Spice Antioxidants Extraction and Activity Assessment Methods
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Transcript of Spice Antioxidants Extraction and Activity Assessment Methods
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Presented By: Ramesh Khadka
M.S. Food Science 1st Year ,2011
ID: 5415001298
Advisor: Asst. Prof. Dr. Wannee Jirapakkul
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` Lipid oxidation
` Antioxidants and their classification
` Spice as a source of natural antioxidants
` Evaluation of antioxidant potential of spices
1. extraction of antioxidants from spices
2.Assessing antioxidant capacity.
`
Summary
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` Initiation: RH + initiator R .............(1)
ROOH + initiator ROO
` Propagation: R + O2 ROO ..................(2)
ROO + RH ROOH + R
` Termination: R + R R-R ...................(3)
ROO+ R ROOR
Initiator: heat, ionizing radiation or UV light(physical)
metal ions, free radicals and metalloproteins (chemical)
ROOH(hydro peroxide) : primary oxidation products
ROOR(hydrocarbons, aldehydes, alcohols and volatile ketones): secondaryoxidation products
(source: Brewer ,2011)3
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` Any substance that when present at low
concentrations compared with those of an oxidizable
substrate significantly delays or prevents oxidation of
that substrate. (Halliwell, 2002)
` Awide array of compounds
` Awide array of mechanism
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1.Primary antioxidants : type I or free radical scavengers(FRS)
HAT: hydrogen atom transferSET :singleelectron transfer to the free radicals.
e.g. phenolic compounds
2.Secondary antioxidants: type II or synergist
` Singlet oxygen quenchers: e.g. -carotene, lycopene
` Oxygen scavengers and reducing agents e.g. ascorbic acid, ascorbylpalmitate
` chelateprooxidant metals e.g. EDTA, citric acid
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` Natural
` Synthetic
Commonly used synthetic antioxidants
` Butylated hydroxyanisole (BHA): Removed from the GRAS
` Butylated hydroxy tolune (BHT)
` Tert-butyl hydooxyquinone(TBHQ): Not allowed in Japan,Canada and Europe
` Propyl Gallate (PG)
(source: Mohdaly et al.2010)
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` Often contain high concentrations of phenolic compounds
with strong H-donating capacity.
` Phenolic compounds (phenolics): a group of approximately
8000 naturally occurring compounds.
` The major antioxidative plant phenolics :4 general groups
1.phenolic acids (gallic, protochatechuic, caffeic and
rosmarinic acids)
2. phenolic diterpenes (carnosol and carnosic acid)
3. flavonoids (quercetin and catechin )and
4. volatile oils (eugenol, carvacrol, thymol, and menthol;
(source: Brewer ,2011)7
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Methods
1. Extraction with Fats and oils
2. Extraction with organic solvents
3. Extraction with supercritical fluid carbon dioxide
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Ground Spices (sage)Cottonseed oil
Mixing &
Heating
Centrifugation
Deodorization
Deodorized
Sage extract
15-20% spice in oil
At 120- 1250c for 2h Withcontinuous stirring
To separate theextract
At 175-185 0 C/30min
Extraction of antioxidants from spices
Extraction with Fats and oils
(source: modified from Pokorny and Korczak,2001)9
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` Hexane, acetone, ethyl acetate and methanol
` Mixtures of organic solvent with water
` solvents of intermediary polarity seemed to be preferable to
either non-polar or highly polar solvents.
` May be assisted with Novel extraction methods: ultrasound-
assistedextraction, microwave-assistedextraction ,
accelerated solvent extraction
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Variety ofzinger
Extractionsource
TP (mg gallic acid/g dry weight)
Methanol Acetone Chloroform
Halia Bentong Leaves 33.11.21b 30.10.22c 28.80.21c
Stems 7.80.89ijk 7.21.05jk 7.070.99k
Rhizomes 10.10.21efg 9.80.22efgh 9.20.66fghi
Halia Bara Leaves 39.061.62a 34.61.88b 33.61.99b
Stems 8.51.02hijk 8.060.92ijk 8.80.82ghij
Rhizomes 13.40.34d 11.10.87e 10.80.75ef
(source: adapted from Ghasemzadeh et al., 2011)
Table 1. phenolic contents in different parts of ginger (Z.officinale) varieties extracted by different solvents
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VarietyExtractionsource
Capabilities of scavenging DPPH free radicals
Inhibition (%)
Methanol Acetone Chloroform
Halia Bentong Leaves 51.120.36d 48.991.07e 47.120.97f
Stems 32.831.02g 30.762.39h 28.950.75i
Rhizomes 51.480.72d 49.220.46e 47.470.64f
Halia Bara Leaves 56.380.23b 54.160.91c 52.530.33d
Stems 31.330.55h 29.111.01i 27.460.62j
Rhizomes 58.210.39a 56.180.51b 54.361.12c
(source: adapted from Ghasemzadeh et al. 2011)
Table 2.Capabilities of scavenging DPPH free radicals from young ginger (Z.
officinale) parts extracted by different solvents.
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2.6 2.2 2.9 1.8
38.8
0
5
10
15
20
25
30
35
40
45
Hexane Benzene Chloroform Methanol Control
peroxidevalue( meq/Kg)of lard in 11days
Fig 1:Antioxidant activity of 0.02 % purified rosemary extracts extracted with
different solvents and applied in lard (aging at 60C; expressed as the peroxide
value [meq/kg])after 11 days( source: Modified from Pokorny and Korczak, 2001)
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` Amodern method
` high operation pressure,
which requires expensive
equipment
` limited use in the
preparation of
natural antioxidants
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` a wide array of assays fordetermination of antioxidant
capacity
` DIRECTMETHODS: assays that test the ability to inhibit
lipid oxidation under accelerated conditions. utilizes
various lipid model systems or lipid containing foods,
superior to the indirect but often time consuming
` INDIRECTMETHODS: assays that evaluate the ability of
antioxidants to scavenge some stable coloured syntheticfree-radicals/ reduce some coloured compounds. little in
common with highly reactive radical oxygen species,
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( Source: Schwarz et al., 2001)
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Real Models
Antioxidant addition
Accelerated tests(Oxidation)
Analysis
Oils, emulsions, lards, Meat
and other lipid containing
Foods
Spice extract or ground
Spice
At elevated Temperature
Assessing antioxidant activity
Direct method for evaluating antioxidant activity ofDirect method for evaluating antioxidant activity ofspicesspices
(source: modified from Pokorny and Korczak,2001)
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1 .peroxide value 2.conjugated dienes
3.anisidine value 4.TBA test.
(Primary oxidation products)
(Secondary oxidation products)
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SET-based assays : measure an antioxidants reducing capacity
` 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)assay/TEAC (Trolox EquivalentAntioxidant Capacity) assay,
`
2,2-d
iphenyl-1-picrylhy
drazyl (D
PPH) assay,
` ferric reducing antioxidant power(FRAP) assay,` Folin-Ciocaltau (FC) assay,
HAT-based assay: quantify hydrogen atom donating capacity.`
oxygen ra
dical absorbanc
ecapacity (OR
AC),
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oxidant (probe) + e(from antioxidant)
(specific colour)
reduced probe + oxidized antioxidant
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( Source: Schwarz et al., 2001)
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Assays oxidant (probe) reduced probe Absorbancemaxima
ABTS ABTS+
(green) ABTS
730 nm
DPPH DPPH ( Nitrogen
radical)dee
p purple
DPPHH 515nm
FRAP Fe3+-TPTZ *complex
yellow
Fe2+_TPTZ(intense
blue)
596nm(reduced
probe)
(FC)
assay
Folin-Ciocaltau reagent
(FCR) (MoVI )(yellow)
FCR (MoV) (blue) 725 nm(reduced
probe)
(*TPTZ = 2,4,6-tripyridyl-s-triazine)
Table: 4 Summary of SET basedAssays
( Source: Prior et al 2005; Huang et al. 2005;Ou et al., 2002)
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AAPH AAPH. +N22, 2-azobis (2-amidino-propane)
Dihydrochloride
AAPH. + O2 peroxy radicals
ROO + FL-H ROOH + FL( fluorescein )
ROO +A-H ROOH +A
loss of fluorescence measured by spectrofluorometer.
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( Source: Prior et al 2005, Huang et al. 2005)
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Fig 3:correlation coefficients (R2) between different antioxidant assays of19
commonly consumed spices in China
Correlation R2
ABTS vs. FRAP 0.9557
ABTS vs. DPPH 0.7609
FRAP vs. DPPH 0.7335
R2 =0.9652
R2 =0.9197
Source: Adapted from Lu et al. 2011
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FC assay
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` Lipid and lipid containing foods are susceptible to oxidationwhich can be retarded by the application of synthetic orNatural antioxidants.
` Growing interests to replace the synthetic antioxidants withnatural ones, which, in general, are supposed to be safer.
` Spices are one of the most important targets to search fornatural antioxidants .
` Evaluation of antioxidant activity of spices includeextractionof antioxidants using suitable methods followed by assessmentof the antioxidant activity .
22Contd
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` Organic solvents of intermediary polarity areeffectivefor theextraction of antioxidants from spice.
` Direct methods forevaluation of antioxidant capacity of
the spice are considered to be superior than indirectmethods but are time consuming.
` Various indirect assays has been used for assessing the
antioxidant activity of the spices But there no anyspecific correlation among the assays although variousresearchers have tried to correlate them.
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FOR YOUR KINDATTENTION
Welcome to Questions and Suggestion
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Yield,rosemary:2.045
& Sage 1.987 %(w/w)
30 MPa and
100C
Yield,rosemary:0.934
& Sage 0.912 %(w/w
30 MPa and
40C
Fig 2:Yield of antioxidative fraction as function of specific amount of solvent (kg CO2/ kg herbaceous material) forSFE from rosemary at 30 MPa and 100C (a) and from rosemary and sage at 30 MPa and 40C (b).
(Source: adapted from Ivanovi et al. 2008)
Mco2/M solid MCo2/M solid
(a)
(b)
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Table1. Critical properties ofvarious solvents (Reidet al., 1987)
Solvent Molecular weight Critical temperature Critical pressure Critical density
g/mol K MPa (atm) g/cm3
Carbon dioxide (CO2) 44.01 304.1 7.38(72.8) 0.469
Water(H2O)
(acc. IAPWS)18.015 647.096 22.064 (217.755) 0.322
Methan
e(CH4) 16.04 190.4 4.60 (45.4) 0.162
Ethane (C2H6) 30.07 305.3 4.87 (48.1) 0.203
Propane (C3H8) 44.09 369.8 4.25(41.9) 0.217
Ethylene (C2H4) 28.05 282.4 5.04 (49.7) 0.215
Propylene (C3H6) 42.08 364.9 4.60 (45.4) 0.232
Methanol (CH3OH) 32.04 512.6 8.09(79.8) 0.272
Ethanol (C2H5OH) 46.07 513.9 6.14 (60.6) 0.276
Acetone (C3H6O) 58.08 508.1 4.70 (46.4) 0.278
Table 2 shows density, diffusivity and viscosity for typical liquids, gases and supercritical fluids.(Non polar)
(Reid et al., 1987)
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Comparison of Gases, Supercritical Fluids and Liquids[1]
Density
(kg/m3)
Viscosity
(Pas)
Diffusivity
(mm/s)
Gases 1 10 110
Supercritical
Fluids 1001000 50100 0.010.1
Liquids 1000 5001000 0.001
Extraction is a diffusion-based process, with the solvent required
to diffuse into the matrix, and the extracted material to diffuse out
of the matrix into the solvent. Diffusivities are much faster in
supercritical fluids than in liquids, and therefore extraction can
occur faster. Also, there is no surface tension and viscosities aremuch lower than in liquids, so the solvent can penetrate into
small pores within the matrix inaccessible to liquids. Both the
higher diffusivity and lower viscosity significantly increase the
speed of the extraction: An extraction using an organic liquid may
take several hours, whereas supercritical fluid extraction can be
completed in 10 to 60 minutes
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where distinct liquid and gas phases do not exist. It
can effuse through solids like a gas, and dissolve materials like a liquid
Propane/butane, methanol, ethanol and other substances may be used as co-solvents, improvingyield or selectivity. Carbon dioxide itself is non-polar, and has somewhat limiteddissolving power,
so cannot always be used as a solvent on its own, particularly for polar solutes.The use of modifiers
increases the range of materials which can beextracted.Food grade modifiers such as ethanol can
often be used, and can also help in the collection of theextracted material, but reduces some of the
benefits of using a solvent which is gaseous at room temperature.
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Ultrasound
Unlikeelectromagnetic waves, sound waves must travel in a matter and they involve
expansion and compression cycles during travel in the medium. Expansion pulls molecules
apart and compression pushes them together.Theexpansion can create bubbles in a liquid
and produce negative pressure.The bubbles form, grow and finally collapse. Close to a
solid boundary, cavity collapse is asymmetric and produces high-speed jets of liquid.The
liquid jets have strong impact on the solid surface
The mechanical effects of ultrasound induce a greater penetration of solvent into
cellular materials and improve mass transfer. Ultrasound in extraction can also disrupt
biological cell walls, facilitating the release of contents.Therefore, efficient cell
disruption andeffective mass transfer are cited as two major factors leading to the
enhancement ofextraction with ultrasonic power
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Microwaves are electromagnetic radiations with a
frequency from 0.3 to 300 GHz. Domestic and industrial
microwaves generally operate at 2.45 GHz, and occasionally
at 0.915 GHz in the USA and at 0.896 GHz in Europe.
Microwaves are transmitted as waves, which can penetrate
biomaterials and interact with polar molecules such as water
in thebiomaterials to create heat. Consequently, microwaves
can heat a whole material to penetration depth
simultaneously.
Microwave-assisted extraction (MAE) offers a rapid
delivery ofenergy to a total volume of solvent and solid
plant matrix with subsequent heating of the solvent and
solid matrix, efficiently and homogeneously. Because
water within theplant matrix absorbs microwave energy,
cell disruption is promotedby internal superheating,
which facilitates desorption of chemicals from the matrix,
improving the recovery of nutraceuticals
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Accelerated solvent extraction (ASE) is a solidliquidextraction process
performed at elevated temperatures, usually between 50 and 200 8C and atpressures between 10 and15MPa.Therefore, accelerated solvent extraction
is a form of pressurized solvent extraction that is quite similar to SFE.
Extraction is carried out under pressure to maintain the solvent in its liquid
state at high temperature.The solvent is still below its critical condition
duringASE. Increased temperature accelerates theextraction kinetics
andelevated pressure keeps the solvent in the liquid state, thus achieving safeand rapidextraction.Also, pressure allows theextraction cell to be filled
faster and helps to force liquid into the solid matrix. Elevated temperatures
enhancediffusivity of the solvent resulting in increasedextraction
kinetics
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Methods Spices References
ABTS sage and basil
19 Chinese spices
Lu et al.,2011
Wang et al., 1998)
DPP
H 9 spices Hinn
eburg et al.,2006
FRAP 19 chinese spices Lu et al.,2011
(FC) assay 9 Spices
19 Chinese spices
Hinneburg et al.,2006
Lu et al.,2011
ORAC sage Zheng andWang, 2001.
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Fig: Example of the use of the antioxidant activity assessing methods in spices
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` also known as TEAC (Trolox EquivalentAntioxidantCapacity) assay
` ABTS - e- potassium persulfate ABTS+ (green)
` ABTS+ + e- (fromAH) ABTS
` Loss in absorbance measured at 730 nm` ABTS+ : soluble in both aqueous and organic solvents and
used to determine both hydrophilic and lipophilic antioxidants
` Simple method
Limitation:
` Poor selectivity ofABTS+ to H- atom donors .It also reactswith OH-groups of hydroxylated aromatics which do notcontribute to the antioxidation
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AAPH AAPH. +N22, 2-azobis (2-amidino-propane)
Dihydrochloride
AAPH. + O2 peroxy radicals
ROO + FL-H ROOH + FL
( fluorescein )
ROO +A-H ROOH +A
loss of fluorescence measured by spectrofluorometer.
uses a controllable source of peroxyl radicals and can detect both
hydrophobic and hydrophilic antioxidants.recommended as a standard method for a routine quality control
and measurement of foodAOC
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(Source: Prior et al 2005, Huang et al. 2005)
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` DPPH+AH DPPHH +A
` (DPPH) : stable organic nitrogen-radical ,a deep purple colour ,
` Colour change measured by monitoring thedecrease in absorbance
at 515 nm` a convenient and popular routine free radical method.
Limitation
` no similarity to the highly reactive and transient peroxyl radicals
` Reduced by some unrelated compounds` Interpretation is complicated when the test compounds have spectra
that overlap DPPH at 515 nm (e.g. carotenoids)
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Source: Prior et al 2005
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` Fe3+-TPTZ complex + e-(AH) Fe2+_TPTZ
(yellow in acetate buffer) (intense blue)
(TPTZ = 2,4,6-tripyridyl-s-triazine)
` Absorbance measured at 593nm
` a simple, rapid, inexpensive and robust assay,
Limitation
` Some antioxidant compounds such as polyphones compoundsreduceFe(III)very slowly, lead to underestimation
` Some colourextract having absorbance at 593 may interfere
` Source: Ou et al., 2002 43
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` oxidation-reduction reaction between theFolin-Ciocaltaureagent (FCR) containing molybdenum (Mo), and a phenoliccompound
` MoVI (yellow)+ e MoV (blue)
` Absobance measured at 725 nm` oldest and commonly accepted assays in food research
laboratories.
Limitation` FCR is nonspecific to phenolic compounds and it can be
reduced by many nonphenolic compounds (e.g.vitamin C,Fe2+, Cu+).
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(Source: Prior et al 2005, Huang et al. 2005)
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AAPH AAPH. +N22, 2-azobis (2-amidino-propane)
Dihydrochloride
AAPH. + O2 peroxy radicals
ROO + FL-H ROOH + FL
( fluorescein )
ROO +A-H ROOH +A
loss of fluorescence measured by spectrofluorometer.
uses a controllable source of peroxyl radicals and can detect both
hydrophobic and hydrophilic antioxidants.recommended as a standard method for a routine quality control
and measurement of foodAOC
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(Source: Prior et al 2005, Huang et al. 2005)
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` Peroxide value: The most commonly used method of assessing oxidative status
is the(hydro) peroxidevalue.This is based on the fact that hydroperoxides react
with potassium iodide to liberate iodine which can be measured by its reaction with
thiosulphate, orelectrochemically.
` Conjgated diens Lipidscontaining methylene-interrupteddienes or polyenes showa shift in theirdoublebondposition during oxidation that is due to isomerization
and conjugated bondformation. Oxidation of polyunsaturated fatty acids is
accompanied by an increase in the ultraviolet absorption of the product.The
resulting conjugateddienes exhibit intense absorption at 234 nm; similarly
conjugated trienes absorb at 268 nm. Shahidi et al. (1994) andWanasundara et al.
(1995) have found that conjugated dienes and PV of edible oilscorrelate wellduring their oxidation.
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` In this assay, theMA is reacted with thiobarbituric acid(TBA) to form a
pinkMA-TBA complex that is measured spectrophotometrically at its
absorption maximum at 530535 nm. During lipid oxidation,
malonaldehyde (MA), a minor component of fatty acids with 3 or more
double bonds, is formed as a result ofthe degradation of polyunsaturated
fatty acids
` It is based on the color reaction of p-methoxyaniline (anisidine) and the
aldehydic compounds.The reaction of p-anisidine reagent with aldehydes
under acidic conditions affords yellowish products that absorb at 350 nm.A
highly signicant correlation between p-AnV and avor scores andPV hasbeen found.
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