CN115772145A

CN115772145A - Citrus fruit extract and preparation method and application thereof

Info

Publication number: CN115772145A
Application number: CN202211509125.8A
Authority: CN
Inventors: 邓张双; 秦烨; 王羿语; 孙梦; 杜姝
Original assignee: China Three Gorges University CTGU
Current assignee: China Three Gorges University CTGU
Priority date: 2022-11-29
Filing date: 2022-11-29
Publication date: 2023-03-10
Also published as: WO2024114248A1

Abstract

The invention relates to the technical field of medicines, in particular to a citrus fruit extract and a preparation method and application thereof. The invention provides a citrus fruit extract, wherein the main component of the extract is polymethoxylated flavone. The present invention is directed to overcoming the problems of natural origin, low toxic side effects and lack of estrogen receptor degradants for endocrine therapy and providing a method for treating breast cancer using citrus fruit extract as estrogen receptor degradant. The citrus fruit extract is used for treating estrogen receptor positive breast cancer, namely, estrogen receptor is degraded through ubiquitin-proteasome approach, and then the growth of the estrogen receptor positive breast cancer is remarkably inhibited.

Description

Citrus fruit extract and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a citrus fruit extract and a preparation method and application thereof.

Background

Cancer is one of the major killers threatening human health, with the first highest incidence of breast cancer patients. Estrogen receptor positive breast cancer is a common type of breast cancer, the condition of which is affected by estrogen, and endocrine therapy is generally an effective treatment. Because the ER signal transduction cannot be completely inhibited, the cancer cell proliferation is difficult to be effectively inhibited, and the clinical medication is limited due to the problem of drug resistance, a new small molecule drug for treating ER positive breast cancer is urgently needed.

Currently, the representative therapeutic agents are tamoxifen and fulvestrant. Clinically, the problem of tamoxifen resistance gradually appears. Although fulvestrant has higher curative effect and lower side effect than tamoxifen and can effectively prolong the life of a patient, due to the high price and the problems of intramuscular injection, poor solubility, lack of oral availability and the like, the fulvestrant has limited wide application. In recent years, the development of estrogen receptor degraders for breast cancer has been increasing, and more has been focused on small molecule synthetic compounds or pharmaceutical compositions (CN 110343101A, CN 111646972a, CN 112041307a, CN 112424205a, cn112638916a, CN 112912078a, CN 113614076a, CN 114302879a, cn114502539a, CN 114656452A and CN 114667147A). The estrogen receptor degradation agent with natural source has less reports, and has important significance for searching the estrogen receptor degradation agent with natural source for the purposes of fully developing abundant plant resources and taking convenience, rapidness and economy as the purposes.

The citrus is the largest category of fruit used as both medicine and food in the world, is an important branch of rutaceae plants, has rich variety resources and wide distribution, is mainly concentrated in southern provinces of Yangtze river drainage areas such as Hubei, hunan, guangdong, fujian and the like, and is the fruit with the largest planting area. The citrus fruits are various in chemical component types, mainly comprise flavonoids, terpenes, coumarins, alkaloids and the like, and mainly comprise flavone components; the biological activity is wide, and the research on the citrus compounds in various aspects such as tumor resistance, inflammation resistance, oxidation resistance, virus resistance, cardiovascular disease prevention and the like is carried out at present. The research of the literature [ Journal of agricultural and Food Chemistry,2011,59 (6): 2314-2323 ] finds that compared with other limonin compounds and synthetic limonin derivatives, the deacetyl nomilin has 400 times higher inhibition rate on estrogen receptor positive breast cancer cells and is the most effective estrogen receptor positive breast cancer cell inhibitor. The research provides a thought for the inventor, and the possibility of searching safer and more efficient estrogen receptor degradation agents from citrus fruits to treat breast cancer becomes possible.

Therefore, aiming at the scientific problems, the active substance components of citrus fruits are further excavated and developed to be used as a new breast cancer treatment scheme, the development of deep processing of citrus to a large health industry is promoted, and the method has important basic and application basic research values.

Disclosure of Invention

The invention aims to overcome the problems of natural sources, low toxic and side effects and lack of estrogen receptor degradants for endocrine treatment, and provides a method for treating breast cancer by using citrus fruit extract as an estrogen receptor degradant. In particular to a citrus fruit extract, a preparation method thereof and application of the extract in treating estrogen receptor positive breast cancer, namely degrading estrogen receptors through a ubiquitin-proteasome way, and further obviously inhibiting the growth of the estrogen receptor positive breast cancer.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a citrus fruit extract, wherein the main component of the extract is polymethoxylated flavone;

the polymethoxyflavone comprises one or more of 5,7,8,4',5' -pentamethoxyflavone, 5,6,7,4',5' -pentamethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone.

The invention also provides a preparation method of the citrus fruit extract, which comprises the following steps:

(1) Mixing citrus fruit with water, pulverizing, centrifuging for the first time to remove residue to obtain material 1, mixing material 1 with hydrochloric acid, standing, cooling to room temperature, centrifuging for the second time, and collecting precipitate to obtain material 2;

(2) Washing the material 2 obtained in the step (1) with water, centrifuging for three times to obtain a precipitate, adding absolute ethyl alcohol to leach for 2-4 times, combining leaching liquor, concentrating under reduced pressure, and drying to obtain the citrus fruit extract.

Preferably, the citrus fruit in the step (1) is mixed with water in a ratio of 8-12 g:1mL; the rotation speed of the primary centrifugation and the rotation speed of the secondary centrifugation are 8000-12000 rpm independently, and the time is 8-12 min independently.

Preferably, the volume ratio of the material 1 to the hydrochloric acid in the step (1) is 1:1.5 to 2.5; the standing time is 12-16 h.

Preferably, the mass-to-volume ratio of the material 2 to the water in the step (2) is 1g: 8-12 mL, washing for 3 times; the rotating speed of the third centrifugation is 8000-12000 rpm, and the time is 8-12 min; for each leaching, the mass-to-volume ratio of the precipitate to absolute ethyl alcohol is 1g: 1.5-2.5 mL, the leaching temperature is 22-28 ℃, and the leaching time is 12-18 min.

Preferably, the citrus fruit comprises tangerine, orange or mandarin orange.

Preferably, the citrus fruit comprises whole fruit, peel or pulp.

The invention further provides the citrus fruit extract, and an application of the citrus fruit extract prepared by the preparation method of the citrus fruit extract in preparation of a medicine for treating breast cancer.

Preferably, the drug is an estrogen receptor degrading agent.

Preferably, the breast cancer is estrogen receptor positive breast cancer.

Compared with the prior art, the invention has the following beneficial effects:

(1) The structural model of the invention uses more than two cell estrogen receptor positive breast cancer cell strains for verification, and simultaneously uses animal experiments to further confirm the curative effect of the anti-tumor drug mixed extract on estrogen receptor positive breast cancer and the degradation effect on estrogen receptor.

(2) The invention provides a citrus fruit extract as an estrogen receptor degradation agent, which is a natural component derived from citrus plants in Rutaceae, can be used as an additive component for preparing foods, health-care products and medicines, and also provides application of the extract as an estrogen receptor degradation agent for treating breast cancer, so that the defect of the estrogen receptor degradation agent of natural sources is made up.

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 embodiments or the prior art descriptions 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 shows the result of the staining of live crystal violet cells after co-incubation of the extract of example 1 with MCF7 cells of human breast cancer;

FIG. 2 is a plot of the inhibition of MCF7 cell growth in human breast cancer by the extract of example 1;

FIG. 3 is a graph of the effect of the extract of example 1 on estrogen receptor protein levels of human estrogen receptor positive breast cancer cells MCF7 over time and concentration gradients;

FIG. 4 is a graph showing the effect of estrogen receptor degrading agents of example 1 on estrogen receptor protein levels of human and murine estrogen receptor positive breast cancer cells MCF7, T47D and SSM2 at different time gradients after inhibiting protein degradation related pathways using MG132 (a proteasome pathway inhibitor) and BAF-A1 (a lysosomal pathway inhibitor), respectively, of example 4.

FIG. 5 is a tumor beat result of the SSM2-129 mouse allograft tumor model in example 5;

FIG. 6 is the statistical results of tumor volumes of the SSM2-129 mouse allograft tumor model in example 5;

FIG. 7 is a statistical result of tumor weights of the SSM2-129 mouse allograft tumor model in example 5;

FIG. 8 is a graph of the effect of tumor estrogen receptor protein levels in the SSM2-129 mouse xenograft tumor model of example 5.

FIG. 9 is a high performance liquid chromatogram and the main chemical components of extract 3-1 of example 1.

The analysis conditions of the high performance liquid chromatography are as follows: the analysis conditions of the high performance liquid chromatography are as follows: the mobile phase is methanol-water, and is gradient eluted from 10% methanol for 5min to 60% methanol, from 60% methanol for 20min to 80% methanol, from 80% methanol for 2min to 100% methanol, and from 100% methanol for 3min; the detection wavelength is 254nm; the flow rate is 1mL/min; the sample size is 10uL; the chromatographic column is InertSustanin AQ-C18,5 μm,4.6 × 250mm (UP); the chromatograph is Shimadzu LC-2030C 3D Plus.

Detailed Description

The invention provides a citrus fruit extract, the main component of the extract is polymethoxylated flavone;

the polymethoxyflavone comprises one or more of 5,7,8,4',5' -pentamethoxyflavone, 5,6,7,4',5' -pentamethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone, and 5,6,7,8,4' -pentamethoxyflavone; preferred are 5,7,8,4',5' -pentamethoxyflavone, 5,6,7,4',5' -pentamethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone.

(2) Washing the material 2 obtained in the step (1) with water, centrifuging for three times to obtain a precipitate, adding absolute ethyl alcohol to extract for 2-4 times, combining the extract, concentrating under reduced pressure, and drying to obtain an extract of citrus fruits; preferably 3 times.

In the invention, the mixing ratio of the citrus fruit in the step (1) to water is 8-12 g:1mL; preferably 9 to 11g:1mL; more preferably 10g:1mL.

In the invention, the rotation speed of the primary centrifugation and the secondary centrifugation in the step (1) is 8000-12000 rpm independently, and the time is 8-12 min independently; preferably, the rotation speed is 9000-11000 rpm independently, and the time is 9-11 min independently; further preferably, the rotation speed is 10000rpm independently and the time is 10min independently.

In the invention, the volume ratio of the material 1 to the hydrochloric acid in the step (1) is 1:1.5 to 2.5; preferably 1:2.

in the invention, the standing time in the step (1) is 12-16 h; preferably 13 to 15 hours; further preferably 14h.

In the invention, the mass-to-volume ratio of the material 2 to water in the step (2) is 1g: 8-12 mL, washing for 3 times; preferably 1g: 9-11 mL; more preferably 1g:10mL.

The rotating speed of the third centrifugation is 8000-12000 rpm, and the time is 8-12 min; preferably, the rotating speed is 9000-11000 rpm, and the time is 9-11 min; more preferably, the rotation speed is 10000rpm and the time is 10min.

In the invention, in each leaching in the step (2), the mass-to-volume ratio of the precipitate to the absolute ethyl alcohol is 1g: 1.5-2.5 mL; preferably 1g:2mL.

In the invention, the leaching temperature in the step (2) is 22-28 ℃; preferably 23 to 27 ℃; further preferably 24 to 26 ℃; more preferably 25 deg.c.

In the invention, the leaching time in the step (2) is 12-18 min; preferably 13-17 min; more preferably 14 to 16min; more preferably 15min.

In the present invention, the citrus fruit includes mandarin orange, orange or mandarin orange; preferably orange.

In the present invention, the citrus fruit includes whole fruit, peel or pulp; preferably whole fruits.

In the present invention, the drug is an estrogen receptor degrading agent.

In the present invention, the breast cancer is an estrogen receptor positive breast cancer.

In the invention, the breast cancer model is an MCF7 and SSM2 cell model and an SSM2-129 mouse tumor-bearing model.

In the present invention, the detection substance selected in the model is an estrogen receptor, specifically expressed as degrading the estrogen receptor via the ubiquitin-proteasome pathway.

In the present invention, MCF7, SSM2, T47D-related estrogen receptor positive breast cancer cell lines were used as model cells.

In the present invention, 129 strain mice were used, and the fourth breast pad on the right was homograft SSM2 cell line as a model animal.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

This example provides a method for preparing an extract from citrus fruit, comprising the steps of:

taking fresh ripe summer oranges, cleaning, airing and weighing 178.8g, adding 18mL of tap water for crushing, centrifuging at 10000rpm for 10min for removing slag to obtain 100mL of liquid, adding 200mL of hydrochloric acid solution with the concentration of 1mol/L, carrying out acidolysis for 14h at 80 ℃, standing, cooling to room temperature, centrifuging at 10000rpm for 10min, collecting precipitate, washing the precipitate with water for 3 times, centrifuging, and mixing according to 1g: adding 5mL of absolute ethanol into the precipitate at a ratio of 2mL, leaching for 3 times, each time for 15min, mixing leaching solutions, concentrating under reduced pressure, drying, and weighing to obtain 189.6mg of citrus fruit extract, which is named as 3-1. The extract mainly comprises the following components: 5,7,8,4',5' -pentamethoxyflavone, 5,6,7,4',5' -pentamethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone and 5,6,7,8,4' -pentamethoxyflavone.

Example 2

The extract in the embodiment 1, MCF7 and SSM2 which are incubated with human estrogen receptor positive breast cancer cells can obviously inhibit the growth of tumor cells. The inhibitory activity of typical human breast cancer MCF7 cells is now described as follows.

The extract of example 1 was incubated with MCF7 cells, human estrogen receptor positive breast cancer cells, for 72 hoursThe method comprises the following steps: the estrogen receptor positive breast cancer cells MCF7 cells were seeded in DMEM medium supplemented with 10% fetal bovine serum along with 100units/mL penicillin and 100. Mu.g/mL streptomycin at 37 ℃ and 5% CO ₂ Culturing in an incubator. Inoculating the cells into a 96-well plate at the density of 5000 cells per well overnight for complete adherence of the cells, and then adding an estrogen receptor degradation agent to incubate with the completely adherent cells for 72 hours respectively. The non-drug-added substances are respectively used as blank control groups, the endocrine treatment drug tamoxifen is respectively used as a positive control group, the cells after the co-incubation of the groups are stained with crystal violet solution to obtain viable cells, and the staining result is shown in figure 1. As can be seen from FIG. 1, the estrogen receptor degrader and the human estrogen receptor positive breast cancer cell MCF7 can both significantly inhibit the growth of tumor cells when incubated together.

The staining results of the crystal violet living cells after the co-incubation of the extract of example 1 and the human estrogen receptor positive breast cancer cells MCF7 were quantified, and the cells were eluted and dissolved for one hour in a shaking table at room temperature using a trisodium citrate solution, and the absorbance was measured at an absorption wavelength of 570 nm. The measurement results are shown in FIG. 2. As shown in FIG. 2, the half inhibitory concentration IC of the estrogen receptor degradation agent and MCF7 of human estrogen receptor positive breast cancer cells ₅₀ The value was 0.06mg/mL.

Example 3

In this embodiment, the extracts in example 1 have the effect on the protein levels of estrogen receptors related to the estrogen receptor positive breast cancer cell line MCF7, and the estrogen receptor degrading agent and the human estrogen receptor positive breast cancer cells incubated with MCF7 and SSM2 can all significantly reduce the protein levels of the estrogen receptors. The following is an example of the typical human breast cancer MCF7 cells down-regulating their estrogen receptor protein levels.

The extract in the example 1 is respectively incubated with human estrogen receptor positive breast cancer cell MCF7 cells for 24 hours, 48 hours and 72 hours to extract protein, and the protein level of the estrogen receptor is detected by Westernblot, wherein the specific method comprises the following steps: the estrogen receptor positive breast cancer cells MCF7 cells in logarithmic growth phase were taken and digested with 0.125% trypsinThen, it was inoculated into 6-well culture plates containing DMEM medium containing 10% fetal bovine serum and 100units/mL penicillin and 100. Mu.g/mL streptomycin at 37 ℃ with 5% CO ₂ Culturing in an incubator. At 2X 10 per hole ⁵ The density of each cell is inoculated to a 96-well plate culture plate overnight to wait for the cells to be completely attached, and then estrogen receptor degradation agents are respectively added to be incubated with the completely attached cells for 24 hours, 48 hours and 72 hours to serve as time gradients. The non-drug-added drugs were used as blank control groups, and the concentration of the extract was determined as a concentration gradient according to half, one, or two times the half inhibitory concentration corresponding to the drug obtained in example 2. After fully adherent cells are incubated for 24, 48 and 72 hours, the supernatant is discarded, the cells are washed by PBS precooled at 4 ℃, an appropriate amount of RIPA cell lysate is added, then cells are scraped by a cell scraper and lysate containing cell debris is sucked, after ice bath is carried out for 30 minutes, the cells are centrifuged at 4 ℃,12000rpm is carried out, the cells are separated for 30 minutes, and the supernatant is taken as a whole-cell protein extraction sample.

The Western blot comprises the following specific methods: the gel is prepared from 10% separating gel and 5% concentrating gel, and SDS-PAGE gel is prepared. Protein was extracted from the cell sample, quantified using BCA Kit (BCAProteinaassay Reagent Kit), and the sample was prepared and denatured at 100 ℃ for 10min. Then, the total amount of 20. Mu.g of the protein was loaded and subjected to SDS-PAGE (constant pressure 120V) for about 2 hours. After electrophoresis, the gel was peeled off and the concentrated gel was cut off. And placing the NC membrane, the filter paper and the gel in a membrane transferring solution for assembly. Stacking the black surface (cathode) to the red surface (anode) on the electric rotating plate in sequence: sponge cushion, 3 pieces of filter paper, gel, NC membrane, 3 pieces of filter paper and finally the sponge cushion. After the gel is sequentially installed, the gel is inserted into an electric rotary tank, newly prepared membrane transferring liquid is poured in, electrodes are inserted, and the membrane is transferred for 90min under the condition of 200mA of steady flow, so that the protein in the gel is transferred onto an NC membrane. After the electrotransfer was completed, the NC membrane was taken out and placed in TBST solution for brief rinsing. The NC membrane was gently blocked with 5% skim milk (in TBST) for 1h on a shaker at room temperature, followed by 3 washes with TBST for 5min each. Incubate primary antibody (rabbit source) overnight on a shaker at 4 ℃. The membrane was washed with TBST for 5 min/time for 3 times. The diluted, HRP-labeled goat anti-rabbit IgG was co-incubated with NC membranes and shake-incubated for 1h at room temperature. The membrane was washed with TBST for 5 min/time for 3 times. The developing solutions a and B were mixed at 1. The results of the Westernblot development are shown in FIG. 3. As can be seen from fig. 3, the estrogen receptor degrading agent down-regulates the estrogen receptor protein level in human estrogen receptor positive breast cancer cells MCF7 as a function of concentration and time.

Example 4

In this embodiment, the mechanism related to the down-regulation of the protein levels of estrogen receptor positive breast cancer cell lines MCF7, T47D, and SSM2 estrogen receptors by the extract in example 1 is further explored. A typical human breast cancer MCF7 cell is described below. The effect of the extract on estrogen receptor protein levels after inhibition of protein degradation-related pathways with MG132 (proteasome pathway inhibitor), BAF-A1 (lysosomal pathway inhibitor), respectively, is shown in fig. 4. After using BAF-A1 (lysosomal pathway inhibitor) to inhibit the lysosomal pathway of protein degradation, the extract can still down-regulate estrogen receptor protein levels at different time gradients. After the MG132 (proteasome pathway inhibitor) is used for inhibiting the proteasome pathway of protein degradation, the extract has obvious back-up effect on the level of estrogen receptor protein. The results are shown in fig. 4, and the down-regulated level of estrogen receptor of the extract on the estrogen receptor positive breast cancer cell line is promoted to be degraded by a proteasome pathway and not by a lysosome pathway. The concrete method is the same as the embodiment 3.

Example 5

In this example, the tumor inhibition rate of the extract (3-1) of example 1 on estrogen receptor positive breast cancer, its effect on estrogen receptor expression in tumor tissues, was evaluated by pharmacodynamics of SSM2-129 breast cancer tumor-bearing mouse model. The tumor inhibition rate of the extract in example 1 on estrogen receptor positive breast cancer after 10 days of administration treatment is about 50%, which shows that the extract has a remarkable inhibition effect on estrogen receptor positive breast cancer, does not have obvious damage to important organs, and can also obviously reduce the protein level of estrogen receptors.

Tumor mouse model constructionVertically: SSM2 cells of human estrogen receptor positive breast cancer cells are divided into 1 × 10 cells ⁶ The density of each mouse was inoculated to the fourth breast pad on the right side of the 129 mice of the same species (18-20 g). The specific method comprises the following steps: SSM2 cells in the logarithmic growth phase were digested with 0.125% trypsin and centrifuged after cell counting, and the cells were resuspended and diluted to the desired density with PBS. SSM2 cell suspension at 1X 10 ⁶ The density of each mouse was inoculated at the fourth breast pad on the right side of 129 mice. Subcutaneous tumor nodules of about 0.5cm in diameter were observed at 2 weeks, indicating successful establishment of the tumor mouse model.

Grouping and administration treatment: mice were divided into 4 groups of 5 mice according to treatment pattern and dosing trials were started 2 weeks after tumor inoculation. The solvent without drug was administered as a solvent control group, and the concentrations of the extract in example 1 were selected to be 25mg/kg, 50mg/kg, and 100mg/kg as drug treatment groups. Mouse body weights were recorded at

days

0,1,3,5,7,9, 11 after the start of the administration, and tumor volumes were recorded using vernier caliper measurement and tumor inhibition rates were calculated, and the results are shown in fig. 6. Tumors were weighed after sacrifice and the results are shown in figures 5 and 7. The tumor and the important organs (heart, liver, spleen, lung and kidney) of each group of mice are fixed by tissue fixing liquid, then paraffin sections are cut, and H & E staining is carried out to observe the influence of the drug on each important organ of the mice. In addition, the protein level of estrogen receptor in tumor tissue was measured by western blot, and the results are shown in fig. 8, where the expression level of estrogen receptor in SSM2 tumor tissue of mice in the drug-treated group was decreased, and the decrease was significant at the 50mg/kg dose. As shown in FIGS. 5-8, the extracts with medium and high concentrations can obviously inhibit the tumor growth of SSM2-129 mouse allograft tumor model, have no obvious damage to important organs, and obviously reduce the estrogen receptor expression level of the tumor part.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims

1. An extract of citrus fruit, wherein the extract comprises polymethoxylated flavones as a major component;

the polymethoxyflavone comprises one or more of 5,7,8,4',5' -pentamethoxyflavone, 5,6,7,4',5' -pentamethoxyflavone, 5,6,7,8,3',4' -hexamethoxyflavone, 3,5,6,7,8,3',4' -heptamethoxyflavone, and 5,6,7,8,4' -pentamethoxyflavone.

2. A process for preparing a citrus fruit extract according to claim 1, comprising the steps of:

(1) Mixing Citrus fruits with water, pulverizing, centrifuging for the first time to remove residue to obtain material 1, mixing material 1 with hydrochloric acid, standing, cooling to room temperature, centrifuging for the second time, and collecting precipitate as material 2;

3. The method according to claim 2, wherein the citrus fruit of step (1) is mixed with water in a ratio of 8 to 12g:1mL; the rotation speed of the primary centrifugation and the rotation speed of the secondary centrifugation are 8000-12000 rpm independently, and the time is 8-12 min independently.

4. The method of claim 2, wherein the volume ratio of the material 1 to the hydrochloric acid in the step (1) is 1:1.5 to 2.5; the standing time is 12-16 h.

5. The method according to claim 2, wherein the mass-to-volume ratio of the material 2 to the water in the step (2) is 1g: 8-12 mL, washing for 3 times; the rotating speed of the third centrifugation is 8000-12000 rpm, and the time is 8-12 min; for each leaching, the mass-to-volume ratio of the precipitate to the absolute ethyl alcohol is 1g: 1.5-2.5 mL, the leaching temperature is 22-28 ℃, and the leaching time is 12-18 min.

6. The method of claim 2, wherein the citrus fruit comprises mandarin orange, or mandarin orange.

7. The method of claim 2, wherein the citrus fruit comprises whole fruit, peel, or pulp.

8. Use of a citrus fruit extract according to claim 1, or of a citrus fruit extract obtained by a method for the preparation of a citrus fruit extract according to any one of claims 2 to 7, for the preparation of a medicament for the treatment of breast cancer.

9. The use of claim 8, wherein the drug is an estrogen receptor degrading agent.

10. The use of claim 8 or 9, wherein the breast cancer is estrogen receptor positive breast cancer.