KR100658154B1 - Active fractions having metastasis inhibitory activity obtained from the dried flower bud of Eugenia caryophyllata - Google Patents

Abstract

The invention is isolated from the as cancer metastases obtained from clove on the inhibitory activity fractions, and more particularly, clove (Eugenia caryophyllata Thunb.) Is the bud Dried clove (Caryophylli Flos) before blooming flowers of cancer metastasis that occurs at the time of The present invention relates to an active fraction having heparinase and heparanase inhibitory activity required for endothelial cell invasion or small blood vessel formation of cancer cells, a method for separating the same, and a cancer metastasis inhibiting composition containing the same as an active ingredient.

Cloves, cancer metastasis inhibition, active fraction, heparanase and heparanase inhibitory activity

Description

Active fractions having metastasis inhibitory activity obtained from the dried flower bud of Eugenia caryophyllata}             

1 is a comparison of the difference of metastasis to the mouse lung of the experimental group in which the active fraction of Example 1 was administered for 10 days to female SPF C57BL / 6 mice injected with tail vein of B16F10 melanoma cells and the control group treated with 0.5% Tween 80 only One picture.

Figure 2 is a graph comparing the number of cancer cells metastasized into the mouse lungs after administration of the active fraction of Example 1 to female S.P.F C57BL / 6 mice injected with tail vein of B16F10 melanoma cells for 10 days.

3 is a graph showing the weight change of the experimental group treated with the active fraction of Example 1 to female S.P.F C57BL / 6 mice for 14 days and the control group treated with 0.5% Tween 80 only.

The invention is isolated from the as cancer metastases obtained from clove on the inhibitory activity fractions, and more particularly, clove (Eugenia caryophyllata Thunb.) Is the bud Dried clove (Caryophylli Flos) before blooming flowers of cancer metastasis that occurs at the time of The present invention relates to an active fraction having heparinase and heparanase inhibitory activity required for endothelial cell invasion or small blood vessel formation of cancer cells, a method for separating the same, and a cancer metastasis inhibiting composition containing the same as an active ingredient.

Invasion of cancer cells or tumor metastasis in mammalian tissues involves the enzymatic degradation of basement membranes by enzymes that exist outside the cell. Heparan sulfate or heparin, together with collagen, laminin and fibronectin, is one of the major components of the basement membrane present in most mammalian cells. In addition, heparin or heparan sulfate proteoglycans (HSPGs) are present as components of the cell surface or extracellular matrix (ECM), which are growth factors or cytokines. It is involved in cell growth, cancer cell migration, cell differentiation, angiogenesis, virus penetration, and anticoagulant by combining or reacting with various molecules including enzymes, enzyme inhibitors, and viral proteins. That is, HPSG such as heparin and heparan sulfate is involved not only in the growth of cancer cells but also in angiogenesis, adhesion by bacteria, protozoa, viruses, and the like. In particular, it mediates vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), heparin binding proteins such as angiogenin and nidkine. It is known to act as.

Heparin and heparan sulfate are complex linear combinations of disaccharide units of N-acetylated or N-sulfated glucosamine and uronic acid. As polysaccharides they are very similar in structure to each other. In other words, heparin and heparan sulfate are sulfur compounds as a repeating unit of disaccharides in which L-iduronic acid or D-glucuronic acid and hexosamine are bound to 1,4 positions. Depending on the location and number of polysaccharides represent various forms. Heparin and heparan sulfate activate the antithrombin III, a serine protease inhibitor, through inactivation of serine proteases such as thrombin and factor Xa. Prevents blood clotting and has been used as an anticoagulant for the last 50 years.

Heparin and heparan sulfate are degraded by heparanase and heparanase enzymes, respectively. Heparinase is an enzyme that hydrolyzes not only heparin but also heparan sulfate. Heparin-binding growth factor is derived from and is involved in the physiological and pathological actions from wound healing to cancer metastasis and is achieved through cleavage of the binding of heparin polysaccharide α-D glucosamine and duuronic acid. Heparanase and heparanase activity has been identified in circulating cells, neutrophils, mast cells, macrophages, activated T lymphocytes, etc., passing through the extracellular grid and basement membrane.

These heparanase and heparanase enzymes are known to be closely related to angiogenesis and cancer metastasis processes [J. Clinic. Invest. 108, 341-347, 2001; FASEB J. 15, 1661-1663, 2001; Science 220, 313-325, 1983]. In order for cancer cells to metastasize to other tissues, new blood vessels must be formed to supply nutrients. Such angiogenesis requires vascular endothelial growth factor or basic fibroblast growth factor. These growth factors are bound to heparin or heparan sulfate and released when jellyfish or heparanase enzymes break down to induce angiogenesis by inducing angiogenesis. Therefore, by inhibiting the activity of these enzymes can inhibit the production of neovascularization can inhibit the growth of cancer cells, in particular the growth of metastasized cancer cells. In addition, it has been reported that inhibitors of these enzymes inhibit cancer metastasis [Leukemia 16, 376-381, 2002; J. Med. Chem. 43, 2591-2600, 2000; Int. J. Cancer 83, 424-431, 1999] The possibility of development as an anticancer agent has been suggested, and several researchers are searching for new inhibitors of these enzymes. To date, polyanionic compounds such as sulfated chitin, laminarin sulfate, calcium spirulin, phosphorothioate DNA oligonucleotides [ Biochem. Biophysic. Acta 1471, M99-M108, 2001] and A-72363C [J. Antibiotics 49, 61-64, 1996] and suramin [J. Biol. Chem. 266, 9661-9666, 1991], PI-88 (phosphomannopentaose sulfate) [Cancer Res. 59, 3433 ~ 3441, 1999] have been reported as inhibitors of heparanase and heparanase enzymes, and suramin and PI-88, which inhibit heparanase activity and inhibit tumor cell metastasis and angiogenesis, are anticancer agents. It is being developed and is undergoing preclinical and clinical trials.

Therefore, there is a demand for the development of novel compounds that can inhibit cancer metastasis by inhibiting heparanase or heparanase enzyme activity as a trace amount, as well as in vivo. In general, among the various methods for the development of drugs with new ingredients, there is a high possibility of finding new active ingredients from natural medicines used in traditional medicine rather than the effort by experimental modification of existing drugs. There is less concern about toxicity due to the drugs developed.

Therefore, the present inventors have searched for drugs that have been used in traditional medicine of Korea, including Dongbobom, for the development of a new type of anticancer drug, showing a strong inhibitory activity against heparanase or heparanase. The present invention was completed by obtaining an active fraction from cloves that exhibits inhibitory activity.

Accordingly, an object of the present invention is to provide a clove active fraction having a distinct effect on heparanase and heparanase inhibitory activity and inhibiting cancer metastasis, a method for separating the same, and a cancer metastasis inhibiting composition containing the same as an active ingredient.

The present invention features heparase and heparanse enzyme activity inhibitory and cancer metastasis inhibiting active fractions obtained from cloves and methods for isolating and purifying them.

The present invention also includes a method for separating the active fractions as follows:

1) crushing the cloves ( Caryophylli Flos , dried flower bud of Eugenia caryophyllata Thunb.) And then solvent extraction with hot water or an aqueous alcohol solution;

2) evaporating the extract filtrate and concentrating, then suspending the concentrate in distilled water, extracting and concentrating with ethyl acetate to obtain an extract;

3) adsorbing the extract onto silica gel, and then eluting with chloroform, methanol, or a mixed solvent thereof; And

4) separating the active fraction by eluting the eluate with methanol by Sephadex LH 20 resin column chromatography.

In addition, it contains a cancer metastasis inhibiting composition containing the active fraction as an active ingredient.

Referring to the present invention in more detail as follows.

The present invention is separated from dried clove ( Caryophylli Flos ) before flowering of Eugenia caryophyllata Thunb. The present invention relates to an active fraction having linase and heparanase inhibitory activity, a separation method thereof, and a cancer metastasis inhibiting composition containing the same as an active ingredient.

Referring to the process for separating the active fraction of the present invention from cloves in detail as follows.

First, after crushing Caryophylli Flos , the solvent is extracted by adding 5 to 10 times the amount of hot water or an aqueous alcohol solution to the contents of the clove. The filtrate is evaporated and concentrated, and the concentrate is distilled with 10 to 15 times the total amount. After suspension, the mixture is extracted with ethyl acetate and concentrated to obtain an extract. The alcohol is preferably at least one selected from methanol, ethanol, butanol and propanol.

The extract is adsorbed onto silica gel, eluted with chloroform, methanol or a mixed solvent thereof to separate and purify the active fraction. Preferably, in the process of separating the active fraction, starting with a solvent in which chloroform and methanol are mixed in a 10: 1, eluting with a solvent in which chloroform and methanol 5: 1 are mixed to separate the active fraction. The active fraction thus obtained is again subjected to column chromatography using Sephadex LH 20 resin, eluted with methanol to separate the active fraction.

In order to remove the trace amount of the solvent that has not been removed from the active fraction, distilled water of 2 to 5 times the total amount of the active fraction is added to the mixture, and then suspended homogeneously and lyophilized to obtain a powdered active fraction.

Thus, it was confirmed for the first time that the active fraction obtained from cloves inhibits heparinase enzyme activity and inhibits cancer metastasis in female S.P.F C57BL / 6 mice injected with tail vein of B16F10 melanoma cells.

Thus, a cancer metastasis inhibiting composition containing the active fraction as an active ingredient can be prepared, and the composition can be administered orally or parenterally during clinical administration, for example, intravenously, subcutaneously, intraperitoneally or topically. It can be used in the form of general medicines and health foods.

The compositions 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. Is 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 composition of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. 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 active ingredient represented by the active fraction may vary depending on the age, physical condition, weight, etc. of the patient, but is generally administered within the range of 1 to 100 mg / kg (weight) per day. In addition, the dose is administered once a day or divided several times a day within the effective dosage range per day.

In addition, the above-mentioned health food is a food prepared by adding the active fraction to food materials, such as beverages, teas, spices, gums, confections, or the like, encapsulated, powdered, suspensions, and the like. Means to bring a certain effect, unlike the general medicine has the advantage that there is no side effect that can occur when using the drug as a raw material for long-term use.

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 Preparation of Active Fractions from Cloves

The crushed cloves were extracted under reflux for 5 hours to allow the active substance to be eluted with 5 times the amount of ethanol, and this process was repeated three times, and the ethanol extract was concentrated under reduced pressure. Then, the concentrate was suspended in 10 times distilled water and solvent fractionated three times with the same amount of ethyl acetate to obtain an extract. The extract was adsorbed onto silica gel, and then eluted with a solvent mixed with chloroform and methanol 5: 1, starting with a solvent in which chloroform and methanol were mixed at 10: 1. Each of the obtained elution fractions was examined for heparinase inhibitory activity to isolate active fractions showing inhibitory activity. The active fraction thus obtained was subjected to silica TLC (Thin Layer Chromatography) using chloroform / methanol / water (10/5/1) as a developing solvent, and was used as a coloring reagent of anisaaldehyde-sulfuric acid (anisaldehyde-H ₂ SO ₄ ). When it confirmed, it developed yellow at _Rf value 0.5.
The eluate thus obtained was again subjected to column chromatography using Sephadex LH 20 resin, and methanol was used as the eluent. Each eluted fraction was examined for heparinase inhibitory activity to isolate the active fraction. At this time, the active fractions were fractions having an R _f value of 0.33 to 0.42 by TLC of RP-18 using a 30% methanol aqueous solution.
Distilled water twice the total amount of the active fraction was added to remove the trace amount of the solvent which was not removed during the process, and then suspended and homogeneously dried to obtain an active fraction in powder form.

Example 2 Measurement of Heparinase Inhibitory Activity

To 17 μl of the reaction solution containing 10 ng of heparin, 3 μl of the sample to be assayed (the active fraction of Example 1 and the control suramin) was added, and 10 μl (0.1 unit) of heparinase enzyme solution was added thereto. Reacted for 15 minutes. Again, 20 μl of antithrombin III solution was added and reacted at room temperature for 2 minutes, followed by 20 μl of factor VIIa solution. One minute after the addition, 20 µl of factor VIIa was added to react for 15 minutes, and 20 µl of glacial acetic acid was added to stop the reaction. The inhibition rate for the heparinase enzyme was calculated as in Equation 1 below, and the IC ₅₀ value was determined as the concentration of the inhibitor whose inhibition rate of the enzyme activity reaches 50%.

A: absorbance after the reaction without the inhibitor

B: Absorbance after the reaction of not adding the enzyme solution

C: absorbance after the reaction with the inhibitor

As a result of measuring the concentration (IC ₅₀ ) which inhibits the heparinase enzyme activity of the isolated active fraction by 50%, it was 2.0 ㎍ / ml, respectively, and in the case of Suramin, which is known as an inhibitor, 50% inhibition in this experiment The concentration (IC ₅₀ ) was found to be 5 μΜ. At this time, heparanase was used in Flavobacterium heparinum, and heparin was isolated from pig viscera mucosa and used from Sigma Co.

Example 3 Measurement of Cancer Metastasis Inhibitory Activity in Animal Model Mice

The dose of the active fraction sample of Example 1 was set to 10 mg / kg based on the sample amount obtained. Methanol was added to the sample to dissolve it, and methanol was evaporated using a dryer, and 0.5% Tween 80 was used as a medium. 0.5% Tween 80 was used as a solvent control.

After culturing B16F10 melanoma cells, the cells were separated using trypsin-EDTA, diluted with 0.85% saline, and adjusted to 2.5 × 10 ⁶ cells / ml. The prepared cell suspension was injected into the tail vein into female SPF C57BL / 6 mice (Daebiolink, 14-20 g) at 0.2 ml per horse. Mice were placed in 7 groups in each group.

Four hours after the cancer cell transplantation, drug administration was started and intraperitoneally administered 0.2 ml of the sample solution per 20 g of the mouse body weight until the 13th day. Weight measurements were taken on days 0, 3, 6, 9, 12 and 14. After 14 days of cancer cell transplantation, mice were necropsied and lungs from which cancer cells had metastasized were taken, and lungs were photographed in each group using a camera. The lungs were soaked in neutral formalin and counted visually for pulmonary metastatic tumor colony. Statistical significance was tested by Student's t-test.

Samples of the active fractions were observed intraperitoneally once daily for 14 days, and normal body weight gain was observed as in the solvent control group. There was no change in body weight due to drug administration during the test period.

On the last day of the experiment (day 14), mice were autopsied and treated with active fractions, and when the control group formed an average of 128 cancer cell colonies, the average of 78 (p <0.01) in the experimental group treated with 10 mg / kg of active fractions. Formation of cancer cell colonies showed significant cancer metastasis inhibitory activity of 39.4% [FIG. 2].

Example 4: Toxicity Test

1) Acute Toxicity

This experiment was carried out to investigate the toxicity to the animal body acutely (within 24 hours) upon ingestion of the active fraction of Example 1 in a short time and to determine the mortality rate.

50 ICR mouse strains, which are general mice, were assigned to 10 control groups and 20 experimental groups. Only DMSO was administered to the control group, and the experimental group was orally administered the active fraction of Example 1 at concentrations of 2,000 times (20 g / kg) and 10,000 times (100 g / kg), respectively, at a concentration of 10 mg / kg. After 24 hours of administration, the mortality rate was 12% in the control group and the experimental group treated with 20 g / kg active fraction. Seemed.

From the above experimental results, the active fraction of Example 1 was about 10,000 times the concentration (10 mg / kg) showing a general efficacy as a single animal lethal dose was about 100 g / kg (100,000 mg / kg), and very little toxicity was observed. It was found to be a safe substance.

2) Organ and Histotoxicity

The active fraction of Example 1 was administered at a concentration of 10 mg / kg for 14 days to test female S.P.F C57BL / 6 mice used in the cancer metastasis animal model. In order to investigate the effect on each organ (tissue) of the animal, 14 days after each animal in the experimental group administered the active fraction of Example 1 and the control group administered only DMSO, heart, lung, pancreas, adrenal gland, liver, kidney Muscles were cut and histological observations were performed with an optical microscope through a conventional tissue fabrication process. As a result, no abnormal abnormalities were observed in histological observations of all organs of the control and experimental animals, so no toxicity was observed for the organ organs of the active fraction of Example 1.

Formulation Example 1 Preparation of Tablet

10 g of active fraction of Example 1

70 g of lactose

15 g of crystalline cellulose

5 g of magnesium stearate

Total amount 100 g

The tablets were prepared by direct tableting method after mixing the ingredients listed above finely. The total amount of each tablet is 100 mg, of which the active ingredient content is 10 mg.

Formulation Example 2: Preparation of Powder

10 g of active fraction of Example 1

50 g of corn starch

40 g of carboxy cellulose

Total amount 100 g

A powder was prepared by crushing and mixing the ingredients listed above. 100 mg of powder was put into the hard capsule to prepare a capsule.

Formulation Example 3: Preparation of Injection

1 g of the active fraction of Example 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.

Formulation Example 4: Beverage Preparation

After dissolving 500 mg of the active fraction of Example 1 in an appropriate amount of water, an appropriate amount of vitamin C, citric acid, sodium citrate, and oligosaccharides were added as auxiliary ingredients, and an appropriate amount of sodium benzoate was added as a preservative, followed by water. 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 clove active fraction according to the present invention is expected to be useful as medicines and health foods with excellent heparanase and heparanase inhibitory activity and cancer metastasis inhibiting activity.

Claims (4)

1) crushing the cloves ( Caryophylli Flos , dried flower bud of Eugenia caryophyllata Thunb.) And then solvent extraction with hot water or an aqueous alcohol solution; 2) evaporating the extract filtrate and concentrating, then suspending the concentrate in distilled water, extracting and concentrating with ethyl acetate to obtain an extract; 3) adsorbing the extract onto silica gel, and then eluting with chloroform, methanol, or a mixed solvent thereof; And 4) separating the eluate with methanol by eluting with Sepadex LH 20 column chromatography; Separation method of a cancer metastasis inhibiting active fraction from cloves comprising a. The method of claim 1, wherein the alcohol is at least one selected from methanol, ethanol, butanol and propanol. An active fraction of cancer metastasis inhibiting, which is obtained by the separation method of claim 1 or 2. Cancer metastasis inhibiting composition comprising the active fraction of claim 3 as an active ingredient.

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