PRESENTATION: Lipid-soluble bioactive substances …...Dimitrios Zabaras & Izabela Konczak CSIRO...
Transcript of PRESENTATION: Lipid-soluble bioactive substances …...Dimitrios Zabaras & Izabela Konczak CSIRO...
Lipid-soluble bioactive substances in Australian avocados
Dimitrios Zabaras & Izabela Konczak
CSIRO Food and Nutritional Sciences
23/07/09
CSIRO. Lipid-soluble bioactives in Australian 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) reportedactivity against prostate cancer cells
(Lu et al., Journal of Nutritional Biochemistry, 16, 23-30, 2005)
Some general information about avocados
CSIRO. Lipid-soluble bioactives in Australian avocados
�To obtain credible information relating to the health-
promoting benefits of lipid-soluble substances found inAustralian-grown avocados (Hass variety)
� This could then be used for:� promotion of the fruit/industry
� education of public/consumers
Our objective
CSIRO. Lipid-soluble bioactives in Australian avocados
� Measurement of selected substances in extracts
�Vitamin E components
�Chlorophylls- responsible for the green pigmentation
�Carotenoids- responsible for the yellow pigmentation
Activities
� Assessment of antioxidant capacity of extracts
� Evaluation of growth-inhibitory activity against varioushuman cancer cells:
� Gastric
� Colon
� Leukaemia
CSIRO. Lipid-soluble bioactives in Australian avocados
Fruit (randomly from 3 farms in QLD)
Homogenise &
extract with acetone
Concentrate
Extract with hexane &
concentrate under vacuum
Crude extract
SPE Diol clean-up
Purified extract
HPLC, LC/MS
Antioxidant assay (DPPH)
Anticancer assay (MTT)
Generation of lipid-soluble extracts
CSIRO. Lipid-soluble bioactives in Australian avocados
Composition of ‘Hass’
extracts
CSIRO. Lipid-soluble bioactives in Australian avocados
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).
Major lipid-soluble substances in ‘Hass’
CSIRO. Lipid-soluble bioactives in Australian avocados
Antioxidant activity of ‘Hass’
extracts(antioxidants prevent and repair damage to our cells)
CSIRO. Lipid-soluble bioactives in Australian avocados
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
1 2 3 4 5 6 7 8 9 10 4 6
Fruit extract
IC5
0 (
mg
/L F
FW
)
Crude
Purified
�Crude fraction showed much stronger
antioxidant activity then purified fraction
Antioxidant activity of ‘Hass’ extracts
(Smaller columns indicate more activity)
better
CSIRO. Lipid-soluble bioactives in Australian avocados
0
1000
2000
3000
4000
5000
crude SPE
Chlorophylls
Vitamin E
Carotenoids
Concentr
ation (
ug
/100g
FF
W)
Crude Purified
What has been identified in the two fractions?
� Purified fraction has less of these substances compared to the
crude extract
CSIRO. Lipid-soluble bioactives in Australian avocados
IC50 (µg/mL)
Su
m o
f m
ajo
r c
om
po
un
ds
(µ
g/1
00
g F
FW
)
y = -29.182x + 7923.5
R2 = 0.8526
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
40 60 80 100 120 140
�Vitamin E, chlorophylls and carotenoids are primarily responsible
for the antioxidant activity of ‘Hass’ extracts
Antioxidant activity of ‘Hass’ extracts
CSIRO. Lipid-soluble bioactives in Australian avocados
Ca
rro
t
Ora
ng
e
Gre
en
Kiw
i
Go
ld K
iwi
Wa
lnu
t
Av
oc
ad
o
Lipid-soluble antioxidants from ‘Hass’: how do they
compare?
�Lipid-soluble antioxidant fraction from ‘Hass’ very strong compared to similar fraction from other sources
IC5
0valu
es a
gain
st D
PP
H
(Smaller columns indicate more activity)
Please note that total activity= lipid-soluble + water soluble components
better
CSIRO. Lipid-soluble bioactives in Australian avocados
Antiproliferative activity of
‘Hass’ extracts(inhibition of growth of cancer cells)
CSIRO. Lipid-soluble bioactives in Australian avocados
0
20
40
60
80
100
120
140
0 1 2 3 4 5
Extract in culture (uL/mL)
Ce
ll S
urv
iva
l (%
)
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.
Colon cancer-cell survival when treated with
‘Hass’ extract in test-tube assays
Lutein
Crude
Purified
CSIRO. Lipid-soluble bioactives in Australian avocados
0
20
40
60
80
100
120
0 1 2 3 4 5
Extract in culture (uL/mL)
Ce
ll s
urv
iva
l (%
)
Assuming complete extraction, 1 µL extract contains phytochemicals found in
approx. 1.5 mg fruit fresh weight
leukaemia
gastric
colon
Survival of various cancer-cells when treated
with ‘Hass’ extract in test-tube assays
CSIRO. Lipid-soluble bioactives in Australian avocados
RT: 0.00 - 18.03
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Tim e (m in)
0
500000000
1000000000
1500000000
2000000000
2500000000
3000000000
3500000000
4000000000
4500000000
5000000000
5500000000
6000000000
6500000000
7000000000
7500000000
8000000000
8500000000
9000000000
9500000000
10000000000
10500000000
11000000000
11500000000
Inte
nsity
2 .74
5.90
4.68
5.52 6.98
8.03
3.18
7.74
8.692.01
3.77
9.80
1.809.24 10.05
10.5612.07
13.30 14.36 14.94 16.41 17.920.70 1.46
NL:
1.18E10
TIC F: + p Q1MS [
200.00-
400.00]
MS 10Bdiol
10Bdiol #602 RT: 4.68 AV: 1 NL: 2.13E8
T: + p Q1MS [ 200.00-400.00]
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
m /z
0
10000000
20000000
30000000
40000000
50000000
60000000
70000000
80000000
90000000
100000000
110000000
120000000
130000000
140000000
150000000
160000000
170000000
180000000
190000000
200000000
210000000
Inte
nsi
ty
329.36065
311.37056
233.32017370.45086
293.24047251.31026
339.44071269.37035 392.43097359.39081207.28004 285.33043222.12012
a)
b)
m/z
311.3
m/z
329.3
� Persin and a few other fatty
acid derivatives are knownbioactives in avocadoes (e.g.
work at the Garvan Institute)
� However, data indicates thatthe active fraction contains
several additional unreportedcomponents
� Further work will hopefully
reveal more about the activephytochemicals
LC-MS TIC: m/z 200-400
Several unreported as yet components?
CSIRO. Lipid-soluble bioactives in Australian avocados
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
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
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
Contact Us
Phone: 1300 363 400 or +61 3 9545 2176
Email: [email protected] Web: www.csiro.au
Thank you
Food and Nutritional Sciences
Dr. Dimitrios Zabaras C.Chem. MRACI
Research Scientist
Phone: 02 9490 8352
Email: [email protected]
Web: www.foodscience.csiro.au/