JP2014101328A - Anti-inflammatory agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide medicament or quasi-drug having action capable of alleviating or inhibiting inflammation, and having high safety.SOLUTION: Methylhesperidin aglycon is contained as an active ingredient.

Description

  The present invention relates to an anti-inflammatory agent.

  Inflammatory reaction is induced by various chemical mediators, then leukocytes are concentrated and infiltrate into the tissue, and various cytokines and lipid mediators are released from infiltrating cells, resulting in vasodilation, increased vascular permeability, leukocytes Tissue reactions such as migration and connective tissue proliferation can lead to unpleasant conditions such as pain, fever, redness, and swelling.

  One factor causing skin irritation is the penetration of ultraviolet rays (particularly UVB) into the skin. When inflammation due to ultraviolet rays occurs, arachidonic acid present in the cell membrane is released within the skin, and various prostaglandins are produced via the cyclooxygenase system.

On the other hand, methyl hesperidin is methylated from hesperidin and solubilized in water, and has physiological functions such as strengthening of capillaries, bleeding prevention, blood pressure adjustment, etc., and as yellow colorant, antioxidant, etc. It is used in foods, pharmaceuticals, cosmetics, etc. In Japan, it is also designated as a pharmaceutical additive and food additive.
Cited Document 1 describes that when methyl hesperidin is taken orally, the skin's resistance to ultraviolet rays is increased.
However, it has not been known so far that methyl hesperidin aglycone has an anti-inflammatory action. There is no report on the pharmacokinetics of methyl hesperidin.

JP 2011-105714 A

  The present invention relates to providing a highly safe pharmaceutical or quasi-drug having an action capable of reducing or suppressing inflammation.

  The present inventors examined materials having an anti-inflammatory action, and found that methyl hesperidin aglycone has an action of significantly suppressing inflammation caused by exposure to ultraviolet rays, and completed the present invention.

That is, the present invention relates to the following 1) to 3).
1) An anti-inflammatory agent containing methyl hesperidin aglycone as an active ingredient.
2) A prostaglandin production inhibitor containing methyl hesperidin aglycone as an active ingredient.
3) Inflammatory cytokine production inhibitor containing methyl hesperidin aglycone as an active ingredient.

  Since the present invention has an excellent anti-inflammatory effect, it is useful as a pharmaceutical, a quasi-drug, a cosmetic, and a food or drink that can exhibit an anti-inflammatory effect, or a material or preparation for blending them.

It is a figure which shows the result of having measured the UVB induction prostaglandin production inhibitory effect. It is a figure which shows the result of having measured the IL-1 induction prostaglandin production inhibitory effect. It is a figure which shows the result of having measured the inflammatory cytokine expression inhibitory effect. It is a figure which shows the result of having measured the UVB induction erythema suppression effect by oral ingestion of methyl hesperidin aglycone. It is a figure which shows the result of having measured the UVB induction epidermal thickening inhibitory effect by oral ingestion of methyl hesperidin aglycone.

  In the present specification, “aglycone” means the entire non-sugar part of a glycoside that is bound to a sugar via an O-glycoside bond or an S-glycoside bond.

  As used herein, “non-therapeutic” is a concept that does not include medical practice, that is, does not include methods for surgery, treatment or diagnosis of humans.

  The methyl hesperidin aglycone as an active ingredient of the present invention is obtained by hydrolyzing methyl hesperidin with an acid such as hydrochloric acid. It is known that such methyl hesperidin aglycone mainly contains flavanone type compounds (A) and chalcone type compounds (B) (Sakiba, Nippon Chemical Journal, 79, 6, 733-740 (1958). ), For example, those having the structure shown below.

(In the formula, R represents a hydrogen atom or a methyl group.)

  The methyl hesperidin aglycone used in the present invention may contain both the chalcone type compound (A) and the flavanone type compound (B) shown above, or may contain only one of them. In particular, since a strong action is observed in the chalcone body, those containing a large amount of the chalcone body are preferable.

Methyl hesperidin aglycone is a known method, for example, hesperidin produced from citrus peel and the like is dissolved in an aqueous sodium hydroxide solution, a corresponding amount of dimethyl sulfate is allowed to act on the alkaline solution, and the reaction solution is neutralized with sulfuric acid. Extraction with n-butyl alcohol, evaporation of the solvent, and recrystallization with isopropyl alcohol produced methyl hesperidin (Sakiburo, Nippon Kagaku Kagaku, 79, 733-6 (1958)), which was converted to hydrochloric acid. Although it can manufacture by hydrolyzing etc., the manufacturing method is not restricted to this.
In addition, commercially available products (for example, those distributed as pharmaceutical additives, food additives, and cosmetic raw materials, or “methyl hesperidin” (Tokyo Chemical Industry Co., Ltd.), “hesperidin methyl chalcone” (Sigma), etc.) It is also possible to use aglycones purchased and produced by acid hydrolysis.

  As shown in the Examples below, methyl hesperidin aglycone has an excellent anti-inflammatory effect, prostaglandin production inhibitory action, or inflammatory cytokine production inhibitory action that methyl hesperidin does not have in an in vitro evaluation system.

  Here, prostaglandin (PG) is known as one of inflammatory mediators, and regulates inflammatory responses by cooperating with various other inflammatory mediators. When tissue is damaged due to trauma or pathogen infection, phospholipids in the cell membrane are converted to arachidonic acid, and cyclooxygenase (COX) acts there to produce prostaglandins. Prostaglandins, together with factors such as inflammatory cytokines, cause vasodilation, increased vascular permeability, pain sensitization, and leukocyte migration, resulting in inflammatory symptoms such as pain, fever, redness, and swelling. Therefore, the inflammatory reaction can be improved by suppressing the production of prostaglandins and inflammatory cytokines.

In particular, prostaglandin E ₂ (PGE ₂ ) and prostaglandin F ₂ α (PGF ₂ α), through their production, promote the pain-inducing action and vascular permeability-increasing action of bradykinin, and inflammation by bradykinin and histamine. Sexual exudation is enhanced. Therefore, by suppressing the production of prostaglandin E ₂ and prostaglandin F ₂ α, the inflammatory reaction can be suppressed by suppressing the action of inflammatory mediators (such as bradykinin and histamine) and inflammatory cytokines. .

  Therefore, the methyl hesperidin aglycone is used as a medicine, quasi-drug, cosmetic, food, or feed for suppressing or suppressing inflammation, for example, skin inflammation induced by UV exposure, or for incorporation in these. It is useful as a raw material or formulation. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

  In addition, the food is based on the concept of suppression of inflammation, for example, reduction or suppression of skin inflammation induced by UV exposure, and food, functional food, food for the sick, specified Health foods are included.

  That is, the methyl hesperidin aglycone can be used as an anti-inflammatory agent, a prostaglandin production inhibitor, or an inflammatory cytokine production inhibitor, and can further be used to produce these agents.

  The above pharmaceutical products (including quasi-drugs) include tablets, capsules, granules, powders, syrups, intravenous injections, intramuscular injections, suppositories, inhalants, transdermal absorption agents, and eye drops. Nasal drops, poultices, poultices, ointments, lotions, creams, tinctures, liniments, suspensions, lotions, bops, pastes, gels, sprays, aerosols or oils, oral preparations (dentifrices, liquids Any of dentifrices, mouthwashes, gum massage creams, oral ointments, gargle tablets, troches, throat lozenges, etc.) may be used. The administration form may be either oral administration (internal use) or parenteral administration (external use, injection).

  In order to prepare such pharmaceutical preparations of various dosage forms, the agent is used alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants, lubricants. Agent, dispersant, buffer, preservative, flavoring agent, flavoring agent, film agent, carrier, diluent, osmotic pressure regulator, fluidity promoter, absorption aid, pH adjuster, emulsifier, preservative, stabilization Agent, antioxidant, colorant, UV absorber, wetting agent, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, flavoring agent, flavoring agent, extender, surfactant, dispersant, A buffer, a preservative, a fixing agent, a fragrance, a film agent, and the like can be used in appropriate combination.

  The content of methyl hesperidin aglycone in oral administration in the preparation is generally preferably 0.1% by mass or more, more preferably 1% by mass or more. And it is 95 mass% or less preferably, More preferably, it is 90 mass% or less. Moreover, Preferably it is 0.1-95 mass%, More preferably, it is 1-90 mass%.

  The content of methyl hesperidin aglycone in parenteral administration in the preparation is generally preferably 0.00001% by mass or more, more preferably 0.0001% by mass or more. And preferably it is 10 mass% or less, More preferably, it is 1 mass% or less. Moreover, Preferably it is 0.00001-10 mass%, More preferably, it is 0.0001-1 mass%.

  The dosage of the above pharmaceuticals and the like may vary according to the condition, body weight, sex, age or other factors of the subject, but in the case of parenteral administration, the above-mentioned methyl hesperidin aglycone per adult is preferably 0. 01 mg / kg or more, particularly preferably 0.1 mg / kg or more. And it is preferably 100 mg / kg or less, particularly preferably 20 mg / kg or less. Moreover, Preferably it is 0.01-100 mg / kg, Most preferably, it is 0.1-20 mg / kg. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to divide once to several times a day, and to administer continuously for several weeks to several months.

  The cosmetics (including quasi-drugs) can be used as external preparations for skin, cleaning agents, makeup cosmetics, etc., and depending on the method of use, lotions, emulsions, gels, creams, ointments, powders, It can be provided in various dosage forms such as granules. Cosmetics of such various dosage forms include the above-described methyl hesperidin aglycone and oily ingredients, moisturizers, powders, pigments, emulsifiers, solubilizers, detergents, ultraviolet absorbers, which can be blended with skin cosmetics. Thickeners, drugs (for example, anti-inflammatory agents, bactericides, antioxidants, vitamins, drugs or natural products known to promote fat metabolism or uncoupled protein expression), fragrances, resins, antibacterial agents It can be prepared by appropriately combining antifungal agents, plant extracts, alcohols and the like.

  The methyl hesperidin aglycone in the total amount of the cosmetic is usually preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, and further preferably 0.01% by mass or more. And preferably it is 20 mass% or less, More preferably, it is 10 mass% or less, More preferably, it is 5 mass% or less. Moreover, Preferably it is 0.0001-20 mass%, More preferably, it is 0.001-10 mass%, More preferably, it is 0.01-5 mass%.

  Examples of the form of the food containing the methyl hesperidin aglycone include soft drinks, tea beverages, coffee beverages, fruit juice beverages, carbonated beverages, juices, jelly, wafers, biscuits, breads, noodles, sausages, and other foods and nutrition In addition to various foods such as foods, further, nutritional supplement compositions in the same form (tablets, capsules, syrups, etc.) as the above-mentioned oral administration preparations can be mentioned.

  The foods of various forms include the above methyl hesperidin aglycone alone or other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, coloring agents, antioxidants, humectants, It can be prepared by appropriately combining thickeners, active ingredients other than the present invention, and the like.

  Examples of the feed include feed for small animals used for rabbits, rats, mice, etc., feed for pet foods used for dogs, cats, small birds, squirrels, etc., and can be prepared in the same form as the above foods.

  The content of methyl hesperidin aglycone in the food or feed is generally preferably 0.0001% by mass or more, more preferably 0.001% by mass or more. And preferably it is 10 mass% or less, More preferably, it is 1 mass% or less. Moreover, Preferably it is 0.0001-10 mass%, More preferably, it is 0.001-1 mass%.

With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> An inflammatory agent comprising methyl hesperidin aglycone as an active ingredient.
<2> A prostaglandin production inhibitor containing methyl hesperidin aglycone as an active ingredient.
<3> Inflammatory cytokine production inhibitor comprising methyl hesperidin aglycone as an active ingredient.
<4> The agent according to any one of <1> to <3>, which reduces or suppresses inflammation induced by ultraviolet exposure.
<5> Use of methyl hesperidin aglycone for producing an anti-inflammatory agent.
<6> Use of methyl hesperidin aglycone for producing a prostaglandin production inhibitor.
<7> Use of methyl hesperidin aglycone for producing an inflammatory cytokine production inhibitor.
<8> Methyl hesperidin aglycone for use in anti-inflammation.
<9> A methyl hesperidin aglycone for use in suppressing prostaglandin production.
<10> A methyl hesperidin aglycone for use in suppressing inflammatory cytokine production.
<11> The use according to any one of <8> to <10>, which is a non-therapeutic use.
<12> An anti-inflammatory method of administering methyl hesperidin aglycone to a human or an animal.
<13> A method for inhibiting prostaglandin production, comprising administering methyl hesperidin aglycone to a human or an animal.
<14> A method for suppressing inflammatory cytokine production, comprising administering methyl hesperidin aglycone to a human or an animal.
<15> The method according to any one of <12> to <14>, which is a non-therapeutic method.
<16> The method according to any one of <12> to <15>, wherein the inflammation induced by ultraviolet exposure is reduced or suppressed.

(Preparation of methyl hesperidin aglycone)
After dissolving 5.00 g of dietary methyl hesperidin (Alps Pharmaceutical Co., Ltd.) in 250 mL of 50% EtOH solution, 15 mL of concentrated hydrochloric acid was added, followed by heating to reflux at 84 ° C. for 16 hours. After cooling to room temperature, the mixture was neutralized with sodium hydroxide and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over MgSO ₄ , filtered and concentrated to obtain 3.32 g of a crude product (methyl hesperidin aglycone). Formation of methyl hesperidin aglycone was confirmed on TLC using hexane-ethyl acetate (1: 1) as a developing solvent.

[Inhibition of UVB-induced prostaglandin production by methyl hesperidin aglycone]
Normal human newborn foreskin epidermal keratinocytes (NHEK) (KURABO) were seeded on a 12-well plate, and when sub-confluent, the culture medium was replaced with a growth factor-free (−) medium, and the test was performed the next day. This medium was replaced with a (−) medium prepared so that the final concentration of methyl hesperidin (MHES) or methyl hesperidin aglycone was 16.5 μg / mL (about 50 μM), and pretreatment was performed for 2 hours. This medium was replaced with phosphate buffered saline (PBS), irradiated with UVB (20 mJ; dose of 1 mW / cm ² for 20 seconds), replaced with medium containing the specimen again, and cell supernatant was collected 24 hours later. . The amount of prostaglandin contained in the cell supernatant was quantified with PGE ₂ EIA Kit (Cayman) or PGF ₂ α EIA Kit (Cayman). The result is shown in FIG.

From the results shown in FIG. 1, the production of PGE ₂ and PGF ₂ α was induced by UVB stimulation, and the production inhibitory effect of PGE ₂ and PGF ₂ α was recognized only with methyl hesperidin aglycone. From this, it was clarified that methyl hesperidin has no anti-inflammatory action and only methyl hesperidin aglycone has an anti-inflammatory action.

[Inhibitory effect of methyl hesperidin aglycone on IL-1β-induced prostaglandin production]
Normal human neonatal foreskin dermal fibroblasts (NHDF) (KURABO) were seeded on a 12-well plate, and when sub-confluent, the medium was changed to a (−) medium containing no growth factor, and the test was performed the next day. This medium was replaced with (−) medium prepared so that the final concentration of methyl hesperidin aglycone was 3.3, 6.6, 16.5 μg / mL (about 10, 20, 50 μM), and pre-treated for 16 hours. Went. Thereafter, IL-1β was added so that the final concentration was 1 ng / mL, and the cell supernatant was recovered after 24 hours. The amount of PGE ₂ contained in the cell supernatant was quantified with PGE ₂ EIA Kit (Cayman). The result is shown in FIG.

From the results shown in FIG. 2, it was confirmed that the production of PGE ₂ and PGF ₂ α was induced by IL-1β stimulation, and this induction was suppressed in a concentration-dependent manner by methyl hesperidin aglycone. This indicates that methyl hesperidin aglycone also has an inhibitory effect on prostaglandin production by stimulating inflammatory cytokines.

[Inhibitory effect of methyl hesperidin aglycone on inflammatory cytokine expression]
Normal human newborn foreskin epidermal keratinocytes (NHEK) (KURABO) were seeded on a 12-well plate, and when sub-confluent, the culture medium was replaced with a growth factor-free (−) medium, and the test was performed the next day. This medium was replaced with (−) medium prepared so that the final concentration of methyl hesperidin aglycone was 3.3, 6.6, 16.5 μg / mL (about 10, 20, 50 μM), and pretreated for 2 hours. Went. The medium was replaced with PBS, irradiated with UVB (20 mJ; dose of 1 mW / cm ² for 20 seconds), replaced with the medium containing the specimen again, and cells were collected after 8 hours. RNA was extracted from the cells using RNeasy Mini Kit (QIAGEN), 500 μg of the cell-extracted RNA was synthesized with High Capacity RNA-to-cDNA Kit (Applied Biosystems), and 20 ng equivalent of total RNA was subjected to real time PCR. IL1B (Hs0155410_m1), IL6 (Hs00985639_m1), and TNF (Hs00174128_m1) were used as TaqMan Gene Expression Assay (Applied Biosystems) probes that specifically detect the target gene. The target gene expression level was corrected by the expression level of the internal standard gene RPLP0 (Hs99999992_m1). For the analysis, 7500 Real Time PCR System (Applied Biosystems) was used. The result is shown in FIG.

  From the results shown in FIG. 3, expression of IL-1β, IL-6, and TNFα was increased by UVB stimulation, and its expression was suppressed in a concentration-dependent manner by methyl hesperidin aglycone aglycone. From this, it was revealed that methyl hesperidin aglycone aglycone also has an inhibitory effect on the expression of inflammatory cytokines.

[Inhibition of UVB-induced erythema by oral ingestion of methyl hesperidin aglycone]
Seven-week-old HR-1 hairless mice (♀) (Japan SLC) were preliminarily raised for one week and then divided into groups so that the average body weights were equal (each group n = 10). The mice were bred under conditions of 23 ± 1 ° C., 12 hours of light period (7: 0 to 19:00), and 3 to 4 animals / cage. From 9 weeks of age, the methyl hesperidin aglycone administration group was administered by oral gavage with 52 mg / kg body weight of methyl hesperidin aglycone daily in an emulsion form for 2 weeks using a stomach tube. In the control group, the one excluding methyl hesperidin aglycone was made into an emulsion and similarly administered orally. Two weeks after the start of administration, an unirradiated site (1 cm × 1.5 cm) and an irradiated site (1 cm × 1.5 cm) were set on the back of the mouse, and under pentobarbital (trade name: Somnopentyl (Kyoritsu Pharmaceutical)) anesthesia The irradiated part was irradiated with UVB at a dose of 1 mW / cm ² for 40 seconds (40 mJ). After 48 hours of UVB irradiation, the skin color (a * value) was measured using a spectroscopic color difference meter SE-6000 (Nippon Denshoku Industries Co., Ltd.). Anesthesia was performed by inhaling 0.8 to 1.2% of isoflurane (trade name: Foren (Abbott Japan)) using an inhalation anesthesia apparatus 400 Anaesthesia Unit (Universe). Unirradiated sites and irradiated sites were measured 5 times or more, and the average value was defined as the skin color value. The result is shown in FIG.

  From the result of FIG. 4, the methyl hesperidin aglycone administration group suppressed the increase of a * value by UVB irradiation compared with the non-administration group. That is, it has been clarified that methyl hesperidin aglycone suppresses skin inflammation caused by ultraviolet rays.

[Inhibition of UVB-induced epidermal thickening by oral ingestion of methyl hesperidin aglycone]
Seven-week-old HR-1 hairless mice (♀) (Japan SLC) were preliminarily raised for one week and then divided into groups so that the average body weights were equal (each group n = 10). The mice were bred under conditions of 23 ± 1 ° C., 12 hours of light period (7: 0 to 19:00), and 3 to 4 animals / cage. From 9 weeks of age, the methyl hesperidin aglycone administration group was administered by oral gavage with 52 mg / kg body weight of methyl hesperidin aglycone daily in an emulsion form for 2 weeks using a stomach tube. In the control group, the one excluding methyl hesperidin aglycone was made into an emulsion and similarly administered orally. Two weeks after the start of administration, an unirradiated site (1 cm × 1.5 cm) and an irradiated site (1 cm × 1.5 cm) were set on the back of the mouse, and under pentobarbital (trade name: Somnopentyl (Kyoritsu Pharmaceutical)) anesthesia The irradiated part was irradiated with UVB at a dose of 1 mW / cm ² for 40 seconds (40 mJ). Dissection was performed 48 hours after UVB irradiation, the skin was fixed in formalin, and a histopathological specimen (HE staining) was prepared (requested from Histochemical Laboratories). The skin thickness of each specimen at five locations was measured, and the average value was taken as the individual skin thickness. The result is shown in FIG.

(result)
From the results shown in FIG. 5, in the methyl hesperidin aglycone administration group, an increase in the skin thickness due to UVB irradiation was suppressed as compared to the non-administration group.

Claims (3)

  An anti-inflammatory agent containing methyl hesperidin aglycone as an active ingredient.   A prostaglandin production inhibitor containing methyl hesperidin aglycone as an active ingredient.   Inflammatory cytokine production inhibitor comprising methyl hesperidin aglycone as an active ingredient.