JP6445567B2 - Drugs containing lignin extract as an active ingredient - Google Patents

Description

  The present specification relates to a drug containing a lignin extract as an active ingredient.

  The lignocellulosic material containing lignin and the black liquor that is the residue obtained by separating cellulose as the pulp raw material from the lignocellulosic material are not always used effectively, and the ratio of disposal as waste is not small. is there.

  On the other hand, a technique for effectively using lignin as a drug has been developed. For example, a wrinkle improving agent and a stain improving agent containing a lignin extract extracted from a lignin-containing material by bringing metal ions into contact with the lignin-containing material are known (Patent Document 1).

JP 2010-309221 A

  In addition to using the lignin extract as an active ingredient of a wrinkle improver or a stain improver, new uses are expected.

  The present specification provides a new use for lignin extracts.

  The present inventor has found that the lignin extract has new activities including antitumor action, antibacterial action and antiviral action. According to the present specification, the following means are provided.

(1) A cell growth inhibitor comprising, as an active ingredient, a lignin extract extracted by promoting decomposition of lignin in the lignin-containing material by bringing iron ions into contact with the lignin-containing material.
(2) The cell growth inhibitor according to (1), wherein the lignin extract is extracted from the lignin-containing material by fermenting the lignin material in the presence of iron ions.
(3) The cell growth inhibitor according to (1) or (2), wherein the fermentation is performed using lactic acid bacteria.
(4) The cell growth inhibitor according to any one of (1) to (3), wherein the lignin-containing material includes herbaceous foliage.
(5) The cell growth inhibitor according to any one of (1) to (4), which suppresses the growth of cancer cells.
(6) The cell growth inhibitor according to (5), wherein the cancer cell is a cell of one or more types of cancer selected from the group consisting of lung cancer, skin cancer, esophageal cancer and liver cancer.
(7) The cell growth inhibitor according to any one of (1) to (4), which inhibits the growth of microbial cells.
(8) The cell growth inhibitor according to (7), which has an antibacterial action against gram-negative and gram-positive bacteria.
(9) The cell growth inhibitor according to any one of (1) to (4), which inhibits the growth of periodontal disease bacteria.
(10) A virus growth inhibitor comprising, as an active ingredient, a lignin extract extracted by accelerating the degradation of lignin in the lignin-containing material by bringing iron ions into contact with the lignin-containing material.

It is a figure which shows the cell survival rate of a lung cancer cell line (A549 cell). The cell viability of A549 cells cultured with lignin extract added to the medium is shown on the upper side, and the cell viability of A549 cells cultured with Deferasirox added to the medium on the lower side. It is a figure which shows the cell viability of the esophageal cancer cell line (TE4 cell) which added and extracted the lignin extract in the culture medium. It is a figure which shows the cell survival rate of the liver cancer cell line (HLE cell) which added and extracted the lignin extract in the culture medium. It is a figure which shows the cell viability of a Deferasirox resistant liver cancer cell line (Huh-7 cell). The cell viability of Huh-7 cells cultured with Deferasirox added to the medium is shown on the upper side, and the cell viability of Huh-7 cells cultured with the lignin extract added to the medium on the lower side. It is a figure which shows the cell survival rate of the skin cancer (HMY-cell) which added and extracted the lignin extract in the culture medium. It is a spectrum figure which shows the result of GC-MS.

  The present specification relates to a drug containing, as an active ingredient, a lignin extract extracted from a lignin-containing material by bringing the lignin-containing material into contact with iron ions to promote the decomposition of the lignin (hereinafter, referred to as the present lignin extract). This lignin extract has cell growth inhibitory action such as antitumor action and antibacterial action, virus growth inhibitory action and the like. That is, the drug disclosed herein is also used as an antitumor agent, antibacterial agent or antiviral agent.

  Although the disclosure of this specification is not theoretically constrained, the mechanism of such action of the present lignin extract is considered as follows.

  Although iron ions are necessary for cells to divide, the present lignin extract consumes iron ions by binding to the iron ions to form a complex. As the lignin extract consumes iron, cell division is suppressed. Thereby, it is thought that a cell growth inhibitory effect is exhibited.

  Cancer cells require more iron because cell division is active. For this reason, this lignin extract can suppress the growth of tumor cells by the action of suppressing cell division. Thereby, it is thought that an anti-tumor effect is exhibited.

  Iron is also required for virus growth. Therefore, similarly, the lignin extract is considered to exhibit a virus growth-inhibiting action.

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

(Lignin extract)
The lignin extract, which is the active ingredient of this drug, is extracted from the lignin-containing material by bringing iron ions and the lignin-containing material into contact with each other in the presence of an aqueous medium to promote the degradation of the lignin in the lignin-containing material. Is done. Hereinafter, the manufacturing method of this lignin extract (henceforth this lignin extract) is demonstrated.

(Method for producing lignin extract)
(Lignin-containing material)
As a lignin containing material, the lignin contained in a plant structure or the derivative | guide_body of the said lignin should just be included. That is, the lignin and the lignin derivative referred to here are substantially free of lignin treated under conditions that promote polycondensation of lignin under strong alkali or strong acid in the pulp production process. Therefore, the lignin-containing material in this specification does not contain black liquor or the like.

  As such a lignin-containing material, a lignocellulosic material-treated product obtained by treating a plant-derived lignocellulosic material as it is or under mild conditions can be mentioned. Examples of lignocellulosic materials include whole or part of plants, processed products having a woody part, and the like. Plants include, but are not limited to, conifers, broad-leaved trees, eucalyptus, and other herbs, as well as rice, wheat, corn, sugarcane, kenaf, ghetto, moonfish, and other herbs and bamboo. . Non-use materials of plant-derived lignocellulose materials can also be used. Non-use materials include thinned wood, pruned plant branches and leaves, non-edible fruits such as pine trees, and stalks and leaves after harvesting edible parts. Among these, herbaceous stems and leaves can be preferably used as the lignocellulosic material. Since these tissues are coarser than woody species, a lignin extract can be efficiently obtained. Moreover, a chlorophyll extract is also contained in a lignin extract by using the leaf part containing a chlorophyll.

  The lignin-containing material is preferably crushed to some extent in consideration of extraction efficiency. For example, the lignocellulosic material derived from herbs is preferably cut appropriately, and the lignocellulosic material of woody materials is preferably in the form of chips or powder.

  Further, the lignin-containing material may be a material obtained by treating a lignocellulosic material under mild conditions. Examples thereof include lignophenol derivatives described in JP-A-2-233701 and JP-A-9-278904. Moreover, vegetable food wastes such as tea gala and cedar liquor are listed. For example, the essential oil is extracted from Kumazasa by steam distillation, and the distilled water after separation of the essential oil contains polyphenols derived from Kumazasa lignin. Such a distillation residue can also be used as a lignin-containing material. Furthermore, the residue after isolate | separating a cellulose from bagasse which is a residue after squeezing sugarcane, lignocellulose, etc. are mentioned. Such mild conditions are preferably 100 ° C. or lower, more preferably 80 ° C. or lower, more preferably 70 ° C. or lower, and even more preferably 60 ° C. or lower. (About 2) is preferable.

(Iron ion)
The iron ion only needs to be present as a divalent or trivalent ion in the presence of water. In any form, degradation of lignin can be promoted as a result in the presence of water. Such iron ion sources include iron (II) sulfate (FeSO ₄ ), ferric sulfate (III) ((Fe) ₂ (SO ₄ ) ₃ ), so-called iron rust (iron oxide (III) and its hydrates, Iron oxide (II, III) and hydrates thereof, iron hydroxide, and various iron oxyhydroxides). Such iron rust becomes an iron ion source that generates iron ions (II) and iron ions (III) in the presence of water. In addition, when metallic iron rusts, iron ions (Fe (II)) are eluted in the presence of water and pass through iron hydroxide. As a result, iron oxyhydroxide such as iron (III) hydrous oxide ( III) is formed, or iron ions (II) are oxidized by air to iron ions (III), and as a result, iron (III) oxyhydroxide such as iron (III) hydrated oxide can be generated. For this reason, metallic iron or a metallic alloy containing iron can also serve as an iron ion source. Therefore, for example, a mixer or a press used in the extraction process has an iron inner surface and an iron rust on the inner surface is also an iron ion source.

  Iron ions (III) are also preferred from the standpoints of suppressing the stability of the extract (anaerobic fermentation (rot)), cost, availability, and the like.

(Extraction process)
The step of generating and extracting the present lignin extract from the lignin-containing material is a step of promoting the oxidation of lignin by bringing the iron ions into contact with the lignin-containing material. By this step, lignin can be degraded, reduced in molecular weight, and / or slowly modified.

  The extraction process is performed in the presence of water. This is because iron ions (II) and (III) are interposed. Water in the extraction step can be supplied from the lignin-containing material itself as a cell fluid or intercellular fluid, or can be supplied even if associated with the lignin-containing material. Moreover, water may be separately supplied to the extraction process. The water in the extraction step is preferably in such a level that the lignin-containing material can be appropriately contacted with oxygen in the air. This is because the decomposition of lignin is promoted by the gas-liquid interface. In particular, when a herbaceous lignocellulosic material is used, it is not always necessary to add an aqueous medium, but water or the like can be appropriately added in consideration of the concentration of the extract and extraction efficiency. On the other hand, when using woody lignocellulosic materials such as wood powder, it is preferable to use an appropriate amount of water.

  In the extraction process, other solvents can be positively added in addition to water. Such a solvent may be one that dissolves or separates water, but a solvent that does not inhibit the decomposition of lignin due to the presence of water can be appropriately selected.

  In the extraction step, the lignin-containing material may be allowed to stand as appropriate in a state where metal ions can exist, or may be mixed. When the lignin-containing material is immersed in an aqueous medium containing metal ions, an extract can be easily obtained. For example, by immersing the lignin-containing material in a metal salt solution used in the extraction process for about 1 to 24 hours, the metal ions and the lignin-containing material can be gently brought into contact with each other. Considering the subsequent fermentation, it is preferable to stand at room temperature or at a temperature lower than that. The lignin-containing material after immersion may be continuously mixed and fermented with the immersion liquid, or the extraction process may be performed by newly bringing the lignin-containing material after immersion into contact with iron ions. The extraction step may be performed, or the lignin-containing material separated from the immersion liquid may be contacted with metal ions again to perform the extraction step.

  In the extraction step, the lignin-containing material can be mixed (by stirring or vibration) under an aqueous medium containing metal ions. Although the time of the extraction process accompanied by mixing etc. is not specifically limited, For example, when obtaining a lignin extract from a lignocellulosic material, it should just be performed to such an extent that the restraint state of the cellulose by a lignin is substantially cancelled | released. Although it does not specifically limit, For example, it can implement in tens of minutes-about several hours. In the extraction step, mixing or the like can be stopped as necessary. Moreover, the extraction step may include adjusting the metal ion concentration by adding a metal salt solution as appropriate.

  Moreover, in an extraction process, depending on the form of a lignocellulosic material, pressing and crushing can also be accompanied.

  Mixing / stirring and pressing / crushing in the extraction process increase the contact opportunity, contact area and contact time between iron ions and lignin-containing materials, as well as contact between iron ions and lignin-containing materials and decomposition of lignin. It is also meaningful to generate at the gas-liquid interface between gas and liquid. This is because, under these conditions, it is considered that the degradation of lignin is promoted by the Fenton reaction based on the presence of iron ions.

  The amount of metal ions present in the extraction process is not particularly limited. When crushed herbaceous foliage is used as a lignin-containing material, the extract is colored due to the presence of a sufficient amount of metal ions. For example, the iron ion is blackened. Therefore, an appropriate metal ion concentration can be set by observing the appearance color of the extract. When iron ions are used as metal ions, a ferric sulfate solution can be typically used. Further, the iron ion concentration is not particularly limited. As an example, a solution of about 0.2 g / l or more and 1 g / l or less can be used as the ferric sulfate solution.

  In order to promote the treatment using iron ions, it is preferable to involve fermentation of the lignin-containing material. Although the reason is not clear, the present lignin extract having an excellent cell growth inhibitory effect can be obtained. For fermentation, resident bacteria may be used, but lactic acid bacteria are preferably used. Although it does not specifically limit as lactic acid bacteria, Well-known lactic acid bacteria, such as Lactobacillus genus, Bifidobacterium genus, Enterococcus genus, Lactococcus genus, Pediococcus genus, Leuconostoc genus, can be selected suitably and can be used. For example, Lactobacillus casei strain shirota, L. casei YIT 9029 and the like are preferable.

  Fermentation is preferably performed by aerobic fermentation. The aerobic conditions in the fermentation are not particularly limited, and may be set as appropriate as long as the aerobic fermentation can be maintained and the decomposition of lignin can be promoted. Moreover, although the temperature conditions for fermentation are not specifically limited, it can be about 25 degreeC or more and 40 degrees C or less. Further, the agitation / aeration conditions are not particularly limited, and may be set as appropriate as long as aerobic fermentation can be maintained and decomposition of lignin can be promoted. For example, the agitated aeration culture may be performed for the first 24 hours, and the stationary culture may be performed for 72 hours thereafter. In addition, various conditions can be appropriately set by those skilled in the art to ensure good aerobic fermentation.

  Prior to the extraction step or prior to the fermentation in the extraction step, various operations for promoting the decomposition of lignin or promoting the separation from cellulose can be performed. For example, lignophenol derivatization described in JP-A-2-233701 and JP-A-9-278904, explosion and steaming used for crushing and extraction of lignocellulosic materials, oxidation treatment, reduction treatment, acid treatment, alkali Examples include treatment, enzyme treatment, microbial treatment, subcritical fluid treatment of water and organic solvents, and supercritical fluid treatment. These treatments are preferably performed prior to the extraction step.

Examples of the oxidizing agent for the oxidation treatment include ozone, Fe ³⁺ -HCl, alkali-H ₂ O ₂ and Ce ⁴⁺ . Examples of the reducing agent for the reduction treatment include lithium tetrahydridoaluminate and sodium tetrahydroborate. Among these, sodium tetrahydroborate that selectively reduces a carbonyl group contained in lignin is preferably used. Examples of the alkali treatment chemical include various basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, and sodium borate. As the chemical for acid treatment, sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, organic acid and the like are preferably used. As the enzyme treatment, a peroxidase that promotes fragmentation of lignin, a saccharifying enzyme that forms a glycoside, a glucuronyltransferase that oxidizes glucuron, and the like are preferably used.

  Prior to the extraction step or prior to fermentation in the extraction step, heating may be performed to promote lignin elution and promote binding to metal ions. For example, it is preferable to heat to about 80 ° C. or higher and 100 ° C. or lower. Preferably, it is 80 degrees C or less. Further, although pressurization is not required, pressurization may be performed. These treatments are preferably performed prior to the extraction step.

  The extract after the extraction process includes a decomposition product of lignin in the presence of iron ions. In the extract, polymer lignin may exist as a solid content, such as when a lignocellulosic material is used as a lignin-containing material. In this case, if necessary, the liquid portion can be separated as a lignin extract by a known solid-liquid separation technique such as centrifugation or filtration. Moreover, you may remove suitably the contaminating component in an extract according to the kind of used lignin containing material, and the use of a lignin extract.

  The extract obtained by the extraction process is usually colored. By adding an extract (typically an extract) extracted with citric acid from acids such as citric acid or other herbs to the extract, the color of the extract can be changed to a lighter color, Brightness can be improved. As such an extract for color tone adjustment, for example, a citric acid extract that exhibits a light pink to light purple color such as perilla leaves, hibiscus petals, cherry leaves, and the like can be used. What is necessary is just to set a citric acid concentration suitably. It is preferable to adjust the color tone at as low a temperature as possible.

(This lignin extract)
The present lignin extract is an extract of lignin in the presence of iron ions, and can typically be obtained by the above-described production method disclosed herein. The lignin extract may be in a solid form such as liquid, slurry, paste, powder. For example, the extract obtained by the above method may be used as it is, or may be diluted or concentrated. Further, the extract may be dried and solidified by freeze drying or the like. This lignin extract has an anti-corruption action and is itself excellent in stability.

  This lignin extract can exert an antitumor effect. Anti-tumor effects include, but are not limited to, for example, tumor growth (eg, slowing tumor growth), modulation of tumor size or metastasis, reduction of toxicity and side effects associated with certain anticancer agents, relief of clinical disorders or symptoms of cancer, or Includes minimization, prolonging the subject's life, and preventing tumor growth in animals that do not have any tumor formation prior to administration, ie, prophylactic administration.

  Therefore, this lignin extract can be used as an active ingredient of an antitumor agent. The tumor that can be treated with the antitumor agent is not particularly limited, and may be a solid cancer or a blood cancer. Examples of solid cancer include colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, stomach cancer, biliary tract cancer, gallbladder / bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, uterus Examples include body cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, skin cancer, brain tumor, testicular tumor and the like. Examples of hematological cancer include leukemia, malignant lymphoma and multiple myeloma. Preferred are lung cancer, esophageal cancer, liver cancer and skin cancer.

  Antitumor agents can take various forms as pharmaceuticals and the like. Specifically, it can be used as capsules, granules, pills, solutions, injections, ointments, extracts, elixirs, suspensions, and the like.

  This lignin extract has an antibacterial action. The antibacterial action includes an action of killing bacteria (bactericidal action) and an action of suppressing bacterial growth (bacteriostatic action). Therefore, this lignin extract can be used as an active ingredient of an antibacterial agent. This lignin extract is considered to have an action of inhibiting the supply of iron ions to cells by binding to iron ions to form a complex. Therefore, the cell in which the present lignin extract exhibits an antibacterial action is not particularly limited, and may be a gram positive bacterium or a gram negative bacterium. Examples of gram positive bacteria include staphylococci, streptococci, pneumococci, diphtheria, and botulinum. Staphylococcus is preferable. Examples of gram-negative bacteria include Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Salmonella, Neisseria pneumoniae, Shigella, Pseudomonas aeruginosa, and periodontal disease. Preferably, it is E. coli. Preferably, it is a periodontal disease bacteria. Periodontal bacteria include Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and the like. More preferred is Porphyromonas gingivalis.

  Antibacterial agents can take various forms as pharmaceuticals, cosmetics, quasi drugs, and the like. Specifically, capsules, granules, pills, solutions, injections, ointments, extracts, elixirs, suspensions, liniments, lotions, etc. for pharmaceuticals, dentifrices for cosmetics, etc. Lotion, cream, milky lotion, funny, cosmetic oil, hair cosmetics, hair dye, hair perfume, powder, pack, foundation, powder perfume, blusher, eyeliner, eye cream, eye shadow, mascara, eyebrow, lipstick, lip balm, It can be used as soap, face wash, shampoo, rinse and the like.

  This lignin extract has an antiviral effect. Antiviral action means the action of inactivating the infectivity of pathogenic viruses that cause human and animal infections, or the action of suppressing the growth of viruses already infected in host cells. Therefore, this lignin extract can be used as an active ingredient of an antiviral agent. In addition, the virus to be used as an antiviral agent is not particularly limited. For example, herpes virus, cytomegalovirus, human papilloma virus, RS (respiratory syncytial virus) virus, influenza virus, HIV (Human Immunodeficiency Virus) virus. , Hepatitis B virus and hepatitis C virus.

  Antiviral agents can take various forms as pharmaceuticals, cosmetics, quasi drugs, and the like. Specifically, capsules, granules, pills, solutions, injections, ointments, extracts, elixirs, suspensions, liniments, lotions, etc. for pharmaceuticals, dentifrices for cosmetics, etc. Lotion, cream, milky lotion, funny, cosmetic oil, hair cosmetics, hair dye, hair perfume, powder, pack, foundation, powder perfume, blusher, eyeliner, eye cream, eye shadow, mascara, eyebrow, lipstick, lip balm, It can be used as soap, face wash, shampoo, rinse and the like.

Moreover, a lignin extract can be used as an antiallergic agent based on an antiallergic action. The antiallergic agent can take various forms such as cosmetics, quasi drugs, and pharmaceuticals. Specifically, cosmetic forms such as lotion, cream, milky lotion, funny, cosmetic oil, hair cosmetics, hair dye, paste perfume, powder, pack, foundation, powder perfume, blusher, eyeliner, eye cream, Eye shadow, mascara, eyebrow, lipstick, lip balm, soap, face wash, shampoo, rinse and the like. Examples of pharmaceuticals include aerosols, solutions, extracts, ointments, eye ointments, suspensions, patches, poultices, liniments, lotions and the like.

The various preparations (external preparations for skin) containing the lignin extract of the present invention may further contain components having various effects other than the lignin extract. For example, moisturizer, keratin improving agent, keratolytic agent, antibiotics, skin permeation enhancer, blood circulation promoter, anti-inflammatory agent, cell activator, anti-inflammatory agent, analgesic agent, emollient, emollient, wound treatment agent And metabolism promoters.

  Various preparations containing the present lignin extract may further contain components having various effects other than the present lignin extract. Antitumor agents include, for example, other antitumor agents for the purpose of multidrug therapy, analgesics, antiemetics, laxatives, antidiarrheals, bronchodilators to prevent or reduce the symptoms associated with cancer and side effects due to treatment Further, it may contain a therapeutic agent for heart disease and the like. The antibacterial agent and antiviral agent may contain, for example, an analgesic agent, an anti-inflammatory agent and the like.

  Various preparations containing the present lignin extract can contain various pharmaceutically acceptable ingredients in these preparations. For example, excipients, binders, disintegrants, lubricants, diluents, solubilizers, suspending agents, tonicity agents, pH adjusters, buffers, stabilizers, colorants, flavoring agents, Examples include flavoring agents and soothing agents.

  In addition, about the addition amount of each component, the addition method, and the formulation method, it can follow a method well-known in this technical field. Those skilled in the art can easily prepare the above-mentioned various preparations containing the lignin extract based on the above general description and the specific disclosure of the examples or by appropriately modifying or modifying them as necessary. Can be manufactured. The lignin extract content in the preparation is not particularly limited, and is appropriately selected according to the use of the preparation.

  The usage of various preparations having the lignin extract of the present invention is not particularly limited. That is, these preparations may be internal preparations, external preparations, or injections as described above. Also, the dosage of these preparations is not particularly limited. For example, it is conceivable to administer 10 to 30 mg per kg of body weight once a day.

  The present invention will be specifically described below with reference to examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

(Preparation of lignin extract)
In this example, as an example of a herbaceous system, an iron crusher having rust as an iron ion source inside the crushed material (3 kilogram units) of the peach (Gentou (sannin)) of the genus Gingeraceae Using a liquid extracted from a plant without adding water and continuously oscillating at 25 ° C. to 35 ° C. for 1 week with continuous bacteria fermentation, 20 to 25 mm filter treatment Thereafter, the color was adjusted with a citric acid shiso solution, and the solution was filtered again through a filter (manufactured by Millipore) having a pore size of 0.45 μm.

(Evaluation of growth inhibitory action on human lung cancer cells, human esophageal cancer cells, human liver cancer cells and human skin cancer)
Using the lignin extract solution prepared in Example 1, antitumor activity against lung cancer cells, esophageal cancer cells and liver cancer cells was evaluated by the following method. Lung cancer cell line (A549 cell), esophageal cancer cell line (TE4 cell), liver cancer cell line (HLE cell) liver cancer cell line (Huh-7 cell) and skin cancer cell line (HYM-1) were treated with 10% FBS. After culturing using MEM containing 1% NEAA and 1 mmol / l sodium pyruvate, cells were collected by trypsin treatment. Huh-7 cells are cells that are resistant to Deferasirox, an iron chelator. The collected cells were diluted with the above medium to a concentration of 2 × 10 ⁵ cells / ml and then seeded at 100 μl per well in a 96-well microplate and cultured overnight. After completion of the culture, the medium was removed, and a lignin extract solution dissolved in MEM containing 0.5% FBS was added to each well, and the mass% concentrations of the lignin extract solution were 10%, 5%, 2%, 1 % And 0.1%. The cells were then cultured for 3 days. After 3 days of culture, the recovered cells were stained with trypan blue, and the cell viability was evaluated from the results. In addition, the same experiment was performed for each cell without adding the lignin extract solution.

  Moreover, it replaced with the lignin extract and experimented similarly using Deferasirox. Specifically, the concentration of Deferasirox was adjusted to 100 μM, 10 μM, and 1 μM, and the cells were cultured for 2 days. Cell viability was assessed after 2 days of culture. Also, the same experiment was performed without adding Deferasirox.

  The cell viability of A549 cells cultured by adding lignin extract to the medium and A549 cells cultured by adding Deferasirox is shown in FIG.

  As shown in the upper part of FIG. 1, the survival rate of A549 cells decreased depending on the lignin extract concentration. That is, it was found that the lignin extract has an antitumor effect on lung cancer cells, and in particular, when the lignin extract has a high concentration, the antitumor effect is increased.

  As shown in the lower part of FIG. 1, the survival rate of A549 cells decreased depending on the Deferasirox concentration. That is, it was found that the antitumor action of the iron chelating agent showed the same behavior as the antitumor action of the lignin extract. This result supports the hypothesis that the lignin extract exhibits an antitumor action by forming a complex with iron.

  The cell viability of TE4 cells cultured with the lignin extract added to the medium is shown in FIG.

  As shown in FIG. 2, the cell viability of TE4 cells decreased depending on the lignin extract concentration. That is, it was found that the lignin extract has an antitumor effect on esophageal cancer cells, and in particular, when the lignin extract has a high concentration, the antitumor effect is increased.

  The cell viability of HLE cells cultured with the lignin extract added to the medium is shown in FIG.

  As shown in FIG. 3, the survival rate of HLE cells decreased depending on the lignin extract concentration. That is, it was found that the lignin extract has an antitumor action against liver cancer, and in particular, when the lignin extract has a high concentration, the antitumor action is increased.

  FIG. 4 shows the cell viability of Huh-7 cells cultured with the lignin extract added in the medium and Huh-7 cells cultured with the Deferasirox added.

  As shown in the upper side of FIG. 4, although the cell viability of Huh-7 cells was reduced by adding 500 μM of Deferasirox, the cells of Huh-7 cells were reduced by adding a lower concentration of Deferasirox. Survival did not decrease. That is, it was confirmed that Huh-7 cells are deferasirox resistant.

  As shown in the lower part of FIG. 4, the cell viability of Huh-7 cells decreased substantially depending on the lignin extract concentration. That is, it was found that the lignin extract also has an antitumor effect on Deferasirox-resistant liver cancer cells, and in particular, when the lignin extract has a high concentration, the antitumor effect is increased.

  FIG. 5 shows the cell viability of HYM-1 cells cultured with the lignin extract added to the medium. As shown in FIG. 5, the lignin extract was found to suppress the growth of human skin cancer cells in a concentration-dependent manner.

  From the above, it was found that the present lignin extract has a growth inhibitory action on various cancer cells.

(Evaluation of cell growth inhibitory effect against periodontal disease bacteria)
Using the lignin extract solution prepared in Example 1, antibacterial action was evaluated by the following method. 15 ml of 5% horse defibrinated blood added to the agar medium so that the lignin extract solution was 5, 10 and 20% by mass was dispensed into a plastic petri dish (diameter 90 mm), solidified, did. Next, 0.1 ml (10 ³ / ml) of a test bacterial solution of Porphyromonas gingivalis, which is an anaerobic gram-negative bacilli and a periodontal disease fungus, was applied to each medium, and anaerobically cultured at 35 ° C. for 7 days. After culturing, the number of villages was counted. The same experiment was performed without adding the lignin extract. The number of settlements at each lignin extract concentration is shown in Table 1.

  As shown in Table 1, the number of settlements of Porphyromonas gingivalis decreased by adding the lignin extract. From the above, it was found that the present lignin extract has an effect of inhibiting the growth of periodontal disease bacteria that are gram-negative bacilli.

(Evaluation of growth inhibitory effect of influenza A virus)
Using the lignin extract solution prepared in Example 1, antiviral activity was evaluated by the following method. To 100 parts by mass of Eagle MEM medium “Nissui” (manufactured by Nissui Pharmaceutical Co., Ltd.), 10 parts by mass of fetal calf serum was added to obtain a cell growth medium. Dog-derived kidney cells ((MDCK (NBL-2) cell ATTC CCL-34 strain (manufactured by Dainippon Pharmaceutical Co., Ltd.)) were cultured in a single layer in a tissue culture flask using a cell growth medium according to a conventional method. After layer culture, the recovered cells were inoculated with Influenza A virus (H1N1) A / PR / 8/34 ATCC (registered trademark) VR-1469 (American Type Culture. Collection).

Cell maintenance at the composition of Eagle MEM medium “Nissui” 1000 mL, 10% NaHCO ₃ 14 mL, L-glutamine (30 g / L) 9.8 mL, 100 × MEM vitamin solution 30 mL, 10% albumin 20 mL and 0.25% trypsin 20 mL A medium was prepared. Subsequently, the cells were cultured for 1 to 5 days in a carbon dioxide incubator (CO ₂ concentration 5%) at 37 ± 1 ° C. using a fine maintenance medium. After culturing, the morphology of the collected cells was observed using an inverted phase contrast microscope, and it was confirmed that morphological changes due to cell degeneration occurred. Next, the collected cells were centrifuged at 3000 rpm for 10 minutes, and the resulting supernatant was used as a virus suspension.

  1 mL of the lignin extract solution prepared in Example 1 was filtered through a membrane filter (pore size 0.45 μm), and 0.1 mL of a virus suspension was added to obtain a working solution. The working solution was allowed to stand at room temperature for 24 hours, and then diluted 10-fold with a cell location medium. As a control experiment, a control solution was prepared using purified water instead of the working solution.

The virus infectivity titer was measured as follows. MDCK cells were monolayer cultured in a 96-well microplate for tissue culture using a cell growth medium according to a conventional method. After monolayer culture, the cell growth medium was removed and 0.1 mL of cell maintenance medium was added. Furthermore, it diluted 10 times with the cell maintenance medium, and prepared the sample dilution liquid. Subsequently, each of the 10-fold diluted working solution and the control solution was serially diluted 10-fold with the sample diluent. 0.1 mL of each diluted solution was inoculated into 4 wells and cultured in a 37 ± 1 ° C. carbon dioxide incubator (CO ₂ concentration 5%) for 4-7 days. After culturing, the morphology of the collected cells is observed using an inverted phase contrast microscope to confirm whether or not morphological changes have occurred due to cell degeneration, and 50% tissue culture infectious dose (TCID ₅₀ ) by the Reed-Muench method. Was calculated and converted to the virus infectivity per mL of working fluid. Table 2 shows the log TCID ₅₀ values per mL of the specimen and the subject.

  As shown in Table 2, according to the present lignin extract, it was found that the virus concentration can be effectively reduced and there is a virus growth inhibitory action.

(Confirmation of components in lignin extract by GC-MS)
The lignin extract (stock solution) obtained in Example 1 was analyzed under general conditions using GC-MS. The results are shown in FIG.

  As shown in FIG. 6, various low molecular weight compounds considered to be derived from lignin could be confirmed in the lignin extract. From the above, it was found that lignin was stabilized by lowering the molecular weight by bringing a metal ion and a lignin-containing material into contact with each other, and a lignin extract having various functions was obtained.

(Evaluation of toxicity of lignin extract)
A lignin extract solution having a mass% concentration of 20% was prepared in the same procedure as in Example 1. The toxicity of the lignin extract was evaluated by the following method using the 20% lignin extract solution. First, the weight of four nude mice was measured. For each nude mouse, a 20% lignin extract solution was appropriately given as drinking water (water bottle type). After a week, the weight of each nude mouse was measured, and changes in skin and color of excrement were observed. Table 3 shows the body weight of each nude mouse before and after administration of the lignin extract and the increase and decrease in body weight.

  As shown in Table 3, the body weight hardly changed before and after administration of the lignin extract in any individual. In addition, no skin change was observed in any individual, and no change in the color of excrement was observed. That is, it was found that the dose of the lignin extract in this example did not show toxicity.

(Evaluation of growth inhibitory action against HIV virus)
Ferric sulfate is added to the suspension of the powder of Matsukasa to prepare a solution of 100 mg / ml of the powder of Matsukasa and 1.0 mg / ml of ferric sulfate, and lactic acid bacteria are added at room temperature and stirred gently. While fermenting. Thereafter, the culture broth was filtered, and CC ₅₀ (50% toxic concentration) and EC ₅₀ (50% effective concentration) of the filtrate were measured. As a result, CC50 was 562 μg / ml and EC50 was 3.74 μg / ml.

(Acquisition of lignin extract from lignin-containing material and antibacterial evaluation)
5 g of Kumazasa pieces are immersed in 100 ml of 5% strength sodium hydroxide solution for 7 days. After adding 0.1 g of ferric (III) sulfate at room temperature of 25 ° C. to 35 ° C., 1 ml of lactic acid bacteria (about 400 million yen) (~ 500 million) was added to conduct fermentation. After solid-liquid separation of the culture solution, it was filtered through a filter with a pore size of 0.25 mm, and half the amount of Shiso citric acid extract was added to adjust the color tone, and after adjusting to a mass concentration of 50% based on the mass of crumbly of Kumazasa, Furthermore, it filtered again with the filter (made by a Millipore company) with the hole diameter of 0.45 micrometer. In addition, the Shiso citric acid extract was prepared by boiling about 300 g of red / blue purple soup in 1.8 L of water for several minutes and then adding 35 g of citric acid.

(2) Suspend 0.5 g of cedar powder in 400 ml of 20% strength aqueous sodium hydroxide solution for 7 days, leave 1/10 cedar powder in 100 ml solution prepared by adding 400 ml of purified water, 25 ° C. After adding 0.1 g of ferric sulfate (III) at room temperature of ˜35 ° C., 1 ml of lactic acid bacteria (about 400 million to 500 million) was added for fermentation. After solid-liquid separation of the culture solution, it was filtered through a filter with a pore size of 0.5 mm, and in order to adjust the color tone, half amount of Shiso citric acid extract was added, Furthermore, it filtered again with the filter (made by Millipore) of the hole diameter 0.45 micrometer.

  About the lignin extract obtained by said (1), the growth inhibitory effect about colon_bacillus | E._coli and Staphylococcus aureus was evaluated. The results are shown in Table 4, respectively.

  As shown in Table 4, any lignin extract was able to confirm the growth-inhibiting action for Escherichia coli and Staphylococcus aureus.

(Preparation of lignin extract)
In this example, the crushed material of the shoot and leaf portion of the genus Ganyoga genus (Gentou (sannin)) is put into an iron crusher having rust as an iron ion source, and at room temperature without adding water To obtain a black-brown lignin extract. A brown supernatant obtained by removing the solid content of the extract by centrifugation was obtained. The obtained supernatant was diluted 3 times by adding 2 times the amount of water by volume. Moreover, it diluted 2 times with 1 time amount of water. In Examples 2, 4 and 5, the 3-fold diluted solution was used as the lignin extract, and in Example 3, the 2-fold diluted solution was used as the lignin extract. For the lignin extract (stock solution) prepared in this example, the gene mutagenicity test (reversion mutation test) was performed for three strains (Salmonella typhimurium TA100, Escherichia coli WP2uvrA strain, Salmonella yphimurium TA198 strain). As a result, mutagenicity was not observed. Moreover, when the irritation and sensitization by the Marzulli-maibach method were evaluated (50 test subjects), it was confirmed that there was no irritation and sensitization and hypoallergenicity.

  Anti-allergic action was evaluated using the lignin extract (3-fold diluted solution) prepared in this example. The test subjects were two latex allergic patients, and the lignin extract was applied in an appropriate amount to the affected area when symptoms and symptoms occurred. As a result, itching was suppressed and improvement of allergic symptoms was confirmed for two subjects. From the above, it was found that the lignin extract has an antiallergic action that particularly suppresses early inflammation.

Claims (8)

An antitumor agent comprising an extract extracted from a fermented liquid obtained by bringing iron ions into contact with stem and leaf portions of moon peach and fermenting with lactic acid bacteria in the presence of iron ions. It consists of lung cancer, skin cancer, esophageal cancer, and liver cancer, which contains extracts extracted from the fermented liquid obtained by contacting ferric ions with the shoots of moon peach and fermenting with lactic acid bacteria in the presence of iron ions. A cell growth inhibitor for one or more cancer cells selected from the group. The cell growth inhibitor according to claim 1 or 2, comprising the fermentation broth . An antiallergic agent comprising an extract extracted from a fermented liquid obtained by bringing iron ions into contact with the stem and leaves of moon peach and fermenting with lactic acid bacteria in the presence of iron ions . The antiallergic agent of Claim 4 containing the said fermented liquor . A growth inhibitor of periodontal disease bacteria, comprising a fermented liquid obtained by bringing iron ions into contact with the stems and leaves of moon peach and fermenting with lactic acid bacteria in the presence of iron ions.   The growth inhibitor according to claim 6, wherein the periodontal disease bacteria is Porphyromonas gingivalis. A type A influenza virus growth inhibitor comprising a fermented liquid obtained by bringing ferric ions into contact with stem and leaf portions of moon peach and fermenting with lactic acid bacteria in the presence of iron ions.