Characterization and Stability Evaluation of Egyptian Propolis Extract Nano Capsules and Their Application

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OPEN Characterization and stability

evaluation of Egyptian propolis
extract nano‑capsules and their
application
Azza A. Amin 1, Khaled F. Mahmoud 1*, Manal F. Salama 1, Vincenzo Longo 2, Luisa Pozzo 2,
Effat I. Seliem 1 & Mona A. Ibrahim 1
The increasing demand for natural products and biotechnological activities from bees facilitate their
widespread use in food preservation and beneficial effects on humans. This study aimed to prepare
and characterize the nano-capsules of Qaluiobia (PQG) governorates propolis extracted with water,
ethanol and supercritical fluid-carbon dioxide at 50 °C with co-solvent. Propolis bioavailability was
analyzed and introduced to prepare crackers to extend their shelf life. Nano-encapsulation was
examined using transmission electron microscopy (TEM), differential scanning calorimetry (DSC) and
antioxidant activity. Ethanol and supercritical fluid-carbon dioxide (SCF-CO2) at 50 °C with ethanol as
co-solvent recorded higher yield, antioxidant activities, total phenolics and total flavonoids. SCF-CO2
extracts had a higher flavonoid concentration. It was revealed that propolis nano-capsules had
high-temperature stability and cytotoxic effects against the three tested human cancer cell lines (i.e.
PC3, MCF7 and HePG2). The higher overall acceptability of crackers fortified with PQG was achieved
with SCF-CO2 at 50 °C and ethanol extract nano-capsules, i.e. 86.57% and 86.29% respectively. The
higher ability to retain antioxidant activity reduces the increase of peroxide value (PV), preventing
rancidity and increasing the shelf life of crackers during the storage period. Practical application: This
study can provide a suitable method for extracting bioactive compounds from propolis, and improve
the biological properties and activities by nano-encapsulation, also reveals the extent of its use as a
natural antioxidant and anticancer and its application in bakery products as a functional food.
Propolis is a complex resinous produced by honey bees and has highly different physical characteristics depend-
ing mainly on the environmental c onditions1. It is well known that the biochemical compounds in propolis
contain phenolic compounds, vitamins, carbohydrates, hydrocarbons and carboxylic acids, mainly responsible
for therapeutic effects and high antioxidant activity2,3.
Raw propolis is not soluble in water and is extracted by solvents. Propolis is purified to preserve its poly-
phenolic fraction. These polyphenolic compounds are essential for healing effects4. The ingredients extracted
from propolis exhibit many biological activities and health benefits and can be used as functional ingredients
in food and m edicine4.
Propolis is complex and contains 30% wax, 50% resin (mainly flavonoids and phenolic acid derivatives), 10%
aromatic oils, 5% other organic residues and 5% pollen5. The chemical composition of propolis, especially related
to its components and their polarity, makes it challenging to use in a free or non-encapsulated form in various
applications. The extraction yield of bioactive components depends on the extraction methods and organic sol-
vents (i.e. methanol, chloroform, ethanol and ethyl-ether). Many researchers have studied propolis processing
using conventional m ethods6,7. However, despite its use, it has some shortcomings, mainly due to the quality of
processing resulting in the presence of solvent residues in the final product.
Supercritical fluid (SCF) extraction is an innovative technology that has gained widespread interest due to
increasingly restrictive environmental regulations (i.e. the use of low temperatures, reduced solvent use and
energy consumption)8–10. This technology has spread in multiple industrial areas due to its unique advantages and
SCF characteristics11,12. EPE has the potential to be used as a natural additive with antimicrobial and antioxidant
1
Department of Food Technology, National Research Centre (NRC), Dokki 12622, Egypt. 2Institute of Agricultural
Biology and Biotechnology (IBBA), National Research Council (IBBA-CNR), Via Moruzzi 1, 56124 Pisa, Italy. *email:
[email protected]
Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 1
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read13. Still, SCF is a promising alternative technique in the fine chemistry, foodstuff
characteristics in toast b
fields and pharmaceuticals14.
Nanotechnology is an innovative technique for various applications in nano-food and nano-medicine. Nano-
particles with encapsulation produce small size, large surface area and protection, with high bioavailability
compared to their bulk counterparts15.
Nano-capsulation can be obtained using emulsification, high-pressure homogenization, evaporation, spray
drying, ultrasonication, high-speed stirring, micro-emulsion and ball m illing16.
This work evaluated Qaluibia (PQG) propolis extraction produced methods using extraction methods (i.e.
water, ethanol 95% and one green technology SCF-CO2 at 50 °C) as the extractants were used. The physico-
chemical properties, nano-capsulation, cytotoxic activity of propolis and their application as functional bakery
products were also studied.
Materials and methods

Materials and chemicals
Egyptian raw honey bee’s propolis was collected from Delta region Qaluiobia Governorate (PQG) and kept in
dark sterile glass containers at room temperature until further use. All chemicals and reagents i.e. Folin–Ciocalteu
reagent, 2,2-diphenyl-1-picryl hydrazyl radical (DPPH), Tween 20 (T20), Ethanol, Aluminum chloride, Potas-
sium acetate, linoleic acid, Gallic acid, Sodium alginate, Butylated hydroxyltoluene (BHT), Sodium Thiocyanate,
Ascorbic acid, Quercetin, Methanol, Phosphate buffer, Ammonium thiocyanate, Ferrous chloride, Potassium
ferricyanide, Trichloroacetic acid, Ferric chloride, Sodium carbonate, Aluminum chloride, Formic acid, Ace-
tonitrile, Phosphotungestic acid, Chloroform, Glacial acetic acid, Sodium thiosulphate were of analytical grade
and purchased from Sigma Aldrich Chemical (Merck KGaA, Darmstadt, Germany).
The work does not involve humans or conduct experiments on live animals.
Methods
Extraction methods
Propolis water extracts (PWE). Honey bees propolis powders (10 g) were suspended in 100 ml distilled water and
shaken for 24 h at 24 °C. The suspension was then centrifuged at 3000 rpm for 20 min at 4 °C, and the supernatants
were collected after several extractions (3 times) under the same conditions, dialyzed against distilled water, and lyo-
philized (LABCONCO, Freeze dryer, Console, 12 L −50 °C, 240 V, Catalog No. 7754030, USA)17. The yield of water
extract was defined as the mass of water extract of propolis obtained by dry mass of raw propolis used in percentage.
Propolis ethanolic extracts (PEE). Ethanolic extracts of propolis (PEE) were obtained using the method of Yong
and Masaharu18 with some modification where, (10 g) propolis powder was mixed with 250 methanol (95%) at
room temperature in a dark place for 24 h. The mixture was centrifuged (3000 rpm, for 10 min) to obtain the
supernatant. The propolis yield was calculated based on the initial amount of dry propolis in percentage.
Propolis supercritical fluid carbon dioxide extracts (PSCF‑CO2). The extracts of propolis were carried out at
temperatures of 50 °C and 250 bar pressure using a laboratory-scale unit in National Research Centre (Speed
TM SFE-2/4, Applied separations, Built in conjunction with the USDA1- USA). Ten grams of propolis powder
were mixed with ethanol 15% (w/w) as co-solvent. The C O2 was pumped into the reactor, which was supported
by two: 300 vessels and kept for 30 min to allow complete contact and guarantee that the operational conditions
of temperature and pressure were stabilized. The CO2 mass flow rate was 1.0 g/min. The samples were collected,
and the process line was washed with the used ethanol to recover the extract deposited. The global extract yield
Xo (%) (extraction + cleaning process) to the initial mass of raw material (drybases)19.
At the end of the extraction methods, the yield of propolis was collected and calculated as follows:
Yield (%) = (W1 /W2 ) × 100 (1)
where: W 1 is the weight of propolis extracts (g) and W2 is the weight of dried raw propolis powder (g).
Preparation of nano‑capsules
Ethanol and SCF-CO2 Propolis extracts were encapsulated into nano-forms using ultrasound and high-speed
homogeniser (PRO, USA) methods, according to Vasiliki and C onstantina20. Nano-emulsion was prepared by
adding one gram propolis extract to 10 ml deionized water (water (1:1) with addition of 0.1% T 20. The water was
gradually added with stirring using a magnetic stirrer (2000 rpm at room temperature) to avoid the formulation
of bubbles during mixing till complete dissolving, then homogenized using high-speed homogenizer (Model:
400ELPC, PRO Scientific Inc., 01-02411ELPC HOMOGENIZER, USA) at 18,000 rpm for 30 min in the pres-
ence of an ice water bath to reduce the temperature of the mixture. The sample was stored at 4 °C for 24 h before
encapsulation. For encapsulation, 50 ml sodium alginate solution (prepared by adding 3 g sodium alginate to
100 ml deionized water and mixed by magnetic stirrer at 2000 rpm for 60 min and left in a refrigerator at 4 °C to
form a gel) were gradually added to 10 ml nano extract emulsion (5:1) using homogenizer (2000 rpm, 10 min)
and treated by ultrasound (Sonics & Materials Inc., Newton, Connecticut, USA) for 30 min at 30 °C then the
encapsulated emulsion was packed in brown packages at 4 °C till use.
Chemical properties of propolis extracts and their nano‑capsules

Total phenolic content (TPC). The total phenolic content (TPC) of three extracts and their nano-capsules
were determined spectrophotometrically using Folin-Ciocalteu reagent according to the method described by
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Ebrahimzadeh et al.21 with some modification. The extracted samples and their nano-capsules (0.5 ml) were
mixed separately with Folin-Ciocalteu reagent (5 ml with distilled water at a rate of 1:10) for 3 min then; 3 ml of
2% sodium carbonate (1 M) was added. The mixture was left for 15 min, and the polyphenols were determined
by an automated UV–VIS spectrophotometer at 765 nm and the results were calculated using a Gallic acid cali-
bration curve (0–100 mg/l). The blank was prepared using the same procedure with 0.5 ml of pure water in place
of the extract. The results are expressed as equivalents to Gallic acid (mg GAE/g dry extract).
Total flavonoid content (TFC). The total flavonoid content (TFC) of three extracts and their nano-capsules
were determined according to the method described by Huang et al.22, and Ebrahimzadeh et al.21, Nabavi et al.23.
The propolis extracts and their nano-capsules (0.5 ml) were mixed separately with 1.5 ml methanol, 0.1 ml of
10% aluminium chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. They then left at room
temperature for 10 min. The absorbance of the mixture was measured at 415 nm on a UV/visible spectropho-
tometer. The quercetin (µg/g) was used as a standard for the calibration curve.
Determination of radical‑scavenging activity by (DPPH). Radical scavenging activity of tested extracts ability was
assayed using the method of Hatano et al.24. Different concentrations of three extracts (i.e.10, 20, 30, 40, 50 and
60 µg/ml) were added to reaction solution DPPH (1 ml) (0.2 mM). The mixture was shaken forcibly and left at room
temperature for 30 min, and then the absorbance of the solution was measured spectrophotometrically at 517 nm.
% DPPH radical scavenging activity = ((Ac − As)/Ac) × 100 (2)
As is the absorbance of the sample; Ac is the absorbance of control in the absence of the sample.
Nano-capsules of ethanol and SCF-CO2 with (60 µg/ml) were determined as above.
Reducing power (RP). The reducing power (RP) of three extracts were determined following the method of
Oyaizu25. Briefly, different concentrations of extracts (10, 20, 30, 40, 50 and 60 µg/ml) were mixed with 5 ml
phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (1%) and compared with the same concen-
tration of BHT and ascorbic acid in ethanol (95%) with 2.5 ml of sodium phosphate buffer (200 mM, pH 6.6).
The reaction mixture was incubated at 50 °C for 20 min. Aliquots of 1% trichloroacetic acid (5 ml) were added
to the mixtures, then centrifuged at 2000 rpm for 10 min, and the absorbance of the pink color mixture was
recorded spectrophotometrically at 700 nm.
Nano-capsules of ethanol and SCF-CO2 with (60 µg /ml) were determined as above.
Total antioxidant activity (TAA). Water, Ethanol and CO2 extracts total antioxidant activity (TAA) were deter-
mined using a linoleic acid s ystem26. The reaction mixture at different extract concentrations (i.e.10, 20, 30, 40,
50 and 60 µg/ml) were separately treated with linoleic acid (0.13 ml), phosphate buffer 0.2 M (pH 7.0, 10 ml) and
ethanol (99.8%). The mixture was adjusted to 25 ml by distilled water. Then incubated at 40 °C for 10 min and
the oxidation rate was measured using thiocyanate m ethod27. The obtained solutions were added to a mixture
of 10 ml ethanol (75%) with 0.2 ml ammonium thiocyanate (30%), 0.2 ml of sample solution and 0.2 ml of fer-
rous chloride solution (20 mM in 3.5% HCl), stirring for 3 min and the peroxide value was measured using a
spectrophotometer (JASCO, Corporation Model V-730, S.N. A112961798, Tokyo, Japan) at 500 nm. The percent
inhibition of linoleic peroxidation is calculated as 100 – (Abs increase of sample/Abs increase of control X100).
The commercial antioxidants butylated hydroxytoluene (BHT) and ascorbic acid were used.
Nano-capsules of ethanol and SCF-CO2 with (60 µg/ml) were determined as above.
Physical properties of nano‑capsules

The nano-capsules formed were tested by their efficiency, yield, TEM, DSC effect as follows:
Encapsulation efficiency (EE). Encapsulation efficiency (% encapsulation) was calculated following Busch
et al.28 based on the total amount of phenolic in encapsulated propolis per total amount of phenolic in propolis
extract. Encapsulation efficiency can be calculated by the following formula:
%Encapsulation = x/y × 100 (3)
where x is the total amount of phenol in encapsulated propolis (%); Y is the total amount of phenol in propolis
extract (%).
Encapsulation yield (EY). The encapsulation yield (EY) is another factor to be considered during an encap-
sulation process. The effectiveness of encapsulation yield (EY) was measured using Paviani et al.19 method and
calculated as follows:
Total mass of the sample after encapsulation
EY (%) = × 100 (4)
Total mass of the sample before encapsulation
Transmission electron microscopy (TEM). Propolis extracts and their nano-capsules’ morphological charac-
teristics were tested by TEM (1400, JEOL, Japan) using Saloka et al.29 method. The nano-particles suspensions
were dripped onto a 400-mesh copper grid coated with Forvar and stained by 2% phosphotungstic a cid30. The
samples were air-dried at room temperature for more than 2 h before analyzing on the TEM.
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Thermal stability (DSC). Thermal stability of propolis extract and their nano- capsules were measured using
a differential scanning calorimeter (DSC) (Mettler Toledo, SWITZERLAND) according to Hazra et al.31 as fol-
lows. Ten milligrams of both samples were placed in aluminium crucibles under a flow of nitrogen gas (40 ml/
min). A dynamic scan was performed at a heating rate of 10 °C/min over a temperature range of −150 to 300 °C.
Evaporation enthalpies were calculated by peak area integration of DSC profiles, and the results were compared
with the estimated vaporization enthalpy of major components.
LC–ESI–MS/MS analysis of propolis forms. The analysis of crude propolis, nano-capsules propolis extracts
by slovent and SCF-CO2 at 50 °C was performed using liquid chromatography–electro-spray ionization–tan-
dem mass spectrometry (LC–ESI–MS/MS) with an Exion LC/AC system for separation and SCIEX Triple Quad
5500+ MS/MS system equipped with an electro-spray ionization (ESI) for detection. The instrument data were
collected and processed using the SCIEX OS 1.6.10.40973 software.
The separation of the targeted analyses was performed with a D iscovery® BIO Wide Pore C18-5 Column
(4.6 × 250 mm, 5 µm). The mobile phases were consisted of two eluents A: 0.1% formic acid in water; B: 0.1%
formic acid in acetonitrile (LC grade). The mobile phase gradient was programmed as follows: 5% B at 0 min,
5–25% B from 0.0 to 60.0 min, 25–5% B from 60 to 65 min, 5% from 65 to 70. The flow rate was 1.0 ml/min and
the injection volume was 5 µl. For MS/MS analysis, negative ionization mode was applied with a scan (EMS-
IDA-EPI) from 150 to 800 Da with the following parameters: curtain gas: 25 psi; ion spray voltage: −4500; source
temperature: 400 °C; ion source gas 1 & 2 were 55 psi.
Cytotoxic effect on human cell lines. Cell viability was assessed by the mitochondrial-dependent reduction of
yellow MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to purple f ormazan32.
Cells from (National Cancer Institute, Egypt) were suspended in DMEM-F12 medium (for HePG2, MCF7,
and PC3) beside one normal cell line (BJ1), 1% antibiotic–antimycotic mixture (10,000 U/ml Potassium Peni-
cillin, 10,000 µg/ml Streptomycin Sulfate and 25 µg/ml Amphotericin B) and 1% l-glutamine at 37 °C under
5% CO2.
Cells were batch cultured for 10 days, then seeded at concentration of 10 × 103 cells/well in fresh complete
growth medium in 96-well microtiter plastic plates at 37 °C for 24 h under 5% C O2 using a water-jacketed
CO2dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without
serum) was added, and cells were incubated either alone (negative control) or with different concentrations of
sample to give a final concentration of (100–50–25–12.5–6.25–3.125–0.78 and 1.56 µg/mL). After 48 h of incuba-
tion, the medium was aspirated, 40 µL MTT salt (2.5 µg/mL) were added to each well and incubated for a further
4 h at 37 °C under 5% CO2. To stop the reaction and dissolve the formed crystals, 200 µL of 10% Sodium dodecyl
sulfate (SDS) in deionized water were added to each well and incubated overnight at 37 °C.
A positive control which composed of 100 µg/ml was used as a known cytotoxic natural agent that gives 100%
lethality under the same conditions33. The absorbance was then measured using a microplate multi-well reader
(Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595 nm and a reference wavelength of
620 nm. The percentage of change in viability was calculated according to the formula:
(5)

[ Absorbance of extract /absorbance of negative control − 1 ] × 100
Using the SPSS 11 program, the IC50 was determined using probit analysis. The degree of selectivity of the
synthetic compounds was expressed as SI = IC50 of the pure compound in a normal cell line/IC50 of the same pure
compound in a cancer cell line, where I C50 is the concentration required to kill 50 percent of a cell population.
Application
Crackers preparation. With slight modification, five types of crackers were prepared according to the method
of Benjakula and Karnjanapratum34. The control crackers were prepared using 62.5% wheat flour (72%), 1.16%
salt, 0.23% sugar, 0.37% baking powder, 15% sunflower oil, 0.187% paprika and 20.25% water to produce the
dough. The ingredients were mixed at a low speed for 3 min. The resulting cracker dough was sheeted to a thick-
ness of a 0.4 mm and cut into a rectangle (2.4 × 7.3 c m2). The shaped cracker dough was baked in an electric oven
at 120 °C for 30 min (SL-Shel-LAB-1370FX). In the same way, the remaining cracker samples were prepared with
the same previous ingredients to produce 1.8 g of four types of crackers containing 0.6 g of nano-capsules of PEE
(Propolis ethanolic extract), PSCF-E (Propolis Supercritical Extract at 50 °C), PNC-EE (Propolis Nano-Capsule
Ethanolic Extract) and PNC-SCFE (Propolis Nano-capsule Supercritical Extract at 50 °C) (1.8 g capsule contain
0.6 g propolis extract of their nano-forms).
Sensory evaluation of crackers. All samples of the crackers were sensory assessed by ten-member panels for
appearance, color, thickness, texture, shrinkage, taste and odor using the method described by S mith35.
Storage stability of crackers

The stability of the stored cracker samples (at 25 °C for 90 days) was determined every month compared to the
control sample using the DPPH and PV previous described methods.
Determination of radical‑scavenging activity using (DPPH). The oils from each cracker sample were extracted
by n-hexane (25 ml) that was evaporated after keeping it in a refrigerator for 24 h using rotary evaporator36. The
antioxidant activity of the crackers’ residue was determined as described previously.
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Determination of peroxide value (PV). Peroxide value of all cracker samples fortified with propolis extracts
and their nano-forms were determined by extracting the oil from samples with n-hexane, and then PV was per-
formed according to A OAC36. Two mg of oil were added and mixed in a solution containing chloroform-glacial
acetic acid (30 ml, 3:2 v/v) and sodium thiosulphate (1 ml, 0.1 M) until the disappearance of yellow color. PV
(meq/kg) was calculated as follows:
(6)

PV meq/kg = C × (V − V0 ) × 12.69 × 78.8/m
where; C is sodium thiosulphate concentration (mol/l), V and VO volumes of sodium thiosulphate blank respec-
tively (ml), and m is the mass of cracker sample extracts (mg).
Statistical analysis
Results were expressed as means ± standard deviation (n = 3) and ANOVA variance analysis with average com-
parison Duncan’s37 Multiple Range set to ˂0.05. All the statistical processes were conducted by the Statistical
Package for Social Science (SPSS, V
21.0) for Windows (SPSS, Inc., Chicago, IL, USA).
Results and discussion

Physical and chemical properties of propolis extracts
Efficiency of extraction yield methods
The yield of the different extraction methods was determined as tabulated in Table 1.
In the above Table, the propolis extraction yield differences were shown as affected by the three extraction
methods. Significant differences among values (p ˂ 0.05) were found in the extracted yields of the brown propolis
(PQG). Water extraction had a lower yield, due to the high polarity of water resulting from polarization of its
molecule what makes this solvent suitable for extraction of lyphylic organic compounds present in propolis.
Although the polarity of water was high, the yield was low because it was not the only factor affecting the extrac-
tion efficiency38. The yield of water extraction of PQG was 14.63 g/100 g dw and agreed with that obtained by
Biscaia and Ferreira39, at 14.3%.
The highest yields obtained with SCF-CO2 with co-sovent at 50 °C and 250 bar for PQG was 24.26%. These
high pressures increase the density and polarity of carbon dioxide which increase its capacity to extract more
polar components19.
TPC and TFC of propolis extracts

The optimum phenolic content varied according to the plant types and active c ompounds40 in which Phenolic
compounds can be classified into polar and weak-polar41. The most crucial feature of propolis extracts was poly-
phenols in their structure, which is an indication for high biological activity. The total contents of polyphenols
and flavonoids in extracts of the propolis obtained using the three extraction methods are shown in Table 2.
Results showed that the highest TPC value for C O2 extract at 50 °C was 395.22 ± 4.1, followed by ethanolic
sample 318.36 ± 3.8, while the water samples had the lowest activity.
When the total flavanoids amount (TFC) was examined, it was found that samples extracted with C O2 extrac-
tor at 50 °C had the highest flavonoid value being 745.42 ± 8. All supercritical extracts presented higher flavonoids
concentration to the initial value found in three extracted method,). Flavonoid level in ethanol extract is lower
than that extracted by CO2 as ethanol belongs to the group of less polar solvent. While, water extract showed
lower value for TFC being 366.51 ± 3.9.
The total concentration of polyphenols or flavonoids was not the only factor responsible for antioxidant
properties but also, the chemical nature of polyphenols and the presence of other compounds contributed to
the overall antioxidant capacity of the extracts42.
Type of extraction Techniques Yield (g/100 g dw) of propolis

Water extract Traditional extract 14.63 ± 0.56b
Ethanol extract Traditional extract 19.21 ± 0.75bc
SCF-CO2 at 50 °C Green extract 24.26 ± 0.82cd
Table 1. Total yield of propolis samples extracted by traditional and green techniques. SCF-CO2 super critical
fluid-carbon dioxide. Different superscripts in the same column or row are significant differences at P ≤ 0.05
level.
Types of extraction TPC(GA/G) TFC((QE/G)

Water extract 162.33 ± 2.7a 366.51 ± 3.9b
Ethanol extract 318.36 ± 3.8c 653.73 ± 6.8e
d
SCF-CO2 extract at 50 ºC 395.22 ± 4.1 745.42 ± 8.4f
Table 2. Total phenolic and flavonoids of propolis extracts. TPC total polyphenolic content, TFC total
flavonoids content, SCF-CO2 super critical fluid-carbon dioxide. Different superscripts in the same column or
row are significant differences at P ≤ 0.05 level.
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Antioxidant activity of propolis extracts

DPPH, reducing power (RP), and total antioxidant activity (TAA). The antioxidant properties determined
using DPPH, reducing power methods and TAA of propolis extracts at different extracting methods in Figs. 1,
2 and 3 have been thoroughly investigated and compared. Propolis extracts were characterized by the high
or similar values of scavenging activity, RP, and TAA when compared to the synthetic BHT and ascorbic acid
in vitro studies. Propolis extracts DPPH scavenging activity ranged from about 51.16% to 96.54% using different
extraction methods. PQG antioxidant activities in CO2 extract at 50 °C were higher than other extracts in Fig. 1.
Water extract Ethanol extract

SCF-CO2 extract at 50 ºC BHT
Ascorbic Acid
120
DPPH Radical Scavenging
100
80
60
40
20
0
0 10 20 30 40 50 60 70
Concentraon (µg/ml) of PQG extract
Figure 1. DPPH scavenging activities (%) of different extracts. BHT and ascorbic acid were used as positive
controls. Where; SCF-CO2 super critical fluid-carbon dioxide.
Water extract Ethanol extract

SCF-CO2 extract at 50 ºC
0.7
Absorbance at 700 nm
0.6
0.5
0.4
0.3
0.2
0.1
0
10 20 30 40 50 60
Concentraon (µg/ml) of PQG extracts
Figure 2. The reducing power of different extracts. BHT and ascorbic acid were used as positive controls.
Where; SCF-CO2 super critical fluid-carbon dioxide.
Crude propolis 2 Water extract Ethanol extract

SCF-CO2 extract at 50 ºC BHT Ascorbic Acid
98.312
93.75
92.17
100
89.017
85.206
85.09
83.65
83.45
77.116
76.63
76.33
72.189
71.55
68.356
80
Total Antioxidant %
65.71
63.53
61.451
53.071
55.63
53.94
54.5
51.23
60
47.631
46.15
41.452
43.88
42.36
40.88
37.892
31.75
40
29.08
27.33
24.203
21.45
19.37
11.63
20
0
10 20 30 40 50 60
Concentra ons (µg/ml) of PQG extracts
Figure 3. Total antioxidant activity (TAA %) of propolis extracts. BHT and ascorbic acid were used as positive
controls. Where; SCF-CO2 super critical fluid-carbon dioxide.
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Reducing power was related to antioxidant activity because antioxidants can give off their electrons to reduce
reactive radicals in Fig. 2. Thus, the reducing power could indicate the antioxidative potential of prospective
antioxidants. The increase in the absorbance values for the method of determining reducing power was due to
the phytochemical components present.
At the same time, RP had a slight increase from 0.624 to 0.692 at ethanolic extract and from 0.472 to 0.484
(Abs. at 700 nm) for SCF-CO2.
SCF-CO2 extraction showed the highest value (92.17%) followed by ethanol being 85.09%. while; sample
extracted by water had lower TAA content. These results were in agreement with those reported by Paviani
et al.19. Generally, water extract showed lower values for TPC, TFC and TAA than the others extracted methods;
therefore it was excluded from the nano procedure.
Antioxidant activity of Propolis nano‑capsule bioactive compounds

Propolis nano-capsules had significant differences among values in Table 3 (p ˂ 0.05) and had the highest TPC
and TFC values for CO2 extract at 50 °C equal to 381.86 GAE/g and736.92 mg PE/g, respectively. This was fol-
lowed by the ethanolic sample (286.12 GAE/g and 561.58 mg PE/g, respectively.
The propolis extract obtained using ethanolic and supercritical fluid CO2 methods differ significantly between
each other in terms of antioxidant activity (Table 3). Sodium alginate-propolis ethanolic extract nano-capsules
significantly decreased by nearly 10%, 14%, 10, 15% and 10% for TPC, TFC, RP, DPPH and TAA, respectively
than ethanolic free extracts. While, the percentage decrease between the SCF-CO2 extract and its nano-capsules
was lower (3.5, 1, 2.5, 4 and 3%), respectively. The nano-capsules of SCF-CO2 extract maintained the stability of
the bioactive compound despite the ethanolic extract in all antioxidants parameter.
Falcão et al.43 proved that European propolis samples (Russia and Italy) had similar polyphenol compo-
nents and antioxidant activity of Brazilian propolis, which contained fewer polyphenols and fewer antioxidant
properties.
In addition, SCF-CO2 presents good yields and nano-particles preserves the physico-chemical characteristics
of the components to be extracted14, and improve the bioavailability of the polyphenolic c ompound44.
Physical properties of nano‑capsules

Nano-capsules formed were tested by their efficiency, yield, TEM, DSC, LC–ESI–MS/MS and cytotoxic effect as follows:
Encapsulation efficiency (%)

The encapsulation efficiency of Propolis ethanol and SCF-CO2 at 50 °C extracts was 84.56% and 95.89%, respec-
tively. This was in agreement with Chen et al.45, who reported an adequate encapsulation efficiency of at least 80%.
Encapsulation yield (%)

The encapsulation yield of Propolis SCF-CO2 extract at 50 °C was significantly higher than that of the ethanolic
extract. The nano-capsules of PQG-SCF-CO2 extract at 50 °C were 95.89% more effective than ethanol extract
(84.56%) based on the basic structure of total flavonoid (736.92). The nano-capsules were also more hydrophobic
than the total phenols (381.86). The possibility that sodium ALg bound the hydroxyl groups found in flavonoids
with hydrogen bonds46 was supported by these data.
Transmission electron microscopy (TEM)

The morphology and particle size of the propolis nano ethanolic and SCF-CO2 at 50 °C extracts were measured
using TEM. These results are shown in Figs. 4A, B. Propolis extracts had a large particle size in the micrometre
range, with an average particle size between 0.03 and 0.09 μm for the ethanol extract and 0.02 to 0.07 μm for the
SCF-CO2 extract at 50 °C. The propolis ethanol extract in sodium-ALg nano-particles was homogenous, had
no aggregation, had a round shape and smooth surface and were discrete, with an average ranging from 18.87
to 39.12 nm, as shown using a TEM image in Fig. 4C. The SCF-CO2 extract at 50 °C had spherical shape, had
no aggregation and homogenous, with an average ranging from 1.49 to 36.14 nm in Fig. 4D, compared to the
encapsulated ethanol e xtract47. This result may be due to the anionic carboxylic groups of sodium-ALg, which
caused strong electrostatic repulsion between the particles48.
Antioxidant activity of propolis

Ethanol extract SCF-CO2 extract at 50 °C
Items Free extract Nano-capsules Free extract Nano-capsules
TPC (mgGA/G) 318.36 ± 3.8a 286.12 ± 3.2ab 395.22 ± 4.1b 381.86 ± 3.9ab
c b d
TFC (QE/G) 653.73 ± 6.8 561.58 ± 7.1 745.42 ± 8.4 736.92 ± 8.0cd
b c a
RP (Abs) 0.624 ± 0.05 0.692 ± 0.05 0.472 ± 0.03 0.484 ± 0.03ab
b a c
DPPH (%) 76.73 ± 2.8 64.88 ± 2.6 96.54 ± 3.1 92.57 ± 2.9c
TAA (%) 85.092 ± 4.62ab 76.183 ± 4.31a 92.173 ± 5.02c 89.584 ± 4.81b
Table 3. Antioxidant activities of propolis nano-capsules bioactive compounds. TPC total polyphenolic
content, TFC total flavonoids content, SCF-CO2 super critical fluid-carbon dioxide, RP reducing power, TAA
total antioxidant activity. Different superscripts in the same column or row are significant differences at
P ≤ 0.05 level.
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A 0.03 – 0.09 µm B 0.02 – 0.07 µm
C 18.87 – 39.12 nm D 1.49 – 36.14 nm
Figure 4. (A–D) TEM image of propolis extracts before and after nano-capsulation. (A) TEM of propolis
ethanol extract; (B) TEM of propolis SCF-CO2 extract at 50 °C. (C) TEM of propolis ethanol extract nano-
capsules; (D) TEM of propolis SCF-CO2 extract nano-capsules at 50 °C.
Differential scanning calorimetry (DSC)

Three steep sorbents at temperatures representing melting points of 90.27, 128.64 and 134.11 °C are shown in
the thermal schematic diagram DSC of Propolis in Fig. 5A. The extracted compounds represented a considerable
area starting at 67.10 °C and ending at 123.62 °C. This sharp heat absorption peak starts from the melting point
of the weak melt transition at 90.27 °C. Therefore, the extract in its free form (un-capsulated) could not be used
in food applications as an additive because could not torelate high t emperature49.
While the DSC thermogram of ethanol extract nano-capsules had four melting points in Fig. 5b (i.e. 107.24,
128.91, 178.05 and 184.82 °C), the Propolis tolerance ranged from 110.04 to 163.84 °C. It was more resistant
to exposure to high temperatures than the un-capsulated Propolis sample, in which a crystalline state was not
formed. This proved the success of the packaging process and the thermal stability against t emperature47.
On the other hand, the SCF-CO2 nano-capsules had higher thermal stability than the other two samples. Three
peaks with thermal exposure from 149.90 to 201.58 °C are shown in Fig. 5C, with a melting point of 167.67 °C.
The ability of SCF-CO2 nano-capsules was proven by these results to be used in food applications, especially in
bakery products.
LC–ESI–MS/MS analysis of propolis nano‑capsules

The polyphenolic profile of different extracts could be determined using LC–ESI–MS/MS50. The LC–ESI–MS/
MS analysis of Propolis nano-capsules of the ethanol and SCF-CO2 extracts at 50 °C are shown in Fig. 6A–C
and Table 4. Heneicosapentaenoic acid, ketotricla-bendazole and heneicosapentaenoic acid were detected in all
three propolis forms.
The highest concentration of flavonoids and phenolics occurred in crude propolis i.e. glycetein, quercetin,
kaemferol, ethyl p-coumarate, and 3,4-dimethoxycinnamic acid were observed in both crude propolis and nano-
capsules SCF-CO2 extracts at 50 °C. The absence of most compounds was indicated in the ethanol extract. The
presence of 30, 49, 50, 52, 56 and 66 derivatives in all propolis samples in Table 4 with a similar retention time
(RT) was evidenced.
The chromatograms (A, B and C) contain the peaks corresponding with the generally expected phenolic
compounds were found in nano-capsules from SCF-CO2 at 50 °C extracts in Fig. 6. The highest component was
kaempferol in the crude propolis at an RT of 56 min, and the lowest component was in the ethanol extract of
PQG (285.1507 and 215.3376 m/z), respectively in Fig. 6.
These compounds were observed in several types of propolis sourced from different parts of the world51.
The biological properties of propolis were confirmed due to its high phenolic and flavonoid content, making
it a food ingredient with high antioxidant activity. These data indicate that propolis may play a critical role in
health beneficial effects.
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Figure 5. DSC profiles of PQG forms. (A) Propolis, (B) ethanol extract nano-capsules, (C) SCF-CO2 extract
nano-capsules.
Cytotoxic effect of human cell lines

Egyptian propolis extracts were examined against the human tumour prostate cell line (PC3), human hepato-
cellular carcinoma cell line (HePG2) and human Caucasian breast adenocarcinoma (MCF7). The antitumoural
effects of propolis on the three cells are documented in Fig. 7A, B.
Results in Fig. 7A showed that SCF-CO2 propolis extract nano-capsule increase in the inhibitory activity for
the three cancer cells was 98.3, 72.3 and 55.4% for PC3, MCF7 and HePG2 respectively compared to ethanol
extract and their nano-capsules being (25.3, 23.5 and 22.3%, respectively). These results indicated that there is
a direct correlation between TPC concentration and cancer cell inhibition due to the presence of highest anti-
oxidant compounds (most notably TPC and TFC)52.
On the other hand, IC50 described the lethal concentration of the sample which causes 50% death of cells in
48 h compared to the positive control (i.e. Doxorubicin, an anticancer drug for hematologic and solid tumors,
was used as the positive control).
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Figure 6. LC–ESI–MS/MS components analysis present in propolis. (A) Propolis, (B) ethanol extract nano-
capsules; (C) SCF-CO2 extract nano-capsules at 50 °C.
Results in Fig. 7B showed that the lower values belonged to SCF-CO2 propolis nano-capsule in the case of
HePG2 (90.8 µg/mL), PC3 (99.2 µg/mL), and MCF7 (124.6 µg/mL) followed by SCF-CO2 extract for HePG2
cells (63.8 µg/mL), MCF7 cells (78.4 µg/mL) and PC3 cells (116.6 mg/mL).
While IC50 for ethanol extract was (44.6 µg/mL) for PC3 cells followed by (47 g/mL) and (75.9 µg/mL) for
MCF7cells and PC3 respectively .The lower was for ethanol nano-capsule extract in all cells.
The recommendation of NCI stated that IC50 of crude extract, if incubated from 48 to 72 h, has cytotoxic
activity in vitro less than 20 and 4 µg/ml pure compounds53.
Different types of honey and propolis extracts have been indicated in many reports to significantly inhibit
cell growth and reduce the differentiation or proliferation of cells from various tumour cell lines54. Cancer cell
inhibition should be evaluated to develop new anticancer drugs. Based on these criteria, nano-capsule C O2
propolis extracts were assessed for their inhibition as candidate anticancer cancer drugs for human hepatocel-
lular, prostate and human Caucasian breast adenocarcinoma.
Application
Sensory evaluation of crackers fortified with propolis extracts
The sensory scores of crackers fortified with propolis extracts and their nano-capsules are shown in Table 5 and
Fig. 8.
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Precursor m/z
Nano-capsulate PQG solvent Nano-capsulate PQG-
N RT Formula structure Molecular weight (MW) Putative compound name Propolis extract SCF-CO2 at 50 °C extract
Heneicosapentaenoic acid
30 36.34 C21H32O2 315.091 315.0914 231.0392 315.0914
(NIST)
31 36.88 HOOC(CH2)7COOH 187.173 Azelaic acid (NIST) 187.1725 – 187.1725
(. + −.)-Camphor-10-sulfonic
32 37.14 C10H16O4S 231.046 – 231.0454 –
acid (NIST)
33 39.49 C16H12O5 283.108 Glycetein 283.1087 – 280.0259
3,4-Dimethoxycinnamic acid
34 39.63 C11H12O 207.160 207.1635 – 201.2361
(NIST EL)
3,4-Dimethoxycinnamic acid
35 39.93 C11H12O 207.113 207.1395 – 200.3567
(NIST EL)
36 40.49 – 245.130 Met-Pro (NIST) 245.1296 – 240.1136
37 43.40 – 271.114 No match 271.1135 – 268.1360
3-Hydroxyoctadecanoic acid
38 44.77 C18H36O3 299.108 299.1035 – 291.2305
(NIST)
3,4,2’,4’,6’-Pentahydroxychal-
39 45.69 C15H14O6 287.102 287.1020 – 280.2504
cone (NIST)
7-Hydroxy-3-(4-
40 46.49 C22H16O4 343.095 methoxyphenyl)-4-phenylcou- 343.0949 – 339.1055
marin
41 46.61 C10H13N5O5 282.123 2-Hydroxyadenosine 282.1246 – 275.2158
42 48.15 C15H10O7 301.118 Quercetin 301.1126 – 298.2531
43 48.33 C27H38O9 329.115 11a-Hydroxyprogesterone 329.1154 – 321.2791
44 48.56 C15H10O6 285.154 Kaempferol 285.1447 – 275.1836
49 53.70 C16H12O7 315.101 Isorhamnetin 315.0980 271.1153 303.6513
50 55.95 C18H24O2 271.201 Estradiol 271.1955 242.2291 264.2894
DL-.beta.-hydroxypalmitic acid
51 56.57 C16H32O3 271.125 271.1403 – 263.0258
(NIST)
52 57.18 C13H7Cl3N2O2 327.121 Ketotriclabendazole 327.1222 269.0904 319.3691
53 57.89 C15H10O5 269.129 Genistein 269.1283 – 191.0703
54 58.07 – 242.332 Gln-Pro (NIST) 242.3310 228.2186 –
55 58.36 C15H12O6 287.162 Funalenone 287.1635 – –
56 58.90 C15H10O6 285.151 Kaempferol 285.1507 215.3376 276.1369
3-Hydroxyoctadecanoic acid
60 60.64 C18H36O3 299.116 299.1158 242.2430 –
(NIST)
7-Hydroxy-6-methoxyisofla-
65 61.53 C16H12O4 267.161 267.1759 – 254.1587
vone (NIST)
66 62.06 C21H32O2 315.151 315.1539 315.0855 304.2510
(NIST)
67 62.55 C21H32O2 315.120 315.0979 – 311.1054
(NIST)
68 62.55 C12H24O11 343.104 Lactitol (NIST) 343.1036 – 331.1364
69 63.08 C11H12O3 191.183 Ethyl p-coumarate (NIST) 191.1837 – 186.4542
70 63.32 C15H12O4 255.180 Pinocembrin (NIST) 255.1788 – 246.2408
71 63.55 C15H10O5 269.166 Genistein 269.1652 – 251.2208
72 63.91 C15H12O4 255.193 Pinocembrin (NIST) 255.2029 – 248.1344
73 64.68 C16H12O6 299.101 Hydroxygenkwanin 299.1002 – 282.2013
74 65.53 C16H12O6 299.086 Hydroxygenkwanin 299.0914 – 289.0045
76 65.59 – 284.136 No match 284.1402 – 272.0523
Table 4. LC–ESI–MS/MS compounds analysis of Egyptian propolis ethanol and SCF-CO2 extracts at 50 °C
nano-capsules. RT retention time, SCF-CO2 super critical fluid-carbon dioxide.
The control sample was characterized by the highest (p ˂ 0.05) total overall acceptability (86.84%) among
the cracker samples tested (Table 5). At the same time, the lower scores of crackers fortified with Propolis etha-
nol extract than the two nano-capsules sample may be due to the gum taste of the propolis extract. The overall
acceptability of crackers fortified with Propolis SCF-CO2 at 50 °C and ethanol extracts nano-capsules was 86.57
and 86.29, respectively.
These crackers (FE-EEN and FP-SCFN) obtained the highest score, probably because they acquired the fragil-
ity and porosity properties of the sodium alginate capsules and their ability to bind water and reduce its content55.
For a long time, propolis bioactive ingredients have been used in many applications. Many new functional food
products have appeared in the markets in recent years to respond to health problems faced by consumers.
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PC3 MCF7 HePG2

98.3
100
90 A
68.3 72.3
80
70
Inhibition %
44.8 55.4
60
50 41.9
25.3 25.6
40 23.5
30 22.3 22.5 21.8
20
10
0
Ethanol extract Nano-capsule CO2 extract Nano-capsule CO2
ethanol
PC3 MCF7 HePG2
140 124.6
B 116.6
120
99.2 90.8
75.9 78.4
100
IC50 (µg/ml)
80 51.2 63.8
47
60 44.6 45 43.6
40 20 20
20
20
0
Ethanol Nano-capsule CO2 extract Nano-capsule Posive
extract ethanol CO2 control
Figure 7. (A,B) Anticancer effect of propolis extracts and their nano-capsules. IC50 lethal concentration of the
sample that causes 50% death of PC3: prostate cell line; MCF7 human Caucasian breast adenocarcinoma, HePG2
human hepatocellular carcinoma cell line, positive control Adriamycin (doxorubicin) after 48 h.
Storage stability of fortified cracker samples

Antioxidant activity of stored crackers. The DPPH scavenging activity of the stored cracker samples fortified
with propolis extracts and their nano-capsules were measured compared with the control samples during the
storage period of 90 days, as shown in Table 6.
A clear and noticeable significant decrease in DPPH (p ˂ 0.05) between the fortified crackers with ethanol and
SCF-CO2 at 50 °C extracts with increasing storage time are shown in Table 6. From zero to 90 days of storage, the
decrease was between 54.58 and 36.18% for the ethanol extract and 76.84% and 48.33% for the SCF-CO2 extract.
This was due to the high sensitivity of the un-capsulated extract to storage period. The cracker samples fortified
with nano-capsules of ethanol and SCF-CO2 at 50 °C extracts had slight decreases, from 62.43 to 55.07% and
from 90.72 to 84.79% after 90 days storage respectively, due to the ability of encapsulation to protect the extract
from exposure to high temperatures.
These results were indications that crackers fortified with nano-capsulate propolis SCF-CO2 extract at 50 °C
had a higher ability to retain antioxidant activity during the storage period, followed by the crackers fortified
with propolis nano-capsulate ethanol extract. The encapsulation process was able to store the propolis extract
for nine months in solid-state form, as mentioned by Ticiano et al.56.
Peroxide value (PV) of the crackers. The changes in PV of the five stored cracker samples that occurred during
the storage period are shown in Table 7. The increase of peroxide values was relatively low in the cracker fortified
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Total overall
Cracker samples Appearance (10) Color (15) Thickness (15) Crispness (15) Shrinkage (15) Taste (15) Odor (15) acceptability (100)
Control 9.62 ± 0.5a 14.31 ± 0.4ab 10.63 ± 0.4a 12.85.25 ± 0.3a 12.95 ± 0.4a 13.23 ± 0.5ab 13.25 ± 0.3ab 86.84
ab b ab ab b b
FP-EE 8.35 ± 0.5 12.03 ± 0.5 10.42 ± 0.5 11.56 ± 0.4 11.31 ± 0.5 12.55 ± 0.6 11.11 ± 0.4b 77.33
FP-SCF 8.94 ± 0.5ab 12.27 ± 0.5b 10.68 ± 0.5a 11.62 ± 0.4ab 11.79 ± 0.5b 12.76 ± 0.5b 12.03 ± 0.4b 80.09
FP-EEN 9.35 ± 0.6b 13.35 ± 0.6bc 11.38 ± 0.5b 13.78 ± 0.6b 12.52 ± 0.6bc 13.09 ± 0.6b 12.82 ± 0.5c 86.29
FP-SCFN 9.71 ± 0.7b 13.40 ± 0.6bc 11.35 ± 0.6bc 13.04 ± 0.7bc 12.88 ± 0.6c 13.11 ± 0.6bc 13.08 ± 0.5cd 86.57
Table 5. Sensory evaluations of fortified crackers with different types of Propolis extracts and nano-capsules.
FP-EE cracker fortified with PQG ethanol extract, FP-SCF cracker fortified with propolis SCF-SO2 extract
at 50 °C, FP-EEN cracker fortified with propolis ethanol extract nano-capsules, FP-SCFN cracker fortified
with propolis SCF-SO2 extract at 50 °C nano-capsules. Different superscripts in the same column or row are
significant differences at P ≤ 0.05 level.
Figure 8. Crackers samples unfortified and fortified with different types of propolis extracts and nano-capsules.
with PQG ethanol and SCF-CO2 at 50 °C extracts nano-capsules, being 0.11 and 0.08, respectively. This result
occurred because of the ability of capsules to prevent the oxidation of fat and rancidity, increasing the shelf life
of crackers.
Conclusions and prospects

Currently, there is a significant interest directed towards the bioactive compounds of plants to build up a robust
immune system. This future vision led researchers to discover a novel technique for extracting these bioactive
compounds facilitate and increase the yield, improving the process without degradation. The functional food
market has expanded over the years due to the demand of consumers for healthy food products, reducing the
use of synthetic preservatives and antioxidants and improving the quality of products57.
Nano-capsulation technologies in the food industry, mainly nano-structured food ingredients, improve solu-
bility, sensory properties and stability during s torage58,59. Controlling food safety and risk assessments of novel
materials added to food encourage many countries to establish powerful p latforms60. However, the toxicological
repercussions of these new materials and ethical issues are still limited and remain to be tested.
We choose nano-capsules of ethanolic and SCF-CO2 extracts at 50 °C in this study. These nanocapsules were
characterized by the high extraction yields and supercritical extracts, which provided higher flavonoid con-
centrations than EEP. Such a result indicates essential compounds in the fractionation of propolis compounds,
as made by several authors before being commercialised. Propolis nano-capsules (SCF-CO2 at 50 °C extracts)
had robust inhibitory effects on human tumour growth, potentially preventing oxidative damage and inducing
apoptosis and immune stimulation. The crackers fortified with these two propolis extract nano-forms proved
their overall acceptability, improved their stability and extended their shelf life. This product could be considered
a new functional food that the food industry could process to improve human health. Finally, numerous futures
expand for nano-capsulation in the food industry will enhance the standard of living of people.
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DPPH radical scavenging activity %

Propolis extract (0.6 g) before cracker
Fortified cracker samples fortification Zero time after baking After 30 days storage After 60 days storage After 90 days storage
FP-EE 76.73 ± 2.8ab 54.58 ± 1.9a 48.33 ± 1.9a 42.51 ± 2.1a 36.18 ± 1.6a
c ab b b
FP-SCF 96.54 ± 3.1 76.84 ± 3.1 68.13 ± 2.5 59.42 ± 2.3 48.33 ± 2.1ab
a b b b
FP-EEN 64.88 ± 2.6 62.43 ± 2.3 60.92 ± 2.2 58.14 ± 2.2 55.07 ± 2.3b
d c c c
FP-SCFN 92.57 ± 2.9 90.72 ± 3.1 88.27 ± 2.8 86.24 ± 2.7 84.79 ± 2.7c
Table 6. Storage stability of cracker samples fortified with various propolis extracts. FP-EE cracker fortified
with propolis ethanol extract, FP-SCF cracker fortified with propolis SCF-SO2 extract at 50 °C, FP-EEN cracker
fortified with propolis ethanol extract nano-capsules, FP-SCFN cracker fortified with propolis SCF-SO2 extract
at 50 °C nano-capsules. Different superscripts in the same column or row are significant differences at P ≤ 0.05
level.
Peroxide value
Days Control FP-EE FP-SCF FP-EEN FP-SCFN
0 0.13 ± 0.01a 0.13 ± 0.01a 0.13 ± 0.01a 0.13 ± 0.01a 0.13 ± 0.01a
b b b ab
30 0.75 ± 0.04 0.62 ± 0.03 0.59 ± 0.02 0.18 ± 0.01 0.16 ± 0.02ab
bc b b ab
60 0.96 ± 0.06 0.71 ± 0.04 0.64 ± 0.03 0.21 ± 0.02 0.19 ± 0.02ab
90 1.23 ± 0.08cd 0.84 ± 0.06c 0.76 ± 0.03bc 0.24 ± 0.02b 0.21 ± 0.02b
Increase T0–T90 1.1 0.71 0.63 0.11 0.08
Table 7. Peroxide values (PV) of cracker samples during incubation period (days). FP-EE cracker fortified
with PQG ethanol extract, FP-SCF cracker fortified with SCF-SO2 at 50 °C extract from PQG, FP-EEN cracker
fortified with PQG ethanol extract nano-capsules, FP-SCFN cracker fortified with PQG-SCF-SO2 at 50 °C
extract nano-capsules. Different superscripts in the same column or row are significant differences at P ≤ 0.05
level.
Data availability
Tip 1: In general, different methods were used to extract the bioactive ingredients in propolis, and the best
extraction method was to use supercritical fluid carbon dioxide as shown in Table 1. The data issued in Table 2
also shows the content of phenols and total flavonoids for those extracts, and Figs. 1 and 2 show the antioxidant
activity of those extracts through reducing power and DPPH, and Fig. 3 confirms the total antioxidant activity of
the extracts. And the comparison between them (free and encapsulated extracts in nano-capsules). Table 3 shows
the antioxidant activity of the extracts before and after nano-encapsulation, which showed significant differences
for the encapsulated extracts higher than the un-encapsulated ones. The physical and chemical properties of
those free and encapsulated extracts were studied, and the encapsulation efficiency was studied. Figure 4 shows
the morphological composition of the particles before and after nano-encapsulation. Figure 5 shows the thermal
stability of the extracts before and after nano-encapsulation to determine the extent to which they can be applied
in the field of foods that are exposed to high temperatures. The toxicity of these extracts on cancer cells of the
liver and prostate was also studied, and their effects on cell vitality and inhibition of cancer cells were studied.
Finally, the applied study was done by adding it to crackers (bakery products) by comparing the alcoholic extract
and the extract with C O2 before and after nano-encapsulation, and studying the extent of storage stability for
those products that contain natural antioxidants, which were favored by consumers. Tip 2: Data is "on demand"
and all data in a study is available to scholars in the same field. Tip 3: All data we create is disclosed. "All the data
mentioned in this paper are available and help in understanding the importance of the study."
Received: 4 March 2023; Accepted: 4 September 2023
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Acknowledgements
The authors wish to thank the Academy of Scientific Research and Technology (ASRT) in Egypt and the National
Research Centre (NRC) Institute of Biology and Agricultural Biotechnology (IBBA) in Italy. These institutions
supported and funded this research throughout the bilateral project between Egypt and Italy in the project
“Preparation and Utilization of Nano-Propolis Active Compounds, their Biological Function and Application”.
Author contributions
A.A.A.: visualization,writing and reviewing, project administration. K.F.M.: methodology, formal analysis,
design. M.F.S.: investigation, writing and reviewing. V.L.: supervision and final approval. L.P.: methodology,
and design. E.I.S.: formal analysis and supervision. M.A.I.: methodology, formal analysis.
Funding
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in coopera-
tion with The Egyptian Knowledge Bank (EKB).
Competing interests
The authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to K.F.M.
Reprints and permissions information is available at www.nature.com/reprints.
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OPEN Characterization and stability

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 1

Materials and methods

Chemical properties of propolis extracts and their nano‑capsules

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 2

Physical properties of nano‑capsules

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 3

Storage stability of crackers

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 4

Results and discussion

TPC and TFC of propolis extracts

Type of extraction Techniques Yield (g/100 g dw) of propolis

Types of extraction TPC(GA/G) TFC((QE/G)

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 5

Antioxidant activity of propolis extracts

Water extract Ethanol extract

Water extract Ethanol extract

Crude propolis 2 Water extract Ethanol extract

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 6

Antioxidant activity of Propolis nano‑capsule bioactive compounds

Physical properties of nano‑capsules

Encapsulation efficiency (%)

Encapsulation yield (%)

Transmission electron microscopy (TEM)

Antioxidant activity of propolis

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 7

A 0.03 – 0.09 µm B 0.02 – 0.07 µm

C 18.87 – 39.12 nm D 1.49 – 36.14 nm

Differential scanning calorimetry (DSC)

LC–ESI–MS/MS analysis of propolis nano‑capsules

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 8

Cytotoxic effect of human cell lines

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 9

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 10

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 11

PC3 MCF7 HePG2

PC3 MCF7 HePG2

Storage stability of fortified cracker samples

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 12

Conclusions and prospects

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 13

DPPH radical scavenging activity %

Received: 4 March 2023; Accepted: 4 September 2023

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 14

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 15

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 16

© The Author(s) 2023

Scientific Reports | (2023) 13:16065 | https://doi.org/10.1038/s41598-023-42025-0 17

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