CN116554142A - A cicada ecdylide acetyldopamine oligomer composition for treating allergic asthma - Google Patents

Abstract

The invention discloses an oligomer composition of cicada slough acetyldopamine for treating allergic asthma. The present invention conducts systematic and in-depth research on the chemical components of cicada slough, uses a silica gel column to carry out systematic separation and purification, and screens out a specific ratio of the cicada slender acetyldopamine compound. Compositions of polymers, trimers, tetramers and pentamers. Pharmacological experiments show that the isolated cicada acetyldopamine oligomer composition of the present invention has a good effect on treating allergic asthma, and its new clinical application can be developed.

Description

A cicada ecdylide acetyldopamine oligomer composition for treating allergic asthma

technical field

The invention relates to a new clinical application of cicada slough acetyldopamine oligomer composition separated from cicada slough, belonging to the technical field of medicine.

Background technique

Cicadas slough into the lungs and liver meridians, and are clinically used to treat asthma with remarkable curative effect, which has been recorded in many herbal works. For example, "Chinese Clinical Chinese Materia Medica" says that it can "relieve cough and relieve asthma, and treat cough and asthma syndromes mainly". Asthma belongs to the category of "asthma" and "asthma syndrome" in traditional Chinese medicine. Cicada slough medicinal materials contain acetyldopamine dimers, trimers, tetramers, pentamers and other oligomer components. Studies have shown that these components have strong The activity of cicada slough is the main active substance.

Allergic asthma (Asthma) is a chronic airway inflammation involving a variety of cells, especially mast cells, eosinophils, and T lymphocytes. In susceptible individuals, this inflammation can cause recurrent wheezing, Symptoms such as shortness of breath, chest tightness and/or cough are prone to diseases in young children and adolescents.

Although some studies have shown that cicada slough has the effect of treating asthma, at present, there is no research report on specific cicada slough acetyldopamine oligomer composition for treating allergic asthma.

Contents of the invention

Purpose of the invention: the purpose of the present invention is to further study the new clinical application of cicada acetyldopamine oligomers, and screen out dimers, trimers, tetramers and pentamers of cicada acetyldopamine in specific proportions through a large number of clinical experiments Novel clinical use of the composition for the treatment of asthma.

Technical scheme: in order to realize above object, the technical scheme that the present invention takes is:

A cicada ecdylide acetyldopamine oligomer composition for treating allergic asthma, which is prepared by the following method:

(1) After taking the cicada slough medicinal material and pulverizing it, add ethanol, heat and reflux for extraction, filter, combine the filtrate, reclaim the ethanol extract, concentrate under reduced pressure, and obtain the cicada slough extract;

(2) Take the cicada slough extract obtained in step (1), put it on the forward silica gel chromatographic column, and use the methanol-chloroform gradient with volume ratio of 20:1, 15:1, 12:1, 10:1, 8:1 successively Elution, 20:1 and 15:1 elution fractions were collected, concentrated under reduced pressure to obtain the cicada acetyldopamine oligomer composition.

As a preferred version, the above-mentioned cicada acetyldopamine oligomer composition for the treatment of allergic asthma is prepared by the following method:

(1) After the cicada slough medicinal material is crushed, add ethanol with a volume concentration of 95% at a solid-to-liquid ratio of 1:10, heat and reflux for extraction 1 to 3 times, each time for 1 to 3 hours; filter, combine the filtrates, and recover the ethanol extract , concentrated under reduced pressure to a relative density of 1.04 to 1.12 cicada slough extract;

(2) Take the cicada slough extract obtained in step (1), put it on the forward silica gel chromatographic column, and use the methanol-chloroform gradient with volume ratio of 20:1, 15:1, 12:1, 10:1, 8:1 successively For elution, the flow rate was 1.5 BV/h, and the elution fractions of 20:1 and 15:1 were collected and concentrated under reduced pressure to obtain the cicada acetyldopamine oligomer composition.

As a preferred version, the above-mentioned cicadacetyl dopamine oligomer composition for the treatment of allergic asthma comprises dimers, trimers and tetramers of cicadacetyldopamine with a weight ratio of 10.7:10.7:3.6:1.0 and pentameric compositions.

The present invention screens out through a large number of clinical experiments that the dimer, trimer, tetramer and pentamer composition of cicadacetyl dopamine with a specific ratio has the effect of treating allergic asthma. The cicada acetyldopamine oligomer composition and pharmaceutically acceptable carriers can be prepared into capsules, tablets, granules, mixtures, pills and powders.

Beneficial effects: the cicada acetyldopamine oligomer composition with anti-allergic asthma activity provided by the present invention has the following advantages compared with the prior art:

The present invention conducts systematic in-depth research on the chemical components of the cicada slough, uses a silica gel column to carry out systematic separation and purification, and screens out the dimer, trimer, tetramer and pentamer composition of the cicada slender acetyldopamine in a specific proportion. The experimental results show that The cicada ecdylide acetyldopamine oligomer composition provided by the invention has the functions of relieving airway inflammation, reducing the level of inflammatory factors in alveolar lavage fluid, etc., has a good effect on treating allergic asthma, and can be used with pharmaceutically acceptable carriers Develop its clinical new pharmaceutical preparations for the treatment of allergic asthma.

Description of drawings

Fig. 1 Dimer, trimer, tetramer and pentamer content in cicada slough oligomer composition.

The influence of Figure 2 cicada slough acetyldopamine oligomer composition on allergic asthma mice cough frequency (A) and asthma latency period ( n=6. ^*** p<0.001, ^** p<0.01, ^* p<0.05).

The influence of Figure 3 cicada slough acetyldopamine oligomer composition on the total cells of alveolar lavage fluid of allergic asthmatic mice ( n=6. ^*** p<0.001, ^** p<0.01, ^* p<0.05).

Fig. 4 cicada ecdylide acetyldopamine oligomer composition HE staining result (A) and inflammation score ( n=6. ^*** p<0.001, ^** p<0.01, ^* p<0.05).

Fig. 5 cicada slough acetyldopamine oligomer composition is to Eotaxin-1 (A), IL-13 (B), IL-4 (C) and IL-5 (D) level in allergic asthma mouse alveolar lavage fluid Influence( n=6. ^*** p<0.001, ^** p<0.01, ^* p<0.05).

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art will readily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims .

Example 1

1. A cicada slough acetyldopamine oligomer composition for the treatment of allergic asthma, which is prepared by the following method:

(1) After the cicada slough medicinal material is crushed, add ethanol with a volume concentration of 95% at a solid-to-liquid ratio of 1:10, heat and reflux for extraction 1 to 3 times, each time for 1 to 3 hours; filter, combine the filtrates, and recover the ethanol extract , concentrated under reduced pressure to a relative density of 1.04 to 1.12 cicada slough extract;

(2) Get the cicada slough extract obtained in step (1), put it on the forward silica gel chromatographic column, and use the volume ratio successively as 20:1, 15:

1. Gradient elution with methanol-chloroform of 12:1, 10:1, and 8:1, with a flow rate of 1.5BV/h, collecting the eluted fractions of 20:1 and 15:1, and concentrating under reduced pressure to obtain the cicada acetyldopamine oligosaccharide polymer composition.

2. Content determination:

Determination of Dimer, Trimer, Tetramer and Pentamer Compositions of Cicadacetyldopamine Oligomer Composition

(1) Preparation of the test solution: take by weighing 1 g of the cicada acetyldopamine oligomer composition powder prepared above, add 10 mL of methanol with a volume concentration of 80% and place it in a stoppered test tube, weigh it, extract it by ultrasonic for 1 h, and again Weigh the weight, make up the lost weight with 80% methanol, centrifuge the extract, take 1mL of the supernatant, dilute with 80% methanol and dilute to 10mL, that is to say;

(2) Preparation of reference substance solution: Accurately weigh 1 mg of each of the reference substances of acetyldopamine dimer A and B, add 1 mL of methanol to prepare 1 mg/mL of acetyldopamine dimer A and B respectively; then take 200 μL each for control Add methanol to make up to 1mL, prepare a mixed reference substance stock solution of acetyldopamine dimer A and B with a concentration of 200 μg/mL, and finally dilute it step by step to 10 μg/mL to obtain a mixed reference solution;

Chromatographic conditions: Waters ACQUITY UPLC SYSTEM was used to analyze small molecular components in cicada slough. Diode matrix detector; autosampler; chromatographic column: ACQUITY HSS C18 column, 100mm×2.1mm, Φ=1.8μm; mobile phase: 0.1% formic acid aqueous solution is phase A-0.1% formic acid acetonitrile solution is phase B; elution Gradient: 0-10min, 15-50%B; 10-10.5min, 50-95%B; 10.5-12.5min, 95%B; flow rate 0.4mL/min; column temperature 40°C; injection volume 2μL;

Mass spectrometry conditions: Synapt G2-S Q-TOF Mass Spectrometer system is used to obtain the mass spectrum information of the compound; electrospray ion source; positive ion scanning mode; mass scanning range m/z 50-1500; capillary voltage 3000V, cone voltage 40V; Ion source temperature 120°C; desolvation temperature 450°C; desolvation gas flow rate 800L/h; cone gas flow rate 50L/h; MS collision energy: 6eV; MS/MS collision energy: 30-60V; MassLynx software was used for analysis.

(3) Take the above-mentioned need testing solution, and inject and analyze according to the above chromatographic and mass spectrometric conditions. The measurement was repeated three times for each sample, the peak area was recorded, and the standard curves for acetyldopamine A (Y=12516X+601.31, r ² =0.9988) and acetyldopamine B (Y=12736X+78.489, r ² =0.9992) were calculated. The absolute content of acetyldopamine A and B was calculated by the standard curve method. Carry out semi-quantitative to total dimer, trimer, tetramer, pentamer in the cicada slough composition by the result of absolute quantification again, calculation formula is as follows:

where Ar is the peak area of acetyldopamine dimer A, Ai is the peak area of the analyte, Cr is the concentration of acetyldopamine dimer A, and Ci is the concentration of the analyte.

(4) Quantitative analysis

Since the mother nuclei are the same, the other components are semi-quantified according to the standard curve of the reference substance acetyldopamine A and B. The quantitative results are shown in the figure. The weight ratio of dimer, trimer, tetramer and pentamer is 10.7:10.7:3.6:1.0.

Example 2

1. Experimental method

1. Preparation and grouping of allergic mouse models

BALB/c mice were randomly divided into normal group (Control), model group (Model), cicada ecdylide acetyldopamine oligomer high dose group (CP-WBT-H) prepared in Example 1, cicada ecdylide acetyldopamine oligomer low Dose group (CP-WBT-L), positive drug dexamethasone group, 10 rats in each group. In the other groups except the normal group, the sensitization solution (containing 20 μg ovalbumin (OVA) and 0.1 mL aluminum hydroxide gel) was injected intraperitoneally on days 0, 7, and 14, and 0.5% OVA was atomized and inhaled from day 21. Solution for 20 minutes, once every two days, for 21 consecutive days. The normal group was given the same volume of normal saline since the 21st day. The cicada acetyldopamine oligomer high and low dose groups were intragastrically administered every day, the model group and the normal group were given the same volume of normal saline, and the positive drug group was injected intraperitoneally with dexamethasone (DEX) 1 mg/kg from the nebulization period. 24 hours after the last OVA nebulization, 4 mice were taken from each group, put into the whole body plethysmography box in turn, and were sprayed with acetylcholine (Ach) at concentrations of 0, 3.13, 6.25, 12.5, 25, and 50 mg/mL. After 1 min, the changes in the number of times of coughing and wheezing and the latency of asthma in mice within 5 min of excitation were measured.

2. Alveolar lavage fluid (BALF) total cell count

24 hours after the last OVA atomization, the mice were plucked from the eyeballs to take blood, and then sacrificed by dislodging the cervical spine. After 3 times, the lavage fluid was collected and mixed evenly, centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was collected and stored at -80°C. The total cells in the alveolar lavage fluid (BALF) were counted with a cell counter.

3. Pathological examination of lung tissue

24 hours after the last OVA atomization, the mice were removed from the eyeballs to take blood, and then the cervical spine was removed to kill. The middle lobe of the right lung of the mouse was opened and soaked in 4% paraformaldehyde for fixation, embedded in paraffin, and the lungs with a thickness of 5 μm were cut. Tissue sections were dewaxed and stained according to the instructions of the hematoxylin-eosin (HE) staining reagent, and the pathological conditions of lung tissue were observed under a microscope.

4. Detection of inflammatory factors

According to the operation method of the ELISA kit, the experiment is divided into 9 steps: standard dilution, sample addition and incubation, addition of detection antibody, plate washing, enzyme incubation, plate washing, color development, termination, and microplate reader measurement. The levels of Eotaxin-1, IL-13, IL-4 and IL-5 inflammatory factors in mouse alveolar lavage fluid were determined.

2. Experimental results

1. The effect of cicada slough on the frequency of cough and asthma latency in allergic asthma mice

The results are shown in Figure 1. Compared with the Control group, the number of coughs in the Model group was significantly increased, and the latency period of asthma was significantly shortened (p<0.001). Compared with the Model group, the number of coughs in the DEX group, CP-WBT-L and CP-WBT-H groups was significantly reduced, and the latency period of asthma was significantly prolonged, indicating that the positive drug dexamethasone, low-dose and high-dose cicada acetyldopamine oligomers were all It has the effect of relieving allergic asthma. Compared with the CP-WBT-L and CP-WBT-H groups, the coughing times of the latter group were significantly reduced, and the incubation period of asthma was significantly shortened (p<0.05), which indicated that the treatment of allergic asthma by cicada slough was dose-dependent.

2. Effect of cicada slough on total cells in alveolar lavage fluid (BALF) of allergic asthma mice

The results are shown in Figure 2, compared with the Control group, the total cell number in the BALF of the Model group was significantly increased (p<0.001). Compared with the Model group, the number of total cells in the BALF of the DEX group, CP-WBT-L and CP-WBT-H groups was significantly reduced, indicating that the positive drug dexamethasone, low-dose and high-dose cicada acetyldopamine oligomers all had Relieve airway inflammation and treat allergic asthma. Compared with CP-WBT-L and CP-WBT-H groups, the number of total cells in BALF of the latter group was significantly reduced (p<0.01), indicating that the treatment of allergic asthma by cicada slough was dose-dependent.

3. Effect of cicada slough on lung tissue of allergic asthma mice

The results are shown in Figure 3. Compared with the Control group, a large number of inflammatory cell infiltrations were seen around the trachea and blood vessels in the lungs of the Model group, the airway and blood vessel walls were significantly thickened, and the pathological score was significantly increased (p<0.001). Compared with the Model group, the infiltration of inflammatory cells in the lungs of the mice in the DEX group and CP-WBT-H group was significantly improved, and the pathological score was significantly reduced, indicating that the positive drug dexamethasone and high-dose cicada can relieve airway inflammation and treat allergies role in asthma.

4. Effect of cicada slough on the levels of inflammatory factors in allergic asthma mice

The results are shown in Figure 4. Compared with the Control group, the contents of Eotaxin-1, IL-13, IL-4, and IL-5 in the BALF of the Model group mice were significantly increased (p<0.001). Compared with the Model group, the contents of Eotaxin-1, IL-13, IL-4, and IL-5 in the BALF of mice in the DEX group, CP-WBT-L group, and CP-WBT-H group were all significantly reduced, indicating a positive Drug dexamethasone, low-dose and high-dose cicada acetyldopamine oligomers can all reduce the levels of inflammatory factors in alveolar lavage fluid. Among them, the effect of high dose of cicada acetyldopamine oligomer and lower dose is better.

The preparation of embodiment 3 tablet

Take the cicada acetyldopamine oligomer composition prepared in the above-mentioned Example 1, add an appropriate amount of pharmaceutical excipients such as starch and magnesium stearate, mix thoroughly, and compress into tablets to make tablets for oral use.

The preparation of embodiment 4 capsules

Take the cicada acetyldopamine oligomer composition prepared in the above-mentioned Example 1, add an appropriate amount of starch as a pharmaceutical adjuvant, mix thoroughly, put into capsules, and make capsules for oral use.

The preparation of embodiment 5 granules

Take the cicada slough acetyldopamine oligomer composition prepared in Example 1 above, add an appropriate amount of starch as a pharmaceutical excipient, mix well, size the granules, and dry them to make granules.

The above embodiments are only to illustrate the technical conception and characteristics of the present invention. All equivalent changes or modifications should fall within the protection scope of the present invention.

Claims (5)

1. The cicada slough-acetyldopamine oligomer composition for treating allergic asthma is characterized by being prepared by the following steps:

(1) Pulverizing periostracum Cicadae, adding ethanol, reflux-extracting under heating, filtering, mixing filtrates, recovering ethanol extractive solution, and concentrating under reduced pressure to obtain periostracum Cicadae extract;

(2) Taking the cicada slough extract obtained in the step (1), and loading the cicada slough extract on a forward silica gel chromatographic column, wherein the volume ratio of the cicada slough extract to the forward silica gel chromatographic column is 20: 1. 15: 1. 12: 1. 10: 1. 8:1, methanol-chloroform gradient elution, collection 20:1 and 15:1, concentrating under reduced pressure to obtain periostracum Cicadae acetyldopamine oligomer composition.

2. The periostracum cicada and acetyldopamine oligomer composition for treating allergic asthma according to claim 1, which is prepared by the following method:

(1) Pulverizing periostracum Cicadae, mixing with 1: adding ethanol with the volume concentration of 95% into the solid-liquid ratio of 10, and carrying out heating reflux extraction for 1-3 times, wherein each time lasts for 1-3 hours; filtering, mixing the filtrates, recovering ethanol extract, concentrating under reduced pressure to obtain periostracum Cicadae extract with relative density of 1.04-1.12;

(2) Taking the cicada slough extract obtained in the step (1), and loading the cicada slough extract on a forward silica gel chromatographic column, wherein the volume ratio of the cicada slough extract to the forward silica gel chromatographic column is 20: 1. 15: 1. 12: 1. 10: 1. 8:1, methanol-chloroform gradient elution at a flow rate of 1.5BV/h, collection 20:1 and 15:1, concentrating under reduced pressure to obtain periostracum Cicadae acetyldopamine oligomer composition.

3. The periostracum cicadae acetyldopamine oligomer composition for treating allergic asthma according to claim 1 or 2, which comprises the following components in weight ratio of 10.7:10.7:3.6:1.0 periostracum Cicadae Acetyldopamine dimer, trimer, tetramer and pentamer compositions.

4. Use of a periostracum cicada acetyldopamine oligomer composition according to any of claims 1-3 for the preparation of a medicament for treating allergic asthma.

5. The use according to claim 4, wherein the periostracum cicada acetyldopamine oligomer composition and a pharmaceutically acceptable carrier are prepared into capsules, tablets, granules, mixture, pills and powder.