Letter pubs.acs.org/JAFC
Determination of Neurotoxic Acetogenins in Pawpaw (Asimina triloba) Fruit by LC-HRMS ABSTRACT: The concentrations of the neurotoxins, annonacin and squamocin, were determined in a lyophilized sample of the fruit pulp of the North American pawpaw (Asimina triloba) by LC coupled to high resolution mass spectrometry or LC-HRMS. The sample was extracted using dry methanol at 100 °C and 10 MPa pressure in a sealed container. The extraction of annonacin and squamocin was optimal at 100 °C with 7.72 and 0.162 mg/g, respectively, being found. Also, several isomers of annonacin and squamocin were separated and detected but not quantified. KEYWORDS: pawpaw, Asimina triloba, Parkinson’s disease, LC-HRMS, annonacin, squamocin
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conditions.5 In contrast, pressurized liquid extraction (PLE) using an accelerated solvent extractor (ASE) can solubilize much more material in many samples.13 This includes açai ́ (Euterpe oleracea), black olives (Olea europaea), elderberries (Sambucus nigra subsp. canadensis), noni (Morinda citrifolia), soursop (Annona muricata), atemoya (A. squamosa x A. cherimola), sugar apple (A. squamosa), biribá (A. mucosa), and pawpaw (Asimina triloba) fruit pulps as well as mangosteen (Garcinia mangostana) mesocarps and peels and mango (Mangifera indica) and Spanish plum (Spondias purpúrea) peels.14 In this paper, lyophilized pawpaw fruit pulp was extracted with dry methanol at 100 °C and 10 MPa pressure in a sealed container and then analyzed for the concentrations of annonacin and squamocin by LC-HRMS. The first aim of this work was to compare the amounts of annonacin and squamocin that could be extracted at 23, 60, and 100 °C and compare those to the amounts found in a previous study in which the methanol extraction was done under ambient conditions.5 The second aim was to use LC-HRMS to separate annonacin and squamocin from their isomers and to quantify them. It is important to improve the existing analytical methods for determining these two neurotoxins, because the previous methods did not extract all the annonacin and squamocin nor separate them from their isomers.5 So, the potential neurotoxicity of pawpaw fruits may have been underestimated.5
INTRODUCTION The fresh fruit pulp of the North American pawpaw (Asimina triloba) is cultivated and consumed in Missouri, Illinois, Indiana, Ohio, Pennsylvania, Delaware, Virginia, West Virginia, Kentucky, Tennessee, Arkansas, Louisiana, Missisippi, Alabama South Carolina, North Carolina, and parts of the Florida panhandle.1 It grows best in areas that have hot summers and cold winters.2 It is in the Annonaceae family, which contains mostly tropical fruits like graviola (soursop).2,3 However, sometimes it is confused with the papaya (Carica papaya),4 which is a completely different fruit. It contains some vitamins and minerals at higher concentrations than apples, grapes, and peaches.1 The color of the fruit changes from “creamy white through bright yellow to shades of orange”.1 It tastes like a mixture of bananas, mangos, and pineapples and has a powerful aroma when ripe.1 Although they are often consumed locally, there are some commercial-sized orchards in Kentucky and Ohio, including one which processed and sold over 400 kg of pulp in 2009.2 Moreover, dietary supplements containing twigs and stems or extracts of pawpaws are sold over the Internet as treatments for cancer because they contain acetogenins that have anticancer properties.3 However, acetogenins are also neurotoxic and may cause tau pathologies, including an atypical form of Parkinson’s disease that does not respond to the standard medication, L-DOPA.5 Pawpaw fruit was reported to contain the known neurotoxin, annonacin, at a concentration of about 30 μg/mL.5 The analysis was done using matrix assisted laser desorption and ionization coupled to time-of-flight mass spectrometry or MALDI-TOF MS.1 It was extracted from the fruit using methanol under ambient conditions and then partitioned into ethyl acetate.6 This ethyl acetate extract was found to be lethal to 50% of the cortical neurons grown in cell cultures at a dose of about 48 μg/ mL2. So, it may be similar to graviola (Annona muricata), which may have caused atypical parkinsonism on the Caribbean island of Guadeloupe and the Pacific islands of Guam and New Caledonia.7−11 However, there were some disadvantages to the method used to quantify annonacin in pawpaw fruit.5 First, no column was used to separate annonacin from its isomers. That is, MALDITOF MS separates analytes based on the time that it takes for them to travel through the flight tube, which depends on their molecular weights.12 So, isomers that have the same molecular weight will appear as one peak in the data. The second problem is that the methanol extraction was done under ambient © 2015 American Chemical Society
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MATERIALS AND METHODS
Pawpaw fruits of the ‘Overleese’ cultivar were harvested Sept. 27, 2013 from a research orchard in southwest Missouri (lat. 37.085845, long. −93.867079). The orchard was established in 2003 on an excellent alluvial soil using both pregrafted trees and seedling trees that were later grafted to improve fruit-producing cultivars. Fruits were harvested at peak ripeness. The skin and seeds were separated from the pulp, which was ground, lyophilized, and provided to the FDA lab in Kansas for analysis. Annonacin and squamocin standards were provided by Pierre Champy at the University of Paris, South, after being analyzed by NMR and mass spectrometry.5,15−17 About 10 g of lyophilized pawpaw fruit pulp were mixed with enough HydroMatrix (SigmaAldrich, St. Louis, MO) to fill the 100 mL stainless steel sample cell used in an Accelerated Solvent Extractor Received: Revised: Accepted: Published: 1053
September 17, 2014 January 7, 2015 January 16, 2015 January 16, 2015 DOI: 10.1021/jf504500g J. Agric. Food Chem. 2015, 63, 1053−1056
Letter
Journal of Agricultural and Food Chemistry
μm particle size and dimensions of 2.1 × 150 mm was used with a gradient elution. It started with CH3OH and water containing 0.1% formic acid at a flow rate of 0.300 mL/min. For the first 25 min of the first gradient, an isocratic mobile phase of 4:1 CH3OH:H2O (v/v) was used. This was changed linearly to 49:1 CH3OH:H2O (v/v) from 25 to 30 min and held at this mixture from 30 to 35 min, followed by a step change back to 4:1 CH3OH:H2O (v/v) that was held from 35 to 45 min. An Orbitrap Exactive HRMS (Thermo Scientific, Sunnyvale, CA) was used with heated electrospray ionization (HESI) at 350 °C and a nitrogen gas sheath flow 45 arbitrary units, auxiliary nitrogen gas flow 12 arbitrary units. For negative ionization, the following parameters were used: spray voltage 2.60 kV, capillary temperature 325 °C, capillary voltage −35.00 V, tube lens voltage −80.00 V, skimmer voltage −18.00 V. For positive ionization, the following parameters were used: spray voltage 3.50 V, capillary temperature 325 °C, capillary voltage 32.50 V, tube lens voltage 100.00 V, skimmer voltage 16.00 V. Its resolution at m/z 400 was 100,000 full width at half-maximum peak height (fwhm). It used an internal mass lock. The mass range was 100−1000 m/z with a target of 1,000,000 charges and a maximum ionization time of 250 ms. It was able to rapidly switch between positive and negative ionization, so H+, Na+, and formate (HCOO−) adducts could be detected. So, for annonacin, masses in the range of 597.47071−597.47549, 619.45251−619.45747, and 641.46397−641.46911 were monitored for [M + H]+, [M + Na]+, and [M+HCOO]−, respectively. For squamocin, masses in the range of 623.48628−623.49126, 645.46808−645.47324, and 667.47985− 667.48519 Da were monitored for [M + H]+, [M + Na]+, and [M +HCOO]−, respectively. Annonacin and squamocin standards from Pierre Champy at the University of Paris were used to identify and quantify the peaks due to annonacin and squamocin that eluted at 19.74 and 22.51, min, respectively. Calibration curves were obtained using 10−1000 ng/mL annonacin and squamocin, assuming that the standards were 100% pure. Samples were diluted until they produced peaks with areas that fell within the range of the calibration standards.
Figure 1. 1H NMR in CD3OD of the methanolic extract of lyophilized pawpaw fruit pulp. Peaks: 1: −CH3 2: -(CH2)n 3: HCO of sugars and acetogenins. (ASE, ThermoFisher Scientific, Sunnyvale, CA). Then, methanol (CH3OH) was added while the temperature and pressure were increased to 23, 60, or 100 °C and 10.3 MPa (1500 psi) over a 3 min time (static time). Next, the solvent was purged into a collection vessel. A total of four cycles were run to statically extract the sample, resulting in a total volume of about 160 mL. The solvent was evaporated off, and the oily residues remaining were weighed. A portion of the residue remaining after the methanolic extraction at 100 °C was redissolved in methanol for LC-HRMS analysis using a Thermo Scientific HPLC with an Accela 1250 pump and Accela Open AS autosampler. A Waters Acquity UPLC C18 BEH column with 1.7
Figure 2. LC-HRMS of methanolic extract of lyophilized whole pawpaw fruits done at 100 °C and 10 MPa pressure in a sealed container. All of the peaks in the top chromatogram are due to [M + H]+ ions that had a mass to charge ratio (m/z) of 597.47, like annonacin. All of the peaks in the bottom chromatogram are due to [M + H]+ ions that had a mass to charge ratio (m/z) of 623.49, like squamocin. 1054
DOI: 10.1021/jf504500g J. Agric. Food Chem. 2015, 63, 1053−1056
Letter
Journal of Agricultural and Food Chemistry
Figure 3. LC-HRMS of methanolic extract of lyophilized whole pawpaw fruits done at 23 °C and 10 MPa pressure in a sealed container. All of the peaks in the top chromatogram are due to [M + H]+ ions that had a mass to charge ratio (m/z) of 597.47, like annonacin. All of the peaks in the bottom chromatogram are due to [M + H]+ ions that had a mass to charge ratio (m/z) of 623.49, like squamocin.
Table 1. Concentrations (in μg/g) of Annonacin and Squamocin Found at Three Different Extraction Temperatures in Lyophilized Whole Pawpaw Fruits from Missouri
Another portion of the residue remaining after the methanolic extraction at 100 °C was redissolved in CD3OD for NMR analysis using an Agilent DD2 600 MHz NMR (Santa Clara, CA). A 30° pulse width and 1 s pulse delay were used for the 1H NMR, while a 30° pulse width and 2 s pulse delay were used for the 13C NMR spectra. Chemical shifts were referenced to the CD3OD peaks at 3.35 and 4.78 ppm (for 1H) and 49.3 ppm (for 13C).
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RESULTS AND DISCUSSION
About 59.3% of the lyophilized pawpaw fruit was solubilized by dry methanol at 100 °C, compared to 40.4 and 50.1% at 23 and 60 °C. This does not refer to the percent recovery, which is a term that refers to the recovery of standards that are spiked into a sample. It refers to the percentage of material in the fruit that was solubilized. Like the NMR spectra of the methanolic extracts of four other fruits in the Annonaceae family,18 the pawpaw spectra are dominated by peaks due to fructose and other carbohydrates, as shown in Figure 1. There are no peaks due to triglycerides, which would be strongly retained on a C18 column. So, this extract was suitable for LC-HRMS analysis without any sample cleanup being required. The LC-HRMS chromatograms of the methanolic extract of pawpaw detected not just annonacin and squamocin but also isomers of them, as shown in Figures 2 and 3. The chromatograms of the extracts obtained at 100 and 23 °C look almost identical, indicating that the same compounds were solubilized in about the same ratios. However, the peaks areas were larger in the chromatogram of the extract at 100 °C, and more material was solubilized at this higher temperature. So, the total concentration found in the 100 °C extract was higher than those found in the 23 and 60 °C extracts. The concentrations of annonacin and squamocin found at each temperature are in Table 1. That is, 7724 and 162 μg/g
temp
annonacin (μg/g)
squamocin (μg/g)
23 °C 60 °C 100 °C
3750 6370 7724
74 128 162
annonacin and squamocin were found when the extraction was done at 100 °C, while only 3750 and 74 were found at 23 °C and 6370 and 128 were found at 60 °C. This compares to 70.1 μg/g annonacin and no squamocin found by others in fresh (not lyophilized) pawpaw fruit using MALDI-TOF MS analysis.5 Since pawpaw fruit contains about 77% moisture,15 this would correspond to about 304 μg annonacin per g of dry weight. There were not enough of the annonacin and squamocin standards to completely validate the method. That is, there was not enough to do the required analyses for percent purity. There was not enough for moisture analysis. Still, each standard produced only one peak in the LC-MS. They were also the same standards used in the previous study that found lower concentrations of them.5 So, even though the exact concentrations were calculated based on the assumption that the standards were 100% pure (no moisture), they can be compared to the previous study.5 The main reason why so much less was found in that study was because the extraction was done under ambient conditions and because there was so much moisture in the fresh pawpaw fruit. Annonacin, squamocin, and other acetogenins are lipophilic,16 so they are extracted best from dry (lyophilized) fruit using dry methanol at 100 °C and 10 MPa pressure. However, if the extraction is 1055
DOI: 10.1021/jf504500g J. Agric. Food Chem. 2015, 63, 1053−1056
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Journal of Agricultural and Food Chemistry
in rats: possible relevance for atypical parkinsonism in Guadeloupe. J. Neurochem. 2004, 88, 63−69. (12) Smith, R. E. Medicinal Chemistry − Fusion of Traditional and Western Medicine; Bentham Science: Sharjah, U.A.E., 2013; pp 212− 215. (13) Richter, B. E.; Jones, B. A.; Ezzell, J. L.; Porter, N. L. Accelerated solvent extraction: A technique for sample preparation. Anal. Chem. 1996, 68, 1033−1039. (14) Richards, K. M.; et al. Improved extraction of soluble solids from some Brazilian and North American fruits. Nat. Prod. J. 2014, 4, 201− 210. (15) 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. 2004, 88, 63−69. (16) Champy, P.; Guérineau, V.; Laprévote, O. MALDI-TOF MS Profiling of annonaceousa acetogenins in Annona muricata products for human consumption. Molecules 2009, 14, 5235−5246. (17) Allegrand, J.; et al. Structural study of acetogenins by tandem mass spectrometry under high and low collision energy. Rapid Commun. Mass Spectrom. 2010, 24, 3602−3608. (18) Luo, R.; et al. NMR Analysis of Potentially Neurotoxic Annonaceous Fruits. Nat. Prod. J. 2013, 230−241.
done under these conditions and then the extract is analyzed by MALDI-TOF MS, all the isomers of annonacin would coelute with each other, since no column was used to separate them. This is also true for isomers of squamocin. As a result, the apparent concentrations of annonacin and squamocin would be overestimated. In conclusion, a combination of high pressure liquid extraction using dry methanol at 100 °C and 10 MPa pressure and LC-HRMS is the preferred method.
Robert A. Levine† Kristy M. Richards† Kevin Tran† Rensheng Luo‡ Andrew L. Thomas§ Robert E. Smith*,† †
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U.S. Food and Drug Administration, Total Diet and Pesticide Research Center, 11510 West 80th Street, Lenexa, Kansas 66214, United States ‡ University of Missouri − St. Louis, One University Drive, St. Louis, Missouri 63121, United States § Southwest Research Center, University of Missouri, Mt. Vernon, Missouri 65712, United States
AUTHOR INFORMATION
Corresponding Author
*Phone: 913-752-2127. Fax: 913-752-2122. E-mail: robert.
[email protected]. Notes
This work should not be taken as reflecting FDA policy or regulations. The authors declare no competing financial interest.
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REFERENCES
(1) Templeton, S. B.; Marlette, M.; Pomper, K. W.; Jones, S. C. Favorable taste ratings for several pawpaw products. Hort. Technol. 2003, 13, 445−448. (2) Ames, G., Greer, L. Pawpaw − A “Tropical” Fruit for Temperate Climates; National Sustainable Agriculture Information Service: Buttte, MT, 2010. (3) Smith, R. E.; Tran, K.; Richards, K. M. Bioactive annonaceous acetogenins. In Studies in Natural Products Chemistry; Atta-ur-Rahman, Ed.; Elsevier: New York, 2014; Chapter 4, pp 93−117. (4) Adeoye, I. B.; Umeh, U. C.; Ajetunmobi, T. Prospects and problems of marketing pawpaw in the urban environment of Ibadan, Nigeria. Libyan Agric. Res. Cent. J. Int. 2012, 3, 80−84. (5) Potts, L. F.; et al. Annonacin in Asimina triloba fruit: Implication for neurotoxicity. Neurotoxicology 2012, 33, 53−58. (6) Caparros-Lefebvre, D.; Elbaz, A. Possible relation of atypical parkinsonism in the French West Indies with consumption of tropical plants: a case-control study. Lancet 1999, 354, 281−285. (7) Escobar-Khondiker, M.; et al. Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured neurons. J. Neurosci. 2007, 27, 7827−7837. (8) Lannuzel, A.; et al. Atypical parkinsonism in Guadeloupe: a common risk factor for two closely related phenotypes? Brain 2007, 130, 816−827. (9) Escobar-Khondiker, M.; et al. Annonacin, a natural mitochondrial complex I inhibitor, causes tau pathology in cultured Neurons. J. Neurosci. 2007, 27, 7827−7837. (10) Hö llerhage, M.; et al. Natural lipophilic inhibitors of mitochondrial complex I are candidate toxins for sporadic neurodegenerative tau pathologies. Exp. Neurol. 2009, 220, 133−142. (11) Champy, P.; et al. Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration 1056
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