阅读说明：本技术 西番莲提取物在制备防治乳腺癌药物中的应用 (Application of passion flower extract in preparation of medicine for preventing and treating breast cancer ) 是由 马伏宁 宋顺 黄东梅 许奕 吴斌 邢文婷 徐兵强 郭刚 王达新 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种西番莲藤叶提取物在制备防治乳腺癌药物中的应用,所述西番莲藤叶提取物中的活性成分包含异荭草素、牡荆素和异牡荆素黄酮。本发明还公开了所述西番莲藤叶提取物的提取方法。本申请所述西番莲藤叶提取物能抑制乳腺癌细胞的增殖,能抑制乳腺癌细胞的迁移,能抑制乳腺癌细胞周期；可以用于制备乳腺癌预防、治疗的药物,以及用于其他抗癌药物。(The invention discloses an application of passion flower vine leaf extract in preparing a medicine for preventing and treating breast cancer. The invention also discloses an extraction method of the passion flower vine leaf extract. The passion flower vine leaf extract can inhibit proliferation of breast cancer cells, migration of the breast cancer cells and the breast cancer cell cycle; can be used for preparing medicine for preventing and treating breast cancer and other anticancer medicines.)

1. Application of Passiflora edulis leaf extract in preparing medicine for preventing and treating breast cancer is provided.

2. The use of the passion flower vine leaf extract of claim 1 in the preparation of a medicament for the prevention and treatment of breast cancer, wherein: the passion flower vine leaf extract is applied to preparation of a medicine for inhibiting breast cancer cell proliferation.

3. The use of the passion flower vine leaf extract of claim 1 in the preparation of a medicament for the prevention and treatment of breast cancer, wherein: the passion flower vine leaf extract is applied to preparation of a medicine for inhibiting breast cancer cell migration.

4. The use of the passion flower vine leaf extract of claim 1 in the preparation of a medicament for the prevention and treatment of breast cancer, wherein: the passion flower vine leaf extract is applied to the preparation of a medicine for inhibiting the cell cycle process of breast cancer.

5. The use of the passion flower vine leaf extract of claim 1 in the preparation of a medicament for the prevention and treatment of breast cancer, wherein: the passion flower is passion fruit passion flower.

6. The use of the passion flower vine leaf extract of claim 1 in the preparation of a medicament for the prevention and treatment of breast cancer, wherein: the active ingredients in the passiflora edulis leaf extract comprise isoorientin, vitexin and isovitexin flavone.

7. The use of passion flower vine leaf extract according to claim 2 in the preparation of a medicament for the prevention and treatment of breast cancer, wherein: IC of passionflower vine leaf extract for inhibiting breast cancer cell proliferation₅₀It was 222.47. mu.g/mL.

8. The use of passion flower vine leaf extract according to claim 3 in the preparation of a medicament for the prevention and treatment of breast cancer, wherein: the concentration of the passion flower vine leaf extract for inhibiting the migration of breast cancer cells is 100-.

9. The method for extracting the passion flower vine leaf extract applied to the medicine for preventing and treating breast cancer as claimed in any one of claims 1 to 8, is characterized in that: the method sequentially comprises the following steps:

(1) collecting Passiflora Caerulea leaf, naturally drying or oven drying at 35-45 deg.C, pulverizing, and sieving with 30-60 mesh sieve to obtain Passiflora Caerulea leaf powder;

(2) soaking passion flower vine leaf powder in ethanol with the mass concentration of 70% -80% for 2-5 days, then performing ultrasonic-assisted extraction, filtering an extracting solution, then performing reduced pressure evaporation to obtain a concentrated extract, and freeze-drying the extract to obtain extract dry powder;

(3) dissolving the dry extract powder into a dry extract powder solution by using water, adding an extracting agent petroleum ether or ethyl acetate for extraction, and performing reduced pressure evaporation on the extract liquid to obtain a passionflower leaf extract; the volume of the extractant is 2-5 times of the extract dry powder solution.

10. The method for extracting the passion flower vine leaf extract applied to the medicine for preventing and treating breast cancer as claimed in claim 9, is characterized in that: the volume ratio of the ethanol to the passion flower vine leaf powder in the step (2) is 5: 1; the ultrasonic-assisted extraction conditions are that the ultrasonic power is 50-200W, the ultrasonic temperature is 20-30 ℃, and the ultrasonic extraction is carried out for 3 times, 30min each time; the temperature of the freeze drying is-50 ℃ to-30 ℃; the reduced pressure evaporation conditions in the steps (2) and (3) are that the vacuum degree is 30-50kPa, and the temperature is 35-45 ℃.

Technical Field

The application relates to the field of plant extract application, in particular to application of passion flower extract in preparation of a medicine for preventing and treating breast cancer.

Background

Passiflora edulis (Passiflora edulis) is also known as "passion fruit" and belongs to the order Tiger (Malpighiales) and family Passiflorae (Passiflorae) plants of the genus Passiflora. Passiflora edulis is native to tropical and subtropical regions in south America and Brazil, and vine leaves have both shading and medicinal functions and are introduced and cultivated all over the world due to beautiful flower colors. There are more than about 500 passionflower plants and about 50 edible plants. In China, there are more than 20 kinds of passionflower plants, which are mainly distributed in Guangdong, Guangxi, Fujian, Guizhou, Yunnan, Hainan and the like. Fruits of passion fruit (p. edulis) can be used for making jam, fruit juice, red wine and the like, are important commercial crops in brazil, ecuador and other countries, and tender leaves of the fruits can be brewed and drunk as tea or eaten as vegetables. The roots, fruits and leaves of passion flower can be used as medicines in Chinese medicine records, and the passion flower has the effects of astringing, stopping diarrhea, quenching thirst, promoting the production of body fluid, promoting the circulation of qi and activating blood circulation in the records of common Chinese herbal medicine color maps. It is indicated for poor body fluid, dyspepsia and traumatic injuries. "pharmaceutically active. Domestic and foreign researches show that passion flower plants contain flavone, phenolic acid, cyclic peptide and triterpenes, and have good pharmacological activity in the aspects of oxidation resistance, virus resistance, depression resistance and the like.

Breast cancer is the most common cancer in women in the world today, and about 50 million women die of breast cancer each year worldwide. MDA-MB-231 is triple negative breast cancer, is a malignant breast cancer cell, accounts for about 15% of breast cancer patients, and has the characteristics of rapid metastasis, high invasiveness, early local recurrence, poor prognosis and the like. Chemotherapy is the only effective drug treatment means for the patients at present, and new treatment drugs and treatment methods are urgently needed to be found. The research of active drugs for treating tumors from plants is always the direction of research of global medicine researchers, and paclitaxel (paclitaxel) from plants is a widely used drug for treating breast cancer and ovarian cancer, but has the defects of low water solubility, large adverse reaction and the like.

The application discovers that the activity of the ethanol extract of the vine leaves of passion fruit (P.edulis) is screened as follows: the extract of the vine leaves of passion fruit (P.edulis) has the activity of inhibiting the proliferation of breast cancer cells, and further research shows that the extract of the vine leaves of passion fruit (P.edulis) can effectively inhibit the migration and invasion of tumor cells; the composition analysis shows that the composition contains flavonoid active ingredients such as isoorientin, vitexin, etc.

Disclosure of Invention

Therefore, the application provides the application of the passion flower extract in preparing the medicine for preventing and treating the breast cancer so as to develop a novel natural product with the activity of inhibiting the proliferation of the breast cancer.

The technical scheme of the application is realized as follows:

application of Passiflora edulis leaf extract in preparing medicine for preventing and treating breast cancer is provided.

The further technical scheme is that the passion flower vine leaf extract is applied to preparation of a medicine for inhibiting breast cancer cell proliferation.

The further technical scheme is that the passion flower vine leaf extract is applied to preparation of a medicine for inhibiting breast cancer cell migration.

The further technical scheme is that the passion flower vine leaf extract is applied to preparation of a medicine for inhibiting the cell cycle process of breast cancer.

The further technical scheme is that the passion flower is passion fruit.

The further technical scheme is that the active ingredients in the passion flower vine leaf extract comprise isoorientin, vitexin and isovitexin flavone.

According to a further technical scheme, the western medicine isIC for inhibiting breast cancer cell proliferation by using senna leaf extract₅₀It was 222.47. mu.g/mL.

The further technical proposal is that the concentration of the passion flower vine leaf extract for inhibiting the migration of the breast cancer cells is 100-2000 mug/mL.

The extraction method of the passion flower vine leaf extract applied to the medicine for preventing and treating breast cancer sequentially comprises the following steps:

(1) collecting Passiflora Caerulea leaf, naturally drying or oven drying at 35-45 deg.C, pulverizing, and sieving with 30-60 mesh sieve to obtain Passiflora Caerulea leaf powder;

(2) soaking passion flower vine leaf powder in 70-80 wt% ethanol at room temperature (20-35 deg.c) for 2-5 days, ultrasonic extracting, filtering the extracted liquid, decompression evaporating to obtain concentrated extract, and freeze drying the extract to obtain dry extract powder;

(3) dissolving the dry extract powder into a dry extract powder solution by using water, adding an extracting agent petroleum ether or ethyl acetate for extraction, and performing reduced pressure evaporation on the extract liquid to obtain a passionflower leaf extract; the volume of the extractant is 2-5 times of the extract dry powder solution.

The further technical scheme is that the volume ratio of the ethanol to the passion flower vine leaf powder in the step (2) is 5: 1; the ultrasonic-assisted extraction conditions are that the ultrasonic power is 50-200W, the ultrasonic temperature is 20-30 ℃, and the ultrasonic extraction is carried out for 3 times, 30min each time; the temperature of the freeze drying is-50 ℃ to-30 ℃; the reduced pressure evaporation conditions in the steps (2) and (3) are that the vacuum degree is 30-50kPa, and the temperature is 35-45 ℃.

Compared with the prior art, the beneficial effects of this application are:

(1) the passion flower vine leaf extract can inhibit proliferation of breast cancer cells, migration of the breast cancer cells and the breast cancer cell cycle.

(2) The passion flower vine leaf extract has good anti-breast cancer activity and has potential application value in the aspect of breast cancer treatment.

(3) The passion flower vine leaf extract can be used for preventing and treating breast cancer and other anti-cancer drugs.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only preferred embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 shows the effect of ethanol extract of passion flower (P. edulis) vine leaves on MDA-MB-231 (human breast cancer cell, the same below) cell proliferation activity.

FIG. 2 is a graph showing the effect of different extractions of the ethanol extract of passion flower (P. edulis) vine leaves on the proliferation activity of MDA-MB-231 cells.

FIG. 3 is a graph showing the effect of passion flower (P. edulis) vine leaf extract on MDA-MB-231 cell migration.

FIG. 4 is a graph showing the effect of passion flower (P. edulis) vine leaf extract on MDA-MB-231 cell cycle.

FIG. 5 is UHPLC-MS/MS analysis total ion flow diagram of passion flower (P.edulis) vine leaf extract of the invention.

Fig. 6 is a mass spectrum of the active ingredients isoorientin, vitexin and isovitexin of the passion flower (p. edulis) vine leaf extract of the present invention.

Detailed Description

For clear and complete description of the technical solutions in the present invention, it is obvious that the inventor combines the embodiments to describe, but the following embodiments describe only some embodiments of the present invention, not all 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 extraction of active ingredients from Passiflora Caerulea leaves

Experimental materials: the vine leaves of passion fruit, passion flower (p. edulis), were collected from chengmai, haikou, hainan province.

Drying collected vine leaves in an oven at 40 ℃, crushing, sieving with a 40-mesh sieve, and mixing according to the weight ratio of 1: soaking in 80% ethanol at volume ratio of 5, and ultrasonic extracting for 30min for 3 times. Concentrating the extracting solution by reduced pressure rotary evaporation to obtain extract, and freeze-drying the extract to obtain dry extract powder; dissolving the dry extract powder into water to obtain a dry extract powder solution, adding an extracting agent petroleum ether for extraction, and performing reduced pressure evaporation on the extract liquid to obtain the passionflower leaf extract.

The volume of the extractant is 2 times of the volume of the dry extract powder solution. The volume ratio of the ethanol to the passion flower vine leaf powder is 5: 1; the ultrasonic-assisted extraction conditions are that the ultrasonic power is 100W, the ultrasonic temperature is 5 ℃, and the ultrasonic extraction is carried out for 3 times, each time for 30 min; the temperature of the freeze drying is-30 ℃; the reduced pressure evaporation conditions are vacuum degree of 30kPa and temperature of 40 ℃.

EXAMPLE 2 CCK-8 assay for inhibition of Breast cancer cell proliferation Activity

The passion flower vine leaf extract of example 1 was dissolved in DMSO solvent to prepare solutions of passion flower vine leaf extract at various concentrations (31, 62.5, 125, 250, 500. mu.g/mL) for cell viability testing, and DMSO content in the medium was less than 0.1%.

About 4000 MDA-MB-231 cells per well were plated in 96-well plates, and after the cells adhered, DMEM medium (containing 0.25% HEPES) containing passion flower vine leaf extract at different concentrations as described above was added, and 0.1% DMSO medium was used as a negative control. After 72h of treatment, CCK-8 was added and CO was added₂After 1h in the incubator, the absorbance at 405nm was read by a microplate reader, and the cell viability was (a blank group-a treatment group)/a blank group × 100%.

The experimental result shows that the ethanol extract of passion flower vine leaves has the activity of inhibiting MDA-MB-231 breast cancer cell proliferation and IC (Integrated Circuit) for inhibiting breast cancer cell proliferation₅₀222.47. mu.g/mL (MDA-MB-231) (FIG. 1).

After the ethanol extract of passion flower vine leaves is sequentially extracted by petroleum ether, ethyl acetate and n-butanol, the proliferation inhibition activity on breast cancer cells shows that the cells are treated for 72 hours under the concentration of 200 mu g/mL, the activity of the ethyl acetate extract phase is better, and then the petroleum ether extract phase is used (figure 2).

Example 3 assay of the Scoring method for inhibiting migration Activity of Breast cancer cells

24-well plate 2X 10 per well⁵Inoculating MDA-MB-231 cells in a logarithmic growth phase in each cell quantity, scratching the cells by using a gun head after the cells adhere to the wall 24 hours, washing floating cells by using PBS, dissolving the passion flower vine leaf extract in the embodiment 1 by using a DMSO solvent to prepare passion flower vine leaf extract extracts (25, 50, 100, 200 and 400 mu g/mL) with different concentrations, staining the cells by using crystal violet after 24 hours, photographing under a microscope, counting the change of scratching areas before and after treatment, and calculating the migration inhibition rate.

The experimental results show that: different concentrations of passion flower vine leaf extract all had the activity of inhibiting MDA-MB-231 cell migration, with the average inhibition of 96.14% for the 400 μ g/mL treatment compared to the control (FIG. 3).

Example 4 flow cytometry Effect on cell cycle

Inoculation of 5X 10 wells with 6-well plates per well⁵MDA-MB-231 cells in logarithmic growth phase are cultured for 24h until the cells are attached to the wall, a blank control group and an extract treatment group are respectively arranged, 0.1% DMSO is added into the blank group, 200 mug/mL passion fruit extract is added into the extract group, the cells are cultured in a DMEM culture medium for 24h, then the cells are subjected to cell wall removal by trypsin without EDTA, centrifugation is carried out for 5min at 1500rpm, the supernatant is discarded and washed for 2 times by precooled PBS, precooled 70% ethanol is used for fixation, and the cells are kept overnight in a refrigerator at 20 ℃. The cells were washed 2 times with pre-cooled PBS before detection, centrifuged to discard the supernatant, added 2. mu.L ribonuclease (RNaseA) at a concentration of 1mg/mL, added 400. mu.L PI (e.g., Cat #537059, Calbiochem, San Diego, Calif.) and reacted in the dark for 30min, and then the effect of the extract on the cell cycle was examined by flow cytometry.

The experimental results show that: 200 ug/mL passion flower vine leaf extract can increase the number of G1 periodic cells from 66.37% to 72.62% of the control, and decrease the number of S phase cells from 22.50% to 17.46%. It was shown that passion flower extract blocked the cell cycle at G1, thereby inhibiting its proliferation (fig. 4).

Example 5 detection of active ingredients of Passiflora Caerulea leaf extract by UHPLC-MS/MS

UHPLC-MS/MS analysis, DAD detector, mobile phase A (methanol), B (0.5% formic acid aqueous solution), procedure of 0 min-40% A, 10 min-60% A, 15 min-80% A, hold 10min, 25 min-40% A. The flow rate is 0.4mL/min, the column temperature is 28 ℃, and the injection volume is 10 mu L. Mass spectrum detection conditions: electrospray ionization (ESI), positive and negative ion mode, and scanning range m/z of 100-500.

According to the analysis of the composition of active components of passion flower vine leaf extracts by PLC-MS/MS mass spectrum information, the components which peak at 13.0 min, 13.8 min and 14.7min are respectively identified as A: isoorientin (C21H20O11), B: vitexin (C21H20O10), C: isovitexin (C21H20O10) (fig. 5). The molecular ion peak M/z is 449.1127[ M + H ] by mass spectrum detection]⁺，433.1175[M+H]⁺And 433.1176[ M + H ]]⁺(FIG. 6).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.