Research Article

Received: 3 March 2020 Revised: 21 May 2020 Accepted article published: 12 June 2020 Published online in Wiley Online Library: 22 July 2020

(wileyonlinelibrary.com) DOI 10.1002/jsfa.10566

Hypoglycemic effect and bioactive

compounds associated with the ripening
stages of the Cucurbita ficifolia Bouché fruit
Araceli Moya-Hernández,a* Elsa Bosquez-Molina,a
José Ramón Verde-Calvo,a Gerardo Blancas-Floresb and
Gloria Maribel Trejo-Aguilara

Abstract
BACKGROUND: The fruit of Cucurbita ficifolia Bouché is known in Mexico as ‘chilacayote’. The scientific interest that C. ficifolia
Bouché has acquired is due to its important hypoglycemic effect. The present research aimed (i) to discover whether this hypo-
glycemic property is present at different stages of development of this fruit, and (ii) to characterize some bioactive compounds
with antioxidant or anti-inflammatory properties. Ethylene production, respiration rate, and maturity indices were determined
during fruit development. The chemical characterization of the aqueous extracts of each stage of maturity studied was deter-
mined and their hypoglycemic effects were bioassayed using groups of normal mice with diabetes induced by streptozotocin at
a dose of 500 mg−1 kg−1 body weight.
RESULTS: Respiration rate and ethylene production showed a typical pattern for non-climacteric fruit and the quality parame-
ters did not show significant changes. Phenolic compounds such as gallic acid and chlorogenic acid were found to have the
highest concentration at 15 days of development. Extracts at 15 days showed a hypoglycemic effect that was 11% greater than
that of glibenclamide in diabetized mice.
CONCLUSION: All stages of development of C. ficifolia fruit had a hypoglycemic effect; however, the aqueous extract from the
fruit at 15 days of development showed a better effect than glibenclamide. This finding highlights the potential of this maturity
stage, and shows that it is appropriate for inclusion in treatments of type 2 diabetes mellitus. The results also indicate that phe-
nolic compounds are mainly responsible for this effect and not D-chiro-inositol as previously thought.
© 2020 Society of Chemical Industry

Keywords: hypoglycemic effect; Cucurbita ficifolia; type 2 diabetes mellitus; phenolic compounds; stages of development

INTRODUCTION ‘tzilacayutli’, which means white pumpkin.7 In the last 24 years,

Type 2 diabetes (T2D) is the main cause of mortality in Mexico.one
This disease occurs when the body does not produce enough
insulin, or the bodyʼs cells do not react to this hormone, resulting
in hyperglycemia.two Currently, the most commonly used drugs to
control this ailment are tolbutamide and glibenclamide, however
these drugs have multiple adverse effects in addition to a high
cost because they have to be administered for life.3. 4 On the other
hand, the World Health Organization (WHO) recognized the value
of the use of plants as medicinal sources in health care and has
recommended their incorporation in public health systems.5 The
benefit of many species is unknown or they have not been
exploited appropriately, hence the relevance of research to
change herbalism into a valuable medicinal resource through
the characterization and identification of beneficial active com-
pounds.6 In this wider context, the complete characterization of
the compounds involved in the hypoglycemic effect of C. ficifolia,
and their mechanism of action, remain unknown.
The fruit of Cucurbita ficifolia Bouché is known in Mexico as ‘chi-
C. ficifolia fruit has acquired an increasing scientific interest due
to its hypoglycemic effect. Since 1995 it has been reported that
the aqueous extract of this fruit has remarkable hypoglycemic
properties in comparison with 11 other species of plants, and
even better than tolbutamide or glibenclamide, which are the
common medications prescribed for treatment of T2D.8–10
This hypoglycemic effect of C. ficifolia has consistently demon-
strated both in experimental conditions in vitro as in vivo. In vivo
studies, with mice and patients, have reported that C. ficifolia
extract significantly lowers blood glucose levels.9
* Correspondence to: A Moya-Hernández, Department of Biotechnology, Univer-
sidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico. E-mail:
aramoyahdez@gmail.com
a Department of Biotechnology, Universidad Autónoma Metropolitana-
Iztapalapa, Mexico City, Mexico
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b Department of Health Sciences, Universidad Autónoma Metropolitana-

lacayote’. This word etymologically comes from Nahuatl Iztapalapa, Mexico City, Mexico

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 www.soci.org A Moya-Hernández et al.

C. ficifolia has also been reported to contain D - chiro inositol
(INN) (an isomer of inositol that has been proposed as a mediator
for insulin action) and flavonoids, suggesting that the hypoglyce-
mic effect could be attributed to DCI.eleven Other studies carried
out by the same authors indicate that the extract of C. ficifolia fruit
reduces blood glucose levels, increases plasma insulin and the
number of beta cells, suggesting the mode of action of this fruit
in the treatment. of diabetes.12
More recent research indicates that this species has antioxidant
and anti-inflammatory effects in diabetic mice in addition to its
hypoglycemic potential. It has been hypothesized that INN acts
in synergy with other compounds such as polyphenols and specif-
ically flavonoids, since the presence of compounds such as gallic
acid and catechins have also been reported.13–15 Regarding anti-
inflammatory properties, studies have indicated that C. ficifolia
extract modulates inflammatory cytokines, suggesting that the
extract may prevent obesity and the development of associated
pathologies, such as glycemia, triglycerides. and cholesterol.
These are factors that increase insulin resistance and lead to type
2 diabetes.16 Moreover, recently it was discovered that C. ficifolia
increases insulin secretion in cells ⊎ pancreatic RINm5F (a pro-
ducer cell line insulin) due to an increase in intracellular calcium
concentration of the endoplasmic reticulum. These findings par-
tially explain the hypoglycemic properties of this species.17
All the research mentioned above highlights the hypoglycemic
potential of C. ficifolia ; however, none of these reports indicates
the variety or stage of fruit maturity; At best, the fruit is only
known as 'ripe' or 'highly ripe'.
In a previous study, the authors found that C. ficifolia extracts
obtained from young fruit also exhibited hypoglycemic effects
(data not shown). Therefore, the precise characterization of the
physiological stage of fruit development was considered as rele-
vant information to determine if the findings can be attributed
to the variety, the stage of maturity or another characteristic. Con-
sequently, a comparative study of the genetic diversity of C. ficifo-
lia in some regions of Mexico was recently performed using
amplified fragment length polymorphism (AFLP).The fruit was
found to possess hypoglycemic properties regardless of the mor-
phological characteristics and the region of Mexico where it is
grown.18On the other hand, respiration rate is known to be an
excellent indicator of the metabolic activity of plant organs and
a very useful guide to the potential life of horticultural products.
Breathing rates vary by stage of development, and their measure-
ment is very important because of their association with cellular
metabolism because the energy derived from respiration drives
all other reactions within a cell. Similarly, there appears to be a
correlation between the level of ethylene produced by the fruit
and its maturation and decomposition after harvest.19–21
Therefore, this research focused on investigating whether the
hypoglycemic effects of this species depend on the stage of
development of the fruit and on characterizing the chemical com-
pounds that could be responsible for this activity.
65% RH for 15 days, with irrigation to maintain soil moisture. Ger-
mination time was approximately 6 days with a germination rate
of 90% of the seeds. During this period, exposure to sunlight was
gradually increased until the seedlings reached about 20-25 cm in
height. The seedlings were then transplanted to a plot on the uni-
versity campus (19 ° 21 0 34.8 00 N, 99 ° 4 0 27.2 00 W). During the 85
days of growth of the crop, the environmental conditions were
23.5 ± 2.2 ° C with 63.5 ± 7.5% Relative Humidity (RH), under
a light / dark photoperiod of 15/9 h, and with capillary irrigation
twice a week. After full flowering (5 days after transplanting) sam-
ples of five fruits were harvested in triplicate at 5, 10, 15, 25, 30, 40,
45, 50 and 55 days of development.
The ethylene production parameters, respiratory rate and qual-
ity were determined for each sample, as indicated in the following
sections. The biological tests and the characterization of the hypo-
glycemic compounds were carried out with the juice extracts of
each stage of maturity. These extracts were frozen and stored at
-80 ° C before analysis.
Ethylene production and respiration rate
These physiological parameters were determined with intact fruit
at each stage of development. The closed system method was
used.22, 23 Depending on the volume and weight of the fruit, sam-
ples of one piece or more were placed in different airtight glass
containers with ambient air as the initial atmosphere (Table one).
A set of three similar containers was prepared for each stage of
fruit development and held at 20 ° C for 1 hr to measure CO 2
concentration.
Carbon dioxide (mg CO 2 kg −1 h −1 ) and ethylene concentra-
tions (ng C 2 H 4 kg −1 s −1 ) extracted from the upper space of
the container were determined using a Varian Star chromato-
graph (Walnut Creek, CA, USA) with two detectors in series: a ther-
mal conductivity detector (TCD) to determine CO 2 and a flame
ionization detector (FID) for ethylene, equipped with a 15 m
megaboro column (Porapak Q, Santa Clara, CA, USA), which uses
helium as a carrier gas. The column temperature was 8 ° C, injec-
tor temperature was 120 ° C, FID detector temperature was 230 °
C, and TCD detector temperature was 210 ° C.
Maturity parameters
The shape of the fruit was controlled by measuring the equatorial
and polar dimensions and reporting them as the sphericity coeffi-
cient expressed as ε = ϕE / ϕP where "ε" is sphericity, "ϕE" equatorial
diameter and "ϕP" polar diameter. Weight (g) and fruit volumes
(cm 3 ) were determined using an Ohaus electronic scale with
Table 1 Average weight and volume of C. ficifolia fruit placed in the
different containers
Age Total
Fruit development weight Total fruit Container
quantity (days) fruit (g) volume (L) capacity (L)

3 10 298.95 0.285 1
MATERIAL AND METHODS 3 15 905.10 1.110 4
Vegetal material 3 25 4002.75 4.635 11
First, 144 selected C. ficifolia seeds, harvested in Mexico (19 ° 19 0 3 30 8088.00 90.70 20
23 00 N, 98 ° 45 0 38 00 W) were sown in a 12 × 6-well seedbed (two in 2 40 9995.70 11.940 20
each well), indentified by the Metropolitan Herbarium of the 1 45 6792.20 8.300 20
Iztapalapa Autonomous Metropolitan University with the number 1 50 7059.00 8.507 20
of voucher 84 029. The soil mixture consisted of 50% soil, 25% ver-
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1 55 7184.00 8.569 20
miculite, and 25% agrolite. The seedbed was kept at 23.1 ° C and

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Hypoglycemic effect in Cucurbita ficifolia
readability of 0.1 g (Parsippany, NY, USA) and by water displace-
ment, respectively. The firmness of the meat was measured on
the peeled opposite sides of each fruit with a penetrometer
(Effegi, Milan, Italy) supplied with an 8 mm diameter tip and
expressed in Newtons (N). Moisture and dry matter were
expressed as percentages and were determined according to
OECD guidelines.24 Total soluble solids (TSS) were measured using
an Atago refractometer (Tokyo, Japan) and expressed as percent-
ages. The pH of the juice was measured with a Hanna E1023 meter
(Mexico City, Mexico). Titratable acidity (TA) was determined as a
percentage of equivalents of malic acid.24
Hypoglycemic effect of C. ficifolia fruit
Preparation of the aqueous extract of C. ficifolia fruit
The exocarp and seeds of each fruit were removed. The mesocarp
was squeezed using a Breville JE95XL Juice Extractor (Gardena,
CA, USA). The juice obtained was vacuum filtered using Whatman
filter paper, with a pore size of 45 μm. The filtrate was poured into
glass trays forming thin layers that were dried under a laminar
flow hood for 48 h. The extract was stored in amber bottles at
25 ° C.
Experimental normoglycemic animals
Used CD-1 normal males (33-40 mice g body weight). They were
born in the UPEAL-Bioterio gene bank of the Autonomous Metro-
politan University-Xochimilco with verification number
BOO.02.03.02.01.281 / 07 Ministry of Agriculture, Livestock, Rural
Development, Fisheries and Food (SAGARPA). The mice were
maintained and cared in the Bioterio with the following condi-
tions; 12/ 12 h light/dark period, 22±1°C, RH of 55 ± 3%, basic diet
for rodents (Harlan type food) and water ad libitum.
Laboratory animals were managed according to the guidelines
of the National Institutes of Health for the care and use of labora-
tory animals.25 and official Mexican rules.26
Biological assay
It is used a single dose of 500 mg -1 kg -1 of body weight for all
experiments following the methodology described by Moya-Her-
nandez et to the. 18 Before starting the study, the mice were
fasted for 8 h, just with water ad libitum, and during the 6 h study
they remained without food but with free access to water. Eight
groups of three mice each were administered intraperitoneally
as follows: Group 1 received isotonic saline solution (ISS),
4 mL−1 kg−1 as a control; group 2 received glibenclamide
(10 mg−1 kg−1) as a positive control; the other six groups received
a single dose of 500 mg−1 kg−1 of the aqueous extract of each of
the different stages of fruit development. All treatments were
vehicled in ISS (4 mL−1 kg−1). All experiments followed the meth-
odology described by Alarcón-Aguilar et al.10
Streptozotocin-induced diabetes and treatment
Seventy-two male CD-1 mice (33-46 g) were randomly divided
into nine groups of eight animals. Diabetes was induced in the
animals in groups 1–8 with a single dose of STZ (Sigma-Aldrich,
St Louis, MO, USA) intraperitoneally (135 mg−1 kg−1 in 0.1 mol L−1
citrate buffer, pH 4.5, prepared at the time of use). Group 9 only
received the same volume of ISS.
After a week of administering the drug, five mice of each group
with glycemia higher than 200 mg−1 dL−1 were included in the
study and 24 h later the following treatments were administered:
groups 1–6 received the aqueous extracts of C. ficifolia at
500 mg−1 kg−1; group 7 received glibenclamide 10 mg−1 kg−1;
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groups 8 and 9 received ISS. All treatments were administered
intraperitoneally and were maintained under the same fasting
conditions mentioned above.
Determination of glucose
The hypoglycemic effect was determined by the glucose dehy-
drogenase method using a glucometer (Accu-Chek Performa,
Roche, Mannheim, Germany) for both tests, with normal glycemic
mice, and streptozotocin-induced diabetes. Blood samples were
obtained from the tail vein of each animal. The basal glucose
levels of each rodent were determined before administering the
treatments. Blood glucose levels were then registered at intervals
of 2 h for each animal until to reach 6 h. The effect on glycaemia
levels was calculated by subtracting the basal glycemic (t = 0)
from the glycemic values at 120, 240, and 360 min.
C. ficifolia extract chemical characterization
Quantitative analysis of the DCI content in the aqueous C.
ficifolia extract
The DCI content in the aqueous C. ficifolia extract was determined
using ultra-fast liquid chromatography (UFLC) with a refractive
index detector (Shimadzu, Tokyo, Japan) and a Spherisorb NH2
column (5 μm, 4.6 × 250 mm) (Kildare, Ireland). The mobile phase
was an isocratic mixture of acetonitrile / water (90:10), and the
flow rate was maintained at 1 mL/min for 30 min at a temperature
of 30 °C.
Aliquots of 20 μL of DCI standard (Sigma-Aldrich, St Louis, MO,
USA) and the samples were injected. The DCI was identified in
the extract by its retention time (13.8 min) and was corroborated
by the aggregated standard.
Analysis of other chemical compounds content
The other chemical compounds were characterized using high-
pressure liquid chromatography (HPLC). The equipment con-
sisted of Agilent 1100 Series separation module (New York, NY,
USA), equipped with a diode array detector. A Zorbax C18 column
(Darmstadt, Germany) of 250 mm × 4.6 mm diameter and a parti-
cle size of 5 μm was used. The mobile phases were (A) water /
acetic acid (CH3CO2H) 96:4 and (B) acetonitrile (CH3CN) 100%.
The elution system had the following gradient: 0 min 5% B,
10 min 25 %B, 30 min 35% B, 40 min 40% B and 45 min 10% B
with a flow of 0.5 mL min−1, in a total of 35 min. For each sample,
20 mg of the extract of C. ficifolia was weighed and dissolved in
5 mL of CH3OH. Catechin, coumaric acid, ferulic acid, quercetin,
gallic acid, coffee acid, epicatechin, syringic acid, sinapic acid,
chlorogenic acid, kaempferol, myricetin, isoramnetin, taxifolin,
and vitexin were obtained from Sigma-Aldrich and were com-
pared with the aqueous extract under these chromatographic
conditions. Aliquots of 20 μL of the standards and the samples
were injected.
Data analysis
For physiological parameters, only the mean and the standard
deviation were calculated for each stage of development, consid-
ering three replicates.
Statgraphics software, version 5.1 (Manugistic Inc., Rockville,
MD, USA) was used to perform an ANOVA with a significance level
of P ≤ 0.05, and a complementary Tukey–Kramer test to deter-
mine the significant differences among the means of each of
the quality parameters. In this case, each fruit was considered an
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experimental unit.
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The blood glucose data were corrected by subtracting the initial
level (basal glycemia) from the glycemic values obtained in the
three times analyzed (120, 240 and 360 minutes). That was real-
ized for each mice to eliminate variations between individuals
and to reflect the actual effect of every treatment. The response
variable was defined as ΔG = glucose in blood at ti – glucose in
blood at t0, where ti is any of the time levels of the study and t0
is initial time of the experiment.27
The same statistic program was used to perform an ANOVA with
a significance level of P < 0.05 and a complementary Tukey–Kra-
mer test to determine the significant differences among the mean
ΔG concentrations of all the treated groups. One ANOVA was per-
formed for each of the experimental times after consumption of
the extracts provided.
RESULTS
Ethylene production and respiration rate
The respiration rate throughout the growth of the C. ficifolia fruit
exhibited a typical pattern for non-climacteric fruit (Fig. one). It
showed a higher CO2 evolution in young fruit (from 131 to
115 mg CO2 kg−1 h−1, at 10 and 15 days, respectively), a steep
decrease towards 30 days, and then a slow but gradual decrease
until the end of its complete development.
Regarding ethylene evolution, C. ficifolia fruit produced little
ethylene during the whole development. The amounts found
were about 0.03 ng to 0.3 ng kg−1 s−1 at 20 °C, which is typical
for non-climacteric fruit (Fig. one).
Maturity parameters
Growth and development pattern of C. ficifolia
The growth patterns of the fruit in terms of weight and volumes
are illustrated in Fig. two; both patterns exhibit a simple sigmoidal
shape showing clearly three stages clearly. The first one shows a
slow-growth phase (5 to 15 days), the second stage shows an
exponential growth rate (embracing from 20 to 45 days after
anthesis) and the last stage showed a declining period of about
10 days until reaching the maximum size. At the lower part of
Figure 1 Respiratory activity during the development of C. ficifolia fruit. Because the standard deviation is very small (0.03-0.27), the bars cannot be
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observed on the graph.
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A Moya-Hernández et al.
the graph, the images of the harvested fruit show a slight change
in the color of the pericarp.
Shape changes during growth
According to the sphericity coefficient (ε) the shape of C. ficifolia
fruit throughout the development showed an oval profile, as ε
values of 1.0 corresponds to a spherical shape and those higher
or lower than this value are considered as oval (Fig. 3).
Physical and chemical parameters
The quality parameters throughout the development of the fruit
show that the main changes were observed in flesh firmness, with
significant differences from 15 to 25 and 40 to 45 days of develop-
ment, whereas the percentage of dry matter, TSS, percentage TA,
and pH showed no significant changes (Table two).
Hypoglycemic effect of C. ficifolia fruit in normal mice
The blood glucose concentration at 120, 240, and 360 min after
administration of the different treatments to the mice is shown
in Table 3.
All extracts of C. ficifolia of the different development stages
behaved in a similar way to glibenclamide. However, the most
notable effect was obtained with the extracts at 10, 15, 25, and
30 days after anthesis, because they worked similarly to glibencla-
mide at the three analyzed times. Only the 15 day extract showed
a significant difference in respect to ISS control in the three
periods; whereas the extracts of the fruit at 40, 45 and 55 days
only showed it at 360 min.
Hypoglycemic effect of C. ficifolia fruit in STZ induced-
diabetic mice
In the acute study of streptozotocin-induced diabetes (Table 4),
the treatment with C. ficifolia extracts at 15 days of fruit develop-
ment showed the best hypoglycemic effect compared with glib-
enclamide; this extract had about 11% greater effect than
glibenclamide at 360 min after administration. The rest of the
extracts were less effective than this medicine.
The extract at 25 days had a similar effect to the sample at
15 days at 120 and 240 min after administration but not at
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Figure 2 Growth pattern of C. ficifolia from fruit set. Bars show standard deviations of the mean.

Figure 3 Shape pattern of C. ficifolia during growth and development. Bars show standard deviations of the mean.

Table 2 Physical and chemical characteristics of C. ficifolia fruit at different stages of growth and development

Days firmness (N) TSS pH % dry material % TA

10 33.7 ± 0.9a 5.6 ± 0.1c 5.4 ± 0.2a 7.4 ± 1.7ab 3.4 ± 0.3c
15 32.8 ± 0.4a 6.0 ± 0.0d 5.9 ± 0.0b 7.8 ± 0.5ab 2.7 ± 0.3b
25 51.1 ± 8.1b 5.8 ± 0.2cd 6.1 ± 0.0bc 8.1 ± 1.3ab 2.1 ± 0.8ab
30 59.4 ± 6.8b 5.7 ± 0.2ab 6.4 ± 0.3d 8.8 ± 0.5ab 1.6 ± 0.3a
40 64.5 ± 4.3b 5.5 ± 0.0a 6.0 ± 0.1bc 8.5 ± 0.0b 1.9 ± 0.1ab
45 81.8 ± 7.8c 5.0 ± 0.0b 6.1 ± 0.0cd 8.6 ± 0.2b 1.7 ± 0.2a
55 81.9 ± 9.2c 5.0 ± 0.2b 6.1 ± 0.3cd 8.5 ± 0.4b 1.8 ± 0.1a

Different letters mean significant difference among the different stages of development (P < 0.05).
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Table 3 Hypoglycemic effect of aqueous extract of C. ficifolia fruit of different stages of development administered intraperitoneally to healthy
mice in fasting

ΔG = Glucose in blood at ti – Glucose in blood at t0 (avg ± std)

ti = 120 min ti = 240 min ti = 360 min

†
ISS −9.6 ± 1.3 b
−14.8 ± 4.1 b
−14.4 ± 3.8b
Glibenclamide ‡ −16.2 ± 6.8a −30.2 ± 7.4a −29.0 ± 4.9a
10 days −12.8 ± 3.7ab −21.2 ± 2.3ab −24.6 ± 5.9ab
15 days −11.6 ± 6.9a −29.0 ± 9.7a −28.0 ± 8.7a
25 days −10.6 ± 5.0a −26.0 ± 1.9a −26.4 ± 4.5ab
30 days −12.0 ± 4.5ab −23.0 ± 3.3ab −27.8 ± 4.3a
40 days −9.0 ± 4.6b −15.2 ± 4.3b −23.2 ± 6.6ab
45 days −7.9 ± 4.6b −13.0 ± 4.1b −21.5 ± 9.4ab

Different letters mean that there is a significant difference between the treatments (P < 0.05).
†
ISS, isotonic saline solution – control; ‡Glibenclamide – positive control.

360 min. According to the degree of hypoglycemic effect, the
extracts could be ordered from highest to lowest as follows:
15 days > 25 days > 30 days > 10 days > 40 days > 45 days.
Quantification of D-chiro-inositol in C. ficifolia extract
Figure 4 shows the chromatograms of (A) the standard DCI and (B)
the aqueous extract of C. ficifolia. Surprisingly this compound was
only found in the sample at 45 days of development with a con-
centration of 42.77 mg per gram of dry extract.
Cucurbita ficifolia extract chemical characterization
The chromatograms obtained by HPLC-DAD showed different
compounds depending on the stages of fruit development. In
the samples from 15 and 25 days, a higher concentration of
chemical compounds was found. Among them, gallic acid, chloro-
genic acid, catechin, epicatechin, syringic acid, and myricetin
were identified (Fig. 5); all of them decreased as development pro-
gressed. Gallic acid was present in the 15, 25, 30, 40, and 45 day
samples, while chlorogenic acid was not detected from day 30.
Of the compounds found, gallic acid and chlorogenic acid were
those that presented the largest areas and since the amount of
a compound is proportional to the area of the peak obtained by
the chromatograph, it was decided to quantify only these two
compounds. Table 5 shows that there was a decrease in the con-
centration of gallic acid as its development increased; the highest
concentration was at 15 days. Chlorogenic acid also presented
the highest concentration in the same sample.
DISCUSSION
The respiration rate and ethylene patterns found in the C. ficifolia
fruit throughout their growth and development indicate that this
species is a non-climacteric fruit because there is no high respira-
tion rate and ethylene production remains at low levels.22, 23
The respiration rates of C. ficifolia (21–131 mg CO2 kg−1 h−1) are
similar to those reported for other members of the same Cucurbi-
taceae family, such as pumpkins, winter squash, or summer
squash, whose values oscillate between 61 to 121 and 153 to
175 mg CO2 kg−1 h-1.28 Likewise, the low ethylene levels of C. fici-
folia are characteristic of this horticultural group, and it has been
reported that winter squash even produces traces of ethylene (i.
e. concentrations below 0.03 ng kg−1 s−1).29 The minimal changes
observed in the quality parameters are also indicative of a non-cli-
macteric fruit.22, 23

Table 4 Hypoglycemic effect of aqueous extract of C. ficifolia fruit at different stages of development administered intraperitoneally to
streptozotocin-induced diabetes mice

ΔG = Glucose in blood at ti – Glucose in blood at t0 (avg ± std)

ti = 120 min ti = 240 min ti = 360 min

ISS† −13.4 ± 5.18a −19.8 ± 5.22d −24.8 ± 7.19d

Glibenclamide‡ −30.6 ± 1.13a −116.4 ± 7.95a −184.8 ± 4.93ab
Diabetics ISS§ −18.4 ± 1.80a −31.0 ± 8.80d −41.6 ± 1.19d
10 days −11.4 ± 2.25a −48.0 ± 2.66bcd −157.4 ± 2.11ab
15 days −22.2 ± 2.55a −107.8 ± 5.71ab −205.6 ± 6.05a
25 days −31.4 ± 3.60a −111.2 ± 9.64a −169.0 ± 7.34ab
30 days −9.4 ± 3.90a −98.2 ± 9.78abc −161.2 ± 3.95ab
40 days −3.8 ± 1.30a −39.0 ± 9.24cd −118.0 ± 3.44bc
45 days −6.8 ± 6.26a −21.6 ± 1.93d −77.4 ± 2.25cd

Different letters mean that there is a significant difference between the treatments (P < 0.05).
5176

†
ISS, isotonic saline solution – control healthy; ‡Glibenclamide – positive control; §Diabetic ISS – control.

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Hypoglycemic effect in Cucurbita ficifolia
Figure 4 (A) The standard DCI; (B) DCI found in the 45 day extract.
Ethylene production also indicates that it is a non-climacteric
fruit because the concentrations found were very low (from
0.03 ng to 0.3 ng kg−1 s−1). These concentration ranges have also
been reported in non-climacteric fruit such as citric fruits30 and
Cucurbitaceae.31–34 It has even been reported that in this type
of fruit, only appear traces of ethylene in concentrations below
0.03 ng kg−1 s−1.29
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Climacteric and non-climacteric fruits may be further differenti-
ated by their response to external ethylene. In non-climacteric
fruits, external ethylene induces a transient increase in their respi-
ration rate and the magnitude of the increase depends on the
concentration of ethylene.2. 3 On the other hand, recent research
evidence suggests that sensitivity to ethylene differs depending
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on the tissue type and/or the stage of development due to
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 www.soci.org A Moya-Hernández et al.

Figure 5 HPLC-DAD chromatograms of extracts of C. ficifolia fruit. (A) 15 days after anthesis and (B) 25 days. 1) gallic acid, 2) chlorogenic acid, 3) catechin,
4) epicatechin, 5) syringic acid, 6) myricetin.

signaling interactions of this phytohormone with other plant hor-
mones, metabolites, and environmental signals. 35 In this context,
it would be interesting to investigate the changes in ethylene sen-
sitivity that might induce physiological changes during the whole
development and ripening of C. ficifolia fruit and the possibility of
influencing the production of compounds with hypoglycemic
activity.
5178
The growth pattern of a commodity is very useful not only for
commercial purposes but also for research studies, as in this case,
because it enables specific maturity stages to be identified. The
sigmoid curve pattern of growth for C. ficifolia corresponds to a
typical pepo berry (a berry with a hard rind, the receptacle
completely enclosing the ovary). From 25 days of development,
fruit started to show a mottled green appearance, which became

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Hypoglycemic effect in Cucurbita ficifolia
Table 5 Gallic and chlorogenic acids found in the extracts of C.
ficifolia
Gallic acid Chlorogenic acid
‡ −1†
DAA Concentration mg g Concentration mg g−1†
10 nd nd§
15 16.97 1.59
25 15.62 0.07
30 12.81 nd
40 1.28 nd
45 0.24 nd
†
g of dry extract; ‡ DAA, days after anthesis; § nd, not detected.
completely defined to the end of the growth. This is a special fea-
ture of this species.18, 36, 37
This physiological characterization of C. ficifolia is considered a
relevant contribution because this fruit is attracting great interest
due to its hypoglycemic effect.
Regarding the hypoglycemic effect, it was found that C. ficifolia
extracts (in all stages of development analyzed) lower glucose
levels in both normoglycemic mice and STZ-induced diabetic
mice, however, the best effects were observed in the latter.
The bioassays with normal mice showed that extracts that
exhibited an effect similar to, but not better than, glibenclamide
corresponded to fruit at 10, 15, 25 and 30 days, meanwhile, fruit
at 40 and 45 days had the hypoglycemic effect only 360 min after
administration.
In similar studies, it has been reported that the extract of C. fici-
folia mature fruit (it means fruit about 40–45 days of develop-
ment) has a better effect than tolbutamide;8, 10 however, this
drug is 100 times less potent than glibenclamide.38 The results
obtained in this investigation therefore demonstrate that the
extracts of younger fruit have a better hypoglycemic property.
In the case of STZ-induced diabetic mice, the 15 day extract was
the only one that overcame the effect of glibenclamide by about
11% 360 min after administration.
It should be noted that if the effect provided by the 15 day
extract (young fruit) is compared with that of 40 and 45 day
extracts (mature fruit), the hypoglycemic response is four, three,
and two times better at 120, 240, and 360 min, respectively.
Hence, fruit at this early stage of development might be a bet-
ter option for future research on lowering glucose levels. The 15
day fruit have 14% more juice than the mature ones (40–
45 days), which is related to a larger amount of raw material
that can be obtained for the future elaboration of
nutraceuticals.
The different responses of the hypoglycemic effect in the differ-
ent groups of mice, that can be explained as follows: In the case of
healthy mice, although if the hypoglycemic effect was observed, it
was not found to have been statistically significant when com-
pared to glibenclamide, these results agree with what was
reported by.12 The slight hypoglycemic effect found occurs
because the pancreas works normally in these animals, so there
is a release of glucagon, a hormone that stimulates the production
of glucose by the liver, thus increasing blood glucose. This action
is opposite to that of insulin, a hormone that lowers glucose levels
in the blood. Then, when glucose levels are low due to the effect
of C. ficifolia, the pancreas releases glucagon, keeping blood
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glucose stable. In the case of STZ-induced diabetic mice it is well
known that the streptozotocin destroys pancreatic islets and
therefore ⊍ and ⊎ cells cannot release insulin, so, as reported by
Miranda-Perez et al.,17 the action of C. ficifolia has an effect as
secretagogue for insulin through an increase in concentration of
intracellular calcium from the calcium reservoir in the endoplas-
mic reticulum, suggesting that its mechanism of action is different
from those of hypoglycemic drugs. It is also reported that C. ficifo-
lia extract may have a role in the renewal of ⊎ cells or may allow
the recovery of partially destroyed cells.12
Regarding DCI, the results obtained in this work showed that
this compound was present only in the extracts of 45 day fruit.
This agrees with the findings of several researchers who have
used mature fruit in their research.15, 39, 40 It should be noted that
we found that the younger C. ficifolia fruit had no DCI but exhib-
ited the best hypoglycemic effect. This result supports that
reported by Xia and Wang,eleven who mentioned that C. ficifolia
extract has a significant hypoglycemic effect in comparison with
synthesized DCI. Hence, DCI may not be the main active hypogly-
cemic compound of the fruit.
Another special feature of our work was the chemical character-
ization of the extracts at each stage of developmental maturity.
The higher concentration of the phenolic compounds identified
in the first days of development of C. ficifolia fruit is consistent
with their hypoglycemic effects and particularly the presence of
gallic and chlorogenic acids (potent antioxidants). It has been
reported that STZ-induced diabetic rats increase the levels of lipid
peroxides, which eventually leads to membrane damage.12 Thus,
the flavonoids present in the fruit extracts in our work act as anti-
oxidant components. It has also been reported that gallic acid
reduces the level of glucose in diabetic rats due to its effect as
an insulin releaser.41
On the other hand, the chlorogenic acid is related to the
increase in insulin sensitivity,42, 43 improves glucose tolerance
by reducing basal hyperglycemia in STZ-induced diabetic rats,44,
45
and increases insulin secretion. Hence, this compound is con-
sidered as multitarget agent that helps in the treatment of
T2D.46 Both gallic and chlorogenic acids have an anti-inflamma-
tory effect.13–16 This is the first report about the chlorogenic acid
content found in C. ficifolia fruit of 15 and 25 days maturity.
In summary, this research shows that the hypoglycemic proper-
ties of fruit extracts of C. ficifolia are higher in young fruit than in
the mature fruit and the phenolic compounds are mainly respon-
sible for this effect and not the DCI as previously thought. These
compounds in addition to providing an antioxidant action are
multitarget agent to the control of diabetes as a secretagogue
and sensitizer insulin and a lipid-lowering agent.
However, further research is required to identify all the com-
pounds present in the extract of C. ficifolia fruit and its molecular
mechanisms. That is a current research challenge because it can
lead to the discovery of new therapeutic targets in the control
of this disease.
ACKNOWLEDGEMENTS
This research was partially carried out with the funding support of
UAM-Iztapalapa. The authors thank CONACyT for the scholarship
of Araceli Moya-Hernández during her PhD (with fellowship num-
ber 302053). We also would like to thank Raziel Estrada-Martinez
for technical support for the quantitative analysis of D-chiro-
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inositol.
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 www.soci.org
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