TWI465237B - Neuroblast cell multiplication promoter and synapse extender - Google Patents

Description

Neuroblast cell proliferation promoter and neurite outstretcher

The invention relates to a neuroblast cell (Neuroblast, also known as neuroblast cell, neuroblast) proliferation promoting agent and a neurite outgrowth agent, and the invention can be widely applied to mammalian medicines, skin external preparations and foods including humans. .

According to the fact that it has been medically proven since ancient times, the fresh stems of the Cistanche herb are quite effective for diseases such as infertility, impotence, constipation (please refer to Patent Document 1), and in addition, the herbaceous plants The fresh stems are quite beneficial for the blood and kidneys. These perennial parasitic herbaceous plants are widely cultivated in northwestern China. In some places, they are called “desert ginseng”, while in the herb family, the most parasitic plant Cistanche tubulosa (Schrenk.) Wigh is the most tailored. Bulk.

Japanese scholars conducted a systematic study on the chemical constituents and pharmacological activities of Cistanche plants. The results showed that the phenylethanoid glycosides in the plants of the family Denko are a major active ingredient (for example, please refer to the non-patent literature). 1-3), while phenylethanoid glycosides can be used as effective antioxidants, metabolism promoters, memory improvers and libido promoters, leading to a series of more in-depth experiments in the medical community on the medical properties of various phenylethanoid glycosides. the study.

In addition, please refer to Patent Document 1, and the results show that the administration of the phenylethanoid glycosides in the brain of the brain can restore the function of the brain of the original lesion; in addition, please refer to Non-Patent Document 4, The addition of a TNFα to the nerve bud cells causes the neuroblast cells to become pathological, and after the addition of the phenylethanoid glycosides, the neurite outgrowth of the nerve bud cells can be restored to the original normal state.

Patent Document 1: Japanese Laid-Open Patent Publication No. 2004-250449

Non-Patent Document 1: Sato T, et al. Yakugaku Zasshi, 1985, 105(12): 1131

Non-Patent Document 2: Jimenez C., et al. Nat. Prod. Rep., 1994, 11 (6): 591

Non-Patent Document 3: Cometa F., et al. Fitoterapia, 1993, 64 (3): 195

Non-Patent Document 4: Deng Min, et al. Pharmacoe Sin, 2004, 25 (10): 1276-1284

However, in the above Patent Document 1, it has been found that in the case of a brain function lesion, the function of the brain can be restored by administering a phenylethanoid glycoside compound, but Patent Document 1 does not describe the phenylethanoid glycoside compound in the brain before the lesion occurs. In addition, in the above-mentioned Non-Patent Document 4, it is described that after the lesion of the neuroblast is caused, the phenylethanide compound can be used to extend the neurites and thereby restore the function of the brain. Non-Patent Document 4 knows nothing about the following conditions: in the case where the nerve bud cells do not have a lesion, the neuroblastic cells can be proliferated by adding a phenylethanoid glycoside compound, and the nerve cells of the nerve bud cells can be made. The protrusions stretch.

It can be seen that there are still many shortcomings in the above-mentioned methods of use, which is not a good method and needs to be improved.

In view of the above-mentioned shortcomings of the above-mentioned applications, the inventors have finally developed and designed a "nerve cell proliferation promoter and a long-term research and experiment with relevant knowledge based on the relevant experience of the subject for many years. Nerve extension device".

In the context of the foregoing information, the inventors have discovered the fact that phenylethanoid glycosides or phenoxyglucosides containing phenylethanoid glycosides are added to neuroblasts (SK-N-SH) where no lesions have occurred. After that, the proliferation of the nerve bud cells can be promoted, and the neurite outgrowth of the nerve bud cells can be promoted, thereby completing the present invention. In other words, the present invention aims to provide a neuroblast cell proliferation promoting agent and a neurite outgrowth agent even in the nerve. When the bud cells do not have lesions, the nerve bud cell proliferation promoter and the neurite outgrowth agent can promote the proliferation of the nerve bud cells, and can promote the stretching of the neurites of the nerve bud cells, thereby promoting the differentiation of the nerve bud cells. For nerve cells, even in the case of degeneration of nerve cells, the neuroblast growth promoter and neurite stretcher can effectively prevent the reduction of nerve cells, thereby effectively preventing aging caused by the brain, and further improving Brain function.

In order to solve the above problems, the neuroblast growth promoting agent according to the first aspect of the invention is characterized in that the neuroblast growth promoting agent has a phenylethanoid glycoside compound as an active ingredient. Further, the neuroblast growth promoter comprises at least one of echinacoside and flavonoid as the phenylethanoid compound, and more importantly, the phenylethanoid compound contains echinacoside And both of the leaves are asparagus.

Further, as a neuroblast cell proliferation promoting agent of the second invention, The neuroblast cell proliferation promoting agent is characterized in that an extract of a family of plants is used as an active ingredient, and the extract contains a phenylethanoid glycoside compound as an active ingredient, and the phenylethanoid glycoside compound contains echinacoside and the like. At least one of, and more importantly, the extract contains both echinacoside and flavonoids as the phenylethanoid glycoside compound.

Further, the neurite stretcher according to the third aspect of the invention is characterized in that the neurite stretcher is an phenylethanoid glycoside compound containing an echinacoside compound and a flavonoid At least one of them, and more importantly, the extract contains both echinacoside and flavonoids as the phenylethanoid glycoside compound.

The neurite outgrowth agent contains at least one of echinacoside and flavonoids as the phenylethanoid glycoside, wherein the neurite outgrowth agent contains both echinacoside and flavonoids as The phenylethanoid glycoside compound, and the neurite outgrowth agent of the fourth invention, characterized in that the neurite outgrowth agent is an extract of a plant of the genus Lepaceae, and the extract contains phenylethanoid glycosides. As the active ingredient, it is important that the extract contains at least one of echinacoside and flavonoids as the phenylethanoid compound, and more importantly, the extract Both echinacoside and flavonoids are contained as the phenylethanoid glycoside compound.

In the above-mentioned first invention and the second invention, the neuroblast cell proliferation promoting agent can promote the proliferation of the neuroblast cells and promote the transdifferentiation of the neuroblast cells even when the neuroblast cells do not have a lesion. For nerve cells, this can further improve memory Functions such as force, computational power, and judgment. In addition, even when nerve cells are degenerated due to aging, etc., by dividing nerve cells into nerve cells, brain function can be maintained, and therefore, the brain can be effectively prevented. The occurrence of brain disorders such as aging or cognitive disorders.

Further, in the third invention and the fourth invention, the neurite stretcher can promote the stretching of the neurites even in the case where the neuroblasts do not have a lesion, and therefore, for the normal brain, the neurites are stretched. The agent can also construct a lot of synapses, so it can further improve brain function. On the other hand, even if the nerve cells are denatured due to aging, the neurite outgrowth can quickly construct a synapse to compensate for the function of the nerve cells, so It can maintain brain function and effectively prevent brain lesions such as brain aging or cognition.

Hereinafter, the present invention will be described in detail: "a neuroblast cell proliferation promoting agent and a neurite outgrowth agent (hereinafter also referred to as "a nerve cell proliferation promoting agent or the like")", which is characterized by a phenylethanoid glycoside compound. As an active ingredient. In addition, in the present specification, "a nerve cell growth promoting agent or the like" means both a "neurocyte growth promoting agent" and a "neurite stretcher".

Here, the "phenylethanoid compound" means a compound represented by the following chemical formula (1):

The phenyl is not particularly limited glycosides, e.g., may include echinacoside, acteoside, 2 '- acetylsalicylic acteoside (2' -acetylacteoside), campneoside I , campneoside II, Cicumubuloside A, Citrus aurantium B1, Cistanche B. sinensis, Cistanche C. C. Cistanche C2, crenatoside, decaffeoylacteoside, heterogeneous It is a component of isoacteoside, rhodioloside, syringalide A 3 ' -α-L-rhamnopyranoside and tubeuloside A. In addition, these compounds may be used alone or in combination. Use two or more.

In addition, the neuroblast growth promoter comprises at least one of echinacoside and flavonoids as a phenylethanoid glycoside, because these may have more significant neuroblast proliferation and neurites. The stretching action, and more importantly, the neurite stretching agent contains at least echinacoside as an active ingredient because it can have a more excellent neurite outgrowth effect.

The method for obtaining the phenylethanoid glycoside compound is not particularly limited, and it can be obtained by synthesis, or can be extracted from plants, and when extracted from plants, the plant as a raw material is not particularly limited, for example, Listed as raw medicine Rehmannia glutinosa, Labiatae, Staphylococcus aureus, olive branch fruit, and Leydaceae herb, etc., and one of the plants may be used alone to extract phenylethanoid glycosides, or two or more kinds of plants may be used in combination. To extract phenylethanoid glycosides, and among these plants, the best choice is from the genus Leucoe, because in the plants of the family Lepaceae, as a phenylethanoid glycoside, in addition to echinacoside and phylloside but also containing 2 '- acetylsalicylic acteoside (2' -acetylacteoside), campneoside I , campneoside II, Cistanche glycosides A (cistantubuloside A), Cistanche glycosides B1, B2 glycosides tubulosa , Cistanche C. sinensis C1, Cistanche C. sinensis, decaffeeoylacteoside, isoacteoside, rhodiolaside syringalide A 3 '-α-L- rhamnopyranoside and flower tube glycosides A (tubuloside A) and the like, having a more significant of neuroblastoma cell proliferation and neurite outgrowth action, and here, no plant raw materials used Orobanchaceae Particularly limited, for example, Cistanche tubulosa , Cistanche salsa , Aeginetia indica , Boschniakia rossica , etc., but not limited to these plants, On the one hand, these plants may be used alone or in combination of two or more. Among these plants, the most preferred is the use of the whole parasitic plant Cistanche tubulosa .

The relationship between the components of the phenylethanoid glycoside compound and the chemical formula (1) is listed below. However, the structural formula of crenatoside is as shown in the following chemical formula (2). The preparation contains substantially only a small amount or a small amount of other components than the echinacoside and the flavonoid in the above components.

The compounds shown in Table 1 above can be passed through high performance liquid chromatography. (HPLC) confirmed that at this time, the conditions of high performance liquid chromatography were as follows, the stationary phase was C18 alkyl decane bonded yttrium, and the mobile phase was acetonitrile-0.05 M phosphoric acid aqueous solution (elution gradient was 4:96→15). : 85), the flow rate was 1 ml/min, and the detection wavelength was 330 nm.

The second neuroblast growth promoter of the present invention is characterized in that an extract extracted from a plant of the genus Lepidoptera is used as an active ingredient.

Here, the plant for use as a raw material is not particularly limited, and examples thereof include the all-parasitic plant Cistanche tubulosa , Cistanche salsa , Aeginetia indica , and Cistanche ( Boschniakia rossica ), etc., but not limited to these plants. Further, these plants may be used alone or in combination of two or more. In addition, among these plants, the best selection is the use of the whole parasitic plant Cistanche tubulosa .

Further, the "extract" preferably contains a phenylethanoid glycoside compound. Here, the "phenylethanoid glycoside compound" is not particularly limited, and examples thereof include echinacoside and phylloside. , 2 '- acetylsalicylic acteoside (2' -acetylacteoside), campneoside I , campneoside II, Cistanche glycosides A (cistantubuloside A), Cistanche glycosides B1, Cistanche glycosides B2, tubulosa Glycoside C1, Cistanche C. sinensis C2, crenatoside, decaffeoylacteoside, isoacteoside, rhodiolaside, syringalide A 3 ' -α-L- Rhamnopyrano side and tubeuloside A and other components. In this case, the extract may contain only one of the above components, or may contain a mixture of two or more components. Among them, at least one of echinacoside and flavonoids is preferably selected.

Further, particularly in the case where the above extract is used as a neurite stretcher, the extract is preferably selected to contain at least echinacoside because it can have a more excellent neurite extension.

In this case, the content of echinacoside is not particularly limited, and when the total quality of the extract of the plant (for example, the whole parasitic plant Cistanche tubulosa ) is 100% by mass, the echinacoside is The content is 10% by mass to 70% by mass, preferably 15% by mass to 50% by mass, more preferably 20% by mass to 40% by mass, and further preferably 23% by mass to 38% by mass. In addition, the content of the leaf gonadoside is not particularly limited, and when the total quality of the extract of the plant is 100% by mass, the content of the flavonoids is 1% by mass to 40% by mass, preferably selected. It is 5% by mass to 30% by mass, more preferably 7% by mass to 20% by mass, and further preferably 8% by mass to 10% by mass.

In this case, the extracted part of the plant is not particularly limited, and leaves, roots, stems, and the like may be used as long as the desired effect can be achieved. Preferably, the extract is extracted from the stems of the plants of the family Levy, particularly fresh stems.

The method for obtaining an extract from a plant which is a raw material is not particularly limited, and examples thereof include a solvent extraction method, a supercritical extraction method, and the like.

On the other hand, when extracting by a solvent extraction method, the solvent to be used is not particularly limited, but it is preferred to use a polar solution. Further, the polar solvent is not particularly limited, and examples thereof include water, methanol, ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene glycol, propylene glycol, glycerin, acetic acid, and ethyl acetate. , ether, etc. These solvents may be used alone or in combination of two or more; preferably, subsequent dilution, concentration, drying, and purification treatments are carried out because a high concentration of the phenylethanoid glycoside compound extract can be obtained by the permeation treatment, and Examples of the purification method include activated carbon treatment, resin adsorption treatment, ion exchange resin, and liquid-liquid countercurrent distribution.

Specific examples of the preparation of the extract are detailed below, but the preparation method of the extract is not limited to the following examples.

The method for producing the extract consists of two stages of extraction and purification; in the first stage, the stem of the plant (for example, the whole parasitic plant Cistanche tubulosa ) is preferred (the plant is preferably selected) The fresh stem is cut into thin pieces or pulverized into fine particles or powder, and then the sheet, or fine particles or powder is immersed in a solvent or a mixed solution such as water or a lower aliphatic alcohol such as methanol or ethanol. The extraction is carried out under temperature. Next, the mixed solution is filtered, and the filtrate is concentrated under reduced pressure or under vacuum to obtain an extract, and in the second stage, after the extract is heated in water, the extract is transferred to be filled with Purification is carried out in an adsorption column of D-101 or AB-8 macroporous adsorption resin. At this time, water, methanol, ethanol, a mixture of water and methanol, or a mixture of water and ethanol is used as a washing solvent. To flush the column. When rinsing, the above rinsing solvent may be washed as a solution of a certain concentration, or the rinsing solvent may be washed according to a concentration gradient, and then the rinsing liquid is collected, and after concentration, drying is performed by a known drying method, to be rinsed After the completion of the drying, the extract is obtained, and the extract contains a phenylethanoid glycoside compound.

The neuroblast cell proliferation promoting agent and the like of the present invention can also be used as a raw material for various foods to be used as a drinking food, and examples thereof include edible oil (salad oil), snacks (chewing gum, candy, caramel, chocolate, biscuits). , snacks, jellies, chewables, small desserts, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, yoghurt, etc.), seasonings (miso, miso, etc.), soup Classes, beverages (juice, coffee, black tea, tea, carbonated drinks, sports drinks, etc.) are general foods, or health foods (tablets, capsules, etc.), nutritional supplements (nutritional drinks, etc.). The neuroblastic cell proliferation agent of the present invention or the like can be suitably contained in these drinking foods.

Depending on the type of food to be consumed, various ingredients may be added thereto, for example, glucose, fructose, sucrose, maltose, sorbitol, Stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, amber may be used. Acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester , acacia, carrageenan (carrageenan), casein, gelatin, pectin, agar, vitamin B, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, spices, preservatives, etc. Further, in the present invention having a health-maintaining function, that is, a neuroblast cell proliferation promoting agent, other auxiliary agents such as an antioxidant substance or a health food raw material such as an antioxidant substance and reduced ascorbic acid may be added. Vitamin C), vitamin E, Reduced glutathione, tocotrienol, vitamin A derivative, lycopene, β-zeaxanthin, astaxanthin, zeaxanthin, fucoidan, uric acid, ubiquinone, coenzyme Q10, folic acid, garlic Extract, allicin, sesamin, lignin, catechin, isoflavones, chalcone, tannins, flavonoids, coumarin, isocoumarin, bilberry extract, arbutin, dan Ning, anthocyanin, apple polyphenol, grape seed extract, ellagic acid, kojic acid, sea buckthorn extract health food raw materials, V. (vitamin) A, VB1, VB2, VB6, VB12, VC, VD, VE, VP, choline, nicotinamide, pantothenic acid, calcium folate, EPA, oligosaccharides, dietary fiber, squalene, soy lecithin, taurine, Dunaliella, protein, octacosanol, DHA , egg yolk lecithin, linoleic acid, lactoferrin, magnesium, zinc, chromium, selenium, potassium, ferrous hemoglobin, oyster meat extract, acetaminophen, chitin oligosaccharide, collagen, cartilage Vegetarian, turmeric, licorice, hazelnut, cinnamon, hawthorn, ginger, ganoderma, mussel extract, turtle, licorice, alfalfa , cinnamon, hawthorn, ginger, ganoderma, plantain, gerbera, chamomile, dandelion, hibiscus, honey, pollen, royal jelly, lime, lavender, rose hip, rosemary, sage, bifidobacteria, bacillus (faecalis), cytosolic lactic acid bacteria, wheat germ oil, castor oil, perilla oil, soybean oil, medium chain fatty acid, Agaricus, Ginkgo biloba extract, turmeric, chondroitin, brown rice germ extract, Ganoderma lucidum, Onion, DHA, EPA, DPA, Sweet Tea, Cordyceps, Garlic, Bee Seed, Papaya, Pu'er Tea, Propolis, Clairvoyia, Hericium, Royal Jelly, Saw Palmetto Extract, Hyaluronic Acid, Collagen, GABA, Seal Oil, shark cartilage, glucosamine, lecithin, phospholipid 醯 serine, notoginseng, mulberry leaves, soy extract, echinacea, siberian ginseng, barley extract, olive leaf, Olive fruit, Gymnema, Lagerstroemia, Salacia, Garcinia, Chitosan, St. John's wort, Jujube, Ginseng, Passiflora, Green cabbage, placenta, alfalfa, grape seed, peanut skin, cranberry, black cohosh, milk thistle, bay laurel, sage, rosemary, apocynum, balsamic vinegar, bitter gourd, malay, safflower, flax, oolong tea Oxytropis, caffeine, capsaicin, xylooligosaccharides, glucosamine, buckwheat, citrus, dietary fiber, protein, prunes, spirulina, barley leaves, nucleic acids, yeast, mushrooms, plum, Amino acids, deep sea shark extract, noni, oyster meat, soft-shelled turtle, mushroom, plantain, cherry, pineapple, banana, peach, apricot, cantaloupe, strawberry, raspberry, orange, fucoidan, mulberry, Heliberry, chondroitin sulfate, zinc, iron, ceramide, silk peptide, glycine, nicotinic acid, vine, neuropterin, L-cysteine, L-meat, red grape leaf, 黍, ask Jing, biotin, Centella asiatica, Lonicera japonica L., Pycnogenol (PYCNOGEN) OL), butterbur, rhubarb, clove, rosemary, catechin, Pu'er tea, citric acid, brewer's yeast, grass rhinoceros, black soybean, ginger, medlar, natto, red peony, tocotrienol , lactoferrin, cinnamon, buckwheat, cocoa powder, grapefruit seed extract, perilla seed extract, litchi seed extract, evening primrose extract, brown rice extract, alpha-lipoic acid, GABA, green coffee bean extract, Butterbur extract, kiwi seed extract, Wenzhou orange extract, ginger extract and astaxanthin.

As a specific manufacturing method, the method of spray drying or freeze-drying can be used to directly form the nerve growth factor promoting agent of the present invention (if the extract is used, the extract together with the dextrin) to form a powder or a granule. , tablets or solutions to easily include them in food (speed In addition to the actual needs, the neuroblast growth promoter or the like of the present invention may be mixed with a binder such as gum arabic to form a powder or granules, and then added to the solid type. In food, on the other hand, in the case of an extract, the extract may be directly added to the beverage, or the extract may be dispersed and dissolved in, for example, water, ethanol, glycerin or a mixture, and then added to the beverage. .

The neuroblast growth promoter of the present invention can also be used as a material for pharmaceuticals (including pharmaceuticals and pharmaceutical products), and can be appropriately added to the raw materials for pharmaceutical preparations by adding the neuroblast growth promoter of the present invention. The drug may be used in humans or in mammals, and may be used as a raw material of a preparation which can be combined with the neuroblast growth promoting agent of the present invention, for example, excipients (glucose, lactose) , white sugar, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, cocoa butter, plant hardening oil, kaolin, talcum powder, etc.), binder (distilled water, physiological saline, ethanol water, white syrup, glucose solution, Starch solution, gelatin solution, carboxymethyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrant (sodium alginate, agar, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, glyceryl monostearate, Starch, lactose, gum arabic powder, gelatin, ethanol, etc.), disintegration inhibitor (white sugar, stearic acid, cocoa butter, hydrogenated oil, etc.), absorption enhancer (quaternary ammonium hydrazine, month Yl sulfate, etc.), adsorbents (glycerin, starch, lactose, kaolin, bentonite, silicic acid, etc.) and a lubricant (purified talc, stearate, polyethylene glycol, etc.) and the like.

As a method of administering the neuroblast cell proliferation promoting agent of the present invention, generally, tablets, pills, soft capsules, hard capsules, and the like can be used. The microparticles, powders, granules and the like are administered orally, and as the liquid preparation, the water-soluble preparation may be administered orally or parenterally. When administered as a parenteral preparation, the neuroblast growth promoter or the like of the present invention may be dispersed in a suitable soluble reagent such as ethanol or water, and then used as a cataplasm, a lotion, an ointment, an elixir, an emulsion, or the like. In addition, the water-soluble preparation of the neuroblast growth promoting agent of the present invention can be used as it is, or a dispersing agent, a suspending agent, a stabilizer, etc. can be added to the water-soluble preparation, and then a cataplasm, Dosage forms such as lotions, ointments, tinctures, and emulsions are used.

The dose to be administered may vary depending on the administration method, the condition, the age of the patient, etc., but in general, the active ingredient administered by an adult may be 5 mg to 400 mg per day, and the active ingredient administered by a child according to one day is 0.5 mg. A dose of about 200 mg is administered.

When the nerve growth factor promoting agent of the present invention is used as a medicine, the ratio of the addition can be appropriately changed depending on the dosage form, and generally, when administered orally or by mucosal absorption, a neuroblast growth promoter or the like is used. The addition ratio may be about 0.01% by weight to 10% by weight, and when it is not administered orally, the addition ratio of the neuroblast growth promoting agent or the like may be about 0.01% by weight to 20% by weight. In addition, since the dose to be administered is adjusted according to various conditions, sometimes it is sufficient even if the dose to be administered is less than the above-mentioned dose range, and sometimes the dose is required to exceed the above dose range, and the pharmaceutical composition contains the above. The nerve bud cell proliferation promoting agent or the like may also contain other compounds known in the field of medicine, and may contain a compound required for molding in a form suitable for oral administration, and examples of the compound include milk. Sugar, starch, hydroxypropyl cellulose, kaolin, talc and calcium carbonate.

In addition, the mammalian animal feed may also contain the neuroblast growth promoter of the present invention, and the neuroblast growth promoter of the present invention may be contained in the animal feed by the same method as the above-described drinking food. The animal to be used for animal feed is not particularly limited. For example, the feed can be used for livestock animals such as cows and pigs, dogs, cats, hamsters, and the like, and flour, meat, and the like can be used. In this case, as the flour, For example, there are wheat flour, rice flour, rye flour, oat flour, buckwheat flour, millet flour, corn flour and soy flour. These flours can also be used in combination of two or more kinds. By using the flour, the pet can be provided with carbohydrates and the like. Among the flours, the wheat flour is optimally selected, and the flour powder, the medium-force powder, and the thin powder may be used singly or in combination, and the wheat flour may also be used in combination with other flours. After heat treatment, the animal feed elasticity can also be used in combination with wheat bran, soybean protein, etc. Network structure of contained bran after heating treatment bulked form a tissue structure, help to improve the taste.

The meat to be used in the present invention is not particularly limited, and chicken, pork, beef, lamb, goat meat, rabbit meat, turkey meat, and horse meat can be used, and the best use of chicken meat can be considered from the viewpoint of taste. The livestock meat is slaughtered and dissected to obtain the edible meat, and the deterioration of the quality of the intermediate moisture product or the low moisture state product is mainly caused by the oxidation of the fat, so the meat is preferably low in fat or has been removed. Lean meat, in addition, containing meat in the feed, not only can provide animals with excellent animal protein, but also Can improve the pet's hobby.

Although various methods can be used to formulate the pet food, a preferred method is as follows: preparing a raw material mixture (hereinafter referred to as "dough") which is mixed with flour (preferably wheat flour) and edible meat, after being formed. Heat treatment is then carried out. The composition of the flour and the meat in the dough is not particularly limited, and generally, it is formulated to be about 5% to 60%, preferably about 10% to 50%, and about 5% to 80%. Preferably, the edible meat consisting of about 20% to 50% of the edible meat and the necessary amount of water is used; and, in the case of using the Aw adjusting agent, the amount of the Aw adjusting agent is about 5% to 30%, Preferably, the amount is about 10% to 20%. Further, the amount of water can be appropriately adjusted according to the mixing and kneading of the dough, the degree of forming, and the amount of flour, meat, Aw conditioner, and the like. The preparation method of the dough is not particularly limited. Preferably, the meat is first made into a meat stuffing by a chopper, a chopper, etc., at this time, in order to make the meat in the meat. The air is sufficiently contained, and it is preferable to finely cut the edible meat, and then add flour, water, and Aw conditioner as needed, and stir well to make bubbles therein, thereby preparing the meat. a dough containing air bubbles, wherein a foaming agent may be added in the preparation of the dough, especially in the case where the flour other than the wheat flour is used as the flour, it is preferred to use a foaming agent through the addition. The foaming agent can uniformly contain fine bubbles in the dough, and can be used as a foaming agent, and various foaming agents can be used. However, from the viewpoint of stability of the bubble, etc., it is preferred to use a soy protein foaming agent. Or enzymatically decomposed soy protein foaming agent.

After the prepared dough is molded and heat treated, it is favored The feed for food, wherein the dough is formed into a suitable shape according to the easiness of the pet when eating and the ease of operation of the keeper, for example, a sheet, a rod, a disc, a ring. In addition, the dough prepared from the same compounding component can be dyed with various pigments, or added to vegetables or fruits, etc., to prepare a variety of doughs having different appearances, and then these doughs can be combined. The heating means for forming the dough after the molding is not particularly limited. For example, the heating means may be oven heating, microwave heating, etc., and these heating methods are generally used. The heat treatment may be carried out according to a usual method, and the moisture content of the feed after heat treatment is generally about 20% to 40%. After the heat treatment, the water is evaporated and the bubbles expand, that is, the dough is puffed, and The water is evaporated in a short time, so the Aw is lowered, thereby improving the preservability. Further, in the case where wheat flour is used as the flour, the network structure of the bran contained in the wheat flour is fixed by heat treatment, thereby improving the mouthfeel. Moreover, in the case of oven heating, there is an advantage of producing a unique hue (fox color) or aroma in the feed, and in the case of microwave heating, since heating is started inside the dough, it is possible The dough is uniformly expanded, and there is an advantage that a feed containing uniform bubbles can be obtained. In the heat treatment, it is preferred to adjust the Aw in the obtained feed to a range of 0.6 to 0.9. As described above, by adjusting Aw within this range, the preservability of the feed can be remarkably improved.

a feed for a pet prepared by the above method, which is a feed having the shape of a bread, since the feed has a soft mouthfeel and a suitable Softness and elasticity, it is suitable for use as feed, snacks, etc. for companion animals with weak dental ability such as puppies, aged dogs or cats. The animal feed described above is contained in a packaging container in an appropriate amount and sealed to form a product. As the packaging container, it is preferred to use an oxygen-impermeable packaging material. Examples of the shape of the package include a vacuum package, a package filled with activated carbon, and the like, but a package filled with an oxygen scavenger (for example, AgelessTM or the like) and activated carbon is preferable. With the relevant packaging form, deterioration of quality due to oxygen and reproduction of microorganisms can be prevented during storage.

The neuroblast growth promoter of the present invention or the like can also be used as an external preparation for skin (including cosmetics, pharmaceuticals, and pharmaceutical products), and it is expected that the external preparation for skin has a neurite stretching action. Further, the external preparation for skin can be used for humans as well as other mammals other than humans. Examples of the skin external preparation which can be used in combination with the neuroblast growth promoting agent of the present invention include, for example, an emulsion, a soap, a face wash, a bathing agent, a skin cream, a lotion, a lotion, a toilet water, and a shaving cream. , shaving lotion, make-up oil, sunscreen lotion, powder, foundation, perfume, pack, nail polish, nail polish, nail polish, eyebrow, blusher, eye cream, eye shadow, dye Mascara, eyeliner, lipstick, lipstick, shampoo, conditioner, hair dye, dispersion, detergent. In addition, as a form of a pharmaceutical product or a pharmaceutical external product which can be used in combination with the neuroblast growth promoting agent of the present invention, an ointment, an emulsion, an external solution, and the like can be mentioned.

In the skin external preparation of the above-mentioned form, in addition to the neuroblast growth promoter of the present invention or the like, the following substances may be added in a range which does not impair the stretching action of the neurites, and the substance may be added. Ingredients in skin external preparations such as cosmetics and pharmaceutical products, including oil components, higher alcohols, fatty acids, ultraviolet absorbers, powders, pigments, surfactants, polyols, sugars, polymers, physiologically active substances, Solvents, antioxidants, perfumes, preservatives, etc. Although examples of these substances are listed below, the present invention is not limited to these examples.

(1) Examples of oil components The oil components of the esters are exemplified by triethyl 2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, and butyl myristate. , isopropyl palmitate, ethyl stearate, octyl palmitate, isohexadecanoyl isostearate, butyl stearate, butyl myristate, ethyl linoleate, isopropyl linoleate Ester, ethyl oleate, isohexadecanol myristate, isostearyl myristate, isostearyl palmitate, octyldodecanol myristate, isohexadecanol isostearate Ester, diethyl sebacate, diisopropyl adipate, isodecyl pentaerythritol, tris(octyldecanoate) glyceride, tris-ethyl 3-ethylhexanoate, Trimethylol propyl triisostearate, pentaerythritol tetraethyl 2-hexanoate, cetyl octyl citrate, decyl laurate, hexyl laurate, decyl myristate, myristyl myristate Ester, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinoleate, isostearyl laurate, isodecyl myristate, nutmeg Isohexadecyl ester, myristic acid Octadecanol ester, isohexadecanol palmitate, isostearyl palmitate, octyl stearate, isohexadecanoyl stearate, isodecyl oleate, octyldodecanol oleate, sub Octyl oleic acid oleate, isopropyl isostearate, cetyl alcohol octadecyl 2-ethylhexanoate, stearyl alcohol 2-ethylhexanoate, hexyl isostearate, dioctanoic acid Ethylene glycol ester, ethylene glycol dioleate, propylene glycol dicaprate, propylene glycol di(octyldecanoate), propylene glycol dicaprylate, Neopentyl glycol dicaprate, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl tris(undecanoate), glyceryl triisopalmitate, glyceryl triisostearate, octyl pivalate Decadiol ester, isostearyl octanoate, octyl isophthalate, hexyl dodecyl neodecanoate, octyl dodecyl neodecanoate, isohexadecyl isostearate, isostearyl Acid isostearyl alcohol ester, octyldodecanol isostearate, polyglyceryl oleate, polyglyceryl isostearate, dipropyl carbonate, dialkyl carbonate (C12-C18) ester, citric acid three Isohexadecanol ester, triisodecyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyl lauryl lactate, triethyl citrate , Ethyl citrate triethyl ester, Ethyl citrate tributyl citrate, Trioctyl citrate, diisostearyl malate, 2-ethylhexyl hydroxystearate, succinic acid 2- 2 Ethylhexyl ester, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, cholesterol stearate, cholesteryl isostearate, cholesteryl hydroxystearate ,oil Cholesterol ester, dihydrocholesteryl oleate, phytosterol ester of isostearic acid, phytosterol oleate, isostearyl ester of 12-stearyl hydroxystearate, 12-stearyl hydroxyl hard Octadecyl fatty acid ester, isostearyl alcohol 12-stearyl hydroxy stearate, and the like. Examples of the oil component of the hydrocarbon include squalene, liquid paraffin, α-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybutene, microcrystalline wax, petrolatum and the like. Examples of animal and vegetable oils and their hardened oils, as well as natural waxes are: tallow, hardened tallow, lard, hardened lard, horse oil, hardened horse oil, oyster sauce, orange linden oil (a fish of the genus Ranunculus) Animal oils such as fish oil, hardened fish oil, egg butter and hardened oil thereof; avocado oil, almond oil, olive oil, korean fat, kiwi seed oil, almond oil, aloe vera oil, sesame oil, wheat germ oil, rice germ oil, Black rice oil, safflower oil, shea butter, soybean oil, evening primrose oil, perilla oil, tea seed oil, camellia oil, corn oil, rapeseed oil, hardened rapeseed oil, palm kernel oil, hardened palm kernel oil, peanut oil, hardened peanut oil, Castor oil, hardened castor oil, sunflower oil, grape seed oil, jojoba oil, hardened jojoba oil, macadamia nut oil, pond flower seed oil, cottonseed oil, hardened cottonseed oil, coconut oil, hardened coconut oil and other vegetable oils and their hardening Oil; beeswax, high acid beeswax, lanolin, reduced lanolin, hardened lanolin, liquid lanolin, carnauba wax, montan wax and other waxes. Examples of the oil component of the decane are: dimethyl polyoxyalkylene, methylphenyl polyoxyalkylene, methylcyclopolyoxyalkylene, octamethylpolyoxyalkylene, decamethylpolyoxyl Alkane, dodecamethyl polyoxyalkylene, methyl hydrogen polyoxyalkylene, polyether modified organopolyoxyalkylene, dimethyloxane. Methyl cetyl hydroxy phthalate copolymer, dimethyl methoxy alkane. Methyl octadecyl hydroxy decane copolymer, alkyl modified organopolyoxy siloxane, terminal modified organopolyoxy siloxane, amino modified fluorenone oil, amino modified organic polyoxy siloxane, dimethyl Dimethiconol, decyl ketone, propenyl ketone, trimethyl methacryloxy decanoic acid, fluorenone RTV gum, and the like. Examples of fluorine-based oil components are: perfluoropolyether, fluorine-modified organopolyoxane, fluorinated pitch, carbon fluoride, fluoroalcohol, fluoroalkyl. Polyhydroxyalkylene co-modified organopolyoxane and the like.

(2) Examples of higher alcohols are: lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl alcohol, isostearyl alcohol, oleyl alcohol, behenyl alcohol, 2-ethylhexanol, hexadecane Alcohol, octyldodecanol, and the like.

(3) Examples of fatty acids are: caprylic acid, capric acid, undecylenic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, flax. Acid, arachidic acid, arachidonic acid, Twenty-two acid, sinapic acid, 2-ethylhexanoic acid, and the like.

(4) Examples of the ultraviolet absorber are: p-aminobenzoic acid, amyl benzoic acid, ethyl dihydroxypropyl p-aminobenzoate, glyceryl p-aminobenzoate, ethyl p-aminobenzoate, p-aminobenzoin Octanoic acid ester, octyl dimethyl benzoate, octyl dimethyl salicylate, octyl salicylate, triethanolamine salicylate, phenyl salicylate, butyl phenyl salicylate, benzyl salicylate , with menthyl salicylate, benzyl cinnamate, octyl p-methoxycinnamate, 2-ethylhexyl p-methoxycinnamate, di-methoxy cinnamic acid mono-2-ethylhexanoate , p-methoxy cinnamic acid isopropyl ester, p-methoxy cinnamic acid diethanolamine, diisopropyl. Diisopropyl cinnamic acid ester mixture, urinary acid, ethyl urate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone sulfonic acid and its salt, dihydroxymethoxybenzophenone, two Sodium hydroxymethoxybenzyl ketone sulfonate, dihydroxybenzophenone, dihydroxydimethoxybenzophenone, hydroxyoctyloxybenzophenone, tetrahydroxybenzophenone, butyl methoxy dibenzoylmethane , 2,4,6-triphenylamino-p-(2-ethylhexyl-1-carboxy)-1,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzo Triazole, methyl o-aminobenzoate, 2-ethylhexyl 2-amino-3,3-diphenylacrylate, phenyl benzimidazole sulfuric acid, 3-(4-methylbenzylidene) camphor , isopropyldibenzoylmethane, 2-(3,4-dimethoxyphenylmethylene)-2,5-dioxo-1-imidazolidinylpropionic acid 2-ethylhexyl ester Etc., and polymer derivatives or decane derivatives of these substances.

(5) Examples of powders and pigments include: red 104, red 201, yellow 4, blue 1, black 401, etc.; yellow 4 AL precipitated color, yellow 203 BA precipitated pigment, etc. pigment; Nylon powder, silk powder, urethane powder, Teflon (registered trademark) powder, fluorenone powder, polymethyl methacrylate powder, cellulose powder, starch, fluorenone globular elastomer powder, polyethylene powder Other polymers; yellow iron oxide, red iron oxide, black iron oxide, chromium oxide, carbon black, ultramarine blue, dark blue and other colored pigments; zinc oxide, titanium oxide, cerium oxide and other white pigments; talcum powder, mica, white lead, Body pigments such as kaolin and platy barium sulfate; pearlescent pigments such as mica titanium; metal salts such as barium sulfate, calcium carbonate, magnesium carbonate, aluminum citrate and magnesium citrate; inorganic powders such as barium and alumina; aluminum stearate and hard Metal soaps such as magnesium sulphate, zinc palmitate, zinc myristate, magnesium myristate, zinc laurate, zinc undecylenate; bentonite, montmorillonite, boron nitride, and the like. The shape (spherical shape, rod shape, needle shape, plate shape, amorphous shape, scaly shape, spindle shape, etc.) and particle diameter of these powders are not particularly limited. In addition, a known surface treatment method (for example, a fluorine compound treatment method, an anthrone treatment method, an anthracene resin treatment method, a suspension treatment method, a decane coupling agent treatment method, a titanium coupling agent treatment method, an oil treatment method, or the like) may be employed. N-thiolated resin treatment method, polyacrylic acid treatment method, metal soap treatment method, amino acid treatment method, lecithin treatment method, inorganic compound treatment method, plasma treatment method, mechanochemical treatment method, etc.) deal with.

(6) Examples of Surfactants Anionic surfactants may, for example, be a fatty acid soap, an α-mercaptosulfonate, an alkylsulfonate, an alkylallylsulfonate or an alkylnaphthalenesulfonate. Alkyl sulfate, POE alkyl ether sulfate, alkyl amino sulfate, alkyl phosphate, POE alkyl phosphate, alkyl amino phosphate, alkyl alkyl alkyl taurate, N-decyl amino acid Salt, POE alkyl ether carboxylate, alkyl sulfosuccinate, Sodium alkyl sulfoacetate, thiolated hydrolyzed collagen peptide salt, perfluoroalkyl phosphate, and the like. The cationic surfactant may, for example, be an alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyltrimethylammonium bromide or an octadecyl hexadecanocarbonate. Trimethylammonium chloride, distearyl dimethyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium chloride, docosaluminyl trimethyl ammonium bromide, chlorinated chlorhexidine, two Dodecanoic acid aminopropyl dimethyl hydroxypropyl ammonium chloride, diethylaminoacetamide stearate, dimethylaminopropionamide stearate, lanolin derivative quaternary ammonium salt, and the like. The amphoteric surfactant may, for example, be a carboxy beet oxime type, a guanyl beet oxime type, a sulfo betain oxime type, a hydroxy sulfo beet oxime type, a guanyl sulfo beetine type, a phospho beet oxime type, an aminocarboxylic acid. A salt type, an imidazoline derivative type, a guanamine type, and the like. Examples of the nonionic surfactant include propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE sorbitan fatty acid ester, POE sorbitol fatty acid ester, and POE. Glycerol fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE. POP copolymer, POE. POP alkyl ether, polyether modified decane decanoic acid alkyl decylamine, alkyl amine oxide, hydrogenated soybean lecithin, and the like. Examples of the natural surfactant include lecithin, saponin, and a saccharide surfactant.

(7) Examples of the polyhydric alcohol and the sugar include ethylene glycol, diethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, polypropylene glycol, glycerin, diglycerin, polyglycerin, and 3-methyl-1. 3-butanediol, 1,3-butanediol, sorbitol, mannitol, raffinose, erythritol, glucose, sucrose, fructose, xylose, lactose, maltose, maltitol, trehalose, alkyl Fucose, mixed isose, sulfated trehalose, amylopectin, and the like. Further, chemical modifications thereof and the like can also be used.

(8) Examples of the polymer include an acrylate/methacrylate copolymer (Plus size, manufactured by Mutual Chemical Co., Ltd.), a vinyl acetate/crotonic acid copolymer (resin 28-1310, manufactured by NSC Corporation), and acetic acid. Vinyl ester/crotonic acid/vinyl neodecanoate copolymer (28-2930, manufactured by NSC Corporation), methyl vinyl ether maleic acid half ester (Gantrez ES, manufactured by ISP), tert-butyl acrylate/acrylic acid Ester/methacrylic acid copolymer (manufactured by Luvimer, BASF Co., Ltd.), vinylpyrrolidone/vinyl acetate/vinyl propionate copolymer (Luviskol VAP, manufactured by BASF Corporation), vinyl acetate/crotonic acid copolymer ( Luvi set CA, manufactured by BASF Co., Ltd.), vinyl acetate/crotonic acid/vinylpyrrolidone copolymer (Luvi set CAP, manufactured by BASF Corporation), vinylpyrrolidone/acrylate copolymer (Luviflex, manufactured by BASF Corporation) , acrylate/acrylamide copolymer (Ultra Hold, manufactured by BASF Corporation), vinyl acetate/butyl maleate/isobornyl acrylate copolymer (Advantage, ISP), carboxyvinyl Polymers (Carbopol, BFGoodrich Co., Ltd.), acrylic acid. An anionic polymer compound such as an alkyl methacrylate copolymer (manufactured by Pemulen, BF Goodrich); or an acetic acid amphoteric compound of a dialkylaminoethyl methacrylate polymer (manufactured by Yukaformer, Mitsubishi Chemical Corporation), octyl acrylate Amphoteric polymer compound such as acrylamide/hydroxypropyl acrylate/butylaminoethyl methacrylate copolymer (manufactured by AMPHOMER, NSC Co., Ltd.); quaternary carbonization of vinylpyrrolidone/dimethylaminoethyl methacrylate (GAFQUAT, manufactured by ISP), methyl ethylene a cationic polymer compound such as a pyridoxazole chloride/vinylpyrrolidone copolymer (manufactured by Luvi Co., Ltd.); polyvinylpyrrolidone (Luviskol K, manufactured by BASF Corporation), vinylpyrrolidone/vinyl acetate copolymer ( Luviskol VA, manufactured by BASF Co., Ltd., vinylpyrrolidone/dimethylaminoethyl methacrylate copolymer (Copolymer 937, manufactured by ISP), vinyl caprolactam/vinylpyrrolidone/dimethylaminoethyl methacrylate A nonionic polymer compound such as an ester copolymer (Copolymer VC713, manufactured by ISP). In addition, the following natural polymer compounds are also suitable: cellulose and its derivatives, keratin and collagen or their derivatives, calcium alginate, pullulan, agar, gelatin, tamarind seed polysaccharide, Xanthan gum, carrageenan, high methoxyl pectin, low methoxyl pectin, guar gum, gum arabic, crystalline cellulose, arabinogalactan, karaya gum, tragacanth, seaweed Acid, albumin, casein, curdlan, gellan gum, dextran, and the like.

(9) Examples of physiologically active ingredients As the physiologically active ingredient, in the case of application to the skin, those which can bring about some physiological activity to the skin can be cited. For example, whitening ingredients, immunostimulating agents, anti-aging agents, anti-UV agents, slimming agents, stretching agents, antioxidants, hair-increasing agents, hair growth agents, moisturizers, blood circulation-reducing agents, antibacterial agents, and fungicides can be cited. , desiccant, cold sensitizer, warming agent, vitamins, amino acids, wound healing enhancers, stimulating mitigators, analgesics, cell activating agents, enzyme components, and the like. Examples of suitable ingredients that are compatible with these physiological activities include, for example, backgrass extract, avocado extract, earthen mountain extract, hollyhock extract, arnica extract, aloe extract, apricot extract. , Apricot kernel extract, Ginkgo biloba extract, fennel extract, turmeric extract, oolong tea extract, commercial extract, Echinacea extract, Astragalus extract, Astragalus extract, Coptis extract, Barley extract , Small forsythia extract, wild sesame extract, cardamom extract, orange extract, seawater dry matter, seaweed extract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed silk, chamomile extract, carrot extract, capillaris Artemisia extract, licorice extract, hibiscus extract, Pyracantha extract, cinchona extract, cucumber extract, guanosine, hazelnut extract, mountain white bamboo extract, Sophora flavescens extract, walnut extract, grapefruit Extract, Hydrangea vine extract, spirulina extract, mulberry extract, gentian extract, black tea extract, yeast extract, burdock extract, black rice fermentation extract , rice germ oil, polymeric grass extract, collagen, bilberry extract, asarum extract, Bupleurum extract, umbilical cord extract, sage extract, saponin extract, bamboo extract, hawthorn extract , pepper extract, shiitake mushroom extract, rehmannia extract, purple root extract, perilla extract, eucalyptus extract, spirea extract, peony extract, calamus root extract, birch extract, wattle extract , Ivy extract, Western hawthorn extract, elderberry extract, yarrow extract, western peppermint extract, sage extract, mallow extract, Chuanxiong extract, sauerkraut extract, soy Extract, jujube extract, thyme extract, tea extract, clove extract, thatch extract, dried tangerine peel extract, angelica extract, calendula extract, peach extract, eucalyptus extract, houttuynia cordata Extract, tomato extract, natto extract, ginseng extract, garlic extract, wild rose extract, hibiscus extract, wheat winter extract, parsley extract, honey, witch hazel extract Chrysanthemum extract, fragrant tea extract, bisabolol, alfalfa extract, dandelion extract, butterbur extract, alfalfa extract, false leaf tree extract, grape extract, propolis, loofah extract, Safflower extract, peppermint extract, linden extract, peony extract, hops extract, pine extract, horse chestnut extract, water plantain extract, sapindus extract, lemon balm extract, peach extract , cornflower extract, eucalyptus extract, saxifrage extract, coix seed extract, mugwort extract, lavender extract, apple extract, lettuce extract, lemon extract, milk vetch extract, rose extract, fan Rosemary extract, Roman chamomile extract, royal jelly extract, and the like. Further, examples thereof include biopolymers such as deoxyribonucleic acid, mucopolysaccharide, sodium hyaluronate, sodium chondroitin sulfate, collagen, elastin, chitin, chitosan, and hydrolyzed eggshell membrane; Amino acid, hydrolyzed peptide, sodium lactate, urea, sodium pyrrolidone carboxylate, beet mash, whey, trimethylglycine and other moisturizing ingredients; neurolipids, neuropterin, phytosphingosine, cholesterol, cholesterol derivatives, phospholipids and other oily ingredients ; ε-aminocaproic acid, glycyrrhizic acid, β-glycyrrhetic acid, chlorinated lysozyme, guaiac blue hydrocarbon, hydrogen hydroxyl adrenocortical hormone and other immune activators; vitamin A, vitamin B2, vitamin B6, vitamin C, vitamins D, vitamin E, calcium pantothenate, biotin, niacinamide, vitamin C ester and other vitamins; allantoin, diisopropylamine diisopropylamine, 4-aminomethylcyclohexanoic acid and other active ingredients; tocopherols, Carotenoids, flavonoids, tannins, lignin, saponins and other antioxidants; α-hydroxy acid, β-hydroxy acid and other cell activators; γ-oryzanol, vitamin E derivatives and other blood loop promoter; retinol Retinol Wound healing agent such as albumin, kojic acid, placenta extract, sulfur, ellagic acid, linoleic acid, tranexamic acid, glutathione, etc.; bellows glycosides, licorice extract, capsaicin, quinone Alcohol, iodinated garlic extract, pyridoxine hydrochloride, DL-α-tocopherol, DL-α-tocopherol acetate, nicotinic acid, nicotinic acid derivative, calcium pantothenate, D-panthenol, ethyl sulfonium Carboxyl ether, biotin, allantoin, isopropylmethylphenol, estradiol, ethinyl estradiol, caprochloroammonium, benzalkonium chloride, diphenhydramine hydrochloride, quaternary ammonium salt, camphor, water Salicylic acid, vanillic acid phthalic acid, phthalic acid, vanillyl amide, pyridone, ethanolamine, pentyl pentanoate, L-menthol, mononitroguaiacol, resorcinol, γ -amino Butyric acid, predopyl ammonium chloride, mexiletine hydrochloride, auxin, estrogen, cantharidin, cyclosporin, zinc pyrithione, hydroxyrepinephrine, minoxidil, polystearate A hair growth agent such as ethylene sorbitan, peppermint oil, or Sasanishiki rice extract.

(10) Examples of the antioxidant include sodium hydrogen sulfite, sodium sulfite, erythorbic acid, sodium erythorbate, dilauryl thiodipropionate, tocopherol, tolylbiguanide, nordihydroguaiaretic acid, Hydroxyanisole, butylhydroxyanisole, dibutylhydroxytoluene, ascorbyl stearate, ascorbyl palmitate, octyl gallate, propyl gallate, carotenoids, flavonoids, tannins Plant extracts having antioxidant effects such as lignin, saponin, apple extract and clove extract.

(11) Examples of the solvent include purified water, ethanol, lower alcohols, ethers, LPG, fluorocarbons, N-methylpyrrolidone, fluorine-substituted alcohols, volatile linear siloxanes, secondary freons, and the like. .

In order to facilitate the review of the technical means and operation of the present invention The process has a further understanding and understanding, and an embodiment is combined with the drawing, which is described in detail below.

The present invention relates to a "neuroblast cell proliferation promoting agent and a neurite outgrowth agent", and the preparation example 1 of the neuroblast cell proliferation promoting agent of the present invention is a Caspanche tubulosa ( Cistanche tubulosa ) The extract of the article is referred to as "Cistanche tubulosa", and the preparation includes the following steps:

Step 1: Extracting: Take 10 kg of a fresh stem of Cistanche tubulosa and immerse it in 8 times the amount of water in the slice. After immersing for 1 hour, the plate is decocted with water for 2 hours to form a mixture, and the mixture is decoctioned. The mixture is filtered to obtain a first filtrate. Further, one of the filtered residue of the mixture is mixed with 4 times the amount of 40% ethanol of the residue, and then boiled for 4 hours, and filtered again to obtain a second filtrate. The residue after filtering the second filtrate was mixed in the same manner as above, and the residue was mixed with 4 times of 40% ethanol of the residue, and then boiled for 4 hours and filtered, and the operation was repeated twice to obtain a third filtrate and a The fourth filtrate was further combined with the first, second, third and fourth filtrates, and concentrated under vacuum to concentrate the specific gravity to 1.05 to obtain a final extract (the final extract had a weight of 6.2 kg).

Step 2: Purification; the above final extract is heated in a half weight of water (50 ° C), dissolved in water to obtain an extract solution, and then the extract solution is placed in a pre-treated D In the adsorption column of the -101 type macroporous adsorption resin, the adsorption column is washed with water to obtain an aqueous solution of the fresh stem twice, and then the adsorption column is washed with a 20% ethanol to obtain a fresh stem twice. First of 20% ethanol In the aqueous solution, the first aqueous solution is again subjected to the aforementioned adsorption-rinsing step to obtain a second aqueous solution of 20% ethanol, and the first and second aqueous solutions are combined, and after a concentration and drying process, a phenylethanoid glycoside is obtained. An extract of the compound (the weight of the extract was 0.865 kg).

The content of echinacoside and flavonoids was determined by high performance liquid chromatography (HPLC). The conditions of the HPLC were as follows: the stationary phase was a C18 alkyl decane bonded yttrium gel, and the mobile phase was a methanol - 0.15%. Aqueous acetic acid (30:70) at a flow rate of 1 ml/min and a detection wavelength of 330 nm.

Each scale was dried in a vacuum at 60 ° C for 24 hours, one of the echinacosides and one type of lutein, dissolved in a 50% methanol to prepare a control solution (the concentration of the control solution is 1 mL of the solution containing 0.1 Mg solute).

While supersonic treatment, 50 mg of the extract containing the phenylethanoid glycoside compound was dissolved in a 25 mL volumetric flask containing an appropriate amount of 50% methanol, and 50% methanol was further added to the above solution, and adjusted to a 25 mL scale. At the same time, accurately measure about 1 mL of the solution, place it in a 10 mL volumetric flask, add 50% methanol to the mark, and finally filter it with a 0.45 μm membrane to prepare a sample solution.

5 μl of the control solution and the sample solution were respectively weighed and injected into a high-performance liquid chromatograph to determine the peak area of the echinacoside and the leaf gonadoside, and finally calculate the content according to the peak area thereof. The results showed that the content of the echinacoside in the extract was 37.5%, and the content of the flavonoid in the extract was 9.0%.

In the second embodiment and the third embodiment, each is an echinacoside (in the present embodiment, the echinacoside is manufactured by Xinghui Pharmaceutical Industry Co., Ltd. And a class of pure lutein (in this example, the pure class of leaf asparagine is manufactured by Xinghui Pharmaceutical Industry Co., Ltd.) for the proliferation of nerve bud cells (please refer to the second figure) Show).

Experiment 1: Evaluation method for proliferation of nerve bud cells, a human nerve bud cell (the nerve bud cell is a SK-N-SH) was suspended in an MEM medium (the MEM medium contained a 10% FCS, one) 100units / mL penicillin and a 100 μ g / mL streptomycin) in each dish of a 6mm was added 5ml of the above suspension, and then formulated into various embodiments of a concentration in the extract was added tubulosa The culture was cultured for 5 days, and then the cell proliferation degree was evaluated by the MTT assay on the 5th day of the culture, and the results were as shown in the first figure, and the extracts of Example 2 and Example 3 were tested in the same manner. The result is shown in the second figure.

As shown in FIG. First, when the degree of proliferation of cells was measured by MTT assay, it was found that the extract of Cistanche tubulosa exhibits concentration-dependent proliferation of neuroblastoma cells when the extract of Cistanche tubulosa concentration of 10 μ g when / mL, the proliferation of neuroblastoma cells was 7%, when the concentration of extract of Cistanche tubulosa of 30 μ g / mL, the proliferation of nerve cells bud 16%, which results demonstrate: even in the nerve When the bud cells are not diseased, the Cistanche tubulosa extract still has the effect of proliferating human neural bud cells (SK-N-SH). In addition, as shown in the second figure, the nerve bud cells are determined by MTT assay. when the degree of proliferation, and found that such echinacoside acteoside exhibits concentration-dependent proliferation of neuroblastoma cells, particularly in echinacoside 1 μ g / mL and 10 μ g / mL concentration The following shows a significant difference, which can also confirm that the phenylethanoid glycosides, especially the echinacoside and the cohosin, also have humans even in the absence of pathological changes in the neuroblasts. The role of proliferation of nerve bud cells (SK-N-SH).

Experiment 2: Evaluation of neurite outgrowth

The human neuroblastoma cells (SK-N-SH) were suspended in a MEM medium (containing an 10% FCS, a 100units / mL penicillin and 100 μ g / mL streptomycin) in each dish in a 6mm 5 ml of the above suspension was added, and the extract of Cistanche tubulosa in Example 1 was added to various concentrations, and the changes of the neurites over time were observed with a microscope, and photographic records were recorded. The results are shown in the third figure, and The extracts of Example 2 and Example 3 were also tested according to the same procedure, and the results are shown in the fourth and fifth figures, respectively.

As shown in the third figure, although the control group showed a cell proliferation phenomenon, the neurite outgrowth was not observed at all, and in contrast, the Cistanche tubulosa extract group was observed to be observed. the multi neurite outgrowth (with reference to the case of the third coil to FIG solid), particularly when the extract of Cistanche tubulosa concentration of 30 μ g / mL, and can be found in the neighboring cell neurites to form a network, the This confirmed that the Cistanche tubulosa extract also had a concentration-dependent neurite outgrowth even in the state where the neuroblasts did not develop lesions.

In addition, in the fourth and fifth figures, although the control group exhibited a cell proliferation effect, substantially no neurite outgrowth was observed, and in contrast, the echinacoside group (the first Four figures) and this class of leaf asparagine group (fifth figure) can observe more neurite outgrowth (please refer to the situation in the dotted circle of the fourth and fifth figures), In addition, in the echinacoside group, it was apparent that the neurites formed a network with adjacent cells at a low concentration (refer to the case of the dotted circle in the fifth figure), thereby confirming that even In the state where the neuroblast cells are not impaired, the phenylethanoid glycoside compound, particularly the echinacoside and the genus cytosine, also promote the neurite outgrowth in a concentration-dependent manner.

The formulation examples of one of the neuroblast growth promoters of the present invention are listed below, but the formulation is not intended to limit the present invention.

As described above, the present invention can provide a neuroblast cell proliferation promoting agent and a neurite outgrowth agent, and the neuroblast cell proliferation promoting agent and the neurite outgrowth agent can promote the nerve bud cells even when the neuroblast cells are not diseased. Proliferation, and can promote the expansion of neurites of nerve bud cells, and promote the differentiation of nerve bud cells into nerve cells. Even in the case of degeneration of nerve cells, the nerve bud cell proliferation promoter and neurite outgrowth agent can be effectively Prevents the reduction of nerve cells, thereby effectively preventing the aging caused by the brain while improving brain function.

The above detailed description is intended to be illustrative of a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Variations and modifications are intended to be included in the scope of the patents covered by the present invention.

The first picture is the extract of Cistanche tubulosa to human neuroblasts. Proliferation map.

The second figure is a graph showing the proliferative effect of echinacoside and flavonoids on human neuroblasts.

The third picture is a photograph of the stretched state of the neurites caused by the extract of Cistanche tubulosa.

The fourth picture is a photograph of the stretched state of the neurites caused by echinacoside.

The fifth picture is a photograph of the stretched state of the neurites caused by asparagine.

Claims (4)

The invention relates to the use of an extract of Cistanche tubulosa as an active ingredient for preparing a nerve bud cell proliferation promoting agent, wherein the method for manufacturing the Cistanche tubulosa extract comprises two stages: in the first small stage, the tube flower is The stem of Cistanche is sliced or pulverized into particles or powder; next, the thus obtained flakes, or microparticles or powder, are immersed in water, allowed to stand for a while, filtered, and the residue after immersion is taken out from the water; And then mixing the residue with 4 times the amount of 40% ethanol, and extracting at room temperature; then, filtering the extraction mixture, and concentrating the filtrate in vacuo to a specific gravity of 1.05; next, in the second stage, The concentrated filtrate obtained in the first stage is heated in water, and then transferred to an adsorption column packed with D-101 type or AB-8 type macroporous adsorption resin for purification, using water, methanol, ethanol, a mixture of water and methanol, or The mixture of water and ethanol is used as a rinsing solvent, and the adsorption column is washed, and the rinsing solvent is washed twice with 20% ethanol of the rinsing solvent; The liquid is concentrated and dried to obtain an extract of Cistanche tubulosa containing echinacoside and acteoside, which has the effect of regenerating nerve bud cells. Use as described in claim 1 of the patent application, characterized in that The adsorption column is an adsorption column filled with a D-101 type macroporous adsorption resin, and the rinsing of the adsorption column includes first rinsing the adsorption column with water to obtain an aqueous solution of twice the amount of the stem, and then a 20% The adsorption column is washed with ethanol to obtain a first aqueous solution of 20% ethanol of the stem twice, and the first aqueous solution is again subjected to the aforementioned adsorption-rinsing step to obtain a second aqueous solution of 20% ethanol, and the first solution is combined. And a second aqueous solution. The use as described in claim 1 or 2, characterized in that in the first stage, the fresh stem of Cistanche tubulosa is extracted. The invention relates to the use of an extract of Cistanche tubulosa as an active ingredient for preparing a nerve bud cell proliferation promoting agent, wherein the method for preparing the Cistanche tubulosa extract comprises the following steps: immersing a slice of fresh stem of Cistanche tubulosa In the water of 8 times the amount of the sheet, the sheet was immersed in water for 1 hour, and then decocted with water for 2 hours, and the thus decocted mixture was filtered to obtain a first filtrate; then, the residue of the decocted mixture was The residue was mixed with 40 times of 40% ethanol, and then boiled for 4 hours, and the mixture after decoction was filtered to obtain a second filtrate, and the residue of the second filtrate was repeated twice in the same manner as above to obtain a third filtrate and a fourth filtrate; the above four filtrates are combined, and concentrated in a vacuum to a specific gravity of 1.05 (50 ° C) to obtain a final extract; the final extract is dissolved in the final extraction under heating Half the weight of water; next, the extract solution is placed in an adsorption column that has been filled with pre-treated D-101 macroporous adsorption resin; Rinse the adsorption column to obtain an aqueous solution of the fresh stem twice, and then rinse the adsorption column with a 20% ethanol to obtain a first aqueous solution of the fresh stem twice the amount of 20% ethanol, and the first aqueous solution is again Performing the aforementioned adsorption-rinsing step to obtain a second aqueous solution of 20% ethanol, combining the first and second aqueous solutions, concentrating and drying to obtain echinacoside and acteoside. The Cistanche tubulosa extract has the effect of regenerating nerve bud cells.