A method for extracting two tocopherol derivatives A and B from epimedium sagittatum and application thereof

Abstract

A method for extracting two tocopherol derivatives A and B from Epimedium sagittatum and application thereof can effectively solve the problem of preparing tocopherol derivatives A and B from Epimedium sagittatum and realize the application in preparing anti-breast tumor drugs. The method is as follows: the aboveground part of Epimedium sagittatum is crushed, extracted by refluxing with ethanol, recovered under reduced pressure, extracted, separated by chromatography, eluted by gradient, recovered under reduced pressure, identified by thin layer spotting, combined, purified, collected in the retention time, dried, and obtained. The raw material is abundant, the preparation method is easy to operate, the obtained product is good in quality and good in use effect, can be effectively used for treating breast cancer, realizes the application in preparing anti-breast cancer drugs, develops the medicinal value and commercial value of Epimedium sagittatum, and has remarkable economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to a method for extracting two tocopherol derivatives A and B from Epimedium sagittatum and their applications. Background Technology

[0002] Epimedium sagittatum, also known as three-branched nine-leaf grass, is a perennial herb belonging to the genus Epimedium in the family Berberidaceae. It is mainly distributed in Zhejiang, Anhui, Fujian, Jiangxi, Hubei, and Hunan provinces of China. Records of Epimedium sagittatum can be traced back to the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), where it is described as having the effects of tonifying kidney yang, strengthening muscles and bones, and dispelling wind and dampness. Modern research shows that Epimedium sagittatum contains various structural compounds, including flavonoids, glycosides, lignans, alkaloids, and phenolic acids, and possesses anti-tumor, antioxidant, anti-osteoporosis, and antidepressant activities.

[0003] Breast cancer is a common and prevalent disease that seriously threatens people's lives and health, especially breast cancer caused by human breast cancer cells MCF-7. Although there are currently therapeutic drugs, their effectiveness is limited. This invention identifies two novel tocopherol derivatives, A (12'-hydroxy-δ-tocopherol) and B (12'-hydroxy-α-tocopherol), from the dichloromethane extract of Epimedium sagittatum. Both derivatives significantly inhibit the cell viability of human breast cancer cells MCF-7 and are non-toxic to normal human breast cells MCF-10A. Therefore, these two compounds may be the pharmacologically active substances of Epimedium sagittatum that exert its anti-breast cancer activity. Furthermore, 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol were found to significantly inhibit sphingosine kinase 1 (Sphk1) in MCF-7 cells, suggesting that these two compounds may exert their anti-breast cancer effect by inhibiting Sphk1 expression levels. They hold promise as lead compounds for anti-breast cancer drugs, providing technical support for their development. However, there are no publicly available reports on how to extract the effective active ingredients 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol from Epimedium sagittatum and use them to prepare drugs for treating breast tumors (cancer). Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for extracting two tocopherol derivatives A and B from Epimedium sagittatum and their applications, which can effectively solve the problem of preparing tocopherol derivatives A and B from Epimedium sagittatum and realizing their application in the preparation of anti-breast tumor drugs.

[0005] The technical solution provided by this invention is: two tocopherol derivatives A and B extracted from Epimedium sagittatum, with the following chemical molecular formulas:

[0006]

[0007]

[0008] The preparation method is as follows:

[0009] (1) The aerial parts of dried Epimedium brevicornu were crushed, extracted by reflux with ethanol, and recovered under reduced pressure to obtain an extract. The extract was suspended in distilled water and extracted sequentially with petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature to obtain extracts of each part. The dichloromethane extract was separated by chromatography and eluted with gradient to obtain the polar fraction.

[0010] (2) The target polar segment was subjected to chromatography, gradient elution, vacuum recovery, thin-layer chromatography, and similar components were combined to obtain the first group of fractions.

[0011] (3) The first target group is separated by stratification, gradient elution, vacuum recovery, thin-layer chromatography, and similar components are combined to obtain the second group.

[0012] (4) Purify the second target group and collect the samples after retention time t. R = 45.0 min fraction, dried to obtain tocopherol derivative B (12'-hydroxy-α-tocopherol);

[0013] (5) Chromatography of the target polar segment, gradient elution, vacuum recovery, thin-layer chromatography, and combination of similar components to obtain the third group;

[0014] (6) The third group was eluted by gradient, recovered under reduced pressure, spotted in thin layer lithography, and similar components were combined to obtain the fourth group;

[0015] (7) The fourth group was eluted by gradient, recovered under reduced pressure, spotted in thin layer lithography, and similar components were combined to obtain the fifth group;

[0016] (8) The fifth group was eluted by gradient, recovered under reduced pressure, spotted in thin layer lithography, and similar components were combined to obtain the sixth group;

[0017] (9) Purify the sixth group and collect the samples with retention time t. R =36.5 min fraction, dried to obtain tocopherol derivative A (12'-hydroxy-δ-tocopherol).

[0018] The application of two tocopherol derivatives A and B extracted from Epimedium sagittatum in the preparation of anti-breast cancer drugs.

[0019] This invention utilizes abundant raw materials and employs an easy-to-operate preparation method. Both compounds significantly inhibit the cell viability of human breast cancer cells MCF-7 and are non-toxic to normal human breast cells MCF-10A. They also significantly inhibit sphingosine kinase 1 (Sphk1) in MCF-7 cells, indicating that these two compounds can exert their anti-breast cancer effect by inhibiting Sphk1 expression levels. They hold promise as lead compounds for anti-breast cancer treatment, providing technical support for the development of anti-breast cancer drugs. The resulting products are of high quality and effective, and can be effectively used to treat breast cancer, realizing their application in the preparation of anti-breast cancer drugs. This expands the medicinal and commercial value of Epimedium sagittatum, yielding significant economic and social benefits. Attached Figure Description

[0020] Figure 1 The chemical molecular structure diagrams of tocopherol derivatives A and B of the present invention are shown.

[0021] Figure 2 This is a diagram showing the lowest energy conformation of tocopherol derivatives A and B generated by molecular docking in this invention and their binding to Sphk1. The compounds are shown in a ball-and-stick model (carbon: yellow, hydrogen: gray, oxygen: red), and the blue line shows the key hydrogen bond interactions with the enzyme.

[0022] Figure 3 The tocopherol derivative A of the present invention 1 1H NMR spectrum (500MHz, deuterated reagent: CDCl3);

[0023] Figure 4 DEPT and the tocopherol derivative A of the present invention 13 C10 NMR spectrum (125 MHz, deuterated reagent: CDCl3);

[0024] Figure 5 The HSQC spectrum (500 MHz, deuterated reagent: CDCl3) of the tocopherol derivative A of the present invention is shown.

[0025] Figure 6 The HMBC spectrum of tocopherol derivative A of the present invention (deuterated reagent: CDCl3);

[0026] Figure 7 The tocopherol derivative A of the present invention 1 H- 1 HCl COSY spectrum (deuterated reagent: CDCl3);

[0027] Figure 8 The NOESY spectrum of tocopherol derivative A of the present invention (deuterated reagent: CDCl3);

[0028] Figure 9The HRMSESI spectrum of tocopherol derivative A of the present invention is shown below.

[0029] Figure 10 The UV spectrum of tocopherol derivative A of the present invention is shown below.

[0030] Figure 11 The IR spectrum of tocopherol derivative A of the present invention is shown below.

[0031] Figure 12 The tocopherol derivative B of the present invention 1 1H NMR spectrum (500MHz, deuterated reagent: CDCl3);

[0032] Figure 13 DEPT and the tocopherol derivative B of the present invention 13 C10 NMR spectrum (125 MHz, deuterated reagent: CDCl3);

[0033] Figure 14 The HSQC spectrum of tocopherol derivative B of the present invention (deuterated reagent: CDCl3);

[0034] Figure 15 The HMBC spectrum of the tocopherol derivative B of the present invention (deuterated reagent: CDCl3);

[0035] Figure 16 The tocopherol derivative B of the present invention 1 H- 1 HCl COSY spectrum (deuterated reagent: CDCl3);

[0036] Figure 17 The NOESY spectrum of tocopherol derivative B of the present invention (deuterated reagent: CDCl3);

[0037] Figure 18 The HRMSESI spectrum of tocopherol derivative B of the present invention is shown below.

[0038] Figure 19 The UV spectrum of tocopherol derivative B of the present invention is shown below.

[0039] Figure 20 The image shows the IR spectrum of tocopherol derivative B of this invention. Detailed Implementation

[0040] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0041] Two tocopherol derivatives, A and B, extracted from Epimedium sagittatum, have the following chemical molecular formulas:

[0042]

[0043] The preparation method is as follows:

[0044] (1) 80 kg of dried Epimedium brevicornu aerial parts were crushed and extracted three times with 70% ethanol at 45°C under reflux, with 120 L of ethanol each time, for 30 min. The extracts were combined and recovered under reduced pressure to obtain 6.5 kg of extract. The extract was suspended in three times its volume of distilled water to obtain a suspension. The suspension was extracted four times with petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature, for 2 h each time, with 15 L of ethanol each time, to obtain petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract, respectively. The extract (2.1 kg) was separated by normal-phase silica gel column chromatography using a 100-200 mesh filter. A gradient elution was performed using petroleum ether:ethyl acetate at volume ratios of 50:1 (12 L), 40:1 (20 L), 35:1 (20 L), 20:1 (40 L), 10:1 (40 L), 5:1 (40 L), 1:1 (20 L), and 0:1 (4 L). Similar fractions were combined based on the TLC results, yielding eight polar fractions: Fr.A, Fr.B…Fr.F…Fr.H.

[0045] (2) The polar fraction Fr.F (13.7 g) was subjected to normal phase silica gel column chromatography with a volume ratio of petroleum ether:ethyl acetate. The gradient ratios were 35:1 (1 L), 20:1 (3 L), 10:1 (3 L), 5:1 (3 L), 1:1 (2 L), and 0:1 (1 L). The eluents were combined in 500 mL increments, and the eluents were recovered under reduced pressure. The fractions were then spotted onto a thin-layer chromatography plate by TLC. The fractions with similar absorption were combined to obtain 15 first-group fractions Fr.F1, Fr.F2…Fr.F12…F15.

[0046] (3) The first group fraction Fr.F12 (1.0 g) was subjected to ODS reversed-phase silica gel column chromatography with gradient elution of MeOH:H2O at volume ratios of 40:60 (1 L), 50:50 (1 L), 60:40 (2 L), 70:30 (2 L), 80:20 (2 L), 90:10 (1 L), and 100:0 (1 L). The eluent was combined in 200 mL increments and the eluent was recovered under reduced pressure to obtain multiple fractions. The fractions were then spotted onto a thin-layer plate by TLC, and the fractions with similar absorption were combined to obtain 10 second group fractions Fr.F12.1…Fr.F12.8…F12.10.

[0047] (4) The second group of Fr.F12.8 (70.0 mg) was purified by semi-preparative high performance liquid chromatography (HPLC) using a RP C18 column with a column volume of 10 ID × 250 mm. The purification solution was acetonitrile:water at a volume ratio of 85.5:14.5, the flow rate was 2 mL / min, and the retention time was collected. R = 45.0 min fraction, dried to obtain tocopherol derivative B (12'-hydroxy-α-tocopherol, 17.6 mg);

[0048] (5) The polar fraction Fr.G (100.0g) was subjected to normal phase silica gel column chromatography with a volume ratio of petroleum ether:ethyl acetate. The gradient ratios were 50:1 (1L), 35:1 (5L), 20:1 (5L), 10:1 (5L), 5:1 (5L), 1:1 (2L), and 0:1 (1L). Each 1L of eluent was combined and the eluent was recovered under reduced pressure to obtain multiple fractions. The fractions with similar absorption on the thin-layer plate were combined by TLC to obtain 18 third groups: Fr.G1, Fr.G2...Fr.G17, Fr.G18.

[0049] (6) The third group fraction Fr.G17 (65.0g) was eluted with a gradient of petroleum ether:ethyl acetate in a volume ratio of 200-300 mesh normal phase silica gel. The gradient ratios were 35:1 (1L), 20:1 (4L), 10:1 (4L), 5:1 (4L), 1:1 (2L), and 0:1 (1L). The eluents were combined in 500mL increments and the eluents were recovered under reduced pressure to obtain multiple fractions. The fractions were then spotted onto a thin-layer chromatography (TLC) plate, and the fractions with similar absorption on the TLC plate were combined to obtain 19 fourth group fractions Fr.G17.1…Fr.G17.15…Fr.G17.19.

[0050] (7) The fourth group fraction Fr.G17.15 (38.9g) was eluted by ODS column chromatography with a gradient of MeOH:H2O at volume ratios of 40:60 (1L); 50:50 (2L); 60:40 (3L); 70:30 (3L); 80:20 (3L); 90:10 (2L); and 100:0 (1L). The eluent was combined in 500mL increments and the eluent was recovered under reduced pressure to obtain multiple fractions. The fractions were then spotted onto a thin-layer plate by TLC and the fractions with similar absorption were combined to obtain 12 fifth group fractions Fr.G17.15.1…Fr.G17.15.8…Fr.G17.15.12.

[0051] (8) The fifth group fraction Fr.G17.15.8 (0.49 g) was eluted by ODS column chromatography with a gradient of MeOH:H2O at a volume ratio of 50:50 (1 L); 60:40 (2 L); 70:30 (3 L); 80:20 (3 L); 90:10 (1 L); and 100:0 (1 L). The eluent was combined in 300 mL increments and the eluent was recovered under reduced pressure to obtain multiple fractions. The fractions with similar absorption on the thin-layer plate were combined by TLC to obtain 14 sixth group fractions Fr.G17.15.8.1…Fr.G17.15.8.14.

[0052] (8) The sixth group of Fr.G17.15.8.14 (8.1 mg) was purified by semi-preparative HPLC RP C18 column with a column volume of 10ID×250 mm. The purification solution was acetonitrile:water = 85:15 (v / v), the flow rate was 2 mL / min, and the retention time was collected. R The fraction was discharged for 36.5 min, dried, and to obtain tocopherol derivative A (12'-hydroxy-δ-tocopherol, 5.1 mg).

[0053] The application of the two tocopherol derivatives A and B prepared by the above method in the preparation of anti-breast cancer drugs.

[0054] The embodiments given above are merely illustrative of specific implementations of the present invention and are not intended to limit the scope of protection of the present invention. Any technical solution that is essentially the same as the technology of this application made by using equivalent substitutions or modifications falls within the scope of protection of the present invention. The tocopherol derivatives A and B can be extracted and prepared in any amount as needed according to the above preparation method.

[0055] This invention utilizes abundant raw materials and an easy-to-operate preparation method to effectively extract tocopherol derivatives A and B from Epimedium sagittatum. These tocopherol derivatives significantly inhibit the cell viability of human breast cancer cells MCF-7 and are non-toxic to normal human breast cells MCF-10A. They also significantly inhibit sphingosine kinase 1 (Sphk1) in MCF-7 cells, indicating that these two compounds can exert anti-breast cancer effects by inhibiting Sphk1 expression levels. They are expected to become lead compounds for anti-breast cancer treatment, providing technical support for the development of anti-breast cancer drugs. The resulting products are of high quality and have good efficacy, effectively treating breast cancer and enabling their application in the preparation of anti-breast cancer drugs. Experiments have yielded very good and beneficial technical results. The relevant experimental materials are as follows:

[0056] I. Instruments and Materials

[0057] 1.1 Instruments:

[0058] High-resolution mass spectrometer Bruker Maxis HD (Bruker, Germany)

[0059] Evolution 300 Ultraviolet Spectrometer (Thermo, MA, USA)

[0060] Nicolet IS10 infrared spectrometer (Thermo Scientific, USA)

[0061] Bruker Avance III 500 nuclear magnetic resonance spectrometer (Bruker, Germany)

[0062] High-performance liquid chromatograph (HPLC) Shimadzu LC-40, equipped with a DAD detector and an RPC18 column (10ID×250mm, Cosmosil 5C18-MS-IIPacked column, Nacalai Tesque, China).

[0063] N-1100 rotary evaporator and N-1111 chilled water circulation system (Shanghai Ailang Instrument Co., Ltd.)

[0064] BT25S Sartorius precision analytical balance (1 / 100,000)

[0065] Real-time label-free cell system (Agilent Technologies)

[0066] Thermo 3111 CO2 Incubator

[0067] Centrifuge-5804R High-Speed ​​Centrifuge (Eppendorf)

[0068] Multiskan MK3 microplate reader (Thermo Fisher)

[0069] Clean bench (Suzhou Cleanroom Group)

[0070] HVA-85 Autoclave (Hirayama)

[0071] 1.2 Materials:

[0072] Reverse silica gel (ODS, 50μm, YMC Group, Kyoto, Japan)

[0073] Normal phase silica gel (100-200 and 200-300 mesh, TOSOHCorp., Tokyo, Japan)

[0074] Chromatographically pure methanol and acetonitrile (Tianjin Siyou Fine Chemicals Co., Ltd.)

[0075] Analytical grade methanol, dichloromethane, ethyl acetate, and petroleum ether (Beijing Chemical Plant and Tianjin No. 3 Chemical Reagent Plant) were tested.

[0076] 16-well plate (Agilent Technologies)

[0077] Culture dishes, 96-well culture plates, cell cryopreservation tubes (Corning)

[0078] Fetal bovine serum (Hangzhou Sijiqing Company), DMEM culture medium (Gibco Invitrogen)

[0079] Thiazole Blue MTT (Beijing Solarbio Technology Co., Ltd.)

[0080] Ampicillin, Streptomycin (Sigma)

[0081] Dimethyl sulfoxide (DMSO) (Shanghai Maclean Biotechnology Co., Ltd.)

[0082] Ham's F12K medium (Pronosei Corporation)

[0083] Docetaxel (Shanghai Yuanye Biotechnology Co., Ltd.)

[0084] Human normal breast cells MCF-10A and human breast cancer cells MCF-7 (Shanghai Cell Bank, Chinese Academy of Sciences)

[0085] Plant material: The Epimedium sagittatum used in this study was collected in September 2020 from Zhumadian, Henan Province, and identified by Professor Chen Suiqing of Henan University of Traditional Chinese Medicine. The specimen is deposited at Henan University of Traditional Chinese Medicine, with the preservation number 20200960.

[0086] II. Structural Identification

[0087] 2.1. Tocopherol Derivative A:

[0088] According to the high-resolution mass spectrometry data of 12'-hydroxy-δ-tocopherol (A), its molecular ion peak is 441.3345 ([M+Na)). + The calculated value is 441.3339, from which the molecular formula of the compound can be deduced to be C. 27 H 46 O3. Based on the NMR data (see attached figure), the structure of the compound can be deduced as follows:

[0089]

[0090] The 12'-hydroxy-δ-tocopherol structure has three chiral carbons, which can be calculated... 13 C NMR can determine its relative configuration as 2S,4'S,8'R.

[0091] Physicochemical data of 12'-hydroxy-δ-tocopherol: colorless oil; [α] 20 D +4(c 0.08, MeOH); UV(MeOH)λ max (logε):203(4.46),297(3.56)nm; IR(ν max ):3409,2941,2843,1684,1471,1377,1220,1152,1029,935,857cm -1 HRESIMS m / z 441.3345 [M+Na] + (calcd.for C 27 H 46 O3Na, 441.3339). 1 H NMR(500MHz,CDCl3,δin ppm,J in Hz): 1.77(m,H-3a),1.74(m,H-3b),2.68(t,J=6.8,H2-4),6.38(d,J=3.0,H-5),6.48(d,J=3.0,H-7),1.25(s, Me-11),2.12(s,Me-12),1.22(s,Me-13'),0.85(d,J=6.6,Me-14'),0.84(d,J=6.6,Me-15'),1.22(s,Me-16'); 13 C NMR (125MHz, CDCl3, δin ppm): 75.7(C-2),31.6(C-3),22.7(C-4),112.7(C-5),147.9(C-6),115.7(C-7),127.4(C- 8),146.1(C-9),121.4(C-10),24.4(C-11),16.2(C-12),39.7(C-1'),20.9(C-2'),37.5(C- 3'),32.7(C-4'),37.4(C-5'),24.6(C-6'),37.4(C-7'),33.0(C-8'),37.7(C-9'),21.9(C- 10'),44.4(C-11'),71.5(C-12'),29.4(C-13'),19.9(C-14'),19.9(C-15'),29.4(C-16').

[0092] 2.2 Tocopherol Derivative B:

[0093] Based on the high-resolution mass spectrometry of 12'-hydroxy-α-tocopherol(B) and13 Based on the C10 NMR data, its molecular formula can be deduced to be C10. 28 H 48 O3. Based on other NMR spectral data (see attached figure), the structure of the compound can be deduced as follows:

[0094]

[0095] The relative configuration of 12'-hydroxy-α-tocopherol was calculated. 13 C NMR can identify it as 2S, 4'R, 8'R.

[0096] Physicochemical data of 12'-hydroxy-α-tocopherol: colorless oil; [α] 20 D +9(c 0.15,MeOH); UV(MeOH)λ max (logε):204(4.68),295(3.75)nm; IR(ν max ):3396,2929,2863,1711,1463,1416,1377,1231,1158,1062,1009,910cm φ1 HRESIMS m / z 455.3495 [M+Na] + (calcd.forC 28 H 48 O3Na, 455.3496). 1 H NMR(500MHz,CDCl3,δin ppm,J in Hz): 1.81(m,H-3a),1.76(m,H-3b),2.60(t,J=6.9,H2-4),6.48(s,H-7),1.21(s,Me-11),2.09(s,Me-12 ),2.10(s,Me-13),1.22(s,Me-13'),0.85(d,J=6.6,Me-14'),0.84(d,J=6.6,Me-15'),1.22(s,Me-16'); 13C NMR (125MHz, CDCl3, δin ppm): 74.6(C-2),31.7(C-3),20.9(C-4),119.4(C-5),145.9(C-6),115.4(C-7),124.1(C-8), 146.0(C-9),120.5(C-10),24.1(C-11),11.1(C-12),16.0(C-13),39.5(C-1'),21.0(C-2'),37 .6(C-3'),32.8(C-4'),37.4(C-5'),24.6(C-6'),37.4(C-7'),33.0(C-8'),37.7(C-9'),21.9 (C-10'),44.4(C-11'),71.5(C-12'),29.4(C-13'),19.8(C-14'),19.9(C-15'),29.4(C-16').

[0097] III. Activity Test

[0098] 3.1 Cell Culture

[0099] Thaw frozen MCF-10A and MCF-7 cells in a 37°C water bath until they reach an ice-water balance, then immediately centrifuge (1000 rpm, 5 min). Discard the supernatant and transfer the cells to DMEM culture dishes containing 10% FBS (ampicillin and streptomycin, both 100 kU / L). Incubate at 37°C in a 5% CO2 incubator until the cells reach 80%–90% confluence. Passage the cells every 24 hours with fresh culture medium.

[0100] 3.2 Effects of monomeric compounds on the viability of MCF-10A and MCF-7 cells based on MTT assay

[0101] MCF-10A and MCF-7 cells were cultured at 37°C in a 5% CO2 incubator until the logarithmic growth phase, and then cultured at a cell density of 2×10⁶ cells / year. 4 Cells were seeded at a density of 200 μL / well in a 96-well plate. After 24 h, the plates were divided into a normal control group (CON), a 12'-hydroxy-δ-tocopherol group (10 μM), and a 12'-hydroxy-α-tocopherol group (10 μM). After 24 h of culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were cultured for another 4 h. The culture medium was carefully aspirated, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min to completely dissolve the blue-purple crystals. The absorbance (OD) of each well was measured at 490 nm using a microplate reader, and cell viability was calculated. The experiment was repeated in triplicate.

[0102] 3.3 Immunofluorescence detection of Sphk1 levels in MCF-7 cells

[0103] MCF-7 cells were divided into 2×10 4 Cells were seeded at a density of 10 cells / mL in wells. After 24 h, they were divided into a CON group and a compound group (10 μM) and cultured for another 24 h. Immobilization was performed in 4% paraformaldehyde for 15 min, followed by permeabilization with 0.25% Triton X-100 for 10 min. Cells were then blocked with 1% BSA for 30 min, and then incubated overnight at 4°C with primary antibody Sphk1. Cells were washed three times with PBST, counterstained with DAPI for 4 min, washed once with PBS, and then imaged using the OperettaCLS high-content imaging system.

[0104] 3.4 Statistical Analysis

[0105] Experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 26.0. One-way ANOVA was used for comparisons between groups. ** P<0.01 indicates a highly significant difference.

[0106] IV. Activity Evaluation

[0107] This experiment used the MTT assay to detect the effects of two tocopherol derivatives, A and B, on the cell viability of human breast cancer cells MCF-7. The results are shown in Table 1. After treatment with 10 μM of the compounds, the cell viability of MCF-7 cells was significantly reduced compared with the control group (CON), and the results were statistically significant (P<0.01). That is, compounds 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol at a concentration of 10 μM can significantly inhibit the cell viability of human breast cancer cells MCF-7. Then, the effects of the compounds on normal human breast cells MCF-10A were detected, and it was found that neither of the two compounds had a significant effect on the cell viability of MCF-10A cells (Table 1). Based on the above results, it can be inferred that compounds 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol may be the pharmacologically active substances in Epimedium sagittatum that exert anti-breast cancer activity.

[0108] Table 1. Effects of compounds at 10 μM concentration on the viability of MCF-7 and MCF-10A cells.

[0109]

[0110] ** This indicates that P < 0.01; docetaxel is a positive control.

[0111] Sphingosine kinase 1 (SphK1), a key rate-limiting enzyme and intracellular signal transduction enzyme in sphingolipid metabolism, plays a crucial role in the regulation of tumor cell proliferation. Studies have shown that inhibiting SphK1 expression can effectively suppress tumor cell proliferation. Therefore, SphK1 is a highly promising target for cancer therapy, and SphK1 inhibitors hold promise as novel anti-tumor lead compounds.

[0112] The compounds 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol were molecularly docked with Sphk1, respectively. Figure 2 12'-hydroxy-δ-tocopherol and amino acid residue ASP-167 and VAL-346 Hydrogen bonds are formed between 12'-hydroxy-α-tocopherol and amino acid residue SER-165. ARG-277 and THR-282 Hydrogen bonds are formed between them. The lowest binding energy of both compounds with Sphk1 is less than -7 kcal / mol, indicating that these two compounds have a strong binding interaction with Sphk1.

[0113] Then, the effects of compounds 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol on the expression level of Sphk1 in MCF-7 cells were detected using immunofluorescence assay. The results are shown in Table 2. The experimental results show that the fluorescence intensity of Sphk1 in MCF-7 cells treated with 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol monomers was significantly different from that in the control group (P<0.01), indicating that both compounds could significantly reduce the expression level of Sphk1 in MCF-7 cells.

[0114] Table 2. Effects of compounds on Sphk1 levels in MCF-7 cells ( n=3)

[0115]

[0116] ** This indicates that P < 0.01

[0117] The above results indicate that compounds 12'-hydroxy-δ-tocopherol and 12'-hydroxy-α-tocopherol likely exert their anti-MCF-7 breast cancer cell activity by inhibiting Sphk1 expression. They hold promise as lead compounds for anti-breast cancer research, providing a basis and technical support for the development of anti-breast cancer drugs. Furthermore, the plant-based raw materials are abundant, and the preparation method is easy to operate. Tocopherol derivatives A and B can be effectively extracted from Epimedium sagittatum for the treatment of breast cancer, realizing their application in the preparation of anti-breast cancer drugs. This expands the medicinal and commercial value of Epimedium sagittatum, yielding significant economic and social benefits.

Claims

1. A method for extracting two tocopherol derivatives A and B from Epimedium sagittatum, characterized in that, The chemical molecular structures of the tocopherol derivatives A and B are as follows: 、 ； Its preparation method is as follows: (1) 80 kg of dried Epimedium brevicornu aerial parts were crushed and extracted three times with 70% ethanol at 45°C, using 120 L of ethanol each time for 30 min. The extracts were combined and recovered under reduced pressure to obtain 6.5 kg of extract. The extract was suspended in three times its volume of distilled water to obtain a suspension. The suspension was then extracted four times each with petroleum ether, dichloromethane, ethyl acetate, and n-butanol at room temperature, with each extraction lasting 2 minutes. h, each time using 15L, petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract were obtained respectively. The dichloromethane extract was separated by normal phase silica gel column chromatography with a 100-200 mesh, using a gradient elution of petroleum ether:ethyl acetate at volume ratios of 50:1 (12L); 40:1 (20L); 35:1 (20L); 20:1 (40L); 10:1 (40L); 5:1 (40L); 1:1 (20L); 0:1 (4L). Based on the TLC colorimetric results, similar fractions were combined to obtain 8 polar fractions Fr.A, Fr.B…Fr.F…Fr.H. (2) The polar fraction Fr. F was subjected to normal phase silica gel column chromatography with a volume ratio of petroleum ether:ethyl acetate. The gradient ratios were 35:1 (1 L), 20:1 (3 L), 10:1 (3 L), 5:1 (3 L), 1:1 (2 L), and 0:1 (1 L). The eluent was combined in 500 mL increments and the eluent was recovered under reduced pressure. The fractions were then spotted onto a thin-layer plate by TLC. The fractions with similar absorption were combined to obtain 15 first-group fractions Fr. F1, Fr. F2…Fr. F12…F15. (3) The first group fraction Fr. F12 was subjected to ODS reversed-phase silica gel column chromatography with gradient elution of MeOH:H2O at a volume ratio of 40:60 (1 L); 50:50 (1 L); 60:40 (2 L); 70:30 (2 L); 80:20 (2 L); 90:10 (1 L); 100:0 (1 L). The eluent was combined in 200 mL increments and the eluent was recovered under reduced pressure to obtain multiple fractions. The fractions with similar absorption on the thin-layer plate were combined by TLC to obtain 10 second group fractions Fr. F12.1…Fr. F12.8…F12.

10. (4) The second group of Fr. F12.8 was purified by semi-preparative high performance liquid chromatography (RP C18 column) with a column volume of 10ID × 250 mm. The purification solution was acetonitrile:water at a volume ratio of 85.5:14.5, the flow rate was 2 mL / min, and the retention time was collected. t R =45.0 min fraction, dried to obtain tocopherol derivative B; (5) The polar fraction Fr. G was subjected to normal phase silica gel column chromatography with a volume ratio of petroleum ether:ethyl acetate for gradient elution. The gradient ratios were 50:1 (1 L), 35:1 (5 L), 20:1 (5 L), 10:1 (5 L), 5:1 (5 L), 1:1 (2 L), and 0:1 (1 L). Each 1 L of eluent was combined together, and the eluent was recovered under reduced pressure to obtain multiple fractions. The fractions with similar absorption on the thin-layer plate were combined by TLC to obtain 18 third-group fractions Fr. G1, Fr. G2...Fr. G17, Fr. G18. (6) The third group Fr. G17 was eluted with a gradient of petroleum ether:ethyl acetate in a volume ratio of 200–300 mesh normal phase silica gel. The gradient ratios were 35:1 (1 L), 20:1 (4 L), 10:1 (4 L), 5:1 (4 L), 1:1 (2 L), and 0:1 (1 L). Each 500 mL eluent was combined and the eluent was recovered under reduced pressure to obtain multiple components. The components with similar absorption on the thin-layer chromatography plate were combined to obtain 19 fourth group Fr. G17.1…Fr. G17.15…Fr. G17.

19. (7) The fourth group fraction Fr. G17.15 was eluted by ODS column chromatography with a gradient of MeOH:H2O at a volume ratio of 40:60 (1 L); 50:50 (2 L); 60:40 (3 L); 70:30 (3 L); 80:20 (3 L); 90:10 (2 L); and 100:0 (1 L). The eluent was combined in 500 mL increments, and the eluent was recovered under reduced pressure to obtain multiple components. The components with similar absorption on the thin-layer plate were combined by TLC to obtain 12 fifth group fractions Fr.G17.15.1…Fr.G17.15.8…Fr.G17.15.

12. (8) The fifth group fraction Fr. G17.15.8 was eluted by ODS column chromatography with a gradient of MeOH:H2O at a volume ratio of 50:50 (1 L); 60:40 (2 L); 70:30 (3 L); 80:20 (3 L); 90:10 (1 L); and 100:0 (1 L). The eluent was combined in 300 mL increments, and the eluent was recovered under reduced pressure to obtain multiple components. The components with similar absorption on the thin-layer plate were combined by TLC to obtain 14 sixth group fractions Fr.G17.15.8.1…Fr.G17.15.8.

14. (8) The sixth group of Fr. G17.15.8.14 was purified by semi-preparative HPLC RP C18 column with a column volume of 10ID × 250 mm. The purification solution was acetonitrile:water = 85:15 (v / v), the flow rate was 2 mL / min, and the retention time was collected. t R The fraction was discharged for 36.5 min, dried, and tocopherol derivative A was obtained.

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