KR102700435B1 - Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same - Google Patents

Abstract

The present invention relates to a novel compound derived from the leaves of the genus Lepidium, a composition having tyrosinase activity inhibition ability including the compound, and a composition having tyrosinase activity inhibition ability including an extract of the genus Lepidium leaf or a fraction thereof. The novel compound derived from the leaves of the genus Lepidium according to one aspect exhibits low cytotoxicity and has tyrosinase inhibition activity, and therefore can be safely used in foods, medicines, cosmetics, etc. having tyrosinase inhibition functionality. The composition having tyrosinase activity inhibition ability according to another aspect can improve diseases caused by tyrosinase or be used for skin beauty purposes by including the novel compound derived from the leaves of the genus Lepidium, the extract of the genus Lepidium leaf, or a fraction thereof.

Description

{Compound extracspted from the leave of Viburnum erosum Thunb and tyrosinase activity inhibitory ability composition comprising the same}

The present invention relates to a chemical derived from the leaves of the lesser pheasant tree, a composition having tyrosinase activity inhibition ability comprising the compound, and a composition having tyrosinase activity inhibition ability comprising an extract of the lesser pheasant tree leaves or a fraction thereof.

Melanin is a black-brown pigment produced by the oxidation polymerization of phenol compounds in the animal body. It determines the color of human skin, hair, and eyes, and protects the skin from damage caused by ultraviolet rays.

However, if melanin is produced excessively, it can cause skin pigmentation, freckles, and blemishes. Tyrosinase is an enzyme involved in melanin production, and it sequentially oxidizes L-tyrosine in melanocytes to produce melanin.

Tyrosinase inhibitors can be developed as skin whitening agents in the cosmetics industry, and are considered key elements for suppressing or treating hyperpigmentation in skin cancer, blemishes, freckles, etc. Representative tyrosinase inhibitors found in natural products include arbutin, kojic acid, and niacinamide.

Viburnum erosum Thunb. is a deciduous broadleaf shrub belonging to the Viburnum family in the Viburnum order and is native to East Asian countries, Korea, China, and Japan. Neuroprotective and anti-inflammatory lignans have been reported in the stem of Viburnum erosum, but the various chemical constituents of Viburnum erosum have not been extensively studied.

Based on the technical background described above, one embodiment of the present invention provides six novel compounds extracted from the leaves of the lesser pheasant tree.

As another example, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by tyrosinase activity, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

As another embodiment, a health functional food composition for preventing or improving a disease caused by tyrosinase is provided, which contains the compound as an effective ingredient.

As another embodiment, a cosmetic composition for improving skin beauty is provided, which comprises the compound as an effective ingredient.

In another embodiment, a pharmaceutical composition for preventing or treating a disease caused by tyrosinase is provided, comprising an extract of the leaves of the lesser pheasant tree or a fraction thereof as an active ingredient.

In another embodiment, a health functional food composition for preventing or improving a disease caused by tyrosinase is provided, which contains an extract of the leaves of the lesser pheasant tree or a fraction thereof as an active ingredient.

As another embodiment, a cosmetic composition for improving skin beauty is provided, which comprises a pheasant tree leaf extract or a fraction thereof as an active ingredient.

One embodiment of the present invention provides a compound represented by the following chemical formulas 1 to 9, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a hydrate thereof, or a solvate thereof.

[chemical formula]

The term "pharmaceutically acceptable" means that it is or has been approved by a governmental or equivalent regulatory agency for use in animals, and more specifically in humans, by avoiding significant toxic effects when used at usual pharmaceutical doses, or is listed in a pharmacopoeia or recognized as being otherwise generally available.

The term "pharmaceutically acceptable salt" means a salt which is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. The salt may include, but is not limited to, hydrochloride salts, sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, and the like.

The term "isomer" may include optical isomers (e.g., essentially pure enantiomers, essentially pure diastereomers, or mixtures thereof), as well as conformation isomers (i.e., isomers which differ only in the angles of one or more chemical bonds), position isomers (particularly tautomers) or geometric isomers (e.g., cis-trans isomers).

The term "essentially pure", when used in reference to, for example, enantiomers or diastereoisomers, can mean that a particular compound is present in an amount of at least about 90% (w/w), preferably at least about 95%, more preferably at least about 97% or at least about 98%, even more preferably at least about 99%, and even more preferably at least about 99.5% (w/w) of the enantiomer or diastereoisomer.

The term “hydrate” refers to a compound that has water bound to it, and can refer to a broad concept that includes inclusion compounds where there is no chemical bond between the water and the compound.

The term “solvate” can mean a higher-order compound formed between molecules or ions of a solute and molecules or ions of a solvent.

The above compound may be extracted from the leaves of the lesser pheasant tree. The above compound may be extracted or isolated from an extract of the lesser pheasant tree leaves or a fraction thereof.

Viburnum erosum Thunb. is a deciduous broadleaf shrub belonging to the Viburnum family in the Viburnum order, native to East Asian countries, Korea, China, and Japan. The leaves are opposite, ovate, and have hairy petioles and pointed stipules.

The above compound may have the ability to inhibit tyrosinase activity.

The term "activity inhibition ability" may mean the ability to inhibit an increase in the level of expression of a gene or protein, or to inhibit the activation of a gene or protein. Specifically, the compound may inhibit or inhibit tyrosinase activity.

Another aspect provides a composition having tyrosinase activity inhibition ability comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The above tyrosinase activity-inhibiting composition can be used for various purposes and applications requiring tyrosinase activity-inhibiting ability, and specifically, can be used as a functional material capable of imparting tyrosinase activity-inhibiting ability to products applied in various industrial fields such as pharmaceuticals, cosmetics, food, and animal feed.

It can also be used as a material for pharmaceutical preservatives, cosmetic preservatives, food preservatives, pharmaceutical additives, cosmetic additives, food additives, and feed additives. Accordingly, the composition having tyrosinase inhibition activity can be a cosmetic, food, or pharmaceutical composition.

Another aspect provides a pharmaceutical composition for preventing or treating a disease caused by tyrosinase, comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In the above pharmaceutical composition, the compound and the pharmaceutically acceptable salt thereof are as described above.

The term "prevention" may include inhibiting the occurrence of a disease. The term "treatment" may include inhibiting, alleviating, or eliminating the development of a disease.

Another aspect provides a health functional food composition for preventing or improving a disease caused by tyrosinase, comprising the compound as an effective ingredient.

In the above health functional food composition, the disease caused by the compound and tyrosinase and prevention thereof are as described above.

The term “improvement” may mean any action that reduces at least the severity of a symptom, for example, a parameter related to the alleviation or treatment of a condition.

Another aspect provides a cosmetic composition for improving skin beauty, comprising the compound as an effective ingredient.

In the above cosmetic composition, the compounds are as described above.

The above skin beauty improvement may be wrinkle improvement or skin whitening.

Another aspect provides a composition having tyrosinase inhibition activity comprising a deciduous tree leaf extract or a fraction thereof as an active ingredient. The composition having tyrosinase inhibition activity can be used for various purposes and applications requiring tyrosinase inhibition activity, and specifically, it can be used as a functional material capable of imparting tyrosinase inhibition activity to articles applied in various industrial fields such as pharmaceuticals, cosmetics, foods, and animal feeds. In addition, it can be used as a material for pharmaceutical preservatives, cosmetic preservatives, food preservatives, pharmaceutical additives, cosmetic additives, food additives, and feed additives. Therefore, the composition having tyrosinase inhibition activity can be a cosmetic, a food, or a pharmaceutical composition.

Another aspect provides a pharmaceutical composition for preventing or treating a disease caused by tyrosinase, comprising an extract of the leaves of the lesser pheasant tree or a fraction thereof as an active ingredient.

In the above pharmaceutical composition, the disease, prevention, and treatment caused by tyrosinase are as described above.

The above-mentioned pheasant tree leaf extract or a fraction thereof may contain the above-mentioned compound. For example, it may contain one or more of the above-mentioned compounds 1 to 6. The description of the above-mentioned compounds 1 to 6 is as described above.

The above-mentioned deciduous tree leaves may be whole or part thereof. The deciduous tree leaves used for extraction may be whole or part thereof, ground or chopped, or suitably dried.

The above extract may be extracted by a method conventional in the art, such as heated extraction, pressurized extraction, ultrasonic extraction, hot water extraction, reflux cooling extraction, subcritical extraction, or supercritical extraction.

A composition according to one specific example may contain a fraction of the extract of the leaves of the above-mentioned lesser pheasant tree as an effective ingredient.

The term "fraction" refers to a material in which the extract of the above-mentioned lesser pheasant leaf is divided into its components, i.e., a fractionated material. The fraction may be obtained by solvent fractionation. The solvent fractionation may be mixing the lesser pheasant leaf extract with a solvent and separating the material present in the solvent. The fraction may be

The pharmaceutical composition may contain the extract or a fraction thereof in an effective amount or as an effective ingredient. The effective amount may be appropriately selected depending on the individual. It may be determined based on factors including the severity of the disease or condition, the age, weight, health, sex of the individual, the sensitivity of the individual to the extract, the time of administration, the route of administration and the excretion rate, the period of administration, other compositions combined or used simultaneously with the composition, and other factors well known in the field of physiology or medicine.

The above pharmaceutical composition may further comprise a pharmaceutically acceptable excipient or carrier. The description of the pharmaceutically acceptable excipient or carrier is as described above.

Another aspect provides a health functional food composition for preventing or improving a disease caused by tyrosinase, comprising an extract of the leaves of the lesser pheasant tree or a fraction thereof as an active ingredient.

In the above health functional food composition, the disease, prevention, and improvement caused by the extract, fraction, and tyrosinase of the leaves of the lesser pheasant tree are as described above.

Another aspect provides a cosmetic composition for improving skin beauty, comprising an extract of the leaves of the lesser pheasant tree or a fraction thereof as an active ingredient.

In the above cosmetic composition, the extract and fraction of the pheasant tree leaves and the skin beauty improvement properties are as described above.

Another aspect provides a method for preventing, ameliorating or treating a disease caused by tyrosinase in a subject, comprising the step of administering to the subject the pharmaceutical composition. The pharmaceutical composition, the disease caused by tyrosinase, the prevention, the amelioration and the treatment are as described above.

The terms "administering," "introducing," and "implanting" are used interchangeably and may refer to placement of a composition according to one embodiment into a subject by a method or route which results in at least partial localization of the composition to a desired site in an embodiment. Administration may be by any suitable route which delivers at least a portion of the compound, extract, or fraction component thereof of the composition according to one embodiment to a desired location in a surviving subject.

The novel compound derived from the leaves of the lesser pheasant tree according to the present invention exhibits low cytotoxicity and has tyrosinase inhibitory activity, and therefore can be safely used in foods, pharmaceuticals, cosmetics, etc. having tyrosinase inhibitory activity functionality.

A composition having tyrosinase inhibition activity according to another aspect can be used to improve diseases caused by tyrosinase or for skin cosmetic purposes by including a novel compound derived from the leaves of the lesser pheasant tree or an extract of the lesser pheasant tree leaves or a fraction thereof.

Figure 1 is a drawing showing the extraction and fractionation of the lesser pheasant tree according to one embodiment of the present invention.
Figure 2 is a drawing showing fractions from the acetylacetate shown in Figure 1.
Figure 3 is a drawing showing the chemical structures of compounds 1 to 9 of the present invention.
Figure 4 is a graph showing the results of comparing the cytotoxicity of fractions extracted from the leaves of the lesser pheasant tree.
Figure 5 is a graph showing the results of comparing the tyrosinase activity of fractions extracted from the leaves of the lesser pheasant tree.
Figure 6 is a graph showing the results of comparing the tyrosinase activities of the compounds shown in Figure 3.
Figure 7 is a table showing the content of compound 9 shown in Figure 3.
Figure 8 is a graph showing the chromatography results of compound 9 shown in Figure 7.

Hereinafter, with reference to the attached drawings, embodiments of the present invention will be described in detail so that a person having ordinary skill in the art to which the present invention pertains can easily implement the present invention.

Extraction and isolation of compounds 1 to 9 from the leaves of the lesser pheasant tree

(1) Extraction and fractionation

Referring to Figures 1 to 3, 2 kg of dried leaves of the lesser deciduous tree (V. erosum) were extracted three times for 3 hours with 80% MeOH. The extract was filtered and concentrated in a reduced pressure evaporator to obtain 356.0 g of the extract. The extract was suspended in distilled water to obtain fractions in the following order: n-Hexane (0.5 g), EtOAc (66.4 g), n-BuOH (123.7 g), and water-soluble (165.0 g).

Of the EtOAc fraction (66.4 g) of the leaves of the lesser deciduous tree, 23.0 g was separated using MPLC and RediSep® Rf RP-C18 column under the solvent condition of MeOH: H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into eight fractions (VEE1 to VEE8). Among them, compounds 1-8 were obtained from VEE2 and compound 9 was obtained from VEE3.

For compound 1, the VEE2 fraction was separated using MPLC and a silica gel column under the solvent condition of DCM:MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH) and divided into nine subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under the solvent condition of MeOH:H2O (6:4→MeOH) and divided into 11 subfractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column with solvent condition of MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into 10 subfractions (VEE2-6-4-1 ~ VEE2-6-4-10). Finally, VEE2-6-4-7 was separated using MPLC and YMC-Actus Triart C18 column with solvent condition of MeOH : H2O (65:35). The purity of 7 subfractions (VEE2-6-4-7-1 ~ VEE2-6-4-7-7) was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 1 (11.7 mg) was obtained from VEE2-6-4-7-5.

For compound 2, VEE2-6 was separated using a Sephadex LH-20 column with a solvent condition of MeOH : H2O (6:4 → MeOH) and divided into 11 fractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and a RediSep® Rf RP-C18 column with a solvent condition of MeOH : H2O (5:5 → 6:4 → 7:3 → 8:2 → 9:1 → MeOH) and divided into 10 fractions (VEE2-6-4-1 to VEE2-6-4-10). Among them, VEE2-6-4-9 was separated using a silica gel column with a solvent condition of DCM : MeOH (9:1 → 8:2 → MeOH) and divided into 9 fractions (VEE2-6-4-9-1 to VEE2-6-4-9-9). Finally, VEE2-6-4-9-5 was purified using preparative HPLC and RP-C18 column with a solvent condition of MeOH: H2O (7:3). The purity was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 2 (0.9 mg) was obtained from VEE2-6-4-9-5-1.

For compound 3, the VEE2 fraction was separated using MPLC and a silica gel column under the solvent condition of DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH) and divided into nine subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under the solvent condition of MeOH : H2O (6:4→MeOH) and divided into 11 subfractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column with solvent condition of MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into 10 subfractions (VEE2-6-4-1 ~ VEE2-6-4-10). Finally, VEE2-6-4-7 was separated using MPLC and YMC-Actus Triart C18 column with solvent condition of MeOH : H2O (65:35). The purity of 7 subfractions (VEE2-6-4-7-1 ~ VEE2-6-4-7-7) was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 3 (19.4 mg) was obtained from VEE2-6-4-7-2.

For compound 4, the VEE2 fraction was separated using MPLC and a silica gel column under the solvent condition of DCM:MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH) and divided into nine subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under the solvent condition of MeOH:H2O (6:4→MeOH) and divided into 11 subfractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column with solvent condition of MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into 10 subfractions (VEE2-6-4-1 ~ VEE2-6-4-10). Finally, VEE2-6-4-7 was separated using MPLC and YMC-Actus Triart C18 column with solvent condition of MeOH : H2O (65:35). The purity of 7 subfractions (VEE2-6-4-7-1 ~ VEE2-6-4-7-7) was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 4 (11.1 mg) was obtained from VEE2-6-4-7-3.

For compound 5, the VEE2 fraction was separated using MPLC and a silica gel column under the solvent condition of DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH) and divided into nine subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column under the solvent condition of MeOH : H2O (6:4→MeOH) and divided into 11 subfractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column with solvent condition of MeOH:H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into 10 subfractions (VEE2-6-4-1 ~ VEE2-6-4-10). Finally, VEE2-6-4-2 was purified using preparative HPLC and RP-C18 column with solvent condition of MeOH:H2O (35:65). The purity of two subfractions (VEE2-6-4-2-1, VEE2-6-4-2-2) was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 5 (6.3 mg) was obtained from VEE2-6-4-2-1.

For compound 6, the VEE2 fraction was separated using MPLC and a silica gel column with the solvent condition of DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH) and divided into nine subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column with the solvent condition of MeOH : H2O (6:4→MeOH) and divided into 11 subfractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and RediSep® Rf RP-C18 column with solvent condition of MeOH:H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into 10 subfractions (VEE2-6-4-1 ~ VEE2-6-4-10). Finally, VEE2-6-4-2 was purified using preparative HPLC and RP-C18 column with solvent condition of MeOH:H2O (35:65). The purity of two subfractions (VEE2-6-4-2-1, VEE2-6-4-2-2) was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 6 (5.2 mg) was obtained from VEE2-6-4-2-2.

For compound 7, the VEE2 fraction was separated using MPLC and a silica gel column with the solvent condition of DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH) and divided into nine subfractions (VEE2-1 to VEE2-9). Among them, VEE2-6 was separated using a Sephadex LH-20 column with the solvent condition of MeOH : H2O (6:4→MeOH) and divided into 11 subfractions (VEE2-6-1 to VEE2-6-11). VEE2-6-4 was separated using MPLC and a RediSep® Rf RP-C18 column with a solvent condition of MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and fractionated into ten fractions (VEE2-6-4-1 ~ VEE2-6-4-10). VEE2-6-4-4 was again separated using MPLC and a RediSep® Rf RP-C18 column with a solvent condition of MeOH : H2O (4:6→5:5→6:4→MeOH) and fractionated into three fractions (VEE2-6-4-4-1 ~ VEE2-6-4-4-3). Finally, VEE2-6-4-4-2 was separated using MPLC and a YMC-Actus Triart C18 column with a solvent condition of MeOH : H2O (4:6). The purity of three fractions (VEE2-6-4-4-2-1 to VEE2-6-4-4-2-3) was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 7 (114 mg) was obtained from VEE2-6-4-4-2-3.

For compound 8, the VEE2 fraction was separated using MPLC and a silica gel column with the solvent condition of DCM : MeOH (9:1→8:2→7:3→6:4→5:5→3:7→MeOH) and divided into nine subfractions (VEE2-1 to VEE2-9). Among them, VEE2-2 was separated using MPLC and a RediSep® Rf RP-C18 column with the solvent condition of MeOH : H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into seven subfractions (VEE2-2-1 to VEE2-2-7). Finally, VEE2-2-5 was purified using preparative HPLC and RP-C18 column with the solvent condition of MeOH : H2O (1:1). The purity of four fractions (VEE2-2-5-1 to VEE2-2-5-4) was confirmed using Thin Layer Chromatography (TLC) identification test, and a single compound 8 (7.3 mg) was obtained from VEE2-2-5-2.

For compound 8, 23.0 g of the EtOAc fraction (66.4 g) of the leaves of the lesser pheasant tree was separated using MPLC and a RediSep® Rf RP-C18 column under the solvent condition of MeOH: H2O (5:5→6:4→7:3→8:2→9:1→MeOH) and divided into eight fractions (VEE1 to VEE8).

For compound 9, a small fraction VEE3-C was obtained by crystallization from VEE3 among eight fractions (VEE1 to VEE8). The purity was confirmed using a thin layer chromatography (TLC) identification test, and a single compound 9 (198 mg) was obtained from VEE3-C.

(2) Structure of a compound isolated from the leaves of the lesser pheasant tree (V. erosum)

NMR and MS analyses were performed on compounds 1 to 9. H was obtained at 600 MHz and C at 150 MHz on a Bruker Avance II 600 (Bruker, Germany) spectrometer using pyridine-d5 as a solvent. The NMR results of compounds 1 to 4 are shown in Table 1 below, and the NMR results of compounds 5 to 7 are shown in Table 2 below.

[Table 1]

[Table 2]

Compound 1 was obtained as a white amorphous powder and its molecular formula was determined to be C22H28O8 by HRESI-MS (m/z 419.1690 [M-H]- calcd for 419.1706).

The 1D NMR and HSQC data of compound 1 show signals expected to be typical β-D-glucosyl groups: δH/δC 4.71 (1H, d, J = 7.4 Hz, H-1) / 103.6 (C-1), 4.51 (1H, dd, J = 2.1, 11.8 Hz, H-6) / 65.1 (C-6), 4.24 (1H, dd, J = 7.2, 11.8 Hz, H-6) / 65.1 (C-6), 3.62 (1H, ddd, J = 2.1, 7.2, 9.5 Hz, H-5) / 75.4 (C-5), 3.43 (H, m, H-3) / 77.9 (C-3), 3.43 (H, m, H-2) / 74.9 (C-2), 3.36 (1H, dd, J = 6.5, 11.7 Hz, H-4) / 71.9 (C-4) signals were confirmed, and the presence of a hydroquinone group with two hydroxyl groups at the para position was confirmed through the signals of 153.9 (C-6′), 152.3 (C-1′), 6.93 (2H, d, J = 8.9 Hz, H-2′, 6′) / 119.5 (C-4′, 6′), 6.66 (2H, d, J = 8.9 Hz, H-3′, 5′) / 116.6 (C-3′, 5′). Based on the correlation peaks of δH 4.71 (1H, d, J = 7.4 Hz, H-1) of the β-D-glucosyl group and δC 152.3 (C-1′) of the hydroquinone group in the HMBC data, it was predicted that compound 1 was a derivative of arbutin.

Furthermore, the 1D NMR and HSQC data of compound 1 showed two olefinic groups: δH/δC 6.82 (1H, td, J = 1.4, 7.3, 7.3 Hz, H-3″) / 143.7 (C-3″), 6.40 (1H, dd, J = 10.9, 17.7 Hz, H-7″) / 139.7 (C-7″), two exomethylene: 5.28 (1H, d, J = 14.3 Hz, H-8″), 5.09 (1H, d, J = 14.3 Hz, H-8″) / 113.9 (C-8″), 5.03 (2H, d, J = 16.2 Hz, H-7″) / 116.9 (C-7″), two The signals of methylene: 2.43 (2H, dd, J = 7.3, 14.5 Hz, H-4″) / 28.4 (C-4″), 2.37 (2H, dd, J = 6.8, 7.9 Hz, H-5″) / 31.1 (C-5″), one methyl group: 1.84 (3H, s, H-9″) / 12.6 (C-9″) were identified. In the 13C NMR data, the signals of one carboxyl group: δC 169.4 (C-1″), two olefinic quaternary carbons: 146.76 (C-6″) and 128.9 (C-2″) were identified. These signals showed correlation peaks between H-3″ and H-4″, H-4″ and H-5″, and H-7″ and H-8″ in the COSY data, and between H-3″ and C-1″, C-9″, between H-4″ and C-2″, C-3″, C-6″, between H-5″ and C-3″, C-6″, C-7″, C-10″, between H-7″ and C-5″, C-6″, C-10″, between H-8″ and C-6″, C-7″, between H-9″ and C-1″, C-2″, and between H-10″ and C-5″, C-7″ in the HMBC data. Based on this, it was predicted that compound 1 contains a 2-methyl-6-methylene-2,7-octadienoyl group.

Finally, compound 1 was determined to be 6-O-(2-methyl-6-methylene-2,7-octadienoyl)-arbutin by identifying the correlation peak between H-6 of the arbutin molecule and C-1″ of the monoterpene molecule in the HMBC data.

Compound 2 was obtained as a white amorphous powder and its molecular formula was determined to be C22H30O9 by HRESI-MS (m/z 437.1834 [M-H]- calcd for 437.1812).

1D and 2D NMR data showed similar patterns to compound 1, except for the monoterpene moiety attached to C-6 of the glucopyranosyl group. 1D NMR and HSQC data for two olefinic groups: δH/δC 6.79 (1H, td, J = 1.4, 7.3 Hz, H-3″) / 143.7 (C-3″), 5.39 (1H, t, J = 6.1 Hz, H-7″) / 125.7 (C-7″), one oxymethylene: 4.08 (2H, d, J = 6.7 Hz, H-8″) / 59.4 (C-8″), two methylenes: 2.36 (2H, dd, J = 7.4, 14.9 Hz, H-4″) / 28.0 (C-4″), 2.16 (2H, t, J = 7.6, 7.6 Hz, H-5″) / 39.1 (C-5″), two methyl groups 1.85 (3H, s, H-9″) / 12.5 (C-9″), 1.70 (3H, s, H-10″) / 16.2 (C-10″) signals were identified. In the 13C NMR data, one carboxyl group: δC 169.4 (C-1″), two olefinic quaternary carbons: 138.5 (C-6″), 128.9 (C-2″) signals were identified. These signals showed correlation peaks between H-3″ and H-4″, H-4″ and H-5″, and H-7″ and H-8″ in the COSY data, and between H-5″ and C-3″, C-6″, C-7″, C-10″, between H-7″ and C-5″, C-10″, between H-8″ and C-6″, C-7″, and between H-10″ and C-6″, C-7″ in the HMBC data. Based on this, it was predicted that compound 2 contains a 2,6-dimethyl-8-hydroxy-2,6-octadienoyl group.

Finally, compound 2 was determined to be 6-O-(2,6-dimethyl-8-hydroxy-2,6-octadienoyl)-arbutin by confirming the correlation peak between H-6 of the arbutin molecule and C-1″ of the monoterpene molecule in the HMBC data.

Compound 3 was obtained as a white amorphous powder and its molecular formula was determined to be C23H32O9 by HRESI-MS (m/z 451.1975[M-H]- calcd for 451.1968).

1D and 2D NMR data showed similar patterns to compound 2 except that the hydroxyl group at C-8″ was replaced by a methoxy group. There were significant changes in the chemical shift values of C-6″ (δC 140.8, +2.3 ppm), C-7″ (δC 122.6, -3.1 ppm), and C-8″ (δC 69.7, +10.3 ppm) compared to compound 2. The location of the methoxy group at C-8″ was confirmed by confirming the correlation peaks at δH 3.28 (3H, s, H-11″) and δC 69.7 (C-8″) in the HMBC data.

Based on this, it was predicted that compound 3 contains a 2,6-dimethyl-8-methoxy-2,6-octadienoyl group. Therefore, compound 3 was determined to be 6-O-(2,6-dimethyl-8-methoxy-2,6-octadienoyl)-arbutin.

Compound 4 was obtained as a white amorphous powder and its molecular formula was determined to be C23H32O9 by HRESI-MS (m/z 451.1975 [M-H] - calcd for 451.1968). The 1D and 2D NMR data showed similar patterns to compound 7 except that the hydroxyl group at C-6″ was replaced by a methoxy group. Compared to compound 7, there were significant changes in the chemical shift values of C-5″ (δC 39.2, -2.5 ppm), C-6″ (δC 78.5, +4.8 ppm), C-7″ (δC 144.3, -1.6 ppm), C-8″ (δC 115.8, +3.3 ppm) and C-10″ (δC 21.9, -6.0 ppm). The methoxy group was confirmed to be located at C-6″ by confirming the correlation peaks of δH 3.17 (3H, s, H-11″) and δC 78.5 (C-6″) in the HMBC data. The absolute structure of the methoxy group in compound 4 was confirmed by acid hydrolysis of compounds 4 and 7. Their acid hydrolysis products had the same Rf values in TLC analysis and their H NMR spectra were consistent. Based on this, it was predicted that compound 4 contained a (6S)-2,6-methyl-6-methoxy-2,7-octadienoyl group. Therefore, compound 4 was determined to be 6-O-[(6S)-2,6-dimethyl-6-methoxy-2,7-octadienoyl]-arbutin.

Compound 5 was obtained as a white amorphous powder and its molecular formula was determined to be C22H32O11 by HRESI-MS (m/z 471.1868[M-H]- calcd for 471.1866). 1D and 2D NMR data showed similar patterns to compound 7 except for the terminal part of the monoterpene. Compared to compound 7, there were significant changes in the chemical shift values of C-5″ (δC 38.6, -3.1 ppm), C-7″ (δC 78.2, -67.7 ppm), C-8″ (δC 64.0, -48.5 ppm) and C-10″ (δC 22.1, -5.8 ppm). Unlike compound 7, one oxymethine: δH/δC 3.50 (1H, dd, J = 3.2, 7.9 Hz, H-7″) / 78.2 (C-7″) and one oxymethylene: 3.55 (1H, m, H-8″), 3.80 (1H, m, H-8″) / 64.0 (C-8″) signal were identified in the 1D NMR and HSQC data. The HMBC data showed correlation peaks between H-7″ and C-6″, C-8″, C-10″, and between H-8″ and C-7″. This means that the C-7″, C-8″ double bond of compound 7 was changed to single bond, and a hydroxyl group was bonded at each C-7″, C-8″ site.

Based on this, it was predicted that compound 5 contains a 2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl group. Therefore, compound 5 was determined to be 6-O-(2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl)-arbutin. The absolute structure of the C-7″,C-8″ hydroxyl group of compound 5 has not been elucidated.

Compound 6 was obtained as a white amorphous powder and its molecular formula was determined to be C22H32O11 by HRESI-MS (m/z 471.1868 [M-H] - calcd for 471.1866). The 1D and 2D NMR data showed very similar patterns to compound 5. Except for slight changes in the chemical shift values of C-4″ (δC 24.0, -0.3 ppm), C-5″ (δC 37.9, -0.7 ppm), C-7″ (δC 78.5, -0.3 ppm), and H-8″ (δH 3.75, -0.05 ppm) compared to compound 5, the 1D and 2D NMR spectra were identical. This suggests that compounds 5 and 6 are stereoisomers.

Therefore, compound 6 was determined to be 6-O-(2,6-dimethyl-6,7,8-trihydroxy-2-octenoyl)-arbutin. The absolute structure of the C-7″,C-8″ hydroxyl group of compound 6 was not elucidated.

Compound 7 was obtained as a white amorphous powder and its molecular formula was determined to be C22H30O9 by HRESI-MS (m/z 437.1834 [M-H]- calcd for 437.1812). 1D and 2D NMR data showed similar patterns to compound 1 except for the monoterpene moiety attached to C-6 of the glucopyranosyl group. 1D NMR and HSQC data for two olefinic groups: δH/δC 6.80 (1H, td, J = 1.4, 7.6 Hz, H-3″) / 144.4 (C-3″), 5.93 (1H, ddd, J = 2.9, 10.8, 17.4 Hz, H-7″) / 145.9 (C-7″), one exomethylene: 5.07 (H, d, J = 14.1 Hz, H-8″), 5.23 (H, d, J = 14.1 Hz, H-8″) / 112.48 (C-8″), two methylenes: 2.25 (2H, m, H-4″) / 24.6 (C-4″), 1.60 (2H, m, The signals of two methyl groups: 1.84 (3H, s, H-9″) / 12.4 (C-9″), 1.29 (3H, s, H-10″) / 27.9 (C-10″) were identified. In the 13C NMR data, one carboxyl group: δC 169.4 (C-1″), two olefinic quaternary carbons: 73.7 (C-6″), 128.5 (C-2″) signals were identified. By comparing the above results with the NMR shift values in the reference, compound 7 was determined to be Phlebotrichin, 6-O-[(6S)-2,6-dimethyl-6-hydroxy-2,7-octadienoyl]-arbutin.

Compound 8 was obtained as a brown amorphous powder and its molecular formula was determined to be C20H22O6 by ESI-MS measurement (m/z 357.18[M-H]-). Twenty carbons were identified from 13C NMR data, and non-oxidized furofuran carbons δC 89.2 (C-7), 83.3 (C-7′), 72.0 (C-9), 70.6 (C-9′), 55.7 (C-8), 51.2 (C-8′) appearing in furofuran lignan were confirmed. In the 1H NMR data, the benzene ring signals of the pinoresinol-type lignan were confirmed at δH 6.89 (2H, t, J = 7.9 Hz, H-2, 2′), 6.84 (2H, dd, J = 5.8, 1.7 Hz, H-5, 5′), and 6.81 (2H, dd, J = 8.2, 1.8 Hz, H-6, 6′). Subsequently, the signal 3.85 (6H, s, H-10, 10′) was confirmed, confirming the presence of methoxy groups on both benzene rings of the lignan.

By comparing the above results with the NMR shift values in the reference literature, compound 8 was determined to be (-)-epipinoresinol.

Compound 9 was obtained as a yellow powder and its molecular formula was determined to be C30H18O10 by ESI-MS (m/z 537.18[M-H]-) measurement. Thirty carbons were identified from 13C NMR data, and conjugated carbonyl carbons δC 181.8 (C-4), 182.2 (C-4″) appearing in flavonoids were confirmed. Typical signals expected for flavonoids were confirmed from 1H NMR data at δH 6.16 (1H, d, J = 2.1 Hz, H-6), 6.44 (1H, d, J = 2.1 Hz, H-8). The two doublet peaks are H-6 and H-8 of the A ring of the aromatic ring. Subsequently, the signal at 6.38 (1H, s, H-6″) was confirmed to additionally confirm the presence of the H-6 position of the flavonoid A ring, and it was predicted that compound 9 was a biflavonoid based on the fact that the H-6″ signal was a singlet and the absence of another signal at the H-8 position of the flavonoid A ring. The signals at 6.77 (1H, s, H-3) and 6.81 (1H, s, H-3″) are signals at the H-3 position of the flavonoid C ring, and show singlet peaks because there are no protons at the adjacent positions.

By comparing the above results with the NMR shift values in the reference literature, compound 9 was determined to be amentoflavone.

Experiment 1: Cytotoxicity of the leaf fraction of the lesser pheasant tree

(1) B16F10, HEP-2, PC3 cell line culture

B16F10, HEP-2, and PC3 cancer cell lines were obtained from KCLB (Korean Cell Line Bank, Seoul, Republic of Korea) and RPMI 1640 medium (added 1% Penicillin and 10% FBS) and DMEM medium (added 1% Penicillin and 10% FBS) were used (WELGENE, Republic of Korea). Each cell line was cultured at 37℃ in an incubator with a continuous supply of 5% CO2.

(2) Confirmation of cytotoxicity

B16F10, HEP-2, and PC3 cell lines were diluted to 5.0 x 103 cells/well and seeded in a 96-well plate, and cultured for 24 hours. The cells were treated with a final concentration of 100νg/mL of the deciduous tree leaf fraction dissolved in DMSO, and cultured for 72 hours. The absorbance was confirmed at 540 nm using an MTT assay. DMSO was used as a negative control, and the experiment was repeated three times for each sample.

(3) Statistical processing

To compare the cytotoxicity of the fractions of the leaves of the lesser pheasant tree, DMSO was used as a negative control and the survival rate was set to 100%. The survival rate after sample treatment was calculated as a % value and compared.

(4) Cytotoxicity results of the leaf fraction of the lesser pheasant tree

To confirm the cytotoxicity of EtOAc fractions VEE1~VEE8 of the leaves of the lesser pheasant tree, the sample concentration was 100νg/mL, and B16F10 (skin cancer cells), HEp-2 (laryngeal cancer cells), and PC-3 (prostate cancer cells) were used as cell lines. The cell viability was calculated as 100% through DMSO, which is a negative control group, and the average value was calculated after each fraction sample was treated three times and compared.

Referring to Figure 4, the cell viability in B16F10 skin cancer cells showed a noticeable difference in each fraction. In VEE1~3 and VEE8, the cell viability was 103%~128%, confirming that there was no cytotoxicity. In contrast, in VEE4~7, the cell viability was 19%~22%, indicating strong cytotoxicity.

* In HEp-2, a laryngeal cancer cell line, cell viability showed a similar trend to that of B16F10. In VEE1~2, cell viability was 112% and 100, respectively, indicating no cytotoxicity. In VEE3 and VEE8, cell viability was 86% and 83%, respectively, indicating very weak cytotoxicity. In VEE4~7, cell viability was 15%~36%, indicating strong cytotoxicity.

In PC-3, a prostate cancer cell line, cell viability showed strong cytotoxicity in all fractions except VEE2. In VEE2, cell viability was 85%, indicating very weak cytotoxicity, but in all other fractions, cell viability was 14% to 39%, confirming the presence of strong cytotoxicity.

Among the fractions VEE1~VEE8, the fractions with relatively high polarity, VEE1~VEE3, showed relatively high cell viability, and among them, the VEE2 fraction, which showed high viability in all cell lines, was used for separation. VEE4~VEE7 showed strong cytotoxicity in all cell lines used in the experiment. It is thought that the fractions can be usefully studied for the isolation of active ingredients with anticancer activity.

Experiment 2: Tyrosinase activity inhibition test of the pheasant tree leaf compound

*(1) Test method

The sample was dissolved in ethanol and the concentration was set to confirm the inhibition of tyrosinase activity. 220νM of 0.1 M phosphate buffer (pH 6.5), 20νL of sample solution, and 20νL of mushroom tyrosinase solution (2000 U/mL) were sequentially treated in a 96-well plate, and then incubated in an incubator at 37°C for 10 minutes. 40νL of 1.5 mM tyrosine solution was treated to this solution, and the reaction was performed at 37°C for 15 minutes, and then the absorbance was measured at 490 nm.

(2) Statistical processing

To compare the tyrosinase inhibition activity of the fractions of the leaves of the lesser deciduous tree and compounds 1-9 isolated from the leaves of the lesser deciduous tree, EtOH was used as a negative control and arbutin was used as a positive control. The tyrosinase activity was calculated using the following method.

Tyrosinase activity (%): [(A1-A0)/(B1-B0)] ×100

A0 is the absorbance of the sample before reaction, A1 is the absorbance of the sample after reaction. B0 and B1 are the absorbance of the negative control before and after reaction.

(3) Tyrosinase activity inhibition ability of the pheasant tree leaf compound

The tyrosinase inhibitory activity of EtOAc fractions VEE1~VEE8 of the leaves of the lesser deciduous tree was confirmed at a sample concentration of 0.2 mg/mL. Mushroom tyrosinase was used as the enzyme, L-tyrosine as the substrate, EtOH as the negative control, and arbutin as the positive control. The tyrosinase activity was calculated as 100% through the negative control, and the average value was calculated after repeating each fraction sample three times and comparing them.

Referring to Figure 5, the positive control, arbutin, showed strong tyrosinase inhibition ability by reducing tyrosinase activity to 21% at a concentration of 0.2 mg/mL. VEE1 and VEE2 showed inhibitory abilities similar to that of the positive control, arbutin, with tyrosinase activities of 29% and 28%, respectively. The tyrosinase activity of VEE3 was 52%, confirming that it had inhibitory ability, although its efficacy was weaker than that of the previous fractions.

For fractions VEE4 to VEE8, it was confirmed that there was no inhibition ability or an adverse effect with tyrosinase activity values of 94% to 124%. Among fractions VEE1 to VEE8, relatively high tyrosinase activity inhibition ability was confirmed in fractions VEE1 to VEE3, which were relatively highly polar.

Compounds 1-8 were obtained from the VEE2 fraction, which showed the highest inhibitory ability among these.

(4) Tyrosinase activity inhibition ability of compounds 1-9 isolated from the leaves of the lesser pheasant tree

The tyrosinase inhibitory activity of compounds 1-9 isolated from the leaves of the lesser deciduous tree was confirmed at a sample concentration of 200 μM. Mushroom tyrosinase was used as the enzyme, L-tyrosine as the substrate, EtOH as the negative control, and arbutin as the positive control. The tyrosinase activity was calculated as 100% through the negative control, and the average value was calculated after three repeated treatments of each fraction sample and compared.

Referring to Figure 6, arbutin, a positive control, showed strong tyrosinase inhibition ability by reducing tyrosinase activity by 30% at a concentration of 200 μM. Compounds 1-6 and compound 9 were confirmed to have no inhibition ability with tyrosinase activity values of 91% to 112%.

Compound 7 was confirmed to have a very weak inhibitory ability with 75% of tyrosinase activity. In the case of compound 8, the tyrosinase activity was 27%, showing strong inhibitory ability together with arbutin, which is a positive control. The IC50 value of the tyrosinase activity inhibition ability of compound 8 is 37.9 μM. In the case of compounds 1-7, although they contain an arbutin structure in their molecular structures, they showed that the inhibitory ability was lost due to the monoterpen bound to the C-6 position.

Experiment 3: Comparison of the content of compound 9 in each fraction of the extract of the leaves of the lesser pheasant tree

(1) Preparation of samples for analysis

The extract of the leaves of the lesser pheasant tree (VET) and the n-Hexane (VEH), EtOAc (VEE), n-BuOH (VEB), and water-soluble (VEW) fractions were each freeze-dried, dissolved at a concentration of 10 mg/mL, and filtered through a 0.45 μm syringe filter to prepare samples for HPLC analysis. The solvent used was 100% MeOH.

(2) Preparation of calibration curve samples

A calibration curve was created by selecting five concentrations of compound 9 in the range of 0.02 to 1 mg/mL. 100% MeOH was used as the solvent.

(3) HPLC-UV analysis method

0.1F.A in water (A) and MeOH (B) were used as the HPLC mobile phase for VET, VEH, VEE, VEB, VEW, and compound 9. The analysis was performed under gradient conditions of 5% B solvent, 5% (B) from 0 to 10 min, 20% (B) from 10 to 20 min, 70% (B) from 20 to 30 min, and 70% (B) from 30 to 40 min. (flow rate: 1 mL/min, column temperature: 30℃, wavelength: 260 nm)

(4) Comparison of the content of compound 9 in each fraction of the extract of the leaves of the lesser pheasant tree

Compound 9, which was extracted in large quantities from the EtOAc fraction of the extract of the leaves of the lesser deciduous tree, was compared in its content in the total extract (VET) of the lesser deciduous tree leaves and the n-Hexane (VEH), EtOAc (VEE), n-BuOH (VEB), and water-soluble (VEW) fractions.

Referring to FIGS. 7 and 8, compound 9 was eluted at 38.572 min in the concentration range of MeOH: Water 70:30, and the detection wavelength was 260 nm. Compound 9 showed a content of 7.39 mg/g in VET, 31.48 mg/g in VEE, 4.61 mg/g in VEB, and was not detected in VEH and VEW.

Compound 9 has been studied as a compound with antioxidant, anti-aging, and anti-inflammatory effects, and is known as a representative natural ingredient for wrinkle improvement in the cosmetics industry. It is thought that the pheasant tree leaf extract with a high content of 7.39 mg/g can be used as an excellent skin improvement material.

Although the embodiments of the present invention have been described above, the spirit of the present invention is not limited to the embodiments presented in this specification, and those skilled in the art who understand the spirit of the present invention will be able to easily propose other embodiments by adding, changing, deleting, or adding components within the scope of the same spirit, but this will also be considered to fall within the spirit of the present invention.

Claims (14)

A compound represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Chemical formula 1]
A compound represented by the following chemical formula 2, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Chemical formula 2]
A compound represented by the following chemical formula 3, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Chemical formula 3]
A compound represented by the following chemical formula 4, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof:
[Chemical formula 4]
In any one of the above clauses 1 to 4,
The above compound is a compound extracted from the leaves of Viburnum erosum Thunb. In any one of the above clauses 1 to 4,
The above compound is a compound characterized by having the ability to inhibit tyrosinase activity. A pharmaceutical composition for preventing or treating a disease caused by tyrosinase, comprising a compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient,
A pharmaceutical composition wherein the disease caused by the above tyrosinase is any one selected from the group consisting of hyperpigmentation diseases. A health functional food composition for preventing or improving a disease caused by tyrosinase, comprising any one of the compounds of claims 1 to 4 as an effective ingredient,
A health functional food composition, wherein the disease caused by the above tyrosinase is any one selected from the group consisting of hyperpigmentation diseases. A cosmetic composition for improving skin beauty comprising any one of the compounds of claims 1 to 4 as an effective ingredient,
The above skin beauty improvement is a cosmetic composition that is a skin wrinkle improvement or whitening agent.
delete delete A pharmaceutical composition for preventing or treating a disease caused by tyrosinase, comprising a compound represented by the following chemical formula 5, or a pharmaceutically acceptable salt thereof, as an active ingredient:
A pharmaceutical composition wherein the disease caused by the above tyrosinase is any one selected from the group consisting of hyperpigmentation diseases.
[Chemical Formula 5]
A health functional food composition for preventing or improving a disease caused by tyrosinase, comprising a compound represented by the following chemical formula 5 as an effective ingredient:
A health functional food composition, wherein the disease caused by the above tyrosinase is any one selected from the group consisting of hyperpigmentation diseases.
[Chemical Formula 5]

A cosmetic composition for improving skin beauty, comprising a compound represented by the following chemical formula 5 as an effective ingredient:
The above skin beauty improvement is a cosmetic composition that is a skin wrinkle improvement or whitening agent.
[Chemical Formula 5]