12TH INTERNATIONAL CONFERENCE FUNCTIONAL FOOD INGREDIENTS AND NUTRACEUTICALS IN CHRONIC DISEASE: SCIENCE AND PRACTICE NOVEMBER 29-DECEMBER2 DALLAS, TX, USA

Teresa Serra, PhD

Researcher

Nutraceuticals & Controlled Delivery Unit - IBET/ITQB-UNL

e-mail: tserra@ibet.pt

is the second most fatal and the third most diagnosed type of cancer worldwide

From 1981 to 2002…

Almost 74% of all drugs approved for cancer therapy were:

(i) natural products

(ii) based on natural products or

(iii) mimicked natural products in one form or another

In 2008: 7.5 million people died from cancer worldwide In 2020: it is estimated that mortality from cancer will increase to more than 10 million

Emerging interest in chemotherapeutic application of

Reverting chemoresistance

Phytochemicals (polyphenols and terpenes)

Anticancer effect

Inhibition of tumour cell mediated

protease activity

Attenuation of tumour angiogenesis Induction of cell cycle

arrest

Induction of apoptosis

…..

Evaluation of the antiproliferative effect and cell cycle modulation of several plant derived natural extracts in human colorectal cancer cells

Correlation of the bioactive effect with phytochemical composition of samples

SWEET CHERRIES OLIVES CACTUS PEAR CORK

• Crop residues Cherry culls (fruit that is not suitable to sell for eating due to small size and weight)

• Olive oil by-products (olive mill semi solid residue)

• Leaves

• Juice prodution residues (peels and seeds)

• Cork industry by-products

Bioactive rich fractions (e.g. polyphenols, carotenoids,

phytosterols, terpenes,...)

Reduces or eliminates the use or generation of

hazardous substances

Green sustainable processes

High pressure and

supercritical fluid

technology

Membrane Filtration

Adsorption process (with macroporous

resins)

Conventional Extraction

(with biocompatible

solvents)

• Total polyphenols content

• Total flavonoids content

• Total phenolic acids content

• Total procyanidin content

Spectrophotometric assays

• Total anthocyanin content

• Total betalain content

• Analysis of carotenoids

• TLC

• UPLC

•HPLC-DAD-FD-ED

•HPLC-MS/MS

• GC-MS

Chromatographic techniques

HT29 cells (Human colon adenocarcinoma)

ANTIPROLIFERATIVE EFFECT

CELL CYCLE ARREST

• Dose-response curves • Effective dose values (ED50)

MTT and MTS assays

by Flow Cytometry

Saco cherry culls

Perillyl alcohol

Chemoprevention of Colon Carcinogenesis by Dietary Perillyl Alcohol Reddy et al., 1997

Chemotherapy of pancreatic cancer with the monoterpene perillyl alcohol Stark et al., 1995

Perillyl Alcohol Inhibits Human Breast Cancer Cell Growth in vitro and in vivo Yuri et al., 2004

Effects of Perillyl alcohol on Melanoma in the Tpras Mouse Model Lluria-Prevatt et al., 2002

High Pressure Extraction with CO2 and ethanol

Higher selectivity

Short extraction times

GRAS solvents

Supercritical CO2 has been shown to be efficient in the extraction of perillyl alcohol from natural sources

SWEET CHERRIES NATURAL EXTRACTS

SFE (Supercritical Fluid Extraction)

CO2; P= 25 MPa;

T= 50ºC; t= 60 min

ESE (Enhanced Solvent Extraction)

CO2+EtOH (10-100%v/v)

P= 25 MPa; T= 50ºC;

t= 90 min

Dried cherry

1st step

2nd step

Methodology:

Fractioned High Pressure Extraction

10% EtOH

20% EtOH

40% EtOH

60% EtOH

75% EtOH

100% EtOH

To remove unwanted compounds

SWEET CHERRIES NATURAL EXTRACTS

0

20

40

60

80

100

120

A B C D E F G

Cel

l via

bilit

y (%

of c

ontro

l)

24h 48h 72h 96h

10% EtOH

20% EtOH

40% EtOH

60% EtOH

75% EtOH

100% EtOH

CO2

Tested concentration: 0.5mg/mL 24h 48h 72h 96h

Control

10% EtOH

20% EtOH

100m

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

• Antiproliferative effect

Serra et al. (2011), J Supercr Fluids,55, 184-191

The extract obtained with 10% EtOH exhibited the highest antiproliferative effect

(ED50=0.20 mg/mL) being 150 times more effective than fresh fruit

SWEET CHERRIES NATURAL EXTRACTS

Cherry extracts (0.5mg/mL) Doxorubicin (125nM) 10%EtOH

• Induction of cell cycle arrest

0

20

40

60

80

100

Control 4h 24h 48h 72h 96h

cell (

%)

G1 S G2/M

0

20

40

60

80

100

Control 4h 24h 48h 72h 96h

cell (

%)

G1 S G2/M

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

Cell cycle arrest in G1 Cell cycle arrest in G2/M

Serra et al. (2011), J Supercr Fluids, 55, 1007-1013

20%EtOH

0

20

40

60

80

100

Control 4h 24h 48h 72h 96h

cell (

%)

G1 S G2/M

SWEET CHERRIES NATURAL EXTRACTS

some studies showed that potent inhibition of tumour survival is achieved when combining drugs

with different cell cycle checkpoints

Cherry extracts (0.5mg/mL) 10%EtOH

• Induction of cell cycle arrest

0

20

40

60

80

100

Control 4h 24h 48h 72h 96h

cell (

%)

G1 S G2/M

Cell cycle modulation of cherry extracts was related with the presence of perillyl alcohol

Cell cycle arrest in G1 20%EtOH

0

20

40

60

80

100

Control 4h 24h 48h 72h 96h

cell (

%)

G1 S G2/M

0

20

40

60

80

100

Control 24h

cell

(%)

G1 S G2/M

Cell cycle arrest in G1

Perillyl alcohol (130 µg/mL)

SWEET CHERRIES NATURAL EXTRACTS

1

2 3

4

OLIVE - BASED NATURAL EXTRACTS

Membrane separation process

Conventional solvent extracton

C=220mg/g extract (dw)

EP1910257, 2008.

Olive natural extract derived from olive oil by-products is rich hydroxytyrosol – it contained 22% w/w of HT Matias, AA (2009), PhD thesis

Olive oil by-products

(olive mill semi solid residue)

Dried leaves

1- Hydroxytyrosol HT rich extract

2- Tyrosol 3- Caffeic acid 4- p-coumaric acid

2- Oleuropein

LT+OL rich

extract

Olive natural extract derived from dried leaves contains mainly luteolin derivatives and oleuropein

1- Luteolin derivatives

Ext. Folhas Oliveira Arbasana MS Scan25-Nov-201216:59:34

Time10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00

AU

0.0

2.5e-2

5.0e-2

7.5e-2

1.0e-1

1.25e-1

1.5e-1

1.75e-1

2.0e-1

2.25e-1

2.5e-1

2.75e-1

3.0e-1

3.25e-1

3.5e-1

3.75e-1

4.0e-1

4.25e-1

IBET_23Novembro012_24 2: Diode Array 280

Range: 4.704e-18.50

88.68

85.45

79.57

59.7392.53 106.61

1 1

1

1 2

OLIVE - BASED NATURAL EXTRACTS

• Antiproliferative effect

Matias, AA (2009), PhD thesis

HT rich extract - HT-rich extract

- Standard

Note: Cells were incubated with extract/compound for 4h and cell proliferation was evatuated after 24h

Olive HT-rich extract was more effective in inhibiting

cancer cell growth and inducing cell cycle arrest in the G1

phase than standard compound

• Cell cycle arrest

HT rich extract- C=25M (HT)

Hydroxytyrosol - C=100 M

OLIVE - BASED NATURAL EXTRACTS

• Antiproliferative effect

Note: Cells were incubated with extract/compound for 24h

LT+OL rich

extract

Oleuropein induced cell cycle arrest of HT29 cells in S phase

Oleuropein C=50 M

LT+OL rich extract

• Cell cycle arrest

LT+OL rich extract induced cell cycle arrest in both S and G2 phases

Luteolin was shown to induce cell cycle arrest in G2/M phase (Lim et al., 2006)

OPUNTIA - BASED NATURAL EXTRACTS

Hidroalcoholic extraction

Pressurized liquid extracton

Opuntia rubusta extract contains betalains pigments whereas Opuntia ficus indica extract present flavonoids (isorhamnetin and quercetin glycosides), terpenes and fatty acids (palmitic acid, octadecadienoic acid)

Opuntia rubusta fruit juice residues (seeds and peels)

Opuntia ficus indica fruit juice residues (seeds and peels)

Minutes

15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5

mAU

0

250

500

750

1000

1250

1500

1750

2000

2250

mAU

0

250

500

750

1000

1250

1500

1750

2000

2250

Detector 1-Scan-535 nm

Betalains

HPLC-DAD-UV 535nm

60% CO2: 40%EtOH v/v P= 20MPa; T= 313K

50% H2O :50%EtOH v/v

Minutes10 20 30 40 50 60 70 80 90 100 110

mAU

0

100

200

300

400

500

600

700

800

900

1000

mAU

0

100

200

300

400

500

600

700

800

900

1000

Detector 1-360nmOpuntia 2Jul2611018.dat

• Flavonoids HPLC-DAD-UV 360nm

Phytochemical composition

• Fatty acids

•palmitic acid • octadecadienoic acid

OPUNTIA - BASED NATURAL EXTRACTS

Both Opuntia extracts inhibited HT29 cell growth in a dose dependent manner.

• Antiproliferative effect

Dose response curves

Note: Incubation time- 24 hours

ED50 values ROS generation

Note: Extracts concentration- 0.5mg/mL

OR Extract OFI Extract OR Extract OFI Extract

Opuntia rubusta extract was the most effective in inhibiting HT29 cell growth – lowest ED50 value.

The antiproliferative effect of Opuntia extracts is probably related with the ROS generation at a cellular level

- Opuntia rubusta extract - Opuntia ficus indica extract

OPUNTIA - BASED NATURAL EXTRACTS

Opuntia extracts exhibited different responses on cell cycle arrest, which could be related with the distinct composition of samples:

• Opuntia robusta extract induced cell cycle arrest into G1

• Opuntia ficus indica extract induced cell cycle arrest in S phase

• Cell cycle arrest

Note: Incubation time- 24 hours; extracts concentration-ED50 value

OR Extract OFI Extract

Effect of betalains on cell cycle arrest

Effect of phytochemicals on cell cycle arrest

• Betanin– G0/G1 phase (myeloid leukemia cell line- K562)

(Sreekanth et al., 2007)

• Isorhamnetin– G2 and S phase (colon cell line, HCT-116) (Jaramillo et al., 2010)

•Quercetin–S phase (colon cell line, SW480) (Richter et al., 1999)

CORK - BASED NATURAL EXTRACTS natural extracts provided by Corticeira AMORIM, S.G.P.S., S.A (NutraCork project, QREN)

Cork extracts are rich phytochemicals

Phytochemical composition

CORK E1

CORK E2 Total Phenolics 116 mg GAE/g

• Cork E1- polyphenols and triterpenes

Triterpenes

Betulinic acid Ursolic acid

Friedelin Lupeol

• Cork E2 – polyphenols (phenolic acids and ellagitanins)

Minutes

10 20 30 40 50 60 70 80 90 100

mAU

0

50

100

150

200

250

300

350

400

450

500

mAU

0

50

100

150

200

250

300

350

400

450

500

Detector 1-280nmÁcido Elágico 45ppmNov 2612003.dat

Detector 1-280nmAmostra 6 Dil. 1:5Nov 2612004.dat

1- Gallic acid

2- Ellagic acid

- Ellagitanins

1

2

Ellagic acid

Minutes

0 10 20 30 40 50 60 70 80 90 100

mAU

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

mAU

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80Detector 1-280nmNUC 183 (Conc.)Set2512010.dat

Total Phenolics 2.9 g GAE/L

Ellagic acid

CORK - BASED NATURAL EXTRACTS

Both cork extracts inhibited cancer cell growth in a dose dependent manner

ED50 = 3 mL extract/mL

• Antiproliferative effect

ED50 = 0.25 mg extract/mL

Note: Incubation time- 24 hours

CORK E1

CORK E2

CORK - BASED NATURAL EXTRACTS

Cork extracts exhibited different responses on cell cycle moduation, which could be related with the distinct composition of samples:

• Cork E1 extract induced cell cycle arrest into G1

• Cork E2 extract induced cell cycle arrest in S and G2/M phases

• Cell cycle arrest

Note: Incubation time- 24 hours; C Cork E1=2.4%; C Cork E2= 0.22mg/mL

Effect of phenolics on cell cycle arrest

Effect of triterpenes on cell cycle arrest

• Betulinic acid – G1 phase

• Ursolic acid- G1 phase

• Ellagic acid– S phase (HT29 cells)

• Gallic acid- S phase (HT29 cells)

• Ellagitanins- G0/G1 and G2/M phases (HT29 cells)

Kasimsetty et al.(2010)

Khan SA (2009)

Preliminary experiments

in HT29

This study demonstrates that cherry, cactus pear, olive and cork-based natural extracts inhibit the growth of HT29 through cell cycle modulation.

Natural extracts Induction of cell

cycle arrest Induction of cell

cycle arrest Compounds

CHERRIES POH-RICH EXTRACT

G1

OLIVE HT-RICH EXTRACT

OLIVE LT-OL-RICH EXTRACT

G1

S and G2/M

Perillyl alcohol (monoterpene)

G1

G1 Hydroxytyrosol (phenolic acid)

S Oleuropein (tyrosol esters)

G2/M Luteolin (flavonoid)

OPUNTIA RUBUSTA EXTRACT

OPUNTIA FICUS EXTRACT

Betanin (betaleins)

G1 G1 ?

S ? Quercetin (flavonoid)

G1 Betulinic acid and Ursolic acid (triterpenes)

Isorhamnetin (flavonoid)

S and G2/M ?

? .....

S

CORK EXTRACT 1

CORK EXTRACT 2

G1

S and G2/M Ellagic and gallic acids (phenolic acids)

S

G0/G1 and G2/M Ellagitannins

The natural extracts used in this study have the potential to improve the research and development of new chemotherapeutic agents for colon cancer.

This study demonstrates that cherry, cactus pear, olive and cork-based natural extracts inhibit the growth of HT29 through cell cycle modulation.

Evaluation of other natural extracts and ingredients

Evaluation of more anticancer effects (induction of apoptosis, 3D cell-based assays, …)

Joana Poejo, Inês Silva

Dr. Ana Matias

Group Leader: Dr. Catarina Duarte

Nutraceuticals and Controlled Delivery

Lab IBET/ITQB-UNL

Financial Support: • FEDEP/COMPETE -QREN project “NUTRACork” (ref. 21597/2011) • Fundação para a Ciência e Tecnologia (FCT) - PTDC/AGR-AAM/099645/2008 project - PEst-OE/EQB/LA0004/2011 grant - REDE/1518/REM/2005

Analytical Chemistry Lab ITQB/IBET

Dr. Rosário Bronze

Polymer Processing and Supercritical

Technology Lab FCT-UC

Dr. Herminio de Sousa

Dr. Mara Braga, Dr. Inês Seabra

CORTICEIRA AMORIM, S.G.P.S., S.A.

(Portugal)

Dr. Susana P. Silva, Patrícia Correia

iBET is a private not for profit research organization in the area of biotechnology and life sciences.

Oeiras, Portugal

The largest Biotechnology Research Organisation in Portugal

Brings together as partners and collaborators, public institutions and private companies

Main

Activities

Health – Pharma

Agro – Industry

Plants – Forestry

Environment

Mission

Foster the competitiveness of its customers

and partners

Create wealth out of

knowledge of Chemistry,

Biochemistry and Biology

Agro – Industry

Processing Chemical

characterization Biological

characterization Formulation

Development of alternative green technologies for the isolation of natural of bioactive compounds with high added-value and application in food, cosmetic and pharmaceutical industries

• Nutraceuticals and Controlled Delivery Lab • Membrane Processes Lab

Development and application of cell-based assays

• Nutraceuticals and Controlled Delivery Lab • Animal Cell Technology Lab

Identification and quantification of several classes of compounds

• Nutraceuticals and Controlled Delivery Lab • Analytical Chemistry Lab

Development of adequate and efficient systems to : -Increase Shelf life and stability of the bioactive -Increase solubility -- Increase bioavailaibity - ….

• Nutraceuticals and Controlled Delivery Lab

e-mail: tserra@ibet.pt

http://www.itqb.unl.pt/labs/nutraceuticals-and-delivery

12TH INTERNATIONAL CONFERENCE FUNCTIONAL FOOD INGREDIENTS AND NUTRACEUTICALS IN CHRONIC DISEASE: SCIENCE AND PRACTICE NOVEMBER 29-DECEMBER2 DALLAS, TX, USA