阅读说明：本技术 翼首草毒素a及其应用和低肝损伤毒性翼首草提取物的制备方法 (Pterocephalus hookeri toxin A, application thereof and preparation method of pterocephalus hookeri extract with low liver injury toxicity ) 是由 陈敏 王睿 董召月 廖志华 于 2019-11-19 设计创作，主要内容包括：本发明公开了翼首草毒素A及其应用和低肝损伤毒性翼首草提取物的制备方法,本发明首次从翼首草中分离鉴定得到的Pterocephtoxin A,研究发现Pterocephtoxin A具有诱导肝损伤的作用,因此在制备翼首草提取物时去除含有Pterocephtoxin A的部分,能够为翼首草临床合理用药提供重要的保障。(The invention discloses pterocephalus hookeri toxin A, application thereof and a preparation method of a pterocephalus hookeri extract with low liver injury toxicity.)

1. Pterocephalus hookeri toxin a, characterized in that its structural formula is as follows:

2. use of pterocephalin a according to claim 1 for the preparation of an agent for liver damage.

3. An extract of pterocephalus hookeri with low liver injury toxicity, which is characterized in that: the pterocephalus hookeri extract does not contain pterocephalus hookeri toxin A or the concentration of the pterocephalus hookeri toxin A is lower than the concentration of liver injury.

4. The extract of pterocephalus hookeri with low liver injury toxicity according to claim 3, wherein: the pterocephalus hookeri extract is prepared by the following method: crushing dried pterocephalus hookeri, extracting with an ethanol solution, recovering a solvent to obtain an extract, adding water to disperse the extract to prepare a suspension, extracting with petroleum ether, ethyl acetate and n-butyl alcohol in sequence, concentrating an extract under reduced pressure to recover the solvent to obtain an n-butyl alcohol extract, passing the obtained n-butyl alcohol extract through an HP-20 macroporous resin column, eluting with an ethanol solution with the volume fraction of less than 40%, and collecting an eluent to obtain the pterocephalus hookeri extract with low liver injury toxicity.

5. The extract of pterocephalus hookeri with low liver injury toxicity according to claim 4, wherein: the extraction is 5 times of extraction by using ethanol solution with volume fraction of 95%.

6. The extract of pterocephalus hookeri with low liver injury toxicity according to claim 4, wherein: the elution is carried out by using a volume ratio of ethanol to water of 0: 100-40: a gradient elution of 60 solution.

7. The preparation method of the pterocephalus hookeri extract with low liver injury toxicity is characterized by comprising the following steps: crushing dried pterocephalus hookeri, extracting with an ethanol solution, recovering a solvent to obtain an extract, adding water to disperse the extract to prepare a suspension, extracting with petroleum ether, ethyl acetate and n-butyl alcohol in sequence, concentrating an extract under reduced pressure to recover the solvent to obtain an n-butyl alcohol extract, passing the obtained n-butyl alcohol extract through an HP-20 macroporous resin column, eluting with an ethanol solution with the volume fraction of ethanol being lower than 40%, and collecting an eluent to obtain the pterocephalus hookeri extract with low liver injury toxicity.

8. The method of claim 7, wherein: the extraction is 5 times of extraction by using ethanol solution with volume fraction of 95%.

9. The method of claim 7, wherein: the elution is carried out by using a volume ratio of ethanol to water of 0: 100-40: a gradient elution of 60 solution.

10. Use of an extract of pterocephalus hookeri with low liver injury toxicity according to any one of claims 3 to 6 for the manufacture of a medicament for the treatment of rheumatoid arthritis.

Technical Field

The invention relates to the field of natural medicinal chemistry, in particular to pterocephalus hookeri toxin A, application of the pterocephalus hookeri toxin A and a preparation method of a pterocephalus hookeri extract with low liver injury toxicity.

Background

Rheumatoid arthritis is a chronic autoimmune disease that manifests clinically primarily as symmetrical joint pain and swelling, accompanied by destruction of cartilage structure and bone mass to varying degrees. Patients with rheumatoid arthritis need to take the medicine for a long time, so that the safety of the medicine is higher. The liver is the largest processing plant of the human body and bears the living thingsSynthesis, metabolic conversion, secretion and excretion. The liver is an important organ for drug metabolism, and most drugs enter the body, are metabolized by the liver and then absorbed by the body. Thus, the liver is one of the major target organs with drug-induced damage in vivo. The liver toxicity of Chinese herbal medicine is mainly reflected in liver injury caused by Chinese herbal medicine. In recent years, reports of the type are gradually increased, and Chinese herbal medicines account for about 45.43% of liver injury induced by medicines. In the world, about 80% of people are used to the treatment of diseases by using Chinese herbal medicines, improper use of Chinese herbal medicines is the main cause of liver injury, and the importance of Chinese herbal medicine hepatotoxicity is an important link for evaluating Chinese herbal medicine toxicity. Pterocephalus hookeri is a common Tibetan medicinal material, and the Tibetan names include aconitum kangarense, cumin kangarensis wu, cumin kangargi wu and the like. 2015 edition "pharmacopoeia of the people's republic of China" (one part) contains Pterocarpus hooker (C.B. Clarke) of Pterocarpus spathulifolius of Dipsacaceae

The dried whole herb is used as a genuine basic source plant, is bitter in taste, cold in nature and low in toxicity, and has the effects of detoxifying, removing plague, clearing heat, stopping dysentery, dispelling wind and removing arthralgia. The folk medicine use history of the pterocephalus hookeri is very long, and various medical classics such as Tibetan medicine record, Tibetan common Chinese herbal medicine record, Jingzhu materia Medica and the like have detailed records on the medicine use of the pterocephalus hookeri, wherein the medicine use is most obvious in rheumatoid arthritis resistance. Meanwhile, there are also records on the "minor toxicity" of the pterocephalus. The pterocephalus hookeri is a common Tibetan medicinal material and has a long history of drug use for resisting rheumatoid arthritis, the invention clarifies that Pterocephtoxin A obtained by separation and identification from pterocephalus hookeri has the effect of inducing liver injury, and the part containing the Pterocephtoxin A is removed in the preparation process, so that important guarantee can be provided for the clinical reasonable drug use of the pterocephalus hookeri.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a pterocephalus toxin a; the second purpose of the invention is to provide the application of the pterocephalus toxin A; the invention also aims to provide a pterocephalus hookeri extract with low liver injury toxicity; the fourth purpose of the invention is to provide a preparation method of the pterocephalus hookeri extract with low liver injury toxicity; the fifth purpose of the invention is to provide the application of the pterocephalus hookeri extract with low liver injury toxicity in preparing the medicine for treating rheumatoid arthritis.

In order to achieve the purpose, the invention provides the following technical scheme:

1. pterocephalus hookeri toxin A, the structural formula of which is as follows:

2. the pterocephalus hookeri toxin A is applied to preparation of a liver injury reagent.

3. A pterocephalus hookeri extract with low liver injury toxicity does not contain pterocephalus hookeri toxin A or the concentration of the pterocephalus hookeri toxin A is lower than the liver injury concentration.

Preferably, the pterocephalus hookeri extract is prepared by the following method: crushing dried pterocephalus hookeri, extracting with an ethanol solution, recovering a solvent to obtain an extract, adding water to disperse the extract to prepare a suspension, sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol, concentrating the extract under reduced pressure to recover the solvent to obtain an n-butyl alcohol extract, passing the obtained n-butyl alcohol extract through an HP-20 macroporous resin column, eluting with an ethanol solution with the volume fraction of ethanol being lower than 40%, and collecting the eluent to obtain the pterocephalus hookeri extract with low liver injury toxicity.

Preferably, the extraction is 5 times using 95% ethanol solution by volume fraction.

Preferably, the elution is performed by using a volume ratio of ethanol to water of 0: 100-40: a gradient elution of 60 solution.

4. The preparation method of the pterocephalus hookeri extract with low liver injury toxicity comprises the following steps: crushing dried pterocephalus hookeri, extracting with an ethanol solution, recovering a solvent to obtain an extract, adding water to disperse the extract to prepare a suspension, extracting with petroleum ether, ethyl acetate and n-butyl alcohol in sequence, concentrating an extract under reduced pressure to recover the solvent to obtain an n-butyl alcohol extract, passing the obtained n-butyl alcohol extract through an HP-20 macroporous resin column, eluting with an ethanol solution with the volume fraction of ethanol being lower than 40%, and collecting an eluent to obtain the pterocephalus hookeri extract with low liver injury toxicity.

Preferably, the extraction is 5 times using 95% ethanol solution by volume fraction.

Preferably, the elution is performed by using a volume ratio of ethanol to water of 0: 100-40: a gradient elution of 60 solution.

5. The pterocephalus hookeri extract with low liver injury toxicity is applied to the preparation of the medicine for treating rheumatoid arthritis.

The invention has the beneficial effects that: the Pterocephtoxin A obtained by separation and identification from pterocephalus hookeri for the first time is found to have the effect of inducing liver injury, so that the part containing the Pterocephtoxin A is removed in the preparation process, and important guarantee is provided for the clinical reasonable medication of the pterocephalus hookeri.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows a high performance liquid phase diagram of n-butanol extract of Pterocephalus hookeri and cytotoxic activity analysis (A: separation chromatogram; B: cytotoxic activity analysis) of each ethanol elution site of n-butanol extract.

FIG. 2 shows a schematic diagram of the preparation of Pterocarptoxin A in the n-butanol extract of Pterocarpus hookeri at 80% ethanol elution site B.

FIG. 3 shows the structural formula of Pterocephtoxin A.

FIG. 4 shows Pterocephtoxin A¹H NMR spectrum.

FIG. 5 shows Pterocephtoxin A¹³C NMR spectrum.

FIG. 6 shows the effect of different doses of Pteroceptoxin A on serum biochemical indicators of mouse liver function, glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) (A: ALT; B: AST; Con in the figure is control group, Pteroceptoxin A-L is low dose group 60mg/kg, Pteroceptoxin A-H is high dose group 120 mg/kg. compared with control group:^*P<0.05，^**P<0.01)。

figure 7 shows the changes in pathological sections of mouse liver tissue for different doses of ptococephtoxin a: in the figure, Con is a control group, Pterocephtoxin A-L is 60mg/kg in a low dose group, and Pterocephtoxin A-H is 120mg/kg in a high dose group.

FIG. 8 shows the effect of different doses of Pteroceptoxin A on the survival of human normal hepatocytes L-02, Lactate Dehydrogenase (LDH) glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) (A: L-02 cell survival; B: LDH; C: ALT; D: AST; Con in the figure is a control group, and the concentration of Pteroceptoxin A is 2, 4 and 8. mu. mol/L, respectively, compared to the control group:^*P<0.05，^**P<0.01)。

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.