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to ‘NC-1’ twig tissue (Table 1). Both frozen and dried pawpaw pulp extracts showed similar bioactivity levels. Extracts of banana and peach, which were included as additional controls, displayed bioactivity only at the highest extract concentration. The LC50 values were <1 ppm for extracts of ‘NC-1’ dried twig and pulp, as well as frozen pulp, indicating high bioactivity in these samples. It is possible that as the frozen pulp thawed and dried, some of the acetogenin compounds present may have been metabolized or degraded as indicated by a trend for higher LC50 values for dried pulp. Using the method of Ratnayake et al. (17, 30), the greatest dried extract weight per gram of dry weight (DW) of pulp or twig material was found for ‘NC-1’ dried pulp and twig (Table 1).

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Table 2. Weight of Extract and Brine Shrimp Mortality (LC50) Values for Extracts from Canned Soursop Pulp and Frozen Peach, Cherimoya, and ‘NC-1’ and ‘Sunflower’ Pawpaw Pulpa

aParentheses 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.

Table 3. Brine Shrimp Mortility (LC50) Values for the Pawpaw Cultivars ‘Mitchell’, ‘NC-1’, ‘Overleese’, ‘Zimmerman’, ‘Wells’, and ‘Sunflower’ from Frozen Pulp of Ripe Fruita

aParentheses enclose standard error.

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fruit contains acetogenin compounds (27, 28); however, acetogenin bioactivity has not been reported in cherimoya fruit. In terms of brine shrimp mortality, extracts obtained from ripe fruit pulp of the pawpaw cultivar ‘NC-1’ were again high in bioactivity; however, pulp from the pawpaw cultivar ‘Sunflower’ displayed lower bioactivity than observed for ‘NC-1’ (Table 2). Soursop displayed lower activity than either pawpaw cultivar, but higher bioactivity than peach or cherimoya. The LC50 values were <1 ppm for extracts of ‘NC-1’ pulp, followed by ‘Sunflower’ and soursop, with cherimoya and peach displaying little bioactivity (Table 2). Using the method of Ratnayake et al. (17, 30), the greatest total weight of extract per gram of FW of material was for ‘NC-1’ pulp, followed by soursop (Table 2).

To determine whether bioactivity of frozen ripe fruit pulp varied by genotype, six commercially available pawpaw cultivars were evaluated for bioactivity. The LC50 values were highest (<1.0 ppm) in the cultivars ‘Mitchell’, ‘NC-1’, and ‘Overleese’, with ‘Wells’ and ‘Sunflower’ displaying lower activity (<7.0 ppm) and the cultivar ‘Zimmerman’ displaying intermediate activity (4.0 ppm; Table 3).

A sample of frozen ripe pawpaw pulp extract was obtained using the enrichment method of Ratnayake et al. (17, 30) and was subjected to HPLC-MS analysis. Three acetogenin compounds were identified at high levels in the sample: asimicin, bullatacin, and bullatalicin (Figures 1 and 2). The acetogenin compounds asimicin, bullatacin, and trilobacin have been reported at high levels in pawpaw twig samples previously by Gu et al. (18), and bullatalicin was reported by Martin et al. (31) in pawpaw twigs.

The methods used for tissue extraction likely yielded a mixture of compounds in addition to acetogenin compounds. Ripe pawpaw fruit pulp contains phenolic and antioxidant compounds that may have influenced extract efficacy in the bioassays (33). However, HPLC-MS analysis identified acetogenin compounds in the fruit pawpaw extracts, and the relative amount of acetogenin compounds in the extracted material was likely reflected in the bioassay. Some hydrophobic substances contained in the extracts could also display bioactivity. Acetogenin compounds have been identified in pawpaw twigs, bark, leaves, and unripe

Figure 1. Ripe pawpaw pulp extract from the cultivar 'NC-1' subjected to HPLC-MS analysis. The three largest peaks correspond to the acetogenin compounds asimicin, bullatacin, and bullatalicin.