LC-MS and NMR Analyses of Neurotoxic Fruits in the Annonaceae Family

Robert E. Smith1*, Robert A. Levine1, Kevin Tran1, Kristy M. Richards1, Sean Ryan1, Rensheng Luo2, Jos Guilherme S. Maia3, Armando A.U. Sabaa-Srur4,
Maria Rosa de Moraes5, Helena T. Godoy5, Ingrid de Moraes6, Flvio L. Schmidt5 and Andrew Thomas7
1 The U.S. Food and Drug Administration, Lenexa, KS 66214
2 University of Missouri St. Louis, One University Drive, St. Louis, MO 63121
Abstract
3 Universidade Federal do Par, Brasil
4 Federal University of Rio de Janeiro, Brazil
Overconsumption of graviola (Annona muricata) caused an atypical form of
5 Campinas State University (UNICAMP)
Parkinsons disease to emerge in the Caribbean island of Guadeloupe and the
6 Embrapa Agroindstria Tropical, Fortaleza, CE, Brazil
Pacific islands of Guam and New Caledonia [1, 2]. Graviola and the North
7 University of Missouri - Southwest Research Center
American pawpaw (Asimina triloba) contain the neurotoxins, annonacin and
* - Author to whom correspondence should be addressed: robert.smith@fda.hhs.gov
squamocin [3, 4], which are in the class of compounds called acetogenins. NMR
analysis of extracts of three Brazilian fruits from the same family (Annonaceae),
atemoia (Annona squamosa x Annona Cherimola), ata (Annona squamosa) and
birib (Annona mucosa), indicated the presence of the toxicophore that is present
Table
in annonacin, squamocin and other known acetogenins [5]. NMR analysis of
extracts of graviola leaves indicated that they also contain this toxicophore. This is
2
a
important because they are used to treat or cure cancer [6]. Analysis by LC-MS
Fruit Graviola Atemoya
Ata
Birib Pawpawb
indicated that not only annonacin and squamocin, but also isomers of them were
Metal
mg/kg
mg/kg
mg/kg mg/kg
mg/kg
present in almost all the samples. Moreover, pressurized liquid extraction with dry
Ca
810
753
1110
815
450
methanol solubilized over 20 times as much annonacin in the pawpaw fruit than
what was reported previously by extracting under ambient conditions [4]. That is,
K
15000
10200
10900
8230
730
in the current study 7724 and 162 g/g annonacin and squamocin were found in
Na
123
22.4
14.3
20.6
10.9
1
lyophilized pawpaw fruit pulp. Even more annonacin (1034 g/g) was found in
Figure
2.
H-NMR
of
lipophilic
portion
of
pawpaw
fruits.
P
1380
1330
1690
1480
1290
one sample of graviola fruit, while much less was found in lyophilized ata and
Peaks: 1: CH2 of THF ring 2: -CHO- of triglycerides 3: H2C=CH- 4: HC=CH
birib fruit pulps (2.70 and 2.21 g/g, respectively). On the other hand, atemoia
Cu
6.12
4.74
2.73
1.75
7.35
5
and
6:

HC=Cof
,-unsaturated
-lactones
(toxicophores)
in
acetogenins
fruit had 76.2 g/g of squamocin and the seeds had 14,201 g/g. Also, graviola
Fe
12.3
6.42
7.44
8.41
11.5
7: CDCl3. Thus, it may contain potentially neurotoxic acetogenins
stems and leaves, as well as pawpaw twigs that are sold over the internet as cures
Mg
980
551
721
750
419
for cancer had annonacin and/or squamocin in them. Finally, the concentrations of
Results and Discussion. The concentrations of annonacin and squamocin in the different
metals in the lyophilized fruit pulps were determined by inductively coupled
Mn
4.70
4.67
1.86
2.60
3.35
samples are listed in Table 1. There were substantial differences in graviola fruits from
plasma atomic emission spectroscopy, or ICP-AES.
Zn
7.14
5.87
5.66
7.32
5.70
different parts of Brazil. However, the North American pawpaw had much more annonacin
Cr
0.587
0.235
0.235
0.220
0.07
than the other fruits. Moreover, over six times as much annonacin was solubilized using
Materials and Methods
o
methanol
at
100
C and 10 MPa (100 atm) pressure than extractions done under ambient
Lyophilized graviola, atemoya (A. squamosa x A. cherimola), ata (A.
Ni
0.234
0.195
0.050
0.046
0.52
conditions in a previous study [4]. The same extraction method was also able to solubilize
squamosa) and birib (Rollinia mucosa) lyophilized fruits were from Rio de
a Avg of two samples
many more phenolic compounds in lyophilized graviola leaves than sonication. That is 84 mg
Janeiro, So Paulo, Pilar do Sul and Belm, Brazil. Paw paw (A. triloba) fruit
b Avg of three samples
GAE/g were solubilized using hot, pressurized methanol, compared to 6 mg GAE/mg when
was from Andrew Thomas at the University of Southwest Missouri. They were
o
o
extracted
at
40
C with 1:1 ethanol:water using ultrasound. The highest concentration of
extracted with methanol at 100 C and 10 Mpascal (100 atm) pressure using an
squamocin was in atemoya seeds. Pictures of the fruits are shown below the Table. The
Accelerated Solvent Extractor (ASE, ThermoFisher, Sunnyvale, CA). A portion
concentrations of metals and total phosphorus are in Table 2. They are all good sources of
was partitioned between water and chloroform (CHCl3). The extracts were
potassium (K), magnesium (Mg), calcium (Ca) and phosphorus (P). The LC-HRMS
analyzed by LC-HRMS and NMR. Another portion of each lyophilized fruit
chromatogram of the methanolic extract of graviola fruit pulp is shown in Fig. 1. Not only
pulp was analyzed for metals by ICP-AES. Total phenolics were quantified
annonacin and squamocin, but also isomers of them were detected. We only had standards of
using the Folin-Ciocalteu reagent [7].
annonacin and squamocin, so only these two acetogenins were quantified. The 1H-NMR
Table
spectrum of the methanolic extract of pawpaw fruit is shown in Fig. 2. Peaks were seen with
Fruit
Annonacin
Squamocin
chemical shifts due to the HC=C in ,-unsaturated -lactones (toxicophores) in
1
(g/g dry weight) (g/g dry weight)
acetogenins. There were only very small peaks due to the CH2O in triglycerides. Finally,
extracts of graviola, atemoya, ata and birib were found to be neurotoxic in a recent study, as
Graviola pulp, Rio de Janeiro, Brazil
323
113
Annonacin C35H64O7
were dietary supplements made from graviola and pawpaw twigs, stems and leaves [8].
Graviola pulp from Belm, Brazil
631
127
Monoisotopic MW 596.4652
References
Graviola pulp from So Paulo, Brazil
1034
6.1
[1] D. Caparros-Lefebvre, A. Elbaz. Possible relation of atypical parkinsonism in the French West Indies with
Atemoya pulp from Belm, Brazil
3.8
68.0
consumption of tropical plants: a case-control study. Lancet vol. 354, pp. 281-286, 1999.
Atemoya seeds from Belm, Brazil
454
14201
[2] Champy P et al. Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal
neurodegeneration in rats: possible relevance for atypical parkinsonism in Guadeloupe. J Neurochem vol. 88, pp.
Atemoya pulp from Pilar do Sul, Brazil 3.68
76.2
6369, 2004.
Ata pulp from Belm, Brazil
2.70
0.85
[3] P. Champy et al. Quantification of acetogenins in Annona muricata linked to atypical parkinsonism in
Guadeloupe. Movement Disorders vol. 20, pp. 16291633, 2005.
Birib pulp from Belm, Brazil
2.21
5.28
[4] L.F. Potts et al. Annonacin in Asimina triloba fruit: Implication for neurotoxicity. Neurotoxicol vol. 33, pp. 53-58,
Pawpaw pulp from Missouri
7724
162
2012.

Figure 1. LC-HRMS of methanolic extract

of lyophilized graviola pulp showing peaks
+
due to Na adducts of acetogenins with
masses of 619.46, 615.42, 617.44, 633.43
and 635.45. Every peak is due to a
neurotoxic acetogenin or one of its isomers.

Squamocin C37H66O7
Monoisotopic MW 622.4808

[5] R. Luo et al. NMR Analysis of Potentially Neurotoxic Annonaceous Fruits. The Natural Products Journal vol. 2,
pp. 230-241, 2013.
[6] D.M. Hansra et al. "Patient with Metastatic Breast Cancer Achieves Stable Disease for 5 Years on Graviola and
Xeloda after Progressing on Multiple Lines of Therapy." Advances in Breast Cancer Research vol. 3, p. 84-87, 2014.
[7] V. L. Singleton, R. Orthofer, R. M. Lamuela-Raventos, Analysis of total phenols and others oxidation substrates
and antioxidants by folin-ciocauteau reagent. Methods in Enzymology, vol. 299, p. 152, 1999.
[8] M. Hllerhage et al. Potential neurotoxicity of dietary supplements sold as cancer treatments. Submitted to
NeuroToxicology, 2014.

Acknowledgements: Annonacin and squamocin standards were provided by Pierre Champy at the
Universit Paris-Sud.