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

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INTRODUCTION
Cancer is a deadly disease caused by abnormal cell growth in certain parts of the body, having the ability to grow improperly, and spread to other parts of the body.According to the World Health Organization, around 4.4 million women died due to cancer in 2020, with 25% being caused by breast cancer (WHO, 2023).In contrast, cervical cancer affects women more frequently than any other form in 23 countries, with a total of 604,000 cases (IARC, 2022).Cancer treatment can be performed through therapy or medical appointments, such as surgery, radiotherapy, and chemotherapy (Hosseinzadeh et al., 2017).This method aims to suppress the growth and even destroy cancer cells, but this therapy will also damage healthy cells.In addition, the high cost of therapeutics and the success is not optimal.Therefore, herbal treatment approaches are in

ABSTRACT
In this study, five biflavonoids (1 to 5) have been isolated from the leaves of the Indonesian plant Araucaria hunsteinii K. Schum.The structures of biflavonoids were determined using various techniques, including one-and two-dimensional nuclear magnetic resonance, infrared, ultraviolet-visible, and mass spectrometric investigations.These compounds were identified as derivatives of cupressuflavone, amentoflavone, and agathisflavone.The cytotoxic potential of the isolated biflavonoids was evaluated against cancer cell lines, the cervical HeLa cancer cells, and the human breast Michigan Cancer Foundation-7 (MCF-7).All compounds were first isolated from this species, and compound 1 was first recognized as a member of the genus Araucaria.Compounds 1, 2, and 3 inhibition concentration 50 (IC 50 of 11.54 ± 3.4, 3.40 ± 0.3, and 2.14 ± 0.6 μM, respectively) showed very active inhibition with 3 having the best activity against inhibition of MCF-7 cells.In addition, 2 and 3 (IC 50 of 1.42 ± 1.1 and 11.03 ± 2.9 μM, respectively) also showed very strong inhibitory effects, with 2 having the best inhibitory activity against HeLa cancer cells.All compounds have a higher IC 50 value compared to epirubicin-HCl and napradox-50 (IC 50 0.55 ± 0.2 and 0.35 ± 0.03 µM, respectively), indicating that all samples have lower activity than the positive controls.The structure-activity relationship analysis of these isolated metabolites shows that cupressuflavone and amentoflavone are the two most promising biflavonoid skeletons for developing anticancer drugs.The methoxy groups of the biflavonoid moiety modify their inhibitory effects on MCF-7 and HeLa cancer cells, and compounds 2 and 3 work best as inhibitors against HeLa cancer cells and MCF-7 cells, respectively, because of their hydroxyl position at C4′, 4ʺ′, and 7ʺ.This study has revealed the potency of cupressuflavone and amentoflavone-derived compounds that potentially developed further as active constituents in cancer medication.

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 great demand in a way that the search for anticancer secondary metabolites derived from plants is fundamental.

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 O n l i n e F i r s t hydroxyl group is more shielded than the methoxy group.In the Cupressuflavone derivatives (1, 2, and 5)
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: 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 1 H NMR and 13 C-NMR data reveal various properties of these compounds.On the other hand, biflavonoids showed two distinct signals in their 1 H-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 160.0-166.0ppm.The protons of the methoxy group of biflavonoids are known from the proton chemical shift value of δH 3.75-3.85ppm with a higher 13 C-NMR shift value than the carbon in the hydroxyl group because the carbon attached to the % Inhibition = Control Absorbans -Sample Absorbans × 100% Control Absorbans O n l i n e F i r s t Cupressuflavone is a flavone dimer binding at C8 and C8ʺ as shown by evidence from heteronuclear singular quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC).The HSQC spectrum gives information about the correlation of a bond between the corresponding proton and carbon.At the same time, the HMBC showed a correlation between proton and carbon separated by two to three bonds.Both C8 and C8ʺ correlate with H6 and H6ʺ respectively, but do not bind with protons (quaternary carbons).HMBC correlation also can determine the location of methoxy groups.The proton of methoxy groups in 1 showed HMBC correlation with C4ʺ′ and C7, while 5 showed correlations with C4′, C4ʺ′, C7, and C7ʺ.Compound 2 showed an HMBC correlation between the proton of the methoxy functional group and C7.HSQC and HMBC correlation of the cupressuflavone group can be seen in Figure 2.
LC-MS/MS analysis confirmed that the NMR data of 1, 2, and 5 are biflavonoids and not flavonoid monomers since the molecular weight of 1, 2, and 5 is twice that of flavonoids.The molecular weights of 1, 2, and 5 are m/z 567.1291 [M+H] + (C32H22O10); 553.3317 [M+H] + (C31H20O10); and 595.1628 [M+H] + (C34H26O10) respectively.In 5 analyses, the NMR data showed that half of the biflavonoids were nearly symmetrical.However, the results of the LC-MS/MS measurement of 5 showed that the compound has a molecular weight twice higher than the flavonoids.Therefore 5 is a large compound consisting of two flavonoids with a symmetrical structure.The fragmentation pathways of the cupressuflavone groups are shown in Figure 3.The flavone C-ring was cleaved at fragment m/z 567 becomes 449 at 1, 553 becomes 391 and 463 becomes 359 at 2, also 549 becomes 403 and 430 becomes 374 at 5. The termination of the functional group occurs in the fragment m/z 553 becomes 463 and 297 becomes 255 at 2, and 521 becomes 463 at 5. Similarly, the cleavage of the hydroxyl group also occurs in fragments m/z 553 becomes 463 at 2 and 521 becomes 463, and 374 becomes 359 at 5 (Nakata et al., 2018;Zhang et al., 2011).

O n l i n e F i r s t
Agathisflavone derivative (4)
Agathisflavone showed a dimer bond at quaternary carbons C6 and C8ʺ from HSQC and HMBC data (Fig. 2).
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 .
The Amentoflavone has a dimer bond at quaternary carbons C3′ and C8ʺ, confirmed by HSQC and HMBC data (Fig. 2).The 3′ carbon showed an HMBC correlation with H5′ while carbon 8ʺ exhibited HMBC correlation with H6ʺ.The proton of methoxy groups in 3 has HMBC correlation with C4′, and 4ʺ′ which indicates methoxy groups at C4′ and 4ʺ′.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. O n l i n e F i r s t 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 notactive if IC50 >500 µM (Damasuri et al., 2020).
Based on the HMBC data, C6 shows HMBC correlation with H8, while C8ʺ has an HMBC correlation with H6ʺ.The proton of methoxy groups in 4 has HMBC correlation with C7 and 7ʺ, indicating methoxy groups at C7 and 7ʺ.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.

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

O n l i n e F i r s t
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 <MCF-7.While for the 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 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.O n l i n e F i r s t C6ʺ linkage, is a potent inhibitor of the DENV-NS5 RdRp (IC50 = 0.33 µM).However, the 7ʺ-O-methylamentoflavone (sotetsuflavone), which has a C3′-C8ʺ 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).

CONCLUSION
Five biflavonoids from the leaves of A. hunsteinii K. Schum.Indonesia have been successfully isolated.These compounds were identified as 7,4ʺ′-di-O-methycupressuflavone (1), 7-O-methylcuppresuflavone (2), 4′, 4ʺ′-di-O-methyl amentoflavone (3), 7,7ʺ-di-O-methylagathisflavone (4) and 7,4′,7ʺ,4ʺ′-tetra-O-methyl-cupressuflavone (5).Compound 1 is the first isolated from the genus Araucaria.At the same time, 2-5 have been reported from several species of the genus Araucaria, but this is the first time it has been identified in A. husteinii.Regarding the SAR analysis, we also suggested that cupressuflavone and amentoflavone, are the most promising building block for developing anticancer drugs among biflavonoids.The number and location of methyl groups on the biflavonoid moiety modulate their inhibition growth of MCF-7 and HeLa cancer cells.A strong relationship was also discovered between patterns of oxygenation and the control of the anticancer potential.The most active compound and potent inhibitor against HeLa cancer cells are compounds 2 and 3, with an IC50 of 1.42 ± 1.1 and 11.03 ± 2.9 μM, respectively.Additionally, 1, 2, and 3 are also the most active compound and are the strongest inhibitor against MCF-7 cells with an IC50 of 11.54 ± 3.4, 3.40 ± 0.3, and 2.14 ± 0.6 μM, respectively.(Agusta et al., 2022).Methylations at position 4ʺ′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 .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).

LIST OF ABBREVIATIONS
Compound 3 belongs to the amentoflavone group, which has two methoxy groups at C4 and 4ʺ′.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′,4ʺ′-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.
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-C8ʺ 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′-C8ʺ 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′-O n l i n e F i r s t

Figure 1 .
Figure 1.Structure of compounds 1-5 isolated from the leaves of A. hunsteinii.

Figure 6 .
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).
a STD: standard deviation; b human breast carcinoma cell line (MCF-7); c human cervical carcinoma cell line (HeLa).