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A new N-oxide benzylisoquinoline alkaloid isolated from the leaves of atemoya (Annona cherimola × Annona squamosa)

  • Suzana V. Rabêlo , Edigênia C. C. Araújo , Emmanoel V. Costa , Raimundo Braz-Filho , Andersson Barison , Maria de F. C. Santos , Gibson G. Oliveira , José C. Tomaz , Larissa A. Rolim , Norberto P. Lopes , Maria F. S. Silva , Manoel O. Moraes , Cláudia do Ó Pessoa , Noureddine El Aouad and Jackson R. G. S. Almeida EMAIL logo
Published/Copyright: June 17, 2021
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Zeitschrift für Naturforschung B
From the journal Zeitschrift für Naturforschung B Volume 76 Issue 6-7

Abstract

Phytochemical investigation of the atemoya aerial parts was carried out by LC-MS-IT and cytotoxic activities were evaluated as well. These results led to the identification of a new N-oxide alkaloid (dehydroanomuricine-N-oxide) and eight other alkaloids: scoulerine, reticuline, isocorydine, norisocorydine, asimilobine, nornuciferine, anonaine, and liriodenine. The new alkaloid dehydroanomuricine-N-oxide and anomuricine were also isolated. The structures of these compounds were determined by spectroscopic and spectrometric techniques. The cytotoxic capacity of crude methanolic extract and the alkaloidal fraction were evaluated, showing moderate cytotoxicity. The isolation and identification of these alkaloids are an important contribution to the chemotaxonomy of the genus Annona and the Annonaceae family.

Keywords: anomuricine; cytotoxicity; LC-MS; NMR; N-oxide

1 Introduction

Annona is a genus of flowering plants in the pawpaw/sugar apple family (Annonaceae). It is the second largest genus in the family after Guatteria. This genus is represented by 175 species and is the main genus of the Annonaceae family [1], which is well known to produce alkaloids as main secondary metabolites. They are considered chemotaxonomic markers of the genus, with very common alkaloids such as anonaine, asimilobine, liriodenine, and reticuline [2]. Annona atemoya Mabb. commonly named Atemoya, is a hybrid resulting from mixing of two species of the genus Annona: cherimoya (Annona cherimola Mill) and sugar apple (Annona squamosa L.) [3]. Previous phytochemical studies investigated alkaloids in atemoya seeds and leaves. Thus, atemoine and cleistofoline were identified in seeds [4], while the aporphines anonaine and asimilobine, the oxoaporphines lanuginosine, liriodenine, lysicamine, the proaporphines pronuciferine, and stepharine were identified in the leaves [5]. The chemical composition of essential oil from fruits and hexane crude extract from aerial parts as well as the cytotoxic and antimicrobial activity were also investigated [6].

Nowadays, hyphenated techniques have been widely used in phytochemical studies as they allow a quick and sensitive identification of the chemical constituents. These analyses, coupled with in vitro biological activity evaluations, provide fast and targeted results in the search for bioactive compounds.

Therefore, this work reports the results of a phytochemical investigation and cytotoxic potential of aerial parts of atemoya, with a detailed description of the identification process of a new N-oxide benzylisoquinoline alkaloid (6,7-dimethoxy-1-[(4-methoxyphenyl)methyl]-2-oxo-3,4-dihydro-2λ5-isoquinolin-5-ol, named dehydroanomuricine-N-oxide) and 1D and 2D NMR data attributed to the benzyltetrahydroisoquinoline alkaloid anomuricine.

2 Experimental section

2.1 Plant material

Aerial parts of atemoya (A. cherimola Mill. × A. squamosa L.) were collected in July 2013 and September 2014, in Petrolina, state of Pernambuco, Brazil. This plant was identified by Prof. José Alves de Siqueira Filho, botanist of the Federal University of San Francisco Valley, Petrolina, Pernambuco, Brazil. A voucher specimen (16310) was deposited in the Herbário Vale do São Francisco (HVASF) of the Federal University of San Francisco Valley (UNIVASF). All procedures for access to genetic patrimony and associated traditional knowledge were carried out and the project was registered in SisGen (#ABD9AA7).

2.2 Extraction, fractionation, and TLC evaluation

The aerial parts of atemoya were dried, powdered, and 2200 g of this sample were extracted with hexane (3 L, three times), then with methanol (3 L, three times), yielding 84.1 and 8.5 g of hexane and methanolic extracts, respectively. The extracts were concentrated under vacuum. The methanol extract was submitted to an acid–base extraction to obtain an alkaloid and a neutral fraction. The total alkaloidal fraction (FAT) was then submitted to a solid–liquid partition with hexane (Hex), chloroform (CHCl3), ethyl acetate (EtOAc), and methanol (MeOH), using a silica gel previously treated with an aqueous solution of NaHCO3, giving the fractions FAT-Hex, FAT-CHCl3, FAT-AcOEt, and FAT-MeOH.

The dried and powdered leaves of atemoya (1163 g) were extracted with hexane and MeOH at the same conditions as described above, yielding 63 and 120 g of hexane and MeOH extracts, respectively. The MeOH extract was submitted to an acid–base extraction to obtain an alkaloidal fraction (1.5 g), which was submitted to a silica gel column chromatography (CC) previously treated with a 10% NaHCO3 solution, and eluted with increasing concentrations of hexane, dichloromethane, ethyl acetate, and methanol, giving 297 fractions (40 mL each). These fractions were evaluated and pooled to smaller groups according to a TLC analysis, yielding 18 groups. Group 7 (66.9 mg) was submitted to a preparative TLC eluted with CH2Cl2–MeOH (95:05, v/v), giving compounds 7 (1.3 mg) and 9 (9.3 mg). Silica gel 60 (230–240 mesh) was used for CC. Silica gel 60 (F254) was used for analytical thin-layer chromatography (TLC). Spots on chromatograms were detected under exposure to UV light (254 and 365 nm). When necessary, Dragendorff’s reagent was used to visualize the spots on the TLC plates.

2.3 LC-MS procedures

The FAT and the fractions FAT-Hex, FAT-CHCl3, FAT-AcOEt, and FAT-MeOH, obtained from FAT, were solubilized in methanol (HPLC grade) at a concentration of 1 mg mL−1 and were individually analyzed by LC-MS using an apparatus type Shimadzu® LC-20 equipped with a quaternary pump system LC-20ADVP model, DGU-20A degasser, CTO-20ASVP model oven, and SIL-20ADVP model automatic injector. An octadecylsilane column (250 × 4.6 mm, 5 µm, Luna® C18, Phenomenex®) was used as the stationary phase and two solvents were used as the mobile phase, consisting of solvent A: 0.1% formic acid in ultrapure water and solvent B: 0.1% formic acid in methanol (HPLC grade), with a flow rate of 1.0 mL min−1, using the following linear gradient over a total run time of 90 min: 10–20% solvent B in 60 min, 100% solvent B maintained until completion of the run (i.e., 20 min), and linearly returning to 10% solvent B in 5 min, 10% solvent B maintained for 5 min. The stationary phase was maintained at 30 °C, the injection volume was 20 μL for the samples in the HPLC-DAD-MS, and the spectra were monitored between 190 and 400 nm and m/z = 50 to 1000. The analyzes were developed with a PDA detector model SPD-20AVP and an SCL-20AVP model controller coupled to an ESI-IT mass spectrometer from Bruker Daltonics, equipped with an electrospray ionization source operating in the analyzer mode, and by trapping positive ions to divide the HPLC eluent, a flow rate of 0.2 mL min−1 being introduced to the source. The parameters used for the mass spectrometer were capillary voltage of 3.5 kV; desolvation temperature 330 °C; gas flow of 10 L min−1; a pressure of 60 psi using nitrogen as drying gas [7].

The fragmentation of compounds 7 and 9 was done in the positive ion mode on a Bruker Daltonics TOF-Q-II Mass Spectrometer with a capillary voltage of 3.5 kV with nitrogen as a vector gas (4 L min−1), a pressure of 0.4 Bar, and a temperature of 180 °C. The equipment was calibrated with NaTFA (200 μg mL−1). The sample was solubilized in methanol at a concentration of 100 μg mL−1, acidified with formic acid and directly injected into the mass spectrometer.

2.4 Chemical characterization by NMR

The NMR data were acquired at T = 293 K in CDCl3 on a Bruker AVANCE III 600 NMR spectrometer operating at 14.1 T, observing 1H and 13C at 600 and 150 MHz, respectively. The spectrometer was equipped with a 5 mm multinuclear inverse detection probe with z gradient. One-bond and long-range 1H–13C correlation from HSQC and HMBC NMR experiments were optimized for average coupling constants 1J(C,H) and LRJ(C,H) of 140 and 8 Hz, respectively. All 1H and 13C NMR chemical shifts were given in ppm related to the TMS signal at 0.0 ppm as internal reference, and the coupling constants (J) are given in Hz.

2.5 Cytotoxic activity

2.5.1 Cytotoxic activity of extracts and fractions

The cytotoxic activity was evaluated using the MTT method as described in ref. [8]. This technique has the capacity to detect the viability and the metabolic activity of the cell. It is a colorimetric analysis based on the conversion of the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide salt (MTT) in blue formazan by mitochondrial enzymes present only in metabolically active cells. The cytotoxic study with the MTT method allows to easily defining cytotoxicity, but not the mechanism of action [9]. For this, the cells were plated at 0.07, 0.1, and 0.3 × 106 cells mL−1 concentrations for the HCT-116 (human colon carcinoma), SF295 (human glioblastoma) and HL60 (promyelocytic leukemia) cell lines, respectively. The plates were incubated with the samples for 72 h at 37 °C and 5% of CO2. At the end, the cells were centrifuged and the supernatant removed. Then, 150 μL of the MTT (tetrazolium salt) solution were added and the plates were incubated for 3 h. After incubation, the plates were centrifuged again for the removal of the MTT solvent. The absorbance was read with a spectrophotometer at 595 nm after dissolution of the formazan precipitate with 150 μL of pure dimethylsulfoxide (DMSO). The single-concentration experiments were analyzed according to the mean ± standard deviation (SD) of the percentage of cell growth inhibition using statistical analysis software. All analyses were performed using GraphPad Prism® 5.0 (Graph Pad Prism Software, Inc., San Diego, CA, USA).

2.5.2 Cytotoxic activity of anomuricine

Anomuricine was diluted in sterile pure DMSO at 50 μg mL−1. The tumor cell lines were provided by the National Cancer Institute (Bethesda, Maryland, USA) and were maintained in RPMI 1640, supplemented with 10% fetal bovine serum and 1% antibiotics. They were incubated at 37 °C and 5% CO2. The cell lines used were plated at concentrations of 0.1 × 106 cells mL−1 for the cell lines PC3 (prostate carcinoma), PC-3M (metastasis-derived variant of PC3), PC9 (lung adenocarcinoma), COLO 205 (colorectal adenocarcinoma), SW620 (colorectal adenocarcinoma), B16F10 (skin carcinoma), and SF-295 (human glioblastoma). Concentrations of 0.3 × 106 and 0.7 × 105 cells mL−1 were applied for the HCT-116 (human colon carcinoma) and cell line HL60 (promyelocytic leukemia), respectively. The plates were incubated for 72 h at 5% of CO2 and 37 °C. At the end of the treatment, MTT (tetrazolium salt) was added and incubated for 3 h. The absorbance was measured at 595 nm. DMSO and doxorubicin were used as negative and positive controls, respectively.

2.6 Spectroscopic data

2.6.1 Spectroscopic data of anomuricine (7)

Brown amorphous solid. – 1H NMR (600 MHz, CDCl3, 20 °C, TMS): δ 2.67 (t, 2H, J = 6.0 Hz, H-4), 2.92 (dd, 1H, J = 13.8 and 9.2 Hz, H-N), 2.94 (dt, 1H, J = 12.1 and 6.0 Hz, H-3), 3.13 (dd, 2H, J = 13.8 and 4.6 Hz, H-9), 3.21 (dt, 1H, J = 12.1 and 6.0 Hz, H-3), 3.78 (s, 3H, OCH3-7), 3.83 (s, 3H, OCH3-13), 3.91 (s, 3H, OCH3-6), 4.11 (dd, 1H, J = 9.2 and 4.6 Hz, H-1), 6.24 (s, 1H, H-8), 6.86 (d, 2H, J = 8.5 Hz, H-12, and H-14), 7.16 (d, 2H, J = 8.5 Hz, H-11, and H-15). – 13C NMR (150 MHz, CDCl3): δ 23.0 (C-4), 39.7 (C-3), 41.2 (C-9), 55.2 (OCH3-13), 55.8 (OCH3-7), 56.9 (C-1), 60.9 (OCH3-6), 101.5 (C-8), 114.0 (C-12 and C-14), 114.9 (C-4a), 130.3 (C-11 and C-15), 130.6 (C-10), 133.7 (C-6), 133.8 (C-8a), 146.8 (C-5), 150.0 (C-7), 158.3 (C-13). – HRMS ((+)-ESI): m/z 330.1665 (C19H24NO4 [M+H]+).

2.6.2 Spectroscopic data of dehydroanomuricine-N-oxide (9)

Yellowish powder. – 1H NMR (600 MHz, CDCl3, 20 °C, TMS): δ 3.07 (t, 2H, J = 7.7 Hz, H-4), 3.76 (s, 6H, OCH3-13 and OCH3-7), 3.90 (s, 3H, OCH3-6), 4.16 (t, 2H, J = 7.7 Hz, H-3), 4.26 (s, 2H, H-9), 6.51 (s, 1H, H-8), 6.81 (d, 2H, J = 8.6 Hz, H-12 and H-14), 7.23 (d, 2H, J = 8.5 Hz, H-11 and H-15). – 13C NMR (150 MHz, CDCl3): δ 21.2 (C-4), 58.8 (C-3), 32.1 (C-9), 55.9 (OCH3-13), 56.0 (OCH3-7), 61.3 (OCH3-6), 101.4 (C-8), 111.4 (C-4a), 114.8 (C-12 and C-14), 126.0 (C-8a), 129.7 (C-10), 130.1 (C-11 and C-15), 136.6 (C-6), 144.5 (C-1), 146.4 (C-5), 151.6 (C-7), 158.9 (C-13). – HRMS ((+)-ESI): m/z 344.1559 (C19H22NO5 [M+H]+).

Chemical structures with numeration, MS, and NMR spectra of compounds 7 and 9 are available as Supporting Information (Figures S1–S23).

3 Results and discussion

3.1 Chemical analysis

The analysis of the chromatogram of the fractions (FAT-Hex, FAT-CHCl3, FAT-AcOEt, and FAT-MeOH) obtained from FAT from atemoya×s aerial parts (FAT) indicated the presence of 11 compounds (Figures 1 and 2). The chromatography-mass spectrometry-ion trap (LC-MS-IT) analysis led to the identification of the benzylisoquinoline derivatives scoulerine (1), reticuline (2), anomuricine (7) and the new N-oxide benzylisoquinoline, here named dehydroanomuricine-N-oxide (9). Further, the aporphines isocorydine (3), norisocorydine (4), asimilobine (5), nornuciferine (6) and anonaine (8) and the oxoaporphines liriodenine (10), and lanuginosine (11) were identified.

Figure 1: Chromatograms of (a) total alkaloid fraction (FAT), (b) hexane (FAT-Hex), (c) chloroform (FAT-CHCl3), (d) ethyl acetate (FAT-AcOEt) and (e) methanolic (FAT-MeOH) fractions obtained from FAT from atemoya’s aerial parts.
Figure 1:

Chromatograms of (a) total alkaloid fraction (FAT), (b) hexane (FAT-Hex), (c) chloroform (FAT-CHCl3), (d) ethyl acetate (FAT-AcOEt) and (e) methanolic (FAT-MeOH) fractions obtained from FAT from atemoya’s aerial parts.

Figure 2: Alkaloids identified in the alkaloidal fraction of atemoya.
Figure 2:

Alkaloids identified in the alkaloidal fraction of atemoya.

The confirmation was made based on the precursor ion, the fragmentation profile of the chemical constituents, and comparison with literature data. Table 1 shows the alkaloids identified in FAT and in its fractions.

Table 1:

LC-MS data of alkaloids from total alkaloid fraction (FAT) and fractions from atemoya.a

Compound identified in FAT (Nº)Molecular formulaRT+MS+MS2–MS–MS2Sample
FAT-HexFAT-CHCl3FAT-AcOEtFAT-MeOH
Scoulerine (1)C19H21NO414.0328.15300.13

178.02		326.20312.15

301.20

248.96

209.86		––++
Reticuline (2)C19H23NO416.7330.16299.08

192.03		328.22313.05

175.92

135.94

121.94		––++
Isocorydine (3)C20H23NO418.3342.17311.13

279.13

165.02		––++
Norisocorydine (4)C19H21NO418.6328.13311.13

279.11		324.19––++–
Asimilobine (5)C17H17NO221.4268.04251.07

219.01		––++++
Nornuciferine (6)C18H19NO223.8282.09265.08280.07252.04–+++
Anomuricine (7)C19H23NO425.5330.18313.14–––++–
Anonaine (8)C17H15NO229.0266.12249.06––++––
Dehydroanomuricine-N-oxide (9)C19H21NO530.7344.15327.22

311.16

279.06		––++––
Liriodenine (10)C17H9NO336.1276.04–––+–++
Lanuginosine (11)C18H11NO441.4306.11–––+++–

  1. aFAT-Hex, hexane fraction; FAT-CHCl3, chloroformic fraction; FAT-AcOEt, ethyl acetate fraction; FAT-MeOH, methanolic fraction.

The leaves extract led to the separation of the group 7 of the fractions (see above). This group was submitted to a preparative TLC, giving compounds 7 and 9. These compounds were identified by their 1D and 2D NMR spectra and mass spectrometry data as is shown in the experimental section (Section 2.6).

Asimilobine (5), anonaine (8), liriodenine (10), and lanuginosine (11) had already been identified in the leaves of atemoya in a study previously conducted by our research group [5]. In this work, we identified other compounds, such as scoulerine (1), reticuline (2), isocorydine (3), norisocorydine (4), nornuciferine (6), anomuricine (7), and dehydroanomuricine-N-oxide (9) in this plant. The new N-oxide alkaloid dehydroanomuricine-N-oxide (9) is an unprecedented compound and its chemical characterization, to the best of our knowledge, is described for the first time in this work.

The compound 7 was obtained as a brown amorphous solid with molecular formula C19H23NO4, determined by HR-ESI-MS (m/z 330.1665 [M+H]+). The mass spectrum (see Supporting Information, Figure S1) showed ionic fragments similar to those described in the literature [10].

The 1H NMR spectrum revealed the presence of two spin systems, one consisting of the signals at δ 2.94 (dt, 1H, J = 12.1 and 6.0 Hz, H-3) and 3.21 (dt, 1H, J = 12.1 and 6.0 Hz, H-3), as well as 2.67 (t, 2H, J = 6.0 Hz, H-4) and other comprising the signals at δ 2.92 (dd, 1H, J = 13.8 and 9.2 Hz, H-N), 3.13 (dd, 2H, J = 13.8 and 4.6 Hz, H-9) and 4.11 (dd, 1H, J = 9.2 and 4.6 Hz, H-1) (Figure S2). These signals are characteristic of a benzyltetrahydroisoquinoline alkaloid. Singlets corresponding to methoxyl groups at δ 3.91 (s, 3H, OCH3-6), 3.78 (s, 3H, OCH3-7), 3.83 (s, 3H, OCH3-13) and one at 6.24 (s, 1H, H-8) corresponding to an aromatic hydrogen were also observed. The signals at δ 7.16 (d, 2H, J = 8.5 Hz, H-11 and H-15) and 6.86 (d, 2H, J = 8.5 Hz, H-12 and H-14) indicated the presence of a p-substituted benzene ring in the structure.

The respective location of the methoxyl groups at the carbons C-6, C-7, and C-13 was defined based on the 1D and 2D NMR data (Figures S2–S7). The HSQC correlation map (Figure S6) allowed establishing the one-bond (1JCH) correlation. The long-range 1H–13C HMBC correlation experiment (Figure S7) allowed observing the correlation of the aromatic hydrogen at δ 6.24 with the carbons at δ 56.9 (C-1), 114.9 (C-4a), 133.7 (C-6) at three bonds (3JHC), and two bonds (2JHC) with the carbon at δ 150.0 (C-7), supporting the assignments the hydrogen in C-8. Likewise, the signal at δ 4.11 showed long-range 1H–13C correlation with the carbons at δ 41.2 (C-9), 101.5 (C-8), 114.9 (C-4a), and 133.8 (C-8a). Correlations between the methylene hydrogen H-9 at δ 3.13 with the carbons in δ 56.9 (C-1) and 130.3 (C-11/C-15) were also observed.

The carbon atoms at δ 23.0 (C-4), 39.7 (C-3), 41.2 (C-9), and 56.9 (C-1) in combination with the other signals confirmed the presence of a benzyltetrahydroisoquinoline alkaloid (Figure S3). The AA′BB′ system was also observed with the signals in δ 114.0 (C-12 and C-14) and 130.3 (C-11 and C-15), which suggested a p-substituted benzene ring. In addition, three signals corresponding to methoxyl carbons were observed at δ 60.9 (H3CO-6), 55.8 (H3CO-7), and 55.2 (H3CO-13). The overall analysis of 1D and 2D NMR experiments enabled the complete and unambiguous attribution of 1H and 13C NMR chemical shifts of compound 7.

Comparison with the literature data allowed us to identify compound 7 as the alkaloid 6,7-dimethoxy-1-(4′-methoxybenzyl)-1,2,3,4-tetrahydroisoquinoline-5-ol, known as anomuricine, isolated for the first time from Annona muricata stem [11]. It is the first time that this molecule is isolated from atemoya leaves and the data of 1D and 2D NMR are pointed out unequivocally in this work.

The new alkaloid 9 was obtained as a yellowish powder with the molecular formula, C19H21NO5, as determined by HR-ESI-MS (m/z 344.1559 [M+H]+). The mass spectrum (Figure S8) showed ionic fragments for this compound.

The NMR data for this compound were very similar to those observed for anomuricine, except by the signal at δ 56.91 (C-1) in the 13C NMR spectrum which was replaced by the signal at δ 144.5, indicating that the methine group was replaced by a quaternary carbon at C-1. This information was supported by the lack of double doublets at δ 4.11 (see experimental Section 2.6).

The 1H NMR spectra (Figures S9–S11) revealed the presence of signals at δ 3.07 (t, 2H, J = 7.7 Hz, H-4) and δ 4.16 (t, 2H, J = 7.7 Hz, H-3), two singlets attributed to methoxy groups at δ 3.76 (s, 6H, OCH3-7 and OCH3-13) and δ 3.90 (s, 3H, OCH3-6), and other signal at δ 4.26 (s, 2H, H-9) characteristic for the presence of a benzyltetrahydroisoquinoline alkaloid.

The 1H NMR spectrum of the compound displays three signals characteristic of an aromatic hydrogen atom, one at δ 6.51 ppm (s, 1H, H-8), shielded by oxygenation in the ortho and para positions, two doublets at δ 6.81 (d, 2H, H-12 and H-14) and δ 7.23 (d, 2H, H-11 and H-15) showed spin-spin interaction in ortho (J = 8.6 Hz), which indicates a p-substituted benzene ring, typically of an AA’BB’ spin system [12].

The complete assignments of the 1H and 13C NMR chemical shifts were established based on 1H–1H correlation map COSY, 1D NOE, and one-bond and long-range 1H–13C correlation maps from HSQC and HMBC experiments (Figures S12–S23). The singlet at δ 4.26 (2H, H-9) showed long-range 1H–13C correlation with the carbons at δ 126.0 (C-8a), 129.7 (C-10), 130.1 (C-11 and C-15), and 144.5 (C-1). The signal at δ 6.51 (1H, H-8) displayed correlation with the carbons at δ 111.4 (C-4a), 136.6 (C-6), 144.5 (C-1), and 151.6 (C-7). The presence of a hydroxyl group in the molecule located in the A ring at C-5 was established based on long-range 1H–13C correlation of the hydrogen H-4 with the carbon δ 146.4, which showed no correlation with the methoxyl groups. However, the singlets at δ 3.76 and 3.90 showed long-range correlation with the carbons at δ 151.6 (C-7) and 136.6 (C-6), respectively, indicating the presence of the two methoxyl groups in the benzene ring.

The p-substituted benzene ring was established in C ring based on direct 1H–13C correlation of hydrogens at δ 7.23 (2H, H-11, and H-15) and 6.81 (2H, H-12, and H-14) with the carbons at δ 114.8 and 130.1, respectively, and long-range 1H–13C correlation with the carbon δ 158.9 supporting the presence of methoxyl group in C-13.

The correlation map of HSQC NMR experiment allowed establishing the direct correlations (1JCH), while the long-range 1H–13C correlation HMBC NMR experiment permitted to establish the location of substituents in the molecule, as well as the methylene carbons C-9, C-4, and the N-linked C-3.

The presence of N-oxide group in the molecule was supported by mass spectrum that revealed a molecular ion m/z 344.1559 (Figure S8). The overall analysis of 1D and 2D NMR experiments enabled the complete and unambiguous assignments of 1H and 13C NMR chemical shifts. These data allowed identifying the compound as 6,7-dimethoxy-1-[(4-methoxyphenyl)methyl]-2-oxo-3,4-dihydro-2λ5-isoquinolin-5-ol, named dehydroanomuricine-N-oxide. 1D-NOE NMR experiments supported the proposed structure.

3.2 Cytotoxic activity

The crude extracts from aerial parts of atemoya were evaluated in vitro against three cancer cell lines: HL-60 (leukemia), HCT-116 (human colon), and SF295 (human glioblastoma). The results showed that the most significant activity was observed toward SF295; the crude hexane extract (FAT-Hex) was highly active with a cell growth inhibition of 97.97% (Table 2).

Table 2:

Mean percentage of cell growth inhibition (CI) of the samples at the single concentration of 50 μg mL−1.

SampleHCT116SDaSF295SDaHL60SDa
CI (%) (mean)CI (%) (mean)CI (%) (mean)
EMB65.773.0186.212.6254.238.72
FAT95.680.2595.652.0595.540.35
FAT-Hex96.980.5997.971.0797.240.92
FAT-CHCl395.320.2587.491.1596.640.50
FAT-AcOEt79.811.4264.081.6490.072.69
FAT-MeOH54.584.9474.747.5444.304.18

  1. aSD, standard deviation. EMB, crude methanolic extract; FAT, total alkaloid fraction; FAT-Hex, hexane fraction; FAT-CHCl3, chloroformic fraction; FAT-AcOEt, ethyl acetate fraction; FAT-MeOH, methanolic fraction; HCT-116, human colon; SF295, human glioblastoma; HL-60, leukemia.

This is not the first time that Annona extracts were tested against cancer cells. Species of this genus are known for having cytotoxicity in a previous study [13], where the extracts from roots of Annona crassiflora showed cytotoxicity against SF95 and HL60 cells. A recent study [14] presented some of the possible in vitro anticancer mechanisms of compounds extracted from A. muricata. The focus of this work was on a group of important secondary metabolites of the Annonaceae family, the acetogenins. However, another class of molecules of this family (alkaloids) is very important in the search for new anticancer medicines. Some work done on alkaloids found in Annonaceae species showed that this compounds presented high cytotoxic activity [15], [16], [17], [18].

To date, a total of 21,685 research publications have studied the alkaloid cytotoxicity. Of these, 411 are related to Annona alkaloids; and 82 of them were studied only between 2017 and 2018. These results show the growing interest in this topic. For this reason, the alkaloidal fraction (FAT) from the crude methanolic extract (EMB) of atemoya was tested, and its hexane, chloroform, ethyl acetate, and methanolic fractions were also tested.

The results presented in Table 2 show that the cytotoxic potential of FAT was higher than 95% for all tested cells, being slightly higher in the hexane and chloroform fractions, and reduced in the ethyl acetate and methanol fractions. Many of the alkaloids identified in the active fractions from atemoya have already been studied for cytotoxicity, such as anonaine and asimilobine [19], and isocorydine and reticuline [20], [21]. Nornuciferine still showed moderate activity against KB cells and was considered an important chemical constituent of the cytotoxically active alkaloid fraction from Annona hypoglauca [22], [23].

In this study, anomuricine (7) was tested against nine cancer cell and the detailed results are summarized in Table 3. Anomuricine showed moderate cytotoxic activity against all evaluated cell lines. The IC50 had large cytotoxic activity against the leukemic cell HL60 with 41.03 µm, for lines of human colon with IC50 values of 78.75, 87.90, and 110.73 µm for SW620, COLO 205, and HCT-116, respectively.

Table 3:

IC50 and selectivity index values for anomuricine.a

Cell lineIC50 (μm)

(interval)		Selectivity index
SF-295 (Glioblastoma)96.26 (71.0–130.49)0.71
PC3 (Prostate)114.62 (75.94–172.98)0.59
HL60 (Leukemia)41.03 (29.42–57.2)1.66
HCT-116 (Colon)110.73 (76.82–160.64)0.61
PC9 (Lung)109.91 (98.33–123.25)0.62
B16F10 (Skin)105.07 (98.27–116.81)0.64
COLO 205 (Colorectal)87.90 (72.46–106.63)0.77
SW620 (Colorectal)78.75 (68.51–90.55)0.86
PC3M (Prostate)>152ND
L929 (Fibroblast)67.99 (59.33–78.21)–

  1. aSelectivity index values IC50 (non-tumor cells)/IC50 (tumor cell). ND = not defined.

The selectivity index (SI) indicates the selectivity of a compound between a neoplastic lineage and a normal lineage, suggesting the potential of its use for future clinical trials. The calculation of this index corresponds to the division between the IC50 values of each test compound in the L929 non-tumor cell line and the neoplastic cell line (SI = IC50 (L929)/IC50) (neoplastic cells) (Table 3).

SI values ≤2.0 indicate that the activity is twice as high in the neoplastic cell line than in non-tumor cells [24]. The SI found for anomuricine ranged from 0.59–1.66, the highest selectivity value being observed with HL60 (leukemia). These results suggest that atemoya can be considered a promising source of substances with cytotoxic potential.

4 Conclusion

This work described the isolation and characterization of a new alkaloid, dehydroanomuricine-N-oxide, and the alkaloids scoulerine, reticuline, anomuricine, isocorydine, norisocorydine, and nornuciferine were reported for the first time in atemoya. These findings contribute to the chemotaxonomy of the Annonaceae family, especially to the genus Annona and show how hyphenated techniques can lead to the rapid discovery of plant compounds. Moreover, atemoya can be considered a promising source of substances useful for cancer treatment.

5 Supporting information

Chemical structures with numeration, MS and NMR spectra of compounds 7 and 9 are available as Supporting Information (Figures S1–S23) (https://doi.org/10.1515/znb-2020-0187).

Corresponding author: Jackson R. G. S. Almeida, Center for Studies and Research of Medicinal Plants (NEPLAME), Federal University of San Francisco Valley, CEP 56.304-205, Petrolina, Pernambuco, Brazil; and Post-Graduate Program in Biotechnology (RENORBIO), Federal Rural University of Pernambuco, Recife, Pernambuco, Brazil, E-mail:

Funding source: CNPq

Award Identifier / Grant number: 470594/2013-6

Award Identifier / Grant number: 442209/2014-2

Award Identifier / Grant number: 303587/2014-8

Funding source: CAPES

Funding source: FACEPE

Funding source: USP

Funding source: UENF

Funding source: UFPR

Funding source: UFAM

Funding source: UNIVASF

Acknowledgments

The authors wish to express their thanks to Centro de Referência para Recuperação de Áreas Degradadas (CRAD) for botanical identification of the plant material.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: The authors are grateful to CNPq (Processes: 470594/2013-6, 442209/2014-2, and 303587/2014-8), CAPES, FACEPE, USP, UENF, UFPR, UFAM, and UNIVASF for financial support, fellowships, and collaboration.

  3. Conflict of interest statement: The authors declare that there are no conflicts of interests and they affirm that this paper consists of original and unpublished work.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/znb-2020-0187).

Received: 2020-11-18
Accepted: 2021-05-14
Published Online: 2021-06-17
Published in Print: 2021-07-27

© 2021 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/znb-2020-0187
Keywords for this article
anomuricine; cytotoxicity; LC-MS; NMR; N-oxide

Articles in the same Issue

  1. Frontmatter
  2. In this issue
  3. Research Articles
  4. Synthesis and structure of the λ6Si-silicate [Cs(18-crown-6)]2[Si(OSO2CH3)6]
  5. Approximate estimation of the critical diameter in Koenen tests
  6. Laves phases forming in the system ScCo2-“InCo2”-TaCo2
  7. Magnesium intermetallics: synthesis and structure of Eu2Pt2Mg and Sr2Pt2Mg
  8. Intermetallic phases in the Sc–Ir–In system – synthesis and structure of Sc1.024Ir2In0.976 and Sc3Ir1.467In4
  9. Crystal structure and physical properties of a new two-dimensional zinc coordination polymer based on 1,4-bis(4-(imidazole-1-yl)benzyl)piperazine and benzophenone-2,4′-dicarboxylate ligands
  10. A new N-oxide benzylisoquinoline alkaloid isolated from the leaves of atemoya (Annona cherimola × Annona squamosa)
  11. One-pot synthesis of 1-(benzothiazolylamino)aryl methyl-2-naphthols and 3-benzothiazolyl 2,3-dihydroquinazolinones using a magnetically recoverable core–shell nanocomposite as catalyst
  12. An efficient click synthesis of chalcones derivatized with two 1-(2-quinolon-4-yl)-1,2,3-triazoles
  13. Facile decoration of CdS nanoparticles on TiO2: robust photocatalytic activity under LED illumination
  14. Book Review
  15. A. Sigel, E. Freisinger and R. K. O. Sigel (Editors and Series Editors): Metal Ions in Bio-Imaging Techniques: Volume 22 of the series Metal Ions in Life Sciences
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