Phenolic compounds and carotenoids in pumpkin fruit and related

traditional products
Gordana M. Zdunić1, Nebojša R. Menković1, Milka B. Jadranin2, Miroslav M. Novaković2,
Katarina P. Šavikin1, Jelena Č. Živković1
1
Institute for Medicinal Plants Research “Dr Josif Pančić”, Belgrade, Serbia
2
Institute for Chemistry, Metallurgy and Technology, University of Belgrade, Belgrade, Serbia

Abstract
Pumpkin fruit is used in a diet since ancient times especially in rural communities. The SCIENTIFIC PAPER
major contributory factors of nutritional and medicinal value of pumpkins are carotenoids,
polysaccharides, vitamins, minerals, and phenolic compounds. Due to a very large fruit UDC 635.621:664:54
that it is not easy to consume a whole as well as short shelf-life of fresh-cut pumpkin, dif-
ferent ways of conserving and processing are performed. In our study, total carotenoids,
total phenolics and individual phenolics in fresh pumpkin and pumpkin traditional products Hem. Ind. 70 (4) 429–433 (2016)
such as sweet in wine, jam and juice, which are typical for northern parts of Serbia, were
studied. Total carotenoids ranged from 27.6 μg/g of pumpkin sweet in wine to86.3 μg/g of doi: 10.2298/HEMIND150219049Z
fresh fruit, while the amount of total phenolics varied between 93.0 μg GAE/g of pumpkin
juice and 905.9 μg GAE/g of fresh fruit. Eight phenolic compounds were identified in the
investigated samples and among them phenolic acids dominated. Among flavonoids,
flavanon glycoside hesperidin was detected.
Keywords: Cucurbita maxima, jam, juice, sweet, phenols, carotenoids, LC/MS.
Available online at the Journal website: http://www.ache.org.rs/HI/

Pumpkin (Cucurbita pepo, C. moschata, C. maxima who showed increased levels of serum insulin, reduced
and C. mixta) is one of the vegetables used in healthy blood glucose levels and improvement of glucose tol-
diets as well as in traditional medicine in many erance in mice by pumpkin polysaccharides. Also, hypo-
countries. Since ancient times, it has been essential in cholesterolemic, antibacterial, antiinflammatory and
the diet of rural communities. Nowadays, it is culti- antitumor activities were reported [11].
vated both, for fruit and seeds that are used in a variety One of the problems with consuming pumpkin is
of ways. Fruit is regarded by consumers due to its that the fruits are big and it is not easy to consume a
sweet and mild taste as well as high nutritive value [1]. whole pumpkin in a day even by a whole family. On the
Also, pumpkin flesh is widely used as a component in a other hand, shelf-life of fresh-cut pumpkin is very
variety of products for children and adults [2]. Pumpkin short, and it tends to deteriorate during storage. So,
seeds have also high nutritional and medicinal value. different ways of conserving and processing of pump-
Cold pressed pumpkin seed oils are rich source of kin are reported among them processing into jams,
phytosterols, tocopherols and squalene [3]. puree, juice pickles and dried products and it is also
The major contributory factors of nutritional and used as a base for soups and desserts [12–14]. There
medicinal value of pumpkin fruit are high total content are a few works about engineering of the pumpkin pro-
of carotenoids with >80% of β-carotene [4,5] as well as cessing [15,16], but the results on the effects of pro-
presence of pectin and non-pectin polysaccharides, cessing on pumpkin phenolic compounds are limited
minerals (potassium, phosphorus, magnesium, iron, [17].
and selenium), vitamins (C, E, K, thiamine (B1) and ribo- The aim of this study was the analysis of total caro-
flavin (B2), piridoxine (B6)), dietary fiber, phenolic com- tenoids, total phenolics and individual phenolic com-
pounds (flavonoids, phenolic acids) and other sub- pounds in traditional products of pumpkin such as
stances beneficial to human health [1,6–8]. sweet in wine, jam and juice, which are typical for
Due to such diverse chemical composition, a lot of northern parts of Serbia, together with analyses in
biological activities are attributed to pumpkin. Jin et al. fresh pumpkin fruit. LC/MS analysis was used for qual-
[9] showed that a pumpkin-rich diet could reduce blood itative analysis of individual phenolic compounds.
glucose. Similar result was obtained by Zhao et al. [10]
EXPERIMENTAL
Correspondence: G. Zdunić, Institute for Medicinal Plants Research
“Dr Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia. Material
E-mail: [email protected]
Traditional pumpkin products (sweet in wine, jam
Paper received: 19 February, 2015
Paper accepted: 1 June, 2015 and juice) were obtained from household, family Stoja-

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novic, Vojvodina, Serbia. These products are unique in and analyzed by LC/MS techniques. Apparatus: liquid
wider area and they are prepared according to the old, chromatograph Agilent 1200 series, Agilent Technol-
traditional recipes and technology that has been ogies, with a degasser, a binary pump, an autosampler,
handed down in the family for generations. Local vari- a thermostated column compartment and a DAD det-
ety of pumpkin (Cucurbita maxima L., Cucurbitaceae), ector, coupled with a 6210 time-of-flight LC/MS system
which is grown in the household was used for products (Agilent Technologies); column: Zorbax Eclipse Plus RR
preparation. The pumpkins were washed with potable C-18, 150 mm×4.6 mm id., (1.8 μm); mobile phase: A −
water, peeled and cut into small pieces. Prior further 0.5% formic acid in water and B − acetonitrile; com-
processing, average sample of fresh pumpkin fruit was bination of gradient and isocratic modes of elution:
used for chemical analysis. After processing, products 0−20 min, 5−16% B, 20−28 min, 16−40% B, 28−40 min,
were stored in cold, dry and dark place. Five samples of 40−90% B, 40−45 min, 90% B, 45−46 min, 90−5% B,
each type of traditional pumpkin products were ran- 46−51 min, 5% B; flow rate: 0.95 mL/min. The injection
domly chosen, mixed and average sample was used volume was 5 μL, and the column temperature 40 °C.
further for analysis. DAD-MSD conditions: spectral data for all the signals
were accumulated in the wavelength range of 190–450
Total phenolics
nm, and chromatograms were recorded at wavelengths
Samples of fresh pumpkin fruit and sweet in wine of 230, 260, 280 and 340 nm. The total ion chromato-
were grained prior extraction and homogenized. A grams were recorded using the following parameters:
mass of 10 g of each sample was extracted with 20 mL ion source (ESI) polarity, negative; capillary voltage,
of solvent mixture H2O/MeOH (1:1) for 30 min using 4000 V; gas temperature, 350 °C; drying gas, 12 L/min;
the ultrasonic bath. From those extracts 3 dilutions nebulizer pressure, 45 psig (310.26 Pa); fragmentor vol-
were made (depends of the sample, experimentally tage, 140 V, mass range 100–1500 m/z. A personal
determined to be in a linear range A = f(c)) and used for computer system running Mass Hunter Workstation
obtaining total phenolics). Total phenolic content was software was used for data acquisition and processing.
determined using the method of Folin–Ciocalteu Identification: compounds were identified by com-
reagent (FCR) with slight modifications [18]. An aliquot paring retention times (tR), UV and MS spectra with
(200 μL) of each extract was mixed with 1000 μL of those of the reference standards (protocatechuic acid,
1:10 dilution of Folin-Ciocalteu reagent. After 6 min 800 p-hydroxybenzoic acid, vanillin, p-coumaric acid and
μL of sodium carbonate (75 g/L) was added. After 2 h of hesperidin), or on the basis of the exact mass measure-
incubation in the dark at room temperature, absorb- ment and literature data (salicylic acid, eriodictyol
ance was measured at 740 nm. Gallic acid (0–100 mg/L) 7-neohesperidoside, and abscisic acid).
was used for the preparation of the calibration curve.
Results are expressed as μg of gallic acid equivalents RESULTS AND DISCUSSION
(GAE) per g of fresh fruit or product, or per mL of juice.
Analyses were performed in triplicates and the results Total carotenoids
are expressed as mean ± standard deviation. Cucurbita species are known as a good source of
Total carotenoids carotenoids [4]. Total carotenoid content was analyzed
in traditional products typically produced in north part
Samples of fresh pumpkin fruit and sweet in wine
of Serbia (Table 1). Fresh pumpkin fruit contained the
were grained prior extraction and homogenized. For all
highest amount of total carotenoids (86.3 μg/g FW),
samples 1 g was extracted with 50 mL of methanol
thus presenting a valuable source of this bioactive com-
which was the solvent that gave the best results for the
pounds. Murkovic et al. [20] investigated a carotenoid
extraction of carotenoids [19]. Total carotenoids were
content in different varieties of three Cucurbita species
determined using earlier developed method and were
grown in Austria (C. pepo, C. maxima and C. moschata)
calculated and expressed in μg/g of the sample. Anal-
yses were performed in triplicates and the results are
expressed as mean ± standard deviation. Table 1. Total carotenoids and total phenolics in fresh pump-
kin fruit and pumpkin traditional products; results are expres-
LC/MS analysis sed on fresh mass basis as mean ± standard deviation of
triplicate analysis
Sample preparation for analysis: Each sample (100
g) was homogenized, diluted with water (100 mL), and Total carotenoids Total phenolics
Sample
then this solution was extracted three times with ethyl μg/g μg GAE/g
acetate (100 mL). The organic phases were combined Pumpkin fruit 86.3±1.9 905.9±13.9
and evaporated to dryness in a rotary vacuum evapor- Pumpkin sweet in wine 27.6±0.8 227.2±8.0
ator. Obtained ethyl acetate extracts (10 mg) were Pumpkin jam 63.9±1.6 769.1±14.1
dissolved in 1 mL of methanol, filtered (0.45 μm filter) Pumpkin juice 28.6±1.1 93.0±6.0

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and it was similar as in our sample. On the other hand, registered reductions were probably due to the ther-
Provesi et al. [21] reported lower amounts of caro- mal processing. Pumpkin jam was the richest product in
tenoids in C. moschata and C. maxima from Brasil (cca. total phenols, pointed that this type of processing pro-
40 and 30 μg/g, respectively). Total carotenoids con- vides a product of high value. The lowest amount of
tent in pumpkin is known to be strongly influenced by total phenolics was detected in juice and it was almost
the cultivar type as well as by the conditions of culti- 2.5 times lower than in jam. Content of total phenols is
vation [22]. important parameter because it is usually correlated
Processing affected the amount of total carot- with antioxidant activity.
enoids, but the smallest decrease was noticed in jam in
LC/MS analysis
which about 70% of carotenoids content was pre-
served. Also, Provesi et al. [21] showed that decrease Eight compounds were identified, and among them
of carotenoids in pumpkin puree (similar product to phenolic acids dominated (Table 2). Protocatechuic and

Table 2. Compounds identified in pumpkin fruit and pumpkin traditional products by LC/MS

tR in LC/ DAD, min tR in ESI ToF, min Molecular formula Exact mass Compound
6.20 6.31 C7H6O4 154.0266 Protocatechuic acid
6.42 6.58 C16H18O9 354.0951 Chlorogenic acid
8.79 8.92 C7H6O3 138.0317 Salicylic acid
9.21 9.36 C7H6O3 138.0317 p-Hydroxibenzoic acid
a
16.58 16.75 C8H8O3 152.0473 Vanillin
17.21 17.46 C9H8O3 164.0473 p-Coumaric acid
22.19 22.47 C27H32O15 596.1741 Eriodictyol-7-neohesperidoside
25.34 25.64 C28H34O15 610.1898 Hesperidin
a
Detected only in pumpkin jam

jam) was less than 50%. On the other hand, decrease of chlorogenic acid are polyphenols which are widely dis-
total carotenoids in pumpkin sweet in wine as well as in tributed in variety of plant species known as the
pumpkin juice was more pronounced (for almost 70%). sources of potent antioxidants such as green tea. Also,
Total carotenoids content in pumpkin products are Dragovic-Uzelac et al. [26] reported the presence of
strongly influenced by the technological processes chlorogenic acid in C. pepo, C. maxima and C. mos-
applied, specifically thermal processes [11,14]. The chata. Among flavonoids, hesperidin, glycoside of hes-
highest retention of carotenoids was obtained when peretin, characteristic for citrus fruits was detected.
vegetables were cooked almost without water and the Hesperidin shows different biological activities such as
lowest retention of carotenoids was associated with antioxidant, anti-inflammatory and anticancer activities
the use of a large amount of water during cooking [23]. [27,28]. Eriodictyol-7-neohesperidoside is also found in
Moreover, Provesi and Amante [14] stated that pro- citrus fruits. It is strong antioxidant since it has catechol
cessing of pumpkin may cause oxidation and/or iso- moiety in B ring and 5-OH group in A ring (capable for
merization of carotenoids, which affect biological acti- chelating metal cations with carbonyl group in position
vity and colour. The most important factors that lead to 3). Vanillin was noticed only in the pumpkin jam pro-
this loss are temperature and contact with oxygen and bably due to its addition according to the recipe.
light.
CONCLUSION
Total phenolics
Also, total phenolic content was determined in Consumption of fruits and vegetables has been inc-
fresh pumpkin fruit as well as in pumpkin products. As reased rapidly due to awareness regarding their health
it was previously shown for total carotenoids, pumpkin benefits for humans. Carotenoids and phenolics are
fruit was the most abundant in phenolics (Table 1). among the phytochemicals, believed to reduce the risk
Obtained result was in accordance with literature data of developing degenerative and chronic diseases. The
as Azizah et al. [24] detected similar amounts of total results of our study indicated that tested pumpkin fruit
phenolics (90 mg GAE/100 g). However, Nawirska- as well as products made from pumpkin fruit, i.e., jam,
-Olsza et al. [25] detected lower amounts of total phe- sweets in wine, and juice, are a valuable source of caro-
nolics in pumpkin (24 mg/100 g FW). As it could be tenoids and phenolics. Although total carotenoids and
expected, in pumpkin products lower but still signific- total phenolic content in pumpkin products are
ant amounts of phenolics were determined and the strongly influenced by the applied technological pro-

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cesses, specifically thermal processes, about 70% of [11] C. Fu, H. Shi, Q. Li, A review on pharmacological
carotenoids and 85% of total phenolics were preserved activities and utilization technologies of pumpkin, Plant
in pumpkin jam compared to fresh pumpkin fruit. Thus, Foods Hum. Nut. 61 (2006) 73−80.
obtained results pointed out possibilities for better util- [12] J.G. Provesi, C.O. Dias, R.D. de Mello Castanho Amboni,
ization of such a nutritionally rich underutilized pump- E.R. Amante, Characterisation and stability of quality
indices on storage of pumpkin (Cucurbita moschata and
kin products especially in the periods with the lack of
Cucurbita maxima) purees, Int. J. Food Sci. Technol. 47
fresh fruits.
(2012) 67–74.
Acknowledgements [13] M.T.M. Assous, E.M. Soheir Saad, A.S. Dyab, Enhance-
ment of quality attributes of canned pumpkin and pine-
The authors acknowledge their gratitude to the apple, Ann. Agric. Sci. 59 (2014) 9–15.
Ministry of Education, Science and Technological Devel- [14] J.G. Provesi, E.R. Amante, Carotenoids and Impact of
opment of Serbia for financial support, project number Processing Treatments and Storage, in: V. Preedy (Ed.),
46013. Processing and Impact on Active Components in Food,
Academic press, London, 2015, pp. 71–80.
REFERENCES [15] H.S. Phanindra-Kumar, K. Radhakrishna, P.K. Nagaraju,
D. Vijaya-Rao, Effect of combination drying on the
[1] S. Sharma, T.V.R. Rao, Nutritional quality characteristics physico-chemical characteristics of carrot and pumpkin,
of pumpkin fruit as revealed by its biochemical analysis, J. Food Proc. Preserv. 25 (2001) 447–460.
Int. Food Res. J. 20 (2013) 2309–2316. [16] C.C. Garcia, M.A. Mauro, M. Kimura, Kinetics of osmotic
[2] T. Rakcejeva, R. Galoburda, L. Cude, E. Strautniece, Use dehydration and air-drying of pumpkins (Cucurbita mos-
of dried pumpkins in wheat bread production, Proc. chata), J. Food Eng. 82 (2007) 284–291.
Food Sci. 1 (2011) 441–447. [17] R. Contador, F. González-Cebrino, J. García-Parra, M.L.
[3] B.B. Rabrenovic, E.B. Dimic, M.M. Novakovic, V.V. Tese- R. Ramírez, Effect of hydrostatic high pressure and ther-
vic, Z.N. Basic, The most important bioactive compo- mal treatments on two types of pumpkin purée and
nents of cold pressed oil from different pumpkin (Cucur- changes during refrigerated storage, J. Food Proc. Pre-
bita pepo L.) seeds, LWT - Food Sci Tech. 55 (2014) 521– serv. 38 (2014) 704–712.
–527. [18] P. Waterman, S. Mole, Analysis of Phenolic Plant Meta-
[4] C.H. Azevedo-Meleiro, D.B. Rodriguez-Amaya, Qualita- bolites, Blackwell Scientific Publication, Oxford, 1994.
tive and quantitative differences in carotenoid compo- [19] S. Dere, T. Gunes, R. Sivaci, Spectrophotometric deter-
sition among Cucurbita moschata, Cucurbita maxima, mination of chlorophyll – A, B and total carotenoid con-
and Cucurbita pepo, J. Agric. Food Chem. 55 (2007) tents of some algae species using different solvents, Tr.
4027–4033. J. Bot. 22 (1998) 13–17.
[5] C. Kurz, R. Carle, A. Schieber, HPLC-DAD-MSn character- [20] M. Murkovic, U. Mülleder, Carotenoid content in differ-
isation of carotenoids from apricots and pumpkins for ent varieties of pumpkin, J. Food Compos. Anal. 15
the evaluation of fruit product authenticity, Food Chem. (2002) 633–638.
110 (2008) 522–530. [21] J.G. Provesi, C.O. Dias, E.R. Amante, Changes in caro-
[6] A. Nawirska-Olszanska, A. Biesiada, A. Sokol-Letowska, tenoids during processing and storage of pumpkin
A. Z. Kucharska, Characteristics of organic acids in the puree, Food Chem. 128 (2011) 195–202.
fruit of different pumpkin species, Food Chem. 148 [22] F.M. Oloyede, G.O. Agbaje, E.M. Obuotor, I.O. Obisesan,
(2014) 415–419. Nutritional and antioxidant profiles of pumpkin (Cucur-
[7] C.L. Zhou, W. Liu, J. Zhao, C. Yuan, Y. Song, D. Chen, Y.Y. bita pepo Linn.) immature and mature fruits as influ-
Ni, Q. H. Li, The effect of high hydrostatic pressure on enced by NPK fertilizer, Food Chem. 135 (2012) 460–
the microbiological quality and physical–chemical char- –463.
acteristics of pumpkin (Cucurbita maxima Duch.) during [23] E. Lešková, J. Kubíková, E. Kováčiková, M. Košická, J.
refrigerated storage, Innov. Food Sci. Emerg. Technol. Porubská, K. Holčíková, Vitamin losses: Retention during
21 (2014) 24–34. heat treatment and continual changes expressed by
[8] M.Y. Kim, E.J. Kim, Y.N. Kim, C. Choi, B.H. Lee, Com- mathematical models, J. Food Compos. Anal. 19 (2006)
parison of the chemical compositions and nutritive 252–276.
values of various pumpkin (Cucurbitaceae) species and [24] A. Azizah, K.C. Wee, O. Azizah, M. Azizah, Effect of
parts, Nutr. Res. Pract. 6 (2012) 21–27. boiling and stir frying on total phenolics, carotenoids
[9] H. Jin, Y.J. Zhang, J.X. Jiang, L.Y. Zhu, P. Chen, J. Li, H.Y. and radical scavenging activity of pumpkin (Cucurbita
Yao, Studies on the extraction of pumpkin components moschato), Int. Food. Res. J. 16 (2009) 45–51.
and their biological effects on blood glucose of diabetic [25] A. Nawirska-Olszanska, A. Biesiada, A. Sokol-Letowska,
mice, J. Food Drug Anal. 21 (2013) 184–189. A.Z. Kucharska, Content of bioactive compounds and
[10] X.H. Zhaoa, L. Qianb, D.L. Yina, Y. Zhoua, Hypolipidemic antioxidant capacity of pumpkin puree enriched with
effect of the polysaccharides extracted from pumpkin japanese quince, cornelian cherry, strawberry and
by cellulase-assisted method on mice, Int. J. Biol. Macro- apples, Acta Sci. Pol. Technol. Aliment. 10 (2011) 51–60.
mol. 64 (2014) 137–138.

432
G.M. ZDUNIĆ et al.: PHENOLIC COMPOUNDS AND CAROTENOIDS IN PUMPKIN FRUIT Hem. ind. 70 (4) 429–433 (2016)

[26] V. Dragovic-Uzelac, K. Delonga, B. Levaj, S. Djakovic, J. [27] J.A. Manthey, K. Grohmann, N. Guthrie, Biological
Pospisil, Phenolic profiles of raw apricots, pumpkins, properties of citrus flavonoids pertaining to cancer and
and their purees in the evaluation of apricot nectar and inflammation, Curr. Med. Chem. 8 (2001) 135–153.
jam authenticity, J. Agric. Food Chem. 53 (2005) 4836– [28] P.K. Wilmsen, D.S. Spada, M. Salvadar, Antioxidant
–4842. activity of the flavonoid hesperidin in chemical and bio-
logical systems, J. Agric. Food. Chem. 53 (2005) 4757–
–4761.

ИЗВОД

ФЕНОЛНЕ КОМПОНЕНТЕ И КАРОТЕНОИДИ У ПЛОДУ ТИКВЕ И ТРАДИЦИОНАЛНИМ ПРОИЗВОДИМА ОД ТИКВЕ

Гордана М. Здунић1, Небојша Р. Менковић1, Милка Б. Јадранин2, Мирослав М. Новаковић2,
Катарина П. Шавикин1, Јелена Ч. Живковић1
1
Институт за проучавање лековитог биља „Др Јосиф Панчић”,Тадеуша Кошћушка 1, 11000 Београд
2
Институт за хемију, технологиј и металургију, Универзитет у Београду, Његошева 12, 11000 Београд
(Научни рад)
Тиква се користи у исхрани од давнина, посебно у руралним срединама. Кључне речи: Cucurbita maxima • Џем •
Главни фактори који доприносе нутритивним и здравственим вредностима Сок • Слатко • Феноли • Каротеноиди
тикве су каротеноиди, полисахариди, витамини, минерали, и фенолна једи- • LC/MS
њења. Због веома великих плодова као и кратког рока трајања свеже сечених
кришки тикве, користе се различити начини чувања и прераде. У нашим
истраживањима испитивали смо садржај укупних каротеноида и укупних
фенола и присуство појединачних фенолних једињења у свежем плоду тикве,
као и у традиционалним производима од тикве који су типични за северне
делове Србије, као што су џем, слатко у вину и сок. Садржај укупних каро-
теноида се кретао од 27,6 μg/g слатка од тикве у вину до 86,3 μg/g свежег
плода тикве, док сe садржај укупних фенола кретао између 93,0 μg GAE/g
сока тикве до 905,9 μg GAE/g свежег плода тикве. Што се тиче појединачних
фенолних једињења у испитиваним узорцима, коришћењем LC/MS технике,
идентификовано је осам једињења, међу којима доминирају фенолне кисе-
лине. Од флавоноида, детектован је флаванонски гликозид хесперидин.

433