CN112263600A

CN112263600A - Carpesium abrotanoides extract, preparation method and application thereof in anti-liver cancer active drugs passing through JAK2/STAT3 channel

Info

Publication number: CN112263600A
Application number: CN202011163501.3A
Authority: CN
Inventors: 沈冰冰; 张水寒; 杨玉佩; 成飞; 蔡媛; 刘雪辉
Original assignee: HUNAN ACADEMY OF CHINESE MEDICINE
Current assignee: HUNAN ACADEMY OF CHINESE MEDICINE
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2021-01-26
Anticipated expiration: 2040-10-27
Also published as: CN112263600B

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and discloses a carpesium abrotanoides extract, a preparation method and application thereof in an anti-liver cancer active medicine through JAK2/STAT3 channels. The method comprises the following operation steps: drying and crushing whole plant of carpesium abrotanoides, soaking and extracting for 24h by using ethanol with the volume percentage concentration of 95%, extracting for three times, and mixing and drying extracting solutions to obtain a total extract; adding NaOH solution into the total extract to dissolve until the pH value is 9, stopping adding the NaOH solution, and then adjusting the pH value to 2 by using concentrated hydrochloric acid to obtain precipitate to obtain the Carpesium abrotanoides total extract; separating the total extract of radix Carpesii by high speed countercurrent chromatography to obtain three compounds respectively with structural formula C₁₅H₂₂O₄Compound 1 of the formula C₁₅H₂₀O₃Compound 2 of the structural formula C₁₅H₂₀O₃Compound 3 of (1).

Description

Carpesium abrotanoides extract, preparation method and application thereof in anti-liver cancer active drugs passing through JAK2/STAT3 channel

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a carpesium abrotanoides extract, a preparation method and application thereof in an anti-liver cancer active medicine through JAK2/STAT3 channels.

Background

The genus carpesium is a genus of the family asteraceae, about 21, and is mainly distributed in southern europe and asia. Wherein, there are 17 kinds in the southwest mountain area of China. Tianming Jing, a famous ethnic medicine, has a long medicinal history in the Tujia, Bai nationality, Miao nationality and other minority nationalities. The research shows that the sesquiterpene lactone mainly contains chemical components such as sesquiterpene lactone, and most of the sesquiterpene lactone contains alpha-methylene-gamma-butyrolactone, and has biological activities such as anticancer, anti-inflammatory and antibacterial activities. Screening of cck-8 activity at the early stage shows that the total Sesquiterpene Lactone Extract (SLEC) has good effect (IC) on HepG-2 cells₅₀4.2 mug/ml), but the effective components and the action mechanism of the medicine for resisting liver cancer are not clear.

The aim of modern Chinese medicine research is to explain which chemical components of Chinese medicine are effective and how to act. Although a complete system is formed by the traditional extraction, separation and analysis method, the defects of time consumption, labor waste, long period and the like still exist, and the traditional extraction, separation and analysis method cannot meet and adapt to the research of modern traditional Chinese medicine.

Among them, high-speed countercurrent chromatography (HSCCC) is a very effective tool for separation and purification of Chinese herbs. Compared with other separation methods, the method does not use a solid carrier as a stationary phase, but disperses a separated sample in an immiscible two-phase liquid to achieve the separation effect. Therefore, the HSCCC has the advantages of high loading capacity, high preparation efficiency, low solvent consumption, good repeatability and the like.

High performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS)^E) Accurate information such as molecular ion peaks and fragment ions can be rapidly acquired, and then different types of chemical components can be analyzed and identified. Therefore, we used HPLC-Q-TOF-MS^EThe strategy identifies the sesquiterpene component in carpesium abrotanoides, guides HSCCC to rapidly separate out compounds with large active parts, and finally uses the separated compounds for anti-liver cancer activity through a JAK2/STAT3 signal channel.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention mainly aims to provide a preparation method of carpesium abrotanoides extract.

The invention also aims to provide the carpesium abrotanoides extract prepared by the preparation method.

The invention further aims to provide application of the carpesium abrotanoides extract prepared by the preparation method.

The purpose of the invention is realized by the following technical scheme:

a preparation method of carpesium abrotanoides extract comprises the following operation steps:

(1) taking whole plant of carpesium abrotanoides, drying, crushing, soaking and extracting for 24 hours by using ethanol with volume percentage concentration of 95% and mass which is 10 times of that of the whole plant of carpesium abrotanoides, extracting for three times, mixing extracting solutions of the three times, concentrating, and drying to obtain a total extract; adding NaOH solution with the mass percentage concentration of 2% into the total extract for dissolving until the pH value is 9, stopping adding the NaOH solution, and then adjusting the pH value to 2 by using concentrated hydrochloric acid with the mass percentage concentration of 1% to obtain a precipitate to obtain the carpesium abrotanoides total extract;

(2) separating the total extract of radix Carpesii by high speed countercurrent chromatography to obtain three compounds respectively with structural formula C₁₅H₂₂O₄Compound 1 of the formula C₁₅H₂₀O₃Compound 2 of the structural formula C₁₅H₂₀O₃Compound 3 of (1);

the parameters of the high-speed counter-current chromatography are as follows: firstly, when an n-hexane-ethyl acetate-methanol-water solvent system with the flow rate of 3.0mL/min, the rotation speed of 835rpm, the sampling amount of 200mg and the volume ratio of 1:9:9:1 is adopted to run for 150min, the pump stops working; then replacing the solvent system with chloroform-methanol-water in a volume ratio of 4:3: 2; after operating for 320min, the pump is closed, so that a peak A is obtained in 60-66min of the first stage, a peak B is obtained in 74-82min, and a peak C is obtained in 80-92min of the second stage, wherein the peak A corresponds to a compound 1, a compound 2 and a compound 3 respectively;

a Carpesium abrotanoides total extract prepared by the above preparation method is provided.

The application of the total extract of carpesium abrotanoides in preparing anti-liver cancer active medicines through JAK2/STAT3 channels.

The compound 1 is applied to the anti-liver cancer active drugs through JAK2/STAT3 channels.

The application of the compound 2 in the anti-liver cancer active drug through JAK2/STAT3 channels.

The application of the compound 3 in anti-liver cancer active drugs through JAK2/STAT3 channels.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention adopts the method of alkali dissolution and acid precipitation to extract the total sesquiterpene component of carpesium abrotanoides for the first time;

(2) the invention adopts high-speed counter-current chromatography to separate the three active substances with the highest content in the carpesium abrotanoides extract, thereby overcoming the defects of time consumption and labor waste of the carpesium abrotanoides extraction process in the prior art;

(3) the carpesium abrotanoides extract extracted by the invention has very obvious anti-liver cancer activity through JAK2/STAT3 channels.

Drawings

FIG. 1 is a positive, negative TIC and UV map of SLEC sites.

Fig. 2 and 3 are chromatograms of compounds prepared by HSCCC.

FIG. 4 is a graph of the effect of compounds 1-3 on JAK2 and STAT3mRNA expression in HepG2 cells.

FIG. 5 is a graph showing the effect of compounds 1-3 on JAK2, p-JAK2, STAT3, and p-STAT3 protein expression in HepG2 cells.

Detailed Description

The following further describes the present invention with reference to specific examples and drawings, but the present invention should not be construed as being limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Example 1:

first, the method used in this embodiment:

1. preparation of carpesium abrotanoides extract

Taking 2Kg of Carex meyeriana whole plant, drying and crushing, soaking for 24h with 95% ethanol with the volume percentage concentration of 10 times of the Carex meyeriana whole plant, extracting for three times, mixing the three extracting solutions, and drying to obtain 458g of Total Extract (TEC). TEC (400g) was dissolved in 2% by weight NaOH solution until pH 9, and then concentrated 1% by weight hydrochloric acid was used to adjust pH to 2 to obtain a precipitate, and total sesquiterpene lactone total extract (SLEC) (42g) was obtained.

2. Screening of antitumor Activity by cck-8 method

Cells in logarithmic growth phase were taken, digested and counted in 3 x 10³The cells were seeded at a density of 100. mu.L per well in 96-well plates. Each group is provided with 3 multiple wells, after adherence culture, TEC parts, SLEC parts and residual samples with different concentrations (50, 100, 150, 200 and 250g/mL) are respectively subjected to medicine adding treatment for corresponding time, according to each well, 10 mu l/well of cck8 is added, complete culture medium is used for preparing cck8 solution (100 ul of cck8 solution is added according to 1mL of complete culture medium), and 100ul of culture medium containing cck8 is added into each well of the medicine-containing culture medium. 37 ℃ and 5% CO₂After further incubation for 4 hours, absorbance (OD) values at 450nm were analyzed on a Bio-Tek microplate reader.

3. Liquid mass analysis of active site

By HPLC-Q-TOF-MS^EThe chemical composition of the SLEC site was analyzed. The liquid phase condition is Agilent high performance liquid chromatography column (Eclipse XDB C)₁₈4.6X 250X 5 μm); mobile phase a was 0.1% formic acid water, mobile phase B was acetonitrile, elution procedure: 0-8min, 2% B; 8-10min, 2% -20% B; 10-50min, 20% -52% B; 50-60min, 52% B; 60-65min, 52% -80% B; 65-68min, 80-98% B; 68-75min, 98% B column temperature 25 deg.C, volume flow 1.0mL/min, injection volume 5 μ L.

The mass spectrum conditions are as follows: mass spectrum condition ionization mode electrospray ionization (ESI), collecting positive and negative ion modes respectively, wherein the temperature of the drying gas is 325 ℃, the volume flow of the drying gas is 6.8 L.min < -1 >, the temperature of the sheath gas is 350 ℃, the voltage of a capillary tube is 4.0kV, the fragment voltage is 130V, and the mass number range is m/z 50-1000.

4. Preparation method of HSCCC

4.1 determination of partition coefficient

The distribution coefficients of the SLEC sites in different solvent systems were calculated by HPLC in this experiment. The specific operation is as follows: preparing a certain volume (20mL) of upper and lower phase solvents according to a solvent system proportion, placing the upper and lower phase solvents with the same volume in the same container (10mL test tube) after phase separation balance, adding a certain amount (0.1g) of sample, shaking violently to dissolve, and standing. And (3) taking upper and lower phase solvents with the same volume, injecting HPLC (high performance liquid chromatography) to measure peak areas AS and AM of the target component in the upper and lower phases, and calculating a distribution coefficient K value according to AS/AM.

4.2HSCCC separation

The solvent system is selected primarily based on the partition coefficient of the target. Five solvent systems: n-hexane-ethyl acetate-methanol-water (1:1:1:1), n-hexane-ethyl acetate-methanol-water (1:9:9:1), n-hexane-ethyl acetate-methanol-water (3:1:1:1), n-hexane-methanol-water (5:4:1), and chloroform-methanol-water (4:3:2) were used for the SLEC site separation experiments, respectively.

For each separation experiment, the separation column was packed with 30.0mL/min of stationary phase, followed by forward rotation (FWD) at 835 rpm. The mobile phase was pumped into the instrument at a rate of 3.0 mL/min. After equilibrium is reached, the sample is injected into the injection valve. Monitoring was continued at 218nm, with a UV detector to monitor the peak composition.

5. In vitro cell experiments

The cytotoxicity of the isolated compound (1-3) against HegG-2 cell line was examined by the cck-8 method.

5.1 quantitative real-time polymerase chain reaction

Total RNA was extracted from HepG-2 cells using TRIzol reagent. Approximately 500ng of total RNA was reverse transcribed per sample using PrimeScript II RTase reverse transcriptase to prepare cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using CFX-Connect 96 and SYBR fast qPCR master batches. The PCR primer sequence is JAK2, ACTAAATGCTGTCCCCC in the forward direction and TTCATGCAGTTGACCGT in the reverse direction; STAT3, forward TGAGGGAGCAGAGATGTG, reverse TGGGGGCTTGGTAAAA; GAPDH, forward CCACTCCTCCACCTTTG, reverse CACCACCCTGTTGCTGT. Relative GAPDH expression calculation for each target Gene 2 was used^-ΔΔCMethod of producing a composite material。

5.2 immunoblot detection

Total protein was extracted from RA-FLS using RIPA lysis buffer and protein concentration was measured using BCA protein assay kit. After the 20mg sample is completely lysed with 200mol/L lysis buffer, the sample is centrifuged at 1200rpm for 20min at 4 ℃, and the supernatant is taken for western blot analysis. The protein concentration of each sample was determined from Bicinchoninic Acid protein Assay kit. The loaded proteins were electrophoresed and separated on a 10% SDS-polyacrylamide gel (SDS-PAGE), and then transferred onto a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were blocked in 5% skim milk for 1 hour at room temperature and incubated with primary antibodies (JAK2, Anti-JAK2, STAT3, Anti-STAT3, GAPDH) overnight at 4 ℃. The membrane was washed 4 times 5 minutes each with PBST. Adding goat anti-rabbit IgG (1:10000), incubating at room temperature for 1h, detecting by ECL, and reading the gray value of the related strip by TANON GIS software.

6. Statistical analysis

The comparisons between groups were expressed as Standard Deviations (SD) using one-way analysis of variance (ANOVA) and t-test. All statistical analyses were significant at p <0.05 or p <0.01 using SPSS 15.0.

Second, experimental conclusion

1. ccK-8 method for treating TEC and SLEC with anti-tumor effect

The CCk8 method was used to evaluate the cytotoxicity of TEC and SLEC sites on tumor cell lines Hela, HepG2, SW620, A549, HL-60, smmc-7721 and PC-3. The results are shown in Table 1. From the table, it can be seen that TEC and SLEC both have some effect on different types of tumor cells. In particular, SLEC sites showed good activity against HepG-2 and Smmc-7721 cells.

Table 1: IC of parts of radix Carpesii on cancer cells₅₀Value of

2. Liquid mass analysis

Based on the fact that SLEC parts have good activity on hepatoma cells, the chemical components of SLEC parts are analyzed by HPLC-Q-TOF-MS/MS. By passing through the liquid phaseAnd mass spectrometry condition optimization, mass spectrometry was performed in positive and negative modes, respectively, as shown in fig. 1 and table 2. It was found that this type of compound (sesquiterpene lactones) has a higher response in the positive mode, mainly with [ M + H ]]⁺The form of the compound exists, the cracking rule of molecular ion peaks and multi-stage mass spectrum is further researched, and a large number of isomers are found. Therefore, the specific compound name needs to be further determined by other methods.

Table 2: chemical composition study of SLEC sites

3. HSCCC fast separation

The selection of a suitable biphasic solvent system is critical to the high performance separation of HSCCC, and typically requires high performance liquid chromatography to detect the appropriate k-value of the target component. In general, the value of K should be between 0.5 and 2.0.

The k values for the three target compounds by HPLC analysis are shown in Table 3. As can be seen from the table, the A and B peaks are well suited for solvent system 2. However, peak C is not suitable for this and is suitable for solvent system 5. Thus, when the mobile phase was running for 150min, the pump was stopped and the solvent system was then replaced with chloroform-methanol-water (v/v/v,4:3: 2). After 320min, the A peak (60-66min), the B peak (74-82min) and the C peak (80-92min) are obtained, as shown in FIG. 3.

Table 3: HSCCC separation of SLEC Compounds in different solvent systems

Besides a solvent system, factors such as the flow speed and the rotating speed of a mobile phase, the sample loading amount and the like are considered in an experiment. Different flow rates (1.5, 2.0, 2.5 and 3.0mL/min), different rotational speeds (700, 800, 835 and 850rpm) and sample loadings (50, 100, 150 and 200mg) were compared. The results show that good separation efficiency can be obtained when the flow rate is 3.0mL/min, the rotation speed is 835rpm, and the sampling amount is 200 mg. The chromatograms are shown in FIGS. 2 and 3.

4. Structural identification of isolated sesquiterpene lactones

The obtained compounds are all prepared by HPLC-Q-TOF-MS,¹H-NMR、¹³C-NMR was identified and compared with relevant literature. Their detailed data are shown below, and it was confirmed that they are each of the structural formula C₁₅H₂₂O₄The compound 1(2 alpha, 5 alpha-dihydroxy-11 alpha H-eudesma-4(15) -en-12,8 beta-olide) has a structural formula of C₁₅H₂₀O₃Compound 2 (Telekin) with structural formula C₁₅H₂₀O₃Compound 3(1-oxoeudesm-11(13) -eno-12,8 a-lactone):

2α,5α-dihydroxy-11αH-eudesma-4(15)-en-12,8β-olide(A):ESI-MS,m/z267.1541[M+H]⁺,C₁₅H₂₂O₄.¹H NMR(600MHz,MeOD)δ:0.89(s,3H,H-14),1.17(d,J＝7.2Hz,3H,H-13),1.41(td,J＝6.2,5.5,1.9Hz,1H,H-1β),1.43(m,1H,H-6β),1.69(dd,J＝13.8,6.1Hz,1H,H-6α),1.80(m,1H,H-1α),1.83(m,1H,H-9β),2.03(dd,J＝15.4,4.7Hz,1H,H-9α),2.40(ddd,J＝12.2,5.4,1.9Hz,1H,H-3β),2.61(m,1H,H-3α),2.83(dtd,J＝12.7,6.4,4.3Hz,1H,H-7),2.97(p,J＝7.1Hz,1H,H-11),3.80(tt,J＝11.3,5.1Hz,1H,H-2),4.59(td,J＝4.5,1.7Hz,1H,H-8),4.80(t,J＝1.5Hz,1H,H-15),4.94(t,J＝1.5Hz,1H,H-15).¹³C NMR(151MHz,MeOD)δ:180.8(C-12),149.7(C-4),108.8(C-15),78.5(C-8),72.1(C-5),66.1(C-2),43.7(C-1),40.9(C-3),40.7(C-11),37.1(C-7),36.5(C-10),35.4(C-9),26.6(C-6),21.5(C-14),8.2(C-13).

Telekin(B):ESI-MS,m/z 249.1418[M+H]⁺,C₁₅H₂₀O₃.¹H NMR(600MHz,CDCl3)δ:0.98(s,3H,H-14),3.37(m,1H,H-7),4.58(td,J＝5.2,1.5Hz,1H,H-8),4.71(t,1H,H-15),4.88(t,1H,H-15),5.61(d,1H,J＝1.1Hz,H-13),6.16(d,1H,J＝1.2Hz,H-13).¹³C NMR(151MHz,CDCl₃)δ:170.8(C-12),150.1(C-4),142.1(C-11),120.3(C-13),109.0(C-15),77.0(C-8),74.3(C-5),37.6(C-7),36.5(C-10),35.6(C-1),35.4(C-2),33.8(C-3),31.8(C-9),21.8(C-14),21.6(C-6).

1-oxoeudesm-11(13)-eno-12,8a-lactone(C):ESI-MS,m/z 249.1423[M+H]⁺,C₁₅H₂₀O₃.¹H NMR(600MHz,CDCl₃)δ:0.96(s,3H,H-14),1.02(d,J＝5.8Hz,3H,H-15),1.35(q,J＝11.9Hz,1H,H-9),1.45(dd,J＝14.8,11.8Hz,1H,H-6),1.55(m,1H,H-2),1.83(m,1H,H-5),1.87(m,1H,H-4),2.04(m,1H,H-6),2.10(m,1H,H-2),2.43(m,1H,H-3),2.39(dd,J＝9.4,4.2Hz,1H,H-9),2.70(m,1H,H-7),4.21(ddd,J＝12.0,9.2,3.1Hz,1H,H-8),5.44(d,J＝3.2Hz,1H,H-13),6.11(d,J＝3.5Hz,1H,H-13).¹³C NMR(151MHz,CDCl₃)δ:222.0(C-1),169.8(C-12),140.2(C-11),120.1(C-13),80.8(C-8),50.0(C-10),48.7(C-5),44.8(C-7),44.1(C-3),35.2(C-2),34.5(C-9),29.6(C-4),24.1(C-6),22.0(C-14),20.0(C-15).

5. activity verification

Experiments show that the SLEC part has obvious inhibition effect on HepG-2 cells. Thus, this experiment was continued with in vitro cell experimental validation of the three isolated compounds. The results show that all three compounds show some inhibitory effect (IC50 ═ 9.83, 2.95, 4.15 μ M).

5.1 Effect of Compounds on HepG-2 cell JAK2 and STAT3mRNA expression

As shown in FIG. 4, the mRNA expression levels of both JAK-2 and STAT-3 were significantly reduced for the positive control paclitaxel compared to the control group (p < 0.01). Given compound-dried prognosis, high and intermediate concentrations of JAK-2mRNA expression levels of

compounds

1 and 2 were significantly reduced compared to the control group (p < 0.01); the JAK-2mRNA expression level of the compound 3 is obviously reduced compared with that of a control group (p <0.05), and the compound has concentration dependence; STAT-3mRNA expression levels were reduced (p <0.01) for

compounds

1, 2 and 3 compared to the control group and were concentration dependent.

5.2 Effect of Compounds on HepG-2 cell JAK2 and STAT3mRNA expression

As shown in figure 4, mRNA expression levels of both JAK2 and STAT3 were significantly reduced for the positive control paclitaxel compared to the control group (p < 0.01). Given compound-dried prognosis, high and intermediate concentrations of JAK 2mRNA expression levels of

compounds

1 and 2 were significantly reduced compared to the control group (p < 0.01); JAK 2mRNA expression level of compound 3 was significantly reduced (p <0.05) compared to control group and was concentration dependent; STAT3mRNA expression levels were reduced (p <0.01) for

compounds

1, 2, and 3 compared to the control group and were concentration dependent.

5.3 Effect of Compounds on expression of JAK2, p-JAK2, STAT3, p-STAT3 proteins by HepG2 cells

As shown in FIG. 5, the p-JAK2 and p-STAT3 protein expression levels of the positive control paclitaxel were significantly reduced (p <0.01) compared to the control group. Given the compound-dried prognosis, the high and medium concentrations of p-JAK2 and p-STAT3 protein expression levels of

compounds

1 and 3 were significantly reduced compared to the control group (p <0.05 or p < 0.01); the expression level of p-JAK2 protein at high concentration of compound 2 was significantly reduced (p <0.05), and the expression level of p-STAT3 protein at high and medium concentration of

compounds

1 and 3 was significantly reduced (p <0.05 or p < 0.01). This suggests that

compounds

1, 2, 3 and the positive drug paclitaxel can significantly inhibit the expression of p-JAK2 and p-STAT3 proteins. Paclitaxel, JAK2 of compounds 1-3, and STAT3 protein expression were not statistically significant on average.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Hunan province institute of traditional Chinese medicine

<120> an radix Carpesii extract, a preparation method and application thereof in anti-liver cancer active drugs through JAK2/STAT3 channels

<130> 1

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

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<212> DNA

<213> Artificial

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Claims

1. The preparation method of the carpesium abrotanoides extract is characterized by comprising the following operation steps of:

the parameters of the high-speed counter-current chromatography are as follows: firstly, when an n-hexane-ethyl acetate-methanol-water solvent system with the flow rate of 3.0mL/min, the rotation speed of 835rpm, the sampling amount of 200mg and the volume ratio of 1:9:9:1 is adopted to run for 150min, the pump stops working; then replacing the solvent system with chloroform-methanol-water in a volume ratio of 4:3: 2; after 320min of operation, the pump was turned off, resulting in peak a at 60-66min in the first stage, peak B at 74-82min, and peak C at 80-92min in the second stage, corresponding to compound 1, compound 2, and compound 3, respectively.

2. A total extract of Carpesium abrotanoides prepared by the preparation method of claim 1.

3. The use of the total extract of carpesium abrotanoides as claimed in claim 2 in the preparation of anti-hepatoma active drugs via JAK2/STAT3 channel.

4. The compound 1 is applied to the anti-liver cancer active drugs through JAK2/STAT3 channels.

5. The application of the compound 2 in the anti-liver cancer active drug through JAK2/STAT3 channels.

6. The application of the compound 3 in anti-liver cancer active drugs through JAK2/STAT3 channels.