# Systemic Pharmacological Approach to Identification and Experimental Verification of the Effect of Anisi Stellati Fructus Extract on Chronic Myeloid Leukemia Cells

Authors: Youn Sook Kim; Su Yeon Suh; Yong Tae Ahn; Chul Won Lee; Sang Yull Lee; Soon Cheol Ahn; Won G. An
Journal: Evidence-Based Complementary and Alternative Medicine
 (2019)

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## Abstract

Anisi stellati fructus (ASF) is the dried fruit of theIllicium verum Hook.f. tree. The aim of this research was to evaluate the antileukemic effect of ASF on chronic myeloid leukemia (CML) cells, which was hypothesized from the systemic pharmacological analysis of ASF, focusing on the combined effect of ASF extract (ASFE) and imatinib (IM). The compounds of ASF were identified using the Traditional Chinese Medicine Systems Pharmacology database and analysis platform. The target gene information was acquired from the UniProt database. The compound and target interaction network was generated from Cytoscape 3.7.1. Using this analysis, 10 compounds effective against CML cells were obtained. ASFE was prepared and analyzed by high-pressure liquid chromatography to provide experimental proof for the relationship between ASF and CML. The anti-p210Bcr-Abl effects of ASFE and ASFE + IM combination were evaluated by western blotting. Either ASFE alone or in combined treatment with IM on K-562 CML cells resulted in a significant reduction of the Bcr-Abl levels. As expected from the systemic analysis results, ASF had antileukemic activity, showing that it is a potential therapy for CML.


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## Body

## 1. Introduction

Anisi stellati fructus (ASF) is fruit that comes from the evergreen aromatic tree,Illicium verum Hook. f. According to The Great Pharmacopoeia written by Li Shi Zhen, published in 1596, the fruit of Illicium verum Hook. f. (Chinese star anise) has been used as a remedy for infectious diseases. ASF has long been used as a food as well as for medical purposes because of its effect on eliminating odor and relieving symptoms such as high fever, diarrhea, and vomiting [1]. Additionally, Tamiflu, a treatment for swine-origin influenza A, was developed based on the components of ASF [2].Chronic myeloid leukemia (CML) is considered to be a myeloproliferative neoplasm characterized by the expansion of a clone of hematopoietic cells that carries the abnormal Philadelphia chromosome and fusedBcr-Abl gene [3]. Bcr-Abl is a constitutively active cytoplasmic tyrosine kinase [4]. Bcr-Abl fusion protein is present in 95% of CML cases and 20%–30% of acute lymphoblastic leukemia cases [5, 6]. For these reasons, p210Bcr-Abl is a major target of tyrosine kinase inhibitors (TKIs) [7]. CML is diagnosed in the chronic phase and is transformed into the acute phase in an average of 2-3 years, resulting in mortality within a few months despite antileukemic combination therapy [8, 9].Imatinib (brand name: Gleevec, Novartis; formerly called STI571) is a relatively specific inhibitor of the Bcr-Abl tyrosine kinase, which has efficacy in CML [10]. Although survival from CML was increased by imatinib (IM), drug resistance by mutated forms of Bcr-Abl led to the development of new drugs such as dasatinib, nilotinib, bosutinib, and ponatinib [11]. The K-562 CML cell line is highly resistant to various stimuli. However, the development of new drugs often causes resistance to these drugs [12].To date, some studies have shown a correlation between infection and cancer, and approximately 2 million emerging cancer cases are due to infections from microorganisms [13–15]. Numerous studies have shown that antimicrobial peptides have cytotoxic effects on cancer cells and have the possibility of being used in anticancer therapy either alone or in combination with other conventional drugs. An increasing number of studies have shown that some of the cationic antimicrobial peptides, which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activities against cancer cells [16, 17]. Our previous study has shown that ASF contains 49 identified compounds, of which 35 compounds were analyzed to have target genes related to antimicrobial activities [18]. However, there has been minimal research on the anticancer effects of ASF. Only ASF has been reported to inhibit the metastasis and angiogenesis of malignant cancer cells [19]. In the present study, the 49 identified compounds of ASF were further investigated for their efficacy against cancer using systemic pharmacological analysis. The results showed that 20% of the total ASF compounds were associated with CML. Based on this result, we characterized the antileukemic potential of ASF against K-562 CML cells. The results showed that the ASF extract (ASFE) either alone or in combination with IM significantly reduced the amount of p210Bcr-Abl protein, suggesting further studies to confirm ASF as an effective antileukemic adjuvant.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

IM was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline (D-PBS) was purchased from Gibco (Grand Island, NY, USA). High-pressure liquid chromatography- (HPLC-) grade methanol and water were obtained from JT Baker (Phillipsburg, NJ, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Rockville, MD, USA). Anti-Bcr and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody specific to beta-actin was purchased from Sigma-Aldrich. The 4-anisaldehyde, 4-prophenylanisole, and other chemicals were purchased from Sigma-Aldrich.


### 2.2. Systemic Pharmacological Analyses of ASF

#### 2.2.1. Identification of ASF Compounds

To search for identified compounds in ASF and to investigate the relationship between the compounds and cancers, the Traditional Chinese Medicine Systems Pharmacology (TCMSP,http://lsp.nwu.edu.cn/) database and analysis platform were used. The 49 compounds were found in ASF, and all diseases related to each compound were recorded. After screening a total of 49 compounds, 33 compounds were found to have relationships with cancer categories. Among them, 10 CML-related compounds were identified. To screen bioactive compounds of these 10 CML-related compounds, five parameters related to absorption, distribution, metabolism, and excretion were used: molecular weight (MW), oral bioavailability (OB), Caco-2 permeability (Caco-2 cells), drug likeness (DL), and drug half-life (HL) [20–22].


#### 2.2.2. Target Collection

Molecular targets of the 10 CML-related compounds and the official gene information were acquired and listed using the UniProt database (http://www.uniprot.org).


#### 2.2.3. Network Construction

To visualize the 10 CML-related compounds and their possible targets, a compound (C)-target (T) network was constructed using Cytoscape 3.7.1, an open-source bioinformatics platform for visualizing molecular interaction networks (https://cytoscape.org/). In the network, the node denoted either compounds or target proteins, and the edges represented compound-target connections.


### 2.3. Preparation of ASF

Dried fruits of ASF (1.0 g) obtained from Kwangmyungdang Medical Herbs (Ulsan, Republic of Korea) were broken into pieces, ground, and mixed with 10 mL of methanol. The mixture was vortexed and sonicated for 30 min at room temperature. The resulting mixture was centrifuged at 3,000 rpm for 30 min at room temperature. The supernatant was carefully collected and filtered through a 0.2μm syringe filter (BIOFACT™, Yuseong-Gu, Republic of Korea). The filtrate was evaporated using a pressured gas blowing concentrator (MGS-2200; EYELA, Chula Vista, CA, USA). The yield of the ASF was 5%.


### 2.4. Chemical Profiling of ASF by HPLC

#### 2.4.1. Chromatography Conditions

Analysis was performed using an Agilent 1290 HPLC system (Agilent Technologies, Palo Alto, CA, USA) consisting of a quaternary pump, an autosampler, a column oven, and a diode-array detector. The data were processed using ChemStation software, rev. B. 03. 02 (Agilent). To separate the sample, an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7μm; Waters, Milford, MA, USA) was used. The mobile phase was comprised of 0.1% formic acid in distilled water (A) and acetonitrile (B). The column temperature was maintained at 40°C. The analysis was conducted at a flow rate of 1.0 mL/min with PDA detection wavelengths of 254 nm and 275 nm. The injection volume was 5 μL.


#### 2.4.2. Preparation of Standard Solutions

The standard stock solutions of 4-anisaldehyde and 4-prophenylanisole were prepared in methanol and stored at 4°C. A working standard solution was prepared by serial dilution of stock solutions with methanol. All calibration curves were obtained from assessments of peak areas of standard solutions in the following concentration ranges: 4-anisaldehyde, 0.10–2.00μg/mL; 4-prophenylanisole, 0.10–2.00 μg/mL.


#### 2.4.3. Preparation of Sample Solution

The resulting solution of ASFE was filtered through a 0.2μm syringe filter and injected into the HPLC instrument.


### 2.5. Cell Culture

Chronic myeloid K-562 leukemia cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI 1640 containing 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich) and 1% (v/v) penicillin/streptomycin (Gibco/BRL, Grand Island, NY) at 37°C in a 5% (v/v) CO2 incubator.


### 2.6. Cell Viability Assay

Cell viability was determined using the CCK-8 assay according to the manufacturer’s instructions. K-562 cells were seeded at a density of 1 × 104/well in a 96-well plate. After incubation for 24 h, the cells were treated with dimethyl sulfoxide or various concentrations of ASFE for 24 h at 37°C in an incubator with a 5% CO2 atmosphere. The cells were then incubated with 10 μL of CCK-8 reagent for 4 h at 37°C. Absorbance was measured at 450 nm using an ELISA microplate reader (Tecan, Mannedorf, Switzerland). Data are presented as the percentage viabilities of untreated cells (100%). The assay was independently repeated three times.


### 2.7. Western Blot Analysis

After treatment with ASFE, IM, and ASFE + IM by either a dose-dependent or time-dependent manner, K-562 cells were harvested by centrifugation at 4°C, washed once with ice-cold D-PBS, resuspended in lysis buffer (50 mM Tris (pH7.5), 2 mM EDTA, 100 mM NaCl, and 1% NP-40) containing protease inhibitor cocktail (Sigma-Aldrich), and incubated for 30 min at 4°C. Following centrifugation for 15 min at 13,000 rpm at 4°C, the protein concentration of the lysate was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. 30μg of total protein was separated on a 6% or 7% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The blots were incubated with a blocking solution (5% skim milk) for 1 h at room temperature, followed by incubation overnight with the primary antibodies (anti-Bcr, 1 : 3,000 dilution; anti-beta-actin, 1 : 1,000 dilution) at 4°C. The blots were then washed three times with Tween-20/Tris-buffered saline (TTBS), followed by incubation with the appropriate horseradish peroxide-conjugated secondary antibodies (1 : 3,000 dilution) for 1 h at room temperature, and then washed with TTBS. Immunoreactive bands were visualized using the Enhanced Chemiluminescence Detection Solution (ECL Plus; Thermo Fisher Scientific). ImageJ software was used to quantitate the band densities.


### 2.8. Quantification of Apoptosis by Annexin V Labeling

Apoptosis was quantified using an annexin V and 7-AAD Kit (Millipore, Hayward, CA, USA) according to the manufacturer’s instructions. Briefly, after 1 × 106 K-562 cells were seeded in a 60 mm cell culture dish, ASFE (2 μg/mL), IM (2.0 μmol), or their combination was added. Following incubation for 24 h at 37°C in a 5% CO2 incubator, the cells were collected and incubated with annexin-V and 7-AAD for 20 min at room temperature in the dark. The events for live, dead, and early (annexin V+/7-AAD+) cells were counted using the Muse cell analyzer (Merck Millipore, Billerica, MA, USA).


### 2.9. Statistical Analysis

Statistical analysis was performed using SPSS statistical software for Windows, version 23 (IBM, Armonk, NY, USA). Data are expressed as the mean ± standard deviation (SD) of triplicate determinations. Differences in means between groups were subjected to one-way analysis of variance followed by the least-significant multiple comparison test and independentt-test. P values <0.05 were considered significant differences.


## 2.1. Chemicals and Reagents

IM was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline (D-PBS) was purchased from Gibco (Grand Island, NY, USA). High-pressure liquid chromatography- (HPLC-) grade methanol and water were obtained from JT Baker (Phillipsburg, NJ, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Rockville, MD, USA). Anti-Bcr and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody specific to beta-actin was purchased from Sigma-Aldrich. The 4-anisaldehyde, 4-prophenylanisole, and other chemicals were purchased from Sigma-Aldrich.


## 2.2. Systemic Pharmacological Analyses of ASF

### 2.2.1. Identification of ASF Compounds

To search for identified compounds in ASF and to investigate the relationship between the compounds and cancers, the Traditional Chinese Medicine Systems Pharmacology (TCMSP,http://lsp.nwu.edu.cn/) database and analysis platform were used. The 49 compounds were found in ASF, and all diseases related to each compound were recorded. After screening a total of 49 compounds, 33 compounds were found to have relationships with cancer categories. Among them, 10 CML-related compounds were identified. To screen bioactive compounds of these 10 CML-related compounds, five parameters related to absorption, distribution, metabolism, and excretion were used: molecular weight (MW), oral bioavailability (OB), Caco-2 permeability (Caco-2 cells), drug likeness (DL), and drug half-life (HL) [20–22].


### 2.2.2. Target Collection

Molecular targets of the 10 CML-related compounds and the official gene information were acquired and listed using the UniProt database (http://www.uniprot.org).


### 2.2.3. Network Construction

To visualize the 10 CML-related compounds and their possible targets, a compound (C)-target (T) network was constructed using Cytoscape 3.7.1, an open-source bioinformatics platform for visualizing molecular interaction networks (https://cytoscape.org/). In the network, the node denoted either compounds or target proteins, and the edges represented compound-target connections.


## 2.2.1. Identification of ASF Compounds

To search for identified compounds in ASF and to investigate the relationship between the compounds and cancers, the Traditional Chinese Medicine Systems Pharmacology (TCMSP,http://lsp.nwu.edu.cn/) database and analysis platform were used. The 49 compounds were found in ASF, and all diseases related to each compound were recorded. After screening a total of 49 compounds, 33 compounds were found to have relationships with cancer categories. Among them, 10 CML-related compounds were identified. To screen bioactive compounds of these 10 CML-related compounds, five parameters related to absorption, distribution, metabolism, and excretion were used: molecular weight (MW), oral bioavailability (OB), Caco-2 permeability (Caco-2 cells), drug likeness (DL), and drug half-life (HL) [20–22].


## 2.2.2. Target Collection

Molecular targets of the 10 CML-related compounds and the official gene information were acquired and listed using the UniProt database (http://www.uniprot.org).


## 2.2.3. Network Construction

To visualize the 10 CML-related compounds and their possible targets, a compound (C)-target (T) network was constructed using Cytoscape 3.7.1, an open-source bioinformatics platform for visualizing molecular interaction networks (https://cytoscape.org/). In the network, the node denoted either compounds or target proteins, and the edges represented compound-target connections.


## 2.3. Preparation of ASF

Dried fruits of ASF (1.0 g) obtained from Kwangmyungdang Medical Herbs (Ulsan, Republic of Korea) were broken into pieces, ground, and mixed with 10 mL of methanol. The mixture was vortexed and sonicated for 30 min at room temperature. The resulting mixture was centrifuged at 3,000 rpm for 30 min at room temperature. The supernatant was carefully collected and filtered through a 0.2μm syringe filter (BIOFACT™, Yuseong-Gu, Republic of Korea). The filtrate was evaporated using a pressured gas blowing concentrator (MGS-2200; EYELA, Chula Vista, CA, USA). The yield of the ASF was 5%.


## 2.4. Chemical Profiling of ASF by HPLC

### 2.4.1. Chromatography Conditions

Analysis was performed using an Agilent 1290 HPLC system (Agilent Technologies, Palo Alto, CA, USA) consisting of a quaternary pump, an autosampler, a column oven, and a diode-array detector. The data were processed using ChemStation software, rev. B. 03. 02 (Agilent). To separate the sample, an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7μm; Waters, Milford, MA, USA) was used. The mobile phase was comprised of 0.1% formic acid in distilled water (A) and acetonitrile (B). The column temperature was maintained at 40°C. The analysis was conducted at a flow rate of 1.0 mL/min with PDA detection wavelengths of 254 nm and 275 nm. The injection volume was 5 μL.


### 2.4.2. Preparation of Standard Solutions

The standard stock solutions of 4-anisaldehyde and 4-prophenylanisole were prepared in methanol and stored at 4°C. A working standard solution was prepared by serial dilution of stock solutions with methanol. All calibration curves were obtained from assessments of peak areas of standard solutions in the following concentration ranges: 4-anisaldehyde, 0.10–2.00μg/mL; 4-prophenylanisole, 0.10–2.00 μg/mL.


### 2.4.3. Preparation of Sample Solution

The resulting solution of ASFE was filtered through a 0.2μm syringe filter and injected into the HPLC instrument.


## 2.4.1. Chromatography Conditions

Analysis was performed using an Agilent 1290 HPLC system (Agilent Technologies, Palo Alto, CA, USA) consisting of a quaternary pump, an autosampler, a column oven, and a diode-array detector. The data were processed using ChemStation software, rev. B. 03. 02 (Agilent). To separate the sample, an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7μm; Waters, Milford, MA, USA) was used. The mobile phase was comprised of 0.1% formic acid in distilled water (A) and acetonitrile (B). The column temperature was maintained at 40°C. The analysis was conducted at a flow rate of 1.0 mL/min with PDA detection wavelengths of 254 nm and 275 nm. The injection volume was 5 μL.


## 2.4.2. Preparation of Standard Solutions

The standard stock solutions of 4-anisaldehyde and 4-prophenylanisole were prepared in methanol and stored at 4°C. A working standard solution was prepared by serial dilution of stock solutions with methanol. All calibration curves were obtained from assessments of peak areas of standard solutions in the following concentration ranges: 4-anisaldehyde, 0.10–2.00μg/mL; 4-prophenylanisole, 0.10–2.00 μg/mL.


## 2.4.3. Preparation of Sample Solution

The resulting solution of ASFE was filtered through a 0.2μm syringe filter and injected into the HPLC instrument.


## 2.5. Cell Culture

Chronic myeloid K-562 leukemia cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI 1640 containing 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich) and 1% (v/v) penicillin/streptomycin (Gibco/BRL, Grand Island, NY) at 37°C in a 5% (v/v) CO2 incubator.


## 2.6. Cell Viability Assay

Cell viability was determined using the CCK-8 assay according to the manufacturer’s instructions. K-562 cells were seeded at a density of 1 × 104/well in a 96-well plate. After incubation for 24 h, the cells were treated with dimethyl sulfoxide or various concentrations of ASFE for 24 h at 37°C in an incubator with a 5% CO2 atmosphere. The cells were then incubated with 10 μL of CCK-8 reagent for 4 h at 37°C. Absorbance was measured at 450 nm using an ELISA microplate reader (Tecan, Mannedorf, Switzerland). Data are presented as the percentage viabilities of untreated cells (100%). The assay was independently repeated three times.


## 2.7. Western Blot Analysis

After treatment with ASFE, IM, and ASFE + IM by either a dose-dependent or time-dependent manner, K-562 cells were harvested by centrifugation at 4°C, washed once with ice-cold D-PBS, resuspended in lysis buffer (50 mM Tris (pH7.5), 2 mM EDTA, 100 mM NaCl, and 1% NP-40) containing protease inhibitor cocktail (Sigma-Aldrich), and incubated for 30 min at 4°C. Following centrifugation for 15 min at 13,000 rpm at 4°C, the protein concentration of the lysate was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. 30μg of total protein was separated on a 6% or 7% sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The blots were incubated with a blocking solution (5% skim milk) for 1 h at room temperature, followed by incubation overnight with the primary antibodies (anti-Bcr, 1 : 3,000 dilution; anti-beta-actin, 1 : 1,000 dilution) at 4°C. The blots were then washed three times with Tween-20/Tris-buffered saline (TTBS), followed by incubation with the appropriate horseradish peroxide-conjugated secondary antibodies (1 : 3,000 dilution) for 1 h at room temperature, and then washed with TTBS. Immunoreactive bands were visualized using the Enhanced Chemiluminescence Detection Solution (ECL Plus; Thermo Fisher Scientific). ImageJ software was used to quantitate the band densities.


## 2.8. Quantification of Apoptosis by Annexin V Labeling

Apoptosis was quantified using an annexin V and 7-AAD Kit (Millipore, Hayward, CA, USA) according to the manufacturer’s instructions. Briefly, after 1 × 106 K-562 cells were seeded in a 60 mm cell culture dish, ASFE (2 μg/mL), IM (2.0 μmol), or their combination was added. Following incubation for 24 h at 37°C in a 5% CO2 incubator, the cells were collected and incubated with annexin-V and 7-AAD for 20 min at room temperature in the dark. The events for live, dead, and early (annexin V+/7-AAD+) cells were counted using the Muse cell analyzer (Merck Millipore, Billerica, MA, USA).


## 2.9. Statistical Analysis

Statistical analysis was performed using SPSS statistical software for Windows, version 23 (IBM, Armonk, NY, USA). Data are expressed as the mean ± standard deviation (SD) of triplicate determinations. Differences in means between groups were subjected to one-way analysis of variance followed by the least-significant multiple comparison test and independentt-test. P values <0.05 were considered significant differences.


## 3. Results

### 3.1. Screening 33 Cancer-Related Compounds

According to the TCMSP database, ASF contained 49 identified compounds. Every compound and its related diseases were investigated, and 33 cancer-related compounds were screened (Table1).Table 1
The 33 compounds and their related cancers.
Active compounds
Related cancers
C09628
Bladder cancerBreast cancerCancer, unspecific Colorectal cancerGenitourinary tumorsLung cancerOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Kaempferol
Prostate cancerAdrenocorticotropic hormone-secreting pituitary tumorsBladder cancerPancreatic cancerRenal cell carcinomaTesticular cancerThyroid follicular carcinomaBreast cancerColorectal cancerGenitourinary tumorsLung cancerHead and neck cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaChronic myelogenous leukemia (CML)Gastrointestinal stromal tumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumorsMalignanciesGliomasColon cancerAcute promyelocytic leukemiaChronic lymphocytic leukemiaProstate cancer (hormone-refractory)Waldenstrom’s macroglobulinemiaSolid tumorCancer (multidrug-resistant)ChondrosarcomaHormone-refractory prostate cancerKaposi’s sarcomaBreast neoplasmsColorectal neoplasmsOvarian neoplasmsUrinary bladder neoplasmsBronchiolar carcinomaCervical cancerRectal neoplasmsChronic myeloid leukemiaGastrointestinal cancersUrological cancersNon-small-cell lung carcinomaGastrointestinal cancersGastrointestinal neoplasmsHead and neck neoplasmsNeoplasmsPrecursor cellLymphoblastic leukemia-lymphoma
ZINC02040970
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ unspecifiedColorectal cancerGenitourinary tumorsLung cancerOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
(1R,5R,7S)-4,7-Dimethyl-7-(4-methylpent-3-enyl)bicyclo[3.1.1]hept-3-ene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
1,8-Cineole
Colon cancerBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerOropharyngeal squamous cell carcinomaProstate cancer
3-O-Feruloylquinic acid
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaPancreatic cancerTumors
Anisketone
GliomasBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaColon cancerPancreatic cancer
Copaene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Terpilene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOesophageal cancerOropharyngeal squamous cell carcinomaPathological angiogenesisGliomasRenal cell carcinomaLeukemia,Myeloid malignanciesProstate cancerSolid tumors
Guaiene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Salicylic acid
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedCarpal tunnel syndromeColorectal cancerGenitourinary tumorsLung cancerMalignant mesothelioma MeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaBronchiolar carcinomaCervical cancerChronic myeloid leukemiaGastrointestinal cancersPancreatic cancerUrological cancersLeukemia, myeloid,AcuteEndometrial carcinomaLeukemia, unspecifiedMesotheliomaTumorsPrecursorCell lymphoblasticLeukemia-lymphomaBreast neoplasmsNon-small-cell lung carcinomaColorectal neoplasmsGastrointestinal neoplasmsHead and neck neoplasmsNeoplasmsOvarian neoplasmsRectal neoplasms
(Z)-Caryophyllene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
(R)-Linalool
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaAcute promyelocytic leukemiaChronic myelogenous leukemia (CML)Gastrointestinal stromal tumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumors
Mairin
Breast cancer
Honokiol
GliomasBreast cancerProstate cancerEndocrine independent cancerAdrenocorticotropic hormone-secreting pituitary tumorsPancreatic cancerBladder cancerRenal cell carcinomaTesticular cancerThyroid follicular carcinomaSolid tumorsCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaRefractory hematological malignanciesOropharyngeal squamous cell carcinomaChronic myelogenous leukemia (CML)MalignanciesMultiple myelomaGastrointestinal stromaltumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaNon-small-cell lung cancerOvarian cancerAcute lymphoblastic leukemia (ALL)Acute myeloid leukemia (AML)Advanced solid tumorsB-cell malignanciesChronic lymphocytic leukemia (CLL)Hepatocellular carcinoma (HCC)Nasopharyngeal cancer (NPC)Non-Hodgkin’s lymphoma leukemiaMyeloidOesophageal cancer
Hemo-sol
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancer
(1S,5S)-1-Isopropyl-4-methylenebicyclo[3.1.0]hexane
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
CHEBI:7
Chronic myeloid leukemiaBreast cancerBladder cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaAcute promyelocytic leukemia
Luteolin
Prostate cancerBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaRenal cell carcinomaChronic myelogenousleukemia (CML)Gastrointestinal stromaltumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumorsMalignanciesHead and neck tumorsPancreatic cancerSolid tumorSquamous cell carcinoma tumorsColorectal neoplasmsBrain cancerHepatocellular carcinomaHormone-refractory prostate cancerKaposi’s sarcomaCancer (multidrug-resistant)Kidney cancerChondrosarcomaBreast neoplasmsNon-small-cell lung carcinomaGastrointestinal neoplasmsHead and neck neoplasmsNeoplasmsOvarian neoplasmsPrecursor cellLymphoblastic leukemia-lymphomaRectal neoplasmsGastric cancerProstate cancer (metastatic)Small-cell lung cancer
HCI
GliomasBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaMalignancies
Beta-selinene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
2-Methyl-N-phenylmaleimide
Cancer, unspecificTumorsBladder cancerBreast cancerCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerProstate cancerRenal cell carcinomaPancreatic cancerChronic myeloid leukemiaOesophageal cancer
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-[(2R,3R,4S,5R)-3,4,5-trihydroxytetrahydropyran-2-yl]oxy-chromone
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaProstate cancerMeningiomaRenal cell carcinomaOropharyngeal squamous cell carcinomaAcute promyelocytic leukemia
Anethole
GlaucomaBreast cancerCancer (multidrug-resistant)Melanoma
(+)-Catechin
Breast cancerEndocrine independent cancerBladder cancerCancer, unspecificColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaChronic myelogenous leukemia (CML)Refractory hematological malignanciesGastrointestinal stromal tumors (GIST)Precursor cellLymphoblastic leukemia-lymphomaHematological malignanciesHER2-positiveMetastatic breast cancerMelanomaNon-small-cell lung cancerOvarian cancerSolid tumors
Shikimic acid
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Astragalin
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerLung cancerGenitourinary tumorsProstate cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaRenal cell carcinomaAcute promyelocytic leukemiaChronic myelogenous leukemia (CML)Gastrointestinal stromal tumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumorsGliomasMalignancies
Cis-beta-farnesene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerMeningiomaGenitourinary tumorsMalignant mesotheliomaLung cancerOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
5,7-Dihydroxy-2-(4-hydroxyphenyl)-3-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one
Acute promyelocytic leukemiaCancer, unspecific
Beta-bisabolene
Bladder cancerProstate cancerBreast cancerCancer, unspecificRenal cell carcinomaColorectal cancerCarcinoma in situ, unspecifiedGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinoma
Quercetin
Prostate cancerBladder cancerPancreatic cancerRenal cell carcinomaAdrenocorticotropic hormone-secreting pituitary tumorsTesticular cancerThyroid follicular carcinomaBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerMalignant mesotheliomaMeningiomaUrinary bladder neoplasmsGenitourinary tumorsLung cancerMelanomaMultiple myelomaNon-small-cell lung cancer Oropharyngeal squamous cell carcinomaOvarian cancerChronic myelogenous leukemia (CML)Solid tumorsHematological malignanciesMalignanciesGastrointestinal stromal tumors (GIST)GliomasHER2-positiveMetastatic breast cancerBrain cancerColon cancerSolid tumorRefractory hematological malignanciesTumorAcute promyelocytic leukemiaKaposi’s sarcomaHead and neck tumorsRectal neoplasmsCancer (multidrug-resistant)Kidney cancerSquamous cell carcinomaColorectal neoplasmsBreast neoplasmsOvarian neoplasmsChronic lymphocytic leukemiaHormone-refractory prostate cancerWaldenstrom’s macroglobulinemiaEndometrial neoplasmsHepatocellular carcinomaChondrosarcomaBronchiolar carcinomaUrological cancersNon-small-cell lung carcinomaGastrointestinal neoplasmsHead and neck neoplasmsCervical cancerChronic myeloid leukemiaGastrointestinal cancersNeoplasmsPrecursor cell lymphoblastic leukemia-lymphoma
Terragon
Cancer, unspecificBladder cancerBreast cancerCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Humulene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaProstate cancerSolid tumor


### 3.2. Collecting 10 CML-Related Active Compounds and Systemic Pharmacological Analyses

Ten CML-related compounds and the analyzed results are shown in Table2. To search bioactive compounds, the criteria of each parameter were suggested. Molecular weights from 180 to 500 Daltons were recognized as comparatively more druggable [23]. Oral bioavailability (OB) ≥30% and drug likeness ≥0.18 were used as ideal drug characteristics [24, 25]. Caco-2 cell permeability was tested using an in vitro experiment with the Caco-2 human intestinal cell line. Drug half-life (t1/2) represented how long it took for the drug to be internalized and reduced by half; a drug half-life ≤4 h was classified as the fast elimination group, between 4 and 8 h as the midelimination group, and ≥8 h as the slow-elimination group [20, 21].Table 2
The 10 CML-related compounds of Anisi stellati fructus.
Molecule name
MW
OB (%)
Caco-2
DL
HL
Astragalin
448.41
14.03
−1.34
0.74
N/A
CHEBI:7
136.26
45.2
1.84
0.04
11.44
(+)-Catechin
290.29
54.83
−0.03
0.24
0.61
2-Methyl-N-phenylmaleimide
187.21
87.36
0.77
0.06
2.93
Kaempferol
286.25
41.88
0.26
0.24
14.74
Honokiol
266.36
60.67
1.43
0.15
2.88
Luteolin
286.25
36.16
0.19
0.25
15.94
Salicylic acid
138.13
32.13
0.63
0.03
12
(R)-Linalool
154.28
39.8
1.33
0.02
6.48
Quercetin
302.25
46.43
0.05
0.28
14.4
MW: molecular weight; OB: oral bioavailability; Caco-2: Caco-2 permeability; DL-2: drug likeness; HL: drug half-life.Among the 10 CML-related compounds, luteolin (MW = 286.25, OB (%) = 36.16, Caco-2 = 0.19, DL = 0.25, and HL = 15.94), kaempferol (MW = 286.25, OB (%) = 41.88, Caco-2 = 0.26, DL = 0.24, and HL = 14.74), and quercetin (MW = 302.25, OB (%) = 46.43, Caco-2 = 0.05, DL = 0.28, and HL = 14.4) largely met the suggested criteria and have been reported as representative antibacterial compounds in previous studies [26–28], including our study [18]. Additionally, luteolin exhibited an antibacterial action that suppressed the activity of bacterial DNA topoisomerases 1 and 2 and reduced the synthesis of nucleic acids and proteins [29] and was also known to have antioxidant, anticancer, anti-inflammatory, and neuroprotective effects [30]. Kaempferol is a flavonoid and possesses anti-inflammatory, antioxidant, anticancer, and antibacterial activities [31–33]. It has been shown to be effective against acne-induced Propionibacterium acnes and Helicobacter pylori found in the stomach [34]. Quercetin is a compound that plays an important role in inflammation, cancer, aging, cell signaling, proapoptotic effects, antiproliferative effects, antioxidant effects, and growth suppression [35, 36]. Astragalin (MW = 448.41, OB (%) = 14.03, Caco-2 = -1.34, DL = 0.74, and HL = N/A) has been reported to inhibit autophagosome formation in airways [37]. Honokiol (MW = 266.36, OB (%) = 60.67, Caco-2 = 1.43, DL = 0.15, and HL = 2.88) has been shown to have antitumorigenic, anti-inflammatory, and antioxidant effects [38–40]. (+)-Catechin (MW = 290.29, OB (%) = 54.83, Caco-2 = −0.03, DL = 0.24, and HL = 0.61) is a well-known phenolic compound found in tea and wine.


### 3.3. Network Construction of 10 CML-Related Compounds and Their Related Targets

To visualize the target genes of 10 CML-related compounds, a compound (C)-target (T) network was established. A total of 313 target proteins were collected from the TCMSP, and the official gene names of the targets were obtained from the UniProt database. Figure1 shows that the C-T network revealed interactions between 10 CML-related compounds and 313 target genes. The nodes showed the 10 CML-related compounds and the target genes, and the edges represented the interaction of compounds and targets. The size of nodes was related to the degrees. Quercetin (degree = 77) had the greatest interaction with targets, followed by luteolin (degree = 56), kaempferol (degree = 36), salicylic acid (degree = 35), 2-methyl-N-phenylmaleimide (degree = 29), R-linalool (degree = 20), honokiol (degree = 18), astragalin (degree = 16), CHEBI:7 (degree = 15), and (+)-catechin (degree = 10).Figure 1
The network of 10 CML-related compounds and their related target genes. Triangle nodes represent compounds and circular nodes represent targets. Node size is relative to the degree, and the edges demonstrate the interaction between nodes.


### 3.4. HPLC Analysis of ASFE

The main components of ASFE were determined using an HPLC system to evaluate the quality of ASFE. Because 4-anisaldehyde and 4-prophenylanisole [(E)-anethole] have been reported to be the main compounds of ASFE, we used them as standards and conducted the analysis. As shown in the representative chromatographic fingerprint of ASFE, the main components of ASFE were 4-anisaldehyde and 4-prophenylanisole (Figure2).Figure 2
Chromatograms of two major components (4-anisaldehyde and 4-prophenylanisole) identified in Anisi stellati fructus extract (ASFE). (a) Chromatograms of the standard mixture. (b) Chromatograms of the major components in ASFE. The chromatograms were obtained at 254.5 and 275 nm.
(a)
(b)


### 3.5. Effect of ASFE on the Cell Viability of K-562 Cells

The possible cytotoxic effect of ASFE (0–8μg/mL) on K-562 cells was assessed (Figure 3). ASFE significantly inhibited the proliferation of K-562 cells in a dose-dependent manner. The IC50 value of ASFE was approximately 4.03 ± 2.3 μg/mL (∗P<0.05; ∗∗P<0.01).Figure 3
Cytotoxicity of the ASFE on K-562 cells. The cells (1 × 104/well) were determined using a CCK-8 assay.


### 3.6. ASFE Treatment on K-562 Cells Decreases the Amount of p210Bcr-Abl in a Time-dependent Manner

ASFE or IM was used to treat K-562 cells, and the p210Bcr-Abl protein steady-state levels in the total lysate were analyzed by western blotting (Figure 4). 2 μmol IM was subjected based on previous studies [41]. The p210Bcr-Abl protein levels were decreased after treatment with ASFE or IM on K-562 cells in a time-dependent manner (∗P<0.05; ∗∗P<0.01).Figure 4
Changes in the p210Bcr-Abl protein levels treated with ASFE or imatinib (IM). K-562 cells were treated with (a) ASFE (2 μg/mL) or (b) IM (2 μmol) for increasing time periods (0–16 h). The p210Bcr-Abl protein steady-state levels in the total lysates were analyzed by western blotting. Relative protein levels versus controls (beta-actin) were determined by densitometry. Data are the mean ± SD of three independent experiments (∗P<0.05; ∗∗P<0.01).
(a)
(b)


### 3.7. ASFE Enhanced IM-Induced Destabilization of p210Bcr-Abl

Figure5 shows that when both ASFE and IM were added together to K-562 cells, the level of p210Bcr-Abl was diminished compared to that of the lanes containing K-562 cells after treatment with IM or ASFE, indicating that the combined treatment of K-562 cells by ASFE and IM was more potent than each one separately.Figure 5
ASFE + IM combined therapy led to a decrease of p210Bcr-Abl. K-562 cells were treated with IM (2 μmol), ASFE (2 μg/mL), and IM + ASFE combination for 12 h at 37°C. The p210Bcr-Abl protein levels in the total lysate were analyzed by western blotting (∗P<0.05; ∗∗P<0.01).


### 3.8. Assessment of ASFE- and IM-Inducing Cell Death Mechanisms on K-562 Cells

Apoptosis, necrosis, and autophagy are well-known cell death mechanisms. To observe apoptosis and necrosis, we conducted flow cytometry using annexin V and 7-AAD staining. After 16 h treatment of IM, ASFE, or IM + ASFE, the cells were stained with both annexin V and 7-AAD. The results showed that, in the control, >85% of the cells survived; the cell population decreased to 82.79% for the IM, 9.54% for the ASFE, and 0% for the IM + ASFE. The late apoptotic/necrotic cell population increased to 13.03% (IM), 36.33% (ASFE), and 42.89% (ASFE + IM). The early apoptotic cell population increased to 0.93% (IM), 0.00% (ASFE), and 0.00% (ASFE + IM) (Figure6). The cell populations of late apoptosis/necrosis significantly increased, but increased cell population during early apoptosis was rare. Based on these results, we postulated that K-562 cells were involved in an alternate pathway and were not involved in apoptosis. These results also showed that K-562 cell killing was significant after treatment with ASFE.Figure 6
Apoptosis assessment of the effects of IM, ASFE, and IM + ASFE on K-562 cells. After 16 h of treatment, the cells were stained with annexin V/7-AAD reagent and cytometric analysis was performed using a MUSE™ cell analyzer. (a) Control, (b) IM, (c) ASFE, (d) IM + ASFE.
(a)
(b)
(c)
(d)


## 3.1. Screening 33 Cancer-Related Compounds

According to the TCMSP database, ASF contained 49 identified compounds. Every compound and its related diseases were investigated, and 33 cancer-related compounds were screened (Table1).Table 1
The 33 compounds and their related cancers.
Active compounds
Related cancers
C09628
Bladder cancerBreast cancerCancer, unspecific Colorectal cancerGenitourinary tumorsLung cancerOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Kaempferol
Prostate cancerAdrenocorticotropic hormone-secreting pituitary tumorsBladder cancerPancreatic cancerRenal cell carcinomaTesticular cancerThyroid follicular carcinomaBreast cancerColorectal cancerGenitourinary tumorsLung cancerHead and neck cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaChronic myelogenous leukemia (CML)Gastrointestinal stromal tumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumorsMalignanciesGliomasColon cancerAcute promyelocytic leukemiaChronic lymphocytic leukemiaProstate cancer (hormone-refractory)Waldenstrom’s macroglobulinemiaSolid tumorCancer (multidrug-resistant)ChondrosarcomaHormone-refractory prostate cancerKaposi’s sarcomaBreast neoplasmsColorectal neoplasmsOvarian neoplasmsUrinary bladder neoplasmsBronchiolar carcinomaCervical cancerRectal neoplasmsChronic myeloid leukemiaGastrointestinal cancersUrological cancersNon-small-cell lung carcinomaGastrointestinal cancersGastrointestinal neoplasmsHead and neck neoplasmsNeoplasmsPrecursor cellLymphoblastic leukemia-lymphoma
ZINC02040970
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ unspecifiedColorectal cancerGenitourinary tumorsLung cancerOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
(1R,5R,7S)-4,7-Dimethyl-7-(4-methylpent-3-enyl)bicyclo[3.1.1]hept-3-ene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
1,8-Cineole
Colon cancerBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerOropharyngeal squamous cell carcinomaProstate cancer
3-O-Feruloylquinic acid
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaPancreatic cancerTumors
Anisketone
GliomasBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaColon cancerPancreatic cancer
Copaene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Terpilene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOesophageal cancerOropharyngeal squamous cell carcinomaPathological angiogenesisGliomasRenal cell carcinomaLeukemia,Myeloid malignanciesProstate cancerSolid tumors
Guaiene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Salicylic acid
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedCarpal tunnel syndromeColorectal cancerGenitourinary tumorsLung cancerMalignant mesothelioma MeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaBronchiolar carcinomaCervical cancerChronic myeloid leukemiaGastrointestinal cancersPancreatic cancerUrological cancersLeukemia, myeloid,AcuteEndometrial carcinomaLeukemia, unspecifiedMesotheliomaTumorsPrecursorCell lymphoblasticLeukemia-lymphomaBreast neoplasmsNon-small-cell lung carcinomaColorectal neoplasmsGastrointestinal neoplasmsHead and neck neoplasmsNeoplasmsOvarian neoplasmsRectal neoplasms
(Z)-Caryophyllene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
(R)-Linalool
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaAcute promyelocytic leukemiaChronic myelogenous leukemia (CML)Gastrointestinal stromal tumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumors
Mairin
Breast cancer
Honokiol
GliomasBreast cancerProstate cancerEndocrine independent cancerAdrenocorticotropic hormone-secreting pituitary tumorsPancreatic cancerBladder cancerRenal cell carcinomaTesticular cancerThyroid follicular carcinomaSolid tumorsCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaRefractory hematological malignanciesOropharyngeal squamous cell carcinomaChronic myelogenous leukemia (CML)MalignanciesMultiple myelomaGastrointestinal stromaltumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaNon-small-cell lung cancerOvarian cancerAcute lymphoblastic leukemia (ALL)Acute myeloid leukemia (AML)Advanced solid tumorsB-cell malignanciesChronic lymphocytic leukemia (CLL)Hepatocellular carcinoma (HCC)Nasopharyngeal cancer (NPC)Non-Hodgkin’s lymphoma leukemiaMyeloidOesophageal cancer
Hemo-sol
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancer
(1S,5S)-1-Isopropyl-4-methylenebicyclo[3.1.0]hexane
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
CHEBI:7
Chronic myeloid leukemiaBreast cancerBladder cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaAcute promyelocytic leukemia
Luteolin
Prostate cancerBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaRenal cell carcinomaChronic myelogenousleukemia (CML)Gastrointestinal stromaltumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumorsMalignanciesHead and neck tumorsPancreatic cancerSolid tumorSquamous cell carcinoma tumorsColorectal neoplasmsBrain cancerHepatocellular carcinomaHormone-refractory prostate cancerKaposi’s sarcomaCancer (multidrug-resistant)Kidney cancerChondrosarcomaBreast neoplasmsNon-small-cell lung carcinomaGastrointestinal neoplasmsHead and neck neoplasmsNeoplasmsOvarian neoplasmsPrecursor cellLymphoblastic leukemia-lymphomaRectal neoplasmsGastric cancerProstate cancer (metastatic)Small-cell lung cancer
HCI
GliomasBladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaMalignancies
Beta-selinene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
2-Methyl-N-phenylmaleimide
Cancer, unspecificTumorsBladder cancerBreast cancerCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerProstate cancerRenal cell carcinomaPancreatic cancerChronic myeloid leukemiaOesophageal cancer
2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-3-[(2R,3R,4S,5R)-3,4,5-trihydroxytetrahydropyran-2-yl]oxy-chromone
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaProstate cancerMeningiomaRenal cell carcinomaOropharyngeal squamous cell carcinomaAcute promyelocytic leukemia
Anethole
GlaucomaBreast cancerCancer (multidrug-resistant)Melanoma
(+)-Catechin
Breast cancerEndocrine independent cancerBladder cancerCancer, unspecificColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinomaChronic myelogenous leukemia (CML)Refractory hematological malignanciesGastrointestinal stromal tumors (GIST)Precursor cellLymphoblastic leukemia-lymphomaHematological malignanciesHER2-positiveMetastatic breast cancerMelanomaNon-small-cell lung cancerOvarian cancerSolid tumors
Shikimic acid
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Astragalin
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerLung cancerGenitourinary tumorsProstate cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaRenal cell carcinomaAcute promyelocytic leukemiaChronic myelogenous leukemia (CML)Gastrointestinal stromal tumors (GIST)Hematological malignanciesHER2-positiveMetastatic breast cancerMelanomaMultiple myelomaNon-small-cell lung cancerOvarian cancerRefractory hematological malignanciesSolid tumorsGliomasMalignancies
Cis-beta-farnesene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerMeningiomaGenitourinary tumorsMalignant mesotheliomaLung cancerOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
5,7-Dihydroxy-2-(4-hydroxyphenyl)-3-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one
Acute promyelocytic leukemiaCancer, unspecific
Beta-bisabolene
Bladder cancerProstate cancerBreast cancerCancer, unspecificRenal cell carcinomaColorectal cancerCarcinoma in situ, unspecifiedGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinoma
Quercetin
Prostate cancerBladder cancerPancreatic cancerRenal cell carcinomaAdrenocorticotropic hormone-secreting pituitary tumorsTesticular cancerThyroid follicular carcinomaBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerMalignant mesotheliomaMeningiomaUrinary bladder neoplasmsGenitourinary tumorsLung cancerMelanomaMultiple myelomaNon-small-cell lung cancer Oropharyngeal squamous cell carcinomaOvarian cancerChronic myelogenous leukemia (CML)Solid tumorsHematological malignanciesMalignanciesGastrointestinal stromal tumors (GIST)GliomasHER2-positiveMetastatic breast cancerBrain cancerColon cancerSolid tumorRefractory hematological malignanciesTumorAcute promyelocytic leukemiaKaposi’s sarcomaHead and neck tumorsRectal neoplasmsCancer (multidrug-resistant)Kidney cancerSquamous cell carcinomaColorectal neoplasmsBreast neoplasmsOvarian neoplasmsChronic lymphocytic leukemiaHormone-refractory prostate cancerWaldenstrom’s macroglobulinemiaEndometrial neoplasmsHepatocellular carcinomaChondrosarcomaBronchiolar carcinomaUrological cancersNon-small-cell lung carcinomaGastrointestinal neoplasmsHead and neck neoplasmsCervical cancerChronic myeloid leukemiaGastrointestinal cancersNeoplasmsPrecursor cell lymphoblastic leukemia-lymphoma
Terragon
Cancer, unspecificBladder cancerBreast cancerCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaOropharyngeal squamous cell carcinomaProstate cancerRenal cell carcinoma
Humulene
Bladder cancerBreast cancerCancer, unspecificCarcinoma in situ, unspecifiedColorectal cancerGenitourinary tumorsLung cancerMalignant mesotheliomaMeningiomaProstate cancerSolid tumor


## 3.2. Collecting 10 CML-Related Active Compounds and Systemic Pharmacological Analyses

Ten CML-related compounds and the analyzed results are shown in Table2. To search bioactive compounds, the criteria of each parameter were suggested. Molecular weights from 180 to 500 Daltons were recognized as comparatively more druggable [23]. Oral bioavailability (OB) ≥30% and drug likeness ≥0.18 were used as ideal drug characteristics [24, 25]. Caco-2 cell permeability was tested using an in vitro experiment with the Caco-2 human intestinal cell line. Drug half-life (t1/2) represented how long it took for the drug to be internalized and reduced by half; a drug half-life ≤4 h was classified as the fast elimination group, between 4 and 8 h as the midelimination group, and ≥8 h as the slow-elimination group [20, 21].Table 2
The 10 CML-related compounds of Anisi stellati fructus.
Molecule name
MW
OB (%)
Caco-2
DL
HL
Astragalin
448.41
14.03
−1.34
0.74
N/A
CHEBI:7
136.26
45.2
1.84
0.04
11.44
(+)-Catechin
290.29
54.83
−0.03
0.24
0.61
2-Methyl-N-phenylmaleimide
187.21
87.36
0.77
0.06
2.93
Kaempferol
286.25
41.88
0.26
0.24
14.74
Honokiol
266.36
60.67
1.43
0.15
2.88
Luteolin
286.25
36.16
0.19
0.25
15.94
Salicylic acid
138.13
32.13
0.63
0.03
12
(R)-Linalool
154.28
39.8
1.33
0.02
6.48
Quercetin
302.25
46.43
0.05
0.28
14.4
MW: molecular weight; OB: oral bioavailability; Caco-2: Caco-2 permeability; DL-2: drug likeness; HL: drug half-life.Among the 10 CML-related compounds, luteolin (MW = 286.25, OB (%) = 36.16, Caco-2 = 0.19, DL = 0.25, and HL = 15.94), kaempferol (MW = 286.25, OB (%) = 41.88, Caco-2 = 0.26, DL = 0.24, and HL = 14.74), and quercetin (MW = 302.25, OB (%) = 46.43, Caco-2 = 0.05, DL = 0.28, and HL = 14.4) largely met the suggested criteria and have been reported as representative antibacterial compounds in previous studies [26–28], including our study [18]. Additionally, luteolin exhibited an antibacterial action that suppressed the activity of bacterial DNA topoisomerases 1 and 2 and reduced the synthesis of nucleic acids and proteins [29] and was also known to have antioxidant, anticancer, anti-inflammatory, and neuroprotective effects [30]. Kaempferol is a flavonoid and possesses anti-inflammatory, antioxidant, anticancer, and antibacterial activities [31–33]. It has been shown to be effective against acne-induced Propionibacterium acnes and Helicobacter pylori found in the stomach [34]. Quercetin is a compound that plays an important role in inflammation, cancer, aging, cell signaling, proapoptotic effects, antiproliferative effects, antioxidant effects, and growth suppression [35, 36]. Astragalin (MW = 448.41, OB (%) = 14.03, Caco-2 = -1.34, DL = 0.74, and HL = N/A) has been reported to inhibit autophagosome formation in airways [37]. Honokiol (MW = 266.36, OB (%) = 60.67, Caco-2 = 1.43, DL = 0.15, and HL = 2.88) has been shown to have antitumorigenic, anti-inflammatory, and antioxidant effects [38–40]. (+)-Catechin (MW = 290.29, OB (%) = 54.83, Caco-2 = −0.03, DL = 0.24, and HL = 0.61) is a well-known phenolic compound found in tea and wine.


## 3.3. Network Construction of 10 CML-Related Compounds and Their Related Targets

To visualize the target genes of 10 CML-related compounds, a compound (C)-target (T) network was established. A total of 313 target proteins were collected from the TCMSP, and the official gene names of the targets were obtained from the UniProt database. Figure1 shows that the C-T network revealed interactions between 10 CML-related compounds and 313 target genes. The nodes showed the 10 CML-related compounds and the target genes, and the edges represented the interaction of compounds and targets. The size of nodes was related to the degrees. Quercetin (degree = 77) had the greatest interaction with targets, followed by luteolin (degree = 56), kaempferol (degree = 36), salicylic acid (degree = 35), 2-methyl-N-phenylmaleimide (degree = 29), R-linalool (degree = 20), honokiol (degree = 18), astragalin (degree = 16), CHEBI:7 (degree = 15), and (+)-catechin (degree = 10).Figure 1
The network of 10 CML-related compounds and their related target genes. Triangle nodes represent compounds and circular nodes represent targets. Node size is relative to the degree, and the edges demonstrate the interaction between nodes.


## 3.4. HPLC Analysis of ASFE

The main components of ASFE were determined using an HPLC system to evaluate the quality of ASFE. Because 4-anisaldehyde and 4-prophenylanisole [(E)-anethole] have been reported to be the main compounds of ASFE, we used them as standards and conducted the analysis. As shown in the representative chromatographic fingerprint of ASFE, the main components of ASFE were 4-anisaldehyde and 4-prophenylanisole (Figure2).Figure 2
Chromatograms of two major components (4-anisaldehyde and 4-prophenylanisole) identified in Anisi stellati fructus extract (ASFE). (a) Chromatograms of the standard mixture. (b) Chromatograms of the major components in ASFE. The chromatograms were obtained at 254.5 and 275 nm.
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## 3.5. Effect of ASFE on the Cell Viability of K-562 Cells

The possible cytotoxic effect of ASFE (0–8μg/mL) on K-562 cells was assessed (Figure 3). ASFE significantly inhibited the proliferation of K-562 cells in a dose-dependent manner. The IC50 value of ASFE was approximately 4.03 ± 2.3 μg/mL (∗P<0.05; ∗∗P<0.01).Figure 3
Cytotoxicity of the ASFE on K-562 cells. The cells (1 × 104/well) were determined using a CCK-8 assay.


## 3.6. ASFE Treatment on K-562 Cells Decreases the Amount of p210Bcr-Abl in a Time-dependent Manner

ASFE or IM was used to treat K-562 cells, and the p210Bcr-Abl protein steady-state levels in the total lysate were analyzed by western blotting (Figure 4). 2 μmol IM was subjected based on previous studies [41]. The p210Bcr-Abl protein levels were decreased after treatment with ASFE or IM on K-562 cells in a time-dependent manner (∗P<0.05; ∗∗P<0.01).Figure 4
Changes in the p210Bcr-Abl protein levels treated with ASFE or imatinib (IM). K-562 cells were treated with (a) ASFE (2 μg/mL) or (b) IM (2 μmol) for increasing time periods (0–16 h). The p210Bcr-Abl protein steady-state levels in the total lysates were analyzed by western blotting. Relative protein levels versus controls (beta-actin) were determined by densitometry. Data are the mean ± SD of three independent experiments (∗P<0.05; ∗∗P<0.01).
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## 3.7. ASFE Enhanced IM-Induced Destabilization of p210Bcr-Abl

Figure5 shows that when both ASFE and IM were added together to K-562 cells, the level of p210Bcr-Abl was diminished compared to that of the lanes containing K-562 cells after treatment with IM or ASFE, indicating that the combined treatment of K-562 cells by ASFE and IM was more potent than each one separately.Figure 5
ASFE + IM combined therapy led to a decrease of p210Bcr-Abl. K-562 cells were treated with IM (2 μmol), ASFE (2 μg/mL), and IM + ASFE combination for 12 h at 37°C. The p210Bcr-Abl protein levels in the total lysate were analyzed by western blotting (∗P<0.05; ∗∗P<0.01).


## 3.8. Assessment of ASFE- and IM-Inducing Cell Death Mechanisms on K-562 Cells

Apoptosis, necrosis, and autophagy are well-known cell death mechanisms. To observe apoptosis and necrosis, we conducted flow cytometry using annexin V and 7-AAD staining. After 16 h treatment of IM, ASFE, or IM + ASFE, the cells were stained with both annexin V and 7-AAD. The results showed that, in the control, >85% of the cells survived; the cell population decreased to 82.79% for the IM, 9.54% for the ASFE, and 0% for the IM + ASFE. The late apoptotic/necrotic cell population increased to 13.03% (IM), 36.33% (ASFE), and 42.89% (ASFE + IM). The early apoptotic cell population increased to 0.93% (IM), 0.00% (ASFE), and 0.00% (ASFE + IM) (Figure6). The cell populations of late apoptosis/necrosis significantly increased, but increased cell population during early apoptosis was rare. Based on these results, we postulated that K-562 cells were involved in an alternate pathway and were not involved in apoptosis. These results also showed that K-562 cell killing was significant after treatment with ASFE.Figure 6
Apoptosis assessment of the effects of IM, ASFE, and IM + ASFE on K-562 cells. After 16 h of treatment, the cells were stained with annexin V/7-AAD reagent and cytometric analysis was performed using a MUSE™ cell analyzer. (a) Control, (b) IM, (c) ASFE, (d) IM + ASFE.
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## 4. Discussion

Traditionally, ASF has been used in Chinese medicine for the treatment of skin inflammation, stomach aches, and rheumatic pain [1]. For decades, herbal medicine has been used as an adjunct in chemotherapy for cancer treatment, demonstrating their synergistic roles in enhancing efficacy, ameliorating side effects, and reducing drug resistance [42]. However, there have been few scientific studies describing the anticancer effects of ASF.CML is a clonal myeloproliferative disorder characterized by the presence of a constitutive tyrosine kinase activity of the fusion oncogene,Bcr-Abl [11]. Bcr-Abl protein induces cellular transformation by activating the signaling molecules, STAT5 and Akt [43]. As a first-line treatment, TKI-targeted Bcr-Abl was expected to treat CML effectively, but it has shown drug resistance, so the next generation of drugs from imatinib has been required [44]. Bcr-Abl T315I is the most frequent resistant mutation to tyrosine kinase inhibitors [45]. Following imatinib, the second-generation inhibitors such as dasatinib, nilotinib, and bosutinib and third-generation inhibitor such as ponatinib have been used to treat resistant cases of CML. Ponatinib has been developed to overcome the T315I mutation. The next candidates of tyrosine kinase inhibitors, including bafetinib, danusertib, rebastinib, tozasertib, HG-7-85-01, GNF-2, and 1,3,4-thiadiazole derivatives are presently undergoing approval as the next generation of therapeutic agents [46]. However, the issue of drug resistance is a rapidly increasing problem, and alternatives that are less toxic are urgently needed. Accordingly, this study aimed at providing the potential for exploring new therapies that are less harmful and more effective.In the present study, we used a systemic pharmacological approach, filtered active compounds of ASF, constructed the compound-target network, and screened cancer-related compounds of ASF. ASF was found to contain 49 identified compounds, and 33 compounds were active in biological pathways related to cancers. Moreover, 10 of the 33 were classified as being associated with CML. Additionally, the compound-target network showed multiple compounds and multiple targeting characteristics of ASF. Multiple compounds and multiple targeting actions of other herbal medicines such as licorice [47], Xiao-Chaihu Decoction, Da-Chaihu Decoction [48], Pulsatillae Radix, Baekduong-tang [49, 50], and Bulsusan [51] have also been studied. Based on our results, we propose that ASF is a potential antileukemic agent.Experiments were conducted to confirm this possibility. First, the active components of ASF were extracted. According to previous studies, the extraction of ASF could be conducted using several extraction techniques such as hydrodistillation, steam distillation, solvent extraction, supercritical fluid CO2 extraction, hydrodistillation-headspace solvent microextraction, and microwave-assisted extraction [52, 53]. In our study, a combination of solvent extraction and sonication, followed by pressured gas blowing concentration, was used. To obtain the maximum extraction ASF, methanol was used as a solvent [54], followed by sonication to increase the extraction efficiency. The 4-prophenylanisole was one of the main compounds of ASF and accounted for 94% of the essential oil [55]. Another major component of ASF was 4-anisaldehyde, which is used as a food fragrance and reported to have antifungal and saliva-enhancing effects [56]. In this study, the main compounds of 4-prophenylanisole and 4-anisaldehyde were identified in the ASFE using HPLC.The ASFE and ASFE + IM combination showed potent antileukemic effects by decreasing the p210Bcr-Abl levels. Because leukemic stem cells do not respond to TKI, when the drug supply is stopped, the cells can continuously multiply to allow the disease to persist. Moreover, the combination of treatment targets results, more efficiently, in alternative surviving mechanisms of cancer cells such as autophagy [16].Our results showed that ASFE combined with IM resulted in the significant death of K-562 cells. The results suggested the feasibility of further research to identify cell death mechanisms following a decrease in Bcr-Abl. In addition, our next phase of research will be to verify the effects of 10 CML-related compounds as well as two representative compounds of ASF, 4-anisaldehyde and 4-prophenylanisole, on CML. The workflow scheme of our study is summarized in Figure7.Figure 7
A workflow scheme illustrating the hypothesis that compounds of ASF having antileukemic potential can be identified using systematic pharmacological approaches; the experimental proof was that ASF was effective in killing of K-562 chronic myeloid leukemia cells.


## 5. Conclusion

We have identified 10 CML-related compounds of ASF using systematic pharmacological analyses. The ASF extract significantly induced cell death of K-562 cells. In addition, combined treatment with ASFE and IM led to a significant decrease of Bcr-Abl protein levels. The results showed that ASF is a promising source of effective chemotherapeutic adjuvants and is a candidate, in combination with other chemotherapeutic drugs, for the treatment of CML patients, to ameliorate the side effects and overcome resistance. Although we do not know the exact mechanisms of the 10 compounds of ASF and their antileukemic effects, this study is the first to report the antileukemic effects of ASF on K-562 CML cells, which should prompt further mechanistic studies of these antileukemic compounds.



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*Source: 6959764-2019-12-12.xml*