Pharmaceutical composition for chronic venous disease

Abstract

This pharmaceutical composition for chronic venous disease includes one or more diosmetin compounds selected from the group consisting of a diosmetin-7-glucoside-cyclodextrin inclusion compound and a diosmetin glycosyltransferase of formula (1). The pharmaceutical composition can be used for the prevention or treatment of chronic venous disease.

Description

Pharmaceutical composition for chronic venous disease

[0001] The present invention relates to a pharmaceutical composition for chronic venous disease and a method for preparing the same.

[0002] When humans walk, leg muscles compress the veins in their legs and feet, helping blood return to the heart. When the leg muscles relax, valves inside the veins close, preventing blood from flowing backward down the legs. This entire process is called the muscle pumping action. However, sitting or standing for long periods of time can lead to chronic venous insufficiency (CVI). CVI, also known as chronic venous disease (CVD), is a condition in which leg veins are damaged and blood does not flow normally, resulting in leg discomfort, swelling, skin rashes, discoloration, and ulcers. It is thought to be caused by varicose veins or deep vein thrombosis.

[0003] Varicose veins are a symptom in which veins swell like lumps, and the number of patients with varicose veins in Japan is estimated to be over 10 million (Non-Patent Document 1), with one in two adult women who have given birth reportedly suffering from the condition (Non-Patent Document 2). Furthermore, a survey report found that 8.6% of 9,123 people surveyed over the age of 40 (average age 62.4 years) had varicose veins (Non-Patent Document 3).

[0004] Furthermore, humans have three types of veins: superficial veins located near the skin, deep veins located in muscle groups, and perforating veins that connect the superficial veins and deep veins. In deep vein thrombosis (DVT), blood coagulates in the deep veins of the lower limbs, forming a blood clot that blocks the blood vessels, causing symptoms such as acute swelling of the lower limbs, pain, and changes in skin color, and the blood clot may become dislodged, causing acute pulmonary thromboembolism (PTE) (Non-Patent Document 4).

[0005] The flavonoid diosmin has antioxidant, antiradical, and anti-inflammatory effects and has been reported to be effective against symptoms associated with "chronic venous insufficiency," such as varicose veins, venous edema, phlebitis, leg swelling, venous hypertension, venous ulcers, venous thrombosis, and hemorrhoids (Non-Patent Documents 5 to 8). In addition, the flavonoid hesperidin has also been reported to have antioxidant effects and, together with diosmin, promote healthy vascular function (Non-Patent Document 8).

[0006] Examples of pharmaceuticals include "Daflon" (diosmin 90%, hesperidin etc. 10%, Servier), which is used worldwide as a treatment for various chronic venous insufficiency (Non-Patent Document 5).

[0007] Although diosmin can be isolated from various citrus plants, its content in citrus fruits is extremely low. Therefore, diosmin used in pharmaceuticals is produced from hesperidin by oxidative dehydration using iodine, bromine, pyridine dimethyl sulfoxide (DMSO), etc. (Patent Document 1, Non-Patent Documents 9 and 10).

[0008] On the other hand, in Patent Document 2, the present inventors have proposed a method for producing a flavonoid inclusion compound, which includes an elimination step of treating a poorly soluble flavonoid having rutinose (a disaccharide consisting of rhamnose and glucose) with an enzyme having rhamnosidase activity in the presence of cyclodextrin to eliminate rhamnose, with the aim of improving the solubility of the poorly soluble flavonoid. However, there is no disclosure of its use in the prevention or treatment of chronic venous diseases.

[0009] WO2010 / 092592A2 Patent No. 6925381

[0010] "You can treat varicose veins and swelling in your lower extremities yourself," Gakken Plus, (2016) "Pregnancy and varicose veins," Phlebology, 255-261, (1997) "Overview of the ELVeS laser, a semiconductor laser device for treating varicose veins in the lower extremities," Journal of the Japanese Society for Laser Surgery and Medicine, 33(1), (2012) THE JOURNAL of JAPANESE COLLEGE of ANGIOLOGY, 49, 195-200, (2009) Phlebolymphology, 23(2), 82-91, (2016) Biomedicines, 10(5), 1076, (2022) Food & function, 11(10), 8472-8492, (2020) Bioscience, Biotechnology, and Biochemistry, 87(7), 771-776, (2023) J.Chem.Soc. 12,19, (1978)Org. Commun, 12, 101-108, (2019)

[0011] The present invention relates to providing novel pharmaceutical compositions for chronic venous disease and methods for their preparation.

[0012] The present invention relates to the following items [1] to [4]: ​​[1] A pharmaceutical composition for chronic venous disease, comprising one or more diosmetin compounds selected from the group consisting of diosmetin-7-glucoside-cyclodextrin inclusion compounds and diosmetin glycosyltransferases represented by the following formula (1): [2] A method for producing a pharmaceutical composition for chronic venous disease, comprising a step of combining one or more diosmetin compounds selected from the group consisting of a diosmetin-7-glucoside-cyclodextrin inclusion compound and a diosmetin glycosyltransferase represented by the following formula (1): [3] A method for preventing or treating chronic venous diseases, using one or more diosmetin compounds selected from the group consisting of diosmetin-7-glucoside-cyclodextrin inclusion compounds and diosmetin glycosyltransferases represented by the following formula (1): [4] A method for producing a diosmetin transglycosylation product of the following formula (1), which comprises a step of subjecting a hesperetin transglycosylation product of the following formula (2) to an oxidative dehydrogenation reaction:

[0013] According to the present invention, a novel pharmaceutical composition for chronic venous disease and a method for producing the same can be provided.

[0014] Diosmetin glycosyltransferase (DIOS-(Glc)) obtained in Test 2 ｎ 1 shows a chromatogram obtained by HPLC of the freeze-dried product of diosmetin glycosyltransferase (DIOS-(Glc) (n=1 to 15)) obtained by β-amylase F treatment in Test 2. １ and DIOS-(Glc) ２ ) is a chromatograph obtained by HPLC.

[0015] As a result of investigating the above-mentioned problems, the present inventors have newly discovered that diosmetin-7-glucoside-cyclodextrin inclusion compounds and specific diosmetin transglycosylation products can be used to prevent or treat chronic venous disease. Furthermore, unexpectedly, it has been found that superior preventive or therapeutic effects can be achieved compared to the use of the conventional therapeutic drug diosmin. While the mechanism behind this is unclear, it is presumed that the inclusion complex with cyclodextrin and transglycosylation improve water solubility and bioavailability.

[0016] The pharmaceutical composition of the present invention is used for the prevention or treatment of chronic venous diseases, and contains as active ingredients diosmetin-7-glucoside-cyclodextrin inclusion compound (DIOSG-CD) and diosmetin glycosyltransferase (DIOS-(Glc)) represented by the following formula (1): ｎ ) (these DIOSG-CD and DIOS-(Glc) ｎ (These may be collectively referred to as "diosmetin compounds"). The diosmetin compounds may be in the form of a hydrate.

[0017] The diosmetin-7-glucoside-cyclodextrin inclusion compound is a compound in which diosmetin-7-glucoside is included in cyclodextrin. Examples of cyclodextrin (CD) include β-cyclodextrin (β-CD), branched β-cyclodextrin (branched β-CD), and γ-cyclodextrin (γ-CD). From the viewpoint of the preventive or therapeutic effect on chronic venous disease, the molar ratio of cyclodextrin to diosmetin-7-glucoside (cyclodextrin / diosmetin-7-glucoside) is preferably 0.9 to 3.0, more preferably 0.9 to 2.0, and even more preferably 0.9 to 1.5.

[0018] The method for producing the diosmetin-7-glucoside-cyclodextrin inclusion compound is not particularly limited, but from the viewpoints of solubility in water and bioavailability, the compound obtained by the method described in Patent Document 2 is preferred. That is, the compound obtained by a elimination step in which diosmin having rutinose (a disaccharide consisting of rhamnose and glucose) is treated with an enzyme having rhamnosidase activity in an aqueous medium at a pH of 3 to 7 in the presence of cyclodextrin to eliminate rhamnose is preferred.

[0019] The amount of diosmin used in the elimination step is not particularly limited, but is preferably 0.1 to 20% by mass, more preferably 1 to 15% by mass, and even more preferably 2 to 14% by mass in the reaction system. Examples of cyclodextrins (CDs) present in the elimination step include β-cyclodextrin (β-CD), branched β-cyclodextrin (branched β-CD), and γ-cyclodextrin (γ-CD). The amount of cyclodextrin present is not particularly limited, but is preferably 0.01 to 60% by mass, more preferably 1 to 50% by mass, and even more preferably 3 to 40% by mass in the reaction system. When two or more types of cyclodextrin are used, the amount refers to the total amount. Enzymes with rhamnosidase activity can be derived from any source, including animals, plants, and microorganisms. Genetically recombinant enzymes are also acceptable. Furthermore, the form of these enzymes is not particularly limited. Specific examples of enzymes with rhamnosidase activity include hesperidinase, naringinase, β-glucosidase, and pectinase. The amount of enzyme with rhamnosidase activity used varies depending on the type of enzyme used, reaction conditions, etc.; for example, in the case of hesperidinase, naringinase, and β-glucosidase, a preferred amount is 0.01 to 1,000 U per 1 g of diosmin. Reaction conditions, such as the reaction temperature and pH of the reaction solution, can be selected according to the characteristics of the enzyme used; however, a pH of 3 to 7 is preferred, and a pH of 3.5 to 6.5 is more preferred. Alternatively, diosmin can be dissolved in an alkaline range and then subjected to the enzymatic reaction at a pH of 7 or lower. Examples of solvents used in the reaction system include aqueous media. Aqueous media refer to water or an aqueous solution of an organic solvent. Examples of water include tap water, distilled water, ion-exchanged water, and purified water. The organic solvent is not particularly limited as long as it is uniformly miscible with water; however, ethanol is preferred from the perspective of its applicability to pharmaceutical compositions. The reaction temperature is preferably 10 to 80° C., more preferably 40 to 75° C. The reaction time is, for example, 1 to 100 hours, or 2 to 24 hours.The desorption step can be carried out in a solvent such as water by standing or stirring, and to prevent oxidation or browning during the reaction, the air in the headspace of the reaction system may be replaced with an inert gas such as nitrogen, or an antioxidant such as ascorbic acid may be added to the reaction system. The desorption step can be completed by a known method, such as heating the reaction solution to inactivate the enzyme.

[0020] This elimination step yields a composition containing a diosmetin-7-glucoside-cyclodextrin inclusion compound and rhamnose (diosmetin-7-glucoside-cyclodextrin inclusion compound-containing composition (DIOSG-CD)). The pharmaceutical composition of the present invention may contain this diosmetin-7-glucoside-cyclodextrin inclusion compound-containing composition as is. That is, the pharmaceutical composition of the present invention may further contain rhamnose. In an embodiment in which the pharmaceutical composition contains rhamnose, the molar ratio of the rhamnose content to the diosmetin compound content (diosmetin-7-glucoside equivalent value) (rhamnose / diosmetin compound) is preferably 0.5 to 3.0, more preferably 0.9 to 2.0, and even more preferably 0.9 to 1.5. Alternatively, rhamnose may be removed from the diosmetin-7-glucoside-cyclodextrin inclusion compound-containing composition to obtain the diosmetin-7-glucoside-cyclodextrin inclusion compound. Rhamnose removal can be achieved by membrane treatment (ultrafiltration membrane treatment, reverse osmosis membrane treatment, zeta potential membrane treatment, etc.), electrodialysis, salting out, acid precipitation, recrystallization, solvent fractionation, etc.

[0021] The diosmetin glycosyltransferase of formula (1) is a compound or composition containing at least one compound in which n, the number of glucose residues, is 1 to 15. That is, the pharmaceutical composition of the present invention may contain a composition containing all of the compounds of formula (1) in which n is 1 to 15, or may contain specific compounds obtained by purifying these compounds, either singly or in combination. From the viewpoint of the preventive or therapeutic effect on chronic venous disease, it is preferable to contain one or more diosmetin glycosyltransferases of formula (1) in which n is 1 to 5. Examples include an embodiment in which a single compound of formula (1) in which n is 1 to 5 is contained, and an embodiment in which two or more compounds in which n is 1 to 5 are contained. In an embodiment in which two compounds in which n is 1 to 5 are contained, the content ratio, in terms of diosmetin-7-glucoside, is preferably 1:10 to 10:1, more preferably 1:5 to 5:1, even more preferably 1:4 to 4:1, and even more preferably 1:3 to 3:1. An embodiment containing a single compound where n is any one of 1 to 5 means an embodiment containing only a compound where n = 1, an embodiment containing only a compound where n = 2, an embodiment containing only a compound where n = 3, an embodiment containing only a compound where n = 4, or an embodiment containing only a compound where n = 5, and an embodiment containing two compounds where n is any one of 1 to 5 means an embodiment containing compounds where n = 1 and n = 2, an embodiment containing compounds where n = 1 and n = 3, an embodiment containing compounds where n = 1 and n = 4, an embodiment containing compounds where n = 1 and n = 5, an embodiment containing compounds where n = 2 and n = 3, an embodiment containing compounds where n = 2 and n = 4, an embodiment containing compounds where n = 2 and n = 5, an embodiment containing compounds where n = 3 and n = 4, an embodiment containing compounds where n = 3 and n = 5, or an embodiment containing compounds where n = 4 and n = 5. Similarly, an embodiment may contain a combination of any three, any four, or any five of n's from 1 to 5.

[0022] From the viewpoint of the preventive or therapeutic effect on chronic venous diseases, the total content of compounds where n is 1 to 5 in the diosmetin transglycosylation product of the above formula (1) (n = 1 to 5 / n = 1 to 15) is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, even more preferably 70% or more, and even more preferably 90% or more, expressed as a molar ratio equivalent to diosmetin-7-glucoside. The upper limit can be, for example, 100% or less, 90% or less, or 80% or less. Furthermore, from the viewpoint of bioavailability, the total content of compounds in which n is 2 to 5 in the diosmetin glycosyltransferase of formula (1) above (n = 2 to 5 / n = 1 to 15) is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, even more preferably 70% or more, and even more preferably 90% or more, expressed as a molar ratio equivalent to diosmetin-7-glucoside. The upper limit can be, for example, 100% or less, 90% or less, or 80% or less.

[0023] In an embodiment comprising a diosmetin-7-glucoside-cyclodextrin inclusion compound and a diosmetin glycosyltransferase of the above formula (1) where n is 1 to 5, the content of the diosmetin-7-glucoside-cyclodextrin inclusion compound:the total content of the diosmetin glycosyltransferase of the above formula (1) where n is 1 to 5 is preferably 1:10 to 10:1, more preferably 1:5 to 5:1, even more preferably 1:4 to 4:1, and even more preferably 1:3 to 3:1, in terms of a molar ratio of diosmetin-7-glucoside.

[0024] Diosmetin glycosyltransferase (DIOS-(Glc)) of the above formula (1) ｎThe method for producing diosmetin-7-glucoside (n = 1 to 15)) is not particularly limited, and it can be prepared, for example, by the method described in Patent Document 2. Specifically, this method includes a step of treating the diosmetin-7-glucoside-cyclodextrin inclusion compound with a glycosyltransferase such as cyclodextrin glucanotransferase (CGTase) to glycoside. The glycosyltransferase is not particularly limited as long as it has the activity of transferring sugars to the diosmetin-7-glucoside-cyclodextrin inclusion compound. Specific examples of glycosyltransferases include cyclodextrin glucanotransferase, glucosyltransferase, α-glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, α-amylase, xylanase, pullulanase, and arabinofuranosidase. The amount of glycosyltransferase used varies depending on the type of enzyme used, the conditions for the glycosyltransferase reaction, the type of sugar, etc.; for example, in the case of cyclodextrin glucanotransferase, 1 to 10,000 U per 1 g of diosmetin-7-glucoside is preferred. From the viewpoints of production efficiency and quality, the enzymatic reaction is preferably performed at a pH of 3 to 7 or a pH of 6 to 6.8, but glycosyltransferase can also be performed in the alkaline range, such as at a pH of 7 to 9. Examples of solvents used in the reaction system include aqueous media. The reaction temperature is preferably 40 to 70°C, more preferably 50 to 65°C. The reaction time varies depending on the type of enzyme, etc., but can be, for example, 0.5 to 120 hours or 1 to 30 hours. From the viewpoint of production efficiency, it is preferable to perform the glycosidation step by adjusting the temperature and pH appropriately and adding the glycosyltransferase continuously after the elimination step. In the glycosidation step, cyclodextrin in the flavonoid clathrate serves as a glycosyl donor, allowing the production of a flavonoid transglycosylated product. However, there is no limitation to the addition of an additional glycosyl donor. Specific examples of glycosyl donors that can be added include starch, dextrin, partial starch hydrolysates such as maltooligosaccharides, xylooligosaccharides, and compounds containing these. As with the desorption step, the glycosidation step can be carried out in a solvent such as water, with standing or stirring. To prevent oxidation or browning during the reaction, the air in the headspace of the reaction system may be replaced with an inert gas such as nitrogen, and an antioxidant such as ascorbic acid may also be added to the reaction system.The glycosidation step can be completed by a known method, such as inactivating the enzyme by heating the reaction solution.

[0025] Diosmetin glycosyltransferase (DIOS-(Glc)) obtained in the glycosidation step ｎ The number of glucose groups bonded (n) in the diosmetin transglycosylation product (n = 1 to 15) can be adjusted as desired. For example, after diosmetin transglycosylation product production, the number of glucose chains in the diosmetin transglycosylation product molecule can be reduced by treating it with various amylases (α-amylase, β-amylase, glucoamylase, α-glucosidase, etc.), either alone or in combination, to obtain diosmetin transglycosylation products with any glucose chain length. Furthermore, the product can be optionally purified by resin treatment processes (adsorption, ion exchange, etc.), membrane treatment processes (ultrafiltration membrane treatment, reverse osmosis membrane treatment, zeta potential membrane treatment, etc.), electrodialysis, salting out, acid precipitation, recrystallization, solvent fractionation, etc. For example, the diosmetin transglycosylation product obtained in the glycosidation process can be adsorbed onto a porous synthetic adsorbent, washed with water, eluted with alcohol, and then spray-dried to obtain a purified powder. Furthermore, after alcohol elution, the composition may contain diluents or other additives as components other than the composition. Thus, diosmetin glycosyltransferase (DIOS-(Glc) ｎ By combining various amylases, resin treatment steps, preparative HPLC, and the like, compounds with the desired number of glucose groups bonded (n number) (compounds with n=1, n=2, n=3, n=4, n=5, etc.) from a soluble saccharide (n=1 to 15) can be isolated and purified, and the composition ratio of each compound can also be adjusted as desired.

[0026] As mentioned above, diosmin, which is used in pharmaceuticals, is produced from hesperidin by oxidative dehydration using iodine, bromine, pyridine, dimethyl sulfoxide (DMSO), etc. It has now been discovered that diosmetin transglycosylation products can also be produced by subjecting hesperetin transglycosylation products to oxidative dehydrogenation in a similar manner. Therefore, the present invention also provides a method for producing diosmetin transglycosylation products of the following formula (1), which comprises the step of subjecting hesperetin transglycosylation products of the following formula (2) to oxidative dehydrogenation.

[0027] Hesperetin glycosyltransferase (HPT-(Glc) ｎ (n=1-15)) is a diosmetin glycosyltransferase (DIOS-(Glc) ｎ (n=1 to 15)), it can be prepared by the method described in Patent Document 2. In addition, hesperetin glycosyltransferase (HPT-(Glc) ｎ Compounds with the desired number of glucose groups (n=1, n=2, n=3, n=4, n=5, etc.) can be isolated and purified from hesperetin diglucoside (HPT-(Glc) (n=1 to 15)) using various amylases, resin treatment steps, and preparative HPLC, and then the isolated compounds can be subjected to an oxidative dehydration reaction. For example, hesperetin diglucoside (HPT-(Glc) ２ ) is subjected to an oxidative dehydrogenation reaction to give diosmetin-diglucoside (DIOS-(Glc) ２ More specifically, the oxidative dehydration reaction can produce HPT-(Glc) ２ with a catalytic amount of iodine (I ２ ) and concentrated sulfuric acid in dimethyl sulfoxide (DMSO) at 100°C for 30 minutes to give DIOS-(Glc). ２ You can get.

[0028] The pharmaceutical composition of the present invention may contain hesperetin-7-glucoside-cyclodextrin inclusion compound (HPTG-CD) or hesperetin glycosyltransferase of the above formula (2) (HPT-(Glc) ｎ (n=1 to 15)) and other compounds (HPTG-CD and HPT-(Glc) ｎ (these may be collectively referred to as "hesperetin-based compounds") may also be included. The hesperetin-based compounds may be in the form of a hydrate.

[0029] The hesperetin-7-glucoside-cyclodextrin inclusion compound in the pharmaceutical composition of the present invention is a compound in which hesperetin-7-glucoside is included in cyclodextrin. Examples of cyclodextrin (CD) include β-cyclodextrin (β-CD), branched β-cyclodextrin (branched β-CD), and γ-cyclodextrin (γ-CD). From the viewpoint of the preventive or therapeutic effect on chronic venous disease, the molar ratio of cyclodextrin to hesperetin-7-glucoside (cyclodextrin / hesperetin-7-glucoside) is preferably 0.9 to 3.0, more preferably 0.9 to 2.0, and even more preferably 0.9 to 1.5.

[0030] The method for producing the hesperetin-7-glucoside-cyclodextrin inclusion compound is not particularly limited, but from the viewpoints of solubility in water and bioavailability, the compound obtained by the method described in Patent Document 2 is preferred. That is, the compound obtained by a elimination step in which hesperidin having rutinose (a disaccharide consisting of rhamnose and glucose) is treated with an enzyme having rhamnosidase activity in an aqueous medium at a pH of 3 to 7 in the presence of cyclodextrin to eliminate rhamnose is preferred. The reaction conditions, etc. are the same as those described above.

[0031] From the viewpoint of the preventive or therapeutic effect on chronic venous diseases, the molar ratio of the content of the hesperetin-based compound (converted to hesperetin-7-glucoside) to the content of the diosmetin-based compound (converted to diosmetin-7-glucoside) (hesperetin-based compound / diosmetin-based compound) is preferably 0.01 to 0.20, more preferably 0.01 to 0.15, and even more preferably 0.02 to 0.10.

[0032] The pharmaceutical composition of the present invention may further contain known ingredients used in the prevention or treatment of chronic venous diseases, such as diosmin, hesperidin, diosmetin, and hesperetin.

[0033] The pharmaceutical composition of the present invention may further contain known ingredients used in pharmaceutical compositions, such as excipients, binders, disintegrants, lubricants, salts, coenzymes, minerals, antioxidants, vitamins, and anticoagulants.

[0034] The excipient is not particularly limited as long as it can be used in pharmaceuticals, quasi-drugs, etc., and examples thereof include lactose hydrate, crystalline cellulose, corn starch, mannitol, potato starch, etc.

[0035] The binder is not particularly limited as long as it can be used in pharmaceuticals, quasi-drugs, etc., and examples thereof include polyvinyl alcohol, polyvinylpyrrolidone (povidone), hydroxypropylmethylcellulose (hypromellose), agar, and gelatin.

[0036] The disintegrant is not particularly limited as long as it can be used in pharmaceuticals, quasi-drugs, etc., and examples thereof include croscarmellose sodium, carboxymethylcellulose calcium, carboxymethylstarch sodium, low-substituted hydroxypropylcellulose, etc.

[0037] The lubricant is not particularly limited as long as it can be used in pharmaceuticals, quasi-drugs, etc., and examples thereof include stearic acid, magnesium stearate, calcium stearate, talc, carnauba wax, L-leucine, macrogol, etc.

[0038] The salts are not particularly limited as long as they can be used in pharmaceuticals, quasi-drugs, etc., and examples thereof include chlorides, citrates, phosphates, fumarates, sulfates, sodium sulfate, and table salt.

[0039] The coenzyme is not particularly limited as long as it can be used in pharmaceuticals, quasi-drugs, etc., and examples thereof include TPP (thiamine diphosphate), 2-oxoacids (pyruvic acid, etc.), FAD, FMN (vitamin B2), PLP (vitamin B6), NAD, NADP (niacin), coenzyme A (pantothenic acid), and coenzyme R (biotin).

[0040] The minerals are not particularly limited as long as they can be used in pharmaceuticals, quasi-drugs, etc., and examples include sodium, potassium, calcium, iron, magnesium, copper, manganese, zinc, selenium, phosphorus, iodine, chromium, molybdenum, etc.

[0041] The antioxidant is not particularly limited as long as it can be used in pharmaceuticals, quasi-drugs, etc., and examples thereof include erythorbic acid, sulfites, tocopherols, dibutylhydroxytoluene (BHT), butylhydroxyanisole (BHA), ethylenediaminetetraacetic acids, gallic acids, licorice oil extract, edible canna extract, clove extract, sage extract, tempeh extract, Houttuynia cordata extract, green coffee bean extract, chlorogenic acid, sunflower seed extract, grape seed extract, blueberry leaf extract, propionyl hydroxybenzoate, benzoyl ... Possible antioxidants include, but are not particularly limited to, squirrel extract, ginkgo extract, bayberry extract, myricitrin, eucalyptus leaf extract, rosemary extract, clove extract, enzyme-treated rutin, enzyme-treated isoquercitrin, isoflavone, taxifolin (dihydroquercetin), catechin, polymerized catechin, tea extract, nobiletin, methoxyflavone, coenzyme Q10, apple extract, enzymatically hydrolyzed apple extract, sesame oil extract, rice bran oil extract, tannin, caffeine, and oxides of these antioxidants.

[0042] The vitamins are not particularly limited as long as they can be used in pharmaceuticals, quasi-drugs, etc., and examples include vitamin A, vitamin B1, vitamin B2, niacin, pantothenic acid, vitamin B6, vitamin B12, biotin, ascorbic acid (vitamin C), vitamin D2, vitamin D3, vitamin E, vitamin K, and folic acid.

[0043] Anticoagulants are not particularly limited as long as they can be used in pharmaceuticals, quasi-drugs, etc., and examples include "heparin" heparin sodium, "low molecular weight heparin" Fragmin (dalteparin sodium) and Clexane (enoxaparin sodium), "heparinoid" Orgaran (danaparoid sodium), "coumarin" warfarin (warfarin potassium), and "direct thrombin inhibitor" Pradaxa (dabigatran ethoxylate methanesulfonate).

[0044] Chronic venous diseases include varicose veins, venous edema, phlebitis, leg swelling, venous hypertension, venous ulcers, venous thrombosis, and hemorrhoids, and the diosmetin compound can be preferably used for the prevention or treatment of varicose veins, venous edema, phlebitis, leg swelling, and hemorrhoids. Therefore, the present invention also provides a method for preventing or treating chronic venous diseases using the diosmetin compound.

[0045] As used herein, the term "pharmaceutical composition" refers to a drug, a quasi-drug, or a manufacturing intermediate thereof. The pharmaceutical composition of the present invention can be administered orally, parenterally, intravenously, topically, and / or transdermally, with oral administration being preferred. Examples of dosage forms include solutions, pills, tablets, powders, fine granules, granules, dry syrup, capsules, vials, ointments, gels, liquids, semi-liquids, solids, and semi-solids. Animal models may be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. The precise effective amount for a human subject will depend on the severity of the disease state, the subject's general health, age, weight, and sex, diet, number and frequency of administration, concurrent drug(s), reaction sensitivities, and tolerance / response to therapy. This amount can be determined by routine experimentation, and the optimal amount is determined by the clinician's judgment through clinical trials, etc. For example, the following embodiments (a) to (c) are exemplified.

[0046] (a) a diosmetin-7-glucoside inclusion compound in a dose of 1 to 3,000 mg (the preferred dose to be taken daily is, in terms of diosmetin-7-glucoside, preferably 1 to 500 mg, more preferably 1 to 100 mg, and even more preferably 1 to 50 mg).

[0047] (b) Diosmetin glycosyltransferase at a dose of 1 to 3000 mg (the preferred daily dose is, in terms of diosmetin-7-glucoside, preferably 1 to 500 mg, more preferably 1 to 100 mg, and even more preferably 1 to 50 mg).

[0048] (c) A combination of a diosmetin-7-glucoside inclusion compound and a diosmin glycosyltransferase (The preferred daily dose, in terms of diosmetin-7-glucoside, is preferably 1 to 500 mg, more preferably 1 to 100 mg, and even more preferably 1 to 50 mg.)

[0049] EXAMPLES The present invention will be specifically explained below with reference to examples and comparative examples, but the present invention is not limited to these.

[0050] (Test 1) Preparation of Diosmetin-7-glucoside Inclusion Compound-Containing Composition (DIOSG-CD) 270 g of diosmin (95% content, manufactured in China) and 850 g of γ-cyclodextrin (manufactured by CycloChem Co., Ltd.) were placed in an 8 L stainless steel vessel, and water was added to bring the total weight to 6 kg, and the temperature was adjusted to 73°C and the pH to 4.5. Thereafter, 60 g of naringinase (Amano Enzyme Co., Ltd., 160 unit / g) was added while stirring with a stirrer (As One Corporation), and the mixture was allowed to react for 24 hours. The mixture was analyzed by HPLC (Nexera XR HPLC system (Shimadzu Co. Ltd, Kyoto, Japan): column, Capcell Pak C18 size 4.6 × 250 mm (Shiseido, Tokyo, Japan); eluent, 35% (v / v) acetonitrile / 0.1% phosphoric acid; detection, 345 nm; flow rate, 0.3 mL / min; column temperature, The solution confirmed to have produced diosmetin-7-glucoside at a conversion rate of 98% or higher (area of ​​diosmetin-7-glucoside × 100 / area of ​​diosmin + area of ​​diosmetin-7-glucoside) was designated reaction solution A. Reaction solution A: 1 kg was returned to room temperature, filtered through a filter paper, and lyophilized to yield 195 g of a composition containing a diosmetin-7-glucoside inclusion compound (DIOSG: 12%, γ-CD: 51%, other sugars, etc.: 37%; molecular weight molar ratio of γ-CD / DIOSG = 1.5). Five kg of the reaction solution was used to prepare diosmetin transglycosylation product (Test 2). This production method demonstrated that rhamnose elimination from diosmin and an inclusion complex of diosmetin-7-glucoside with γ-cyclodextrin were efficiently obtained.

[0051] (Test 2) Diosmetin glycosyltransferase (DIOS-(Glc) ｎ (n=1-15), DIOS-(Glc) １ , DIOS-(Glc) ２ , DIOS-(Glc) ３ , DIOS-(Glc) ４ , DIOS-(Glc) ５ Preparation of Diosmetin-7-Glucoside (DIOS-(GlcNAc)-7-Glucoside) : A small amount of alkali was added to reaction solution A (70°C, pH 4.5, 5 kg) prepared in Test 1 to adjust the temperature to 62°C and pH 6.2, and then 80 g of cyclodextrin glucanotransferase (CGTase: Amano Enzyme Co., Ltd., trade name "Contizyme", 600 U / ml) was added to initiate the reaction, which was then maintained for 24 hours. 1 kg of the resulting reaction solution B was heat-treated to inactivate the enzyme, and then passed through a column packed with an adsorption resin (Diaion HP-20, Mitsubishi Chemical Corporation) to adsorb the diosmetin-7-glucoside transfer product. The column was then washed with twice the resin volume of water to remove sugars such as rhamnose. The diosmetin-7-glucoside transfer product (DIOS-(GlcNAc)-7-Glucoside) was then obtained by elution with 70% (v / v) ethanol. ｎ (n = 1 to 15)) was obtained as a freeze-dried product (60 g, Figure 1). In addition, β-amylase (β-amylase F, Amano Enzyme Co., Ltd.) was added to approximately 4 kg of the reaction solution B obtained in the preparation of Test 2, and after maintaining the mixture at 60°C and pH 5.8 for a certain period of time, a portion of the reaction solution was periodically sampled (reaction solution C). As an example, the solution treated with β-amylase F for 24 hours contained DIOS-(Glc) １ :DIOS-(Glc) ２The resulting reaction mixture had a molar ratio of 1:1 (Figure 2). The enzyme in reaction mixture C was inactivated by heat treatment, and then the mixture was passed through an adsorption resin column (Diaion SP-207, Mitsubishi Chemical Corporation). The adsorption resin was thoroughly washed with water to remove carbohydrates and other substances. Subsequently, the column was gradually eluted with ethanol at 10-70% (v / v) concentrations. Using a combination of gel filtration resins, Sephadex LH-20, G-10, and G-15 (Sigma-Aldrich), and preparative HPLC (preparative purification LC), 10 g or more of each diosmetin transglycosylation product was isolated and purified. The purity of each diosmetin transglycosylation product was confirmed to be 98% or higher by HPLC analysis (Nexera XR HPLC system (Shimadzu Co. Ltd, Kyoto, Japan): column, Capcell Pak C18 size 4.6 × 250 mm (Shiseido, Tokyo, Japan); eluent, 20% (v / v) acetonitrile / 0.1% phosphoric acid; detection, 345 nm, flow rate, 0.3 mL / min; column temperature, 70°C). Furthermore, each diosmetin glycosylation product was identified as diosmetin-7-glucoside (DIOS-(Glc)) by nuclear magnetic resonance (NMR). １ ) and DIOS-(Glc) １ A compound in which 1 to 4 glucose units are α-1,4-linked to DIOS-(Glc) ２ , DIOS-(Glc) ３ , DIOS-(Glc) ４ , DIOS-(Glc) ５ It was confirmed that this is the case.

[0052] (Test 3) Evaluation of the Solubility of Diosmin (DSN), Diosmetin-7-glucoside-Cyclodextrin Inclusion Compound, and Each Diosmetin Glycosylated Product Diosmin (DSN) and the dried samples prepared in Tests 1 and 2 were added to a 100 ml beaker containing 50 ml of water (pH 4.0, citric acid-Na citrate) at 50°C with stirring until the solution was dissolved and precipitated. After allowing to stand at room temperature (25°C), 1 ml of the supernatant was filtered, and the solubilities of each sample were compared using HPLC analysis based on the molar equivalent of diosmetin-7-glucoside. The results are shown in Table 1.

[0053]

[0054] As is clear from Table 1, the solubility of diosmin (Comparative Example 1) was extremely low, at 0.00927 mM. On the other hand, the solubility of Examples 1 to 7 was higher than that of Comparative Example 1, and in particular, Examples 1, 2, 4, 5, 6, and 7 had a solubility ratio of 5,000 times or more.

[0055] (Test 4) Evaluation of bioavailability Five-week-old Sprague-Dawley (SD) rats (male) were fed MF (Oriental Yeast Co., Ltd.) as a feed for 7 days and then fasted from the day before administration of the test substance. Diosmin (DSN), each sample prepared in Tests 1 and 2, and a composition combining each sample prepared in Tests 1 and 2 were administered orally in a single dose (oral gavage via probe, 300 μmol / kg (equivalent to diosmetin-7-glucoside)). Blood samples were collected from the tail vein of the rats with heparin at 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, and 24 hours, and then centrifuged to obtain plasma. The amount of diosmetin in the collected plasma samples was analyzed by high performance liquid chromatography (SHIMADZU) using a photodiode array detector (SPD-M30A, SHIMADZU) according to the method described in Non-Patent Document 8. The results are shown in Table 2. Table 2 also shows the area under the blood concentration-time curve (AUC) of each sample. ０－２４ (μM·h) are shown.

[0056]

[0057] As shown in Table 2, all of Examples 8 to 18 were superior in terms of absorbability in the body compared to Diosmin (Comparative Example 2). １ and DIOS-(Glc) ２ ,DIOS-(Glc) ２ and DIOS-(Glc) ３ ,DIOS-(Glc) ３ and DIOS-(Glc) ４、 and DIOSG-CD and DIOS-(Glc) ２ The combined composition showed higher bioavailability than when used alone.

[0058] Venous insufficiency results in inadequate blood flow, which increases intravenous pressure in the anorectal region, causing the veins in the anus to swell and form hemorrhoids, accompanied by bleeding, prolapse, swelling, discharge, and pain. Daflon (90% diosmin, 10% hesperidin, etc., manufactured by Servier), a micronized (MPFF) diosmin drug, is used as a treatment for hemorrhoids. Daily ingestion of 1000 mg to 3000 mg / day for several days to several months (high intake, long-term) prevents and improves hemorrhoid symptoms (bleeding, prolapse, swelling, discharge, pain, etc.). Therefore, the effectiveness of each sample in preventing and improving hemorrhoids in a hemorrhoids model rat was compared with that of diosmin.

[0059] (Test 5) Prevention and Improvement of Hemorrhoids in a Rat Hemorrhoid Model To evaluate the reduction of rectal and anal swelling in a rat croton oil-induced hemorrhoid model, male SD rats (8 weeks old, approximately 220 g) were purchased from Charles River Laboratories Japan Inc. and allowed to acclimate for one week. Rats were maintained in a pathogen-free facility in accordance with the Animal Welfare and Management Act (1973, Law No. 105, revised 2017) and the Guidelines for Proper Conduct of Animal Experiments (June 1, 2006, Science Council of Japan) guidelines for handling and use. Each experiment was performed using age-matched rats aged 9-10 weeks. A croton oil-induced hemorrhoid model in rats was performed with reference to the methods published by Dubey et al. (Pharmacognosy Magazine, 09731296231170936 (2023)) and Nishiki et al. (Japanese Pharmacology Journal 92 (4) 215-225 (1988), Japanese Pharmacology Journal 92 (4) 227-240 (1988)). Specifically, each sample was added to 1 mL of saline at a concentration equivalent to 100 μM / kg of diosmetin-7-glucoside, and the resulting solution was orally administered to the rats once daily for five days. For comparison, a suspension of diosmin (100 μmol / kg: diosmin equivalent) was orally administered. On the fifth day, 0.16 mL of inducer (deionized water: pyridine: diethyl ether: 6% croton oil / diethyl ether (1:4:5:10)) was applied to the rat anus using a 4 mm diameter cotton swab for 12 seconds. The final concentration of croton oil was 3%. Edema developed linearly up to 7-8 hours after application, and the severity of edema persisted for more than 24 hours. After 24 hours, the rats were euthanized, and anorectal tissues (approximately >10 mm) were isolated. The rat body weight and anorectal weight were measured. The anorectal index (RAC) was calculated using the formula: anorectal weight (mg) / body weight (g). The results are shown in Table 3.

[0060]

[0061] Although not shown in the table, the DIOSG-CD:HPTG-CD molar ratios in Table 3(1) were similar to those in Table 3(1) at 8:2 and 9.5:0.5. Furthermore, when the inclusion compound was replaced with a diosmetin transglycosylation product:hesperetin transglycosylation product at molar ratios of 9:1, 8:2, or 9.5:0.5, the same level of effect was observed. In Tables 3(2) to (5), the same level of effect was observed not only at a molar ratio of 1:1, but also at 1:2, 1:3, 2:1, and 3:1.

[0062] As shown in Table 3, each Example was able to suppress swelling of the anus and rectum more effectively than Diosmin (Comparative Example 4), demonstrating an excellent preventive effect against hemorrhoids. １ ,DIOS-(Glc) ２ ,DIOS-(Glc) ３ ,DIOS-(Glc) ４ In the case of compositions combining these (Examples 27 to 30), extremely high preventive effects were observed in a hemorrhoid model. Furthermore, if pre-administration is performed before edema induction and the anorectal index (RAC) is at an appropriate value after edema formation, not only hemorrhoid preventive effects but also ameliorative effects can be expected. Therefore, the samples of each Example are also expected to be effective in treating hemorrhoids. Although not shown in the results in Table 3, no difference in effect was observed between the micronized (MPFF) diosmin product "Daflon" (90% diosmin, 10% hesperidin, etc., manufactured by Servier) 100 μmol / kg (diosmin molar equivalent) and the diosmin used in Table 3 above.

[0063] The pharmaceutical composition of the present invention can be used for the prevention or treatment of chronic venous diseases.

Claims

1. A pharmaceutical composition for chronic venous disease, comprising one or more diosmetin compounds selected from the group consisting of diosmetin-7-glucoside-cyclodextrin inclusion compounds and diosmetin glycosyltransferases represented by the following formula (1):

2. The pharmaceutical composition according to claim 1, wherein the diosmetin-7-glucoside-cyclodextrin inclusion compound is obtained through an elimination step in which diosmin is treated with an enzyme having rhamnosidase activity in the presence of cyclodextrin in an aqueous medium at a pH of 3 to 7 to eliminate rhamnose.

3. The pharmaceutical composition according to claim 1, wherein the cyclodextrin in the diosmetin-7-glucoside-cyclodextrin inclusion compound is one or more selected from the group consisting of β-cyclodextrin, branched β-cyclodextrin, and γ-cyclodextrin.

4. The pharmaceutical composition according to claim 1, comprising one or more diosmetin glycosyltransferases in which n in formula (1) is 1 to 5.

5. The pharmaceutical composition according to claim 1, comprising two or more diosmetin glycosyltransferases in which n in formula (1) is 1 to 5.

6. The pharmaceutical composition according to claim 1, comprising two types of diosmetin glycosyltransferases in which n in formula (1) is 1 to 5, and the content ratio of the two types of diosmetin glycosyltransferases is 1:10 to 10:1 in terms of a molar ratio of diosmetin-7-glucoside.

7. The pharmaceutical composition according to claim 1, comprising the diosmetin-7-glucoside-cyclodextrin inclusion compound and a diosmetin glycosyltransferase in formula (1) where n is 1 to 5, wherein the content of the diosmetin-7-glucoside-cyclodextrin inclusion compound:the total content of the diosmetin glycosyltransferase in formula (1) where n is 1 to 5 is 1:10 to 10:1 in terms of diosmetin-7-glucoside molar ratio.

8. The pharmaceutical composition of claim 1, further comprising rhamnose.

9. The pharmaceutical composition according to claim 8, wherein the molar ratio of the rhamnose content to the diosmetin compound content (converted to diosmetin-7-glucoside) (rhamnose / diosmetin compound) is 0.5 to 1.

5.

10. The pharmaceutical composition according to claim 1, further comprising one or more hesperetin-based compounds selected from the group consisting of hesperetin-7-glucoside-cyclodextrin inclusion compounds and hesperetin glycosyltransferases of the following formula (2):

11. The pharmaceutical composition according to claim 10, wherein the hesperetin-7-glucoside-cyclodextrin inclusion compound is obtained via an elimination step in which hesperidin is treated with an enzyme having rhamnosidase activity in the presence of cyclodextrin in an aqueous medium at a pH of 3 to 7 to eliminate rhamnose.

12. The pharmaceutical composition according to claim 10, wherein the cyclodextrin in the hesperetin-7-glucoside-cyclodextrin inclusion compound is one or more selected from the group consisting of β-cyclodextrin, branched β-cyclodextrin, and γ-cyclodextrin.

13. The pharmaceutical composition according to claim 10, wherein the molar ratio of the amount of the diosmetin compound (converted to diosmetin-7-glucoside) to the amount of the hesperetin compound (converted to hesperetin-7-glucoside) (hesperetin compound / diosmetin compound) is 0.01 to 0.

2.

14. The pharmaceutical composition of claim 1, further comprising one or more selected from the group consisting of excipients, binders, disintegrants, lubricants, salts, coenzymes, minerals, antioxidants, vitamins, and anticoagulants.

15. The pharmaceutical composition of claim 1, wherein the chronic venous disease comprises one or more diseases selected from the group consisting of varicose veins, venous edema, phlebitis, leg swelling, venous hypertension, venous ulcers, venous thrombosis, and hemorrhoids.

16. The pharmaceutical composition of claim 1, which is administered orally, parenterally, intravenously, topically, and / or transdermally.

17. The pharmaceutical composition of claim 1, which is administered orally.

18. A method for producing a pharmaceutical composition for chronic venous disease, comprising the step of combining one or more diosmetin compounds selected from the group consisting of a diosmetin-7-glucoside-cyclodextrin inclusion compound and a diosmetin glycosyltransferase represented by the following formula (1):

19. A method for preventing or treating chronic venous disease, using one or more diosmetin compounds selected from the group consisting of diosmetin-7-glucoside-cyclodextrin inclusion compounds and diosmetin glycosyltransferases represented by the following formula (1):

20. A method for producing a diosmetin glycosyltransferase of the following formula (1), which comprises a step of subjecting a hesperetin glycosyltransferase of the following formula (2) to an oxidative dehydrogenation reaction:

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