TW201225970A - A pharmaceutical composition for preventing or treating gout - Google Patents

Abstract

A pharmaceutical composition for preventing or treating gout is provided, including an extract of Anisomeles indica as an active ingredient, in which the extract comprises ovatodiolide, anisomelic acid, or the combination thereof.

Description

201225970 VI. Description of the Invention: [Technical Field] The present invention relates to a pharmaceutical composition for preventing or treating gout, particularly relates to a pharmaceutical composition for preventing or treating gout using fish needle grass (extract as an active ingredient). Prior Art Gout is one of the common metabolic diseases characterized by deposition of uric acid crystals on the toes, ankles, knees, wrist joints, fingers, elbows, etc., causing arthritis. The diagnosis of gout is defined as blood. The urate concentration exceeds 6.8 mg/dl, which is called hyperuricemia. When the urate concentration in the blood exceeds the above-mentioned threshold of renal metabolism, excessive urate crystals, or trophi, are formed. Accumulated in the joints. Accumulation of tophi in the joints can cause acute arthritis and inflammation around the joints and joints, including activation of complement and release of inflammatory cytokines, such as purine, which is easily absorbed from beer. Will promote the excessive formation of uric acid. However, some studies have shown that the cause of gout may come from multiple factors, such as Such as genetics, consumption of large amounts of alcohol, decreased glomerular rate, drugs (such as diuretics, aspirin, niacin, etc.). * In more detail, the metabolism and elimination of uric acid is from The hypoxanthine of the nucleoside acid (hyp. read e) is catalyzed by uric acid by the yellow sputum oxidase (the brain). After the uric acid enters the blood of the circulatory system, it is delivered to the kidney of the kidney. Glomeruli, in the proximal tubule (10) tubule) for urate reabsorption and secretion. The reabsorbed urate returns to the blood, and the secreted urate is excreted in the urine. The drug currently used for the treatment of gout has a <亍 inhibitory effect on the above-mentioned gout metabolic pathway 201225970. For example, allopurinol and its metabolite 〇Xypurjn〇i inhibit the formation of uric acid by inhibiting the activity of xanthine oxidase upstream. The action of Febuxostat, such as the selective xanthine oxidase inhibitor, also blocks the formation of uric acid from upstream of the uric acid metabolic pathway. The uricosuric drug, such as Probenecid and Benzbromarone, acts to inhibit the reabsorption of urate by endothelial cells that inhibit the renal proximal tubules. However, the administration of the above-mentioned uric acid-lowering medication is not compliant, including the patient's dietary pattern is not changed, the lack of education in taking the drug, and the partial reduction of uric acid in vivo clearance is poor and the valley is easily accumulated in the body. Intra- or when other diseases (such as diabetes) coexist, the medications taken may reduce the efficacy of the uric acid-lowering drug (Update on gout: New therapeutic strategies and options. Nat

Rev Rheumatol. 2010 Jan;6(l):30-8) 〇Inflammation caused by urate crystallization, at this stage with non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids (glucocorticosteroids) and colchicine (c 〇ichicine) is the main drug. However, these drugs are known to have the risk of serious side effects and interactions between drugs', especially for elderly people and patients taking both gout and ‘13⁄4 kidney disease (CKD) or diabetes. The new generation of gout treatment drug, Illonacept, is designed to neutralize the cytokine "globin-1 (IL-1) fusion protein' to block iL-1 attachment to the cell surface and trigger an inflammatory response (The interleukin 1 inhibitor rilonacept In treatment of chronic gouty arthritis: results of a placebo-controlled, monosequence crossover, non-randomised, single-blind pilot study. Ann Rheum Dis. 201225970 2009 Oct;68(10):1613-7. Epub 2009 Jul 26). The acute inflammatory symptoms associated with gout are known to be caused by a variety of inflammatory cytokines and chemokines induced by urate crystallization, such as tumor necrosis factor (TNF-α) and interleukin-1/3 (IL-l). /3), IL-6, CXCL8 (IL-8), and CXCL1 (growth-related oncogene a) (The role of interleukin-1 and the inflammasome in gout: implications for therapy. Arthritis Rheum. 2007 Oct;56( 10): 3183-8.). Moreover, IL-1 is considered to be a key cytokine in gout inflammation. In view of the above previous studies, the inventors of the present invention actively sought alternative medicines for treating gout with low side effects, easy availability, and simple process, and completed the present invention. SUMMARY OF THE INVENTION An object of the present invention is to provide a pharmaceutical composition for preventing or treating gout, which comprises an extract of the needle grass as an active ingredient and a pharmaceutically acceptable carrier. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating gout, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises ovatodiolide and anisomelic acid. ), or a combination thereof. [Embodiment] The "fish needle grass" described in this case is also known as the wind grass, Benhuxiang or the guest wiping grass. The needle grass is mainly distributed in the subtropical zone and the tropics. It is an annual or overage herb. It has been previously documented that the fish needle plant is distilled into an essential oil for the treatment or prevention of allergic diseases (US2006/0003030A1), and there is also a literature documenting that the extract of the needle grass is resistant to skin aging, inhibiting melanin formation and capturing free radicals in 201225970. Efficacy on (US2(8)4/0028643A1). However, there has not been any literature on the application of fish needle grass in the prevention or treatment of gout. ^ "Aquilaria sinensis extract" used in this case may include roots, stems, leaves, flowers, or whole plants of the needle grass, extracted with an organic solvent. The polar solvent used in the present invention includes c "c12 alcohol, c2_c5 acetic acid_, c5_c6 burning or a combination thereof, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, 2-butanol, second butanol, 1,3-butanediol, L4-butanediol, pentanol, isoamyl alcohol, 2,3-pentanediol, 2,4-pentanediol, cyclopentanol, hexanol, cyclohexanol, heptanol , octanol, decyl alcohol, decyl alcohol, undecyl alcohol, dodecanol, ethyl acetate, propyl acetate, amyl acetate, n-pentane, cyclopentane, n-hexane, cyclohexane, or the like Combination, but not limited to. In the present invention - ethanol is used as an extraction solvent in the embodiment. The concentration of ethanol may be from 5 to 95%, or from 75% to 95%. The extraction temperature and time may be determined by conditions such as solvent properties. Decided that there is no special restriction 'the temperature can be 50~80 ° C, or 70~80 ° C, the extraction time can be 2~4 hours. The extract in this case can be further purified to obtain higher purity. The extraction step may be column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography, ion exchange, etc. The column chromatography is filled with oxidized granules. The extract of the present invention can also be obtained by repeating the extraction step and the purification step to obtain a higher purity extract. Milk (10) / Lie, dm · such as me / es ma / aMZ / ce and other plants, can be isolated from the following formula 之) fish needle grass s (ovatodiolide) and the following formula (2) shown as oxalic acid (anisomeHc 201225970 Acid) diiterpenes (PubChem Compound at NCBI; http://pubchem.ncbi.nlm.nih.gov/) ° 〇,

Formula (1)

Equation (2) 0

The inventors of the present invention further purified the crude crystals obtained by extracting the above-mentioned fish needle grass/W/ca) extract according to the prior literature, and analyzing and aligning the crystals by NMR spectroscopy to confirm that the crystals obtained were the fish needle lactone and the oxalic acid. Otatodiolide, chemically simple C2〇H24 04, full name 3,7,11,15(17)-cetamidinetetraene-16,2:19,6-lactone 3484-37- 5(3Ε,12Ε)-3,12·didecyl-8-anthracene-6,18-dioxo-tricyclic φ [14.2.1.0.5,9]19-院-3,12,16( 19)-Tris-7,17-dione) (3,7,11,15(17)-Cembratetraene-16,2:19,6-diolide 3484-37-5(3E,12E)-3,12 -Dimethyl-8-methylene-6,l 8-dioxa-t ricyclo[14.2.1.0.5,9]nonadeca-3,12,16(19)-triene-7,17-dione) Studies have confirmed several species Human tumor cell lines are cytotoxic and can induce oral squamous cell carcinoma death (apoptosis) (Hou YY, et al) The nature diterpenoid ovatodiolide induces cell cycle arrest and apoptosis in human oral squamous cell carcinoma Ca9 -22 cells, Life Sci. 2009, 201225970 8591-2): 26-32.) and biological activity with anti-HIV virus (Shahidul

Alam M., et al., HIV-inhibitory diterpenoid from Anisomeles indica. Fitoterapia. 2000 Sep;71(5):574-6.). Analsomelic acid, chemically simple C2〇H26〇4, full name (6E,10Z,14Z)-6,14-dimercapto-3-indolyl-2-sidedoxy 3a,4, 5,8,9,12,13,15a-octahydrocyclotetraindole [b]furan_10_carboxylic acid ((6E,10Z,14Z)-6,14-dimethyl-3-methylidene-2-oxo-3a, 4,5,8, 9,12,13,15 a-octahydrocyclotetradeca [b]furan-10-carboxylic acid), no relevant physiological activity report. However, studies or literature related to the prevention or treatment of glucurolide and methic acid have been reported to date. The present inventors further carried out the cell test and the animal model test on the above-mentioned extract of the needlefish and the fish of the fish, and the animal model test, and found that the cytokines IL-1 and IL which inhibit gout are present. -6. Secretion of TNF-α, etc., it is believed that the extract of the needle grass, the needle of the needle and the tartaric acid have the effects of preventing and treating gout. The "pharmaceutically acceptable carrier" as used in the present invention means a carrier, such as an additive, an excipient, a preservative, a bridge, etc., which can be used in pharmaceutical technology. Specifically, for example, starch, corn starch, lactose, dextrin, cyclodextrin, thioglycolic cellulose, retinoic cellulose, thioglycolate, gelatin, gum, acacia, gum ( Guar), pectin, gum arabic, tragacanth, carrageenan, or similar additives. The medical composition described in the present invention may be appropriately designed according to the administration route, and may be, for example, a key agent, a capsule, a film-coated tablet, a powder, a granule, a 201225970 syrup, a suspension, an injection, a suppository, a patch, and the like. The administration route may be, for example, oral, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, anal administration, inhalation administration, topical administration, or transdermal administration. The dosage of the pharmaceutical composition described in the present invention can be appropriately formulated according to the conditions of the patient's body weight, age, symptoms of the affected part, physiological condition, and administration route, depending on the condition of the patient or the deacon. The documents cited herein are hereby incorporated by reference. The specific embodiments of the present invention are described in detail below. However, the following embodiments are only used to further disclose the technical contents of the present invention, and should not limit the scope of the invention. [Example 1] Extraction and Purification of Needle Grass The dried whole plant of the needle grass was ground into a powder of 2 kg, added with 95% B, heated under reflux for 3 hours, and the alcohol extract was concentrated under reduced pressure to obtain a needle grass. Ethanol extract 200g. Take 200g of the above-mentioned ethanol extract of the needlefish, add a ceria-filled color column (1 〇 x 15 cm), and the extract is hexanol / acetic acid ethoxylate = 10/1, 5/1, 3/ 1, 1 / 1 and n-hexane / ethyl acetate / methanol = 6 / 4 / 1, 3 / 2 / 1 and 曱 • alcohol each 1200ml gradient extraction initial separation, to obtain 140g of the initial separation liquid. Thereafter, 140 g of the initial separation liquid was further divided into a ruthenium dioxide-filled column color layer (10×15 cm), and dichlorosilane, dichloromethane, decyl alcohol = 10/1, 5/1, 7/3, and Each 1000 ml of sterol was washed and separated to obtain two concentrated fractions. The two concentrated fractions were further recrystallized by a n-hexane-ethyl acetate solvent to obtain two crystals, respectively. The chemical structure of the two crystals was identified by H]-NMR, c, 3-nmr spectroscopy as ovatodiolide (Fig. 1B) and anisomelic acid (Fig. 2A, 2B). Diterpenoids. 201225970 [Example 2] Cellular activity evaluation of the extract of Semena serrata L. The ethanol extract of the squid, the liquid extract of the fish, and the inhibitory effect of the oil on the secretion of IL_3 by human mononuclear THP-1 cells. The cells (Ccrc 68023) were implanted into a 96-well plate, the number of cells implanted was 1.5 X i〇5ceils/weu, and Phorbol 12 Myristate 13 acetate (PMA) was added to the cell culture medium (Sigma). , Cat. No. P-8139), the final concentration of PMA was 5 ng/ml. The 96-well plate was placed in a 37 ° C incubator overnight. The cell culture medium containing PMA was removed every other day and added with 2%. Serum fresh cell culture medium (RPMI-1640, Invitrogen, Cat. No. 31800-022), and then added different concentrations of the ethanol extract of fish needle grass obtained from Example 1, the fish needle lactone or the wind oxalic acid, The 96-well plate was reacted at 37 ° C for 60 minutes. After the reaction, sodium urate crystals (Mos) (Sigma) were added to give a final concentration of MSU of 0.25 mg/ml. The 96-well plate was placed at 37°. The reaction was carried out for 4 hours at C, and then the supernatant was collected by centrifugation, and the IL-Ιβ content was measured using an ELISA kit (DY201, R&D Systems). The activity of lactose dehydrogenase (LDH) was measured by CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, Cat. No. Gl781) to evaluate cell viability, and the IC5 value was calculated to be 32±5. // g/ml. The results are shown in Figure 3 and Table 1. According to Figure 3, the control group is a blank test without MSU stimulation. The horizontal coordinate indicates that 0, 4, 11, 33, 100/zg/ is administered. The test group of the ethanol extract of the fish needle grass of ml concentration, the ordinate indicates the percentage of IL-1 /5 secretion of the test group of the above test group relative to the ethanol extract of the fish needle grass of 0 #g/ml desert. As shown in Fig. 3, the ethanol extract of fish needle grass obtained in Example 1 201225970 can inhibit the secretion of IL-lyS from human mononuclear THP-1 cells stimulated by MSU. The needles and the oxalic acid against humans Inhibition of secretion of TNF-α by mononuclear ball U937 cells U937 cells (ATCC: CRL-1593.2) were cultured in a cell culture medium containing 50 ng/ml PMA for 24 hours, and then replaced with a cell culture medium containing no PMA to continue culture. Hour. Activated U937 cells were seeded in 96-well plates at 1.6 X 105 cells. s/well. Thereafter, the same concentration of luterolactone or oxalic acid and fresh cell culture solution (RPMI-1640, Invitrogen, Cat. No. 31800-022) were added and reacted at 37 ° C for 30 minutes. After completion of the reaction, a lipopolysaccharide (LPS) having a concentration of 0.1 pg/ml was added to a total volume of 200 μM per well. The 96-well plate was placed at 37 ° C for 4 hours, then the supernatant was collected by centrifugation, and the TNF-α content was measured using an ELISA kit (DY210, R&D Systems), and MTT (3-(4,5-dimethylthiahiazo-) was used. 2-yl)-2,5-di-phenyl tetrazolium bromide) (SIGMA, Cat. No. M-2128) was used to measure the cell survival rate, and the calculated IC5G values are shown in Table 1. Inhibition of lipophyllin on the secretion of IL-6 by human monocyte U937 cells U937 cells (ATCC: CRL-1593.2) were cultured in cell culture medium containing 50 ng/ml PMA for 24 hours, then replaced with The cell culture medium containing no PMA was further cultured for 48 hours. Activated U937 cells were seeded into 96-well plates at a dose of 1.6 X 105 cells/well. Different concentrations of luterolactone and rubic acid and fresh cell culture medium were separately added and reacted at 37 ° C for 30 minutes. After the reaction was completed, a concentration of 1 μl was added to 2 (^g/ml of 201225970).

The LPS has a total volume of 2 〇〇 μ1 per well. The % well plate was placed overnight and centrifuged to collect the supernatant, using an ELISA kit (DY4〇6, R&D

Systems) detected IL_6 levels and measured cell viability using Μττ, calculated as IC5 〇 values as shown in Table 1. [Table 1] Cytokine g/mn Fish-to-grass West-lead oxalic acid IL-Ιβ 0,8 2.2 TNF-a 12.0 10.4 IL-6 1.7 ------ 6.5 According to the data in Table 1, the fish needle grass The secretion of IL-β from human mononuclear ball THpj cells stimulated by MSU and the secretion of IL-6 and TNF-α from human mononuclear ball u937 cells stimulated by LPS in extracts Both have obvious inhibitory effects. [Example 3] Activity evaluation of fish needle extract in animal model Urine sodium salt crystal (MSU)-induced mouse peritonitis animal model # MSU-induced animal model of mouse peritonitis Reference Resident macrophages initiating and driving inflammation in A monosodium urate monohydrate crystal-induced murine peritoneal model of acute gout. Arthritis Rheum. 2009 Jan; 60(l): 281-9. This document is incorporated by reference in its entirety in this specification. Mice (purchased from the National Animal Center) were randomly divided into body weights before the experiment and fasted about 2 hours before the experiment. 3 mg (0.5 ml) of monosodium urate (MSU) per 12 201225970 mice were administered by intraperitoneal injection, and the clinical symptoms of the mice after intraperitoneal injection were observed and recorded. One hour after the injection of MSU, the ethanol extract of the squid of Example 1 was administered orally with 500 mg/kg or 0-25 ml of 5% ethanol (EtOH) in 30% polyoxyethylene sesame oil polymer (CrEL) as Carrier, observe and record the abnormality of the clinical symptoms of the mice. Three hours after the injection of MSU, the mice were euthanized with an excess of CO 2 , 1.5 ml of phosphate buffered saline (PBS) was injected into the peritoneal cavity, exudate was collected, and the secretion was analyzed using an ELISA kit (R&D Reference Systems). The content of IL-Ιβ in the liquid. The result is shown in Figure 4. The oral extract of S. chinensis ethanol 500 mg/kg inhibited the secretion of IL-Ιβ by 39% and reached a statistically significant difference (t-test, p < 0.05). False Synovial Cavity Mouse Model (Role of S100A8 and S100A9 in neutrophil recruitment in response to monosodium urate monohydrate crystals in the air-pouch model of acute gouty arthritis· Arthritis Rheum 2003 Aug; 48(8): 2310-20. • Experimental methods are carried out. This document is incorporated by reference in its entirety in this specification. Mice (purchased from the National Animal Center) were randomly divided into body weights by subcutaneous injection before administration of the needles. The mice were injected with 0.22 μιη microfilter (Millipore) on the 0th day (Day 0) by subcutaneous injection. Filtered sterile air (2 ml/mouse) to form a pseudosynovial cavity. On the third day (Day 3), sterile air (3 ml/mouse) was injected again to keep the pseudo-sliding cavity. On day 6 (Day 6), sodium pseudo sodium urate (MSU) (3 mg/2 ml/mouse) was injected into the pseudosynovial cavity. Two hours after the injection of 201225970 MSU, 5 mg/kg of ripoxil or 0.25 ml of 3% dimercaptopurine (DMSO) was administered as a vehicle by subcutaneous injection, and the mice were observed and recorded. Whether the clinical symptoms are abnormal. Three hours after the injection of MSU, the mice were euthanized with an excess of C〇2, and physiological saline (2 ml/mouse) was injected into the pseudosynovial cavity to collect the exudates in the pseudosynaptic cavity, using ELISA. The kit (R&D Systems) analyzed the amount of IL-6 in the secretion. The result is shown in Figure 5. According to Fig. 5, it was shown that subcutaneous injection of fish needle lactone 50 mg/kg inhibited 47% of IL-6 secretion and reached a statistically significant difference (t test, p < 〇.05). Mice (purchased from the National Animal Center) were randomly divided into body weights by oral administration of the oxalic acid test and subcutaneously injected on day 0 (Day 0), and 0.24 μm microfilter filtered sterile air was injected on the back of the mouse (2 Ml/mouse) to form a pseudosynovial cavity. Day 3 (Day 3) was re-injected with sterile air (3 ml/mouse) to keep the pseudo-sliding cavity round. On day 6 (Day 6), sodium sulphate crystals (MSU) (3 mg/2 ml/mouse) were injected into the pseudosynaptic cavity. Two hours after the injection of MSU, 50 mg/kg of oxalic acid or 3%25 ml of 3% DMSO was orally administered to 30% CrEL as a vehicle control group. Three hours after the injection of MSU, the mice were euthanized with an excess of CO 2 , and physiological saline (2 ml/mouse) was injected into the pseudosynovial cavity to collect the exudate in the pseudosynaptic cavity, using an ELISA kit. (R&D Systems) analyzed the amount of IL-6 in the secretion. The result is shown in Figure 6. According to Figure 6, it was shown that oral administration of 50 mg/kg of oxalic acid inhibited the secretion of IL-6 201225970 in the pseudo-smooth model of the air pouch, and reached a statistically significant difference (t test). , p < 0.05). While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application. 201225970 [Simple description of the diagram] Figure 1A shows the H^NMR spectrum analysis of one of the compounds in the extract of the needlefish, which is ovatodiolide. Fig. 1B shows a C13-NMR spectrum analysis of one of the compounds in the extract of S. needles, which is ovatodiolide. Figure 2A shows a H^NMR spectrum analysis of one of the compounds in the extract of A. sinensis. Figure 2B shows a C13-NMR spectral analysis of one of the compounds in the extract of S. chinensis which is anisomelic acid. Figure 3 shows the inhibition of IL-1/3 secretion by MSU-stimulated human mononuclear THP-1 cells by ethanol extract of S. chinensis. Figure 4 shows the inhibitory effect of the ethanol extract of S. chinensis on the secretion of IL-1 /3 induced by MSU-induced mouse models. Figure 5 shows the inhibitory effect of subcutaneous injection of fishlactone on IL-6 secretion in a pseudosynaptic mouse model. Figure 6 shows the inhibitory effect of oral methic acid on IL-6 secretion in a rat model of pseudosynaptic cavity. [Main component symbol description] None. 16

Claims (1)

201225970 VII. Scope of application: L A pharmaceutical conjugate for the prevention or treatment of gout The extract is active in two or two including fish needles. And the pre-drug composition as described in the patent application scope of the patent application, wherein the fish needle extract is composed of organic dissolved wind. 3. The pre-drug described in claim 2 A composition wherein the organic solvent comprises ethanol. H. Gout of the doctor 4. The pharmaceutical composition according to claim 3, wherein the ethanol is 50 to 95% aqueous solution or the treatment of the gout Or a therapeutic composition for treating gout, wherein the temperature of the extraction is 50 to 803⁄4. The medicine for preventing or treating gout 2, and wherein the fish needle extract is applied for prevention or treatment of gout composition according to column chromatography In the pot, the 裟 裟 — 瓜 - Guaxi /, Zhong ° Xuan ... needle grass extracts including fish needle lactone (〇 〇 〇 〇 〇 ) lide), wind oxalic acid (anis 〇 mdic a 8. (4) The L7 item--the t-pre-treatment of the medicine (four) compound, wherein the pharmaceutical composition has the function of reducing the cytokine secretion, and the cytokines include JL] stone, river _6, _ combinations thereof. ^ 9. A pharmaceutical composition for preventing or treating gout, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises vinegar (t〇di〇〗 ide) and oxalic acid (anis〇nie) The invention relates to a pharmaceutical composition for preventing or treating gout according to claim 9 of the invention, wherein the fish needle lactone and the wind oxalic acid are derived from the extract of the needle grass. 11. The pharmaceutical composition for preventing or treating gout according to claim 10 of the patent application scope Wherein the fish needle extract comprises a pharmaceutical composition for preventing or treating gout as described in claim 5, wherein the organic solvent comprises ethanol. The prophylactic or therapeutic pharmaceutical composition according to the item 12, wherein the ethanol is an aqueous solution of 5 G to 95%, and the method for preventing or treating gout, as described in the patent application (4) n, is 5, wherein The extraction temperature is 50~8〇t. 15· The composition of the prevention or treatment of gout according to the scope of the patent scope 帛1(), the potted medium is poorer than the previous one ^ 'purification. ,, m4 Strokes through the column chromatography analysis method A patent scope 9-15, any one, ten, ^ treatment of gout medical composition, the prevention or treatment of secretion of Wei, heart _ 2 1 · The substance has a reduced cell stimuli or its combination of elements including ΙΜΠ6, Τ··α,