TWI682777B - Use of tanaka bark extracts for reducing skin aging - Google Patents

Abstract

The present invention relates to applications of a Tanaka bark extract in manufacture of composition for reducing skin aging. The Tanaka bark extract is prepared by extracting Tanaka barks using water, alcohols, or mixtures of water and alcohols as solvents. This extract is effective in increasing resistance of skin cells to ultraviolet irradiation, reducing damage to nucleic acids in skin cells, improving skin texture, and reducing brown spots.

Description

Uses of Tenaka bark extract for preventing skin aging

The invention relates to the use of a plant extract, in particular to the use of a Tenka bark extract for preparing a composition for preventing skin aging.

The skin provides the first-stage protection of individuals against environmental factors such as ultraviolet (UV) radiation, pathogens, friction in sunlight. The skin sequentially includes the epidermal layer, the dermis layer mainly composed of connective tissue, and the subcutaneous tissue from outside to inside. The epidermis contains the outermost cuticle and is constantly updated. There are continuously dividing cells between the epidermis and the dermis, such as skin fibroblasts, keratinocytes, and melanocytes. The dermis layer contains collagen, elastin, and hyaluronic acid and other molecules, which give the skin elasticity and support. With age, the skin will show aging phenomena such as wrinkles, fine lines, sagging, depression, enlarged pores, and melanin precipitation. The formation of these skin aging phenomena is related to many factors. For example, the content of molecules such as collagen, elastin, and hyaluronic acid in the dermis layer decreases with age. Exposure to high amounts of ultraviolet light (mainly ultraviolet A) can cause skin cells Death or nucleic acid molecule damage.

Methods available on the market to improve the aforementioned skin aging phenomenon include direct injection of collagen or hyaluronic acid into the dermis layer, oral supplementation of collagen or hyaluronic acid, and use of sunscreen to reduce skin damage caused by ultraviolet rays. However, collagen or hyaluronic acid injected into the skin is easily broken down by enzymes in the body over time, which results in the need to regularly apply these substances, which is very expensive. Oral supplementation of collagen or hyaluronic acid will cause these large molecules to be digested into small molecules of amino acids or monosaccharides in the gastrointestinal tract. Although the body can use these amino acids or monosaccharides to synthesize proteins or polysaccharides, they are not necessarily formed Collagen or hyaluronic acid, so the substantial effect of supplementing collagen or hyaluronic acid is limited. In addition, the sunscreen contains Chemicals may cause photosensitivity, so that the combination of these chemicals and ultraviolet light has an adverse effect on the skin, such as a rash or more severe sunburn.

In view of this, it is necessary to develop a composition with natural ingredients that can be directly applied to the skin and can effectively slow down skin aging to maintain skin health and reduce the occurrence of skin lesions.

Therefore, an object of the present invention is to provide a use of Tanaka bark extract for preparing a composition for preventing skin aging, wherein the Tenaka bark extract is a solvent to extract a Tenka bark And get.

In one embodiment of the present invention, the solvent is water, alcohol, or an alcohol-water mixture, the weight ratio of the solvent to the tenacar bark is 20:1 to 1:1, and the extraction step is at 50°C to At 80°C.

In one embodiment of the present invention, the Tenaka bark extract is a water extract of Tenaka bark, the concentration of which is at least 0.5 mg/ml.

In one embodiment of the present invention, the Tenaka bark extract enhances the resistance of a skin cell to ultraviolet rays, reduces the damage of a nucleic acid molecule in a skin cell, or improves the skin texture and reduces spots.

In one embodiment of the present invention, the composition of the present invention is in the form of powder, granule, liquid, gel or paste.

In view of the effects of Tenaka bark extract on effectively inhibiting environmental factors that contribute to skin aging, such as ultraviolet-induced skin fibroblast death and nucleic acid molecule damage due to oxidative stress, Tenka bark extract can be used to prepare a combination to prevent skin aging Thing.

The following will further illustrate the embodiments of the present invention with reference to the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than to limit the scope of the present invention. Anyone who is familiar with this skill will not deviate from it. Within the spirit and scope of the present invention, some changes and modifications can be made, so the scope of protection of the present invention shall be deemed as defined by the scope of the attached patent application.

Figure 1 shows that Tenka bark extract enhances the resistance of skin fibroblasts to ultraviolet A (UVA) irradiation.

Figure 2 shows that the extract of Tenaka bark reduces the accumulation of reactive oxygen species in skin fibroblasts caused by hydrogen peroxide (H ₂ O ₂ ).

Figure 3 shows that the extract of Tenaka bark reduces the damage of nucleic acid molecules in skin fibroblasts caused by hydrogen peroxide (H ₂ O ₂ ).

Figure 4 shows the improvement rate of the skin texture after applying the essence or placebo containing 1% Tenaka bark extract to the skin of the subject's face for four consecutive weeks.

Figure 5 shows the change in the relative amount of brown spots on the skin of the subject's skin after applying the essence or placebo containing 1% Tenaka bark extract to the skin of the subject's face for four consecutive weeks.

definition

The numerical values used in this article are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.

The invention provides a use of Tenaka bark extract for preparing a composition for preventing skin aging. In the following examples, it is disclosed that the Tenaka bark extract or the composition containing the extract can reduce the death of skin fibroblasts caused by ultraviolet radiation, reduce the damage of nucleic acid molecules in skin cells, and improve skin texture and reduce spots.

Materials and Methods material

Purchase Eagle's minimum essential medium (MEM medium), Fetal bovine serum (FBS), non-essential amino acids, sodium bicarbonate, pyruvate from Thermo Fisher Scientific Sodium, and phosphate buffered saline (PBS). Purchase 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2 -yl)-2,5-diphenyltetrazolium bromide (MTT). Purchase dimethyl sulfoxide (DMSO) from ECHO Chemical. Purchase hydrogen peroxide and dichlorodihydrofluorescence from Sigma Diacetate (2,7-dichloro-dihydro-fluorescein diacetate, DCFH-DA). The DCFH-DA is dissolved in DMSO before use to prepare a 5 mg/ml DCFH-DA solution.

Cell culture

The following examples used human skin fibroblast CCD-966SK (BCRC 60153) purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research Institute for in vitro experiments. Human skin fibroblasts are cultured in MEM medium supplemented with 10% FBS, 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, and 1 mM sodium pyruvate at 37°C and 5% carbon dioxide, hereinafter referred to as cell culture medium .

MTT analysis

(formazan)結晶。最終，使用ELISA讀盤機(enzyme-linked immunosorbent assay reader；BioTek)測量該細胞混合物在570nm的吸光值(O.D.570)。 Cell survival rate was determined by MTT analysis. Briefly, MTT solution (4 mg/ml MTT dissolved in PBS solution) was added to cells in a 96-well dish at 15 μl/well, and reacted at 37° C. for 4 hours. After removing the reaction solution, add 50 μl/well of DMSO to the cells and shake the reaction for 10 minutes to dissolve the generated formazan.

(formazan) crystallization. Finally, the absorbance at 570 nm (OD570) of the cell mixture was measured using an ELISA disk reader (Enzyme-linked immunosorbent assay reader; BioTek).

Statistical Analysis

The statistically significant differences in the experimental data are determined by the Student t test of Excel software.

Example 1 Preparation of Tenaka Bark Extract

This example illustrates the preparation method of the extract of Tenacar bark. First, the Tenaka bark (Tanaka, also known as Limonia acidissima ) is washed and dried, and the bark is coarsely crushed into flakes, granules, or powder by cutting, grinding, or other similar methods. Secondly, the flake, granular, or powdered Tenaka bark is extracted with water as the solvent, the weight ratio of the solvent to the Tenaka bark is 20:1 to 1:1, and the extraction temperature is between At 50°C to 80°C, preferably 60°C to 70°C. The extraction time is about 0.5 to 3 hours. After the extract of the Tenaka bark obtained through this extraction step was cooled to room temperature, it was filtered through a 400 mesh filter to remove residual solids. The filtered Tenaka bark extract can be further concentrated under reduced pressure at 45°C to 70°C to obtain a concentrated product. In order to obtain the solid Tenaka bark extract, the aforementioned reduced-pressure concentrated Tenaka bark extract may be spray-dried to remove the solvent, thus obtaining Tenka bark extract powder.

Example 2 Tenaka bark extract inhibits skin fibroblast death caused by ultraviolet radiation

To test the effect of Tenaka bark extract on protecting the skin against ultraviolet radiation, cell survival analysis (MTT analysis) was used to evaluate the cell survival of human skin fibroblast CCD-966SK after ultraviolet A irradiation after Tenka bark extract treatment rate. Briefly, 5×10 ³ CCD-966SK cells were seeded into each well of a 96-well dish containing 200 μL of cell culture medium, and after culturing at 37° C. for 24 hours, the cell culture medium was removed, and a solution containing 1 mg/mL Turner was added. Cell culture medium of card bark water extract was used as the experimental group, which was incubated at 37°C for another 24 hours. After that, the cells were placed in an ultraviolet irradiation box (Vilber) to receive 12J/cm ² ultraviolet A (wavelength 315-400mn) for 1 hour. This radiation dose caused half of the cells to die. For comparison, a group of cells irradiated with the aforementioned dose of ultraviolet A but treated with a cell culture medium that does not contain the water extract of Tenacar bark was used as a negative control group, and a group of cells that were not irradiated with ultraviolet A and only Cells treated with cell culture medium containing water extracts of Tenaka bark were used as blank control group. Finally, perform MTT analysis and calculate the cell survival rate of each group of cells (quadruple experiment) according to the following formula: cell survival rate = OD570 of each group/OD570×100% of blank control group

Figure 1 shows the cell survival rate of CCD-966SK cells after the aforementioned different treatments; * and *** in the figure represent p <0.05 and p <0.001, respectively, relative to the negative control group. According to Figure 1, the negative control group had a significantly reduced cell survival rate compared to the blank control group, showing that ultraviolet A irradiation would cause a large number of skin fibroblast death. However, compared with the negative control group, the treatment of Tenaka bark water extract increased the survival rate of cells in the experimental group by 16.7%, indicating that Tenka bark extract can increase the resistance of skin cells to ultraviolet rays.

Example 3 Tenaka bark extract reduces the accumulation of reactive oxygen species in skin fibroblasts

To investigate the regulatory effect of Tenaka bark extract on oxidative stress in skin cells, fluorescent probe DCFH-DA was used in conjunction with flow cytometry (Beckman) to determine human skin fibroblast CCD-966SK via Turner After the card bark extract treatment, the content of reactive oxygen species (ROS) under oxidative stimulation changes. Briefly, 2×10 ⁵ CCD-966SK cells were seeded into each well of a 6-well culture dish containing 2 ml of cell culture medium, and after culturing at 37° C. for 24 hours, the medium was removed. Thereafter, the cells were sequentially treated with 2 mL of cell culture medium containing 0.5 or 1 mg/mL Tenaka bark water extract at 37°C for 1 hour, cells were treated with 5 μg/ml DCFH-DA solution for 15 minutes, and then treated with 1 mM Hydrogen oxide treatment for 1 hour. After washing the cells twice with PBS solution, the cells were treated with 200 μl trypsin in the dark for 5 minutes, and then the cells and the medium were collected and the supernatant was removed by centrifugation (400×g, 10 minutes). The cell pellet thus obtained was washed once with PBS solution and suspended again, and the fluorescence intensity of the cells was detected using a flow cytometer. The excitation wavelength for fluorescence detection is 450-490nm, and the emission wavelength is 510-550nm. Since DCFH-DA enters the cell, it will be hydrolyzed to DCFH (dichlorodihydrofluorescein), and then oxidized by reactive oxygen species to DCF (dichlorofluorescein), which can emit green fluorescence, and then treated by DCFH-DA The fluorescence intensity of the cells can reflect the content of reactive oxygen species in the cells. For comparison, another set of cells that have been treated with hydrogen oxide and cell culture medium without Tenka bark water extract as a negative control group, and one set without hydrogen peroxide but only without Turner Cells treated with cell culture medium of card bark water extract were used as blank control group.

Figure 2 shows the relative content (%) of active oxygen species of CCD-966SK cells after the different treatments described above, defined as the active oxygen species content of cells in the negative control group as 100%; *** in the figure represents p relative to the negative control group <0.001. According to Fig. 2, the negative control group showed a significantly higher content of reactive oxygen species than the blank control group, showing that hydrogen peroxide treatment would result in the accumulation of reactive oxygen species in the cells. However, compared with the negative control group, the treatment of 0.5 or 1 mg/mL Tenka bark water extract reduced the intracellular active oxygen species by approximately 60%, showing that Tenka bark water extract has the ability to scavenge active oxygen species.

Example 4 Tenaka bark extract reduces intracellular nucleic acid molecule damage

To test the protective effect of Tenka bark extract on nucleic acid molecules in skin cells, human skin fibroblast CCD-966SK was treated in a similar manner as described in Example 3, and single cell gel electrophoresis (SCGE) was used Analyze the damage of chromosomal nucleic acid molecules after different treatments. Briefly, three groups of CCD-966SK cells were set up. The first group of cells was first treated with 1 mM hydrogen peroxide for 1 hour, and then treated with cell culture medium containing 0.5 or 1 mg/mL Tenaka bark water extract for 1 hour; second The cells in the group were treated with hydrogen oxide and cell culture medium without the extract of Tenacar bark (negative control group); the cells in the third group were treated with cell culture medium without the extract of Tenacar bark without the use of hydrogen peroxide. (Blank control group). Afterwards, the three groups of cells were collected for single-cell colloid electrophoresis and measured The length of the tail of the nucleic acid molecule in the stained colloid. Since the broken nucleic acid molecule will show a tailing phenomenon after cell electrophoresis, the length of the tailing can be used to quantify the degree of damage of the nucleic acid molecule.

Figure 3 shows the relative length (%) of nucleic acid tailing of each group of CCD-966SK cells after electrophoresis in the above groups. The length of nucleic acid molecule tailing of the blank control group is defined as 100%; *** in the figure represents the relative length of the blank control group p <0.001, ^### represents a negative control group with respect to the p <0.001. According to Figure 3, the negative control group had significantly longer nucleic acid tails than the blank control group, showing that hydrogen peroxide treatment would seriously damage the nucleic acid molecules of the cells. However, compared with the negative control group, treatment with 0.5 or 1 mg/mL Tenka bark water extract shortened the nucleic acid tail by approximately 62.5%, indicating that Tenka bark extract can reduce the damage of nucleic acid molecules in skin cells.

Example 5 Tenaka bark extract improves skin texture and reduces spots

Tenaka bark extract can be prepared for topical application to the skin, which can be in the form of, but not limited to, solution, lotion, cream or gel. In one embodiment, the composition is an essence containing 1% w/w Tenaka bark water extract (containing a base solvent, the ingredients of which include water, xanthanone, hexanediol, 1,3-butane Diol, three xanthan gum, thickener, and triethanolamine). To assess the effect of this composition on the skin condition, the essence and the base solvent as a placebo were applied to 9 cleaned and cleaned male and female subjects aged 25 to 40 years old, one time each day The left and right half faces were measured before and after four weeks of continuous use with a full-face skin quality tester (7th Generation VISIA Complexion Analysis System; Canfield, USA) to determine the full-face skin texture and the number of brown spots on these subjects.

Fig. 4 shows the average value of the skin texture improvement rate (%) of the aforementioned subjects; the skin roughness of the subject before application is defined as 100%, and the reduction in skin roughness of the same subject before and after application is taken as the skin texture Improvement rate. According to Figure 4, the improvement in skin texture compared to placebo is about 10.5%, and the application of essence containing Tenaka bark water extract for four consecutive weeks significantly improves skin texture by 17.4%.

Fig. 5 shows the average value of the relative amount (%) of brown spots of the aforementioned subject, with the number of brown spots of the subject's skin before application being 100%. According to Figure 5, the use of a placebo did not reduce the brown spots on the skin, but applying the essence containing Tenaka bark water extract for four consecutive weeks reduced the brown spots by 7.7%. This result confirms the effect of Tenaka bark extract to slow down skin aging.

In summary, Tenka bark extract or a composition containing the extract can enhance the resistance of skin cells to ultraviolet light, reduce the damage of nucleic acid molecules in skin cells, improve skin texture and reduce spots, and ultimately prevent skin Aging goals. Therefore, Tenka bark extract can be used to prepare a composition for preventing skin aging.

Claims (5)

A use of Tenka bark extract for preparing a composition for improving skin texture, wherein the Tenka bark extract is obtained by extracting one Tenka bark with water. The use as described in item 1 of the patent application scope, wherein the weight ratio of the water to the tenacar bark is 20:1 to 1:1. The use as described in item 1 of the patent application scope, wherein the water extraction is carried out at 50 to 80°C. The use as described in item 1 of the patent application scope, wherein the concentration of the extract of Tenacar bark is at least 0.5 mg/ml. The use as described in item 1 of the scope of the patent application, wherein the composition is in the form of powder, granule, liquid, gel or paste.

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