Heat Stability of Annonaceous Acetogenin Activity in Pawpaw (Asimina triloba) Fruit Pulp

De'Anna Craycroft*, Jeremiah D. Lowe, Kirk W. Pomper, and Sheri B. Crabtree, Land Grant Program, Kentucky State University, Atwood Research Facility, Frankfort, KY 40601-2355

Introduction

•The North American pawpaw [Asimina triloba (L.) Dunal] is a tree fruit that is native to Kentucky which contains Annonaceous acetogenins in the twigs and ripe fruit (Pomper and Layne, 2005; Pomper et al., 2007). •Annonaceous acetogenins are a large class of unique structurally homogenous polyketide (C32 or C34 fatty acid) compounds found in the Annonaceae family (McLaughlin, 2008).

•These compounds are potent inhibitors of mitochondrial (complex I) as well as cytoplasmic (anaerobic) production of adenosine triphosphate (ATP) and related nucleotides (McLaughlin, 2008).

•The acetogenins have antitumor, pesticidal, antimalarial, antiviral, and antimicrobial activity, suggesting many potentially useful applications.

•There has been a concern that these acetogenin compounds may not be stable with heating above 50circC; heating of extracts with sugars from the fruit could also lead to problems of caramelization.

•Our working hypothesis was that heating pawpaw fruit extracts above 50circC will reduce acetogenin activity in the extracts.

•Brine shrimp mortality can be used to assess the activity of pharmaceutical compounds and can be used to assess acetogenin activity (Ratnayake et al., 1993).

Figure 1. A) The author (DeAnna Craycroft) adding extract solution and brine shrimp to vials. B) Frozen pawpaw fruit pulp. C) Brine shrimp larvae as seen in petri dish under microscope. D) Chemical structure of the acetogenin Asimicin.

Objective

Results

•Ripe pawpaw pulp from the pawpaw cultivar Shenandoah was extracted with either Protocol A using 95% ethanol or Protocol B using 95% ethanol followed by a long enrichment protocol to increase acetogenin concentration and reduce sugar content; thereby reducing caramelization.

•In Protocol A, 10 g of thawed pulp was extracted with 25 mL of 95% ethanol and filtered to remove solids. In Protocol B, 10 g of thawed pawpaw pulp was extracted with 25 mL of 95% ethanol and filtered. Then the pulp extract was re-extracted with distilled water and chloroform, the water removed, and the chloroform dried down. Then 20 mL each of methanol and hexane was added to the tubes, the hexane layer was removed, and the methanol evaporated. The total mg of extract obtained was then determined and resuspended in methanol.

•The extracts were heated to 30, 65, or 100circC for 16 or 96 hours in controlled heat blocks.

•Following quantification a 2.5 mg/ml solution was made using 95% ethanol, and extract added to dram vials to correspond to 0, 1, 5, 10, and 50 parts per million (ppm).

•After the ethanol was evaporated, ten brine shrimp larvae, taken 48 h after initiation of hatching in artificial sea water, were added to each vial, and the final volume of each vial was adjusted to 5 ml using the artificial sea water.

• After 24 h, survivors were counted and standard deviation was measured from three replicate extracts (Ratnayake et al., 1993) (Figure 1).

•LC_{50} (ppm) values were computed using probit regression analysis and log10 transformation for extract concentration using the statistical software SPSS (SPSS Inc, Chicago).

Table 1. The effect of heating pawpaw fruit extracts on extract biological activity as measured using the brine shrimp assay.

• Brine shrimp mortality increased with increasing extract concentration in all treatments (Fig. 2).
• Heating of enriched extracts for 16 hours at 65circC or 100circC resulted in a 1.2 or 1.6 fold increase in LC50 or decline in potency, respectively (Table 1).
• Heating of enriched extracts for 96 hours at 65circC or 100circC resulted in a 1.1 fold decrease or 1.9 fold increase in LC50 or potency, respectively.
• Heating of ethanol extracts for 16 hours at 65circC or 100circC resulted in a 2.4 or 3.5 fold increase in LC50 or decline in potency, respectively.
• Heating of enriched extracts for 96 hours at 100circC resulted in a 2.3 fold increase in LC50 or decreased potency.

Discussion

•Heating of all extracts to 100circC decreased the acetogenin activity in both ethanol only and enriched extracts. •Heating of enriched extracts to 100circC resulted in a lower decline of extract potency than in ethanol only extracts, suggesting that caramelization (browning was noted) did result in a reduction in acetogenin activity with heating. •Our working hypothesis was that heating pawpaw fruit extracts above 50circC will reduce acetogenin activity in the extracts. Heating samples to 65circC resulted in only a small decline in acetogenin activity.

•Ripe pawpaw pulp may be heated at temperatures less than 65circC and dried with little loss of activity for later extraction and product development.

Conclusions

•Acetogenin compounds did show greater stability to heating than had been previously suggested. •Ripe pawpaw pulp may be heated at temperatures less than 65circC and dried with little loss of activity for later extraction and product development.

Figure 2. An example of brine shrimp mortality with increasing extract concentration

Literature Cited

Materials and Methods

Results

• McLaughlin, J.L. 2008. Paw Paw and Cancer: Annonaceous Acetogenins from Discovery to Commercial Products. Journal of Natural Products, 71:1311-1321.
• Pomper, K.W., J. D. Lowe, S.B. Crabtree, and W. H. Keller. 2007. Assessment of Annonaceous Acetogenin Activity in Pawpaw (Asimina triloba) Fruit. HortScience 42:939.
• Pomper, K.W. and D.R. Layne. 2005. The North American pawpaw: botany and horticulture. Hort. Rev. 31:351-384.

pawpaw: botany and horticulture. Hort. Rev. 31:351-384.

• Ratnayake, S., J.K. Rupprecht, W.M. Potter, and J.L. McLaughlin. 1993. Evaluation of the pawpaw tree, Asimina triloba (Annonaceae), as a commercial source of the pesticidal annonaceous acetogenins. p. 644-648. In: J. Janick and J.E. Simon (eds.), New crops. Wiley, New York.