JP5097356B2 - New anticancer drug - Google Patents

Description

  The present invention relates to the field of anticancer agents and anti-AIDS agents. Furthermore, the present invention relates to the field of DNA polymerase inhibitors.

  Conventionally, it is known that DNA polymerase is involved in cell proliferation, division, differentiation and the like. In addition, a DNA polymerase inhibitor has an inhibitory effect on cancer cell growth, has an inhibitory effect on HIV-derived reverse transcriptase in the case of AIDS, and suppresses specific antibody production against an antigen in the case of an immunosuppressive effect. It is considered to have an action. Therefore, DNA polymerase inhibitors are expected as pharmaceuticals and foods having a preventive effect on cancer, AIDS and immune diseases.

As DNA polymerase inhibitors, dideoxy TTP (ddTTP), N · methylmylimide, butylphenyl · dGTP and the like are currently known (Non-patent Document 1).
Wang TS. "Eukaryotic DNA polymers", Annual Review of Biochemistry, USA, Annual Reviews, 1991, Vol. 60, 513-552.

  As the above-mentioned DNA polymerase inhibitor, dideoxy TTP (ddTTP), N · methylmylimide, butylphenyl · dGTP, and the like are used, but the problem is that the production is not easy. Furthermore, anticancer agents generally have a problem of having strong side effects.

  In view of the above points, the present inventors made it an object of the present invention to provide a DNA polymerase inhibitor composition, an anticancer agent, and an anti-AIDS agent that are inexpensive, easy and safe to produce.

  The present inventors have found that a substance that inhibits DNA polymerase activity is contained in the extract of guava leaves and agaricus koji. Furthermore, the present inventors have found that these extracts have high anticancer action and anti-AIDS action, and that the guava leaf extract and the agaricus koji extract have a synergistic effect, thereby completing the present invention. did.

Accordingly, the present invention provides the following:
(Item 1) A pharmaceutical composition for treating and / or preventing cancer, comprising a guava leaf extract,
The guava leaf extract contains polyphenols and the following:
A pharmaceutical composition having an inhibitory activity against an enzyme selected from the group consisting of DNA polymerase α, DNA polymerase γ, DNA polymerase ε, DNA polymerase λ, and HIV reverse transcriptase.
(Item 2) The pharmaceutical composition according to item 1, wherein the guava leaf extract has a DNA polymerase α inhibitory activity.
(Item 3) The pharmaceutical composition according to item 1, wherein the guava leaf extract has a DNA polymerase λ inhibitory activity.
(Item 4) The pharmaceutical composition according to item 1, further comprising an agaricus koji extract.
(Item 5) A food composition for treating and / or preventing cancer, comprising a guava leaf extract,
The guava leaf extract contains polyphenols and the following:
A composition for food having inhibitory activity against an enzyme selected from the group consisting of DNA polymerase α, DNA polymerase γ, DNA polymerase ε, DNA polymerase λ, and HIV reverse transcriptase.
(Item 6) The food composition according to Item 5, wherein the guava leaf extract has a DNA polymerase α inhibitory activity.
(Item 7) The food composition according to Item 5, wherein the guava leaf extract has a DNA polymerase λ inhibitory activity.
(Item 8) The food composition according to Item 5, further comprising an Agaricus koji extract.
(Item 9) A DNA polymerase α-inhibiting composition comprising polyphenol extracted from guava leaves.
(Item 10) A DNA polymerase λ-inhibiting composition comprising polyphenol extracted from guava leaves.
(Item 11) A DNA polymerase α-inhibiting composition comprising a component having a molecular weight of 16,000 to 130,000 extracted from Agaricus koji.
(Item 12) A DNA polymerase λ-inhibiting composition comprising a component having a molecular weight of 16,000 to 130,000 extracted from Agaricus koji.
(Item 13) A DNA polymerase α-inhibiting composition comprising a polyphenol extracted from guava leaves and a component having a molecular weight of 16,000 to 130,000 extracted from Agaricus koji.
(Item 14) A DNA polymerase λ-inhibiting composition comprising a polyphenol extracted from guava leaves and a component having a molecular weight of 16,000 to 130,000 extracted from Agaricus koji.
(Item 15) A pharmaceutical composition for treating and / or preventing AIDS, comprising a guava leaf extract,
The pharmaceutical composition, wherein the guava leaf extract contains polyphenols and has inhibitory activity against HIV reverse transcriptase.
(Item 16) The pharmaceutical composition according to item 15, further comprising an agaricus koji extract.
(Item 17) A food composition for treating and / or preventing AIDS, comprising a guava leaf extract,
The pharmaceutical composition, wherein the guava leaf extract contains polyphenols and has inhibitory activity against HIV reverse transcriptase.
(Item 18) The food composition according to item 17, further comprising an agaricus koji extract.

  Polyphenols extracted and purified from guava leaves are used as food materials such as α-amylase inhibitory activity and whitening agents, so there is no problem in terms of safety. By applying such a safe extract as a DNA polymerase and reverse transcriptase inhibitory composition, a cancer cell growth inhibitory composition and an anti-AIDS drug, the food composition is safe and has no side effects. And pharmaceutical compositions can be provided.

  The component with a molecular weight of 16,000 to 130,000 extracted and purified from Agaricus koji is used as a food material for immunostimulatory action, so there is no problem in terms of safety. By applying such a safe extract as a DNA polymerase inhibitor composition, an anticancer agent and an anti-AIDS agent, it is possible to provide a pharmaceutical composition and a food composition that are highly safe and can be continuously taken without side effects.

  Furthermore, guava leaf extract and agaricus koji extract synergistically exhibit DNA polymerase inhibitory activity, anticancer activity and anti-AIDS activity, and therefore a mixture of these extracts can be used to produce a safe and highly effective anticancer agent and anticancer agent. It becomes possible to provide AIDS drugs.

  The components of the DNA polymerase inhibitor composition and anticancer agent / anti-AIDS agent of the present invention are extracted and purified from guava leaves or Agaricus koji. Similarly, the mixture of each extract acts as a DNA polymerase inhibitor composition and an anticancer agent / anti-AIDS agent. The origin and production area of guava leaves and Agaricus mushrooms are not particularly limited. The extraction and purification method is not particularly limited, but the following method is particularly desirable.

(Preparation and purification of guava leaf extract)
Guava as a starting material for preparing guava leaf extract in the present invention is typically Osidium guajava L. However, it is not limited to this. Guava leaf extract is not limited, but can be prepared, for example, by the following method:
A dried product of guava (Osidium guajava L., also known as bunjiro, banzakuro, etc.) leaves is cut, and the polyphenol-containing fraction is eluted from the dried product using a water-containing organic solvent by chromatography.

  In a particularly preferred embodiment, a polyphenol-containing fraction is eluted by chromatography using guava leaves as a raw material and a hydrous organic solvent having a concentration of 30 to 80% by weight as an elution solvent.

  Preferably, a preliminary extract is once obtained from guava leaves, the preliminary extract is dissolved in a solvent, and this solution is subjected to chromatography using a water-containing organic solvent as an elution solvent.

  Here, in the pre-extraction process for obtaining an extract from guava leaves, the extraction method is not particularly limited. For example, water, a water-containing organic solvent, or a water-soluble organic solvent can be used as the extraction solvent. Among these, a water-containing organic solvent is particularly preferable from the viewpoint of obtaining a high concentration polyphenol-containing fraction.

  In the preliminary extraction step, the organic solvent that can be used as the water-containing organic solvent or the water-soluble organic solvent is not particularly limited, but alcohols such as ethanol and methanol, ketones such as acetone are preferable, ethanol, methanol, and acetone are preferable. Is most preferred. Moreover, the mixture of two or more types of organic solvents may be sufficient.

  In the preliminary extraction step, the weight ratio of the organic solvent in the water-containing organic solvent (the total ratio when a plurality of organic solvents are used) is not limited, but it is 30 weight from the viewpoint of obtaining a high concentration polyphenol-containing fraction. % To 80% by weight, more preferably 40% to 60% by weight.

  Although the extraction temperature in a preliminary extraction process is not specifically limited, In order to improve the density | concentration of the active ingredient in a polyphenol containing fraction, 40 degreeC or more is preferable. Moreover, although there is no upper limit in temperature, from the viewpoint of improving the concentration of the active ingredient, it can be set at, for example, 130 ° C. or less, and more preferably 100 ° C. or less. Further, it is most preferable to perform reflux extraction with a water-containing organic solvent.

  The extraction time in the preliminary extraction stage is not particularly limited, but is preferably 30 minutes or more from the viewpoint of improving the concentration of polyphenol. Moreover, since the yield of an active ingredient does not necessarily increase even if it extracts for a long time, it is preferable to make extraction time into 180 minutes or less from this viewpoint.

  In a preferred embodiment, the pre-extract obtained in the pre-extraction step is dissolved in a solvent to obtain a solution. This solution is subjected to the main extraction step using chromatography.

  Here, the solvent used for dissolving the preliminary extract is not particularly limited as long as the preliminary extract can be dissolved, but water or a water-containing organic solvent is preferable. Examples of the organic solvent that can be used as the water-containing organic solvent include the organic solvents for pre-extraction described above.

  The type of chromatography used in this extraction step is not particularly limited. For example, after the pre-extract is adsorbed on a chromatographic medium, the polyphenol-containing fraction can be eluted using a water-containing organic solvent. However, in a preferred embodiment, the pre-extract is adsorbed to the chromatographic medium, then water is flowed to wash unadsorbed material, and water-containing organic solvent is then flowed to the medium to elute the polyphenol-containing fraction. Let

  Examples of the types of chromatography include so-called ion exchange chromatography, gel filtration, hydrophobic chromatography, and reverse phase chromatography. Hydrophobic chromatography is particularly excellent in terms of obtaining a high concentration polyphenol-containing fraction. .

  The filler constituting such hydrophobic chromatography is not particularly limited, but examples of the synthetic resin include polystyrene synthetic adsorbents, modified polystyrene synthetic adsorbents, and methacrylic synthetic adsorbents. Moreover, when modifying a synthetic resin, a bromine group can be illustrated as a modified functional group. For example, “Diaion HP-20” manufactured by Mitsubishi Chemical Corporation is particularly preferable.

  In this method, a water-containing organic solvent is used as an elution solvent at the time of extraction using chromatography. The type of the organic solvent constituting the water-containing organic solvent is not particularly limited, but alcohols such as ethanol and methanol, ketones such as acetone are preferable, ethanol, methanol and acetone are preferable, and ethanol is most preferable. Moreover, the mixture of two or more types of organic solvents may be sufficient.

  At the time of extraction using chromatography, the weight ratio of the organic solvent in the water-containing organic solvent (the total ratio when a plurality of organic solvents are used) is preferably 30% by weight or more and 80% by weight or less. Thereby, a polyphenol-containing fraction having an antioxidant property or a high α-amylase inhibitory activity is obtained. From this viewpoint, the weight ratio of the organic solvent in the water-containing organic solvent is more preferably 40% by weight or more, and further preferably 60% by weight or less.

(Preparation and purification of Agaricus koji extract)
Agaricus koji as a starting material for preparing an Agaricus koji extract in the present invention is typically Agaricus blazei Murill, but is not limited thereto. The extract of Agaricus koji is not limited, but can be prepared, for example, by the following method:
Water or water-containing alcohol is used as a processing solvent by using a dried fruit body of Agaricus moth (also referred to as Agaricus blazei). Pre-extraction is performed at a low temperature of 50 ° C. or lower in advance, and then main extraction at 50 ° C. or lower is performed. The temperature of the leaching process and the extraction process is a low temperature extraction of 50 ° C. or less. Preferably, it is −20 ° C. to 40 ° C. When the extraction temperature is 60 ° C. or higher, the amount of extract obtained increases, but the DNA polymerase inhibitory activity decreases conversely. On the other hand, when the extraction temperature is −20 ° C. or lower, the amount of extract obtained is too low, which is not suitable for mass production.

  That is, water or hydrous alcohol is added to dried agaricus koji, subjected to low temperature leaching treatment at 50 ° C. or lower and subjected to solid-liquid separation, and then water or hydrous alcohol is added again to the residue to perform low temperature extraction at 50 ° C. or lower. Preferably, it is −20 ° C. to 40 ° C. as described above.

  Examples of the alcohol include ethanol, methanol, and propanol, but are not particularly limited, and may be a mixture of two or more. Ethanol is preferable.

  As the processing time, the pre-soaking process is within 6 hours, and the extraction process is 12 hours or more. The extract obtained by the extraction treatment has a lower yield than the eluate obtained by the pretreatment, but the immunostimulatory effect is significantly higher. When the pretreatment is performed for 6 hours or more, the yield of the extract obtained by the main extraction is further reduced, and the component that becomes the activity is dispersed and the immunostimulatory effect is not enhanced.

  The composition obtained by the extraction treatment according to the above-mentioned conditions already exhibits a remarkable DNA polymerase inhibitory activity, but the activity can be further increased by precipitation in a hydroalcoholic solvent, and the inhibitory composition is purified. The

  The composition obtained by the extraction treatment is measured for sugar content using a saccharimeter and concentrated to 20% Brix, and then half to twice the amount of alcohol is added to precipitate a precipitate. Although the kind of alcohol is not specifically limited, Preferably it is ethanol.

  Furthermore, it has been found that the above-described DNA polymerase inhibitor composition is further purified by anion exchange treatment and becomes a composition having extremely high activity.

  The anion exchange treatment is not particularly limited, such as chromatography and batch treatment, but is preferably chromatography. The filler constituting the anion exchange chromatography is not particularly limited, but DEAE-650M manufactured by Tosoh Corporation is preferable as an example of the trade name.

  A sodium chloride solution was used as a desorption solution at the time of extraction using an anion exchange resin, and fractionation was performed using the concentration gradient. That is, a solution that dissolves the DNA polymerase inhibitor composition is added to an anion exchange resin that has been substituted with water, and the non-adsorbed fraction is first washed out, and then the desorbed fraction is recovered using a 0.5 M sodium chloride solution as the desorbed solution. . A purified inhibitory composition can be obtained by dialysis treatment of sodium chloride in the desorption fraction.

(Measurement of polyphenols)
As used herein, the term “polyphenol” refers to a compound in which two or more hydrogens of an aromatic hydrocarbon are substituted with a hydroxyl group. Those having two hydroxyl groups are called divalent phenols, and those having three hydroxyl groups are called trivalent phenols. The polyphenol of the present invention refers to a compound whose content can be analyzed by, for example, the foreign dennis method, but is not limited thereto.

  The “Foreign Dennis method” is typically a phenol reagent (for example, 25 g of sodium molybdate, 100 g of sodium tungstate, 85% phosphoric acid 50 ml, concentrated hydrochloric acid 100 ml, water 700 ml, and heated for 10 hours under reflux. Next, 150 g of lithium sulfate, 50 ml of water and a few drops of bromine were added and boiled for 15 minutes to expel excess bromine, then water was added and diluted to 1 L.) was added to the sample. This is a method for measuring polyphenol content by color reaction. Variations of this exemplary method can also be used in the present invention.

(Pharmaceutical composition and food composition)
The anti-cancer agents of the invention can also be formulated in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier). When an anti-cancer agent formulated with the extract used in the present invention is used as a therapeutic agent, it takes into account individual solids, administration methods, administration schedules and other factors known to those skilled in the art, and the medical practice standard (GMP = good medical) prescription and dosing in a manner consistent with practice. Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.

  The therapeutic agents of the present invention can be oral, rectal, parenteral, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, gel, drops, or transdermal patch), intraoral Alternatively, it can be administered as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation adjuvant. As used herein, “parenteral” refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and indirect injections and infusions.

  The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained-release therapeutic agents are oral, rectal, parenteral, intravascular, intravaginal, intraperitoneal, topical (powder, ointment, gel, infusion, or transdermal patch) Etc.), or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulant, or any type of formulation aid. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions. Suitable examples of sustained release therapeutic agents include suitable polymer materials (eg, semipermeable polymer matrices in the form of molded articles (eg, films or microcapsules)), suitable hydrophobic materials (eg, in acceptable quality oils). Emulsified) or ion exchange resins, and poorly soluble derivatives (eg, poorly soluble salts).

  Sustained release matrices include polylactide (US Pat. No. 3,773,919, EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sindman et al., Biopolymers 22: 547-556 (1983)). ), Poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Lager, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl. Acetate (Lager et al., Ibid.) Or poly-D-(-)-3-hydroxybutyric acid (EP133,988).

  Sustained release therapeutic agents also include the therapeutic agents of the present invention encapsulated in liposomes (generally, Langer, Science 249: 1527-1533 (1990); Treat et al., Liosomes in the Therapeutic Disease and Cancer-Lopez, Lopez). (See Berestin and Fielder (eds.), Liss, New York, pages 317-327, and pages 353-365 (1989)). Liposomes containing therapeutic agents can be prepared by methods known per se. : DE 3,218, 121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; Japanese Patent Application No. 83-118008; US Patent No. 4,485,045 and No. 4,544,545 and EP No. 102,324. Usually, liposomes are small (about 200-800 cm) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol, and the selected proportion is adjusted for optimal therapeutic agents.

  Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

  For parenteral administration, in one embodiment, in general, the therapeutic agent is toxic to the recipient in the desired degree of purity, in a pharmaceutically acceptable carrier, i.e. the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is not and compatible with the other ingredients of the formulation. For example, the formulation is preferably free of oxidation and other compounds known to be harmful to therapeutic agents.

  Generally, the formulation is prepared by contacting the therapeutic agent uniformly and intimately with liquid carriers or finely divided solid carriers or both. Next, if necessary, the product is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vectors include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

  The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipient at the dosage and concentration used, such as phosphate, citrate, succinate, acetic acid and other organic acids, or their Buffers such as salts; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); such as serum albumin, gelatin, or immunoglobulin Proteins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, including cellulose, or derivatives thereof, glucose, mannose, or dextrin, and others Hydrocarbons, chelating agents such as EDTA; mannitol or sol Counterions such as sodium; sugar alcohols such as tall and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

  The therapeutic agent is typically formulated in such a vehicle at a pH of about 6-8 at a concentration of about 0.01 mg / ml to 10 mg / ml, preferably 0.1-5 mg / ml. It is understood that by using the specific excipients, carriers or stabilizers described above, salts are formed.

  Any drug to be used for therapeutic administration can be free of organisms or viruses, ie, sterile. This is easily accomplished by filtration through a sterile filtration membrane (eg, a 0.2 micron membrane filter). Generally, the therapeutic agent is placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be punctured with a hypodermic syringe.

  The therapeutic agents are usually stored in unit dose or multi-dose containers such as sealed ampoules or vials as aqueous solutions or lyophilized formulations for reconstitution. As an example of a lyophilized formulation, a 10 ml vial is filled with 5 ml of a sterile filtered 1% (W / V) aqueous therapeutic agent and the resulting mixture is lyophilized. The lyophilized therapeutic agent is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention. A notice of the form prescribed by a government agency for the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice shall be issued by the government agency regarding the manufacture, use or sale for human administration. Represents approval. In addition, the therapeutic agent may be used in combination with other therapeutic compounds.

  The therapeutic agents of the present invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered in combination with the therapeutic agents of the present invention include other anti-cancer agents, chemotherapeutic agents, antibiotics, steroid and non-steroidal anti-inflammatory agents, conventional immunotherapeutic agents, other cytokinins, and / or Examples include but are not limited to growth factors. The combination can be administered, for example, either as a mixture simultaneously (simultaneously or concurrently but separately) or over time. This includes the presentation that the combined agents are administered together as a therapeutic mixture, and also the procedure where the combined agents are administered separately but simultaneously, eg, through separate intravenous lines to the same individual. Administration in combination further includes separate administration of one of the compounds given first, followed by the second, or drug.

  In certain embodiments, the therapeutic agents of the invention are administered in combination with an antiretroviral agent, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, and / or a protease inhibitor.

  In a further embodiment, the therapeutic agent of the invention is administered in combination with an antibiotic. Antibiotics that can be used include aminoglycoside antibiotics, polyene antibiotics, penicillin antibiotics, cephem antibiotics, peptide antibiotics, macrolide antibiotics, tetracycline antibiotics. It is not limited.

  In a further embodiment, the therapeutic agents of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that can be administered with the therapeutic agents of the present invention include glucocorticoids and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, Thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzidoamine, bucolome, difenpyramide, ditazole, emorfazoline, guaiazulene, nabumetone, nimesulide , Orgothein, oxaceptol, paraniline, perizoxal, pifoxime, proxazoline, proxazole, and tenidap. Not a constant.

  In further embodiments, the therapeutic agents of the invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation therapy).

  In this invention, the composition for foodstuffs containing a guava leaf extract and / or an agaricus koji extract is provided. That is, the extract or pharmaceutical composition or food / beverage composition obtained as described above is a liquid, gel or solid food such as juice, soft drink, coffee, tea, Japanese tea, oolong tea. , Vegetable juice, natural fruit juice, milk drink, milk, soy milk, sports drink, near-water drink, nutritional drink, coffee drink, cocoa, soup, dressing, mousse, jelly, yogurt, pudding, sprinkle, infant formula, processing Milk, sports drink, energy drink, cake mix, bread, pizza, pie, crackers, biscuits, cake, cookies, spaghetti, macaroni, pasta, udon, soba, ramen, candy, soft candy, gum, chocolate, rice cake, potato chips , Snacks, ice cream, shah Of powdered or liquid dairy products such as tart, cream, cheese, powdered milk, condensed milk, milk beverages, buns, oysters, rice cakes, oysters, soy sauce, sauce, noodle soup, sauce, dashi stock, stew base, soup Ingredients, compound seasonings, curry sauce, mayonnaise, ketchup, retort curry, retort stew, retort soup, retort bowl, canned, ham, hamburger, meatballs, croquettes, dumplings, pilaf, rice balls, frozen and refrigerated foods, chikuwa , Sushi, bento rice, sushi, baby milk, baby food, baby food, sports food, nutritional supplements, supplements, health foods, etc., and if necessary, excipients such as dextrin, lactose, starch Can be processed into pellets, tablets, granules, etc. together with fragrances, pigments, etc. It can be used as food and dietary supplements, and the like. The blending amount of the extract in these foods or food compositions is difficult to define uniformly depending on the type and state of the food or composition, but the blending amount is about 10 to 50% by weight, more preferably 25. ~ 50% by weight. If the blending amount is less than 10% by weight, the desired effect by oral intake is small, and if it exceeds 50% by weight, depending on the type of food, the flavor may be impaired or the food may not be prepared.

  For example, the DNA polymerase-inhibiting composition of the present invention can be used as a food, food additive, health functional food, and drug for the purpose of preventing cancer and the like. Examples thereof include foods and beverages. Specifically, in addition to tableting, capsule tablets, granules, etc., they can be in the form of bread, cookies, strawberries, jelly, yogurt, noodles, sprinkles, tea, soup and the like. Depending on their processing application, the inhibitory composition of the present invention can also be mixed with an excipient such as dextrin and used as a powder. Note that the inhibitory composition of the present invention may be used for food as it is.

  Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited thereto.

  The DNA polymerase inhibitor composition of the present invention will be specifically described below.

(Example 1: Production of purified product derived from guava leaves)
A polyphenol-containing composition was purified from raw guava leaves. First, 100 g of dried guava leaf was shredded, 1000 ml of 50% aqueous ethanol was added, and extraction was performed twice at 60 ° C. under reflux. After filtration of the extract, concentration was performed under reduced pressure to obtain 21 g of a guava leaf crude product.

  Next, the obtained roughly purified product was dissolved in water and poured into 150 ml of a hydrophobic chromatography resin (Diaion HP-20, manufactured by Mitsubishi Chemical Corporation). Thereafter, unadsorbed substances were washed and removed with water, and fractionated into fraction I (polyphenol-containing composition) eluted with 50% aqueous ethanol.

  The polyphenol purified product was analyzed for polyphenol content by the foreign dennis method. As a result, the purified guava leaf product was 42% (polyphenol content), and the purified product was 80% (polyphenol content).

(Example 2: Production of purified Agaricus koji)
600 mL of water was added to 50 g of dried Agaricus koji, and leaching was performed at 40 ° C. for 4 hours. 500 mL of water was added to the residue after solid-liquid separation, and extraction was performed at 40 ° C. for 24 hours. After the extraction, the extract was recovered by solid-liquid separation, concentrated under reduced pressure about 5 times, and then freeze-dried to obtain 10.1 g of powder.

  After adding ethanol 69mL to the composition 60mL (Brix 21%) before freeze-drying obtained by the above-mentioned treatment and allowing it to stand at 4 ° C overnight, the deposited precipitate was centrifuged (5000 rpm x 10 minutes). The supernatant and the precipitate were collected separately, and freeze-dried to obtain 10.4 g and 1.4 g (Agaricus soot precipitate), respectively.

To 1.01 g of Agaricus soot precipitate obtained by the above method, 10 mL of water was added and dissolved again in water, and the solution was passed through a column filled with 76 mL of anion exchange resin DEAE 650M (manufactured by Tosoh Corporation). Thereafter, 100 mL of water was passed through the column and recovered as a non-adsorbed fraction. Thereafter, 0.5 M sodium chloride aqueous solution was passed through a 200 mL column and recovered as a desorption fraction. The collected non-adsorbed fraction and desorbed fraction were each concentrated under reduced pressure about 5 times, filled in a dialysis membrane BioDesignDialysis Tubing ^TM (manufactured by BioDesign Inc. of New York), and 24 times in a water bath stirred with a magnetic stirrer. Dialyzed by floating for a period of time. The membrane solution after the dialysis treatment was collected, concentrated under reduced pressure approximately twice, and freeze-dried to obtain powders of a non-adsorbed fraction 140 mg and a desorbed fraction 210 mg (purified agaricus), respectively.

(Example 3: Molecular weight distribution analysis of agaricus koji purified product (Example 2))
After weighing 10 mg of the purified Agaricus koji obtained above, 1 mL of pure water was added and dissolved, and the mixture was filtered through a disk filter having a pore size of 0.45 μm and subjected to gel filtration analysis. The conditions of the gel filtration analysis were as follows: column: SHODEX ASAHIPAK GS-710 7G + GS-310 7G 50 cm long × 7.6 mm inner diameter (made by Showa Denko), column temperature: room temperature, mobile phase: 0.1 mol / L NaCl aq . Flow rate: 0.7 mL / min, detection: suggested refractive index detection (RI) and UV 280 nm, injection volume: 20 μL. A standard pullulan with a known molecular weight (made by Showa Denko) is used as a molecular weight marker for RI detection, and a protein with a known molecular weight (made by Oriental Yeast) is used as a molecular weight marker for UV detection. And the molecular weight of the sample was determined. Both peaks were confirmed, and the molecular weight of each peak vertex was measured.

（実施例４：アガリクス茸精製物（実施例２）の中性糖分析）
上記で得られたアガリクス茸精製物中の多糖を分析する目的で、酸加水分解−中性糖の分析を高速液体クロマトグラフィー（ＨＰＬＣ）法で実施した。サンプルの前処理としては、サンプル１．２０ｍｇに２Ｎトリフルオロ酢酸（ＴＦＡ）５００μＬ添加し、１００℃で６時間加水分解し、加水分解液（２Ｎ ＴＦＡ）を回収し減圧乾固した。乾固した残渣は、精製水５００μＬに溶解し、０．２２μｍのフィルターで濾過し、ろ液５０μＬをＨＰＬＣ分析に供した。ＨＰＬＣ分析の条件は、カラム：ＴＳＫ−ｇｅｌ Ｓｕｇａｒ ＡＸＧ １５ｃｍ×４．６ｍｍ Ｉ．Ｄ．（東ソー製）、カラム温度：７０℃、流速：０．４ｍＬ／分、検出：蛍光検出（ＥＸ．：３２０ｎｍ ＥＭ．：４３０ｎｍ）、移動相：０．５Ｍホウ酸カリウム緩衝液ｐＨ８．７で行った。また、ポストカラム標識は、反応試薬：１％アルギニン／３％ホウ酸、反応試薬流速：０．５ｍＬ／分、反応温度：１５０℃で行った。

(Example 4: Neutral sugar analysis of purified Agaricus koji (Example 2))
For the purpose of analyzing polysaccharides in the purified Agaricus koji obtained above, acid hydrolysis-neutral sugars were analyzed by high performance liquid chromatography (HPLC). As a sample pretreatment, 500 μL of 2N trifluoroacetic acid (TFA) was added to 1.20 mg of sample, and hydrolyzed at 100 ° C. for 6 hours. The hydrolyzed solution (2N TFA) was recovered and dried under reduced pressure. The dried residue was dissolved in 500 μL of purified water, filtered through a 0.22 μm filter, and 50 μL of the filtrate was subjected to HPLC analysis. The conditions for HPLC analysis were as follows: Column: TSK-gel Sugar AXG 15 cm × 4.6 mm D. (Manufactured by Tosoh Corporation), column temperature: 70 ° C., flow rate: 0.4 mL / min, detection: fluorescence detection (EX .: 320 nm EM .: 430 nm), mobile phase: 0.5 M potassium borate buffer, pH 8.7 It was. Further, post-column labeling was performed at a reaction reagent: 1% arginine / 3% boric acid, a reaction reagent flow rate: 0.5 mL / min, and a reaction temperature: 150 ° C.

（実施例５：アガリクス茸精製物（実施例２）のアミノ酸分析）
上記で得られたアガリクス茸精製物中の蛋白量を測定する目的で、酸加水分解−アミノ酸分析を行った。サンプルの前処理は、サンプル０．６３ｍｇに６Ｎ塩酸５００μＬ添加し、１００℃で２２時間加水分解し、加水分解液を減圧乾固した。乾固した残渣は、精製水５００μＬに溶解し、０．２２μｍのフィルターで濾過し、ろ液５０μＬをアミノ酸分析に供した。アミノ酸分析の条件は、日立Ｌ−８５００形アミノ酸分析計を用い、特殊アミノ酸分析／ニンヒドリン発色で行った。

(Example 5: Amino acid analysis of purified Agaricus koji (Example 2))
In order to measure the amount of protein in the purified Agaricus koji obtained above, acid hydrolysis-amino acid analysis was performed. The sample was pretreated by adding 500 μL of 6N hydrochloric acid to 0.63 mg of the sample, hydrolyzing at 100 ° C. for 22 hours, and drying the hydrolyzed solution under reduced pressure. The dried residue was dissolved in 500 μL of purified water, filtered through a 0.22 μm filter, and 50 μL of the filtrate was subjected to amino acid analysis. The amino acid analysis was carried out using Hitachi L-8500 type amino acid analyzer with special amino acid analysis / ninhydrin color development.

（実施例６：ＤＮＡポリメラーゼの調製）
ほ乳類のＤＮＡポリメラーゼのうち、複製型の代表としてＤＮＡポリメラーゼαを、修復型の代表としてＤＮＡポリメラーゼλを使用した。ＤＮＡポリメラーゼαは子牛胸腺抽出液から抗ＤＮＡポリメラーゼα抗体を樹脂に結合させたカラムを用い、アフィニティー精製した。ＤＮＡポリメラーゼλは，ヒトＤＮＡポリメラーゼλの遺伝子を大腸菌へ導入して、培養・発現させてから、組換えタンパク質を精製した。

(Example 6: Preparation of DNA polymerase)
Among mammalian DNA polymerases, DNA polymerase α was used as a representative replication type, and DNA polymerase λ was used as a representative repair type. DNA polymerase α was affinity purified from a calf thymus extract using a column in which an anti-DNA polymerase α antibody was bound to a resin. For DNA polymerase λ, the gene of human DNA polymerase λ was introduced into E. coli, cultured and expressed, and then the recombinant protein was purified.

(Example 7: DNA polymerase activity measurement)
The DNA polymerase activity was measured according to a conventional method. As template DNA and nucleotide as substrates, poly (dA) / oligo (dT) _12-18 and [ ³ H] -dTTP (2′-deoxythymidine 5′-triphosphase) (both GE Healthcare) are used, respectively. Thus, the amount of nucleotide polymerized on the template DNA was determined by radioactivity to determine the activity. DNA polymerases α and λ were adjusted to 0.05 units and used for the test (1 unit was defined as the activity to polymerize 1 nmol nucleotides at 37 ° C. for 60 minutes). The crude and purified guava leaves and agaricus were dissolved in DMSO, preincubated with the enzyme, and then reacted by adding a substrate. Enzyme activity to which no extract was added was taken as 100%. The results are shown in the following table.

上記表に示される結果から、グァバ葉の粗精製物および精製物、ならびにアガリクス茸精製物のいずれもが、ＤＮＡポリメラーゼαおよびλに対する阻害活性を示した。ＤＮＡポリメラーゼαに対しては、アガリクス茸精製物の阻害活性は、低く１００ｎｇ／ｍｌ程度の濃度では、阻害活性が検出されなかった。しかしながら、１００ｎｇ／ｍｌのアガリクス茸精製物と、１０ｎｇ／ｍｌのグァバ葉精製物または１００ｎｇ／ｍｌのグァバ葉粗精製物とを組み合わせた場合、相乗的阻害作用が確認された。

From the results shown in the above table, all of the crude and purified guava leaves and the purified Agaricus koji showed inhibitory activity against DNA polymerases α and λ. For DNA polymerase α, the inhibitory activity of the purified Agaricus koji was low, and no inhibitory activity was detected at a concentration of about 100 ng / ml. However, when a 100 ng / ml Agaricus koji purified product was combined with a 10 ng / ml guava leaf purified product or a 100 ng / ml guava leaf crude product, a synergistic inhibitory action was confirmed.

(Example 8: Inhibitory activity against various enzymes) Using the same conditions as in Example 7, inhibitory activities against various enzymes shown in Table 10 were measured.

以上の結果から、本発明の抽出物は、ＤＮＡポリメラーゼαおよびλのみならず、いくつかの他の酵素に対しても阻害活性を有することが実証された。特にＨＩＶの逆転写酵素に対する阻害活性は、本発明の組成物が、抗エイズ薬として使用可能であることを実証する。

From the above results, it was demonstrated that the extract of the present invention has inhibitory activity not only for DNA polymerases α and λ but also for several other enzymes. In particular, the inhibitory activity of HIV against reverse transcriptase demonstrates that the composition of the present invention can be used as an anti-AIDS agent.

(Example 9: Measurement of human cancer cell growth inhibitory activity)
Human leukemia cells (HL-60 cells) were purchased from the Human Science Research Resource Bank. Using RPMI 1640 liquid medium [10% heat-treated fetal bovine serum, 100 U / mL penicillin / streptomycin] (Gibco Laboratories), the cells were cultured in a 37 ° C., 5% CO ₂ incubator. A 96-well flat bottom plate (FALCON) was dispensed at 3 × 10 ⁵ cells / well, and an extract dissolved in DMSO was added so that the final concentration was 0.5%. After culturing for 24 hours, proliferation reaction was measured by the MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolbromide) method. The results are shown below.

上記の表から明らかなように、アガリクス茸精製物と、グァバ葉精製物または抽出物との組み合わせによって、相乗的な抗癌作用が確認された。本実施例において使用した抽出物の濃度は、食品等として摂取した場合において安全であることが確認されている濃度である。従って、この結果は、アガリクス茸抽出物と、グァバ葉抽出物とを含有する抗癌剤が非常に有効かつ安全な薬物であることを実証する。

As is clear from the above table, a synergistic anticancer effect was confirmed by the combination of the purified Agaricus koji and the purified guava leaf extract or extract. The concentration of the extract used in this example is a concentration that has been confirmed to be safe when ingested as food or the like. Thus, this result demonstrates that anticancer agents containing Agaricus koji extract and guava leaf extract are very effective and safe drugs.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  As described above, the DNA polymerase inhibitor composition of the present invention has a useful physiological activity such as a cancer cell growth inhibitory effect, and thus can be used as a cancer preventive and therapeutic agent, and is particularly suitable for functional foods. be able to.

Claims (5)

A pharmaceutical composition for treating and / or preventing cancer, comprising guava leaf extract and agaricus koji extract,
The guava leaf extract contains polyphenols and the following:
A pharmaceutical composition having an inhibitory activity against an enzyme selected from the group consisting of DNA polymerase α, DNA polymerase γ, DNA polymerase ε, DNA polymerase λ, and HIV reverse transcriptase ,
Wherein the Agaricus koji extract contains a component having a molecular weight of 16,000-130,000 and the following steps:
(1) Step of obtaining a water extract of Agaricus koji:
(2) adding alcohol to the water extract of (1) to precipitate a precipitate;
(3) a step of dissolving the precipitate of (2) above in an aqueous solution; and
(4) A step of subjecting the lysate of (3) to anion exchange treatment and collecting a desorption fraction;
A pharmaceutical composition which is an extract prepared by a method comprising: The pharmaceutical composition according to claim 1, wherein the guava leaf extract has DNA polymerase α inhibitory activity. The pharmaceutical composition according to claim 1, wherein the guava leaf extract has DNA polymerase λ inhibitory activity. A composition for inhibiting the activity of DNA polymerase α, comprising a polyphenol extracted from guava leaves and a component having a molecular weight of 16,000 to 130,000 extracted from Agaricus koji ,
Here, the component extracted from the Agaricus koji has the following steps;
(1) Step of obtaining a water extract of Agaricus koji:
(2) adding alcohol to the water extract of (1) to precipitate a precipitate;
(3) a step of dissolving the precipitate of (2) above in an aqueous solution; and
(4) A step of subjecting the lysate of (3) to anion exchange treatment and collecting a desorption fraction;
A composition prepared by a method comprising: A composition for inhibiting the activity of DNA polymerase λ, comprising a polyphenol extracted from guava leaves and a component having a molecular weight of 16,000 to 130,000 extracted from Agaricus koji ,
Here, the component extracted from the Agaricus koji has the following steps;
(1) Step of obtaining a water extract of Agaricus koji:
(2) adding alcohol to the water extract of (1) to precipitate a precipitate;
(3) a step of dissolving the precipitate of (2) above in an aqueous solution; and
(4) A step of subjecting the lysate of (3) to anion exchange treatment and collecting a desorption fraction;
A composition prepared by a method comprising: