TWI777096B - Use of extract of ginkgo biloba callus tissue for improving skin redness - Google Patents

Abstract

The present disclosure provides a use of an extract of the Ginkgo biloba callus tissue for inducing expression of Tgm1 gene, KRT gene, AQP3 gene, FLG-F gene, GBA gene, HAS gene, CCT gene, Pink1 gene, Atg gene, SIRT1 gene, NADSYN gene, MRPS5 gene or Ubl-5 gene, enhancing the moisture-retaining capacity of the skin, and anti-aging. The present disclosure also provides a method for inducing and proliferating the Ginkgo biloba callus tissue, and a medium for culturing the Ginkgo biloba callus tissue.

Description

Use of Ginkgo biloba callus extract to improve skin redness

The present invention relates to a kind of Ginkgo biloba callus extract for improving Tgm1 gene, KRT gene, AQP3 gene, FLG-F gene, GBA gene, HAS gene, CCT gene, Pink1 gene, Atg gene, SIRT1 gene Gene, expression level of NADSYN gene, MRPS5 gene or Ubl-5 gene, use for improving skin moisturizing ability and anti-aging, method for inducing and proliferating ginkgo callus, and medium for cultivating ginkgo callus.

Skin tissue is composed of epidermis, dermis and subcutaneous tissue. The dermis contains a lot of collagen and hyaluronic acid, which are closely related to the water retention and elasticity of the skin. Human skin will experience aging, rough skin or wrinkles with age, physiological factors or environmental factors. For example, the skin of normal young people has a certain elasticity and tension. When the facial muscles are relaxed, the skin will quickly recovery, so that wrinkles disappear; but after entering middle age, the skin begins to age significantly, the skin becomes thinner, stiffer, dry, and the tension is reduced; the dermal collagen is reduced, the elastic fibers are degenerated and broken, and the tension and elasticity of the skin are reduced. Therefore, When the facial muscles relax, the skin cannot recover quickly, and wrinkles are formed over time; and with the increase of age, the skin and subcutaneous tissue become more slack, coupled with the atrophy or loss of facial support tissues, and the softness of the muscles, the skin will Slips under the action of gravity, forming deeper wrinkles. Rough skin is caused by external factors such as dryness, ultraviolet rays, irritating substances such as detergents or chemicals, or internal factors such as hormone balance disorders. Decreased moisture content, hypersensitivity of epidermal metabolism, the production of scales and keratin roughening and so on. Therefore, if the cells on the skin lose their elasticity and moisturizing function, it will cause skin wrinkles, dryness and loss of luster.

In recent years, the demand for moisturizing the skin is increasing day by day, because once the moisturizing ability of the skin is improved, the anti-aging effect can be achieved. However, it is now commonly used to Most of the ways to increase skin moisturizing ability are to use cosmetics, skin care products applied to the skin surface, or oral health food that claims to have skin moisturizing effect. However, conventional cosmetics, skin care products and health foods are mostly made of chemical ingredients. Long-term use is not only harmful to human health, but also these products are often expensive and not affordable for ordinary users.

In addition, since ancient times, human beings have always dreamed of pursuing eternal youth and immortality. In recent years, with the development and progress of medicine and biotechnology, not only can front-end medical technology be used to fight diseases, but many products that demand anti-aging are also available. Research and development have come out one after another. In recent years, the trend of anti-aging has slowly spread to the world. Taking Japan as an example, more than 70% of the people have anti-aging awareness, and Taiwanese people's concern for anti-aging has also continued to increase. The anti-aging trend will drive the market sales of anti-aging related products, and the global market is also expected to continue to expand.

On the other hand, the mitochondria (mitochondria) is also known as the power station of the cell, because it is the main site for the synthesis of adenosine triphosphate (ATP) (a molecule that transmits energy) in the cell, providing various activities of the cell. chemical energy. If mitochondria are damaged, the impact on cells and individual organisms is huge. During the process of synthesizing ATP, mitochondria will generate a lot of free radicals. The activity of free radicals is extremely strong, and they will have strong oxidation reactions with any substances in the body and destroy their normal functions. Free radicals damage enzymes and DNA in mitochondria over time, gradually reducing their function and thus the function of various organs and tissues. Therefore, how to enhance the mitochondrial activity of cells to achieve the effect of anti-aging has become an important topic in the field.

In order to solve the above problems, those skilled in the art are in urgent need of developing novel pharmaceuticals, food products or skin care products with improved skin moisturizing ability and anti-aging effect, so as to benefit the vast population with such needs.

In view of this, the purpose of the present invention is to provide a kind of Ginkgo biloba ( Ginkgo biloba ) callus extract for preparing a transglutaminase 1 (transglutaminase 1, Tgm1 ) gene, keratin (keratin, KRT ) gene , aquaporin 3 ( AQP3 ) gene, filaggrin ( FLG-F ) gene, Glucosylceramidase ( GBA ) gene, hyaluronan synthase ( HAS ) gene, containing T-complex protein 1 subunit alpha (TCP1) complex chaperonin (chaperonin containing T-complex protein 1 subunit alpha(TCP1) complex, CCT ) gene, PTEN-induced kinase 1 (PTEN-induced kinase 1, Pink1 ) gene, Autophagy-related ( Atg ) gene, Sirtuin 1 ( SIRT1 ) gene, Glutamine-dependent NAD(+) synthase synthetase, NADSYN ) gene, mitochondrial ribosomal protein S5 (mitochondrial ribosomal protein S5, MRPS5 ) gene or ubiquitin-like protein 5 (Ubiquitin-like protein 5, Ubl-5 ) The use of the composition of the expression amount of the gene, wherein The extract of the ginkgo callus is prepared by extracting the ginkgo callus with water, alcohol, water-containing alcohol or a combination thereof as an extraction solvent.

In one embodiment of the present invention, the KRT gene is KRT1 gene, KRT10 gene or KRT14 gene.

In one embodiment of the present invention, the HAS gene is the HAS2 gene or the HAS3 gene.

In one embodiment of the present invention, the CCT gene is a chaperonin containing TCP1 subunit 2 ( CCT2 ) gene and a chaperonin containing TCP1 subunit 5 ( CCT5 ) gene , the chaperonin containing TCP1 subunit 6A (chaperonin containing TCP1 subunit 6A, CCT6A ) gene, the chaperonin containing TCP1 subunit 7 (chaperonin containing TCP1 subunit 7, CCT7 ) gene, or the chaperonin containing TCP1 subunit 8 (chaperonin containing TCP1 subunit 8, CCT8 ) gene.

In one embodiment of the present invention, the Atg gene is the Atg1 gene or the Atg8 gene.

Another object of the present invention is to provide the use of a Ginkgo biloba callus extract for preparing a composition for improving skin moisturizing ability and anti-aging, wherein the Ginkgo biloba callus extract is water , alcohols, water-containing alcohols or their combination as an extraction solvent to extract the ginkgo callus.

In an embodiment of the present invention, the ginkgo callus is cultured through a medium supplemented with methyl jasmonic acid (MeJA).

In one embodiment of the present invention, the effective concentration of the Ginkgo biloba callus extract is at least 0.25% (v/v).

In an embodiment of the present invention, the composition is a medicine, a food product or a skin care product.

Another object of the present invention is to provide a method for culturing a ginkgo callus, comprising using a medium supplemented with methyl jasmonic acid (MeJA) to cultivate the ginkgo callus, wherein the methyl jasmine flower The concentration of acid is at least 0.1 mM.

To sum up, the effect of the extract of the ginkgo callus of the present invention is that: by enhancing the Tgm1 gene, KRT gene, AQP3 gene, FLG-F gene, GBA gene, HAS gene, CCT gene, Pink1 gene, Atg gene , SIRT1 gene, NADSYN gene, MRPS5 gene or Ubl-5 gene expression to improve the skin's moisturizing ability and anti-aging effect, and human experiments have also proved effective. On the other hand, the present invention utilizes the addition of methyl jasmonic acid in the culturing process to generate a large amount of polyphenols and flavonoid equivalents for moisturizing and anti-aging, so that the extract of the ginkgo callus of the present invention is more conducive to industrial utilization .

The embodiments of the present invention will be further described below. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Anyone who is familiar with this technique, without departing from the spirit and scope of the present invention, Some changes and modifications can be made, so the protection scope of the present invention should be determined by the scope of the appended patent application.

FIG. 1A is a data graph of the efficacy of the extract of Ginkgo biloba callus of the present invention in increasing the content of total polyphenols.

FIG. 1B is a data graph of the efficacy of the extract of the ginkgo callus of the present invention in increasing the content of total flavonoids.

Fig. 2 is the expression of Tgm1 , KRT1 , KRT10 , KRT14, AQP3 , FLG-F , GBA , HAS2 and HAS3 genes related to skin cell moisturization enhanced by the extract of Ginkgo biloba callus of the present invention at 6 or 24 hours of action The data graph of efficacy, in which * means compared with the control group, p <0.05; ** means compared with the control group, p <0.01; *** means compared with the control group, p < 0.001.

Figure 3 is a data graph of the efficacy of the extract of the ginkgo callus of the present invention in promoting the secretion of hyaluronic acid by keratinocytes, wherein *** indicates that compared with the control group, p < 0.001.

4A is a data graph showing the efficacy of the extract of the ginkgo callus of the present invention in enhancing the expression of CCT2 , CCT5 , CCT6A , CCT7 , CCT8 and Pink1 genes related to anti-aging after 48 hours of action, wherein * represents the comparison with the control group Comparison, p <0.05; ** indicates comparison with control group, p <0.01; *** indicates comparison with control group, p < 0.001.

Figure 4B is a data graph showing the efficacy of the extract of the ginkgo callus of the present invention in enhancing the expression of Atg1 , Atg8 , SIRT1 , NADSYN , MRPS5 and Ubl-5 genes related to anti-aging at 24 or 48 hours, wherein * Indicates the comparison with the control group, p <0.05; ** indicates the comparison with the control group, p <0.01; *** indicates the comparison with the control group, p < 0.001.

Figure 5 is a data graph and a photograph showing the efficacy of the Ginkgo callus extract of the present invention in reducing the formation of fine lines, wherein * means compared with the control group, p < 0.05.

FIG. 6 is a data diagram of the efficacy of the extract of the ginkgo callus of the present invention in increasing skin water retention.

7 is a data graph and an image graph showing the efficacy of the extract of Ginkgo biloba callus of the present invention in improving facial redness.

definition

Numerical values used herein are approximations and all experimental data are expressed within 20%, preferably within 10%, and most preferably within 5%.

According to the present invention, Ginkgo biloba , also known as Gongsun tree or Duck's foot tree, is a deciduous tree of the Ginkgo family ( Ginkgoaceae ). Ginkgo is native to southern China, and its existence can be traced back to 200 million years ago. It is the only extant species in the Ginkgo family, and is known as a living fossil in the plant kingdom. The branches of ginkgo will not die in the fire, and they will regenerate in every difficulty. Nagasaki and Hiroshima, where Araki was dead after World War II, were also the first to sprout new buds of ginkgo, showing its strong life. Ginkgo biloba is a tree species with excellent adaptability and strong resistance to stress. It can generate special substances to resist adversity in extreme environments, so it is resistant to cold, heat and is not easily infected by pests or bacteria.

According to the present invention, the callus is the original undifferentiated state of the plant, which is derived from the apical meristem or somatic cell of the stem and root. Callus has epigentic function and totipotent ability, which can differentiate into plant embryo cells to form new plants, and help cells to metabolize and regenerate, delay aging and impart vitality. In modern times, using plant tissue culture methods, a set of techniques for plant callus proliferation has been successfully developed. However, for different varieties of plants, it is necessary to find out a suitable culture formula through exhaustive experiments in order to produce valuable plant callus for industrial use.

As used herein, the terms "extract of ginkgo callus" and "ginkgo stem cells" are used interchangeably.

As used herein, the term "anti-aging" means preventing, slowing down the appearance of aging phenomena of human skin, such as the development of wrinkles and loss of elasticity. The extent to which this is assessed will depend upon a number of factors known to those skilled in the art, such as the general condition of the consumer, age, gender, and the like.

According to the present invention, pharmaceutical products can be manufactured into a dosage form suitable for parenterally, orally or topically, using techniques well known to those skilled in the art, including, but not limited to: injection [eg, sterile aqueous solution or dispersion], sterile powder, tablet, troche, oral Lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixirs (elixir), slurry, external preparation and the like.

According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents 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 agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.

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

According to the present invention, the medicinal product may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intradermal injection Intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection and intralesional injection.

According to the present invention, the medicinal product may be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to: emulsions, gels Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serum, paste, foam, drop, suspension, salve and bandage.

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

According to the present invention, the substrate may comprise one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons (such as petroleum, jelly) and white petrolatum], waxes [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers ( absorption enhancers), stabilizing agents, gelling agents [such as Carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose and carboxymethylcellulose], Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants and propellants (propellants) etc. The selection and quantity of these additives are within the professional and routine skills of those skilled in the art.

According to the present invention, the skincare product may further comprise an acceptable adjuvant which is widely used in skincare product manufacturing techniques. 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 agents, thickening agents, fillers, fragrances and odor absorbers. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.

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

According to the present invention, the skin care product may also be used in combination with one or more external use agents with known activity selected from the group consisting of whitening agents (such as tretinoin, catechins) (catechin), koji acid, arbutin and vitamin C], humectants, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts[ such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, anti-inflammatory agents antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, Antiseborrheic agents, wound-healing agents, corticosteroids and hormones. The selection and quantity of these topical preparations fall within the scope of the professionalism and routine skills of those skilled in the art.

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

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

Example 1. Preparation of Ginkgo biloba callus extract

First, take a bud of Ginkgo biloba as a culture body, and sterilize the ginkgo biloba to remove the microorganisms on the surface of the plant, so that the ginkgo biloba can be wounded in any way, for example, can be cut, torn or sheared. Ginkgo biloba with a wound, and a callus is formed on the wound.

Methyl jasmonic acid (MeJA) was dissolved in dimethyl sulfite to prepare a molecular weight of 0.1 micromolar for use. Next, 5 mL of methyl jasmonic acid solution (concentration: 0.1 mM) was uniformly dropped on the medium containing ginkgo callus (MS medium (Murashige and Skoog), and 0.5 mg/L of 1-naphthaleneacetic acid was added. acid, NAA), 0.1 mg/L 6-Benzylaminopurine (6-Benzylaminopurine), 3% sucrose and 0.8% agar, adjusted to pH 5.8), and harvested one week after culture for use. After that, the cultured ginkgo callus was harvested for subsequent extraction. Then, homogenize the harvested ginkgo callus, and then extract the homogenized ginkgo callus with water, alcohol, water-containing alcohol or a combination thereof, wherein water is the best solvent as an extraction solvent For 20 minutes, the volume ratio of the homogenized ginkgo callus to the extraction solvent is 0.8-1.2:8-12 (preferably 1:10), and the extraction temperature is between 30°C and 50°C. After that, it was cooled to room temperature, and then the obtained product was filtered with a 400-mesh filter to obtain the extract of the ginkgo callus of the present invention.

Example 2. Evaluation of the effect of extracts from Ginkgo biloba callus cultured in a medium supplemented with methyl jasmonic acid in increasing the content of total polyphenols and total flavonoids

First, a standard solution was prepared by dissolving 10 g of gallic acid in water and adding a volume of 10 mL to a measuring flask. Next, standard solutions of 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL were prepared, respectively, and then 100 μL of each standard solution was taken into a centrifuge tube with a volume of 10 mL. After that, 500 μL of Folin-Ciocalteu’s phenol reagent was added, mixed and stood upright for 3 minutes, followed by 400 μL of 7.5% sodium carbonate (sodi μM carbonate), mix and stand for 30 minutes. Next, 200 μL of each reaction solution was transferred to a 96-well plate, and the absorbance was measured at 750 nm.

In addition, the Ginkgo biloba callus extract obtained in Example 1 was used as the experimental group, and the Ginkgo biloba callus cultured without addition of methyl jasmonic acid was used as the comparison group. The experimental group and the comparison group were diluted with water and 100 mL were taken into microcentrifuge tubes. After that, 500 μL of verschol reagent was added, mixed and stood upright for 3 minutes, followed by 400 μL of 7.5% sodium carbonate, mixed and stood upright for 30 minutes. Next, 200 μL of each group of reaction solutions were transferred to a 96-well plate, and the absorbance was measured at 750 nm. The results for total polyphenol content are shown in Figure 1A.

FIG. 1A is a data graph of the efficacy of the extract of Ginkgo biloba callus of the present invention in increasing the content of total polyphenols. As can be seen from Figure 1A, compared with the comparison group, the total polyphenol content of the experimental group was significantly increased by 1.6 times. The results of this example show that the extract of Ginkgo biloba callus obtained by culturing in a medium supplemented with methyl jasmonic acid of the present invention can effectively increase the total polyphenol content.

In addition, the experimental procedure for the detection of total flavonoid content is as follows: the total flavonoid content is detected with the equivalent of rutin (ChromaDex ASB-00018440) as the expression of the relative content of total flavonoids. Preparation materials include 10% aluminum nitrate (aqueous solution) (Alfa Aesar 12360), 5% sodium citrate (aqueous solution) (Sigma 31443), 4% sodium hydroxide (aqueous solution) (Macron 7708-10) and 200 μg/mL rutin (in methanol).

Take 0, 200 μL, 400 μL, 600 μL, 800 μL, 1000 μL and 1200 μL of the above rutin standard solution into test tubes, respectively, add 1200 μL, 1000 μL, 800 μL, 600 μL, 400 μL, 200 μL and 0 μL of water in sequence, shake and mix evenly; take 200 μL Rutin solution of each concentration; add 200 μL of 5% sodium citrate respectively, mix well and let stand for 6 minutes; add 200 μL of 10% aluminum nitrate, mix well and let stand for 6 minutes; then add 2 mL of 4% sodium hydroxide After mixing evenly, add 1.4 mL of H ₂ O to mix evenly; take 200 μL of the above reaction solution in a 96-well reaction plate, detect the absorbance at 500 nm with a spectrophotometer, and draw a standard curve.

The Ginkgo biloba callus extract obtained in Example 1 was used as the experimental group, and the Ginkgo biloba callus cultured without the addition of methyl jasmonic acid was used as the comparison group. After the experimental group or the comparison group is properly diluted, take 200 μL of the experimental group or the comparison group sample and place it in a test tube; add 200 μL of 5% sodium citrate, mix well and let stand for 6 minutes; add 200 μL of 10% aluminum nitrate, mix well After that, let stand for 6 minutes; add 2 mL of 4% sodium hydroxide and mix well, then add 1.4 mL of H ₂ O and mix well; take 200 μL of the above reaction solution in a 96-well reaction plate, and detect the absorbance at 500 nm with a spectrophotometer . The results for total flavonoid content are shown in Figure IB.

FIG. 1B is a data graph of the efficacy of the extract of the ginkgo callus of the present invention in increasing the content of total flavonoids. As can be seen from Figure 1B, compared with the comparison group, the total flavonoid content of the experimental group was significantly increased by 2.3 times. The results of this example show that the extract of Ginkgo biloba callus obtained by culturing in a medium supplemented with methyl jasmonic acid of the present invention can effectively increase the content of total flavonoids.

Example 3. Efficacy evaluation of Ginkgo biloba callus extract in enhancing skin moisturizing ability

In this example, it is investigated whether Ginkgo biloba callus extract can improve the skin's moisturizing ability by enhancing the expression of genes related to skin cell moisturizing.

Human epidermal keratinocytes (HPEK-50; purchased from CELLnTEC) were cultured in a 6-well plate in a serum-free medium for keratinocytes (Keratinocyte-SFM; purchased from Thermo, product number: 17005042), and the cell concentration of 2 mL of the medium was 1 ×10 ⁵ cells/well.

After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental groups 1 and 2). The extracts of Ginkgo biloba callus were diluted with culture medium to have dilutions of 0.25% (v/v) and 0.5% (v/v) concentrations, and then 0.25% dilutions were added to the cells of experimental group 1, respectively, and The 0.5% dilution was added to the cells of the experimental group 2, and the cells of the control group (ie, HPEK-50) were only added with the medium. Next, the cells of each group were cultured in an incubator for 6 or 24 hours, and then the cell cultures of each group were harvested and used for gene expression analysis.

In this example, the genes used to analyze skin cell moisturizing include transglutaminase 1 ( Tgm1 ) gene, keratin 1 ( KRT1 ) gene, KRT10 gene, KRT14 gene, water Channel protein 3 (aquaporin 3, AQP3 ) gene, filaggrin (filaggrin, FLG-F ) gene, glucosylceramidase (Glucosylceramidase, GBA ) gene, hyaluronan synthase 2 (hyaluronan synthase 2, HAS2 ) gene and HAS3 Gene.

Each group of cell cultures obtained above was subjected to RNA extraction with an RNA extraction kit (Geneaid). 2,000 ng of each group of RNA thus obtained was taken and the extracted RNA was reverse transcribed into cDNA with SuperScript ^® III reverse transcriptase (Invitrogen). Next, using cDNA as a template, and using primer pairs used to amplify target genes, including Tgm1 , KRT1 , KRT10 , KRT14, AQP3 , FLG-F , GBA , HAS2 , HAS3 and TBP (as an internal control), their The nucleotide sequences are shown in Table 1 below. Quantitative real-time PCR was performed in the StepOne Plus Real-Time PCR System (ABI) using the KAPA CYBR FAST qPCR Kit (2x) (KAPA Biosystems) to amplify and quantify the target gene. . Melting curves of PCR products are confirmed during quantitative real-time PCR reactions.

The relative expression level of the target gene was derived from Equation 2 ^-ΔΔCt , and the relative fold change was calculated using the cycle threshold of the TBP gene (as an internal control) and the benchmark gene and by standard deviation, where ΔCt=Ct _{target gene /baseline gene} -Ct _TBP , △△Ct=△Ct _target gene-△Ct _{reference gene} , fold change=2- ^△△Ct _mean . The expression level of the target gene in the control group was used as a comparison benchmark of 1. Statistically significant differences between groups were determined by one-tailed Student's t-test. The results of this example are shown in FIG. 2 .

Fig. 2 is the expression of Tgm1 , KRT1 , KRT10 , KRT14, AQP3 , FLG-F , GBA , HAS2 and HAS3 genes related to skin cell moisturization enhanced by the extract of Ginkgo biloba callus of the present invention at 6 or 24 hours of action Data graph of efficacy. As can be seen from Figure 2, whether it is Tgm1 gene, KRT14 gene, FLG-F gene, GBA gene, HAS2 gene or HAS3 gene, compared with the control group (ie HPEK mock), the experimental group 1 and the experimental group 2 are in the effect of 6 Or the relative expression level of genes at 24 hours was significantly improved. In terms of KRT1 gene, compared with the control group, except the experimental group 2 acted for 6 hours, the relative gene expression of the experimental group 1 at 6 and 24 hours and the experimental group 2 at 24 hours There is a significant improvement. In terms of KRT10 gene, compared with the control group, except the experimental group 2 acted for 24 hours, the relative gene expression of the experimental group 1 at 6 and 24 hours and the experimental group 2 at 6 hours There is a significant improvement. In terms of AQP3 gene, compared with the control group, except the experimental group 2 acted for 24 hours, the relative gene expression of the experimental group 1 at 6 and 24 hours and the experimental group 2 at 6 hours There is a significant improvement. The results of this example show that the extract of the ginkgo callus of the present invention can maintain keratinocytes by enhancing the expression of Tgm1 , KRT1 , KRT10 , KRT14, AQP3 , FLG-F , GBA , HAS2 and HAS3 genes related to skin cell moisturization The cells are arranged to make the skin keratinous tissue complete, increase the skin's water retention, increase the synthesis of hyaluronic acid by the keratinocytes, effectively lock the moisture for the skin, fill the lipids between the cells of the skin's stratum corneum, and achieve the effect of improving the skin's moisturizing ability.

Example 4. Evaluation of the efficacy of Ginkgo biloba callus extract in promoting the secretion of hyaluronic acid by keratinocytes

This example further tests the effect of the Ginkgo biloba callus extract of the present invention on promoting the secretion of hyaluronic acid by keratinocytes, because it is known that keratinocytes secrete substances such as hyaluronic acid as the intercellular substance to maintain the integrity of the epidermal barrier and prevent skin moisture Lost and form complete protection, therefore. First, 200 μL of serum-free medium for keratinocytes (Keratinocyte-SFM; purchased from Thermo, product number: 17005042) was added to each well in a 96-well culture plate, and 1×10 ⁴ human epidermal keratinocytes (HPEK-50; purchased) were implanted. from CELLnTEC)/well and then incubated overnight at 37°C.

After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental groups 1 and 2). The extract of Ginkgo biloba callus was diluted into a dilution with a concentration of 0.5% (v/v) and 1% (v/v) with a medium, and then the 0.5% dilution was added to the cells of the experimental group 1, and The 1% dilution was added to the cells of the experimental group 2, and the cells of the control group were left untreated. Incubate at 37°C for 24 hours, and collect 100 μL of medium per well without disturbing the attached cells.

Next, the ELISA analysis kit (purchased from Cusabio Biotech, China, No. CSB-E04805h) of human hyaluronic acid (HA, also known as human hyaluronic acid) was used for analysis. First, add 100 μL of the culture medium collected from each well, or a standard solution dissolved in phosphate buffered solution containing 1% bovine serum albumin, in a 96-well culture dish covered with a layer of human hyaluronic acid capture antibody at the bottom, at 37°C Binding with the capture antibody for 2 hours, remove the liquid after the time is up, and directly add 100 μL of detection antibody (biotin-antibody (1X)) to each well, then detect at 37°C Capture antibodies for 1 hour. Next, each well was aspirated and washed, repeating the process twice for a total of three washes. Afterwards, wash each well by filling each well with wash buffer (200 μL) using a multi-channel pipette and let stand for 2 min before complete removal of liquid at each step is essential for good performance of. After the last wash, any remaining wash buffer was removed by aspiration or decanting. Invert the culture plate and dry with a clean paper towel. After that, 100 μL of horseradish peroxidase-avidin (HRP-avidin) was added to each well for 1 hour at 37°C, and then the suction/wash procedure was repeated 5 times. Next, add 90 μL of TMB substrate coloring solution, act at 37°C for 15-30 minutes, and Protect from light, then add 50 μL of stop solution to each well to stop the reaction, tap the culture plate lightly to ensure adequate mixing, and finally measure the absorbance at 450 nm within 5 minutes with an enzyme immunoassay analyzer (BioTek). The Student's t-test was then performed with Excel software to determine the coefficient of variation and whether there was a statistically significant difference. The experimental results are shown in FIG. 3 .

Fig. 3 is a data graph of the efficacy of the extract of the ginkgo callus of the present invention in promoting the secretion of hyaluronic acid by keratinocytes. It can be seen from Figure 3 that compared with the control group, the production of hyaluronic acid in both experimental group 1 and experimental group 2 has been significantly increased, of which experimental group 1 increased by 30.8%, and experimental group 2 increased by 27.5%. The results of this example show that the extract of the ginkgo callus of the present invention can effectively promote the secretion of hyaluronic acid by keratinocytes, can effectively complete the structure of the stratum corneum of the skin, improve the skin barrier function, and improve the water retention capacity of the skin.

Example 5. Evaluation of the anti-aging effect of the extract of Ginkgo biloba callus

In this example, it is investigated whether Ginkgo biloba callus extract can achieve anti-aging effect by enhancing the expression of genes related to anti-aging.

First, with Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco), DMEM) cultured human bone marrow neuroblastoma SHSY-5Y (TCC ^® CRL-2266 ^TM ) in a 6-well dish, and the cell concentration of 2 mL of medium was 1×10 ⁵ cells/well.

After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental groups 1 and 2). The extract of Ginkgo biloba callus was diluted into a dilution with a concentration of 0.5% (v/v) and 1% (v/v) with a medium, and then the 0.5% dilution was added to the cells of the experimental group 1, and The 1% dilution was added to the cells of the experimental group 2, and the culture medium was added to the cells of the control group. Next, the cells of each group were cultured in an incubator for 24 or 48 hours, and then the cell cultures of each group were harvested and used for gene expression analysis.

In this example, the genes used to analyze anti-aging include the chaperonin containing TCP1 subunit 2 ( CCT2 ) gene, the chaperonin containing TCP1 subunit 5 (chaperonin containing TCP1 subunit 5, CCT5 ) gene, chaperonin containing TCP1 subunit 6A ( CCT6A ) gene, chaperonin containing TCP1 subunit 7 ( CCT7 ) gene, chaperonin containing TCP1 subunit 8 (chaperonin containing TCP1 subunit 8, CCT8 ) gene, PTEN-induced kinase 1 (PTEN-induced kinase 1, Pink1 ) gene, autophagy-related protein 1 (Autophagy-related 1, Atg1 ) gene, autophagy-related protein 8 (Autophagy-related 8, Atg8 ) gene, sirtuin 1 (Sirtuin 1, SIRT1 ) gene, glutamine-dependent NAD(+) synthetase (Glutamine-dependent NAD(+) synthetase, NADSYN ) gene, Mitochondrial ribosomal protein S5 ( MRPS5 ) gene and ubiquitin-like protein 5 ( Ubl-5 ) gene, among which NADSYN gene assists in the synthesis of NAD, provides mitochondrial energy source and maintains vitality ; SIRT1 gene assists mitochondria to repair damaged DNA and slow down aging; Atg1 gene is a gene related to mitochondrial rejuvenation, rejuvenation and anti-aging; Atg8 gene removes mutant DNA to rejuvenate mitochondria and restore youthful vitality; MRPS5 gene assists Mitochondrial protein synthesis, providing energy source; CCT2 , CCT5 , CCT6A , CCT7 and CCT8 genes make old mature cells back to young cells; Pink1 gene restores aging mitochondria to youthful state; Ubl-5 gene restores mitochondrial activity , animal experiments confirmed that the old mice returned to the state of youth.

Each group of cell cultures obtained above was subjected to RNA extraction with an RNA extraction kit (Geneaid). 2,000 ng of each group of RNA thus obtained was taken and the extracted RNA was reverse transcribed into cDNA with SuperScript ^® III reverse transcriptase (Invitrogen). Next, use cDNA as a template, and use primer pairs for amplifying target genes, including CCT2 , CCT5 , CCT6A , CCT7 , CCT8 , Pink1 , Atg1 , Atg8 , SIRT1 , NADSYN , MRPS5 , Ubl-5 and GAPDH (as internal control group), their nucleotide sequences are shown in Table 2 below, quantitative real-time PCR was performed in the StepOne Plus real-time PCR system (ABI) using the KAPA CYBR FAST qPCR kit (2x) (KAPA Biosystems) to compare the target Gene amplification and quantification. Melting curves of PCR products are confirmed during quantitative real-time PCR reactions.

The relative expression of the target gene was derived from Equation 2 ^-ΔΔCt , and the relative fold change was calculated using the cycle threshold of the GAPDH gene (as an internal control) and the benchmark gene and by standard deviation, where ΔCt=Ct _{target gene /baseline gene} - _CtGAPDH , △△Ct=△Ct _target gene-△Ct _{reference gene} , fold change=2- ^△△Ct _mean . The expression level of the target gene in the control group was used as a comparison benchmark of 1. Statistically significant differences between groups were determined by one-tailed Student's t-test. The results of this example are shown in FIGS. 4A and 4B .

Figure 4A is a data graph showing the efficacy of the Ginkgo callus extract of the present invention in enhancing the expression of CCT2 , CCT5 , CCT6A , CCT7 , CCT8 and Pink1 genes related to anti-aging at 48 hours. As can be seen from Figure 4A, whether it is CCT2 , CCT5 , CCT6A , CCT8 or Pink1 gene, compared with the control group, the relative expression of the genes in the experimental group 1 and the experimental group 2 was significantly increased at 48 hours. As for the CCT7 gene, compared with the control group, the relative expression of the gene in the experimental group 1 was significantly increased at the 48-hour period, except that the experimental group 2 acted for 48 hours.

Figure 4B is a data graph of the efficacy of the extract of the ginkgo callus of the present invention in enhancing the expression of Atg1 , Atg8 , SIRT1 , NADSYN , MRPS5 and Ubl-5 genes related to anti-aging at 24 or 48 hours. As can be seen from Figure 4B, whether it is Atg1 , SIRT1 or MRPS5 gene, compared with the control group, the relative expression levels of the genes in experimental group 1 and experimental group 2 were significantly increased at 24 or 48 hours. As for the Atg8 gene, compared with the control group, except that the experimental group 1 acted for 24 hours, the relative gene expression of the experimental group 1 at 48 hours and the experimental group 2 at 24 hours or 48 hours. There is a significant improvement. In terms of NADSYN gene, compared with the control group, except that the experimental group 1 acted for 48 hours, the relative gene expression of the experimental group 1 at 24 hours and the experimental group 2 at 24 hours or 48 hours There is a significant improvement. In terms of Ubl-5 gene, compared with the control group, except that the experimental group 1 acted for 24 hours, the gene of the experimental group 1 at 48 hours and the experimental group 2 at 24 hours or 48 hours were relative. Performance has improved significantly. The results of this example show that the Ginkgo biloba callus extract of the present invention can enhance anti-aging related CCT2 , CCT5 , CCT6A , CCT7 , CCT8 , Pink1 , Atg1 , Atg8 , SIRT1 , NADSYN , MRPS5 and Ubl-5 Gene expression enhances mitochondrial activity in multiple directions, strengthens the source of skin vitality, and achieves anti-aging effects.

Example 6. Human efficacy test of the extract of Ginkgo biloba callus

In this example, by using the extract of ginkgo callus prepared in Example 1 to test whether it has the effect of improving human skin.

First, 8 subjects were recruited, and the left face of each subject was used as a control group, and the right face was used as an experimental group. After cleansing the face every morning and evening, a placebo was applied to the skin of the control group. , and apply 1% ginkgo callus extract as ginkgo stem cell essence to the skin of the experimental group, massage with finger pulp to promote absorption, and before use (week 0) and 4 weeks or 15 minutes after use test. The test items include skin wrinkles, skin moisture content and skin redness. Skin wrinkles and skin moisture content are detected at the 4th week after use, and skin redness is detected at 15 minutes after use. VISIA Complexion Analysis System (VISIA Complexion Analysis) System) (Canfield scientific, USA) for detection. The results of this example are shown in FIGS. 5 to 7 .

FIG. 5 is a data graph and a photo of the efficacy of the extract of the ginkgo callus of the present invention in reducing the formation of fine lines. FIG. 6 is a data diagram of the efficacy of the extract of the ginkgo callus of the present invention in increasing skin water retention. 7 is a data graph and an image graph showing the efficacy of the extract of Ginkgo biloba callus of the present invention in improving facial redness. As can be seen from Figure 5, compared with the 0th week, the skin fine lines in the experimental group will be significantly reduced with time, but not in the control group; compared with the 0th week, the experimental group in the 4th week after use skin fine lines were reduced by 13%. As can be seen from Figure 6, compared with the 0th week, the skin water retention of the experimental group will increase with time. Compared with the 0th week, the skin water retention of the experimental group in the 4th week after use An increase of 13.3%. As can be seen from Figure 7, compared with before use, the redness of the face in the experimental group was improved 15 minutes after use, but not in the control group; compared with before use, the skin redness of the experimental group 15 minutes after use was improved. An improvement of 10.3%. The results of this example show that the extract of the ginkgo callus of the present invention can improve human skin to achieve moisturizing and anti-aging effects.

To sum up, the extract of Ginkgo biloba callus of the present invention can improve Tgm1 gene, KRT gene, AQP3 gene, FLG-F gene, GBA gene, HAS gene, CCT gene, Pink1 gene, Atg gene, SIRT1 gene, The expression of NADSYN gene, MRPS5 gene or Ubl-5 gene can improve the skin's moisturizing ability and anti-aging effect, and human experiments have also proved effective. On the other hand, the present invention utilizes the addition of methyl jasmonic acid in the culturing process to generate a large amount of polyphenols and flavonoid equivalent substances, which are used for moisturizing and anti-aging, so that the extract of the ginkgo callus of the present invention is more conducive to industrial utilization .

The above description is exemplary only, not limiting. Any equivalent modifications or changes that do not depart from the spirit and scope of the present invention shall be included in the appended patent application scope.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of Ginkgo biloba callus extract for improving skin redness

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Claims (4)

Use of a Ginkgo biloba callus extract for preparing a composition for improving skin redness, wherein the Ginkgo biloba callus extract uses water as an extraction solvent on the Ginkgo callus at 30 It is prepared by extracting at ℃ to 50 ℃, and the extract of the ginkgo callus contains polyphenols. The use according to claim 1, wherein the ginkgo callus is cultured through a medium supplemented with methyl jasmonic acid (MeJA). The use according to claim 1, wherein the effective concentration of the extract of the ginkgo callus is at least 0.25% (v/v). The use according to claim 1, wherein the composition is a medicine, a food product or a skin care product.

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