BIOACTIVE COMPOUNDS AND ANTIOXIDANT BIOACTIVE COMPOUNDS AND ANTIOXIDANT CAPACITIES OF FRESH AND CANNED FRUITCAPACITIES OF FRESH AND CANNED FRUIT

OF PINEAPPLEOF PINEAPPLE

Supervisor; Shahzad Ali Shahid ChathaSupervisor; Shahzad Ali Shahid Chatha

Presenter;Presenter;Muhammad WaqasMuhammad Waqas

Roll No. Roll No. 209 209

M.Sc Industrial ChemistryM.Sc Industrial Chemistry

Department of Industrial ChemistryDepartment of Industrial ChemistryGC University, Faisalabad,GC University, Faisalabad,

PakistanPakistan..

CONTENTSCONTENTS

IntroductionIntroduction

Aims and ObjectivesAims and Objectives

Materials and MethodsMaterials and Methods

Results and DiscussionsResults and Discussions

ConclusionConclusion

INTRODUCTIONINTRODUCTION

Antioxidant compounds in food play an important role as Antioxidant compounds in food play an important role as a health protecting factora health protecting factor

The word “antioxidants” means to compounds that can The word “antioxidants” means to compounds that can avoid the oxidation of lipids or other molecules by avoid the oxidation of lipids or other molecules by inhibiting the commencement of oxidative chain reaction inhibiting the commencement of oxidative chain reaction

Antioxidants are substances' that may protect cells from Antioxidants are substances' that may protect cells from the damage caused by unstable molecules known as free the damage caused by unstable molecules known as free radicalsradicals

Types of Antioxidants Types of Antioxidants Antioxidants are classified into two groups on the Antioxidants are classified into two groups on the basis of their origin basis of their origin

(1) Natural Antioxidants(1) Natural Antioxidants

The antioxidants which is naturally present in fruits The antioxidants which is naturally present in fruits and vegetables grains etc are called Natural and vegetables grains etc are called Natural Antioxidants e.g. Flavonoids, Carotenoids, Pectin Antioxidants e.g. Flavonoids, Carotenoids, Pectin etc.etc.

(2) (2) Synthetic AntioxidantsSynthetic Antioxidants

Synthetic antioxidants are those, which are prepared Synthetic antioxidants are those, which are prepared in laboratory e.g. Butylated hydroxyl toluene in laboratory e.g. Butylated hydroxyl toluene (BHT), butylated hydroxyanisole (BHA) etc.(BHT), butylated hydroxyanisole (BHA) etc.

SYNTHETIC ANTIOXIDANTSSYNTHETIC ANTIOXIDANTSExamplesExamples

BHTBHT

Molecular Formula: CMolecular Formula: C1515HH2424OO

Appearance: White Crystal or PowderAppearance: White Crystal or Powder

Uses: As a food additive because of it Uses: As a food additive because of it antioxidant propertiesantioxidant properties

Structure: Structure:

SYNTHETIC ANTIOXIDANTS SYNTHETIC ANTIOXIDANTS ExampleExample

BHABHA

Molecular Formula: CMolecular Formula: C1111HH1616OO22

Appearance: Appearance: Waxy solidWaxy solid

Solubility: Solubility: Soluble in waterSoluble in water

Uses: Uses: Antioxidants in food because Antioxidants in food because it can slow down the rate of autoxidation that changes it can slow down the rate of autoxidation that changes the food taste and colourthe food taste and colour

Structure: Structure:

NATURAL ANTIOXIDANTSNATURAL ANTIOXIDANTS ExampleExample

Cinnamic AcidCinnamic Acid

Molecular Formula: CMolecular Formula: C99HH88OO22

Appearance: Monoclinic crystalAppearance: Monoclinic crystal

Solubility: Slightly soluble in waterSolubility: Slightly soluble in water

Uses: In food and pharmaceutical Uses: In food and pharmaceutical industriesindustries

Structure: Structure:

NATURAL ANTIOXIDANTS NATURAL ANTIOXIDANTS ExampleExample

CyanidinCyanidin

Molecular Formula: Molecular Formula: CC1515HH1111OO66

Appearance: Appearance: Reddish orange colourReddish orange colour

Solubility: Solubility: Water solubleWater soluble

Uses: Uses: Antioxidant/ Health benefits Antioxidant/ Health benefits anticancer, skin protectionanticancer, skin protection

Structure: Structure:

NATURAL ANTIOXIDANTSNATURAL ANTIOXIDANTS ExampleExample

AnthocyaninsAnthocyanins

Molecular Formula:Molecular Formula: C C1515HH1111OO55

Appearance: Appearance: Red, Purple or Blue Red, Purple or Blue

Solubility: Solubility: Water solubleWater soluble

Uses: Uses: Food coloring Food coloring

Structure: Structure:

INTRODUCTIONINTRODUCTIONPineapplePineapple

Pineapple belongs to the bromeliaceae familyPineapple belongs to the bromeliaceae family The pineapple is the leading edible member of The pineapple is the leading edible member of

bromoliaceae which embraces about 2,000 speciesbromoliaceae which embraces about 2,000 species

It have a wide cylindrical shape, brown or yellow skin and It have a wide cylindrical shape, brown or yellow skin and a regal crown of spiny, blue green leaves a regal crown of spiny, blue green leaves

Pineapple is a rich source of natural antioxidants e.g. Pineapple is a rich source of natural antioxidants e.g. Flavonoids, Carotenoids etc.Flavonoids, Carotenoids etc.

To investigate the antioxidant activity of To investigate the antioxidant activity of Ananas Ananas comosuscomosus fresh and cannned fruit extracts fresh and cannned fruit extracts

To evaluate the effect of different extraction procedures To evaluate the effect of different extraction procedures on the extraction efficiency of antioxidantson the extraction efficiency of antioxidants

To evaluate the total phenolics and total flavonoids in To evaluate the total phenolics and total flavonoids in fruit extracts by using in vitro assaysfruit extracts by using in vitro assays

AIMS AND OBJECTIVESAIMS AND OBJECTIVES

EXPERIMENTAL PROTOCOLEXPERIMENTAL PROTOCOL

Preparation of Sample

Preparation of Extracts

Collection of Pineapple

Antioxidants Activity

Estimation of total flavonoids

DPPH radical scavenging assay

Estimation of Total

Phenolic Content

Determination of %age inhibition of peroxidation of linoleic acid

Determination of reducing power

MATERIALS AND METHODMATERIALS AND METHOD Total Phenolic ContentsTotal Phenolic Contents

0.5g Extracts was mixed with 0.5mL of Folin-0.5g Extracts was mixed with 0.5mL of Folin-Ciocalteu reagent and 7.5 mL of deionized waterCiocalteu reagent and 7.5 mL of deionized water

After 10min add 1.5 ml of 20% sodium carbonateAfter 10min add 1.5 ml of 20% sodium carbonate

The mixture was heated in a water bath at 40cThe mixture was heated in a water bath at 40c° for ° for 20min and cooled in ice bath20min and cooled in ice bath

Absorbance was taken at 750nm by Absorbance was taken at 750nm by spectrophotometer using a calibration curve of Gallic spectrophotometer using a calibration curve of Gallic acidacid

MATERIALS AND METHODMATERIALS AND METHOD Total Flavonoid ContentsTotal Flavonoid Contents

0.01g Extracts was mixed with 5mL distilled water 0.01g Extracts was mixed with 5mL distilled water followed by 0.3 mL of 5% NaNOfollowed by 0.3 mL of 5% NaNO22

After 5min add 0.6mL of 10% AlClAfter 5min add 0.6mL of 10% AlCl33

Again after 5min add 2mL of 1M NaOH and make the Again after 5min add 2mL of 1M NaOH and make the volume up to 10ml with distilled watervolume up to 10ml with distilled water

Absorbance was taken at 510nm Absorbance was taken at 510nm

The amounts of TFC express as Catechin equivalentsThe amounts of TFC express as Catechin equivalents

MATERIALS AND METHODMATERIALS AND METHODDPPH scavenging assayDPPH scavenging assay

The sample (from 6.25 to 1oomg/mL) were mixed The sample (from 6.25 to 1oomg/mL) were mixed with 1mL OF 90with 1mL OF 90µM DPPH solution and filled up µM DPPH solution and filled up with 95% Methanol, to a final volume of 4mL.with 95% Methanol, to a final volume of 4mL.

The absorbance of the resulting solution and the The absorbance of the resulting solution and the blank were recorded after 1 hourblank were recorded after 1 hour

BHT was used as positive controlBHT was used as positive control Inhibition of free radical DPPH in percent was Inhibition of free radical DPPH in percent was

calculated ascalculated as 1%=100 x ( Ablank – Asample)/ Ablank1%=100 x ( Ablank – Asample)/ Ablank

MATERIALS AND METHODMATERIALS AND METHODReducing PowerReducing Power

Dry matter extracts 8-12mg was mixed with 5.0 mLof Dry matter extracts 8-12mg was mixed with 5.0 mLof sodium phosphate buffer (0.2M, Ph =6.6) and 5.0 mL sodium phosphate buffer (0.2M, Ph =6.6) and 5.0 mL of 1% potassium ferricyanide was addedof 1% potassium ferricyanide was added

The mixture was incubated at 50The mixture was incubated at 50°°cc for 20 mintues and for 20 mintues and then 5.0 Ml of 10% trichloro acetic acid was added then 5.0 Ml of 10% trichloro acetic acid was added and centrifuged at 980g for 10 mintues at 5°and centrifuged at 980g for 10 mintues at 5°c in c in refrigerator centrifugerefrigerator centrifuge

Upper layer was diluted with 5.0 Ml distilled water Upper layer was diluted with 5.0 Ml distilled water and 1mL of 0.1% ferric chloride was addedand 1mL of 0.1% ferric chloride was added

Finally absorbance at 700 nm was measured with Finally absorbance at 700 nm was measured with spectrophotometerspectrophotometer

MATERIALS AND METHODMATERIALS AND METHOD%Inhibition of peroxidation of linoleic acid%Inhibition of peroxidation of linoleic acid

5mg of extracts were added to a solution of lineolic 5mg of extracts were added to a solution of lineolic acid (0.13 mL) in 25mL volumetric flaskacid (0.13 mL) in 25mL volumetric flask

Then 99.8% methanol (10mL) and 10mL of 0.2 M Then 99.8% methanol (10mL) and 10mL of 0.2 M Sodium phosphate buffer Ph =7 was added and dilute Sodium phosphate buffer Ph =7 was added and dilute up to 25mL with distilled water up to 25mL with distilled water

After 3 mintues of shaking the absorbance of mixture After 3 mintues of shaking the absorbance of mixture measured with spectrophotometer at 500 nmmeasured with spectrophotometer at 500 nm

BHT was used as positive controlBHT was used as positive control Maximum oxidation was observed after 360 hourMaximum oxidation was observed after 360 hour

RESULTS AND DISCUSSIONSRESULTS AND DISCUSSIONS

Yields (g/100g) of fresh and canned fruit extracts of Pine Apple (Ananas comosus L.)

Values are mean ± standard deviation of three separate experiments.

0

10

20

30

40

50

60

Fresh Canned

Yie

ld o

f E

xtr

ac

ts (

%)

Values are mean ± standard deviation of three separate experiments.

Total phenolic contants (g/100g as GAE) in fresh and canned fruit extracts of Pine apple (Ananas comosus L.)

0

1

2

3

4

5

6

7

8

Fresh Canned

TP

C (

g/1

00

g a

s G

AE

)

Values are mean ± SD of three citrus peel extract analyzed individually in triplicate

Total flavonoids (g/100g as CE) in fresh and canned fruit extracts of Pine apple (Ananas comosus L.)

0

10

20

30

40

50

60

Fresh Canned

TF

C (

g/1

00

g a

s C

E)

Percentage Inhibition oxidation of linoleic acid system

0

10

20

30

40

50

60

70

80

90

100

Fresh Canned BHT

% In

hib

itio

n

Values are mean ± SD of three citrus peel extract analyzed individually in triplicate

y = 5.9976Ln(x) + 73.574

R2 = 0.9332

0

10

20

30

40

50

60

70

80

90

0.0001 0.001 0.01 0.1 1

Concentration (mg/mL)

% S

cave

ngi

ng

DPPH free radical scavenging activity of methanolic extracts of fresh fruit of Pine apple (Ananas comosus L.)

Values are mean ± SD of three citrus peel extract analyzed individually in triplicate

y = 3.3015Ln(x) + 86.342

R2 = 0.8443

0

10

20

30

40

50

60

70

80

90

100

0.0001 0.001 0.01 0.1 1

Concentration (mg/mL)

% S

cave

ngi

ng

DPPH free radical scavenging activity of methanolic extract of canned fruit of Pine apple (Ananas comosus L.)

Reducing power of fresh and canned fruit extracts of Pine apple (Ananas comosus L.)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 2.5 5 7.5 10

Concentration (mg/mL)

Abs

orba

nce

(700

nm)

Fresh Extract

Canned Extract

Canned Extract