Phytochemical characterization of pumpkin seed with

antiparasitic action
I.R. Maldonade1,a, G.B. Amaro2, R.F.A. Luengo1, R.L.V. Ribeiro1, M.I.O. Lozada3, L.L. Oliveira3 and
E.R. Machado4,5
1Laboratory of Food Science & Technology, Brazilian Agricultural Research Corporation (Embrapa Vegetables), Rod

BR060 km 09, CP 218, CEP 70275-970, Brası́lia, Brazil; 2Department of Breeding and Genetic Resources, Brazilian
Agricultural Research Corporation (Embrapa Vegetables), Rod BR060 km 09, CP 218, CEP 70275-970, Brası́lia,
Brazil; 3Department of Nutrition, University of Brası́lia (UnB), Asa Norte, CEP 70904-970, Brası́lia, Brazil;
4Laboratory of Medical Parasitology and Vector Biology, Faculty of Medicine, Pathology Area, University of Brası́lia

(UnB), Asa Norte, CEP 70904-970, Brası́lia, Brazil; 5Department of Biomedicine and Pharmacy, Anhanguera School
of Brası́lia, University Kroton, Taguatinga, Brazil.

Abstract
The indiscriminate use of antibiotics, vermifuges and antifungals by individuals
has been causing resistance in some parasites and microorganisms. Pumpkin seeds
have been used in traditional medicine because they have action against human
parasites. In the agroindustry, these seeds are discarded as by-products, although they
present bioactive compounds such as tocopherol, fatty acids (omega oils), carotenoids
and amino acids, which make them a promising approach to use as an alternative to
treat helminth infections, as well as to be used in human feeding to improve the
immune system. Thus, the objective of the present study was to determine the chemical
composition of Cucurbita maxima seeds and to evaluate in tests in vitro the potential of
their extract against Strongyloides venezuelensis larvae. Aqueous extracts of pumpkin
seeds were prepared, and the chemical composition of the seeds was characterized by
AOAC methods. Oil stability was measured by rancimat. Tocopherol and carotenoids
were quantified by high-performance liquid chromatography (HPLC), while
antioxidant capacity has been determined by spectrophotometer methods. The
antiparasitic action against larvae of S. venezuelensis was evaluated in vitro, where the
extract was added into assay tubes varying in volume from 0 to 400 μL, containing S.
venezuelensis larvae (1500), in which they were incubated at 25°C for 48 h. Samples
were withdrawn every 12 h, centrifuged and the larvae were suspended in aqueous
solution and counted by microscopic method. Ivermectin and water have been used as
positive and negative controls, respectively. Chemical analysis (HPLC) of the seeds
revealed a predominant presence of tocopherol. The seed extract showed antioxidant
activity, probably due to bioactive substances, mainly tocopherol and fatty acids. After
12 h, volume greater than 200 μL (33.3 mg mL-1) eliminated 92% of the larvae.
Increasing the incubation period up to 48 h, low concentrations of extract (<10 mg
mL-1) were sufficient to eliminate more than 90% of the larvae, while ivermectin killed
90% and in the water control sample, around 80% of the larvae remained alive. This is
the first study to evaluate the potential of C. maxima seed extract to eliminate S.
venezuelensis larvae in tests in vitro. Our results suggest that the low dose of pumpkin
seeds’ extract has anthelmintic properties to eliminate infective larva of Strongyloides
venezuelensis, being a promising alternative as a medicinal plant and phytotherapeutic
product for the elimination of helminths.

Keywords: parasites, Cucurbita maxima, trolox, helminth, tocopherol, rancimat

INTRODUCTION
Pumpkin is a vegetable that belongs to the family of Cucurbitaceae, Cucurbita genus,
native to the Americas. This plant is cultivated on a large scale in tropical regions mainly in

aE-mail: iriani.maldonade@embrapa.br

Acta Hortic. 1287. ISHS 2020. DOI 10.17660/ActaHortic.2020.1287.17 127

XXX IHC – Proc. Int. Symp. on Advances in Production and Processing of
Medicinal and Aromatic Plants
Eds.: B. Patil et al.
Brazil, where it is widely used in Brazilian popular medicine and composes the diet of the
population when in the mature phase (Naves et al., 2010).
Pumpkin seeds contain compounds such as lipids, proteins, phytosterol, vitamins and
minerals. In addition, they are a good source of the elements K, P, Fe and β-carotene (Seo et al.,
2005). Recently they have received attention for potential human health benefits, due to their
antimicrobial, hypoglycemic, anticancer, anti-inflammatory effects, and for their vermifuge
action that goes back many years (Matthews et al., 2016). Its antioxidant action is important
because of its usage in the food and pharmaceutical industry as well it can prevent diseases
related to aging and many pathological diseases, such as cancer, atherosclerosis, inflammation
and neurodegenerative disorders caused by oxidative stress (Yin et al., 2011). In addition to
the decrease in costs from its use as a natural antioxidant because they come from a renewable
source, they also are a by-product of agro-industry that are generally discarded. Several
studies have shown that the oil, protein and polysaccharides present in the seeds may present
antioxidant, hyperlipidemic, hyperglycemic and antibacterial activities (Wang et al., 2017;
Zhang et al., 2014). Anthelmintics and toxicological tests in rats show a good effect of
phytochemical seeds’ flour and do not induce acute toxicity and can be used with a good safety
margin in the treatment of the patients.
In regions where the population has a low income, people generally use plants, such as
pumpkin seeds, as alternative medicine. Thus, strongyloidiasis is a medical and social problem
of worldwide distribution, which in its disseminated form becomes severe, and may lead the
patient to death (Morais, 2012). Literature data show that the species Strongyloides
venezuelensis has been used to standardize new immunological and therapeutic techniques,
in order to improve the diagnosis of human strongyloidosis (Braga et al., 2011). Other studies
also showed that pumpkin seed was successfully used as an anthelmintic by many native
people (Fujimoto et al., 2012). In this way, considering the aspects presented, this research
seeks to show the efficacy of new natural drugs for the control of strongyloidiasis, considering
the ineffectiveness of many vermifuges due to the resistance developed by this nematode
(Rosa et al., 2011).
In this study the objective was to determine the bioactive compounds in the seeds and
evaluate the potential of C. maxima seed extract to eliminate S. venezuelensis larvae in tests in
vitro.

MATERIAL AND METHODS

Plant material and flour

The pumpkins (Cucurbita maxima) were cultivated in the experimental field of
production of Embrapa Vegetables, Gama-DF. The pumpkin seeds were washed and dried in a
ventilated oven at 40°C for 18 h, stored in a refrigerator at 2°C. Afterwards, the samples were
crushed in a blender, sieved until the flour is obtained.

Determination of the centesimal composition of the seeds

The centesimal composition (moisture, ash, lipid and protein) of flour sample was
determined by AOAC methods (AOAC, 2005). The carbohydrate content was determined by
difference.

Aqueous extract
Pumpkin seeds were washed and dried in an oven at 40°C for 18 h, stored in a
refrigerator at 4°C for later use. The seeds were crushed in a blender and sieved. After that,
500 g of flour was diluted in 1 L of distilled water and then the aqueous extract was filtered
and freeze-dried to obtain the extracts for further analyses. The extract samples were diluted
again with water and used in in vitro assays to evaluate the anthelmintic action and
antioxidant activities.

Oil extraction
For the extraction of the oil, the seeds were mixed with petroleum ether at 40-60°C

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using the Soxhlet apparatus for 6 h. After extraction, the oil solvent mixture was filtered
through a glass funnel containing anhydrous sodium sulfate. Then the solvent was evaporated
under reduced pressure at 60°C. The oil was stored in dark amber bottles.

Determination of carotenoids
The carotenoid content was determined according to procedures described by
Maldonade et al. (2012). Dried samples were macerated with Hyflosupercel using a mortar
and pestle then they were exhaustively extracted with acetone. The carotenoids were
transferred to petroleum ether in a separator funnel by the addition of water and washed free
of acetone. The pigment solution was dried with anhydrous sodium sulfate and the volume
was adjusted in a volumetric flask. The total carotenoid concentration was determined by
measuring the absorbance at 450 nm and using the formula provided by Rodriguez-Amaya
(1999). The absorption coefficient of β-carotene (A1%1 cm =2592) was used because it was the
major carotenoid, then results were expressed as carotenoid production (μg g-1).

Determination of tocopherol
Samples of oil extract were added into methanol and the volume was adjusted to 100
mL. Samples of 20 μL aliquots were injected into the high efficiency liquid chromatograph
(HPLC, SHIMADZU), column C18 (250×5 mm). The mobile phase used was
hexane:isopropanol (99:01 v v-1) at a flow rate of 1.3 mL min-1. Fluorescence detector was set
at 290 and 330 nm for tocopherols and quantification of the contents was determined by
constructing a standard curve.

Antioxidant activity (AA)

AA was determined using Folin-Ciocalteu reagent (Rumbaoa et al., 2009), which 0.300
g of seed sample was extracted for 1 h with 20 mL of 80% methanol at room temperature on
an orbital shaker set at 200 rpm. The mixture was centrifuged at 10,000×g for 15 min.
Supernatants were added into assay tubes and the volume adjusted to 50 mL. Then, 200 μL of
extract was mixed with 1.40 mL of water and 100 μL of Folin-Ciocalteu reagent. After at least
30 s (but not exceeding 8 min), 300 μL of 20% Na2CO3 solution was added and the mixture
was allowed to stand in room temperature for 2 h. The absorbance was measured at 765 nm
with UV-Vis Spectrophotometer (HP8453, Agilent Technologies, USA). Standard solutions of
trolox were used as standard (15 μM). The calculation of the percentage of inhibition of the
radical was carried out from the following formula:
𝐴𝐴𝐴𝐴
% inhibition of the radical = 100 (1 − )
𝐴𝐴0

where, “Ao” is the initial absorbance and “Af” is the final absorbance. The results were
expressed as μmol TEAC g-1 (Trolox equivalent antioxidant capacity).

Oxidative stability: Rancimat

The oxidative stability was determined by through the Rancimat test, according to the
EN 14112 method, using Rancimat (Metrohm®). The analyses were carried out weighing 3 g
of the oil samples, which have been added into a sealed glass vessel reaction. The equipment
was set at 100 and 110°C under a constant air flow rate of 10 L h-1 passing through the samples.
The conductivity was measured as a function of the time.

Maintenance of Strongyloides venezuelensis

For the maintenance of the Strongyloides venezuelensis L-2 lineage, male and female
Rattus novergicus Wistar rats weighing approximately 40-45 g were used. These animals were
infected subcutaneously with about 9,000 infective filarioid larvae (L3). On the seventh day
after infection, the feces were collected and the cultures were harvested in mineral coal, which
were left for three days in a greenhouse 28°C (Machado et al., 2011) to obtain the infective
larvae L3. These larvae were recovered using the parasitic method of Rugai (Rugai et al., 1954).

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After quantification of the larvae, new rats were infected with 9,000 L3. The other larvae were
used in this experiment.

Antiparasitic action against larvae of S. venezuelensis evaluated in vitro

The extracts were added into assays tubes, varying the concentrations of seeds extracts
from 0 to 400 μL, adjusting to the final volume of 600 μL with water, containing S. venezuelensis
larvae (1500). Then, the tubes were incubated at 25°C for 48 h. Samples were withdrawn
every 12 h, centrifuged and the larvae were suspended in aqueous solution and counted by a
microscopic method. Ivermectin and water have been used as positive and negative controls,
respectively.
Mortality rate was calculated as follows:
(𝑀𝑀𝑜𝑜𝑜𝑜𝑜𝑜−𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀)
% mortality = 100
(1500−𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀)

where, “Mobs” is the number of larvae’s death and “Mwater” is the number of larvae’s death
treated only with water at a period of time (h).

Statistical analysis
The analyses were performed in triplicate and the results expressed in means ± SD. The
results were submitted to analysis of variance by F-test and the means compared by Tukey
test at 5% probability.

RESULTS AND DISCUSSION

The composition of whole pumpkin seeds showed that it is rich in protein, lipid and
carbohydrate.
The centesimal composition of seeds obtained in the extracts had higher content of lipid
and protein than carbohydrate. Comparing to the values presented by Lima et al. (2011), the
lipid and protein showed higher contents. In general, seeds have high content of lipids, and
pumpkin seeds are rich in fatty acids found as oleic (47%) and linoleic (41%) and some
secondary constituents such as α-tocopherol, which are substances responsible for the
increase of nutritional quality and enhance its chemical stability due to its resistance to
oxidation, according to Arriel et al. (2007).
In relation to pumpkin seed, in this study, protein concentrations of 35.29 g 100 g-1
(Table 1) were determined, with a higher concentration of those (21.43 g 100 g-1) presented
by Sant’Anna (2005) and Pumar et al. (2008), who found in their study that the chemical
composition of the pumpkin seed meal presented protein content between 26.79 and 26.77 g
100 g-1. In relation to the use of pumpkin seed as a source of vegetable protein, it can be used
in fortification of foods like Mansour et al. (1999) and El-Soukkary (2001), who obtained 320
g 100 kg-1 of protein and 450 g 100 kg-1 of oil, increasing the protein concentration in food
preparations and reducing the costs of not being used for this purpose.

Table 1. Centesimal analysis of pumpkin seed on dry basis (100 g).

Sample Protein (%) Lipid (%) Carbohydrate (%) Moisture (%) Ash (%)
C. maxima seeds 35.29 34.65 21.11 5.46 3.80
Literaturea 26.68 31.60 32.42 3.80 5.50
aLima et al. (2011).
Chemical analysis (HPLC) of the seeds revealed a predominant presence of tocopherol
but low concentration of carotenoids (data not shown). The seed extract showed antioxidant
activity (AA), probably due to bioactive substances, mainly tocopherol and fatty acids. In Table
2, the AA was 43.50 µmoL g-1 and showed 16.43% of inhibition of free radical. In addition, the
phytosterols and carotenoids play an important role on the prevention of oxidation of oil, not
only capturing the free radical but also not allowing the formation of free radical.

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Table 2. Antioxidant activity (AA) of seed of C. maxima.
Sample ATT (µmol Trolox g-1 DW) Inhibition of free radical (%)
Seeds 43.50±0.5a 16.43±3.01a
aValues represent means ± standard deviation.
The results of oxidative stability (Figures 1 and 2) showed that the pumpkin seed oil
had as good as soya oil stability at 100°C, used as control, once the induction time determined
was 17.11 and 17.07 h, respectively. The induction period (IP) is the time that the oil is
oxidized at determined temperature. Increasing temperature the IP is reduced, significantly.
At 110°C, the induction period (IP) for pumpkin seeds was 10.94 h and for soya 7.97 h. The
presence of polyunsaturated fatty acids and tocopherol in the pumpkin seeds probably act as
antioxidant and might improve the oil quality. The more polyunsaturated bonds the oil are,
the more oxidative stability the oil has. Furthermore, the findings suggest than pumpkin oil
could be used for culinary purpose.

Figure 1. Induction time (h) of oxidative stability soya oil measured at 100°C.

Figure 2. Induction time (h) of oxidative stability of pumpkin seed oil measured at 100°C.

After 12 h, concentrations greater than 33.3 mg mL-1 eliminated 92% of the larvae
(Figure 3). Low concentrations of extract (>10 mg mL-1) were sufficient to eliminate more
than 80% of the larvae (Table 3), while ivermectin killed 90% and in the water control sample,
80% of the larvae remained alive (1385 larvae). This is the first study to evaluate the potential
of C. maxima seed extract to eliminate S. venezuelensis larvae in tests in vitro.
The pumpkin seed aqueous extract had a significantly (p<0.05) higher antihelminthic
effect at concentrations >8.33 mg mL-1 when compared with dexamethasone (15.81%) while
its vermifuge effect was satisfactory from that of ivermectin (Table 3). Ivermectin had a worm
mortality of 90.25% at 12 h and increased with time of exposure. Aziz et al. (2018) determined
that 28.6% of mortality in worms when studying anthelminthic effect of ethanolic extract of
pumpkin seed against Ascaridia galli.

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Figure 3. The effect of concentration (mg mL-1) of pumpkin seed extract on the death rate of
Strongyloides’ larvae after 12 and 48 h of incubation in vitro.

Table 3. Mortality rate (%) of S. venezuelensis larvae after 12 h of treatment with pumpkin
seeds aqueous extract.
Assays/treatment time Mortality rate (%)
Pumpkin seeds aqueous extract (>8.33 mg mL-1) 80.0
Ivermectin 90.25
Dexametasone 15.81
Pumpkin seeds can be used as herbal medicine to treat strongyloidiasis, where pumpkin
seed acts on the larvae of S. venezuelensis and causes no health damage. Moreover, it is
important to show that the search for alternatives in medicinal plants is an option, because of
the resistance of the parasite to the drugs.

CONCLUSIONS
The results indicated that pumpkin seed acts on the evolution of strongyloidiasis in the
larvae in tests in vitro, reducing the number of worms, significantly compared to other
infected and treated groups. Based on the aspects, it is understood that the role of the
biomedical professional is to investigate the parasitological and phytotherapeutic analyses, to
know the helminthiasis in question and to explore the benefits of pumpkin seed, as well as to
carry out detailed studies about the action of the aqueous extract of Cucurbita maxima seed
on the control of strongyloidiasis.
In this work, we have shown that phytotherapy is an important and viable alternative
for the treatment of strongyloidiasis, due to the high resistance developed by nematodes.
However, it is important to highlight the effect and the use of the seed, a very popular herbal
medicine, but until then little studied. Thus, it can be said that the aqueous extract of seed
Cucurbita maxima has an effect on the evolution of strongyloidiasis.

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