage, placing the water phase 53% -77% obtained in the step (1) into a beaker, stirring the emulsifying agent and the auxiliary emulsifying agent for 10min by using a magnetic stirrer to completely and uniformly mix, adding the oil phase, continuously stirring until the oil phase is uniformly mixed, slowly dripping the water phase into a mixed system, and stirring for 20min to obtain clear and transparent microemulsion;

(3) And taking 98-99% of the obtained microemulsion according to the mass percentage, adding 1-2% of gel matrix, stirring and fully swelling for 12-16h, and regulating the pH value by triethanolamine to 4-5 to obtain the microemulsion gel preparation.

The weight ratio of the emulsifier to the auxiliary emulsifier in the step (2) is 1:2.

The active ingredients of the invention are the traditional named formula Sanhuang heart-purging decoction: the radix scutellariae, the rheum officinale and the coptis root are compatible with the liquorice, so that the synergistic effect of the components can be effectively exerted, and compared with the effect of singly serving as an active component, the effect is obvious. The liquorice is added into the Sanhuang Xixin decoction, the liquorice plays a role in harmonizing the drug property, and the Sanhuang Xixin decoction is used for treating symptoms such as excessive fire of triple energizer or invisible heat evil internal force in clinical application, and can treat acne. In addition, the microemulsion is a thermodynamic stability system consisting of a water phase, an oil phase, an emulsifier and an auxiliary emulsifier, has the particle size of between 10 and 100nm, is favorable for skin absorption, improves the utilization rate of medicines, has strong fluidity, can change the current situation by reasonably adding a gel, has good quoting value for skin diseases, replaces the water phase in the microemulsion with an flavored Sanhuangxiexin decoction extract, improves the drug loading rate, has good coating property of the obtained microemulsion gel, has small skin irritation, is convenient and fast to use by patients, has high safety, and is suitable for skin-sensitive acne patients.

The beneficial effects of the invention are as follows:

1) Provides a medicinal preparation for treating acne, which has the advantages of convenient use, definite curative effect and small toxic and side effects.

2) The invention has good biocompatibility, the matrix material in the microemulsion gel is usually natural or synthetic polymer or biological macromolecule with high biocompatibility, and negative charge of the flavored Sanhuang Xixin decoction microemulsion gel tape is known through Zeta potential, which shows that the invention has good adsorption capacity and can be absorbed by skin well.

3) The invention has higher stability, the gel structure in the microemulsion gel can ensure that the particle size of the microemulsion is between 1 and 100nm under the conditions of different temperatures, pH values and the like, the PDI dispersion index is smaller than 0.3, the particle size is uniform, the obtained microemulsion has higher stability, and the preparation process is simple and can be used for large-scale industrial production.

4) The invention has good drug delivery and controlled release characteristics, the microemulsion has small drop size, large drug-loading capacity and strong adsorptivity, and the gel is added to enable the drug to achieve the slow release effect.

5) The invention verifies that the flavored Sanhuang Xixin decoction microemulsion gel has obvious treatment effect on common acne from three aspects of in vitro, animals and human bodies, and widens the application prospect of the flavored microemulsion gel. The in vitro antibacterial rates of the microemulsion gel on propionibacterium acnes and staphylococcus aureus are 91.22 +/-1.01% and 95.85+/-2.10% respectively, and the antibacterial effect is obvious, so that the prescription has good in vitro antibacterial effect on main pathogenic bacteria of acnes; the Kligman method is adopted to model the mice, the repairing condition of the flavored Sanhuang Xixin decoction microemulsion gel on the acne model is examined, and the results show that the skin horny layer of the mice is normal after administration, and the inflammation is eliminated, so that the traditional Chinese medicine compound microemulsion gel can treat inflammation acne, and has obvious effect; the method has the advantages that the proper volunteer is selected for trial, compared with other medicines, the number of white comedones, blackheads, papules, cyst nodules and pustules on the face is obviously reduced, the secretion of grease is reduced, and the inflammation of the skin is relieved, so that the method can well inhibit the propionibacterium acnes from propagating, inhibit the secretion of skin grease, inhibit the diffusion of acne and improve the influence of the acne on the face.

Drawings

FIG. 1 is a pseudo-ternary phase diagram of a microemulsion at different Km;

FIG. 2 is a microemulsion particle size distribution and Zeta potential diagram;

FIG. 3 is a graph of the tyndall effect of blank and medicated microemulsions;

FIG. 4 is a transmission electron microscope image of a blank microemulsion and a medicated microemulsion;

FIG. 5 is a graph of the tyndall effect for blank microemulsions and microemulsion gels;

FIG. 6 is a graph of an antibacterial test of the present invention;

FIG. 7 is a bar graph showing the sterilization rate of propionibacterium acnes and staphylococcus aureus according to the invention,

FIG. 8 is a H & E staining pattern of mouse tissue;

FIG. 9 is a H & E staining chart of mouse skin;

fig. 10 is a graph of a guinea pig stimulation experiment, in which a blank matrix is left and a microemulsion gel is right.

Detailed Description

Example 1

(1) The components in parts by weight: 10 parts of rheum officinale, 10 parts of coptis chinensis, 5 parts of scutellaria baicalensis and 1 part of liquorice, adding 75% ethanol according to the weight-volume ratio of 1:10 feed liquid, carrying out ultrasonic extraction twice, each time for 1h, combining the two extracting solutions, removing excessive ethanol by using a rotary evaporator, and using distilled water to fix the volume until the crude drug content is 1g/mL to serve as a water phase for standby;

(2) Taking 19.95% of oil phase, 1.65% of emulsifier and 1.4% of auxiliary emulsifier according to weight percentage, and 77% of water phase obtained in the step (1), placing the emulsifier and the auxiliary emulsifier into a beaker, stirring for 10min by using a magnetic stirrer to completely and uniformly mix, adding the oil phase, continuously stirring until the oil phase is uniformly mixed, slowly dripping the water phase into a mixed system, and stirring for 20min to obtain clear and transparent microemulsion;

wherein the oil phase is ethyl oleate, the emulsifying agent is tween-80 and polyoxyethylene hydrogenated castor oil CO-40, and the auxiliary emulsifying agent is polyethylene glycol 400 and 1,2 propylene glycol;

(3) And taking 98% of the obtained microemulsion according to the mass percentage, adding 2% of a gel matrix, wherein the gel matrix is carbomer 940, stirring and fully swelling for 12 hours, and regulating the pH value to 4 by triethanolamine to obtain the microemulsion gel preparation.

Example 2

(1) 15 parts of rheum officinale, 15 parts of coptis chinensis, 7.5 parts of scutellaria baicalensis and 3 parts of liquorice are taken according to parts by weight, 75% of ethanol is added according to the weight-volume ratio of 1:10 feed liquid, ultrasonic extraction is carried out twice, each time for 1h, the two extraction solutions are combined, redundant ethanol is removed by a rotary evaporator, distilled water is used for constant volume until the content of crude drugs is 1g/mL, and the obtained solution is used as a water phase for standby;

(2) Taking 5% of oil phase, 14% of emulsifier and 28% of auxiliary emulsifier according to mass percentage, placing the water phase 53% obtained in the step (1) into a beaker, stirring for 10min by using a magnetic stirrer to completely and uniformly mix the emulsifier and the auxiliary emulsifier, adding the oil phase, continuously stirring until the oil phase and the auxiliary emulsifier are uniformly mixed, slowly dripping the water phase into a mixed system, and stirring for 20min to obtain clear and transparent microemulsion;

the oil phase is isopropyl myristate, the emulsifier is tween-80, and the auxiliary emulsifier is polyethylene glycol 400;

(3) Taking 98.5% of the obtained microemulsion according to the mass percentage, adding 1.5% of a gel matrix, wherein the gel matrix is carbomer 940, stirring and fully swelling for 14 hours, and regulating the pH value to be 4.5 by triethanolamine to obtain the microemulsion gel preparation.

Example 3

(1) Taking 20 parts of rheum officinale, 20 parts of coptis chinensis, 10 parts of scutellaria baicalensis and 5 parts of liquorice according to the weight-to-volume ratio of 1:10, adding 75% ethanol, carrying out ultrasonic extraction twice, each time for 1h, combining the two extracting solutions, removing excessive ethanol by using a rotary evaporator, and using distilled water to fix the volume until the crude drug content is 1g/mL, and taking the mixture as a water phase for standby;

(2) Taking 15% of oil phase, 7% of emulsifier and 15% of auxiliary emulsifier according to mass percentage, placing the water phase 63% obtained in the step (1) into a beaker, stirring for 10min by using a magnetic stirrer to completely and uniformly mix the emulsifier and the auxiliary emulsifier, adding the oil phase, continuously stirring until the oil phase and the auxiliary emulsifier are uniformly mixed, slowly dripping the water phase into a mixed system, and stirring for 20min to obtain clear and transparent microemulsion;

the oil phase is isopropyl myristate, the emulsifier is polyoxyethylene ether castor oil-40, and the auxiliary emulsifier is isopropanol;

(3) And taking 99% of the obtained microemulsion according to the mass percentage, adding 1% of a gel matrix, wherein the gel matrix is carbomer 940, stirring and fully swelling for 16 hours, and regulating the pH value to 5 by triethanolamine to obtain the microemulsion gel preparation.

The microemulsion is O/W type microemulsion.

The average particle diameter of the microemulsion gel is 56.32+/-3.55-97.19 +/-2.52 nm, and the polydispersity PDI is below 0.31+/-0.01.

The invention is further illustrated by the following specific experimental examples.

Experimental example 1 selection of oil phase, emulsifier, co-emulsifier

Examination of solubility of each phase, preparing the aqueous solution of the compound extract into lyophilized powder, and respectively analyzing the solubility of the compound extract in oil phase (isopropyl myristate, ethyl oleate, and olive oil), emulsifier (Tween-80, polyoxyethylene hydrogenated castor oil CO-40, polyoxyethylene ether castor oil-40) and coemulsifier (polyethylene glycol 400, 1,2 propylene glycol, and isopropanol). At room temperature, adding 1mL of solvent (oil phase, emulsifier and co-emulsifier) into a centrifuge tube with a capacity of 5mL, adding 1g of flavored Sanhuangxiuxin decoction compound freeze-dried powder into the solvent, oscillating each solvent for 30s in a water bath at the room temperature of 25+/-2 ℃ for 30s every 5min, repeatedly carrying out for 30min, and observing whether solute particles exist within 30 min. I.e. the solute is completely dissolved in the solution and after standing there is a partial precipitation of solids. The mixed EP tubes were then placed in a water bath shaker at room temperature for overnight shaking for 48h, and whether supersaturation was absorbed was observed. After confirming the supersaturation, the respective EP tubes containing the solvent were centrifuged at 12000g/min for 10min to obtain supernatants, and after dilution by the same factor, concentration analysis was performed on an ultraviolet spectrophotometer at an absorption wavelength of 280nm, and each sample was tested three times.

Light transmittance measurement

Analysis was performed based on the above solubility results. The light transmittance of each oil phase, emulsifier and co-emulsifier was measured separately, and the oil phase obtained was mixed with the emulsifier and co-emulsifier, respectively, in a ratio of 1:1. The most soluble emulsifier was then mixed with the co-emulsifier separately (1:1), and the mixture was tested for light transmittance (T%) using an ultraviolet spectrophotometer (TU-1901) over the entire wavelength range of 600-190 nm. When the light transmittance is less than 90%, it is indicated that the two-phase agent is not miscible, and that the inclusion of emulsifiers and co-emulsifiers in the mixture of less than 90% is not suitable as an alternative agent. All samples were measured three times individually.

Table 1 oil phase emulsifier and co-emulsifier light transmittance and solubility magnitudes

TABLE 2 schematic table of light transmittance after mixing oil phase with each emulsifier, co-emulsifier

TABLE 3 schematic table of light transmittance after mixing the emulsifier and co-emulsifier

Determination of Km ratio and ratio of oil phase to mixed emulsifier by pseudo ternary phase diagram

The pseudo ternary phase diagram is an important basis for prescription screening. The solubility of the flavored Sanhuangxiex decoction extract in the oil phase, the surfactant and the cosurfactant is determined by screening, isopropyl myristate and ethyl oleate are used as the oil phase, tween-80 and CO-40 are used as emulsifying agents, and polyethylene glycol and 1, 2-propylene glycol are used as the cosurfactants to perform titration of the microemulsion formula. A pseudo-ternary phase diagram of oil, emulsifier, co-emulsifier and water is established at normal temperature by adopting a water titration method. Referring to fig. 1, the mixture is fully stirred for 10min after the mass ratio (Km) of the emulsifier to the auxiliary emulsifier is 3:1, 2:1, 1:1, 1:2 and 1:3, and then the mixture is fully stirred for 10min after the mass ratio (Km) of the oil phase to the auxiliary emulsifier is 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1. The compound water extract of the traditional Chinese medicine is added into each mixture drop by drop under the stirring of continuous mechanical force 600r/min at the room temperature of 25 ℃. The mixture system was observed visually from clear to turbid, and from turbid to clear critical point at different ratios, and the amount of water added at the critical point was recorded. The clear and transparent mixture was identified as microemulsion. The oil phase, km and distilled water were accurately weighed and recorded, 3 vertices of a pseudo ternary phase diagram were respectively made with the oil phase, km and water phase, and the pseudo ternary phase diagram was drawn using OriginPro 9.0 software to determine the microemulsion region. Conclusion: and selecting and obtaining the optimal oil phase Km according to the size of the microemulsion area of the pseudo ternary phase diagram. Conclusion: mixing tween-80 and polyethylene glycol 1:2, and mixing oil phase isopropyl myristate with mixed emulsifier 5:42, a clear and transparent microemulsion was obtained.

Experimental example 2, examination of physicochemical Properties of microemulsion

The obtained microemulsion (without medicine) is colorless, clear and transparent solution, has good fluidity and uniform dispersion, and the medicated microemulsion is brown, and has particle diameter, PDI and Zeta potential

TABLE 4 microemulsion light transmittance, particle size, PDI and Zeta potential

And (3) checking to obtain: the particle size of the microemulsion is between 1 and 100nm, and the PDI dispersion index is smaller than 0.3, so that the obtained microemulsion is stable, uniform in particle size and low in Zeta potential, and has good adsorption capacity and can be well absorbed by skin. (see FIG. 2)

The tyndall effect of the microemulsion is shown in figure 3.

The tyndall effect reveals interactions and kinetic behavior between tiny droplet micelles composed of emulsifier and co-emulsifier molecules and dispersed phase water, indicating that stable dispersions of micelles in microemulsions can form highly dispersed colloids between oil and water.

The pH value of the microemulsion is measured to be between 4 and 6, and the microemulsion can be used for skin administration.

The form of the microemulsion is observed through a transmission electron microscope (HITACHI HT7800/HT 7700), the particle size of the microemulsion is observed to be uniform through the transmission electron microscope, and is below 100nm, as shown in figure 4, after the water phase is replaced by the traditional Chinese medicine extract, the medicine is wrapped by the microemulsion, and the microemulsion has small particle size, can be well absorbed by the skin after being administrated to the skin, and improves the medicine availability and the treatment effect.

Experimental example 3 microemulsion stability examination

Light stability test

Placing the prepared microemulsion in a transparent glass bottle, sealing and preserving under a fluorescent lamp, sampling at 10d, 20d and 30d respectively, observing appearance morphology change, measuring particle size and PDI by using a Markov particle size meter, and not finding any change, thus indicating that the prepared microemulsion has light stability.

Temperature versus microemulsion stability test

Placing the prepared microemulsion into a dark glass bottle, respectively preserving at 4 ℃ and 37 ℃ for 30d, sampling, observing appearance morphology change, measuring particle size and PDI by a Markov particle size meter, and not finding any change, thus indicating that the prepared microemulsion has temperature stability.

After the gel is added into the microemulsion, the dosage form is not changed through the Tyndall effect, and various stability in the system is proved. As in fig. 5.

Experimental example 4 evaluation of in vitro antibacterial action of the invention

Propionibacterium acnes (ATCC 6919 Propionibacterium acnes), staphylococcus aureus (ATCC 6538Staphylococcus aureus) were purchased from northlasis. Gram-positive staining, facultative anaerobic, culture temperature 37 ℃.

Preparation of the culture medium:

LB liquid medium: 1g of tryptone, 1g of NaCl, 0.5g of yeast extract are weighed into a 200mL conical flask, 100mL of distilled water is added, 1.5g of agar is added to a solid medium, and the mixture is autoclaved at 121 ℃ for 15min.

Preparation of BHI culture medium: 3.85g of BHI culture medium dry powder is weighed, placed in a 200mL conical flask, 100mL of distilled water is added, 1.5g of agar is added to the solid culture medium, and the mixture is autoclaved at 121 ℃ for 15min.

Bacterial culture

Taking 10 mu L of frozen staphylococcus aureus, inoculating to 50mL of liquid culture medium, placing in a constant temperature incubator at 37 ℃ for 120r/min, culturing for 12h, adding anaerobic gas producing bag into an anaerobic bag, and culturing at 180r/min at 37 ℃ in the constant temperature incubatorTaking a proper amount of bacterial suspension before the test for 12 hours, diluting with sterile PBS, and regulating the concentration of bacterial liquid at OD600nm by an ultraviolet spectrophotometer to ensure that the concentration of the test bacteria is 1 multiplied by 10 ⁷ cfu/mL。

Determination of the Sterilization Rate

1g of the invention is weighed and added into 4.9mL of sterile PBS, then 0.1mL of the bacteria to be tested is added into the mixed system, after being uniformly mixed, 0.1mL of the bacteria to be tested is placed into a flat plate and is cultured for 12 hours in a constant temperature incubator at 37 ℃, the metronidazole gel is used as positive control according to the same method, physiological saline is used as blank control, the colony number in each flat plate is recorded, the sterilization rate of the traditional Chinese medicine compound is calculated, and the test is repeated for three times. See fig. 6.

As shown in figure 7, the invention has the inhibition effect on propionibacterium acnes and staphylococcus aureus, the bacterial quantity is obviously reduced after administration, the compound has obvious inhibition effect on propionibacterium acnes and staphylococcus aureus, and the sterilization rates are 91.22 +/-1.01% and 95.85+/-2.10% respectively, which indicates that the prescription has good inhibition effect.

Minimum Inhibitory Concentration (MIC) determination

Taking 8 sterilized test tubes, diluting the medicines to the concentration of 500, 250, 125, 62.5, 31.25 and 15.625mg/mL by a double dilution method, placing filter paper sheets in solutions with different concentrations for 8h, sterilizing at 121 ℃ for 15min under high pressure, taking 100 mu L of bacteria liquid to be tested as blank control, uniformly coating the bacteria liquid on an agar plate, airing, flatly attaching the filter paper sheets on the agar plate, placing propionibacterium acnes in an anaerobic culture box, adding an anaerobic gas production bag for culture, culturing in a constant-temperature incubator at 37 ℃ for 16-18h, observing the diameter of a bacteria inhibition zone, wherein the concentration of the liquid medicine corresponding to the minimum bacteria inhibition zone is the minimum bacteria inhibition concentration, and repeating the test three times.

Table 5 MIC test results of microemulsion gels on propionibacterium acnes and staphylococcus aureus (n=3, mg/mL)

As can be seen from the table, the MIC values of the flavored three-yellow heart disease decoction micro-emulsion gel group of the invention on propionibacterium acnes and staphylococcus aureus are lower than those of the traditional three-yellow heart disease decoction micro-emulsion gel group, and the MIC values of the flavored three-yellow heart disease decoction micro-emulsion gel group of the invention on propionibacterium acnes and staphylococcus aureus are respectively 62.5mg/mL and 31.25mg/mL.

Safety evaluation of the invention

And (3) performing stability inspection according to the quality requirements of the gel in the pharmacopoeia of the people's republic of China (2020 edition), inspecting the heat-resistant and cold-resistant layering phenomenon of the gel, and judging the stability of the gel.

The invention is safe and pharmacodynamic

Dividing 24 ICR mice (license number SCXK 2018-0007) into blank group, model group, administration group and toxicological group, establishing acne model by Kligma method, dehairing the mice, smearing oleic acid on the back of the mice, and subcutaneously injecting 1×10 every other day ⁸ 20 mu L of Propionibacterium acnes, stopping molding after 14 consecutive days, and observing skin morphology change of mice after continuous administration for 20 days with a dosage of 1 g/dose each time in the morning and evening, wherein the skin roughness of mice is observed, and the mice are marked by H&E staining observed pathological changes, toxicological groups were not treated and were dosed in the same manner.

Morphological observation

The acne part was visually observed 12 hours after the last administration, and the determination was made based on the preparation standard (draft) of the acne animal model and the actual conditions. The visual inspection shows that the back skin of the normal group mice is thinner, the surface is smooth and soft, white villus is clearly visible, the hair follicle is smooth, and no skin damage phenomenon occurs. The back skin of the model group mice is seriously red and swollen, the hand touch hardness and thickness are obviously higher than those of the normal group mice, the skin has red spots, the surface hair follicle has serious cornification, and the skin is scattered on micro acne. Compared with the model group, the red swelling of the back skin of the mice in the other groups is reduced, the hardness and thickness of hand touch are improved to different degrees, and the acne is basically disappeared, as shown in figure 8.

According to pharmacodynamics evaluation, H & E staining is carried out on the tissue of the toxicological group mice, no pathological change is seen, and the traditional Chinese medicine compound microemulsion gel has no toxicity to the mice, is safe and reliable, and can be applied to acne treatment.

As shown in fig. 9, the sebum secretion of the model group is increased, the cutin is blocked and shaped compared with the administration group, the skin is inflamed due to bacterial infection, and after administration, the skin cutin layer is normal and the inflammation is eliminated compared with the blank group, so that the traditional Chinese medicine compound microemulsion gel can treat inflammatory acne, and the effect is obvious.

Irritation test

10 clean guinea pigs (body weight: 200-300 g) were obtained and purchased from Liaoning long biotechnology Co., ltd (SCXK 2020-0002). Shaving off the back hair, spreading gel, spreading blank matrix, recording the phenomena of guinea pig such as screaming, bending body, escaping, etc., removing gel layer after 6 hr, and observing erythema and edema.

10 guinea pigs have no symptoms such as screaming, countering, escaping and the like in the smearing process, have no abnormal symptoms such as itching, dysphoria and the like in the test period, have no obvious change of dehaired skin, and have no abnormal conditions such as bleeding, erythema, allergy and the like which are harmful to skin health in the blank matrix group and the dehaired skin of the microemulsion gel group, and are shown in figure 10.

Experimental example 5 evaluation of clinical anti-acne Effect of the invention

Volunteers with acne on the faces (19 men and 21 women) were enrolled in 4 groups, 10 persons/group, respectively using flavored Sanhuang Xixin soup microemulsion gel group, blank control group and metronidazole gel control group. Each group was evaluated using the scoring method of the comprehensive grading system for acne (GAGS) proposed by Doshi et al in 1997, using 3 days, 7 days, 14 days, and 21 days before use, and specifically operated to divide the acne-promoting area into 6 zones (factor scores for different skin zones in brackets) according to the density, distribution and general area of the pilo-sebaceous units: zone I: forehead (2), zone ii: right cheek (2), region iii: left cheek (2), region iv: nose (1), zone v: chin region (1), vi region: chest and upper back (3). The skin loss score for each region is: 0 = no skin loss; 1. 1 acne or more; 2.1 pimple or more; 3. more than or equal to 1 pustule; 4. more than or equal to 1 nodule or cyst. The total score = factor score x skin damage score, the sum of the total scores of different skin regions is the comprehensive score, the comprehensive score takes the average number of each group, the lower the comprehensive score is, the better the acne cure rate and cure speed are indicated, and the result is shown in table 6.

Table 6 clinical anti-acne effect statistics

The result shows that after the flavored Sanhuang Xixin decoction microemulsion gel group added with the antibacterial composition is used, the comprehensive score of each period is lower than that of a positive control group and a traditional Sanhuang Xixin decoction microemulsion gel group without liquorice, which indicates that the acne cure rate and cure speed of the flavored Sanhuang Xixin decoction microemulsion gel group added with the antibacterial composition are obviously superior to those of other groups, the antibacterial effect is obvious, and adverse reactions such as allergy and the like are avoided.

Claims (10)

1. A compound microemulsion gel preparation for treating acne vulgaris, which is characterized in that: the material consists of the following raw materials in percentage by mass: 98-99% of microemulsion and 1-2% of gel matrix, wherein the microemulsion is prepared from the following raw materials in percentage by mass: 5% -19.95% of oil phase, 0.7% -14% of emulsifying agent, 1.4% -28% of auxiliary emulsifying agent and 53% -77% of water phase, wherein the water phase is an aqueous solution prepared from ethanol extracts of rheum officinale, coptis chinensis, scutellaria baicalensis and liquorice.

2. A compound microemulsion gel formulation for treating acne vulgaris according to claim 1, wherein: the microemulsion is O/W type microemulsion.

3. A compound microemulsion gel formulation for treating acne vulgaris according to claim 1, wherein: the water phase is prepared from the following raw materials in parts by weight: 10-20 parts of rheum officinale, 10-20 parts of coptis chinensis, 5-10 parts of scutellaria baicalensis and 1-5 parts of liquorice, adding 75% ethanol for ultrasonic extraction, removing excessive ethanol by a rotary evaporator, and fixing the volume by distilled water until the crude drug content is 1g/mL.

4. A compound microemulsion gel formulation for treating acne vulgaris according to claim 1, wherein: the oil phase is ethyl oleate or isopropyl myristate.

5. A compound microemulsion gel formulation for treating acne vulgaris according to claim 1, wherein: the emulsifier is one or more of Tween-80, polyoxyethylene hydrogenated castor oil CO-40 and polyoxyethylene ether castor oil-40.

6. A compound microemulsion gel formulation for treating acne vulgaris according to claim 1, wherein: the auxiliary emulsifier is one or more of polyethylene glycol 400, 1,2 propylene glycol and isopropanol.

7. A compound microemulsion gel formulation for treating acne vulgaris according to claim 1, wherein: the gel matrix is carbomer 940.

8. A compound microemulsion gel formulation for treating acne vulgaris according to claim 1, wherein: the average particle diameter of the microemulsion gel is 56.32+/-3.55-97.19 +/-2.52 nm, and the polydispersity PDI is below 0.31+/-0.01.

9. A method for preparing a compound microemulsion gel formulation for treating acne vulgaris according to any one of claims 1 to 8, comprising the steps of:

(1) 10-20 parts of rheum officinale, 10-20 parts of coptis chinensis, 5-10 parts of scutellaria baicalensis and 1-5 parts of liquorice are taken according to parts by weight, 75% ethanol is added according to the weight-volume ratio of 1:10 feed liquid, ultrasonic extraction is carried out twice, each time for 1h, the two extraction solutions are combined, redundant ethanol is removed by a rotary evaporator, distilled water is used for constant volume until the content of crude drugs is 1g/mL, and the mixture is used as a water phase for standby;

(2) Taking 5% -19.95% of oil phase, 0.7% -14% of emulsifying agent and 1.4% -28% of auxiliary emulsifying agent according to mass percentage, placing the water phase 53% -77% obtained in the step (1) into a beaker, stirring the emulsifying agent and the auxiliary emulsifying agent for 10min by using a magnetic stirrer to completely and uniformly mix, adding the oil phase, continuously stirring until the oil phase is uniformly mixed, slowly dripping the water phase into a mixed system, and stirring for 20min to obtain clear and transparent microemulsion;

(3) And taking 98-99% of the obtained microemulsion according to the mass percentage, adding 1-2% of gel matrix, stirring and fully swelling for 12-16h, and regulating the pH value by triethanolamine to 4-5 to obtain the microemulsion gel preparation.

10. The method for preparing a compound microemulsion gel formulation for treating acne vulgaris according to claim 9, wherein the weight ratio of the emulsifier to the co-emulsifier in the step (2) is 1:2.