KR20050093090A - Galla rhois extract with anti-metastasis activity - Google Patents

Abstract

The present invention relates to a gall bladder extract that inhibits cancer cell metastasis, and more particularly, gallola (Halla Rhois) extract and the compound of formula (I) isolated from the ethyl acetate solubility (chromatography) This cancer cell metastasis inhibiting activity can be used in medicines and health foods, and relates to a method for extracting the compound of formula (1) from the gall bladder.

Description

Galla Rhois extract with anti-metastasis activity

The present invention relates to a gall bladder extract that inhibits cancer cell metastasis, and more specifically, gall bladder extract and the compound of formula (1) isolated from the ethyl acetate soluble part by chromatography from inhibiting cancer cell metastasis It can be used in medicines and health foods by having activity, and relates to a method for extracting the compound of formula

The main cause of death of cancer patients is not due to early tumors, but to metastasis of cancer cells to other tissues. Therefore, studies on the mechanism of cancer cell invasion and metastasis and the method of inhibiting it are being actively conducted. In the metastasis process of cancer cells, first, metastatic cancer cells are separated from the primary tumor, infiltrate into supporting structures such as epilepsy and basement membrane of normal tissue, attach to capillary walls of other target tissues, and decompose extracellular matrix and basement membrane. Outflow into the blood vessels, new tissue is accompanied by angiogenesis, secondary tumors are formed. Therefore, the metastasis process consists of three important stages: adhesion, infiltration, and angiogenesis, and if one of them can be prevented, cancer metastasis can be suppressed. Invasion of cancer cells plays an important role in matrix degraded metalloproteinases (MMPs) secreted by adherent cells. MMPs are enzymes that break down collagen and proteoglycan, and include interstitial collagen enzymes, MMP2, and stromelsin, all of which are metal enzymes with Zn in the active center, secreted in the form of zymogens, to break down other proteins. Activity is inhibited by TIMP (tissue inhibitor of metalloproteinase), which is activated by enzymes or organophosphates and secreted with MMP, and is classified as MMPs because it has homology in cDNA sequence. In fact, cancer cells with active metastasis have higher activity of MMP2 or MMP9 than normal cells or non-metastatic cancer cells, and substances that inhibit them have been shown to inhibit cancer metastasis.

Natural herbal medicines having such cancer metastasis inhibitory effects are known as polysaccharides isolated from citrus fruits, saponin compounds of ginseng, and polysaccharides of tangui.

On the other hand, the medicinal herb used in the present invention is known as an insect worm caused by the spawning of the Mallow (Melaphis chinsis) on the leaves of Rhus javanica. It has been prescribed for diarrhea, prolapse, gastric ulcer, duodenal ulcer, sweating, oil well, blood stool, hematuria and stomatitis. In addition, gall bladder contains 50 to 60% of tannin components and is used as a raw material for hair dyes and inks including tannins. However, studies on the inhibitory efficacy of gall bladder cancer cell metastasis have not been reported.

Accordingly, the present inventors have completed the present invention by confirming the cancer cell metastasis inhibiting activity in the compound of formula 1 separated from the methanol extract obtained from the gall bladder during the study to develop a useful cancer metastasis inhibitor from the herbal medicine.

[Formula 1]

Accordingly, an object of the present invention is to provide a cancer metastasis inhibitor composition containing a gall bladder extract.

It is another object of the present invention to provide a cancer metastasis inhibitor composition containing the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

In addition, the present invention has another object to provide a method for extracting cancer cell metastasis inhibiting active fraction from the gall bladder.

The present invention is characterized by cancer metastasis inhibitor composition and health food containing the gall extract as an active ingredient.

In addition, the present invention is characterized by another cancer metastasis inhibitor composition and health food containing a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Formula 1]

In addition, the present invention

1) extracting the acquaintances, refluxing with methanol, filtering, and concentrating the filtrate under reduced pressure to obtain a methanol extract;

2) suspending the methanol extract in distilled water and shaking with ethyl acetate to concentrate the extract under reduced pressure to obtain an ethyl acetate soluble part; And

3) a method of extracting the cancer cell metastasis inhibiting active fraction from the gall bladder comprising the step of dissolving the ethyl acetate soluble part in ethyl acetate and then performing chromatography to obtain an active fraction.

Referring to the present invention in more detail as follows.

The present invention is isolated from the gall bladder extract inhibiting cancer cell metastasis, and chromatograph of the ethyl acetate soluble portion from the following compound of formula 1 to be used in medicine and health foods in connection with the inhibition of cancer cell metastasis It can be, and relates to a method for extracting the compound of formula (1) from the gall bladder.

Compounds having inhibitory effect on cancer cell metastasis according to the present invention are extracted from the gall bladder through the following process.

The magnificent was extracted under reflux for 6 to 24 hours with 100% methanol, and the filtrate was concentrated under reduced pressure at 40 to 60 ° C. to obtain a methanol extract.

The methanol extract was suspended in the same amount of water and ethyl acetate, and the supernatant obtained by shaking extraction in an aliquot was collected and concentrated under reduced pressure at 40-60 ° C. to obtain an ethyl acetate soluble portion.

The ethyl acetate soluble part was dissolved in ethyl acetate, dried over silica gel, and subjected to silica gel column chromatography to collect active fractions. As the eluent, the composition of hexane and ethyl acetate in 1: 1 is preferable. The collected active fractions are concentrated under reduced pressure at 40 to 60 ° C. to obtain a white powder.

As a result of analyzing the structure of the powder component thus obtained, it was confirmed that it is 3,4,5-trihydroxy-benzoic acid methyl ester (methylated methacrylate), and the compound is excellent in inhibiting cancer cell metastasis, which is very useful for cancer metastasis inhibitors or health foods. I am expected to.

In addition, the methylated molar acid is pharmacologically methylated with an organic or inorganic acid, such as acid, such as hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, fumaric acid, lactic acid, maleic acid, succinic acid and tartaric acid. All of the pharmaceutically acceptable salts of the methylated molar assets may be formed in the present invention because they may form an acceptable salt, or may react with alkali metal ions such as sodium or potassium or ammonium ions to form a pharmaceutically acceptable salt. Included.

In addition, the compound is an active ingredient exhibiting a cancer metastasis inhibitory effect, it can be useful for suppressing cancer recurrence and metastatic cancer in the treatment of various cancer diseases such as gastric cancer, lung cancer, liver cancer and blood cancer.

Therefore, the pharmaceutical composition containing the compound as an active ingredient can be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration, and used in the form of general medicines and health foods. Can be.

The pharmaceutical composition may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Formulated). Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above. In addition, the anticancer composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection. To formulate into a parenteral formulation, the composition is prepared in solution by mixing in water with stabilizers or buffers and formulated into unit dosage forms of ampoules or vials.

The effective dose of the methylated molar asset according to the invention is generally 1-30 mg / day per kg of adult patient weight, preferably 3-20 mg / day, several times a day at regular time intervals, as determined by the physician. It can be administered in divided doses 2-3 times a day.

In addition, the present invention includes a health food containing the compound represented by the formula (1) as an active ingredient.

The health food is a food prepared by adding the compound represented by Chemical Formula 1 to food materials, such as beverages, teas, spices, gums, confectionery, or the like, encapsulated, powdered, or suspension, and when ingested, have a specific health effect. Means to bring, but unlike the general medicine has the advantage that there is no side effect that can occur during long-term use of the drug as a raw material of the food.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation of Cancer Metastasis Inhibitors from Mice

3 kg of gallnuts were refluxed with 100% methanol for 24 hours, and the filtrate was concentrated under reduced pressure in a 60 ° C. water bath, and the concentrated methanol concentrate was suspended in 2 L of distilled water. The methanol extract suspended in distilled water was shaken and extracted with 2 L of ethyl acetate in a separatory filter to extract a butanol soluble part. The extract was further concentrated under reduced pressure in a 60 ° C. water bath to obtain an ethyl acetate extract. The ethyl acetate extract was dissolved in 20 ml of ethyl acetate and subjected to silica gel column chromatography as a mixed solvent of hexane / ethyl acetate (1/1) to obtain an active fraction. The white powder obtained by purifying the active fraction was dissolved in heavy methanol (CD ₃ OD) to determine the chemical shift of hydrogen and carbon of Table 1 through ¹ H-NMR, ¹³ C-NMR and two-dimensional NMR. Could.

Carbon number Chemical shift of hydrogen NMR (ppm) Chemical shift of carbon NMR (ppm) One 121.5 2 7,03 110.1 3 146.5 4 139.7 5 146.5 6 7.03 110.1 7 169.0 CH3 3.80 52.6

The white powder was identified as 3,4,5-trihydroxy-benzoic acid methyl ester (gallic acid methylester), and the structural formula is represented by the following Chemical Formula 1.

[Formula 1]

Example 2 Measurement of MMP9 Expression Inhibitory Activity of Methylated Molecular Assets

HT1080 human fibrosarcoma cells at a concentration of 1 × 10 ⁵ cells / ml were incubated in 96-well microplates using a medium containing 10% FBS for one day, and then treated with the methylated glutamate (sample) of the present invention. After changing to serum-free medium, 5 ng / ml of tumor necrosis factor α (TNF-α) was treated 3 hours after sample treatment and incubated for 17 hours. After incubation, the shape and cytotoxicity of the cells were examined, and only the culture medium was taken and mixed with the filling buffer containing glycerol and the coloring reagent, respectively, and electrophoresed, and MMP9 expression inhibitory activity was compared with the control. The control group used tumor necrosis factor-alpha as an inducer for activating cancer cells, and visually confirmed the induction pattern by dividing into treated and non-treated groups and confirming the expression pattern of MMP-9 in the sample added culture by SDS-PAGE [FIG. 1].

Example 3: Determination of Methylation-inhibited Inhibitory Activity of C57BL / 6 Mice in C57BL / 6 Mice

After culturing B16F10 melanoma cells, the cells were separated using trypsin-EDTA, and diluted with physiological saline at a concentration of 2.5 × 10 ⁶ cells / ml. The prepared cell suspension was injected into the tail vein in 0.2 ml per head of SPF C57BL / 6 mice (♀, 17-20 g). Mice were placed in 10 groups in each group.

Experiment 1) SPF C57BL / 6 mouse (♀, 17-20g) used as an experimental animal was intraperitoneally administered with methylated glutamate for 2 weeks at 100 mg / kg concentration and then injected into the tail vein. After 14 days of transplantation, the lungs were excised and the number of tumors formed was counted. As a result, as shown in Table 2, after saline administration for 2 weeks, saline showed about 17% of cancer cell metastasis inhibition effect compared to the animal group transplanted with cancer cells.

Experiment 2) Treatment of cancer cells taken by the above method with a concentration of 30 μg / ml of methylated glutathione for 8 hours, followed by experiment 1 in SPF C57BL / 6 mice (♀, 17-20 g), which were used as experimental animals. In the same manner, cancer cells were transplanted, and after 8 hours, 100 mg / kg of methylated glutamate was intraperitoneally administered for 4 days, and after 14 days, the lungs were excised to count the number of tumors formed. As a result, as shown in the following Table 2, the treatment of cancer cells with physiological saline for 8 hours and after cancer cell transplantation showed about 60% of cancer cell metastasis suppression ability compared to the animals group intraperitoneally administered with saline for 2 weeks.

division Number of metastatic cells (mean ± standard deviation, p value) Experiment 1) Experiment 2) Methylated Monomeric Assets Methylated Monomeric Assets menstruum 291.5 ± 124.75 71.2 ± 38.63 Methylated Monomeric Assets 242.7 ± 78.76, 0.390 29.3 ± 11.77, 0.00061

Example 4 Oral Administration of Rats Acute Toxicity

Acute toxicity test was performed using 6-week-old SPF SD rats as follows.

Two animals per group were dissolved in distilled water for injection and administered once orally at a dose of 1 g / kg / ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. Therefore, the novel compounds according to the present invention do not show toxicity changes up to 500 mg / kg in all rats, and the minimum lethal dose (LD ₅₀ ) for oral administration was determined to be a safe substance of 500 mg / kg or more.

Formulation Example 1 Preparation of Syrup

Syrup containing the compound of formula 1 according to the present invention or a pharmaceutically acceptable salt thereof as an active ingredient 2% (weight / volume) was prepared by the following method.

Acid addition salts, saccharin and sugars of the compound of formula 1 were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Compound of the formula (1), hydrochloride 2 g

Saccharin 0.8 g

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·······················

Formulation Example 2 Preparation of Tablet

A tablet containing 15 mg of active ingredient was prepared by the following method.

250 g of the compound of Formula 1 were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

Formulation Example 3: Preparation of Injection

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of the compound of formula 1, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C for 30 minutes.

The components of the injection solution are as follows.

Compound of Formula 1 ··············· 10

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ·····················

Formulation Example 4: Beverage Preparation

After dissolving 500 mg of the novel compound according to the present invention in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharide as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, followed by adding water to the whole amount. 100 ml was prepared to prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

As described above, the gall bladder extract and methylated molar asset according to the present invention is excellent as a cancer drug metastasis inhibitory activity is very useful as a medicine or health food.

Figure 1 shows the expression of MMP-9 expression of the methylated molar asset by SDS-PAGE.

Claims (6)

Cancer metastasis suppressor composition characterized in that it contains a Galla Rhois extract as an active ingredient. Health food containing Galla Rhois extract as an active ingredient. The cancer metastasis inhibitor composition comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1] A health food containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1] 1) collecting Galla Rhois and refluxing with methanol, filtering and filtrating the filtrate under reduced pressure to obtain a methanol extract; 2) suspending the methanol extract in distilled water and shaking with ethyl acetate to concentrate the extract under reduced pressure to obtain an ethyl acetate soluble part; And 3) dissolving the ethyl acetate soluble part in ethyl acetate and then performing chromatography to obtain an active fraction; Method for extracting cancer cell metastasis inhibiting active material from a gall bladder comprising a. The method of claim 5, wherein the active fraction of step 3) comprises the compound represented by the following formula (1). [Formula 1]