TWI687237B - Use of extract of victoria cruziana for inducing expression of keratin gene and hyaluronan synthase 2 gene, and enhancing moisture-retaining capacity of skin - Google Patents

Abstract

The present disclosure provides a use of an extract of Victoria cruziana for inducing expression of keratin gene and hyaluronan synthase 2 gene, and enhancing the moisture-retaining capacity of the skin.

Description

The extract of King Cruz is used to enhance keratin gene and hyaluronic acid synthesis Gene expression level of genase 2 gene and use for enhancing skin's moisturizing ability

The present invention relates to a use of the extract of King Lutzia to enhance the expression of keratin gene and hyaluronic acid synthase 2 gene and enhance the skin's moisturizing ability.

The skin is the largest barrier to protect human individuals, it has the function to resist water loss, pathogenic bacteria and various environmental damage. Exposure to a large amount of ultraviolet (UV), ionizing radiation, drugs or xenobiotics will cause the skin to generate reactive oxygen species (ROS) and free radicals. When the accumulated amount of reactive oxygen species and free radicals exceeds the antioxidant capacity of the cell or tissue itself, oxidative stress will be formed. Next, the reactive oxygen species and free radicals will react with intracellular components (including DNA, proteins, lipids, etc.), which will undesirably affect the skin.

In recent years, human demand for skin moisturizing has been increasing day by day. However, the most common ways to improve skin moisturizing ability at present are to use cosmetics, skin care products applied to the skin surface, or oral health foods that claim to enhance skin moisturizing effect. . However, the 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 general users.

In order to solve the above-mentioned problems, those skilled in the art urgently need to develop novel medicines, food products, or skin care products that enhance the moisturizing ability of the skin, so as to benefit the broad ethnic groups in need.

In view of this, the object of the present invention is to provide an extract of Victoria cruziana for preparing a keratin ( KRT ) gene and hyaluronan synthase 2 ( HAS2 ) gene The use of the expressive composition, in which the extract of King Cruz is made by extracting King Lotus from water, alcohol, water-containing alcohol or a combination thereof as an extraction solvent.

Another object of the present invention is to provide a use of the extract of Cruz Royale for the preparation of a composition for enhancing the moisturizing capacity of the skin, wherein the extract of Royal Lotus is water, alcohols, hydrous alcohols or The combination is used as an extraction solvent to extract the Cruz royal lotus.

In an embodiment of the invention, the volume ratio of the Cruz King Lotus to the extraction solvent is between 1 and 5: 10 and 20.

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

In an embodiment of the present invention, the effective concentration of the extract of King Cruz Cruz is at least 0.03125 mg/mL.

In an embodiment of the invention, the extract of King Cruz is a flower extract of King Lotus blooming on the first day.

In an embodiment of the invention, the composition is a pharmaceutical product, a food product or a maintenance product.

To sum up, the effect of the extract of the Royal Lotus of Cruz of the present invention is that the effect of enhancing the moisturizing ability of the skin can be achieved by enhancing the expression of the keratin gene and hyaluronic acid synthase 2 gene.

The embodiments of the present invention will be further described below. The examples listed below are used to clarify the present invention, and are not intended to limit the scope of the present invention. Anyone who is familiar with this art, without departing from the spirit and scope of the present invention, Some changes and retouching can be done, so the scope of protection of the present invention shall be deemed as defined by the scope of the attached patent application.

FIG. 1 is a data graph of the detection of total polyphenol content of the extract of the Royal Lotus of Cruz of the present invention, where “***” indicates comparison with the comparison group, p <0.001.

FIG. 2 is a data graph showing the efficacy of the extract of Kreuz king lotus of the present invention in enhancing the KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization after 6 hours of action, where “*” represents the control group For comparison, p <0.05;"**" indicates comparison with the control group, p <0.01.

FIG. 3 is a data graph showing the efficacy of the extract of the King of Lotus of the present invention in enhancing the KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization for 24 hours, where “*” represents the control group For comparison, p <0.05;"**" means comparison with the control group, p <0.01;"***" means comparison with the control group, p <0.001.

definition

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

According to the present invention, Victoria cruziana ( Victoria cruziana ) is aliased as Paraguay king lotus or Argentine king lotus, which is an aquatic plant of Nymphaeaceae (Nymphaeaceae) King lotus ( Victoria ), native to tropical South America. King Cruz has underground stems, rhizomes erect and massive, thick, thorny, with thick and strong lateral roots, soft and brittle texture, single leaf alternate.

According to the present invention, the flowers of King Lotus can have different flowering stages. Preferably, the flowers of King Cruz are blooming on days 1 and 2. More preferably, the flowers of King Cruz Cruz bloom on the first day.

According to the present invention, pharmaceutical products can be manufactured into a dosage form suitable for parenterally or topically administration using techniques well known to those skilled in the art, including, but not limited to: injections (injection) [for example, sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.

According to the present invention, the pharmaceutical product may further include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, and decomposers ), 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 professional qualities and routine techniques of those who are familiar with this technology.

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

According to the present invention, the medicinal product can be administered via parenteral routes selected from the group consisting of subcutaneous injection, intraepidermal injection, and intradermal injection Intradermal injection and intralesional injection.

According to the present invention, pharmaceutical products can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, which include, but are not limited to: emulsion, gel Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serums, pastes, foams, drops, suspensions, salves and bandages.

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

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

According to the present invention, the maintenance product may further include an acceptable adjuvant widely used in the maintenance product manufacturing technology. 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 agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the professional qualities and routine techniques of those who are familiar with this technology.

According to the present invention, skin care products can be manufactured into a form suitable for skincare or makeup using techniques well known to those skilled in the art. This includes, but is 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 Glue, ointment, cream, mask, patch, pack, wipe, powder, aerosol, spray, emulsion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, foundation remover products, makeup remover products, soap, and other body cleansing products.

According to the present invention, the skin care product 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, catechins (catechin), kojic acid, arbutin and vitamin C], moisturizer, anti-hair Anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [such as aloe extract], skin nutrients, anesthetics (anesthetics), anti-acne agents, antipruritics, analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin 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 external agents fall within the professional qualities and routine technologies of those who are familiar with this technology.

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

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

Example 1. Preparation of the extract of King Cruz

First, homogenize the flowers of King Cruz Cruz (from Lianhe United Leisure Garden in Baihe District, Tainan City), which is not blooming, blooming on the first day or blooming on the second day, and then use water, alcohol, water-containing alcohol or The combination is used as an extraction solvent to extract the homogenized Cruz king lotus for 0.5~3 hours, wherein the extraction temperature is between 50°C and 100°C, and the volume ratio of the extraction solvent to the Cruz king lotus is between 10-20: 1~5. Next, the obtained product was cooled to room temperature, and the product was filtered with a 400-mesh filter to obtain a filtrate, and then the filtrate was concentrated under reduced pressure at 45 to 70°C to obtain a concentrated product. After that, the concentrated product was filtered with a filter core with a pore size of 5 μm to obtain an extract of the King Cruz of the present invention.

Example 2. Determination of total polyphenol content in the extract of King Lotus of Cruz

First, prepare a standard solution and draw a standard curve, dissolve 10 mg of gallic acid (Sigma G7384) in water and add a volume of 10 mL to a measuring flask, and then store at -20°C as a stock solution. After that, dilute the stock solution 10 times to a concentration of 100 μg/mL, and the unused liquid can be stored at -20°C. Next, prepare 0 μg/mL, 20 μg/mL, and 40 in glass test tubes, respectively The standard solutions of gallic acid for μg/mL, 60μg/mL, 80μg/mL and 100μg/mL are shown in Table 1.

After that, add 500 μL of Folin-Ciocalteu's phenol reagent (Merck 1.09001.0100), mix well and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate (Sigma 31432, dissolved in (In water), mix well and react for 30 minutes. Next, after ensuring no bubbles through vortex, 200 μL of the standard solution was taken and the absorbance was measured at 750 nm, and a standard curve was drawn.

In addition, the extract of the Royal Lotus of Cruzia that bloomed on the first day obtained in Example 1 was taken as the experimental group 1, and the extract of the Royal Lotus of Cruzia that bloomed on the second day was the experimental group 2, and the extract of the Royal Lotus of Cruzia unpublished As a comparison group. The experimental group 1, the experimental group 2 and the comparative group were respectively diluted with water and a volume of 100 μL was taken into a glass test tube. The samples of each group were repeated three times. Afterwards, add 500 μL of flavanol reagent, mix well and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix well and react for 30 minutes to 1 hour. Next, after vortexing to ensure that there are no bubbles, 200 μL of each group of reaction solution was taken to measure the absorbance at 750 nm, and then the concentration was calculated by the internal difference method, and then the dilution factor was multiplied to obtain the original extract concentration. The results of this example are shown in Figure 1.

FIG. 1 is a data graph of the detection of the total polyphenol content of the extract of the Royal Lotus of Cruz. As can be seen from Figure 1, compared with the comparison group, the total polyphenol content of the experimental group 1 and the experimental group 2 has a significant increase, and compared with the comparison group, the total polyphenol content of the experimental group 1 increases by 7.1 times . The results of this example show that the extract of Kreuz Wanglian of the present invention will release a large amount of total polyphenols. Since the extract of the flowering Kr. Cruze on the first day had the highest total polyphenol content, the extract of the flowering Kr. Cruze on the first day was used for subsequent experiments.

Example 3. Evaluation of the effectiveness of the extract of Cruz Royale in enhancing the moisturizing ability of the skin

In this embodiment, by exploring whether the extract of King Cruz Cruz can regulate the gene expression related to skin cell moisturization, the effect of enhancing the moisturizing capacity of the skin can be achieved.

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 dish, with a cell concentration of 1.5 in 2 mL of medium ×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). Dilute the extract of the King Cruz lotus flower blooming on Day 1 with the medium to a dilution with a concentration of 0.0625 mg/mL and 0.03125 mg/mL, and then add the dilution of 0.0625 mg/mL to the cells of Experimental Group 1, respectively, and A dilution of 0.03125 mg/mL was added to the cells of experimental group 2, and the cells of the control group were added with culture medium. Next, cells of each group were cultured in an incubator for 6 hours, and then cell cultures of each group were collected and used for gene expression analysis.

In this embodiment, the genes used to analyze skin cell moisturization include keratin 1 (keratin 1, KRT1 ) gene, keratin 10 (keratin 10, KRT10 ) gene, keratin 14 (keratin 14, KRT14 ) gene and Hyaluronan synthase 2, HAS2 gene.

RNA extraction was performed on the cell cultures of each group obtained above with an RNA extraction kit (Geneaid). Take 2,000 ng of each set of RNA thus obtained and reverse transcribe the extracted RNA into cDNA with SuperScript ^® III reverse transcriptase (Invitrogen). Next, using cDNA as a template and using primer pairs to amplify the target gene, including KRT1 , KRT10 , KRT14 , HAS2, and TBP (as internal control groups), their nucleotide sequences are shown in Table 2 below, in StepOne Plus real-time PCR system (ABI) uses KAPA CYBR FAST qPCR kit (2x) (KAPA Biosystems) 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 cycle threshold of the TBP gene (as an internal control group) and the reference gene and the relative fold change are calculated by standard deviation, where △Ct=Ct _{target gene / Reference gene-} Ct _TBP , △△Ct = △Ct _target gene-△Ct _{reference gene} , fold change = 2- ^△△Ct _average . The target gene expression amount of the control group was used as the comparison criterion of 1. The statistically significant differences between the groups were determined by the one-tailed Stein's t-test. The results of this example are shown in FIG. 2.

FIG. 2 is a data graph of the effect of the extract of the king lotus of Cruz of the present invention on the performance of KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization at 6 hours of action. As can be seen from Figure 2, whether it is the KRT1 gene, KRT10 gene, KRT14 gene or HAS2 gene, compared with the control group, the relative performance of the gene in the experimental group 1 has been significantly improved. In terms of KRT14 gene and HAS2 gene, compared with the control group, the relative performance of the gene in the experimental group 2 has been significantly improved. The results of this example show that the extract of Kreuz king lotus of the present invention can maintain the arrangement of keratinocytes by enhancing the expression levels of KRT1 gene, KRT10 gene and KRT14 gene to complete the skin keratinous tissue and prevent the skin from losing water. On the other hand, the extract of King Cruz of the present invention can promote the production of endogenous hyaluronic acid by enhancing the expression level of the HAS2 gene, support the skin by adsorbing moisture, and maintain skin elasticity and gloss.

In addition, the present invention utilizes the extract of King Cruz Cruz with a concentration of 0.03125 mg/mL to explore whether it can enhance the performance of the KRT1 gene, KRT10 gene and KRT14 gene. The experimental procedure is the same as above, the difference is that the extract is added to After the cells in the experimental group, the action time was replaced by 24 hours for 6 hours. The results of this example are shown in Figure 3.

FIG. 3 is a data graph of the effect of the extract of the Royal Lotus of Cruz of the present invention on enhancing the expression of the KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization when acting for 24 hours. As can be seen from Figure 3, whether it is the KRT1 gene, KRT10 gene or KRT14 gene, compared with the control group, the relative expression of the gene in the experimental group has significantly improved. The results of this example show that the extract of Kreuz King Lotus of the present invention can maintain the arrangement of keratinocytes by increasing the expression levels of KRT1 gene, KRT10 gene and KRT14 gene in 24 hours, so that the skin keratinous tissue is intact and prevents skin water loss .

To sum up, the extract of Kreuz King Lotus of the present invention can enhance the expression of keratin genes (including KRT1 gene, KRT10 gene and KRT14 gene) and hyaluronic acid synthase 2 ( HAS2 ) gene by maintaining skin cells Arrange and maintain constant moisture in the skin, increase the production of hyaluronic acid, enhance the overall performance of keratinocyte moisturizing genes, and achieve the effect of enhancing the moisturizing ability of the skin.

The above is only exemplary, and not restrictive. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention shall be included in the scope of the attached patent application.

<110> Dajiang Biomedical Co., Ltd.

<120> The use of the extract of Cruz Royale to enhance the expression of keratin gene and hyaluronic acid synthase 2 gene and enhance the moisturizing ability of the skin

<130> 107B0592-I1

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Claims (9)

The use of a flower extract of Victoria cruziana to prepare a pharmaceutical composition that enhances the expression of keratin ( KRT ) gene and hyaluronan synthase 2 ( HAS2 ) gene, Wherein the extract of King Cruz Cruz is prepared by extracting King Lotus Cruz with water, alcohol, water-containing alcohol or a combination thereof as an extraction solvent. The use as described in Item 1 of the patent application scope, wherein the volume ratio of the Cruz King Lotus to the extraction solvent is between 1 and 5: 10 and 20. The use as described in item 1 of the patent application range, wherein the KRT gene is a KRT1 gene, a KRT10 gene or a KRT14 gene. The use as described in item 1 of the scope of the patent application, wherein the effective concentration of the extract of King Cruz Cruz is at least 0.03125 mg/mL. The use as described in item 1 of the scope of the patent application, wherein the extract of King Cruz is a flower extract of King Lotus blooming on the first day. The use of a flower extract of Cruzia crassifolia to prepare a medicinal composition that enhances the skin's moisturizing ability, wherein the extract of Cruzia crassifolia is based on water, alcohols, water-containing alcohols or a combination thereof as an extraction solvent The Cruz King Lotus is prepared by extraction. The use as described in Item 6 of the patent application scope, wherein the volume ratio of the Cruz King Lotus to the extraction solvent is between 1 and 5: 10 and 20. The use as described in item 6 of the patent application range, wherein the effective concentration of the extract of Kreuz king lotus is at least 0.03125 mg/mL. The use as described in Item 6 of the patent application range, wherein the extract of King Cruz is a flower extract of King Lotus blooming on the first day.

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