KR20130050751A - Novel compound from ganoderma lucidum and use of the same - Google Patents

Abstract

PURPOSE: A Ganoderma lucidum-derived novel compound, and an application thereof are provided to suppress tyrosinase activity and melanogenesis, and to be used in a cosmetic product or a pharmaceutical composition. CONSTITUTION: A compound or a salt thereof is denoted by chemical formula 1. The compound suppresses tyrosinase activity. A cosmetic composition for whitening contains the compound denoted by the chemical formula 1 as an active ingredient. A pharmaceutical composition for whitening contains the compound denoted by the chemical formula 1 as an active ingredient.

Description

Novel compound from Ganoderma lucidum and use of the same}

The present invention relates to a novel compound derived from ganoderma lucidum mushroom, more specifically, ganoderma lucidum extract or ergosterol peroxide compound and ganadomanandiol that inhibit tyrosinase activity and melanin biosynthesis. (ganodermanondiol) relates to a cosmetic composition comprising a compound. The Ganoderma lucidum extract obtained according to the present invention and the compounds isolated therefrom have inhibitory activity against tyrosinase, and in particular, show a significant inhibitory effect on tyrosinase activity and melanin biosynthesis in melanoma cells, which are associated with whitening. It can be usefully used as a cosmetic composition.

Due to environmental pollution, UV exposure of the skin is increasing, resulting in severe skin color deposition due to skin aging (JM Wood, et al. Exp. Dermatol. 8. 153-164 (1999), Kim C. H, et al. J. Kor. Acupuncture, 5, 69-78 (2004)). In addition, there is a growing interest in skin pigmentation for cosmetic reasons, and researches to find more stable and effective whitening materials are being actively conducted (Hwang JS et al. J. Soc. Cosmet. Sic. Kor. 28 (1). ), 135-149 (2002)).

Human skin color is mainly determined by the amount of melanin and is affected by pigment components such as hemoglobin and carotene, blood vessel distribution and thickness of the stratum corneum. In particular, melanocytes distributed in the epidermis not only play an important role in pigmentation of skin by synthesizing melanin, but also act as a photoprotective effect that the generated melanin migrates to keratinocytes to absorb and block excessive UV rays (M. Rozanowska). , et al. Free Radic. Bio.Med. 26. 518-525 (1999)). However, melanin excess pigmentation is known to form spots, freckles, blotch and promote skin aging (HZ Hill, et al. Pigment Cell Res. 10, 158 (1997), JH Kway. Et al. Korean J. Biotechnol Bioeng. 19 (6), (2004).

Melanin production in the skin is achieved through the action of several enzymes in the melanosomes in melanocytes (R. Cui, et al. Cell, 128, 853 (2007)). Tyrosine, a type of amino acid, is oxidized by tyrosinase and becomes dopa and dopaquinone, which in turn is 5,6-dihydroxy indole and indole. 5,6-quinone) and finally melanin polymer by polymerization (Ken-ichi. Y. et al. J. Proteome Research. 2 (1). 69-7 (2002)). In addition, melanin synthesis includes -MSH, cholera toxin and forskolin, cAMP inducers, tyrosinase-related proteins (TRP-1 and TRP-2), melanogenic enzymes, various cytokines involved in inflammatory reactions, and melananocyste gene expression. factors such as MITF, Pax-3 and others (Parvze et al. Phytotherapy Research. 20, 921-934 (2006)).

Related studies show the study of tyrosinase activity inhibitory material (arbutin, kojic acid, licorice extract, extract of mulberry, etc.) for regulating tyrosinase activity, the main enzyme of melanin synthesis, the study of the material having the effect of removing the stratum corneum (AHA, BHA, amino acid, salicylic acid, etc.), sunscreen material research (titanium dioxide, gamma-olizanol, oxybenzone, etc.), melanocytes that are toxic to melanocytes, which are melanocyte biosynthesis sites Materials research (vitamin C, etc.), active oxygen removal materials (vitamin E, vitamin C), etc. (KT Lee, et al. J. Cosmet. Sic., 54, 133 (2003), K Sato , et al. Biol. Pharm. Bull., 31, 33 (2008), Battaini. G., et al. J. Biol. Inorg.Chem. 5, 262-268 (2000).

 However, most of them have insufficient effects or incomplete formulations, their use is limited in terms of stability to the skin, and their dependence on imports for raw materials has recently led to the development of safer and more effective melanin inhibitors. Many studies are required (Lee. H. B et al. J. Kor. Soc. Food Sci. Nutr. 34 (9), 1325-1329 (2005)). In particular, kojic acid has been reported to be ineffective in inhibiting melanin production in cells or in vivo compared with in vitro tyrosinase inhibitory effects and also induce liver cancer in long-term use (Cabanes, JS et al. J. Pharm.Pharmacol. 46. 982-985 (1994)). In addition, arbutin (arbutin) is a problem due to the cytotoxicity and weak activity of the hydroquinone (hydroquinone) series, vitamin C is also difficult for long-term storage because of problems with chemical stability. Therefore, research on functional whitening materials using various natural product extracts as active ingredients for whitening has been actively conducted, and whitening skin, cheonhwamin extract, yulpi, and green beans are used as whitening raw materials, and the development of excellent alternative whitening agents is urgently needed (Park, SS et al. J. Life Sci. 17, 816-821 (2007)).

As a related prior patent, Republic of Korea Patent Publication No. 1020100130109 relates to a 'cosmetic composition for skin whitening containing edible mushroom fruiting body extract as an active ingredient', selected from the group consisting of Pleurotus eryngii, Enoki mushroom and Manny gang mushroom It describes a cosmetic composition for skin whitening containing an edible mushroom fruiting body extract selected from the combination of

As another related prior patent, Korean Patent Publication No. 1020050034833 relates to 'whitening cosmetics containing chaga mushroom extract', and the mixture of chaga mushroom fruit body and mycelium with water, hydrophilic organic solvent, and a mixture thereof Extract the mushroom fruiting body and mycelium extract, and the extract is described in the whitening cosmetics containing chaga extract, characterized in that contained 0.01 to 10% by weight relative to the cosmetic.

The present invention has been made by the above necessity, and an object of the present invention is to provide a tyrosinase inhibitor comprising a compound isolated from Ganoderma lucidum.

In order to achieve the above object, the present invention provides an egosterol peroxide compound of the formula (1).

[Formula 1]

In another aspect, the present invention provides a kanadomanediol compound of the formula (2).

[Formula 2]

In an embodiment of the present invention, the compound is preferably derived from Ganoderma lucidum, but is not limited thereto, and the compound prepared by chemically synthesizing is also included in the scope of the present invention.

In one embodiment of the present invention, the compounds preferably have tyrosinase inhibitory activity, but are not limited thereto.

The present invention also provides a whitening cosmetic composition comprising the compound of the present invention as an active ingredient.

In another aspect, the present invention provides a whitening pharmaceutical composition comprising the compound of the present invention as an active ingredient.

As used herein, a "pharmaceutical composition" may include a pharmacologically effective amount of a tyrosinase inhibitor and a pharmaceutically acceptable carrier. As used herein, "pharmacologically effective amount", "therapeutically effective amount" or simply "effective amount" refers to the amount of tyrosinase inhibitory substance effective to achieve the intended pharmacological, therapeutic or prophylactic result.

The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavors, colorants, and preservatives. no. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. The binder may include starch and gelatin, while the lubricant will generally be magnesium stearate, stearic acid or talc. In some cases, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

The present invention provides a pharmaceutical composition for skin whitening comprising a tyrosinase inhibitor as described above, and a pharmaceutically acceptable carrier.

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery.

The pharmaceutical composition of the present invention is administered at a dose sufficient to inhibit tyrosinase. In general, the dosage will range from 0.01 to 5.0 mg / kg body weight of the recipient per day, generally from 1 μg to 1 mg / kg body weight per day. The pharmaceutical compositions may be administered once per day, or the compounds of the present invention may be administered at appropriate intervals in small doses of two, three or more times per day, or using continuous infusion or delivery via a controlled release formulation.

The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and on the site to be treated. Administration may be by topical administration, pulmonary administration, for example by pulmonary administration by inhalation or aeration of a powder or aerosol comprising administration by means of a sprayer; Intravenous, intranasal, epithelial and transdermal, oral or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; Or intracranial, eg, epidural or intraventricular administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be required. Preferred topical formulations include formulations in which the compounds of the invention are mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline), negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. : Dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). The compounds of the invention may be encapsulated in liposomes or complexed thereto, in particular for cationic liposomes. Alternatively, the compounds may be complexed with lipids, in particular cationic lipids. Preferred fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolee Phosphorus, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acrylcarnitine, acylcholine or C1-10 alkyl esters (e.g. isopropylmyristate IPM), monoglycerides, Diglycerides or pharmaceutically acceptable salts thereof, including but not limited to.

Compositions and formulations for oral administration include suspensions or solutions, capsules, gel capsules, sachets, tablets or mini tablets (either as powders or granules, fine granules, nanoparticles, water or non-aqueous media) ). Thickeners, flavors, diluents, emulsifiers, dispersion aids or binders may be preferred. Preferred oral formulations are those in which the compounds of the invention are administered in combination with one or more penetration enhancers, surfactants and chelating agents. In addition, the compounds of the present invention may be orally delivered or complexed in the form of granules comprising spray dried particles to form microparticles or nanoparticles. Compositions and formulations for parenteral, intramedullary, intraventricular or intrahepatic administration may contain other suitable additives such as, but not limited to, buffers, diluents and penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Sterile aqueous solution may be included.

In addition, pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of ingredients including, but not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semi-solids. Pharmaceutical formulations of the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with the pharmaceutical carrier (s) or excipient (s). In general, the formulations are prepared by homogeneously and intimately blending the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then optionally molding the product.

In addition, the compositions of the present invention may be formulated in any of a number of possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suppositories in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran.

The present invention also provides a cosmetic composition for skin whitening comprising a tyrosinase inhibitor.

In the cosmetic composition for skin whitening of the present invention, the tyrosinase inhibitor is preferably contained in an amount of 0.005 to 50% by weight based on the total weight of the composition. It may be used above or below. In addition, the cosmetic composition for skin whitening of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the tyrosinase inhibitor.

Cosmetics prepared with the cosmetic composition for skin whitening of the present invention can be prepared in the form of a general emulsion formulation and solubilized formulation. Cosmetics of emulsified form include nutrition lotion, cream, essence, etc., and cosmetics of solubilized form have softening longevity. In addition, the cosmetic composition for skin whitening of the present invention can be prepared in the form of adjuvants that can be applied topically or systemically, which is commonly used in the field of dermatology by containing a dermatologically acceptable medium or base in addition to cosmetics.

Suitable cosmetic formulations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionics (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase. It may be provided in the form of a vesicle dispersant, in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick.

It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the cosmetic composition for skin whitening of the present invention, in addition to the tyrosinase inhibitors, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfaces Common to active agents, water, ionic or nonionic emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics It may contain adjuvants conventionally used in the cosmetic or dermatological field, such as any other ingredients used as. And the above ingredients may be introduced in amounts generally used in the field of dermatology.

Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, eye shadows and the like.

Hereinafter, the present invention will be described.

The present invention is a compound containing Ganoderma lucidum extract or egosterol peroxide or Ganadomanediol isolated from it has a tyrosinase inhibitory activity, in particular in tyrosinase activity and melanin biosynthesis in melanoma cells Significant inhibitory effect was shown, and these results can be usefully provided as a cosmetic composition related to the Ganoderma lucidum-derived egosterol peroxide or kanadomanediol compound.

The present invention will be described in detail.

The present invention provides a tyrosinase inhibitor or whitening cosmetic composition comprising the Ganoderma lucidum extract. In addition, it provides a tyrosinase inhibitor or a whitening cosmetic composition comprising a compound represented by the following formula (1) and (2).

[Formula 1]

Ganoderma lucidum extract according to the present invention and the active ingredient isolated therefrom can be obtained by a general method for separation, commercially available reagents can be obtained from plants and herbal medicines including fungi. In the present invention, the Ganoderma lucidum extract and the active ingredient separated therefrom are prepared by extracting and separating in the following manner.

Specifically, the dried Ganoderma lucidum fruiting body 10 times water, or an organic solvent, or a mixed solvent thereof at 20 to 85 ℃ 3 hours to 7 days, preferably 10 days to 15 days by ultrasonic extraction or reflux cooling extraction method Extract 1 to 3 times. The extract can be concentrated and / or dried. The Ganoderma lucidum fruiting body extract was suspended in distilled water and extracted three times with ethyl acetate to obtain an ethyl acetate fraction and a water fraction.

The ethyl acetate fraction was subjected to silica gel column chromatography using hexane / acetate and / or chloroform / methanol as mobile phases to obtain ten active fractions (Fr1 to Fr10). The 5th and 6th fractions were combined and concentrated, and four active fractions were obtained using silica gel column chromatography as a elution solvent (10: 1 to 5: 1) as a mixed solvent of chloroform / ethyl acetate. Among the second active fractions, reverse phase chromatography (RP C-18) was purified by separating acetonitrile: water mixed solvent (50:50 ~ 70:30) with the elution solvent, and then purified ergosterol peroxide (compound). 1), and the fourth fraction was purified by reverse phase chromatography (RP C-18) using acetonitrile: water mixed solvent (50:50 ~ 70:30) as an eluting solvent and purified as a pure compound ganodermanondiol. And compound 2) were obtained.

[Formula 2]

Ganoderma lucidum extract according to the present invention and Compound 1 and Compound 2 isolated therefrom have a significant inhibitory activity against tyrosinase in melanoma cells.

In addition, the Ganoderma lucidum extract comprising Compound 1 and Compound 2 having tyrosinase inhibitory activity according to the present invention effectively inhibited melanin production in melanoma cells. Therefore, Ganoderma lucidum extract comprising Compounds 1 and 2 according to the present invention acts as an inhibitor to tyrosinase, which plays a key role in the melanin biosynthesis process, thereby reducing the oxidative reaction of dopaquinone generated from tyrosine to dopaquinone. Since it inhibits melanin biosynthesis in melanocytes, it can be effectively used in cosmetic compositions including whitening.

Therefore, the extract of Ganoderma lucidum mushroom and Compound 1 and Compound 2 isolated therefrom exhibit a significant inhibitory activity against tyrosinase activity and melanin biosynthesis in melanoma cells, thereby inhibiting melanin production and thus whitening function. It can be used as an excellent whitening agent as an active ingredient in a cosmetic composition having.

As can be seen through the present invention, Ganoderma lucidum mushroom extract and the egosterol peroxide and Ganadomanediol compounds isolated therefrom have tyrosinase inhibitory activity, in particular the extracts and compounds of the compounds in melanoma cells By inhibiting tyrosinase activity and melanin biosynthesis by the effect, it can be used as a cosmetic or pharmaceutical composition involved in skin whitening.

1 is a diagram describing the chemical formula of ergosterol peroxide, compound 1.
FIG. 2 is a diagram describing the chemical formula of ganodermanondiol, compound 2. FIG.
Figure 3 shows the effect of compounds on cytotoxicity in melanoma B16F10 cells. This figure is an experiment on the presence or absence of Ganoderma lucidum extract and melanoma mushroom isolated compounds against melanoma B16F10 cells, the color of MTT is decomposed by the activity of the intracellular mitochondria, which develops purple color. As can be seen from the drawings, Compounds 1 and 2 isolated from Ganoderma lucidum extract were treated with concentrations of 0.1 to 1 uM and 0.1 to 10 uM, respectively, and the absorbance was measured. Did.
Figure 4 shows the inhibitory effect of tyrosinase activity by treatment with -MSH and ergosterol peroxide, Compound 1 in melanoma B16F10 cells. This figure is to determine the degree of inhibition of tyrosinase activity when treated with Ganoderma lucidum extract and Compound 1 to melanoma B16F10 cells, the result of the treatment of tyrosinase of Compound 1 when treated with Arbutin positive control The activity was significantly inhibited, and about 33.10% 4.91% at 10 uM concentration.
Figure 5 is a figure showing the inhibitory effect of tyrosinase activity by the treatment of α-MSH and ganodermanondiol, compound 2 in melanoma B16F10 cells. 4 shows the inhibition of tyrosinase activity when the Ganoderma lucidum extract and Compound 2 were treated to melanoma B16F10 cells by the same method as in the experiment of FIG. 4, and as a result, Compound 2 was treated as compared to Arbutin, a positive control. In the case of treatment, the concentration-dependently inhibited the activity of tyrosinase significantly, and at a concentration of 10 uM was about 57.18% 0.71%.
Figure 6 shows the inhibitory effect of ergosterol peroxide, compound 1 on α-MSH induced melanin production in melanoma B16F10 cells. Melanin is biosynthesized from melanocytes of melanocytes in the epidermal basal layer, and there are dark brown eumelanin and red-yellow pheomelanin, which are related to melanogenesis-related proteins or melanocytes and melanocytes. It is triggered by α-MSH (-melanocyte stimulating hormone). In this figure, melanoma B16F10 cells were treated with α-MSH to induce melanin production, and then the inhibition of melanin production by the treatment of Ganoderma lucidum extract and Compound 1 was attempted. As a result, treatment with Compound 1 inhibited melanin production in a concentration-dependent manner, compared with treatment with arbutin, which was a positive control, and showed about 39.60% 0.51% at 10 uM.)
Figure 7 is a diagram showing the inhibitory effect of ganadermanondiol, compound 2 on α-MSH induced melanin production in melanoma B16F10 cells. This figure is to induce melanin production by treating α-MSH in melanoma B16F10 cells, and then to determine the degree of inhibition of melanin production by treatment of Ganoderma lucidum extract and Compound 1, as a result of the treatment of arbutin as a positive control When Compound 1 was treated, it inhibited melanin production in a concentration-dependent manner, and it was about 50.98% and 1.52% at 10 uM concentration.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example 1 Preparation of Ganoderma lucidum Extract

4 kg of ganoderma lucidum mushrooms, which were purchased from a ganoderma lucidum farm, were sliced and immersed in 30 L of ethanol, and left to filter at room temperature for 7 days. The extract was concentrated under reduced pressure and dried to obtain 90 g of the total extract.

Example 2. Preparation of Ganoderma lucidum ethyl acetate soluble extract

90 g of the total extract was suspended in 2 L of distilled water, and then extracted three times by adding 3 L of ethyl acetate. An ethyl acetate fraction having high tyrosinase inhibitory activity and melanin production inhibitory activity was obtained. The ethyl acetate fractions were concentrated under reduced pressure to obtain 70 g of an ethyl acetate soluble part.

Example 3. Isolation of Chemical 1 from Ganoderma lucidum ethyl acetate soluble extract

70 g of the ethyl acetate soluble part of Example 2 was adsorbed onto silica gel (230-400 mesh, Merck) and silica gel column chromatography was performed. In this case, the hexane / ethyl acetate mixed solvent (20: 1) was used as the developing solvent, and the hexane / ethyl acetate mixed solvent (1: 1) was adjusted to adjust the polarity. Then, the solvent composition was mixed with the chloroform / methanol mixed solvent. Conversion to (20: 1) was performed to increase the polarity to the chloroform / methanol mixed solvent (1: 1) to perform fractionation. As a result, 10 active fractions were obtained (Fr1 ~ Fr10). Secondary silica gel column chromatography (70-230 mesh, 5th fraction) with high tyrosinase inhibitory activity and melanin inhibitory activity were combined and concentrated. Merck Co., Ltd. obtained four active fractions using a mixed solvent of chloroform / ethyl acetate (10: 1 to 5: 1) as an eluting solvent. The second active fraction was purified by reverse phase chromatography (RP-C-18) and purified by acetonitrile / water mixed solvent (50:50 ~ 70:30) as the elution solvent. Compound 1) was obtained (FIG. 1).

Example 4. Isolation of Compound 2

The fourth fraction of the active fraction obtained through the second silica gel chromatography of Example 3 was subjected to reverse phase chromatography (RP C-18) to acetonitrile / water mixed solvent (50:50 to 70:30). Purified and separated to obtain a pure compound gana derma diol (ganodermanondiol, Compound 2) (Fig. 2).

Example 5 Physicochemical Properties of Compounds Represented by Formula 1

The physicochemical properties of ergosterol peroxide (Formula 1) are as follows:

Colorless needles, C ₂₈ H ₄₆ O ₃ ;

¹ H-NMR (CDCl ₃ , 300 MHz): δ 0.81 (3H, s, H-18), 0.82 (3H, d, J = 4.5 Hz, H-26), 0.88 (3H, s, H-19) , 0.90 (3H, d , J = 6.6 Hz, H-28), 0.915 (2H, s, H-15), 0.99 (3H, d, J = 6.6 Hz, H-21), 3.96 (1H, m, H-3), 5.13 (1H, dd, J = 8.1, 15 Hz, H-22), 5.21 (1H, dd, J = 7.5 Hz, 15.36 Hz H-23), 6.24 (1H, d, J = 8.4 Hz, H-6), 6.51 (1H, d, J = 8.4 Hz, H-7). ¹³ C-NMR (75 MHz, CDCl3): 12.84 (C-18), 17.53 (C-28), 18.15 (C-19), 19.61 (C-27), 19.92 (C-26), 20.60 (C- 15), 20.85 (C-21), 23.37 (C-11), 28.61 (C-16), 30.08 (C-2), 33.04 (C-25), 34.67 (C-1), 36.89 (C-10 ), 36.94 (C-4), 39.32 (C-12), 39.7 (C-20), 42.75 (C-24), 44.53 (C-13), 51.06 (C-9), 51.65 (C-14) , 56.17 (C-17), 66.43 (C-3), 79.40 (C-8), 82.13 (C-5), 130.72 (C-7), 132.28 (C-23), 135.17 (C-22), 135.39 (C-6)

Example  6. Physicochemical Properties of Compounds Represented by Formula 2

The physicochemical properties of Ganodermanondiol (Formula 2) are as follows:

Colorless crystals, C ₃₀ H ₄₈ O ₃ ;

¹ H-NMR (CDCl ₃ , 300 M Hz): δ 0.59 (3H, s, H-18), 0.87 (3H, s, H-28), 0.91 (3H, d J = 6.3 Hz, H-21), 1.08 (3H, s, H-19), 1.12 (3H, s, H-29), 1.16 (3H, s, H-26), 1.19 (3H, s, H-27), 2.77 (1H, m, H-1), 3.30 (1H, bd, J = 8.1 Hz, H-24), 5.38 (1H, bd, J = 6.3 Hz, H-7), 5.50 (1H, bd, J = 6.3 Hz, H- 11). ¹³ C-NMR (75 MHz, CDCl ₃ ): 15.70 (C-18), 18.61 (C-21), 22.02 (C-19), 22.43 (C-30), 23.19 (C-28), 23.64 (C -6), 25.32 (C-27), 25.42 (C-26), 26.54 (C-29), 27.83 (C-16), 28.69 (C-15), 31.44 (C-22), 33.45 (C- 23), 34.83 (C-2), 36.51 (C-20), 36.51 (C-20), 36.60 (C-1), 37.17 (C-12), 37.80 (C-10), 43.73 (C-13 ), 47.45 (C-4), 50.28 (C-5), 50.69 (C-14), 50.95 (C-17), 73.22 (C-25), 79.57 (C-24), 117.23 (C-11) , 119.90 (C-7), 142.83 (C-8), 144.49 (C-9), 216.83 (C-3)

Example  7. Effects on Cell Culture and Cell Viability of Melanoma B16F10 Cell Line

Melanoma B16F10 cell line was used from the ATCC (American Type Culture Collection, 6475). The cells were cultured at 37 ° C., 5% CO _{2 in} a medium mixed with 10% FBS, 100U / ml penicillin and 100ug / ml streptomycin in DMEM. Lt; / RTI > All cells in the experiment were tested at 80-90% confluency.

MTT assay was performed to confirm the presence of Ganoderma lucidum extract and compounds against melanoma B16F10 cells. Cells were placed in DMEM containing 10% FBS and 100ul each of 3x10 ³ cells / well in 96 well plate at 37 ℃, 5% CO ₂ Incubate for 24 hours under conditions. After incubation, the medium is removed, and the compound is treated with triplicate for each group, and then incubated for 72 hours at 37 ° C. and 5% CO ₂ . After incubation, 10 μl of 5 mg / ml MTT reagent was dispensed into the medium containing the test solution, and then incubated for 4 hours in a 37 ° C., 5% CO ₂ incubator by blocking the light of a 96 well plate. When the culture was completed, the medium containing the MTT reagent was removed, and 100ul of DMSO (dimethyl sulfoxide) was added to dissolve the MTT-formazan crystal, and the cell viability was confirmed by measuring the absorbance at 570 nm.

Cell viability (%) = (absorbance of control group / absorbance of experimental group) X 100

As shown in Figure 3, Ganoderma lucidum extract of the present invention and compounds 1 and 2 isolated therefrom did not show cytotoxicity against melanoma B16F10 cells in the concentration range of 5 ~ 50ug / ml, 0.1 ~ 10uM, respectively.

Example  8. Inhibitory effect of tyrosinase activity on melanoma B16F10 cell line

Tyrosinase activity was shown as a measure of DOPA oxidase activity. Melanoma B16F10 cells were aliquoted in 6 well plates with DMEM containing 10% FBS at a concentration of 1x10 ⁵ cells / well and incubated for 24 hours at 37 ° C and 5% CO ₂ . After incubation, the medium was removed, and different concentrations of Ganoderma lucidum extract and Compounds 1 and 2 were added to a DMEM culture medium containing 10% FBS, and α-MSH (100 nM) was added thereto as an inducer, and 37 ° C and 5% CO ₂ Incubated for 72 hours under conditions. After culturing the cells, the medium is removed, washed twice with PBS, and then 1 ml of PBS is collected per well to recover the cell pellet. The recovered pellet was centrifuged at 14,000 rpm for 20 minutes, the supernatant was removed, and 500ul of lysis buffer (67mM sodium phosphate, pH 6.8, 1% Triton-X100, 0.2mM PMSF) was added to pulverize the cells with an ultrasonic grinder. Cell lysate was stored on ice for 1 hour and centrifuged at 14,000 rpm for 20 minutes to obtain supernatant, and the protein was quantified for use in tyrosinase activity analysis. 40ul of 4mM L-DOPA and 40ug of supernatant were mixed in 67mM sodium phosphate (pH6.8) and added to a 96ul plate to 200ul, followed by reaction at 37 ° C for 2 hours. Dopa chrome produced after the reaction was measured by absorbance at 475nm and expressed as relative tyrosinase activity.

% Inhibition of tyrosinase activity = 1- (absorbance of control / absorbance of experimental group) X 100

sample density Inhibition rate (%) sample density Inhibition rate (%) sample density Inhibition rate (%) No treatment 0 0 Compound 1
(uM) 0 0 Compound 2
(uM) 0 0 Arbutin
(ug / ml) 100 12.29 0.1 - 0.1 16.62 500 33.87 0.5 - 0.5 15.75 Ganoderma lucidum extract
(ug / ml) 5 - One - One 13.40 25 1.94 5 34.94 5 19.74 50 19.10 10 33.41 10 57.18

Table 1 is a table showing the inhibitory effect of melanoma B16F10 intracellular tyrosinase.

As shown in Figures 4 and 5, Compounds 1 and 2 isolated from the Ganoderma lucidum extract of the present invention was found to significantly inhibit intracellular tyrosinase activity (tyrpsinase) significantly compared to the negative control, Inhibitors of tyrosinase activity were more strongly inhibited at lower concentrations than the arbutin treatment group, which is currently used as a whitening agent. Tyrosinase activity was inhibited by 33.10% 4.91% and 57.18% 0.71%, respectively, at the concentrations treated with Compounds 1 and 2 (10 uM) (Table 1).

Example  9. Melanin Inhibitory Effect of Melanoma B16F10 Cell Line

Melanoma B16F10 cells were dispensed in DMEM containing 10% FBS at a concentration of 1x10 ⁵ cells / well in 6 well plates and 37, 5% CO ₂ Incubate for 24 hours under conditions. After incubation, the medium was removed, and different concentrations of Ganoderma lucidum extracts and compounds were added to DMEM culture medium containing 10% FBS, and α-MSH (100 nM) was added thereto as an inducer. 37, 5% CO ₂ Incubated for 72 hours under conditions. After incubation, the medium is removed, washed twice with PBS, and 1 ml of PBS is dispensed per well to recover the cell pellet. The recovered pellet is centrifuged at 14,000 rpm for 20 minutes and the supernatant is removed. The cell pellet was then dried at 60 hours for 1 hour, and then 150 μl of 1M NaOH containing 10% DMSO was added for 1 hour in a 60 degree thermostat to dissolve the melanin in the cells. 100ul of this solution was put into a 96 well plate and the absorbance was measured at 405nm with an ELISA reader, and the melanin content was quantified by comparing with the standard curve using the melanin standard solution.

Inhibition rate of melanin content (%) = 1- (Melanin content of control group / Melanin content of test group) X 100

sample density Inhibition rate (%) sample density Inhibition rate (%) sample density Inhibition rate (%) No treatment 0 0 Compound 1
(uM) 0 0 Compound 2
(uM) 0 0 Arbutin
(ug / ml) 100 12.37 0.1 6.77 0.1 3.84 500 23.12 0.5 4.10 0.5 13.81 Ganoderma lucidum extract
(ug / ml) 5 - One 12.44 One 20.13 25 15.61 5 40.58 5 40.43 50 37.14 10 39.60 10 50.98

 Table 2 is a table showing the melanin content inhibitory effect in melanoma B16F10 cells.

 As shown in Figure 6 and 7, compounds 1 and 2 isolated from the Ganoderma lucidum extract of the present invention was confirmed to significantly inhibit the melanin content produced by the induction of α-MSH. In addition, compounds 1 and 2 inhibited melanin production at a lower concentration than the arbutin treatment group, which is a positive control currently used as a whitening agent. They inhibited melanin production at 10 uM concentrations of 39.60% 0.51% and 50.98% 1.52%, respectively, compared to controls (Table 2).

Claims (8)

A compound of formula 1 or a salt thereof.

[Formula 1] According to claim 1, wherein the compound is a compound having a tyrosinase inhibitory activity or a salt thereof A cosmetic composition for whitening comprising the compound of claim 1 or 2 as an active ingredient. A whitening pharmaceutical composition comprising the compound of claim 1 or 2 as an active ingredient. A compound of formula (2) or a salt thereof.

(2) The compound or salt thereof according to claim 5, wherein the compound has tyrosinase inhibitory activity A cosmetic composition for whitening comprising the compound of claim 5 or 6 as an active ingredient. A whitening pharmaceutical composition comprising the compound of claim 5 or 6 as an active ingredient.

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