JP5634066B2 - Salivary secretion promoter - Google Patents

Description

  The present invention relates to a saliva secretion promoter, particularly a saliva secretion promoter having a form used in the oral cavity (form for oral cavity) or a form used by oral administration (oral administration form).

  Saliva plays an important role in maintaining the oral environment and oral function. Examples of oral functions include functions that facilitate conversation and vocalization and food intake functions. Particularly in the food intake function, saliva is digestive activity such as bolus formation and digestive enzyme action, solubilization of taste substances, and carbonates.・ It is deeply involved in the taste maintaining effect by dehydrogenase secretion. In the oral environment maintenance function, saliva is deeply involved in the self-cleaning action of teeth and oral cavity, tooth remineralization action, antibacterial action, immune action, anti-inflammatory action, and tissue repair promoting action by growth factors.

  However, various diseases such as emotional and stress disorders, neuropathy, organ disorders, encephalitis, tumors, cerebrovascular disorders, hypertension, Graves' disease, and diabetes, side effects of drugs, radiation therapy, and aging, etc. It is known that the amount of secretion decreases. In particular, in addition to the decrease in salivary gland function due to aging, many elderly people complain of dryness in the oral cavity due to a decrease in salivary secretion due to multiple chronic diseases and their therapeutic agents.

  The decrease in saliva secretion causes dryness in the oral cavity, resulting in difficulty in chewing and swallowing and a decrease in digestive function. In addition, it may cause discomfort in the oral cavity and generation of bad breath. Further progression of symptoms may cause periodontal diseases and oral infections such as stomatitis. Therefore, it is required to increase the saliva secretion amount of the patient by some means.

As means for increasing the secretion amount of saliva, a method of giving a taste taste or a method of giving a sense of smell using a sour taste has been proposed (see, for example, Patent Documents 1 and 2). In addition, a method using a plant extract having a salivary secretion promoting action (see, for example, Patent Documents 3 to 6), a drug targeting muscarinic receptors (Patent Document 7), and a drug targeting PAR-2 A method using (Patent Document 8) has also been proposed.
Japanese Patent Laid-Open No. 7-101856 JP 2003-40752 A JP 11-71253 A Japanese Patent Laid-Open No. 10-182392 JP 2002-265375 A JP 2005-162633 A JP-A-8-12575 JP 2001-64203 A

  An object of this invention is to provide a salivary secretion promoter. Preferably, an object of the present invention is to provide a saliva secretion promoter that can selectively promote saliva secretion when saliva secretion is low. More preferably, an object of the present invention is to provide a salivary secretion promoter capable of promoting saliva secretion for a long time and healing dry mouth.

  The inventors of the present invention have made extensive studies to solve the above problems, and that the black bean solvent extract has a salivary secretion promoting action. In particular, the black bean solvent extract has a saliva amount as described above. We found that there is an ideal action to selectively promote saliva secretion when saliva secretion is low rather than when it is high, and by further using potassium in combination with the black bean extract, promotion of saliva secretion of black bean extract It has been found that the action is sustained and a high saliva secretion amount can be maintained over a long period of time. In addition, the present inventors have conducted extensive studies aiming to isolate and identify an active ingredient having a salivary secretion-promoting action. As a result, the effective ingredient is Bowman-Birk Protease Inhibitor (Bomann-Birk protease protease) having a molecular weight of about 8 kDa. Inhibitor) (hereinafter also referred to as “BBI”).

  Based on these findings, the present inventors have found that a composition containing such BBI and a plant extract fraction containing the BBI (for example, a fraction having a molecular weight of 6-13 kDa), or a composition containing a combination of these in combination with potassium is saliva. It was confirmed that the present invention was completed by confirming that it is useful as a salivary secretion promoter that suppresses oral dryness caused by a decrease in the secretion amount and a decrease in oral function and oral environment maintenance function due to this. .

That is, the present invention includes the following embodiments:
(I) Salivary secretion promoter (I-1 ) A salivary secretion promoter containing Bowman-Birk Protease Inhibitor (BBI) as an active ingredient.

(I-2) The salivary secretion promoter according to (I-1), wherein BBI is a protein represented by the following (a) or (b):
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1,
(B) A protein having one or more amino acids deleted, substituted or added in SEQ ID NO: 1, and having trypsin inhibitory activity, chymotrypsin inhibitory activity, and salivary secretion promoting action.

  (I-3) The salivary secretion promoter according to (I-1), comprising a plant extract containing BBI.

  (I-4) The salivary secretion promoter according to (I-3), wherein the plant extract is a plant extract containing a fraction in the range of 6 to 13 kDa centered on a molecular weight of 8 kDa.

  (I-5) Extraction of a plant belonging to any one of the above-mentioned plant extracts selected from the group consisting of legumes, eggplants, gramineae, scallops, pineapples, leeks, buttercups, and crucifers The salivary secretion promoter described in (I-3) or (I-4), which is a product.

  (I-6) The salivary secretion promoter according to (I-3) or (I-4), wherein the plant extract is derived from seeds of a plant belonging to the leguminous family.

  (I-7) The salivary secretion promoter according to (I-3) or (I-4), wherein the plant extract is derived from seeds of a plant belonging to the leguminous soybean genus.

  (I-8) The salivary secretion promoter according to any one of (I-1) to (I-7), further containing potassium.

  (I-9) The salivary secretion promoter according to any one of (I-1) to (I-8), which has a form for oral administration or oral administration.

  (I-10) Syrup, gel, spray, chewable tablet, film, troche, tablet, capsule, pill, granule, powder, toothpaste, salmon, jelly, gum or beverage The salivary secretion promoter according to any one of (I-1) to (I-8).

(II) Method for preparing saliva secretion promoter (II-1) Saliva secretion promoter according to any one of (I-1) to (I-10), which comprises the following steps (1) and (3): Preparation method:
(1) A step of collecting a fraction containing BBI from a plant containing BBI, and (3) a step of processing the BBI-containing product obtained above into a form for oral or oral administration.

(II-2) A method for preparing a salivary secretion promoter according to any one of (I-8) to (I-10), which comprises the following steps (1) to (3):
(1) collecting a fraction containing BBI from a plant containing BBI;
(2) A step of preparing potassium-containing BBI-containing material obtained in step (1) to prepare a composition containing BBI and potassium, and (3) oral or oral use of the composition obtained above. Process to form for administration.

  (II-3) The method according to (II-1) or (II-2), wherein the fraction containing BBI is a fraction in the range of 6 to 13 kDa centered on a molecular weight of 8 kDa.

  (II-4) The plant is a plant belonging to any family selected from the group consisting of legumes, eggplants, gramineae, scallops, pineapples, leeks, buttercups, and crucifers, The method according to any one of II-1) to (II-3).

  (II-5) The method according to any one of (II-1) to (II-3), wherein the plant is a seed of a plant belonging to the leguminous family.

  (II-6) The method according to any one of (II-1) to (II-3), wherein the plant is a seed of a plant belonging to the leguminous soybean genus.

(III) Method for promoting saliva secretion (III-1) having an effective amount of the saliva secretion promoter described in any of (I-1) to (I-10), orally ingesting the subject, A method of promoting saliva secretion in the subject.

  (III-2) The method according to (III-1), wherein the subject is a patient with hyposalivation or xerostomia.

(IV) Use as a saliva secretion agent (IV-1) Use of BBI or a plant extract containing BBI as a saliva secretion promoter.

  (IV-2) Use of a composition containing potassium in addition to BBI or a plant extract containing BBI as a salivary secretion promoter.

  (IV-3) The use according to (IV-1) or (IV-2), wherein the plant extract is a plant extract containing a fraction in the range of 6 to 13 kDa centered on a molecular weight of 8 kDa.

  (IV-4) Extraction of plants belonging to any one of the above-mentioned plant extracts selected from the group consisting of legumes, cucurbitaceae, gramineae, scallops, pineapples, leeks, buttercups, and crucifers The use according to any one of (IV-1) to (IV-3), which is a product.

  (IV-5) The use according to any one of (IV-1) to (IV-3), wherein the plant extract is derived from a seed of a plant belonging to the leguminous family.

  (IV-6) The use according to any one of (IV-1) to (IV-3), wherein the plant extract is derived from seeds of a plant belonging to the leguminous soybean genus.

  According to the saliva secretion-promoting agent of the present invention, by promoting the secretion of saliva, the self-cleaning action in the oral cavity is enhanced, and various adverse effects caused by the decrease in the secretion of saliva (for example, dryness in the oral cavity, intestinal defects) Pleasure, taste disorder, bad breath, dental caries, periodontal diseases, oral dysfunction such as mucosal infection, difficulty in speaking and speaking, etc.) can be prevented. In particular, the saliva secretion-promoting agent of the present invention does not exhibit much effect when the amount of saliva secreted is large, and has an ideal action that selectively exerts an effect when the amount of saliva secreted decreases.

  Further, the salivary secretion promoting agent of the present invention containing potassium is improved in the sustainability of the salivary secretion promoting action by using potassium in combination with BBI.For this reason, the promotion of salivary secretion is maintained for a long time. Occurrence of the above adverse effects can be prevented over a long period of time.

  Moreover, since the salivary secretion promoter of the present invention can be used as a material for plants having experience of eating such as soybeans, especially black beans, it is highly safe and is used continuously in the oral cavity or taken orally. Or a composition for oral administration (including pharmaceuticals, quasi-drugs, and foods). The salivary secretion promoter of the present invention can also be used in medicine as a pharmaceutical, but can be suitably used in alternative medicine or complementary and alternative medicine as a functional food or health food.

(I) Bowman-Birk Protease Inhibitor
Bowman-Birk Protease Inhibitor (hereinafter referred to as “BBI”) is a soluble protein known as a serine protease inhibitor, and it is known that 14 cysteine residues form disulfide bonds to form a total of 7 loops. (Journal of Biology, 2001, Vol.3, pp164-173). BBI has trypsin inhibitory activity and chymotrypsin inhibitory activity, and these active sites are known to be located in amino acid regions of different loops (J. Biol Chem. (1992) 267, 1990- 1994; Eur. J. Biochem. (1993) 212,549-555; Eur. J. Biochem. 242, 122-131).

  Such BBI is known to be contained in the plants listed below.

(1) Leguminosae (Leguminosae) plant legume genus (Canavalia lineata), soybean genus (Glycine max, Glycine microphylla), cowpea genus (mung bean: Vigna radiata var. Radiata, Vigna trilobata, red cowpea: Vigna vexillata 、 Azuki bean: Vigna angularis 、 Black-breasted bean: Vigna mungo 、 Alaska: Vigna marina, Cowpea: Vigna unguiculata) : Phaseolus polyanthus, Tepary bean: Phaseolus acutifolius, Phaseolus filiforms, Phaseolus wrightii, Phaseolus glabellus, Phaseolus microcarpus, Phaseolus augusti, Phaseolus oligosperus, Phaseolus maculatus, Phaseolus hintonii, Phaseolus colinus, Phaseolus zimapanus, Phaseolus zimapanus, Phaseolus zimapanus ), Genus (Apios: Apios americana), genus Umanago (Medicago scutellata, Medicago sativa), lentil (Lens culinaris, Lens culinaris subsp. Tomentosus, Lens culoderis subs. .orientalis, Lens nigricans, Lenservoides, Lens lamottei), broad bean genus (broad bean: Vicia faba, yellow pea: Vicia sativa subsp. nigra), deigo (deigo: Erythrina variegata), Dioclea genus (Fuji bean: Dolichos glabra, Dioclea) Plants belonging to the genus Amburana (Amburana acreana, Amburana cearensis), the genus Ronchocarpus capassa, or the genus Lupinus albus.

(2) Plants belonging to the family Gramineae (rice: Oryza sativa), barley (barley: Hordeum vulgar), wheat (Wheat: Triticum aestivum), corn (Zea mays), butter sorghum (Zea diploperennis), A plant belonging to the genus Sphagnum (Staria italica), or the genus Coix lacryma-jobi.

(3) A plant belonging to the genus Solanum tuberosum belonging to the family Solanum.

(4) A plant belonging to the genus Musa acuminata belonging to the family Salamaceae.

(5) Plants belonging to the genus Ananas comosus belonging to the pineapple family .

(6) A plant belonging to the genus Allium cepa belonging to the family Allium.

(7) A plant belonging to the genus Oren (Coptis japonica) belonging to the family Ranunculaceae .

(8) Plants belonging to the genus Arabidopsis thaliana belonging to the family Brassicaceae.

  Among the above-mentioned plants, the amino acid sequence of BBI derived from soybean which is a plant belonging to the leguminous soybean genus is shown in SEQ ID NO: 1. The soybean-derived BBI is an acidic protein having a molecular weight of about 8 kDa and an isoelectric point of about 4.5. Such BBI is first translated as a precursor consisting of 110 amino acids (SEQ ID NO: 2), but is cleaved at the N-terminus in the process of being transported to each organ to become a mature protein (mature protein). The amino acid sequence shown in SEQ ID NO: 1 is a mature BBI present in soybean seeds. The amino acid sequence of the BBI precursor immediately after translation and the base sequence of the gene encoding it are registered in SWISS-Prot access number P01055 and NCBI GeneID548083, respectively.

  As described above, BBI is contained in various plants, and its amino acid sequence is known to have a homology of usually about 20 to 60% between biological species. In particular, a cysteine-rich region from the first cysteine residue to the 14th last cysteine residue has about 50 to 60% homology between species. Regarding BBI amino acid sequences contained in plant species other than soybean, for example, the amino acid sequence of BBI derived from azuki bean is SWISS-Prot access number P01058, and the amino acid sequence of BBI derived from peanut is SWISS-Prot access number P01066. It is registered.

  BBI is known to have a plurality of isoforms. Specifically, in such an isoform, in the mature BBI amino acid sequence shown in SEQ ID NO: 1, at least one of the N-terminal and C-terminal amino acid residues is at most 10, preferably at most, Those in which the amino acid residues from the N-terminal or C-terminal to the first disulfide bond site are cleaved are included. For example, it is known that 9-10 amino acid residues on the C-terminal side of BBI are removed by drying soybean seeds. In addition, since the trypsin inhibitory active site and chymotrypsin inhibitory active site of BBI exist on the loop, the cleavage of amino acid residues as described above does not affect the trypsin inhibitory activity and chymotrypsin inhibitory activity of BBI at all.

  BBI can be prepared by using the above-mentioned plant as a raw material and isolating and purifying it from the above using trypsin inhibitory activity, chymotrypsin inhibitory activity, molecular weight (about 8 kDa), isoelectric point (pI about 4.5), and the like. For example, as a method for isolating and purifying BBI from black beans, Experimental Example 2 described later can be referred to. BBI can also be produced by genetic engineering techniques based on the genetic information (see, for example, JP-A-2007-528212).

(II) Salivary secretion promoter The salivary secretion promoter of the present invention is characterized by containing the aforementioned BBI as an active ingredient.

  Here, the origin of BBI is not particularly limited, and plants containing BBI, such as the above-mentioned legumes, gramineous plants, solanaceous plants, squash plants, pineapple plants, leek plants, buttercups, etc. It may be derived from either a plant or a cruciferous plant. BBI derived from legumes, more preferably plants belonging to the leguminous soybean genus, particularly soybeans and black soybeans. As shown in Experimental Example 1, these legumes contain a large amount of BBI in their seeds.

  The saliva secretion-promoting agent of the present invention preferably contains BBI derived from soybean seeds, more preferably BBI derived from black bean seeds.

Here, black beans are black seeds (grains) (black soybeans) of short-day annual plants belonging to the leguminous soybean species Glycine max (L.) Merrill. Black beans include, for example, varieties such as Meiko Koguro, Tokachikuro, Iwakuro, Tamadaguro, Tamba Black, Shinano Black, and Goku, but seeds of any variety may be used as long as they are black beans.

  Specific examples of the BBI include a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

  As described above, since BBI has a plurality of isoforms, the BBI used in the present invention may be an isoform of the above protein. Examples of such isoforms include those in which one or more amino acids are deleted in SEQ ID NO: 1, have trypsin inhibitory activity and chymotrypsin inhibitory activity, and have a salivary secretion promoting action. As described above, the region in which one or a plurality of amino acids are deleted is a region consisting of a maximum of 10 amino acids from the N-terminus and C-terminus of BBI, preferably the N-terminus or C-terminus. The amino acid region from the terminal to the first disulfide bond site.

  As described above, since both the trypsin inhibitory active site and chymotrypsin inhibitory active site of BBI exist on the loop formed by disulfide bonds, amino acid residues in the amino acid region from the N-terminal or C-terminal to the first disulfide bond site Substitution of amino acids or addition of amino acid residues does not affect the trypsin inhibitory activity and chymotrypsin inhibitory activity of BBI. Therefore, BBI used in the present invention has one or more amino acids substituted or added in the amino acid region of SEQ ID NO: 1, has trypsin inhibitory activity and chymotrypsin inhibitory activity, and has a salivary secretion promoting action. It may be a thing.

  Here, the trypsin inhibitory activity of BBI is that trypsin mixed with BBI in advance using BAEE (N-Benzoyl-L-Arginine Ethyl Ester), BAPNA (N-Benzoyl-L-Arginine 4-nitroanilide), or casein as a substrate. It can be evaluated from the reaction inhibition rate due to BBI when the enzyme reaction is carried out (see, for example, Sigma's recommended protocol SIGMA QUALITY CONTROL TEST, Enzymatic Assay of TRYPSIN INHIBITOR). Moreover, chymotrypsin inhibitory activity can be evaluated from the reaction inhibition rate by BBI when BTEE (N-Benzoyl-L-Tyrosine Ethyl Ester) is used as a substrate and trypsin is mixed with BBI in advance. (For example, see literature name Planta (2007) 226: 73-85). Furthermore, the salivary secretion promoting action of BBI can be evaluated by any of the methods described in Experimental Examples 1 to 3 described later.

  The salivary secretion promoter of the present invention is desirably prepared so that the BBI intake amount is 0.02 to 0.25 mg, preferably 0.036 to 0.18 mg. In addition, the saliva secretion promoter of this invention can be used suitably when the amount of saliva falls or when it is desired to increase saliva secretion. For this reason, the number of times per day of the saliva secretion promoter of the present invention is not limited, and the saliva secretion promoter containing the above-mentioned amount of BBI is taken once or a plurality of times a day, continuously or intermittently. be able to.

  The salivary secretion promoter of the present invention may contain BBI in a purified state, but is not limited thereto, and may contain BBI in a crudely purified state. Examples of such crudely purified BBI include BBI-containing extracts prepared from the above-mentioned plants containing BBI (hereinafter referred to as “BBI-containing extracts”). In addition, it contains the above BBI as an indicator of BBI activity (trypsin inhibitory activity, chymotrypsin inhibitory activity), molecular weight (about 8 kDa), isoelectric point (about pI4.5), etc. so as to contain more BBI of higher purity A processed product obtained by further purifying the extract (hereinafter referred to as “BBI-containing processed product”) can also be used. The “plant extract containing BBI” targeted by the present invention in the claims includes both the BBI-containing extract and the BBI-containing treated product. BBI-containing extracts or BBI-containing processed products prepared from legume seeds, more preferably seeds of plants belonging to the leguminous soybean genus, particularly preferably soybean or black bean seeds are preferred.

  Examples of the extraction solvent used for the preparation of the BBI-containing extract include water, a polar organic solvent, or a mixture thereof. Examples of the polar organic solvent include alcohol, acetone, ethyl acetate and the like. Among these, water, lower alcohol, or a mixture thereof can be used as the extraction solvent from the viewpoint of safety to the human body and handleability. Examples of the lower alcohol include lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropyl alcohol and butanol, preferably ethanol. The extraction solvent is preferably water or hydrous alcohol (particularly hydrous ethanol). In addition, when using hydrous alcohol (especially hydrous ethanol) as a solvent, it is preferable that the amount of alcohol (ethanol) contained in it is 40 volume% or less.

  The extraction solvent may be prepared in any of acidic, neutral and alkaline ranges.

  As the extraction method, a generally used method can be employed. Although it is not limited, for example, a method of immersing a plant (preferably soybean seed) that has been raw or dried in a solvent (as it is, or a crude powder, shredded product) under low temperature, normal temperature, warming or boiling conditions; Examples of the method include extraction with stirring under low temperature, normal temperature, heating, or boiling conditions; a supercritical extraction method using carbon dioxide gas; or a percolation method. Preferably, a method of extracting a dried plant, preferably soybean seeds (as-is, or crude powder, shredded product) in a solvent under high temperature or boiling conditions and stirring as necessary (Heat treatment extraction method).

  The obtained extract can remove solids (residues) unnecessary for the solvent by various solid-liquid separation means such as filtration, coprecipitation, centrifugation, and pressing as required. Further, the obtained extract can be further freeze-dried or concentrated to prepare a powdery or pasty extract (concentrated extract).

  Although not limited, the extract obtained above can be further subjected to an enzyme treatment or a treatment for fractionating a desired molecular weight fraction (molecular weight fractionation treatment). These processes can be performed in combination. In the present invention, the desired molecular weight fraction is a fraction having a molecular weight in the range of 6000 to 13000 Da centering on a molecular weight of 8000 Da.

  Here, examples of the enzyme used for the enzyme treatment include an enzyme having an action of degrading a sugar or / and a protein. Specifically, for example, amylases such as Christase Y (manufactured by Yamato Kasei) and Sumiteam S (manufactured by Shin Nippon Chemical Industry); cellulase A (manufactured by Amano Enzyme), and cellulosin AC40 ( Cellulases such as HBI); Sucrase S (manufactured by Sankyo) and hemicellulases such as Sumiteam X (manufactured by Shin Nippon Chemical Industry) can be used. Examples of the latter proteolytic enzymes include acidase proteases such as Newase A (manufactured by Amano Enzyme) and Protease YP-SS (manufactured by Yakult Pharmaceutical Co., Ltd.); Neutral proteases such as Tokasei Kogyo Co .; enzymes having proteolytic activity such as alkaline proteases such as Novozymes FM (Novozymes) and Biolase SP-4FG (Nagase ChemteX) can be used.

  Such an enzyme having a saccharide-degrading activity and an enzyme having a proteolytic activity may be used singly or in combination. The enzyme to be used may be a single enzyme, but various enzymes may be used in combination. A preferred enzyme is an enzyme having a saccharide-degrading activity, and in particular, an enzyme having an activity of degrading a polysaccharide such as the above-mentioned cellulase, amylase, hemicellulase or the like can be suitably used.

  The enzyme treatment can be carried out under predetermined conditions suitable for each enzyme used. The temperature condition for performing the enzyme treatment is not particularly limited, but a range of 30 to 80 ° C. can be usually used. Preferably it is 35-60 degreeC.

  Examples of the fractionation treatment with a molecular weight of 6000 to 13000 Da include a gel filtration method, a membrane separation method, and an adsorption treatment method. A membrane separation method is preferred. The membrane separation method used here is not particularly limited as long as it can obtain a fraction having a molecular weight of 6000 to 13000 Da centering on a molecular weight of 8000 Da as a result from a solvent extract of a plant. BBI has been reported to form a dimer by polymerization or reassociation (literature name: The Journal of Biological Chemistry, Vol. 279, No. 29, Issue of July 16, pp. 30425- 30432, 2004). For this reason, in order to efficiently recover even such a dimer, it is preferable to employ a fractionation processing method for obtaining a fraction having a molecular weight of 6,000 to 26,000 Da as a result. In this case, specifically, a membrane in which the set cutoff value of the molecular weight cut-off is, for example, in the range of 26,000 Da ± 10%, preferably in the range of 18,000 Da ± 10%, more preferably in the range of 13,000 Da ± 10%. And a treatment method for separating and removing a high molecular weight compound and a treatment method for separating and removing a low molecular weight compound using a membrane having a set cutoff value in the range of 6,000 Da ± 10%, for example. preferable.

  As a method for separating and removing the former polymer compound, specifically, NTU-3150 membrane, NTU-3250 membrane, NTU-3550 membrane, NTU-3800 UF membrane (Nitto Denko) ); Cefilt-UF (manufactured by NGK); AHP-2013 membrane, AHP-3013 membrane, AHP-1010 membrane (above, manufactured by Asahi Kasei); . As a method for separating and removing the latter low molecular weight compounds, specifically, NTR-7250 membrane, NTR-7410 membrane, NTR-7430 membrane, NTR-7450 membrane (Nitto Denko) ), AIP-3013 membrane, ACP-3013 membrane, ACP-2013 membrane, AIP-2013 membrane, AIO-1010 membrane (above, manufactured by Asahi Kasei) and other reverse osmosis (RO) membrane treatment A filtration (UF) membrane treatment can be mentioned.

  Examples of the membrane material used for the membrane separation method include cellulose, cellulose diacetate or triacetate, polyamide, polysulfone, polystyrene, polyimide, polyacrylonitrile and the like, regardless of whether they are natural, synthetic, or semi-synthetic. . In addition, as a membrane used for the fractionation treatment, a microfiltration (MF) membrane or the like can be used in addition to the ultrafiltration membrane and the reverse osmosis membrane.

  The above enzyme treatment and molecular weight fractionation treatment can be carried out alone or in combination with respect to the aforementioned plant extract (BBI-containing extract) or its treated product (BBI-containing treatment). Moreover, when performing combining both, an enzyme process and a molecular weight fractionation process can be performed combining arbitrarily arbitrarily continuously or discontinuously. The order of the combination is not particularly limited, and any of the order of molecular weight fractionation treatment following the enzyme treatment and the order of molecular weight fractionation treatment followed by the enzyme treatment may be used, but the former method is preferred. In addition to the above enzyme treatment and fractionation treatment with a molecular weight of 6000 to 13000 Da, the plant extract or its treatment product is further subjected to normal purification such as acid treatment, extraction treatment, adsorption treatment, ion exchange treatment or membrane treatment. It is also possible to perform the processing used in These treatments can be performed on a plant extract (BBI-containing extract) or a treated product thereof (BBI-containing treatment product), either alone or in combination of two or more.

  The salivary secretion-promoting agent of the present invention is 0.126 to 1.55 g, preferably 0.22 to 1.12 g, as a water-soluble solid content of the starting plant of the BBI-containing extract or BBI-containing processed product prepared according to the above method. It is desirable to adjust so that it can be administered (taken) at a rate. At this time, it is desirable to adjust so that BBI can be administered (taken) at a rate such that BBI is 0.02 to 0.25 mg, preferably 0.036 to 0.18 mg per time in terms of the amount of BBI.

  In addition to the BBI or BBI-containing extract or BBI-containing treatment product, potassium can be added to the salivary secretion promoter of the present invention. The proportion of potassium compounded in the saliva secretion promoter is not particularly limited, but is 1 to 100 mg, preferably 1.5 to 50 mg as the amount of potassium ion per saliva secretion promoter (per dosage form) taken once. Can be mentioned.

  Here, the blending of potassium is usually performed by using a potassium-containing material. Examples of the potassium-containing material include those containing a potassium salt. Examples include inorganic compounds such as potassium chloride, potassium carbonate, dipotassium phosphate, tripotassium phosphate, potassium gluconate, tripotassium citrate, potassium glutamate, and potassium hydrogen tartrate; and whey salt, seaweed ash extract And compositions such as buckwheat ash extract, crude seawater potassium chloride, brine lake water sodium chloride and the like.

  The saliva secretion-promoting agent of the present invention can also be blended with other components such as a fragrance, a coloring agent, a refreshing agent, an antioxidant, a sweetener, or an antibacterial agent, as long as the effects of the present invention are not impaired.

  The saliva secretion promoter of the present invention is not particularly limited in its shape, and can be prepared, for example, in a liquid form and a semi-solid or solid form.

  A salivary secretion promoter having a liquid form can be prepared using water (purified water, ionic water, distilled water, physiological saline, etc.) and a pharmaceutically acceptable solvent such as ethanol or a food solvent. If necessary, a viscosity imparting agent is used to adjust the viscosity to 50 mPa · s or more, preferably 50 to 36,000 mPa · s. More preferably, it is 60-10000 mPa * s, More preferably, it is 90-8000 mPa * s.

  Here, the viscosity is a BL type rotational viscometer (manufactured by Toki Sangyo Co., Ltd., rotor No. 1 up to a viscosity of 450 mPa · s, viscosity 450-2250 mPa · s is rotor No. 2, viscosity 2250 under the condition of 20 ° C. ˜9000 mPa · s rotor No. 3, viscosity of 9000 mPa · s or higher means the viscosity obtained when measured at a rotational speed of 12 rpm using rotor No. 4). In the entire specification, “viscosity” means a viscosity measured under such conditions.

  Here, the viscosity-imparting agent is not limited, but the viscosity of the liquid composition containing a water-soluble solid content derived from the raw material plant in a proportion of 4 to 20% by weight is 50 mPa · s or more, preferably 50 to 36,000 mPa · s. Those that can be adjusted to s are preferred.

  For example, locust bean gum, tamarind seed gum, pullulan, xanthan gum, guar gum, tara gum, rhamzan gum, mannan and other gums; starch (hereinafter collectively referred to as “polysaccharide”); monosaccharides such as glucose and fructose Disaccharides such as sucrose, lactose and trehalose; oligosaccharides consisting of three or more sugars; polysaccharides such as inulin and polydextrose; chickenpox; sugar alcohols such as sorbitol, mannitol, maltitol, erythritol, lactitol, xylitol, glycerin; A mixture of sugars such as molasses, honey, fructose dextrose liquid sugar, reduced maltose starch syrup, reduced starch hydrolyzate, starch syrup, etc. (these are also collectively referred to as “saccharides”). These can be used alone or in any combination of two or more.

  Preferably, in polysaccharides such as locust bean gum, tamarind seed gum, pullulan, guar gum, inulin, and polydextrose; and sugars such as molasses, honey, fructose dextrose sugar, reduced maltose starch syrup, reduced starch hydrolyzate and starch syrup Yes, more preferably sugars.

  Further, the pH of the liquid saliva secretion promoter is not limited, but is usually preferably in the range of pH 5 to 9, more preferably pH 5 to 8, and further preferably pH 5 to 7.5.

  These liquid saliva secretion promoters are in the form of solutions, sprays, drops, or discharges (oral compositions such as mouthwashes, liquid toothpastes, gargles, and oral sprays, and oral administration of syrups, etc. The composition can be prepared in a conventional beverage form. The salivary secretion promoter having the above various forms can be used in a state filled in various containers such as bottles, pumps, sprays, tubes, cans, and bottles according to the form.

  The salivary secretion promoter having a semi-solid or solid form is not particularly limited as long as it has a form used in the oral cavity (oral form) or a form used by oral administration (oral administration form). For example, it can be in the form of powder, tablet, granule, pill, troche, syrup, chewable tablet, film, gel, dry syrup and the like. Moreover, you may have the form of a candy (a hard candy, a soft candy, a gummy is included), jelly, or gum. A semi-solid or solid composition having such a form can be prepared according to conventional methods in the art.

  The saliva secretion-promoting agent of the present invention may be formulated with various carriers and additives that are pharmaceutically acceptable for oral administration in order to prepare the oral form or oral dosage form as described above and to stabilize it. (For example, refer to the Pharmacopoeia or “Pharmaceutical Additives Encyclopedia” (published by Yakuji Nippo).)

  Carriers or additives for oral administration (or ingestion) include lactose, sucrose, glucose, mannitol, sorbitol, corn starch, partially pregelatinized starch, crystalline cellulose, low-substituted hydroxypropylcellulose, carmellose sodium, talc, etc. Excipients; binders such as starch, α-starch, agar, gelatin, gum arabic, dextrin, methylcellulose, ethylcellulose, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose or salts thereof, crystalline cellulose; calcium carbonate , Crospovidone, starch, carboxymethylcellulose calcium, low substituted hydroxypropylcellulose, carboxymethyl starch, etc .; Lubricants such as gnesium, talc, polyethylene glycol and silicic acid; suspending agents such as polysorbate 80, polyoxyethylene hydrogenated castor oil, and pluronic; sucrose, talc, precipitated calcium carbonate, gelatin, gum arabic, pullulan, Examples thereof include coating agents such as carnauba wax and hydroxypropylmethylphthalate; flavoring agents such as sucrose, glucose, sodium saccharin, sorbitol, citric acid, and aspartame. In addition to the above components, as long as the effects of the present invention are not impaired, stabilizers, emulsifiers, dispersants, fluidizing agents, buffering agents, wetting agents, interfaces that are usually acceptable as additives for pharmaceuticals or foods Optional components such as an activator, a thickener, a preservative, a pH adjuster, a colorant, a solvent, and a solubilizer can be added as desired.

  These salivary secretion promoters having a semi-solid or solid form are preferably prepared so that the pH is usually in the range of 5 to 9 in the oral cavity after eating or taking. The pH in the oral cavity is preferably pH 5-8, more preferably pH 5-7.5.

  As shown in the experimental examples to be described later, the salivary secretion promoter of the present invention thus prepared has an action of promoting saliva secretion. Especially, the salivary secretion promoter containing potassium in addition to BBI or BBI-containing extract or BBI-containing processed product is excellent in sustainability, and can exert the salivary secretion promoting effect for a long time.

  The saliva secretion-promoting agent of the present invention can be prepared and administered or ingested in the solid, semi-solid or liquid oral form or oral administration form described above. The administration or ingestion method and the timing thereof are not particularly limited, and can be used as needed when the oral cavity is dried or before drying. In addition, although the amount used is not particularly limited, it is administered (taken) at a rate such that BBI is 0.036 to 0.18 mg per dose (taken) in terms of the amount of BBI contained in the salivary secretion promoter. Or it is preferable to administer (ingest) the water-soluble solid content of the raw material plant at a rate of 0.22 to 1.12 g per time. In addition, the number of administration (ingestion frequency) per day of the saliva secretion promoter of the present invention is not limited, and the saliva secretion promoter containing the above-mentioned amount of BBI is once or more times a day or continuously as described above. Alternatively, it can be administered (taken) intermittently.

  In addition, the saliva secretion promoter of this invention can be used suitably with respect to the test subject who the saliva secretion fell or the test subject who feels the saliva secretion fall. As a subject whose saliva secretion has decreased, a patient who preferably has hyposalivation or xerostomia can be mentioned.

(III) Method for Promoting Saliva Secretion in Subjects From the above, according to the saliva secretion promoting agent of the present invention, saliva secretion can be promoted for subjects who have decreased saliva secretion or subjects who feel a decrease in saliva secretion. it can. Therefore, the present invention provides a method for promoting saliva secretion for the subject.

  Such a method can be carried out by applying the above-described salivary secretion promoter of the present invention to the oral cavity of the subject or by oral administration. Application to the oral cavity can be appropriately selected according to the form of the saliva secretion promoter, for example, when the saliva secretion promoter has a liquid form such as a solution type, a spray type, a dripping type, or a discharge type, Examples include mouthwash, toothpaste, gargle (including gargling), and spray into the oral cavity. In addition, when the saliva secretion promoter has a solid form such as gum, chewing can be mentioned. For oral administration, liquid, semi-solid or solid forms (eg powders, tablets, granules, pills, troches, syrups, films, chewable tablets and dry syrups) , Drinks, strawberries, jellies, gummi, edible films, etc.) can be orally ingested (administered) according to the form.

  The time of ingestion (administration) is not particularly limited, and can be used as needed when the oral cavity is dried or before drying. In addition, the amount used is not particularly limited, but it is administered (ingested) at a rate such that BBI is 0.036 to 0.18 mg per time in terms of the amount of BBI contained in the salivary secretion promoter, or the raw material It is preferable to administer (ingest) the water-soluble solid content of the plant at a rate of 0.22 to 1.12 g per time. In addition, the number of administration (ingestion frequency) per day of the salivary secretion promoter is not limited, and the salivary secretion promoter containing the above-mentioned amount of BBI is once or more times a day, as described above, continuously or intermittently. Can be administered (ingested).

  Examples of the subject whose salivary secretion is reduced targeted by the method of the present invention include preferably patients having hyposalivation or xerostomia. The methods for diagnosing the decrease in saliva secretion include (1) a method for measuring the amount of saliva at rest (vomiting saliva method, saliva watter method), (2) a method for measuring the amount of stimulated saliva (Saxon method), (3 ) Methods for measuring moisture retention of oral mucosa (periotron method, filter paper method, oral moisture meter), (4) methods for measuring saliva physical properties (viscosity test, string drawing test), and (5) questionnaire method. Any one of these methods or a combination of two or more methods can be used to evaluate the presence or absence of decreased salivary secretion in the subject.

  In order to clarify the configuration and effects of the present invention, experimental examples and formulation examples are described below. However, the present invention is not affected by these experimental examples.

Experimental example 1
(1) Preparation of black bean concentrated extract (water-soluble solid content 70% by weight) 10 kg of leguminous plant (black soybean: Glycine max) seeds (including seed coat) (hereinafter referred to as “black beans”) are put into 70 L of water. And left overnight at room temperature, followed by boiling for 2 hours. The hot water extract thus obtained was subjected to solid-liquid separation, and the obtained extract was mixed with a filter aid and diatomaceous earth and subjected to suction filtration to obtain about 55 L (pH 5.8) of filtrate. 10 g of enzyme / cellulase (Cellulosin AC40, titer: 4000 units / g, manufactured by HI) was added to 55 L (pH 5.8) of the hot water extraction filtrate, and the mixture was slowly stirred at 40 to 55 ° C. for 2 hours for enzyme reaction treatment. went. Next, the reaction solution was heated to reach 95 ° C. to inactivate the enzyme to obtain an enzyme treatment solution. The obtained enzyme-treated solution was concentrated under reduced pressure to obtain 2.5 kg of a concentrated black bean extract (water-soluble solid content 70 wt%, containing BBI 0.197 g).

(2) Evaluation of saliva secretion promoting effect The syrup form shown in Table 1 using the concentrated extract of black beans prepared in (1) (water-soluble solid content 70% by weight) and varicella and reduced maltose varicella as a viscosity-imparting agent. The compositions (test samples 1 and 2) were prepared.

(2-1) Preparation of test sample Warm starch syrup and reduced malt starch syrup are heated to 60 ° C. and slowly stirred with black bean concentrate extract (water-soluble solid content 70% by weight). In addition, it was mixed until uniform. Then, the fragrance | flavor was added and the syrup-like composition which contains the water-soluble solid content of a black bean in the ratio of 7.98 weight% was prepared. Next, this was sterilized at 90 ° C. for 20 minutes, cooled to room temperature, and used as test samples 1 and 2. The viscosity of this composition (test samples 1 and 2) is a BL type rotational viscometer (manufactured by Toki Sangyo Co., Ltd.) (uses rotor NO.1 up to a viscosity of 450 mPa · s, and the viscosity is greater than 450 mPa · s). Use rotor NO.2 up to 2250mPa · s, use rotor NO.3 for viscosity greater than 2250mPa · s up to 9000mPa · s, and use rotor NO.4 for viscosity over 9000mPa · s) at 12rpm Measured at 20 ° C.

(2-2) Evaluation of salivary secretion promoting effect (a) Selection of subjects Using healthy subjects (six men) with the same rhythm in daily life, closing their mouths with no foreign bodies in the mouth, and resting While maintaining, saliva was collected every 5 minutes, the saliva weight for 30 minutes was measured, and the daily fluctuation of saliva secretion was observed. As a result of examining the diurnal variation of the saliva secretion amount of 6 healthy subjects, 3 individuals having relatively close diurnal variation were selected and used as subjects of the following experiment.

(B) Evaluation of salivary secretion promoting effect Salivary stimulating effect was evaluated for the test samples 1 and 2 prepared above, using the three persons selected by the above method as panelists. Specifically, from 5 minutes after ingesting 4 g of each test sample, saliva was collected every 5 minutes according to the method described above, and the total amount for 30 minutes was measured to evaluate the daily fluctuation of saliva secretion. . Saliva collection started at 9:30 in the morning and continued until 6 in the evening. The test sample was ingested by placing it on the back of the tongue and swallowing it at once.

  FIG. 1 shows a result of comparing the daily fluctuation of saliva secretion due to the intake of each test sample and the daily fluctuation of saliva secretion when not ingesting (control).

  As can be seen from this result, it was found that ingestion of the test sample 1 containing the black bean extract significantly increased salivation. Moreover, the state where the amount of saliva secretion increased was maintained over a long time by using potassium (KCl) together with black bean extract (test sample 2). From this, it was considered that the saliva secretion promoting action of the black bean extract is continuously maintained by using the black bean extract and potassium together.

Experimental Example 2 Isolation and identification of salivary secretion-promoting components The salivary secretion-promoting effect was evaluated for each fraction of the black bean extract, and active ingredients that exerted the salivary secretion-promoting action were isolated and identified.

(1) Evaluation method for the effect of promoting salivation Secretion was evaluated in the same manner as in Experimental Example 1, and three subjects (one male in their 50s and two males in their 30s) with relatively close daily fluctuations in saliva secretion were selected. And it implemented by performing the following experiment.

  First, the mouth was closed with no foreign substance in the oral cavity, and while maintaining a rest, saliva was collected every 5 minutes and the saliva weight was measured for 15 minutes (the amount of saliva before ingestion. “Normal saliva amount”) Say). Next, after each test sample was ingested and stabilized for 15 minutes, saliva was collected every 5 minutes and the saliva weight was measured for 15 minutes while maintaining a resting state from the 15th minute (amount of saliva after ingestion) ). The experiment was conducted so that each subject did not know what the test sample was (blinding).

(2) Salivary secretion promoting effect for each black bean part (2-1) Preparation of test sample The seed coat is removed from 4.6 kg of black soybean (light black soybean), and 0.37 kg of black bean seed coat (hereinafter referred to as “black bean seed coat”) It was divided into 4.23 kg of black bean seeds (hereinafter referred to as “black bean seeds”) from which the seed coat had been removed.

  360 g of the obtained black bean seed coat was immersed in 1.5 L of ion-exchanged water and extracted at 100 ° C. for 2 hours. The hot water extract thus obtained was subjected to solid-liquid separation, and the recovered extract was filtered using filter paper to recover 870 mL of Brix 0.6 extract. Next, the extract was concentrated and freeze-dried to obtain 5.5 g of a dried black bean seed coat extract (referred to as “black bean seed coat extract”).

  On the other hand, apart from the black bean seed coat, 840 mL of ion-exchanged water was added to 300 g of black bean seeds, allowed to stand overnight at room temperature to swell, and then extracted under boiling conditions for 2 hours. After solid-liquid separation of the hot water extract thus obtained, 0.3 g of enzyme / cellulase (cellulosin AC40, titer: 4000 units / g, manufactured by HI) was added to the obtained extract. The reaction was carried out at ˜55 ° C. with slow stirring for 2 hours. The reaction solution is heated to 95 ° C. to inactivate the enzyme, and then the obtained enzyme treatment solution is mixed with a filter aid and diatomaceous earth and suction filtered, and 1.16 kg of Brix 4.4 extract is added. Obtained. Next, the obtained extract was concentrated and freeze-dried to obtain 43.8 g of a dried black bean seed extract (referred to as “black bean seed extract”).

  Using the black bean seed coat extract or black bean seed extract thus prepared and purified honey, a syrup-like composition (test samples 3 and 4) having the formulation shown in Table 2 was prepared.

(2-2) Evaluation of salivary secretion promoting effect Three subjects took 3.5 g of each of test samples 3 and 4 (black bean seed coat extract 20 mg, black bean seed extract 245 mg). The amount of saliva secretion at rest for 15 minutes and 15 minutes after ingestion was measured to evaluate the rate of increase in saliva secretion.

  The results are shown in FIG. FIG. 2 shows the amount of saliva (ml) before ingestion of the test sample on the horizontal axis, that is, the amount of normal saliva (ml) in which nothing is ingested (normal amount of saliva (ml)). The vertical axis shows the rate of increase in saliva secretion due to the intake of the test sample. The rate of increase is shown as a relative ratio of the amount of saliva (ml) after ingesting the test sample when the amount of normal saliva (ml) is 1. It is judged that the saliva promoting effect of the test sample is higher as the relative ratio is larger than 1. However, this saliva promoting effect is preferably increased when the amount of normal saliva is small, and it is preferable that the effect is not high when the amount of normal saliva is large. For this reason, in FIG. 2, when a high rate of increase is shown when the amount of normal saliva is small, that is, when a downward-sloping curve with a large slope is obtained, the test sample has an ideal saliva promoting effect. Judgment can be made.

  2A shows the rate of increase in saliva secretion after ingestion of test sample 3 (black bean seed extract), FIG. 2B shows the rate of increase in saliva secretion after ingestion of test sample 4 (black bean seed extract), and FIG. The rate of increase in saliva secretion after ingestion of purified honey) is shown respectively. From the results shown in FIGS. 2A and 2B, it was found that the active ingredient exhibiting the salivary secretion promoting action is contained in the black bean seeds more than the black bean seed coat. Moreover, from the result of FIG. 2B, it was determined that the extract of black bean seed coat significantly promotes saliva secretion when the amount of saliva during normal times is small, and thus exhibits an ideal saliva promoting effect.

(3) Salivary secretion promoting effect of each fraction of black bean seeds (3-1) Preparation of test sample In 100 L of water, 7.4 kg of black bean seeds from which seed coats were removed from black soybeans (light black soybeans) was added at room temperature. After being left overnight, boiling was performed for 2 hours. The hot water extract thus obtained is subjected to solid-liquid separation, and the obtained extract is mixed with a filter aid and diatomaceous earth and suction filtered to obtain about 100 L (pH 5.8) of hot water extraction filtrate. It was. 7.4 g of enzyme / cellulase (Cellulosin AC40, titer: 4000 units / g, manufactured by HI) was added to 100 L of the hot water extraction filtrate (pH 5.8), and the mixture was slowly stirred at 40 to 55 ° C. for 2 hours to carry out the enzyme treatment. Went. Next, this reaction solution was heated to reach 95 ° C. to inactivate the enzyme to obtain an enzyme treatment solution. The obtained enzyme-treated solution was concentrated under reduced pressure to obtain 3.7 kg (water-soluble solid content 37.8% by weight) of a black bean seed concentrate extract.

  Next, the obtained black bean seed concentrated extract was subjected to an ultrafiltration membrane device (manufactured by Asahi Kasei Co., Ltd.) to (a) a fraction having a molecular weight of less than 6 kDa (fractional molecular weight of less than 6 kDa), and (b) a molecular weight of 6 kDa or more. Fractions were fractionated into a fraction in the range of 13 kDa or less (fraction molecular weight 6-13 kDa) (recovery rate 1%), and (c) a fraction having a molecular weight greater than 13 kDa (fraction molecular weight greater than 13 kDa). These were freeze-dried and powdered, and purified honey was added thereto as a carrier, and syrup-shaped test sample 5 (fractional molecular weight of less than 6 kDa), test sample 6 (fractionated molecular weight of 6-13 kDa), and test sample 7 (fractional molecular weight greater than 13 kDa) was prepared.

(3-2) Evaluation of salivary secretion promoting effect Each of the three subjects ingested the black bean seed concentrated extract at a rate of 245 mg in terms of solid content, and according to the method described above, 15 minutes before ingestion And the amount of saliva at rest for 15 minutes after ingestion was measured to evaluate the rate of increase in saliva secretion after ingestion.

  As a result, the test sample 6 (fractionated molecular weight: 6-13 kDa) showed a strong salivary secretion promoting action. The result is shown in FIG. The “rate of increase in saliva secretion after ingestion of test sample (times)” shown on the vertical axis of the figure is the same as in FIG. It is the value computed from the relative ratio of the amount of saliva (ml) after ingesting the test sample. As can be seen from the results in FIG. 3, the test sample 6 (molecular weight cut off 6-13 kDa), like the black bean seed extract of (2), exhibits a significantly high salivary secretion promoting effect when the amount of normal saliva is small. In addition, an ideal saliva promoting effect is shown in which the effect decreases as the amount of saliva increases during normal times. From this, it was estimated that the active ingredient which exhibits salivary secretion promoting action is a component having a molecular weight of 6-13 kDa contained in black bean seeds.

(4) Isolation and identification of active ingredient having salivary secretion promoting action (4-1) Isolation of active ingredient (i) Anion exchange column chromatography Molecular weight of black bean seed extract collected in (3) above 6- A 13 kDa fraction (hereinafter referred to as “black bean extract 6-13 kDa”) (dry weight 3.63 g) was dissolved in 20 mM Tris-HCl buffer (pH 8.5) and subjected to anion exchange column chromatography under the following conditions.

<Conditions for anion exchange column chromatography>
Column: 30 cm x 2.5 cm id
Stationary phase: DEAE-TOYOPEARL 650M (manufactured by Tosoh)
Mobile phase: 20mM Tris-HCl pH8.5 containing 20mM Tris-HCl pH8.5 / 1M NaCl
Gradient: After linear gradient gradient from 0 to 0.7M NaCl, stepwise to 1M NaCl (shown in dotted line in FIG. 4)
Flow rate: 3ml / min
Column temperature: Room temperature (25 ° C)
Detector: UV280nm absorption is measured for each fraction.

  The results are shown in FIG. Each fraction A to F shown in the chromatogram of FIG. 4 was collected, concentrated after removing salts by dialysis against ion-exchanged water. Purified honey was added to these as a carrier to prepare syrup-like test samples A to J. Similarly to the above (3) and (3-2), each of the three subjects ingested the black bean seed concentrated extract at a rate of 245 mg in terms of solid content. The rate of increase was evaluated. As a result, the test sample J (fraction J) was confirmed to have a salivary secretion promoting action.

(Ii) Hydrophobic interaction column chromatography Ammonium sulfate is added to the fraction J in which the salivary secretion promoting action is recognized as above to prepare a 40% saturated ammonium sulfate aqueous solution, and this is subjected to hydrophobic interaction column chromatography under the following conditions. It was used for.

<Conditions for hydrophobic interaction chromatography>
Column: 20 cm x 2.5 cm id
Stationary phase: Phenyl-TOYOPEARL 650M (manufactured by Tosoh)
Mobile phase: 40% saturated ammonium sulfate / ion-exchanged water gradient: Reversed linear gradient from 40% saturated ammonium sulfate to 0% (ion-exchanged water), then passed with ion-exchanged water (then gradient to 40% EtOH) (Indicated by dotted lines in FIG. 5)
Flow rate: 3ml / min
Column temperature: Room temperature (25 ° C)
Detector: UV280nm absorption is measured for each fraction.

  The results are shown in FIG. Fractions JA to JE shown in the chromatogram of FIG. 5 were collected, concentrated after removing salts by dialysis against ion-exchanged water. Purified honey was added as a carrier to each obtained concentrate to prepare syrup-like test samples JA to JE. Similarly to the above (3) and (3-2), each of the three subjects ingested the black bean seed concentrated extract at a rate of 245 mg in terms of solid content. The rate of increase was evaluated.

  As a result, as shown in FIG. 6, it was confirmed that the test sample JD (fraction JD) has an ideal salivary secretion promoting action.

(4-2) Identification of active ingredient Fraction JD in which the salivary secretion promoting action was observed was concentrated and subjected to SDS-PAGE according to the following method.

  SDS-PAGE was performed by applying slab electrophoresis for 2 hours at a voltage of 20 mA using an ATTO precast gel (gel concentration: 15%) and Tris / Tricine buffer. An analytical sample was prepared by adding a sample buffer containing dithiothreitol (DTT) to fraction JD, followed by heat treatment with boiling water for 5 minutes, and then cooling. The analysis sample thus prepared was subjected to electrophoresis simultaneously with a molecular weight marker (for protein molecular weight marker low molecule, manufactured by Wako Pure Chemical Industries, Ltd.). After electrophoresis, the gel is stained with Coomassie brilliant blue staining (using CBB R-250), and the molecular weight of the stained band (the molecular weight of the protein contained in fraction JD) is determined from a calibration curve based on the migration distance of the molecular weight marker. It was.

  The result of SDS-PAGE is shown in FIG. As shown in FIG. 7, a substantially single band was observed around 8 kDa. This was blotted on a PDVF membrane and then subjected to N-terminal amino acid analysis. As a result, the N-terminal amino acid sequence was DESSKPXXDQ (SEQ ID NO: 3). In this amino acid sequence, a small proline peak was detected for X, but no other amino acids were detected. Therefore, the possibility of a cysteine residue was considered high. When the homology search was performed using BLAST using such an amino acid sequence as an index, the above N-terminal amino acid sequence was a mature protein of Bowman-Birk Protease Inhibitor (abbreviated as “BBI”, hereinafter simply referred to as “BBI”). It was very similar to the N-terminal sequence “DDESSKPCCDQ” (SEQ ID NO: 4). BBI is a protein known as a serine protease inhibitor having an action to inhibit trypsin activity and chymotrypsin activity (Krishnan, HB, et al, Planta, 217 (3), 523-427 (2003); Egyptian Journal of Biology, 2001, Vol. 3, pp.164-173).

  Incidentally, the amino acid sequence of the mature protein of BBI is shown in SEQ ID NO: 1, and the amino acid sequence of its precursor is shown in SEQ ID NO: 2. As can be seen, BBI has 14 cysteine residues, resulting in 7 disulfide bonds in the structure. The mature protein has a theoretical molecular weight of about 7.9 kDa and an isoelectric point (pI) of 4.51.

Experimental example 3
(1) Preparation of black bean extract (BBI-containing fraction) Into 70 L of water, 10 kg of leguminous plant (black soybean: Glycine max) seeds (black beans) are added and left overnight at room temperature, followed by boiling 2 Went for hours. The obtained hot water extract was subjected to solid-liquid separation, and the recovered extract was mixed with a filter aid and diatomaceous earth and subjected to suction filtration to obtain about 55 L of hot water extraction filtrate. To the obtained hot water extraction filtrate 55 L (pH 5.8), 10 g of enzyme / cellulase (Cellulosin AC40, titer: 4,000 units / g, manufactured by HI) was added and slowly stirred at 40 to 55 ° C. for 2 hours for enzyme treatment. To obtain an enzyme-treated solution. Next, the enzyme-treated solution was concentrated under reduced pressure to obtain 2.5 kg of a black bean concentrated extract (aqueous solution solid content: 70% by weight).

  Take 1.78 kg from the extract thus prepared, and apply to an ultrafiltration membrane device (manufactured by Asahi Kasei Co., Ltd.) as described in Experimental Example 2 (3) to obtain a fraction having a molecular weight in the range of 6-13 kDa. (Fraction molecular weight 6-13 kDa) is collected to obtain the above-mentioned fraction containing BBI (molecular weight of about 8 kDa), and then concentrated and freeze-dried to obtain 14.0 g of black bean extract (treated product containing BBI). ) Was obtained (containing 0.14 g of BBI).

(2) Preparation of test sample A test sample was prepared according to the formulation shown in Table 3 below. Specifically, first, syrup and reduced malt syrup are heated to 60 ° C., and while slowly stirring, the BBI-containing treated product prepared above and test sample 8 are further added with KCl and mixed until uniform. did. Then, a fragrance | flavor was added and the syrup-like composition which contains a BBI containing processed material in the ratio of 0.45 weight% or 0.25 weight% was prepared (test sample 7 and 8). This was then sterilized at 90 ° C. for 20 minutes and cooled to room temperature. The viscosities of these test samples were measured at 20 ° C. at a rotational speed of 12 rpm using a BL type rotational viscometer (manufactured by Toki Sangyo Co., Ltd.) (see Experimental Example 1).

(3) Evaluation of salivary secretion promoting effect (3-1) Selection of test subjects Using healthy subjects (six men) with no foreign matter in the oral cavity and similar rhythms of daily life, gauze (pure sterilized gauze M 7.5cm X7.5 cm (made by Tamagawa Eizo Co., Ltd.) was included in the mouth, and continued to chew accurately at intervals of 1 second for 2 minutes, and the weight of the gauze was measured (Saxon method). The weight of a new gauze was measured in advance, and the difference was taken as the salivary secretion weight for 2 minutes, and the diurnal variation of saliva secretion was observed. From these results, three persons with relatively close daily fluctuations were selected and used as subjects for the following experiment.
(3-2) Evaluation of saliva secretion promoting effect The subjects selected by the above method were asked to ingest 4 g each of test sample 7 and test sample 8 prepared in (2), and the saliva secretion promoting effect was evaluated. . Specifically, in order to measure the amount of saliva in each subject under non-ingestion conditions on the test day, the weight (g) of saliva for 2 minutes was measured at 9:25 according to the method described above (Saxon method). Thereafter, 4 g of the test sample was ingested at 9:30, and the saliva weight (g) was measured 30 minutes later. The saliva weight was measured from 10:30 to 18:30 at 1 hour intervals. In addition, as a control test (control), the saliva weight (g) was measured every hour from 9:30 to 18:30 on another day (control sample), and the test sample Circadian variation of saliva secretion under the condition of not ingesting.

  Fig. 8 shows the diurnal variation in saliva secretion when the test sample 7 (-▲-) and the test sample 8 (-■-) are ingested, and the diurnal variation in saliva secretion in the non-intake (control) (-●-). Shown in

  From this result, it was confirmed that by ingesting a test sample containing a BBI-containing extract of black beans, the amount of saliva secretion increased and the increased state persisted. In addition, by using potassium in combination with black bean extract (BBI-containing treatment) (test sample 8), even if the amount of black bean extract (BBI-containing treatment) is reduced by half, the double amount of black bean extract It was observed that (BBI-containing product) (test sample 7) had almost the same effect.

  From the above, the BBI contained in the black bean extract has a salivary secretion promoting action (BBI is an active ingredient of the salivary secretion promoting action), and the action is enhanced by using potassium in combination with this. It has been found. In consideration of the results of Experimental Example 1, it is considered that the use of potassium in combination with BBI imparts persistence to the BBI salivary secretion promoting action.

Experimental Example 4
(1) Preparation of test sample Tablets (1 tablet 0.3 g) were prepared according to the formulation shown in Table 4 using the black bean extract (BBI-containing product) prepared in Experimental Example 3 (1). Using this as a test sample 9, the following experiment was conducted.

(2) Evaluation of salivary secretion promoting effect (2-1) Test method Healthy subjects (1 male, 58 years old) were used as subjects. Prior to the test, one piece of gauze (pure sterilized gauze M 7.5cm x 7.5cm Tamagawa Sanyo Co., Ltd.) was put in the mouth, and continued to be chewed accurately at intervals of 1 second, and the weight of the gauze was measured (Saxon method) . The weight of a new gauze was measured in advance, and the difference was taken as the salivary secretion weight for 2 minutes, and the daily fluctuation of salivary secretion was observed (n = 1).
(2-2) Evaluation of saliva secretion promoting effect The saliva secretion promoting effect of the test sample 9 prepared in (1) was evaluated.

  Specifically, in order to measure the control (normal saliva amount) of the subject on the test day, the saliva weight for 2 minutes was measured at 9:25 according to the method described above. Thereafter, the test sample 9 (1 tablet, 0.3 g) was taken with water without chewing at 9:30, and the saliva weight was measured 30 minutes later. The same test was conducted from 10:30 to 18:30 at 1 hour intervals. This test was continued for 6 days.

  Figure 9 shows daily fluctuations in saliva secretion on day 3 (-◆-) and day 6 (-▲-), and non-intake (control) (-●-). Shows daily fluctuations in saliva secretion.

  As can be seen from this result, even when the test sample containing the black bean extract (BBI-containing treatment product) is orally ingested without chewing, when it is continuously ingested, it is contacted as it is in the oral cavity (for example, As in Experimental Examples 1 to 3, it was confirmed that the salivary secretion promoting effect was significantly exhibited.

Formulation Examples 1-4
In accordance with the formulations described in Tables 5 and 6, a liquid saliva secretion promoter (drink type: formulation examples 1 and 2) and a solid saliva secretion promoter (chewable type: formulation example 3, troche type: formulation example 3) Prepared.

(1) Formulation Examples 1-2 (Liquid salivary secretion promoter)
In accordance with the formulation shown in Table 5, 57.2 g of black bean concentrated extract (70% water-soluble solid content) prepared in Experimental Example 1 (1), 2.1 g of K ₃ PO ₄ , and 939.7 g of water were blended at 80 ° C. 1 g of a fragrance was added, and autoclaved at 121 ° C. for 20 minutes to prepare 1 kg of a liquid saliva secretion promoter of Formulation Example 1 having a viscosity of 3 mPa · s and pH 7.6. This was filled into a bottle so as to have a volume of 50 ml to prepare a drink.

Moreover, the liquid saliva secretion promoter (pH 6) of the formulation example 2 is the same as the liquid saliva secretion promoter of the formulation example 1 according to the formulation of Table 5 except that KCl is used instead of the K ₃ PO ₄ described above. Prepared by method. This was filled into a bottle so as to have a volume of 50 ml to prepare a drink.

(2) Formulation Examples 3 to 4 (solid saliva secretion promoter)
Ingredients other than fragrance are mixed according to the formulation described in Table 6, granulated with a fluid bed granulator, fragrance is added and mixed uniformly, and tableting is performed with a rotary tableting machine (rotation speed 20 rpm). did. Thus, two types of tablets (one tablet, 0.9 g) of a chewable type having a hardness of 10 kg / cm ² (formulation example 3) and a troche type having a hardness of 19 kg / cm ² (formulation example 4) could be obtained.

The black bean extract FD powder was prepared as follows.
First, a black bean concentrated extract (water-soluble solid content 20%) was prepared, and 140 kg of the extract was freeze-dried at 0.8 Torr or less for 24 hours and crushed into blocks. Thereafter, it was passed through a 30 mesh sieve to obtain 30.5 kg of black bean extract FD powder (3.4 g of BBI contained in 30.5 kg of FD powder).

  When two types of tablets (0.9 g) prepared by the above method were orally ingested, 1 chewable tablet (0.9 g) can be dissolved by chewing about 1 g of saliva, and 1 lozenge (0.9 g) It was possible to lick and dissolve with about 5 g of saliva. At this time, the pH in the oral cavity was pH 6.6 and 7.1, respectively.

Formulation Examples 5-8 Salivary secretion promoter (acid beverage type)
According to the formulation described in Table 7, concentrated black bean extract (70% water-soluble solid content) prepared in Experimental Example 1 (1), fructose glucose solution, citric acid, and water (for Formulation Examples 6-8, further chlorination) Potassium) was mixed and dissolved by heating and stirring at 80 ° C. Thereafter, 2 g of a fragrance was added, sterilized at a temperature of 93 ° C., and 1 kg of a salivary secretion promoter (acid beverage type) of prescription examples 5 to 8 of pH 3.1 and Brix 9.0 was prepared. The reference intake amount at one time was 200 ml.

Formulation Examples 9-12 Salivary secretion promoter (neutral beverage type)
According to the formulation described in Table 8, concentrated black bean extract (water-soluble solid content 70%) prepared in Experimental Example 1 (1), green tea extract powder, sodium ascorbate, and water (for formulation examples 10-12, Potassium chloride) was added and dissolved by heating and stirring at 80 ° C. Then, fragrance | flavor 0.25g was added, it filled with the can, and autoclave sterilization was performed at 121 degreeC for 20 minutes, and 1 kg of saliva secretion promoter (neutral drink type) of pH6.8 and Brix0.7 was prepared. A single intake was 200 ml.

Formulation Examples 13 to 16 Salivary secretion promoter (jelly type)
According to the formulation described in Table 9, 10 g of agar and 500 g of water were blended, dissolved by heating and stirring at 80 ° C., and then a black bean concentrated extract (water-soluble solid content 70%) prepared in Experimental Example 1 (1). 150 g of sugar, 2 g of citric acid, 2.5 g of fragrance, and the remaining water (for formulation examples 14 to 16, further potassium chloride) are blended, sterilized at 85 ° C. for 30 minutes, pH 3. 1 kg of 8 salivary secretion promoter (jelly type) was prepared. A single intake was 100 g.

Formulation Examples 17-20 Salivary secretion promoter (gum type)
According to the formulation described in Table 10, black bean extract (BBI-containing product) prepared in Experimental Example 3 (1) and chewing gum base 24 g, granulated sugar, starch syrup 15 g, softener 5 g, chlorophyll 0.1 g, flavor 0.1 g (For Formulation Examples 18 to 20, potassium chloride) was further blended and kneaded well, and then molded to prepare 100 g of a saliva secretion promoter (plate gum type). One intake was 5 g (one sheet).

Formulation Examples 21-24 Salivary secretion promoter (飴 type)
According to the prescription described in Table 11, the black bean extract (BBI-containing product) prepared in Experimental Example 3 (1), 45 g of granulated sugar, 43.6 g of enzymatically saccharified starch syrup, and 30 g of water are blended and boiled to 190 ° C. After cooling to 160 ° C., 0.1 g of a fragrance (for example 22 to 24, further potassium chloride) was blended, mixed, and then molded to prepare 100 g of a salivary secretion promoter (acupuncture type). In addition, the intake amount per time was 5 g (1 grain).

Formulation Examples 25-28 Liquid Saliva Secretion Promoter (Mouth Wash)
According to the formulation described in Table 12, black bean extract (BBI-containing treated product) prepared in Experimental Example 3 (1), 0.1 g of 1-menthol, mint oil 0.1 g, polyglycerin fatty acid ester 4 g, ethanol 45 g, saccharin sodium 0.01 g and an appropriate amount of a pH adjusting agent (for example, potassium chloride for formulation examples 26 to 28) were blended and adjusted to 100 g with purified water. Note that the amount used once was 10 ml.

Formulation examples 29-32 Liquid saliva secretion promoter (oral gel)
According to the formulation described in Table 13, the black bean extract (BBI-containing product) prepared in Experimental Example 3 (1), glycerin 20 g, sorbitol 25 g, xylitol 10 g, carboxymethylcellulose 1 g, carrageenan 0.3 g, flavor 0 0.1 g, methylparaben 0.1 g, and an appropriate amount of a pH adjuster (for formulation examples 30 to 32, further potassium chloride) were added to prepare 100 g with purified water. A single intake was 1 g.

Formulation Examples 33 to 36 Liquid saliva secretion promoter (oral spray)
According to the formulation described in Table 14, the black bean extract (BBI-containing product) prepared in Experimental Example 3 (1), 35 g of glycerin, 7.5 g of sorbitol, 0.1 g of fragrance, 0.1 g of methylparaben, an appropriate amount of pH adjuster (For formulation examples 34 to 36, further potassium chloride) was prepared to 100 g with purified water. In addition, the intake amount per time was 2 g (1 g amount per time was sprayed twice).

Formulation Examples 37 to 40 Liquid saliva secretion promoter (toothpaste)
According to the formulation described in Table 15, black bean extract (treated product containing BBI) prepared in Experimental Example 3 (1), calcium carbonate 35 g, sorbitol 20 g, carboxymethyl cellulose 1.0 g, sodium lauryl sulfate 1.0 g, saccharin 0. 1 g, 1.0 g of fragrance, 0.01 g of allantoin, and 0.01 g of dipotassium glycyrrhizinate (for formulation examples 38 to 40, potassium chloride) were added and prepared to 100 g with purified water. One use amount was 1 g.

It is the figure which contrasted the daily fluctuation | variation of the saliva secretion amount when ingesting the saliva secretion promoter (test sample 1 and 2) of this invention with the daily fluctuation | variation (control) at the time of non-ingestion. The vertical axis represents the amount of saliva (g) collected for 30 minutes, from which the change in saliva secretion over time can be seen. 2A shows the rate of increase in saliva secretion after ingestion of test sample 3 (black bean seed extract), FIG. 2B shows the rate of increase in saliva secretion after ingestion of test sample 4 (black bean seed extract), and FIG. The rate of increase in saliva secretion after ingestion of purified honey) is shown respectively. The horizontal axis represents the amount of saliva (ml) before ingestion of the test sample (normal amount of saliva (ml)), and the vertical axis represents the rate of increase in the amount of saliva secreted by ingestion of the test sample. The rate of increase is shown as a relative ratio of the amount of saliva (ml) after ingesting the test sample when the amount of normal saliva (ml) is 1. In the figure, the solid line indicates the result of obtaining a linear regression line by the least square method for each result. (Experimental example 2 (2)). The rate of increase in salivary secretion after ingestion of test sample 6 (fractional molecular weight of 6-13 kDa) is shown. The horizontal axis represents the amount of saliva (ml) before ingestion of the test sample (normal amount of saliva (ml)), and the vertical axis represents the rate of increase in the amount of saliva secreted by ingestion of the test sample. The rate of increase is shown as a relative ratio of the amount of saliva (ml) after ingesting the test sample when the amount of normal saliva (ml) is 1. In the figure, the solid line indicates the result of obtaining a linear regression line by the least square method for each result. (Experimental example 2 (3)). The chromatogram obtained by the anion exchange column chromatography performed in Experimental example 2 (4) (4-1) (i) is shown. The chromatogram obtained by the hydrophobic interaction column chromatography performed in Experimental example 2 (4) (4-1) (ii) is shown. The rate of increase in saliva secretion after ingestion of test sample JD is shown. The horizontal axis represents the amount of saliva (ml) before ingestion of the test sample (normal amount of saliva (ml)), and the vertical axis represents the rate of increase in the amount of saliva secreted by ingestion of the test sample. The rate of increase is shown as a relative ratio of the amount of saliva (ml) after ingesting the test sample when the amount of normal saliva (ml) is 1. In the figure, the solid line indicates the result of obtaining a linear regression line by the least square method for each result. (Experimental example 4 (4) (4-2)). The result of having applied test sample JD to SDS-PAGE is shown (Experimental Example 4 (4) (4-2)). Daily variation of saliva secretion when test sample 7 (-▲-) and test sample 8 (-■-) are ingested, non-ingestion (control) (-●-) shows diurnal variation of saliva secretion (experiment) Example 3). Daily fluctuations in the amount of saliva secreted on day 3 (-◆-) and day 6 (-▲-), and non-ingested (control) (-●-) salivary secretion The daily fluctuation of the amount is shown (Experimental Example 4).

Claims (14)

A salivary secretion-promoting agent applied to a subject who has reduced saliva secretion or who feels a decrease in saliva secretion, comprising Bowman-Birk Protease Inhibitor as an active ingredient. The salivary secretion promoter according to claim 1, wherein the Bowman-Birk Protease Inhibitor is a protein represented by the following (a) or (b):
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1,
(B) in SEQ ID NO: 1, has 10 deletions in any least-way amino acid residues of the N-terminal and C-terminal up and trypsin inhibitory activity, chymotrypsin inhibitory activity, and a salivary secretion promoting action protein. The saliva secretion promoter of Claim 1 or 2 containing the plant extract containing Bowman-Birk Protease Inhibitor. The salivary secretion promoter according to claim 3, wherein the plant extract is a plant extract containing a fraction in the range of 6 to 13 kDa centered on a molecular weight of 8 kDa. The salivary secretion promoter according to claim 3 or 4 , wherein the plant extract is derived from seeds of a plant belonging to the leguminous family. Furthermore, the salivary secretion promoter in any one of Claims 1 thru | or 5 containing potassium. Salivation accelerator of any one of claims 1 to 6 having a form for oral or parenteral administration. Syrups, dry syrups, films, gels, sprays, chewable tablets, lozenges, tablets, capsules, pills, granules, powders or any one of claims 1 to 7 having a toothpaste Kino form, Saliva secretion promoter described in 1.   The salivary secretion promoter according to any one of claims 1 to 8, wherein Bowman-Birk Protease Inhibitor is administered or ingested at a rate of 0.0036 to 0.18 mg per day. The method for preparing a salivary secretion promoter according to claim 1 or 2 , which comprises the following steps (1) and (3):
(1) a step of collecting a fraction containing Bowman-Birk Protease Inhibitor from a plant containing Bowman-Birk Protease Inhibitor; and (3) oral or oral use of the Bowman-Birk Protease Inhibitor-containing product obtained above. Process to form for administration. The method for preparing a salivary secretion promoter according to claim 6, which comprises the following steps (1) to (3):
(1) collecting a fraction containing Bowman-Birk Protease Inhibitor from a plant containing Bowman-Birk Protease Inhibitor;
(2) A step of preparing a composition containing Bowman-Birk Protease Inhibitor and potassium by adding potassium to the Bowman-Birk Protease Inhibitor-containing material obtained in step (1), and (3) obtained above. Processing the prepared composition into a form for oral or oral administration. The method according to claim 10 or 11 , wherein the fraction containing Bowman-Birk Protease Inhibitor is a fraction in the range of 6 to 13 kDa centered on a molecular weight of 8 kDa. The preparation method according to any one of claims 10 to 12 , wherein the plant is a seed of a plant belonging to the leguminous family. Use of a plant extract containing Bowman-Birk Protease Inhibitor or Bowman-Birk Protease Inhibitor to produce a salivary secretion-promoting agent that is applied to subjects who have decreased salivation or who feel decreased salivation.

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