CN106606507B

CN106606507B - Application of flavone diglucuronide compound in preparation of cyclooxygenase inhibitor

Info

Publication number: CN106606507B
Application number: CN201510689649.3A
Authority: CN
Inventors: 胡晓; 杨义芳; 许慧; 牛莉鑫; 黄春跃; 刘欣
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2015-10-21
Filing date: 2015-10-21
Publication date: 2021-06-25
Anticipated expiration: 2035-10-21
Also published as: CN106606507A

Abstract

The invention discloses an application of a flavone diglucuronide compound (a compound shown in a formula I) in preparation of a cyclooxygenase inhibitor, and particularly discloses the flavone diglucuronide compound (the compound shown in the formula I) which has anti-inflammatory activity and cyclooxygenase inhibition activity, so that the flavone diglucuronide compound can be developed into a COX inhibitor and an anti-inflammatory drug and has a great application prospect.

Description

Application of flavone diglucuronide compound in preparation of cyclooxygenase inhibitor

Technical Field

The invention relates to the field of medicines, and in particular relates to an application of a flavone diglucuronide compound in preparation of a cyclooxygenase inhibitor.

Background

The flavone di-glucuronide component can be separated from plant (such as herba Medicaginis), structurally two molecules of glucuronide are connected via 1-2 glycosidic bond, and then glycosidated with flavone aglycone. The structural activity is rarely reported, and the structural activity is mainly focused on antioxidant and anti-aging activity.

Cyclooxygenase (COX), also known as Prostaglandin (PG), endoperoxidase, is a membrane-bound protein that is the rate-limiting enzyme in the body that catalyzes the conversion of arachidonic acid to prostaglandin. Current studies indicate that COX exists in at least two isomers, namely COX-1 and COX-2. COX-1 is structurally expressed in most tissue cells and is involved in maintaining physiological functions. Currently, COX inhibitors are important targets for anti-inflammatory drug development, and for example, the familiar non-steroidal anti-inflammatory drugs indomethacin, ibuprofen and the like are COX inhibitors. In addition, recent research also shows that COX inhibitors are possible to become new targets for drug development of serious diseases such as tumors, Alzheimer's disease and atherosclerosis.

Currently, most cyclooxygenase inhibitors are selective inhibitors, i.e., they inhibit COX-1 or COX-2 only, however, such selective inhibitors often have adverse gastrointestinal, cardiovascular effects.

Therefore, there is an urgent need in the art to develop a COX inhibitor with less side effects and excellent inhibitory effect on cyclooxygenase.

Disclosure of Invention

The invention aims to provide a COX inhibitor with small side effect and good cyclooxygenase inhibiting effect.

The present invention provides in a first aspect the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of (I) a composition or formulation against inflammatory diseases; and/or (ii) a Cyclooxygenase (COX) inhibitor,

in the formula, R₁Is H, methoxy or hydroxy; r₂Is a hydroxyl group.

In another preferred embodiment, the Cyclooxygenase (COX) inhibitor includes a COX-1 inhibitor and a COX-2 inhibitor.

In another preferred embodiment, the inflammatory disease is selected from the group consisting of: neurogenic inflammation, gynecological inflammation, rheumatoid arthritis, or a combination thereof.

In another preferred embodiment, the inflammatory disease is selected from the group consisting of: cervicitis, vaginitis, or a combination thereof.

In another preferred embodiment, the compound of formula I has one or more of the following characteristics:

(1) anti-inflammatory activity;

(2) inhibiting the activity of cyclooxygenase.

In another preferred embodiment, the cyclooxygenase is selected from the group consisting of: COX-1, COX-2, or a combination thereof.

In another preferred embodiment, said inhibition of cyclooxygenase activity comprises co-inhibiting COX-1 and COX-2 activity.

In another preferred embodiment, the composition comprises: a pharmaceutical composition, nutraceutical composition, or food composition, or dietary supplement composition.

In another preferred embodiment, the composition is a pharmaceutical composition.

In another preferred embodiment, the food composition comprises a beverage composition.

In another preferred embodiment, the pharmaceutical composition comprises (a) the compound represented by formula I and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the component (a) is 0.1-99.9 wt%, preferably 10-99.9 wt%, more preferably 70-99.9 wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the component (a) accounts for 60.0-99.5 wt%, preferably 70.0-99.5 wt%, more preferably 80.0-99.5 wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition is a liquid, solid, or semi-solid.

In another preferred embodiment, the dosage form of the pharmaceutical composition comprises tablets, granules, capsules, oral liquid or injection.

In another preferred embodiment, the composition is an oral formulation.

In another preferred embodiment, the composition (e.g., pharmaceutical composition) is administered to the mammal by: oral, intravenous, topical administration.

In another preferred embodiment, the mammal comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes a rodent, such as a mouse, a rat.

In another preferred embodiment, the compound of formula I is a flavone glucuronide compound.

In another preferred embodiment, the compound of formula I is a flavone di-glucuronide compound.

In another preferred embodiment, the compound of formula I is a plant extract.

In another preferred embodiment, the plant is selected from the group consisting of: alfalfa, beautyberry, dichroa febrifuga, or combinations thereof.

In a second aspect, the present invention provides a pharmaceutical composition for anti-inflammation, comprising:

(a1) a first active ingredient which is a compound of formula I or a pharmaceutically acceptable salt thereof, for use as an anti-inflammatory; and

(a2) a second active ingredient for anti-inflammatory, the second active ingredient being a non-steroidal anti-inflammatory ingredient;

(b) a pharmaceutically acceptable carrier, and a pharmaceutically acceptable carrier,

wherein the definition of the compound of formula I is as described in the first aspect of the invention.

In another preferred embodiment, the non-steroidal anti-inflammatory component is selected from the group consisting of: indomethacin, aspirin, or a combination thereof.

In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100: 1, preferably 1:10 to 10: 1.

in a third aspect, the invention provides a method of non-therapeutic inhibition of cyclooxygenase enzyme activity comprising:

culturing the cells in vitro in the presence of a compound of formula I, thereby inhibiting the activity of a cyclooxygenase enzyme in the cells.

In another preferred embodiment, the cell expresses cyclooxygenase.

In another preferred embodiment, the cyclooxygenase enzyme includes COX-1 and COX-2.

In a fourth aspect, the invention provides a non-therapeutic anti-inflammatory method comprising:

(a) administering to a subject in need thereof a compound of formula I or a pharmaceutical composition thereof, wherein the compound of formula I is as described in the first aspect of the invention.

In another preferred embodiment, the subject comprises a non-human mammal.

In another preferred embodiment, the subject comprises a rodent, such as a rat, a mouse.

In another preferred embodiment, the method further comprises the steps of: (b) the subject is sacrificed and the level or activity of the cyclooxygenase in the subject is detected.

In a fifth aspect, the present invention provides a method of screening for anti-inflammatory drug candidates, the method comprising the steps of:

(a) providing a test compound and a positive control compound, wherein the positive control compound is a compound of formula I or a pharmaceutically acceptable salt thereof;

(b) detecting the influence of the compound to be detected on the cyclooxygenase in a test group, and comparing the test result with corresponding experimental results in a positive control group and a negative control group, wherein the influence of the positive control compound on the cyclooxygenase is detected in the positive control group;

wherein, if the inhibition degree of the tested compound on the cyclooxygenase is obviously higher than that of a negative control group, the tested compound is suggested to be a candidate drug for anti-inflammation.

In another preferred example, in step (b), the test group is compared with the positive control group, and the ratio of I1 to I2 is compared, wherein I1 is the inhibition degree of cyclooxygenase I1 of the test compound, and I2 is the inhibition degree of cyclooxygenase of the positive control compound, and if I1/I2 is greater than or equal to 80%, the test compound is suggested to be an anti-inflammatory drug candidate.

In another preferred embodiment, the method further comprises the step (c): further determining the anti-inflammatory effect of the test compound selected in step (b).

In another preferred embodiment, the expression "significantly higher" means that I1/I0 is ≥ 2, preferably ≥ 3, more preferably ≥ 4,

wherein I1 is the inhibition degree of the tested compound cyclooxygenase; and I0 is the degree of inhibition of cyclooxygenase in the negative control group.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In a sixth aspect the present invention provides a method of treating an inflammatory disease comprising:

administering to a mammal in need thereof a compound of formula I or an acceptable salt thereof, wherein the compound of formula I is as described in the first aspect of the invention.

In another preferred embodiment, the mammal comprises a human.

In another preferred embodiment, the mammal comprises a non-human mammal.

In another preferred embodiment, the mammal comprises a rodent, such as a rat, a mouse.

In another preferred embodiment, the dosage is 1-100 mg/kg/day, preferably 10-100 mg/kg/day, more preferably 50-100 mg/kg/day.

In another preferred embodiment, the frequency of application is 1-5 times/day, preferably 1-2 times/day.

In another preferred embodiment, administration comprises one or more cycles, each cycle being 2-30 days, preferably 3-7 days.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows an ESI-MS spectrum of Compound 1 obtained in example 1.

FIG. 2 shows the preparation of Compound 1 from example 1¹H-NMR spectrum.

FIG. 3 shows the preparation of Compound 1 from example 1¹³C-NMR。

FIG. 4 shows the ESI-MS spectrum of Compound 2 obtained in example 1.

FIG. 5 shows Compound 2 prepared in example 1¹H-NMR spectrum.

FIG. 6 shows Compound 2 prepared in example 1¹³C-NMR spectrum.

FIG. 7 shows an ESI-MS spectrum of Compound 3 obtained in example 1.

FIG. 8 shows Compound 3 prepared in example 1¹H-NMR spectrum.

FIG. 9 shows Compound 3 prepared in example 1¹³C-NMR spectrum.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a first surprise, found that flavone diglucuronide compounds can inhibit the activities of both cyclooxygenase COX-1 and cyclooxygenase COX-2 and also have remarkably excellent anti-inflammatory activity, and thus, can be developed into anti-inflammatory agents. The present invention has been completed based on this finding.

As used herein, the terms "anti-inflammatory", "anti-inflammatory disease" are used interchangeably. In the present invention, the compounds of formula I can treat and prevent anti-inflammatory diseases.

As used herein, the terms "cyclooxygenase inhibitor" and "COX inhibitor" are used interchangeably and refer to a substance (e.g., a compound) that inhibits the activity of cyclooxygenase.

Active ingredient

As used herein, the "active ingredient", "compound of formula I", "flavone diglucuronide compound of the present invention", "flavone diglucuronide compound" may be used interchangeably, having the structure of formula I,

wherein R is₁Is H, methoxy or hydroxy; r₂Is a hydroxyl group.

The compounds of formula I may be chemically synthesized and may be extracted from plants such as Medicago truncatula.

The active ingredients of the present invention include compounds of formula I, and may also include pharmaceutically acceptable salts or esters of compounds of formula I.

The active ingredients of the invention have the following characteristics: (i) simultaneously inhibiting the activity of cyclooxygenase COX-1 and COX-2; (ii) anti-inflammatory activity, and can be used for preparing COX inhibitor and anti-inflammatory medicine.

Cyclooxygenase inhibitors

Cyclooxygenase (COX), also known as Prostaglandin (PG), endoperoxidase, is a membrane-bound protein that is the rate-limiting enzyme in the body that catalyzes the conversion of arachidonic acid to prostaglandin. Cyclooxygenase enzymes exist in at least two isomers, COX-1 and COX-2.

The cyclooxygenase inhibitor is a broad-spectrum inhibitor, namely can inhibit COX-1 and COX-2 simultaneously, has better inhibition effect on cyclooxygenase and has small side effect.

Composition and application thereof

The flavone diglucuronide compound and the composition containing the compound as the main active ingredient can be used as a cyclooxygenase inhibitor for treating, preventing and relieving inflammatory diseases (such as neurogenic inflammation, gynecological inflammation, rheumatoid arthritis and the like).

The compositions of the present invention include (but are not limited to): pharmaceutical compositions, food compositions, health care compositions, dietary supplements, beverage compositions and the like.

Take the pharmaceutical composition as an example. The pharmaceutical composition comprises the flavone diglucuronide compound or the pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier within a safe and effective dose range. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000mg of flavone diglucuronide compounds/dose, more preferably 5-200mg of flavone diglucuronide compounds/dose, most preferably 10-100mg of flavone diglucuronide compounds/dose. Preferably, said "dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration, the preferred mode of administration being oral.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (e.g., indomethacin, aspirin, etc.).

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically acceptable effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 5 to 500mg, and more preferably 10 to 100 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The main advantages of the invention include:

(1) the invention discovers that the flavone diglucuronide compound has obvious anti-inflammatory activity for the first time.

(2) The invention discovers for the first time that the flavone diglucuronide compound can simultaneously inhibit the activities of cyclooxygenase COX-1 and cyclooxygenase COX-2.

(3) The flavone diglucuronide compound is extracted from plants, and has the characteristic of small side effect.

(4) The flavone di-glucuronide compound can be extracted from various plants, and has diversified sources and convenient acquisition.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight. The following examples all statistically treated significant differences with P < 0.05.

Instrument and reagent

Nuclear magnetic resonance apparatus: bruker DRX 400 (germany), TMS internal standard; FTIR-8201 PC type infrared spectrophotometer (Shimadzu, Japan); WATERS ZQ2000 mass spectrometer (usa); medium and low pressure column chromatography (YMC corporation, japan).

Silica gel (200-300, 300-400 meshes), wherein the thin-layer chromatography analysis prefabricated plate and the prefabricated plate preparation prefabricated plate are produced in Qingdao ocean factories; c18 filler (japan YMC). All the reagents used were analytical grade (national pharmaceutical group chemical reagents, Inc.).

Example 1 preparation of chrysoeriol-7-O- [ beta-D-glucuronic acid- (1 → 2) ] -beta-D-glucuronide, apigenin-7-O- [ beta-D-glucuronic acid- (1 → 2) ] -beta-D-glucuronide and luteolin-7-O- [ beta-D-glucuronic acid- (1 → 2) ] -beta-D-glucuronide

The first experimental method comprises the following steps:

(1) extraction: pulverizing herba Medicaginis, sieving with 24 mesh sieve, reflux extracting with 80% methanol water solution with 10(v/m) times of the amount of the medicinal materials for 3 times, each for 1 hr, filtering the extractive solutions, mixing, and drying to obtain herba Medicaginis dry extract. Suspending the extract in 4 times of water, subjecting the supernatant to AB-8 macroporous adsorbent resin, statically adsorbing for 2 hr, removing impurities with 3 times of resin volume of water, eluting with 20% ethanol, collecting eluate, and concentrating under reduced pressure to obtain total flavone glucuronide fraction.

(2) Column chromatography: the obtained total flavonoid glucuronide site was subjected to ODS C18 normal pressure column chromatography (10 times volume of ODS to the sample mass, i.e., 30ml of ODS C-18/1g sample, 100cm by 20cm glass column), and eluted with methanol aqueous solution (10% → 15% → 20% → 25% → 30%) in a gradient, the amount of mixed solvent used for each gradient being 3 times the column volume, and collected in order for each column volume to obtain 8 fractions in total.

(3) HPLC preparation: and (3) further refining the components, separating the components by using HPLC (high performance liquid chromatography), and identifying the obtained components, wherein a compound corresponding to one component is a compound 2: apigenin-7-O- [ beta-D-glucuronic acid- (1 → 2)]-beta-D glucuronide (t)_R＝18.3min)。

And (2) a second experimental method:

1) extraction: pulverizing folium Callicarpae Formosanae (sieved with 24 mesh sieve), extracting with 60% ethanol under reflux for 3 times (2 hr each time) with the amount of 12(v/m) times of the amount of the medicinal materials, filtering the extractive solutions, mixing, and drying to obtain dry extract. Suspending the extract in 4 times of water, subjecting the supernatant to AB-8 macroporous adsorbent resin, statically adsorbing for 2 hr, removing impurities with 3 times of resin volume of water, eluting with 20% ethanol, collecting eluate, and concentrating under reduced pressure to obtain total flavone glucuronide fraction.

2) Column chromatography: the obtained total flavonoid glucuronide site was subjected to ODS C18 normal pressure column chromatography (10 times volume of ODS to the sample mass, i.e., 30ml of ODS C-18/1g sample, 100cm by 20cm glass column), and eluted with methanol aqueous solution (5% → 10% → 15% → 20% → 25%) in a gradient manner, the amount of mixed solvent used for each gradient being 3 times the column volume, and collected in order for each column volume to obtain 15 fractions in total. Collecting 15% methanol to elute a second column volume, namely a component 8, and concentrating under reduced pressure to obtain an elution part I; collecting 20% methanol to elute the first column volume, namely the component 10, and concentrating under reduced pressure to obtain an elution part II; the first column volume, fraction 13, was eluted with 25% methanol and concentrated under reduced pressure to give elution fraction III.

3) HPLC preparation: HPLC preparation of site I (14% formazan)Alcohol aqueous solution, detection wavelength 210nm, flow rate 4ml/min) to give compound 3: luteolin-7-O- [ beta-D-glucuronic acid- (1 → 2)]-beta-D glucuronide (t)_R20.5 min); HPLC preparation of the above fraction II (17% methanol in water, 210nm detection wavelength, 4ml/min flow) gave compound 2: apigenin-7-O- [ beta-D-glucuronic acid- (1 → 2)]-beta-D glucuronide (t)_R18.3 min); HPLC preparation of fraction III above (23% aqueous methanol, 210nm detection wavelength, 4ml/min flow) provided Compound 1: Halloween-7-O- [ beta-D-glucuronic acid- (1 → 2)]-beta-D glucuronide (t)_R＝16.8min)。

Identification results

Compound 1 (chrysoeriol-7-O- [ beta-D-glucuronic acid- (1 → 2)]- β -D glucuronide) as a pale yellow amorphous powder, slightly soluble in water, insoluble in methanol, soluble in DMSO. ESI-MS M/z651[ M-H ]]^-1The molecular weight of the compound was determined to be 652 (see FIG. 1).¹H-NMR(DMSO-d₆，400MHz)δ：12.87(1H，s，5-OH)，7.56(1H，dd，J＝8.4，2.0Hz，H-6′)，7.49(1H，d，J＝2.0Hz，H-2′)，7.11(1H，d，J＝8.4Hz，H-5′)，6.69(1H，d，J＝1.6Hz，H-8)，6.69(1H，s，H-3)，6.43(1H，d，J＝1.6Hz，H-6)，5.39(1H，d，J＝6.4Hz，H-1″)，4.56(1H，d，J＝8.0Hz，H-1″′)，4.02(1H，d，J＝9.6Hz，H-5″)，3.88(3H，s，OCH₃-3 '), 3.65(1H, d, J ═ 9.2Hz, H-5 ' "), 3.58(1H, t, J ═ 8.8Hz, H-3"), 3.55(1H, t, J ═ 4.8Hz, H-2 "), 3.47(1H, t, J ═ 8.8Hz, H-4"), 3.29(1H, t, J ═ 8.8Hz, H-4 ' "), 3.27(1H, t, J ═ 9.2Hz, H-3 '"), 3.05(1H, t, J ═ 8.0Hz, H-2 ' "), see fig. 2 above.¹³The C-NMR data are shown in Table 1 and FIG. 3.

Compound 2 (apigenin-7-O- [ beta-D-glucuronic acid- (1 → 2)]- β -D glucuronide) as a pale yellow amorphous powder, slightly soluble in water, insoluble in methanol, soluble in DMSO. ESI-MS M/z 621[ M-H ]]^-1The molecular weight of the compound was determined to be 622 (see FIG. 4).¹H-NMR(DMSO-d₆，400MHz)δ：12.98(1H，s，5-OH)，7.94(1H，dd，J＝6.8，1.8Hz，H-2′)792(1H, dd, J ═ 6.8, 1.8Hz, H-6 '), 6.96(1H, dd, J ═ 6.8, 2.0Hz, H-3'), 6.94(1H, dd, J ═ 6.8, 2.0Hz, H-5 '), 6.81(1H, d, J ═ 2.0Hz, H-8), 6.79(1H, s, H-3 "), 6.44(1H, d, J ═ 2.0Hz, H-6), 5.31(1H, d, J ═ 5.6Hz, H-1"), 4.56 "(1H, d, J ═ 7.6Hz, H-1'"), 3.93(1H, d, J ═ 8.4Hz, H-5 "), 3.71(1H, d, J ″, 8, t ″, 3.5H, t ″, 3.6 Hz, H-6 ″), 3.5H, H-6 ″, 3.5H, and 3.6H, H-6 ″, 1H, J ″, 1 ″, 6, H, 3.28(1H, t, J ═ 8.4Hz, H-4 ' "), 3.24(1H, t, J ═ 8.4Hz, H-3 '"), 3.05(1H, t, J ═ 7.6Hz, H-2 ' "), and the data above are shown in fig. 5.¹³The C-NMR data are shown in Table 1, FIG. 6 (Compound 2 from Medicago truncatula and from Callicarpa purpurea is the same compound).

Compound 3 (luteolin-7-O- [ beta-D-glucuronic acid- (1 → 2)]- β -D glucuronide), a pale yellow amorphous powder, slightly soluble in water, insoluble in methanol, soluble in DMSO. ESI-MS M/z637[ M-H ]]^-1The molecular weight of the compound was determined to be 638, as shown in FIG. 7.¹H-NMR(DMSO-d₆400MHz) δ 12.96(1H, s, 5-OH), 7.44(1H, dd, J ═ 8.4, 2.0Hz, H-6 '), 7.42(1H, dd, J ═ 8.4, 2.0Hz, H-2'), 6.91(1H, d, J ═ 8.8Hz, H-5 '), 6.74(1H, d, J ═ 2.0Hz, H-8), 6.71(1H, s, H-3), 6.42(1H, d, J ═ 1.6Hz, H-6), 5.41(1H, d, J ═ 6.8Hz, H-1 "), 4.56(1H, d, J ═ 7.6Hz, H1'), 4.06(1H, d, J ═ 9.2, H-5 ″, H-1 ″, 5H ″, 5H ″, H, 5 ″, H ″, H, 5 ″, H ″,3 ″, 1H, H ″, 1H, H ″, 6H, H ″, 1H, t, J ═ 8.0Hz, H-4 "), 3.29(1H, t, J ═ 8.8Hz, H-4 '"), 3.27(1H, t, J ═ 9.2Hz, H-3 ' "), 3.05(1H, t, J ═ 8.0Hz, H-2 '"), see fig. 8.¹³The C-NMR data are shown in Table 1 and FIG. 9.

TABLE 1 preparation of Compounds 1-3¹³C-NMR data sheet

Example 2 COX inhibitory Activity of flavone diglucuronide Compounds of the present invention

1. Instrument for measuring the position of a moving object

Microplate reader Labsystems, Wellscan, MK.2

Centrifuge KA-1000 Shanghai flying pigeon

Double super clean bench, Suzhou clean

CO₂Incubator, PHARMA SCIENTIFIC

Adjustable pipettor (Finland, 5-40ul, 40-200ul, 200-1000ul)

2. Material

Cox Fluorescent Inhibitor Screening Assay Kit(Cayman,USA)；

96-well blackboards (Corning, USA);

an inlet lance head (Axygen, USA);

indomethacin batch No.: 080401 Sigma;

SC－560：

NS－398：

comparative compound 1:

3. method of producing a composite material

1) Buffer solution (1X), heme, fluorogenic substrate ADHP, COX-1/COX-2 and the compound to be detected are mixed in sequence in a reaction system of 200 mul, and incubated for 5 minutes at 25 ℃.

2) The cyclooxygenase substrate arachidonic acid was added.

3) Carefully shake the plate for a few seconds and incubate at 25 ℃ for 2min

4) Ex 530 nM 540 nM; em:585-590nM detection.

4. Method for processing sample to be tested and positive medicine

The sample to be tested and indometacin are accurately weighed and diluted to working concentration by using detection buffer solution before the experiment.

COX-1 selective inhibitor SC-560(CAS 188817-13-2), 15 μ l of which is added with 985 μ l of DMSO to dilute into working solution according to the instruction; COX-2 selective inhibitor DuP-697(CAS88149-94-4)), diluted to working solution by pipetting 50. mu.l and adding 550. mu.l DMSO as required by the instructions.

5. Computing

6. Results of the experiment

6.1 preliminary screening

TABLE 2 inhibition ratio prescreening table

6.2IC₅₀Value determination

TABLE 3 Compounds 1-3 cyclooxygenase IC₅₀Watch (A)

The results are shown in tables 2 and 3. The result shows that the flavone diglucuronide compound (I) has stronger COX-1 and COX-2 inhibitory activities which are obviously higher than the inhibitory activity of the comparative compound 1. And, IC of Compound 3 against COX-1 and COX-2₅₀All reach nM level, wherein IC is opposite to COX-2₅₀IC for COX-1 over NS-398, a COX-2 selective inhibitor₅₀Is in the same order of magnitude as indomethacin. Therefore, the flavone diglucuronide (I) can provide a new way for preventing or treating inflammatory diseases such as cervicitis, vaginitis and the like, and can be developed into a new cyclooxygenase inhibitor.

Example 3 pharmacodynamic Effect of the Flavodiglucuronide Compounds of the present invention on acute inflammation in rats caused by Carrageenan

1 laboratory animal

1.1 name: SD rat

1.2 sources: shanghai Sphall-Bikai laboratory animals Co., Ltd

1.3 weight: 130-150 g

1.4 sex: male sex

1.5 qualification: SCXK (Shanghai) 2008-0016

2. Experimental dose design

Two drug effect dose groups of low and high are designed, and the doses are respectively as follows: 5mg/kg, 25 mg/kg.

3 preparation of the drug

The low dose group dissolved 5mg drug to 10ml and the high dose group dissolved 25mg drug to 10 ml.

4 route and volume of administration

4.1 pathway: is administered orally

4.2 Capacity: 100g of rats: 1ml of

4, positive medicine:

4.1 name Indometacin

4.2 units are provided: shanghai Xinyi yellow river pharmaceutical Co Ltd

4.3 batch number: 091201

4.4 dose: 5mg/kg

5 Experimental preparation and apparatus

5.1 inflammatory agents: carrageenan, sigma product, batch number: 1408463.

0.1g of carrageenan was made up to 10ml with double distilled water to 1%. Each rat was inflamed by subcutaneous injection of 0.1ml into the middle of the right hind paw.

5.2 measuring instrument: italy UGO company limb volume measuring instrument 7140-Plutysmemeter

Production unit: biological Research Apparatus 21025 Commerio VA Italy

6 Experimental methods

6.1 grouping and administration

Rats were randomly divided into a normal group, a model group, and a high-low dose group of samples, and each dose of drug was administered in groups on the day of the experiment.

6.2 Molding

Rats in each group were inflamed 1% by subcutaneous injection of carrageenan 0.1ml into the plantar region of the right hind limb 1 hour after dosing, resulting in an acute inflammation model in the rats.

6.3 index measurement

The volume of the foot pad of the inflammation-causing foot is respectively measured at different times (0, 1, 2 and 3 hours) before and after inflammation causing, a clear marked line is made on the front surface of the foot, and the measurement and the record are carried out by using a toe volume measuring instrument. The swelling rate was calculated from the swelling value, and the inhibition rate was calculated from the swelling rate of the toes of the rats in the model group and the administration group.

Percent swelling ═ En-Eo)/Eo 100%

En: swelling values Eo at different times after inflammation: pre-inflammatory swelling values

Inhibition%

Zn: swelling rate Zo of each administration group: swelling Rate of model group

7 results of the experiment

The experimental data are shown in Table 4. After inflammation, the swelling rate of the model group is obviously increased, which indicates that the model is successfully made and the peak is reached in 2h after inflammation. Compared with the model group, the swelling rate of the indometacin serving as the positive drug at each time point is reduced, the inhibition rates of 1h, 2h and 3h after inflammation are respectively 25.79%, 45.50% and 45.71% (P <0.05, P <0.01 and P <0.01), and the better treatment effect is shown.

The inhibition rate of compound 3 low dose group at 2h was 26.31% (P <0.05) compared to model group; the inhibition rate of the high-dose group in 1h and 2h is 33.25% (P <0.01) and 43.25% (P <0.01), respectively; the inhibition rate of the compound 1 high-dose group at 1h is 37.77% (P < 0.01); the inhibition rate of the compound 2 high-dose group at 2h is 37.40% (P < 0.01); whereas the low dose group of comparative compound 1 (corresponding to the high dose group of compound 1/2/3) showed only 16.78% (P <0.01) inhibition at 2h, which was significantly lower than the level of inhibition of compound 1/2/3.

The results showed that the high dose groups of compounds 1-3 all showed anti-inflammatory effects, with the most significant effect in the high dose group of compound 3. The compounds 1-3 can obviously reduce the toe swelling rate of rats with acute inflammation caused by carrageenan, and have certain pharmacodynamic action of preventing acute inflammation of rats, so the compounds have better development potential of anti-inflammatory drugs.

TABLE 4 pharmacodynamic Effect of Compounds 1-3 on acute inflammation in Carrageenan induced rats

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the preparation of (I) a composition or formulation against an inflammatory disease; and/or (ii) a Cyclooxygenase (COX) inhibitor,

in the formula, R₁Is methoxy or hydroxy; r₂For hydroxyl, the Cyclooxygenase (COX) inhibitor includes a COX-1 inhibitor and/or a COX-2 inhibitor.

2. The use of claim 1, wherein the inflammatory disease is selected from the group consisting of: neurogenic inflammation, gynecological inflammation, rheumatoid arthritis, or a combination thereof.

3. The use of claim 1, wherein the compound of formula I is a flavone glucuronide compound.

4. The use of claim 1, wherein the compound of formula I is a flavone diglucuronide.

5. A pharmaceutical composition for anti-inflammatory use, comprising:

wherein the definition of the compound of formula I is as described in claim 1.

6. The pharmaceutical composition of claim 5, wherein the non-steroidal anti-inflammatory component is selected from the group consisting of: indomethacin, aspirin, or a combination thereof.

7. The pharmaceutical composition of claim 5, wherein the weight ratio of the first active ingredient to the second active ingredient is from 1:100 to 100: 1.

8. the pharmaceutical composition of claim 7, wherein the weight ratio of the first active ingredient to the second active ingredient is from 1:10 to 10: 1.

9. a method of non-therapeutic inhibition of cyclooxygenase enzyme activity, comprising:

culturing cells in vitro in the presence of a compound of formula I, thereby inhibiting the activity of cyclooxygenase, including COX-1 and/or COX-2, in the cells; wherein the compound of formula I is as described in claim 1.

10. The method of claim 9, wherein the cell expresses a cyclooxygenase enzyme.

11. A method of screening for anti-inflammatory drug candidates, the method comprising the steps of:

(b) detecting the influence of the compound to be detected on the cyclooxygenase in a test group, and comparing the test result with corresponding experimental results in a positive control group and a negative control group, wherein the influence of the positive control compound on the cyclooxygenase is detected in the positive control group; the cyclooxygenase comprises COX-1 and/or COX-2;

wherein, if the inhibition degree of the tested compound on the cyclooxygenase is obviously higher than that of a negative control group, the tested compound is suggested to be an anti-inflammatory candidate drug; the compound of formula I is as described in claim 1.

12. The method of claim 11, wherein the method further comprises step (c): further determining the anti-inflammatory effect of the test compound selected in step (b).