Characterization and stability evaluation of Egyptian propolis extract nano-capsules and their application

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.


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 lyophilized (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 Masaharu 18 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-CO 2 ).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 CO 2 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 CO 2 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: where: W 1 is the weight of propolis extracts (g) and W 2 is the weight of dried raw propolis powder (g).

Preparation of nano-capsules
Ethanol and SCF-CO 2 Propolis extracts were encapsulated into nano-forms using ultrasound and high-speed homogeniser (PRO, USA) methods, according to Vasiliki and Constantina 20 .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 presence 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 (1) Yield (%) = (W 1 /W 2 ) × 100 www.nature.com/scientificreports/Ebrahimzadeh et al. 21with 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 calibration 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 spectrophotometer.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.
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-CO 2 with (60 µg/ml) were determined as above.
Reducing power (RP).The reducing power (RP) of three extracts were determined following the method of Oyaizu 25 .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 concentration 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-CO 2 with (60 µg /ml) were determined as above.
Total antioxidant activity (TAA).Water, Ethanol and CO 2 extracts total antioxidant activity (TAA) were determined using a linoleic acid system 26 .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 method 27 .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 ferrous 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-CO 2 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: 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 encapsulation process.The effectiveness of encapsulation yield (EY) was measured using Paviani et al. 19 method and calculated as follows: Transmission electron microscopy (TEM).Propolis extracts and their nano-capsules' morphological characteristics 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 acid 30 .The samples were air-dried at room temperature for more than 2 h before analyzing on the TEM.
(2) % DPPH radical scavenging activity = ((Ac − As)/Ac) × 100 www.nature.com/scientificreports/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 follows.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-CO 2 at 50 °C was performed using liquid chromatography-electro-spray ionization-tandem 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 1.6.10.40973software.
The separation of the targeted analyses was performed with a Discovery ® 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.
Cells were batch cultured for 10 days, then seeded at concentration of 10 × 10 3 cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37 °C for 24 h under 5% CO 2 using a water-jacketed CO 2 dioxide 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 (100-50-25-12.5-6.25-3.125-0.78and 1.56 µg/mL).After 48 h of incubation, 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% CO 2 .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 conditions 33 .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: Using the SPSS 11 program, the IC 50 was determined using probit analysis.The degree of selectivity of the synthetic compounds was expressed as SI = IC 50 of the pure compound in a normal cell line/IC 50 of the same pure compound in a cancer cell line, where IC 50 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 Karnjanapratum 34 .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 thickness of a 0.4 mm and cut into a rectangle (2.4 × 7.3 cm 2 ).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 Smith 35 .

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 evaporator 36 .The antioxidant activity of the crackers' residue was determined as described previously.www.nature.com/scientificreports/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 performed according to AOAC 36 .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: where; C is sodium thiosulphate concentration (mol/l), V and VO volumes of sodium thiosulphate blank respectively (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 comparison Duncan's 37 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).

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 extraction efficiency 38 .The yield of water extraction of PQG was 14.63 g/100 g dw and agreed with that obtained by Biscaia and Ferreira 39 , at 14.3%.
The highest yields obtained with SCF-CO 2 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 components 19 .

TPC and TFC of propolis extracts
The optimum phenolic content varied according to the plant types and active compounds 40 in which Phenolic compounds can be classified into polar and weak-polar 41 .The most crucial feature of propolis extracts was polyphenols 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 CO 2 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 CO 2 extractor 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 CO 2 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 extracts 42 .www.nature.com/scientificreports/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-CO 2 .
SCF-CO 2 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 and736.92mg PE/g, respectively.This was followed by the ethanolic sample (286.12GAE/g and 561.58 mg PE/g, respectively. The propolis extract obtained using ethanolic and supercritical fluid CO 2 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-CO 2 extract and its nano-capsules was lower (3.5, 1, 2.5, 4 and 3%), respectively.The nano-capsules of SCF-CO 2 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 components and antioxidant activity of Brazilian propolis, which contained fewer polyphenols and fewer antioxidant properties.
In addition, SCF-CO 2 presents good yields and nano-particles preserves the physico-chemical characteristics of the components to be extracted 14 , and improve the bioavailability of the polyphenolic compound 44 .

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-CO 2 at 50 °C extracts was 84.56% and 95.89%, respectively.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-CO 2 extract at 50 °C was significantly higher than that of the ethanolic extract.The nano-capsules of PQG-SCF-CO 2 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 bonds 46 was supported by these data.

Transmission electron microscopy (TEM)
The morphology and particle size of the propolis nano ethanolic and SCF-CO 2 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-CO 2 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-CO 2 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 extract 47 .This result may be due to the anionic carboxylic groups of sodium-ALg, which caused strong electrostatic repulsion between the particles 48 .Table 3. Antioxidant activities of propolis nano-capsules bioactive compounds.TPC total polyphenolic content, TFC total flavonoids content, SCF-CO 2 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.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 temperature 49 .

Items Antioxidant activity of propolis
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 temperature 47 .
On the other hand, the SCF-CO 2 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-CO 2 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/MS 50 .The LC-ESI-MS/ MS analysis of Propolis nano-capsules of the ethanol and SCF-CO 2 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 nanocapsules SCF-CO 2 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-CO 2 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 world 51 .
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.

Cytotoxic effect of human cell lines
Egyptian propolis extracts were examined against the human tumour prostate cell line (PC3), human hepatocellular 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-CO 2 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 antioxidant compounds (most notably TPC and TFC) 52 .
On the other hand, IC 50 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).Results in Fig. 7B showed that the lower values belonged to SCF-CO 2 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-CO 2 extract for HePG2 cells (63.8 µg/mL), MCF7 cells (78.4 µg/mL) and PC3 cells (116.6 mg/mL).
While IC 50 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 IC 50 of crude extract, if incubated from 48 to 72 h, has cytotoxic activity in vitro less than 20 and 4 µg/ml pure compounds 53 .
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 lines 54 .Cancer cell inhibition should be evaluated to develop new anticancer drugs.Based on these criteria, nano-capsule CO 2 propolis extracts were assessed for their inhibition as candidate anticancer cancer drugs for human hepatocellular, prostate and human Caucasian breast adenocarcinoma.

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.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 ethanol 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-CO 2 at 50 °C and ethanol extracts nano-capsules was 86.57and 86.29, respectively.
These crackers (FE-EEN and FP-SCFN) obtained the highest score, probably because they acquired the fragility and porosity properties of the sodium alginate capsules and their ability to bind water and reduce its content 55 .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.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-CO 2 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-CO 2 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-CO 2 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-CO 2 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

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 products 57 .Nano-capsulation technologies in the food industry, mainly nano-structured food ingredients, improve solubility, sensory properties and stability during storage 58,59 .Controlling food safety and risk assessments of novel materials added to food encourage many countries to establish powerful platforms 60 .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-CO 2 extracts at 50 °C in this study.These nanocapsules were characterized by the high extraction yields and supercritical extracts, which provided higher flavonoid concentrations 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-CO 2 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.

Figure 1 .
Figure 1.DPPH scavenging activities (%) of different extracts.BHT and ascorbic acid were used as positive controls.Where; SCF-CO 2 super critical fluid-carbon dioxide.

Figure 2 .
Figure 2. The reducing power of different extracts.BHT and ascorbic acid were used as positive controls.Where; SCF-CO 2 super critical fluid-carbon dioxide.

Figure 3 .
Figure 3.Total antioxidant activity (TAA %) of propolis extracts.BHT and ascorbic acid were used as positive controls.Where; SCF-CO 2 super critical fluid-carbon dioxide.

Figure 7 .
Figure 7. (A,B) Anticancer effect of propolis extracts and their nano-capsules.IC 50 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.

Figure 8 .
Figure 8. Crackers samples unfortified and fortified with different types of propolis extracts and nano-capsules.

Table 1 .
69 × 78.8/mTotal yield of propolis samples extracted by traditional and green techniques.SCF-CO 2 super critical fluid-carbon dioxide.Different superscripts in the same column or row are significant differences at P ≤ 0.05 level.

Table 2 .
TotalAntioxidant 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 CO 2 extract at 50 °C were higher than other extracts in Fig.1.

Table 4 .
LC-ESI-MS/MS compounds analysis of Egyptian propolis ethanol and SCF-CO 2 extracts at 50 °C nano-capsules.RT retention time, SCF-CO 2 super critical fluid-carbon dioxide.

Table 7 .
The increase of peroxide values was relatively low in the cracker fortified

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-SO 2 extract at 50 °C, FP-EEN cracker fortified with propolis ethanol extract nano-capsules, FP-SCFN cracker fortified with propolis SCF-SO 2 extract at 50 °C nano-capsules.Different superscripts in the same column or row are significant differences at P ≤ 0.05 level.

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-SO 2 extract at 50 °C, FP-EEN cracker fortified with propolis ethanol extract nano-capsules, FP-SCFN cracker fortified with propolis SCF-SO 2 extract at 50 °C nano-capsules.Different superscripts in the same column or row are significant differences at P ≤ 0.05 level.

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-SO 2 at 50 °C extract from PQG, FP-EEN cracker fortified with PQG ethanol extract nano-capsules, FP-SCFN cracker fortified with PQG-SCF-SO 2 at 50 °C extract nano-capsules.Different superscripts in the same column or row are significant differences at P ≤ 0.05 level.