TW201536337A - Use of mandelic acid for whitening - Google Patents

Abstract

The invention relates to a use of mandelic acid for whitening by applying 10 wt.% ~ 30 wt.% of the mandelic acid to skin for inhibiting mushroom tyrosinase activity and melanin synthesis, wherein the pH value of the mandelic acid is more than or equal to 3.5. Accordingly, the mandelic acid can be further utilized in manufacturing cosmetic compositions for whitening.

Description

Use of bitter almond acid for whitening

The present invention relates to the use of a mandelic acid for whitening, in particular to a mandelic acid having a tyrosinase activity and a decrease in melanin production in skin cells, which is used for preparing a cosmetic material composition or medicine for whitening and whitening. The composition can achieve the effect of whitening.

According to the basic needs of modern people, in addition to the basic needs of food and clothing, the demand for beauty care has gradually gained attention. Not only do women pay attention to beauty care, but even men are eager for it. Therefore, the beauty care products used by the human body are the current manufacturers. The focus of development. The skin covering the surface of the human body is the first layer of protection between humans and the external environment. It is most vulnerable to external stimuli and ultraviolet rays. In order to protect the skin from the ultraviolet rays in the sun, melanocytes in the skin will synthesize melanin. (melanin) to defend against the ultraviolet rays in the sun, when the human body is constantly attacked by harmful chemicals, excessive light or free radicals generated in the body, it is easy to cause cell damage aging or a large amount of melanin production. Therefore, how to maintain the skin to slow down the skin aging rate and melanin production has become the direction pursued by consumers. For example, people with freckles or age spots want to improve their skin quality (blackening), or other people who want beauty can ask for white. More white skin and so on.

At present, natural substances having a whitening effect such as vitamin C (L-ascorbic acid), kojic acid, acelacic acid, and arbutin have been confirmed by research, although these substances are Whitening does have significant effects, but each has its own use. The upper limit, for example, vitamin C is easily oxidized in the sun or in contact with air, and is not heat-resistant. When the concentration exceeds 5%, the skin will be irritating and red and swollen; the tannic acid is very unstable and easily oxidized to cause discoloration and cause skin. Allergies, and long-term excessive use of tannic acid products can cause cytotoxicity and pathological changes; in addition, some whitening agents are used to directly destroy melanocytes, which is easy to cause cell toxicity. Therefore, it is urgent to pay attention to the safety of the composition of the melanin composition and its concentration.

Mandelic acid, also known as phenylglycolic acid, is known as 2-Hydroxy-2-phenylacetic acid. It has good chemical structure and antibiotics, and is good for both Escherichia coli and streptococci. The antibacterial effect is therefore mainly used in the early treatment of urinary tract infections and as an oral antibiotic. Until the famous American skin specialist Dr. James E. Fulton began to apply bitter almond acid in the field of dermatology and beauty, as a facial rejuvenation. Mandelic acid is a lipophilic fruit acid. It has strong ability to penetrate the stratum corneum. It is mild and non-irritating. It is not easy to produce side effects such as redness, burns and crusting of common fruit acid peeling, so it is more effective than other skin rejuvenating ingredients. However, there is no specific research to confirm the exact effect of bitter almond acid on whitening.

Nowadays, the inventor is still in the light of the tireless spirit of the above-mentioned existing whitening composition in actual practice, and is improved by its rich professional knowledge and years of practical experience. And based on this, the present invention was developed.

The main object of the present invention is to provide a use of amygdalin in whitening, which has bitumenic acid which inhibits tyrosinase activity and reduces melanin production in skin cells, and further, uses mandelic acid to prepare a whitening blemish. Material composition or pharmaceutical composition, up to To the effect of whitening.

In order to achieve the above-mentioned object, the present invention provides a method for the application of bitter almond acid to whitening, which is applied to a skin by a concentration of 10% to 30% by weight of mandelic acid to inhibit mushroom tyrosinase activity and inhibit melanin. The pH of the mandelic acid is 3.5 or more, and the pH is preferably 3.5; thereby, it can be used to inhibit mushroom tyrosinase activity and inhibit melanin production.

The present invention also provides a composition of a cosmetic material containing mandelic acid for whitening, which is applied to an effective amount of a cosmetic material composition for inhibiting mushroom tyrosinase activity and melanin production; wherein the cosmetic material is composed of The content is from 10% to 30% by weight of mandelic acid (preferably, for example, 10% of mandelic acid), and 0.5% to 1.5% of 1,3-butanediol and The residual weight percentage concentration of sterile water is composed, and the pH value of the bitter almond acid is 3.5 or more, and the optimum pH is 3.5.

In one embodiment of the invention, it may be preferred to administer a concentration of 10% by weight of mandelic acid to the skin to inhibit mushroom tyrosinase activity and inhibit melanin production.

In one embodiment of the present invention, administration of a 10% by weight concentration of picramic acid to the skin further increases the collagen content.

According to the use of the balsamidonic acid of the present invention, it can be further used for preparing a cosmetic material composition for whitening or increasing the collagen content to provide a better whitening and wrinkle-removing skin care product selection for the user.

First: Determination of inhibition of mushroom tyrosinase activity by amygdalin

Figure 2: Determination of melanin content in the inhibition of amygdalin

Figure 3: Determination of collagen content in the increase of amygdalin

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

The invention relates to the use of mandelic acid (MA) in whitening, which is applied to a concentration of 10% to 30% by weight of mandelic acid (preferably, for example, 10% by weight) Almondic acid is applied to the skin to inhibit mushroom tyrosinase activity and inhibit melanin production; wherein the pH of the bitter almond acid is 3.5 or more (the optimum is pH 3.5), and a weight of one is administered. Mandelic acid with a concentration of 10% can further increase the collagen content.

Furthermore, the present invention also discloses the use of a cosmetic composition containing a bitter almond acid for whitening, which is applied to an effective dose of a cosmetic material composition to inhibit the activity of mushroom tyrosinase and melanin production; The composition of the cosmetic material is from 10% to 30% by weight (preferably, for example, 10% by weight) of mandelic acid, and 0.5% to 1.5% of 1,3-butanediol (1,3- Butanediol) and the remaining weight percent concentration of sterile water, the pH value of mandelic acid is 3.5 or more, the optimum pH is 3.5, and the application of a 10% by weight concentration of mandelic acid can be further increased. Collagen content.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

I. <Pharmaceutical Preparation>

The bitter almond acid of the present embodiment is commercially available from Guangde Keyuan Chemical Co., Ltd. Weigh 1 g of bitter almond acid powder, add 1 ml of sterile water and 50 μl of 1,3-butanediol, then heat to The solution was dissolved at 90 ° C for about 10 minutes, and the concentration was 100%, and it was adjusted to pH 1.9 or pH 3.5, and then filtered through a 0.22 μm filter membrane for the following test.

II. <Preparation of culture solution and buffer solution>

(1) Preparation of DMEM medium (for B16 and Hs68 cells): Weigh 13.4g of DMEM powder, add 1.5g of sodium bicarbonate, 4.5g of glucose, dissolve in 250ml of deionized water, then add 1 ml/L gentamicin, then quantified to 1 liter in deionized water, filtered through a 0.22 μm pore size filter, and stored at 4 ° C until use.

(2) Preparation of Phosphate-buffered saline (PBS): 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of Na ₂ HPO ₄ ‧7H ₂ O and 0.24 g of KH ₂ PO _{4 were} weighed and dissolved in 980 ml of deionized After the water, the pH was adjusted to 7.4, and deionized water was added to 1 liter. After high temperature sterilization, it was stored at room temperature.

(3) Prepare 10 times Trypsin-EDTA solution: weigh 0.5g of trypsin and 0.2g of 2-[2-(bis(carboxymethyl)amino)ethyl-(carboxymethyl)amino]acetic acid (EDTA), add PBS to 100ml Thereafter, it was filtered through a 0.22 μm filter and stored at -20 °C.

III. <Cell culture and preservation>

Mouse melanoma cells (B16 cells) and human skin fibroblast (Hs68 cells) were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37 ° C, 5% carbon dioxide. Culture in a cell culture incubator and change the medium 2-3 times a week.

When subculture, remove the old culture medium and wash the cell surface with PBS buffer. After the layer is 1-2 times, add an appropriate amount of Trypsin-EDTA solution and put it in the incubator for 37 minutes. After the cells are detached from the culture bottle wall, immediately add fresh cell culture medium and evenly disperse the cells with a sterile pipette. , dispense appropriate cell numbers into new culture flasks and continue to culture.

Furthermore, the cells to be cryopreserved should have a high growth rate and a high survival rate of about 80-90%. The cells which grew well were collected by cell subculture, and the cells were collected into a 15 ml centrifuge tube containing an appropriate amount of cell culture medium, and centrifuged at 1,200 rpm for 10 minutes. After centrifugation, the supernatant was removed, and a cell culture medium containing 0.5% DMSO was additionally prepared as a cell cryopreservation solution. The frozen preservation solution was added to the centrifuged cells, mixed and evenly dispersed, and the appropriate amount of cell liquid was dispensed to the frozen. In the tube, put it in the freezer box. The freezer box was placed in a -80 ° C refrigerator and moved into a liquid nitrogen drum for storage for a long time.

In addition, in order to prevent ice crystals from harming cells, the principle of thawing cells is rapid thawing. The cell cryotube was taken out from the liquid nitrogen drum and quickly transferred to a 37 ° C water bath for rapid thawing. The frozen tube was gently shaken to melt as soon as possible to a small portion of the ice, and then the cell cryopreservation solution (10% FBS, 0.5%) DMSO, medium) was pipetted into a sterile 15 ml centrifuge tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was removed, and the precipitated cells were dispersed in a fresh cell culture medium and transferred to a 75 cm ² cell culture flask. The cells were grown in a 5% CO ₂ cell culture incubator at 37 ° C, and the culture medium was changed every 2 days on average. .

IV. <Cell Survival Test>

Detection by MTT assay. 1×10 ⁴ /100 μl of mouse melanoma cells (B16) were cultured in a 96-well plate and cultured in a 37 ° C and 5% CO ₂ incubator for at least 24 hours. Thereafter, the respective concentrations (0.1%, 1%, 10%, 20%, 30%) of the mandelic acid to be detected were added, and the mixture was reacted at 37 ° C in a 5% CO ₂ incubator until the action time. After the reaction time, the old culture solution was removed, washed once with PBS, and replaced with a new medium, and 10 μl of MTT (3-(4,5-cimeth-ylthiazol-2-yl)-2,5- was added. The reaction was carried out in a diphenyl tetrazolium bromide solution, and the culture solution was removed after reacting at 37 ° C, 5% CO ₂ for four hours, 100 μl of DMSO was added to dissolve the formazan precipitate, and finally, the absorbance was measured at a wavelength of 570 nm. The results show that more than 30% of mandelic acid causes a large amount of cell death, so the present invention uses less than 30% of mandelic acid.

Experiment 1: Analysis of inhibition of mushroom tyrosinase activity by bitter almond

<Inhibition of mushroom tyrosinase activity assay>

Take different concentrations (0.1%, 1%, 10%, 20%, 30%) of mandelic acid samples and vitamin C (Ascorbic acid) for mushroom tyrosinese activity test, in which different concentrations of vitamins Line C was used as a control standard. In a 96-well multiplate, 20 μL of the sample was added and 25 μL of tyrosinase (50 unit) was mixed, and then allowed to stand at room temperature for 10 minutes, and the background value of the absorbance at 492 nm was measured by a spectrophotometer. the reaction was then taken 155μl, 2.5mM of L- dopa (L-dopa) was added 96 well plates to a spectrophotometer ^{(BioTek, Synergy TM 2, USA} ) detected in each well of a mixture of absorbance change at 492nm. The inhibition rate (%) of tyrosinase activity was calculated as [1-(absorbance of sample at 492 nm / absorbance at 492 nm of control group without added sample)] × 100%.

For the experimental results, please refer to the first figure. The bitter mandelic acid at pH 3.5 inhibits the activity of mushroom tyrosinase activity under the action of different concentrations of 0.01%, 1%, 10%, 20% and 30%. The efficacy increased with the increase in the concentration of the works, and the inhibition rates were 13%, 17%, 91%, 93%, and 99%, respectively. The results confirmed that mandelic acid has the effect of inhibiting the activity of mushroom tyrosinase in vitro, especially that the concentration of 10%-30% of mandelic acid has the effect of inhibiting the activity of mushroom tyrosinase.

Experiment 2: Analysis of the inhibition of melanin content by amygdalin

1×10 ⁵ /mL mouse melanoma cells (B16 cells) were cultured in a 96-well multiplate and grown in a 37 ° C, 5% CO ₂ incubator for 24 hours using a medium containing α-MSH. the above. Phenol red free medium and 10% mandelic acid were added, and after 72 hours, an equal amount of supernatant was taken, and the absorbance was measured at 405 nm.

For the results of the experiment, please refer to the second figure, after the addition of bittenic acid at pH 3.5 and pH 7.0 to B16 cells at a concentration of 10% for 72 hours, the production of melanin from bitumic acid at pH 3.5 and pH 7.0 The inhibition rates were 42.6% and 39.7%, respectively. From the above results, it was found that the mandelic acid at pH 3.5 and pH 7.0 did have an ability to inhibit melanin production on B16 cells at a concentration of 10%.

Experiment 3: Analysis of the content of collagen in the cells of amygdalin

1.5×10 ⁵ cell/ml of Hs68 cells were cultured in a 3 cm diameter dish (3 cm-dish) for 24 hours, and the supernatant was removed and then added with different concentrations (5% or 10%) of mandelic acid. The serum culture medium was cultured for 48 hours, in which different concentrations of glycolic acid were used as control standards; after washing with PBS, the cells were scraped off, centrifuged at 1,200 rpm for 5 minutes, and the supernatant was removed. In this test, a collagen assay was performed using a Sircol soluble collagen assay kit. First, 100 μl of the cell liquid was mixed with 1 ml of sircol dye reagent, followed by shaking at room temperature for 30 minutes, and then centrifuged at 1, 2000 rpm for 10 minutes, and the supernatant was directly poured out, and then 750 μl of ice-cold acid-salt wash reagent was added. The sircol dye reagent was completely removed by centrifugation at 1, 2000 rpm for 10 minutes. Then, 250 μl of the alkali reagent was added and mixed uniformly, and 100 μl was taken to a 96-well disk, and the absorbance was measured at 555 nm.

For the experimental results, please refer to the third figure. Different concentrations (5% and 10%) of mandelic acid were applied to human fibroblasts Hs68 cells for 48 hours, and bitter almond was used at 10%. The collagen content is 17% more, and the test confirms that more than 10% bitter almond Acid can effectively increase the content of collagen.

In summary, the present invention demonstrates that mandelic acid has the effects of inhibiting tyrosinase, inhibiting melanin production, and increasing collagen content, so that mandelic acid can be used as a cosmetic material composition for whitening and increasing collagen. A skin care product or a pharmaceutical composition, and the pharmaceutical composition can be used in combination with a skin external preparation. The above-mentioned "skin external preparation" means a topical ingredient which is usually used in cosmetics or pharmaceuticals, including, but not limited to, other whitening agents, moisturizers, antioxidants, ultraviolet absorbers, surfactants, thickeners. The coloring material, the skin nutrient and the like can be used, for example, as a component of the concentrated essence or added to the mask, and the user can be applied to the skin in an appropriate amount to protect the skin from ultraviolet rays and whitening.

It is worth noting that the acidity and alkalinity value of the bitter almond of the present invention conforms to the legal pH value of 3.5 or more, and the use of bitter almond acid having a pH of less than 3.5 (for example, pH 1.9) can inhibit melanin production; however, skin may be caused when applied to human skin. Stimulation, redness and other issues. In addition, when the concentration of bitter almond acid is less than 10%, it can not achieve the effect of inhibiting melanin production; if the concentration is more than 30%, although it can inhibit melanin production, it may also cause skin irritation and redness of the user. Allergies and other issues. Therefore, in the present invention, the optimum concentration of pH 3.5 mandelic acid is 10% to 30%.

It can be seen from the above description that the present invention is compared with the prior art, and the present invention demonstrates by specific experiments that mandelic acid has an activity of inhibiting mushroom tyrosinase activity and inhibiting melanin production, and is further applied as a skin for application. Make-up materials, skin care products or pharmaceutical compositions will achieve whitening and anti-wrinkle effects.

In summary, the use of the bitter almond acid of the present invention in whitening can indeed achieve the intended use efficiency by the above-exemplified embodiments, and the present invention has not been disclosed before the application, and has completely complied with the patent law. Regulations and requirements.提出Proposing an invention patent in accordance with the law,恳 Please give it a review and grant a patent.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

Claims (8)

A use of bitter almond acid for whitening, which is applied to a skin by a concentration of 10% to 30% by weight of mandelic acid to inhibit mushroom tyrosinase activity and melanin production; wherein the pH of the bitter almond acid The value is 3.5 or more. The use according to claim 1, wherein the bitter almond acid has a pH of 3.5. The use according to claim 1, wherein a concentration of 10% by weight of mandelic acid is applied to the skin to inhibit mushroom tyrosinase activity and melanin production. The use according to claim 1, wherein the bitter almond acid having a concentration of 10% by weight further increases the collagen content. A composition of a cosmetic material containing bitter almond acid for whitening, which is applied to an effective amount of a cosmetic material composition for inhibiting mushroom tyrosinase activity and melanin production; wherein the cosmetic material composition is composed of weight Percentage of 10% to 30% of mandelic acid, 0.5% to 1.5% of 1,3-butanediol and 3% by weight of sterile water, the bitter almond acid The pH is 3.5 or more. The use according to claim 5, wherein the bitter almond acid has a pH of 3.5. The use according to claim 5, wherein a concentration of 10% by weight of mandelic acid is applied to the skin to inhibit mushroom tyrosinase activity and melanin production. The use according to claim 5, wherein the bitter almond acid having a concentration of 10% by weight further increases the collagen content.