Molecules 2014, 19(2), 2629-2636; doi:10.3390/molecules19022629

Article
Three New Tetranorditerpenes from Aerial Parts of Acerola Cherry (Malpighia emarginata)
Jie-Qing Liu 1,, Yuan-Yuan Deng 1,2,, Ting-Zhao Li 3, Qiang Han 3, Yan Li 1 and Ming-Hua Qiu 1,2,*
1
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, China; E-Mails: liujieqing@mail.kib.ac.cn (J.-Q.L.); yuanyuan_deng@yeah.net (Y.-Y.D.); liyanb@mail.kib.ac.cn (Y.L.)
2
University of Chinese Academy of Science, Beijing 100049, China
3
Amway (China) Botanical Research Center, Wuxi 214115, China; E-Mails: teric.li@amway.com (T.-Z.L.); johnson.han@amway.com (Q.H.)
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: mhchiu@mail.kib.ac.cn; Tel.: +86-871-6522-3257; Fax: +86-871-6522-3255.
Received: 15 January 2014; in revised form: 19 February 2014 / Accepted: 20 February 2014 /
Published: 24 February 2014

Abstract

: 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 (13) 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.
Keywords:
acerola; Malpighia emarginata; Malpighiaceae; tetranorditerpenes; cytotoxicity

1. 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 (13) 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.

Molecules 19 02629 g001 200
Figure 1. Structures of compounds 13 from the aerial parts of acerola.

Click here to enlarge figure

Figure 1. Structures of compounds 13 from the aerial parts of acerola.
Molecules 19 02629 g001 1024

2. Results and Discussion

Compound 1 was obtained as yellow power and had a molecular formula C16H14O4 by HREI-MS ion at m/z 270.0900 [M]+ with 10 degrees of unsaturation. 13C-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 δH 1.44 (H3-18 and H3-19) and δC 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 δH 7.27 and 12.08. Correlations from δH 7.27 to 133.6 (C-5), 141.0 (C-6), and 184.6 (C-7), from δH 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 δH 7.41 (1H, d, J = 6.0 Hz) and δH 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 1H-1H COSY spectrum and by the HMBC correlations from δH 7.41 to C-13, C-8, and C-10, from δH 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 δH 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 Table 1. 1H- and 13C-NMR data for compounds 13 (in CDCl3, 600 MHz for 1H and 150 MHz for 13C, δ in ppm).

Click here to display table

Table 1. 1H- and 13C-NMR data for compounds 13 (in CDCl3, 600 MHz for 1H and 150 MHz for 13C, δ in ppm).
No.123
δCδH (mult, J in Hz)δCδH (mult, J in Hz)δCδH (mult, J in Hz)
1125.9, d6.77 (s)125.2, d6.76 (s)184.3, s
3209.4, s 209.9, s 205.6, s
446.2, s 46.3, s 43.2, s
5133.6, s 131.0, s 163.4, s
6141.0, s 141.3, s 98.7, d6.80 s
7184.6, s 180.1, s 164.6, s
8112.9, s 130.7, s 133.2, s
9128.5, s 131.9, s 128.8, s
10118.4, s 156.3, s 125.2, s
11155.6, d 7.41 (d, 6.0)106.5, d7.25 (s)124.8, d9.05 (d, 8.4)
12136.7, d7.45 (d, 6.0)162.1, s 133.1, d7.62 (d, 8.4)
13131.5, s 122.8, s 137.9, s
14160.9, s 129.8, s8.02 (s)122.4, d8.12 (s)
1516.1, q2.35 (s)16.8, q2.33 (s)22.1, q2.56 (s)
1821.6, q1.44 (s)21.7, q1.44 (s)25.0, q1.50 (s)
1921.6, q1.44 (s)21.7, q1.44 (s)25.0, q1.50 (s)
-OMe 56.0, q4.00 (s)56.6, q4.20 (s)
6-OH; 7.27 (s) 7.01 (s)
14-OH 12.08 (s)
Molecules 19 02629 g002 200
Figure 2. The key HMBC (H→C) and 1H-1H COSY correlations ( Molecules 19 02629 i001) of 13.

Click here to enlarge figure

Figure 2. The key HMBC (H→C) and 1H-1H COSY correlations ( Molecules 19 02629 i001) of 13.
Molecules 19 02629 g002 1024

Compound 2 was obtained as a light yellow power and had a molecular formula C17H16O4 as evidenced by HREI-MS at m/z 284.1056 [M]+. The 1D NMR data (Table 1) of 2 showed high similarities to those of 1 except that there was one methoxyl group (δC 56.0 and δH 4.00) in 2 instead of one hydroxyl group in 1. The methoxyl group was determined to be attached to C-12 by the following evidence: HMBC (Figure 2) correlations from δH 2.33 (15-CH3) to δC 129.8 (C-14), 162.1 (C-12), and 122.8 (C-13), from δH 4.00 (MeO-12) to δC 162.1 (C-12); Furthermore, the fact that δH 7.25 (H-11, 1H) and 8.02 (H-14, 1H) were singlets in the 1H-NMR demonstrated they were not in the ortho-position as in compound 1. Further detailed study of the HMBC and 1H-1H COSY data (Figure 2) determined the other parts of the structure of 2. Therefore, the structure of compound 2 was elucidated as shown in Figure 1. The compound was named acerolanin B.

Acerolanin C (3) was obtained as colorless monoclinic crystals from CHCl3/MeOH (1:3). The molecular formula C17H16O3 was established by the positive HREI-MS (found [M]+ at m/z 268.1097, calcd for C17H16O3 at m/z 268.1099), corresponding to 10 degrees of unsaturation. The skeleton of 3 was the same as 2 according to its 1D NMR data (Table 1). The difference between 3 and 2 was the absence of one hydroxyl group in 3 according to comparison of their formula and 13C-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 1H-1H 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 δH 1.50 correlated with a carbonyl-carbon signal at δC 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.

Molecules 19 02629 g003 200
Figure 3. X-Ray crystal structure of 3.

Click here to enlarge figure

Figure 3. X-Ray crystal structure of 3.
Molecules 19 02629 g003 1024

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 13 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 13 showed moderate cytotoxicity against above five cell lines with IC50 values from 10 to 40 μM.

Table Table 2. Cytotoxicity data of compounds 13 with IC50 values (μM) a.

Click here to display table

Table 2. Cytotoxicity data of compounds 13 with IC50 values (μM) a.
CompoundsHL-60SDSMMC-7721SDA-549SDMCF-7SDSW480SD
110.230.3212.200.4212.320.4516.220.7218.120.86
214.110.6116.540.6318.270.7222.081.1224.321.21
322.17 1.8020.101.0731.65 1.2228.04 1.47>40-
Cisplatin1.86 0.106.13 0.347.27 0.4215.27 0.6516.23 0.76

a Data were obtained from triplicate experiments, and cisplatin was used as positive control. SD = standard deviation.

3. Experimental

3.1. General Procedures

1H- and 13C-NMR spectra were measured on Bruker AVANCE III-600 instruments (Bruker, BioSpin International AG, Karlsruhe, Germany) with trimethylsilane (TMS) as the internal standard. ESIMS were recorded on a VG Auto Spec-3000 mass spectrometer (VG, Manchester, UK), while HREIMS was measured on an AutoSpec Premier P776 mass spectrometer (Water Corporation, Billerica, MA, USA). IR spectra were obtained on a Bio-Rad FTS-135 spectrometer (Bio-Rad Laboratories Inc., Richmond, CA, USA).

TLC was performed on precoated TLC plates (200–250 μM thickness, F254 Si gel 60, Qingdao Marine Chemical, Inc., Qingdao, China) with compounds visualized by spraying the dried plates with 10% aqueous H2SO4 followed by heating until dryness. Silical gel (200–300 mesh, Qingdao Marine Chemical, Inc.) and Lichroprep RP-18 (40–63 μm, Merck, Darmstadt, Germany) were used for column chromatography. Methanol, trichloromethane, n-hexane, and acetone were purchased from Tianjing Chemical Reagents Co. (Tianjing, China).

3.2. Plant Material

The aerial parts of acerola (Malpighia emarginata) were collected in May 2012 from Nanning, Guangxi province, China. The samples were identified by Prof. Zongyu Wang, Kunming Institute of Botany, Chinese Academy of Science. A voucher specimen (No. KIB 2012-04-20) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

3.3. Extraction and Isolation

The powder and dried aerial parts of M. emarginata (10 kg) were extracted with acetone at room temperature (20 L × 3, 3 days each time) and concentrated in vacuo to give a crude extract (400 g), which was then partitioned in succession between H2O and CHCl3. The CHCl3 fraction (85 g) was chromatographed on silica gel with CHCl3/MeOH gradient elution (100:0→20:1) to afford four fractions: Fr.1 (35 g, 100:0), Fr. 2 (12 g, 100:1), Fr.3 (10 g, 50:1), and Fr.4 (25 g, 10:1). By reverse-phase silica gel (MeOH/H2O, step gradients), Fr.2 was divided into three parts (MeOH/H2O: 40%, 60%, 80%), and 60% part was on chromatography over silica gel with n-hexane/CHCl3 (2:1) to yield acerolanin C (3, 7 mg). Using reverse-phase silica gel (MeOH/H2O, step gradient), Fr.3 was also divided into three fractions (MeOH/H2O: 40%, 60%, 80%). 60% fraction was further chromatographed on a silica gel column gradient eluting with CHCl3/acetone (15:1) to obtain acerolanin B (2, 5 mg), while 40% fraction was subjected to silica gel column chromatography (CHCl3/MeOH, 30:1) to give acerolanin A (1, 8 mg).

3.4. Characteristic Data of Compounds 13

Acerolanin A (1). Yellow powder; UV in CHCl3 λmax (log ε): 444 (4.86), 326 (4.98), 239 (4.88). IR (KBr) vmax 3450, 1680, 1601, 1311, 1236 cm−1; 1H- and 13C-NMR data: see Table 1; positive ESI-MS: m/z 293 [M + Na]+; positive HREIMS: m/z 270.0900 [M]+ (calcd for C16H14O4, 270.0892).

Acerolanin B (2). Light yellow powder; UV in CHCl3 λmax (log ε): 345 (4.52), 275 (4.51), 230 (4.23). IR (KBr) vmax 3461, 1683, 1637, 1597, 1383, 1270, 1247 cm−1; 1H- and 13C-NMR data: see Table 1; positive ESI-MS: m/z 307 [M + Na]+; positive HREIMS: m/z 284.1056 [M]+ (calcd for C17H16O4, 284.1049).

Acerolanin C (3). Colorless Monoclinic crystals from CHCl3/MeOH (1:3); UV in CHCl3 λmax (log ε): 389 (3.98), 274 (4.11), 239 (4.06). IR (KBr) vmax 1755, 1682, 1570, 1463, 1231 cm−1; 1H- and 13C-NMR data: see Table 1; positive ESI-MS: m/z 291 [M + Na]+; positive HREIMS: m/z 268.1097 [M]+ (calcd for C17H16O3, 268.1099).

Crystal data for 3: C17H16O3, M = 268.30, monoclinic, a = 8.8643(9) Å, b = 13.7477(14) Å, c = 11.3957(12) Å, α = 90.00°, β = 104.141(2)°, γ = 90.00°, V = 1346.6(2) Å3, T = 100(2) K, space group P21/c, Z = 4, μ(MoKα) = 0.090 mm−1, 14026 reflections measured, 3824 independent reflections (Rint = 0.0282). The final R1 values were 0.0423 (I > 2σ(I)). The final wR(F2) values were 0.1020 (I > 2σ(I)). The final R1 values were 0.0620 (all data). The final wR(F2) values were 0.1157 (all data). The goodness of fit on F2 was 1.026. The crystal structure of 3 was solved by direct method SHELXS-97 (Sheldrich, G.M. University of Gottingen: Gottingen, Germany, 1997) and the full-matrix least-squares calculations. Crystallographic data for the structure of 2 have been deposited with the Cambridge Crystallographic Data Centre (deposition number: CCDC 939851). CCDC 939851 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via the Internet at www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk.

3.5. Cytotoxicity Assay

The cytotoxicity of compounds 13 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% CO2. 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 × 105 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 (%) = (ODsample/ODcontrol) × 100%. The IC50 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).

4. Conclusions

Phytochemical study of the aerial parts of acerola (M. emarginata) has resulted in the isolation of three new degraded diterpenes 13. As far as we know, this is the first report of this class of degraded diterpenes from the Malpighiaceae family. In addition, compounds 13 showed cytotoxic activities.

Supplementary Materials

1D and 2D NMR spectra of compounds 13 (S1–S15) are available as Supporting Information, Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/2/2629/s1.

Acknowledgments

The project was financially supported by the General Program of NSFC (No. 81172940) and Knowledge Innovation Program of the CAS (Grant No. KSCX2-YW-G-038, KSCX2-YW-R-194), as well as Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ14).

Author Contributions

The isolation and structural elucidations were accomplished by Jie-Qing Liu, Yuan-Yuan Deng, Ting-Zhao Li, Qiang Han and Ming-Hua Qiu. The cytotoxic assay was performed by Yan Li. All the authors had known about this manuscript and had no objection to submitting it.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. De Assis, S.A.; Lima, D.C.; de Faria Oliveira, O.M.M. Activity of pectinmethylesterase, pectin content, and vitamin C in acerola fruit at various stages of fruit development. Food Chem. 2001, 74, 133–137, doi:10.1016/S0308-8146(01)00104-2.
  2. Galvao de Lima, V.L.A.; Pinheiro, I.O.; Silva do Nascimento, M.; Gomes, P.B.; Guerra, N.B. Identification of anthocyanidins in acerola fruits from Active Germplasm Bank of the Rural Federal University of Pernambuco. Food Sci. Technol. 2006, 26, 927–935.
  3. Vera de Rosso, V.; Hillebrand, S.; Montilla, E.C.; Bobbio, F.O.; Winterhalter, P.; Mercadante, A.Z. Determination of anthocyanins from acerola (Malpighia emarginata DC.) and acai (Euterpe oleracea Mart.) by HPLC-PDA-MS/MS. J. Food Compos. Anal. 2008, 21, 291–299, doi:10.1016/j.jfca.2008.01.001.
  4. Vendramini, A.L.A.; Trugo, L.C. Phenolic compounds in acerola fruit (Malpighia punicifolia, L.). J. Braz. Chem. Soc. 2004, 15, 664–668, doi:10.1590/S0103-50532004000500009.
  5. Hanamura, T.; Hagiwara, T.; Kawagishi, H. Structural and functional characterization of polyphenols isolated from acerola (Malpighia emarginata DC.) fruit. Biosci. Biotechnol. Biochem.  2005, 69, 280–286, doi:10.1271/bbb.69.280.
  6. Rufino, M.S.M.; Fernandes, F.A.N.; Alves, R.E.; de Brito, E.S. Free radical-scavenging behaviour of some north-east Brazilian fruits in a DPPH system. Food Chem. 2009, 114, 693–695, doi:10.1016/j.foodchem.2008.09.098.
  7. Delva, L.; Goodrich-Schneider, R. Antioxidant activity and antimicrobial properties of phenolic extracts from acerola (Malpighia emarginata DC) fruit. Int. J. Food Sci. Tech. 2013, 48, 1048–1056, doi:10.1111/ijfs.12061.
  8. Nagamine, I.; Fujita, M.; Hongo, I.; Nguyen, H.T.T.; Miyahara, M.; Parkanyiova, J.; Pokorny, J.; Dostalova, J.; Sakurai, H. Hepatoprotective effects of acerola cherry extract powder against d-galactosamine-induced liver injury in rats and its bioactive compounds. Czech J. Food Sci. 2004, 22, 159–162.
  9. Hanamura, T.; Mayama, C.; Aoki, H.; Hirayama, Y.; Shimizu, M. Antihyperglycemic effect of polyphenols from acerola (Malpighia emarginata DC.) fruit. Biosci. Biotechnol. Biochem. 2006, 70, 1813–1820, doi:10.1271/bbb.50592.
  10. Liu, J.-Q.; Peng, X.-R.; Li, X.-Y.; Li, T.-Z.; Zhang, W.-M.; Shi, L.; Han, J.; Qiu, M.-H. Norfriedelins A–C with acetylcholinesterase inhibitory activity from acerola tree (Malpighia emarginata). Org. Lett. 2013, 15, 1580–1583, doi:10.1021/ol4003702.
  11. Seephonkai, P.; Pyne, S.G.; Willis, A.C.; Lie, W. Fimbricalyx A, a novel phenanthrenone derivative having a rare 2H-benz[e]inden-2-one substructure. Tetrahedron Lett. 2013, 54, 2085–2088, doi:10.1016/j.tetlet.2013.02.025.
  12. Tchinda, A.T.; Tsopmo, A.; Tene, M.; Kamnaing, P.; Ngnokam, D.; Tane, P.; Ayafor, J.F.; Connolly, J.D.; Farrugia, L.J. Diterpenoids from Neoboutonia glabrescens (Euphorbiaceae). Phytochemistry 2003, 64, 575–581, doi:10.1016/S0031-9422(03)00158-4.
  13. Tene, M.; Tane, P.; de Dieu Tamokou, T.; Kuiate, J.-R.; Connolly, J.D. Degraded diterpenoids from the stem bark of Neoboutonia mannii. Phytochem. Lett. 2008, 1, 120–124, doi:10.1016/j.phytol.2008.06.003.
  14. Tang, G.-H.; Zhang, Y.; Yuan, C.-M.; Li, Y.; Gu, Y.-C.; Di, Y.-T.; Wang, Y.-H.; Zuo, G.-Y.; Li, S.-F.; Li, S.-L.; et al. Trigohowilols A–G, degraded diterpenoids from the stems of Trigonostemon howii. J. Nat. Prod. 2012, 75, 1962–1966, doi:10.1021/np3006315.
  • Sample Availability: Samples of the compounds 13 are available from the authors.
Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert