TWI784229B - Lonicera japonica thunb ferment, preparation method thereof and use thereof for improving skin appearance and anti-aging - Google Patents

Abstract

The present invention provides a Lonicera japonica Thunb ferment, preparation method thereof and use thereof for improving skin appearance and anti-aging. The extraction process of the present invention can improve the content of total polyphenols, increase the reduce activity and antioxidant activity, increase the ability to remove the final glycation end product, and can significantly increase the gene expression level of SOD3, Parkin, Atg1 and Ubl-5, and simultaneously reduce the gene expression level of PARP2 in skin cells. The Lonicera japonica Thunb ferment is prepared by fermenting the Lonicera japonica Thunb extract to yeast, lactic acid bacteria, and acetic acid bacteria for three-stage fermentation.

Description

Preparation method of honeysuckle fermented product and its use for improving skin appearance and anti-aging

The present invention relates to a preparation method and application of honeysuckle fermented product, especially a honeysuckle fermented product prepared by the three-stage fermentation process of the present invention, and its use for improving skin appearance and anti-aging uses.

The epidermis is the outermost layer of the skin. From the outside to the inside, it is divided into stratum corneum, granular layer, spinous layer and basal layer. The epidermis is mainly formed by continuous upward differentiation of undifferentiated cylindrical keratinocytes in the basal layer. This process called keratinization. The water content in the corneocytes is high. As the cells metabolize and differentiate upward, the shape of the corneocytes will gradually become flat, and the nucleus and organelles will begin to degenerate and shrink, and dead cells without nuclei and organelles will form in the stratum corneum. The main function of the epidermis is to keep the skin water and form a skin barrier to resist various external injuries. The outermost layer of the epidermis is composed of a weakly acidic sebum membrane and a brick-like stratum corneum. This barrier can lock the skin Moisture and oil, resisting the invasion of germs on the surface of the skin, and resisting damage from external foreign bodies and ultraviolet light have very important protective effects on the human body.

In the stratum corneum of the epidermis, although the keratinocytes are dead cells, its main component is keratin. Keratin can absorb water to keep the skin moist, and keratinocytes also secrete substances such as hyaluronic acid as intercellular substance. To maintain the structural integrity of the epidermis skin barrier to prevent skin moisture loss and form a complete protection. When the skin is exposed to excessively cold or overheated environments and irradiated by ultraviolet light, it will cause keratinocytes to fail to maintain normal metabolic cycle, and the skin's water retention capacity will also decrease, and It leads to damage to the epidermal barrier of the skin, making the skin rough, dry and desquamated, fragile and easily irritated, sensitive and red. Therefore, the health and water retention capacity of the stratum corneum is really very important to resist external damage.

However, as we get older, the skin will gradually age (Ageing). The causes and process of skin aging are very complicated and involve countless physiological phenomena, including ultraviolet damage, free radical damage, collagen reduction, cell renewal slowdown, The appearance of abnormal cells, reduction of skin fat, lack of intercellular substance, cessation of cell growth, and decline of hormones are more common factors. But so far, most of the skin anti-aging products on the market can only start to increase the antioxidant activity, and cannot directly and effectively improve or delay the occurrence of skin aging.

Glycation end products (Advanced Glycation End Products, AGEs) are a group of highly oxidized compounds, so they are considered to be a kind of glycotoxin (Glycotoxin). Studies have shown that glycation end products will bind to receptors on the cell surface to change its structure and function. It also increases the oxidative stress and inflammatory response of cells, and is inseparable from the formation of diabetes, arteriosclerosis and chronic kidney diseases. In addition to the end products of glycation produced during the normal metabolic process of the human body, many processed foods also contain end products of glycation. Studies have also pointed out that avoiding the intake of end products of glycation from food can help delay the progress of chronic diseases and aging.

Based on the above, in order to improve the skin’s fragility and sensitivity caused by damaged keratinocytes, decreased water retention capacity, and skin aging, it is necessary to develop a method that can directly and effectively improve or delay the occurrence of skin aging, and at the same time effectively It is really necessary to maintain the normal physiological metabolism of keratinocytes, maintain the integrity of the composition of the stratum corneum structure, and increase the active ingredients of anti-glycation activity.

Therefore, one object of the present invention is to provide a honeysuckle fermented product for delaying aging, which is a honeysuckle extract obtained by extracting a honeysuckle through a solvent, and then passing the honeysuckle water extract through a yeast ( Saccharomyces cerevisiae ), a lactic acid bacterium ( Lactobacillus plantarum ), and an acetic acid bacterium ( Acetobacter aceti ) sequentially undergo three-stage fermentation.

Another object of the present invention is to provide a honeysuckle fermented product as described above for the preparation of superoxide dismutase 3 (Superoxide dismutase 3, SOD3 ) gene, Parkinson's disease juvenile protein (Parkinson juvenile disease protein) in skin cells , Parkin ) gene, autophagy-related gene 1 (Autophagy-related gene 1, Atg1 ) gene, Ubl gene is ubiquitin-like protein 5 (Ubiquitin-like protein 5, Ubl-5 ) gene, and inhibit protein aggregation in skin cells (ADP-ribose) polymerase 2 (Poly (ADP-Ribose) Polymerase 2, PARP2 ) gene expression composition.

Another object of the present invention is to provide a fermented product of honeysuckle for preparing a composition for improving anti-aging ability.

Another object of the present invention is to provide a method for producing a honeysuckle fermented product, which includes: extracting a honeysuckle extract obtained by extracting a honeysuckle with a solvent, and then passing the honeysuckle extract through a yeast ( Saccharomyces cerevisiae ) , a lactic acid bacterium ( Lactobacillus plantarum ), and an acetic acid bacterium ( Acetobacter aceti ) were sequentially subjected to three-stage fermentation.

In one embodiment of the present invention, according to the colorimetric method of reducing iron oxidation (Ferric reducing antioxidant power, FRAP), the ability of the fermented product of honeysuckle to reduce iron ion (Fe3 ⁺ ) is equivalent to 300-400 μg/mL ascorbic acid The ability to reduce iron ions (Fe3 ⁺ ).

In yet another embodiment of the present invention, the honeysuckle fermented product further enhances antioxidant activity and anti-glycation activity.

In yet another embodiment of the present invention, the honeysuckle fermented product promotes the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene; and suppresses the expression of PARP2 gene.

In yet another embodiment of the present invention, the honeysuckle fermented product increases the concentration of NAD ⁺ in skin cells, maintains the normal function of decomposing old waste proteins in cells, promotes the degradation of waste in cells, and promotes the normal function of autophagy in cells. function, enhance the ability to repair DNA damage, and/or promote the activity of mitochondrial metabolism.

In yet another embodiment of the present invention, the honeysuckle fermented product is enhanced in antioxidant activity, reducing activity, anti-glycation activity, and/or in total polyphenol content.

In yet another embodiment of the present invention, the effective concentration of the honeysuckle fermented product is at least 0.5% (v/v).

In another embodiment of the present invention, the honeysuckle extract is obtained by extracting the honeysuckle with water as a solvent; and the honeysuckle and water are mixed at a ratio of 1:10-20 (w/w); the yeast The added amount of bacteria is 0.01-0.5% (w/w); the added amount of the lactic acid bacteria is 0.01-0.25% (w/w); the added amount of the acetic acid bacteria is 3-10% (w/w); and the The fermentation time ratio of the yeast, the lactic acid bacteria, and the acetic acid bacteria is 1-2.5:1-3:3-10.

In the present invention, the honeysuckle fermented product obtained by performing three-stage fermentation of the honeysuckle extract with yeast, lactic acid bacteria, and acetic acid bacteria can significantly increase the content of total polyphenols in the honeysuckle fermented product of the present invention through the microbial fermentation process, Significantly improve the reducing activity of the honeysuckle fermented product of the present invention, significantly improve the antioxidant activity of the honeysuckle fermented product of the present invention in skin cells, significantly improve the ability of the honeysuckle fermented product of the present invention to remove saccharification end products, and significantly improve the The flower fermentation product increases the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in cells, and at the same time reduces the ability of PARP2 gene expression; in order to achieve the effect of enhancing anti-oxidation and anti-glycation, it helps to delay chronic diseases and the progress of aging, and can increase the concentration of NAD ⁺ in cells, maintain the normal function of decomposing old waste proteins in cells, promote the degradation of waste in cells, and promote the normal operation of autophagy in cells, and can enhance the repair of DNA damage , and promote the activity of mitochondrial metabolism to prolong the life of cells and achieve anti-aging effects. Therefore, the honeysuckle fermented product of the present invention can be used to prepare a composition for improving skin appearance and anti-aging, and the composition is a medicine, a skin care product, or a food, and can be administered by oral administration, smearing, etc. a body.

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 bar graph showing the effect of improving the total polyphenols in honeysuckle fermented product by a three-stage fermentation method according to an embodiment of the present invention.

Fig. 2 is a bar graph showing the effect of reducing activity of honeysuckle fermented product according to one embodiment of the present invention.

Fig. 3 is a bar graph showing the efficacy of honeysuckle fermented product in enhancing the antioxidant activity of skin cells according to an embodiment of the present invention. **p<0.01.

Fig. 4 is a bar graph showing the anti-glycation activity of honeysuckle fermented product according to one embodiment of the present invention.

Fig. 5 is a bar graph showing the effect of the honeysuckle fermented product on enhancing the expression of SOD3 gene according to one embodiment of the present invention. *p<0.05;**p<0.01.

Fig. 6 is a bar graph showing the effect of the honeysuckle fermented product on enhancing the expression of Parkin gene according to one embodiment of the present invention. *p<0.05;**p<0.01.

Fig. 7 is a bar graph showing the effect of the honeysuckle fermented product on enhancing the expression of Atg1 gene according to one embodiment of the present invention. *p<0.05;**p<0.01.

Fig. 8 is a bar graph showing the effect of the honeysuckle fermented product on reducing the PARP2 gene and increasing the expression level of the Ubl-5 gene according to an embodiment of the present invention. *p<0.05;**p<0.01.

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 are analyzed by student's t -test .

Honeysuckle ( Lonicera japonica Thunb. ) is a perennial evergreen winding woody vine plant of the genus Lonicera in the family Caprifoliaceae . It is native to Taiwan, China, and Japan. Alias, vine, honey jue vine, psychic grass, sweet vine, etc. The flowers of honeysuckle grow in pairs, the ovaries are connected, and they bloom in summer and autumn. The flowers are white at first, and then turn yellow, and will gradually turn yellow as time goes by. Because there are white and yellow mixed in it, it is called honeysuckle. Its buds are dried in the sun, which is the honeysuckle of Chinese medicinal materials, commonly known as honeysuckle. It has the effects of detoxification, annealing and anti-inflammatory.

As used herein, the term "honeysuckle fermented product" refers to the honeysuckle extract obtained by extracting honeysuckle and solvent at a ratio of 1:10-20 (w/w) for a specific time and temperature, and then adding yeast Bacteria, lactic acid bacteria, and acetic acid bacteria are obtained by a three-stage fermentation in sequence, wherein the addition of the yeast is 0.01-0.5% (w/w); the addition of the lactic acid bacteria is 0.01-0.25% (w/w ); the added amount of the acetic acid bacteria is 3-10% (w/w).

The "effective concentration" mentioned in this article means that it can effectively improve the reducing activity, antioxidant activity, the ability to remove glycation end products, increase the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in cells, and at the same time reduce The concentration of the honeysuckle fermented product of the present invention required for the expression of PARP2 gene. The effective concentration may vary depending on the target, but the effective concentration can be determined experimentally by, for example, a dose escalation test (dose escalation).

According to the present invention, the operation procedures and parameter conditions related to the extraction fall within the professional quality and routine technical scope of those skilled in this technology.

According to the present invention, the operating procedures and parameter conditions related to the microbial fermentation reaction fall within the professional accomplishment and routine technical scope of those skilled in this technology.

According to the present invention, pharmaceuticals can be manufactured into a dosage form suitable for parenterally or topically administered by techniques well known to those skilled in the art, including, but not limited to: injectable products (injection) [for example, sterile aqueous solution (sterile aqueous solution) or dispersion liquid (dispersion)], sterile powder (sterile powder), external preparation (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, glue 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 subcutaneous injection, intraepidermal injection, intradermal injection, Intradermal 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), preserving agents, antioxidants, surfactants, absorption enhancers (absorption enhancers), stabilizing agents, gelling agents (such as carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose, and carboxymethylcellulose] , active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusives occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants Agents (propellants), etc. 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.

The present invention provides a honeysuckle fermented product for improving skin appearance and anti-aging. The honeysuckle fermented product of the present invention is made by mixing honeysuckle and solvent at a ratio of 1:10-20 (w/w) for a specific time and temperature The extracted honeysuckle extract is obtained by three-stage fermentation in sequence with yeast, lactic acid bacteria, and acetic acid bacteria, wherein the yeast is a strain of BCRC20271, the lactic acid bacteria is a strain of BCRC910805, and the acetic acid bacteria is BCRC11688 strains. The honeysuckle fermented product of the present invention can effectively improve reducing activity, antioxidant activity, ability to remove glycation end products, increase the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in cells, and reduce the expression of PARP2 gene at the same time quantity.

At the same time, the composition of the present invention for improving skin appearance and anti-aging may also include an effective amount of honeysuckle fermented product and a pharmaceutically acceptable carrier. The composition is a medicine, a skin care product, or a food.

The detailed preparation method of the honeysuckle fermented product of the present invention, the test of improving the efficacy of total polyphenols in the fermented product of honeysuckle by the three-stage fermentation method of the present invention, and the improvement of the fermented product of honeysuckle by the three-stage fermentation method of the present invention will be described in detail below The test of the effect of reducing activity, the test of the effect of the honeysuckle fermented product on enhancing the antioxidant activity of skin cells, the test of the effect of the three-stage fermentation method of the present invention on improving the anti-glycation activity of the honeysuckle fermented product, and the honeysuckle fermented product Regulate the effect of SOD3 gene, Parkin gene, Atg1 gene, PARP2 gene, and Ubl-5 gene expression to confirm that the honeysuckle fermented product of the present invention has the ability to improve reducing activity, antioxidant activity, and scavenging glycation end products, Increase the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in cells, and reduce the expression of PARP2 gene at the same time, which helps to delay the progress of chronic diseases and aging, and can increase the concentration of NAD ⁺ in cells , Maintain the normal function of decomposing old and waste proteins in cells, promote the degradation of waste in cells, and promote the normal operation of autophagy in cells, and can improve the activity of repairing DNA damage and promoting mitochondrial metabolism to prolong the life of cells Life expectancy, to achieve the effect of anti-aging.

Embodiment 1 The preparation method of honeysuckle fermented product of the present invention

In one embodiment of the present invention, the flower of honeysuckle is uniformly mixed with water, alcohol, or an extraction solvent of a mixture of alcohol and water in a ratio of 1:10-20 (w/w), the extraction solvent is preferably water, and After simultaneous sterilization and extraction at 50-100°C for 0.5-1.5 hours, add 5-10% (w/w) glucose according to the total weight to obtain a honeysuckle extract, and then cool the honeysuckle extract to room temperature for Subsequent three-stage fermentation is used, and the following three bacteria are fermented in sequence: yeast, lactic acid bacteria, and acetic acid bacteria. The fermentation order of these three bacteria cannot be reversed, and the fermentation time ratio is 1-2.5:1-3:3- 10. In a preferred embodiment of the present invention, first implant 0.01-0.5% (w/w) yeast ( S'accharomyces cerevisiae ) in the extraction solution, purchased from the Biological Resource Conservation and Research Center, Taiwan, number BCRC20271 ), carry out pre-fermentation at 25-35°C for 1-2.5 days, the actual time varies depending on the fermentation state. Then directly implant 0.01-0.25% (w/w) lactic acid bacteria ( Lactobacillus plantarum TCI028, patent deposited in Biological Resource Conservation and Research Center, Taiwan, No. BCRC910805), and carry out post-fermentation at 25-35°C for 1-3 days , the actual time varies depending on the state of fermentation. Then directly implant 3-10% (w/w) acetic acid bacteria ( Acetobacter aceti , purchased from Biological Resource Conservation and Research Center, Taiwan, No. BCRC11688), and carry out submerged fermentation at 25-35°C for 3-10 days , the actual time varies depending on the fermentation state; among them, the lactic acid bacteria TCI028 series has completed the patent deposit in the Republic of China Patent Application No. 106145146. Finally, without removing these three types of bacteria, use the set sugar content range of 2-4°, pH<4, alcohol<5% and other specifications. If the inspection meets the specifications, it is judged that the fermentation is complete and the fermentation broth is obtained. Next, the fermentation broth was concentrated under reduced pressure at 45-70°C, filtered through a 200-400 mesh sieve, and 40-70% isomaltooligosaccharide was added to adjust the specification, and then heated at 95-105°C for 70 Sterilize in -90 minutes to obtain the honeysuckle fermented product of the present invention.

Example 2 The three-stage fermentation method of the present invention improves the efficacy of total polyphenols in the honeysuckle fermented product

One embodiment of the present invention is a test experiment for improving the efficacy of total polyphenols in honeysuckle fermented products by the three-stage fermentation method of the present invention. Therefore, the Folin-Ciocalteu colorimetric method is used to measure the total polyphenol content in the proxies; This assay is carried out by utilizing the antioxidant properties of polyphenols. Phosphotungstic acid and phosphomolybdic acid in the reagent are reduced by polyphenols (from Mo ⁶⁺ to Mo ⁵⁺ ) to produce A blue compound (absorbance value at 750nm), and the depth of the blue compound is directly proportional to the total polyphenol content, so it can be used to quantify the total polyphenol content in the analyte.

First, gallic acid (Gallic acid, purchased from Sigma, the United States, No. G7384) was used as a standard product to prepare a standard curve, and 10.0 g of gallic acid was accurately weighed and placed in a 10 mL volumetric flask, and then ddH ₂ O until the total volume reaches 10mL, and complete the standard product of gallic acid (Gallic acid stock, 1000μg/mL), then first dilute the standard product 10 times to a concentration of 100μg/mL (100μL of Gallic acid stock+900μL ddH ₂ O, the unused Gallic acid stock was stored at -20°C), and then the 100 μg/mL gallic acid was serially diluted to 0, 20, 40, 60, 80, and 100 μL/mL gallic acid (as shown in Table 1), and take 100 μL of standard solutions of various concentrations into 10 mL glass test tubes, and then add 500 μL of Folin-Ciocalteu’s phenol reagent (purchased from Merck, Germany, No. 1.09001. 0100) and mix well and let it stand for 3 minutes, add 400 μL of 7.5% sodium carbonate (Sodium carbonate, quantify 7.5 g of anhydrous sodium carbonate to 100 mL of ddH ₂ O, wherein the anhydrous sodium carbonate is purchased from Sigma, the U.S. , No. 31432,) after mixing evenly, let it stand for 30-60 minutes, preferably 30 minutes, then mix it with Vortex and let it stand until no bubbles are confirmed, then take out 200 μL of the reaction solution from each tube and place it in a 96-well culture plate, and measure its absorbance at 750nm to draw the regression curve formula of the standard solution.

Next, take 100 μL of the honeysuckle extract described in Example 1 and the honeysuckle fermented product of the present invention in 10 mL glass test tubes, then add 500 μL of Folin-Ciocalteu phenolic reagent, mix well and let stand for 3 minutes Finally, add 400 μL of 7.5% sodium carbonate and mix evenly, then let it stand for reaction for 30-60 minutes, preferably 30 minutes, then mix it with Vortex and let it stand until no bubbles are confirmed, then take out 200 μL of the reaction solution of each tube and place In a 96-well culture plate, measure its absorbance at 750nm, and use the regression curve formula of the above-mentioned standard solution to calculate the concentration by the internal difference method, and then multiply the dilution factor to obtain the original honeysuckle extract and the present invention. Concentration of total polyphenols in honeysuckle ferment.

The test results of the three-stage fermentation method of the present invention to improve the efficacy of total polyphenols in the honeysuckle fermented product of the present invention are shown in FIG. 1 . The honeysuckle water extract contains only 853.75 μg/mL of total polyphenol content, but the total polyphenol content in the honeysuckle fermented product of the present invention is significantly as high as 1131.92 μg/mL, which is the highest polyphenol content of honeysuckle. 1.34 times of flower water extract. The results show that the three-stage fermentation method of the present invention can significantly increase the content of total polyphenols in the honeysuckle fermented product of the present invention, so as to achieve the purpose of improving functional components and enhancing the health-care and antioxidant effects.

Example 3 The effect of the three-stage fermentation method of the present invention on improving the reducing activity of honeysuckle fermented products

One embodiment of the present invention is to carry out the test experiment of the effect of the three-stage fermentation method of the present invention to improve the reducing activity of the honeysuckle fermented product of the present invention, so the colorimetric method of reducing iron oxidation (Ferric reducing antioxidant power, FRAP) is used method to determine the antioxidant capacity of the analyte; wherein, the use of the analyte with reducing activity can reduce iron ions (Fe3 ⁺ ) to ferrous ions (Fe2 ⁺ ), and the color of the solution changes from reddish brown to Green, and the depth of the green compound is proportional to the reducing ability, so it can be used to quantify the reducing activity of the analyte.

First, a standard curve was prepared using ascorbic acid, which is known to have reducing activity. Precisely weigh 10mL of L-ascorbic acid (L-Ascorbic acid, Vit C, vitamin C, purchased from Sigma, the United States, No. A5960-25g), put it in a 10mL volumetric flask, and then add ddH ₂ O to set the amount to 10mL, Configured as 1 mg/mL L-ascorbic acid. Next, take 1mL of prepared L-ascorbic acid and place it in a 10mL volumetric flask, then add ddH ₂ O to make the volume to 10mL, configure as 1μg/mL L-ascorbic acid, and make the 100μg/mL L-ascorbic acid serial Dilute to 0, 10, 20, 40, 60, 80, and 100 μL/mL of L-ascorbic acid (as shown in Table 2), and take 250 μL of the standard solution of each concentration into the test tube, and then add 250 μL of phosphoric acid Salt buffer solution (anhydrous sodium dihydrogen phosphate (NaH ₂ PO ₄ , purchased from JTBaker, No. 3828-01) and disodium hydrogen phosphate (Na ₂ HPO ₄ , purchased from Sigma, No. 04270) were mixed at a ratio of 1:1. prepared) was mixed evenly with a vibrator (Vortex), and 250 μL of 1% red blood salt (Potassium ferricyanide, K ₃ Fe (CN), purchased from Sigma, No. 244023) was added. Heat in a water bath at ℃ for 20 minutes, then add 250 μL of 10% trichloroacetic acid (Trichloroacetic acid, TCA, CCl ₃ COOH, purchased from JT Baker, No. 0414-01) and mix evenly with a shaker, then centrifuge at 300 g for 10 minutes , and be careful not to shake when taking it out, then take out 300 μL of the supernatant of the reaction solution in each tube, add 300 μL of ddH ₂ O and 120 μL of 1% ferric chloride (FeCl ₃ , purchased from Alfa Aesar, number A16231) to After mixing evenly with a vibrator, react for 10 minutes, and measure the absorbance value at 700nm to draw the regression curve formula of the standard solution.

Next, take 25 μL of the honeysuckle extract described in Example 1 and the honeysuckle fermented product of the present invention in a 10 mL glass test tube, dilute ten times with ddH ₂ O (1:10 dilution), and then add 250 μL After mixing the phosphate buffer solution with a shaker, add 250 μL of 1% red blood salt and mix evenly with a shaker, heat it in a water bath at 50°C for 20 minutes, and then add 250 μL of 10% trichloroacetic acid to shake After the instrument is mixed evenly, centrifuge at 300g for 10 minutes, and be careful not to shake when taking it out, then take out 300 μL of the supernatant of the reaction solution in each tube, add 300 μL of ddH ₂ O and 120 μL of 1% ferric chloride to the shaker After mixing evenly, react for 10 minutes, and measure its absorbance at 700nm, then use the regression curve formula of the above-mentioned standard solution to calculate the concentration by the internal difference method, and then multiply the dilution factor to obtain the original honeysuckle extract and the present invention. The reducing ability of honeysuckle fermented product.

The test results of the effect of the three-stage fermentation method of the present invention on improving the reducing activity of the honeysuckle fermented product of the present invention are shown in FIG. 2 . The honeysuckle water extract only contains 138.19 units of reducing activity, but the reducing activity of the honeysuckle fermented product of the present invention is significantly as high as 376.67 units, which is 2.73 times that of the honeysuckle water extract. The results show that the three-stage fermentation method of the present invention can significantly increase the reducing activity of the honeysuckle fermented product of the present invention, so as to achieve the purpose of improving functional components and enhancing the health-care and antioxidant effects.

Example 4 The effect of the honeysuckle fermented product of the present invention on enhancing the antioxidant activity of skin cells

In one embodiment of the present invention, human skin fibroblasts (Human skin fibroblast, CCD-966sk) are used to test the effect of the honeysuckle fermented product of the present invention on enhancing the antioxidant activity of skin cells. Among them, the human dermal fibroblasts were purchased from the American Type Culture Collection (ATCC®), the cell number is CRL-1881, and the cell line was cultured in 10% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, U.S.) and 90% of Minimum Essential Medium (MEM, purchased from Gibco, U.S.), which contained 1 mM sodium pyruvate (Sodium pyruvate, purchased from Gibco, U.S.) and 1% Penicillins/streptomycin (purchased from Gibco, USA).

First, 1× ¹⁰⁵ human dermal fibroblasts were planted in a 6-well culture dish containing 2mL of the above culture medium, and cultured at 37°C for 24 hours, and the cell culture medium was removed without disturbing the cells , and washed with 1 times phosphate buffered saline (1xPhosphate buffered saline, 1xPBS) and then removed the cells, and then divided the cells into the following four groups: (1) the blank control group treated with only the cell culture medium for 2 hours, (2) 500 μM H ₂ O ₂ (purchased from Sigma, USA) was added to the control group treated for 2 hours, (3) the comparison group was pretreated with 2 mg/mL honeysuckle extract for 1 hour and then treated with 500 μM H ₂ O ₂ for 1 hour , and (4) an experimental group pretreated with 2 mg/mL honeysuckle fermented product of the present invention for 1 hour and then treated with 500 μM H ₂ O ₂ for 1 hour. Then, add 5 μg/mL DCFH-DA to each group to treat at 37°C for 15 minutes, then treat the cells with 500 μM H ₂ O ₂ at 37°C for 1 hour, then wash the cells with 1 mL of 1-fold phosphate buffer solution Twice, add 200 μL of trypsin (Trypsin) and react in the dark for 5 minutes to make the cells fall off the culture plate, add appropriate culture medium to terminate the reaction, collect the cells together with the culture medium into a 1.5mL centrifuge tube, and centrifuge at 400g Remove the supernatant after 5 minutes, wash the cells once with 1mL of 1xPBS, centrifuge at 400g for 10 minutes and remove the supernatant, resuspend the cells with 1mL of 1xPBS, and add 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA, purchased from Sigma, the United States, No. SI-D6883-50MG, 5 mg/mL stored in DMSO) labeled cells, and using flow cytometry to detect cells at an excitation wavelength of 450-490nm and an emission wavelength of 510-550nm Fluorescence value. Then use Excel software to conduct student t-test to determine whether there is a statistically significant difference between the two sample groups.

2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is a stable non-polar compound that can freely permeate the cell membrane. When DCFH-DA enters the cell, it will be hydrolyzed by intracellular lipase to form a polar 2' ,7'-dichlorodihydrofluorescin (DCFH), stays in the cell and cannot come out, while the reactive oxygen species (Reactive Oxygen Species, ROS) in the cell A redox reaction with DCFH forms 2',7'-dichlorofluorescin (DCF). After excitation at a wavelength of 450-490nm, the green fluorescence generated can be detected at a wavelength of 510-550nm, so the detection is performed by DCFH- The fluorescence intensity of DA-treated cells can reflect the content of reactive oxygen species in the cells.

The test results of improving the effect of the honeysuckle fermented product of the present invention on enhancing the antioxidant activity of skin cells by the three-stage fermentation method of the present invention are shown in FIG. 3 . In the blank control group and control group treated with H ₂ O ₂ , 9.9% and 15.1% of human dermal fibroblasts contained ROS, which showed that H ₂ O ₂ could indeed induce oxidative stress in skin cells; After treatment with the flower water extract, the human skin fibroblasts containing ROS can only be reduced to 2.8%; while the honeysuckle fermented product of the present invention can significantly reduce the human skin fibroblasts containing ROS to 1.1% . The results show that the three-stage fermentation method of the present invention can significantly increase the antioxidant activity of the honeysuckle fermented product of the present invention in skin cells, so as to achieve the purpose of improving functional ingredients and enhancing the health-care and antioxidant effects.

Example 5 The effect of the three-stage fermentation method of the present invention on improving the anti-glycation activity of honeysuckle fermented products

One embodiment of the present invention is to carry out the test experiment of the effect of the three-stage fermentation method of the present invention to enhance the anti-glycation activity of honeysuckle fermented products to inhibit D-fructose (D-fructose) from making bovine serum albumin (Bovine serum albumin, BSA) The efficiency of glycosylation is used to quantify glycosylation activity; anti-glycation can inhibit non-enzymatic browning to avoid denaturation of functional proteins in the body. First, take 0.25mL honeysuckle water extract diluted to 20% (v/v) or honeysuckle fermented product of the present invention respectively, the two are respectively the control group and the experimental group, and 0.25mL of water as the control group, then Each of the three groups was added to 0.25 mL of 60 mg/mL BSA solution containing 0.06% NaN ₃ (configured with 200 mM sodium phosphate buffer, pH 7.4), and mixed with 0.25 mL of 1.5 M D-fructose (with Prepare 200mM sodium phosphate buffer solution, pH7.4) and mix evenly, then take 0.1mL of the mixed solution to measure the fluorescence value under excitation light 360nm, radiation light 460nm, take this as the zero point before the reaction, and take 0.45mL of the mixed solution After the mixed solution was incubated at 50°C for 24 hours, 0.1 mL was taken out to measure the fluorescence value, which was regarded as the end point of the reaction. And with an equal amount of 3mM aminoguanidine (Aminoguanidine, AG, configured with 200mM phosphate buffer, pH 7.4) back to the solvent to an equal volume as a positive control group, where it is known that Aminoguanidine (AG) has an inhibitory effect on glycation effect. Finally, the efficiency of the ability to remove glycation end products (AGEs) was calculated by the following formula to represent its anti-glycation activity.

The test results of the effect of the three-stage fermentation method of the present invention on improving the anti-glycation activity of the honeysuckle fermented product of the present invention are shown in Figure 4, wherein the control group is 100%. The honeysuckle water extract can only reduce the formation of 2.1% saccharification end products, while the honeysuckle fermented product of the present invention can significantly reduce the formation of 33.61% saccharification end products, which is 16 times that of the honeysuckle water extract. The results show that the honeysuckle fermented product of the present invention has a strong activity of inhibiting saccharification reaction, and the three-stage fermentation method of the present invention can significantly improve the ability of the fermented product of honeysuckle of the present invention to remove the end products of saccharification, which is helpful for delaying the saccharification reaction. Progression of disease and aging.

Example 6 The efficacy of the honeysuckle fermented product of the present invention in regulating gene expression

In one embodiment of the present invention, human primary skin keratinocytes HPEK-50 are used to test the effectiveness of the honeysuckle fermented product of the present invention in regulating the expression of SOD3 gene, Parkin gene, Atg1 gene, PARP2 gene, and Ubl-5 gene; wherein, The HPEK-50 cell line was purchased from CELLnTEC (Switzerland) and cultivated in serum-free keratinocyte culture medium (Keratinocyte-SFM). Cultured in a 37°C cell incubator containing 5% _CO2 .

First, ^1.5x105 human primary skin keratinocytes were cultured in a 6-well culture dish containing 2mL of the above-mentioned culture solution in each well, and cultured at 37°C for 16-18 hours, and then the cells were divided into the following three groups: (1) Add only The control group of the cell culture medium, (2) the comparison group adding 0.125% honeysuckle water extract, and (3) the experimental group adding 0.125% honeysuckle fermented product of the present invention, and the three groups were respectively heated at 37°C After 48 hours of action, the expression level of the target gene in the primary human skin keratinocytes of each group was tested. First, after the cells of the three groups were recovered with cell lysate (RB buffer, purchased from Geanaid Company, Taiwan, Cat No. RBD300), the RNA extraction reagent set (purchased from Geneaid Company, Taiwan, Cat No. RBD300 ) to collect the total RNA in the three groups of cells, and then use SuperScript ^® III reverse transcriptase (purchased from Invitrogene, the United States, No. 18080-051) to use 2000ng of extracted RNA as a template, and use the combined primers and reverse transcriptase in Table 3 Transcriptase carried out reverse transcription to produce the cDNA products corresponding to the mRNAs of these genes, and then used ABI StepOnePlus ^TM Real-Time PCR system (Thermo Fisher Scientific Company, USA), and KAPA SYBR FAST (purchased from Sigma Company, USA) , No. 38220000000) the three groups of reverse-transcribed products were subjected to quantitative real-time polymerase chain reaction (qPCR) test with the combined primers in Table 3. The conditions were 95°C for 1 second and 60°C for 1 second. 20 seconds for a total of 40 loops. Used to quantify the mRNA expression of SOD3 gene, Parkin gene, Atg1 gene, PARP2 gene, and Ubl-5 gene in the two groups of human primary skin keratinocytes, wherein the quantitative value is taken by the threshold cycle number (Ct), and the target The relative amount of mRNA of a gene is derived from Equation 2 ^-ΔΔCt , where ΔC _T = _{CT target gene in comparison group or experimental group/control group target gene} -C _TTBP (TATA-binding protein, TATA-binding protein); ΔΔC _T =C _T The target gene of the _{comparison group or the experimental group -CT target gene of the control group} ; the fold change of each gene in each group is 2 ^-ΔΔCt . Then, use Excel software to determine whether the coefficient of variation and whether there is a statistically significant difference (* p value <0.05; ** p value <0.01; *** p value <0.001).

The protein encoded by the Parkin gene is an enzyme present in the Ubiquitin-proteasome system (Ubiquitin-proteasome system) and acts as a regulator of protein breakdown. It is known that the mutation of this gene is related to Parkinson's disease, and previous studies have also It is pointed out that increasing the expression of this gene can delay the aging effect of cells, so it is considered to be related to the aging effect of cells.

Atg gene is a gene related to autophagy. The protein encoded by it will participate in the degradation of waste in cells and autophagy in circulation. Previous studies have pointed out that the overexpression of this gene can prolong the lifespan of mice, so it is considered to be related to related to cellular aging.

Previous studies have pointed out that compared with old mice, young mice contain more NAD ⁺ in their bodies; and increasing the concentration of NAD ⁺ in old mice can make their physiological conditions younger, so NAD ⁺ is It is believed that it is related to delaying the aging of individuals, and the results of the research have known that the proteins encoded by the Ubl-5 gene and the SOD3 gene are related to the regulation of NAD ⁺ .

The protein poly(ADP-ribose) polymerase 2 (Poly(ADP-Ribose) Polymerase 2) encoded by the PARP2 gene is NAD ⁺ as a substrate, and its main function is to catalyze adenosine diphosphate-ribose (ADP-ribose) to form Poly(ADP-ribose) polymer (referred to as PAR). When the DNA in the cell is structurally damaged, PARP-2 will be activated due to DNA single-strand or double-strand breaks, so it is regarded as a sensor of DNA damage, and the activation of PARP-2 will affect mitochondrial activity , and then regulate cellular energy metabolism, therefore, PARP and mitochondrial-related diseases and aging phenomena are also important.

The three-stage fermentation method of the present invention improves the honeysuckle fermented product of the present invention to increase the expression level of the SOD3 gene. The test results are shown in FIG. 5 . After the primary human skin keratinocytes were treated with honeysuckle water extract, the expression level of SOD3 gene could only be increased by 1.1 times that of the control group; however, the honeysuckle fermented product of the present invention could significantly increase the expression level of SOD3 gene by 1.5 times that of the control group. This result shows that the three-stage fermentation method of the present invention can significantly improve the ability of the honeysuckle fermented product of the present invention to increase the expression level of the SOD3 gene in skin cells, so as to increase the concentration of NAD ⁺ in skin cells and achieve the effect of skin anti-aging.

The three-stage fermentation method of the present invention improves the expression level of the Parkin gene by improving the honeysuckle fermented product of the present invention. The test results are shown in FIG. 6 . After the primary human skin keratinocytes were treated with honeysuckle water extract, the expression of Parkin gene could only be increased by 1.2 times that of the control group; however, the fermented product of honeysuckle of the present invention could significantly increase the expression of Parkin gene by 1.4 times that of the control group. This result shows that the three-stage fermentation method of the present invention can significantly improve the ability of the honeysuckle fermented product of the present invention to enhance the expression of Parkin gene in skin cells, maintain the normal function of protein decomposition in cells, and have the effect of preventing cell aging .

The three-stage fermentation method of the present invention improves the expression level of Atg1 gene of the honeysuckle fermented product of the present invention, and the test results are shown in FIG. 7 . After the primary human skin keratinocytes were treated with honeysuckle water extract, the expression level of Atg1 gene could only be increased by 1.0 times that of the control group; however, the honeysuckle fermented product of the present invention could significantly increase the expression level of Atg1 gene by 1.3 times that of the control group. This result shows that the three-stage fermentation method of the present invention can significantly improve the ability of the honeysuckle fermented product of the present invention to enhance the expression of the Atg1 gene in skin cells, and can enhance the degradation of waste in the cells and the autophagy of the cycle to prolong the life of the cells. Life expectancy, to achieve the effect of cell anti-aging.

The three-stage fermentation method of the present invention improves the honeysuckle fermented product of the present invention in reducing the PARP2 gene and increasing the expression level of the Ubl-5 gene. The test results are shown in FIG. 8 . After the first generation of human skin keratinocytes were treated with honeysuckle water extract, the expression of PARP2 gene could only be reduced to 0.85 times that of the control group, and the expression of Ubl-5 gene could only be increased to 1.0 times that of the control group; while the gold and silver of the present invention The flower fermentation product can significantly reduce the expression level of PARP2 gene to 0.75 times that of the control group, and can significantly increase the expression level of Ubl-5 gene to 1.1 times that of the control group. This result shows that the three-stage fermentation method of the present invention can effectively improve the ability of the honeysuckle fermented product of the present invention to reduce the expression of PARP2 gene in skin cells and increase the expression of Ubl-5 gene, which can increase the concentration of NAD ⁺ in skin cells , and enhance the activity of repairing DNA damage and promoting mitochondrial metabolism, so as to achieve the effect of skin anti-aging.

In summary, the honeysuckle fermented product obtained by the three-stage fermentation of the honeysuckle extract in the present invention with yeast, lactic acid bacteria, and acetic acid bacteria can significantly increase the total amount of the honeysuckle fermented product of the present invention through the microbial fermentation process. content of polyphenols, significantly improve the reducing activity of the honeysuckle fermented product of the present invention, significantly improve the antioxidant activity of the honeysuckle fermented product of the present invention in skin cells, significantly improve the ability of the honeysuckle fermented product of the present invention to remove saccharification end products, and can Significantly improve the ability of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene expression in the cells of the honeysuckle fermented product of the present invention, and reduce the expression of PARP2 gene; to achieve the effect of enhancing anti-oxidation and anti-glycation, there is Helps delay the progress of chronic diseases and aging, and can increase the concentration of NAD ⁺ in cells, maintain the normal function of decomposing old waste proteins in cells, promote the degradation of waste in cells, and promote the normal operation of autophagy in cells, and can Improve the activity of repairing DNA damage and promoting mitochondrial metabolism to prolong the life of cells and achieve anti-aging effects. Therefore, the honeysuckle fermented product of the present invention can be used to prepare a composition for improving skin appearance and anti-aging, and the composition is a medicine, a skin care product, or a food, and can be administered by oral administration, smearing, etc. a body.

【Biological Material Storage】

Institute of Food Industry Development (Taiwan); December 20, 2016; No. BCRC910805.

<110> Dajiang Biomedical Co., Ltd.

<120> Preparation method of honeysuckle fermented product and its use for improving skin appearance and anti-aging

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<170> PatentIn version 3.5

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Claims (8)

A method for producing a honeysuckle fermented product, comprising: extracting a honeysuckle flower extract obtained by extracting a honeysuckle flower with water as a solvent, mixing the honeysuckle flower and water in a ratio of 1:10-20 (w/w), and then The honeysuckle extract is obtained by sequentially fermenting a yeast ( Saccharomyces cerevisiae ), a lactic acid bacteria ( Lactobacillus plantarum ) and an acetic acid bacteria ( Acetobacter aceti ) respectively, wherein the added amount of the yeast is 0.01-0.5 %(w/w); the added amount of the lactic acid bacteria is 0.01-0.25% (w/w); the added amount of the acetic acid bacteria is 3-10% (w/w), the yeast, the lactic acid bacteria, and the acetic acid The fermentation time ratio of bacteria is 1-2.5:1-3:3-10. A use of the honeysuckle fermented product as described in claim 1 for preparing a composition for delaying aging. The use as described in claim 2, wherein according to the colorimetric method of reducing iron oxidation (Ferric reducing antioxidant power, FRAP), the ability of the honeysuckle fermented product to reduce iron ions (Fe3 ⁺ ) is equivalent to 300-400 μg/mL The ability of ascorbic acid to reduce iron ions (Fe3 ⁺ ). The use as described in claim 2, wherein the honeysuckle fermented product further enhances antioxidant activity and anti-glycation activity. The use as described in claim 2, wherein the honeysuckle fermented product promotes superoxide dismutase 3 (Superoxide dismutase 3, SOD3 ) gene, Parkinson's disease juvenile protein (Parkinson juvenile disease protein, Parkin ) gene, autophagy The expression level of the function-related gene 1 (Autophagy-related gene 1, Atg1 ) gene, and the Ubl gene is the ubiquitin-like protein 5 (Ubiquitin-like protein 5, Ubl-5 ) gene; and it is the inhibitory protein poly(ADP-ribose) Poly(ADP-Ribose) Polymerase 2, PARP2 gene expression. The use as described in claim 2, wherein the honeysuckle fermented product increases the concentration of NAD ⁺ in skin cells, maintains the normal function of decomposing old waste proteins in cells, promotes the degradation of waste in cells, and promotes the normal function of autophagy in cells function, enhance the ability to repair DNA damage, and/or promote the activity of mitochondrial metabolism. A use of the honeysuckle fermented product as described in claim 1 for preparing a composition for improving anti-aging ability, wherein improving the anti-aging ability is to improve skin appearance, wherein the honeysuckle fermented product enhances the anti-aging ability of human skin fibers Oxidative activity, enhanced reducing activity, enhanced anti-glycation activity, and/or increased total polyphenol content. The use as described in any one of claims 2 to 7, wherein the effective concentration of the honeysuckle fermented product is at least 0.5% (v/v).

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