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Table 1. Dried Weight of Extract and Brine Shrimp Mortality (LC50) Values for Extracts for the Pawpaw Cultivar ‘NC-1’ for Frozen Pulp, Dried Pulp, Dried Twig Tissue, and Frozen Peach and Fresh Banana Pulpa

a Parentheses enclose standard error; any two means within a column not followed by the same letter are significantly different at P ≤ 0.05 using Fisher's protected LSD mean separation.

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acetogenins serve as slow-acting toxins and are particularly effective against chewing insects (3). Acetogenin compounds are toxic to several pest insects including cockroaches, Colorado potato beetle, blowfly larvae, Mexican bean beetles and their larvae, bean leaf beetle, mosquito larvae, two-spotted spider mites, striped cucumber beetles, European corn borers, melon or cotton aphids, and a nematode (9–12).

The North American pawpaw [Asimina triloba (L.) Dunal] is a tree fruit in the initial stages of commercial production in the United States (13, 14). There are about 40 commercially available clonal pawpaw cultivars with excellent fruit quality that are propagated clonally by grafting or budding with scionwood from superior trees onto common seedling rootstock of diverse genetic origin (15). Previous studies have identified over 40 other bioactive acetogenins and other compounds in the crude extracts of twigs, unripe fruits, seeds, root, and bark of the North American pawpaw (16–18). Pawpaw twigs show seasonal variation in acetogenin bioactivity, displaying the highest bioactivity during the growing season, in May and June, with increased concentrations of asimicin, bullatacin, and trilobacin (18).

The identification of a larger biomass source of acetogenin compounds would be helpful in developing commercial products based on acetogenin compounds. Fruit biomass represents a potentially larger, about 35 lb of fruit fresh weight (9.9 lb of dry weight)/mature tree/year versus about 5 lb of fresh weight (1.8 lb of dry weight) of young twig tissue/mature tree/year, source for extractable acetogenin compounds than twig tissue for use as a botanical pesticide (19). Ripe pawpaw fruit has not been investigated as a source of these compounds. Recent scientific papers have suggested a possible link between the long-term frequent consumption of Annonaceae family fruit and tea in Guadeloupe (20–24), New Caledonia (25), and people of Caribbean origin (26) and a higher rate of atypical Parkinsonism in later life. Acetogenin compounds present in the fruit and leaves of these pawpaw relatives in the genus Annona [e.g., soursop (Annona muricata)] may be responsible for the development of atypical Parkinsonism in later life (27,28). However, sensitivity to acetogenin compounds may be limited to those with a specific genetic predisposition (29). If the North American pawpaw also contains these compounds, issues of bioavailability of acetogenin compounds in fruit pulp and varietal differences in acetogenin concentration would become important research areas. Therefore, the objectives of this study were (1) to determine if extracts of ripe pawpaw fruit pulp displayed acetogenin activity, (2) to identify the major acetogenin compounds in the fruit tissue if acetogenin activity was present, and (3) to determine if the acetogenin activity varied in diverse pawpaw genotypes and closely related Annona species.

bags, and stored in a freezer at -15circC until extraction. Three replicate bags of five ripe fruits each were collected. Fruit weight averaged 100 g, and approximately 50 g of pulp was extracted from each fruit prior to homogenization; there was a total of about 250 g of pulp per replicate bag. About 50 g of the well-homogenized pulp from each replicate bag was thawed and dried at 50circC for 4 days prior to being extracted. Actively growing twig tissue was collected in June from primeNC-1, dried at 50circC for 4 days, and milled for extraction. Three ripe cherimoya (Annona cherimola) and peach fruits were purchased at a local supermarket, and the pulp was cut into small pieces and frozen until extracted. Three ripe yellow bananas were also purchased, and the pulp was cut into small pieces and the fresh tissue extracted. Canned soursop fruit pulp in syrup (La Fe Foods Inc., Moonachie, NJ) was extracted. Ripe fruits were also collected from the pawpaw cultivars ‘Mitchell’, ‘Overleese’, ‘Zimmerman’, ‘Wells’, ‘Sunflower’, and ‘NC-1’, and the material was frozen as described above for later extraction. All pawpaw fruit were produced from the orchards located at the Kentucky State University Research and Demonstration Farm (Frankfort, KY) and produced without the application of any pesticides.

Extraction and Brine Shrimp Testing (BST). Tissues were extracted using either dried or thawed pulp or dried twig tissue with 25 mL of 95% ethanol and concentrated through a series of chloroform/water and methanol/hexane partitions as described by Ratnayake et al. (17, 30) with the exception that only one chloroform water partition was performed. Frozen pawpaw fruit tissue contains approximately 28% dry matter, and fresh current season growth twig tissue contains about 36% dry matter (unpublished data); therefore, a correspondingly larger amount of fresh tissue was used in relation to dried tissue for extraction. Extracts were obtained from 9.0 g of frozen pulp from the pawpaw cultivar ‘NC-1’, 2.5 g of dried pulp and dried twig tissue, and 9.0 g of peach and banana pulp. In a second experiment, extracts were obtained from 9.0 g of thawed peach and cherimoya fresh pulp, canned soursop pulp, and ‘NC-1’ and ‘Sunflower’ pawpaw thawed pulp. Twenty-five milligrams of extract, which was between 40 and 60% of the total dried extract weight, was removed from the final concentrated fraction and dissolved in 5 mL of methanol; 10, 100, and 1000 μL amounts were transferred to 20 mL liquid scintillation vials corresponding to 10, 100, and 1000 ppm concentrations. Methanol was completely evaporated from the vials before 20 brine shrimp larvae, taken 48 h after initiation of hatching in artificial seawater, were added to each vial, and the final volume of each vial was adjusted to 5 mL using artificial seawater. After 24 h, survivors were counted and LC50 values with standard error were computed using probit regression analysis and a log10 transformation for extract concentration using the statistical software SPSS (SPSS Inc., Chicago, IL). At least three replicate extractions from individual zip-lock bags of pulp were examined for each treatment.

To evaluate the genetic variation in ripe fruit pulp for bioactivity among pawpaw cultivars, the LC50 values were calculated for 95% ethanol extracts from 9 g of frozen pulp from the cultivars ‘Mitchell’, ‘NC-1’, ‘Overleese’, ‘Zimmerman’, ‘Wells’, and ‘Sunflower’ on the basis of a response curve of 0, 10, 100, and 1000 ppm from three replicate extractions.

HPLC-MS Analysis. HPLC-MS was completed on the basis of the method of Gu et al. (18) at Nature's Sunshine Products (Spanish Fork, UT) for determining the presence of acetogenin compounds in extracts from frozen pulp of ripe fruit of the pawpaw cultivar 'NC-1'.

MATERIALS AND METHODS

Plant Material. Ripe pawpaw fruit was collected from the cultivar 'NC-1' in September 2005. Pulp from at least five ripe fruits were separated from skin and seeds, homogenized in a food processor, placed in zip-lock bags, and stored in a freezer at -15circC until extraction. Three replicate bags of five ripe fruits each were collected. Fruit weight averaged 100 g, and approximately 50 g of pulp was extracted from each fruit prior to homogenization; there was a total of about 250 g of pulp per replicate bag. About 50 g of the well-homogenized pulp from each replicate bag was thawed and dried at 50circC for 4 days prior to being extracted. Actively growing twig tissue was collected in June from 'NC-1', dried at 50circC for 4 days, and milled for extraction. Three ripe cherimoya (Annona cherimola) and peach fruits were purchased at a local supermarket, and the pulp was cut into small pieces and frozen until extracted. Three ripe yellow bananas were also purchased, and the pulp was cut into small pieces and the fresh tissue extracted. Canned soursop fruit pulp in syrup (La Fe Foods Inc., Moonachie, NJ) was extracted. Ripe fruits were also collected from the pawpaw cultivars 'Mitchell', 'Overleese', 'Zimmerman', 'Wells', 'Sunflower', and 'NC-1', and the material was frozen as described above for later extraction. All pawpaw fruit were produced from the orchards located at the Kentucky State University Research and Demonstration Farm (Frankfort, KY) and produced without the application of any pesticides.

RESULTS AND DISCUSSION

Extracts of ripe fruit of the pawpaw cultivar ‘NC-1’ had similar bioactivity or brine shrimp mortality, as indicated by LC50 values,