Diterpene derivatives exhibiting epidermal cell growth promoting action via fibronectin activation

Abstract

To provide a diterpene derivative exhibiting an epidermal cell-proliferating action via a fibronectin-activating action.SOLUTION: A diterpene derivative exhibiting an epidermal cell proliferating action via a fibronectin activation action is composed of one molecule of diterpene and one molecule of L-tyrosine. Since they are bound by covalent bonds, they are less likely to accumulate in the body and are safe. A method of producing the same is either a fermentation method in which Porphyrum as a raw material is fermented with Lactobacillus or a cell culture method. Since it shows the proliferation of human epidermal cells through the activation of fibronectin, it is utilized as a cosmetic field or a cosmetic material. It is also used as emulsified oil of human keratinocyte-conditioned medium, emulsified oil of human fibroblast conditioned medium, or emulsified oil of human adipocyte conditioned medium.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a diterpene derivative exhibiting an epidermal cell growth promoting action through a fibronectin activating action.

Background Art

[0002] Fibronectin is a glycoprotein that constitutes one of the extracellular matrices and is involved in cell functionality and cell proliferation. Fibronectin was discovered as a blood coagulation factor and is also expressed on the surface of cancer cells, and thus is considered to have diversity in its functions. In addition, since reactivity with cell growth factor receptors has been recognized, it has been studied as a factor that regulates cell growth.

[0003] In particular, since the activation of fibronectin is related to enhanced cell function and cell proliferation, research has also been conducted in the field of anti-aging and beauty.

[0004] As an invention related to the activation of fibronectin, there is an invention of a fibronectin fragment used for the production of stem cells. Here, a method for producing stem cells that retain the ability to differentiate into various cells using fibronectin is described. (For example, see Patent Document 1). However, specific substances are not disclosed, and it is in the area of production methods.

[0005] Also, for example, there is an invention of an isolated fibronectin type III (FN3) domain, wherein the FN3 domain specifically binds to domain I or III of human serum albumin, and the serum half-life of the FN3 domain is at least 10 times higher than the serum half-life of the Tencon25 protein having the amino acid sequence of SEQ ID NO: 67. Here, a search regarding the detection of fibronectin in serum is made (for example, see Patent Document 2). Thus, many studies and inventions regarding fibronectin have been made. However, inventions regarding the utilization of fibronectin for enhancing cell function and normal cell function and cell proliferation are not recognized.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] However, the epidermal cell proliferation effect through the fibronectin activation effect by existing plant extracts is extremely mild, and there is a problem that its industrial use is limited. On the other hand, chemically synthesized substances have problems with safety, and their use is limited.

[0008] Therefore, a naturally derived substance that exhibits an epidermal cell proliferation effect through a fibronectin activation effect with weak side effects is desired.

Means for Solving the Problems

[0009] In order to achieve the above object, the invention according to claim 1 relates to a diterpene derivative that exhibits an epidermal cell proliferation effect through the fibronectin activation effect represented by the following formula (1).

[0010]

Chemical Formula

Effects of the Invention

[0011] Since this invention is configured as described above, it has the following effects.

[0012] According to the diterpene derivative described in claim 1, a naturally derived substance with few side effects and exhibiting an epidermal cell proliferation effect through an excellent fibronectin activation effect can be obtained.

Mode for Carrying Out the Invention

[0013] Hereinafter, embodiments embodying the present invention will be described in detail.

[0014] The diterpene derivative exhibiting an epidermal cell growth promoting action via fibronectin activation action has a structure represented by the following formula (1).

[0015]

Chemical formula

[0016] The diterpene derivative shown here exhibits an epidermal cell growth promoting action via fibronectin activation action. EGF receptors are present in epidermal cells, and when the EGF receptors receive EGF, epidermal cell growth is shown. Fibronectin is present in the vicinity of the EGF receptors, and the activation of fibronectin activates the EGF receptors, which is the mechanism of action.

[0017] That is, when the diterpene derivative shown here binds to fibronectin, which is a ligand, it decreases the km value, which is the binding constant of the binding. This causes hypersensitivity in which the reactivity increases. In addition, since the diterpene derivative also increases the Vmax, which is the maximum binding value, it acts through the mechanisms of action of both the binding center and the surrounding support portion.

[0018] This diterpene derivative is composed of one molecule of diterpene and one molecule of L-tyrosine as shown in formula (1). The molecular formula is C28H43N1O5, which is composed of 28 carbon atoms, 43 hydrogen atoms, 1 nitrogen atom, and 5 oxygen atoms.

[0019] This diterpene derivative can be synthesized organochemically using diterpene and L-tyrosine as raw materials and can be used for the purpose of structural analysis as a standard product. That is, by using terpene and tyrosine and combining organic chemical synthesis steps such as esterification coupling reaction, elimination reaction, hydrolysis reaction and condensation reaction, the target diterpene derivative is produced. The derivative synthesized in this way is purified by Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation), XAD-2 or XAD-4 (manufactured by Rohm and Haas), Sephadex LH-20 (manufactured by Amersham Pharmacia), ion exchange carrier IRA-410 (manufactured by Rohm and Haas), and reversed-phase carrier DM1020T (manufactured by Fuji Silysia), and a purified product with a purity of 95% or more can be obtained.

[0020] The structure of this diterpene derivative is analyzed by a nuclear magnetic resonance apparatus (for example, NMR manufactured by Bruker) through the analysis of 1H-NMR and 13C-NMR in CDCL3. This structure shows peaks at 0.95, 1.37, 1.38, 1.39, 1.56, 2.36, 2.52, 2.63, 3.16, 3.41, 3.60, 3.85, 4.09, 4.72, 4.83, 5.93, 5.99, 6.62, 6.70, 7.49 and 7.92 ppm by 1H-NMR analysis at 500 MHz.

[0021] Furthermore, by the analysis of 13C-NMR in CDCL3, peaks are observed at 14.1, 22.6, 25.6, 28.3, 29.3, 29.7, 31.8, 33.3, 34.0, 35.8, 36.4, 45.5, 51.2, 56.2, 56.5, 80.7, 93.7, 117.6, 127.1, 127.9, 131.4, 134.4, 146.0, 164.8, 170.0, 172.8 and 173.8 ppm.

[0022] The diterpene and tyrosine, which are components, are bonded by a covalent bond. That is, the meta position of the benzene ring of tyrosine and the terpene are covalently bonded via a methylene group. Generally, diterpenes exist in nature and have high safety. The diterpene derivative shown here has high safety because it is decomposed and excreted in the living body when an excessive amount is ingested. In addition, no accumulation is observed in the body or the environment, and its safety to the environment is also high.

[0023] The diterpene derivative shown here has a double bond in the terpene moiety and shows a state rich in electrons, so it exhibits a reducing action. Due to this reducing action, fibronectin is stabilized, and furthermore, the potential of the cell membrane is also stabilized. Due to this reducing action, the cell membrane of epidermal cells is stably maintained. In addition, this diterpene derivative exhibits both water solubility and fat solubility, and in particular, it has the property of existing in an emulsified state in fat-soluble solvents such as oils and fats. Since it is in an emulsified state, it is preferable that it is easily incorporated into the cell membrane of skin cells located at the boundary between fat solubility and water solubility.

[0024] This diterpene derivative binds directly to fibronectin to stabilize fibronectin. Fibronectin is involved in cell adhesion and intercellular information transmission as a cell membrane matrix. It is preferable that the action of genes is also stabilized by the activation of fibronectin.

[0025] The method of activating fibronectin by this diterpene derivative is an improvement in sensitivity, so-called hypersensitivity, which enhances the sensitivity of fibronectin. Separately from that, this diterpene derivative suppresses the degranulation of histamine in mast cells, thereby suppressing inflammatory and allergic reactions. On the other hand, an excessive amount of this diterpene derivative is decomposed by non-specific esterases present in the liver and kidneys, producing diterpene and tyrosine.

[0026] Since diterpene, aspartic acid, and tyrosine all exist in nature and their safety has been confirmed, the safety of this diterpene derivative is high.

[0027] In addition, this diterpene derivative does not accumulate in fat and is not concentrated in the body, so it has little cumulative toxicity. Furthermore, it has no accumulation in the environment and is safe.

[0028] Furthermore, this diterpene derivative activates fibronectin present extracellularly in epithelial cells other than the skin, increases the extracellular matrix, and protects tissues. For example, it also stabilizes nerve cells, muscle cells, intestinal cells, and alveolar epithelial cells.

[0029] As a method for producing this diterpene derivative, there is a method of synthesizing it organically. In addition, it can be extracted from plants, vegetables, algae, and animals, and can also be obtained by fermenting plants, etc. In particular, since it is also contained in the culture supernatant obtained by culturing human skin epidermal cells, human skin fibroblasts, or human adipocytes using royal jelly fermentation extract as a medium, the method of emulsifying the culture supernatant is preferable as a method for producing the diterpene derivative.

[0030] Among these, producing from plants by fermentation as a method for producing the diterpene derivative is preferable because of its high safety and high production efficiency. Suitable plants for fermentation include beans such as green tea and mung beans containing diterpenes, herbs such as lavender, and citrus fruits such as bergamot and mandarin oranges. In particular, algae rich in diterpenes, especially red algae such as Chondrus ocellatus (scientific name Porphyridium purpureum), are preferable as raw materials. Furthermore, it is preferable to specify the diterpene derivative for the purpose of limiting the production method by using a production method that purifies the fermentation broth obtained by fermenting Chondrus ocellatus with Lactobacillus to obtain a diterpene derivative exhibiting an epidermal cell proliferation effect through a fibronectin activation effect. This production method by fermentation is different from the production method by chemical synthesis and is a naturally occurring production method. Since impurities are also natural products, it is also preferable in terms of safety.

[0031] As a method for producing this fermentation, there is a method of fermenting chinolimo with lactic acid bacteria using chinolimo as a raw material. This method produces a large amount of the target diterpene derivative and is a natural method. Since it is not a chemical synthesis, in addition to being used as a cosmetic raw material, it is also used as a food raw material.

[0032] For example, chinolimo and lactic acid bacteria are added to a clean fermentation tank and fermented under aerobic conditions.

[0033] Chinolimo as a raw material may be from any origin such as Japan, China, the United States, Russia, etc. However, chinolimo collected in Japan is preferred because it has a high diterpene content.

[0034] When using chinolimo, after washing and drying, it is ground by grinders such as the Free Mill, Super Free Mill, Sample Mill, Goblin, Super Clean Mill, Micros manufactured by Nara Machinery Co., Ltd., and the small vacuum dryer manufactured by Toyo Riko Co., Ltd., the small vacuum heat transfer dryer DPTH-40 manufactured by Matsui Co., Ltd., the Clean Dry VD-7, VD-20 manufactured by AQM Kyushu Technos Co., Ltd., and the DM-6 manufactured by Nakayama Technical Research Institute. This grinding is preferred because it facilitates the efficient progress of the fermentation process.

[0035] The lactic acid bacteria used belong to the genus Lactobacillus of the true bacteria Lactobacillaceae with the scientific name Lactobacillus, and are useful bacteria used in the production of food and cosmetic raw materials. They are used in dairy products such as cheese and yogurt, and in vegetables such as pickled bran.

[0036] This lactic acid bacteria is highly safe and has a usage experience. Among them, the lactic acid bacteria handled by Akita Konno Shoten Co., Ltd. are also used for sake, create a lactic acid environment, and are preferred because they can efficiently carry out fermentation.

[0037] Regarding the respective addition amounts for the above fermentation, it is preferable that the amount of lactic acid bacteria is 0.002 to 0.01 weight based on 1 weight of chinolimo. Pre-culturing the lactic acid bacteria before fermentation is preferable because it shortens the initial fermentation time and the fermentation time is shortened.

[0038] The above fermentation is carried out in a clean culture tank, and it is preferable to mix the above materials with sterilized tap water.

[0039] Also, this fermentation is heated to 38 - 44°C and carried out for 5 to 18 days. After fermentation, the lactic acid bacteria are killed by heating to about 90°C and the fermentation is stopped. The target diterpene derivative is produced by this fermentation process.

[0040] In addition to being obtained by such a fermentation process, it can also be produced by a cell culture method. That is, since it is also contained in the culture supernatant obtained by culturing human skin epidermal cells, human skin fibroblasts or human adipocytes using royal jelly fermentation extract as a medium, the method of emulsifying the culture supernatant is preferable as a method for producing a diterpene derivative. Also, these human cell-conditioned culture medium emulsified oils are classified into human keratinocyte-conditioned culture medium emulsified oil, human fibroblast-conditioned culture medium emulsified oil or human adipocyte-conditioned culture medium emulsified oil according to the cell origin, and each is used as a cosmetic raw material.

[0041] Since the diterpene derivative produced by the above fermentation method or cell culture method exhibits both lipophilic and hydrophilic properties, extraction with hydrous ethanol is preferable because the product can be efficiently recovered and the next step can be easily carried out. Also, ultrasonic disruption treatment of the obtained diterpene derivative is preferable because the product is easily separated. Also, concentration by freeze-drying or the like is preferable because the following steps can be carried out in a short time.

[0042] Separating and purifying the above diterpene derivative is preferable because the intake amount can be reduced as a highly pure substance. As this purification method, it is preferable to use a purification operation such as a separation resin.

[0043] For example, it is preferable to be separated and fractionated by a separation carrier or resin. As the separation carrier or resin, porous polysaccharides, silicon oxide compounds, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymers, etc. whose surfaces are coated as described below are used. Those having a particle size of 0.1 to 300 μm are preferable. The finer the particle size, the higher the accuracy of separation, but there is a drawback that the separation time is long.

[0044] For example, those whose surfaces are coated with a hydrophobic compound as a reverse phase carrier or resin are used for the separation of highly hydrophobic substances. Those coated with a cationic substance are suitable for the separation of anionic substances. Also, those coated with an anionic substance are suitable for the separation of cationic substances. When coated with a specific antibody, it is used as an affinity carrier or resin that separates only specific substances.

[0045] The affinity carrier or resin is used for the specific preparation of an antigen by utilizing the antigen-antibody reaction. The partitioning carrier or resin is used for the isolation of those substances when there is a difference in the partition coefficient between the substance and the separation solvent, such as silica gel (manufactured by Merck).

[0046] Among these, from the viewpoint of being able to reduce the manufacturing cost, an adsorptive carrier or resin, a partitioning carrier or resin, a carrier or resin for molecular sieving, and an ion exchange carrier or resin are preferable. Furthermore, from the viewpoint of a large difference in the partition coefficient with respect to the separation solvent, a reverse phase carrier or resin and a partitioning carrier or resin are more preferable.

[0047] When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used. Also, a carrier or resin used in pharmaceutical manufacturing or food manufacturing is preferable.

[0048] From these aspects, Diaion (HP-20 type or HP21 type, manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Rohm and Haas), Sephadex LH-20 (manufactured by Amersham Pharmacia) as a carrier for molecular sieve, silica gel as a carrier for partition, IRA-410 (manufactured by Rohm and Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silysia) as a reverse phase carrier are more preferable as the adsorptive carrier.

[0049] Among these, Diaion HP-20 type, Sephadex LH-20 and DM1020T are more preferable.

[0050] The obtained extract is dissolved in a solvent for swelling the separation carrier or resin before separation. The amount is preferably 1 to 35 times, more preferably 4 to 25 times the weight of the extract from the viewpoint of separation efficiency. As the separation temperature, 4 to 30 °C is preferable, and 10 to 25 °C is more preferable from the viewpoint of the stability of the substance.

[0051] As the separation solvent, water, or a lower alcohol, a hydrophilic solvent, or a lipophilic solvent containing water is used. As the lower alcohol, methanol, ethanol, propanol, or butanol is used, and ethanol used for food is preferable.

[0052] When using Sephadex LH-20, a lower alcohol is preferable as the separation solvent. When using silica gel, chloroform, methanol, acetic acid, or a mixture thereof is preferable as the separation solvent.

[0053] When using Diaion HP-20 type and DM1020T, a lower alcohol such as methanol or ethanol, or a mixture of a lower alcohol and water is preferable as the separation solvent.

[0054] In addition, collecting the fraction containing the activity and removing the solvent by drying or vacuum drying to obtain a powder or a concentrated solution is preferable because the influence of the solvent can be excluded.

[0055] In addition, it is preferable to perform the final extraction using fats and oils for edible oils or cosmetics, since the resulting active portion is stably maintained in the oil. For example, it is preferable to extract with soybean oil, rice bran oil, grape seed oil, olive oil, or jojoba oil.

[0056] It is used as a parenteral preparation such as an injection, oral preparation, or coating agent for pharmaceuticals, and as quasi-drugs, it is used by being formulated into tablets, capsules, drink preparations, soaps, coating agents, gel preparations, toothpastes, etc.

[0057] Examples of oral preparations include tablets, capsules, powders, syrups, drink preparations, etc. When mixed with the above-mentioned tablets and capsules, they can be used together with binders, excipients, swelling agents, lubricants, sweeteners, flavoring agents, etc. The above-mentioned tablets can also be coated with shellac or sugar.

[0058] In addition, in the case of the above-mentioned capsules, materials such as fats and oils can be further contained in the above-mentioned materials. In the case of the above-mentioned syrups and drink preparations, sweeteners, preservatives, coloring flavoring agents, etc. can be added.

[0059] Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams, and aqueous solutions. As the base material for external preparations, petrolatum, paraffin, fats and oils, lanolin, macrogol, etc. are used, and they can be made into ointments, creams, etc. by ordinary methods.

[0060] Injections include solutions and, in addition, lyophilized agents. These are aseptically dissolved in distilled water for injection, physiological saline, etc. and used at the time of use.

[0061] It is used for beauty foods for anti-inflammatory and beauty purposes, foods for anti-inflammatory and beauty purposes, diet foods, tonics for maintaining liver cells, etc. as food preparations. In addition, it is preferably used for nutritional functional foods and foods for specified health uses as health functional foods.

[0062] When the obtained food preparation is used for pets or livestock animals such as dogs and cats, it is used as feed or supplements for the purpose of maintaining the overall health of the animal, including the epithelial cell proliferation effect.

[0063] It can be used as a cosmetic product together with a surfactant, a solvent, a thickener, an excipient, etc. according to a conventional method. For example, it can be in the form of a cream, a hair gel, a facial cleanser, a beauty essence, a lotion, etc., and is a cosmetic product that promotes an anti-inflammatory effect via fibronectin activation. The cosmetic product can be in any form, and can be used in the form of a solution, a cream, a paste, a gel, a gel, a solid, or a powder. In addition, it is obtained by culturing human skin epidermal cells, human skin fibroblasts, or human adipocytes using the royal jelly fermented extract as a medium, and is used as a cosmetic raw material as a human keratinocyte-conditioned culture emulsion oil, a human fibroblast-conditioned culture emulsion oil, or a human adipocyte-conditioned culture emulsion oil, respectively.

[0064] It can also be used as a plant activator to promote plant growth through cell wall and cell membrane components. It can also be used on beans, grains, rice, root vegetables and flowers, activating plant fibronectin and increasing cell reactivity, improving yield and quality, and increasing plant growth and lifespan. It can also be used to preserve cut flowers.

[0065] The above embodiment will be specifically described below using examples and test examples. Note that these are merely examples, and conditions can be changed within the bounds of common sense depending on differences in materials, raw materials, and samples. EXAMPLES

[0066] Chinorimo (scientific name: Porphyridium purpureum) was used. Dried Chinorimo sold by Kurakon Holdings Co., Ltd. (Osaka Prefecture) is produced in Hokkaido and Tohoku and is preferred due to its high quality. 1 kg of dried Chinorimo was mixed in a mixer.

[0067] The resulting crushed chinorimo was placed into a clean fermentation tank (manufactured by Owaki Engineers, 150 kg capacity).

[0068] This was suspended in 8 L of tap water. This suspension of chinolimo was placed in an autoclave (manufactured by Tomy Seiko, SR-240) and sterilized at 121°C.

[0069] Separately, lactic acid bacteria purchased from Akita Konno Shoten Co., Ltd. were suspended in sterilized water and fermented at 37°C for 1 day to obtain a preculture solution.

[0070] 0.1 kg of the preculture solution was added to the chinolimo suspension, and fermentation was carried out in a fermentation tank at 39°C to 42°C for 7 days. The degree of fermentation was observed by analyzing diterpene derivatives, the generation of bubbles, and the change in the color of the fermentation broth. After 7 days of fermentation, the fermentation broth was placed in a boiling water bath at 98°C and heated for 20 minutes to be sterilized. After cooling this, it was filtered to obtain 3.3 L of filtrate. This was used as an extract containing diterpene derivatives. This was stored in a cool and dark place until use.

[0071] 1 L of purified water containing 6% ethanol was added to 3 L of the above-mentioned extract containing diterpene derivatives. This was filtered through filter paper, and the filtrate was applied to a column filled with 200 g of Diaion HP-20 type (manufactured by Mitsubishi Chemical) suspended in a 6% ethanol solution.

[0072] 3 L of 10% ethanol solution was added thereto for cleaning, and further, 2 L of 20% ethanol solution was added for washing. After that, 60% ethanol was applied to elute the target diterpene derivative. The purified diterpene derivative was subjected to vacuum distillation to remove the ethanol portion and made into an aqueous solution. This purification process was repeated 3 times. After the aqueous solution of the third time was dried under reduced pressure, it was dried to obtain a powder, which was designated as Specimen 1.

[0073] The identification test of the diterpene derivative will be described below. (Test Example 1)

[0074] Specimen 1, which is the diterpene derivative of Example 1 obtained as described above, was analyzed with a nuclear magnetic resonance apparatus (NMR, manufactured by Bruker) using CDCL3 as a solvent.

[0075] As a result, 1H-NMR analysis at 500 MHz in CDCl3 revealed peaks at 0.95, 1.37, 1.38, 1.39, 1.56, 2.36, 2.52, 2.63, 3.16, 3.41, 3.60, 3.85, 4.09, 4.72, 4.83, 5.93, 5.99, 6.62, 6.70, 7.49 and 7.92 ppm.

[0076] Furthermore, 13C-NMR analysis in CDCl3 revealed peaks at 14.1, 22.6, 25.6, 28.3, 29.3, 29.7, 31.8, 33.3, 34.0, 35.8, 36.4, 45.5, 51.2, 56.2, 56.5, 80.7, 93.7, 117.6, 127.1, 127.9, 131.4, 134.4, 146.0, 164.8, 170.0, 172.8 and 173.8 ppm.

[0077] The following shows the chart of the 13C-NMR analysis results. (The horizontal axis unit is ppm, and the vertical axis unit indicates peak intensity.) JPEG0007687973000003.jpg2094

[0078] That is, Specimen 1 of Example 1 showed the same analysis results as the standard product synthesized and purified organochemically. These analyses identified it as the target diterpene derivative. That is, it was composed of one molecule of diterpene and tyrosine and was identical to the structure shown by Formula (1). Also, the target diterpene derivative showing a single peak was identified by high performance liquid chromatography, and its purity was 98.2%.

[0079] The following describes the confirmation test of the human epidermal cell proliferation effect through fibronectin activation. (Test Example 2)

[0080] Human epidermal cells (product name: Epidermal Keratinocytes) purchased from Funakoshi Co., Ltd. and a dedicated medium (CnT-PR) were used in the test. The purchased human epidermal cells were suspended in the dedicated medium and seeded in a culture dish (Falcon, 35 mm diameter) and cultured at 37°C under 5% carbon dioxide. After confirming the cell proliferation, the cells were suspended in a PBS solution containing trypsin and 1000 cells were seeded together with 1 mL of the medium in a new culture dish. This was cultured for 1 day, and the above Specimen 1 and EGF (human recombinant / animal-derived free type, manufactured by Fujifilm Wako Pure Chemical Corporation) were dissolved in PBS and added at a final concentration of 0.1 mg / mL. The same amount of PBS was added as a solvent control. This was cultured for 48 hours.

[0081] After 48 hours, the number of living cells was counted under a microscope by the trypan blue dye method. As a result, the addition of 0.1 mg / ml of Specimen 1 increased the cell number by 230% on average compared to the solvent control group. Also, in the case of the addition of the control EGF, the cell number was 188% compared to the control group. That is, Specimen 1 showed an epidermal cell proliferation effect superior to that of EGF.

[0082] Furthermore, the activation state of fibronectin in the above specimen-treated epidermal cells was quantified by the BIA (manufactured by GE, model T200) method. BIA stands for Biophysical Interaction Analysis. That is, it is a biophysical interaction analysis method and a method for measuring intermolecular interactions in vivo. The base was coated with fibronectin (manufactured by Fujifilm Wako Pure Chemical Corporation, fibronectin solution, derived from human plasma), and the reactivity of the cell suspension with this fibronectin was quantified.

[0083] As a result, the km value of the binding of the epidermal cells added with Specimen 1 to fibronectin decreased by 79% on average compared to the solvent control group, and the Vmax increased by 186%. The decrease in this km value indicates that fibronectin binds even at low concentrations. Vmax is the maximum binding number, and the high Vmax means that the maximum value of the binding is high, indicating that the binding was promoted.

[0084] From these results, it can be concluded that Specimen 1 activated fibronectin and exhibited an epidermal cell growth-promoting effect. On the other hand, in the EGF-added group used as a control, the Km value of the binding affinity with fibronectin was 101% on average and the Vmax was 102% compared to the solvent control group. It was found that EGF did not activate fibronectin and did not promote cell growth.

[0085] The apoptosis induction test of human cancer cells will be described below. This test method is a reproducible conventional method that can biochemically and molecularly verify the function of the active ingredient, and there are abundant test results and high reliability. The fibronectin activation effect is also related to apoptosis induction. (Test Example 3)

[0086] A human breast cancer cell line (MCF-7, No. 300273, Human Breast Adenocarcinoma) purchased from Cosmo Bio Co., Ltd. was used. 1000 cells cultured using a dedicated culture medium as the culture solution were seeded in a 35 mm culture dish and cultured at 37 °C under 5% carbon dioxide.

[0087] Here, Specimen 1 obtained in Example 1 above and mitomycin C, an anticancer agent, as a positive control (manufactured by Nacalai Tesque, 1 mg / ml) were both added at a final concentration of 10 mg / ml. This was cultured for 48 hours.

[0088] After the culture was completed, the number of breast cancer cells was counted microscopically. Furthermore, fibronectin of breast cancer cells was measured by the same method as above. Also, after PI staining as an index of apoptosis, fragmented deformation of the nucleus (apoptotic image) was counted under a fluorescence microscope.

[0089] As a result, the addition of 10 mg / ml of Specimen 1 decreased the number of breast cancer cells to 56% on average compared to the solvent control group. On the other hand, mitomycin C was 69%. From this result, Specimen 1 exhibited a more excellent inhibitory effect on breast cancer cells than mitomycin C.

[0090] In addition, as a result of measuring the fibronectin activation effect by BIA, when specimen 1 was added compared to the control group, Vmax increased to 188%, indicating the activation effect of fibronectin. In the count of apoptotic cells, a 222% increase in apoptosis was observed compared to the control with solvent addition. This result shows that specimen 1 enhances the cancer cell inhibitory effect.

[0091] On the other hand, in a skin irritation experiment using EpiSkin (manufactured by SkinEthic), which is artificial skin, as part of the safety test, no irritation was observed upon addition of specimen 1, confirming its safety. This method has been established as a method that does not use animals for evaluating skin irritation test using cells.

[0092] Furthermore, the target diterpene derivative was also produced by a human cell culture method in the same way as obtained in the fermentation process described above. That is, the culture supernatant obtained by culturing human skin epidermal cells, human skin fibroblasts, or human adipocytes using royal jelly fermentation extract as a medium was extracted and purified with a fat-soluble solvent. As a result, a target diterpene derivative with a purity of 98.3% at the same level as the fermentation process could be produced. The obtained diterpene derivative showed the same physiological activity as the diterpene derivative obtained by the fermentation method.

Industrial Applicability

[0093] The diterpene derivative obtained in the present invention shows the growth promoting effect of epidermal cells through the activation effect of fibronectin, and has few side effects. Therefore, it can improve the QOL of the people, increase the healthy working population, and reduce medical expenses.

[0094] Since the diterpene derivative obtained in the present invention has the effect of reducing cancer cells through the activation effect of fibronectin, it can be used for the purpose of cancer treatment.

[0095] Since the diterpene derivative obtained in the present invention can also be used as a food, it contributes to the development of the food industry.

Claims

【Claim 1】 A diterpene derivative that exhibits an epidermal cell growth promoting effect via a fibronectin activation effect represented by the following formula (1). 【Chemical 1】

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