JP5942185B2 - Whitening composition and anticancer agent having melanin suppression and MITF suppression action - Google Patents

Description

  The present invention relates to a physiologically active composition comprising an alcohol extract component of Cyprus and / or Inudouna and capable of effectively suppressing the production of melanin pigment in a living body.

  In recent years, interest in health and beauty has increased, and for example, many studies on skin whitening have been conducted. The cause of darkening of the skin is due to the production of melanin, but the production mechanism of melanin in the skin is usually explained as follows. That is, as shown in FIG. 1, when stimulated by ultraviolet light or melanin-stimulating hormone in the body, tyrosinase, which is a melanin-producing enzyme, is activated, and dopachrome is activated by an enzyme reaction using tyrosine in the melanocytes of the skin as a substrate. Or indole-5,6-quinone, which gradually becomes black due to the oxidative polymerization reaction, producing a melanin pigment.

  Among the above, melanin production inhibitors include those that inhibit the activity of tyrosinase. For example, “Arbutin”, a glycoside with one glucose bonded to hydroquinone, has the effect of inhibiting the activity of tyrosinase in melanocytes (pigmenting cells) that produce melanin pigment. Suppresses generation. In addition, the inhibitory effect on pigmentation induced by irradiating human skin with ultraviolet rays has been investigated by a double-blind method, and it has been shown that application of an arbutin-containing emulsion is effective. Thus, many components that inhibit the activity of tyrosinase have been found, but no matter how much they have an inhibitory action, they cannot be applied if they are harmful to humans. From such a point of view, many techniques for finding a melanin production inhibitor component in edible plants and the like that are highly safe for humans and extracting and using them in cosmetics (for example, Patent Documents 1 to 5 and Non-patent Documents) 1).

JP 2011-88856 A JP 2011-37851 A JP 2010-173964 A JP 2010-111645 A JP 2010-43041 A

Miyazawa et al. “Character Impact Odorants of Wild Edible Plant-Cacalia hastate L. var. Orientalis-Used in Japanese Traditional Food”, Journal of Oleo Science 59, (10) 527-533 (2010)

  As described above, many melanin production inhibitors have an inhibitory effect on melanin pigment-producing enzymes themselves such as tyrosinase. Alternatively, oxidative polymerization at the time of melanin production is also suppressed by an antioxidant component. However, suppression of melanin production may not be sufficient only by suppressing the enzyme reaction of tyrosinase or suppressing oxidative polymerization.

  For example, microphthalmia-related transcription factor (MITF) is known to promote gene expression of melanin pigment producing enzyme, and if MITF can be suppressed, melanin production can be suppressed from the root. It is done.

  The present invention includes a physiologically active composition that includes an edible plant-derived component, exhibits an MITF inhibitory action, and has an excellent melanin production inhibitory action, and a method for producing the same, an MITF inhibitor, a melanin production inhibitor, and a cosmetic composition It is an object to provide a product and an anticancer agent.

  The present inventor has collected various natural resources, prepared various extracts based on differences in polarity using various solvents, and examined their physiological activities to construct a systematic library. As a result of intensive research on the melanin production-inhibiting action of natural resource extracts, it was found that alcohol extracts of naturally-occurring wild plants such as Jobus mushrooms and Inudouna were combined with tyrosinase inhibitory components and antioxidant components to suppress MITF. It was found that the ingredients were included. Yeosummasou and Inudouna are wild plants that grow widely in cool areas and have high food safety. And by including each said component, it is also possible to suppress the tyrosinase gene expression by MITF, to inhibit the enzyme reaction by tyrosinase, and to suppress the oxidative polymerization reaction after the enzyme reaction. It was found that they can be effectively acted at each stage from the root to the final generation.

The present invention has been made based on the above findings. That is,
The first aspect of the present invention is a bioactive composition obtained by alcohol-extracting Cyprus and / or Inudouna.

  In the present invention, “yobus masou” and “inuuna” mean the genus Parasenecio, inuuna. “Alcohol extraction” means that a part of the components contained in Cyprus and / or Inudouna is extracted into a solvent containing alcohol depending on the difference in polarity.

  The first aspect of the present invention includes a microphthalmia-related transcription factor-suppressing component derived from Candida and / or Inudouna, can suppress tyrosinase gene expression by MITF, and can suppress melanin production from the root.

  In the first invention, the molecular weight of the microphthalmia-related transcription factor inhibitory component is 3000 or less.

  The second aspect of the present invention is a method for producing a physiologically active composition, comprising a step of alcohol extraction of Cyprus and / or Inudouna.

  In the second aspect of the present invention, the alcohol concentration in the alcohol extraction is preferably 25% or more and 80% or less. The “alcohol concentration” means the proportion of the alcohol volume occupying when the volume of the entire extraction solvent is 100%. Within the said range, the bioactive composition containing said MITF suppression component can be obtained efficiently. If water (hot water) or pure alcohol is used as a solvent, the bioactive composition has a poor melanin production inhibitory effect.

  The third aspect of the present invention is a microphthalmia-related transcription factor inhibitor comprising the bioactive composition according to the first aspect of the present invention.

  4th this invention is a melanin production inhibitor containing the bioactive composition which concerns on 1st this invention.

  The fifth aspect of the present invention is a cosmetic composition containing the bioactive composition according to the first aspect of the present invention.

  The sixth invention is an anticancer agent comprising the bioactive composition according to the first invention.

  The physiologically active composition according to the present invention comprises an extract component of edible wild plants that is highly safe for humans, suppresses tyrosinase gene expression by MITF, inhibits tyrosinase enzyme reaction, and enzyme reaction It is also possible to suppress the subsequent oxidative polymerization reaction. That is, according to this invention, while including the component derived from an edible plant, the bioactive composition which shows the inhibitory action with respect to MITF, and was excellent in the melanin production inhibitory action conventionally, and its manufacturing method can be provided.

It is a figure for demonstrating the melanin production | generation mechanism of skin. It is a figure for demonstrating the effect of the bioactive composition which concerns on this invention. It is a data which shows the influence which acts on the mushroom tyrosinase activity of a Jobus masou extract. It is a data which shows the influence which it has on the melanin production of an Aedes masou extract. It is data which shows the influence which it has on the mushroom tyrosinase activity of a commercial product. It is a data which shows the influence which it has on the melanin production of a commercial thing of Jobus masou. It is the data which shows the antioxidant ability of the refined product of Aedes japonicum. It is the data which concern on the fixed_quantity | quantitative_assay of the mRNA expression of the cell by a commercial thing of a Jobsaw millet (fraction 3). It is the data which concerns on the melanin production inhibitory effect of a purified job (Fraction 3). It is the data which compared the influence which it has on a gene expression about the refined material (fractions 3 and 4) and the existing melanin inhibitor. It is the data which compared the influence which it has on the melanin production about the refined material (Fraction 3, 4) and the existing melanin inhibitor. It is the data which compared the influence which it has on the gene expression of the refined product of Yorca masou by the difference of various stimulants. It is the data which compared the influence which the difference in various irritants has on the melanin production of the refined material of C. communis. It is the data which shows the anticancer effect | action of a purified job. It is a figure for demonstrating the anticancer effect | action of the bioactive composition which concerns on this invention. It is the data which compared the difference of the anticancer activity by a cell density about the fraction 3. It is a data which shows the influence which acts on the mushroom tyrosinase activity of a Jobus masou extract and a Japanese maple extract. It is the data which shows the antioxidant ability of an extract of Aedes masou and a maple extract. It is a data which shows the influence which it has on the melanin production of a Giant moth extract and a Japanese maple extract. It is a data which shows the influence which acts on the MITF mRNA expression of an extract of Aedes masou and a maple extract.

<Bioactive composition>
The physiologically active composition according to the present invention is obtained by alcohol-extracting Yorca and / or Indouna.

  Naturally occurring Asteraceae of the genus Asteraceae genus Batsoma and Indouna can be used as Aedesa and Indouna. Or what was artificially grown and harvested by the promotion cultivation etc. may be used.

  The physiologically active composition according to the present invention contains an MITF-suppressing component derived from Jobsaw and / or Inudouna. The said MITF suppression component is a highly fat-soluble substance having a molecular weight of 3000 or less.

  Moreover, the physiologically active composition according to the present invention may include a tyrosinase inhibitory component derived from Candida and / or Inudouna, and an antioxidant component in addition to the above-mentioned MITF suppressing component. The tyrosinase-inhibiting component contained in the physiologically active composition is a component having a higher water solubility than the MITF suppressing component, and the antioxidant component is a component having the same or higher water-solubility than the tyrosinase-inhibiting component. In addition, the ratio of said various components contained in a bioactive composition is not specifically limited, It can change with extraction operation. Specifically, such a physiologically active composition can be produced by, for example, the following production method.

<Method for producing bioactive composition>
The method for producing a physiologically active composition according to the present invention includes a step of alcohol extraction of Jobsaw and / or Inudouna. The process may be any process that can extract a part of the components contained in Candacea and / or Inudouna according to the difference in polarity, and the extraction operation can be performed by a known method. In addition, when extracting, it is good to cut | disconnect Jobus and / or Inudouna finely using a blade etc. Thereby, a content component can be extracted efficiently.

  In the method for producing a physiologically active composition according to the present invention, the alcohol concentration in the extraction solvent is preferably 25% or more and 80% or less. More preferably, it is 50% or more and 80% or less, and particularly preferably 60% or more and 80% or less. In the present invention, the use of alcohol having a concentration of 25% or more and 80% or less can extract the above-mentioned various active ingredients more efficiently than using water (hot water) or pure alcohol as an extraction solvent. . The temperature of alcohol is not specifically limited, For example, it can extract at normal temperature. The type of alcohol is not particularly limited, such as methanol, ethanol, propanol, butanol and the like, but ethanol is particularly preferable.

  The method for producing a physiologically active composition according to the present invention may include a step of drying the physiologically active composition obtained by alcohol extraction under reduced pressure or freeze drying. Thereby, the preservability and handleability of a physiologically active composition can be improved.

  The bioactive composition according to the present invention thus obtained contains an MITF suppressing component derived from an edible plant, and further contains a tyrosinase inhibiting component and an antioxidant component. That is, as shown in FIG. 2, tyrosinase that suppresses the gene expression of tyrosinase and suppresses melanin production from the root by the MITF suppressing component (first effect), and the expression cannot be completely suppressed, is suppressed by the tyrosinase inhibiting component. The enzyme reaction is inhibited to suppress the production of dopachrome and indole-5,6-quinone (second effect), and even if they are produced, the oxidation polymerization can be suppressed by the antioxidant component (third effect). ). That is, it can be said that the physiologically active composition according to the present invention can suppress melanin production in three stages and is more excellent in melanin production inhibitory action than before.

<Microphthalmia-related transcription factor inhibitor, melanin production inhibitor>
As described above, the bioactive composition according to the present invention contains a MITF inhibitor component, a tyrosinase inhibitor component, and an antioxidant component, and thus is suitable as a microphthalmia-related transcription factor inhibitor or a melanin production inhibitor. Is available. In the case of a microphthalmia-related transcription factor inhibitor, after obtaining the above physiologically active composition, an extract in which the MITF inhibitor component is further concentrated using a highly lipophilic solvent such as ethyl acetate is obtained. Good.

  In the case of producing a melanin production inhibitor containing the physiologically active composition according to the present invention, the melanin production inhibitor can contain a known gelling agent, preservative, etc. in addition to the above-mentioned extract. It can be used as a liquid or cosmetic cream. In such a case, there is no particular limitation on the concentration of the extract obtained by alcohol-extracting Yorca masou and / or Inudouna contained in the cosmetic liquid or cosmetic cream. When the extract is dried by lyophilization or the like, if 1 to 5 mg, preferably 1 to 5 mg, of the extract after drying is contained per 1 ml of cosmetic liquid or cosmetic cream, sufficient melanin is produced. An inhibitory effect can be obtained. As described above, the physiologically active composition according to the present invention suppresses tyrosinase gene expression by the MITF suppressing component to suppress melanin production from the root (first effect), and tyrosinase that has not been able to suppress the expression. The enzyme reaction is inhibited by the tyrosinase inhibiting component to suppress the production of dopachrome and indole-5,6-quinone (second effect), and even if they are produced, the oxidation component can be suppressed by the antioxidant component. (Third effect) Therefore, a sufficient melanin production suppressing effect can be obtained even at a low concentration.

  Hereinafter, although the Example demonstrates further in detail about the melanin production inhibitory effect of the bioactive composition which concerns on this invention, this invention is not limited to the specific form described in the following Examples.

1. Mushroom tyrosinase inhibitory action of A. japonicus extract As an extract, a solvent ((1) hot water, (2) 70% ethanol, (3) 100% methanol) was added to 88 g of Cyprus and finely pulverized with a mixer. In the case of (1), it was then heated at 100 ° C. for 10 minutes. Centrifugation was performed at 3000 rpm for 10 minutes, and the upper layer was collected by filtration through filter paper. Thereafter, (1) was lyophilized, and (2) and (3) were dried under reduced pressure using a rotary evaporator to remove the solvent. After measuring the weight of the solid extract, it was adjusted to 0.1 g / ml.

  Take 96 μl of L-tyrosine-containing solution (2 mM L-tyrosine, solvent: 50 mM phosphate buffer pH 6.8) in 100 μl, add 50 μl of 50 mM phosphate buffer pH 6.8, and 20 μl of the extract prepared to 1 mg / ml. It was. Then, 20 μl of mushroom tyrosinase (300 U) was added and stirred, placed in a hybrid oven set at 25 ° C., incubated for 60 minutes, and the absorbance at 490 nm was measured. The results are shown in FIG.

From FIG. 3, it was found that Cyprus has a tyrosinase inhibitory action and is best extracted with 70% ethanol. The concentration at which 70% ethanol extract of C. communis inhibits tyrosinase by 50% was IC ₅₀ = 100 μg / ml.

2. Inhibitory effect on melanin production by A. japonicus extract Using mouse-derived cell line (B16 melanoma 4A5), 10% fetal bovine serum, 100 μg / ml streptomycin, 100 μg / ml penicillin were added using DMEM as a basal medium at 37 ° C., 5 The cells were cultured for 3 to 4 days in a% CO ₂ atmosphere. In measuring melanin production, the cells were cultured for 24 hours, the medium was changed, 100 μM IBMX, an extract and a purified product were added, and then cultured at 37 ° C. in a 5% CO ₂ atmosphere for 3 to 4 days. Melanin production was measured.

  For melanin measurement, after culturing the cells, PBS was added, the cells were scraped twice, centrifuged at 3000 rpm for 5 minutes, the supernatant was taken, and 1N NaOH was added and treated at 60 ° C. for 60 minutes. After the treatment, cells were measured at 450/655 nm and the medium at 450 nm with a microplate reader using synthetic melamine as a standard substance. The results are shown in FIGS. 4 (A) and (B).

From FIG. 4, 70% ethanol extract showed the strongest inhibition of IBMX-induced melanin production among the extracts of Cyprus. When the melanin content of the cells and the amount of melanin released into the medium were combined, the concentration at which melanin pigment production was inhibited by 50% was IC ₅₀ = 8 to 10 μg / ml. This was a low concentration compared to the tyrosinase inhibitory concentration.

3. Purification of active ingredients from 70% ethanol extract of Jojoba Soma 1362 g of Jobsamaso was shredded, 5.5 L of 70% ethanol was added and ground with a Waring blender. This was centrifuged at 2000 rpm for 5 minutes, and the upper layer was collected by filtration through filter paper. The residue was recovered, 2.2 L of 70% ethanol was added, and centrifuged in the same manner. The upper layer was recovered by filtration through filter paper, and then added to the first recovery. Thereafter, the mixture was dried under reduced pressure using a rotary evaporator to distill off ethanol, and then lyophilized to completely remove the solvent. The weight of the solid extract was 44.5 g.

  A reverse-phase resin Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) was used to purify a 70% ethanol extract based on the difference in polarity. 45 g of HP-20 was dissolved in methanol and packed into a column. This was equilibrated with 30% methanol, and 21 g of C. 70% ethanol extract dissolved in 245 ml of 30% ethanol was injected into the column. The fraction eluted here was designated as “Fraction 1” (30% methanol eluate). Next, “fraction 2” was obtained by injecting 300 ml of 75% methanol. Further, the fraction eluted by injecting 300 ml of 100% methanol was designated as “Fraction 3”. Finally, “Fraction 4” was obtained by injecting 300 ml of ethyl acetate. The same operation was performed twice to fractionate a total of 42 g of Jobus 70% ethanol extract. For each fraction, the solvent was removed by vacuum drying and lyophilization using a rotary evaporator. The yield is as described in Table 1 below.

4). Characterization of Jobbill fractions Mushroom tyrosinase inhibitory action and melanin pigment inhibitory action were evaluated for each fraction.

FIG. 5 shows the tyrosinase inhibitory action of each fraction purified with HP-20 from the 70% ethanol extract of Jojoba. The concentration at which fraction 2 inhibits tyrosinase by 50% was IC ₅₀ = 50 μg / ml. On the other hand, fractions 3 and 4 showed almost no tyrosinase inhibitory action at the measured concentrations.

  FIGS. 6A and 6B show the effect of each fraction on melanin pigment production by B16 melanoma 4A5. Fraction 2 having a high tyrosinase inhibitory action had a weak melanin pigment suppressing effect. On the other hand, a strong melanin inhibitory effect was observed in fractions 3 and 4, which showed almost no tyrosinase inhibitory action.

5). Measurement of antioxidant capacity In order to suppress melanin pigment production, in addition to the action of suppressing tyrosinase as shown in Fig. 2, it may be possible to inhibit oxidative polymerization reaction, so measure the antioxidant capacity of each fraction did. Antioxidant ability was evaluated by measuring radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH). 100 μM DPPH (ethanol solution) was placed in a 1 ml test tube, 20 μl of a fraction adjusted to each concentration was added, stirred for 30 seconds, and allowed to stand at room temperature in the dark for 30 minutes. Thereafter, the disappearance of radicals was measured by measuring the absorbance at 517 nm with a spectrophotometer. The results are shown in FIG.

  As shown in FIG. 7, Fraction 2 having a strong tyrosinase inhibitory action had the strongest antioxidant ability, and then Fraction 1 showed a strong activity. On the other hand, the antioxidative ability of fractions 3 and 4, which had the strongest effect of suppressing melanin production by cells, was weak. From the above, it is considered that the mechanism in which fractions 3 and 4 suppress melanin production is separate from suppression of tyrosinase and inhibition of oxidative polymerization reaction.

6). Suppression of MITF and tyrosinase gene expression by a purified product of Falcon (Fraction 3) Fraction 3 of a purified product of Cyprus, which showed a strong melanin production inhibitory effect, was thought to have actions other than tyrosinase inhibitory activity and antioxidant capacity. The effect of the MITF gene, which is a pigment cell-specific transcription factor, on mRNA expression was examined. MITF (microphthalmia-related transcription factor) is a master transcription factor that controls the differentiation and proliferation of pigment cells (melanin producing cells). The target genes are melanin synthase tyrosinase, tyrosinase amino acid sequence and 40% These are tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) / dopachrome tautomerase (DCT), which have similarities, and specifically promote expression thereof. In recent years, it has been discovered that a gene encoding MITF is greatly amplified in melanoma cells, suggesting that it may function as a melanoma oncogene, and has attracted attention as a new strategy for cancer treatment. Yes.

  Cells cultured in a φ60 mm dish for 12 hours to 3 days were washed once with 5 ml of PBS, 1 ml of TRIzol (Gibco) was added, and RNA was extracted according to a conventional method. From this, cDNA was synthesized using PrimeScript RT Master Mix (manufactured by Takara Bio Inc.), and mRNA was quantified by Real-time PCR method using SYBR Premix Ex Taq II (manufactured by Takara Bio Inc.). The base sequence of Primer used in the experiment is shown in Table 2 below.

  FIGS. 8 (A) to (D) show the effect of the purified product (Fraction 3) on MITF and its downstream mRNA expression when cells were treated with IBMX for 12 hours. As shown in FIG. 8 (A), Jobsaw fraction 3 strongly inhibited the induction of MITF mRNA expression by IBMX at a concentration of 1.5 μg / ml. Moreover, as shown to FIG.8 (B)-(D), the expression of the tyrosinase, TRP-1, and DCT of the downstream was also inhibited.

  In FIG. 9, the verification result of the melanin production inhibitory effect of the cell by the fraction 3 is shown. As shown in FIG. 9, in the fraction 3, a sufficient melanin inhibitory effect was observed at a concentration of 0.75 μg / ml, which is lower than 1.5 μg / ml.

7). Comparison of Acer japonicum and existing melanin inhibitor Cultured for 24 hours, medium change, cells treated with IBMX, fractions 3, 4 (Fr. 3, 4) 1.5 μg / ml, arbutin Kojic acid was added at a concentration of 15 and 30 μg / ml, followed by culturing for 12 hours to extract RNA. Then, it was examined whether the expression of MITF, tyrosinase, TRP-1, and DCT genes was suppressed using a real-time PCR method. The results are shown in FIGS. 10 (A) to (D).

  As shown in FIG. 10, the expression of all the genes was suppressed in fractions 3 and 4. The expression of arbutin was suppressed at 30 μg / ml compared to 15 μg / ml, but the expression was activated with kojic acid. Thus, fractions 3 and 4 were found to have an MITF inhibitory effect not found in existing melanin inhibitors, and at the same time, suppressed gene expression downstream thereof.

  The effect of B16 melanoma cells on melanin production by IBMX stimulation was compared. The results are shown in FIGS. 11 (A) and (B). As shown in FIG. 11, fractions 3 and 4 suppressed melanin production at a concentration of 1/10 or less as compared with arbutin and kojic acid, which are existing melanin inhibitors.

8). Comparison of the effect of purified cassava by different stimulants 24 hours of culture, medium change, and addition of fraction 3 at a concentration of 1.5 μg / ml to cells treated with IBMX, α-MSH, Forskolin After that, the cells were cultured for 12 hours to extract RNA. Then, it was investigated whether or not to suppress the expression of MITF, tyrosinase, TRP-1, and DCT genes using a real-time PCR method. The results are shown in FIGS.

  As shown in FIG. 12, in any stimulant, fraction 3 most strongly suppressed the expression of MITF. From these results, it is considered that fraction 3 has a strong MITF inhibitory effect, and subsequently suppresses the expression of tyrosinase, TRP-1, and DCT, which are downstream genes.

  Furthermore, the effects of various stimulants on B16-melanoma cells on melanin production were compared. The results are shown in FIGS. 13 (A) and 13 (B). As shown in FIG. 13, fraction 3 was strongly suppressed regardless of the melanin produced by any stimulation of IBMX, α-MSH, and Forskolin.

  As described above, the alcoholic extract of Cyprus contains antioxidant components (fractions 1 to 3), tyrosinase inhibitory components (fractions 1 and 2), and MITF inhibitory components (fractions 3 and 4). It has been found that melanin production can be suppressed at a lower concentration than kojic acid and arbutin contained in conventional whitening agents. These fractions 1 to 4 can be purified with various solvents depending on the difference in polarity. For example, fractions 3 and 4 can be purified and used as MITF inhibitors.

9. Cell growth inhibitory action (anti-cancer action) of purified Prussian perforatum (Fraction 3)
The proliferation of various cancer cell lines was measured using cells cultured with the addition of fraction 3. The results are shown in FIG. As shown in FIG. 14, strong cell growth suppression was confirmed for fraction 3 against B16 melanoma 4A5 and human lung cancer cell line A549, which are malignant melanomas. On the other hand, the cell growth of mouse-derived mesenchymal stem cells C3H / 10T1 / 2, human liver cancer cell line HepG2, human leukemia cell line U937, and mouse-derived adipose progenitor cell 3T3-L1 is almost affected in the same concentration range. It did not reach. As described above, it was found that fraction 3 selectively shows an anticancer action against malignant melanoma and lung cancer.

  For example, Nature. 480 (7375), 94-98 (2011), A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma, Nature. 480 (7375), 99-103 (2011), A novel recurrent mutation in As disclosed in MITF predisposes to familial and sporadic melanoma, recently, it has been found that MITF is a causative gene of malignant melanoma. As shown conceptually in FIG. It is considered that the bioactive composition according to (Fraction 3) exhibited an anticancer action by suppressing this MITF. No anticancer agent targeting MITF has been obtained so far, and the present invention is considered to be extremely effective as a novel anticancer agent.

  FIG. 16 shows the result of comparison of the difference in anticancer activity depending on the cell density. As is clear from FIG. 16, it was found that fraction 3 has a stronger anticancer effect when cells are cultured at a high density.

10. Supplementary experiment (comparison with other Cacaria plants)
Below, it shows about having the whitening effect which the bioactive composition obtained from the armyworm which concerns on this invention has the extremely superior whitening effect from the composition obtained from Momijigasa which is a kind of the same Cacaria plant.

10.1. Mushroom tyrosinase inhibitory action As an extract, 88 g of Jobus mushroom was added with a solvent ((1) hot water, (2) 70% ethanol, (3) 100% methanol) and finely pulverized with a mixer. In the case of (1), it was then heated at 100 ° C. for 10 minutes. Centrifugation was performed at 3000 rpm for 10 minutes, and the upper layer was collected by filtration through filter paper. Thereafter, (1) was lyophilized, and (2) and (3) were dried under reduced pressure using a rotary evaporator to remove the solvent. After measuring the weight of the solid extract, it was adjusted to 0.1 g / ml. On the other hand, Momijigasa was similarly prepared to 0.1 g / ml.

  Take 96 μl of L-tyrosine-containing solution (2 mM L-tyrosine, solvent: 50 mM phosphate buffer pH 6.8) in 100 μl, add 50 μl of 50 mM phosphate buffer pH 6.8, and 20 μl of the extract prepared to 1 mg / ml. It was. Then, 20 μl of mushroom tyrosinase (300 U) was added and stirred, placed in a hybrid oven set at 25 ° C., incubated for 60 minutes, and the absorbance at 490 nm was measured. The results are shown in FIG.

  From FIG. 17, the extract of Aedes japonicum inhibited tyrosinase most strongly with the 70% ethanol extract, but the extract of Momijigasa showed no inhibitory effect even when extracted with any solvent.

10.2. Measurement of Antioxidant Capacity As described above, the antioxidant capacity was evaluated by measuring radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH). 100 μM DPPH (ethanol solution) was placed in a 1 ml test tube, 20 μl of the above extract adjusted to a concentration of 10 mg / ml was added, stirred for 30 seconds, and allowed to stand at room temperature in the dark for 30 minutes. Thereafter, the disappearance of radicals was measured by measuring the absorbance at 517 nm with a spectrophotometer. The results are shown in FIG.

  As shown in FIG. 18, the extract from any solvent showed stronger antioxidant ability than the extract of Momijigasa. In particular, the extract of 70% ethanol was strong in antioxidant capacity.

10.3. Melanin production inhibitory effect Using mouse-derived cell line (B16 melanoma 4A5), 10% fetal bovine serum, 100 μg / ml streptomycin, 100 μg / ml penicillin were added using DMEM as a basal medium, 37 ° C., 5% CO ₂ atmosphere The cells were cultured for 3 to 4 days. In measuring melanin production, the cells were cultured for 24 hours, the medium was changed, 100 μM IBMX, an extract and a purified product were added, and then cultured at 37 ° C. in a 5% CO ₂ atmosphere for 3 to 4 days. Melanin production was measured.

  For melanin measurement, after culturing the cells, PBS was added, the cells were scraped twice, centrifuged at 3000 rpm for 5 minutes, the supernatant was taken, and 1N NaOH was added and treated at 60 ° C. for 60 minutes. After the treatment, cells were measured at 450/655 nm and the medium at 450 nm with a microplate reader using synthetic melamine as a standard substance. The results are shown in FIGS. 19 (A) and (B).

  From FIG. 19, it was shown that the extract of Yobusuma soo has a stronger inhibitory effect on the melanin production by IBMX stimulation in B16 melanoma cells than the extract of Momijigasa. In particular, 70% ethanol extract of Jojoba masou showed strong inhibitory action on melanin production.

10.4. Effect on MITF mRNA expression Cells cultured for 12 hours to 3 days in a φ60 mm dish were washed once with 5 ml of PBS, 1 ml of TRIzol (Gibco) was added, and RNA was extracted according to a conventional method. From this, cDNA was synthesized using PrimeScript RT Master Mix (manufactured by Takara Bio Inc.), and mRNA was quantified by Real-time PCR method using SYBR Premix Ex Taq II (manufactured by Takara Bio Inc.).

  FIG. 20 shows the influence of the extract on MITF and its downstream mRNA expression when cells were treated with IBMX for 12 hours. As shown in FIG. 20, the job of 70% ethanol extract showed a strong inhibitory effect on the increase in MITF mRNA expression induced by IBMX stimulation in B16 melanoma cells, whereas the extract of 70% ethanol did not have an inhibitory effect. It was. This difference is considered to be a difference in suppression of melanin production.

  Although the present invention has been described with reference to the most practical and preferred embodiments at the present time, the invention is not limited to the embodiments disclosed herein. And can be appropriately changed without departing from the spirit or idea of the invention that can be read from the claims and the entire specification, and a bioactive composition accompanying such a change, a method for producing the bioactive composition, and microphthalmia-related Transcription factor inhibitors, and melanin production inhibitors, cosmetic compositions, anti-cancer agents should also be understood as being included within the scope of the present invention.

  The physiologically active composition according to the present invention contains an antioxidant component, a tyrosinase inhibitor component and a MITF inhibitor component (particularly MITF inhibitor component), and has a lower concentration than kojic acid and arbutin contained in conventional whitening agents. Can suppress the production of melanin and can be suitably used for cosmetics, drugs and the like.

Claims (4)

A method for producing a microphthalmia-related transcription factor inhibitor, comprising the step of: alcohol extraction of cynomolgus and / or inu-ununa to obtain the microphthalmia-related transcription factor-suppressing component derived from said petratum and / or inu-ununa . A method for producing a melanin production inhibitor comprising a step of alcohol-extracting Jobsaw and / or Inuduna to obtain a microphthalmia-related transcription factor inhibitory component derived from the Jobsaw and / or Inuduna . A method for producing a cosmetic composition for whitening , comprising a step of alcohol-extracting Jobsoma and / or Inudouna to obtain a microphthalmia-related transcription factor inhibitory component derived from the Jobsaw and / or Inudouna . The manufacturing method of any one of Claims 1-3 whose alcohol concentration in the said alcohol extraction is 25% or more and 80% or less .