阅读说明：本技术 一种光茎栓果菊提取物的制备方法及其应用 (Preparation method and application of coreopsis glabra extract ) 是由 张颖 李嘉 巫凯 杨海船 张赟赟 陈锋 何春欢 于 2021-01-18 设计创作，主要内容包括：本发明公开了一种光茎栓果菊提取物的制备方法及其应用,属于医药技术领域。本发明公开的一种光茎栓果菊提取物在制备治疗肺癌药物中的应用,首次将光茎栓果菊提取物用于制备抗肺癌药物,为中药提取物抗肺癌作用的研究与开发提供了新的思路。(The invention discloses a preparation method and application of a coreopsis glabra extract, and belongs to the technical field of medicines. The application of the echinacea phoma extract in preparing the medicine for treating lung cancer disclosed by the invention is to use the echinacea phoma extract in preparing the anti-lung cancer medicine for the first time, and provide a new thought for the research and development of the anti-lung cancer effect of the traditional Chinese medicine extract.)

1. Application of Echinacea glabra extract in preparing medicine for treating lung cancer is disclosed.

2. The application of the echinacea phoma extract in preparing the medicine for treating the lung cancer according to claim 1, wherein the echinacea phoma extract is prepared by the following method:

(1) drying and crushing the helichrysum medicinal material, adding 80% ethanol according to the ratio of the material to the liquid of 1:8, heating and refluxing for 3 times, and extracting for 2 hours each time; filtering, recovering ethanol from the filtrate until no ethanol smell is produced, and concentrating to obtain ethanol extract I;

(2) adding water into the ethanol extract I to prepare a suspension, extracting with petroleum ether of the same volume, removing the petroleum ether solution, and taking the rest part as an extract II;

(3) extracting the extract II with equal volume of ethyl acetate, recovering ethyl acetate from the extractive solution, and concentrating to obtain extract.

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method and application of a coreopsis glabra extract.

Background

The lung cancer is a malignant tumor with high morbidity at present, and the morbidity and mortality of the lung cancer are on a trend of obviously increasing along with the aggravation of environmental pollution, thereby seriously threatening the life health of people. Currently, the main methods for clinical treatment of lung cancer include surgery, radiotherapy, chemotherapy, immunotherapy, and the like. Although these techniques have contributed greatly to prolonging the life of patients, reducing their suffering, and improving their quality of life, they also have certain limitations. Traditional Chinese medicine has achieved considerable success in treating tumors, especially in developing effective anticancer components in Chinese herbal medicines.

The Calligonum Launea acaulis is a plant of Echinacea of Compositae, also called as taraxacum officinale and Szechwan lovage, and is mainly distributed in Guangxi, Guangdong, Guizhou, Yunnan and the like. The whole herb or root is used as the medicine, and has the efficacies of clearing away heat and toxic material and promoting urination. Can be used for treating carbuncle, cellulitis, furuncle, and urinary tract infection. However, the research on chemical components, pharmacological effects and the like of the Dendranthema phocarpum is still blank, and the basis of medicinal substances is unclear, so that the research cannot guide the clinical application of the Dendranthema phocarpum.

Therefore, it is an urgent need to solve the problems of the art to provide a preparation method and application of a coreopsis glabra extract.

Disclosure of Invention

In view of the above, the invention provides a preparation method and application of a coreopsis glabra extract, and the coreopsis glabra extract is used for preparing a medicament for treating lung cancer, has fewer side effects and better clinical curative effect, and has the characteristics of multiple directions and multiple targets.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of Echinacea glabra extract in preparing medicine for treating lung cancer is disclosed.

Further, the preparation method of the coreopsis glabra extract comprises the following steps:

(1) drying and crushing the helichrysum medicinal material, adding 80% ethanol according to the ratio of the material to the liquid of 1:8, heating and refluxing for 3 times, and extracting for 2 hours each time; filtering, recovering ethanol from the filtrate until no ethanol smell is produced, and concentrating to obtain ethanol extract I;

(2) adding water into the ethanol extract I to prepare a suspension, extracting with petroleum ether of the same volume, removing the petroleum ether solution, and taking the rest part as an extract II;

(3) extracting the extract II with equal volume of ethyl acetate, recovering ethyl acetate from the extractive solution, and concentrating to obtain extract.

According to the technical scheme, compared with the prior art, the invention discloses the preparation method and the application of the coreopsis glabra extract, and lays a foundation for researching and developing a novel high-efficiency low-toxicity medicament for treating lung cancer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the morphological changes of different cells after the action of different concentrations of Echinacea glabra extract according to the present invention; wherein, A: blank control, final DMSO concentration 0.1%; b: positive control drug with final concentration of 1 μm; c: the final concentration of the extract is 5 mug/ml; d: the final concentration of the extract is 10 mug/ml; e: the final concentration of the extract was 20. mu.g/ml.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A preparation method of a coreopsis glabra extract comprises the following specific steps:

drying the helichrysum glabrum medicinal material, crushing, adding 80% ethanol in a mass-volume (kg/L) ratio of material-liquid ratio of 1:8, heating, refluxing and extracting for 3 times (2 h each time), filtering, recovering ethanol from the filtrate until no ethanol smell exists, and concentrating to obtain an ethanol extract I;

adding water into the ethanol extract I to prepare a suspension, extracting with petroleum ether of the same volume, removing the petroleum ether solution, and taking the rest part as an extract II;

extracting the extract II with equal volume of ethyl acetate, recovering ethyl acetate from the extractive solution, and concentrating to obtain extract.

Example 2

(1) MTS method for detecting activity of lung adenocarcinoma A549 cells

Inoculating cells: preparing lung adenocarcinoma A549 cells into single cell suspension by using culture solution (DMEM or RMPI1640) containing 10% fetal calf serum, inoculating 3000-15000 cells per hole to a 96-hole plate, wherein the hole volume is 100 mu l, and the cells are inoculated and cultured 12-24 h in advance.

② adding an extract solution: the extract was dissolved in DMSO and sieved at final concentrations of 100. mu.g/ml, 20. mu.g/ml, 4. mu.g/ml, 0.8. mu.g/ml, 0.16. mu.g/ml, with a final volume of 200. mu.l per well, with 3 wells for each treatment.

③ developing color: after culturing for 48h at 37 ℃, removing culture solution in the adherent cells, and adding 20 mul of MTS solution and 100 mul of culture solution containing 10% fetal calf serum into each well; discarding 100 mul of culture supernatant from the suspension cells, and adding 20 mul of MTS solution into each well; setting 3 blank multiple wells (mixed solution of 20 mul MTS solution and 100 mul culture solution), and continuing incubation for 2-4 h to ensure that the light absorption value is measured after the reaction is fully performed.

And fourthly, color comparison: selecting 492nm wavelength, reading the light absorption value of each hole by a multifunctional microplate reader (MULTISKAN FC), recording the result, drawing a cell growth curve by taking the concentration as an abscissa and the cell survival rate as an ordinate after data processing, and calculating the IC50 value of the extract by using a two-point method (Reed and Muench method).

Positive control compound: two positive compounds of cisplatin (DDP) and paclitaxel (Taxol) are set in each experiment, a cell growth curve is drawn by taking the concentration as the abscissa and the cell survival rate as the ordinate, and the IC50 value of the compound is calculated by using a two-point method (Reed and Muench method).

After 0.16 mu g/ml, 0.8 mu g/ml, 4 mu g/ml, 20 mu g/ml and 100 mu g/ml of the chamomile extract respectively acts on the lung adenocarcinoma A549 cells for 48 hours, the survival rate of the lung adenocarcinoma A549 cells is detected by adopting an MTS method, and the results are shown in Table 1.

TABLE 1 inhibition of A549 cell Activity by Echinacea phoma extracts

The results in Table 1 show that the inhibition of lung adenocarcinoma A549 cell activity by Echinacea phoma extract is concentration-dependent, and that the median lethal concentration (IC50) of Echinacea phoma extract is about 11.75 μ g/ml.

(2) MTS method for detecting activity of normal lung epithelial cell BEAS-2B of human

The MTS method is adopted to detect the inhibition of the activity of the echinacea extract on human normal lung epithelial cells BEAS-2B, and the IC50 value of the extract is calculated. The process is the same as the step (1).

After 0.16. mu.g/ml, 0.8. mu.g/ml, 4. mu.g/ml, 20. mu.g/ml and 100. mu.g/ml of the extract of Dendranthema glabrum was applied to human normal lung epithelial cell BEAS-2B for 48 hours, the survival rate of human normal lung epithelial cell BEAS-2B was examined by MTS method, and the results are shown in Table 2.

TABLE 2 inhibition of human Normal Lung epithelial cell BEAS-2B Activity by Echinacea Pholiota extract

The results in Table 2 show that the concentration of Echinacea phoma extract below 20 μ g/ml had no significant inhibitory effect on human normal lung epithelial cell BEAS-2B activity, and that the median lethal concentration (IC50) of Echinacea phoma extract was about 66.54 μ g/ml.

(3) Detection of cell death patterns by annexin V-PE/7-AAD

(ii) cells in logarithmic growth phase A549 (lung adenocarcinoma cells) were collected at 3 × 10 per well⁵The cells were inoculated into 6-well plates, each having a volume of 2ml, and the cells were cultured in a culture medium containing 10% fetal bovine serum 24 hours in advance.

② adding the extract after 24h, the final concentration of the extract (dissolved by DMSO) is 10 mug/ml, 20 mug/ml and 30 mug/ml; doxorubicin (Doxorubicin) was used as a positive control drug at a final concentration of 1 μm; DMSO was used as a blank control at a final concentration of 0.1%.

③ 24h, collecting cells, centrifuging for 5min at 300g, discarding the supernatant, resuspending with precooled PBS, and centrifuging and washing for 2 times at 300 g.

Mu.l of 1 XBinding Buffer is added to gently resuspend the cells.

Fifthly, adding 5 mul PE Annexin V or 5 mul 7-AAD into the single dyeing tube respectively, adding 5 mul PE Annexin V and 5 mul 7-AAD into the sample tube, mixing evenly, and incubating for 15min at room temperature in a dark place.

Annexin V-PE/7-AAD fluorescence double-staining apoptosis detection kit purchased from BD Pharmingen;

sixthly, performing flow cytometry detection within 1 hour, adjusting the voltage of the FSC, the SSC and the fluorescence channel by using a blank tube during detection, adjusting the compensation of the fluorescence channel by using a single staining tube under the voltage condition, and sequentially collecting data of an experimental group. The results are shown in FIG. 1.

The A549 cells are pretreated by the extracts of the callicarpa pholiota of 5 mu g/ml, 10 mu g/ml and 20 mu g/ml respectively, and then are stained by Annexin V-PE/7-AAD double staining method, and finally the flow cytometry result is as follows: the number of viable cells, i.e., PE Annexin V negative/7-AAD negative, is significantly reduced (P < 0.001); apoptotic cells (PE Annexin V positive/7-AAD negative) did not show significant changes (P ═ 0.1780); however, the number of necrotic cells showing PE Annexin V positive/7-AAD positive and PE Annexin V negative/7-AAD positive was significantly increased compared to the positive control (P < 0.001).

From the data analysis of the control group, the proportion of mechanical damage was almost zero, so it was concluded that the chamomile extract had the effect of causing necrosis of lung cancer cells a 549. Echinacea pholidoides extract induced a549 cell death by necrosis.

In conclusion, when the concentration of the inula phoma extract is 20 mug/mL, the inula phoma extract can cause the lung adenocarcinoma cells A549 to be necrotic by about 71.0 percent compared with a positive control group, and has little influence on the activity of the human normal lung epithelial cells BEAS-2B. It was concluded that the echinacea phoma extract induced death of a549 cells in a necrotic manner.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.