JP2014111664A - Dried plant tissue and plant tissue extract for ameliorating central nervous system degenerative diseases accompanied by learning/memory disorders, movement disorders and the like, and pharmaceutical agent and food comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a dried plant tissue or a plant tissue extract having a nonconventional significant ameliorating effect on central nervous system degenerative diseases, particularly Alzheimer's disease and Parkinson's disease.SOLUTION: The present invention provides a dried plant tissue and a plant tissue extract each having a nonconventional significant anti-Alzheimer's disease activity and/or a nonconventional significant anti-Parkinson's disease activity. The present invention also provides a pharmaceutical agent and a food for ameliorating central nervous system degenerative diseases, particularly for enhancing the ability of acquiring, maintaining and recalling memory, which comprises the dried plant tissue or the plant tissue extract.

Description

  The present invention relates to a dried plant tissue and a plant tissue extract that improve central neurodegenerative diseases associated with learning / memory disorders and movement disorders. The present invention also relates to pharmaceuticals and foods containing the dried plant tissue or plant tissue extract.

  In the modern aging society, the number of patients with central neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease has increased and has become a social problem. Among them, Alzheimer's disease is a progressive central neurodegenerative disease accompanied by cognitive dysfunction, learning / memory impairment, etc. As a cause thereof, the function of N-methyl-D-aspartate (NMDA) receptor, which is important for learning / memory, is reduced. Neurodegeneration due to polymerization and accumulation of amyloid β peptide is considered (see Non-Patent Document 1).

で示されるノビレチンが知られており（特許文献１または非特許文献１もしくは２を参照のこと）、ノビレチンはまた、神経細胞に対して神経突起伸長作用を有することも知られている（特許文献２を参照のこと）。ノビレチンは種々の柑橘類植物の果皮などに含まれるが、通常極めて微量しか含まれていない。そのため、柑橘類植物の果皮由来の生薬の一つである陳皮は通常、健胃作用、去痰作用または鎮咳作用などを示し、これらの作用にはノビレチンは関与していないと考えられる。これまでにノビレチン特有の薬効を凌駕する、つまりノビレチンを上回る中枢神経変性疾患改善作用を示す柑橘類の乾燥植物組織およびその抽出物は知られていない。 In general, memory consists of three processes: memory acquisition, retention, and recall, and the mechanisms of these three processes are considered different. It is understood that learning and memory impairment, which is a core symptom of Alzheimer's disease, is particularly markedly impaired in memory retention and recall processes. Among these, as one of natural flavonoids having the action of improving the recall ability, the formula (I):

(See Patent Document 1 or Non-Patent Document 1 or 2), and nobiletin is also known to have a neurite outgrowth action on nerve cells (Patent Document 1). 2). Nobiletin is contained in the skin of various citrus plants, but is usually contained in a very small amount. Therefore, Chen, which is one of the herbal medicines derived from the peels of citrus plants, usually exhibits a gastric action, expectorant action, antitussive action, etc., and it is considered that nobiletin is not involved in these actions. To date, no dried citrus plant tissue and its extract that surpass the medicinal effects peculiar to nobiletin, that is, has an action to improve central neurodegenerative diseases over nobiletin.

  Moreover, although the functional food which shows the learning and memory disorder improvement effect containing the squeezed liquid derived from the whole fruit of citrus fruits or its extract is known (refer patent document 3), the citrus fruits used here The whole fruit contains only a small amount of nobiletin, and its medicinal effect does not exceed the effect of enhancing the recall ability of nobiletin alone.

  On the other hand, Parkinson's disease, one of the central neurodegenerative diseases, is characterized by four major motor signs such as tremor, slow movement, muscle rigidity, and postural reflex disorder, and the dopaminergic nervous system degenerates due to some factor. This is because of motor dysfunction. As one of the treatment methods, dopamine replacement therapy is currently performed. Many drugs that increase the transcriptional activity of the enzyme in the dopamine biosynthetic pathway, that is, the expression level of its mRNA, are known, but few drugs that increase the production of dopamine itself are known. In addition, a dried citrus plant tissue exhibiting a dopamine synthesis ability promoting action and a dopamine secretion promoting action and an extract thereof are not known.

International Publication No. 2005/082351 JP 2002-60340 A JP 2007-61028 A

Pharmacology Journal (Folia Pharmacol. Jpn.) 132, 155-159 (2008) The Journal of Pharmacology and Experimental Therapeutics, 2007, Vol. 321, No. 2, pp.784-790

  Under such circumstances, if a dry plant tissue or plant tissue extract having an effect of improving central neurodegenerative diseases, particularly Alzheimer's disease and Parkinson's disease, is developed, the development of a promising fundamental therapeutic agent in central neurodegenerative diseases will be realized. Can be expected. As a result, we can make significant progress toward overcoming intractable progressive neurological diseases such as dementia in Japan's aging society and solving problems in the medical insurance system. Furthermore, if a dry plant tissue or plant tissue extract is developed that enhances memory acquisition and retention ability as well as memory recall ability in learning and memory disorders, it will be more useful for pharmaceutical and food applications It becomes.

  As a result of intensive studies, the present inventors have found a dried citrus plant tissue and a plant tissue extract having a remarkable effect of improving central nervous degenerative diseases. In particular, the present inventors have found dried plant tissues and plant tissue extracts of citrus fruits that can be applied as a fundamental therapeutic agent for Alzheimer's disease and Parkinson's disease among central neurodegenerative diseases. The inventors have further discovered dried citrus plant tissues and plant tissue extracts that enhance not only memory recall but also memory acquisition and retention in memory impairment, a core symptom of Alzheimer's disease.

Specifically, the present invention includes the following embodiments:
(1) Dry plant tissue of citrus peel for improving central neurodegenerative diseases, containing 0.4% by weight or more of nobiletin with respect to 100% by weight of dry plant tissue;
(2) Dry plant tissue of citrus leaves for improving central neurodegenerative diseases, containing 0.3% by weight or more of nobiletin with respect to 100% by weight of dry plant tissue;
(3) The dry plant tissue according to any one of (1) and (2), wherein the weight ratio of nobiletin / nariltin is 1.0 or more;
(4) The dried plant tissue according to any one of (1) and (2), wherein the central neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease;
(5) Improvement of central neurodegenerative diseases is increased cAMP response element (CRE) -dependent transcriptional activity, memory acquisition, retention and enhanced recall ability, tyrosine hydroxylase (TH) transcriptional activity, TH expression level The dried plant tissue according to any one of (1) and (2), which is brought about by an increase, promotion of dopamine synthesis ability, or promotion of dopamine secretion;

(6) Citrus plant tissue extract for improving central neurodegenerative diseases, containing 0.6% by weight or more of nobiletin with respect to 100% by weight of plant tissue extract;
(7) The plant tissue extract according to (6), wherein the content weight ratio of nobiletin / nariltin is 2.0 or more;
(8) The plant tissue extract according to (6), which is obtained from the dried plant tissue according to (1) or (2) by water extraction, preferably 60 to 100 ° C water extraction;
(9) The plant tissue extract according to (6), wherein the central neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease;
(10) Improvement of central neurodegenerative diseases may increase CRE-dependent transcriptional activity, acquire memory, enhance retention and recall ability, promote TH transcriptional activity, increase TH expression, promote dopamine synthesis, or dopamine secretion The plant tissue extract according to (6), which is caused by the promotion of

(11) The dried plant tissue according to any one of (1) and (2), wherein the citrus fruit is Citrus reticulata Blanco (Rutaceae);
(12) The plant tissue extract according to (6), wherein the citrus fruit is Citrus reticulata Blanco (Rutaceae);
(13) The dried plant tissue according to any one of (1) and (2), wherein the citrus fruit is Tachibana, preferably Japanese Tachibana;
(14) The plant tissue extract according to (6), wherein the citrus fruit is Tachibana, preferably Japanese Tachibana;

(15) The dried plant tissue according to any one of (1) and (2), wherein the citrus fruit is a giant orange.
(16) The plant tissue extract according to (6), wherein the citrus fruit is Obenimikan;

(17) 10% to 100% by weight of the dry plant tissue according to (1) or (2) or 10% to 90% by weight of the plant tissue extract according to (6) is effective with respect to 100% by weight of the pharmaceutical product A drug for treating Alzheimer's disease as an ingredient;
(18) 10% to 100% by weight of the dry plant tissue according to (1) or (2) or 10% to 90% by weight of the plant tissue extract according to (6) is effective with respect to 100% by weight of the pharmaceutical product A drug for treating Parkinson's disease as an ingredient;
(19) Extraction with water from a blend obtained by blending the dry plant tissue according to any one of (1) and (2) and a pharmaceutically acceptable crude drug component in a weight ratio of 1: 5 to 1:10. A medicament for treating a central neurodegenerative disease, which preferably comprises an extract obtained by water extraction at 60 to 100 ° C. as an active ingredient;
(20) 10 to 100% by weight of the dry plant tissue according to (1) or (2) or 10 to 90% by weight of the plant tissue extract according to (6) with respect to 100% by weight of food Food for ameliorating central neurodegenerative diseases;
(21) The food according to (20), wherein the central neurodegenerative disease is Alzheimer's disease and / or Parkinson's disease;
(22) The food according to (20), wherein the improvement of the central neurodegenerative disease is brought about by an enhancement mechanism of memory acquisition, retention and recall ability;

(23) The content of nobiletin is 0.3 to 2.0% by weight with respect to 100% by weight of the dried plant tissue, and the weight ratio of nobiletin / nariltin is 1.0 to 23.0, (4) Dry plant tissue of;
(24) The dried plant tissue according to (4), which is obtained by heating and drying a citrus plant tissue at 50 to 100 ° C for 1 to 3 hours, and the yield is 20 to 50%;
(25) 0.6 to 3.0% by weight of nobiletin with respect to 100% by weight of plant tissue extract, and the weight ratio of nobiletin / nariltin is 2.0 to 14.0, (9) Plant tissue extracts as described;
(26) The pharmaceutical according to (19), which is in the form of a fine granule, tea, decoction, capsule, tablet, granule, jelly, powder, liquid, syrup or extract preparation.

  The citrus dried plant tissue and plant tissue extract of the present invention show a significant central nervous degenerative disease improving effect as compared with the conventional citrus dried plant tissue.

  The dried plant tissue and plant tissue extract of the present invention also has the effect of activating PKA / ERK / CREB signaling and improving learning / memory impairment, which is one of the symptoms of Alzheimer's disease. The dried plant tissue and plant tissue extract of the present invention particularly show the effect of acquiring, maintaining and recalling memory among the learning / memory impairment improving effects. The dried plant tissue and plant tissue extract of the present invention further have a strong phosphorylation promoting effect and a PKA / ERK signal transduction promoting effect as compared with nobiletin alone having the same amount of nobiletin contained therein. In addition to these, the dried plant tissue and plant tissue extract of the present invention exhibit an improvement effect on Parkinson's disease by CRE transcription activity and TH transcription activity, TH expression level increasing action, dopamine synthesis ability promoting action or dopamine secretion promoting action. .

  The dried plant tissue and plant tissue extract of the present invention further has a central neurodegenerative disease ameliorating effect exceeding that predicted from its nobiletin-containing concentration, in particular, an effect of increasing CRE-dependent transcriptional activity, memory acquisition, retention and recall ability. In addition to the effect of enhancing NO, the effect of promoting TH transcriptional activity and the effect of increasing the TH expression level, the effect of promoting dopamine biosynthesis and the effect of promoting dopamine secretion are shown. These effects are considered to be due to a synergistic effect of nobiletin and other components contained in the dried plant tissue and plant tissue extract.

  Since nobiletin alone has no memory acquisition and retention enhancing effect among the above-mentioned memory acquisition, retention and recall enhancing effects, the dried plant tissue and plant tissue extract of the present invention are completely different from nobiletin alone It is thought that the above effect is achieved by a novel mechanism.

FIG. 1A shows a three-dimensional chromatogram of flavonoids contained in the skin of the present invention. FIG. 1B shows a three-dimensional chromatogram of flavonoids contained in a conventional skin. FIG. 2A shows CREB phosphorylation promoting activity of a skin extract in hippocampal neurons. FIG. 2B shows the PKA substrate phosphorylation promoting activity of the skin extract in hippocampal neurons. FIG. 2C shows the ERK1 / 2 phosphorylation promoting activity of the skin extract in hippocampal neurons. FIG. 2D shows the PKA signal transduction promoting effect of the skin extract in hippocampal neurons. FIG. 2E shows the ERK signal transduction promoting effect of the skin extract in hippocampal neurons. FIG. 2F shows the CRE-dependent transcriptional activity of extracts from the present skin 1 and conventional skin 7 and nobiletin in hippocampal neurons. FIG. 2G shows the CRE-dependent transcriptional activity of extracts from nodules 2-4 of the present invention and conventional nodules 8-10 and nobiletin in hippocampal neurons. FIG. 3A shows the effect of improving the learning / memory impairment of crustacean extract, that is, the effect of acquiring, retaining, and recalling memory. FIG. 3B shows the effect of nobiletin on MK801-induced learning / memory impairment. FIG. 4A shows the TH transcriptional activity promoting effect of the skin extract in PC12D cells. FIG. 4B shows the effect of increasing the TH expression level of the crust extract in PC12D cells. FIG. 4C shows the effect of increasing the dopamine content of the skin extract in PC12D cells.

FIG. 5A shows CRE transcriptional activity of Tachibana peel extract in hippocampal neurons. FIG. 5B shows the improvement effect of learning impairment of Tachibana peel extract. Two-way ANOVA including post Bonferroni test for all pairs of learning-promoting effects. Numbers in the figure show mean ± standard error; n = 5 for all tests. ** p <0.01 compared to the control (■), *** p <0.001, ### p <0.01 compared to MK-801 (▲), * p <0.05. FIG. 5C shows the effect of improving the memory impairment of Tachibana peel extract. One-way ANOVA including post Tukey test for all pairs of memory improvement effects. Numbers in the figure show mean ± standard error; n = 5 for all tests. *** p <0.001 compared to control, #p <0.05 and ### p <0.01 compared to MK-801. FIG. 5D shows CRE transcriptional activity of Tachibana peel extract in PC12D cells. FIG. 5E shows TH transcriptional activity of Tachibana peel extract in PC12D cells. FIG. 5F shows the GCH I expression level increasing effect of Tachibana bark extract in PC12D cells. One-way ANOVA including post Tukey test for all pairs. Numbers in the figure show mean ± standard error; n = 3 for all tests. * P <0.05, ** p <0.01, *** p <0.001 compared to control. # P <0.05, ## p <0.01, ### p <0.001 compared to the crushed extract.

FIG. 6A shows CRE transcriptional activity of the extract derived from the leaf of Tachibana in hippocampal neurons. FIG. 6B shows CRE transcriptional activity of Tachibana leaf-derived extract in PC12D cells. FIG. 7A shows the CRE transcriptional activity of the extract derived from citrus pericarp in hippocampal neurons. FIG. 7B shows the CRE transcriptional activity of the extract derived from the citrus pericarp in PC12D cells. FIG. 7C shows the TH transcriptional activity of the extract derived from the citrus pericarp in PC12D cells.

  The citrus fruits used in the present invention include: Citrus tachibana, C. nipponokoreana, Hanayyu, Shikitsu, Pheasant, Daidai, Mediterranean Mandarin, Dancy Tangerine, Obanimikan, Kobenimikan, Nucleated Kishu, Fukurimita, Capuchi, Ponkan, Hirakishu, Sankitsu, Cleopatra, Koji, Giri Mikan, Ichan Lemon, Unshuu Mikan (Citrus unshiu Markovich), Citrus reticulata Blanco (Rutaceae), Sikhwasa, Yuzu, Bungtan, Hyuganatsu, Ponkan, Natsumikan, Naburiyo, Hakubo Among plants selected from the group consisting of those having a high nobiletin content, preferably Citrus reticulata Blanco (Rutaceae), Citrus tachibana, C. nipponokoreana The other is Oobenimikan. As used herein, the term “tachibana” means Japanese tachibana or Koraitachibana.

  In the dried plant tissue of the present invention, the yield of the citrus peel or leaf (mass after drying / mass before drying) is 20 to 50% in the case of the peel and 20 to 50% in the case of the leaf. It means a substance dried under such conditions. As heat drying conditions, 1-3 hours are mentioned at the temperature of 50-100 degreeC, Preferably it is 2 hours at the temperature of 60 degreeC. Preferred dry plant tissues of the present invention are Chen, Tachibana and Tachibana leaves. Chen means the mature pericarp of citrus plants (for example, Citrus reticulata Blanco (Rutaceae), Obenimikan, etc.), and Tachibana persimmon means the mature pericarp of Japanese tachibana or Korai Tachibana, especially tachibana. The leaf means a leaf of Tachibana such as Japanese Tachibana or Kourai Tachibana.

  The plant tissue extract of the present invention is an extract from the dried plant tissue, preferably an extract obtained by water extraction. The preferable water extraction in this specification is extraction with water at 60 to 100 ° C.

  Examples of components contained in the dried plant tissue and plant tissue extract of the present invention include flavonoids such as nobiletin, nariltin, tangeretin, and hesperidin. The activity of the dried plant tissue and plant tissue extract of the present invention is remarkable as compared with the medicinal effect of nobiletin alone due to the synergistic effect due to the content and content ratio of the components contained therein.

The dry plant tissue of the present invention has a nobiletin content of 0.3 or 0.4% by weight or more, preferably 0.3 to 2.0% by weight, more preferably 0.4 to 2.0% by weight, based on the total weight. 0.7% by weight or less, preferably 0.4% by weight or less of nariltin, 0.1 to 0.8% by weight of tangeretin and 0.4 to 12% by weight of hesperidin. The dry plant tissue of the present invention also has a nobiletin / nariltin content weight ratio of 1.0 or more, preferably 1.5 to 23.0, more preferably 2.0 to 15.0, and still more preferably 5.0 to 15.0.
The dry plant tissue of the present invention can be derived from any plant as long as the proportion of each component contained in the dry tissue is within the above range, that is, the drug effect of the present invention, that is, the central nervous degenerative disease improving effect (anti-antigen). Alzheimer's disease activity, anti-Parkinson disease activity, etc., learning / memory impairment improvement effect, phosphorylation promotion effect and PKA / ERK signaling promotion effect, CRE transcription activity and TH transcription activity, TH expression level increase effect, dopamine synthesis ability promotion effect Or show dopamine secretion promoting action.

The plant tissue extract of the present invention is 0.6 to 2.0% by weight or more, preferably 0.6 to 3.0% by weight of nobiletin, 0.7% by weight or less, preferably 0. Contains no more than 4% by weight nariltin, 0.1-1.0% by weight tangeretin and 1.8-6.0% by weight hesperidin. The plant tissue extract of the present invention also has a nobiletin / nariltin content weight ratio of 2.0 or more, preferably 2.0 to 14.0, more preferably 2.5 to 10.0.
The plant tissue extract of the present invention can be derived from any plant as long as the ratio of each component contained therein is within the above-mentioned range. Anti-Alzheimer's disease activity, anti-Parkinson's disease activity, etc., learning / memory impairment improvement effect, phosphorylation promotion effect and PKA / ERK signaling promotion effect, CRE transcription activity and TH transcription activity, TH expression level increasing effect, dopamine synthesis ability promotion An action or a dopamine secretion promoting action is shown.

  As used herein, the term “central neurodegenerative disease” includes Alzheimer's disease, particularly learning / memory disorders, and Parkinson's disease.

Examples of the anti-Alzheimer's disease activity and / or anti-Parkinson's disease activity of the dried plant tissue and plant tissue extract of the present invention include improvement of learning / memory impairment related to the onset mechanism of central neurodegenerative diseases or promotion of dopamine synthesis. . Specifically, synaptic plasticity promoting activity and CRE (cAMP responsive element) -dependent transcription activity, or tyrosine hydroxylase (TH) transcription activity promoting activity, TH expression level increasing activity, dopamine content increasing activity, dopamine secretion promoting activity, etc. It is.
More specifically, the dried plant tissue and plant tissue extract of the present invention may exhibit different activities depending on the type of plant tissue. For example, with regard to the activity associated with dopamine, the skin or skin extract may exhibit a dopamine content increasing activity, and the tachibana skin or citrus skin extract may exhibit a dopamine secretion promoting activity.

  As used herein, a learning / memory disorder is a disorder that reduces the following three abilities in the memory process: memory acquisition, retention, and recall. In this specification, the term “acquisition” refers to capturing information into memory, “holding” refers to storing captured information, and “recalling” refers to recalling stored information. .

  By treating the dried plant tissue or plant tissue extract of the present invention according to a method commonly used in the art, a therapeutic agent for central neurodegenerative diseases can be produced. As used herein, therapeutic agents for central neurodegenerative diseases include anti-Alzheimer's disease agents and / or anti-Parkinson's disease agents, that is, pharmaceuticals having anti-Alzheimer's disease activity and / or anti-Parkinson disease activity.

  As used herein, the term “pharmaceutical” means a substance for diagnosis, treatment or prevention of diseases in humans and animals, and includes pharmaceuticals including herbal medicines, such as herbal medicines and herbal medicines. A preferred pharmaceutical is an oral dosage form, more preferably a fine granule, tea, decoction, capsule, tablet, granule, jelly, powder, liquid, syrup or extract.

  As used herein, the term “herbal medicine” is a general term for substances used for improving a subject's constitution without purifying an active ingredient from a product having a natural medicinal effect, and includes one or more herbal ingredients. As used herein, the term “Chinese medicine” means a medicine prescribed on the basis of Chinese medicine theory.

  In this specification, the term “food” refers to so-called health foods and health functional foods such as foods for specified health use and functional nutritional foods that are distinguished by the presence or absence of permission from the authorities and the purpose and function of the food. Including. Preferred foods herein are fine granules, jelly sauce, peel, jam or tea, more preferably fine granules or tea.

  The amount of each component added to the pharmaceutical and food in the present specification can be appropriately adjusted according to the intended purpose of use, gender, symptoms, etc. For example, the dry plant tissue of the present invention is 10 to 100% by weight or 10 to 90% by weight of tissue extract and 0 to 90% by weight of other carriers or additives. The daily intake amount of the dried plant tissue or plant tissue extract of the present invention per adult is preferably 3 to 50 g or 1 to 20 g, and more preferably 5 to 30 g or 2 to 10 g.

  Other carriers and additives herein include those commonly used in the art, such as pharmaceutically or food hygienically acceptable herbal ingredients, ascorbic acid, aspartame, apple flavor, orange flavor, carrageenan. , Caramel, carnauba wax, carmellose, carmellose calcium, reduced maltose liquid sugar, reduced maltose starch syrup, hydrous silicon dioxide, xylitol, citric acid, trisodium citrate, granulated sugar, light anhydrous silicic acid, gelling agent (FG-2266, Nitta Gelatin Co., Ltd.), synthetic aluminum silicate, hydroxypropyl starch, crystalline cellulose, honey beeswax, titanium oxide, salt, sucrose fatty acid ester, magnesium stearate, cellulose, talc, dextrin, corn starch, lactose, honey, hydroxy Cypropyl methylcellulose, fine silicon dioxide, pullulan, pectin, maltose, magnesium aluminate metasilicate, methylcellulose, lecithin and locust bean gum.

  The pharmacologically or food hygienically acceptable herbal medicine components in this specification include those commonly used in the art. For example, Ireisen, Uyaku, Engosaku, Ogi, Ogon, Oubak, Onji, Cuckoo, Kakon, Kankyo , Licorice, kyouko, kikuka, pheasant, kyounin, keihi, kou, kobushi, kokuboku, goshtsu, goshyu, garbage, psycho, sashishi, jiou, peonies, gyoza, ginger, shinkiku, gypsum, senkou, sekibo, sakuju Soyo, Daiou, Taiso, Daifukuhi, Takusha, Chikujo, Chimo, Butterflyfish, Tenma, Tenmondo, Toki, Tonin, Carrot, Bakuga, Bakumondou, Hange, Bikaku, Beak Jutsu, Areca, Bakuryo, Bowie, Bow ® U, windproof, moutan bark, ephedra, Mokutsuu, woodworking, motherwort, include Ryuutan and Wakyoukatsu.

  In the following examples, the skins 1 to 6, the tachibana skins 1 to 6, the leaves of tachibana 1 to 3 and the giant beetle 1 (pericarp) are the dry plant tissue of the present invention, and the skins 7 to 12 are conventional. Moreover, the plant tissue of the same degree can be obtained from Kotaro Hanpo Pharmaceutical Co., Ltd. from the viewpoint of the samples and the components used in the following examples.

[Example 1] Production of dried plant tissue In this example, the peels and leaves collected from various citrus fruits were shade-dried, sun-dried or heat-dried to obtain dried bodies of the peel, tachibana peels, tachibana leaves, and the fruit of periwinkle. Manufactured. Heat drying is carried out until the yield (ratio of mass after drying / mass before drying) is 20 to 50% for Chen and Tachibana bark, 20 to 50% for Tachibana leaves, and 20 to 50% for persimmon skins. Specifically, it was dried by heating at a temperature of 60 ° C. for 2 hours.

[Example 2] Production of plant tissue extract 400 mL of pure water was added to about 10 g of the cut skin, tachibana bark, leaves of tachibana or persimmon bark obtained in Example 1 and heated (tachibana bark) For 6), 1000 mL of pure water was added to about 20 g of the minced and heated. After the mixture boiled, it was extracted at 100 ° C. for 1 hour. Subsequently, it filtered through gauze (2 sheets), and the filtrate was freeze-dried to obtain a plant tissue extract. The production results of the plant tissue extract are shown in Table 1 below.

[Example 3] Analysis of contained components The contained components of the dried plant tissues and plant tissue extracts obtained in Examples 1 and 2 were measured as follows. Below, the analysis method about a crust and a crust extract is shown.

(Quantification of nobiletin and tangeretin)
A standard solution used for quantification of nobiletin and tangeretin was prepared as follows. Nobiletin (Wako Pure Chemical Industries, Ltd.) was dried with a desiccator (silica gel) for 24 hours or more. About 5 mg of dried nobiletin was dissolved in a methanol / water mixture (7: 3) to make 100 mL, and used as a nobiletin standard solution. Further, tangeretin (Wako Pure Chemical Industries, Ltd.) was dried with a desiccator (silica gel) for 24 hours or more. About 3 mg of dry tangeretin was dissolved in a methanol / water mixture (7: 3) to make 100 mL, and used as a tangeretin standard solution.

As a sample solution of crust, about 0.3 g of crust powder was placed in a 50 mL stoppered centrifuge tube, and 50 mL of methanol was added thereto. The mixture was extracted with ultrasonic waves (UT-305HS, Sharp Corporation) and then centrifuged (KUBOTA KN-70, Kubota Corporation). Thereafter, it was filtered to obtain a sample solution for skin.
As a sample solution of the peel extract, about 0.3 g of the peel extract was placed in a 50 mL stoppered centrifugal tube, and 50 mL of a methanol / water mixture (7: 3) was added thereto. The mixture was extracted with ultrasonic waves (UT-305HS, Sharp Corporation) and then centrifuged (KUBOTA KN-70, Kubota Corporation). Then, it filtered and it was set as the sample solution of the crust extract.

The standard solution and sample solution prepared as described above were quantified under the following analytical conditions.
Detector: UV absorptiometer (measurement wavelength: 338 nm)
Column: Mightysil RP-18 4.6 mm x 15 cm (Kanto Chemical Co., Inc.)
Mobile phase: Water / acetonitrile mixture (3: 2)
Flow rate: 1.0 mL / min (Nobiletin retention time was approximately 10 minutes)
Column temperature: 40 ° C
Injection volume: 10μL

(Quantitative determination of hesperidin and nariltin)
A standard solution used for quantification of hesperidin and nariltin was prepared as follows. Hesperidin (Wako Pure Chemical Industries, Ltd.) was dried with a desiccator (silica gel) for 24 hours or more. About 10 mg of dry hesperidin was dissolved in 50% methanol to make 50 mL, and used as a hesperidin standard solution. Also, Nariltin (Wako Pure Chemical Industries, Ltd.) was dried with a desiccator (silica gel) for 24 hours or more. About 10 mg of dry narilutin was dissolved in 50% methanol to make 500 mL, which was used as a narilutin standard solution.

As a skin sample solution, 30 mL of methanol was added to about 0.1 g of skin powder. The mixture was irradiated with ultrasonic waves for 20 minutes (UT-305HS, Sharp Corporation), extracted, and then centrifuged (KUBOTA KN-70, Kubota Corporation) to collect the supernatant. 20 mL of methanol was added to the residue, and extraction was performed by irradiating with ultrasonic waves for 20 minutes, followed by centrifugation. This was combined with the collected supernatant to obtain a skin sample solution.
As a sample solution of the peel extract, about 0.1 g of the peel extract was placed in a 50 mL stoppered centrifugal sedimentation tube, and 50 mL of a methanol / water mixture (1: 1) was added thereto. The mixture was extracted with ultrasonic waves (UT-305HS, Sharp Corporation) and then centrifuged (KUBOTA KN-70, Kubota Corporation). Then, it filtered and it was set as the sample solution of the crust extract.

The standard solution and sample solution prepared as described above were quantified under the following analytical conditions.
Detector: UV spectrophotometer (measurement wavelength: 285 nm)
Column: L-column2 ODS 4.6 mm x 25 cm (Research Organization for Chemical Evaluation)
Mobile phase: Water / acetonitrile / acetic acid mixture (40: 10: 1)
Flow rate: 0.8 mL / min (Hesperidin retention time was about 15 minutes)
Column temperature: 30 ° C
Injection volume: 10μL

  Table 2 below shows the analysis results of the peel, tachibana peel, leaves of Tachibana and pericarp obtained in Examples 1 and 2, and Table 3 shows the analysis results of these plant tissue extracts. In addition, FIGS. 1A and 1B show three-dimensional chromatograms of flavonoids contained in the skin of the present invention and the conventional skin, respectively.

*: 各成分の含量は、用いる柑橘類の植物体の採取場所や採取時期などによって変化することがある。例えば、橘皮の青色果皮では約1.60重量%のノビレチン含量のものもあったが、黄色果皮になると約1.1重量%であった。

*: The content of each component may vary depending on the sampling location and timing of the citrus plants used. For example, the blue peel of tachibana has some nobiletin content of about 1.60% by weight, but about 1.1% by weight of yellow peel.

*: 各成分の含量は、用いる柑橘類の植物体の採取場所や採取時期などによって変化することがある。

*: The content of each component may vary depending on the sampling location and timing of the citrus plants used.

[Test example]
The following in vitro and in vivo tests were conducted on the skin extract obtained in the above examples.

[General test procedure 1]
The general test procedure used in Test Example 1 below is as follows.
(Culture of rat fetal primary hippocampal neurons)
Pregnant Sprague-Dawley (SD) rats were bred by feeding and watering in a 12-hour light / dark cycle. The uterus was aseptically removed from the rat (E18) on day 18 of gestation by a midline abdominal incision under deep ether anesthesia. Under a stereomicroscope, remove the fetal hippocampus in ice-cold phosphate buffered saline (PBS), disperse the tissue with papain enzyme (SUMILON), and centrifuge at 1000 rpm for 4 minutes. Removed. Next, the cell pellet was dispersed in a dispersion liquid (SUMILON), and then the removal liquid (SUMILON) was added to the cells sufficiently dispersed by pipetting, followed by centrifugation at 900 rpm for 5 minutes, and then the supernatant was removed. .
Next, the pellet is suspended using Neurobasal Medium (Neurobasal Medium 500 mL / Phenol Red-free, 50 x B27 Supplement 10 ml, 0.5 mM L-glutamine, 0.005% penicillin-streptomycin) and added with poly-L-lysine. Seeded on coated dishes or plates. After 1 day of culture, the medium was changed, and then half of the medium was changed every 3 to 4 days, and cultured in a medium containing 10 μM AraC at 37 ° C. in a 5% CO ₂ incubator for 14 days.
In addition, Neurobasal Medium containing 10 μM AraC was used as a test medium for drug treatment experiments.

(SDS-PAGE and Western blot analysis 1)
Rat fetal primary hippocampal neurons were seeded at 1 × 10 ⁶ cells / dish in a 35 mm dish, cultured in a growth medium for 14 days in a 5% CO ₂ incubator, and then treated with the drug for 10 minutes.
Each cell was treated with drugs and then washed with ice-cold PBS. Cell lysate (1 mM EDTA, 1% SDS, 10 mM NaF, 10 nM caliculin, 320 nM okadaic acid, 1 mM sodium orthovanadate, 1 mM p-APMSF, 10 μg / mL pepstatin A, 10 μg / mL Antipine, 10 μg / mL leupeptin, 10 μg / mL chymostatin, 10 μg / mL phosphoramidon, 10 mM HEPES, pH 7.5). Thereafter, it was immediately boiled at 95 ° C. for 5 minutes to denature the protein, and then the DNA was sonicated to prepare a sample for SDS-PAGE.

  12.5% polyacrylamide gel was used as a separation gel for SDS-PAGE. After electrophoresis at 35 V and 10 mA, the protein was transferred from the gel to a PVDF membrane, and TBST containing 5% skim milk (10 mM Tris-HCl, 100 Blocking was performed at room temperature for 1 or 2 hours using mM NaCl, 0.05% Tween 20, pH 7.4; Samples were then washed with TBST and incubated overnight at 4 ° C. with primary antibody diluted 1000-fold with blocking buffer. Further, the sample was washed with TBST, incubated with HRP-labeled IgG antibody diluted 2000 times with blocking buffer at room temperature for 2 hours, and washed with TBST. The ECL method was used to detect antibody-positive bands. For reprobing, the PVDF membrane is placed in a stripping buffer (62.5 mM Tris-HCl, 2% SDS, 100 mM β-mercaptoethanol, pH 7.4) pre-warmed to 50 ° C, and incubated at 50 ° C for 30 minutes. Was removed and washed with TBST. Thereafter, the internal standard protein was detected by reprobing.

(Statistical analysis 1)
The experimental results were evaluated using a one-way ANOVA (Student-Newman-Keuls). The significance level was tested at 5% on both sides, and p <0.05 was considered significant.

[Test Example 1] Improvement of learning and memory impairment by the skin extract (in vitro test)
In this test example, the effect of the skin extract of the present invention on learning / memory impairment was examined. In vitro study, we investigated the phosphorylation-promoting action of nuclear protein CREB in hippocampal neurons, and the effect of skin extract on PKA / ERK signaling suppression by NMDA receptor blocker (MK801) in primary cultured hippocampal neurons It was investigated.
MK801 used in the present specification is a non-selective antagonist of NMDA receptor, which is one of the subtypes of glutamate receptor, and is also referred to as dizocilpine.
The skin extract used in this test example was prepared as follows. That is, in the in vitro test, 7.2 g of the skin extract from the skin 1 was weighed and placed in a 50 ml centrifuge tube, and dimethyl sulfoxide (DMSO) was added thereto to make 30 mL. Then, it was sonicated for 30 minutes and centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and used as a skin extract.

[Test Example 1-1] CREB phosphorylation-promoting effect of skin extract in primary cultured hippocampal neurons The effect of various concentrations of skin extract on CREB phosphorylation was examined using Western blotting. E18 rat hippocampal neurons grown at a density of 1 × 10 ⁶ cells / 35-mm plastic dish were cultured for 14 days. Cells were treated with crust extract (30, 60, 120, 240 and 480 μg / ml) for 10 minutes. Western blotting was performed using anti-phosphorylated CREB antibody. The blot was then stripped and tested again for anti-14-3-3-β antibody to confirm that an equal amount of protein was electrophoresed in each lane.
As shown in FIG. 2A, the peel extract promoted CREB phosphorylation in a concentration-dependent manner.

[Test Example 1-2] PKA substrate phosphorylation promoting effect of skin extract in primary cultured hippocampal neurons The effects of various concentrations of skin extract on PKA substrate phosphorylation were examined using Western blotting. E18 rat hippocampal neurons seeded at a density of 1 × 10 ⁶ cells / 35-mm plastic dish were cultured for 14 days. Cells were treated with different concentrations of crust extract for 10 minutes. Western blotting was performed using anti-phosphorylated PKA substrate antibody. The blot was then stripped and tested again for anti-14-3-3-β antibody to confirm that an equal amount of protein was electrophoresed in each lane.
As shown in FIG. 2B, the peel extract promoted PKA substrate phosphorylation in a concentration-dependent manner.

[Test Example 1-3] Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation-promoting effect of skin extract in primary cultured hippocampal neurons About the effect of various concentrations of skin extract or nobiletin on ERK1 / 2 phosphorylation Blotting was used (contains nobiletin 1 μM in 60 μg / ml of skin extract). E18 rat hippocampal neurons grown at a density of 1 × 10 ⁶ cells / 35-mm plastic dish were cultured for 14 days. Cells were treated for 10 minutes with crushed extract (30, 60, 120, 240 and 480 μg / ml) or nobiletin (4, 8 and 30 μM). Western blotting was performed using anti-phosphorylated ERK1 / 2 antibody. The blot was then stripped and tested again for anti-ERK1 / 2 antibodies to confirm that equal amounts of protein were electrophoresed in each lane.
As shown in FIG. 2C, the peel extract promoted ERK1 / 2 phosphorylation in a concentration-dependent manner. Moreover, Chen extract showed a strong effect compared with nobiletin. That is, it was suggested that the phosphorylation promoting effect of the present skin extract 240 μg / ml containing nobiletin 4 μM is stronger than the effect of nobiletin 4 μM depending on the size of the blot.

[Test Example 1-4] PKA / ERK signal transduction promoting effect of skin extract in primary cultured hippocampal neurons The effect of skin extract on PKA / ERK signaling suppression by MK801 in primary cultured rat hippocampal neurons was examined. Primary cultured rat hippocampal neurons were pretreated for 1 hour with skin extract or nobiletin, then with MK801 for 30 minutes, and then with N-methyl-D-aspartic acid (NMDA) for 15 minutes. Western blotting was performed using anti-phosphorylated PKA substrate antibody. The blot was then stripped and retested for anti-14-3-3-β antibody to confirm that equal amounts of protein were electrophoresed in each lane (see FIG. 2D). Further, Western blotting was performed using an anti-phosphorylated ERK1 / 2 antibody. The blot was then stripped and retested for anti-ERK1 / 2 antibodies to confirm that equal amounts of protein were electrophoresed in each lane (see FIG. 2E).
As a result, MK801 suppressed PKA / ERK signaling by NMDA. However, the skin extract showed an antagonistic effect on the signal transduction suppression by MK801 in a concentration-dependent manner, and the effect of the skin extract (containing 8 μM nobiletin in the skin extract 480 μg / ml) was stronger than that of 8 μM nobiletin .

(Discussion 1)
From the results of the above test examples, CREB, a nuclear protein known to be deeply involved in synapse formation and long-term memory formation, as well as phosphorylation of PKA substrate and ERK1 / 2 upstream thereof, It was revealed that the peel extract has a significant phosphorylation promoting action. The skin extract of the present invention also exhibited a stronger phosphorylation promoting effect than nobiletin at ERK1 / 2.
Furthermore, the skin extract of the present invention exhibited a significant antagonistic effect on the suppression of PKA / ERK signaling by MK801, and the effect was stronger than nobiletin. Thereby, it was shown that the skin extract of this invention has a synergistic effect | action with components other than nobiletin contained therein and nobiletin.

[Test Example 2] CRE-dependent transcriptional activity of a crust extract in hippocampal neurons (in vitro test)
In this test example, the CRE-dependent transcriptional activity of the skin extract of the present invention was compared with the conventional skin extract and nobiletin.

As described in General Test Procedure 1 above, rat hippocampal neurons were primarily cultured and then a reporter gene assay was performed. Hippocampal neurons were seeded in a 48-well plate at 8 × 10 ⁴ cells / well and cultured in Neurobasal medium for 10-14 days. Reporter plasmid (0.1 μg / well) and Renilla pRG-TK plasmid (0.01 μg / well) were transfected by lipofection and cultured for 16 hours. It was diluted with Neurobasal medium without AraC (containing B-27 Supplement, L-glutamine, penicillin-streptomycin) and treated with a crust extract at a concentration of 300 μg / ml for 8 hours. The skin extract was a 150 mg / ml DMSO solution as a stock solution, dispensed, and stored at -20 ° C. Transcriptional activity was measured using Promega's Dual-Luciferase (registered trademark) Reporter Assay System. Hippocampal neurons were solubilized with Passive Lysis Buffer (Promega), then mixed with Luciferase Assay Regent II (Promega) and Stop & Glo (registered trademark) Reagent (Promega), and the fluorescence value was measured with a luminometer.
For statistical analysis, the reporter gene assay and RT-PCR results were evaluated using one-way ANOVA (Tukey). The significance level was tested at 5% on both sides, and p <0.05 was considered significant.

(Discussion 2)
As shown in FIGS. 2F and 2G, the concentration of nobiletin in the skin extract used in this test example is 2.5 μM, and its CRE-dependent transcriptional activity is 1 / 12th that of 30 μM nobiletin. More than 30 μM nobiletin. That is, it was found that in the multi-component system of the skin extract of the present invention, the presence of components other than nobiletin significantly and synergistically increases the CRE-dependent transcription promoting activity of nobiletin than the activity expected from nobiletin alone. In addition, the skin extract of the present invention showed a CRE-dependent transcriptional activity stronger than that of conventional skin extracts, so it showed an action to improve learning and memory impairment and more effectively improved the recognition function of Alzheimer's disease. It has been shown.

[Test Example 3] Learning and memory impairment improving action of the skin extract (in vivo test)
Next, in the in vivo test, the action of the skin extract of the present invention on behavioral pharmacological learning / memory impairment was examined using MK801-induced memory impairment model mice.
The skin extract used in this test example was prepared as follows. That is, 5.54 g of skin extract from skin 1 was weighed and placed in a 50 ml centrifuge tube, and physiological saline was added thereto to make 30 mL. Then, it was sonicated for 30 minutes.
The administration dose of the skin extract to mice was prepared based on the nobiletin content. Here, the nobiletin content was measured in advance by the absolute calibration curve method of the HPLC method using a nobiletin standard substance.

We investigated the effects of chronic administration of Chen extract on MK801-induced learning and memory impairment. Mice were divided into 4 groups, each with a skin extract of 1.48 g / kg or 3.69 g / kg (nobiletin content: 10 mg / kg or 25 mg / kg, respectively), and 2 groups with physiological saline for 7 consecutive days Orally. On the 7th day, 90 minutes after administration of these, MK801 (0.08 mg / kg) or physiological saline was administered subcutaneously. Thirty minutes later, a fear conditioning learning test was conducted.
In the horror learning test, mice were placed in a transparent box and allowed to explore freely for 2 minutes, followed by 0.7 mA, 2 s electrical stimulation. This was repeated three times and learning trials were performed to evaluate memory acquisition and retention ability. After 24 hours, place the mouse in the transparent box again, perform a confirmation test using the mouse's freezing action, i.e., all movements other than breathing as an index, and keep it as a learning / memory action by measuring for 5 minutes. Recall ability was assessed. The results are shown in FIG. 3A.

[Comparative Test 1] Examination of learning / memory impairment improving effect of nobiletin In Comparative Example 1-1, a comparative test was conducted by replacing oral administration of crust extract with intraperitoneal administration of nobiletin (10, 50 mg / kg). It was. The results are shown in FIG. 3B.

  As shown in FIGS. 3A and B, the freezing action was significantly reduced by the treatment with MK801. However, the administration of the crustacean extract showed an improvement effect on the reduction in a dose-dependent manner in both the learning trial and the confirmation trial. It was. In particular, in the learning trial, the mice administered with the skin extract of the present invention received the MK801 treatment, and the learning / memory function was hindered and easily forgotten. The ratio was about 1.5 times that of the control mouse and about 4.4 times that of the MK801-treated mouse skin extract-untreated mouse (FIG. 3A). Such a result was not recognized by administration of nobiletin (Comparative Test 1, FIG. 3B). That is, it has been found that the skin extract of the present invention has an effect of acquiring memory and enhancing retention ability, which is not recognized by administration of nobiletin alone.

  Furthermore, in the verification trial, in the test with the skin extract of the present invention, no significant difference was observed compared with the result of intraperitoneal administration of nobiletin, despite oral administration.

(Discussion 3)
From the results of the above test examples, it is clear that, in vivo, chronic administration of the skin extract of the present invention significantly improves learning and memory impairment, and also enhances the ability to acquire and retain memory that is not observed with nobiletin. It became.
From the above, it was shown that the skin extract of the present invention improves learning / memory impairment and further enhances the ability to acquire memory by promoting synaptic plasticity and protecting PKA / ERK. That is, it is shown that the skin extract of the present invention improves learning / memory impairment, which is a core symptom of Alzheimer's disease, and enhances and / or improves the ability to acquire and retain memory, which is not found in nobiletin, more than nobiletin. It was done.

[General test procedure 2]
The general test procedure used in Test Example 4 below is as follows.
(PC12D cell culture)
Dulbecco's modified Eagle medium (DMEM) (9.5 g) was dissolved in MilliQ water (800 mL), and glucose (3.5 g) was added thereto to make 1 L with MilliQ water. 830 mL of the product sterilized by autoclave was taken, and 17 mL of 10% aqueous sodium hydrogen carbonate solution and 16.5 mL of 3% L-glutamine aqueous solution were added. Next, equine serum (HS) to 10% and fetal calf serum (FCS) to 5% were prepared as growth media and cultured in a 5% CO ₂ incubator at 37 ° C. .
The test medium used was hDMEM containing 2% HS and 1% FCS.

(Plasmid introduction and reporter gene assay)
PC12D cells were seeded in a 48-well plate at 8 × 10 ⁴ cells / well and cultured in a growth medium for 24 hours. Then, pRTH plasmid was 0.1 μg / well, and Renilla pRG-TK plasmid was 0.01 μg / well lipofected. The medium was replaced with a test medium in which the compound was dissolved 19 hours after transfection. After incubation for 24 hours, the medium was aspirated and collected with Passive Lysis Buffer.
Firefly shiitake and Renilla luciferase were measured with a luminometer using Dual-Luciferase Reporter Assay System (Promega).

(SDS-PAGE and Western blot analysis 2)
PC12D cells were seeded at 0.67 × 10 ⁶ cells / dish in a 35 mm dish, cultured in a growth medium for 2 days in a 5% CO ₂ incubator, and then treated with the drug for 24 hours.
Each cell was treated and then washed with ice-cold PBS. Cell lysate (1 mM EDTA, 1% SDS, 10 mM NaF, 10 nM caliculin, 320 nM okadaic acid, 1 mM sodium orthovanadate, 1 mM p-APMSF, 10 μg / mL pepstatin A, 10 μg / mL Antipine, 10 μg / mL leupeptin, 10 μg / mL chymostatin, 10 μg / mL phosphoramidon, 10 mM HEPES, pH 7.5). Thereafter, the mixture was immediately boiled at 95 ° C. for 5 minutes to denature the protein, and then the DNA was sonicated and the centrifuged supernatant was prepared as a sample for SDS-PAGE.

  Using a 12.5% gel as a separation gel for SDS-PAGE and running at 35 V, 10 mA, the protein was transferred from the gel to a PVDF membrane, and TBST containing 5% skim milk (10 mM Tris-HCl, 100 mM NaCl) , 0.05% Tween 20, pH 7.4; hereinafter referred to as blocking buffer) for 1 hour at room temperature. Samples were then washed with TBST and incubated overnight at 4 ° C. with primary antibody diluted 1000-fold with blocking buffer. Further, the sample was washed with TBST, incubated with an HRP-labeled IgG antibody diluted 2000 times with a blocking buffer at room temperature for 1 hour, and washed with TBST. The ECL method was used to detect antibody-positive bands. For reprobing, the antibody was peeled off using a stripping buffer (62.5 mM Tris-HCl, 2% SDS, 100 mM β-mercaptoethanol, pH 7.4), the blot was washed with TBST, and then the internal standard protein was detected.

(Statistical analysis 2)
The experimental results were evaluated using a one-way ANOVA (Bonferroni). The significance level was tested at 5% on both sides, and p <0.05 was considered significant.

[Test Example 4] Dopamine synthesis promoting effect of skin extract In this test example, the effect of the skin extract of the present invention on improving the symptoms of Parkinson's disease was examined using the effect on the dopamine biosynthesis system as an index. Specifically, the influence of the cinnamon extract on tyrosine hydroxylase (TH) transcriptional activity was examined by introducing a TH gene promoter region DNA reporter gene and measuring luciferase activity in PC12D cells, which are dopaminergic cells. In order to examine the influence on TH protein, the expression of TH protein in PC12D cells treated with crushed extract was examined by Western blotting. Furthermore, in order to investigate the medicinal effects of crustacean extract or nobiletin on the reduction of dopamine content in the striatum due to degeneration of substantia nigra-striatal dopamine nerve in Parkinson's disease model mice, It was measured.

[Test Example 4-1] Promotion of TH transcriptional activity of skin extract in PC12D cells
PC12D cells grown at a density of 8 × 10 ⁴ cells / 48 well plate were cultured for 24 hours. Cells were transfected with a luciferase reporter construct for 19 hours, then control, a peel extract of the present invention (30, 60 and 120 μg / ml) from skin 1 and a conventional skin 7 extract (120 μg / ml) or nobiletin (2 , 10 and 30 μM) for 24 hours.
As shown in FIG. 4A, in PC12D cells, the skin 1 extract of the present invention markedly increased TH transcriptional activity compared to the control and conventional skin 7 extract. Moreover, it was shown that the extract 1 of skin 1 of 2 μM in terms of nobiletin 120 μg / ml promotes TH transcriptional activity more strongly than 2 μM and 10 μM nobiletin.

[Test Example 4-2] Increased TH expression level of skin extract in PC12D cells
In PC12D cells, TH protein expression promoting effect was examined. PC12D cells grown at a density of 0.67 × 10 ⁶ cells / dish were cultured for 48 hours. Cells were treated with control or skin extract from skin 1 (60, 120, 240 and 480 μg / ml) for 24 hours. Western blot analysis was performed using anti-TH and anti-PKAα antibodies. The blot was first examined for anti-TH antibody, then stripped and retested with anti-PKAα antibody to confirm that an equal amount of protein was electrophoresed in each lane.
As shown in FIG. 4B, in PC12D cells, the crust extract increased the expression of TH protein in a concentration-dependent manner.

[Test Example 4-3] Elevating dopamine content of skin extract in PC12D cells
Since TH is the rate-determining enzyme for dopamine synthesis and PC12D cells are dopamine-synthesizing cells, we investigated the effect of dopamine on the increase.
PC12D cells were seeded at 1.0 × 10 ⁶ cells / dish in a 35 mm dish and cultured in a 5% CO ₂ incubator on a growth medium for 24 hours, and then the cell culture solution supplemented with skin 1 and skin 7 was further added for 24 hours. Cultured. Subsequently, a low K ⁺ buffer (140 mM NaCl, 4.7 mM KCl, 1.2 mM KH ₂ PO ₄ , 2.5 mM CaCl ₂ , 1.2 mM MgSO ₄ , 11 mM glucose and 15 mM HEPES-Tris, pH 7 heated to 37 ° C. The cells were washed in .4), a perchloric acid solution was added so that the final concentration was 0.4 N, and the cells were disrupted with ultrasonic waves for 1 minute. The obtained solution was centrifuged, and dopamine was separated and quantified by high performance liquid chromatography using an electrochemical detector.
As shown in FIG. 4C, the skin 1 extract of the present invention increased the dopamine content at both 24 and 48 hours treatment compared to the control and conventional skin 7 extract.

(Discussion 4)
In the above test examples, the skin extract of the present invention increases the TH transcriptional activity, TH expression level and dopamine content in PC12D cells in a concentration-dependent manner, and its action is unexpected and exceptional compared to nobiletin at an equal concentration. It was powerful.
That is, it was revealed that the skin extract of the present invention exhibits a dopamine synthesis promoting activity more markedly than nobiletin. Since the activity is not recognized in the conventional extract derived from dermis, it is considered that the dermis extract of the present invention exhibited a dopamine synthesis promoting activity due to a synergistic effect of a high content of nobiletin and other components other than nobiletin. As described above, the skin extract of the present invention is expected to have a medicinal effect as a therapeutic agent for Parkinson's disease.

Test Example 5 CRE Transcriptional Activity of Tachibana Skin Extract in Hippocampal Nerve Cells Using the extract extracted from Tachibana Skin 3 obtained in Example 2 by the method described herein, CRE transcriptional activity in hippocampal neurons is controlled And compared with nobiletin. The test procedure was the same as in Test Example 2. The result is shown in FIG. 5A.
The Tachibana peel 3 extract 300 μg / mL used in this test example contained about 9.4 μM nobiletin.
As shown in FIG. 5A, the extract of Tachibana peel 3 showed significant CRE transcriptional activity in hippocampal neurons compared to control and nobiletin. From this, the tachibana peel extract of the present invention is expected to have a medicinal effect as a therapeutic agent for Alzheimer's disease.

[Test Example 6] Tachibana peel extract learning / memory impairment improving effect (in vivo test)
Using the extract extracted from Tachibana bark 6 obtained in Example 2 by the method described in this specification, the effect of the Tachibana bark extract of the present invention on learning / memory impairment was examined. The test procedure was the same as in Test Example 3. The results are shown in FIGS. 5B and 5C.
Tachibana peel extract 1.87 g / kg or 3.73 g / kg (nobiletin content: 25 mg / kg or 50 mg / kg, respectively) was orally administered to each group continuously for 7 days.
As shown in FIG. 5B and FIG. 5C, in addition to significantly improving learning / memory impairment by chronic administration of the tachibana peel extract of the present invention, it is possible to enhance the ability to acquire and retain memory that is not observed with nobiletin. It became clear.

(Discussion 5)
From the results of the above test examples, the tachibana peel extract of the present invention is expected to have a medicinal effect as a therapeutic agent for Alzheimer's disease.

[Test Example 7] CRE transcription activity of Tachibana peel extract in PC12D cells Using the extract extracted from Tachibana peel 3 obtained in Example 2 by the method described herein, CRE transcription activity in PC12D cells was compared with control and nobiletin. And compared. The test procedure was the same as in Test Example 2. The result is shown in FIG. 5D.
The Tachibana peel 3 extract 300 μg / mL used in this test example contained about 9.4 μM nobiletin.
As shown in FIG. 5D, the extract of Tachibana peel 3 showed significant CRE transcriptional activity in PC12D cells compared to control and nobiletin.

[Test Example 8] TH transcription activity of Tachibana peel extract in PC12D cells Using the extract extracted from Tachibana peel 3 obtained in Example 2 by the method described in this specification, the TH transcription activity in PC12D cells was compared with control and nobiletin. And compared. The test procedure was the same as in Test Example 4-1. The result is shown in FIG. 5E.
As shown in FIG. 5E, Tachibana peel 3 extract showed significant TH transcriptional activity in PC12D cells compared to control and nobiletin.

[Test Example 9] Increase in GTP cyclohydrolase I (GCH I) content of Tachibana bark extract in PC12D cells
In PC12D cells, the effect of increasing the amount of GCH I was examined. GCH I is a rate-limiting enzyme in the biosynthesis of the coenzyme tetrahydrobiopterin (BH ₄ ) essential for TH.
PC12D cells were treated with Tachibana peel extract, Chen peel extract and DMSO (control) at a concentration of 120 μg / mL for 48 hours, washed with PBS, and cell lysate (1 mM EDTA, 1% SDS, 10 mM HEPES, 10 mM NaF) , 1 mM p-APMSF, 1 mM sodium orthovanadate, 10 nM calyculin, 320 μM okadaic acid, 10 μg / mL pepstatin, 10 μg / mL antipine, 10 μg / mL leupeptin, 10 μg / mL chymostatin, 10 μg / mL phosphoramidon, 240 pM cypermethrin). Thereafter, the mixture was boiled at 95 ° C. for 5 minutes, sonicated, centrifuged at 10000 × g for 10 minutes, and SDS-PAGE was performed using the supernatant. A 10 μg sample was applied to a gel with a gel concentration of 12.5%. Thereafter, transcription was performed on a PVDF membrane at 36 V for 2 hours, and the membrane was blocked with 5% skim milk for 1 hour. After that, GCH I antibody (ABBIOTEC, diluted 1000 times) was used to react overnight at 4 ° C, washed and further reacted with peroxidase-labeled anti-rabbit secondary antibody (CST, diluted 2000 times), and the band was detected by ECL method. did. The results are shown in FIG.
The Tachibana peel extract 120 μg / mL used in this test example contained about 3.76 μM nobiletin, and the cuticle extract 120 μg / mL contained about 2.02 μM nobiletin.
As shown in FIG. 5F, in PC12D cells, Tachibana peel extract and Chen peel extract significantly increased the expression level of GCH I compared to the control. Furthermore, Tachibana peel extract significantly increased the expression level of GCH I compared to Chen peel extract.

(Discussion 6)
Coenzyme BH4, which is essential for the expression of TH activity, increases the amount of TH protein, increases the TH enzyme activity, and promotes dopamine synthesis when the intracellular BH4 content is higher than the amount indicated by the Michaelis constant. .
From the results of the above test examples, the Tachibana peel extract of the present invention significantly increases the expression level of GCH I, and therefore increases the amount of GCH I, which is the rate-determining enzyme for coenzyme BH4 synthesis, which is essential for the expression of TH activity. As a result, TH activity is increased, and as a result, production and secretion of dopamine are presumed to be promoted, and a medicinal effect as a Parkinson's disease therapeutic agent is expected.

[Test Example 10] CRE transcription activity of Tachibana leaf-derived extract in hippocampal nerve cells Using extracts extracted from Tachibana leaves 1 to 3 obtained in Example 2 by the method described in this specification, CRE transcriptional activity was compared with control and nobiletin. The test procedure was the same as in Test Example 2. The results are shown in FIG. 6A.
In addition, nobiletin is about 4.9 μM for Tachibana leaf 1 extract 300 μg / mL used in this test example, nobiletin is about 8.5 μM for Tachibana leaf 2 extract 300 μg / mL, Tachibana leaf 3 extract 300 μg / mL mL contained about 6.9 μM nobiletin.
As shown in FIG. 6A, Tachibana leaf-derived extract showed significant CRE transcriptional activity in hippocampal neurons compared to control and nobiletin.

[Test Example 11] CRE transcriptional activity of Tachibana leaf-derived extract in PC12D cells CRE transcription in PC12D cells using extracts extracted from Tachibana leaves 1 to 3 obtained in Example 2 by the method described herein The activity was compared with the control and nobiletin. The test procedure was the same as in Test Example 2. The result is shown in FIG. 6B.
In addition, nobiletin is about 4.9 μM for Tachibana leaf 1 extract 300 μg / mL used in this test example, nobiletin is about 8.5 μM for Tachibana leaf 2 extract 300 μg / mL, Tachibana leaf 3 extract 300 μg / mL mL contained about 6.9 μM nobiletin.
As shown in FIG. 6B, Tachibana leaf-derived extract showed significant CRE transcriptional activity in PC12D cells compared to control and nobiletin.

(Discussion 7)
From the results of the above test examples, the extract derived from the leaf of Tachibana of the present invention shows a remarkable CRE transcriptional activity that exceeds the activity predicted from the content of nobiletin, and is effective as a therapeutic agent for Alzheimer's disease and Parkinson's disease. There is expected.

Test Example 12 CRE Transcriptional Activity of Citrus Citrus Skin Extract in Hippocampal Nerve Cells Using the extract extracted from Citrus mandarin 1 obtained in Example 2 by the method described herein, CRE transcriptional activity in hippocampal neurons is controlled And compared with nobiletin. The test procedure was the same as in Test Example 2. The results are shown in FIG. 7A.
In addition, 100 μg / mL of the extract derived from 1 pericarp used in this test example contained about 3.4 μM nobiletin, and about 10.1 μM nobiletin in 300 μg / mL.
As shown in FIG. 7A, the extract derived from the citrus pericarp showed significant CRE transcriptional activity in hippocampal neurons as compared to the control and nobiletin.

(Discussion 6)
From the results of the above-mentioned test examples, the extract derived from the fruit peel of the present invention shows a remarkable CRE transcriptional activity exceeding the activity predicted from the content of nobiletin, and is expected to be effective as a therapeutic agent for Alzheimer's disease.

[Test Example 13] CRE transcriptional activity of the extract derived from Citrus unshiu peel in PC12D cells Using the extract extracted from Citrus unshimi 1 obtained in Example 2 by the method described herein, CRE transcriptional activity in PC12D cells was compared with control and nobiletin. And compared. The test procedure was the same as in Test Example 2. The result is shown in FIG. 7B.
In addition, about 3.4 μM of nobiletin was contained in 100 μg / mL of the extract derived from the fruit cuttlefish used in this test example.
As shown in FIG. 7B, the extract derived from the citrus pericarp showed significant CRE transcriptional activity in PC12D cells compared to the control and nobiletin.

[Test Example 14] TH transcriptional activity of Citrus pericarp peel-derived extract in PC12D cells Using the extract extracted from Citrus mandarin 1 obtained in Example 2 by the method described herein, TH transcriptional activity in PC12D cells was compared with control and nobiletin. And compared. The test procedure was the same as in Test Example 4-1. The result is shown in FIG. 7C.
In addition, about 3.4 μM of nobiletin was contained in 100 μg / mL of the extract derived from the fruit cuttlefish used in this test example.
As shown in FIG. 7C, the extract derived from Papilio mandarin peel showed significant TH transcriptional activity in PC12D cells compared to control and nobiletin.

(Discussion 7)
From the results of the above-mentioned test examples, the extract derived from Papilio mandarin peel of the present invention exhibits remarkable CRE transcription activity and TH transcription activity exceeding the activity predicted from the content of nobiletin, and is expected to be effective as a treatment for Parkinson's disease. Is done.

[Production Example] Production of Drug and Food of the Present Invention A drug or food containing the skin, tachibana peel or tachibana leaf of the present invention was produced by the following procedure.

[Production Example 1] Fine granules A
A total of 200 to 200 g in a ratio of 3.0 g of leather, 3.0 g of touki, 3.0 g of butterfly 3.0 g, 3.0 g of cucumber, 4.0 g of peanut, 4.0 g of peony, 2.0 g of psycho, 1.5 g of licorice and 5.0 g of hanger (total 28.5 g) Herbal medicine was blended to 800 kg and extracted (extracted can) with water at 2000 to 8000 L at 60 to 100 ° C. for 30 to 180 minutes. After filtration (centrifugation) at a rotational speed of 1000 to 5000 rpm for solid-liquid separation, the solution was concentrated (coil rotation type concentrator) to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less. The concentrate was spray-dried (spray dryer) at a rotational speed of 10,000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract preparation (Yokukansan Kasatsu Hanshin extract). . In the ratio of 2.9 g of an additive consisting of magnesium stearate, corn starch, lactose, pullulan and magnesium aluminate metasilicate to 6.1 g of the obtained yokukansan kachu semi-summer extract, the total amount is 50 to 400 kg. Mix in the range, mix at 4 rpm for 20 minutes (container rotating type mixer), mold at roll pressure 490-2500 Pa, size control (dry granulator), particles between sieves 30-50 Classification (cassette screen) was performed to obtain fine granules A.

  The obtained fine granule A 9.0 g contains 6.1 g of Yokukansanka-chukan semi-summer extract containing 3.0 g of dermis and other herbal medicines. Take 2 to 3 times a day.

[Production Example 2] Fine granule B
Tachibana bark 3.0 g, Betel loaf 4.0 g, Koboku 3.0 g, Keihi 3.0 g, Soyo 1.5 g, Licorice 1.0 g, Daio 1.0 g, Pepper 1.0 g, Mokko 1.0 g, Goshuyu 1.0 g and Bukkyo 3.0 g (total 22.5 g) Herbal medicines were blended so that the total amount was 200 to 800 kg, and extracted (extracted can) with water at 2000 to 8000 L at 60 to 100 ° C. for 30 to 180 minutes. After filtration (centrifugation) at a rotational speed of 1000 to 5000 rpm for solid-liquid separation, the solution was concentrated (coil rotation type concentrator) to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less. The concentrate was spray-dried (spray dryer) at a rotational speed of 10,000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract formulation (Kumisoto extract). In the ratio of 2.3 g of additive consisting of magnesium stearate, corn starch, lactose, pullulan and magnesium aluminate metasilicate to 3.7 g of the resulting Kumi miso extract, the total amount is in the range of 50 to 400 kg Mix at 4 rpm for 20 minutes (container rotation type mixer), mold at roll pressure of 490-2500 Pa, adjust particle size (dry granulator), and classify particles between sieves 30-50 (cassette Screen) to obtain fine granule B.

  The resulting fine granule B 6.0 g contains 3.7 g of Kumiso-to extract containing 3.0 g of Tachibana peel and other herbal medicines. Take 2 to 3 times.

[Production Example 3] Fine granule C
Chestnut 2.4 g, Butterflyfish 2.4 g, Hange 2.4 g, Bakumondou 2.4 g, Bukkuri 2.4 g, Carrot 1.6 g, Bow Fu 1.6 g, Kikuka 1.6 g, Licorice 0.8 g, Pepper 0.8 g and Gypsum 4.0 g (total 22.4 g) Herbal medicines were blended so that the total amount was 200 to 800 kg, and extracted (extracted can) with water at 2000 to 8000 L at 60 to 100 ° C. for 30 to 180 minutes. After filtration (centrifugation) at a rotational speed of 1000 to 5000 rpm for solid-liquid separation, the solution was concentrated (coil rotation type concentrator) to a concentration of about 10 to 40% under a reduced pressure of 8 kPa or less. The concentrated solution was spray-dried (spray dryer) at a rotational speed of 10,000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract formulation (Chitosan extract). The ratio of 1.52 g of additive consisting of hydrous silicon dioxide, magnesium stearate and corn starch is blended in the total amount of 50-400 kg to 4.48 g of the obtained Chotosan extract, 4 rpm, 20 minutes Mix (container rotating type mixer), mold at roll pressure 490-2500 Pa, adjust size (dry granulator), classify particles between sieves 30-50 (cassette screen), fine granule C was obtained.

  The resulting fine granule C 6.0 g contains 4.48 g of Chotosan extract containing 2.4 g of crust and other herbal medicines. As a guide, fine granule C 6.0 g 3 times a day for adults Take in divided doses.

[Production Example 4] Tea preparation The skin or tachibana peel was cut into 850 to 4750 μm squares, and 3 to 7 g of cut cut skin or tachibana peel was placed in a paper or cloth bag to obtain a tea preparation.
The obtained tea is leached 3 times a day (1 bag / time) for adults.

[Production Example 5] Herbal powder-fine granules D
Crude or tachibana peel was crushed (pulverizer) at a rotational speed of 2000-3500 rpm and an aperture of 0.5-3.0 mm, and further pulverized (pulverizer) at a rotational speed of 5000-8000 rpm, an aperture of 150 μm to obtain a crude drug powder. . The ratio of 9.2 g of the additive consisting of hydrous silicon dioxide, magnesium stearate and corn starch to 10 g of the crude or powdered citrus peel, and the total amount is in the range of 50 to 400 kg. Mix at 4 rpm for 20 minutes (container rotation type mixer), mold at roll pressure 490-2500 Pa, adjust the size (dry granulator), and classify particles between sieves 30-42 (cassette screen) As a result, a fine granule D was obtained.

  The obtained fine granule D 19.2 g contains 10 g of skin or tachibana peel, and as a guideline, the fine granule D 19.2 to 38.4 g is taken 2 to 3 times per day for an adult.

[Production Example 6] Decoction 10-30 g of decoction or tachibana bark was decocted with 200-600 mL of 20 times the amount of water at 100 ° C. for 1 hour to make half the water, and filtered to obtain a decoction.
The obtained decoction (100-300 mL) contains 10-30 g of decoction or tachibana peel, and as a guideline, 100-300 mL of decoction is divided into three doses per day for an adult.

[Production Example 7] Fine granule E
50 kg of peel or tachibana peel was extracted (extracted can) with water of 500 to 2000 L at 60 to 100 ° C. for 30 to 180 minutes. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. To 7.2 g of the obtained extract, blended in a ratio of 1.8 g of additive consisting of fine silicon dioxide and sucrose fatty acid ester in a total amount of 50 to 400 kg, and mixed for 20 minutes at 4 rpm Rotating mixer), molded at roll pressure of 490-2500 Pa, sized (dry granulator), classified between sieves 30-42 (cassette screen), and obtained fine granules E It was.

  The resulting fine granule E 9.0 g contains 7.2 g of a crust or tachibana peel extract equivalent to 20 g of crust or tachibana peel, and as a guideline, 2 g of fine granule E 9.0 g per day for adults. Take 3 divided doses.

[Production Example 8] Capsule 50 kg of peel or tachibana peel was extracted (extracted can) with 60 to 100 ° C for 30 to 180 minutes with 500 to 2000 L of water. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. 4.75 g of an additive consisting of magnesium aluminate metasilicate, synthetic aluminum silicate / hydroxypropyl starch / crystalline cellulose, corn starch, light anhydrous silicic acid, magnesium stearate and carmellose calcium per 10 g of the resulting extract Mix in a ratio of 50 to 400 kg, mix at a rotation speed of 4 rpm for 20 minutes (container rotation type mixer), mold at a roll pressure of 490 to 2500 Pa and size control (dry granulator) And particle classification (cassette screen) between sieves Nos. 30 to 42 to obtain fine granules. 400 mg of this fine granule was filled into a commercially available capsule usually used in this field (capsule filling machine) to obtain a capsule.

  The resulting 13 capsules (470-480 mg / piece) contain 3.6 g of a peel or tachibana peel extract equivalent to 10 g of peel or tachibana peel. As a guideline, 13 to 26 capsules are included. Take 2 to 3 times a day for adults.

[Production Example 9] Coated tablet 50 kg of peel or tachibana peel was extracted (extracted can) with 60 to 100 ° C for 30 to 180 minutes with 500 to 2000 L of water. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. In the ratio of 9.1 g of an additive consisting of maltose, lactose, magnesium aluminate metasilicate and magnesium stearate to 10 g of the obtained extract, the total amount is blended in the range of 50 to 400 kg, the rotation speed is 4 rpm, Mix for 20 minutes (container rotation type mixer), tablet at a rotational speed of 20-55 rpm, primary compression 0.6 mm or more, secondary compression 0.4 mm or more (high-speed rotary tableting machine), and 350 mg of 1 tablet I got a tablet. Dissolve or suspend 1.1 g of coating agent consisting of hydroxypropylmethylcellulose, talc, titanium oxide and caramel in water / ethanol mixture (3: 7-10: 0), exhaust temperature 40-55 ° C, flow rate 200-400 g / mL , Coating (coating machine) at a rotational speed of 4-6 rpm, polishing (coating machine) at a rotational speed of 1 rpm, exhaust temperature of 20-30 ° C using a small amount of carnauba wax and white beeswax, 1 tablet 370 mg Coated tablets were used.

  The obtained 20 coated tablets contain 3.6 g of crust or extract of Tachibana peel corresponding to 10 g of crust or tachibana peel, and as a guideline, 20 to 40 coated tablets 2 to 3 times a day for adults Take in divided doses.

[Production Example 10] Fine granule F
50 kg of peel or tachibana peel was extracted (extracted can) with water of 500 to 2000 L at 60 to 100 ° C. for 30 to 180 minutes. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. 10 g of the obtained extract is blended in a ratio of 15 g of an additive consisting of corn starch, maltose, hydrous silicon dioxide and magnesium stearate in a total amount in the range of 50 to 400 kg. Mix for a minute (container rotating type mixer), mold at roll pressure 490-2500 Pa, adjust the size (dry granulator), classify particles between sieves 30-42 (cassette screen), fine particles Agent F was obtained.

  The obtained fine granule F 4.5 g contains 1.8 g of a cinnamon extract or a citrus peel extract equivalent to 5 g of cinnamon or tachibana peel. Take 2 to 3 times.

[Production Example 11] Granule 50 kg of peel or tachibana peel was extracted (extracted can) with 60 to 100 ° C for 30 to 180 minutes with 500 to 2000 L of water. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. 10 g of the obtained extract is blended in a ratio of 40 g of additive consisting of calcium carmellose, hydrous silicon dioxide, magnesium aluminate metasilicate and magnesium stearate in a total amount of 50 to 400 kg and rotated. Mixing at several 4 rpm for 20 minutes (container rotating type mixer), molding at roll pressure 490-2500 Pa, sizing (dry granulator), particle classification between sieves 12-42 (cassette screen ) To obtain granules.

  18 g of the obtained granule contains 3.6 g of a crust or extract of tachibana which is equivalent to 10 g of crust or tachibana. As a guideline, 18 to 36 g of granule 2 to 3 times a day for adults Take in divided doses.

[Production Example 12] Jelly Agent 50 kg of peel or tachibana peel was extracted (extracted can) with 60 to 100 ° C. for 30 to 180 minutes with 500 to 2000 L of water. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. 10 g of the resulting extract, carrageenan, locust bean gum, reduced maltose liquid sugar, xylitol, apple fragrance, lecithin and citric acid additive 40 g in a total amount of 10-30 kg The mixture was mixed at a rotational speed of 50 to 150 rpm (mixing stirrer), and an equal amount of hot water of 80 ° C. or higher was added with stirring, and cooled to 15 ° C. or lower to obtain a jelly agent.

  18 g of the obtained jelly preparation contains 3.6 g of a crust or citrus peel extract equivalent to 10 g of crust or tachibana peel. As a guideline, 18 to 36 g of jelly preparation 2 to 3 times a day for adults Take in divided doses.

[Production Example 13] 50 kg of fine-grained peel or tachibana peel was extracted (extracted can) at 60 to 100 ° C. for 30 to 180 minutes with 500 to 2000 L of water. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. To the obtained extract 3.6 g, blended in a ratio of 0.9 g of additive consisting of fine silicon dioxide and sucrose fatty acid ester in a total amount of 50-400 kg and mixed for 20 minutes at 4 rpm Rotating mixer), molded at a roll pressure of 490-2500 Pa, sized (dry granulator), and classified between particles No. 30-42 (cassette screen) to obtain fine particles.

  The obtained 4.5 g of fine granules contain 3.6 g of a crust or extract of tachibana which is equivalent to 10 g of crust or tachibana, and as a guideline, ingest 4.5 g of fine per day for adults.

[Production Example 14] 50 kg of jelly skin or tachibana peel was extracted (extracted can) at 60 to 100 ° C for 30 to 180 minutes with 500 to 2000 L of water. After filtration (centrifugation) at a rotational speed of 1000 to 2500 rpm and solid-liquid separation, the solution was concentrated to a concentration of about 10 to 40% under reduced pressure of 8 kPa or less (coil rotation type concentrator). The concentrate was spray-dried (spray dryer) at a rotational speed of 10000 to 20000 rpm, an air supply temperature of 130 to 180 ° C., and an exhaust temperature of 60 to 120 ° C. to obtain an extract of crust or tachibana peel. Ratio of 15.9 g of additive consisting of lecithin, reduced maltose starch syrup, aspartame, ascorbic acid, trisodium citrate, gelling agent (FG-2266, Nitta Gelatin Co., Ltd.) and orange flavor to 10 g of the obtained extract Then, the total amount was blended in the range of 50 to 400 kg, and mixed at a rotational speed of 4 rpm for 20 minutes (container rotating type mixer) to obtain a jelly base.

  The resulting 9.3 g of jelly contains 3.6 g of cinnamon extract or tachibana peel equivalent to 10 g of cinnamon or tachibana skin. As a guideline, take 9.3 to 18.6 g of jelly element per day for adults. .

[Production Example 15] Tea Tachibana leaves were cut into 850 to 4750 μm squares, and 3 to 7 g of the cut tachibana leaves were put in a paper or cloth bag or used as tea.
As a guideline, drink the tea obtained in hot water in adults 3 to 5 times a day (1 bag or 3 to 7 g / time).

[Production Example 16] Peel 200 g of peel or tachibana peel and 2 g of salt were placed in water that was sufficiently immersed, covered and boiled for 15 minutes, then the hot water was discarded and cold water was passed through. Into a mixed solution in which 200 g of granulated sugar and 100 mL of water were mixed and boiled, the moistened skin or tachibana bark was added and boiled with occasional mixing until the skin became clear. It was boiled with occasional mixing so that it would not burn for 40-50 minutes on a very low heat without a lid. After removing the broth and thoroughly draining the water, it was taken out while it was hot, spread and dried. The obtained skin was coated with 20 g of granulated sugar to make a peel.
The obtained peel 21.1 g contains 10 g of cinnamon or tachibana peel, and as a guideline, ingest 21.1 to 42.2 g of peel per day for adults.

[Production Example 17] 100 mL hot water (100 ° C) was applied to 100 g jam jam or tachibana peel, steamed in a container overnight, and pulverized with a mixer. 1 L of water was added and simmered on high heat for 1 hour 30 minutes. After adding 25-30 g of granulated sugar and simmering for 10-15 minutes on medium heat, it was set to low heat and 25-30 g of granulated sugar was added and dissolved. Furthermore, 25-30 g of granulated sugar was added and dissolved, and after cooling to 70 ° C. or lower, 60 g of honey, 1.7 g of pectin and 1.7 g of ascorbic acid were added and stirred to form a jam.
100 g of the obtained jam contains 20 g of crust or tachibana bark and takes 25-50 g of jam per adult as a guide.

[Production Example 18] Tea 30 kg of leaf of Tachibana was extracted (extracted can) with 300 L of water at 60-100 ° C for 5-30 minutes. Filtration (centrifugation) was performed at a rotational speed of 1000 to 2500 rpm for solid-liquid separation, and 0.2 g / L ascorbic acid was added to the filtrate to obtain tea.
The obtained tea 500 mL contains 5 g of Tachibana leaves, and as a guideline, ingest 500 mL of tea per day for adults.

  The dried plant tissue or plant tissue extract of the present invention is useful as a pharmaceutical or food for improving central neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and is also used as a component of a promising fundamental therapeutic agent for the disease Is possible. The dried plant tissue or plant tissue extract of the present invention is particularly useful for memory acquisition, retention and enhanced recall ability.

Claims (7)

Nobiletin is contained in an amount of 0.6 to 3.0% by weight, naryltin is contained in an amount of 0.4% by weight or less, tangeretin is contained in an amount of 0.1 to 1.0% by weight, and hesperidin is contained in 100% by weight of the plant tissue extract. Of citrus plant tissue extract containing 2.0 to 14.0% by weight and nobiletin / nariltin content ratio of 2.0 to 14.0. A pharmaceutical product for enhancing or promoting dopamine synthesis in Parkinson's disease, increasing dopamine content or promoting dopamine secretion,
A pharmaceutical product wherein the citrus fruit is Citrus reticulata blanco (Lutacy), Tachibana or Giant orange.   The pharmaceutical product according to claim 1, wherein the citrus fruit is Tachibana or Obenimikan.   3. A pharmaceutical product according to claim 1 or 2 for enhancing the ability to acquire memory.   The pharmaceutical product according to any one of claims 1 and 2, for promoting dopamine synthesis ability, increasing dopamine content, or promoting dopamine secretion.   The citrus plant tissue extract is obtained by water extraction from a blend obtained by blending dried citrus plant tissue and a pharmaceutically acceptable herbal medicine component in a weight ratio of 1: 5 to 1:10. The pharmaceutical according to any one of 1 to 4.   The pharmaceutical product according to any one of claims 1 to 5, which is in the form of a fine granule, tea, decoction, capsule, tablet, granule, jelly, powder, solution, syrup or extract.   Herbal medicine ingredients are Ireisen, Uyaku, Engosaku, Ogi, Ogon, Oubak, Onji, Cuckoo, Cuckoo, Kyokyo, Kanzo, Kyoku, Kikuka, Kijitsu, Kyonin, Keihi, Koka, Koubushi, Kokuboku, Goshitsu, Goshiyu, Ganoderma , Jiou, peonies, ginger, ginger, ginkgo, gypsum, senkou, zenko, suzutsu, soboku, soyo, diou, taisou, daifukuhi, takusha, chikujo, chimo, butterfly, tenma, tenmondo, touki, tonin, carrot, cucumber , Bakumondo, Hange, Scotch, Sandalwood, Betel Shrimp, Bukryo, Bowie, Bowshaw, Bowfish, Buttonpi, Maou, Mokutsu, Mokko, Yakumosou, Ryutan and Wakakatsu Is one or more herbal components selected from the group, according to claim 5 pharmaceuticals according.

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