JP7687973B2 - Diterpene derivatives that promote epidermal cell proliferation through fibronectin activation - Google Patents

Description

This invention relates to diterpene derivatives that exhibit epidermal cell proliferation activity via fibronectin activation.

Fibronectin is a glycoprotein that constitutes part of the extracellular matrix 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, so it is thought to have diverse functions. In addition, because it has been shown to be reactive with cell growth factor receptors, it has been studied as a factor that regulates cell growth.

In particular, since fibronectin activation is involved in cell function enhancement and cell proliferation, it has also been studied in the field of anti-aging and beauty.

An invention related to fibronectin activation is an invention of a fibronectin fragment used in the production of stem cells. This invention describes a method of using fibronectin to produce stem cells that retain the ability to differentiate into various cells. (See, for example, Patent Document 1). However, no specific substances are disclosed, and this falls within the realm of production methods.

Also, for example, there is an invention of an isolated fibronectin type III (FN3) domain, which specifically binds to domain I or III of human serum albumin and has a serum half-life at least 10 times higher than the serum half-life of Tencon25 protein having the amino acid sequence of SEQ ID NO:67, and a search is conducted for the detection of fibronectin in serum (see, for example, Patent Document 2). Thus, many studies and inventions have been made on fibronectin. However, no inventions have been found that relate to the use of fibronectin to enhance cell function and for normal cell function and cell proliferation.

Patent application No. 2018-530429 Patent application No. 2018-562935

However, the epidermal cell proliferation effect of existing plant extracts via fibronectin activation is extremely mild, limiting their industrial use. On the other hand, chemically synthesized substances have safety issues, limiting their use.

Therefore, there is a demand for naturally occurring substances that have minimal side effects and exhibit excellent epidermal cell proliferation effects via fibronectin activation.

In order to achieve the above object, the invention described in claim 1 relates to a diterpene derivative that exhibits epidermal cell proliferation activity via fibronectin activation activity, as shown in the following formula (1).

As this invention is configured as described above, it has the following advantages:

The diterpene derivative described in claim 1 provides a naturally derived substance that has few side effects and exhibits excellent epidermal cell proliferation activity via fibronectin activation.

The following is a detailed description of an embodiment of the invention.

The diterpene derivative that exhibits epidermal cell proliferation activity via fibronectin activation activity has a structure represented by the following formula (1).

The diterpene derivatives shown here have an epidermal cell proliferation effect mediated by fibronectin activation. EGF receptors are present in epidermal cells, and the EGF receptors receive EGF, resulting in epidermal cell proliferation. Fibronectin is present in the vicinity of the EGF receptor, and the mechanism of action is that the EGF receptor is activated by activating fibronectin.

In other words, when the diterpene derivatives shown here bind to the ligand fibronectin, they lower the km value, which is the binding constant of the bond. This leads to hypersensitivity, which means increased reactivity. Furthermore, since the diterpene derivatives also increase Vmax, which is the maximum binding value, they work through the mechanism of both the binding center and the surrounding support moieties.

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, and it is composed of 28 carbons, 43 hydrogens, 1 nitrogen, and 5 oxygens.

The diterpene derivative can be synthesized organically using diterpenes and L-tyrosine as raw materials, and can be used as a standard for structural analysis. In other words, the desired diterpene derivative is produced by combining organic chemical synthesis steps such as esterification bond reaction, elimination reaction, hydrolysis reaction and condensation reaction using terpenes and tyrosine. The derivative synthesized in this way is purified using Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) and 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 reverse phase carrier DM1020T (manufactured by Fuji Silysia Pharmaceuticals), and a purified product with a purity of 95% or more can be obtained.

The structure of this diterpene derivative is analyzed by 1H-NMR and 13C-NMR in CDCL3 using a nuclear magnetic resonance apparatus (e.g., Bruker NMR). 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 in 500 MHz 1H-NMR analysis.

Furthermore, 13C-NMR analysis of CDCL3 showed 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.

The diterpene and tyrosine components are covalently bonded. In other words, the meta position of the benzene ring of tyrosine is covalently bonded to the terpene via a methylene. Diterpenes generally exist in nature and are highly safe. The diterpene derivatives shown here are highly safe because they are broken down and excreted in the body when ingested in excess. In addition, they have no observed tendency to accumulate in the body or the environment, making them highly safe for the environment.

The diterpene derivatives shown here have a double bond in the terpene moiety and are electron-rich, exerting a reducing effect. This reducing effect stabilizes fibronectin and also stabilizes the potential of the cell membrane. This reducing effect maintains the stability of the cell membrane of epidermal cells. In addition, this diterpene derivative is both water-soluble and fat-soluble, and has the property of existing in an emulsified state in fat-soluble solvents such as oils and fats. Because it is in an emulsified state, it is advantageous in that it is easily taken up into the cell membranes of skin cells, which are located at the boundary between fat-soluble and water-soluble.

This diterpene derivative binds directly to fibronectin and stabilizes it. Fibronectin is involved in cell-cell junctions and intercellular information transmission as a cell membrane matrix. Activation of fibronectin also favorably stabilizes gene function.

The method of fibronectin activation by this diterpene derivative is to improve sensitivity, or so-called hypersensitivity, which enhances the sensitivity of fibronectin. Apart from that, this diterpene derivative inhibits the degranulation of histamine in mast cells, thereby suppressing inflammatory and allergic reactions. On the other hand, excess amounts of this diterpene derivative are broken down by non-specific esterases present in the liver and kidneys, producing diterpenes and tyrosine.

Diterpene, aspartic acid, and tyrosine all exist in nature and their safety has been confirmed, so this diterpene derivative is highly safe.

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 does not accumulate in the environment and is safe.

Furthermore, this diterpene derivative activates fibronectin present outside the cells of epithelial cells other than the skin, increasing the extracellular matrix and protecting tissues. For example, it also stabilizes nerve cells, muscle cells, intestinal cells, and alveolar epithelial cells.

The diterpene derivatives can be produced by organic chemical synthesis, extraction from plants, vegetables, algae or animals, or by fermenting plants. In particular, they are contained in the culture supernatant obtained by culturing human skin epidermal cells, human skin fibroblasts or human adipocytes in a medium containing royal jelly fermented extract, so the method of emulsifying the culture supernatant is preferred as a method for producing diterpene derivatives.

Among these, the method of producing diterpene derivatives from plants by fermentation is preferred because it is safe and highly efficient. Suitable plants for fermentation include green tea, beans such as mung beans, herbs such as lavender, and citrus fruits such as bergamot and mandarin oranges, all of which contain diterpenes. In particular, algae rich in diterpenes, particularly red algae such as Porphyridium purpureum, are preferred as raw materials. Furthermore, it is preferred to specify the diterpene derivatives that exhibit epidermal cell proliferation activity via fibronectin activation activity by using a limited production method, which is produced by fermenting Porphyridium purpureum with lactobacillus and purifying the fermented liquid. This fermentation production method is different from the production method by chemical synthesis, and is a naturally occurring production method, and impurities are also natural products, so it is also preferred in terms of safety.

One method for producing this fermentation is to use Lactobacillus subtilis as the raw material, which produces a large amount of the desired diterpene derivatives and is a natural method. As it is not a chemical synthesis, it is used as an edible ingredient in addition to being a cosmetic ingredient.

For example, chinorimo and lactobacillus are added to a clean fermentation tank and fermented under aerobic conditions.

The raw material, Chinorimo, can be from anywhere, including Japan, China, America, and Russia, but Chinorimo harvested in Japan is preferred due to its high diterpene content.

When used, Chinorimo is washed and dried, and then crushed in a crusher such as the Jiyu Mill, Super Jiyu Mill, Sample Mill, Goblin, Super Clean Mill, or Micros manufactured by Nara Machinery Co., Ltd., a small vacuum dryer manufactured by Toyo Riko Co., Ltd., a small vacuum heat transfer dryer DPTH-40 manufactured by Matsui Co., Ltd., Clean Dry VD-7 and VD-20 manufactured by ACM Kyushu Technos Co., Ltd., or a DM-6 manufactured by Nakayama Technical Research Institute. This type of crushing is preferable as it allows the fermentation process to proceed more efficiently.

The lactobacillus bacteria used are microorganisms with the scientific name Lactobacillus that belong to the Lactobacillaceae family of eubacteria, and are useful bacteria that are used in the manufacture of edible and cosmetic ingredients. They are used in dairy products such as cheese and yogurt, and in vegetable pickles such as bran pickles.

This lactobacillus is highly safe and has a history of use. Of these, the lactobacillus handled by Akita Konno Shoten Co., Ltd. is also used for sake brewing, and is preferred because it creates an acidic lactic acid environment and efficiently carries out fermentation.

The amount of each of the above fermentation additives is preferably 0.002 to 0.01 weight of lactobacillus per 1 weight of Chinese cucumber. It is preferable to pre-culture the lactobacillus before fermentation, as this shortens the initial fermentation time and shortens the fermentation time.

The fermentation is preferably carried out in a clean culture tank and the materials are mixed with sterilized tap water.

Furthermore, this fermentation is heated to 38-44°C and continues for 5 to 18 days. After fermentation, the lactobacillus is killed by heating to about 90°C, and the fermentation is stopped. The desired diterpene derivatives are produced by this fermentation process.

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 fermented extract as a medium, a method of emulsifying the culture supernatant is a preferred method for producing diterpene derivatives. Furthermore, these human cell-conditioned culture emulsion oils are classified into human keratinocyte-conditioned culture emulsion oil, human fibroblast-conditioned culture emulsion oil, and human adipocyte-conditioned culture emulsion oil depending on the origin of the cells, and each is used as a cosmetic ingredient.

The diterpene derivatives produced by the above-mentioned fermentation method or cell culture method are both fat-soluble and water-soluble, so extraction with aqueous ethanol is preferred because the product can be efficiently recovered and the next step can be easily carried out. Ultrasonic disruption of the obtained diterpene derivative is also preferred because the product can be easily separated. Concentration by freeze-drying or the like is also preferred because the following steps can be carried out in a short time.

It is preferable to separate and purify the diterpene derivatives, since they are highly pure substances and the amount ingested can be reduced. As a method for this purification, it is preferable to use a purification procedure such as a separation resin.

For example, it is preferable to separate and fractionate using 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 with a particle size of 0.1 to 300 μm are preferable, and the finer the particle size, the more accurate the separation, but the disadvantage is that the separation time is long.

For example, reversed-phase carriers or resins whose surfaces are coated with hydrophobic compounds are used to separate highly hydrophobic substances. Those coated with cationic substances are suitable for separating anionically charged substances. And those coated with anionic substances are suitable for separating cationically charged substances. When coated with a specific antibody, they are used as affinity carriers or resins that separate only specific substances.

Affinity carriers or resins are used to specifically prepare antigens by utilizing antigen-antibody reactions. Partitioning carriers or resins, such as silica gel (Merck), are used to isolate substances when there is a difference in the partition coefficient between the substances and the separation solvent.

Among these, adsorption carriers or resins, distribution carriers or resins, molecular sieve carriers or resins, and ion exchange carriers or resins are preferred because they can reduce production costs. Furthermore, reverse phase carriers or resins and distribution carriers or resins are more preferred because they have a large difference in distribution coefficient with respect to the separation solvent.

When using an organic solvent as the separation solvent, a carrier or resin that is resistant to the organic solvent is used. In addition, carriers or resins used in pharmaceutical or food manufacturing are preferred.

From these viewpoints, more preferred are Diaion (HP-20 or HP21, manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Rohm and Haas) as adsorption carriers, Sephadex LH-20 (manufactured by Amersham Pharmacia) as molecular sieve carriers, silica gel as distribution carriers, IRA-410 (manufactured by Rohm and Haas) as ion exchange carriers, and DM1020T (manufactured by Fuji Silysia) as reverse phase carriers.

Of these, Diaion HP-20 type, Sephadex LH-20 and DM1020T are more preferred.

The obtained extract is dissolved in a solvent to swell the separation carrier or resin before separation. From the viewpoint of separation efficiency, the amount is preferably 1 to 35 times the weight of the extract, and more preferably 4 to 25 times. From the viewpoint of substance stability, the separation temperature is preferably 4 to 30°C, and more preferably 10 to 25°C.

The separation solvent is water, or a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent. Examples of lower alcohols that can be used include methanol, ethanol, propanol, and butanol, with ethanol being preferred as it is used for food.

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

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

In addition, it is preferable to collect the fraction containing the activity, remove the solvent by drying or vacuum drying, and obtain it as a powder or concentrated liquid, since this eliminates the effects of the solvent.

In addition, it is preferable to carry out the final extraction using edible oils or oils and fats used in cosmetics, since the active moieties obtained remain stable in the oil. For example, it is preferable to extract using soybean oil, rice bran oil, grapeseed oil, olive oil, or jojoba oil.

As a medicine, it is used as an injection, oral preparation, or parenteral preparation such as a topical application, and as a quasi-drug, it is used by being incorporated into tablets, capsules, drinks, soaps, topical applications, gels, toothpaste, etc.

Oral preparations include tablets, capsules, powders, syrups, drinks, etc. When mixed with the tablets and capsules, they can be used together with binders, excipients, bulking agents, lubricants, sweeteners, flavoring agents, etc. The tablets can also be coated with shellac or sugar.

In the case of the capsules, the ingredients mentioned above can further contain other materials such as oils and fats. In the case of the syrups and drinks, sweeteners, preservatives, colorants, flavorings, etc. can be added.

Parenteral preparations include external preparations such as ointments, creams, and solutions, as well as injections. Vaseline, paraffin, oils and fats, lanolin, macrogold, etc. are used as base materials for external preparations, and they can be made into ointments, creams, etc. by conventional methods.

Injectables include liquid formulations and freeze-dried formulations. When used, these are dissolved aseptically in distilled water for injections or physiological saline solution.

As a food preparation, it is used in beauty foods for anti-inflammatory and beauty purposes, foods for anti-inflammatory and beauty purposes, diet foods, and tonics for maintaining liver cells. It is also preferable to use it as a health functional food in nutritional functional foods and foods for specified health uses.

When the resulting food preparation is used for pets or livestock animals such as dogs and cats, it is used as feed or supplements to maintain the overall health of the animal, including promoting epithelial cell proliferation.

As a cosmetic, it can be used together with surfactants, solvents, thickeners, excipients, etc. in the usual manner. For example, it can be in the form of cream, hair gel, face wash, beauty essence, lotion, etc., and is a cosmetic that promotes anti-inflammatory action via fibronectin activation. The cosmetic can be in any form, and can be used in the form of a solution, cream, paste, gel, gel, solid, or powder. In addition, it is obtained by culturing human skin epidermal cells, human skin fibroblasts, or human adipocytes in a medium using royal jelly fermented extract, 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.

It can also be used as a plant activator to invigorate plants by promoting proliferation through plant 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 yields and quality, and enhancing plant growth and lifespan. It can also be used to preserve cut flowers.

The above embodiment will be specifically explained below using examples and test examples. Note that these are only examples, and conditions can be changed within the bounds of common sense depending on the materials, raw materials, and specimens.

Porphyridium purpureum (scientific name) was used. Dried porphyridium purpureum 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 porphyridium purpureum was mixed in a mixer.

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

This was suspended in 8 L of tap water. The chinorimo suspension was placed in an autoclave (Tomy Seiko, SR-240) and sterilized at 121°C.

Separately, lactobacillus bacteria purchased from Akita Konno Shoten Co., Ltd. was suspended in sterilized water and fermented at 37°C for one day to prepare a preculture solution.

0.1 kg of preculture liquid was added to the Chinese moss suspension and fermented in a fermentation tank at 39°C to 42°C for 7 days. The degree of fermentation was observed by analysis of diterpene derivatives, the generation of bubbles, and color changes in the fermented liquid. After 7 days of fermentation, the fermented liquid was placed in a boiling water bath at 98°C and heated for 20 minutes to sterilize. After cooling, it was filtered to obtain 3.3 L of filtrate. This was used as an extract containing diterpene derivatives. It was stored in a cool, dark place until use.

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

3 L of 10% ethanol solution was added to this to clean it, and then 2 L of 20% ethanol solution was added to wash it. After this, 60% ethanol was added to elute the desired diterpene derivative. The purified diterpene derivative was distilled under reduced pressure to remove the ethanol portion, and an aqueous solution was obtained. This purification process was repeated three times. The aqueous solution from the third run was dried under reduced pressure, and then dried and powdered, which was designated as sample 1.

The identification test for diterpene derivatives is described below.
(Test Example 1)

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

As a result, 500 MHz 1H-NMR analysis 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.

Furthermore, 13C-NMR analysis of 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.

The chart showing the results of 13C-NMR analysis is shown below. (The horizontal axis indicates ppm, and the vertical axis indicates peak intensity.)

In other words, sample 1 in Example 1 showed the same analytical results as the standard product synthesized and purified by organic chemistry. These analyses identified it as the desired diterpene derivative. That is, it was composed of one molecule of diterpene and tyrosine, and was identical to the structure shown in formula (1). In addition, the desired diterpene derivative was identified as showing a single peak by high performance liquid chromatography, and its purity was 98.2%.

The following describes a test to confirm the effect of activating fibronectin on the proliferation of human epidermal cells.
(Test Example 2)

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 (35 mm diameter, made by Falcon) and cultured at 37°C under 5% carbon dioxide. After confirming the proliferation of the cells, the cells were suspended in a trypsin-containing PBS solution and 1000 cells were seeded together with 1 mL of medium in a new culture dish. This was cultured for one day, and the above-mentioned sample 1 and EGF (human recombinant/animal-derived free type, made by Fujifilm Wako Pure Chemical Industries) were dissolved in PBS and added to the dish 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.

After 48 hours, the number of live cells was counted under a microscope using the trypan blue dye method. As a result, the addition of 0.1 mg/ml of sample 1 increased the cell count to an average of 230% compared to the solvent control group. Furthermore, the addition of EGF as a control increased the cell count to 188% compared to the control group. In other words, sample 1 demonstrated a superior epidermal cell proliferation effect to EGF.

Furthermore, the activation state of fibronectin in the epidermal cells treated with the above sample was quantified by the BIA method (GE, T200 model). BIA stands for Biophysical Interaction Analysis. In other words, it is a biophysical interaction analysis method that measures intermolecular interactions in vivo. A substrate was coated with fibronectin (manufactured by Fujifilm Wako Pure Chemical Industries, fibronectin solution, derived from human plasma), and the reactivity of this fibronectin with the cell suspension was quantified.

As a result, the km value of the binding affinity of epidermal cells to fibronectin added with sample 1 decreased to an average of 79% compared to the solvent control group, and Vmax increased to 186%. This decrease in km value indicates that fibronectin binds even at low concentrations. Vmax is the maximum number of bindings, and a high Vmax means a high maximum value of binding, and binding was promoted.

These results indicate that sample 1 activated fibronectin and exerted the effect of proliferating epidermal cells. On the other hand, in the control group to which EGF was added, the average km value of binding to fibronectin was 101% and the Vmax was 102% compared to the solvent control group. It was found that EGF does not activate fibronectin but rather causes cells to proliferate.

The apoptosis induction test for human cancer cells is described below. This test method is a reproducible standard method that can verify the function of active ingredients biochemically and molecular biologically, has a wealth of test results, and is highly reliable. Fibronectin activation is also involved in apoptosis induction.
(Test Example 3)

A human breast cancer cell line (MCF-7, No. 300273, Human Breast Adenocarcinoma) purchased from Cosmo Bio Co., Ltd. was used. 1,000 cells cultured in a dedicated culture medium were seeded on a 35 mm culture dish and cultured at 37°C under 5% carbon dioxide gas.

To this, sample 1 obtained in Example 1 above and the anticancer drug mitomycin C (Nacalai Task, 1 mg/ml) as a positive control were added at a final concentration of 10 mg/ml. This was then cultured for 48 hours.

After the culture was completed, the number of breast cancer cells was counted microscopically. In addition, fibronectin in the breast cancer cells was measured using the same method as above. In addition, after PI staining as an indicator of apoptosis, the fragmented deformation of the nucleus (apoptotic image) was counted under a fluorescent microscope.

As a result, the addition of 10 mg/ml of sample 1 reduced the number of breast cancer cells to an average of 56% compared to the solvent control group. On the other hand, the number was reduced to 69% with mitomycin C. As a result, sample 1 exhibited a superior inhibitory effect on breast cancer cells than mitomycin C.

Furthermore, the results of measuring fibronectin activation by BIA showed that the addition of sample 1 increased Vmax by 188% compared to the control group, indicating fibronectin activation. Counting the number of apoptotic images showed a 222% increase in apoptosis compared to the solvent-added control. These results indicate that sample 1 enhances the cancer cell suppression effect.

On the other hand, in a skin irritation experiment using EpiSkin (manufactured by SkinEthic), an artificial skin, as part of a safety test, no irritation was observed with the addition of Sample 1, confirming its safety. This method has been established as a method for evaluating skin irritation using cells without using animals.

Furthermore, the desired diterpene derivatives were also produced by human cell culture, in the same way as those obtained by the fermentation process described above. That is, human skin epidermal cells, human skin fibroblasts, or human adipocytes were cultured in royal jelly fermentation extract as a medium, and the culture supernatant obtained was extracted with a fat-soluble solvent and purified. This enabled the production of the desired diterpene derivatives with a purity of 98.3%, the same level as the fermentation process. The diterpene derivatives obtained showed physiological activity similar to that of the diterpene derivatives obtained by the fermentation method.

The diterpene derivative obtained by this invention promotes the proliferation of epidermal cells through the activation of fibronectin, and has few side effects, improving the quality of life of the nation, increasing the healthy working population, and reducing medical costs.

The diterpene derivatives obtained in the present invention have the effect of reducing cancer cells through the activation of fibronectin, and can therefore be used for anticancer purposes.

The diterpene derivatives obtained by this invention can also be used as food, contributing to the development of the food industry.

Claims (1)

A diterpene derivative having an epidermal cell proliferation effect via fibronectin activation action, as shown in the following formula (1).