WO2018088516A1

WO2018088516A1 - Anti-tumor agent

Info

Publication number: WO2018088516A1
Application number: PCT/JP2017/040575
Authority: WO
Inventors: 国昭根路銘; 雅山本; 拓呉羽; 令子根路銘
Original assignee: 有限会社生物資源研究所
Priority date: 2016-11-10
Filing date: 2017-11-10
Publication date: 2018-05-17
Also published as: JP2018188480A; JPWO2018088516A1; JP6443872B2

Abstract

The present invention addresses the problem of providing: an anti-tumor agent and a food, each of which comprises a plant-derived component and has a higher anti-tumor activity and less adverse side effects; and an interleukin-12 inducer or activator, a cell necrosis factor α inducer, a DNA synthesis inhibitor and an autophagy inducer, each of which contains a plant-derived component as an active ingredient. The present invention relates to an anti-tumor agent and a food, each of which comprises (a) a plant belonging to the lily family or an extract thereof and (b-1) a plant belonging to the Amaryllidaceae family or an extract thereof and/or (b-2) a plant belonging to the Meliaceae family or an extract thereof, and also relates to an interleukin-12 inducer or activator, a cell necrosis factor α inducer, a DNA synthesis inhibitor and an autophagy inducer, each of which contains a plant belonging to the Meliaceae family or an extract thereof.

Description

Antitumor agent

The present invention relates to an antitumor agent, in particular, an antitumor agent containing a plant-derived component and having an excellent effect on human colon cancer, stomach cancer, lung cancer, brain tumor and kidney cancer, and food.

Cancer continues to exist as a serious disease as medical technology advances and aging is renewed. Of course, cancer treatment technology has greatly advanced, and many medicines based on synthetic medicines, Chinese medicines, extracts from natural products and the like have been developed. In contrast to synthetic drugs, extracts from natural products are excellent in terms of side effects. For example, taxol has been discovered from western yew and is used as an active ingredient of anticancer agents (Japanese Patent Laid-Open No. 63-30478 and However, the side effects are still large.
On the other hand, the present inventor pays attention to plant components, finds that the components contained in a specific plant have excellent antitumor, and contains an antitumor agent containing a Sendanid plant or an extract thereof ( JP-A-2004-256426), Amaryllidaceae plant, Hydrangeaceae plant, or an antitumor agent containing the extract thereof (JP-A-2004-300082), Oleander plant, Lily family plant or an extract thereof. Containing an antitumor agent (WO2005087246), and an antitumor agent containing a Sendamaceae plant or an extract thereof, an Amaryllidaceae plant or an extract thereof, and / or an Oleander plant or an extract thereof (JP 2008-031071 A) No. publication).

An object of the present invention is to provide an antitumor agent which is a plant component and has higher antitumor activity and less side effects.
Another object of the present invention is to provide a food containing plant components.
Another object of the present invention is to provide an interleukin 12 inducer or activator, a cell necrosis factor α inducer, a DNA synthesis inhibitor and an autophagy inducer containing a plant component as an active ingredient.

Among the plant components having antitumor activity found so far, the present invention is a combination of a liliaceae plant or an extract thereof, and an amaryllidaceae plant or an extract thereof, and / or a Sendai plant or an extract thereof. This is based on the knowledge that the antitumor effect is further improved by the synergistic effect.
That is, the present invention is characterized by containing (a) a lily family plant or an extract thereof, and (b-1) an Amaryllidaceae plant or an extract thereof, or (b-2) a Sendai family plant or an extract thereof. An antitumor agent is provided.
The present invention is also characterized in that it contains (a) a lily family plant or an extract thereof, (b-1) an Amaryllidaceae plant or an extract thereof, and (b-2) a Sendai family plant or an extract thereof. An antitumor agent is provided.
The present invention is also characterized by containing (a) a lily family plant or an extract thereof, (b-1) an Amaryllidaceae plant or an extract thereof, and / or (b-2) a Sendai family plant or an extract thereof. And provide food.
Among the plant components having antitumor activity that have been found so far, the present invention is that the Sendanid plant or an extract thereof has an interleukin 12 inducing action, an activator action, a cell necrosis factor α inducing action, a DNA synthesis inhibiting action. And found to have an autophagy-inducing action.
That is, the present invention provides an interleukin-12 inducer or activator characterized by containing a ginseng plant or an extract thereof.
The present invention also provides a cell necrosis factor α-inducing agent characterized in that it contains a Sendanid plant or an extract thereof.
The present invention also provides a DNA synthesis inhibitor characterized by containing a Sendanid plant or an extract thereof.
The present invention also provides an autophagy induction characterized in that it contains a Cypridaceae plant or an extract thereof.

The expression level of IL-12 mRNA in macrophages treated with PBS or Sendan extract (MLE) is shown. The protein expression level of TNF-α in macrophages treated with PBS or Sendan extract (MLE) is shown. It is the figure which showed the ratio of each period of the cell cycle in the MKN1 cell (gastric cancer cell) processed with Sendan extract or mitomycin C. It is the figure which showed the result of the Western blot analysis of the cutting | disconnection type | mold PARP and cleaving type | mold caspase (TM) 3 (apoptosis parameter | index) in the MKN1 cell processed by Sendan extract (MLE) or mitomycin C treatment. It is a photograph of the transmission electron microscope (TEM) which shows the change of the intracellular structure of the cell processed with the sendan extract (MLE). The left figure shows MKN1 cells not treated with Sendan extract (control), the middle figure shows MKN1 cells treated with Sendan extract, and the right figure is an enlarged view of the square part in the middle figure. It is the figure which showed the result of the Western blot analysis of LC3 * I and LC3 * II (index of autophagy) in MKN1 cell processed with Sendan extract (MLE) or mitomycin C.

The liliaceae plant used in the present invention is a plant belonging to the order Lilyceae (Liliaceae), and examples thereof include Yucca Gloriosa L., Chionoqraphis, and lily of the valley (Convallaria). Of these, Yucca Gloriosa L. is preferred.
In the present invention, leaves, flowers, berries, stems and (bulb) roots of these plants can be used. Of these, stems or leaves are preferably used. When using the plant itself as an active ingredient of an antitumor agent, it is preferable to dry these and then finely pulverize them.
In the present invention, the leaves, flowers, berries, stems and (bulb) roots of the lily family plants, for example, after being dried and pulverized, or in an undried raw state, are water, such as distilled water or ion-exchanged water. Alternatively, the liquid itself extracted with a hydrophilic or hydrophobic organic solvent or a dried product thereof can be used.

Amaryllidaceae used in the present invention is a plant belonging to the order of Amaryllidales, and includes Agapanthus, Allium, Galanthus, Amarylilis Crinum, Zephyranthes, Haemanthus), Hymenocallis, Lycoris, Amaryllis (Hippeastrum) and Narcissus (Narcissus). More specifically, the narcissus (Narcissus tazetta L.), the Japanese daffodil (Lycoris traubii Hayward), the Japanese amber (Lycoris radiata Herb.), The Sakuya yuri (Hymenocallis americana Roem.), The tamasuda (Zephyranthes candida Herb.) Hippeastrum reticulatum Herb. Var striatifolium Herb. tuberosa L., Agave attenuata Salm-Dyck, Agave horrida Lam., Filius anusifolia Haw. cv. Marginata, Agave geminiflora Ker-Gaul Agave americana L.). Among these, narcissus (Narcissus tazetta L.), daffodil (Lycoris traubii Hayward), and Japanese Aedes (Lycoris radiata Herb.) Are preferable, and daffodil is particularly preferable.
In the present invention, leaves, flowers, stems and (bulb) roots of these plants can be used. When using the plant itself as an active ingredient of an antitumor agent, it is preferable to dry these and then finely pulverize them.
In the present invention, the leaves, flowers, stems and (bulb) roots of the Amaryllidaceae are dried, crushed, or in an undried raw state, with water, for example, distilled water or ion exchange water, Alternatively, the liquid itself extracted with a hydrophilic or hydrophobic organic solvent or a dried product thereof can be used.

The Sendai family used in the present invention is a plant belonging to the family Medeliaceae (Meliaceae), which is mainly an evergreen or deciduous tall tree having a feathery compound leaf at a thick branch tip and having a conical inflorescence. There are also. Of these, it is particularly preferable to use Sendan (Melia Azedarach L. or Melia Azedarach var. Subtripinnata). In the present invention, leaves, stems, branches, bark, and fruits of the family Sendanidae can be used. A root bark can also be used. In the case of using the Sendanid plant itself as an active ingredient of an antitumor agent, it is preferable to dry these and then finely pulverize them.
In the present invention, the leaves, stems, branches, bark, or berries of the ginseng plant are dried with water, for example, distilled water or ion-exchanged water, after being dried, pulverized, or in an undried raw state. Alternatively, the liquid itself extracted with a hydrophilic or hydrophobic organic solvent or a dried product thereof can be used.

Examples of the organic solvent used for extraction include ethyl acetate, carbon tetrachloride, chloroform, dichloromethane, methanol, ethanol, (iso) propyl alcohol, butanol, acetone, or DMSO. Here, the hydrophilic solvent can also be used in a water-containing form. The amount of water or solvent to be used can be arbitrarily determined, but it is preferably used in an amount of 1/5 to 5 times, particularly preferably in an equivalent amount. The extraction is preferably performed at a temperature of 60 ° C. or lower, more preferably at room temperature, and particularly with stirring by a mixer or the like.
The molecular weight of the active ingredient in the extract is preferably less than 300,000, more preferably 100,000 or less, and most preferably 10,000 or less. In particular, the active ingredient in the extract of the family Sendanidae preferably has a molecular weight of 10,000 or less, more preferably a molecular weight of 4,000 to 10,000 or 3,000, most preferably about 5,000.
The water or solvent extract can be used in the liquid state as it is, but it can also be dried and used in a solid form such as powder or granule.
In the present invention, it is particularly preferable to use water or a solvent extract, more preferably purified with a molecular sieve membrane, and particularly preferably purified with an ultrafiltration membrane having a molecular weight of 10,000. This is because the sendan component having a molecular weight of 10,000 or less retains high antitumor activity despite its low toxicity.
In the present invention, the component (a) and the component (b-1) and / or (b-2) can be combined in any proportion, but the component (a) and the component (b-1) or (b-2) ) Is preferably set to 1 / 0.01 to 1/100, more preferably 1 / 0.1 to 1/10 (mass ratio).
Further, it is preferable that the component (a) / component (b-1) / (b-2) is from 1 / 0.01 / 0.01 to 1/100/100, more preferably 1 / 0.1 / 0.1 to 1/10/10 (mass ratio).

In addition, in the case of using an antitumor agent or interleukin-12 inducer or activator containing the above plant or an extract thereof, a cell necrosis factor α inducer, a DNA synthesis inhibitor and an autophagy inducer, Various pharmaceutically acceptable pharmaceutical substances such as excipients, diluents, disintegrants, binders, coatings, lubricants, lubricants, lubricants, flavors, sweeteners, solubilizers, etc. It can be included as an adjuvant. Specific examples include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol, glycerol and the like.
The antitumor agent or the like of the present invention is preferably administered orally, but is not limited thereto. The antitumor agent of the present invention is preferably used in an amount of about 0.25 to 2 g in terms of dry weight per kg of body weight.
The antitumor agent of the present invention can be applied to a wide range of animals such as mammals, for example, humans, domestic animals such as cows and pigs, pets such as dogs and cats.
The antitumor agent of the present invention can be applied to a wide range of animals such as mammals, for example, humans, domestic animals such as cows and pigs, pets such as dogs and cats.
Moreover, when it is set as the foodstuff containing the said plant or its extract, it can be set as the health food or the functional food which has antitumor activity. The plant or the extract thereof in the food is not particularly limited, but is preferably contained in an amount of about 0.01 to 5% by mass.
According to the present invention, an excellent antitumor agent is provided for various tumors, particularly colon cancer, stomach cancer, lung cancer, brain tumor, kidney cancer and the like.
EXAMPLES Next, an Example demonstrates this invention in detail.

<Extraction of plant components>
(a) The leaves and stems of the liliaceae that grow naturally on the main island of Okinawa Prefecture were weighed in a dry state and chopped to a width of several millimeters with a knife. An equal weight of distilled water at room temperature was added thereto, and the mixture was treated at 10,000 rpm for 10 minutes using a commercially available large mixer. This mixture was dispensed into a 50 ml plastic centrifuge tube and centrifuged twice at 7,000 rpm for 20 minutes using a Beckman centrifuge (GS-15R). After passing through the filter paper, it was further subjected to a test after sterilization by filtration with Sartorius minisart (0.45 μm). In addition to component extraction with distilled water, solvents such as chloroform, isopropyl alcohol, ethyl acetate, ethanol, methanol, and dichloromethane were also used in the extraction experiment.
(b-1) The bulbs of Lycoris traubii Hayward that grows naturally on Kumejima, Okinawa Prefecture were weighed in a raw state and cut into a few millimeters wide with a knife. An equal weight of distilled water at room temperature was added thereto, and the mixture was treated at 10,000 rpm for 10 minutes using a commercially available large mixer. This mixture was dispensed into a 50 ml plastic centrifuge tube and centrifuged twice at 7,000 rpm for 20 minutes using a Beckman centrifuge (GS-15R). After passing through the filter paper, the obtained supernatant was used as a crude fraction total component of Daffodil. In addition to component extraction with distilled water, solvents such as chloroform, isopropyl alcohol, ethyl acetate, ethanol, methanol, and dichloromethane were also used in the extraction experiment.
(b-2) The leaves of Sendai (Melia azedarach L.) native to the main island of Okinawa were collected, washed with tap water, and dried overnight at 65 ° C. To the obtained 1.1 kg dried dry leaves, 5.5 L of hot water at 60 ° C. was added and immersed for 3 hours for extraction treatment. The extract was then dispensed into a plastic centrifuge tube and centrifuged at 9,000 rpm for 30 minutes using a centrifuge, and the resulting supernatant was used as the crude crude total component. In addition to component extraction with hot water, solvents such as chloroform, isopropyl alcohol, benzene and ether were also used in the extraction experiment.

<Purification from total components of crude image>
In order to remove the toxic components from the crude fractions of Daffodil and Sendang obtained by the above method, purification was performed using a molecular sieve membrane. For purification, a Pellicon 2 mini holder (MILLIPORE) is equipped with an ultrafiltration membrane (MILLIPORE) with a molecular weight of 10,000, and the total components of the crude fraction are adjusted with the peristaltic pump (MILLIPORE) within the operating conditions of the operating manual. The liquid was fed while adjusting the pressure (inlet pressure: 0.5-4 kg / cm ² , outlet pressure: 0.2-1 kg / cm ² ), and the liquid discharged from the permeate side outlet was collected. Furthermore, after filter sterilization with Sartorius mini-sarts (0.22 μm), it was used as a test sample in the following tests.

<Measurement of anticancer activity>
<Comparison>
As a comparative control, commercially available mitomycin C (Kyowa Hakko), which is already widely used as an anticancer agent, was dissolved in a commercially available RPMI 1640 (GIBCO) medium at 1 mg per ml.
≪Cell and culture≫
Human colon cancer cells (HT-29), gastric cancer cells (MKN1) and lung cancer cells (A549) were purchased from JCRB (Japanese Collection of Research Bioresources) cell bank. Each cell was maintained in a 25 cm ² flask (IWAKI) using a commercially available RPMI 1640 (GIBCO) medium containing 5% fetal bovine serum.
≪Culture of cells for evaluation≫
The HT-29, MKN1 and A549 cells are cultured in a 25 cm ² flask. After 3 days, the cells are washed with minus phosphate buffered saline (-PBS), and trypsin-EDTA (GIBCO) is added to the cells. And then suspended in RPMI 1640 medium containing 5% fetal calf serum to count the number of cells. The above three cell concentrations were determined to be 10 ⁵ cells / ml, and 0.1 ml was dispensed per well of a 96-well (well) plastic cell culture plate (IWAKI). For example, in each plate, row A and row H, number plate 1 and row 2 are medium only, and each test cell is dispensed from row 2 to row 11, row B to row G at 24 ° C. Incubate for hours.

[Example 1: Evaluation of activity of antitumor agent according to the present invention for each cancer cell (in vitro)]
Using each of the extracted plant fluids (Atsubatakigayoran extract, Daffodil extract and Sendan extract), Sendai extract 50 μl, Daffodil extract 50 μl, Atsubikigayoran extract 50 μl, Sendai extract 25 μl and Dendrobium extract An equal volume mixture of 25 μl of the extract, 25 μl of Sendan extract and 25 μl of the extract of Atsumi kigayoran, 50 μl of Sendan extract, 25 μl of the daffodils extract, and 25 μl of Atsumi kigayoran were prepared. Each prepared extract was diluted with RPMI 1640 medium without serum from 10 ^-1 to 10 ^-4 and used for evaluation. For each dilution point of each specimen, 100 μl per well was added to two wells of the plastic plate in which the cells were cultured on the previous day. The sample was dispensed and cultured for 2 days, 50 μl of 50% trichloroacetic acid (TCA) was added to each well, and the mixture was allowed to stand at 4 ° C. for 1 hour. Next, after washing with tap water, the plate was dried, and 50 μl of sulforadamine staining solution dissolved in 1% acetic acid was added to each well and allowed to stand for 10 minutes. Finally, 10 mM Tris [hidroxymethyl] aminomethane solution 150 μl was added, shaken at 750 rpm for 20 minutes, and the absorbance was measured at a wavelength of 525 nm. Cell viability was calculated as follows.
Cell viability = 100 × (average absorbance of subject at each dilution point−average absorbance of medium control corresponding to each dilution point) / (average absorbance of cell control−average absorbance of medium control corresponding to each dilution point)

Colorectal cancer of anticancer component contained in Sendan extract alone, Equivalent mixture of Daffodil extract and Sendan extract, and Equivalent mixture of Atsumiki Migayoran extract and Sendan extract, Table 1 summarizes the killing effect on gastric cancer and lung cancer cells. Table 2 shows the killing effect of mitomycin C as a comparative control. In Tables 1 and 2, “anticancer activity” was evaluated using as an index the dilution factor that kills 50% of cancer cells.

From the results of Table 1, Sendan alone had a high 50% cell killing effect on colorectal cancer, but the 50% cell killing effect on stomach cancer and lung cancer was not as high as the lethal effect on colorectal cancer. However, it has been shown that the effect on gastric cancer and lung cancer is enhanced to about 2.9-fold and about 23.0-fold, respectively, when adding Sekikisen to Sendan. In addition, it was shown that when Atsuba Kimigayoran was added to Sendang, the effect on gastric cancer and lung cancer was enhanced to about 4.7 times and about 18.9 times, respectively. When compared with the results of mitomycin C as a control shown in Table 2, it was shown that the effect on gastric cancer was particularly remarkably enhanced when S. edulis or Atsuba kigayoran was added to Sendan.

Table 3 shows the killing effect on colon cancer, gastric cancer, and lung cancer cells of anti-cancer components contained in an equivalent mixture of a daffodil extract alone or a mixture of sendan extract and daffodil extract from which side-effect substances are excluded. It summarizes and shows. In Table 3, “anticancer activity” was evaluated using as an index the dilution factor at which 50% of cancer cells were killed.

From the results in Table 3, the effect on colorectal cancer and gastric cancer is about 13.2 times and about 2.2 times, respectively, when Sendang is added to Sekizuisen, compared with the case of Sekizuisen alone. The activity was shown to be enhanced. Compared with the results of mitomycin C as a control shown in Table 2, it was shown that the effect on gastric cancer was particularly remarkably enhanced when Sendang was added to Drosophila.

Anti-cancer component contained in Atsuba Kimigayoran extract alone, Atsuba Kimigayoran extract and Sendan extract, and a mixture of Sendan extract, Daffodil extract and Atsuba Migayoran extract excluding side effect substances Table 4 summarizes the killing effect on colon cancer, stomach cancer, and lung cancer cells. In Table 4, “anticancer activity” was evaluated using as an index the dilution factor at which 50% of cancer cells were killed.

From the results in Table 4, it was shown that the effect on gastric cancer was enhanced by about 12.9 times when Sendang was added to Atsubakimigayoran compared to the case of Atsubakimigayoran alone. Moreover, it was shown that when Sendan and Duckweed are added to Atsumikimigayoran, the activity for gastric cancer is enhanced up to about 8.1 times. Compared with the results of mitomycin C as a control shown in Table 2, it was shown that the effect on gastric cancer was particularly remarkably enhanced when S. oryzae and / or Sendan was added to Atsumikimigayoran.

Table 5 summarizes the killing effect of the anticancer component contained in the mixture of Atsuba Kimigayoran extract, which is free of side-effect substances, and an equivalent mixture of Atsuba Kimigayoran extract and Daffodil extract, on colon cancer and gastric cancer cells. In Table 5, “anticancer activity” was evaluated using as an index the dilution factor that kills 50% of cancer cells.

From the results of Table 5, it was shown that the effect on gastric cancer was enhanced to about 2.2 times when adding Shochu daffodil to Atsuba Kimigayoran compared to the case of Atsuba Kimigayoran alone. Moreover, it was shown that the effect on colorectal cancer is enhanced up to about 1.4 times in the case of adding daffodils to Atsumikigayoran. Compared with the results of mitomycin C as a control shown in Table 2, it was shown that the effect on colorectal cancer was particularly enhanced when Scots narcissus was added to Atsumikimigayoran.

Table 6 summarizes the killing effect of anticancer components on colon cancer and gastric cancer cells contained in an equivalent mixture of daffodil extract alone, excluding admixtures of duckweed extract and Atsuba migayoran extract. . In Table 6, “anticancer activity” was evaluated using as an index the dilution factor at which 50% of cancer cells were killed.

From the results of Table 6, it was shown that the effect on colorectal cancer is slightly enhanced when adding Atsubakimigayoran to duckweed compared to the case of duckweed alone.

<Analysis of in vivo normalization mechanism by sendan component>
As described in Japanese Patent Application Laid-Open No. 2008-031071, which is a related application by the present inventor, the present inventor conducted a morphological examination of a tumor of a mouse killed by treatment with a sendan component. Many lymphoid cells accumulate in the vicinity of, and as a result of examination by an electron microscope, it is revealed that most of these cells are natural killer (NK) cells. Japanese Patent Application Laid-Open No. 2008-031071 also clarifies that the sendan component prevents nuclear division of cancer cells.
Therefore, in order to clarify the in vivo mechanism caused by Sendan, the present inventor (i) induction of interleukin (IL) 12 and tumor necrosis factor (TNF-α) by the Sendan component, (ii) DNA synthesis Experiments were performed to evaluate inhibition and (iii) autophagy that caused cell death.

Here, the background of this experiment will be described just in case.
(1) Overview of cytokines Cytokines are now applied to elucidation of etiology and treatment. In that sense, controlling cytokines has led to the treatment of certain diseases, such as viral diseases and cancer. One example is tumor necrosis factor (TNF-α), interleukin, which is also used for rheumatism and infectious diseases. Therefore, it is necessary to sort out how cytokines and interleukins are different. As the main explanation, those whose biological functions are clarified by one molecule are called interleukin (IL) and are currently classified from IL-1 to IL-29. Therefore, interleukin and TNF-α are “broadly defined cytokines”. The living body has a protective function against foreign microorganisms. Its contents correspond to the skin and mucous membrane that surround the surface of the body, various cellular factors distributed there, and their metabolites. These non-specific representative factors are macrophages that eat and swallow foreign factors and phagocytes called polymorphonuclear leukocytes (neutrophilic granulocytes). In addition to these phagocytes, there is a natural killer (NK) that is classically well known. These NK cells are a type of lymphocyte that kills and eliminates its own cancer cells and infected cells.

(2) Interleukin 12
Interleukin 12 (IL-12) is a type of substance that promotes activation of NK cells, called Natural Killer Stimulatory Factor (NKSF), and has come to be called interleukin. In other words, it was positioned as a new cytokine, determined as a protein molecule with a molecular weight of 70,000 (70 kDa), and this substance was called interleukin 12 (IL-12). Cells that produce it are macrophages, dendric cells, and polymorphonuclear leukocytes. This IL-12 promotes an inflammatory response and stimulates an immune response through the division and proliferation of T lymphocytes and NK cells. As a result, IL-12 was confirmed to have an antitumor effect on melanoma, reticulosarcoma cells, gastric cancer and the like.
Furthermore, IL-12, which is useful for the prevention and treatment of various diseases, is considered to have a significant effect on the treatment of parasitic diseases, viral diseases and bacterial diseases. In fact, it helps in the treatment and prevention of influenza viruses.
(3) Tumor Necrosis Factor (TNF-α)
TNF-α literally causes tumor necrosis, and the basis for this is that when a solid tumor-bearing mouse is inoculated with BCG and then inoculated with Gram-negative lipopolysaccharide (Lipopolysacharide: LPS), the center of the solid tumor Is called tumor necrosis factor because it causes bleeding and necrosis, and the tumor disappears completely. Subsequent induction of functional changes in many types of cells revealed a broad range of significant effects of TNF as a multifunctional cytokine. In other words, TNF has come to be understood as a cytokine widely involved in biological defense mechanisms and the constant prevention of the body during stress through inflammation.
TNF is a cytokine secreted from macrophages, but is a lymphotoxin (LT) secreted by liquefied lymphocytes and causes cancer cell injury. As a result, TNF-α has been used as a cancer treatment and a preventive agent for viral infections.

≪Immunological analysis≫
Since interleukin 12 and TNF-α are secreted from macrophages, mouse-derived macrophages (Sigma-Aldrich) J774A strain cells cultured in DMEM (Corning — 10-013-CVR) containing 10% fetal calf serum are 3 × 10 ⁵ Seeded in 6-well plastic plates at cell / well concentration. The next day, 10 μL of a 100-fold diluted solution of Sendan leaf extract (MLE), a 200-fold diluted solution, and LPS (Lipopolysacharide) derived from E. coli (E. coli: O55, Wako) were added. LPS was used as a positive control. RNA was extracted after 1-2 hours. This reaction solution was subjected to RTP-PCR using Prime Script RT reaction kit (Takara). The expression level of the gene was quantified using TaqMan Gene Expression Master Mix (Thermo Fisher) in this system. Propidium iodide staining was used for cell cycle analysis.
≪Immunoblot analysis≫
Cells were lysed with lysis buffer (containing 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, Roche complete protease inhibitor cocktail). Protein concentration was measured with a Bio-Rad-protein assay. Cell lysates were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon P, Merck Millipore). Next, the protein on the membrane was reacted with the primary antibody. Immunoreactive protein was reacted with horseradish peroxidase labeled anti-rabbit or anti-mouse IgG (GE Healthcare) and visualized by ECL detection system (GE Healthcare).

≪Cell cycle analysis≫
Cell cycle experiments were performed using the human cell line MKN1. MKN1 cells in RPMI 1640 medium at a density of 2 × 10 ⁵ cells / cell, with or without Sendan leaf extract (100-fold dilution, 1,000-fold dilution, 10,000-fold dilution), or presence of mitomycin C Seeded under (1 mg / ml of medium). Viable cells remaining 1 and 2 days after cell seeding were counted. For cell cycle analysis, cells were fixed and stained with PBS containing propidium iodide and RNase A. Cells were analyzed using FACS alibur and Cell Quest software (BD Bioscience). Apoptotic cells were evaluated by cell appearance using the amount of DNA in sub-G1 (amount of DNA less than 2N).
≪Transmission electron microscope (TEM) analysis≫
Cells were washed and fixed with 2.5% glutaraldehyde for 30 minutes at room temperature, then treated with 2% osmium tetroxide, dehydrogenated with a graded ethanol bath, and embedded in resin. A 50 nm ultrathin section was cut with a Leica EM UC6 Ultramicrotome, stained with 4% uranyl acetate and 2% lead citrate, and examined with a transmission electron microscope JEOL JEM-1230R (TEM) at an acceleration voltage of 100 kV.

[Example 2: Evaluation of induction of IL12 and TNF-α in cancer cells by Sendan component]
To verify the induction of IL-12 and TNF-α activity, mouse macrophage cells were treated with the sendan component in vitro as described above. In FIG. 1A, the expression level of IL-12 mRNA in PBS-treated macrophages (control) is shown. Lot. 28 and Lot. 29 are extracts obtained from Sendan collected in August 2013 and 2015, respectively. The IL-12 mRNA expression level in macrophages treated with an extract obtained from Sendang collected in August of 2012 is shown. From FIG. 1A, it can be confirmed that the mRNA expression level of IL-12 is increased by the sendan component. Similarly, TNF-α secretion activity was also confirmed in mouse macrophage cells treated with the sendan component in the culture medium. In FIG. 1B, lane 1 shows the protein expression level of TNF-α in PBS-treated macrophages (control), and lanes 2 to 4 correspond to Lot. 28, Lot. 29 and Lot. 29-1, respectively. Specifically,

lanes

2 and 3 are respectively in macrophages treated with an extract obtained from a sendan collected in August 2013 and an extract obtained from a sendan collected in August 2015. The protein expression level of TNF-α is shown, and lane 4 shows the protein expression level of TNF-α in macrophages treated with an extract subdivided from Lot. As can be seen from FIG. 1B, the activity of TNF-α increased by the sendan component was conspicuous compared to PBS treated macrophages (control).

[Example 3: Evaluation of inhibition of DNA synthesis in cancer cells by Sendan component]
In order to test whether apoptosis occurred in MKN1 cells by treatment with Sendan extract, the cell cycle was analyzed (FIG. 2). Mitomycin C, which is a representative substance for inhibiting DNA synthesis, is a positive control. The treatment with Sendan extract clearly stagnated the cell cycle progression of MKN1 cells, and the proportion of sub-G1 fraction increased in a dose-dependent manner. The sendan component also inhibited DNA synthesis in the G1 phase of the cell cycle and interfered with the mitosis process in the G2 / M phase. From this result, it was confirmed that the DNA synthesis in the cells was inhibited by the sendan component. Surprisingly, Western blot analysis revealed that no increase in truncated PARP and truncated caspase 3 (apoptosis index) was observed in Sendan-treated cells (FIG. 3). On the other hand, these truncated proteins were detected in cells treated with mitomycin C. This suggests that Sendan cell death occurs by a mechanism different from apoptosis.

[Example 4: Verification of autophagy causing cell death by Sendan component]
It was confirmed using a transmission electron microscope (TEM) whether the intracellular structure in the cells was affected by the treatment with the sendan component (FIG. 4). As a result, it was found that treatment with the sendan component induces autophagy in MKN1 cells, but does not show autophagosomes for MKN1 cells not treated with the control sendan component. It was also confirmed that LC3 I and LC3 II, which are autophagy markers, were significantly expressed in Sendan-treated cells (FIG. 5). These data suggest that cell death induced by treatment with the sendan component is due to autophagy.

From the above experimental results, it has been confirmed that the ginseng plant or its extract is useful as an interleukin 12 and / or cell necrosis factor α inducer or activator. In addition, it was confirmed that the Sendidae plant or an extract thereof is useful as a DNA synthesis inhibitor or an autophagy inducer.
Since autophagy has been suggested to be associated with cancer and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, the plant family or its extract is an effective means of treating these diseases. It can be. Here, examples of target mammals for the above diseases include a wide range of animals such as humans, domestic animals such as cows and pigs, and pets such as dogs and cats.
The present invention may also include the following aspects.
1. An interleukin 12 and / or a cell necrosis factor α inducer or activator containing a plant of the family Sendanidae or an extract thereof.
2. A DNA synthesis inhibitor containing a plant of the family Sendanid or an extract thereof.
3. An autophagy-inducing agent containing a Sendai family plant or an extract thereof.
4). The inducer or activation according to any one of the above aspects 1 to 3, wherein the extract of the plant family is an aqueous extract or organic solvent extract of leaves, stems, berries, branches, or bark of the plant family Agent or inhibitor.
5). The inducer, activator or inhibitor according to any one of the above embodiments 1 to 4, wherein the extract is purified by a molecular sieve membrane.

Claims

(a) a lily family plant or an extract thereof;
An antitumor agent comprising (b-1) an Amaryllidaceae plant or an extract thereof, or (b-2) a Sendamaceae plant or an extract thereof.
An antitumor agent comprising (a) a lily family plant or an extract thereof, (b-1) an amaryllidaceae plant or an extract thereof, and (b-2) a ginger family plant or an extract thereof.
3. The antitumor agent according to claim 1 or 2, wherein the lily family plant extract is a leaf, stem, fruit, (bulb) root, or flower aqueous extract or organic solvent extract of the lily family plant.
3. The antitumor agent according to claim 1 or 2, wherein the extract of the Amaryllidaceae plant is an aqueous extract or an organic solvent extract of leaves, stems, berries, (bulb) roots, or flowers of the Amaryllidaceae plant.
3. The antitumor agent according to claim 1 or 2, wherein the extract of the plant family is an aqueous extract or organic solvent extract of leaves, stems, fruits, branches or bark of the plant family.
The antitumor agent according to claim 1, 2 or 3, wherein the lily family plant is Atsuba Kimigayoran.
The antitumor agent according to claim 1, 2 or 4, wherein the Amaryllidaceae plant is Drosophila.
The antitumor agent according to claim 1, 2, or 5, wherein the plant of the family Sendanidae is Sendan.
The antitumor agent according to any one of claims 1 to 8, wherein the extract is purified by a molecular sieve membrane.
The antitumor agent according to any one of claims 1 to 9, which is used for treating colon cancer, stomach cancer, lung cancer, brain tumor or renal cancer.
A food comprising (a) a lily family plant or an extract thereof, and (b-1) an Amaryllidaceae plant or an extract thereof and / or (b-2) a Sendanid plant or an extract thereof.
An interleukin-12 inducer or activator characterized by containing a herbaceous plant or an extract thereof.
A cell necrosis factor α-inducing agent characterized by containing a Sendai family plant or an extract thereof.
A DNA synthesis inhibitor characterized by containing a Sendai family plant or an extract thereof.
An autophagy-inducing agent characterized by containing a Sendai family plant or an extract thereof.
The inducer according to any one of claims 12 to 15, wherein the extract of the family Sendanaceae is an aqueous extract or an organic solvent extract of leaves, stems, fruits, branches or bark of the family Sendanaceae, Activator or inhibitor.
16. The inducer, activator or inhibitor according to any one of claims 12 to 15, wherein the extract is purified by a molecular sieve membrane.