Inhibition of DNA Topoisomerase Ι by Flavonoids and Polyacetylenes Isolated from Bidens pilosa L.

Human DNA topoisomerase I (Topo I) is an essential enzyme in regulating DNA supercoiling during transcription and replication, and it is an important therapeutic target for anti-tumor agents. Bidens pilosa L. is a medicinal herb that is used as a folk medicine for cancers in China. A new flavonoid (1) and a new polyacetylene (20), along with eighteen flavonoids (2–19) and nine polyacetylenes (21–29), were isolated and identified from the methanol extract of the whole plant of B. pilosa, and some of the compounds (4, 5, 6 and 7) exhibited potent cytotoxicity against a panel of five human cancer cell lines. The DNA relaxation assay revealed that some flavonoids and polyacetylenes exerted inhibitory activities on human DNA Topo I, among them compounds 1, 2, 5, 6, 7, 8, 15, 19, 20, 22, and 24 were the most active ones, with IC50 values of 393.5, 328.98, 145.57, 239.27, 224.38, 189.84, 89.91, 47.5, 301.32, 178.03, and 218.27 μM, respectively. The structure–activity analysis of flavonoids was performed according to the results from the Topo I inhibition assay. The DNA content analysis revealed that 5, 6, and 7 potently arrested cell cycle at the G1/S and G2/M phases in human colon cancer cell DLD-1 depending on the concentration of the inhibitors. The levels of protein expression related to the G1/S and G2/M cell cycle checkpoints were in accordance with the results from the DNA content analysis. These findings suggest that flavonoids are one of the key active ingredients accounting for the anti-tumor effect of B. pilosa.


Introduction
Bidens pilosa L. is an annual herb of the Asteraceae; it is edible and has been traditionally used for the treatment of many diseases in many countries.For its anti-hyperglycemic, antihypertensive, and anti-tumor effects, B. pilosa has been widely used as a folk herbal medicine in China for treating various disorders, and in Brazil, it has been included in the official list of medicinal plants by the public health system [1].Modern phytochemical studies revealed that B. pilosa contains flavonoids, polyacetylenes, phenolic acids, terpenoids, steroids, and other components [2], of which flavonoids and polyacetylenes are the main constituents [3].A wide range of biological activities has been reported about B. pilosa and the compounds isolated from it, including antioxidative, anti-inflammatory, antibacterial, anticancer, antidiabetic, and immunomodulatory activities, etc. [4,5].Flavonoids are a kind of polyphenols that broadly exist in plants and exhibit a wide range of bioactivities; to date, more than one hundred flavonoids have been isolated from B. pilosa, which were reported to have antioxidant, hepatoprotective, and cytotoxic activities, etc. [6].Acetylenes are a kind of compounds containing one or more carbon-carbon triple bond(s) in their backbones and were found regularly in only five families, namely Campanulaceae, Asteraceae, Araliaceae, Pittosporaceae, and Umbelliferae.To date, more than fifty acetylenes have been isolated from B. pilosa, which are reported to play an important role in the plant with anti-cancer, anti-malaria, and anti-diabetes activities [7,8].
DNA topoisomerases (Topos) are the key enzymes that control DNA topology through the passage of DNA strands [9], which is important for DNA replication and transcription.According to whether they make transient single-or double-stranded breaks in the DNA, Topos are classified as Topo I and II, respectively [10].Because of the overexpression and higher activity in tumor cells compared to normal cells, Topos have become important therapeutic targets for anti-tumor drugs [11].Many natural compounds isolated from plants, such as camptothecin (CPT) and podophyllotoxin, exhibit potent inhibitory activities in Topos [12], and their derivatives are used as important anti-tumor drugs in the clinic, such as topotecan, irinotecan, and etoposide [13].
Currently, many studies have been conducted on the anti-tumor potential of natural products against Topos.As far as we know, no description of the inhibitory effect of polyacetylenes on Topos has been found, and the mechanism of the B. pilosa compounds' activity has not been elucidated.Regarding the good anticancer activity of B. pilosa, the chemical and pharmacological constituents of the plant were investigated by us [14].In this study, a new flavonoid and a new polyacetylene, together with 27 known flavonoids and polyacetylenes, were isolated from B. pilosa, of which some showed potent cytotoxicity and inhibitory activities on DNA Topo I.In this paper, we report the identification of the new compounds, the cytotoxicity, the inhibitory activities on Topo I, and the cytotoxic mechanism in cancer cells.To the best of our knowledge, this is the first time that the inhibition of flavonoids and polyacetylenes isolated from B. pilosa against DNA Topo I is reported.

Compounds 1-29 Showed Cytotoxicities and Topo I Inhibitory Activities
The cytotoxicities of 1-29 were tested by an SRB assay on a panel of five human cancer cell lines and one human hepatocyte cell line.The results from Table 3 showed that compounds 4, 5, 6, and 7 exhibited potent cytotoxicities against A549, HCT116, and DLD-1 cells, and their IC 50 values were at the same levels compared to those of CPT, while, in MDA-MB-231 and HepG2 cells, they are less active than CPT.Compared to 4, 5, 6, and 7  A DNA relaxation assay was performed to determine the inhibitory effect of compounds 1-29 on Topo I. To remove false positive results, a preliminary assay of DNA cleavage was performed, and no cleavage effect was detected in any of the compounds (Figure S20).As the results show in  50 values were over the maximum tested concentration, the percentage inhibition values of these compounds on Topo I activity were different at 400 µM.The results from Figure 4 show that 3, 11, 12, 16, 13, 18, 21, and 23 exhibited inhibitory activities on Topo I at the concentration of 400 µM and the percentage inhibition values were 24.63%, 28.56%, 26.41%, 10.92%, 38.61%, 46.66%, 23.04%, and 15.18%, respectively, which indicated that these compounds showed weak inhibitory activity against DNA Topo I in vitro.For compounds 4, 9, 10, 14, 17, and 25~29, no inhibition at 400 µM was detected.
(Figure S20).As the results show in Table 3 As for the other compoun though the IC50 values were over the maximum tested concentration, the percentage in bition values of these compounds on Topo I activity were different at 400 µM.The resu from Figure 4 show that 3, 11, 12, 16, 13, 18, 21, and 23 exhibited inhibitory activities Topo I at the concentration of 400 µM and the percentage inhibition values were 24.63 28.56%, 26.41%, 10.92%, 38.61%, 46.66%, 23.04%, and 15.18%, respectively, which in cated that these compounds showed weak inhibitory activity against DNA Topo I in vit For compounds 4, 9, 10, 14, 17, and 25~29, no inhibition at 400 µM was detected.

Compounds 5-7 Arrested Cell Cycle in Cancer Cells
The effect of flavonoids 5, 6, and 7 on the DLD-1 cell cycle was examined by DN content analysis.The results show that all the compounds inhibited the cell growth in cell cycle-nonspecific way, in which a potent G2/M-phase arrest was observed after tre ment with a higher concentration, and the obvious cell cycle arrest in the G1 or S pha was monitored after treatment with a lower concentration.After treatment with 2 µM compounds 5, 6, and 7 for 24 h, the percentages of the cell population in the G2/M pha significantly increased from 20.12% to 86.05%, 76.49%, and 75.75%, and the cells in t G1/S (combined) phase remarkably decreased from 79.53% to 13.46%, 22.91%, and 24.26 respectively, which means a potent G2/M cell cycle arrest caused by the treatment (p 0.0001).In addition, after treatment with 1 and 0.5 µM of compounds 5, 6, and 7 for 24 the percentages of cells in the G1/S phase increased from 79.53% to 76.83%, 88.65% (p 0.0001), and 86.07%(p < 0.001) and from 79.53% to 85.30% (p < 0.01), 82.61%, and 81.16 (p < 0.01), respectively, which indicates that an apparent G1or S-phase arrest was induc after the treatment with a lower compound concentration (Figure 5).

Compounds 5-7 Arrested Cell Cycle in Cancer Cells
The effect of flavonoids 5, 6, and 7 on the DLD-1 cell cycle was examined by DNA content analysis.The results show that all the compounds inhibited the cell growth in a cell cycle-nonspecific way, in which a potent G 2 /M-phase arrest was observed after treatment with a higher concentration, and the obvious cell cycle arrest in the G 1 or S phase was monitored after treatment with a lower concentration.After treatment with 2 µM of compounds 5, 6, and 7 for 24 h, the percentages of the cell population in the G 2 /M phase significantly increased from 20.12% to 86.05%, 76.49%, and 75.75%, and the cells in the G 1 /S (combined) phase remarkably decreased from 79.53% to 13.46%, 22.91%, and 24.26%, respectively, which means a potent G 2 /M cell cycle arrest caused by the treatment (p < 0.0001).In addition, after treatment with 1 and 0.5 µM of compounds 5, 6, and 7 for 24 h, the percentages of cells in the G 1 /S phase increased from 79.53% to 76.83%, 88.65% (p < 0.0001), and 86.07%(p < 0.001) and from 79.53% to 85.30% (p < 0.01), 82.61%, and 81.16% (p < 0.01), respectively, which indicates that an apparent G 1 -or S-phase arrest was induced after the treatment with a lower compound concentration (Figure 5).

Compound 5 Regulated Cell Cycle-Related Protein Expression in Cancer Cells
As a compound that showed potent inhibitory activity on DNA Topo I and cytotoxicity on cancer cell lines, the effect of 5 on cell cycle-related protein expression levels was investigated by Western blot in DLD-1 cells after treatment for 24 h.The results show that compound 5 potently decreased the expression levels of cyclin A, cyclin B, and CDK6 at 2 µM; meanwhile, 5 caused a potent downregulation of cylcin D, cyclin E, and CDK6 at 1 and 0.5 µM (Figure 6).The results indicate that, at different concentration of the inhibitor treated, flavonoid 5 caused G 1 /S-or G 2 /M-phase cell cycle arrests by regulating the expression of different cell cycle-related proteins.

Compound 5 Regulated Cell Cycle-Related Protein Expression in Cancer Cells
As a compound that showed potent inhibitory activity on DNA Topo I and cytotoxicity on cancer cell lines, the effect of 5 on cell cycle-related protein expression levels was investigated by Western blot in DLD-1 cells after treatment for 24 h.The results show that compound 5 potently decreased the expression levels of cyclin A, cyclin B, and CDK6 at 2 µM; meanwhile, 5 caused a potent downregulation of cylcin D, cyclin E, and CDK6 at 1 and 0.5 µM (Figure 6).The results indicate that, at different concentration of the inhibitor treated, flavonoid 5 caused G1/Sor G2/M-phase cell cycle arrests by regulating the expression of different cell cycle-related proteins.

Plant Material
The samples of B. pilosa L. were collected from the Liangwangshan mountain, Kunming, Yunnan province, China, in June 2020 and authenticated by Kunming Plant Branch Biotechnology Co., Ltd.(Zhang Jun) (Kunming, China), and a voucher specimen (YMU-ZF20200624) was deposited at the Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan Minzu University.

Plant Material
The samples of B. pilosa L. were collected from the Liangwangshan mountain, Kunming, Yunnan province, China, in June 2020 and authenticated by Kunming Plant Branch Biotechnology Co., Ltd.(Zhang Jun) (Kunming, China), and a voucher specimen (YMU-ZF20200624) was deposited at the Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan Minzu University.

Cytotoxicity Assay
The cytotoxic activities of the compounds against cancer cells were examined with a sulforhodamine B (SRB) colorimetric assay.Cells were seeded at a density of 5 × 10 3 /well in 96-well plates for 24 h; various concentrations of the tested compounds, dissolved in DMSO and diluted with the complete medium, were added in the wells in triplicate and incubated for 48 h.After that, the cell viability was assessed with the SRB assay as we described before [43].The IC 50 values were determined using the Reed and Muench method and are expressed as the mean ± SD of at least three independent measurements.Camptothecin (CPT) (Energy Chemical, Shanghai, China) was used as a reference compound for the positive control.

Topo I Inhibition Assay
The DNA relaxation assay was adopted to investigate the compounds' inhibitory activities on Topo I. Different concentrations of the compounds, dissolved and diluted in DMSO, were added into the relaxation buffer (50 mM Tris, pH 7.5, 50 mM KCl, 10 mM MgCl 2 , 0.1 mM EDTA, 0.5 Mm dithiothreitol, and 0.05 mg/mL bovine serum albumin) containing 0.2 U Topo I (ThermoFisher, Waltham, MA, USA) and incubated at room temperature for 10 min.After that, 500 ng supercoiled pBR322 plasmid DNA (ThermoFisher, Waltham, MA, USA) was added and then incubated at 37 • C for another 30 min.Finally, the reactions were stopped with a stopping solution (5% sodium dodecyl sulfate, 0.0025% bromophenol blue, and 25% glycerol), and the mixtures were applied onto 0.8% agarose gel and subjected to electrophoresis for 1.5 h in Tris-Acetate-EDTA buffer.After being stained with GelRed (Biotium, Fremont, CA, USA), the gel was imagined with the imaging system (Biotop, Shanghai, China).CPT (Energy Chemical, Shanghai, China) was used as a reference compound for the positive control.The band density of the supercoiled DNA was quantified by the ImageJ software (version 1.53a, National Institutes of Health, Bethesda, MD, USA), and the inhibition activity is expressed as the percentage inhibition and calculated by formula: Inhibition % = [(A − B)/(C − B)] × 100%, where A is the band density of the tested samples, B is the band density of the negative control, and C is the band density of the blank control.IC 50 values were determined using the Reed and Muench method and are expressed as the mean ± SD of at least three independent measurements in duplicate.

DNA Content Analysis
Cells were seeded in 6-well plates and allowed to adhere for 24 h at 37 • C.After treatment with different concentrations of the samples for another 24 h, the cells were harvested and washed with PBS, and then, the DNA content analysis was conducted with a cell cycle staining kit (Multi sciences, Hangzhou, China), according to the manufacturer's instructions.The fluorescence intensity of the cells was measured by flow cytometry (Beckman CytoFlex, Miami, FL, USA).Topotecan (TPT) (Solarbio, Beijing, China) was used as a reference compound for the positive control.

Western Blot Analysis
Cells treated with the compounds were harvested, and the total protein was isolated with lysis buffer (72 mM Tris pH 6.8, 2% SDS, 10% glycerol, and 0.1% bromophenol blue).The proteins were subjected to SDS-PAGE and transferred to a 0.45 µm PVDF membrane (Millipore, Burlington, USA).After being blocked with 5% non-fat milk in TBST, the membranes were washed and then incubated with specific primary antibodies (Cdk2 and Cdk6 were purchased from BBI Life Sciences Corporation, Shanghai, China; cyclin A, cyclin B, cyclin D, cyclin E, Cdk1, and Cdk4 were purchased from Proteintech, Rosemont, IL, USA) in TBST containing 2% non-fat milk overnight at 4 • C.After washing, the membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies at room temperature for 1 h.The blot was visualized using an enhanced chemiluminescence assay kit (Meilunbio, Dalian, China) under a chemiluminescence detector (Tanon, Shanghai, China).The band density of the blot was quantified by the ImageJ software with at least three independent experiments.

Statistical Analysis
All the results were calculated by at least three independent experiments and are presented as mean ± S.D. values.A one-way ANOVA or the Student's t-test was used for the statistical analysis.A p value < 0.05 was considered statistically significant.

Discussion
Cancer is commonly characterized by uncontrolled cell division and proliferation, which are directly related to DNA replication.Targeting Topos disrupts DNA replication and transcription, which leads to the inhibition of cell division, thereby stopping the growth of cancer cells [12].In a wide range of human solid tumors, the intracellular level of Topo I is higher than that in normal tissues, especially in metastatic ovarian cancer, cervical cancer, small-cell lung cancer, metastatic colon cancer, and pancreatic cancer [11], suggesting that controlling the Topo I level is essential in treating cancers.Several studies have revealed that specific flavonoids in natural products inhibit the activity of DNA Topo I [44].Licochalcones A, a chalcone isolated from Glycyrrhiza inflata, and luteolin, a flavone isolated from the leaves of Vitex negundo, were reported to show cytotoxicity by inhibiting DNA Topo I [45,46]; therefore, flavonoids hold therapeutic prospects in the treatment of cancers.
B. pilosa L. is a herb that is used as a folk medicine for cancer treatment in China.In this study, nineteen flavonoids and ten polyacetylenes (Figure 3) were isolated from the plant, including one flavone (15), eleven flavonols (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), two flavanones (16)(17), three chalcones (1-3), two aurone (18,19), and ten polyacetylenes (20)(21)(22)(23)(24)(25)(26)(27)(28)(29), and their inhibitory activities on DNA Topo I and cytotoxicities on cancer cell lines were tested (Table 3).The new compounds 1 and 20 and the polyacetylenes 22 and 24 were found to be the inhibitors of DNA Topo I for the first time.The structure-activity relationship of flavonoids against Topo I was analyzed.Based on the results from Table 3 and Figure 4, it was demonstrated that, compared to chalcones, flavones, flavonols, and aurones, flavonoids showed a strong inhibitory activity against DNA Topo I in vitro.Compared to flavanones, flavones and flavonols are more active, indicating that the double bond between C2 and C3 is important to the inhibitory activity, which was verified by the observation of the decreased inhibitory activities of flavanones (16 and 17), which lack the C2-C3 unsaturation bond on the C-ring.By comparing the activity of 4 with those of 5, 6, and 7, we deduced that the substitution of the C-6 position on the A-ring of flavonols was essential for Topo I inhibition, and if there was no substitution, the activity potently decreased.Additionally, if the substitution at the C-6 position was glycosylated, the activities of the compounds also decreased, which was verified by comparing 5, 6, 7, and 8 with 9.The C-7 position is also important for the inhibitory activity, which was enhanced by the substitution of small groups (-OH and -OCH 3 ), and if the substitution was glycosylated, the activities of the compounds (10, 12, 13, 14, and 17) potently decreased, which was also confirmed by comparing 14 with its aglycone 5.As for the B-ring of flavonols, substitutions at C-3 ′ and C-4 ′ are important for the inhibitory activity (5, 6, and 7), and comparing 5 with 7, the activity of the hydroxy substitution at the C-3 ′ position is better than that of the methoxy substitution.Furthermore, the inhibition results from compounds 12 and 14 indicates that the hydroxyl group substitution at C-3 ′ and C-4 ′ is superior to that of methoxy group, which is correlated with what has been described in previous publications [47,48].As for the cytotoxicity of compounds, only 5, 6, 7, 15, and 22 showed good activity in cancer cell lines, as well as in the DNA Topo I assay, which indicates that their cytotoxicity was partly caused by DNA Topo I inhibition.As for compound 4, though inactive against Topo I, a good cytotoxicity on cancer cells was observed (not as good as those of 5, 6, and 7), indicating that DNA Topo I is only one of the cytotoxic targets of flavonoids, which intervenes in the characteristics of multi-targets for natural products [44].As the most active inhibitor of Topo I, 15, a more hydrophilic compound compared to 5, 6, and 7, shows a weaker cytotoxicity, which is attributed to the lipid bilayer of cell membranes that prevents the entrance of 15 into the cytosol, leading to less cytotoxic activity.Comparing aurones 18 and 19, the esterification of the glucose of 19 enhanced the inhibitory effect on Topo I.Although 19 showed a good inhibitory activity, its cytotoxicity was weak, which may be attributed to the metabolized difference between cellular experiments and non-cellular ones.
Topoisomerase I-targeting drugs exert a cytotoxic effect by producing enzyme-mediated DNA damage, which leads to cell cycle arrest.Thus, the cell cycle analysis of DLD-1 cells treated with 5, 6, and 7 was conducted by flow cytometry.The results from the DNA content (Figure 5) analysis show that all flavonoids caused potent G 2 /M arrests at a higher concentration and significant G 1 /S arrests at a lower concentration, which indicates that flavonoids 5, 6, and 7 induced double-strand DNA breaks by the inhibition of DNA Topo I; then, the activated DNA damage response accumulated cells in different phases depending on the concentration of the inhibitor used.The cell cycle-nonspecific arrest by flavonoids is in agreement with what was reported for DNA damaging agents in previous publications [49,50].The eucaryotic cell cycle is regulated by the periodic synthesis and destruction of cyclins that complex with cyclin-dependent kinases (Cdks); thus, cyclin/Cdk complexes are the key regulators for governing the checkpoints in a cell division cycle.In order to verify the results of the DNA content analysis, the levels of different cyclins and Cdks in the cell cycle were investigated by Western blot (Figure 6).The results exhibit that compound 5 caused cell cycle arrest by downregulating the expression of different cyclins or Cdks at different concentrations.As the main regulator for controlling the G 2 /M checkpoint, the cyclin B/Cdk1 complex is the key regulator of cell mitosis, and treatment with 2 µM of compound 5 caused a significant decrease in cyclin B, and because the periodic synthesis and destruction of cyclins control the activation of the Cdks and the cyclin/Cdk complexes [51], it suggests that the reduction in cyclin B induced by 5 blocked the cells' entry into mitosis and led to the potent G 2 /M arrest in DLD-1 cells.In the normal eucaryotic cell cycle process, the activity of the cyclin D/Cdk4/6 and cyclin E/Cdk2 complexes controls G 1 -phase progression, and the cyclin A/Cdk2 complex controls the S phase.In this study, the results from the Western blot show that the treatment with lower concentrations of compound 5 decreased the expression of cyclin D, cyclin E, and Cdk6 (Figure 6), which indicates that 5 inhibits the activity of the cyclin D/Cdk4/6, cyclin A/Cdk2, and cyclin E/Cdk2 complexes at a lower concentration and arrests the G 1 /S cell cycle checkpoint in DLD-1 cells.The results from the Western blot are consistent with that from the DNA content analysis.Additionally, as it was shown in DLD-1 cells, compound 5 also caused potent G 2 /M arrests at 2 µM and significant G 1 /S arrests at 0.5 µM and regulated the expression of cell cycle-related proteins in the HCT116 cell line (Figure S21).All the results indicate that the flavonoids and polyacetylenes that exist in B. pilosa are the cytotoxic components responsible for its anti-tumor activity by inhibiting DNA Topo I.

Conclusions
Flavonoids broadly exist in plants and exhibit a wide range of bioactivities.B. Pilosa is a traditional herb that is used as anti-tumor folk medicine in China and many other countries.However, its main constituents with anti-tumor activities have not been identified.In this study, a new chalcone and a new polyacetylene, together with twenty-seven known flavonoids and polyacetylenes, were isolated from B. pilosa, and their inhibitory activities against DNA Topo I were reported for the first time, except for compound 15, which was isolated from the leaves of Vitex negundo L. and has been reported to show good activity [46].The study of the structure-activity relationship analysis and cytotoxicity mechanism indicated that Topo I-targeting flavonoids and polyacetylenes are two kinds of active ingredients that lead to the anti-tumor effect of B. pilosa, which is of great importance for the R&D of flavonoids and the traditional herb B. pilosa.

Figure 4 .
Figure 4.The inhibitory effect of flavonoids 3, 11, 12, 16, 13, 18, 21, and 23 on DNA Topo I. ( Representative images of the Topo I inhibition assay; the tested concentration of the flavonoids 400 µM.(B) The percentage inhibition of the compounds on Topo I activity at the concentration 400 µM.

Figure 4 .
Figure 4.The inhibitory effect of flavonoids 3, 11, 12, 16, 13, 18, 21, and 23 on DNA Topo I. (A) Representative images of the Topo I inhibition assay; the tested concentration of the flavonoids is 400 µM.(B) The percentage inhibition of the compounds on Topo I activity at the concentration of 400 µM.

Figure 5 .
Figure 5. Results of flavonoid (5, 6, and 7)-induced cell cycle arrests in DLD-1 cells.(A) Representative images of flow cytometry analysis of the DNA content.(B) Statistic analysis of cells in the G1, S, and G2/M phases after treatment with 5, 6, and 7. ** p < 0.01 and **** p < 0.0001 mean significant differences from the vehicle control.Data are expressed as mean ± SD values from three independent experiments.

Figure 5 .
Figure 5. Results of flavonoid (5, 6, and 7)-induced cell cycle arrests in DLD-1 cells.(A) Representative images of flow cytometry analysis of the DNA content.(B) Statistic analysis of cells in the G 1 , S, and G 2 /M phases after treatment with 5, 6, and 7. ** p < 0.01 and **** p < 0.0001 mean significant differences from the vehicle control.Data are expressed as mean ± SD values from three independent experiments.

Figure 6 .
Figure 6.Effects of flavonoid 5 on cell cycle-relevant proteins in DLD-1 cells.(A) Representative immunoblots of the protein expression.(B) Histograms for the quantified results of protein levels, which were adjusted with corresponding α-tublin/GAPDH protein levels and expressed as folds of the control.Experiments were independently repeated at least three times.* p < 0.05, ** p < 0.01 and *** p < 0.001 mean significant differences from the vehicle group.

Figure 6 .
Figure 6.Effects of flavonoid 5 on cell cycle-relevant proteins in DLD-1 cells.(A) Representative immunoblots of the protein expression.(B) Histograms for the quantified results of protein levels, which were adjusted with corresponding α-tubulin/GAPDH protein levels and expressed as folds of the control.Experiments were independently repeated at least three times.* p < 0.05, ** p < 0.01 and *** p < 0.001 mean significant differences from the vehicle group.
Figure S19: IR spectrum of compound 20. Figure S20: The effects of compounds on DNA cleavage.
Figure S21: Results of flavonoid 5 induced cell cycle arrests in HCT116 cells.And spectra data of compounds 2-19 and 21-29.

Table 3 .
The cytotoxicities against five cancer cell lines and Topo I inhibitory activities of compounds 1-29.
a the reference compounds of the positive control.