KR20030084175A - Active fractions showing inhibitory effects on heparinase activity and cancer metastasis from the root bark of Morus alba L. - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing an active fraction separated from a Mori Cortex Radicis extract as an effective ingredient is provided. It has an inhibition effect on the endothelial invasion of cancer cells generated during cancer metastasis and on the activity of heparinase which is necessary during the formation of a small blood vessel as well as on the activity of cancer metastasis. CONSTITUTION: An active fraction for inhibition of heparinase activity or cancer metastasis is obtained by the steps of: grinding Mori Cortex Radicis and extracting with an aqueous alcohol solution; suspending the extract in distilled water and concentrating an active material with ethyl acetate; absorbing the extract in silica gel and eluting with chloroform or methanol to separate an active fraction; and suspending the active fraction in distilled water and then freeze drying. The effective dose of the active fraction is in the range of 1 to 100mg/kg(body weight)/one day.

Description

Active fractions showing inhibitory effects on heparinase activity and cancer metastasis from the root bark of Morus alba L.}

The present invention relates to an active fraction that inhibits heparinase (heparinase) enzyme activity and cancer metastasis activity obtained from lettuce skin, and more specifically, cancer warfare from Morie Cortex Radicis, the root bark of Morus alba L. The active fraction which inhibits the activity of heparinase required for endothelial cell invasion or small blood vessel formation of cancer cells at the same time and at the same time inhibits cancer metastasis activity, a method for efficiently separating and purifying it, and the active fraction It relates to a drug for inhibiting heparinase enzyme activity and cancer metastasis activity contained as an active ingredient.

Invasion of cancer cells or tumor metastasis in mammalian cells involves enzymatic degradation of basement membranes by enzymes 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. Heparin, heparan sulfate, and heparin / heparan sulfate proteoglycans (HSPGs) are present on the cell surface or in the extracellular matrix (ECM), which are growth factors, cytokines, enzymes, enzyme inhibitors, and viruses. It is involved in cell growth, cancer cell migration, cell differentiation, angiogenesis, viral infiltration, and anticoagulant by combining or reacting with various molecules including proteins. In other words, heparin, heparan sulphate, and polysaccharide-binding protein (HPSG) are involved in not only cancer cell growth but also angiogenesis, adhesion by bacteria, protozoa, viruses, etc., and thus vascular endothelial growth factors. growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin (angiogenin), and heparin binding proteins such as nidkine are known to act as a medium.

Heparin is mainly present in mast cells, basophilic granulocytes, heparan sulphate on the cell surface or extracellular grid, and heparin and heparan sulphate are N-acetylation. Or a complex linear polysaccharide in which di-saccharide units of N-sulfated glucosamine and uronic acid are continuously combined, and are very similar in structure. In other words, heparin and heparan sulphate are a repeating unit of disaccharides in which L-iduronic acid or D-glucuronic acid and hexosamine are bonded in 1,4 positions. Depending on the position and number, various forms of polysaccharides are shown. Heparin and heparan sulfate activate the antiserumbin III, a serine protease inhibitor, to inactivate serine proteases such as thrombin and factor Xa. It has been used as an anticoagulant for the last 50 years by preventing blood clotting.

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. It is a growth factor that is involved in the physiological and pathological processes from wound healing to cancer metastasis and cleavage of α-D glucosamine and uronic acid in heparin polysaccharides. Heparinase activity has been identified in circulating cells, extracellular grids and basal membranes, neutrophils, mast cells, macrophages and activated T lymphocytes.

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 the growth of angiogenic cells. Therefore, inhibiting the activity of these enzymes can inhibit angiogenesis and 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. Polyanionic compounds such as sulfated chitin, laminarin sulfate, calcium spirulin, phosphorothioate DNA oligonucleotides to date [ 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] are 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 undergoing preclinical and clinical trials.

Therefore, there is a demand for the development of novel compounds capable of inhibiting heparanase or heparanase enzyme activity as a trace amount, as well as inhibiting cancer metastasis by inhibiting them 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.

Thus, the present inventors have long researched to develop a substance showing a strong inhibitory activity against heparanase as part of the development of a new type of anticancer agent. Thus, we observed cancer metastasis inhibitory activity in mice injected with heparanase enzyme and B16F10 melanoma cells in order to search for cancer metastasis inhibitors in drugs that have been used in traditional Korean medicine, including Dongbogam. The present invention was completed by obtaining an active fraction showing specific inhibitory activity from the epidermis.

Accordingly, the present invention provides a method for separating the baekryepi active fraction having a distinct effect on heparinase enzyme activity and cancer metastasis inhibiting activity in an animal model and the pharmaceutical composition containing the extract as an active ingredient. The purpose is.

1 compares the difference of metastases to the mouse lungs of a control group treated with 0.5% Tween 80 only for 10 days with an active fraction isolated from epithelium on female SPF C57BL / 6 mice injected with tail vein injection of B16F10 melanoma cells. One picture.

FIG. 2 is a bar graph comparing the number of cancer cells metastasized to mouse lungs after 10 days administration of active fraction SBC100 isolated from epithelium to female S.P.F C57BL / 6 mice injected with tail vein of B16F10 melanoma cells.

FIG. 3 is a bar graph comparing the number of cancer cells metastasized to mouse lungs after 10 days of active fraction SBCM101 isolated from epithelium on female S.P.F C57BL / 6 mice injected with tail vein injection of B16F10 melanoma cells.

The present invention is characterized by the activity of inhibiting heparinase (heparinase) enzyme activity and cancer metastasis activity obtained from the epidermis and the method for separating and purifying it.

In addition, it comprises the use of heparinase enzyme activity and cancer metastasis inhibitors, etc., using the extract from the epidermis active fraction isolated according to the separation and purification method as an active ingredient.

Referring to the present invention in more detail as follows.

The present invention relates to an active fraction isolated from epithelium, which inhibits heparinase enzyme activity and cancer metastasis inhibiting activity in mice injected with tail vein injection of B16F10 melanoma cells.

That is, the composition was observed for the first time that the active fraction obtained by fractionation from epidermis was inhibited heparinase enzyme activity and inhibited cancer metastasis in female SPF C57BL / 6 mice injected with tail vein of B16F10 melanoma cells. We have developed an active fraction with inhibitory effect on heparanase enzyme activity and cancer metastasis activity.

In addition, after grinding the Mori Cortex Radicis, the alcohol solution was extracted three times by adding 5 times the amount of alcohol to the content of the lettuce, and the filtrate obtained was evaporated and concentrated, and then the concentrate was suspended in distilled water of 10 times the total amount. The active material is extracted and concentrated with an organic solvent of ethyl acetate to obtain an extract. The extract is adsorbed onto silica gel, eluted with chloroform or methanol to separate and purify the active fraction.

Preferably, the active fractions obtained by eluting with chloroform or a solvent mixed with chloroform and methanol in a ratio of 10: 1 in the process of separating the active fraction are measured for heparinase inhibitory activity and cancer metastasis inhibitory activity in an animal model. do.

In order to remove the trace amount of the solvent that has not been removed from the active fraction, distilled water twice the total amount of the active fraction is added to homogeneously suspend and freeze-dried to prepare an active fraction in powder form.

In addition to the active fraction thus prepared, a pharmaceutically acceptable carrier or excipient may be used in the form of a unit dosage form or a multiple dosage form for tablet, powder, granule, capsule, suspension, emulsion, or parenteral administration.

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.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by Examples.

Example 1 Preparation of Active Fraction from Morus Cortex

The pulverized baekbaekpi was extracted by refluxing for 5 hours so that the active substance was eluted using 5 times the amount of alcoholic solvent, and the alcoholic solvent extract was concentrated under reduced pressure. Then, the concentrated active fraction was suspended in 10 times distilled water and solvent fractionated three times with the same amount of an organic solvent of ethyl acetate to obtain a crude extract containing the active substance. The crude extract obtained above was adsorbed onto silica gel, eluted with chloroform to elute the active fraction SBC100, and eluted with a solvent mixed with chloroform and methanol in a ratio of 10: 1 to separate the active fraction SBCM101. Distilled water twice the total amount of the active fraction was added thereto to homogeneously suspend and freeze-dried to remove a trace amount of the solvent that was not removed in the solvent used to prepare a powdered active fraction.

Example 2 Measurement of Heparinase Inhibitory Activity

3 µl of the sample solution to be assayed was added to 17 µl of the reaction solution containing 10 ng of heparin, 10 µl (0.1 unit) of heparinase enzyme solution was added thereto, and the reaction was performed at room temperature for 15 minutes. Again, 20 μl of antisrombin 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, and then absorbance was measured at 410 nm. 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 heparanase enzyme activity of the isolated active fraction SBC100 and the active fraction SBCM101 by 50%, it was found to be 4 ㎍ / ml and 1 ㎍ / ml, respectively. In the case of lamin, the 50% inhibitory concentration (IC ₅₀ ) was 5 μM in this experiment. At this time, heparinase was used in Flavoacterium 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 doses of the two samples, the SBC100 active fraction and the SBCM101 active fraction, were set to 10 mg / kg and 10 mg / kg, respectively, based on the sample volume obtained. Methanol was added to each 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 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, 0.2 ml of the sample solution per 20 g of the mouse body weight was administered 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. The lungs were soaked in neutral formalin and counted visually for pulmonary metastatic tumor colony. Statistical significance was tested by Student's t-test.

In the SBM100 and SBCM101 active fractions, the normal weight gain was observed in the abdominal administration once daily for 14 days, as in the solvent control group, and there was no change in weight due to drug administration during the test period.

On the last day of the experiment (day 14), mice were autopsied and treated with SBC100 active fractions, and when the control group formed an average of 129 cancer cell colonies, the average of 89 (p <0.05) in the experimental group treated with 10 mg / kg SBC100 active fractions. ) Form a cancer cell colony showing a significant in vivo metastasis inhibiting activity of 31% (Fig. 2). In the SBCM101 active fraction treatment, the solvent control group formed about 135 cancer cell colonies, whereas the experimental group treated with SBCM101 active fraction 10 mg / kg formed about 87 (p <0.01) cancer cell colonies, resulting in 36% significant biomarkers. It showed internal metastasis inhibitory activity [FIG. 3].

Example 4: Preparation of Tablets

10 g of active ingredients

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.

Example 5: Preparation of Powder

10 g of active ingredients

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.

Example 6: Toxicity Test

Acute Toxicity

This experiment was conducted to investigate the toxicity of animal fractions isolated from baekpibaek skin in acute (within 24 hours) 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 fractions isolated from the epidermis 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.

The experimental results showed that the epidermis fraction was a very safe substance with almost no toxicity, with one animal lethal dose of about 100 g / kg (100,000 mg / kg) and about 10,000 times the concentration (10 mg / kg) showing general efficacy. .

2. Organ and tissue toxicity

Active fractions isolated from epidermis were administered for 14 days at a concentration of 10 mg / kg and tested in female S.P.F C57BL / 6 mice used in a cancer metastasis animal model. To investigate the effects on the organs (tissues) of the animals, the heart, lung, pancreas, adrenal gland, liver, kidney and muscles were examined after 14 days from each animal in the experimental group administered with the epidermal active fraction and the control group administered only DMSO. Histopathological observations were made by optical microscopy after cutting through a conventional tissue fabrication process. As a result, no abnormality was observed in all histological observations of the control and experimental animals, and no toxicity to the animal organs of the active extract of the epidermis was found.

As described above, the active fraction obtained from the baekryepi skin according to the present invention is excellent in inhibiting heparinase enzyme activity and cancer metastasis activity can be used as a drug containing it as an active ingredient.

Claims (5)

An active fraction for heparanase enzyme activity or cancer metastasis obtained from Morus Cortex Radicis, root bark of Morus alba L. (1) pulverizing Morri Cortex Radicis and then extracting a solvent by adding an aqueous alcohol solution; (2) evaporating and concentrating the extract filtrate, suspending the concentrate in distilled water and extracting and concentrating the active substance with ethyl acetate to obtain an extract; (3) adsorbing the extract onto silica gel and then eluting with chloroform or methanol to separate the active fraction; And (4) separating and purifying the heparanase enzyme activity and cancer metastasis inhibiting active fraction obtained from lettuce skin, comprising adding the distilled water to the active fraction and suspending it homogeneously, followed by freeze drying. According to claim 2, Heparanase enzyme activity and cancer metastasis inhibiting active fractions obtained from lettuce skin, characterized in that eluted with a solution of chloroform or a mixture of chloroform and methanol in a 10: 1 step (3), separation, purification How to. Heparinase enzyme inhibitor, characterized in that the active fraction of claim 1 contained as an active ingredient. Cancer metastasis inhibitor, characterized in that the active fraction of claim 1 contained as an active ingredient.