JP2014237635A - Production method and nerval protective agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To propose a novel application of an extract in order to utilize the extract from Angelica shikokiana Makino effectively, and to propose reviewing Angelica shikokiana Makino as a raw material which produces a novel physiologically active agent.SOLUTION: Disclosed is a production method for producing a neuroprotective substance, produced by extracting an extract from Angelica shikokiana Makino. Particularly an acetylcholine esterase inhibition effect of Isoepoxypteryxin, β-amyrin, α-glutinol, Quercetin, Kaempferol-3-O-glucoside, or Kaempferol-3-O-rutinoside is utilized for a nerval protective agent and the like. And a cytotoxic preventive effect by active oxygen or β-amyloid of extracts containing other extracts is utilized for a nerval protective agent and the like.

Description

  The present invention relates to a production method and a neuroprotective agent, and more particularly to a production method for producing a substance having a neuroprotective effect.

  There are two kinds of plants belonging to the genus Sisiudo, known as Japanese genus Ginseng, which are two kinds of scientific names: Intoukiki (dog season: Angelica shikokiana) and Hyugatouki (Hinata season: Angelica tenuisecta var furcijuga). Nihonsan ginseng is sometimes referred to as the collective term for Inu and Hyugatoki and their similar plants. Collectively, in addition to Inu and Hyugatouki, those with the same names of Toki, Hokaitouki, Seiyo, Karatouki, Shishiudo, Ashitaba are included.

  Conventionally, isoepoxypterixin extracted from Scotch is a vasodilator, improving peripheral circulation, improving diabetes symptoms, preventing liver fat accumulation, improving allergic symptoms, alleviating symptoms such as dermatitis, improving diabetes, hypertension It is said to be effective for improvement (see, for example, Patent Document 1 and Non-Patent Document 1).

  On the other hand, with regard to Hyugatoki, the physiological activity of the extract has been clarified by various studies (for example, see Non-Patent Documents 2 and 3). Hyugatouki was certified as a medicinal product (herbal medicine) by the Ministry of Health, Labor and Welfare in November 2002.

JP-A-7-53560

Taniguchi M, 4 authors, The 115th Annual Meeting of the Pharmaceutical Society of Japan, Sendai, March 1995, Abstract of Papers II, p.194. Yoichi Matsushita, 3 other authors, Separation and analysis of Angelica furcijuga Kitagawa leaves and stems, Memoirs of the Faculty of Engineering, Miyazaki University, 38: 87-90, 2009-09-30. Dr. Fujiwara and 6 other authors, Effects of Angelica furcijuga on melanin production, Jpn Phaimacok Ther (Pharmacology and Treatment), vol.38, no.12, 2010.

  However, in contrast to Hyugatouki, the physiological activity of the extract has hardly been studied, and only the tip of the iceberg has been clarified in terms of pharmacological effects. As a result, Inuyuki has not been certified as a medicinal product (herbal medicine), and a product using an extract from Inutouki has not practically been put to practical use.

  Therefore, the present invention proposes a new use of the extract in order to effectively use the extract extracted from Inu Touki, and proposes to reconsider Inu Touki as a raw material for producing a new bioactive agent. For the purpose.

  A first aspect of the present invention is an extraction method for extracting an extract from Inu Touki, a selection step for selecting a site of Inu Touki, and promoting or suppressing a physiological activity effect from the selected Inutou site An extraction step for extracting the extract to be performed.

  According to a second aspect of the present invention, in the extraction method of the first aspect, in the extraction step, when extracting an extract that promotes the physiological activity effect, the first extraction solvent is used to In the case of extracting an extract and extracting an extract that suppresses the physiological activity effect, the extract is extracted from the ginseng using a second extraction solvent different from the first extraction solvent.

  A third aspect of the present invention is the extraction method according to the second aspect, wherein one of the first extraction solvent and the second extraction solvent is a nonpolar solvent and the other is a polar solvent.

  A fourth aspect of the present invention is the extraction method according to any one of the first to third aspects, wherein, in the selection step, the portion of the ginseng is part of or all of a root, a stem, a seed, and a leaf. The physiologically active effect is at least one of melanin biosynthesis, lipase inhibitory activity, antibacterial activity, antiallergic activity and antioxidant activity.

  A fifth aspect of the present invention is the extraction method according to any one of the first to fourth aspects, wherein the physiologically active effect is melanin biosynthesis, and the first extraction solvent is the second extraction solvent. Are more polar.

  According to a sixth aspect of the present invention, there is provided an extract extracted from Inuzuki, a physiologically active effect that promotes or suppresses at least one of melanin biosynthesis, lipase inhibitory activity, antibacterial activity, antiallergic activity, and antioxidant activity. It is characterized by exhibiting.

  A seventh aspect of the present invention is the extract according to the sixth aspect, which is extracted from the stem of the above-mentioned Inuzuki.

  An eighth aspect of the present invention is a product including daily necessities using the extract of the sixth or seventh aspect.

  A ninth aspect of the present invention is the product of the eighth aspect, wherein the extract exhibits a bioactive effect of suppressing melanin biosynthesis by being extracted using ethanol as an extraction solvent. The daily necessities are whitening cosmetics.

  A tenth aspect of the present invention is the product according to the eighth aspect, wherein the extract exhibits a bioactive effect of promoting melanin biosynthesis by being extracted using water as an extraction solvent. The daily necessities are hair care products.

  An eleventh aspect of the present invention is a production method for producing a substance having a neuroprotective effect, the method comprising the step of extracting the substance having a neuroprotective effect from Scotch.

  A twelfth aspect of the present invention is the production method according to the eleventh aspect, wherein the neuroprotective effect is an acetylcholinesterase inhibitory effect.

  A thirteenth aspect of the present invention is the production method according to the twelfth aspect, wherein the substance having an inhibitory effect on acetylcholinesterase is Quercetin, Kaempferol-3-O-glucoside, Kaempferol-3- O-rutinoside, Isoepoxypteryxin.

  A fourteenth aspect of the present invention is the production method according to the eleventh aspect, wherein the neuroprotective effect is a cytotoxic effect.

  A fifteenth aspect of the present invention is the production method according to the fourteenth aspect, wherein the cytotoxicity is cytotoxicity due to active oxygen.

  A sixteenth aspect of the present invention is the production method according to the fifteenth aspect, wherein the substance having an effect of preventing the cytotoxicity caused by active oxygen is 3-O-trans-caffeoyl-quinic acid shown in FIG. -1-methyl ester, 3-O-trans-caffeoyl-quinic acid, Quercetin, Adenosine, Kaempferol, Luteolin, Kaempferol-3-O-glucoside, Kaempferol-3-O-rutinoside, or isoepoxypterixin (Isoepoxypteryxin ).

  A seventeenth aspect of the present invention is the production method according to the fourteenth aspect, wherein the cytotoxicity is cytotoxicity caused by β-amyloid.

  An eighteenth aspect of the present invention is the production method according to the seventeenth aspect, wherein the substance having the effect of preventing the cytotoxicity by β-amyloid is Isoepoxypteryxin, Kaempferol-3-O-glucoside shown in FIG. , Kaempferol-3-O-rutinoside, Kaempferol, β-amyrin, α-glutinol, 3-O-trans-caffeoyl-quinic acid-1-methyl ester, 3-O-trans-caffeoyl-quinic acid, Adenosine, Luteolin, Or Quercetin.

  A nineteenth aspect of the present invention is a production method for producing 3-O-trans-caffeoyl-quinic acid-1-methyl ester shown in FIG. Including production methods.

  A twentieth aspect of the present invention is a production method for producing α-glutinol or Kaempferol-3-O-rutinoside shown in FIG. 10, which comprises a step of extracting an extract from a plant of the genus Sisius Is the method.

  The twenty-first aspect of the present invention is as shown in FIG. 10, Palmitic acid, α-glutinol, β-amyrin, 3-O-trans-caffeoyl-quinic acid, Kaempferol, Luteolin, Quercetin, Kaempferol-3-O-glucoside. Or it is a production method which produces Adenosine, Comprising: It is a production method including the step which extracts an extract from Inuyuki.

  A twenty-second aspect of the present invention is a neuroprotective agent containing isoepoxypteryxin shown in FIG.

  A twenty-third aspect of the present invention is the neuroprotective agent according to the twenty-second aspect, comprising the isoepoxypteryxin as an acetylcholinesterase inhibitor or an inhibitor of cytotoxicity caused by active oxygen or β-amyloid. .

  Examples of products include hair care products (for example, shampoos, rinses, hair dyes, etc.) from the viewpoint of melanin biosynthesis, toothpastes, dishwashing detergents, bath additives, laundry detergents, etc. from the viewpoint of antibacterial and antiallergy. There is. In addition, there are food and drink, supplements and other tablets, drinks, daily goods. In addition, for example, from the viewpoint of anti-allergy, effects such as clean skin such as atopy are expected. In addition, it is expected to be used as a medicine such as a neuroprotective agent having an effect on Alzheimer's disease and the like.

  According to each aspect of the invention of the present application, an extract that suppresses or promotes a specific physiological activity effect can be extracted from each part of Inuzuki.

  It has been clarified by the inventors that the extract of Inu touki has specific functionalities depending on each site, and in particular, the stem (stem) also has good physiological activity. Nintouki is not related to “carrot” in the classification, but it has been referred to as “Yamasan ginseng”, including Hyugatouki. Therefore, the site attracting attention is a root generally considered to have high medicinal effects, and then a leaf. In particular, stems are supposed to be discarded and are not utilized. However, the inventors have clarified that the specific functionality differs depending on the site of Inuzuki, and in particular, the stem also exhibits good physiological activity. Therefore, it is important to specify the site for the functionality of the extract of Inutoki. Thus, regarding the functionality of the extract, it is something that a person skilled in the art could not have expected to focus on the site of Intochiki, in particular, that the stem can be utilized.

  Furthermore, the inventors, for example, when comparing an extract with ethanol and an extract with water, for example, for melanin biosynthesis, the ethanol extract suppresses this and the water extract promotes this. Revealed. Here, ethanol is an example of a solvent with low polarity, and water can be an example of a solvent with high polarity. As in the second aspect of the present invention, the inventors clearly show that the extract from each part of Inu touki promotes or suppresses a specific physiological activity effect depending on the solvent. It has been done. Therefore, even if it is the site of the same Inuzuki, it becomes possible to use the extract which promoted or suppressed the specific bioactivity effect with the solvent. As described above, it has not been anticipated by those skilled in the art that an extract that promotes or suppresses the same physiological activity effect can be obtained by a solvent.

  As described above, it is not easily guessed by those skilled in the art that the extracted components are different and the functionality is different depending on the site of Inuyuki and the extraction solvent.

  In addition, according to the eleventh aspect of the present invention, it is possible to extract a substance having a neuroprotective effect from Inuto. Until now, it has not been known that the extract from Intouki has a neuroprotective effect, and it becomes possible to utilize Intouki as a raw material for a new bioactive agent.

  According to the twelfth to eighteenth aspects of the present invention, it becomes possible to specifically utilize an extract from Inuzuki as an inhibitor of acetylcholinesterase or a cytotoxicity inhibitor by active oxygen or β-amyloid. .

  The inventors have found out which substance has a remarkable neuroprotective effect among the extracted substances of Intochiki. Therefore, it becomes easy to efficiently produce a neuroprotective agent from an extract derived from Inuyuki.

It is a flowchart which shows an example of the extraction process which concerns on embodiment of this invention. It is a table | surface which shows the test result of a melanin biosynthesis inhibition test. It is a graph which shows the test result of a lipase inhibition test. It is a graph which shows the test result of an antibacterial activity test. It is a graph which shows the test result of an antiallergic activity test. It is a graph which shows the test result of an antioxidant activity test. It is a table | surface which shows a fractionation condition. It is a table | surface which shows the fraction of the chromatography of a total extraction. It is a table | surface which shows the fraction of the chromatography of F10 of FIG. It is a figure which shows the structural formula of the substance isolated from F2, F5, and F10. It is a graph which shows the result of an acetylcholinesterase inhibition test. It is a graph which shows the test result of the cytotoxicity test using hydrogen peroxide. It is a graph which shows the test result of the cytotoxicity test using the substance and hydrogen peroxide which were specified from the extract of Inuyuki. It is a graph which shows the test result of the cytotoxicity test using the substance and β-amyloid specified from the extract of Inuyuki.

  FIG. 1 is a flowchart showing an example of extraction processing according to the embodiment of the present invention. First, the site of Inuyuki is selected (step ST1 in FIG. 1). For example, roots, stems, leaves and seeds. Subsequently, an extraction solvent is selected (step ST2 in FIG. 1). The extraction solvent is, for example, water, ethanol or the like. Subsequently, the part selected in step ST1 is made into powder, for example, and an extract is prepared using the extraction solvent selected in step ST2 (step ST3). Subsequently, a product containing the extract prepared in step ST3 is manufactured (step ST4).

  Below, first, the experimental result regarding physiological activity is demonstrated about the water extract and the ethanol extract about the root, the stem, the leaf, and the seed of Inuto. According to the results of this experiment, it was clarified that Inuzuki has various physiological activities (melanin biosynthesis inhibition, anti-allergy, anti-lipase, antibacterial, antioxidant) for each site. For specific experiments, see, for example, the Food Functional Science Editorial Board, “Food Functional Science”, 2008, etc.

  First, preparation of a plant sample will be described.

  20 g of each part (root, stem, leaf, seed) of Inuto was prepared, and each was extracted three times with 150 mL of ethanol. Three extractions were performed by stirring at room temperature and occasionally filtering each day and night. The solvent was removed from the ethanol extract using a rotary evaporator. Thereby, an ethanol extract was obtained. The ethanol extract was dissolved in dimethylsulfoxide (DMSO) and adjusted to 160, 40, 20, 10, 5, 2.5, 1.25 mg / mL, and used for the subsequent experiments.

  In addition, 20 g of each part (root, stem, leaf, seed) of Inuto was prepared, and each was extracted three times with 100 mL of water. The extraction method is the same as above. The solvent was removed from the aqueous extract using a freeze dryer TAITEC (Freeze Dryer) VD-250R. This gave a water extract. The water extract was dissolved in a solution of water: DMSO = 1: 1 and adjusted to 160, 40, 20, 10, 5, 2.5, 1.25 mg / mL, and used for the subsequent experiments.

  Table 1 shows the extraction rate (weight% of extract per solvent weight) for each site.

  Subsequently, the melanin biosynthesis test will be described. The effect of each extract on the amount of melanin biosynthesis in B16 melanoma cells was evaluated. In addition, when commercializing cosmetics and the like, it is important that cells do not die. Therefore, cell viability was also evaluated.

10 μL of B16 melanoma cell culture medium (Eagle's Minimum Essential Medium Alpha (EMEM)) cultured on a petri dish was added to the blood cell counter, and the number of cells was measured using a microscope. Thereafter, EMEM medium was added to the cell culture medium to prepare a cell concentration of 1.0 × 10 ⁵ cells / mL. 1 mL of this was inoculated into each well of a 24-well plate and statically cultured at 5 mol% CO ₂ and 37 ° C. for 24 hours. After the culture, the medium in the 24-well plate was removed, and 998 μL of EMEM medium and 2 μL of the sample solution were newly added. This was incubated at 5 mol% CO ₂ at 37 ° C. for 48 hours. After the culture, the medium was removed again, 998 μL of EMEM medium and 2 μL of the sample solution were added, and the mixture was statically cultured at 5 mol% CO ₂ and 37 ° C. for 24 hours. After the culture, cell viability and melanin biosynthesis were measured by the following methods.

50 μL of an MTT staining solution (5 mg / mL PBS (phosphate buffered saline)) was added to each well, followed by stationary culture at 5 mol% CO ₂ and 37 ° C. for 4 hours. The medium was removed, 1 mL of hydrochloric acid-isopropanol solution was added to each well, protected from light, and allowed to stand at room temperature for 4 hours. Absorbance at 570 nm was measured with a microplate reader and used as an indicator of cell viability.

  The medium was removed, and each well was washed with 300 μL of PBS (−) (ie, containing no calcium). PBS (−) was removed, and 1 mL of 1M NaOH aqueous solution was added to each well to lyse the cells. Shielded from light at room temperature and left for 4 hours. Absorbance at 405 nm was measured with a microplate reader and used as an indicator of melanin biosynthesis.

  Subsequently, the melanin biosynthesis inhibition test will be described. FIG. 2 is a table showing experimental results. MC is melanin biosynthesis (%) and CV is cell viability (%). For Type, A is a sample given MC45% (or less), B is a sample given MC45-60%, C is a sample given MC60-80%, D is a melanin synthesis. Samples accelerated by more than 10%, E is otherwise. Also, * is a sample with CV> 80%, and ** is CV = 70-80%.

  Root ethanol extract (RE) inhibited melanin biosynthesis in a concentration-dependent manner. The most potent inhibitory activity was the root ethanol extract (40 μg / mL) with an inhibition rate of 73.57%. However, strong cytotoxicity (cell mortality 41.47%) was also confirmed. Even at low concentrations (20, 10 μg / mL), melanin biosynthesis inhibitory activity (62.77%, 45.05%) was exhibited, and cytotoxicity was 14.63% and 7.32%, respectively. This value indicates that the inhibitory activity of melanin biosynthesis is stronger than that of arbutin used as a positive control and the cytotoxicity is low (arbutin: melanin biosynthesis inhibition rate 49.7%, cytotoxicity: 14.53%).

  The ethanol extract of stem (STE) showed strong melanin biosynthesis inhibitory activity (65.1%) at 40 μg / mL. Even at 20 μg / mL, strong melanin biosynthesis inhibitory activity (56.61%) and low cytotoxicity (4.36%) were exhibited. At a lower concentration (10 μg / mL), the melanin biosynthesis inhibitory activity decreased (28.38%), and no cytotoxicity was detected.

  Leaf ethanol extract (LE) showed weak melanin biosynthesis inhibitory activity (29.2%, 22.53%) and cytotoxicity (27.65%, 20.48%) at 40, 20 μg / mL. At 10 μg / mL, the activity of the extract was low, with a melanin biosynthesis inhibitory activity of 15.62% and a cytotoxicity of 18.23%.

  The seed ethanol extract (SE) showed strong melanin biosynthesis inhibitory activity (53.6%) and weak cytotoxicity (29.81%) at 40 μg / mL. At 20 and 10 μg / mL, weak cytotoxicity (16.3%, 15.0%) was exhibited as well as weak melanin biosynthesis inhibition activity (26.58%, 22.98%).

  On the other hand, no inhibitory effect on melanin biosynthesis was observed in most water extracts (RW, STW, LW, SW). Stems (10 μg / mL) promoted melanin biosynthesis at 21.6%.

  Thus, the ethanol extract exhibits melanin biosynthesis inhibitory activity, while the water extract exhibits melanin biosynthesis promoting activity. In order to commercialize cosmetics and the like, it is difficult to use a toxic solvent such as methanol as a solvent. Therefore, ethanol and water are promising candidates for commercialization. Water can be evaluated as a highly polar solvent (polar solvent). Ethanol can be evaluated as a low polarity solvent (nonpolar solvent). The nature of the extract obtained by a polar solvent and a nonpolar solvent (especially water and ethanol) is a matter that cannot be predicted by those skilled in the art. Thereby, the extract by a nonpolar solvent (especially ethanol) can be applied to whitening cosmetics, for example, and the polar solvent (especially water) extract can be used for, for example, hair care products such as suntan agents and white hair prevention ( For example, application to shampoo, rinse, hair restorer, etc. is possible.

  Subsequently, the lipase activity inhibition test will be described. The effect of each extract on lipase activity was evaluated.

Each extract was made into 3% DMSO (3 ml DMSO / 97 ml aqueous solution (volume%)) and made up to 25 μL with 13 mM Tris-HCl, 150 mM NaCl, 1.3 mM CaCl ₂ buffer (lipase buffer: pH 7.98). To this solution, add 50 μL of 0.05 mM 4-methylumbelliferyl oleate (4-MUO) dissolved in lipase buffer, and then dissolve 25 UL of 50 U / mL, 150 μg / mL pancreatic lipase in the same buffer as above. To start the reaction. After reacting at 25 ° C. for 30 minutes and adding 100 μL of 0.1 M citrate buffer (pH 4.2) to stop the reaction, fluorescence intensity was measured (Em: 355 nm, Ex: 460 nm). The effect on lipase activity is evaluated by measuring the fluorescence intensity of 4-methylumbelliferone released by the action of lipase on 4-MUO. The lipase activity (%) was calculated according to the formula (1). Here, S is the fluorescence intensity after the reaction of the sample addition system, S _Blank is the fluorescence intensity of only the sample and 4-MUO, C is the fluorescence intensity after the reaction of the sample addition system, C _Blank is the fluorescence intensity of 4-MUO only. In addition, orlistat used as a lipase inhibitor was used for positive control.

  Then, the test result of a lipase inhibition test is demonstrated. FIG. 3 is a graph showing the effect of each extract on lipase activity. The final concentration is μg / mL. All values are the average of 4 replicas. * Indicates a sample in which a significant difference from the control group was observed. P <0.01 was used for the P value, which is a measure showing the possibility that a difference between groups occurred by chance during the experiment. P <0.01 means that this result occurs less than once in 100. The smaller the P value, the more the difference between the control group and the sample group is due to the effect of the sample. Usually, 0.05 or less is considered effective. Orlistat (1 μg / mL) was used as a positive control.

  Strong lipase inhibitory activity (81.2%, 76.41%, 57.09%) was confirmed in the ethanol extract (400 μg / mL) of stems, roots and leaves (RE400, STE400, LE400). At 200 μg / mL, the lipase inhibitory activity decreased to 49.65%, 45.61%, and 40.26%, respectively.

  In the water extract, the species showed inhibitory activity (31.7%) at 400 μg / mL (SW400). For other water extracts, only 15.09-4.44% weak inhibitory activity was detected.

  Thus, the nonpolar solvent (ethanol) exhibits inhibitory activity, whereas the polar solvent (water) hardly exhibits inhibitory activity. Thus, it was discovered for the first time by the inventors that the properties of the extract differ depending on the solvent.

  Subsequently, the antibacterial activity test will be described. The effect of each extract on the growth of Escherichia coli (NBRC13276) and Staphylococcus aureus (NBRC3301) was evaluated.

A colony of bacteria cultured on a Nutrient agar (NA) medium was added to 5 mL of Nutrient broth (NB) medium and cultured at 37 ° C. for 18 hours with shaking (160 rpm) to obtain stationary cells. The obtained stationary-phase culture solution was diluted with NB medium to prepare OD ₆₆₀ = 0.4 (E. coli: 10 ⁹ CFU / mL, S. aureus: 10 ⁸ CFU / mL). Further, this was diluted with sterilized water, and the bacterial concentration was 10 ⁵ CFU / mL, which was used for the antibacterial test.

  The antibacterial activity was measured as follows. 445 μL of NB medium, 50 μL of bacterial solution, and 5 μL of sample solution were added to the Eppendorf tube and mixed well. 150 μL of this solution was dispensed into 96-well plates (n = 3), and cultured with shaking (1150 rpm) at 37 ° C. for 18 hours. After culturing, the turbidity at 630 nm was measured and used as an index of antibacterial activity.

  Then, the test result of an antimicrobial activity test is demonstrated. FIG. 4 is a graph showing the antibacterial activity of each extract against E. coli and S. aureus. The value of the result was shown by the average value +/- standard deviation in 3 repetition experiment. The final concentration was 400 μg / mL for the ethanol extract and 1600 μg / mL for the water extract. As a statistical process, the student's t-test was performed to test whether there was a difference from the extract-free group (control group). * Is indicated when P <0.05, and ** is indicated when P <0.01.

  Antimicrobial activity against E. coli could not be confirmed through the whole extract. On the other hand, ethanol extract of roots and stems showed strong antibacterial activity against S. aureus at 400 μg / mL. The ethanol extract of leaves showed weak antibacterial activity against S. aureus at 400 μg / mL.

  Thus, for E. coli as a whole, no antibacterial activity is shown, and for S. aureus, the non-polar solvent (ethanol) extract shows good antibacterial activity as a whole, and polar solvent (water ) The extract as a whole exhibits weak antimicrobial activity. Thus, it was discovered for the first time by the inventors that the antibacterial activity is exhibited only for some of the bacteria, and that the properties of the extract differ depending on the solvent. The invention of the present application may be regarded as an ethanol extract of the leaves and / or stems of Scotch and exhibiting antibacterial activity against S. aureus.

  The present invention may be regarded as an ethanol extract of a part or all of the leaves, roots, and stems of Inuki, and exhibiting antibacterial activity against Staphylococcus aureus.

  Subsequently, the antiallergic activity test will be described. In order to evaluate the antiallergic activity of ingredients in foods, the presence or absence of degranulation inhibitory activity was examined using the rat mast cell-like cell line RBL-2H3.

A 96-well plate was inoculated with 100 μL (1 × 10 ⁶ cells / well) of the RBL-2H3 cell solution and cultured at 37 ° C. under 5 mol% CO ₂ conditions for 24 hours. After culture, 1 μL of 1 μg / mL anti-DNP-IgE antibody was added. After further culturing under the same conditions for 24 hours, the medium was removed, and 100 μL of Tyroid buffer and 1 μL of the extract solution were added. After culturing for 30 minutes, the medium was removed, and 1 μL of 100 μL Tyroid buffer and 20 μL / mL DNP-BSA was added. After further incubation for 30 minutes, 50 μL was taken from each well and transferred to another 96-well plate. Here, 50 μL of 1 mM p-nitrophenyl-N-acetyl-β-glucosaminide was added as a substrate, and the mixture was allowed to stand at room temperature for 1 hour. Finally, 100 μL of 100 mM Na ₂ CO ₃ aqueous solution (pH = 10) was added to stop the reaction, and the absorbance at 405 nm was measured.

The effect of the extract on the cell viability was evaluated by the MTT method. A 96-well plate was inoculated with 100 μL (1 × 10 ⁶ cells / well) of the RBL-2H3 cell solution and cultured at 37 ° C. under 5 mol% CO ₂ conditions for 24 hours. After culture, 1 μL of the extract solution was added. After further culturing for 24 hours, 10 μL of 5 mg / mL MTT solution was added to each well, and cultured for 4 hours at 37 ° C. under 5 mol% CO ₂ conditions. After culturing, the medium was removed, the cells were lysed with 100 μL of isopropanol, protected from light and left overnight. Absorbance at 570 nm was measured with a plate reader and used as an index of viable cell count.

  Then, the test result of an antiallergic activity test is demonstrated. FIG. 5 is a graph showing the effect of ionophore A23187 on RBL-2H3 cells and β-hexamidase activity induced by IgE. The value of the result was shown by the average value +/- standard deviation in five repetition experiment. As statistical processing, the student's t-test was performed to test whether there was a difference from the extract-free group (control group). In the case of P <0.01, * is described. Gallic acid (100 μM) was used as a positive control.

  The results of the MTT test showed that the extract at the test concentration was not cytotoxic to RBL-2H3 cells. Experiments using immunoglobulins and ionophores showed some correlation. The leaf ethanol extract showed the strongest inhibitory activity on Β-hexamidase secretion (95.68%: IgE, 69%: A23187), followed by the seed water extract (63%: IgE, 77.6%: A23187) ), An aqueous extract of leaves (61%: IgE, 77.6%: A23187). Other extracts also showed mild inhibitory activity in both tests. From this result, it was found that secretion of β-hexamidase produced by IgE or ionophore in RBL-2H3 cells can be inhibited by the tested extract.

  Subsequently, the antioxidant activity test by the ORAC (Oxygen Radical Absorbance Capacity) method will be described. The peroxy radical scavenging ability of each extract was evaluated by ORAC method.

20 μL of Trolox standard sample solution (75 mM) or 20 μL of extract solution (Blank is 75 mM phosphate buffer) was placed in a 96-well plate, and 200 μL of 94.4 nM fluorescein solution (dissolved in phosphate buffer) was added. This was heated in a 37 ° C. water bath for 10 minutes. Furthermore, 31.7 mM AAPH (2,2′-azobis (2-amidino-propane) -dihydrochloride) solution heated at 37 ° C. for 10 minutes was added in an amount of 75 μL by an 8-unit pipette. The apparatus was started 30 seconds after AAPH was added, and the fluorescence intensity was measured for 90 minutes at 30 second intervals while reacting at 37 ° C. Using the calculation formula of Equation 2, the AUC of each sample was calculated from the measurement result of the fluorescence intensity in each obtained well. Here, AUC is the area under the curve of fluorescence intensity (AUC: Area Under the Curve), f ₀ is the fluorescence intensity at 0 seconds (at the start of measurement), and f _i is after i seconds. Of the fluorescence intensity.

In addition, the data of _Trolox 0μM was blank, and the increase in AUC (AUC _Trolox- AUC _Blank ) of the data obtained at each Trolox standard concentration (6.33, 3.16, 1.58, 0.79μg / mL) was netAUC . A linear regression equation (Y = aX + b) was created by taking the average value of Trolox netAUC on the X axis and the Trolox concentration on the Y axis. The relative ORAC value was calculated using the regression equation of Equation 3.

  Then, the test result of an antioxidant activity test is demonstrated. FIG. 6 is a diagram showing the ORAC value of each extract. The horizontal axis indicates the type of extract for each part, and the vertical axis indicates the Trolox equivalent ORAC value per 100 grams of dry weight of the part.

  The ORAC value is shown in μM TE / 100 g (raw weight), and has already been reported (Wu, 5 other authors, Lipophilic and hydrophilic antioxidant capacities of common foods in the United States, J. Agric. Food Chem., 52, 4026− 4037 (2004)) compared with food ORAC values. The leaf water extract and stem ethanol extract showed the highest ORAC values, 11181.1 and 10831.6, respectively. These values are comparable to 100 g of elderberry (10655). The ORAC values of the root and stem water extracts were slightly lower, 8673.1 and 8064.8. These values correspond to 100 g of apple (7781). The ORAC value of the seed water extract was 6650.6, a value close to 100 g of pomegranate (5923). Other extracts had low ORAC values (3809.4-2141.5), similar to 100 g of broccoli. Thus, the extract as a whole exhibits antioxidant activity.

  Subsequently, the isolation of physiologically active substances from the above-ground part of Nihonsan Ginseng will be described.

  First, mass extraction will be described. 10 kg of A. furcijuga aerial powder was extracted with methanol at room temperature (14 L × 4 times). The extract was concentrated under reduced pressure to obtain 700 g of a methanol extract.

  Subsequently, the fractionation of methanol extract will be described. 650 g of methanol extract was dissolved in methanol, mixed with 700 g of silica gel and then ground. Then, it was dried at room temperature and packed in a silica gel column (50 cm × 19 cm i.d., 3.5 kg). Then, elution was performed with hexane-ethyl acetate (100: 0-0: 100, gradient), ethyl acetate-methanol (100: 0-0: 100, gradient), and methanol-water (1: 1). The eluent was collected in 20 L portions, concentrated, and analyzed by silica gel TLC.

  FIG. 7 is a table showing fractionation conditions.

  Next, isolation of a neuroprotective substance will be described. The extract of ginseng has been reported to have various physiological activities such as blood pressure regulation, appetite enhancement, immunostimulatory effect, skin cell metabolism regulation, recovery from fatigue, and carbohydrate metabolism regulation.

  Alzheimer's disease is a brain degenerative disorder that develops in advanced, especially elderly people in developed countries. Alzheimer's disease has been suggested to be associated with a loss of the cholinergic mechanism due to a decrease in acetylcholine in brain regions that handle learning, memory, behavior, and emotional behavior. Neuropathologically, Alzheimer's disease is characterized by the presence of β-amyloid plaques, neurofibrillary tangles, and cholinergic degeneration and atrophy of the forebrain basal ganglia. Loss of forebrain basal ganglia cholinergic cells diminishes acetylcholine availability at synapses and causes cognitive impairment in Alzheimer's disease. Therefore, the most effective method for alleviating the symptoms of Alzheimer's disease is to increase acetylcholine in the synapse by inhibiting acetylcholinesterase which hydrolyzes acetylcholine.

  Therefore, in the following, an acetylcholinesterase inhibitor is isolated from Japanese ginseng and the possibility of a novel therapeutic agent for Alzheimer's disease is examined.

In addition, oxidative damage caused by reactive oxygen species such as free radicals such as superoxide anion (O ^-2 ) and non-free radical species such as hydrogen peroxide (H ₂ O ₂ ) causes neurosis. Therefore, we will investigate the anti-hydrogen peroxide activity of neuro-2A cells in the ground part of Ginseng.

  The experimental method will be described. First, prepare a sample. 10kg of ground powder of Japanese ginseng was extracted with methanol at room temperature (14Lx4). The extract was immersed in a hot water bath of less than 45 ° C. and subjected to a rotary evaporator to remove the solvent to obtain 700 g of a methanol extract. The methanol extract was a green viscous substance. The methanol extract was subjected to silica gel column chromatography. Open column (50x19cm) packed with silica gel 4.2kg, methanol extract, n hexane and ethyl acetate (100: 0-0: 100, gradient), ethyl acetate and methanol (100: 0-0: 100, gradient), Methanol and water (1: 1) were flowed in this order. From the TLC analysis results of the fractions, the fractions containing similar components were collected together and arranged.

  Among the fractions, in particular, the second, fifth, and tenth fractions (Fr.2, Fr.5, Fr.10) showed strong acetylcholinesterase inhibitory effect and also used hydrogen peroxide. Since the cell viability was also effectively increased in the cytotoxicity test, these fractions were focused on.

  Fr.2 (F2, 13.2 g) was further fractionated by silica gel column chromatography. As a solvent, n-hexane and ethyl acetate were used. The substance of the obtained fraction was crystallized using chloroform and methanol as solvents to obtain Palmitic acid (compound 1) 88 mg, α-glutinol (α-glutinol) (compound 2) 68 mg, β-amyrin (β-amylin) (compound 3) 10 mg was obtained.

  Fr. 5 (F5, 12.5 g) was subjected to silica gel column chromatography using methanol and water as solvents to obtain 4 mg of Isoepoxypteryxin (isoepoxypterixin) (Compound 4).

  Fr. 10 (F10, 200 g) was further fractionated by silica gel column chromatography. An open column (30 × 8 cm) was filled with 800 g of silica gel and F10, and chloroform and methanol (100: 0-0: 100, gradient) were allowed to flow. Each elution fraction was collected on silica gel TLC and contained similar components.

  The extracted substance was identified by, for example, comparison with NMR data described in a previously reported document or comparison of NMR data with a commercially available sample.

  The test results will be described. FIG. 8 is a table showing chromatographic fractions of total extraction. As shown in FIG. 8, fractionation was carried out into a total of 12 fractions by silica gel column chromatography. Since F1 was a very small amount of 0.003 mg, it was not used for later tests. FIG. 9 is a table showing chromatographic fractions of F10. In the F10 fraction, as shown in FIG. 9, the fraction was fractionated into a total of seven fractions. Of the obtained seven fractions, the fourth fraction, 14.587 g, was further fractionated.

  Of the 11 fractions obtained at this time, the second fraction 40 mg was subjected to PTLC using n-hexane and ethyl acetate as solvents, and 5- (hydroxy methyl) -furan-2-caraldehyde (compound 5) 12 mg. Got. In addition, 6 mg of Kaempferol (compound 6) was obtained from 223 mg of the fourth fraction through gel filtration chromatography using chloroform and methanol as solvents. Further, 328 mg of the fifth fraction was crystallized to obtain 6 mg of Luteolin (Compound 7). Further, from 2 g of the sixth fraction, fractionation was performed using chloroform and methanol as solvents, and the first fraction 303 mg was further subjected to column chromatography using chloroform and methanol as solvents to give 3-O-trans-caffeoyl- 35 mg of quinic acid-1-methyl ester (Compound 8) was obtained. Further, from 2 g of the sixth fraction, fractionation was carried out using chloroform and methanol as solvents, and the second fraction 185 mg was further subjected to gel filtration chromatography using water and methanol as solvents, and then 3-O-trans-caffeoyl. -Quinic acid (Compound 9) 16 mg, Quercetin (Compound 10) 6 mg, and Kaempferol-3-O-glucoside (Compound 11) 61 mg were obtained. Further, fractionation was carried out from 3.1 g of the ninth fraction using chloroform and methanol as solvents, and 43 mg of the first fraction was further subjected to gel filtration chromatography using water and methanol as solvents, followed by Kaempferol-3-O- 6 mg of rutinoside (Compound 12) was obtained. In addition, fractionation was carried out from 3.1 g of the ninth fraction using chloroform and methanol as solvents, and 1.9 g of the second fraction was further subjected to gel filtration chromatography using water and methanol as solvents to obtain Adenosine (compound 13). 10 mg was obtained.

  FIG. 10 shows the structural formula of the isolated substance.

  Subsequently, the acetylcholinesterase inhibition test (Ellman's Method) will be described.

  The acetylcholinesterase enzyme hydrolyzes the substrate acetylcholine to produce thiocholine. Thiocholine reacts with Ellman's reagent 5,5-dithiobis [2-nitrobenzoic acid] (DTNB) to give 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate. These show absorbance at 405 nm.

  Tris buffer (50 mM, pH = 8.0, 0.1% BSA for stabilizing the enzyme) 100 μL, 0.25 U / mL acetylcholinesterase 10 μL, 10 mM DTNB 10 μL, fraction F2 fractionated by silica gel column chromatography 10 μL or 5 μL of F12 (2,10 mg / mL ethanol, final concentration; 76.92, 384.6 μg / mL) was added, mixed, and incubated at 30 ° C. for 5 minutes. Next, 5 μL of 75 mM acetylcholine was added as a substrate and incubated again at 30 ° C. for 10 minutes. After incubation, the absorbance was measured at 405 nm.

The calculation method of the inhibition rate (%) is based on the equation (4). Here, A _Control is the absorbance of the system to which ethanol is added instead of the sample, A _Blank is the absorbance of the system to which the same amount of buffer solution is added instead of the sample and the enzyme, and A _Sample is The absorbance of the system to which the sample was added, and A _{Sample Blank} is the absorbance of the system to which a buffer solution was added instead of the sample and the substrate. Galantamine was used as a positive control. Significant differences were examined using T-test.

  FIG. 11 shows the results of an acetylcholinesterase inhibition test. The vertical axis shows the inhibition rate of acetylcholinesterase (n = 4). In F2, 9, 10, 11, and 12, almost no inhibition was observed at a low concentration (76.92 μg / mL), but significant inhibition was observed at a high concentration (384.6 μg / mL). In F3, F4, F5, and F6, high inhibition was observed at both low and high concentrations.

Table 2 lists the IC ₅₀ values for the acetylcholinesterase inhibition test. Among the substances specified by the inventors as components contained in the extract of Inutoki, Quercetin, Kaempferol-3-O-glucoside, Kaempferol-3-O-rutinoside, and Isoepoxypteryxin are the enzyme activities of acetylcholinesterase in the amounts shown in Table 2. Over 50%. Isoepoxypteryxin has been suggested to be less reactive than Quercetin, Kaempferol-3-O-glucoside and Kaempferol-3-O-rutinoside. However, considering that moderate reactivity is preferable in the body, it can be said that a moderate acetylcholinesterase inhibitory effect is rather preferable as a pharmaceutical product.

  Subsequently, the cytotoxicity test (MTT test) of euro 2A cells and the cytotoxicity test of hydrogen peroxide will be described.

First, the cytotoxicity test (MTT test) will be described. Neuro-2A cells were seeded in a 96-well plate (1 × 10 5 cells / well) and incubated for 24 hours in a CO ₂ incubator (5 mol% CO ₂ , 37 ° C.). The medium was changed and 99 μL was added, and samples of F2 to F12 dissolved in 1 μL of DMSO were added, respectively, and incubated again for 24 hours. 20 μL of MTT solution (5 mg / mL phosphate buffer) was added. After incubation for 4 hours, precipitated formazan was dissolved by adding 150 μL of DMSO. After an additional 4 hours under dark conditions, the absorbance at 570 nm was measured with a microplate reader. Significant differences were calculated using t-test.

Next, the cytotoxicity test of hydrogen peroxide will be described. Neuro-2A cells were seeded in a 96-well plate (1 × 10 5 cells / well) and incubated for 12 hours in a CO ₂ incubator (5 mol% CO ₂ , 37 ° C.). Samples of F2 to F12 dissolved in 1 μL of DMSO were added to the medium, respectively, and incubated again for 1 hour. One plate was replaced with 99 μL of fresh medium and 1 μL of H ₂ O ₂ was added. To the other plate, except for the control well, 1 μL of H ₂ O ₂ was added without changing the medium. Then, after further incubation for 24 hours, the cell viability was measured. Cell viability was measured in the same manner as the MTT method. Significant difference was calculated using t-test.

  FIG. 12 is a graph showing experimental results. FIG. 12 (a) shows the results of a cytotoxicity test in neuro-2A cells. F2, F8, F9, F10, F11, and F12 showed a cell viability of 80% or more at 400 μg / mL. Overall, F4, F5 and F7 showed a cell viability of 80% or more at 200 μg / mL and F3 and F6 at 100 μg / mL. These concentrations were used for the cytotoxicity test of the following hydrogen peroxide. 90 μM hydrogen peroxide causes a 40% to 50% reduction in cell viability. However, the addition of F2, F3, F4, and F5 improved cell viability from 86% to 94%. Cell viability in the system without hydrogen peroxide was 81% to 100%. Therefore, it was suggested that these fractions can not only pass through the nerve cell membrane but also reduce intracellular oxidative stress and react with hydrogen peroxide generated by extracellular radicals. On the other hand, F7, F9, F10, and F11 increased CV from 66% to 78% in the system without hydrogen peroxide. Therefore, these fractions have a low ability to pass through the cell membrane, but have been found to have a function of strongly reducing the oxidative stress caused by extracellular hydrogen peroxide.

  FIG. 13 is a graph showing experimental results after specifying a substance. The vertical axis represents cell viability. Cell viability is indicated by five bar graphs for each substance, indicating that 6.25 mM, 12.5 mM, 25 mM, 50 mM, and 100 mM substances were added in order from the left. Cell viability is particularly high when 3-O-trans-caffeoyl-quinic acid-1-methyl ester, 3-O-trans-caffeoyl-quinic acid, and Quercetin are added among the compounds obtained from Inuyuki There was a trend. In addition, cell viability increased significantly when Adenosine, Kaempferol, Luteolin, Kaempferol-3-O-glucoside, Kaempferol-3-O-rutinoside, and Isoepoxypteryxin were added.

Furthermore, a cytotoxicity test using β-amyloid represented by Aβ _25-35 will be described. First, as in the cytotoxicity test using hydrogen peroxide, the concentration of Aβ _25-35 that reduces cells by 50% was determined. The inventors have found that incubation of 19 mM Aβ _25-35 at 37 ° C. for 3 days shows 50% ± 3% cytotoxicity at 72 hours of incubation. Therefore, each compound isolated was changed in concentration (6.25 mM, 12.5 mM, 25 mM, 50 mM, 100 mM) and incubated with cells for 3 hours, followed by addition of the above Aβ _25-35 for 72 hours. Incubated. Cell viability was then determined.

  FIG. 14 is a graph showing experimental results. The vertical axis represents cell viability. Cell viability is indicated by five bar graphs for each substance, indicating that 6.25 mM, 12.5 mM, 25 mM, 50 mM, and 100 mM substances were added in order from the left. Among the compound groups obtained from Inuyuki, high cell viability was shown in the order of Isoepoxypteryxin, kaempferol glucoside, rutinoside, and kaempferol. Compared to the case where these substances were not added, the cell viability increased by 37 ± 4%, 36 ± 3%, 31 ± 4%, and 26 ± 3%, respectively. Others, β-amyrin, α-glutinol, methyl ester of chlorogenic acid (3-O-trans-caffeoyl-quinic acid-1-methyl ester), chlorogenic acid (3-O-trans-caffeoyl-quinic acid), Adenosine, When Luteolin and Quercetin were added, cell viability increased significantly.

Claims (13)

A production method for producing a substance having a neuroprotective effect,
A method of production comprising the step of extracting the substance having a neuroprotective effect from Scotch.   The production method according to claim 1, wherein the neuroprotective effect is an acetylcholinesterase inhibitory effect. The substance having an inhibitory effect on acetylcholinesterase includes Quercetin represented by formula (1), Kaempferol-3-O-glucoside represented by formula (2), and Kaempferol-3-O- represented by formula (3). The production method according to claim 2, which is rutinoside or isoepoxypteryxin represented by the formula (4).
  The production method according to claim 1, wherein the neuroprotective effect is a cytotoxicity-preventing effect.   The production method according to claim 4, wherein the cytotoxicity is cytotoxicity due to active oxygen. Substances having the effect of preventing the cytotoxicity due to active oxygen are 3-O-trans-caffeoyl-quinic acid-1-methyl ester represented by the formula (5) and 3-O- represented by the formula (6). trans-caffeoyl-quinic acid, Quercetin represented by formula (7), Adenosine represented by formula (8), Kaempferol represented by formula (9), Luteolin represented by formula (10), (11) Kaempferol-3-O-glucoside represented by the formula, Kaempferol-3-O-rutinoside represented by the formula (12), or isoepoxypteryxin represented by the formula (13): The production method according to claim 5.
  The production method according to claim 4, wherein the cytotoxicity is cytotoxicity caused by β-amyloid. Substances having the effect of preventing the cytotoxicity caused by β-amyloid include Isoepoxypteryxin represented by the formula (14), Kaempferol-3-O-glucoside represented by the formula (15), and Kaempferol represented by the formula (16). -3-O-rutinoside, Kaempferol represented by formula (17), β-amyrin represented by formula (18), α-glutinol represented by formula (19), 3 represented by formula (20) -O-trans-caffeoyl-quinic acid-1-methyl ester, 3-O-trans-caffeoyl-quinic acid represented by formula (21), Adenosine represented by formula (22), represented by formula (23) The production method according to claim 7, which is Luteolin or Quercetin represented by the formula (24).
(25) A production method for producing 3-O-trans-caffeoyl-quinic acid-1-methyl ester represented by the formula:
A method of production comprising the step of extracting an extract from a plant of the family Apiaceae.
A production method for producing α-glutinol represented by the formula (26) or Kaempferol-3-O-rutinoside represented by the formula (27),
A production method comprising the step of extracting an extract from a plant of the genus Sisius.
Palmitic acid represented by formula (28), α-glutinol represented by formula (29), β-amyrin represented by formula (30), 3-O-trans-caffeoyl represented by formula (31) -quinic acid, Kaempferol represented by formula (32), Luteolin represented by formula (33), Quercetin represented by formula (34), Kaempferol-3-O-glucoside represented by formula (35), Or a production method for producing Adenosine represented by formula (36),
A production method comprising the step of extracting an extract from Inuto.
(37) A neuroprotective agent containing isoepoxypteryxin represented by the formula:
  The neuroprotective agent of Claim 12 which contains the said isoepoxypterixin (Isoepoxypteryxin) as an acetylcholinesterase inhibitor or a cytotoxicity inhibitor by active oxygen or (beta) -amyloid.