CN113244357A

CN113244357A - Micro-emulsion gel of anti-HPV plant extract and preparation method and application thereof

Info

Publication number: CN113244357A
Application number: CN202110601643.1A
Authority: CN
Inventors: 李斯文; 李钰
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2021-08-13
Anticipated expiration: 2041-05-31
Also published as: CN113244357B

Abstract

The invention discloses a micro-emulsion gel of an anti-HPV plant extract, a preparation method and an application thereof, wherein the micro-emulsion gel comprises the plant extract and auxiliary materials, and the plant extract comprises one or more of the following components: zedoary turmeric extract, folium Eucalypti Globueli extract, radix Sophorae Flavescentis extract, fructus Cnidii extract, radix Stemonae extract, and cortex Phellodendri extract. The invention utilizes natural plant extracts as the components of a carrier, and simultaneously as active ingredients to enhance the improvement of drug effect, takes carbomer as a substrate to prepare a local external preparation with bacteriostasis, inflammation diminishing and virus resistance, is microemulsion gel for HPV infection and prevention, bacterial and fungal vaginitis, cervical erosion and daily nursing of female private parts, and particularly can reduce the mRNA expression level of key oncogenes E6 and E7 in Hela cells. The microemulsion gel prepared by the invention has high safety, good stability, convenient administration and strong patient compliance; simple processing technology, low cost and convenient large-scale production.

Description

Micro-emulsion gel of anti-HPV plant extract and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a micro-emulsion gel of an anti-HPV plant extract, a preparation method thereof and anti-HPV application thereof.

Background

HPV, namely human papilloma virus, has obvious characteristics of being addicted to human skin and mucous epithelial cells, and HPV pathogenic mechanisms are mainly related to immune escape: the virus is integrated into particle molecules in epithelial cells, so that the whole life cycle of the virus is in a state that the virus cannot be dissolved by intracellular lytic enzymes, and the immune response of an organism is reduced; after infecting the organism, the HPV virus can integrate self genes into host genes, thereby further avoiding the monitoring of an immune system. The HPV is divided into a plurality of subtypes, wherein the high-risk type of the mucous membrane is mainly HPV16/18, the expression of related proteins is mainly regulated and controlled by genes E6 and E7, the persistent infection of the high-risk type HPV is the main cause of canceration, the probability of infecting the high-risk type HPV (HR-HPV) in the lifetime of women is as high as 80 percent, the early symptoms are local pruritus and pain, and few patients have no obvious symptoms. The main growing parts are vulva, vagina, cervix, perianal and the like, the two parts commonly occur simultaneously, the local part shows pale red or gray small papule which is warty, the wart is always fused to form vegetable-shaped neoplasm, the infectivity is strong, and therefore, the important significance on the prevention and control of HPV virus on female health is taken into consideration. One of the keys to HPV treatment is that the drug can effectively enter the target tissue and target cells, killing the HPV virus.

The microemulsion is a liquid dispersion system of a uniform, transparent and thermodynamically stable body formed by a water phase, an oil phase, a surfactant and a cosurfactant under certain conditions, can increase the solubility of fat-soluble medicines and water-soluble medicines, can improve the stability of substances, is convenient to store and transport, masks the unpleasant odor of the medicines, improves the appearance of products, and relieves the problems of embarrassment of patients in use and the like. More importantly, the nano-scale structure (10-100nm) formed by the microemulsion is beneficial to enhancing the cellular uptake, but the problems of poor adhesion and the like exist when the microemulsion is applied to the skin and the mucous membrane surface independently. Although no gland exists in the vagina, mucus is generated to form a mucus layer, so that the contact between the medicine and a target tissue is hindered, and the gel can rapidly swell in a vaginal aqueous environment and is tightly bonded with the vaginal mucosa, so that the problem is remarkably improved by combining the gel and the microemulsion, the detention time of the medicine in the vagina is prolonged, the treatment effect is improved, the administration frequency is reduced, and the medication compliance of a patient is improved. Under normal physiological conditions, the vagina is maintained in an acidic environment (pH 3.8-4.2) for lactic acid bacteria to resist external attack, and under pathological conditions, the pH of the vagina rises to 5.5-6.5. The gel of the invention takes carbomer as a substrate and triethanolamine as a pH regulator to adjust the micro-emulsion gel to a proper pH value.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the microemulsion gel of the anti-HPV plant extract, the plant extract is directly used as the oil phase of the microemulsion, and the microemulsion gel can be directly used as the composition component of a carrier, simplifies the raw material, increases the solubility of insoluble components, improves the stability of the medicament, covers the unpleasant odor of the medicament, is also used as an active component to enhance the improvement of the medicament effect, and effectively solves the problem that the conventional vaginal external medicament has poor effect on preventing and treating HPV.

The invention also provides a preparation method and application of the micro-emulsion gel of the anti-HPV plant extract.

The technical scheme is as follows: in order to achieve the purpose, the microemulsion gel of the anti-HPV plant extract comprises the plant extract and auxiliary materials, wherein the plant extract comprises one or more of the following raw materials in parts by weight: 1-8 parts of curcuma zedoary extract, 1-8 parts of eucalyptus leaf extract, 1-4 parts of sophora flavescens extract, 1-4 parts of fructus cnidii extract, 1-4 parts of radix stemonae extract and 1-4 parts of cortex phellodendri extract, wherein the auxiliary materials comprise auxiliary materials for preparing microemulsion and gel.

Wherein, the auxiliary materials comprise 0.25 to 3 weight portions of carbomer required for preparing the gel and 0.5 to 2.5 weight portions of borneol for promoting the permeation; 1.5-12 parts by weight of surfactant required for preparing the microemulsion, wherein the mass ratio of the plant extract to the surfactant is (1-9) to 10.

Preferably, the carbomer is any one of carbomer 934, carbomer 940 and carbomer 941.

Preferably, the surfactant is one or more of tween 80, tween 20, span 80, span 20 and polyoxyethylene hydrogenated castor oil.

The zedoary turmeric extract in the invention is represented by zedoary turmeric oil which is volatile oil obtained by steam distillation of zedoary turmeric, contains various anticancer active ingredients, such as beta-elemene, curcumenol, zedoary turmeric ketone, germacrone and the like, and has biological activities of broad-spectrum pathogenic microorganism resistance, tumor resistance, virus resistance, organism immunity improvement and the like.

The folium Eucalypti Globueli extract is represented by folium Eucalypti Globueli oil which is volatile oil obtained by steam distillation of folium Eucalypti Globueli, and contains various terpenes such as 1, 8-cineole, camphene, phellandrene, terpineol, geraniol, isovaleraldehyde, citronellal and piperonyl ketone, and has strong biological activities of resisting inflammation, sterilizing, promoting osmosis, repairing skin barrier function, etc.

The radix Sophorae Flavescentis extract is derived from dried root of radix Sophorae Flavescentis of Leguminosae, and is harvested in spring and autumn, cleaned, and dried, and has heat clearing, dampness eliminating, parasite killing, and antibacterial effects. It is used topically to treat leucorrhea with red and white discharge, pudendal swelling, pudendal pruritus, and trichomonas vaginitis.

The fructus Cnidii extract is obtained from dried mature fruit of cnidium monnieri (L.) DC of Umbelliferae, and collected in summer and autumn. It is pungent in flavor and capable of dispersing wind, bitter in flavor and drying dampness to kill parasites, and good for external use to treat strange itching of female pudendum and yellow leucorrhea due to damp-heat, and often combined with Ku Shen, Zao jiao, Bai Bu and Huang Bai to be decocted for fumigation and washing, and for external use to treat trichomonas vaginitis.

The radix Stemonae extract is derived from dried root tuber of Stemonae plant, has effects of expelling parasite, killing bacteria, and resisting virus, and has obvious curative effect on gynecological diseases such as pubic louse, trichomonas vaginitis, and colpitis mycotica caused by Candida albicans infection, and nonspecific vaginitis.

The cortex Phellodendri extract is derived from dried bark of phellodendron amurense belonging to Rutaceae. Is bitter in taste and cold in nature, contains alkaloids as main components, has pharmacological effects of clearing heat and drying dampness, resisting inflammation and bacteria, stopping bleeding, resisting oxidation and the like, and has obvious inhibition effect on trichomonas vaginitis.

Borneol is prepared by extracting fresh branches and leaves of Lauraceae plants, has pungent and bitter taste, has effects of resisting inflammation, killing bacteria, promoting drug penetration, etc., has fragrant smell, and can be used as taste masking agent and penetration enhancer.

The plant extract of the invention can be used not only as an active drug effect component, but also as a carrier component of the microemulsion.

The preparation method of the micro-emulsion gel of the anti-HPV plant extract comprises the following steps:

(1) preparing gel: weighing carbomer powder according to the weight parts, uniformly dispersing on the surface of the purified aqueous solution, swelling at room temperature overnight, uniformly stirring, adding borneol, and adjusting the pH value by using triethanolamine to form gel;

(2) preparing a microemulsion: uniformly mixing one or more of curcuma zedoary extract, eucalyptus leaf extract, sophora flavescens extract, fructus cnidii extract, radix stemonae extract and phellodendron bark extract with a mixed surfactant according to parts by weight, stirring under the condition of water bath, and dropwise adding purified water until the system is changed from turbid viscous to transparent to form micro-emulsion; the amount of purified water at which the system changes from cloudy, viscous to clear was recorded and represents the critical point for microemulsion formation.

(3) Preparing a microemulsion gel: mixing the prepared microemulsion with the gel, and stirring uniformly to obtain the microemulsion gel for resisting the HPV plant extract.

Wherein, the pH value of the step (1) is adjusted to 5.5-6.5 by using triethanolamine.

Wherein the temperature of the water bath condition in the step (2) is 25-60 ℃, and the stirring speed is 40-100 rpm.

Wherein the mass ratio of the microemulsion to the gel in the step (3) is 1:3-3: 1.

Further, the water used is purified water. The purified water is treated by water purifying equipment to remove various substances in the water, and can be applied to the pharmaceutical industry.

The invention relates to an application of micro-emulsion gel of anti-HPV plant extract in preparing medicine for preventing and treating HPV infection, bacteria and gynecological diseases of mycotic vaginitis.

The design principle of the invention is as follows: the HPV virus has obvious epitheliophilic property, and one of the key points of HPV treatment is that the medicine can effectively enter target tissues and target cells so as to kill the HPV virus, so that the medicine needs to be retained in the genital tract firstly, and then the medicine needs to enter the epithelial cells.

According to the invention, the active substance plant extract is loaded in the microemulsion, the active ingredients of the plant extract are effectively promoted to enter cells by utilizing the nanostructure of the microemulsion, the anti-HPV curative effect is enhanced, and the microemulsion is combined with the gel, so that the retention of the medicine in the genital tract is prolonged. The microemulsion is characterized in that one or more of plant extracts such as curcuma zedoary extract, eucalyptus leaf extract, sophora flavescens extract and the like are used as the oil phase of the microemulsion, so that the microemulsion can be directly used as a component of a carrier, simplifies raw materials, can increase the solubility of insoluble components, improves the stability of a medicament and covers the bad smell of the medicament, and simultaneously is used as an active component to enhance the medicinal effect, so that the 'drug-adjuvant-in-one' effect of the Chinese medicinal components is reflected.

The invention utilizes natural plant extracts and carbomer as a matrix to prepare a local external preparation with bacteriostasis, inflammation diminishing and virus resisting functions, and is microemulsion gel for HPV infection and prevention, bacterial and fungal vaginitis, cervical erosion and daily nursing of female private parts. The prepared microemulsion gel has high safety, good stability, convenient administration and strong patient compliance; simple processing technology, low cost and convenient large-scale production.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention is a brand-new therapeutic drug for HPV infection by combining multiple plant extracts and microemulsion gel, can achieve synergistic effect, high-efficiency drug delivery, mild nourishing, cooling and bacteriostasis, and solves the problems of poor prevention and treatment effect on HPV infection and the like of the existing vaginal external drug.

1. The plant extract is simultaneously used as a carrier component and an active drug effect component, the prepared microemulsion has a nano-scale structure, the plant extract and microemulsion gel formed by other raw materials have synergistic effect, compared with a solution of the plant extract, the mRNA expression level of key oncogenes E6 and E7 in Hela cells can be obviously reduced, the nano-structure is proved to effectively promote the uptake of the active plant extract by cervical epithelial cells while the contact area of the active plant extract and the cervical epithelial cells is obviously increased, so that the active plant extract has the opportunity to be directly contacted with HPV virus in the cells to play an anti-HPV role, and in addition, the synergistic effect among multiple effective components of the plant extract can further enhance the anti-HPV curative effect;

2. the microemulsion prepared by adopting the active ingredient plant extract does not need to be added with other oil phases such as oleic acid and the like; meanwhile, the plant extract has anti-inflammatory and bacteriostatic effects, so that chemical substances such as preservatives and the like do not need to be additionally added;

3. the added plant extracts such as curcuma zedoary, eucalyptus leaf, sophora flavescens, radix stemonae, fructus cnidii and cortex phellodendri have various pharmacological activities of resisting inflammation, resisting virus, enhancing the immune function of an organism and the like, assist HPV to transform negative and simultaneously effectively resist inflammation and bacteria, and restore the normal physiological environment and the immune function of the vagina;

4. the microemulsion gel has the advantages of high viscosity, high drug loading capacity, rapid absorption and the like, can effectively permeate into vaginal mucosa folds after administration, prolongs the detention time of the drug at a patient part, is easy to coat, has no foreign body sensation, has strong water solubility and is easy to clean, and can improve the compliance of a patient;

5. the plant herbal formula effectively reduces the potential toxic and side effects of western medicine components after long-term application;

6. the preparation process is simple, has no chemical additives such as preservative, essence and pigment, and is safe and efficient.

Drawings

FIG. 1 is a schematic representation of the nano-scale structure of the microemulsion by transmission electron microscopy.

FIG. 2 is a graph showing the effect of plant extract microemulsion on animal skin irritation.

FIG. 3 is a graph showing the effect of plant extract microemulsion on the irritation of vaginal mucosa in animals.

FIG. 4 shows the verification of positive expression of HPV18 in Hela cells.

FIG. 5 shows the effect of microemulsion gel of plant extracts on the morphology of Hela cells.

FIG. 6 is a graph showing the effect of microemulsion gel of plant extracts on the expression of HPV18 mRNA from Hela cells.

Detailed Description

In order that the present invention may be more readily understood, the present invention is further described without limiting in any way the invention by reference to the following specific examples which are intended only to illustrate the invention and are not intended to limit the scope of the invention, which are intended to be within the scope of the claims of the invention without departing from the technical solution of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.

Curcumae rhizoma extract (yakwool is more than or equal to 8%), Borneolum Syntheticum, and polyoxyethylene hydrogenated castor oil CO-40 (Cas: 61788-85-0), which are obtained from Shanghai leaf Biotech limited; eucalyptus extract, available from Shanghai Michelin Biotech, Inc.; radix Sophorae Flavescentis extract, fructus Cnidii extract, radix Stemonae extract, and cortex Phellodendri extract, which are available from Beijing Tongrentang.

Example 1

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(1) weighing carbomer 934 powder (1g), and swelling in 50g of purified water for 12 h;

(2) adding 0.5g of borneol into the step (1), uniformly stirring, and regulating the pH value to 6.5 by using triethanolamine to obtain gel;

(3) respectively weighing Curcumae rhizoma extract (1.5g), folium Eucalypti Globueli extract (1.5g) and radix Sophorae Flavescentis extract (1.5g), mixing, adding Tween 80(10g), and adding purified water at 25 deg.C and 40rpm until the system turns from turbid to clear to obtain microemulsion;

(4) and (3) mixing the gel obtained in the step (1) with the microemulsion obtained in the step (3) according to a mass ratio of 1:3, and uniformly stirring to obtain the microemulsion.

The sensory indexes and stability of the plant extract microemulsion gel prepared in the embodiment are examined and shown in table 1.

TABLE 1 comprehensive evaluation of sensory index and experimental index of plant extract microemulsion gel

As shown in Table 1, the microemulsion gel prepared by the invention has good stability and is convenient to store and use.

Example 2

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(1) weighing carbomer 940 powder (1g), and swelling in 50g of purified water for 12 h;

(2) adding borneol (1g) into the step (1), uniformly stirring, and regulating the pH value to 5.5 by using triethanolamine to obtain gel;

(3) weighing folium Eucalypti Globueli extract (8g), adding Tween 80(8g) and Tween 20(2g), mixing, and adding purified water dropwise at 25 deg.C and 60rpm until the system turns from turbid to clear to obtain microemulsion;

Example 3

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(1) weighing carbomer 941 powder (1.5g), and swelling in 50g of purified water for 12 h;

(2) adding borneol (1g) into the step (1), uniformly stirring, and regulating the pH value to 6.5 by using triethanolamine to obtain gel;

(3) weighing Curcumae rhizoma extract (1.5g) and radix Stemonae extract (1.5g), adding span 80(6g), mixing, adding dropwise purified water at 25 deg.C and 60rpm until the system turns from turbid to clear to obtain microemulsion;

(4) and (3) mixing the gel obtained in the step (1) with the microemulsion obtained in the step (3) according to a mass ratio of 1:2, and uniformly stirring to obtain the microemulsion.

Example 4

The microemulsion gel for resisting the HPV plant extract is characterized by comprising the following steps:

(1) weighing carbomer 941 powder (3g), and swelling in 50g of purified water for 12 h;

(2) adding borneol (1.5g) into the step (1), uniformly stirring, and regulating the pH value to 6.5 by using triethanolamine to obtain gel;

(3) weighing Curcumae rhizoma extract (1.5g) and radix Sophorae Flavescentis extract (4g), adding span 20(12g), mixing, adding purified water dropwise at 40 deg.C and 60rpm until the system turns from turbid to clear to obtain microemulsion;

Example 5

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(3) weighing Curcumae rhizoma extract (4g) and folium Eucalypti Globueli extract (2g), adding Tween 80(6g) span 20(4g), mixing, and dripping purified water at 40 deg.C and 80rpm until the system turns from turbid to clear to obtain microemulsion;

(4) and (3) mixing the gel obtained in the step (1) with the microemulsion obtained in the step (3) according to a mass ratio of 1:1, and uniformly stirring to obtain the microemulsion.

Example 6

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(1) weighing carbomer 941 powder (2g), and swelling in 50g of purified water for 12 h;

(2) adding borneol (2g) into the step (1), stirring uniformly, and regulating the pH value to 6.5 by using triethanolamine to obtain gel;

(3) weighing a curcuma zedoary extract (2g), a sophora flavescens extract (2g), a stemona extract (2g), a phellodendron extract (2g) and a cnidium fruit extract (2g), adding span 80(4g) and polyoxyethylene hydrogenated castor oil (8g), uniformly mixing, and dropwise adding purified water at 60 ℃, 80rpm until the system is clarified from turbid to obtain microemulsion;

Example 7

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(1) weighing carbomer 941 powder (2g), and swelling in 50g of purified water solution for 12 h;

(3) weighing Curcumae rhizoma extract (6g) and folium Eucalypti Globueli extract (1g), adding polyoxyethylene hydrogenated castor oil (12g), mixing, and dripping purified water at 60 deg.C and 100rpm until the system turns from turbid to clear to obtain microemulsion;

(4) and (3) mixing the gel obtained in the step (1) with the microemulsion obtained in the step (3) according to a mass ratio of 2:1, and uniformly stirring to obtain the microemulsion.

The microemulsion gel prepared in the same way as in example 1 has good stability and is convenient to store and use in the study on the sensory index and stability of the microemulsion gel of the plant extract in example 7.

Example 8

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(3) weighing Curcumae rhizoma extract (8g), adding polyoxyethylene hydrogenated castor oil (12g), mixing, and dripping purified water at 40 deg.C and 100rpm until the system turns from turbid to clear to obtain microemulsion;

(4) and (3) mixing the gel obtained in the step (1) with the microemulsion obtained in the step (3) according to a mass ratio of 3:1, and uniformly stirring to obtain the microemulsion.

Example 9

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(2) adding borneol (2.5g) into the step (1), uniformly stirring, and regulating the pH value to 6.5 by using triethanolamine to obtain gel;

(3) weighing Curcumae rhizoma extract (2g) and radix Sophorae Flavescentis extract (4g), adding polyoxyethylene hydrogenated castor oil (8g) and span 20(4g), mixing well, dripping purified water at 40 deg.C and 100rpm until the system turns from turbid to clear to obtain microemulsion;

Example 10

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(1) weighing carbomer 940 powder (0.25g), and swelling in 50g of purified water for 12 h;

(3) weighing Curcumae rhizoma extract (1g), adding polyoxyethylene hydrogenated castor oil (1.5g), mixing, and dripping purified water at 25 deg.C and 100rpm until the system turns from turbid to clear to obtain microemulsion;

Example 11

A microemulsion gel of anti-HPV plant extract comprises the following steps:

(1) weighing carbomer 934 powder (3g), and swelling in 50g of purified water for 12 h;

(3) weighing Curcumae rhizoma extract (8g), adding polyoxyethylene hydrogenated castor oil (10g), mixing, and dripping purified water at 60 deg.C and 40rpm until the system turns from turbid to clear to obtain microemulsion;

Test example 1

The microemulsion preparation method of example 7 was used to study the effect of the oil phase to adjuvant surfactant ratio on microemulsion formation in the microemulsion preparation method.

The proportions of the components are the same as example 7, the oil phase is zedoary extract and eucalyptus leaf extract, the adjuvant surfactant is polyoxyethylene hydrogenated castor oil, and the difference is that:

group 1: oil phase: surfactant 2:8 (w/w);

group 2: oil phase: surfactant 4:6 (w/w);

group 3: oil phase: surfactant 5:5 (w/w);

group 4: oil phase: surfactant 6:4 (w/w);

group 5: oil phase: surfactant 8:2 (w/w).

And taking samples prepared by each group, diluting the samples by 50 times with purified water, and measuring the particle size of each group of samples by using a Malvern particle sizer. The measurement results are shown in Table 2.

Taking a microemulsion sample, diluting the microemulsion sample by 50 times with purified water, then spotting the microemulsion sample on a special copper net for natural drying, dyeing the microemulsion sample with 1% phosphotungstic acid, and shooting the morphological structure of the microemulsion by using a transmission electron microscope, wherein the result is shown in figure 1.

TABLE 2 Effect of oil phase to surfactant ratio on microemulsion formation

Group of	Particle size (nm)	Polydisperse coefficient (PDI)	Status of state
				Group
1	23.41±0.11	0.23±0.01	Light yellow clear liquid
				Group
2	39.01±0.69	0.12±0.01	Light yellow clear liquid
				Group
3	>1μm	1	Milky white semi-turbid liquid
				Group
4	>1μm	1	White milky liquid
				Group 5	>1μm	1	White milky liquid

As can be seen from Table 2, the clear liquids obtained from

groups

1 and 2 meet the microemulsion particle size range, and the polydispersion coefficients are less than 0.3, so that the prepared sample has uniform particle size distribution, and the characterization result of the transmission electron microscope on the microemulsion morphology is shown in figure 1, so that the microemulsion has a nanoscale structure; the emulsion liquids obtained from groups 3-5 do not conform to the microemulsion particle size range, and the microemulsion cannot be formed when the oil phase proportion exceeds 50% under the condition.

Test example 2

Experimental animals: SD male rats (200. + -.20 g) purchased from Nanjing Qinglongshan animal farm.

The invention researches the influence of plant extract microemulsion gel on the irritation of animal skin.

Healthy male SD rats with intact skin are selected, 10% chloral hydrate anesthesia (dose is 3.5mL/kg) is adopted, abdominal hair is removed, the hair removal area is 4cm multiplied by 4cm, and four limbs are fixed on a rat board. 1g of the microemulsion gel prepared in the embodiment 7 of the invention is applied to the surface of the unhaired skin on one side, and blank gel is applied to the unhaired skin on the other side as a control (blank gel without drug microemulsion). Removing the cover after 12h, cleaning the skin surface with warm water, observing the skin reaction at the application part, killing and taking down the skin at the administration part, carefully removing adipose tissues, HE staining, and observing the erythema and edema conditions under an optical microscope.

Compared with the blank control, no erythema, redness, swelling and inflammation phenomenon is observed on the skin surface of the test animal (figure 2), which shows that the plant extract microemulsion gel of the invention is non-irritant to the skin of the test animal.

Test example 3

Experimental animals: 10 ICR male mice (20. + -.2 g) were purchased from the animal farm of Qinglongshan, Nanjing.

The invention researches the influence of the plant extract microemulsion gel on the irritation of mouse vaginal mucosa.

10 healthy ICR mice were selected and randomly divided into a blank control group and a plant extract microemulsion gel administration group, and each group had 5 mice. Taking 0.1g of the microemulsion gel prepared in example 7, delivering the microemulsion gel into the vagina of a test animal by using a special vagina syringe, taking physiological saline as a blank control, contacting with the vagina mucosa for 12h, recording the conditions of diet, spirit, excretion and the like, continuously administering for 7d, killing the animal at 8d, visually observing whether the administration part has swelling, slicing vaginal tissue, HE staining, and observing the conditions of erythema and edema under an optical microscope.

Within one week of administration, the animal activities were normal, and no abnormality such as red swelling or death occurred at the administration site.

Compared with a blank control, the animal vaginal tissue has no erythema and edema reaction, the tissue is complete and compact, the cells are arranged orderly, and inflammatory cell infiltration is not seen (figure 3), so that the microemulsion gel provided by the invention is proved to have no irritation to the tested animal vaginal mucosa.

Test example 4

Main test materials: hela cells, purchased from shanghai ATCC cell bank; trizol kit, available from Solambio.

The invention researches the in vitro anti-HPV effect of the plant extract micro-emulsion gel.

Hela cells of human cervical carcinoma were inoculated in a six-well plate at 37 ℃ with 5% CO₂The microemulsion gel of example 7 was diluted to 600. mu.g/mL with DMEM medium containing 1% DMSO, 2mL of the solution of the plant extract applied to the cells at an equal concentration per well was used as a control (the plant extract of example 7 in DMEM medium containing 1% DMSO in 2mL of the solution applied to the cells) and the untreated cells were used as a blank (the medium was DMEM medium containing 1% DMSO) at 37 ℃ with 5% CO₂Culturing for 24h in an incubator, observing cell morphology under an optical microscope, collecting cells, suspending in 1mL PBS buffer solution, extracting total RNA in the cells according to a Trizol kit method, carrying out reverse transcription to obtain a cDNA template, measuring changes of HPV 18E 6 and E7 mRNA on gene expression levels after different groups act on Hela cells by adopting a real-time quantitative fluorescence detection technology (q-PCR) and determining the inhibition effect on HPV18 virus by taking E6 and E7 as target genes and GAPDH as reference genes.

Hela cells are one kind of cervical cancer cells, but the HPV positive expression of the Hela cells may be reduced with the increase of the number of passages, so the experimental example verifies the HPV positive expression of the Hela cells (FIG. 4, the method is the same as the above), and proves that the Hela cells used by the invention are HPV18 positive.

Compared with untreated cells, the solution group and the microemulsion gel group of the plant extract can change the cell morphology, causing cell death to varying degrees (FIG. 5), but the solution group of plant extracts did not significantly reduce the mRNA expression levels of the key oncogenes E6, E7 in Hela cells (FIG. 6), the microemulsion-gel group of plant extracts significantly reduced the mRNA expression levels of the related genes, meanwhile, the microemulsion gel prepared by replacing the curcuma zedoary extract and the eucalyptus leaf extract in the embodiment 7 with oleic acid with equal mass does not influence the mRNA expression level of key oncogenes E6 and E7 in Hela cells, and the microemulsion gel prepared by the method provided by the invention is proved to have good inhibition effect on HPV virus, compared with a solution group of the plant extract, the microemulsion gel prepared by the plant extract can improve the effect of reducing the mRNA expression level of key oncogenes E6 and E7 in Hela cells.

Although both the solution group and the microemulsion-gel group of the plant extract in the experimental example 3 can cause the death of Hela cells to a certain extent, compared with the microemulsion-gel group, the solution form does not have a significant influence on the mRNA level of HPV, so that the plant extract microemulsion-gel prepared by the invention can not only promote the shrinkage death of cervical cancer cells, but also can effectively enter cells to inhibit the expression of HPV viruses. The combination of the plant extract and the microemulsion gel is used for resisting HPV, and cell experiments prove that the plant extract can obviously reduce the expression level of HPV mRNA genes after acting on Hela cells.

Claims

1. The microemulsion gel for resisting the HPV plant extract is characterized by comprising the plant extract and auxiliary materials, wherein the plant extract comprises one or more of the following raw materials in parts by weight: 1-8 parts of curcuma zedoary extract, 1-8 parts of eucalyptus leaf extract, 1-4 parts of sophora flavescens extract, 1-4 parts of fructus cnidii extract, 1-4 parts of radix stemonae extract and 1-4 parts of cortex phellodendri extract; the auxiliary materials comprise auxiliary materials for preparing the micro-emulsion gel.

2. The microemulsion gel of anti-HPV plant extract of claim 1, wherein the adjuvant comprises 0.25-3 parts by weight of carbomer, 0.5-2.5 parts by weight of borneol; 1.5-12 parts by weight of surfactant required for preparing the microemulsion.

3. The microemulsion gel of anti-HPV plant extract of claim 2, wherein the mass ratio of the plant extract to the surfactant is (1-9): 10.

4. the microemulsion gel of anti-HPV plant extract according to claim 2, wherein the carbomer is preferably any one of carbomer 934, carbomer 940 and carbomer 941.

5. The microemulsion gel of anti-HPV plant extract of claim 2, wherein the surfactant is one or more selected from tween 80, tween 20, span 80, span 20, and polyoxyethylene hydrogenated castor oil.

6. A method for preparing a microemulsion gel of an anti-HPV plant extract of claim 1, comprising the steps of:

(2) preparing a microemulsion: uniformly mixing one or more of curcuma zedoary extract, eucalyptus leaf extract, sophora flavescens extract, fructus cnidii extract, radix stemonae extract and phellodendron bark extract with a mixed surfactant according to parts by weight, stirring under the condition of water bath, and dropwise adding purified water until the system is changed from turbid viscous to transparent to form micro-emulsion;

7. The method of preparing a microemulsion gel of anti-HPV plant extract of claim 6, wherein the step (1) is adjusting pH to 5.5-6.5 using triethanolamine.

8. The method of preparing a microemulsion gel of anti-HPV plant extract of claim 6, wherein the temperature of the water bath condition of the step (2) is 25-60 ℃ and the stirring speed is 40-100 rpm.

9. The method for preparing a microemulsion gel of an anti-HPV plant extract according to claim 6, wherein the mass ratio of the microemulsion to the gel in the step (3) is 1:3-3: 1.

10. Use of the microemulsion gel of anti-HPV plant extract of claim 1 in the preparation of a medicament for the prevention and treatment of HPV infections, bacterial and mycotic vaginitis gynecological diseases.