STUDIES ON EXTRACTION, ISOLATION AND

APPLICATIONS OF LYCOPENE

Sanjay Metkar et al., IAJPR. 2014; volume4(10): 5017-5028.

Published under www.iajpr.com (1.38 Impact factor) ISSN NO: 2231-6876

Copy right © 2014

INTRODUCTION

What is lycopene ?

Lycopene is part of the family of pigments called

carotenoids, which are natural compounds that create

the colors of fruits and vegetables.

Lycopene is a pigment principally responsible for the

characteristic deep-red color of ripe tomato fruits.

Lycopene is a lipophilic compound that is insoluble in

water, but soluble in organic solvents.

What are the importance of lycopene ?

Lycopene is a powerful antioxidant that may help to

protect cells from damage.

This is why there is a lot of research interest in

lycopene’s role, if any, in preventing cancer.

LYCOPENE STRUCTURE

Lycopene belongs to the family of carotenoids. It has

Structure that consist of a long chain of conjugated

double bonds with two open rings.

The structure of lycopene is the longest of all

carotenoids.

Lycopene ([C40H56],M.Wt.-536.85) is an unsaturated

hydrocarbon containing 13 carbon-carbon double bonds,

11 of which are conjugated and arranged in a linear

array.

These conjugated double bonds are responsible for the

vibrant red color of lycopene.

Lycopene Structure

What is the source of Lycopene ?

• The best sources of lycopene are tomatoes and tomato

products.

•Lycopene can also be found in guava, watermelon, and

pink grapefruit.

OBJECTIVE AND SCOPE

The aim of this study was to investigate antioxidant

property of lycopene.

MATERIALS AND METHODOLOGY

Plant Materials

Fresh fruits

Processed fruits

Riped fruits

EMS mutagenesis fruits

All Tomato fruits were collected from markets in

Aurangabad. It was identified as Lycopersicum

esculentum (Solanaceae).

METHODOLOGY

A.ISOLATION OF LYCOPENE BY USING LIQUID -

LIQUID EXTRACTION

For the isolation of lycopene we employed four different

methods and studied the comparative yield of lycopene.

By using standard formula calculated the yeild of

lycopene by using std. formula.

The std. formula ;

Mg lycopene in 100g sample = 31.206 X Absorbance at 503nm

Weight of sample (g)

The comparative yield of lycopene in which the more amount of lycopene was found in first procedure, in this method methanol:carbon tetrachloride was used as an organic solvent for extraction of lycopene.

Therefore further isolation of lycopene was carried out by only this method.

ISOLATION PROCEDURE 1

This liquid-liquid extraction method comprises

minimum organic solvent and also lycopene yield was

better compare to other procedure described in

articles.

In this method;

50gm of tomato was dehydrated by adding 65ml

methanol.

After 2 hour, the thick suspension was filtered; the

dark red cake was shaken for another 15 min with

75 ml mixture of equal volume of methanol and

carbon tetrachloride and separated by filtration.

The carbon tetrachloride phase was transferred to a

separatory funnel; added one volume of water and

shaked well.

After phase separation, the carbon tetrachloride phase

was evaporated and the residue was diluted with

about 2ml of benzene.

1 ml of boiling methanol was added in portion, then

crystals of crude lycopene were appeared immediately

and the crystallization was completed by keeping the

liquid at room temperature and ice bath, respectively.

The crystals were washed 10 times using benzene and

boiling methanol.

liquid-liquid Extraction usually can be performed by using separatory funnels.

It is an extraction of a substance from one liquid into another liquid phase.

i.e. here from water to carbon tetra chloride.

By using two phase extraction ,lycopene was isolated from;

Fresh fruits

Processed fruit

Riped fruits

EMS mutagenesis fruits

Two-Phase Extraction

Seeds of fresh tomatoes were treated with ethyl metanesulfonate (0.5 percent EMS for 12 h; LD15) causing primarily C/G to T/A transitions.

PROCEDURE ;

Imbided seeds on wet Whatman paper (8-10 hrs).

Transfered seeds to an Erlenmeyer bottle containing dH2O (100ml for 10,000 seeds).

Added 0.5% EMS solution.

Mixed and incubated for 12 hrs with gentle shaking in hood at room temperature.

Removed the EMS (puted EMS solution into 1M NaOH overnight, then disposed as regular waste.Also washed all dishes and pipettes with NaOH).

Rinsed the seeds extensively under running water for 5-10 minutes.

Dried the seeds in incubator.

Sowed the seeds, preferably on the same day.

B.EMS MUTAGENESIS

Control EMS treated

The tomato paste was obtained from the Control

tomato plants i.e. untreated with EMS and EMS

treated tomato plants.

0.9677 4.Riped

• To avoid Deviations of Beer’s lambert’s Law, solution of

Lycopene is diluted up to 100x.

The graph showed the highest yield of lycopene was

found in no 4 sample i.e. riped tomato fruits sample.

Sample no

m g lycopene/ml

C. COLUMN CHROMATOGRAPHY Chromatography is an effective and very useful method

for separation and purification of organic compounds that can be used even for complex mixtures.

For the purification of lycopene the stationary phase was solid inert material that contains a polar functional group, and therefore polar compounds have a greater affinity for the stationary phase.

Silica gel was employed in this study as a stationary phase and the mobile phase was organic solvent i.e Acetone:Hexane (10:90)

To increase elution rate the conc. of mobile phase was varied in this study.

Actually the lycopene extract contains high level beta carotene and lycopene.

To remove beta carotene from lycopene column chromatography technique was used in this study.

PROCEDURE Column was packed with neutral silica 1.0 to 2.0 gm as an

absorbent.Then for the washing of column and to remove impurity in stationary phase used hexane several times to drained impurities of column.

After several washing of Hexane added lycopene extract via pasteur pipette to the top of the column. after this addition immediately filled all column with hexane.

When the first yellow band starts to drain out of the column, added second eluent (10:90 volume acetone:hexane) to the top of the column and kept the eluent level constant as before.

When the lycopene layer (orange-red) begins to leave the column, collected the orange-red layer into the Erlenmeyer flask.

when the band is almost completely off the column, removed the sample vial and replaced it with the waste beaker that used earlier.

Lycopene contain 13 carbon carbon double bond, that was strongly attracted towards the silica or aluminia absorbent compare to beta carotene and related carotene which have 11or12 bonds.

Therefore the yellow carotene band moved down faster than orange red lycopene.

After the addition of second eluent the bonding

between silica and lycopene became somewhat low so

the orange red lycopene moves down that was collected

in separate flask.

After column chromatography solution of lycopene was

so concentrated so it was diluted with hexane for

quantification of lycopene using std. formula.

Sample no

L

Y

C

O

P

E

N

E

C

O

N

T

E

N

T

Sample No

D.HYDROGEN PEROXIDE SCAVENGING ASSAY

The quantitative determination of hydrogen peroxide is

important in numerous studies since H2O2 is involved

in oxidative cellular damages as well as in signalling

processes.

In biological systems incomplete reduction of O2 during

respiration produces superoxide anion (O2-·), which is

spontaneously or enzymatically dismutated by

superoxide dismutase to H2O2.

The ability of the lycopene to scavenge hydrogen

peroxide was assessed by the method of Ruch et al.

(1989).

Phosphate buffer (0.1M, pH 7.4) and H2O2 (40mM) in

phosphate buffer reagents were used in this assay.

PROCEDURE A solution of H2O2 (40mM) was prepared in phosphate buffer.

Lycopene of various concentration from stock 5mg/ml were added to H2O2 solution (0.6ml) and the total volume was made up to 3ml.

The absorbance of the reaction mixture was recorded at 230nm in a spectrophotometer.

A blank solution containing phosphate buffer, without H2O2 was prepared.

The extent of H2O2 scavenging of the plant extracts was calculated as;

Graph showed up to 60% inihibition of radical scavenging capacity of lycopene sample.

Sr.No. Lycopene

mg/ml

Phosphate buffer O.D. at 230 nm % of inihibition

1. 0.1 2.9 1.250 0.15

2. 0.2 2.8 0.914 26.99

3. 0.3 2.7 0.702 34.26

4. 0.4 2.6 0.620 34.50

5. 0.5 2.5 0.528 43.92

6. 0.6 2.4 0.820 50.47

7. 0.7 2.3 0.823 52.07

8. 0.8 2.2 0.600 57.82

9. 0.9 2.1 0.512 59.10

10. 1.0 2.0 0.512 59.10

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10

% of inihibition

Sample No Graph of H2O2 Assay

%

o

f

I

n

i

h

i

b

i

t

i

o

n

E.ESTIMATION OF RNA DAMAGE Overproduction of free radicals can damage cellular

components resulting in progressive physiological

dysfunction, which has been implicated in many human

diseases.

Oxidative modification to RNA results in disturbance of the

translational process and impairment of protein synthesis,

which can cause cell deterioration or even cell death.

The method described by Chang et al. (2002) was used to

assess the RNA damage.

In this method FeCl3 and H2O2 react with each other

resulting in the generation of hydroxyl radicals.

The most prevalent oxidized base in RNA is 8-

hydroxyguanosine (8-OHG). Guanosine (a RNA nucleoside)

can be oxidized by highly reactive hydroxyl radicals to form a

C8-OH adduct radical, which then loses an electron (e−) and a

proton (H+) to form 8-OHG (an oxidized RNA nucleoside)

PROCEDURE

The method described by Chang et al. (2002) was used

to assess the RNA damage.

The reaction was carried out in tris buffer (pH 7.4) at

37°C.

Each reaction mixture contained 5µl of tris buffer in

RNA(2µg) and 5µl of tris buffer in lycopene. FeCl3 (5µl)

and 10µl of H2O2 were added to test samples and

incubated at 37°C for 15 minutes for RNA.

To the reaction mixture,0.06 ml of gel loading dye was

added and electrophoreses in 1% agarose gel containing

3µg/ml EtBr, at 100V for 15 minutes.

Gels were viewed under transilluminating UV light

photographed and analysed the band intensity.

(H2O2+RNA) (H2O2+RNA+Lycopene sample) Control(Std.RNA)

•The band intensity was found more in the sample contain

lycopene and free radical that it suggest lycopene may be prevent

RNA Damage again oxidative insult.

F.LYCOPENE AS A FOOD COLORANTS The FDA in the United States has approved only

lycopene sourced from tomatoes as a colour additive in foods.

Lycopene, like other carotenes, may provide health benefits due to its antioxidant properties.

In this study lycopene as a food colorants was used against the standard orange red color i.e.(Caramoisine and Nacl-15%).

Simply appropriate conc. of lycopene and std. orange red (i.e control) was applied on sugar cubes and wrapped in aluminium foil and in polythene bags.

And observed the color efficiency and effect of light on lycopene against the control sample.

Lycopene as a food colorants showed good color efficiency as compare to standard color(Caramoisine and Nacl-15 %) that normally used as in a food color.

Standard(Caramoisine) food color Spreaded on Sugar cubes Lycopene color spreaded on sugar cubes.

Lycopene color spreaded on sugar cubes

DISCUSSION

Combination of recrystalization and column

chromatographic method gave completely pure

lycopene as a colorless substance was seen under

microscope.

The amount of pure lycopene was also good (2.313 mg

per 100 g tomato paste)compare to those obtained from

other studies.

Lycopene yield was improved by the treatment of EMS

mutagen to the seed of tomato.

Lycopene has a great ability for radical scavenging up

to 60% inihibition was seen in radical scavenging

assay.

Lycopene has deep red color and its antioxidants

property makes it to use as a food colorants.

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Total Antioxidant Properties and Other Quality Factors in Wild Tomato (Solanum pimpinellifoliumL). International Journal of PharmTech Research CODEN (USA): IJPRIF ISSN : 0974-4304,Vol.5, No.4, pp 1655-1663.

Aghel N et al., (2011). Isolation and Quantification of lycopene from tomato cultivated in dezofoul,IRAN. Jundishapur Journal of Natural Pharmaceutical Products 6(1): 9-15.

Jasmina M et al.,(2013) Antioxidant Capacity and Contents of Phenols, Ascorbic Acid, β-Carotene and Lycopene in Lettuce. Faculty of Agronomy, University of Kragujevac, Serbia.

R. Bunghez et al.,(2011) Lycopene Determination In Tomatoes By Different Spectral Techniques (Uv-Vis, Ftir And Hplc) Digest Journal Of Nanomaterials And Biostructures Vol. 6, No 3, P. 1349-1356.

Rajamanikandan S. et al.,(2011) Radical Scavenging and Antioxidant Activity of Ethanolic Extract of Mollugo nudicaulis by Invitro Assays. Indian Journal of Pharmaceutical Education and Research.

Kin-Weng Kong et al.,(2012) Revealing the Power of the Natural Red Pigment Lycopene. Advance Journal of Food Science and Technology 4(4): 182-188.

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