CN-120004842-B - Method for extracting two tocopherol derivatives A, B from Epimedium sagittatum and application thereof

Abstract

A process for extracting two tocopherol derivatives A, B from epimedium, which can effectively solve the problem of preparing tocopherol derivative A, B from epimedium, includes such steps as pulverizing aerial parts of epimedium, reflux extracting with alcohol, vacuum recovering to obtain extract, extracting, chromatographic separation, gradient eluting, vacuum recovering, thin-layer spot plate detection, merging, purifying, collecting the fractions in retention time and drying. The invention has the advantages of rich raw materials, easy operation of the preparation method, good quality of the obtained product and good use effect, can be effectively used for treating breast cancer, realizes the application in preparing the breast cancer resistant medicament, exploits the medicinal value and the commercial value of the epimedium with arrow leaves, and has remarkable economic and social benefits.

Inventors

• XIE SHUANGSHUANG
• YU XIANG
• LIU YANLING
• ZHANG JINGKE
• HAO ZHIYOU
• ZHENG XIAOKE
• FENG WEISHENG

Assignees

• 河南中医药大学

Dates

Publication Date

20260508

Application Date

20250214

Claims (1)

1. 1. A method for extracting two tocopherol derivatives A, B from epimedium, which is characterized in that the chemical molecular structural formulas of the tocopherol derivatives A, B are respectively as follows: 、 ; The preparation method comprises the following steps: (1) Crushing the aerial parts of dried epimedium, reflux-extracting with 70% ethanol at 45 ℃ for 3 times, extracting with 120L of ethanol each time, mixing the extracts, recovering under reduced pressure to obtain extract 6.5 kg, suspending with 3 times of distilled water to obtain suspension, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature for 4 times, extracting with 2h each time, obtaining petroleum ether extract, dichloromethane extract, ethyl acetate extract and n-butanol extract each time, separating the dichloromethane extract by 100-200 mesh normal phase silica gel column chromatography, gradient eluting with petroleum ether/ethyl acetate with a gradient ratio of 50:1, 12L, 40:1, 20L, 35:1, 20L, 20:1, 40L, 10:1, 40L, 5:1, 40L, 1:1, 20L, 0:1 and 4L, mixing the polar parts according to the color development results of thin layer chromatography, and obtaining 8 pieces of TLC, fr.B.Fr.F.; (2) Subjecting the polar section Fr. F to 200-300 mesh normal phase silica gel column chromatography, carrying out gradient elution with petroleum ether and ethyl acetate according to a volume ratio, wherein the gradient ratio is 35:1, the dosage is 1L, 20:1, 3L, 10:1, 3L, 5:1, 3L, 1:1, 2L, 0:1 and 1L, combining the eluents every 500: 500mL, recovering the eluent under reduced pressure, and combining similar components absorbed on the thin layer plate through a TLC (thin layer chromatography) plate to obtain 15 first component components Fr. F1, fr. F2... Fr. F12..F15; (3) Subjecting the first fraction Fr. F12 to ODS reverse phase silica gel column chromatography, subjecting to gradient elution with MeOH: H 2 O in a volume ratio of 40:60, 1L, 50:50, 1L, 60:40, 2L, 70:30, 2L, 80:20, 2L, 90:10, 1L, 100:0, 1L, combining the eluents every 200 mL together, recovering the eluents under reduced pressure to obtain a plurality of fractions, combining the similar absorbed fractions on the thin layer plate by TLC (thin layer chromatography) plates to obtain 10 second fractions Fr. F12.1.. Fr. F12.8..F 12.10; (4) Purifying the second component Fr. F12.8 with semi-preparative high performance liquid chromatography RP C18 column with volume of 10ID× mm, and acetonitrile with volume ratio of water=85.5:14.5 and flow rate of 2mL/min, collecting fraction with retention time t R =45.0 min, and drying to obtain tocopherol derivative B; (5) Subjecting the polar section Fr. G to 100-200 mesh normal phase silica gel column chromatography, carrying out gradient elution by using petroleum ether and ethyl acetate according to the volume ratio, wherein the gradient ratio is 50:1, the dosage is 1L, 35:1, the dosage is 5L, 20:1, the dosage is 5L, 10:1, the dosage is 5L, 5:1, the dosage is 5L, 1:1, 2L, 0:1 and 1L, merging the eluent with each 1L, decompressing and recovering the eluent to obtain a plurality of components, merging the components which are similar to each other absorbed on the thin layer plate through a TLC point plate, and obtaining 18 third component Fr. G1, fr. G2.. (6) Subjecting third fraction Fr. G17 to gradient elution with 200-300 mesh normal phase silica gel using petroleum ether/ethyl acetate in a volume ratio of 35:1, 1L, 20:1, 4L, 10:1, 4L, 5:1, 4L, 1:1, 2L, 0:1, 1L, combining each 500 mL eluate, recovering the eluate under reduced pressure to obtain multiple fractions, and combining similar components absorbed on the thin layer plate by thin layer chromatography dot plates to obtain 19 fourth fraction Fr. G17.1.. Fr. G17.15.. Fr. G17.19; (7) Subjecting the fourth fraction Fr. G17.15 to ODS column chromatography with a gradient of MeOH: H 2 O in a volume ratio of 40:60, 1L, 50:50, 2L, 60:40, 3L, 70:30, 3L, 80:20, 3L, 90:10, 2L, 100:0, 1L, combining the eluents every 500 mL together, recovering the eluents under reduced pressure to obtain a plurality of fractions, combining the similar absorbed fractions on the thin layer plate through TLC spot plates to obtain 12 fifth fractions Fr.G17.15.1..Fr.G17.15.8..Fr.G17.15.12; (8) Subjecting the fifth fraction Fr. G17.15.8 to ODS column chromatography with a gradient of MeOH: H 2 O in a volume ratio of 50:50, 1L, 60:40, 2L, 70:30, 3L, 80:20, 3L, 90:10, 1L, 100:0, 1L, combining the eluents together every 300 mL, recovering the eluents under reduced pressure to obtain a plurality of fractions, combining the similar absorbed fractions on the thin layer plate by TLC (thin layer chromatography) to obtain 14 sixth fractions Fr.G17.15.8.1. (8) Purifying the sixth component Fr. G17.15.8.14 by semi-preparative HPLC RP C18 column with volume of 10ID× mm, collecting the fraction with retention time t R 36.5.5 min and drying to obtain tocopherol derivative A, wherein the volume ratio of the purified solution is acetonitrile with water=85:15 and flow rate of 2 mL/min.

Description

Method for extracting two tocopherol derivatives A, B from Epimedium sagittatum and application thereof Technical Field The invention relates to the field of medicines, in particular to a method for extracting two tocopherol derivatives A, B from epimedium herb serving as a arrow leaf and application thereof. Background Herba Epimedii of berberidaceae is perennial herb of Epimedium, and is mainly distributed in Zhejiang, anhui, fujian, jiangxi, hubei, hunan, etc. The Chinese medicinal herb of Epimedium sagittatum can be traced to Shennong Ben Cao Jing, and has the effects of tonifying kidney yang, strengthening tendons and bones and dispelling wind-dampness. Modern researches have shown that the epimedium has flavone and its glucoside, lignan, alkaloid and phenolic acid compounds, and has antitumor, antioxidant, osteoporosis and depression resisting activities. Breast cancer is a common frequently-occurring disease, seriously threatens the life safety and physical health of people, and particularly has limited effect due to the fact that breast cancer is caused by human breast cancer cells MCF-7, and the existing therapeutic drugs are limited. The 2 new tocopherol derivatives A (12 '-hydroxy-delta-tocopheryl) and B (12' -hydroxy-alpha-tocopheryl) identified from the dichloromethane extraction part of the epimedium sagittifolium have remarkable inhibition effect on the cell viability of human breast cancer cells MCF-7 and have no toxicity on human normal breast cells MCF-10A. Therefore, the two compounds are possible drug effect substances of the epimedium arrowhead for exerting the anti-breast cancer activity, and meanwhile, the 12 '-hydroxy-delta-tocopheryl and the 12' -hydroxy-alpha-tocopheryl are also found to show remarkable inhibition effect on sphingosine kinase 1 (Sphk 1) in MCF-7 cells, which suggests that the two compounds possibly exert the anti-breast cancer effect by inhibiting the Sphk1 expression level and are expected to become lead compounds for resisting breast cancer, thereby providing technical support for development of anti-breast cancer drugs. However, how to extract effective active ingredients 12 '-hydroxy-delta-tocopherol and 12' -hydroxy-alpha-tocopherol from epimedium, and to prepare medicaments for resisting breast tumors (cancers) has not been reported so far. Disclosure of Invention Aiming at the situation, the invention aims to overcome the defects of the prior art and provide a method for extracting two tocopherol derivatives A, B from the epimedium, and application thereof, which can effectively solve the problem of application in preparing the tocopherol derivatives A, B from the epimedium, and realize the preparation of the anti-breast tumor medicines. The technical scheme is that two tocopherol derivatives A, B extracted from Epimedium sagittatum have chemical molecular structural formulas: The preparation method comprises the following steps: (1) Pulverizing aerial parts of herba Epimedii, reflux-extracting with ethanol, recovering under reduced pressure to obtain extract, suspending with distilled water, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol at room temperature to obtain extract of each part, separating dichloromethane extract by chromatography, and gradient eluting to obtain polar section; (2) Subjecting the target polar section to chromatography, gradient elution, decompression recovery, thin layer spotting, and combining similar components to obtain a first component; (3) Subjecting the target first fraction to chromatography, gradient elution, recovering under reduced pressure, spotting on a thin plate, and combining similar components to obtain a second fraction; (4) Purifying the target second fraction, collecting the fraction with retention time t R =45.0 min, and drying to obtain tocopherol derivative B (12' -hydroxy- α -tocopherol); (5) Subjecting the target polar section to chromatography, gradient elution, decompression recovery, thin layer spotting, and combining similar components to obtain a third component; (6) Gradient eluting the third group, recovering under reduced pressure, spotting on a thin layer plate, and combining similar components to obtain a fourth group component; (7) Gradient eluting the fourth fraction, recovering under reduced pressure, spotting on a thin layer plate, and combining similar components to obtain a fifth fraction; (8) Gradient eluting the fifth fraction, recovering under reduced pressure, spotting on a thin layer plate, and combining similar components to obtain a sixth fraction; (9) The sixth fraction was purified, and the fraction with retention time t R =36.5 min was collected and dried to give tocopherol derivative a (12' -hydroxy- δ -tocopherol). An application of two tocopherol derivatives A, B extracted from herba Epimedii in preparing medicine for treating breast cancer is provided. The invention has rich raw materials and easy operation of the preparation method,