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




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): secondary oxidation products (source: Brewer ,2011)


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) A wide array of compounds A wide array of mechanism




1.Primary antioxidants : type I or free radical scavengers(FRS)
HAT: hydrogen atom transfer SET :single electron transfer to the free radicals. e.g. phenolic compounds

2.Secondary antioxidants: type II or synergist

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Singlet oxygen quenchers: e.g. -carotene, lycopene Oxygen scavengers and reducing agents e.g. ascorbic acid, ascorbyl palmitate chelate prooxidant 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)




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)

Methods 1. Extraction with Fats and oils

Extraction with organic solvents Extraction with supercritical fluid carbon dioxide



Extraction of antioxidants from spices

Extraction with Fats and oils
Cottonseed oil Mixing & Heating Ground Spices (sage) 15-20% spice in oil At 120- 125 0c for 2h With continuous stirring To separate the extract



At 175-185 0 C/30min

Deodorized Sage extract
(source: modified from Pokorny and Korczak,2001)


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: ultrasoundassisted extraction, microwave-assisted extraction , accelerated solvent extraction





Table 1. phenolic contents in different parts of ginger (Z. officinale) varieties extracted by different solvents Variety of zinger Halia Bentong TP (mg gallic acid/g dry weight) Extraction source Leaves Stems Rhizomes Halia Bara Leaves Stems Rhizomes Methanol 33.1±1.21b 7.8±0.89ijk 10.1±0.21efg 39.06±1.62a 8.5±1.02hijk 13.4±0.34d Acetone 30.1±0.22c 7.2±1.05jk 9.8±0.22efgh 34.6±1.88b 8.06±0.92ijk 11.1±0.87e Chloroform 28.8±0.21c 7.07±0.99k 9.2±0.66fghi 33.6±1.99b 8.8±0.82ghij 10.8±0.75ef

(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.

Capabilities of scavenging DPPH free radicals Inhibition (%) Variety Halia Bentong Extraction source Leaves Stems Rhizomes Halia Bara Leaves Stems Rhizomes Methanol
51.12±0.36d 32.83±1.02g 51.48±0.72d 56.38±0.23b 31.33±0.55h 58.21±0.39a

48.99±1.07e 30.76±2.39h 49.22±0.46e 54.16±0.91c 29.11±1.01i 56.18±0.51b

47.12±0.97f 28.95±0.75i 47.47±0.64f 52.53±0.33d 27.46±0.62j 54.36±1.12c

(source: adapted from Ghasemzadeh et al. 2011)

45 40 35 30 25 20 15 10 5 0

38.8 peroxide value ( meq/Kg) of lard in 11 days





Hexane Benzene Chloroform Methanol Control Fig 1:Antioxidant activity of 0.02 % purified rosemary extracts extracted with different solvents and applied in lard (aging at 60°C; expressed as the peroxide value [meq/kg])after 11 days
( source: Modified from Pokorny and Korczak, 2001)


A modern method


high operation pressure, which requires expensive equipment limited use in the preparation of natural antioxidants





a wide array of assays for determination of antioxidant capacity DIRECT METHODS: 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
INDIRECT METHODS: assays that evaluate the ability of


antioxidants to scavenge some stable coloured synthetic free-radicals/ reduce some coloured compounds. little in common with highly reactive radical oxygen species,
( Source: Schwarz et al., 2001)

Assessing antioxidant activity

Direct method for evaluating antioxidant activity of spices
Real Models Antioxidant addition Accelerated tests(Oxidation) Analysis 1.peroxide value 3.anisidine value 2.conjugated dienes 4.TBA test.
(Primary oxidation products)
Oils, emulsions, lards, Meat and other lipid containing Foods Spice extract or ground Spice

At elevated Temperature

(Secondary oxidation products)

(source: modified from Pokorny and Korczak,2001)

SET-based assays : measure an antioxidant¶s reducing capacity
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2¶-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay/TEAC (Trolox Equivalent Antioxidant Capacity) assay, 2,2¶-diphenyl-1-picrylhydrazyl (DPPH) assay, ferric reducing antioxidant power (FRAP) assay, Folin-Ciocaltau (FC) assay,

HAT-based assay: quantify hydrogen atom donating capacity. ` oxygen radical absorbance capacity (ORAC),


oxidant (probe) + e (from antioxidant) (specific colour) reduced probe + oxidized antioxidant

( Source: Schwarz et al., 2001)

Table: 4 Summary of SET based Assays
Assays oxidant (probe) ABTS DPPH ABTS·+ (green) DPPH· ( Nitrogen radical) deep purple reduced probe Absorbance maxima 730 nm ABTS DPPHH 515nm

FRAP (FC) assay

Fe3+-TPTZ *complex yellow Folin-Ciocaltau reagent (FCR) (MoVI )(yellow)

Fe2+_TPTZ(intense blue) FCR (MoV) (blue)

596nm(reduced probe) 725 nm(reduced probe)


= 2,4,6-tripyridyl-s-triazine)
( Source:
Prior et al 2005; Huang et al. 2005;Ou et al., 2002)


2, 2¶-azobis (2-amidino-propane) Dihydrochloride AAPH. + O2

AAPH. +N2 peroxy radicals ROOH + FL ROOH + A·

( fluorescein

ROO· + A-H

loss of fluorescence measured by spectrofluorometer.

( Source: Prior et al 2005, Huang et al. 2005)

Correlation R2 =0.9197 ABTS vs. FRAP ABTS vs. DPPH R2 =0.9652 FRAP vs. DPPH

R2 0.9557 0.7609 0.7335

FC assay

Fig 3:correlation coefficients (R2) between different antioxidant assays of 19 commonly consumed spices in China
Source: Adapted from Lu et al. 2011


Lipid and lipid containing foods are susceptible to oxidation which can be retarded by the application of synthetic or Natural antioxidants. Growing interests to replace the synthetic antioxidants with natural ones, which, in general, are supposed to be safer. Spices are one of the most important targets to search for natural antioxidants . Evaluation of antioxidant activity of spices include extraction of antioxidants using suitable methods followed by assessment of the antioxidant activity .







Organic solvents of intermediary polarity are effective for the extraction of antioxidants from spice. Direct methods for evaluation of antioxidant capacity of the spice are considered to be superior than indirect methods but are time consuming. Various indirect assays has been used for assessing the antioxidant activity of the spices But there no any specific correlation among the assays although various researchers have tried to correlate them.





Welcome to Questions and Suggestion









Yield,rosemary:2.045 & Sage 1.987 %(w/w)

Yield,rosemary:0.934 & Sage 0.912 %(w/w

30 MPa and 100°C

30 MPa and 40°C

Mco2/M solid

MCo2/M solid

Fig 2:Yield of antioxidative fraction as function of specific amount of solvent (kg CO2/ kg herbaceous material) for
SFE from rosemary at 30 MPa and 100°C (a) and from rosemary and sage at 30 MPa and 40°C (b).

(Source: adapted from Ivanovi

et al. 2008)

Table 2 shows density, diffusivity and viscosity for typical liquids, gases and supercritical fluids.(Non polar)

Table 1. Critical properties of various solvents (Reid et al., 1987) Solvent Molecular weight g/mol Carbon dioxide (CO2) Water (H2O) (acc. IAPWS) Methane (CH4) Ethane (C2H6) Propane (C3H8) Ethylene (C2H4) Propylene (C3H6) Methanol (CH3OH) Ethanol (C2H5OH) Acetone (C3H6O) 44.01 18.015 16.04 30.07 44.09 28.05 42.08 32.04 46.07 58.08 Critical temperature K 304.1 647.096 190.4 305.3 369.8 282.4 364.9 512.6 513.9 508.1 Critical pressure MPa (atm) 7.38 (72.8) 22.064 (217.755) 4.60 (45.4) 4.87 (48.1) 4.25 (41.9) 5.04 (49.7) 4.60 (45.4) 8.09 (79.8) 6.14 (60.6) 4.70 (46.4) Critical density g/cm3 0.469 0.322 0.162 0.203 0.217 0.215 0.232 0.272 0.276 0.278

(Reid et al., 1987)


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 are much 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

Comparison of Gases, Supercritical Fluids and Liquids[1] Density (kg/m3) Gases 1 Supercritical 100±1000 Fluids Liquids 1000 Viscosity (µPa·s) 10 50±100 500±1000 Diffusivity (mm²/s) 1±10 0.01±0.1 0.001


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, improving yield or selectivity. Carbon dioxide itself is non-polar, and has somewhat limited dissolving 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 be extracted. Food grade modifiers such as ethanol can often be used, and can also help in the collection of the extracted material, but reduces some of the benefits of using a solvent which is gaseous at room temperature.


Ultrasound Unlike electromagnetic 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. The expansion 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 and effective mass transfer are cited as two major factors leading to the enhancement of extraction with ultrasonic power


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 the biomaterials to create heat. Consequently, microwaves can heat a whole material to penetration depth simultaneously. Microwave-assisted extraction (MAE) offers a rapid delivery of energy 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 the plant matrix absorbs microwave energy, cell disruption is promoted by internal superheating, which facilitates desorption of chemicals from the matrix, improving the recovery of nutraceuticals


Accelerated solvent extraction (ASE) is a solid±liquid extraction process performed at elevated temperatures, usually between 50 and 200 8C and at pressures between 10 and 15 MPa. 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 during ASE. Increased temperature accelerates the extraction kinetics and elevated pressure keeps the solvent in the liquid state, thus achieving safe and rapid extraction. Also, pressure allows the extraction cell to be filled faster and helps to force liquid into the solid matrix. Elevated temperatures enhance diffusivity of the solvent resulting in increased extraction kinetics


Fig: Example of the use of the antioxidant activity assessing methods in spices Methods Spices References


sage and basil 19 Chinese spices 9 spices 19 chinese spices 9 Spices 19 Chinese spices sage

Lu et al.,2011 Wang et al., 1998) Hinneburg et al.,2006 Lu et al.,2011 Hinneburg et al.,2006 Lu et al.,2011 Zheng and Wang, 2001.

DPPH FRAP (FC) assay



also known as TEAC (Trolox Equivalent Antioxidant Capacity) assay potassium persulfate ` ABTS eABTS·+ (green) ` ABTS·+ + e- (from AH) 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 of ABTS·+ to H- atom donors .It also reacts with OH-groups of hydroxylated aromatics which do not contribute to the antioxidation


2, 2¶-azobis (2-amidino-propane) Dihydrochloride

AAPH. +N2 peroxy radicals ROOH + FL

AAPH. + O2 ROO· + FL-H
( 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 food AOC
( Source: Prior et al 2005, Huang et al. 2005)




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(DPPH·) : stable organic nitrogen-radical ,a deep purple colour , Colour change measured by monitoring the decrease 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)
Source: Prior et al 2005

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 compounds reduce Fe (III) very slowly, lead to underestimation ` Some colour extract having absorbance at 593 may interfere
Source: Ou et al., 2002




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oxidation-reduction reaction between the Folin-Ciocaltau reagent (FCR) containing molybdenum (Mo), and a phenolic compound 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+).
( Source: Prior et al 2005, Huang et al. 2005)

2, 2¶-azobis (2-amidino-propane) Dihydrochloride

AAPH. +N2 peroxy radicals ROOH + FL

AAPH. + O2 ROO· + FL-H
( 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 food AOC
( Source: Prior et al 2005, Huang et al. 2005)


Peroxide value: The most commonly used method of assessing oxidative status is the (hydro) peroxide value. This is based on the fact that hydroperoxides react with potassium iodide to liberate iodine which can be measured by its reaction with thiosulphate, or electrochemically. Conjgated diens Lipidscontaining methylene-interrupted dienes or polyenes show a shift in their doublebond position 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 conjugated dienes exhibit intense absorption at 234 nm; similarly conjugated trienes absorb at 268 nm. Shahidi et al. (1994) and Wanasundara et al. (1995) have found that conjugated dienes and PV of edible oils correlate well during their oxidation.




In this assay, the MA is reacted with thiobarbituric acid (TBA) to form a pink MA-TBA complex that is measured spectrophotometrically at its absorption maximum at 530±535 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 signi¿cant correlation between p-AnV and Àavor scores and PV has been found.



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