阅读说明：本技术 野马追倍半萜内酯b在制备抗肝癌药物中的应用及一种抗肝癌药物 (Application of lindley eupatorium herb sesquiterpene lactone B in preparation of anti-liver cancer medicine and anti-liver cancer medicine ) 是由 张永慧 魏勇 陈地龙 刘桂元 张昊洋 牟金阿哥 韩美月 潘李安 曹志豪 王婷婷 于 2020-11-26 设计创作，主要内容包括：本发明涉及野马追倍半萜内酯B在制备抗肝癌药物中的应用及一种抗肝癌药物,属于医药技术领域。野马追倍半萜内酯B在体内、体外对人源肝癌均有一定的抑制作用,能够显著抑制肝癌细胞生长,具体机制为将细胞周期阻滞在S期、下调肝癌细胞周期相关蛋白CDK2和Cyclin E1表达。野马追倍半萜内酯B抑制肝癌细胞迁移,具体机制为下调Vimentin表达和上调E-cadherin表达。同时,野马追倍半萜内酯B使肝癌细胞线粒体皱缩,膜密度增加,可以增加肝癌细胞活性氧并诱导细胞铁死亡信号通路,从而发挥抗肝癌的作用。(The invention relates to application of lindley eupatorium herb sesquiterpene lactone B in preparation of an anti-liver cancer medicament and an anti-liver cancer medicament, and belongs to the technical field of medicines. The Eupatorium eupatorium sesquiterpene lactone B has certain inhibition effect on human liver cancer in vivo and in vitro, can obviously inhibit the growth of liver cancer cells, and has the specific mechanism of retarding the cell cycle in the S phase and reducing the expression of liver cancer cell cycle related proteins CDK2 and Cyclin E1. The Eupatorium adenophorum sesquiterpene lactone B can inhibit the migration of liver cancer cells by down-regulating Vimentin expression and up-regulating E-cadherin expression. Meanwhile, the lindley eupatorium sesquiterpene lactone B enables mitochondria of the liver cancer cells to shrink, increases the membrane density, can increase active oxygen of the liver cancer cells and induce cell iron death signal pathways, thereby playing a role in resisting liver cancer.)

1. The application of the lindley eupatorium sesquiterpene lactone B in preparing the anti-liver cancer medicine is characterized in that the chemical structural formula of the lindley eupatorium sesquiterpene lactone B is as follows:

2. the use of claim 1, wherein the medicament has the effect of inhibiting the growth of liver cancer cells.

3. The use of claim 2, wherein said inhibition of growth of liver cancer cells is achieved by arresting the cell cycle in S phase and/or down-regulating expression of liver cancer cell cycle-associated proteins; the related protein is CDK2 and/or Cyclin E1.

4. The use of claim 1, wherein the medicament has the effect of reducing the migratory capacity of liver cancer cells.

5. The use of claim 4, wherein the reduction of the migration capacity of liver cancer cells is achieved by down-regulating the expression of a protein Vimentin associated with liver cancer cell migration and/or up-regulating the expression of a protein E-cadherin associated with liver cancer cell migration.

6. The use of claim 1, wherein the medicament has the effect of inducing iron death signaling in hepatoma cells.

7. The use of claim 6, wherein said induction of the iron death signaling pathway in hepatoma cells is achieved by down-regulating the expression of the iron death-related protein GPX4 in hepatoma cells.

8. The use of claim 1, wherein the medicament has the effect of increasing active oxygen in liver cancer cells.

9. The use of claim 1, wherein the medicament has the effect of affecting the morphology of the mitochondria of the hepatoma cells, shrinking the mitochondria of the hepatoma cells and increasing the membrane density.

10. An anti-liver cancer drug is characterized in that the drug takes lindley eupatorium sesquiterpene lactone B as the only active ingredient, or takes lindley eupatorium sesquiterpene lactone B and other ingredients with anti-liver cancer effect as the active ingredients.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of lindley eupatorium sesquiterpene lactone B in preparation of an anti-liver cancer medicine and an anti-liver cancer medicine.

Background

The primary liver cancer is one of the clinically common malignant tumors, and most liver cancers (75-85%) are primary liver cancers according to histological analysis. The high morbidity and mortality of liver cancer imposes a huge economic burden on society, and therefore, the exploration of effective treatment schemes for liver cancer is still a clinical problem which needs to be solved urgently.

Currently, liver cancer treatment protocols mainly include surgical treatment and non-surgical treatment. Although the most effective method for treating primary liver cancer is surgical treatment at present, the primary liver cancer is hidden, asymptomatic or unobvious in early stage, most patients have metastasized to middle and late stages or at distant sites when the diagnosis is confirmed, and the chance of surgery is lost, so that the systemic drug therapy plays an important role in the liver cancer treatment. The liver cancer treatment drugs comprise molecular targeted drugs (sorafenib) and traditional cytotoxic drugs (adriamycin, cisplatin, fluorouracil and the like), but the sorafenib is only effective for partial patients, and the traditional cytotoxic drugs have low efficiency and large toxic and side effects. The traditional Chinese medicine treatment is used as an important component of the comprehensive treatment of the liver cancer, and has unique advantages in the aspects of preventing the occurrence of the liver cancer, reducing the recurrence, improving the life quality, prolonging the life cycle and the like. The traditional Chinese medicine theory carries out syndrome differentiation treatment on liver cancer from the holistic concept, and considers that tumors, namely abdominal mass, carbuncle, cellulitis, sore and swelling, and the like, have pathogenesis generated by internal and external interaction and are closely related to weakness of internal organs, qi and blood stasis and internal generation of cancer toxins. Therefore, the traditional Chinese medicine compound can be used for treating liver cancer by regulating balance of yin and yang, qi and blood stasis, playing roles of tonifying qi, reducing phlegm and removing blood stasis and eliminating pathological factors. The commonly used Chinese herbal compound comprises a double-lotus prescription, small bupleurum decoction, particles for strengthening body resistance and inhibiting tumor, detoxifying and eliminating mass, and the like. However, in general, the traditional Chinese medicine treatment has many action targets, complex medicine components, difficult mechanism definition and lack of high-level evidence of sufficient support to prove the action. The traditional Chinese medicine extract not only can fully exert the advantages of low toxic and side effects and good tolerance of the traditional Chinese medicine, but also can combine modern subject theory, clinical practice basis and perfect experimental method. Therefore, screening more efficient active ingredients from the traditional Chinese medicines, exploring the anti-liver cancer effect and the specific anti-cancer mechanism of the traditional Chinese medicine extract has important clinical significance, and provides more effective means for the comprehensive treatment of liver cancer.

Eupatorium fortunei is a whole plant of Eupatorium apetalum of Eupatorium of Compositae, and is mainly produced in Jiangsu, Gansu, Shandong, Hunan, etc. Herba Eupatorii Lindleyani has effects of eliminating phlegm, relieving cough, clearing heat, detoxicating, inducing diuresis, relieving swelling, and lowering blood pressure, and can be used for treating chronic tracheitis, bronchitis, hypertension, relieving exterior syndrome, eliminating dampness, regulating the middle warmer, and eliminating dampness. So far, the chemical components extracted from the lindley eupatorium herb are mainly volatile oil, organic acid, flavonoid, triterpenes, sesquiterpenes and other components, the research on the anti-tumor effect of the lindley eupatorium herb extract is less, the studies show that the lindley eupatorium herb extract has the effect of resisting breast cancer and lung cancer, and the lindley eupatorium herb extract can have the cytotoxicity effect on a mouse leukemia cell line P-388 and a human lung cancer cell line A-549 which are discovered by Shanghai pharmaceutical research institute of Chinese academy of sciences in 2004, but subsequent verification experiments and mechanism researches are not carried out. In 2016, research of Zhao Huajun subject group of Zhejiang Chinese medicine university shows that: the lindley eupatorium herb extract lindley eupatorium lactone O (EO) can obviously inhibit the activity of breast cancer cells, and the mechanism of the lindley eupatorium herb extract lindley eupatorium lactone O (EO) is that the breast cancer cells are blocked in the G2/M stage and caspase-dependent apoptosis signal paths can be induced by reducing the levels of cell cycle related proteins CyclinB1 and CDC 2. The subject group is separated and identified with Eupalinolide J (EJ) and F1012-2, wherein Eupalinolide J can reduce STAT3 luciferase activity, is a natural Michael reaction receptor, F1012-2 can activate autophagy, induce apoptosis and inhibit cell cycle, and both compounds can inhibit triple-negative breast cancer. However, the studies on liver cancer and mechanism of lindley eupatorium are few.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of lindley eupatorium sesquiterpene lactone B in preparing anti-liver cancer drugs; the other purpose is to provide an anti-liver cancer drug.

In order to achieve the purpose, the invention provides the following technical scheme:

1. the application of the lindley eupatorium sesquiterpene lactone B in preparing the anti-liver cancer medicine is as follows:

preferably, the medicament has the effect of inhibiting the growth of liver cancer cells.

Preferably, the inhibition of the growth of the liver cancer cells is realized by blocking the cell cycle in the S phase and/or down regulating the expression of the liver cancer cell cycle related protein; the related protein is CDK2 and/or Cyclin E1.

Preferably, the medicament has the function of reducing the migration capacity of the liver cancer cells.

Preferably, the reduction of the migration capacity of the liver cancer cells is realized by down-regulating the expression of a liver cancer cell migration related protein Vimentin and/or up-regulating the expression of a liver cancer cell migration related protein E-cadherin.

Preferably, the medicament has the effect of inducing iron death signaling pathway of the hepatoma cells.

Preferably, the induction of the iron death signaling pathway of the liver cancer cell is realized by down-regulating the expression of a liver cancer cell iron death-related protein GPX 4.

Preferably, the medicament has the effect of increasing active oxygen of the liver cancer cells.

Preferably, the medicament has the effect of influencing the morphology of the mitochondria of the liver cancer cells, so that the mitochondria of the liver cancer cells shrink and the membrane density is increased.

2. An anti-liver cancer medicine contains lindley eupatorium sesquiterpene lactone B as the only active component, or lindley eupatorium sesquiterpene lactone B and other components with anti-liver cancer effect as the active components.

The invention has the beneficial effects that: the invention provides application of lindley eupatorium sesquiterpene lactone in preparing an anti-liver cancer medicament and the anti-liver cancer medicament, wherein the lindley eupatorium sesquiterpene lactone B (EB) has certain inhibition effect on human liver cancer in vivo and in vitro, can obviously inhibit the growth of liver cancer cells, and has the specific mechanism of blocking the cell cycle in the S phase and reducing the expression of liver cancer cell cycle related proteins CDK2 and Cyclin E1. The Eupatorium adenophorum sesquiterpene lactone B can inhibit the migration of liver cancer cells by down-regulating Vimentin expression and up-regulating E-cadherin expression. Meanwhile, the lindley eupatorium sesquiterpene lactone B enables mitochondria of the liver cancer cells to shrink, increases the membrane density, can increase active oxygen of the liver cancer cells and induce cell iron death signal pathways, thereby playing a role in resisting liver cancer.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.

Drawings

For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 is a graph showing the results of the growth inhibition test of Eupatorium Adenophorum hand-Mazz sesquiterpene lactone B on hepatoma cell lines (in FIG. 1, A is a graph showing the growth inhibition test of Eupatorium Adenophorum hand-Mazz sesquiterpene lactone B on SMMC-7721 human hepatoma cell lines, and B is a graph showing the growth inhibition test of Eupatorium Adenophorum hand-Mazz lactone B on HCCLM3 human hepatoma cell lines);

FIG. 2 is a diagram showing the results of liver cancer cell cycle arrest tests with Eupatorium lappaconitine B (in FIG. 2, A is a diagram showing the results of cell cycle arrest tests with Eupatorium lappaconitine B on an SMMC-7721 human hepatoma cell line, and B is a diagram showing the results of cell cycle arrest tests with Eupatorium lappaconitine B on an HCCLM3 human hepatoma cell line);

FIG. 3 is a graph showing the results of a liver cancer cell cycle-associated protein expression down-regulation test using Eupatorium sesquiterpene lactone B;

FIG. 4 is a graph showing the test results of the decrease of migration ability of Eupatorium lindleyanum sesquiterpene lactone B to hepatoma cell lines;

FIG. 5 is a graph showing the results of a protein expression test for Eupatorium sesquiterpene lactone B affecting migration of liver cancer cells;

FIG. 6 is a graph showing the results of the test of the sesquiterpene lactone B downregulated expression of proteins associated with iron death of hepatoma cells;

FIG. 7 is a graph showing the results of the test of increasing the active oxygen of hepatoma cells with Eupatorium sesquiterpene lactone B;

FIG. 8 is a graph showing the results of the effect of Eupatorium sesquiterpene lactone B on the mitochondrial morphology of hepatoma cells;

FIG. 9 is a graph showing the results of the test of the volume inhibition of Eupatorium sesquiterpene lactone B on transplanted tumor in nude mice;

FIG. 10 is a graph of the results of the test of the inhibition of eupatorium sesquiterpene lactone B on the weight of transplanted tumor in nude mice.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Example 1

Research on growth inhibition effect and mechanism of Eupatorium Adenophorum sesquiterpene lactone B on hepatoma cell line

Culturing two human liver cancer cells SMMC-7721 and HCCLM3 by CCK8 method to density of about 90%, respectively, counting cells, and inoculating to 96-well culture plate according to experimental groups, wherein the number of cells per well is 5 × 10³Each group is provided with 6 multiple holes, and the inoculated mixture is placed at 37 ℃ and 5% CO₂The culture in the incubator is respectively carried out for 24 h. Respectively adding Eupatorium Adenophorum hand-Mazz lactone B with final concentration of 6, 12, 24 μmol/L into the administration group, culturing for 24h, 48h and 72h, adding 10 μ L CCK8 solution, incubating at 37 deg.C for 4h, measuring OD value at 450nm on microplate reader, and determining the result as shown in FIG. 1, wherein A in FIG. 1 is the test result of Eupatorium Adenophorum hand-Mazz lactone B on growth inhibition of SMMC-7721 human liver cancer cell line, and B in FIG. 1 is the test result of Eupatorium Adenophorum hand-Mazz lactone B on HCCLM3 human liver cancer cell lineTest result chart of cell strain growth inhibition. Data analysis was performed, and the cell growth inhibition rate was (control OD value-test OD value)/control OD × 100%. Statistical treatment was analyzed using SPSS15.0 statistical software, and the mean comparisons between the two groups were performed using the t-test. It is known that the Eupatorium adenophorum sesquiterpene lactone B has inhibition effect on the growth of liver cancer cell strains SMMC-7721 and HCCLM3, and the inhibition effect is increased along with the increase of the concentration of the Eupatorium adenophorum sesquiterpene lactone B.

(1) Eupatorium eupatorium sesquiterpene lactone B blocks the cell cycle of liver cancer

Two human liver cancer cells of SMMC-7721 and HCCLM3 are cultured, and the experiment groups are as follows: control group (DMSO), lindley eupatorium sesquiterpene lactone B medium dose group (12 μ M), and lindley eupatorium sesquiterpene lactone B high dose group (24 μ M), were treated with each drug for 48h, when both cells were in logarithmic growth phase, as follows: digesting with trypsin for 1-2min, collecting cells, washing cells with PBS, centrifuging at 2000rpm for 5min, and adjusting cell concentration to 1 × 10⁶1mL of single cell suspension was taken. Centrifuging the prepared single cell suspension, removing supernatant, adding 500 μ L70% cold ethanol into cells, fixing, and storing at 4 deg.C; adding 500 μ L of PI/RNaseA dyeing working solution prepared in advance, and keeping out of the sun at room temperature for 30-60 min; and (4) detecting by using a computer, recording red fluorescence at the 488nm excitation wavelength, collecting data and analyzing by using ModFit software. The results are shown in fig. 2, a in fig. 2 is a graph of the test result of the influence of eupatorium lappaconite B on the cell cycle of SMMC-7721 human hepatoma cell line, B in fig. 2 is a graph of the test result of the influence of eupatorium lappaconite B on the cell cycle of HCCLM3 human hepatoma cell line, and it can be seen from fig. 2 that after being treated by eupatorium lappaconite B, the cell cycles of both cells are arrested in S phase, and the arresting effect is increased along with the increase of the concentration of eupatorium lappaconite B.

(2) Eupatorium eupatorium sesquiterpene lactone B down-regulated expression of liver cancer cell cycle related protein

Two human liver cancer cells of SMMC-7721 and HCCLM3 are cultured, and the experiment groups are as follows: control group (DMSO), lindley eupatorium sesquiterpene lactone B low dose group (6 μ M), lindley eupatorium sesquiterpene lactone B medium dose group (12 μ M), and lindley eupatorium sesquiterpene lactone B high dose group (24 μ M), respectively, the drug treatment was carried out for 48h, and when the two cells were in logarithmic growth phase, the culture medium was poured out, and the following operations were carried out: washing with PBS for 2 times, adding RIPA, phosphatase inhibitor, and protease inhibitor (100:10:1), scraping adherent cells with cell scraper, sucking into centrifuge tube, centrifuging at 3000rpm and 4 deg.C for 5 min. Centrifuging, collecting supernatant, and extracting total protein; protein concentration was determined by BCA method, and the amount of sample was calculated and the samples denatured at 100 ℃ for 5min and rapidly placed on ice. Separating proteins by 8-12% lauryl sodium sulfate polyacrylamide gel electrophoresis, wet-transferring to a PVDF membrane, shaking for 2h at room temperature in a confining liquid containing 5% skimmed milk powder, gently rinsing the confined PVDF membrane for 5min by using a PBS buffer solution, incubating by using different primary antibodies according to different target proteins, and incubating overnight in a shaking table at 4 ℃. After the primary antibody incubation was completed, the cells were washed 4 times with TBS-T buffer for 5min each time. Incubating the fluorescent secondary antibody for 45min at room temperature in a dark place, washing for 4 times by TBS-T buffer solution for 5min each time, and finally performing ECL luminescence color development and result analysis. As shown in fig. 3, it is evident from fig. 3 that the expression of the Cyclin-related proteins CDK2 and Cyclin E1 was significantly reduced in both cells after treatment with tremoline B, and the inhibitory effect increased with the increase in tremoline B concentration.

Example 2

Research on migration capacity reduction effect and mechanism of Eupatorium Adenophorum sesquiterpene lactone B on liver cancer cell strain

Inoculating SMMC-7721 and HCCLM3 two human liver cancer cells in logarithmic growth phase into 6-well plate at 5 × 10%⁵Individual cells/well, 2mL medium/well. Adding 5% CO at 37 deg.C₂The cells were incubated in an incubator for 24 h. The cells were spread over the bottom wall, streaked evenly on the bottom of the dish using a 20 μ L pipette tip, the cells were washed 3 times with PBS, the experimental cells were treated with serum-free medium containing 6 μ M, 12 μ M, and 24 μ M of eupatorium sesquiterpene lactone B, and the control cells were treated with serum-free medium containing 0.1% DMSO, respectively. Using an inverted microscope, samples were taken at 0, 6, 24, and 48h time points and photographed, and the scratch wound healing rate was calculated as (0h scratch width-post-incubation scratch width)/0 h scratch width × 100% using Image J statistical data. The results are shown in FIG. 4, which is a graph of FIG. 4It is known that the Eupatorium adenophorum sesquiterpene lactone B has inhibitory effect on the migration of liver cancer cell lines SMMC-7721 and HCCLM3, and the inhibitory effect increases with the increase of the concentration of Eupatorium adenophorum sesquiterpene lactone B.

(1) Eupatorium eupatorium sesquiterpene lactone B affecting expression of liver cancer cell migration related protein

Two human liver cancer cells of SMMC-7721 and HCCLM3 are cultured, and the experiment groups are as follows: control group (DMSO), lindley eupatorium sesquiterpene lactone B low dose group (6 μ M), lindley eupatorium sesquiterpene lactone B medium dose group (12 μ M), and lindley eupatorium sesquiterpene lactone B high dose group (24 μ M), respectively, the drug treatment was carried out for 48h, and when the two cells were in logarithmic growth phase, the culture medium was poured out, and the following operations were carried out: washing with PBS for 2 times, adding RIPA, phosphatase inhibitor, and protease inhibitor (100:10:1), scraping adherent cells with cell scraper, sucking into centrifuge tube, centrifuging at 3000rpm and 4 deg.C for 5 min. Centrifuging, collecting supernatant, and extracting total protein; protein concentration was determined by BCA method, and the amount of sample was calculated and the samples denatured at 100 ℃ for 5min and rapidly placed on ice. Separating proteins by 8-12% lauryl sodium sulfate polyacrylamide gel electrophoresis, wet-transferring to a PVDF membrane, shaking for 2h at room temperature in a confining liquid containing 5% skimmed milk powder, gently rinsing the confined PVDF membrane for 5min by using a PBS buffer solution, incubating by using different primary antibodies according to different target proteins, and incubating overnight in a shaking table at 4 ℃. After the primary antibody incubation was completed, the cells were washed 4 times with TBS-T buffer for 5min each time. Incubating the fluorescent secondary antibody for 45min at room temperature in a dark place, washing for 4 times by TBS-T buffer solution for 5min each time, and finally performing ECL luminescence color development and result analysis. The results are shown in fig. 5, and it can be seen from fig. 5 that after treatment with eupatorium sesquiterpene lactone B, the expression of cyclin-related protein Vimentin of both cells was significantly decreased, the expression of E-cadherin was significantly increased, and the regulation effect was increased with the increase of the concentration of eupatorium sesquiterpene lactone B.

Example 3

Research on mechanism of action of Eupatorium lindleyanum sesquiterpene lactone B induced hepatoma cell iron death signal pathway

(1) Eupatorium eupatorium sesquiterpene lactone B down-regulates expression of hepatoma cell iron death-related protein GPX4

Two human liver cancer cells of SMMC-7721 and HCCLM3 are cultured, and the experiment groups are as follows: control group (DMSO), and high-dose group (24. mu.M) of Eupatorium sesquiterpene lactone B, were treated with each drug for 48h, and the medium was decanted when both cells were in logarithmic phase, all as follows: washing with PBS for 2 times, adding RIPA, phosphatase inhibitor, and protease inhibitor (100:10:1), scraping adherent cells with cell scraper, sucking into centrifuge tube, centrifuging at 3000rpm and 4 deg.C for 5 min. Centrifuging, collecting supernatant, and extracting total protein; protein concentration was determined by BCA method, and the amount of sample was calculated and the samples denatured at 100 ℃ for 5min and rapidly placed on ice. Separating proteins by 8-12% lauryl sodium sulfate polyacrylamide gel electrophoresis, wet-transferring to a PVDF membrane, shaking for 2h at room temperature in a confining liquid containing 5% skimmed milk powder, gently rinsing the confined PVDF membrane for 5min by using a PBS buffer solution, incubating by using different primary antibodies according to different target proteins, and incubating overnight in a shaking table at 4 ℃. After the primary antibody incubation was completed, the cells were washed 4 times with TBS-T buffer for 5min each time. Incubating the fluorescent secondary antibody for 45min at room temperature in a dark place, washing for 4 times by TBS-T buffer solution for 5min each time, and finally performing ECL luminescence color development and result analysis. As shown in fig. 6, it is clear from fig. 6 that the expression of GPX4, a protein involved in iron death, was significantly reduced in both cells after treatment with lindley eupatorium sesquiterpene lactone B.

Example 4

Eupatorium eupatorium sesquiterpene lactone B with effect of increasing active oxygen of liver cancer cells

Two human liver cancer cells of SMMC-7721 and HCCLM3 are cultured, and the experiment groups are as follows: control group (DMSO), and high-dose group (24. mu.M) of Eupatorium sesquiterpene lactone B, were treated with each drug for 48h, when both cells were in logarithmic growth phase, as follows: DCFH-DA was diluted in serum-free medium at a ratio of 1:1000 to a final concentration of 10. mu. mol/L. After cell collection, the cells were suspended in diluted DCFH-DA and incubated in a cell incubator at 37 ℃ for 20 min. The mixture was inverted every 3-5min to bring the probe into full contact with the cells. Cells were washed 3 times with serum-free cell culture medium to remove DCFH-DA well without entering the cells. The yield of active oxygen radicals in the cells was expressed by measuring the fluorescence intensity (excitation wavelength 488nm, emission wavelength 525nm) in the cells by flow cytometry. As shown in FIG. 7, it is clear from FIG. 7 that more active oxygen was produced in both cells after treatment with Eupatorium sesquiterpene lactone B.

Example 5

Eupatorium eupatorium sesquiterpene lactone B affecting morphology of liver cancer cell mitochondria

Two human liver cancer cells of SMMC-7721 and HCCLM3 are cultured, and the experiment groups are as follows: control group (DMSO), and high-dose group (24. mu.M) of Eupatorium sesquiterpene lactone B, were treated with each drug for 48h, when both cells were in logarithmic growth phase, as follows: digesting, blowing and centrifuging cells by pancreatin without EDTA, discarding supernatant, centrifuging again by using complete culture medium, discarding supernatant, keeping cell masses in a 1mL EP tube, slowly adding 1mL of 2.5% glutaraldehyde stationary solution (4 ℃) along the tube wall, and using 1% OsO at room temperature₄Postfixed in PBS for 1 h. Dehydrated with ethanol and embedded with epoxy resin and sectioned. Ultrathin (60nm) sections were collected on a grid and stained with uranyl acetate and lead citrate and images were obtained using transmission electron microscopy. The change in mitochondrial morphology was also one of the features of iron death, and as a result, as shown in fig. 8, it can be seen from fig. 8 that after treatment with lindley eupatorium sesquiterpene lactone B, the mitochondria of the cells of both cells appeared to shrink significantly, and the membrane density increased.

Example 6

Research on in-vitro anti-liver cancer effect of eupatorium lindleyanum sesquiterpene lactone B

Mouse subcutaneous tumor model experiment: female BALB/C athymic immunodeficient nude mice of about 4 weeks old are bred in an IVC system of SPF-level animal room for 7 days, and the growth conditions of the athymic immunodeficient nude mice are observed. Two kinds of human liver cancer cells of SMMC-7721 and HCCLM3 in logarithmic growth phase are subcultured according to a ratio of 1:3, and are respectively inoculated into 6 culture dishes of 10cm, when the density of the cells is 80% of the area of the culture dishes, pancreatin is digested and counted. The 6 disks of cells were collected in the same sterile centrifuge tube, washed once with PBS, and 1.2mL of precooled PBS was added to resuspend the cells, respectively, and placed on an ice box for future use. Taking three 1mL syringes, respectively marking DMSO group, Eupatorium Adenophorum sesquiterpene lactone B low dose group (25mg/kg) and Eupatorium Adenophorum sesquiterpene lactone BTerpene lactone B high dose group (50mg/kg), prepared cell suspension group was aspirated, air bubbles were carefully removed, and each nude mouse was injected subcutaneously with 1X 10⁶And (4) cells. Three groups of mice were individually placed in standard ICV cages for further rearing. When the mice were observed to develop macroscopic tumors subcutaneously, tumor diameters were measured using a vernier caliper and changes in tumor size were recorded. Continuously measuring for about 3 weeks, and observing the influence of Eupatorium Adenophorum sesquiterpene lactone B on tumor growth. After the last measurement is completed, the mouse is killed by removing the neck, the subcutaneous tumor of the mouse is taken out, the tumor weight is weighed, pictures are taken, the data are analyzed, and the growth curve of the tumor is mapped, as shown in fig. 9 and 10, fig. 9 is a test result diagram of the volume inhibition effect of the lindley eupatorium sesquiterpene lactone B on the nude mouse transplanted tumor, and fig. 10 is a test result diagram of the weight inhibition effect of the lindley eupatorium sesquiterpene lactone B on the nude mouse transplanted tumor. The tumor tissue is put into a prepared cryopreservation tube containing tissue cryopreservation liquid and preserved at the temperature of minus 80 ℃ for later use. As can be seen from fig. 9 and 10, the volume and weight of nude mouse transplanted tumor after treatment with lindley eupatorium sesquiterpene lactone B was significantly reduced compared to the control group.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.