Spice Antioxidants Extraction and Activity Assessment Methods

8/3/2019 Spice Antioxidants Extraction and Activity Assessment Methods

1/47

Presented By: Ramesh Khadka

M.S. Food Science 1st Year ,2011

ID: 5415001298

Advisor: Asst. Prof. Dr. Wannee Jirapakkul


2/47

` 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

2


3/47

` 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


4/47

` 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

4


5/47

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

5


6/47

` 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)

6


7/47

` 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


8/47

Methods

1. Extraction with Fats and oils

2. Extraction with organic solvents

3. Extraction with supercritical fluid carbon dioxide

8


9/47

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


10/47

` 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

10


11/47

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

11


12/47

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.

12


13/47

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)

13


14/47

` Amodern method

` high operation pressure,

which requires expensive

equipment

` limited use in the

preparation of

natural antioxidants

14


15/47

` 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,

15

( Source: Schwarz et al., 2001)


16/47

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)

16

1 .peroxide value 2.conjugated dienes

3.anisidine value 4.TBA test.

(Primary oxidation products)

(Secondary oxidation products)


17/47

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),

17


18/47

oxidant (probe) + e(from antioxidant)

(specific colour)

reduced probe + oxidized antioxidant

18

( Source: Schwarz et al., 2001)


19/47

19

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)


20/47

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.

20

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


21/47

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

21

FC assay


22/47

` 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


23/47

` 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.

23


24/47

FOR YOUR KINDATTENTION

Welcome to Questions and Suggestion

24


25/47

25


26/47

26


27/47

27


28/47

28


29/47

29


30/47

30


31/47

31


32/47

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)

32


33/47

33

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)


34/47

34

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


35/47

35

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.


36/47

36

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


37/47

37

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


38/47

38

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


39/47

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.

39

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


40/47

` 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

40


41/47


Dihydrochloride


ROO + FL-H ROOH + FL

( fluorescein )

ROO +A-H ROOH +A


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

41

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


42/47

` 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)

42

Source: Prior et al 2005


43/47

` 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


44/47

` 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+).

44



45/47


Dihydrochloride


ROO + FL-H ROOH + FL

( fluorescein )

ROO +A-H ROOH +A


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

45



46/47

` 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.

46


47/47

` 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.

47

Documents

Spice Antioxidants Extraction and Activity Assessment Methods