JP2009280514A - Organic acid peptide-bonded luteolin derivative exhibiting anti-oxidizing action and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an organic acid peptide-bonded luteolin derivative exhibiting an excellent anti-oxidizing action and weak side effects, and to provide a method for producing the organic acid peptide-bonded luteolin derivative. <P>SOLUTION: Provided is the organic acid peptide-bonded luteolin derivative exhibiting the excellent anti-oxidizing action and the weak side effects, wherein two molecules of tripeptide comprising cysteine, phenylalanine and tyrosine and two molecules of caffeoylquinic acid as an organic acid are bonded to one luteolin molecule. Provided is the method for producing the same, characterized by comprising a process for alkali-reducing a fermentation solution obtained by adding and fermenting Monascus prepreus to the ground product of chrysanthemum flowers, gold powder and soybean powder. The fermentation process and the reduction process are main processes. In the fermentation process, the tripeptide is bonded to the luteolin by the fermentation reaction due to the Monascus prepreus and by the catalytic action of the gold, and the bond is stabilized by the alkali reduction to sustain the anti-oxidizing action. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to an organic acid peptide-bonded luteolin derivative exhibiting an antioxidant action and a method for producing the same.

Cancer is the leading cause of death among Japanese people, and approximately 300,000 people die from cancer in one year, causing medical expenses. For this reason, doctors, the pharmaceutical industry, the Ministry of Health, Labor and Welfare, the health and health industry, etc. are implementing various measures and research and development in order to solve cancer, and in particular, the search and development of substances that exhibit antioxidant effects are awaited. .

In addition, various anti-cancer drugs have been developed for pharmacotherapy for cancer, and drugs that suppress cancer cell proliferation, differentiate cancer, restore immunity, and suppress angiogenesis have been developed. It is also used in medical settings such as hospitals.

Of these, substances having an action that works specifically for cancer cells are desired because they have few side effects. Many of the chemically synthesized anticancer agents also act on normal cells that proliferate, causing serious side effects. For example, although cisplatin and 5-FU have strong anticancer effects, their side effects are severe, and side effects such as vomiting, anemia, decreased immunity, and infectious diseases appear. As a result, the amount and period of use of anticancer agents are limited. Yes.

On the other hand, it has been found that many carcinogenic processes are caused by oxidation, and an action that prevents this oxidation, that is, a substance exhibiting an antioxidant action, is considered to be effective for carcinogenesis or treatment of cancer itself.

In addition, a substance exhibiting an antioxidant action has a high industrial utility value in terms of protecting the skin from oxidizing substances.

Although there are chemically synthesized vitamin C derivatives also in substances exhibiting an antioxidative action, they are poorly degradable and have problems such as accumulation in the living body. Therefore, an antioxidant effect derived from a natural product having high safety and high effectiveness is desired.

Chrysanthemum flowers, which are also used for food, contain various polyphenols such as luteolin and tannins, and several inventions have been made for its use. It has been reported (for example, see Patent Document 1).

In the field of use as food, there is an invention of a chrysanthemum flower-containing drink, which is characterized by containing chrysanthemum flower or an extract thereof (for example, see Patent Document 2).

As a cosmetic, there is an invention of a whitening cosmetic characterized by blending a herbal medicine extract of chrysanthemum (kica), and here it is reported that the extract of chrysanthemum exhibits an excellent melanin production inhibitory action at a low concentration. (For example, see Patent Document 3).

Further, regarding the inhibitory action on inflammatory cytokines derived from chrysanthemums, the invention of terpenoid derivatives has been recognized, and as a production method thereof, chrysanthemums are fermented with natto bacteria together with fish (for example, see Patent Document 4).

Luteolin is an example of a flavonoid that exhibits an excellent antioxidant action. As an invention related to luteolin, there is a novel flavonoid compound, a method for producing the same, and an invention of an antioxidant containing the compound as an active ingredient (see, for example, Patent Document 5).

Moreover, as a thing using fermentation in the manufacturing method regarding a luteolin, there exists an example of a lipoxygenase inhibitor, and here, Japanese charcoal is fermented with lactic acid bacteria, yeast, and Bacillus subtilis (for example, refer patent document 6).

However, regarding the components related to the above invention, there is a problem that, in particular, a substance exhibiting an antioxidant action has not been identified, and the action thereof is also mild.

Therefore, this time, invented an organic acid peptide-bonded luteolin derivative exhibiting an antioxidant action, and in order to produce this substance, a production method characterized by fermentation technology using chrysanthemum flowers as a raw material and gold as a catalyst. Since it invented, it demonstrates below.
JP-A-9-37737 JP-A-8-322526 JP 2002-68958 JP2007-176816 JP 2006-265249 A JP 2005-89385 A

As described above, the chemically synthesized anticancer agents such as cisplatin and 5-FU have serious side effects, and there is a problem that the QOL of the patient is lowered and the amount used and the period of use are limited.

In addition, chemically synthesized vitamin C derivatives and polyphenol derivatives are subject to bioaccumulation and side effects, and their use is limited.

On the other hand, although the safety | security is high about the substance which exhibits an antioxidant effect derived from nature, there exists a problem that an antioxidant effect | action is mild. Therefore, a natural product-derived substance having a weak side effect and an excellent antioxidant action is desired.

In order to achieve the above object, the invention described in claim 1 relates to an organic acid peptide-bonded luteolin derivative exhibiting an antioxidant action.

The invention according to claim 2 is an organic acid peptide having an antioxidant action, characterized in that it comprises a step of alkali reduction of a fermented liquor fermented by adding koji molds to ground chrysanthemums, gold powder and soybean powder. The present invention relates to a method for producing a bound luteolin derivative.

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

According to the organic acid peptide-bonded luteolin derivative of claim 1, side effects are weak and an excellent antioxidant action is exhibited.

According to the manufacturing method of Claim 2, an organic acid peptide bond luteolin derivative can be manufactured efficiently.

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

First, an organic acid peptide-bonded luteolin derivative having an antioxidant action represented by the following formula (1) will be described.

The organic acid peptide-bonded luteolin derivative referred to here has two tripeptide molecules ester-bonded to one molecule of luteolin. The tripeptide is in the order of N-terminal to tyrosine, phenylalanine, and cysteine, and the C-terminal is cysteine. All are peptide-bonded.

Furthermore, the carboxyl group of the C-terminal cysteine is ester-bonded to the hydroxyl group of luteolin.

Furthermore, the organic acid of the organic acid peptide-bonded luteolin derivative is two molecules of caffeoylquinic acid, and the carboxyl group of caffeoylquinic acid is ester-bonded to the hydroxyl group of luteolin.

That is, two molecules of tripeptide and two molecules of caffeoylquinic acid are ester-bonded to one molecule of luteolin.

All of the amino acids forming these tripeptides are in L form.

In this organic acid peptide-bonded luteolin derivative, the SH group of the cysteine moiety of the two tripeptides maintains a reduced state, and thus a strong antioxidant action is maintained. The structure of luteolin is maintained by the ester bond between luteolin and these two tripeptide sites.

Further, the hydroxyl group of the tyrosine residue of the tripeptide has the property of reducing the oxidized substance.

In caffeoylquinic acid bonded to luteolin, the hydroxyl group of the caffeoyl group exhibits an antioxidant action. In particular, the hydroxyl group of quinic acid and caffeic acid is ionized and reacts with the nucleophilic molecule of the oxidizing substance to reduce the oxidizing substance.

Furthermore, the quinic acid and caffeic acid moieties share double-bonded electrons between the benzene rings of luteolin and the electron cloud is stabilized, so that a wide-ranging and highly durable antioxidant action is exhibited.

By this antioxidant action, the cell membrane of cancer cells is stably maintained, and the functions of growth factor receptors necessary for cancer cells, such as epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor, Suppresses the growth of cancer cells.

This organic acid peptide-bound luteolin derivative suppresses vascular smooth muscle cells necessary for angiogenesis of cancer cells by its antioxidant power, and suppresses the formation of new blood vessels.

This organic acid peptide-bound luteolin derivative peptide exhibits chelating action due to the presence of a pair of cysteine and tyrosine residues, and produces toxic chemicals such as benzpyrene and dioxin as well as diesel particles and asbestos. By wrapping in microscopic particles, it has the feature that it can detoxify harmfulness.

Furthermore, for toxic metals such as arsenic, lead, mercury, cadmium, the two SH groups express chelating action, trap the toxic metal, and wrap the toxic metal so that the peptide folds, Eliminate adverse effects.

In addition, when the organic acid peptide-bound luteolin derivative is ingested in excess, it is degraded by esterases in the gastrointestinal tract, lungs, blood, or organs to be decomposed into tripeptides, luteolin and organic acids.

The constituting tripeptide is decomposed into amino acids, further decomposed into sulfur dioxide and carbon dioxide, and excreted from the kidney, which is highly safe and more preferable. Also, luteolin and organic acids are decomposed and then metabolized in the liver and kidneys, decomposed, and excreted from the body as urine via the kidneys.

Furthermore, since this organic acid peptide-binding luteolin derivative suppresses production of inflammatory cytokines such as interleukin (IL) -1 and tumor necrosis factor (TNF) alpha and collagenase, it suppresses various inflammations.

In addition, this organic acid peptide-bonded luteolin derivative enhances the regenerative power of the skin by promoting the exfoliation of the skin cells against the keratinocytes, and further, atopy and contact by harmful substances through anti-inflammatory action It is preferable because it improves sexual allergy.

In addition, this organic acid peptide-bound luteolin derivative has a reducing action of the peptide SH group, assisting the action of vitamin C, decomposing the melanin oxidation product, eliminating the causative agent of the stain, and promoting collagen production To do.

The organic acid peptide-bound luteolin derivative is produced by fermenting and reducing plants, soybeans and rice bran as raw materials. In order to accelerate the fermentation process, a metal having a catalytic action such as gold or silver is used.

That is, in the process of producing the organic acid peptide-bonded luteolin derivative, a substance that donates electrons is required, and gold atoms are most suitable for a stable catalyst for this reaction. That is, it is necessary to catalyze the bond between the peptide and luteolin and to maintain the reducing power by maintaining the hydroxyl group.

As a source of luteolin, chrysanthemum flowers and marigold flowers having a high content are excellent and easy to process. Chrysanthemum flowers are preferable because they are edible and cultivated and used in Yamagata and Akita in Japan.

The gold powder used as a catalyst in this production process becomes a gold atom in a solvent, and is positively ionized to donate the electron of the gold atom to the luteolin derivative and maintain the peptide bond. Gold atoms act only as a catalyst, are not incorporated into luteolin derivatives, and are excluded during the manufacturing process.

The organic acid peptide-bound luteolin derivative is obtained by fermentation with gold powder and plant cells, animal cells, yeast or microorganisms, and can be obtained by biosynthesis. Since the gold powder is recovered, electrically reduced, and regenerated, a part of it can be used repeatedly.

This organic acid peptide-bound luteolin derivative is obtained as a liquid or powder. This can be used as a pharmaceutical preparation as an anticancer agent, a cancer inhibitor or an anti-inflammatory agent.

When used as a pharmaceutical material, it is preferable to separate and purify the target organic acid peptide-bonded luteolin derivative because the purity of the target organic acid peptide-bonded luteolin derivative is increased and impurities can be removed.

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 contained.

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.

Food preparations are used as raw materials for raw material processing and food production for the purpose of preventing cancer based on antioxidant activity. 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 suppressing pet cancer and oxidation.

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 oil-soluble cream, hair gel, facial cleanser, beauty essence, 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 obtained cosmetic promotes keratin improvement and skin regeneration of atopic patients through antioxidant and anti-inflammatory effects. Moreover, since it exhibits a whitening effect through an antioxidant effect, it is also used as a whitening cosmetic or a quasi-drug.

In addition, it is used on farms for the purpose of removing harmful substances in fields and fields as a soil conditioner that applies antioxidant action. It is also used in chemical factories for the purpose of improving the surrounding soil contamination and working environment.

The organic acid peptide having an antioxidant action according to claim 1, wherein the fermented liquid obtained by adding red yeast to fermented chrysanthemum flowers, gold powder and soybean powder is subjected to alkali reduction. A method for producing a bound luteolin derivative will be described.

The organic acid peptide-bonded luteolin derivative exhibiting an antioxidative action here is the aforementioned organic acid peptide-bonded luteolin derivative.

The chrysanthemum flower used as a raw material is a sweet chrysanthemum flower, citrus or yeokiku flower represented by the scientific name Chrysanthemum morifolium, and other chrysanthemum flowers belonging to the chrysanthemum genus Chrysanthemum, such as a chrysanthemum flower, a scented chrysanthemum flower, or a mulberry flower. The color of the chrysanthemum flower may be any of white, yellow, purple, orange and the like.

As for the chrysanthemum flower here, any of Japanese, Chinese, American and African chrysanthemum flowers is used.

Of these, chrysanthemum flowers in the Tohoku region such as Yamagata and Akita that are used for food are preferred. Also, those made in Japan or China are preferable because the use history of agricultural chemicals can be traced, the quality is stable, and the price is low.

The chrysanthemum flower may be obtained by any of the cultivation of outdoor cultivation, house cultivation, hydroponics, and lighting cultivation.

Chrysanthemum flowers can be fresh or dried.

The collected chrysanthemum flowers are preferably washed with tap water.

Chrysanthemum flowers are crushed. For pulverization, free mill, super free mill, sample mill, goblin, super clean mill, micros, manufactured by Nara Machinery Co., Ltd. as a pulverizer, a small vacuum dryer manufactured by Toyo Riko as a vacuum dryer, manufactured by Matsui Co., Ltd. A small decompression heat transfer type dryer DPTH-40, clean dry VD-7, VD-20, etc. manufactured by AKM Kyushu Technos Co., Ltd. are used.

Gold powder used as a raw material can be obtained by pulverizing a lump of pure gold, gold bullion, gold dust, gold foil, or the like. In particular, pure gold having a purity of 99% or more is preferable as a raw material because it has few impurities. The gold ingot is roughly ground and then powdered with a stone mortar or pulverizer. A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process. A gold bullion made by Mitsubishi Materials purchased from US Corporation is preferred because of its high purity and high reactivity.

Moreover, you may add rice bran as a raw material. Addition of rice bran is preferable because fermentation is promoted and the production amount of the target compound is increased.

Although the rice bran used here can be used in any production area, it is preferable to use Japanese rice that has reliable traceability and a clear producer. Of these, rice bran obtained from organically cultivated rice or rice cultivated with no pesticides is preferred because it does not contain harmful pesticides or metals.

The soybean powder used as a raw material can be used in soybeans of any origin such as Japanese, Chinese, American, and Russian, but is preferably Japanese because of its reliable traceability and clear producers. Of these, organically cultivated and soybeans cultivated without pesticides are more preferable because they do not contain harmful pesticides and metals.

When using soybeans, Nara Machinery Co., Ltd. free mill, super free mill, sample mill, goblin, super clean mill, micros, small vacuum dryer manufactured by Toyo Riko as vacuum dryer, small size manufactured by Matsui Co., Ltd. Fermentation is efficient by being 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, etc. It is preferable because it easily proceeds.

Further, chrysanthemum flowers, gold powder and soybeans are preferably sterilized after being crushed and then sterilized by an autoclave or the like because they can prevent the propagation of various bacteria.

The koji mold used is a useful edible fungus, and here, bacterial species originating in Japan such as Okinawa and Kagoshima, and originating in Southeast Asia such as China and Taiwan are used. Of these, the red koji mold, which is the Monacaseae native name in China, which is the manufacture of the red mushroom main office, is preferable because it exhibits high reactivity.

In the presence of gold atoms, these red koji molds ferment soybeans and chrysanthemum at the same time to produce conjugated enzymes and ester-linked enzymes that bind proteases and esters.

The respective addition amounts relating to the fermentation are 0.0001 to 0.01 wt. For gold powder, 0.5 to 3 wt. 0.02 weight is preferred.

It is preferable to pre-culture the koji mold before it is fermented because it accelerates the initial fermentation and shortens the fermentation time.

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 30-53 degreeC, and fermentation is performed for 14 days from 2 days. The process control of the fermentation is carried out by quantifying the target organic acid peptide-bound luteolin derivative by HPLC or TLC and confirming the growth of the cells.

By this fermentation process, luteolin binds to the tripeptide using a gold atom as a catalyst, but the bond is unstable and easily degraded by an enzyme such as esterase. In order to solve this, it is necessary to carry out the following alkaline reduction.

It is preferable that the fermented product produced by the fermentation is extracted with water-containing ethanol containing 20% to 80% ethanol because the product can be efficiently recovered and the next step can be easily performed.

It is preferable to ultrasonically treat the obtained fermented product because the products are easily separated. Concentration by freeze-drying or the like is preferable because the following steps can be efficiently performed in a short time.

This fermented product is alkali reduced. The alkali reduction is preferably performed by an alkali reduction device or an alkali reduction water conditioner. For example, a continuous generator of alkaline reduced water and strong oxidized water “Protech ATX-501” manufactured by Zemaitis, an alkali reduced water production apparatus “Techno Super 502” manufactured by NCI, “Mineria CE-212” manufactured by Marthaka, manufactured by Crescent It is more preferable to use an apparatus such as “Acura Blue” or “Mineral Reduction Water Conditioner“ Genki no Water ”” manufactured by Nippon Kosen Research Co., Ltd.

As the solvent for carrying out the alkali reduction, a hydrous ethanol solution containing 20% to 80% ethanol is preferable in order to efficiently carry out the alkali reduction.

This reduction step stabilizes the binding of the target organic acid peptide-bonded luteolin derivative.

Separating and purifying the target organic acid peptide-bonded luteolin derivative from the reduction reaction product is preferable 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.

The desired organic acid peptide-bound luteolin derivative is obtained by separation with a separation carrier or resin and separation. 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 30 times, more preferably 3 to 20 times the weight of the extract from the viewpoint of separation efficiency. The separation temperature is preferably 4 to 35 ° 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.

Since the fraction containing the organic acid peptide-bound luteolin derivative is collected, the solvent is removed by drying or vacuum drying, and obtaining the desired organic acid peptide-bound luteolin derivative as a powder or concentrated solution can eliminate the influence of the solvent, preferable.

The organic acid peptide-bonded luteolin derivative thus obtained is obtained as a liquid or a powder.

Hereinafter, the embodiment will be specifically described with reference to examples and test examples. These are examples, and are used in the actual industry by changing conditions within the range of common sense due to differences in materials, raw materials and specimens.

9 kg of sweet chrysanthemum flowers cultivated in Yamagata Prefecture, Japan with no pesticides were collected, washed thoroughly with tap water, and dried with a dryer.

The 2 kg of this sweet chrysanthemum flower was subjected to a pulverizer (manufactured by Sanriki Seisakusho Co., Ltd., a three-purpose universal pulverizer) and pulverized to obtain a crushed flower of chrysanthemum.

100 g of gold bullion purchased from US Trading Corporation was pulverized with a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 90 g of pulverized gold having a particle size of 3 to 4 micrometers.

Soybeans from Hokkaido were used in a mixer (Cuisinate) to obtain 1 kg of soybean pulverized product. Further, the above crushed chrysanthemum flower, pulverized soybean powder and gold powder were subjected to autoclaving 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.

A culture solution pre-cultured at 40 ° C. for 1 day with 50 g of soybean powder sterilized with 10 g of powdered red koji mold (manufactured by Kurisu Honpo) and 1 L of purified water was prepared, and this was used as a pre-culture solution of koji mold.

Add the pre-cultured red koji mold solution to the fermentation tank containing the crushed chrysanthemum flower, gold powder, and pulverized soybean powder, and after heating, heat within a temperature range of 40-45 ° C. And fermented.

In the fermentation process, sampling was performed while bubbling and stirring by aeration, and the target organic acid peptide-bound luteolin derivative product was measured.

In the method, the fermented liquid was subjected to high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation) and analyzed to confirm the production of the target substance.

As a result, after 8 days of fermentation, a sufficient amount of the desired organic acid peptide-bound luteolin derivative was produced, so that the fermentation was terminated. After stopping the fermentation, 5 kg of ethanol at 30 ° C. was added to the fermentation broth and stirred.

The fermented liquid was supplied to a filter with diatomaceous earth and filtered. The obtained filtrate was used for Cellar Kiss (manufactured by Zenon Co., Ltd.).

The obtained filtrate was subjected to alkali reduction by subjecting to an alkali reduction device (alkaline reduced water / strongly oxidized water continuous generator “Protech ATX-501” manufactured by Zemaitis).

The alkali reduced product thus obtained was subjected to a lyophilizer manufactured by Nippon FD Co., Ltd., and further sterilized at a low temperature using an ultraviolet sterilizer to obtain 139 g of the desired organic acid peptide-bound luteolin derivative as a powder. This was used as a specimen 1 for the following test.

Below, the test method regarding a structural analysis of an organic acid peptide bond luteolin derivative and a result are demonstrated.
(Test Example 1)

The specimen 1 obtained as described above was dissolved in an extraction medium 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 structural analysis, a target conjugate in which a tripeptide consisting of tyrosine, phenylalanine, tyrosine, luteolin and caffeoylquinic acid was bound from the N-terminal was identified.

The two hydroxyl groups of this luteolin and the tripeptide were ester-linked. It was also found that caffeoylquinic acid as an organic acid was ester-bonded to the side chain of luteolin.

Hereinafter, the redox potential test of the organic acid peptide-bonded luteolin derivative will be described.
(Test Example 2)

0.1 g of the sample 1 was dissolved in 100 ml of tap water (Tokyo). The oxidation-reduction potential was measured with an oxidation-reduction potentiometer (manufactured by Sato Shoji Co., Ltd., OPR Pro) to examine the antioxidant effect.

As a result, the oxidation-reduction potential of specimen 1 one minute after lysis was minus 329 mV, and the minus potential was maintained until 24 hours after lysis. On the other hand, the redox potential of tap water was plus 435 mV. As a result, it was concluded that Sample 1 exhibits an antioxidant effect.

Below, the confirmation test of an antioxidant effect is described.
(Test Example 3)

Antioxidant action was measured using DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity and superoxide scavenging activity as indicators. DPPH radical scavenging activity was measured according to the method of Shinohara et al. The DPPH radical scavenging activity and superoxide scavenging activity used have been established as a method for investigating the antioxidant action and have abundant test results.

A 0.1M solution of DPPH (50% ethanol) was mixed with Sample 1 or a solution obtained by dissolving vitamin C (manufactured by Sigma) as a control substance, and the DPPH radical scavenging activity was measured for absorbance at 520 nm. The activity with respect to 1 micromole was defined as 1 DPPH unit using Torox (manufactured by Sigma) as a standard substance.

Superoxide scavenging activity was measured by electron spin resonance (ESR) after superoxide was generated in a hypoxanthine / xanthine oxidase (SOD) system, a solution of vitamin C serving as specimen 1 or a control substance was added. Activity was measured using superoxide scavenging enzyme (SOD) as a standard.

As a result, for DPPH radical scavenging activity, the activity of Sample 1 was 26,000 units / g. On the other hand, the activity of vitamin C was 6,000 units / g, and Sample 1 showed a four times or more stronger activity than vitamin C.

As for superoxide scavenging activity, the activity of Sample 1 was 2.4 million SOD units / g, and vitamin C was 400,000 units / g. Specimen 1 showed 6 times stronger activity than vitamin C.

The anticancer test using human cancer cells is described below.
(Test Example 4)

HeLa cells, which are human-derived uterine cancer cells purchased from ATCC, were used. As a culture solution, 10000 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, cisplatin was added at a final concentration of 0.01 mg / ml and 0.1 mg / ml, respectively, as the sample 1 obtained in Example 1 and the control anticancer drug, and further cultured for 48 hours.

Furthermore, apoptosis of cancer cells was also observed. That is, after detaching the cancer cells, the number of cells was counted and fluorescently stained with Hoechst 33352. This was subjected to a fluorescence microscope, and the number of apoptotic cells in which the nucleus was divided was counted. In addition, the petri dish calculated the average value using five sheets. As a result, when 0.01 mg / ml of Specimen 1 was added, the number of cancer cells decreased to an average of 23% compared to the control group, and the appearance rate of apoptosis was 45%. It was.

When 0.1 mg / ml of Specimen 1 was added, the number of cancer cells decreased to 15% compared to the control group, and the appearance rate of apoptosis was 67%.

As a result, when 0.01 mg / ml of cisplatin as a control drug was added, the number of cancer cells was 90% compared to the value of the control group, and the appearance rate of apoptosis was 10%.

Further, when 0.1 mg / ml of cisplatin as a control drug was added, the number of cells was 81% compared to the value of the control group, and the appearance rate of apoptosis was 13%.

From the above results, the specimen 1 was observed to have a cancer cell destruction action due to apoptosis. Its strength was more than 10 times that of cisplatin, an anticancer drug as a control.

As a result of the same test with normal human-derived dermal fibroblasts, the addition of the sample 1 did not change the number of cells, and safety against normal cells was confirmed. However, with cisplatin as a control drug, destruction of normal cells was observed and strong toxicity was observed.

Hereinafter, a mouse cancer transplantation test using mouse-derived melanoma cells S-100 will be described.
(Test Example 5)

Mouse-derived melanoma cells S-100 purchased from ATCC were used. The cells were cultured in a 5% fetal calf serum-containing MEM medium (manufactured by Sigma), and 100,000 cells were injected subcutaneously into the back of ICR mice.

To this mouse, the sample 1 obtained in Example 1 was dosed 0.1 mg / kg and 1 mg / kg, and orally administered once a day for 28 days. In addition, 10 animals were used per group, and distilled water was administered as a control.

As a control drug, cisplatin was intraperitoneally administered at doses of 0.1 mg / kg and 1 mg / kg.

The average value of tumor weight of animals administered with distilled water was 10.56 g. On the other hand, the average value of the tumor weight of Sample 1 administered at 0.1 mg / kg was 3.35 g. Furthermore, the average value of the tumor weight of Sample 1 administered at 1 mg / kg was 1.03 g.

As a control, the tumor weight of an animal administered with 0.1 mg / kg cisplatin was 8.21 g on average. Furthermore, the tumor weight of the animals administered with 1 mg / kg of cisplatin was 6.19 g on average.

As a result, it was confirmed that tumor administration and tumor weight reduction were caused by administration of specimen 1, and specimen 1 was confirmed to have a tumor reducing action. The reduction action was more than 10 times that of cisplatin, which is an anticancer drug.

In addition, no deaths or side effects were observed in the animals to which Sample 1 was administered. On the other hand, 3 of 10 cases died after administration of cisplatin, and hematuria, kidney damage and anemia were observed in all cases, and side effects were confirmed.

Examples of cosmetics comprising organic acid peptide-bonded luteolin derivatives will be described below.

In a cosmetic mixer, 1 g of polyethylene glycol monostearate, 1 g of glyceryl monostearate, 2 g of horse oil ester and 2 g of oleic acid were heated and dissolved.

30 g of the sample 1 obtained in Example 1 was added to the obtained solution, and 0.1 g of α-tocopherol and 60 g of purified water were further added. These were stirred and mixed, suspended, and then cooled to obtain an emulsion as a cosmetic. This was designated as Sample 2 of Example 2. As a test control cosmetic, an emulsion excluding only the powder of Sample 1 obtained in Example 1 was prepared.

Below, it shows about the test which evaluated the effect and side effect of the said cosmetics.
(Test Example 6)

Ten healthy females aged 33 to 69 years old applied 10 mL of the emulsion obtained in Example 2 to the right half of the face for 14 days. The left half of the face was coated with an emulsion excluding specimen 1 alone.

Before application and on the 14th day of application, the moisture retention power (manufactured by Integral, CM825) and skin elasticity (manufactured by Integral, shock wave measuring device, RVM600) were measured on the left and right sides of the face.

As a result, compared with the left half of the face, the moisture retention and skin elasticity of the right half of the face to which the cosmetic of Example 2 was applied were 136% and 161%, respectively, and both values were improved. Since skin elasticity represents exfoliation of the skin and regeneration of the skin, cosmetics containing organic acid peptide-bonded luteolin derivatives were considered effective for beauty.

As a result, it was confirmed that the cosmetic of Example 2 has improved moisture retention and elasticity. In addition, side effects and abnormal feeling of use due to the application of the cosmetic were not recognized.

The organic acid peptide-bonded luteolin derivative obtained in the present invention exhibits an antioxidant action, and further has an action of destroying cancer cells. Therefore, it has an effect on cancer and has few side effects. From the perspective of preventing cancer, it can improve the QOL of the people, increase the healthy workforce, and contribute to reducing medical costs.

Since the organic acid peptide-bonded luteolin derivative obtained in the present invention has an action of improving skin cells, it contributes to those suffering from atopy and skin trouble, and contributes to the development of the cosmetics industry.

Since the organic acid peptide-bonded luteolin derivative obtained in the present invention can be used as a food, it contributes to the development of the food industry using an antioxidant action.

The organic acid peptide-bonded luteolin derivative obtained in the present invention is used as a pharmaceutical preparation and contributes to the development of the pharmaceutical industry.

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

Claims (2)

The organic acid peptide bond luteolin derivative which shows the antioxidant effect | action shown by following formula (1).

  2. The organic acid peptide-bound luteolin derivative having antioxidant activity according to claim 1, wherein the fermented liquor is fermented by adding koji mold to fermented chrysanthemum flowers, gold powder and soybean powder. Manufacturing method.