Moranoline derivatives exhibiting epidermal cell proliferation activity via stem cell factor activity and their production method

Abstract

To provide a moranoline derivative exhibiting epidermal cell expanding effect via stem cell factor action and methods of making thereof.SOLUTION: The moranoline derivative of the invention is an amphiphatic low molecular weight compound constituted from three molecules of moranoline and one molecule of phenylalanine, which acts as a stem cell factor-like ligand and exhibits epidermal cell-proliferating activity. Combination use of the moranoline derivative with EGF is synergistic. The moranoline derivative can be used as a water soluble or oil soluble cosmetic as well as a treating agent for the nerve. A production method comprises the steps of culturing pig dental pulp cells in a culture medium prepared by fermenting mulberry leaf with Bacillus natto to obtain a pig dental pulp cell-conditioned medium, and purifying the conditioned medium.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a moranoline derivative which exhibits epidermal cell proliferation activity via stem cell factor activity, and a method for producing the same. [Background technology]

[0002] Stem cells are involved in the regeneration of cells and tissues. For this reason, stem cell activation is being studied to prevent aging. Stem cells exist in tissues throughout the body and are involved in the regeneration of tissues. However, stem cells require stem cell factor, a growth factor, for self-proliferation. This stem cell factor contains components specific to organs and active components common to the entire body.

[0003] Stem cells have stem cell factor receptors, and when they bind to these receptors, signal transduction is activated in the cells, ultimately amplifying genes required for proliferation. In other words, activation of stem cell factor receptors is essential for stem cell proliferation.

[0004] As a means of activating stem cell factors, it is possible to use both hypersensitization, which is an increase in the sensitivity of the stem cell factor receptor binding site, and upregulation, which is an increase in the number of stem cell factor receptors. If stem cell factors can be activated by both of these methods, a synergistic effect on stem cell proliferation and regeneration can be expected.

[0005] There are few examples of inventions related to stem cell factors. For example, there is an invention related to genetic marking of cells and its use for the prevention and treatment of diseases. Although there is a mention of stem cell factor receptors, it is not an invention that activates stem cell factor receptors (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special table 2002-529080 Summary of the Invention [Problem to be solved by the invention]

[0007] The epidermal cell proliferation effect of existing substances via stem cell factor activity is mild, which limits their industrial use, and chemically synthesized substances have safety issues, which limits their use. Furthermore, their manufacturing methods have not been established.

[0008] Therefore, there is a demand for natural products that have few side effects and exhibit excellent epidermal cell proliferation effects via stem cell factor activity, and for production methods using natural products. [Means for solving the problem]

[0009] In order to achieve the above-mentioned object, the invention described in claim 1 relates to a moranoline derivative that exhibits epidermal cell proliferation activity via stem cell factor activity, as shown in the following formula (1).

[0010] [ka] In order to achieve the above object, the invention described in claim 2 relates to a method for producing a moranoline derivative represented by formula (1) described in claim 1 using natural products. Effect of the Invention

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

[0012] The moranolin derivative described in claim 1 has excellent epidermal cell proliferation activity via stem cell factor activity.

[0013] The method for producing a moranoline derivative according to claim 2 is superior because it utilizes natural products. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail.

[0015] A moranolin derivative that exhibits epidermal cell proliferation activity via stem cell factor activity has a structure represented by the following formula (1).

[0016] [ka]

[0017] As shown in formula (1) above, the moranoline derivative, which exhibits epidermal cell proliferation activity via stem cell factor activity, is composed of three molecules of moranoline and one molecule of phenylalanine. All of these molecules and their bonds are natural types that exist in nature, and the molecules are bonded together via ether bonds or ester bonds. Its chemical formula is C27H42O14N4, and it is formed from 27 carbon atoms, 42 hydrogen atoms, 14 oxygen atoms, and 4 nitrogen atoms.

[0018] This moranoline derivative can be obtained by chemical ester synthesis or ether synthesis processes using moranoline and phenylalanine as raw materials. For example, moranoline and phenylalanine can be commercially available chemical products or purified products. They can be chemically synthesized by conventional organic synthesis methods while protecting the functional groups.

[0019] On the other hand, its chemical synthesis results in a large loss of raw materials and high production costs, limiting its industrial use. The chemically synthesized moranoline derivatives with high purity are used to obtain standards for quantitative and qualitative analysis.

[0020] Analyzing the structure of this moranolin derivative is preferable because it allows identification of the target active ingredient, and is also preferable because it can be used as an index of the content of the active ingredient when used in products or formulations for sale.

[0021] As an example of the structural analysis of this moranoline derivative, when a chemically synthesized high-purity (96% or higher) standard substance was used for analysis by 600 MHz H-NMR (1H-NMR) in deuterated dimethyl sulfoxide, peaks were observed at 0.98, 0.99, 1.00, 1.02, 1.03, 2.11, 2.12, 2.13, 2.15, 2.22, 2.25, 2.31, 2.66, 2.76, 3.72, 3.79, 4.74, 4.84, 4.97, 5.03, 6.64, and 6.68 ppm.

[0022] In addition, C-NMR (13C-NMR) analysis showed peaks at 17.5, 18.9, 21.1, 22.4, 22.5, 22.6, 25.9, 26.0, 30.2, 41.1, 43.4, 43.5, 50.1, 54.7, 62.4, 70.8, 77.7, 80.4, 80.9, 90.2, 113.9, 146.0, 171.4, 172.2, 172.3 and 174.8 ppm.

[0023] Furthermore, this moranolin derivative is analyzed by high performance liquid chromatography etc. to identify its structure. The component moranolin is a naturally occurring compound, and is found in insects such as silkworms, plants and microorganisms, and its safety has been confirmed.

[0024] In particular, mulberry and mulberry leaves naturally contain moranolin, and it is possible to produce the desired moranolin derivatives by fermenting mulberry and mulberry. It is preferable to use these vegetables, algae, fruits, and grains as raw materials for producing the desired moranolin derivatives.

[0025] Originally, moranolin itself has functions such as protecting skin cells, anti-inflammatory effects, antitumor effects, fat burning effects, and neuroprotective effects. However, moranolin itself is not sufficiently absorbed, pharmacokinetically, or pharmacodynamically, and has little industrial application. Therefore, a substance with better absorption and stronger effects was desired.

[0026] This moranolin derivative is preferred because it has excellent structural characteristics in terms of both absorption and action. That is, this moranolin derivative has a hydrophobic region inside, while on the outside it has multiple hydroxyl groups, which gives it excellent water solubility and antioxidant properties, and it can be both water soluble and fat soluble. Therefore, it dissolves in an aqueous solution and easily passes through fat soluble parts such as cell membranes. Due to this amphiphilic nature, it penetrates the cell membrane near the SCF receptor, activating the stem cell factor receptor and causing cell proliferation.

[0027] Since this molanolin derivative exhibits amphiphilicity, it is preferable that it easily passes through the mucous layer of the intestine and is absorbed into the body. Furthermore, it easily penetrates and infiltrates the barrier tissue between keratinocytes in the stratum corneum of the skin, and easily reaches the skin cells in the epidermis layer, that is, the granular layer, the spinous layer, and the basal layer. Since it exhibits amphiphilicity, it is preferable that it can be used in a wide range of aqueous solutions and oils.

[0028] In addition, the benzene ring of phenylalanine also contributes to hydrophobicity, and the amino group and carboxylic acid have a pH buffering effect. When consumed, it provides resistance to gastric acid. In other words, it is preferable to use this pH buffering effect to mitigate gastric acid and prevent decomposition by gastric acid.

[0029] All of the hydroxyl groups of this molanolin derivative are free, and it exhibits a strong antioxidant effect. It is preferable that this antioxidant effect exerts a protective effect against oxidative stimuli. For example, when applied to the skin, it has the function of preventing oxidation of the skin caused by ultraviolet rays, and it is preferable that it protects the skin. In addition, it is preferable that this derivative is powdered and then reacted with a water-soluble solvent to generate hydrogen gas.

[0030] The molanolin derivative itself acts as a ligand for the stem cell factor receptor, acting at the active center of the stem cell factor receptor and exhibiting stem cell factor activity. In addition, the abundant hydroxyl groups stop the antioxidant effect, aid the receptor activity on the cell membrane, and are favorable for their stabilizing effect.

[0031] Furthermore, this moranolin derivative exhibits an epidermal cell proliferation effect via stem cell factor action. Its action is twofold: it modifies the stem cell factor receptor, and it acts directly on the receptor as a ligand. In other words, this moranolin derivative reacts with the vicinity of the active center of the stem cell factor receptor, causing a change in its three-dimensional structure. This change in structure increases its ability to bind to stem cell factors.

[0032] Phenylalanine, a component of this moranolin derivative, is a natural L-type phenylalanine that is abundant in nature and has been confirmed to be safe. Moranolin also exists in nature as a component of amino acids and is highly safe.

[0033] Furthermore, it is preferable that the stem cell factor action enhances the action of EGF (epithelial-derived growth factor), IL-3 (interleukin-3), and IGF-1 (insulin-like growth factor-1) in addition to the stem cell factor itself. Moranolin derivatives also show synergistic action with EGF.

[0034] Stem cell factor receptors are present in the cells of each organ, and their reactivity is organ-specific. Differences are also observed due to age, sex, and genetic polymorphisms.

[0035] However, because the stem cell factor action of this moranolin derivative is expressed through two different mechanisms, it is possible for it to act on all conditions and all tissues, including genetic polymorphisms and pathological conditions.

[0036] In particular, it is preferable that the moranolin derivative has high reactivity with stem cell factor receptors in basal layer cells of the skin and nerve cells, and therefore has a proliferation effect on the skin and nerves, and is excellent in the treatment and prevention of skin diseases and nerve diseases.

[0037] Moranolin derivatives are used as plant activators. These moranolin derivatives are amphiphilic, so they pass through the cell membranes of animals and the cell walls of plants and yeasts, and are easily absorbed into cells. Among these, plant hormones are stem cell factors for plants. These moranolin derivatives are preferably used to promote plant growth by increasing the reactivity of auxins, cytokinins, abscisic acid, gibberellins, etc.

[0038] The moranolin derivatives act as plant growth enhancers to promote the overall growth of plants, including germination, growth, flowering, fruiting, and harvesting, and are therefore favorable for contributing to the development of the agricultural sector and increased food production. In particular, they are favorable for use in the growth of precious flowers, such as orchids and pine orchids, and bonsai.

[0039] Moranolin derivatives are used as microbial activators. In addition, because they can promote the growth of useful microorganisms, they can be used to shorten and streamline fermentation processes. They are advantageous in that they can be used to increase production and shorten production times in the brewing of sake, miso, soy sauce, natto, and other products.

[0040] This moranolin derivative easily passes through the stratum corneum of the skin, and maintaining the barrier function of the stratum corneum is desirable in terms of skin health and beauty.

[0041] Furthermore, since this moranolin derivative is amphiphilic and exhibits a pH buffering effect, it is advantageous in that it can be incorporated into both water-soluble lotions and creams.

[0042] It is preferable that this molanolin derivative promotes cell proliferation and collagen and elastin production, thereby promoting skin cell function. Furthermore, it is preferable that a synergistic effect is obtained when used in combination with EGF (Epidermal Growth Factor). Furthermore, when used in combination with EFG, the proliferation of skin cells is increased by about 5 times.

[0043] It also acts as a stem cell factor in nerve cells, activating their proliferation. Neurons have a weakness in that their cellular functions are reduced by reactive oxygen species and amyloid beta protein in dementia and Alzheimer's disease. Therefore, restoring the function of nerve cells through the stem cell factor action of this moranolin derivative is desirable for the purpose of preventing and recovering from neurological diseases.

[0044] It is also preferable that the release of neurotransmitters from nerve endings is promoted to enhance neurotransmission. It is also preferable that a synergistic effect is obtained by combining with NGF. It is even more preferable that about three times the proliferation of nerve cells is obtained by combining with NGF.

[0045] It is desirable to increase nerve and muscle activity by enhancing muscle contraction through increased release of acetylcholine from the nerve endings of motor neurons.

[0046] It is preferable that this moranoline derivative activates stem cell factor receptors in cardiac myocytes, even in the case of myocardial infarction, when the coronary artery is infarcted or in an ischemic state, thereby causing cardiac myocyte proliferation and promoting the regeneration of the damaged area.

[0047] In addition, when athletes or those exercising wish to build muscle, it is preferable that the moranolin derivative acts as a stem cell factor in muscle cells to proliferate skeletal muscle cells and thereby build muscle tissue. In particular, it is preferable that the moranolin derivative regenerates muscle cells after the loss of muscle cells due to muscle tears or muscle cuts.

[0048] In the body, this moranolin derivative is decomposed by esterases in the kidney and liver and excreted in the urine. It is decomposed into its constituent components, moranolin and phenylalanine, which are highly safe. Therefore, this moranolin derivative does not accumulate in the body, is decomposed by enzymes in the body, and is highly safe because the decomposition products are natural products.

[0049] Producing the desired moranolin derivatives using mulberry leaves (scientific name: Morus Alba), algae, toadflax fruits, citrus fruits such as grapefruit and Satsuma mandarins, and buckwheat seeds as raw materials through fermentation or the cultivation of pig dental pulp cells is preferable from the standpoint of safety, as the raw materials are natural products.

[0050] It is possible to extract the moranolin derivatives from the above-mentioned plants or cell culture acclimatization solutions by purification, but the purification requires a large amount of raw material, which is costly, and the use of organic solvents limits industrial use.

[0051] Fermentation and cell culture methods are preferred because they are efficient in that they can break down proteins and remove dietary fiber, and are therefore suitable for producing this moranolin derivative. When using the resulting moranolin derivative as a pharmaceutical ingredient, it is preferred to purify the desired moranolin derivative, since this increases the purity of the desired moranolin derivative and allows impurities to be removed.

[0052] As pharmaceuticals, they are used as injections, oral preparations, or parenteral preparations such as topical applications, and as quasi-drugs, they are incorporated into tablets, capsules, drinks, soaps, topical applications, gels, toothpastes, etc.

[0053] Oral preparations include tablets, capsules, powders, syrups, drinks, etc. When mixed with the tablets and capsules, they can be used together with binders, excipients, swelling agents, lubricants, sweeteners, flavoring agents, etc. The tablets can also be coated with shellac, polysaccharides, sugars, etc.

[0054] In the case of the capsules, the above-mentioned materials may further contain liquid carriers such as oils and fats. In the case of the syrups and drinks, sweeteners, preservatives, colorings, flavorings, etc. may be added.

[0055] Examples of parenteral preparations include injections as well as external preparations such as ointments, creams, and solutions. Vaseline, paraffin, oils and fats, lanolin, macrogold, and the like are used as base materials for external preparations, and they can be made into ointments, creams, and the like by a conventional method.

[0056] Injections include liquid formulations and freeze-dried formulations, which are dissolved in distilled water for injections or physiological saline under sterile conditions before use.

[0057] In addition, it can be used as a food preparation for tissue regeneration such as skin regeneration, nerve regeneration, muscle regeneration, etc. It is preferable to use it as a health functional food such as a nutrient functional food or a food for specified health uses.

[0058] When the obtained food preparations are used for pets or livestock animals such as dogs and cats, they are used as feed or pet supplements for the purpose of recovering skin disorders and nerve disorders, slowing down aging, and improving athletic ability.

[0059] It can be used as a cosmetic material together with a surfactant, a solvent, a thickener, an excipient, etc. in the usual manner. For example, it can be in the form of a cream, a hair gel, a facial cleanser, a beauty essence, a lotion, etc. Since it exhibits amphiphilicity, it is preferable that the range of use can be expanded as an aqueous solution or an oil.

[0060] The cosmetic preparation may be in any form, and may be used in the form of a solution, cream, paste, gel, solid or powder.

[0061] The cosmetics produced here are used for the purposes of repairing damaged skin, increasing collagen and elastin, and maintaining healthy skin.

[0062] In addition, this moranolin derivative can be used in toothpastes, mouthwashes, toothpastes, etc., with the aim of maintaining the proliferation and function of gingival cells that decrease with age.

[0063] As a method for producing this moranolin derivative, a fermentation method using mulberry leaves or a method for sterilizing the filtered liquid after treating mulberry leaves with a protease is preferred. In particular, it is preferred to specify the desired moranolin derivative by a limited production method, which is produced by using a fermentation liquid obtained by fermenting mulberry leaves and soybeans with Bacillus subtilis natto as a medium to produce an acclimated culture liquid using porcine dental pulp cells and purifying the culture liquid. This fermentation production method is different from the production method by chemical synthesis, and is a production method that exists in nature, and impurities are also natural products, so it is also preferred from the viewpoint of safety.

[0064] Next, we will describe a method for producing moranolin derivatives that exhibit epidermal cell proliferation activity via stem cell factor activity as shown in the above formula (1), which involves using a fermentation liquid obtained by fermenting mulberry leaves and soybeans with Bacillus subtilis natto as a medium and purifying the porcine dental pulp cell-conditioned culture medium obtained by culturing porcine dental pulp cells.

[0065] First, mulberry leaves, which are the raw material for the fermented liquid obtained by fermenting mulberry leaves and soybeans with Bacillus subtilis natto as a medium, are cultivated all over Japan, and organically cultivated mulberry leaves are preferable because they are less likely to be contaminated by agricultural chemicals. In addition, the fact that they have a track record of being used as raw materials for cosmetics and food products makes them preferable from the standpoint of safety.

[0066] Mulberry leaves can be purchased from the Sakurae Town Mulberry Tea Producers Association. Organically grown mulberry leaves are particularly preferred. Proteases for food processing can be used. For example, Amano Enzyme's Protease N is preferred as it has a long track record in food processing and is highly safe.

[0067] Mulberry leaves are placed in a clean tank and dispersed in purified water. After heating this to 37-43°C, Protease N is added and the protein is decomposed for 3 to 6 hours with stirring. The weight of mulberry leaves is preferably 300g-1kg and the weight of Protease N is preferably 10-30g per 10L of purified water.

[0068] After the reaction, it is preferable to collect the filtrate by filtration through filter paper in order to remove undigested mulberry leaf residue, and it is preferable to boil the filtrate at a temperature of 95°C or higher in order to inactivate the protease and to sterilize the mixture.

[0069] Furthermore, it is preferable to ferment mulberry leaves, for example, with soybeans using Bacillus subtilis natto, due to the wealth of technical knowledge and experience involved.

[0070] In addition to mulberry leaves, soybeans can be used as the raw material from anywhere, including Japan, China, the United States, and Russia, but Japanese soybeans are preferable because they have reliable traceability and the producer is clearly identified.

[0071] Among these, soybeans grown organically or without pesticides are more preferable since they do not contain harmful pesticides or metals.

[0072] When soybeans are used, they are crushed using grinders such as the Jiyu Mill, Super Jiyu Mill, Sample Mill, Goblin, Super Clean Mill, and Micros manufactured by Nara Machinery Co., Ltd., the small vacuum dryer manufactured by Toyoriko Co., Ltd., the small vacuum heat transfer dryer DPTH-40 manufactured by Matsui Co., Ltd., the Clean Dry VD-7 and VD-20 manufactured by ACM Kyushu Technos Co., Ltd., and the DM-6 manufactured by Nakayama Technical Research Institute. This makes it easier for the fermentation process to proceed efficiently.

[0073] The natto bacteria used is Bacillus subtilis, a useful microorganism used in the manufacture of natto. The powdered natto bacteria from Takahashi Yuzo Laboratory Ltd. and Nattomoto Honpo are preferred because they are of good quality and suitable for fermentation. First, mulberry leaves are crushed using a grinder or mixer. By being crushed, they become easier to process. Soybeans are crushed using a grinder and suspended in clean water. 60 to 200 g of soybeans are added to 100 g of mulberry leaves, and the mixture is stirred in a clean container with 3 to 10 liters of water, such as purified water.

[0074] Mulberry leaves and soybeans are boiled and sterilized before being added to the fermentation tank. Sterilization prevents the intrusion of unwanted bacteria, allowing fermentation by the natto bacteria to proceed.

[0075] Fermentation may be performed by either the static or agitation method, but the agitation method is preferred since fermentation can be performed in a short time. Fermentation is preferably performed at 40 to 44°C for 22 to 76 hours. If the temperature is low and the time is short, fermentation will not proceed, and if the temperature is high and the time is long, the product may be decomposed. The fermented liquid thus obtained is sterilized in an autoclave at 121°C for 10 minutes and used as the following medium.

[0076] It is preferable to collect porcine dental pulp cells from the teeth of Japanese pigs from the viewpoint of hygiene and freshness of the cells. For example, pigs processed by pig processing companies or livestock corporations are preferable because they are fresh.

[0077] Any breed of pig can be used, but Nagara York or Sangen pigs, which show stable growth and are resistant to viral infection, are preferable. Pigs used should be those that have completed quarantine. The dental pulp is collected by collecting teeth from the head and mouth, cleaning and disinfecting them, crushing them with a hammer, and removing the dental pulp inside.

[0078] The collected dental pulp is immediately dispersed in a medium made of the mulberry leaf fermentation liquid and cultured. The collection of dental pulp cells is performed aseptically in a clean clean bench. The dental pulp is dispersed in the medium and the cells are suspended in the medium. It is preferable to add 1 / 20th the amount of diluted sterile trypsin solution and treat at 37°C for 5 minutes to break the cells into single cells.

[0079] The resulting dispersed dental pulp cells are collected and precipitated by centrifugation (1500g, 5 minutes). The cells are dispersed in fresh medium and seeded in a culture flask (FALCON) with 50 mL to 120 mL of medium. A cell count in the range of 10,000 to 30,000 cells is preferable because it is suitable for proliferation and easy to acclimate.

[0080] The culture is carried out in a carbon dioxide incubator under 5% carbon dioxide gas at 37°C for 5 to 10 days. The culture status is monitored by observing the cells under a microscope, and the culture is terminated before the cells become confluent.

[0081] After the culture is completed, the culture medium is collected, centrifuged, and the supernatant is collected and filtered to obtain the porcine dental pulp cell conditioned culture medium. The dental pulp cells remaining in the culture flask are detached with a diluted trypsin solution, and after adjusting the cell number, the cells are dispersed in the medium again and cultured, allowing the porcine dental pulp cell conditioned culture medium to be repeatedly collected.

[0082] This porcine dental pulp cell conditioned culture medium is purified. As a purification method, for example, separation is performed 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 having a particle size of 0.1 to 300 μm are preferable, and the finer the particle size, the more accurate the separation is, but there is a drawback in that the separation time is long.

[0083] 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. 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.

[0084] Affinity carriers or resins are used for specific preparation of antigens by utilizing antigen-antibody reactions. Partitioning carriers or resins, such as silica gel (Merck), are used for isolating substances when there is a difference in the partition coefficient between the substance and the separation solvent. It is preferable to repeat the purification procedure to obtain a highly pure product.

[0085] Among these, the adsorbent carrier or resin, the distributive carrier or resin, the molecular sieve carrier or resin, and the ion exchange carrier or resin are preferred from the viewpoint of reducing the production cost, and the reversed phase carrier or resin and the distributive carrier or resin are more preferred from the viewpoint of a large difference in the distribution coefficient with respect to the separation solvent.

[0086] When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used, and carriers or resins used in pharmaceutical or food manufacturing are preferred.

[0087] From these viewpoints, more preferred are Diaion HP20 (manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (manufactured by Rohm and Haas) as adsorptive carriers, Sephadex LH-20 (manufactured by Amersham Pharmacia) as molecular sieve carriers, silica gel as partition carriers, IRA-410 (manufactured by Rohm and Haas) as ion exchange carriers, and DM1020T (manufactured by Fuji Silysia Pharmaceuticals) as reverse phase carriers.

[0088] Among these, Diaion HP20, Sephadex LH-20 and DM1020T manufactured by Mitsubishi Chemical are more preferable.

[0089] The obtained extract is dissolved in a solvent for swelling the separation carrier or resin before separation. The amount of the solvent is preferably 2 to 40 times the weight of the extract, more preferably 4 to 20 times the weight, from the viewpoint of separation efficiency. The temperature for separation is preferably 10 to 30°C, more preferably 12 to 25°C, from the viewpoint of the stability of the substance.

[0090] The separation solvent is water or a water-containing lower alcohol, a hydrophilic solvent, or a lipophilic solvent. Examples of the lower alcohol include methanol, ethanol, propanol, and butanol, and ethanol, which is used for food, is preferred.

[0091] When Sephadex LH-20 is used, the separation solvent is preferably a lower alcohol, and when silica gel is used, the separation solvent is preferably chloroform, methanol, acetic acid or a mixture thereof.

[0092] When Diaion HP20 and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol, or a mixture of a lower alcohol and water.

[0093] It is preferable to collect the fraction containing the moranoline derivative, remove the solvent by drying or vacuum drying, and obtain the target moranoline derivative as a powder or a concentrated liquid, since this eliminates the influence of the solvent.It is also preferable that the amphiphilicity of the moranoline derivative expands the range of use as an aqueous solution or oil.

[0094] On the other hand, the final extraction can be carried out using edible oils or oils and fats used in cosmetics. For example, it is possible to extract using soybean oil, rice bran oil, grape seed oil, olive oil, and jojoba oil. In other words, by making it into an oil form, the range of applications can be expanded.

[0095] It is also preferable to powderize the molanolin derivative by spray drying or freeze drying for the purposes of preservation and extending the storage period.

[0096] It is preferable to perform structural analysis of the moranoline derivatives produced in this manner by HPLC or NMR, since this allows the provision of high-quality moranoline derivatives.

[0097] 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

[0098] The porcine dental pulp, which is the source of the porcine dental pulp cells, was collected from the teeth of a three-way crossbreed pig produced by crossbreeding between pedigree pigs using Nagara York, sold by Gifu Prefectural Livestock Corporation. The pigs were raised in a pig farm with strict hygiene management, and no abnormalities were found in virus or bacterial tests. The pig used was a male 180 days old, weighing 118 kg.

[0099] Dental pulp cells were collected from the teeth of these pigs. The surfaces of the teeth were disinfected with 70% alcohol, crushed with an iron hammer, and dental pulp was collected from the inside of the crushed teeth. The dental pulp was immediately dispersed in a medium. The medium used was the fermented mulberry leaf liquid shown below.

[0100] The mulberry leaf fermentation liquid was prepared as follows: 1 kg of organically grown mulberry leaves was purchased from the Matsuzakura-cho Mulberry Tea Producers Association. After washing, the leaves were suspended in a grinder (Super Jiyu Mill manufactured by Nara Machinery Co., Ltd.) together with purified water to obtain 0.9 kg of suspension.

[0101] The mulberry leaves were analyzed for pesticides in advance to confirm that they were free of pesticides, and were stored frozen until use.

[0102] In addition, 1 kg of soybeans from Hokkaido was purchased from Miwa Bussan Co., Ltd. and used. After washing, these were crushed in a grinder to obtain about 950 g of crushed material.

[0103] 500 g of the mulberry leaf suspension and 500 g of soybean powder were transferred to a clean stainless steel stockpot, and 5 liters of purified water was added to suspend the mixture.

[0104] The mixture was sterilized by boiling at 95 to 99° C. for 1 hour. The mixture was transferred to a 100 kg capacity stirred fermentation tank (FP211) manufactured by Yokogawa Electric Corporation, and 20 liters of sterilized purified water was added.

[0105] For this, 3 g of powdered natto bacteria manufactured by Takahashi Yuzo Laboratory, Ltd. was purchased. This natto bacteria was suspended in 100 g of sterilized water, to which 5 g of powdered soybean flour was added, and after stirring, the mixture was heated at 37°C for 1 hour for pre-culture.

[0106] This natto bacteria liquid was added to the above-mentioned stirred fermentation tank and fermented for 36 hours at 41 to 43° C. The state of fermentation was monitored by measuring the decomposition rate of soybean powder and the amount of dissolved protein (Biuret method).

[0107] After fermentation, the supernatant of the fermented broth was roughly filtered through a filter cloth to obtain a filtrate. This filtrate was used as a mulberry leaf fermented broth for the culture medium of porcine dental pulp cells. The mulberry leaf fermented broth was sterilized by autoclaving before use.

[0108] The porcine dental pulp cells dispersed in the mulberry leaf fermentation solution were centrifuged (1500g, 10 minutes) to collect the cell fraction. Fresh medium was added to the dental pulp cells, and the cells were dispersed and counted. 10,000 cells were seeded in a 150mL culture flask (FALCON) with 100mL of medium and cultured at 37℃ under 5% carbon dioxide.

[0109] After 7 days of culture, the culture was stopped and only the culture solution was collected. The culture solution was boiled at 100°C for 10 minutes. After cooling, it was suction filtered through a membrane filter (manufactured by ADVANTEC, pore size 0.1 μm), and the filtrate was used as a culture solution for conditioned porcine dental pulp cells. The dental pulp cells remaining in the culture flask were detached with diluted trypsin solution, and after adjusting the cell number, they were dispersed in the medium again and cultured.

[0110] 10 L of the conditioned culture medium of the porcine dental pulp cells obtained from several experiments was freeze-dried using a freeze dryer (Freeze Trap VA-140S manufactured by Taitec Co., Ltd.) to obtain 36 g of the desired powder. This was designated as Sample 1.

[0111] The obtained sample 1 was subjected to a purification process. That is, 30 g of this powder was suspended in 400 mL of purified water and subjected to 500 g of Diaion HP20 (manufactured by Mitsubishi Chemical) that had been swollen with 7% ethanol. After washing with 900 mL of 7% ethanol, it was further washed with 1 L of 55% ethanol.

[0112] To this, 700 mL of 80% ethanol was added to obtain the desired moranoline derivative. This purification process was repeated three times to increase the purity. The final fraction obtained from the three purification processes was dried in a vacuum dryer (manufactured by Japan Biocon Co., Ltd.) to obtain 4 g of powder. This powder was designated as sample 2.

[0113] The test methods and results for the structural analysis of moranoline derivatives are described below. (Test Example 1)

[0114] Sample 2 obtained as described above was dissolved in ethanol and analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation).

[0115] Furthermore, the samples were analyzed using a nuclear magnetic resonance spectrometer (600 MHz, H-NMR and C-NMR, manufactured by Bruker), and the desired moranoline derivatives were identified in Sample 1 and Sample 2.

[0116] That is, as a result of H-NMR measurement of sample 2 in deuterated dimethyl sulfoxide, the peak positions were observed at 0.98, 0.99, 1.00, 1.02, 1.03, 2.11, 2.12, 2.13, 2.15, 2.22, 2.25, 2.31, 2.66, 2.76, 3.72, 3.79, 4.74, 4.84, 4.97, 5.03, 6.64, and 6.68 ppm.

[0117] In addition, as a result of C-NMR measurement of sample 2, peaks were observed at 17.5, 18.9, 21.1, 22.4, 22.5, 22.6, 25.9, 26.0, 30.2, 41.1, 43.4, 43.5, 50.1, 54.7, 62.4, 70.8, 77.7, 80.4, 80.9, 90.2, 113.9, 146.0, 171.4, 172.2, 172.3 and 174.8 ppm.

[0118] The chart below shows the results of the C-NMR analysis. (The horizontal axis indicates ppm, and the vertical axis indicates peak intensity.) JPEG0007627398000003.jpg1885

[0119] The above analysis results showed that it had the same structure as the chemically synthesized standard. In other words, it was confirmed that sample 2 was the desired moranoline derivative, consisting of three moranoline molecules and one phenylalanine molecule. In addition, HPLC analysis of sample 2 showed one peak, with a purity of 99.7%. The purity of sample 1 was 80.3%.

[0120] The following describes an epidermal cell proliferation test using human skin epidermal cells via stem cell factor action. This test method is a reproducible standard method that can biochemically verify the effects of ingredients. (Test Example 2)

[0121] Human epidermal cells (epidermal derived, Epider Cell) purchased from Kurabo were used. 1000 cells cultured in MEM medium (Sigma) containing 5% fetal bovine serum as the culture medium were seeded on a 35 mm culture dish (FALCON) and cultured at 37°C under 5% carbon dioxide. The cells were irradiated with 280 nm ultraviolet light using an ultraviolet irradiation device (Quark Technology). After that, the above-mentioned sample 1, sample 2, or EGF (Funakoshi, epidermal growth factor) and recombinant SCF protein (R&D systems) as a positive control were added at a final concentration of 0.1 mg / ml. At the same time, the effect of combined use with EGF (Funakoshi, product code NO20) was examined. The cells were cultured for 48 hours and tested.

[0122] After collecting the culture medium, the survival rate of the epidermal cells was counted by the trypan blue method. Then, a suspension of the epidermal cells was prepared. The activation state of the stem cell factor receptor was measured using this cell suspension. The binding affinity between the stem cell factor receptor and the sample was measured by measuring the binding affinity with the above-mentioned recombinant SCF protein. For the measurement, a BIA (GE, T200 type) was used. BIA stands for Biophysical Interaction Analysis. In other words, it is a biophysical interaction analysis method, and is a method for measuring molecular interactions in vivo.

[0123] Furthermore, mRNA was extracted from the cell suspension using a nucleic acid extraction kit (Funakoshi). The amount of stem cell factor receptor mRNA was quantified by RT-PCR (Takara Bio, microRNA Ready-to-Use PCR Panel) using Takara Bio primers according to standard methods.

[0124] A human-derived recombinant SCF protein (No. 255-SC-050) purchased from R&D systems was used as a ligand for the stem cell factor receptor.

[0125] The binding affinity to the stem cell factor receptor was measured by measuring the binding affinity to the recombinant SCF protein using the BIA method described above.

[0126] The average value was calculated using five petri dishes and compared with the solvent control group to which the solvent was added.

[0127] As a result, the addition of 0.1 mg / ml of sample 1 increased the number of human-derived epidermal cells to an average of 180% compared to the solvent control group. The number increased to 229% in the sample 2 addition group. Meanwhile, the number increased to 130% with the addition of EGF and 170% with the addition of SCF. As a result, samples 1 and 2 exhibited superior human-derived epidermal cell proliferation effects to EGF and SCF. Furthermore, the combined use of sample 2 and EGF increased the number to 808% compared to the solvent control group, demonstrating a synergistic effect with EGF.

[0128] Measurement of binding affinity with stem cell factor receptors using the BIA method showed that the average binding affinity increased to 189% when sample 1 was added compared to the solvent control group. It also increased to 366% when sample 2 was added. On the other hand, the binding affinity decreased to 109% when EGF was added, and to 148% when SCF was added. As a result, samples 1 and 2 showed superior binding affinity to stem cell factor receptors compared to EGF and SCF. This means that the affinity to stem cell factor receptors was increased. Furthermore, the combined use of sample 2 with EGF increased the binding affinity to 669%, demonstrating a synergistic effect when combined with EGF.

[0129] The expression level (copy number) of stem cell factor receptor mRNA in the above cells was 21 copies in the solvent control group, 43 copies in the sample 1 treatment group, 304 copies in the sample 2 treatment group, 32 copies in the EGF treatment group, and 41 copies in the SCF treatment group. Furthermore, the combined use of sample 2 and EGF increased the expression level to 841 copies. This combined effect indicates that sample 2 and EGF acted synergistically.

[0130] The expression level of stem cell factor receptor mRNA was significantly higher in Sample 1 and Sample 2 than in EGF and SCF. This indicates that Sample 1 and Sample 2 have an effect of inducing stem cell factor receptor mRNA. In addition, a synergistic effect was observed when Sample 2 was used in combination with EGF.

[0131] On the other hand, as part of the safety test, a skin irritation test was conducted using EpiSkin (manufactured by SkinEthic), an artificial skin. As a result, no irritation was observed due to the addition of Samples 1 and 2, and the safety of Samples 1 and 2 was confirmed. This safety test method using artificial skin is a skin irritation test evaluation method using cells, which has been established as an alternative method to animal testing that does not use animals, and is a highly reliable method.

[0132] The following describes a test for damage using a human nerve cell damage model. This test method is a reproducible and standard method for verifying the function of active ingredients biochemically and molecular biologically, and has a wealth of test results and is highly reliable. (Test Example 3)

[0133] Human neurons (HN) purchased from Cosmo Bio were used. 1,000 cells cultured in a dedicated culture medium (neuron growth medium) were seeded on a 35 mm culture dish and cultured at 37°C under 5% carbon dioxide. A 1% aqueous solution of acrylamide, a neurotoxin, was added to stimulate the neurons.

[0134] To this were added sample 1 and sample 2 obtained in Example 1 above, and as a positive control, NGF (Funakoshi Co., Ltd., human type) and human-derived SCF, all at a final concentration of 0.1 mg / ml. This was cultured for 48 hours. Furthermore, an experiment was performed using NGF (Funakoshi Co., Ltd., β type, product code 450-01) and sample 2 in combination.

[0135] After the culture was completed, the number of neurons was counted under a microscope. In addition, the activity of stem cell factor receptors (BA method) and the expression level of mRNA were measured by RT-PCR (Takara Bio Inc., microRNA Ready-to-Use PCR Panel) in the same manner as above.

[0136] As a result, the addition of 0.1mg / ml of sample 1 increased the number of nerve cells to an average of 138% compared to the solvent control group. In the sample 2 addition group, the number increased to 209%. Meanwhile, in the NGF addition group, the number was 110%, and in the SCF addition group, the number was 130%. As a result, samples 1 and 2 exhibited superior cell activation effects to NGF and SCF. Furthermore, the combined use of NGF and sample 2 increased the number of cells to 471%, demonstrating a synergistic proliferation effect of the combined use of sample 2 and NGF.

[0137] Measurement of binding affinity with stem cell factor receptors by BIA showed that the addition of sample 1 increased the binding affinity to 166% on average compared to the solvent control group. The addition of sample 2 increased the binding affinity to 243%. Meanwhile, the addition of NGF reduced the binding affinity to 113%, and the addition of SCF reduced the binding affinity to 128%. As a result, samples 1 and 2 showed stronger binding affinity with stem cell factor receptors than NGF and SCF. This shows that samples 1 and 2 increased the affinity with stem cell factor receptors. Furthermore, the combined use of sample 2 with NGF increased the binding affinity to 556%, demonstrating a synergistic effect of sample 2.

[0138] The expression level (copy number) of stem cell factor receptor mRNA in the above cells was 19 copies in the solvent control group, 70 copies in the sample 1 treatment group, 145 copies in the sample 2 treatment group, 110 copies in the NGF treatment group, and 127 copies in the SCF treatment group. In addition, the combined use of sample 2 and NGF increased the expression level to 641 copies, demonstrating a synergistic effect with NGF.

[0139] The expression level of stem cell factor receptor mRNA was significantly higher in Sample 1 and Sample 2 than in NGF and SCF. This indicates that Sample 1 and Sample 2 have an effect of inducing stem cell factor receptor mRNA. In addition, a synergistic effect was observed when used in combination with NGF. [Industrial Applicability]

[0140] The moranolin derivative obtained by the present invention exhibits stem cell factor activity and promotes skin cells, thereby improving the QOL of the skin. In addition, when used in cosmetics, it contributes to improving the skin of people who suffer from skin problems such as wrinkles and sagging, and contributes to the development of the cosmetics industry.

[0141] The moranolin derivative obtained by the present invention increases nerve cells and can be used to treat dementia and Alzheimer's disease, thereby contributing to the maintenance of public health.

[0142] In addition, the method for producing the moranoline derivative obtained by the present invention utilizes discarded pig dental pulp, and therefore can be used to promote the development of the livestock industry by recycling waste materials.

Claims

1. A moranolin derivative having an epidermal cell proliferation effect mediated by stem cell factor action, as shown in the following formula (1). 【Chemistry 1】

2. A method for producing a moranoline derivative that exhibits epidermal cell proliferation activity via stem cell factor activity as shown by formula (1) of claim 1, comprising the steps of using a fermentation liquid obtained by fermenting mulberry leaves and soybeans with Bacillus subtilis natto as a medium, and purifying a porcine dental pulp cell-conditioned culture medium obtained by culturing porcine dental pulp cells in the fermentation liquid.

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