TWI743393B - Use of extract of rhinacanthus communis callus tissue for promoting collagen production - Google Patents

Abstract

The present disclosure provides a use of an extract of the Rhinacanthus communis callus tissue for inducing expression of collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene, TGM gene and AQP gene, promoting collagen production, and anti-oxidation. The present disclosure also provides a method for inducing and proliferating the Rhinacanthus communis callus tissue, and a medium for culturing the Rhinacanthus communis callus tissue.

Description

White Crane Ganoderma lucidum callus extract is used to promote collagen proliferation way

The present invention relates to an extract of white crane ganoderma (Rhinacanthus communis ) callus used to enhance collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene, TGM The expression of genes, AQP genes and GBA genes, the use of promoting collagen production and anti-oxidation, a method for inducing and proliferating Baihe Ganoderma lucidum callus, and a medium for cultivating Baihe Ganoderma lucidum 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 suffer from aging, rough skin or wrinkles with age, physiological factors or environmental factors. For example, the skin of normal young people has a certain degree of elasticity and tension. When the expression muscles relax, the skin will quickly become Recover and make wrinkles disappear; but after entering middle age, the skin begins to age significantly, the skin becomes thinner, harder, dry, and tension reduced; dermal collagen decreases, elastic fibers degeneration, and breakage, which reduces the tension and elasticity of the skin. Therefore, When the expression muscles relax, the skin cannot recover quickly, and wrinkles are formed over time. With the increase of age, the skin and subcutaneous tissues become more relaxed, coupled with the atrophy or loss of facial support tissues, and the softness of muscles, the skin will become weaker. Slippage occurs under the action of gravity, forming deeper wrinkles. Rough skin is caused by externally important factors such as dryness, ultraviolet rays, cleansers, chemical substances and other irritating substances, or internal important factors such as hormonal balance disorder, and is accompanied by a decline in the barrier function of the stratum corneum. Phenomenon such as the decrease of water content, the increase of epidermal metabolism, the production of scales and the roughening of keratin. Therefore, if the cells on the skin lose their elasticity and moisturizing function, it will cause skin wrinkles, dryness, and loss of luster.

At present, most common methods used to solve skin problems are to use cosmetics and skin care products applied to the surface of the skin, or oral health foods with antioxidant effects. However, most of the conventional cosmetics, skin care products and health foods are 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 order to solve the above-mentioned problems, those skilled in the art urgently need to develop novel medicines, cosmetics or skin care products with the effects of promoting collagen production, anti-oxidation, moisturizing, and anti-aging, so as to benefit the majority of people in need.

In view of this, the purpose of the present invention is to provide an extract of white crane ganoderma lucidum (Rhinacanthus communis ) callus for preparing a collagen type I (collagen type I) gene, elastin (ELN) gene, hyaluronic acid Synthase 3 (hyaluronan synthase 3, HAS3) gene, X-ray repair cross complementary protein 1 (XRCC1) gene, MSH gene, superoxide dismutase 1 (superoxide dismutase 1, SOD1) gene , Glutathione peroxidase 1 (glutathione peroxidase 1, GPX1) gene, chloramphenicol acetyltransferase (CAT) gene, transglutaminase (TGM) gene, aquaporin (aquaporin, AQP) gene, and the use of GBA gene expression in medicine, cosmetics or skin care products. Among them, the extract of Siberian Ganoderma lucidum callus uses water or alcohol as the extraction solvent to extract Siberian Ganoderma lucidum callus. In the preparation, the volume ratio of the white crane ganoderma lucidum callus to the extraction solvent is between 1:5 and 1:10, and the extraction temperature is between 60°C and 80°C.

Another object of the present invention is to provide a white crane ganoderma lucidum callus extract for the preparation of a medicine, cosmetics or skin care products that promote collagen production and antioxidant, wherein the white crane ganoderma lucidum callus extract is based on Water or alcohol is used as the extraction solvent to extract the callus of Baihe Ganoderma lucidum. The volume ratio of the callus of Baihe Ganoderma lucidum to the extraction solvent is between 1:5 and 1:10, and the extraction temperature is between 60°C and 80°C. ℃.

Another object of the present invention is to provide a method for inducing and proliferating the callus of a white crane Ganoderma lucidum, which comprises using an addition of 2,4-dichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid) and 6-aniline purine ( 6-benzyladenine) MS medium (Murashige and Skoog medium) to cultivate Baihe Ganoderma lucidum callus.

Another object of the present invention is to provide a culture medium for cultivating callus of a white crane ganoderma lucidum, which contains 2,4-dichlorophenoxyacetic acid and 6-aniline purine.

In an embodiment of the present invention, Ganoderma lucidum callus is cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-aniline purine.

In an embodiment of the present invention, the MS medium contains 3% sucrose and 0.8% agar.

In an embodiment of the present invention, the collagen type I gene is collagen type I, α 1 (collagen type I, alpha 1, COL1A1) gene or collagen type I, α 2 (collagen type I, alpha 1, 2, COL1A2) gene.

In an embodiment of the present invention, the MSH gene is MSH2 gene or MSH6 gene.

In an embodiment of the present invention, the TGM gene is TGM1 gene.

In an embodiment of the present invention, the AQP gene is AQP3 gene.

In an embodiment of the present invention, the concentration of 2,4-dichlorophenoxyacetic acid is 0.5 mg/L to 5 mg/L, and the concentration of 6-aniline purine is 0.1 mg/L to 1 mg/L.

In an embodiment of the present invention, the callus of Baihe Ganoderma lucidum has a growth amount of at least 4 times.

In an embodiment of the present invention, the medium is an MS medium.

In summary, the effect of the white crane Ganoderma lucidum callus extract of the present invention is that it can enhance the collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene. , TGM gene, AQP gene and GBA gene expression level to achieve moisturizing and anti-aging effect; promote skin cell collagen production; and achieve anti-oxidation effect by reducing skin damage caused by oxidation. On the other hand, the present invention uses a changed MS medium formula to achieve the effect of cultivating a large number of Siberian Crane Ganoderma lucidum callus, making the Siberian Crane Ganoderma lucidum callus extract of the present invention more favorable for industrial use.

The following will further explain the embodiments of the present invention. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with this technique will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the appended patent scope.

Fig. 1 is a data diagram of the effectiveness of the extract of Baihe Ganoderma lucidum callus of the present invention in promoting the production of collagen, where "*" means compared with the control group, p <0.05.

Figure 2 is a data diagram of the anti-oxidation effect of the white crane Ganoderma lucidum callus extract of the present invention on skin cells, where "***" means compared with the hydrogen peroxide group, p <0.001.

Fig. 3 is a data diagram showing the efficacy of the Ganoderma lucidum callus extract of the present invention in regulating the expression of genes related to skin cell elasticity (including COL1A1 gene, COL1A2 gene, ELN gene and HAS3 gene).

Figure 4 is the data on the efficacy of the Ganoderma lucidum callus extract of the present invention in regulating the expression of genes related to skin cell resistance to photoaging (including XRCC1 gene, MSH2 gene, MSH6 gene, SOD1 gene, GPX1 gene and CAT gene) picture.

Fig. 5 is a data chart of the efficacy of the callus extract of Siberian Crane Ganoderma lucidum in regulating the expression of TGM1 gene of the present invention.

Fig. 6 is a data chart of the efficacy of the extract of the callus of the white crane ganoderma lucidum of the present invention in regulating the expression of the AQP3 gene.

Fig. 7 is a data diagram showing the efficacy of the extract of the callus of the white crane ganoderma lucidum of the present invention in regulating the expression of the GBA gene and the HAS3 gene.

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%.

According to the present invention, Rhinacanthus communis , also known as Agrimonia or Ganoderma lucidum, is a shrub plant of Acanthaceae and Rhinacanthus . It is a perennial and is also a precious Chinese medicinal material. It is native to China. The Yunnan-Guizhou Mountains are mostly distributed in plains and wastelands, suitable for warm and humid environments. Wild Siberian Ganoderma lucidum contains many active ingredients, including lupeol, β-sitosterol, stigmasterol and Rhinacanthin A. Baihe Lingzhi is known to be used to treat hypertension, diabetes, liver disease, and tuberculosis.

According to the present invention, callus is the original undifferentiated state of the plant, derived from apical meristem or somatic cells of stems and roots. Callus has epigentic effects and totipotent differentiation ability (totipotent), which can differentiate into plant embryo cells to form new plants, and has the functions of helping cell metabolism to regenerate, delaying aging, and imparting vitality. In modern times, plant tissue culture methods have been used to successfully develop a set of plant callus proliferation techniques. However, for different varieties of plants, detailed experiments have to be conducted to find suitable cultivation formulas before they can produce useful plant calluses. Injury organizations and provide industrial use.

As used herein, the term "anti-aging" means to prevent and slow down the appearance of aging of human skin, such as the generation of wrinkles and loss of elasticity. The assessment of the extent to which this goal is achieved will be determined based on many factors known to those familiar with the art, such as the general state of the consumer, age, gender, etc.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally or topically by using techniques well known to those skilled in the art. This includes, but is not limited to: injections (injection) [e.g., sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.

According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents 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 agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

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

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection Injection (intradermal injection) and intralesional injection (intralesional injection).

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well-known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation 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 medicine of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and white Vaseline (white petrolatum), wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], the activity of Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occluding agents occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants ( propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine technology of those who are familiar with this technology.

According to the present invention, the cosmetic may further include a cosmetically acceptable adjuvant which is widely used in cosmetic manufacturing technology. For example, the cosmetically 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, and surfactants. , Coloring agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, cosmetics can be manufactured into a form suitable for skincare or makeup by using techniques well known to those skilled in the art, which include, but are not limited to: aqueous solution (aqueous solution), water- Aqueous-alcohol solution or oily solution, oil-in-water type, water-in-oil type or compound emulsion, gel , Ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, emulsion, paste, foam, dispersion, drops, mousse ), sunblock, tonic water, foundation, makeup remover products, soap, and other body cleansing products.

According to the present invention, cosmetics can also be used in combination with one or more external use agents of known activity selected from the following: whitening agents (such as tretinoin, catechin ( catechin), kojic 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-dermatitis ( antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, anti-wrinkle agents Antiseborrheic agents, wound-healing agents, corticosteroids and hormones. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

Example 1. Preparation of extract of Baihe Ganoderma lucidum callus

First, take Baihe Ganoderma lucidum as a culture body and sterilize Baihe Ganoderma lucidum to remove microorganisms on the surface of the plant. The white crane Ganoderma lucidum can be wounded in any way. For example, it can be cut, teared or cut to obtain White Crane Ganoderma in the wound.

Next, using MS medium, add 0.5~5mg/L of 2,4-dichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid), 0.1~1mg/L of 6-benzyladenine, 3% sucrose And 0.8% agar to MS medium, and mix evenly. Afterwards, adjust the pH value to 5.7~5.8 with 0.1N sodium hydroxide solution, and then carry out the callus proliferation culture of Baihe Ganoderma lucidum. After a 30-day culture cycle, 4 times the growth of the callus can be obtained.

Then, the cultured Siberian Crane Ganoderma lucidum callus was dried with a freeze dryer at -40°C for 16 hours, and then the product was collected to ensure that the moisture content was less than 5%, to obtain a dried Siberian Crane Ganoderma lucidum callus. Afterwards, the dried Siberian Ganoderma lucidum callus (the weight ratio of Siberian Ganoderma lucidum callus to ethanol is between 1:5 to 1:10) is extracted with 75% ethanol at 70°C for 20-40 minutes (preferably 30 Minutes) to obtain the extract of the callus of the white crane ganoderma lucidum of the present invention.

Example 2. Evaluation of the effectiveness of Baihe Ganoderma lucidum callus extract in promoting collagen production

Firstly, human skin fibers are cultured with minimal essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1mM sodium pyruvate (Gibco) Human skin fibroblast CCD-966SK (ATCC CRL-1881) in a 24-well plate, 500μL of culture medium with a cell concentration of 2×10 ⁴ cells/well, then cultured at 37°C for 24 hours, and washed with PBS . Next, the cultured cells were divided into 2 groups, including a control group and an experimental group. The white crane Ganoderma lucidum callus extract obtained in Example 1 was diluted with a serum-free medium to a dilution with a concentration of 2%, and then 500 μL of the dilution was added to the cells in the experimental group, and 500 μL of the cells in the control group Serum-free medium. After that, 1,000 μL of culture medium was collected and transferred to a 1.5 mL microcentrifuge tube, and then 200 μL of collagen isolation & concentration reagent was added, and the centrifuge tube was turned over 6-8 times. Then, culture was performed overnight at 4°C. After that, the centrifuge tube was taken out and centrifuged at 12,000 rpm for 10 minutes, and then 1,000 μL of the supernatant was removed. Next, soluble collagen Sircol ^TM Analysis kit ^{(Sircol TM Soluble Collagen Assay Kit)} (Biocolor Life Science Assays, Northern Ireland, UK) to analyze the collagen relative percentage of each group. Add 1,000 μL of Sircol ^® dye to the centrifuge tube, and gently shake for 30 minutes, then centrifuge at 12,000 rpm for 10 minutes, and remove the supernatant. Next, 750 μL of acid-salt washing reagent was added, and centrifuged at 12,000 rpm for 10 minutes. After that, remove the supernatant and turn over the centrifuge tube, and then use a cotton swab to remove the liquid in the centrifuge tube. Then, add 250μL of alkaline reagent and mix thoroughly, then measure the absorbance at 555nm with a spectral signal sensor to calculate the collagen secretion rate. The collagen secretion rate of the control group is taken as the reference of 100%. . The results of this example are shown in Fig. 1.

Fig. 1 is a data diagram showing the effectiveness of the extract of the callus of Siberian Crane Ganoderma lucidum of the present invention in promoting the production of collagen. It can be seen from Figure 1 that compared with the control group, the relative percentage of collagen measured in the experimental group has increased. The results of this example show that the extract of Siberian Crane Ganoderma lucidum callus of the present invention does have the effect of promoting collagen production.

Example 3. Evaluation of the effect of Baihe Ganoderma lucidum callus extract on anti-oxidation of skin cells

Firstly, human skin fibers are cultured with minimal essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1mM sodium pyruvate (Gibco) The parent cell CCD-966SK (BCRC 60153) was placed in a 6-well plate, and the cell concentration of 2 mL of medium was 2×10 ⁵ cells/well. Then, it was cultured at 37° C. for 24 hours, and the medium was removed. Next, the cultured cells were divided into 4 groups, including a control group, a hydrogen peroxide group, and two experimental groups (ie, experimental groups 1 and 2). The white crane Ganoderma lucidum callus extract obtained in Example 1 was diluted with a culture medium into a diluent with a concentration of 2% and 1%, and then 2% diluent and 1 mM hydrogen peroxide (Sigma) were added to the experimental group 1 In the cells, and 1% dilution and 1mM hydrogen peroxide were added to the cells of experimental group 2. The cells in the hydrogen peroxide group were supplemented with 1 mM hydrogen peroxide, and the cells in the control group were supplemented with culture medium. After that, add 5μg/mL dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Sigma/SI-D6883-50MG) (stock solution is 5mg/mL dissolved in DMSO) and add React at 37°C for 1 hour, then wash each well twice with 1 mL of 1X PBS (Gibco). Then, 200 μL of trypsin was added and reacted for 5 minutes in a dark environment, and then the cell culture was collected into a centrifuge tube with a volume of 1.5 mL, and centrifuged at 400×g for 10 minutes. After that, the supernatant was removed and washed once with 1X PBS, followed by centrifugation at 400×g for 10 minutes. Next, remove the supernatant and resuspend the cell pellet in 1 mL of 1X PBS. After that, a flow cytometer (BD Accuri) was used to detect the fluorescence signal of DCFH-DA by excitation wavelength 450~490nm and emission wavelength 510~550nm, thereby calculating the active content The percentage of cells damaged by reactive oxygen species (ROS). The statistically significant differences between the groups were determined by Student's t-test. The results of this example are shown in FIG. 2.

Fig. 2 is a data diagram showing the effect of the extract of Ganoderma lucidum callus on the antioxidant effect of skin cells of the present invention. It can be seen from Figure 2 that compared with the control group, the percentage of cells damaged by ROS measured in the hydrogen peroxide group has a significant increase, which means that hydrogen peroxide will produce a large amount of ROS damage to the cells; compared with the hydrogen peroxide group In contrast, the percentage of cells damaged by ROS measured in experimental group 1 and experimental group 2 was significantly reduced. The results of this example show that the extract of the callus of Siberian Crane Ganoderma lucidum of the present invention does have an antioxidant effect.

Example 4. Evaluation of the anti-aging effect of Baihe Ganoderma lucidum callus extract 4.1 Analysis of gene expression related to skin cell elasticity

Firstly, human skin fibers are cultured with minimal essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1mM sodium pyruvate (Gibco) The parent cell CCD-966SK (BCRC 60153) was placed in a 6-well plate, and the cell concentration of 2 mL of medium was 2×10 ⁵ cells/well. Then, it was cultured at 37° C. for 24 hours, and the medium was removed.

After that, the cultured cells were divided into 2 groups, including a control group and an experimental group. The white crane Ganoderma lucidum callus extract obtained in Example 1 was diluted with a culture medium to a dilution with a concentration of 2%, and then added to the cells of the experimental group 1 and allowed to stand for 24 hours, and the cells of the control group were added Medium. Then, the cell cultures of each group were collected and used for gene expression analysis.

In this example, the genes used to analyze skin cell elasticity include collagen type I, α 1 (collagen type I, alpha 1, COL1A1) gene, collagen type I, α 2 (collagen type I, alpha 2, COL1A2) gene, elastin (ELN) gene, and hyaluronan synthase 3 (HAS3) gene.

RNA extraction kit (Geneaid) was used to extract RNA from the cell cultures obtained above. Thereafter, SuperScript ^® III reverse transcriptase ^{(SuperScript ® III Reverse Transcriptase) (} Invitrogen) The extracted RNA was reverse transcribed to cDNA. Next, use cDNA as a template, and use the primer pair used to amplify the target gene [including COL1A1, COL1A2, ELN, HAS3, and GAPDH (as an internal control), and their nucleotide sequences are shown in the table below 1], use the KAPA CYBR FAST qPCR kit (2x) (KAPA Biosystems) to perform quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction, hereinafter referred to as quantitative real-time PCR), in order to amplify the target gene and Quantitative. The melting curve of the PCR product is confirmed during the quantitative real-time PCR reaction.

The relative expression level of the target gene is derived from Equation 2 ^-△△Ct , and uses the cycle threshold (C _t )value of the GAPDH gene (as an internal control group) and the reference gene (cycle threshold (C t) value) and standard deviation (standard deviation, STDEV) to calculate the relative fold change, where △Ct=Ct _{target gene/reference gene-} Ct _GAPDH , △△Ct=△Ct _target gene-△Ct _{reference gene} , fold change=2 ^-△△Ct _average . The target gene expression level of the control group was used as the comparison benchmark of 1.00. The statistically significant differences between the groups were determined by the single tailed Student's t-test. The results of this example are shown in FIG. 3.

Fig. 3 is a data diagram showing the efficacy of the Ganoderma lucidum callus extract of the present invention in regulating the expression of genes related to skin cell elasticity (including COL1A1 gene, COL1A2 gene, ELN gene and HAS3 gene). It can be seen from Figure 3 that whether it is COL1A1 gene, COL1A2 gene, ELN gene, or HAS3 gene, compared with the control group, the relative expression of genes related to skin cell elasticity in the experimental group has been significantly improved. This experimental result shows that the white crane Ganoderma lucidum callus extract of the present invention can achieve anti-aging effects by regulating gene expression related to skin cell elasticity.

4.2 Analysis of gene expression related to skin cell anti-photoaging

Firstly, human skin fibers are cultured with minimal essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1mM sodium pyruvate (Gibco) The parent cell CCD-966SK (BCRC 60153) was placed in a 6-well plate, and the cell concentration of 2 mL of medium was 2×10 ⁵ cells/well. Then, it was cultured at 37° C. for 24 hours, and the medium was removed.

After that, the cultured cells were divided into 3 groups, including a control group, a UVA group, and an experimental group. The white crane Ganoderma lucidum callus extract obtained in Example 1 was diluted with a culture medium to a dilution with a concentration of 2%, and then added to the cells of the experimental group and allowed to stand for 24 hours. The cells of the UVA group were treated with 5J /cm ² UVA, and the cells in the control group are not treated. Then, the cell cultures of each group were collected and used for gene expression analysis.

In this example, the genes used to analyze skin cell resistance to photoaging include X-ray repair cross complementary protein 1 (X-ray repair cross complementary protein 1, XRCC1) gene, MSH2 gene, MSH6 gene, and superoxide dismutation. Enzyme 1 (superoxide dismutase 1, SOD1) Gene, glutathione peroxidase 1 (glutathione peroxidase 1, GPX1) gene, and chloramphenicol acetyltransferase (CAT) gene.

Use the RNA extraction kit to extract RNA from the cell cultures obtained above. Take 2,000ng of each group of RNA thus obtained and reverse transcribed the extracted RNA into cDNA ^{with SuperScript ® III reverse transcriptase.} Next, use cDNA as a template, and use the primer pair used to amplify the target gene [including XRCC1, MSH2, MSH6, SOD1, GPX1, CAT, and GAPDH (as an internal control group). Their nucleotide sequences are shown below Table 2], the KAPA SYBR FAST qPCR kit (2x) is used in the StepOne Plus Real Time PCR system (ABI) to perform quantitative real-time PCR to amplify and quantify the target gene. The melting curve of the PCR product is confirmed during the quantitative real-time PCR reaction.

The relative expression level of the target gene is derived from Equation 2 ^-△△Ct , and the GAPDH gene (as an internal control group) and the cycle threshold of the reference gene are used to calculate the relative fold change by standard deviation, where △Ct=Ct _{target gene /Reference gene-} Ct _GAPDH , △△Ct=△Ct _target gene-△Ct _{reference gene} , fold change=2 ^-△△Ct _{average value} . The target gene expression level of the control group was used as the comparison benchmark of 1.00. The statistically significant differences between the groups were determined by the one-tailed Studen's t-test. The results of this example are shown in FIG. 4.

Figure 4 is the data on the efficacy of the Ganoderma lucidum callus extract of the present invention in regulating the expression of genes related to skin cell resistance to photoaging (including XRCC1 gene, MSH2 gene, MSH6 gene, SOD1 gene, GPX1 gene and CAT gene) picture. It can be seen from Figure 4 that whether it is XRCC1 gene, MSH2 gene, MSH6 gene, SOD1 gene, GPX1 gene, or CAT gene, compared with the control group and the UVA group, the experimental group measured the anti-photoaging related skin cells The relative expression of genes has been significantly improved. This experimental result shows that the white crane Ganoderma lucidum callus extract of the present invention can achieve the anti-aging effect by regulating the gene expression related to the anti-photoaging of skin cells.

Example 5. Evaluation of the effectiveness of Baihe Ganoderma lucidum callus extract in moisturizing

In this example, the applicant explored whether the White Crane Ganoderma lucidum callus extract can achieve the moisturizing effect by regulating the gene expression related to skin cell moisturizing.

Culture human epidermal keratinocytes (HPEK-50; purchased from CELLnTEC) in a serum-free medium for keratinocytes (Keratinocyte-SFM; purchased from Thermo, product number: 17005042) in a 6-well plate. The cell concentration of 2 mL of the medium is 1 ×10 ⁵ cells/well, followed by culturing at 37°C for 24 hours, and removing the medium.

After that, the cultured cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental groups 1 and 2). The white crane Ganoderma lucidum callus extract obtained in Example 1 was diluted with culture medium into diluents with concentrations of 0.5 mg/mL and 1 mg/mL, and then 0.5 mg/mL diluents were added respectively To the cells of experimental group 1, and add 1mg/mL dilution to the cells of experimental group 2, as for the cells of the control group, add culture medium. Then, the cell cultures of each group were collected and used for gene expression analysis.

In this example, the genes used to analyze skin cell moisture retention include transglutaminase 1 (TGM1) gene, aquaporin 3 (AQP3) gene, GBA gene, and HAS3 gene .

Use the RNA extraction kit to extract RNA from the cell cultures obtained above. Take 2,000ng of each group of RNA thus obtained and reverse transcribed the extracted RNA into cDNA ^{with SuperScript ® III reverse transcriptase.} Next, use cDNA as a template, and use the primer pair used to amplify the target gene [including TGM1, AQP3, GBA, HAS3, and TBP (as an internal control group), their nucleotide sequences are shown in Table 3 below], Use the KAPA SYBR FAST qPCR kit (2x) in the StepOne Plus real-time PCR system to perform quantitative real-time PCR to amplify and quantify the target gene. The melting curve of the PCR product is confirmed during the quantitative real-time PCR reaction.

The relative expression of the target gene is derived from Equation 2 ^-△△Ct , and the TBP gene (as an internal control group) and the cycle threshold of the reference gene are used to calculate the relative fold change by the standard deviation, where △Ct=Ct _{target gene /Reference gene-} Ct _GAPDH , △△Ct=△Ct _target gene-△Ct _{reference gene} , fold change=2 ^-△△Ct _{average value} . The target gene expression level of the control group was used as the comparison benchmark of 1.00. The statistically significant differences between the groups were determined by the one-tailed Studen's t-test. The results of this example are shown in Figs. 5-7.

Figure 5 is a data chart of the efficacy of the callus extract of Siberian Crane Ganoderma lucidum in regulating the expression of TGM1 gene of the present invention; Figure 6 is a data chart of the efficacy of the extract of Ganoderma lucidum callus in regulating the expression of AQP3 gene of the present invention; Fig. 7 is a data diagram showing the efficacy of the extract of the callus of the white crane ganoderma lucidum of the present invention in regulating the expression of the GBA gene and the HAS3 gene. It can be seen from Figures 5 to 7, whether it is TGM1 gene, AQP3 gene, GBA gene, or HAS3 gene, compared with the control group, the relative performance of the genes related to skin cell moisturization measured in the experimental group 1 and the experimental group 2 The amount has increased significantly. The results of this example show that the extract of Siberian Crane Ganoderma lucidum callus of the present invention can achieve moisturizing effect by regulating gene expression related to skin cell moisturizing.

To sum up, the extract of Ganoderma lucidum callus of the present invention can improve collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene, TGM gene, The expression of AQP gene and GBA gene can achieve moisturizing and anti-aging effects; promote skin cell collagen production; and achieve anti-oxidation effect by reducing skin damage caused by oxidation. On the other hand, the present invention uses a changed MS medium formula to achieve the effect of cultivating a large number of Siberian Crane Ganoderma lucidum callus, making the Siberian Crane Ganoderma lucidum callus extract of the present invention more favorable for industrial use.

The above descriptions are merely illustrative and not restrictive. Any equivalent modifications or alterations that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent application.

<110> Dajiang Biomedical Co., Ltd.

<120> The use of white crane ganoderma lucidum callus extract to promote collagen proliferation

<130> N/A

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<170> PatentIn version 3.5

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Claims (7)

The use of an extract of white crane ganoderma lucidum ( Rhinacanthus communis ) callus for preparing a composition for promoting collagen proliferation, wherein the extract of white crane ganoderma lucidum callus promotes collagen type I (collagen type I) The expression level of the gene can achieve the effect of promoting the production of collagen type I (collagen type I). The use described in item 1 of the scope of patent application, wherein the collagen type I gene is collagen type I, α 1 (collagen type I, alpha 1, COL1A1) gene or collagen type I, α 2( collagen type I, alpha 2, COL1A2) gene. The use as described in the first scope of the patent application, wherein the white crane ganoderma lucidum callus extract enhances the expression of transglutaminase (TGM) gene. The use described in item 3 of the scope of patent application, wherein the TGM gene is the TGM1 gene. The use described in item 1 of the scope of patent application, wherein the white crane ganoderma lucidum callus extract is prepared by extracting the white crane ganoderma lucidum callus with water or alcohol as the extraction solvent, and the white crane ganoderma callus The volume ratio to the extraction solvent is between 1:5 and 1:10, and the extraction temperature is between 60°C and 80°C. The white crane Ganoderma lucidum callus is obtained by adding 2,4-dichlorophenoxyacetic acid (2 , 4-dichlorophenoxyacetic acid) and 6-benzyladenine (6-benzyladenine) were cultured in MS medium (Murashige and Skoog medium). The use described in item 5 of the scope of patent application, wherein the extract of the callus of the white crane ganoderma lucidum is extracted with water or alcohol as the extraction solvent for the callus of the white crane ganoderma lucidum for 20-40 minutes. The use described in item 5 of the scope of patent application, wherein the MS medium contains 3% sucrose and 0.8% agar.

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