阅读说明：本技术 达玛烷型三萜皂苷类化合物及其制备方法和在制备抗炎药物中的应用 (Dammarane type triterpenoid saponin compound, preparation method thereof and application thereof in preparation of anti-inflammatory drugs ) 是由 李俊 刘威 黄锡山 邓胜平 李陈国 黄艳 张艳军 于 2019-11-14 设计创作，主要内容包括：本发明公开了达玛烷型三萜皂苷类化合物及其制备方法和在制备抗炎药物中的应用,所述达玛烷型三萜皂苷类化合物从青钱柳叶中分离得到,所述制备方法包括：S1.将青钱柳叶用醇溶剂提取,其中,所述青钱柳叶与醇溶剂的质量比为1：(10-15),得到浸膏；S2.将步骤S1制备的浸膏依次采用硅胶柱色谱、MCI凝胶柱色谱、反相柱色谱、凝胶柱色谱和制备型高效液相色谱进行分离,得到化合物。用这种方法制备的该类新化合物具有抑制LPS诱导的RAW264.7细胞中NO、TNF-<I>α</I>、PGE<Sub>2</Sub>和IL-6的释放,能降低iNOS、COX-2和NF-κB/p65蛋白的表达,显示良好的抗炎活性,可用于开发抗炎的相关药物。(The invention discloses dammarane type triterpenoid saponin compounds, a preparation method thereof and application thereof in preparing anti-inflammatory drugs, wherein the dammarane type triterpenoid saponin compounds are separated from cyclocarya paliurus leaves, and the preparation method comprises the following steps: s1, extracting cyclocarya paliurus leaves by using an alcohol solvent, wherein the mass ratio of the cyclocarya paliurus leaves to the alcohol solvent is 1: (10-15) obtaining an extract; s2, sequentially separating the extract prepared in the step S1 by using silica gel column chromatography, MCI gel column chromatography, reversed phase column chromatography, gel column chromatography and preparative high performance liquid chromatography to obtain the compound. The novel compound prepared by the method has the function of inhibiting NO and TNF- α 、PGE 2 And IL-6 release, can reduce the expression of iNOS, COX-2 and NF-kB/p 65 proteins, shows good anti-inflammatory activity, and can be used for developing anti-inflammatory related medicaments.)

1. A dammarane-type triterpene saponin compound is characterized by having a structure shown in a formula (I):

2. a method for preparing dammarane-type triterpene saponin compounds according to claim 1, which comprises the steps of:

s1, extracting cyclocarya paliurus leaves by using an alcohol solvent, wherein the mass ratio of the cyclocarya paliurus leaves to the alcohol solvent is 1: (10-15) obtaining an extract;

s2, sequentially separating the extract prepared in the step S1 by adopting silica gel column chromatography, MCI gel column chromatography, reversed phase column chromatography, gel column chromatography and preparative high performance liquid chromatography to obtain the compound shown in the formula (I).

3. The preparation method according to claim 2, wherein the eluent used for the silica gel column chromatography is one or a mixture of different solvents selected from petroleum ether, ethyl acetate, dichloromethane, chloroform, acetone, ethanol and methanol.

4. The method according to claim 2, wherein the eluting solution used in the MCI gel column chromatography is an alcohol solvent.

5. The preparation method according to claim 2, wherein the reverse phase column chromatography is performed by using a low pressure preparative chromatography, a medium pressure preparative chromatography, a high performance liquid chromatography or a dynamic axial compression chromatography, wherein the mobile phase used in the reverse phase chromatography is an aqueous solution of an organic solvent, and the organic solvent is methanol or acetonitrile.

6. The method of claim 2, wherein the elution of the reverse phase column chromatography is a gradient elution, and the gradient elution is performed to the extent that: 0-20min, 10% -30% of water solution of organic solvent; 20-60min, 30-60% organic solvent water solution; 60-80min, 60% -90% organic solvent water solution; 80-100min, 100% organic solvent water solution.

7. The method according to claim 2, wherein the gel in the gel column chromatography is Sephadex LH-20, Sephadex G15 or Sephadex G50.

8. The method according to claim 2, wherein the mobile phase of preparative high performance liquid chromatography is methanol-water solution or acetonitrile-water solution.

9. Use of one or more dammarane-type triterpene saponin compounds of claim 1 or prepared according to any one of claims 2-8 in preparing anti-inflammatory drugs.

10. A pharmaceutical preparation comprising one or more dammarane-type triterpene saponin compounds according to claim 1 or one or more dammarane-type triterpene saponin compounds prepared according to any one of claims 2 to 8 and a pharmaceutically acceptable carrier.

Technical Field

The invention relates to chemical synthesis, in particular to a dammarane type triterpenoid saponin compound extracted and separated from cyclocarya paliurus leaves, a preparation method and application thereof.

Background

Cyclocarya paliurus (Batal) Iljinsk (Juglandaceae) belongs to Juglandaceae, has pungent, slightly bitter and mild properties, has the effects of dispelling wind and relieving itching, is produced in Jiangxi, Guangxi, Guizhou, Hunan, Hubei, Sichuan, Fujian, Jiangxi, Zhejiang and Anhui, has long medicine application history, and is a common Chinese medicinal material in China.

The main chemical components of cyclocarya paliurus are triterpenoids, mainly including dammarane type triterpenoid saponins, flavonoids, phenolic acid compounds and the like. Modern pharmacological studies show that the cyclocarya paliurus crude extract has the effects of reducing blood sugar, blood pressure and blood fat, resisting inflammation, tumors, oxidation and bacteria and the like.

Macrophages are an important immune cell in the body, are the main cells for starting the production of inflammatory mediators in the body, and have the functions of resisting infection, resisting tumor and regulating immunity. Macrophages are activated by a variety of inflammatory factors, such as cytokines, bacterial Lipopolysaccharide (LPS), extracellular matrix proteins, and other chemical mediators. LPS is an important inflammation-causing factor, and can stimulate macrophages in vivo to synthesize and release various endogenous active factors, thereby inducing inflammation. The use of LPS to treat macrophages is a common in vitro model of inflammation.

There are two main classes of anti-inflammatory drugs currently used clinically: non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs. Although both anti-inflammatory drugs have good clinical anti-inflammatory effects, a series of adverse reactions and tolerance, such as gastric mucosa injury, liver injury, kidney injury and the like, can be generated after long-term use of the anti-inflammatory drugs in large quantities. In order to solve the tolerance and adverse reaction of the drugs, the search for new anti-inflammatory drugs and related drugs with novel skeleton types becomes a hotspot in the research field of anti-inflammatory drugs.

The natural product is an important source of a drug lead compound, and metabolites are rich and diverse and are always important sources of drug screening. The secondary metabolite of the plant has wide physiological activities, such as multiple activities of antibiosis, tumor resistance, immunoregulation, anti-inflammation, enzyme inhibition and the like. The search for new drug source molecules from medicinal plants is a hot spot of research at home and abroad.

Disclosure of Invention

The invention aims to provide a dammarane type triterpenoid saponin compound, a preparation method thereof and application thereof in preparing anti-inflammatory drugs aiming at the defects of the prior art, and the novel compound prepared by the method has the function of inhibiting NO, TNF- α and PGE in RAW264.7 cells induced by LPS₂And IL-6 release, can reduce the expression of iNOS, COX-2 and NF-kB/p 65 proteins, shows good anti-inflammatory activity, and can be used for developing anti-inflammatory related medicaments.

The technical scheme for realizing the purpose of the invention is as follows:

the difference of the dammarane type triterpenoid saponin compound in the prior art is that the structure of the dammarane type triterpenoid saponin compound is shown as a formula (I):

the preparation method of the dammarane type triterpenoid saponin compound comprises the following steps:

s1, extracting cyclocarya paliurus leaves by using an alcohol solvent, wherein the mass ratio of the cyclocarya paliurus leaves to the alcohol solvent is 1: (10-15) obtaining an extract;

s2, sequentially separating the extract prepared in the step S1 by adopting silica gel column chromatography, MCI gel column chromatography, reversed phase column chromatography, gel column chromatography and preparative high performance liquid chromatography to obtain the compound shown in the formula (I).

Preferably, the eluent used for the silica gel column chromatography is one solvent or a mixture of different solvents of petroleum ether, ethyl acetate, dichloromethane, trichloromethane, acetone, ethanol or methanol.

Preferably, the eluent used for the MCI gel column chromatography is an alcohol solvent.

Preferably, the apparatus used for the reverse phase column chromatography is low pressure preparative chromatography, medium pressure preparative chromatography, high performance liquid chromatography or dynamic axial compression chromatography, wherein the mobile phase used for the reverse phase chromatography is an aqueous solution of an organic solvent, and the organic solvent is methanol or acetonitrile.

Preferably, the elution of the reversed phase column chromatography is gradient elution, and the gradient elution degree is as follows in volume fraction: 0-20min, 10% -30% of water solution of organic solvent; 20-60min, 30-60% organic solvent water solution; 60-80min, 60% -90% organic solvent water solution; 80-100min, 100% organic solvent water solution.

Preferably, the gel in the gel column chromatography is Sephadex LH-20, Sephadex G15 or Sephadex G50.

Preferably, the mobile phase of preparative high performance liquid chromatography is methanol-water solution or acetonitrile-water solution.

The dammarane type triterpenoid saponin compound prepared by the method is applied to preparation of anti-inflammatory drugs.

The technical scheme provides a pharmaceutical preparation which comprises one or more of the dammarane type triterpenoid saponin compounds and a pharmaceutically acceptable carrier.

The new compound prepared by the method has the function of inhibiting NO, TNF- α and PGE in RAW264.7 cells induced by LPS₂And IL-6 release, can reduce the expression of iNOS, COX-2 and NF-kB/p 65 proteins, shows good anti-inflammatory activity, and can be used for developing anti-inflammatory related medicaments.

Drawings

FIG. 1 is a graph showing the effect of compounds IT-1, 2, 3, 4, 5 and 6 in the examples on TNF- α production in LPS-stimulated RAW264.7 cells;

FIG. 2 is a graph showing the effect of compounds IT-1, 2, 3, 4, 5 and 6 in the examples on the production of PGE2 in LPS-stimulated RAW264.7 cells;

FIG. 3 is a graph showing the effect of compounds 7, 8, 10 and 11 in the examples on IL-6 production in LPS-stimulated RAW264.7 cells;

FIG. 4 is a graph showing the effect of Compound 7 in example on the expression of iNOS, COX-2 and NF-. kappa.B/p 65 in lipopolysaccharide-induced RAW264.7 cells at concentrations of 5, 10 and 20. mu.M.

Detailed Description

The present invention will be further illustrated with reference to the following examples, but is not limited thereto.