Carotenoids in yellow- and red-fleshed papaya (Carica papaya L)

Abstract

Vitamin A deficiency is a disorder of public health importance in Sri Lanka. A recent national survey revealed that 36% of preschool children in Sri Lanka have vitamin A deficiency (serum retinol <0.2 µg ml−1). In view of its well-established association with child morbidity and mortality, this is a reason for concern. One of the main fruits which has been recommended for prevention of vitamin A deficiency in Sri Lanka is papaya (Carica papaya L). In this study the carotenoid profiles of yellow- and red-fleshed papaya were analysed by medium-pressure liquid chromatography (MPLC) and UV-vis spectrophotometry. A section of yellow-fleshed papaya showed small carotenoid globules dispersed all over the cell, whereas in red-fleshed papaya the carotenoids were accumulated in one large globule. The major carotenoids of yellow-fleshed papaya were the provitamin A carotenoids β-carotene (1.4 ± 0.4 µg g−1 dry weight (DW)) and β-cryptoxanthin (15.4 ± 3.3 µg g−1 DW) and the non-provitamin A carotenoid ζ-carotene (15.1 ± 3.4 µg g−1 DW), corresponding theoretically to 1516 ± 342 µg kg−1 DW mean retinol equivalent (RE). Red-fleshed papaya contained the provitamin A carotenoids β-carotene (7.0 ± 0.7 µg g−1 DW), β-cryptoxanthin (16.9 ± 2.9 µg g−1 DW) and β-carotene-5,6-epoxide (2.9 ± 0.6 µg g−1 DW), and the non-provitamin A carotenoids lycopene (11.5 ± 1.8 µg g−1 DW) and ζ-carotene (9.9 ± 1.1 µg g−1 DW), corresponding theoretically to 2815 ± 305 µg kg−1 DW mean RE. Thus the carotenoid profile and organisation of carotenoids in the cell differ in the two varieties of papaya. This study demonstrates that carotenoids can be successfully separated, identified and quantified using the novel technique of MPLC. Copyright © 2003 Society of Chemical Industry

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Journal of the Science of Food and Agriculture J Sci Food Agric 83:1279–1282 (online: 2003)
DOI: 10.1002/jsfa.1533
Carotenoids in yellow- and red-fleshed papaya
(Carica papaya L)
U Gamage Chandrika,1∗Errol R Jansz,1SMD Nalinie Wickramasinghe1and
Narada D Warnasuriya2
1Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka
2Department of Paediatrics, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka
Abstract: Vitamin A deficiency is a disorder of public health importance in Sri Lanka. A recent
national survey revealed that 36% of preschool children in Sri Lanka have vitamin A deficiency
(serum retinol <0.2µgml
−1). In view of its well-established association with child morbidity and
mortality, this is a reason for concern. One of the main fruits which has been recommended for
prevention of vitamin A deficiency in Sri Lanka is papaya (Carica papaya L). In this study the
carotenoid profiles of yellow- and red-fleshed papaya were analysed by medium-pressure liquid
chromatography (MPLC) and UV-vis spectrophotometry. A section of yellow-fleshed papaya showed
small carotenoid globules dispersed all over the cell, whereas in red-fleshed papaya the carotenoids were
accumulated in one large globule. The major carotenoids of yellow-fleshed papaya were the provitamin
A carotenoids β-carotene (1.4±0.4µgg
−1dry weight (DW)) and β-cryptoxanthin (15.4±3.3µgg
−1DW)
and the non-provitamin A carotenoid ζ-carotene (15.1±3.4µgg
−1DW), corresponding theoretically to
1516 ±342 µgkg
−1DW mean retinol equivalent (RE). Red-fleshed papaya contained the provitamin A
carotenoids β-carotene (7.0±0.7µgg
−1DW), β-cryptoxanthin (16.9±2.9µgg
−1DW) and β-carotene-5,6-
epoxide (2.9±0.6µgg
−1DW), and the non-provitamin A carotenoids lycopene (11.5±1.8µgg
−1DW) and
ζ-carotene (9.9±1.1µgg
−1DW), corresponding theoretically to 2815 ±305 µgkg
−1DW mean RE. Thus the
carotenoid profile and organisation of carotenoids in the cell differ in the two varieties of papaya. This
study demonstrates that carotenoids can be successfully separated, identified and quantified using the
novel technique of MPLC.
2003 Society of Chemical Industry
Keywords: Carica papaya; carotenoids; structure; dispersion
INTRODUCTION
Vitamin A deficiency is a disorder of public health
importance in Sri Lanka. A recent national sur-
vey revealed that 36% of preschool children in Sri
Lanka have vitamin A deficiency (serum retinol
<0.2µgml
−1). In view of its well-established asso-
ciation with child morbidity and mortality, this is a
reason for concern.
Vitamin A is available from animal sources in the
form of retinol, retinal, retinoic acid or esters, and
from plant sources, particularly fruits and vegetables,
in the form of provitamin A carotenoids. There
are approximately 50 known active provitamin A
carotenoids, of which β-carotene makes the largest
contribution to vitamin A activity in plant foods.
Recent findings suggest that the bioavailability of
carotenoids in fruits and vegetables may be much lower
than previously estimated.1,2Research is currently
under way to revise the previously established
conversion factors.
In addition to this traditional role, carotenoids with
or without vitamin A activity are known to be involved
in immunoenhancement,3treatment and prevention
of cancer4and antioxidant capacity.5
According to a survey carried out by the Medical
Research Institute,6although mothers’ awareness and
children’s consumption of vitamin A-rich foods are
good, vitamin A deficiency persists. This implies one
or more of several possibilities.
(a) The wrong impression is held that yellow fruits
(Carica papaya species, etc) contain provitamin
A (the colour may be due to non-provitamin
A carotenoids or compounds of other chemi-
cal origin).
(b) Food preparation can affect biological activ-
ity and/or bioavailability. Recent studies have
indicated that several factors (eg heat) affect
the biological activity and bioavailability of
carotenoids.7
∗Correspondence to: U Gamage Chandrika, Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura,
Gangodawila, Nugegoda, Sri Lanka
E-mail: chandri@sjp.ac.lk
Contract/grant sponsor: IPICS; contract/grant number: SRI 07
Contract/grant sponsor: University of Sri Jayewardenepura; contract/grant number: ASP/6/PR/2000/13
(Received 12 March 2002; accepted 12 February 2003)
2003 Society of Chemical Industry. J Sci Food Agric 0022–5142/2003/$30.00 1279

UG Chandrika et al
(c) Some factors (eg lycopene) can inhibit β-carotene
15, 15-dioxygenase enzyme which is responsible
for the conversion of β-carotene to vitamin A.
These factors can be present in the food or food
preparation materials (eg tomatoes contain large
amounts of lycopene).
The main strategy for prevention of vitamin A
deficiency in Sri Lanka has been the promotion of
general consumption of provitamin A, especially as
carotenoids from plant sources. Papaya is one of the
main fruits recommended for vitamin A deficiency in
Sri Lanka. There are different varieties (eg red- and
yellow-fleshed) of C papaya. The carotenoid compo-
sitions of red- and yellow-fleshed papaya fruits found
in Japan have been studied by Yamamoto8using
traditional separation techniques (thin layer chro-
matography) and common identification methodology
(chemical reactions). Papaya fruit carotenoid compo-
sition has also been studied using high-performance
liquid chromatography (HPLC)9–12 andopencolumn
chromatography.13 To our knowledge, there has not
been a single study on the carotenoid composition of
C papaya found in Sri Lanka.
This may be due the fact that the capital and
maintenance costs of HPLC equipment are high and
often beyond the budgets of laboratories in developing
countries such as Sri Lanka. Furthermore, many
carotenoids cannot be identified owing to the lack of
reference standards. This study was undertaken with a
view to identifying and quantifying provitamin A and
non-provitamin A carotenoids of two main varieties
(red- and yellow-fleshed) of C papaya grown in Sri
Lanka, using medium-pressure liquid chromatography
(MPLC). The MPLC technique was selected because
it is a closed column method and can minimise
oxidation of carotenoids.
MATERIALS AND METHODS
Yellow- and red-fleshed papaya (Carica papaya
L) fruits were bought from a local market. The
yellow-fleshed fruits selected were of the same
skin colour, shape (globular) and ripeness level
(pH 4.5–5.0). Likewise, the red-fleshed fruits
selected for this study were of the same skin
colour, shape (elongated) and ripeness level (pH
4.5–5.0).
Chemicals
β-Carotene, lycopene and apo-8-carotenal were
obtained from Sigma Chemical Co (St Louis, MO,
USA). All other chemicals used were of analyti-
cal grade.
Sample preparation
A freeze-dried, homogeneous representative sample of
fruit pulp (5 g) was ground with methanol (3 ×50 ml)
using a mortar and pestle. The three extracts were
combined, filtered and portions (30 ml of each at a
time) were added to hexane (50 ml) in a separat-
ing funnel, mixed well and allowed to separate. The
lower aqueous layer was re-extracted into another
50 ml of hexane and this was repeated until the entire
colour was transferred to hexane. The combined
hexane extracts (150 ml) were saponified for 16 h
(dark, room temperature) by adding 0.15 g of buty-
lated hydroxytoluene (10 g l−1in hexane) and 150 ml
of potassium hydroxide (100 g l−1in methanol), then
concentrated to 2 ml in a rotary evaporator (30 ◦C)
andusedforMPLC.
Medium-pressure liquid chromatography
The MPLC set-up consisted of a solvent pump (FMI
model QD-O-SSY lab pump, Fluid Metering Inc,
Oyster Bay, NY, USA; J.125 inch piston diameter,
pressure up to 100, flow rate range 0–100 ml min−1)
and a SEPARO column (10 cm ×1.5 cm; Baeck-
strom SEPARO AB, Lindigo, Sweden). Teflon tubes
between the pump and the column were intersected
with Luerlock (Baeckstrom, SEPARO AB, Lindigo,
Sweden) connectors to make sample injection possi-
ble with a Luerlock syringe (a constant-volume mixing
chamber combined with solvent reservoirs to create
a continuous gradients). The column was dry packed
with Merck silica gel 60A (Kebo Lab, Uppsala, Swe-
den) of particle size 40– 63 µm and compressed by
axial compression (pressure 8 bar) in a quick-grip
carpenter’s vice.
The carotenoid sample (1 ml) in hexane was injected
at a rate of 15 ml min−1into the MPLC silica gel
column equilibrated with hexane. The fractions were
eluted successively with 100 ml portions of 0:100,
3.125:96.875, 6.25:93.75, 12.5:87.5, 25:75 and 50:50
CH2Cl2/hexane. Separation of the carotenoids was
monitored visually and each separated fraction was
collected as it left the column. Components seperate
from the extracts were concentrated to dryness using
nitrogen gas and dissolved in light petroleum, ethanol
and chloroform. The visible spectra of carotenoid
bands were recorded using a 1 cm cuvette from 350 to
600 nm on a Shimadzu UV-1601 spectrophotometer
(Kyoto, Japan). Purity of the bands was checked by
reverse phase HPLC (RP-HPLC).
Carotenoids were identified by comparison of
their absorption spectra in light petroleum, ethanol
and chloroform with data in the literature. Quan-
tification was accomplished using molar extinction
coefficients.14
Microscopic studies
Cells of yellow- and red-fleshed papaya were exam-
ined using an Olympus B ×50 research microscope
(Olympus Optical Co, Ltd, Tokyo, Japan) at ×40
magnification.
RESULTS
The major carotenoids found in ripe fruits of yellow-
and red-fleshed papaya and their observed λmax values
are shown in Table 1.
1280 J Sci Food Agric 83:1279–1282 (online: 2003)

Carotenoids in papaya
Table 1. Major provitamin A and non-provitamin A carotenoids in fruit pulp of yellow- and red-fleshed papaya (Carica papaya L)
Carotenoid and observed λmax values Dry weight (µgg
−1)
(nm) in light petroleum Yellow-fleshed (n=10) Red-fleshed (n=10) pvalue
Provitamin A carotenoids
β-Carotene (474.5, 448.5, 420.5) 1.4±0.47.0±0.7<0.0001
β-Cryptoxanthin (473, 448.5, 421.5) 15.4±3.316.9±2.9
β-Carotene-5,6-epoxide (423, 444.5, 474) ND 2.9±0.6
Calculated retinol equivalent (µgkg
−1DW) 1516 ±342 2815 ±305
Non-provitamin A carotenoids
Lycopene (503, 472, 446.5, 363) ND 11.5±1.8
ζ-Carotene (449, 426.5, 402.5, 381) 15.1±3.49.9±1.1 0.018
ND, not detected (detection limit 0.08 µgg
−1).
(a)
(b)
Figure 1. Microscopic view of sections of (a) yellow-fleshed and (b)
red-fleshed papaya (×40 magnification).
Carotenoids of yellow-fleshed papaya were dis-
persed all over the cell in small globules (Fig 1(a)),
whereas those of red-fleshed papaya were accumulated
in one large globule (Fig 1(b)). This difference may
have a bearing on the bioavailability of carotenoids
from the two types of papaya.
DISCUSSION
Separation and quantification of the carotenoids in
two major varieties of C papaya growninSriLanka
indicated that red- and yellow-fleshed varieties had
different carotenoid profiles. Yellow-fleshed papaya
contained three major carotenoids, ie β-carotene, β-
cryptoxanthin and ζ-carotene. In addition to these
three carotenoids, red-fleshed papaya also contained
lycopene and β-carotene-5,6,-epoxide. It is interesting
to note that the lycopene content was fairly high in
the red-fleshed variety. The β-carotene content in red-
fleshed papaya was significantly higher (p<0.0001)
than that in yellow-fleshed papaya. ζ-Carotene was
the second most abundant carotenoid in yellow-
fleshed papaya, and its content was significantly higher
(p=0.018) than in red-fleshed papaya.
Red-fleshed fruits contained a higher proportion
of provitamin A carotenoids than yellow-fleshed
fruits. Hence the calculated mean retinol equivalent
(RE) was 1516 ±342 µgkg
−1DW in yellow-fleshed
papaya, whereas in red-fleshed papaya it was 2815 ±
305 µgkg
−1DW. Studies should be carried out to
determine if the bioavailability of vitamin A is
also higher in the red-fleshed type which contains
lycopene, an inhibitor of 15,15-dioxygenase enzyme
(which is responsible for the cleavage of provitamin
A carotenoids to give vitamin A).15 Lycopene is
an antioxidant beneficial to cardiovascular ailments.5
Excessive dietary intake of papaya has been observed
on occasion to cause a ‘yellow–orange’ discoloration of
the skin of the palm among the Sri Lankan population.
This is not caused by any other yellow fruits in Sri
Lanka, eg mango, and may be due to lycopene, which
is present in high concentration in red-fleshed papaya.
The condition is known as lycopenaemia.16
The different carotenoid dispersion patterns (one
large globule in red-fleshed and many small globules
in yellow-fleshed papaya) may have a bearing on the
absorption of carotenoids in the gastrointestinal tract
and therefore on bioavailability.
J Sci Food Agric 83:1279 – 1282 (online: 2003) 1281

UG Chandrika et al
CONCLUSIONS
The carotenoid profile and organisation of carotenoids
in the cell differ in yellow- and red-fleshed varieties
of papaya. This study demonstrates that carotenoids
can be successfully separated, identified and quantified
using the novel technique of MPLC. Studies using ani-
mal models and humans will be required to determine
the nutritional significance of these differences.
ACKNOWLEDGEMENTS
Financial assistance from the IPICS (research grant
SRI 07) and the University of Sri Jayewardenepura
(research grant ASP/6/PR/2000/13) is gratefully
acknowledged.
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