TWI740096B - Use of tussilago farfara extract for enhancing the gene expression of tgm, krt, aqp, flg, gba, and has - Google Patents

Abstract

The present invention provides a use of Tussilago farfara extract for enhancing the gene expression of TGM, KRT, AQP, FLG, GBA, and HAS. The Tussilago farfara extract can form more moisturizing factors on the skin and maintain the integrity of the stratum corneum to enhance the skin barrier function. The Tussilago farfara extract is prepared by extracting Tussilago farfara using water, alcohols, or mixtures of water and alcohols as solvents.

Description

The use of coltsfoot extract to enhance the expression of TGM gene, KRT gene, AQP gene, FLG gene, GBA gene, and HAS gene

The present invention relates to the use of a winter flower extract, in particular to the use of a butterbur flower extract to enhance the expression of TGM genes, KRT genes, AQP genes, FLG genes, GBA genes, and HAS genes.

The epidermal layer is the outermost layer of the skin. From the outside to the inside, it is the stratum corneum, the granular layer, the spinous layer and the basal layer. The epidermal layer is mainly formed by the undifferentiated cylindrical keratinocytes in the basal layer. This process It is called keratinization. The water content in keratinocytes is high. As the cells are metabolized and differentiated, the shape of keratinocytes will gradually become flat, and the nucleus and organelles will begin to degenerate and shrink, and dead cells without nuclei and organelles will be formed in the stratum corneum. The main function of the epidermis is to keep the skin water and form a skin barrier to resist various external damages. The outermost layer of the epidermis is composed of a weakly acidic sebum membrane and a stratum corneum like a brick wall. This barrier can lock the skin Moisture and grease, resistance to the invasion of bacteria on the skin surface, and resistance to damage from foreign bodies and ultraviolet light, have a very important protective effect on the human body.

In the stratum corneum in the epidermis, although the keratin cells are dead cells, its main component is keratin. Keratin can absorb water to keep the skin moist, and keratinocytes also secrete substances such as hyaluronic acid as intercellular substance. To maintain the integrity of the skin barrier of the epidermis to prevent skin moisture Disperse and form a complete protection. When the skin is exposed to a cold or hot environment and irradiated with ultraviolet light, it will cause the keratinocytes to fail to maintain the normal metabolic cycle, and the skin's water retention capacity will also decrease, and cause damage to the skin's epidermal barrier, making the skin rough, Dry desquamation, fragility and irritation, and sensitive redness. Therefore, the health and water retention capacity of the stratum corneum are very important to resist external damage.

Based on the above, in order to solve the problem of skin becoming fragile and sensitive due to the damage of keratinocytes and the decrease in water retention capacity, we have developed a method that can effectively make keratinocytes secrete more moisturizing factors, and maintain the arrangement of keratinocytes and maintain the stratum corneum. It is really necessary to have a complete structure to enhance the barrier function of the skin.

For this reason, one purpose of the present invention is to provide a coltsfoot flower extract for preparing transglutaminase ( TGM ) genes, keratin ( KRT ) genes, and filaggrin ( FLG) genes. ) Gene, aquaporin ( AQP ) gene, glucocerebrosidase ( GBA ) gene, and/or hyaluronan synthase ( HAS ) gene expression composition, wherein the coltsfoot flower The extract is obtained by extracting a winter flower with a solvent, the solvent is water, alcohol, or a mixture of alcohol and water; the composition may further include a pharmaceutically acceptable carrier.

In an embodiment of the present invention, the weight ratio of the solvent to the coltsfoot flower is 10-20:1-5; and the extraction temperature is 50-100°C.

In another embodiment of the present invention, the transglutaminase gene is the transglutaminase 1 (Transglutaminase 1, TGM1 ) gene; the aquaporin is the Aquaporin 3 (AQP 3) gene .

In another embodiment of the present invention, the keratin gene is a keratin 1 (Keratin 1, KRT1 ) gene, a keratin 14 (Keratin 14, KRT14 ) gene, or any combination thereof.

In another embodiment of the present invention, the HAS gene is a Hyaluronan synthase 2 (Hyaluronan synthase 2, HAS2 ) gene, a Hyaluronan synthase 3 (Hyaluronan synthase 3, HAS3 ) gene or a combination thereof.

In another embodiment of the present invention, the concentration of the Coltsfoot flower extract is at least 0.25 mg/mL.

In another embodiment of the present invention, the coltsfoot extract maintains the integrity of the stratum corneum structure of the skin and/or improves the moisturizing ability of the skin.

The coltsfoot extract of the present invention can effectively enhance the TGM1 gene, KRT1 gene, KRT14 gene, AQP3 gene, FLG gene, GBA gene, HAS2 gene, and/or HAS3 related to moisturizing in skin keratinocytes within 6 hours. The expression of genes can effectively maintain the arrangement of skin keratinocytes, complete the structure of the skin stratum corneum, enhance the skin barrier function, and prevent skin water loss; and can also effectively regulate the skin's water retention state and maintain the constant skin water retention; It can also make the skin form more moisturizing factors, increase the water retention capacity of the skin, and maintain skin elasticity. Therefore, the coltsfoot extract of the present invention can be used to prepare a composition for enhancing skin moisturizing ability, wherein the composition is a medicine, a food, or a skin care product, and can be taken orally, applied to the skin, etc. Give a body.

Fig. 1 is a bar graph showing the effect of a coltsfoot extract of one embodiment of the present invention on increasing the expression levels of TGM1 , KRT1 , KRT14 , FLG , GBA , HAS2 , and HAS3 genes after 6 hours of action. * p<0.05; ** p<0.01; *** p<0.001.

Fig. 2 is a bar graph showing the effect of a coltsfoot extract of one embodiment of the present invention on increasing the expression levels of TGM1 , KRT1 , AQP3 , GBA , HAS2 , and HAS3 genes after 24 hours of action. * p<0.05; ** p<0.01; *** p<0.001.

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, the pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline (normal saline), phosphate buffered saline (PBS), Aqueous solution containing alcohol and combinations thereof.

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well-known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation Gel, ointment, cream, liniment, powder, spray, lotion, foam, suspension, etc.

According to the present invention, the external preparation is prepared by mixing the medicine of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and White petrolatum (white petrolatum,)], wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers (absorption enhancers), stabilizers (stabilizing agents), gelling agents, etc. The selection and quantity of these additives fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the skin care product may further include an acceptable adjuvant that is widely used in skin care product manufacturing technology. For example, the acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, dyes Coloring agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, the skin care product can be manufactured into a form suitable for skincare or makeup by using techniques well known to those skilled in the art. This includes, but is not limited to: aqueous solution, water -Aqueous-alcohol solution or oily solution, oil-in-water type, water-in-oil type or compound emulsion, gel Glue, ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, makeup remover products, soap, and other body cleansing products.

According to the present invention, skin care products can also be used in combination with one or more external use agents with known activity selected from the following: whitening agents, moisturizers, anti-inflammatory agents ), bactericides, and UV absorbers (ultraviolet absorbers) and so on. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and formulated with any edible material for supply Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, health foods, and dietary supplements.

The present invention provides a use of coltsfoot flower extract for preparing a composition for enhancing the expression of TGM gene, KRT gene, FLG gene, AQP gene, GBA gene, and HAS gene. The coltsfoot flower extract of the present invention is extracted with a solvent Obtained from a winter flower, the solvent is water, alcohol, or a mixture of alcohol and water, which can be used to maintain the integrity of the skin stratum corneum structure and/or enhance skin moisturization.

At the same time, the present invention is used to prepare a composition for enhancing the expression level of TGM gene, KRT gene, FLG gene, AQP gene, GBA gene, and HAS gene, and may also include an effective amount of coltsfoot extract and a pharmaceutically acceptable The carrier, the composition is a medicine, a food or a skin care product.

Hereinafter, the detailed extraction method of the coltsfoot flower extract of the present invention will be described in detail, and the coltsfoot flower extract can regulate the TGM1 gene, KRT1 gene, KRT14 gene, AQP3 gene, FLG gene, GBA gene, HAS2 gene, and/ Or HAS3 gene expression test to confirm that the coltsfoot extract can effectively complete the skin stratum corneum structure to enhance the skin barrier function, and can effectively maintain the constant skin water retention and form more moisturizing factors to make the skin water retention capacity rise.

Example 1 The preparation method of the coltsfoot extract of the present invention

Tussilago farfara (Tussilago farfara) is a perennial herbaceous plant in the genus Tussilago of the Asteraceae family (Asteraceae), and is also called winter flower, coltsfoot, lantern flower, or wormwood. The stems are scape-shaped, clustered and upright, without branching between each other, and the surface is covered with dense brown pubescence and spider silk-like wool; the leaves are divided into root leaves and stem leaves, and the root leaves are clustered, heart-shaped to kidney-shaped, The leaf margins are serrated, and the stem leaves are oblong-lanceolate to ovate-oblong. Its flower bud, also known as Coltsfoot, is a kind of commonly used Chinese medicinal materials, which can be used to relieve cough and relieve phlegm, treat asthma or bronchitis.

In an embodiment of the present invention, the buds of the coltsfoot flower are washed, and the extracted solvent of the washed buds of the coltsfoot flower and water, alcohol, or a mixture of alcohol and water is taken. The extraction solvent is preferably water, with a ratio of 1-5:10. Mix at a weight ratio of -20, homogenize and extract in a solvent at 50-100°C for 0.5-3 hours, then cool the crude extract to room temperature, and filter the crude extract with a 400 mesh filter to obtain a filtrate. Finally, the filtrate is concentrated under reduced pressure at 45-70°C, and then filtered through a filter with a pore size of 5 μm. The obtained filtrate is the coltsfoot extract of the present invention.

In this embodiment, preferably, the washed coltsfoot flower buds are mixed with water in a weight ratio of 1:10, homogenized and extracted in a solvent at 100°C for 0.5 hours, and then the crude extract is cooled to the room Warm, and filter the crude extract with a 400 mesh filter to obtain a filtrate. Finally, the filtrate was concentrated under reduced pressure at 70°C, and then filtered through a filter with a pore size of 5 μm, and the filtrate obtained was the coltsfoot extract of the present invention. The "coltsfoot extract of the present invention" used in the experiments of subsequent examples was prepared under the extraction conditions described in this paragraph.

Example 2 The effect of the coltsfoot extract of the present invention in regulating gene expression of keratinocytes

One embodiment of the present invention uses human primary epidermal keratinocytes (HPEK) to carry out the coltsfoot extract of the present invention to regulate the TGM1 gene, KRT1 gene, KRT14 gene, AQP 3 gene, The efficacy test of FLG gene, GBA gene, HAS2 gene, and HAS3 gene expression level. The HPEK-50 cell line was purchased from CELLnTEC (Switzerland), and was cultured in serum-free keratinocyte-SFM (Gibco company, number #17005042, USA), and contained in 5% CO ₂ 37 Cultivate in a cell incubator at ℃.

Firstly, ^{1.5x10 5} HPEK-50 cells were cultured in a 6-well culture dish containing 2 mL of the above culture medium per well, and cultured at 37°C for 16-18 hours, and then the cells were divided into the following two groups: (1) Only containing cells The control group of the culture solution, (2) the experimental group added 0.25 mg/mL of the coltsfoot extract of the present invention, and acted for 6 hours respectively; wherein the coltsfoot extract was extracted with water as a solvent. Then, HPEK-50 cells were recovered with cell lysate (purchased from Genaid Company, Taiwan, code RBD300), and the RNA extraction reagent kit (purchased from Geneaid Company, Taiwan, code RBD300) was used to collect the cells in the two groups. Then use SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) with 2000ng of extracted RNA as a template and primers to generate the corresponding cDNA product of mRNA reverse transcription, and then use ABI StepOnePlus ^TM Real- Time PCR system (Thermo Fisher Scientific Company, USA), and KAPA SYBR FAST (purchased from Sigma Company, USA, No. 38220000000) The two sets of reverse transcription products were respectively used for quantitative real-time polymerase chain reaction ( Quantitative real-time polymerase chain reaction test, the conditions are 95°C for 1 second, 60°C for 20 seconds, a total of 40 cycles. It is used to quantify the mRNA expression level of TGM1 gene, KRT1 gene, KRT14 gene, AQP3 gene, FLG gene, GBA gene, HAS2 gene, and HAS3 gene. The quantitative value is taken from the threshold cycle number (Ct), and the mRNA of the target gene The relative quantity is derived from the equation 2-△Ct, where △Ct=Ct _{target gene-} Ct _TBP (TATA-binding protein), and then use Excel software to perform unpaired one-tailed student t-test to determine the coefficient of variation And whether there is a statistically significant difference (*p value<0.05; **p value<0.01; ***p value<0.001).

Previous studies have pointed out that TGM will form a strong bond between the cell membrane of keratinocytes and structural proteins, and increase the strength and stability of the epidermal layer; KRT will form keratin filaments, and FLG will help keratin filaments to assemble into a strong The network provides strength and elasticity to the skin. Therefore, the expression levels of keratinocyte structural genes- TGM1 gene, KRT1 gene, and KRT14 gene are increased, which can maintain the arrangement of keratinocytes, make the structure of skin keratinous tissue intact, and prevent skin water loss. The increased expression of the FLG gene, the moisturizing fiber gene of skin cells, can promote the production of natural moisturizing factor (NMF) and continue to maintain the skin in a water-retaining state.

AQP will increase the water permeability of keratinocytes to increase the water content of keratinocytes; GBA is related to maintaining the integrity of the keratinocyte structure. Therefore, the expression levels of the AQP3 gene and GBA gene, which are the moisture regulating genes of skin cells, are increased, and the moisture state of the skin can be stably regulated to maintain the constant skin moisture.

HAS promotes the ability of keratinocytes to secrete hyaluronic acid and promotes the production of endogenous hyaluronic acid in skin cells. Hyaluronic acid can absorb water to improve the water retention capacity of the skin, and can maintain the integrity of the skin to enhance the skin barrier function, and can support the skin to maintain Skin elasticity. Therefore, the expression levels of the hyaluronic acid secretion genes-HAS2 gene and HAS3 gene of skin cells increase, which can increase the water retention capacity of the skin and maintain the integrity of the skin stratum corneum structure to enhance the skin barrier function.

The results of the coltsfoot extract of the present invention enhancing the expression levels of TGM1 gene, KRT1 gene, KRT14 gene, FLG gene, GBA gene, HAS2 gene, and HAS3 gene in keratinocytes after acting for 6 hours are shown in FIG. 1. After human primary keratinocytes were treated with the coltsfoot extract of the present invention for 6 hours, the expression level of TGM1 gene was 1.8 times that of the control group, KRT1 gene was 2.9 times, KRT14 gene was 1.3 times, FLG gene was 1.3 times, and GBA gene was 1.2 times. , HAS2 gene is 1.9 times, HAS3 gene is 1.2 times, and the AQP gene is not much different from the control group (not shown in the figure). This result shows that the coltsfoot extract of the present invention can effectively increase the expression of TGM1 gene, KRT1 gene, KRT14 gene, FLG gene, GBA gene, HAS2 gene, and HAS3 gene under the action of a short time, and can effectively maintain the skin The arrangement of keratinocytes completes the structure of the skin stratum corneum to enhance the skin barrier function and prevent skin moisture loss; it can also effectively regulate the skin's water retention state and maintain the skin's water retention constant; it can also make the skin more moisturized Factors to increase the water retention capacity of the skin and maintain skin elasticity.

Example 3 The effect of the coltsfoot extract of the present invention in regulating gene expression in keratinocytes

An embodiment of the present invention uses human primary skin keratinocyte HPEK-50 to perform the coltsfoot extract of the present invention to regulate TGM1 gene, KRT1 gene, KRT14 gene, AQP 3 gene, FLG gene, GBA gene, HAS2 gene for a long time , And the test of the efficacy of HAS3 gene expression. Firstly, ^{1.5x10 5} HPEK-50 cells were cultured in a 6-well culture dish containing 2 mL of the above culture medium per well, cultured at 37°C for 16-18 hours, and then the cells were divided into the following three groups: (1) Containing only cells The control group of the culture solution, (2) the experimental group added 0.25 mg/mL of the coltsfoot extract of the present invention, and acted for 24 hours respectively; wherein the coltsfoot extract was extracted with water as a solvent. Then, after the HPEK-50 cells were recovered with the cell lysate, the RNA in the three groups of cells was collected according to the steps described in Example 2, and reverse transcriptase was performed to produce the corresponding cDNA product of mRNA reverse transcription, and then proceed Quantitative real-time reverse transcription polymerase chain reaction test to quantify the mRNA expression of TGM1 gene, KRT1 gene, KRT14 gene, AQP3 gene, FLG gene, GBA gene, HAS2 gene, and HAS3 gene. The quantitative value is taken from the threshold cycle The relative amount of target gene mRNA is derived from the equation 2-△Ct, where △Ct=Ct _{target gene-} Ct _GAPDH , and then use Excel software to perform unpaired one-tail student t-test to determine the coefficient of variation And whether there is a statistically significant difference (*p value<0.05; **p value<0.01; ***p value<0.001).

After the coltsfoot extract of the present invention acts for 24 hours, the results of increasing the expression levels of TGM1 gene, KRT1 gene, AQP 3 gene, GBA gene, HAS2 gene, and HAS3 gene in keratinocytes are shown in FIG. 2. After human primary keratinocytes were treated with 0.25 mg/mL coltsfoot extract of the present invention for 24 hours, the expression level of TGM1 gene was 1.3 times that of the control group, KRT1 gene was 1.4 times, AQP 3 gene was 2.3 times, and GBA gene was 1.2 times. , HAS2 gene is 2.4 times, HAS3 gene is 2.7 times, but KRT14 gene and FLG gene are not much different from the control group (not shown in the figure). These results show that the coltsfoot extract of the present invention can effectively increase the expression level of TGM1 gene, KRT1 gene, AQP 3 gene, GBA gene, HAS2 gene, and HAS3 gene, and can effectively maintain the arrangement of skin keratinocytes and make skin stratum corneum. The structure is complete to enhance the skin barrier function and prevent skin water loss; and it can also effectively regulate the skin in a water-retaining state, and maintain the constant water retention of the skin; it can also make the skin form more moisturizing factors and increase the skin's water retention capacity. And maintain skin elasticity.

In summary, the coltsfoot extract of the present invention can effectively enhance the moisturizing-related TGM1 gene, KRT1 gene, KRT14 gene, AQP3 gene, FLG gene, GBA gene, and skin keratinocytes within 6-24 hours. The expression level of HAS2 gene and/or HAS3 gene can effectively maintain the arrangement of skin keratinocytes, complete the structure of the skin stratum corneum, enhance the skin barrier function, and prevent skin moisture loss; and can also effectively regulate the skin in a state of water retention, It also maintains the skin's water retention constant; it can also make the skin form more moisturizing factors, increase the skin's water retention capacity, and maintain skin elasticity. Therefore, the coltsfoot extract of the present invention can be used to prepare a composition for enhancing skin moisturizing ability, wherein the composition is a medicine, a food, or a skin care product, and can be taken orally, applied to the skin, etc. Give a body.

<110> Dajiang Biomedical Co., Ltd.

<120> The use of coltsfoot extract to enhance the expression of TGM gene, KRT gene, AQP gene, FLG gene, GBA gene, and HAS gene

<130> 107B0528-I1

<160> 18

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Claims (8)

A coltsfoot flower extract is used to prepare a skin barrier function enhancing composition, wherein the composition achieves the skin barrier function by maintaining the integrity of the keratinocyte structure, wherein the coltsfoot flower extract is used to extract a winter flower with a solvent Obtained, the solvent is water, alcohol, or a mixture of alcohol and water, and the concentration of the coltsfoot extract is at least 0.25 mg/mL. For the use described in item 1 of the scope of patent application, the weight ratio of the solvent to the coltsfoot is 10-20:1-5. The use as described in item 1 of the scope of patent application, wherein the extraction temperature is 50-100°C. The use described in item 1 of the scope of the patent application, wherein the composition is improved by enhancing the transglutaminase ( TGM ) gene, keratin (Keratin, KRT ) gene, and/or glucocerebrosidase ( Glucocerebrosidase ( GBA ) gene expression level to achieve the function of maintaining the integrity of the keratinocyte structure. The use described in item 4 of the scope of patent application, wherein the keratin gene is a keratin 1 (Keratin 1, KRT1 ) gene, a keratin 14 (Keratin 14, KRT14 ) gene, or any combination thereof. For the use described in item 1 of the scope of patent application, the extraction time is 0.5-3 hours. The use described in item 1 of the scope of the patent application, wherein the composition further comprises a pharmaceutically acceptable carrier. The use described in item 1 of the scope of patent application, wherein the composition is a medicine, a food or a skin care product.

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