JP5403638B2 - Composition exhibiting collagen producing action - Google Patents

Description

The present invention relates to a method for producing a composition exhibiting a collagen producing action.

Collagen is an important protein that builds skin and tissues, and is mainly composed of hydroxyproline obtained by hydroxylating proline. The reaction requires vitamin C.

Collagen is synthesized mainly by fibroblasts, and fibroblast growth factor is involved in its regulation.

A method for synthesizing collagen in the body in the skin region has been developed, but has not been completed. There is a problem that a natural product has a mild collagen synthesis action.

As an invention relating to collagen production, for example, there is a cosmetic for moisturizing (see, for example, Patent Document 1). Here, korinai ginseng is used, but its application is limited.

  Japanese Patent Application No. 2008-329517

Collagen production action by existing substances is mild, and there is a problem that the industrial use is limited. Also, chemically synthesized substances have a problem in safety and are limited in use.

Therefore, a production method for efficiently producing a terpene derivative exhibiting an excellent collagen production action with weak side effects is desired.

In order to achieve the above object, the invention described in claim 1 relates to a method for producing a composition exhibiting a collagen producing action.

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

According to the manufacturing method of Claim 1, the composition containing a terpene derivative can be manufactured efficiently.

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

The manufacturing method of the composition which exhibits the collagen production effect which consists of the process which protease-processes the fermented liquor which added and fermented the sanshuyu fruit, rice bran powder, and Benikouji bacteria is demonstrated.

The composition of the present invention comprises a terpene derivative. The terpene derivative here is a derivative in which a terpene and a peptide are peptide-bonded. Since this terpene derivative is decomposed and excreted by an enzyme in vivo, it is highly safe.

The terpenes of this terpene derivative are monoterpenes such as kadinene and pinene, and diterpenes, and these terpenes are preferable because of their safety.

This derivative acts on the fibroblasts of skin and joints, penetrates into the tissue, and exerts a collagen producing action by directly activating the intracellular collagen producing enzyme.

When the obtained terpene derivative is used as a pharmaceutical material, it is preferable to separate and purify the target terpene derivative from the viewpoint of increasing the purity of the target terpene derivative and removing impurities.

Used as pharmaceuticals, parenteral preparations such as injections, oral preparations and coatings, and quasi-drugs used in tablets, capsules, drinks, soaps, coatings, gels, toothpastes, etc. Is done.

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, liquid carriers, 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.

It is used as a food preparation for health food and beauty food for collagen production and beauty. 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 and livestock animals such as dogs and cats, it is used as feed or supplements for the purpose of maintaining fat decomposition and 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, gel for hair, facial cleanser, cosmetic liquid, lotion and the like.

The form of the cosmetic is arbitrary, and can be used as a solution, cream, paste, gel, gel, solid or powder.

The resulting cosmetic has a function of promoting collagen production and improving elasticity and gloss.

In addition, by forming a collagen film, the stratum corneum of atopic patients is strengthened by utilizing the action of suppressing the entry of bacteria, stimulants, inflammatory substances, and oxides.

This production method comprises a step of subjecting a fermented liquid fermented with the addition of sanshuyu fruit, rice bran powder and benichow fungus to protease treatment.

The raw materials are sanshuyu fruit, rice bran powder, benichow fungus and protease.

Sanshuyu here is a tree of the family of the dogwood with the scientific name Cornus officinalis, and its beauty is used as food. It is also called the foot of the mountain, Harukoganebana, Axango or Yamaguchi.

Sunshuyu fruit is preferable as a raw material for this derivative because it contains abundant terpenes.

Sanshuyu fruit may come from any country such as Japan, China, Taiwan, USA. In addition, those produced with low pesticides or reduced pesticides are preferred.

It is preferable that the sanshuyu fruit is dried and pulverized, and it is preferable to sterilize by autoclave before fermentation because the fermentation is performed smoothly.

A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.

The rice bran powder used as a raw material can be used in rice bran from any origin, such as from Japan, China, the United States, and Russia. However, it is preferable that the powder be produced in Japan because the traceability is reliable and the producer is clear.

Among these, rice bran cultivated 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. Thereby, the process of fermentation tends to advance efficiently.

Furthermore, it is preferable to sterilize the sunshu fruit and rice bran after pulverization by autoclaving or the like because it can prevent the propagation of various bacteria.

The fungus used is a filamentous fungus of the scientific name Monasuc purpureus and has long been used in fermented foods such as red sake and tofu in Japan, China and Taiwan.

In addition, bacterial species from Japan such as Okinawa and Kagoshima, and from Southeast Asia in China and Taiwan are used.

The terpenes and protein are combined by fermenting the terpene and protein consisting of sanshuyu fruit and rice bran at the same time.

As for each addition amount regarding the said fermentation, 0.1 to 2 weights of rice bran powder and 0.0001 to 0.005 weights of B. niger are preferable with respect to 1 weight of dry powder of samyu fruit. It is preferable to pre-cultivate B. subtilis 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 37-46 degreeC, and fermentation is performed for 14 days from 2 days. The process control of fermentation is carried out by quantifying the target terpene derivative by HPLC or TLC and confirming the growth of the bacterial cells.

Although the protein binds to the terpene by this fermentation process, its molecular weight is large, so that it is degraded by protease and reduced in molecular weight for the purpose of promoting absorption and penetration from the skin.

Proteases are hydrolytic enzymes that break down proteins to produce peptides and amino acids, and are also used as food. Amano's protease N is preferred because of its high enzyme activity.

By adding a protease to the fermented product and heating, the terpene and protein are decomposed into a peptide-type terpene derivative.

The amount of protease added is preferably 0.0001 to 0.03 weight per 1 weight of the fermented product. The heating temperature is preferably 30 to 45 degrees. The heating time is preferably 1 to 6 hours.

It is preferable that the protease-treated decomposition product is extracted with water-containing ethanol because the product can be efficiently recovered, the protease can be deactivated, and the next step can be easily performed.

Moreover, since the product is easy to isolate | separate, it is preferable to ultrasonically treat 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.

Separating and purifying the target terpene derivative from the reduction reaction product is preferable because 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, the desired terpene derivative can be obtained by separation with a separation carrier or resin and fractionation. 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 (Mitsubishi Chemical Co., Ltd.) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH-20 (Amersham Pharmacia) is used as the molecular sieve carrier. Silica gel as the distribution carrier, IRA-410 (Rohm and Haas) as the ion exchange carrier, and DM1020T (Fuji Silysia) as the reverse phase carrier are more preferable.

Of these, Diaion, 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 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.

It is preferable to collect the fraction containing the terpene derivative and remove the solvent by drying or vacuum drying to obtain the target terpene derivative as 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 terpene derivative is stably maintained. For example, extraction with rice bran oil, rice bran oil, grape seed oil, olive oil or jojoba oil is preferred.

In addition, it is preferable to powder this terpene derivative for the purpose of preserving.

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.

Sunshuyu fruit grown in Okinawa Prefecture with reduced pesticides was used. After collecting the fruit, it was washed with tap water, dried in the sun, and pulverized with a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 1.1 kg of dried powder pulverized product of Sangshuyu fruit. .

Rice-derived rice bran from Mie Prefecture was used in a mixer (Cuisinate) to obtain 1.1 kg of rice bran pulverized material. The pulverized sanshuyu fruit and rice bran were put in an autoclave and sterilized at 121 ° C. for 20 minutes.

These were put into a clean fermentation tank (sterilized round 40-liter tank for fermentation), and 10 kg of sterilized tap water was added and stirred.

Separately from this, 10 g of powdered Benikouji fungus (manufactured by Natto Honpo) was subjected to a small fermentation tank to prepare a sterilized rice bran powder and a pre-cultured culture solution.

The pre-cultured solution of B. cerevisiae was added to the fermentation tank containing the dried powder of sanshuyu fruit and rice bran, and after stirring, the mixture was heated in a temperature range of 40 to 43 ° C. and fermented.

In the fermentation process, the fermentation liquor was sampled while bubbling and stirring by aeration.

10 g of Amano Protease N was added to 1 kg of the obtained fermented product and heated at 40 degrees for 3 hours.

This treated product was heated, and ethanol was added to obtain 190 g of a composition containing the desired terpene derivative .

Below, the test method regarding the structural analysis of a terpene derivative and a result are demonstrated.
(Test Example 1)

The specimen 1 obtained as described above was dissolved in ethanol and analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation).

Furthermore, it analyzed with the nuclear magnetic resonance apparatus (NMR, the Bruker make, AC-250). As a result of the structural analysis, terpene, peptide and conjugate thereof were detected from specimen 1.

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

Human skin fibroblasts 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 was added Specimen 1 obtained in Example 1 above and FGF as a control at a final concentration of 0.1 mg / ml. This was cultured for 48 hours.

After detaching the cells, the number of cells was counted, the cell suspension was adjusted, and the amount of collagen in the cells was measured by ELISA (Wako Pure Chemical Industries). In addition, the petri dish calculated the average value using five sheets.

As a result, the addition of 0.1 mg / ml of Specimen 1 increased the number of fibroblasts to an average value of 305% compared to the control group. On the other hand, the increase in FGF was 210%, and Sample 1 was superior in work.

The amount of collagen increased to 420% by Sample 1 compared to the control group. On the other hand, FGF was 277%, and the function of Sample 1 was remarkable.

The composition obtained in the present invention brings about an increase in collagen in the skin and joints and maintains the health of the skin. Therefore, the QOL of the people can be improved, the healthy working population can be increased, and the medical cost can be reduced.

Since the composition obtained by the present invention has the effect of improving the elasticity of the skin, it contributes to the improvement of those who suffer from atopy and skin trouble as a cosmetic, and contributes to the development of the cosmetic industry.

Since the composition obtained by this invention can be utilized also as a foodstuff, it contributes to development of the food industry.

By using dry powder of sanshuyu fruit, waste can be effectively used and agricultural resources can be effectively used to develop agriculture and its secondary processing industry.

This production method uses advanced fermentation technology and contributes to the improvement of new fermentation technology and the advancement of the fermentation industry.

Claims (1)

Cornus fruit, compositions exhibiting collagen production effect obtained by protease treatment of the fermentation broth obtained by fermenting a mixture of rice bran and Monascus fungus.