Biochemical Systematics and Ecology 39 (2011) 872–875

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Biochemical Systematics and Ecology

journal homepage: www.elsevier.com/locate/biochemsyseco

Chemical constituents from the bark of Annona salzmannii (Annonaceae)

Pedro Ernesto Oliveira da Cruz a, Emmanoel Vilaça Costa a, *, Valéria Regina de Souza Moraes a,
Paulo Cesar de Lima Nogueira a, Mayara Evelyn Vendramin b, Andersson Barison b,
Antonio Gilberto Ferreira c, Ana Paula do Nascimento Prata d
a
Laboratório de Pesquisa em Química Orgânica de Sergipe (LABORGANICS), Departamento de Química, Universidade Federal de Sergipe, Rosa Elze, São Cristóvão,
SE 49100-000, Brazil
b
Departamento de Química, Universidade Federal do Paraná, Centro Politécnico, Jardim das Américas, PO Box 19081, Curitiba, PR 81531-990, Brazil
c
Departamento de Química, Universidade Federal de São Carlos, PO Box 676, São Carlos, SP 13565-905, Brazil
d
Departamento de Biologia, Universidade Federal de Sergipe, Rosa Elze, São Cristóvão, SE 49100-000, Brazil

a r t i c l e i n f o
.
Article history:
Received 11 March 2011
Accepted 4 June 2011
Available online 19 July 2011

Keywords:
Annonaceae
Annona salzmannii
Alkaloids
Sesquiterpenes
Steroids

1. Subject and source

Annona salzmannii A. DC. (Annonaceae) is a tree of 6–20 m tall popularly known as “araticum-da-mata” and “araticum-
apé” (Pontes et al., 2004). It is very common in Brazil, mainly in the States of Bahia, Pernambuco, and Paraíba (Pontes et al.,
2004). Some of its organs (leaves, roots, and seeds) are used in folk medicine to treat several human ailments such as ver-
minosis, dysentery, ulcers, and inflammatory conditions (Agra, 1977).
The bark of A. salzmannii was collected from “Mata do Crasto”, Santa Luzia do Itanhy city [coordinates: 11
230 08”
S 037
250 10” W], Sergipe State, Brazil, in August 2009. Its identity was confirmed by Dr. Ana Paula do Nascimento
Prata, a plant taxonomist of the Department of Biology of the Federal University of Sergipe (UFS), and a voucher
specimen (#15438) has been deposited in the Herbarium of the Federal University of Sergipe (ASE/UFS), Sergipe,
Brazil.

2. Previous work

Previous phytochemical studies on this species described the isolation and identification of alkaloids (Paulo et al., 1992),
acetogenins (Queiroz et al., 1999, 2003), and essential oils (Costa et al., 2011).

* Corresponding author. Tel.: þ55 79 2105 6657; fax: þ55 79 2105 6651.
E-mail address: emmanoelvilaca@yahoo.com.br (E.V. Costa).

0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bse.2011.06.008
 P.E.O. da Cruz et al. / Biochemical Systematics and Ecology 39 (2011) 872–875 873

3. Present study

The dried and powdered bark (1800 g) was extracted with hexane (4 L, five times), followed by MeOH (4 L, five times), to give
hexane (8.69 g) and MeOH (143.29 g) extracts after removal of each solvent. The hexane extract (5.0 g) was subjected to silica gel
column chromatography (CC) using a petroleum ether-CH2Cl2 gradients (100:0 to 10:90), followed by a CH2Cl2-EtOAc gradients
(100:0 to 10:90) and an EtOAc-MeOH gradients (100:0 to 80:20) as eluent, yielding 159 fractions of 25 mL each that were
evaluated and pooled according to TLC analysis, to give twenty six fractions. Fraction 12 (513.8 mg) was further purified on
a silica gel CC using petroleum ether with increasing amounts of CH2Cl2 (0, 10, 20, 40, 60, and 80%), followed by CH2Cl2 with
increasing amounts of EtOAc (0, 10, and 20%), affording caryophyllene oxide (1, 23.9 mg; Ragasa et al., 2003). Fraction 14
(421.7 mg) was subjected to silica gel CC using the same methodology above, to give fourteen subfractions. Subfraction 14.7
(103.5 mg) was further purified on preparative TLC eluted with petroleum ether-EtOAc (90:10, v/v, two times) to yield spa-
thulenol (2, 6.9 mg; Ragasa et al., 2003) and selin-11-en-4a-ol (3, 11.8 mg; Chanotiya et al., 2005). Subfraction 14.8 (82.6 mg) was
further purified on preparative TLC eluted with CH2Cl2-EtOAc (90:10, v/v) to yield stigmast-4-en-3-one (4, 10.0 mg; Della Greca
et al., 1990) and b-sitosterol (5, 11.8 mg; Della Greca et al., 1990). Fraction 15 (871.0 mg) was subjected to silica gel CC using the
same methodology above, to give nineteen subfractions. Subfraction 15.6 (209.1 mg) was further purified on preparative TLC
eluted with CH2Cl2-EtOAc (90:10, v/v) to yield again stigmast-4-en-3-one (4, 15.2 mg; Della Greca et al., 1990) and b-sitosterol
(5, 71.7 mg; Della Greca et al., 1990).
TLC investigations indicated a high concentration of alkaloids in the MeOH extract. Therefore, an aliquot of the MeOH
extract (129.0 g) was initially subjected to an acid-base extraction (Costa et al., 2006) to give the alkaloid fraction (2.22 g) and
neutral fraction (2.72 g). An aliquot of the alkaloid fraction (1.73 mg) was initially subjected to silica gel column chroma-
tography (CC) previously treated with a 10% NaHCO3 solution (Costa et al., 2006), and eluted with increasing concentrations of
CH2Cl2 in hexane (100:0 to 10:90), followed by EtOAc in CH2Cl2 (100:0 to 20:80), and MeOH in EtOAc (100:0 to 50:50) as
eluents, giving 225 fractions of 20 mL each that were evaluated and pooled according to TLC analysis to yield twenty-one
fractions.

O
H
H

H
H
H HO
HO H

1 2 3

O CH3

N
O HO O
4 5 6

5 4
O R2 H3CO
6 4a 3
7
O N NH 8a
N CH
R1 HO 8 1
2 3
15
HO 9
O 14 10
13
11
R H3CO 12
R3
7R=H 12
8 R = OCH3
9 R1 - R2 = OCH2O; R3 = H
10 R1 - R2 = OCH2O; R3 = OCH3
11 R1 = OCH3; R2 = OH; R3 = H

Fig. 1. Chemical constituents isolated from the bark of Annona salzmannii.

874 P.E.O. da Cruz et al. / Biochemical Systematics and Ecology 39 (2011) 872–875

Additional chromatography separation of fraction 13 (67.0 mg) by preparative TLC eluted with CH2Cl2-MeOH (90:10, v/v),
resulted in cleistopholine (6, 5.0 mg; Waterman and Muhammad, 1985; Tadic et al., 1987), liriodenine (7, 7.2 mg; Chang et al.,
2000; Costa et al., 2009a), anonaine (9, 10.6 mg; Chang et al., 2000), and a mixture of anonaine 9 and xylopine 10 (7.7 mg;
Chang et al., 2000). Subfraction 14 (40.8 mg) yielded oxolaureline or 10-methoxyliriodenine (8, 2.5 mg; Harrigan et al., 1994),
after being purified by preparative TLC eluted with CH2Cl2-MeOH (90:10, v/v). Fraction 15 (67.8 mg) was subjected to
preparative TLC eluted with CH2Cl2-MeOH (90:10, v/v), giving asimilobine (11, 6.6 mg; Chang et al., 2000). Fraction 16
(272.9 mg) was further purified to successive silica gel CC using the same conditions described as above, and posterior
preparative TLC eluted with CH2Cl2-MeOH (8.5:1.5, v/v), resulting in reticuline (12, 20.6 mg; Leboeuf et al., 1982; Janssen et al.,
1990). Although reticuline has been described a long time ago, the NMR dataset published for this compound is incomplete
(Leboeuf et al., 1982; Janssen et al., 1990) and shows ambiguities (Fechine et al., 2002). Corrected 1H and 13C NMR chemical
shifts assignments using 2D methods (HSQC and HMBC) are presented in this work. 1H NMR (400 MHz, CDCl3 þ drops of
CD3OD) d 6.74 (1H, d, J ¼ 8.2 Hz, H-12), 6.70 (1H, d, J ¼ 2.0 Hz, H-15), 6.54 (1H, dd, J ¼ 8.2 and 2.0 Hz, H-11), 6.55 (1H, s, H-5),
6.20 (1H, s, H-8), 3.84 (3H, s, 13-OCH3), 3.83 (3H, s, 6-OCH3), 3.72 (1H, dd, J ¼ 7.0 and 5.2 Hz, H-1), 3.17 (1H, m, H-3pseudoeq),
3.07 (1H, dd, J ¼ 13.8 and 5.2 Hz, H-9b), 2.86 (1H, m, H-4pseudoax), 2.80 (1H, m, H-3pseudoax), 2.74 (1H, dd, J ¼ 13.8 and
7.0 Hz, H-9a), 2.64 (1H, m, H-4pseudoeq), 2.47 (3H, s, N-CH3); 13C NMR (100 MHz, CDCl3 þ drops of CD3OD) d 146.0 (C-6), 145.7
(C-13), 145.6 (C-14), 143.5 (C-7), 132.5 (C-10), 129.1 (C-8a), 124.1 (C-4a), 121.0 (C-11), 116.1 (C-15), 114.2 (C-8), 111.1 (C-12), 111.0
(C-5), 64.6 (C-1), 55.9 (13-OCH3), 55.8 (6-OCH3), 46.4 (C-3), 42.0 (N-CH3), 40.6 (C-9), 24.6 (C-4). All isolated compounds (Fig. 1)
were identified by a series of spectrometric methods, such as MS, NMR (1D and 2D), as well as comparison with data reported
in the literature.

4. Chemotaxonomic significance

The present work reports the isolation and identification of three sesquiterpenes (1–3), two steroids (4–5), and seven
alkaloids (6–12): one azaanthracene (6), two oxoaporphine (7–8), three aporphine (9–11), and one benzyltetrahy-
droisoquinoline (12) from the bark of A. salzmannii (Fig. 1). All isolated compounds, except 9 and 12 are reported from this
plant for the first time. Caryophyllene oxide (1) and spathulenol (2) has been found as a component of essential oils from the
leaves of several genera of Annonaceae, including Annona (Costa et al., 2009b, 2011), Duguetia (Maia et al., 2006), Guatteria
(Costa et al., 2008; Maia et al., 2005a), Hexalobus (Boyom et al., 2003), Pachypodanthium (Boyom et al., 2003), and Xylopia
(Boyom et al., 2003; Maia et al., 2005b). Selin-11-en-4a-ol (3) is reported for the first time in the Annona, and its occurrence in
the Annonaceae is observed only in the essential oils. Stigmast-4-en-3-one (4) and b-sitosterol (5) are two compounds
reported to occur in many plants. The presence of aporphine (anonaine 9, xylopine 10, and asimilobine 11), oxoaporphine
(liriodenine 7), and benzyltetrahydroisoquinoline (reticuline 12) alkaloids in A. salzmannii revealed the presence of typical
annonaceous constituents. Anonaine, asimilobine, liriodenine, and reticuline have been described in several species of
Annona (Chang et al., 2000; Costa et al., 2006; Campos et al., 2008), and could be considered as chemotaxonomic markers of
this genus. Cleistopholine is an azaanthracene alkaloid type that has been found in several unrelated genera in Annonaceae,
including Annona (Rasamizafy et al., 1987; Rios et al., 1989; Dos Santos et al., 2003; Wu et al., 2005), Duguetia (Pérez et al.,
2004), Guatteria (Goulart et al., 1986), Cleistopholis (Waterman and Muhammad, 1985; Seidel et al., 2000), Hornschuchia
(Fechine et al., 2003), and Porcelia (Chaves et al., 2001), but it is very common in the genera Annona and Cleistopholis.

Acknowledgements

The authors are grateful to FAPITEC/SE (Editais No 07/2009 and 10/2009), and CNPq, CAPES, FAPESP, UFPR, and FINEP, for
financial support.

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