CHEMICAL CONSTITUENTS FROM THE STEM BARK OF Annona pickelii (Annonaceae)

Abstract

The phytochemical investigation of the stem bark of Annona pickelii yielded four steroids (β-sitostenone, β-sitosterol, stigmasterol and campesterol), three lignans (eudesmin, magnolin and yangambin), twelve alkaloids (liriodenine, lysicamine, atherospermidine, anonaine, analobine, asimilobine, discretamine, stepholidine, coclaurine, orientaline, juziphine and stepharine), and a benzenoid (2-methoxybenzoic acid). These compounds support their recent reclassification from Rollinia to Annona, and that it is a typical species of the family Annonaceae. Significant antifungal and antioxidant activities were found for the methanol crude extract as well as for the lignans eudesmin, magnolin, yangambin and the alkaloid discretamine. In addition, several items of NMR data for the alkaloids were reviewed and unequivocally described in this work.

Keywords
Annonaceae; Annona pickelii ; steroids; lignans; alkaloids; NMR

INTRODUCTION

Annona L. belongs to the family Annonaceae and comprises approximately 175 species of trees and shrubs, found predominantly in lowland tropical regions.¹ Economically, this genus is the most important of the family Annonaceae due to its edible fruits and medicinal properties.² Previous chemical and pharmacological investigations on some species of this genus revealed the presence of important bioactive compounds, exhibiting several pharmacological activities, including cytotoxicity against tumor cell lines,^3-5antimicrobial,^6-8 antioxidant,^6-8 antiparasitic properties, mainly against Leishmania sp. and Trypanosoma cruzi,^5-11 and analgesic and anti-inflammatory activities.¹² These activities generally are attributed to alkaloids, acetogenins, and terpenes.

In this context, Annona pickelii (Diels) H. Rainer [synonym: Rollinia pickelii Diels] is a small tree endemic to Brazil, popularly known as 'araticum-do-mato', 'araticum-da-mata', 'jaquirinha-do-mato', and 'jussara' found in Paraíba, Pernambuco and recently in Sergipe, Brazil,¹³ where this species is endemic and commonly found in Atlantic forest remainders. Previous phytochemical and pharmacological investigations on this species described the isolation of terpenes, lignans and alkaloids.^6-11,13,14

In our continuous search for bioactive natural products from Sergipe annonaceous plants, four steroids (1-4), three lignans (5-7), twelve alkaloids (8-19) and one benzenoid (20) (Figure 1) were obtained according to a bio-guided systematic investigation from the stem bark of A. pickelii. Their structures were established on basis of spectrometric data, including 1D (¹H and ¹³C) and 2D (HSQC and HMBC) NMR experiments, as well as 1D NOE and MS analysis. Moreover, many of these compounds were isolated a long time ago, and their ¹H and ¹³C NMR chemical shifts were performed only on basis of general chemical shifts arguments. The NMR chemical shift assignments based on two-dimensional NMR experiments have not been performed. Therefore, their previous ¹³C assignments contain some ambiguities. In the same way, their previous ¹H NMR chemical shifts as well as the scalar coupling constants values have not been specifically assigned and/or obtained. In this work, the complete and unequivocal assignments of ¹H and ¹³C NMR chemical shifts are described for several alkaloids. Additionally some biological activities were demonstrated for the pure compounds. This is the first report on the phytochemical investigation and biological activities of the stem bark of this plant.

Figure 1
Chemical constituents from the stem bark of Annona pickelii

EXPERIMENTAL

General procedures

GC-MS analyses were performed on a Shimadzu QP5050A GC-MS system equipped with an AOC-20i auto-injector, and an Rtx×-5Sil MS fused capillary chromatography column (30 m x 0.25 mm x 0.25 µm film thickness) coated with 5%-diphenyl-95%-dimethylpolysiloxane. MS analysis were taken at 70 eV with a scan interval of 0.5 s and fragments from 40-500 Da. LR-ESI-MS were obtained in the positive ion mode on an ultra-high performance Waters Acquity UHPLC-TQD LC-MS system, equipped with an ESI source. 1D and 2D NMR data were acquired at 303 K in CDCl₃ or CDCl₃ plus some drops of CD₃OD on a Bruker AVANCE III 400 NMR spectrometers, operating at 9.4 Tesla, observing ¹H and ¹³C at 400.13 and 100.61 MHz, respectively. The spectrometer was equipped with a 5-mm multinuclear direct detection probe with z-gradient. One-bond and long-range ¹H-¹³C correlation from HSQC and HMBC NMR experiments were optimized for an average coupling constant ¹J_H,C and ^LRJ_H,C of 140 and 8 Hz, respectively. All ¹H and ¹³C NMR chemical shifts (δ) are given in ppm related to the TMS signal at 0.00 ppm as internal reference, and the coupling constants (J) in Hz. Silica gel 60 (70-230 mesh) was used for column chromatography, while silica gel 60 F₂₅₄ was used for analytical (0.25 mm), and preparative (1.00 mm) TLC. Compounds were visualized by exposure under UV_254/365 light and spraying of p-anisaldehyde reagent followed by heating on a hot plate, and Dragendorff's reagent.

Botanical material

The stem bark of Annona pickelii were collected at 'Mata do Crasto', in the city of Santa Luzia do Itanhy [coordinates: 11º 23' 01" S, 37º 25' 13" W], Sergipe, Brazil, in March 2010. The identity of the plant was confirmed by Dr. A. P. N. Prata, Department of Biology, Sergipe Federal University (UFS), Brazil, and a voucher specimen (#15442) has been deposited in the Herbarium of Sergipe Federal University (ASE/UFS).

Extraction and isolation

Dried at room temperature and powdered stem bark of A. pickelii(790 g) was successively extracted with n-hexane (2.5 L, five times) followed by MeOH (2.5 L, five times), yielding hexane (5.53 g) and MeOH (129.32 g) extracts, after solvent removal under reduce pressure. An aliquot of the hexane extract (5.0 g) was initially subjected to silica gel column chromatography (CC) eluted with increasing concentrations of CH₂Cl₂ in n-hexane (0 to 100, v/v), followed by EtOAc in CH₂Cl₂ (0 to 100, v/v), and MeOH in EtOAc (0 to 70, v/v), affording 177 fractions (25 mL each), that were pooled in 23 groups (G1 to G23), according to TLC analysis. Group G17 (446.0 mg) eluted with CH₂Cl₂-EtOAc (80:20, v/v), was submitted to a new silica gel CC eluted with increasing concentrations of CH₂Cl₂ in n-hexane (0 to 100, v/v) followed by EtOAc in CH₂Cl₂ (0 to 100, v/v), giving 201 fractions (each 20 mL) that were pooled in 31 groups (G17.1 to G17.31), according to TLC analysis. Groups G17.6 to G17.8 were also pooled (213.0 mg) and submitted to preparative TLC eluted with n-hexane-EtOAc (80:20, v/v, two times), affording 1 (5.3 mg) and a mixture of 2, 3 and 4 (48.9 mg). Group G18 (346.0 mg) eluted with CH₂Cl₂-EtOAc (70:30, 60:40 and 50:50, v/v) was submitted to a new silica gel CC eluted with the same eluent system as described for initial CC, yielding 78 fractions (25 mL each), that were subsequently pooled into 14 groups (G18.1 to G18.14). Group G18.10 (76.1 mg) was submitted to preparative TLC eluted with n-hexane-EtOAc (95:05, v/v, four times), giving 5 (4.3 mg), 6 (27.4 mg), and 7(20.1 mg).

TLC investigations indicated a high concentration of alkaloids in the MeOH extract. Therefore, an aliquot of this extract (120.0 g) was initially subjected to an acid-base extraction to give alkaloid (0.68 g) and neutral (3.04 g) fractions.¹⁵ An aliquot of alkaloid fraction (0.60 g) was submitted to silica gel CC previously treated with a 10% NaHCO₃ solution,¹⁵ and eluted with increasing concentrations of CH₂Cl₂ in n-hexane (0 to 100, v/v), followed by EtOAc in CH₂Cl₂ (0 to 100, v/v), and MeOH in EtOAc (0 to 50, v/v), giving 130 fractions (15 mL each), that were pooled in 12 groups (G1 to G12), according to TLC analysis. Group G4 (195.0 mg) eluted with CH₂Cl₂ (100%) and CH₂Cl₂-EtOAc (95:05 and 90:10, v/v) was submitted to a new silica gel CC eluted with increasing concentrations of CH₂Cl₂ in n-hexane (0 to 100, v/v) followed by MeOH in CH₂Cl₂ (0 to 50, v/v), affording 27 fractions (25 mL each) that were subsequently pooled into 5 groups (G4.1 to G4.5). Group G4.2 (29.9 mg) was submitted to preparative TLC eluted with CH₂Cl₂-MeOH (95:05, v/v, two times) yielding a mixture of 8 and 20 (3.1 mg) and 11 (18.3 mg). Group G6 (27.3 mg) eluted with EtOAc (100%) was also submitted to preparative TLC eluted with CH₂Cl₂-MeOH (95:05, v/v, two times) giving 14 (4.8 mg) and 15 (9.5 mg), respectively. Group G7 (128.8 mg) eluted with EtOAc-MeOH (95:05, v/v) was submitted to a new silica gel CC eluted with increasing concentrations CH₂Cl₂ in n-hexane (0 to 100, v/v) followed by MeOH in CH₂Cl₂ (0 to 100, v/v) resulting in 38 fractions (25 mL each), that were subsequently pooled into 7 groups (G7.1 to G7.7). Group G7.2 (58.8 mg) was submitted to preparative TLC eluted with CH₂Cl₂-MeOH (90:10, v/v, three times) yielding a mixture of 12 and 16 (9.9 mg), and a mixture of 17 and 18 (6.1 mg). Group G8(50.2 mg) eluted with EtOAc-MeOH (95:05 and 90:10, v/v) was subjected to a new silica gel CC eluted with increasing concentrations of CH₂Cl₂ in n-hexane (0 to 100, v/v) followed by MeOH in CH₂Cl₂ (0 to 100, v/v) resulting in 30 fractions (20 mL each) that were pooled into 7 groups (G8.1 to G8.7). Groups G8.3 and G8.4 were also pooled (21.0 mg) and submitted to preparative TLC eluted with CH₂Cl₂-MeOH (90:10, v/v, two times) yielding a mixture of 8, 9 and 10 (7.4 mg), 8 (2.7 mg), 12 (5.1 mg), and 19 (1.7 mg), respectively.

β-sitostenone (1) and Mixture of β-sitosterol (2), stigmasterol (3) and campesterol (4): white needles; identified by comparison with literature data (co-TLC, mp, ¹H NMR and ¹³C NMR);^16,17 EI-MS m/z 412, 414, 412, and 400 [M]^+., respectively.

Eudesmin (5), Magnolin (6) and Yangambin (7): Yellow amorphous powder (CHCl₃); identified by comparison with literature data (¹H NMR and ¹³C NMR);^18,19 EI-MS m/z 386, 416 and 446 [M]^+., respectively.

Liriodenine (8), Atherospermidine (9) and lysicamine (10): Orange crystals (CH₂Cl₂:MeOH 3:1); identified by comparison with literature data (¹H NMR and ¹³C NMR).^9-11

Anonaine (11): Brown amorphous powder (MeOH); ¹H and ¹³C NMR data, see Table 1; LR-ESI-MS [M+H]⁺m/z 266.5.

Table 1
NMR data (400 and 100 MHz ¹H for and ¹³C, respectively) for the aporphine alkaloids 11-13

Position	Anonaine (11)			Asimilobine (12)			Analobine (13)
	δC mult.a,c	δH mult. (J in Hz)a		δC mult.b,c	Sh mult. (J in Hz)b		δC mult.b,c	δH mult. (J in Hz)b
1	142.5, C			143.5, C			143.0, C
1a	116.1, C			125.9, C			115.7, C
2	146.8, C			148.9, C			148.3, C
3	107.9, CH	6.57 s		115.1, CH	6.68 s		107.6, CH	6.50 s
3a	126.4, C			129.2, C			127.2, C
3b	128.1, C			127.4, C			127.3, C
4pseudoeq	29.0, CH2	3.01 m		28.3, CH2	3.01 m		29.4, CH2	2.67 m
4pseudoax		2.67 m			2.70 m			2.30 m
5pseudoeq	43.1, CH2	3.41 m		42.8, CH2	3.35 m		43.9, CH2	3.33 m
5pseudoax		3.02 m			3.02 m			2.96 m
6a	53.3, CH	3.99 dd (14.2; 4.9)		53.4, CH	3.81 dd (13.8; 4.6)		54.5, CH	3.89 m
7pseudoeq	36.8, CH2	2.96 dd (14.2; 4.9)		36.9, CH2	2.86 dd (13.8; 4.6)		37.4, CH2	2.84 m
7pseudoax		2.83 dd (14.2; 14.2)			2.76 brd (13.8)			2.74 m
7a	134.9, C			135.7, C			137.6, C
8	128.1, CH	7.25 m		127.3, CH	7.31 ddd (6.7, 2.0, 0.6)		115.8, CH	6.70 d (2.7)
9	127.5, CH	7.22 m		127.6, CH	7.22 m		158.0, C
10	127.00 CH	7.31 m		128.0, CH	7.22 m		114.9, CH	6.71 dd (8.3, 2.7)
11	127.03, CH	8.07 brd (7.7)		127.6, CH	8.32 dd (8.1, 0.6)		129.5, CH	7.91 d (8.3)
11a	131.1, C			132.9, C			123.9, C
1-OCH2O-2	100.6, CH2	5.94 d (1.4)					101.7, CH2	5.90 d (1.2)
		6.09 d (1.4)						6.07 d (1.2)
H3CO-1				60.2, CH3	3.60 s

• ^a
  The experiments were acquired at 293 K with TMS as internal reference (0.00 ppm) in CDCl₃
• ^b
  or CDCl₃ with some drops of CD₃OD.
• ^c
  Multiplicities determined by DEPT 135, HSQC e HMBC experiments.

Asimilobine (12): Brown amorphous powder (MeOH); ¹H and ¹³C NMR data, see Table 1. LR-ESI-MS [M+H]⁺ m/z 268.4.

Analobine (13) and coclaurine (16): Brown amorphous powder (MeOH); ¹H and ¹³C NMR data, see Table 1 and 3, respectively.

Discretamine (14): Yellow amorphous powder (MeOH); ¹H and ¹³C NMR data, see Table 2. LR-ESI-MS [M+H]⁺m/z 328.6.

Table 2
NMR data (400 and 100 MHz for 'H and ¹³C, respectively) for tetrahydroprotoberberine alkaloids 14 and 15

Position	Discretamine (14)				Stepholidine (15)
	δC mult.a,b	δH mult. (J in Hz)a	NOE		δC mult.a,b	δH mult. (J in Hz)a	NOE
1	108.3, CH	6.71 s	H3CO-2; H- 13pgeudoeq		112.0, CH	6.77 s
2	145.O, C				144.6, C
3	144.4, C				146.3, C
4	114.7, CH	6.64 s	H-5pseudoeq		111.4, CH	6.61 s	H3CO-3; H-5pseudoeq
4a	127.0, C				125.5, C
5pseudoeq	28.4, CH2	3.08 ddd (17.0; 12.0;5.0)			28.7, CH2	3.11 m
5pseudoax		2.67 m				2.69 m
6pseudoeq	51.6, CH2	3.19 ddd (10.9; 5.0;1.6)			52.0, CH2	3.21 m
6pseudoax		2.63 m (11.9; 10.8; 2.9)				2.67 m
8pseudoeq	53.9, CH2	4.20 d (15.5)	H-8pseudoax		54.1, CH2	4.20 d (15.6)	H-8pseudoax
8pseudoax		3.56 d (15.5)				3.55 d (15.6)
8a	127.8, C				127.9, C
9	143.6, C				143.8, C
10	147.2, C				147.5, C
11	115.1, CH	6.77 d (8.2)			115.4, CH	6.76 d (8.3)
12	124.7, CH	6.81 d (8.2)			124.7, CH	6.80 d (8.3)
12a	126.5, C				126.4, C
13pseudoeq	35.9, CH2	3.28 dd (16.0; 3.8)			35.8, CH2	3.28 dd (16.0; 3.9)
13pseudoax		2.82 dd (16.0; 11.4)				2.78 dd (16.0; 11.4)
13a	59.6, CH	3.59 dd (11.4; 3.8)			59.6, CH	3.57 dd (11.4; 3.9)
13b	128.7, C				129.9, C
H3CO-2	56.2, CH3	3.88 s	H-1
H3CO-3					56.0, CH3	3.86 s	H-4
H3CO-9	60.3, CH3	3.82 s	H-8pseudoeq		60.2, CH3	3.83 s	H-8pseudoeq

• ^a
  The experiments were acquired at 293 K with TMS as internal reference (0.00 ppm) in CDCl₃ + drops of CD₃OD.
• ^b
  Multiplicities determined by DEPT 135, HSQC e HMBC experiments.

Stepholidine (15): Yellow amorphous powder (MeOH); ¹H and ¹³C NMR data, see Table 2; LR-ESI-MS [M+H]⁺m/z 328.6.

Mixture of orientaline (17) and juziphine (18): Brown amorphous powder (MeOH); ¹H and ¹³C NMR data, see Table 3.

Table 3
NMR data (400 and 100 MHz for 'H and ¹³C, respectively) for the benzyltetrahydroisoquinoline alkaloids 16-18

Position	Coclaurine (16)			Orientaline (17)			Juziphine (18)
	δC mult.a,b	δH mult. (J in Hz)a		δ>C mult.a,b	δH mult. (J in Hz)a		δC mult.a,b	δH mult. (J in Hz)a
1	57.8, CH	4.13 dd (9.4; 4.4)		65.8, CH	3.82 m		61.9, CH	4.39 dd (8.9; 2.8)
3pseudoeq	41.2, CH2	3.22 m		47.4, CH2	3.22 m		46.0, CH2	3.31 m
3pseudoax		2.93 m			2.84 m			2.82 m
4pseudoeq	28.8, CH2	2.78 m		25.5, CH2	2.88 m		24.2, CH2	2.91 m
4pseudoax		2.78 m			2.73 m			2.63 m
4a	126.1, C			124.6, C			126.5, C
5	112.7, CH	6.65 s		112.4, CH	6.64 s		120.1, CH	6.61 d (8.4)
6	148.0, C			148.0, C			111.4, CH	6.83 d (8.4)
7	145.7, C			145.2, C			146.6, C
8	114.0, CH	6.69 s		115.5, CH	6.14 s		144.2, C
8a	129.6, C			129.3, C			124.0, C
9	41.7, CH2	3.17 dd (14.2, 4.4)		40.9, CH2	3.07 m		38.8, CH2	3.08 m
		2.84 dd (14.2, 9.4)			2.77 m			2.93 m
10	128.8, C			132.9, C			132.9, C
11	131.3, CH	7.07 d (8.5)		121.8, CH	6.54 dd (8.2; 2.1)		131.2, CH	7.11 d (8.6)
12	116.6, CH	6.77 d (8.5)		112.7, CH	6.80 d (8.2)		116.0, CH	6.70 d (8.6)
13	157.2, C			147.3, C			156.6, C
14	116.6, CH	6.77 d (8.5)		147.5, C			116.0, CH	6.70 d (8.6)
15	131.3, CH	7.07 d (8.5)		117.4, CH	6.63 d (2.1)		131.2, CH	7.11 d (8.6)
H3CO-6	56.4, CH3	3.83 s		56.3, CH3	3.81 s
H3CO-7							56.6, CH3	3.86 s
H3CO-14				56.4, CH3	3.83 s
h3c-n				42.3, CH3	2.54 s		42.7, CH3	2.43 s

• ^a
  The experiments were acquired at 293 K with TMS as internal reference (0.00 ppm) in CDCl₃ + drops of CD₃OD.
• ^b
  Multiplicities determined by DEPT 135, HSQC e HMBC experiments.

Stepharine (19): Brown amorphous powder (CHCl₃:MeOH 3:1); ¹H and ¹³C NMR data, see Table 4. EI-MS [M]^+.m/z 297.

Table 4
NMR data (400 and 100 MHz for ¹H and ¹³C, respectively) for the proaporphine alkaloid 19

Position		Stepharine (19)
	δC mult.a,b	δH mult. (J in Hz)a
1	144.3, C
2	153.4, C
3	112.7, CH	6.66 s
3a	128.8, C
4pseudoeq	26.1, CH2	2.79 m
4pseudoax		2.79 m
5pseudoeq	44.8, CH2	3.44 ddd (12.7; 6.6; 2.3)
5pseudoax		3.14 ddd (12.7; 10.3; 6.6)
6a	57.9, CH	4.30 dd (10.8; 6.3)
6b	134.7, C
7pseudoeq	48.2, CH2	2.41 dd (12.1; 6.3)
7pseudoax		2.21 dd (12.1; 10.8)
8	150.8, CH	7.06 dd (10.0; 2.9 )
9	127.6, CH	6.30 dd (10.0; 1.9)
10	186.7, C
11	128.4, CH	6.42 dd (10.0; 1.9)
12	154.3, CH	6.93 dd (10.0; 2.9)
12a	51.4, C
12b	137.0, C
H3CO-1	61.4, CH3	3.60 s
H3CO-2	56.6, CH3	3.81 s

• ^a
  The experiments were acquired at 293 K with TMS as internal reference (0.00 ppm) in CDCl₃.
• ^b
  Multiplicities determined by DEPT 135, HSQC e HMBC experiments.

Mixture of 2-Methoxybenzoic acid (20) and liriodenine (8): Mixture of yellow and white needles (CH₂Cl₂:MeOH 3:1); identified by comparison with literature data (¹H and ¹³C NMR).^9-11,20-22

Antifungal and antibacterial activities (in vitro)

Crude extracts, fractions and isolated compounds were evaluated for antifungal and antibacterial activities using the broth microdilution method (96-well microtiter plates), as previously described by Salvador et al.²³ in the concentration of 12 to 5000 µg mL^-1. The minimal inhibitory concentration (MIC) was the lowest concentrations that completely inhibit a tested strain. In these assays, chloramphenicol and ketoconazole were used as positive controls, while a DMSO solution in distilled water (5:95, v/v) was the negative control. Each assay was performed in duplicate for each microorganism evaluated and repeated three times. The strains of microorganisms utilized are shown in Table 5.

Table 5
Biological activities of the crude extracts, fractions and pure compounds

Microorganisms	Antifungal and antibacterial activities expressed as MICa (μg mL-1)
	Hexane extract	Methanolic extract	Alkaloidal fraction	Neutral fraction	Eudesmin (5)	Magnolin (6)	Yangambin (7)	Discretamine (14)	Stepholidine (15)	Positive controlsb
Bacillus subtilis (Bs)d	250	250	500	500	-	-	-	-	-	50
Candida albicans (ATCC 10231)c	5000	5000	500	1000	125	125	125	>500	>500	12.5
Candida albicans (ATCC 1023)c	5000	5000	1000	1000	125	125	125	>500	-	12.5
Candida parapsilosis (ATCC 22019)c	5000	500	500	500	-	-	-	125	-	12.5
Candida tropicalis (ATCC 157)c	5000	5000	5000	1000	250	500	-	>500	-	12.5
Candida tropicalis (CT)d	5000	5000	5000	1000	250	-	-	>500	-	12.5
Candida glabrata (ATCC 30070)c	5000	5000	250	1000	-	-	-	-	-	12.5
Candida dubliniensis (ATCC 778157)c	5000	5000	1000	1000	250	250	-	125	-	12.5
Enterobacter aerogenes (Ea)d	500	500	250	500	-	-	-	-	-	50
Escherichia coli (ATCC 10538)c	> 5000	> 5000	1000	> 5000	-	-	-	-	-	50
Escherichia coli (ATCC 10799)c	1000	1000	1000	1000	-	-	-	-	-	50
Proteus vulgaris (Pv)d	500	1000	500	500	500	-	-	>500	-	50
Pseudomonas aeruginosa (ATCC 27853)c	> 5000	1000	1000	1000	-	-	-	-	-	850
Staphylococcus aureus (ATCC 14458)c	500	500	500	500	500	500	500	-	-	25
Staphylococcus aureus (ATCC 6538)c	> 5000	> 5000	500	> 5000	500	500	500	-	-	25
Staphylococcus epidermidis (ATCC 1228)c	1000	500	1000	500	-	-	500	>500	-	50
Staphylococcus epidermidis (6epi)d	1000	500	1000	500	-	-	500	>500	-	50
Antioxidant activity (ORAC-FL)
	352.73	1925.82	4545.04	862.60	-	-	-	2.10 (1.35)f	0.78 (1.57)f	*
	(3.60)eμ	(1.37)e	(2.46)e	(7.01)e

• ^a
  MIC (minimum inhibitory concentration with 100% of microorganism inhibition) in μg mL^-1;
• ^b
  positive controls (chloramphenicol for bacteria strains and ketoconazole for yeast strains);
• ^c
  standard strain; dfield strain.
• ^d
  field strain.
• ^e
  ORAC data expressed as mmol of TE g^-1 of extract or fraction, mean (%RSD, relative standard deviation) of triplicate assays;
• ^f
  ORAC data expressed as Trolox equivalent relative, mean (%RSD, relative standard deviation) of triplicate assays;
• ^*
  Positive controls (Caffeic acid:^f, quercetin:^f and isoquercitrin:^f).

Antioxidant assay by ORAC-FL kinetic assay (in vitro)

The antioxidant capacities of the extracts, fractions and pure compounds were assessed through the Oxygen Radical Absorbance Capacity (ORAC) assay (Table 5). This technique measures the scavenging activity against the peroxyl radical [Azobis (2-amidinopropane) dihydrochloride (AAPH), using fluorescein as the fluorescent probe. The ORAC assays were carried out on a Biotek Synergy 2 multidetection microplate reader system. The incubator temperature was set at 37 ºC. The procedure was carried out according to the method established by Ou et al.²⁴ with modifications Salvador et al.²⁵ The data are expressed as µmol of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents (TE) per gram of extracts and fractions on a dry basis (µmol of TE g^-1) and as the relative Trolox equivalent for pure compounds. In these evaluations, quercetin, isoquercitrin, and caffeic acid were used as positive controls and the assays were performed in triplicate.

RESULTS AND DISCUSSION

Once a time the crude extracts of the stem bark of A. pickelii were found to have antimicrobial and antioxidant activities (Table 5), they were subjected to successive chromatographic separations as described in the Experimental leading to the isolation and identification of 20 chemical constituents, being four steroids (1-4), three lignans (5-7), six aporphinoid alkaloids (8-13), two tetrahydroprotoberberine alkaloids (14-15), three benzyltetrahydroisoquinoline alkaloids (16-18), one proaporphine alkaloid (19), and one benzenoid (20) (Figure 1). Compounds 2, 5-8, 10 and 13 were recently described in the leaves of this species,¹³ while the other compounds are described here for the first time in this species.

All compounds were identified by comparing their spectrometric data with those reported in the literature, as well as, extensive analysis of NMR data as β-sitostenone (1),^16,17β-sitosterol (2),^16,17 stigmasterol (3),^16,17 campesterol (4),^16,17 eudesmin (5),^13,18 magnolin (6),^13,18 yangambin (7),^13,19 liriodenine (8),^9-11,13 atherospermidine (9),^9-11 lysicamine (10),^26-30 and 2-methoxybenzoic acid (20).²⁴Although, as the alkaloids 11-19 have been described a long time ago, their ¹H and ¹³C NMR data are incomplete and showed ambiguities according to literature data. In this work, the complete and unequivocal ¹H and ¹³C NMR data were revised according to 1D and 2D NMR experiments (Tables 1-4).

Compound 11 displayed two spin system in the ¹H NMR spectrum which is characteristic of aporphine alkaloids, one consisting of the signals at δ 2.67 and δ 3.01 (1H each, m, H-4) and δ 3.02 and δ 3.41 (1H each, m, H-5), and the other comprising the signals at δ 3.99 (1H, m, H-6a) and δ 2.83 and δ 2.96 (1H each, m, H-7). The singlet at δ 6.57 (1H, H-3) indicated a 1,2,3,4,5-pentasubstituted benzene ring, while the other aromatic signals revealed any substitution on the D ring (Table 1). The location of the methylenedioxy at C-1 and C-2 were established on the basis of the long-range ¹H-¹³C correlation map from HMBC NMR experiment. In this, the hydrogens at δ 6.57 (H-3) as well as the methylenedioxy hydrogens showed correlation with the carbons at δ 142.5 (C-1) and δ 146.8 (C-2). The overall analysis of 1D and 2D NMR data enable the complete and unequivocal ¹H and ¹³C NMR chemical shifts assignments (Table 1).

Compound 12 presented ¹H and ¹³C{¹H} NMR spectra very similar to those of 11. However, in this case, the methylenedioxy signals were replaced by signals suggesting the presence of a methoxyl and hydroxyl groups at C-1 and C-2. The location of the methoxyl group at C-1 was established on the basis of the long-range ¹H-¹³C correlation map from HMBC NMR experiment. In this, the hydrogen at δ 6.68 (H-3) as well the methoxyl hydrogens at δ 3.60 showed strong correlation with the carbon at δ 143.5 (C-1) (Table 1).

Compound 14 showed several signals in the aliphatic region of ¹H NMR spectrum that along with ¹H-¹H correlation map from COSY NMR experiment revealed three spin systems, which are typical for tetrahydroprotoberberine alkaloids (Table 2). The signals at δ 3.82 and δ 3.88 (3H each, s) indicated the presence of two methoxyl groups in the structure. Furthermore, the ¹H NMR spectrum exhibited two signals at δ 6.71 (1H, H-1) and d 6.64 (1H, H-4), indicating a 1,2,4,5-tetrasubstituted benzene ring and two doublets at δ 6.81 (1H, J 8.2 Hz, H-12) and δ 6.77 (1H, J 8.2 Hz, H-11) suggesting a 1,2,3,4-tetrasubstituted benzene ring. The complete assignments of the ¹H and ¹³C NMR chemical shifts were established on basis of one-bond and long-range ¹H-¹³C NMR correlation experiments and 1D experiments (Table 2). In this, the hydrogen at δ 6.64 (H-4) and the methoxyl hydrogens at δ 3.88 showed long-range ¹H-¹³C correlation with the carbon at δ 145.8 (C-2), while the hydrogen at δ 6.77 (H‑11) and the methoxyl hydrogens at δ 3.82 showed long-range ¹H-¹³C correlation with the carbon at δ 143.6 (C-9). The hydroxyl groups at C-3 and C-10 were established on the basis of the long-range ¹H-¹³C correlations of hydrogen at δ 6.71 (H-1) with the carbon at δ 144.4 (C-3) and the hydrogen at δ 6.81 (H-12) with the carbon at δ 147.2 (C-10), that showed any correlation with the methoxyl groups (Table 2). Moreover, the selective irradiation of the resonance frequency of the methoxyl hydrogens at δ 3.88 caused a NOE enhancement in the signal at δ 6.71 (H-1), while the selective irradiation of the resonance frequency of the methoxyl hydrogens at δ 3.82 caused a NOE intensification in the signal at δ 4.20 (H-8 pseudoequatorial) (Table 2).

Compound 15 showed ¹H and ¹³C{¹H} NMR spectra very similar to those of 14. However, in this case, the hydrogen at δ 6.77 (H-1) as well as the methoxyl hydrogens at δ 3.86 showed ¹H-¹³C long-range correlation with the same carbon at δ 146.3 (C-3), while the hydrogen at δ 6.61 (H-4) showed ¹H-¹³C long-range correlation with the carbon at δ 144.6 (C-2), that showed any correlation with methoxyl groups. Thus, the methoxyl group at C-1 in 14 was replaced by a hydroxyl group in 15. In addition, the selective irradiation of the resonance frequency of the methoxyl hydrogens at δ 3.86 showed a NOE intensification in the signal of H-4 at δ 6.61 and any enhancement in the signal of H-1 at δ 6.77. The overall NMR data are described in (Table 2).

Compounds 13 and 16 were obtained in a mixture once its NMR data indicated the presence of two set of signals with different ratios. The first set of signals in the ¹H NMR spectrum displayed two spin system characteristic of aporphine alkaloids at δ 2.30 and δ 2.67 (H-4), δ 2.96 and δ 3.33 (H-5), δ 3.89 (H-6a) and δ 2.84 and δ 2.74 (H-7) (Table 1). The singlet at δ 6.50 (1H, H-3) indicated a 1,2,3,4,5-pentasubstituted benzene ring and the spins system at δ 6.70 (1H, d, J 2.7 Hz, H-8), 6.71 (1H, dd, J 8.3 and 2.7 Hz, H-10) and 7.91 (1H, d, J 8.3 Hz, H-11), indicating a 1,2,4-trisubstituted benzene ring. The location of the hydroxyl and the methylenedioxy at C-9 and C-1 and C-2 were established on the basis of the long-range ¹H-¹³C correlation map from HMBC NMR experiment. In this, the hydrogens at δ 6.50 (H-3) as well as the methylenedioxy hydrogens showed correlation with the carbons at δ 143.0 (C-1) and δ 148.3 (C-2). On the other hand, the hydrogen at 7.91 (H-11) showed ¹H-¹³C long-range correlation with the carbon at 158.0 (C-9), that showed no correlation with hydrogens from methoxyl group. The overall analysis of 1D and 2D NMR experiments enable the complete and unequivocal ¹H and ¹³C NMR chemical shifts assignments (Table 1).

The ¹H NMR spectrum showed also a second NMR dataset consisting of two spin system characteristic of a benzyltetrahidroisoquinoline alkaloid at δ 4.13 (H-1), δ 2.93 and δ 3.22 (H-3), δ 2.78 (H-4) and δ 2.84 and δ 3.17 (H-9) (Table 3). The signals at δ 6.65 (1H, s, H-5) and δ 6.69 (1H, s, H-8) indicated a 1,2,4,5-tetrasubstituted benzene ring, while the signals at δ 7.07 (2H, d, J 8.5 Hz, H-11 and H-15) and δ 6.77 (2H, d, J 8.5 Hz, H-12 and H-14) indicted presence of a p-substituted benzene ring in the structure. The location of the two hydroxyl groups at C-7 and C-13 and the methoxyl group at C-6, were established on the basis of the long-range ¹H-¹³C correlation map from HMBC NMR experiment. In this, the hydrogens at δ 6.69 (H-8) as well as the methoxyl hydrogens showed correlation with the same carbon at δ 148.0 (C-6). The hydroxyl groups at C-7 and C-13, were established through the ¹H-¹³C long-range correlations of hydrogens at δ 6.65 (H-5) and δ 7.07 (H-11 and H-15) with the carbons at δ 145.7 (C-7) and δ 157.2 (C-13), respectively, that showed no correlation with hydrogens from methoxyl groups. The overall analysis of 1D and 2D NMR experiments enable the complete and unequivocal ¹H and ¹³C NMR chemical shifts assignments (Table 3).

Compounds 17 and 18 presented NMR data indicating the presence of two set of signals from two benzyltetrahydroisoquinoline alkaloids. The first set of signals in the ¹H NMR spectrum displayed two spin system at δ 3.82 (H-1) and δ 2.77 and δ 3.07 (H-9) and δ 2.84 and δ 3.22 (H-3) and δ 2.73 and δ 2.88 (H-4) (Table 3). The signal at 2.54 (3H, s) revealed the presence of an N-CH₃ group. The singlet at δ 6.64 (1H, s, H-5) and δ 6.14 (1H, s, H-8) indicated a 1,2,4,5-tetrasubstituted benzene ring and the spins system at δ 6.54 (1H, dd, J 8.2 and 2.1 Hz, H-11), δ 6.80 (1H, d, J 8.2 Hz, H-12) and δ 6.63 (1H, d, J 2.1 Hz, H-15), indicating a 1,2,4-trisubstituted benzene ring. The overall analysis of 1D and 2D NMR experiments enable to locate the methoxyl and hydroxyl groups in the structure as well as to perform the complete and unequivocal ¹H and ¹³C NMR chemical shifts assignments (Table 3).

The second NMR dataset in the ¹H NMR spectrum showed two very similar spin system (Table 3). However, for this compound only one signal for a methoxyl group was observed at δ 3.86 (3H, s), while the signal at δ 2.43 (3H, s) revealed the presence of an N-CH₃ group, as well. The main difference was the two doublets at δ 6.61 (H-5) and δ 6.83 (H-6) (1H each, J 8.4 Hz) that indicated a 1,2,3,4-tetrasubstituted benzene ring and the two doublets at δ 7.11 (H-11 and H-15) and δ 6.70 (H-12 and H-14) (2H each, J 8.4 Hz) indicted the presence of a p-substituted benzene ring in the structure. The overall analysis of 1D and 2D NMR experiments enable to locate the methoxyl and hydroxyl groups in the structure as well as to perform the complete and unequivocal ¹H and ¹³C NMR chemical shifts assignments (Table 3).

Compound 19 showed a ¹H NMR spectrum and HSQC correlation map consistent with six typical signals for a proaporphine alkaloid at δ 2.21 (H-7 pseudoaxial), δ 2.41 (H-7 pseudoequatorial), δ 2.79 (H-4 pseudoaxial/pseudoequatorial), δ 3.14 (H-5 pseudoaxial), δ 3.44 (H-5 pseudoequatorial), and δ 4.30 (H-6a). Furthermore, the ¹H NMR spectrum exhibited one singlet at δ 6.66 indicated a 1,2,3,4,5-pentasubstituted benzene ring, two double doublets at δ 6.30 and δ 6.42 (each 1H, dd, J 10.0 and 1.9 Hz), and two other double doublets were observed at δ 6.93 and δ 7.06 (each 1H, dd, J 10.0 and 2.9 Hz). The HSQC and HMBC correlations maps showed 14 signals, being one at δ 186.7 from a carbonyl group, ten between δ 154.3 and δ 112.7, two methoxyl at δ 56.6 and δ 61.4, three methylenes at δ 48.2, δ 44.8 and δ 26.1, one methine at δ 57.9, and a quaternary carbon at δ 51.4 (Table 4). Moreover, the hydrogen at δ 6.66 (H-3) as well as the methoxyl hydrogens at δ 3.60 and δ 3.81 showed the long-range ¹H-¹³C correlations with the same carbons at δ 144.3 (C-1) and δ 153.4 (C-2). It showed a strong correlation with the carbon at δ 144.3 and a weak correlation with de carbon at δ 153.4 revealing ³J_H,C and ²J_H,C correlations, respectively. The presence of a proaporphine moiety was also established on basis of long-range ¹H-¹³C correlation of the hydrogens at δ 7.06 and δ 6.93 (H-8 and H-12) with the carbon at δ 186.7 (C-10). The overall analysis of 1D and 2D NMR experiments enabled its complete and unambiguous ¹H and ¹³C NMR chemical shift assignments (Table 4).

Among the compounds found in A. pickelli, the aporphinoids alkaloids liriodenine (8), anonaine (11), and asimilobine (12),^20,22,26-30 are the most representative of the family Annonaceae, once they are found in most genera of this family being considered as chemotaxonomic markers.^13,20,21 On the other hand, the lignans eudesmin (5), magnolin (6), and yangambin (7) have been found only in the genus Annona. Therefore, this finding is very important from the chemotaxonomic point of view, once this species belonging to the genus Rollinia was recently reclassified as Annona.¹ Thus, these lignans may be useful as chemotaxonomic markers of the genus.

The hexane and methanol crude extracts, as well as the alkaloidal and neutral fractions from methanol extract showed antifungal and antibacterial activities (Table 5). Therefore, the major compounds eudesmin (5), magnolin (6), yangambin (7), discretamine (14), and stepholidine (15), mixture of β-sitosterol (2), stigmasterol (3) and campesterol (4), and β-sitostenone (1) were also evaluated. Among them, only the lignans and the tetrahydroprotoberberine alkaloid discretamine (14) showed significant activities (Table 5). The alkaloids liriodenine (8), anonaine (11) and asimilobine (12) has been previously investigated.^6-8 The lignans were active against C. albicans (ATCC 10231 and ATCC 1023), all with MIC of 125 µg mL^-1. Eudesmin (5) and magnolin (6) were also active against C. dubliniensis(ATCC 778157), while only eudesmin (5) was active against C. tropicalis (ATCC 157 and CT), both with MIC of 250 µg mL^-1 (Table 5). The tetrahydroprotoberberine alkaloid, discretamine (14) was active against C. parapsilosis (ATCC 22019) and C. dubliniensis (ATCC 778157) with MIC of 125 µg mL^-1. On the other hand, all the steroids were inactive until 500 µg mL^-1. MIC values lower than 100 µg mL^-1 were considered as good activity, between 100 and 1000 µg mL^-1 moderate activity and small activity to MIC value higher than 1000 µg mL^-1for extracts. On the other hand, for pure compounds MIC value lower than 100 µg mL^-1 was considered as good activity, between 100 and 500 µg mL^-1 moderate activity and small activity to value higher than 500 µg mL^-1.²⁵

Among crude extracts evaluated, methanol shows the high antioxidant activity which ORAC of 1925.82 µmol of TE g^-1 (Table 5). After acid-base extraction, the antioxidant activity was revealed only in the alkaloidal fraction with ORAC value of 4545.04 µmol of TE g^-1. In a previous work, the aporphinoid alkaloids liriodenine (8), anonaine (11), and asimilobine (12) were evaluated on ORAC assay and asimilobine (12) was the most active. In this work the tetrahydroprotoberberine alkaloids discretamine (14) and stepholidine (15) were investigated, although only discretamine was active with ORAC of 2.10 trolox equivalents relative (Table 5). According to these results, the antioxidant activity of the methanol extract can be attributed in part to the alkaloids asimilobine (12) and discretamine (14). Those extracts and pure compounds that shown ORAC values higher than 1000.00 µmol of TE g^-1 and 1.00 Trolox equivalent relative were considered to have good antioxidant capacity.^5,23

CONCLUSION

This is the first phytochemical and biological investigation of the stem bark of A. pickelii. The lignans found in this species support their recent reclassification from Rollinia to Annona. In this way, the alkaloids liriodenine, anonaine, and asimilobine support that A. pickelii is a typically species of the family Annonaceae. Significant antifungal and antioxidant activities were found to the lignans and the alkaloids from A. pickelii, and then it can be a promising source for bioactive compounds. In addition, several NMR data for the alkaloids were reviewed and are unequivocally described in this work.

• SUPPLEMENTARY MATERIAL
  NMR data including spectra and correlation maps for compounds 11-19 are available free of charge at http://quimicanova.sbq.org.br as PDF file.

ACKNOWLEDGMENTS

The authors are grateful to FAPITEC/SE (Editais # 07/2009 and 10/2009), CNPq, CAPES, FINEP, UFPR and FAPESP for financial support and fellowships, and Professor Dr. Ana Paula do Nascimento Prata for botanical identification.

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Publication Dates

• Publication in this collection
  July 2015

History

• Received
  23 Oct 2014
• Accepted
  05 Mar 2015