TWI789553B - Use of hemerocallis fulva linn. extract for manufacturing of a composition for promoting skeletal muscle basal metaboilc rate - Google Patents

Abstract

The present disclosure provides a use of a Hemerocallis fulva Linn. extract for manufacturing of a composition for improving skin condition, wherein the improving skin condition include improving skin elasticity, whitening skin, maintaining skin structure improving UV resistance of the skin,.

Description

Golden Needle Flower Extract Used to Prepare a Composition for Improving Basal Metabolic Rate of Skeletal Muscle the use of

The present invention relates to a kind of golden needle flower extract used to regulate XRCC1 gene, UNG gene, OGG1 gene, MPG gene, ERCC1 gene, ERCC6 gene, GPX1 gene, MLH1 gene, MSH6 gene, SOD gene, MKP-1 gene, and DDC Gene expression, increase anti-glycation activity, inhibit cell fat accumulation, promote fat decomposition, increase skeletal muscle basal metabolic rate, anti-depression, inhibit melanin production, improve skin anti-ultraviolet ability, increase SOD activity, reduce ROS expression, increase grain Cellular activity and the purpose of enhancing antioxidant capacity.

Fat is an essential component in the human body, but excessive fat content will cause damage to the human body. In countries where the national economy is taking off more and more, the problem of obesity (also known as metabolic syndrome) will only increase. Therefore, from the perspective of the Asian region, China will be the country where the next wave of obesity problems will increase significantly.

In addition, Taiwan has now become the "fattest country in Asia". Among citizens over the age of 18, 51.1% of men are overweight, and 35.8% of women are overweight, and the proportion is still rising, and even overweight The age group is also declining year by year, and the proportion of overweight primary and middle school students has risen to more than 25%, ranking the eighth highest in the world. Therefore, obesity is an urgent problem to be solved.

In addition, glycation is related to skin and internal aging, and it is also related to diabetes, which is very harmful to the health of Chinese people. The essence of the saccharification reaction is the Maillard reaction (Maillard reaction) or carbonyl-amine browning reaction (carbonyl-amine browning), which is the aldehyde (ketone) group of sugar and amine-containing substances (such as protein, peptide, amine group A nonenzymatic glycosylation (glycosylation) reaction between the amino groups of acids, phospholipids, nucleic acids, or derivatives of the above. Chronic hyperglycemia Under this condition, it will cause glucose auto-oxidation and protein glycation to form advanced glycation end products (AGEs). And AGEs can cause a variety of aging diseases, such as skin wrinkles, cataracts, atherosclerosis, kidney failure and so on.

In recent years, the number of people suffering from depression is increasing. In addition, when the so-called pseudodepressants are included, the number increases to a considerably high proportion.

The cause of depression is still not clearly understood, but it is not limited to biological factors and personality factors. For example: the drastic changes in the modern social environment, the social structure of abandoning the weak, the trend of being dominated by a single value and excluding heresies, etc. are considered to be inseparable from the increase in depression patients.

The treatment of depression can be, for example: rest more first, medication second, or a combination of the two. However, in reality, it is not easy to allow rest, etc., but in fact it is mostly covered up with antidepressants.

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 large amounts of ultraviolet rays (ultraviolet, UV), ionizing radiation, drugs or xenobiotics can promote the generation of reactive oxygen species (reactive oxygen species, ROS) and free radicals (free radicals) in the skin. When the amount of accumulated reactive oxygen species and free radicals exceeds the antioxidant capacity of cells or tissues, oxidative stress will be formed. Then, reactive oxygen species and free radicals will react with intracellular components (including DNA, protein and lipids, etc.), and then have unwanted effects on the skin.

Melanogenesis (melanin synthesis) refers to when skin melanocytes (dermal melanocytes) are subjected to environmental factors (such as ultraviolet (ultraviolet, UV)) or physiological factors (such as fatigue (fatigue), stress ( After the induction of stress), chronic inflammation (chronic inflammation) and abnormal release of α-melanocyte stimulating hormone (α-MSH) in the body), tyrosine (tyrosine) in melanocytes The process of being converted into melanin through the catalysis of tyrosinase (which is the rate-limiting step of melanin production) and a series of redox reactions. Melanin can protect the hypodermis of the skin from photodamage caused by ultraviolet rays, but when melanin is accumulated in large amounts on the skin or distributed abnormally, it may cause skin disorders such as freckles (lentigines), freckles, melasma, age spots, and hyperpigmentation.

In order to achieve the purpose of skin whitening, many melanogenesis inhibitors have been used to lighten or remove melanin or dark spots accumulated on the skin, and most of these melanogenesis inhibitors are through the following The mechanism of action to regulate the production of melanin: (1) Before melanin production: such as inhibiting the mRNA transcription of tyrosinase (mRNA transcription) (such as C2-ceramide (C2-ceramide), vitamin A acid (tretinoin) and vanillic acid) and glycosylation (such as calcium D-pantetheine-S-sulfonate (PaSSO3Ca)); (2) during melanin production: such as inhibition of tyrosinase activity (such as hydroquinone (hydroquinone), arbutin (arbutin) and kojic acid (kojic acid)), accelerated degradation of tyrosinase (such as linoleic acid (linoleic acid) and phenylthiourea ( phenylthiourea)), and promote the reduction of dopaquinone (reduction) (such as ascorbic acid (ascorbic acid)); and (3) after melanin production: for example, promote the decomposition of melanin (such as linoleic acid (linoleic acid)), Inhibit melanosome transfer (such as niacinamide and serine protease inhibitor), and accelerate skin turnover (such as liquiritin and glycolic acid) acid)).

In recent years, human needs for skin whitening and maintenance are increasing day by day. However, the melanin synthesis inhibitors currently used are still not satisfactory in lightening or removing melanin. In addition, most of the currently common ways to solve the problem of melanin production are to use medicines or skin care products that are applied to the skin surface.

Cerebrovascular disease currently accounts for the second highest death rate in Taiwan. Even if the disease is mild, the patient and his family will be financially and mentally burdened. Yuan died of hypoxia due to ischemia. At present, it is known that the pathological mechanism of some central nervous system injury death is that after cerebral ischemia/hypoxia, the energy supply system of neurons is disturbed, and a large amount of neurotransmitter (glutamic acid) is released, which affects calcium through activation of NMDA receptors. The ion stabilization system produces free radicals and nitric oxide, causing secondary damage to cells. Research in the last few years has found that free radicals produced by oxidation are also an important cause of nerve cell death, and many antioxidant genes and reaction mechanisms have also been discovered.

On the other hand, mitochondria (mitochondria) is also known as the cell's power station, because it is the main site for the synthesis of adenosine triphosphate (ATP) (a molecule that transmits energy) in the cell, providing energy for various activities of the cell. chemical energy. If the mitochondria are damaged, the impact on cells and individual organisms is huge. Mitochondria will produce a lot of free radicals in the process of synthesizing ATP, free radicals The activity is extremely strong, and it will have a strong oxidation reaction with any substance in the body and destroy its normal function. Free radicals accumulatively damage the enzymes and DNA in the mitochondria, gradually reducing their function and further weakening the function of various organs and tissues. Therefore, how to improve the mitochondrial activity of nerve cells, and then enhance the antioxidant capacity of nerve cells to achieve the effect of protecting nerves, has become an important topic in this field.

However, most of the medicines, food products or skin care products currently used to solve the above problems are made of chemical ingredients. Long-term use is not only harmful to human health, but also these products are often expensive and cannot be afforded by ordinary users. In order to solve the above problems, those skilled in the art urgently need to develop anti-glycation activity, inhibit cell fat accumulation, promote lipolysis, increase skeletal muscle basal metabolic rate, anti-depression, inhibit melanin production, improve skin anti-ultraviolet ability, enhance skin Novel medicines, food products or skin care products with superoxide dismutase (superoxide dismutase, SOD) activity, reduction of ROS expression, improvement of mitochondrial activity, and anti-oxidation ability to benefit the majority of groups in need.

In view of this, the purpose of the present invention is to provide a kind of golden needle flower ( Hemerocallis fulva Linn.) extract to be used for preparing a regulation X-ray repair cross complementary protein 1 (X-ray repair cross complementary protein 1, XRCC1 ) gene, uracil DNA Glycosidase (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 (ERCC excision repair 1, endonuclease non-catalytic subunit, ERCC1 ) gene, ERCC excision repair 6, endonuclease non-catalytic subunit (ERCC excision repair 6 , endonuclease non-catalytic subunit, ERCC6 ) gene, glutathione peroxidase 1 ( GPX1 ) gene, mutL homolog 1 (mutL homolog 1, MLH1 ) gene, melanocyte-stimulating hormone 6 (melanocyte- stimulating hormone 6, MSH6 ) gene, superoxide dismutase (superoxide dismutase, SOD ) gene, mitogen-activated protein kinase dephosphorylase-1 (mitogen-activated protein kinase phosphatase 1, MKP-1 ) gene, and multiple The use of the expression level composition of dopa decarboxylase (dopa decarboxylase, DDC ) gene, wherein the daylily flower extract is obtained by extracting a daylily flower with a solvent, and the solvent is water, alcohol, or a mixture of alcohol and water.

In one embodiment of the present invention, the SOD gene is a SOD1 gene or a SOD2 gene.

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 is upregulated and the MKP-1 gene is downregulated.

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

Another object of the present invention is to provide a use of Hemerocallis fulva Linn. extract for preparing a composition for enhancing anti-glycation activity, wherein the Hemerocallis fulva Linn. extract is obtained by extracting Hemerocallis fulva Linn. with a solvent, The solvent is water, alcohol, or a mixture of alcohol and water.

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

Another object of the present invention is to provide a use of Hemerocallis fulva Linn. extract for preparing a composition that inhibits cell fat accumulation, promotes lipolysis and improves skeletal muscle basal metabolic rate, wherein the Hemerocallis fulva Linn. It is obtained by extracting a lily lily flower with a solvent, the solvent is water, alcohol, or a mixture of alcohol and water.

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

Another object of the present invention is to provide a kind of golden needle flower ( Hemerocallis fulva Linn.) extract to be used for preparing to inhibit melanin production, improve skin anti-ultraviolet ability, promote superoxide dismutase (superoxide dismutase, SOD) activity, and reduce activity The application of the reactive oxygen species (reactive oxygen species, ROS) expression composition, wherein the daylily flower extract is obtained by extracting a daylily flower with a solvent, and the solvent is water, alcohol, or a mixture of alcohol and water.

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

Another object of the present invention is to provide a use of Hemerocallis fulva Linn. extract for preparing a composition for enhancing mitochondrial activity and enhancing antioxidant capacity, wherein the Hemerocallis fulva Linn. extract is extracted with a solvent A lily lily is obtained, and the solvent is water, alcohol, or a mixture of alcohol and water.

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

Another object of the present invention is to provide a use of Hemerocallis fulva Linn. extract for preparing an antidepressant composition, wherein the Hemerocallis fulva Linn. extract is obtained by extracting Hemerocallis fulva Linn. with a solvent, and the solvent For water, alcohol, or alcohol-water mixture.

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

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

In summary, the efficacy of the extract of Daylily flower of the present invention lies in: it can regulate XRCC1 gene, UNG gene, OGG1 gene, MPG gene, ERCC1 gene, ERCC6 gene, GPX1 gene, MLH1 gene, MSH6 gene, SOD gene, MKP-1 Gene and DDC gene expression can improve anti-glycation activity, inhibit cell fat accumulation, promote fat decomposition and increase skeletal muscle basal metabolic rate, anti-depression, inhibit melanin production, improve skin anti-ultraviolet ability, increase SOD activity, and reduce The expression of ROS, the improvement of mitochondrial activity and the effect of enhancing antioxidant capacity.

Embodiments of the present invention will be further described below. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with the art will not depart from the spirit and scope of the present invention Some changes and modifications can be made, so the scope of protection of the present invention should be defined by the scope of the appended patent application.

Fig. 1 is a data graph of the anti-glycation effect of the golden needle flower extract of the present invention.

Fig. 2 is a data graph of the efficacy of the extract of daylily flower of the present invention in inhibiting fat accumulation.

Fig. 3 is a data graph of the effect of the extract of Golden Needle Flower of the present invention on promoting lipolysis, wherein "***" means compared with the control group, p <0.001.

Fig. 4 is a data graph of the effect of the extract of Daylily Flowers of the present invention on increasing the basal metabolic rate of skeletal muscle, where "***" means compared with the control group, p <0.001.

Fig. 5 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on inhibiting the expression of the MKP-1 gene related to depression, wherein "**" means compared with the control group, p <0.01.

Fig. 6 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on enhancing and promoting the expression of DDC genes related to dopamine, wherein "*" means compared with the control group, p <0.05.

Fig. 7 is a data graph of the efficacy of the extract of Golden Needle Flower of the present invention on the ability to resist and defend against UVA, wherein "*" means compared with the UVA group, p <0.05.

Fig. 8 is a data graph of the effect of the extract of Golden Needle Flower of the present invention on inhibiting melanin production, wherein "*" means compared with the control group, p <0.05.

Fig. 9 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on enhancing the SOD activity of skin fibroblasts.

Fig. 10 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on reducing the expression of ROS in dermal fibroblasts, wherein "***" means p <0.001.

Fig. 11 is a data graph showing the effect of the extract of Golden Needle Flowers of the present invention on enhancing the mitochondrial activity of nerve cells, where "***" means compared with the control group, p <0.001.

Fig. 12 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on enhancing the antioxidant capacity of nerve cells, wherein "***" means p <0.001.

Figure 13 is a data graph showing the effect of the extract of Golden Needle Flowers of the present invention on enhancing the expression of genes related to the anti-oxidation of nerve cells, where "*" means compared with the control group, p <0.05;"**" means compared with the control group , p <0.01;"***" means compared with the control group, p <0.001.

definition

The numerical values used herein are approximate values, and all experimental data are expressed within the range of 20%, preferably within the range of 10%, and most preferably within the range of 5%.

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

According to the present invention, Daylily ( Hemerocallis fulva Linn.), also known as Hemerocallis, Forget Worry or Daylily, is a perennial herb of Liliaceae (Liliaceae) Hemerocallis ( Hemerocallis ), with a plant height of about 60 to 80 cm and a fleshy underground root. ; Leaves grow in clusters from the roots, linear and long, up to 80cm. Daylily flower can be used for food and medicine, and has the effects of treating edema, dysuria, stranguria, leukorrhea, jaundice and blood in the stool.

As used herein, the terms "inhibition of melanogenesis" and "inhibition of melanin synthesis", "depigmenting", "lightening the melanin", " Whitening", "skin color lightening", "bleaching", "whitening", "brightening", "darkening" and "blackening" can be used interchangeably .

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

According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier (pharmaceutically acceptable carrier) which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may contain 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 quantities of these reagents are within the professionalism and routine skill 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 solution 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 Intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

In accordance with the present invention, pharmaceuticals may be formulated as 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 base may contain one or more additives (additives) selected from the group consisting of water, alcohols, glycols, hydrocarbons [such as petroleum jelly (petroleum, jelly) and white petrolatum], waxes (such as paraffin and yellow wax), preservatives, antioxidants, surfactants, absorption enhancers ( absorption enhancers), stabilizing agents, gelling agents (such as carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose, and carboxymethylcellulose (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) and so on. The selection and amounts of these additives are within the professionalism and routine skill of those skilled in the art.

According to the present invention, the skin care product may further include an acceptable adjuvant widely used in the skin care 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 agents, thickening agents, fillers, fragrances and odor absorbers. The selection and quantities of these reagents are within the professionalism and routine skill of those skilled in the art.

According to the present invention, skin care products can be manufactured into a form suitable for skin care or makeup by utilizing techniques well known to those skilled in the art, including, but not limited to: aqueous solution, water - alcohol solution (aqueous-alcohol solution) or oily solution (oily solution), in the form of oil-in-water type (oil-in-water type), oil-in-water type (water-in-oil type) or composite type emulsion, gel Gels, ointments, creams, masks, patches, packs, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, dispersions, drops, mousses ( mousse), sunblock, tonic water, foundation, makeup remover products, soap and other body cleansing products.

According to the present invention, the skin care product can also be combined with one or more known active external use agents (external use agents) selected from the following: whitening agents (such as tretinoin, catechin (catechin, kojic acid, arbutin and vitamin C], moisturizers, anti-inflammatory agents (anti-inflammatory agents), bactericides (bactericides), ultraviolet absorbers (ultraviolet absorbers), plant extracts (plant extracts)[ Such as aloe extract (aloe extract)], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, anti-inflammatory agents (antidermatitis agents), antihyperkeratolytic agents, anti-dry skin agents, antiperspirants (antipsoriatic agents), antiaging agents (antiaging agents), antiwrinkle agents (antiwrinkle agents), Antiseborrheic agents, wound-healing agents, corticosteroids and hormones. The selection and amount of these topical agents are within the professionalism and routine skill of those skilled in the art.

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

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

Embodiment 1. Preparation of Lily Flower Extract

First, homogenize the dry material of daylilies (planted by farmers in Yuli, Hualien), and then extract the homogenized daylilies with an extraction solvent at a volume ratio of 5~20:1~5 at 50~100°C 0.5 to 3 hours to obtain a crude extract, wherein the extraction solvent is water, alcohols, water-containing alcohols or a combination thereof, and the preferred extraction solvent is water. Next, cool to room temperature, and then filter the crude extract with a 400-mesh filter to obtain a filtrate, and then concentrate the filtrate under reduced pressure at 45-70° C. to obtain a daylily flower extract.

Example 2. Efficacy Evaluation of Golden Needle Flower Extract on Anti-Glycation

This example is to test the anti-glycation activity of the golden needle flower extract of the present invention. Therefore, the glycation activity is quantified by inhibiting the efficiency of D-fructose from glycosylation of collagen. First, take 0.2mL of water as the control group, or take 0.2mL of the 100mg/mL golden needle flower extract of the present invention as the experimental group, add 0.2mL of 60mg/mL collagen containing _0.06 % NaN solution (prepared with 200mM sodium phosphate buffer, pH 7.4), and mixed evenly with 0.2mL of 1.5M D-fructose (prepared with 200mM sodium phosphate buffer, pH 7.4), and took out 0.1mL of the mixed solution as the origin product, and react the remaining mixed solution at 50°C for 24 hours, take out 0.1 mL as the end product, and then measure the fluorescence values of the original product and the end product at excitation wavelength 360nm and emission wavelength 460nm. Finally, the efficiency of the ability to remove advanced glycation end-products (Advanced glycation end-products, AGEs) was calculated by the following formula to represent its anti-glycation activity, wherein the less advanced glycation end-products produced, the higher the anti-glycation activity.

The results of this example are shown in Figure 1, which inhibits non-enzymatic browning and prevents denaturation of functional proteins in vivo. Fig. 1 is a data graph of the anti-glycation effect of the golden needle flower extract of the present invention. It can be seen from Figure 1 that compared with the control group, the percentage of saccharification in the experimental group was significantly reduced. Among them, compared with the control group, the percentage of saccharification in the experimental group was reduced by about 56%. The results of this example show that the extract of the golden lily flower of the present invention can effectively improve its ability to remove highly glycated end products, and has excellent anti-glycation activity.

Example 3. Efficacy Evaluation of Golden Needle Flower Extract in Inhibiting Fat Accumulation

In this example, mouse bone marrow stromal cells were used to test the efficacy of the extract of Golden Needle Flower of the present invention in inhibiting fat accumulation. The mouse bone marrow stromal cell line was purchased from the American Type Culture Collection (ATCC ^® ), under the number CRL-2749 ^TM . The cells were cultured in pre-adipocyte expansion medium (Pre-adipocyte Expansion Medium) before differentiation, which contained 90% of the minimum essential medium Alpha medium (minimum essential medium alpha medium, purchased from Gibco, the United States, 12100-046) , 20% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, U.S.), and add 1% penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, U.S.); and use differentiation medium (Differentiation Medium) Mouse bone marrow stromal cells were differentiated with 90% minimal essential medium Alpha medium, 20% fetal bovine serum, and 1% penicillin/streptomycin. The lipids in the cells were stained with Oil Red O reagent (purchased from Sigma, USA), in which a 3 mg/mL oil red O stock solution was prepared with 100% isopropanol, and the stock solution was prepared with ddH ₂ O to 60% the action solution.

In order to confirm that the golden needle flower extract of the present invention has the effect of inhibiting fat accumulation, firstly, the mouse bone marrow stromal cells were differentiated into adipocytes. First, 8×10 ⁴ mouse bone marrow stromal cells OP9 and 500 μL of preadipocytes were expanded and cultured The medium was inoculated into 24-well culture plates and cultured at 37°C for 7 days, with fresh differentiation medium replaced every 3 days. Next, use a microscope (ZEISS) to observe the formation of lipid droplets to ensure that the cells are fully differentiated.

Afterwards, the OP9 cells were divided into two groups, including a control group and an experimental group. Add 0.25 mg/mL Golden Needle Flower extract to the cells in the experimental group, and do not do any treatment to the cells in the control group. Next, the cells in each group were cultured for 7-10 days, and the medium was replaced every 3 days.

Then, use Oil Red O to stain the lipids in the cells to evaluate whether the extract of Daylily Flowers of the present invention can really reduce fat accumulation. ) to wash the cells twice, then add 1 mL of 10% formaldehyde (purchased from Echo chemical, Taiwan, Cat.TG1794-4-0000-72NI) and react at room temperature for 30 minutes to fix the cells, then remove the formaldehyde and add 1 mL Gently wash the cells twice with PBS, then add 1 mL of 60% isopropanol (purchased from Echo chemical, Taiwan, PH-3101) to the cells in each well for 1 minute, remove the isopropanol and add 1 mL of The Oil Red O working solution was reacted at room temperature for 1 hour, then the Oil Red O working solution was removed and quickly decolorized with 1 mL of 60% isopropanol for 5 seconds, and then photographed and quantified using a microscope. Then, add 100% isopropanol to the stained cells, and place it on a shaker for 10 minutes to dissolve the oil droplets, then take 100 μL into a 96-well culture plate, and use the ELISA reader (BioTek) to read each group The OD _510nm reading value was used to quantify oil red O.

The results of this example are shown in FIG. 2 . Fig. 2 is a data graph of the efficacy of the extract of daylily flower of the present invention in inhibiting fat accumulation. It can be seen from Figure 2 that, compared with the control group, the relative amount of fat oil droplets in the experimental group decreased. Among them, compared with the control group, the relative amount of fat oil droplets in the experimental group was reduced by about 15%. The results of this example show that the extract of the lily lily flower of the present invention has the effect of inhibiting fat accumulation in cells.

Example 4. Efficacy Evaluation of Golden Needle Flower Extract in Promoting Lipolysis

First, mix 8×10 ⁴ mouse bone marrow stromal cells (bone marrow stromal cell) OP9 (ATCC ^® CRL-2749 ^TM ) with 500 μL of pre-adipocyte expansion medium (supplemented with 90% minimum essential Medium Alpha medium (minimum essential medium alpha medium) (Gibco), 20% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco)) were inoculated in 24-well culture plates and cultured at 37°C for 7 days, and replaced with fresh differentiation medium (supplemented with 90% minimum essential medium Alpha medium, 20% fetal bovine serum and 1% penicillin/streptomycin) every 3 days. Next, the formation of lipid droplets was observed using a microscope (ZEISS) to confirm complete differentiation of the cells.

Afterwards, the OP9 cells were divided into two groups, including the control group and the experimental group. Add 0.0625 mg/mL Lily lily extract to the cells of the experimental group, while the cells of the control group were not treated. Next, the cells in each group were cultured for 7-10 days, and the medium was replaced every 3 days. Afterwards, with cytosolic Glycerol cell-based assay kit (Cayman) was used to perform glycerol assay on cells in each group.

Collect the cell culture supernatant from each well, and then transfer 25 μL of the cell culture supernatant in each well and standard to a new 96-well culture plate. Next, 100 μL of reconstituted free glycerol assay reagent (reconstituted Free Glycerol Assay Reagent) was added to each well, and then reacted at room temperature for 15 minutes. Afterwards, the OD _540nm readings of each group were read with an ELISA reader (BioTek).

The results of this example are shown in FIG. 3 . Fig. 3 is a data graph of the effect of the extract of daylily flower of the present invention on promoting lipolysis. It can be seen from Figure 3 that compared with the control group, the percentage of lipolysis in the experimental group was significantly increased. Among them, compared with the control group, the percentage of lipolysis in the experimental group increased by about 10%. The results of this example show that the extract of the lily lily of the present invention has the effect of promoting fat decomposition.

Example 5. Efficacy Evaluation of Golden Needle Flower Extract in Improving Basal Metabolic Rate of Skeletal Muscle

In this example, mouse skeletal muscle cells C2C12 were used to test the efficacy of the extract of daylily flower of the present invention in increasing the basal metabolic rate of skeletal muscle. The mouse skeletal muscle cell C2C12 was purchased from the American Type Culture Collection (ATCC ^® ), number CRL-1772 ^TM .

First, cells were cultured in Dubacil supplemented with 3.7 g/L sodium bicarbonate (sodium bicarbonate), 10% fetal bovine serum (FBS) (Gibco), 100 I.U. penicillin and 100 μg/mL streptomycin. Cells were seeded in 6-well cell culture dishes in DMEM. When the cells were nearly 100% confluent, the cells were cultured with conditioned medium (DMEM containing 1% FBS and 1% horse serum) to differentiate the cells into myotubes.

Afterwards, the differentiated cells were divided into two groups, including a control group and an experimental group. Add 0.0625 mg/mL Lily lily extract to the cells of the experimental group, while the cells of the control group were not treated. Next, the cells of each group were cultured for 48 hours. Thereafter, the cells were lysed with 100 μL of pyruvate assay buffer per well, centrifuged at 10,000 g for 10 minutes at 4° C., and the supernatant was collected. Next, 20 μL of lysate was added to the 96-well culture plate (three replicates), and then the volume was adjusted to 50 μL/well with pyruvate assay buffer. Afterwards, to make a standard curve for the pyruvate colorimetric assay (BioVision), dilute the pyruvate standard to 1 nmol/μL, then prepare 0, 2, 4, 6, 8, 10 nmol/well, a total of 50 μL/μL hole. Next, add 50 μL of reaction mixture to each well Mixture (containing 46 μL of pyruvate assay buffer, 2 μL of pyruvate probe and 2 μL of enzyme mixture), mixed well and reacted for 30 minutes. Afterwards, the absorbance was measured at 570 nm, and then the pyruvate content was calculated using linear interpolation. Finally, the total protein concentration of each sample was normalized (Bradford assay) (Bio-Rad).

The results of this example are shown in FIG. 4 . Fig. 4 is a data graph of the effect of the extract of daylily flower of the present invention on increasing the basal metabolic rate of skeletal muscle. It can be seen from Figure 4 that, compared with the control group, the relative concentration of pyruvate in the experimental group was significantly increased. Among them, compared with the control group, the relative concentration of pyruvate in the experimental group increased by about 80%. The results of this example show that the extract of daylily flower of the present invention has the effect of increasing the basal metabolic rate of skeletal muscle.

Example 6. Efficacy Evaluation of Golden Needle Flower Extract on Anti-depression

This example is to explore whether the extract of golden needle flower can achieve the effect of anti-depression by regulating and inhibiting the expression of genes related to depression and promoting dopamine.

Culture human neuroma blasts (SH-SY5Y; purchased from (ATCC ^® CRL-2266 ^TM )) in DMEM medium (containing 10% fetal bovine serum and 1% penicillin/streptomycin) in 6-well plates, 2 mL The cell concentration of the culture medium was 1.5×10 ⁵ cells/well.

Afterwards, the cells were divided into two groups, including a control group and an experimental group. Add 0.5mg/mL Golden Needle flower extract to the cells in the experimental group, and do not do any treatment to the cells in the control group. Next, each group of cells was cultured in an incubator for 48 hours, and then the cell cultures of each group were harvested and used for gene expression analysis.

In this example, the gene used to analyze the inhibition of depression is the mitogen-activated protein kinase dephosphorylase-1 (mitogen-activated protein kinase phosphatase 1, MKP-1 ) gene, and the gene associated with the promotion of dopamine is Dopa decarboxylase (dopa decarboxylase, DDC ) gene.

RNA was extracted from each group of cell cultures obtained above with an RNA extraction kit (Geneaid). 2,000 ng of each group of RNA thus obtained was taken, and the extracted RNA was reverse-transcribed into cDNA with SuperScript ^® III reverse transcriptase (Invitrogen). Next, use cDNA as a template, and use the primer pairs used to amplify the target genes, including MKP-1, DDC and GAPDH (as an internal control group), their nucleotide sequences are shown in Table 1 below, in StepOne Plus instant The KAPA SYBR FAST qPCR Kit (2x) (KAPA Biosystems) was used in the PCR system (ABI) for quantitative real-time PCR to amplify and quantify the target gene. The melting curve of the PCR product was confirmed during the quantitative real-time PCR reaction.

The relative expression of the target gene was derived from Equation 2 ^{- ΔΔCt} , and the relative fold change was calculated using the GAPDH gene (as an internal control group) and the cycle threshold of the reference gene and the standard deviation, where ΔCt=Ct _{target gene /reference gene} -Ct _GAPDH , △△Ct=△Ct _{target gene-} △Ct _{reference gene} , fold change=2- ^△△Ct _mean . The expression level of the target gene in the control group was used as the comparison benchmark of 1. Statistically significant differences between groups were determined by one-tailed Student's t-test. The results of this example are shown in FIGS. 5 and 6 .

Fig. 5 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on inhibiting the expression of the MKP-1 gene related to depression. It can be seen from Figure 5 that, compared with the control group, the relative gene expression of the experimental group was significantly reduced, and compared with the control group, the relative gene expression of the experimental group was reduced by about 16.5%. Fig. 6 is a graph showing the effect of the extract of Golden Needle Flower of the present invention on enhancing the expression of DDC genes related to the promotion of dopamine. It can be seen from Figure 6 that, compared with the control group, the relative gene expression of the experimental group has significantly increased, and compared with the control group, the relative gene expression of the experimental group has increased by about 10%. The results of this example show that the extract of the lily lily of the present invention can achieve the effects of anti-depression and improving mood by regulating and inhibiting the expression of genes related to depression and promoting dopamine.

Example 7. Efficacy Evaluation of Golden Needle Flower Extract on Resistance and Defense Against UVA

First, culture human skin fibroblast CCD-966Sk (corresponding to ATCC ^® CRL-1881) in minimum essential medium (added with 10% fetal bovine serum (FBS) , 1% penicillin/streptomycin and 1 mM sodium pyruvate (Gibco). Add 200 μL of medium to each well of a 96-well culture plate, so that each well has 5 x 10 ³ human dermal fibroblasts. After 24 hours of incubation at 37°C, the medium was removed.

Afterwards, the human dermal fibroblasts were divided into 3 groups, including 1 experimental group, 1 UVA group and 1 control group. The cells in the experimental group and the UVA group were irradiated with UVA for 1 hour by using an ultraviolet radiation chamber at 15J/cm ² . This condition would cause 50% lethal dose (lethal dose 50%, LD ₅₀ ), which means that 50% Radiation exposure dose for cell death. Next, 1 mg/mL Lily lily extract was added to the cells in the experimental group, while the cells in the UVA group were not treated. As for the control group, the cells were not irradiated with UVA.

After each group of cell cultures were cultured in a 37°C incubator for 24 hours, 15 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide was added to each well {3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide} (MTT, 4 mg/mL in PBS) (AMERSCO/0793-5G) was incubated at 37°C for 4 hours. Afterwards, the medium in each well was removed, and then 50 μL of DMSO (ECHO/DA1101-000000-72EC) was added to each well to decompose formazan crystals, and then the culture plate was placed on a shaker and incubated for 10 minutes, and then read the absorbance value (OD ₅₇₀ ) of each well with an ELISA reader (BioTek) at a wavelength of 570 nm.

Cell viability (%) was calculated by substituting the measured absorbance value (OD ₅₇₀ ) into the following formula (1): Cell viability (%)=(OD ₅₇₀ absorbance value measured in each group/control OD ₅₇₀ absorbance value measured by group)×100% (1)

Statistically significant differences between groups were determined by Student's t-test. The results of this example are shown in FIG. 7 .

Fig. 7 is a data graph of the efficacy of the extract of daylily flower of the present invention in resisting and defending against UVA. It can be seen from FIG. 7 that compared with the control group, the viability (%) of the cells in the UVA group decreased, which indicated that irradiating human dermal fibroblasts with UVA would cause cell death. Compared with the UVA group, the viability (%) of the cells in the experimental group was significantly increased (compared with the UVA group, the viability of the cells in the experimental group was increased by about 8%). The results of this example show that the extract of the lily lily of the present invention has the ability to resist and defend against UVA damage, and enhance the ability of skin cells to resist ultraviolet rays.

Example 8. Efficacy Evaluation of Golden Needle Flower Extract in Inhibiting Melanin Production

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)]. Add 3 mL of culture medium to each well of a 6-well culture plate, so that each well has 1.5 x 10 ⁵ B16F10 cells. After 24 hours of incubation at 37°C, the medium was removed.

Afterwards, the B16F10 cells were divided into two groups, including an experimental group and a control group. Add 0.5mg/mL Golden Needle flower extract to the cells in the experimental group, and do not do any treatment to the cells in the control group.

After each group of cell cultures was incubated at 37°C for 48 hours, the medium was removed and washed twice with 1xPBS (Gibco). After that, trypsin was added to treat the cells for 3 minutes and the suspended cells were collected in a centrifuge tube with a volume of 15 mL, followed by spinning at 400×g/5 minutes to pellet the cells. After washing twice with 1xPBS, the cell pellet was resuspended in 200 μL of 1XPBS. Next, the cell solution was placed in liquid nitrogen for 10 minutes, and then allowed to stand at room temperature for 30 minutes to thaw. After complete thawing, spin at 12,000 xg for 30 minutes, followed by removal of supernatant and addition of 120 μL of 1 N NaOH (in ddH ₂ O). After mixing uniformly, it was left to stand in a dry bath at 60° C. for 1 hour. Afterwards, a volume of 100 μL was put into a 96-well culture plate and the absorbance value (OD ₄₅₀ ) of each well was read with an ELISA reader at a wavelength of 450 nm.

Melanin content (%) is calculated by substituting the measured absorbance value (OD ₄₅₀ ) into the following formula (2): Melanin content (%)=(OD ₄₅₀ absorbance value measured in each group/control group Measured OD ₄₅₀ (absorbance value) × 100% (2)

Statistically significant differences between groups were determined by Student's t-test. The results of this example are shown in FIG. 8 .

Fig. 8 is a data graph of the efficacy of the extract of Golden Needle Flower of the present invention on inhibiting melanin production. As can be seen from Figure 8, compared with the control group, the melanin content of the experimental group was significantly reduced (compared with the control group, the melanin content of the experimental group was reduced by about 11%). The results of this example show that the extract of the lily lily of the present invention has the effect of inhibiting melanin production.

Example 9. Efficacy Evaluation of Golden Needle Flower Extract in Improving Superoxide Dismutase (superoxide dismutase, SOD) Activity of Skin Fibroblasts

Human dermal fibroblasts were purchased from Taiwan Bioresource Collection and Research Center (BCRC), number BCRC 60153. The cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS) (GIBCO Company, No. 10438-026, U.S.), 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate (Sigma Company, No. S5761, the United States), 1mM sodium pyruvate (GIBCO company, No. 11360-070, the United States) minimum essential medium (Minimum essential medium, MEM) (Eagle) (mixed with Earle's Balanced Salt Solution (Earle's Balanced Salt Solution, Earle's BSS)) (GIBCO Corporation, No. 41500-034, USA).

Inoculate 2 × ¹⁰⁵ human dermal fibroblasts into each well of a 6-well culture plate containing 2 mL of the above medium, and then culture overnight at 37 °C. After removing the medium, the cells were divided into 2 groups, including 1 control group and 1 experimental group. Add 0.5mg/mL Golden Needle flower extract and 1mM H ₂ O ₂ to the cells of the experimental group and act at 37°C for 6 hours, while the cells of the control group were not treated. Next, each group of cells was cultured at 37° C. for 24 hours. Afterwards, the medium was removed and washed once with 1X PBS (Gibco), and then trypsin was added to treat the cells for 3 minutes, followed by medium to stop trypsin activity and transferred to a 1.5 mL centrifuge tube. Next, centrifuge at 300xg for 5 minutes, then wash with PBS, and then centrifuge at 300xg for 5 minutes. Afterwards, 30 μL of 1X cell extraction buffer was added, and then the cell suspension was reacted on ice for 30 minutes, vortexed every 10 minutes to mix. Next, the broken cell suspension was centrifuged at 10,000 xg for 10 minutes at 4°C to remove insoluble material, and then 25 μL of 1X SOD buffer was added to the bottom of the active control well. After that, add 25 μL of prepared SOD standards (S1~S7, see Table 2).

Afterwards, 25 μL of the sample was added to the bottom of the appropriate well, and then 150 μL of Master Mix was added to each well. Next, 25 μL of 1X Xanthine solution was added to all wells using a multichannel pipet to initiate the reaction, and the plate was immediately transferred to a microtiter plate reader , and read the absorbance at 450 nm every minute for 10 minutes at room temperature.

Statistically significant differences between groups were determined by Student's t-test. The results of this example are shown in FIG. 9 .

Fig. 9 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on enhancing the SOD activity of skin fibroblasts. As can be seen from Figure 9, compared with the control group, the SOD activity of the experimental group was significantly increased (compared with the control group, the SOD activity of the experimental group was increased by about 30.7%). The results of this example show that the extract of the lily lily of the present invention has the effect of enhancing the SOD activity of dermal fibroblasts.

Example 10. Efficacy Evaluation of Golden Needle Flower Extract in Reducing the Expression of Reactive Oxygen Species (ROS) in Skin Fibroblasts

First, human skin fibers were cultured in Minimal Essential Medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1 mM sodium pyruvate Mother cells CCD-966SK (BCRC 60153) were placed in a 6-well plate with a cell concentration of 2×10 ⁵ cells/well in 2 mL of medium, then cultured at 37° C. for 24 hours, and the medium was removed. Then, the cultured cells were divided into 3 groups, including a control group, a hydrogen peroxide group, and an experimental group. Add 1 mg/mL Golden Needle flower extract and 1 mM H ₂ O ₂ to the cells of the experimental group. The cells in the hydrogen peroxide group were added with 1 mM H ₂ O ₂ , while the cells in the control group were not treated. Afterwards, 5 μg/mL dichloro-dihydro-fluorescein diacetate (Dichloro-dihydro-fluorescein diacetate, DCFH-DA) (Sigma/SI-D6883-50MG) was added (the stock solution was 5 mg/mL dissolved in DMSO) and dissolved in React at 37°C for 1 hour, and then wash each well twice with 1 mL of 1X PBS (Gibco). Next, 200 μL of trypsin was added and reacted in a dark environment for 5 minutes, and then the cell culture was collected into a centrifuge tube with a volume of 1.5 mL, and centrifuged at 400×g for 10 minutes. Thereafter, the supernatant was removed and washed once with 1X PBS, followed by centrifugation at 400×g for 10 minutes. Next, remove the supernatant and resuspend the cell pellet with 1 mL of 1X PBS. Afterwards, use a flow cytometer (BD Accuri) to detect the fluorescent signal of DCFH-DA with an excitation wavelength of 450-490nm and an emission wavelength of 510-550nm, so as to calculate the damage received by ROS percentage of cells. Statistically significant differences between groups were determined by Student's t-test. The results of this example are shown in FIG. 10 .

Fig. 10 is a data graph showing the effect of the extract of Golden Needle Flower of the present invention on reducing the expression of ROS in dermal fibroblasts. It can be seen from Figure 10 that, compared with the control group, the percentage of cells damaged by ROS measured in the hydrogen peroxide group (i.e. cells with high oxidative stress) was significantly increased, which indicates that hydrogen peroxide can cause a large amount of ROS damage to the cells ; and compared with the hydrogen peroxide group, the percentage of cells damaged by ROS measured in the experimental group was significantly reduced (the ROS expression was reduced by about 47%). The results of this example show that the extract of the lily lily of the present invention has the effect of reducing the ROS expression level of dermal fibroblasts, thereby improving skin quality.

Example 11. Efficacy Evaluation of Golden Needle Flower Extract in Improving Mitochondrial Activity of Nerve Cells

In this example, mouse brain neuroblastoma cells (Brain neuroblastoma) Neuro-2a were used to analyze the activity of mitochondrial mitochondria of nerve cells, and the flow cytometry mitochondrial membrane potential detection kit (Flow cytometry Mitochrondrial membrane potential detection kit, BD) for experiments. Mouse brain neuroblastoma cells Neuro-2a were purchased from the American Type Culture Collection (ATCC ^® ), number CCL-131 ^TM . The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (GIBCO Company, No. 10438-026, USA), 1% penicillin/streptomycin (Gibco).

Inoculate 1× ¹⁰⁵ brain neuroblasts (n=2) in each well of a 6-well culture dish containing 2 mL of the above medium. Afterwards, the cells were divided into two groups, including a control group and an experimental group. 1 mg/mL Golden Needle flower extract was added to the cells of the experimental group, and the culture medium was added to the cells of the control group. Next, each group of cells was cultured at 37°C for 24 hours, and then pre-warmed 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 stain (BD ^TM MitoScreen (JC-1) Reagent Set) to prepare JC-1 stock solution, which can be stored at -20°C for 6 months. Next, prepare a working solution with JC-1 mitochondrial stain and 1X assay buffer at a ratio of 1:100, then remove the medium and rinse twice with 1XPBS. After that, trypsin/EDTA was added to treat 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, resuspend the cells in 1 mL of 1X PBS, transfer to a 1.5 mL centrifuge tube, and centrifuge at 400 g for 5 minutes. After removing the supernatant, add 100 μL of JC-1 working solution, mix well and act for 15 minutes in the dark. 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. The cells were resuspended in 500 μL of 1X PBS containing 2% FBS, and then analyzed by flow cytometry (BD Accuri) to observe the change of mitochondrial membrane potential during cell apoptosis, and the Statistical analysis was performed by Studen's t-test with Excel Statistical significance of differences between sample populations.

Fig. 11 is a data graph showing the effect of the extract of daylily flower of the present invention on enhancing the mitochondrial activity of nerve cells. It can be seen from Figure 11 that, compared with the control group, the mitochondrial activity (ie, the relative JC-1 aggregate (aggregate)) of the neuroblasts in the experimental group was significantly improved (compared with the control group, the experimental group increased neuroblast activity). Cellular glandular activity is about 13%). The results of this example show that the extract of the lily lily of the present invention has the effect of enhancing the mitochondrial activity of nerve cells.

Example 12. Efficacy Evaluation of Golden Needle Flower Extract in Improving the Antioxidative Ability of Nerve Cells

In this example, mouse brain neuroblastoma cells (Brain neuroblastoma) Neuro-2a were used to analyze the antioxidant capacity of nerve cells. Mouse brain neuroblastoma cells Neuro-2a were purchased from the American Type Culture Collection (ATCC ^® ), number CCL-131 ^TM . Brain neuroblast cells Neuro-2a were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (GIBCO Company, No. 10438-026, USA), 1% penicillin/streptomycin (Gibco) in 6-wells plate, the cell concentration of 2mL medium was 1×10 ⁵ cells/well, followed by culturing at 37°C for 24 hours, and the medium was removed. Then, the cultured cells were divided into 3 groups, including a control group, a hydrogen peroxide group, and an experimental group. Add 1 mg/mL Golden Needle flower extract and 1 mM H ₂ O ₂ to the cells of the experimental group and act at 37° C. for 1 hour. The cells in the hydrogen peroxide group were added with 1 mM H ₂ O ₂ and reacted at 37° C. for 1 hour, while the cells in the control group were not treated. Afterwards, 5 μg/mL dichloro-dihydro-fluorescein diacetate (Dichloro-dihydro-fluorescein diacetate, DCFH-DA) (Sigma/SI-D6883-50MG) was added (the stock solution was 5 mg/mL dissolved in DMSO) and dissolved in React at 37°C for 15 minutes, and then wash each well twice with 1 mL of 1X PBS (Gibco). Next, 200 μL of trypsin was added and reacted in a dark environment for 5 minutes, and then the cell culture was collected into a centrifuge tube with a volume of 1.5 mL, and centrifuged at 400×g for 10 minutes. Thereafter, the supernatant was removed and washed once with 1X PBS, followed by centrifugation at 400×g for 10 minutes. Next, remove the supernatant and resuspend the cell pellet with 1 mL of 1X PBS. Afterwards, use a flow cytometer (BD Accuri) to detect the fluorescent signal of DCFH-DA with an excitation wavelength of 450-490nm and an emission wavelength of 510-550nm, so as to calculate the damage received by ROS percentage of cells. Statistically significant differences between groups were determined by Student's t-test. The results of this example are shown in FIG. 12 .

Fig. 12 is a data graph showing the effect of the extract of daylily flower of the present invention on enhancing the antioxidant capacity of nerve cells. As can be seen from Figure 12, compared with the control group, the percentage of cells damaged by ROS measured in the hydrogen peroxide group (i.e. the relative amount of ROS produced) was significantly increased, which indicated that hydrogen peroxide would cause a large amount of ROS damage to the cells; Compared with the hydrogen peroxide group, the percentage of cells damaged by ROS measured in the experimental group was significantly reduced (the relative ROS production was reduced by about 73%). The results of this example show that the extract of the lily lily of the present invention has the effect of enhancing the antioxidant capacity of nerve cells.

Example 13. Efficacy Evaluation of Golden Needle Flower Extract in Improving the Expression of Genes Related to Nerve Cell Antioxidation

This example is to explore whether the extract of lily lily can improve the antioxidant capacity of nerve cells by increasing the expression of genes related to the antioxidant function of nerve cells.

Mouse brain neuroblastoma cells (Neuro-2a, purchased from the American Type Culture Collection (ATCC ^® ), No. CCL-131 ^TM ) in a 6-well plate, the cell concentration in 2 mL of culture medium was 1.5×10 ⁵ cells/well.

Afterwards, the cells were divided into two groups, including a control group and an experimental group. 1 mg/mL Golden Needle Extract and 200 μM H ₂ O ₂ were added to the cells in the experimental group, while the cells in the control group were added with 200 μM H ₂ O ₂ . Next, each group of cells was cultured in an incubator for 6 hours, and then the cell cultures of each group were harvested and used for gene expression analysis.

In this example, the genes used to analyze the anti-oxidation 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 cut-out repair 1, Endonuclease non-catalytic subunit (ERCC excision repair 1, endonuclease non-catalytic subunit, ERCC1 ) gene, ERCC excision repair 6, endonuclease non-catalytic subunit (ERCC excision repair 6, endonuclease non-catalytic subunit, ERCC6 ) gene, glutathione peroxidase 1 ( GPX1 ) gene, mutL homolog 1 (mutL homolog 1, MLH1 ) gene, melanocyte-stimulating hormone 6 ( MSH6 ) gene, Superoxide dismutase 1 (superoxide dismutase 1, SOD1 ) gene and superoxide dismutase 2 (superoxide dismutase 2, SOD2 ) gene.

RNA was extracted from each group of cell cultures obtained above with an RNA extraction kit (Geneaid). 2,000 ng of each group of RNA thus obtained was taken, and the extracted RNA was reverse-transcribed into cDNA with SuperScript ^® III reverse transcriptase (Invitrogen). Next, using cDNA as a template and using primer pairs for amplifying target genes, including XRCC1, UNG, OGG1, MPG, ERCC1, ERCC6, GPX1, MLH1, MSH6, SOD1, SOD2 and GAPDH (as an internal control group), Their nucleotide sequences are shown in Table 3 below. Quantitative real-time PCR was performed using the KAPA SYBR FAST qPCR Kit (2x) (KAPA Biosystems) in the StepOne Plus Real-Time PCR System (ABI) to amplify the target gene and quantitative. The melting curve of the PCR product was confirmed during the quantitative real-time PCR reaction.

The relative expression of the target gene was derived from Equation 2 ^{- ΔΔCt} , and the relative fold change was calculated using the GAPDH gene (as an internal control group) and the cycle threshold of the reference gene and the standard deviation, where ΔCt=Ct _{target gene /reference gene} -Ct _GAPDH , △△Ct=△Ct _{target gene-} △Ct _{reference gene} , fold change=2- ^△△Ct _average . The expression level of the target gene in the control group was used as the comparison benchmark of 1. Statistically significant differences between groups were determined by one-tailed Student's t-test. The results of this example are shown in FIG. 13 .

Fig. 13 is a data graph showing the effect of the extract of daylily flower of the present invention on enhancing the expression of genes related to the anti-oxidation of nerve cells. It can be seen from Figure 13 that, compared with the control group, the relative expression of genes in the experimental group was significantly increased (the expression of various nerve cell antioxidant-related genes was increased by about 12.5-195%). The results of this example show that the extract of the lily lily of the present invention can enhance the antioxidant capacity of nerve cells by increasing the expression of genes related to the antioxidant properties of nerve cells.

In summary, the extract of Golden Needle Flower of the present invention can regulate XRCC1 gene, UNG gene, OGG1 gene, MPG gene, ERCC1 gene, ERCC6 gene, GPX1 gene, MLH1 gene, MSH6 gene, SOD gene, MKP-1 gene, and DDC Gene expression can improve anti-glycation activity, inhibit cell fat accumulation, promote fat decomposition and increase skeletal muscle basal metabolic rate, anti-depression, improve mood, inhibit melanin production, improve skin anti-ultraviolet ability, increase SOD activity, and reduce ROS performance Quantity, improve skin quality and condition, enhance mitochondrial activity, enhance antioxidant capacity, and protect nerve cells.

The above descriptions are illustrative only, not restrictive. Any equivalent modification or change made without departing from the spirit and scope of the present invention shall be included in the scope of the appended patent application.

<110> Dajiang Biomedical Co., Ltd.

<120> Application of the extract of lily lily flower to prepare a composition for improving the basal metabolic rate of skeletal muscle

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<170> PatentIn version 3.5

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Claims (6)

A use of Hemerocallis fulva Linn. extract for preparing a composition for increasing basal metabolic rate of skeletal muscle, wherein the extract is obtained by extracting Hemerocallis fulva Linn. with a solvent, the solvent being water. The use as described in claim 1, wherein the extraction temperature of the lily lily extract is 50-100°C. The use as described in Claim 1, wherein the volume ratio of the daylily flower extract to the extraction solvent is 1:5~1:20. The use as described in claim 1, wherein the extraction time of the lily lily extract is 0.5 to 3 hours. The use as claimed in item 1, wherein the composition is a food product or a skin care product. The use as claimed in item 1, wherein the composition is a medicine.

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