CN114224929A

CN114224929A - Erigeron multiradiatus extract and preparation method and application thereof

Info

Publication number: CN114224929A
Application number: CN202111502269.6A
Authority: CN
Inventors: 张志锋; 李丽; 苏学燕
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2021-12-10
Filing date: 2021-12-10
Publication date: 2022-03-25
Anticipated expiration: 2041-12-10
Also published as: LU501349B1; CN114224929B

Abstract

The invention provides a preparation method of a multiradiate fleabane extract, a UPLC (ultra performance liquid chromatography) multi-component simultaneous detection and quantification method for the multiradiate fleabane extract based on the method, and new medical application. The detection method can realize the identification and accurate detection of more than 8 reference substances, and meanwhile, the research of the invention also discovers the pharmacological activity of the erigeron multiradiatus extractive in resisting the injury of vascular endothelial cells and provides a new scheme of natural plant sources for the treatment of pulmonary embolism.

Description

Erigeron multiradiatus extract and preparation method and application thereof

Technical Field

The invention relates to the fields of national medicines, quality detection, medical application and the like.

Background

Erigeron multiradiatus (academic name) is a plant of Erigeron genus of Compositae family. Is distributed in Afghanistan, Nipol, India and Tibet, Sichuan and Yunnan of China, grows in the area with the altitude of 2,500-4,600 m, mostly grows in hillside, subalpine, high mountain grassland and forest edge, and the whole grass can relieve exterior syndrome, dispel cold and help digestion.

At present, researches show that the erigeron multiradiatus contains various flavonoid and caffeic acid ester compounds, is mainly used for treating traumatic injury, rheumatic pain, toothache, stomachache, cold and other symptoms, has the effects of relieving exterior syndrome, dispelling cold, diminishing inflammation, relieving pain, promoting blood circulation, removing blood stasis and the like, and has the activity of resisting myocardial ischemia.

In the '2015 version of the processing standard of Sichuan Chinese medicines', a quality control method of erigeron multiradiatus is disclosed, which uses high performance liquid chromatography, uses C18 as a stationary phase, and uses methanol-0.2% phosphoric acid 45:55 for isocratic elution, the detection wavelength is 335nm, the erigeron is mainly detected, and the content of the erigeron breviscapus is required to be not less than 0.5%.

Disclosure of Invention

The invention aims to provide a method for better detecting the quality of a multiradiate flea and more indications.

The invention actually provides a preparation method of the erigeron multiradiatus extractive, wherein the erigeron multiradiatus is extracted by 60-80% ethanol, the ethanol is recovered from the extracting solution, the obtained product is loaded on a nonpolar or low-polarity macroporous adsorption resin, the obtained product is sequentially eluted by 10-20% ethanol and 60-80% ethanol, and the 60-80% ethanol eluent is collected, concentrated and dried to obtain the erigeron multiradiatus extractive.

Wherein the concentration of ethanol used for extraction is 70%; eluting with 15% and 70% ethanol sequentially.

In the invention, the ethanol concentration is measured in V/V.

Wherein the nonpolar macroporous adsorption resin is selected from HPD-100, HPD-300, D-101, X-5 or H103; the weak polar macroporous adsorbent resin is selected from AB-8, DA-201, and HPD-400.

In order to better perform quality detection on multiradiate fleabane, the invention hopes to identify and detect more components, the inventor examines chromatographic conditions in the experimental process, but finds that the chromatographic detection result is significantly influenced by mobile phase elution gradient, extraction conditions of a test sample and the like, and the detection on more than 8 components can be realized by using specific gradient conditions and extraction conditions of the test sample:

in the preliminary experiment of the invention, a plurality of mobile phase gradient conditions are used successively, but most conditions can not effectively separate and detect chromatographic peaks, such as:

gradient condition 1: gradient elution with acetonitrile (a) -0.2% aqueous formic acid (B): 0-10 min, 5% -18% A; 10-12 min, 18% -19% A; 12-17 min, 19% A; 17-19 min, 19% -20% A; 19-21 min, 20% -55% A; 21-26 min, 55-70% A.

Gradient condition 2: gradient elution with acetonitrile (a) -0.2% aqueous formic acid (B): 0-10 min, 5% -18% A; 10-12 min, 18% -19% A; 12-19 min, 19% A; 19-21 min, 19% -20% A; 21-23 min, 20% -22% A; 23-30 min, 22% -60% A.

Gradient condition 3: gradient elution with acetonitrile (a) -0.2% aqueous formic acid (B): 0-5 min, 10% -12% A; 5-19 min, 12% -16% A; 19-23 min, 16% -21% A; 23-28 min, 21% -23% A; 28-29 min, 23% -24% A; 29-30 min, 24% A.

Gradient condition 4: gradient elution with acetonitrile (a) -0.2% aqueous formic acid (B): 0-5 min, 10% -16% A; 5-18 min, 16% -18% A; 18-26 min, 18-24% of A.

According to the chromatogram under the conditions, only the chromatogram under the condition 4 has good separation effect and small peak-shaped tailing, and more components are easy to identify and accurately detect.

In addition, the invention also comprehensively compares different extraction methods (ultrasonic and heating reflux), extraction solvents (ethanol, methanol and 70% methanol), different extraction times (1 time and 2 times) and different extraction time (15min, 30min and 45min), so that the sample extracted by 70% methanol for 30min through ultrasonic is more in components and relatively higher in content of each component. The final preparation method of the selected test sample comprises precisely weighing 0.1g crude extract of erigeron multiradiatus, placing in 50mL conical flask, adding 10mL 70% methanol solution, shaking, ultrasonic extracting for 30min, filtering, and filtering the filtrate with 0.22 μm microporous membrane to obtain test sample solution. The present invention can compare the effect of different extraction solvents on the chromatographic assay results by means of FIGS. 5-7.

According to a large number of screening experiments, the invention finally provides a method for detecting multiradiate fleabane, which comprises the following steps:

taking multiradiate fleabane, preparing the multiradiate fleabane extract by adopting the method, and detecting by adopting a UPLC method, wherein the chromatographic conditions are as follows:

stationary phase: c18

Mobile phase is gradient eluted by acetonitrile A-0.1-5% acid water solution B: 0-5 min, 10% -16% A; 5-18 min, 16% -18% A; 18-26 min, 18-24% of A.

In the present invention, the type of the column used for UPLC is selected from 2.1X 100mm and 1.8 μm.

Wherein the concentration of the aqueous acid solution is selected from 0.1-0.3%.

Wherein the acid aqueous solution is selected from aqueous solutions of formic acid, acetic acid and phosphoric acid.

The UPLC further comprises one or more of the following conditions:

(1) the flow rate is 0.1-0.3 ml/min;

(2) the column temperature is 30-40 ℃;

(3) the detection wavelength is 320-330 nm.

In the invention, one or more of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, scutellarin and isochlorogenic acid C are used as reference substances.

The detection method can realize simultaneous identification and content detection of the 8 chemical components.

The invention also provides application of the erigeron multiradiatus extractive prepared by the method in preparing a product for resisting vascular endothelial cell injury.

The invention also provides application of the erigeron multiradiatus extractive prepared by the method in preparing a product for preventing or treating pulmonary embolism.

Pulmonary embolism refers to the pathological victory clinical syndrome of pulmonary circulatory disturbance caused by endogenous or exogenous emboli reaching the lung through systemic circulation to block the pulmonary artery or branches thereof. Liu Zhong jin et al show that vascular endothelial cell injury is the first element to trigger pulmonary embolism, and repair of vascular endothelial cell injury is helpful for treating pulmonary embolism, and drugs with new action targets can be found through endothelial injury mechanism, repair mechanism and the like, and have irreplaceable effects in treating pulmonary embolism (Liu Zhong Cheng jin, electronic journal of Chinese Severe medicine, 8 months 2017, Vol3, No. 3). According to the invention, through a zebra fish vascular endothelial cell injury resisting model, the erigeron multiradiatus extractive has a good effect of inhibiting vascular endothelial cell injury, so that a scheme with a new natural source can be provided for treating pulmonary embolism.

Drawings

FIG. 1 chromatogram of gradient Condition 1

FIG. 2 chromatogram of gradient Condition 2

FIG. 3 chromatogram of Condition 3

FIG. 4 chromatogram of Condition 4

FIG. 5 chromatogram of methanol extraction

FIG. 6 chromatogram of ethanol extraction

FIG. 770 chromatogram of methanol extraction

FIG. 8 UPLC chromatograms of a mixed control solution (A) and a multiradiate fleabane test solution (B), 1-neochlorogenic acid; 2-chlorogenic acid; 3-cryptochlorogenic acid; 4-scutellarin; 5-isochlorogenic acid B; 6-isochlorogenic acid A; 7-breviscapine; 8-Isochlorogenic acid C

FIG. 9 is a typical graph of the staining intensity of red blood cells of zebra fish treated with the sample (anti-vascular endothelial cell damage), wherein A is a normal control group B, a model control group C, 50.0. mu.g/mL aspirin enteric-coated tablets, 50.0. mu.g/mL erigeron multiradiatus, 250. mu.g/mL erigeron multiradiatus, F, 1000. mu.g/mL erigeron multiradiatus (yellow frame line is heart of analysis site)

FIG. 10 intensity of cardiac red blood cell staining (anti-vascular endothelial cell damage) of zebrafish after sample treatment, compared to model control group,. about.p <0.001

Detailed Description

Example 1

Weighing 300g of erigeron multiradiatus, adding 1000ml of 70% ethanol, heating, refluxing and extracting for 3 times, each time for 1h, and combining filtrates. Concentrating the filtrate under reduced pressure to recover ethanol, wherein the concentration is 1g/ml of crude drug. Dissolving the concentrated solution with 2 times of distilled water, adding onto treated D101 macroporous adsorbent resin column, eluting with 15% ethanol to remove pigment, discarding, eluting with 70% ethanol, collecting eluate, concentrating, and freeze drying to obtain 14.84g of herba Erigerontis crude extract.

Example 2

Instrument and reagent

Waters

A CLASS ultra-high performance liquid chromatograph,

chromatography workstations (Waters corporation); ACQUITY

HSS C18 Column (2.1X 100mm Column, 1.8 μm) (Waters Corp.); a METTLER TOLEPO ME55 electronic balance, a METTLER TOLEPO ME104 electronic balance (mettess-toledo instruments (shanghai) ltd); KQ-300DE type numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); HWS-12 model electric heating constant temperature water bath (Shanghai-Hengscientific instruments Co., Ltd.); FD-1A-50 Freeze dryer (Beijing Bo Yi kang laboratory instruments Co., Ltd.).

Chlorogenic acid (batch No. 2101082), chlorogenic acid (batch No. 21040904), scutellarin (batch No. 21050603), and breviscapine (batch No. 21033102) were obtained from VICKQI Biotech, Inc., of Sichuan province; cryptochlorogenic acid (batch No. 20052601), isochlorogenic acid B (batch No. 20032602) were purchased from Dolpheder Biotech Ltd; isochlorogenic acid A (batch No. 15100702) and isochlorogenic acid C (batch No. 15100816) were obtained from Kyoho Biotech, Inc.; drochen water (guangzhou drochen food and beverage limited); chromatographic grade acetonitrile (Beijing Dike Mackyo Co., Ltd.); chromatographic grade formic acid (Tianjin, Kemiou Chemicals, Inc.).

Extraction of crude extract of erigeron multiradiatus

Chromatographic conditions

Gradient elution with acetonitrile (a) -0.2% aqueous formic acid (B): 0-5 min, 10% -16% A; 5-18 min, 16% -18% A; 18-26 min, 18% -24% A; the flow rate is 0.2 ml/min; the column temperature is 35 ℃; the sample injection volume is 1 mu L; the detection wavelength was 325 nm.

Preparation of control solutions

Respectively and precisely weighing appropriate amount of chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, breviscapine and isochlorogenic acid C, adding methanol, and ultrasonically dissolving to obtain mixed reference stock solutions with mass concentrations of 0.098mg/mL, 0.109 mg/mL, 0.101mg/mL, 1.090mg/mL, 0.104mg/mL, 0.102mg/mL, 0.520mg/mL and 0.103 mg/mL.

Preparation of test solution

Precisely weighing 0.1g of crude extract of erigeron multiradiatus, placing in a 50mL conical flask, adding 10mL of 70% methanol solution, shaking, ultrasonically extracting for 30min, filtering, and filtering the filtrate with 0.22 μm microporous membrane to obtain the sample solution, wherein the chromatogram of the sample is shown in FIG. 8.

Methodology investigation

Taking the mixed reference solution of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, scutellarin and isochlorogenic acid C through a 0.22 mu m organic microporous filter membrane according to the linear relation, respectively injecting samples of 0.1, 0.2, 0.3, 0.5 and 1 mu L according to the chromatographic conditions, and recording the peak area of each reference. The results of linear regression analysis using the chromatographic peak area as ordinate (y) and the mass concentration as abscissa (x) are shown in Table 1, and the linear relationship among the 8 components in the corresponding ranges is good.

Table 18 linear regression equation, linear range, correlation coefficient of index components

The precision test takes the mixed reference solution, samples are continuously injected for 6 times according to the chromatographic conditions, peak area values of chromatographic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, scutellarin A and isochlorogenic acid C are measured, and the RSD value is calculated. As a result, the RSDs were 0.87%, 0.78%, 0.98%, 0.63%, 0.77%, 0.90%, 0.96%, and 0.85%, respectively, indicating good precision of the instrument.

Repeatability test 6 parts of multiradiate fleabane sample, each part is about 0.1g, the sample is processed according to the preparation method of the sample solution, and sample injection is carried out according to the chromatographic condition to determine the RSD value of each peak area of chromatographic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, breviscapine and isochlorogenic acid C. Results RSDs were 0.91%, 1.04%, 0.98%, 0.86%, 1.06%, 0.93%, 1.07%, 0.92%, respectively, indicating good reproducibility of the method.

Stability test sample solution is taken, sample injection is carried out for 0, 2, 4, 8, 12 and 24 hours after preparation according to chromatographic conditions, peak area values of chromatographic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, breviscapine and isochlorogenic acid C are measured, and RSD of the sample solution is calculated. Results RSDs were 0.78%, 1.01%, 1.04%, 0.93%, 0.98%, 1.20%, 1.12%, 1.17%, respectively, indicating good stability of the test solution over 24 hours.

Sample adding recovery rate test 6 portions of multiradiate fleabane sample with known content are precisely weighed, each portion is 0.05g, a proper amount of reference solution is respectively added, a sample solution to be tested is prepared according to the preparation method of the sample solution, sample injection is carried out according to chromatographic conditions, peak areas of chromatographic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, breviscapine and isochlorogenic acid C are measured, and the sample adding recovery rate and the RSD value of each measured component are calculated, and the results are shown in Table 2.

Table 2 sample recovery rate measurement results (n ═ 6)

Sample assay

Accurately weighing 0.1g of crude extract of erigeron multiradiatus, pretreating according to the preparation of a test solution to prepare the test solution, injecting sample according to chromatographic conditions, and calculating the content of each component in the sample according to a regression equation, wherein the result is shown in Table 3.

Table 3 multiradiate fleabane content measurement results (n ═ 3)

Example 3

1 detection Material

1.1 sample preparation information

And (3) testing the sample: the erigeron multiradiatus extract prepared in example 1 was prepared into a 2.00mg/mL stock solution with standard dilution water and used as it is.

Positive control: aspirin enteric-coated tablets, white tablets, lot number BJ54728, bayer pharmaceutical health limited, stored in the shade and in the dark. Prepared into 50.0mg/mL stock solution with DMSO and stored at-20 ℃.

1.2 Experimental animals

The zebra fish are all raised in water for fish culture at 28 ℃ (water quality: 200mg of instant sea salt is added in per 1L of reverse osmosis water, the conductivity is 450-550 mu S/cm, the pH is 6.5-8.5, and the hardness is 50-100 mg/L CaCO₃) The license number for experimental animals is as follows: SYXK (Zhe) 2012-0171. The feeding management meets the requirements of the international AAALAC certification (certification number: 001458).

Melanin allele mutant translucent Albino strain zebrafish, in a natural pairwise mating breeding mode. Zebrafish aged 5 days after fertilization (5dpf) were used for evaluation of the efficacy of erigeron multiradiatus for vascular endothelial cell damage.

1.3 instruments, consumables and reagents

Dissecting microscopes (SZX7, OLYMPUS, Japan); a CCD camera (VertA1, shanghai geosson vision science and technology ltd, China); precision electronic balances (CP214, OHAUS, USA); 6-well plates (Nest Biotech, China).

Dimethyl sulfoxide (DMSO, lot No. BCCD8942, Sigma, Switzerland); arachidonic acid (batch No. C2123090, shanghai alatin biochem technologies ltd., China); o-dianisidine (batch No. MKCC7501, Sigma, USA); phenylhydrazine (batch No. YH0170509, ashore ihh biotechnology limited, China); ponatinib (batch No. 13771, MedChemExpress, USA).

2. Efficacy evaluation method

Randomly selecting 5dpf melanin allele mutant type translucent Albino strain zebra fish in a 6-well plate, and treating 30 zebra fish in each well. Dissolving in water respectively, and administering different concentrations of erigeron multiradiatus extract, wherein the positive control is aspirin enteric-coated tablet. A normal control group and a model control group were set at the same time, and the volume per well was 3 mL. After treatment for 3h, 6h and 21h at 28 ℃, the other experimental groups except the normal control group are respectively dissolved in water and are given with ponatinib to establish a zebra fish vascular endothelial cell injury model. And after the treatment is carried out for 90min, 18h and 21h at the temperature of 28 ℃, dyeing o-dianisidine, randomly selecting 10 zebra fish from each experimental group after the dyeing is finished, placing the zebra fish under a dissecting microscope for photographing, collecting data by NIS-Elements D3.20 advanced image processing software, analyzing the dyeing intensity of the heart of the zebra fish, and evaluating the effect of the sample on resisting vascular endothelial cell damage according to the statistical significance of the index. Statistical analysis was performed with SPSS software and p <0.05 indicated significant differences.

3 results of detection

3.1 anti-vascular endothelial cell injury MTC

Under the experimental condition, the MTC of the multiradiate fleabane on the model zebra fish is 1000 mug/mL. The results are shown in Table 4.

TABLE 4 concentration of the efficacy of multiradiate fleabane on vascular endothelial cell injury (n ═ 30)

3.2 evaluation of efficacy against vascular endothelial cell injury

Under the experimental conditions, the erigeron multiradiatus has the efficacy of resisting the injury of vascular endothelial cells, and the results are shown in table 5, figure 9 and figure 10.

Table 5 evaluation of efficacy of samples against vascular endothelial cell injury (n ═ 10)

P <0.05, p <0.001 compared to model controls.

Claims

1. The preparation method of the erigeron multiradiatus extractive comprises the following steps: extracting erigeron multiradiatus with 60-80% ethanol, recovering ethanol from the extractive solution, loading onto nonpolar or low-polarity macroporous adsorbent resin, sequentially eluting with 10-20% ethanol and 60-80% ethanol, collecting 60-80% ethanol eluate, concentrating, and drying to obtain erigeron multiradiatus extract.

2. The method of claim 1, wherein: the concentration of ethanol used for extraction is 70%; eluting with 15% and 70% ethanol sequentially.

3. The method of claim 1, wherein: the nonpolar macroporous adsorption resin is selected from HPD-100, HPD-300, D-101, X-5 or H103; the weak polar macroporous adsorbent resin is selected from AB-8, DA-201 or HPD-400.

4. The method for detecting the multiradiate fleabane is characterized by comprising the following steps: taking erigeron multiradiatus, preparing an erigeron multiradiatus extract by using the method of any one of claims 1 to 3, and detecting by using a UPLC method, wherein the chromatographic conditions are as follows:

stationary phase: c18

Mobile phase is gradient eluted by acetonitrile A and 0.1-5% acid water solution B: 0-5 min, 10% -16% A; 5-18 min, 16% -18% A; 18-26 min, 18% -24% A;

the preparation method of the test sample comprises the following steps: extracting herba Erigerontis extract with 60-80% methanol.

5. The detection method according to claim 4, characterized in that: in the preparation method of the test sample, 70% methanol is adopted for ultrasonic extraction for 30 min.

6. The detection method according to claim 4 or 5, characterized in that: the acid aqueous solution is selected from aqueous solutions of formic acid, acetic acid, phosphoric acid.

7. The detection method according to claim 4, characterized in that: in the UPLC, one or more of the following conditions are also included:

(1) the flow rate is 0.1-0.3 ml/min;

(2) the column temperature is 30-40 ℃;

(3) the detection wavelength is 320-330 nm.

8. The detection method according to claim 4, characterized in that: one or more of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, scutellarin, isochlorogenic acid B, isochlorogenic acid A, scutellarin A, and isochlorogenic acid C are used as reference.

9. Use of an erigeron multiradiatus extract prepared by the method of any one of claims 1-3 for the preparation of a product for combating vascular endothelial cell damage.

10. Use of an erigeron multiradiatus extract prepared by the process of any one of claims 1 to 3 for the preparation of a product for the prevention or treatment of pulmonary embolism.