Itching relieving gel containing 3-hydroxydecanoic acid microemulsion and preparation method thereof

Abstract

The invention discloses an itching-relieving gel containing 3-hydroxydecanoic acid microemulsion and a preparation method thereof, the gel combines natural itching-relieving components 3-hydroxydecanoic acid and peppermint essential oil, the natural itching-relieving components 3-hydroxydecanoic acid and peppermint essential oil are prepared into a microemulsion form, nanoscale liquid drops are compounded with a skin-promoting absorbent, and the gel is slowly released, so that the itching-relieving effect is achieved. Therefore, the percutaneous absorption efficiency is improved, the residence time in the skin is prolonged, and a lasting itching relieving effect is realized; 3-hydroxydecanoic acid exerts an itching relieving effect through an anti-inflammatory effect, blocking of an itching signal channel and reduction of mast cell degranulation, and the itching relieving gel is stable at room temperature, convenient to use and suitable for relieving mosquito bites and skin itch problems caused by histamine or non-histamine substances and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of antipruritic drugs and preparations thereof, and particularly relates to an antipruritic gel containing 3-hydroxydecanoic acid microemulsion and a preparation method thereof. Background Art

[0002] As a natural barrier of the human body, the skin effectively prevents foreign substances from entering the body and protects internal organs and blood vessels from damage. However, the skin barrier function also makes transdermal drug delivery face challenges, and it is difficult for topical drugs to effectively penetrate the skin to exert their effects. Since the skin is composed of multiple layers of cells, compounds with smaller size and certain lipophilicity are more likely to pass through the skin barrier. At present, there are many methods to promote transdermal absorption, such as adding penetration enhancers, preparing drugs as nanoparticles or emulsions, and using physical means such as ultrasound or microneedles. Microemulsion is an emulsion with a particle size of less than 20 nanometers. It is transparent, has good heat and cold stability, and can be stored for a long time without stratification. Therefore, it is regarded as an ideal transdermal drug delivery method. However, the preparation methodology of establishing microemulsions is relatively complicated, and it is necessary to screen suitable surfactants, co-surfactants and their ratios through experiments.

[0003] Itch is a discomfort that triggers the desire to scratch, and chronic itching is also accompanied by dermatitis or systemic diseases, which seriously affects the quality of life. The occurrence of itching is closely related to a variety of cells and factors, such as keratinocytes, neurons, immune cells, as well as interleukins and chemokines (Du et al., Neuroscience 495, 2022: 74-85). Currently, pure natural antipruritic ingredients used for mosquito bites, such as comfrey, menthol, eugenol, camphor oil, etc., have very limited antipruritic effects. Many antipruritic products for mosquito bites still need to add chemically synthesized antipruritic components such as dexamethasone and methyl salicylate. Commonly used oral antipruritic drugs in clinical practice include antihistamines (such as loratadine and diphenhydramine), and topical drugs are mostly glucocorticoids (such as dexamethasone and hydrocortisone), etc., which may cause serious side effects with long-term use, and have poor effects on non-histamine-mediated itching.

[0004] 3-Hydroxydecanoic acid is a natural compound, widely present in glycolipids and polyhydroxyalkanoates secreted by bacteria (Applied Microbiology and Biotechnology, 2000, 53: 167-172), and is rich in sources and safe. Although there is no report on the antipruritic effect of 3-hydroxydecanoic acid, we accidentally found that it has a strong anti-inflammatory effect in cell experiments, indicating that it may produce an antipruritic effect by inhibiting inflammation. And as a lipophilic compound, 3-hydroxydecanoic acid may have transdermal absorption ability. However, 3-hydroxydecanoic acid is more insoluble in water, and it is inconvenient to directly apply it as a solid, and it needs to be prepared into a preparation that is easy to use. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides an antipruritic gel containing 3-hydroxydecanoic acid microemulsion and a preparation method thereof.

[0006] The core principle of the present invention is that 3-hydroxydecanoic acid reduces skin itching caused by inflammation by inhibiting the production of inflammatory factors in immune cells such as macrophages and degranulation of mast cells. In addition, 3-hydroxydecanoic acid can also inhibit the TRP neural pathway associated with itching. Compared with conventional chemical synthetic antipruritic agents, 3-hydroxydecanoic acid has a mild effect and has both specific and nonspecific dual inhibitory effects. Therefore, it is not only effective for histamine-mediated allergic itching, but also can effectively relieve itching caused by non-histamine.

[0007] The technical solution of the present invention is as follows:

[0008] An antipruritic gel containing 3-hydroxydecanoic acid microemulsion, which is composed of 20-50% of 3-hydroxydecanoic acid microemulsion and 50-80% of carbomer gel;

[0009] The formula of the 3-hydroxydecanoic acid microemulsion is: 5-10% 3-hydroxydecanoic acid, 1-2% peppermint essential oil, 15-30% surfactant, 15-30% co-surfactant, and the balance is pure water; wherein the surfactant is preferably alkyl glycoside, and the co-surfactant is preferably glycerol;

[0010] The carbomer gel is obtained by mixing carbomer 940 and pure water in a mass ratio of 2:98, and then adjusting the pH value to 7.4 with triethanolamine.

[0011] The preparation method of the antipruritic gel containing 3-hydroxydecanoic acid microemulsion of the present invention is as follows:

[0012] (1) Preparation of 3-hydroxydecanoic acid microemulsion

[0013] Mix 3-hydroxydecanoic acid and peppermint essential oil, add surfactant, co-surfactant and pure water in sequence, heat to 40-50° C., stir and mix at 5000-10000 rpm for 5-30 minutes to obtain 3-hydroxydecanoic acid microemulsion;

[0014] (2) Preparation of Carbomer Gel

[0015] Carbomer 940 was mixed with pure water, and the pH was adjusted to 7.4 with triethanolamine under stirring to prepare a transparent carbomer gel;

[0016] (3) Preparation of antipruritic gel

[0017] The carbomer gel obtained in step (2) is added to the 3-hydroxydecanoic acid microemulsion obtained in step (1), and stirred for 5 to 30 minutes to obtain an antipruritic gel.

[0018] The antipruritic gel containing the 3-hydroxydecanoic acid microemulsion of the present invention can be used to prepare medicines for treating mosquito bites and skin itching caused by histamine or non-histamine substances (such as quinoa pod hairs).

[0019] The beneficial effects of the present invention are:

[0020] The present invention uses 3-hydroxydecanoic acid as an antipruritic for the first time. It has a small molecular weight and lipophilic properties, can effectively penetrate the skin, inhibit multiple inflammatory factors and block the itch signal pathway, thereby achieving a good antipruritic effect. In addition, 3-hydroxydecanoic acid is derived from microbial fermentation, has high safety, few side effects, and is suitable for long-term use. Through preparations in the form of microemulsions and gels, the particle size of 3-hydroxydecanoic acid droplets is reduced to less than 10 nm, and synergistically acts with a surfactant that promotes penetration, thereby improving transdermal absorption and prolonging the residence time in the skin, thereby enhancing the antipruritic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Appearance of antipruritic gel containing 3-hydroxydecanoic acid microemulsion and its stability after centrifugation, high temperature and low temperature treatment.

[0022] Figure 2 : Skin surface condition of volunteers bitten by mosquitoes 1 hour after applying anti-itch gel.

[0023] Figure 3 :Mouse macrophages RAW264.7 secrete three inflammatory factors after being stimulated by lipopolysaccharide (LPS), while the secretion of inflammatory factors is reduced after the action of 3-hydroxydecanoic acid (3-HDA).

[0024] Figure 4 :TRPV1 protein immunofluorescence staining (red fluorescence), cell nuclei are stained with DAPI (blue fluorescence). Capsaicin activates the TRPV1 pathway, while 3-hydroxydecanoic acid can inhibit the activation of this pathway.

[0025] Figure 5 : Degranulation of mast cells (RBL-2H3), arrows indicate degranulated mast cells. DETAILED DESCRIPTION

[0026] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following implementation is only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0027] In the following embodiments,

[0028] 3-Hydroxydecanoic acid was provided by Huzhou Zijin Biotechnology Co., Ltd. with a purity of 90%, and the main impurity was 3-hydroxylauric acid;

[0029] Peppermint essential oil was purchased from Anhui Taihe Peppermint Base and is 100% natural peppermint extract;

[0030] Surfactant alkyl glycoside was provided by Jiangsu Wanqi Biotechnology Co., Ltd.;

[0031] Carbomer 940 was purchased from Wuhan Desheng Biochemical Technology Co., Ltd.

[0032] Example 1: Preparation of 3-hydroxydecanoic acid microemulsion, carbomer gel and antipruritic gel

[0033] 3-Hydroxydecanoic acid is almost insoluble in water. After mixing with peppermint essential oil, it forms an oily liquid. After adding surfactants, co-surfactants, and water, it is heated to 40-50°C and fully stirred to form a light yellow transparent microemulsion.

[0034] Table 1 Formula of 3-hydroxydecanoic acid microemulsion

[0035]

[0036] Carbomer 940 and pure water were mixed in a mass ratio of 2:98, and then triethanolamine was added while stirring to adjust the pH to 7.4 to obtain a transparent gel. Carbomer gel was added to the hydroxydecanoic acid microemulsion numbered 2 in the above table, and stirring was continued for 5-30 minutes after the addition. The formulation parameters are shown in Table 2. In the end, a uniform and transparent gel was obtained. Its morphology is as follows Figure 1 shown.

[0037] Table 2 Mixing ratio of 3-hydroxydecanoic acid microemulsion and carbomer gel

[0038]

[0039] Example 2: Particle size and stability analysis of antipruritic gel

[0040] The particle size of the microemulsions in the gels No. 1-3 in Table 2 was measured using a nanoparticle size analyzer. The stability of the microemulsions in the gels No. 1-3 in Table 2 was also measured under centrifugation (1000 rpm for 10 minutes), low temperature (-5°C for 7 days), and high temperature (45°C for 7 days). The results are shown in Table 3.

[0041] Table 3 Particle size and stability analysis of microemulsion gel

[0042]

[0043] The results showed that the particle size of the microemulsion of the antipruritic gel was less than 10 nm, and the gel had good stability and could withstand high and low temperature storage environments without stratification (appearance Figure 1 shown).

[0044] Example 3: Evaluation of the transdermal effect of 3-hydroxydecanoic acid in antipruritic gel

[0045] Fresh pigskin (skin near the nipple) was obtained from a local slaughterhouse, placed in PBS buffer, refrigerated and transported back to the laboratory within 1 hour. The pigskin was cut into small pieces of 1 cm×1 cm, and antipruritic gel (product numbered 2# configured in Example 1) and 3-hydroxydecanoic acid microemulsion (formula numbered 2 in Table 1) were applied on the surface, 0.1 g each. The permeation rate was then measured every 1 hour. Three parallel groups were set up at each time point. The permeation rate % = the total amount of hydroxydecanoic acid remaining on the surface of the pigskin in mg / the amount of hydroxydecanoic acid initially applied in mg×100%. The results are shown in Table 4.

[0046] Table 4 Comparison of the skin penetration rate of antipruritic gel (2#), 3-hydroxydecanoic acid solid and 3-hydroxydecanoic acid microemulsion on fresh pig skin

[0047]

[0048] As can be seen from the above table, 3-hydroxydecanoic acid, as a waxy solid, is difficult to apply to the skin surface and is almost impossible to penetrate the skin. The preparation of microemulsion significantly improves the transdermal rate of 3-hydroxydecanoic acid, thereby increasing its utilization rate, and the transdermal rate reaches more than 70% in 2 hours. This may be because the microemulsion has a nanoscale and contains a large amount of surfactants that can help transdermal penetration, so the formula of the antipruritic gel shows a good transdermal effect. However, as a liquid preparation, the microemulsion is easy to lose from the skin surface and has a poor skin feel. Although the antipruritic gel slows down the transdermal rate of 3-hydroxydecanoic acid to a certain extent, the gel form is conducive to the residence of 3-hydroxydecanoic acid on the skin surface, has a better skin feel, and is easier to maintain a long-term antipruritic effect.

[0049] Example 4: Experiment on the inhibition of histamine-induced skin pruritus in mice by microemulsion gel

[0050] Experimental method (reference: Yang et al. Journal of Ethnopharmacology 298, 2022, 115543): 20 mice, half male and half female, were selected and randomly divided into a blank group, a model group, a positive control group and an antipruritic gel treatment group, with 5 mice in each group. One day before the experiment, the hair on the back of the mouse's neck was removed with a shaver, with an area of ​​approximately 2cm×2cm. The mice were kept in a single cage for 24 hours to adapt to the environment. Each group of mice was treated with local medication: the blank group and the model group were smeared with sterile saline, the control group was smeared with a commercially available brand of antipruritic water, and the treatment group was smeared with the antipruritic gel numbered 2# in Example 1, and each group was smeared twice.

[0051] After medication, except for the blank group, mice in other groups were intradermally injected with 100 μL of histamine in the hair removal area on the back of the neck, and then the mice were immediately placed in the experimental observation cage. In a quiet environment, the number of times the mice scratched the injection site with their hind paws within 30 minutes was recorded. The formula for calculating the itch inhibition rate is:

[0052] Itch inhibition rate (%) = (average scratching times in the model group - average scratching times in the treatment group) / average scratching times in the model group × 100%.

[0053] The average scratching times of mice in each group within 30 minutes after histamine injection are shown in Table 5. Histamine injection triggered an itch reaction in mice. Compared with the model group, the scratching times of mice in the treatment group were significantly reduced, indicating that the antipruritic gel of the present invention has a good inhibitory effect on histamine-induced skin itch in mice.

[0054] Table 5 Itching conditions of animals in each group in the histamine-induced pruritus model experiment in mice

[0055]

[0056] Example 5: Experiment on the inhibition of non-histamine substances-induced itching of mouse skin by antipruritic gel

[0057] Experimental method (reference: W.-J.Lee et al.J Ginseng Res 42 (2018) 470-475): Referring to Example 4, only the subcutaneously injected irritant histamine was replaced with a subcutaneously injected 4ng of quinoa pod hair extract (reference: The Journal of Neuroscience, 2008, 28: 4331-4335), and the rest of the experimental operations remained the same. The treatment group was smeared with the antipruritic gel numbered 2# in Example 1. The average number of scratching of mice in each group within 30 minutes after the injection of quinoa pod hair was recorded (see Table 6). Compared with the model group, the number of scratching of mice in the treatment group was significantly reduced, indicating that the antipruritic gel of the present invention has a good inhibitory and antipruritic effect on skin itching caused by the non-histamine pathway induced by quinoa pod hair.

[0058] Table 6 Itch conditions of each group of animals in the mouse non-histamine itch pathway model experiment

[0059]

[0060] Example 6: Volunteer testing of the antipruritic effect of the antipruritic gel on mosquito bites

[0061] Observation subjects and grouping: This experiment selected 60 volunteers bitten by mosquitoes. The clinical manifestations were red papules, swelling, and bulges at the bite site, accompanied by local itching and heat pain. The subjects were randomly divided into a treatment group and a control group. There were 30 cases in the treatment group, including 10 males and 20 females, with an age range of 25 to 60 years old; there were 30 cases in the control group, including 12 males and 18 females, with an age range of 22 to 55 years old. There was no statistically significant difference in age and gender between the two groups, and they were comparable.

[0062] Experimental method (refer to CN106138241B method): The treatment group used the antipruritic gel numbered 2# in Example 1, and the control group used a certain brand of antipruritic floral water, which were applied to the mosquito bite sites, 2 to 4 times a day.

[0063] Efficacy evaluation criteria:

[0064] - Significantly effective: Clinical symptoms completely disappeared within 30 minutes, red spots and swelling completely subsided, and itching and heat pain disappeared.

[0065] - Effective: Symptoms basically disappeared or were significantly relieved within 60 minutes, red spots basically disappeared, redness, swelling and bulges were significantly reduced, and itching and heat pain were significantly reduced.

[0066] - Ineffective: No significant improvement in symptoms within 4 hours, red spots did not disappear, redness and swelling did not change, and itching and heat pain did not improve.

[0067] The experimental results show that the total effective rate of the antipruritic gel prepared by the present invention on mosquito bites is 97%, and the itching disappears within an average of 10 minutes, and the redness and swelling subside within 30 minutes. The total effective rate of the control group of floral water is 77%. The results show that the antipruritic gel of the present invention is effective in treating red spots, redness, swelling, bulges, itching and heat pain caused by mosquito bites, and is better than ordinary floral water on the market. The treatment effects of the two groups are shown in Table 7. The pictures of the bite sites of typical volunteers are shown in Figure 2 As shown, it shows that the itching sensation subsided about 3 minutes after applying the anti-itch gel, and the redness and swelling subsided significantly within 1 hour.

[0068] Table 7 Comparison of clinical efficacy of antipruritic gel in the treatment of mosquito bites.

[0069]

[0070] Example 7: Analysis of the antipruritic mechanism of microemulsion gel containing 3-hydroxydecanoic acid

[0071] The antipruritic gel of the present invention uses 3-hydroxydecanoic acid as the main active ingredient, and its antipruritic effect is discovered for the first time, so it is necessary to conduct an in-depth analysis of its antipruritic mechanism. Through in vitro cell experiments, the study was conducted from three aspects: inhibiting the secretion of inflammatory factors, blocking the itching signal pathway, and inhibiting mast cell degranulation.

[0072] 1. Experiment on inhibiting the secretion of inflammatory factors (reference Xu et al. Molecules 2022, 27, 5037): The experiment used mouse RAW264.7 cells as a model and was divided into a control group, a model group, and an experimental group. The control group was cultured in normal culture medium for 12 hours, the model group was cultured with 1 mg / L LPS (lipopolysaccharide), and the experimental group was cultured with 10 mg / L 3-hydroxydecanoic acid on the basis of LPS for 12 hours. The secretion level of inflammatory factors such as TNF-α in the cell supernatant was detected by enzyme-linked immunosorbent assay. Figure 3 As shown, compared with the control group, the inflammatory factor TNF-α in the model group was significantly increased; 3-hydroxydecanoic acid in the experimental group significantly inhibited the increase of inflammatory factors, indicating that it may exert an antipruritic effect by inhibiting the secretion of inflammatory factors.

[0073] 2. Signal pathway influence experiment (reference Yang et al. International Immunopharmacology 96, 2021, 107620): RAW264.7 cells were inoculated in 96-well plates and divided into blank control group, model group and experimental group. HBSS buffer was added to the blank control group and the model group, and HBSS buffer containing 10 mg / L 3-hydroxydecanoic acid was added to the experimental group. After 2 hours of pretreatment, 100 μM capsaicin was added to the model group and the experimental group for stimulation. After incubation for 24 hours, TRPV1 protein was immunofluorescently stained and DAPI was used to mark the cell nucleus. Observation results under laser confocal microscopy showed that 3-hydroxydecanoic acid significantly reduced the expression of TRPV1 protein, indicating that it reduces the conduction of itch signals by inhibiting TRPV1 channels ( Figure 4 ).

[0074] 3. Mast cell degranulation experiment (reference WO2018115295A1): Mast cells (RBL-2H3) were used as a model and divided into a control group, a model group and an experimental group. The control group was cultured in normal culture medium for 2 hours, the model group was stimulated by adding 10 μM capsaicin, and the experimental group was cultured for 2 hours by adding 10 mg / L 3-hydroxydecanoic acid on the basis of capsaicin. After staining with toluidine blue, the changes in purple-red granules in the cytoplasm were observed under an optical microscope. The results showed that compared with the model group, 3-hydroxydecanoic acid in the experimental group significantly inhibited the degranulation of mast cells, and its effect was equivalent to that of dexamethasone ( Figure 5 ). Inhibiting mast cell degranulation can reduce the subsequent production of histamine substances, thereby achieving an antipruritic effect.

[0075] Based on the above mechanism research results, the antipruritic mechanism of 3-hydroxydecanoic acid is mainly achieved through three aspects: inhibiting the secretion of inflammatory factors, blocking the TRPV1 signaling pathway, and reducing mast cell degranulation. These research results provide a theoretical basis for the antipruritic gel of the present invention and verify the scientificity and rationality of its antipruritic effect.

[0076] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. An antipruritic gel containing 3-hydroxydecanoic acid microemulsion, characterized in that: It is composed of 20-50% 3-hydroxydecanoic acid microemulsion and 50-80% carbomer gel.

2. The antipruritic gel containing 3-hydroxydecanoic acid microemulsion according to claim 1, characterized in that: The formula of the 3-hydroxydecanoic acid microemulsion is: 5-10% of 3-hydroxydecanoic acid, 1-2% of peppermint essential oil, 15-30% of surfactant, 15-30% of co-surfactant and the balance of pure water.

3. The antipruritic gel containing 3-hydroxydecanoic acid microemulsion according to claim 2, characterized in that: The surfactant is an alkyl glycoside.

4. The antipruritic gel containing 3-hydroxydecanoic acid microemulsion according to claim 2, characterized in that: The co-surfactant is glycerol.

5. The antipruritic gel containing 3-hydroxydecanoic acid microemulsion according to claim 1, characterized in that: Carbomer gel is prepared by mixing Carbomer 940 and pure water in a mass ratio of 2:98, and then adjusting the pH value to 7.4 with triethanolamine.

6. The method for preparing the antipruritic gel containing 3-hydroxydecanoic acid microemulsion according to claim 1, characterized in that: The preparation method is as follows: (1) Preparation of 3-hydroxydecanoic acid microemulsion Mix 3-hydroxydecanoic acid and peppermint essential oil, add surfactant, co-surfactant and pure water in sequence, heat to 40-50° C., stir and mix at 5000-10000 rpm for 5-30 minutes to obtain 3-hydroxydecanoic acid microemulsion; (2) Preparation of Carbomer Gel Carbomer 940 was mixed with pure water, and the pH was adjusted to 7.4 with triethanolamine under stirring to prepare a transparent carbomer gel; (3) Preparation of antipruritic gel The carbomer gel obtained in step (2) is added to the 3-hydroxydecanoic acid microemulsion obtained in step (1), and stirred for 5 to 30 minutes to obtain an antipruritic gel.

7. Use of the antipruritic gel containing 3-hydroxydecanoic acid microemulsion as claimed in claim 1 in the preparation of medicines for treating mosquito bites and skin itching caused by histamine or non-histamine substances.

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