New polyphenolic compounds

Abstract

The present invention provides novel active ingredients derived from carob and their uses. The present invention is a compound represented by the following formula (I), a salt thereof, or a solvate thereof,

Description

Technical Field

[0001] The present invention relates to a novel polyphenol compound isolated and purified from locust bean (Ceratonia siliqua L.), a leguminous plant, and uses thereof, and further relates to a method for producing the compound. Background Art

[0002] Locust bean (Ceratonia siliqua L.) is a legume native primarily to the Mediterranean region. The pods and pulp of locust beans, known as carobs, have been eaten and used as a food ingredient since ancient times. Mature pods are approximately 10 to 25 cm long and have a sweet taste. Locust bean pods contain large amounts of polysaccharides, cellulose, and minerals, as well as small amounts of protein and non-carbohydrate low-molecular compounds. In recent years, research into new functions of locust beans has been ongoing, with reports indicating that locust bean pod extracts have preventive and therapeutic effects on digestive diseases such as ulcerative colitis and gastric ulcers (Non-Patent Documents 1 and 2). Furthermore, Patent Document 1 describes that locust bean seed extracts have α-glucosidase inhibitory activity and are effective in suppressing weight gain (Patent Document 1).

[0003] Collagen, on the other hand, is a vital protein that accounts for approximately 20% of the body's total protein. Collagen is primarily found in connective tissue, providing strength, elasticity, and stretchability to numerous tissues, including cartilage, bone, tendons, ligaments, dermis, and the whites of the eyes. Furthermore, collagen is a major component of the extracellular matrix. Collagen exists in dozens of different forms depending on its structure. Type I collagen is found in high concentrations in the dermis of the skin, contributing to its strength and elasticity. The COL1A1 gene plays a crucial role in the expression and production of type I collagen. Type I collagen is composed of two α1 chains encoded by the COL1A1 gene and one α2 chain encoded by the COL1A2 gene. Because collagen maintains skin elasticity, it is known that collagen levels decrease with aging, contributing to the appearance of wrinkles. Therefore, promoting collagen gene expression and thereby increasing collagen production in the body is considered an effective way to replenish this aging-related collagen loss.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 2005-119999

[0007] Non-patent literature

[0008] Non-patent Document 1: Rtibi K, Jabri M A, Selmi S, et al., "Preventive effect of carob (Ceratonia siliqua L.) in dextran sulfate sodium-induced ulcerative colitis in rat", RSC Advances, 2016, Vol.6, p.19992 - 20000.

[0009] Non-patent Document 2: Rtibi K, Selmi S, Grami D, et al., "Chemical constituents and pharmacological actions of carob pods and leaves (Ceratonia siliqua L.) on the gastrointestinal tract: A review", Biomedicine & Pharmacotherapy, 2017, Vol.93, p.522 - 528. Summary of the Invention

[0010] Problems to be Solved by the Invention

[0011] However, in the above Non-patent Documents 1 and 2 and Patent Document 1, although it is reported that the carob extract has a therapeutic effect on digestive organ diseases such as ulcers and an inhibitory effect on weight gain, the specific active ingredients have not been specified.

[0012] Furthermore, there has been no research on using carob for promoting collagen gene expression so far, and its effectiveness remains completely unclear.

[0013] Therefore, the present invention has been completed in view of the above matters, and its object is to provide: a new active ingredient derived from carob and its uses.

[0014] Furthermore, another object of the present invention is to provide: a new collagen production promoter that can promote collagen gene expression, which is derived from carob. And the object is to provide: a skin external preparation, a cosmetic, and a food or drink for promoting collagen production containing the collagen production promoter.

[0015] Means for Solving the Problems

[0016] The inventors isolated a new polyphenol compound from the extract of carob pods and found that this new compound has the effect of increasing the expression level of collagen genes. The present invention has been completed based on this finding.

[0017] In order to solve the above-mentioned problems, the present invention provides a compound represented by the following formula (I) or a salt thereof, or a solvate thereof.

[0018] [Chemistry 1]

[0019]

[0020] The compound represented by formula (I) is a novel polyphenol compound isolated and purified from a locust bean (Ceratonia siliqua) pod extract, and has an excellent collagen production-promoting effect.

[0021] Furthermore, the collagen production promoter of the present invention comprises the compound represented by formula (I) or a salt thereof, or a solvate thereof. Administration of the compound represented by formula (I) promotes the expression of collagen genes, thereby promoting collagen production.

[0022] Furthermore, the collagen production promoter of the present invention preferably has a concentration of the compound represented by formula (I) or a salt thereof, or a solvate thereof of 0.0001 mM to 1 mM. This allows selection of an active ingredient concentration that exhibits an excellent collagen production promoting effect.

[0023] Furthermore, the collagen production promoter of the present invention is preferably a skin external preparation or cosmetic. Thus, a skin external preparation or cosmetic that can promote collagen production in the skin can be obtained.

[0024] Furthermore, the food or beverage for promoting collagen production of the present invention contains the compound represented by the above formula (I) or a salt thereof, or a solvate thereof, thereby providing a food or beverage capable of promoting collagen production in vivo.

[0025] Furthermore, the present invention provides a method for producing the compound represented by formula (I) comprising: obtaining a hydroalcoholic extract from locust bean (Ceratonia siliqua) pods; sequentially subjecting the hydroalcoholic extract to liquid-liquid extraction with petroleum ether and ethyl acetate to recover an ethyl acetate fraction; and isolating the compound represented by formula (I) from the recovered ethyl acetate fraction. This method provides a novel polyphenolic compound having a collagen production-promoting effect.

[0026] Effects of the Invention

[0027] According to the present invention, there can be provided a novel polyphenol compound, a collagen production promoter, a skin external preparation, a cosmetic, and a food or drink having the following excellent effects.

[0028] (1) Increase the expression of collagen genes and promote collagen production.

[0029] (2) Since it contains a compound derived from the pods of carob beans, which have been consumed since ancient times, it is highly safe for the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a graph showing the high-resolution ESI mass spectrum of the compound of the present invention.

[0031] Figure 2 It is a graph showing the ultraviolet absorption spectrum of the compound of the present invention.

[0032] Figure 3 It is a graph showing the infrared absorption spectrum of the compound of the present invention.

[0033] Figure 4 It is a graph showing the 1 1H-NMR spectrum (CD3OD, 600 MHz) of the compound of the present invention.

[0034] Figure 5 It is a graph showing the 13 13C-NMR spectrum (CD3OD, 150 MHz) of the compound of the present invention.

[0035] Figure 6 It is a graph showing the HSQC spectrum of the compound of the present invention.

[0036] Figure 7 It is a graph showing the HMBC spectrum of the compound of the present invention.

[0037] Figure 8 It is a graph showing the NOESY spectrum of the compound of the present invention.

[0038] Figure 9 It is a graph summarizing the 1 1H-NMR signals and 13 13C-NMR signal summary of the compound of the present invention.

[0039] Figure 10 It is a graph showing the HMBC correlations and NOESY correlations of the compound of the present invention.

[0040] Figure 11 It is a graph showing the mRNA expression level of the collagen gene (COL1A1) induced by the compound of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0041] Hereinafter, the new polyphenol compound and collagen production promoter of the present invention, a topical skin preparation, a cosmetic, a food or drink for promoting collagen production, and a method for producing the compound will be described.

[0042] The novel polyphenol compound represented by the following formula (I) of the present invention is a compound in which isoferulic acid and gallic acid are ester-bonded to glucose. This compound is named "OLANDU" (registered trademark).

[0043] [Chemistry 2]

[0044]

[0045] The novel polyphenol compound of the present invention may be in the form of a salt, preferably a pharmacologically acceptable salt. The pharmacologically acceptable salt of the novel polyphenol compound is not particularly limited as long as it is a salt formed with an acid or a base. Furthermore, the novel polyphenol compound or its salt may be in the form of a solvate, which is not particularly limited and includes, for example, hydrates and organic solvates such as ethanol.

[0046] The novel polyphenol compound "OLANDU" of the present invention has a collagen production-promoting effect and can be used as a collagen production promoter. The collagen to be promoted is preferably type I collagen or type III collagen, which are abundant in the skin, and more preferably type I collagen.

[0047] In the present invention, promotion of collagen production refers to promoting collagen gene expression or collagen (protein) expression, with the expression of the collagen gene or collagen protein being enhanced compared to a control group to which the novel polyphenol compound of the present invention is not added or administered. More specifically, the expression level of the collagen gene is preferably 1.5 times or more, more preferably 1.7 times or more, and particularly preferably 2 times or more of that of the control group. Collagen gene expression levels can be measured using known methods such as real-time PCR (qPCR) and microarrays, while collagen protein expression levels can be measured using known methods such as immunostaining and Western blotting.

[0048] The novel polyphenol compound "OLANDU" of the present invention can be obtained by isolating and purifying the pods of locust beans. The locust beans used in this invention are scientifically known as Ceratonia siliqua, a plant of the genus Ceratonia in the subfamily Caesalpinioideae of the family Fabaceae. Although native to the Mediterranean coast, the present invention does not specifically limit the origin or cultivation environment; locust beans from any origin and cultivation environment can be used.

[0049] A method for separating the novel polyphenol compound "OLANDU" of the present invention will be described. First, an aqueous alcohol extract is obtained from the pods of carob. The so-called aqueous alcohol extract of carob pods in the present invention refers to an extract obtained by adding an aqueous alcohol as an extraction solvent to the pods of carob and performing an extraction treatment. The so-called pods of carob refer to the pods and pulp of the pod-bearing fruit of carob, and either the pod or the pulp can be used as an extraction material, but it is more preferable to use both the pod and the pulp. The extraction treatment is performed on the collected state, that is, on the raw state of carob pods or the dried state of carob pods. However, in order to improve the extraction efficiency or for easy handling, the extraction treatment may also be performed on carob pods that have been subjected to various pretreatment processes. As the pretreatment, there is no particular limitation, and examples include drying treatment, crushing treatment, or pulverization treatment, etc. The extraction treatment may also be performed on the pods of carob that have been subjected to these pretreatment processes to obtain an extract.

[0050] Regarding the alcohol that constitutes the aqueous alcohol used as the extraction solvent, as long as it can extract the polyphenol compound of the present invention, there is no particular limitation, and examples include ethanol, methanol, propanol, isopropanol, butanol, or isobutanol, etc. Among them, from the viewpoints of safety to the human body and extraction efficiency, etc., the extraction solvent is preferably aqueous ethanol. And the alcohol concentration of the aqueous alcohol is preferably 50% - 99%, more preferably 60% - 97%, and particularly preferably 70% - 95%. And in the extraction solvent, other components may also be contained within a range that does not hinder the extraction of the compounds of the present invention.

[0051] Extraction using aqueous alcohol involves adding aqueous alcohol as an extraction solvent to locust bean pods and immersing them in the pods. For example, when the locust bean pods are dried and crushed to a moisture content of less than 10%, 5 to 10 parts by weight of the extraction solvent are preferably used per 1 part by weight of the plant. While extraction can be performed using any method, including room temperature extraction, heated extraction, pressurized heated extraction, or subcritical extraction, heated extraction using reflux is preferred for efficiency. Furthermore, to improve extraction efficiency, multiple extractions are preferably performed, more preferably by varying the alcohol concentration in the extraction solvent. While not particularly limited, a specific example involves performing one to five reflux extractions using 95% ethanol, followed by one to five reflux extractions using 70% ethanol. The extraction time can be set depending on the extraction method, the form of the extracting material, the type of extraction solvent, the extraction temperature, and other factors. For example, when reflux extraction is performed using 70% to 95% ethanol, the extraction time per extraction is preferably set to approximately 1 to 3 hours, particularly preferably approximately 1.5 hours. After the extraction, the residue is removed by decantation, centrifugation, or filtration to obtain a hydroalcoholic extract of locust bean pods. The obtained extract can also be concentrated into a liquid or solid form by subjecting it to treatment such as vacuum distillation.

[0052] The hydroalcoholic extract of locust bean pods obtained in the above manner is believed to contain carbohydrates and other substances, which are abundant in locust bean pods. Therefore, to remove these unwanted components, separation using an ion exchange resin can be performed. Specifically, 1 to 10 parts by weight of water is added to 1 part by weight of the hydroalcoholic extract of locust bean pods to disperse the extract. The extract is then passed through a column packed with an ion exchange resin, such as a macroporous adsorption resin, to adsorb the polyphenol compounds of the present invention, removing unwanted components such as carbohydrates. Subsequently, the extract is eluted with, for example, 95% ethanol to recover a fraction containing the polyphenol compounds of the present invention.

[0053] Next, the separation process performed on the hydrous alcohol extract of locust bean pods or the recovered fraction separated using the ion exchange resin described above will be described. The hydrous alcohol extract or the recovered fraction is dispersed in an aqueous solvent and then subjected to solvent extraction using petroleum ether and then ethyl acetate, sequentially. The aqueous solvent is not particularly limited as long as it can disperse the polyphenol compound of the present invention; 50% aqueous methanol is suitable. Multiple liquid-liquid extractions are performed using petroleum ether / aqueous solvents, followed by multiple liquid-liquid extractions using ethyl acetate / aqueous solvents. The ethyl acetate fraction recovered from these solvent extractions contains the novel polyphenol compound of the present invention. The number of liquid-liquid extractions using each solvent system is preferably about 2 to 10, and particularly preferably about 5.

[0054] The novel polyphenol compound "OLANDU" of the present invention can be isolated by purifying the methyl acetate fraction obtained in the above manner according to conventional methods. Examples of purification methods include normal phase chromatography, reverse phase chromatography, thin layer chromatography, gel filtration chromatography, and high performance liquid chromatography. Purification can be performed using any one of these methods alone or in combination. The carrier, elution solvent, and other factors used in each chromatography method can be appropriately selected depending on the method.

[0055] It should be noted that the polyphenol compound of the present invention separated and purified from locust bean pods in the above manner may not be isolated as a pure substance, but may be used as a mixture containing other components derived from the locust bean raw material.

[0056] The novel polyphenol compound "OLANDU" of the present invention can be used as a collagen production promoter that promotes collagen gene expression and thus promotes collagen production in vivo. The compound of the present invention promotes collagen production, thereby increasing collagen in target cells.

[0057] The collagen production promoter containing the novel polyphenol compound "OLANDU" of the present invention can be used as a topical skin preparation for increasing the amount of collagen contained in skin cells, preventing or improving skin aging, and maintaining a stable skin state. Furthermore, the collagen production promoter of the present invention can be used as a cosmetic product that increases the amount of collagen contained in skin cells, preventing or improving skin wrinkles and sagging, and maintaining healthy skin.

[0058] The dosage of the collagen production promoter of the present invention varies depending on the desired collagen production promoting effect, preventive or therapeutic effect, method of administration, age, etc., and therefore cannot be generally specified. However, when used as an external preparation, the polyphenol compound of the present invention is preferably administered parenterally daily in an amount of 0.02 μg to 30 mg, more preferably 0.2 μg to 3 mg, and even more preferably 2 μg to 300 μg. Furthermore, when used as an internal preparation, the polyphenol compound of the present invention is preferably administered orally daily in an amount of 0.2 μg to 1000 mg, more preferably 2 μg to 200 mg.

[0059] There are no particular limitations on the dosage forms of the collagen production promoter, external skin preparation, and cosmetics of the present invention. Examples include: low-viscosity liquids, liquid preparations such as lotions; emulsions, gels, pastes, creams, foams, masks, ointments, powders, sprays, or patches; and tablets, granules, capsules, or oral liquid preparations. It should be noted that the collagen production promoter of the present invention can also be applied to any of cosmetics, quasi-drugs, or drugs. There are no particular limitations on the specific products, and examples include: lotion, makeup cream, makeup emulsion, beauty liquid, makeup mask, makeup cleanser, soap, hair conditioner, bath agent, or color cosmetics.

[0060] In the collagen production promoter, external skin preparation, and cosmetics of the present invention, the compounding concentration of the novel polyphenol compound of the present invention is preferably 0.0001 mM to 1 mM, more preferably 1 μM to 100 μM, and further preferably 5 μM to 20 μM. By setting the compounding amount of the novel polyphenol compound within this range, this compound can be stably compounded, the safety for the skin can be improved, and a high collagen production promoting effect can be exerted.

[0061] The collagen production promoter of the present invention can be prepared into various forms by conventional methods used in the past. In this case, carriers, excipients, etc. commonly used in formulations can be used as additives acceptable as pharmaceutical additives for formulation. Also, in order to improve the bioavailability and stability of this compound, a drug delivery system using formulation techniques such as inclusion in microcapsules, micronization, and inclusion using cyclodextrin can also be adopted.

[0062] Furthermore, in the collagen production promoter, external skin preparation, and cosmetics of the present invention, components such as water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, vitamins, water-soluble polymers, surfactants, metal soaps, alcohols, polyols, pH regulators, preservatives, fragrances, powders, thickeners, pigments, or chelating agents, which are commonly used as components of external skin preparations and cosmetics, can be appropriately compounded. Furthermore, within the range that does not impair the action effects of the present invention, various commonly used functional components can be used in combination, such as one or more functional components selected from humectants, whitening agents, anti-inflammatory agents, cell activators, anti-ultraviolet agents, blood circulation promoters, and antioxidants.

[0063] Furthermore, the food or drink for promoting collagen production of the present invention contains the novel polyphenol compound "OLANDU" of the present invention as an active ingredient. The food or drink for promoting collagen production of the present invention can be made into: supplements such as tablets, capsules, granules, syrups, etc.; beverages such as soft drinks, fruit juices, alcoholic beverages, etc.; confectioneries such as candies, chewing gums, cookies, crackers, chocolates, etc.; all forms such as bread, porridge, cereal flakes, noodles, jellies, soups, dairy products, seasonings, etc. When used as a food or drink in this way, various combinations can also be made with other active ingredients, vitamins, minerals, or nutrients such as amino acids within the range that does not affect the efficacy of the active ingredient of the present invention. The food or drink of the present invention includes: supplements, health foods, functional foods, foods for specified health uses, etc. Moreover, the daily intake of the food or drink of the present invention is preferably set at 0.2 μg to 1000 mg, more preferably 2 μg to 200 mg per day for the polyphenol compound of the present invention.

[0064] Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited by any of these examples.

[0065] Examples

[0066] [Example 1]

[0067] 1. Preparation of the hydroalcoholic extract of carob pods

[0068] After collection, the seeds were removed from the pod-bearing fruits of dried carob (Ceratonia siliqua). The pods of the carob were crushed using a crusher to obtain a crushed product with a particle size of 2 mm or less. To 20 kg of the crushed product, 140 kg (7-fold amount) of 95% aqueous ethanol was added, and after performing two 1.5-hour reflux extraction operations, the residue was further subjected to 1.5-hour reflux extraction using 140 kg (7-fold amount) of 70% aqueous ethanol. After combining the obtained reflux extracts, the solvent was removed by vacuum distillation to obtain 12.4 kg of the hydroalcoholic extract of carob pods.

[0069] [Example 2]

[0070] 2. Separation and purification of the hydroalcoholic extract of carob pods

[0071] The water-containing ethanol extract of carob pods obtained in Example 1 was dispersed in 1 to 10 times the amount of water and adsorbed onto an ion exchange resin (macroporous adsorption resin D101, Cangzhou Bon Adsorber Technology Co., Ltd.). After washing with distilled water in an amount 3 times the column volume to remove impurities such as saccharides, it was eluted with 95% ethanol containing water in an amount 3 times the column volume, and the solvent was removed by distillation under reduced pressure to obtain 462.7 g of an ethanol eluate fraction (non-carbohydrate low-molecular compound fraction). Next, the obtained ethanol eluate fraction was dispersed in 1.0 L of 50% methanol containing water, and liquid-liquid extraction was performed 5 times each with petroleum ether and ethyl acetate in turn. The solvents were removed by distillation under reduced pressure to obtain 28.4 g of a petroleum ether fraction, 139.4 g of an ethyl acetate fraction, and 290.2 g of an aqueous fraction, respectively.

[0072] Next, for 135.0 g of the ethyl acetate fraction, normal-phase silica gel column chromatography (column packing material: 200 - 300 mesh, product of Qingdao Ocean Chemical Factory) was performed, and separation was carried out using two developing solvents, petroleum ether (P) / ethyl acetate (E) and dichloromethane (C) / methanol (M). As a result, 108 eluate fractions were obtained. For the 108 eluate fractions obtained, identification was performed by thin-layer chromatography, and similar fractions were combined to obtain 10 eluate fractions A - J.

[0073] Next, for eluate fraction F (8.5 g) and eluate fraction G (9.6 g), reverse-phase ODS column chromatography (column packing material: 40 - 63 μm, product of Merck & Co., Inc.) was performed, and separation was carried out using a gradient elution of methanol:water = 15:85 → 100:0. For the obtained eluate fractions, identification was performed by thin-layer chromatography, and similar fractions were combined to obtain 6 eluate fractions F1 - F6.

[0074] Next, for eluate fraction F4 (4.2 g), gel filtration chromatography (column packing material: Sephadex LH-20) was performed, and elution was carried out using dichloromethane:methanol = 1:1 to obtain 5 eluate fractions F4a - F4e.

[0075] Next, semi-preparative HPLC was performed on the elution fraction F4c (1.1 g) (Column I: YMC-Pack ODS-A, 250×20 mm, 5 μm), and separation was carried out using methanol:water = 40:60 and a detection wavelength of 217 nm to obtain three fractions F4c1, F4c2, and F4c3. Among them, semi-preparative HPLC was performed on fraction F4c2 (Column II: YMC-Pack ODS-A, 250×10 mm, 5 μm), and separation was carried out using methanol:water = 36:64 and a detection wavelength of 217 nm to obtain 23.8 mg of the compound of the present invention (hereinafter, this compound is referred to as “OLANDU”) (retention time tR = 37.14 minutes).

[0076] [Example 3]

[0077] 3. Structure Analysis of Compound (OLANDU)

[0078] Structure analysis was performed on the compound obtained in Example 2, “OLANDU”. During the structure analysis, high-resolution mass spectrometry (HR-ESI-MS, negative ion mode), ultraviolet absorption spectroscopy, infrared absorption spectroscopy, 1 1H-NMR, 13 13C-NMR, HMBC, HSQC, and NOESY analyses were performed. The apparatuses used for these analyses are as described below.

[0079] · High-resolution mass spectrometry: Electrospray ionization quadrupole time-of-flight mass spectrometer (product of Bruker Daltonics)

[0080] · Ultraviolet absorption spectroscopy: Ultraviolet-visible spectrophotometer (UV-2401PC, product of Shimadzu Corporation)

[0081] · Infrared absorption spectroscopy: FT-IR (NEXUS470, product of Thermo nicolet)

[0082] · NMR analysis: 600 MHz nuclear magnetic resonance apparatus (AVANCE III 600, product of Bruker)

[0083] The physical properties of OLANDU are as described below. The spectrum of high-resolution mass spectrometry (HR-ESI-MS) is shown in Figure 1 , the results of ultraviolet absorption spectroscopy are shown in Figure 2 , and the results of infrared absorption spectroscopy are shown in Figure 3 .

[0084] · Appearance: Yellow powder

[0085] HR-ESI-MS (negative) m / z: 507.1180 [MH] -

[0086] UV absorption spectrum: λmax(MeOH)nm(logε):192.2(3.82),218.0(4.26),291.0(4.02),322.4(3.95)

[0087] Infrared absorption spectrum (KBr, cm -1 ): νmax: 3378.33,1699.52,1630.21,1603.89,1515.90,1451.32,1351.12,1326.00,1270.16,1 214.57,1186.83,1126.01,1070.65,1031.88,816.60,765.83,624.09,580.17,537.28,522.40

[0088] High-resolution mass spectrometry ( Figure 1 ) results, according to the quasi-molecular ion peak is m / z507.1180[MH] - , the inferred composition formula is C 23 H 24 O 13 , the degree of unsaturation is 12. And, according to the infrared absorption spectrum ( Figure 3 ) It is speculated that there are hydroxyl groups in the molecule (3378.33cm -1 ), benzene ring (1603.89cm -1 、1515.90cm -1 ), and ester bonds (1699.52cm -1 、1214.57cm -1 ).

[0089] Furthermore, according to the use 1 The spectrum obtained by H-NMR analysis (solvent: CD3OD, observation frequency: 600 MHz) Figure 4 ), confirmed that: a group of trans olefin proton signals [δ H :7.62(1H,d,J=15.6Hz,H-7),6.39(1H,d,J=15.6Hz,H-8)], a group of benzene ring ABX series signals [δ H :7.18 (1H, s, H-2), 7.06 (1H, d, J=7.8 Hz, H-6), 6.80 (1H, d, J=7.8 Hz, H-5)], hydrogen signals of 1,3,4,5-substituted benzene ring [δ H:7.14 (2H, s, H-2', H-6')], 1 methoxy signal [δ H :3.88(3H,s,OCH3-3)], a group of hydrogen signals of glucose residues [δ H :5.68(1H,d,J=5.4Hz,H-1"),4.52(1H,d,J=11.4Hz,H-6α"),4.31(1H,m, H-6β"),3.70(1H,m,H-5"),3.51(2H,m,H-2",H-3"),3.46(1H,m,H-4")]( Figure 9 ). And, according to the use 13 The spectrum obtained by C-NMR analysis (solvent: CD3OD, observation frequency: 150 MHz) Figure 5 ), 23 carbon signals were confirmed ( Figure 9 Specifically, there are two carbonyl carbon signals of ester (δ C :169.1,166.9), 14 aromatic or olefinic carbon signals (δ C :150.6,149.3,147.2,146.5×2,140.4,127.7,124.2,120.6,116.4,115.2,111.6,110.6×2), 1 methoxy carbon signal (δ C :56.4), and a group of carbon signals of glucose residues (δ C Based on these analysis results, it was estimated that the compound "OLANDU" is composed of one glucose residue unit, one ferulic acid unit, and one gallic acid unit.

[0090] Furthermore, according to the spectrum obtained by HMBC analysis ( Figure 7) Distant correlation signals were confirmed at H-2 and C-4 / C-6, H-5 and C-1 / C-3, H-6 and C-4, H-7 and C-2 / C-6 / C-9, H-8 and C-1, and OCH3-3 and C-3. Based on this result, it indicates that there is a ferulic acid unit in the structure of this compound. Also, since distant correlation signals were confirmed at H-2', H-6' and C-4” / C-3' / C-7', it indicates that there is a gallic acid unit in the structure of this compound. And, since a distant correlation signal was confirmed at H-6" and C-9, it indicates that ferulic acid is bonded to C-6" of the glucose residue via an ester bond. Also, based on the distant correlation signal of H-1" and C-7', it indicates that gallic acid is bonded to the terminal carbon of the glucose residue via an ester bond. The coupling constant of the terminal proton of glucose is 5.4 Hz, and it is known that the relative configuration of the glucose terminal is the α-form. From these analysis results, the structure of this compound "OLANDU" is determined to be as follows formula (I). Figure 9 Shows a summary of 1 δ of H-NMR H and 13 δ of C-NMR C comparison table. And, Figure 10 Shows the analysis results of NOESY and HMBC of OLANDU.

[0091] [Chemical formula 3]

[0092]

[0093] [Example 4]

[0094] 4. Investigation of the promoting effect on the expression of collagen gene of OLANDU

[0095] Using the OLANDU obtained in Example 2, the promoting effect on the expression of the collagen gene (COL1A1 gene) was investigated in HaCaT cells, which are keratinocyte cell lines derived from human epidermis.

[0096] First, HaCaT cells were cultured using DMEM medium (containing: 10% FBS, 100 U / mL penicillin, 100 μm / mL streptomycin) under the conditions of 37 °C, 5% CO2 and saturated humidity. Then, in each well of a 6-well cell culture dish, cells were seeded to make 5×10 5HaCaT cells with the cell concentration adjusted in the manner of cells / mL were cultured in a CO2 incubator (5% CO2, 37°C) for 24 hours. Then, OLANDU isolated in Example 2 was added in such a way that different final concentrations were obtained in each well. Specifically, the final concentrations of OLANDU in the HaCaT cell culture medium were made to be 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM, respectively. And the well without the addition of OLANDU was set as a control (0 μM). After the addition, the cells were cultured for 24 hours.

[0097] After the culture was completed, HaCaT cells were recovered from each well, and total RNA was extracted using an RNA extraction reagent (RNAzol (registered trademark), product of Molecular Research Center, Inc.). The concentration of the extracted total RNA was confirmed using a ultra-micro spectrophotometer (NanoDrop, product of ThermoFisher Scientific, Inc.). To this total RNA, oligo dT primer, dNTP mixture (dNTP Mix), 5× first strand buffer, RNase inhibitor, DTT, and M-MLV reverse transcriptase were added, and cDNA was synthesized from the total RNA. Using this cDNA, the expression level of the collagen gene was measured by real-time PCR (QPCR).

[0098] QPCR was performed using a commercially available QPCR kit and a QPCR measurement device (Light Cycler (registered trademark) 480, product of Roche Diagnostics Corporation), and SYBR (registered trademark) Green, a fluorescent dye that binds to double-stranded DNA, was used for detection. The target gene was the COL1A1 gene encoding the α1 chain of type I collagen, and the QPCR primers used were the primers with sequence numbers 1 and 2 shown in Table 1 below.

[0099] [Table 1]

[0100] Forward primer 5’-TCTGCGACAACGGCAAGGTG-3’ Sequence ID No. 1 Reverse primer 5’-GACGCCGGTGGTTTCTTGGT-3’ Sequence ID No. 2

[0101] The results are shown in Figure 11 . The mRNA expression level of the collagen gene (COL1A1 gene) is represented as a relative value when the expression level of the control (control group) is set to 100. As Figure 11As shown, by adding the compound of the present invention, OLANDU, compared with the control without addition, it is known that the expression level of the collagen gene has increased significantly. Even when the concentration of OLANDU is 1 μM, the expression level of the collagen gene still increases by about 2 times, and when it is 10 μM, the expression level of the collagen gene increases by more than 3 times, indicating that the expression promoting effect is the highest.

[0102] The present invention is not limited to the above embodiments or examples, and various design change schemes within the scope not departing from the gist of the invention described in the claims are all covered within the technical scope.

[0103] Industrial Applicability

[0104] The new polyphenol compound of the present invention can be used as a collagen production promoter, a topical skin agent, and a cosmetic, and is widely used in the medical and beauty fields.

[0105] Sequence Listing Free Text

[0106] Sequence No. 1: Forward primer for QPCR of target gene: COL1A1

[0107] Sequence No. 2: Reverse primer for QPCR of target gene: COL1A1 Sequence Listing <110> Nippon Garlic Co., Ltd. <120> New Polyphenol Compound <130> PCT22 - 001 <150> JP 2021 - 066340 <151> 2021 - 04 - 09 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer <400> 1 tctgcgacaa cggcaaggtg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer <400> 2 gacgccggtg gtttcttggt 20

Claims

1. A compound represented by the following formula (I) or a salt thereof, 。 2. A collagen production promoter that contains only the compound represented by the following formula (I) or a salt thereof, 。 3. A collagen production promoter that contains the compound represented by the following formula (I) or a salt thereof, and optionally contains components selected from water, oils and fats, waxes, hydrocarbons, fatty acids, esters, plant extracts, vitamins, water-soluble polymers, surfactants, metal soaps, alcohols, pH adjusters, preservatives, fragrances, powders, thickeners, pigments or chelating agents, The concentration of the compound represented by the formula (I) or a salt thereof is 0.0001 mM to 1 mM.

4. A collagen production promoter that contains the compound represented by the following formula (I) or a salt thereof, and optionally contains hydrous alcohol, The concentration of the compound represented by the formula (I) or a salt thereof is 0.0001 mM to 1 mM.

5. A topical skin agent for promoting collagen production, which contains the collagen production promoter according to any one of claims 2 to 4 as an active ingredient, wherein, Excluding a skin external preparation for promoting collagen production containing an aqueous alcohol extract of the pods of locust bean as an active ingredient.

6. A cosmetic for promoting collagen production, which contains the collagen production promoter according to any one of claims 2 to 4 as an active ingredient, wherein, Excluding a cosmetic for promoting collagen production containing an aqueous alcohol extract of the pods of locust bean as an active ingredient.

7. A food or drink for promoting collagen production that contains the compound represented by the following formula (I) or a salt thereof, 。

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