JP2010031033A - Food and drink for improving rheumatic symptom - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide foods and drinks which prevent rheumatism of mammals including humans, can be orally taken everyday, and quickly improves and treats developed rheumatic symptoms without side effects. <P>SOLUTION: The foods and drinks for improving rheumatic symptoms comprises flavonoid and/or flavonoid glycoside. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a food or drink for preventing or ameliorating rheumatic symptoms.

  Rheumatoid arthritis (RA) is a chronic inflammatory disease centering on joints throughout the body, and autoimmune phenomena are deeply involved in the pathological state. Clinical symptoms mainly include chronic polyarthritis, general malaise / slight fever / muscular pain, and subcutaneous nodules, which progress chronically with repeated remissions and hatreds. I will come and will eventually have impaired motor function. Thus, RA patients suffer a lifetime of pain that is immeasurable physically and mentally.

  On the other hand, the number of RA patients in Japan is about 60 to 1.2 million, accounting for 0.5 to 1.0% of the population, the male-female ratio is 1: 4, which is more common among women, and the age may develop between 20 and 60 years old. Many. Unfortunately, however, current medicine cannot completely cure or prevent RA. Therefore, the current therapeutic goal is to diagnose RA early and to suppress RA inflammation as quickly and maximally as possible to prevent or prevent the development of irreversible changes, To improve physical, mental and social quality of life (QOL).

  The cause of RA is unknown, but the mechanism of its onset is gradually becoming clear as research on the immune mechanism progresses, and various treatments have been developed based on these findings. That is, production suppression of lymphocytes and cytokines, neutralization of activity, inhibition of binding to receptors, and the like.

  Cytokines are glycoproteins having a molecular weight of 8000 to 100,000 that are produced from cells and regulate cell-cell interactions through signal transduction between cells. Therefore, it is very important for maintaining the homeostasis of the living body. On the other hand, if they are produced excessively or the production balance is lost, the immune system is affected and chronic inflammation such as RA is caused. In particular, what are called inflammatory cytokines that are strongly involved in inflammation include interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and macrophage inflammation. Protein (MIP-1) and the like are known. In other words, improvement of inflammation can be expected by suppressing the expression of these cytokines.

  Among these cytokines, TNF-α is involved in many inflammatory conditions, and when it is produced in vivo, IL-1, IL-6, IL-8, etc. are subsequently produced and the immune system is enhanced. Happens. It has also been clarified that a large amount of TNF-α is produced from the RA synovium. Furthermore, TNF-α transgenic mice are also known to develop arthritis that closely resembles RA (see, for example, Non-Patent Document 1). Thus, antibody therapy that neutralizes TNF-α with an antibody has been proposed (see, for example, Non-Patent Documents 2 and 3), and cA2 (Centocor Inc.) has been developed.

  However, since these mouse monoclonal antibodies are heterologous proteins to humans, allergies and anti-mouse antibodies may occur. For this reason, there are a lot of problems to be solved, such as verification of efficacy in long-term administration and the presence or absence of side effects including effects on infectious diseases and other immune diseases.

  In addition, as a therapeutic method for RA at present, direct administration to an affected area is common, and in oral administration, proteins such as TNF-α antibodies and IL-1 receptor antagonists that have recently attracted attention In some cases, there are effects such as low pH in the stomach, hydrolysis by digestive enzymes, etc., and even with ordinary drugs, the therapeutic effect is low compared to direct administration to the affected area due to low absorption of therapeutic drugs and drug dilution The current situation is almost impossible to expect. However, since treatment by injection or infusion imposes various burdens on patients as compared with oral administration, development of a drug that can exert its effect by oral administration is desired.

  On the other hand, flavonoids are polyphenol compounds having a C6-C3-C6 (A ring, C ring, and B ring, respectively) skeleton, which is a secondary metabolic component widely present in plants. They are classified into flavones, flavonols, isoflavones, catechins, chalcones, etc., depending on the bonding position of the B ring, the presence or absence of a double bond in the C ring, the opening and closing of the C ring, and the number and position of hydroxyl groups bonded to each ring. These flavonoids can be easily purified by extraction from plants.

  Flavonoids are contained in plants such as citrus fruits, fruits and vegetables, and are taken into the body by eating a normal meal. In addition, in Europe, a report of ingesting 25.9 to 26.6 mg / day of flavonoids (see, for example, Non-Patent Document 4), and in the United States, a report of ingesting 115 mg / day of flavonoids (for example, Non-Patent Document 5), and it is clear that it is safe from past eating experience, so it can be used safely as a food or drink or medicine.

  For example, hesperidin has long been known as the major flavonoid of Wenzhou mandarin oranges. Until now, hesperidin is known as a cloudy substance for canned mandarin oranges and fruit juice products, and research for preventing cloudiness has been repeated (for example, see Non-Patent Documents 6, 7 and 8).

  Recently, however, attention has been focused on its physiological activity, and the use as a functional material has been developed along with an increase in health consciousness. These physiological actions include vitamin P action that has been known for a long time: strengthening of capillaries (for example, see Non-Patent Document 9), anti-inflammatory action (for example, see Non-Patent Documents 10 and 11), and reduction of cholesterol. Effects (for example, see Non-Patent Documents 12 and 13), neutral lipid reducing action (for example, see Non-Patent Document 14), carcinogenesis-inhibiting action (see Non-Patent Documents 15, 16, 17 and 18), etc. However, there are currently no known findings regarding the improvement of rheumatic symptoms.

Keffer, J. et al., “EMBO J.”, 1991, Vol. 10, p. 4025-4031 Elliot, M. J. et al., “Lancet”, 1994, 344, p. 1105-1140 Maini, RN, “Arthritis Rheum.”, 1997, Vol. 40, S126 Hertog, M.G.L., "Lancet", 1993, 342, p. 1007-1011 Kuhnau, J., “World Rev. Nutr. Diet”, 1976, Vol. 24, p. 117-191 Sugano et al., “The Journal of the Japan Food Industry Association”, 1982, Volume 29, p. 225-258 Misaki et al., “Journal of the Japan Food Industry Association”, 1982, Vol. 29, p. 228-231 Misaki, “Starch Science”, 1984, Vol. 31, p. 98-106 Kamiya, "New Vitaminology (Japan Vitamin Society)", 1969 Matsuda et al., “Pharmaceutical Journal”, 1991, Vol. 111, p. 193-198 Da Silva Emim J. A. et al., “J. Pharm. Pharmacol.”, 1994, 46, p. 118-122 Monforte M. T. et al., “Il Farmaco”, 1995, Volume 50, p. 595-599 Bok S-H. Et al., “J. Nutr.”, 1999, Vol. 129, p. 1182-1185 Kawaguchi K. et al., “Biosci. Biotech. Biochem.”, 1997, Vol. 61, p. 102-104 Tanaka T. et al., “Cancer Research”, 1994, Vol. 54, p. 4653-4659 Tanaka T. et al., “Cancer Research”, 1997, Vol. 57, p. 246-252 Tanaka T. et al., “Carcinogenesis”, 1997, Vol. 18, p. 761-769 Tanaka T. et al., “Carcinogenesis”, 1997, Vol. 18, p. 957-965

  An object of the present invention is to solve the above-mentioned problems, and to provide a food and drink that can be taken orally daily and that can prevent or rapidly improve rheumatic symptoms in mammals including humans without side effects. And

  In view of the above situation, as a result of intensive studies, flavonoids widely present in citrus fruits and / or glycosides in which sugars are further bound to these flavonoids are administered to living bodies to prevent rheumatic symptoms and After that, it was found that rheumatic symptoms were remarkably improved, and foods and drinks using them were developed to complete the present invention.

  That is, this invention relates to the food-drinks for rheumatic symptom improvement containing a flavonoid and / or a flavonoid glycoside.

  The present invention relates to a rheumatic symptom-improving food or drink containing hesperidin glycoside, naringin glycoside, diosmin glycoside and / or rutin glycoside.

  The present invention relates to a food or drink for suppressing the expression of TNF-α containing hesperidin glycoside, naringin glycoside, diosmin glycoside and / or rutin glycoside.

  1 to 3 show that the onset of CIA can be prevented and suppressed by oral administration of flavonoids and / or flavonoid glycosides (for example, hesperidin, naringin, hesperidin glycoside). 4 and 5, it was shown that even when flavonoid administration was started after the onset of CIA, the symptoms could be improved and treated. This suggested the possibility of new preventive and therapeutic methods for RA in the clinic. That is, by orally administering a flavonoid and / or a flavonoid glycoside, rheumatism can be significantly prevented, and symptoms of rheumatism after the onset can be suppressed / improved.

  FIGS. 6 and 7 show that hesperidin and a mixture of hesperidin glycosides are sufficiently effective at a dose of about 1/10 compared to hesperidin.

  In addition, since flavonoids and flavonoid glycosides are safe, they can be taken orally daily to prevent rheumatism in mammals including humans, or to improve the symptoms of rheumatism, and have no side effects. It can be a food and drink that can be quickly improved and treated.

It is a figure which shows the clinical score of a hesperidin administration group. It is a figure which shows the clinical score of a naringin administration group. It is a figure which shows the clinical score of a hesperidin glycoside administration group, and a black triangle shows the negative control which has not performed type II collagen treatment. It is a figure which shows a clinical score at the time of administering hesperidin after CIA onset. It is a figure which shows a clinical score at the time of administering naringin after CIA onset. It is a figure which shows the clinical score with respect to the dose of hesperidin. It is a figure which shows the clinical score with respect to hesperidin (3 mg) administration and equimolar mixture of hesperidin and hesperidin glycoside (mole number equivalent to 0.3 mg of hesperidin). It is a HE-stained micrograph of the joint slice of the positive control group (a) and the hesperidin administration group (b), and Y in (a) is an enlarged view of the X region. It is an electrophoresis photograph which shows the expression suppression of TNF- (alpha) mRNA in a joint tissue cell. It is a figure which shows the histopathological observation result of a joint tissue of the mouse | mouth which started administration of hesperidin or naringin after CIA onset.

  The present invention will be described in detail below.

  The flavonoid of the present invention is not particularly limited as long as it has an effect of alleviating symptoms or preventing the onset of rheumatic symptoms. Specifically, for example, hesperidin, naringin, diosmin, rutin and their aglycones such as hesperetin, naringenin, diosmethine, quercetin and the like can be used alone or in combination. Among these, hesperidin or naringin is more preferable.

  Next, the present invention will be specifically described using hesperidin and naringin.

Hesperidin is sparingly soluble in water, but dissolves very well in alkali, so it can be efficiently extracted from Wenzhou oranges (Miyake et al., Journal of the Japan Food Industry Association, 37, 631-636 (1990), Inaba et al., Journal of the Japan Food Industry Association, 40, 833-840 (1993)). Specifically, after squeezing mandarin oranges with a squeezing machine, adding 0.1 to 1.0% calcium hydroxide to the discharged squeezed rice cake, the mixture is squeezed at 30 to 40 kg / cm ^2. To do. To this, for example, a carbohydrate hydrolase such as pectinase, cellulase, hemicellulase or the like derived from a microorganism such as Aspergillus niger is added and allowed to act at 25 to 50 ° C. for 10 to 36 hours. 1 to 4 times as much water is added to the centrifugal supernatant of this enzyme solution, adjusted to pH 11.0 to 12.5 using calcium hydroxide or sodium hydroxide, stirred for about 1 hour, and then centrifuged to obtain a supernatant. As a result, a hesperidin extract is obtained. This is adjusted to pH 4.5-6.0 with concentrated hydrochloric acid, heated to 50-80 ° C., and the precipitated crystals are collected by centrifugation to obtain crude hesperidin. In the present invention, this crude hesperidin can be used as it is, but it can be recrystallized and used with higher purity.

  Hesperidin powder has a pale yellow color and a unique flavor, but it is weak and can be eaten without problems by adding to other food ingredients and foods and various processing .

  Naringin is also abundant in citrus grapefruit, summer orange, Daidai, pomelo fruit and pericarp, especially immature fruits, and can be usually extracted from grapefruit, summer tangerine and Daidai pericarp. Naringin's bitterness is very strong, it has a bitter bitterness than quinine, and even when diluted 50,000 times with water, it feels bitter, but it is a good quality bitterness that stimulates appetite as you can feel when you eat citrus fruits etc. Presents.

  As for the preparation of naringin, if a grapefruit, summer tangerine or the like is used in place of Wenzhou tangerine, it is possible to obtain naringin in substantially the same manner as in the case of hesperidin.

  Naringin powder has a pale yellow color and is bitter, but it is of good quality (clean and refreshing bitterness) and can be added to other food materials and foods and various processing can cause problems. Can be taken without.

  In the present specification, the “flavonoid glycoside” means an artificial glycosylation of the flavonoid. The flavonoid contains 1 to 20 sugars such as glucose, xylose, sorbose, galactose, preferably 1 to The one with 6 attached. When the number is more than 20, amylose-like properties appear, and in an aqueous solution, they tend to age easily and cause precipitation. In the present invention, the sugar to be bound to the flavonoid is preferably glucose from the viewpoint of daily intake as a constituent of starch. Furthermore, the flavonoid glycoside used in the present invention may be monoglucoside or diglucoside. Furthermore, the same effect is exhibited by a mixture of monoglucoside and oligoglucoside.

  As such flavonoid glycosides, hesperidin glycosides, naringin glycosides, glycosides such as diosmin and rutin, and glycosides such as hesperetin, naringenin, diosmethine and quercetin are preferable, alone or in combination. Can be used. Among these, hesperidin glycoside and naringin glycoside are more preferable.

  The glycosyl transfer reaction for the production of glycoside can be performed under normal conditions. For example, as long as the glycosyltransferase is a glycosyltransferase, any of cyclodextrin synthase, amylase, etc. can be used (T. Kometani, et al, Biosci. Biochem., 58, 517-520 (1994)). T. Kometani, et al, Biosci. Biotechem., 58, 1990-1994 (1994), T. Kometani, et al, Biosci. Biotech. Biochem., 60, 645-649 (1996)). At this time, the origin of the enzyme does not matter. In addition, hesperidin and naringin can be glycosylated by organic chemistry or glycosylation using plant tissue cells (E. Lewinsohn, et al., Phytochemistry, 25, 2531-2535). (1986)).

  In addition, when an enzyme derived from rat small intestine is allowed to act on a flavonoid glycoside solution, it is easily decomposed into flavonoids. Therefore, flavonoid glycosides are considered to be very safe substances like flavonoids.

  A flavonoid glycoside, for example, a glycoside having about 1 to 20 glucoses in hesperidin is prepared, for example, under the following conditions described in JP-A-5-256141.

  To a substrate solution having a pH of 8.0 to 10.5 containing 0.5 to 10% by weight of soluble starch as a sugar donor and 0.1 to 5.0% by weight of hesperidin as an acceptor, for example, an alkalophilic Bacillus species ( Alkalophilic Bacillus sp.) A2-5a (National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession No. FERM P-13864) produced by alkali-resistant cyclodextrin glucanotransferase (hereinafter referred to as “CGTase”), that is, 1,4-α -D-glucan; 4-α-D- (1,4-glucano) -transferase (EC 2.4.4.19) was added to 0.1 to 10 units / ml, and 25 Start the reaction at ~ 50 ° C. After reacting for 4 to 48 hours, the reaction is stopped by heating at 90 to 100 ° C. for 10 to 30 minutes. After performing the enzyme reaction, it can be pulverized by operations such as freeze-drying and spray-drying, and a crude hesperidin glycoside in which 70 to 80% of hesperidin added to the reaction solution is glycosylated is obtained.

  The purity of the hesperidin glycoside in this crude hesperidin glycoside varies depending on the amount of enzyme to be added, the sugar donor and acceptor concentrations, etc., but is usually about 2 to 50%. Unreacted hesperidin and dextrin Contains. The hesperidin glycoside thus produced is a mixture in which about 1 to 20 glucoses are bound to one molecule of hesperidin.

  In the enzyme reaction, the reaction is carried out in the alkaline region of pH 9.5. In addition to this method, (a) β-cyclodextrin (hereinafter referred to as β-cyclodextrin) is used as a method for increasing the solubility of hesperidin and increasing the productivity. CD)) is added to the reaction solution to include β-CD and hesperidin, and (b) a thickening polysaccharide such as methylcellulose is added to solubilize hesperidin. Moreover, the solubility of hesperidin can also be increased by combining these methods.

  The mixture thus obtained may be used as it is as the hesperidin glycoside of the present invention, and may be purified by column chromatography using a hydrophobic resin or the like, if necessary.

  As a purification method, for example, the reaction solution after completion of the enzyme reaction is passed through column chromatography using Amberlite XAD-16 resin, etc., and sufficiently washed with water to remove non-adsorbed fractions such as unreacted dextrin. Fractions eluting with 50% ethanol are obtained. It can be concentrated to a liquid form or dried to a powder form. The obtained sample contains about 20% unreacted hesperidin, and the purity as a hesperidin glycoside is about 80%.

  The hesperidin glycoside thus obtained has about 1 to 20 glucose bound to the original hesperidin, and is further treated with β-amylase and the like, and then separated using a gel filtration column such as Sephadex LH20. Thus, hesperidin having one, two and three glucoses bonded thereto can be obtained. Their purity is 100%.

  In the mixture of hesperidin glycoside and hesperidin used in the present invention, the content of hesperidin glycoside is 10% or more, preferably 20% or more, more preferably 50% or more, based on the entire mixture.

  The color of the sample is powdery and slightly yellowish, but almost tasteless and odorless. Moreover, it is stable between pH 3-13. Neutral to heating, stable at 120 ° C. for 15 minutes.

  In addition, when an enzyme derived from the rat small intestine is allowed to act on a hesperidin glycoside solution, hesperidin glycoside is easily decomposed into hesperidin. Thus, hesperidin glycoside is considered to be a very safe substance like hesperidin.

Furthermore, there are no deaths in acute toxicity tests on rats, and no abnormalities are observed in biochemical blood tests and histopathological examinations. Therefore, the hesperidin glycoside of the present invention is non-toxic. . Further, LD ₅₀ of hesperidin glycoside is 2000 mg / kg rat, not observed even mutagenicity.

  Naringin glycosides are also produced in the same manner as hesperidin glycosides, and the structure is also similar to that obtained by binding about 1 to 20 glucoses to one naringin molecule. Furthermore, since the property expected as a glycoside is similar to that of hesperidin glycoside, it can be used in the same manner as hesperidin glycoside when used for the purpose of the present invention.

  The dosage of flavonoids and / or flavonoid glycosides as the active ingredient varies depending on the type of flavonoid and / or flavonoid glycoside used, when two or more are used, the combination thereof, and the type and degree of the target disease. However, it is desirable to use in the range of 1-1000 mg / kg / day, more preferably 10-500 mg / kg / day. If the dose is less than 1 mg / kg / day, the effect tends to be difficult to appear, and if it is more than 1000 mg / kg / day, there is no concern about side effects. There is a tendency to require intake of a high concentration or a large intake. When the concentration is high, the flavor unique to the flavonoid and / or flavonoid glycoside may appear, and it tends to be difficult to drink.

  When the flavonoid and / or flavonoid glycoside is contained in a food or drink, the content of the flavonoid and / or flavonoid glycoside is preferably 1 to 100% by weight, more preferably 10 to 80% by weight. It is desirable to do. If the content is less than 1% by weight, a slightly larger amount is required to ingest the necessary amount, and if it is greater than 80% by weight, its unique flavor may appear.

  Moreover, in this invention, it is preferable to coexist a flavonoid and a flavonoid glycoside from the point of solubilizing a slightly soluble flavonoid. In this case, an additive or synergistic effect can be obtained as compared with the case where flavonoids are used alone, and the dose can be greatly reduced to reduce the burden on the living body.

  Since flavonoid glycosides solubilize poorly soluble flavonoids, the absorption is improved when a mixture of flavonoid glycosides and flavonoids is administered. That is, it is absorbed faster and the absorption efficiency is higher than the flavonoid single administration. For example, a mixture of hesperidin and hesperidin glycoside can exhibit an effect equal to or greater than 1/10 of the amount compared to single administration of hesperidin. Furthermore, a mixture of flavonoid glycosides and flavonoids that have not been absorbed in the small intestine migrates to the large intestine, where they are broken down into aglycones by the intestinal flora and absorbed. In this case, flavonoids are more advantageous than flavonoid glycosides. Therefore, it is considered that when these mixtures are used, absorbability to a living body is improved and physiological effects are also improved.

  When the flavonoid and flavonoid glycoside are used in combination, the ratio of the flavonoid glycoside to the total weight of the flavonoid and flavonoid glycoside is preferably 5% by weight or more, and more preferably 20% by weight or more. When the proportion of the flavonoid glycoside is less than 5% by weight, a hardly soluble flavonoid tends to precipitate.

  In addition, the flavonoid and / or flavonoid glycoside as an active ingredient can be administered alone in any form such as a solution, powder granule, tablet, emulsion, jelly, etc., but it should be added to various foods and drinks. Can do.

  Furthermore, since flavonoids and / or flavonoid glycosides have high thermal stability and pH stability as described above, they can be used in a wide variety of foods and drinks.

  The food and drink in the present invention may be anything as long as it can be taken orally, and specifically, Japanese confectionery, Western confectionery, ice confectionery, beverages, spreads, pastes, seasonings, pickles, canned bottles, processed meat storage products , Processed fish / fishery products, processed milk / eggs, processed vegetables, processed fruits, processed cereals, and the like. Of these, processed vegetables, fruit processed products, foods with vegetable and fruit flavors, and hesperidin glycosides have the effect of improving bitterness and taste, in terms of familiarity with hesperidin glycosides. As it has been clarified, reducing the blue odor, acidity or astringency of vegetable beverages, reducing the bitterness, astringency or odor of foods and drinks containing herbal medicines, reducing the acidity or astringency of cocoa products To add to the health-oriented foods and drinks that have a health image such as fruits, vegetables, cacao, and honey, but have a slightly disgusting taste and are difficult to drink, with the aim of reducing the taste and taste of honey products It is also possible to improve the flavor.

  Next, the present invention will be described in more detail based on examples, but the present invention is not limited to such examples.

Production Example 1 <Production of Hesperidin Glycoside>
A tris-HCl buffer solution of pH 9.5 containing 5% by weight of soluble starch (manufactured by Merck), 3% by weight of hesperidin (manufactured by Sigma) and 0.1% of methylcellulose (manufactured by Wako Pure Chemical Industries, Ltd.) CGTase (Method JP-A-7-107972) purified from Bacillus cells by the method was added to 2 units / ml and reacted at 40 ° C. for 16 hours. Subsequently, it heated at 100 degreeC for 10 minute (s), and the enzyme reaction was stopped. The resulting reaction mixture was purified by column chromatography using Amberlite XAD-16 resin (Falzbauer) to remove unreacted dextrin and methylcellulose. Subsequently, spray drying was performed to obtain a powder of hesperidin glycoside. The purity of the obtained powder sample was 80% as a hesperidin glycoside.

  The color of the sample was powdery and slightly yellowish, but almost tasteless and odorless. Moreover, it was stable between pH 3-13. Neutral to heating was stable at 120 ° C. for 15 minutes.

  The hesperidin glycoside thus produced was a mixture in which 1 to 10 or more glucoses were bonded to one molecule of hesperidin.

  The hesperidin glycoside mixture powder sample obtained here was used as the hesperidin glycoside in the following examples.

Production Example 2 <Production of Naringin Glycoside>
A reaction was carried out in the same manner as in Production Example 1 except that 3% by weight of hesperidin was changed to 3% by weight of naringin (manufactured by Sigma) to obtain a powder of naringin glycoside.

  The naringin glycoside thus produced was a mixture in which 1 to 10 or more glucoses were bonded to one naringin molecule. The purity of the obtained powder sample was about 80%.

  The color of the sample is powder and slightly yellowish. It was almost odorless, and the original refreshing bitterness was retained even after glycosylation. Moreover, it was stable between pH 3-11. Neutral to heating was stable at 120 ° C. for 15 minutes.

  The naringin glycoside mixture powder sample obtained here was used as naringin glycoside in the following examples.

Examples 1-10
9-week-old male DBA / 1J mice were used as experimental animals. Breeding conditions were a humidity of 50 ± 10%, a room temperature of 23 ± 2 ° C., and a light / dark cycle of 12 hours, and drinking water and basic food (trade name: CE-2, manufactured by Clea Japan) were freely fed. The composition of CE-2 is 45.5% carbohydrate, 24.8% protein, 4.6% fat, 7.2% ash, 4.2% cellulose, 3.9% mineral, 1% vitamin mixture, 8% water. 8% and does not contain any flavonoids or flavonoid glycosides.

  Collagen-induced arthritis (hereinafter referred to as CIA), which is generally used as a model of RA, was induced as follows. 8 mg of type II collagen (manufactured by Sigma, derived from chicken sternall cartilage) was dissolved in 4 ml of 10 mM acetic acid. An equal amount of this solution and complete Freund's adjuvant were mixed to prepare a W / O emulsion, and 100 μl was administered to the tail base of a mouse preliminarily raised for 1 week (this is defined as the following days as day 0). . Similar treatment was performed again on day 21.

  Thus, oral administration of the composition formulated in the composition of Table 1 to the mice in which CIA was induced was started on the 21st day, and was performed 3 times a week at a dose of 3 mg / mouse (body weight 20 g). For example, the administration was performed 6 times over 2 weeks in Examples 1 and 2 and 18 times over 6 weeks in Example 4. Only distilled water for injection (Otsuka Pharmaceutical Co., Ltd.) was orally administered to the positive control group. Arthritis symptoms appearing in mice were monitored and clinical scores were measured three times a week. Depending on the symptoms, the clinical score was 0 = normal, 1 = redness, 2 = slight swelling, 3 = moderate swelling, 4 = severe swelling, 5 = joint stiffness / loss of function. A score was given to each of the four limbs, and the total was used as the clinical score.

  In each administration group of Examples 1 to 10, the clinical score was significantly suppressed compared to the CIA positive control group. The results for Examples 1, 2, and 4 are shown in FIGS. The CIA positive control group was confirmed to have a marked increase in clinical score after day 23. On the other hand, in the hesperidin, naringin and hesperidin glycoside administration groups, the clinical score was significantly reduced compared to the positive control group (FIGS. 1 to 3). In the CIA positive control group, the onset group was 100%, whereas hesperidin administration and naringin administration were 50% (Examples 1 and 2).

Example 11
CIA-derived mice were produced in the same manner as in Example 1.

  Hesperidin was orally administered to 5 mice induced with CIA from the 28th day when the onset was observed, and was administered 3 times a week at a dose of 3 mg / mouse (body weight 20 g) for a total of 9 times over 3 weeks. In the positive control group, physiological saline alone was orally administered to 6 mice in which CIA was induced. Arthritis symptoms appearing in the mice were monitored, and clinical scores were measured three times a week in the same manner as in Example 1. The results are shown in FIG.

  In the administration group, immediately after the start of administration, the clinical score was the same as that of the CIA positive control group, but showed a decreasing tendency from the vicinity of the 32nd to 35th days with respect to the positive control group (FIG. 4).

Example 12
CIA-derived mice were produced in the same manner as in Example 1.

  Oral administration of naringin to five mice induced with CIA was started from the 31st day when the onset was observed, and was administered 3 times a week at a dose of 3 mg / mouse (body weight 20 g) for a total of 8 times over 3 weeks. Five animals in the positive control group were orally administered with distilled water for injection only. Arthritis symptoms appearing in the mice were monitored, and clinical scores were measured three times a week in the same manner as in Example 1. The results are shown in FIG.

  In the administration group, the clinical score was the same as that of the CIA positive control group immediately after the start of administration, but showed a decreasing tendency from the CIA positive control group from around 35 days (FIG. 5). Furthermore, a significant difference was observed after 40 days (FIG. 5).

Example 13
CIA-derived mice were produced in the same manner as in Example 1.

  Hesperidin was orally administered at the same time as the second type II collagen administration (day 21), and each dose (0.3 mg, 1 mg, 3 mg / mouse (body weight 20 g)) was administered three times a week for a total of 11 weeks. Performed once. The number of mice was 5, 5 and 9 for each dose. Only 18 distilled water for injection was orally administered to 18 animals in the positive control group. The symptoms of arthritis appearing in the mice were monitored, and clinical scores were measured three times a week in the same manner as in Example 1. In the administration group, the clinical score decreased in a dose-dependent manner relative to the CIA positive control group (FIG. 6).

Example 14
CIA-derived mice were produced in the same manner as in Example 1.

  Oral administration of hesperidin (3 mg / 20 g body weight) or an equimolar mixture of hesperidin and hesperidin glycoside (equivalent to 0.3 mg of hesperidin / 20 g body weight) occurred in CIA-induced mice (6 mice in each group), respectively. The test was started on the 28th day when the above was observed, and was performed 3 times a week for a total of 12 times over 4 weeks. Six animals in the positive control group were orally administered with distilled water for injection only. Arthritis symptoms appearing in the mice were monitored, and clinical scores were measured three times a week in the same manner as in Example 1. The results are shown in FIG.

  The hesperidin administration group and the equimolar mixture administration group of hesperidin and hesperidin glycoside showed a marked decrease in clinical scores compared to the CIA positive control group, but further administration of an equimolar mixture of hesperidin and hesperidin glycoside The group showed a lower clinical score than the 10-fold hesperidin administration group (FIG. 7).

Examples 15 and 16
A juice obtained by adding 0.1% hesperidin or 0.2% hesperidin glycoside to orange juice (100% fruit juice) prepared through normal squeezing, sterilization, and filling steps was prepared. With the addition of hesperidin or hesperidin glycoside, it was possible to produce orange juice with little effect on taste and aroma.

Examples 17 and 18
Concentrated reduced orange juice obtained by reducing frozen concentrated orange juice (trade name: Valencia Orange 5-fold concentrated fruit juice # 4000N, importer: Oyama Corporation), 0.2% hesperidin or 0.2% hesperidin glycoside The body was added to make juice. With the addition of hesperidin or hesperidin glycoside, it was possible to produce orange juice with little effect on taste and aroma.

Examples 19 and 20
A candy containing 70 parts of granulated sugar, 30 parts of starch syrup, 20 parts of water, 1 part of acidulant, flavoring and coloring was prepared. The granulated sugar was heated to 110 ° C. while completely dissolved in water, and the temperature was raised to 125 ° C. by adding waterpox. Further, after simmering at 145 ° C., it was poured on a cooling plate and mixed with acidulant, followed by flavor and colorant, 0.2 part of hesperidin or 0.2 part of hesperidin glycoside, and molded as a candy. Even if hesperidin or hesperidin glycoside was added, a candy having almost no influence on taste and aroma could be produced.

Examples 21 and 22
300 parts whole egg, 200 parts soft flour, 200 parts sugar, 80 parts edible oil and fat, 30 parts emulsified oil and fat, 0.6 parts salt, 4 parts baking powder, 0.5 part hesperidin or 0.5 part hesperidin glycoside The dough was mixed by the method, and the dough was adjusted to a dough specific gravity of 0.50. This was fired at 180 ° C. for 30 minutes to produce a sponge cake. Even if hesperidin or hesperidin glycoside was added, a cake having almost no influence on taste and aroma could be made.

Examples 23 and 24
75 parts of salad oil, 25 parts of vinegar, 1 part of salt, and a proper amount of spice were well stirred at 1000 rpm to obtain a normal French dressing. On the other hand, 0.3 parts of hesperidin or 0.3 part of hesperidin glycoside was added to this formulation to obtain a French dressing to which hesperidin or hesperidin glycoside was added.

  When eaten over a vegetable salad, the same taste, texture and physical properties as the conventional product were obtained.

Examples 25 and 26
A normal pudding was prepared according to a conventional method by blending 5 yolks, 4 eggs, 150 g granulated sugar and 700 g milk. A hesperidin or hesperidin glycoside-added purine was prepared by adding 2 g of hesperidin or hesperidin glycoside to the formulation.

  As a result of examining the taste of the normal purine and hesperidin or hesperidin glycoside-added pudding for 15 panelists, the purine added with hesperidin or hesperidin glycoside also had the same taste as the normal purine.

Test Example 1 <Digestibility of Hesperidin Glycoside>
The enzyme was extracted from 1 mg of acetone powder (manufactured by Sigma) of rat small intestine with 1 ml of distilled water, the extract was centrifuged at 5000 g for 5 minutes, and the supernatant was used as a digestive enzyme solution. As the hesperidin glycoside, a mixture containing approximately 1: 1 hesperidin monoglucoside and hesperidin diglucoside dissolved in distilled water at 20 mg / ml was used. 50 μl of hesperidin glycoside solution was added to 50 μl of the enzyme solution, incubated at 37 ° C., and analyzed by HPLC over time. As a result, hesperidin monoglucoside was completely degraded in 1 hour and hesperidin diglucoside was completely degraded in 4 hours.

  Thus, when an enzyme derived from rat small intestine was allowed to act on a hesperidin glycoside solution, hesperidin glycoside was easily decomposed into hesperidin, so that hesperidin glycoside is considered to be a very safe substance like hesperidin.

Test example 2
The mice in the hesperidin administration group treated in Example 1 and the mice in the CIA positive control group were dissected on the 35th day after the first type II collagen administration. The blood obtained by heart blood collection was centrifuged at 10,000 rpm for 1 minute, and the supernatant obtained by centrifuging again at 10000 rpm for 1 minute was used for (1) measurement of anti-type II collagen IgG titer. Also, lymph nodes were collected, and the obtained cells were suspended in RPMI 1640 medium, adjusted to 2 × 10 ⁶ cells / ml, dispensed at 1 ml / well into a 24-well plate, and dissolved in 10 mM type II Collagen 50 μg / ml or purified tuberculin (PPD) 50 μg / ml prepared from defatted cells of Mycobacterium tuberculosis (H37Ra) was added and stimulated for 72 hours. The culture supernatant was collected, and this was used as a test sample, and (2) TNF-α concentration was measured. Furthermore, the joint tissue was used for (3) pathological analysis and (4) mRNA analysis. Each experimental operation is as follows.

(1) Measurement of anti-type II collagen IgG titer
A serially diluted test sample was added to an ELISA plate coated with type II collagen and allowed to stand for 1 hour, and then reacted with an HRP (horse radish peroxidase) -labeled anti-mouse IgG antibody for 1 hour. Color was developed using TMB (3,3 ′, 5,5′-tetramethylbenzidine) as a substrate, and then the absorbance at 450 nm was measured. When the anti-type II collagen IgG titer in plasma was compared, the anti-type II collagen IgG titer was slightly suppressed in the hesperidin administration group, but this was not a significant difference.

(2) Measurement of TNF-α concentration L929 (C5F6) cells were dispensed into a 96-well plate and cultured for 4 hours. Actinomycin D and a diluted test sample were added, followed by further incubation for 16 hours. Using recombinant mouse TNF-α as a standard sample, TNF-α of the test sample was calculated from the dilution rate of the test sample showing 50% cytotoxic activity and the concentration of the standard sample. TNF-α production was hardly suppressed relative to the CIA positive control group.

(3) Pathological analysis of joint tissue The rear knee joint was fixed with 4% paraformaldehyde buffer, decalcified with EDTA (pH 7.4), sliced, stained with HE (hematoxylin-eosin), and observed with a microscope. . The results are shown in FIGS. 8 (a) and (b). Regarding the joint tissue, in the CIA positive control group, the proliferation 2a and multi-layering of the synovial tissue are remarkable, and cell infiltration of lymphocytes 6, neutrophils, epithelioid cells 8 and the like is remarkable in the subsynovial tissue. In addition, precipitation of fibrin 7 was also observed (FIG. 8 (a)). On the other hand, such findings were suppressed in the hesperidin administration group (FIG. 8 (b)).

(4) Analysis of TNF-α mRNA After extracting total RNA from joint tissue cells and synthesizing cDNA, PCR was performed using TNF-α specific primers. The PCR product was confirmed by 2% agarose gel electrophoresis. The results are shown in FIG. Expression of TNF-α mRNA in the joint tissue of the hesperidin administration group was suppressed compared to the CIA positive control group.

  From the results of the experiments (1) to (4) above, it was revealed that CIA can be suppressed by oral administration of hesperidin. In the hesperidin administration group, cell infiltration and synovial inflammation were suppressed in the joint tissue. Moreover, the expression of TNF-α mRNA in the joint tissue was suppressed. There was no difference between the CIA positive control group and the TNF-α production ability and anti-type II collagen IgG titer of lymph node cells in vitro. From these results, it is considered that administration of hesperidin did not act on T cells, but acted on macrophages infiltrating the inflammatory site to suppress TNF-α production, thereby suppressing the onset of CIA.

Test example 3
Mice treated with Examples 11 and 12 and mice in the CIA positive control group were dissected 49 days after the first type II collagen administration. The blood obtained by heart blood collection was centrifuged at 10,000 rpm for 1 minute, and the supernatant obtained by centrifuging again at 10000 rpm for 1 minute was used for (1) measurement of anti-type II collagen IgG titer. The joint tissue was used for (2) histopathological observation, (3) analysis of TNF-α mRNA, and (4) measurement of COX-2 (cyclooxygenase-2). Each experimental operation is as follows.

(1) Measurement of anti-type II collagen IgG titer The measurement was performed in the same manner as the measurement of anti-type II collagen IgG titer in Test Example 2. The anti-type II collagen IgG titer in plasma was slightly suppressed in the hesperidin and naringin group compared with the positive control group, but it was not a significant difference.

(2) Histopathological observation of joint tissue <Sample preparation and staining>
The posterior knee joint was fixed with 4% paraformaldehyde / phosphate buffer and then decalcified with an EDTA-sodium aqueous solution (pH 7.4) to prepare a continuous section having a thickness of 3 μm. For staining, HE (hematoxylin-eosin) staining, fibrin staining PTAH (phosphotungstic acid hematoxylin) staining, and collagen fiber staining AZAN staining were performed and observed under a microscope. In order to examine the localization of TNF-α producing cells, immunohistochemical analysis was performed using the ABC (avidin-biotin complex) method. Methyl green staining was performed as a counterstaining.

<Observation results>
Determination of CIA onset was performed based on the histological findings of each mouse. The results were scored 0-4 according to the following histological criteria.
(A) Articular cartilage damage: frequency of articular cartilage destruction and collagen fiber hyperplasia (b) synovial hyperplasia: scope and extent of synovial hyperplasia (c) inflammatory cell infiltration: frequency of inflammatory cell appearance and formation of lymphoid follicles ( d) Pannus growth: degree of pannus formation, adipose tissue loss

  The results are shown in FIG.

  Compared to the CIA positive control group, hesperidin and naringin administration group often had adipose tissue remaining at the synovial fold site, articular cartilage damage, synovial hyperplasia, synovial surface inflammatory cell infiltration, pannus hyperplasia All were suppressed. In particular, in the hesperidin administration group, synovial hyperplasia (P <0.05), inflammatory cell infiltration (P <0.05) and pannus proliferation (P <0.001) were significantly suppressed. Similarly, in the naringin administration group, suppression of cartilage damage, inflammatory cell infiltration, and pannus growth (P <0.05) was significant (FIG. 10).

  In the staining of TNF-α-producing cells by the ABC method, TNF-α-producing cells were observed in the periphery of the lymphoid follicle formation of the joint capsule in the CIA positive control group, but in the heparidin and naringin administration group in the same part, inflammatory cell infiltration was observed. Lymphoid follicle formation was not observed, and TNF-α producing cells could not be confirmed.

(3) Analysis of TNF-α mRNA The analysis was performed in the same manner as the analysis of TNF-α mRNA in Test Example 2.

  The expression of TNF-α mRNA in the joint tissues of the hesperidin administration group and naringin administration group was suppressed compared to the CIA positive control group.

(4) COX-2 measurement After crushing the ankle joint (approximately 200 mg) of each administration group and positive control group, the mice were washed twice with 20 μl heparin-containing Tris buffer (pH 7.4), and 1 ml cold buffer ( 0.1M Tris-hydrochloric acid, pH 7.8, 1 mM ethylenediaminetetraacetic acid (EDTA), 250 mM mannitol, 0.3 mM diethyldithiocarbamic acid) was added, and the mixture was homogenized with a polytron homogenizer. The supernatant after centrifugation at 13,000 rpm for 15 minutes at 4 ° C. was concentrated with Microcon 30 (Amicon), and the level of COX-2 was measured using a COX-2 activity assay kit (Cayman Chemical).

  There was no significant difference in COX-2 levels in day 49 tissues.

  From the results of the experiments (1) to (4) above, it has been clarified that CIA can be treated by oral administration of hesperidin or naringin after CIA has developed. In the hesperidin administration group, cell infiltration and synovial inflammation were suppressed in the joint tissue. Moreover, the expression of TNF-α mRNA in the joint tissue was suppressed. There was no difference in anti-type II collagen IgG titer in vitro with the CIA positive control group. In addition, there was no significant difference in COX-2 levels in the tissues. From these facts, it was speculated that hesperidin and naringin suppress inflammation by a mechanism different from that of normal anti-inflammatory drugs such as aspirin.

Test example 4
BALB / c mice (13 mice) were used, and 5 ml of air sterilized with a 0.22 μm filter was injected into the back. From this day, hesperidin was orally administered to 7 mice at a daily dose of 3 mg / mouse for 7 days. The positive control group (6 mice) was orally administered with distilled water for injection. Subsequently, 3 ml of sterilized air was injected again 3 days after the injection of air. After 3 days, stimulation with 0.3 ml of 1% (w / v) Zymosan A (manufactured by Wako Pure Chemical Industries, Ltd.) suspension / saline solution has infiltrated into the air-pouch after 12 and 24 hours. A part of the cells was collected using a 3 mM EDTA-containing RPMI1640 medium, the number of cells was stained with Turk's solution and measured with a hemocytometer, and the cell structure was examined by staining smears with Maygrunwald Giemsa stain. Similarly, the cell structure of peripheral blood leukocytes obtained by cardiac blood sampling was examined with a smear.

The cell composition of peripheral blood leukocytes 12 hours after stimulation with Zymosan was slightly decreased in monocytes, but there was no significant difference (positive control group n = 3: 22.3 ± 4.2%, hesperidin administration group n = 3: 13.3 ± 4.5%). The number of cells infiltrating into the air-pouch was almost the same in the positive control group (6.72 × 10 ⁷ ) and the hesperidin administration group (6.98 × 10 ⁷ ), but the ratio of macrophages was hesperidin administration. The group was significantly less than the positive control group (positive control group n = 3: 31.3 ± 4.2%, hesperidin administration group n = 3: 20.0 ± 2.6%; P <0.02 ).

  It should be noted that there was a significant difference in the cell configuration of peripheral blood leukocytes, the number of cells infiltrating into Air-pouch and the cell configuration in the positive control group (n = 3) and the hesperidin administration group (n = 4) 24 hours after Zymosan stimulation Was not recognized.

  From the results of the Air-pouch experiment and the pathological analysis of the joint tissue, it was inferred that administration of hesperidin or naringin suppresses macrophage infiltration into the inflamed area of arthritis. Since the infiltration of macrophages was specifically suppressed, it was speculated that the production of chemokines that specifically act on macrophages such as MCP-1 was suppressed.

DESCRIPTION OF SYMBOLS 1 Synovial surface 2 Synovial tissue 2a Proliferated synovial tissue 3 Synovial fluid 4 Cartilage 5 Bone tissue 6 Lymphocyte infiltration 7 Fibrin 8 Neutrophil, epithelioid cell infiltration 9 Joint capsule

Claims (4)

A food and drink for improving symptoms of rheumatism containing hesperidin and hesperidin glycoside. The food or drink according to claim 1, wherein the dose is 1-1000 mg / kg / day in terms of the amount of hesperidin and hesperidin glycoside. A food or drink for suppressing the expression of TNF-α containing hesperidin and hesperidin glycoside. The food or drink according to claim 3, wherein the dose is 1 to 1000 mg / kg / day in terms of the amount of hesperidin and hesperidin glycoside.

Images