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



Madonna of the Pomegranate by Sandro Botticelli

Galleria degli Uffizi in Florence

Use of natural products !!!

http://en.wikipedia.org/wiki/Madonna_of_the_Pomegranate

http://en.wikipedia.org/wiki/Sandro_Botticelli



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.



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



Nutraceutical Drug Food

Nutraceutical occupies position between food and drug



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



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?



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



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



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!



Squalene and Tocopherols – Olive Oil



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



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:



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



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



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



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



Braga et al, Journal of Supercritical Fluid, 34, 149-156, 2005.

Lippia alba leaves

Solubility of compounds: carvone and limonene



Curcuma longa roots

Cosolvent

Braga et al. Journal of Agricultural and Food Chemistry. 51, 6604-6611, 2003.



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



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



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



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



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



Talansier et al. J. of Supercritical Fluids 47, 200–208, 2008.

Vetiveria zizanioides roots Pressure and temperature

Cosolvent

Kinetic study



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.



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.



Serra et al. J. of Supercritical Fluids 54, 2010.

Prunus avium fruits

Flow rate and Fractionation



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



Impregnation of phytochemicals

into polymeric matrices for

biomedical applications



Supercritical Fluid Impregnation

(SSI)

Bioactive Wound Dressing

Biopolymers

foams/films

Quercetin/Thymol

Biomolecules

“Tunable” loaded

materials

Quercetin

Thymol


(SSI)


Biopolymers

foams/films

Quercetin/Thymol

Biomolecules


materials


(SSI)


Biopolymers

foams/films

Quercetin/Thymol

Biomolecules


materials


(SSI)


Biopolymers

foams/films

Quercetin/Thymol

Biomolecules


materials


(SSI)


Biopolymers

foams/films

Quercetin/Thymol

Biomolecules


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.



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.



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;



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…



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

separation and characterization of extracted bioactive ... · extracted bioactive phytochemicals =...

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