Application of acer palmatum 'Jinling Huangfeng' extract in preparation of medicine for preventing and treating breast cancer

Abstract

The invention discloses application of an acer palmatum 'Jinling Huangfeng' extract in preparation of a medicine for preventing and treating breast cancer, and belongs to the technical field of natural product extraction. The invention further discloses an extraction method of the acer palmatum 'Jinling Huangfeng' extract and a separation method of active ingredients of the acer palmatum 'Jinling Huangfeng' extract. Scratch repair experiments directly prove that the acer palmatum 'Jinling Huangfeng' extract can significantly inhibit the migration ability of highly metastatic breast cancer cells MDA-MB-231. The separation method of the active components of the acer palmatum 'Jinling Huangfeng' extract is closely linked with anti-breast cancer metastasis activity screening, so that the experimental process has definite pharmacological guidance quality, the efficiency and success rate of finding the active components are greatly improved, and a high-value candidate resource is provided for developing anti-metastasis drugs.

Description

Application of extracts from Japanese maple 'Jinling Yellow Maple' in the preparation of drugs for the prevention and treatment of breast cancer Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to the application of an extract of Japanese maple 'Jinling Yellow Maple' in the preparation of drugs for the prevention and treatment of breast cancer. Background Technology

[0002] Breast cancer is a malignant tumor that occurs in the glandular epithelial tissue of the human breast. It seriously endangers the health of women in my country, with the highest incidence rate, accounting for about 10%. MDA-MB-231 is a triple-negative breast cancer cell, accounting for about 15% of breast cancer patients. It is characterized by rapid metastasis, high invasiveness, early local recurrence, and poor prognosis.

[0003] Discovering active drugs for treating tumors from natural plants has always been a focus of global pharmaceutical research. Therefore, finding new natural plants that inhibit the proliferation and migration of breast cancer cells and induce apoptosis, and discovering active drugs from them, is of great clinical need and has important practical significance for the treatment of breast cancer.

[0004] Japanese maple (Acer palmatum Thunb.) is a deciduous small tree belonging to the genus Acer in the family Sapindaceae. As a world-renowned colorful-leaved plant, it also has a history of use in traditional medicine. Modern pharmacological studies have shown that extracts from Japanese maple possess various biological activities such as anti-inflammatory and antioxidant effects, suggesting that it may contain active ingredients with medicinal value. 'Jinling Yellow Maple' is a new Japanese maple variety independently bred by the Jiangsu Academy of Agricultural Sciences, with a yellow leaf period lasting over 160 days, filling a gap in the domestic market for yellow-colored Japanese maples.

[0005] Currently, there are no reports on the application of extracts from Japanese maple (Acer palmatum 'Jinling Huangfeng') in the treatment of breast cancer.

[0006] The isolation and purification of plant active ingredients is crucial for studying their pharmacodynamic material basis. As a plant with potential medicinal value, the study of the active ingredients of *Acer palmatum* typically relies on traditional column chromatography methods (such as silica gel columns and macroporous adsorption resin columns). The core principle of this technique is to separate the target components based on the difference in adsorption capacity between the solid stationary phase and the mobile phase. Although this method is well-established, it has significant inherent limitations:

[0007] Firstly, solid stationary phases may lead to irreversible adsorption of target components, resulting in the loss of trace or highly active components, low sample recovery rate, and significant losses.

[0008] Secondly, the separation process is lengthy, involving multiple column packing, elution, and fraction collection, and the entire process takes several days or even weeks, resulting in low separation efficiency.

[0009] Third, it requires a large amount of organic solvents, which is costly and environmentally unfriendly.

[0010] Furthermore, current research on Japanese maple primarily focuses on its basic activities such as antioxidant activity, while targeted isolation studies targeting its key pharmacological activities, particularly anti-tumor activity and anti-breast cancer metastasis, are almost nonexistent. This leads to a severe disconnect between the isolation process and the screening of target activities. Consequently, the traditional strategy of "comprehensive isolation first, then activity verification" suffers from unclear objectives, significant haphazardness in the isolation process, and low success rates, making it difficult to efficiently target and lock onto effective components from complex plant matrices. Moreover, the large number of isolated compounds requires tedious secondary screening, making it easy for many trace components effective against metastasis to be missed or overlooked during the lengthy isolation process.

[0011] Therefore, developing a targeted, efficient, and integrated method for the isolation and identification of anti-breast cancer metastasis active components in Acer palmatum, and elucidating its mechanism of action, has become an urgent problem to be solved in this field.

[0012] Compared with traditional techniques, high-speed countercurrent chromatography (HSCCC), as a novel liquid-liquid partition chromatography technique, exhibits unique advantages. It completely abandons the solid stationary phase, relying on a two-phase immiscible solvent system under the action of centrifugal force, in which one phase of liquid is retained as the stationary phase, thereby achieving rapid and efficient separation of compounds in the liquid-liquid two-phase system based on the partition coefficient.

[0013] Applying the efficient and green preparation and separation technology HSCCC to the targeted discovery of the anti-breast cancer metastasis active ingredient of Acer palmatum 'Jinling Huangfeng' is expected to overcome many bottlenecks of traditional methods and provide strong technical support for rapidly identifying its active substances and elucidating its mechanism of action. Summary of the Invention

[0014] Purpose of the invention: The purpose of this invention is to provide the application of extract of Acer palmatum 'Jinling Huangfeng' in the preparation of drugs for the prevention and treatment of breast cancer.

[0015] This invention uses Acer palmatum 'Jinling Huangfeng' as the test material to analyze the active ingredients in the leaves and the anti-breast cancer metastasis activity of Acer palmatum 'Jinling Huangfeng' extract.

[0016] Technical solution: The objective of this invention is achieved through the following technical solution:

[0017] This invention provides the application of an extract of Japanese maple 'Jinling Yellow Maple' in the preparation of drugs for the prevention and treatment of breast cancer.

[0018] The present invention provides a preferred technical solution, which is the application of the extract of Acer palmatum 'Jinling Huangfeng' in the preparation of a drug for inhibiting breast cancer cell migration.

[0019] The present invention provides a preferred technical solution, which is the application of the extract of Acer palmatum 'Jinling Huangfeng' in the preparation of a drug for inhibiting the invasion of breast cancer cells.

[0020] Preferably, the Japanese maple 'Jinling Yellow Maple' extract is an ethanol extract with a mass concentration of 80%.

[0021] Preferably, the active ingredients in the Japanese maple 'Jinling Yellow Maple' extract include vitexin, vitexin and isovitexin.

[0022] Preferably, the concentration of the extract of Acer palmatum 'Jinling Huangfeng' that inhibits breast cancer cell migration is 1-10 μg / mL.

[0023] This invention also provides a method for extracting the above-mentioned extract of *Acer palmatum* 'Jinling Huangfeng' used in the prevention and treatment of breast cancer, comprising the following steps:

[0024] (1) Take the dried leaves of Japanese maple 'Jinling Yellow Maple', crush them and pass them through a 60-mesh sieve to obtain dried leaf powder;

[0025] (2) The dried leaf powder obtained in step (1) was ultrasonically extracted using an 80% ethanol aqueous solution, filtered to obtain an extract, and the solvent was evaporated to obtain a crude extract, namely the extract of Acer palmatum 'Jinling Huangfeng'.

[0026] Preferably, 25-30 ml of 80% ethanol aqueous solution is added to every 1 g of dried leaf powder.

[0027] Preferably, in step (2), the ultrasonic extraction power is 200-260W, the temperature is 55-60℃, and the time is 2-2.5h.

[0028] This invention also provides a method for isolating the active ingredients of the above-mentioned extract of *Acer palmatum* 'Jinling Huangfeng' used in the prevention and treatment of breast cancer, comprising the following steps:

[0029] (1) Preparation of crude extract: Take the dried leaves of Japanese maple 'Jinling Huangfeng', crush them and pass them through a 60-mesh sieve to obtain dried leaf powder; use 80% ethanol aqueous solution to ultrasonically extract the obtained dried leaf powder, filter to obtain the extract, evaporate the solvent to obtain crude extract, which is the Japanese maple 'Jinling Huangfeng' extract.

[0030] (2) Separation of active ingredients: The crude extract obtained in step (1) was separated by high-speed countercurrent chromatography using an eluent according to an elution program, and the target active fraction was collected;

[0031] The eluent is selected from an ethyl acetate-n-butanol-water solvent system, an ethyl acetate-water solvent system, or an ethyl acetate-methanol-water solvent system;

[0032] The separation conditions include: main unit rotation speed of 800-1000 rpm; mobile phase flow rate of 1.5-2.5 mL / min; stationary phase flow rate of 15-20 mL / min;

[0033] (3) Concentrate and dry to obtain three active ingredients.

[0034] Preferably, in step (2), the eluent is selected from the ethyl acetate-n-butanol-water solvent system, and the volume ratio of ethyl acetate, n-butanol and water is 4:1:5; the separation conditions are: main unit speed 900 rpm; mobile phase flow rate 2 mL / min; stationary phase flow rate 15 mL / min.

[0035] Preferably, in step (2), the stationary phase of the high-speed countercurrent chromatography is an organic phase and the mobile phase is an aqueous phase.

[0036] More preferably, in high-speed countercurrent chromatography, the injection volume of the crude extract is 50 mg of crude extract dissolved in a 10 mL solvent system.

[0037] Preferably, in step (2), the elution procedure is set as follows:

[0038] (1) Pump the stationary phase into the high-speed countercurrent chromatograph;

[0039] (2) Then start the host and set it to forward rotation mode, with the speed stabilizing at 800-1000 rpm;

[0040] (3) Pump the mobile phase into the column at a flow rate of 1.5-2.5 mL / min;

[0041] (4) After the two phases reach dynamic equilibrium in the column, the crude extract dissolved in the solvent system is injected.

[0042] (5) Continue elution by pumping the mobile phase at a flow rate of 1.5-2.5 mL / min;

[0043] (6) Collect the fraction based on the signal from the ultraviolet detector at a wavelength of 254 nm.

[0044] The method for separating the active ingredients of the extract of Acer palmatum 'Jinling Huangfeng' in this invention fundamentally avoids sample loss caused by adsorption, and the sample recovery rate is close to 100%. The operation process is simplified, and high-purity monomers can be obtained from the crude extract in one separation, which greatly shortens the experimental cycle. Moreover, it has a large preparation capacity and good reproducibility.

[0045] The method for separating the active ingredients of the extract of Acer palmatum 'Jinling Huangfeng' of this invention can establish a comprehensive technical solution that can simultaneously achieve efficient and high-recovery separation and can be closely linked with the screening of anti-breast cancer metastasis activity, so as to overcome the shortcomings of traditional methods in terms of speed, efficiency and the ability to target specific active ingredients.

[0046] The present invention also provides a specific preferred embodiment, wherein the method for separating the active ingredient of the extract of *Acer palmatum* 'Jinling Huangfeng' used in the prevention and treatment of breast cancer includes the following steps:

[0047] (1) Take the dried branches and leaves of Japanese maple 'Jinling Yellow Maple', crush them and pass them through a 60-mesh sieve to obtain dried leaf powder; add 25-30 ml of 80% ethanol aqueous solution to each 1 g of dried leaf powder, and ultrasonically extract at 55℃ for 2 h; after extraction, filter to obtain the extract, concentrate to remove the solvent, and obtain crude extract.

[0048] (2) The crude extract obtained in step (1) was preparatively separated using a high-speed countercurrent chromatograph. The solvent system used was ethyl acetate-n-butanol-water with a volume ratio of 4:1:5. 1000 mL of the mixed solvent was placed in a separatory apparatus, shaken thoroughly, and allowed to stand for separation. The upper organic phase was used as the stationary phase, and the lower aqueous phase was used as the mobile phase.

[0049] (3) First, pump the stationary phase into the high-speed countercurrent chromatograph at a flow rate of 15 mL / min, then start the main unit, set it to forward rotation mode, and stabilize the rotation speed at 900 rpm; pump the mobile phase into the column at a flow rate of 2 mL / min; after the two phases reach dynamic equilibrium in the column, inject 50 mg of crude extract dissolved in 5 mL of upper organic phase and 5 mL of lower aqueous phase; continue to pump the mobile phase into the column at a flow rate of 2 mL / min for elution, and collect the fraction according to the signal of the ultraviolet detector at a wavelength of 254 nm;

[0050] (4) Concentrate and dry to obtain three active ingredients.

[0051] Beneficial effects:

[0052] 1. This invention directly demonstrates through scratch repair experiments that the extract of *Acer palmatum* 'Jinling Huangfeng' significantly inhibits the migration ability of highly metastatic breast cancer cells MDA-MB-231, and this inhibitory effect exhibits a clear dose-dependent relationship. Compared with the solvent control group, the cell scratch healing ability significantly decreased with increasing extract concentration, providing direct and powerful evidence that the extract of *Acer palmatum* 'Jinling Huangfeng' possesses excellent anti-tumor metastasis potential.

[0053] 2. This invention, through systematic verification at the cellular and molecular levels, has for the first time identified the extract of *Acer palmatum* 'Jinling Huangfeng'.

[0054] Its clear efficacy in inhibiting breast cancer metastasis and its unique mechanism of action provide valuable lead compounds and scientific evidence for its further development into a novel, natural anti-tumor metastasis drug or health product.

[0055] 3. Significantly improved separation efficiency and purity, with a clear objective: This invention employs high-speed countercurrent chromatography (HSCCC) technology.

[0056] This technique overcomes the problem of irreversible adsorption of active ingredients by traditional silica gel column chromatography, and enables the efficient and high-recovery preparation of active components from the extract of Japanese maple 'Jinling Huangfeng'.

[0057] 4. The method for isolating the active ingredients of the extract of Acer palmatum 'Jinling Huangfeng' in this invention is closely linked with the screening of anti-breast cancer metastasis activity, which makes the experimental process have clear pharmacological guidance, greatly improves the efficiency and success rate of discovering active ingredients, and provides high-value candidate resources for the development of anti-metastasis drugs. Attached Figure Description

[0058] Figure 1 shows the scratch inhibition experiment results of the extract of Acer palmatum 'Jinling Huangfeng' in this invention on MDA-MB-231 cells; wherein, Figure 1A shows the scratch experiment of Acer palmatum 'Jinling Huangfeng' extract on MDA-MB-231 cells, scale bar = 100μm; Figure 1B shows the quantitative results of the scratch experiment; n = 3, x ± SD;

[0059] Figure 2 shows the effect of the extract of *Acer palmatum* 'Jinling Huangfeng' from this invention on the expression of E-cadherin, N-cadherin, Vimentin, and Twist1 proteins in MDA-MB-231 cells; wherein, Figure 2A shows the Western blot experiment of the extract of *Acer palmatum* 'Jinling Huangfeng' on MDA-MB-231 cells; Figure 2B shows the quantitative results of the Western blot experiment; n = 3, x±SD;

[0060] Figure 3 shows the high performance liquid chromatography detection results of the active ingredients in the extract of Acer palmatum 'Jinling Huangfeng' in Example 3 of the present invention;

[0061] Figure 4 is a chromatogram of crude extract of Acer palmatum 'Jinling Huangfeng' separated by high-speed countercurrent chromatography in Example 4 of the present invention. Detailed Implementation

[0062] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0063] In this embodiment of the invention, the Japanese maple 'Jinling Yellow Maple' was obtained from the Jiangsu Academy of Agricultural Sciences.

[0064] Example 1: Preparation of extract from Japanese maple 'Jinling Yellow Maple'

[0065] (1) Take the dried leaves of Japanese maple 'Jinling Yellow Maple', crush them and pass them through a 60-mesh sieve to obtain dried leaf powder;

[0066] (2) Add 200 mL of 80% ethanol aqueous solution to 8 g of dried leaf powder and extract by ultrasonication (ultrasonic frequency 250 W) at 55 °C for 2 h. After extraction, filter to obtain the extract, remove the solvent by vacuum rotary evaporator, and finally obtain 343 mg of crude extract, namely the extract of Japanese maple 'Jinling Yellow Maple'.

[0067] Example 2: Cell scratch assay to determine the ability of the extract to inhibit cell migration

[0068] Human breast cancer MDA-MB-231 cells in logarithmic growth phase (Chinese Academy of Sciences Cell Bank, Shanghai) were used at a density of 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μL in 6-well plates. When the cells adhered and grew to 90% confluence, a straight line was drawn on the cell monolayer in each well using a 200 µL sterile pipette tip. The cells were then gently washed three times with PBS to remove any detached cells.

[0069] Subsequently, the culture medium was replaced with experimental medium containing 5 μg / mL and 10 μg / mL of *Acer palmatum* 'Jinling Huangfeng' extract (the crude extract of *Acer palmatum* prepared in Example 1, dissolved in DMEM culture medium (Grace Biotech, Shanghai)), and control medium containing only an equal volume of solvent (DMEM culture medium) (i.e., *Acer palmatum* extract concentration 0 μg / mL). Cells were cultured for another 24 h at 37°C in a 5% CO2 incubator. Observations and photographs were taken under an inverted microscope at 0 h and 24 h after scratching.

[0070] Use ImageJ software to measure the scratch width and calculate the scratch healing rate using the following formula:

[0071] Scratch healing rate = (0h scratch width - 24h scratch width) / 0h scratch width × 100%.

[0072] The experimental results are shown in Figure 1. Compared with the solvent control group, cells treated with 5 μg / mL and 10 μg / mL *Acer palmatum* extract showed significantly reduced scratch healing ability, and the inhibitory effect was dose-dependent (Figure 1A, Figure 1B). Statistical analysis showed that compared with the solvent control group, p < 0.01, *p < 0.001. These results demonstrate that the extract of *Acer palmatum* 'Jinling Huangfeng' can significantly inhibit the migration ability of MDA-MB-231 cells.

[0073] Example 3: Western blot experiment to elucidate the mechanism of anti-metastasis.

[0074] To explore its mechanism of action at the molecular level, we performed a Western blotting experiment. MDA-MB-231 cells (Chinese Academy of Sciences Cell Bank) were digested and seeded in six-well plates at a density of 500,000 per well. After adhesion, extracts of *Acer palmatum* 'Jinling Huangfeng' (crude extract of *Acer palmatum* prepared in Example 1) at concentrations of 0, 5, and 10 μg / mL (dissolved in DMEM medium) were added, and the plates were incubated at 37°C for 24 h.

[0075] After processing, cells were collected and lysed on ice for 30 minutes with RIPA lysis buffer. The cells were then centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using the BCA method. An equal volume of protein sample was mixed with loading buffer and heated at 100°C for 10 minutes to denature the protein. SDS-PAGE electrophoresis was then performed, followed by transfer to a PVDF membrane.

[0076] After transfer, the PVDF membrane was blocked with 5% skim milk for 2 hours, and then incubated overnight at 4°C with specific primary antibodies (antibodies E-cadherin (4A2), N-cadherin, Vimentin (D21H3), Twist1, and internal control β-actin antibody (Abcam)). The next day, after washing with TBST, it was incubated at room temperature for 2 hours with the corresponding horseradish peroxidase-labeled secondary antibody (Horseradish Peroxidase, HRP (Abcam)). Finally, it was developed and quantitatively analyzed using ECL chemiluminescence reagent on a ChemiDOC™ XRS+ imaging system.

[0077] The experimental results are shown in Figure 2. Compared with the control group containing an equal amount of solvent (i.e., the concentration of Acer palmatum extract was 0 μg / mL), the protein expression level of the epithelial marker E-cadherin was significantly upregulated with increasing concentration of Acer palmatum extract; while the protein expression levels of the mesenchymal markers N-cadherin, Vimentin, and the transcription factor Twist1 were downregulated in a concentration-dependent manner (Figure 2A, Figure 2B). These results clearly indicate that the extract of Acer palmatum 'Jinling Huangfeng' exerts its activity in inhibiting breast cancer cell migration and invasion by inhibiting the key process of epithelial-mesenchymal transition.

[0078] This invention further reveals the molecular mechanism of *Acer palmatum* 'Jinling Yellow Maple' through Western blotting experiments.

[0079] Mechanism of the anti-metastasis effect of the extract. Experimental results showed that the extract (i.e., crude extract) upregulated the expression of the epithelial marker E-cadherin in a concentration-dependent manner, while downregulating the expression of mesenchymal markers N-cadherin and Vimentin, as well as the key transcription factor Twist1.

[0080] This series of evidence conclusively demonstrates that the *Acer palmatum* 'Jinling Huangfeng' extract provided by this invention exerts its effect of inhibiting the invasion and migration of breast cancer cells by inhibiting the epithelial-mesenchymal transition (EMT), a key process in tumor metastasis, thus providing solid theoretical support for its efficacy.

[0081] Example 3: Detection of active ingredients in the extract of Japanese maple 'Jinling Yellow Maple'

[0082] The active ingredients of the extract of Acer palmatum 'Jinling Huangfeng' prepared in Example 1 were detected by high performance liquid chromatography.

[0083] The specific method is as follows:

[0084] An Agilent 1260 high-performance liquid chromatograph was used with an Agilent 20RBAX SB-C18 column (4.6 mm × 150 mm × 5 μm), a detection wavelength of 365 nm, a mobile phase of methanol-water (0-20 min 10%-100% methanol), a column temperature of room temperature, a flow rate of 1 mL / min, and an injection volume of 10 μL.

[0085] Detection results: Liquid chromatography analysis revealed three main active ingredients in the extract (crude extract) of *Acer palmatum* 'Jinling Huangfeng': compound i, compound ii, and compound iii. The results are shown in Figure 3.

[0086] Example 4 Screening of solvent systems for high-speed countercurrent chromatography

[0087] Example 3, based on high-performance liquid chromatography (HPLC) analysis, revealed three main active ingredients in the *Acer palmatum* 'Jinling Huangfeng' extract prepared in Example 1. This invention employs high-speed countercurrent chromatography (HSCLC) for preparative separation. This example also includes screening of the HSCLC solvent system.

[0088] In this embodiment, the screening process of the high-speed countercurrent chromatography solvent system for the extract of Acer palmatum 'Jinling Huangfeng' consists of two steps:

[0089] 1. Initial screening of polar systems: The ethyl acetate-methanol-water system was tested. It was found that with increasing methanol content, the K values ​​of all components increased sharply (from 1.67 to >10, see Table 1), indicating that in this system, the three compounds exhibited excessively strong affinity for the methanol-rich polar upper phase, resulting in K values ​​far exceeding the applicable window of HSCCC (generally, K>2 is considered undesirable). Therefore, this system was abandoned because it could not achieve effective elution within a reasonable time.

[0090] 2. Optimization of the hydrophobic system: An ethyl acetate-n-butanol-water system was adopted. The proportion of the more hydrophobic n-butanol was gradually increased (from 0 to 1 and then to 2), while the proportion of ethyl acetate was correspondingly decreased. When the ratio was adjusted to 4:1:5, the K values ​​of all three compounds (0.25, 1.36, 3.29, see Table 1) were all within the adjustable range. The K value of compound ii (1.36) was already in the golden range (1~1.5), and the K values ​​of compounds i and iii also approached the ideal range, showing good separation potential. However, when the n-butanol ratio was further increased to 3:2:5, the K values ​​again showed a "surge" trend similar to the first system (Ki=2.80, Kii=6.21, Kiii>10, see Table 1), indicating that the system was too hydrophobic at this point, causing excessive partitioning of compounds to the upper phase.

[0091] Table 1. Partition coefficients K of i-iii in extracts of Acer palmatum 'Jinling Huangfeng' under different solvent systems a value

[0092] Solvent system (v / v) K i K ii K iii Ethyl acetate-methanol-water (10:4:6) 1.67 8.38 9.02 Ethyl acetate-methanol-water (10:5:5) 3.22 9.89 >10 Ethyl acetate-methanol-water (10:6:4) 6.90 >10 >10 Ethyl acetate-n-butanol-water (5:0:5) 0.02 0.27 1.01 Ethyl acetate-n-butanol-water (4:1:5) 0.25 1.36 3.29 Ethyl acetate-n-butanol-water (3:2:5) 2.80 6.21 >10 surface

[0093] a K = A U / A L , where A U and A L These represent the absorption peak areas of the compounds in the upper and lower phases, respectively.

[0094] Based on the above data, ethyl acetate-n-butanol-water (4:1:5, v / v) was identified as the solvent system with the greatest application potential. Under these conditions, compound ii had an ideal K value (1.36), indicating that it could achieve separation with good peak shape and moderate elution time. Although the K values ​​of compounds i (K=0.25) and iii (K=3.29) were not entirely within the core range of 0.5-2, they were on the edge of acceptable ranges, and the differences in K values ​​among the three compounds were significant, resulting in a sufficiently large separation factor (α). This laid a solid foundation for subsequent elution using forward rotation mode to achieve baseline separation.

[0095] Example 5: Extraction and separation of active ingredients from *Acer palmatum* 'Jinling Huangfeng' extract

[0096] In this embodiment, high-speed countercurrent chromatography was used to preparatively separate the three main active components present in the extract of *Acer palmatum* 'Jinling Huangfeng' obtained in Example 1. The specific steps are as follows:

[0097] (1) Preparative separation of the extract (i.e., crude extract) of Acer palmatum 'Jinling Huangfeng' was performed using a TBE-300C high-speed countercurrent chromatograph. The selected solvent system was ethyl acetate-n-butanol-water with a volume ratio of 4:1:5. 1000 mL of this mixed solvent was placed in a separatory funnel, shaken thoroughly, and allowed to stand for separation. The upper organic phase was used as the stationary phase, and the lower aqueous phase was used as the mobile phase.

[0098] (2) The separation process is as follows: First, the stationary phase is pumped into the main separation column at a flow rate of 15 mL / min. Then, the main unit is started and set to forward rotation mode, with the rotation speed stabilized at 900 rpm. Next, the mobile phase is pumped into the column at a flow rate of 2 mL / min. After the two phases reach dynamic equilibrium in the column (i.e., a stable mobile phase flows out of the pipeline outlet), 50 mg of crude extract (dissolved in a mixed solvent of 5 mL upper phase and 5 mL lower phase) is injected through a six-way valve. Elution is continued by pumping the mobile phase at a flow rate of 2 mL / min, and the fraction is collected according to the signal of the ultraviolet detector at a wavelength of 254 nm.

[0099] The chromatogram of crude extract of *Acer palmatum* 'Jinling Huangfeng' separated by high-speed countercurrent chromatography is shown in Figure 4. Based on the chromatogram, three major fractions were collected at 240-300 min (fraction 1), 300-400 min (fraction 2), and 400-500 min (fraction 3). Mass spectrometry and nuclear magnetic resonance identification confirmed that fractions 1, 2, and 3 were vitexin, vitexin, and isovitexin, respectively.

[0100] Identification of the active ingredient structure:

[0101] Component 1: Herba viridissin, its structural formula is:

[0102] ;

[0103] 1H NMR (500 MHz, DMSO) δ 13.17 (s, 1H), 10.81 (s, 1H), 10.04 (s, 1H), 9.05 (s, 1H), 7.5364 (dd, J = 8.3, 2.2 Hz, 1H), 7.4827 (d, J = 2.2 Hz, 1H), 6.8658 (d, J = 8.4 Hz, 1H), 6.6438 (s, 1H), 6.2701 (s, 1H), 3.2606 (d, J =9.2 Hz, 2H).

[0104] 13 C NMR (126 MHz, DMSO) δ 182.31, 164.83, 162.38, 156.29, 149.88,146.09, 122.33, 119.66, 115.95, 104.85, 104.34, 102.71, 98.41, 82.28, 79.08,73.69, 71.08, 61.94.

[0105] The above data are consistent with the data on arbutin in the literature (Fahmy MI, Sadek MA, Abdou K, El-Dessouki AM, El-ShiekhRA, Khalaf SS. Orientin: A Comprehensive Review of A Promising Bioactive Flavonoid. Inflammopharmacology. 2025 Apr;33(4):1713-1728.), therefore the compound was identified as arbutin.

[0106] Component 2: Vitexin, its structural formula is:

[0107] ;

[0108] 1 H NMR (500 MHz, DMSO) δ 13.1653 (s, 1H), 10.8189 (s, 1H), 10.3226(s, 1H), 8.0201 (d, J = 8.7 Hz, 2H), 6.7649(s, 1H), 6.2712 (s, 1H).

[0109] 13C NMR (126 MHz, DMSO) δ181.89, 163.75, 162.34, 160.92, 160.19, 155.79, 128.75 , 121.41, 115.60, 104.42, 103.85, 102.24, 97.93, 81.64, 78.48, 73.18, 70.64, 70.36, 61.10.

[0110] The above data are consistent with the data on vitexin in the literature (Wu JY, Wang TY, Ding HY, Zhang YR, Lin SY, Chang TS. Enzymatic Synthesis of Novel Vitexin Glucosides. Molecules. 2021 Oct 16;26(20):6274), therefore the compound was identified as vitexin.

[0111] Component 3: Isovitilione, its structural formula is:

[0112] ;

[0113] 1 H NMR (500 MHz, DMSO) δ 13.5506 (s, 1H), 10.5614 (s, 1H), 10.3435(s, 1H), 7.9569 (d, J = 8.7 Hz, 2H), 6.7853 (d, J = 8.7 Hz, 2H), 6.5098 (s,1H) , 4.8576 (d, J = 9.9 Hz, 1H).

[0114] 13 CNMR (126 MHz, DMSO) δ181.59, 163.16, 162.86, 160.80, 160.28, 155.85, 128.09, 120.75, 115.61 , 108.52, 103.06, 102.44, 93.26, 81.18, 78.57, 72.69, 70.24, 69.87, 61.10.

[0115] The above data is consistent with the data on isovitexin in the literature (Yan W, Cheng J, Xu B. Dietary Flavonoids Vitexin and Isovitexin: New Insights into Their Functional Roles in Human Health and Disease Prevention. Int J Mol Sci. 2025 Jul 21;26(14):6997.), therefore the compound was identified as isovitexin.

[0116] Example 6: Purity Identification of Three Main Active Ingredients in Extracts of Japanese Maple 'Jinling Yellow Maple'

[0117] The purity of the three main active ingredients in the isolated Acer palmatum 'Jinling Huangfeng' extract was identified using high-performance liquid chromatography (HPLC), with the instruments and detection parameters as described in Example 3. The results showed that the purity of the three main active ingredients in the Acer palmatum 'Jinling Huangfeng' extract obtained using the high-speed countercurrent chromatography parameters described in Example 5 could reach over 93%, with the purity of purslane and isovitexin reaching 96%.

[0118] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Application of an extract of Japanese maple 'Jinling Huangfeng' in the preparation of drugs for the prevention and treatment of breast cancer.

2. The application according to claim 1, characterized in that, The application of the extract of Acer palmatum 'Jinling Huangfeng' in the preparation of drugs that inhibit breast cancer cell migration.

3. The application according to claim 1, characterized in that, The extract of the Japanese maple 'Jinling Yellow Maple' is an ethanol extract with a mass concentration of 80%.

4. The application according to claim 1, characterized in that, The active ingredients in the extract of the Japanese maple 'Jinling Yellow Maple' include vitexin, vitexin and isovitexin.

5. The application according to claim 1, characterized in that, The concentration of the extract of the Japanese maple 'Jinling Yellow Maple' that inhibits the migration of breast cancer cells is 1-10 μg / mL.

6. A method for extracting the extract of *Acer palmatum* 'Jinling Huangfeng' as described in any one of claims 1-5, applicable to the prevention and treatment of breast cancer, characterized in that, The steps include: (1) taking dried leaves of Japanese maple 'Jinling Yellow Maple', crushing them and passing them through a 60-mesh sieve to obtain dried leaf powder; (2) using 80% ethanol aqueous solution to ultrasonically extract the dried leaf powder obtained in step (1), filtering to obtain the extract, evaporating the solvent to obtain crude extract, which is the Japanese maple 'Jinling Yellow Maple' extract.

7. The extraction method according to claim 6, characterized in that, Add 25-30 ml of 80% ethanol aqueous solution to every 1 g of dried leaf powder.

8. The extraction method according to claim 6, characterized in that, In step (2), the ultrasonic extraction power is 200-260W, the temperature is 55-60℃, and the time is 2-2.5h.

9. A method for separating the active ingredient from the extract of *Acer palmatum* 'Jinling Huangfeng' as described in any one of claims 1-5, used in the prevention and treatment of breast cancer, characterized in that... The process includes the following steps: (1) Preparation of crude extract: Take the dried leaves of Acer palmatum 'Jinling Huangfeng', crush them and pass them through a 60-mesh sieve to obtain dried leaf powder; use 80% ethanol aqueous solution to ultrasonically extract the obtained dried leaf powder, filter to obtain the extract, evaporate the solvent to obtain crude extract, which is the Acer palmatum 'Jinling Huangfeng' extract; (2) Separation of active ingredients: Use high-speed countercurrent chromatography to separate the crude extract obtained in step (1) using an eluent according to the elution program, and collect the target active fraction; the eluent is selected from ethyl acetate-n-butanol-water solvent system, ethyl acetate-water solvent system or ethyl acetate-methanol-water solvent system; The separation conditions include: main unit speed 800-1000 rpm; mobile phase flow rate 1.5-2.5 mL / min; stationary phase flow rate 15-20 mL / min; (3) concentration and drying to obtain three active ingredients.

10. The separation method according to claim 9, characterized in that, In step (2), the eluent is selected from the ethyl acetate-n-butanol-water solvent system, and the volume ratio of ethyl acetate, n-butanol and water is 4:1:5; the separation conditions are: main unit speed 900 rpm; mobile phase flow rate 2 mL / min; stationary phase flow rate 15 mL / min.

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