separations

Article
Cucumis metuliferus L. Fruits Extract with Antioxidant,
Anti-Inflammatory, and Antidiabetic Properties as Source of
Ursolic Acid
Anna Cazanevscaia Busuioc 1 , Giorgiana Valentina Costea 2 , Andreea Veronica Dediu Botezatu 1 ,
Bianca Furdui 1, * and Rodica Mihaela Dinica 1, *

1 Department of Chemistry, Physics and Environment, “Dunărea de Jos” University of Galati, 111 Domnească
Street, 800201 Galati, Romania; anna.cazanevscaia@ugal.ro (A.C.B.); andreea.botezatu@ugal.ro (A.V.D.B.)
2 Department of Food Science, Food Engineering and Applied Biotechnology, “Dunărea de Jos” University of
Galati, 111 Domnească Street, 800201 Galati, Romania; giorgiana.costea@ugal.ro
* Correspondence: bianca.furdui@ugal.ro (B.F.); rodica.dinica@ugal.ro (R.M.D.)

Abstract: To identify healthy, nutritious, and sustainable plant-based products rich in biologically
active compounds, this present study was conducted, and the phytochemical composition and bio-
logical properties of the hydroethanolic ultrasound-assisted extract of the fruits of Cucumis metuliferus
were investigated. Cucumis metuliferus is an unexplored fruit of a climbing plant in the Cucurbitaceae
family, widely distributed in the tropical and subtropical regions of sub-Saharan Africa and whose
nutritional and medicinal benefits are well known in African countries, especially. Therefore, its
cultivation in other regions could influence chemical composition. The structural identification of
the compounds from the hydroethanolic extract from Cucumis metuliferus fruits grown in Romania
was carried out by chromatographic techniques (HPLC). The main compounds identified were
catechin, oleanolic acid, ursolic acid, p-coumaric acid, and epicatechin. Subsequently, a method
was proposed to isolate and characterize ursolic acid, one of the major compounds. The obtained
results show that the hydroethanolic extract is rich in antioxidant compounds evaluated using the
DPPH radical inhibition method (IC50 = 32.74 ± 0.02 µg/mL) and ABTS cation radical inhibition
Citation: Busuioc, A.C.; Costea, G.V.; method (IC50 = 11.37 ± 0.07 µg/mL). It also demonstrate in vitro anti-inflammatory activities, such
Botezatu, A.V.D.; Furdui, B.; Dinica,
as anti-lipoxygenase (IC50 = 32.90 ± 0.05 µg/mL) and anti-proteinase (IC50 = 16.34 ± 0.07 µg/mL),
R.M. Cucumis metuliferus L. Fruits
and antidiabetic properties by inhibiting α-amylase (IC50 = 429.541 ± 0.25 µg/mL) and β-glucosidase
Extract with Antioxidant,
activity (IC50 = 385.685 ± 0.76 µg/mL). Therefore, C. metuliferus fruits could be effectively used in
Anti-Inflammatory, and Antidiabetic
the development of various health-promoting products, being not only appetizing, with spectacular
Properties as Source of Ursolic Acid.
Separations 2023, 10, 274. https://
appearance and with extended storage life, but also curative and healthy.
doi.org/10.3390/separations10050274
Keywords: C. metuliferus; ursolic acid; separation; antioxidant; anti-inflammatory activities
Academic Editors: Nguyen Van
Quan and Tran Dang Xuan

Received: 31 March 2023

Revised: 20 April 2023 1. Introduction
Accepted: 21 April 2023 In recent years, healthy food and the high prevalence of malnutrition have become
Published: 23 April 2023 a topic of interest as diet affects human health and the sustainability of the planet [1,2].
Increasingly, people around the world are recognizing the importance of promoting healthy
and sustainable food systems in order to ensure food security for the current global popula-
tion as well as for generations to come [3,4]. Today’s society’s high interest in swapping
Copyright: © 2023 by the authors.
some foods for healthier versions and also improving the nutritional profile has led to major
Licensee MDPI, Basel, Switzerland.
This article is an open access article
changes in the food industry [5,6]. In this context, due to a rich composition in a variety
distributed under the terms and
of essential nutrients, vitamins, minerals, antioxidant compounds, and good adaptation
conditions of the Creative Commons
to climate changes, Cucumis metuliferus can be an important ingredient for improving dif-
Attribution (CC BY) license (https:// ferent food products for healthy diets with high nutritional potential, essential for human
creativecommons.org/licenses/by/ health and sustainable food for the future. This aspect is especially important for people
4.0/). with special dietary restrictions due to certain medical conditions [7–11]. Different parts

Separations 2023, 10, 274. https://doi.org/10.3390/separations10050274 https://www.mdpi.com/journal/separations

Separations 2023, 10, 274 2 of 15

of this plant (the fruit’s pulp and seeds) can be used for their nutritional and medicinal
properties [12,13], rapid growth, and long shelf life (over 6–7 months without cold stor-
age) [14], alongside other nutritious vegetables and fruits, to achieve the most benefit from
a healthy diet.
Cucumis metuliferus is a species of the cucurbit (Cucurbitaceae) family with ellipsoid
fruits of green color that change to orange color during ripening [15]. The rind is covered
with cone-shaped protuberances with pointed tips. Inside, the fruit contains a juicy, green,
slimy mass with many white seeds [16]. The fruits come in two forms, the bitter form
containing mainly cucurbitacins (triterpenoids), and the non-bitter form, which is more
cultivated [17]. This plant is native to Africa and has been mentioned in other studies as
jelly melon, horned cucumber, spiked melon, horned melon, or kiwano. It is internationally
spread due to its rapid growth and adaptation to various climate conditions. Its history
began approximately in 1982 with a family from New Zealand who started to cultivate
it for commercial purposes. In just two years, they managed to export the “phenomenal”
fruit to Japan, where it was received with curiosity and interest. Later, it became a fruit of
interest among American farmers and then easily entered other markets. Under the name
of horned melon, this plant soon reached Europe [12,18]. Currently, this plant is cultivated
in many countries, and opinions about its taste and appearance are divided.
It is well known that the chemical compounds of each species may vary due to various
factors such as variety, maturity, growing conditions, and soil [16]. Moreover, the variation
in the chemical composition can lead to differences in therapeutic properties [19]. Therefore,
different parts of medicinal plants can potentially be excellent sources of phytochemical
compounds with biological activity. In general, fruits contain a combination of important
nutrients, such as proteins, carbohydrates, fats, vitamins, and minerals. The seeds are
rich in linoleic and oleic acids, which are important for human health. In particular, some
studies show that oleic acid can help lower blood pressure [17,20,21]. γ-tocopherol and α-
tocopherol identified in considerable quantities in kiwano fruits are two types of vitamin E
known for their antioxidant capacity [22]. The fruit of C. metuliferus has a wide list of health
benefits due to its varied phytochemical composition. The pulp contains considerable
amounts of β-carotene, vitamin A, and vitamin C, which are very important for strengthen-
ing the immune system, night vision, or the curative effect of the skin [23,24]. In addition, a
wide range of other secondary metabolites has been identified, such as glycosides, alkaloids,
flavonoids, saponins, steroids, tannins, and terpenoids [25]. The fruits are also characterized
by a high content of minerals such as iron, potassium, phosphorus, zinc, magnesium, cop-
per, calcium, and sodium [15]. Among the numerous secondary metabolites contained by
C. metuliferus species, ursolic acid (UA) (3β-3-hydroxy-urs-12-en-28-oic-acid) is a compound
of great interest. This pentacyclic triterpenoid compound has low toxicity and is known for
its beneficial therapeutic properties, including antioxidant, antimicrobial, hepatoprotective,
immunomodulatory, antiviral, antitumor, chemopreventive, anti-inflammatory, cardiopro-
tective, antihyperlipidemic, and hypoglycemic properties [26,27]. Thus, some studies have
shown that it can help prevent and treat obesity by modulating fat and glucose metabolism
and reducing fat storage in cells [28]. In addition, ursolic acid can help to relieve diabetes
symptoms by lowering blood sugar levels and stimulating insulin secretion. In the case
of the inflammatory process, UA can help to reduce it by modulating the activity of in-
flammatory enzymes and blocking molecules that trigger inflammation [26,27,29]. Various
technologies are known for UA extraction and purification, both traditional (maceration,
Soxhlet, heat reflux) and modern (microwaves, ultrasound, and supercritical fluid) [27,30].
In recent years, researchers have explored structural modifications of ursolic acid intending
to produce novel derivatives that possess enhanced biological activities and increased
bioavailability [31–34].
For the purification of various compounds from extracts of various plants, different
methods and different auxiliary substances were used [35,36]. Other methods for the pu-
rification of UA are based on chromatographic separation of crude extracts, on the column
with macroporous adsorbent resins or silica gel [37], by thin-layer chromatography [38],
Separations 2023, 10, 274 3 of 15

or by fractioned extraction with solvents. Ursolic acid is almost non-toxic and is used
in various cosmetics and health products either as an active ingredient or in a mixture
with other active substances. In addition, it can be used as a natural compound in the
semi-synthesis of numerous new bioactive molecules [39].
Our study aimed to assess the phytochemical profile and the antioxidant, anti-inflammatory,
and antidiabetic activities of ultrasound-assisted extracts from C. metuliferus “Tempus”
fruits grown in Romania (Southeastern Europe). Furthermore, the study proposes a new
method for the separation and purification of one of the major compounds, ursolic acid,
from C. metuliferus “Tempus” fruit extracts, which will be further explored in future studies
as a lead compound for the development of new derivatives with therapeutic properties.

2. Materials and Methods

2.1. Reagents, Plant Materials, and Apparatus
All reagents and solvents used for isolation, purification, and analysis were purchased
from Sigma-Aldrich and Merck (Darmstadt, Germany). The ethanol used for extraction was
of analytical grade. The standard compound ursolic acid (≥95%, HPLC grade) was also pur-
chased from Merck. All optical density measurements for antioxidant, anti-inflammatory,
and antidiabetic activities were performed with a multiplate reader (Tecan Trading AG,
Tecan Pro 200, Männedorf, Switzerland). The fruits used in this study were collected
at the ripening stage from the Station of Vegetable Research and Development (SCDL),
Buzau, Muntenia Region, Romania (Southeastern Europe, 45◦ 090 30.100 N 26◦ 490 39.200 E).
An ultrasonic bath was used for the extraction with solvents (Bandelin Sonorex Ultrasonic,
Berlin, Germany, with a digital timer, temperature control, and a frequency of 35 kHz).

2.2. Preparation of Crude Exact of C. metuliferus

Fruits of C. metuliferus “Tempus” were examined for integrity, absence of dust, and
insect contamination, then cleaned in the first stage with ultrapure water as a first step.
Then, the fresh fruits were washed with deionized water, cut into small pieces, and dried at
50 ◦ C in a hot air oven for 48 h. The dried fruits were ground in an electronic mill, and the
resulting powder was passed through a 60-mesh sieve. The obtained powdered and dried
material was stored in a dark and dry place until use.
The dry material was successively extracted three times, with hexane, under reflux,
filtered, and dried, followed by a repetitive extraction with 70% ethanol in an ultrasonic
bath. The optimal time and temperature conditions for ultrasonic extraction were selected
from the literature [40]: 40 ± 5 ◦ C and 60 min. The final step consisted of the evaporation
of the solvent with a rotary evaporator. The obtained extract was kept at 2–4 ◦ C and
further used in the following analyses and for the isolation and purification of the ursolic
acid [41,42].

2.3. HPLC-DAD Analysis of the Extract

High-performance liquid chromatography studies using photodiode arrays (HPLC-
DAD) were performed with equipment (Rigol Technologies Inc., Beijing, China), with an
autosampler, binary pump, vacuum degasser, and photodiode array detector. A Synergi
Polar-RP C18 (250 × 4.6 mm, 4 µm) analytical column (Phenomenex, Chesire, UK), preceded
by a security cartridge, was used for chromatographic separation. Quantification of the
different analytes was performed according to the validated method as described in other
studies [43]. Quantification curves were generated by injecting 1 to 50 mg/L of the standard
compound solutions at five different concentrations. Thus, correlation coefficients (R2 )
greater than 0.99 were obtained, indicating a good linearity of the HPLC-DAD method.
The mobile phase was a mixture of 0.1% formic acid in water (v/v) and 0.1% formic acid
in methanol (v/v) [13]. UV-visible spectra (210–520 nm) for the analyzed compounds
were recorded so that each compound was analyzed at the corresponding wavelength; the
data are presented in Table 1. The organic compounds were identified by combining the
Separations 2023, 10, 274 4 of 15

following data: comparison with standard compounds, retention time, and elution order,
and quantified by using quantification curves of the analyzed compounds.

Table 1. Chemical composition of the hydroethanolic extract of the fruit of C. metuliferus from
HPLC-DAD analysis.

HPLC-DAD UHPLC-MS
Compound λ, [M-H]−
RT * µg/g of Extract SD ** RSD % Exact Mass
nm Ion (m/z)
Hydroxybenzoic acids
Gallic acid 5.9 272.00 401.39 1.46 0.36 170.02152 169.01302
Catechin 17.6 280.00 6576.76 3.86 0.06 290.07904 289.071
Epicatechin 23.9 280.00 2016.71 3.59 0.18 302.04265 289.071
Procianidin B2 24.3 230.00 35.88 0.22 0.61 578.14242 577.1558
Procianidin A2 29.9 230.00 756.10 1.86 0.25 578.14242 577.1558
Flavonols
Rutin 31.4 265.00 39.42 0.23 0.60 610.15338 609.14613
Quercetin 35.8 365.00 5.98 0.56 9.43 302.04265 301.23813
Quercetin-3-D-galactoside 32.0 265.00 11.62 0.28 2.45 464.09548 463.0876
Kampferol-3-glucoside 33.6 265.00 21.90 0.70 3.20 448.10056 447.09331
Kampferol 37.8 365.00 5.14 1.49 28.88 286.04774 285.13422
Hydrocinnamic acids
Neochlorogenic acid 10.5 325.00 22.89 0.53 2.31
Chlorogenic acid 22.3 325.00 77.39 0.15 0.19 354.09508 354.09508
Caffeic acid 22.9 325.00 58.65 0.57 0.96 180.04226 179.03501
p-Coumaric acid 28.9 325.00 116.11 1.46 1.26 164.04734 163.03954
trans-Ferulic acid 30.5 325.00 34.27 1.86 5.42 194.05791 193.05066
Triterpenes
Oleanolic acid 45.8 210.00 551.83 3.25 0.59 456.36034 455.35309
Ursolic acid 45.9 210.00 577.31 3.19 0.55 456.36034 455.35309
* RT—retention time; ** SD—standard deviation.

2.4. Isolation and Purification of Ursolic Acid from C. metuliferus Extract

The isolation and purification of ursolic acid were carried out from the hydroethanolic
extract of C. metuliferus species, obtained as previously described, using silica gel column
chromatography, then analyzed by HPLC-DAD. The ethanolic extract was subjected to
column chromatography using a silica gel column (30 × 2.0 cm) preconditioned with
chloroform. For elution, a solution of 2 to 4% methanol in chloroform was used. The eluent
was collected at every 3–5 mL fraction. After, all the fractions that were collected during the
elution process were subjected to TLC testing on silica gel 60 F254 plates (Merck, Darmstadt,
Germany). A mixture of chloroform: methanol (95:5, v/v) was used as the mobile phase,
and UV light was used for revealing (254 nm, 365 nm, and white light). The fractions with
the same Rf were combined and subjected to solvent evaporation [41,44].

2.5. Structure Elucidation and Identification of the Isolated Compound

2.5.1. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy
(FTIR-ATR) Analysis
The obtained fractions were analyzed using a Nicolet iS50 FT-IR spectrometer (Thermo
Scientific, Waltham, MA, USA) assembled with an ATR accessory. The analysis was performed
with a resolution of 4 cm−1 by coaddition of 32 scans in the range 4000 cm−1 –400 cm−1 . Air
was carried out as a reference for the background spectrum before each sample analysis,
and the diamond crystal was cleaned with alcohol after each sample. Analyses were
performed in a temperature-controlled room (21 ± 2 ◦ C) [45].

2.5.2. Analysis of Fraction by UHPLC-MS

In order to certify the presence of ursolic acid in the obtained fraction, the analysis
using ultrahigh-performance liquid chromatography coupled with mass spectrometry
Separations 2023, 10, 274 5 of 15

(UHPLC-MS) was performed. A method similar in certain parameters was used, with
a mobile phase similar to that previously described, while maintaining the parameters
for MS. For ionization, a HESI (Heated ElectroSpray Ionization) ion source in negative
mode was used. The calibration solution was used for external calibration in both negative
and positive modes. Full scan HRMS (high-resolution mass spectrometry) analysis of the
compounds was accomplished using a mass spectrometer from Q-Exactive. Full scan data
in negative mode were recorded at 70,000 FWHM resolving power, at m/z 200. A scan
range of m/z 100–1000 Da was chosen. The temperature of the heat source and capillary
was set at 300 ◦ C [46,47].

2.5.3. NMR Analysis

Samples for NMR (nuclear magnetic resonance) experiments were performed in
deuterated solvent, CD3 OD, and analyzed using a Varian mercury-plus spectrometer,
400 MHz for 1 H. 1 H chemical shifts were reported in parts per million (ppm), while the
coupling constant (J) was Hertz (Hz) (Figures S1–S3, Supplementary Materials) [48].

2.6. Antioxidant Assay

2.6.1. DPPH Scavenging Activity
The antioxidant activity of the extract was analyzed by the DPPH (1,1-diphenyl-2-
picrylhydrazyl) radical scavenging test [49]. This method is based on the change of purple
color to yellow, resulting in a reduced structure of the DPPH radical. The method was
performed in a 96-well plate, and the absorbance was recorded at 517 nm after 30 min
using a microplate reader. Calculations were made according to the literature [46,50]. The
standard compound Trolox was used as a positive control due to its known antioxidant
properties [51].

2.6.2. ABTS Radical Cation Decolorization Assay

For this antioxidant activity, a previously prepared solution of ABTS•+ (2,20 -azino-
bis (3-ethylbenzothiazoline-6-sulfonic acid) was used [52,53]. The assay was performed
according to the descriptions in the literature, with minor modifications [54,55]. The
absorbance of the solution was measured at 734 nm in a 96-well plate using the microplate
reader. Samples were prepared by mixing 100 µL of the kiwano extract with 100 µL of
ABTS•+ radical cation solution, and after 30 min, the absorbance was measured. Results
were conveyed using a Trolox calibration curve.

2.7. In Vitro Anti-Inflammatory Activities

2.7.1. Anti-Lipoxygenase Activity
To perform the test, a reaction mixture (200 µL) was obtained by adding 160 µL of
sodium phosphate buffer (pH 8.0) to 10 µL of the sample dissolved in Tris buffer (pH 7.4) at
various concentrations, and also 20 µL of lipoxygenase. The reaction mixture was incubated
at 25 ◦ C for 10 min. To initiate the reaction, 10 µL of the linoleic acid solution was added
as substrate and incubated at 25 ◦ C for 6 min, and the absorbance at 234 nm was read in
the microplate reader [46,56,57]. Indomethacin (250–255 µg/mL) was used as a positive
control due to its known anti-lipoxygenase properties. The percentage of inhibition was
calculated using the following formula:

% Inhibition = (AControl − ASample )/(AControl ) × 100 (1)

2.7.2. Anti-Proteinase Action

In this method, samples dissolved in 20 mM Tris-HCl buffer (pH 7.4)) were mixed with
trypsin solution. The reaction mixture was incubated for 5 min at 37 ◦ C and then 0.3 mL of
1.5% casein (w/v) was added. Then, the obtained mixture was again incubated for 20 min.
The final step to complete the reaction was performed by adding 200 µL of 70% perchloric
acid. Prior to spectrophotometric analysis of the samples, consisting of absorbance mea-
Separations 2023, 10, 274 6 of 15

surement at 210 nm, the mixture was centrifuged for 5 min at 5000 rpm [58,59]. Aspirin
(10–500 µg/mL), a known inhibitor of proteinase activity, was used as a positive control.
The percentage of inhibition was calculated according to the following equation:

% Inhibition = (AControl − ASample )/(AControl ) × 100 (2)

2.8. In Vitro Evaluation of the Antidiabetic Activity

The α-amylase inhibition test was assessed with the 3,5-dinitrosalicylic acid (DNS)
method [60], with minor modifications. Briefly, 80 µL of each sample or positive control
was added to 40 µL of α-amylase solution (2 U/mL, α-Amylase from Aspergillus oryzae, EC
Number: 232-588-1, Sigma-Aldrich® ) and incubated for 20 min at 37 ◦ C. After incubation,
140 µL of 1% starch solution was added to each tube and incubated again for 30 min. The
reaction was terminated by adding 0.40 mL of DNS 1% and heating it in a boiling water
bath for 10 min. The reaction mixture was cooled to room temperature and then diluted
with 0.80 mL phosphate buffer when the absorbance at 540 nm was measured. The blank
sample was prepared by replacing the plant extract samples with 0.20 mL of phosphate
buffer. A positive control sample was prepared using acarbose (0.025–1 mg/mL), a known
amylase inhibitor [61]. The α-amylase inhibitory activity was expressed as a percentage of
inhibition and calculated according to the following formula:

% α-amylase Inhibition = (AControl − ASample )/(AControl ) × 100 (3)

The β-glucosidase inhibition test was assessed as previously described [62]. Briefly, a
reaction mixture was obtained from 15 µL phosphate buffer (pH 5.0), 20 µL p-nitrophenyl-
β-D-glucopyranoside (1mg/mL, Sigma-Aldrich® ) and 20 µL sample or positive control
at different concentrations. The 96-well plates were incubated in a microplate reader for
10 min at 37 ◦ C. Then, 5 µL β-glucosidase (from almonds, Sigma-Aldrich® ) solution was
added (2.5 mg/mL) to each well and incubated again for 30 min at 37 ◦ C. The reaction was
terminated by addition of 0.140 mL buffer (pH = 10). Absorbance was measured at 410 nm
using a microplate reader. As positive control, acarbose was used, being a known inhibitor
of glucosidase [62]. The β-glucosidase inhibitory activity was expressed as a percentage of
inhibition and calculated according to the Formula (4).

% β-glucosidase Inhibition = (AControl − ASample )/(AControl ) × 100 (4)

2.9. Statistical Analysis

The carried experiments were achieved in triplicate, and the presented data represent
the average of the three individual determinations ± standard deviation (SD). Statistical
evaluation of the obtained data was estimated by one-way analysis of variance (ANOVA)
to find out significant differences (p ≤ 0.05). The inhibitory concentration IC50 values were
calculated from linear regression analysis of the percent activity depending on concentration
by using the GraphPad Prism software (vs. 5.0).

3. Results and Discussion

3.1. Preparation of Crude Exact of C. metuliferus
The current study presented an adapted method for obtaining a hydroethanolic
ultrasound-assisted extract of C. metuliferus. Further, for the purification and characteriza-
tion of ursolic acid, one of the major terpenoid compounds, two steps were proposed, using
two solvents with different polarities. The first step was to remove the other compounds.
The combined filtrate obtained after extraction was concentrated under reduced pressure
to obtain, from 1 kg of plant material, 183 g of crude extract, which has been used for
the following analyses and also for the isolation and purification of the major terpenoid
compound, ursolic acid. Figure 1 shows the scheme for obtaining the crude extract starting
from the fresh fruits of C. metuliferus.
 tion of ursolic acid, one of the major terpenoid compounds, two steps were pr
using two solvents with different polarities. The first step was to remove the oth
pounds. The combined filtrate obtained after extraction was concentrated under r
pressure to obtain, from 1 kg of plant material, 183 g of crude extract, which h
used for the following analyses and also for the isolation and purification of th
Separations 2023, 10, 274 7 of 15
terpenoid compound, ursolic acid. Figure 1 shows the scheme for obtaining th
extract starting from the fresh fruits of C. metuliferus.

Figure
Figure 1. Separation 1. Separation
scheme scheme
starting from starting
the fruits from
of C. the fruits of C. metuliferus.
metuliferus.

3.2. Identification3.2.
of Bioactive Compounds
Identification by HPLC
of Bioactive Compounds by HPLC
The results of thisThe results of this studychemical
study concerning the concerningcomposition
the chemicalof the hydro-ethanolic
composition of the hydro-e
extract from theextract C. metuliferus,
fruit offrom obtained by HPLC-DAD analysis, are presented
the fruit of C. metuliferus, obtained by HPLC-DAD analysis, are prese
in Table 1. Thus,Table
for the
1. compound
Thus, for theclass of flavan-3-ols,
compound theflavan-3-ols,
class of highest amount of catechin
the highest amount of c
(6576.76 ± 3.89 µg/g dry extract) and epicatechin (2016.71 ±
(6576.76 ± 3.89 µg/g dry extract) and epicatechin (2016.71 ± 3.59 µg/g and
3.59 µg/g dry extract) dry extract)
the lowest amount of procyanidin
lowest B2 (35.88 µg/g
amount of procyanidin B2 dry extract)
(35.88 wereextract)
µg/g dry determined.
were determined.
In the case of hydroxycinnamic
In the case of acids compounds, p-coumaric
hydroxycinnamic acid (116.11
acids compounds, ± 1.46 µg/g
p-coumaric acid (116.11
dry extract) was identified in the highest concentration, followed by chlorogenic acid
µg/g dry extract) was identified in the highest concentration, followed by chlo
(77.39 ± 0.15 µg/g dry extract). Several representative flavonoids, such as rutin
acid (77.39 ± 0.15 µg/g dry extract). Several representative flavonoids, such as ruti
(39.42 ± 0.23 µg/g dry extract) and kaempferol-3-glucoside (21.90 ± 0.70 µg/g dry extract),
± 0.23 µg/g dry extract) and kaempferol-3-glucoside (21.90 ± 0.70 µg/g dry extrac
were identified in much smaller amounts compared to other compounds. Additionally, the
identified in much smaller amounts compared to other compounds. Additiona
pentacyclic triterpenoids, ursolic acid (577.31 ± 3.19 µg/g dry extract), and oleanolic acid
pentacyclic triterpenoids, ursolic acid (577.31 ± 3.19 µg/g dry extract), and oleano
(551.831 ± 3.25 µg/g dry extract) were identified in high concentrations. A composition
(551.831 ± 3.25 µg/g dry extract) were identified in high concentrations. A comp
rich in the biologically active triterpenoid ursolic acid of the extract was highlighted, which
rich in the biologically active triterpenoid ursolic acid of the extract was high
was further investigated by isolation and characterization in the next step of our study.
According to other studies from the literature, fruits of C. metuliferus grown in an
ecological farm on the Fruška gora Mountain (Pannonia plain, Novi Sad, Republic of
Serbia) showed considerable amounts of polyphenols, expressed as gallic acid (GAE), in
different parts of the plant, such as 58.22–74.90 mg GAE/100 g in pulp, between 1728.94
and 1923.52 mg GAE/100 g in peel extracts samples, and 141.25 mg GAE/100 g in seed
extracts [23]. Another study that demonstrated the neuroprotective action of this plant pre-
sented the chemical composition of the fruits from Hby local farm located at Cinfães, Douro,
Portugal, with smaller concentrations of gallic acid (11.7 ± 0.6 mg/100 g), p-coumaric
acid (2.86 ± 0.14 mg/100 g), protocatechuic acid (7.69 ± 0.38 mg/100 g), (+)-catechin
(4.86 ± 0.24 mg/100 g), and (-)-epicatechin (4.58 ± 0.23 mg/100 g) [63]. Several studies
on the African species of C. metuliferus also highlighted the presence of a wide range of
vitamins (vitamin A 198.51 mg/mL, B6 835.00 mg/mL, B12 0.10 mg/mL, C 682.00 mg/mL,
D 5.28 mg/mL, E 0.42 mg/mL) [64,65] and minerals (Ca, Al, Mg, Zn, K) [22,64,66].

3.3. Analysis and Identification of Ursolic Acid

All the obtained fractions after the separation of the phytochemical compounds from
the hydroethanolic extract of C. metuliferus fruits by using column chromatography were
Separations 2023, 10, 274 8 of 15

characterized through FT-IR, and the corresponding fraction of ursolic acid was identified.
Ursolic acid was isolated from the ultrasound-assisted extract from kiwano fruit (Table 2)
and further characterized by IR spectroscopy, mass spectroscopy, and 1 H NMR, and the
results were confirmed by comparing them with other studies [67–69].

Table 2. Characterization of the isolated compound.

Physical Properties Ursolic Acid Fraction

Color white
Melting Point 291–293 ◦ C
Solubility soluble in ethanol, DMSO
Rf Value
Separations 2023, 10, x FOR PEER REVIEW 0.41 9 of 15
Solvent system: chloroform: methanol (95:5, v/v)

3.3.1. FT-IR Analysis

νs (COO
All the; fractions
− C=O) w
obtained after1405.15 1406.03
the separation of the phytochemical compounds from
δ (CH ) w 1385.72 1386.45
C. metuliferus fruit’s ethanolic extract using column chromatography were characterized
s 3

through βs (OH)
FT-IRmanalysis, and the functional
1374.72 groups were identified1375.79 based on data in the
ν(C-OH)
literature. m
The fraction 1029.87
corresponding to ursolic acid was of interest,1030.96
and in this study, the
ATR-FTIR
γ (−C = C−,spectrum
CH) m identified as ursolic
999.56 acid was analyzed in a range of 4000 to 400 cm−1
999.59
with a resolution − 1
ν (C-C, C-O, C-H)ofw4 cm . Table971.08 3 shows the vibration bands of ursolic
972.12 acid and their
characteristic
δs (CH3) w functional groups. Interpretation
807.15 and assignment of IR bands (Figure 2) were
808.14
made based on data in the literature concerning ursolic acid [67–70].
and intensities: s—strong, w—weak, m—medium; νass—asymmetric stretch; νs—symmetric stretch.

UA
105 ursolic acid

100

95

90

85

80
%T

3523.22

828.84
1244.06

75
885.87
1359.65
1291.16

563.23

νas(COO-)
1181.69

923.83
1374.79

70 ν (OH)
1092.18

732.97

509.97
971.12
1453.80

ν(OH)
2954.34

606.14
763.69
999.59

65
650.44
807.15
1029.82

ν (C-H) β as (OH)
660.58
1122.12

60
ν (C=O)
1161.14

δs(CH3)
βs(OH) ν (OH)
1714.54

55
(C-OH) δs(CH3)

4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers (cm-1)

Figure
Figure 2.
2. The
The FTIR-ATR
FTIR-ATR spectra
spectra of
of pure
pure ursolic
ursolic acid
acid (purple
(purple spectra)
spectra) and
and isolated
isolated ursolic
ursolic acid
acid (UA,
(UA,
red spectra).
red spectra).

3.3.2. Sample Analysis by UPLC-MS

The UHPLC-MS chromatographic data demonstrated that the isolated compound
was the ursolic acid (supplementary material) by using the mass spectra.

3.3.3. NMR Analysis

The ursolic acid was identified using 1H-NMR spectroscopy by comparing the UA
assignments with data in the literature. The 1H-NMR spectra of the isolated UA revealed
the presence in the higher field region of five methyl groups by a corresponding singlet
(0.77, 0.88, 0.95. 0.97, and 1.10 ppm), two methyl groups by a doublet (0.87 and 0.99 ppm),
and the peaks were characteristic for the skeleton of ursane-type triterpenes [71,72]. Two
Separations 2023, 10, 274 9 of 15

Table 3. The main characteristic experimental FTIR-ATR bands (cm−1 ) of standard commercial UA
and isolated UA from C. metuliferus fruits extract and their assignments.

ATR-IR Bands (cm−1 )

Assignments
Standard Commercial UA Isolated UA from C. metuliferus
νass (OH) w 3523.33 3523.22
ν (O–H) w 2965.41 2966.61
ν (CH) m 2953.90; 2918.75 2954.34; 2918.32
νass (C=O) s 1714.66 1715.54
νas (COO− ) w 1553.56 1543.80
βas (OH) m 1453.78 1454.54
νs (COO− ; C=O) w 1405.15 1406.03
δs (CH3 ) w 1385.72 1386.45
βs (OH) m 1374.72 1375.79
ν(C–OH) m 1029.87 1030.96
γ (−C=C−, CH) m 999.56 999.59
ν (C–C, C–O, C–H) w 971.08 972.12
δs (CH3 ) w 807.15 808.14
and intensities: s—strong, w—weak, m—medium; νass —asymmetric stretch; νs —symmetric stretch.

3.3.2. Sample Analysis by UPLC-MS

The UHPLC-MS chromatographic data demonstrated that the isolated compound was
the ursolic acid (supplementary material) by using the mass spectra.

3.3.3. NMR Analysis

The ursolic acid was identified using 1 H-NMR spectroscopy by comparing the UA
assignments with data in the literature. The 1 H-NMR spectra of the isolated UA revealed
the presence in the higher field region of five methyl groups by a corresponding singlet
(0.77, 0.88, 0.95. 0.97, and 1.10 ppm), two methyl groups by a doublet (0.87 and 0.99 ppm),
and the peaks were characteristic for the skeleton of ursane-type triterpenes [71,72]. Two
singlet peaks at δH 1.10 ppm, characteristic for the methyl group of oleanane triterpene
skeleton, and at δH 1.29 ppm for the methylene protons –CH2 - were also observed in the
isolated UA. The peak at δH 3.3 ppm from the lower field region, representing H, may be
due to the hydroxyl group, and at δH 4.94 ppm, an olefinic proton peak was assigned to
the H of a triterpene.
Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid): powder, white, 1 H-NMR (CD3 OD)
(supplementary material); 1 H-NMR (CD3 OD): 1.63 & 1.69 (H-1,2H, m), 1.65 (H-2, 2H, m),
3.13 (H-3, 2H), 0.74 (H-5, 1H, d), 1.43 & 1.29 (H-6, 2H, m), 1.29 (H-7, 2H, m), 1.56 (H-9, 1H,
m), 1.91 (H-11, 2H, q), 5.21 (H-12, 1H, t), 1.62 (H-15, 2H, m), 1.58 & 1.63 (H-16, 2H, m),
2.20 (H-18, 1H), 1.38 (H-19, 1H, m), 1.36 (H-20, 1H, m), 1.32 (H-21, 2H, m), 1.53(H-22, 2H,
m), 0.95 (H-23, 3H, s), 0.77 (H-24, 3H, s), 0.97 (H-25, 3H, s), 0.84 (H-26, 3H, s), 1.10 (H-27,
3H, s), 0.87 (H-29, 3H, d), 0.95 (H-30, 3H, d).

3.4. Evaluation of the Antioxidant Activity

It is known that oxidative stress leads to the production of free radicals, and this can
affect various cellular events, some of which are cancer proliferation, inflammation, and
aging [73,74]. Various in vitro and in vivo studies have been performed to demonstrate
the antioxidant of C. metuliferus extracts and ursolic acid, but only a few investigated the
anti-inflammatory properties [65,75,76]. Table 4 shows the results of the two methods
applied by us for the evaluation of the antioxidant activity data compared with Trolox and
ascorbic acid, known for their antioxidant properties.
The purpose of this present research was to analyze the antioxidant activity of the hy-
droethanolic extract and also of the purified fraction of ursolic acid obtained from C. metuliferus.
The obtained fraction of ursolic acid presented IC50 values (IC50 DPPH = 4.27 ± 0.009 µg/mL
and IC50 ABTS = 6.96 ± 0.014 µg/mL) close to those reported by data in the literature
Separations 2023, 10, 274 10 of 15

for the pure compound [77–79], greater than that of ascorbic acid and Trolox. The extract
demonstrated good antioxidant activity, greater than Trolox and ascorbic acid in an ABTS
assay (IC50 = 11.37 ± 0.071 µg/mL). The antioxidant activity of C. metuliferus extract can
be due to its rich composition in various active compounds, such as catechin, epicatechin,
oleanolic acid, ursolic acid, and gallic acid.

Table 4. Evaluation of the antioxidant activity.

Samples IC50 DPPH (µg/mL) IC50 ABTS (µg/mL)

Fruit extract 32.74 ± 0.022 d 11.37 ± 0.071 b
Isolated ursolic acid 4.27 ± 0.009 a 6.96 ± 0.014 a
Trolox 10.57 ± 0.002 b 32.56 ± 0.002 c
Ascorbic acid 20.34 ± 0.034 c 13.76 ± 0.044 b
The values succeeded by the same letters (a, b, c, d) in the same column show no statistically significant differences
(p < 0.05) according to the analysis of variance (ANOVA).

Various studies on the extracts from C. metuliferus fruits cultivated in different ge-
ographic areas and also extracts from various parts of the plant showed different re-
sults [22]. Thus, other studies presented that the antioxidant activity of the extracts
from the pulp by the DPPH method was 77.98 ± 2.13 µmol TE/100 g, by the ABTS
method was 2508.69 ± 23.14 µmol TE/100 g, and that the peel extracts recorded higher
activity for DPPH, 211.53 ± 7.95 µmol TE/100 g and 7845.91 ± 57.13 µmol TE/100 g for
ABTS, respectively [22]. Seeds have weak antioxidant activity, as evaluated by the DPPH
method (49.21 ± 1.15 µmol TE/100 g) and by the ABTS method (1425.196 ± 21.96 µmol
TE/100 g) [22]. Another study showed that the DPPH and ABTS radical scavenging capac-
ity of the methanolic extract from kiwano whole fruit was 20.88 µmol Trolox, equivalent
per g, and, 185.36 µmol Trolox equivalent per g, respectively [23]. According to some stud-
ies, ursonic acid, which can be semi-synthesized by the oxidation of UA, has therapeutic
potentials that are similar to or stronger than UA in some aspects. In particular, ursonic
acid showed better anticancer, antiproliferative, and antiprotozoal effects [77]. Therefore,
UA can be a good lead compound for the development of new derivatives with various
therapeutic properties.

3.5. Evaluation of the in Vitro Anti-Inflammatory Activities

It is known that inflammation is liable for many diseases, such as diabetes, multiple
sclerosis, arthritis, Parkinson’s, and Alzheimer’s among others, and is also associated with
the progression of cancer cells [19,80].
Table 5 shows the results of this study, from which it appears that the proteinase
inhibition activity of the C. metuliferus extract is mostly due to the presence of ursolic
acid in its composition. The obtained results demonstrate that the isolated compound has
a good anti-lipoxygenase activity, even if the values are lower compared to the known
standards used, i.e., indomethacin and aspirin, so the C. metuliferus extract can be used as
an alternative or as an adjuvant in suppressing inflammation.

Table 5. Evaluation of the anti-inflammatory and antidiabetic activities.

Anti-Lipoxygenase Anti-Proteinase β-glucosidase

α-amylase Inhibition
Samples Activity Action Inhibition IC50
IC50 (µg/mL) *
IC50 (µg/mL) * IC50 (µg/mL) * (µg/mL) *
Hydroethanolic extract 32.90 ± 0.045 b 16.34 ± 0.067 b 429.541 ± 0.252 c 385.685 ± 0.758 c
Ursolic acid fraction 18.61 ± 0.086 a 12.53 ± 0.044 a 394.264 ± 0.143 b 322.412 ± 0.517 b
Aspirin 160.20 ± 0.020c
Indomethacin 45.12 ± 0.014 c
Acarbose 341.577 ± 0.398 a 308.474 ± 0.296 a
* The values succeeded by the same letters (a, b, c) in the same column show no statistically signifcant differences
(p < 0.05) according to the analysis of variance (ANOVA).
Separations 2023, 10, 274 11 of 15

Terpenoids are one of the classes of compounds present in plants that have demon-
strated anti-inflammatory potential, ursolic acid being one of the representatives of this
class [81]. The ability of ursolic acid to effectively inhibit aggregation of human platelet
induced by arachidonic acid (IC50 = 0.26 mM) was also demonstrated through in vivo
studies, compared to the activity of the known compounds quercetin (IC50 = 4.41 µM)
and aspirin (28–71% inhibition at 150–300 mg/kg) [76]. Ursolic acid has been shown to
exhibit weaker inhibitory activity (IC50 = 10 µM) than patuletin, but stronger activity than
quercetin [75].

3.6. In Vitro Evaluation of the Antidiabetic Activity

The present research was designed to also evaluate the possible antidiabetic activity
of C. metuliferus hydroethanolic extract by using in vitro enzyme assays. To investigate
the pharmacological potential of hydroethanolic extract and isolated UA as amylase and
glucosidase inhibitors in the development of new drugs to keep off and treat diabetes,
they underwent specific tests. The results of the in vitro inhibition tests of α-amylase and
α-glucosidase (Table 5) displayed that C. metuliferus extract had a similar inhibitory effect
(IC50 α-amylase = 429.541 ± 0.252 µg/mL and IC50 βglucosidase = 385.685 ± 0.758 µg/mL)
with that of the standard drug acarbose (IC50 α-amylase = 341.577 ± 0.398 µg/mL and
IC50 βglucosidase = 308.474 ± 0.296 µg/mL). Several other studies demonstrated that triter-
penoids and their derivatives could efficaciously inhibit the activity of glucosidase and
amylase to diminish the absorption of carbohydrates [34,82]. These findings suggest that
C. metuliferus extract could decrease the postprandial glucose level by inhibiting the ac-
tivities of β-glucosidase or α-amylase, which are key enzymes in the digestion process of
complex carbohydrates from food into adsorbable monosaccharides.

4. Conclusions
In this present work, the HPLC analysis was used to identify the chemical composition
of the hydroethanolic extract of Cucumis metuliferus. The obtained results highlight the
presence of 17 known phytochemical compounds in different amounts, including ursolic
acid. A method of identification and isolation of ursolic acid, which presents antioxidant
and anti-inflammatory activity, from the fruits of Cucumis metuliferus was proposed. The
structure of the isolated compound was determined by IR spectroscopy, mass spectroscopy,
and 1 H NMR, and the data were confirmed by comparison with previously reported data.
The obtained fraction showed antioxidant, anti-inflammatory, and antidiabetic activities,
as well as the hydro-ethanolic extract. C. metuliferus exhibited considerable enzymatic
inhibitory activity on key enzymes involved in diabetes and encourages the use of its
extracts and phytochemical constituents, such as ursolic acid, in the prevention and therapy
of diabetes mellitus. The results demonstrate the good activity of the hydro-ethanolic
extracts obtained from fruits of Cucumis metuliferus “Tempus” grown in Romania due to
the bioactive compounds identified in the extract, with major potential for various uses in
disease management, as an alternative medicine to improve the overall health and wellness.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/separations10050274/s1, Figure S1: MS Spectra of fraction of
C. metuliferus spicies with ursolic acid (negative 16 mode) performed on mass spectrometry; Figure S2:
Proposed fragmentation pattern of ursolic acid, Figure S3. The 1 H NMR spectrum of the ursolic
acid fraction.
Author Contributions: Conceptualization, R.M.D. and A.C.B.; Data curation, A.V.D.B., A.C.B. and
B.F.; Funding acquisition, R.M.D. and B.F.; Investigation A.C.B., G.V.C. and A.V.D.B.; Methodology,
R.M.D., A.V.D.B., A.C.B. and B.F.; Project administration R.M.D. and A.C.B.; Resources R.M.D. and
B.F. Software A.V.D.B.; Supervision, R.M.D. and B.F.; Visualization A.C.B., A.V.D.B. and G.V.C.;
Writing—original draft, A.C.B., A.V.D.B. and R.M.D.; Writing—review and editing, R.M.D., A.V.D.B.,
A.C.B. and B.F. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Separations 2023, 10, 274 12 of 15

Data Availability Statement: The data presented in this study are available in the figures and
supplementary materials.
Acknowledgments: The authors are grateful for the technical support provided by the MoRAS and
ECEE Centers, and by “Dunarea de Jos” University, to Costel Vînătoru (Plant Genetic Resources
Bank for Vegetables, Floriculture, Aromatic, and Medicinal Plants Buzău, 56 Nicolae Bălcescu Street,
120187, Buzau, Romania), and the Research-Development Station for Vegetable Growing Buzău for
the plant material that was used in this study.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Fischer, N.M.; Pallazola, V.A.; Xun, H.; Cainzos-Achirica, M.; Michos, E.D. The evolution of the heart-healthy Ddet for vascular
health: A walk through Time. Vasc. Med. 2020, 25, 184–193. [CrossRef] [PubMed]
2. Dzah, C.S.; Asante-Donyinah, D.; Letsyo, E.; Dzikunoo, J.; Adams, Z.S. Dietary olyphenols and obesity: A Review of polyphenol
effects on lipid and glucose metabolism, mitochondrial homeostasis, and starch digestibility and absorption. Plant Foods Hum.
Nutr. 2023, 78, 1–12. [CrossRef] [PubMed]
3. Lutz, M. Healthy sustainable food patterns and systems: A planetary urgency. Medwave 2021, 21, e8436. [CrossRef] [PubMed]
4. Dinu, M.; Pagliai, G.; Sofi, F. A heart-healthy diet: Recent insights and practical recommendations. Curr. Cardiol. Rep. 2017, 19, 95.
[CrossRef]
5. Van Raaij, J.; Hendriksen, M.; Verhagen, H. Potential for improvement of population diet through reformulation of commonly
eaten foods. Public Health Nutr. 2009, 12, 325–330. [CrossRef]
6. Herforth, A.; Arimond, M.; Álvarez-Sánchez, C.; Coates, J.; Christianson, K.; Muehlhoff, E. A global review of food-based dietary
guidelines. Adv. Nutr. 2019, 10, 590–605. [CrossRef]
7. Ekstrand, B.; Scheers, N.; Rasmussen, M.K.; Young, J.F.; Ross, A.B.; Landberg, R. Brain Foods—The role of diet in brain
performance and health. Nutr. Rev. 2021, 79, 693–708. [CrossRef]
8. Mozaffarian, D. Dietary and policy priorities for cardiovascular disease, diabetes, and obesity. Circulation 2016, 133, 187–225.
[CrossRef]
9. Pistollato, F.; Iglesias, R.C.; Ruiz, R.; Aparicio, S.; Crespo, J.; Lopez, L.D.; Manna, P.P.; Giampieri, F.; Battino, M. Nutritional
patterns associated with the maintenance of neurocognitive functions and the risk of dementia and Alzheimer’s disease: A focus
on human studies. Pharmacol. Res. 2018, 131, 32–43. [CrossRef]
10. Opie, R.S.; Itsiopoulos, C.; Parletta, N.; Sanchez-Villegas, A.; Akbaraly, T.N.; Ruusunen, A.; Jacka, F.N. Dietary Recommendations
for the prevention of depression. Nutr. Neurosci. 2017, 20, 161–171. [CrossRef]
11. Wang, X. Healthy diet during pregnancy—Navigating the double-ddged Sword. Am. J. Clin. Nutr. 2021, 114, 414–415. [CrossRef]
[PubMed]
12. Omokhua-Uyi, A.G.; Van Staden, J. Phytomedicinal relevance of South African Cucurbitaceae species and their safety assessment:
A review. J. Ethnopharmacol. 2020, 259, 112967. [CrossRef] [PubMed]
13. Busuioc, A.C.; Botezatu, A.V.D.; Furdui, B.; Vinatoru, C.; Maggi, F.; Caprioli, G.; Dinica, R.M. Comparative study of the chemical
compositions and antioxidant activities of fresh juices from Romanian Cucurbitaceae varieties. Molecules 2020, 25, 5468. [CrossRef]
[PubMed]
14. Murthy, H.N.; Paek, K.-Y. Bioactive Compounds in Underutilized Vegetables and Legumes; Murthy, H.N., Paek, K.Y., Eds.; Reference
Series in Phytochemistry; Springer International Publishing: Cham, Switzerland, 2021; ISBN 978-3-030-57414-7.
15. Usman, J.G.; Sodipo, O.A.; Kwaghe, A.V.; Sandabe, U.K. Uses of Cucumis metuliferus: A review. Cancer Biol. 2015, 5, 24–34.
16. Bisognin, D.A. Origin and evolution of cultivated Cucurbits. Ciência Rural 2002, 32, 715–723. [CrossRef]
17. Hartman, J.L.; Wehner, T.C.; Ma, G.; Perkins-Veazie, P. Citrulline and arginine content of taxa of Cucurbitaceae. Horticulturae 2019,
5, 22. [CrossRef]
18. Mallick, M.F.R.; Masui, M. Origin, Distribution and taxonomy of melons. Sci. Hortic. 1986, 28, 251–261. [CrossRef]
19. González, R.; Ballester, I.; López-Posadas, R.; Suárez, M.D.; Zarzuelo, A.; Martínez-Augustin, O.; Sánchez de Medina, F. Effects of
flavonoids and other polyphenols on inflammation. Crit. Rev. Food Sci. Nutr. 2011, 51, 331–362. [CrossRef]
20. Innih, S.O.; Eze, I.G.; Omage, K. Cardiovascular benefits of Momordica charantia in cholesterol-fed wistar rats. Clin. Phytosci. 2021,
7, 65. [CrossRef]
21. De Melo, M.L.S.; Narain, N.; Bora, P.S. Characterisation of some nutritional constituents of melon (Cucumis melo hybrid AF-522)
seeds. Food Chem. 2000, 68, 411–414. [CrossRef]
22. Šovljanski, O.; Šeregelj, V.; Pezo, L.; Šaponjac, V.T.; Vulić, J.; Cvanić, T.; Markov, S.; Ćetković, G.; Čanadanović-Brunet, J. Horned
melon pulp, peel, and seed: New insight into phytochemical and biological properties. Antioxidants 2022, 11, 825. [CrossRef]
[PubMed]
23. Šeregelj, V.; Šovljanski, O.; Šaponjac, V.T.; Vulić, J.; Ćetković, G.; Markov, S.; Čanadanović-Brunet, J. Horned melon (Cucumis metuliferus
E. Meyer Ex. Naudin)—Current knowledge on its phytochemicals, biological benefits, and potential applications. Processes 2022,
10, 94. [CrossRef]
24. Ferrara, L. The dietary importance of a tropical fruit: The kiwano. Ingred. Aliment. 2006, 5, 14–17.
Separations 2023, 10, 274 13 of 15

25. Anyanwu, A.; Jimam, N.; Wannang, N. Assessment of the effects of Cucumis metuliferus fruits alkaloids against Newcastle disease
virus-LaSota. Environ. Dis. 2016, 1, 130. [CrossRef]
26. Seo, D.Y.; Lee, S.R.; Heo, J.W.; No, M.H.; Rhee, B.D.; Ko, K.S.; Kwak, H.B.; Han, J. Ursolic acid in health and disease. Korean
J. Physiol. Pharmacol. 2018, 22, 235–248. [CrossRef]
27. Ramos-Hryb, A.B.; Pazini, F.L.; Kaster, M.P.; Rodrigues, A.L.S. Therapeutic potential of ursolic acid to manage neurodegenerative
and psychiatric diseases. CNS Drugs 2017, 31, 1029–1041. [CrossRef] [PubMed]
28. Iqbal, J.; Abbasi, B.A.; Ahmad, R.; Mahmood, T.; Kanwal, S.; Ali, B.; Khalil, A.T.; Shah, S.A.; Alam, M.M.; Badshah, H. Ursolic acid
a promising candidate in the therapeutics of breast cancer: Current status and future implications. Biomed. Pharmacother. 2018,
108, 752–756. [CrossRef]
29. Venugopal, R.; Liu, R.H. Phytochemicals in diets for breast cancer prevention: The importance of resveratrol and ursolic acid.
Food Sci. Hum. Wellness 2012, 1, 1–13. [CrossRef]
30. Ramirez, C.N.; Li, W.; Zhang, C.; Wu, R.; Su, S.; Wang, C.; Gao, L.; Yin, R.; Kong, A.N.T. In vitro-in vivo dose response of ursolic
acid, sulforaphane, PEITC, and curcumin in cancer prevention. AAPS J. 2018, 20, 19. [CrossRef]
31. Gu, W.; Hao, Y.; Zhang, G.; Wang, S.F.; Miao, T.T.; Zhang, K.P. Synthesis, in vitro antimicrobial and cytotoxic activities of new
carbazole derivatives of ursolic acid. Bioorg. Med. Chem. Lett. 2015, 25, 554–557. [CrossRef]
32. Shao, J.W.; Dai, Y.C.; Xue, J.P.; Wang, J.C.; Lin, F.P.; Guo, Y.H. In vitro and in vivo anticancer activity evaluation of ursolic acid
derivatives. Eur. J. Med. Chem. 2011, 46, 2652–2661. [CrossRef]
33. Mlala, S.; Oyedeji, A.O.; Gondwe, M.; Oyedeji, O.O. Ursolic acid and its derivatives as bioactive agents. Molecules 2019, 24, 2751.
[CrossRef]
34. Wu, P.P.; Zhang, K.; Lu, Y.J.; He, P.; Zhao, S.Q. In vitro and in vivo evaluation of the antidiabetic activity of ursolic acid derivatives.
Eur. J. Med. Chem. 2014, 80, 502–508. [CrossRef] [PubMed]
35. Yang, P.; Li, Y.; Liu, X.; Jiang, S. Determination of free isomeric oleanolic acid and ursolic acid in Pterocephalus hookeri by capillary
zone electrophoresis. J. Pharm. Biomed. Anal. 2007, 43, 1331–1334. [CrossRef] [PubMed]
36. Wang, J.; Liu, L.; Qiu, H.; Zhang, X.; Guo, W.; Chen, W.; Tian, Y.; Fu, L.; Shi, D.; Cheng, J.; et al. Ursolic acid simultaneously targets
multiple signaling pathways to suppress proliferation and induce apoptosis in colon cancer cells. PLoS ONE 2013, 8, e63872.
[CrossRef] [PubMed]
37. Xu, X.H.; Su, Q.; Zang, Z.H. Simultaneous determination of oleanolic acid and ursolic acid by RP-HPLC in the leaves of
Eriobotrya japonica Lindl. J. Pharm. Anal. 2012, 2, 238–240. [CrossRef]
38. Shanmuga Sundaram, R.; Ramanathan, M.; Rajesh, R.; Satheesh, B.; Saravanan, D. LC-MS quantification of rosmarinic acid and
ursolic acid in the Ocimum sanctum Linn. leaf extract (Holy Basil, Tulsi). J. Liq. Chromatogr. Relat. Technol. 2012, 35, 634–650.
[CrossRef]
39. Fan, J.P.; Kong, T.; Zhang, L.; Tong, S.; Tian, Z.Y.; Duan, Y.H.; Zhang, X.H. Solubilities of ursolic acid and oleanolic acid in four
solvents from (283.2 to 329.7) K. J. Chem. Eng. Data 2011, 56, 2723–2725. [CrossRef]
40. Fan, J.P.; Liao, D.D.; Zhang, X.H. Ultrasonic assisted extraction of ursolic acid from apple pomace: A novel and facile technique.
Sep. Sci. Technol. 2016, 51, 1344–1350. [CrossRef]
41. Wang, X.; Sun, W.; Cao, J.; Qu, H.; Bi, X.; Zhao, Y. Structures of new triterpenoids and cytotoxicity activities of the isolated major
compounds from the fruit of Momordica charantia L. J. Agric. Food Chem. 2012, 60, 3927–3933. [CrossRef]
42. Tian, S.; Shi, Y.; Yu, Q.; Upur, H. Determination of oleanolic acid and ursolic acid contents in Ziziphora clinopodioides Lam. by
HPLC method. Pharmacogn. Mag. 2010, 6, 116–119. [CrossRef]
43. Wandjou, J.G.N.; Mevi, S.; Sagratini, G.; Vittori, S.; Dall’acqua, S.; Caprioli, G.; Lupidi, G.; Mombelli, G.; Arpini, S.; Allegrini, P.;
et al. Antioxidant and enzyme inhibitory properties of the polyphenolic-rich extract from an ancient apple variety of central Italy
(Mela rosa dei Monti Sibillini). Plants 2020, 9, 9. [CrossRef]
44. Verma, S.C.; Jain, C.L.; Kumari, A.; Padhi, M.M.; Devalla, R.B. Microwave-assisted extraction and rapid isolation of ursolic acid
from the leaves of eucalyptus × Hybrida maiden and its quantification using HPLC-diode array Technique. J. Sep. Sci. 2013, 36,
1255–1262. [CrossRef]
45. Kaur, P.; Gupta, R.C.; Dey, A.; Kumar Pandey, D. Simultaneous quantification of oleanolic acid, ursolic acid, betulinic acid and
lupeol in different populations of five Swertia species by using HPTLC-densitometry: Comparison of different extraction methods
and solvent selection. Ind. Crops Prod. 2019, 130, 537–546. [CrossRef]
46. Zongo, E.; Busuioc, A.; Meda, R.N.-T.; Botezatu, A.V.; Mihaila, M.D.; Mocanu, A.-M.; Avramescu, S.M.; Koama, B.K.; Kam, S.E.;
Belem, H.; et al. Exploration of the antioxidant and anti-inflammatory potential of Cassia sieberiana DC and Piliostigma
thonningii (Schumach.) Milne-Redh, traditionally used in the treatment of hepatitis in the Hauts-Bassins region of Burkina Faso.
Pharmaceuticals 2023, 16, 133. [CrossRef] [PubMed]
47. Costea, L.; Chit, escu, C.L.; Boscencu, R.; Ghica, M.; Lupuliasa, D.; Mihai, D.P.; Deculescu-ionit, ă, T.; Dut, u, L.E.; Popescu, M.L.;
Lut, ă, E.A.; et al. The polyphenolic profile and antioxidant activity of five vegetal extracts with hepatoprotective potential. Plants
2022, 11, 1680. [CrossRef]
48. Fouedjou, R.T.; Nguelefack, E.P.; Ponou, B.K.; Nguelefack, T.B.; Barboni, L.; Tapondjou, L.A. Antioxidant activities and chemical
constituents of extracts from Cordyline fruticosa (L.) A. Chev. (Agavaceae) and Eriobotrya japonica (Thunb) Lindl, (Rosaceae).
Pharmacologia 2016, 7, 103–113. [CrossRef]
Separations 2023, 10, 274 14 of 15

49. Kaur, N.; Chahal, K.; Singh, R.U. Phytochemical screening and antioxidant activity of Anethum graveolens L. seed extracts. Pharma
Innov. J. 2018, 7, 324–329.
50. Matsusaka, Y.; Kawabata, J. Evaluation of antioxidant capacity of non-edible parts of some selected tropical fruits. Food Sci.
Technol. Res. 2010, 16, 467–472. [CrossRef]
51. Sirivibulkovit, K.; Nouanthavong, S.; Sameenoi, Y. Paper-Based DPPH assay for antioxidant activity analysis. Anal. Sci. 2018, 34,
795–800. [CrossRef]
52. Dinica, R.M.; Sandu, C.; Botezatu, A.V.D.; Busuioc, A.C.; Balanescu, F.; Mihaila, M.D.I.; Dumitru, C.N.; Furdui, B.; Iancu, A.V.
Allantoin from valuable Romanian animal and plant sources with promising anti-inflammatory activity as a nutricosmetic
ingredient. Sustainability 2021, 13, 170. [CrossRef]
53. Saravanakumar, K.; Park, S.J.; Mariadoss, A.V.A.; Sathiyaseelan, A.; Veeraraghavan, V.P.; Kim, S.J.; Wang, M.H. Chemical compo-
sition, cntioxidant, and anti-diabetic activities of ethyl acetate fraction of Stachys riederi Var. Japonica (Miq.) in streptozotocin-
induced Type 2 diabetic mice. Food Chem. Toxicol. 2021, 155, 112374. [CrossRef]
54. Wojdyło, A.; Oszmiański, J.; Czemerys, R. Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem. 2007,
105, 940–949. [CrossRef]
55. Bălănescu, F.; Botezatu, A.V.; Marques, F.; Busuioc, A.; Marincaş, O.; Vînătoru, C.; Cârâc, G.; Furdui, B.; Dinica, R.M. Bridging the
chemical profile and biological activities of a new variety of Agastache foeniculum (Pursh) Kuntze extracts and essential oil. Int.
J. Mol. Sci. 2023, 24, 828. [CrossRef]
56. Okpoko, C.; Ezenyi, I.; Adzu, B.; Salawu, O. Evaluation of two medicinal plants used for arthritis in Northern Nigeria with focus
on Terminalia avicennioides Guill. & Perr. and its mechanism of action. Sci. African 2020, 8, e00357. [CrossRef]
57. Fofana, S.; Gnoula, C.; Draogo, M.O.E.; Eacute, E.P.; Eacute, R.H.N.E.B.; Nikiema, J.B.; Guissou, I.P.; Simpore, J. DPPH radical
scavenging and lipoxygenase inhibitory effects in extracts from Erythrina senegalensis (Fabaceae) DC. Afr. J. Pharm. Pharmacol.
2016, 10, 185–191. [CrossRef]
58. Naz, R.; Ayub, H.; Nawaz, S.; Islam, Z.U.; Yasmin, T.; Bano, A.; Wakeel, A.; Zia, S.; Roberts, T.H. Antimicrobial activity, toxicity and
anti-inflammatory potential of methanolic extracts of four ethnomedicinal plant species from Punjab, Pakistan. BMC Complement.
Altern. Med. 2017, 17, 302. [CrossRef]
59. Oyedepo, O.O.; Femurewa, A.J. Anti-protease and membrane stabilizing activities of extracts of Fagra zanthoxiloides, Olax
subscorpioides and Tetrapleura tetraptera. Int. J. Pharm. 1995, 33, 65–69. [CrossRef]
60. Wan, L.S.; Chen, C.P.; Xiao, Z.Q.; Wang, Y.L.; Min, Q.X.; Yue, Y.D.; Chen, J.C. In vitro and in vivo anti-diabetic activity of
Swertia kouitchensis extract. J. Ethnopharmacol. 2013, 147, 622–630. [CrossRef]
61. Sales, P.M.; Souza, P.M.; Simeoni, L.A.; Magalhães, P.O.; Silveira, D. α-Amylase inhibitors: A Review of raw material and isolated
compounds from plant source. J. Pharm. Pharm. Sci. 2012, 15, 141. [CrossRef]
62. Ngenge Tamfu, A.; Mfifen Munvera, A.; Veronica Dediu Botezatu, A.; Talla, E.; Ceylan, O.; Tagatsing Fotsing, M.; Tanyi Mbafor, J.;
Shaheen, F.; Mihaela Dinica, R. Synthesis of benzoyl esters of β-amyrin and lupeol and evaluation of their antibiofilm and
antidiabetic activities. Results Chem. 2022, 4, 100322. [CrossRef]
63. Vieira, E.F.; Podlasiak, M.; Moreira, M.M.; Grosso, C.; Rodrigues, F.; Fernandes, V.C.; Delerue-Matos, C. New insights of
phytochemical profile and in vitro antioxidant and neuroprotective activities from optimized extract of horned melon fruit. J. Food
Meas. Charact. 2022, 16, 1847–1858. [CrossRef]
64. Maluleke, M.K.; Moja, S.J.; Nyathi, M.; Modise, D.M. Nutrient concentration of African horned cucumber (Cucumis metuliferus L.)
fruit under different soil types, environments, and varying irrigation water levels. Horticulturae 2021, 7, 76. [CrossRef]
65. Bogopa, J. P2 Exploring the functional components of the African horned cucumber (Cucumis metuliferus), Mokapana (Tswana)—
Characterisation by phenolic content and antioxidant activity in Botswana. Biochem. Pharmacol. 2017, 139, 125. [CrossRef]
66. Ani, O.N.; Achikanu, C.E.; Onyishi, C.K. Comparative analysis of minerals, heavy metals and amino acids compositions of the
seeds and juice of Cucumis metuliferus. Asian J. Res. Biochem. 2020, 54, 31–42. [CrossRef]
67. Pai, S.R.; Upadhya, V.; Hegde, H.V.; Joshi, R.K.; Kholkute, S.D. Determination of betulinic acid, oleanolic acid and ursolic acid
from Achyranthes aspera L. using RP-UFLC-DAD analysis and evaluation of various parameters for their optimum Yield. Indian
J. Exp. Biol. 2016, 54, 196–202.
68. Zhou, Z.; Tong, H.H.Y.; Li, L.; Shek, F.L.Y.; Lv, Y.; Zheng, Y. Synthesis, characterization and thermal analysis of ursolic acid solid
forms. Cryst. Res. Technol. 2015, 50, 538–548. [CrossRef]
69. Gómez-Pulido, L.D.M.; González-Cano, R.C.; Domínguez, E.; Heredia, A. Structure determination of oleanolic and ursolic acids:
A combined density functional theory/vibrational spectroscopy methodology. R. Soc. Open Sci. 2021, 8, 210162. [CrossRef]
70. Samsonowicz, M.; Kalinowska, M.; Gryko, K. Enhanced Antioxidant activity of ursolic acid by complexation with copper (II):
Experimental and theoretical study. Materials 2021, 14, 264. [CrossRef]
71. Sang, S.; Lapsley, K.; Rosen, R.T.; Ho, C.T. New prenylated benzoic acid and other constituents from almond hulls (Prunus amygdalus
Batsch). J. Agric. Food Chem. 2002, 50, 607–609. [CrossRef]
72. Somantri, A.D.; Kurnia, D.; Zainuddin, A.; Dharsono, H.D.; Satari, M.H. Action mode of ursolic acid as a natural antioxidant and
inhibitor of superoxide dismutase: In vitro and in silico study. J. Adv. Pharm. Technol. Res. 2021, 12, 389–394. [CrossRef]
73. Krishnaiah, D.; Sarbatly, R.; Nithyanandam, R. A Review of the antioxidant potential of medicinal plant species. Food Bioprod.
Process. 2011, 89, 217–233. [CrossRef]
Separations 2023, 10, 274 15 of 15

74. Alam, M.N.; Bristi, N.J.; Rafiquzzaman, M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi
Pharm. J. 2013, 21, 143–152. [CrossRef]
75. Jedinák, A.; Mučková, M.; Košt’álová, D.; Maliar, T.; Mašterová, I. Antiprotease and antimetastatic activity of ursolic acid isolated
from Salvia officinalis. Z. Fur Nat.-Sect. C J. Biosci. 2006, 61, 777–782. [CrossRef]
76. Checker, R.; Sandur, S.K.; Sharma, D.; Patwardhan, R.S.; Jayakumar, S.; Kohli, V.; Sethi, G.; Aggarwal, B.B.; Sainis, K.B. Potent
anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression of NF-KB, AP-1 and
NF-AT. PLoS ONE 2012, 7, e31318. [CrossRef]
77. Son, J.; Lee, S.Y. Therapeutic potential of ursonic acid: Comparison with ursolic acid. Biomolecules 2020, 10, 1505. [CrossRef]
78. Jabeen, M.; Ahmad, S.; Shahid, K.; Sadiq, A.; Rashid, U. Ursolic acid hydrazide based organometallic complexes: Synthesis,
characterization, antibacterial, antioxidant, and docking studies. Front. Chem. 2018, 6, 55. [CrossRef] [PubMed]
79. Ramachandran, S.; Prasad, N.R. Effect of ursolic acid, a triterpenoid antioxidant, on ultraviolet-B radiation-induced cytotoxicity,
lipid peroxidation and DNA damage in human lymphocytes. Chem. Biol. Interact. 2008, 176, 99–107. [CrossRef]
80. Pradhan, B.; Patra, S.; Behera, C.; Nayak, R.; Jit, B.P.; Ragusa, A.; Jena, M. Preliminary investigation of the antioxidant, anti-diabetic,
and anti-inflammatory activity of Enteromorpha intestinalis extracts. Molecules 2021, 26, 1171. [CrossRef] [PubMed]
81. Woźniak, Ł.; Skapska,
˛ S.; Marszałek, K. Ursolic acid—A pentacyclic triterpenoid with a wide spectrum of pharmacological
activities. Molecules 2015, 20, 20614–20641. [CrossRef] [PubMed]
82. Ali, H.; Houghton, P.J.; Soumyanath, A. α-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with
particular reference to Phyllanthus amarus. J. Ethnopharmacol. 2006, 107, 449–455. [CrossRef] [PubMed]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.