Food Chemistry 122 (2010) 1129–1138

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Food Chemistry
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Acetogenins from Annona cornifolia and their antioxidant capacity

Luciana A.R. Santos Lima, Lúcia P.S. Pimenta, Maria Amélia D. Boaventura *
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-970 Belo Horizonte – MG, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Seed ethanol extract, fractions and acetogenins from Annona cornifolia A. St.-Hil were tested for their anti-
Received 10 July 2009 oxidant activities against DPPH. Acetogenins showed a strong DPPH radical-scavenging activity, similar
Received in revised form 20 January 2010 to that of ascorbic acid. Ten acetogenins were isolated in their pure form and two were isolated as a mix-
Accepted 24 March 2010
ture. Five are new acetogenins and are described here for the first time. Their structures were elucidated
by NMR and ESI-MS analysis, and relative configurations bearing THF rings were established. The abso-
lute configuration at C-36 of two acetogenins was defined by circular dichroism. This is, to the best of our
Keywords:
knowledge, the first description of the antioxidant activity of acetogenins.
Annona cornifolia
Acetogenins
Ó 2010 Elsevier Ltd. All rights reserved.
Antioxidant capacity
DPPH

1. Introduction associated with a decreased risk of development of several chronic

Several Annona species of the Annonaceae family furnish edible
fruits, some of them very much appreciated in Brazil, such as Anno-
na crassiflora (‘‘araticum”), Annona squamosa (‘‘fruta do conde”) and
Annona muricata (‘‘graviola”). The fruits are usually consumed ‘‘in
natura” or used in juices, desserts or ice-cream preparations.
Annona cornifolia A. St.-Hil. is an annual perennial small tree
found in the Brazilian savannah and popularly known as ‘‘arati-
cum-mirim”. The ripe orange fruit pulp is sweet and aromatic.
The green fruit is popularly used against ulcer (Correa, 1974).
Annonaceous acetogenins are long chain fatty acid derivatives
isolated, until 2008 (Pettit et al., 2008), exclusively from plants
belonging to the Annonaceae family. These natural products exhi-
bit a broad range of biological properties, such as cytotoxic, immu-
nosuppressive, pesticidal, antiparasitic and antimicrobial activities,
and their potential to inhibit cells that are multiple drug-resistant
has attracted increasing interest (Bermejo et al., 2005). Our group
have already investigated acetogenins from seeds of A. crassiflora
(Pimenta, Pinto, Takahashi, Silva, & Boaventura, 2003; Santos,
Boaventura, Sun, Cassady, & Oliveira, 1996) and A. cornifolia (Lima,
Pimenta, & Boaventura, 2009; Santos, Boaventura, & Pimenta,
2006; Santos, Pimenta, & Boaventura, 2007) and leaves of Rollinia
laurifolia (Pimenta, Nascimento, Assunção, & Boaventura, 2001;
Pimenta, Nascimento, & Boaventura, 2005).
The search for natural antioxidants, especially of plant origin,
has greatly increased in recent years. Epidemiological studies have
revealed that an increased consumption of fruits and vegetables is
* Corresponding author. Tel.: +55 3134995760; fax: +55 3134995700.
E-mail address: dianadb@netuno.lcc.ufmg.br (M.A.D. Boaventura).
and degenerative diseases. A positive correlation was also de-
scribed for elevated b-carotene plasma levels and a reduced risk
of lung and stomach cancer (Stahal et al., 1997). Besides vitamins
A, C and E, the most important naturally occurring plant sub-
stances showing antioxidant activity are carotenoids, flavonoids
and other simple phenolic compounds, which, in different propor-
tions and quantities, are found in cereals, fruits and vegetables
(Arnao, Cano, & Acosta, 2001).
Some natural antioxidants have a hydrophilic nature (e.g. ascor-
bic acid) and others are clearly lipophilic (e.g. carotenoids). Vita-
min C (ascorbic acid), with four hydroxyl groups, is an important
hydrophilic antioxidant; it is found in cytosolic, mitochondrial
and nuclear aqueous compartments of the cell, and scavenges reac-
tive oxygen and nitrogen species; this hypothesis was confirmed
by ESR data (Wagner et al., 2008). The antioxidant activity of
carotenoids is also related to their ability to quench reactive oxy-
gen species such as singlet molecular oxygen and peroxyl radicals,
thus acting as deactivators of excited molecules or as chain-break-
ing agents, respectively (Stahal et al., 1997). A study with carote-
noids revealed that the scavenging ability towards DPPH was
increased by the length of the effective conjugated double-bond
system and was modulated by the addition of hydroxyl groups
on the terminal rings (Jimenez-Escrig, Jimenez-Jimenez, Sanchez-
Moreno, & Saura-Calixto, 2000).
The most widely used methods for measuring antioxidant activ-
ity are those that involve the generation of radical species, and the
presence of antioxidants determines the disappearance of these
radicals. From the methodological point of view, the widespread
use of the stable 2,2-diphenyl-picrylhydrazyl (DPPH) radical-scav-
enging model is recommended as a fast, easy and accurate

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.03.100
1130 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138
methodology for measuring the antioxidant activity. This test sys-
tem can be used for the primary characterisation of the scavenging
potential of extracts and pure compounds. It is well accepted that
the DPPH radical-scavenging by antioxidants is attributable to
their hydrogen donating ability (Krings & Berger, 2001).
Scavenging activity of polar components of A. crassiflora pulp,
peel and seed ethanol extracts was evaluated against the DPPH
radical and the IC50 showed values ranging between 1.9 lg/ml, re-
lated to caffeic acid and 9.9 lg/ml, related to ferulic acid, both
present in these extracts (Roesler, Catharini, Malta, Eberlin, & Pas-
tore, 2007).
In the present communication, the DPPH-scavenging activities
of seed ethanol extract, fractions and isolated acetogenins from
A. cornifolia A. St.-Hil were evaluated. This is the first time, to the
best of our knowledge, that the antioxidant activity of acetogenins
has been evaluated.
2. Materials and methods
2.1. Instrumentations
IR spectra of ethanol extract and fractions were recorded on a
Shimadzu 400 spectrometer (Shimadzu, Kyoto, Japan), sampling
mode KBr, number of scans 16, resolution 4 cm1, spectral range
400–4000 cm1 and those of the pure acetogenins were recorded
on a Shimadzu FTIR-8400, sampling mode KBr, number of scans
32, resolution 4 cm1, spectral range 400–4000 cm1. The 1D and
2D NMR spectra were run on Bruker Avance DRX 200 and on Bruc-
ker Avance DRX 400 spectrometers (Ettlingen, Germany) in CDCl3
containing 0.1% tetramethylsilane as the internal chemical shift
standard. Electrospray Ionisation Mass Spectrometry (ESI-MS)
was performed using a Waters MICROMAS Q-TOF (Milford, Massa-
chusetts, USA), equipped with an electrospray ion (ESI) source.
Solutions of the compounds at 200 lg ml1 in ACN–H2O (1:1) were
infused at 25 ll min1, and the positive mass spectra were ac-
quired with m/z range between 50 and 1000 daltons. The cone
voltages were optimised for positive ion analysis in the range be-
tween 35 and 50 V. In the MS/MS experiments, the parent ion iso-
lation width was 3.8 daltons and the normalised collision energy
was set at 30% for the compounds. Fifty scans, from 50 to 700 dal-
tons, were collected to generate the averaged spectra. Optical rota-
tions were measured on a Perkin Elmer 341 polarimeter (Waltham,
Massachusetts, USA) the circular dichroism spectrum was obtained
on a Jasco J720 spectropolarimeter (Great Dunmow, Essex, UK) at
20 °C in methanol solution. Final purification was performed on a
Waters 501 apparatus (Milford, Massachusetts, USA), with a 486
UV-detector and 746 integrator. Absorbances for the antioxidant
test were measured in a Hitachi 2010 spectrophotometer (Schaum-
burg, Illinois, USA).
2.2. Plant material
Seeds of A. cornifolia A. St.-Hil. (Annonaceae) were collected in
the Curvelo region, Minas Gerais, Brazil, and identified by Dr. Rena-
to Mello-Silva. A voucher specimen (No. 68114) was deposited at
the Herbarium of the Natural History Museum of UFMG, Belo Hor-
izonte, Minas Gerais, Brazil.
2.3. Chemicals
DPPH (1,1-diphenyl-2-picrylhydrazyl), BHT (2,6-di-tert-butyl-
4-methylphenol) and ascorbic acid were purchased from Sigma
(St. Louis, MO, USA). Butenolide was kindly furnished by Dr. Luiz
Claudio de Almeida Barbosa, Departamento de Química, UFV, Bra-
zil. Silica gels from Merck (Darmstadt, Germany), 100–200 and
200–425 mesh, were used for column chromatography and silica
gel Merck 60G, for thin-layer chromatography. All solvents used
were of PA and HPLC grade and purchased from Vetec (Brazil)
and Sigma, respectively.
2.4. Isolation and extraction procedure
Extract and fractions were evaporated to dryness before column
chromatography and HPLC. Extraction of the dried and powdered
seeds (850.0 g), by percolation (EtOH, 7 l, 50 h) gave a brown resi-
due (120 g, F01), that was dissolved in MeOH/H2O (3:7) and suc-
cessively extracted with C6H14 and CHCl3, resulting in 79.5, 7.3
and 21.1 g of hexane (F02), chloroform (F03) and hydroalcoholic
(F04) fractions, respectively.
F02 was chromatographed (6.4  57.0 cm column, 900 g of SiO2
170–230 mesh, with C6H14/CH2Cl2/EtOAc/MeOH/H2O, as eluents,
either neat or in mixtures of increasing polarity) to produce 340
fractions of 200 ml each, combined according to their TLC patterns,
yielding 12 groups of fractions (G-1 to G-12).
The group of fractions 6 (G-6, 730 mg, positive to Kedde re-
agent) underwent column chromatography (1.5  25 cm column,
100 g of SiO2 230–400 mesh, with CH2Cl2/EtOAc/MeOH, as eluents,
either neat or in mixtures of increasing polarity), yielding 16 sub-
groups of fractions. Subgroup 3 (192.0 mg) was purified by RP-
HPLC (Shim-pack C18 5.0 lm, 20  250 mm cartridge column, flow
rate 16 ml/min, MeCN/H2O 80:20, detection 220 nm), leading to
60.5 mg of 1. Subgroup 11 (98.9 mg) was submitted to a prepara-
tive-layer chromatography (C6H14/EtOAc 1:1), leading to 15.1 mg
of a mixture of acetogenins 2 and 3.
The group of fractions 9 (G-9, 596.0 mg, positive to Kedde re-
agent), from F02, by RP-HPLC (same conditions as above) gave 16
subgroups of fractions. Subgroups 9, 13 and 14 gave acetogenins
4 (54.0 mg, impure), 5 (38.0 mg) and 6 (14.7 mg), respectively; 4
was further purified by recrystalization using AcOEt (38.7 mg).
Part of the group of fractions 11 (G-11, from F02, 150.0 mg, po-
sitive to Kedde reagent), was purified by RP-HPLC (Shim-pack C18
5.0 lm, 20  250 mm cartridge column, flow rate 19 ml/min,
MeCN/H2O 90:10, detection 220 nm), leading to 11 subgroups of
fractions. Subgroup 9 gave acetogenin 7, a white solid (40.0 mg).
The chloroform fraction (F03, 2.0 g) was fractionated by normal
phase MPLC (using as eluents C6H14/UCH3/EtOAc/MeOH in mix-
tures of increasing polarity) to produce 54 fractions of 75 ml each,
combined according to their TLC patterns, yielding 10 groups of
fractions (G-1 to G-10).
The group of fraction 7 (G-7, 620.0 mg, positive to Kedde re-
agent) underwent column chromatography (1.5  25 cm column,
50.0 g of SiO2, 230–400 mesh, with CH2Cl2/MeOH 97:3 as eluents),
yielding 52 fractions of 15 ml each, combined according to their
TLC patterns in 19 subgroup of fractions. Subgroup 2 (45.4 mg)
was submitted to a RP-HPLC (Shim-pack C18 5.0 lm, 20  250
mm cartridge column, flow rate 15 ml/min, MeCN/H2O 70:30,
detection 220 nm), leading to acetogenin 8 (6.0 mg). Subgroup 7
(90.1 mg), from G-7, led to 15 subfractions, after being submitted
to purification by RP-HPLC (Supelco SPLC-18 5.0 lm, 10  250
mm cartridge column, flow rate 2.5 ml/min, MeCN/H2O 60:40,
detection 220 nm). Subfractions 13 and 15 gave 10.3 and 3.5 mg
of pure acetogenins 9 and 10, respectively. Subgroup 11
(84.7 mg), from G-7, by HPLC (same conditions as above) gave ace-
togenins 11 (30.4 mg, after recrystalization with EtOAc) and 12
(12.0 mg).
2.5. Determination of DPPH radical-scavenging capacity
Radical-scavenging activities of extract, fractions and pure ace-
togenins were determined according to the method described by
Burda and Oleszek (2001). Ascorbic acid (AA), butenolide and
 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138 1131

BHT (2,6-di-tert-butyl-4-methylphenol) were used as reference
compounds. Samples, AA, butenolide and BHT (750 ll) were pre-
pared in triplicate for each concentration (1.0, 10.0 and 100 lg/
ml) and, in each flask, the volume was adjusted to 2.0 ml by adding
1.5 ml of a 0.002% w/v DPPH-methanol solution. The solutions
were shaken vigorously and kept in the dark for 30 min. The con-
trol was prepared as above without any extract or substance.
Absorbances were measured at 517 nm and methanol was used
for the baseline correction.
Radical-scavenging activity was expressed as the inhibition per-
centage and was calculated as:
ðAbscontrol  Abssample Þ=Abscontrol  100
where Abscontrol = absorbance of DPPH radical in methanol and
Abssample = absorbance of the extract, fractions and pure substances
in methanol + DPPH. Scavenging activities were expressed in lg/
ml. IC50 values (in lg/ml) expressed the concentration of samples
necessary to scavenge 50% of DPPH free radicals.
IC50 values were calculated by Probit analysis (Finney, 1980).
3. Results and discussion
3.1. General
All 12 acetogenins (Fig. 1) had their planar structures deter-
mined by IR, 1H and 13C NMR spectra (Tables 1 and 2) and ESI-
MS or ESI-MS/MS (Fig. 3). IR spectra of F02, F03, G-9 and G-11, from
F02, folianin B (6) and annofolin (7) are presented in Fig. 2.
3.2. Acetogenins from F02 (hexane fraction)
3.2.1. General
Twelve groups of fractions were obtained, by submission of F02
to column chromatography. Seven acetogenins, either pure or in
mixtures, were isolated: 9-hydroxyfolianain (1), 4-desoxylongimi-
cin (2), folianin A (3), squamocin M (4), squamocin L (5), folianin B
(6) and annofolin (7).
3.2.2. Characterisation of acetogenin 1
9-Hydroxyfolianin (1, 60.5 mg), an optically active white wax,
was isolated by using RP-HPLC, from G-6, of the hexane fraction
(F02) and had its absolute configurations around THF rings and
at C-36 determined by Mosher ester methodology and by circular
dichroism, respectively (Lima et al., 2009).
3.2.3. Characterisation of acetogenins 2 and 3
A mixture of 4-desoxylongimicin B (2) and folianin A (3), two
new acetogenins, was also isolated from G-6. This mixture pre-
sented as a yellow wax, m.p. 65.9–66.8 °C and the ESI-MS showed
two [M+NH4]+ (at m/z 596.62 and 624.62) and two [M+H]+ adducts
(at m/z 579.01and 607.61). The m/z 596.62 and 579.01 adducts
(acetogenin 2) suggested the molecular formula C35H62O6 (Mr

threo threo threo

erythro threo
erythro
3

34 12 (CH2)2 9 (CH2)5 15 (CH2)11 37

CH3 (CH2)11 21 O CH3 (CH2)8 24 O
34
O O
OH 1 O OH
1 O
OH O O
OH
HO
trans trans

9-Hydroxyfolianin (1) Squamocin L (5)

threo threo threo
erythro threo
threo
3

34 12 (CH2)8 37 12 (CH2)8 37
CH3 (CH2)11 21 O CH3 (CH2)11
O 21 O
34 O
OH 1 O 1 O
O OH
OH O
trans HO
cis

Folianin A ( 2) Folianin B (6)

threo threo
threo
threo erythro
threo
3

32 11 (CH2)7 35
11
9 (CH2)5
CH3 (CH2)10 20 O CH3 (CH2)11 26 O
O O
OH 1 O 1 O
O OH OH O
OH
OH
trans trans

4-Desoxylongimicin B (3) Annonfolin (7)

threo
erythro threo
threo threo
threo
3 32 (CH2)6 3
11
37
CH3 (CH2)10 O 35
15 (CH2)11 20
CH3 (CH2)8 O
24 O OH
34 O 1 O OH 1 O
OH O OH O
trans
HO cis
trans
Isolongimicin B (8)
Squamocin M (4)

Fig. 1. Structures of acetogenins 1–12, isolated from seed ethanol extract of A. cornifolia, of ascorbic acid and butenolide.
1132 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138

threo threo
erythro

(CH2)10 3
15
CH3 (CH2)8 O
37
24
O
OH
OH OH 1 O
O
trans

Bulatacin (9)

threo threo
threo

(CH2)10 3
15
CH3 (CH2)8 37
24 O
O
OH
OH 1 O
OH O
trans

Asimicin (10)

erythro threo
threo
OH
3
34 17 (CH2)8
CH3 (CH2)6 7 (CH2)3
26 O 37
O
OH 1 O
OH O
trans

Cornifolin (11)

threo threo
erythro OH

(CH2)3 3
34 13 37
CH3 (CH2)10 O 8 (CH2)4
22
O
OH 1 O
O
OH trans
cis

Annotacin (12)

OH
OH
HO
OH
O
O O
O
Butenolide
Ascorbic Acid

Fig. 1 (continued)

578) and the latter (m/z 624.62 and 607.61 adducts) suggested the
molecular formula C37H66O6 (Mr 606, acetogenin 3). Along with a
positive Kedde test (Alali, Liu, & McLaughlin, 1999) and IR absorp-
tions, the presence of the a,b-unsaturated c-lactone moiety, for
both acetogenins, was confirmed by the signals in the 1H NMR (Ta-
ble 1) at dH 6.96 (H-33 and H-35), 4.98 (H-34 and H-36), 1.40 (H-35
and H-37), 2.26 (H-3) and 1.50-1.56 (H-4). In the 13C NMR spec-
trum (Table 2), the corresponding signals appeared at dC 173.88
(C-1), 134.42 (C-2), 25.20 (C-3), 27.45 (C-4), 148.82 (C-33 and C-
35), 77.23 (C-34 and C-36) and 19.23 (C-35 and C-37). The 13C
NMR spectrum also showed, for 2 and 3, eight signals related to
oxygenated carbons at dC 71.51, 74.12, 81.78, 82.25, 82.50, 82.85,
83.19 and 83.23. The signals at dC 71.51, 74.12 are characteristic
of hydroxylated methine carbons adjacent to THF rings; the other
six signals were attributed to hydroxylated carbons of an adjacent
bis-THF ring system (Chang & Wu, 2001). The previous assign-
ments were confirmed by the HMQC spectrum. The location of
the two THF rings, for each acetogenin, was based on ESI-MS
analysis.
Acetogenin 2 [M+H]+, adduct at m/z 579.01 (Mr 578, C35H62O6),
gave, by fragmentation, ions at m/z 211 (cleavage C-11/C-12–CO),
307 (cleavage C-15/C-16–H2) 359 (cleavage C-19/C-20–H2O–H2),
indicating the rings locations between C-11 and C-20 (Fig. 3A).
The relative configuration around THF rings of compound 2 was
established as threo/trans/threo/trans/threo on the basis of the C-
12 to C-21 chemical shifts of the proton and carbon signals and
by comparison of the 1H and 13C NMR data with those of models
of known configurations (Alali et al., 1999; Sahai et al., 1994.
Acetogenin 3 showed mass fragment patterns similar to acetog-
enin 6, described below (Lima et al., 2009), and the same molecular
formula, C37H66O6. Comparison of the NMR data, for both 3 and 6
acetogenins, revealed that the chemical shifts attributable to H-
21 and C-21 were significantly different from each other (dH
3.82–3.94 and dC 71.51, for 3; dH 3.38 and dC 73.86, for 6), clearly
indicating different stereochemistries. Based on comparison of
these NMR data with those of model compounds (Alali et al.,
1999; Sahai et al., 1994), we established, between C-20/C-21, an er-
ythro relationship for 3 (and a threo relationship for 6). Thus, the
 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138 1133

Table 1
1
H NMR [400 MHz, CDCl3, d, J (Hz)] data of 4-desoxylongimicin B (2), folianin A (3), annofolin (7), isolongimicin B (8) and annotacin (12).

H 2 3 7 8 12
3 2.26 tt (J3/4 = 7.70; 2.26 tt (J3/4 = 7.70; 2.26 tt (J3/4 = 7.58; 2.52 dtds (Jgem = 15.04; J3a/4 = 3.28; J3a/33 = J3a/34 = 1.24); 2.27 tt (J3/4 = 7.69;
J3/33 = J3/34 = 1.56) J3/35 = J3/36 = 1.56) J3/35 = J3/36 = 1.54) 2.40 dtd (Jgem = 15.04; J3b/4 = 8.32; J3b/33 = J3b/34 = 1.24) J3/35 = J3/36 = 1.58)
4 1.50–1.56 m 1.50–1.56 m 1.54 m 3.86 m 1.53 m
5 1.26 s 1.26 s 1.26 s 1.47–1.52 m 1.26 s
6 1.26 s 1.26 s 1.26 s 1.26 s 1.61 m
7 1.26 s 1.26 s 1.26 s 1.26 s 1.39–1.43 m
8 1.26 s 1.26 s 1.39–1.42 m 1.26 s 3.60 m
9 1.26 s 1.26 s 3.58 m 1.26 s 1.39–1.43 m
10 1.38–1.43 m 1.26 s 1.39–1.42 m 1.37–1.42 m 1.26 s
11 3.39 m 1.38–1.43 m 3.91 m 3.39 m 1.26 s
12 3.82–3.94 m 3.39 m 3.79–3.93 m 3.86 m 1.39–1.43 m
13 1.62 m/1.84–2.01 m 3.82–3.94 m 1.54 m /1.9 5 m 1.63 m /1.96 m 3.39 m
14 1.62 m/1.84–2.01 m 1.62 m/1.84–2.01 m 1.54 m /1.95 m 1.63 m /1.96 m 3.85 m
15 3.82–3.94 m 1.62 m/1.84–2.01 m 3.79–3.93 m 3.86 m 1.61 m /1.98 m
16 3.82–3.94 m 3.82–3.94 m 3.79–3.93 m 3.94 m 1.61 m /1.98 m
17 1.62 m/1.84–2.01 m 3.82–3.94 m 1.54 m /1.95 m 1.85 m /1.96 m 3.85 m
18 1.62 m/1.84–2.01 m 1.62 m/1.84–2.01 m 1.54 m /1.95 m 1.85 m/1.96 m 3.92 m
19 3.82–3.94 m 1.62 m/1.84–2.01 m 3.79–3.93 m 3.94 m 1.85 m /1.98 m
20 3.39 m 3.82–3.94 m 3.39 q 3.86 m 1.85 m /1.98 m
(J19/20 = J20/21 = 5.74)
21 1.38–1.43 m 3.82–3.94 m 1.32–1.35 m 1.37–1.42 m 3.92 m
22 1.26 s 1.38–1.43 m 1.26 s 1.26 s 3.85 m
23 1.26 s 1.26 s 1.26 s 1.26 s 1.39–1.43 m
24–31 1.26 s 1.26 s 1.26 s 1.26 s 1.26 s
32 0.87 t (J31/32 = 6.76) 1.26 s 1.26 s 0.88 t (J31/32 = 6.74) 1.26 s
33 6.96 dt 1.26 s 1.26 s 7.18 dt 1.26 s
(J3/33 = J33/34 = 1.56) (J3a/33 = J3b/33 =J33/34 = 1.24)
34 4.98 dq (J34/35 = 6.80; 0.87 t (J33/34 = 6.76) 0.88 t 5.05 dq (J34/35 = 6.84; J33/34 = 1.24) 0.88 t
J33/34 = 1.56) (J33/34 = 6.32) (J33/34 = 6.79)
35 1.40 d (J34/35=6.80) 6.96 dt 7.01 dt 1.43 d (J34/35 = 6.84) 6.99 dt
(J3/35 = J35/36 = 1.56) (J3/36 = J35/36 = 1.54) (J3/35 = J35/36 = 1.58)
36 – 4.98 dq (J36/37 = 6.80; 5.99 dq (J36/37 = 6.79; – 5.00 dq (J36/37 = 6.78;
J35/36 = 1.56) J35/36 = 1.54) J35/36 = 1.58)
37 – 1.40 d (J36/37 = 6.80) 1.41 d (J36/37 = 6.79) – 1.40 d (J36/37 = 6.78)

relative configuration around THF rings for 3 was found to be
threo/trans/threo/trans/erythro, from C-12 to C-21.
3.2.4. Characterisation of acetogenins 4, 5 and 6
G-9, from F02, was purified by using RP-HPLC, giving squamocin
M (4, 38.7 mg), squamocin L (5, 38.0 mg) and folianin B (6,
14.7 mg). Compound 6 was concluded to be a novel acetogenin
(Lima et al., 2009), whereas squamocins M (4) and L (5) have al-
ready been isolated from A. squamosa (Sahai et al., 1994) and from
A. cornifolia (Santos et al., 2007).
3.2.5. Characterisation of acetogenin 7
G-11, from F02, gave 40 mg of acetogenin 7 by RP-HPLC purifi-
cation (m.p. 30.8–33.8 °C; ½a25D ¼ 2:0 (c 0.185, CHCl3). The molec-
ular formula C37H66O7 (Mr 622) was proposed on the basis of the
[M+Na]+, [M+NH4]+ and [M+H]+ adducts, at m/z 645.62, 640.57
and 623.53, respectively, from ESI-MS. The characteristic signals
in the 1H and 13C NMR spectra (Tables 1 and 2), along with positive
Kedde test and IR absorptions (Fig. 2), suggested the a,b-unsatu-
rated c-lactone moiety. 1H and 13C NMR spectra (Tables 1 and 2)
also showed characteristic signals related to the presence of an
adjacent bis-THF ring system with two flanking hydroxyl groups
(Chang & Wu, 2001). This system was located between C-11 and
C-20, as indicated by fragment ions at m/z 209, 279 and 347, in
ESI-MS (Fig. 3B). The signals at dY 3.58 and dC 71.54 suggested
the presence of a third hydroxyl group along the hydrocarbon
chain (Liaw et al., 1999) and its position at C-9 was evidenced by
ESI-MS (peaks at m/z 181 and 387, Fig. 3). The relative configura-
tion for 7 from C-11 to C-20 was found to be erythro/trans/threo/
trans/threo (Alali et al., 1999; Sahai et al., 1994). The absolute con-
figuration of C-36 in compound 7 was determined according to
Gawronski and Wu (1999) methodology, by circular dichroism,
which showed a negative n–p* Cotton effect at 235 nm (De =
0.1744) and a positive p–p* Cotton effect at 213 nm
(De = 1.7194), clearly indicating a S configuration at this stereocen-
tre of the c-lactone. Based on the C-36 configuration and relative
stereochemistry, it was concluded that 7 is a novel acetogenin,
which was named annofolin.
3.3. Acetogenins from F03 (chloroform fraction)
3.3.1. General
The chloroform fraction (F03) was fractionated by normal phase
MPLC and the group of fractions 7, was further purified by using
flash chromatography and RP-HPLC, giving five acetogenins: iso-
longimicin B (8), bullatacin (9), asimicin (10), cornifolin (11) and
annotacin (12).
3.3.2. Characterisation of acetogenin 8
Isolongimicin B (8), a novel C-35 acetogenin was isolated as a
grey solid, m.p. 43.8–45.1 °C; ½a25D ¼ þ3:0 (c 0.300, CHCl3). The
molecular formula of 8 was established as C35H62O7, from the
[M+Na]+, [M+NH4]+ and [M+1]+ adducts at m/z 617.47, 612.49
and 595.49, respectively, in the ESI-MS spectrum. A positive Kedde
test (Alali et al., 1999) and IR absorptions suggested that 8 is an
annonaceous acetogenin bearing an a,b-unsaturated c-lactone
moiety. The oxymethine carbon signal at dC 70.02, in the 13C
NMR spectrum (Table 2), was reminiscent of C-4 hydroxylated ace-
togenins (Kim et al., 2001). The C-4 hydroxy function was also sup-
ported by the 1H NMR spectrum of 8 (Table 1), in which the C-3
methylene protons were observed as part of a characteristic ABX
system at dH 2.40 and 2.52. These assignments were confirmed
by the HMQC spectrum. The location of an adjacent bis-THF ring
system with two flanking OH groups, confirmed by 1H and 13C
1134 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138

Table 2
13
C NMR (100 MHz, CDCl3, d) data of 4-desoxylongimicin B (2), folianin A (3), annofolin (7), isolongimicin B (8) and annotacin (12).

C 2 3 7 8 12
1 173.88 173.88 173.84 174.60 173.82
2 134.42 134.42 134.11 131.24 134.47
3 25.20 25.20 25.02 33.37 25.22
4 27.45 27.45 27.25 70.02 27.49
5 29.12–29.79 29.12–29.79 29.04–29.64 37.42 29.21–29.77
6 29.12–29.79 29.12–29.79 29.04–29.64 25.56 22.06
7 29.12–29.79 29.12–29.79 29.04–29.64 29.34–29.71 37.35
8 29.12–29.79 29.12–29.79 37.11 29.34–29.71 71.85
9 25.67 29.12–29.79 71.54 25.61 37.58
10 33.49 25.67 37.33 33.41 29.21–29.77
11 74.12 33.51 71.26 74.12 25.00
12 83.19 74.12 83.27 83.21a 33.49
13 28.43 83.23 25.55 28.43b 74.19
14 28.96 28.43 28.88 28.89b 83.29
15 81.78 28.96 82.45 82.25a 28.47
16 81.78 82.25 82.05 82.53a 28.89
17 28.96 82.50 28.88 28.93b 82.17
18 28.43 24.62 28.36 24.55 82.48
19 83.19 24.93 82.73 82.85a 28.89
20 74.12 82.85 74.08 71.38 25.68
21 33.49 71.51 33.30 32.47 82.87
22 25.67 32.53 25.55 26.08 71.61
23 29.12–29.79 26.06 29.04–29.64 29.34–29.71 32.62
24 29.12–29.79 29.12–29.79 29.04–29.64 29.34–29.71 25.68
25–29 29.12–29.79 29.12–29.79 29.04–29.64 29.34–29.71 29.33–29.77
30 31.93 29.12–29.79 29.04–29.64 31.92 29.33–29.77
31 22.69 29.12–29.79 29.04–29.64 22.69 29.33–29.77
32 14.10 31.93 31.74 14.11 31.88
33 148.82 22.69 22.50 151.78 22.64
34 77.23 14.10 13.98 77.97 14.06
35 19.23 148.82 148.87 19.13 148.78
36 – 77.23 77.34 – 77.33
37 – 19.23 19.08 – 19.24
a,b
Assignments may be interchanged within the column.

NMR data (Tables 1 and 2), between C-11 and C-20, was based on
the peaks at m/z 199 (cleavage C-19/C-20), 375 (cleavage C-19/C-
20–H2O–H2) in the ESI-MS spectrum. The peaks at m/z 121 (cleav-
age C-4/C-5–H2O–H2) and m/z 453 (cleavage C-4/C-5) are consis-
tent with the hydroxyl at C-4 (Fig. 3C). The stereochemistry of
the bis-THF portion was determined to be threo/trans/threo/cis/ery-
thro (Alali et al., 1999; Sahai et al., 1994). The cis stereochemistry
for C-16/C-19 was established on the basis of the chemical shifts
of H-17 and H-18, in the 1H NMR spectrum (Fujimoto et al., 1994).
3.3.3. Characterisation of acetogenins 9, 10 and 11
Two known acetogenins, 9 and 10, were reported, for the first
time, from A. cornifolia (Lima et al., 2009), although they had al-
ready been isolated from Annona bullata and Asimina triloba,
respectively (Rupprecht, Hui, & McLaughlin, 1990). Cornifolin
(11) was only previously isolated from A. cornifolia (Santos et al.,
2006).
3.3.4. Characterisation of acetogenin 12
Annotacin (12), a yellow wax, m.p. 39.5–41.1 °C, ½a25 D ¼ þ12:3
(c 0.4, CHCl3) showed a molecular formula C37H66O7 (Mr 622), sug-
gested by the adduct molecular ions [M+H]+, at m/z 623.47, and
[M+NH4]+, at m/z 640.50, in the ESI-MS spectrum. The positive
response to Kedde reagent and the IR carbonyl absorption,
at 1752 cm1, indicated the presence of a c-methyl-a,b-unsatu-
rated c-lactone, confirmed by signals in the 1H and 13C NMR spec-
tra (Tables 1 and 2). The signals in these spectra also suggested the
presence of an adjacent bis-THF ring system with two flanking OH
groups (Chang & Wu, 2001) and the additional signals at dY 3.60
and dC 71.85 are characteristic of the presence of a third hydroxyl
group, along with the hydrocarbon chain. Fragment ions at m/z
247, 265, 335, 387 and 181, in the ESI-MS/MS spectrum, indicated
that the bis-THF system is located between C-13 and C-22 and frag-
ment ions at m/z 167, 177 and 195 evidenced that the third hydro-
xyl is located at C-8 (Fig. 3D). The relative threo/trans/threo/cis/
erythro stereochemistries from C-13 to C-22 of 12 were supported
by comparison of 1H and 13C NMR data of model compounds (Alali
et al., 1999; Sahai et al., 1994). The absolute configuration of C-36
in acetogenin 12 was also determined according to Gawronski and
Wu (1999) methodology, by circular dichroism, which showed a
negative n–p* Cotton effect at 238 nm (De = 0.6581) and a posi-
tive p–p* Cotton effect at 209 nm (De = 5.9210), clearly indicating
an S configuration at this stereocentre of the c-lactone. Based on
the C-36 configuration and relative stereochemistry, it was con-
cluded that annotacin (12) is a novel acetogenin.
3.4. DPPH radical-scavenging activity
The ethanol extract, fractions and acetogenins obtained from A.
cornifolia were examined for their radical-scavenging activity to-
ward the stable free radical DPPH and the results are presented
in Fig. 4. Their IC50 values are presented in Table 3. Acetogenins
presented a better radical-scavenging activity and IC50 than did
BHT, a commercial antioxidant, and IC50 values similar to those
of ascorbic acid (AA) and butenolide, all three used as positive con-
trols. Analysis of Fig. 4 showed an antiradical scavenger effect of
the acetogenins, less dose-dependent than those presented by AA
and BHT. Thus, despite the fact that the scavenging activity of AA
was greater than those of acetogenins, at 100 and 10 lg/ml, their
IC50 values were similar. The ethanol extract (F01), as well as the
chloroform fraction, F03, showed a very good antioxidant activity
at 100 lg/ll and low IC50, contrasting with the hexane fraction
 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138 1135

Hexane fraction (F02) Chloroform fraction (F03)

Group of fractions 9, from F02 Group of fractions 11, from F02

106,0 105,0
100
100
95 3595

95
90
3424
1172
90 85 3423 1398
1372 1202 813 745
1145

85 1415 80 1464 911 840

1373 789 1318 928 785
1319
75 1119 889 723
%T 80 1462 1267
875 721
%T 949
861

70
75 1116

65 2850
2853
70 929
60 1017

1180 2919 1050

1027
65 55 1744

2923

60 50
1752 1066

45
55,0 43,0
4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650,0 4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 650,0
cm-1 cm-1

Folianin B (6) Annofolin (7)

Fig. 2. IR spectra of hexane fraction (F02), chloroform fraction (F03), G-9 and G-11, from F02, folianin B (6) and annofolin (7).

(F02), which presented low activity (and high IC50). G-6, G-9 and G- in this fraction (ca. 2.9% of the total extract). However, the chlo-
11 were much more active than was F02, while G-7 showed a roform fraction (F03), with 6.7% of acetogenins in the total ex-
slight decrease of activity compared to F03. The low IC50 observed tract, was one hundred times more active than was F02 and this
for F01 may be explained by the presence of several phenolic com- fact could also be attributed, as for F01, to the presence of phenolic
pounds in the total ethanol extract, and the high IC50 observed for compounds in this fraction. In confirmation, G-6, G-9, G-11 and G-
F02 may be associated with the low concentration of acetogenins 7 showed similar activities.
1136 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138

- H2O
A (379) 359
- H2
- H2O +
(269) 249
- H2 - CO
(239) 211
3

32 11 (CH2)7 35
CH3 (CH2)10 20 O
O
1 O
OH O
H+
OH
- 2 H2O - CO
(409) 373 345
- H2O - H2
289 307 (309)

B 279 (297) (325)

- H2O
307
- H2O

(227) +
209 - H2O
- H2O (255) 237
OH
34
20
11 (CH2) 9 (CH2)5 37
CH3 (CH2)12 O
O H+
OH 1 O
HO O
- H2O
(395) 377
- H2
347 - H O (367)
2

- 3 H2O
387 (441) 181

- CO2
C - H2O
(255) 211

249 +
- H2 (269) 453
199

32 (CH2)6 3
11
CH3 (CH2)10 O 35
20
O
OH H+
OH 1 O
OH O

- H2O - H2O - H2O

(395)
- H2 375 357 (141)
- H2
121

- H2O - H2O
(325) 305 287
- H2

D (353)
- H2O
335
- H2O
317

+
181 (199)
- H2O
OH
34
CH3 (CH2)10 13 (CH2)4 8 (CH2)4 37
22 O
O H+
1 O
OH O
HO
-2 H2O 167
(423) 387
(197) 195 177
- H2 - H2O

(283) 265 247

- H2O - H2O

Fig. 3. Diagnostic ESI-MS/MS fragment ions (m/z). (A) 4-Desoxylongimicin B (2); (B) annofolin (7); (C) isolongimicin B (8); (D) annotacin (12). Peaks in parentheses were not
observed.
 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138 1137

75 Table 3
1 µg/mL IC50 values on DPPH-scavenging activities of extract, fractions and pure acetogenins
70 10 µg/mL of A. cornifolia A. St.-Hil. and reference compounds.
100 µg/mL
65
Extract, fractions and IC50 (lg/ Extract, fractions IC50 (lg/
60
acetogenins ml)a and acetogenins ml)a
55
EtOH extract (F01) 33.9 ± 6.26 Folianin B (6) 1.54 ± 0.37
% Antioxidant activity

50 Hexane fraction (F02) 1104 ± 31.5 Annofolin (7) 1.12 ± 0.16

45 Chloroform fraction (F03) 14.7 ± 3.05 Isolongimicina B 1.66 ± 0.33
(8)
40 G-6 from F02 26.6 ± 4.10 Bullatacin (9) 1.29 ± 0.23
35 G-9 from F02 36.4 ± 8.71 Asimicin (10) 0.99 ± 0.18
30 G-11 from F02 22.8 ± 3.80 Cornifolin (11) 1.36 ± 0.29
G-7 from F03 24.2 ± 4.32 Anotacin (12) 1.76 ± 0.42
25 9-Hydroxyfolianin (1) 1.23 ± 0.21 BHT 16.36 ± 3.63
20 4-Desoxilongimicin B (2) 1.95 ± 0.34 Ascorbic acid 1.62 ± 0.35
and Folianin A (3)
15
Squamocin M (4) 1.73 ± 0.39 Butenolide 2.21 ± 0.43
10 Squamocin L (5) 1.29 ± 0.25
5 a
Values are means ± SD of triplicate determinations.
0
F01 F02 F03 G-6 G-9 G-11 G-7

80
1 µg/mL proposed for AA establishes a reaction of the conjugated base AH
75 10 µg/mL (the form in which the ene-diol structure of AA exists, at physio-
100 µg/mL
70 logical pH) as a one-electron donor in free radical-scavenging,
65 transforming into an ascorbyl radical AH (AH + X ? AH + X).
60 This radical deprotonates to A, which is highly stabilized via elec-
55 tron delocalization (Wagner et al., 2008). However, the in vitro
% Antioxidant activity

50 mechanism of action of AA should be more similar to that of the

45 lipid peroxidation, in which allylic hydrogens are involved. Acetog-
40 enins should act in the same way, since they also possess allylic
35 hydrogens, as well as the stabilization via electron delocalization
30 in the a,b-insaturated lactone ring moiety. A piece of evidence to
25 support these observations is ascorbigen, which naturally occurs
20 in Brassica vegetables, and contains hydroxyl groups attached to
1
15
a saturated lactone ring. This compound exhibited very little
scavenging activity towards DPPH free radicals (Wagner et al.,
10
2008).
5
The results found in the present work are also interesting con-
0
1 6 8 cerning the report on the antiulcer activity of A. cornifolia fruits
2+3 4 5 7
(Correa, 1974), since vitamin C and E supplementations lead to
100 some short-term protective effects on Helicobacter pylori-induced
95 1 µg/mL
10 µg/mL gastritis in guinea pigs (Sjunnesson, Sturegard, Willén, & Wads-
90 100 µg/mL
85
tröm, 2001).
80
75 4. Conclusion
% Antioxidant activity

70
65
This work presented the isolation and identification of twelve
60
55 acetogenins, pure or in mixtures, from seed ethanol extract of A.
50 cornifolia A. St.-Hil. Five are novel acetogenins, not yet described.
45 Their structures were determined using IR, 1D and 2D 1H and 13C
40 NMR and ESI-MS experiments. The absolute configuration at C-
35
36 was established for two novel acetogenins by means of circular
30
25
dichroism. The DPPH radical-scavenging activity of the ethanol ex-
20 tract, fractions and pure acetogenins revealed that they present
15 antioxidant capacity, with IC50 at the same level as ascorbic acid.
10 This is the first time, to the best of our knowledge, that this activity
5 is described for this class of natural compounds and encourages
0
additional studies on species of this family, in order to evaluate
9 10 11 12 BHT AA Bu
the possibilities of using them as natural sources for the develop-
Fig. 4. DPPH radical-scavenging activities of ethanol extract (F01), fractions (F02, ment of dietary supplements.
F03, G-6, G-9, and G-11) and acetogenins 1–12, from A. cornifolia, BHT, ascorbic acid
(AA), and butenolide (Bu), at three different concentrations. Acknowledgements

Thanks go to FAPEMIG, for financial support and CNPq, for

The DPPH antiradical activity of the acetogenins may be related LARSL and MADB grants and to Adriano M.C. Pimenta and Marcos
to the a,b-insaturated lactone ring moiety, also present in ascorbic Eberlin, for the ESI Mass spectra and to Anita Marsaioli, for the CD
acid (AA) and butenolide. The intracellular antioxidant mechanism spectra.
1138 Luciana A.R. Santos Lima et al. / Food Chemistry 122 (2010) 1129–1138

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