Cytotoxic Activities of Flavonoids from Centaurea scoparia

Phytochemical studies on the ethanolic extract of the aerial parts of Centaurea scoparia led to the isolation of two new flavonoids, 3′,4′-dihydroxy-(3′′,4′′-dihydro-3′′-hydroxy-4′′-acetoxy)-2′′,2′′-dimethylpyrano-(5′′,6′′:7,8)-flavone-3-O-β-D-glucopyranoside (1) and 3,3′,4′-trihydroxy-(3′′,4′′-dihydro-3′′,4′′-dihydroxy)-2′′,2′′-dimethylpyrano-(5′′,6′′:7,8)-flavone (2), along with eight known flavonoids isolated for the first time from this plant, cynaroside (3), Apigetrin (4), centaureidin (5), oroxylin A (6), 5,7-dihydroxy-3′,4′,5′-trimethoxyflavone (7), atalantoflavone (8), 5-hydroxy-3′,4′,8-trimethoxy-2′′,2′′-dimethylpyrano (5′′,6′′:6,7)-flavone (9), and 3′,4′,5,8-tetramethoxy-2′′,2′′-dimethylpyrano (5′′,6′′:6,7)-flavone (10). The structures of the isolated compounds were elucidated by means of spectroscopic tools including 1D and 2D NMR, UV, IR, and mass spectroscopy. Cytotoxic activities of the isolated compounds were evaluated against human cervical carcinoma HeLa, human hepatocellular carcinoma HepG2, and human breast carcinoma MCF-7. Compound 2 was the most potent cytotoxic agent against HeLa cells with an IC50 0.079 μM.


Experimental
2.1. General. 1 H (500 MHz) and 13 C (125 MHz) NMR were recorded in DMSO-6 on a Bruker AV-500 NMR spectrometer using TMS as internal standard. Chemical shifts ( ) are expressed in ppm and coupling constants ( ) are reported in Hz. Optical rotations were measured with Perkin-Elmer 341 polarimeter. UV data were obtained from Shimadzu UV-vis 160i spectrophotometer. HREIMS and EIMS mass spectroscopic analysis was measured on a Finnigan MAT TSQ 700 mass spectrometer. IR spectra were recorded on KBr pellets on a Shimadzu FTIR-8400 instrument.

Cell
Culture. All cell lines were purchased from the American Type Culture Collection (ATCC). Three cell lines were used in this study: HepG2 cells (human cell line of a well-differentiated hepatocellular carcinoma isolated from a liver), HeLa (cervical carcinoma cells), and MCF-7 (breast carcinoma cells). Cells were grown or maintained upon arrival at 37 ∘ C in a humidified incubator with 5% CO 2 and 95% atmosphere as recommended by ATCC. All media were supplemented with penicillin (100 U/mL), streptomycin (100 g/mL), and 10% heat-inactivated fetal bovine serum (FBS).

Results and Discussion
Compound 1 was obtained as a pale yellow amorphous powder. The UV spectrum showed maxima at 258 and 335 nm, suggesting a flavone moiety [28]. The IR spectrum displayed strong bands for OH (3371 cm −1 ) and conjugated carbonyl (1691 cm −1 ). The HREIMS mass spectrum revealed a [M + H] + ion at / 590.5124 corresponding to the molecular formula C 28 H 30 O 14 . The 1 H and 13 C NMR spectrum of 1 (Table 1) cyclized with C-2 to form a dioxo-substituted pyran ring. The presence of an acetoxy group was detected by a signal in the 1 H NMR at H 1.95 (3H, s) and two signals in the 13 C NMR at C 171 and C 20.7 (OAc-4 ). Two methyl groups were detected from signals at H 1.42 and 1.39 (3H, s each) for Me 1 -2 and Me 2 -2 , respectively, confirmed by two signals in the 13 C NMR at C 26.6 (Me 1 -2 ) and 21.5 (Me 2 -2 ). An oxygenated quaternary carbon (C-2 ) was detected by a signal at C 79.6. Searching the literature, we have found that compound 1 was similar to 5-methoxy-(3 ,4 -dihydro-3 ,4 -diacetoxy)-2 ,2 -dimethylpyrano-(7,8:5 ,6 )-flavone [29]. By comparison of the NMR Spectral data of the two compounds, the differences were the presence of only one acetoxy group in compound 1, two ortho-coupled protons at C-5 and C-6, trisubstituted B-ring in compound 1 as previously referred to, and the presence of a sugar moiety which was indicated by the signals of the anomeric proton at H 4.95 (1H, , = 6.9) and C 102.4, this coupling constant diagnostic for the -anomer. We have observed an upfield shift in the C of C-3 and a downfield shift in that of C-2 and C-4 which is diagnostic for the linkage of the sugar moiety to C-3. In the 1 H NMR spectrum, signals at H 4.95 (1H, , = 6.9); H 3.11-3.45 (m) for the rest of the sugar protons and in the 13 C NMR spectrum the signals at C 102.4, 74.8, 77.9, 70.1, 76.7, and 61.3 suggesting the sugar to be -glucopyranose. Furthermore, the sugar moiety was confirmed to be D-glucose by TLC analysis and coPC after hydrolysis. Besides the molish test the ESIMS fragment ions at 428 [M + H-162] + also indicated the presence of a sugar moiety. The presence of a hydroxyl group at C-3 was indicated from the chemical shift in the 13 C NMR C 69.7 (C-3 ) and the HREIMS data. The 1D NMR data suggests that compound 1 is a flavone with acetylated pyran ring and a sugar moiety. In the HMBC correlation as illustrated in Figure 2 and tabulated in Table 1, the signal at H 7.51 (1H, , = 8.9 Hz, H-5) indicated three key correlations with carbon signals at C 178.2 (C-4), 158.6 (C-7), and 155.7 (C-8a), the correlation between H 7.51 (H-5) and C 178.2 (C-4) indicated that the A-ring was unsubstituted at C-5 and C-6 positions. The proton signals at H 6.95 (1H, , = 8.9 Hz, H-6) correlated with two quaternary carbons at C 118.9 (C-4a) and C 108.4 (C-8) as well as one oxygenated carbon at C 158.6 (C-7). The correlation from the signal of H-3 (  with C-3 and that of H-4 ( H 6.55) with the carbonyl of the acetoxy group is a proof for the location of the acetoxy group to be at position C-4 . The configuration of C-3 and C-4 was determined to be cis based on the value of the coupling constant ( 3 ,4 = 4.8) and the difference in the values of the chemical shifts of the 2 -geminal dimethyl groups (0.03 ppm) [29]. The HMBC correlation was further utilized to identify the linkage between the aglycone and sugar moiety. The correlation between H-1 ( H 4.95, glc) and C-3 ( C 133.7) is a confirmation for the linkage of the sugar moiety to C-3 of the aglycone. Based on these assignments, the structure of compound 1 was elucidated as 3 ,4 -dihydroxy-(3 ,4dihydro-3 -hydroxy-4 -acetoxy)-2 ,2 -dimethylpyrano-  (Table 1), with the differences being the absence of the signals of the acetoxy group and the sugar moiety (negative molish test). The 1D NMR data of 2 (Table 1), recorded in DMSO-6 , showed a signal for the conjugated carbonyl carbon (C-4) at a chemical shift lower than that of compound 1 ( C 173.7), which is suggestion for the absence of the sugar moiety at C-3. The 1 H NMR spectrum exhibited two signals at H 7.55 and 6.97 (1H, , , which is diagnostic for the cyclization of the oxygen atom at C-7 with the C-2 to form the dihydroxy substituted pyran ring. The value of the chemical shift of the proton at C-4 in compound 1 is higher than that in compound 2 due to the replacement of the acetoxy group in 1 with a hydroxy group in 2 which makes this proton more deshielded in 1 than in 2. The two methyl groups at C-2 were confirmed by signals at ( H 1.41, s, 3H; C 24.1, Me 1 -2 ) and ( H 1.38, s, 3H; C 21.2, Me 2 -2 ). An oxygenated quaternary carbon at C-2 was assigned from a signal at C 78.3. A confirmation for the absence of the sugar moiety at C-3 is the higher C value of C-3 in compound 1 ( C 133.7) than in 2 ( C 141.2) and the decreasing of the C of C-2 and C-4 to be 154.2 and 173.7, respectively. In the HMBC correlation (Table 1; Figure 3), the H-5 ( H 7.55) proton correlated with C-4 (173.7), C-7 (158.2), and C-8a (155.1) diagnostic for orthocoupled aromatic protons at C-5 and C-6, confirmed by the proton signals at H 6.97 (H-6) correlated to C 117.8 (C-4a), C 107.7 (C-8), and C 158.2 (C-7). The attachment of the pyran ring to C-7 and C-8 was estimated from the correlation between the signal of H-3 ( H 3.81) with C-8 (107.7) and the signal of H-4 ( H 5.24) with C-8 ( C 107.7), C-7 ( C 158.2), and C-8a ( C 155.1). In comparing the HMBC correlation of compound 1 and compound 2, we have found that the correlation of H-1 with C-3 and H-4 with the carbonyl carbon of the acetoxy is not present in compound 2, which confirms the structure of compound 2 to be the same as compound 1 with the replacement of the acetoxy group and the sugar moiety with hydroxyl groups. Accordingly, the structure of compound 2 was elucidated to be 3,3 ,4 -trihydroxy-(3 ,4 -dihydro-3 ,4 -dihydroxy)-2 ,2 -dimethylpyrano-(5 ,6 : 7,8)-flavone.
Cytotoxic activity evaluating models provide effective preliminary data to help in selecting plant extracts with potential antineoplastic properties for future work [30]. In view of the present data, Cytotoxic activity of the isolated flavonoids was evaluated by the MTT assay. As shown in Table 2, the pyranoflavones was shown to exhibit a potent cytotoxic activity against the three cancer cell lines while the flavonoid glycosides (2,3) showed a weak activity. This indicated that the cyclization of the oxygen atom at C-7 to form the pyran ring increases the cytotoxic activity of the flavone. Compound 2 was the most potent cytotoxic agent against HeLa cells with an IC 50 0.079 M. Centaureidin (5) showed a very strong inhibition against HepG2 and MCF-7 cells with an IC 50 0.25 and 0.14, respectively.

Conclusion
In the present study, the cytotoxicity of ten flavonoids (1-10) isolated from C. scoparia was evaluated by the MTT assay against three human cancer cell lines. All the tested compounds displayed a different inhibition in a dose-dependent manner. Compound 2 was the best cytotoxic agent against HeLa cell line with an IC 50 0.079 M, while Centaureidin (5) is a better cytotoxic agent against HepG2 and MCF-7 cells with an IC 50 0.25 and 0.14, respectively.