阅读说明：本技术 一种土牛膝抗炎活性提取物及其制备方法 (Anti-inflammatory active extract of achyranthes aspera and preparation method thereof ) 是由 罗懿钒 欧阳文 张云坤 李顺祥 唐纯玉 王雄龙 唐代凤 周华荣 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种土牛膝抗炎活性提取物及其制备方法,其技术方案要点是包括以下步骤：A、选取粗毛牛膝的根及根茎作为植物原料,将植物阴干粉碎后,通过20目筛网得到细粉,将细粉用70％～100％醇溶液,回流提取2h后,重复提取3次,在60℃下减压浓缩,在阴暗低温及通风较好的地方晾干,制得甲醇中间提取物；B、将步骤A得到的甲醇中间提取物130g用甲醇充分溶解,与反相硅胶搅拌,水浴锅挥干溶剂,后经ODS柱层析(4*10cm),利用醇类-水溶液梯度洗脱,其中水、10％醇溶液、30％醇溶液、50％醇溶液、70％醇溶液、90％醇溶液、纯醇溶液、丙酮分别洗脱2.0L,并合并30％-70％醇溶液洗脱组分得到土牛膝抗炎活性提取物,经试验验证对急慢性咽炎具有明显疗效。(The invention discloses an anti-inflammatory active extract of achyranthes aspera and a preparation method thereof, and the technical scheme is characterized by comprising the following steps: A. selecting roots and rhizomes of crude achyranthes bidentata as plant raw materials, drying and crushing the plants in the shade, passing the plants through a 20-mesh screen to obtain fine powder, performing reflux extraction on the fine powder for 2 hours by using 70-100% alcohol solution, repeatedly extracting for 3 times, performing reduced pressure concentration at 60 ℃, and drying in the shade at low temperature and in a well ventilated place to obtain a methanol intermediate extract; B. and B, fully dissolving 130g of the intermediate methanol extract obtained in the step A with methanol, stirring with reverse phase silica gel, volatilizing the solvent in a water bath kettle, performing ODS column chromatography (4 x 10cm), performing gradient elution by using alcohol-water solution, wherein water, 10% alcohol solution, 30% alcohol solution, 50% alcohol solution, 70% alcohol solution, 90% alcohol solution, pure alcohol solution and acetone are respectively eluted for 2.0L, and the elution components of the 30% to 70% alcohol solution are combined to obtain the achyranthes aspera anti-inflammatory active extract, which has obvious curative effect on acute and chronic pharyngitis.)

1. A preparation method of achyranthes aspera anti-inflammatory active extract is characterized by comprising the following steps:

A. selecting roots and rhizomes of crude achyranthes bidentata as plant raw materials, drying and crushing the plants in the shade, passing the plants through a 20-mesh screen to obtain fine powder, performing reflux extraction on the fine powder for 2 hours by using 70-100% alcohol solution, repeatedly extracting for 3 times, performing reduced pressure concentration at 60 ℃, and drying in the shade at low temperature and in a well ventilated place to obtain an alcohol intermediate extract;

B. and B, fully dissolving 130g of the alcohol intermediate extract obtained in the step A by using methanol, stirring the solution and reversed phase silica gel, volatilizing the solvent in a water bath kettle, performing ODS column chromatography (4 x 10cm), performing gradient elution by using alcohol-water solution, wherein water, 10% alcohol solution, 30% alcohol solution, 50% alcohol solution, 70% alcohol solution, 90% alcohol solution, pure alcohol solution and acetone are respectively eluted for 2.0L, and combining elution components of the 30% to 70% alcohol solution to obtain the achyranthes aspera anti-inflammatory active extract.

2. The method for preparing an anti-inflammatory active extract of achyranthes aspera as claimed in claim 1, wherein: the alcohol or alcohol solution is methanol or ethanol, and the extraction process is ultrasonic or reflux.

3. The method for preparing an anti-inflammatory active extract of achyranthes aspera as claimed in claim 1, wherein: the final anti-inflammatory extract of radix Achyranthis bidentatae is used at a concentration of 6.25-200 μ g/ml.

4. The method for preparing an anti-inflammatory active extract of achyranthes aspera as claimed in claim 1, wherein: the solvent volatilizing process in the water bath is drying at the temperature of less than or equal to 60 ℃.

5. The method for preparing an anti-inflammatory active extract of achyranthes aspera as claimed in claim 1, wherein: and the alcohol intermediate extract is separated by using a fully hydrophilic material ODS-AQ reverse phase column (the dosage is 0.9-1.5 times of the extract ratio, based on drying).

6. The method for preparing an anti-inflammatory active extract of achyranthes aspera as claimed in claim 1, wherein: selecting roots and rhizomes of crude achyranthes bidentata as plant raw materials, drying and crushing the plants in the shade, passing the plants through a 20-mesh screen to obtain fine powder, extracting 1.17kg of the fine powder by 6 times of methanol in volume under reflux for 2 hours, repeatedly extracting for 3 times, concentrating under reduced pressure at 60 ℃, and drying in the air at a dark low temperature and a place with good ventilation to obtain an alcohol intermediate extract, wherein the extract is a dark brown solid, 130.33g and the yield is 11.15%; dissolving 130g of the methanol intermediate extract with methanol, stirring with 117.5g of ODS-AQ full hydrophilic reverse phase silica gel, evaporating the solvent in a water bath, performing ODS column chromatography (4 x 10cm), and performing gradient elution with methanol-water solution, wherein water, 10% methanol water solution, 30% methanol water solution, 50% methanol water solution, 70% methanol water solution, 90% methanol water solution, pure methanol, and acetone elute 2.0L, respectively, and concentrating the fractions under reduced pressure to obtain water-washed matter 110.5g, 10% methanol eluate 1.3g, 30% methanol eluate 2.42, 50% methanol eluate 2.99g, 70% methanol eluate 4.37g, 90% methanol eluate 4.31g, 100% methanol eluate 3.39g, and acetone eluate 1.27g, respectively. The activity of each part was measured and combined with 30% -70% methanol elution fractions to give 9.78g, with a final yield of 0.83%.

7. An anti-inflammatory active extract of achyranthes aspera, which is characterized in that: the extract contains eight effective components of ecdysterone, R-achyranthis bidentatae ketosteroid, S-achyranthis bidentatae ketosteroid, N-trans feruloyltyramine, N-cis feruloyltyramine, N-trans feruloyl-3-methoxytyramine, N-cis feruloyl-3-methoxytyramine and panax japonicus saponin IVa.

8. An application of anti-inflammatory active extract of radix Achyranthis in treating pharyngitis is provided.

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of medicines, and particularly relates to an anti-inflammatory active extract of achyranthes aspera and a preparation method thereof.

[ background of the invention ]

Pharyngitis is nonspecific inflammation of the pharynx and is generally classified into acute pharyngitis and chronic pharyngitis. Acute pharyngitis is acute inflammation of pharyngeal mucosa and tissues under the mucosa, pharyngeal lymphatic tissues are often involved, early inflammation can be limited, the whole pharyngeal cavity can be involved with the progress of the disease, and the acute pharyngitis is more common in autumn, winter and spring. It is mainly manifested as dry throat, burning sensation, pain, obvious pain in swallowing, congestion and swelling of throat, etc. The chronic pharyngitis can be divided into chronic simple pharyngitis, chronic hypertrophic pharyngitis and atrophic pharyngitis, wherein the chronic simple pharyngitis is more common, the pathological changes are mainly in mucous membrane layers, the mucous membrane layers are manifested by chronic congestion of pharyngeal mucous membranes, hyperplasia of mucous membranes and connective tissues under the mucous membranes, the mucous glands can be enlarged, the secretion function is enhanced, and the secretion of mucous is increased. The patient often expectorates phlegm in the pharynx, or feels foreign body sensation in the pharynx, unable to expectorate and unable to swallow. The disease course is long, the disease is easy to recur, and the symptoms are stubborn. In recent years, along with the diversification development of life and diet, the bad living habits are increasingly increased, and the pharyngitis, which is a common disease in the department of respiration and the department of otorhinolaryngology, has a long course of disease, is easy to repeat symptoms, is difficult to cure, causes symptoms such as dry cough, pharyngalgia, pharynx itch and the like, and affects the sleeping and the working quality.

Radix Achyranthis bidentatae (Achyranthus aspera L.) is a plant of the genus Achyranthus of the family Amaranthaceae, also called Tu niu xi, and its medicinal parts mainly include roots and rhizomes. The Chinese medicinal preparation is mainly distributed in a hillside sparse forest with the elevation of 200-1750m, which is mainly distributed in the north of China from the great wall, Taiwan province, Fujian province, West to Yunnan and Sichuan, and Nanda Guangdong and Guangxi. According to records of book records of Chinese materia medica and national Chinese herbal medicine compilation and the like, achyranthes aspera has the effects of activating blood and dissolving stasis, purging fire and removing toxicity, and inducing diuresis for treating stranguria, and is mainly used for treating traumatic injury, rheumatic arthralgia, sore throat, diphtheria and edema. The systematic components, the drug effect, the toxicology and the like of the achyranthes aspera are researched, and the achyranthes aspera is proved to have the effect of resisting respiratory tract and urinary system infection.

The preparation and research of the anti-inflammatory components of the achyranthes aspera, which are prepared by the invention, have simple process and low cost, and have important significance for the development of achyranthes aspera resources and the prevention and treatment of pharyngitis.

[ summary of the invention ]

The achyranthes aspera anti-inflammatory active extract has obvious curative effect on acute and chronic pharyngitis through test verification.

The invention also aims to provide a preparation method of the achyranthes aspera anti-inflammatory active extract.

The invention is realized by the following technical scheme:

a method for preparing anti-inflammatory active extract of radix achyranthis bidentatae comprises the following steps:

A. selecting roots and rhizomes of crude achyranthes bidentata as plant raw materials, drying and crushing the plants in the shade, passing the plants through a 20-mesh screen to obtain fine powder, performing reflux extraction on the fine powder for 2 hours by using 70-100% alcohol solution, repeatedly extracting for 3 times, performing reduced pressure concentration at 60 ℃, and drying in the shade at low temperature and in a well ventilated place to obtain a methanol intermediate extract;

B. and B, fully dissolving 130g of the intermediate methanol extract obtained in the step A with methanol, stirring with reverse phase silica gel, volatilizing the solvent in a water bath kettle, performing ODS column chromatography (4 x 10cm), performing gradient elution by using alcohol-water solution, wherein water, 10% alcohol solution, 30% alcohol solution, 50% alcohol solution, 70% alcohol solution, 90% alcohol solution, pure alcohol solution and acetone are respectively eluted for 2.0L, and combining elution components of 30% -70% alcohol solution to obtain the achyranthes aspera anti-inflammatory activity extract.

Preferably, the alcohol or alcohol solution is methanol or ethanol, and the extraction process is ultrasonic or reflux.

Preferably, the final anti-inflammatory extract of achyranthes aspera is used at a concentration of 6.25-200. mu.g/ml.

Preferably, the solvent volatilizing process in the water bath is drying at 60 ℃ or lower.

Preferably, the methanol intermediate extract is separated by using a fully hydrophilic material ODS-AQ reverse phase column (the dosage is 0.9-1.5 times of the extract ratio, based on drying).

Preferably, roots and rhizomes of the crude achyranthes bidentata are selected as plant raw materials, the plants are dried in the shade and crushed, fine powder is obtained through a 20-mesh screen, the fine powder is extracted for 2 hours in a refluxing mode through 70% -100% of alcohol solution, repeated extraction is carried out for 3 times, reduced pressure concentration is carried out at the temperature of 60 ℃, air drying is carried out in places with low temperature in the shade and good ventilation, and a methanol intermediate extract is prepared, wherein the extract is a dark brown solid, 130.33g and the yield is 11.15%; dissolving 130g of the methanol intermediate extract with methanol, stirring with 117.5g of ODS-AQ full-hydrophilic reverse phase silica gel, evaporating the solvent in a water bath, performing ODS column chromatography (4 x 10cm), and performing gradient elution with methanol-water solution, wherein water, 10% methanol water solution, 30% methanol water solution, 50% methanol water solution, 70% methanol water solution, 90% methanol water solution, pure methanol, and acetone elute 2.0L, and the fractions are concentrated under reduced pressure to obtain 110.5g of water-washed matter, 1.3g of 10% methanol eluate, 2.42 g of 30% methanol eluate, 2.99g of 50% methanol eluate, 4.37g of 70% methanol eluate, 4.31g of 90% methanol eluate, 3.39g of 100% methanol eluate, and 1.27g of acetone eluate, respectively. The activity of each part was measured and combined with 30% -70% methanol elution fractions to give 9.78g, with a final yield of 0.83%.

An anti-inflammatory active extract of achyranthes aspera comprises eight effective components of ecdysterone, R-achyranthes aspera, S-achyranthes aspera, N-trans feruloyltyramine, N-cis feruloyltyramine, N-trans feruloyl-3-methoxytyramine, N-cis feruloyl-3-methoxytyramine and panax japonicus saponin IVa. The monomer obtained by separation is confirmed by chromatographic analysis and identification and comparison with literature, and the above 8 traditional Chinese medicine effective components are proved to have the function of inhibiting pharyngitis.

An application of anti-inflammatory active extract of radix Achyranthis in treating pharyngitis is provided.

Compared with the prior art, the invention has the following advantages:

1. the anti-inflammatory active extract of achyranthes aspera can effectively show obvious inhibition activity on NO generated by RAW264.7 cells induced by LPS, has obvious anti-inflammatory effect, and is used for purifying and impurity-removing anti-inflammatory substances of achyranthes aspera under the guidance of an active model; LPS is used for stimulating a mouse mononuclear macrophage strain RAW264.7, cells can be induced to generate a plurality of related mediators and cytokines, wherein NO is one of important factors in a plurality of inflammatory mediators and plays an important role in regulating a plurality of physiological functions, such as vasodilatation, neurotransmission, inflammatory response and the like, the RAW264.7 cell strain releases the generated NO into a cell culture solution through LPS stimulation, and the concentration of the NO can be used for evaluating the strength of inflammatory response; the invention is established in an in vitro inflammation model of LPS-induced RAW264.7 mouse macrophage, and whether NO generated by mouse macrophage stimulated by LPS is interfered by prepared medicament to generate inhibition or not is detected by a NO detection kit;

2. according to the achyranthes aspera anti-inflammatory active extract, the extract is further analyzed to obtain that the extract contains main eight pharmacodynamic monomer components, and each monomer is qualitatively and quantitatively determined through liquid chromatography analysis, so that the validity of the achyranthes aspera anti-inflammatory active extract is proved, and reference is provided for further product improvement;

3. according to the preparation method of the achyranthes aspera anti-inflammatory active extract, through the steps of purification and concentration, the anti-inflammatory extract containing 8 pharmacodynamic components is extracted to serve as main pharmacodynamic components for anti-inflammation and anti-inflammation, the effect of the achyranthes aspera anti-inflammatory active extract is further explained, the extraction and concentration methods are efficient, the extraction methods under different conditions are compared, the basis is provided for improving the yield, the extraction method is relatively stable and controllable in quality, and the reference is provided for wide popularization of batch production.

[ description attached drawings ]

FIG. 1: liquid chromatogram of extract

[ detailed description ] embodiments

The preparation method of the anti-inflammatory active extract of achyranthes aspera of the invention is described by combining the specific examples: