Journal of Functional Foods 66 (2020) 103821

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Journal of Functional Foods

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Calabash (Lagenaria siceraria) potency to ameliorate hyperglycemia and T

oxidative stress in diabetes
⁎
Lana Y.M. Juee , Alaadin M. Naqishbandi
Department of Pharmacognosy, Pharmacy College, Hawler Medical University, Erbil, Kurdistan Region, Iraq

A R T I C LE I N FO A B S T R A C T

Keywords: Calabash fruit is traditionally used as an antidiabetic remedy in the Kurdistan region. Different extracts (400 mg/
Antidiabetic kg) were investigated for antihyperglycemic potency on normal and alloxan-induced diabetic mice. The sig-
HepG2 nificant potency exhibited by the aqueous extract produced a decrease in fasting blood glucose level (52.685%,
2-NDBG P ≤ 0.05) in diabetic mice with no apparent effect using extracts in the normoglycemic model. The anti-
Protocatechuic acid
hyperglycemic extract was evaluated for in vitro antidiabetic efficacy based on HepG2 cells, and the 2-NDBG
Alloxan
Glucose uptake assay
indicator showed a significant glucose uptake (164.87%, P ≤ 0.05), whereas the in vitro antioxidant study
showed high ABTS free radical scavenging activity (EC50 64.4 ± 2.12 µg/mL). The extract phytochemical study
recorded a high phenolic content with the identification of two compounds, protocatechuic acid, and luteolin-7-
glucoside, using liquid chromatography-tandem mass spectrometry. The results revealed the benefits of the fruit
in the reduction of hyperglycemia and attenuating oxidative stress associated with diabetes.

1. Introduction
Diabetes mellitus (DM) is a chronic metabolic disease characterized
by hyperglycemia and is associated with various complications, ranging
from chronic to acute cases, causing considerable damage affecting
different organs of the body, such as the renal, retinal, cardiovascular,
and neurological systems, resulting in microvascular complications.
The main cause that underlines hyperglycemia is either an absolute or
relative lack of insulin secretion and/or its action on the target tissue
(Alberti & Zimmet, 1998; Skyler et al., 2017). In 2017, the global
prevalence of DM was 11.4% with a treatment rate of 48.2% and a
mortality rate of 2.7% (Ge, Chen, & Chen, 2017). The International
Diabetes Federation report recorded 425 million people aged
20–79 years with diabetes in 2017, with an expected increase to 629
million by 2045 (International Diabetes Federation, 2017).
Acute as well as chronic elevation in blood glucose levels can result
in the release of reactive oxygen species (ROS) via autoxidation side
reactions. ROS, such as superoxide anion radicals, hydrogen peroxide,
and hydroxyl radicals, can produce more serious complications, such as
lipid peroxidation, enhancing glucose resistance when the body’s an-
tioxidants are unable to scavenge the ROS (Lien, Hua, & Chuong, 2008;
Maritim, Sanders, & Watkins, 2003; Yang, Jin, Lam, & Yan, 2011). In
vitro, cell-based studies have revealed microvascular complications that
are mediated via oxidative stress and ROS production in elevated
glucose conditions (Barthelmes, Zhu, Shen, Gillies, & Irhimeh, 2014;
Wolff, 1993; Xu, Tappia, Neki, & Dhalla, 2014).
The beneficial usage of medicinal herbs in the ethnomedicine of
many cultures has been extensively recorded. Numerous plants have
been used as adjuvants and for the treatment of various diseases
without a full understanding of their appropriate functions and con-
stituents. These actions could be assigned to the unaffordable costs and
adverse effects of conventional hypoglycemic drugs. Many synthetic
drugs have been developed for DM management, but the production of
prototype antidiabetic drugs has yet not been achieved (Patel, Kumar,
Laloo, & Hemalatha, 2012).
Calabash is botanically known as Lagenaria siceraria (Molina)
Standley (Cucurbitaceae), which is a climbing or trailing herb, with a
bottle- or dumb-bell-shaped fruits. Both its aerial parts and fruits are
commonly consumed as a vegetable containing many essential com-
ponents that are required for a normal quality of human life (Shah &
Seth, 2010), such as primary metabolites including carbohydrates,
proteins, minerals (phosphorus, potassium, zinc, magnesium, copper,
sodium, calcium, and manganese), and vitamins (vitamin C, B6, and E,
thiamin, riboflavin, niacin, pantothenic acid, and choline) (Lim, 2012;
Meenal, Khadabadi, Farooqui, & Deore, 2010; Milind & Satbir, 2011;
Rahman, 2003; Shah & Seth, 2010).
L. siceraria is characterized by the presence of cucurbitacin.
Cucurbitacin B, D, G, and H can be detected in different parts of a plant

⁎
Corresponding author.
E-mail address: lana.juee@hmu.edu.krd (L.Y.M. Juee).

https://doi.org/10.1016/j.jff.2020.103821
Received 25 October 2019; Received in revised form 21 January 2020; Accepted 25 January 2020
Available online 12 February 2020
1756-4646/ © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
L.Y.M. Juee and A.M. Naqishbandi
such as fruit (as aglycon), roots, and leaves, (Dhiman, Gupta, Sharma,
Gill, & Goyal, 2012; Hideki et al., 2007) Other phytoconstituents that
have been recorded in the plant include sterols, oleanolic acid, tri-
terpenoids, tannins, saponins, flavone C-glycosides, and flavonoids
(Gangwal, Parmar, & Sheth, 2010; Shirwaikar & Sreenivashan, 1996;
The Wealth of India, 2004).
Traditionally, this plant has been used as a cardiotonic, diuretic, and
general tonic in many European countries, as well as in India and China.
Many pharmacological properties have been studied, such as the anti-
hepatotoxic, analgesic, anti-inflammatory, hypolipidemic, im-
munosuppressive, immunomodulatory, cytotoxic, diuretic, cardiopro-
tective, antihyperlipidemic, antiobesity, and antioxidant properties of
different parts of the plant (Ahmed, Fatima, & Saeed, 2014; Aslam &
Najem, 2014; Ghule, Ghante, Saoji, & Yeole, 2006; Ghule, Ghante,
Upganlawar, & Yeole, 2006; Kota, Sharma, & Tahsildar, 2016;
Maqsood, Ahmed, Atique, & Malik, 2017; Sankari, Jubilee, Janaki, &
Raju, 2010). The antidiabetic activity of the L. siceraria plant has been
recorded using different techniques and models were recorded by
(Bhattacharya & Das, 2012; Deshpande et al., 2008; Dheeraj et al.,
2019).
In this study, we evaluated the potency of calabash fruit extracts to
ameliorate hyperglycemia and oxidative stress in diabetes. This fruit is
used as an antidiabetic remedy traditionally in the Kurdistan region of
Iraq, and a phytochemical bioassay-guided identification is provided for
its main active constituents.
2. Material and methods
2.1. Plant material
Fruits of Calabash (L. siceraria) were collected during June 2016
from different areas of the Kurdistan region. The plant was authenti-
cated by assistant professor Alaadin Naqishbandi. The fruits were
cleaned, dried in shade for 4–6 days, and kept in closed containers at
25 °C. The voucher specimen (L-22) was stored at the Pharmacognosy
Department, Pharmacy College, Hawler Medical University, Erbil,
Kurdistan region\Iraq.
2.2. Plant extract preparation
An ultrasonic-assisted extraction technique was applied for extrac-
tion, as described by Siriangkhawut and Kaewboo (2013), and used for
extraction of powdered plant material for 1 h at 40 °C by solvents with
increasing polarity (petroleum ether, chloroform, methanol, and water)
at a ratio of 1:10 (w/v), as adapted from Mona et al. (2012). The liquid
extracts obtained from petroleum ether, chloroform, and methanol
were dried using a rotary vapor machine at 40-–50 °C (Buchi Rota-
vator®, Switzerland), and the water extract was dried using a freeze
dryer (Martin Christ Alpha 1–2 LD plus, Germany) to afford the cor-
responding extracts LSPEF, LSCF, LSMF, and LSAF with the following
yields: 0.81, 0.73, 32.91, and 0.74% w/w, respectively. All extracts
were maintained at 4 °C until further use.
For acute toxicity and in vivo study, petroleum ether and chloro-
form extracts were reconstituted in tween 80 (20%) and normal saline
used as a solvent for reconstitution of methanol and aqueous extracts.
2.3. In vivo antidiabetic study
2.3.1. Animals
Adult Swiss albino mice (mean weight 25 g/mouse) were obtained
from the animal house of the Medicine College, Hawler Medical
University, Erbil, Kurdistan region\Iraq. They were housed in groups of
six animals per polypropylene cage at a temperature of 22 ± 3 °C
under a 12 h light/12 h dark cycle. The animals were allowed to access
commercial food and tap water ad libitum. The experimental procedure
was approved by the Pharmacy College, Hawler Medical University
2
Journal of Functional Foods 66 (2020) 103821
ethic committee (code number 160302/20).
2.3.2. Acute toxicity test
The experiment was conducted in accordance with the guidelines of
the Organization for Economic Cooperation and Development (OECD)
for the testing of chemicals (OECD, 2001). We divided 24 female albino
mice into groups of six mice and were administered a high dose
(2000 mg/kg BW) of each of the plant extracts. A single oral dose of
plant extracts was administered to all animals, who were kept under
close observation for 24 h. Monitoring continued for 7 days until any
changes either in general behavior or physical activity were noticed.
2.3.3. Experimental protocol
The antidiabetic test methods described by Khalid et al. (2013) were
used, with slight modification. Diabetes was induced by two sub-
cutaneous injections of alloxan (Sigma Aldrich Chemical Co., U.K.)
using an initial dose of 200 mg/kg BW, followed by 150 mg/kg BW as
the second dose after (72 h) to ensure the appearance of diabetic signs.
After 16 h fasting, the mice were evaluated for hyperglycemia using a
counter blood glucose meter (Bayer). Mice with blood glucose levels
higher than 200 mg/dL were considered diabetic.
2.3.4. Antidiabetic test
Forty two mice from the normoglycemic and equal number of al-
loxan-induced diabetic mice were randomly selected and divided into 7
groups each (n = 6). Groups 1–4 received 0.5 mL (400 mg/kg BW) each
of extracts LSPEF, LSCF, LSMF, and LSAF, respectively. Group 5 were
received 0.5 mL metformin (500 mg/kg BW in normal saline) as posi-
tive controls. Groups 6 and 7 were given 0.5 mL each of normal saline
and Tween 80 (20%), respectively served as negative controls.
In all groups, oral administration was performed with a specific
mouse feeding needle. Blood was collected from the tail vein at 0, 1, 2,
3, 5, and 7 h for glucose level measurements.
2.4. In vitro antidiabetic study
We performed an in vitro antidiabetic study on the anti-
hyperglycemic extract (LSAF) through a glucose uptake assay using the
fluorescence glucose analog 2-(n-(7-nitrobenz-2-oxa-1,3-diazol- 4-yl)
amino)-2-deoxyglucose (2-NBDG) and liver carcinoma cell HepG2 ob-
tained from the tumor bank of the German Cancer Research Center
(Heidelberg, Germany).
2.4.1. Cell culture
Human liver carcinoma cells were maintained in Dulbecco’s mod-
ified Eagle’s medium (DMEM) with Gluta Max (Invitrogen, Germany)
containing 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/
streptomycin (Invitrogen), incubated in a 5% CO2 atmosphere at 37 °C.
2.4.2. Cytotoxicity assay
A resazurin reduction assay was conducted to evaluate the cytotoxic
effect of the antihyperglycemic active extract of L. siceraria (LSAF) on
HepG2 cells. In the process of the conversion of resazurin to its reduced
form resorufin via viable cells, the blue color intensity was detected in
nonviable cells (O’Brien, Wilson, Orton, & Pognan, 2000). In brief, an
aliquot of 100 µL containing 5 × 103 cells per well was seeded in a 96-
well plate incubated for 24 h at 37 °C, 5% CO2. After the incubation
period, 100 µL of each tested extract (at 50 µg/mL and 100 µg/mL),
dimethyl sulfoxide (DMSO) at a final concentration (0.3%), and media
were added to the cells and further incubated for 72 h at 37 °C, with 5%
CO2. Resazurin (Sigma-Aldrich) solution was added to each well at a
volume of 20 µL, the plates were incubated for 4 h, and then staining
was measured using an Infinite M2000 Pro TM plate reader (Tecan,
Germany) at wavelengths of 544 nm (excitation) and 590 nm (emis-
sion). The assay was independently performed three times with six
replicates according to the reported protocol (Kuete et al., 2016).
L.Y.M. Juee and A.M. Naqishbandi
2.4.3. Glucose uptake assay
2.4.3.1. Experimental design. The maintained HepG2 cells were divided
into three groups. Group I included cells of the control cultured with the
maintenance media (DMEM, glucose-free, serum-free). Group II
included cells that were cultured with metformin 100 µM/mL and
reconstituted in glucose-free and serum-free media. Group III included
cells that were cultured with LSAF 50 µg/mL and reconstituted in
glucose-free and serum-free media.
2.4.3.2. 2-NBDG uptake. Categorized groups of cells were evaluated for
their uptake of fluorescent D-glucose derivative (2-deoxyglucose, 2-(N-
(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)-2-deoxy-d-glucose; 2-
NDBG) (Zou, Wang, & Shen, 2005). The seeded cells in 96-well plates
with categorized groups were aspirated after incubation for 24 h using
phosphate buffer saline (PBS; 200 µL). The cells were incubated for an
additional 2 h with 2-NDBG for a final concentration of 100 µM and
reconstituted in glucose-free and serum-free media. After treatment, the
free 2-NDBG was washed out, and cells were washed with 200 µL PBS
twice, and 200 µL of DMSO was added to each well. An Infinite M2000
Proplate reader (Tecan, Germany) was used to measure the
fluorescence at 544 nm (excitation) and 590 nm (emission)
wavelengths. The glucose uptake of the control set was 100 (2-NBDG)
uptake, and the 2-NBDG of the sample treated groups was calculated
according to the following equation:
2‐NBDG uptake (%) = (Asample /Acontrol ) × 100
where A is absorbance.
2.5. Antioxidant study
Antioxidant activity indicates the free radical scavenging and re-
ducing power activity of the antihyperglycemic extract LSAF, which
was evaluated using the 2,2-diphenyl-1,1-picrilhydrazil (DPPH) free
radical, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)
free radical, and cupric ions.
2.5.1. Free radical scavenging activity
The DPPH assay was conducted according to the method previously
described by Blois (1958). We dissolved 10 mg of the anti-
hyperglycemic extract (LSAF) in 10 mL ethanol, and stock solutions
were prepared. Aliquots of 2, 5, 10, and 20 µL of these stock solutions
were taken up to 40 µL with ethanol, and 160 µL of 0.1 mM DPPH
solution was added to produce a final concentration of the extract (10,
25, 50, or 100 µg/mL). The control sample contained ethanol instead of
the extract solution. After incubation in the dark for 30 min, the ab-
sorbance was measured at 517 nm.
The ABTS assay was completed using the method reported by Re
et al. (1999). We dissolved 10 mg of the antihyperglycemic extract
(LSAF) in 10 mL ethanol to prepare stock solutions. Aliquots of 2, 5, 10,
and 20 µL of stock solutions were completed to a 40 µL volume with
ethanol, and 160 µL of 7 mM ABTS cation radical solution was added to
produce a final concentration of the extract (10, 25, 50, or 100 µg/mL).
After 6 min in the dark, the absorbance was measured at 734 nm. The
control sample contained ethanol instead of the extract solution.
The free radical removal activities, expressed as a percentage of
inhibition, were calculated using the following equation:
% Inhibition = (A control − Asample )/ A control × 100
where A is absorbance. Three parallel measurements were performed
for each sample, and butylated hydroxytoluene (BHT) and α-tocopherol
(α-TOC) were used as standards.
EC50, the effective concentration of the extract for the scavenging of
50% of the free radicals, is expressed as µg/mL ± standard error of the
mean (SEM), which was calculated from the linear regression analysis
equation obtained from the graph plotting the percentage of radical
scavenging activity versus sample concentration.
3
Journal of Functional Foods 66 (2020) 103821
2.5.2. Reducing power
The reducing power of the antihyperglycemic extract (LSAF) was
measured using 2,9-dimethyl-1,10-phenanthroline (neocuproin) and a
copper chloride compound via the method reported by Apak, Güçlü,
Özyürek, and Karademir (2004). Samples and standards were prepared,
Cu(II), Neosporin, and NH4CH3COO buffer were added to produce a
final concentration of 10, 25, 50, or 100 μg/mL, and the absorbance
was measured after 1 h incubation. Three parallel studies were per-
formed for each sample. BHT and α-TOC were used as standards. The
reducing power of the extract is expressed as EC50, and the effective
concentration at an absorbance of 0.5 was measured in µg/mL ± SEM,
obtained from the interpolation of the linear regression analysis.
2.6. Phytochemical study
Biologically active extracts were introduced to phytochemically
study the total phenolic and flavonoid contents to bioassay-guided
identification of the active constituents.
2.6.1. Total phenolic and flavonoid contents
The total phenolic content (TPC) of antihyperglycemic extract LSAF
were determined using Folin–Ciocalteu reagent to be equivalent to
pyrocatechol (Slinkard & Singleton, 1977 using the equation
(y = 0.0347x + 0.0384, determination coefficient (R2) = 0.9916)
obtained from the pyrocatechol calibration curve (0–4 µg/mL) and
expressed as the mean values ( ± SEM) of pyrocatechol (µg) per 1 g
extract.
Total flavonoid content (TFC) was determined using the aluminum
nitrate method (Moreno, Isla, Sampietro, & Vattuone, 2000), with the
equation obtained from the calibration curve of quercetin
(y = 0.0445x + 0.1352, R2 = 0.9962) expressed as mean values
( ± SEM) of µg quercetin per 1 g extract.
2.6.2. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Shimadzu Nexera model ultra-high performance liquid chromato-
graphy (UHPLC) was combined with a Shimadzu LCMS 8040 model
triple quadrupole mass spectrometer to evaluate the physiologically
active plant extract using a method adapted from Akdeniz (2018). The
liquid chromatography components were an LC-30 AD gradient pump,
a DGU-20A3R degasser, a CTO-10ASvp column oven, and a SIL-30AC
autosampler. The chromatographic separation was performed on an
Inertsil ODS-4 C18 column (100 × 2.1 mm, 2 µm). The column tem-
perature was maintained at 35 °C during the analysis. The mobile phase
A consisted of water. We added 10 mM ammonium formate–0.1%
formic acid to the water phase to facilitate chromatographic separation
and ionization. We used ethanol as the mobile phase B. The applied
gradient profile was optimized as 5–20% B (0–10 min), 20% B
(10–22 min), 20–50% B (22–36 min), 95% B (36–40), and 5% B
(40–50 min). The flow rate of the mobile phase was 0.25 mL/min, and
the injection volume was 4 µL.
A triple quadrupole mass spectrometer was supplied with an elec-
trospray ionization (ESI) source that works in both negative and posi-
tive mode. LC-ESI-MS/MS data were collected and processed by
LabSolutions software (Shimadzu, Kyoto, Japan).
We used multiple reaction monitoring (MRM) mode for the quan-
titative determination of the analytes and the molecular (parent) ions
and (product ions). The first one was used for quantitative purposes,
and the other was used for qualitative purposes. The other parameters
optimized in the spectrometer were 250 °C desolvation line temperature
(DL) temperature, 350 °C interface temperature, and 400 °C heat block
temperature. We used 15 L/min and 3 L/min as the drying gas (N2) and
nebulizer flow rates, respectively.
2.6.3. Validation parameters
The method performance was evaluated using standard spiked and
non-spiked samples. For method validation, linearity, trueness
L.Y.M. Juee and A.M. Naqishbandi Journal of Functional Foods 66 (2020) 103821

(recovery), precision (repeatability and reproducibility), limits of de-

0.0215
0.0086
Uf
tection (LODs) and quantification (LOQs), and relative standard un-
Inter days certainty (U% at 95% confidence level (k) = 2) are provided in Table 1
and Fig. 1.
0.9988
1.0072
Recovery (%)

2.7. Statistical analysis

Intra day

1.0096
1.0014

In vivo antidiabetic data are expressed as the mean ± SEM (n = 6)

for animal groups. The comparison was performed using one-way
analyses of variance (ANOVA). For intergroup comparisons, a post-hoc
Inter days

Dunnett's test was used with SPSS software version 23 for data analysis.
0.0060
0.0037

At least three independent experiments were conducted for each in

vitro biological and phytochemical aspect evaluated unless specified
otherwise. The results are expressed as the mean ± SEM calculated by
Intra day
(%RSDd)

0.0060
0.0052

Microsoft Excel 2007. P ≤ 0.05 was considered statistically significant.

Standards calibration curves were considered to be linear if R2 ˃ 0.95.
Student’s t-test was applied for the comparison of data in the glucose
e
LOD/LOQ (µg/L)

uptake assay. Pearson’s correlation coefficient test was applied to de-

termine the relationship between antioxidant activities using different
4.26/5.32
2.30/3.02

chemical tests and phytochemical constituents with total phenolic and

flavonoid contents.
LOD/LOQ (μg/L): Limit of determination/Limit of quantification, Uf (%): Percent relative uncertainty at 95% confidence level (k = 2).

3. Results
Linearity (µg/L)

100–3200

3.1. In vivo antidiabetic study

75–2400

3.1.1. Acute toxicity test

The oral administration of high doses (up to 2000 mg/kg) of L. si-
0.9909
0.9939

ceraria fruit extracts did not produce significant changes in terms of the
c 2
R

physical activity and general behavior of the mice, including motor

activity, alertness, convulsion, restlessness, coma, breathing, diarrhea,
y = 215.412x + 36852.1
y = 590.460x + 120226

and the general appearance of the animals. No mortality was recorded

within the 24 h observation period after oral administration of the plant
extracts. At the end of the observation period (seven days), the mice
looked normal in their physical activity, which revealed that the doses
Equation

of plant extracts used did not exert any adverse effect on the animals
tested.
Ionization mode

3.1.2. Antidiabetic study

The mean values ( ± SEM) of blood glucose levels in the normal
mice models are shown in Table 2. Fasting followed by treatment with
various L. siceraria extracts showed insignificant activity (P > 0.05) in
Neg
Neg

Precursor ion(m/z): Molecular ions of standard compounds (m/z rate).

comparison to metformin.
The diabetic mice’s blood glucose levels (mean ± SEM) are listed
Fragment ion

109.1–108.0
285.1–284.1

in Table 3. Blood glucose levels decreased after administration of the

different extracts of L. siceraria. The highest activity expressed as a
percentage of reduction from fasting blood glucose level was recorded
for LSAF (52.685%) at the fifth hour after drug administration, which
Precursor ion (m/z)b

reached significantly higher values than the positive control (40.91%,

P ≤ 0.05).
Analytical parameters of LC-MS/MS study.

3.2. In vitro antidiabetic study

153.4
447.0

RSD: Relative standard deviation.

3.2.1. Resazurin assay

13.20

Antihyperglycemic LSAF, which was evaluated for its cytotoxic ef-

7.00
RTa

R2: Correlation coefficient.

fects on HepG2 cells using a resazurin assay, showed that a 50 µg/mL

concentration is safe for use in a glucose uptake assay due to its higher
Luteolin-7-glucoside
Protocatechuic acid

RT: Retention time.

percentage of viability (65.107%) compared to 100 µg/mL at

(41.632%).
Analyte

3.2.2. Glucose uptake assay

A significant elevation in the 2-NDBG uptake (P ≤0.05) was re-
Table 1

corded for the HepG2 cells in the presence of the LSAF in comparison to
No

d
b
a

e
1
2

the control and metformin (Fig. 2).

4
L.Y.M. Juee and A.M. Naqishbandi Journal of Functional Foods 66 (2020) 103821

Fig. 1. Standard chromatogram for (1) Protocatechuic acid, (2) luteolin-7-glycoside.

Table 2
Antidiabetic test of L. siceraria fruit extracts in normal mice (n = 6).
1
No Treatment groups Blood glucose level mg/dl(mean ± SEM)

0h 1h 2h 3h 5h 7h

1 LSPEF 57.5 ± 1.545 78.167 ± 2.211 70.333 ± 1.855 74.667 ± 4.036 70 ± 4.998 57.333 ± 3.709
2 LSCF 92.167 ± 3.38 126.5 ± 5.508 98 ± 2.772 115.5 ± 4.274 123.667 ± 5.892 96.667 ± 3.816
3 LSMF 81.5 ± 10.368 140.667 ± 2.665 129.167 ± 1.711 134.833 ± 1.284 108.167 ± 1.017 106.167 ± 1.199
4 LSAF 69.833 ± 5.41 164 ± 4.459 133.667 ± 2.486 109.5 ± 7.0616 118 ± 1.821 101.333 ± 1.409
5 Control I (NS) 99.167 ± 1.27 119.167 ± 1.83 119.67 ± 0.79 109.67 ± 1.86 115.167 ± 2.02 98.5 ± 0.98
6 Control II (Tween 80) 102.67 ± 1.12 120.167 ± 2.51 118.83 ± 3.07 115 ± 2.24 104 ± 3.68 101.67 ± 3.51
7 Metformin 95 ± 1.28 93.83 ± 3.09 61.667 ± 2.19 45.167 ± 1.11 86.167 ± 1.22 82.67 ± 1.23

1
PEF petroleum ether extract, CF chloroform extract, MF methanol extract, AF aqueous extract, and NS normal saline.

3.3. Antioxidant activity 3.4. Phytochemical study

Antioxidant activity was evaluated for the LSAF extract at con-
centrations of 10–100 µg/mL and for a standard antioxidant in the same
concentration range. A dose-dependent effect was observed for the
samples and standards in all investigated tests (Figs. 3–5). L. siceraria
aqueous extract showed a high scavenging activity of ABTS radicals
(EC50 64.4 ± 2.12 µg/mL). The tested extracts showed lower anti-
oxidant activity than the tested standards within the experiment en-
vironment (Table 4).
The total phenolic and flavonoid contents in the LSAF anti-
hyperglycemic extract was 43.53 ± 7.2, which is equivalent to one
microgram pyrocatechol per one gram extract for the phenolic content,
with a lower concentration of flavonoids (16.36 ± 0.674 equivalent to
1 µg quercetin/1 g extract), expressed as the mean ± SEM.
The LC-MS/MS analysis revealed the presence of two analytes. The
quantification of analytes showed the highest quantity for luteolin-7-
glucoside (16.83 µg/g extract) and a lower amount of protocatechuic
acid (4.79 µg/g extract; Fig. 6).

Table 3
Antidiabetic test of L. siceraria fruit extracts in alloxan-induced diabetic mice (n = 6).
No. Treatment groups1 Blood glucose level mg/dl(mean ± SEM)

0h 1h 2h 3h 5h 7h

1 LSPEF 409.5 ± 8.354 345.833 ± 2.572 341.333 ± 1.52 291 ± 10.478 256.333 ± 3.74 265.5 ± 6.832
(15.547%) (16.646%) (28.937%)2 (37.403%) (35.164%)
2 LSCF 504.5 ± 2.609 368.5 ± 4.018 391.5 ± 7.603 366.5 ± 7.454 360.667 ± 4.615 338.833 ± 4.98
(17.0465%) (22.398%) (27.354%) (28.51%) (32.837%)
3 LSMF 309.833 ± 3.86 330.667 ± 4.444 304.5 ± 8.251 285.5 ± 5.354 201.667 ± 3.138 212.667 ± 3.165
(1.721%) (7.854%) (34.911%) (31.361%)
4 LSAF 412.83 ± 1.163 369.5 ± 9.437 261.5 ± 1.129 251.333 ± 1.261 195.333 ± 10.089 172.833 ± 8.971
(10.495%) (36.657%) (39.119%) (52.685%)3 (58.135%)
5 Control I 444.67 ± 1.28 427.17 ± 4.53 430.5 ± 0.67 421 ± 5.96 461 ± 3.38 439.33 ± 0.6
(NS)
6 Control II (Tween 80) 382.5 ± 1.12 389.33 ± 0.78 393.83 ± 0.61 393 ± 0.95 377.33 ± 1.62 368.83 ± 1.19
7 Metformin 423.67 ± 1.3 364.5 ± 5.4 307 ± 3.786 280.833 ± 5.58 250.33 ± 2.3 174. ± 1.32
(13.965%) (27.54%) (33.713%) (40.912%) (58.93%)

1
PEF petroleum ether extract, CF chloroform extract, MF methanol extract, AF aqueous extract, and NS normal saline.
2
Percentage of blood glucose reduction calculated on fasting blood glucose level.
3
Significant vs metformin (P ≤ 0.05).

5
L.Y.M. Juee and A.M. Naqishbandi Journal of Functional Foods 66 (2020) 103821

200 4.5
* 4
% of 2-NBDG uptake

150 3.5
3

Absorbance
100 2.5
LSAF
2
50 -TOC
1.5
BHT
1
0
LSAF (μg/mL) Metformin (μM/mL) Control 0.5
0
Fig. 2. 2-NBDG uptake of L. siceraria fruit aqueous extract (LSAF) at (50 µg/ 10 25 50 100
mL), Metformin at (100 µM/mL), control untreated group using HepG2 cell
Concentration (μg/mL)
line. Results expressed as (mean ± SEM). The bar with (*) symbol indicated a
significant elevation of the 2-NBDG uptake (P ≤ 0.05) in comparison to met- Fig. 5. Cupric reducing power of L. siceraria aqueous extract (LSAF), α-toco-
formin and control groups using student t-test analysis. pherol and BHT at concentration range (10–100 µg/mL) expressed as absor-
bance at 450 nm (n = 3).
90
Free radical scavenging activity (%)

80 Table 4
70 Antioxidant activity of L. siceraria fruit aqueous extract (LSAF) (n = 3).

60 Samples EC50 (µg/ml)1

50
LSAF DPPH Free Radical2 ABTS Cation Radical2 CUPRAC(A0.5)3
40
-TOC LSAF 410.00 ± 4.31 64.4 ± 2.12 108.95 ± 6.12
30
BHT α-TOC 13.10 ± 0.62 12.08 ± 0.33 10.31 ± 0.22
20 BHT 58.15 ± 1.35 11.60 ± 0.18 6.40 ± 0.06
10 1
Values expressed as mean ± SEM.
0 2
EC50 is an effective concentration for scavenging 50% of the free radicals.
10 25 50 100 3
EC50 is effective concentration when absorbance is equal to 0.5.
Concentration (μg/mL)

Fig. 3. DPPH radical scavenging activity of L. siceraria aqueous extract (LSAF), (r = −0.91). Both phytochemical contents showed a positive re-
α-tocopherol and BHT at concentration range (10–100 µg/mL) expressed as a lationship with glucose uptake with greater contribution for TPC in
percentage (n = 3). enhancing glucose uptake.

100
4. Discussion
90
80 Calabash (L. siceraria) fruit extracts showed insignificant activity
70 (P > 0.05) in normoglycemic mice at an evaluated concentration
Absorbance

60 (400 mg/kg BW). Using an antidiabetic test in comparison with met-

50
40
30
20
10
0 ging from weak to strong, and rapid to delayed activities. LSAF showed
10 25 50
Concentration (μg/mL)
Fig. 4. ABTS radical scavenging activity of L. siceraria aqueous extract (LSAF),
α-tocopherol and BHT at concentration range (10–100 µg/mL) expressed as a
percentage (n = 3).
3.5. Correlation of in vitro antidiabetic and antioxidant evaluation
parameters and phytochemical contents
The correlation was examined among all evaluated variables for
total phenolic and total flavonoid contents and in vitro biological ac-
tivities (free radical scavenging activity (EC50) and glucose uptake). The
analysis was performed using Pearson’s correlation test with the aver-
aged values of each variable’s results, as shown in Table 5. A strong
negative correlation (r = −0.998) was observed between TPC and
ABTS with a lower negative correlation (r = −0.871) for cupric re-
ducing power. TFC showed a weak positive correlation with both ABT
scavenging activity (r = 0.121) and cupric reducing power (r = 0.221)
and a strong negative relationship with DPPH scavenging activity
LSAF formin showed the absence of hypoglycemia; the main adverse effects
-TOC were similar to those known for conventional antidiabetic drug ad-
BHT ministration. Hyperglycemic amelioration was recorded for different
extracts at the tested concentration in alloxan-induced diabetic mice.
Variant extracts showed variation in their corresponding activity, ran-
100 a strongly significant blood glucose reduction activity from fasting
blood glucose level expressed as percentage (P ≤ 0.05, 52.685%) at the
fifth hour after extract administration in comparison with the positive
control metformin, with a similar decrease percentage at the seventh
hour after extract administration to reach 58.135%, resembling met-
formin 58.93%. Other extracts of L. siceraria fruit showed more than
25% blood glucose reduction but a statistically insignificant potency
(P > 0.05) compared with metformin at the tested concentration.
Khan and Shechter (1991) suggested that a 25% reduction in blood
glucose levels can be considered significant hypoglycemic activity.
Most rapid insignificant hyperglycemic lowering activities
(P > 0.05) were recorded for LSAF and LSCF in alloxan-induced dia-
betic mice. This activity started at the second hour of drug adminis-
tration, concomitant with the start of metformin activity, whereas
LSPEF activity was noted at the third hour and LSMF at the fifth hour
after extract administration. Physiologically significant and insignif-
icant active extracts showed continuous activity, maintaining blood
glucose level amelioration until the seventh hour of evaluation after
drug administration. The antidiabetic activity of different parts of L.
siceraria extracts using in vivo and in vitro models were previously

6
L.Y.M. Juee and A.M. Naqishbandi
Fig. 6. LC-MS/MS chromatogram for L. siceraria fruit aqueous extract (LSAF). (1) Protocatechuic acid, (2) luteolin-7-glycoside.
Table 5
Pearsons correlation among total phenolic (TPC), total flavonoid (TFC) and
antioxidant activity (DPPH scavenging activity, ABTS scavenging activity, and
cupric reducing power) and glucose uptake.
Variables TPC TFC
DPPH scavenging activity (EC50) 0.138 −0.91
ABTS scavenging activity (EC50) −0.998 0.121
Cupric reducing power (EC50) −0.871 0.221
Glucose uptake (%) 0.982 0.463
recorded in the literature (Attar & Ghane, 2019; Deshpande et al., 2008;
Saha, Mazumder, Haldar, Sen, & Naskar, 2011; Sulaiman & Ooi, 2013).
In vitro antidiabetic studies based on cells were conducted to il-
lustrate the mechanism of LSAF’s significant antidiabetic activity. A
significant elevation in glucose uptake (P ≤ 0.05) was exhibited by
HepG2 cells in the presence of LSAF in comparison with metformin.
These findings reveal a metformin imitator mechanism of action for L.
siceraria antidiabetic activity. Metformin is an oral hypoglycemic agent
that belongs to the biguanide class of antidiabetic drugs. Metformin
produces antidiabetic activity through the activation of AMP-activated
protein kinase (AMPK) in the liver, leading to a sequence of pharma-
cological effects, including inhibition of glucose production and im-
proves hepatic sensitivity to insulin and lipid synthesis (Foretz &
Viollet, 2011; Viollet et al., 2012). LSAF activity may be linked to ac-
tivation of the insulin signaling cascade, stimulating glucose transporter
2 (GLUT 2), which facilitates glucose translocation in the cells.
Our in vitro antioxidant study revealed the efficacy of the anti-
hyperglycemic extract LSAF for improving oxidative stress. This effect
has indicated the extract potency for free radical reduction and at-
tenuating the corresponding cellular destructive effects of ROS asso-
ciated with diabetes. The analysis showed the high antioxidant capacity
of the extract for scavenging the ABTS radicals with a lower scavenging
efficacy of DPPH radicals and a cupric reducing power. Different studies
have been conducted on L. siceraria fruit extracts using a different
solvent system, and the concentrations showed an agreement with our
findings for antioxidant activity (Attar & Ghane, 2019; Deshpande,
Mishra, Meghre, Vadodkar, & Dorle, 2007; Sulaiman & Ooi, 2013).
In our phytochemical study of LSAF, we recorded the high phenolic
content with lower flavonoid concentrations. To confirm the relation-
ship between phytochemical constituents and biological activities, we
conducted a correlation analysis between phytochemical variables (TPC
and TFC) and biological activities (antioxidant and glucose uptake).
This analysis showed a strong negative correlation between TPC with
ABT scavenging (r = −0.99) and cupric reducing power (r = −0.871),
as well as a positive weak relationship with DPPH scavenging
(r = 0.138), indicating that phenolic compounds are responsible for
quenching the ABTS free radical and reducing cupric ions, rather than
7
Journal of Functional Foods 66 (2020) 103821
scavenging the DPPH free radical. TFC expressed a strong negative
correlation with DPPH (r = −0.91) and a positive correlation with
ABTS (r = 0.121) and cupric reducing potency (r = 0.221), revealing
that TFC minorly contributes to the scavenging of ABTS and reducing
the cupric ions with efficacy in extinguishing DPPH free radicals. Both
components (TPC and TFC) participated in free radical scavenging ac-
tivities and reducing oxidative stress with varying extents of efficacy in
diabetes. The association between TPC and glucose uptake showed a
positive strong relationship with TPC (r = 0.982) and a weak to a
moderate relationship with TFC (r = 0.463). Phenolics and flavonoid
compounds are known for their ability to lower blood glucose levels
and their antidiabetic efficacy, which may be attributed to the potent
antidiabetic activity of LSAF (Lu et al., 2011; Song, Manson, Buring,
Sesso, & Liu, 2005; Tadera, Minami, Takamatsu, & Matsuoka, 2006).
The LC-MS/MS results revealed the presence of luteolin-7-glucoside
(16.83 µg/g) and lower quantities of protocatechuic acid (4.79 µg/g).
Both components play roles in the antidiabetic potency of LSAF, as
shown in the activity of the detected constituents recorded in the lit-
erature (Harini & Pugalendi, 2011), Yanqing, Kiharu, & Yu, 2016).
Future studies should clarify whether the full activity of the extract is
due to a single phytochemical component or a synergistic effect of a
mixture of diverse compounds present in the extract.
5. Conclusion
We have provided a scientific basis for the traditional usage of ca-
labash as an antidiabetic remedy via experimental studies of the in vivo
antidiabetic potency of different fruit extracts in normal and alloxan-
induced diabetic mice. This was further confirmed by the significant
cell-based in vitro antidiabetic activity of the physiologically active
extract, with speculation as to the activation of insulin cascades. The
high phenolic content of calabash fruit could contribute to the ameli-
oration of the oxidative stress associated with diabetes.
Ethical statement
(1) This material is the authors' own original work, which has not been
previously published elsewhere.
(2) The paper is not currently being considered for publication else-
where.
(3) The paper reflects the authors' own research and analysis in a
truthful and complete manner.
(4) The paper properly credits the meaningful contributions of co-au-
thors and co-researchers.
(5) The results are appropriately placed in the context of prior and
existing research.
(6) All sources used are properly disclosed (correct citation). Literally
copying of text must be indicated as such by using quotation marks
L.Y.M. Juee and A.M. Naqishbandi
and giving a proper reference.
(7) All authors have been personally and actively involved in sub-
stantial work leading to the paper and will take public responsi-
bility for its content.
CRediT authorship contribution statement
Lana Y.M. Juee: Software, Data curation, Writing - original draft,
Visualization, Investigation, Validation, Writing - review & editing.
Alaadin M. Naqishbandi: Conceptualization, Methodology,
Supervision, Writing - review & editing.
Acknowledgments
The authors would like to thank Prof. Thomas Efferth, the chair of
the department of pharmaceutical biology at the Institute of Pharmacy
and Biochemistry Johannes Gutenberg University Mainz, for support
with the in vitro study, and the animal house staff of Medicine College
at Hawler Medical University for their assistance with the in vivo study.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jff.2020.103821.
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Further reading
Shah, B. N., Seth, A. K., & Desai, R. V. (2010). Phytopharmacological profile of Lagenaria
siceraria: a review. Asian Journal of Plant Science, 9, 152–157. https://scialert.net/
abstract/?doi=ajps.2010.152.157.