Three New Tetranorditerpenes from Aerial Parts of Acerola Cherry (Malpighia emarginata)

Acerola cherry is a world famous fruit which contains abundant antioxidants such as vitamin C, anthocyanins, flavonoids, and phenolics. However, studies concerning bioactivity components from aerial parts of acerola (Malpighia emarginata) are scarce. In view of this, we have examined the constituents of aerial parts of acerola, and three new tetranorditerpenes acerolanins A–C (1–3) with a rare 2H-benz[e]inden-2-one substructure were isolated. Their structures were determined on the basis of spectral studies and acerolanin C was confirmed by X-ray crystallographic analysis. Furthermore, three new compounds have been studied for their cytotoxic activity.


Introduction
Acerola (Malpighia emarginata DC.) is a shrub grown in tropical and subtropical areas. It has been introduced into many provinces of China including Guangxi, Guangdong, and Yunnan, etc. Acerola fruits are mainly utilized by the supplement, pharmaceutical, and fruit-juices industries as a rich source of vitamin C [1]. However, recent research showed that besides vitamin C, acerola fruits may be also a good source of phytochemicals such as anthocyanins [2,3], flavonoids and phenolic acids [4], and polyphenols [2,5]. With respect to bioactivities, acerola showed antioxidant [6], antimicrobial [7], hepatoprotective [8], and anti-hyperglycemic [9] effects. Nevertheless, there is seldom report on the bioactivity constituents from aerial parts of acerola.
In our previous research, three norfriedelins with acetylcholinesterase inhibitory activity were found in acerola tree (M. emarginata) [10]. As continuation of this work, we have examined the lipophilic constituents of aerial parts of acerola collected in Nanning, Guangxi Province, China. Three new tetranorditerpenes (1-3) with a rare 2H-benz[e]inden-2-one substructure were obtained ( Figure 1). This report describes the isolation and structural determination of the compounds, as well as their cytotoxic activities.

Results and Discussion
Compound 1 was obtained as yellow power and had a molecular formula C 16 H 14 O 4 by HREI-MS ion at m/z 270.0900 [M] + with 10 degrees of unsaturation. 13 C-DEPT (Table 1) revealed sixteen resonances consisting of two carbonyls, ten olefinic carbons, one quaternary carbon and three methyl groups. Seven out of ten degrees of unsaturation were occupied by two carbonyls and five double bonds and the remaining three indicated that compound 1 was tricyclic. The 1D NMR data of 1 was similar to those of substructure B in fimbricalyx A [11]. In the HMBC spectrum ( Figure 2), cross-peaks between δ H 1.44 (H 3 -18 and H 3 -19) and δ C 209.4 (C-3), 46.2 (C-4), and 133.6 (C-5) suggested that the carbonyl was located at C-3. According to the HSQC spectrum, two hydroxyl proton signals were identified at δ H 7.27 and 12.08. Correlations from δ H 7.27 to 133.6 (C-5), 141.0 (C-6), and 184.6 (C-7), from δ H 12.08 to 112.9 (C-8), 131.5 (C-13), and 160.9 (C-14) in the HMBC spectrum suggested that two hydroxyl groups were attached to C-6 and C-14, respectively. Two aromatic proton signals at δ H 7.41 (1H, d, J = 6.0 Hz) and δ H 7.45 (1H, d, J = 6.0 Hz) were attributed to H-11 and H-12, respectively, by their correlations with each other in the 1 H-1 H COSY spectrum and by the HMBC correlations from δ H 7.41 to C-13, C-8, and C-10, from δ H 7.45 to C-14 and Me-15. The formation of an intramolecular hydrogen bond between 14-OH with 7-(C=O) shifted the proton signal of 14-OH to lower field at δ H 12.08, which further confirmed the C-7 location of the carbonyl group. Thus, the structure of compound 1, named acerolanin A, was identified as shown in Figure 1.   Table 1). The difference between 3 and 2 was the absence of one hydroxyl group in 3 according to comparison of their formula and 13 C-DEPT data (four =CH in 3 and three =CH in 2). As supported by the HMBC correlations from Me-15 to C-12, C-13, and C-14 and by the 1 H-1 H COSY between H-11 (9.05, d, J = 8.4 Hz) with H-12 (7.62, d, J = 8.4 Hz), there was no substituent at C-11, C-12, and C-14 Thus, the only methoxyl group could be located at C-7 by the HMBC correlations from H-6 to C-4, C-8, and C-10, from MeO-7 to C-7. In the HMBC spectrum, two methyl-proton signals at δ H 1.50 correlated with a carbonyl-carbon signal at δ C 205.6 suggested one carbonyl at C-3. Therefore, the other carbonyl should be located at C-1. In order to confirm its structure, the X-ray crystallography of 3 was completed and the result (Figure 3) allowed unambiguous assignment of its planar structure. Acerolanins A-C are a class of tetranorditerpenoids possessing a rare 2H-benz[e]inden-2-one substructure. Analogues have been isolated previously from four species of the Euphorbiaceae, namely, Neoboutonia glabrescens [12], Neoboutonia mannii [13], Trigonostemon howii [14], and Strophioblachia fimbricaly [11]. Some compounds of this class were reported to have cytotoxic and antimicrobial activities, so compounds 1-3 were evaluated for cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human tumor cell lines using the MTS method. Cisplatin was used as a positive control. Results are summarized in Table 2. As can be observed, compounds 1-3 showed moderate cytotoxicity against above five cell lines with IC 50 values from 10 to 40 μM.

Plant Material
The

Cytotoxicity Assay
The cytotoxicity of compounds 1-3 was tested against human breast cancer (MCF-7), hepatocellular carcinoma (SMMC-7721), myeloid leukemia (HL-60), lung cancer (A-549) and colon cancer (SW480) cell lines using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) assay, with cisplatin (Sigma-Aldrich, St. Louis, MO, USA) as the positive control. All the cell lines were obtained from Shanghai cell bank in China and were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 . The viability of cells was determined by performing colorimetric measurements of soluble formazan formed through the reduction of MTS in living cells. In brief, 100 μL medium containing 5,000 cells were plated in each wells in 96 well plates and allowed to adhere for 24 h before drug treatment, while suspension cells were seeded just before drug addition at a concentration of 1 × 10 5 cells/mL. Cells were exposed to the test compound dissolved in dimethyl sulfoxide (DMSO) at different concentrations in triplicates at 37 °C for 48 h. At the end of the incubation, the medium were replaced with MTS medium (317 μg/mL), and then the incubation was continued for 4 h at 37 °C. The optical densities of the cell lysates were measured at 490 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The cell viability was calculated by the following formula: cell viability (%) = (OD sample /OD control ) × 100%. The IC 50 value of each compound was calculated by Reed and Muench's method based on the corresponding dose response curve, and data were obtained from triplicate experiments. Statistical analysis was performed using the commercially available statistical software (SPSS 11.5 for Windows, SPPS Incorporation, Chicago, IL, USA).

Conclusions
Phytochemical study of the aerial parts of acerola (M. emarginata) has resulted in the isolation of three new degraded diterpenes 1-3. As far as we know, this is the first report of this class of degraded diterpenes from the Malpighiaceae family. In addition, compounds 1-3 showed cytotoxic activities.