Novel phenylpropanoid compounds

Abstract

The present invention provides a novel active ingredient derived from carob and its use. The present invention is a compound represented by the following formula (I) or a salt thereof or a solvate thereof.

Description

Technical Field

[0001] The present invention relates to a novel phenylpropanoid compound obtained by separation and purification from carob (Ceratonia siliqua L.) as a leguminous plant and its use, and also relates to a method for preparing the compound. Background Art

[0002] Ceratonia siliqua L. is a leguminous plant mainly native to the Mediterranean region. The pods of carob, i.e., the pods and pulp of carob are called carob pods, and have been used as food or food ingredients since ancient times. The length of mature pods is about 10 to 25 cm, and they are sweet. The pods of carob contain a large amount of polysaccharides, cellulose and minerals, and a small amount of protein, non-carbohydrate low molecular weight compounds, etc. In recent years, research related to the new functions of carob is constantly advancing, and it is reported that the water extract of carob pods has a variety of pharmacological effects such as antidiarrheal, antioxidant, antibacterial, anti-ulcer and anti-inflammatory effects in the digestive tract, and has preventive and therapeutic effects on digestive diseases such as ulcerative colitis and gastric ulcers (Non-patent Documents 1, 2). In addition, Patent Document 1 records that the seed extract of carob has α-glucosidase inhibitory activity, which has the effect of inhibiting weight gain (Patent Document 1).

[0003] On the other hand, free radicals are intermediate products generated during the metabolic process in the body. They are atoms, atomic groups or molecules with very active chemical properties that contain unpaired electrons. Excessive free radicals generated in the body damage the cell membranes of normal cells, modify proteins, cause loss of enzyme function and damage to DNA and extracellular matrix. The damage caused by free radicals is closely related to aging, carcinogenesis, Alzheimer's disease, cardiovascular, cerebrovascular diseases and skin diseases. Although the human body has an antioxidant mechanism for preventing damage caused by free radicals, it is difficult to completely eliminate the damage caused by excessive free radicals due to continuous aging, illness, etc. Therefore, in order to maintain the normal function of the human body, it is very effective to use antioxidants to remove free radicals. As the antioxidants currently used, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), etc. can be cited, but these have problems such as high price and poor safety.

[0004] If the extracellular matrix is ​​damaged due to the strong oxidative power of free radicals, the skin may lose its elasticity. In addition, due to aging, the skin becomes loose, rough, and dull, so the skin color becomes darker. At the same time, since the human skin color mainly depends on the amount of melanin produced in the body, the accumulation of melanin in the skin is also one of the important reasons for the darkening of the skin color, loss of elasticity and glossiness.

[0005] According to recent research results, substances with antioxidant activity can reduce damage caused by free radicals by removing free radicals in the body, thereby slowing down the aging process of the skin, and as a result, can bring about a whitening effect. Therefore, substances that inhibit melanin production and have antioxidant activity can exert a synergistic whitening effect.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2005-119999

[0009] Non-patent literature

[0010] Non-patent document 1: Rtibi K, Jabri MA, Selmi S, et al., "Preventive effect of carob (Ceratonia siliqua L.) in dextran sulfate sodium-induced ulcerativecolitis in rat", RSC Advances, 2016, Vo1.6, p.19992-20000.

[0011] Non-patent literature 2: Rtibi K, Selmi S, Grami D, et al., "Chemicalconstituents and pharmaceutical actions of carob pods and leaves (Ceratonia siliqua L.) on thegastrointestinal tract: A review", Biomedicine&Pharmacotherapy, 2017, Vol.93, p.522-528. Summary of the invention

[0012] Problem that the invention aims to solve

[0013] However, although the above-mentioned non-patent documents 1 and 2 report that the pod extract of carob has pharmacological effects such as antidiarrheal, antioxidant, antibacterial, anti-ulcer and anti-inflammatory effects in the digestive tract and has a therapeutic effect on digestive diseases, and patent document 1 reports that the seed extract of carob has an effect of inhibiting weight gain, the specific active ingredient has not yet been identified.

[0014] Additionally, the use of carob as a skin-lightening agent has not been studied to date, and its effectiveness is completely unclear.

[0015] Therefore, the present invention has been made in view of the above aspects, and an object of the present invention is to provide a novel active ingredient derived from carob and use thereof.

[0016] In addition, another object of the present invention is to provide a new whitening agent having a melanin production inhibitory effect and an antioxidant activity, which is derived from carob, which is a natural product. In addition, an object is to provide a skin external preparation, a cosmetic, and a skin whitening composition containing the whitening agent.

[0017] Technical solutions to the problem

[0018] The present inventors have found that novel phenylpropanoid compounds can be isolated from carob pod extracts, and that the novel compounds have not only melanin production inhibitory effects but also antioxidant activity. Based on this finding, the present invention has been completed.

[0019] 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.

[0020] [Chemical formula 1]

[0021]

[0022] The compound represented by formula (I) is a novel phenylpropanoid compound separated and purified from the pod extract of Ceratonia siliqua, has melanin production inhibition and antioxidant activity, and has a whitening effect.

[0023] In addition, the whitening agent of the present invention contains the compound represented by the above formula (I) or its salt or their solvate. By administering the compound represented by formula (I), melanin production in the skin can be inhibited, and free radicals can be removed to inhibit skin aging, thereby exerting a synergistic whitening effect.

[0024] In the whitening agent of the present invention, the concentration of the compound represented by the above formula (I) or its salt or solvate thereof is preferably 0.001 mM to 1 mM. This allows selection of a concentration of the active ingredient that provides excellent effects.

[0025] The melanin production inhibitor of the present invention contains the compound represented by the above formula (I) or a salt thereof or a solvate thereof. By administering the compound represented by the formula (I), melanin production in the skin can be inhibited.

[0026] In addition, the antioxidant of the present invention contains the compound represented by the above formula (I) or a salt thereof or a solvate thereof. By administering the compound represented by the formula (I), free radicals can be removed and damage caused by free radicals can be reduced.

[0027] In addition, the whitening agent, melanin production inhibitor or antioxidant of the present invention is also preferably a skin external preparation or cosmetic. Thus, a skin external preparation or cosmetic having a skin whitening effect, a melanin production inhibitory effect in the skin or an anti-aging effect on the skin can be obtained.

[0028] In addition, the skin whitening composition of the present invention contains the compound represented by the above formula (I) or its salt or solvate thereof. Thus, a composition that inhibits melanin production in the skin and removes free radicals in the body to slow down the aging process of the skin, thereby exerting a synergistic whitening effect, can be obtained.

[0029] In addition, the method for producing the compound represented by the above formula (I) of the present invention comprises the following steps: a step of obtaining a hydroalcohol extract of the pods of Ceratonia siliqua; a step of subjecting the hydroalcohol extract to liquid-liquid extraction with petroleum ether and ethyl acetate in that order to recover the ethyl acetate fraction; and a step of separating the compound represented by formula (I) from the recovered ethyl acetate fraction. Thus, a novel phenylpropanoid compound having an antioxidant activity and a melanin production inhibitory effect and having a whitening effect can be obtained.

[0030] Effects of the Invention

[0031] According to the present invention, there are provided a novel phenylpropanoid compound having the following excellent effects, and a whitening agent, a melanin production inhibitor, an antioxidant, a skin external preparation, a cosmetic, and a skin whitening composition containing the compound.

[0032] (1) It has melanin production inhibition and antioxidant activity, and has a synergistic whitening effect.

[0033] (2) Since the active ingredient contains a compound derived from the pods of carob beans, which have been used as food for a long time, it is highly safe for the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a figure which shows the high-resolution ESI mass spectrum of the compound of the present invention.

[0035] Figure 2 It is a graph showing the ultraviolet absorption spectrum of the compound of the present invention.

[0036] Figure 3 This is a diagram showing the infrared absorption spectrum of the compound of the present invention.

[0037] Figure 4 The compounds of the present invention are shown 1 H-NMR spectrum (CD3OD, 600 MHz).

[0038] Figure 5 The compounds of the present invention are shown 13 C-NMR spectrum (CD3OD, 150 MHz).

[0039] Figure 6 It is a figure which shows the HSQC spectrum of the compound of this invention.

[0040] Figure 7 It is a figure which shows the HMBC spectrum of the compound of this invention.

[0041] Figure 8 The compound of the present invention 1 H-NMR signal and 13 C-NMR signals are summarized in one overview graph.

[0042] Fig. 9 This is a diagram showing the HMBC correlation of the compound of the present invention.

[0043] Fig.10 This is a graph showing the DPPH radical scavenging activity of the compound of the present invention in Example 4.

[0044] Fig.11 This is a graph showing the results of a cytotoxicity test on HepG2 cells of the compounds of the present invention in Example 5.

[0045] Fig.12 This is a graph showing the ROS production inhibitory effect in HepG2 cells brought about by the compound of the present invention in Example 5.

[0046] Fig.13 This is a graph showing the results of a cytotoxicity test of the compound of the present invention on B16 melanoma cells in Example 6. DETAILED DESCRIPTION

[0047] Hereinafter, the novel phenylpropanoid compound of the present invention, and the whitening agent, melanin production inhibitor, antioxidant, external preparation for skin, cosmetic and skin whitening composition containing the same, and the method for producing the compound will be described.

[0048] The novel phenylpropanoid compound represented by the following formula (I) of the present invention is a compound in which glucose and 2-methylpropionic acid are ester-bonded to sinapyl alcohol.

[0049] [Chemical formula 2]

[0050]

[0051] The novel phenylpropanoid compound of the present invention may be a salt, preferably a pharmacologically acceptable salt. The pharmacologically acceptable salt of the novel phenylpropanoid compound is not particularly limited as long as it is a salt formed with an acid or a base. In addition, the novel phenylpropanoid compound or its salt may be a solvate, which is not particularly limited, and examples thereof include organic solvates such as hydrates and ethanol.

[0052] The novel phenylpropanoid compounds of the present invention have excellent melanin production inhibitory effects and also have antioxidant effects, and therefore can be used as whitening agents that can obtain whitening effects based on multiple mechanisms. That is, since the novel phenylpropanoid compounds of the present invention can inhibit melanin production in the skin and remove free radicals through antioxidant effects, darkening of the skin caused by skin aging due to damage caused by free radicals can be inhibited, and a synergistic whitening effect can be exerted.

[0053] In the present invention, the melanin production inhibitory effect refers to an effect in which the amount of melanin produced in melanin-producing cells such as B16 melanoma cells is reduced compared to a control group in which the novel phenylpropanoid compound of the present invention is not added or administered. More specifically, the amount of melanin produced is preferably 80% or less of that in the control group, and more preferably 70% or less.

[0054] In the present invention, the antioxidant effect refers to an effect such as a high DPPH radical elimination rate or a reduction in intracellular ROS, compared to a control group to which the novel phenylpropanoid compound of the present invention is not added or administered.

[0055] The novel phenylpropanoid compounds of the present invention can be obtained by separation and purification from the pods of carob. The scientific name of the carob used in the present invention is Ceratonia siliqua, which is a plant of the genus Ceratonia of the subfamily Caesalpinioideae of the family Leguminosae. It is a plant native to the Mediterranean coastal area, but in the present invention, the origin and cultivation environment are not particularly limited, and carob from all origins and cultivation environments can be used.

[0056] The method for separating the novel phenylpropanoid compounds from carob beans of the present invention is described. First, a hydrous alcohol extract is obtained from the pods of carob beans. The hydrous alcohol extract of the pods of carob beans in the present invention refers to an extract obtained by adding hydrous alcohol as an extraction solvent to the pods of carob beans and performing an extraction treatment. The pods of carob beans refer to the pods and pulp of the podded fruits of carob beans. Either the pods or the pulp can be used as the extraction material, but the pods and the pulp are more preferably used. Regarding the extraction treatment, the carob pods in the state after collection, that is, the unprocessed state, or the carob pods in the dry state are subjected to extraction treatment, but in order to improve the extraction efficiency or to make the operation easier, the carob pods subjected to various pretreatments can also be subjected to extraction treatment. As a pretreatment, there is no particular limitation, and examples include: drying treatment, crushing treatment or pulverization treatment, and the extract can also be obtained by subjecting the pods of carob beans subjected to these pretreatments to extraction treatment.

[0057] As alcohol constituting the aqueous alcohol used as the extraction solvent, there is no particular limitation as long as it can extract the phenylpropanoid compound of the present invention, and examples thereof include ethanol, methanol, propanol, isopropanol, butanol or isobutanol. Among them, from the viewpoints of safety to the human body and extraction efficiency, aqueous ethanol can be preferably selected as the extraction solvent. In addition, the alcohol concentration of the aqueous alcohol is preferably 50 to 99%, more preferably 60 to 97%, and particularly preferably 70 to 95%. In addition, the extraction solvent may also contain other components within the range that does not hinder the extraction of the compound of the present invention.

[0058] As an extraction method using aqueous alcohol, aqueous alcohol as an extraction solvent is added to the pods of carob and immersed to perform extraction. For example, when the pods of carob are made into a dry crushed product with a moisture content of less than 10%, it is preferred to use 5 to 10 parts by weight of the extraction solvent relative to 1 part by weight of the plant body. In addition, as an extraction method, it can be performed by any method such as extraction at room temperature, heating extraction, pressurized heating extraction or subcritical extraction, but from the viewpoint of extraction efficiency, heating extraction using reflux operation is preferred. In addition, in order to improve the extraction efficiency, it is preferred to perform the extraction operation several times, and it is further preferred to perform the extraction operation several times while changing the alcohol concentration in the extraction solvent. Although not particularly limited, specifically, it can be cited as an extraction method in which reflux extraction using 95% ethanol is performed 1 to 5 times, followed by reflux extraction using 70% ethanol 1 to 5 times. The extraction time can be set in various ways depending on the extraction method, the type of extraction material, the type of extraction solvent, the extraction temperature, etc. For example, when 70-95% ethanol is used for reflux extraction, the extraction time per time is preferably set to about 1-3 hours, and particularly preferably about 1.5 hours. After the above extraction treatment, the residue is removed by decantation, centrifugal separation or filtration, thereby obtaining a hydroalcohol extract of carob pods. It also includes a concentrate or a solid obtained by subjecting the obtained extract to a treatment such as reduced pressure distillation.

[0059] The hydroalcohol extract of carob pods obtained as described above may be subjected to separation using an ion exchange resin in order to remove unnecessary components, since sugars and the like are present in large quantities in the carob pods. Specifically, 1 to 10 parts by weight of water are added to 1 part by weight of the hydroalcohol extract of carob pods to disperse the extract, and the extract is passed through a column equipped with an ion exchange resin such as a macroporous adsorption resin to adsorb the phenylpropanoid compound of the present invention, and the unnecessary components such as sugars are removed by elution. Thereafter, the extract is eluted with 95% ethanol or the like, thereby recovering a fraction containing the phenylpropanoid compound of the present invention.

[0060] Next, further separation operation of the hydrous alcohol extract of carob pods or the recovered fraction separated by the above-mentioned ion exchange resin is described. The hydrous alcohol extract or the recovered fraction is dispersed in an aqueous solvent, and solvent extraction is performed in the order of petroleum ether and ethyl acetate. As the aqueous solvent, there is no particular limitation as long as the phenylpropanoid compounds of the present invention can be dispersed, and 50% aqueous methanol is preferably used. After performing liquid-liquid extraction using petroleum ether / aqueous solvent several times, liquid-liquid extraction using ethyl acetate / aqueous solvent is performed several times. The ethyl acetate fraction recovered by this solvent extraction operation contains the novel phenylpropanoid compounds of the present invention. The number of liquid-liquid extractions using each solvent system is preferably about 2 to 10 times, and particularly preferably about 5 times.

[0061] The methyl acetate fraction obtained as described above is purified based on a conventional method, thereby the novel phenylpropanoid compound of the present invention can be isolated separately. As purification methods, normal phase chromatography, reverse phase chromatography, thin layer chromatography, gel filtration chromatography and high performance liquid chromatography can be cited, and one or more of these can be combined for purification. The carrier, elution solvent, etc. used in various chromatography methods can be appropriately selected according to various chromatography methods.

[0062] It should be noted that the phenylpropanoid compounds of the present invention separated and purified from carob pods as described above may not be isolated as completely pure substances, but may contain a part of other components derived from the carob raw material.

[0063] Furthermore, the novel phenylpropanoid compounds of the present invention may be synthesized by known methods.

[0064] The novel phenylpropanoid compound of the present invention has an excellent melanin production inhibitory effect and also has an antioxidant effect, so it can be used as a whitening agent that can obtain whitening effects based on various mechanisms. In addition, the phenylpropanoid compound can be used as a melanin production inhibitor or an antioxidant.

[0065] Whitening agents and melanin production inhibitors containing the novel phenylpropanoid compounds of the present invention can be used as skin topical agents or cosmetics, which are used to inhibit melanin production in the skin and to inhibit skin darkening caused by skin aging due to damage caused by free radicals. In addition, antioxidants containing the novel phenylpropanoid compounds of the present invention can prevent skin aging caused by damage caused by free radicals and are used as skin topical agents for keeping the skin in a stable state. In addition, the antioxidants of the present invention can prevent skin aging caused by damage caused by free radicals, prevent skin sagging, rough skin texture, and dull skin, and are used as cosmetics that have the effect of maintaining skin health.

[0066] The dosage of the whitening agent, melanin production inhibitor and antioxidant of the present invention varies depending on the target effect, therapeutic effect, method of administration or age, etc., and therefore cannot be stipulated in general terms, but the usual non-oral dosage per day when used as an external preparation is preferably 0.2 μg to 30 mg, more preferably 1 μg to 3 mg, and further preferably 5 μg to 500 μg, based on the phenylpropanoid compound of the present invention. In addition, the oral dosage when used as an internal preparation is preferably 1 μg to 1000 mg, more preferably 5 μg to 500 mg, based on the phenylpropanoid compound of the present invention.

[0067] The dosage forms of the whitening agent, melanin generation inhibitor and antioxidant of the present invention, as well as the skin external preparation and cosmetics are not particularly limited, and for example, low-viscosity liquid, liquid preparations such as coatings, emulsions, gels, slurries, creams, foams, facial masks, ointments, powders, mists or patches, and tablets, granules, capsules or oral liquid preparations, etc. It should be noted that the whitening agent, melanin generation inhibitor and antioxidant of the present invention can be applied to any of cosmetics, quasi-drugs or pharmaceuticals. As specific products, there are no particular limitations, and examples include: lotion, cosmetic cream, cosmetic emulsion, beauty serum, cosmetic mask, cosmetic cleanser, soap, hair care agent, bath agent or makeup cosmetics, anti-acne skin care cosmetics, etc.

[0068] In the whitening agent, melanin production inhibitor, antioxidant, skin external preparation, and cosmetic of the present invention, the novel phenylpropanoid compound of the present invention is preferably used at a concentration of 0.001 mM to 1 mM, more preferably 5 μM to 100 μM, and even more preferably 10 μM to 100 μM. By setting the amount of the novel phenylpropanoid compound to be used within this range, the compound can be stably used, the safety to the skin is also high, and a high whitening effect, melanin production inhibitory effect, and antioxidant effect can be exerted.

[0069] The whitening agent, melanin production inhibitor and antioxidant of the present invention can be made into various forms by conventional methods. In this case, the additives allowed as additives of pharmaceuticals such as carriers and excipients for conventional preparations can be used for formulation. In addition, in order to improve the bioavailability and stability of the present compound, a drug delivery system including microcapsules, micronization, inclusion of cyclodextrins and the like can also be used.

[0070] In addition, water, oils, 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 ingredients in skin external preparations and cosmetics, can be appropriately formulated in the whitening agent, melanin production inhibitor and antioxidant of the present invention, as well as skin external preparations and cosmetics. Furthermore, it can be used in combination with various commonly used functional ingredients, such as one or more functional ingredients selected from moisturizers, anti-inflammatory agents, cell activators, ultraviolet protection agents, blood circulation promoters and other whitening agents, antioxidants, etc., within the scope of not impairing the effects of the present invention.

[0071] Furthermore, the skin whitening composition of the present invention contains the novel phenylpropanoid compound of the present invention as an active ingredient. The skin whitening composition of the present invention can be prepared in all forms such as tablets, capsules, granules, syrups, etc. Regarding the daily intake, it is usually preferably set to 1μg to 1000mg per day, and more preferably set to 5μg to 500mg, based on the phenylpropanoid compound of the present invention.

[0072] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited by these examples in any way.

[0073] Example

[0074] [Example 1]

[0075] 1. Preparation of Hydroalcoholic Extract of Ceratonia sibiricum Fruit

[0076] After collection, the seeds are removed from the dried carob (Ceratonia siliqua) pods. The carob pods are crushed by a grinder to obtain a crushed product with a particle size of less than 2 mm. For 20 kg of the crushed product, 140 kg (7 times the amount) of 95% ethanol containing water is added, and reflux extraction is performed for 1.5 hours. After the above operation is performed twice, the residue is further reflux extracted with 140 kg (7 times the amount) of 70% ethanol containing water for 1.5 hours. After combining the obtained reflux extracts, the solvent is removed by reduced pressure distillation to obtain 12.4 kg of a hydrous ethanol extract of carob pods.

[0077] [Example 2]

[0078] 2. Isolation and purification of the hydroalcoholic extract of carob pods

[0079] The hydrous ethanol extract of carob pod obtained in Example 1 was dispersed in 1 to 10 times the amount of water and adsorbed on an ion exchange resin (macroporous adsorption resin D101, Cangzhou Baoen Adsorption Material Technology Co., Ltd.). It was dissolved with distilled water 3 times the volume of the column to remove impurities such as sugars, and then dissolved with 95% ethanol containing water 3 times the volume of the column. The solvent was removed by vacuum distillation to obtain 462.7 g of ethanol elution fraction (non-carbohydrate low molecular weight compound fraction). Next, the obtained ethanol elution fraction was dispersed in 1.0 L of 50% methanol containing water, and liquid-liquid extraction was performed 5 times in the order of petroleum ether and ethyl acetate. The solvents were removed by vacuum distillation to obtain 28.4 g of petroleum ether fraction, 139.4 g of ethyl acetate fraction and 290.2 g of aqueous fraction.

[0080] Next, 135.0 g of the ethyl acetate fraction was subjected to normal phase silica gel column chromatography (column filling material: 200-300 mesh, product of Qingdao Ocean Chemical Plant), and eluted using two developing solvents of petroleum ether (P) / ethyl acetate (E) and dichloromethane (C) / methanol (M) to distinguish them. As a result, 108 elution fractions were obtained. The 108 elution fractions obtained were identified by thin layer chromatography (silica gel GF254 plate, product of Qingdao Ocean Chemical Plant), and similar fractions were combined to obtain 10 elution fractions A to J.

[0081] Next, the elution fraction I (20.0 g) was subjected to reverse phase ODS column chromatography (column filling material: 40-63 μm, Merck product) and fractionated by gradient elution of methanol: water = 15: 85 → 100: 0. The obtained elution fractions were identified by thin layer chromatography (silica gel GF254 plate, Qingdao Ocean Chemical Factory product), and similar fractions were combined to obtain 5 elution fractions I1-15.

[0082] Next, fraction I5 (2.7 g) was subjected to gel filtration chromatography (column filling material: Sephadex LH-20), and eluted with dichloromethane:methanol=1:1 to obtain five fractions I5a to I5e.

[0083] Next, the eluted fraction I5b (0.5 g) was subjected to semi-preparative HPLC (column I: YMC-Pack ODS-A, 250×20 mm, 5 μm), and separated under the conditions of methanol:water=55:45 and detection wavelength of 220 nm to obtain fraction I5b1. This fraction I5b1 was subjected to semi-preparative HPLC (column II: YMC-Pack ODS-A, 250×10 mm, 5 μm), and separated under the conditions of methanol:water=55:45 and detection wavelength of 267 nm to obtain 48.3 mg of the compound of the present invention (hereinafter referred to as "Ceratonia siliqua B") (retention time tR=27.27 minutes).

[0084] [Example 3]

[0085] 3. Structural analysis of the compound (Ceratonia siliqua B)

[0086] The structure of the compound "Ceratonia siliqua B" obtained in Example 2 was analyzed. When analyzing the structure, high-resolution mass spectrometry (HR-ESI-MS; positive ion mode), ultraviolet absorption spectrum analysis, infrared absorption spectrum analysis, 1 H-NMR, 13C-NMR, HMBC and HSQC analysis: The apparatus used in these analyses are shown below.

[0087] High-resolution mass spectrometry: Electrospray ionization quadrupole time-of-flight mass spectrometry (BrukerDaltonics)

[0088] Ultraviolet absorption spectrum analysis: UV-visible spectrophotometer (UV-2401PC, Shimadzu Corporation)

[0089] Infrared absorption spectrum analysis: FT-IR (NEXUS470, product of Thermo Nicolet)

[0090] NMR analysis: 600 MHz nuclear magnetic resonance device (AVANCE III 600, a product of Bruker)

[0091] The physical properties of Ceratonia siliqua B are shown below. The spectra of high resolution mass spectrometry (HR-ESI-MS) are shown in Figure 1 The results of the ultraviolet absorption spectrum analysis are shown in Figure 2 The results of infrared absorption spectrum analysis are shown in Figure 3 .

[0092] ·Appearance: Yellow powder

[0093] ·HR-ESI-MS(positive)m / z:465.1983[M+Na]+

[0094] UV absorption spectrum: λmax(MeOH)nm(logε): 193.6(3.73), 211.6(4.32), 269.2(3.97)

[0095] Infrared absorption spectrum (KBr, cm -1 ): vmax: 3393.18, 2933.52, 1731.29, 1587.20, 1506.71, 1464.86, 1421.12, 1388.81, 1336.38, 1243.07, 1158.97, 1129.16, 1070.11, 625.20, 609.78, 595.24, 581.17, 566.35, 536.59, 523.24

[0096] High resolution mass spectrometry ( Figure 1 ) results in a quasi-molecular ion peak of m / z 465.1983 [M+Na] + , so the composition formula is inferred to be C 21 H 30 O10 , the unsaturation is 7. In addition, according to the ultraviolet absorption spectrum ( Figure 2 ), with maximum absorption at 211nm and 269nm, thus suggesting the possibility that Ceratonia siliqua B is a phenylpropanoid compound. In addition, according to the infrared absorption spectrum ( Figure 3 ), indicating that the molecular structure contains hydroxyl groups (3393.18 cm -1 ), benzene ring (1587.20cm -1 、1506.71cm -1 ) and ester bond (1731.29cm -1 、1129.16cm -1 ) and other functional groups.

[0097] Furthermore, according to 1 The spectrum obtained by H-NMR analysis (solvent: CD3OD, observation frequency: 600 MHz) Figure 4 ), confirmed: 1 group of 1,3,4,5-substituted benzene ring hydrogen signals [δ H :6.76 (2H, s, H-2, H-6)], 1 group of trans-olefin proton signals [δ H : 6.61 (1H, d, J = 16.2 Hz, H-7), 6.30 (1H, dt, J = 15.6 Hz, J = 6.0 Hz, H-8)], 1 methylene hydrogen signal [δ H :4.71(2H, d, J=6.0Hz, H-9)], 2 methoxy signals [δ H :3.86 (6H, s, OCH3-3, OCH3-5)], 1 methine signal [δ H :2.60(1H,m,H-2′)], 2 methyl signals [δ H :1.18 (6H, d, J=7.2 Hz, H3-1′ / H3-4′)], a group of hydrogen signals of glucose residues [δ H : 4.89 (1H, d, J = 7.8Hz, H-1”), 3.78 (1H, dd, J = 12.0Hz, J = 2.4Hz, H-6α”), 3.67 (1H, dd, J = 12.0Hz, J=5.4Hz, H-6β”), 3.48 (1H, m, H-2”), 3.43 (1H, m, H-3”), 3.42 (1H, m, H-4”), 3.22 (1H, m, H-5”)] ( Figure 8 ). In addition, according to 13 The spectrum obtained by C-NMR analysis (solvent: CD3OD, observation frequency: 150 MHz) Figure 5 ), 21 carbon signals were confirmed ( Figure 8). These include: 1 carbonyl carbon signal of ester (δ c :178.5), 8 aromatic or olefinic carbon signals (6 C :154.3×2,136.2,134.8,134.5,124.4,105.6×2),1 methylene signal (δ c :66.0), 2 methoxy signals (6 C :57.0×2)、1 methine signal (δ C :35.2), 2 methyl signals (δ C :19.3×2), a group of carbon signals of glucose residues (δ C :105.2, 78.3, 77.8, 75.7, 71.3, 62.5). According to these analysis results, the NMR data of the present compound are similar to those of syringin, but the present compound also contains a 2-methylpropionic acid unit, and in this respect, the present compound is a compound different from syringin.

[0098] In addition, according to the spectrum obtained by HMBC analysis ( Figure 7 ), long-range correlation signals were confirmed between H-2 / H-6 and C-4, H-7 and C-2 / C-6, H-8 and C-1, H-9 and C-7, and H-1″ and C-4. This result showed that a syringin unit existed in the structure of the present compound. In addition, since long-range correlation signals were confirmed between H3-1′ and C-3' / C-4′, and H-2′ and C-3' / C-4′, it was shown that a 2-methylpropionic acid unit existed in the structure of the present compound. Furthermore, since long-range correlation signals were confirmed between H2-9 and C-3′, it was shown that a 2-methylpropionic acid unit was bonded to the C-9 position. It was found that the coupling constant of the proton at the glucose terminal was 7.8 Hz, and the relative configuration of the glucose terminal was β-type. Based on these analytical results, the structure of the present compound Ceratonia siliqua B was determined to be as shown in the following formula (I).

[0099] Figure 8 The summary based on HSQC spectrum and HMBC spectrum analysis is shown as follows 1 H-NMR δ H and 13 C-NMR δ C In addition, Fig. 9 The results of HMBC analysis of Ceratonia siliqua B are shown.

[0100] [Chemical formula 3]

[0101]

[0102] [Example 4]

[0103] 4. Antioxidant activity of Ceratonia siliqua B (1)

[0104] Using the Ceratonia siliqua B obtained in Example 2, the DPPH radical scavenging activity of Ceratonia siliqua B was measured.

[0105] First, the Ceratonia siliqua B obtained in Example 2 was accurately weighed and dissolved in methanol to prepare a sample solution with a concentration of 100 μM. The sample solution with a concentration of 100 μM was further diluted with methanol to obtain sample solutions of different concentrations of 1 μM, 5 μM, 10 μM and 50 μM. Next, 2 mg of DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in 50 mL of methanol to prepare a 100 μM DPPH solution.

[0106] Place 190 μL of DPPH solution in the wells of a 96-well sample plate, and quickly add 10 μL of the sample solution. The total amount of the reaction solution in the well is 200 μL. Slowly shake the sample plate and react in a dark room at room temperature for 30 minutes. Then, measure the absorbance (A) at 517 nm using an ELISA reader. 样品 In addition, as a blank sample, the reaction solution to which methanol was added was used instead of the sample solution, and the absorbance (A 空白样品 ). Then, as a sample blank, the original coloring absorbance (A 样品空白 ). For each concentration of the sample solution, blank sample and sample blank, the test was performed 3 times (3 wells) (n=3).

[0107] The free radical elimination rate (%) was calculated by the following formula.

[0108] Free radical elimination rate (%) = [A 空白样品 -(A 样品 -A 样品空白 )] / A 空白样品 ×100

[0109] A 空白样品 is the absorbance of the blank sample, A 样品 is the absorbance of the sample solution, A 样品空白 is the absorbance of the sample blank.

[0110] The results are shown in Fig.10 This indicates that the compound of the present invention, Ceratonia siliqua B, eliminates DPPH radicals in a concentration-dependent manner. This indicates that Ceratonia siliqua B has free radical scavenging activity, that is, antioxidant activity.

[0111] [Example 5]

[0112] 5. Antioxidant activity of Ceratonia siliqua B (2)

[0113] Using Ceratonia siliqua B obtained in Example 2, the effect of Ceratonia siliqua B on the generation of reactive oxygen species (ROS) in living cells was examined.

[0114] (a) Living cells and cell culture conditions

[0115] In this experiment, HepG2 cells, a tumor cell line derived from human liver, were used as living cells. HepG2 cells were cultured in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 μM / mL streptomycin) at 37°C, 5% CO2, and saturated humidity.

[0116] (b) Preparation of sample solution

[0117] Ceratonia siliqua B was diluted with DMEM medium to obtain sample solutions of different concentrations of 1 μM, 5 μM, 10 μM, 50 μM and 100 μM.

[0118] (c) Cytotoxicity study on HepG2 cells

[0119] First, the MTT test was used to confirm whether Ceratonia siliqua B had cytotoxicity to HepG2 cells. HepG2 cells subcultured for more than 3 generations were treated with trypsin to prepare a single cell suspension at a concentration of 1×105 cells / mL. The cell suspension was inoculated into each well of a 96-well sample plate at 100 μL per well and cultured in a CO2 incubator for 24 hours. After culture, the supernatant was removed and the cells were divided into a control (blank control) group and a test group. 200 μL of DMEM culture medium was added to the control group, and 200 μL of the sample solution (b) was added to each well of the test group. After 48 hours of culture in a CO2 incubator, the supernatant was removed and 0.5 mg / mL of MTT was added at 100 μL per well. After 4 hours, the supernatant was removed, DMSO was added at 150 μL per well, and the absorbance at 570 nm was measured by an enzyme reader. The test was performed three times (three wells) for each sample solution of each concentration in the control group and the test group (n=3).

[0120] The results are shown in Fig.11The cell survival rate of the test group was calculated by setting the value of the control (blank control) group to 100. It was found that Ceratonia siliqua B, the compound of the present invention, had no cytotoxicity to HepG2 cells at a concentration of 1 to 100 μM.

[0121] (d) Determination of ROS in HepG2 cells

[0122] A HepG2 cell suspension at a concentration of 1×105 cells / mL was prepared, and 100 μL of the cell suspension was inoculated into each well of a 96-well sample plate, and cultured in a CO2 incubator for 24 hours. The cells were divided into a blank control group, a positive control group, and a test group. 100 μL of DMEM medium was added to the blank control group, 100 μL of a solution obtained by dissolving L-ascorbic acid in DMEM medium was added to the positive control group (final concentration of L-ascorbic acid: 100 μM), and 100 μL of a solution obtained by appropriately diluting Ceratonia siliqua B in DMEM medium was added to the test group in different concentrations (final concentrations: 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM). After culturing in a CO2 incubator for 24 hours, the culture solution was removed from each well, 100 μL of serum-free medium was added, and the cells were washed, and the above operation was repeated 3 times. Next, 10 μM DCFH-DA (2′, 7′-dichlorofluorescein diacetate) was added to each well at 100 μL per well, and after culturing in a CO2 incubator for 60 minutes, DCFH-DA was removed from each well, and 100 μL of serum-free medium was added and washed, and the above operation was repeated 3 times. Finally, 400 μM hydrogen peroxide (H202) diluted with serum-free medium was added to each well at 100 μL per well and cultured for 90 minutes. Thereafter, the culture medium in the well was removed, 100 μL of PBS was added, and the fluorescence intensity at 535 nm was measured with an excitation wavelength of 485 nm. The fluorescence intensity of the blank control group was set to 1.0 (base), and the ROS generation of the positive control group and the test group was calculated. For each concentration of sample solution in the blank control group, the positive control group, and the test group, 3 tests (3 wells) were performed (n=3).

[0123] The results are shown in Fig.12 Vit.C represents 100 μM L-ascorbic acid as a positive control. It is shown that Ceratonia siliqua B, a compound of the present invention, inhibits the increase of ROS induced by hydrogen peroxide in HepG2 cells in a concentration-dependent manner. This shows that Ceratonia siliqua B has an antioxidant activity that reduces the amount of ROS generated in living cells.

[0124] [Example 6]

[0125] 6. Melanin production inhibitory effect of Ceratonia siliqua B

[0126] Using the Ceratonia siliqua B obtained in Example 2, the melanin production inhibitory effect of Ceratonia siliqua B was examined.

[0127] (a) Cell culture conditions

[0128] In this experiment, B16 melanoma cells were used. B16 melanoma cells are a cell line derived from a melanoma tumor of mouse skin and have the ability to produce melanin. B16 melanoma cells were cultured in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 μM / mL streptomycin) at 37°C, 5% CO2, and saturated humidity.

[0129] (b) Preparation of sample solution

[0130] Ceratonia siliqua B was diluted with DMEM medium to obtain sample solutions of different concentrations of 1 μM, 5 μM, 10 μM, 50 μM and 100 μM.

[0131] (c) Cytotoxicity study on B16 melanoma cells

[0132] First, the MTT test was used to confirm whether Ceratonia siliqua B had cytotoxicity to B16 melanoma cells. B16 melanoma cells that had been subcultured for more than 3 generations were treated with trypsin to prepare a single cell suspension at a concentration of 3×104 cells / mL. The cell suspension was inoculated into each well of a 96-well sample plate at 100 μL per well and cultured overnight in a CO2 incubator. The supernatant was removed from each well and divided into a control (blank control) group and a test group. 200 μL of DMEM culture medium was added to the control group, and 200 μL of the sample solution (b) was added to each well of the test group. After culturing in a CO2 incubator for 48 hours, the supernatant was removed, and 0.5 mg / mL of MTT was added at 100 μL per well. After 4 hours, the supernatant was removed, DMSO was added at 150 μL per well, and the absorbance at 570 nm was measured by an enzyme reader. The test was performed three times (three wells) for each sample solution of each concentration in the control group and the test group (n=3).

[0133] The results are shown in Fig.13 The cell survival rate of the test group was calculated by setting the value of the control (blank control) group to 100. It was found that Ceratonia siliqua B, a compound of the present invention, had no cytotoxicity to B16 melanoma cells at a concentration of 1 to 100 μM.

[0134] (d) Determination of melanin production in B16 melanoma cells

[0135] To prepare a single cell suspension of B16 melanoma cells, 4 × 10 4 B16 melanoma cells were inoculated into each well of a 12-well sample plate. After culturing overnight in a CO2 incubator, the supernatant was removed and the cells were divided into a blank control group, a positive control group, and a test group. 1 mL of DMEM culture medium was added to the blank control group, and 1 mL of a solution obtained by dissolving L-ascorbic acid in DMEM culture medium at different concentrations (L-ascorbic acid concentration: 0.1 μM, 5 μM, 10 μM) was added to the positive control group. In the test group, 1 mL of the sample solution (b) was added to each well. After culturing for 48 hours, the supernatant was removed from each well, washed twice with PBS buffer, and then treated with trypsin, and 1.2 mL of PBS buffer was added to prepare a single cell suspension. 200 μL of the cell suspension was taken and the protein content was determined. In addition, the remaining cell suspension was centrifuged at 3000 rpm for 5 minutes to remove the supernatant, and then 250 μL of 1 M NaOH solution containing 1% DMSO was added and dissolved in a water bath heated to 80°C for 30 minutes. 200 μL of the dissolved solution was taken and the absorbance at 405 nm was measured (A test group, A 阳性对照组 , A 空白对照组 ). For each concentration of the sample solution in the blank control group, positive control group and test group, the test was performed 3 times (3 wells) each (n=3).

[0136] The melanin production inhibition rate (%) in cells was calculated by the following formula.

[0137] Melanin production inhibition rate of the test group (%) = 1-[(A 聪 Protein content of group / experimental group) / (A 空白对照组 / protein content of blank control group)].

[0138] Melanin production inhibition rate of the positive control group (%) = 1-[(A 阳性对照组 / protein content of positive control group) / (A 空白对照组 / protein content of blank control group)].

[0139] Here, A 试验组 is the absorbance of the test group, A 阳性对照组 is the absorbance of the positive control group, A 空白对照组 is the absorbance of the blank control group.

[0140] The results are shown in Table 1 below. L-ascorbic acid, which is a positive control, showed a significant inhibitory effect on melanin production at a concentration of 10 μM (P < 0.01), and the inhibition rate was 32.8%. On the other hand, for Ceratonia siliqua B, which is a compound of the present invention, a significant inhibitory effect on melanin production in B16 melanoma cells was observed at a concentration of 10 to 100 μM (P < 0.05). The melanin production inhibitory effect of Ceratonia siliqua B increased in proportion to its concentration, and at a concentration of 100 μM, a melanin production inhibitory effect of more than 30% was shown.

[0141] [Table 1]

[0142]

[0143] ( n=3)

[0144] **: P<0.01.*: P<0.05

[0145] The present invention is not limited to the above-described embodiments or examples, and various forms in which the design is modified within the scope of the gist of the invention described in the scope of claims are also included in the technical scope.

[0146] Industrial Applicability

[0147] The novel phenylpropanoid compounds of the present invention can be used as whitening agents, melanin production inhibitors, antioxidants, skin external preparations and cosmetics, and can be widely used in the medical and beauty fields.

Claims

1. A compound or a salt thereof, wherein the compound is represented by the following formula (I): 。 2. A whitening agent comprising a compound represented by the following formula (I) or a salt thereof: 。 3. The whitening agent according to claim 2, wherein The concentration of the compound represented by the formula (I) or a salt thereof is 0.001 mM to 1 mM.

4. A melanin production inhibitor, comprising a compound represented by the following formula (I) or a salt thereof: 。 5. An antioxidant comprising a compound represented by the following formula (I) or a salt thereof: 。 The agent according to any one of claims 2 to 5, which is an external preparation for skin.

7. The agent according to any one of claims 2 to 5, which is a cosmetic.

8. A skin whitening composition comprising a compound represented by the following formula (I) or a salt thereof: 。

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