Pomegranate peel polyphenol nano-emulsion temperature-sensitive gel as well as preparation method and application thereof

Abstract

The invention relates to pomegranate peel polyphenol nanoemulsion temperature-sensitive gel as well as a preparation method and application thereof, and the temperature-sensitive gel is prepared from pomegranate peel polyphenol, plant essential oil, grease, an emulsifier, a co-emulsifier, a gel matrix, a humectant, gel, purified water and a pH regulator by combining a water titration method with an improved freezing method. The obtained nano-emulsion temperature-sensitive gel has the advantages of both nano-emulsion and temperature-sensitive gel, and the nano-emulsion can significantly improve the permeability of the drug and improve the stability of the drug; the selected plant essential oil is used as a natural bacteriostatic agent, not only has a synergistic bacteriostatic effect with pomegranate peel polyphenol, but also can play a role of a penetration enhancer to promote the absorption of medicines in vaginal mucosa, and has the application characteristic of combining medicines with auxiliary medicines. The drug has the advantages of good stability of physical properties and chemical properties, obvious antibacterial effect, lasting action time, good biocompatibility and convenient administration mode, and is suitable for treating vulvovaginal candida as a vaginal drug for treating vulvovaginal candida. The method is mild in reaction condition, simple in experimental operation and suitable for expanded production.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine nanoemulsion gel, and particularly relates to a pomegranate peel polyphenol nanoemulsion thermosensitive gel and a preparation method and application thereof. Background Art

[0002] Vulvovaginal candidiasis (VVC) is an inflammation of the vulva and vagina caused by fungal infection, among which Candida albicans is the main pathogenic species of VVC. Although vulvovaginal candidiasis does not cause high mortality, it has a persistent negative impact on the patient's quality of life, including irritation, itching, dysuria, dyspareunia, cheese-like vaginal discharge, vulvar burning and rupture, accompanied by mucosal lesions and minor bleeding. According to surveys, 70%-75% of women will be infected with VVC at least once in their lives, especially during their childbearing years, and 40%-50% of women will experience one or more recurrences after the symptoms of the first infection are significantly relieved. Therefore, research on therapeutic drugs for vulvovaginal candidiasis has received widespread attention. However, due to the widespread use of azole antifungal drugs, Candida albicans has increased its resistance to azole drugs, and clinical infections are difficult to cure. Therefore, it is particularly important to find new therapeutic drugs from Chinese herbal extracts to replace the frequent use of azole antifungal drugs.

[0003] Pomegranate has a long history of use in Uyghur medicine. Pomegranate peel polyphenols are polyphenol components extracted from the dried peel of pomegranate (Punica granatum L.), a plant of the Punicaceae family. They have antibacterial, antioxidant, antiviral, and antitumor pharmacological activities, and are of great significance for the prevention and treatment of chronic diseases. The polyphenol components in pomegranate peel mainly include punicalagin, punicalin, gallic acid, ellagic acid, epicatechin, kaempferol, caffeic acid, quercetin, etc. In recent years, pomegranate peel polyphenols have made good progress in the treatment of vaginitis. Pomegranate peel polyphenols have obvious antibacterial activity against Candida albicans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, etc., and are quite effective in the treatment of vaginitis. In particular, pomegranate peel polyphenol extracts have a certain restrictive effect on the growth of Candida albicans, the formation of its biofilm, and the release of specific autoinducers.

[0004] Plant essential oil is a substance with aromatic smell obtained from natural plants. It can be used as a natural broad-spectrum antibacterial agent to inhibit or kill harmful bacteria, and often has a synergistic antibacterial effect with other natural ingredients. At the same time, plant essential oils not only have good permeability themselves, but can also act as penetration enhancers for other drugs, thereby achieving better therapeutic effects. Pomegranate seed essential oil, oregano essential oil, schizonepeta microphylla essential oil, peppermint essential oil, etc., as Xinjiang specialty plant essential oils, not only have a synergistic antibacterial effect with pomegranate peel polyphenols, but can also replace penetration enhancers to promote the absorption of pomegranate peel polyphenols in the vaginal mucosa, achieving the application effect of combining medicine with adjuvant.

[0005] Nanoemulsion thermosensitive gel has the dual advantages of nanoemulsion and thermosensitive gel. As a new drug delivery carrier, nanoemulsion has the advantages of increasing drug solubility, increasing drug permeability, improving drug stability, and prolonging drug action time. Nanoemulsion can simultaneously load lipophilic and hydrophilic drugs in the oil phase and the water phase respectively, so that drugs with different physical and chemical properties can enter the lesion at the same time to play a role, thereby improving the therapeutic efficiency of the drug; the temperature-sensitive in situ gel (thermosensitive gel) is in a liquid state at room temperature, which is convenient for the injection of the gel in the vagina and the spreading of the gel to the deep vaginal cavity. After administration, it can be quickly transformed into a semisolid gel at body temperature (37°C), solidify in the vaginal cavity and adhere closely to the vaginal mucosa to form a local drug reservoir, realizing sustained drug release. Therefore, nanoemulsion thermosensitive gel can be used as a new drug delivery strategy for vaginal mucosal administration. Summary of the invention

[0006] The present invention aims to provide a pomegranate peel polyphenol nanoemulsion thermosensitive gel and a preparation method and application thereof, wherein the thermosensitive gel is made of pomegranate peel polyphenol, plant essential oil, grease, emulsifier, co-emulsifier, gel matrix, moisturizer, gel, purified water and pH regulator by water titration combined with improved freezing method, and the nanoemulsion thermosensitive gel has the dual advantages of nanoemulsion and thermosensitive gel, and the nanoemulsion can significantly improve the permeability of the drug and improve the drug stability; the selected plant essential oil not only plays a synergistic antibacterial effect with pomegranate peel polyphenol as a natural antibacterial agent, but also plays a role of a penetration enhancer, promotes the absorption of the drug in the vaginal mucosa, and has the application characteristics of drug-adjuvant integration. The thermosensitive gel is in a liquid state at room temperature, which facilitates the push injection of the gel in the vagina and the spreading of the gel to the deep part of the vaginal cavity. After administration, it is rapidly converted into a semisolid gel at body temperature (37° C.), solidifies in the vaginal cavity and adheres closely to the vaginal mucosa to form a local drug reservoir, and realizes sustained release of the drug. The nanoemulsion thermosensitive gel prepared by the present invention has good stability of physical and chemical properties, obvious antibacterial effect, long-lasting action time, good biocompatibility, convenient administration, and is suitable for vaginal administration preparation to treat vulvovaginal candidiasis. The method has mild reaction conditions, simple experimental operation, and is suitable for expanded production.

[0007] The pomegranate peel polyphenol nanoemulsion thermosensitive gel of the present invention is prepared from the following raw materials, in parts by weight: 30-50 parts of pomegranate peel polyphenol, 1-20 parts of plant essential oil, 20-50 parts of fat, 50-100 parts of emulsifier, 25-200 parts of auxiliary emulsifier, 300-500 parts of gel matrix, 10-25 parts of gel, pH regulator, and 2000-5000 parts of purified water, wherein the content of punicalagin, a main component of pomegranate peel polyphenol, is 40-45%, and the content of ellagic acid is 3-5%; the plant essential oil is one or three of pomegranate seed essential oil, oregano essential oil, thyme essential oil, and peppermint essential oil; the fat is ethyl oleate, oil Acid, catmint oil, coconut oil, myristic acid isopropyl ester or medium chain triglyceride or soybean oil; emulsifier is Tween-80, Span-80, caprylic acid capric acid macrogol glyceride or polyoxyethylene hydrogenated castor oil; gel matrix is ​​poloxamer F127 or poloxamer F68; gel is sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, deacetylated chitosan, water-soluble chitosan or carbomer 934; pH adjuster is hydrochloric acid, phosphoric acid, pectin, triethanolamine, salbutamol, sodium hydroxide or potassium hydroxide; auxiliary emulsifier is n-butanol, anhydrous ethanol, polyethylene glycol-400, glycerol, 1,2-propylene glycol or diethylene glycol monoethyl ether. The specific operation is carried out according to the following steps:

[0008] a. Mix 20-50 parts of fat and 1-20 parts of plant essential oil to obtain a multi-component oil phase, add 50-100 parts of emulsifier to the multi-component oil phase, add 25-200 parts of co-emulsifier, and stir evenly to obtain a clarified mixed oil phase;

[0009] b. Place 30-50 parts of pomegranate peel polyphenols in 2000-5000 parts of purified water, dissolve them by ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring, stirring until the solution is clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion;

[0010] c. Add 2000-5000 parts of purified water to dilute the nanoemulsion obtained in step b for later use, add 300-500 parts of gel matrix and 10-25 parts of gel adhesive to the diluted nanoemulsion at 4° C., stir until completely dissolved, refrigerate at 4° C. overnight, then add a pH adjuster to obtain a nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0011] A method for preparing a pomegranate peel polyphenol nanoemulsion thermosensitive gel, based on parts by weight, is specifically performed according to the following steps:

[0012] a. Mix 20-50 parts of oils such as ethyl oleate, oleic acid, nepeta oil, coconut oil, myristic acid isopropyl ester or medium chain triglyceride or soybean oil with 1-20 parts of plant essential oils such as one or three of pomegranate seed essential oil, oregano essential oil, thyme essential oil and peppermint essential oil to obtain a multi-electrolyte oil phase, then add 50-100 parts of emulsifiers such as Tween-80, Span-80, caprylic acid capric acid macrogol glyceride or polyoxyethylene hydrogenated castor oil, and 25-200 parts of auxiliary emulsifiers such as n-butanol, anhydrous ethanol, polyethylene glycol-400, glycerol, 1,2-propylene glycol or diethylene glycol monoethyl ether, and stir evenly to obtain a clear mixed oil phase;

[0013] b. Place 30-50 parts of pomegranate peel polyphenols in 2000-5000 parts of purified water, dissolve them by ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring, stirring until the solution is clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion;

[0014] c. The nanoemulsion obtained in step b is diluted with 2000-5000 parts of purified water for standby use, 300-500 parts of gel matrix such as poloxamer F127 or poloxamer F68 and 10-25 parts of gel adhesive such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, deacetylated chitosan, water-soluble chitosan or carbomer 934 are added to the diluted nanoemulsion at a temperature of 4° C., stirred until completely dissolved, refrigerated at a temperature of 4° C. overnight, and then a pH regulator such as hydrochloric acid, phosphoric acid, pectin, triethanolamine, salbutamol, sodium hydroxide or potassium hydroxide is added to obtain a nanoemulsion thermosensitive gel having a pH of 3.5-4.5.

[0015] Application of the pomegranate peel polyphenol nanoemulsion thermosensitive gel in preparing a medicine for treating vulvovaginal candidiasis.

[0016] The pomegranate peel polyphenol nanoemulsion thermosensitive gel of the present invention has the following beneficial effects:

[0017] The present invention adopts a water titration method combined with an improved freezing method to prepare a pomegranate peel polyphenol nanoemulsion thermosensitive gel, improves the problems of poor membrane permeability and low bioavailability of pomegranate peel polyphenol, short gel action time and easy leakage, characterizes the pomegranate peel polyphenol nanoemulsion thermosensitive gel from multiple aspects such as appearance, morphology, particle size, Zeta potential, in vitro release and in vitro penetration, and evaluates the antibacterial activity of the pomegranate peel polyphenol nanoemulsion thermosensitive gel. The research results show that the pomegranate peel polyphenol nanoemulsion thermosensitive gel has stable physical and chemical properties, good penetration and absorption effects in vivo, obvious antibacterial effects, long-lasting action time, good biocompatibility, and convenient administration, and is suitable for use as a vaginal preparation to treat vulvovaginal candidiasis.

[0018] The pomegranate peel polyphenol nanoemulsion thermosensitive gel prepared by the present invention is a temperature-responsive in-situ gel, which is in liquid state at room temperature, is easy to administer, and is convenient for the flow and spreading of drugs in the vagina; under a body temperature environment of 37°C, the gel changes from a liquid state to a semisolid state, which is convenient for the gel to adhere to the vaginal mucosa and prolong the retention time of the drug at the lesion.

[0019] Pomegranate peel polyphenol nanoemulsion thermosensitive gel significantly increased the mucosal permeability of pomegranate peel polyphenol.

[0020] Pomegranate peel polyphenol nanoemulsion thermosensitive gel has a significant ability to inhibit the growth of Candida albicans. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the process of the present invention;

[0022] Figure 2 This is the particle size distribution diagram of the pomegranate peel polyphenol nanoemulsion of the present invention;

[0023] Figure 3 This is the Zeta potential distribution diagram of the pomegranate peel polyphenol nanoemulsion of the present invention;

[0024] Figure 4 This is the appearance of the pomegranate peel polyphenol nanoemulsion thermosensitive gel of the present invention;

[0025] Figure 5 This is the in vitro release diagram of the pomegranate peel polyphenol nanoemulsion thermosensitive gel of the present invention;

[0026] Figure 6 This is the in vitro permeation diagram of the pomegranate peel polyphenol nanoemulsion thermosensitive gel of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below based on examples, but the scope of the present invention is not limited by these examples.

[0028] Example 1 (based on a volume of 20 ml)

[0029] a. Mix 20 parts of ethyl oleate and 1 part of pomegranate seed essential oil to obtain a polybasic oil phase, then add 50 parts of polyoxyethylene hydrogenated castor oil as an emulsifier and 60 parts of glycerol as an auxiliary emulsifier, and stir evenly to obtain a clear mixed oil phase;

[0030] b. Place 30 parts of pomegranate peel polyphenols, the main components of which are 40-45% punicalagin and 3-5% ellagic acid, in 2000 parts of purified water, dissolve them under ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring conditions, stirring until the solution is clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion;

[0031] c. Add 2000 parts of purified water to dilute the 30 parts of nanoemulsion obtained in step b for later use, add 300 parts of gel matrix poloxamer F127 and 5 parts of gel adhesive sodium carboxymethyl cellulose to the diluted nanoemulsion at 4° C., stir until completely dissolved, refrigerate at 4° C. overnight, then add pectin as a pH adjuster to obtain a pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0032] The average particle size of the obtained pomegranate peel polyphenol nanoemulsion thermosensitive gel is 39.2 nm, the dispersion coefficient is 0.332, the Zeta potential is -0.224 mV, and the gel phase transition temperature is 36.1°C.

[0033] Example 2 (based on a volume of 20 ml)

[0034] a. Mix 25 parts of medium-chain glycerides with 2 parts of pomegranate seed essential oil and 1 part of oregano essential oil to obtain a polybasic oil phase, then add 40 parts of emulsifier polyoxyethylene hydrogenated castor oil and 60 parts of co-emulsifier 1,2-propylene glycol, and stir evenly to obtain a clear mixed oil phase;

[0035] b. Place 40 parts of pomegranate peel polyphenols, the main components of which are 40-45% punicalagin and 3-5% ellagic acid, in 2500 parts of purified water, and slowly add the mixture dropwise to the mixed oil phase in step a under ultrasonic dissolution and stirring conditions, and stir until the solution is clear and transparent with a light blue opalescence to obtain a nanoemulsion;

[0036] c. Add 30 parts of nanoemulsion to 2500 parts of purified water for dilution and set aside. Add 350 parts of gel matrix poloxamer F127 and 10 parts of gel adhesive sodium carboxymethyl cellulose to the diluted nanoemulsion at 4°C, stir until completely dissolved, refrigerate at 4°C overnight, then add pH adjuster hydrochloric acid to obtain pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0037] The average particle size of the obtained pomegranate peel polyphenol nanoemulsion thermosensitive gel is 37.6 nm, the dispersion coefficient is 0.427, the Zeta potential is -0.265 mV, and the gel phase transition temperature is 36.4°C.

[0038] Example 3 (based on a volume of 20 ml)

[0039] a. Mix 30 parts of isopropyl myristate with 5 parts of pomegranate seed essential oil and 2 parts of thyme oil to obtain a polybasic oil phase, then add 40 parts of emulsifier Tween-80 and 80 parts of co-emulsifier polyethylene glycol-400, and stir evenly to obtain a clarified mixed oil phase;

[0040] b. Place 50 parts of pomegranate peel polyphenols, the main components of which are 40-45% punicalagin and 3-5% ellagic acid, in 5000 parts of purified water, dissolve them in ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring, and stir until the solution is clear and transparent with a light blue opalescence to obtain a nanoemulsion;

[0041] c. Add 30 parts of nanoemulsion to 4500 parts of purified water for dilution and set aside. Add 400 parts of gel matrix poloxamer F127 and 14 parts of gel adhesive hydroxypropyl methylcellulose to the diluted nanoemulsion at 4°C, stir until completely dissolved, refrigerate at 4°C overnight, then add pH adjuster triethanolamine to obtain pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0042] The average particle size of the obtained pomegranate peel polyphenol nanoemulsion thermosensitive gel is 36.8 nm, the dispersion coefficient is 0.234, the Zeta potential is -0.735 mV, and the gel phase transition temperature is 36.0°C.

[0043] Example 4 (based on a volume of 20 ml)

[0044] a. Mix 35 parts of ethyl oleate with 6 parts of pomegranate seed essential oil and 6 parts of peppermint essential oil to obtain a polybasic oil phase, then add 45 parts of emulsifier caprylic acid capric acid macrogol glyceride and 60 parts of co-emulsifier ethanol, and stir evenly to obtain a clarified mixed oil phase;

[0045] b. Place 50 parts of pomegranate peel polyphenols, the main components of which are 40-45% punicalagin and 3-5% ellagic acid, in 3000 parts of purified water, dissolve them under ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring, and stir until the solution is clear and transparent with a light blue opalescence to obtain a nanoemulsion;

[0046] c. Add 30 parts of nanoemulsion to 4000 parts of purified water for dilution and set aside. Add 450 parts of gel matrix poloxamer F127 and 15 parts of gel adhesive carbomer 934 to the diluted nanoemulsion at 4°C, stir until completely dissolved, refrigerate at 4°C overnight, then add phosphoric acid as a pH adjuster to obtain a pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0047] The average particle size of the obtained pomegranate peel polyphenol nanoemulsion thermosensitive gel is 42.68 nm, the dispersion coefficient is 0.392, the Zeta potential is -0.867 mV, and the gel phase transition temperature is 35.9°C.

[0048] Example 5 (based on a volume of 20 ml)

[0049] a. Mix 45 parts of coconut oil with 10 parts of pomegranate seed essential oil, 5 parts of peppermint essential oil and 5 parts of oregano essential oil to obtain a polybasic oil phase, then add 60 parts of emulsifier caprylic acid capric acid macrogol glyceride and 100 parts of co-emulsifier glycerol, and stir evenly to obtain a clarified mixed oil phase;

[0050] b. Place 45 parts of pomegranate peel polyphenols, the main components of which are 40-45% punicalagin and 3-5% ellagic acid, in 3000 parts of purified water, dissolve them in ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring, and stir until the solution is clear and transparent with a light blue opalescence to obtain a nanoemulsion;

[0051] c. Add 45 parts of nanoemulsion to 4000 parts of purified water for dilution and set aside, add 450 parts of gel matrix poloxamer F127 and 20 parts of gel adhesive carbomer 934 to the diluted nanoemulsion at 4°C, stir until completely dissolved, refrigerate at 4°C overnight, add 5 parts of moisturizer propylene glycol, and then add pH adjuster triethanolamine to obtain pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0052] The average particle size of the obtained pomegranate peel polyphenol nanoemulsion thermosensitive gel is 47.86 nm, the dispersion coefficient is 0.222, the Zeta potential is -1.340 mV, and the gel phase transition temperature is 35.7°C.

[0053] Example 6 (based on a volume of 20 ml)

[0054] a. Mix 50 parts of isopropyl myristate with 8 parts of pomegranate seed essential oil, 3 parts of peppermint essential oil and 1 part of oregano essential oil to obtain a polybasic oil phase, then add 80 parts of emulsifier Tween-80, add 165 parts of co-emulsifier diethylene glycol monoethyl ether, stir evenly to obtain a clarified mixed oil phase;

[0055] b. Place 46 parts of pomegranate peel polyphenols whose main components are 40-45% punicalagin and 3-5% ellagic acid in 4000 parts of purified water, dissolve them by ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring conditions, and stir them with a constant temperature magnetic force at 5000 r / min at a temperature of 25° C. until the solution becomes clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion;

[0056] c. Add 30 parts of nanoemulsion to 4500 parts of purified water for dilution and set aside. Add 100 parts of gel matrix poloxamer F127 and 25 parts of gel adhesive carbomer 934 to the diluted nanoemulsion at 4°C, stir until completely dissolved, refrigerate at 4°C overnight, then add pH adjuster sodium hydroxide to obtain pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0057] The average particle size of the obtained pomegranate peel polyphenol nanoemulsion thermosensitive gel is 27.66 nm, the dispersion coefficient is 0.276, the Zeta potential is -1.009 mV, and the gel phase transition temperature is 36.2°C.

[0058] Example 7 (based on a volume of 20 ml)

[0059] a. Mix 35 parts of nepeta oil, 10 parts of pomegranate seed essential oil and 5 parts of peppermint oil to obtain a polybasic oil phase, then add 50 parts of emulsifier caprylic acid capric acid macrogol glyceride, add 150 parts of auxiliary emulsifier anhydrous ethanol, and stir evenly to obtain a clarified mixed oil phase;

[0060] b. Place 35 parts of pomegranate peel polyphenols whose main components are 40-45% punicalagin and 3-5% ellagic acid in 3000 parts of purified water, dissolve them by ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring conditions, and stir them with a constant temperature magnetic force at 3500 r / min at 25° C. until the solution becomes clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion;

[0061] c. Add 3500 parts of purified water to dilute 30 parts of nanoemulsion for later use, add 500 parts of gel matrix poloxamer F127 and 25 parts of gel adhesive sodium carboxymethyl cellulose to the diluted nanoemulsion at 4°C, stir until completely dissolved, refrigerate at 4°C overnight, then add phosphoric acid as a pH adjuster to obtain a pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0062] The average particle size of the obtained pomegranate peel polyphenol nanoemulsion thermosensitive gel is 42.68 nm, the dispersion coefficient is 0.392, the Zeta potential is -0.867 mV, and the gel phase transition temperature is 35.9°C.

[0063] Example 8 (based on a volume of 20 ml)

[0064] a. Mix 35 parts of isopropionate with 8 parts of pomegranate seed essential oil and 2 parts of oregano essential oil to obtain a multi-element oil phase, then add 80 parts of emulsifier Span-80, add 200 parts of co-emulsifier n-butanol, and stir evenly to obtain a clarified mixed oil phase;

[0065] b. Place 50 parts of pomegranate peel polyphenols whose main components are 40-45% punicalagin and 3-5% ellagic acid in 5000 parts of purified water, dissolve them by ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring conditions, and stir them with a constant temperature magnetic force at 4000 r / min at 25° C. until the solution becomes clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion;

[0066] c. Add 30 parts of nanoemulsion to 5000 parts of purified water for dilution and set aside, add 25 parts of gel adhesive hydroxypropyl methylcellulose to the diluted nanoemulsion at 4°C, stir until completely dissolved, add 500 parts of gel matrix poloxamer F127 to the solution, stir until completely dissolved, refrigerate at 4°C overnight, then add pH adjuster potassium hydroxide to obtain pomegranate peel polyphenol nanoemulsion thermosensitive gel with a pH of 3.5-4.5.

[0067] The average particle size of the pomegranate peel polyphenol nanoemulsion thermosensitive gel obtained in Example 8 is 35.69 nm, the dispersion coefficient is 0.209, the Zeta potential is -1.337 mV, and the gel phase transition temperature is 35.8°C.

[0068] Example 9

[0069] Determination of particle size, dispersibility and zeta potential:

[0070] Any of the pomegranate peel polyphenol nanoemulsions described in Examples 1-8 was diluted 5 times with water, and its particle size, dispersion coefficient and Zeta potential were measured using a Malvern nanoparticle size and Zeta potential analyzer. The particle size distribution and Zeta potential were shown in Table 1. Figure 2 and Figure 3 .

[0071] Example 10

[0072] Appearance of Nanoemulsion Thermosensitive Gel:

[0073] When the pomegranate peel polyphenol nanoemulsion thermosensitive gel described in Example 1 was irradiated with laser, it was observed that the gel was uniform and bubble-free, clear and transparent, and the Tyndall phenomenon occurred. Figure 4 ;

[0074] Embodiment 11

[0075] Stability test:

[0076] (1) Lighting test:

[0077] The pomegranate peel polyphenol nanoemulsion and the pomegranate peel polyphenol nanoemulsion thermosensitive gel described in Example 1 were placed under 4500±500Lx light conditions for 10 days, and their appearance was observed, and the particle size, dispersion coefficient and Zeta potential were measured to evaluate their stability. The results showed that under 4500±500Lx light conditions, the pomegranate peel polyphenol nanoemulsion maintained a clear and transparent appearance, uniform state, no stratification, demulsification and flocculation occurred, the average particle size was 39.7nm, the dispersion coefficient was 0.364, and the Zeta potential was -0.227mV. The pomegranate peel polyphenol nanoemulsion thermosensitive gel maintained a clear and transparent appearance, uniform state, no stratification, demulsification and flocculation occurred, and the gel phase transition temperature was 36.1°C.

[0078] (2) Accelerated testing:

[0079] The pomegranate peel polyphenol nanoemulsion and the pomegranate peel polyphenol nanoemulsion thermosensitive gel described in Example 1 were placed under accelerated conditions of temperature (30±2)°C and relative humidity (60±5)% for 6 months, and their appearance was observed, and the particle size, dispersion coefficient and Zeta potential were measured to evaluate their stability. The results showed that under accelerated conditions of temperature (30±2)°C and relative humidity (60±5)%, the pomegranate peel polyphenol nanoemulsion maintained a clear, transparent, uniform state, no stratification, demulsification and flocculation occurred, the average particle size was 42.1nm, the dispersion coefficient was 0.378, and the Zeta potential was -0.254mV. The pomegranate peel polyphenol nanoemulsion thermosensitive gel maintained a clear, transparent, uniform state, no stratification, demulsification and flocculation occurred, and the gel phase transition temperature was 36.4°C.

[0080] (3) Long-term testing:

[0081] The pomegranate peel polyphenol nanoemulsion and the pomegranate peel polyphenol nanoemulsion thermosensitive gel described in Example 1 were placed in a temperature of (25±2)°C and a relative humidity of (60±5)% for a long-term test for 18 months, and their appearance was observed, and the particle size, dispersion coefficient and Zeta potential were measured to evaluate their stability. The results showed that under the accelerated conditions of a temperature of (30±2)°C and a relative humidity of (60±5)%, the appearance of the pomegranate peel polyphenol nanoemulsion remained clear and transparent, and was in a uniform state, without stratification, demulsification or flocculation. The average particle size was 45.2 nm, the dispersion coefficient was 0.428, and the Zeta potential was -0.176 mV. The appearance of the pomegranate peel polyphenol nanoemulsion thermosensitive gel remained clear and transparent, and was in a uniform state, without stratification, demulsification or flocculation. The gel phase transition temperature was 36.0°C.

[0082] Example 12

[0083] Fluid dynamics determination:

[0084] The pomegranate peel polyphenol nanoemulsion and the pomegranate peel polyphenol nanoemulsion thermosensitive gel described in Example 1 were placed in a rotational rheometer, respectively, and the fluid mechanics properties of the nanoemulsion and the nanoemulsion thermosensitive gel were measured; the results showed that the pomegranate peel polyphenol nanoemulsion and the pomegranate peel polyphenol nanoemulsion thermosensitive gel both had non-Newtonian fluid properties and had a viscosity suitable for drug administration.

[0085] Embodiment 13

[0086] Gel phase transition assay:

[0087] The pomegranate peel polyphenol nanoemulsion thermosensitive gel described in Example 1 was placed in a constant temperature magnetic stirrer, and the gel phase transition temperature was determined using a rotor method. The rotor was placed in the pomegranate peel polyphenol nanoemulsion thermosensitive gel and heated at a constant speed of 500 r / min until the gel was in a semi-solid state. The temperature at which the rotor no longer rotated was the gel phase transition temperature.

[0088] Embodiment 14

[0089] Adhesion performance test:

[0090] The pomegranate peel polyphenol nanoemulsion thermosensitive gel described in Example 1 was placed in a texture analyzer to measure the separation force and adhesion work between the gel and the pig gastric mucin disk to evaluate its adhesion. The results showed that the separation force of the pomegranate peel polyphenol nanoemulsion thermosensitive gel was -8.025 kgf, and the adhesion work was -22.71 J / m 2 It can form good adhesion with the vaginal mucosa, is suitable for vaginal administration, and is not easy to leak.

[0091] Embodiment 15

[0092] HPLC method establishment:

[0093] The content of punicalagin (α+β) of pomegranate peel polyphenol in the pomegranate peel polyphenol nanoemulsion thermosensitive gel described in any one of Examples 1-8 was determined by high performance liquid chromatography (HPLC), chromatographic column: Thermo Scientific Syncronis C18 reverse analytical chromatographic column (250mm×4.6mm, 5μm,); mobile phase: methanol-0.3% phosphoric acid aqueous solution (5-100%); volume flow rate: 1.0mL / min; column temperature: 30°C; detection wavelength: 254nm; injection volume: 10μL, punicalagin α peak time is 12.8min, punicalagin β peak time is 28.1min, and blank solution has no interference; methodological experimental results show that punicalagin (α+β) has a good linear relationship in the concentration range of 1.0-120.0μg / mL, and the RSDs of repeatability, stability test and sample recovery test are all less than 2.0%.

[0094] Example 16

[0095] In vitro release test of pomegranate peel polyphenol nanoemulsion thermosensitive gel:

[0096] The in vitro release characteristics of pomegranate peel polyphenol nanoemulsion thermosensitive gel were investigated by dialysis bag method. 5 ml of fresh pomegranate peel polyphenol nanoemulsion thermosensitive gel was placed in a dialysis bag with a molecular weight cutoff of 12000-14000. The dialysis bag was suspended in a beaker containing 200 ml of simulated vaginal physiological fluid with a pH value of 4.2, and the sample was shaken in a constant temperature water bath at 37°C. 1.0 ml of sample was drawn at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, respectively, and 1.0 ml of simulated vaginal physiological fluid at a temperature of 37°C was added at the same time. The sample was filtered through a 0.45 μm filter membrane, and the filtrate was taken to determine the concentration of punicalagin by HPLC method. The cumulative release rate of punicalagin was calculated by formula (I), as follows: Figure 5 As shown;

[0097]

[0098] E in the formula r : Cumulative drug release; V e : replacement volume of PBS; V0: total volume of release medium; C i : the concentration of the release liquid during the ith replacement sampling; m drug : total mass of drugs carried by nanoparticles; n: number of times PBS was replaced.

[0099] Embodiment 17

[0100] In vitro permeation test of pomegranate peel polyphenol nanoemulsion thermosensitive gel:

[0101] The in vitro permeation characteristics of pomegranate peel polyphenol nanoemulsion thermosensitive gel were determined by the Franz diffusion cell method. The TP-6A intelligent transdermal tester was fixed, and a stirring bar and a water bath were added to the receiving chamber at a constant temperature of 37°C for 30 minutes. The freshly prepared isolated bovine vaginal mucosa was taken out from the physiological saline, with the mucosal surface facing up, and fixed on the diffusion cell. After 30 minutes of adaptive equilibrium, 5 g of pomegranate peel polyphenol nanoemulsion thermosensitive gel was placed in the supply chamber, and the receiving chamber was filled with a 30% ethanol solution prepared with physiological saline as The diffusion medium and the outer layer were kept warm in a circulating water bath at 37°C and stirred at 300 r / min by a magnetic stirrer. 1 ml of receiving solution was drawn from the sample receiving chamber at 0.5, 1, 2, 4, 6, 8, 10, and 12 h after the start of the experiment, and an equal volume of blank receiving solution at the same temperature was added to the receiving chamber. The drawn receiving solution was filtered with a 0.22 μm microporous filter membrane, and the filtrate was taken to determine the concentration of punicalagin by HPLC. The cumulative permeation amount (Q) was calculated according to the following formula (II): n ),like Figure 6 ;

[0102]

[0103] In the formula, Q n: Cumulative area permeation at the nth time point μg / cm 2 ; V: The total volume of the receiving liquid in the receiving chamber is 17ml; V i :The sampling volume each time is 1ml; C n : drug mass concentration measured at the nth sampling point μg / ml; A: effective diffusion area cm 2 .

[0104] Embodiment 18

[0105] Antibacterial activity test:

[0106] The agar dilution method was used to add pomegranate peel polyphenol nanoemulsion thermosensitive gel to the agar medium cooled to 50°C after sterilization, so that the final concentrations were 4, 2, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 mg / mL, respectively, and each was poured into two culture dishes for parallel experiments; after cooling and solidification, 2 μL of Candida albicans (ATCC10231) diluted bacterial suspension was dripped on the agar surface, 3 drops were dripped on each plate, and after drying, the plates were placed in a constant temperature incubator at 37°C and inverted for 24 hours to observe the bacterial growth; a blank plate without addition was used as a positive control, and the lowest dilution concentration for sterile growth was determined as the minimum inhibitory concentration; the experiment measured that when the concentration of pomegranate peel polyphenol nanoemulsion thermosensitive gel was 0.3 mg / ml, there was no bacterial growth, that is, the minimum inhibitory concentration was 0.3 mg / ml.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A pomegranate peel polyphenol nanoemulsion thermosensitive gel, characterized in that: The invention is prepared from the following raw materials by weight: 30-50 parts of pomegranate peel polyphenol, 1-20 parts of plant essential oil, 20-50 parts of fat, 50-100 parts of emulsifier, 25-200 parts of auxiliary emulsifier, 300-500 parts of gel matrix, 10-25 parts of gel, pH regulator, and 2000-5000 parts of purified water, wherein the content of punicalagin, a main component of pomegranate peel polyphenol, is 40-45%, and the content of ellagic acid is 3-5%; the plant essential oil is one or three of pomegranate seed essential oil, oregano essential oil, thyme essential oil, and peppermint essential oil; the fat is ethyl oleate, oleic acid, nepeta oil, coconut oil, Myristic acid isopropyl ester or medium chain triglyceride or soybean oil; emulsifier is Tween-80, Span-80, caprylic acid capric acid macrogol glyceride or polyoxyethylene hydrogenated castor oil; gel matrix is ​​poloxamer F127 or poloxamer F68; gel is sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, deacetylated chitosan, water-soluble chitosan or carbomer 934; pH regulator is hydrochloric acid, phosphoric acid, pectin, triethanolamine, salbutamol, sodium hydroxide or potassium hydroxide; auxiliary emulsifier is n-butanol, anhydrous ethanol, polyethylene glycol-400, glycerol, 1,2-propylene glycol or diethylene glycol monoethyl ether. The specific operation is carried out according to the following steps: a. Mix 20-50 parts of fat and 1-20 parts of plant essential oil to obtain a multi-component oil phase, add 50-100 parts of emulsifier to the multi-component oil phase, add 25-200 parts of co-emulsifier, and stir evenly to obtain a clarified mixed oil phase; b. Place 30-50 parts of pomegranate peel polyphenols in 2000-5000 parts of purified water, dissolve them by ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring, stirring until the solution is clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion; c. Add 2000-5000 parts of purified water to dilute the nanoemulsion obtained in step b for later use, add 300-500 parts of gel matrix and 10-25 parts of gel adhesive to the diluted nanoemulsion at 4° C., stir until completely dissolved, refrigerate at 4° C. overnight, then add a pH adjuster to obtain a nanoemulsion thermosensitive gel with a pH of 3.5-4.

5.

2. A method for preparing a pomegranate peel polyphenol nanoemulsion thermosensitive gel, characterized in that: The specific operation is carried out according to the following steps: a. Mix 20-50 parts of oils such as ethyl oleate, oleic acid, nepeta oil, coconut oil, myristic acid isopropyl ester or medium chain triglyceride or soybean oil with 1-20 parts of plant essential oils such as one or three of pomegranate seed essential oil, oregano essential oil, thyme essential oil and peppermint essential oil to obtain a multi-electrolyte oil phase, then add 50-100 parts of emulsifiers such as Tween-80, Span-80, caprylic acid capric acid macrogol glyceride or polyoxyethylene hydrogenated castor oil, and 25-200 parts of auxiliary emulsifiers such as n-butanol, anhydrous ethanol, polyethylene glycol-400, glycerol, 1,2-propylene glycol or diethylene glycol monoethyl ether, and stir evenly to obtain a clear mixed oil phase; b. Place 30-50 parts of pomegranate peel polyphenols in 2000-5000 parts of purified water, dissolve them by ultrasonication, and slowly add them dropwise to the mixed oil phase in step a under stirring, stirring until the solution is clear and transparent with a light blue opalescence, thereby obtaining a nanoemulsion; c. The nanoemulsion obtained in step b is diluted with 2000-5000 parts of purified water for standby use, 300-500 parts of gel matrix such as poloxamer F127 or poloxamer F68 and 10-25 parts of gel adhesive such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, deacetylated chitosan, water-soluble chitosan or carbomer 934 are added to the diluted nanoemulsion at a temperature of 4° C., stirred until completely dissolved, refrigerated at a temperature of 4° C. overnight, and then a pH regulator such as hydrochloric acid, phosphoric acid, pectin, triethanolamine, salbutamol, sodium hydroxide or potassium hydroxide is added to obtain a nanoemulsion thermosensitive gel having a pH of 3.5-4.

5.

3. Use of the pomegranate peel polyphenol nanoemulsion thermosensitive gel as claimed in claim 1 or 2 in the preparation of a medicament for treating vulvovaginal Candida.

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