阅读说明：本技术 一种羊耳菊提取物及其制备和应用 (Chrysanthemum indicum extract and preparation and application thereof ) 是由 杨建波 阮国永 刘光军 钟正胜 张旭 于 2020-07-21 设计创作，主要内容包括：本发明涉及是一种羊耳菊提取物及其制备和应用。所述羊耳菊提取物的制备方法,包括以下步骤：(1)预处理；(2)膨化处理：预处理后的羊耳菊粉碎物加到真空膨化罐中,进行压差膨化处理,获得羊耳菊膨化处理物；(3)提取：将羊耳菊膨化处理物添加到离子液体中进行超声提取,过滤,除去滤液中的离子液体,制得提取液；(4)纯化：将提取液采用柱层析分离,减压浓缩,去除洗脱剂,制得纯化液；(5)固定化。本发明提供的制备方法,整个制备方法在低温下进行,能降低活性成分提取过程中的损失,提高活性成分提取率,更能富聚药物活性成分。本发明所制得的羊耳菊提取物对吸入性颗粒物引起的肺部损伤、肺纤维化具有良好的治疗作用。(The invention relates to a inula cappa extract and a preparation method and application thereof. The preparation method of the inula cappa extract comprises the following steps: (1) pre-treating; (2) puffing: adding the pretreated inula cappa crushed material into a vacuum puffing tank, and carrying out differential pressure puffing treatment to obtain an inula cappa puffed treated material; (3) extraction: adding the expanded product of the inula cappa into ionic liquid for ultrasonic extraction, filtering, and removing the ionic liquid in the filtrate to obtain an extracting solution; (4) and (3) purification: separating the extractive solution by column chromatography, concentrating under reduced pressure, and removing eluent to obtain purified solution; (5) and (4) immobilizing. The preparation method provided by the invention is carried out at low temperature, can reduce the loss in the extraction process of the active ingredients, improves the extraction rate of the active ingredients, and can enrich the active ingredients of the medicine. The inula cappa extract prepared by the invention has good treatment effect on lung injury and pulmonary fibrosis caused by inhalation particles.)

1. A preparation method of a inula cappa extract is characterized by comprising the following steps:

(1) pretreatment: pulverizing herba Inulae Cappae, adding 0.1-0.15% pretreatment agent, mixing, and adjusting water content to 20-50%;

(2) puffing: adding the pretreated inula cappa crushed material into a vacuum puffing tank, and carrying out differential pressure puffing treatment to obtain an inula cappa puffed treated material;

(3) extraction: adding the expanded product of the inula cappa into ionic liquid for ultrasonic extraction, filtering, and removing the ionic liquid in the filtrate to obtain an extracting solution;

(4) and (3) purification: separating the extractive solution by column chromatography, concentrating under reduced pressure, and removing eluent to obtain purified solution;

(5) immobilization: and uniformly stirring and mixing the purified solution and a fixing agent, and performing vacuum drying to obtain the inula cappa extract.

2. The method for preparing an inula cappa extract according to claim 1, wherein the inula cappa is pulverized to 60-80 mesh; the pretreating agent is sodium bicarbonate.

3. The method of preparing an inula cappa extract of claim 1, wherein the differential pressure puffing process conditions are: heating to make the temperature in the expansion tank be 30-40 ℃, firstly treating for 15-20min under 0.2-0.4MPa, and then treating for 20-30min under-0.01-0.03 MPa.

4. The method for preparing the inula cappa extract as claimed in claim 1, wherein the ionic liquid is an aqueous solution of 1-dodecyl-3-methylimidazole chloride with a concentration of 0.5 mol/L; the addition amount is 10 times of the quality of the expanded treatment of the inula cappa.

5. The method for preparing an inula cappa extract as claimed in claim 1, wherein the ultrasonic extraction conditions are: the ultrasonic power is 200W, and the ultrasonic time is 35-40 min.

6. The method for preparing an inula cappa extract as claimed in claim 1, wherein the column chromatography separation conditions are: 100-200 mesh silica gel column, and the eluent is composed of 70% ethanol and acetone according to the volume ratio of 1: 1.

7. The method of preparing an inula cappa extract of claim 1, wherein the fixative is nanocellulose and is added in an amount of 1-1.5 times the mass of the purified liquid.

8. An extract of tanacetum vulgare prepared by the method of preparing an extract of tanacetum vulgare as claimed in any one of claims 1 to 7.

9. Use of an extract of tanacetum vulgare according to claim 8 in a medicament for the treatment of impaired lung function caused by inhaled particulate matter.

10. The use of claim 9, wherein the inula cappa extract is used in the treatment of pulmonary fibrosis induced by inhaled particulates.

Technical Field

The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to a inula cappa extract and preparation and application thereof.

Background

Herba Inulae Cappae is root and whole plant of herba Inulae Cappae of Compositae, also called herba Elephantopi scaberis, DALIWANG, radix bupleuri, cacumen Securinegae Suffruticosae, and radix Angelicae Dahuricae, and is a herb of southern folk medicine. The chemical components of the inula cappa root and the whole plant comprise hemiterpene lactone, triterpene, catalpol, flavone, aromatic compound, organic acid and the like, and have the effects of dispelling wind and cold, promoting qi circulation and inducing diuresis, reducing phlegm and relieving cough, and relieving swelling and pain. It is commonly used for common cold due to wind-cold, cough, nervous headache, stomach ache, rheumatic lumbago and skelalgia, traumatic injury, menoxenia, leucorrhea, and schistosomiasis.

Air particulate refers to various dispersed ions in an aerosol system composed of various solid or liquid particles in the air, i.e., a general term for various solid and liquid particulate materials. Particulate matter is the main pollutant in the air and has very complicated physicochemical characteristics. Particulate matter is generally classified into total suspended particulate matter (particle size 100 μm or less), PM10 (particle size 10 μm or less), PM2.5 (particle size 2.5 μm or less) according to its aerodynamic equivalent diameter. The air particles have complex components, including various metal elements, inorganic salts, organic matters, various pathogenic microorganisms adsorbed by the particles and the like. The PM2.5 has small particle size, can enter the deep lung tissue of a human body along with breathing, and generates serious harm to the lung tissue of the human body. According to related researches, the inhaled particles enter the lung through the phagocytosis of alveolar macrophages, and are treated by the macrophages to activate specific immune response, so that the fibroblast is activated, the tissue is proliferated, and fibrosis is formed. The particulate matter can also cause inflammatory reactions in the lungs through oxidative stress; oxidative stress can directly damage lung tissue, trigger inflammatory injury, cytokine release, inflammatory gene expression, lung fibroblast proliferation through a series of mechanisms, matrix collagen deposition, lung compliance decline, restrictive ventilation disorder, result in fibrosis generation, and the like.

At present, the research on treating lung injury and lung fibrosis caused by air particles by the active ingredients of the inula cappa is reduced.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a preparation method of a inula cappa extract by researching active ingredients and an extraction method of the inula cappa extract, and the prepared inula cappa extract has good prevention and treatment effects on lung injury and lung fibrosis caused by air particles, and is realized by the following technical scheme.

A preparation method of a inula cappa extract comprises the following steps:

(1) pretreatment: pulverizing herba Inulae Cappae, adding 0.1-0.15% pretreatment agent, mixing, and adjusting content to 20-50%;

(2) puffing: adding the pretreated inula cappa crushed material into a vacuum puffing tank, and carrying out differential pressure puffing treatment to obtain an inula cappa puffed treated material;

(3) extraction: adding the expanded product of the inula cappa into ionic liquid for ultrasonic extraction, filtering, and removing the ionic liquid in the filtrate to obtain an extracting solution;

(4) and (3) purification: separating the extractive solution by column chromatography, concentrating under reduced pressure, and removing eluent to obtain purified solution;

(5) immobilization: and uniformly stirring and mixing the purified solution and a fixing agent, and performing vacuum drying to obtain the inula cappa extract.

Preferably, the inula cappa is crushed to 60-80 meshes; the pretreating agent is sodium bicarbonate.

Preferably, the pressure difference puffing treatment conditions are as follows: heating to make the temperature in the expansion tank be 30-40 ℃, firstly treating for 15-20min under 0.2-0.4MPa, and then treating for 20-30min under-0.01-0.03 MPa.

Preferably, the ionic liquid is a chlorinated 1-dodecyl-3-methylimidazole aqueous solution with the concentration of 0.5 mol/L; the addition amount is 10 times of the quality of the expanded treatment of the inula cappa.

Preferably, the ultrasonic extraction conditions are: the ultrasonic power is 200W, and the ultrasonic time is 35-40 min.

Preferably, the column chromatography separation conditions are as follows: 100-200 mesh silica gel column, and the eluent is composed of 70% ethanol and acetone according to the volume ratio of 1: 1.

Preferably, the fixing agent is nano-cellulose, and the addition amount of the fixing agent is 1-1.5 times of the mass of the purified liquid.

The invention also aims to provide the inula cappa extract prepared by the preparation method.

The invention also provides application of the inula cappa extract in a medicine for treating lung injury caused by inhaled particles.

Preferably, the inula cappa extract is applied to the medicine for treating pulmonary fibrosis caused by inhalation particulate matters.

The invention has the beneficial effects that:

according to the invention, the inula cappa is extracted after being treated by adopting pressure difference puffing, the cell walls of plant cells can be effectively destroyed by adopting the pressure difference puffing, the leaching rate of effective components in the subsequent extraction process is improved, and the loss of volatile components of the inula cappa is basically avoided. The ionic liquid is adopted to assist ultrasonic extraction for extraction, so that water-soluble components and fat-soluble components of the inula cappa can be extracted simultaneously, and active components of the inula cappa can be enriched. The preparation process provided by the invention has the advantages that the whole extraction process is carried out at low temperature, so that the loss in the extraction process of the active ingredients can be reduced, the extraction rate of the active ingredients is improved, and the active ingredients of the medicine can be enriched; the extracted extract is treated by adopting nano microcrystalline cellulose, so that the stability and biocompatibility of active ingredients of the medicine can be enhanced, and the medicine effect of the medicine can be improved. The inula cappa extract prepared by the invention has good treatment effect on lung injury and pulmonary fibrosis caused by inhalation particles.

In order to better illustrate the advantageous effects of the present invention, the following test examples are also presented in the present invention, which are intended to illustrate the advantageous effects of the present invention, but are by no means limited to the scope of protection of the present invention.

Test example 1 study of extraction Process

1. Screening an extraction mode: in the process of technological research, in order to utilize the active ingredients of all medicines to the maximum extent, the inula cappa is dried and crushed to 60-80 meshes, and the modes of ultrasonic-assisted water extraction, water decoction extraction, ethanol reflux extraction, ultrasonic-assisted ethanol extraction, ionic liquid extraction, ultrasonic-assisted ionic liquid extraction and the like are respectively examined, and the result shows that the mode of ultrasonic-assisted ionic liquid extraction is adopted, so that the extraction efficiency of the active ingredients of the medicinal materials in the formula is high, the contents of the hemiterpene lactones, the quercetin, the chlorogenic acid and the like are high, and the dissolved substances are more. Factors such as production time, production cost and the like in large-scale batch production are integrated, and the power and the extraction time of ultrasonic extraction are further investigated.

By adopting 5 ultrasonic powers (100W, 200W, 300W, 400W and 500W) and 5 extraction times (20-30min, 30-40min, 40-50min, 50-60min and 60-70min), the results of the examination show that when the 200W ultrasonic extraction is adopted for 50-60min, the extract can better enrich the active ingredients of the medicine, and the detection shows that the crude drugs with the volatile oil content of more than or equal to 0.37 percent, the crude drugs with the polysaccharide content of more than or equal to 0.86 percent, the crude drugs with the flavone content of more than or equal to 7.2 percent and the crude drugs with the total extract of more than or equal to 53 percent can exert the maximum medicine effect. Wherein, the differential pressure puffing degree treatment is carried out before the extraction, the effect is optimal, the content of active ingredients is ideal, and the extraction time can be shortened to 35-40 min. Due to the destructive effect of differential pressure puffing on the plant cell wall of the inula cappa, the dissolution of effective components can be promoted, and the extraction time is shortened. Wherein, column chromatography is adopted for purification after extraction, so that impurities in the extracting solution can be removed, and active ingredients in the extracting solution can be enriched.

Test example 2 pharmacodynamic study

1. Research on protective effect of respiratory system injury of rat caused by inhalation particles

1.1 Experimental animals: SD rats, SPF grade. The number of the animals is 50, the animals are half male and female, the animals are 6-8 weeks old when the animals are ordered, and the weight of the animals is 180-200 g. All rats are raised uniformly for one week and then used for the test; during the raising period, the padding and the cages are replaced 1 time per week, the special feed for the rats is added every day for the animals to eat, and the drinking water bottles are used for filling water for the animals to drink, so that the free diet and activity of the animals are kept in the whole raising process.

1.2 Experimental methods

SD rats were randomly divided into 5 groups of 10 animals each, each female half. The tanacetum vulgare extract prepared in example 1 was prepared by adding water to a solution having a concentration of 0.1 g/ml.

Control group: rats were exposed daily to a clean inhalation contamination device inhaling clean air for 30min in the mouth and nose.

Model group: exposing the mouth and nose of a rat to an air fine particle aerosol toxicant exposure device every day, and inhaling air haze for 30 min;

test group 1 (low dose group): exposing the mouth and nose of a rat to an air fine particle aerosol toxicant exposure device every day, and inhaling air haze for 30 min; 1ml/100g (mouse weight) of the liquid medicine is administered by gavage 1 hour before infection.

Test group 2 (medium dose group): exposing the mouth and nose of a rat to an air fine particle aerosol toxicant exposure device every day, and inhaling air haze for 30 min; the drug solution was administered 1 hour before the infection by gavage at a concentration of 2.5ml/100g (mouse body weight).

Trial group 3 (high dose group): exposing the mouth and nose of a rat to an air fine particle aerosol toxicant exposure device every day, and inhaling air haze for 30 min; the drug solution was administered 1 hour before infection by gavage at a concentration of 5ml/100g (mouse body weight).

The lung function of a conscious rat is measured by adopting an EMKA animal lung function monitoring system, the EMKA animal lung function monitoring system is placed in a quiet laboratory, the system is corrected, calibrated and ventilated before formal measurement, the data acquisition frequency is automatically acquired once every 5 seconds, and the measurement result is displayed once every minute. The rats are placed in a whole body scanning meter box to adapt for 5-10min, the recording time is 3-8min, and the average value of the results of each animal is taken as the measurement result of the lung function of the animal. The measured indices include inspiration time (Ti), expiration time (Te), Tidal Volume (TV), Expiration Volume (EV), Relaxation Time (RT), and Minute Ventilation (MV). The respiratory function of the animals was measured 8 weeks after the exposure of the rats to the virus, and the rats were placed in a whole plethysmograph box and connected to a respiratory function measuring instrument for measurement, and the results are shown in table 1.

TABLE 1

Ti(msec)

Te(msec)

TV(ml)

EV(ml)

RT(msec)

MV(ml)

Control group

96.8±16.2

168.7±16.4

0.97±0.22

1.02±0.38

105.4±34.2

297.3±38.4

Model set

101.3±17.6

192.4±19.2

0.72±0.28

0.85±0.42

118.3±32.5

229.4±32.1

Test group 1

99.8±20.3

183.7±22.7

0.86±0.32

0.93±0.41

109.6±35.6

287.4±36.4

Test group 2

98.4±18.2

178.7±21.2

0.90±0.36

0.97±0.34

106.2±34.4

290.2±38.2

Test group 3

97.1±16.7

173.4±21.8

0.92±0.31

0.99±0.32

104.8±35.3

295.8±40.2

The Ti value of the model group rats inhaled and infected by virus is obviously higher than that of the control group (p is less than 0.05), and the RT value of the SD rat infected by virus is obviously higher than that of the control group (p is less than 0.05), which indicates that when the model group rats are obviously inhibited from lung function indexes related to flow and volume. The rats TV, EV and MV in the model group all decreased to different degrees during the observation period, indicating that the volume of breath was significantly decreased and the lung ventilation function reserve was also decreased. Compared with the model group, the experimental group 1-3 can obviously improve and improve the lung function damage of the rat caused by continuous fine particulate matter inhalation, and has certain improvement effect.

2. Research on influence of inula cappa extract on pulmonary fibrosis of mice caused by particles

2.1 materials of the experiment

Experimental animals: 50 SPF grade ICR healthy mice with the body mass of 18-20 g are used for the test after 1.1 all the mice are uniformly bred for one week; during the raising period, the padding and the cages are replaced 1 time per week, the special feed for the rats is added every day for the animals to eat, and the drinking water bottles are used for filling water for the animals to drink, so that the free diet and activity of the animals are kept in the whole raising process.

PM 2.5: PM2.5 collected near a coal-fired power plant in Guizhou province is added with normal saline to prepare a particle suspension with the concentration of 30 mg/ml.

2.2 Experimental methods

Mice were randomly divided into 5 groups of 10 mice each, female halves. The tanacetum vulgare extract prepared in example 1 was prepared by adding water to a solution having a concentration of 0.1 g/ml.

Control group: on days 1, 6, 11, 16 and 21 from the start of the experiment, 0.9% physiological saline (20. mu.l/mouse) was added dropwise through the nasal cavity.

Model group (model): mice were titrated nasally with 30mg/ml of the pellet suspension (20. mu.l/mouse) on days 1, 6, 11, 16, 21 of the start of the experiment;

test group 1 (low dose group): mice were titrated nasally with 30mg/ml of the pellet suspension (20. mu.l/mouse) on days 1, 6, 11, 16, 21 of the start of the experiment; on days 1 to 21, 1ml/100g (mouse body weight) of the liquid medicine was administered by gavage every day.

Test group 2 (medium dose group): mice were titrated nasally with 30mg/ml of the pellet suspension (20. mu.l/mouse) on days 1, 6, 11, 16, 21 of the start of the experiment; on days 1 to 21, 2.5ml/100g (mouse body weight) of the liquid medicine was administered by gavage each day.

Trial group 3 (high dose group): mice were titrated nasally with 30mg/ml of the pellet suspension (20. mu.l/mouse) on days 1, 6, 11, 16, 21 of the start of the experiment; on days 1 to 21, 5ml/100g (mouse body weight) of the liquid medicine was administered by gavage each day.

Material obtained on day 22 (lung tissue): the model mice of each experimental group were sacrificed on day 22, bilateral lung tissues were taken, labeled with tinfoil paper, placed in liquid nitrogen for rapid freezing, and then stored in a 80 ℃ refrigerator for testing.

Preparation of tissue homogenate: taking 0.1g of lung tissue in a centrifuge tube, adding 0.9g of normal saline, placing the test tube in an ice box, breaking the tissue by adopting an ultrasonic breaking method, subpackaging the tissue in two centrifuge tubes, centrifuging the centrifuge tube 1 at 1500r/min for 10min, taking supernatant, storing in a refrigerator at 4 ℃ for SOD and MDA measurement. Centrifuging the centrifuge tube 2 at 5000r/min for 5min, collecting supernatant, storing in refrigerator at 4 deg.C, and determining IL-6 and TNF-alpha. The measurement results are shown in Table 2.

TABLE 2

	SOD(U/ml)	MDA(nmol/L)	IL-6(pg/ml)	TNF-α(ml)
					Control group	99.43±7.12	5.13±0.26	76.14±5.68	551.96±70.05
Model set	74.18±7.34	16.43±1.68	134.28±21.26	813.46±64.58
					Test group 1	80.23±7.38	14.39±2.24	126.48±18.21	769.89±69.34
Test group 2	89.16±6.42	10.89±2.05	93.43±16.23	628.16±50.12
					Test group 3	99.71±9.43	7.36±1.18	86.32±15.18	589.92±60.16

The results in the table show that after mice are induced by injecting PM2.5 suspension liquid through nasal cavities, the SOD activity in lung tissue homogenate of the model group is obviously lower than that of the control group, and the MDA content, the IL-6 content and the TNF-alpha content are obviously higher than those of the control group; compared with the model group, the test groups 1-3 have obviously reduced SOD activity, MDA content, IL-6 content and TNF-alpha content compared with the model group, which shows that the inula cappa extract can obviously improve the collagen deposition and fibrosis sample change of the lung tissue of mice caused by PM2.45 particles.

Detailed Description

The technical solution of the present invention is further limited by the following specific embodiments, but the scope of the claims is not limited to the description.