Supercritical Fluid Extraction of Bioactive Compounds from Natural Products

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Prof. Sandra R.S. Ferreira
LATESC/EQA - CTC – UFSC
Chapecó – November, 2011

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Supercritical Fluid Extraction of Bioactive Compounds from Natural Products

  1. 1. 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
  2. 2. Summary èNatural products èSFE vs low pressure èBiological activities v Extraction yield v Solute composition èExtraction methods v Antioxidant activity èSFE v Antimicrobial activitySRSFerreira - Chapecó, November, 2011 2
  3. 3. Natural Productsè Naturally occurring compounds or group of substancesèSources: v Plants, fruits, animals, microorganisms, fungus, etc. v Biological activitiesèRelated areas: v Medicine, food, flavoring and nutritional supplementèDrugs 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 syntheticSRSFerreira - Chapecó, November, 2011 3
  4. 4. 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 SRSFerreira - Chapecó, November, 2011 4
  5. 5. Biological activityè 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).SRSFerreira - Chapecó, November, 2011 5
  6. 6. Few sources: 2008 Property - action Raw material (source) – substance ReferenceAnti-HIV Hypericum H. chinese L. salicifolium, biyouyanagin A Nicolaou, et al., 2008.Antimalarial/AM Manzamines: class of marine natural products Shilabin, et al., 2008Melanoma 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.SRSFerreira - Chapecó, November, 2011 6
  7. 7. Few sources: 2011 Property - Raw material (source) – substance Reference actionAntitumor glycans from green tea Fan et al., 2011.Antitumor polysaccharides extracted from Zhao et al., 2011. Asparagus officinaliAntimicrobial Eucalyptus globulus oil Tyagi & Malik, 2011Antimicrobial Mentha piperita oil Tyagi & Malik, 2011Antioxidant Food-derived peptidic antioxidants Samaranayaka et al, 2011Antioxidant Marine Food Ngo et al., 2011SRSFerreira - Chapecó, November, 2011 7
  8. 8. Bioactive extractsèNatural 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 agentsèAmazon: huge natural reservoir v 55,000 vegetable species cataloged from a total of 350,000 to 550,000SRSFerreira - Chapecó, November, 2011 8
  9. 9. Important groupsè Alkaloids: high biological activity v Anti-tumor, anti-spasmodic.è Flavonoids: Present in flowers and fruits v Blood vessels protectors, anti-inflammatory. Alkaloid: caffeineè Essential oils: Aromatic compounds v Anti-septic and stimulant.è Tannins: Phenolic compounds v Ad stringent, bactericide and cicatrisation. Flavonoid SRSFerreira - Chapecó, November, 2011 9
  10. 10. 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) 253–261SRSFerreira - Chapecó, November, 2011 10
  11. 11. Extraction methodsèHydrodistillationèCold pressed extractionèSteam distillationèSolvent extraction v Soxhlet v Maceration/fractionation v Percolation Accelerated solvent extraction Microwave assisted extraction Ultrasound assisted extraction Supercritical Fluid ExtractionSRSFerreira - Chapecó, November, 2011 11
  12. 12. Hydrodistillationè Clevenger type apparatusè Solvent: waterè Raw material: v Soaked into waterè Essential oilsè High temperature http://pagesperso-orange.fr/guy.chaumetonSRSFerreira - Chapecó, November, 2011 12
  13. 13. Cold pressed extractionè Used for vegetable and essential oils from natural productsè Screw pressed extractionè Low heat technique http://www.abchansenafrica.co.zaSRSFerreira - Chapecó, November, 2011 13
  14. 14. 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 pipeSRSFerreira - Chapecó, November, 2011 14
  15. 15. Solvent extractionè Raw material and solvent: dissolve soluteè Solvent selection: v Hydrocarbons, alcohols, ketone, acetic acid v Polarity, solubility, solute interactionsè Solute/solvent separation http://www.armfield.co.uk/ SRSFerreira - Chapecó, November, 2011 15
  16. 16. Maceration and soxhletè Raw matter soaked in solvent, è Reflux heated and strained è High temperatureè Variables: time and temperature è Time consuming è Solvent consuming http://www.albrigi.co.uk http://www.rsc.org/chemistryworldSRSFerreira - Chapecó, November, 2011 16
  17. 17. Conventional extractionsèProblems: vHigh temperatures vSolvent contamination: reduces product quality vInflammability or explosion risks vTime, solvent and energy consuming vComposition varies with solvent and extraction techniqueSRSFerreira - Chapecó, November, 2011 17
  18. 18. New extraction methodsèASE: accelerated solvent extraction (Jaques et al., 2008) v Pressurized extraction: high pressure (3000 psi) and temperature v Good performance: break of solute–matrix interactionsèMAE: 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.SRSFerreira - Chapecó, November, 2011 18
  19. 19. SFE: alternativeèClassical methods: v Limitations: time and solvent consuming and energy costsèSFE: v Product quality: Ø Solvent free Ø Thermal degradation free v Energy saving: Ø Extraction + separation http://www.nature.com SRSFerreira - Chapecó, November, 2011 19
  20. 20. www.chem.leeds.ac.ukSRSFerreira - Chapecó, November, 2011 20
  21. 21. 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 ratesSRSFerreira - Chapecó, November, 2011 21
  22. 22. Basic components SOLVENT PUMP EXTRACTOR CO2: most used solvent (304.2K and 72.3bar) SEPARATOR SOLUTESRSFerreira - Chapecó, November, 2011 22
  23. 23. SFE: aspectsè Solvent power control (T/P)è Fractionationè Selectivityè Extraction and separation: v One stepSRSFerreira - Chapecó, November, 2011 23
  24. 24. SFE: applicationsè Alternative process for high aggregated value productsè 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…SRSFerreira - Chapecó, November, 2011 24
  25. 25. SFE: industrial units http://www.vtt.fi/pro www.separex.frSRSFerreira - Chapecó, November, 2011 25
  26. 26. 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)SRSFerreira - Chapecó, November, 2011 26
  27. 27. LATESC: raw materialsè Marigold (Calendula officinalis) è Apple pomaceè Horsetail (Equisetum giganteum) è Propolisè Rice bran oil (Oryza sativa L.) è Peach almondè Avenca-da-praia (Polygala cyparisias) è Shrimp shellsè Menthe (Mentha spicata) è Banana peelè Rosemary (Rosmarinus officinalis L.) è Orange pomaceè Shiitake (Lentinula edodes) è Eucalypt leavesè Grape pomace (Vitis vinífera) è Plumb v Shiraz, Merlot, Cabernet sauvignon è Fishery by-productsè Erva baleeira (Cordia verbenacea) è Pecan nutsè Spent Coffee ground and coffee husk è Shrimp residueSRSFerreira - Chapecó, November, 2011 27
  28. 28. Extraction resultsèProcess aspects: v Extraction yield: cross-over influence v Use of co-solvents in SFE: increase yieldèProduct quality: v Extract composition: chromatographic methods v Biological activities: antioxidant and antimicrobial activitiesSRSFerreira - Chapecó, November, 2011 28
  29. 29. 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 SRSFerreira - Chapecó, November, 2011 29
  30. 30. 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 ± 0.3SRSFerreira - Chapecó, November, 2011 30
  31. 31. SFE: co-solventsèCO2: 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 selectivitySRSFerreira - Chapecó, November, 2011 31
  32. 32. SFE: ModifierèImprove the SFE performance: yield and selectivityèSelection: Ability to dissolve compounds of interest;èCommon examples: v EtOH, EtAc, DCM, BtOHLATESC 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.SRSFerreira - Chapecó, November, 2011 32
  33. 33. Extract qualityè Product quality as important as process yieldè Results (LATESC): v Different raw materials v Different extraction methods Ø SFE (CO2 and CO2 + CS) and classical extraction methodsè Quality evaluation: 1. Chemical composition profile 2. Antioxidant activity 3. Antimicrobial activitySRSFerreira - Chapecó, November, 2011 33
  34. 34. 1. CompositionèChromatographic methods v Gas Chromatography: mostly for non-polar substances Ø adequate for CO2 extracts v HPLC: detect substances with higher polarityèMass spectrometry: v Components identification: Ø Standard Reference Data Series of the National Institute of Standard and Technology.èComposition: v Retention time and standard curvesSRSFerreira - Chapecó, November, 2011 34
  35. 35. Grape pomaceè Miolo winery Ltda. (Vintage 2003) v Residue from wine production v Cabernet Sauvignon, Merlot and Shirazè Compounds remain in pomace v Essential oil and pigments v Resveratrol, linoleic acidè 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.SRSFerreira - Chapecó, November, 2011 35
  36. 36. GC-MS: grape pomaceNo 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/minSRSFerreira - Chapecó, November, 2011 36
  37. 37. 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)SRSFerreira - Chapecó, November, 2011 37
  38. 38. HPLC: Merlot, Syrah, C. sauvignonExtraction 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 ± 7250bar/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 SRSFerreira - Chapecó, November, 2011 38
  39. 39. HPLC: coffee husk and coffee spent Identificaçãode 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 PRESSSRSFerreira - Chapecó, November, 2011 39
  40. 40. 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 PRESSSRSFerreira - Chapecó, November, 2011 40
  41. 41. 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 0.149SRSFerreira - Chapecó, November, 2011 25 1,16-hexadecanediol 258 41 0.257
  42. 42. 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-MSSRSFerreira - Chapecó, November, 2011 42
  43. 43. GC-MS: horsetailNo00 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 549SRSFerreira - Chapecó, November, 2011 43
  44. 44. 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 andBioproc. Tech. .IN PRESS.SRSFerreira - Chapecó, November, 2011 44
  45. 45. 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 compoundsè 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.SRSFerreira - Chapecó, November, 2011 45
  46. 46. Antioxidant methodsè Methods for AA%: Espectrophotometric and fluorescence methods v Free radical scavenging methods v Redox potential of antioxidantsè DPPH: (MENSOR et al. 2001) v Radical (2,2-difenil-1-picrilhidrazil): electron capture from antioxidantè 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 oxidationè 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-phenolsSRSFerreira - Chapecó, November, 2011 46
  47. 47. 3. Antimicrobial activityèAntimicrobial substances: v Prevent or inhibit microorganisms growthèResistant bacteria: v Challenge to infection treatments v Patient sensibility to traditional antimicrobials v New substances are necessaryèAntimicrobial compounds from plant material: v Not well exploited v Studies are very incipient.SRSFerreira - Chapecó, November, 2011 47
  48. 48. Antimicrobial activityè 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, : fungusè Minimum inhibition concentration (MIC) v Positive result (Duarte et al., 2007): Ø < 500 mg/mL: strong inhibitors Ø 600 – 1500 mg/mL: moderate inhibitor.SRSFerreira - Chapecó, November, 2011 48
  49. 49. 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 concentrationSRSFerreira - Chapecó, November, 2011 49
  50. 50. Grape Pomace: antimicrobial activity ADM (mm)SRSFerreira - Chapecó, November, 2011 50
  51. 51. 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)SRSFerreira - Chapecó, November, 2011 51
  52. 52. Antimicrobial activity: MICExtraction 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
  53. 53. 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 20.3SRSFerreira - Chapecó, November, 2011 53
  54. 54. 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 activityè Shiitake extract: v 60 pills 500 mg = US$ 9.95 (http://www.vitacost.com)SRSFerreira - Chapecó, November, 2011 54
  55. 55. 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.SRSFerreira - Chapecó, November, 2011 55
  56. 56. 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. SRSFerreira - Chapecó, November, 2011 56
  57. 57. %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.SRSFerreira - Chapecó, November, 2011 57
  58. 58. 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 CSRSFerreira - Chapecó, November, 2011 58
  59. 59. AM: Propolismethod E. coli S. aureus B. cereus SFE (P/T) E. coli S. aureus B. cereus 100/30 13 19 16Sox-EtOH 0 16 13 ADM (mm) 100/40 14 15 15 100/50 12 12 14Sox-EtAc 0 14 13 150/30 0 15 16Sox-CHCl3 0 14 12 150/40 13 17 20Sox-Hex 0 18 14 150/50 0 21 20 200/30 0 16 21Sox-H2O 0 14 10 200/40 0 15 15COSE-EtOH 0 18 13 200/50 0 18 17COSE-EtH2O 70% 0 14 14 250/40 0 16 18COSE-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 0.5000SRSFerreira - Chapecó, November, 2011 59
  60. 60. Cordia verbenaceaè 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 treatmentsè Essential oils (aroma): v a-humulene, b-caryophylene, pineneè Flavonoids: v quercetine, artemetin www.jardimdeflores.com.brSRSFerreira - Chapecó, November, 2011 60
  61. 61. 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/mLSRSFerreira - Chapecó, November, 2011 61
  62. 62. 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±0.9CO2 +2%EtAc 73±3 35±2 Sox 25%EtOH 187.3±0.9 81±2CO2 +5%EtAC 76±7 43±1 COSE EtAc 358±7 81.5±0.6CO2 +8%EtAc 85.7±0.3 39±1 COSE Hx 63±4 27±0.5CO2 +2%EtOH 84±3 32±1 COSE DCM 114±7 47.1±0.3CO2 +5%EtOH 64.5±0.3 30±2 COSE water 84±2 77.1±0.3CO2 +8%EtOH 77.8±0.8 46±3 SRSFerreira - Chapecó, November, 2011 62
  63. 63. AM: C. verbenaceaExtract 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 00 ADM (mm)SFE300bar/30 C 16 13 00 11SFE300bar/40 C 15 13 00 10CO2+ 5% EtAc 17 14 00 10CO2+ 5% EtOH 15 12 00 09 All extracts presentCOSE EtAc 19 16 00 11 AM against Gram +COSE Water 20 13 00 00 bacteriaSox 25% EtOH 25 15 16 21Sox 50% EtOH 18 11 00 00Sox EtOH 16 13 00 00Sox Water 20 10 12 16Sox EtAc 21 16 12 00SRSFerreira - Chapecó, November, 2011 63
  64. 64. AM: C. verbenacea Extract S. aureus B. cereus E. coli Pseudomona aeruginosaSFE 100/50 °C 1.000 0.125 NT >4.000 MIC (mg/mL)SFE 200/40 °C 0.500 0.0468 NT NTSFE 300/30 °C 0.375 < 0.0078 NT >4.000CO2 + 5% EtAc 0.250 0.0156 NT >4.000 SFE: excellentCO2 + 5% EtOH 0.250 0.03125 NT 1.000 method for highlyMac EtAc 0.250 0.0468 NT 1.500 effective AMSox 25% EtOH 2.000 1.000 2.000 1.000Sox Water 2.000 2.000 2.000 1.000Sox Hx NT NT 2.000 >4.000Sox EtAc 0.500 0.0468 2.000 NT MIC (Positive result): < 0.5 mg/mL: strong inhibitorsSRSFerreira - Chapecó, November, 2011 64
  65. 65. Economic issuesè Last 15 years (natural products): v More than 100 plants (pilot and industrial scales) in operationè Small to medium units: v 100 to 1000 ton/year at 3 to 4 EUR/kg productè Industrial units: v 10.000 ton/year at 0.5 EUR/kg productè Natural products (3% extract) v Cost: 100 to150 EUR/kg product v Continuous process: cost reduction in 5 timesSRSFerreira - Chapecó, November, 2011 65
  66. 66. ConclusionèExtraction method and process conditions affect: v Extraction yield v Chemical profile v Biological activityèSFE: relevant technology for biological active extracts v Screening v Process optimization v Product standardize v Costs Good idea!SRSFerreira - Chapecó, November, 2011 66
  67. 67. 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.brSRSFerreira - Chapecó, November, 2011 67
  68. 68. Referencesè 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) 253–261è 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. SRSFerreira - Chapecó, November, 2011 68
  69. 69. Acknowledgements Thank you!SRSFerreira - Chapecó, November, 2011 69

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