Studies on Isolation and Quantification of Lycopene from Tomato and Papaya and

its Antioxidant and Antifungal Properties

Pooja R. Desai1, Payal D. Holihosur2, Sangeeta V. Marathe2, Shivaprakash V. Hiremath2,Bhushan B.
Kulkarni2*
1 Mahatma Gandhi Mission's Institute of Biosciences and Technology, MGM Deemed University, N-6,
CIDCO, Aurangabad-431003, Maharashtra, India .
2 P. C Jabins Science College, Department of Biotechnology, Vidyanagar. Hubli.580021. India.
*

Correspondence: Phone: +918362372285; eMail: [email protected]

Abstract
Lycopene is a plant carotenoid that imparts the red pigment to the fruits and
vegetables like tomatoes, watermelon, papaya, pink guava, apricots, carrot and red
bell peppers. With respect to antioxidant property, lycopene has the highest singlet
oxygen quenching activity, thus making it one of the best dietary constituent in
combating the ill effects caused by reactive oxygen species like singlet oxygen. In the
present study investigation was made to evaluate the lycopene content in local
varieties of tomato and papaya available in North Karnataka region of Indian origin.
Studies were also carried out further to find out antioxidant and antifungal properties
of the lycopene. The result showed that the Namadhari variety of tomato contains
highest amount of lycopene (14.04mg/100g) followed by Rupali and Pusa ruby
variety with 12.79mg/100g and 10.92mg/100g respectively and Solo variety of
papaya contains 10.29mg/100g of lycopene. As far as antioxidant activity of tomato
and papaya, is concerned, it was observed that the papaya contains higher activity
compared to tomato varieties studied. However, the extract of tomato and papaya
when tested for antifungal activity, both did not show any inhibiting activity against
the growth of the fungus Candida albicans.
Keywords: Lycopene; Antioxidant; Antifungal activity
Introduction
Lycopene is a plant carotenoid that imparts red pigment to the fruits and vegetables
like tomatoes, watermelon, papaya, pink guava, apricots, carrot and red bell peppers.
It is naturally synthesized by plants 1,2. This pigment was first discovered by a French
botanist Alexis Millardet in 1876 in the tomato fruit 4. It was later named as lycopene
by chemist C. A. Schunck in5. It is a tetraterpene (terpenes consisting of eight isoprene
units) and have the molecular structure C40H643. Lycopene is found in both cis (human
blood)and trans (plants)forms. It is water insoluble, but soluble in organic solvents
like chloroform, hexane, benzene, methylene chloride, acetone, and petroleum ether 3,
6
.Among carotenoids, lycopene is of the great interest due to its high antioxidant
activity29. Reactive oxygen species (ROS) are the unstable compounds like singlet
oxygen, hydrogen peroxide, superoxide anion, hydroxyl radical, hydroxyl ion, and

nitric oxide. The imbalance of excess of oxidants is referred as oxidative stress which
leads to chronic diseases such as, cardiovascular disease13-17, cancer8-12,28,
diabetes29,26 eye diseases and aging associated ailments 7and skin damage18, 19.
Lycopene due to its potential antioxidant activity encounters these singlet oxygen or
peroxy radicals and transfers energy between these molecules 7.Lycopene also exerts
potent antifungal activity on Candida albicans by causing significant damage to the
cell membranes of the yeast. The antifungal mode of action of lycopene was
understood by examining the action of lycopene against fungal cell membranes by
Fluorescence Activated Cell Sorter (FACS) scan analysis and glucose and trehaloserelease test1. The killing property of lycopene was assessed by conducting a killingcurve assay against S.aureus and C. albicans, and the results showed that lycopene
has antimicrobial activity. Lycopene due to its strong color and non-toxicity can be
used as a food coloring agent and also as a preservative in place of toxic artificial
coloring agents like nitrites, monosodium glutamate (MSG)20,21,22. Apart from fruits
and vegetables, lycopene is also found in photosynthetic bacteria, fungus
(Blakesleatrispora) and algae 1,2,3. Processed tomato products such as pasteurized
tomato juice, soup, sauce, and ketchup contain the highest concentrations of
bioavailable lycopene. All fruits and vegetables mentioned above form a staple part of
day to day diet; hence it calls for a study to assay the lycopene content in the tomato
and papaya varieties available in this region.
Material and Methods
Samples of tomatoes were selected from local market of Hubli-Dharwad (North
Karnataka) region with distinct morphological characteristics depending on its size,
shape, color and locules and they were identified by Horticulture Department of
University of Agriculture Science, Dharwad (fig.1). One variety of papaya was
collected from University of Agriculture Science, Dharwad. Pure culture of Candida
albicans ATCC 10231, NCIM No: 3471 was procured from National Chemical
Laboratory (NCL), Pune. Antibiotics, ketoconazole and fluconazole were used as
standard.
Lycopene assay:
One mole of Lycopene when dissolved in one liter light petroleum ether and measured
in a spectrophotometer at 503 nm in one cm light path gives an absorbance of
17.2104. Hence, a concentration 3.1206g lycopene/ml gives unit absorbance.
Preparation of extract for Lycopene assay23
The samples were cleaned and wiped dry. The weight of the whole sample was noted.
The sample was blended using blender to form smooth pulp. Homogenizer was used
for complete extraction of lycopene from the weighed amount of sample. 10g of this
pulp was weighed and homogenized with 20ml of acetone till the tissue becomes
colorless. During the process of homogenization ice cold water was used to maintain
temperature. The acetone solution was taken in centrifuge tube and was spin for 1min

at 5000rpm. Supernatant obtained was collected in a brown bottle. The pellet was resuspended into 20ml acetone and the homogenization and centrifugation process was
repeated for complete extraction of lycopene from the tissue. Pool the acetone
extracts. Separating funnel was set up. Acetone extracts were transferred to a
separating funnel and 20ml of petroleum ether was added to the separating funnel;
mix the contents of the funnel gently. The solution in the separating funnel was kept
undisturbed for a while. 20ml of 5% sodium sulphate (Na 2SO4) was then added to
obtain clear solution. The volume of petroleum ether was reduced by evaporation
hence 20ml of more petroleum ether was added to the separating funnel. Majority of
the color will be observed in the upper petroleum ether organic layer. Two layers were
separated and the lower aqueous phase was re-extracted with additional 20ml of
petroleum ether till the aqueous phase was colorless. Petroleum ether extracts were
washed with 10ml of distilled water to remove debris. These petroleum ether extracts
were collected in brown bottle containing 10g anhydrous sodium sulphate (Na 2SO4)
and was kept for 30minutes. The extract was filtered to a volumetric flask containing
cotton wool.
Lycopene estimation using UV visible spectrophotometer23
Absorbance was measured at 503nm by using petroleum ether as blank and the
readings were noted (1ml of lycopene solution was diluted ten times with petroleum
ether).
Absorbance (I unit) =
Antioxidant assay:
Potassium permanganate assay (KMnO4)24
The assay is based on the redox reactions between the antioxidant sample and the
potassium permanganate in the sulfuric acid media that is leading to the discoloration
of the potassium permanganate. Ten clean and dry test tubes of 50 ml were arranged
in test tube stand. KMnO4 solution was prepared of 0.01M. Dilutions of KMnO4
(0.01M) were prepared in series ranging from 0, 0.2, 0.4 till 2ml. 3.5ml of H 2SO4
(2M) was added and was followed by 20ml of distilled water addition. The unknown
was prepared by using previously extracted petroleum ether lycopene solutions that
were diluted in 1:10 ratio. 2ml of this unknown was mixed with 1.5ml KMnO 4, 3.5ml
H2SO4 and 20ml distilled water. The contents of tubes were mixed and immediately
the absorbance was measured at 535nm. Ascorbic acid of 0.001M was used as
standard.
Antifungal activity: 3
The procured pure culture of Candida albicans was sub cultured. Sabourauds
medium was prepared and autoclaved at 120C for 15 minutes. The slants were
prepared under aseptic conditions. With the help of sterile nicrome wire loop the pure

culture of C. albicans was streaked on slants. These slants were incubated at 37C for
48hours.
Preparation of extract for antibiotic assay:
Tomato samples were blended using blender and were refrigerated at -20C for
storage purpose. The previously stored tomato samples were brought to room
temperature. 10g of each tomato sample was weighed and was mixed with 10ml of
DMSO (dimethyl sulfoxide). The mixture was homogenized using homogenizer to
obtain clear solution. 1g of standard antibiotic was dissolved in 10ml distilled water to
give the concentration of 0.1g/ml. Solutions of antibiotic of subsequent concentrations
were prepared in distilled water as follows:
Sabourauds agar medium was prepared and autoclaved at 120C for 15 minutes. Petri
plates were sterilized by wiping it with alcohol and then autoclaving at 120C for
15minutes. Approximately 20ml of sterile Sabouraudss media was poured to each
Petri plates to give thickness of 3-4 mm. This was performed under aseptic
conditions. The agar was allowed to solidify. 200l of overnight activated candida
broth was spread on solidified medium with help of glass spreader. Well of 1.5-2mm
diameter were made using sterile borer. Precaution was taken that well did not touch
the bottom of plate. Plates were marked with different concentration ranging from 5,
10, 20, 40, 80, 100 g/ml. 40l samples and antibiotics of above prepared dilutions
were poured to each well as per the markings made on plate. Fluconazole and
Ketoconazole were used as a standard. The plates were incubated for 37C for 72
hours.
Results and Discussion
The varieties of tomatoes were identified as Rupali, Pusa ruby and Namadhari based
on the fruit shape, size, color and locules at Horticulture Department of University of
Agriculture Science, Dharwad. The papaya variety selected was Solo (fig.1).

Rupali

Pusa ruby

Namadhari

Solo

Fig 1: Varieties of tomato and papaya obtained from the local market
Tomato and papaya are the most prominent source of lycopene and also easily
available2,25,26. Hence an attempt was made to evaluate the lycopene content of
tomatoes and papaya variety available in Hubli-Dharwad region. In this study
lycopene content was found to be highest among Namadhari followed by Rupali and
Pusa ruby whereas Solo variety of papaya had least content of lycopene (table 1). Our
results are in accordance with reports of other workers indicating tomato has highest
nutritional content of lycopene in it 21, 27.

Table 1: Lycopene contents in different varieties of tomatoes and papaya

Sample

Lycopene (mg/100g)

1. Namadhari

14.04

2. Rupali

12.79

3. Pusa ruby

10.92

4. Solo

10.29

The highest antioxidant activity was seen in Solo variety of papaya. The tomato
varieties Namadhari, Rupali and Pusa ruby showed lesser antioxidant activity than
Solo variety of papaya. These results are not in accordance with reports of other
workers24 where tomato showed higher antioxidant activity. However when this was
compared to standard ascorbic acid, it was seen that all extracts had lesser antioxidant
activity than standard ascorbic acid.

The antioxidant assay of lycopene was carried out by using potassium permanganate
method.

Fig.2:KMnO4calibration curve, indicating antioxidant activity of Lycopene

Lycopene has also shown its promising antibacterial effect on E. coli, S.aureus and
antifungal effect on C. albicans. The antifungal assay carried out for C. albicans using
fluconazole and ketoconazole as control did not show inhibition effect on growth C.
albicans. Antifungal activity was assessed in lycopene rich food sources like tomato
and papaya extract. However the extract containing approximately 100g of lycopene
failed to show antifungal effect. On contrary standard antibiotic showed effective
antifungal activity against C. albicans. This lack of reactivity may be due presence of
other compounds in the preparation of samples which may hinder the effect of
lycopene on C. albicans (fig.2).

Fig 2: Well diffusion plates inoculated with C. albicans and various concentration of
Lycopene extracts. Left plate shows no growth after 24 hrs of incubation and right
plate showing growth of fungus after 48 hrs of incubation.
Considering beneficial effects of lycopene in regulating oxidative stress due to its
antioxidant ability and ability to prevent bacterial and fungal contamination it is
clearly evident that lycopene rich sources should be incorporated in the staple diet of
individuals. Hence studies to assess lycopene content in various food sources should
be done to estimate nutritional significance in them.
Conclusions
Lycopene has an efficient antioxidant property which helps in minimizing the risk
associated with the ailments of oxidative stress. No studies have been done to estimate
the lycopene content from any source in Karnataka. Hence, this study was carried out
to assess the estimation of lycopene, and antioxidant and antifungal activity of
lycopene. The results point out clearly that tomato is a rich source of lycopene, and
also papaya has considerable amount of lycopene. Though lycopene content is less in
papaya, it has shown highest antioxidant activity than tomato. But however this
antioxidant property was lesser when compared to vitamin C. This calls for using few
more methods of antioxidant assay among these food sources. Tomato and papaya
extract did not show any antifungal activity against C. albicans even at a
concentration of 100g/ml. However, the antibiotics showed its effect on C. albicans
at 100g concentration. Antimicrobial effect can also be studied on other fungal
species apart from C. albicans. In future, such studies are required to assess the
lycopene content in other sources such as processed tomato products and other
lycopene rich food sources.

Acknowledgements
The authors are thankful to University of Agricultural Sciences Dharwad, Karnataka
for assisting us in the sample identification and nomenclature. We are equally grateful
to the Secretary, Mahatma Gandhi Mission (MGM) Trust, and the Director In-charge,
Institute of Biosciences & Technology, Aurangabad for providing necessary facilities
in bringing out this manuscript. Our sincere thanks to Dr A D Diwan, Former Asstt
Director General, ICAR, New Delhi for providing the necessary technical guidancefor
the manuscript.
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