Green synthesis of nano-propolis and nanoparticles (Se and Ag) from ethanolic extract of propolis, their biochemical characterization: A review

2.1.1 Encapsulation via casein micelle with a homogenizer

The pH of cow milk obtained from a market store is adjusted to 6.4 by incubating it at 30°C for 1 h with hydrochloride acid of 1 N. After adding rennet and agitating it for 15 min at 30°C, the casein was aggregated. To increase the particle size, the aliquot was incubated at the same temperature for 15 min. Filtration was used to separate the casein and other proteins. After inactivating the rennet with hot water (70°C) for 5 min, the casein and water were separated by filtration. Using 12% SDS PAGE, the molecular weight of casein was examined [43].

2.1.2 Synthesis of nanopropolis

5 g casein was taken and diluted in 50 mL of 10 mM phosphate buffer with pH = 10. In ethanol, 5 mL propolis was added while stirring. Every 5 min, 1 mL of 10% CaCl₂ was added to the mixture. The pH of the mixture was adjusted to 7 using 0.1 N HCl or 0.1 N NaOH. For 5 min, the mixture was also sonicated. Microfiltration (Whatmann paper No. 42) was used to separate the mixture. Cut off the permate at 10 kDa and ultrafiltrate it. Phosphate buffer was used to dilute the retentante of micro and ultra filtration. The total of polyphenols and total flavonoids were assessed in the ultrafiltration permeate, which contained unencapsulated propolis [43].

2.2.1 Preparation of NIPAAM/VP/PEG-A copolymeric nanoparticles

NIPAAM + VP + PEG-A + MBA were dissolved in water in molar ratios of 90:5:5, they took 90 mg NIPAAM, 5 μL of freshly distilled VP, 500 μL of PEG-A (1% w/v), 10 mL of water. To cross link polymer chains, 30 μL of MBA was used. Polymerization was done using 30 μL of APS, 20 μL of TEMED, 20 μL of FAS (0.5% w/v) in N₂ atmosphere at 30°C. After that dialysis for 2–3 min was done in aqueous medium for co-polymeric nanoparticles containing unreacted monomers as given in Figure 2. Pure co-polymeric nanoparticles in aqueous medium were employed for lyophilization to obtain dry powder of co-polymeric nanoparticles. To remove any inhibitors, NIPAAM was recrystallized using hexane, VP was freshly distilled before use, and PEG-A was washed three times with n-hexane [76].

Figure 2

Procedure studied in the preparation of co-polymeric nanoparticles. NIPAAM – N-isopropylacrylamide; VP – N-vinyl-2-pyrrolidone; PEG-A – poly(ethyleneglycol) monoacrylate; MBA – N,N′-methylene bis-acrylamide; APS – ammonium persulfate; TEMED – tetramethyl ethylene diamine; FAS – ferrous ammonium sulfate [76].

2.2.2 Preparation of propolis loaded polymeric nanoparticles with polycaprolactone-pluronic polymeric matrix

Nanoprecipitation with poly-caprolactone and pluronic combination is another approach for the synthesis of propolis loaded polymeric nanoparticles. The preformed polymer interfacial deposition method, also known as nanoprecipitation preformed polymer, was used to create colloidal suspensions of nanoparticles. The organic and aqueous phase components were weighed and placed in separate containers, then subjected to sonication until finished evaporation. The organic phase (500 μL) was then added into the aqueous phase (50 mL) with vortexing for 1 min following the freeze drying of nanoparticles. Polymeric nanoparticle suspensions with loaded propolis were submitted to the assays for characterization [75].

2.2.3 Extraction of propolis

Various authors prepared propolis extract by dissolving dry propolis. 35 g of dried propolis powder was dissolved in 250 mL of deionized water. The extraction was carried out by placing the flask on an orbital shaker at room temperature for 72 h and agitating it at 100 rpm. The aqueous solution was filtered with Whatman filter paper No. 1 after extraction. Crude extract was prepared by concentrating the extract under reduced pressure [9].

2.2.4 Loading of propolis

Loading of the propolis in the polymeric nanoparticles was done using a post-polymerization method. For this, in 10 mL of distilled water, 100 mg of the lyophilized powder was added and stirred to reconstitute the micelles. In Chloroform, free propolis was dissolved (CHCl₃; 10 mg‧mL⁻¹). With constant vortexing and mild sonication, the propolis solution in CHCl₃ was slowly added to the polymeric solution. By physical entrapment, propolis was directly loaded into the hydrophobic core of the nanoparticles. Then, these propolis-loaded nanoparticles were lyophilized to dry powder for subsequent use [75].

2.3.1 From ethanol extract of propolis

For the synthesis of SeNPs, three main methodologies can be applied, including physical methods which involves pyrolysis, physical vapor deposition, crushing, lithography, grinding, attrition, and ball milling [10,78,79], chemical methods including solvothermal, sol gel, thermal decomposition, microwave-assisted synthesis, ultrasonic-assisted, and electrochemical methods [80,81,82,83] and biosynthetic processes [10,18]. The most prevalent method for preparing SeNPs is reduction, which includes chemical reduction [18,84], γ-radiolytic reduction [85], and bacterial reduction [86].

Main drawback of these methods is that they have long synthesis time, requires high temperature, have high production cost, low yield, and also involves some hazardous chemicals affecting human health and the environment as they might be genotoxic, cytotoxic, or carcinogenic. As a result green chemistry methods have been developed and have become popularized which involve the use of biological systems such as microorganisms (bacteria, fungi, algae, and yeast) and plant extracts [87]. Use of biogenic synthesis of SeNPs is more advantageous as it is economical, non-toxic, uses non-hazardous materials, produces stable nanomaterials, also have natural organic covering to avoid the nanoparticles aggregation. SeNPs have exceptional applications such as significant biocidal role, capability to provide protection against free radicals, least toxicological effects, and prominent biological activities.

To 30 mM sodium selenite solution, 10 mL of freshly prepared ethanol extract was added along with 40 mM ascorbic acid. Mixture was placed at room temperature for 24 h until the light brown color changed to orange color. Centrifugation was done for newly biosynthesized SeNPs at 10,000 rpm for 30 min at 4°C. Washing was done for the separated nanoparticles thrice with double distilled water and then washed by using ethanol. After that SeNPs were dried overnight and their powdered form was used for further characterization and analysis [88]. Cycloheximide, a common fungicide, was employed as a positive control, and ethanol was utilized as a negative control. Schematic representation is given in Figure 3.

Figure 3

Schematic representation for the synthesis of selenium nanoparticles from ethanolic extract of propolis.

2.3.2 Other techniques for fabrication of SeNPs with propolis

It has been revealed in many studies that there are also some other techniques such as microwave irradiation, UV radiation, hydrothermal, ultrasonication, self-assembling, and conventional heating for the fabrication of SeNPs with propolis extract [77]. Initially propolis extract was added to the prepared selenium salt solution and this combination was used as sample for the different methods. In hydrothermal technique, colloidal solution was autoclaved at 121°C for 15 min with 1.5 atm. By using 800 W microwave device, the mixture solution was heated for 30 s in microwave radiation technique. In the ultrasonication method, the probe of the equipment was engrossed in the colloidal solution and further by using a laboratory ultrasound device it was treated with a frequency and power of 20 kHz and 300 W, respectively. From a 6 W UV lamp, UV light of 365 nm was used for the exposure of mixture solution at room temperature for a maximum of 1 h. Colloidal solution was kept overnight at room temperature (33°C) in the self-assembling technique [77].

2.3.3 From watery extract of propolis

150 mL of sodium selenite solution was poured gently drop by drop to a conical flask containing 300 mL of propolis watery extract solution. To avoid the effect of light on the nanoparticle composition process, a sheet of aluminum foil was placed over the flask. By adding drops of 1 N NaOH to the mixture, the pH was adjusted to 8, and it was agitated for 6 h at 37–40°C. Centrifugation at 12,000 rpm for 30 min in 20°C separated the produced nanoparticles [10].

3.1.1 Determination of total encapsulated polyphenols or flavonoids

where A – total polyphenols or flavonoids added initially, and B – total unencapsulated polyphenols or flavonoids [43].

3.1.2 Particle size analysis

The diameters of the nano/micro particles were measured using a laser light scattering granularity analyzer (DelsaTM Nano, Germany) Beckman Coulter. A sample was taken and triplicate analyses were performed [43].

3.1.3 TEM analysis

TEM was used to study the microstructure of particles which was operated at 80 kV. The sample was obtained by placing one preparation drop on a collodion support on grids.

3.2.1 UV-Vis spectrum analysis

For the identification and characterization of propolis, best used technique was UV-Vis spectrum. By using Genesys 10S UV-Vis spectrophotometer, the wavelength of biosynthesized SeNPs from 200–800 nm was recorded and analyzed for the maximum absorption peaks [48]. Shimadzu UV-1600, Japan, was used by authors of ref. [10] to study the spectrum with wave range of 190–1,100 nm, scan speed of sampling interval was 0.5 min.

3.2.2 Fourier transform infrared spectroscopy analysis (FT-IR)

Shimadzu – 00463 model spectrophotometer was used to obtain FT-IR spectrum. It was obtained in the spectral region of 4,000–600 cm⁻¹ with a resolution of 4 cm⁻¹ and 64 co-added scans [88]. FT-IR spectroscopy (ABB/spectrolab/MB3000/UK) was used to examine the produced SeNP. The FTIR characterization was done using the conventional KBr pellet technique, which measures infrared intensity vs wavelength (wave number) of light from 400 to 4,000 cm⁻¹ [10]. Organic functional group responsible for stabilization of nanoparticles, nature of associated functional groups, structural features of biological extracts with nanoparticles and surface chemistry was confirmed by using FT-IR spectrum [10,88].

3.2.3 XRD analysis

XRD was used to investigate the composition of the synthesized particles [10], and also for the structure of nanoparticles and phase identification [88]. Bruker D8 Advanced Eco X-ray diffractometer having Bragg-Brentano focusing geometry was used to take the XRD readings and then this XRD data were analyzed for the identification of the crystalline phases by using X’pert high score software [88].

3.2.4 SEM analysis

To illustrate the morphological characteristics and size of biosynthesized SeNPs, scanning electron microscope (Hitachi, Model SU3500) with ultrahigh resolution field emission was used [88]. To analyze the morphology and form of the particles, samples were mildly pressed into pellets at 0.5 ton-load to achieve the best picture under SEM [10,91].

3.2.5 Antimicrobial assay

3.3.1 Analysis of photocatalytic activity of synthesized AgNPs

Under sunlight irradiation, the degradation of malachite green was observed to determine the photocatalytic activity of the synthesized AgNPs. In 50 mL of malachite green solution (10 mg‧L⁻¹), 10 mg AgNPs were added, and the mixture was magnetically swirled for 45 min in the dark. After that, the colloidal suspension was exposed to sunlight at room temperature with steady stirring under constant irradiation. The color shift was tracked over time, and the samples were obtained and centrifuged every 30 min. In the region of 400–800 nm, the supernatant was scanned. Meanwhile, a control containing only the dye was tested at the same conditions, as was a sample comprising AgNPs suspended in dye solution, constantly swirled in the dark [20].

3.3.2 Phagocytosis activity assay

Phagocytosis activity assay was performed to check the effectiveness of nanoparticles in stimulation of phagocytic cells. Suliman’s protocol (with some modifications) was used to assess macrophage phagocytic activity. Fresh human blood sample was isolated in tubes containing EDTA. 200  μ L of nanoparticles with concentrations of 10 g‧mL⁻¹ were added to 200 μ L of blood samples. For 30 min, the samples were incubated in a water bath at 37°C. After incubation, each sample was given 20  μ L of S. aureus bacterial suspension and again incubated for 30 min at 37°C. The blood samples and bacterial suspensions were mixed and utilized as a positive control. Then, slide smear was prepared by taking one drop from each blood sample with control. Slides were dried at room temperature. Staining was done with the help of Leishman stain. Five drops of Leishman stain were used, and the slides were left for 10 min before observing them under light microscope (100×) after washing with distilled water [9].

3.3.3 Biochemical assay

4.1.1 Result for the determination of total encapsulated polyphenols and flavonoids

High flavonoid and moderate polyphenol capacities were exhibited by nanopropolis [1,43]. It was reported by authors of ref. [7] that encapsulation efficiency of total polyphenols and total flavonoids was 67% and 94%, respectively.

4.1.2 Particle size analysis

Particle size analyzer was used to determine the size of particles, variation was reported in nanoparticle size between 252 and 530 nm [43].

4.2.1 UV-vis spectrum analysis

In the range of 200–800 nm, the absorption spectrum was recorded for biosynthesized SeNPs. For selenium in the samples, a high absorption peak was recorded between 250 and 280 nm [88].

4.2.2 FT-IR analysis

FT-IR analysis confirmed the presence of different functional groups like alcohol and polyphenols. It was reported that the presence of these functional groups was mainly involved in the reduction of selenium ion to SeNPs. Peak values were observed between 2,950 and 2,850 cm⁻¹ which were due to the presence of organic polymer lignin. It was evident from different research papers that absorption peak was present between 1,750 and 1,470 cm⁻¹ which corresponded to the carboxyl group peak, also at 1,030 and 730 cm⁻¹, another peak was observed which corresponded to phenolic group [88].

4.2.3 XRD analysis

The diffraction intensity was observed from 2 θ angles varying from 10° to 100° when XRD pattern of SeNP’s was studied. It was reported from different papers that crystalline nature of SeNPs was determined by the distinct peaks at 23.51°, 29.50°, 41.30°, 45.35°, and 48.10°. With lattice constants a = 4.3662 Å and c = 4.9536 Å, all the diffraction peaks recorded in the 2θ range related to the hexagonal structure of Se. The sample’s amorphous/hexagonal nano-crystalline nature was revealed by the sharpness and broad diffraction of the peaks at low angle [10,88].

4.2.4 SEM analysis

For the assessment of the physical dimensions, morphology, and shape of the nanoparticles, widely used technique is SEM. Researchers revealed that the structure and size of biosynthesized nanoparticles get influenced by the concentration of polyphenol in propolis extract. Biosynthesized SeNPs by propolis extract were found to have spherical shape with a smooth surface and size range between 30 and 36 nm [10].

4.3.1 Determination of MIC and MBC

It was noticed that S. aureus and P. aeruginosa were amenable to AgNPs effect. Gram-positive bacteria (S. aureus) exhibited highest antibacterial activity that was most probably due to the differences in molecular makeup of cell walls [29,93]. AgNPs could be a source of reactive oxygen species (ROS), which can damage bacteria proteins and DNA, causing cell membrane permeability to change and bacterial membrane destruction [20,90]. MIC values for antibacterial activity of propolis was reported to be 4,162, 8,325, and 16,650 μg‧mL⁻¹ for S. aureus, S. epidermidis, and P. aeruginosa, respectively. Different results had been reported in the literature: some show that AgNPs are more effective against Gram-positive bacteria than Gram-negative bacteria [20,56], while others lead to the opposite conclusion [20,24] given in Table 4. Furthermore, Gram-negative bacteria had an exterior lipidic membrane with a negative surface charge. The AgNPs linked with propolis (AgNP-P) share the same surface charge, which can cause electrostatic repulsion between them, making it difficult to fix and penetrate AgNPs into bacterial cells and necessitating a greater concentration to kill this type of bacteria.

4.3.2 Visual and UV-Vis analysis

AgNPs’ biosynthesis was confirmed by observing the change in color of the reaction mixture which is due to the excitation of surface plasmon vibrations in AgNPs [7,20,94]. The color of the reaction mixture changed from white-yellowish to brown [20,94]. After continuous stirring, the UV-Vis spectra of the mixture extract, silver nitrate, was recorded in the 350–600 nm wavelength region, revealing the surface plasmon resonance [20]. Due to the formation of small spherical nanoparticles, an absorption band at 480 nm was recorded. AgNPs derived from aqueous propolis extract were active at a shorter wavelength [20]. Many other studies revealed the presence of peak at 420 nm [7], and in the propolis samples from Tamilnadu, absorption spectra was observed between 260 and 290 nm [95].

4.3.3 FT-IR analysis

FT-IR analysis was performed to determine the biomolecules identified in the propolis extract that are responsible for the reduction, capping, and stabilization of AgNPs. From the propolis extract the functional groups were recorded at different wavelengths [20]. The carboxylic group was detected in the FT-IR spectrum of the propolis aqueous extract without AgNO₃ with an intense peak at 1,716 and 1,100 cm⁻¹ [7]. The stretching vibrations of OH from phenolic compounds were observed at 3,403 cm⁻¹ and stretching vibration of C–H were recorded at 2,919 and 2,849 cm⁻¹. 1,635 cm⁻¹ mainly corresponded to stretching vibration of C═C and C═O groups from flavonoids and asymmetric bending vibration of N–H from amino acids. Due to stretching C═C of aromatic ring, bending vibration of C–H from CH₃, CH₂, and the stretching vibration of aromatics from flavonoids and aromatic rings’ peak were recorded at 1,514 and 1,449 cm⁻¹, respectively. 1,264 cm⁻¹ corresponded to C–O group of polyols (hydroxyflavonoids), 1,082 cm⁻¹ due to C–O stretching ester group, 815 cm⁻¹ due to aromatic ring vibration, and 698 cm⁻¹ corresponded to phenyl group [20,96]. The majority of the FT-IR bands in propolis aqueous extract were due to triterpenoids, flavonoids, furanoids, sugars, coumarins, quinines, tannins, phenols, and acids [7]. Some peaks in the FT-IR spectra of AgNPs shifted or vanished as a result, indicating that particular polyphenols, flavonoids, or amino acids are involved in the formation of nanoparticles [20].

4.3.4 Particle size and TEM analysis

The majority of AgNPs were spherical in shape [7,20] and ranged in size from 10 to 50 nm, with an average size of about 15 nm [20], 9–30 nm [7]. The TEM pictures revealed a weak aggregation tendency, which could be due to the extract’s contribution to AgNPs’ stabilization [20]. In a study, it was reported that the size of nanoparticles with ethanolic extract of propolis at pH = 10.62 was 50 nm and at same pH for water extract of propolis, it was 20 nm [97]. It was revealed in the field emission SEM analysis that the size of synthesized AgNPs was 62, 41, and 82 nm for the samples from different regions of Tamilnadu [95].

4.3.5 Determination of zeta potential

AgNPs coated with polyphenolic chemicals found in propolis extract had a negative zeta potential of −21.36 mV. This suggested that the suspension of colloidal nanoparticles was stable [20,24]. The AgNPs’ negatively charged surface reduces particle agglomeration by preventing particle rejection [20,57].

4.3.6 Determination of elemental silver in AgNPs

The existence of elemental silver was confirmed by the EDX spectra of AgNPs, which showed a peak pattern at 3 keV in the silver region [7,20,93]. Apart from silver, the most prominent elements in the chemical composition of AgNPs were carbon, which made up 53.26%, oxygen – 14.06%, and nitrogen – which made up 03.12%, showing the presence of compounds connected to nanoparticles [20]. After 4 h, a spectroscopic signal indicating the production of AgNPs was seen around 480 nm. In the extract and silver nitrate spectra, this peak does not appear. AgNPs are polydispersed, as seen by the spreading contour of the peak [5,295]. Atomic % reported via EDX spectrum of AgNPs was 91.23, 1.71, 0.66, 0.53, 0.96, and 4.91 for elements C-K, Na-K, Mg-K, Si-K, Ca-K, and Ag-L, respectively [95].

4.3.7 Analysis of photocatalytic activity

Malachite green (colorant) degradation was analyzed to determine the photocatalytic activity of the synthesized AgNPs. Malachite green, as well as its reduced form, can be found in both aquatic and terrestrial systems, posing a risk to human health [20,98]. Initially, the photocatalytic degradation was analyzed by color change, a color shift from dark blue to light blue was noticed. Thereafter, the diminution in color was measured spectrophotometrically by recording the absorbance at 617 nm. It could be possible to get a significant decline in absorbance over time, indicating a decrease in dye concentration owing to degradation. The control containing merely the dye did not change color when exposed to sunshine, while the color and absorbance of the sample mixed in the dark dropped.

Dye degradation was calculated by the formula:

where C _o is the initial concentration of the malachite green, and C _t is the concentration of the malachite green after each period of exposure time to sunlight [20,81]. It was illustrated that the obtained AgNPs were highly photocatalytically active under light radiation as when the sample was exposed to sunlight, dye degradation obtained was 90%, for the control degradation it was 5%, and 30% for the sample mixed in dark. Photons of visible light hitting AgNPs are absorbed via the surface plasmon resonance phenomenon, which causes electrons to be excited to a higher energy state during sunlight exposure.

4.3.8 Determination of phagocytosis activity assay

Phagocytosis is a process in which phagocytic cells engulf, destroy, and digest bacteria or foreign substances [9,99]. Significant increase was noticed in the activity of phagocytic cells. This increased activity of phagocytic cells to engulf bacteria could be due to certain chemical components present in the AgNPs of propolis which acted as immune modulators [9].