TWI772723B - Use of viola mandshurica extract for anti-aging - Google Patents

Abstract

The present disclosure provides a use of a Viola mandshurica extract for anti-aging.

Description

Anti-aging use of Radix Radix et Rhizoma extract

The present invention relates to an anti-aging application of an extract of Viola vulgaris.

The skin is the largest barrier to protect the human individual, it has the function of resisting water loss, pathogenic bacteria and various environmental damages. Exposure to a large amount of ultraviolet (ultraviolet, UV), ionizing radiation (ionizing radiation), drugs or xenobiotics (xenobiotics) can stimulate the skin to generate reactive oxygen species (ROS) and free radicals (free radicals). When the amount of accumulated reactive oxygen species and free radicals exceeds the antioxidant capacity of cells or tissues, oxidative stress is formed. Next, reactive oxygen species and free radicals react with intracellular constituents (including DNA, proteins, lipids, etc.), thereby having undesired effects on the skin.

In recent years, there has been an increasing demand for enhancing skin activity (for example, by promoting dermal fibroblast proliferation), because once skin activity is enhanced, anti-aging effects can be achieved. However, at present, most common ways to enhance skin activity are to use cosmetics, skin care products applied to the skin surface, or oral health foods that claim to enhance skin activity. However, conventional cosmetics, skin care products and health foods are mostly made of chemical ingredients. Long-term use is not only harmful to human health, but also these products are often expensive and not affordable for ordinary users.

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

In order to solve the above problems, those skilled in the art urgently need to develop novel medicines, food products or skin care products with the functions of promoting skin fibroblast proliferation, enhancing skin cell activity, enhancing mitochondrial activity of cells and anti-aging effects for the benefit of A large group of people with this need.

In view of this, the purpose of the present invention is to provide the use of a Viola mandshurica extract for preparing an anti-aging composition, wherein the Viola mandshurica extract is obtained by extracting a Viola mandshurica with a solvent , the solvent is water, alcohol, or mixture of alcohol and water.

In one embodiment of the present invention, the anti-aging comprises promoting skin fibroblast proliferation, enhancing skin cell activity, and enhancing cellular mitochondrial activity.

In one embodiment of the invention, the cell is a dermal fibroblast.

In one embodiment of the present invention, the effective concentration of the extract of Radix Radix et Rhizoma is at least 0.03125 mg/mL.

In an embodiment of the present invention, the extraction is performed at a temperature ranging from 50°C to 100°C.

In an embodiment of the present invention, the volume ratio of the solvent to the violetidine is 10-20:1-5.

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

In one embodiment of the present invention, the pharmaceutical product comprises a pharmaceutically acceptable carrier.

In one embodiment of the present invention, the medicinal product is in a dosage form for topical administration.

In one embodiment of the present invention, the skin care product is in a form for topical application.

To sum up, the effect of the extract of the present invention is: by promoting the proliferation of skin fibroblasts and improving the mitochondrial activity of the cells, it can maintain cell vitality and normal metabolism, improve skin cell activity and anti-aging. effect.

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

Fig. 1 is a data graph of the efficacy of the extract of the present invention in promoting the proliferation of skin fibroblasts after 24 hours of action, wherein "**" means compared with the control group, p < 0.01.

Figure 2 is a data graph of the effect of the extract of the present invention on the improvement of mitochondrial activity of skin fibroblasts after 24 hours of action, wherein "**" indicates that compared with the control group, p < 0.01.

definition

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

Statistical analysis was performed using Excel software. Data were expressed as mean ± standard deviation (STDEV), and differences between them were analyzed by Student's t -test .

According to the present invention, Viola mandshurica is a perennial herb of the Violaceae family. In the field of traditional Chinese medicine, the whole herb is used as medicine, which is cold in nature, bitter in taste, and has the functions of clearing away heat and detoxifying, cooling blood and reducing swelling.

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

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

According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.

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

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

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

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

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

According to the present invention, the skincare product may further comprise an acceptable adjuvant which is widely used in skincare product manufacturing techniques. For example, the acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, dyes Coloring agents, thickening agents, fillers, fragrances and odor absorbers. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.

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

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

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

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

Example 1. Preparation of Radix Viola extract

First, the Viola mandshurica ( Viola mandshurica ), sourced from Viola mandshurica produced in Shaanxi, China, purchased from: Xinda Agricultural Production Co., Ltd. in Taiwan, was subjected to homogenization treatment, and then homogenized with a solvent at 50~100 ° C. The treated violacean is extracted at a volume ratio of 10-20:1-5 for 0.5-2 hours to obtain a crude extract, wherein the solvent is water, alcohol, or a mixture of alcohol and water. Next, it was cooled to room temperature, and then the crude extract was filtered through a 400 mesh filter to obtain a filtrate, and then the filtrate was concentrated under reduced pressure at 45-70° C. to obtain the extract of Radix Radix et Rhizoma.

Example 2. Efficacy evaluation of the extract of Radix Viola in promoting the proliferation of dermal fibroblasts, enhancing the activity of skin cells and anti-aging

First, add 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate (90%), and 10% fetal bovine serum (FBS) ( Gibco) minimum essential medium (Minimum essential medium, MEM) (Eagle) (mixed with Earle's Balanced Salt Solution, Earle's BSS) (GIBCO Company, No. 41500-034, USA) to culture human skin fibroids Cells CCD-966SK (purchased from Taiwan Bioresource Collection and Research Center, BCRC, No. BCRC 60153) were placed in a 6-well plate at a cell concentration of 1×10 ⁵ cells/well.

After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental group 1 and experimental group 2). 0.03125 mg/mL Radix radix extract was added to the cells of experimental group 1, and 0.0625 mg/mL Radix Radix Radix was added to the cells of experimental group 2 and acted for 24 hours. As for the cells of the control group, no treatment was performed. Next, 10 μM EdU (component A, 5-ethynyl-2'-deoxyuridine, a thymidine analog that can be incorporated into dividing cells) was added to Cells are grown in medium for 1 to 2 hours, and then harvested. After that, the cells were washed once with 1% Bovine serum albumin (BSA) (in phosphate buffered saline (PBS, GIBCO, No. 14200-075, USA)), and then removed supernatant. Next, 100 μL of Click-iT ^TM fixative ⁽ Component D, containing 4% paraformaldehyde in PBS) was added and mixed well, and allowed to act in the dark at room temperature for 15 minutes . Afterwards, cells were washed once with 1% BSA (in PBS) and the supernatant was removed. Next, 100 μL of 1X Click-iT ^TM saponin-based permeabilization and wash reagent (Component E ⁾ was added and allowed to act in the dark at room temperature for 15 minutes. Afterwards, cells were washed once with 1% BSA (in PBS) and the supernatant was removed. Next, cells were resuspended in 100 μL of 1X Click-iT ^™ Saponin-based Permeabilization and Washing Reagent prior to Click-iT ^™ reaction.

The Click-iT ^™ reaction scheme is as follows: First prepare 1X Click-iT ^™ EdU buffer additive (component G) (dilute 10X buffer with 2 mL of deuterium depleted water (DDW) at -20°C ). Next, click-iT ^™ Plus reaction cocktails were prepared, including PBS (or Dulbecco's phosphate-buffered saline (D-PBS) or TriS-Hcl Buffered saline (D-PBS) Saline, TBS)), copper protectant (component F), fluorescent dye picolyl azide (component B), and reaction buffer additives (prepared within 15 minutes). After that, 0.1 mL of Click-iT ^™ Plus reaction mixture (total volume 200 μL) was added to each test tube and allowed to react in the dark at room temperature for 30 minutes. Next, cells were washed with 1X Click-iT ^™ Saponin-based Permeabilization and Washing Reagent, and the supernatant was removed. Afterwards, cells were resuspended in 500 μL of 1X Click-iT ^™ Saponin-based Permeabilization and Washing Reagent. Next, the analysis was performed by flow cytometry, the excitation wavelength was 488 nm, the emission wavelength was 530/30 nm, and the detection was performed by logarithmic amplification. The results of this example are shown in FIG. 1 .

Fig. 1 is a data graph of the efficacy of the extract of the present invention in promoting the proliferation of skin fibroblasts after 24 hours of action. It can be seen from FIG. 1 that compared with the control group, the relative EdU incorporation multiples of experimental group 1 and experimental group 2 were significantly increased. The results of this example show that the extract of the present invention can promote the proliferation of skin fibroblasts and enhance the activity of skin cells after 24 hours, thereby achieving the effects of enhancing skin cell activity and anti-aging.

Example 3. Efficacy evaluation of Radix radix extract in enhancing the mitochondrial activity of cells

In this example, human skin fibroblasts were used to analyze the mitochondrial activity of skin cells, and the flow cytometry Mitochrondrial membrane potential detection kit (BD) was used to conduct experiments. Human skin fibroblasts were purchased from Taiwan Bioresource Collection and Research Center (BCRC), number BCRC 60153. The cells were cultured in supplemented with 10% fetal bovine serum (FBS) (GIBCO, No. 10438-026, USA), 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate (Sigma, No. S5761, USA), 1 mM sodium pyruvate (GIBCO, No. 11360-070, USA) minimum essential medium (Minimum essential medium, MEM) (Eagle) (in Earle's Balanced Salt Solution, Earle's BSS) (GIBCO, No. 41500-034, USA) .

¹ x 105 human dermal fibroblasts (n=3) were seeded per well in a 6-well culture dish containing 2 mL of the above medium. After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental group 1 and experimental group 2). 0.03125 mg/mL Radix radix extract was added to the cells of experimental group 1, and 0.0625 mg/mL Radix Radix radix extract was added to the cells of experimental group 2. As for the cells of the control group, culture medium was added. Next, groups of cells were incubated at 37°C for 24 hours and then pre-warmed with 10X assay buffer at 37°C. Afterwards, prepare 1X assay buffer with sterile 1X PBS, mix well and place at 37°C, then add 130 μL of Dimethyl sulfoxide (DMSO) to lyophilized JC-1 mitochondrial dye to prepare JC- 1 stock solution, the stock solution can be stored at -20°C for 6 months. Next, a working solution of JC-1 mitochondrial stain and 1X assay buffer was prepared at a ratio of 1:100, then the medium was removed and rinsed twice with 1X PBS. Then, after adding trypsin/EDTA for 3 minutes, the suspended cells were pipetted into a 1.5 mL microcentrifuge tube, and the precipitated cells were collected by centrifugation at 400 g for 5 minutes.

After removing the supernatant, cells were resuspended in 1 mL of IX PBS, then transferred to a 1.5 mL centrifuge tube and centrifuged at 400 g for 5 min. After removing the supernatant, add 100 μL of the JC-1 working solution, mix well, and act in the dark for 15 minutes. Afterwards, centrifuge at 400g for 5 minutes, wash with 1 mL of 1X wash buffer and centrifuge at 400g for 5 minutes, then wash with 1 mL of 1X wash buffer and centrifuge at 400g for 5 minutes. Cells were resuspended with 500 μL of 1X PBS containing 2% FBS, and then the mitochondrial membrane potential changes during apoptosis were observed by flow cytometry (Beckman) analysis, and t -test (student t -test) statistics were performed with Excel Statistical significance of differences between sample populations was analyzed.

Fig. 2 is a data graph showing the effect of the extract of the present invention on the mitochondrial activity of dermal fibroblasts after acting for 24 hours. It can be seen from Figure 2 that, compared with the control group, the mitochondrial activity (ie, the relative JC-1 aggregate) of the skin fibroblasts in the experimental group 1 and the experimental group 2 was significantly improved. The results of this example show that, after stimulating human skin fibroblasts for 24 hours, the extract of the present invention can significantly enhance the mitochondrial activity of cells, maintain cell vitality and normal metabolism, and achieve anti-aging potential.

To sum up, the extract of the present invention can achieve the effects of maintaining cell vitality and normal metabolism, enhancing skin cell activity and anti-aging by promoting skin fibroblast proliferation and enhancing mitochondrial activity of cells.

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

Claims (8)

The purpose of an extract of Radix Radix is used to prepare a composition for promoting the proliferation of skin fibroblasts, enhancing the activity of skin cells, and improving the mitochondrial activity of cells. Obtained from ditin, the solvent is water, and the volume ratio of the solvent to the ditin is between 10-20: 1-5. The use according to claim 1, wherein the cell is a dermal fibroblast. The use as described in item 1 of the claimed scope, wherein the effective concentration of the extract of Radix Radix et Rhizoma is at least 0.03125 mg/mL. The use as described in claim 1, wherein the extraction is performed at a temperature between 50 and 100°C. The use as described in item 1 of the scope of the application, wherein the composition is a medicine, a food product or a skin care product. The use as described in item 5 of the claimed scope, wherein the medicinal product comprises a pharmaceutically acceptable carrier. The use as described in item 5 of the claimed scope, wherein the medicinal product is in a dosage form for topical administration. The use as described in claim 5, wherein the skin care product is in a form for topical application.

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