JP2008088119A - Anti-neovascular composition comprising marine organism phospholipid and genistein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition enhanced in the anti-neovascular activity of genistein as its component. <P>SOLUTION: The anti-neovascular composition comprises a marine organism phospholipids and genistein. This composition has a synergistic anti-neovascular activity. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an anti-angiogenic composition comprising marine phospholipids and genistein.

  The present invention also relates to a method for treating or preventing a disease or psychosomatic dysfunction that is effective in suppressing angiogenesis, using the anti-angiogenic composition.

Angiogenesis means the proliferation and growth of new blood vessels. Angiogenesis plays an important role in various physiological and pathological conditions.
In healthy bodies, angiogenesis occurs during the proliferative phase of cells in wound healing, to restore blood flow to tissues after injury, and during menstrual periods and pregnancy in women.
In a healthy body, the promotion and suppression of angiogenesis is controlled by switching between “on” and “off”. The “on” switch is an angiogenesis stimulating factor, and the “off” switch is an angiogenesis inhibiting factor. is called.
In a normal healthy body, an angiogenesis factor and an angiogenesis inhibitory factor are balanced in order to control the promotion and suppression of angiogenesis.

  The lack of control of angiogenesis causes many serious diseases. Many diseases caused by so-called “excessive neovascularization” are known. These diseases include psoriasis, arthritis, retinopathy, macular degeneration and cancer. For example, cancer has high metabolic efficiency compared to normal growth and organs. Thus, because cancer requires more blood to obtain a higher nutrient supply, inhibition of angiogenesis can be a mechanism for inhibiting or controlling tumor growth. Thus, angiogenesis inhibitors can delay the progression of these diseases.

  Many anti-angiogenic compositions and formulations (Non-Patent Documents 1 and 2) are known, including lipid-based formulations. One of these is derived from various types of shark muscle tissue (Patent Document 1).

日本をはじめアジアの人々は性別特有のガン（乳ガン、前立腺ガンなど）や大腸ガン、更年期障害、骨粗鬆症などの罹患率が低いことが疫学的調査から知られている。アジア人は大豆食品を日常的に多く摂取するため、その中に含まれるイソフラボンがその健康に大きく関わっていると考えられてる。イソフラボンの中でも特に、ゲニステインは血管内皮細胞増殖と生体内の血管新生形成を抑制することが知られている。ゲニステインは生体内の血管新生形成の抑制作用機序としては、それぞれvascular endothelial growth factor 165, platelet-derived growth factor, matrix metalloprotease-2 やmatrix metalloprotease- 9 などの活性抑制を示す。（非特許文献３−１３）。 Genistein (Genistein, 4 ', 5,7-trihydroxyisoflavone) is a kind of soy isoflavone. Molecular structure C ₁₅ H ₁₀ O ₅ , molecular weight 270.24 daltons.

It is known from epidemiological studies that Asians including Japan have a low incidence of gender-specific cancers (breast cancer, prostate cancer, etc.), colon cancer, menopause, and osteoporosis. Since Asians consume a large amount of soy food on a daily basis, it is thought that the isoflavones contained in them are greatly related to their health. Among isoflavones, genistein is known to suppress vascular endothelial cell proliferation and angiogenesis in vivo. Genistein inhibits the activities of vascular endothelial growth factor 165, platelet-derived growth factor, matrix metalloprotease-2, matrix metalloprotease-9, etc. (Nonpatent literature 3-13).

However, since the action of genistein is not sufficient, it is desired to enhance the action.
Special table 2004-516272 Scappaticci, Expert Opinion on Investigational Drugs, 12: 923-32. Caceres and Gonzalez, Puerto Rico Health Sciences Journal, 22: 149-51. Polkowski K, et al. Acta Pol Pharm. 57 (2): 135-55. (2000) S. Kapiotis, et al. Arteriosclerosis, thrombosis, and vascular biology. 17: 2868-2874 (1997). Setchell, K, et al. J. Nutr., 131, 1362S-1375S (2001). King, R. A, et al. J. Nutr., 126, 176-182 (1996). Slavin, J. et al. Am. J. Clin. Nutr., 68 (suppl), 1492S-1495S (1998). Xu, X. et al. J. Nutr., 125, 2307-2315 (1995). T Akiyama, et al. J. Biol. Chem. 262: 12, 5592-5595, (1987) Y Matsukawa, et al. Cancer Research, 53: 6 1328-1331, (1993) J Markovits, et al. Cancer Research, 49:18, 5111-5117, (1989) Y. Li, et al. J. Nutr. 132: 3623-3631, (2002) Fotsis T, et al. J Nutr. 125 (3 Suppl): 790S-797S. (1995)

  An object of the present invention is to enhance the effect of genistein.

The inventor has achieved the present invention by finding that a mixture of marine phospholipids and genistein enhances the anti-angiogenic effect of genistein.
First, the present invention provides an anti-angiogenic composition containing marine phospholipids and genistein.
In a first embodiment of the present invention, the marine organism phospholipid is a shark meat extract and / or a mussel meat extract. The mussel extract is preferably an extract of New Zealand green mussel (Perma canaliculus) meat.

Desirably, the anti-angiogenic composition of marine phospholipids and genistein further comprises an edible oil such as olive oil. The anti-angiogenic composition preferably contains vitamin E.
The ratio based on the weight of marine phospholipid and genistein contained in the anti-angiogenic composition may be any weight ratio that provides a synergistic effect, but marine phospholipid: genistine is 10: 0. It is in the range of 1-10: 10, preferably 10: 0.5-10: 5. The optimum is 10: 0.7 to 10: 1.

  Secondly, the present invention provides a method for treating or preventing a disease or disorder in which it is beneficial to suppress angiogenesis by administering an anti-angiogenic composition to a mammal.

  The present invention also provides an anti-angiogenic composition for preventing or treating a disease or disorder considered to be beneficial for inhibiting angiogenesis.

  Diseases and disorders useful for inhibiting angiogenesis according to the present invention are arthritis, retinopathy, macular degeneration, and cancer. The target of treatment or prevention is a mammal, for example, a pet such as a human, a dog, or a cat, or a livestock animal such as a cow, pig, or chicken, and is preferably a human.

  The present invention also provides the use of an anti-angiogenic composition for the manufacture of a medicament for the prevention and treatment of diseases and disorders for which it is thought beneficial to inhibit angiogenesis.

  Surprisingly, according to the present invention, a mixture of marine phospholipids and genistein, such as shark meat extract and mussel meat extract, remarkably enhances the anti-angiogenic effect compared with genistein alone. .

  Thus, by combining marine phospholipids with genistein, the anti-angiogenic effect can be enhanced compared to when each is used alone. In particular, the combination of marine phospholipids and genistein produced from shark meat extract and / or mussel meat extract can enhance the anti-angiogenic effect of these extracts than when each is used alone. .

  The present invention provides an anti-angiogenic composition comprising marine phospholipids and genistein. In particular, it relates to an anti-angiogenic composition containing marine phospholipids and genistein from shark meat extract and / or mussel meat. The anti-angiogenic action means an angiogenesis inhibitory action, that is, an action that inhibits or suppresses the proliferation and growth of new blood vessels.

  The term “marine organism phospholipid” in the present invention means a phospholipid extracted from marine organisms, and examples thereof include phospholipids extracted from shark meat and phospholipids extracted from mussel meat. . Here, marine organisms mean organisms such as animals, plants, and microorganisms that inhabit the sea, and marine organisms may be processed or unprocessed.

  The term “extraction” in the present invention means a solvent fraction after mixing a solid and / or liquid with a specific solvent or a substance after removing the solvent from the solvent fraction. The extract includes those that have been fractionated and isolated by ordinary methods such as chromatography and filtration.

  Extraction methods include solvent extraction methods, but are not necessarily limited thereto. Any suitable solvent may be used, and the solvent may be an organic solvent, an aqueous solvent, or a mixed solvent thereof. Ethanol is a typical organic solvent (or aqueous ethanol mixed solvent), but other organic solvents (for example, methanol, ethyl acetate, dichloromethane, or a mixed solvent thereof) may be used. . In addition, a supercritical fluid such as supercritical carbon dioxide can be used as the extraction solvent.

The term “shark meat extract” in the present invention means a phospholipid obtained from shark meat using the above-mentioned solvent. In particular, it means a phospholipid extracted from shark meat disclosed in Patent Document 1. The term “shark meat” as used herein refers to all fish meat parts including edible parts, but in order to avoid misunderstanding, the internal organs parts are not included.
The shark meat extract of the present invention is generally an oily substance, such as an oil or paste, at ambient temperatures that generally range from about 15 ° C to 25 ° C.

  It is known that the main component of phospholipid extracted from shark meat is phospholipid such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) (Patent Document 1). The phospholipid content is usually in the range of 20-40% by weight. Furthermore, it usually contains phospholipid fatty acids such as docosahexaenoic acid (DHA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and the like. Compared to other fish extracts or products, shark meat extract contains a high concentration (20-35% by weight) of DHA relative to total fatty acids. DHA samples are known to be potent inhibitors of angiogenesis (Patent Document 1). Since the DHA concentration in the shark meat extract is high, it is presumed that DHA is a component that plays a part in the ability of this extract to suppress angiogenesis. The shark meat extract is also clearly different from other fish extracts and products in that the triglyceride concentration is low (less than 10% by weight).

  In a typical extraction method, one or more pieces of shark meat are minced using a blender, freeze dried (or air dried), and then mixed with an extraction solvent (eg, ethanol). Usually, the mixture is stirred at room temperature for 1 to 24 hours, and then the extract and the meat pieces are separated by filtration. Mixing of meat pieces and solvent is optional, but repeated extraction is performed. Then, the solvent is removed by concentrating at as low a temperature as possible (preferably 50 ° C. or less), and the shark meat extract can be obtained as an oil or oily substance. This oil or oily substance usually contains various free fatty acids and glycerol-bound fatty acids.

  The term “shark meat” in the present invention is not limited to those described herein, but includes rig (lemonfish), school shark, ghost shark (mako), blue shark (blue) Means meat from various sharks or shark-like organisms, including shark, elephant fish, salmon shark, and blacktip reef shark.

  The term “mussel meat extract” in the present invention means a phospholipid extracted from mussel meat using the above-mentioned solvent. The “mussel meat” mentioned here refers to all shellfish parts including edible parts, and also includes internal organs parts. The mussel extract of the present invention is generally an oily substance, such as an oil or paste, at ambient temperatures that generally range from about 15 ° C to 25 ° C.

  In a typical extraction method, one or more mussel pieces are minced using a blender, freeze dried (or air dried), and then mixed with an extraction solvent (eg, ethanol). Usually, the mixture is stirred at room temperature for 1 to 24 hours, and then the extract and the meat pieces are separated by filtration. Mixing of meat pieces and solvent is optional, but repeated extraction is performed. Thereafter, the solvent is removed by concentrating at as low a temperature as possible (preferably 50 ° C. or less), and the mussel extract can be obtained as an oil or oily substance. This oil or oily substance usually contains various free fatty acids and glycerol-bound fatty acids.

  The term “mussel” in the present invention refers to various mussel meats, including but not limited to those described herein, including green mussels (Perna canaliculus) and purple mussels (Mytilus edulis).

  The term “genistein” in the present invention means a commercially available genistein product or composition. Preferably, it means a genistein product or composition comprising genistein in an amount of 90% by weight, preferably 95% by weight, based on the total composition. The origin of isoflavones including genistein is not particularly limited, and refers to all isoflavones obtained from plants (eg, soybean). For example, soy genistein may be a commercially available isoflavone product or composition that includes isoflavones made from soy.

  The “anti-angiogenic composition” in the present invention means a composition having an anti-angiogenic action.

  The anti-angiogenic mixture of the present invention can be used alone or in a mixture with edible oil for oral administration and encapsulated. It is also possible to mix the anti-angiogenic composition with a solid carrier such as cyclodextrin to form a tablet, granule, powder or the like. The granules, powders, and similar shapes may be included or mixed with other substances such as food for oral administration. It is also conceivable that the anti-angiogenic composition can be dissolved in a solvent corresponding to the injection. In addition, the addition of ingredients such as vitamin E may also be included in the formulation. Alternatively, it is considered that the anti-angiogenic composition can be used as an additive component in cosmetics.

  Edible oils include olive oil, sesame oil, cottonseed oil, peanut oil, palm oil, rapeseed oil, corn oil, linseed oil, wheat germ oil, soybean oil, castor oil, rice oil and the like. These may be used alone or in combination. These edible oils may be commercially available or prepared by themselves. Preferably, it is olive oil.

  Olive oil means oil extracted from crushed or squeezed olive fruit. Similarly, sesame oil, cottonseed oil, peanut oil, palm oil, rapeseed oil, corn oil, linseed oil, wheat germ oil, soybean oil, castor oil, and rice oil are obtained by grinding or squeezing each seed and fruit. means.

  Vitamin E means α-, β-, γ-, δ-tocopherol and α-, β-, γ-, δ-tocotrienol, and is used for antioxidant purposes.

  The anti-angiogenic composition of the present invention may further contain sweetening agents, coloring agents, flavoring agents, antioxidants, and / or preservatives well known to those skilled in the art.

  Angiogenesis is involved in a wide range of diseases and disorders. Therefore, the anti-angiogenic composition of the present invention is useful for the treatment and prevention of diseases and disorders related to angiogenesis. Diseases and disorders associated with angiogenesis include, but are not limited to, cancer, retinopathy, macular degeneration, psoriasis and arthritis.

  Therefore, the “disease or disorder in which it is beneficial to suppress angiogenesis” in the present invention refers to a disease in which progression of the disease or disorder is suppressed, or symptoms are improved or reduced by inhibiting angiogenesis. Or it means a disorder, for example, cancer, retinopathy, macular degeneration, psoriasis and arthritis.

  “Treatment” in the present invention means to suppress the progression of a disease or disorder that has already developed, or to improve or reduce symptoms. Also, “prevention” means preventing or delaying the onset of a disease or disorder.

Psoriasis means, for example, psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis, psoriatic arthritis, psoriatic psoriasis, and the like.
Arthritis means, for example, osteoarthritis, temporomandibular disorder and rheumatoid arthritis. Preferably, it means rheumatoid arthritis with synovitis.
Retinopathy means, for example, diabetic retinopathy and hypertensive retinopathy.
Macular degeneration means, for example, age-related macular degeneration. Preferably, cancer meaning wet age-related macular degeneration means a malignant tumor, such as lung cancer, breast cancer, stomach cancer, colon cancer, uterine cancer, ovarian cancer, laryngeal cancer, pharyngeal cancer, tongue cancer, Examples include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, leukemia, malignant lymphoma, myeloma and the like.

  The anti-angiogenic composition of the present invention can be used for the manufacture of a medicament for the treatment or prevention of these diseases or disorders. The medicament containing the anti-angiogenic composition may be administered orally by injecting the anti-angiogenic composition of the present invention into tablets, capsules, granules, powders, syrups, etc. Or it may be administered transdermally as a suppository. Therefore, the medicament containing the anti-angiogenic composition may further contain a pharmaceutically acceptable carrier or adjuvant well known to those skilled in the art.

  The invention is further illustrated by the following examples. However, it should be understood that the present invention is not limited to the examples shown below.

Anti-angiogenic action of marine organism phospholipid + genistein

Preparation of Blue Shark Meat Extract Frozen blue shark meat is sliced to a thickness of 2 cm and placed in a vacuum oven set at 35 ° C. and less than 2 mbar. After 24 hours, the meat pieces are taken out, crushed into fine pieces, and placed in a vacuum oven again for 3-4 days. Shark meat weight is usually reduced to the original 20% during the lyophilization process. The freeze-dried meat pieces are further finely divided by hand and then pulverized by a walling blender to form a mixture of powder and fibers.

  Shark meat powder (900 g) and solvent (ethanol 4.5 L) are stirred for 12 hours or more. In order to effectively stir, it was necessary that the ratio of solvent (volume): powder (weight) was 5: 1. The container was shielded from light by covering it with aluminum foil. The mixture was filtered using Schleicher & Schuell 595 filter paper and the filter residue was stirred overnight with fresh solvent (3 L) and filtered. The filtrates from the two filtrations were mixed and the solvent was removed by rotary evaporation to give a Blue Shark meat extract as an oily substance (yield 13.7%).

  As the genistein, “Genistein 95%” manufactured by JF-NATURAL R & D Co., Ltd. China was used. “Genistein 95%” is genistein extracted from natural soybean (non-genetically modified soybean), and its composition is 95% of genistein and the remaining 5% is genistin (Genistin) which is a glycoside.

Measurement of anti-angiogenic activity Anti-angiogenic activity was measured by aortic ring assay. The experimental method is based on the description of Nicosia and Ottinetti (Lab Invest. 63: 115-122 (1990)) and the description of Brown et al. (Lab Invest. 75: 539-555 (1996)).

  After removing the fat and fibrous tissue around the blood vessels, the aorta of rats (Lewis rat, Charles River Laboratories, Wilmington, Mass., USA) is sliced into 2 mm thick rings. Fibrinogen solution 0.4 mL (Fibrinogen solution is 1 mg / ml in 3 mL of 50 mL MCDB131 culture solution (Sigma Cat No. M-8537, Batch 064K8305) in which 150 mg fibrinogen (Sigma Cat No. F-9754, Batch 041K7604) is dissolved. 10 ml of thrombin (prepared by adding Serva Cat No. 36402. Batch 09309) to a 24-well culture plate (Costar® 24 Well TC-Treated Microplates, Individually Wrapped (Product # 3524) Corning Incorporated, USA) in each well to make a fibrin gel. The sliced aortic ring is placed in the central part of each well and covered by pouring another fibrin gel (0.4 mL of fibrinogen solution) over it. Furthermore, the gel was covered with MCDB131 culture medium (1.5 mL) and cultured under conditions of 37 ° C. and 3% CO 2/97% air. Originally produced marine organism phospholipids or genistein alone or independently produced marine organism phospholipids + genistein are added as supplements to the culture solution. Three samples were prepared for each of the above samples and analyzed. Moreover, the sample of only the culture solution was used as a control.

  It should be possible to confirm that microvessels have grown from around the ring (both outside and inside the ring) after approximately 5 days. From day 5 to day 14, images of each well were recorded at regular intervals. A digital camera (Pixera PVC-100C digital camera) attached to an inverted microscope (OLYMPUS CK-12 microscope) was used for photography. The growth range of microvessels around the ring was determined for each image using NIH Image 1.59 software (http://rsb.info.nih.gov/nih-image/about.html.). At each time point, average the growth rate (specimen A / control A × 100 = growth rate, A = angiogenic area extending from the outer ring of the ring + angiogenic area extending to the inner ring of the ring) ÷ area of the ring itself) Were determined. Microvessel growth rates were determined for marine phospholipids or genistein alone or independently produced marine phospholipids + genistein, and angiogenic growth rates were calculated.

  Table 1 shows the anti-angiogenic action of marine organism phospholipid (MPL) or genistein (Gen) alone or marine organism phospholipid + genistein (MPL + Gen). Based on the results of calculation assuming that the growth rate of the control is 100%, the angiogenesis growth rate of marine phospholipid alone is 57.19%, the angiogenesis growth rate of genistein alone is 54.7%, and marine phospholipid is genistein. When added to, the angiogenic growth rate is more than twice the anti-angiogenic action of each single substance (angiogenic growth rate is 24.67%). This indicates that marine phospholipid + genistein has a synergistic effect on the anti-angiogenic effect.

  Although the present invention has been described with reference to examples, various applications or improvements are possible without departing from the scope of the invention. Furthermore, substances known to have similar characteristics are to be treated in the same manner as detailed in this specification.

The anti-angiogenic composition of the present invention is useful for the treatment and prevention of a wide variety of diseases. These diseases include diseases involving angiogenesis such as psoriasis, arthritis, retinopathy, macular degeneration, psoriasis, and cancer.

Claims (10)

  An anti-angiogenic composition comprising marine phospholipids and genistein.   The anti-angiogenic composition according to claim 1, wherein the marine phospholipid is a shark meat extract and / or a mussel meat extract.   The anti-angiogenic composition according to claim 2, wherein the mussel meat is New Zealand green mussel meat.   Furthermore, the anti-angiogenic composition of any one of Claims 1-3 containing edible oil.   The anti-angiogenic composition according to claim 4, wherein the edible oil is olive oil.   The anti-angiogenic composition according to any one of claims 1 to 5, wherein the weight ratio of marine organism phospholipid and genistein is in the range of 0.5 to 10 genistein with respect to marine organism phospholipid 10.   The anti-angiogenic composition according to any one of claims 1 to 6, for treating or preventing a disease or disorder for which it is beneficial to suppress angiogenesis.   8. The anti-angiogenic composition according to claim 7, wherein the disease or disorder is selected from the group consisting of psoriasis, arthritis, retinopathy, macular degeneration and cancer.   Use of the anti-angiogenic composition according to any one of claims 1 to 8 for the manufacture of a medicament for treating or preventing a disease or disorder beneficial for inhibiting angiogenesis.   It is selected from the group consisting of psoriasis, arthritis, retinopathy, macular degeneration, and cancer, comprising administering the anti-angiogenic composition according to any one of claims 1 to 8 to a mammal. A method of treating or preventing a disease or disorder.