JP2009280513A - Beta-glucan peptide-calcium conjugate and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a beta-glucan peptide-calcium bonded product, and to provide a method for producing the beta-glucan peptide-calcium bonded product. <P>SOLUTION: Provided is the beta-glucan peptide-calcium bonded product wherein a tripeptide comprising tyrosine, cysteine and phenylalanine is ester-bonded to the hydroxy groups of beta-glucan comprising three 1,3-bonded beta-glucan molecules. Two beta-glucan peptide molecules used as base molecules are bonded to each other, and the hydroxy groups of the tyrosine groups of the two molecules are bonded to one calcium atom. The beta-glucan peptide-calcium bonded product exhibits a cancer cell-reducing action and an action for adsorbing harmful substances and environmental substances. Provided is a method for producing the same, comprising a process for alkali-reducing a fermentation product obtained by adding and fermenting Monascus prepreus to the dry powder of lettuce roots, gold powder and soybean powder. The fermentation process and the reduction process are main processes. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a beta glucan peptide calcium conjugate and a method for producing the same.

Cancer is the leading cause of death among Japanese people, and about 300,000 people die from cancer or related diseases in one year, causing an increase in medical expenses. For this reason, doctors, pharmaceutical industries, the Ministry of Health, Labor and Welfare, and the health and medical industry are implementing various measures and research and development to reduce the incidence of cancer.

In addition, various anticancer drugs have been developed as drug treatments for cancer, and drugs that suppress cancer cell growth, differentiate cancer, restore immunity, and suppress cancer 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. 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 a strong anticancer effect, their side effects are also severe, and as a result of the occurrence of vomiting, anemia, decreased immunity, infection, etc., the amount and period of use of anticancer agents are limited.

Apoptosis is a mechanism that specifically destroys cancer cells. Apoptosis is a phenomenon in which a cancer cell's DNA is broken into pieces and the cancer cell is killed spontaneously so that the cancer cell itself commits suicide, and there are few side effects. Therefore, search for drugs and natural products that induce apoptosis that specifically destroys cancer cells is in progress.

The inventions derived from natural products, plants and herbs that induce apoptosis include polycoic acid A (compound 1), polycoic acid B (compound 2), polycoic acid G (compound 3), polycoic acid H ((compound 4), dehydro An active ingredient containing at least one triterpene compound selected from the group consisting of ebliconic acid (compound 5), tumrosic acid (compound 6), dehydrotumulosinic acid (compound 7) and 3-epidehydrotumulosinic acid (compound 8) There is an invention relating to a DNA synthesizing enzyme and a DNA topoisomerase-inhibiting composition (see, for example, Patent Document 1).

In addition, an invention relating to a drug capable of inducing apoptosis of synovial cells that has caused abnormal proliferation or the like and suppressing the abnormal proliferation has been reported (for example, see Patent Document 2).

As an invention relating to apoptosis, there are a diterpene compound and a composition comprising an effective amount of a diterpene compound (see, for example, Patent Document 3).

Also, a mixture comprising one or more isolated triterpene glycosides, a) can be isolated from tissue of Acacia victoriae, b) a tritenpene glycoside having a molecular weight in the range of about 1800-2600 There are inventions of c) the ability to induce cytotoxicity against Jurkat cells, and d) the ability to induce apoptosis against Jurkat cells (see, for example, Patent Document 4).

Furthermore, for terpene derivatives, an apoptosis inducer containing as an active ingredient a compound selected from the group consisting of maslinic acid, erythrodiol, ubaol, betulin, physiologically acceptable salts thereof and derivatives thereof has been reported. However, industrial use is limited (for example, refer to Patent Document 5).

There is a relationship between environmental pollution and the development of cancer, and there are already inventions of anti-fading food, anti-fading method and anti-fading food manufacturing method for detoxifying substances, but this is limited to konjac food. Therefore, there is no description of the new component (see, for example, Patent Document 6).

This time, we have invented beta-glucan that exhibits a cancer-suppressing action, a pollutant-removing action, and a skin-improving action, and a substance characterized by the binding of peptide and calcium, and is characterized by fermentation and reduction treatment. Now that the method has been invented, it will be described.
JP 2005-89328 A JP 2004-168713 A Special table 2006-515292 Special table 2002-515430 WO2003 / 057224 JP 2004-215570 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. Moreover, there is a problem that a chemically synthesized environmental improving agent remains in the natural world.

On the other hand, although the safety | security is high about the naturally derived substance, there exists a problem that the destruction effect | action of a cancer cell and the removal effect | action of a harmful substance are mild. Therefore, a natural product-derived substance that has weak side effects and excellent cancer cell destruction action and harmful substance removal action is desired.

In order to achieve the above object, the invention according to claim 1 relates to a beta-glucan peptide calcium conjugate that exhibits a cancer suppressing action, a contaminant removing action, a skin improving action, and the like.

The invention according to claim 2 is a beta-glucan peptide calcium conjugate comprising a step of adding red koji molds to dry powder of lettuce root, gold powder and soybean powder and subjecting the fermented liquid fermented to alkali to reduction. It relates to a manufacturing method.

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

According to the beta-glucan peptide calcium conjugate of claim 1, side effects are weak, and excellent cancer cell destruction action, harmful substance removal action and skin improvement action are exhibited.

According to the manufacturing method of Claim 2, a beta glucan peptide calcium conjugate | bonded_body can be manufactured efficiently.

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

First, the beta glucan peptide calcium conjugate represented by the following formula (1) will be described.

The beta-glucan peptide calcium conjugate as used herein refers to a tripeptide tyrosine carboxyl group bound to beta-type glucose formed by binding three molecules of beta 1-3 in the order of tyrosine, cysteine, and phenylalanine from the C-terminal. The 4-glucose hydroxyl group of beta glucose is ester-bonded.

The beta glucan peptide structure is bound to one calcium atom. That is, two molecules of the above-mentioned beta glucan peptide structure are required, and a structure in which the hydroxyl group of the benzene ring of each tyrosine is bonded to one atom of calcium is shown.

Glucose constituting this structure is D-type, and all amino acids are L-type.

In this beta-glucan peptide calcium conjugate, the beta-glucan peptide moiety reacts with the surface antigen of the cancer cell to target the cancer cell. Calcium and tripeptide attack the targeted cancer cell gene, destroy the gene, and exhibit apoptosis.

This beta glucan peptide calcium conjugate has an SH group due to one cysteine in the tripeptide. This SH group is preferable because it exhibits a reducing action and suppresses an oxidation reaction caused by cancer cells, so that the structure is stably maintained.

Calcium in the beta-glucan peptide calcium-bound body is a process of intracellular activation after 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. That is, the intracellular signal transduction pathway via calcium in the growth of cancer cells is suppressed, and cancer cells are suppressed.

This beta-glucan peptide calcium conjugate has 3 carbohydrates as a beta-glucan and is a short chain, but has the properties of a beta-glucan, and is an immune cell such as a natural killer cell or a killer T cell. Activates and promotes destruction of cancerous tissue. In addition, the glucan moiety takes up toxic metals and detoxifies.

In addition, the peptide part of this beta-glucan peptide calcium conjugate acts on newly formed vegetative blood vessels in cancer tissue, suppresses the growth of angiogenesis by suppressing the intracellular signal transduction system via calcium, and shrinks cancer tissue. . Instead of calcium, it adsorbs and detoxifies harmful metals such as mercury, arsenic and chromium.

Further, the beta glucan moiety has a cyclic structure, and has characteristics that it can suppress emission to the environment and detoxify the environment by enclosing fine particles such as diesel particles and asbestos.

Two SH groups exert chelating action against dioxins and pesticides, trap dioxins and pesticides, and wrap the dioxins and pesticides so that the cyclic structure of beta-glucan folds, eliminating harmful effects And improve the environment.

In addition, when this beta-glucan peptide calcium conjugate is ingested in excess, it is degraded by esterases in the digestive tract, lungs, blood, and organs, and is degraded into tripeptides, beta-glucan and calcium atoms.

Tripeptides become amino acids, and are further decomposed into sulfur dioxide and carbon dioxide gas and excreted from the kidney, so that the safety is high and more preferable. The decomposed calcium binds to albumin in the blood and is excreted from the body as urine via the kidney.

Furthermore, this beta glucan peptide calcium conjugate is preferable because the beta glucan moiety exhibits an anti-inflammatory action, and thus inflammation caused by carcinogens and harmful substances is also suppressed.

In addition, this beta glucan peptide calcium conjugate enhances skin regenerative power by promoting exfoliation of skin cells from keratinocytes. Furthermore, by inhibiting the intracellular signal transduction system via calcium for inflammatory cells, it is preferable because it exerts an anti-inflammatory action and improves atopy and contact allergy.

Furthermore, this beta-glucan peptide calcium conjugate has a reducing action on the SH group of the peptide, assists in the action of vitamin C and vitamin E, suppresses the production of melanin, an oxidation product of tyrosine, and eliminates the causative agent of stains. Eliminate and increase the utilization rate of proline to promote collagen production. Furthermore, the glucan moiety promotes the production of chondroitin and hyaluronic acid.

This beta glucan peptide calcium conjugate is produced by an alkali reduction or ion reduction device. In particular, by using a gold atom as a catalyst, the reducibility is enhanced and the structure is maintained.

This beta-glucan peptide calcium conjugate is produced by vegetables such as lettuce and cabbage, plant cells and animal cells in the presence of gold powder, and useful yeast such as baker's yeast, useful yeast such as red yeast and natto It can also be obtained by a fermentation process using microorganisms, biosynthesized in the body of microorganisms, and extracted. In particular, lettuce root, cabbage root, and Chinese cabbage root are preferable as raw materials because sugars and peptides as materials are present.

This beta glucan peptide calcium conjugate is obtained as a liquid or powder. Moreover, sterilization is also easy and it can utilize also as an injection of an anticancer agent and a cancer inhibitor as a sterile pharmaceutical formulation.

When used as a pharmaceutical material, it is preferable to separate and purify the target beta-glucan peptide calcium conjugate because the purity of the target beta-glucan peptide calcium conjugate 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 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.

Food preparations are used as raw materials for raw material processing and food production for the purpose of preventing cancer and maintaining skin health. 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 cleaning the skin.

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 obtained cosmetic promotes keratin improvement and skin regeneration of atopic patients through immunoregulatory action and anti-inflammatory action. Moreover, since it exhibits a whitening effect, it is also used as a whitening cosmetic or a quasi-drug.

Dioxins, PCBs, asbestos, chemical substances, radiation substances and environmental pollutants are used as soil conditioners for the purpose of removing harmful substances from agricultural land. In addition, it is used to improve the surrounding air environment, soil contamination, and work environment in chemical factories, main roads and houses with a lot of exhaust gas. It is also used as an environmental improver to remove pollutants from rivers and oceans.

Next, a method for producing a beta-glucan peptide calcium conjugate, characterized by adding red koji molds to lettuce root dry powder, gold powder and soybean powder and alkali-reducing the fermented fermentation broth, will be described.

The beta glucan peptide calcium conjugate referred to here is the aforementioned beta glucan peptide calcium conjugate.

The dry powder of lettuce root used as a raw material is a dry powder of lettuce root cultivated for food. The scientific name for lettuce is Lactuca sativa, an annual or biennial plant belonging to the genus Asteraceae, which is used as an edible vegetable. The Japanese name is Chisha (both 萵苣 / 苣 and Chisa). Some of the roots are used as food, but most are used as fertilizers, and their eating experience and safety have been confirmed.

Examples of lettuce include head lettuce, leaf lettuce, standing lettuce, cutting lettuce, and stem lettuce, and any lettuce is used as a raw material here.

Lettuce roots have growth points, are rich in growth factors, and microorganisms symbiotic to the roots produce growth factors consisting of proteins that regulate the growth of lettuce. Aspergillus niger acts on this growth factor and is fermented and catabolized, thereby generating a peptide portion of a beta glucan peptide calcium conjugate.

Lettuce roots are used in any of Japan, China, Korea, and the United States. Of these, reduced pesticide-free or pesticide-free lettuce roots with limited use of pesticides are preferred because the action of microorganisms is promoted. Moreover, since the lettuce seeds are hydroponically cultivated, the cultured main roots and whiskers are soft and easy to process.

Lettuce roots are used as either main roots or whiskers. Lettuce roots are washed with tap water and dried to reduce impurities.

Lettuce roots are not used for food as vegetables but are mainly used as fertilizers. When grown in large quantities, they are discarded as waste. Can contribute to the reduction of waste. Cabbage root, Chinese cabbage root, Komatsuna and spinach root are also used as a substitute for lettuce root.

Here, the gold powder used as a raw material is 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. Gold bullion made by Mitsubishi Materials and Sumitomo Metal Mining purchased from US Trading Corporation is preferred because of its high purity and high reactivity.

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, soybeans cultivated organically or without agricultural chemicals are more preferred because they do not contain harmful agricultural chemicals or metals.

Soybeans and lettuce roots are used when using free mill, super free mill, sample mill, goblin, super clean mill, micros, vacuum dryer from Toyo Riko Co., Ltd., Matsui Co., Ltd. It is fermented by being pulverized by a pulverizer such as a small decompression heat transfer dryer DPTH-40 manufactured by AKM, Kyushu Technos Co., Ltd., Clean Dry VD-7, VD-20, Nakayama Technical Research Institute DM-6, etc. This step is preferable because it easily proceeds efficiently.

Furthermore, it is preferable to sterilize the gold powder and the dried powder of soybean and lettuce root by autoclaving after pulverization because it can prevent the propagation of various bacteria. 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.

This koji mold ferments a dry powder of lettuce root, a glucan consisting of soybeans and a peptide component at the same time, and a gold atom induces a conjugated enzyme and an ester-linked enzyme that bind this ester to catalyze the reaction.

The respective addition amounts relating to the fermentation are 0.0001 to 0.003 weight for gold powder, 0.4 to 4 weight for soybean powder, and 0.001 to 0.001 for soybean powder with respect to 1 weight of dry powder of lettuce root. 0.04 weight is preferred. It is preferable to pre-culture the koji mold before fermentation because it shortens the initial time of 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 target beta glucan peptide calcium conjugate is quantified by HPLC or TLC, and the growth control of the bacterial cells is confirmed, thereby controlling the fermentation process.

In this fermentation process, beta-glucan binds to the tripeptide in the presence of gold powder, but the bond is unstable and easily degraded by enzymes such as esterase. is necessary.

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 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.

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 / strongly oxidized water “Protech ATX-501” manufactured by Zemaitis, an alkali reduced water manufacturing 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 ethanol having a concentration of 20% to 80% is preferable in order to carry out the alkali reduction efficiently. This reduction step stabilizes the binding of the target beta glucan peptide calcium conjugate.

Separating and purifying the target beta-glucan peptide calcium conjugate 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.

The desired beta glucan peptide calcium conjugate 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.

Collecting fractions containing beta-glucan peptide calcium conjugate, removing the solvent by drying or vacuum drying, and obtaining the desired beta-glucan peptide calcium conjugate as a powder or concentrate can eliminate the effect of the solvent, preferable.

The beta glucan peptide calcium conjugate 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 merely examples, and conditions can be changed within the range of common sense according to differences in materials, raw materials, and specimens.

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.

A dry powder of lettuce root grown with reduced pesticides in the Amami region of Aichi Prefecture was used. After collecting leaves, only lettuce roots are collected, washed with tap water, dried in the sun, pulverized with a crusher (Super Free Mill manufactured by Nara Machinery Co., Ltd.), and dried lettuce roots. 1 kg of the pulverized powder was obtained.

Soybeans from Hokkaido were used in a mixer (Cuisinate) to obtain 1 kg of soybean pulverized product. The lettuce root and soybean grind 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.

Separately, 12 g of powdered red koji mold (manufactured by Kurisu Honpo) was subjected to a small fermentation tank, and a sterilized soybean powder and a precultured culture solution were prepared.

The pre-cultured red koji mold solution is added to the fermented tank containing the gold pulverized product, dried lettuce root powder and soybeans, and after stirring, warmed in a temperature range of 45 to 46 ° C. and fermented. It was.

In the fermentation process, the fermented liquor was sampled while bubbling and stirring by aeration, and the production of the intended beta glucan peptide calcium conjugate 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, a sufficient amount of the intended beta glucan peptide calcium conjugate was produced 5 days after the fermentation, and thus the fermentation was terminated. After stopping the fermentation, 3 kg of ethanol was added to the fermentation broth.

This fermentation broth was subjected 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 FP Japan, and 248 g of the intended beta glucan peptide calcium conjugate was obtained as a powder. This was used as a specimen 1 for the following test.

Below, the test method regarding the structural analysis of a beta glucan peptide calcium conjugate 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 structural analysis, a target conjugate obtained by binding a tripeptide composed of tyrosine, cysteine, and phenylalanine, beta-glucan, and calcium from specimen 1 was identified.

In this beta glucan, glucose was bound to beta 1,3. One molecule of calcium atom was bonded to the tyrosine hydroxyl group of two molecules of beta-glucan peptide.

Hereinafter, the redox potential test of the beta glucan peptide calcium conjugate 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 Corporation, OPR Pro).

As a result, the oxidation-reduction potential before addition was plus 525 mV, but the oxidation-reduction potential one minute after dissolution of Sample 1 was minus 249 mV, and the minus potential was maintained until 24 hours after dissolution. On the other hand, the redox potential of tap water was maintained at +531 mV.

Hereinafter, an apoptosis induction test using human cancer cells will be described.
(Test Example 3)

HeLa cells, which are human-derived uterine cancer cells purchased from ATCC, were used. As a culture solution, 10,000 cells cultured using a 5% fetal bovine serum-containing MEM medium (manufactured by Sigma) were seeded in a 35 mm culture dish and cultured at 37 ° C. in 5% carbon dioxide gas. To this, the specimen 1 obtained in Example 1 and cisplatin used as a control pharmaceutical were added at final concentrations of 0.01 mg / ml and 0.1 mg / ml, and further cultured for 48 hours.

After detaching the 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 cells decreased to an average value of 43% compared to the control group, and the appearance rate of apoptosis was 48%.

When 0.1 mg / ml of Specimen 1 was added, the number of cells decreased to 30% compared to the control group, and the appearance rate of apoptosis was 76%.

As a result, when 0.01 mg / ml of cisplatin as a control drug was added, the number of cells was 87% compared to the control group, and the appearance rate of apoptosis was 9%.

In addition, when 0.1 mg / ml of cisplatin as a control pharmaceutical was added, the number of cells was 77% compared to the control group, and the appearance rate of apoptosis was 10%.

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 as a control drug.

On the other hand, as a result of the same test using normal human-derived skin 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, normal cell damage was observed and toxicity was observed.

Hereinafter, a mouse cancer transplantation test using mouse-derived melanoma cells S-100 will be described.
(Test Example 4)

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

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

In addition, cisplatin was administered at the same dose as a control drug.

The average value of the tumor weight in the distilled water administration group was 9.67 g. On the other hand, the average value of the tumor weight of Sample 1 administered at 0.1 mg / kg was 3.56 g. Furthermore, the average value of the tumor weight of Sample 1 administered at 1 mg / kg was 1.89 g.

By administration of 0.1 mg / kg of cisplatin as a control drug, the average value of the tumor weight was 6.34 g. Furthermore, the average value of the tumor weight by the administration of 1 mg / kg of cisplatin was 3.26 g.

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

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

Hereinafter, examples of cosmetics comprising a beta glucan peptide calcium conjugate will be described.

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.

20 g of the specimen 1 obtained in Example 1, 0.1 g of α-tocopherol and 60 g of purified water were added to the obtained solution. These were dissolved and then cooled to obtain a milky lotion as a cosmetic. This was designated as Sample 3 of Example 3. As a control cosmetic, an emulsion was prepared in which only the powder of Sample 1 obtained in Example 1 was excluded.

Below, the test example evaluated about the effect and side effect of cosmetics is shown.
(Test Example 5)

Ten healthy females aged 24 to 58 years old applied 10 mL of the emulsion obtained in Example 2 to the right half of the face for 14 days. An emulsion excluding the specimen 1 was applied to the left half of the face.

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 to the left half of the face, the water retention and skin elasticity of the right half of the face to which the emulsion of Example 2 was applied were improved to 167% and 189%, respectively. Since skin elasticity depends on skin exfoliation and skin regeneration, cosmetics containing a beta-glucan peptide calcium conjugate were considered effective for beauty.

From these results, it was confirmed that the emulsion of Example 2 has improved water retention and elasticity. In addition, no side effects were observed due to the application of this cosmetic.

The adsorption test for heavy metals, cyanides, pesticides, dioxins and asbestos of beta-glucan peptide calcium conjugates is described below.

(Test Example 6)
As heavy metals, mercury chloride (Wako Pure Chemical), arsenic (Tokyo Chemical), lead chloride (Wako Pure Chemical), cadmium chloride (Tokyo Chemical), organic cyanide (potassium cyanide) (Wako Pure Chemical), pesticide (acetoate) TCDD and asbestos (Sigma Aldrich) were used as dioxins and Pirimiphosmethyl (manufactured by Kaeda Takeda Horticultural Co., Ltd.) and pyrimiphos methyl (manufactured by Sumika Takeda Horticultural Co., Ltd.).

These were dissolved or suspended in dimethyl sulfoxide to prepare liquids containing 100 ppm each. 0.01 mg of the beta glucan peptide calcium conjugate obtained in Sample 1 was added to each solution and left for 1 hour. This solution was subjected to ultrafiltration and separated into a beta glucan peptide derivative component (not filtered) and a filtrate. Mercury chloride, arsenic, lead, and cadmium contents of the filtrate were measured by an atomic absorption measuring device (Shimadzu Corporation, AA-6800).

The cyanide content of the filtrate was measured by the HPLC method using a Ponal kit CN · T manufactured by Dojin Chemical. Dioxins and asbestos were also analyzed by HPLC.

As a result, the mercury chloride, arsenic, lead and cadmium contents of the filtrate were 3 ppm, 2 ppm, 4 ppm and 5 ppm, respectively. The cyan content of the filtrate was 1 ppm. Furthermore, the concentrations of acephate and pyrimifosmethyl were 3 ppm and 1 ppm, respectively.

The residual amount of dioxin was 1 ppm, and the residual amount of asbestos was 2 ppm.

As a result, it was found that the beta-glucan peptide calcium conjugate of Sample 1 adsorbs and removes mercury chloride, arsenic, lead, cadmium, cyanide, pesticide, dioxin and asbestos.

Since the beta-glucan peptide calcium conjugate obtained in the present invention has an action of destroying cancer cells, it has an effect on cancer and has few side effects, thereby improving QOL of cancer patients and the public, and health. Increase the labor force and reduce medical costs.

Since the beta-glucan peptide calcium conjugate obtained in the present invention has an action of improving skin cells based on an antioxidant action, it contributes to those who suffer from atopy and skin trouble as a cosmetic, and contributes to the development of the cosmetic industry.

Since the beta glucan peptide calcium conjugate obtained in the present invention can be used as a food, it contributes to the development of the food industry.

The beta glucan peptide calcium conjugate obtained in the present invention is used as a pharmaceutical preparation for cancer treatment or cancer prevention and contributes to the development of the pharmaceutical industry.

The beta-glucan peptide calcium conjugate obtained in the present invention absorbs and excretes pesticides, chemical substances, toxic metals and environmental pollutants such as dioxin and asbestos, and improves soil, air, and water quality. Contribute to.

Lettuce root as a raw material is not used for food, becomes waste, and part of it is incinerated, causing an increase in carbon dioxide. By using the dry powder of lettuce root, it is possible to effectively use waste and agricultural resources, and 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 (2)

A beta glucan peptide calcium conjugate represented by the following formula (1).

  The method for producing a beta-glucan peptide calcium conjugate according to claim 1, wherein a step of adding fermented koji molds to lettuce root dry powder, gold powder and soybean powder and alkali-reducing the fermented fermented liquid.