Supercritical Fluid Extraction of Bioactive Compounds from Natural Products

Supercritical Fluid Extraction of
Bioactive Compounds from
Natural Products

Prof. Sandra R.S. Ferreira
sandra@enq.ufsc.br

LATESC/EQA - CTC – UFSC
Chapecó – November, 2011
SRSFerreira - Chapecó, November, 2011

Summary

èNatural products èSFE vs low pressure

èBiological activities v Extraction yield
v Solute composition
èExtraction methods
v Antioxidant activity
èSFE v Antimicrobial activity

SRSFerreira - Chapecó, November, 2011 2

Natural Products

è Naturally 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 connection
Ø 18% are biologics
Ø 30% purely synthetic


Anticancer drugs

• 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.

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

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


Biological activity

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

è Bioactivities:
v Anti-microbial, antioxidant, antifungal, antibacterial, cytotocxic (toxic to cells:
used as anti-cancer), enzymatic, antiviral, anti-inflammatory, anesthetic,
allelopathic (interfering herbs).

è Chemical 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 - Raw material (source) – substance Reference
action
Antitumor glycans from green tea Fan et al., 2011.
Antitumor polysaccharides extracted from Zhao et al., 2011.
Asparagus officinali
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:
v Total or partial extracts, essential oil, oilresin...

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

èAmazon: huge natural reservoir
v 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.
Alkaloid: caffeine

è Essential oils: Aromatic compounds
v Anti-septic and stimulant.

è Tannins: Phenolic compounds
v Ad stringent, bactericide and cicatrisation. Flavonoid


Raw
material Extraction vs extract

Pre- Separation /
treatment Extraction concentration

Separation steps used for isolation of plant metabolites
G. Romanik et al. J. Biochem. Biophys. Methods, 70 (2007) 253–261

Extraction methods

èHydrodistillation
èCold pressed extraction
èSteam distillation
èSolvent extraction
v Soxhlet
v Maceration/fractionation
v Percolation

Accelerated solvent extraction
Microwave assisted extraction
Ultrasound assisted extraction
Supercritical Fluid Extraction


Hydrodistillation

è Clevenger type apparatus
è Solvent: water
è Raw material:
v Soaked into water
è Essential oils
è 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

http://everestherbs.com.np
/

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


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, è Reflux
heated and strained è High temperature
è Variables: time and temperature è Time consuming
è Solvent consuming

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

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 temperature
v Good performance: break of solute–matrix interactions

èMAE: microwave assisted extraction (Wang et al., 2008)
v Less solvent consumption, shorter times, higher yields,
v Microwave plus solvent extraction: enhances efficiency.

èUSE: ultrasonic solvent extraction (Cuoco et al., 2008)
v Efficient contact (sample/solvent): increases efficiency
v 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

www.chem.leeds.ac.uk

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

SCF
Pc; Tc 0.2 – 0.5 0.7 x 10-3 (1–3) x 10-5
4Pc; Tc 0.4 – 0.9 0.2 x 10-3 (3–9) x 10-5
Liquid 0.6 – 1.6 (0.2–2) x 10-5 (0.2–3) x 10-3

High mass transfer rates


Basic components

SOLVENT
PUMP

EXTRACTOR CO2: most used
solvent (304.2K
and 72.3bar)

SEPARATOR

SOLUTE


SFE: aspects

è Solvent power control (T/P)

è Fractionation

è Selectivity

è Extraction and separation:
v One step


SFE: applications

è Alternative process for high aggregated value products

è When quality is determinant
v Fine chemistry and pharmacy (active principles)
v Food (caffeine, hop, essential oils, aromas...)

è Sources:
v Plants, microorganisms, food, sea organisms, fungus…

è Examples:
v Food, colorants, drugs, vitamins, phyto-hormones
v Spices, coffee, aromas, oil seeds, hop, tobacco
v Wax, polymers, cleaning products…


SFE: industrial units

http://www.vtt.fi/pro

www.separex.fr

P/T conditions

Extraction
Raw material Product
P (bar) T (oC)
Oily plants (crops) Soy oil Up to 700 Up to 50
Corn oil
Black pepper Black pepper oil 90 40
Piperine 200 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 Apple pomace
è Horsetail (Equisetum giganteum) è Propolis
è Rice bran oil (Oryza sativa L.) è Peach almond
è Avenca-da-praia (Polygala cyparisias) è Shrimp shells
è Menthe (Mentha spicata) è Banana peel
è Rosemary (Rosmarinus officinalis L.) è Orange pomace
è Shiitake (Lentinula edodes) è Eucalypt leaves
è Grape pomace (Vitis vinífera) è Plumb
v Shiraz, Merlot, Cabernet sauvignon è Fishery by-products
è Erva baleeira (Cordia verbenacea) è Pecan nuts
è Spent Coffee ground and coffee husk è Shrimp residue


Extraction results

èProcess aspects:

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

èProduct quality:

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


Peach almond

Peach

è Peach almond:
Products
v Industrial residue
v 32-55 % of oil
v Rich in fatty acids
Residue 20 % (oleic and linoleic)

Animal feed Oil extraction


Yield: peach almond
Higher than
COSE and HD

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 Co-solvent HD 0.13 ± 0.02
200/50 11.1 ± 0.5
300/50 17.4 ± 0.3


SFE: co-solvents

èCO2: non-polar è Ethanol, methanol, propane...
v Dissolves non-polar v EtOH: legally accepted
v From 1% to 5% (w/w)

èCo-solvents: èInfluence:
v Volatility between solute/SCF v Composition
v Increase solubility v Biological activity
v Target compounds v Extraction yield
v Affect selectivity


SFE: Modifier

èImprove the SFE performance: yield and selectivity
èSelection: 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% EtOH
è Biscaia & Ferreira (2009): SFE from propolis
v Increased yield from 8.6% to 24.8% by using 5% of EtOH
è Michielin et al. (2009): SFE from C. verbenacea
v Increased yield from 5.0% to 7.7% by using 5% of EtOH
è 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 methods
Ø 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 methods
v 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 production
v Cabernet Sauvignon, Merlot and
Shiraz

è Compounds remain in pomace
v Essential oil and pigments
v Resveratrol, linoleic acid

è Extract quality:
v Composition Grape pomace
v Fruit origin, harvest,
v Wine process, Campos, L.M.A.S.; Leimann, F.V., Curi, R.P.;
v Extraction/solvent method. Ferreira, S.R.S. Bioresource Technology,
99(17), 2008: 8413-8420.


GC-MS: grape pomace

No Components
For cosmetic products,
1 Phenyl ethyl alcohol synthesis vit. E
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 GC-MS from grape pomace extract obtained by SFE
with pure CO2 at 150 bar, 40ºC and 3.33gCO2/min

HPLC: grape pomace
Phenolic compounds
1 Epicatechin Reduces the risk of
2 Galic acid cardiovascular disease
3 Tannic acid (Schroeter et al., 2006)
4 p-OH-benzoic acid
5 Vanillic acid
6 Caffeic acid

Antioxidant and
antimicrobial activity Salicylic acid isomer
(Rosso, 2005) Antimicrobial activity
(Naz et al., 2006)

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

HPLC: Merlot, Syrah, C. sauvignon
Extraction Grape Process p-OH- Protocatecuic
Epicatechin Gallic acid Tannic acid Vanillic acid
method varietal conditions benzoic acid acid

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
Merlot 50°C
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
150bar ND 121.6 ± 0.7 ND ND 33.7 ±0.1 ND
SFE 200bar ND 44 ± 1 ND 4.6 ± 0.1 10.6 ± 0.1 ND
Merlot 60°C
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.
60°C 250bar 87.99 ± 0.04 18.7 ± 0.1 ND 207.5 ± 0.3 58 ± 1 ND
sauvignon
SFE CO2 + 12.5% 119 ± 1 123.09 ± 0.09 ND 500.5 ± 0.5 3.85 ± 0.07 ND
EtOH Merlot 15.0% ND ND ND ND ND 537 ± 7
250bar/60°C 17.5% 200.8 ± 0.3 162.4 ± 0.4 26.3 ± 0.2 65.9 ± 0.3 15.21 ± 0.06 ND
Syrah 5.3 ± 0.1 10.6± 0.3 20.3 ± 0.2 22.8 ± 0.5 ND ND
SOX
Merlot ND ND ND ND ND 9864 ± 4
EtOH
Syrah - - - - - -
UME
Merlot 121.9 ± 0.6 51.0 ± 0.5 62.1 ± 0.1 505.73± 0.06 ND ND

Antimicrobial compounds

HPLC: coffee husk and coffee spent
Identificação
de Compostos Condition/ Clorogenic
Fenólicos Extraction Galic Ac. Tanic Ac. Cafeic Ac.
Solvent Ac.
Spent EtOH - 0.7 - -
US Spent EtAc 14.3 - 0.3 -
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 -
SFE Husk 200 bar/40°C 0.9 - 174.5 -
Husk 300 bar/60°C - - 942.8 -
SFE + Spent 100 bar/60°C/15% - - 19.6 -
EtOH Husk 200 bar/50°C/8% - - 232.3 -
Clorogenic ac.: potent antioxidant, hepatoprotection,
hipoglicemiant and antiviral (DUARTE et al., 2010).
Andrade et al. TALANTA. IN PRESS

HPLC: coffee husk and coffee spent

Teobromine Caffeine
Extraction Condition/Solvent
(µg/mgextract) (µg/mgextra
Hx - 0.734
Identificação de Spent DCM - 38.2
Metilxantinas EtOH - 25.7
US
Hx - 5.54
Husk
DCM 0.66 139.2
EtOH - 71.1
Hx - 3.27
Spent DCM - 25.9
EtOH - 11.8
Soxhlet
Hx - 2.1
Husk DCM 0.745 189.9
EtOH - 129.6
200 bar/60°C - 27.2
Spent
300 bar/60°C - 41.3
SFE
200 bar/40°C - 185.7
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

Andrade et al. TALANTA. IN PRESS

GC-MS: marigold oil
SFE: 200 bar/20oC
Peak Component MOL % Peak
200 bar/20oC (g/gmol) 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
L. Danielski, et al. Chem. Eng. 17 Octacosane 394 9.952
Proc., 46 (2). 2007: 99-106. 19 Eicosane-7-hexyl 366 0.449
20 Eicosane-9-octyl 394 0.612

Phytosterol (against breast 21 Canescegenine 420 0.792

cancer, reduces cholesterol) 22 Cholest-4-en-3-one-
14-methyl
398 0.831
Absent in other extracts 23 Taraxasterol 426 1.186
24 1-octadecanol 270 0.149
SRSFerreira - Chapecó, November, 2011 25 1,16-hexadecanediol 258
41
0.257

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


GC-MS: horsetail
No00 Compound Mol

1 Diphenyl carbonate 214

2 Dodecanoic acid 212

3 3-noninoic methyl ester 168 Phyto-sterol: absent
in other extracts
4 3,6-dimetil decane 170

5 Heneicosane 296 0.6
313K/12MPa
6 26-Hydroxicholesterol 402 0.5 313K/20MPa
0.4
303K/12MPa
7 Ergosta-4,7,22-trien-3-one 394

Molar fraction
303K/20MPa
276 0.3
8 8,12-Dimethyl-4Z,8E,12E-
octadecatriene 0.2

9 Methenolone 302 0.1

10 2,6,10,14-hexadecatet.-1- 332 0
Compound 1 Compound 3 Compound 6 Compound 8
ol,3,7,11,15-tetrametilacetato
Horsetail components
11 Z-13-Octadecenal 266
Michielin, et al. Composition Profile Of Horsetail
12 Heneicosane-11-decil 436 Oleoresin: Comparing SFE And Organic Solvent
13 Eicosane-10-heptyl-10-octil 492 Extraction. J. Supercritical Fluids. 2005. V. 33: 131.

14 17,21-dimetil-heptatriacontano 549


GC-MS: Mentha spicata L

èGC-MS: menthe
v Carvone: antifungal and antimicrobial
v Pulegone: muscle reliever and for indigestion
v Phytol: diterpene alcohol used for vitamin E
synthesis and regulates metabolic process.

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


2. Antioxidant activity (AA)

è Ability to avoid or reduce oxidative rancidity (food deterioration)

è Synthetics (carcinogenic effect):
v BHA, BHT, TBHQ (phenols)

è Naturals (non toxic):
v From food products and plant material
v Tocopherols, ascorbic acid, phenolic compounds

è Several 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 methods
v Free radical scavenging methods
v 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 extracts
v For poly-phenols and mono-phenols


3. Antimicrobial activity

èAntimicrobial substances:
v Prevent or inhibit microorganisms growth

èResistant bacteria:
v Challenge to infection treatments
v Patient sensibility to traditional antimicrobials
v New substances are necessary

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


Antimicrobial activity

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

è Minimum inhibition concentration (MIC)
v Positive result (Duarte et al., 2007):
Ø < 500 mg/mL: strong inhibitors
Ø 600 – 1500 mg/mL: moderate inhibitor.


Yield and DPPH: grape pomace

10 25
Yield AA %
8 20
Yield [%]

AA [%]
6 15

4 10

2 5

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

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)


Grape Pomace – ADM results
ADM (mm)

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 MIC (µg/mL)
Grape Process conditions
Method S. aureus B. cereus E. coli P. aeruginosa C. albicans

150 bar 750 ± 250 2000 >2000 >2000 -

200 bar 1500 ± 500 1000 >2000 >2000 500
Merlot 50 °C
250 bar 2000 1500 ± 500 >2000 >2000 -

300 bar 625 ± 375 1000 1000 1000 -

ESC CO2 150 bar 1000 1000 2000 2000 >2000

200 bar 1000 2000 2000 2000 -
Merlot 60 °C
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 - - -

SRSFerreira - Chapecó, November, 2011

AA% (DPPH): menthe
High AA for SFE
with co-solvent

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


Shiitake (Lentinula edodes)

è 2° most consumed mushroom:

è Mushrooms: nutraceutic food
v Source of active compounds
v Cancer protection: Lentinan
v Anti-cholesterolemic: Eritadenina
v Antioxidant activity
v Antimicrobial activity

è Shiitake extract:
v 60 pills 500 mg = US$ 9.95
(http://www.vitacost.com)


AA%: Shiitake

IC50 (DPPH): Inhibition concentration SFE: AA%
ETA (equivalent of tanic acid): Total Phenolic Content similar to DCM
200 2.5
IC50
ETA

ETA (g/100 g extract)
160 2

120 1.5
IC50

80 1

40 0.5

0 0
Cose:DCM Cose:EtAc SF:EtOH 5% SF:EtOH SF:EtOH
10% 15%
Shiitake extract

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


AM: Shiitake
ADM (mm)
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
MIC (Positive result):
< 0.5 mg/mL: strong inhibitors
40/15 0 I.H. 14 0 12

40/20 0 I.H. N.T. N.T. N.T. MIC (mg/mL)
40/30 I.H. 0 19 12 0 Microrganismos
SFE (°C/MPa)
M. luteus B. cereus C. albicans
50/15 I.H. I.H. 16 14 0
30/15 N.T. N.T. 2.0
50/20 0 I.H. 12 10 0
30/20 0.5 N.T. N.T.
40/20 + EtAc 15% N.T. N.T. 0 12 0
30/30 1.0 0.25 N.T.
Low pressure extracts: 40/15 1.0 N.T. 2.0
no antimicrobial activity 40/30 1.0 0.5 N.T.
50/15 0.5 0.5 N.T.
Kitzberger et al., Journal of Food Engineering, 50/20 1.0 N.T. N.T.
80(2): 631-638. 2007.
40/20+EtAc15% >2.0 0.5 N.T.

%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).

http://www.natucentro.com.br/
è Component: artepilin C


AM: Propolis
method E. coli S. aureus B. cereus SFE (P/T) E. coli S. aureus B. cereus
100/30 13 19 16
Sox-EtOH 0 16 13 ADM (mm) 100/40 14 15 15
100/50 12 12 14
Sox-EtAc 0 14 13 150/30 0 15 16
Sox-CHCl3 0 14 12 150/40 13 17 20
Sox-Hex 0 18 14 150/50 0 21 20
200/30 0 16 21
Sox-H2O 0 14 10 200/40 0 15 15
COSE-EtOH 0 18 13 200/50 0 18 17
COSE-EtH2O 70% 0 14 14 250/40 0 16 18
COSE-EtH2O 50% 0 18 19
S. B.
Method E. coli aureus cereus
Sox-EtOH
Sox-Hx
NT
NT
0.2500
0.0625
0.5000
0.5000
MIC (mg/mL)
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 SFE: excellent method for
SFE200/30 NT 0.0625 1.0000 antimicrobial extracts
SFE250/40 NT 0.0625 0.5000
SFE150/40/5% NT 0.0625 0.5000


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

1200
C. verbenacea:
DPPH (EC50 g/mL)

800
Extracts with
excellent AA

400

0

Ac
H

t

x
Ac

CM

H

SE M
x

er

er
H

in

in
Ke

H
H
O

O

O

C

ut
at

et
at
Et
Et

SE
Et

D

Et
s

D
Et
x

rc
R
w

w
So

So

SE
x

x

%

SE

ue
x

O
x
So

%
So
So

So

50

C
O
25

Q
O

O
C

C

C
Extracts

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

TPC & ABTS: C. verbenacea
SFE TPC ABTS Sox and COSE:
P/T (bar/°C) mgEAG /g extract % inhibition Better for AA
100/30 47±1 30.4±0.2
100/40 63±2 19.1±0.2 Extract TPC ABTS
mgEAG/gextract % inhibition
100/50 46±2 21±1
Sox EtAc 72±2 39.3±0.1
200/30 105±3 24±1
Sox EtOH 97±3 46.7±0.4
200/40 112±3 33±3
Sox Hx 42±2 27.7±0.1
200/50 67±1 31±1
Sox DCM 82±3 32.9±0.3
300/30 61±1 29±1
Sox water 111±3 77±3
300/40 63±3 27.5±0.7
Sox ketone 82±2 42.1±0.5
300/50 71±1 29.4±0.3
Sox 50%EtOH 102±9 43.2±0.9
CO2 +2%EtAc 73±3 35±2
Sox 25%EtOH 187.3±0.9 81±2
CO2 +5%EtAC 76±7 43±1
COSE EtAc 358±7 81.5±0.6
CO2 +8%EtAc 85.7±0.3 39±1
COSE Hx 63±4 27±0.5
CO2 +2%EtOH 84±3 32±1
COSE DCM 114±7 47.1±0.3
CO2 +5%EtOH 64.5±0.3 30±2
COSE water 84±2 77.1±0.3
CO2 +8%EtOH 77.8±0.8 46±3

AM: C. verbenacea

Extract S. aureus B. cereus E. coli P. aeruginosa
SFE100bar/30 C 09 11 00 12
SFE100bar/50 C 15 15 00 11
SFE200bar/30 C 15 13 00 00
SFE200bar/40 C 31 12 00 00 ADM (mm)
SFE300bar/30 C 16 13 00 11
SFE300bar/40 C 15 13 00 10
CO2+ 5% EtAc 17 14 00 10
CO2+ 5% EtOH 15 12 00 09 All extracts present
COSE EtAc 19 16 00 11 AM against Gram +
COSE Water 20 13 00 00 bacteria
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 Pseudomona
aeruginosa
SFE 100/50 °C 1.000 0.125 NT >4.000
MIC (mg/mL)
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
SFE: excellent
CO2 + 5% EtOH 0.250 0.03125 NT 1.000
method for highly
Mac EtAc 0.250 0.0468 NT 1.500
effective AM
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 (Positive result): < 0.5 mg/mL: strong inhibitors

Economic issues

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

è Small 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

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


Conclusion

èExtraction method and process conditions affect:
v Extraction yield
v Chemical profile
v Biological activity

èSFE: relevant technology for biological active extracts

v Screening
v Process optimization
v Product standardize
v Costs Good idea!

Federal University of Santa Catarina

Chemical and Food
Engineering Department

EQA/CTC – UFSC, P.O. Box 476
Florianópolis, SC – BRAZIL
ZIP Code 88040-900
Phone: + 55 48 3721.9448
FAX: + 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.


Acknowledgements

Thank you!


Supercritical Fluid Extraction of Bioactive Compounds from Natural Products

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (20)

Similar to Supercritical Fluid Extraction of Bioactive Compounds from Natural Products

Similar to Supercritical Fluid Extraction of Bioactive Compounds from Natural Products (20)

More from senaimais

More from senaimais (20)

Recently uploaded

Recently uploaded (20)

Supercritical Fluid Extraction of Bioactive Compounds from Natural Products