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Dimitrios Zabaras Presentation Transcript

  • 1. Lipid-soluble bioactive substances in Australian avocados Dimitrios Zabaras & Izabela Konczak CSIRO Food and Nutritional Sciences 23/07/09
  • 2. Some general information about avocados Avocados are known to contain high amounts of phytonutrients: monounsaturated fatty acids phytosterols carotenoids vitamins B, C and E polyphenols A recent study (using Californian-grown fruit) reported activity against prostate cancer cells (Lu et al., Journal of Nutritional Biochemistry, 16, 23-30, 2005) CSIRO. Lipid-soluble bioactives in Australian avocados
  • 3. Our objective To obtain credible information relating to the health- promoting benefits of lipid-soluble substances found in Australian-grown avocados (Hass variety) This could then be used for: promotion of the fruit/industry education of public/consumers CSIRO. Lipid-soluble bioactives in Australian avocados
  • 4. Activities Measurement of selected substances in extracts Vitamin E components Chlorophylls- responsible for the green pigmentation Carotenoids- responsible for the yellow pigmentation Assessment of antioxidant capacity of extracts Evaluation of growth-inhibitory activity against various human cancer cells: Gastric Colon Leukaemia CSIRO. Lipid-soluble bioactives in Australian avocados
  • 5. Generation of lipid-soluble extracts Fruit (randomly from 3 farms in QLD) Homogenise & extract with acetone Concentrate Extract with hexane & concentrate under vacuum Crude extract HPLC, LC/MS SPE Diol clean-up Antioxidant assay (DPPH) Anticancer assay (MTT) Purified extract CSIRO. Lipid-soluble bioactives in Australian avocados
  • 6. Composition of ‘Hass’ extracts CSIRO. Lipid-soluble bioactives in Australian avocados
  • 7. Major lipid-soluble substances in ‘Hass’ Compound Concentration range (µg/100g FFW) a-Tocopherol 1197-2151 γ-Tocopherol 118-232 δ-Tocopherol 83-260 Chlorophyll a 1073-1465 Chlorophyll b 1231-2215 Lutein (all trans-) 160-273 Similar variation has been reported for Californian-grown fruit (Lu et al., Journal of Nutritional Biochemistry, 16, 23-30, 2005). CSIRO. Lipid-soluble bioactives in Australian avocados
  • 8. Antioxidant activity of ‘Hass’ extracts (antioxidants prevent and repair damage to our cells) CSIRO. Lipid-soluble bioactives in Australian avocados
  • 9. Antioxidant activity of ‘Hass’ extracts 700.0 Purified 600.0 Crude fraction showed much stronger 500.0 antioxidant activity then purified fraction IC50 (mg/L FFW) 400.0 (Smaller columns indicate more activity) 300.0 Crude 200.0 100.0 better 0.0 1 2 3 4 5 6 7 8 9 10 4 6 Fruit extract CSIRO. Lipid-soluble bioactives in Australian avocados
  • 10. What has been identified in the two fractions? Concentration (ug/100g FFW) 5000 4000 3000 Chlorophylls 2000 Vitamin E Carotenoids 1000 0 Crude crude Purified SPE Purified fraction has less of these substances compared to the crude extract CSIRO. Lipid-soluble bioactives in Australian avocados
  • 11. Antioxidant activity of ‘Hass’ extracts Sum of major compounds (µg/100g FFW) 7000 6500 6000 5500 5000 4500 y = -29.182x + 7923.5 4000 R2 = 0.8526 3500 3000 2500 2000 40 60 80 100 120 140 IC50 (µg/mL) Vitamin E, chlorophylls and carotenoids are primarily responsible for the antioxidant activity of ‘Hass’ extracts CSIRO. Lipid-soluble bioactives in Australian avocados
  • 12. Lipid-soluble antioxidants from ‘Hass’: how do they compare? Lipid-soluble antioxidant fraction from ‘Hass’ very strong compared to similar fraction from other sources IC50 values against DPPH (Smaller columns indicate more activity) better Walnut Avocado Gold Kiwi Green Kiwi Orange Carrot Please note that total activity= lipid-soluble + water soluble components CSIRO. Lipid-soluble bioactives in Australian avocados
  • 13. Antiproliferative activity of ‘Hass’ extracts (inhibition of growth of cancer cells) CSIRO. Lipid-soluble bioactives in Australian avocados
  • 14. Colon cancer-cell survival when treated with ‘Hass’ extract in test-tube assays 140 120 Cell Survival (%) 100 80 Lutein 60 40 20 Crude 0 Purified 0 1 2 3 4 5 Extract in culture (uL/mL) 1 µL extract contained substances found in approx. 1.5 mg fruit FW. The concentration of lutein was 25 times higher than found in fruits. CSIRO. Lipid-soluble bioactives in Australian avocados
  • 15. Survival of various cancer-cells when treated with ‘Hass’ extract in test-tube assays 120 100 Cell survival (% ) 80 60 leukaemia 40 20 gastric 0 colon 0 1 2 3 4 5 Extract in culture (uL/mL) Assuming complete extraction, 1 µL extract contains phytochemicals found in approx. 1.5 mg fruit fresh weight CSIRO. Lipid-soluble bioactives in Australian avocados
  • 16. Several unreported as yet components? R T: 0.00 - 1 8.03 2 .7 4 NL : 1.18 E10 11 50 00 00 00 0 TIC F: + p Q1 MS [ 11 00 00 00 00 0 20 0.00 - 5 .9 0 10 50 00 00 00 0 4.68 a) 40 0.00 ] MS 10 Bdiol 10 00 00 00 00 0 5 .5 2 6 .9 8 95 00 00 00 00 90 00 00 00 00 8 .0 3 LC-MS TIC: m/z 200-400 85 00 00 00 00 80 00 00 00 00 75 00 00 00 00 3 .1 8 70 00 00 00 00 7.74 65 00 00 00 00 Intensity 60 00 00 00 00 55 00 00 00 00 50 00 00 00 00 45 00 00 00 00 2 .0 1 3 .7 7 8.69 Persin and a few other fatty 40 00 00 00 00 35 00 00 00 00 acid derivatives are known bioactives in avocadoes (e.g. 30 00 00 00 00 9.80 25 00 00 00 00 20 00 00 00 00 work at the Garvan Institute) 1.80 15 00 00 00 00 9.24 10 .0 5 10 00 00 00 00 10 .5 6 12 .0 7 50 00 00 00 0 1 3.30 14 .3 6 14 .9 4 16 .4 1 17 .9 2 0 .7 0 1.46 0 1 0 Bd iol # 60 2 R T:04 .6 8 AV: 1 1 N L: 2 .1 3E 8 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 T: + p Q1MS [ 20 0 .0 0-40 0 .0 0] Tim e (m in ) 3 2 9.3 6 06 5 2 1 0 0 00 0 0 0 2 0 0 0 00 0 0 0 1 9 0 0 00 0 0 0 b) However, data indicates that 1 8 0 0 00 0 0 0 1 7 0 0 00 0 0 0 the active fraction contains 1 6 0 0 00 0 0 0 1 5 0 0 00 0 0 0 1 4 0 0 00 0 0 0 several additional unreported 1 3 0 0 00 0 0 0 1 2 0 0 00 0 0 0 m/z m/z components 311.3 3 1 1.3 7 05 6 329.3 ity 1 1 0 0 00 0 0 0 Intens 1 0 0 0 00 0 0 0 9 0 0 0 00 0 0 8 0 0 0 00 0 0 7 0 0 0 00 0 0 Further work will hopefully reveal more about the active 6 0 0 0 00 0 0 5 0 0 0 00 0 0 4 0 0 0 00 0 0 3 0 0 0 00 0 0 2 0 0 0 00 0 0 23 3 .3 20 1 7 25 1 .31 0 2 6 2 93 .2 4 04 7 3 7 0.4 5 0 86 phytochemicals 1 0 0 0 00 0 0 2 6 9.3 7 0 35 3 3 9 .4 4 0 71 3 59 .3 90 8 1 3 9 2.4 3 09 7 2 07 .2 8 00 4 2 2 2 .1 2 0 12 2 8 5 .33 0 4 3 0 2 10 220 2 30 240 250 260 2 70 280 29 0 300 310 320 33 0 3 40 350 3 60 370 380 390 40 0 m /z CSIRO. Lipid-soluble bioactives in Australian avocados
  • 17. Conclusions Lipid-soluble extracts from ‘Hass’ fruit were found, as expected, to be rich in Vitamin E, chlorophylls and carotenoids Extracts exhibited strong antioxidant and antiproliferative activity against various human cancer cells Different components were responsible for each activity: Carotenoids, chlorophylls, vitamin E - antioxidants Others (e.g. persin, unidentified) - antiproliferative CSIRO. Lipid-soluble bioactives in Australian avocados
  • 18. Significance of the work? This information can now be used to promote avocado consumption and educate consumers (For example, retail surveys show that health-minded consumers are more likely to purchase avocados than other consumers) (Hughes, D. Infocado Summit 2008) CSIRO. Lipid-soluble bioactives in Australian avocados
  • 19. Acknowledgements This project was funded by Horticulture Australia Limited (HAL) using the avocado levy and matched funding from the Australian Government Thank you to the ANZAGC09 organising committee for the opportunity to present the work Thank you to all of you for your time CSIRO. Lipid-soluble bioactives in Australian avocados
  • 20. Food and Nutritional Sciences Dr. Dimitrios Zabaras C.Chem. MRACI Research Scientist Phone: 02 9490 8352 Email: dimitrios.zabaras@csiro.au Web: www.foodscience.csiro.au/ Thank you Contact Us Phone: 1300 363 400 or +61 3 9545 2176 Email: enquiries@csiro.au Web: www.csiro.au