KR960004026B1 - Angiogenesis inhibitor composition comprising ursolic acid - Google Patents

Abstract

The angiogenesis inhibitor composition contains 1˜10 microgram of an ursolinic acid. The ursolinic acid is produced by drying and crushing an eriobotrya japonica L., extracting it with a methanol, pressure reduction concentrating it to obtain a methanol extract, fractionating the extract with a polar solvent i.e. hexane, chloroform, ethyl acetate, butanol or water, purifying the fractionated material by the chromatography, and recrystallizing the purified material in the ethanol.

Description

Angiogenesis Inhibitor Composition Containing Ursolic Acid

FIG. 1 is a photograph showing that angiogenesis is inhibited in CAM when treated with ursolic acid and retinoic acid.

The present invention relates to an angiogenesis inhibitor composition containing ursolic acid obtained from Eriobotrya japonica L. and the like. More specifically, ursolic acid, an angiogenesis-inhibiting factor, exhibits the effect of inhibiting blood vessel formation when treated with chorioallantoic membranes (weakly referred to as CAM and used in the specification) that relate to respiration during fertilization. An angiogenesis inhibitor composition is included.

Blood vessel formation involves a series of actions leading to the migration and proliferation of vascular endothelial cells and differentiation into angiogenic cells, including embryonic development, wound healing, and chronic inflamation. It is also closely related to various physiological and pathological phenomena.

Abnormal blood vessel formation is an important prerequisite for cancer growth and metastasis, as well as diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, and corneal transplantation. ocular diseases such as cornealgraft neovascularization, trachoma, rheumatoid arthritis, hemangiomas, angiofibroma, psoriasis, pyogenic granuloma, etc. It is also associated with the induction of various diseases due to excessive angiogenesis.

Therefore, screening of angiogenesis inhibitors for the development of effective angiogenesis inhibitors will enable new treatments for these diseases.

In particular, when the disease death is related to the cancer which enters the upper level, as the growth of cancer cells, the surrounding host tissues form new blood vessels, and thus angiogenesis is indispensably involved in the proliferation and metastasis of cancer or tumors. Therefore, inhibition of blood vessel formation is associated with inhibition of cancer growth and metastasis, and these angiogenesis inhibitors are important subjects to develop anti-transduction factors in cancer treatment.

Methods for screening angiogenesis inhibitors include methods using rabbit corneas (Brem H. & Folkman J., J. Exp. Med., 141, 427, 1975; Langer et. Al. Science, 193, 70, 1976; Lee A. & Langer R., Science, 221, 1185, 1983), but recently use a more convenient and simpler fertilized egg CAM (Oikawa et. Al., Cancer Lett., 48, 157, 1989; Ausprunk et. al., Issues Biomed., 14, 93, 1991).

The CAM is produced on the fourth day of embryonic development in fertilized eggs, and can be clearly distinguished from capillaries and other blood vessels, thereby analyzing the movement and formation patterns of blood vessels. In the present invention, instead of using an artificial membrane containing angiogenesis-inhibiting factor among the methods of Oikawa et al., Instead of slightly modifying this method, angiogenesis-inhibiting factor is applied as a simpler method of applying angiogenesis-inhibiting factor to cover slip. Screened.

Known angiogenesis inhibitors include protamine sulfate, tumor necrosis factor, factors derived from cartilage tissue, antibiotics such as herbimycin A and eponmycin and angiogenesis such as cortisone. It has been reported that there are angiostatic steroids, and that angiogenesis inhibitors are also shown to be potent when using acetic acid-medroxy progesteroneacetate alone and also hydrocortisone with heparin (Takigawa et. Al. Biochem). Inf., 14, 357, 1987; Oikawa et. Al. Cancer Lett., 43, 85, 1988). Recently, retinoids, known as cell differentiation inducers, and synthetic derivatives including active metabolites of vitamin D ₃ have also been found to significantly inhibit angiogenesis. However, these substances also have a toxic effect (Kamm, jjet al., 13. Preclinical and clinical Toxicology of selected Retinoids. Edited by Sporn, MB ets al., The Retinoids. Vol 2. p 287 (1984)).

Therefore, the inventors of the present invention, while conducting research to develop safer angiogenesis inhibitors, have found that ursolic acid obtained from non-lobal leaves exhibits an effective angiogenesis inhibitory effect and has no side effects.

Ursolic acid is a component extracted from anti-leaflet, an herbal medicine with anti-inflammatory effect, which inhibits the deterioration of cancer in the skin of rats, and is a lymphoblastoid cell having the genome of Epstein-Barr virus (EBV). Although it has an effect of inhibiting EBV activation in phosphorus Raji cells, and its toxicity is reported to be weaker than retinoic acid, it has been found for the first time by the present inventors to have an angiogenesis inhibitory effect.

That is, the present invention relates to an angiogenesis inhibitor composition containing ursolic acid.

Hereinafter, the present invention will be described in more detail.

Ursolic acid according to the present invention is obtained from non-leaflets. After drying and grinding the non-leaflets, the methanol extract is extracted with methanol, and the methanol extract is fractionated with a polar solvent and purified by chromatography or the like to purify ursolic acid. Ursolic acid may be administered in an amount effective to inhibit angiogenesis, for example, from 0.1 to 100 μg, preferably from 1 to 10 μg. Angiogenesis inhibitor compositions containing ursolic acid according to the present invention may be administered orally or parenterally and contain pharmaceutically acceptable excipients.

Ursolic acid has been reported to be very effective in hepatoprotective and hepatocellular survival tests (B. Shukla et al., Phytotherapy Research, Vol. 6, 74-79 (1992)) and is more toxic than conventional angiogenesis inhibitor retinoic acid. It is weak and can be used safely in vivo.

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

Example 1

[Purified Ursolic Acid from Eriobotya japonica L.]

The dried loquat (Eriobotrya japonica L.) was pulverized and extracted with methanol three times for 3 hours in a water bath, and concentrated under reduced pressure to obtain a methanol extract.

The methanol extract of the experimental material was systematically fractionated with hexane, chloroform, ethyl acetate, butanol, and water, and each fraction was dried and used as a sample.

Liebermann-Burchard reaction was performed on each of the fractions to determine the presence of triterpenoid (Triterpenoid) and ethyl acetate fraction in the butanol fraction. , E. Merk, Germany), was subjected to thin layer chromatography using benzene-ether (mixing cost 4: 1 → 2: 1) as a solvent and divided into four subfractions. The fractions were purified several times to identify a single spot on TLC and recrystallized in ethanol to give colorless needles (Compound I) (780 mg).

The structure of the isolated compound I was confirmed by melting point and ¹ H-NMR, ¹³ C-NMR, IR spectral data, and the instrument was analyzed by ¹ H-, ¹³ C-NMR spectroscopy (Brucker 300, Germany, 300 MHz), mass spectrometer (Geol). , double focus, JMSDX 303). As a result, it was confirmed that Compound I is consistent with ursolic acid standard and all physical and chemical properties.

Example 2

[CAM assay]

Seven fertilized eggs of the purchased Abaica broiler chicken were placed at 18 ° C. for 45 hours, and then placed in a 37 ° C. incubator maintained at 60% humidity, and cultured at 0-fold. After 3 days, 2 ml of albumin is extracted from the lower part of the fertilized egg, and after 4 days, the upper part of the fertilized egg air bag is sterilized with iodine tincture, a window is used to make a window, and the tweezers under the air bag Removed.

A round coverslip with a diameter of 13 mm, which was dried for 40 minutes by applying 100 μg of 5 μl solution of ursolic acid, was contacted on a 4 mm fold of 3-5 mm in size and covered with a glass tape. After two days of incubation in the incubator, 10% emulsion (Intralipid, Green Cross) was injected into the CAM and photographed the CAM based on the white color produced by the emulsion. The results are shown in FIG. 1 is a picture of the control group treated with nothing and 2 is a picture of CAM treated with ursolic acid. It can be determined that the angiogenesis-inhibiting effect is when the site where the angiogenesis is suppressed is 3 mm or more in comparison with the control group treated with nothing, and as shown in FIG. 1, the angiogenesis was significantly suppressed as compared with the control group. In addition, 1/8 of the control group was inhibited, and the group treated with ursolic acid was 100% inhibited to 7/7.

Example 3-7

As in the method of Example 2, when different concentrations of ursolic acid were administered at 1, 5, 10, 50, and 100 µg, 5/25 (20%), 13/26 (50%), and 16 / 21 (76.8%), 17/19 (89.5%), and 24/25 (96%) inhibited angiogenesis.

Therefore, the effect was increased according to the concentration, 50% angiogenesis inhibitory effect (ID ₅₀ ) was found to be 5㎍ / fertilized egg, that is 11nmol / fertilized egg.

On the other hand, when retinoic acid 0.01, 0.1, 1 ㎍ was treated, 1/5 (20%), 4/9 (44%) and 7/8 (87.5%), respectively.

In the above, ursolic acid obtained from the non-leaflet was observed using CAM to inhibit angiogenesis, and the 50% angiogenesis inhibitory effect (ID ₅₀ ) was 5 µg / fertilized egg, that is, 11 nmol / fertilized egg.

Claims (2)

An angiogenesis inhibitor composition comprising ursolic acid as an active ingredient in an amount of 1 to 10 µg. The composition of claim 1, wherein the ursolic acid is obtained from Eriobotrya japonica L ..