JP2013139432A - Angiogenesis inhibitor comprising ethanol extract or red coloring matter of fruit of serenoa repens, and manufacturing method for red coloring matter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an angiogenesis inhibitor comprising ethanol extract or red coloring matter of a fruit of Serenoa repens, and to provide crystallized red coloring matter of a fruit of Serenoa repens, and an angiogenesis inhibitor (anti-angiogenesis agent) containing the crystallized red coloring matter, and a manufacturing method for the same.SOLUTION: The angiogenesis inhibitor comprises ethanol extract or red coloring matter of a fruit of Serenoa repens. A cosmetic comprising the angiogenesis inhibitor is also provided.

Description

  The present invention relates to an angiogenesis inhibitor containing an ethanol extract or red pigment of saw palmetto fruit. The present invention also relates to a method for producing the red pigment.

  Saw palmetto has long been used as a therapeutic agent for urological diseases (Chinese medicine) in China, and has been suggested to have effects on tonicity, diuresis and benign prostatic hyperplasia. These effects reside in the fruit or its lipid component (80 to 90% of which is fatty acid) (Non-patent Documents 1 and 2).

  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.

  It is known that thicker capillaries related to wrinkle formation increase abnormally in skin exposed to excessive ultraviolet rays, and the use of angiogenesis inhibitors for preventing or improving wrinkles has been studied (Non-Patent Documents). 3-6).

  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, glaucoma, macular degeneration, periodontal disease 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 suppressing or controlling cancer growth. Thus, angiogenesis inhibitors can delay the progression of these diseases.

Plosker GL, Brogden RN. Serenoa repens (Permixon). A review of its pharmacology and therapeutic efficacy in benign prostatic hyperplasia. Drugs Aging. 1996 Nov; 9 (5): 379-95. http://www.sawpalmetto.com/lib/datasheet/index.html Chung JH, Eun HC. Angiogenesis in skin aging and photoaging. J Dermatol. 2007; 34 (9): 593-600. Yano K, Kajiya K, Ishiwata M, Hong YK, Miyakawa T, Detmar M. Ultraviolet B-induced skin angiogenesis is associated with a switch in the balance of vascular endothelial growth factor and thrombospondin-1 expression.J Invest Dermatol. 2004; 122 (1): 201-8. Shigeo Kawada, Masaru Ohtani, and Naokata Ishii. Increased oxygen tension attenuates acute ultraviolet-B-induced skin angiogenesis and wrinkle formation. Am J Physiol Regul Integr Comp Physiol 2010; 299: R694-R701. Kiichiro Yano, Hajimu Oura, and Michael Detmar.Targeted overexpression of the angiogenesis Inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol 2002; 118: 800-805. Invest Dermatol 118: 800-805, 2002.

  An object of the present invention is to provide an anti-angiogenic (anti-angiogenic) agent containing an ethanol extract or a red pigment. Moreover, this invention makes it a subject to provide the angiogenesis inhibitor containing the manufacturing method of the red pigment | dye from the ethanol extract of a saw palm fruit, and this red pigment | dye.

  The present inventor has found that an ethanol extract of saw palm fruit has a remarkable angiogenesis inhibitory action, and further established a method for purifying red pigment of saw palm fruit from the ethanol extract, and the angiogenesis remarkable to the red pigment. The inventors found that there is an inhibitory action and completed the present invention.

Therefore,
(1) The present invention relates to an angiogenesis inhibitor containing a red ethanol extract or red pigment of saw palmetto fruit.
(2) The present invention relates to a cosmetic containing the angiogenesis inhibitor described in (1) above.
(3) The present invention relates to a medicine containing the angiogenesis inhibitor described in (1) above.
(4) The present invention relates to the cosmetic according to (2) above for improving or preventing skin wrinkles.
(5) The present invention relates to the medicament according to (3) above for treating or preventing a disease or disorder for which it is beneficial to suppress angiogenesis.
(6) The present invention includes an angiogenesis inhibitor according to (1), a cosmetic according to (2) or (4), or (3) or above, which comprises extracting a saw palm fruit with ethanol. The present invention relates to a method for producing the pharmaceutical according to (5).
(7) The present invention relates to a crystallized red pigment derived from a saw palmetto fruit.
(8) The present invention relates to the crystallized red pigment according to (7) above, which is obtained by extracting a saw palm fruit with ethanol and precipitating the red pigment in the extract.
(9) The present invention relates to a composition comprising the crystallized red pigment described in any one of (7) to (8) above.
(10) The present invention relates to an angiogenesis inhibitor comprising the crystallized red pigment according to any one of (7) to (8) above.
(11) The present invention contains the crystallization red pigment described in any one of (7) to (8) above, the composition described in (9) above, or the angiogenesis inhibitor described in (10) above. To the food.
(12) The present invention contains the crystallization red pigment described in any one of (7) to (8) above, the composition described in (9) above, or the angiogenesis inhibitor described in (10) above. , Regarding cosmetics.
(13) The present invention contains the crystallization red pigment described in any one of (7) to (8) above, the composition described in (9) above, or the angiogenesis inhibitor described in (10) above. , Relating to medicine.
(14) The present invention relates to the cosmetic described in (12) above for improving or preventing skin wrinkles.
(15) The present invention relates to the medicament according to the above (13), for treating or preventing a disease or disorder that is beneficial to suppress angiogenesis.
(16) The present invention provides:
Extracting the saw palm fruit with ethanol;
Separating the extract from the solid residue;
Concentrating the extract;
Mixing the concentrate with water;
Irradiating the mixture with ultrasonic waves to separate it into an oil layer and an aqueous phase; and collecting a red pigment precipitated from the aqueous phase;
Optionally washing the red pigment with water;
The crystallized red pigment according to any one of (7) to (8) above.
(17) The present invention includes the method for producing a crystallized red pigment according to (16) above, the composition according to (9) above, the angiogenesis inhibitor according to (10) above, (11) above Or the cosmetic according to (12) or (14) above, or the pharmaceutical production method according to (13) or (15) above.
(18) The present invention relates to the crystallized red pigment according to (7) or (8) above, wherein the crystallized red pigment contains proanthocyanidins derived from saw palmetto fruit.
(19) The present invention relates to the angiogenesis inhibitor described in (1) above, wherein the ethanol extract or red pigment of saw palm fruit is the crystallized red pigment described in (18) above.
(20) The present invention relates to a composition comprising the crystallized red pigment described in (18) above.
(21) The present invention relates to a food containing the crystallized red pigment described in (18) above.
(22) The present invention relates to a cosmetic containing the crystallized red pigment described in (18) above.
(23) The present invention relates to a medicine containing the crystallized red pigment described in (18) above.
(24) The present invention
Extracting the saw palm fruit with ethanol,
Separating the extract from the solid residue,
A step of concentrating the extract,
Mixing the concentrate with water,
Irradiating the mixture with ultrasonic waves, separating the oil layer into an aqueous phase, and collecting the red pigment precipitated from the aqueous phase;
The method for producing a crystallized red pigment described in (18) above.
(25) The present invention includes the composition according to (20), the food according to (21) above, the cosmetic according to (22) above, or the ( The method for producing a medicine according to 23).

  Surprisingly, the present invention has found that an ethanol extract of saw palm fruit has a remarkable angiogenesis inhibitory action, and further, a red pigment of saw palm fruit purified from the ethanol extract has a remarkable angiogenesis inhibitory action. Found that there is.

Photograph of tube formation in the presence of test substance against HUVEC on Matrigel. (A): shows tube formation when treated with 100 μg / mL red pigment. Little tube formation is observed. (B), (c), (d), (e): shows tube formation when treated with 10, 1, 0.1, 0.01 μg / mL red pigment. (F): Shows the tube formed when treated with vehicle control. The total length of the tube formed was measured from the photograph. (G) shows tube formation when treated with 30 μM scramine. Little tube formation is observed. (H), (i), (j), (k): shows tubes formed when treated with 15, 10, 3, 1 μM scramin. (L): Shows tube formation when treated with vehicle control. The total length of the tube formed was measured from the photograph. Inhibition of tube formation of red pigment in saw palmetto fruit. (A): The total length of the tube formed when treated with the red pigment of each concentration, where 100 is the total length of the tube formed in the vehicle control (FIG. 1 (f)). (E)) is expressed as a percentage (%). The total length of the tube formed when treated with 100 μg / mL red dye is about 20% of the total length of the tube formed with the vehicle control. The total length of the tube formed when treated with 10 μg / mL red dye is approximately 80% of the total length of the tube formed with the vehicle control. (B): The total length of the tube formed when treated with each concentration of scramine, where 100 is the total length of the tube formed in the vehicle control (FIG. 1 (l)) ((g) to ( k)) is expressed as a percentage (%). The total length of the tube formed when treated with 30 μM scramin is approximately 0% of the total length of the tube formed with the vehicle control. Little tube formation is seen. *: Significant inhibition of tube formation. Antitumor action of saw palmetto red pigment. (A) shows the tumor growth curve of human liver cancer cells in a xenograft nude mouse model. "NYG-1 0.1 mg / kg" and "NYG-1 1 mg / kg" indicate that saw palmetto pigment was administered intraperitoneally in amounts of 0.1 mg and 1 mg per kg body weight using physiological saline, respectively. . “Control” is only saline. “Days post injection” indicates the number of days after the last administration (day 0). “Tumor growth (%)” was calculated on the basis of 100 tumor sizes (Control, NYG-1 0.1 mg / kg and NYG-1 1 mg / kg) on the measurement start date on the seventh day after administration. (B) Inhibition of VEGF expression in human liver cancer in a xenograft nude mouse model. “VEGF expression (%)” indicates the amount of vascular endothelial growth factor (VEGF) contained in the supernatant after the tumor is homogenized and centrifuged. TLC analysis of purified saw palmetto red pigment. Lane 1: Before hydrolysis of purified saw palmetto red pigment, Lane 2: After hydrolysis of purified saw palmetto red pigment, Lane 3: Cyanidine (standard), Lane 4: Delphinidin (standard), Lane 5: Peonidin (standard), Lane 6 : Petunidin (standard), Lane 7: Malvidin (standard). In a closed container, at room temperature, it was developed to the top of the plate using acetic acid-hydrochloric acid-water (30: 3: 10) on a TLC glass plate (Merck) of cellulose, and then detected visually. As an example of the structure of proanthocyanidins (condensed tannin), the structure of grape proanthocyanidins (condensed tannin) is shown. The structure of a typical anthocyanidin is shown.

  Saw palm fruit means the fruit of a plant belonging to the genus Arecaceae (Trachycarpus). Preferably, it is the fruit of Serenoa serrulata or Serenoa repens. Saw palm fruit can be obtained from the market.

  The saw palm fruit is preferably a dried saw palm fruit. Dried saw palm fruit has a moisture content of, but not limited to, 10 wt% or less, preferably 5 wt% or less. The dried saw palm fruit can be obtained by dehydrating and drying raw saw palm fruit by any method (for example, sun drying, heat drying, vacuum drying, freeze drying, etc.). The dried saw palm fruit is preferably crushed and in the form of crushed material or powder.

  Ethanol is not limited as long as it is a solvent containing ethanol, but is preferably aqueous ethanol containing 30% by volume or more of ethanol. More preferably 80 to 100% by volume, still more preferably 90 to 95% by volume ethanol.

  Extraction of saw palm fruit with ethanol is performed by adding ethanol to saw palm fruit and then stirring at room temperature (15 ° C. to 28 ° C.) or under heating for a certain period of time.

  The heating temperature can also be a temperature below the boiling point of ethanol, or a temperature below 120 ° C. in a sealed container. A preferable extraction temperature is 90 to 110 ° C. More preferably, it is 95-105 degreeC. The heating method is not limited, but it is preferably performed in an oil bath.

  The extraction time is not limited, but is 5 minutes to 24 hours, preferably 15 minutes to 3 hours, and more preferably 30 minutes to 2 hours.

  Although extraction is not limited, it can be performed once or twice or more. Extraction is not limited, but is preferably performed with stirring. Although the stirring method is not limited, it is preferable to use a combination of a magnetic stirrer and a stirrer or a motor type stirrer. The stirring speed is not limited, but is 100 to 600 rpm, preferably 200 to 500 rpm.

  Although the usage-amount of ethanol with respect to the saw palm fruit in extraction is not limited, ethanol is 300 weight part or more with respect to 100 weight part of saw palm, Preferably, it is 500-2000 weight part, More preferably, it is 750-1500 weight part.

  Or the moisture content in the mixture which mixed the saw palm fruit and ethanol in extraction is 70 weight% or less, Preferably it is 20 weight% or less, More preferably, it is 10 weight% or less. For example, when 100 g of saw palmetto fruit with a water content of 10% by weight is extracted with 1000 mL of 90% ethanol by volume (90% ethanol density at 15 ° C. of 0.8222 (based on the International Legal Metrology Organization (OIML)), the water content at the time of extraction (%) Is calculated as [moisture weight in saw palm 10 g + water weight in ethanol 100 g] / [saw palm weight 100 g + ethanol weight 822 g] × 100 = 12%.

  After ethanol extraction, it is separated into an ethanol extract and a solid residue to obtain an ethanol extract. The separation method is not limited, but filtration or centrifugation is preferable.

  Filtration of the ethanol extract is not limited, but it is preferable to use a filter membrane (nylon mesh, filter paper, etc.), a filter (Nucche filter, Buchner funnel, etc.) and the like. A combination of coarse and fine filtration is preferred.

  The pore size of the coarse filtration is not limited, but is 500 to 50 μm, preferably 300 to 100 μm.

  The pore size of the fine filtration is 10 to 1 μm, preferably 7.5 to 2.5 μm, more preferably 6 to 4 μm. Further, it is preferable to perform the process by spreading celite or diatomaceous earth on a filter paper as a filter aid.

  The filtration method is not limited, and examples include natural fall, pressure filtration, and vacuum (suction) filtration. Filtration may be performed once or more.

  The filtrate is not limited but is preferably concentrated. It is more preferable to concentrate under reduced pressure. Concentration is not limited, but is 2 times concentration to dryness, preferably 3 to 30 times concentration, more preferably 5 to 20 times concentration.

  The ethanol extract means an extract obtained by extracting the saw palm fruit with ethanol and separating the solid residue, or a concentrate thereof. When the separation is performed by filtration, the filtration may be only rough filtration.

  To the concentrate, add water, mix and stir. The method of stirring the concentrate and water is not limited, but it is preferable to use a combination of a magnetic stirrer and a stirrer or a motor type stirrer. The stirring speed is not limited, but is 100 to 600 rpm, preferably 200 to 500 rpm. The stirring time is not limited, but is 1 to 60 minutes, preferably 3 to 30 minutes, and more preferably 5 to 15 minutes. The temperature at the time of stirring is 15-30 degreeC, Preferably it is 20-25 degreeC.

  The mixing ratio of the concentrate and water is 100 parts by weight or more, preferably 200 to 1000 parts by weight, more preferably 300 to 500 parts by weight with respect to 100 parts by weight of the concentrate.

  Ultrasound is irradiated to the mixture of the concentrate and water after stirring. By applying ultrasonic waves to the mixed solution, the mixed solution is separated into an oil layer and an aqueous layer, and a red red pigment crystallizes and precipitates in the aqueous layer. Therefore, the ultrasonic frequency, intensity, irradiation time, irradiation method, and ultrasonic generator are not limited.

  The frequency is 20 kHz to 1 MHz, preferably 20 kHz to 100 kHz, and more preferably 25 kHz to 50 kHz. The irradiation time is 10 seconds to 1 hour, preferably 20 seconds to 30 minutes, and more preferably 30 seconds to 5 minutes. The intensity is not limited, but an initial set value of the ultrasonic generator can be used.

  The ultrasonic generator is not limited, and examples thereof include an ultrasonic oscillator equipped with a vibrator and an ultrasonic cleaner. When an ultrasonic oscillator is used, it is preferable to provide an ultrasonic wave by installing a vibrator in the mixed solution. When an ultrasonic cleaner is used, it is preferable that the container containing the mixed solution is placed in an ultrasonic cleaner filled with water to give ultrasonic waves. A preferable irradiation method is a method using an ultrasonic cleaner.

  The aqueous layer is separated from the oil layer and collected. The separation method is not limited, but it is preferable to collect the aqueous layer using a separatory funnel or the like.

  Crystals are recovered from the aqueous layer. The recovery method is not limited, but filtration or centrifugation is preferred.

  The filtration for recovering the crystals is performed using a filter membrane (nylon mesh, filter paper, etc.) and a filter (Nutsche filter, Buchner funnel, etc.). Although the pore size of filtration will not be limited if it is a size which can collect | recover crystals, it is 10-1 micrometer, Preferably, it is 7.5-2.5 micrometers, More preferably, it is 6-4 micrometers. The filtration method is not limited, and examples include natural fall, pressure filtration, and vacuum (suction) filtration. Preferably, it is reduced pressure (suction) filtration.

  The recovered crystals are preferably washed with water for oils and fats remaining after filtration.

The crystallized red pigment refers to a red pigment purified by the above steps. The purified red pigment includes, but is not limited to, 5% by weight or more, preferably 10% to 70% by weight, more preferably 20% to 50% by weight of proanthocyanidins (condensed tannins). Alternatively, the crystallized red pigment is proanthocyanidin (condensed tannin) having a purity of 5% by weight or more, preferably 10% by weight to 70% by weight, and more preferably 20% by weight to 50% by weight.
Further, preferably, the crystallized red pigment is substantially free of sugar (sugar chain) and / or carotenoid.
Crystallization means a crystalline form. As long as the red pigment is a red substance of saw palmetto fruit, the purification method is not limited, but a red substance containing anthocyan, more preferably a red substance containing proanthocyanidins (condensed tannins), and even more preferably the crystallization described above. It is a red pigment.
Proanthocyanidins and condensed tannins are used interchangeably. Proanthocyanidins (condensed tannins) are a wide variety of catechins, epicatechins, epigallocatechins, afuzeletins and the like, which are widely contained in plants, or flavan oligomers (preferably 2 to 10) To polymers (11 or more, preferably 11 to 50). Proanthocyanidins (condensed tannins) generate anthocyanidins (aurantinidin, cyanidin, delphinidin, europinidine, luteolinidine, pelargonidin, malvidin, peonidin, petunidin, rosinidine, etc.) when hydrolyzed with hydrochloric acid.
Anthocyanidin means a part (aglycone) other than sugars and sugar chains in anthocyan, which is widely present in the plant kingdom.
Proanthocyanidins (condensed tannins) in the present invention include substances that generate at least one selected from cyanidin, delphinidin, peonidin, petunidin, and malvidin when hydrolyzed with hydrochloric acid. That is, it includes at least one selected from procyanidins, prodelphinidins, propeonidins, propetunidins, and promalvidins.

  The obtained crystals are dried. It is preferable to heat and dry under reduced pressure. Although heating temperature is not limited, It is room temperature-80 degreeC, Preferably, it is 40-70 degreeC. By drying by heating, the crystal is in a tar state. The obtained crystals are easily dissolved in ethanol to give a red ethanol solution.

  The form of the red pigment of the saw palm fruit is not limited, but may be a crystalline state, a heat-dried oil state, or a state dissolved in ethanol. The red pigment is preferably stored at 4 degrees for light shielding. Although the form of the ethanol extract of saw palm fruit is not limited, it may be a dried oil state or a state dissolved in ethanol.

  The ethanol extract or red pigment of saw palmetto fruit may be, but is not limited to, food, cosmetics, and medicine. Further, it may be a food additive, a cosmetic additive, or a pharmaceutical additive. Similarly, the ethanol extract of saw palm fruit or a composition containing red pigment may be, but is not limited to, a food, a cosmetic, or a pharmaceutical. Further, it may be a food additive, a cosmetic additive, or a pharmaceutical additive.

  The amount of saw palm fruit ethanol extract or red pigment in the composition is not limited, but is 0.1 to 99.9% by weight, preferably 1 to 99%. The remaining part is a conventional carrier, excipient, additive or the like.

  The composition may further contain a conventional carrier, excipient, additive or the like. Conventional carriers, excipients, or additives include solvents, vegetable oils, mineral oils, fatty oils, liquid paraffin, buffers, preservatives, wetting agents, chelating agents, antioxidants, stabilizers, emulsifiers, suspensions. Suspending agents, gel forming agents, ointment bases, suppository bases, penetration enhancers, fragrances, sweeteners, colorants, fragrances, skin protection agents and the like can be mentioned.

  Examples of the solvent include, but are not limited to, water, alcohol, BG (1,3-butylene glycol), polyethylene glycol, propylene glycol, glycerol, liquid polyalkylsiloxane, and mixtures thereof.

  Vegetable oil includes almond oil, castor oil, cacao butter, coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppy seed oil, rapeseed oil, sesame oil, soybean oil, sunflower oil, wheat germ oil , Rice oil and tea seed oil, and mixtures thereof, but are not limited thereto. These edible oils may be commercially available or prepared by themselves. Preferably, it is olive oil.

Buffering agents include, but are not limited to, citric acid, acetic acid, tartaric acid, lactic acid, hydrogen phosphate, diethylamine, and mixtures thereof.
Wetting agents include, but are not limited to, glycerin, propylene glycol, pentylene glycol, sorbitol, lactic acid, urea, BG (1,3-butylene glycol), soy sterol, and mixtures thereof. .

  Chelating agents include, but are not limited to sodium EDTA, citric acid and mixtures thereof.

  Antioxidants include butylated hydroxyanisole (BHA), ascorbic acid and its derivatives, α-, β-, γ-, δ-tocopherol and its derivatives, α-, β-, γ-, δ- Examples include, but are not limited to, tocotrienol and its derivatives, cysteine and mixtures thereof.

  Emulsifiers include natural rubber (eg, acacia gum), tragacanth gum, xanthan gum; natural phosphatide (eg, soy lecithin); sorbitan monooleate; wool oil; wool alcohol; sorbitan ester; monoglyceride; fatty alcohol (eg behenyl alcohol); Fatty acid esters (eg, triglycerides of fatty acids such as glyceryl tri (capryl / capric acid), glyceryl stearate (SE)); and mixtures thereof, but are not limited to these.

  Suspending agents include cellulose and its derivatives (such as carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.), carrageenan, acacia gum, gum arabic, tragacanth and mixtures thereof. There is no limit.

  Gel bases and thickening ingredients include liquid paraffin, polyethylene, fatty oil, colloidal silica or aluminum, zinc soap, glycerol, propylene glycol, tragacanth, carboxyvinyl polymer, magnesium silicate-aluminum, hydrophilic polymers (eg starch Cellulose derivatives such as carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives), water-swellable hydrocolloids, carrageenan, hyaluronic acid salts (eg hyaluronic acid gels selectively containing sodium chloride), alginate esters (eg propylene alginate) Glycol) and mixtures thereof, but are not limited thereto.

  Ointment base includes beeswax, paraffin, cetanol, cetyl palmitate, cetearyl alcohol, polyglyceryl-10 stearate, stearic acid, PEG-150 stearate, vegetable oil, fatty acid sorbitan ester, polyethylene glycol, fatty acid sorbitan ester and oxidized Examples include, but are not limited to, condensation products with ethylene (for example, polyoxyethylene sorbitan monooleate) and mixtures thereof.

  Hydrophobic ointment bases include, but are not limited to paraffins, vegetable oils, animal fats, synthetic glycerides, waxes, lanolin, liquid polyalkylsiloxanes and mixtures thereof.

  The hydrophilic ointment base includes, but is not limited to, solid macrogol (polyethylene glycol). Furthermore, conventional carriers, excipients, or additives include squalane, lecithin, hydrogenated lecithin, ascorbyl tetrahexyldecanoate, allantoin, glycyrrhizic acid 2K, glycosyl trehalose, hydrolyzed hydrogenated starch, hydrolyzed collagen, Examples include rose extract, dimethicone, caprylyl glycol, betaine, sodium stearoyl glutamate, rose oil, batyl alcohol, hydroproxyproline, and the like.

  Angiogenesis suppression means the action of inhibiting or suppressing the formation and growth of new blood vessels. An angiogenesis inhibitor means a composition having an angiogenesis inhibitory action. The angiogenesis inhibitor may be a food, a cosmetic, or a medicine.

  A disease or disorder that is beneficial to suppress angiogenesis means a disease or disorder in which progression of the disease or disorder is suppressed, or symptoms are ameliorated or reduced by inhibiting angiogenesis, for example, Cancer, retinopathy, glaucoma, macular degeneration, periodontal disease, psoriasis and arthritis. Preferred are cancer, psoriasis and arthritis.

  Psoriasis is preferably psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis, psoriatic arthritis, psoriatic psoriasis and the like.

  The arthritis is preferably osteoarthritis, temporomandibular disorder or rheumatoid arthritis. More preferred is rheumatoid arthritis with synovitis.

  The retinopathy is preferably diabetic retinopathy, hypertensive retinopathy and the like.

  The glaucoma is preferably primary glaucoma, congenital glaucoma, secondary glaucoma, normal-tension glaucoma and the like.

  The macular degeneration is preferably age-related macular degeneration or the like. More preferred is exudative age-related macular degeneration.

  Periodontal disease is preferably gingivitis and periodontitis.

  Cancer means a malignant tumor, preferably skin cancer, lung cancer, breast cancer, stomach cancer, colon cancer, uterine cancer, ovarian cancer, laryngeal cancer, pharyngeal cancer, tongue cancer, osteosarcoma, chondrosarcoma, striated muscle Tumor, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, leukemia, malignant lymphoma, myeloma, liver cancer, brain tumor, kidney cancer, esophageal cancer, prostate cancer, testicular cancer, bladder cancer, oral cancer, thyroid cancer, pancreas Cancer, biliary tract cancer, head and neck cancer, etc. More preferred are skin cancer such as melanoma, breast cancer, and liver cancer.

  Treatment means inhibiting the progression of a disease or disorder that has already developed, or improving or reducing symptoms. Prevention also means preventing or delaying the onset of a disease or disorder.

  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.

  Administration may be systemic administration, local administration, systemic administration, local administration is transdermal administration, sublingual administration, oral administration, enteral administration, intramuscular administration, subcutaneous administration, intravenous administration, nasal administration, Any dosage form such as eye drops may be used. Preferable are transdermal administration, sublingual administration, oral administration, subcutaneous administration and intravenous administration.

  The effective dose for treating or preventing a disease or disorder varies depending on the degree of the disease or disorder and the administration method, and is not limited as long as it is an effective amount for suppressing angiogenesis, but an ethanol extract of saw palmetto fruit Alternatively, the amount of red pigment is 0.01 to 1000 mg / kg body weight · day, 0.1 to 1000 mg / kg body weight · day, preferably 1 to 500 mg / kg body weight · day, more preferably 10 to 100 mg / kg body weight · day. Day.

  The ethanol extract or red pigment of saw palmetto fruit can be used for the manufacture of a medicament for the treatment or prevention of these diseases or disorders. Examples of pharmaceutical forms include creams, sublinguals, massadi oil, solutions, suspensions, lotions, ointments, gels, tablets, capsules, granules, powders, syrups, and suppositories. Creams, sublinguals, massadi oil, solutions, tablets, capsules, granules, and powders are preferable.

  The composition may further contain another agent having an anti-angiogenic action.

  The present invention is directed to treating or preventing a disease or disorder associated with a disease or disorder in which angiogenesis is beneficial, comprising administering to a mammal an effective amount of an ethanol extract or red pigment of saw palmetto fruit. Is the method.

  In skin exposed to excessive ultraviolet rays, thicker capillaries (wrinkle vessels) that are involved in wrinkle formation increase abnormally. Therefore, the angiogenesis inhibitor can exert an effect of preventing and improving wrinkles. The angiogenesis inhibitor may be a cosmetic for preventing or improving wrinkles. Therefore, the ethanol extract or red pigment of saw palmetto fruit can be used for the production of cosmetics for preventing and improving the generation of wrinkles.

  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.

Manufacture of refined saw palm red pigment 1000 grams of saw palm fruit (Saw Palmetto Berries Co-Op of Florida Inc. (SPB), 1206 Kings Way, Naples, Florida 34112, USA) was ground with a mixer (Panasonic Corporation, Fiber Mixer) Dried. The weight of the dried saw palmetto fruit was 886 g.

  An oil bath at 100 ° C. on a magnetic stirrer with a hot plate was charged with 100 g of saw palm fruit pulverized and dried with a mixer and a stir bar in 1000 mL of 90 vol% ethanol in a stainless steel pressurized container (Azwan Corporation). A stainless steel pressure vessel was placed inside and stirred at 400 rpm for 1 hour. The internal pressure in the container was 0.1 MPa on the basis of atmospheric pressure.

  Remove the container from the oil bath, cool it down to normal pressure (temperature dropped to about 60 ° C), and then separate the residue with 100 mesh nylon cloth (Kitamura Fabric Co., Ltd., nylon filter cloth, pore size 150 μm). Further, 950 mL of the obtained extract was further filtered using a filter aid (Celite, Celite 503) as a precoat. The precoat was prepared by spreading 5 g of Celite on a filter paper (Advantech Toyo Co., No. 2) spread on a Nutsche filter (5 μm, Sansho Co., Ltd.).

  After filtration of the extract, it was further filtered with 50 mL of 90% by volume ethanol, and 1000 mL of the filtrate thus obtained was concentrated to 100 mL under reduced pressure using a rotary evaporator (Tokyo Rika Kikai Co., Ltd.).

  100 mL of the obtained concentrated solution was transferred to a 500 mL beaker (Shibata Kagaku Co., Ltd.), 300 mL of water and a stirrer were added, and the mixture was stirred at 400 rpm for 10 minutes at room temperature (23 ° C.).

  Next, the beaker containing the solution was immersed in an ultrasonic cleaner (As One Co., Ltd.) filled with water and irradiated at a frequency of 40 kHz for 1 minute to separate the oil layer (upper layer) and the aqueous layer (lower layer). Red crystals (precipitation) were observed in the lower layer.

  The solution divided into the oil layer and the water layer is transferred to a separatory funnel, and the water layer is collected in a beaker and filtered under a reduced pressure (8 kPa on an absolute pressure basis) with filter paper (Advantech Toyo Co., Ltd., No. 2). Using a vessel (5 μm, Sansho Co., Ltd.), the crystals were obtained on a filter paper. After the remaining fat and oil were washed away with water, the crystals were transferred to another container and dried under reduced pressure at 60 ° C. (Advantech Toyo Co., Ltd., vacuum constant temperature dryer) to obtain 1.0 g of saw palmetto red pigment.

1. Measurement of Antiangiogenic Activity of Purified Saw Palmetto Red Pigment The following biological test results were sent to Ricerca Bioscience, LLC (http: // www. Ricerca.com) for the crystallized red pigment prepared in Example 1. The anti-angiogenic activity was obtained by requesting a test (Assay # 368000 Tumor, Angiogenesis, Tube Formation).

  Normal human umbilical vein endothelial cells (HUVEC) differentiate into capillary-like structures on Matrigel in the presence of endothelial cell proliferation supplements, forming a tubular network. The anti-angiogenic activity of a test compound can be assessed by observing the continuity of the formed vascular network compared to an untreated control. The test compound (crystallized red dye prepared in Example 3) was tested for anti-angiogenic effects at final concentrations of 100, 10, 1, 0.1, 0.01 μ / mL.

2. Preparation of Test Compound (Crystalline Red Dye Prepared in Example 1) The crystallized red dye prepared in Example 1 is dissolved in 100% DMSO, diluted with sterile water, and 1, 10, 100, 1000, A 40% DMSO solution of 10,000 μg / mL was prepared. Furthermore, final concentrations of 100, 10, 1, 0.1, and 0.01 μg / mL were prepared by 100-fold dilution with a medium.

3. Method for culturing normal human umbilical vein endothelial cells (HUVEC) HUVEC was purchased from American Type Culture Collection (ATCC CRL-1730). HUVEC cells were cultured at 37 ° C. in a 5% CO ₂ air atmosphere. The cells were cultured using an endothelial cell growth medium containing 10% by volume fetal bovine serum (FBS) and 1% antifungal antibiotic.

4). Reagents: Antifungal antibiotics (GIBCO BRL, USA), dimethyl sulfoxide (Merck, Germany), endothelial cell growth medium (CELL APPLICATIONS, USA), fetal calf serum (HyClone, USA), Matrigel (BD Biosciences, ), Suramin (Sigma, USA).

5. Equipment and instruments: 96 microwell culture plate (NUNC, USA), Centrifuge 5810R (Eppendorf, Germany), digital camera (Nikon, Japan), hematocytometer (Hausser Scientific Horsham, USA), Inverted microscope CK-40 (Olympus, Japan), System microscope E-400 (Nikon, Japan), Biological safety cabinet (NuAire, USA).

6). Method The Matrigel matrix was thawed and placed on ice at 4 ° C., and 50 μL of the matrix was transferred to each well of a 96 microwell culture plate. Plates are incubated at 37 ° C for at least 1 hour to allow the matrix solution to solidify before processing.

A 200 μL aliquot of the HUVEC suspension (approximately 1.5 × 10 ⁴ cells / well) was placed in a 96-well Matrigel plate. Test compound or vehicle (40% DMSO) was added at 2 μL per well and incubated for 18 hours at 37 ° C., 5% CO ₂ (made in duplicate). The final concentration of DMSO is 0.4%. Test compounds were evaluated at concentrations of 100, 10, 1, 0.1, 0.01 μg / mL. At the end of the 18 hour culture, the tube network formed by the endothelial cells in individual wells was evaluated and photographed with a microscope (40X magnification). The total tube length of the tube network was measured from each photo (FIG. 2).

A decrease in tube length of the tube network in test compound treatment relative to the tube length of the vehicle treatment control tube network represents anti-angiogenic activity. The minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC ₅₀ ) were determined to evaluate the effect of the test substance on anti-angiogenesis.

Grant, DS, Kinsella, JL, Fridman, R., Auerbach, R., Piasecki, BA, Yamada, Y., et al. Interaction of endothelial cells with a laminin A Chain peptide (SIKVAV) in vitro and induction of angiogenetic behavior in vivo. J. Cell Physiol. 153: 614-625, 1992.
Belotti, D., Vergani, V., Drudis, T., Borsotti, P., Pitelli, MR, Viale, G., Giavazzi, R. And Taraboletti, G. The microtubule-affecting drug paclitaxel has antiangiogeneic activity. Clin. Cancer Res. 2: 1843 1-1849, 1996.

7. Results 100 μg / mL of the red pigment of the present invention showed significant inhibition of tube formation relative to the vehicle-treated control. The standard reference reagent, Suramin, showed significant inhibition at 30 and 15 μM (Table 2, FIG. 1, FIG. 2). IC ₅₀ values are shown in Table 3.

Table 2 Inhibition of tube formation by red pigment of saw palmetto fruit

a: Significant inhibition of tube formation (≧ 70%) b: Minimum inhibitory concentration (MIC)
Inhibition of tube formation by more than 70% relative to the vehicle-treated control indicates significant anti-angiogenic activity.

Table 3 Summary of Minimum Inhibitory Concentration (MIC) and IC ₅₀

A composition containing the red pigment of saw palmetto was prepared with the following formulation.
Ingredient name Cream content (wt%)
Ethanol extract or red pigment of saw palmetto 2.0
Olive oil 10.0
Tri (capryl / capric acid) glyceryl 10.0
Tocopherol 1.0
Purified water 77.0
Total 100.0

Measurement of VEGF-Induced Vascular Endothelial Cell Growth Inhibitory Activity with Purified Saw Palmetto Red Dye The following biological test results show that the crystallized red dye prepared in Example 1 was converted to Ricerca Bioscience, LLC (http: // www. Ricerca .com) and tested for cell growth inhibitory activity.

Test substances and doses Test substances NYG-1, DS-SMO, SMO and AK were used for in vitro VEGF proliferation studies. These were dissolved in 100% DMSO, diluted with DMSO, and adjusted to concentrations of 2.63 mg / mL and 26.3 mg / mL. The final concentrations in the test give 10 μg / mL and 100 μg / mL, respectively.

NYG-1: Crystallized red pigment prepared in Example 1 DS-SMO: Fat-soluble fraction obtained by extracting deep-sea salmon meat with ethanol SMO: Fat-soluble fraction obtained by extracting salmon meat with ethanol AK: Agate liver oil Concentrated product of alkoxyglycerol (AKG)

Cells and Medium Normal human umbilical vein endothelial cells (HUVEC) were purchased from American Type Culture Collection (ATCC CRL-1730). HUVEC cells were cultured at 37 ° C. in a 5% CO ₂ air atmosphere. The cells were cultured using an endothelial cell growth medium containing 10% by volume fetal bovine serum (FBS). The assay medium is M199 containing 10% FBS and 10 μg / mL heparin.

Reagents Dimethyl sulfoxide (Merck, Germany), endothelial cell growth medium (CELL APPLICATIONS, USA), fetal calf serum (HyClone, USA), heparin (Sigma, USA), SU5416 (Calbiochem, USA), VEGF ₁₆₅ (Calbiochem, USA) HBSS (Gibco, USA).

Equipment / Equipment 96 Microwell culture plate (NUNC, USA), Centrifuge CT6D (Hitachi, Japan), Nucleocounter (ChemoMetec, Denmark), Inverted microscope CK-30 (Olympus, Japan), Biological safety cabinet (NuAire, USA).

Methods HUVEC were pre-seeded at 1.1 × 10 ⁵ / ml in 96-well TC plates in a 37 ° C. incubator containing 5% CO _{2 the} day before the assay. Test substances and vehicle (DMSO) were preincubated with cells at 37 ° C. for 20 minutes. VEGF ₁₆₅ (0.2 nM) was then added and cultured for 48 hours. After 48 hours, the cells were washed once with HBSS buffer. Fluorescent reagent Calcein AM dye (BD Biosciences, USA) 5 μg / mL was added, and further incubated for 50 minutes. The fluorescence intensity was read with a microplate reader. An inhibition of 50% or more on VEGF _165- induced cell proliferation indicates a significant antagonist activity of the test substance.

Table 4: Summary of methods

Results The results are shown in Table 5 below. NYG-1 (saw palm red pigment) showed antagonist activity. Significant antagonistic activity (140%) was observed at 100 μg / mL (final concentration).

Table 5: Summary of activities of AK, DS-SMO, NYG-1, and SMO

IC ₅₀ / EC ₅₀ are shown in Table 6 below. The IC ₅₀ / EC ₅₀ of saw palmetto red pigment was calculated to be 0.12 μM. SU5416 is a known angiogenesis inhibitor (antagonist).

Table 6: IC ₅₀ / EC ₅₀

  Thus, it was shown that saw palmetto red pigment can suppress VEGF-induced cell proliferation, activation and angiogenesis in vascular endothelial cells.

Anti-tumor activity of saw palmetto red pigment (A) Saw palmetto red pigment inhibited human liver cancer in a xenograft nude mouse model.

Materials Saw palm red pigment NYG-1 (crystallized red pigment prepared in Example 1)
Cancer cell line: human liver cancer cell line MHCC-97L
Xenograft model: nude mouse

Method MHCC97L (human liver cancer cell line) 1 × 10 ⁶ was transplanted subcutaneously into the right flank of nude mice. One week later, NYG-1 (0.1 mg / kg and 1 mg / kg) was injected into the peritoneal cavity of the mice. The same injection was treated once a week x 3 times. Thereafter, on day 25, the mice were sacrificed and the tumors were removed. Tumor size was measured with calipers every 2-3 days after the third injection to generate a tumor growth curve. The tumor size was calculated by measuring the minor axis and major axis of the tumor with a caliper and calculating the minor axis x minor axis x major axis / 2.

MHCC97L cells were treated with 10% by volume non-immobilized fetal bovine serum (FBS), 100 IU / mL penicillin G, 100 μg / mL streptomycin (Sigma-Aldrich) in DME medium (GIBCO) at 37 ° C. and 5% CO ₂ air atmosphere. And cultured.

Results As shown in FIG. 3 (A), the NYG-1 treatment group suppressed tumor growth in xenograft mice in a concentration-dependent manner. In particular, the treatment group of NYG-1 (1 mg / kg) significantly suppressed tumor growth (about 50% of the control) (P <0.01).

(B) Saw palm red pigment suppressed the expression of VEGF in human liver cancer in a xenograft nude mouse model.

  The tumor after measuring the size of (A) was homogenized as it was, centrifuged, and the amount of vascular endothelial growth factor (VEGF) in the supernatant was measured by ELISA.

Results As shown in FIG. 3B, the NYG-1 treatment group suppressed the expression of vascular epidermal growth factor (VEGF) in human liver cancer tumors in xenograft mice in a concentration-dependent manner. In particular, the NYG-1 (1 mg / kg) treatment group significantly suppressed tumor vascular epidermal growth factor (VEGF) (about 40% of control) (P <0.01).

A composition containing the red pigment of saw palmetto was prepared with the following formulation.
Ingredient name Formulated amount (mg) / tablet Ethanol extract or red pigment of saw palmetto 50
Lactose 110
Microcrystalline cellulose 30
Silicon dioxide 5
Talc 5
Final weight 200

  A test was conducted to identify the pigment component of the purified saw palmetto red pigment of Example 1.

  Information related to the pigment component of Serenoa repens to date includes β-carotene and tannin by HANDBOOK of Phytoehemical Constituents of GRAS Herbs and Other Economic Plants (1992, JAMES A, DUKE). Therefore, in addition to the above two types of compounds, it was considered that there is a possibility of polyphenol compounds such as flavonoids, which are general pigment components, and substances were identified by conducting confirmation tests of these three types of compounds.

A) Confirmation test of carotenoid compound No carotenoid compound was detected by TLC and HPLC.

B) Confirmation of tannins (1) Confirmation test with iron chloride (III) test solution The addition of the test solution (9 g of iron (III) chloride hexahydrate dissolved in water and adjusted to 100 mL) gave a greenish brown color. This suggested the possibility of being condensed tannin (proanthocyanidins). In this test, condensed tannin (proanthocyanidin) exhibits green to green brown, and hydrolyzed tannin exhibits blue to dark blue.
(2) Confirmation test with vanillin hydrochloric acid test solution It was possible to be tannin because it showed orange color by adding the test solution (0.5 mg vanillin dissolved in 0.5 mL ethanol (95) and adding 0.5 mL water and 3 mL hydrochloric acid). Sex was shown. This test exhibits a red-orange color when tannin is present.
(3) Confirmation of condensed tannin (proanthocyanidins) by TLC-1
In TLC conditions where catechin and oligoproanthocyanidins (2 to 4 mer) can be detected, only the origin was detected as a spot. This suggests that it is a condensed tannin (proanthocyanidin) having a high degree of polymerization.
(4) Confirmation of condensed tannin (proanthocyanidins) by TLC-2
An anthocyanidin was considered to be formed because it became bright red by acid hydrolysis (heated in an 80 ° C. water bath for 30 minutes after addition of hydrochloric acid). As a result of analysis of this hydrolyzed solution in TLC, spots corresponding to anthocyanidins were confirmed, and thus the component before decomposition was considered to be proanthocyanidins (FIG. 4).

Results From the above test results, it was suggested that the purified saw palmetto red pigment is a condensed tannin (proanthocyanidin) containing no carotenoid and having a high degree of polymerization.

Measurement of polyphenol content The total polyphenol content was measured using a foreign thiocult reagent.
Measuring method 100 ml of 50% ethanol was added to 25 mg of the purified saw palmetto red pigment of Example 1, and 2 ml of a solution diluted four times with distilled water was dispensed into a test tube. To this test tube, 2 ml of a foreign thiocult reagent diluted twice with distilled water was added and stirred. After 3 minutes, 2 ml of 10% aqueous sodium carbonate solution was added and stirred. The test tube was held at 25 ° C. for 60 minutes, and then the absorbance at 655 nm was measured with a spectrophotometer. At this time, a calibration curve was prepared using an aqueous catechin solution, and the total polyphenol content was calculated as an equivalent amount of catechin (mg / 100 g).

Result As a result of the analysis, the total polyphenol content of the purified saw palmetto red pigment was 30 to 40% by weight.
As components other than polyphenols in the red pigment, the presence of remaining oily components was considered.

  The ethanol extract or red pigment of saw palmetto fruit of the present invention has angiogenesis inhibitory activity and is useful for treatment and prevention of a wide variety of diseases. These diseases include diseases involving angiogenesis such as psoriasis, arthritis, retinopathy, glaucoma, macular degeneration, periodontal disease, and cancer. It is also beneficial for wrinkle prevention or improvement.

Claims (25)

  An angiogenesis inhibitor comprising an ethanol extract or red pigment of saw palmetto fruit.   Cosmetics containing the angiogenesis inhibitor of Claim 1.   A medicament comprising the angiogenesis inhibitor according to claim 1.   The cosmetic according to claim 2, for improving or preventing skin wrinkles.   The medicament according to claim 3, for treating or preventing a disease or disorder in which it is beneficial to suppress angiogenesis.   The method for producing an angiogenesis inhibitor according to claim 1, the cosmetic according to claim 2 or 4, or the pharmaceutical according to claim 3 or 5, comprising extracting a saw palm fruit with ethanol.   Crystallized red pigment derived from saw palmetto fruit.   The crystallized red pigment according to claim 7, which is obtained by extracting a saw palm fruit with ethanol and precipitating a red pigment in the extract.   The composition containing the crystallized red pigment | dye as described in any one of Claims 7-8.   An angiogenesis inhibitor comprising the crystallized red pigment according to any one of claims 7 to 8.   The foodstuff containing the crystallization red pigment | dye as described in any one of Claims 7-8, the composition of Claim 9, or the angiogenesis inhibitor of Claim 10.   Cosmetics containing the crystallization red pigment | dye as described in any one of Claims 7-8, the composition of Claim 9, or the angiogenesis inhibitor of Claim 10.   The pharmaceutical containing the crystallization red pigment | dye as described in any one of Claims 7-8, the composition of Claim 9, or the angiogenesis inhibitor of Claim 10.   The cosmetic according to claim 12, for improving or preventing skin wrinkles.   14. The medicament according to claim 13, for treating or preventing a disease or disorder for which it is beneficial to suppress angiogenesis. Extracting the saw palm fruit with ethanol,
Separating the extract from the solid residue,
A step of concentrating the extract,
Mixing the concentrate with water,
Irradiating the mixture with ultrasonic waves, separating the oil layer into an aqueous phase, and collecting the red pigment precipitated from the aqueous phase;
The manufacturing method of the crystallized red pigment | dye of Claim 7 or 8 containing these.   The composition according to claim 9, the angiogenesis inhibitor according to claim 10, the food according to claim 11, the food according to claim 11, and the food according to claim 12 or 14, comprising the method for producing a crystallized red pigment according to claim 16. The method for producing a pharmaceutical according to claim 13 or 15, or a cosmetic according to claim 13 or 15.   The crystallized red pigment according to claim 7 or 8, wherein the crystallized red pigment contains proanthocyanidins derived from saw palmetto fruit.   The angiogenesis inhibitor according to claim 1, wherein the ethanol extract or red pigment of saw palm fruit is the crystallized red pigment according to claim 18.   A composition comprising the crystallized red pigment of claim 18.   A food comprising the crystallized red pigment according to claim 18.   A cosmetic comprising the crystallized red pigment according to claim 18.   A medicament comprising the crystallized red pigment according to claim 18. Extracting the saw palm fruit with ethanol,
Separating the extract from the solid residue,
A step of concentrating the extract,
Mixing the concentrate with water,
Irradiating the mixture with ultrasonic waves, separating the oil layer into an aqueous phase, and collecting the red pigment precipitated from the aqueous phase;
The manufacturing method of the crystallized red pigment | dye of Claim 18 containing this.   A method for producing the composition according to claim 20, the food according to claim 21, the cosmetic according to claim 22, or the medicine according to claim 23, comprising the production method according to claim 24.