阅读说明：本技术 具有神经细胞保护活性的透骨草木脂素制备方法及其应用 (Preparation method and application of tuberculate speranskia herb lignan with nerve cell protection activity ) 是由 刘学贵 张立新 高品一 李丹琦 岳丹丹 于 2020-05-20 设计创作，主要内容包括：具有神经细胞保护活性的透骨草木脂素制备方法及其应用,涉及一种天然药物制备方法及其应用,本发明透骨草木脂素的提取、分离和纯化等工艺的简要过程：首先从干燥的透骨草全草中得到醇提浸膏,经水分散后,依次用石油醚、乙酸乙酯萃取,萃取液经减压浓缩后得到石油醚萃取物,乙酸乙酯萃取物,水层萃取物。取乙酸乙酯萃取物,经硅胶柱色谱、MCI柱色谱、开放ODS柱色谱、凝胶柱色谱以及高效液相色谱分离纯化后,收集得到4个木脂素化合物1-4。上述木脂素均具神经细胞保护活性,其中化合物2和4相比较阳性对照药维生素E具有显著的神经细胞保护活性。本发明为透骨草木脂素治疗神经退行性疾病的药品开发奠定了基础。(The invention discloses a preparation method and application of tuberculate speranskia herb lignan with nerve cell protection activity, and relates to a preparation method and application of a natural medicine, wherein the simple processes of extraction, separation, purification and other processes of the tuberculate speranskia herb lignan comprise the following steps: firstly, an alcohol extract is obtained from dried garden balsam stem whole grass, after water dispersion, petroleum ether and ethyl acetate are used for extraction in sequence, and after the extract liquid is decompressed and concentrated, a petroleum ether extract, an ethyl acetate extract and a water layer extract are obtained. Separating and purifying the ethyl acetate extract by silica gel column chromatography, MCI column chromatography, open ODS column chromatography, gel column chromatography and high performance liquid chromatography, and collecting to obtain 1-4 lignan compounds. The lignans all have nerve cell protective activity, wherein the compounds 2 and 4 have obvious nerve cell protective activity compared with the positive control medicament vitamin E. The invention lays a foundation for the development of the medicine for treating neurodegenerative diseases by using the speranskia tuberculata lignan.)

1. The preparation method of the garden balsam stem lignan with the nerve cell protection activity is characterized by comprising the following preparation processes:

taking dried garden balsam stem whole grass, carrying out reflux extraction for 2-6 times by using an ethanol-water solution, carrying out reflux extraction for 4-10 hours each time, combining extracting solutions, and carrying out reduced pressure concentration to obtain a total extract;

dispersing the obtained extract in water, sequentially extracting with petroleum ether and ethyl acetate, and concentrating the extractive solution under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and water layer extract;

separating and purifying the ethyl acetate extract by silica gel column chromatography, MCI column chromatography, open ODS column chromatography, gel column chromatography and high performance liquid chromatography to obtain 4 lignan compounds 1-4.

2. The method for preparing speranskia tuberculata lignan having a neuroprotective activity according to claim 1, wherein the ethyl acetate extract is subjected to silica gel column chromatography and gradient elution with dichloromethane-methanol (100: 1-0: 1) system to obtain 6 fractions (fr.1-fr.6).

3. The method for preparing ajugamarin having neuroprotective activity according to claim 2, wherein the Fr.4 fraction is separated and purified by silica gel reduced pressure column chromatography, MCI column chromatography, open ODS column chromatography, gel column chromatography and high performance liquid chromatography to obtain compound 1, compound 2, compound 3 and compound 4.

4. Application of tuberculate speranskia herb lignan with nerve cell protection activity is characterized in that the tuberculate speranskia herb lignan uses H₂O₂Induced SH-SY5Y cells are used as a model for biological activity determination, and the nerve cell protection activity of the speranskia herb lignans is evaluated by using an MTT determination method; the speranskia tuberculata lignan is suitable for the development of medicines for treating neurodegenerative diseases.

5. The use of tuberculate speranskia herb lignans having neuroprotective activity according to claim 4, wherein the comparison of the neuroprotective activity indicates that compounds 2 and 4 have significant neuroprotective activity compared to the control drug vitamin E.

6. The use of speranskia tuberculata lignan having neuroprotective activity according to claim 5, wherein the survival rate of SH-SY5Y cells is 81.68% and 93.19% when the concentration of compound 2 and compound 4 is 50 μmol/L, respectively.

Technical Field

The invention relates to a preparation method and application of a natural medicine, in particular to a preparation method and application of tuberculate speranskia herb lignan with nerve cell protection activity.

Background

Garden balsam stem (Tu Ji) (Phryma leptostachyaL.) also known as Gnaphalium japonicum, muscardine fly, is a perennial herb of the genus Phryma of the family Phryptospermaceae. The garden balsam stem is bitter and astringent in taste, is fond of yin, has strong adaptability, is easy to survive after transplantation, has rapid growth, and is widely planted in northeast, north China and all parts of Yangtze river drainage areas. The garden balsam stem can be used as a medicine for killing cabbage caterpillar, has the functions of anti-inflammation and sterilization, and is suitable for common sore, tinea and pyogenic infections. Herba speranskiae tuberculatae can also be used for treating rheumatalgia, early stage of toxic swelling, and rheumatic arthritis.

The speranskia tuberculata lignan is extracted from the whole plant of speranskia tuberculata, has been proved to have the effects of oxidation resistance, tumor resistance and inflammation resistance, and has very small adverse reaction on organisms. Therefore, the method deeply develops the lignan resource in the garden balsam stem, systematically studies the structure and the pharmacological activity of the garden balsam stem, and has great economic and social benefits for the deep processing of the garden balsam stem and the wide application of the garden balsam stem in the fields of medicine and the like.

Disclosure of Invention

The invention aims to provide a preparation method and application of tuberculate speranskia herb lignan with nerve cell protection activity.

The purpose of the invention is realized by the following technical scheme:

a preparation method of garden balsam stem lignans with nerve cell protection activity comprises the following preparation processes:

taking dried garden balsam stem whole grass, carrying out reflux extraction for 2-6 times by using ethanol-water solution, carrying out reflux extraction for 4-10 h each time, combining extracting solutions, and carrying out reduced pressure concentration to obtain a total extract. And (3) dispersing the obtained extract in water, extracting the extract by using petroleum ether and ethyl acetate in sequence, and concentrating the extract under reduced pressure to obtain a petroleum ether extract, an ethyl acetate extract and a water layer extract. Separating and purifying the ethyl acetate extract by silica gel column chromatography, MCI column chromatography, open ODS column chromatography, gel column chromatography and high performance liquid chromatography to obtain 4 lignan compounds 1-4.

The preparation method of the speranskia tuberculata lignan with the nerve cell protection activity comprises the steps of taking an ethyl acetate extract, separating by silica gel column chromatography, and carrying out gradient elution by using a dichloromethane-methanol (100: 1-0: 1) system to obtain 6 fractions (Fr.1-Fr.6).

The preparation method of the garden balsam stem lignan with the nerve cell protection activity comprises the step of separating and purifying the Fr.4 fraction by silica gel reduced pressure column chromatography, MCI column chromatography, open ODS column chromatography, gel column chromatography and high performance liquid chromatography to obtain a compound 1, a compound 2, a compound 3 and a compound 4.

Application of tuberculate speranskia herb lignan with nerve cell protection activity, wherein the tuberculate speranskia herb lignan is H₂O₂Induced SH-SY5Y cells are used as a model for biological activity determination, and the nerve cell protection activity of the speranskia herb lignans is evaluated by using an MTT determination method; the speranskia tuberculata lignan is suitable for the development of medicines for treating neurodegenerative diseases.

The application of the garden balsam stem lignan with the nerve cell protection activity shows that the compounds 2 and 4 have obvious nerve cell protection activity compared with a control drug vitamin E.

According to the application of the garden balsam stem lignan with the nerve cell protection activity, when the concentration of the compound 2 and the compound 4 reaches 50 mu mol/L, the survival rates of SH-SY5Y cells are 81.68% and 93.19% respectively.

The invention has the advantages and effects that:

the invention adopts a chromatographic separation method to separate the whole herb of the garden balsam stem to obtain the garden balsam stem lignan, and analyzes the nerve cell protection activity of the garden balsam stem lignan, and the result shows that the garden balsam stem lignan has the nerve cell protection activity. The invention lays a foundation for the development of the medicine for treating neurodegenerative diseases by using the speranskia tuberculata lignan.

Drawings

FIG. 1 is a drawing of Compound 1¹An H-NMR spectrum;

FIG. 2 is a drawing of Compound 1¹³A C-NMR spectrum;

FIG. 3 is a drawing of Compound 2¹An H-NMR spectrum;

FIG. 4 is a drawing of Compound 2¹³A C-NMR spectrum;

FIG. 5 is a drawing of Compound 3¹An H-NMR spectrum;

FIG. 6 is a drawing of Compound 3¹³A C-NMR spectrum;

FIG. 7 is a drawing of Compound 4¹An H-NMR spectrum;

FIG. 8 is a drawing of Compound 4¹³A C-NMR spectrum;

FIG. 9 shows the combination of 1-4 pairs of H of tuberculate speranskia herb lignan compounds₂O₂Induced SH-SY5Y cell neuroprotective activity.

Detailed Description

The present invention will be described in detail with reference to the embodiments shown in the drawings.

The present invention is illustrated by the following specific examples. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available.