JP2007008907A - Vascularization inhibitor and external preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vascularization inhibitor that has the action to inhibit vascularization and provide an external preparation including the vascularization inhibitor. <P>SOLUTION: The vascularization inhibitor includes at least one selected from the group consisting of the extract of Isodon japonicus, the extract of Rubus suavissimus, the extract of Raeonia suffrutcosa and the extract of Daeonia lactiflora. An external preparation including the vascularization inhibitor. In an preferred embodiment, the vascularization inhibitor at least includes the extract of Isodon japnicus. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a natural angiogenesis inhibitor and an external preparation containing the angiogenesis inhibitor, which have an excellent angiogenesis inhibitory effect and are effective for the prevention and treatment of various diseases associated with angiogenesis.

  Angiogenesis means a phenomenon in which new blood vessels are formed from existing blood vessels. The outer envelope (basement membrane) of blood vessels is degraded by proteases produced by vascular endothelial cells, and migrates, settles, and proliferates there. It occurs when vascular endothelial cells differentiate to form vascular cavities. In addition to being observed under physiological conditions such as luteinization, ovulation, embryogenesis, and placenta formation, the angiogenesis plays an important role in the development of living organisms and the recovery of tissues, such as occurring during wound healing and inflammation repair processes. On the other hand, solid tumors (gastric cancer, colon cancer, lung cancer, pancreatic cancer, etc.) growth and metastasis, various inflammatory diseases (diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, etc.), atheromatous It is known to be related to pathological conditions such as arteriosclerosis and myocardial infarction (see Non-Patent Documents 1 to 5).

  In recent years, ligands such as Vascular Endothelial Growth Factor (VEGF) and angiopoietin, which specifically act on vascular endothelial cells, and their receptor systems have been discovered one after another, and the relationship between angiogenesis and the disease has become clearer Has been. Therefore, suppression of angiogenesis is thought to lead to the treatment and prevention of the disease.For example, the growth of the solid tumor requires major angiogenesis to supply oxygen and nutrients. If angiogenesis is inhibited and the solid tumor is brought into a so-called “military attack” state, the tumor can be expected to shrink as a result. Also, for diabetic retinopathy, it is thought that the formation of microcapillaries is promoted due to lack of oxygen, which eventually ruptures and bleeds to form scar tissue, leading to retinal detachment Therefore, the suppression of the angiogenesis can be expected to suppress the seriousness of the retinopathy.

Therefore, many methods for suppressing the angiogenesis have been studied for the purpose of treating and preventing the disease, and for example, changes in the skin vasculature due to long-term ultraviolet exposure have been reported (Non-patent Document 6).
In addition, many substances having an anti-angiogenic activity have been searched, and extracts derived from basidiomycetes (Patent Document 1) and citrus genus plants are derived from natural products that are advantageous in terms of safety. Effectiveness has been confirmed for components (Patent Document 2) and the like.

  Therefore, to date, no angiogenesis inhibitors that are readily available, inexpensive, highly safe natural products, and that can be suitably used for the treatment and prevention of various diseases have yet to be provided. However, there is a strong demand for prompt provision.

JP 2004-307453 A JP 2004-331567 A Folkman et al., Nature, Vol. 288, 551-556, 1980 Hitoshi Takagi, Cell Engineering, Vol. 19, no. 8,1160-1165,2000 Satoshi Terano, Cell Engineering, Vol. 14, no. 4,426-430, 1995 Richard D Connell, Exp. Opin. Ther. Patens, 10 (6), 767-786, 2000 Masafumi Shibuya, Protein Nucleic Acid Enzyme, Vol. 45, no. 6,1182-1187,2000 Kiichiro Yano, 23rd IFSCC Orlando Conference Proceedings, 18-22, November 2004

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the first object of the present invention is to provide a naturally-occurring angiogenesis inhibitor that has an excellent angiogenesis-inhibiting action, is highly safe, and is readily available as a raw material.
A second object of the present invention is to provide an external preparation containing the angiogenesis inhibitor.

  In order to solve the above-mentioned problems, the present inventors have conducted intensive studies, and as a result, the extract of enamel, extract of tencha, extract of button and extract of peonies have an excellent anti-angiogenic action, The present inventors have found that it is effective as an angiogenesis inhibitor for various diseases requiring angiogenesis inhibition.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> An angiogenesis inhibitor characterized by containing at least one selected from an extract of enamel, an extract of tencha, an extract of buttons, and an extract of peonies.
<2> The angiogenesis inhibitor as described in <1> above, which contains an extract of enamel.
<3> An external preparation comprising the angiogenesis inhibitor according to any one of <1> to <2>.

According to the present invention, there is provided an angiogenesis inhibitor that can solve the above problems and has an excellent angiogenesis inhibitory action. In addition, the angiogenesis inhibitor of the present invention is naturally derived and is excellent in the feeling of use and safety, and therefore is suitable for blending into an external preparation.
Since the external preparation of this invention mix | blends the angiogenesis inhibitor of this invention, it has the outstanding angiogenesis inhibitory effect.

(Angiogenesis inhibitor)
The angiogenesis inhibitor of the present invention contains at least one selected from an extract of enamel, an extract of tencha, an extract of buttons and an extract of peonies, and further contains other components as necessary. It contains. Among these, it is particularly preferable to contain an extract of enamel from the viewpoint of high angiogenesis inhibitory action.

The Enmezo ( Isodon japonicus ) is a perennial herb belonging to the genus Lamiaceae that is distributed from the southwestern part of Hokkaido to Kyushu. The enamel is known as a healthy stomach medicine and can be used for indigestion, anorexia, mild stomach pain, and the like.

The tencha ( Rubus suavissimus ) is a shrub with a height of 2-3 m in the genus Rosaceae, distributed from Guangxi to Guangdong Province in southwestern China. Tencha is said to be effective in promoting appetite, expectoration, coughing, antipyretic, etc. Recently, anti-allergic effects, bad breath prevention effects, good mineral balance, etc. have been found one after another in the course of research, and its taste is good. It is also widely known as a healthy tea.

Said button (peony, Paeonia suffruticosa), the northwest China origin unit of origin, also known Hatsukagusa, Fukamigusa a deciduous shrub peony called Natorigusa etc., are generally planted garden as an ornamental. Paeoniflorin, the main component of the skin, has effects such as anti-inflammatory, analgesic, blood purification, drainage, etc., and is used as a medicine for gynecological diseases, constipation, hemorrhoids, etc.

The peony (a glaze Paeonia lactiflora ) is a perennial herbaceous plant that grows naturally in the northwestern part of China, North China, the northwest region, East Siberia, and the Korean Peninsula. The peonies were not native to Japan and were said to have come as drugs during the Nara period, and many varieties were created for ornamental use during the Edo period. The peony has effects such as blood circulation promotion, analgesia, antipyretic, diuresis, and astringency, and is used as a medicine for various gynecological diseases in the Chinese medicine as well as the button.

  The extract of enamel, tencha extract, button extract and peonies extract have been used for various purposes since ancient times and have high safety, but has an excellent anti-angiogenic action and angiogenesis. It is not known at all to be useful as an inhibitor, and this is a new finding by the present inventors.

  The details of the substance having an anti-angiogenic activity of the extract of the gypsophila, the extract of the tencha, the extract of the button and the extract of the peony are unknown, but these active ingredients are generally used for the extraction of plants. The extraction method can be obtained. The extract includes an extract, a dried product obtained by drying a diluted solution of the extract, and a crude product or a purified product thereof.

There are no particular restrictions on the parts used for extraction of the enamel, tencha, button and peonies that are the extraction raw materials to be subjected to the extraction treatment, such as leaves, branches, roots, bark parts, stem parts, fruit parts, etc. Can be used.
Each of the extraction raw materials is preferably dried and crushed in advance, or may be used after degreasing with a nonpolar solvent such as hexane. In addition, the extraction process by the polar solvent of each said extraction raw material can be performed efficiently by performing pre-processing, such as degreasing.

As the solvent used for the extraction, it is preferable to use water, a hydrophilic organic solvent, or a mixture thereof at room temperature or a temperature not higher than the boiling point of the solvent.
Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol, Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, 1-90 mass parts with respect to 10 mass parts of water in the case of the said lower alcohol, and 10 mass of water in the case of the said lower aliphatic ketone. In the case of the polyhydric alcohol, 1 to 90 parts by mass is preferably added to 10 parts by mass of water.

  In the present invention, it is not necessary to employ a special extraction method for extracting a substance having an angiogenesis inhibitory action from each of the extraction raw materials, and the extraction can be performed using an arbitrary apparatus at room temperature or under reflux heating.

Specifically, each extraction raw material is put into a treatment tank filled with an extraction solvent, and is allowed to stand for 30 minutes to 2 hours with occasional stirring as necessary to elute soluble components, and then filtered and solidified. An extract is obtained by removing a thing, distilling an extraction solvent off from the obtained extract, and drying. The amount of the extraction solvent is preferably 5 to 15 times the mass of the extraction raw material (mass ratio), and the extraction conditions are usually about 50 to 95 ° C. for about 1 to 4 hours when water is used as the extraction solvent. It is. Moreover, when using the mixed solvent of water and ethanol as an extracting solvent, it is about 30 minutes-about 4 hours at 40-80 degreeC normally.
In order to obtain the obtained enamel extract, tencha extract, button extract or peony extract, a diluted or concentrated solution of the extract, a dried product of the extract, or a crude or purified product thereof. Treatments such as dilution, concentration, drying and purification may be performed according to conventional methods.

  Each extract obtained can be used as an active ingredient of the angiogenesis inhibitor of the present invention as long as the extraction solvent is highly safe, but it can be used as a concentrated solution or a dried product thereof. Things are easier to use. In obtaining a dried product of the extract, carriers such as dextrin and cyclodextrin may be added to improve hygroscopicity.

  In addition, although the obtained extract of enamel, extract of tencha, extract of button or extract of peony has a unique smell, it is not unpleasant and can be used as an angiogenesis inhibitor as it is. It is. However, if necessary, purification for the purpose of improving the angiogenesis inhibitory effect and decolorization / deodorization may be carried out, or it may be mixed with an optional auxiliary agent. The angiogenesis inhibitor containing the extract can be made into any dosage form such as powder, granule, tablet, etc. by formulation. In addition, each extract may be used individually by 1 type, and may use 2 or more types together.

  Since the angiogenesis inhibitor of the present invention has an excellent angiogenesis inhibitory effect, it is useful for the prevention and treatment of various diseases associated with angiogenesis, and may be applied directly to the skin, etc. It is preferable to mix and use, and it can be used particularly suitably for the following external preparations of the present invention.

(External preparation)
The external preparation of the present invention contains the angiogenesis inhibitor of the present invention, and further contains other components appropriately selected as necessary.

  The said external preparation means various chemical | medical agents, For example, cosmetics, a quasi-drug, a pharmaceutical, etc. are contained. Specific examples of external preparations may include ointments, pops, creams, emulsions, essences, lotions, packs, jellies, foundations, etc. for the skin, and tonics, rinses, shampoos, astringents, hair creams for the scalp. , Hair foam, hair spray, hair gel and the like, but are not limited thereto.

Examples of the other components include auxiliary raw materials or additives usually used in producing the external preparation.
The raw material or additive may be selected within a range that does not impair the effects of the present invention. Examples include astringents, bactericides / antibacterial agents, whitening agents, ultraviolet absorbers, cell activators, anti-inflammatory / antiallergic agents, Examples include oxidizing / active oxygen scavengers, purified water, lower alcohols, polyhydric alcohols, fats and oils, powders, surfactants, dyes, preservatives, and fragrances.

The external preparation of the present invention can be prepared by blending with various forms of bases known as ordinary external preparations.
The amount of the angiogenesis inhibitor of the present invention added to the external preparation is preferably in the range of 0.001 to 10 mass%, more preferably 0.01 to 5 mass% as the dry solid content with respect to the total amount of external preparation. Range. Within this range, an anti-angiogenic effect can be expected.

  The external preparation of the present invention has high safety, and effectively suppresses the progression of angiogenesis by the action of an extract of enamel which is an active ingredient, an extract of tencha, an extract of a button or an extract of peony. Thus, various diseases associated with angiogenesis can be prevented and treated.

  The angiogenesis inhibitor and the external preparation of the present invention described above are preferably applied to humans, but may be applied to animals other than humans as long as the effects of the present invention are exhibited. May be.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Production Example 1)
-Manufacture of 50% ethanol extract of Enmeiso-
2,000 ml of 50% ethanol (mass ratio of water and ethanol: 1: 1) as an extraction solvent is added to 200 g of the dried portion of the dried portion of the above-ground Amaranthus and heated at 90 ° C for 2 hours with a reflux extractor. Extract and filter hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and dried to obtain a 50% ethanol extract of enamel. The yield of the extract was as shown in Table 1.

(Production Example 2)
-Production of 50% ethanol extract of Tencha-
2,000 ml of 50% ethanol (mass ratio of water and ethanol: 1: 1) as an extraction solvent is added to 200 g of the dried leaves of tencha leaves and heated at 90 ° C for 2 hours with a reflux extractor. Extract and filter hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and dried to obtain a 50% ethanol extract of tencha. The yield of the extract was as shown in Table 1.

(Production Example 3)
-Production of 50% ethanol extract of buttons-
2,000 ml of 50% ethanol (mass ratio of water and ethanol: 1: 1) as an extraction solvent is added to 200 g of the dried bark portion of the button, and heated at 90 ° C. for 2 hours with a reflux extractor. Extract and filter hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and dried to obtain a 50% ethanol extract of a button. The yield of the extract was as shown in Table 1.

(Production Example 4)
-Production of 50% ethanol extract of peony-
2,000 ml of 50% ethanol (mass ratio of water and ethanol: 1: 1) as an extraction solvent is added to 200 g of dried peony roots, and extracted with a reflux extractor at 90 ° C. for 2 hours. And filtered while hot. The same extraction process was further performed on the residue. The obtained extracts were combined, concentrated under reduced pressure, and dried to obtain a 50% ethanol extract of peony. The yield of the extract was as shown in Table 1.

Example 1
-Angiogenesis inhibitory action test-
Each extract obtained in Production Examples 1 to 4 was tested for angiogenesis inhibitory effect by the following method. For angiogenesis, a method of culturing a rat artery piece in a collagen gel was used.

-Test animal-
Wistar male rats 6-8 weeks old (body weight 160-240 g) purchased from CLEA Japan were subjected to the following angiogenesis inhibitory action test.

--Angiogenesis inhibitory action test method--
The test animal was anesthetized with diethyl ether and bleeded by cutting the right femoral artery, and then the thoracic aorta was taken out and cut to a length of 1 to 1.5 mm. The arterial piece was transferred to a 6-well culture plate, embedded with a collagen gel solution, and gelled at 37 ° C. Thereafter, 2 ml of RPMI 1640 medium (Gifco) containing 1% ITS + (Becton Dickinson Bioscience) was added. Next, each extract obtained in Production Examples 1 to 4 was added to a concentration of 100 μg / ml, and cultured in a 5% CO ₂ incubator for 7 days. For the control, the same operation was performed except that no extract was added.
On the 7th day from the culture, the microvessel hairs generated from the arterial piece were photographed under an inverted microscope, the image was taken into a computer, and the length of the microvessel was measured. From the obtained results, the ratio of the average microvessel length of the sample to which each extract was added relative to the average microvessel length of the control was calculated, and was used as the inhibition rate (%). The inhibition rate (%) and standard deviation (SD) are shown in Table 2.
The collagen solution is composed of a collagen stock solution (cell matrix type Ia, manufactured by Nitta Gelatin), 10 × Eagle's MEM medium (Gifco), and a reconstitution buffer (made by Nitta Gelatin) in a volume ratio. A mixture prepared to be 8: 1: 1 and stored at 4 ° C. was used.

表２の結果から、製造例１〜４の各抽出物が血管新生抑制作用を有することが確認できた。特にエンメイソウの抽出物に優れた血管新生抑制作用が認められた。

From the results in Table 2, it was confirmed that the extracts of Production Examples 1 to 4 have angiogenesis-inhibiting action. In particular, an anti-angiogenic action was recognized for the extract of Enmeiso.

(Formulation Example 1) -Ointment-
An ointment having the following composition was produced by a conventional method.
50% ethanol extract of Enmeiso (Production Example 1) 5.00 g
White petrolatum 24.00g
Stearyl alcohol 20.00g
Propylene glycol 12.00g
Indomethacin 1.00g
Fragrance 0.10g
0.05 g of methyl paraoxybenzoate
Purified water balance
Total 100.00g

The angiogenesis inhibitor of the present invention suppresses angiogenesis, for example, growth and metastasis of solid tumors (gastric cancer, colon cancer, lung cancer, pancreatic cancer, etc.), and various inflammatory diseases (diabetic retinopathy, macular degeneration) Rheumatoid arthritis, psoriasis, etc.), atherosclerosis, myocardial infarction and other various diseases can be prevented and treated, and it is preferably used as an external preparation.
Since the external preparation of the present invention containing the angiogenesis inhibitor has an excellent anti-angiogenesis action and is excellent in safety, it can be used, for example, in various external preparations such as cosmetics, quasi drugs, and pharmaceuticals. Widely used.

Claims (3)

  An angiogenesis inhibitor comprising at least one selected from an extract of enamel, an extract of tencha, an extract of buttons, and an extract of peony.   The angiogenesis inhibitor according to claim 1, comprising an extract of enamel. An external preparation comprising the angiogenesis inhibitor according to claim 1.