Two New Flavonoids from the Nuts of Areca catechu

Two new flavonoids, calquiquelignan M (1), calquiquelignan N (2), along with nine known compounds (3–11), were isolated from the nuts of Areca catechu (Palmae). The new structures, including absolute configurations, were established by a combination of spectroscopic data and electronic circular dichroism (ECD) calculation. The known compounds were identified by comparing their spectroscopic data with reported in the literature. The flavonoids compounds (1–8) were evaluated for their cytotoxicity activities against three human cancer cell lines. Compounds 1 and 2 exhibited a moderate cytotoxic activity against HepG2 cell lines with IC50 values of 49.8 and 53.6 μM, respectively.


Structural Elucidation
Calquiquelignan M (1) was isolated as a yellow powder. The molecular formula of 1 was assigned as C21H24O9 based on its HRESIMS at m/z 443.1310 [M + Na] + (calcd for C21H24O9Na, 443.1313), indicating 10 degrees of unsaturation. The IR absorption showed the characteristic absorptions for hydroxyl (3477 cm −1 ), carbonyl (1660 cm −1 ) groups, and aromatic ring (1535 and 1450 cm −1 ). The UV spectrum exhibited absorption maxima at 287 and 208 nm.  13 C-NMR signals of 1 were assigned as shown in Table 1.

Structural Elucidation
Calquiquelignan M (1) was isolated as a yellow powder. The molecular formula of 1 was assigned as C 21 Table 1.
The above data of 1 resembled those of dihydrotricin (3) [12], except for the presence of one methoxy and one glycerin unit carbon signals. The 1 H-1 H COSY correlations between δ H 3.85 (H 2 -8 ) -4.08 (H-7 )-3.77 (H 2 -9 ) confirmed the glycerin unit existence, and the HMBC correlations between H-7 and C-4 allowed the linkage between dihydroflavone fragment and glycerin fragment through the C-4 -O-C-7 bond. The HMBC correlations between δ H 3.82 (methoxy) and C-7 led to the increased methoxy substitution position at C-7 ( Figure 2). Finally, the absolute configuration of 1 was deduced using the computational calculation method. The experimental ECD spectrum of 1 showed positive Cotton effects at 342 (∆ε + 6.6), 260 (∆ε + 29.3) nm and negative Cotton effects at 299 (∆ε − 7.0), 216 (∆ε − 18.2) nm, which were similar to those in the quantum chemical ECD calculation in Gaussian 09 software ( Figure 3) [20]. Accordingly, the absolute configuration of 1 was determined as 2S. (Δε − 18.2) nm, which were similar to those in the quantum chemical ECD calculation in Gaussian 09 software (Figure 3) [20]. Accordingly, the absolute configuration of 1 was determined as 2S.   . The 13 C-NMR spectrum displayed 28 carbon signals, including a chelated phenolic ketone carbon, eighteen olefinic carbons, three methines, two methylenes, and four methoxyls ( Table 1). The above-NMR of 2 resembled those of 1, except for the absence of a methylene, and the present of a 1 ,3 ,4 -trisubstitution phenyl ring and a methine at (δ C 74.5). The signal at δ C 74.5 was assigned to C-8 indicated the 1 ,3 ,4 -trisubstitution phenyl ring substituted there, which was supported by the HMBC correlations between H-8 and C-2 /C-6 . Furthermore, the HMBC correlations between H-7 and C-4 confirmed the C-4 -O-C-7 bond existence, and correlations between methoxy (δ H 3.84) and C-3 , led to the methoxy substitution position at C-3 ( Figure 4). Thus, the planar structure of 2 was determined, as shown in Figure 4.  . The 13 C-NMR spectrum displayed 28 carbon signals, including a chelated phenolic ketone carbon, eighteen olefinic carbons, three methines, two methylenes, and four methoxyls ( Table 1). The above-NMR of 2 resembled those of 1, except for the absence of a methylene, and the present of a 1′′,3′′,4′′-trisubstitution phenyl ring and a methine at (δC 74.5). The signal at δC 74.5 was assigned to C-8′ indicated the 1′′,3′′,4′′-trisubstitution phenyl ring substituted there, which was supported by the HMBC correlations between H-8′ and C-2′′/C-6′′. Furthermore, the HMBC correlations between H-7′ and C-4′ confirmed the C-4′-O-C-7′ bond existence, and correlations between methoxy (δH 3.84) and C-3′′, led to the methoxy substitution position at C-3′′ ( Figure 4). Thus, the planar structure of 2 was determined, as shown in Figure 4. Generally, adjacent protons of the erythro type have been reported to have smaller coupling constants (2.8-5.6 Hz) than those of the threo type (6.0-8.6 Hz) in different d-solvents [21][22][23]. Likewise, compound 2 was identified as a threo-configuration due to the 6.7 Hz coupling constants between H-7′ and H-8′ in CD3OD. Furthermore, the agreement of the ECD spectrum of 2 with those of 1 allowed the absolute configuration of 2 to be determined as 2S (Figure 3).
The pharmacological study of flavonoids and their derivatives showed that they possess cytotoxic activity [24]. Thus, the flavonoid compounds (1-8) were tested for their cytotoxic activity Generally, adjacent protons of the erythro type have been reported to have smaller coupling constants (2.8-5.6 Hz) than those of the threo type (6.0-8.6 Hz) in different d-solvents [21][22][23]. Likewise, compound 2 was identified as a threo-configuration due to the 6.7 Hz coupling constants between H-7 and H-8 in CD 3 OD. Furthermore, the agreement of the ECD spectrum of 2 with those of 1 allowed the absolute configuration of 2 to be determined as 2S (Figure 3).
The pharmacological study of flavonoids and their derivatives showed that they possess cytotoxic activity [24]. Thus, the flavonoid compounds (1-8) were tested for their cytotoxic activity against three human cancer cells MCF-7, HepG2, and A-549 using MTT assay. Compounds 1 and 2 exhibited a moderate cytotoxic activity against HepG2 cell lines with IC 50 values of 49.8 and 53.6 µM, respectively (Table 2).

Plant Material
The areca nuts were collected in Sanya, Hainan province, P. R. China, in October 2014, and identified by Professor Guang-Xiong Zhou (College of Pharmacy, Jinan University). A voucher specimen (No. CP2014101403) was deposited at the herbarium of the College of Pharmacy, Jinan University, Guangzhou, P. R. China.

Extraction and Isolation
The air-dried and powdered areca nuts (30.0 kg) were extracted at room temperature with 95% EtOH to afford a residue (1.1 kg), which was then suspended in H 2 O and treated with 0.5% hydrochloric acid to adjust the pH to 2 to 3. After extraction with CHCl 3 , the CHCl 3 extract (60.0 g) was subjected to silica gel column chromatography and eluted with CHCl 3 -CH 3

Computational Calculation
Conformational searches were performed in the Sybyl 8.1 software by using the MMFF94S molecular force field, which afforded 12 conformers for 1, with an energy cutoff of 10 kcal/mol. The ECD calculation for the optimized conformers was carried out using time-dependent DFT (TDDFT) methods at the B3LYP/6-31+G(d) level in the gas phase by using Gaussian 09 software. The overall ECD curves of 1 were weighted by Boltzmann distribution of each conformer (with a half-bandwidth of 0.3 eV). The calculated ECD spectra of 1 were subsequently compared with the experimental ones. The ECD curves were produced by SpecDis 1.6 software (University of Wuerzburg, Bavaria, Germany).

Cytotoxicity Assay
The MTT assay for the determination of the cytotoxicity was performed as described previously [25]. Briefly, cancer cells were plated into 96-well plates. After 48 h of preculture, the cells were treated with compounds at various concentrations for 72 h and then stained with MTT. Absorbance at 570 nm was measured on a microplate reader.
Author Contributions: M.Y., Y.A., and J.X. isolated and identified the structure of the compounds. N.Y. and D.Z carried out the cytotoxicity assay. J.Z. and X.Z. designed and supervised the study, and wrote and revised the manuscript. W.Y. revised the manuscript.