KR19990085642A - Cancer metastasis inhibiting composition - Google Patents

Abstract

The present invention relates to a composition having anti-cancer activity and anti-cancer metastasis, 30-40% by weight of Crestarae Appendiculatae Tuber, 30-40% by weight of Percicae Semen, 10-20% by weight of Coicis Semen , 1-8% by weight of Trichoicis Carapax, 1-8% by weight of Hippocampus and 1-8% by weight of Zedoariae Rhizoma, the herbal composition of the present invention is a collagen that plays a crucial role in metastasis of cancer. By effectively inhibiting intracellular production and activity of nausea (collagenase) can be usefully used as a prophylactic and therapeutic agent of various cancers.

Description

Cancer metastasis inhibiting composition

The present invention relates to a composition having an anticancer action and a cancer metastasis inhibiting action, and more particularly, 30-40 wt% of Crestarae Appendiculatae Tuber, 30-40 wt% of Percicae Semen, Coicis Semen 10 Regarding a composition having anticancer activity and cancer metastasis inhibiting activity, comprising 20% by weight, 1-8% by weight of Trichoicis Carapax, 1-8% by weight of Hippocampus and 1-8% by weight of Zedoariae Rhizoma will be.

Cancer is a genetic disease caused by mutations in genes such as proto-oncogenes and anti-oncogenes, and is caused by cell level. to be. Proto-oncogene products play a role in regulating the signaling system of cell division or differentiation by forming networks in cells. In other words, when their regulation is abnormal, the cell differentiation process is interrupted and indefinite division is continued. If the signal transduction pathway can be reduced normally using the herbal composition, excellent treatment with fewer side effects is possible. It is expected to be effective.

One of the most important biological properties of cancer is that it can cause metastasis, which is considered the biggest obstacle to treating cancer. In fact, about 60% of all solid tumor patients already have cancers that have been clinically metastasized at the time of primary tumor diagnosis, and it is well known that the major cause of death in most cancer patients is cancer metastasis. In metastasis, the invasion of local tissues of cancer is angiogenesis, which involves tumor vasculature, and the blood vessels newly formed by the tumor are easily invaded by cancer cells. . The process of cancer infiltration and metastasis also includes receptors on numerous cancer cell surfaces, such as, for example, laminin receptors required to adhere to the tissue matrix and basement membrane; Numerous enzymes required to dissolve normal tissue epilepsy, such as type IV collagenase, plasminogen activator and cathepsin B; Growth factors; Autocrine motility factor; And expression of cancer genes.

Meanwhile, MMP2 and MMP9, a type of collagenase, play an important role in metastasis of cancer cells, and their promoters have binding sites for AP-1 such as c-fos and c-jun. And jun increases expression of matrix metalloproteinase (MMP). Therefore, if one can inhibit the activity of MMPs, a kind of collagenase, or inhibit the expression of c-fos and c-jun, cancer metastasis can be prevented.

Studies on the mechanism of cancer development and cancer metastasis have been mainly conducted in the pure scientific aspects, and in recent years, researches using molecular biological methods have been actively conducted. There is a method for treating, preventing and inhibiting cancer from a molecular biological point of view, but there are methods for using antibodies or peptides, but the effects of these are being questioned, and the problem of effective drug delivery and in the body during administration There is a lot of research that needs to be solved, such as the problem of producing antibodies.

In real clinical trials, the combination of East-West medicine improves the 5-year survival rate of tumor patients and the relatively long survival of patients with cancer, suggesting that herbal medicines (ie, herbal medicines) act to prevent and delay cancer recurrence and metastasis. To explain. However, research on the prevention of cancer metastasis through Chinese medicine has not been actively conducted at home and abroad.

Therefore, the inventors of the present invention have been to select a herbal composition having an anti-metastasis effect to identify its effect and to analyze the specific mechanism of action, to explore the anti-cancer metastasis effect of the composition of the present invention, The present invention was completed by finding that the composition of the present invention has excellent anticancer action and anticancer action.

It is an object of the present invention to provide a composition having an anticancer action and a cancer metastasis inhibiting action.

1 shows the results of experiments inhibiting the collagenase of the composition of the present invention,

2 is a graph showing the results of a c-fos expression inhibition experiment of the composition of the present invention,

3 is a graph showing the results of inhibition test of TRE CAT of the composition of the present invention,

Figure 4 is a graph showing the results of experiments in vivo toxicity using the composition of the present invention,

5 is a graph showing the results of in vivo anti-cancer metastasis experiments of the composition of the present invention.

In order to achieve the above object, in the present invention, 30-40% by weight, 30-40% by weight of phosphorus, 10-20% by weight of Euiin, 1-8% by weight of Tortoiseshell, 1-8% by weight of hippocampus and 1-8% by weight of seahorse It provides a composition consisting of.

Hereinafter, the present invention will be described in detail.

The composition having the anticancer action and the cancer metastasis inhibiting effect of the present invention is 30-40% by weight, 30-40% by weight, 10-20% by weight of Euiin, 1-8% by weight of seaweed, 1-8% by weight of hippocampus It consists of 1-8 weight%. At this time, instead of living algae, Oledenlandiae Diffusae Herba or Lonicerae Flos can be used in the same amount as live mountain algae. Can be used.

Each component of the composition of the present invention is as follows.

Sanjago is a clearing detoxification drug belonging to the Orchidaceae family. It has the effect of clearing detoxification and sintering, so that it can be bitten by scabies, nagami, pharyngeal pain, poisonous snakes, insects or dogs. It has been used to treat patients.

Doin (활 仁) belongs to the rosacea (활 科) belonging to the vigor of the blood (Activ 血 祛瘀 藥), the blood-sucked up, yunjangtong stool (효능 通 便) efficacious in the menstruation, dysmenorrhea, It is mainly used to treat gonggaebi, jealousy injuries or constipation.

Ui-in is an Isusam medicinal herb belonging to the genus Poaceae.脚氣), urinary deficiency (小 便 不 利), lung 肺 癰 (옹) and janggong (치료) can be treated.

Tortoiseshell is an animal supplement medicine belonging to Trionychidae, which has the effect of consonant yangyang and yeonyanggyeon, so that fever caused by yin-hu Treat Zonga and others.

Hippocampus is an animal rejuvenating drug belonging to the group Syngnathidae. ), It is used to treat gonggai and changchang.

The extract is an active blood pill belonging to the Zingiberaceae family, and it is effective in swelling and dropping pain due to gonggai and vaginal injury. Treat pain

As can be seen from the above, each component of the composition of the present invention is used as a therapeutic agent for various diseases, but their anti-cancer action and cancer metastasis use has not been reported.

Most preferably, the composition of the present invention is composed of 36.2% by weight of acid, 36.2% by weight, 14.5% by weight of Euiin, 4.4% by weight of Tortoiseshell, 4.4% by weight of hippocampus and 4.4% by weight of seaweed.

The composition of the present invention is added to a solvent (80% methanol, 80% ethanol or water) by weighing each of the dried herbs described above, and chilled for one week, or extracted for 3-6 hours at a temperature of 100 ° C. in a water bath equipped with a reflux condenser. Then, this process is repeated 3 to 5 times, the extracts are combined, and then the solvent is completely removed from the vacuum distillation. A small amount of water is added to the dried product from which the solvent is completely dissolved, and then frozen at -80 ° C. and then freeze-dried.

The composition of the present invention may be administered orally, intravenously, intramuscularly, parenterally or rectally, and may be in the form of injections, capsules, tablets, dragees, suppositories, granules, solutions, suspensions, emulsions, suppositories, and the like.

The compositions of the present invention can be used in combination with pharmaceutically acceptable carriers such as organic or inorganic, solid, semisolid or liquid excipients in addition to the respective essential components, and if necessary, auxiliary substances, stabilizers, wetting or emulsifying agents, buffers, Other commonly used additives may be included in these pharmaceutical compositions.

The dosage of the composition of the present invention is 1 to 10 mg / kg for injection, and 10 to 100 mg / kg for oral administration.

Toxicity at the time of oral administration to the composition of the present invention as a result of oral administration up to 50 mg / ml was not toxic, but when administered 100 mg / ml showed weight loss due to toxicity from day 4.

As a result of testing the inhibitory effect of cancer metastasis using the composition of the present invention, the composition of the present invention has the effect of inhibiting the activity of collagenase that plays a crucial role in the metastasis of cancer, expression of c-fos and TRE CAT It can also be seen that the composition can inhibit the activity of cancer very effectively inhibit metastasis.

Hereinafter, the present invention will be described in detail by way of examples. However, the following Examples are only for illustrating the present invention and the present invention is not limited by the Examples.

<Example 1>

The composition of the present invention was prepared in the following composition.

Sanjago ----------------- 36.2 g

Doin -------------------- 36.2 g

Euiin ------------------ 14.5 g

Tortoiseshell -------------------- 4.4 g

Hippocampus -------------------- 4.4 g

Ejection -------------------- 4.4 g

Each dry herbal medicine described above was weighed and added to 500 ml of solvent (80% methanol, 80% ethanol, 100 water) and cooled for one week, or extracted at a temperature of 100 ° C. for 5 hours in a water bath equipped with a reflux condenser. The procedure was repeated three times to combine the extracts, and then the solvent was completely removed from the vacuum distiller. A small amount of water was added thereto to dissolve it, frozen at −80 ° C., and freeze-dried. This powder was dissolved in water or dissolved in 30-70% DMSO as a dissolution aid, and used as a liquid in the following experimental example.

<Example 2>

White flower vinegar -------------- 36.2 g

Doin -------------------- 36.2 g

Euiin ------------------ 14.5 g

Tortoiseshell -------------------- 4.4 g

Hippocampus -------------------- 4.4 g

Ejection -------------------- 4.4 g

The compositions of the present invention were prepared using the same method as Example 1 with the above-described herbals.

<Example 3>

Gold and Silver Coins ------------------ 36.2 g

Doin -------------------- 36.2 g

Euiin ------------------ 14.5 g

Tortoiseshell -------------------- 4.4 g

Hippocampus -------------------- 4.4 g

Ejection -------------------- 4.4 g

The compositions of the present invention were prepared using the same method as Example 1 with the above-described herbals.

<Example 4>

Live altitude ------------------ 36.2 g

Sweet Ginseng -------------------- 36.2 g

Euiin ------------------ 14.5 g

Tortoiseshell -------------------- 4.4 g

Hippocampus -------------------- 4.4 g

Ejection -------------------- 4.4 g

The compositions of the present invention were prepared using the same method as Example 1 with the above-described herbals.

Example 5

Live altitude ------------------ 36.2 g

Samsung -------------------- 36.2 g

Euiin ------------------ 14.5 g

Tortoiseshell -------------------- 4.4 g

Hippocampus -------------------- 4.4 g

Safflower -------------------- 4.4 g

The compositions of the present invention were prepared using the same method as Example 1 with the above-described herbals.

As described above, since the content of the composition of the example may be changed or the acidic acid of Example 1 may be replaced with baekrye-saengcho or gold and silver coins, or the phosphorus or sesame seeds may be replaced with sweet ginseng, safflower, or tritium, In addition to the compositions, a wide variety of combinations can be prepared.

In order to determine the anticancer action and cancer metastasis inhibiting effect of the composition of the present invention was carried out the following experiment.

Cell culture

HT1080 human fibrosarcoma cells were cultured in an incubator controlled at 5% carbon dioxide concentration in culture with 1% penicillin, streptomycin and 10% fetal bovine serum in DMEM medium.

Experimental Example 1 Collagenase Inhibition Experiment of Composition

Since high cancer metastasis is associated with high collagenase secretion of the cells, the collagenase secretion inhibitory ability of the composition of the present invention was tested. 6 well plates were seeded with 1 × 10 ⁶ cells per well and then changed to fetal calf-free DMEM medium 24 hours later. The composition of the present invention was added to 100, 200 and 300 μg / ml, and 10 hours later, PMA (Phorbol 12-Myristate 13-Acetate) was added to 100 ng / ml, followed by further incubation for 12 hours to induce collagenase. . 20 μl of the culture supernatant was then electrophoresed on 8% SDS-PAGE containing 400 μg / ml gelatin B, and the gel was applied twice with 80 ml of 2.5% Triton X-100 for 15 minutes. Washed. The gel was immersed in an enzyme reaction solution (0.05 M Tris, HCl pH 7.5, 0.15 M NaCl, 0.10 M CaCl ₂ , 1 μM ZnCl ₂ , 0.02% NaN ₃ ) and incubated at 37 ° C. for 24 hours. The gel was stained with 0.25% Comasie Blue stain (0.25 g of Coomassie Blue, 45 ml of methanol, 45 ml of water, 10 ml of glacial acetic acid) and photographed after destaining (see Figure 1). As can be seen in FIG. 1, induction of collagenase by PMA was completely inhibited at 200 μg / ml for 92 kDa and at 300 μg / ml for 72 kDa. This is a result showing that the composition of the present invention has an effect of inhibiting the activity of collagenase that plays a crucial role in the metastasis of cancer.

Experimental Example 2 Invasion Inhibition Experiment

Using the HT1080 cell line, we experimented to create an environment that penetrates the extracellular matrix when cells metastasize in cancer tissues. Using a filter with a constant pore size (8 μm pore size), cancer cells were grown in the upper layer, and the rate of cell movement through the pores was measured. The larger the passage rate for this hole, the better the transition, and the lower the ratio when the composition of the present invention is added is an inhibitory effect.

Based on this theory, experiments on infiltration of extracellular matrix of HT1080 cells were performed. Cells were plated in 6 well plates at 1 × 10 ⁶ and after 48 hours the composition of Example 1 was added to 200 μg / ml in fetal bovine serum-free DMEM medium. After 10 hours, PMA was added, and after 17 hours, cells were trypsinized and injected into the upper layer of the filter at 5 × 10 ⁴ / well. The upper layer was incubated with Matrigel and the original layer was added to the collagen-coated filter (transwell) for 24 hours. After hematoxylin and eosin staining of the cells that passed through the filter, the cells that did not pass through the upper layer were removed with a cotton swab, and only the cells passed through the bottom were measured under a microscope (400 times). It was. The addition of PMA increased the invasion activity of 34.4%. When PMA and the composition of the present invention were added together, the infiltration was suppressed on average 66.4%, and the inhibitory effect was very large.

Control + PMA + PMA + composition Infiltrated cell number (625 μm ² ) 38.5 +/- 2.5 58.7 +/- 2.0 19.7 +/- 3.4 + Cell% for PMA 65.6 +/- 4.3 100 +/- 3.4 33.6 +/- 5.7

Experimental Example 3 Inhibition of c-fos Expression and TRE CAT Activity of Composition

Cancer cells that metastasize well are reported to induce more fos. High cancer metastasis is also associated with high expression of the collagenase gene by c-fos. Since c-fos and c-jun proteins act on the TPA responsive element (hereinafter abbreviated as "TRE") in the promoter of collagenase, the degree of action was measured by TRE CAT activity. The present inventors used a plasmid to which the CAT gene was attached after the regulatory site of -450 to +50 of c-fos in order to examine whether the composition of the present invention inhibits the expression of fos. cFos protein is reported to be involved in the induction of collagenase I and 92 kDa collagenase. 10 μg of CAT plasmid containing flanking regions in the 5 'direction from -450 to +50 of the c-fos promoter were transfected into HT1080 cells, and sampled was added to the c-fos gene promoter. The change in activity was measured. As shown in FIG. 2, when the composition of 300 μg / ml was added, the activity was significantly decreased, and the activity of SV40 CAT was also reduced, but the vector was not affected. These results suggest that the composition of the present invention directly inhibited the collagenase synthesis by inhibiting c-fos transcription of cells. In addition, as shown in Figure 3 TRE CAT activity was also reduced when the composition of the present invention is added.

Experimental Example 4 Tripan Blue Dye Exclusion Test

Inject cells into a plate with 24 wells at 3.3 × 10 ⁴ / well and incubate for 48 hours, then subtract fetal bovine serum, add the composition of the invention at 100, 200 and 300 μg / ml and add 6, 12 and 24 hours After the culture was discarded, 0.4% Trypan Blue Dye was added thereto and left for 20 minutes, and then the dye was completely discarded. The number of dead cells dyed was counted as% of the total cell number. Shown in Only 24% of the cells died and 96.6% of the cells survived for 24 hours at 300 μg / ml, indicating that the test result was not the result of cell death.

0 μg / ml 100 μg / ml 200 μg / ml 300 μg / ml 6 hours 0.5% 0.0% 1.2% 1.1% 12 hours 0.1% 1.0% 2.0% 3.2% 24 hours 0.7% 0.8% 2.9% 3.4%

Experimental Example 5 In Vivo Toxicity

When the composition of the present invention was administered in vivo, the degree of toxicity was tested. Ten ICR male mice were grouped and each group was orally administered with an aqueous solution of the composition for 10 consecutive days. When administered at 10 and 50 mg / Kg as shown in Figure 4, the change in weight was not as toxic as the control group, but when added to 100 mg / Kg, weight was continuously decreased from the 6th day to 17 g on the 10th day Decreased. However, intraperitoneal administration of cisplatin 4 mg / Kg continued to lose weight from day 4 and was consistent with previous literature.

Test Example 6 In vivo anticancer metastasis

Lung cancer cell line (Pulmonary adenocarcinoma) (3LL) was passaged and activated, and 10 CDF1 mice were transplanted into a group with 3LL pulmonary adenocarcinoma cells transplanted to the paws of the mice to induce metastasis to the lung and development of lung cancer. The composition was then administered at 50 mg / Kg daily. As shown in FIG. 5, all rats died of cancer metastasis after 19 days of administration of distilled water. However, the rats to which the composition of the present invention was administered survived until 18 days, and mortality gradually increased until 25 days. Since this dose is non-toxic, it was found that cancer metastasis was suppressed, and the dead rats were able to identify black spots on the lung surface.

As described above, the composition of the present invention not only inhibits the intracellular production and activity of collagenase that plays a crucial role in cancer metastasis, but also inhibits the transcription of the c-fos promoter, which plays an important role in the expression of collagenase. Therefore, since the synthesis of collagenase can be directly inhibited, the composition has excellent anticancer action and cancer metastasis suppression effect. In addition, the composition of the present invention is a new type of cancer treatment agent having fewer side effects, because it contains a herbal medicine, and at the same time it may be usefully used to prevent the occurrence of cancer caused by chemicals in everyday life.

Claims (3)

30-40% by weight of Crestarae Appendiculatae Tuber, 30-40% by weight of Percicae Semen, 10-20% by weight of Coicis Semen, 1-8% by weight of Tropicones Carapax, Hippocampus 1 Composition having anti-cancer action and cancer metastasis action consisting of -8% by weight and 1-8% by weight of Zedoariae Rhizoma The composition according to claim 1, wherein the composition has anticancer activity and cancer metastasis inhibiting activity, comprising 36.2% by weight of acid, 36.2% by weight, 14.5% by weight of Euiin, 4.4% by weight of Tortoiseshell, 4.4% by weight of hippocampus and 4.4% by weight of sequestration The method according to claim 1 or 2, wherein the mountain litter is replaced by Oldenlandiae Diffusae Herba or Lonicerae Flos; A composition having anticancer action and cancer metastasis action, characterized by being replaced with Salviae Miltiorrhizae, Safflower (Carthami Flos), or Sparganii Rhizoma.