阅读说明：本技术 一种环烯醚萜苷类化合物及其制备方法和应用 (Iridoid glycoside compound and preparation method and application thereof ) 是由 肖伟 李海波 邓奕 葛雯 胡晗绯 杨彪 曹亮 王振中 于 2020-05-11 设计创作，主要内容包括：本发明公开了一种环烯醚萜苷类化合物,其是在栀子中发现的新化学成分。本发明还通过理化性质和现代波谱学手段,对上述方法分离得到的化合物进行了结构鉴定。本发明还运用LPS诱导RAW 264.7细胞炎症模型等活性筛选体系进行活性评价,发现该化合物对小鼠巨噬细胞系RAW 264.7有一定的保护作用,可以显著抑制PGE<Sub>2</Sub>,显示出较强的抗炎作用。(The invention discloses an iridoid glycoside compound which is a new chemical component found in gardenia. The invention also carries out structural identification on the compound separated by the method through physicochemical properties and modern spectral means. The invention also utilizes an activity screening system such as an LPS (LPS) -induced RAW264.7 cell inflammation model and the like to carry out activity evaluation, and finds that the compound has a certain protection effect on a mouse macrophage system RAW264.7 and can obviously inhibit PGE (platelet-rich antigen) 2 Showing strong anti-inflammatory action.)

1. An iridoid glycoside compound or pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule and metabolite thereof, wherein the structure of the compound is shown as formula I:

2. a process for the preparation of a compound according to claim 1, comprising the steps of:

step 1: taking gardenia medicinal materials, carrying out reflux extraction by 50-70% ethanol, and removing a solvent to obtain a total extract;

step 2: dissolving the total extract in water, separating by macroporous adsorption resin column chromatography, eluting with water, 25-35% ethanol, 45-55% ethanol, 65-75% ethanol and 90-100% ethanol in sequence, collecting eluates respectively, and concentrating under reduced pressure to obtain a water elution part, a 25-35% ethanol elution part, a 45-55% ethanol elution part, a 65-75% ethanol elution part and a 90-100% ethanol elution part;

and step 3: separating the 25-35% ethanol elution part by using a silica gel column chromatography, performing gradient elution and collection by using ethyl acetate-methanol-water to obtain 8 fractions of A-H, performing gradient elution by using ODS column chromatography methanol-water to obtain 9 fractions of D1-D9, performing Sephadex LH-20 column chromatography to obtain 7 fractions of D6A-D6G, and performing gradient elution by using methanol-water to obtain 7 fractions of D6F by using semi-preparative liquid chromatography.

3. The preparation method according to claim 2, wherein the step 1 is to take gardenia medicinal materials, reflux-extract the gardenia medicinal materials for 1 to 3 times by 3 to5 times of 50 to 80 percent ethanol for 1 to 3 hours each time, combine the extract, and remove the solvent under reduced pressure to obtain the total extract.

4. The preparation method according to claim 2, wherein the step 2 comprises eluting with water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol in sequence, collecting eluates, respectively, and concentrating under reduced pressure until no alcohol smell is produced to obtain water eluate, 30% ethanol eluate, 50% ethanol eluate, 70% ethanol eluate, and 95% ethanol eluate.

5. The method according to claim 2, wherein the ethyl acetate-methanol-water gradient elution of step 3 is a gradient elution performed at a volume ratio of 95:5:0 to 0:100: 0; the methanol-water gradient elution is that gradient elution is carried out according to the volume ratio of 30:70 to50: 50; the methanol-water isocratic elution is performed at a volume ratio of 50: 50.

6. The preparation method according to claim 2, wherein the macroporous adsorbent resin is selected from the group consisting of D101 type macroporous adsorbent resin, HP-20 type macroporous adsorbent resin, HPD-100A type macroporous adsorbent resin, and HPD-300 type macroporous adsorbent resin.

7. The method of claim 2, wherein the semi-preparative liquid chromatography conditions include: the mobile phase is acetonitrile-water-formic acid with the volume ratio of 15-25:72-92:0.05-0.5, the detection wavelength is 240-260nm, and the flow rate is 1-5 mL/min.

8. The method according to claim 7, wherein the step 1 is 2 times of reflux extraction with 60% ethanol for 2 hours;

the semi-preparative liquid chromatography conditions include: the mobile phase is acetonitrile-water-formic acid with the volume ratio of 18:82:0.1, the detection wavelength is 254nm, and the flow rate is 4 mL/min.

9. Use of a compound according to claim 1 for the preparation of an anti-inflammatory medicament.

10. A medicament comprising a compound according to claim 1.

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a novel compound and a preparation method and application thereof.

Background

Gardenia jasminoides Ellis is derived from dried mature fruits of Gardenia jasminoides Ellis of Gardenia of Rubiaceae (Rubiaceae), belongs to the first batch of medicinal and edible resources issued by Ministry of health, and has the effects of protecting liver, promoting bile flow, lowering blood pressure, tranquilizing, stopping bleeding, reducing swelling and the like. Is used for treating icteric hepatitis, sprain, contusion, hypertension, diabetes and other diseases in traditional Chinese medicine clinic.

Anti-inflammatory drugs in clinical treatment are the second largest class of drugs next to anti-infective drugs, including steroidal anti-inflammatory drugs (SAID) and non-steroidal anti-inflammatory drugs (NSAID). However, because of the strong toxic and side effects of many synthetic drugs, people pay more and more attention to the development of anti-inflammatory drugs from natural drugs.

Disclosure of Invention

The invention aims to carry out more intensive research on active ingredients of gardenia and find out active ingredients which play certain roles.

In view of the above, the present invention provides an iridoid glycoside compound or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, prodrug molecule, metabolite thereof, wherein the compound has the following structure, and the structure of the compound is shown in formula I:

another object of the present invention is to provide a method for preparing the above compound, which comprises the steps of:

step 1: taking 40kg of gardenia dry medicinal materials, carrying out reflux extraction by 50-70% ethanol, and removing a solvent to obtain a total extract;

step 2: dissolving the total extract in water, separating by macroporous adsorption resin column chromatography, eluting with water, 25-35% ethanol, 45-55% ethanol, 65-75% ethanol and 90-100% ethanol in sequence, collecting eluates respectively, and concentrating under reduced pressure until no alcohol smell exists to obtain a water elution part, a 25-35% ethanol elution part, a 45-55% ethanol elution part, a 65-75% ethanol elution part and a 90-100% ethanol elution part;

and step 3: separating the 25-35% ethanol elution part by silica gel column chromatography, performing gradient elution by using ethyl acetate-methanol-water, collecting 8 fractions A-H, performing gradient elution by using ODS column chromatography methanol-water to fraction D to obtain 9 fractions D1-D9, performing Sephadex LH-20 column chromatography to fraction D6, performing isocratic elution by using methanol-water to obtain 7 fractions D6A-D6G, and performing semi-preparative liquid chromatography to fraction D6F.

Further, the step 1 is as follows: reflux-extracting fructus Gardeniae dried material with 3-5 times of 50-80% ethanol for 1-3 times (each for 1-3 hr), mixing extractive solutions, and removing solvent under reduced pressure to obtain the total extract.

Preferably, the step 2 comprises sequentially eluting with water, 30% ethanol, 50% ethanol, 70% ethanol, and 95% ethanol, collecting eluates, respectively, and concentrating under reduced pressure until no ethanol smell exists to obtain water eluate, 30% ethanol eluate, 50% ethanol eluate, 70% ethanol eluate, and 95% ethanol eluate.

Preferably, the ethyl acetate-methanol-water gradient elution of step 3 is performed by gradient elution with (95:5:0 to 0:0:100, v/v/v); the methanol-water gradient elution is performed by gradient elution with (30: 70-50: 50, v/v); the methanol-water isocratic elution is carried out by isocratic elution with (50:50, v/v).

Specifically, the macroporous adsorption resin comprises D101 type macroporous adsorption resin, HP-20 type macroporous adsorption resin, HPD-100A type macroporous adsorption resin or HPD-300 type macroporous adsorption resin.

Further, the semi-preparative liquid chromatography conditions include: acetonitrile-water-formic acid with the volume ratio of 15-25:72-92:0.05-0.5 is taken as a mobile phase, the detection wavelength is 240-260nm, and the flow rate is 1-5 mL/min.

Preferably, the step 1 is 2 times of reflux extraction with 60% ethanol for 2 hours each time. The conditions of the semi-preparative liquid chromatography are preferably: acetonitrile-water-formic acid with the volume ratio of 18:82:0.1 is used as a mobile phase, the detection wavelength is 254nm, and the flow rate is 4 mL/min.

The invention also aims to provide the application of the compound in preparing anti-inflammatory drugs. The compound of the invention induces RAW264.7 cells to generate PGE by LPS₂Has obvious inhibiting effect.

The invention also provides a medicament for treating inflammation, which comprises the compound shown in the formula (I).

Further, the medicament comprises a therapeutically effective amount of a compound of formula (I) as described above, together with one or more pharmaceutically acceptable carriers.

Specifically, the medicament can be any one of the dosage forms in pharmaceutics, including tablets, capsules, soft capsules, gels, oral preparations, suspensions, granules, patches, ointments, pills, powders, injections, infusion solutions, freeze-dried injections, intravenous emulsions, liposome injections, suppositories, sustained-release preparations or controlled-release preparations.

Further, the pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as: diluents, excipients, and water, and the like, fillers such as starch, sucrose, lactose, microcrystalline cellulose, and the like; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as sodium carboxymethyl starch, hydroxypropyl cellulose, crosslinked carboxymethyl cellulose, agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol, sodium lauryl sulfate; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium and magnesium stearate, micronized silica gel, polyethylene glycol, and the like. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

The iridoid glycoside compound is a new chemical component found in gardenia by researchers, and is found to exist stably in gardenia of each batch. The inventor utilizes physicochemical properties and modern wave spectrum means (MS, B, C, D, C,¹H-NMR、¹³C-NMR, etc.), and carrying out structural identification on the compound obtained by the separation method to confirm that the compound is a novel compound with the structure shown in the formula (I). The invention also utilizes an activity screening system such as an LPS (LPS) -induced RAW264.7 cell inflammation model and the like to carry out activity evaluation, and finds that the compound has a certain protection effect on a mouse macrophage system RAW264.7 and can obviously inhibit PGE (platelet-rich antigen)₂Showing strong anti-inflammatory action. Has good research and development prospect.

Drawings

FIG. 1 is a drawing of a compound of the present invention¹H-NMR spectrum;

FIG. 2 is a drawing of a compound of the present invention¹³C-NMR spectrum;

FIG. 3 is a DEPT-135 spectrum of a compound of the invention;

FIG. 4 is H of a compound of the present invention¹-H¹A COSY spectrum;

FIG. 5 is an HSQC spectrum of a compound of the present invention;

FIG. 6 is an HMBC spectrum of a compound of the present invention;

FIG. 7 is HR-ESI-Q-TOF-MS of a compound of the present invention;

FIG. 8 shows the main HMBC correlation and H for the compounds of the present invention¹-H¹COSY is related.

Detailed Description

The following will specifically describe the contents of the experimental examples.

It is specifically noted that similar alternatives and modifications will be apparent to those skilled in the art, which are also intended to be included within the present invention. It will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately combining the methods and applications described herein without departing from the spirit, scope, and content of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention.

If the specific conditions are not indicated, the method is carried out according to the conventional conditions or the conditions suggested by manufacturers, and the used raw material medicines or auxiliary materials and the used reagents or instruments are the conventional products which can be obtained commercially.