The document summarizes a study examining the lipid-soluble bioactive compounds in Australian avocados. The study found that Australian avocado extracts were high in antioxidants like vitamin E, chlorophylls, and carotenoids. It also found that the extracts inhibited the growth of several cancer cell lines, indicating their potential anticancer properties. Further analysis revealed the presence of additional unreported bioactive compounds beyond those already known. The study aims to provide information about the health benefits of avocados to support the fruit's promotion and increase consumer education.

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
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