LC-MS and NMR Analyses of Neurotoxic Fruits in the Annonaceae Family Robert E. Smith 1* , Robert A. Levine 1 , Kevin Tran 1 , Kristy M. Richards 1 , Sean Ryan 1 , Rensheng Luo 2 , José Guilherme S. Maia 3 , Armando A.U. Sabaa-Srur4 , Maria Rosa de Moraes 5 , Helena T. Godoy 5 , Ingrid de Moraes 6 , Flávio L. Schmidt 5 and Andrew Thomas 7 1 The U.S. Food and Drug Administration, Lenexa, KS 66214 2 University of Missouri – St. Louis, One University Drive, St. Louis, MO 63121 3 Universidade Federal do Pará, Brasil 4 Federal University of Rio de Janeiro, Brazil 5 Campinas State University (UNICAMP) 6 Embrapa Agroindústria Tropical, Fortaleza, CE, Brazil 7 University of Missouri - Southwest Research Center* - Author to whom correspondence should be addressed: [email protected]. gov Abstract Overconsumption of graviola ( Annona muricata ) caused an atypical form of Parkinson’s disease to emerge in the Caribbean island of Guadeloupe and the Pacific islands of Guam and New Caledonia [1, 2]. Graviola and the North American pawpaw ( Asimina triloba) contain the neurotoxins, annonacin and 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 squamosax Annona Cherimola ), ata ( Annona squamosa ) and biribá ( Annona mucosa), indicated the presence of the toxicophore that is present in annonacin, squamocin and other known acetogenins [5]. NMR analysis of extracts of graviola leaves indicated that they also contain this toxicophore. This is important because they are used to treat or cure cancer [6]. Analysis by LC-MS indicated that not only annonacin and squamocin, but also isomers of them were present in almost all the samples. Moreover, pressu rized liquid extraction with dry 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, in the current study 7724 and 162 µg/g annonacin and squamocin were found in lyophilized pawpaw fruit pulp. Even more annonacin (1034 µg/g) was found in one sample of graviola fruit, while much less was found in lyophilized ata and biribá fruit pulps (2.70 and 2.21 µg/g, respectively). On the other hand, atemoia fruit had 76.2 µg/g of squamocin and the seeds had 14,201 µg/g. Also, graviola stems and leaves, as well as pawpaw twigs that are sold over the internet as cures for cancer had annonacin and/or squamocin in them. Finally, the concentrations of metals in the lyophilized fruit pulps were determined by inductively coupled plasma atomic emission spe ctroscopy, or IC P-AES. Materials and Methods Lyophilized graviola, atemoya ( A. squamosax A. cherimola), ata ( A. squamosa) and biribá ( Rollinia mucosa) lyophilized fruits were from Rio de Janeiro, São Paulo, Pilar do Sul and Belém, Brazil. Paw paw (A. triloba) fruit was from Andrew Thomas at the University of Southwest Missouri. They were extracted with methanol at 100 o C and 10 Mpascal (100 atm) pressure using an Accelerated Solvent Extractor (ASE, ThermoFisher, Sunnyvale, CA). A portion was partitioned between water and chloroform (CHCl 3 ). The extracts were analyzed by LC-HRMS and NMR. Another portion of each lyophilized fruit pulp was analyzed for metals by ICP-AES. Total phenolics were quantified using the Folin-Ciocalteu reagent [7]. References [1] D. Caparros-Lefebvre, A. Elbaz. “Possible relation of atypical parkinsonism in the French West Indies with consumption of tropical plants: a case-control study.” Lancet vol. 354, pp. 281-286, 1999. [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. 63–69, 2004. [3] P. Champy et al. ”Quantification of acetogenins in Annona muricatalinked to atypical parkinsonism in Guadeloupe”. Movement Disorders vol. 20, pp. 1629–1633, 2005. [4] L.F. Potts et al. “Annonacin in Asimina trilobafruit: Implication for neurotoxicity”. Neurotoxicol vol. 33, pp. 53-58, 2012. [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. Höllerhage et al. “Potential neurotoxicity of dietary supplements sold as cancer treatments”. Submitted to NeuroToxicology, 20 14. Acknowledgements:Annonacin and squamocin standards were provided by Pierre Champy at the UniversitéParis-Sud. 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. Figure 2. 1 H-NMR of lipophilic portion of pawpaw fruits. Peaks: 1: –CH 2 of THF ring 2: -C HO- of triglycerides 3: –H 2 C=CH- 4: HC=CH 5 and 6: – HC=C- of α,β-unsaturated γ-lactones (toxicophore s) in acetogenins 7: CDCl 3 . Thus, it may contain potentially neurotoxic acetogenins Annonacin C 35 H 64 O 7 Monoisotopic MW 596.4652 Fr ui t Gr aviola a Atemoya Ata Biribá Pawpaw b Metal mg/kg mg/kg mg/kg mg/kg mg/kg Ca 810 753 1110 815 450 K15000 10200 10900 8230 730 Na 123 22.4 14.3 20.6 10.9 P 1380 1330 1690 1480 1290 Cu 6.12 4.74 2.73 1.75 7.35 Fe 12.3 6.42 7.44 8.41 11.5 Mg 980 551 721 750 419 Mn 4.70 4.67 1.86 2.60 3.35 Zn 7.14 5.87 5.66 7.32 5.70 Cr 0.587 0.235 0.235 0.220 0.07 Ni 0.234 0.195 0.050 0.046 0.52 a Avg of two samples b Avg of three s amples Fruit Annonacin (µg/g dry weight) Squamocin (µg/g dry weight) Graviola pulp, Rio de Janeiro, Brazil 323 113 Graviola pulp from Belém, Brazil 631 127 Graviola pulp from São Paulo, Brazil 1034 6.1 Atemoya pulp from Belém, Brazil 3.8 68.0 Atemoya seeds from Belém, Brazil 454 14201 Atemoya pulp from Pilar do Sul, Brazil 3.68 76.2 Ata pulp from Belém, Brazil 2.70 0.85 Biribá pulp from Belém, Brazil 2.21 5.28 Pawpaw pulp from Missouri 7724 162 Squamocin C 37 H 66 O 7 Monoisotopic MW 622.4808 Results and Discussion. The concentrations of annonacin and squamocin in the different samples are listed in Table 1. There were substantial differences in graviola fruits from different parts of Brazil. However, the North American pawpaw had much more annonacin than the other fruits. Moreover, over six times as much annonacin was solubilized using methanol at 100 o C and 10 MPa (100 atm) pressure than extractions done under ambient conditions in a previous study [4]. The same extraction method was also able to solubilize many more phenolic compounds in lyophilized graviola leaves than sonication. That is 84 mg GAE/g were solubilized using hot, pressurized methanol, compared to 6 mg GAE/mg when extracted at 40 o C with 1:1 ethanol:water using ultrasound. The highest concentration of squamocin was in atemoya seeds. Pictures of the fruits are shown below the Table. The concentrations of metals and total phosphorus are in Table 2. They are all good sources of potassium (K), magnesium (Mg), calcium (Ca) and phosphorus (P). The LC-HRMS chromatogram of the methanolic extract of graviola fruit pulp is shown in Fig. 1. Not only annonacin and squamocin, but also isomers of them were detected. We only had standards of annonacin and squamocin, so only these two acetogenins were quantified. The 1 H-NMR spectrum of the methanolic extract of pawpaw fruit is shown in Fig. 2. Peaks were seen with chemical shifts due to the – HC=C– in α,β-unsaturated γ-lactones (toxicophores) in acetogenins. There were only very small peaks due to the –CH 2 O– in triglycerides. Finally, extracts of graviola, atemoya, ata and biribá were found to be neurotoxic in a recent study, as were dietary supplements made from graviola and pawpaw twigs, stems and leaves [8]. Table 2 Table 1
