JP2013184923A - Extract derived from paecilomyces tenuipes, astrocyte growth promoter including the same, and method of producing the astrocyte growth promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an astrocyte growth promoter derived from P.tenuipes.SOLUTION: An extract as an active ingredient is obtained by solid phase extraction using a carrier, wherein a solution containing a dried product of a water extract fraction separated by two phase partition between an aqueous solution containing a hot-water extract derived from P.tenuipes and an organic solvent is charged, and a liquid mixture of water and an organic solvent.

Description

  TECHNICAL FIELD The present invention relates to an extract derived from Prunus japonica, an astrocyte growth promoter containing the same, and a method for producing an astrocyte growth promoter.

  Astrocytes (astrocytes), a type of glial cell, account for about half of all cells in the brain. Conventionally, from the viewpoint that information processing functions are carried out by nerve cells, the functions of astrocytes existing around nerve cells have been considered to support, protect, and supply nutrients to nerve cells.

  On the other hand, in recent years, astrocytes have not only an indirect neural circuit formation assisting mechanism such as a neural circuit formation function (Non-Patent Documents 1-4) and a transmitter concentration control function (Non-Patent Documents 5 and 6), but also from nerve cells. And the subsequent calcium propagation between astrocytes (Non-patent Documents 7-9), synaptic vesicle-like vesicles can be output to neurons (Non-patent Documents 10 and 11), etc. Has been suggested to play a role in information processing cells.

  In addition, studies on the role of astrocytes in memory formation have begun, suggesting that higher brain functions such as memory are regulated through interactions between neurons and astrocytes. Yes. For example, it has been reported that the number of astrocytes increases in the hippocampus after memory formation (Non-Patent Document 12), and that memory formation is inhibited when the function of astrocytes is suppressed (Non-Patent Document 13). .

  In addition, as a common neuroanatomical abnormality in psychiatric disorders such as schizophrenia, bipolar disorder, and depression, macroscopically, brain hypertrophy, hippocampus, and cerebral cortex size are reduced. Levels were known to reduce neuronal cell size, decrease dendritic spine density, shorten dendritic length, and decrease synaptic protein content. These have been thought to be direct abnormalities of neurons, but recently it has been reported that a decrease in the number of astrocytes is also common, an indirect neuronal state based on a decrease in the number of astrocytes The possibility of abnormalities has been studied (Non-Patent Document 14).

  On the other hand, the present inventors have been carrying out research on the pharmacological effects of cordyceps.

Cordyceps is one of the entomopathogenic fungi that stick to insects, and in a narrow sense, the insect net (Insecta), Lepidoptera, Hepialoidea, and Hepatidae (Hepialidiae) It refers to Cordyceps Sinennsis that lives in alpine areas of 3,000 to 4,000 meters above sea level in Nepal and Bhutan, including Tibet Autonomous Region in China, Qinghai, Sichuan, Guizhou, Gansu and Yunnan. There are many types of host insects, including Hemiptera, Lepidoptera, Coleoptera, Hymenoptera, Grasshopper (Orthoptera), Dragonfly (Odonata), and Flyptia (Diptera) And so on.
On the other hand, in a broad interpretation, the whole fungus that parasitizes adult insects and larvae is also called cordyceps.

  Although there are still few scientific findings regarding Chinese caterpillars as ingredients for herbal medicines and health supplements, Cordyceps and its products are examples of research on the physiological activity of Cordyceps soybeans. It is widely used as a nutrient that is effective in preventing cancer and metabolic diseases or delaying the progression of those diseases (Non-patent Document 15). In addition, antioxidant activity (Non-patent Document 16), immunoregulatory effect (Non-patent Document 17), decrease in insulin resistance in vivo and increase in insulin secretion (by non-patent document 16) by water extract of Cordyceps militaris ( Non-patent document 18), antihyperlipidemic effect (Non-patent document 19), anti-tumor activity (non-patent document 20), anti-inflammation by hot water extraction of Cordyceps Sinensis (hereinafter C. sinensis), a Tibetan cordyceps In addition to the action (Non-patent document 21), antitumor activity (Non-patent document 22) by a proteoglycan complex of Phellinus linteus has been reported. C. sinensis, a Tibetan cordyceps, is becoming expensive and difficult to obtain due to such overexploitation due to a sudden increase in demand due to the increasing popularity of cordyceps.

  In addition, as a broad interpretation, a kind of cordyceps (Paecilomyces tenuipes, hereinafter referred to as P. tenuipes) belongs to the genus Cordyceps of the family Ascomycete, and is a fungus that parasitizes silkworm larvae. Because of this, artificial cultivation of cordyceps in combination with silkworm cocoons has recently been commercialized in Japan. However, many commercially available Cordyceps and Paecilomyces Cordyceps products have been prepared from mycelial cultures of asexual species, and research reports on the pharmacological effects of P. tenuipes are overwhelming compared to Cordyceps. Very few.

  So far, as a physiologically active component of P. tenuipes, the cyclic hexadepsipeptide Beauvericin isolated from the ethyl acetate extract of the mixed powder of the host (Silkworm) and fruiting body has the effect of inhibiting the growth of rat liver cancer cells ( Non-patent document 23) and hanasanagin (3,4-diguanidinobuta) obtained from 60% ethanol extraction, 5% methanol extraction, and hot water extraction process using the fruiting body isolated from the host (Silkoptera). Noyl-DOPA) is known to have free radical (DPPH) scavenging activity and superoxide anion scavenging ability (Non-patent Documents 24 and 25).

  In addition, artificial cultivation methods for these cordyceps have been proposed (Patent Documents 1 and 2).

Japanese Patent No. 2676502 Japanese Patent No. 3865735

Le Roux PD, Esquenazi S: Astrocytes mediatecerebral cortical neuronal axon and dendrite growth, in part, by release of fibroblast growth factor.Neurol Res, 24: 81-92, 2002. Biran R, Noble MD, Tresco, PA: Directed nerve outgrowth is enhanced by engineered glial substrates.Exp Neurol, 184: 141-152, 2003. Slezak M, Pfieger FW: New roles for astrocytes: Regulation of CNS synaptogenesis.Trends Neurosci, 26: 531-535, 2003. Ullian EM, Christopherson KS, Barres BA: Role for glia in synaptogenesis.Glia, 47: 209-216, 2004. Schousboe A, Sarup A, Bak LK, Waagepetersen HS, Larsson OM: Role of astrocytic transport processes in glutamatergic and GABAergic neurotransmission.Neurocem Int, 45: 521-527, 2004. Huang YH, Bergles DE: Glutamate transporters bring competition to the synapse. Curr Opin Neurobiol, 14: 346-352, 2004. Fellin T, Carmingnoto G: Neurone-to-astrocyte signaling in the brain represents a distinct multifunctional unit.J Physiol, 559: 3-15, 2004. Araque A, Perea G: Glial modulation of synaptic transmission in culture.Glia, 47: 241-248, 2004. Volterra A, Steinhauser C: Glial modulation of synaptic transmission in the hippocampus.Glia, 47: 249-257, 2004. Parpura V, Scemes E, Spray DC: Mechanisms of glutamate release from astrocytes: gap junction "hemichannels", purinergic receptors and exocytotic release. Neurochem Int, 45: 259-264, 2004. Bezzi P, Gundersen V, Galbete JL, Seifert G, Steinhauser C, et al .: Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate. Nat Neurosci, 7: 613-620, 2004. Jahanshahi, M., Sadeghi, Y., Hosseini, A. et al .: The effect of spatial learning on the number of astrocytes in the CA3 subfield of the rat hippocampus. Singapore Med. J., 49, 388−391 (2008 ) Gibbs, M. E., O'Dowd, B. S., Hertz, E. et al .: Astrocytic energy metabolism consolidates memory in young chicks. Neuroscience, 141, 9-13 (2006) Cotter DR, Pariante CM, Everall IP: Glial cell abnormalities in major psychiatric disorders: The evidence and implications.Brain Res Bull, 55: 585-595, 2001. Paterson, R.R.M. (2008) Cordyceps-A traditional Chinese medicine and another fungal therapeutic biofactory Phytochemistry, 69, 1469-1495. Yamaguchi, Y., Kagota, S., Nakamura, K., Shinozuka, K. and Kunimoto, M. (2000) Antioxidant activity of the extracts from fruiting bodies of cultured Cordyceps sinensis. Phytother. Res., 14, 647-649 . Kuo, Y. C., Tsai, W. J., Wang, J. Y., Chang, S. C., Lin, C. Y. and Shiao, M. S. (2001) Regulation of bronchoalveolar lavage fluids cell function by the immunomodulatory agents from Cordyceps sinensis.Life Sciences, 68, 1067-1082. Choi, SB, Park, CH, Choi, MK, Jun, DW and Park, S. (2004) Improvement of insulin resistance and insulin secretion by water extracts of Cordyceps militaris, Phellinus linteus, and Paecilomyces tenuipes in 90% pancreatectomized rats. Biotechnol. Biochem. 68, 2257-2264. Koh, J.- H., Kim, J. -M., Chang, U.- J. and Suh, H.- J. (2003) Hypocholesterolemic effect of hot-water extract from mycelia of Cordyceps sinensis. Biol. Pharm Bull. 26, 84-87. Bok, J. W., Lermer, L., Chilton, J., Klingeman, H. G. and Neil Towers, G. H., (1999) Antitumor sterols from the mycelia of Cordyceps sinensis.Phytochemistry, 51, 891-898. Rao, Y. K., Fang, S. H. and Tzeng, Y. M. (2007) Evaluation of the anti-inflammatory and anti-proliferation tumoral cells activities of Antrodia camphorate, Cordyceps sinensis, and Cinnamomum osmophloeum bark extracts.J. Ethnopharmacology, 114, 78-85. Nakamura, T., Matsugo, S., Uzuka, Y., Matsuo, S. and Kawagishi, H. (2004) Fractionation and anti-tumor activity of the mycelia of liquid-cultured Phellinus linteus. Biosci. Biotechnol. Biochem. 68 868-872. Junpei Fujita (2008) Functional and structural analysis of physiologically active substances from the silkworm Cordyceps. Pp. 2-5, Master's thesis, Department of Agricultural Life Sciences, Graduate School of Agriculture, Iwate University. Sakakura, A., Suzuki, K., Katsuzaki, H., Komiya, T. Imamura, T., Aizono, Y. and Imai, K. (2005) Hanasanagin: a new antioxidative pseudo-di-peptide, 3,4 -diguanidinobutanoyl-DOPA, from the mushroom, Isaria japonica.Tetrahedron Letters, 46, 9057-9059. Sakakura, A., Shioya, K., Katsuzaki, H., Komiya, T., Imamura, T., Aizono, Y. and Imai, K. (2009) Isolation, structural elucidation and synthesis of a novel antioxidative pseudo-di -peptide, Hanasanagin, and its biogenetic precursor from the Isaria japonica mushroom.Tetrahedron, 65, 6822-6827.

  And while the present inventors are proceeding with research on P. tenuipes, which is easier to obtain than C. sinensis and is superior in terms of cost and stable supply, P. tenuipes The extraction fraction derived from the powder was found to have a remarkable astrocyte growth-promoting activity, leading to the present invention.

  This invention is made | formed in view of the above situations, and makes it a subject to provide the astrocyte proliferation promoter derived from P. tenuipes.

In order to solve the above-mentioned problems, the present invention provides the following extract, astrocyte growth promoter and a method for producing an astrocyte growth promoter.
<1> a carrier charged with (added to) a solution containing a dried product of a water-extracted fraction separated by two-layer distribution of an aqueous solution containing a hot water extract from P. tenuipes and an organic solvent; An extract obtained by solid phase extraction with a mixture of water and an organic solvent.
<2> An astrocyte growth promoter comprising the extract of <1> above.
<3> The following steps: (1) a step of drying a hot water extract of P. tenuipes to obtain a hot water extract; (2) a hot water extract obtained in step (1) above. Separating the water-extracted fraction and the organic-solvent-extracted fraction by performing two-layer partitioning with the aqueous solution and the organic solvent, and obtaining a dried product of the water-extracted fraction; and (3) obtained in the step (2) A step of charging (adding) a solution containing a dried product of the obtained water-extracted fraction to a carrier and then bringing the mixture into contact with a mixed solution of water and an organic solvent to obtain a liquid extract by solid phase extraction. A method for producing an astrocyte growth promoter.
<4> In the step (3), before solid phase extraction with a mixed solution of water and an organic solvent, a solution containing a dried product of the water extraction fraction obtained in the step (2) is charged (added). The method for producing an astrocyte growth promoter according to claim 3, further comprising a step of performing solid-phase extraction with a carrier and water.
<5> The method for producing an astrocyte growth promoter according to claim 3, wherein the organic solvent in the mixed solution in the step (3) is methanol or ethanol.
<6> The method for producing an astrocyte growth promoter according to any one of claims 3 to 5, wherein the concentration of the organic solvent in the mixed solution in the step (3) is 20% to 80%.

  According to the present invention, an astrocyte growth promoter derived from P. tenuipes is provided. Since it is made from easily available P. tenuipes and exhibits an excellent astrocyte growth promoting effect, it is also excellent in terms of cost and stable supply.

It is the microscope picture which observed the state of the nerve cell which passed for 48 hours after each sample addition with the phase-contrast microscope. It is the figure which showed the numerical value of the total process length of the neuron in 72 hours after seeding before the sample addition, and the total process length of the neuron after 48 hours after the addition of each sample. It is the microscope picture which observed the state of the astrocyte 48 hours after each sample addition with the phase-contrast microscope. It is the figure which showed the proliferation rate of the astrocyte 48 hours after each sample addition. It is the figure which showed the proliferation rate of the astrocyte for every time in F5.

The astrocyte growth promoter of the present invention contains an extract derived from P. tenuipes. And the manufacturing method of the astrocyte growth promoter of this invention is the following processes:
(1) A step of drying a hot water extract of P. tenuipes to obtain a hot water extract;
(2) The aqueous extract containing the hot water extract obtained in the step (1) and the organic solvent are separated into a water extract fraction and an organic solvent extract fraction by performing two-layer partitioning. A step of obtaining a dried product; and (3) a solution containing the dried product of the water-extracted fraction obtained in the step (2) is charged (added) to a carrier, and then the mixture is mixed with water and an organic solvent. And obtaining a liquid extract by solid-phase extraction by contacting.

  Hereinafter, each step will be described.

  In the step (1), a hot water extract of P. tenuipes is dried to obtain a hot water extract.

  P. tenuipes are widely distributed in Japan, Taiwan, China, Nepal, etc., parasitizing moth moths, larvae, silkworm moths, larvae, etc. A pale yellow fruit body is generated. P. tenuipes used as a material for the astrocyte growth promoter of the present invention may be self-generated, but is preferably artificially cultivated using a silkworm as a host. As compared with C. sinensis, it is possible to suppress the cost and supply it stably.

  Various methods for artificial cultivation of cordyceps have been proposed. For example, the method of Patent Document 2, that is, the silkworm larvae before the formation of the cocoons are boiled and dried, and the silkworm dried powder 50 to 90 is dried. Mix the food dry powder consisting of one or more of dry powders of weight percent, beans, cereals, seaweeds or mushrooms in the balance, add the culture solution to this, knead, and mix this with the bottom of the growth box A method of cultivating a medium by preparing a medium by enclosing the medium, encapsulating the medium in an inoculation bag and sterilizing by heat, then inoculating the medium with fungus of Cordyceps sinensis.

  In the present invention, it is preferable to use, for example, hanasagitake powder obtained by freeze-drying and then pulverizing hanasagitake (P. tenuipes) cultivated by the above method. Here, the powder of the bamboo bud used in the present invention may be a powder of only the fruit body of the flower bud, but is preferably a powder of the fruit body and a host (for example, silkworm).

  Furthermore, the hot water extraction method for this flower is not particularly limited. For example, a hot water extract can be obtained by solid-liquid extraction in which water is added to this flower and heated in an autoclave or the like. The heating conditions can be appropriately designed. For example, a hot water extract of P. tenuipes powder can be obtained by heating at 80 to 120 ° C. for about 60 minutes. Further, the hot water extract is centrifuged, the supernatant is filtered and collected, and the extraction process is repeated to obtain a hot water extract from P. tenuipes powder with higher yield. be able to. Then, a hot water extract (PTE) of the flower of a bamboo shoot can be obtained by recovering the supernatant of the hot water extract of the flower of a bamboo shoot and lyophilizing it.

  In the step (2), the aqueous solution containing the hot water extract obtained in the step (1) and the organic solvent are separated into a water extraction fraction and an organic solvent extraction fraction by water extraction. A dry fraction is obtained.

  In this step, the hot water extract of the Japanese bamboo shoot powder can be separated into a water extraction fraction and an organic solvent extraction fraction by performing two-layer distribution (liquid-liquid extraction) using water and an organic solvent. Specifically, for example, a hot water extract (PTE) of Japanese bamboo shoot powder is dissolved in water, and this solution and an organic solvent are placed in a separatory funnel and shaken to exchange substances at the interface between the two liquids. The organic matter contained in the aqueous layer can be removed. In this case, examples of the organic solvent include organic solvents such as n-hexane, ethyl acetate, and acetone. Among them, it is preferable to use ethyl acetate.

  And the dried body of a water extraction fraction can be obtained by performing the water extraction fraction obtained by such a method, for example by a nitrogen air drying process or a freeze-drying process.

  In step (3), the solution containing the dried product of the water extraction fraction obtained in step (2) is charged (added) to a carrier, and then the carrier is brought into contact with a mixed solution of water and an organic solvent. An extract is obtained by solid phase extraction.

  In this step, a conventionally known purification method can be employed. For example, column (silica gel) chromatography can be exemplified, and among these, reverse phase flash column chromatography can be preferably exemplified. Reversed-phase flash column chromatography uses a fine column (silica gel) and develops by applying pressure to the inside of the column, and thus has excellent purification ability.

  As a preferred form, for example, solid phase extraction is performed by flowing water (ultra pure water) to a carrier charged (added) with a solution containing a dried product of the water extraction fraction obtained in the step (2). An example is a method in which an extraction liquid is obtained, and then a sequential extraction is performed by performing extraction a plurality of times while sequentially increasing the concentration of a mixed liquid of water and an organic solvent. In the water extraction fraction, solid phase extraction is performed by flowing water (ultra-pure water) through a carrier charged (added) with a solution containing a dried product of the water extraction fraction obtained in the step (2). Can be eluted, and the active ingredient can be more reliably extracted by solid phase extraction with a mixture of water and an organic solvent.

  The concentration of the organic solvent in the mixed solution is preferably 20% to 80%, more preferably 40% to 70%, and particularly preferably about 60%. When the concentration of the organic solvent in the mixed solution is within such a range, a component having astrocyte growth activity can be reliably purified. Further, when performing sequential extraction, after flowing solid (ultra pure water) and performing solid phase extraction, for example, the concentration of the organic solvent in the mixed solution is 10%, 20%, 40%, 60%,.・ ・ Execution is performed while increasing sequentially, so that the active ingredient can be reliably extracted. In this case, the component having an astrocyte growth activity is contained in the extract having a concentration of the organic solvent in the mixed solution of 40% to 70%, particularly about 60%.

  Moreover, the organic solvent used at this process can illustrate methanol, ethanol, acetonitrile, acetone, dioxane, tetrahydrofuran, isopropyl alcohol, etc. Especially, methanol and ethanol can be used preferably. By using methanol and ethanol, an active ingredient can be extracted reliably.

  Further, the extract obtained through this step can be appropriately dried and concentrated to dryness to obtain a solid extract.

  Since the extract obtained through this step has astrocyte growth promoting activity, it can be used as an astrocyte growth promoter by using this as an active ingredient.

  The astrocyte growth promoter of the present invention can contain the extract obtained by the above method as an active ingredient. That is, the extract is a dried product of a water-extracted fraction separated by a two-layer distribution of an aqueous solution containing a hot-water extract obtained by drying a hot-water extract of P. tenuipes powder and an organic solvent. It can be obtained by solid phase extraction of a carrier charged with a solution containing water and a mixture of water and an organic solvent.

  In vitro or in vitro, astrocytes can be proliferated by allowing the astrocyte growth promoter of the present invention to act on brain cells. Therefore, the astrocyte growth promoter of the present invention can be used for the treatment and prevention of various diseases and disorders.

  For example, since astrocytes account for about half of all cells in the human brain, the astrocyte growth promoter of the present invention can be used as a therapeutic agent for brain contusions and the like. In addition, since astrocytes have a function of forming a neural circuit, they can be used as therapeutic agents for brain diseases that cause cognitive dysfunction, such as Alzheimer's disease and Parkinson's disease. Furthermore, since astrocytes are involved in memory formation, they can be used to improve memory and learning abilities such as spatial patterns and information supplementation. And an astrocyte proliferation promoter can be utilized also as a therapeutic agent of mental diseases, such as schizophrenia, bipolar disorder, and depression.

  The astrocyte growth promoter of the present invention can be administered orally or parenterally.

  For oral administration, take the form of tablets, pills, powders, troches, packaging, oblates, elixirs, suspensions, emulsions, solutions, syrups, aerosols, and sterile packaging powders, for example. Can do. In this case, conventional excipients, wetting agents, emulsifiers, dispersants, preservatives, sweeteners, fragrances and the like can be added as appropriate as additives. Examples include lactose, dextrose, sucrose, sorbitol, mannitol starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. . In the case of oral administration, the dose can be appropriately determined in consideration of the formulation method, the administration method, the age, weight, etc. of the administration subject. Furthermore, the astrocyte growth promoter of the present invention can be used not only for pharmaceuticals but also for health supplements.

  In addition, in the case of parenteral administration, direct administration into the brain is possible in addition to intravenous administration, subcutaneous administration, transdermal absorption, and the like. In the case of direct administration into the brain, a region in the brain can be appropriately selected according to treatment, symptoms to be improved, and the like.

  Furthermore, the astrocyte growth promoter of the present invention can be administered not only to humans but also to various mammals.

  The astrocyte growth promoter of the present invention has an excellent astrocyte growth effect and can be effectively used for various applications as described above. In addition, it is superior in terms of cost and stable supply, because it is made of P. tenuipes, which is easier to obtain than C. sinensis.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples at all.

Example 1 Partial Purification of Silkworm Cordyceps Extract 1. Test sample As a cordyceps, which is a test sample, P. tenuipes powder was used for the experiment. As for P. tenuipes, those harvested at Mt. Yachizo across Fukushima and Ibaraki Prefectures were cultured by the same method as that described in Patent Document 1 (Japanese Patent No. 2667502). This strain is deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the name of microorganism identification name Paecilomyces tenuipes, IU070255, and accession number FERM P-22011.

  This was infected with a silkworm of a dried silkworm practical variety (Nishiki x Kanwa) obtained in the process of psoriasis and cultured in a dark room at 25 ° C. for 65 days. The fruiting bodies and host cocoons obtained were dried and pulverized.

2. Preparation of P. tenuipes hot water extract (PTE)
Add 8 times amount (W / V) (640 ml) of ultrapure water (hereinafter referred to as MQ) to P. tenuipes powder (80 g) and autoclave at 105 ° C for 60 min (BS-245, Tommy Seiko) To obtain a hot water extract. The extract was centrifuged at 10,000 × g at 4 ° C. for 10 min, and the supernatant was collected by filtration through qualitative filter paper (No. 2 ADVANTEC). To the residue, 8 times the amount of MQ was added again, and a second extraction was performed under the same conditions. The supernatant was collected, mixed with the supernatant obtained in the first extraction, and the filtrate was collected by suction filtration. The powder obtained by freeze-drying this with a freeze-dryer (EYELA FDU-2100, Tokyo Rika Instrument Co., Ltd.) was used as a P. tenuipes hot water extract (hereinafter referred to as PTE) and kept at -80 ° C until use. Stored in a cryogenic bath).

  Table 1 shows the dry weight and yield of PTE.

3. Preparation of PTE bilayer partition extract MQ was dissolved in PTE (10.1273 g) in a beaker, and the solution was transferred to a 200 ml separatory funnel. The concentration gradient of PTE was made uniform by shaking the separatory funnel, and 100 ml of ethyl acetate was added thereto. Then, after shaking and degassing for 5 min, the separatory funnel was allowed to stand for 15 min and separated into a completely transparent two-liquid layer. The lower MQ layer (MQ-1) was collected, 100 ml of MQ was newly added to the separatory funnel, and the same operation was repeated.

  Collect the lower MQ layer (MQ-2) and the upper ethyl acetate layer (EA-1), put 100 ml of the first collected MQ layer (MQ-1) and ethyl acetate into the separatory funnel, and repeat the same The operation of was repeated. The lower MQ layer (MQ-1) and the upper ethyl acetate layer (EA-2) were recovered. EA-1 and EA-2 were mixed to form an ethyl acetate fraction, and MQ-1 and MQ-2 were mixed to form an MQ fraction. Using a rotary evaporator set (CCA-1100, DPE-1220, SB-1000, N-1000, EYELA; DTU-20, ULVAC), nitrogen air drying and freeze dryer (EYEZA, FDU-2100) After concentration and drying, the yield and yield were calculated and stored in a low temperature bath at −80 ° C.

  Table 2 shows the yield and yield of the ethyl acetate layer (Ethyl acetate-Phase) and the MQ layer (MQ-Phase).

4. Purification by reverse phase flash column chromatography In order to further purify the two-layer partition extract MQ layer obtained in step 1, reverse phase flash column chromatography was performed. The column was produced by a dry packing method. Place silica gel (Wakosil 40C18, Wako Pure Chemical Industries, Ltd.) as a carrier in a chromatographic tube up to a height of 10 cm so that the column volume is 100 cm ³ , swell with methanol, and then chromatograph MQ as the first developing solvent. The upper part of the tube was inserted, and the solvent and the bubbles in the carrier were pushed out and replaced by pressurizing the pump (HIBLOW AIR POMP, model SPP-6EBS, Techno Takagi Co., Ltd.).

  Thereafter, the above 3. 4.0 mg of the two-layer partition extract MQ layer obtained in (1) was dissolved in MQ, centrifuged at 1,000 × g 1 min, and only the supernatant was charged on the surface of the carrier. 500 ml of MQ, 10% methanol (methanol / MQ water (1/9, v / v)), 20% methanol (methanol / MQ water (1/4, v / v)), 40% methanol while pumping (Methanol / MQ water (2/3, v / v), 60% methanol (methanol / MQ water (3/2, v / v)), 80% methanol (methanol / MQ water (4/1, v / v) )), 100% methanol was flowed in an amount of 3 times the column volume (300 ml) sequentially, and each eluted fraction was collected in an Erlenmeyer flask.

  Each fraction collected was concentrated and dried using a rotary evaporator, an aspirator, or a freeze dryer, and after yield and yield were calculated, they were subjected to the following biological activity test.

  Hereinafter, each fraction is divided into F1: MQ extraction fraction, F2: 10% methanol extraction fraction, F3: 20% methanol extraction fraction, F4: 40% methanol extraction fraction, F5: 60% methanol extraction fraction, F6: 80% methanol extraction fraction, F7: 100% methanol extraction fraction.

  Table 3 shows the dry weight and yield of each fraction (F1 to F7).

<Example 2> Primary culture of mouse fetal cerebral neurons 1. Experimental animals Pregnant ICR mice were purchased from Japan SLC, and mouse embryos at the 16th day of pregnancy were used for the following experiments.

2. Primary culture of mouse cerebral nerve (1) Preparation of cerebral nerve cell An ICR mouse of 16 days of gestation was euthanized by cervical dislocation after anesthesia with diethyl ether. The mouse was laparotomized and the fetus was removed together with the uterus, thoroughly disinfected with 70% ethanol, then immersed in 30 ml of PBS (-) in a petri dish for cell culture (diameter 100 mm, Orange Scientific) and carried into a clean bench . All subsequent operations were performed in a clean bench.

The fetus was removed from the uterus and transferred to a petri dish pre-dispensed with 20 ml of PBS (-), and then the fetus was opened and the entire brain was removed. The resulting brain was transferred into 10 ml of high glucose Dulbecco's modified Eagle medium (HG-D-MEM, Wako Pure Chemical Industries), and the olfactory bulb, medial ridge, meninges were removed from the medium using tweezers, and the cerebrum including the hippocampus After that, it was finely chopped with scissors to be less than 1 mm square. The minced cerebrum and the medium were transferred to a 50 ml conical tube (TPP), allowed to stand for 2 min, and the supernatant was removed using an electric pipette. Add 4 ml of HG-D-MEM to the minced brain, add 400 μl of 2.5% trypsin (SIGMA), and 40 μl of 1% DNase I (SIGMA), and then occasionally stir in a 37 ° C thermostatic chamber. Incubated for 10 min. After incubation, add 10 ml of HG-D-MEM (10% FBS) to the conical tube to stop the trypsin reaction, and then centrifuge at 1,000 × g for 3 min with a centrifuge (TOMY, LC-220) . The supernatant was blotted with an electric pipette, 10 ml of HG-D-MEM (10% FBS) was added, and pipetting was performed several times with a sterile glass pipette until no cell clumps were visible. In order to remove excess cell mass, the cell solution was passed through a cell strainer (pore diameter: 100 μm, BD Falcon ^™ ).

(2) Observation of cerebral neurite length (1) Count the number of cells in the cell solution using a cell counter, and use HG-D-MEM (10%) to obtain 5.0 × 10 ⁵ cells / ml. The density was adjusted with FBS). 500 μl per well was seeded on a 24-well Poly-D-Lysine Coated Plate (BD Falcon ^™ ). The cells were cultured for 72 hours under humidified conditions of 5% CO ₂ and 37 ° C., and at the same time as medium change, 0.5 μl each of F1 to F7 obtained in Example 1 was added as a sample, and further cultured under the same conditions for 48 hours. . Further, PBS (−) and DMSO used for sample dissolution were used as controls, and resveratrol (Res. Final concentration 10 μM), which is known to have a nerve cell proliferation effect, was used as a positive control. 4 wells were added per sample.

  Each well of the 24-well plate is observed using a phase-contrast microscope (ECLIPSE Ti-S, Nikon). A 50 μm square field is randomly extracted, and 72 h after cell seeding and 48 h after cell addition (cells) The total process length of cerebral neurons at the time 120 h) after seeding was observed and measured. Neurite outgrowth was measured using NIS-Elements Ar 3.0 (Nikon), which is image analysis software.

3. Statistical analysis JMP 8 (registered trademark) (SAS Institute Inc.) was used as statistical analysis software. Analysis of variance (ANOVA) was performed on the total projection length of cerebral neurons measured in 2. (2) above. As a result, only when a significant difference was found, a post-hoc test was performed using the Tukey-Kramer method. If P <0.05, it was considered statistically significant.

4). Results We observed and measured with a phase-contrast microscope based on the morphological features ascertained by immunostaining of neurons. The values of total cerebral neuron projection lengths 72 h after seeding before sample addition were side by side in all groups, but 48 h after sample addition were all after seeding in the group to which F1 to F7 were added. The value was significantly increased compared to 72 h (FIG. 1).

  At 48 h after the addition of the sample, the values of PBS and DMSO as controls were 374.89 μm and 374.38 μm, respectively, and the value was greatly increased to 608.59 μm with resveratrol as the positive control. Although F1 was 535.07 μm, F2 was 530.73 μm, and F6 was 502.37 μm, there was a tendency for the numerical values to increase greatly, though not as good as the positive control (FIG. 2). However, no statistically significant difference from the control group was observed in all the sample addition groups (F1 to F7).

<Example 3> Astrocyte growth promoting activity experiment (1) Neonatal mouse cerebral neuron primary culture
A new ICR mouse (day 0) is thoroughly disinfected with 70% ethanol, then immersed in 30 ml of PBS (-) in a 100 mm dish for cell culture (diameter 100 mm, Orange Scientific) and transported to a clean bench The pups were euthanized after cervical dislocation. The pups are decapitated, the entire brain is removed, and they are transferred to a 100 mm dish for cell culture containing 15 ml of high glucose Dulbecco's modified Eagle medium (HG-D-MEM, Wako Pure Chemical Industries). After removing the olfactory bulb, medial protuberance, and meninges to make only the cerebrum including the hippocampus, transfer it to a 100 mm dish for cell culture containing 10 ml of HG-D-MEM, and use a scalpel to measure about 1 mm square. I cut it finely. The cut cerebrum together with the medium was transferred to a 50 ml conical tube (TPP), allowed to stand for 2 min, and then the supernatant was removed. 4 ml of HG-D-MEM was newly added, and 400 μl of 2.5% trypsin (SIGMA) and 40 μl of 1% DNase I (SIGMA) were further added, followed by incubation for 10 min with occasional agitation in a 37 ° C. water bath. After incubation, add 10 ml of HG-D-MEM (10% FBS) to the conical tube to stop the trypsin reaction, and then centrifuge at 1,000 × g for 3 min in a centrifuge (H-9R, Kokusan) . The supernatant was sucked up with an electric pipetter, 10 ml of HG-D-MEM (10% FBS) was added, and pipetting was performed several times with a sterile pipette until the cell mass was not visible. To remove excess cell mass, the cell solution was passed through a cell strainer (pore size 100 μm, BD Falcon ^™ ), then the number of cells was counted with a cell counter, and HG- was adjusted to 6.0 × 10 ⁵ cells / ml. Adjusted with D-MEM (10% FBS). After adjusting the cell number, 7 ml each of the cell solution was seeded on Poly-D-Lysine Cellware 100 mm Dish (PDL 100 mm dish, BD Falcon ^™ ). Further, 96 hours after seeding, the medium was once removed with an aspirator, the inside of the PDL 100 mm dish was gently washed with 10 ml of PBS (−), and the medium was exchanged by newly adding 7 ml of HG-D-MEM.

(2) Preparation of astrocytes 72 hours after medium exchange, the PDL 100 mm dish seeded with the cell solution was taken out from the incubator, the lid was sealed with parafilm, and 3-4 dishes were stacked and fixed. This was shake-cultured on a bioshaker (BR-40LF, TAITEC) under the conditions of 37 ° C., 100 rpm, and 20 h to release nerve cells, cell fragments, dead cells, and the like. After shaking, transfer the PDL 100 mm dish into a clean bench, remove the supernatant with an aspirator, wash with 10 ml of PBS (-), add an appropriate amount of 2.5% trypsin (SIGMA) with a Pasteur pipette, and leave it in the incubator for 10 min. I put it. Return the PDL 100 mm dish to the clean bench, add 10 ml of Dulbecco's modified Eagle's medium (D-MEM, Wako Pure Chemicals) (10% FBS) to stop the trypsin reaction, and put the cell solution in a 50 ml conical tube. collected. Thereafter, the number of cells was counted with a cell counting plate and adjusted with D-MEM (10% FBS) so as to be 6.0 × 10 ⁵ cells / ml. This is designated as astrocyte cell fluid.

Then, 7 ml of this astrocyte cell solution was seeded in a PDL 100 mm dish. At this time, only astrocytes, which are neurons in the central nervous system other than neurons, are deposited in the PDL 100 mm dish. (Hereafter referred to as cultured astrocytes)
(3) Passage of cultured astrocytes The cultured astrocytes prepared in the above (2) were subjected to medium exchange 96 hours after seeding and further to medium replacement after 72 hours. The operation was the same as the medium exchange in the experiment of (1) above, but D-MEM (10% FBS) was used as the medium for both medium exchanges. Further, the medium was exchanged according to the above procedure until the culture astrocytes reached confluence in a PDL 100 mm dish. After 10 to 14 days, shaking culture in a bioshaker was not performed, and then the number of cultured astrocytes was adjusted according to the adjustment method in (2), and subculture was performed. In the following experiments, cultured astrocytes after passage (2nd generation) were used.

(4) Experiments for promoting the growth of cultured astrocytes Adjust the astrocyte cell solution from the cultured astrocytes (2nd generation) according to the procedure in (2), and remove the anti-detachment coated slide glass (MAS-GP typeA, MATSUNAMI, Size 76 × 26 mm , Thickness 0.9-1.2 mm) is spread on 5 pieces of square petri dish (sterilized No. 2 petri dish, Size 140 x 100 mm, Height 14.5 mm, Eiken Equipment Co., Ltd.) 96 hours after the culture astrocytes were sufficiently established, the glass slides were transferred one by one to a 100 mm dish for cell culture, 10 ml of D-MEM (10% FBS) was added, and the cells were cultured in an incubator.

  Thereafter, the medium was changed at 72 hours, and further 72 hours later, the medium was changed and the sample was added, and changes in the number and morphology of cultured astrocytes were observed 48 hours after the addition of the sample.

  The sample added was F1-F7 obtained in Example 1, DMSO used as a solvent in dissolving the sample as a control group, and resveratrol used as a positive control in Example 2, In this experiment, it was used as a negative control.

(5) Observation method Observation is performed using a phase-contrast microscope (ECLIPSE Ti-S, Nikon). Three fields of 140 x 140 mm ² are randomly determined, and the number of astrocyte nuclei in the field is counted. The value was defined as the number of astrocyte cells. In addition, NIS-Elements Ar 3.0 (Nikon), which is image analysis software, was used for counting.

(6) Statistical analysis JMP 8 (registered trademark) (SAS Institute Inc.) was used as statistical analysis software. Analysis of variance (ANOVA) was performed on the number of cells counted in (5) above. As a result, only when a significant difference was recognized, a post-hoc test was performed using Dunnett's method. If P <0.05, it was considered statistically significant.

(7) Results As shown in FIG. 3, the growth of astrocytes was confirmed at F1 to F7 48 hours after the addition of the sample. Among them, the astrocyte proliferation promoting activity was significantly confirmed in F4 and F5, and in particular, the most remarkable astrocyte proliferation promoting activity was confirmed in F5.

  Moreover, as shown in FIG. 4, in the added control group (DMSO, resveratrol) and each sample (F1-F7) group, the average number of cultured astrocytes per area observed was DMSO group: 11.67 cells, resveratrol group: 7.00 cells, F1: 12.67 cells, F2: 11.67 cells, F3: 18.33 cells, F4: 25.67 cells, F5: 32.33 cells, F6: 14.67 cells, F7: 7.67 cells . From the number of proliferated astrocytes, it was confirmed that F4 and F5 have strong astrocyte growth promoting activity, and in particular, F5 has the most prominent astrocyte growth promoting activity.

  When F5 was observed over time after 0 h, 3 h, and 48 h, as shown in FIG. 5, after 0 h: 5.67 cells, after 3 h: 21.67 cells, after 48 h : 32.33 cells were obtained, and it was confirmed that the cultured astrocytes supplemented with F5 proliferated explosively.

Claims (6)

  Mixing of water and organic solvent with a carrier charged with a solution containing a dried body of a water-extracted fraction separated by two-layer partitioning between an aqueous solution containing a hot water extract from P. tenuipes and an organic solvent. Extract obtained by solid phase extraction with liquid.   An astrocyte growth promoter comprising the extract according to claim 1. The following steps:
(1) A step of drying a hot water extract of P. tenuipes to obtain a hot water extract;
(2) The aqueous extract containing the hot water extract obtained in the step (1) and the organic solvent are separated into a water extract fraction and an organic solvent extract fraction by performing two-layer partitioning. A step of obtaining a dried product; and (3) a solution containing the dried product of the water-extracted fraction obtained in the step (2) is charged to a carrier, and then a mixed solution of water and an organic solvent is brought into contact with the carrier. And a step of obtaining an extract by solid phase extraction.   In the step (3), before the solid phase extraction with a mixed solution of water and an organic solvent, the solution containing the dried product of the water extraction fraction obtained in the step (2) is charged with a carrier and water. The method for producing an astrocyte growth promoter according to claim 3, further comprising a step of performing solid phase extraction.   The method for producing an astrocyte growth promoter according to claim 3, wherein the organic solvent in the mixed solution in the step (3) is methanol or ethanol.   The method for producing an astrocyte growth promoter according to any one of claims 3 to 5, wherein the concentration of the organic solvent in the mixed solution in the step (3) is 20% to 80%.