The document discusses the development of nutraceuticals targeting cancer, specifically focusing on natural extracts rich in compounds like perillyl alcohol (poh) from sweet cherries and polymethoxylated flavones (pmf) from citrus peels. It highlights the potential of these extracts to inhibit cancer cell growth and induce apoptosis in colorectal cancer cell lines (HT29) when used in conjunction with chemotherapy drugs. Several processes for extraction and characterization of these compounds are described, utilizing high-pressure technology and supercritical fluid methods.

1. Development of Nutraceuticals
targeting cancer
Catarina Duarte
Food & Health Unit
Nutraceuticals and controlled Delivery Lab
IBET/ ITQB-UNL
Oeiras, Portugal

2. Green Processes for developing health promoting products
Extraction /Fraccionation of Natural Compounds
Research interests:
Formulation – particle formation, encapsulation and
impregnation
Characterization and pre-clinical Bioactivity evaluation of bioproducts
Chemical, enzymatic and cell-based assays
Food&Health Unit
Nutraceuticals and Controlled Delivery Lab

3. POH was detected in
10%EtOH and 20%EtOH
extracts
Serra et al. (2010) J Supercri. Fluids, 55, 184-191
Sweet cherries
Development of POH-rich extracts
Application of High Pressure Technology for the development of natural extracts
rich in perillyl alcohol (POH)
Raw material:
Sweet cherry culls
(Saco variety)
POH

4. 96h
Only two extracts (10%EtOH and 20%EtOH) inhibited cancer cell growth in a
time dependent manner and this effect is related with the presence of Perillyl
Alcohol
Cherry extracts induced cell cycle arrest in a different cell cycle checkpoint
than doxorrubicin suggesting that they can be used in combination with the
drug in chemotherapy
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Control 4h 24h 48h 72h 96h
cell(%)
G1 S G2/M
Cherry extract-
10% EtOH
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Control 4h 24h 48h 72h 96h
cell(%)
G1 S G2/M
Doxorubicina
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Cellviability(%ofcontrol)
24h 48h 72h 96h
10%
EtOH
20%
EtOH
40%
EtOH
60%
EtOH
80%
EtOH
100%
EtOH
CO2
Antiproliferative effect on HT29 cells (human colorectal cancer cell line)
• Cell cycle analysis
Controlo
10% EtOH
20% EtOH
• Cell viability after 24, 48, 72 and 96h of proliferation
Serra et al. (2010) J Supercrit. Fluids, 55, 184-191
Serra et al. (2011) J Supercrit. Fluids,55 1007-1013
1st prize: “Inovation and Develo+ment of Products and Services” category
Sweet cherries
Development of POH-rich extracts

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Detector 1-280nm
Laranja (2) TS
Ago2913075.dat
Detector 1-280nm
Laranja (3) TS
Ago2913076.dat
Detector 1-280nm
Laranja (4) TS
Ago2913077.dat
Detector 1-280nm
Laranja Dil 1:10
Jul2413094.dat
Sinensetin
Nobiletin
Tangeretin
Process: CO2+ EtOH (80:20 v/v); P=25MPa; T=323K; t=60min.
Tangeretina
Nobiletin
Sinensetin
Inês Silva (2013), Tese de mestrado
Citrus peels
Development of PMF-rich extracts
Application of Supercritical Fluid Technology for the development of natural extracts
rich in polymetoxylated flavones (PMF)
Sinensetin, tangeritin and nobiletin were
identified in citrus pell extracts.
Raw material:
Orange peels
(by-products of fruit juice
production)

6. Induction of apoptosis on HT29 cell spheroids
Control Citrus extract
(Red: viable cells; Green: apoptotic cells)
Cell viability
• 2D model
Orange extract inhibited the proliferation of
HT29 cells (antiproliferative effect was
dependent on spheroid size).
Orange extract induced apoptosis in HT29
spheroids(apoptotic cells are mainly located
on the surface of the spheroid).
• 3D model
Cell monolayer Cell spheroids with different sizes
Antiproliferative effect on HT29 cells (human colorectal cancer cell line)
Inês Silva (2013), Master thesis
Citrus peels
Development of PMF-rich extracts

7. Opuntia ficus-indica
Development of terpene-rich extracts
Raw material:
Opuntia ficus-indica
(by-products of fruit juice production)
Process: CO2+ EtOH+ H2O mixtures; P=20MPa; T=313K; t=180min.
Application of Pressurized Liquid Extracton for the development
of natural extracts rich in terpenes
fried lup #1 #2 #3 #4 #5 #6 #7 #8 #9 #10fried lup #1 #2 #3 #4 #5 #6 #7 #8 #9 #10
Terpene
detection by TLC
Opuntia extracts obtained with mixtures of CO2 and
EtOH presented high terpene content

8. Opuntia extract obtained with CO2:EtOH (60:40v/v) mixture (PLE2) presented the highest
antiproliferative effect. This effect is probably due to the presence of terpenes.
PLE-2 extracts increased drug sensitivity on HT29 dx cell population cells .
Antiproliferative effect on HT29 cells (human colorectal cancer cell line)
• Cell viability after 24 h of proliferation
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Cellularviability
16 mg/mL 20 mg/mL 24 mg/mL
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Cellviability(%ofcontrol)
Doxorubicin (µM)
Doxorubicin in HT29 dx
Doxorubicin in HT29
PLE-2 + Doxo in HT29 dx
• Chemosensitization effect in a drug resistant cell sub-
population (HT29 dx)
Joana Poejo(2014), Master thesis
Opuntia ficus-indica
Development of terpene-rich extracts

9. e-mail: cduarte@itqb.unl.pt;
http://www.itqb.unl.pt/labs/nutraceuticals-and-delivery
www.ibet.pt
Laboratório de Nutracêuticos e
Libertação Controlada
Oeiras, Portugal