JP2018070559A - Dehydroretinol derivative exhibiting keratin increasing action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dehydroretinol derivative exhibiting keratin increasing action.SOLUTION: The dehydroretinol derivative exhibiting kerating increasing action is a natural substance in which two molecules of L-cysteine are ester-bonded to one molecule of dehydroretinol and exhibits keratin increasing action of skin cells. A production method therefor involves the step of fermenting caviar using rice bran as a base material with Bacillus subtilis var. natto and Monascus purpureus. When used as a cosmetic, excellent wrinkle improvement due to an increase in keratin is exhibited. The dehydroretinol derivative can be used for greening of the desert, promoting the growth of plants, and exhibits a plant activation effect to increase the harvest.SELECTED DRAWING: None

Description

The present invention relates to a dehydroretinol derivative exhibiting a keratin increasing action.

Keratin is a protein that forms an intracellular skeleton and is a hard protein that constitutes tissues such as skin and blood vessels, and plays an important role in maintaining bone and skin tissues. Keratin is biosynthesized in the body by epithelial cell stimulation and cell regeneration.

The keratin contained in the skin is involved in the protection of the stratum corneum, the arrangement of epithelial cells, and cosmetically affects the alignment and arrangement of the skin dermis and skin groove, so it has a relationship with wrinkles and sagging skin. strong. In addition, maintenance and increase of keratin are essential for maintaining hair.

Further, keratin plays an important role as a tissue constituent protein even in non-keratinized epithelial cells, and the sheet-like structure of epithelial tissue maintains mechanical strength by keratin fibers. A highly safe natural product that causes an increase in keratin is desired.

For example, there is a use of a heterocyclic quaternary polyammonium polymer as a protective agent for keratin fibers and a cosmetic composition (see, for example, Patent Document 1). However, the range of use is limited, and no strong action that directly acts on skin cells has been shown.

Patent No. 3823025

The effect of increasing keratin by existing plant extracts and chemical substances is mild, and there is a problem that their industrial use is limited.

In addition, chemically synthesized substances have safety problems and their use is limited.

Therefore, a natural product that exhibits an excellent keratin-increasing action with weak side effects is desired.

In order to achieve the above object, the invention described in claim 1 relates to a dehydroretinol derivative exhibiting a keratin increasing action represented by the following formula (1).

Since this invention is comprised as mentioned above, there exist the following effects.

According to the dehydroretinol derivative according to claim 1, a natural product exhibiting a keratin increasing action with few side effects can be obtained.

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

The dehydroretinol derivative exhibiting a keratin production action has a structure represented by the following formula (1).

The dehydroretinol derivative shown here acts directly on human skin cells and hair cells to increase keratin production. Its action involves the proliferation of skin epithelial cells and the induction of keratinogenic enzymes.

This dehydroretinol derivative is composed of hydroxyretinol and L-cysteine. The molecular formula is C26H38O3N2S2 and is composed of 26 carbons, 38 hydrogens, 3 oxygens, 2 nitrogens and 2 sulfurs. That is, two molecules of L-cysteine are bonded to one molecule of hydroxyretinol.

This dehydroretinol derivative can be synthesized organically from retinol and cysteine, and can be used as a standard product for the purpose of structural analysis. Diaion (Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (Rohm and Haas), Sephadex LH-20 (Amersham Pharmacia), ion exchange carrier IRA-410 (Rohm and) Purified product by reverse phase carrier DM1020T (manufactured by Fuji Silysia), and a purified product having a purity of 95% or more can be obtained.

This structure is analyzed by analyzing 1H-NMR and 13C-NMR in CD3OD with a nuclear magnetic resonance apparatus (for example, NMR, manufactured by Bruker). 1.469, 1.514, 1.661, 1.887, 1.955, 2.079, 2.102, 2.124, 2.192, 2.215, 2.600 by 1H-NMR analysis at 600 MHz. 305, 2.407, 2.565, 2.599, 2.644, 2.667, 2.712, 2.893, 3.040, 3.254, 3.367, 3.763, 4.023, Peaks are observed at 4.068, 5.119, 6.147, 6.283, 7.006, and 7.379 ppm.

Furthermore, by analysis of 13C-NMR in CD3OD, 23.0, 26.7, 27.1, 27.3, 28.7, 30.0, 34.1, 39.9, 41.6, 46.7 49.4, 51.6, 55.3, 58.8, 71.8, 72.6, 78.8, 129.6, 135.1, 141.0, 142.5 and 176.2 ppm. Is recognized.

Dehydroretinol is a kind of carotenoid, a natural product that has an antioxidant effect and an action on the optic nerve, and also works on the proliferation and maintenance of cells on the skin. In nature, dehydroretinol is highly safe because it is produced from retinol by an enzyme, decomposed in vivo, and excreted. In addition, accumulation in the body and environment is not recognized, and safety to the environment is high.

The dehydroretinol derivative shown here has an ester bond between the hydroxyl group of dehydroretinol and the carboxyl group of L-cysteine. Moreover, since it is easily ionized, it is excellent in absorption into the skin and absorption from the intestinal tract. In addition, since this dehydroretinol derivative contains L-cysteine, which is a constituent component of keratin, it is also useful as a raw material for keratin.

In addition, this dehydroretinol derivative activates the epidermal growth factor receptor in skin epithelial cells. The activation method is an improvement in sensitivity, which is so-called hypersensitivity, works on the active center of the enzyme, and increases the sensitivity as a catalyst.

The excessive amount of the dehydroretinol derivative is degraded by non-specific esterase present in the liver and kidney, and is degraded into dehydroretinol and L-cysteine.

Since dehydroretinol and L-cysteine both exist in nature and safety has been confirmed, the safety of this dehydroretinol derivative is high.

In addition, this dehydroretinol derivative is not accumulated in fat and is not concentrated.

Further, this dehydroretinol derivative proliferates epithelial cells and hair matrix cells of the skin and increases the production of keratin, and also has an effect of improving blood flow, detoxification, excretion and metabolic activation.

As a method for producing this dehydroretinol derivative, caviar, vegetables, and algae can be fermented in addition to the organic chemical synthesis method.

Among these, caviar is preferable because it contains abundant retinol and further contains an epidermal growth factor that promotes the growth of skin cells and hair matrix cells. Caviar is a sturgeon egg with the scientific name Acipeser medirostris. Since it does not affect retinol even in the salted state, it can also be used in salted caviar.

This fermentation process consists of adding natto and red yeast and fermenting them.

Next, it is fermented with natto and red yeast. That is, caviar, rice bran, natto and red yeast are added to a clean fermentation tank and fermented under aerobic conditions.

The rice bran that can be used as a raw material can be rice bran obtained from brown rice in any production area such as Japanese, Chinese, American and Russian. However, it is preferable that it is traceable and has a clear producer. Among these, rice bran derived from brown rice grown organically or without agricultural chemicals is more preferable because it does not contain harmful agricultural chemicals or metals.

In use, KOMENUKA is a free-form mill, super-free mill, sample mill, goblin, super-clean mill, micros, vacuum dryer from Toyo Riko Co., Ltd. It is pulverized by a pulverizer such as a vacuum heat transfer dryer DPTH-40, clean dry VD-7, VD-20 manufactured by AKM Kyushu Technos Co., Ltd., DM-6 manufactured by Nakayama Technical Research Institute. This pulverization is preferable because the fermentation process easily proceeds efficiently.

The Bacillus subtilis used is the scientific name Bacillus subtilis, which is widely used in the production of natto and food processing in Japan, and is a useful edible fungus with abundant food experience. Bacteria species from Japan such as Okinawa and Kagoshima, and from Southeast Asia such as China and Taiwan are used. Among these, natto bacteria manufactured by Natto Honpo are preferable because they exhibit high fermentability.

The red koji mold used is the scientific name Monascacee, and is an edible fungus that is rich in food experience and useful. Bacteria species from Japan such as Okinawa and Kagoshima, and from Southeast Asia such as China and Taiwan are used. Among these, the red koji mold made by Kurisu Honpo is preferable because it exhibits high fermentability.

As for each addition amount related to the fermentation, 0.4 to 5 wt. For rice bran, 0.002 to 0.04 wt. For Bacillus natto, and 0.001 to 0.05 wt. . It is preferable to pre-cultivate natto and red yeast before fermentation, because the initial time of fermentation is shortened and the fermentation time is shortened.

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

Moreover, this fermentation is heated at 35-48 degreeC, and fermentation is performed for 14 days from 4 days.

After fermentation, natto and red yeast are killed by heating at about 90 ° C., and the fermentation is stopped. Dehydroretinol is liberated by this fermentation process. Subsequently, hydroxyproline is released from rice bran.

It is preferable that the fermented product produced by the fermentation is extracted with water-containing ethanol because the product can be efficiently recovered and the next step is easily performed. Moreover, since the product is easy to isolate | separate, it is preferable to ultrasonically crush the obtained fermented material. Moreover, it is preferable to concentrate by freeze drying or the like because the following steps can be performed in a short time.

It is preferable to separate and purify the fermented product from the viewpoint that the intake can be reduced as a highly pure substance. As a purification method, it is preferable to use a purification operation such as a separation resin.

For example, it is preferably separated and separated 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 later, are used. Those having a particle size of 0.1 to 300 μm are preferred. The finer the particle size, the higher the accuracy of the separation, but the longer the separation time.

For example, a reverse phase carrier or resin whose surface is coated with a hydrophobic compound is used for separation of a highly hydrophobic substance. Those coated with a cationic substance are suitable for the separation of anionically charged substances. Also, those coated with an anionic substance are suitable for separating a cationically charged substance. When a specific antibody is coated, it is used as an affinity carrier or resin for separating only a specific substance.

The affinity carrier or resin is used for specific preparation of an antigen using an antigen-antibody reaction. A partitionable carrier or resin is used for isolation of a substance such as silica gel (manufactured by Merck) if there is a difference in partition coefficient between the substance and the solvent for separation.

Among these, an adsorbent carrier or resin, a dispersible carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable from the viewpoint of reducing production costs. Furthermore, the reverse phase carrier or resin and the dispersible carrier or resin are more preferable because the difference in the distribution coefficient with respect to the separation solvent is large.

When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used. Moreover, the carrier or resin used for pharmaceutical manufacture or food manufacture is preferable.

From these points, Diaion (HP-20 or HP21, Mitsubishi Chemical Co., Ltd.) and XAD-2 or XAD-4 (Rohm and Haas) are used as adsorptive carriers, and Sephadex LH is used as a molecular sieve carrier. More preferred is -20 (manufactured by Amersham Pharmacia), silica gel as a carrier for distribution, IRA-410 (manufactured by Rohm and Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silysia) as a reverse phase carrier.

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

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

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

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

When Diaion HP-20 type and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol or a mixed solution of lower alcohol and water.

In addition, it is preferable to collect a fraction containing the activity and remove the solvent by drying or vacuum drying to obtain a powder or a concentrated liquid because the influence of the solvent can be excluded.

Moreover, it is preferable to carry out the final extraction with fats and oils used for edible oils and cosmetics because the obtained active part is stably maintained in the oil. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil or jojoba oil is preferred.

It is used as an injectable or parenteral agent such as an oral agent or a coating agent as a pharmaceutical, and as a quasi-drug, it is used in a tablet, capsule, drink, soap, coating agent, gel, toothpaste, etc. The

Examples of oral preparations include tablets, capsules, powders, syrups, and drinks. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, excipient, swelling agent, lubricant, sweetener, flavoring agent and the like. The tablets can also be coated with shellac or sugar.

Moreover, in the case of the said capsule, raw materials, such as fats and oils, can be further contained in said material. In the case of the above syrup and drink, sweeteners, preservatives, pigment flavoring agents and the like can be added.

Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams, and liquids. Vaseline, paraffin, fats and oils, lanolin, macro gold, etc. are used as a base material for external preparations, and can be made into ointments, creams, and the like by ordinary methods.

Injections include liquids, and other lyophilization agents. This is used aseptically dissolved in distilled water for injection or physiological saline at the time of use.

As a food preparation, it is used in beauty foods for beauty purposes, foods for beauty purposes, and nourishing tonics for the purpose of maintaining liver cells. Moreover, as a health functional food, it is preferable to use it for a nutrition functional food or a food for specified health.

When the obtained food preparation is used for pets such as dogs and cats and livestock animals, it is used as feed or supplement for the purpose of maintaining skin health.

As a cosmetic, it can be used together with a surfactant, a solvent, a thickener, an excipient and the like according to a conventional method. For example, it can be in the form of cream, hair gel, facial cleanser, cosmetic liquid, lotion, etc., and becomes a cosmetic that promotes keratin production. The form of the cosmetic is arbitrary, and can be used as a solution, cream, paste, gel, gel, solid or powder.

Moreover, it can be used also for the use which activates a plant energetically by the mechanism which increases the keratin which forms the skeleton of a plant as a plant activator. It can also be used in beans, cereals, rice, root vegetables and flowers, increasing plant keratin to increase yield and quality and increase plant growth and life. Can also be used to hold cut flowers.

Hereinafter, the embodiment will be specifically described with reference to examples and test examples. These are merely examples, and conditions can be changed within the range of common sense according to differences in materials, raw materials, and specimens.

Caviar collected from cultured sturgeon from Okayama Prefecture was purchased and used. 450 g of this was stirred with a mixer.

The obtained caviar suspension was put into a clean fermentation tank (Owaki Engineer, 150 kg capacity).

1.1 kg of rice bran obtained from brown rice with reduced agricultural chemicals produced in Akita Prefecture was purchased and suspended in 9 L of tap water. This rice bran suspension was placed in an autoclave (Tomy Seisakusho, SR-240) and sterilized at 121 ° C.

After sterilization, the cooled rice bran liquid was transferred to a fermentation tank, and 8 g of natto bacteria and 10 g of red koji molds manufactured by Natto Honpo were added and fermented at 37 ° C. for 4 days.

The fermented liquid was put into a 91 degreeC heating tank, was heated for 14 minutes, and was sterilized. This was cooled and then filtered to obtain 8.1 L of filtrate. This was designated as a dehydroretinol derivative-containing extract. Stored at 4 ° C. until use.

3 L of 5% ethanol-containing purified water was added to 4 L of the above-described dehydroretinol derivative-containing extract, and 200 g of Diaion HP-20 type (Mitsubishi Chemical) was suspended in a 4% ethanol solution and packed in a column.

To this, 3 L of 4% ethanol solution was added for cleaning, and then 2 L of 40% ethanol solution was added to elute the desired dehydroretinol derivative. The ethanol portion was removed from the purified dehydroretinol derivative by distillation under reduced pressure to obtain an aqueous solution. Further, this purification process was repeated three times to obtain a sample 1 of a dehydroretinol derivative having a purity of 97.3%. This specimen 1 exhibited a single peak by HPLC analysis.

The identification test of the dehydroretinol derivative will be described below.
(Test Example 1)

The specimen 1 which is the dehydroretinol derivative of Example 1 obtained as described above was dissolved in purified ethanol, analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation), and further a nuclear magnetic resonance apparatus (NMR , Manufactured by Bruker).

As a result, by 1H-NMR analysis at 600 MHz in CD3OD, 1.469, 1.514, 1.661, 1.887, 1.955, 2.079, 2.102, 2.124, 2.192, 2.215, 2.305, 2.407, 2.565, 2.599, 2.644, 2.667, 2.712, 2.893, 3.040, 3.254, 3.367, 3. Peaks were observed at 763, 4.023, 4.068, 5.119, 6.147, 6.283, 7.006 and 7.379 ppm.

Furthermore, by analysis of 13C-NMR in CD3OD, 23.0, 26.7, 27.1, 27.3, 28.7, 30.0, 34.1, 39.9, 41.6, 46.7 49.4, 51.6, 55.3, 58.8, 71.8, 72.6, 78.8, 129.6, 135.1, 141.0, 142.5 and 176.2 ppm. Was recognized.

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

That is, the sample of Example 1 showed the same analysis results as a standard product synthesized and purified organically. The target dehydroretinol induction was identified.

The confirmation test using human skin epithelial cells is described below.
(Test Example 2)

Human skin epithelial cells purchased from Kurabo Industries Co., Ltd. were used. As a culture solution, 1000 cells cultured using 5% fetal calf serum-containing MEM medium (manufactured by Sigma) were seeded in a 35 mm culture dish, and cultured at 37 ° C. under 5% carbon dioxide gas. To this, specimen 1 obtained in Example 1 and skin epidermal growth factor (EGF) as a control were added at a final concentration of 0.1 mg / ml. This was cultured for 48 hours. EGF is used as a physiological substance for proliferating skin epithelial cells and producing keratin. After detaching the cells, the number of cells was counted. In addition, the petri dish calculated the average value using five sheets.

As a result, the addition of 0.1 mg / ml of Sample 1 increased the number of skin epithelial cells to 255% as an average value compared to the solvent control group. Moreover, as a result of quantifying the amount of keratins using an ELISA kit (manufactured by Wako Pure Chemical Industries, Ltd.), the average value was 344% compared to the solvent control group.

In the EGF as a control, the number of cells was 181% compared to the control group. The amount of keratin produced was 210%, and Sample 1 was superior in increasing the amount of keratin.

On the other hand, in a skin irritation experiment using EpiSkin (manufactured by SkinEthic), which is an artificial skin, as part of the safety test, no irritation was observed due to the addition of specimen 1, and safety was confirmed. This method has been established as a method for evaluating skin irritation using cells without using animals.

Below, the confirmation test as a plant activator using perilla seed and soybean seed is described.
(Test Example 3)

Perilla seeds and soybean seeds from Nagano Prefecture were purchased and used. Perilla seeds and soybean seeds were placed on a vat covered with gauze sufficiently soaked in a 1000-fold diluted aqueous solution of Sample 1. This was placed in an incubator at 30 ° C. and cultured for 7 days, and bud spread was measured. The average value of 10 seeds was determined. Only tap water was used as a solvent control. At the same time, the culture was immersed in a 1000-fold solution of HB-101 (Flora Co., Ltd.) as a control.

As a result, perilla seed buds were 146% when soaked in specimen 1, and 131% when soaked in HB-101, assuming that the solvent control was 100%. When soybean seeds were immersed in specimen 1, they extended to 150% compared to the solvent control. On the other hand, when immersed in HB-101, it was 126% of the solvent control. From these results, it was concluded that Specimen 1 exhibits a plant activating action superior to HB-101.

Since the dehydroretinol derivative obtained by the present invention exhibits keratin production and has few side effects, it can improve the national QOL, increase the healthy working population, and reduce medical costs.

Since the dehydroretinol derivative obtained by the present invention has an action of producing keratin and improving skin cells, it contributes to the improvement of those suffering from atopy and skin trouble as a cosmetic and contributes to the development of the cosmetic industry.

Since the dehydroretinol derivative obtained by the present invention can be used as a food, it contributes to the development of the food industry.

The dehydroretinol derivative obtained in the present invention increases the elongation and growth of plants, fruit set and yield as a plant activator, and is also used in desert greening countermeasures and agricultural fields.

Claims (1)

A dehydroretinol derivative exhibiting a keratin producing action represented by the following formula (1).