TW202042787A - 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 method for preparing a honeysuckle fermented product and its use, in particular to a honeysuckle fermented product prepared by the three-stage fermentation process of the present invention with a honeysuckle extract, and its use for improving skin appearance And anti-aging purposes.

The epidermal layer is the outermost layer of the skin. From the outside to the inside, it is the stratum corneum, the granular layer, the spinous layer and the basal layer. The epidermal layer is mainly formed by undifferentiated cylindrical keratinocytes in the basal layer. It is called keratinization. The water content in keratinocytes is high. As the cells metabolize and differentiate, the shape of keratinocytes will gradually become flat, and the nucleus and organelles will begin to degenerate and shrink, and dead cells without nuclei and organelles will be formed 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 stratum corneum like a brick wall. This barrier can lock the skin Moisture and grease, resistance to the invasion of bacteria on the skin surface, and resistance to damage from foreign bodies and ultraviolet light, have a very important protective effect on the human body.

In the stratum corneum in the epidermis, although the keratinocytes are dead cells, the 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 integrity of the skin barrier of the epidermal layer, to prevent skin water loss and form a complete protection. When the skin is exposed to a cold or hot environment and irritation such as ultraviolet light, the keratinocytes will not be able to maintain the normal metabolic cycle, and the skin's water retention capacity will also decrease. The skin's epidermal barrier is damaged, and the skin becomes rough, dry and desquamated, fragile and easily irritated, and sensitive to redness. Therefore, the health and water retention capacity of the stratum corneum is very important to resist external damage.

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

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 the glycation end products will bind to receptors on the cell surface to change its structure and function. It also promotes the increase of oxidative stress and inflammation of cells, and is inseparable from the formation of diabetes, arteriosclerosis and chronic kidney diseases. In addition to the glycation end products produced during the normal metabolism of the human body, many processed foods also contain glycation end products. Studies have also pointed out that avoiding the intake of glycation end products from food can help delay the progression of chronic diseases and aging.

Based on the above, in order to improve the skin becoming fragile and susceptible due to damaged keratinocytes, decreased water retention capacity, and skin aging, we have developed a method that can directly and effectively improve or delay the occurrence of skin aging, while being effective It is indeed necessary for the composition to maintain the normal physiological metabolism of keratinocytes, maintain the integrity of the stratum corneum structure, and increase the anti-glycation activity.

For this reason, one object of the present invention is to provide a honeysuckle fermentation 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 bacteria ( Lactobacillus plantarum ), and an acetic acid bacteria ( Acetobacter aceti ) are obtained by sequentially performing three-stage fermentation.

Another object of the present invention is to provide a honeysuckle fermented product as described above for preparing superoxide dismutase 3 ( SOD3 ) genes and Parkinson juvenile disease protein (Parkinson juvenile disease protein) in skin cells. , Parkin ) gene, Autophagy-related gene 1, Atg1 gene, Ubl gene line 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 use of honeysuckle fermented product for preparing a composition for enhancing anti-aging ability.

Another object of the present invention is to provide a method for manufacturing honeysuckle fermented product, which comprises: extracting a honeysuckle extract obtained by extracting a honeysuckle with a solvent, and then passing the honeysuckle extract through a yeast ( Saccharomyces cerevisiae ) , Lactobacillus plantarum , and Acetobacter aceti are obtained through three-stage fermentation in sequence.

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

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

In another embodiment of the present invention, the honeysuckle fermentation system promotes the expression level of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene; and inhibits the expression level of PARP2 gene.

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

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

In another embodiment of the present invention, the effective concentration of the fermented honeysuckle 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 amount of bacteria added is 0.01-0.5% (w/w); the amount of lactic acid bacteria added is 0.01-0.25% (w/w); the amount of acetic acid bacteria added is 3-10% (w/w); and 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 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 fermentation 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 the end product of saccharification, and can significantly improve the gold and silver of the present invention Flower ferment enhances the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in cells, and reduces the expression of PARP2 gene at the same time; in order to enhance the effect of anti-oxidation and anti-glycation, help 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 products in cells, and promote the normal operation of autophagy in cells, and improve the repair of DNA damage , And promote the activity of mitochondrial metabolism to extend 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, which can be administered by oral, smearing, etc. A body.

The following will further explain the embodiments of the present invention. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone 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 protection scope of the present invention shall be subject to the scope of the attached patent application.

Fig. 1 is a bar graph showing the effect of improving the total polyphenols in honeysuckle fermentation by a three-stage fermentation method according to an embodiment of the present invention.

Fig. 2 is a bar graph showing the reducing activity of the fermented honeysuckle in one embodiment of the present invention.

Fig. 3 is a bar graph showing the effect of fermented honeysuckle 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 fermented honeysuckle according to an embodiment of the present invention.

Fig. 5 is a bar graph showing the effect of a fermented honeysuckle in an embodiment of the present invention on increasing the expression of SOD3 gene. * p<0.05; ** p<0.01.

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

Fig. 7 is a bar graph showing the effect of a fermented honeysuckle in an embodiment of the present invention on increasing Atg1 gene expression. * p<0.05; ** p<0.01.

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

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

Use Excel software for statistical analysis. Data mean ± standard deviation (SD) represented by the difference between the two herein by Student t test (student's t -test) analysis.

Honeysuckle (Lonicera japonica Thunb.) Is Caprifoliaceae (Caprifoliaceae) Lonicera (Lonicera) perennial evergreen twining woody vine, native to Taiwan, China, and Japan, which is also called honeysuckle, silver rattan, two treasure Vine, honey vine, psychic grass, sweet vine and other aliases. The flowers of honeysuckle grow in pairs, the ovary is connected, and blooms in summer and autumn. The flowers bloom white at first, then turn yellow, and gradually turn yellow with the increase of time. They are called honeysuckle because of the mixture of white and yellow. The buds are dried to form the honeysuckle of Chinese medicine, commonly known as honeysuckle. It has detoxification, annealing and anti-inflammatory effects.

As used herein, the term "fermented honeysuckle" means 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 using yeast Bacteria, lactic acid bacteria, and acetic acid bacteria are obtained by sequentially performing a three-stage fermentation, wherein the addition amount of the yeast is 0.01-0.5% (w/w); the addition amount of the lactic acid bacteria is 0.01-0.25% (w/w) ); The addition amount of the acetic acid bacteria is 3-10% (w/w).

The "effective concentration" mentioned in this article means that it can effectively increase the reduction activity, antioxidant activity, the ability to eliminate the end-products of glycation, increase the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in cells, and reduce at the same time 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.

According to the present invention, the operating procedures and parameter conditions related to extraction fall within the professional quality and routine technical scope of those who are familiar with the technology.

According to the present invention, the operating procedures and parameter conditions related to the microbial fermentation reaction fall within the scope of professionalism and routine technology of those who are familiar with the technology.

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

According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, decomposers ), 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 quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

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

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection Injection (intradermal injection) and intralesional injection (intralesional injection).

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well-known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation 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 pharmaceutical product of the present invention with a base well known to those skilled in the art.

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

According to the present invention, the skin care product may further include an acceptable adjuvant that is widely used in 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 agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

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

According to the present invention, skin care products can also be used in combination with one or more external use agents selected from the following known active agents: whitening agents (such as tretinoin, catechins) (catechin), koji acid, arbutin and vitamin C], moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [ Such as aloe extract, skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, and anti-dermatitis agents (antidermatitis agents), antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, Antiseborrheic agents, wound-healing agents, corticosteroids and hormones. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

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

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

The present invention provides a use of honeysuckle fermented product for improving skin appearance and anti-aging. The honeysuckle fermented product of the present invention is honeysuckle and a solvent at a ratio of 1:10-20 (w/w) for a specific time and temperature The honeysuckle extract is obtained by three-stage fermentation with yeast, lactic acid bacteria, and acetic acid bacteria in sequence, wherein the yeast is a strain of BCRC20271, the lactic acid bacteria is a strain of BCRC910805, and the acetic acid bacteria is BCRC11688 The strain. The honeysuckle fermented product of the present invention can effectively improve the reduction activity, the antioxidant activity, the ability to remove the final glycation product, 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 the amount.

At the same time, the composition for improving skin appearance and anti-aging of the present invention can 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 following will describe in detail the detailed preparation method of the honeysuckle fermentation product of the present invention, the test of the three-stage fermentation method of the present invention to improve the total polyphenols in the honeysuckle fermented product, and the three-stage fermentation method of the present invention to improve the honeysuckle fermented product. Test of the efficacy of reducing activity, test of the efficacy of the honeysuckle fermented product in enhancing the antioxidant activity of skin cells, test of the efficacy of the three-stage fermentation method of the present invention for enhancing the anti-saccharification activity of the honeysuckle fermented product, and the test of the honeysuckle fermented product Testing the efficacy of regulating SOD3 gene, Parkin gene, Atg1 gene, PARP2 gene, and Ubl-5 gene expression level to confirm that the honeysuckle fermented product of the present invention has the ability to improve reducing activity, antioxidant activity, and eliminate the end product of glycation. The effect of increasing the expression of SOD3 , Parkin , Atg1 , and Ubl-5 genes in cells, while reducing the expression of PARP2 genes, helps delay the progression 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 the cell, promote the degradation of waste in the cell, and promote the normal operation of autophagy in the cell, and can improve the activity of repairing DNA damage and promoting mitochondrial metabolism to extend the cell’s Longevity, achieve anti-aging effect.

Example 1 Preparation method of honeysuckle fermented product of the present invention

In an embodiment of the present invention, the extraction solvent of honeysuckle flower and water, alcohol, or alcohol-water mixture is uniformly mixed in a ratio of 1:10-20 (w/w). The extraction solvent is preferably water, and After sterilizing and extracting at 50-100℃ for 0.5-1.5 hours, adding 5-10% (w/w) glucose according to the total weight to obtain a honeysuckle extract, and then cooling the honeysuckle extract to room temperature for supply The subsequent three-stage fermentation is used, and the following three bacteria are used for fermentation in sequence: yeast, lactic acid bacteria, and acetic acid bacteria. The fermentation sequence 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, 0.01-0.5% (w/w) yeast ( Saccharomyces cerevisiae , purchased from the Biological Resources Conservation and Research Center, Taiwan, numbered BCRC20271) is first implanted in the extraction solution, The pre-fermentation is carried out at 25-35°C for 1-2.5 days, the actual time depends on the fermentation state. Then directly implant 0.01-0.25% (w/w) lactic acid bacteria ( Lactobacillus plantarum TCI028, patent deposited at the Biological Resources Conservation and Research Center, Taiwan, number BCRC910805), and post-fermentation at 25-35°C for 1-3 days , The actual time varies depending on the fermentation state. Then directly implant 3-10% (w/w) acetic acid bacteria ( Acetobacter aceti , purchased from the Biological Resources Conservation and Research Center, Taiwan, numbered BCRC11688), and carry out deep 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 system has been patented in the Republic of China Patent Application No. 106145146. Finally, without removing these three bacteria, use the set sugar content range of 2-4°, pH<4, alcohol<5% and other specifications. If the test meets the specifications, the fermentation is determined to be completed 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, 40-70% isomalt-oligosaccharides were added to adjust the specifications, and then heated at 95-105°C for 70 -Sterilize for 90 minutes to obtain the honeysuckle fermented product of the present invention.

Example 2 The three-stage fermentation method of the present invention enhances the effect of total polyphenols in honeysuckle fermentation

One embodiment of the present invention is to conduct a test experiment for the three-stage fermentation method of the present invention to improve the efficacy of total polyphenols in honeysuckle fermentation, so the Folin-Ciocalteu colorimetric method is used to determine the total polyphenol content in the substitute test; This assay is based on the antioxidant properties of polyphenols. Phosphotungstic acid and Phosphomolybdic acid in the reagent are reduced by polyphenols (from Mo ⁶⁺ to Mo ⁵⁺ ). Blue compound (absorbance value at 750nm), and the depth of the blue compound is proportional to the total polyphenol content, so it can be used to quantify the total polyphenol content in the test substance.

First, use gallic acid (purchased from Sigma, USA, numbered G7384) as a standard product to prepare a standard curve, accurately weigh 10.0 g of gallic acid and place it in a 10 mL volumetric flask, and then add ddH ₂ O to a total volume of 10mL, and complete the gallic acid standard (Gallic acid stock, 1000μg/mL), then first dilute the standard 10 times to a concentration of 100μg/mL (100μL of Gallic acid stock+900μL DdH ₂ O, the unused Gallic acid stock is stored at -20℃), and then the 100μg/mL gallic acid is serially diluted to 0, 20, 40, 60, 80, and 100μL/mL Gallic acid (as shown in Table 1), and respectively take 100μL of the standard solution of each concentration into a 10mL glass test tube, and then add 500μL of Folin-Ciocalteu's phenol reagent (purchased from Merck, Germany, number 1.09001. 0100) After mixing uniformly and standing 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, where the anhydrous sodium carbonate is purchased from Sigma, USA , No. 31432,) After evenly mixing, let it stand for 30-60 minutes, preferably 30 minutes, and then mix with Vortex until it is confirmed that there are no bubbles, then take out 200μL of each tube of reaction solution and place it in 96-well culture In the plate, and measure its absorbance at 750nm to draw the regression curve formula of the standard solution.

Then, respectively take 100 μL of the honeysuckle extract described in Example 1 and the honeysuckle fermented product of the present invention into a 10 mL glass test tube, and then add 500 μL of Folin-Ciocalteu's phenol reagent, mix well and let stand for 3 minutes Then, add 400μL of 7.5% sodium carbonate and mix it evenly and let it stand for 30-60 minutes, preferably 30 minutes, and then mix with Vortex until it is confirmed that there are no bubbles, then take out 200μL of each tube of reaction solution and place Measure the absorbance at 750nm in a 96-well culture plate, and use the regression curve formula of the above standard solution to calculate the concentration by the inner difference method, and then multiply the dilution factor to obtain the original honeysuckle extract and the present invention The concentration of total polyphenols in honeysuckle fermentation.

The test result of the three-stage fermentation method of the present invention for improving the efficacy of the total polyphenols in the fermentation of honeysuckle of the present invention is shown in FIG. 1. The water extract of honeysuckle contains only 853.75μg/mL of total polyphenol content, while the total polyphenol content in the fermented honeysuckle of the present invention is significantly as high as 1131.92μg/mL, which is gold and silver 1.34 times of flower water extract. This result shows that the three-stage fermentation method of the present invention can significantly increase the content of total polyphenols in the honeysuckle fermentation of the present invention, so as to achieve the purpose of enhancing the functional components and enhancing the health-care antioxidant effect.

Example 3 The three-stage fermentation method of the present invention enhances the effect of honeysuckle fermentation product reduction activity

One embodiment of the present invention is to carry out the test experiment of the three-stage fermentation method of the present invention to improve the reducing activity of the honeysuckle fermented product of the present invention. Therefore, the colorimetry of Ferric reducing antioxidant power (FRAP) is used. Method to determine the antioxidant capacity of the analyte; among them, the analyte with reducing activity can reduce iron ions (Fe3 ⁺ ) to ferrous ions (Fe2 ⁺ ), and the color of the solution turns from reddish brown to Green, and the intensity of the green compound is directly proportional to the reducing power, so it can be used to quantify the reducing activity of the analyte.

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

Next, take 25 μL of the honeysuckle extract described in Example 1 and the fermentation product of the present invention in a 10 mL glass test tube, dilute ten times with ddH ₂ O (1:10 dilution), and add 250 μL respectively After mixing the phosphate buffer solution with a shaker, add 250μL of 1% red blood salt and mix it with a shaker. Heat it in a water bath at 50℃ for 20 minutes, then add 250μL of 10% trichloroacetic acid to shake After the instrument is evenly mixed, centrifuge at 300g for 10 minutes, and be careful not to shake when taking it out. Then take out 300μL of each tube reaction solution supernatant, and add 300μL of ddH ₂ O and 120μL of 1% ferric chloride to shake the instrument. After mixing uniformly, react for 10 minutes, and measure its absorbance at 700nm, then use the regression curve formula of the above 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 fermented honeysuckle.

The test result of the three-stage fermentation method of the present invention for improving the reducing activity of the honeysuckle fermented product of the present invention is 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. This result shows that the three-stage fermentation method of the present invention can significantly improve the reducing activity of the honeysuckle fermented product of the present invention, so as to achieve the purpose of enhancing the functional components and enhancing the health-care and antioxidant effect.

Example 4 The effect of the honeysuckle fermented product of the present invention to enhance the antioxidant activity of skin cells

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

First, plant 1×10 ⁵ human dermal fibroblasts in a 6-well culture dish containing 2 mL of the above-mentioned culture medium, and culture it at 37°C for 24 hours. Without disturbing the cells, remove the cell culture medium. , And rinse the cells with 1x Phosphate buffered saline (1xPhosphate buffered saline, 1xPBS) and remove them. Then divide the cells into the following four groups: (1) add only cell culture solution to the blank control group for 2 hours, (2) The control group was treated with 500μM H ₂ O ₂ (purchased from Sigma, USA) for 2 hours, (3) the control group was treated with 2mg/mL honeysuckle extract for 1 hour and then treated with 500μM H ₂ O ₂ for 1 hour. And (4) the experimental group pretreated with the fermented honeysuckle of the present invention at 2 mg/mL for 1 hour and then treated with 500 μM H ₂ O ₂ for 1 hour. Then, add 5μg/mL DCFH-DA to each group and treat them at 37°C for 15 minutes, and then treat the cells with 500μM H ₂ O ₂ at 37°C for 1 hour, then wash the cells with 1 mL of 1 times phosphate buffer solution Twice, add 200μL trypsin and react for 5 minutes in the dark to make the cells fall off the culture plate, add appropriate culture medium to stop the reaction, and collect the cells together with the culture medium into a 1.5mL centrifuge tube, and centrifuge at 400g Remove the supernatant after 5 minutes, rinse the cells once with 1 mL of 1xPBS, centrifuge at 400g for 10 minutes and remove the supernatant, resuspend the cells in 1mL of 1xPBS, and add 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA, purchased from Sigma, USA, number SI-D6883-50MG, 5mg/mL stored in DMSO) Label the cells, and use a flow cytometer to detect cells at excitation wavelengths of 450-490nm and emission wavelengths of 510-550nm Fluorescence value. Then use Excel software to perform student t-test to determine whether there are statistically significant differences 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 lipid enzymes in the cell to form a polar 2' ,7'-dichlorodihydrofluorescin (DCFH), stays in the cell and cannot come out, and the reactive oxygen species (ROS) in the cell The redox reaction with DCFH forms 2',7'-dichlorofluorescin (DCF). After excitation at 450-490nm wavelength, the green fluorescence produced can be detected at 510-550nm wavelength. Therefore, the detection by DCFH- The fluorescence intensity of DA-treated cells can reflect the content of reactive oxygen species in the cells.

The test result of the three-stage fermentation method of the present invention for enhancing the efficacy of the honeysuckle fermented product of the present invention in enhancing the antioxidant activity of skin cells is shown in FIG. 3. In the blank control group and the control group treated with H ₂ O ₂ , 9.9% and 15.1% of human skin fibroblasts contained ROS, respectively, indicating that H ₂ O ₂ can indeed induce oxidative stress in skin cells; After the flower water extract is processed, it can only reduce the human skin fibroblasts containing ROS to 2.8%; and the honeysuckle fermented product of the present invention can significantly reduce the human skin fibroblasts containing ROS to 1.1% . This result shows that the three-stage fermentation method of the present invention can significantly improve the antioxidant activity of the honeysuckle fermented product of the present invention in skin cells, so as to achieve the purpose of enhancing the functional ingredients and enhancing the antioxidant effect of health care.

Example 5 The three-stage fermentation method of the present invention enhances the anti-saccharification activity of the honeysuckle fermented product

One embodiment of the present invention is to carry out the test experiment of the three-stage fermentation method of the present invention to improve the anti-glycation activity of honeysuckle fermented product to inhibit D-fructose (D-fructose) to make bovine serum albumin (Bovine serum albumin, BSA) Produce glycation efficiency and quantify glycation activity; anti-glycation can inhibit non-enzymatic browning to avoid denaturation of functional proteins in the body. First, take 0.25 mL of the water extract of honeysuckle diluted to 20% (v/v) or the fermented product of honeysuckle of the present invention, which are the control group and the experimental group, and 0.25 mL of water as the control group. The three groups were each added to 0.25mL of 60mg/mL BSA solution containing 0.06% of NaN ₃ (configured with 200mM sodium phosphate buffer, pH7.4), and combined with 0.25mL of 1.5M D-fructose (with 200mM sodium phosphate buffer solution, pH7.4) The solution is uniformly mixed, and then 0.1mL of the mixed solution is measured under excitation light 360nm and emission light 460nm to measure the fluorescence value, which is the zero point before the reaction, and take 0.45mL 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 the end of the reaction. And with the same amount of 3mM aminoguanidine (Aminoguanidine, AG, 200mM phosphate buffer configuration, pH7.4) to re-dissolve the solvent to the same volume as a positive control group, in which Aminoguanidine (AG) is known to inhibit glycation The efficacy. Finally, use the following formula to calculate the efficiency of the ability to eliminate AGEs to represent its anti-glycation activity.

The test result of the three-stage fermentation method of the present invention for enhancing the anti-glycation activity of the honeysuckle fermented product of the present invention is shown in Figure 4, where the control group is 100%. The honeysuckle water extract can only reduce the formation of the final saccharification product by 2.1%, while the honeysuckle fermented product of the present invention can significantly reduce the formation of the final saccharification product by 33.61%, which is 16 times that of the honeysuckle water extract. This result shows that the honeysuckle fermented product of the present invention has a strong activity to inhibit saccharification reaction, and the three-stage fermentation method of the present invention can significantly improve the ability of the honeysuckle fermented product of the present invention to remove the end product of saccharification and help delay the slowness The progression of disease and aging.

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

An embodiment of the present invention uses human primary skin keratinocyte HPEK-50 to test the efficacy 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 was cultured in serum-free keratinocyte culture medium (Keratinocyte-SFM). The cell culture medium was purchased from Gibco (USA), numbered #17005042, and Cultivation is carried out in a 37℃ cell incubator containing 5% CO ₂ .

First, culture 1.5x10 ⁵ human primary skin keratinocytes in a 6-well culture dish containing 2 mL of the above culture medium per well, and incubate at 37°C for 16-18 hours, and then divide the cells into the following three groups: (1) Add only The control group of cell culture solution, (2) the comparison group with 0.125% honeysuckle water extract, and (3) the experimental group with 0.125% honeysuckle fermented product of the present invention, and the three groups were placed at 37°C. After 48 hours of treatment, the expression level of the target gene in the primary human skin keratinocytes of each group was tested. First, the three groups of cells were recovered with cell lysate (RB buffer, purchased from Geanaid Company, Taiwan, Cat No. RBD300), and then RNA extraction reagent kit (purchased from Geneaid Company, Taiwan, Cat No. RBD300) was used to recover the cells. ) Collect the total RNA in the three groups of cells, and then use SuperScript ^® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) with 2000ng of extracted RNA as a template, and use the combination primers and reverse The transcriptase performs reverse transcription to produce the cDNA products corresponding to the mRNA of these genes, and then uses the 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 transcription products were respectively subjected to the quantitative real-time polymerase chain reaction (qPCR) test with the combination primers in Table 3, and the conditions were 95℃ for 1 second and 60℃ for reaction. 20 seconds, a total of 40 cycles. It is used to quantify the mRNA expression levels of SOD3 gene, Parkin gene, Atg1 gene, PARP2 gene, and Ubl-5 gene in the two groups of human primary skin keratinocytes. The quantitative value is taken from the threshold cycle number (Ct), and the target The relative amount of mRNA of the gene is derived from the equation 2 ^-△△Ct , where △C _T =C _{T comparison group or experimental group target gene/control group target gene-} C _TTBP (TATA-binding protein); △△ C _T =C _{T comparison group or experimental group target gene-} C _{T control group target gene} ; the fold change of each gene in each group is 2 ^-△△Ct . Then, use Excel software to determine whether the coefficient of variation is statistically significant (* p value <0.05; ** p value <0.01; *** p value <0.001).

The protein encoded by the Parkin gene is an enzyme that exists in the Ubiquitin-proteasome system (Ubiquitin-proteasome system) and acts as a regulator of protein breakdown. It is known that mutations in this gene are 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 is involved in waste degradation and circulating autophagy in cells. Previous studies have pointed out that overexpression of this gene can prolong the lifespan of mice. The aging effect of cells is related.

Previous studies have pointed out that compared with old mice, young mice contain more NAD ⁺ ; and increasing the NAD ⁺ concentration in old mice can make their physiological conditions younger, so NAD ⁺ is It is believed to be related to the delay of individual aging. The results of the study have shown that the proteins encoded by the Ubl-5 gene and 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 the substrate, and its main function is to catalyze the formation of adenosine diphosphate-ribose (ADP-ribose) Poly (ADP-ribose) polymer (abbreviated 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 PARP-2 activation will affect mitochondrial activity , And then regulate cell energy metabolism. Therefore, PARP and mitochondrial-related diseases and aging are also importantly related.

The test result of the three-stage fermentation method of the present invention for enhancing the expression of SOD3 gene in the honeysuckle fermented product of the present invention is shown in FIG. 5. After the first generation of human skin keratinocytes are treated with the honeysuckle water extract, the expression of SOD3 gene can only be increased by 1.1 times of the control group; and the honeysuckle fermentation product of the present invention can significantly increase the expression of SOD3 gene by 1.5 times 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 of SOD3 gene in skin cells, so as to increase the concentration of NAD ⁺ in skin cells and achieve skin anti-aging effect.

The test result of the three-stage fermentation method of the present invention for improving the expression of Parkin gene in the honeysuckle fermentation product of the present invention is shown in FIG. 6. After the human primary skin keratinocytes are treated with the honeysuckle water extract, the Parkin gene expression can only be increased by 1.2 times that of the control group; and the honeysuckle fermentation product of the present invention can significantly increase the Parkin gene expression 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 increase the expression of Parkin gene in skin cells, can maintain the normal function of protein decomposition in cells, and have the effect of preventing cell aging .

The test result of the three-stage fermentation method of the present invention for improving the expression of Atg1 gene in the honeysuckle fermented product of the present invention is shown in FIG. 7. After human primary skin keratinocytes were treated with honeysuckle water extract, the expression of Atg1 gene could only be increased by 1.0 times of the control group; while the honeysuckle fermentation product of the present invention could significantly increase the expression of Atg1 gene by 1.3 times 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 of Atg1 gene in skin cells, and can enhance the degradation of waste products in cells and the autophagy effect of circulation, so as to prolong cell Longevity, achieve the effect of anti-aging cells.

The test results of the three-stage fermentation method of the present invention for improving the honeysuckle fermented product of the present invention in reducing the PARP2 gene and increasing the expression of the Ubl-5 gene are shown in FIG. 8. After treatment with honeysuckle water extract, human primary skin keratinocytes can only reduce the expression of PARP2 gene by 0.85 times of the control group, and can only increase the expression of Ubl-5 gene by 1.0 times of the control group; and the gold and silver of the present invention Flower fermentation product can significantly reduce the expression of PARP2 gene by 0.75 times of the control group, and can significantly increase the expression of Ubl-5 gene by 1.1 times 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, and 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, in the present invention, the honeysuckle fermented product obtained by three-stage fermentation of the honeysuckle extract 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. The content of polyphenols significantly improves the reducing activity of the honeysuckle fermented product of the present invention, significantly improves the antioxidant activity of the honeysuckle fermented product of the present invention in skin cells, significantly improves the ability of the honeysuckle fermented product of the present invention to remove the final glycation product, and can Significantly improve the ability of the honeysuckle fermented product of the present invention to enhance the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in cells, and at the same time reduce the expression of PARP2 gene; to achieve the effects of enhancing anti-oxidation and anti-glycation, there is Helps delay the progression 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 waste degradation 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 extend the life of cells and achieve the effect of anti-aging. 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, which can be administered by oral, smearing, etc. A body.

【Biological Material Deposit】

Food Industry Development Institute (Taiwan); December 28, 2006; No. BCRC910808.

<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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Claims (14)

A honeysuckle fermentation 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 bacteria ( Lactobacillus plantarum) ), and Acetobacter aceti are obtained by sequentially performing three-stage fermentation. The honeysuckle fermented product described in item 1 of the scope of patent application, wherein according to the colorimetric method of Ferric reducing antioxidant power (FRAP), the honeysuckle fermented product has the same ability to reduce iron ions (Fe3 ⁺ ) The ability of ascorbic acid to reduce iron ions (Fe3 ⁺ ) at 300-400μg/mL. The honeysuckle fermented product described in item 1 of the scope of patent application, wherein the honeysuckle fermented product further enhances the antioxidant activity and the anti-glycation activity. The honeysuckle fermented product described in item 1 of the scope of patent application, wherein the honeysuckle fermented product promotes superoxide dismutase 3 ( SOD3 ) gene, Parkinson juvenile disease protein (Parkinson juvenile disease protein, Parkin gene, Autophagy-related gene 1, Atg1 gene, and Ubl gene are Ubiquitin-like protein 5 (Ubiquitin-like protein 5, Ubl-5 ) gene expression levels; and are inhibitory proteins Poly(ADP-Ribose) Polymerase 2 ( PARP2 ) gene expression level. The honeysuckle fermented product described in item 1 of the scope of patent application, wherein the honeysuckle fermented product increases the concentration of NAD ⁺ in skin cells, maintains the normal function of decomposing old and waste proteins in the cells, promotes the degradation of waste in the cells, and enhances the cells The normal operation of autophagy, the ability to repair DNA damage, and/or the activity of promoting mitochondrial metabolism. A kind of honeysuckle fermented product described in item 1 of the scope of patent application for preparing a composition that increases the expression of SOD3 gene, Parkin gene, Atg1 gene, and Ubl-5 gene in skin cells, and inhibits the expression of PARP2 gene use. The use described in item 6 of the scope of patent application, wherein the honeysuckle fermented product increases the concentration of NAD ⁺ in skin cells, maintains the normal function of decomposing old waste proteins in the cells, promotes the degradation of waste in the cells, and promotes autophagy in the cells The normal operation of the function, the improvement of the ability to repair DNA damage, and/or the activity of promoting mitochondrial metabolism. A honeysuckle fermented product is used to prepare a composition for improving the anti-aging ability. The use as described in item 8 of the scope of the patent application, wherein the honeysuckle fermented product improves the antioxidant activity, the reduction activity, the anti-glycation activity, and/or the total polyphenol content. The use according to any one of items 6 to 9 of the scope of patent application, wherein the effective concentration of the fermented honeysuckle is at least 0.5% (v/v). A method for manufacturing honeysuckle fermented product, comprising: extracting a honeysuckle extract obtained by extracting a honeysuckle through a solvent, and then passing the honeysuckle extract through a yeast ( Saccharomyces cerevisiae ), a lactic acid bacteria ( Lactobacillus plantarum ), And Acetobacter aceti is obtained by three-stage fermentation in sequence. The manufacturing method described in item 11 of the scope of patent application, wherein the honeysuckle extract is obtained by extracting the honeysuckle with water as a solvent; and the honeysuckle and the water are in a ratio of 1:10-20 (w/w) mixing. The manufacturing method described in item 11 of the scope of patent application, wherein the addition amount of the yeast is 0.01-0.5% (w/w); the addition amount of the lactic acid bacteria is 0.01-0.25% (w/w); the acetic acid bacteria The addition amount is 3-10% (w/w). The manufacturing method described in item 11 of the scope of patent application, wherein the fermentation time ratio of the yeast, the lactic acid bacteria, and the acetic acid bacteria is 1-2.5:1-3:3-10.

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