Separation and characterization of extracted bioactive ... · extracted bioactive phytochemicals =...
Transcript of Separation and characterization of extracted bioactive ... · extracted bioactive phytochemicals =...
University of Coimbra
Universidade de Coimbra
Separation and characterization of
extracted bioactive phytochemicals =
Production of phytochemicals through
extraction with supercritical fluids
Mara Elga Medeiros Braga
GSP - Green and Sustainable Processes Lab
CIEPQPF, Chemical Engineering Department,
University of Coimbra, Coimbra, Portugal
University of Coimbra
Universidade de Coimbra
Madonna of the Pomegranate by Sandro Botticelli
Galleria degli Uffizi in Florence
Use of natural products !!!
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The World Health Organization has found that 80% of the population in some Asian
and African countries depend on traditional medicine for their most basic health care
needs. In the developed world, the WHO has found that between 70-80% of the
population in these countries have used such traditional medicinal practices in some
form. Of these, herbal treatments have proven the most sought after, and thus, the
most profitable: in Western Europe, revenues total around $5 billion; in Brazil, $160
million; and in China, $14 billion.
WHO and its member states have identified the following
problems in the current culture of traditional medicine:
• international diversity;
• national policy and regulation;
• safety, effectiveness and quality;
• knowledge and sustainability;
• patient safety and use.
WHO has compiled three volumes (to date) which establish a
list of the most widely-used plants, their proper preparations
and uses, and adverse effects/precautions to be taken.
Among these 90 plants, the WHO has counted 119 pharmaceutical products, 74% of which are used (in
modern medicine) in ways that are linked to their original uses in indigenous cultures.
The trend today, however, is for medicinal plants to be used not in whole form, but in terms of their
individual components. With over 750,000 known plants in the world, the relative number of medicinal
herbs that have been scientifically studied is very small. And until the value of many medicinal plants
has been confirmed in major industrialized nations, traditional herbal medicine will not be deemed as
important as chemically manufactured drugs.
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Desenvolvimento de um novo fármaco à base de produto natural
FDA (2003) :
21 novos fármacos foram colocados no mercado no
período de 1981 a 2002.
De 877 novos fármacos desenvolvidos:
• 6% eram compostos por produtos naturais,
• 27% eram derivados de produtos naturais e
• 16% eram medicamentos sintéticos desenvolvidos a
partir de um modelo de um produto natural (Newman
et al, 2003).
Custos de desenvolvimento de um novo fármaco:
• 800 a 1000 milhões de Euros
Um novo fármaco:
• são testados/estudados de 10.000 a 100.000
compostos (Verpoorte et al, 2006)
Média:
15 anos
M.E.M. Braga, Produção de fitoquímicos bioactivos com fluidos supercríticos e suas aplicações, in Biomateriales
aplicados al diseño de sistemas terapéuticos avanzados. Coimbra University Press, in press., 2013
University of Coimbra
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University of Coimbra
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Nutraceutical Drug Food
Nutraceutical occupies position between food and drug
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Extraction methodology Having established the products to be separated and
the purity and recovery desired for each, the next
essential step is to determine which separation
methods are capable of accomplishing the separation.
In order for two components to be separable, there
must be some difference in properties between them.
The objective of design of the separation process is to
exploit the property differences in the most
economical manner to accomplish the separation.
Functional groups of natural compounds
Functional groups Properties
R C
OH
O
RR
C
O
Hydroxyl Carboxilic acid Ketones
Increase the water solubility
CH3 CH2 CH2 CH3
CH3 C CH3
CH3
Cl
Hydrocarbons Halogens
Reduce the water solubility
R C
OH
O
R C
NH2
O
Hydroxyl Carboxilic acid Amino
Non-volatile
CH2 CH2 O, N, S and P
Aromatic ring Double bond Heteroatoms
Capable of hydrogen bonding and polar
interactions
R OH
R OH
The following properties are used as bases for separation
processes:
Equilibrium properties, Vapor pressure,
Solubility, Distribution between immiscible liquid phases,
Melting point, Chemical reaction equilibrium,
Electric charge (isoelectric point), Surface sorption,
Rate properties, Diffusivity, Ionic mobility,
Molecular size, Molecular shape
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CO2
Tc = 31.1 ºC and Pc = 7.4MPa
S.M. Howdle, B. Wong, http://www.nottingham.ac.uk/~pczctg/Index.htm, (22/05/2003)
SCF’s: properties and applications
- What is a SCF?
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P
T
Solid
Liquid
Gas
Supercritical
Region
P
P
t
c
c
t
TT
A one-component fluid is called a SUPERCRITICAL FLUID
when its temperature and pressure exceeds its critical
temperature and pressure, respectively.
Supercritical Fluid based technologies can be advantageous
alternatives to traditional methods to extract natural compounds
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Comparison of some physical and transport properties of gases, liquids and SCF’s
Property Gas SCF Liquid
Density (kg/m3) 1.0 100-800 1000
Viscosity (cP) 0.01 0.05-0.10 0.5-1.0 Diffusivity (mm
2/s) 1.0-10.0 0.01-0.10 0.001
Intermediate properties
between liquid and vapor
Not intermediate properties
between liquid and vapor
Isobaric heat capacity
Isothermal compressibility
Thermal expansion coefficient
Thermal conductivity
Joule-Thompson coefficient
Density
Dynamic viscosity
Dielectric constant / ionic product
Self-Diffusion coefficient
Enthalpy
Entropy
Supercritical carbon dioxide – scCO2
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Supercritical Carbon Dioxide:
It’s non-toxic, non-flammable, non-expensive and abundant;
Has a low critical temperature (31.05 ºC) and doesn’t degrade thermal-labile
substances, like drugs and/or polymers;
Leaves no residues and dry solid products are easily obtained;
Therefore, they can be used to replace organic solvents and do not leave
any residues or produce dangerous effluents. Supercritical processes are
often referred as “Green” and “Environmental friendly” processes
Can be recovered and reused, not contributing to the greenhouse effect;
SCF’s may be an excellent alternative proposal to traditional solvent
extraction!
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Squalene and Tocopherols – Olive Oil
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Transport of substances in solids depends also on:
Initial distribution of extractable substances within the solid matrix:
Adsorbed on the outer surface;
Adsorbed on the surface of pores;
Uniformly distributed within the solid or plant cells.
Size distribution of particle bed;
Geometry of particle bed (which can vary during the extraction
process);
Bed stirring/fluidization;
Transport Mechanism in solids
Mint tea Cherry conserve L. Ruetsch et al. Lat. Am. Appl. Res. 33 (2003)
- Diffusion of SCF solvent into the
pores and adsorption of the SCF
solvent on the solid surface
- Transport of solute to the outer
layer and formation of a thin liquid
film around the solid particles
- Dissolution of solute in the SCF
solvent
- Convective transport of the solute
to the bulk of the fluid
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Process variables
Pressure
Temperature
Solvent composition
Solvent density – transport properties, viscosity,
Solvent selectivity - polarity and solubility
Solvent contact time - static or dynamic extraction
Flow rate
Solid/solvent ratio
Extraction time – to reach the diffusional extraction period
Fractionation (combination of variables):
Pressure (pressure-gradients)
Temperature (temperature-gradients)
Cosolvents (cosolvent-gradients)
To obtain a high performance extraction or a high yield of target compounds in a short
process time, it is necessary to choose a selective solvent with a high solubility of the
target compounds, and then the solvent properties is the main factor affecting the
extraction process. Other factors which influence mass transfer are:
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How to choose a raw material ?
High value compounds – “non-synthesizable”
Minimal concentrations – high biological activity
Solvent free extracts – pure fractions or extracts
Instable molecules (thermo and enzymatic degradations)
Medical/Pharmaceutical and cosmetic industries
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Costs
Rosa and Meireles, Rapid estimation of the manufacturing cost of extracts obtained by supercritical fluid extraction.
Journal of Food Engineering, 67, 235-240, 2005.
Ginger oil - gingerols and shogaols
Clove bud oil - eugenol
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Residues
Sambucus nigra - elderberry
Malus domestica - apple Prunus avium - cherry
Pinus pinaster –
maritime pine
Juglans regia - walnut
and new products
Caesalpinea spinosa – tara
Cynara candunculus -
cardo Spilantes oleraceae - jambu Caesalpinea ferrea - jucá
Punica granatum –
romã
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Results and Applications
Residues
New products Bioactive compounds
Dyes
Antioxidant
Anti-inflammatory, Analgesic
Bactericide, fungicide
Cytocidal/Cytostatic
Flavors and fragrances
• Phenolic: Proanthocyanidins, quinones,
anthocyanins
• Terpenes: Carotenoids
• Alkaloids
• Triglycerides
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Braga et al, Journal of Supercritical Fluid, 34, 149-156, 2005.
Lippia alba leaves
Solubility of compounds: carvone and limonene
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Braga et al, Journal of Supercritical Fluid, 34, 149-156, 2005.
Lippia alba leaves
Solubility of compounds: carvone and limonene
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Curcuma longa roots
Cosolvent
Braga et al. Journal of Agricultural and Food Chemistry. 51, 6604-6611, 2003.
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Braga, M. E. M.; Meireles, M. A. A. Journal of Food Process Engineering, 30, 501-521, 2007.
HB/DB rates
Fractionation and cosolvent
Static period
Curcuma longa roots
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Seabra et al. Fractioned High Pressure Extraction of
Anthocyanins from Elderberry (Sambucus nigra L.) Pomace.
Sambucus nigra pomace
Raw material
Extraction method: SFE, ESE and CSE
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University of Coimbra
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Caesalpinea spinosa – seed coats
Representation of the assayed solvent mixtures with
diverse CO2, EtOH and H2O molar fractions
Ternary solvent composition
Durling et al. Fluid Phase Equilibria 252 (2007) 103–113
Seabra et al, J. of Supercritical Fluids 64 (2012) 9– 18
yield phenols
Antioxidant
activity
Anti-inflammatory
activity
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Dias et al. Jambu (Spilanthes oleraceae) global extraction yields obtained from flowers, leaves and stems
when using different extraction methods and solvent mixtures. J.Supercritical Fluids, 61 (2012) 62– 70
Spilanthes oleraceae Solvent composition
Parts of plant
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Dias et al. Jambu (Spilanthes oleraceae) global extraction yields obtained from flowers, leaves and stems
when using different extraction methods and solvent mixtures. J.Supercritical Fluids, 61 (2012) 62– 70
Spilanthes oleraceae Solvent composition
Parts of plant
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Dias et al. Jambu (Spilanthes oleraceae) global extraction yields obtained from flowers, leaves and stems
when using different extraction methods and solvent mixtures. J.Supercritical Fluids, 61 (2012) 62– 70
Spilanthes oleraceae Solvent composition
Parts of plant
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Anti-inflammatory activities synergism among compounds
Dias et al. Jambu (Spilanthes oleraceae) global extraction yields obtained from flowers, leaves and stems
when using different extraction methods and solvent mixtures. J.Supercritical Fluids, 61 (2012) 62– 70
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Talansier et al. J. of Supercritical Fluids 47, 200–208, 2008.
Vetiveria zizanioides roots Pressure and temperature
Cosolvent
Kinetic study
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Braga et al. Journal of Supercritical Fluids, 47, 37-48, 2008.
Pinus Pinaster bark Fractionation and flow rate
Fig. 2. Pine bark FSFE kinetics results. Experiments at 40 ◦C and at ∼20MPa:
(□) 1st step CO2 extraction and (■) 2nd step CO2 + EtOH (10%) extraction.
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Braga et al. Journal of Supercritical Fluids, 47, 37-48, 2008.
Pinus Pinaster bark Fractionation and flow rate
solvent flow rate: low medium high
Seabra et al. Journal of Supercritical Fluids, 62 (2012) 135– 148.
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Serra et al. J. of Supercritical Fluids 54, 2010.
Prunus avium fruits
Flow rate and Fractionation
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Serra et al. J. of Supercritical Fluids 54, 2010.
Prunus avium fruits
Flow rate and Fractionation
Antiproliferative activity of cherry extracts in human colon cancer cells.
Category: Products’ development and innovation
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Impregnation of phytochemicals
into polymeric matrices for
biomedical applications
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Supercritical Fluid Impregnation
(SSI)
Bioactive Wound Dressing
Biopolymers
foams/films
Quercetin/Thymol
Biomolecules
“Tunable” loaded
materials
Quercetin
Thymol
Supercritical Fluid Impregnation
(SSI)
Bioactive Wound Dressing
Biopolymers
foams/films
Quercetin/Thymol
Biomolecules
“Tunable” loaded
materials
Supercritical Fluid Impregnation
(SSI)
Bioactive Wound Dressing
Biopolymers
foams/films
Quercetin/Thymol
Biomolecules
“Tunable” loaded
materials
Supercritical Fluid Impregnation
(SSI)
Bioactive Wound Dressing
Biopolymers
foams/films
Quercetin/Thymol
Biomolecules
“Tunable” loaded
materials
Supercritical Fluid Impregnation
(SSI)
Bioactive Wound Dressing
Biopolymers
foams/films
Quercetin/Thymol
Biomolecules
“Tunable” loaded
materials
Thymol
Quercetin
Dias et al, Development of Natural-based Wound Dressings Impregnated with Bioactive Compounds Using Supercritical Carbon Dioxide, International Journal Pharmaceutics, 408 (2011) 9-19.
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Non-loaded
dressings
Jucá - loaded
dressings
Bioactive Wound Dressing: comparison among commercial membranes and CBC
Hyalofill® Promogran® N-carboxybutylchitosan (CBC)
TNF-a
Dias, et al. Wound dressings loaded with an anti-inflammatory jucá (Libidibia ferrea) extract using supercritical carbon dioxide technology. The Journal of Supercritical Fluids, 74 (2013) 34– 45.
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Conclusions
SCF’s have real advantages and can be successfully used for
development of several industrial applications, namely of pharmaceutical
and food applications and when classical methods are no more
employable or have important disadvantages.
Different SCF technological processes can be used and the choice largely
depends on raw materials to be treated and on the final end-use required;
SCF’s may be an excellent alternative proposal to traditional solvent
extraction methods;
Most of SCF techniques (including SFE) are solvent-free and leave dry,
dispersed end-products;
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Most of SCF’s of interest have low critical temperatures (like CO2), thus
do not degrade thermo-labile and bio-labile substances;
Economic evaluation of SCF processes shows that, for high added-value
products like nutraceuticals and pharmaceuticals, they can be profitable,
in spite of their higher initial investments and higher operational costs;
However, they are clearly “environmentally” profitable: SC Processes are
often referred as “Green”, “Clean”, “Sustainable Technology” and
“Environmental Friendly” processes;
Other SCF processes can be combined: SSI, particle and emulsion
production, chemical modifications in SCF, scCO2 sterilization, protein
precipitation, fractionation and purification, SCF chromatography…
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Collaborations team:
Portugal :
Faculdade de Farmácia – Universidade de Coimbra
Instituto de Tecnologia Química e Biológica – ITQB, UNL
Escola Superior Agrária de Coimbra -ESAC
Cooperativa do Vale de Varosa
Curtumes J.B. Salgueiro
Brazil, Spain and Colombia:
Universidade de Sao Paulo - USP
Universidade Federal do Pará – UFPA
Universidade Federal da Bahia – UFBA
Embrapa Agroindústria Tropical
Universidad de Santiago de Compostela, Facultad de Farmacia
Universidad Nacional de Colombia, Departamento de Farmacia
Financial support: FCT-MCTES and CYTED