CN110559334A - May tea and fruit extract and application thereof in preventing and treating breast cancer - Google Patents
May tea and fruit extract and application thereof in preventing and treating breast cancer Download PDFInfo
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- CN110559334A CN110559334A CN201911011260.8A CN201911011260A CN110559334A CN 110559334 A CN110559334 A CN 110559334A CN 201911011260 A CN201911011260 A CN 201911011260A CN 110559334 A CN110559334 A CN 110559334A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
Abstract
The invention discloses application of extracts of leaves and fruits of mountain May tea (Antidesma montanum) in inhibiting proliferation, migration and invasion of MCF-7 and MDA-MB-231 breast cancer cells, and belongs to the technical field of biological medicines. May tea is a plant of May tea genus of Euphorbiaceae family, and its mature fruit and leaf ethanol extract have the ability to inhibit proliferation of MCF-7 and MDA-MB-231 breast cancer cells. The anti-cancer active ingredients of May tea mainly concentrate ethyl acetate extract phase, contain amentoflavone active ingredients, and can prevent MDA-MB-231 cell migration and invasion. The fruit and leaf of May tea have anticancer potential, and are expected to be developed into health products related to tea and fruit.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a plant extract, in particular to an extract of May tea leaves and fruits and application thereof in prevention and treatment of breast cancer.
background
In the modern society, chronic diseases such as cancer, cardiovascular and cerebrovascular diseases and the like become main diseases threatening the survival of human beings, and the incidence rate of the chronic diseases such as cancer, cardiovascular and cerebrovascular diseases and the like can be obviously reduced by taking a high proportion of fruits every day. Breast cancer is the disease with the highest incidence rate of female malignant tumors, and accounts for 30 percent of all new female malignant tumors. About 50 ten thousand women die of breast cancer every year all over the world, and the number of breast cancer attacks is increasing year by year.
MCF-7 is a classical cell model for studying breast cancer, and MDA-MB-231 is a triple-negative breast cancer cell (TNBC). TNBC refers to breast cancer that is negative for all of the Estrogen Receptor (ER), the Progesterone Receptor (PR), and the human epidermal growth factor receptor 2(HER2), and accounts for approximately 15% of the total number of breast cancer patients. TNBC has the characteristics of high invasiveness, early local recurrence, fast systemic metastasis, poor overall prognosis and the like, and chemotherapy is the only effective drug treatment means for TNBC patients at present, so that new treatment drugs and effective treatment methods are urgently needed to be searched. The development of active drugs for treating tumors from plants has been the research direction of medical researchers all over the world.
The fruit, leaf and root of May tea are traditional medicinal components. The tender leaf can be used as tea, and the fruit has dual-purpose of medicine and food, and can be used for preparing jam and fruit wine. The method is found by screening the activity of ethanol extracts of various tropical wild fruits: the extract of the May tea leaves and the fruit has good activity of inhibiting the proliferation of MCF-7 and MDA-MB-231 breast cancer cells, and further research finds that the extract of the May tea leaves can effectively inhibit the migration and invasion of the MDA-MB-231 breast cancer cells. The May tea fruit and leaf extract is expected to be a potential medicine for preventing and treating breast cancer.
Disclosure of Invention
The invention aims to provide an extract of May tea leaves and fruits capable of inhibiting proliferation, migration and invasion of breast cancer cells, and the extract can be used as a traditional Chinese medicine component for preventing and treating breast cancer.
In order to achieve the purpose, the technical scheme of the invention is as follows: the specific preparation process of the May tea and fruit extract is as follows:
(1) Collecting leaves or fruits of mountain May tea (Antidesma montanum), naturally drying indoors or drying in an oven at 35-45 deg.C, and pulverizing;
(2) Soaking and extracting by using ethanol with the concentration of 60-80%, performing ultrasonic-assisted extraction, filtering an extracting solution, and removing a solvent by using a reduced-pressure rotary evaporator to obtain a concentrated extract;
(3) Dissolving the extract with distilled water, sequentially adding petroleum ether and ethyl acetate with the volume of 2-5 times of that of the extract respectively for extraction, and performing reduced pressure rotary evaporation on an ethyl acetate extract phase to obtain the phase extract.
The extract is amentoflavone as active ingredient.
another object of the present invention is to provide the use of May tea leaves and fruits for preventing and treating breast cancer, comprising:
May tea and fruit are used for inhibiting breast cancer cell proliferation;
May tea and fruit are used for inhibiting breast cancer cell migration;
May tea and fruit are used for inhibiting breast cancer cell invasion.
The extract is dissolved in MCF-7 and MDA-MB-231 for proliferation effect experiment of breast cancer cells.
Dissolving the above May tea extract in MDA-MB-231 breast cancer cell migration, invasion, and flow cell cycle influence experiment.
The tea leaves contain amentoflavone active ingredients through HPLC-MS/MS detection.
The extract of the May tea leaves and the fruit can obviously inhibit the proliferation of breast cancer cells, and the extract of the May tea leaves can inhibit the migration and invasion of high-migration-capacity MDA-MB-231 breast cancer cells. The results show that the extract of the May tea leaves and the May fruit has good anti-breast cancer activity and has potential application value in the prevention and treatment of breast cancer.
The extract of May tea and fruit is used for treating breast cancer and preparing anticancer drugs.
The active extract of leaves and fruits with the activity of inhibiting the proliferation of breast cancer cells obtained by the invention can be used as an anti-cancer drug.
Drawings
FIG. 1 shows the effect of ethanol extract of May tea (A. montanum) leaves on the proliferation activity of MCF-7 and MDA-MB-231 cells.
FIG. 2 shows the effect of ethanol extract of May tea (A. montanum) fruit on the proliferation activity of MCF-7 and MDA-MB-231 cells.
FIG. 3 shows the effect of ethyl acetate extraction of May tea (A. montanum) leaves on MDA-MB-231 cell migration.
FIG. 4 is a graph showing the effect of ethyl acetate extraction of May tea (A. montanum) leaves on MDA-MB-231 cell invasion in the mountain area of the present invention.
FIG. 5 is a graph showing the effect of ethyl acetate extraction of May tea (A. montanum) leaves on the cell cycle of MDA-MB-231.
Fig. 6 is a general ion flow diagram of the HPLC extract phase of mountain May tea (a. montanum) leaf ethyl acetate of the present invention.
FIG. 7 is a mass spectrum of amentoflavone as an active ingredient.
FIG. 8 shows the effect of amentoflavone on the proliferation activity of MCF-7 and MDA-MB-231 cells.
Detailed Description
example 1 extraction of active ingredients of May tea leaves and fruits
Experimental materials: fresh leaves and fruits of mountain May tea (A. montanum) are collected from Xinglong of Wanning city, Hainan province.
Drying and crushing collected fresh leaves and fresh fruits, sieving the crushed fresh leaves and fresh fruits by a 40-mesh sieve according to a ratio of 1: 2-1: adding ethanol with the concentration of 60-80% according to the volume ratio of 5, soaking, and performing ultrasonic extraction for 3 times, 30min each time. The extracting solution is subjected to reduced pressure rotary evaporation concentration and near drying, after the extracting solution is dissolved by ultrapure water, petroleum ether (with a boiling range of 60-90 ℃), ethyl acetate and n-butyl alcohol are sequentially used for extraction, each extracting phase is subjected to reduced pressure rotary evaporation concentration and near drying, the extracting phase is dissolved by DMSO to prepare a corresponding concentration for cell activity test, and the content of the DMSO in the culture medium is smaller than 0.1%.
example 2 CCK-8 assay for inhibition of Breast cancer cell proliferation Activity
A96-well plate was seeded with 4000 MCF-7 cells and 2000 MDA-MB-231 cells per well, and after the cells adhered, culture medium (containing 0.25% HEPES) of different concentrations of extracts was added, with 0.1% DMSO medium as a negative control. After 72h of treatment, CCK-8 was added and CO was added2After 1h in the incubator, the absorbance at 450nm was read by a microplate reader, and the cell viability was ═ a blank-a treated group)/a blank group.
Experimental results show that the extracts of the leaves and the fruits of the mountain May tea have the activity of inhibiting the proliferation of two breast cancer cells. IC (Integrated Circuit) for inhibiting proliferation of two breast cancer cells by using ethanol extract of mountain May tea50282.2. mu.g/mL (MCF-7), 137.6. mu.g/mL (MDA-MB-231), respectively (FIG. 1); IC inhibition of ethanol extracts of mountain May tea fruit50302.2. mu.g/mL (MCF-7), 483.3. mu.g/mL (MDA-MB-231) (FIG. 2); after the ethanol extract of the mountain May tea is sequentially extracted by petroleum ether, ethyl acetate and n-butanol, the cell proliferation activity is inhibited, and the ethyl acetate phase activity is the best.
Example 3 assay of inhibition of Breast cancer cell migration Activity by the Scoring method
24-well plate 2X 10 per well5Inoculating MDA-MB-231 cells in a logarithmic growth phase in each cell quantity, scratching with a gun head after the cells adhere to the wall for 24 hours, washing floating cells with PBS, adding extracts with different concentrations, staining the cells with crystal violet after 24 hours, taking a picture under a microscope, counting the change of the scratch area before and after treatment, and calculating the mobility inhibition rate.
the experimental results show that: the 100 μ g/mL ethyl acetate extract of May tea leaves had activity in inhibiting MDA-MB-231 cell migration, with an average inhibition of 86% compared to the control (FIG. 3).
Example 4 Transwell assay for Activity in inhibiting invasion of Breast cancer cells
Treating MDA-MB-231 cells with ethyl acetate extract culture medium of May tea of different concentrations for 24 hr, removing cell wall by trypsinization, and preparing into 4 × 10 culture medium without bovine serum4the method comprises the steps of adding Transwell (placed in a 24-well plate) into a cell suspension per mL, adding complete culture medium into the lower layer of the Transwell, culturing for 24 hours in an incubator at 37 ℃, fixing by 75% ethanol, staining by crystal violet, randomly selecting 5 visual fields under a microscope to take pictures, counting the number of cells, and taking the average number of the 5 visual fields to represent the in vitro migration and invasion number of tumor cells.
The experimental results show that: 25. 50, 100 μ g/mL of ethyl acetate phase extract of May tea leaves treated MDA-MB-231 cells 24, the amount of cells crossing the transwell membrane compared to the control was: 69.9%, 37.6% and 34.3%, the ethyl acetate phase extract of May tea has cell invasion inhibiting activity (FIG. 4).
Example 5 flow cytometry Activity for inducing apoptosis
Inoculation of 5X 10 wells with 6-well plates per well5MDA-MB-231 cells in logarithmic phase are cultured for 24h until the cells adhere to the wall, a blank control group and an extract treatment group are respectively arranged, after 24h of treatment, the cells are detached by trypsin without EDTA, centrifuged at 1500rpm for 5min, supernatant is discarded and washed by precooled PBS for 2 times, and the cells are fixed by precooled 70% ethanol and kept in a refrigerator at the temperature of minus 20 ℃ overnight. The cells were washed 2 times with pre-chilled PBS before detection, centrifuged to remove the supernatant, added 2. mu.L ribonuclease (RNaseA) at 1mg/mL concentration, added 400. mu.L PI, and reacted in the dark for 30min before the influence of the extract on the cell cycle was detected by flow cytometry.
The experimental results show that: may tea can prolong the S cycle of cells from 25.6% to 32.0% of the control, and reduce the number of cells from 14.5% to 6.5% in the G2 phase, thereby inhibiting the proliferation (FIG. 5).
Example 6 HPLC-MS/MS analysis of active ingredients
HPLC-MS/MS analysis, DAD detector, mobile phase A (methanol), B (0.5% formic acid aqueous solution), procedure 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 source (ESI), positive and negative ion mode, scanning range m/z 100 ~ 1500.
the HPLC peak time of the amentoflavone standard product is the same as that of the component of the ethyl acetate extract phase of May tea (figure 6). The same compound was confirmed from the mass spectrum information. The amentoflavone has molecular weight of 538.4579, and molecular formula: C30H18O10, and the molecular ion peak M/z 539.2 is [ M + H ] through actual mass spectrum detection]+(FIG. 7).
Example 7 Activity of amentoflavone against Breast cancer cell proliferation
The CCK-8 method is used for detecting the proliferation effect of amentoflavone on MCF-7 and MDA-MB-231 breast cancer cells in the same way as the example 2, and the result shows that the inhibition IC of 72h of the cells treated by the amentoflavone50628.5 μ g/mL (MCF-7), 192.6 μ g/mL (MDA-MB-231), respectively, amentoflavone is one of the active ingredients in May tea (FIG. 8).
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.
Claims (6)
1. An extract of May tea leaves and fruits, which is characterized by being obtained by the following preparation steps:
(1) Collecting leaves or fruits of mountain May tea, naturally drying indoors or drying in an oven at 35-45 ℃, and crushing;
(2) Soaking and extracting by using ethanol with the concentration of 60-80%, performing ultrasonic-assisted extraction, filtering an extracting solution, and removing a solvent by using a reduced-pressure rotary evaporator to obtain a concentrated extract;
(3) Dissolving the extract with distilled water, sequentially adding petroleum ether and ethyl acetate with the volume of 2-5 times of that of the extract respectively for extraction, and performing reduced pressure rotary evaporation on an ethyl acetate extract phase to obtain the phase extract.
2. The May tea and fruit extract of claim 1, wherein: the active component of the extract is amentoflavone.
3. The May tea and fruit extract according to claims 1 and 2, characterized in that: application of May tea and fruit in preventing and treating breast cancer is provided.
4. The May tea and fruit extract of claim 3, wherein: may tea and fruit are used for inhibiting breast cancer cell proliferation.
5. The May tea and fruit extract of claim 3, wherein: may tea and fruit are used for inhibiting breast cancer cell migration.
6. The May tea and fruit extract of claim 3, wherein: may tea and fruit are used for inhibiting breast cancer cell invasion.
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Cited By (1)
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| CN113599403A (en) * | 2021-08-30 | 2021-11-05 | 中国热带农业科学院海口实验站 | Application of passion flower extract in preparation of medicine for preventing and treating breast cancer |
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Cited By (2)
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| CN113599403A (en) * | 2021-08-30 | 2021-11-05 | 中国热带农业科学院海口实验站 | Application of passion flower extract in preparation of medicine for preventing and treating breast cancer |
| CN113599403B (en) * | 2021-08-30 | 2022-07-26 | 中国热带农业科学院海口实验站 | Application of passion flower extract in preparation of medicine for preventing and treating breast cancer |
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