JP2008031071A - Antitumor agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antitumor agent as a plant component, having a higher antitumor activity and less adverse effects. <P>SOLUTION: This antitumor agent contains (a) a plant of Maliaceae or its extract, (b-1) a plant of Amarylidaceae or its extract and/or (b-2) a plant of Apocynaceae or its extract. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an antitumor agent, particularly 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. Extracts from natural products are superior to synthetic drugs in terms of side effects. For example, taxol has been discovered from Western yew and is used as an active ingredient of anticancer agents (Patent Documents 1 and 2). Side effects are still significant.
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 ( Patent Document 3), Antitumor Agent Containing Amaryllidaceae Plant, Hydrangeaceae Plant or Extract thereof (Patent Document 4) and Antitumor Agent Containing Oleander Plant, Lily Family Plant or Extract thereof (Patent Document) A patent application has been filed for 5).

JP 63-30478 A Japanese Patent Laid-Open No. 7-233064 JP 2004-256426 A JP 2004-300082 A WO2005087246

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.

Among the plant components having antitumor activity found so far, the present invention is synergistic when combined with an Agrobiaceae plant or an extract thereof and / or an Oleander plant or an extract thereof. It was made based on the knowledge that the antitumor effect is further improved by the effect.
That is, the present invention is characterized in that it comprises (a) a plant belonging to the family Sendanidae or an extract thereof, and (b-1) a plant belonging to the family Amaryllidaceae or an extract thereof, or (b-2) an oleander plant or an extract thereof. An antitumor agent is provided.
The present invention is also characterized in that it comprises (a) a plant belonging to the family Sendanidae or an extract thereof, (b-1) a plant belonging to the family Amaryllidaceae or an extract thereof, and (b-2) an oleander plant or an extract thereof. An antitumor agent is provided.
The present invention also comprises (a) a plant belonging to the family Sendanidae or an extract thereof, (b-1) a plant belonging to the family Amaryllidaceae or an extract thereof and / or (b-2) an oleander family plant or an extract thereof. And provide food.

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 and berries of the gypsidaceae plant, for example, after being dried and pulverized, or in an undried raw state, with water, such as distilled water or ion-exchanged water, or 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, Narcissus tazetta L., Daffodil (Lycoris traubii Hayward), Lycaris radiata 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 bulbs 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 bulbs of the Amaryllidaceae plants are, for example, dried, crushed, or in an undried raw state, with water, for example, distilled water or ion-exchanged water, or hydrophilic. Alternatively, the liquid itself extracted with a hydrophobic organic solvent or a dried product thereof can be used.

Apocynaceae used in the present invention is a plant belonging to the order of Gentianales, Apocynales, and includes Nerium Oleander L., Allamanda Oenotheraefolia Pohl, Plumeria Mexicana, Plumeria Mexicana Periwinkle (Lochnera), Nerium olender L. cv Variegatum, Acon, Cerbera manghas L., Thevetia peruviana L., Mandevilla splendens Woodson, Mandevilla splendens Woodson (Apocynum). Of these, oleander (Nerium Oleander L.) is preferred. Also, iricho oleander, acon, okinawa oleander, and kibana oleander are preferable because they have an activity of 50% cell lethality of log10 ^4.5 or more.
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 oleanders, for example, after drying, pulverizing, or in an undried raw state, 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.

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.

In addition, when it is set as the antitumor agent containing the said plant or its extract, in addition to these, various pharmacologically acceptable substances for formulation, for example, an excipient | filler, a diluent, a disintegrating agent, binder, coating | covering Agents, lubricants, lubricants, lubricants, flavors, sweeteners, solubilizers and the like can be included as adjuvants. 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 of the present invention is preferably by oral administration, 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 per 1 kg body weight.
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 anti-tumor activity. Although the said plant in food or its extract is not specifically limited, It is good to contain about 0.01-5 mass%.
According to the present invention, an excellent antitumor agent is provided for various tumors, particularly colon cancer, stomach cancer, lung cancer, brain tumor and renal cancer.
EXAMPLES Next, an Example demonstrates this invention in detail.

Example 1
Extraction of plant components
(a) The leaves of Sendai (Melia azedarach L.) were collected and weighed in an undried raw state, and then dried overnight in a dryer at 60 to 65 ° C. The dried leaves were placed in a dashi bag and 11 times (by weight) hot water was added and immersed for 3 hours. Thereafter, the bag was taken out and dehydrated with a commercially available washing machine to obtain an extract. This extract was dispensed into a plastic centrifuge tube, and centrifuged at 6,500 rpm for 30 minutes using a centrifuge, and the resulting supernatant was used as the total crude crude 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.
(b) After rhizomes of Lycoris traubii Hayward were weighed in a raw state, they were cut into 2 to 3 mm and dried with a dryer at 60 to 65 ° C. The dried leaves were placed in a dashi bag and 11 times (by weight) hot water was added and immersed for 3 hours. Thereafter, the bag was taken out and dehydrated with a commercially available washing machine to obtain an extract. This extract was dispensed into a plastic centrifuge tube, and centrifuged at 6,500 rpm for 30 minutes using a centrifuge, and the resulting supernatant was used as the crude fraction of shochu daffodil. In addition to component extraction with hot water, solvents such as chloroform, isopropyl alcohol, benzene and ether were also used in the extraction experiment.
(c) Nerium indicum Mill leaves were collected and weighed in a dry state, and then dried overnight in a dryer at 60 to 65 ° C. The dried leaves were placed in a dashi bag and 11 times (by weight) hot water was added and immersed for 3 hours. Thereafter, the bag was taken out and dehydrated with a commercially available washing machine to obtain an extract. This extract was dispensed into a plastic centrifuge tube and centrifuged at 6,500 rpm for 30 minutes using a centrifuge, and the resulting supernatant was used as a crude fraction component of oleander. 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 the crude fraction total component In order to remove the toxic component from each crude fraction total component 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). The liquid was fed while being adjusted to (inlet pressure: 0.5-4 kg / cm ² , outlet pressure: 0.2-1 kg / cm ² ), and the liquid coming out 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
Comparative Control As a comparative control, commercially available mitomycin C (Kyowa Hakko) that is already widely used as an anticancer agent was dissolved in PBS so as to be 1 mg per ml.
Cells and cultured human colorectal cancer cells (HT-29), gastric cancer cells (MKN1) and lung cancer cells (A549) received a grant from Dr. Takao Yamori of Molecular Chemistry Department, Cancer Chemotherapy Center, Cancer Research Society, respectively. The cells were maintained in a 25 cm ² flask (IWAKI) using a commercially available RPMI 1640 (GIBCO) medium containing 5% fetal calf serum.
HT-29, MKN1 and A549 cells were cultured in RPMI 1640 containing 5% fetal calf serum in a plastic petri dish (IWAKI) having a culture diameter of 100 mm for evaluation. After 2 days, the cells were washed with minus phosphate buffered saline (-PBS). ), And trypsin-EDTA (GIBCO) was added thereto to disperse the cells. Then, the cells were suspended in RPMI 1640 containing 5% fetal calf serum and the number of cells was calculated. Prepare the above three cell concentrations to 1 x 10 ⁵ cells / ml, dispense 0.1 ml per well of a 96-well (well) plastic cell culture plate (IWAKI), and incubate at 37 ° C for 24 hours did.

Evaluation methods
In vitro evaluation Sendenda, Drosophila and Oleander components and control mitomycin C diluted with GIBCO's Minimum Essential Medium (MEM) without serum to evaluate from 10 ^-1 to 10 ^-8 Used for. 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, add 200 μl of tap water to wash and dry the plate, add 50 μl of sulfodamine solution to each well, let stand for 10 minutes, and then wash by adding 100 μl of 1% acetic acid to dry the plate. It was. Finally, 150 μl of 10 mM Tris [Tridr [hidroxymethyl] aminomethane) was added, shaken at 750 rpm for 5 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)

Evaluation in Experimental Animals (In Vivo) An in vivo antitumor effect test of a test subject on three types of human cancer cells (HT-29, MKN1 and A549) was performed using experimental animals. The experimental animals were 4-5 week old female nude mice (BALB / c nu / nu) purchased from Japan SLC.
Preparation and transplantation of transplanted cells The above three types of cultured cells were prepared to 2 × 10 ⁷ cells / ml, and 0.05 ml was transplanted subcutaneously into the right back of mice. After transplantation, when the tumor reached a size that could be confirmed, the tumor volume was measured with a digital caliper, and those that reached 50 to 150 cm ³ were divided into groups so as to reduce variation, and administration of the subject was started. Tumor volume was calculated as follows.
Tumor volume = 1/2 x major axis x minor axis ²
Administration of subject and tumor measurement The dosage was 0.1 ml per 10 g of mouse body weight, and oral administration was performed daily for 28 days using an oral sonde. Tumor volume was measured once every 4 days.
Evaluation of effect The degree of tumor growth was evaluated using the tumor growth rate. Tumor growth rate was calculated as follows.
Tumor growth rate = tumor volume on day X / tumor volume on day 0 The antitumor effect was determined by determining the ratio of the subject administration group to the tumor growth rate of the control group.

Table 1 summarizes the killing effects of the anticancer components contained in Sendan, Dermatitis and Oleander, which excludes side-effect substances, on colon cancer, stomach cancer and lung cancer cells.

  From the results in Table 1, in the case of Sendan, the 50% cell lethal effect (log10-n) on colon cancer (HT-29), stomach cancer (MKN1), and lung cancer (A549) was 3.43, 2.31, and <2, respectively. . The value of each activity varied slightly during sample preparation, but in any case the degree of action on the three cancer cells was never high in this experimental system. On the other hand, with cellulose alone, the cell death rate for colorectal cancer is 4.11, gastric cancer is 3.66 and lung cancer cells are 3.88, but when used together, the cell death rate is 4.41, gastric cancer is 3.94 and lung cancer. The number of cells was 4.10, indicating that the effect on cancer cells was enhanced from 10 times to nearly 100 times that of Sendan alone. It was also interesting to note that the activity of Schizophyllum alone increased the effect on colon cancer and gastric cancer cells slightly by adding the sendan component. This is because it was expected that the activity of daffodil was reduced by dilution with the sendan component, but it was significant that an upward trend was recognized. This is considered to be a synergistic effect of the two components. Furthermore, when oleander components were added to the sendan, the same effect was observed, and the activity increased from nearly 100 times to over 1,000 times. It was also confirmed that the activity was enhanced compared to the oleander component alone. Considering this, the enhancement of activity by mixing three kinds of sendan, daffodil, and oleander is, for example, a high value of 4.70 for colorectal cancer and 4.67 for gastric cancer is due to the synergistic effect of anticancer components. it is obvious.

The anticancer spectrum is shown in Table 2.

  From Table 2, it can be seen that the anticancer spectrum is expanded by mixing anticancer components. That is, the action range of each component of Sendan on each cancer cell is 3 out of 5 types of colorectal cancer (KM-12, HT-29, HCT-116), 2 out of 5 types of lung cancer (DMS273, DMS114), It was shown to have a narrow spectrum such as 1 of 6 types of gastric cancer (MKN45), 1 of 2 types of brain tumors (SF-295), and 1 of 2 types of kidney cancer (RXF-631L) It was found that the addition of the Daffodil component killed all 20 cancer cells used. In addition, it was shown that when the sendan component and oleander component were mixed, they acted on 19 of 20 types of cancer cells. From the above, it is clear that the anti-cancer effect not only strengthens the activity by changing the combination of the three anti-cancer components, but also significantly increases the spectrum of anti-cancer effect. It was.

Pathological analysis of anticancer mechanisms
At the end of the in vivo evaluation test, nude mice used in the experiment were euthanized under anesthesia, and the transplanted tumor was collected, fixed with formalin or glutaraldehyde, and used for pathological analysis. The material fixed in formalin for 3 to 4 days was washed with water, dehydrated and permeable, embedded in paraffin, sliced with a microtome, stained with hematoxylin and eosin, and observed with an optical microscope. If necessary, immunostaining with anti-Ki67 antibody or TUNEL staining for apoptosis detection was performed. The material fixed with glutaraldehyde was embedded with Epon resin, sliced with an ultramicrotome, and used for electron microscope observation. The obtained electron micrographs are shown in FIGS.
FIG. 1 is a morphological examination of a tumor of a mouse that was killed by treatment with the sendan component, and a number of lymphoid cells accumulated around the killed tumor. Most of these cells were found to be natural killer (NK) cells. FIG. 2 is a morphological examination of cancer cells treated with Sendan components. As is clear from the figure, it became clear that the nuclear division of cancer cells was stopped, and that these cells finally had the fate of apoptosis (cell death). FIG. 3 shows human gastric cancer formed in a mouse treated with an oleander component, but it was found that the dead cancer cells had the DNA completely destroyed as indicated by the arrows.
From these results, it became clear that the combined use of several partially purified plant anti-cancer components enhanced the anti-cancer activity and greatly expanded the anti-cancer spectrum. It became clear that

It is an electron micrograph of the NK cell observed in the transplanted gastric cancer cell (MKN1) of the Sendan administration group. It is an electron micrograph showing the fission inhibition (arrow) observed in the transplanted colon cancer cells (HT-29) in the Sendan administration group. It is an electron micrograph which shows the apoptosis (arrow) observed in the transplanted gastric cancer cell (MKN1) of the oleander administration group.

Claims (11)

  An antitumor agent comprising: (a) a plant belonging to the family Sendanidae or an extract thereof; and (b-1) a plant belonging to the family Amaryllidaceae or an extract thereof or (b-2) an oleander plant or an extract thereof.   An antitumor agent comprising (a) a plant belonging to the family Sendanidae or an extract thereof, (b-1) a plant belonging to the family Amaryllidaceae or an extract thereof, and (b-2) an oleander plant or an extract thereof.   3. The antitumor agent according to claim 1 or 2, wherein the extract of the plant family is an aqueous extract or an organic solvent extract of leaves, fruits, branches or bark of the plant family.   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, fruits, branches or bark of the Amaryllidaceae plant.   The antitumor agent according to claim 1 or 2, wherein the extract of the oleander plant is an aqueous extract or an organic solvent extract of leaves, berries, branches or bark of the oleander plant.   4. The antitumor agent according to claim 1, 2 or 3, wherein the plant family is Sendang.   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 oleander family is Oleander.   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 for treating colorectal cancer, stomach cancer, lung cancer, brain tumor or renal cancer.   A food comprising (a) a plant belonging to the family Sendanidae or an extract thereof, (b-1) a plant belonging to the family Amaryllidaceae or an extract thereof and / or (b-2) an oleander plant or an extract thereof.

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