Collagen production promoter and its use

Abstract

We provide a technology that can promote collagen production. [Solution] The collagen production promoter contains α-isomaltosylglycerol.

Description

[Technical Field]

[0001] This disclosure relates to a collagen production promoter. [Background technology]

[0002] α-glucosylglycerol has been known for its skin moisturizing effect. For example, Patent Document 1 proposes a method for producing α-D-glucopyranosylglycerols, characterized by dissolving sugars in glycerol at a weight-to-volume ratio of 10-50%, and then reacting the mixture with α-glucosidase derived from Aspergillus niger to introduce an α-D-glucopyranosyl group into the glycerol. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Patent No. 3569432 [Overview of the project] [Problems that the invention aims to solve]

[0004] The inventors of this application have found that the technology described in Patent Document 1 has room for improvement in terms of promoting collagen production. Therefore, other technologies that can promote collagen production are needed. [Means for solving the problem]

[0005] This disclosure can be implemented in the following forms:

[0006] (1) According to one embodiment of the present disclosure, a collagen production promoter is provided. This collagen production promoter comprises α-isomaltosylglycerol. According to this embodiment of the collagen production promoter, collagen production can be promoted.

[0007] (2) In the collagen production promoter described in (1) above, the production of type I collagen may be promoted. This form of collagen production promoter can promote the production of type I collagen.

[0008] (3) In other forms of the present disclosure, a cosmetic composition comprising the collagen production promoter described in (1) or (2) above is provided. This form of cosmetic composition can promote collagen production.

[0009] (4) In other forms of the present disclosure, a food or beverage containing the collagen production promoter described in (1) or (2) above is provided. This form of food or beverage can promote collagen production.

[0010] (5) In other forms of the present disclosure, a pharmaceutical or quasi-drug comprising the collagen production promoter described in (1) or (2) above is provided. This form of pharmaceutical or quasi-drug can promote collagen production.

[0011] (6) According to other forms of the present disclosure, a UV protectant for fibroblasts is provided. This fibroblast protectant comprises α-isomaltosylglycerol. According to this form of fibroblast protectant, damage to fibroblasts by UV can be suppressed, and thus collagen production can be promoted.

[0012] Furthermore, this disclosure can be realized in various forms. For example, it can be realized in forms such as a method for producing a collagen production promoter, a method for producing a cosmetic containing a collagen production promoter, a method for producing a topical skin preparation containing a collagen production promoter, a method for producing food and beverages containing a collagen production promoter, a method for producing a pharmaceutical or quasi-drug containing a collagen production promoter, a method for producing a fibroblast protectant, a method for promoting collagen production, a method for protecting fibroblasts, the use of α-isomaltosylglycerol for producing a collagen production promoter, the use of α-isomaltosylglycerol for producing a cosmetic, the use of α-isomaltosylglycerol for producing a topical skin preparation, the use of α-isomaltosylglycerol for producing food and beverages, the use of α-isomaltosylglycerol for producing a pharmaceutical or quasi-drug, the use of α-isomaltosylglycerol for producing a fibroblast protectant, the use of α-isomaltosylglycerol to promote collagen production, and the use of α-isomaltosylglycerol to protect fibroblasts. [Brief explanation of the drawing]

[0013] [Figure 1] This is an explanatory diagram showing the results of LC-MS analysis of the product. [Figure 2] This is an explanatory diagram showing the elution results of in vitro reaction products using an activated carbon column. [Figure 3] This is an explanatory diagram showing the results of purifying the 50% ethanol fraction by HPLC. [Figure 4] This is an explanatory diagram showing an NMR spectrum. [Figure 5] This is an explanatory diagram showing the HSQC and DEPT spectra. [Figure 6] This is an explanatory diagram showing the DQF-COSY and HMBC spectra. [Figure 7] This is an explanatory diagram showing the chemical structure of G2-GL (α-isomaltosylglycerol) and its major HMBC correlations. [Figure 8] This is an explanatory diagram showing the HPLC chromatogram of the purified product. [Figure 9] It is an explanatory diagram showing the effect of adding G2-GLs (62.5 to 1200 μM) on the viability of ASF-4-1 cells. [Figure 10] It is an explanatory diagram showing the effect of adding G2-GLs (62.5 to 1000 μM) on the viability of ASF-4-1 cells. [Figure 11] It is an explanatory diagram showing the effect of adding G2-GLs (0.48 to 12 mM) on the viability of ASF-4-1 cells. [Figure 12] It is an explanatory diagram showing the growth effect of ASF-4-1 cells by adding G2-GLs. [Figure 13] It is an explanatory diagram showing the effect of adding G2-GLs on the total collagen production of ASF-4-1 cells (without UVA irradiation). [Figure 14] It is an explanatory diagram showing the effect of adding G2-GLs on the total collagen production of ASF-4-1 cells (with UVA irradiation). [Figure 15] It is an explanatory diagram showing the effect on the mRNA expression of G2-GLs (without UVA irradiation). [Figure 16] It is an explanatory diagram showing the effect on the mRNA expression of G2-GLs (with UVA irradiation).

Mode for Carrying Out the Invention

[0014] The inventors of the present application have identified α-isomaltosyl glycerol as a glycoside contained in sake, and have found that this α-isomaltosyl glycerol has an effect of promoting the production of collagen, and have thus completed the present invention.

[0015] According to one embodiment of the present disclosure, a collagen production promoter is provided. This collagen production promoter contains α-isomaltosyl glycerol. The IUPAC name of α-isomaltosyl glycerol is 2-O-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl glycerol. The structural formulas of α-isomaltosyl glycerol are shown in the following formulas (1) and (2).

[0016] [ka]

[0017] [ka]

[0018] The α-isomaltosylglycerol contained in the collagen production promoter of this disclosure may consist of only one of the structural isomers shown in formulas (1) and (2) above, or it may contain both structural isomers. The collagen production promoter of this disclosure can promote collagen production by containing α-isomaltosylglycerol. The collagen whose production is promoted is not particularly limited, and examples include type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, etc., but type I collagen is particularly preferred. The α-isomaltosylglycerol contained in the collagen production promoter of this disclosure is a substance also found in sake, and is therefore preferred from the viewpoint of safety, and also has added value as a functional ingredient derived from fermented products.

[0019] The collagen production promoter of this disclosure may contain other components in addition to α-isomaltosylglycerol. Other components are not limited to, but include, anionic surfactants, nonionic surfactants, cationic surfactants, amphoteric surfactants, cationic polymers, anionic polymers, amphoteric polymers, thickeners, plant extracts, polysaccharides or their derivatives, hydrolysates and derivatives of proteins derived from plants, animals, and microorganisms, amino acids, vitamins, humectants, lower alcohols, higher alcohols, oils and fats, silicones, various dyes and pigments, preservatives, fragrances, chelating agents, and the like. The collagen production promoter preferably contains 0.01% by mass or more of α-isomaltosylglycerol among all the components contained in the collagen production promoter, more preferably 0.05% by mass or more, even more preferably 0.1% by mass or more, even more preferably 0.5% by mass or more, even more preferably 1% by mass or more, particularly preferably 5% by mass or more, especially preferably 10% by mass or more, and especially especially preferably 50% by mass or more. The upper limit for the α-isomaltosylglycerol content in the collagen production promoter is 100%.

[0020] The method for producing α-isomaltosylglycerol contained in the collagen production promoter of this disclosure is not particularly limited, and may be produced, for example, by enzymatic reaction of isomaltose and glycerol using α-glucosidase. Alternatively, it may be produced by chemical synthesis, or by separation and purification from fermented products such as sake.

[0021] The collagen production promoter described herein can be used in a wide range of applications, including cosmetics, foods and beverages, pharmaceuticals, and quasi-drugs, but is preferably used as a topical skin preparation. The form of the topical skin preparation is not particularly limited, but examples include creams, lotions, gels, ointments, patches, and sprays.

[0022] Cosmetics containing the collagen production promoter of this disclosure are not particularly limited and include, for example, lotions, emulsions, moisturizers, face masks, foundations, eyeshadows, lip balms, hand creams, facial cleansers, shampoos, hair rinses, conditioners, and treatments. In addition to the collagen production promoter, the cosmetics of this disclosure may also contain optional ingredients commonly used in cosmetics. Optional ingredients are not particularly limited but include, for example, humectants, lower alcohols, polyhydric alcohols, sugars, hydrocarbons, esters, triglycerides, surfactants, buffers, emulsifiers, stabilizers, thickeners, antioxidants, preservatives, antibacterial agents, chelating agents, pH adjusters, fragrances, pigments, UV absorbers, UV scatterers, vitamins, amino acids, anti-inflammatory agents, water, peptides, sugar alcohols, enzymes, plant extracts, antioxidants, talc, and the like.

[0023] The above humectants are not particularly limited, but examples include polyethylene glycol, polypropylene glycol, glycerin, propylene glycol, sorbitol, acidic mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, amino acids, collagen, elastin, etc. The above lower alcohols are not particularly limited, but examples include ethanol, propanol, isopropanol, etc. The above polyhydric alcohols are not particularly limited, but examples include glycerin, pentaerythritol, dipentaerythritol, ethylene glycol, propylene glycol, polypropylene glycol, 1,3-butylene glycol, etc. The above sugars are not particularly limited, but examples include glucose, maltose, lactose, D-glucuronic acid, D-sorbitol, sorbitan, cellulose, starch, oligosaccharides, polysaccharides, and derivatives thereof.

[0024] The form of food and beverages containing the collagen production promoter of this disclosure is not particularly limited, but examples include processed foods, frozen foods, and health supplements. Food and beverages containing the collagen production promoter of this disclosure are not particularly limited, but examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (including concentrated stocks and powders for adjusting these beverages); frozen desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba, udon, vermicelli, Chinese noodles, and instant noodles; confectionery such as candy, chewing gum, candy, gum, chocolate, tablets, snacks, biscuits, jelly, jam, cream, and baked goods; processed seafood and livestock products such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and processed oils such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and dips; and soups, stews, salads, prepared foods, and pickles.

[0025] Pharmaceuticals or quasi-drugs containing the collagen production promoter of this disclosure are not particularly limited, but regardless of their efficacy, include, for example, oral preparations such as tablets, capsules, granules, powders, and liquids, as well as ointments, plasters, suppositories, etc.

[0026] In other forms of the present disclosure, a fibroblast protectant comprising α-isomaltosylglycerol is provided. Fibroblasts are one of the main cells that are damaged during the aging process of the skin, and are mainly located in the dermis layer of the skin, producing extracellular matrix components. Since the collagen-producing capacity of fibroblasts is significantly reduced in aging skin, it is important to protect fibroblasts from UVA-induced damage. According to the fibroblast protectant of the present disclosure, damage to fibroblasts can be suppressed by including α-isomaltosylglycerol.

[0027] The collagen production promoters, cosmetics, topical skin preparations, foods and beverages, pharmaceuticals and quasi-drugs disclosed herein are suitably applied to humans, but may also be applied to animals other than humans, insofar as the effects are achieved. [Examples]

[0028] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0029] 1. Method (1) LC-MS and LC-MS / MS analytical conditions A HILICpak VG-50 4E column (4.6 × 250 mm, particle size 5 μm, manufactured by Showa Denko Corporation) was used, with the column oven temperature at 40°C, flow rate at 1.0 mL / min, and injection volume of 3 μL. The mobile phase consisted of water (A) containing 50 μM lithium chloride and acetonitrile (B). Gradient elution was performed at 0-5 min, 65% (B); 5-40 min, 65%-5% (B); 40-45 min, 5% (B); 45-46 min, 5%-65% (B); and 46-60 min, 65% (B). LC-MS and LC-MS / MS spectra were obtained using an ACQUITY UPLC H-Class system (Waters Corporation) and a Xevo G2-XS high-performance quadrupole time-of-flight mass spectrometer (LC-qTOF / MS, Waters Corporation) equipped with a positive ion mode electrospray ionization (ESI) source. The source parameters were set to a desolvation gas flow of 800 L / h, a desolvation temperature of 450 °C, a cone gas flow of 50 L / h, a source temperature of 120 °C, and a capillary voltage of 2 kV. The collision energy for generating fragment ions was set to 50 eV. The m / z values ​​were corrected using m / z 120 and m / z 278 fragment ions of the standard reagent leucine-enkephalin.

[0030] (2) Preparation of recombinant α-glucosidase A (AgdA) Aspergillus oryzae RIB40, a wild strain of Aspergillus oryzae distributed by the National Research Institute of Brewing, was cultured in YPM medium [0.5% (w / v) yeast extract, 0.5% (w / v) polypeptone, 2.0% (w / v) maltose] at 180 rpm at 30°C for 2 days with shaking. Total RNA was extracted according to the manufacturing method of Isogen (manufactured by Nippon Gene Co., Ltd.). cDNA was synthesized using Superscript III Reverse Transcriptase (manufactured by Thermo Fisher Scientific) with oligo(dT) primers. The coding region of agdA was amplified using the primer AgdA1 (5' -AATGGACAAAAACTCATCTCAGAAG-3') shown in SEQ ID NO: 1 and the primer AgdA2 (5' CCGGCAGCTTCAGCCTCTCTTTTCT-3') shown in SEQ ID NO: 2. The PCR conditions were denaturation at 94°C for 20 seconds, followed by 30 cycles of 98°C for 10 seconds and 68°C for 2 minutes.

[0031] The above agdA cDNA, amplified by PCR, was linearized by PCR using the primers PICZαA1 (5'-AATGGACAAAAACTCATCTCAGAAG-3') shown in SEQ ID NO: 3 and PICZαA2 (5'-CCGGCAGCTTCAGCCTCTTTTCT-3') shown in SEQ ID NO: 4. The PCR conditions were denaturation at 94°C for 20 seconds, followed by denaturation at 98°C for 10 seconds, and then 68°C for 5 minutes, repeated for 30 cycles.

[0032] Linearized pPICzαA (Thermo Fisher Scientific) was cloned using the In-Fusion HD Cloning Kit (Takara Bio Co., Ltd.) according to the protocol to create the agdA cDNA expression vector pAGDA. This vector was introduced into Pichia pastoris X-33 (Thermo Fisher Scientific) by electroporation using a MicroPulser electroporator (Bio-Rad). AgdA was expressed in the resulting transformants. Protein expression was performed according to the method described in Weidner, M., Taupp, M. and Hallam, SJ: Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris, Journal of Visualized Experiments, 36, e1862 (2010). The culture medium was concentrated using an ultrafiltration column (Amicon Ultra-15 Centrifugal Filter Units, NMWL 50kDa, Merck KGaA), and the supernatant was used in the reaction as recombinant AgdA solution.

[0033] (3) In vitro synthesis Recombinant AgdA (0.18 U / mL) was incubated with 20 mM acetate buffer (pH 5.0) along with 20% (v / v) glycerol and 10% (w / v) maltose at 37°C for 18 hours. The resulting reaction solution was then processed using a 3 kDa cutoff ultrafiltration system (Amicon Ultra-15 mL Centrifugal Filters Ultracel-3K, Merck KGaA) to recover the synthesized product from the reaction mixture into a filtrate.

[0034] (4) Purification of in vitro synthesized products The reaction mixture was introduced into an activated carbon column (65 × 400 mm, liquid chromatography grade), and glucose and glycerol were eluted from the column with 10% ethanol. Subsequently, the target compound adsorbed on the column was extracted with 50% ethanol. For the separation and detection of the target compound, an HPLC system consisting of a SIL-20A autosampler, SCL-10Avp controller, LC-20AD pump, and RID-20A refractive index detector (Shimadzu Corporation) was used, and separation was performed using an NH2P-50 4E column. The column temperature was maintained at 40°C using a forced-air-cooled column oven (CTO-20A, Shimadzu Corporation).

[0035] Under isocratic conditions with 60% acetonitrile, 5 μL of sample was introduced into the HPLC system at a flow rate of 0.5 mL / min. The target compound from the 50% ethanol fraction was further fractionated by preparative HPLC using two columns. First, using an Asahipak NH2P-90 20F column (20 × 300 mm, particle size 9 μm, Showa Denko K.K.), 100 μL was introduced under isocratic conditions with 60% acetonitrile, 7.5 mL / min, for 30 minutes, at an oven temperature of 40°C. Next, using a SUGAR SZ5532 column (6.0 × 150 mm, particle size 6 μm, Showa Denko K.K.), 10 μL was introduced under isocratic conditions with 80% acetonitrile, 0.9 mL / min, for 30 minutes, at an oven temperature of 70°C.

[0036] (5)NMR analysis One-dimensional and two-dimensional NMR spectra were recorded using an AVANCE-III (400 MHz) spectrometer (Bruker BioSpin), and chemical shifts were expressed in ppm. The target compound was dissolved in 1 mL of D2O (99.8%), and replaceable protons were replaced with deuterium using a smart evaporator (BioChromato). Signal assignment was as follows: 1 H, 13The analysis was based on 1C and 2D NMR spectra, and chemical shifts were clearly identified using heteronocuar multiple bond coherence (HMBC), heteronocuar single quantum coherence (HSQC), and correlation spectroscopy (COSY). Acetone (δH: 2.225 ppm, δC: 30.50 ppm) was used as an internal standard.

[0037] (6) Cell viability assay The experiment was conducted over seven days. On day 1, human dermal fibroblasts ASF-4-1 were sampled at 2.0 × 10⁶ times. 3 Cells were diluted in low-glucose DMEM medium (with L-glutamine and phenol red) supplemented with 10% (v / v) fetal bovine serum (FBS, Merck) and 1% (v / v) penicillin / streptomycin (penicillin-streptomycin solution (×100)) to achieve a cell / well density. 180 μL of each solution was seeded into a 96-well culture plate and incubated at 37°C at 5% CO2 for 24 hours. On day 2, 20 μL of various concentrations of G2-GL (4.8-1200 μM) were added to each well, and the cells were incubated at 37°C at 5% CO2 for 96 hours. On day 7, cell viability was evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Dojindo Laboratories). 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for 3 hours to induce a color reaction. After removing the staining solution, dimethyl sulfoxide (DMSO) was added to decompose the resulting formazan, and the absorbance at 535 nm [A] was measured. Cell viability was calculated by substituting this value into the following formula. The same procedure was repeated for ASF-4-1 cells numbering 8.0 × 10⁶. 3 The procedure was performed by changing the cell / well and the amount of G2-Gls added from 0.48 to 12 mM.

[0038]

number

[0039] (7) MTT-based cell proliferation assay The test was conducted over seven days. On the first day, ASF-4-1 was administered at a rate of 1.6 × 10⁻¹⁶. 4 The cells were diluted in 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin-supplemented low-glucose DMEM medium to achieve a cell / well density, seeded in 180 μL portions into 96-well culture plates, and incubated at 37°C and 5% CO2 for 24 hours. On day 2, 20 μL of various concentrations of G2-GLs (0.48-12 mM) were added to each well, and incubated at 37°C and 5% CO2 for 24 hours. On day 3, the medium was removed and replaced with phosphate-buffered saline (PBS), and UVA (10 J / cm²) was applied. 2 The cells were irradiated with UVA. UVA irradiation was performed under a UVA fluorescent lamp using a UVB cut filter (WACOM). After removing PBS, the cells were replaced with 180 μL / well of low-glucose DMEM supplemented with 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin, and G2-GLs (19.2-480 μM) was added again. The cells were cultured at 37°C and 5% CO2 for 96 hours. Cell proliferation was evaluated on day 7 using MTT.

[0040] (8) Sirius Red Collagen Detection Assay Cells were cultured using the same procedure as in the MTT-based cell proliferation assay. During this time, 0.048 μM-12 mM G2-GLs were added on day 2, and the same amount was added after UVA irradiation on day 3. After removing the culture medium, the cells were washed with 200 μL of PBS, 200 μL of 3.75% formalin solution was added, and the cells were fixed at room temperature for 15 minutes. After washing again with 200 μL of PBS, the cells were stained with 50 μL of 0.1% Sirius Red (Direct Red 80, Tokyo Chemical Industry Co., Ltd.) solution dissolved in 1% acetic acid. After incubating the plate at room temperature for 1 hour, the wells were washed with 400 μL of 0.1 M hydrochloric acid. Next, 100 μL of 0.1 M NaOH solution was added to each well to elute the binding dye from the collagen bound to the cells. The absorbance of the solution was measured at 540 nm using a microplate reader. Pig skin-derived cell matrix type IC (Nitta Gelatin Co., Ltd.) was used as the collagen standard solution. Furthermore, the same procedure was performed on plates that were not irradiated with UVA, and cell viability was determined using the MTT assay to measure collagen production per surviving cell.

[0041] (9) Gene expression analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) Cells were cultured for 5 days using the same method as in the MTT-based cell proliferation assay. During this time, the amount of G2-GLs added was 0.048-48 μM. This total RNA was reverse transcribed into cDNA using the CellAmp Direct TB Green® RT-qPCR Kit (Takara Bio Inc.), and a genome removal reaction was performed. For qRT-PCR, the following gene-specific primers were used: for the GAPDH gene, 5'-GTCTCCTCTGACTTCAACAGCG-3' (SEQ ID NO: 5) and 5'-ACCACCCTGTTGTGTAGCCAA-3' (SEQ ID NO: 6); for the COL1A1 gene, 5'-AGCCTCTCCATCTTTGCCAGCA-3' (SEQ ID NO: 7) and 5'-GATTCCCTGGACCTAAAGGTGC-3' (SEQ ID NO: 8); and for the COL1A2 gene, 5'-CCTGGTGCTAAAGGAGAAAGAGG-3' (SEQ ID NO: 9) and 5'-ATCACCACGACTTCCAGCAGGA-3' (SEQ ID NO: 10). The qRT-PCR was performed using TB Green Fast qPCR Mix (Takara Bio Inc.), with 40 cycles of initial denaturation at 95°C for 30 seconds, followed by 5 seconds at 95°C and 10 seconds at 60°C. The transcription levels of COL1A1 and COL1A2 mRNA were determined relative to the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0042] 2.Results (1) In vitro synthesis of glycosides Figure 1 is an explanatory diagram showing the results of LC-MS analysis of the product. Figure 1(A) shows the mass chromatogram (m / z 423.16), and Figures 1(B) to (D) show the MS / MS fragmentation at the peaks (a to c) at 11.53 min, 12.87 min, and 13.67 min, respectively. As shown in Figure 1, the LC-MS analysis of the product revealed that peaks with matching MS / MS fragments were clearly detected as peaks at 11.53 min, 12.87 min, and 13.67 min in the mass chromatogram. In all three peaks (a to c), the parent fragment had a m / z of 423.16, and it was presumed to be a structure formed by the dehydration condensation of glycerol and two glucose molecules. Since common fragment peaks of m / z 169.03 and 261.10 were obtained, these were expected to be structural isomers. In the following explanation, the substance with a peak at 11.53 min shown in Figure 1(B) will also be referred to as "G2-GL-a", the substance with a peak at 12.87 min shown in Figure 1(C) will also be referred to as "G2-GL-b", the substance with a peak at 13.67 min shown in Figure 1(D) will also be referred to as "G2-GL-c", and a mixture of these will also be referred to as "G2-GLs".

[0043] (2) Purification of G2-GL-c To purify G2-GL-c, the in vitro synthesized product was passed through an activated carbon column. Figure 2 is an explanatory diagram showing the elution results of the in vitro reaction product using an activated carbon column. Figure 2(A) shows the elution results of the standard, Figure 2(B) shows the 10% ethanol fraction, and Figure 2(C) shows the 50% ethanol fraction. As shown in Figure 2, glucose, glycerol, α-glucosylglycerol, and some maltose were eluted from the column with a 10% ethanol solution. Subsequently, G2-GL was eluted with 50% ethanol to obtain a fraction containing G2-GL, maltose, and panose.

[0044] Figure 3 is an explanatory diagram showing the results of purifying the 50% ethanol fraction by HPLC. Figure 3(A) shows the results using an NH2P-90 20F column, and Figure 3(B) shows the results using a subsequent SUGAR SZ5532 column. In Figure 3, the results of the mass chromatogram m / z 423.17 are shown. Finally, 2.5 mg of G2-GL-c was obtained.

[0045] (3) NMR analysis The structure of G2-GL-c was determined using NMR spectroscopy. 1 H and 13 C NMR data are shown in Table 1.

[0046] [Table 1]

[0047] Figure 4 is an explanatory diagram showing the NMR spectrum. Figure 4(A) shows the 1 H NMR spectrum, and Figure 4(B) shows the 13 C NMR spectrum. Figure 5 is an explanatory diagram showing the HSQC and DEPT spectra. Figure 5(A) shows the HSQC spectrum, and Figure 5(B) shows the DEPT spectrum. Figure 6 is an explanatory diagram showing the DQF-COSY and HMBC spectra. Figure 6(A) shows the DQF-COSY spectrum, and Figure 6(B) shows the HMBC spectrum. Figure 7 is an explanatory diagram showing the chemical structure of G2-GL (α-isomaltosyl glycerol) and the main HMBC correlations.

[0048] According to the results shown in Figures 4 to 6, it was found that G2-GLc is a mixture of the R and S forms of 1-(α-isomaltosyl) glycerol, as shown in Figure 7. Also, from these structures, G2-GLb was estimated to be 2-(α-isomaltosyl) glycerol.

[0049] (4) Purification of G2-GLs G2-GLs were synthesized in vitro and purified again for use in cell studies. Purification by preparative HPLC yielded 220.91 mg of G1-GLs from 15 mL of reaction solution and 42.15 mg of G2-GLs from 120 mL of reaction solution.

[0050] Figure 8 is an explanatory diagram showing the HPLC chromatogram of the purified product. Analysis of the obtained G2-GLs using LC-MS revealed that, in addition to four G2-GLs containing α-isomaltosylglycerol, G1-GLs and panose were also present. More specifically, G1-GLs:G2-GLs:panose were mixed in a ratio of 3:27:1 (area ratio). Since both G1-GLs and G2-GLs are non-reducing sugars, the amount of G2-GL was calculated to be 37.47 mg based on the measured amount of non-reducing sugar (42.15 mg) and the area ratio. Based on the calculated quantitative value of G2-GLs, the sample to be used in the cell test was prepared.

[0051] (5) Cytotoxicity test The effect of G2-GLs on the proliferation of fibroblast ASF-4-1 was estimated by adding G2-GLs in a concentration range of 4.8–1200 μM.

[0052] Figure 9 is an explanatory diagram showing the effect of G2-GLs addition (62.5-1200 μM) on ASF-4-1 cell viability. In Figure 9, the viability is calculated with the absorbance of the control group (no sample added) set to 100%, and the values ​​are shown as the mean ± SD of three experiments. As shown in Figure 9, G2-GLs is 2.0 × 10⁻⁶ 3 No cytotoxicity was observed even at a dosage of 1200 μM per cell / well.

[0053] Figure 10 is an explanatory diagram showing the effect of G2-GLs addition (62.5-1000 μM) on ASF-4-1 cell viability. In Figure 10, the viability is calculated with the absorbance of the control group (no sample added) set to 100%, and the values ​​are shown as the mean ± SD of three experiments. Figure 10 also shows the significance (*p<0.05) obtained by one-way ANOVA and Dunnett's test. As shown in Figure 10, 8.0 × 10 3 When fibroblasts were cultured in DMEM containing G2-GLs (62.5, 1000 μM) relative to cell / well cells, proliferation was 128.9% (p<0.05) and 130.0% (p<0.05), respectively, compared to control cells (no sample added). Therefore, 8.0 × 10 3 The amount of additive was increased relative to the number of cells per well, until the concentration reached 0.48–12 mM.

[0054] Figure 11 is an explanatory diagram showing the effect of G2-GLs addition (0.48–12 mM) on ASF-4-1 cell viability. In Figure 11, the viability is calculated with the absorbance of the control group (no sample added) set to 100%, and the values ​​are shown as the mean ± SD of three experiments. As shown in Figure 11, no cytotoxicity was observed even at 12 mM. Therefore, it was shown that this substance is non-toxic to fibroblasts at these concentrations.

[0055] (6) Cell activation effect test The protective effect of G2-GLs against cell damage caused by UVA irradiation was investigated.

[0056] Figure 12 is an explanatory diagram showing the proliferation effect of ASF-4-1 cells upon addition of G2-GLs. In Figure 12, the values ​​are shown as the mean ± SD of three experiments. Figure 12 also shows the statistical significance (*p<0.05) obtained by one-way ANOVA and Dunnett's test. As shown in Figure 10, when G2-GLs were added to ASF-4-1 cells at concentrations of 2.4, 4.8, and 12 mM on day 2 of culture, the cell viability was 283.4% (p<0.01), 284.7% (p<0.01), and 461.4% (p<0.001), respectively, compared to control cells. This suggests that G2-GLs have an activating effect on human fibroblasts damaged by UVA, or a cytoprotective effect that prevents damage.

[0057] (7) Measurement of total collagen content The effect of G2-GLs addition on the total collagen content of ASF-4-1 cells was investigated.

[0058] Figure 13 is an explanatory diagram showing the effect of G2-GLs addition on total collagen production in ASF-4-1 cells (without UVA irradiation). In Figure 13, the values ​​are shown as the mean ± SD of three experiments. As shown in Figure 13, cells without UVA irradiation showed a tendency to produce more collagen, although there was no significant difference compared to control cells at 48 μM.

[0059] Figure 14 is an explanatory diagram showing the effect of G2-GLs addition on total collagen production in ASF-4-1 cells (with UVA irradiation). In Figure 14, the values ​​are shown as the mean ± SD of three experiments. Figure 14 also shows the significance (*p<0.05) obtained by one-way ANOVA and Dunnett's test. As shown in Figure 14, for UVA-irradiated cells, collagen production changed by 139.5% (p<0.01), 139.4% (p<0.01), 126.4% (p<0.05), 135.8% (p<0.01), and 138.1% (p<0.01) compared to control cells with G2-GLs addition of 0.48, 1, 2.4, 4.8, and 12 mM, respectively.

[0060] (8) RT-qPCR of collagen-producing genes Since 80% of collagen in young people is type I collagen, to investigate the effect of G2-GLs on type I collagen production, mRNA for type I collagen genes (COL1A1 and COL1A2) was synthesized from total RNA and quantified by qRT-PCR.

[0061] Figure 15 is an explanatory diagram showing the effect of G2-GLs on mRNA expression (without UVA irradiation). Figure 15(A) shows the relative expression level of COL1A1, and Figure 15(B) shows the relative expression level of COL1A2. In Figure 15, the values ​​are shown as the mean ± SD of three experiments. Figure 15 also shows the significance level (*p<0.05) calculated by one-way ANOVA and Dunnett's test. As shown in Figure 15, when UVA irradiation was not performed, the addition of 48 μM of G2-GLs resulted in a COL1A2 expression level 1.50 times higher than the control (p<0.05). Furthermore, it was found that the expression level of COL1A1 also tended to increase as the G2-GLs concentration increased.

[0062] Figure 16 is an explanatory diagram showing the effect of G2-GLs on mRNA expression (with UVA irradiation). Figure 16(A) shows the relative expression level of COL1A1, and Figure 16(B) shows the relative expression level of COL1A2. In Figure 16, the values ​​are shown as the mean ± SD of three experiments. As shown in Figure 16, the same trend was observed even with UVA irradiation as with no UVA irradiation (Figure 15). From these results, it was estimated that G2-GLs have the effect of promoting collagen production by inducing the expression of COL1A1 and COL1A2.

[0063] The present invention is not limited to the embodiments described above, and can be realized in various configurations without departing from its spirit. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the summary of the invention can be replaced or combined as appropriate in order to solve some or all of the above-described problems, or to achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. A collagen production promoter containing α-isomaltosylglycerol.

2. In the collagen production promoter described in claim 1, Promotes the production of type I collagen. Collagen production promoter.

3. A cosmetic comprising the collagen production promoter described in claim 1 or claim 2.

4. Food and beverages comprising the collagen production promoter described in claim 1 or claim 2.

5. A pharmaceutical or quasi-drug comprising the collagen production promoter described in claim 1 or claim 2.

6. A fibroblast protective agent containing α-isomaltosylglycerol.

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