TWI740107B - Use of unripe fig fruit extracts for reducing skin collagen loss - Google Patents

Abstract

The present invention provides use of an unripe fig fruit extract for manufacture of composition for reducing skin collagen loss. The unripe fig fruit extract is prepared by extraction of unripe fig fruit using water, alcohols, or mixtures of water and alcohols as solvents. This extract not only enhances collagen production by skin fibroblasts, but also prevents loss of collagen with normal structure and function due to glycation, whereby helping to maintain youthful skin.

Description

The use of fig young fruit extract to reduce the loss of skin collagen

The present invention relates to the use of a plant extract, in particular to the use of a fig young fruit extract to prepare a composition for reducing skin collagen loss.

Collagen is the most abundant protein in mammals. It accounts for about one-third of the total protein in the human body. It is widely found in human skin, cartilage, cornea, blood vessel walls, and internal organs. It mainly exists in the extracellular matrix of connective tissue and can provide the mechanical support and strength required by connective tissue. The collagen in the human body can be divided into different types due to the difference in the composition of multiple peptides. Among them, the first type, the third type, and the fourth type collagen are most related to the skin structure. The dermis contains fibrous type I and type III collagen, and the non-fibrous basement membrane under the epithelial tissue contains type IV collagen. Current studies have pointed out that individual aging and environmental adverse factors (such as ultraviolet light exposure) will reduce the ability of skin fibroblasts to produce collagen. In addition, glycation is also one of the main reasons for the destruction of collagen in the skin. When the rate of collagen loss is greater than the rate of its production, the skin will have wrinkles, sagging and other phenomena.

Glycation refers to the slow chemical reaction of sugar molecules attached to proteins or lipids in the absence of enzymes, which ultimately produces advanced glycated end products (AGEs). Saccharification affects the aggregation of collagen to form fibers, and the saccharified collagen fibers become rigid and brittle. In addition, the accumulation of glycation end products in the extracellular matrix of the skin can interfere with the proliferation, differentiation, migration, or gene expression of surrounding cells, and even promote the production of matrix metalloproteinases that can break down collagen and elastin, and can cause inflammation. Secretion of cytokines in response. These factors ultimately contribute to skin aging.

Therefore, one of the ways to prevent or slow down skin aging is to replenish collagen or enhance the collagen production capacity of cells. At the same time, inhibiting the glycation of collagen can ensure the elasticity of collagen fibers, which not only keeps the skin firm, but also prevents diseases derived from the accumulation of glycated protein, such as hardening of the blood vessels. Currently commercially available collagen products mainly rely on the direct replenishment of collagen. However, the molecular weight of collagen is large, and the application method can hardly make the collagen penetrate the stratum corneum of the skin to reach the dermis where it acts; if it is directly replenished by injection Although collagen can reduce the fine lines of the skin immediately, the collagen injected into the body is easily decomposed by enzymes in the body and requires regular administration. The process is complicated and costly. In addition, the immune rejection reaction that may be caused by collagen injection is even greater. It is a big risk. If the collagen is supplemented by eating foods rich in collagen, since the protein is digested into small molecular peptides or amino acids after entering the gastrointestinal tract, the body will use these amino acids to synthesize protein, but it is not limited to collagen. The effect of supplementing collagen with edible methods is limited.

In view of this, it is necessary to develop a novel composition that not only enhances the collagen production capacity of skin fibroblasts but also inhibits collagen glycation.

For this reason, one object of the present invention is to provide a ficus ( Ficus carica ) young fruit extract for the preparation of a composition for reducing skin collagen loss, wherein the fig fruit extract is extracted from a fig young fruit with a solvent. Obtained, wherein the young fig fruit line is a fig fruit 2 to 3 months after the fruit is formed.

In an embodiment of the present invention, the weight ratio of the solvent to the young fig fruit ranges from 20:1 to 1:1, and the extraction is performed at 50°C to 100°C.

In an embodiment of the present invention, the solvent is water, alcohol, or a mixture of alcohol and water, and the concentration of the young fig fruit extract is at least 1 mg/mL.

In one embodiment of the present invention, the young fig fruit extract promotes collagen production in a skin fibroblast.

In one embodiment of the present invention, the young fig fruit extract inhibits collagen glycation.

The present invention discloses that the fig young fruit extract can not only promote the production of collagen by skin fibroblasts, but also prevent the loss of collagen with normal structure and function due to glycation reaction. Given that collagen is one of the main proteins supporting the skin structure, its stable content can maintain the skin’s youthful appearance In this way, the aforementioned fig young fruit extract can simultaneously solve the problems of insufficient collagen production and structural destruction of the skin with age, and can be used to prepare a composition that reduces the loss of skin collagen and maintains the skin's youthful state. The composition can be powder, granule, solution, colloid or paste, and can be made into food, drink, medicine, or nutritional supplement, and administered to a body by oral administration, skin application, etc.

The following will further illustrate the embodiments of the present invention in conjunction with 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 familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent scope.

Fig. 1 shows the effect of the young fig fruit extract or the mature fig fruit extract of an embodiment of the present invention on the production of collagen glycation end products.

Fig. 2 shows the relative production of collagen of human skin fibroblasts treated with a fig young fruit extract or a fig ripe fruit extract according to an embodiment of the present invention.

The present invention provides a use of fig young fruit extract for preparing a composition for reducing skin collagen loss. The young fig fruit extract of the present invention is obtained by extracting a young fig fruit with a solvent, wherein the young fig fruit is a fig fruit 2 to 3 months after the fruit is formed; the solvent can be water, alcohol, or alcohol Water mixture, the weight ratio of the solvent to the young fig fruit is 20:1 to 1:1, and the extraction is performed at 50°C to 100°C. The application of the fig young fruit extract (concentration of 1 mg/mL to 10 mg/mL) can effectively inhibit collagen glycation through the following examples. In addition, the young fig fruit extract can still promote collagen production in human skin fibroblasts.

definition

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%.

Materials and Methods Material

Eagle's minimum essential medium (Gibco Eagle's minimum essential medium, MEM), fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin containing Earle's balanced salt solution (EBSS) Penicillin/streptomycin) and phosphate buffered saline (PBS solution) were purchased from Thermo Fischer Scientific.

Cell culture

The following examples used human skin fibroblast CCD-966SK (ATCC CRL-1881) purchased from the American Type Culture Collection (ATCC) for experiments. The cell line was cultured in MEM medium supplemented with 10% FBS, 1 mM sodium pyruvate, and 1% penicillin/streptomycin under the condition of 37°C and 5% carbon dioxide, which is hereinafter referred to as cell culture medium.

Collagen production test

The amount of collagen produced by skin fibroblasts was measured using the soluble collagen analysis kit (Sircol Soluble Collagen Assay Kit; Bicolor) according to the manufacturer's instructions. The steps are briefly described as follows. Collect 1 mL of cell culture medium and 0.2 mL of collagen separation/concentration reagent from the cell culture that has undergone the designated treatment, and mix it with 0.2 mL of collagen separation/concentration reagent. After leaving overnight at 4°C, the mixture is centrifuged at 12000 rpm for 10 minutes, and about 1 mL of the supernatant is removed. Next, mix the remaining pellet with 1 mL of Sircol dye for 30 minutes, and then centrifuge the dye mixture at 12000 rpm for 10 minutes to form a collagen precipitate. After pouring the supernatant and removing the excess liquid around the collagen precipitate with filter paper, add 1 mL of alkaline reagent to dissolve the collagen precipitate and let it stand for 5 minutes to 3 hours, and then use an ELISA reader (enzyme- The linked immunosorbent assay reader; BioTek) measured the absorbance of the collagen solution at a wavelength of 570nm. Compared with the standard curve of water-soluble collagen standards, the collagen content in the cell culture medium to be tested can be calculated. The statistical analysis department uses the student t test of Excel software to make the judgment.

Example 1 Preparation of fig young fruit extract

Figs, also known as sun berries or milk berries, are deciduous shrubs or small trees of the moraceae family. They are native to western Asia and the Mediterranean coast. They are now cultivated in Taiwan. The plant fruit is rich in dietary fiber, calcium, copper, magnesium, manganese, potassium and other substances beneficial to human health. Generally, fig fruits can be divided into different stages of development Young fruit and mature fruit. Young fruit refers to the fruit that swells to a green and hairy surface after the fruit is formed; the mature fruit refers to the fruit whose surface color turns red, the hair is almost removed, and the volume increases sharply.

In order to obtain the young fig fruit extract, first, the young figs 2 to 3 months after the formation of the fruit are washed and ground using a homogenizer. Thereafter, the homogenous product of the young fig fruit is extracted with water, alcohol, or alcohol-water mixture as a solvent, and the solvent can be added with a mixed acid containing 0.1% to 5% organic acid and hydrochloric acid, wherein the solvent and The weight ratio of the homogeneous mass of the young figs is 20:1 to 1:1. The extraction temperature is between 50°C and 100°C, preferably between 75°C and 95°C. The young fig fruit extracts in the following Examples 2-3 are all extracted with an aqueous solution containing 0.1% acetic acid, citric acid and hydrochloric acid, and the extraction time is 0.5 to 3 hours.

After the young fig fruit extract obtained by the above extraction step is cooled to room temperature, it can be further centrifuged at 800 to 1000 rpm at 15°C to 25°C for 5 to 10 minutes to obtain a supernatant, and the supernatant is adjusted to 400 meshes. (mesh) filter to remove residual solids. The filtered young fig fruit extract can be further concentrated under reduced pressure at 50°C to 60°C to obtain a concentrated product. In order to obtain a solid young fig fruit extract, the aforementioned concentrated young fig fruit extract may be dried to remove the solvent by a drying method such as freeze drying, spray drying, etc., thereby obtaining a powdered young fig fruit extract.

Example 2 Fig young fruit extract inhibits collagen glycation

In order to explore the effect of the young fig fruit extract of the present invention on the glycation of collagen, the anti-glycation analysis was used to determine the young fig fruit extract described in Example 1 (hereinafter referred to as fig young fruit extract) (10mg/mL) or similar The inhibitory effect of the fig mature fruit extract (10 mg/mL) prepared by the method of Example 1 on the glycation reaction of porcine collagen. In short, a 60 mg/mL collagen solution (containing 0.06% sodium azide) and a 1.5 M fructose solution were prepared using 200 mM phosphate buffer solution (pH 7.4). In order to carry out the collagen saccharification reaction, the mixture of 0.2mL collagen solution and 0.2mL fructose solution and 0.2mL of fig young or mature fruit extract sample or deionized water (blank control group) were evenly mixed and reacted at 50℃ for 24 After hours, aminoguanidine (AG, purchased from Sigma) was added to stop the saccharification reaction. Use a spectrofluorometer (FLx 800, BioTek) to measure the fluorescence intensity of the reaction solution (0.1mL) at 0 hours and 24 hours (excitation wavelength 360nm, detection wavelength 460nm), and calculate collagen glycation according to the following formula End product generation rate and anti-glycation activity: collagen glycation end product generation rate = (sample fluorescence intensity for _{24 hours} -sample fluorescence intensity for _{0 hours} ) / (blank control group fluorescence intensity for _{24 hours} -blank control group fluorescence intensity _{0 Hours} )

Anti-glycation activity=(1-generation rate of final glycation product)×100%.

Figure 1 shows the effect of fig young fruit extract or fig mature fruit extract on the production of collagen glycation end products. According to this figure, compared with the blank control group, the anti-glycation activity of the young fig fruit extract is about 67%, while the anti-glycation activity of the mature fig fruit extract is only about 47%. This result shows that young figs have different chemical compositions at different developmental stages, and that the young fig extracts are more effective than mature fruit extracts in inhibiting the glycation reaction of collagen in the body, so it is more conducive to maintaining the normal structure and elasticity of collagen. .

Example 3 Fig young fruit extract promotes collagen production

In order to test the effect of the fig young fruit extract of the present invention on the collagen production of skin cells, in this example, the collagen production test was used to determine the human skin fibroblast CCD-966SK after the fig young fruit extract described in Example 1 (below The change in the amount of collagen produced after treatment. In short, CCD-966SK cells ^{were seeded on a 24-well plate at 2×10 4} cells/well, and each well contained 500 μL of cell culture medium. After culturing the cells at 37°C for 24 hours, remove the cell culture medium and treat the cells in each well in the following manner: (a) apply 500 μL of 1 mg/mL fig young fruit extract and 500 μL of cell culture medium without FBS; (b) Apply 500 μL of 1 mg/mL fig mature fruit extract and 500 μL cell culture medium without FBS; (c) apply 500 μL cell culture medium without FBS (blank control group). After the aforementioned three groups of cells were cultured at 37°C for 48 hours, the cell culture medium was collected and the collagen content was measured.

Figure 2 shows the relative production of collagen in the aforementioned three groups of skin fibroblasts. According to this figure, compared with the blank control group, the administration of fig young fruit extract significantly increased the collagen content in the cell culture medium by about 25%. Conversely, the application of fig mature fruit extract slightly reduced collagen production. This result once again confirmed that the young figs have significantly different chemical compositions at different developmental stages, and that only the young fig extract has the effect of promoting the production of collagen by skin fibroblasts.

In summary, fig young fruit extract can not only promote the production of collagen by skin fibroblasts, but also prevent the loss of collagen with normal structure and function due to glycation reaction. In view of the fact that the young fig fruit extract can solve the problems of insufficient collagen production and structural variation of the skin with age in a more comprehensive way, it can be used to prepare a composition that reduces the loss of skin collagen. The composition can be powder, granule, solution, colloid or paste, and can be made into food, drink, medicine, or nutritional supplement, and administered to a body by oral administration, skin application, etc.

Claims (8)

A use of a fig young fruit extract for preparing a composition for reducing skin collagen loss, wherein the fig young fruit extract is obtained by extracting a fig young fruit with a solvent, wherein the fig young fruit is after the fruit is formed. Figs up to 3 months old, and the solvent is an aqueous solution containing 0.1% acetic acid, citric acid and hydrochloric acid. The use as described in item 1 of the scope of patent application, wherein the weight ratio of the solvent to the young fig fruit is in the range of 20:1 to 1:1. The use as described in item 1 of the scope of patent application, wherein the extraction is carried out at 50°C to 100°C. The use as described in item 1 of the scope of patent application, wherein the concentration of the young fig fruit extract is at least 1 mg/mL. The use described in item 1 of the scope of patent application, wherein the young fig fruit extract promotes collagen production of a skin fibroblast. The use described in item 1 of the scope of patent application, wherein the young fig fruit extract inhibits collagen glycation. The use described in item 1 of the scope of patent application, wherein the composition is a food, a drink, or a nutritional supplement. The use described in item 1 of the scope of the patent application, wherein the composition has a powder, granule, solution, colloid, or paste dosage form.

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