Supercritical Fluid Extraction of Bioactive Compounds from Natural Products

SRSFerreira - Chapecó, November, 2011

Supercritical Fluid Extraction of Bioactive Compounds from

Natural Products

LATESC/EQA - CTC – UFSCChapecó – November, 2011

Prof. Sandra R.S. [email protected]

SRSFerreira - Chapecó, November, 2011SRSFerreira - Chapecó, November, 2011 2

Summary

èNatural products

èBiological activities

èExtraction methods

èSFE

èSFE vs low pressure

v Extraction yieldv Solute compositionv Antioxidant activityv Antimicrobial activity


Natural Products

èNaturally occurring compounds or group of substances

èSources:v Plants, fruits, animals, microorganisms, fungus, etc.v Biological activities

èRelated areas:v Medicine, food, flavoring and nutritional supplement

èDrugs and natural products? Newman & Cragg, 2007 v 1184 new chemicals approved (1981 to 2006)

Ø 52% natural product connectionØ 18% are biologicsØ 30% purely synthetic


Anticancer drugs

All available anticancer drugs, 1940s-06/2006.

• B (Biological): isolated from organism/cell or produced biotechnologically

• N: Natural product • ND: Derived from a natural

product • S: Synthetic drug.• S*: Synthetic, but similar to natural

product • V: Vaccine.

Newman and Cragg (2007). J. Nat. Prod., 70 (3), 461-477


Biological activity

èBA: properties and reactions of drugs related to their medicinal value (Webster’s, 1993)

èBioactivities:v Anti-microbial, antioxidant, antifungal, antibacterial, cytotocxic (toxic to cells:

used as anti-cancer), enzymatic, antiviral, anti-inflammatory, anesthetic, allelopathic (interfering herbs).

èChemical profile:v GC, GC-MS, HPLC, spectroscopy, X-ray difratometry, fluorescence,

spectrometry, chemometric methods (principal component analysis – PCA).


Few sources: 2008

Property - action Raw material (source) – substance Reference

Anti-HIV Hypericum H. chinese L. salicifolium, biyouyanagin A Nicolaou, et al., 2008.

Antimalarial/AM Manzamines: class of marine natural products Shilabin, et al., 2008

Melanoma cell reduction Dextrin Duncan et al., 2008.

Antibiotic Moenomycins, a potent family of natural product Yuan et al., 2008.

Antiproliferative activity Epothilones: macrocyclic bacterial natural products Feyen et al., 2008.

AM Latarcin from Spider venon Shlyapnikov et al., 2008.

AA Aminothiaxole (alkaloid from Dendrodoa grossularia). Strayo et al., 2008.

AA and AM Cynara cardunculus extracts Kukić et al., 2008.

AA Combine lycopene, b-carotene, vitamin E, vitamin C. Liu et al., 2008.

Antitumor antibiotics Lactimidomycin, iso-migrastatin and migrastatin Ju et al., 2008. In Press.

Cytotoxicity/genotoxicity Marine sponges: sources of alkaloid ingenamine G. Cavalcanti et al., 2008.


Few sources: 2011

Property -action

Raw material (source) – substance Reference

Antitumor glycans from green tea Fan et al., 2011.

Antitumor polysaccharides extracted from Asparagus officinali

Zhao et al., 2011.

Antimicrobial Eucalyptus globulus oil Tyagi & Malik, 2011

Antimicrobial Mentha piperita oil Tyagi & Malik, 2011

Antioxidant Food-derived peptidic antioxidants Samaranayaka et al, 2011

Antioxidant Marine Food Ngo et al., 2011


Bioactive extracts

èNatural products applied as extracts:vTotal or partial extracts, essential oil, oilresin...

èPhytotherapy: v Study of the use of plants or plant extracts as health-promoting agents

èAmazon: huge natural reservoirv 55,000 vegetable species cataloged from a total of 350,000 to 550,000


Important groups

è Alkaloids: high biological activity

v Anti-tumor, anti-spasmodic.

è Flavonoids: Present in flowers and fruits

v Blood vessels protectors, anti-inflammatory.

è Essential oils: Aromatic compounds

v Anti-septic and stimulant.

è Tannins: Phenolic compounds

v Ad stringent, bactericide and cicatrisation.

Alkaloid: caffeine

Flavonoid


Extraction vs extract

G. Romanik et al. J. Biochem. Biophys. Methods, 70 (2007) 253–261

Separation steps used for isolation of plant metabolites

Pre-treatment Extraction

Separation / concentration

Raw material


Extraction methods

èHydrodistillationèCold pressed extractionèSteam distillationèSolvent extraction

v SoxhletvMaceration/fractionationv Percolation

Accelerated solvent extractionMicrowave assisted extractionUltrasound assisted extractionSupercritical Fluid Extraction


Hydrodistillation

è Clevenger type apparatusè Solvent: waterè Raw material:

v Soaked into water

è Essential oilsè High temperature

http://pagesperso-orange.fr/guy.chaumeton


Cold pressed extraction

è Used for vegetable and essential oils from natural products

è Screw pressed extraction

è Low heat technique

http://www.abchansenafrica.co.za


Steam distillation

è Material placed into a still, pressurized steam passes è Heat: globules of oil burst and oil evaporatesè Essential oil condensates in water cooled pipe

http://everestherbs.com.np/


Solvent extraction

è Raw material and solvent: dissolve solute

è Solvent selection:v Hydrocarbons, alcohols, ketone, acetic acid

v Polarity, solubility, solute interactions

è Solute/solvent separation

http://www.armfield.co.uk/


Maceration and soxhlet

è Raw matter soaked in solvent, heated and strained

è Variables: time and temperature

è Refluxè High temperatureè Time consumingè Solvent consuming

http://www.rsc.org/chemistryworldhttp://www.albrigi.co.uk


Conventional extractions

èProblems:

vHigh temperatures

vSolvent contamination: reduces product quality

vInflammability or explosion risks

vTime, solvent and energy consuming

vComposition varies with solvent and extraction technique


New extraction methods

èASE: accelerated solvent extraction (Jaques et al., 2008)

v Pressurized extraction: high pressure (3000 psi) and temperaturev Good performance: break of solute–matrix interactions

èMAE: microwave assisted extraction (Wang et al., 2008)

v Less solvent consumption, shorter times, higher yields,vMicrowave plus solvent extraction: enhances efficiency.

èUSE: ultrasonic solvent extraction (Cuoco et al., 2008)

v Efficient contact (sample/solvent): increases efficiencyv Accelerates extraction due to disruption of cell walls.


SFE: alternative

èClassical methods:v Limitations: time and solvent

consuming and energy costs

èSFE:v Product quality:

Ø Solvent free

Ø Thermal degradation free

v Energy saving: Ø Extraction + separation

http://www.nature.com

SRSFerreira - Chapecó, November, 2011SRSFerreira - Chapecó, November, 2011 20www.chem.leeds.ac.uk

20


High mass transfer rates

SFE: characteristics

SCF: intermediate properties between liquid and gas phases

State r x 103 [kg/m3] DAB x 104 [m2] m [kg/m.s]

Gas (0.6–2) x 10-3 0.1 – 0.4 (1–3) x 10-5

SCFPc; Tc

4Pc; Tc0.2 – 0.50.4 – 0.9

0.7 x 10-3

0.2 x 10-3(1–3) x 10-5

(3–9) x 10-5

Liquid 0.6 – 1.6 (0.2–2) x 10-5 (0.2–3) x 10-3


PUMP

SEPARATOR

SOLUTE

SOLVENT

EXTRACTOR

Basic components

CO2: most used solvent (304.2K

and 72.3bar)


SFE: aspects

è Solvent power control (T/P)

è Fractionation

è Selectivity

è Extraction and separation: v One step


SFE: applications

èAlternative process for high aggregated value products

èWhen quality is determinantv Fine chemistry and pharmacy (active principles)v Food (caffeine, hop, essential oils, aromas...)

èSources: v Plants, microorganisms, food, sea organisms, fungus…

èExamples:v Food, colorants, drugs, vitamins, phyto-hormones

v Spices, coffee, aromas, oil seeds, hop, tobaccov Wax, polymers, cleaning products…


http://www.vtt.fi/pro

SFE: industrial units

www.separex.fr


P/T conditions

Extraction Raw material Product P (bar) T (oC)

Oily plants (crops) Soy oil Corn oil

Up to 700 Up to 50

Black pepper Black pepper oil Piperine

90 200

40 40

Coffee Caffeine 300 80 Jojoba Jojoba oil 700 60

Green pepper Dye (color) 35 60 Hop Hop extract Up to 400 Up to 50

Chamomile Matricine ∼ 100 ∼ 40 Chrysanthemum Piretrine 250 40

Graichen & Hubert, (1994)


LATESC: raw materials

è Marigold (Calendula officinalis)è Horsetail (Equisetum giganteum)è Rice bran oil (Oryza sativa L.)è Avenca-da-praia (Polygala cyparisias)è Menthe (Mentha spicata)è Rosemary (Rosmarinus officinalis L.)è Shiitake (Lentinula edodes)è Grape pomace (Vitis vinífera)

v Shiraz, Merlot, Cabernet sauvignon

è Erva baleeira (Cordia verbenacea)è Spent Coffee ground and coffee husk

è Apple pomaceè Propolisè Peach almondè Shrimp shells è Banana peelè Orange pomaceè Eucalypt leavesè Plumbè Fishery by-products è Pecan nutsè Shrimp residue


Extraction results

èProcess aspects:

v Extraction yield: cross-over influencev Use of co-solvents in SFE: increase yield

èProduct quality:

v Extract composition: chromatographic methodsv Biological activities: antioxidant and antimicrobial activities


Peach almond

è Peach almond:v Industrial residue

v 32-55 % of oil

v Rich in fatty acids (oleic and linoleic)

Oil extractionAnimal feed

Residue 20 %

Peach

Products


Yield: peach almond

SFE (CO2) Soxhlet COSE

(bar/ºC) Yield (%) Solvent Yield (%) Solvent Yield (%)

100/30 10.5 ± 0.3 Hx 19 ± 1 Mac-EtOH 6.6 ± 0.3

200/30 15.6 ± 0.8 DCM 32.2 ± 0.6 Mac-Hx 1.4 ± 0.1

300/30 16.2 ± 0.5 EtAc 26 ± 1 Mac-DCM 2.3 ± 0.4

100/40 9.6 ± 0.6 EtOH 32 ± 2 Mac-EtOAc 0.3 ± 0.1

200/40 13.9 ± 0.9 Hx/DCM 50% 26 ± 2 Mac-H2O 0.8 ± 0.2

300/40 17.0 ± 0.5 EtOH/H2O 50 % 12 ± 1

100/50 2.81± 0.06 HD 0.13 ± 0.02

200/50 11.1 ± 0.5

300/50 17.4 ± 0.3

Higher than COSE and HD

Co-solvent


SFE: co-solvents

èCO2: non-polarv Dissolves non-polar

èCo-solvents:v Volatility between solute/SCFv Increase solubilityv Target compoundsv Affect selectivity

è Ethanol, methanol, propane...v EtOH: legally accepted

v From 1% to 5% (w/w)

èInfluence:v Compositionv Biological activityv Extraction yield


SFE: Modifier

èImprove the SFE performance: yield and selectivityèSelection: Ability to dissolve compounds of interest;èCommon examples:

v EtOH, EtAc, DCM, BtOH

LATESC group:è Campos et al. (2008): SFE from C. sauvignon grape pomace

v Increased yield from 2.7% to 9.2% by using 15% EtOHè Biscaia & Ferreira (2009): SFE from propolis

v Increased yield from 8.6% to 24.8% by using 5% of EtOHè Michielin et al. (2009): SFE from C. verbenacea

v Increased yield from 5.0% to 7.7% by using 5% of EtOHè Mezzomo et al. (2010): SFE from peach almond

v Increased yield from 22% to 24% by using 5% of EtOH.


Extract quality

è Product quality as important as process yield

è Results (LATESC): v Different raw materials

v Different extraction methodsØ SFE (CO2 and CO2 + CS) and classical extraction methods

è Quality evaluation:1. Chemical composition profile

2. Antioxidant activity

3. Antimicrobial activity


1. Composition

èChromatographic methodsv Gas Chromatography: mostly for non-polar substances

Ø adequate for CO2 extracts

v HPLC: detect substances with higher polarity

èMass spectrometry:v Components identification:

Ø Standard Reference Data Series of the National Institute of Standard and Technology.

èComposition: v Retention time and standard curves


Grape pomace

è Miolo winery Ltda. (Vintage 2003)v Residue from wine productionv Cabernet Sauvignon, Merlot and

Shiraz

è Compounds remain in pomacev Essential oil and pigmentsv Resveratrol, linoleic acid

è Extract quality: v Compositionv Fruit origin, harvest, v Wine process, v Extraction/solvent method.

Grape pomace

Campos, L.M.A.S.; Leimann, F.V., Curi, R.P.; Ferreira, S.R.S. Bioresource Technology,99(17), 2008: 8413-8420.


GC-MS: grape pomace

GC-MS from grape pomace extract obtained by SFE with pure CO2 at 150 bar, 40ºC and 3.33gCO2/min

No Components

1 Phenyl ethyl alcohol

2 Capric acid

3 Lauric acid

4 Palmitic acid

5 Tridecanoic acid

6 Phytol

7 Linoleic acid

8 Ethyl linoleate

9 Oleic acid

10 Octadecanoic acid

11 Palmitaldehyde

12 Long chain linear acid

For cosmetic products, synthesis vit. E


Merlot pomace (SFE: 250 bar, 60 C, 17.5 % EtOH)

Phenolic compounds

1 Epicatechin 2 Galic acid 3 Tannic acid 4 p-OH-benzoic acid 5 Vanillic acid 6 Caffeic acid

Reduces the risk of cardiovascular disease (Schroeter et al., 2006)

Antioxidant and antimicrobial activity

(Rosso, 2005)

Salicylic acid isomerAntimicrobial activity

(Naz et al., 2006)

HPLC: grape pomace


Extraction method

Grape varietal

Process conditions Epicatechin Gallic acid Tannic acid p-OH-

benzoic acid Vanillic acid Protocatecuic acid

SFE

Merlot 50°C

150bar 35.5 ± 0.1 10.82 ± 0.09 ND 49.8 ± 0.7 31.5 ± 0.5 ND

200bar ND 115.9 ± 0.8 ND 11.6 ± 0.1 18.9 ± 0.4 ND

250bar 5.4 ±0.1 395.6 ± 0.6 387.0 ± 0.5 121.8 ± 0.7 461.1 ± 0.3 ND

300bar 6.7 ± 0.7 55.0 ± 0.1 50.1 ± 0.5 233 ± 1 14.6 ± 0.9 ND

Merlot 60°C

150bar ND 121.6 ± 0.7 ND ND 33.7 ±0.1 ND

200bar ND 44 ± 1 ND 4.6 ± 0.1 10.6 ± 0.1 ND

250bar ND 14.20 ± 0.09 30.3 ± 0.7 88.81 ± 0.08 50.5 ± 0.3 ND

300bar 9.55 ± 0.05 44.5 ±0.7 38.4 ± 0.1 71.4 ± 0.3 58.8 ± 0.5 ND

Syrah 60°C 250bar ND 64.3 ± 0.9 ND 26.3 ± 0.1 ND ND C.

sauvignon 60°C 250bar 87.99 ± 0.04 18.7 ± 0.1 ND 207.5 ± 0.3 58 ± 1 ND

SFE CO2 + EtOH

250bar/60°C Merlot

12.5% 119 ± 1 123.09 ± 0.09 ND 500.5 ± 0.5 3.85 ± 0.07 ND

15.0% ND ND ND ND ND 537 ± 7

17.5% 200.8 ± 0.3 162.4 ± 0.4 26.3 ± 0.2 65.9 ± 0.3 15.21 ± 0.06 ND

SOX Syrah

EtOH

5.3 ± 0.1 10.6± 0.3 20.3 ± 0.2 22.8 ± 0.5 ND ND

Merlot ND ND ND ND ND 9864 ± 4

UME Syrah - - - - - -

Merlot 121.9 ± 0.6 51.0 ± 0.5 62.1 ± 0.1 505.73± 0.06 ND ND

Antimicrobial compounds

HPLC: Merlot, Syrah, C. sauvignon


HPLC: coffee husk and coffee spent

Andrade et al. TALANTA. IN PRESS

Clorogenic ac.: potent antioxidant, hepatoprotection, hipoglicemiant and antiviral (DUARTE et al., 2010).

Identificação de Compostos

Fenólicos Extraction Condition/ Solvent Galic Ac. Tanic Ac.

Clorogenic Ac. Cafeic Ac.

Spent EtOH - 0.7 - -

Spent EtAc 14.3 - 0.3 - US

Husk EtOH - - - - Husk EtAc 113.8 - 0.5 -

SOX Husk EtOH - 80.3 - - Spent 200 bar/60°C - - 41.3 0.1 Spent 300 bar/60°C - - 27.3 - Husk 200 bar/40°C 0.9 - 174.5 - SFE

Husk 300 bar/60°C - - 942.8 -

Spent 100 bar/60°C/15% - - 19.6 - SFE + EtOH Husk 200 bar/50°C/8% - - 232.3 -


HPLC: coffee husk and coffee spent

Andrade et al. TALANTA. IN PRESS

Extraction Condition/Solvent Teobromine (µg/mgextract)

Caffeine (µg/mgextra

Hx - 0.734 DCM - 38.2 Spent EtOH - 25.7 Hx - 5.54 DCM 0.66 139.2

US Husk

EtOH - 71.1 Hx - 3.27 DCM - 25.9 Spent EtOH - 11.8 Hx - 2.1 DCM 0.745 189.9

Soxhlet

Husk EtOH - 129.6 200 bar/60°C - 27.2

Spent 300 bar/60°C - 41.3 200 bar/40°C - 185.7

SFE Husk

300 bar/60°C 1.13 684.2 Spent 100 bar/60°C/15% - 23.4

SFE + EtOH Husk 200 bar/50°C/8% 0.655 87.8

Identificação de Metilxantinas


GC-MS: marigold oil

200 bar/20oC Peak Component MOL (g/gmol)

% Peak area

1 Acetyl eugenol 206 7.436

2 Phenol-4-octyl 206 1.074

3 Guaiol 222 5.071

4 Cedrol 222 1.944

6 Octadecane 254 4.138

7 Tetradecanoic acid 228 0.951

8 Nonadecane 268 0.479

9 Eicosane 282 3.061

10 Heneicosane 296 0.211

11 Docosane 310 4.875

12 Tricosane 324 0.504

13 Tetracosane 338 10.468

14 Pentacosane 352 1.035

15 Hexacosane 366 18.226

16 Heptacosane 380 1.440

17 Octacosane 394 9.952

19 Eicosane-7-hexyl 366 0.449

20 Eicosane-9-octyl 394 0.612

21 Canescegenine 420 0.792

22 Cholest-4-en-3-one-14-methyl

398 0.831

23 Taraxasterol 426 1.186

24 1-octadecanol 270 0.149

25 1,16-hexadecanediol 258 0.257

Phytosterol (against breast cancer, reduces cholesterol)

Absent in other extracts

L. Danielski, et al. Chem. Eng. Proc., 46 (2). 2007: 99-106.

SFE: 200 bar/20oC


Horsetail (Equisetum giganteum)

è Horsetail = cola de caballo.

è Actions:v Anti-inflammatory, diuretic, anti-

hemorrhagic, skin regenerator

è Extraction:v SFE, soxhlet and COSE

è Composition: v GC-MS


No00 Compound Mol

1 Diphenyl carbonate 214

2 Dodecanoic acid 212

3 3-noninoic methyl ester 168

4 3,6-dimetil decane 170

5 Heneicosane 296

6 26-Hydroxicholesterol 402

7 Ergosta-4,7,22-trien-3-one 394

8 8,12-Dimethyl-4Z,8E,12E-octadecatriene

276

9 Methenolone 302

10 2,6,10,14-hexadecatet.-1-ol,3,7,11,15-tetrametilacetato

332

11 Z-13-Octadecenal 266

12 Heneicosane-11-decil 436

13 Eicosane-10-heptyl-10-octil 492

14 17,21-dimetil-heptatriacontano 549

GC-MS: horsetail

0

0.1

0.2

0.3

0.4

0.5

0.6

Compound 1 Compound 3 Compound 6 Compound 8

Horsetail components

Mol

ar fr

actio

n

313K/12MPa313K/20MPa303K/12MPa303K/20MPa

Michielin, et al. Composition Profile Of Horsetail Oleoresin: Comparing SFE And Organic Solvent Extraction. J. Supercritical Fluids. 2005. V. 33: 131.

Phyto-sterol: absent in other extracts


GC-MS: Mentha spicata L

Compound SFE (CO2) Sox-EtOH SFE CO2+EtOH

Carvone 36.16 41.61 49.76

Pulegone 8.22 9.44 6.9

Phytol 8.11 4.61 11.13

èGC-MS: menthev Carvone: antifungal and antimicrobialv Pulegone: muscle reliever and for indigestionv Phytol: diterpene alcohol used for vitamin E

synthesis and regulates metabolic process.

Almeida et al. Food and Bioproc. Tech. .IN PRESS.


2. Antioxidant activity (AA)

èAbility to avoid or reduce oxidative rancidity (food deterioration)

èSynthetics (carcinogenic effect):v BHA, BHT, TBHQ (phenols)

èNaturals (non toxic): v From food products and plant material

v Tocopherols, ascorbic acid, phenolic compounds

èSeveral classes with diverse chemical behavior:v AA result is dependant of the method

v One method: do not detect all mechanisms that characterize an AA.


Antioxidant methods

è Methods for AA%: Espectrophotometric and fluorescence methodsv Free radical scavenging methodsv Redox potential of antioxidants

è DPPH: (MENSOR et al. 2001) v Radical (2,2-difenil-1-picrilhidrazil): electron capture from antioxidant

è ABTS: v AA compounds: Ability to reduce radical ABTS•+ (600-750 nm)

è β-carotene/linoleic acid bleaching method (Matthäus, 2002)v Suitable for lipophilic fractions: Ability to protect the lipid fraction from oxidation

è Total phenolic content (TPC):v Folin-Ciocalteu colorimetric method [Singleton & Rossi, 1965]v TPC was expressed as gallic acid equivalent (GAE)/mg of extractsv For poly-phenols and mono-phenols


3. Antimicrobial activity

èAntimicrobial substances: v Prevent or inhibit microorganisms growth

èResistant bacteria: v Challenge to infection treatmentsv Patient sensibility to traditional antimicrobialsv New substances are necessary

èAntimicrobial compounds from plant material:v Not well exploitedv Studies are very incipient.


Antimicrobial activity

è Agar diffusion method (ADM): inhibition ≥ 9mm selected for MICv S. Aureus, B. Cereus, M. luteus: Gram positivev E. Coli, P. Aeruginosa: Gram negativev C. Albicans, : fungus

èMinimum inhibition concentration (MIC)v Positive result (Duarte et al., 2007):

Ø < 500 mg/mL: strong inhibitorsØ 600 – 1500 mg/mL: moderate inhibitor.


Yield and DPPH: grape pomace

0

2

4

6

8

10

SFE 0% EtOHSFE 10% EtOHSFE 15% EtOHSFE 20% EtOH

Yie

ld [

%]

0

5

10

15

20

25

AA

[%

]

Yield AA %

ESC at 150 bar, 40ºC: AA by DPPH (sample at 250 µg/mL)

SFE: yield and AA increase with EtOH

concentration


Grape Pomace: antimicrobial activity

ADM (mm)


ADM (mm)

Grape Pomace – ADM results

SRSFerreira - Chapecó, November, 2011 51

Other low pressure extracts:no antimicrobial activity

ADM: positive results: selected for MIC

Baydar et al. (2004): grape peel extracts (not active); grape seed extracts (highly active)


Antimicrobial activity: MIC

Extraction

Method Grape Process conditions

MIC (µg/mL)

S. aureus B. cereus E. coli P. aeruginosa C. albicans

ESC CO2

Merlot 50 °C

150 bar 750 ± 250 2000 >2000 >2000 -

200 bar 1500 ± 500 1000 >2000 >2000 500

250 bar 2000 1500 ± 500 >2000 >2000 -

300 bar 625 ± 375 1000 1000 1000 -

Merlot 60 °C

150 bar 1000 1000 2000 2000 >2000

200 bar 1000 2000 2000 2000 -

250 bar 2000 2000 2000 2000 -

300 bar 1500 ± 500 1000 2000 2000 -

Syrah 60 °C 250 bar 1500 ± 500 2000 >2000 >2000 -

C. sauvignon 60 °C 250 bar 500 1000 >2000 >2000 -

Soxhlet Syrah hexane - 2000 - - -


AA% (DPPH): menthe

SFE: CO2 SFE: CO2 + co-solvent Soxhlet

(bar/ºC) AA(%) (bar/ºC/EtOH%) AA(%) Solvent AA (%)

100/30 15 200/40/10% 70.3 Hx 30.5

100/40 26.7 200/40/15% 49.3 DCM 86.3

100/50 18.7 200/40/20% 71.4 EtAc 92.9

200/30 20.9 150/40/20% 38.2 BtOH 94.0

200/40 28.7 150/50/20% 84.4 EtOH 95.2

200/50 14.2 200/50/20% 24.5

300/30 22.6 230/40/20% 78.6

300/40 24.9 230/50/20% 35.3 HD AA (%)

300/50 24.2 Water 20.3

High AA for SFE with co-solvent


Shiitake (Lentinula edodes)

è 2° most consumed mushroom:

è Mushrooms: nutraceutic foodv Source of active compoundsv Cancer protection: Lentinanv Anti-cholesterolemic: Eritadeninav Antioxidant activityv Antimicrobial activity

è Shiitake extract:v 60 pills 500 mg = US$ 9.95

(http://www.vitacost.com)


AA%: Shiitake

0

40

80

120

160

200

Cose:DCM Cose:EtAc SF:EtOH 5% SF:EtOH10%

SF:EtOH15%

Shiitake extract

IC50

0

0.5

1

1.5

2

2.5

ET

A (

g/1

00 g

ext

ract

)

IC50ETA

IC50 (DPPH): Inhibition concentration

ETA (equivalent of tanic acid): Total Phenolic Content

Kitzberger et al., Journal of Food Engineering, 80(2): 631-638. 2007.

SFE: AA% similar to DCM


AM: Shiitake

SFE (°C/MPa) S.aureus E.coli M.luteus B.cereus C.albicans

30/15 0 I.H. 0 0 12

30/20 0 I.H. 14 10 0

30/30 0 9 12 10 0

40/15 0 I.H. 14 0 12

40/20 0 I.H. N.T. N.T. N.T.

40/30 I.H. 0 19 12 0

50/15 I.H. I.H. 16 14 0

50/20 0 I.H. 12 10 0

40/20 + EtAc 15% N.T. N.T. 0 12 0

Microrganismos SFE (°C/MPa) M. luteus B. cereus C. albicans

30/15 N.T. N.T. 2.0

30/20 0.5 N.T. N.T.

30/30 1.0 0.25 N.T.

40/15 1.0 N.T. 2.0

40/30 1.0 0.5 N.T.

50/15 0.5 0.5 N.T.

50/20 1.0 N.T. N.T.

40/20+EtAc15% >2.0 0.5 N.T.

Low pressure extracts:no antimicrobial activity

Kitzberger et al., Journal of Food Engineering, 80(2): 631-638. 2007.

MIC (mg/mL)

ADM (mm)

MIC (Positive result):< 0.5 mg/mL: strong inhibitors


%AA Orange pomace: β-carotene/linoleic ac.

Extraction Solvent % AA (120 min)

ESC 40 ºC/100 bar CO2 84.4bcdefg ± 0.3

ESC 50 ºC/150 bar CO2 95b ± 4

ESC 40 ºC/200 bar CO2 82cdefgh ± 4

ESC 50 ºC/200 bar CO2 110a ± 3

ESC 50 ºC/250 bar CO2 90bcd ± 3

ESC 40 ºC/300 bar CO2 88bcdef ± 3

ESC 50 ºC/300 bar CO2 88bcde ± 2

ESC 50 ºC/250 bar CO2 + EtOH 92.5bc ± 0.2

BHT - 113a ± 7

Benelli et al. Journal of Supercritical Fluids, v. 55, p. 132-141, 2010.


Propolis

è Natural product used as medicine purpose for centuries;

è Complex mixture:

v Resinous material from plant sources, transferred by enzymes (bee) and wax (MARCUCCI et al., 2001).

è Component: artepilin Chttp://www.natucentro.com.br/


AM: Propolis

method E. coli S. aureus B. cereus

Sox-EtOH 0 16 13 Sox-EtAc 0 14 13 Sox-CHCl3 0 14 12 Sox-Hex 0 18 14 Sox-H2O 0 14 10 COSE-EtOH 0 18 13 COSE-EtH2O 70% 0 14 14 COSE-EtH2O 50% 0 18 19

SFE (P/T) E. coli S. aureus B. cereus 100/30 13 19 16 100/40 14 15 15 100/50 12 12 14 150/30 0 15 16 150/40 13 17 20 150/50 0 21 20 200/30 0 16 21 200/40 0 15 15 200/50 0 18 17 250/40 0 16 18

ADM (mm)

Method E. coli S.

aureus B.

cereus

Sox-EtOH NT 0.2500 0.5000

Sox-Hx NT 0.0625 0.5000

COSE:EtOH50% NT 1.0000 0.2500

SFE100/30 1.0000 0.1250 1.0000

SFE100/40 0.5000 0.2500 1.0000

SFE150/40 0.5000 0.1250 1.0000

SFE200/30 NT 0.0625 1.0000

SFE250/40 NT 0.0625 0.5000

SFE150/40/5% NT 0.0625 0.5000

MIC (mg/mL)

SFE: excellent method for antimicrobial extracts


Cordia verbenacea

è SC and SP shore; v erva baleeira, salicilina (RAMOS et al., 2005, CARVALHO et al., 2004 ).

è Activities:

v anti-inflammatory and cicatrizing

v Rheumatism and attrite treatments

è Essential oils (aroma):v a-humulene, b-caryophylene, pinene

è Flavonoids:v quercetine, artemetin

www.jardimdeflores.com.br


DPPH: C. verbenacea

COSE EtAc EC50= 9.2±0.4 mg/mL Soxhlet 50% EtOH EC50= 29±2 mg/mLRutin Ec50= 6.2±0.3 mg/mL

0

400

800

1200

Sox E

tAc

Sox E

tOH

Sos H

x

Sox D

CM

Sox w

ater

Sox K

et

50%

EtOH

25%

EtO

H

COSE EtA

c

COSE Hx

COSE DCM

COSE wat

er

Rutin

Querc

etin

Extracts

DP

PH

(EC

50

g/m

L) C. verbenacea: Extracts with excellent AA


TPC & ABTS: C. verbenacea

SFEP/T (bar/°C)

TPCmgEAG /g extract

ABTS% inhibition

100/30 47±1 30.4±0.2

100/40 63±2 19.1±0.2

100/50 46±2 21±1

200/30 105±3 24±1

200/40 112±3 33±3

200/50 67±1 31±1

300/30 61±1 29±1

300/40 63±3 27.5±0.7

300/50 71±1 29.4±0.3

CO2 +2%EtAc 73±3 35±2

CO2 +5%EtAC 76±7 43±1

CO2 +8%EtAc 85.7±0.3 39±1

CO2 +2%EtOH 84±3 32±1

CO2 +5%EtOH 64.5±0.3 30±2

CO2 +8%EtOH 77.8±0.8 46±3

Extract TPCmgEAG/gextract

ABTS% inhibition

Sox EtAc 72±2 39.3±0.1

Sox EtOH 97±3 46.7±0.4

Sox Hx 42±2 27.7±0.1

Sox DCM 82±3 32.9±0.3

Sox water 111±3 77±3

Sox ketone 82±2 42.1±0.5

Sox 50%EtOH 102±9 43.2±0.9

Sox 25%EtOH 187.3±0.9 81±2

COSE EtAc 358±7 81.5±0.6

COSE Hx 63±4 27±0.5

COSE DCM 114±7 47.1±0.3

COSE water 84±2 77.1±0.3

Sox and COSE:Better for AA


AM: C. verbenacea

All extracts present AM against Gram +

bacteria

ADM (mm)

Extract S. aureus B. cereus E. coli P. aeruginosaSFE100bar/30 C 09 11 00 12SFE100bar/50 C 15 15 00 11SFE200bar/30 C 15 13 00 00SFE200bar/40 C 31 12 00 00SFE300bar/30 C 16 13 00 11SFE300bar/40 C 15 13 00 10

CO2+ 5% EtAc 17 14 00 10

CO2+ 5% EtOH 15 12 00 09

COSE EtAc 19 16 00 11

COSE Water 20 13 00 00

Sox 25% EtOH 25 15 16 21

Sox 50% EtOH 18 11 00 00

Sox EtOH 16 13 00 00

Sox Water 20 10 12 16

Sox EtAc 21 16 12 00


AM: C. verbenacea

Extract S. aureus B. cereus E. coli Pseudomonaaeruginosa

SFE 100/50 °C 1.000 0.125 NT >4.000

SFE 200/40 °C 0.500 0.0468 NT NT

SFE 300/30 °C 0.375 < 0.0078 NT >4.000

CO2 + 5% EtAc 0.250 0.0156 NT >4.000

CO2 + 5% EtOH 0.250 0.03125 NT 1.000

Mac EtAc 0.250 0.0468 NT 1.500

Sox 25% EtOH 2.000 1.000 2.000 1.000

Sox Water 2.000 2.000 2.000 1.000

Sox Hx NT NT 2.000 >4.000

Sox EtAc 0.500 0.0468 2.000 NT

MIC (mg/mL)

SFE: excellent method for highly

effective AM

MIC (Positive result): < 0.5 mg/mL: strong inhibitors


Economic issues

èLast 15 years (natural products): v More than 100 plants (pilot and industrial scales) in operation

èSmall to medium units:v 100 to 1000 ton/year at 3 to 4 EUR/kg product

è Industrial units:v 10.000 ton/year at 0.5 EUR/kg product

èNatural products (3% extract)v Cost: 100 to150 EUR/kg productv Continuous process: cost reduction in 5 times


Conclusion

èExtraction method and process conditions affect:v Extraction yieldv Chemical profilev Biological activity

èSFE: relevant technology for biological active extracts

v Screeningv Process optimizationv Product standardize

v Costs Good idea!


Federal University of Santa Catarina

Chemical and Food Engineering Department

EQA/CTC – UFSC, P.O. Box 476Florianópolis, SC – BRAZIL

ZIP Code 88040-900Phone: + 55 48 3721.9448FAX: + 55 48 3721.9687

www.enq.ufsc.br


References

è MENSOR, et al. Phitoterapy Research. v. 15, p. 27-130.è SAUCEAU, M. J.of Supercritical Fluids 31 (2004) 133-140.è SOVOVÁ, H. J. Chem. Data 2001, 46, 1255-1257.è CHAFER et al., J. of Supercritical Fluids 32 (2004) 89-96.è BOHN, J.; BEMILLER, J.N. Carbohydrate Polymers, USA, 28, 3-14, 1995.è Campos, L.M.A.S.; Leimann, F.V., Curi, R.P.; Ferreira, S.R.S. Bioresource Technology, 99(17), 2008: 8413-8420.è C. M. P. Sarmento, S. R. S. Ferreira; H. Hense. BJChE (IN PRESS). BJChE, 23(2): 243 – 249. 2006.è Nicolaou, K. C.; Wu, T. R.; Sarlah, D.; Shaw, D. M.; Rowcliffe, E.; Burton, D. R. J. Am. Chem. Soc.; 2008; In Press.è Shilabin, A. G.; Kasanah, N.; Tekwani, B. L.; Hamann, M. T. J. Nat. Prod.; (Article); 2008; 71(7); 1218-1221.è Duncan, R.; Gilbert, H. R. P.; Carbajo, R. J.; Vicent, M. J. Biomacromolecules; (Article); 2008; 9(4); 1146-1154.è Yuan Y. ; Fuse S. ; Ostash B. ; Sliz P. ; Kahne D. ; Walker S. ACS Chem. Biol.; (Article); 2008; 3(7); 429-436. è Feyen, F.; Cachoux, F.; Gertsch, J.; Wartmann, M.; Altmann, K. Acc. Chem. Res.; (Article); 2008; 41(1); 21-31.è Shlyapnikov et al. Protein Expression and Purification, 60(1) 2008: 89-95.è Strayo et al. Devasagayam. Chemico-Biological Interactions, 173(3). 2008: 215-223.è Kukić et al. Food Chemistry, 107 (2). 2008: 861-268.è Liu et al. LWT - Food Science and Technology, 41(7) 2008: 1344-1349.è Ju et al. Bioorg. & Medicinal Chemistry Letters, 2008.è Cavalcanti et al. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 147(4). 2008 409-415.

è G. Romanik et al. J. Biochem. Biophys. Methods 70 (2007) 253–261

è Michielin, E.M.Z.; Bresciani, L. F.V.; Danielski, L.; Yunes, R.A.; Ferreira, S.R.S. J. Supercritical Fluids. 2005. V 33: 131.

è Kitzberger et al., Journal of Food Engineering, 80(2): 631-638. 2007.

è Danielski et al. Chem. Eng. Proc., 46 (2). 2007: 99-106.


Thank you!

Acknowledgements

Education

Supercritical Fluid Extraction of Bioactive Compounds from Natural Products