Use of fritillaria extract for skin care

Abstract

The present invention relates to the field of plant extracts, and in particular to the use of Fomes fomentarius extract for skin care. The present invention provides the use of Fomes fomentarius extract for enhancing anti-glycation activity, inhibiting melanin production, enhancing skin's ability to resist ultraviolet rays, enhancing SOD activity, or reducing ROS expression, wherein the Fomes fomentarius extract is obtained by solvent extraction of Fomes fomentarius, and the solvent is water, alcohol, or a mixture of alcohol and water.

Description

[0001] This application is a divisional application filed by the applicant based on the parent application (application number: 202010088439.X, application date: February 12, 2020, invention title: use of daylily extract for weight loss and increasing basal metabolic rate). Technical Field

[0002] This invention relates to the field of plant extracts, and more particularly to the use of daylily extract for skincare. Background Technology

[0003] Fat is an essential component of the human body, but excessive fat intake can be harmful. In countries where the economy is booming, obesity (also known as metabolic syndrome) is on the rise. Therefore, in Asia, China is likely to be among the countries experiencing the next significant increase in obesity rates.

[0004] Therefore, obesity is a problem that urgently needs to be addressed.

[0005] In addition to its role in skin and internal aging, glycation is also linked to diabetes, a disease that poses a significant threat to the health of many people. The essence of glycation is the Maillard reaction, also known as carbonyl-amine browning. This is a non-enzymatic glycosylation (glycation) reaction between the aldehyde (ketone) group of sugar and the amine group of an amino-containing substance (such as proteins, peptides, amino acids, phospholipids, nucleic acids, or their derivatives). Prolonged exposure to high blood sugar levels can lead to the auto-oxidation of glucose and the glycation of proteins, forming advanced glycation end products (AGEs). AGEs can cause various age-related diseases, such as wrinkles, cataracts, atherosclerosis, and kidney failure.

[0006] In recent years, the number of people suffering from depression has been increasing. Furthermore, if we include so-called pseudo-depression, which does not present with psychotic symptoms, the number of people with this condition increases to a considerably high proportion.

[0007] The causes of depression are still not fully understood, but are not limited to biological or personality factors. For example, drastic changes in the modern social environment, social structures that abandon the weak, and the trend of excluding dissent under the dominance of a single value system are believed to be closely related to the increase in the number of people suffering from depression.

[0008] Treatment for depression can include, for example, rest and recuperation, medication, or a combination of both. However, in reality, rest and recuperation are not readily available, and in practice, antidepressants are often used as a guise for treatment.

[0009] The skin is the greatest protective barrier for the human body, functioning to combat moisture loss, pathogens, and various environmental damages. Exposure to large amounts of ultraviolet (UV) radiation, ionizing radiation, drugs, or xenobiotics stimulates the skin to produce reactive oxygen species (ROS) and free radicals. When the accumulated amount of ROS and free radicals exceeds the antioxidant capacity of the cells or tissues themselves, oxidative stress is created. Subsequently, ROS and free radicals react with intracellular components (including DNA, proteins, and lipids), thereby producing undesirable effects on the skin.

[0010] Melanogenesis (also known as melanin synthesis) refers to the process by which tyrosine in skin melanocytes is converted into melanin after being induced by environmental factors (such as ultraviolet (UV)) or physiological factors (such as fatigue, stress, chronic inflammation, and the release of abnormal α-melanocyte stimulating hormone (α-MSH) in the body). This process involves the catalysis of tyrosinase (which is the rate-limiting step in melanin production) and a series of redox reactions. Melanin protects the hypodermis from photodamage caused by ultraviolet radiation. However, when melanin accumulates excessively on the skin or is abnormally distributed, it can lead to skin disorders such as lentigines, freckles, melasma, age spots, and hyperpigmentation.

[0011] To achieve skin whitening, many melanogenesis inhibitors are currently used to lighten or remove melanin or dark spots accumulated on the skin. These melanogenesis inhibitors mostly regulate melanin production through the following mechanisms: (1) before melanin production: for example, inhibiting tyrosinase mRNA transcription (such as C2-ceramide, tretinoin, and vanillic acid) and glycosylation (such as calcium D-pantetheine-S-sulfonate, PaSSO3Ca); (2) during melanin production: for example, inhibiting tyrosinase activity (such as hydroquinone, arbutin, and kojic acid). (2) acid), accelerate the degradation of tyrosinase (such as linoleic acid and phenylthiourea), and promote the reduction of dopaquinone (such as ascorbic acid); and (3) after melanin production: for example, promote melanin decomposition (such as linoleic acid), inhibit melanosome transfer (such as niacinamide and serine protease inhibitor), and accelerate skin turnover (such as liquiritin and glycolic acid).

[0012] In recent years, the demand for skin whitening and care has been increasing. However, the melanin synthesis inhibitors currently used are still not ideal in lightening or removing melanin. Furthermore, most common methods for addressing melanin production involve applying topical medications or skincare products to the skin.

[0013] Cerebrovascular disease currently ranks second in mortality in Taiwan. Even mild strokes place a tremendous economic and emotional burden on patients and their families. The main cause of stroke is cerebral ischemia due to vascular embolism, leading to local neuronal hypoxia and death. The pathological mechanism of some central nervous system injury-related deaths is now understood to be that cerebral ischemia / hypoxia disrupts the neuronal energy supply system, resulting in the release of large amounts of neurotransmitters (glutamate). This release activates NMDA receptors, affecting the calcium homeostasis system and generating free radicals and nitric oxide, leading to secondary cell damage. Recent studies have further discovered that oxidative free radicals are also a significant cause of neuronal death, and many antioxidant genes and response mechanisms have been identified.

[0014] 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 organism as a whole. 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 nerve cells, thereby improving their antioxidant capacity to protect nerves, has become an important research topic in this field.

[0015] However, most pharmaceuticals, food products, and skincare products currently used to address these problems are made from chemical ingredients. Long-term use is not only harmful to human health but also often prohibitively expensive for the average user. To solve these problems, those skilled in the art urgently need to develop novel food products or skincare products that enhance anti-glycation activity, inhibit cellular fat accumulation, promote fat breakdown, increase skeletal muscle basal metabolic rate, combat depression, inhibit melanin production, enhance skin's UV resistance, increase superoxide dismutase (SOD) activity, reduce ROS levels, enhance mitochondrial activity, and improve antioxidant capacity to benefit the vast population with these needs. Summary of the Invention

[0016] In view of this, the purpose of this invention is to provide a daylily (Hemerocallis fulva Linn.) extract for the preparation of a gene regulating X-ray repair cross complementary protein 1 (XRCC1), uracil DNA glycosylase (UNG), 8-oxoguanine DNA glycosylase (OGG1), N-methylpurine DNA glycosylase (MPG), ERCC excision repair 1 (endonuclease non-catalytic subunit, ERCC1), ERCC excision repair 6 (endonuclease non-catalytic subunit, ERCC6), and glutathione peroxidase 1. The use of a composition of expression levels of the following genes: peroxidase1 (GPX1), mutL homolog 1 (MLH1), melanocyte-stimulating hormone 6 (MSH6), superoxide dismutase (SOD), mitogen-activated protein kinase phosphatase 1 (MKP-1), and dopa decarboxylase (DDC), wherein the daylily extract is obtained by extracting one daylily flower with a solvent, the solvent being water, alcohol, or a mixture of alcohol and water.

[0017] In one embodiment of the present invention, the SOD gene is an SOD1 gene or an SOD2 gene.

[0018] In one embodiment of the present invention, the XRCC1 gene, the UNG gene, the OGG1 gene, the MPG gene, the ERCC1 gene, the ERCC6 gene, the GPX1 gene, the MLH1 gene, the MSH6 gene, the SOD gene, and the DDC gene are upregulated, and the MKP-1 gene is downregulated.

[0019] In one embodiment of the present invention, the effective concentration of the daylily extract is at least 1 mg / mL.

[0020] Another object of the present invention is to provide the use of a daylily (Hemerocallis fulva Linn.) extract for preparing a composition with enhanced anti-glycation activity, wherein the daylily extract is obtained by extracting a daylily with a solvent, the solvent being water, alcohol, or a mixture of alcohol and water.

[0021] In one embodiment of the present invention, the effective concentration of the daylily extract is at least 100 mg / mL.

[0022] Another object of the present invention is to provide the use of a daylily (Hemerocallis fulva Linn.) extract for preparing a composition that inhibits cellular fat accumulation, promotes fat breakdown and increases skeletal muscle basal metabolic rate, wherein the daylily extract is obtained by extracting a daylily with a solvent, the solvent being water, alcohol, or a mixture of alcohol and water.

[0023] In one embodiment of the present invention, the effective concentration of the daylily extract is at least 0.0625 mg / mL.

[0024] Another object of the present invention is to provide the use of a daylily (Hemerocallis fulva Linn.) extract for preparing a composition that inhibits melanin production, enhances skin's UV resistance, increases superoxide dismutase (SOD) activity, and reduces the expression of reactive oxygen species (ROS), wherein the daylily extract is obtained by extracting a daylily with a solvent, the solvent being water, alcohol, or a mixture of alcohol and water.

[0025] In one embodiment of the present invention, the effective concentration of the daylily extract is at least 0.5 mg / mL.

[0026] Another object of the present invention is to provide the use of a daylily (Hemerocallis fulva Linn.) extract for preparing a composition that enhances mitochondrial activity and antioxidant capacity, wherein the daylily extract is obtained by extracting a daylily with a solvent, the solvent being water, alcohol, or a mixture of alcohol and water.

[0027] In one embodiment of the present invention, the effective concentration of the daylily extract is at least 1 mg / mL.

[0028] Another object of the present invention is to provide the use of a daylily (Hemerocallis fulva Linn.) extract for the preparation of an antidepressant composition, wherein the daylily extract is obtained by extracting a daylily with a solvent, the solvent being water, alcohol, or a mixture of alcohol and water.

[0029] In one embodiment of the present invention, the effective concentration of the daylily extract is at least 0.5 mg / mL.

[0030] In one embodiment of the invention, the composition is a food product or a health care product.

[0031] In summary, the efficacy of the daylily extract of this invention lies in its ability to regulate the expression levels of XRCC1, UNG, OGG1, MPG, ERCC1, ERCC6, GPX1, MLH1, MSH6, SOD, MKP-1, and DDC genes, thereby enhancing anti-glycation activity, inhibiting cellular fat accumulation, promoting fat breakdown, increasing skeletal muscle basal metabolic rate, anti-depression, inhibiting melanin production, enhancing skin's UV resistance, increasing SOD activity, reducing ROS expression, enhancing mitochondrial activity, and improving antioxidant capacity.

[0032] The following will further illustrate the embodiments of the present invention. The examples listed below are for illustrative purposes only 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

[0033] Figure 1 This is a data graph showing the efficacy of the daylily extract in anti-glycation.

[0034] Figure 2 This is a data graph showing the efficacy of the daylily extract in inhibiting fat accumulation.

[0035] Figure 3 This is a data graph showing the efficacy of the daylily extract of this invention in promoting fat decomposition, where "***" indicates a comparison with the control group, p<0.001;

[0036] Figure 4 This is a data graph showing the efficacy of the daylily extract of this invention in increasing the basal metabolic rate of skeletal muscle. In the graph, "***" indicates a comparison with the control group, p<0.001.

[0037] Figure 5This is a graph showing the efficacy of the daylily extract of this invention in inhibiting and suppressing the expression of the depression-related MKP-1 gene. In the graph, "**" indicates a comparison with the control group, p<0.01.

[0038] Figure 6 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing and promoting the expression of dopamine-related DDC genes. In the graph, "*" indicates a comparison with the control group, p<0.05.

[0039] Figure 7 This is a data graph showing the efficacy of the daylily extract of the present invention in resisting and defending against UVA, where "*" indicates a comparison with the UVA group, p<0.05;

[0040] Figure 8 This is a data graph showing the efficacy of the daylily extract of this invention in inhibiting melanin production, where "*" indicates a comparison with the control group, p<0.05;

[0041] Figure 9 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing the SOD activity of skin fibroblasts;

[0042] Figure 10 This is a data graph showing the efficacy of the daylily extract of the present invention in reducing the ROS expression level of skin fibroblasts, where "***" indicates p<0.001;

[0043] Figure 11 This is a data graph showing the efficacy of the daylily extract of the present invention in enhancing the mitochondrial activity of nerve cells, where "***" indicates a comparison with the control group, p<0.001;

[0044] Figure 12 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing the antioxidant capacity of nerve cells, where "***" indicates p<0.001;

[0045] Figure 13 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing the expression of genes related to the antioxidant capacity of nerve cells. In the graph, "*" indicates a comparison with the control group, p<0.05; "**" indicates a comparison with the control group, p<0.01; and "***" indicates a comparison with the control group, p<0.001. Detailed Implementation

[0046] definition

[0047] The values ​​used in this paper are approximate, and all experimental data are expressed within a 20% range, a better range of 10%, and a best range of 5%.

[0048] Statistical analysis was performed using Excel software. Data are expressed as mean ± standard deviation (SD), and differences between them were analyzed using Student's t-test.

[0049] According to this invention, daylily (Hemerocallis fulva Linn.), also known as daylily, forget-me-not, or golden needle flower, is a perennial herbaceous plant belonging to the genus Hemerocallis in the family Liliaceae. It grows to a height of approximately 60-80 cm, with fleshy underground roots. The leaves grow in clusters from the base, are linear and narrow, and can reach up to 80 cm in length. Daylily can be used for both food and medicine, and is effective in treating edema, difficulty urinating, urinary tract infections, leukorrhea, jaundice, and hematochezia.

[0050] As used in this article, the terms "inhibition of melanogenesis" and "inhibition of melanin synthesis", "depigmenting", "lightening the melanin", "whitening", "skin colorlightening", "bleaching", "brightening", "removing melanin", and "removing dark spots" can be used interchangeably.

[0051] 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 reagents 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 reagents fall within the scope of professional competence and routine practice for those skilled in the art.

[0052] 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 complex 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.

[0053] 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, and antiwrinkle agents. Topical agents include antiseptic agents, wound-healing agents, corticosteroids, and hormones. The selection and dosage of these topical agents fall within the scope of the professional competence and routine techniques of those skilled in this field.

[0054] 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.

[0055] 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.

[0056] Example 1. Preparation of daylily extract

[0057] First, daylily flowers (cultivated by farmers in Yuli Village, Hualien City) were homogenized from dry material. Then, at 50–100°C, the homogenized daylily flowers were extracted with an extraction solvent at a volume ratio of 5–20:1–5 for 0.5–3 hours to obtain a crude extract. The extraction solvent was water, alcohol, hydrous alcohol, or a combination thereof, with water being the preferred solvent. Next, the mixture was cooled to room temperature, and the crude extract was filtered through a 400-mesh filter to obtain a filtrate. The filtrate was then concentrated under reduced pressure at 45–70°C to obtain the daylily flower extract.

[0058] Example 2. Evaluation of the anti-glycation efficacy of daylily extract

[0059] This embodiment aims to test the anti-glycation activity of the daylily extract of the present invention. Therefore, the efficiency of inhibiting D-fructose-induced collagen glycation is used to quantify the glycation activity. First, 0.2 mL of water is taken as the control group, or 0.2 mL of the daylily extract of the present invention with a concentration of 100 mg / mL is taken as the experimental group. 0.2 mL of a collagen solution with a concentration of 60 mg / mL containing 0.06% NaN3 (prepared with 200 mM sodium phosphate buffer, pH 7.4) is added and mixed evenly with 0.2 mL of a 1.5 MD-fructose solution (prepared with 200 mM sodium phosphate buffer, pH 7.4). 0.1 mL of the mixed solution is taken as the origin product. After reacting the remaining mixed solution at 50°C for 24 hours, 0.1 mL is taken as the endpoint product. The fluorescence values ​​of the origin product and the endpoint product at an excitation wavelength of 360 nm and an emission wavelength of 460 nm are measured respectively. Finally, the efficiency of the ability to remove advanced glycation end-products (AGEs) is calculated using the following formula to represent its anti-glycation activity, where a lower amount of advanced glycation end-products indicates higher anti-glycation activity.

[0060]

[0061] The results of this embodiment are shown in Figure 1 It inhibits non-enzymatic browning and prevents denaturation of functional proteins in the body. Figure 1 This is a data graph showing the anti-glycation efficacy of the daylily extract of this invention. (From...) Figure 1 As can be seen, the percentage of glycation in the experimental group was significantly lower than that in the control group. Specifically, the percentage of glycation in the experimental group was reduced by approximately 56% compared to the control group. The results of this embodiment show that the daylily extract of the present invention can effectively enhance its ability to scavenge highly glycated end products and has excellent anti-glycation activity.

[0062] Example 3. Evaluation of the efficacy of daylily extract in inhibiting fat accumulation

[0063] This embodiment uses mouse bone marrow stromal cells to test the efficacy of the daylily extract of this invention in inhibiting fat accumulation. The mouse bone marrow stromal cells were purchased from the American Center for Type Culture Collection (ACC). ), number CRL-2749 TM The cells were cultured before differentiation in pre-adipocyte expansion medium, which contained 90% minimum essential medium (alpha medium, purchased from Gibco, USA, 12100-046), 20% fetal bovine serum (FeBV), and 1% penicillin-streptomycin (PST). Mouse bone marrow stromal cells were then differentiated using differentiation medium, which contained 90% minimum essential medium (alpha medium), 20% fetal bovine serum, and 1% penicillin-streptomycin. Cellular lipids were stained with Oil Red O (Sigma, USA), with a 3 mg / mL Oil Red O stock solution prepared in 100% isopropanol, and the stock solution was further prepared into a 60% reaction solution using ddH2O.

[0064] To demonstrate that the daylily extract of this invention has the effect of inhibiting fat accumulation, mouse bone marrow stromal cells were first differentiated into adipocytes. First, 8 × 10 4 Mouse bone marrow stromal cells (OP9) were seeded with 500 μL of preadipocyte expansion culture medium in 24-well culture dishes and cultured at 37°C for 7 days, with fresh differentiation medium replaced every 3 days. Subsequently, lipid droplet formation was observed using a ZEISS microscope to ensure complete cell differentiation.

[0065] Next, the OP9 cells were divided into two groups: a control group and an experimental group. 0.25 mg / mL of daylily extract was added to the cells in the experimental group, while the cells in the control group received no treatment. The cells were then cultured for 7–10 days, with the culture medium changed every 3 days.

[0066] Next, Oil Red O was used to stain the lipids in the cells to assess whether the daylily extract of the present invention could indeed reduce lipid accumulation. First, the culture medium was gently removed and the cells were washed twice with 1 mL of phosphate buffered saline (PBS). Then, 1 mL of 10% formaldehyde (purchased from Echo Chemical, Taiwan, Cat.TG1794-4-0000-72NI) was added and reacted at room temperature for 30 minutes to fix the cells. After removing the formaldehyde, the cells were gently washed twice with 1 mL of PBS. Then, 1 mL of 60% isopropanol (purchased from Echo Chemical, Taiwan, PH-3101) was added to each well of the cells and reacted for 1 minute. After removing the isopropanol, 1 mL of Oil Red O reaction solution was added and reacted at room temperature for 1 hour. Then, the Oil Red O reaction solution was removed and the cells were quickly destained with 1 mL of 60% isopropanol for 5 seconds. Finally, the cells were photographed and quantified using a microscope. Next, 100% isopropanol was added to the stained cells, and the mixture was placed on a shaker for 10 minutes to dissolve the oil droplets. Then, 100 μL was transferred to a 96-well culture dish, and the OD values ​​of each group were measured using an ELISA reader (BioTek). 510 nm Read the value to quantify the oil red O.

[0067] The results of this embodiment are shown in Figure 2 . Figure 2 This is a data graph showing the efficacy of the daylily extract in inhibiting fat accumulation, as described in this invention. Figure 2 As can be seen, the relative amount of fat droplets in the experimental group was reduced compared to the control group. Specifically, the relative amount of fat droplets in the experimental group was reduced by approximately 15% compared to the control group. The results of this embodiment show that the daylily extract of the present invention has the effect of inhibiting cellular fat accumulation.

[0068] Example 4. Evaluation of the efficacy of daylily extract in promoting lipolysis

[0069] First, 8×10 4 Mouse bone marrow stromal cells OP9 ( CRL-2749 TMCells were seeded in 24-well culture dishes with 500 μL of pre-adipocyte expansion medium (containing 90% minimum essential medium alpha medium (Gibco), 20% fetal bovine serum (Gibco), and 1% penicillin / streptomycin (Gibco)) and cultured at 37°C for 7 days, with fresh differentiation medium (containing 90% minimum essential medium alpha medium, 20% fetal bovine serum, and 1% penicillin / streptomycin) added every 3 days. Then, droplet formation was observed under a ZEISS microscope to confirm complete cell differentiation.

[0070] Next, OP9 cells were divided into two groups: a control group and an experimental group. 0.0625 mg / mL of daylily extract was added to the cells in the experimental group, while the cells in the control group received no treatment. The cells in each group were then cultured for 7–10 days, with the culture medium changed every 3 days. Subsequently, glycerol assays were performed on the cells in each group using a glycerol cell-based assay kit (Cayman).

[0071] Cell culture supernatant was collected from each well, and then 25 μL of cell culture supernatant from each well, along with the standard solution, was transferred to a new 96-well culture dish. Next, 100 μL of reconstituted free glycerol assay reagent was added to each well, and the mixture was incubated at room temperature for 15 minutes. Afterward, the OD values ​​for each group were read using an ELISA reader (BioTek). 540 nm Read value.

[0072] The results of this embodiment are shown in Figure 3 . Figure 3 This is a data graph showing the efficacy of the daylily extract in promoting fat breakdown, as described in this invention. Figure 3 As can be seen, the percentage of fat breakdown in the experimental group was significantly increased compared to the control group. Specifically, the percentage of fat breakdown in the experimental group increased by approximately 10% compared to the control group. The results of this embodiment demonstrate that the daylily extract of the present invention has the effect of promoting fat breakdown.

[0073] Example 5. Evaluation of the efficacy of daylily extract in increasing skeletal muscle basal metabolic rate

[0074] This embodiment uses mouse skeletal muscle cells C2C12 to test the efficacy of the daylily extract of the present invention in increasing the basal metabolic rate of skeletal muscle. The mouse skeletal muscle cells C2C12 were purchased from the American Center for Type Culture Collection (USA). ), number CRL-1772 TM .

[0075] First, cells were cultured in Dubco's modified IgM medium (DMEM) supplemented with 3.7 g / L sodium bicarbonate, 10% fetal bovine serum (FBS) (Gibco), 100 IU penicillin, and 100 μg / mL streptomycin. Then, the cells were seeded into 6-well cell culture dishes. Once the cells reached near 100% confluence, they were co-cultured with conditioned medium (DMEM containing 1% FBS and 1% horse serum) to allow differentiation into myotubes.

[0076] The differentiated cells were then divided into two groups: a control group and an experimental group. 0.0625 mg / mL of daylily extract was added to the cells in the experimental group, while the cells in the control group received no treatment. The cells were then cultured for 48 hours. Cells were then lysed in each well with 100 μL of pyruvate analysis buffer, followed by centrifugation at 10,000 g for 10 minutes at 4°C, and the supernatant was collected. Next, 20 μL of lysis buffer (triple replicate) was added to each 96-well culture dish, and the volume was adjusted to 50 μL / well with pyruvate analysis buffer. A standard curve was then prepared for colorimetric assay (BioVision). Pyruvate standards were diluted to 1 nmol / μL, and then prepared at concentrations of 0, 2, 4, 6, 8, and 10 nmol / well, for a total of 50 μL / well. Next, 50 μL of the reaction mixture (containing 46 μL of pyruvate analysis buffer, 2 μL of pyruvate probe, and 2 μL of enzyme mixture) was added to each well, thoroughly mixed, and reacted for 30 minutes. Afterward, the absorbance was measured at 570 nm, and the pyruvate content was calculated using linear interpolation. Finally, the total protein concentration of each sample was normalized (Bradford assay) (Bio-Rad).

[0077] The results of this embodiment are shown in Figure 4 . Figure 4 This is a data graph showing the efficacy of the daylily extract of this invention in increasing the basal metabolic rate of skeletal muscle. (From...) Figure 4 As can be seen, the relative pyruvate concentration in the experimental group was significantly increased compared to the control group. Specifically, the relative pyruvate concentration in the experimental group increased by approximately 80% compared to the control group. The results of this embodiment demonstrate that the daylily extract of the present invention has the effect of increasing the basal metabolic rate of skeletal muscle.

[0078] Example 6. Evaluation of the antidepressant efficacy of daylily extract

[0079] This embodiment explores whether daylily extract can achieve antidepressant effects by regulating and inhibiting depression and promoting the expression of dopamine-related genes.

[0080] Human neuroblastoma cells (SH-SY5Y; purchased from DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin) were cultured. CRL-2266 TM The cells were placed in 6-well plates with 2 mL of culture medium at a concentration of 1.5 × 10⁻⁶ cells / well. 5 Cells / pores.

[0081] The cells were then divided into two groups: a control group and an experimental group. 0.5 mg / mL of daylily extract was added to the cells in the experimental group, while the cells in the control group received no treatment. The cells were then cultured in an incubator for 48 hours, and the cell cultures were collected for gene expression analysis.

[0082] In this embodiment, the gene used to analyze the inhibition of depression is the mitogen-activated protein kinase phosphatase 1 (MKP-1) gene, and the gene used to analyze the promotion of dopamine is the dopa decarboxylase (DDC) gene.

[0083] RNA was extracted from the cell cultures obtained above using the Geneaid RNA extraction kit. 2,000 ng of each RNA group was then extracted and... 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 MKP-1, DDC, and GAPDH (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 SYBR 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.

[0084] Table 1

[0085]

[0086] 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 means of the 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. Statistical significance between groups was determined using a one-tailed Stuttgart t-test. The results of this embodiment are shown in... Figure 5 and Figure 6 .

[0087] Figure 5 This is a graph showing the efficacy of the daylily extract of this invention in inhibiting and suppressing the expression of the depression-related MKP-1 gene. Figure 5 It can be seen that, compared with the control group, the relative gene expression level of the experimental group was significantly reduced, with the relative gene expression level of the experimental group decreasing by about 16.5% compared with the control group. Figure 6 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing and promoting the expression of the dopamine-related DDC gene. (From...) Figure 6 As can be seen, compared with the control group, the relative gene expression level of the experimental group was significantly increased, with an increase of about 10% compared with the control group. The results of this embodiment show that the daylily extract of the present invention can achieve the effects of antidepressant and mood improvement by regulating and inhibiting depression and promoting the expression of dopamine-related genes.

[0088] Example 7. Evaluation of the efficacy of daylily extract in resisting and defending against UVA.

[0089] First, human skin fibroblasts (CCD-966Sk, corresponding to...) CRL-1881 was cultured in minimum essential medium (Gibco) [containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 1 mM sodium pyruvate]. 200 μL of medium was added to each well of a 96-well culture dish to achieve a bacterial count of 5 x 10⁻⁶ cells / well. 3 Human skin fibroblasts were cultured at 37°C for 24 hours, after which the culture medium was removed.

[0090] Subsequently, human skin fibroblasts were divided into three groups: an experimental group, a UVA group, and a control group. Cells in the experimental and UVA groups were treated with ultraviolet radiation at 15 J / cm². 2 Irradiating cells with UVA for 1 hour under these conditions will result in a 50% lethal dose (LD50). 50 The value () indicates the radiation dose that causes 50% cell death. Next, 1 mg / mL of daylily extract was added to the cells in the experimental group, while the cells in the UVA group received no treatment. The cells in the control group were not exposed to UVA.

[0091] After culturing cell cultures at 37°C for 24 hours, 15 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 4 mg / mL, prepared in PBS) (AMERSCO / 0793-5G) was added to each well, and the cells were incubated at 37°C for 4 hours. Afterward, the culture medium was removed from each well, and 50 μL of DMSO (ECHO / DA1101-000000-72EC) was added to each well to decompose formazan crystals. The culture dishes were then placed on a shaker and incubated for 10 minutes. The absorbance (OD) of each well was then read at 570 nm using an ELISA reader (BioTek). 570 ).

[0092] Cell viability (%) is determined by measuring the absorbance (OD) value. 570 Substituting into the following formula (1), we can calculate:

[0093] Cellular activity (%) = (OD measured in each group) 570 Absorbance / OD measured in the control group 570 (Absorbance) × 100% (1)

[0094] Statistical significance between groups was determined using Stuttgart's t-test. The results of this embodiment are shown in... Figure 7 .

[0095] Figure 7 This is a data graph showing the efficacy of the daylily extract in resisting and defending against UVA. (From...) Figure 7 As can be seen, compared with the control group, the cell viability (%) in the UVA group was reduced, indicating that UVA irradiation of human skin fibroblasts causes cell death. In contrast, the cell viability (%) in the experimental group was significantly increased (approximately 8% increase compared to the UVA group). The results of this embodiment demonstrate that the daylily extract of the present invention has the ability to resist and defend against UVA damage and enhances the skin cells' resistance to ultraviolet radiation.

[0096] Example 8. Evaluation of the efficacy of daylily extract in inhibiting melanin production

[0097] First, the mouse skin melanoma cell line B16F10 (corresponding to ATCC CRL-6475) was cultured in Dulbecco's Modified Eagle's Medium (DMEM) [supplemented with 1% penicillin / streptomycin (Gibco) and 10% FBS (Gibco)]. 3 mL of the medium was added to each well of a 6-well culture dish to achieve a surface temperature of 1.5 x 10⁻⁶ cells / well. 5 B16F10 cells were cultured at 37°C for 24 hours, and then the culture medium was removed.

[0098] The B16F10 cells were then divided into two groups: an experimental group and a control group. 0.5 mg / mL of daylily extract was added to the cells in the experimental group, while the cells in the control group received no treatment.

[0099] After culturing cell cultures at 37°C for 48 hours, the culture medium was removed and the cells were washed twice with 1xPBS (Gibco). Then, trypsin was added to treat the cells for 3 minutes, and the suspended cells were collected in 15 mL centrifuge tubes. The cells were then spin-dried at 400xg for 5 minutes to pellet the cells. After washing twice with 1xPBS, the cell pellet was resuspended in 200 μL of 1xPBS. The cell solution was then placed in liquid nitrogen for 10 minutes, followed by thawing at room temperature for 30 minutes. After complete thawing, the cells were spin-dried at 12,000xg for 30 minutes, the supernatant was removed, and 120 μL of 1N NaOH (prepared with ddH2O) was added. After thorough mixing, the cells were incubated in a dry bath at 60°C for 1 hour. Next, 100 μL was transferred to a 96-well culture dish, and the absorbance (OD) of each well was read at a wavelength of 450 nm using an ELISA reader. 450 ).

[0100] Melanin content (%) is determined by measuring the absorbance (OD) value. 450 Substituting into the following formula (2), the result is calculated as follows:

[0101] Melanin content (%) = (OD measured in each group) 450 Absorbance / OD measured in the control group 450 (Absorbance) × 100% (2)

[0102] Statistical significance between groups was determined using Stuttgart's t-test. The results of this embodiment are shown in... Figure 8 .

[0103] Figure 8 This is a data graph showing the efficacy of the daylily extract in inhibiting melanin production, as described in this invention. Figure 8 As can be seen, compared with the control group, the melanin content in the experimental group was significantly reduced (approximately 11% lower than that in the control group). The results of this embodiment demonstrate that the daylily extract of the present invention has the effect of inhibiting melanin production.

[0104] Example 9. Evaluation of the efficacy of daylily extract in enhancing superoxide dismutase (SOD) activity in dermal fibroblasts.

[0105] Human skin fibroblasts were purchased from the Bioresource Collection and Research Center (BCRC), Taiwan, catalog number BCRC 60153. These cells were cultured in minimum essential medium (MEM) (Eagle) (prepared with Earle's Balanced Salt Solution (Earle's BSS)) (GIBCO, catalog number 41500-034, USA), supplemented with 10% fetal bovine serum (FBS) (GIBCO, catalog number 10438-026, USA), 0.1 mM non-essential amino acids, 1.5 g / L sodium bicarbonate (Sigma, catalog number S5761, USA), and 1 mM sodium pyruvate (GIBCO, catalog number 11360-070, USA).

[0106] In a 6-well culture dish containing 2 mL of the above-described culture medium, inoculate each well with 2 × 10⁶ cells / well. 5 Human skin fibroblasts were cultured overnight at 37°C. After removing the culture medium, the cells were divided into two groups: a control group and an experimental group. 0.5 mg / mL of daylily extract and 1 mM H2O2 were added to the experimental group cells and incubated at 37°C for 6 hours, while the control group cells received no treatment. All cells were then cultured at 37°C for 24 hours. Afterward, the culture medium was removed, and the cells were washed once with 1X PBS (Gibco). Trypsin was then added to the cells for 3 minutes, followed by rinsing with culture medium to stop trypsin activity, and the cells were transferred to 1.5 mL centrifuge tubes. The cells were then centrifuged at 300 x g for 5 minutes, washed with PBS, and then centrifuged at 300 x g for 5 minutes. 30 μL of 1X cell extraction buffer was added, and the cell suspension was incubated on ice for 30 minutes, vortexed every 10 minutes. Next, the lysed cell suspension was centrifuged at 10,000 x g for 10 minutes at 4°C to remove insoluble matter, and then 25 μL of 1X SOD buffer was added to the bottom of the active control wells. After that, 25 μL of the prepared SOD standards (S1 to S7, see Table 2) were added.

[0107] Table 2

[0108]

[0109] Next, 25 μL of sample was added to the bottom of the appropriate well, followed by 150 μL of Master Mix to each well. Then, 25 μL of 1X xanthine solution was added to all wells using a multichannel pipette to initiate the reaction. The culture dish was then immediately transferred to a microtiter plate reader, and absorbance was read at 450 nm per minute at room temperature for 10 minutes.

[0110] Statistical significance between groups was determined using Stuttgart's t-test. The results of this embodiment are shown in... Figure 9 .

[0111] Figure 9 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing the SOD activity of dermal fibroblasts. Figure 9 As can be seen, the SOD activity in the experimental group was significantly increased compared with the control group (the SOD activity in the experimental group increased by approximately 30.7% compared with the control group). The results of this embodiment show that the daylily extract of the present invention has the effect of increasing the SOD activity of skin fibroblasts.

[0112] Example 10. Evaluation of the efficacy of daylily extract in reducing the expression of reactive oxygen species (ROS) in dermal fibroblasts.

[0113] First, human skin fibroblasts CCD-966SK (BCRC 60153) were cultured in 6-well plates in minimum essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 1 mM sodium pyruvate. The cell concentration was 2 × 10⁶ cells / mL in 2 mL of medium. 5Cells / wells were then cultured at 37°C for 24 hours, and the culture medium was removed. Next, the cultured cells were divided into three groups: a control group, a hydrogen peroxide group, and an experimental group. 1 mg / mL of daylily extract and 1 mM H2O2 were added to the cells in the experimental group. Cells in the hydrogen peroxide group were added with 1 mM H2O2, while cells in the control group were left untreated. Subsequently, 5 μg / mL of dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Sigma / SI-D6883-50MG) (stock solution was 5 mg / mL dissolved in DMSO) was added and the reaction was carried out at 37°C for 1 hour, followed by washing each well twice with 1 mL of 1X PBS (Gibco). Next, 200 μL of trypsin was added and the mixture was reacted in the dark for 5 minutes. The cell culture was then collected into 1.5 mL centrifuge tubes and centrifuged at 400 × g for 10 minutes. The supernatant was then removed, and the cells were washed once with 1X PBS, followed by centrifugation at 400 × g for 10 minutes. The supernatant was then removed, and the cell pellet was resuspended in 1 mL of 1X PBS. The fluorescence signal of DCFH-DA was then detected using a flow cytometer (BD Accuri) at excitation wavelengths of 450–490 nm and emission wavelengths of 510–550 nm to calculate the percentage of cells damaged by ROS. Statistically significant differences between groups were determined using Stuttgart's t-test. The results of this example are shown in... Figure 10 .

[0114] Figure 10 This is a data graph showing the efficacy of the daylily extract of this invention in reducing ROS expression levels in dermal fibroblasts. (From...) Figure 10 As can be seen, compared with the control group, the percentage of cells damaged by ROS (i.e., cells exhibiting high oxidative stress) was significantly increased in the hydrogen peroxide group, indicating that hydrogen peroxide causes significant ROS damage to cells. Conversely, compared with the hydrogen peroxide group, the percentage of cells damaged by ROS was significantly reduced in the experimental group (a reduction of approximately 47% in ROS expression). The results of this embodiment demonstrate that the daylily extract of the present invention has the effect of reducing ROS expression in dermal fibroblasts, thereby improving skin texture.

[0115] Example 11. Evaluation of the efficacy of daylily extract in enhancing mitochondrial activity of nerve cells

[0116] This embodiment uses mouse neuroblastoma cells (Neuro-2a) to analyze mitochondrial activity and employs a flow cytometry mitochondrial membrane potential detection kit (BD) for experiments. The mouse neuroblastoma cells (Neuro-2a) were purchased from the American Center for Type Culture Collection (ACC). ), number CCL-131 TM The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (GIBCO, catalog number 10438-026, USA) and 1% penicillin / streptomycin (Gibco).

[0117] In a 6-well culture dish containing 2 mL of the above-described culture medium, inoculate each well with 1 × 10⁻⁶ cells / well. 5 Two neuroblastoma cells (n=2) were collected. The cells were then divided into two groups: a control group and an experimental group. 1 mg / mL of daylily extract was added to the cells in the experimental group, while the cells in the control group were added to culture medium. The cells were then cultured at 37°C for 24 hours, followed by preheating of the 10X analysis buffer at 37°C. Next, a 1X analysis buffer was prepared using sterile 1X PBS, mixed thoroughly, and incubated at 37°C. Then, 130 μL of dimethyl sulfoxide (DMSO) was added to the lyophilized JC-1 mitochondrial dye (BD). TM The JC-1 stock solution was prepared using the MitoScreen (JC-1) reagent kit and can be stored at -20°C for up to 6 months. Next, a working solution was prepared by mixing JC-1 mitochondrial stain with 1X analysis buffer at a ratio of 1:100. The culture medium was then removed, and the cells were washed twice with 1X PBS. Afterward, the cells were treated with trypsin / EDTA for 3 minutes, and the suspended cells were transferred to a 1.5 mL microcentrifuge tube and centrifuged at 400 g for 5 minutes to collect the precipitated cells.

[0118] After removing the supernatant, cells were resuspended in 1 mL of 1X PBS and transferred to 1.5 mL centrifuge tubes, centrifuged at 400 g for 5 minutes. After removing the supernatant, 100 μL of JC-1 working solution was added, mixed thoroughly, and incubated in the dark for 15 minutes. Cells were then centrifuged at 400 g for 5 minutes, washed with 1 mL of 1X washing buffer, and centrifuged at 400 g for 5 minutes, followed by another wash with 1 mL of 1X washing buffer and centrifuged at 400 g for 5 minutes. Cells were resuspended in 500 μL of 1X PBS containing 2% FBS, and changes in mitochondrial membrane potential during apoptosis were observed using flow cytometry (BD Accuri). Stuttgart's t-test was used in Excel to statistically analyze the differences between sample populations.

[0119] Figure 11 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing mitochondrial activity in nerve cells. (From...) Figure 11 As can be seen, compared with the control group, the mitochondrial activity (i.e., the relative JC-1 aggregate) of neuroblasts in the experimental group was significantly increased (the experimental group increased neuronal mitochondrial activity by approximately 13% compared with the control group). The results of this embodiment show that the daylily extract of the present invention has the effect of enhancing the mitochondrial activity of neuroblasts.

[0120] Example 12. Evaluation of the efficacy of daylily extract in enhancing the antioxidant capacity of nerve cells

[0121] This embodiment uses mouse neuroblastoma cells (Neuro-2a) to analyze the antioxidant capacity of nerve cells. Mouse neuroblastoma cells (Neuro-2a) were purchased from the American Center for Type Culture Collection (ACC). ), number CCL-131 TM Neuro-2a brain cells were cultured in 6-well plates in DMEM supplemented with 10% fetal bovine serum (FBS) (GIBCO, catalog number 10438-026, USA) and 1% penicillin / streptomycin (Gibco). The cell concentration was 1×10⁶ cells / mL in 2 mL of medium. 5Cells / wells were then cultured at 37°C for 24 hours, and the culture medium was removed. Next, the cultured cells were divided into three groups: a control group, a hydrogen peroxide group, and an experimental group. 1 mg / mL of daylily extract and 1 mM H2O2 were added to the cells in the experimental group and incubated at 37°C for 1 hour. Cells in the hydrogen peroxide group were added with 1 mM H2O2 and incubated at 37°C for 1 hour, while the control group cells were left untreated. Subsequently, 5 μg / mL of dichloro-dihydro-fluoresceindiacetate (DCFH-DA) (Sigma / SI-D6883-50MG) (stock solution: 5 mg / mL dissolved in DMSO) was added and reacted at 37°C for 15 minutes, followed by washing each well twice with 1 mL of 1X PBS (Gibco). Next, 200 μL of trypsin was added and the mixture was reacted in the dark for 5 minutes. The cell culture was then collected into 1.5 mL centrifuge tubes and centrifuged at 400 × g for 10 minutes. The supernatant was then removed, and the cells were washed once with 1X PBS, followed by centrifugation at 400 × g for 10 minutes. The supernatant was then removed, and the cell pellet was resuspended in 1 mL of 1X PBS. The fluorescence signal of DCFH-DA was then detected using a flow cytometer (BD Accuri) at excitation wavelengths of 450–490 nm and emission wavelengths of 510–550 nm to calculate the percentage of cells damaged by ROS. Statistically significant differences between groups were determined using Stuttgart's t-test. The results of this example are shown in... Figure 12 .

[0122] Figure 12 This is a data graph showing the efficacy of the daylily extract in enhancing the antioxidant capacity of nerve cells. Figure 12 As can be seen, compared with the control group, the percentage of cells damaged by ROS (i.e., relative ROS production) in the hydrogen peroxide group was significantly increased, indicating that hydrogen peroxide causes a large amount of ROS damage to cells; while compared with the hydrogen peroxide group, the percentage of cells damaged by ROS in the experimental group was significantly reduced (reduced relative ROS production by approximately 73%). The results of this embodiment show that the daylily extract of the present invention has the effect of enhancing the antioxidant capacity of nerve cells.

[0123] Example 13. Evaluation of the efficacy of daylily extract in enhancing the expression of genes related to antioxidant activity in nerve cells.

[0124] This embodiment explores whether daylily extract can enhance the antioxidant capacity of nerve cells by increasing the expression of genes related to nerve cell antioxidant activity.

[0125] Mouse neuroblastoma cells (Neuro-2a, purchased from the American Center for Type Culture Collection) were cultured in DMEM medium (containing 10% fetal bovine serum and 1% penicillin / streptomycin). ), number CCL-131 TM The cells were placed in 6-well plates with 2 mL of culture medium at a concentration of 1.5 × 10⁻⁶ cells / well. 5 Cells / pores.

[0126] The cells were then divided into two groups: a control group and an experimental group. 1 mg / mL of daylily extract and 200 μM H2O2 were added to the cells in the experimental group, while the cells in the control group were added with 200 μM H2O2. The cells were then cultured in an incubator for 6 hours, and the cell cultures were collected for gene expression analysis.

[0127] In this embodiment, the genes used to analyze those related to the antioxidant capacity of nerve cells include X-ray repair cross complementary protein 1 (XRCC1) gene, uracil DNA glycosylase (UNG) gene, 8-oxoguanine DNA glycosylase (OGG1) gene, N-methylpurine DNA glycosylase (MPG) gene, ERCC excision repair 1 (endonuclease non-catalytic subunit, ERCC1) gene, ERCC excision repair 6 (endonuclease non-catalytic subunit, ERCC6) gene, glutathione peroxidase 1 (GPX1) gene, and mutL homolog 1 (mutL... The genes involved include homolog1 (MLH1), melanocyte-stimulating hormone 6 (MSH6), superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2).

[0128] RNA was extracted from the cell cultures obtained above using the Geneaid RNA extraction kit. 2,000 ng of each RNA group was then extracted and... 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 XRCC1, UNG, OGG1, MPG, ERCC1, ERCC6, GPX1, MLH1, MSH6, SOD1, SOD2, and GAPDH (as an internal control), their nucleotide sequences are shown in Table 3 below. Quantitative real-time PCR was performed in the StepOne Plus Real-Time PCR System (ABI) using the KAPA SYBRFAST 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.

[0129] Table 3

[0130]

[0131] 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 means of the 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. Statistical significance between groups was determined using a one-tailed Stuttgart t-test. The results of this embodiment are shown in... Figure 13 .

[0132] Figure 13 This is a data graph showing the efficacy of the daylily extract of this invention in enhancing the expression of genes related to antioxidant activity in nerve cells. Figure 13 As can be seen, compared with the control group, the experimental group showed a significant increase in the relative expression of genes (an increase of approximately 12.5% ​​to 195% in the expression of various nerve cell antioxidant-related genes). The results of this embodiment show that the daylily extract of the present invention can enhance the antioxidant capacity of nerve cells by increasing the expression of genes related to nerve cell antioxidant activity.

[0133] In summary, the daylily extract of this invention can regulate the expression levels of the XRCC1, UNG, OGG1, MPG, ERCC1, ERCC6, GPX1, MLH1, MSH6, SOD, MKP-1, and DDC genes, thereby enhancing anti-glycation activity, inhibiting cellular fat accumulation, promoting fat breakdown, increasing skeletal muscle basal metabolic rate, combating depression, improving mood, inhibiting melanin production, enhancing skin's UV resistance, increasing SOD activity, reducing ROS expression, improving skin texture and condition, enhancing mitochondrial activity, enhancing antioxidant capacity, and protecting nerve cells.

[0134] 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. The use of a daylily extract in the preparation of a composition for inhibiting melanin production and / or enhancing the skin's UV resistance, characterized in that, The daylily extract was obtained by extracting daylily (Hemerocallis fulva Linn.) with water at 50-100℃ for 0.5-3 hours at a volume ratio of 5-20:1-5.

2. The use according to claim 1, characterized in that, The effective concentration of the daylily extract is at least 0.5 mg / mL.

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