Uses of Ginkgo callus extract

Abstract

This invention provides the use of a ginkgo callus extract in the preparation of a composition that reduces the formation of fine lines, wherein the ginkgo callus extract is obtained by extracting the ginkgo callus with water as the extraction solvent, and wherein the ginkgo callus is obtained by taking ginkgo buds as a culture medium.

Description

[0001] This application is a divisional application filed by the applicant based on the parent application (application number: 202010078500.2, invention title: use of ginkgo callus extract and method for culturing ginkgo callus). Technical Field

[0002] This invention relates to an extract of Ginkgo biloba callus tissue, particularly its use in reducing the formation of fine lines. Background Technology

[0003] Skin tissue is composed of the epidermis, dermis, and subcutaneous tissue. The dermis contains a large amount of collagen and hyaluronic acid, which are closely related to the skin's water retention and elasticity. Human skin ages, becomes rough, or develops wrinkles due to age, physiological factors, or environmental factors. For example, the skin of a normal young person has a certain degree of elasticity and tension. When facial muscles relax, the skin recovers quickly, and wrinkles disappear. However, after entering middle age, the skin begins to age significantly, becoming thinner, harder, drier, and less elastic. Dermal collagen decreases, elastic fibers degenerate and break, reducing the skin's tension and elasticity. Therefore, when facial muscles relax, the skin cannot recover quickly, and over time, wrinkles form. Furthermore, with increasing age, the skin and subcutaneous tissue become more relaxed. Coupled with the atrophy or loss of facial supporting tissues and the softening of muscles, the skin will sag under the influence of gravity, forming deeper wrinkles. Rough skin is a skin problem caused by external factors such as dryness, ultraviolet rays, and irritants like cleansers or chemicals, or internal factors such as hormonal imbalances. It is accompanied by a decline in the function of the stratum corneum barrier, a decrease in the moisture content of the stratum corneum, an increased epidermal metabolism, and the production of scales, resulting in roughened skin. Therefore, if skin cells lose their elasticity and moisturizing function, it will cause wrinkles, dryness, and a loss of luster.

[0004] In recent years, the demand for skin moisturizing has been increasing, as improving the skin's moisturizing ability can achieve anti-aging effects. However, most common methods for improving skin moisturizing currently involve applying cosmetics and skincare products to the skin's surface, or taking health supplements that claim to improve skin moisturizing. However, most cosmetics, skincare products, and health supplements are made of chemical ingredients, and long-term use can be harmful to health. Furthermore, these products are often expensive and unaffordable for the average user.

[0005] Furthermore, with the development and progress of medicine and biotechnology in recent years, not only can diseases be combated through cutting-edge medical technology, but many anti-aging products have also been developed and launched. In recent years, the anti-aging trend has gradually spread to the world. For example, in Japan, more than 70% of the population is aware of anti-aging, and the concern of the people of Taiwan for anti-aging has also continued to rise. This anti-aging trend will drive the sales of anti-aging related products, and the global market is expected to continue to expand.

[0006] On the other hand, mitochondria are also known as the cell's power plants because they are the main site of adenosine triphosphate (ATP) synthesis (an energy-transferring molecule) within the cell, providing chemical energy for various cellular activities. Damage to mitochondria has a significant impact on cells and the individual organism. During ATP synthesis, mitochondria generate numerous free radicals. These highly reactive free radicals can react violently with any substance in the body, disrupting its normal function. Over time, free radicals damage enzymes and DNA within mitochondria, gradually impairing their function and leading to the decline of various organ and tissue functions. Therefore, how to enhance mitochondrial activity in cells to achieve anti-aging effects has become an important research topic in this field.

[0007] To address the aforementioned issues, those skilled in the art urgently need to develop novel pharmaceuticals, food products, or skincare products that enhance skin's moisturizing ability and anti-aging effects to benefit the vast population with such needs. Summary of the Invention

[0008] In view of this, the object of the present invention is to provide an extract of Ginkgo biloba callus for use in preparing a composition to reduce the formation of fine lines, wherein the extract of Ginkgo biloba callus is obtained by extracting the Ginkgo biloba callus with water as the extraction solvent, wherein the Ginkgo biloba callus is obtained by taking Ginkgo buds as culture media.

[0009] In one embodiment of the present invention, the extract of Ginkgo callus tissue is used to enhance the following genes: transglutaminase 1 (Tgm1), keratin (KRT), aquaporin 3 (AQP3), filaggrin (FLG-F), glucosylceramidase (GBA), hyaluronan synthase (HAS), chaperonin containing T-complex protein 1 subunit alpha (TCP1) complex (CCT), PTEN-induced kinase 1 (Pink1), autophagy-related (Atg) gene, silencing regulator protein 1 (Sirtuin 1 (SIRT1)) gene, and glutamine-dependent NAD(+) synthase. The use of a combination of expression levels of the NAD(+) synthetase (NADSYN) gene, the mitochondrial ribosomal protein S5 (MRPS5) gene, or the ubiquitin-like protein 5 (Ubl-5) gene, wherein the extract of the Ginkgo callus is prepared by extracting the Ginkgo callus using water, alcohols, hydrous alcohols, or combinations thereof as extraction solvents.

[0010] In one embodiment of the present invention, the KRT gene is the KRT1 gene, the KRT10 gene, or the KRT14 gene.

[0011] In one embodiment of the present invention, the HAS gene is either the HAS2 gene or the HAS3 gene.

[0012] In one embodiment of the present invention, the CCT gene is a chaperon containing TCP1 subunit 2 (CCT2) gene, a chaperon containing TCP1 subunit 5 (CCT5) gene, a chaperon containing TCP1 subunit 6A (CCT6A) gene, a chaperon containing TCP1 subunit 7 (CCT7) gene, or a chaperon containing TCP1 subunit 8 (CCT8) gene.

[0013] In one embodiment of the present invention, the Atg gene is either the Atg1 gene or the Atg8 gene.

[0014] Another object of the present invention is to provide an extract of Ginkgo biloba callus tissue for use in preparing a composition that enhances the skin's moisturizing ability and anti-aging properties, wherein the extract of Ginkgo biloba callus tissue is obtained by extracting the Ginkgo biloba callus tissue with water, alcohols, hydrous alcohols or combinations thereof as extraction solvents.

[0015] In one embodiment of the present invention, the ginkgo callus is obtained by culturing in a medium supplemented with methyljasmonic acid (MeJA).

[0016] In one embodiment of the present invention, the effective concentration of the extract of the ginkgo callus tissue is at least 0.25% (v / v).

[0017] In one embodiment of the invention, the composition is a pharmaceutical product, a food product, or a health care product.

[0018] Another object of the present invention is to provide a method for culturing Ginkgo callus, comprising culturing the Ginkgo callus using a culture medium supplemented with methyl jasmonic acid (MeJA), wherein the concentration of methyl jasmonic acid is at least 0.1 mM.

[0019] In summary, the efficacy of the Ginkgo callus extract of this invention lies in its ability to enhance skin's moisturizing capacity and anti-aging effects by increasing the expression levels of genes such as Tgm1, KRT, AQP3, FLG-F, GBA, HAS, CCT, Pink1, Atg, SIRT1, NADSYN, MRPS5, or Ubl-5. Human trials have also confirmed its effectiveness. Furthermore, this invention utilizes the addition of methyljasmonic acid during the cultivation process to generate abundant polyphenols and flavonoids, which are used for moisturizing and anti-aging, making the Ginkgo callus extract of this invention more suitable for industrial application.

[0020] The following will further illustrate the embodiments of the present invention. The examples listed below are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone skilled in the art can make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims. Attached Figure Description

[0021] Figure 1A This is a data graph showing the efficacy of the extract of Ginkgo callus tissue from the present invention in increasing the total polyphenol content.

[0022] Figure 1B This is a data graph showing the efficacy of the extract of Ginkgo callus tissue from the present invention in increasing the total flavonoid content.

[0023] Figure 2 This is a graph showing the efficacy of the extract of Ginkgo callus tissue of the present invention in enhancing the expression of Tgm1, KRT1, KRT10, KRT14, AQP3, FLG-F, GBA, HAS2 and HAS3 genes related to skin cell hydration after 6 or 24 hours of treatment. In the graph, * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group.

[0024] Figure 3 This is a graph showing the efficacy of the extract of Ginkgo callus tissue from the present invention in promoting the secretion of hyaluronic acid by keratinocytes, where *** indicates a comparison with the control group, p<0.001.

[0025] Figure 4A This is a graph showing the efficacy of the extract of Ginkgo callus tissue 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 treatment. In the graph, * indicates p<0.05 compared with the control group; ** indicates p<0.01 compared with the control group; *** indicates p<0.001 compared with the control group.

[0026] Figure 4B This is a graph showing the efficacy of the extract of Ginkgo callus tissue of the present invention in enhancing the expression of Atg1, Atg8, SIRT1, NADSYN, MRPS5 and Ubl-5 genes related to anti-aging after 24 or 48 hours of treatment. In the graph, * indicates p < 0.05 compared with the control group; ** indicates p < 0.01 compared with the control group; *** indicates p < 0.001 compared with the control group.

[0027] Figure 5 The figures and photographs show the efficacy of the extract of Ginkgo callus tissue in reducing the formation of fine lines, where * indicates a comparison with the control group, p<0.05.

[0028] Figure 6 This is a data graph showing the efficacy of the extract of ginkgo callus tissue from the present invention in increasing skin hydration.

[0029] Figure 7 These are data graphs and images showing the efficacy of the extract of Ginkgo callus tissue from this invention in improving facial redness. Detailed Implementation

[0030] definition

[0031] The values ​​used in this article are approximate. All experimental data are expressed within 20%, the better range is within 10%, and the optimal range is within 5%.

[0032] According to this invention, Ginkgo biloba, also known as maidenhair tree or duck's foot tree, is a deciduous tree belonging to the genus Ginkgo in the family Ginkgoaceae. Native to southern China, Ginkgo has existed for over 200 million years and is the only extant species in the Ginkgo phylum, thus being called a living fossil in the plant kingdom. Ginkgo branches and trunks are resilient, surviving fires and regenerating after adversity. In post-World War II desolate Nagasaki and Hiroshima, Ginkgo trees were the first to sprout new buds, demonstrating its tenacious vitality. Ginkgo is an extremely adaptable and resilient tree species, capable of producing special substances to resist adversity in extreme environments, thus being cold-resistant, heat-resistant, and less susceptible to pests or diseases.

[0033] According to this invention, callus is the primitive, undifferentiated state of a plant, originating from the apical meristem or somatic cells of the stem and root. Callus possesses epigentic activity and totipotency, capable of differentiating into plant embryonic cells to form new plants. It also aids in cell metabolism and regeneration, delays aging, and enhances vitality. Modern plant tissue culture methods have successfully developed a technique for callus proliferation; however, for different plant varieties, detailed experiments are still needed to determine suitable culture formulas to produce commercially viable plant callus for industrial use.

[0034] As used in this article, the terms "extract of ginkgo callus tissue" and "ginkgo stem cells" can be used interchangeably.

[0035] As used in this article, the term "anti-aging" refers to preventing and slowing down the signs of aging in human skin, such as wrinkles and loss of elasticity. The degree to which this goal is achieved will be assessed based on numerous factors known to those skilled in the art, such as the consumer's overall health, age, and gender.

[0036] According to the present invention, pharmaceutical products can be manufactured using techniques known to those skilled in the art into dosage forms suitable for parenterally, orally, or topically administration. These include, but are not limited to, injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, external preparations, and the like.

[0037] According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may comprise one or more reagents selected from the following: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents fall within the scope of professional competence and routine practice of those skilled in the art.

[0038] According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of: water, normal saline, phosphate buffered saline (PBS), an aqueous solution containing alcohol, and combinations thereof.

[0039] According to the present invention, the pharmaceutical product can be administered via a non-parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0040] According to the present invention, pharmaceutical products can be manufactured using techniques known to those skilled in the art into external preparations suitable for topical application to the skin, including, but not limited to: emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, salves, and bandages.

[0041] According to the present invention, the external formulation is prepared by mixing the pharmaceutical product of the present invention with a base known to those skilled in the art.

[0042] According to the present invention, the base material may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white petrolatum], waxes [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers, stabilizing agents, and gelling agents [such as carbomer]. ® 974P (carbopol ®The additives include 974P, microcrystalline cellulose, and carboxymethyl cellulose; active agents; humectants; odor absorbers; fragrances; pH adjusting agents; chelating agents; emulsifiers; occlusive agents; emollients; thickeners; solubilizing agents; penetration enhancers; anti-irritants; colorants; and propellants. The selection and quantity of these additives fall within the scope of professional expertise and routine techniques of those familiar with this technology.

[0043] According to the present invention, the skincare product may further comprise an acceptable adjuvant that is widely used in skincare product manufacturing techniques. For example, the acceptable adjuvant may comprise one or more agents selected from the following: solvents, gelling agents, surfactants, preservatives, antioxidants, screening agents, chelating agents, surfactants, coloring agents, thickening agents, fillers, fragrances, and odor absorbers. The selection and quantity of these agents fall within the scope of professional competence and routine practice of those skilled in the art.

[0044] According to the present invention, skincare products can be manufactured in a form suitable for skincare or makeup using techniques known to those skilled in the art. This includes, but is not limited to: aqueous solutions, aqueous-alcohol solutions or oily solutions, oil-in-water type, water-in-oil type or compound emulsions, gels, ointments, creams, masks, patches, packs, liniments, powders, aerosols, sprays, lotions, emulsions, pastes, foams, dispersions, drops, mousses, sunblocks, tonic water, foundations, makeup remover products, soaps and other body cleansing products.

[0045] According to the present invention, the skincare product may also be used in combination with one or more external use agents selected from the following known active ingredients: whitening agents [such as tretinoin, catechin, kojic acid, arbutin, and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, and antiseborrheic agents. Topical agents, wound-healing agents, corticosteroids, and hormones. The selection and quantity of these topical agents fall within the scope of the professional competence and routine techniques of those skilled in this field.

[0046] According to the present invention, food products can be used as food additives, added during the preparation of raw materials or during the production of food by conventional methods, and formulated with any edible material to form food products for human and non-human animal consumption.

[0047] According to this invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

[0048] Example 1. Preparation of extracts from Ginkgo callus tissue

[0049] First, ginkgo buds are used as culture media, and the ginkgo is sterilized to remove microorganisms from the plant surface. Wounds can be created in the ginkgo in any way, such as by cutting, tearing, or slicing, to obtain ginkgo with wounds, and callus tissue is formed on the wounds.

[0050] Methyl jasmonic acid (MeJA) was dissolved in dimethyl sulfoxide to prepare a molecular weight of 0.1 μmol for later use. Next, 5 mL of the 0.1 mM methyl jasmonic acid solution was uniformly dropped onto a culture medium containing Ginkgo callus (MS medium (Murashige and Skoog), supplemented with 0.5 mg / L 1-naphthaleneacetic acid (NAA), 0.1 mg / L 6-benzylaminopurine, 3% sucrose, and 0.8% agar, adjusted to pH 5.8 or higher). After one week of cultivation, the cultured Ginkgo callus was harvested for subsequent extraction. Next, the harvested ginkgo callus tissue was homogenized, and then extracted with water, alcohol, hydrous alcohol, or a combination thereof, with water being the preferred solvent, for 20 minutes. The volume ratio of the homogenized ginkgo callus tissue to the extraction solvent was 0.8-1.2:8-12 (preferably 1:10), and the extraction temperature was between 30°C and 50°C. Afterward, the mixture was cooled to room temperature, and then filtered through a 400-mesh filter to obtain the extract of ginkgo callus tissue of this invention.

[0051] Example 2. Evaluation of the efficacy of Ginkgo callus extract obtained by culturing in a medium supplemented with methyljasmonic acid in increasing the content of total polyphenols and total flavonoids.

[0052] First, standard solutions were prepared by dissolving 10 g of gallic acid in water and adding 10 mL to a volumetric 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, and 100 µL of each standard solution was transferred to a 10 mL centrifuge tube. Then, 500 µL of Folin-Ciocalteu's phenol reagent was added, mixed, and incubated upright for 3 minutes. Following this, 400 µL of 7.5% sodium carbonate was added, mixed, and incubated upright for 30 minutes. Finally, 200 µL of each reaction solution was transferred to a 96-well plate, and the absorbance was measured at 750 nm.

[0053] In addition, the extract of Ginkgo callus obtained in Example 1 was used as the experimental group, and Ginkgo callus cultured without the addition of methyljasmine acid was used as the control group. Both the experimental and control groups were diluted with water, and 100 mL volumes were transferred to microcentrifuge tubes. Then, 500 µL of florfenicol reagent was added, mixed, and incubated upright for 3 minutes. Next, 400 µL of 7.5% sodium carbonate was added, mixed, and incubated upright for 30 minutes. Then, 200 µL of each reaction solution was transferred to a 96-well plate, and the absorbance was measured at 750 nm. The results of the total polyphenol content are shown in [data missing]. Figure 1A .

[0054] Figure 1A This is a data graph showing the efficacy of the extract from Ginkgo biloba callus tissue in increasing the total polyphenol content. Figure 1A As can be seen, the total polyphenol content of the experimental group was significantly increased by 1.6 times compared with the control group. The results of this embodiment show that the extract of Ginkgo callus obtained by culturing in a medium supplemented with methyljasmonic acid can effectively increase the total polyphenol content.

[0055] In addition, the experimental procedure for detecting total flavonoid content is as follows: the total flavonoid content is expressed as the rutin equivalent (ChromaDex ASB-00018440). The prepared 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 (methanol solution).

[0056] Take 0, 200 μL, 400 μL, 600 μL, 800 μL, 1000 μL, and 1200 μL of the above rutin standard solution and add them to test tubes respectively. Then, add 1200 μL, 1000 μL, 800 μL, 600 μL, 400 μL, 200 μL, and 0 μL of water sequentially, and shake to mix thoroughly. Take 200 μL of each concentration of rutin solution, add 200 μL of 5% sodium citrate, mix thoroughly, and let stand for 6 minutes; add 200 μL of 10% aluminum nitrate, mix thoroughly, and let stand for 6 minutes; then add 2 mL of 4% sodium hydroxide, mix thoroughly, and finally add 1.4 mL of H2O and mix thoroughly. Take 200 μL of the above reaction solution in a 96-well reaction dish, detect the absorbance at 500 nm using a spectrophotometer, and plot a standard curve.

[0057] The extract of Ginkgo callus obtained in Example 1 was used as the experimental group, and Ginkgo callus cultured without the addition of methyljasmine acid was used as the control group. After appropriate dilution, 200 μL of the sample from either the experimental or control group was placed in a test tube, and 200 μL of 5% sodium citrate was added. After mixing thoroughly, the mixture was allowed to stand for 6 minutes; then 200 μL of 10% aluminum nitrate was added, mixed thoroughly, and allowed to stand for 6 minutes; finally, 2 mL of 4% sodium hydroxide was added and mixed thoroughly, followed by 1.4 mL of H2O and mixing thoroughly. 200 μL of the above reaction solution was placed in a 96-well reaction dish, and the absorbance was measured at 500 nm using a spectrophotometer. The results of the total flavonoid content are shown below. Figure 1B .

[0058] Figure 1B This is a data graph showing the efficacy of the extract from Ginkgo biloba callus tissue in increasing the total flavonoid content. (From...) Figure 1B As can be seen, the total flavonoid content in the experimental group was significantly increased by 2.3 times compared to the control group. The results of this embodiment show that the extract of Ginkgo callus obtained by culturing in a medium supplemented with methyljasmonic acid can effectively increase the total flavonoid content.

[0059] Example 3. Evaluation of the efficacy of Ginkgo callus extract in enhancing skin's moisturizing ability.

[0060] This embodiment explores whether extracts from ginkgo callus tissue can enhance the skin's moisturizing ability by increasing the expression of genes related to skin cell moisturization.

[0061] Human epidermal keratinocytes (HPEK-50; purchased from CELLnTEC) were cultured in serum-free keratinocyte-specific medium (Keratinocyte-SFM; purchased from Thermo, product number: 17005042) in 6-well plates. The cell concentration was 1 × 10⁶ cells / well in 2 mL of medium. 5 Cells / pores.

[0062] The cells were then divided into three groups: a control group and two experimental groups (groups 1 and 2). Extracts from Ginkgo callus tissue were diluted with culture medium to 0.25% (v / v) and 0.5% (v / v) concentrations. The 0.25% dilution was added to cells in group 1, and the 0.5% dilution was added to cells in group 2, respectively. The control group cells (HPEK-50) were added only with culture medium. The cells were then cultured in an incubator for 6 or 24 hours. Cell cultures were then collected for gene expression analysis.

[0063] In this embodiment, the genes used to analyze skin cell moisturizing include transglutaminase 1 (Tgm1) gene, keratin 1 (KRT1) gene, KRT10 gene, KRT14 gene, aquaporin 3 (AQP3) gene, filaggrin (FLG-F) gene, glucosylceramidase (GBA) gene, hyaluronan synthase 2 (HAS2) gene, and HAS3 gene.

[0064] RNA was extracted from the cell cultures obtained above using an RNA extraction kit (Geneaid). 2,000 ng of each RNA sample was then processed using SuperScript. ® III reverse transcriptase (Invitrogen) reverse transcribed the extracted RNA into cDNA. Then, using the cDNA as a template, and primer pairs for amplifying the target gene, including Tgm1, KRT1, KRT10, KRT14, AQP3, FLG-F, GBA, HAS2, HAS3, and TBP (as an internal control), whose 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. The melting curve of the PCR product was confirmed during the quantitative real-time PCR reaction.

[0065] Table 1

[0066]

[0067] The relative expression level of the target gene is derived from Equation 2. -△△Ct The relative fold change was calculated using the cycle thresholds of the TBP gene (as an internal control group) and the baseline gene, as well as the standard deviation, where ΔCt = Ct. 目标基因 / 基准基因 -Ct TBP , △△Ct=△Ct 目标基因 -△Ct 基准基因 The multiple change = 2 -△△Ct 平均值 The target gene expression level in the control group was used as the baseline for comparison. Statistically significant differences between groups were determined using a one-tailed Student's t-test. The results of this embodiment are shown in... Figure 2 .

[0068] Figure 2 This is a graph showing the efficacy of the extract from Ginkgo biloba callus tissue of the present invention in enhancing the expression of Tgm1, KRT1, KRT10, KRT14, AQP3, FLG-F, GBA, HAS2, and HAS3 genes related to skin cell hydration after 6 or 24 hours of treatment. Figure 2 As can be seen, compared with the control group (i.e., HPEK mock), the relative expression levels of Tgm1, KRT14, FLG-F, GBA, HAS2, and HAS3 genes in experimental groups 1 and 2 were significantly increased at 6 or 24 hours of treatment. Regarding the KRT1 gene, compared with the control group, except for experimental group 2 which was treated for 6 hours, the relative expression levels of experimental group 1 were significantly increased at 6 and 24 hours of treatment, and experimental group 2 at 24 hours of treatment. Regarding the KRT10 gene, compared with the control group, except for experimental group 2 which was treated for 24 hours, the relative expression levels of experimental group 1 were significantly increased at 6 and 24 hours of treatment, and experimental group 2 at 6 hours of treatment. Regarding the AQP3 gene, compared with the control group, except for experimental group 2 which was treated for 24 hours, the relative expression levels of experimental group 1 were significantly increased at 6 and 24 hours of treatment, and experimental group 2 at 6 hours of treatment. The results of this embodiment show that the extract of Ginkgo callus tissue of the present invention can maintain the arrangement of keratinocytes by increasing the expression of Tgm1, KRT1, KRT10, KRT14, AQP3, FLG-F, GBA, HAS2 and HAS3 genes related to skin cell moisturization, thereby maintaining the integrity of the keratinocyte tissue, increasing skin hydration, enhancing the synthesis of hyaluronic acid by keratinocytes, effectively locking in moisture for the skin, filling the lipid gaps between cells of the stratum corneum, and thus improving the skin's moisturizing ability.

[0069] Example 4. Evaluation of the efficacy of Ginkgo callus extract in promoting hyaluronic acid secretion by keratinocytes

[0070] This embodiment further tests the effect of the extract of Ginkgo callus tissue of the present invention on promoting the secretion of hyaluronic acid by keratinocytes. Since keratinocytes are known to secrete substances such as hyaluronic acid as intercellular matrix to maintain the integrity of the epidermal barrier, prevent skin moisture loss, and form a complete protective layer, therefore... First, in a 96-well culture dish, 200 μL of serum-free keratinocyte culture medium (Keratinocyte-SFM; purchased from Thermo, product number: 17005042) was added to each well, and 1x10 cells were implanted... 4 Human epidermal keratinocytes (HPEK-50; purchased from CELLnTEC) / well, then at 37°C o C culture overnight.

[0071] The cells were then divided into three groups: a control group and two experimental groups (i.e., experimental groups 1 and 2). Extracts from Ginkgo callus tissue were diluted with culture medium to 0.5% (v / v) and 1% (v / v) concentrations. The 0.5% dilution was added to cells in experimental group 1, and the 1% dilution was added to cells in experimental group 2, respectively. The control group cells were left untreated. (37) o After culturing at C for 24 hours, 100 μL of culture medium was collected from each well without disturbing the attached cells.

[0072] Next, analysis was performed using an ELISA kit for human hyaluronic acid (HA) (purchased from Cusabio Biotech, China, product number CSB-E04805h). First, 100 μL of culture medium collected from each well was added to a 96-well culture dish containing a layer of human hyaluronic acid capture antibody at the bottom. Alternatively, a standard dissolved in phosphate buffer solution containing 1% bovine serum albumin was added. The mixture was incubated at 37°C. o The antibody was incubated at C for 2 hours to bind with the capture antibody. After the incubation period, the liquid was removed, and 100 μL of the detection antibody (biotin-antibody (1X)) was added directly to each well. The mixture was then incubated at 37°C. o Detect and capture antibodies at C for 1 hour. Then, aspirate from each well and wash, repeating this process twice, for a total of three washes. Afterward, use a multi-channel pipette to fill each well with washing buffer (200 μL) and let it stand for 2 minutes. Complete removal of liquid at each step is essential for good performance. After the final wash, remove any remaining washing buffer by aspiration or decanting. Invert the culture dish and dry with a clean paper towel. Then, add 100 μL of horseradish peroxidase-avidin (HRP-avidin) to each well at 37°C. o Incubate at C for 1 hour, then repeat the aspiration / washing procedure 5 times. Next, add 90 μL of TMB acceptor colorimetric solution and incubate at 37°C. o Incubate at C for 15-30 minutes in the dark, then add 50 μL of stop solution to each well to halt the reaction. Gently tap the culture dish to ensure thorough mixing. Finally, measure the absorbance at 450 nm within 5 minutes using an enzyme immunoassay analyzer (BioTek). Perform a Student's t-test using Excel software to determine if the coefficient of variation is statistically significant. Experimental results are shown in... Figure 3 .

[0073] Figure 3This is a data graph showing the efficacy of the extract from Ginkgo biloba callus tissue in promoting hyaluronic acid secretion by keratinocytes. Figure 3 As can be seen, compared with the control group, the hyaluronic acid production in both experimental groups 1 and 2 was significantly increased, with an increase of 30.8% in experimental group 1 and 27.5% in experimental group 2. The results of this embodiment show that the extract of Ginkgo callus tissue of the present invention can effectively promote the secretion of hyaluronic acid by keratinocytes, effectively maintain the integrity of the stratum corneum structure, enhance the skin barrier function, and improve the skin's water retention capacity.

[0074] Example 5. Evaluation of the anti-aging efficacy of extracts from Ginkgo callus tissue.

[0075] This embodiment explores whether extracts from Ginkgo callus tissue can achieve anti-aging effects by increasing the expression of genes related to anti-aging.

[0076] First, human myeloma SHSY-5Y (TCC) was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin / streptomycin (Gibco). ® CRL-2266 ™ The cells were placed in a 6-well plate with 2 mL of culture medium at a concentration of 1 × 10⁻⁶ cells / well. 5 Cells / pores.

[0077] The cells were then divided into three groups: a control group and two experimental groups (groups 1 and 2). Extracts from Ginkgo callus tissue were diluted with culture medium to 0.5% (v / v) and 1% (v / v) concentrations. The 0.5% dilution was added to cells in group 1, and the 1% dilution was added to cells in group 2, while the control group cells were simply added to the culture medium. The cells were then cultured in an incubator for 24 or 48 hours. Cell cultures were then collected and used for gene expression analysis.

[0078] In this embodiment, the genes used to analyze anti-aging related genes include the following: chaperonin containing TCP1 subunit 2 (CCT2), chaperonin containing TCP1 subunit 5 (CCT5), chaperonin containing TCP1 subunit 6A (CCT6A), chaperonin containing TCP1 subunit 7 (CCT7), chaperonin containing TCP1 subunit 8 (CCT8), PTEN-induced kinase 1 (Pink1), autophagy-related protein 1 (Atg1), autophagy-related protein 8 (Atg8), and silencing regulator protein 1 (Sirtuin 1). 1. The SIRT1 gene, glutamine-dependent NAD(+) synthetase (NADSYN) gene, mitochondrial ribosomal protein S5 (MRPS5) gene, and ubiquitin-like protein 5 (Ubiquitin-like protein 5) gene. The Ubl-5 gene contains several genes, including the NADSYN gene, which assists in the synthesis of NAD, providing energy to the mitochondria and maintaining their vitality; the SIRT1 gene, which assists in the repair of damaged DNA in the mitochondria and slows down aging; the Atg1 gene, which is related to mitochondrial rejuvenation and anti-aging; the Atg8 gene, which clears mutated DNA to rejuvenate the mitochondria and restore their youthful vitality; the MRPS5 gene, which assists in the synthesis of mitochondrial proteins and provides energy; the CCT2, CCT5, CCT6A, CCT7, and CCT8 genes, which reverse the aging process of mature cells back to young cells; the Pink1 gene, which restores aging mitochondria to a youthful state; and the Ubl-5 gene, which restores mitochondrial activity. Animal experiments have shown that it can restore aged mice to a youthful state.

[0079] RNA was extracted from the cell cultures obtained above using an RNA extraction kit (Geneaid). 2,000 ng of each RNA sample was then processed using SuperScript. ®III reverse transcriptase (Invitrogen) reverse transcribed the extracted RNA into cDNA. Then, using the cDNA as a template, and primer pairs for amplifying the target gene, including CCT2, CCT5, CCT6A, CCT7, CCT8, Pink1, Atg1, Atg8, SIRT1, NADSYN, MRPS5, Ubl-5, and GAPDH (as an internal control), whose 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 amplify and quantify the target gene. The melting curve of the PCR product was confirmed during the quantitative real-time PCR reaction.

[0080] Table 2

[0081]

[0082]

[0083] The relative expression level of the target gene is derived from Equation 2. -△△Ct The relative fold change was calculated using the cycle thresholds of the GAPDH gene (as an internal control group) and the baseline gene, and by standard deviation, where ΔCt = Ct 目标基因 / 基准基因 -Ct GAPDH , △△Ct=△Ct 目标基因 -△Ct 基准基因 The multiple change = 2 -△△Ct 平均值 The target gene expression level in the control group was used as the baseline for comparison. Statistically significant differences between groups were determined using a one-tailed Student's t-test. The results of this embodiment are shown in... Figure 4A and Figure 4B .

[0084] Figure 4A This is a graph showing the efficacy of the extract from Ginkgo biloba callus tissue 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 treatment. Figure 4A It is evident that, compared to the control group, the relative expression levels of CCT2, CCT5, CCT6A, CCT8, and Pink1 genes in experimental groups 1 and 2 were significantly increased at 48 hours of treatment. Regarding the CCT7 gene, compared to the control group, except for experimental group 2, experimental group 1 showed a significant increase in relative expression at 48 hours of treatment.

[0085] Figure 4B This is a graph showing the efficacy of the extract from Ginkgo biloba callus tissue of the present invention in enhancing the expression of Atg1, Atg8, SIRT1, NADSYN, MRPS5, and Ubl-5 genes related to anti-aging after 24 or 48 hours of treatment. Figure 4B As can be seen, for the Atg1, SIRT1, and MRPS5 genes, compared with the control group, the relative expression levels of experimental groups 1 and 2 were significantly increased at 24 or 48 hours of treatment. Regarding the Atg8 gene, compared with the control group, except for experimental group 1 which was treated for 24 hours, the relative expression levels of experimental group 1 were significantly increased at 48 hours of treatment, and experimental group 2 at 24 or 48 hours of treatment. Regarding the NADSYN gene, compared with the control group, except for experimental group 1 which was treated for 48 hours, the relative expression levels of experimental group 1 were significantly increased at 24 hours of treatment, and experimental group 2 at 24 or 48 hours of treatment. Regarding the Ubl-5 gene, compared with the control group, except for experimental group 1 which was treated for 24 hours, the relative expression levels of experimental group 1 were significantly increased at 48 hours of treatment, and experimental group 2 at 24 or 48 hours of treatment. The results of this embodiment show that the extract of Ginkgo callus tissue of the present invention can enhance mitochondrial activity in multiple ways by increasing the expression of genes related to anti-aging, such as CCT2, CCT5, CCT6A, CCT7, CCT8, Pink1, Atg1, Atg8, SIRT1, NADSYN, MRPS5 and Ubl-5, thereby strengthening the source of skin vitality and achieving anti-aging effects.

[0086] Example 6. Human efficacy test of extracts from Ginkgo callus tissue

[0087] In this embodiment, the extract of ginkgo callus tissue prepared in Example 1 was used to test whether it has the effect of improving human skin.

[0088] First, eight participants were recruited. Each participant used their left cheek as a control group and their right cheek as an experimental group. After cleansing their face morning and evening, a placebo was applied to the control group's skin, while a 1% extract of ginkgo callus tissue was applied as a ginkgo stem cell essence to the experimental group's skin. The skin was gently massaged with fingertips to promote absorption. Tests were performed before use (week 0) and at week 4 or 15 minutes after use. Tests included skin wrinkles, skin hydration, and redness. Skin wrinkles and hydration were measured at week 4 after use, while redness was measured 15 minutes after use. The results of this embodiment are shown in [the table / image / etc.]. Figures 5 to 7 .

[0089] Figure 5 The data and photographs show the efficacy of the extract of Ginkgo callus tissue in reducing the formation of fine lines. Figure 6 This is a data graph showing the efficacy of the extract of ginkgo callus tissue from the present invention in increasing skin hydration. Figure 7 These are data graphs and images showing the efficacy of the extract of Ginkgo biloba callus tissue from this invention in improving facial redness. Figure 5 As can be seen, compared to week 0, the experimental group showed a significant reduction in fine lines over time, while the control group showed no reduction; compared to week 0, the experimental group experienced a 13% reduction in fine lines by week 4 after use. Figure 6 As can be seen, compared to week 0, the skin hydration level of the experimental group increased over time. Specifically, compared to week 0, the skin hydration level of the experimental group increased by 13.3% at week 4 after use. Figure 7 As can be seen, compared with before use, the facial redness in the experimental group improved within 15 minutes after use, while the control group did not; compared with before use, the skin redness in the experimental group improved by 10.3% within 15 minutes after use. The results of this embodiment show that the extract of Ginkgo callus tissue of the present invention has the effect of improving human skin to achieve moisturizing and anti-aging effects.

[0090] In summary, the extract from Ginkgo callus tissue of this invention can enhance skin's moisturizing ability and anti-aging effects by increasing the expression levels of genes such as Tgm1, KRT, AQP3, FLG-F, GBA, HAS, CCT, Pink1, Atg, SIRT1, NADSYN, MRPS5, or Ubl-5, and human experiments have also confirmed its effectiveness. Furthermore, this invention utilizes the addition of methyljasmonic acid during the cultivation process to generate a large amount of polyphenols and flavonoids, which are used for moisturizing and anti-aging, making the extract from Ginkgo callus tissue of this invention more suitable for industrial application.

[0091] The above description is illustrative only and not restrictive. Any equivalent modifications or alterations made without departing from the spirit and scope of this invention should be included in the appended claims.

Claims

1. Use of an extract of Ginkgo biloba callus for preparing a composition to reduce the formation of fine lines, wherein the extract of Ginkgo biloba callus is obtained by extracting the Ginkgo biloba callus with water as the extraction solvent, wherein the Ginkgo biloba callus is obtained by taking Ginkgo buds as a culture medium.

2. The use as described in claim 1, wherein the extract of the ginkgo callus tissue is used to promote the secretion of hyaluronic acid.

3. The use as described in claim 1, wherein the ginkgo callus is obtained by culturing in a medium supplemented with methyljasmonic acid.

4. The use as described in claim 3, wherein the concentration of methyljasmine acid in the culture medium of methyljasmine acid is at least 0.1 mM.

5. The use as described in claim 4, wherein the culture medium is prepared by adding 0.5 mg / L of 1-naphthacetic acid, 0.1 mg / L of 6-benzylaminopurine, 3% sucrose and 0.8% agar to MS medium.

6. As described in claim 3 or 4, the culture time of the Ginkgo callus is one week.

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