CN115607510A

CN115607510A - High-concentration oleanolic acid nanoemulsion composition and preparation method thereof

Info

Publication number: CN115607510A
Application number: CN202110419745.1A
Authority: CN
Inventors: 林振放
Original assignee: Quanzhou Daxun Biotechnology Co ltd
Current assignee: Quanzhou Daxun Biotechnology Co ltd
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2023-01-17
Anticipated expiration: 2041-04-19
Also published as: CN115607510B

Abstract

A high-concentration oleanolic acid nanoemulsion composition and a preparation method thereof belong to the technical field of medicines and cosmetics. The oleanolic acid nanoemulsion composition comprises 0.01-8% of oleanolic acid; 1 to 40 percent of surfactant; 1% -40% of cosurfactant; 0.2 to 20 percent of essential oil containing terpene; 0 to 2 percent of baicalin; 0 to 5 percent of oleuropein; a proper amount of additive; the balance being water. Adding cosurfactant and essential oil containing terpene into a container A, and mixing and stirring uniformly; adding a surfactant and mixing uniformly; then adding oleanolic acid and other active ingredients, and stirring until the oleanolic acid and the other active ingredients are completely dissolved; adding water and additives into the container B to obtain a water phase; slowly adding the mixture in the container A into the water phase in the container B while stirring to obtain primary emulsion; shearing and emulsifying at high speed, and fully emulsifying for the second time. Can be used in preparing cosmetics, medicines for treating skin diseases, and skin and hair care products.

Description

High-concentration oleanolic acid nanoemulsion composition and preparation method thereof

Technical Field

The invention belongs to the technical field of medicines and cosmetics, and particularly relates to a high-concentration oleanolic acid nanoemulsion composition and a preparation method thereof.

Background

Oleanolic acid (Oleanolic acid), also known as terraangelic acid, is a natural pentacyclic triterpenoid, widely exists in plants, and exists in various plants as free bodies and glycoside bodies. Generally, the content is 0.2-2%. The powder obtained after the oleanolic acid purification is white crystal, odorless and tasteless. Is unstable to acid and alkali. Melting point 308-310 deg.C, [ alpha ]20D +73.3 deg. (c =0.15, chloroform), and is insoluble in water.

Oleanolic acid has wide pharmacological actions, such as antibacterial, antiinflammatory, antioxidant, antitumor, liver protecting, antidiabetic, hair growth promoting, wound healing promoting, scar formation resisting, collagen formation promoting, and antiallergic effects. At present, oleanolic acid is clinically used for the auxiliary treatment of acute and chronic hepatitis, and is safe and has no toxic or side effect. The development and utilization of oleanolic acid except liver protection are rare, and the clinical preparations of the oleanolic acid mainly comprise two types, namely tablets and capsules.

It is worth noting that some herbal plants rich in oleanolic acid, such as the traditional Chinese medicine fructus ligustri lucidi, are traditionally used in china for the purpose of treating alopecia, promoting hair regeneration and blacking hair; it has been demonstrated that oleanolic acid can upregulate the Wnt/β -Catenin signaling pathway to promote hair growth and extend the anagen phase of hair follicles in vitro (Liu B, et al, β -Catenin is secreted in oleanolic acid-dependent promotion of promotion in human hair matrix cells in an in vitro organic culture model [ J ]. Fitolerapia, 2017, 121; oleanolic acid has been shown to promote the proliferation of human hair follicle stem cells cultured in vitro (Liuben, old swallow, easy atlantoan, etc.; oleanolic acid's influence on the proliferation of human hair follicle stem cells cultured in vitro [ J ]. J. Dermatology, 2017,24 (2): 68-71.); oleanolic ACID has been shown to down-regulate GENE EXPRESSION OF 5A REDUCTASE AND androgen receptor IN hair follicles cultured IN VITRO AND to up-regulate the EXPRESSION levels OF insulin-like growth factor (IGF-1), vascular Endothelial Growth Factor (VEGF) (Cao C, liu Y, et al, EFFECT OF OLEANOLIC ACID ON 5A-REDUCTASE ACTIVITY, DPCS PROLIFERATION AND GENE EXPRESSION CORRELATED WITH DROGENIC ALOPECEIA IN VITRO [ J ]. ACTA MEDIERA, 2019,35 (4): 2159-2165); these studies indicate that oleanolic acid has broad application prospects as a product for promoting hair growth and reducing hair loss.

Oleanolic acid has good supplement or substitute effect on existing hair growth or alopecia treatment; the united states Food and Drug Administration (FDA) has approved only two drugs for the treatment of male androgenetic alopecia (AGA): topical minoxidil (minoxidil) and oral finasteride (finasteride); finasteride is an inhibitor of type 2 alpha-reductase, which prevents DHT-induced hair follicle miniaturization by inhibiting the conversion of testosterone hormone (T) to Dihydrotestosterone (DHT), resulting in a decrease in DHT formation; for AGA patients, finasteride is generally more effective than minoxidil; however, finasteride has the potential risk of causing male sexual side effects and causing anxiety, depression; minoxidil is a nonspecific hair growth promoting drug, the mechanism of promoting hair growth is not completely clear, minoxidil is a potassium channel opener, and more blood and nutrients can enter hair follicles by expanding blood vessels, so that hair growth is promoted; the common adverse effects of minoxidil are mild dermatitis, itching, redness or irritation of the scalp and hirsutism, even leading to an exacerbation of hair loss, "Rogaine" manufacturers indicate that minoxidil causes hair loss as a common side effect and describe the process as "shedding phase"; and the minoxidil is easy to be removed to the level before the minoxidil is used after the minoxidil is stopped; minoxidil inhibits collagen production, possibly accelerating skin aging; serious allergic reactions including rash, chest distress, chest pain, dizziness, tachycardia and the like may occur; existing therapeutic agents are not very successful and often have undesirable side effects, profoundly afflicting both patients and dermatologists; therefore, the development of effective oleanolic acid external preparations is very important.

Oleanolic acid is almost insoluble in water (solubility in water: 4.61mg/L at 25 ℃), has poor cell permeability, and belongs to class VI drugs, i.e., low-solubility and low-permeability drugs, according to the classification system of biopharmaceuticals. Furthermore, the solubility and the dissolution efficiency of the oleanolic acid in common alcohols, fatty acids, surfactants and other fat-soluble solvents are poor, so that the application of the oleanolic acid is greatly limited. The selection of the proper formulation or solvent is critical to increase the solubility of oleanolic acid, since only the dissolved drug can penetrate the skin.

The patents on oleanolic acid formulations have been disclosed to improve the dispersibility of oleanolic acid in water, but there are still some problems that the industrial production and use in large scale are hindered, such as low content of oleanolic acid, the involvement of organic solvents harmful to human skin or health, or complicated processing steps.

For example, chinese patent CN101991532B discloses a non-aqueous self-microemulsion composition, said self-microemulsion composition contains oleanolic acid 0.4-20 mg/ml, said oil phase is one or more of the following: soybean oil, isopropyl myristate, isopropyl palmitate, ethyl laurate, ethyl myristate, glyceryl oleate, glyceryl linoleate, corn oil, sesame oil, olive oil and caprylic capric glyceride, wherein the content of the oil phase is 30-56%; oleanolic acid is solubilized by said co-surfactant/solvent and is selected from the group consisting of glycerol, polyethylene glycol, propylene glycol, ethylene glycol monoethyl ether, absolute ethanol, dimethylacetamide and N-methylpyrrolidone; in example 22, in which the oleanolic acid content is up to 20mg/mL, the total surfactant content is up to 66%, the content of the main solvent N-methylpyrrolidone (NMP) is 25%, but N-methylpyrrolidone (NMP) has the hazards of irritation, reproductive toxicity and the like, and dimethylacetamide, which is a more efficient solvent, has obvious irritation and toxicity to human bodies and skins, is teratogenic and is not suitable for long-term use in high concentration.

Chinese patent application No. CN03125480.2 discloses an emulsion for injection of oleanolic acid and a preparation method thereof, comprising: 0.08-2 g/l of oleanolic acid, 5-30 g/l of phospholipid for injection, 50-200 g/l of soybean oil for injection, 22-25 g/l of glycerol and the balance of water for injection. The preparation method comprises mixing oleanolic acid and soybean oil to obtain oil phase; mixing phospholipid, glycerol and water for injection, and stirring to obtain water phase; adding the oil phase into the water phase under stirring, adjusting the pH value to 6.5-7.0, and adding water for injection to obtain coarse emulsion; homogenizing the crude milk with a high pressure homogenizer to obtain emulsion. The patent adopts the traditional method of mixing and homogenizing oil phase and water phase to prepare emulsion, the grain diameter of the prepared emulsion is larger and is about 1 mu m; and the oleanolic acid content is low, the dissolving process is time-consuming, and the process is complex.

Chinese patent CN2011800558173A relates to a soluble cosmetic composition, which uses nano-liposomes to embed oleanolic acid to enhance the percutaneous absorption of oleanolic acid and promote the synthesis of collagen, thereby improving the anti-wrinkle effect; the preparation method comprises mixing surfactant such as hydrogenated lecithin, PEG-60 hydrogenated castor oil, etc. with polyalcohol component, heating to 80 deg.C for dissolving, adding 0.05% oleanolic acid and other additives, and mixing; the resulting mixture was cooled to 65 ℃ and then ethanol was added thereto; the ingredients of the aqueous part are mixed homogeneously and heated to 65 ℃ and mixed with the previously obtained mixture; the main disadvantages are that the preparation process is complex, multiple heating and cooling are needed, the cost is high, and the liposome stability is poor; in addition, no matter how the ratio of the surfactant, the polyol and the water is adjusted, the concentration of oleanolic acid is generally low, and the content is only 0.05% due to the dissolving capacity of the solvent.

In order to solve the problem of insufficient application of oleanolic acid, the present invention uses a pharmaceutical carrier nano emulsion (nanoemusion) in pharmacy. Nanoemulsions are defined as emulsion systems consisting of an oil phase with a surfactant and water, having an isotropic, transparent (or translucent) appearance. The droplet size is defined as less than 200nm. However, if the emulsion has a low surfactant content and is kinetically stable, then a size of less than 500nm may be accepted as a nanoemulsion. These nanocarriers are transparent, low viscosity, thermodynamically stable, easy to prepare, and high surface area making them effective transdermal and topical drug delivery systems; moreover, they have substantial advantages over other nanocarriers (e.g., microemulsions, liposomes), including the use of less surfactant, low skin irritation, high permeability and high drug loading, allowing topical treatment. The nanoemulsions can be used as starting materials for the preparation of nanocapsules and nanospheres, and can be formulated into various topical dosage forms, such as foams, gels, creams, liquids and sprays. Although the size of microemulsions and nanoemulsions is similar, their preparation methods are different. Both of them require energy input, but nanoemulsions mostly use mechanical shear, while microemulsions use a spontaneous emulsification process. Microemulsions also require high surfactant concentrations compared to nanoemulsions.

The nanometer emulsion gel is prepared by adding water soluble polymer materials such as carbomer, poloxamer, gelatin, cellulose, etc. into nanometer emulsion to form transparent and stable colloid with network structure. The nanoemulsion gel can improve the viscosity of the nanoemulsion, improve the adhesion and the spreadability of the nanoemulsion on the skin, and can ensure that the drug can keep longer action time because the diffusion of the drug in the gel is hindered, the effective diffusion coefficient is reduced and the volatilization of components is reduced due to the increase of the viscosity of the system. Is beneficial to the full absorption of the medicine, thereby improving the bioavailability of the medicine, and has convenient and sanitary administration, difficult pollution to clothes and better patient compliance.

Disclosure of Invention

The invention aims to provide a high-concentration oleanolic acid nanoemulsion composition and a preparation method thereof aiming at the defects in the prior art.

Another object of the present invention is to provide the use of said oleanolic acid nanoemulsion composition.

The oleanolic acid nanoemulsion composition comprises the following components in percentage by weight:

0.01-8% of oleanolic acid, 1-40% of surfactant, 1-40% of cosurfactant, 0.2-20% of essential oil containing terpene, 0-2% of baicalin, 0-5% of oleuropein, a proper amount of additive and the balance of water.

Preferably, the oleanolic acid nanoemulsion composition comprises the following components in percentage by weight:

0.5-2% of oleanolic acid, 10-30% of surfactant, 10-25% of cosurfactant, 1-10% of terpene-containing essential oil, 0.1-1% of baicalin, 0.5-2% of oleuropein, a proper amount of additive and the balance of water.

The oleanolic acid can be pure product or plant extract containing oleanolic acid; the plant extract contains at least 2% of oleanolic acid, and can be selected from fructus Ligustri Lucidi extract, herba Swertiae Bimaculatae extract, herba Swertiae Mileensis extract, and Olea Europaea extract.

The surfactant is preferably a nonionic surfactant; the HLB value of the surfactant can be 8-18, preferably 10-15; the surfactant may be at least one selected from PEG-6 caprylic/capric glyceride, PEG-8 caprylic/capric glyceride, tween 80, tween 20, polyoxyethylene 40 hydrogenated castor oil, polyoxyethylene castor oil, glyceryl monostearate, span 80, glyceryl monostearate, hydrogenated lecithin, steareth, polyethylene glycol stearate, polyglycerol fatty acid ester, and the like.

The cosurfactant can be at least one of diethylene glycol monoethyl ether, dipropylene glycol, 1, 3-butanediol, ethanol, 1, 2-pentanediol, 1, 2-hexanediol, propylene glycol, glycerol, polyethylene glycol and the like; diethylene glycol monoethyl ether is preferred.

Wherein the terpene-containing essential oil contains terpenes structurally belonging to the group of monoterpenes, sesquiterpenes, diterpenes or triterpenes in an amount of at least 1% by weight of the terpene-containing essential oil, more preferably at least 10% by weight of the terpene-containing essential oil. Terpene-containing essential oils include, but are not limited to, cedar essential oil, eucalyptus essential oil, mint essential oil, tea tree essential oil, lavender essential oil, clary sage essential oil, clove bud essential oil, cedar essential oil, geranium essential oil, lemon grass essential oil, sage essential oil, turmeric essential oil, wintergreen essential oil, rosemary essential oil, fennel essential oil, chamomile essential oil, guaiacum essential oil, fennel essential oil, basil essential oil, camphor essential oil, cananga essential oil, citronella essential oil, cinnamon essential oil, ginger essential oil, perilla essential oil, rose essential oil, patchouli essential oil, sandalwood essential oil, bay leaf essential oil, carrot seed essential oil, bergamot essential oil, grapefruit essential oil, lemon essential oil, citrus essential oil, orange flower essential oil, oregano essential oil, sweet orange essential oil, thyme essential oil, and other suitable essential oils.

The terpene-containing essential oil is preferably at least one selected from eucalyptus oil, tea tree essential oil, cedar essential oil, peppermint essential oil, lavender essential oil, rosemary essential oil, etc.; the terpene-containing essential oil comprises, by weight, 0.2% -10% of eucalyptus oil, 0.2% -10% of tea tree essential oil, 0.3% -5% of cedar essential oil, 0.8% -3.2% of mint essential oil, 0.6% -2.4% of lavender essential oil and 0.5% -3.2% of rosemary essential oil, and preferably: eucalyptus oil 0.3-12%, tea tree essential oil 0.3-10%, cedar essential oil 0.3-5%, peppermint essential oil 0.8-3.2%, lavender essential oil 0.6-2.4%, rosemary essential oil 0.5-3.2%; more preferably: eucalyptus oil 0.5-2%, tea tree essential oil 0.5-2%, cedar essential oil 0.3-1%, peppermint essential oil 0.8-2.0%, lavender essential oil 0.5-1.2%, rosemary essential oil 0.5-1.6%.

The additives may include thickeners, humectants, pH adjusters, preservatives, antioxidants, conditioning agents, chelating agents, uv absorbers, nutrients, or other suitable additives. The other suitable additives include, but are not limited to, at least one of an emollient, an anti-androgen agent, a hair growth agent, and the like.

The thickener is selected from carbomer, hydroxyethyl cellulose, sodium carboxymethylcellulose, sodium alginate, polycarbophil, poloxamer, xanthan gum, aloe vera gum, guar gum, polyquaternium Q-10, acrylic acid, and acrylate; carbomers are preferred; the usage amount of the thickener is preferably 0.1 to 5 percent of the total weight of the nanoemulsion;

the humectant can be at least one selected from Astragalus polysaccharides, sodium hyaluronate, dipotassium glycyrrhizinate, glycerol, polyethylene glycol, sorbitol, maltitol, aloe gel, sodium lactate, squalane, etc.; preferably astragalus polysaccharides and sodium hyaluronate; the usage amount of the humectant is preferably 0.5-5% of the total weight of the nanoemulsion.

The pH modifier may be selected from citric acid, malic acid, lactic acid, phosphoric acid, triethanolamine, and combinations thereof, preferably triethanolamine; the usage amount of the pH regulator is preferably 0.1-1% of the total weight of the nanoemulsion.

The preservative can be selected from p-hydroxyacetophenone, phenoxyethanol, benzoic acid, caprylhydroxamic acid, caprylyl glycol, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, dimethylaminoethanol, benzalkonium chloride, benzalkonium bromide and the like, and the usage amount of the preservative is preferably 0.1-0.6% of the total weight of the nanoemulsion.

The antioxidant is selected from one or more of sodium bisulfite, vitamin E, vitamin C, lauryl gallate, propyl gallate, caffeic acid phenethyl ester, arbutin, ferulic acid, ascorbic acid magnesium phosphate, ascorbic acid sodium phosphate, and ascorbic acid ethyl ether.

The conditioner is one or more of allantoin, dipotassium glycyrrhizinate, tocopherol acetate, baicalein, olive leaf extract, scutellaria baicalensis extract, lavender extract, chamomile extract, witch hazel extract, purslane extract, gentian root extract and rosemary extract.

The chelating agent is selected from EDTA, EDTA-2Na or EDTA-4Na.

The ultraviolet absorbent is at least one selected from baicalin derivatives, quercetin derivatives, p-aminobenzoic acid derivatives, salicylic acid derivatives, benzophenone derivatives, diphenyl acrylate, cinnamic acid derivatives, etc.

The nutritional agent is at least one selected from lysine, cystine, arginine, collagen, biotin, chondroitin sulfate, keratan sulfate, heparin sulfate, polypeptide, vitamin A, B1, B2, B6, E, C and vitamin C derivatives.

The emollient is selected from cetostearyl alcohol, lanolin, isopropyl isostearate, vaseline, liquid paraffin, polydimethylsiloxane, dimethiconol, dimethicone, shea butter, cetyl isooctanoate, natural squalane, octyl methoxycinnamate.

The antiandrogen agent is selected from at least one of finasteride, dutasteride, epristeride, flutamide, bicalutamide, safflor yellow, equol, soybean isoflavone, ligustilide, hinokiflavone, saw palmetto extract, sophora flavescens extract, salvia miltiorrhiza extract, lavender extract, genistein, turmeric extract, emblic leafflower fruit extract, nettle extract, speranskia herb extract, ganoderma lucidum extract, dittany bark extract, azelaic acid, quercetin, myricetin, magnolol, luteolin, kaempferol, rutin, tea polyphenol, curcumin, emodin, baicalein, tea tree extract, pueraria radix extract, genistein, scutellaria root extract, spironolactone, cimetidine, cyproterone, hexestrol, 11-alpha-hydroxyprogesterone, diosolone, levonorgestrel, norethindrone, etc.

The pilatory can be one or more selected from Polygoni Multiflori radix extract, fructus Ligustri Lucidi extract, carthami flos extract, chinese arborvitae extract, herba Rosmarini officinalis extract, ginseng radix extract, notoginseng radix extract, green tea extract, herba Centellae extract, herba Swertiae Bimaculatae extract, semen Pisi Sativi extract, ginsenoside, radix Angelicae sinensis extract, olea europaea extract, radix astragali extract, rhizoma Ligustici Chuanxiong extract, rhizoma Zingiberis recens extract, semen Sesami Niger extract, semen Cuscutae extract, ecliptae herba extract, folium Mori extract, epigallocatechin gallate (EGCG), cedrol, stilbene glucoside, adenosine, caffeine, and minoxidil.

The water may be selected from distilled water, purified water and/or deionized water, preferably distilled water.

The additive of the oleanolic acid nanoemulsion composition of the present invention is usually such that the fat-soluble component is dissolved in the oil phase of the emulsification system and the hydrophilic component is dissolved in the water phase component of the emulsification system.

When the dosage form of the present invention is an ointment, paste, cream or gel, as a carrier ingredient, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or a mixture thereof may be used.

When the formulation of the present invention is a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof can be used as a carrier component, and particularly when the formulation is a spray, a propellant such as chlorofluorocarbon, propane/butane or dimethyl ether may be further included.

When the dosage form of the present invention is an emulsion, a liquid diluent such as water, ethanol or propylene glycol may be used as a carrier component.

The preparation method of the oleanolic acid nanoemulsion composition comprises the following steps of:

1) Adding the cosurfactant and the essential oil containing terpene into a container A according to the formula amount, and mixing and stirring uniformly; adding the surfactant with the formula amount, and uniformly mixing; then adding oleanolic acid with formula amount, and further adding other fat-soluble components (such as baicalin) and stirring to completely dissolve;

2) Adding water with the formula amount into the container B, and further adding a proper amount of additives to obtain a water phase;

3) Slowly adding the mixture in the container A into the water phase in the container B while stirring to obtain primary emulsion;

4) Carrying out high-speed shearing emulsification on the primary emulsion prepared in the step 3), carrying out secondary emulsification by using a high-pressure homogenizing emulsification device, and fully emulsifying to obtain the oleanolic acid nanoemulsion composition.

In the step 1), the mass ratio of the cosurfactant to the terpene-containing essential oil can be 4: 1-1: 1; the dissolution can be carried out at the normal temperature of 25-40 ℃ by stirring until the dissolution is complete, or the solution is heated and stirred under the condition of 40-80 ℃ water bath until the dissolution is complete;

in the step 4), the secondary emulsification is carried out by using a high-pressure homogenizing and emulsifying device, the homogenizing speed is 4000-10000 rpm, the homogenizing time is 3-10 min, the high-pressure homogenizing pressure is 800-1000 bar, and the homogenization is carried out for 3-5 times; the average particle size of liquid drops in the prepared oleanolic acid nanoemulsion composition can be 10-200 nm, and is preferably less than 100nm; the secondary emulsification can also adopt micro-jet emulsification or ultrasonic emulsification.

The composition of the present invention is used as a nanoemulsion, and the average particle diameter of the liquid droplet is preferably 10 nm to 200nm. The emulsification process can be selected from the following processes besides the preferred high-pressure homogenization: self-emulsification, ultrasonic emulsification or microfluidization, etc.; if self-emulsification is used, the primary emulsion is stirred and uniformly mixed to obtain the nano emulsion; if ultrasonic emulsification is used, the primary emulsion is subjected to ultrasonic treatment at normal temperature, the power is 0.1-75 KW, the frequency is 20 KHz-500 MHz, the interval is 3s when the ultrasonic treatment is carried out for 3-5 s, and the whole ultrasonic treatment time is 15-300 s; if the micro-jet is emulsified, the primary emulsion is added into a micro-jet high-pressure homogenizer, and is treated for three times by increasing the pressure, wherein the pressure is 1000bar, 1200bar and 1400bar in sequence, and then is filtered by an ultramicro filter membrane with the aperture of 100nm.

The oleanolic acid nanoemulsion composition can be applied to the preparation of cosmetics. The cosmetic includes, but is not limited to, hair tonic, hair growth lotion, hair conditioner essence, skin care gel, anti-aging preparation, shampoo, skin foundation, essence emulsion, lotion, toner, acne lotion, emulsion, microemulsion, shampoo, gel, perfume, hair wax, hair conditioner, hair mask, mascara, hair oil, massage oil, hair conditioner, hair-blacking agent, hair dye, hair spray, hair mousse, etc.

The oleanolic acid nanoemulsion composition is used in a medicine or a cosmetic for improving or preventing alopecia, hair thinning, canities, acne, seborrheic dermatitis, folliculitis, skin aging or other unhealthy skin conditions. The alopecia includes androgenetic alopecia, telogen effluvium, alopecia areata, or other types of alopecia.

The oleanolic acid nanoemulsion composition can be used for skin, hair, facial skin care and the like.

The oleanolic acid nanoemulsion composition can be added with an excipient to be used as a dosage form for skin or hair care, and the oleanolic acid nanoemulsion composition is used alone as a product, or is mixed with other cosmetic or pharmaceutical raw materials to be prepared into nanoemulsion gel, ointment, cream, emulsion, spray, lotion, liniment, patch or liniment.

In the embodiment of the invention, besides oleanolic acid and essential oil containing terpene as active ingredients, other functional ingredients such as baicalin can be added. Baicalin has effects of resisting skin allergy, resisting inflammation, inhibiting bacteria, preventing corrosion, and resisting oxidation, and can be used for preventing and treating skin allergy. Baicalin can absorb ultraviolet ray, remove oxygen free radical, and inhibit melanin generation, and has effects of antagonizing Androgen Receptor (AR), reducing gene expression of AR, and reducing alopecia and acne. Baicalin is extremely difficult to dissolve in various inorganic and organic solvents, has poor water solubility and lipid solubility, and can realize better dissolution and improve bioavailability and biological efficacy in the nanoemulsion system.

Embodiments of the nanoemulsion of the present invention as a cosmetic and/or pharmaceutical composition further comprise oleuropein (oleuropein). Oleuropein is a glycosylated terpenoid compound, is a main effective component of olive leaf extract, and has strong antibacterial and antiviral properties and strong antioxidant ability. It has been proved that topical application of oleuropein promotes collagen growth, and expression of VEGF, IGF-1, KGF and HGF is increased, and these growth factors are closely related to skin healing and hair growth, thus promoting hair growth, accelerating wound healing, soothing and calming the skin.

The present invention may further comprise astragalus polysaccharides as an example of a cosmetic and/or pharmaceutical composition. The astragalus polysaccharide has the functions of oxidation resistance and aging resistance, and simultaneously has good hygroscopicity, thereby playing the role of moisturizing and skin moistening; polysaccharides also have thickening effect, so that astragalus polysaccharides can be used as a thickening agent; in addition, the astragalus polysaccharide has the function of promoting hair regeneration, and has good synergistic effect with oleanolic acid.

Compared with the prior art, the invention has the outstanding advantages and technical effects that:

1. the particle size of the liquid drops in the oleanolic acid nanoemulsion is nano-scale, is less than 200nm, is spherical and is very uniform in distribution, and the defects that the particle size of the traditional emulsion is large and the thermodynamic stability is poor are overcome: the dispersibility of the oleanolic acid in water is remarkably improved, the solubility of the oleanolic acid is improved by more than 10000 times, and the bioavailability of the oleanolic acid is improved. The oleanolic acid nanoemulsion composition disclosed by the invention can greatly improve the solubility and the dissolution rate of oleanolic acid, can improve the drug loading rate after being prepared into a dosage form, improves the treatment or improvement effect, and is beneficial to the application of oleanolic acid in cosmetics and external medicinal preparations.

2. The positive characteristics (namely physiological activity) of the oleanolic acid are combined with the essential oil containing terpene, which has good transdermal absorption capacity and nutrition or healing effect, and the combination of the two has excellent synergistic effect; the two are combined by intermolecular forces, the essential oil containing terpene enhances dissolution and transdermal absorption of oleanolic acid, the oleanolic acid reduces volatilization of the essential oil, and reduces or prevents sensitization of the essential oil to individual skin sensitive individuals.

3. The oleanolic acid nanoemulsion of the invention is a dispersion system which is isotropic, good in fluidity, clear, transparent or semitransparent and slightly opalescent under the combined action of the essential oil, the surfactant and the cosurfactant, can still keep clear after being diluted by water, has good organoleptic property, can reduce viscosity and greasiness when the essential oil is used as an oil phase, has natural plant fragrance, and is excellent in actual use comfort.

4. The anti-corrosion capability of the product is improved, and the anti-corrosion additive is reduced or even not added. The essential oil used by the nanoemulsion has the antibacterial and antiseptic effect, and the oleanolic acid also has the strong antibacterial effect, so that the product can be prevented from deteriorating without adding a chemical preservative, the irritation to the skin is reduced, and the safety is improved.

5. Oleanolic acid is actually able to penetrate the skin to be effective, and must be able to penetrate the stratum corneum barrier of the skin to reach the relevant site of action and maintain the effect at these sites for a certain period of time. The nano-emulsion has small particle size, is beneficial to the transdermal absorption of functional components and plays a role; one or more natural plant essential oils with permeation promoting effect are used, and no chemical permeation promoter is needed, so that the percutaneous absorption capability of oleanolic acid and other active ingredients in the composition is improved, and the safety is better; the nano-emulsion contains water, has better skin hydration property and is more beneficial to absorption.

6. The product has simple preparation process and is beneficial to environmental protection. The traditional emulsion cosmetics or external medical preparations need to use a plurality of raw materials, the oil phase and the water phase have complex components, a plurality of additives, complicated process flow and longer production period, or toxic and harmful solvents are used for improving the dissolving amount of the oleanolic acid; the preparation process can be carried out at room temperature, has stable compatibility with active ingredients, can contain essential oil and other additives without denaturation, and has the advantages of simple preparation components, low cost, simple preparation process, easy industrial production and reduced environmental pollution.

Drawings

Fig. 1 is a product appearance diagram of the oleanolic acid nanoemulsion compositions prepared in examples 1-6.

Fig. 2 is a comparative graph of groups after experimental effect of oleanolic acid nanoemulsion on androgen alopecia model mice.

Wherein A is a blank group; b is a model group; c is minoxidil group; d is 1% oleanolic acid group (group 4); e is oleanolic acid + oleuropein + baicalin group (group 5).

Detailed Description

The following examples further illustrate the invention in conjunction with the drawings. The exemplary compositions are illustrative only and do not limit the scope of the invention. The proportions in the examples and other parts of the specification are by weight unless otherwise indicated. The following formulations are directed to the nanoemulsion, gel embodiments; however, similar techniques may also be used with the formulation in other types of cosmetic or toiletry products, including but not limited to shampoos, creams, sprays, conditioners, and styling products.

The invention relates to a nano-emulsified carrier of oleanolic acid. Terpenoids refer to derivatives with the general formula of (C5H 8) n and their oxygen-containing and different saturation levels, which are widely found in nature, monoterpenes and sesquiterpenes, which are widely found in plant essential oils, oleanolic acid (oleanolic acid) is a pentacyclic triterpenoid, which is a terpene. Based on the principle of similarity and compatibility, the solubility of the essential oil aligned with the oleanolic acid arouses the interest of the inventor; many terpene-containing plant essential oils, oleanolic acid, have surprising solubilizing effects as determined by the present invention, such as eucalyptus oil, tea tree essential oil, cedar essential oil, lavender essential oil, rosemary essential oil, mint essential oil, lemon essential oil, carrot seed oil, and the like; in addition, liquid terpene monomers contained in a large amount in some essential oils, such as Eucalyptol (Eucalyptol) of eucalyptus oil and rosemary oil, terpin-4-ol (terpin-4-ol) of tea tree oil, linalool (linalool) of lavender essential oil, geraniol (Geraniol) of rose essential oil, and the like, have excellent solubility of oleanolic acid.

Applicants have found that nanoemulsions of terpene containing essential oils as the oil phase are suitable bases for oleanolic acid. Thus, the positive properties of oleanolic acid are combined with a nanoemulsion comprising terpene containing essential oils. In particular, nanoemulsions with high oleanolic acid drug loading can be produced, which combine the positive properties of oleanolic acid (i.e. physiological activity) with terpene containing essential oils with good transdermal absorption capacity and nutritional or healing efficacy (if any). This makes the nanoemulsion formulation particularly suitable for cosmetic or dermatological formulations of triterpenoids.

The preparation method is characterized in that a mixture of a cosurfactant and terpene-containing essential oil in a mass ratio of 4: 1-1: 1 is used for solubilizing the oleanolic acid: not only can prevent coagulation in the middle of dissolution, but also can greatly improve the concentration of oleanolic acid in the preparation.

In embodiments of the present invention, novel utilities and/or advantages may be provided when essential oils are both the carrier and solvent as well as the active ingredient; the invention encapsulates essential oil in a nano system to be used as an oil phase, can improve the solubility of oleanolic acid, and can play a synergistic role with active ingredients in application.

The essential oils of the examples of the present invention have a number of valuable properties which can be used as a combination therapy; plant essential oils are useful in dermatological patients, and they generally have antibacterial, antifungal, anti-inflammatory, astringent and regenerative properties; can be used for aromatherapy, and for treating dandruff, psoriasis, eczema, acne, seborrheic dermatitis, etc.; have a very beneficial synergistic effect as active ingredient and/or enhancer in the intended application, in particular as androgen antagonist and hair growth promoter; essential oils such as eucalyptus oil, peppermint oil, tea tree oil, and the like have been widely studied and reported as permeation enhancers for enhancing transdermal drug delivery; in addition, the encapsulation of the nanosystems can provide controlled release of bioactive ingredients such as oleanolic acid, and provide better stability of essential oils and other volatile compounds, enhancing bioavailability and bioefficacy of the formulation.

And from a toxicological point of view, essential oils are considered safer and less irritating than other chemical penetration enhancers; essential or non-volatile oils extracted from aromatic plants are widely used in food, cosmetics and pharmaceuticals, and many are listed in the General Recognized As Safe (GRAS) list of the FDA in the united states, i.e., the "Recognized Safe compounds", essential oils, oleoresins (without solvents) and natural extracts (including distillates) are Generally considered Safe for the intended use (food, cosmetics), such as menthol, peppermint, rosemary, sage, orange blossom, lemon GRASs, thyme, orange leaf, citronella, ylang-ylang, etc., and are not dangerous and suitable for long-term use.

Eucalyptus oil is also called eucalyptus essential oil, has the functions of antibiosis, antioxidation, anti-inflammation, permeation promotion, disinsection, mosquito repelling, itching relieving and corrosion prevention, is a good raw material of medicine and perfume, and is widely used in food processing and cosmetics. Eucalyptus oil is also used to improve the blood circulation of the scalp, thereby promoting hair growth.

The tea tree essential oil has strong cleaning, antibacterial, anti-inflammatory and antiseptic properties, is suitable for oily and acne skin, and can be used for treating suppurative wound, burn, sunburn, psoriasis, dandruff, etc. In addition, the tea tree essential oil has weak estrogen-like activity, and in vitro researches prove that the expression of androgen receptor can be reduced, so that androgenetic alopecia can be reduced.

It is well known that peppermint essential oil produces a cool feeling and promotes blood circulation, helps to reduce skin inflammation and itching, and is confirmed to promote hair growth.

Cedrol, a main component of cedar essential oil, is also a main active component of cacumen biotae, and has a vasodilating effect on blood vessels, so that new hair growth can be stimulated by hair follicles, and meanwhile, the natural shedding process is inhibited. In addition, the cedar essential oil has strong antifungal and antibacterial properties, and the addition of the cedar essential oil in the treatment of acne is helpful for relieving and reducing stubborn acne outbreaks.

Lavender essential oil has been shown to have weak estrogenic activity and very strong anti-inflammatory properties, and may play a key role in helping users relieve itching and swelling of the skin, increasing the speed of wound healing, and thus providing a good environment for skin repair and hair growth.

Rosemary essential oil has strong astringent effect, and can improve greasy desquamation skin, promote blood circulation, and stimulate hair regeneration.

In some embodiments, the composition may include other additives, such as thickeners, humectants, pH adjusters, stabilizers, preservatives, antioxidants, conditioning agents, emollients, colorants, skin protectants, skin and hair nutrients such as collagen, elastin, keratin and other suitable additives.

In some embodiments, the composition comprises a humectant selected from one or more of astragalus polysaccharides, sodium hyaluronate, dipotassium glycyrrhizinate, glycerin, aloe vera gel, more preferably astragalus polysaccharides and sodium hyaluronate;

in some embodiments, the composition may further comprise a thickening agent, such as a naturally derived thickening agent, for example carbomer, hydroxyethyl cellulose, sodium carboxymethyl cellulose, sodium alginate, poloxamer 407, xanthan gum, guar gum, more preferably carbomer.

In some embodiments, the composition may further comprise a pH adjusting agent, which may be selected from citric acid, malic acid, lactic acid, phosphoric acid, triethanolamine, and combinations thereof, preferably triethanolamine.

In some embodiments, no preservative may be added to the composition, for example, the essential oil and 1, 3-butanediol, ethanol, 1, 2-pentanediol, 1, 2-hexanediol all have preservative and bacteriostatic effects; in some embodiments, small amounts of preservatives including p-hydroxyacetophenone and phenoxyethanol may optionally be used.

The compositions of the present invention are hydrophilic in nature. The water may be distilled, purified and/or deionized water, preferably distilled water. In some embodiments, the composition comprises about 1% to 80% by weight water.

The average particle size of liquid drops in the oleanolic acid nanoemulsion composition is 10-200 nm, and the particle size of the nanoemulsion is preferably less than 100nm. The application process can be one or more of high-pressure homogenization in high-energy emulsification, micro-jet or ultrasonic, and preferably high-pressure homogenization.

The obtained nanoemulsion can be used alone, or can be further added with other excipients to be prepared into nanoemulsion gel, cream, emulsion, spray, transdermal patch, paste dosage forms and the like.

According to the preparation method of the present invention, terpene-containing essential oils such as eucalyptus oil, tea tree oil, cedar oil and the like have a high solubility to oleanolic acid, but when the amount of oleanolic acid dissolved in the essential oils exceeds 40mg/mL, white colloidal coagulation is easily formed, which seriously hinders further dissolution.

The preparation method is characterized in that a mixture of a cosurfactant and terpene-containing essential oil in a mass ratio of 4: 1-1: 1 is used for solubilizing oleanolic acid: not only can prevent condensation during dissolution, but also can greatly improve the concentration of oleanolic acid in the preparation;

in embodiments of the present invention, novel utilities and/or advantages may be provided when essential oils are both the carrier and solvent as well as the active ingredient; the invention encapsulates essential oil in a nano system to be used as a lipophilic phase, can improve the solubility of oleanolic acid, and can play a synergistic effect with active ingredients in application.

The oleanolic acid nanoemulsion composition of the present invention may further include other hydrophilic or lipid-soluble active ingredients, thereby providing a cosmetic or external preparation with stronger activity. Generally, a fat-soluble active ingredient is dissolved in an oil phase of an emulsification system, and a hydrophilic ingredient is dissolved in an aqueous phase of the emulsification system. It should be noted that the active ingredients include baicalin with antiandrogen effect, and other active ingredients such as oleuropein and Astragalus polysaccharides. The formulation of the nano-emulsion composition of the present invention is embodied in the form of nano-emulsion, essence, gel or shampoo, etc., and thus, the nano-emulsion of the present invention further comprises auxiliary components of these forms, such as thickener, humectant, preservative, etc.

In the embodiment of the invention, in addition to oleanolic acid and terpene-containing essential oil as active ingredients, other active ingredients and/or enhancers such as baicalin may be added. Other active ingredients and/or enhancers such as baicalin may also be added in embodiments of the present invention. Baicalin is known to have anti-skin allergy, anti-inflammatory, bacteriostatic, antiseptic, and antioxidant effects, and can be used for preventing and treating skin allergy. Baicalin can absorb ultraviolet ray, remove oxygen free radical, and inhibit melanin generation, and has effects of antagonizing Androgen Receptor (AR), reducing gene expression of AR, and reducing alopecia and acne. Baicalin is extremely insoluble in various inorganic and organic solvents, has poor water solubility and lipid solubility, and can be well dissolved and improve bioavailability and biological efficacy in the nanoemulsion system.

Embodiments of the nanoemulsion of the present invention as a cosmetic and/or pharmaceutical composition further comprise oleuropein (oleuropein). Oleuropein is a glycosylated terpenoid compound, is a main effective component of olive leaf extract, and has strong antibacterial and antiviral properties and strong antioxidant capacity. It has been proved that topical application of oleuropein promotes collagen growth, and expression of VEGF, IGF-1, KGF and HGF is increased, and these growth factors are closely related to skin healing and hair growth, thus promoting hair growth, accelerating wound healing, soothing and calming the skin.

The present invention may further comprise astragalus polysaccharides as an example of a cosmetic and/or pharmaceutical composition. The astragalus polysaccharide has the functions of oxidation resistance and aging resistance, and also has good hygroscopicity, thereby playing the role of moisturizing and skin moistening; polysaccharides also have thickening effect, so that astragalus polysaccharides can be used as a thickening agent; in addition, the astragalus polysaccharide has the function of promoting hair regeneration, and has good synergistic effect with oleanolic acid. Other plant extracts having hair growth promoting and antiandrogen effects, such as ginsenoside, ginseng extract, polygonum multiflorum extract, rosemary extract, safflower extract, biota orientalis extract, scutellaria baicalensis extract, and the like, may be added to the preparation of the present invention.

Because the quality standards of the same functional ingredients and raw materials are not completely the same at present, and especially the solubility of the active ingredients is influenced by the difference of the content and the purity of essential oil ingredients, great care must be taken to the quality and the source of the raw materials when the product is prepared. In the embodiment of the invention, the content of eucalyptol in eucalyptus oil is more than or equal to 80 percent, the content of terpinen-4-ol (terpin-4-ol) in tea tree oil is more than or equal to 42 percent, the content of linalool in lavender essential oil is more than or equal to 31 percent, the content of eucalyptol in rosemary essential oil is more than or equal to 26 percent, the content of cedrol in cedar essential oil is more than or equal to 11 percent, the mint essential oil is peppermint essential oil, and the content of menthol is more than or equal to 36 percent; the purity of diethylene glycol monoethyl ether is more than or equal to 99.5 percent, is in cosmetic grade or pharmaceutical grade, and is preferably Transcutol CG or Transcutol HP.

Specific examples are given below.

Example 1:

a1% oleanolic acid nanoemulsion comprises the following raw material components in percentage by weight, as shown in Table 1:

TABLE 1

Components	Weight percent (%)
		Oleanolic acid	1
Eucalyptus oil	1
		Tea tree oil	2
Rosemary essential oil	1.2
		Mint essential oil	2
Lavender essential oil	0.8
		Diethylene glycol monoethyl ether (Transcutol)	16
Dipropylene glycol	5
		Tween 20	25.00
Polyoxyethylene 35 castor oil	8
		Distilled water	37.8

The preparation method comprises the following steps:

(1) weighing eucalyptus oil, tea tree oil, rosemary essential oil, mint essential oil and lavender essential oil according to the formula ratio, and uniformly mixing the eucalyptus oil, the tea tree oil, the rosemary essential oil, the mint essential oil and the lavender essential oil with diethylene glycol monoethyl ether and dipropylene glycol in a container A.

(2) Weighing the Tween 20 and the polyoxyethylene 35 castor oil according to the formula amount, adding the Tween 20 and the polyoxyethylene 35 castor oil into the mixture in the container A, uniformly mixing, adding the oleanolic acid according to the formula amount, and stirring until the oleanolic acid is completely dissolved.

(3) Adding distilled water into the container B, and then adding and uniformly mixing.

(4) Adding the mixture in the container A into the container B under the stirring state to obtain primary emulsion, and then carrying out secondary emulsification by using a high-pressure homogenizing and emulsifying device, wherein the homogenizing speed is 5000rpm, the homogenizing time is 5min, the high-pressure homogenizing pressure is 800bar, and the homogenizing is carried out for 3-5 times.

(5) And (3) closing the high-pressure homogenizer, taking a small amount of finished products for observation, and obtaining the oleanolic acid nanoemulsion if the appearance is transparent or semitransparent, the fluidity and the dispersibility are good, and obvious visible opalescence is available.

Example 2:

a1% oleanolic acid nanoemulsion comprises the following raw material components in percentage by weight as shown in Table 2:

TABLE 2

Components	Weight percent (%)
		Oleanolic acid	1
Eucalyptus oil	2
		Tea tree oil	2
Lavender essential oil	1.2
		Mint essential oil	1.8
Dipropylene glycol	5
		Ethanol	3
1, 2-hexanediol	8
		PEG-6 caprylic/capric glycerides	16
Polyoxyethylene 35 castor oil	12
		Biotin	0.5
Glycyrrhizic acid dipotassium salt	0.5
		Distilled water	47

The preparation method comprises the following steps:

(1) weighing eucalyptus oil, tea tree oil, mint essential oil, lavender essential oil, 1, 2-hexanediol, ethanol, dipropylene glycol, PEG-6 caprylic/capric glyceride and polyoxyethylene 35 castor oil according to the formula ratio, and uniformly mixing in a container A.

(2) Weighing oleanolic acid with the formula ratio, adding the oleanolic acid into the mixture in the container A, and heating and stirring the mixture in a water bath at 80 ℃ until the oleanolic acid is completely dissolved.

(3) Adding distilled water into the container B, adding biotin and dipotassium glycyrrhizinate, and stirring until the mixture is dissolved.

(4) Adding the mixture in the container A into the container B under the stirring state to obtain primary emulsion, and then carrying out secondary emulsification by using a high-pressure homogenizing and emulsifying device, wherein the homogenizing speed is 4000rpm, the homogenizing time is 5min, the high-pressure homogenizing pressure is 800bar, and the homogenizing is carried out for 3-5 times.

(5) And (3) closing the high-pressure homogenizer, taking a small amount of finished products for observation, and obtaining the oleanolic acid nanoemulsion if the appearance is colorless, and the finished products have good fluidity and dispersibility and obvious visible opalescence.

Example 3:

the 5% oleanolic acid nanoemulsion comprises the following raw material components in percentage by weight as shown in table 3:

TABLE 3

Components	Weight percent (%)
		Oleanolic acid	5
Eucalyptus oil	12
		Tea tree oil	8
Diethylene glycol monoethyl ether (Transcutol)	36
		PEG-8 caprylic/capric glycerides	36
Distilled water	3

The preparation method comprises the following steps:

(1) the eucalyptus oil, the tea tree oil, diethylene glycol monoethyl ether (Transcutol) and PEG-8 caprylic/capric glyceride with the formula ratio are weighed and added into a container A to be uniformly mixed.

(2) Adding oleanolic acid into the mixture according to the formula amount, placing the mixture into a water bath at 60 ℃, heating and stirring until the oleanolic acid is completely dissolved.

(3) Distilled water was added to vessel B, and then the mixture of vessel a was slowly added to vessel B with stirring to obtain a primary emulsion.

(4) And then, carrying out secondary emulsification by using a high-pressure homogenizing and emulsifying device, wherein the homogenizing speed is 4000rpm, the homogenizing time is 3min, the high-pressure homogenizing pressure is 800bar, and the homogenizing is carried out for 3-5 times.

Example 4:

the oleanolic acid nanoemulsion for promoting hair growth comprises the following raw material components in percentage by weight as shown in table 4:

TABLE 4

Components	Percentage by weight (%)
		Oleanolic acid	1
Oleuropein	1.6
		Baicalin	0.5
Tea tree oil	2
		Eucalyptus oil	1
Rosemary essential oil	1.2
		Mint essential oil	1.6
Lavender essential oil	0.4
		Cedar essential oil	0.2
1,2 pentanediol	5
		Diethylene glycol monoethyl ether (Transcutol)	23
Astragalus polysaccharides	1
		EDTA-4Na	0.1
P-hydroxyacetophenone	0.3
		Arginine	0.6
Polyoxyethylene 30 castor oil	7
		Tween 80	12
Polyoxyethylene 40 hydrogenated castor oil	16
		Distilled water	25.5

The preparation method comprises the following steps:

(1) respectively weighing eucalyptus oil, tea tree oil, peppermint essential oil, lavender essential oil, rosemary essential oil, cedar essential oil, dipropylene glycol and diethylene glycol monoethyl ether (Transcutol) according to the formula ratio, and uniformly mixing in a container A.

(2) Weighing polyoxyethylene 30 castor oil, tween 80 and polyoxyethylene 40 hydrogenated castor oil according to the formula ratio, uniformly mixing, adding into the mixture in the container A, adding oleanolic acid and baicalin, and heating and stirring in a water bath at 80 ℃ until the oleanolic acid and the baicalin are completely dissolved.

(3) Adding distilled water into container B, adding oleuropein, astragalus polysaccharides, EDTA-4Na, arginine and p-hydroxyacetophenone, and stirring to dissolve.

(5) And (3) closing the high-pressure homogenizer, taking a small amount of finished products for observation, and obtaining the oleanolic acid nanoemulsion if the appearance is brown, semitransparent, good in fluidity and dispersibility and obvious in visible opalescence.

Example 5:

the oleanolic acid skin care nanoemulsion comprises the following raw material components in percentage by weight as shown in table 5:

TABLE 5

Components	Weight percent (%)
		Oleanolic acid	0.5
Oleuropein	1
		Baicalin	0.2
Tea tree oil	0.7
		Eucalyptus oil	0.5
Mint essential oil	1.2
		Mint essential oil	0.5％
Lemon essential oil	0.2％
		1, 2-hexanediol	3
Dipropylene glycol	3
		Diethylene glycol monoethyl ether (Transcutol)	8
PEG-8 caprylic/capric glycerides	12
		Polyoxyethylene 40 hydrogenated castor oil	15
Glyceryl monostearate	3
		Hyaluronic acid sodium salt	1
Vitamin C	0.7
		Aloe gel	0.3
Phenoxyethanol	0.3
		EDTA-2Na	0.1
Distilled water	57

The preparation method comprises the following steps:

(1) respectively weighing tea tree oil, eucalyptus essential oil, lemon essential oil, 1, 2-hexanediol, dipropylene glycol and diethylene glycol monoethyl ether (Transcutol) according to the formula amount, and uniformly mixing in a container A.

(2) Weighing polyoxyethylene 30 castor oil, tween 80 and polyoxyethylene 40 hydrogenated castor oil according to the formula ratio, adding into the mixture in the container A, adding oleanolic acid and baicalin, and heating and stirring in a water bath at 60 ℃ until the mixture is completely dissolved.

(3) Adding distilled water into container B, adding oleuropein, phenoxyethanol, EDTA-2Na, and vitamin C, stirring to dissolve, adding sodium hyaluronate and aloe gel, and standing for 24 hr to swell completely.

(4) Adding the mixture in the container A into the container B under stirring to obtain primary emulsion, and then performing secondary emulsification by using a high-pressure homogenizing and emulsifying device, wherein the homogenizing speed is 6000rpm, the homogenizing time is 5min, the high-pressure homogenizing pressure is 800bar, and the homogenizing is performed for 3-5 times.

(5) And (3) closing the high-pressure homogenizer, taking a small amount of finished products for observation, and obtaining the oleanolic acid nanoemulsion if the appearance is light yellow, semitransparent, good in fluidity and dispersibility and has obvious visible opalescence.

Example 6:

the oleanolic acid acne-removing nanoemulsion gel comprises the following raw material components in percentage by weight as shown in table 6:

TABLE 6

The preparation method comprises the following steps:

(1) respectively weighing tea tree oil, peppermint essential oil, rosemary essential oil, cedar essential oil, 1, 3-butanediol and diethylene glycol monoethyl ether (Transcutol) according to the formula ratio, and uniformly mixing in a container A.

(2) Weighing polyoxyethylene 35 castor oil and tween 80 in the formula amount, adding into the mixture in the container A, adding oleanolic acid and baicalin, and heating and stirring in a water bath at 80 ℃ until completely dissolving.

(3) Adding distilled water into the container B, adding phenoxyethanol, EDTA-2Na and dipotassium glycyrrhizinate, and stirring until the materials are dissolved.

(4) And adding the mixture in the container A into the container B under the stirring state to obtain primary emulsion, and then carrying out secondary emulsification by using a high-pressure homogenizing and emulsifying device, wherein the homogenizing speed is 4000rpm, the homogenizing time is 6min, the high-pressure homogenizing pressure is 800bar, and the nano-emulsion is obtained after 3-5 times of homogenization.

(5) Slowly adding carbomer 940 into the nanoemulsion in a stirring state, adding triethanolamine, and standing at normal temperature for 24h to fully swell carbomer 940 to obtain transparent or semitransparent oleanolic acne removing nanoemulsion gel.

The product appearance of the oleanolic acid nanoemulsion compositions prepared in examples 1-6 is shown in fig. 1.

Test example 1: experiment of effect of oleanolic acid nanoemulsion on androgen alopecia model mice

1.1 Experimental animals

40 male C57BL/6 mice were selected, with a body weight of 16-18g and an age of 3-4 weeks. All rats are placed in an animal room with appropriate temperature and humidity for breeding, and ventilation is carried out for 1-2 times every day, and each time lasts for 1 hour. Each 12h day and night was guaranteed to alternate daily while remaining quiet and avoiding other disturbances. Ensure that the mouse has enough activity space. The mice are fed with the maintenance feed, the purified water is used as the drinking water of the rats, and the drinking water and the feed are replaced every day to keep the ventilation, sanitation and hygiene of the living environment of the animals. Lighting in day (12 +/-2) h, indoor temperature (20 +/-2) DEG C and relative humidity of 40-60%.

1.2 grouping

40 rats were randomly divided into 5 groups, group 1: blank control group 8; group 2: 8 models are selected; group 3: 8 minoxidil groups; group 4: 8 oleanolic acids; group 5: oleanolic acid + Oleuropein + baicalin group 8.

1.3 Experimental drugs

The minoxidil group used a commercially available 5% minoxidil solution; the group of oleanolic acid used the nanoemulsion prepared in example 1, and the group of oleanolic acid + oleuropein + baicalin used the nanoemulsion prepared in example 4.

1.4 Molding

All mice were injected subcutaneously in the back with testosterone propionate at a dose of 5 mg/kg/day 1 time per day for 4 weeks, except for a blank group, which was injected with an equal amount of physiological saline at the same site in the back of the mice.

1.5 administration of drugs

After 4 weeks of successful molding, the placebo group was injected subcutaneously with normal saline, and the remaining groups were continuously injected subcutaneously with testosterone propionate for 4 weeks to maintain androgen levels in the mice. The model group was continuously injected with testosterone propionate only at 5 mg/kg/day, group 3 was evenly coated with 5% minoxidil after injecting testosterone propionate for 1 hour each time, group 4 was coated with 1% oleanolic acid nanoemulsion prepared in example 1, and group 5 was coated with the oleanolic acid nanoemulsion composition prepared in example 4.

1.6 Experimental animal observations

1.6.1 general State Observation

The unhairing degree of each group of rats is observed every day, and the local skin reactions such as red swelling, edema, effusion and the like do not appear in the medicine smearing area.

1.6.2 preparation of skin tissue sections

After 8 weeks of the experiment, the dorsal depilation region of each group of mice was selected, the material was taken parallel to the spinal column and was approximately 1X 1m2, and 3 skin tissue sections of substantially the same area were taken from each mouse. Preparing a specimen according to a conventional skin histopathological process, embedding the specimen in paraffin, slicing, HE staining, observing morphological change of hair follicles of a mouse under a microscope, counting the total number of the hair follicles in a 100-field view, taking the average value of three slices, and carrying out related statistical analysis, wherein data are expressed by X +/-SD. When p is less than 0.05, the difference is significant and has statistical significance.

1.7 results of the experiment

1.7.1 section Hair follicle Observation and Hair follicle enumeration

Observing the cross section of the skin hair follicle, and after the external medicine is applied for 4 weeks, the hair of the blank control group is complete and thick, and is black and glossy; compared with the blank group, the model group has obviously increased alopecia area; the hair loss area of the minoxidil group and the mice of the group 3 and the group 4 is obviously reduced compared with that of the model group, and the figure is shown in figure 2.

TABLE 7 comparison of Hair follicle counts for each group

Group of	Total number of hair follicles
		Blank control group (group 1)	38.75±4.12
Model set (set 2)	13.31±3.42*
		Minoxidil group (group 3)	30.38±8.32 ^#
Oleanolic acid group (group 4)	31.63±6.73 ^&
		Oleanolic acid + Oleuropein + baicalin group (group 5)	34.12±8.85 ^#$

Note comparison of p with blank control group<0.05； ^# In comparison with the modeling group, p<0.05；&In comparison with minoxidil group, p>0.05； ^$ In comparison with minoxidil group, p<0.05。

As can be seen from table 7, the oleanolic acid group (group 4) had a clear therapeutic effect on androgenetic alopecia model mice, and the effect was not statistically significantly different (P > 0.05) compared to the 5% minoxidil group; the group 4, oleanolic acid, oleuropein and baicalin, has better comprehensive effects of promoting hair growth and reducing alopecia than the minoxidil group on androgenetic alopecia mice (P <0.05, table 7).

Test example 2 stability study of Oleanolic acid nanoemulsion formulations

1) Effect of centrifugal acceleration test on nanoemulsion stability

The prepared nano emulsion is centrifuged at 20000r/min, and the appearance of the nano emulsion is still clear and transparent after being observed for 15min, and the phenomenon of oil-water stratification is not generated.

2) Light stability test

The nano-emulsion is put into a glass bottle in proper amount, sealed and placed at room temperature under the condition of 5000 +/-500 lx illumination, and sampled and observed at 1d, 3d and 7d respectively. The results show that the nanoemulsion keeps clear and transparent appearance, and the phenomena of layering, emulsion breaking and the like are not seen.

3) Observation test of retained sample

The emulsion is subpackaged in glass bottles, sealed and respectively placed at 4 ℃, 25 ℃ and 40 ℃ for sample observation for 6 months, and sampled and observed at intervals of 14 days. The results show that the emulsion keeps clear and transparent appearance under the four temperature conditions, and the phenomena of layering, emulsion breaking and the like are not seen.

Test example 3: animal skin irritation study of Oleanolic acid nanoemulsion formulations

New Zealand white rabbits are more sensitive to the response of stimulating substances than humans. The skin irritation test was performed on examples 4 to 6 using 12 healthy New Zealand white rabbits, each half of which was divided into 3 groups of 4 rabbits each, each half of which was female and male. The two sides of the spine of the animal are unhaired by an electric shaver 24 hours before administration, and the area of the unhaired area is 5 multiplied by 5cm. The skin is externally applied for 2 times every day, the skin is continuously applied for 14d as a test period, the animal skin irritation test result is non-irritant according to skin irritation intensity classification of 'cosmetic hygiene Specifications' (2002), and the oleanolic acid essential oil nanoemulsion and the nanoemulsion gel have no obvious irritation to New Zealand white rabbits.

Test example 4: solubilization of essential oil with oleanolic acid

Adding appropriate amount of oleanolic acid into 5mL centrifuge tube, adding 1g of each adjuvant, respectively, performing ultrasonic treatment for 30min, maintaining the temperature at 60 deg.C, oscillating at 100r/min for balancing for 48h, centrifuging at 10000r/min for 10min, collecting supernatant, diluting the filtrate with methanol by appropriate times, performing sample HPLC, and calculating oleanolic acid content in each adjuvant.

The solubility of oleanolic acid in different solvents is shown in table 8, and it can be seen from table 8 that the solubility of essential oil oleanolic acid is much higher than that of common solvents, and the combination of essential oil and cosurfactant has better solubilization and hydrotropy effects.

Table 8: oleanolic acid solubility (mg/ml) of different solvents

Experiments have shown that the topical compositions of the present invention can be used in the preparation of products for treating and controlling the adverse effects of skin conditions including, but not limited to, androgenetic alopecia, telogen effluvium, alopecia areata, or other types of alopecia, acne, seborrheic dermatitis, folliculitis, skin aging, and other skin conditions.

The oleanolic acid nanoemulsion composition of the present invention is applied to cosmetics, and can be used for skin care, hair care, and the like.

The oleanolic acid nanoemulsion composition can be used as a dosage form for caring skin or hair by adding an excipient into the oleanolic acid nanoemulsion composition, and the oleanolic acid nanoemulsion composition is used alone as a product, or is mixed with other cosmetic or pharmaceutical raw materials to be prepared into nanoemulsion gel, ointment, cream, emulsion, spray, lotion, liniment, patch or liniment.

The oleanolic acid nanoemulsion composition of the present invention can be prepared into products including, but not limited to, hair growth lotions, hair care essences, skin care gels, shampoos, hair conditioners, mascaras, masks, creams, and lotions.

The above-described embodiments are merely preferred embodiments of the present invention, and should not be considered as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Claims

1. A high-concentration oleanolic acid nanoemulsion composition is characterized by comprising the following components in percentage by weight:

0.01 to 8 percent of oleanolic acid, 1 to 40 percent of surfactant, 1 to 40 percent of cosurfactant, 0.2 to 20 percent of essential oil containing terpene, 0 to 2 percent of baicalin, 0 to 5 percent of oleuropein, a proper amount of additive and the balance of water.

2. The high concentration oleanolic acid nanoemulsion composition of claim 1, comprising the following components in percentage by weight:

3. The high concentration oleanolic acid nanoemulsion composition of claim 1 or 2, wherein said oleanolic acid is pure or a plant extract containing oleanolic acid; the plant extract contains at least 2% of oleanolic acid, and can be selected from fructus Ligustri Lucidi extract, herba Swertiae Bimaculatae extract, and Olea europaea extract;

the surfactant is preferably a nonionic surfactant; the HLB value of the surfactant can be 8-18, preferably 10-15; the surfactant can be selected from at least one of PEG-6 caprylic/capric glyceride, PEG-8 caprylic/capric glyceride, tween 80, tween 20, polyoxyethylene 40 hydrogenated castor oil, polyoxyethylene castor oil, span 80, glyceryl monostearate, hydrogenated lecithin, steareth, polyethylene glycol stearate and polyglycerol fatty acid ester;

the cosurfactant can be at least one of diethylene glycol monoethyl ether, dipropylene glycol, 1, 3-butanediol, ethanol, 1, 2-pentanediol, 1, 2-hexanediol, propylene glycol, glycerol and polyethylene glycol; preferably diethylene glycol monoethyl ether;

the terpene-containing essential oil can be at least one selected from eucalyptus oil, tea tree essential oil, cedar essential oil, peppermint essential oil, lavender essential oil, and rosemary essential oil; the terpene comprises at least one of monoterpene, sesquiterpene and diterpene.

4. The high concentration oleanolic acid nanoemulsion composition as claimed in claim 1 or 2, wherein the terpene-containing essential oil comprises, by weight, eucalyptus oil 0.2% -10%, tea tree essential oil 0.2% -10%, cedar essential oil 0.3% -5%, peppermint essential oil 0.8% -3.2%, lavender essential oil 0.6% -2.4%, rosemary essential oil 0.5% -3.2%, preferably: eucalyptus oil 0.3-12%, tea tree essential oil 0.3-10%, cedar essential oil 0.3-5%, peppermint essential oil 0.8-3.2%, lavender essential oil 0.6-2.4%, rosemary essential oil 0.5-3.2%; more preferably: eucalyptus oil 0.5-2%, tea tree essential oil 0.5-2%, cedar essential oil 0.3-1%, peppermint essential oil 0.8-2.0%, lavender essential oil 0.5-1.2%, rosemary essential oil 0.5-1.6%.

5. The high concentration oleanolic acid nanoemulsion composition of claim 1 or 2, wherein said additive comprises at least one of a thickener, a moisturizer, a pH adjuster, a preservative, an antioxidant, a conditioner, a chelating agent, an ultraviolet absorber, a nutrient, an emollient, an antiandrogen, a hair restorer, etc.

6. The high concentration oleanolic acid nanoemulsion composition of claim 5, wherein said thickener is selected from the group consisting of carbomer, hydroxyethyl cellulose, sodium carboxymethyl cellulose, sodium alginate, polycarbophil, poloxamer, xanthan gum, aloe vera gum, guar gum, polyquaternium Q-10, acrylic acid, acrylates; carbomers are preferred; the using amount of the thickening agent is preferably 0.1-5% of the total weight of the nanoemulsion;

the humectant can be at least one selected from Astragalus polysaccharides, sodium hyaluronate, dipotassium glycyrrhizinate, glycerol, polyethylene glycol, sorbitol, maltitol, aloe gel, sodium lactate, and squalane; preferably astragalus polysaccharides and sodium hyaluronate; the usage amount of the humectant is preferably 0.5 to 5 percent of the total weight of the nano-emulsion;

the pH modifier may be selected from citric acid, malic acid, lactic acid, phosphoric acid, triethanolamine, and combinations thereof, preferably triethanolamine; the usage amount of the pH regulator is preferably 0.1-1% of the total weight of the nanoemulsion;

the preservative can be selected from p-hydroxyacetophenone, phenoxyethanol, benzoic acid, caprylhydroxamic acid, caprylyl glycol, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, dimethylaminoethanol, benzalkonium chloride and benzalkonium bromide, and the usage amount of the preservative is preferably 0.1-0.6% of the total weight of the nanoemulsion;

the antioxidant is selected from one or more of sodium bisulfite, vitamin E, vitamin C, lauryl gallate, propyl gallate, phenethyl caffeate, arbutin, ferulic acid, magnesium ascorbyl phosphate, sodium ascorbyl phosphate and ascorbyl ethyl ether;

the conditioner is one or more of allantoin, dipotassium glycyrrhizinate, tocopherol acetate, baicalein, olive leaf extract, scutellaria baicalensis extract, lavender extract, chamomile extract, witch hazel extract, purslane extract, gentian root extract and rosemary extract;

the chelating agent is selected from EDTA, EDTA-2Na or EDTA-4Na;

the ultraviolet absorbent is at least one selected from baicalin derivatives, quercetin derivatives, p-aminobenzoic acid derivatives, salicylic acid derivatives, benzophenone derivatives, diphenyl acrylate, and cinnamic acid derivatives;

the nutrient is selected from at least one of lysine, cystine, arginine, collagen, biotin, chondroitin sulfate, keratan sulfate, heparin sulfate, polypeptide, vitamin A, B1, B2, B6, E, C and vitamin C derivatives;

the emollient is selected from cetostearyl alcohol, lanolin, isopropyl isostearate, vaseline, liquid paraffin, polydimethylsiloxane, dimethiconol, dimethicone, shea butter, cetyl isooctanoate, natural squalane, octyl methoxycinnamate;

the antiandrogen agent is selected from at least one of finasteride, dutasteride, epristeride, flutamide, bicalutamide, safflor yellow, equol, soy isoflavone, ligustilide, hinokiflavone, saw palmetto extract, sophora flavescens extract, salvia miltiorrhiza extract, lavender extract, genistein, turmeric extract, emblic leafflower fruit extract, nettle extract, speranskia herb extract, ganoderma lucidum extract, dittany bark extract, azelaic acid, quercetin, myricetin, magnolol, luteolin, kaempferol, rutin, tea polyphenol, curcumin, emodin, baicalein, tea tree extract, kudzu root extract, genistein, scutellaria root extract, spironolactone, cimetidine, cyproterone, hexestrol, 11-alpha-hydroxyprogesterone, diosolone, levonorgestrel, and norethindrone;

the hair restorer is at least one selected from Polygoni Multiflori radix extract, fructus Ligustri Lucidi extract, carthami flos extract, cacumen Platycladi extract, herba Rosmarini officinalis extract, ginseng radix extract, notoginseng radix extract, green tea extract, herba Centellae extract, herba Swertiae Bimaculatae extract, semen Pisi Sativi extract, ginsenoside, radix Angelicae sinensis extract, olea europaea extract, radix astragali extract, rhizoma Ligustici Chuanxiong extract, rhizoma Zingiberis recens extract, semen Sesami Niger extract, semen Cuscutae extract, ecliptae herba extract, folium Mori extract, epigallocatechin gallate, cedrol, stilbene glucoside, adenosine, caffeine and minoxidil.

7. The high concentration oleanolic acid nanoemulsion composition of claim 1 or 2, wherein said water is selected from distilled water, purified water and/or deionized water, preferably distilled water.

8. A method for preparing an oleanolic acid nanoemulsion composition as claimed in claim 1 or 2, characterized by comprising the steps of:

1) Adding the cosurfactant and the essential oil containing terpene into a container A according to the formula amount, and uniformly mixing and stirring; adding the surfactant with the formula amount, and uniformly mixing; then adding oleanolic acid with formula amount, and further adding other liposoluble components (such as baicalin) and stirring to dissolve completely;

9. The method for preparing the oleanolic acid nanoemulsion composition of claim 8, wherein in step 1), the mass ratio of the co-surfactant to the terpene-containing essential oil is 4: 1 to 1: 1; the dissolution can be carried out at the normal temperature of 25-40 ℃ by stirring until the dissolution is complete, or the solution is heated and stirred under the condition of 40-80 ℃ water bath until the dissolution is complete;

in the step 4), the secondary emulsification is carried out by using a high-pressure homogenizing and emulsifying device, the homogenizing speed is 4000-10000 rpm, the homogenizing time is 3-10 min, the high-pressure homogenizing pressure is 800-1000 bar, and the homogenization is carried out for 3-5 times; the average particle size of the liquid drops in the prepared oleanolic acid nanoemulsion composition can be 10-200 nm, and is preferably less than 100nm; the secondary emulsification can also adopt micro-jet emulsification or ultrasonic emulsification.

10. The oleanolic acid nanoemulsion composition according to claim 1 or 2, which can be used for the preparation of cosmetics; the cosmetics include, but are not limited to, hair tonic, hair growth lotion, hair conditioner essence, skin care gel, anti-aging preparation, shampoo, skin foundation, essence emulsion, lotion, toner, acne lotion, emulsion, microemulsion, shampoo, gel, perfume, hair wax, hair conditioner, hair mask, mascara, hair oil, massage oil, hair conditioner, hair-blacking agent, hair dye, hair spray, hair mousse;

the oleanolic acid nanoemulsion composition is used in a medicine or a cosmetic for improving or preventing alopecia, hair thinning, white hair, acne, seborrheic dermatitis, folliculitis, skin aging or other unhealthy skin conditions; the alopecia comprises androgenetic alopecia, telogen effluvium, alopecia areata or other types of alopecia;

the oleanolic acid nanoemulsion composition can be used for caring skin, hair and facial skin;

the oleanolic acid nanoemulsion composition can be added with an excipient to be used as a dosage form for skin or hair care, and the oleanolic acid nanoemulsion composition is used alone as a product, or is mixed with other cosmetic or pharmaceutical raw materials to be prepared into oleanolic acid nanoemulsion gel, ointment, cream, emulsion, spray, lotion, liniment, patch or liniment.