The cytotoxicity and SAR analysis of biflavonoids isolated from Araucaria hunsteinii K. Schum. leaves against MCF-7 and HeLa cancer cells

Material, reagents, and instrumentation

The materials used in this research are acetone fractions of A. hunsteinii leaves free from chlorophyll and tannins that is resulted by Agusta et al. (2022), the technical grade organic solvent, such as acetone, methanol (MeOH), ethanol (EtOH), dichloromethane (CH2Cl2), and chloroform (CHCl3). The separation process used a chromatography method with various support solids such as Sephadex LH-20 (GE Healthcare), silica gel 60 Merck 60 (0.040–0.063 mm) and (0.063–0.2 mm), and GF254 Merck thin layer chromatography (TLC-preparative). The isolated compounds were characterized according to the method described by Agusta et al. (2022). In addition, MCF-7 (ATTC HTB 22) and HeLa cancer cells (ATTC CCL 2) cancer cells were subjected to the cytotoxic anticancer test using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent. Both cells were grown in 24 well microplates at a concentration of 5,000 cells in 100 μl of growth medium (D-MEM, Roswell Park Memorial Institute 1640, 5% fetal bovine serum, and Penicillin 100 U/ml, Streptomycin 100 ug/ml). These two cells were from the IPB University’s Primate Research Center. HeLa (ATTC CCL-2) cell is obtained from adenocarcinoma-affected human uterine and cervix epithelial cells, whereas MCF-7 (ATTC HTB 22) cell is derived from human breast and mammary gland epithelial cells. They were kept in liquid nitrogen’s vapor phase under frozen storage conditions.

Fractionation and isolation of A. hunsteinii leaves fractions

The 5.0 and 10.0-g acetone fractions were separated using Sephadex LH-20, yielding 31 fractions (F1–F31) and 17 fractions (G1–G17), respectively. Biflavonoids are expected to be present in the F1–F14 and G1–G11 fractions of the TLC analysis using the eluent of CHCl3: MeOH (19:1). The yellow dots that show up on TLC after exposure to Ce(SO4)2 solution are biflavonoids. All fractions were purified using silica gel column chromatography (CC) and TLC-preparative. The purification in CC silica gel was performed on the F3, F8, F10, G5, G6, G7, and G8. The F8 fraction was eluted with CH2Cl2:MeOH (65:1) eluent and further purified with CH2Cl2:EtOH (50:1) eluent to obtain 1 (16.8 mg). The F3 fraction (91.9 mg) was eluted with CH2Cl2 eluent with increasing polarity and obtained 5 (26.5 mg). The combined fractions F10 (75.0 mg), G5 and G6 (243 mg), and G7–G8 (174.5 mg) were eluted with CH2Cl2:EtOH (30:1) eluent and afforded 2 (35.3 mg), 3 (20.4 mg) and 4 (21.8 mg). The elusion results were observed under ultraviolet (UV) light at 254 and 366 nm, and the fraction was sprayed with a solution of Ce(SO4)2. Schematic diagrams of the biflavonoids isolation can be seen in Figures S1 and S2. All compounds were characterized by using spectrophotometers of 1D and 2D nuclear magnetic resonance (NMR), UV-visible, infrared (IR), and liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS/MS), and identified as 7,4?′-di-O-methylcupressuflavone (1), 7-O- methylcupressuflavone (2), 4′, 4?′-di-O-methyl amentoflavone or isoginkgetin (3), 7,7?-di-O- methylagathisflavone (4) and 7,4′,7?,4?′-tetra-O-methylcupressuflavone (5). The structure of the five compounds is shown in Figure 1.

Cytotoxicity assay

The MTT method against MCF-7 cell lines is described in the protocol by Sasikala et al. (2020) to perform the cytotoxicity test. After 50% confluency, the cells were grown with 5,000 cells each containing well in 100 ml of the growth medium, the different concentrations of isolated compounds (Table S1) and 10 μl of MTT reagent (5 mg/ml) was added, and then 4 hours at incubated at room temperature (37°C) until the formazan crystal was formed.

The MTT method against HeLa cell lines is described in the protocol by Nawaz et al. (2021). The cells were grown with a concentration of 5,000 cells, each containing well in 100 ml of growth medium after 50% confluency. The different concentrations of isolated compounds (Table S2) and 10 μl of MTT reagent (5 mg/ml) were added, and then incubated for 4 hours at room temperature (37°C). The formed formazan crystals were dissolved in EtOH and then monitored with a spectrophotometric plate reader at 595 nm. The assay had been conducted in three replicates. The percentage of cell inhibition was calculated using the formula:

$\%\mathrm{Inhibition}=\frac{\mathrm{Control}\mathrm{Absorbans}\mathrm{–}\mathrm{Sample}\mathrm{Absorbans}}{\mathrm{Control}\mathrm{Absorbans}}\times 100\%$

The IC50 value of the sample is obtained from the linear regression equation y = a× + b, where x is the concentration of the sample and y is the % inhibition of the sample. The statistical evaluation was given in all IC50 values. The morphology of the conserved MCF-7 and HeLa cells was observed under an inverted microscope with 400 times magnification.

Identification of biflavonoids

The basic structure of biflavonoids has 30 carbon atoms. The ¹H NMR and ¹³C-NMR data reveal various properties of these compounds. On the other hand, biflavonoids showed two distinct signals in their ¹H-NMR spectra at δH 13.00 ppm, indicating the presence of chelation between protons and carbons of hydroxyl groups and carbonyl groups. ¹³C-NMR spectra of biflavonoids showed 2 peaks at δC 182.00 ppm, and biflavonoids also have 10 characteristic peaks at δC 160.0–166.0 ppm. The protons of the methoxy group of biflavonoids are known from the proton chemical shift value of δH 3.75–3.85 ppm with a higher ¹³C-NMR shift value than the carbon in the hydroxyl group because the carbon attached to the hydroxyl group is more shielded than the methoxy group. In the ¹H-NMR spectrum, 1 has 9 signals, 3 has 14 signals, and 4 has 12 signals representing 22 protons. In addition, 2 has 11 signals of ¹H-NMR representing 20 protons, while 5 has 7 signals representing 26 protons. Moreover, the ¹³C-NMR spectrum of 1, 3, and 4 has 28 signals representing 32 carbons, while 2 has 26 signals representing 31 carbons and 5 has 15 signals representing 34 carbons.

Figure 1. Structure of compounds 1–5 isolated from the leaves of A. hunsteinii. [Click here to view]

Cupressuflavone derivatives (1, 2, and 5)

Amentoflavone derivative (3)

4′,4?′-Di-O-methylamentoflavone (Isoginkgetin, 3)

Compound 3 was afforded as pale-yellow amorphous powder with the Rf value of 0.27 (CHCl3:MeOH = 19:1), The UV data (CH3OH) showed the absorbance at λmax 270 nm (benzoyl chromophore) and 331 nm (cinnamoyl chromophore) which indicates 3 has a flavonoid structure The IR data (KBr) showed 3 have several functional groups such as carbonyl groups at νmax 1,653 cm⁻¹, C=C aromatic at 1,599 cm⁻¹, and ether group at 1,279–1,261 cm⁻¹.

Figure 2. HSQC and HMBC key correlation of 1 (cupressuflavone), 3 (amentoflavone), and 4 (agathisflavone). [Click here to view]

Figure 3. Proposed MS fragmentation scheme of cupressuflavones (1, 2, and 5). [Click here to view]

The ¹H NMR (DMSO-d6, 500 MHz) data of 3 showed two signals of hydroxy groups chelated carbonyl δ 13.18 (1H, 5?-OH), 12.84 (1H, 5-OH); six doublet signals of two aromatics rings which para substituted δ , 8.11 (1H, dd, J = 2.4, 8.7 Hz, H-6′), 8.01 (1H, d, J = 2.4 Hz, H-2′), 7.44 (1H, d, J = 8.8 Hz, H-3?′ and -5?′), 7.29 (1H, d, J = 9.0 Hz, H-5′), 6.41 (1H, d, J = 2.0 Hz, H-8), 6.12 (1H, d, J = 2.0 Hz, H-6); four singlet aromatic signals δ 6.85 (1H, s, H-3), 6.77, (1H, s, H-3?) 6.64 (1H, d, J = 8.8 Hz, H-2?′ and -6?′), 6.59 (1H, s, C-6?); and two signals represent two methoxy groups at δ 3.76 (3H, s, 4?′-OCH3), 3.71 (3H, s, 4′-OCH3). The ¹³C NMR (DMSO-d6, 126 MHz) data displayed: two signals of hydroxy groups chelated carbonyls δ 182.4 (C, C-4?), 181.8 (C, C-4); nine signals of oxyaril carbons δ 164.4 (C, C-7), 164.0 (C, C-2?), 163.3 (C, C-2), 162.6 (C, C-5), 161.5 (C, C-5? and -7?), 161.3 (C, C-4?′), 160.4 (C, C-4′), 157.5 (C, C-9), 153.5 (C, C-9?); 16 signals of sp² aromatic carbons δ 130.9 (CH, C-2′), 128.4 (CH, C-2?′ and -6?′), 128.2 (CH, C-6′), 122.6 (C, C-1’), 121.3 (C, C-1?′), 121.2 (C, C-3’), 115.9 (CH, C-3?′ and -5?′), 111.9 (CH, C-5′), 104.7 (C, C-10?), 104.1 NMR spectra also the LC-MS chromatogram of compound 3 can be seen in Figures S19–S26.

Amentoflavone has a dimer bond at quaternary carbons C3′ and C8?, confirmed by HSQC and HMBC data (Fig. 2). The 3′ carbon showed an HMBC correlation with H5′ while carbon 8? exhibited HMBC correlation with H6?. The proton of methoxy groups in 3 has HMBC correlation with C4′, and 4?′ which indicates methoxy groups at C4′ and 4?′. HSQC and HMBC correlation of 3 can be seen in Figure 2. LC-MS/MS fragmentation analysis confirms the NMR data of 3 is biflavonoid. The molecular weight of 3 is m/z 567.1318 [M+H]⁺ (C32H22O10). Cleavage of dimer bond in amentoflavone-type biflavonoids occurs at fragment m/z 567 to 297. The flavone C-ring was cleaved at fragment m/z 521 becomes 403 (Nakata et al., 2018; Zhang et al., 2011), the MS fragmentation of 3 can be seen in Figure 4.

Agathisflavone derivative (4)

7,7?-Di-O-methylagathisflavone (4)

Compound 4 was obtained as yellow amorphous powder with the Rf value of 0.35 (CHCl3:MeOH = 19:1), The UV data (CH3OH) showed the absorbance at λmax 273 nm (benzoyl chromophore) and 330 nm (cinnamoyl chromophore) which indicates 4 has a flavonoid structure. The IR data (KBr) showed 4 have several functional groups such as hydroxyl groups at νmax 3,329 cm⁻¹, C=C aromatic at 1,446 cm⁻¹, and ether group at 1,240 cm⁻¹.

The ¹H NMR (Acetone-d6, 500 MHz) data of 4 showed: two signals of hydroxy groups chelated carbonyl δ 13.29 (1H, s, 5-OH), 13.26 (1H, s, 5?-OH); five doublet signals of two aromatics rings which para substituted δ 8.03 (1H, d, J = 8.8 Hz, H-2′ and -6′), 7.61 (1H, d, J = 8.8 Hz, H-2?′ and H-6?′), 7.07 (1H, d, J = 8.3 Hz, H-3′ and -5′), 6.99 (1H, d, J = 2.0 Hz, H-8) 6.85 (1H, d, J = 8.9 Hz, H-3?′ and -5?′); three singlet aromatic signals δ 6.77 (1H, s, H-3), 6.66 (1H, s, H-3?), 6.57 (1H, s, H-6?); and a signal represent a methoxy group at δ 3.88 (3H, 7- and 7?-OCH3). The ¹³C NMR (Acetone-d6, 126 MHz) data displayed: two signals of hydroxy groups chelated carbonyls δ 182.7 (C, C-4?), 182.4 (C, C-4); 10 signals of oxyaril carbons δ 164.5 (C, C-2), 164.3 (C, C-2?), 163.8 (C, C-7?), 163.7 (C, C-7), 162.4 (C, C-5?), 161.4 (C, C-4′), 161.2 (C, C-4?′), 159.5 (C, C-9), 157.9 (C, C-5), 154.4 (C, C-9?); 13 signals of sp² aromatic carbons δ 128.5 (CH, C-2′ and -6′), 128.1 (CH, C-2’’’ and -6?′), 122.2 (C, C-1′ and -1?′), 116.1 (CH, C-3′ and -5′), 115.9 (CH, C-3?′ and -5?′), 105.1 (C, C-10), 104.6 (C, C-10?), 104.2 (C, C-6), 103.5 (CH, C-3), 102.7 (CH, C-3?), 100.3 (C, C-8?), 95.2 (CH, C-6?), 90.3 (CH, C-8); and a signal of methoxy carbons δ 55.9 (CH3, 7 and 7?-OCH3). LC rt 10.34 minutes, ESI/MS m/z calculated for C32H22O10: 567.1371[M+H]⁺, 521.0929, 447.1125, 431.0812, 405.1014, 389.0701, 361.0741, 332.0716, 285.0437, 242.0253, 186.0343, 121.0308. The UV, IR, and NMR spectra also the LC-MS chromatogram of compound 4 can be seen in Figures S27–S34.

Agathisflavone showed a dimer bond at quaternary carbons C6 and C8? from HSQC and HMBC data (Fig. 2). Based on the HMBC data, C6 shows HMBC correlation with H8, while C8? has an HMBC correlation with H6?. The proton of methoxy groups in 4 has HMBC correlation with C7 and 7?, indicating methoxy groups at C7 and 7?. The HSQC and HMBC correlation of 4 can be seen in Figure 2. LC-MS/MS analysis confirmed the NMR data of 4 is biflavonoid, and the flavone C-ring cleaved at fragment m/z 567 becomes 447. The cleavage of dimer bond in amentoflavone-type biflavonoids occurs at fragment m/z 567 becomes 297 and 361 becomes 285. The termination of the functional group occurs in the fragment m/z 447 becomes 405 and 431 becomes 361, and the cleavage of the hydroxyl group also occurs in fragments m/z 447 becomes 431 (Nakata et al., 2018; Zhang et al., 2011). The MS fragmentation scheme of 4 can be seen in Figure 5.

Figure 4. Proposed MS fragmentation scheme of amentoflavone (3). [Click here to view]

Cytotoxicity activity of biflavonoids against MCF-7 and HeLa cancer cells

The cytotoxicity assay against MCF-7 cancer cells

The cytotoxicity test for MCF-7 and HeLa cancer cells was performed at 1-5. The data can be Table S1 and S2. The cytotoxicity test of biflavonoids against MCF-7 and HeLa cancer cells respectively was evaluated qualitatively by observing cell morphology with an inverted microscope using a spectrophotometric plate reader at a wavelength of 595 nm. Figure 6 shows the morphology of MCF-7 cells with 400× magnification in the negative control, the positive control (Epirubicin-HCl) with a concentration of 31.25 ppm, and 2 with a concentration of 31.25 ppm. The cell morphology in the negative control (Fig. 6N1) showed cells growing on the surface of the plate (confluency close to 100%). The degree of confluency is defined as the percentage of the surface area covered by the cells (Haenel and Garbow, 2014). Living cells have well-defined membranes with clear, light- colored, and epithelial-shaped media. MCF-7 cells given a positive control (Fig. 6N2) and 2 at the same concentration (Fig. 6N3) showed confluent cell death, cell membrane boundaries were not visible, and the cell shape was irregular. However, based on this qualitative analysis, it needs to be ascertained whether the sample added to the cell enters entirely and thoroughly into the cell. According to Yeap et al. (2021), MCF-7 cells have polygonal cell shapes with clear and distinctive cell membrane boundaries, while dead cells have distorted figures. This is different from the characteristics of living cells shown in the negative control (Fig. 6N1), so the addition of compound 2 indicates a cell response.

This test was carried out using the MTT method, which is relatively faster, more sensitive, and accurate, and can be applied to various samples (van Tonder et al., 2015). Table 1 shows the IC50 values of cytotoxicity of the five compounds against the MCF-7 cancer cells. Moreover, the cytotoxicity test showed that 1, 2, and 3 are classified as very active, 4 as moderately active, and 5 as weakly active in inhibiting the growth of MCF-7 cells. According to the National Cancer Institute (NCI) of the United States, the cytotoxicity of the sample tested can be categorized as very active if the IC50 < 20 μM, moderate activity if IC50 ranged between 21 and 200 μM, weak activity if IC50 ranged between 201 and 500 μM and not-active if IC50 >500 μM (Damasuri et al., 2020).

Figure 5. Proposed MS fragmentation scheme of agathisflavone (4). [Click here to view]

Figure 6. The morphology of MCF-7 cells with 400× magnification on the negative control (N1), positive control at a concentration of 31.25 ppm (N2), and the addition of 2 at a concentration of 31.25 ppm (N3). The HeLa c cancer cells ell morphology of negative control (P1), positive control at a concentration of 31.25 ppm (P2), and the addition of 3 at a concentration of 31.25 ppm (P3). [Click here to view]

Table 1. IC50 value of isolated compounds 1–5 against MCF-7 and HeLa cancer cells. [Click here to view]

The cytotoxicity assay against HeLa cancer cells

Qualitative analysis of HeLa cancer cells was carried out by observing the morphology of cancer cells with an inverted microscope. The morphology of HeLa cancer cells in the negative control, positive control (Napradox-50), and compound 3 at a concentration of 31.25 ppm, respectively are shown in Figure 6. The cell morphology in the negative control (Fig. 6P1) shows that HeLa cancer cells containing viable organisms appear confluent, oval in shape, flat, light in color and have clear cell membrane boundaries. Cells supplemented with Napradox-50 at a concentration of 31.25 ppm (Fig. 6P2) showed that the cells were dead, indicated by irregular shape and black cells, but viable HeLa cancer cells were still visible. HeLa cancer cells with the addition of 3 at the same concentration (Fig. 6P3) only showed dead cells that were small and scattered. This suggests that adding 2 to MCF-7 cells and 3 to HeLa cancer cells indicates a cell response, which causes death in these cells.

The quantitative analysis using MTT assay against HeLa cancer cells showed that 2 and 3 are classified as very active, 1 and 4 compounds are moderately active, and 5 are inactive. The activity was categorized according to the NCI of the United States (Damasuri et al., 2020).

Comparison of biflavonoid activity against MCF-7 and HeLa cancer cells

As shown in Table 1, 2 and 3 are the most active compound to inhibit MCF-7 and HeLa cancer cells, respectively. The IC50 value of 2 was observed in the order of HeLa cancer cells 3, the order of IC50 value was MCF-7 < HeLa. Compound 2 is the most sensitive cell on HeLa, while 3 is against MCF-7. Li et al. (2019) reported the cytotoxic activity of 3 on HeLa cancer cells and MCF-7 cells at 3 to 100 μM for 48 hours. Compound 3 showed antiproliferative activities, the order of IC50 value was HeLa < MCF-7. Therefore, the inhibitory effect of 2 and 3 on cancer cell proliferation is dependent on the cell type.

Many pathways have been proposed based on the literature review for the mechanism that prevents the action of three in cancer cell invasion. According to Yoon et al. (2006), 3 can control the synthesis of the metalloproteinase-9 matrix in B16F10 melanoma, MDA-MB-231 breast carcinomas, and HT1080 human fibrosarcoma cells by inhibiting NF-B (factor kappa B) and Akt (protein kinase B). In another study, Tsalikis et al. (2019) found that 3 directly inhibited caspase, trypsin, and chymotrypsin-like activities connected to the beta-1, 2, and 5 subunits of the 20S proteasome. Additionally, O’Brien et al. (2008) reported that 3 also inhibits pre-mRNA splicing in HeLa cancer cells. The pre-mRNA splicing is essential for the emergence and development of different types of cancer.

The amount and position of methoxy groups regulate the biological activity of compounds, as demonstrated by the methylated derivatives of the cupressuflavone 7,4′,7?-tri-O- methylcupressuflavone (A1) (Agusta et al., 2022), 1, 2, and 3 on MCF-7 and HeLa cancer cells (Table 1). A compound with mono-, di- and/or trimethyl groups (A1, 1, and 2) has a more excellent activity (respectively, IC50 of 91.74 ± 5.6, 11.54 ± 3.4, and 3.40 ± 0.3 μM) on MCF-7 than 5 (IC50 = 397.89 ± 28.6 μM). The same case for the cellular compounds HeLa cancer cells A1 and 2 shows a higher activity high with IC50 35.59 ± 1.3 and 1.42 ± 1.1 μM, respectively, than 5 (IC50 = 528.78 ± 40.0 μM). In addition, 5 shows a weaker activity for both cells. The existence of methoxy groups at C4′, C7?, and/or C4?′ positions appears to slightly reduce the activity. Methylations at position C7 on the cupressuflavone skeleton appear to be the most beneficial for inhibiting the MCF-7 and HeLa cancer cells’ activity because 2 were more active than A1, 1, and 5. According to a previous study, the minor difference in activity caused by the position and number of methoxy groups may be due to the ability to form hydrogen bonds to MCF-7 or HeLa cancer cells and changing polarity (Molfetta et al., 2004). Agathisflavone derivatives (4) are slightly more active inhibitors on both cells MCF-7 (115.39 ± 36.5 μM) and HeLa cancer cells (IC50 = 107.63 ± 37.3 μM) than 7,7?,4?′- tri-O- methylagathisflavone (A2) for MCF-7 (IC50 = 314.44 ± 25.0 μM) and HeLa cancer cells (IC50 = 337.05 ± 26.7 μM) (Agusta et al., 2022). Methylations at position 4?′on the agathisflavone skeleton appear to be the least likely to suppress both cell activity, as the IC50 value of A2 was observed to be greater than 4. A comparison of the MCF-7 and HeLa cancer cells inhibitory activities of cupressuflavone derivatives (A1, 1, and 2) was shown to be a strong inhibitor of both cells MCF- 7 and HeLa cancer cells (IC50 between < 20 and 21–200 μM). On the contrary, agathisflavone derivatives (A2, and 4) were significantly less active (IC50 in ranging of 21–200 and 201–500 μM) (Damasuri et al., 2020).

Compound 3 belongs to the amentoflavone group, which has two methoxy groups at C4 and 4?′. It is very active in inhibiting the growth of both cell MCF-7 and HeLa cancer cells with IC50 values of 2.14 ± 0.6 and 11.03 ± 2.9 μM, respectively. The results of this study are slightly different from those of Li et al. (2019), who reported that 3 has an IC50 value of 8.38 ± 0.63 μM for HeLa cancer cells and 91.19 ± 0.5 μM for MCF-7 cells. The difference in the IC50 value is believed to occur due to differences in the condition (health) of the cancer cells used and the condition of the environment. Furthermore, Li et al. (2019) also reported that the amentoflavone derivative, 7,4′,4?′-tri-O-methyl amentoflavone with three methoxy groups shows proliferative activities on both cancer cells, respectively, the IC50 of 14.79 ± 0.64 μM for HeLa cancer cell and 57.62 ± 2.11 μM for MCF-7 cell. In general, the IC50 value is still very active for inhibiting HeLa cancer cells (IC50 < 20 μM), but it is less active in inhibiting MCF-7 cells (IC50 ranging from 21–200 μM). This suggests that the methoxy group at position C7 of the amentoflavone may have reduced its inhibitory effects on both cancer cells.

Compounds 1, 2, and 3 showed very active inhibition with the IC50 value of 11.54 ± 3.4, 3.40 ± 0.3, and 2.14 ± 0.6 μM, respectively. Compound 3 has the best activity against MCF-7 cells. In addition, compound 2 (IC50 of 1.42 ± 1.1 μM) having the best inhibitory activity against HeLa cancer cells, compared to 3 (IC50 11.03 ± 2.9 μM). All compounds have a higher IC50 value compared to epirubicin-HCl and napradox-50 (IC50 0.55 ± 0.2 and 0.35 ± 0.03 μM, respectively), indicating that all samples have lower activity than the positive controls.

The different substituents showed specific interactions between corresponding receptors/target molecules. As an anticancer agent, biflavonoids are known for their ability to control cell proliferation, apoptosis, invasion, metastasis, autophagy, transcription, and drug resistance in various types of cancers, including breast and cervical cancer cells. Biflavonoids may bind to the respective target and inhibit their activity to support cancer cells to grow. Moreover, the substituents attached also have an important role in controlling the energy of those interactions (Xiong et al., 2021).

Compound 2, which has a C8-C8? bond, would be the most potent inhibitor of the HeLa cancer cells (IC50 = 1.42 1.1 μM), in contrast to 3, having a C3′-C8? bond and is the strongest inhibitor of the MCF-7 cell (IC50 = 2.14 0.6 μM). Inhibitory studies using cupressuflavone and amentoflavone derivatives on proliferating cells are necessary to fully assess the anticancer potential of these drugs. Coulerie et al. (2013) reported that the robustaflavone, which has a C3′-C6? linkage, is a potent inhibitor of the DENV-NS5 RdRp (IC50 = 0.33 μM). However, the 7?-O-methylamentoflavone (sotetsuflavone), which has a C3′-C8? linkage, is the strongest inhibitor with IC50 = 0.16 μM. Based on SAR analysis, the skeleton of a biflavonoid provides a compelling template for the production of an anticancer compound. The number and position of -OCH3 groups, as well as the type of biflavonoid, all affect its anticancer effectiveness. Furthermore, a link was also discovered between patterns of oxygenation substitution and the control of the anticancer potential (Laishram et al., 2015; Tchimene et al., 2016; Wu et al., 2016). Biflavonoids are dimers of flavonoids through a C-O-C or C-C connection. The fundamental functional property of flavonoids is their capacity to act as antioxidants, which provide protection against cancer and heart disease. Their activity is strongly correlated with the position and number of hydroxyls that attached to their aromatic rings, the physicochemical properties of another substituent, and the capacity for intramolecular hydrogen bond formation (Çetinkaya et al., 2022; Scotti et al., 2013).