JP7340197B2 - Antitumor and antiviral agents - Google Patents

Description

TECHNICAL FIELD The present invention relates to antitumor agents and antiviral agents.

In recent years, biomass (renewable organic resources obtained from plants and animals) has attracted attention as a carbon-neutral resource from the perspective of environmental issues. An example is the production of bioethanol using edible biomass that can also be used as food, such as carbohydrate raw materials or starch raw materials, but in this case, competition with food is a problem. On the other hand, non-edible biomass is attracting attention because it does not compete with food. For example, lignocellulose biomass, which is a type of non-edible biomass, includes unused thinned wood, leftover wood from sawmills, and wood generated from demolishing houses. The use of lignocellulosic biomass is expected to reduce waste and be used as an energy resource, and is important from an environmental perspective.

Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin. Lignin is an adhesive molecule that holds cellulose together. Because its structure is a polymerized phenylpropane skeleton, it is attracting attention as a resource for aromatic compounds.

Until now, technology for reducing the molecular weight of lignin has been developed mainly in the paper manufacturing industry. Techniques for reducing the molecular weight of lignin in the paper manufacturing process include kraft cooking, sulfite cooking, and alkaline cooking. In kraft cooking, lignin is mainly used as a heat source, and in sulfite cooking, in addition to using lignin as a heat source, sulfonated lignin has also been used as a dispersant. However, lignin obtained through conventional papermaking processes has undergone modification, making it difficult to further reduce the molecular weight. For this reason, lignin obtained through conventional papermaking processes is limited to use as fuel, dispersant, etc., and is difficult to use as a raw material for chemical industrial products.

Patent Document 1 produces a lignin decomposition product by heating lignin in a mixed solvent of hydrocarbon and alcohol in the presence of an acid catalyst, but does not describe its medical use.

Non-Patent Document 1 discloses the antitumor effect of purified lignin sulfonic acid, and Non-Patent Document 2 discloses the antitumor effect of phenolic compounds contained in the alkaline extract of bamboo leaf lignin. Both lignin sulfonic acid and the phenolic compound in the alkaline lignin extract, which have been confirmed to have antitumor activity in 1 and 2, have broken lignin structures, and the bioactivity of lignin decomposition products that retain the lignin structure is unknown.

Japanese Patent Application Publication No. 2016-50200

Kyushu University Faculty of Agriculture Curatorial Magazine, 28(4), pp.215-221 (1974) Kyushu University Faculty of Agriculture Curatorial Magazine, 23(3), pp.103-111 (1968)

The present invention aims to provide novel antitumor and antiviral agents.

The present invention provides the following antitumor agents and antiviral agents.
Item 1. An antitumor agent containing as an active ingredient a lignin decomposition product containing a lignin-derived unit containing a benzene ring having OH and/or OCH3 as a substituent as a partial structure.
Item 2. An antiviral agent containing a lignin decomposition product containing a lignin-derived unit containing a benzene ring having OH and/or OCH3 as a substituent as an active ingredient, and which is effective against viruses of the family Caliciviridae or the family Orthomyxoviridae.
Item 3. Item 2. The antiviral agent according to item 2, which is effective against norovirus of the Caliciviridae family and influenza viruses (types A, B, and C) of the Orthomyxoviridae family.

The antitumor agent and antiviral agent of the present invention contain a lignin decomposition product derived from a natural product as an active ingredient, and have the advantage of low toxicity to normal cells.

Effect of 25 MASLs on LLC cell viability. LLC was treated with the indicated concentrations of MASL for 24 hours. Cell viability was assessed using a water-soluble tetrazolium salt assay. Significant differences in DMSO only treatment versus control were determined using repeated measures analysis of variance (AVNOVA). *p<0.05 MASL reduced tumor cell viability. LLC, A549, HT1080 and HDF were treated with the indicated MASL at a concentration of 0.1 mg/mL. After 12 and 24 hours, cell viability was measured using a water-soluble tetrazolium salt assay. Data were expressed as mean±SD (n=4). Significant differences in DMSO only treatment versus control were determined using repeated measures analysis of variance (AVNOVA). *p<0.05, **p<0.01 YM CL1T administration significantly inhibited LLC proliferation in mice. LLC tumor-bearing mice were injected intraperitoneally with MASL. Control mice were injected with DMSO. Mice were euthanized 14 days after the start of MASL administration. A. Tumor size (mean ± SD) and B. mouse weight (mean ± SD) on day 14 are shown. N = 5 (control and YM E2T groups) or N = 6 (YM CL1T group). Analysis of variance (ANOVA) was used to test for significant differences between groups. *p<0.05, **p<0.01, N.S., not significant. Inhibitory effects of 12 types of MASL on influenza A virus. Anti-feline calicivirus inhibitory effect of each lignin decomposition product. Feline Calicivirus F9 strain (A) 2000 TCID ₅₀ /100 μl, (B) 20000 TCID ₅₀ /100 μl

The lignin decomposition product, which is an active ingredient of the antitumor agent and antiviral agent of the present invention, is expressed by the following formula (1).

(In the formula, R ¹ represents OH or O-. R ² and R ³ are the same or different and represent a hydrogen atom, a single bond, or OCH _3. ).

Note that "O-" means that an oxygen atom (O) is single-bonded to another benzene ring or an aliphatic carbon atom.

The lignin decomposition product of the present invention is a mixture, and one lignin decomposition product has an average of preferably 1 to 15, more preferably 2 to 12, and even more preferably 3 to 10 partial structures of formula (1). Including pcs. For example, alkaline lignin obtained by solubilizing lignin with an alkali contains more than 15 benzene rings having OH and/or OCH ₃ as a substituent on average, and therefore is not included in the lignin decomposition product of the present invention.

The average number of partial structures of formula (1) contained in one lignin decomposition product is determined by the reaction conditions of the decomposition reaction, and for example, the average number tends to become smaller due to higher temperature, longer reaction time, stronger acidic conditions, etc. There is.

The lignin decomposition product of the present invention does not contain lignin sulfonic acid, which is a polymeric substance produced as a by-product during the production of wood pulp.

The lignin decomposition product used as an active ingredient in the present invention consists of lignin as a solvent (at least one organic solvent selected from the group consisting of water-miscible organic solvents and water-immiscible organic solvents) and an acid catalyst (sulfuric acid, It can be produced by heating in the presence of an inorganic acid such as hydrochloric acid, nitric acid, or phosphoric acid; or an organic acid such as acetic acid, citric acid, malic acid, oxalic acid, tartaric acid, succinic acid, or methanesulfonic acid. The amount of acid catalyst used is 0.01% to 5% by mass, preferably 0.01% by mass based on the solvent used, because if the amount is too small, the reaction will be difficult to proceed, and if it is too large, it will be difficult to remove after the reaction. 05% to 2% by mass. The preferred reaction temperature is 40 to 230°C, and the preferred reaction time is 5 to 100 minutes.

Water-miscible organic solvents include alcohols (linear or branched aliphatic alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, 2-butanol, isobutanol, isopentanol, cyclohexanol, methyl Alicyclic alcohols such as cyclohexanol, alkylene glycol monoalkyl ethers such as ethylene glycol monomethyl ether and ethylene glycol monoethyl ether), glycols (ethylene glycol, propylene glycol, etc.), glycerin, ketones (acetone, methyl ethyl ketone, etc.), tetrahydrofuran, Examples include acetonitrile, DMF, DMSO, and the like. Water-immiscible organic solvents include aromatic hydrocarbons (benzene, toluene, xylene, xylene gasoline, ethylbenzene, chlorobenzene, dichlorobenzene, petroleum ether, petroleum naphtha, petroleum benzine, mineral spirits, limonene, etc.), aliphatic or alicyclic hydrocarbons (hexane, heptane, octane, cyclohexane, methylcyclohexane, gasoline, etc.), chlorinated hydrocarbons (methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, etc.), ethyl acetate, diethyl ether Examples include.

Lignin is an aromatic polymer compound that exists in lignified plants in an amount of about 15 to 35%. The raw materials for lignin in the present invention include wood chips such as cedar, cypress, spruce, pine, eucalyptus, beech, willow, and bamboo, wood flour, wheat straw, rice straw, rice husk, sugarcane residue, sugar beet residue, cassava, Examples include rapeseed residue, soybean residue, corn stover, oil palm fruit husk, tobacco residue, napier grass, and Erianthus.

As the lignin used as a raw material for producing the lignin decomposition product, lignocellulosic biomass itself in which lignin is not separated from cellulose may be used, or lignin separated from biomass may be used. In addition to wood, lignocellulosic biomass includes herbaceous biomass such as sugarcane bagasse and napier grass. The lignin separated from biomass in the present invention is lignin that has been separated from biomass in advance apart from the process of the present invention, and depending on the separation method, lignin may be sulfuric acid lignin, hydrochloric acid lignin, periodic acid lignin, dioxane lignin, or alcohol lignin. , thioglycolic acid lignin, kraft lignin, alkaline lignin, Brauns natural lignin, ground lignin, cellulose saccharification residue lignin, hydrothermal lignin, steam blasted lignin, and the like. The lignin decomposition product of the present invention does not contain high molecular weight lignin separated from these biomass; for example, alkaline lignin is not included in the lignin decomposition product of the present invention.

The lignin decomposition product of the present invention preferably does not contain sulfur atoms.

Examples of tumors that can be treated by administering antitumor agents include, in the case of malignant tumors, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gallbladder/cholangiocarcinoma, pancreatic cancer, lung cancer, breast cancer, and ovarian cancer. , cervical cancer, endometrial cancer, renal cancer, bladder cancer, prostate cancer, testicular tumor, bone/soft tissue sarcoma, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor, etc.

Viruses that cause viral infections that can be treated with antiviral drugs include viruses of the Caliciviridae family, such as norovirus and feline calicivirus, and viruses of the Somyxoviridae family, including influenza viruses (types A, B, and C). Examples include viruses.

The dose of lignin decomposition product, which is the active ingredient of antitumor or antiviral drugs, varies depending on the symptoms of patients infected with tumors or viruses, the dosage form, etc., but in general, it is about 1 to 1 for oral drugs per dosage unit form. 1000 mg, preferably about 0.1 to 500 mg for injections. The daily dose for an adult adult of the lignin decomposition product varies depending on the patient's symptoms, weight, age, sex, etc., and cannot be determined unconditionally, but it is usually about 0.1 to 5000 mg, preferably 1 to 1000 mg. It is preferable to administer this once a day or in divided doses of about 2 to 4 times a day.

Examples are shown below, but the present invention is not limited to these examples.

Example 1
(1) Lignin decomposition product samples (No.1 to No.25)
Manufacture of No. 1 (YMC1T): Add 6 mL of sulfuric acid aqueous solution (sulfuric acid content 0.75 g, 0.07 mL/mL), 6 mL of ethanol, and 8 mL of toluene to 1 g of cedar wood flour, and use Biotage's initiator ⁺ to incubate at 180 mL. Microwave heating was performed at ℃ for 30 minutes. After the reaction, the upper organic layer was dispensed, and about 10 mL of toluene was added to the lower aqueous layer and stirred to perform an extraction operation. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and then concentrated under reduced pressure to obtain sample No. 1.
Production of No. 2 (YMC1E): Approximately 10 mL of ethyl acetate was added to the aqueous layer containing solids obtained in the extraction operation of No. 1, and an extraction operation was performed. This operation was repeated three times, and the collected organic layer was dehydrated with sodium sulfate and concentrated under reduced pressure to obtain sample No. 2.
Production of No. 3 (YMC1A): The aqueous layer containing solids obtained in the extraction operation of No. 2 was filtered through cotton, and the residue was extracted three times with acetone. Sample No. 3 was obtained from the acetone solution by concentration under reduced pressure.
Manufacture of No. 4 (YME1T): Add 6 mL of sulfuric acid aqueous solution (sulfuric acid content 0.75 g, 0.07 mL/mL), 6 mL of ethanol, and 8 mL of toluene to 1 g of eucalyptus wood flour, and use Biotage's ^initiator Microwave heating was performed at ℃ for 30 minutes. After the reaction, the upper organic layer was dispensed, and about 10 mL of toluene was added to the lower aqueous layer and stirred to perform an extraction operation. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 4.
Production of No. 5 (YME1E): Approximately 10 mL of ethyl acetate was added to the aqueous layer containing solids obtained in the extraction operation of No. 4, and an extraction operation was performed. This operation was repeated three times, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 5.
Production of No. 6 (YME1A): The aqueous layer containing solids obtained in the extraction operation of No. 5 was filtered through cotton, and the residue was extracted three times with acetone. Sample No. 6 was obtained from the acetone solution by concentration under reduced pressure.
Manufacture of No.7 (YMC2T): Add 6 mL of sulfuric acid aqueous solution (sulfuric acid content 0.25 g, 0.023 mL/mL), 6 mL of ethanol, and 8 mL of toluene to 1 g of cedar wood flour, and use Biotage's initiator ⁺ to incubate at 180 mL. Microwave heating was performed at ℃ for 30 minutes. After the reaction, the upper organic layer was dispensed, and about 10 mL of toluene was added to the lower aqueous layer and stirred to perform an extraction operation. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and concentrated under reduced pressure to obtain sample No. 7.
Production of No. 8 (YMC2E): Approximately 10 mL of ethyl acetate was added to the aqueous layer containing solids obtained in the extraction operation of No. 7, and an extraction operation was performed. This operation was repeated three times, and the collected organic layer was dehydrated with sodium sulfate and concentrated under reduced pressure to obtain sample No. 8.
Production of No. 9 (YMC2A): The aqueous layer containing solids obtained in the extraction operation of No. 8 was filtered through cotton, and the residue was extracted three times with acetone. Sample No. 9 was obtained from the acetone solution by concentration under reduced pressure.
Manufacture of No. 10 (YME2T): Add 6 mL of sulfuric acid aqueous solution (sulfuric acid content 0.25 g, 0.023 mL/mL), 6 mL of ethanol, and 8 mL of toluene to 1 g of eucalyptus wood powder, and heat at 180 °C using Biotage's initiator ⁺ . , microwave heating was performed for 30 minutes. After the reaction, the upper organic layer was dispensed, and about 10 mL of toluene was added to the lower aqueous layer and stirred to perform an extraction operation. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 10.
Production of No. 11 (YME2E): Approximately 10 mL of ethyl acetate was added to the aqueous layer containing solids obtained in the extraction operation of No. 10, and an extraction operation was performed. This operation was repeated three times, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 11.
Production of No. 12 (YME2A): The aqueous layer containing solids obtained in the extraction operation of No. 11 was filtered through cotton, and the residue was extracted three times with acetone. The acetone solution was concentrated under reduced pressure to obtain sample No. 12.
Manufacture of No. 13 (YMCL1T): Sugi alkaline lignin (acid precipitate from black liquor obtained by cooking cedar chips with 23% sodium hydroxide at 170 °C and Hf1500. Details are provided separately) 5 g of sulfuric acid aqueous solution 60 mL (sulfuric acid content: 7.5 g, 0.07 mL/mL), methanol 60 mL, and toluene 80 mL were added, and microwave heating was performed at 180 °C for 30 minutes using Milestone's StartSYNTH. After the reaction, toluene was added and an extraction operation was performed. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 13.
Production of alkaline lignin: Cedar chips are alkali-cooked using sodium hydroxide at 170°C and Hf 1500, the resulting black liquor is neutralized and filtered, the insoluble part is collected and suspended in water, The precipitate produced by acidification with sulfuric acid was separated by filtration and used as alkaline lignin.
Manufacture of No. 14 (YMCL1E): The aqueous layer containing solids obtained in the extraction operation of No. 13 was subjected to an ethyl acetate extraction operation. This operation was repeated three times, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 14.
Production of No. 15 (YMCL1A): The aqueous layer containing solids obtained in the extraction operation of No. 14 was filtered through cotton, and the residue was extracted three times with acetone. The acetone solution was concentrated under reduced pressure to obtain sample No. 15.
Manufacture of No. 16 (YMCL2T): Sugi alkaline lignin (acid precipitate from black liquor obtained by cooking cedar chips with 23% sodium hydroxide at 170 °C and Hf1500. Details in another section) 5 g and 60 g of sulfuric acid aqueous solution mL (sulfuric acid content: 7.5 g, 0.07 mL/mL), 60 mL of ethanol, and 80 mL of toluene were added, and microwave heating was performed at 180 °C for 30 minutes using Milestone's StartSYNTH. After the reaction, toluene was added and an extraction operation was performed. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 16.
Production of No. 17 (YMCL2E): The aqueous layer containing solids obtained in the extraction operation of No. 13 was subjected to an ethyl acetate extraction operation. This operation was repeated three times, and the collected organic layer was dehydrated with sodium sulfate and then concentrated to obtain sample No. 17.
Production of No. 18 (YMCL2A): The aqueous layer containing solids obtained in the extraction operation of No. 17 was filtered through cotton, and the residue was extracted three times with acetone. The acetone solution was concentrated under reduced pressure to obtain sample No. 18.
Production of No. 19 (MYE1): 20 mL of 1% acetic acid peracetic acid solution was added to 3 g of ball-milled eucalyptus wood flour, and microwave heating was performed at 50° C. for 10 minutes using Biotage's initiator ⁺ . After the reaction, 20 mL of acetone was added to perform an extraction operation. The extraction operation was performed three times in total, and the collected acetone fractions were concentrated under reduced pressure to obtain sample No. 19.
Production of No. 20 (MYE2): 20 mL of 1% acetic acid peracetic acid solution was added to 3 g of ball-milled eucalyptus wood flour, and microwave heating was performed at 100 °C for 10 minutes using Biotage's initiator ⁺ . After the reaction, 20 mL of acetone was added to perform an extraction operation. The extraction operation was performed three times in total, and the collected acetone fractions were concentrated under reduced pressure to obtain sample No. 20.
Manufacture of No. 21 (MYE3): 20 mL of 1% acetic acid peracetic acid solution was added to ball-milled eucalyptus wood flour, and microwave heating was performed at 140 °C for 10 minutes using Biotage's initiator ⁺ . After the reaction, 20 mL of acetone was added to perform an extraction operation. The extraction operation was performed three times in total, and the collected acetone fractions were concentrated under reduced pressure to obtain sample No. 21.
Production of No. 22 (MKEL1): 318 mg of paratoluenesulfonyl chloride and 15 mL of heavy water were added to 0.3 g of eucalyptus alkaline lignin, and microwave heating was performed at 160 °C for 30 minutes using Biotage's initiator or initiator ⁺ . . After the reaction, the mixture was filtered, washed with methanol and ethyl acetate, adjusted to pH=3 with sodium hydrogen carbonate, and extracted with ethyl acetate. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and concentrated under reduced pressure to obtain sample No. 22.
Manufacture of No. 23 (MKEL2): 318 mg of paratoluenesulfonyl chloride and 15 mL of heavy water were added to 0.3 g of lignin left after the eucalyptus cellulase treatment, and microwave heating was performed at 160° C. for 60 minutes using StartSYNTH from Milestone. After the reaction, the mixture was filtered, washed with methanol and ethyl acetate, adjusted to pH=3 with sodium hydrogen carbonate, and extracted with ethyl acetate. The extraction operation was performed three times in total, and the collected organic layer was dehydrated with sodium sulfate and concentrated under reduced pressure to obtain sample No. 23.
Manufacture of No.24 (SSE1-4):
Sugarcane bagasse was used as a raw material and digested with dilute sulfuric acid between 150°C and 200°C, and the resulting digested product was saccharified using cellulase. For saccharified bagasse, sugar was converted to ethanol by ethanol fermentation, and then the solution fraction was separated by vibrating sieving with a mesh size of 0.4 mm. Ethanol was separated from the ethanol solution by distillation, and solid and liquid were separated from the remaining distillation waste by centrifugation. The obtained supernatant was concentrated under reduced pressure to obtain sample No. 24.
Manufacture of No. 25 (SSE2-4): Napier grass was used as a raw material and digested with dilute sulfuric acid between 150°C and 200°C, and the resulting digested product was saccharified using cellulase. For the saccharified Napier grass, sugar was converted to ethanol by ethanol fermentation, and then the solution fraction was separated by vibrating sieving with a mesh size of 0.4 mm. Ethanol was separated from the ethanol solution by distillation, and solid and liquid were separated from the remaining distillation waste by centrifugation. The obtained supernatant was concentrated under reduced pressure to obtain sample No. 25.

(2) Cell line and culture conditions Lewis lung cancer cell line (LLC) was obtained from RIKEN BRC (RCB0558). Human alveolar carcinoma cell line A549 and human fibrosarcoma cell line HT1080 were purchased from ATCC (Numbers CCL-185 and CCL-121). aHDFs (healthy adult dermal fibroblasts) were purchased from ScienCell Research Laboratories (cat no. 2320). All cells were grown in DMEM medium containing 10% fetal bovine serum (FBS; Equitech-Bio), 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 mM nonessential amino acids at 37°C in 5% CO2/95% humidified air. It was cultured in Cells were trypsinized before passaging.

(3) Cell viability assay
Cell viability was determined by water-soluble tetrazolium salt assay. Four types of cells were used: LLC, A549, HT1080, and HDFs. Cells of any of the four types were seeded in 96-well plates at a density of 1 x 10 ³ cells per well, and the next day lignin digest was added to each well. In experiments using all Nos. 1 to 25 as lignin decomposition products, LLC was used as the cell, and three concentrations of lignin decomposition products were tested: 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL. (Figure 1). In experiments using No. 10 (YME2T), No. 13 (YMCL1T), and No. 16 (YMCL2T) as lignin decomposition products, four types of cells, LLC, A549, HT1080, and HDFs, were used (Figure 2). The concentration of lignin degradation product was tested at 0.1 mg/mL. After 12 or 24 hours of incubation after addition of the lignin decomposition product, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium monosodium salt (WST-8) solution (Nacalai Tesque) was added. After culturing at 37°C for 1 hour, the culture supernatant was transferred to a new 96-well plate. The absorbance of each well was measured using a microplate reader (Emax; Molecular Devices). The results are shown in FIGS. 1 and 2.

(4) LLC tumor mouse model animal experiments were approved by the Laboratory Animal Committee of Kyoto Prefectural University of Medicine (Code No M29-137), and all methods followed the NIH guidelines for the care and use of laboratory animals. Seven-week-old female C57BL/6 mice were purchased from Shimizu Experimental Materials Co., Ltd. All mice were injected with 1 x 10 ⁵ LLC cells into the flank.
After the tumor grew to a size of approximately 100 ^mm3 , lignin degradation products (YM E2T (■), YM CL1T (◆)) were added at 20 mg/kg body weight or DMSO was added at 20 mg/kg body weight as a control (○). Mice were injected intraperitoneally every other day. Tumor volume calculations were performed as follows: Tumor volume = d ² × D/2, where d and D are the shortest and longest diameters, respectively. After measuring body weight on day 14, all mice were euthanized and tumor size was measured.

(5) Inhibition of influenza virus infection
Influenza A virus (A/Puerto Rico/8/1934 H1N1; PR8 strain) 12 lignin degradation product samples were added to ¹⁰⁴ PFU/100 μl at a concentration of 2 mg/ml, and incubated on ice for 10 minutes. placed. The virus infectivity of this mixture was measured by plaque assay. Plaque assay was performed according to the following procedure. Dilute the mixture solution in 10 steps with PBS(-), and spread the virus solution at each dilution step at 100 μl/well to confluent Madin-Darby Canine Kidney (MDCK: derived from canine renal tubular epithelial cells) cells6. -well plate. Virus adsorption was performed for 1 hour at 37°C and 5% _CO2 , DMEM medium supplemented with 0.8% agarose, 2.5μg/ml trypsin, and 0.2% albumin was added, and cultured at 37°C and 5% _CO2 for 48 hours. Fixed with 5% glutaraldehyde. After removing the agar medium, it was stained with crystal violet and the number of plaques that appeared was counted. A control in which only a medium containing DMSO was added to the virus solution was used as a control, and the proportion of plaques in each sample was calculated, assuming that the number of plaques in this control was 1.

The results are shown in Figure 4.

(6) Inhibition of feline calicivirus infection: Add the lignin degradation product sample shown in Figure 5 to feline calicivirus F9 strain 2000 TCID ₅₀ /100 μl or 20000 TCID ₅₀ /100 μl to a concentration of 1 or 0.25 mg/ml. and placed on ice for 10 minutes. This mixed solution was added at 100 μl/well to a 96-well plate on which CRFK cells (feline kidney-derived cells) were spread to confluence. Virus adsorption was performed for 1 hour at 37°C and 5% CO ₂ , and after removing the supernatant, 100 μl/well of DMEM medium containing 2% FBS was added, and the mixture was left standing at 37°C and 5% CO ₂ for 4 days. . Cell viability was determined by water-soluble tetrazolium salt assay. Add 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) solution (Nacalai) to the well. After reaction at 37°C for 1 hour, the absorbance of each well was measured using a microplate reader (Emax; Molecular Devices). The results are shown in Figure 5.

(7) Statistical analysis All experimental data were expressed as mean ± standard deviation (SD). Two-tailed Student's t-test and parametric one-way or two-way analysis of variance (ANOVA) were used to analyze differences between groups. The Tukey-Kramer post-hoc test was used to determine specific differences between groups. In all analyses, P<0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc.).

(8) Results As shown in Figure 1, lignin degradation product No. 1-25 has an antitumor effect on cancer cell lines (LLC).

As shown in Figure 2, lignin degradation products No. 10 (YME2T), No. 13 (YMCL1T), and No. 16 (YMCL2T) have antitumor effects on cancer cell lines (LLC, A549, HT1080). However, it was revealed that the cytotoxicity against normal cells (HDFs) is low.

As shown in Figure 3, there was no significant difference in weight gain between No. 10 (YME2T) and No. 13 (YMCL1T) lignin degradation products (Figure 3B), and they were able to reduce tumor volume (Figure 3A). It was revealed.

As shown in Figures 4 and 5, lignin degradation products have anti-inflammatory properties against viruses of the Orthomyxoviridae family, such as influenza viruses (types A, B, and C), and viruses of the Caliciviridae family, such as feline calicivirus and norovirus. It was revealed that the virus showed viral activity.

Claims (4)

A method for producing an antitumor agent comprising a lignin decomposition product containing at least one partial structure represented by the following formula (1),
The lignin decomposition product is
Cedar or eucalyptus wood flour,
Alkaline lignin of cedar or eucalyptus, or
Residual lignin after cellulase treatment of eucalyptus
(1) a solvent containing water, a water-miscible organic solvent, and a water-immiscible organic solvent; and (2) step A of performing microwave treatment while heating in the presence of an acid catalyst, or
Eucalyptus alkaline lignin, or
Residual lignin after cellulase treatment of eucalyptus
Step A' of performing microwave treatment while heating in the presence of (1') heavy water and (2) para-toluenesulfonyl chloride;
Step B of recovering lignin decomposition products using a water-immiscible organic solvent during microwave treatment
generated by a method including
Production method:

(In the formula, R ¹ represents OH or O-. R. ² and R ³ are the same or different, hydrogen atom, single bond or OCH ₃ shows. "O-" means that the oxygen atom (O) is single bonded to another benzene ring or aliphatic carbon atom). A method for producing an antiviral agent effective against viruses of the Caliciviridae or Orthomyxoviridae family, comprising a lignin decomposition product containing at least one partial structure represented by the following formula (1),
The lignin decomposition product is
Cedar or eucalyptus wood flour,
Alkaline lignin of cedar or eucalyptus, or
Residual lignin after cellulase treatment of eucalyptus
(1) a solvent containing water, a water-miscible organic solvent, and a water-immiscible organic solvent; and (2) step A of performing microwave treatment while heating in the presence of an acid catalyst, or
Eucalyptus alkaline lignin, or
Residual lignin after cellulase treatment of eucalyptus
Step A' of performing microwave treatment while heating in the presence of (1') heavy water and (2) para-toluenesulfonyl chloride;
Step B of recovering lignin decomposition products using a water-immiscible organic solvent during microwave treatment
generated by a method including
Production method:

(In the formula, R ¹ represents OH or O-. R. ² and R ³ are the same or different, hydrogen atom, single bond or OCH ₃ shows. "O-" means that the oxygen atom (O) is single bonded to another benzene ring or aliphatic carbon atom). A method for producing an antiviral agent effective against viruses of the Caliciviridae or Orthomyxoviridae family, comprising a lignin decomposition product containing at least one partial structure represented by the following formula (1),
The lignin decomposition product is
a step of digesting raw material sugarcane bagasse or napier grass with dilute sulfuric acid at a temperature between 150°C and 200°C;
Step b of performing saccharification treatment on the digested product obtained in step a using cellulase;
Step c of converting sugar into ethanol by performing ethanol fermentation on the saccharified product obtained in step b;
step d of fractionating a solution fraction from the ethanol fermentation product obtained in step c;
Step e of separating the distillation residue from which ethanol has been removed from the solution fraction obtained in Step d into solid and liquid;
Step f of concentrating the supernatant obtained by solid-liquid separation in Step e.
generated by a method including
Production method:

(In the formula, R ¹ represents OH or O-. R. ² and R ³ are the same or different, hydrogen atom, single bond or OCH ₃ shows. "O-" means that an oxygen atom (O) is single bonded to another benzene ring or aliphatic carbon atom). The manufacturing method according to claim 2 or 3, which is effective against norovirus of the Caliciviridae family and influenza viruses (types A, B, and C) of the Orthomyxoviridae family.