JP2009084170A - Terpene derivative exhibiting apoptosis-inducing activity and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a terpene derivative providing weak side effects and exhibiting excellent apoptosis-inducing activity, and to provide a method for producing the terpene derivative. <P>SOLUTION: The terpene derivative providing the weak side effects and exhibiting the excellent apoptosis-inducing activity has -1 mV to -600 mV oxidation-reduction potential. The derivative has a tripeptide consisting of cysteine, glycine and cysteine bonded to a hydroxy group of a terpene by an ester bond. The cysteine, the glycine and the cysteine are peptide-bonded. The method for producing the derivative includes steps of adding soybean powder and Bacillus natto to a powder of a persimmon leaf or a powder of a pine leaf, fermenting the resultant mixture, filtering the fermented liquid, and reducing the resultant filtrate using an alkali, the main steps therein being the fermentation step and the alkali reduction step. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a terpene derivative exhibiting an apoptosis-inducing action having an oxidation-reduction potential of minus 1 mV to minus 600 mV, 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 to solve 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 acts on 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. Accordingly, the search for drugs and natural products that specifically induce apoptosis in 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, the raw materials are not bamboo leaves or pine needles, and no mention is made of reduction action or oxidation-reduction potential (for example, see Patent Document 5).

There is an invention of a composition for preventing or treating inflammation involving type IV allergic reaction, and terpenes derived from bamboo leaves have an allergy-improving effect, but this invention does not mention an effect on cancer. (For example, refer to Patent Document 6).

On the other hand, terpene derivatives derived from bamboo leaves or pine needles, which have an apoptosis-inducing action, are characterized by a reducing action, are structurally characterized by binding to tripeptides, and are characterized by fermentation and reduction treatment in production. Since it invented about the manufacturing method to perform, it demonstrates below.
JP 2005-89328 A JP 2004-168713 A Special table 2006-515292 Special table 2002-515430 WO2003 / 057224 JP-A-2004-10531

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.

On the other hand, a naturally occurring substance has a high safety, but has a problem that its apoptotic action is mild. Therefore, a natural product-derived substance having a weak side effect and an excellent apoptotic action is desired.

The present invention has been made paying attention to the problems existing in the prior art as described above. The object is to provide a terpene derivative that has low side effects and exhibits an excellent apoptosis-inducing action.

Another object of the present invention is to provide a method for producing a terpene derivative that exhibits an efficient apoptosis-inducing action from the step of adding soybean powder and natto bacteria to bamboo leaf powder or pine needle powder and alkali-reducing the fermented fermentation broth. .

In order to achieve the above object, the invention described in claim 1 relates to a terpene derivative exhibiting an apoptosis-inducing action represented by the following formula (1) having an oxidation-reduction potential of minus 1 mV to minus 600 mV.

The invention according to claim 2 is a terpene exhibiting an apoptosis-inducing action according to claim 1, which comprises a step of adding soybean powder and natto bacteria to bamboo leaf powder or pine needle powder and alkali reducing the fermented fermented liquid. The present invention relates to a method for producing a derivative.

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

According to the terpene derivative according to claim 1, the side effect is weak and an excellent apoptosis-inducing action is exhibited.

According to the manufacturing method of Claim 2, a terpene derivative can be obtained efficiently.

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

First, a terpene derivative exhibiting an apoptosis-inducing action represented by the following formula (1) having an oxidation-reduction potential of minus 1 mV to minus 600 mV will be described.

The term “terpene derivative” as used herein refers to a structure that belongs to triterpenes and has a structure in which a tripeptide is ester-bonded to a hydroxyl group, and is represented by the above formula (1).

Further, the terpene derivative represented by the above formula (1) has a feature of having an SH group.

Further, tripeptides bonded to a hydroxyl group are cysteine, glycine, and cysteine, which are bonded by peptide bonds. Furthermore, the carboxyl group of cysteine and the hydroxyl group of the terpene skeleton are ester-bonded.

This terpene derivative has both fat-soluble properties due to the skeleton as a terpenoid and water-soluble properties by tripeptides, so it has high absorption from the intestinal tract and skin, and is easily transferred to the blood. Is preferable.

This terpene derivative works on the cell membrane, has both fat-soluble and water-soluble properties, can enter the cell nucleus and mitochondria, and can act on the target site, so it has excellent absorption and dynamics. To preferred.

This terpene derivative, when ingested in excess, is degraded by esterase into tripeptides and terpenoids, which are further decomposed into amino acids, sulfur dioxide, and carbon dioxide and excreted from the kidneys. Highly preferred.

This terpene derivative is safe from the point that the mechanism of stimulating mitochondria and inducing apoptosis via caspases is clear to cancer cells, and the safety of its action has been confirmed.

Moreover, this terpene derivative accelerates | stimulates peeling with respect to the keratinocyte of a skin cell, and promotes the reproduction | regeneration of skin. In addition, it acts on melanocytes, which are melanin-producing cells, induces apoptosis, destroys melanocytes, and reduces spots and spot production.

Further, since this terpene derivative has a redox potential of minus 1 mV to minus 600 mV, it has a strong reducing action, decomposes melanin, which is an oxidation product, and eliminates the causative substance of stains.

This oxidation-reduction potential of minus 1 mV to minus 600 mV is created by alkali reduction or an ion reduction device. On the other hand, when the oxidation-reduction potential is positive, there is a possibility that the tissue may be damaged by oxidation. On the other hand, it is preferable that the oxidation-reduction potential is negative from the viewpoint of suppressing oxidation and protecting the tissue.

Since this terpene derivative has a negative oxidation-reduction potential, it reduces oxidative stress such as skin oxidation and ultraviolet rays, and makes apoptosis more effective.

The terpene derivative exhibiting the apoptosis-inducing action here can be chemically synthesized, and can be obtained by fermentation using plant cells, animal cells, yeasts or microorganisms and biosynthesized.

It can also be obtained by fermenting and extracting a plant. Furthermore, it can be obtained by extraction from a plant body such as persimmon leaf, persimmon fruit, stem or root, pine needle, green tea leaf and the like.

Further, the terpenic acid derivative can be fermented with bamboo leaf, pine leaf, green tea leaf, ginger garlic, onion, garlic and alkali reduced. For example, bamboo leaves, pine needles, garlic garlic, onion or garlic pulverized product, soybean pulverized product and fermented product fermented by adding Bacillus natto are obtained by alkali reduction. It is preferable because pine needles can be used effectively.

In addition, when extracting from plants, bamboo leaves, pine leaves, green tea leaves, dokudami, garlic garlic, onions, garlic, soybeans, scallops, licorice, vermicelli, barley leaves, barley leaves, kuzu flowers, capsicum, pears, chestnuts, cod, Wasabi, bracken, rice, wheat, corn, radish, rape blossoms, cherry, pine, aoki, akane, akamegashiwa, akebi, amachazul, amadokororo, aloe, licorice, itadori, wild boar, lobster, maggot, radish, radish , Ebisugusa, Auren, Psyllium, Okera, Okra, Hypericum, Onamomi, Ominae, Kakidooshi, Calla Lily, Callas Bisaku, Kawaraketsumei, Kawaranadeshiko, Kanaoi, Jerusalem artichokes, Yellowfin, Yellowfin, Yellowfin Hiki, Kusaiso, Cuco, Bamboo, Kudzu, Gardenia, Kohonone, Kobushi, Saikachi, Sabonso, Sartorii Rose Bucket, Sanshuyu, Janohige, Silane, Honeysuckle, Seri, Assembly, Tamashiba, Taranoki, Dandelion, Chigaya, Thrips Carcinus, Tochiban carrot, Nanten, Neubara, Octopus, Hazel, Hahakogusa, Hikikoshi, Hishi, Hitotsuba, Biwa, Fuki, Fukujusou, Fuji, Matabi, Mehajiki, Yamanoimo, Yukinoshita, Mugwort, Gentian, Gentian, Gentian Flowers or roots are preferred because they are readily available.

When extracting from algae, Aosa, Aonori, Amanori, Alame, Iwanori, Egonori, Ogonori, Kawanori, Enagaonikombu, Gagomecombu, Nagacombu, Hosomecombu, Maconbu, Mitsuishikonbu, Rishibune, Azalea, Azalea The leaves, stems, or roots of squirrel, mukadenori, okinawa mozuku, mozuku, wakame, kukiwakame, mekabuwakame are preferred because they are easily available.

It can be obtained by fermenting lactic acid bacteria, brewer's yeast, Bacillus subtilis or Bacillus subtilis together with persimmon leaves, persimmon seeds, stems or roots, pine needles, pine bark, green tea leaves and soybeans. In addition, fermented products obtained by fermenting lactic acid bacteria, brewer's yeast, Bacillus subtilis or Bacillus subtilis together with soybeans from bamboo leaves, bamboo berries, stems or roots, pine needles, and pine bark can be obtained by alkaline reduction it can. By doing in this way, a product is obtained in a state with strong reducing power and antioxidant power, and it is preferable because it has high resistance to oxidation and storage.

The terpene derivative exhibiting the apoptosis-inducing action here is obtained as a liquid or powder and can be used as a pharmaceutical material, food material, or cosmetic material.

When used as a pharmaceutical material, it is preferable to separate and purify the target terpene derivative because the purity of the target terpene derivative is increased and impurities can be removed.

The desired terpene derivative is obtained by separation with a separating 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 50 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 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.

Collecting the fraction containing the desired terpene derivative using apoptosis-inducing action as an index, removing the solvent by drying or vacuum drying, and obtaining the desired terpene derivative as a powder or concentrated liquid can eliminate the effects of the solvent ,preferable.

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.

0.1-20 weight% is preferable, as for content of the said terpene derivative in these pharmaceuticals, 1-15 weight% is more preferable, and 5-10 weight% is further more preferable.

When the content of the terpene derivative is less than 0.1% by weight, the action cannot be sufficiently exhibited because the content of the terpene derivative is too small. On the other hand, when it exceeds 20% by weight, the content of the component contributing to the stability of the preparation is relatively lowered.

The above pharmaceutical products can be used in combination with other pharmaceutical products. For example, the combined use with cisplatin is preferable because a synergistic apoptosis inducing action and anticancer effect can be obtained by different action mechanisms.

The food preparation is taken orally in several divided doses per day. The daily intake is preferably 0.1 to 10 g, more preferably 0.3 to 5 g, and even more preferably 0.5 to 3 g. If the daily intake is less than 0.1 g, sufficient effects may not be exhibited. If the daily intake exceeds 10 g, the cost may increase. In addition to the above, it can be used in the form of rice cake, rice crackers, cookies, beverages, powders and the like.

The obtained food preparation is preferably used as a health functional food as a nutrition functional food or a food for specified health use.

When the obtained food preparation is used for pets such as dogs and cats, the extract of bamboo leaves and pine needles is preferable because it has a deodorizing action and an antibacterial action.

It can be used together with surfactants, solvents, thickeners, excipients and the like according to conventional methods as cosmetics. 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.

As a cosmetic, it is applied, wiped or sprayed several times a day. The daily usage is preferably from 0.01 to 5 g, more preferably from 0.05 to 3 g, even more preferably from 0.1 to 1 g. If the daily usage is less than 0.01 g, sufficient effects may not be exhibited. If the daily usage exceeds 5 g, the cost may increase.

The resulting cosmetic promotes skin exfoliation improvement and skin regeneration. Moreover, since it exhibits a whitening effect by destroying melanin-producing cells, it is also used as a whitening cosmetic or a quasi-drug.

Next, a method for producing a terpene derivative exhibiting an apoptosis-inducing action comprising a step of adding soybean powder and natto bacteria to bamboo leaf powder or pine needle powder and subjecting the fermented fermentation broth to alkali reduction will be described.

The terpene derivative exhibiting the apoptosis-inducing action here is the terpene derivative described above, belongs to triterpenes, shows a structure in which a tripeptide is ester-bonded to a hydroxyl group, and has an SH group. Has characteristics.

Furthermore, tripeptides bonded to hydroxyl groups are cysteine, glycine and cysteine. The carboxyl group of cysteine and the hydroxyl group of the terpene skeleton are ester-bonded.

As the pulverized product of bamboo leaves used as a raw material, any of Japanese, Chinese, American and African bamboo leaves is used.

The cocoon referred to here may be any of the genus Fuyuri, Jiro, Hiramu Mutsu, Koshu Hyakume, Shizomi, and Dogami Bee.

The pine needles used as the raw material are red pine, wax grove, Maurice Arboretum witches bloom, Janome red pine, weeping pine, tanyosho, tagyosho, and black pine leaves. It is used, has abundant food experience, and has been confirmed to be safe.

The production area of pine needles may be any of Japan, China, Europe, America, Canada, etc., and pine needles cultivated with no agricultural chemicals are preferred.

Bamboo leaves and pine needles can be either fresh or dried.

The collected bamboo leaves and pine needles are preferably washed with tap water.

The bamboo leaves or pine needles are crushed. That is, it is preferable that the bamboo leaves or pine needles are dried and then pulverized by a pulverizer because the reaction can be carried out effectively.

CD dryers manufactured by Nishimura Corporation as dryers, Vibrons and funnels manufactured by Okawara Manufacturing Co., Ltd., swirling air dryers manufactured by Nara Machinery Co., Ltd., tornesh dryers, fluidized bed dryers, and crushers Nara Machinery Free Mill, Super Free Mill, Sample Mill, Goblin, Super Clean Mill, Micros, Toyo Riko's Small Vacuum Dryer as Vacuum Dryer, Matsui's Small Vacuum Heat Transfer Dryer DPTH -40, clean dry VD-7, VD-20, etc. manufactured by AKM Kyushu Technos Co., Ltd. are used.

The soybean powder used as a raw material can be used in soybeans of any origin such as Japanese, Chinese, American or Russian, and is pulverized with a pulverizer such as DM-6 manufactured by Nakayama Technical Research Laboratory.

The Bacillus natto used as a raw material is a kind of Bacillus subtilis used for natto and food processing. Natto bacteria manufactured by Natto Motopo are preferred because they are suitable for fermentation.

As for each addition amount regarding said fermentation, 0.5 to 3 weight is preferable for soybean powder, and 0.001 to 0.03 weight is preferable for Bacillus natto with respect to 1 weight of bamboo leaf powder or pine needle powder.

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

Moreover, this fermentation is heated at 30-53 degreeC, and fermentation is performed for 24 to 90 hours. After fermentation, in order to carry out the extraction efficiently, it is diluted with tap water.

Through this fermentation process, a peptide obtained by decomposing a protein derived from soybean binds to a terpene derived from bamboo leaves or pine needles. However, depending on the fermentation, it is in an oxidized state and is structurally unstable. Therefore, the structure is stabilized by being reduced and maintaining the oxidation-reduction potential low.

It is preferable that the fermented product produced by the above fermentation is extracted with hot water of 30 to 60 ° C. because the product can be efficiently recovered and the next step can be easily performed.

It is preferable to concentrate the obtained fermented product by freeze-drying or the like.

This fermented product is alkali reduced. The alkali reduction step 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 Devices such as “Acura Blue” and “Mineral Reduction Water Conditioner“ Genki no Water ”” manufactured by Nippon Kosen Research Co., Ltd. are preferable.

By this reduction, the redox potential is reduced to minus 1 mV to minus 600 mV, and the target terpene derivative is stabilized.

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.

As the separation carrier or resin, porous polysaccharide, silicon oxide compound, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymer, etc. whose surface is coated are used. It is preferable to obtain the desired terpene derivative by separation with an appropriate separation solvent, purification, and removal of the organic solvent.

As the solvent for separation, methanol, ethanol, chloroform, hexane, ethyl acetate, benzene, ether, and the like are used. Of these, ethanol for food processing or ethanol containing water is preferable because of its high use range.

The terpene 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.

We collected the leaves of Fuyu persimmon grown in pesticide-free in Gifu Prefecture. This was washed with water, dried with a dryer, and pulverized with a pulverizer (Cuisinate) to obtain 1 kg of bamboo leaf pulverized material.

Moreover, the soybeans from Hokkaido were supplied to a mixer (Cuisinate) to obtain 1 kg of soybean pulverized product. 1 kg of each was put into a container of a clean fermentation tank (sterilized round 20 liter tank for fermentation), and 5 kg of tap water was added and stirred.

To this, 50 g of powdered natto bacteria (manufactured by Natto Motopo) was supplied to a fermentation tank, and after stirring, fermented in a temperature range of 38 to 40 ° C.

Stir well 5 times in the middle of the fermentation process. For the determination of the end of fermentation, the production of the target terpene derivative or its oxide was used as an index.

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 desired terpene derivative was produced after 48 hours of fermentation, so that the fermentation time was 48 hours and the fermentation was terminated. 5 kg of warm water of 40 ° C. was added to the fermentation broth.

This fermentation broth was subjected to a filter with diatomaceous earth and filtered. The obtained filtrate was subjected to Cellular Kiss (manufactured by Zenon Co., Ltd.), and subjected to alkali reduction by being subjected to an alkali reduction device (alkaline reduced water / strongly oxidized water continuous generator “Protech ATX-501” manufactured by Zemitis).

The alkali reduced product thus obtained was subjected to a freeze dryer manufactured by Nippon FD, and 768 g of the intended terpene derivative was obtained as a powder. This was designated as Sample 1.

The terpene derivatives obtained from pine needle powder, soybean powder and Bacillus natto are described below.

We collected black pine leaves grown in pesticide-free in Shizuoka Prefecture. This was washed with water, dried with a dryer, and pulverized with a pulverizer (Cuisinate) to obtain 1 kg of pine needles.

In addition, Hokkaido soybean was used in a mixer to obtain 1 kg of soybean ground material. 1 kg of each was put into a clean fermentation tank, and 4 kg of tap water was further added and stirred.

For this, 40 g of powdered natto bacteria (manufactured by Natto Motopo) was supplied to a fermentation tank, and after stirring, fermented in a temperature range of 39 to 42 ° C.

Stir well three times in the middle of the fermentation process. For the determination of the end of fermentation, the production of the target terpene derivative or its oxide was used as an index. 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 desired terpene derivative was produced after 39 hours of fermentation, so that the fermentation time was 48 hours and the fermentation was terminated. 4 kg of warm water at 41 ° C. was added thereto.

This fermentation broth was subjected to a filter with diatomaceous earth and filtered. The obtained filtrate was subjected to Cellular Kiss (manufactured by Zenon Co., Ltd.), and subjected to alkali reduction by being subjected to an alkali reduction device (alkaline reduced water / strongly oxidized water continuous generator “Protech ATX-501” manufactured by Zemitis).

The alkali reduced product thus obtained was subjected to a lyophilizer manufactured by FP Japan, and 550 g of the desired terpene derivative was obtained as a powder. This was designated Sample 2.

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

Sample 1 and sample 2 obtained as described above were dissolved in an extraction medium, analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation), and further a nuclear magnetic resonance apparatus (NMR, manufactured by Bruker, AC- 250). As a result of structural analysis, terpene derivatives bound to the target tripeptide were identified from Sample 1 and Sample 2.

Hereinafter, an apoptosis induction test using human cancer cells will be described.
(Test Example 2)

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 the specimen 2 obtained in Example 2 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 68% on average as compared with the control group, and the appearance rate of apoptosis was 29%.

In addition, when 0.1 mg / ml of Specimen 1 was added, the number of cells decreased to 34% compared to the control group, and the appearance rate of apoptosis was 76%.

As a result, when 0.01 mg / ml of Sample 2 was added, the number of cells decreased to 71% compared to the control group, and the appearance rate of apoptosis was 26%.

When 0.1 mg / ml of Specimen 2 was added, the number of cells decreased to 34% compared to the control group, and the appearance rate of apoptosis was 60%.

From the above results, destruction of cancer cells due to apoptosis was observed in Sample 1 and Sample 2. On the other hand, as a result of the same test using normal human-derived skin fibroblasts, the addition of Sample 1 and Sample 2 did not change the number of cells, and safety against normal cells was confirmed.

Hereinafter, a mouse cancer transplantation test using mouse-derived melanoma cells S-100 will be described.
(Test Example 3)

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 sample 1 obtained in Example 1 and Sample 2 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.

The average value of the tumor weight in the distilled water administration group was 6.63 g. In contrast, the average value of the tumor weight of Sample 1 administered at 0.1 mg / kg was 2.11 g. Furthermore, the average value of the tumor weight of Sample 1 administered at 1 mg / kg was 0.56 g.

By administration of 0.1 mg / kg of Specimen 2, the average value of the tumor weight was 3.56 g. Furthermore, the average value of the tumor weight by 1.0 mg / kg administration of the sample 2 was 1.02 g.

As a result, tumor shrinkage and weight reduction were observed by administration of Sample 1 and Sample 2, and Sample 1 and Sample 2 were confirmed to have a tumor shrinking effect via apoptosis.

Hereinafter, examples of cosmetics composed of terpene derivatives exhibiting an apoptosis-inducing action 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.

30 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. After dissolving these, it cooled and obtained the emulsion as cosmetics. This was designated as Sample 3 of Example 3. As a control cosmetic, an emulsion excluding only the powder of Sample 1 obtained in Example 1 was prepared.

Below, the test example evaluated about the effect and side effect of cosmetics is shown.
(Test Example 4)

Ten healthy females aged 31 to 57 years old applied 10 mL of the emulsion obtained in Example 3 to the right half of the face for 14 days. An emulsion excluding the powder of the terpene derivative of Example 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 moisture retention and skin elasticity of the right half of the face to which the cosmetic product of Example 3 was applied were improved by 134% and 145%, respectively. Since skin elasticity depends on skin exfoliation and skin regeneration, apoptosis of skin cells by terpene derivatives was considered effective.

From these results, it was confirmed that the cosmetic of Example 3 has improved moisture retention and elasticity. In addition, no side effects were observed due to the application of this cosmetic product.

According to the terpene derivative exhibiting an excellent apoptosis-inducing action with weak side effects obtained in the present invention, a therapeutic effect for improving cancer, a preventive effect for onset, and a skin improving effect are expected. The terpene derivative of the present invention can be used in pharmaceuticals, food preparations, and cosmetics, and can improve the lives of people suffering from cancer and skin health. Therefore, the present invention is an invention that can contribute to the development of the pharmaceutical industry, the food industry, and the cosmetics industry.

Furthermore, bamboo leaves and pine leaves are incinerated as waste, leading to environmental destruction. By effectively using bamboo leaves and pine leaves, environmental destruction can be prevented. It also leads to the development of agricultural resources and the development of industry.

Claims (2)

A terpene derivative exhibiting an apoptosis-inducing action represented by the following formula (1) having an oxidation-reduction potential of minus 1 mV to minus 600 mV.

  The method for producing a terpene derivative exhibiting an apoptosis-inducing action according to claim 1, comprising a step of adding soybean powder and natto bacteria to bamboo leaf powder or pine leaf powder and subjecting the fermented fermented liquor to alkali reduction.