阅读说明：本技术 二聚体环烯醚萜苷及其制备方法与用途 (Dimer iridoid glycoside and preparation method and application thereof ) 是由 高慧敏 王智民 陈两绵 孙玉 于 2021-09-26 设计创作，主要内容包括：本发明公开了一种下式I的二聚体环烯醚萜苷类化合物或其药学上可接受的盐、酯、外消旋体、R-异构体、S-异构体、同位素标记物、溶剂化物、代谢产物或前药,其制备工艺简单、重现性好、纯度高,具有较好的抗炎活性。(The invention discloses a dimer iridoid glycoside compound shown in the following formula I or pharmaceutically acceptable salt, ester, raceme, R-isomer, S-isomer, isotope label, solvate, metabolite or prodrug thereof, and the dimer iridoid glycoside compound has the advantages of simple preparation process, good reproducibility, high purity and better anti-inflammatory activity.)

1. A dimer iridoid glycoside compound of the following formula I or pharmaceutically acceptable salt, ester, raceme, R-isomer, S-isomer, isotope label, solvate, metabolite or prodrug thereof,

wherein the 6', 7 double bond configuration is Z formula or E formula, R₁And R₂Are identical or different and are each independently selected from the group consisting of-COOH, -COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-CH₂COOCH₃、-CH₂COOC₂H₅、-CH(CH₃)COOCH₃、-CH(CH₃)COOC₂H₅、-CH₂COOCH₂COCH₃、-OCH₃or-CHO.

2. The dimeric iridoid glycosides of claim 1, wherein the 6 ", 7 double bond configuration is Z or E, R₁And R₂Are identical or different and are each independently selected from the group consisting of-COOH, -COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-OCH₃or-CHO.

3. The dimeric iridoid glycosides of claim 2, wherein the 6 ", 7 double bond configuration is Z or E, R₁And R₂Are identical or different and are each independently selected from-COOH or-COOCH₃。

4. The dimeric iridoid glycosides of claim 3, wherein the 6 ", 7 double bond configuration is Z or E, R₁And R₂Are different and are selected from-COOH or-COOCH₃。

5. The dimeric iridoid glycosides compound according to any one of claims 1 to 4, wherein the 6 ", 7 double bond configuration is Z formula or E formula, R₁is-COOCH₃，R₂is-COOH or R₁is-COOH, R₂is-COOCH₃。

6. A pharmaceutical preparation comprising the dimeric iridoid glycosides of any one of claims 1 to 5 and a pharmaceutically acceptable excipient;

preferably, the dosage form of the pharmaceutical formulation is a liquid dosage form or a solid dosage form;

more preferably, the liquid dosage form is a syrup, injection solution, suspension or emulsion;

particularly preferably, the solid dosage form is a tablet, lozenge, capsule, drop pill, granule, powder, cream or suppository;

particularly preferably, the auxiliary material is selected from one or more of the following: flavoring agents, disintegrating agents, preservatives, lubricants, humectants, binders, thickeners, and solubilizers.

7. The pharmaceutical formulation of claim 6, wherein the pharmaceutical formulation further comprises one or more anti-inflammatory agents;

preferably, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent;

preferably, the non-steroidal anti-inflammatory drug is selected from one or more of the following: aspirin, acetaminophen, indomethacin, naproxen, naproxone, diclofenac, ibuprofen, nimesulide, rofecoxib, and celecoxib.

8. A process for preparing the dimeric iridoid glycosides of any one of claims 1 to 5, wherein said process comprises the steps of:

(1) taking a proper amount of honeysuckle as a raw material, and carrying out cold leaching, percolation, reflux or ultrasonic extraction by using 40-95% ethanol water solution to obtain an extracting solution;

(2) concentrating the percolate to obtain a concentrated solution;

(3) separating the concentrated solution by using macroporous adsorption resin, sequentially eluting with water, 35-45% ethanol water solution, 65-75% ethanol water solution and about 95% ethanol water solution, collecting 35-45% ethanol water solution eluate, and performing polyamide column chromatography to obtain total iridoid extract; and

(4) and carrying out silica gel column chromatography and semi-preparative HPLC separation on the total iridoid extract to obtain the dimer iridoid glycoside compound shown in the formula I.

9. The method according to claim 8, wherein the honeysuckle flower medicinal material in the step (1) is a honeysuckle flower cultivation product Jiufeng I;

preferably, the method of extraction in step (1) is percolation;

preferably, the concentration of the aqueous ethanol solution in step (1) is about 50%;

preferably, the method of concentration in the step (2) is reduced pressure rotary evaporation;

preferably, the eluent in the step (3) is water, about 40% ethanol water solution, about 70% ethanol water solution and about 95% ethanol water solution in sequence;

preferably, the macroporous adsorbent resin in the step (3) is HPD100 macroporous adsorbent resin;

preferably, the silica gel column chromatography in the step (4) adopts forward silica gel with the particle size of 200 to 300 meshes;

preferably, the silica gel column chromatography in the step (4) adopts a Flash medium-pressure preparation method;

particularly preferably, the elution mobile phase of the Flash medium-pressure preparation method is a mixed solvent with a volume ratio of dichloromethane-methanol-water of 9:2: 0.2;

still preferably, the mobile phase of the semi-preparative HPLC separation step is acetonitrile-0.1% formic acid (17: 83) and/or acetonitrile-0.1% formic acid (15: 85);

still preferably, the detection wavelength of the semi-preparative HPLC separation step is 239 nm;

still preferably, the flow rate of the semi-preparative HPLC separation step is 3 mL-min^-1。

10. Use of a dimeric iridoid glycoside compound according to any one of claims 1 to 5 or a pharmaceutical preparation according to claim 6 or 7 for the preparation of a medicament for the treatment and/or prevention of inflammation;

preferably, the inflammation is a nitric oxide-mediated inflammatory response;

more preferably, the nitric oxide is released by macrophages;

particularly preferably, the nitric oxide is released from bacterial lipopolysaccharide-induced macrophages.

Technical Field

The invention relates to the field of traditional Chinese medicine chemistry, relates to a dimer iridoid glycoside compound and a preparation method thereof, and also relates to application of the dimer iridoid glycoside compound in treating inflammatory diseases, symptoms and/or injuries.

Background

Inflammatory responses are efforts to restore and maintain homeostasis after the body has been attacked by infectious agents, antigenic attacks, or physical, chemical, or traumatic injuries. Localized inflammation is localized to a specific site and may exhibit different symptoms including redness, swelling, heat, and pain. Inflammation is often a protective attempt by organisms to clear harmful stimuli and initiate the tissue healing process. However, poorly controlled inflammation can lead to a variety of diseases, regardless of the age of the subject.

Anti-inflammatory drugs currently used clinically are mainly non-steroidal anti-inflammatory drugs (NSAIDs) which reduce inflammation by blocking the action of cyclooxygenase-2 (COX-2), but at the same time, such drugs also block the action of COX-1. The main effect of COX-1 is to protect the gastric mucosa, so that blockade of COX-1 action produces gastric irritation. Therefore, NSAIDs have antipyretic, analgesic, anti-inflammatory, antirheumatic effects, but their side effects are mainly in the gastrointestinal tract, and secondly in the liver and kidney, and they are prone to drug resistance, addiction, and immunosuppressive side effects. In the research and research on anti-inflammatory drugs in recent years, the traditional Chinese medicine has more and more prominent advantages of good efficacy, no addiction, less adverse reaction and the like, and attracts attention of people.

The honeysuckle has the traditional effects of clearing heat and removing toxicity and mainly treats heat-toxin and bloody dysentery, carbuncle and furuncle, pharyngitis, various infectious diseases and the like. Modern pharmacological studies show that the honeysuckle has outstanding anti-inflammatory activity, but the researches mainly take phenolic acid and flavonoid components in the honeysuckle as main research, and the anti-inflammatory activity of dimer iridoid glycoside components in the honeysuckle is less reported.

According to the invention, researches show that the dimeric iridoid glycoside compounds with brand-new chemical structures in honeysuckle have better anti-inflammatory effects and may have wide prospects in preparation of drugs for preventing or/and treating inflammatory diseases and diseases. The dimeric iridoid glycoside compounds in the honeysuckle have wide pharmacological activity, come from natural plants, are safe and reliable, and do not have related reports of the dimeric iridoid glycoside compounds for treating or/and preventing inflammatory diseases and diseases at present.

Disclosure of Invention

The invention mainly aims to provide a medicament for treating inflammatory diseases and symptoms, which has definite curative effect, less adverse reaction and natural safety, and solves the problems that the common medicaments in the prior art have obvious gastrointestinal tract reaction, are easy to generate drug resistance and addiction and have immunosuppressive side effect.

In order to achieve the above objects, according to one aspect of the present invention, there is provided a dimeric iridoid glycoside compound of the following formula I or a pharmaceutically acceptable salt, ester, racemate, R-isomer, S-isomer, isotopic label, solvate, metabolite or prodrug thereof,

wherein the 6', 7 double bond configuration is Z formula or E formula, R₁And R₂Are identical or different and are each independently selected from the group consisting of-COOH, -COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-CH₂COOCH₃、-CH₂COOC₂H₅、-CH(CH₃)COOCH₃、-CH(CH₃)COOC₂H₅、-CH₂COOCH₂COCH₃、-OCH₃or-CHO.

Further, the 6', 7 double bond configuration is Z formula or E formula, R₁And R₂Are identical or different and are each independently selected from the group consisting of-COOH, -COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-OCH₃or-CHO.

Further, the 6', 7 double bond configuration is Z formula or E formula, R₁And R₂Are identical or different and are each independently selected from-COOH or-COOCH₃。

Further, the 6', 7 double bond configuration is Z formula or E formula, R₁And R₂Are different and are selected from-COOH or-COOCH₃。

Further, the 6', 7 double bond configuration is Z formula or E formula, R₁is-COOCH₃，R₂is-COOH or R₁is-COOH, R₂is-COOCH₃。

According to another aspect of the invention, a pharmaceutical preparation is provided, which comprises the dimeric iridoid glycoside compounds and pharmaceutically acceptable auxiliary materials.

Further, the dosage form of the pharmaceutical preparation is a liquid dosage form or a solid dosage form.

Further, the liquid dosage form is a syrup, an injection solution, a suspension or an emulsion.

Further, the solid dosage form is a tablet, lozenge, capsule, drop pill, granule, powder, cream or suppository.

Further, the adjuvant is selected from one or more of the following: flavoring agents, disintegrating agents, preservatives, lubricants, humectants, binders, thickeners, and solubilizers.

Further, the pharmaceutical formulation further comprises one or more anti-inflammatory agents.

Further, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent.

Further, the non-steroidal anti-inflammatory drug is selected from one or more of the following: aspirin, acetaminophen, indomethacin, naproxen, naproxone, diclofenac, ibuprofen, nimesulide, rofecoxib, and celecoxib.

According to another aspect of the present invention, there is provided a method for preparing the above dimer iridoid glycoside compound, wherein the method comprises the steps of:

(1) taking a proper amount of honeysuckle as a raw material, and carrying out cold leaching, percolation, reflux or ultrasonic extraction by using 40-95% ethanol water solution to obtain an extracting solution;

(2) concentrating the percolate to obtain a concentrated solution;

(3) separating the concentrated solution with macroporous adsorbent resin, eluting with water, 35% to 45% ethanol water solution, 65% to 75% ethanol water solution and about 95% ethanol water solution, collecting 35% to 45% ethanol water solution eluate, and subjecting to polyamide column chromatography to obtain total iridoid extract; and

(4) the total iridoid extract is subjected to silica gel column chromatography and semi-preparative HPLC separation steps to obtain the dimer iridoid glycoside compound shown in the formula I.

Further, the honeysuckle medicinal material in the step (1) is a honeysuckle cultivation product Jiufeng I; further, the extraction method in the step (1) is a percolation method; further, the concentration of the ethanol aqueous solution in the step (1) is about 50%; further, the method of concentration in the step (2) is a reduced pressure rotary evaporation method; further, the eluent in the step (3) is water, about 40% ethanol water solution, about 70% ethanol water solution and about 95% ethanol water solution in sequence; further, the macroporous adsorbent resin in the step (3) is HPD100 macroporous adsorbent resin.

Further, the silica gel column chromatography in the step (4) adopts forward silica gel with the particle size of 200 to 300 meshes;

further, the silica gel column chromatography in the step (4) adopts a Flash medium-pressure preparation method;

further, an elution mobile phase of the Flash medium-pressure preparation method is a mixed solvent with a volume ratio of dichloromethane-methanol-water of 9:2: 0.2;

further, the mobile phase of the semi-preparative HPLC separation step is acetonitrile-0.1% formic acid (17: 83) and/or acetonitrile-0.1% formic acid (15: 85).

Further, the detection wavelength of the semi-preparative HPLC separation step was 239 nm.

Further, the flow rate of the semi-preparative HPLC separation step was 3 mL-min^-1。

According to another aspect of the present invention, there is provided a use of the above dimer iridoid glycoside compound or the above pharmaceutical preparation for the preparation of a medicament for the treatment and/or prevention of inflammation.

Further, the inflammation is an inflammatory reaction mediated by nitric oxide.

Further, the nitric oxide is released by macrophages.

Further, the nitric oxide is released from bacterial lipopolysaccharide-induced macrophages.

The invention has the beneficial effects that:

earlier researches compare the quality of all the cultivated varieties of honeysuckle, and find that the cultivated variety Jiufeng I of honeysuckle has obvious chemical component difference compared with other cultivated varieties, and samples of Jiufeng I of different producing areas all contain rich iridoid glycoside components. In order to determine the types of iridoid glycoside components in Jiufeng I, the research adopts silica gel column chromatography, semi-preparative HPLC and other means to separate and purify monomer compounds from the total iridoid extract of Jiufeng I, and performs structural identification of the compounds based on the spectrum technology, thereby enriching the chemical components of honeysuckle. The research finds that dimer iridoid glycoside compounds with new chemical structures extracted and separated from the honeysuckle flower cultivation product Jiufeng I have obvious anti-inflammatory effects, the preparation process is simple, the reproducibility is good, the purity is high, the application range of honeysuckle flowers can be enlarged by developing the dimer iridoid glycoside compounds into medicines for treating inflammatory diseases and symptoms, and the utilization value of honeysuckle flower plant resources is improved.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without exceeding the protection scope of the present application.

FIG. 1 is a flow chart of the total iridoid extract extracted and separated from the honeysuckle flower cultivation product Jiufeng I.

FIG. 2 is a schematic diagram of the process for isolating compounds from total iridoid extracts.

FIG. 3 is the structural formula of Compound 3.

FIG. 4 is the structural formula of Compound 4.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some, but not all, embodiments of the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The present invention is intended to cover all alternatives, modifications and equivalents, which may be included in the field of the present invention as defined by the appended claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein which can be used in the practice of the present invention. The present invention is in no way limited to the description of methods and materials.

The invention described herein may include one or more numerical ranges (e.g., particle size, concentration, etc.). A range of values is understood to include all values within the range, including the values that define the range, as well as values adjacent to the range that produce the same or substantially the same result as the values immediately adjacent to the value defining the boundary of the range.

To the extent that the terms "includes," including, "and" having "or variants thereof are used in either the detailed description and/or the claims, these terms are intended to be inclusive in a manner similar to the term" comprising.

As described in the background section, the common drugs currently available for the treatment of inflammatory diseases and conditions are gastrointestinal, resistant, addictive, and have immunosuppressive side effects. In order to solve the above problems, the present invention provides a dimeric iridoid glycoside compound of the following formula I or a pharmaceutically acceptable salt, ester, racemate, R-isomer, S-isomer, isotopic label, solvate, metabolite or prodrug thereof,

wherein the 6', 7 double bond configuration is Z formula or E formula, R₁And R₂Are identical or different and are each independently selected from the group consisting of-COOH, -COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-CH₂COOCH₃、-CH₂COOC₂H₅、-CH(CH₃)COOCH₃、-CH(CH₃)COOC₂H₅、-CH₂COOCH₂COCH₃、-OCH₃or-CHO.

In a preferred embodiment, the pharmaceutically acceptable salt comprises an inorganic acid salt or an organic acid salt.

In a preferred embodiment, the pharmaceutically acceptable salt is selected from the group consisting of: hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, bisulfate, formate, acetate, adipate, benzoate, benzenesulfonate, tosylate, mesylate, citrate, camphorate, fumarate, gluconate, maleate, oxalate, salicylate, tartrate, or a combination thereof.

In a preferred embodiment, the term "ester" as used herein refers to an ester of a compound of formula I of the present invention which is hydrolysed in vivo (in vivo) and includes esters which readily decompose in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, especially alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety preferably has 6 or fewer carbon atoms.

In a preferred embodiment, the compounds of formula I according to the invention have asymmetric centers, chiral axes and chiral planes and can be present in the form of racemates, R-isomers or S-isomers. The person skilled in the art is able to obtain the R-isomer and/or the S-isomer by resolution of the racemate by means of customary technical measures.

In a preferred embodiment, the isotopic label of the present invention is²H、³H、¹⁰B、¹³C、¹⁴C、¹³N、¹⁵N、¹⁸O、²⁶Mg、³²P、³⁴S、³⁵S、³⁷Cl、⁴¹K、⁴⁵Ca、⁵⁴Fe、⁵⁷Fe and⁶⁵a water-soluble compound of at least one of Cu.

In a preferred embodiment, the term "solvate" as used herein refers to an association of one or more solvent molecules with a compound of formula I of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol.

In a preferred embodiment, the term "metabolite" as used herein refers to the product of the metabolism of a particular compound of formula I or a salt thereof in vivo. Metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by assay methods as described herein. Such products may be obtained by administering the compound by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting a compound of the present invention with a mammal for a sufficient period of time.

In a preferred embodiment, the term "prodrug", as used herein, represents a compound that is converted in vivo to a compound of formula I. Such conversion is effected by hydrolysis of the prodrug in the blood or by enzymatic conversion to the parent structure in the blood or tissue. The prodrug compound of the invention can be ester, and in the prior invention, the ester can be used as the prodrug and comprises phenyl ester and aliphatic (C)_1-24) Esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters.

In a preferred embodiment, the 6', 7 double bond configuration is Z or E, R₁And R₂Are identical or different and are each independently selected from the group consisting of-COOH, -COOCH₃、-COOC₂H₅、-COOC₃H₇、-COOC₄H₉、-OCH₃or-CHO.

In a preferred embodiment, the 6', 7 double bond configuration is Z or E, R₁And R₂Are identical or different and are each independently selected from-COOH or-COOCH₃。

In a preferred embodiment, the 6', 7 double bond configuration is Z or E, R₁And R₂Are different and are selected from-COOH or-COOCH₃。

In a preferred embodiment, the 6', 7 double bond configuration is Z or E, R₁is-COOCH₃，R₂is-COOH.

According to another aspect of the invention, a pharmaceutical preparation is provided, which comprises the dimeric iridoid glycoside compounds and pharmaceutically acceptable auxiliary materials.

In a preferred embodiment, the dosage form of the pharmaceutical formulation is a liquid dosage form or a solid dosage form.

In a preferred embodiment, the liquid dosage form is a syrup, an injectable solution, a suspension or an emulsion.

In a preferred embodiment, the solid dosage form is a tablet, lozenge, capsule, drop pill, granule, powder, cream or suppository.

In a preferred embodiment, the adjuvant is selected from one or more of the following: flavoring agents, disintegrating agents, preservatives, lubricants, humectants, binders, thickeners, and solubilizers.

Among these, non-limiting examples of flavoring agents include, but are not limited to, sugar alcohols, such as sorbitol, xylitol; monosaccharides, including sucrose, glucose, fructose, and lactose; sorbitol, mannitol, glycerol, xylitol, erythritol, maltitol syrup, isomalt, lactitol; and mixtures of sugars, including glucose syrup; invert syrup, high sugar content syrup; and malt or malt extract; disintegrants may include, but are not limited to, one or more of the following: croscarmellose sodium, carboxymethylcellulose calcium, crospovidone, alginic acid, sodium alginate, potassium alginate, calcium alginate, ion exchange resins, effervescent systems based on food acid and alkaline carbonate components, clays, talc, starches, pregelatinized starches, sodium starch glycolate, cellulose floc, carboxymethylcellulose, hydroxypropylcellulose, calcium silicate, metal carbonates, sodium bicarbonate, calcium citrate and calcium phosphate; preservatives can be potassium sorbate, methyl paraben, propyl paraben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butyl paraben, alcohols such as ethanol or benzyl alcohol, phenolic chemicals such as phenol, or quaternary compounds such as benzalkonium chloride; non-limiting examples of lubricants include, but are not limited to, calcium stearate, canola oil, glyceryl palmitostearate, hydrogenated vegetable oil, poloxamer, polyethylene glycol, polyvinyl alcohol, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, sterilized corn starch, talc, and zinc stearate; humectants may include, but are not limited to, one or more of the following: corn starch, potato starch, corn starch, and modified starches, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof; the binder may include, but is not limited to, one or more of the following: a variety of celluloses, crosslinked polyvinylpyrrolidone, and microcrystalline cellulose; the thickening agent may be polyacrylate and polyacrylate copolymer resins, such as polyacrylic acid and polyacrylic acid/methacrylic acid resins; or cellulose and cellulose derivatives including alkyl celluloses such as methyl-, ethyl-, and propyl-cellulose; the solubilizing agent may include, but is not limited to, one or more of the following: acetonitrile, dimethylformamide, dimethylacetamide, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidinone, vitamin E and vitamin E derivatives.

In a preferred embodiment, the pharmaceutical formulation further comprises one or more anti-inflammatory agents.

In a preferred embodiment, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent.

In a preferred embodiment, the non-steroidal anti-inflammatory drug is selected from one or more of the following: aspirin, acetaminophen, indomethacin, naproxen, naproxone, diclofenac, ibuprofen, nimesulide, rofecoxib, and celecoxib.

Wherein, aspirin belongs to cyclooxygenase inhibitor, which can block the conversion of prostaglandin into arachidonic acid, thereby reducing the generation of inflammation mediators and playing an anti-inflammatory role; acetaminophen inhibits central nervous system prostaglandin synthesis similar to aspirin, but has a weaker anti-inflammatory effect; indomethacin acts as an anti-inflammatory by reducing prostaglandin synthesis through inhibition of cyclooxygenase.

According to another aspect of the present invention, there is provided a method for preparing the above dimer iridoid glycoside compound, wherein the method comprises the steps of:

(1) taking a proper amount of honeysuckle as a raw material, and carrying out cold leaching, percolation, reflux or ultrasonic extraction by using 40-95% ethanol water solution to obtain an extracting solution;

(2) concentrating the percolate to obtain a concentrated solution;

(3) separating the concentrated solution with macroporous adsorbent resin, eluting with water, 35% to 45% ethanol water solution, 65% to 75% ethanol water solution and about 95% ethanol water solution, collecting 35% to 45% ethanol water solution eluate, and subjecting to polyamide column chromatography to obtain total iridoid extract; and

(4) the total iridoid extract is subjected to silica gel column chromatography and semi-preparative HPLC separation steps to obtain the dimer iridoid glycoside compound shown in the formula I.

In a preferred embodiment, the honeysuckle flower in step (1) is the cultivated product of honeysuckle flower jiufeng yi; in a preferred embodiment, the method of extraction in step (1) is percolation; in a preferred embodiment, the concentration of the aqueous ethanol solution in step (1) is about 50%.

The term "about" or "approximately" means within an acceptable error range for the particular value determined by one skilled in the art, which error range depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can be within a standard deviation of 1 or greater than 1, in accordance with practice in the art. Alternatively, "about" may refer to a range of up to 10%, preferably up to 5%, and more preferably up to 1% of a given value. Where particular values are described herein, unless otherwise stated, it can be assumed that the term "about" refers to within an acceptable error range for the particular value.

In a preferred embodiment, the method of concentration in the step (2) is reduced pressure rotary evaporation;

in a preferred embodiment, the eluent in the step (3) is water, about 40% aqueous ethanol, about 70% aqueous ethanol and about 95% aqueous ethanol in this order.

In a preferred embodiment, the macroporous adsorbent resin in step (3) is HPD100 macroporous adsorbent resin.

In a preferred embodiment, the silica gel column chromatography in the step (4) is performed using a forward silica gel having a particle size of 200 to 300 mesh;

in a preferred embodiment, the silica gel column chromatography in the step (4) adopts a Flash medium pressure preparation method;

in a preferred embodiment, the elution mobile phase of the Flash medium-pressure preparation method is a mixed solvent with a volume ratio of dichloromethane-methanol-water of 9:2: 0.2;

in a preferred embodiment, the mobile phase of the semi-preparative HPLC separation step is acetonitrile-0.1% formic acid (17: 83) and/or acetonitrile-0.1% formic acid (15: 85).

In a preferred embodiment, the detection wavelength of the semi-preparative HPLC separation step is 239 nm.

In a preferred embodiment, the semi-preparative HPLC separation step has a flow rate of 3mL min^-1。

The eluent in the silica gel Flash column chromatography step and the mobile phase in the semi-preparative HPLC separation step are selected according to actual conditions, and can be a combination of different mobile phases.

According to another aspect of the present invention, there is provided a use of the above dimer iridoid glycoside compound or the above pharmaceutical preparation for the preparation of a medicament for the treatment and/or prevention of inflammation.

The invention relates to dimer iridoid glycoside compounds 1-5 extracted and separated from a honeysuckle flower cultivation product Jiufeng I and analogues thereof, which have the inhibition rate range of about 25 percent to about 75 percent on NO released by RAW264.7 cells induced by Lipopolysaccharide (LPS) under the concentration of 12.5 mu M to 25 mu M, and have NO cytotoxicity on the RAW264.7 cells when the concentration is less than or equal to 100 mu M.

As used herein, the term "inflammation" refers to any disease caused by inflammation or those diseases whose symptoms include inflammation. For example, an inflammation resulting from inflammation may be a septic shock, while an inflammation whose symptoms include inflammation may be rheumatoid arthritis. Inflammation of the present invention includes, but is not limited to: cardiovascular disease, rheumatoid arthritis, multiple sclerosis, Crohn's disease, inflammatory bowel disease, systemic lupus erythematosus, polymyositis, septic shock, graft versus host disease, asthma, rhinitis, cancer-associated cachexia of psoriasis and eczema.

In a preferred embodiment, the inflammation is a nitric oxide mediated inflammatory response.

Wherein, under normal condition, the endothelial source NO has the function of inhibiting inflammatory reaction, and under pathological condition, the inducible NO synthetase synthesizes a large amount of NO and has the cytotoxic function, thus aggravating the inflammatory reaction.

In a preferred embodiment, the nitric oxide is released by macrophages.

In a preferred embodiment, the nitric oxide is released from bacterial lipopolysaccharide-induced macrophages.

Among them, LPS is a major component of the cell wall of gram-negative bacteria and is also the major material basis for its pathogenesis. LPS is a main substance for inducing activation and maturation of mononuclear/macrophage, and can participate in acute phase response of organisms by stimulating mononuclear/macrophage to generate and release a large amount of inflammatory factors such as Nitric Oxide (NO), IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and the like, so as to cause inflammatory injury of the organisms.

The present invention also relates to a method for the treatment and/or prevention of inflammation, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition or pharmaceutical formulation as described above.

As used herein, the term "treatment" refers to any regimen beneficial to a human or non-human animal. Thus, the treatment may be with respect to an existing condition or may be prophylactic (prophylactic treatment). Treatment may include curative, palliative or prophylactic effects; the term "effective amount" refers to the dose or amount of drug administered to a patient, as well as the frequency of administration to a patient, which can be readily determined by one of ordinary skill in the art based on prior art applications and by observing results obtained under similar conditions effective to treat a disease or condition. To determine an effective amount or dose, a skilled diagnostician considers a number of factors, including but not limited to: the effect and duration of action of the compound employed; the characteristics and severity of the condition being treated, as well as the sex, age, weight and general health and individual response of the patient being treated; and related conditions known to those skilled in the art. Generally, it will be present in an amount in the range of about 0.01mg/kg to about 100mg/kg of body weight, typically in the range of about 1mg/kg to about 20mg/kg of body weight.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed herein.

Examples

1 materials of the experiment

BrukerAvance 600MHz NMR spectrometer (Bruker, Switzerland), WatersACQUITY UPLC H-CLASS ultra-high liquid chromatography System (Waters, USA), Xevo G2-S QTOF quadrupole high-resolution time-of-flight mass spectrometer (Waters, USA), Flash Rapid purification preparative chromatograph (Biotage, Switzerland), semi-preparative high-efficiency spectrometerLiquid chromatograph (Alltech, USA), Shimadzu LC-20A high performance liquid chromatograph (Shimadzu, Japan), GCK3308 full automatic air source (Hewlett packard analytical technical research institute in Beijing), BSA2245-CW analytical balance (Sidolisi scientific instruments Co., Ltd.), EYELA rotary evaporator (SB-1100 Shanghai Elang instruments Co., Ltd.), SK10GT type ultrasonic cleaner (Shanghai Kedao ultrasonic instruments Co., Ltd.), Sigma 1-14 small bench centrifuge (Sigma, Germany), vacuum drying oven (DZF-6053 Shanghai Hengscientific instruments Co., Ltd.), LGL-25C freeze dryer (Beijing four ring Co., Ltd.). A chromatographic column: agilent C₁₈Column (5 μm, 9.4X 250mm) (Agilent, USA), Kromasil C₁₈Column (5 μm, 4.6X 250mm) (AKZO NOBEL, Sweden), Waters ACQUITY UPLC C₁₈Column (1.7 μm, 2.1X 100mm) (Waters, USA). Thin layer chromatography silica gel plate, 200-mesh forward silica gel (Qingdao ocean chemical plant), the reagent used is chemical purity or analytical purity.

2 method of experiment

Percolating flos Lonicerae cultivated product JIUFENG I medicinal material 2Kg with 50% ethanol, concentrating the extractive solution by rotary steaming under reduced pressure, loading the concentrated solution onto HPD100 macroporous adsorbent resin, eluting with water, 40% ethanol, 70% ethanol and 95% ethanol respectively, and performing 30-60 mesh polyamide column chromatography to obtain JIUFENG I total iridoid extract (130 g). As shown in particular in figure 1.

Dissolving 30.0g of total iridoid extract with methanol, performing Flash medium-pressure preparation and separation by using 200-300 mesh silica gel, eluting with dichloromethane-methanol-water (9-2-0.2) in an elution system, detecting by TLC, collecting and combining similar fractions to obtain different components, wherein the component A (0.15g) is dissolved with 40% methanol, and is subjected to semi-preparative HPLC separation, the mobile phase is acetonitrile-0.1% formic acid water (17: 83), the detection wavelength is 239nm, and the flow rate is 3 mL/min^-1To give Compound 1(7.63mg) (t)_R30min) and Compound 2(22.52mg) (t)_R43 min); dissolving component B (1.89g) in 40% methanol, separating by semi-preparative HPLC with acetonitrile-0.1% formic acid (15: 85) as mobile phase, detecting wavelength of 239nm, and flow rate of 3 mL/min^-1To give Compound 4(31.04mg) (t)_R40min) and Compound 5 (C)13.74mg)(t_R34 min); lyophilizing the semi-prepared fraction of the component B into powder, dissolving in 40% methanol, separating by semi-preparative HPLC with acetonitrile-0.1% formic acid (17: 83) as mobile phase, detection wavelength of 239nm, and flow rate of 3 mL/min^-1To give Compound 3(2.44mg) (t)_R37 min). A schematic diagram of the process for isolating compounds from total iridoid extract is shown in FIG. 2.

3 structural analysis of Compound

3.1 information on Compounds

By passing¹H-NMR、¹³C-NMR、HSQC、HMBC、TOCSY、¹H-¹Spectroscopic techniques such as H-COSY and LC-MS, etc., and 5 dimer iridoid glycoside compounds are identified, and specific compounds are shown in Table 1.

TABLE 1 dimer iridoid glycosides isolated from Lonicera japonica Thunb et al, cultivated product Jiufeng I

Note: a novel compound;^△is separated from flos Lonicerae for the first time

Wherein compound 1: (Z) -aldosecoogloganin; compound 2: (E) -an aldoescologenin; compound 3: (Z) -demethyl-aldosecocologanin; compound 4: (E) -demethyl-aldoescologenin; compound 5: strychosides A.

3.2 structural analysis of novel Compound

The compounds 3 and 4 are white amorphous powder, are easily soluble in water and methanol, have maximum absorption wavelength of 239nm, and have the same molecular formula C₃₃H₄₄O₁₉。¹³C-NMR and DEPT spectra showed that compounds 3 and 4 had 34 carbons (1 XCH)₃,5×CH₂,22×CH,5×C)。¹³The C-NMR spectrum showed 2 ester carbonyl groups (Compound 3: delta C171.1,169.7, Compound 4: delta C170.6,169.2) and 1 ester carbonyl groupMethoxy (compound 3: deltaC 52.6, compound 4: deltaC 52.1) and group 2 glucose signals (deltaC 62.8-101.0), with the remaining C signals appearing in multiple pairs.¹H-¹The H-COSY spectrum shows that the olefinic proton H-7 (delta 6.42) on the double bond of the compound 3 is coupled with H-6 (delta 3.00,2.70), and the olefinic proton H-7 (delta 6.71) on the double bond of the compound 4 is coupled with H-6 (delta 3.09,2.47), so that the compound is an iridoid dimer.¹The H-NMR spectrum shows that two compounds respectively have terminal signals (8,8 '-2H, 10, 10' -2H) and olefin (3,3 '-2H) in two typical secoiridoid glycosides, and meanwhile, an aldehyde signal (7', -1H) a carbohydrate terminal group hydrocarbon signal (1 ', 1' -2H) and two groups of glucose signals (delta 3.19-4.72) further show that the molecular frameworks of the compounds 3 and 4 are dimers of secologenin (secologanin), only lack an aldehyde group and a methoxy group, and simultaneously have one more double bond, and the double bond (C) on the alpha, beta-unsaturated aldehyde structure is presumed from a chemical shift value₇＝C₆″)。

Compound 3 at¹H and¹³aldehyde group signals [ delta H-7 '10.04(s), delta C-7' 192.8 are observed in the C-NMR spectrum]And substituted alkenes [ δ H-76.42(1H, t, J ═ 8.0Hz), δ C-7151.2 and δ C-6 "141.3]Of the signal of (1). In the HMBC spectra, the aldehyde group H-7' (Δ 10.04) was observed to correlate with C-7 (. delta.151.2); similarly, the double bond linkage between the C-7 and C-6 "positions, similar in structure to Compound 1 (Z-aldosecocolagenin), was fully demonstrated by the correlation of H-7 (. delta.6.42) with the aldehyde groups C-7 '(Δ 192.8) and C-6' (Δ 141.3), respectively. However, only one methoxy group is present in Compound 3, and the HMBC correlation of the methoxy signal Δ 3.73(3H, s) with C-11 ' (Δ 169.7), the olefin signal C-3 ' (Δ 7.51) places the carbomethoxy group on C-4 '.

Compound 4 at¹H and¹³the C-NMR spectrum was similar to that of Compound 3, except that the chemical shifts induced by 7 '-CHO (delta C197.1, delta H9.26) and H-7 (delta C156.5, delta H6.71), the HMBC interrelationships between olefins H-7 (delta H6.71) and C-5, C-7' indicated similarity to Compound 5 (Strychosides A). The difference is that the HMBC correlation of the methoxy signal delta 3.74 with C-11 (delta 169.2), the olefin signal C-3 (delta 7.57) places the carbomethoxy group on C-4, an isomer of compound 3. Comprehensive analysis¹H-¹H COSY, HSQC, HMBC and the like, and the rest carbon and proton signals are subjected to full attribution, and attribution data are shown in a table 2.

The structural formulas of the compounds 3 and 4 are determined according to the physicochemical properties and the spectrum data as shown in the figures 3 and 4, and the compounds 3 and 4 are determined to be new compounds through systematic literature and Scifinder query and are named as (Z) -demethyl-aldoseologenin and (E) -demethyl-aldoseologenin respectively.

TABLE 2 Compounds 3 and 4¹H-NMR and¹³C-NMR data (600and 150 MHz. delta. in ppm, CD)₃OD)

3.3 other known Compounds

Compound 1: white amorphous powder (methanol), readily soluble in water, methanol. The maximum ultraviolet absorption wavelength is 239 nm. HR-ESI-MS negative ion shows: ESI-MS (m/z): 757.2584[ M-H]^-，803.2653[M+HCOO]^-And the molecular formula is presumed to be C by integrating mass spectrum data₃₄H₄₆NO₁₉。¹H-NMR(600MHz,CD₃OD)δ:5.47(1H,s,H-1)，5.35(1H,s,H-1″)，7.66(1H,s,H-3)，7.53(1H,s,H-3″)，3.00(1H,m,H-5)，4.14(1H,m,H-5″)，2.63(1H,m,H-6a)，3.24(1H,m,H-6b)，6.43(1H,s,H-7)，10.08(1H,s,H-7″)，5.83–5.73(1H,m,H-8)，5.55(1H,m,H-8″)，2.72(1H,s,H-9)，2.66(1H,s,H-9″)，5.31(1H,d,J＝10.4Hz,H-10a)，5.35(1H,m,H-10b)，5.12(1H,d,J＝10.7Hz,H-10″a)，5.16(1H,m,H-10″b)，3.76(3H,s,H-12)，3.71(3H,s,H-12″)，4.74(1H,d,J＝5.5Hz,H-1′)，4.71(1H,d,J＝7.4Hz,H-1″′)，3.22-3.40(8H,m,H-glc)，3.93(2H,t,J＝10.7Hz,H-6′a,6″′a)。¹³C-NMR(150MHz,CD₃OD)δ:98.1(C-1)，97.9(C-1″)，154.9(C-3)，154.3(C-3″)，110.7(C-4)，110.4(C-4″)，34.4(C-5，5″)，26.8(C-6)，141.4(C-6″)，151.1(C-7)，192.4(C-7″)，136.0(C-8)，135.6(C-8″)，46.2(C-9)，45.5(C-9″)，120.8(C-10)，120.0(C-10″)，169.3(C-11)，169.1(C-11″)，52.5(C-12)，52.3(C-12″)，100.7(C-1′)，100.4(C-1 '), 75.0 (C-2'), 74.3(C-2 '), 78.4 (C-3', 3 '), 72.0(C-4), 71.9 (C-4'), 78.8(C-5 '), 78.7 (C-5'), 63.2(C-6 '), 63.1 (C-6'). The above data are substantially consistent with (Z) -aldosecologanin reported in the literature (Liu Z X, Liu C T, Liu Q B, et al. journal of functional Foods,2015,18: 512-.

Compound 2: white amorphous powder (methanol), readily soluble in water, methanol. The maximum ultraviolet absorption wavelength is 239 nm. HR-ESI-MS negative ion shows: ESI-MS (m/z): 757.2552[ M-H]^-，803.2617[M+HCOO]^-And the molecular formula is presumed to be C by integrating mass spectrum data₃₄H₄₆O₁₉。¹H-NMR(600MHz,CD₃OD)δ:5.60(1H,s,H-1)，5.51(1H,s,H-1″)，7.56(1H,s,H-3)，7.49(1H,s,H-3″)，3.12(1H,m,H-5,6b)，4.08(1H,m,H-5″)，2.46(1H,s,H-6a)，6.73(1H,s,H-7)，9.24(1H,s,H-7″)，5.80(1H,m,H-8)，5.63(1H,m,H-8″)，2.80(1H,s,H-9)，2.61(1H,m,H-9″)，5.38(1H,d,J＝17.2Hz,H-10a)，5.31(1H,d,J＝9.8Hz,H-10b)，5.08(1H,d,J＝17.3Hz,H-10″a)，5.09(1H,d,J＝8.6Hz,H-10″b)，3.74(3H,s,H-12)，3.61(3H,s,H-12″)，4.70(2H,t,J＝8.0Hz,H-1′,1″′)，3.19-3.40(8H,m,H-glc)，3.90(2H,t,J＝10.3Hz,H-6′a,6″′a)，3.69(2H,m,H-6′b,6″b)。¹³C-NMR(150MHz,CD₃OD) δ 98.6(C-1), 98.3(C-1 '), 155.1(C-3), 153.0(C-3 '), 111.4(C-4), 110.2(C-4 '), 34.5(C-5), 31.9(C-5 '), 30.6(C-6), 144.2(C-6 '), 157.8(C-7), 197.9(C-7 '), 136.5(C-8), 136.3(C-8 '), 47.3(C-9), 46.3(C-9 '), 121.2(C-10), 120.3(C-10 '), 170.1(C-11, 11 '), 53.2(C-12), 52.9(C-12 '), 101.7(C-1 '), 100.7(C-1 '), 75.5(C-2 '), 74.7(C-2 '), 9.9 ', 3 (C-3 '), 78.8(C-3 '), 72.5(C-4), 72.4 (C-4'), 79.2(C-5 '), 79.1 (C-5'), 63.6(C-6 '), 63.5 (C-6'). The above data are substantially consistent with (E) -aldosecologanin reported in the literature (Liu Z X, Liu C T, Liu Q B, et al. journal of Functional Foods 2015,18: 512-.

Compound 5: white amorphous powder (methanol), readily soluble in water, methanol. The maximum ultraviolet absorption wavelength is 239 nm. HR-ESI-MSAnd (3) negative ion display: ESI-MS (m/z): 743.2419[ M-H]^-And the molecular formula is presumed to be C by integrating mass spectrum data₃₃H₄₄O₁₉。¹H-NMR(600MHz,CD₃OD)δ:5.56(1H,s,H-1)，5.51(1H,s,H-1″)，7.62(1H,s,H-3)，7.46(1H,s,H-3″)，3.2(2H,m,H-5,6a)，3.90(1H,m,H-5″)，2.36(1H,m,H-6a)，6.77(1H,d,J＝4.5Hz,H-7)，9.24(1H,s,H-7″)，5.67-2.60(2H,m,H-8,8″)，2.80(1H,s,H-9)，2.53-2.70(1H,m,H-9″)，5.39(1H,d,J＝17.3Hz,H-10a)，5.31(1H,d,J＝9.5Hz,H-10b)，5.06(1H,d,J＝17.1Hz,H-10″a)，5.02(1H,d,J＝9.7Hz,H-10″b)，3.69(3H,s,H-12″)，4.70(1H,d,J＝7.8Hz,H-1′)，4.67(1H,d,J＝7.6Hz,H-1″′)，3.28-3.39(8H,m,H-glc)，3.90(2H,t,J＝11.2Hz,H-6′a,6″′a)，3.70(1H,m,H-6′b)，3.60(1H,m,H-6″b)。¹³C-NMR(150MHz,CD₃OD) delta 98.0(C-1), 97.2(C-1 '), 155.1(C-3), 152.1 (C-3'), 110.5(C-4), 110.2(C-4 '), 31.1(C-5), 31.0 (C-5'), 27.6(C-6), 144.1(C-6 '), 156.9(C-7), 197.6 (C-7'), 136.3(C-8), 136.1(C-8 '), 47.2(C-9), 45.9 (C-9'), 120.5(C-10), 119.7(C-10 '), 172.3(C-11), 169.7 (C-11'), 52.0(C-12 '), 100.5 (C-1', 1 '), 74.8 (C-2'), 74.6(C-2 '), 78.4'), 3 ', 3.4'), 3, 4 '), 4-3'), 72.0(C-4 '), 78.8(C-5 '), 78.7(C-5 '), 63.2(C-6 '), 63.1(C-6 '). The above data are substantially identical to Strychosides A reported in the literature (Atsuko, Itoh, Naoko, et al. journal of Natural Products,2005,68(9): 1434-.

4 in vitro anti-inflammatory Activity Studies of monomeric Compounds

4.1 Experimental materials

SZX16 model microscope and DP2-BSW image acquisition System (Olympus, Japan Co., Thermal CO)₂Incubator (Thermal, USA), microplate reader (Molecular Devices, USA), carbon dioxide cell incubator, biosafety cabinet (Thermo Fisher, USA), BSA2245-CW analytical balance (Sadolis scientific instruments, Inc.). CCK8, NO detection kit (Griess reagent) (Shanghai Biyuntian biotechnology Co., Ltd.), 1% penicillin/streptomycin, DMEM high sugar medium (USA)Hyclone), FBS fetal bovine serum (Gibco, USA), bacterial lipopolysaccharide (LPS, Escherichia coli 055: B5), indomethacin, dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA), data using GraphPad Prism 6.0 software for statistical analysis. Mouse mononuclear macrophage RAW264.7 was purchased from the cell bank of the chinese academy of sciences.

Experimental 5 dimer iridoid glycoside monomeric compounds (compounds 1, 2, 3, 4 and 5) are extracted and separated from the total iridoid extract of Jiufeng I medicinal material of honeysuckle flower cultivation product according to 2 'experimental method', and the structural formula is shown in Table 1.

4.2 Experimental methods

4.2.1 toxic Effect of Compounds on Raw264.7 cells

Taking a proper amount of 5 dimer iridoid glycoside monomeric compounds, adding DMSO to prepare a solution with the concentration of 100 mu M, and sequentially diluting to obtain compounds with the concentrations of 50 mu M, 25 mu M, 12.5 mu M and 6.25 mu M respectively.

RAW264.7 cells were cultured in DMEM medium containing 10% FBS and double antibody at 37 ℃ with 5% CO₂Culturing in an incubator, and subculturing every 2-3 days. During passage, the cell culture medium of RAW264.7 cells in a culture bottle is removed on an operation table under aseptic condition, fresh 3mL DMEM is added, the mixture is blown and uniformly mixed, 100 mu L of RAW264.7 cells are taken and distributed at 1X 10⁴Cell mL^-1The culture medium is inoculated into a 96-well plate at a concentration and cultured in a cell culture box for 24 hours. And after 24h, adding 100 mu L of compound solution with different concentrations to treat cells (5 groups), carrying out culture for 24h after 6 multiple wells in each group, adding 10 mu L of CCK8 reagent into each well, carrying out culture for 4h, detecting the OD value of each well under the wavelength of 450nm by using an enzyme labeling instrument, and calculating the cell survival rate. The formula is calculated as OD average of experimental group/OD average of control group × 100%.

4.2.2 Effect of Compounds on LPS-induced NO Release from RAW264.7 cells

RAW264.7 cells were plated at 1X 10 per well⁴Inoculating the cells in 96-well plate at a certain density, adding different drugs, pre-incubating for 1 hr, and adding 300 ng/ml^-1LPS in 5% CO₂Culturing in an air incubator for 24 h. Wherein the different groups are set as: the culture medium was used as a control group, 300 ng.ml^-1LPS was used as a model group, compounds at concentrations of 12.5. mu.M and 25. mu.M were used as drug groups, and indomethacin at a concentration of 25. mu.M was used as a positive drug group, with 5 duplicate wells per group. After being treated in a cell culture box at 37 ℃ for 24 hours, 50 mu L of cell supernatant is taken to be put in a 96-hole cell culture plate, 50 mu L of Griess Reagent I and 50 mu L of Griess Reagent II are sequentially added, mixed evenly and detected at 540nm by a microplate reader. The NO content was measured by Griess reaction to determine the concentration of NO in the medium and calculate the NO inhibition rate.

The foregoing detailed description of the embodiments of the present application has been presented to illustrate the principles and implementations of the present application, and the description of the embodiments is only intended to facilitate the understanding of the methods and their core concepts of the present application. Meanwhile, a person skilled in the art should, according to the idea of the present application, change or modify the embodiments and applications of the present application based on the scope of the present application. In view of the above, the description should not be taken as limiting the application.