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


�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


� 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


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


Composition of ‘Hass’

extracts


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’


Antioxidant activity of ‘Hass’

extracts(antioxidants prevent and repair damage to our cells)


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


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


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


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


Antiproliferative activity of

‘Hass’ extracts(inhibition of growth of cancer cells)


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


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


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?


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


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)


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/

presentation: lipid-soluble bioactive substances …...dimitrios zabaras & izabela konczak csiro...

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