IT201800002660A1 - USE OF A COMPOSITION OF LACTOBACILLUS PLANTARUM GMNL-6 FOR SKIN TREATMENT - Google Patents

Description

Description

USE OF A COMPOSITION OF LACTOBACILLUS PLANTARUM GMNL-6

FOR SKIN TREATMENT

Technical field

The present invention relates to an isolated lactobacillus for use in the field of skin treatment.

Known art

Currently, probiotic products targeted for use on the skin are relatively limited. Probiotic applications on the skin, as indicated in the literature, are characterized by two methods: one consists in directly applying probiotics to the area to be treated, which can affect the local immune reaction or directly inhibit the growth of harmful bacteria in the skin; the other consists of oral administration to influence the intestinal bacterial phase and the immune reaction, and to influence changes in the skin in the extremities after the probiotics have reached the intestinal tract (Jeong JH et al, 2016).

For example, the application to improve or prevent atopic dermatitis (AD) through oral administration of probiotics is widespread. Previous studies (Baquerizo Nole KL et al, 2014, Fuchs-Tarlovsky V et al, 2016, Jeong JH et al, 2016) have shown that some lactobacillus produce metabolic secretions, e.g. acetic acid, lactic acid, diacetyl, sphingomyelin phospholipase, acid hyaluronic acid, etc., and / or emit substances that have the effect of promoting skin health. Specifically, probiotics used for the skin mainly have effects which include inhibition of acne (Kim J, Ko Y et al, 2010), prevention of UV radiation damage (Ra J et al, 2014), promotion of healing of wounds (Peral MC et al, 2009), and inhibition of melanin formation (Chan CF et al, 2014).

There are few examples in the literature of clinical trials of probiotics for improving skin health, and these tests have been conducted mainly through oral administration, while relatively few studies have been conducted on the application of external use on the skin. In one of these examples, an emulsion prepared from Bifidobacterium longum extract had a significant effect on moistening sensitive skin after removal for 29 days (N = 66, females) (Gueniche A et al, 2010), while examples clinical applications of Lactobacillus strains are still missing.

The main function of collagen in the skin is to provide a complete integration of water for the skin, increase skin elasticity and surface shine. Ceramide is a lipid naturally present in the body in the stratum corneum cells of the skin, and is the main component with the highest proportion in stratum corneum lipids. The main function of ceramide is to bind tightly to maintain the integrity of the lipids in the stratum corneum in order to prevent the evaporation of moisture and resist germs and external damage.

The present invention primarily uses in vitro modalities to select probiotics that stimulate collagen secretion and ceramide production in human skin fibroblasts, and also uses skin testing equipment to confirm the health promoting effects of the skin lotion. probiotics on the skin.

Presentation of the invention

Since the aforementioned probiotics have not been extensively studied in the field of skin treatment, there is a need to research probiotics that provide effective improvement. Since collagen and ceramide have been used extensively in the discussion of skin conditions and mechanisms, this test uses collagen and ceramide to probe the effects of probiotics in improving skin treatment, and the ability to improve the condition of the skin; moreover, it detects the skin parameters of subjects with a high-order VISIA digital detector.

The object of the present invention is to provide a probiotic use for the preparation of a skin treatment composition in which the probiotic comprises Lactobacillus plantarum (GMNL-6) with preservation number BCRC 910777 or CCTCC M 2017765.

To achieve the aforementioned purpose of the invention, Lactobacillus plantarum (GMNL-6) is a dead thallus.

To achieve the aforementioned purpose of the invention, the dead thallus is obtained by treating a liquid containing bacteria at high temperatures and pressure.

To achieve the aforementioned purpose of the invention, the composition is a medicine for external application, a drug for external use, a product for external use or a cosmetic.

To achieve the aforementioned object of the invention, the composition can further comprise a pharmaceutically or cosmetically acceptable carrier.

To achieve the aforementioned purpose of the invention, the product for external use is a tonic, a lotion, a skin cream, a moisturizer, a lip balm, a fluid beauty mask or a facial cleansing product.

To achieve the aforementioned purpose of the invention, skin treatment refers to improving the condition or aesthetic appearance of the skin.

To achieve the aforementioned purpose of the invention, the improvement of skin conditions occurs through the promotion of the secretion of collagen or the expression of ceramide synthase.

To achieve the aforementioned purpose of the invention, the treatment of the skin occurs by decreasing the number of spots, pores, wrinkles, UV spots, or porphyrins in the skin.

The invention in question provides improved condition or aesthetic appearance of the skin including: (1) treating, reducing and / or preventing fine lines or wrinkles; (2) shrink the size of skin pores; (3) improve the thickness, fullness and / or firmness of the skin; (4) improve the softness and / or flexibility of the skin; (5) improve the color, luster and / or clarity of the skin; (6) increase native collagen and / or collagen generation; (7) improve the maintenance and remodeling of elastin; (8) improve skin texture and / or promote restructuring; (9) improve the restoration and / or barrier functionality of the skin; (10) improve the appearance of the skin profile; (11) restore the luster and / or radiance of the skin; (12) restore essential nutrients and / or skin components; (13) improve the unsatisfactory appearance of the skin caused by menopause; (14) improve moist skin retention; (15) increase the resilience and / or elasticity of the skin; (16) treat, reduce and / or prevent sagging of the skin; (17) reduce pigmented spots; (18) treat, reduce and / or prevent acne; or any combination of the above.

To achieve the aforementioned purpose of the invention, the composition contains 1 × 10 <7> ~ 1 × 10 <11> dead bacteria of GMNL-6 per gram.

To achieve the aforementioned purpose of the invention, the composition preferably contains 1 × 10 <8> ~ 1 × 10 <10> dead bacteria of GMNL-6 per gram.

Brief description of the drawings

Fig. 1A is a protein expression of collagen

Fig. 1B is a genetic expression of ceramide synthase

Fig. 2 is the number of spots of the subject of the VISIA high order digital skin detector

Fig. 3 is the number of pores of the subject of the VISIA high order digital skin detector

Fig. 4 is the number of wrinkles of the subject of the VISIA high order digital skin detector

Fig. 5 is the number of UV spots of the subject of the VISIA high order digital skin detector

Fig. 6 is the number of porphyrins of the subject of the VISIA high order digital skin detector.

Detailed description of the preferred embodiment

All technical and scientific terms used in this description, except where otherwise specified, are commonly understood by a person skilled in the art.

The object of the present invention is to provide a use of a probiotic for the preparation of a skin treatment composition, wherein the probiotic comprises Lactobacillus plantarum (GMNL-6) with filing number BCRC 910777 or CCTCC M 2017765. Furthermore , the present invention relates to a new use for the treatment of the skin of the composition, or of Lactobacillus plantarum; its mechanism is to promote the secretion of collagen and the expression of ceramide to achieve the effects of skin treatment.

Among them, Lactobacillus plantarum also includes descendants of its subculture or mutant strains, which however have the same strain, genetic or use characteristics (for the inhibition of enteroviruses) in accordance with the present invention.

The composition described herein may include, but is not limited to: foods, beverages, health food, animal drinking water additives, animal feed additives, medical compositions for animal and human use, food additives, beverage additives which are suitable for use in the present invention.

The expression "pharmaceutically or cosmetically acceptable carrier" means that the substances or compositions must be compatible with other ingredients of the formulation and not be harmful to patients.

The term "extract (cell lysate)" can be a single extract obtained from self-determined extraction steps or batch extraction steps, or the extract can also be a combination of several independent extraction steps. These combined extracts are therefore also included in the term "extract".

Among them, the pharmaceutically or cosmetically acceptable carrier may comprise one or more agents selected from the following: solvent, emulsifier, suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, surfactant and other similar or suitable carriers for the present invention. One or more additives, buffers, preservatives, dyes, spices, flavorings and the like, commonly used in the field of the aforesaid compositions, can also be added, as necessary, to the compositions mentioned above. Furthermore, the new strain object of the present invention can also be contained in a composition in combination with other traditional strains.

For the composition object of the present invention and the use thereof for the treatment of the skin, the dosage can be established according to the need, in a not limited way, preferably in the form of external use on the skin.

The present invention is illustrated, for example and in a not limited manner, by the following embodiments. The drugs and biological materials used in the present invention are commercially available and widely available, and the following are only examples of the means available.

The present invention is illustrated, for example and in a not limited manner, by the following embodiments.

Lactobacillus plantarum, effective for skin treatment, is selected from the strain database, and the filing number, filing date and name of the Lactobacillus plantarum strain are shown in Table 1.

Table 1. Lactobacillus according to the present invention in Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute, Hsinchu, Taiwan and China Center for Type Culture Collection, Wuhan, China

Strain name Filing number Filing date Lactobacillus plantarum GMNL-6 BCRC 910777 14 April 2017

Lactobacillus plantarum GMNL-6 CCTCC M 2017765 November 3, 2017

First embodiment. GMNL-6 is able to promote the secretion of collagen and the expression of ceramide synthase.

1-1 Stages of preparation of dead bacteria:

Lactobacillus plantarum (GMNL-6) is inoculated with 1 ml of MRS broth from cryogenic tubes, and is incubated at 37 ° C for 20 hours under aerobic conditions. The next day, 15 μl of the culture fluid formed overnight is inoculated with 1.5 ml of MRS broth (1% secondary activation) and incubated at 37 ° C for 20 hours under aerobic conditions.

Subsequently, washing with PBS is carried out once and the number of bacteria with OD 600 nm is estimated; then the number is adjusted to 1 × 10 <10> cells / ml, and sterilization is carried out at high temperature and pressure, at 121 ° C for 15 minutes per preparation.

1-2 Human Skin Cell Fibroblast Modes (Hs68):

Steps in the analysis of the collagen of a cell culture fluid: Hs68 cells are inoculated in 6 plates (2 × 10 <5> cells / plate) overnight, then washed twice with PBS, and the replacement with a serum-free medium by adding fluid from dead GMNL-6 bacteria with a concentration of 1 × 10 <9> cells / ml for 24 hours, then the supernatant is collected.

Subsequently, the supernatant is diluted 10 times, and a Procollagen type I C-peptide EIA kit (TaKaRa, # MK101) is used for the analysis.

Phases of the analysis of ceramide synthase genes in the cell (Serine palmitoyltransferase small subunit A, SPTSSA):

Hs68 cells are inoculated in 6 plates (2 × 10 <5> cells / plate), washed with PBS twice and replaced with serum-free medium by adding fluid from dead GMNL-6 bacteria with a concentration of 1 × 10 <9> cells / ml, then the cell extracts are collected after a 24 hour process. Subsequently the RNA is extracted and converted into cDNA, and a quantitative PCR analysis is performed on the ceramide synthase genes (SPTSSA) and on the constitutive genes (β-actin).

Experimental results:

Previously, human skin fibroblasts were in vitro models to select Lactobacillus plantarum (L. Plantarum ,, GMNL-6), which offers skin treatment benefits and the ability to promote collagen secretion, from dozens of strains of Lactobacillus. Furthermore, in the same way, all experimental results (Fig.1A and 1B) have shown that GMNL-6 has both the ability to promote collagen secretion and the ability to stimulate the generation of ceramide synthase (SPTSSA) in the cell, and ceramide synthase is an essential molecule for the synthesis of ceramide in the human body, and its increase also represents an increase in ceramide production in the body.

Second embodiment: the probiotic emulsion of Lactobacillus plantarum (GMNL-6) has the function of improving the skin of the face

2-1 Experimental phases:

1. Number of people subjected to the experiments: five females (30-40 years of age).

2. Evaluation site: Experimental Research Center for the Development of Functional Cosmetics, Jiaonan University, Pharmacy.

3. Before carrying out the experiments, a facial cleansing product is used to clean the face, then the entire face is analyzed using a high-order VISIA digital skin detector, and the data is recorded (day 0).

4. During the experiments, the test samples are applied after the face has been cleaned in the morning and evening every day (in addition to the emulsion, the other conditions for left and right side care remain the same):

- applied on the left side: control group emulsion (matrix);

- applied on the right side: probiotic emulsion (GMNL-6 dead bacteria matrix).

5. The skin of the entire face is analyzed after four weeks (day 28), the facial cleansing product is first used to clean the face, then the entire face is analyzed and the data are recorded.

2-2 Ingredients of the emulsion and concentration of probiotics:

1. All ingredients of the emulsion (matrix): Water, Polyglyceryl-3 / Methyl Glucose / Distearate, Glyceryl Trioctanoin, Macadamia Ternifolia Seed Oil, Propanediol, PEG / PPG / Polybutylene Glycol-8/5/3 Glycerin, Cetyl alcohol , Myristyl myristate, Glycerin, Glycosyltrealose / Hydrogenated Starch Hydrolyzate, Filtered Rice Ferment (Sake), Saccharomyces / Filtered Daylily Flower Ferment, Bis-PEG / PPG-20/15 PEG / PPG-20/5 Dimethicone / Methoxy PEG / PPG-25/4 Caprylic / Capric Dimethicone Triglyceride, Sodium polyacrylate, Phenoxyethanol, o-Cymen-5-ol, Xanthan gum, Potassium hydroxide.

2. GMNL-6 emulsion concentration: 1 × 10 <9> cells / g of emulsion.

2-3 Evaluation method:

1. Skin detection is performed by high-order VISIA digital skin detector, and high resolution images of the face are taken by a camera with 12 million pixels and three different light sources (ordinary light, UV light, polarized light) , then the skin of certain areas is analyzed by software, in order to accurately derive parameters of the skin such as spots, pores, wrinkles, textures, sealing marks and porphyrins to evaluate the potential conditions on the base layer of the skin. In addition, previous skin problems are examined, to evaluate changes in the data relating to before and after the use of the emulsion. A comparison of qualified data in normal areas obtained from experimental data in terms of gender and age can serve as an effective tool for product evaluation.

2. The difference in score between the left side (control group emulsion) and the right side (probiotic emulsion) of each person before use (day 0) and after use (day 28). The calculation formula for the score changes is shown in the following table:

※, the percentage represents the degree of difference between the scores after use and before use and is compared to determine if there is an improvement between the same left and right sides of the faces, a decrease in score variations representing a better skin condition.

Experimental results:

The five subjects (NO.1 ~ 5) used control group emulsion (left side) and probiotic emulsion (right side) for 28 days, then the detection was performed, by means of high order VISIA digital skin detector, of the skin parameters of the five subjects, including: spots (fig.2), pores (fig.3), wrinkles (fig. 4), sealing marks (fig. 5) and porphyrins (fig.6). After comparing each person with their left side, the probiotic emulsion group use test had better skin improvement results, in each assessment set at least three or more of them had improvement, especially for blemishes and pores in 80% to 90% of people had significant improvements. Therefore, on the whole, probiotics have the effect of improving the texture of the skin, especially in reducing blemishes and reducing pores.

Collagen and ceramide in the skin are gradually lost due to aging or poor lifestyle habits, therefore the integration and restoration of collagen and ceramide content is relatively important for skin treatment, resistance to wrinkles, UV damage contrast and age signs contrast. Through in vitro experiments, the tests conducted on the present invention have found that Lactobacillus plantarum GMNL-6 can promote the secretion of collagen and the expression of ceramide synthase, highlighting that GMNL-6 has special effects on the skin. It can be demonstrated in human clinical trials using high order VISIA digital skin detector that the probiotic emulsion of Lactobacillus plantarum GMNL-6 has the function of improving the entire face, including: spots, pores, wrinkles, marks sealing and porphyrins, in which at least three or more are improved, especially in reducing blemishes and shrinking pores, between 80% and 100% of people have experienced efficacy; in addition, the decrease in porphyrins means that the growth of acne is prevented, resulting in an improvement in boils and acne.

Probiotics are mainly applied, in clinical studies on human skin, via oral administration, and there are few clinical studies involving the addition of dead Lactobacillus bacteria to the emulsion. The latter application employs the test method of comparing both left and right sides at the same time, reducing variability due to other skin care products and other individual differences. After four weeks of using GMNL-6 probiotic emulsion by five female subjects, no allergic reactions or other unpleasant effects appeared. Therefore, the application of Lactobacillus plantarum (GMNL-6) for skin care products is safe and has no side effects, and should be used as the optimal choice for promoting skin health and preventing aging. of the skin in the future.

The many effects mentioned above make it possible to satisfy the requisites envisaged by the patent law in terms of novelty and inventive step. The inventor therefore filed a patent application according to the law, and requests the Office to approve the patent application relating to this invention.

Claims (12)

Claims 1) Use of a probiotic for the preparation of a skin treatment composition, where the probiotic includes Lactobacillus plantarum (GMNL-6) with preservation number BCRC 910777 or CCTCC M 2017765. 2) Use according to claim 1, wherein Lactobacillus plantarum (GMNL-6) is a dead bacterium. 3) Use according to claim 1, wherein the dead bacterium is obtained by treating a liquid containing bacteria under conditions of high temperature and pressure. 4) Use according to claim 1, wherein the composition is a drug for external use, a drug for external use, a treatment product for external use or a cosmetic. 5) Use according to claim 1, wherein the composition comprises a pharmaceutically or cosmetically acceptable carrier. 6) Use according to claim 1, wherein the treatment product for external use is a tonic, a lotion, a skin cream, a moisturizer, a lip balm, a fluid beauty mask or a cleaning product of the face. 7) Use according to claim 1, in which the treatment of the skin refers to the improvement of the condition of the skin or its aesthetic appearance. 8) Use according to claim 7, in which the improvement of skin conditions occurs through the promotion of collagen secretion or the expression of ceramide synthase. 9) Use according to claim 1, wherein the skin treatment refers to the decrease in the number of spots, pores, wrinkles, UV spots or porphyrins of the skin. 10) Use according to claim 1, where the improvement of the skin condition or aesthetic appearance includes: (1) treating, reducing and / or preventing fine lines or wrinkles; (2) shrink the size of the skin pores; (3) improve the thickness, fullness and / or firmness of the skin; (4) improve the softness and / or flexibility of the skin; (5) improve the color, luster and / or clarity of the skin; (6) increase native collagen and / or collagen generation; (7) improve the maintenance and remodeling of elastin; (8) improve skin texture and / or promote restructuring; (9) improve the restoration and / or barrier functionality of the skin; (10) improve the appearance of the skin profile; (11) restore the luster and / or radiance of the skin; (12) restore essential nutrients and / or skin components; (13) improve the unsatisfactory appearance of the skin caused by menopause; (14) improve moist skin retention; (15) increase the resilience and / or elasticity of the skin; (16) treat, reduce and / or prevent sagging of the skin; (17) reduce pigmented spots; (18) treat, reduce or prevent acne; or any combination of the above. 11) Use according to claim 1, wherein the composition contains 1 × 10 <7> ~ 1 × 10 <11> of GMNL-6 dead bacteria per gram. 12) Use according to claim 1, wherein the composition preferably contains 1 × 10 <8> ~ 1 × 10 <10> dead bacteria of GMNL-6 per gram.