www.nature.

com/scientificreports

OPEN Biosynthesis and characterization

of gold nanoparticles using
Brazilian red propolis
and evaluation of its antimicrobial
and anticancer activities
C. E. A. Botteon1, L. B. Silva1, G. V. Ccana‑Ccapatinta2, T. S. Silva3, S. R. Ambrosio3,
R. C. S. Veneziani3, J. K. Bastos2 & P. D. Marcato1*

Gold nanoparticles (AuNPs) are highlighted due to their low toxicity, compatibility with the human
body, high surface area to volume ratio, and surfaces that can be easily modified with ligands.
Biosynthesis of AuNPs using plant extract is considered a simple, low-cost, and eco-friendly
approach. Brazilian Red Propolis (BRP), a product of bees, exhibits anti-inflammatory, anti-tumor,
antioxidant, and antimicrobial activities. Here, we described the biosynthesis of AuNPs using BRP
extract ­(AuNPextract) and its fractions ­(AuNPhexane, ­AuNPdichloromethane, ­AuNPethyl acetate) and evaluated
their structural properties and their potential against microorganisms and cancer cells. AuNPs showed
a surface plasmon resonance (SPR) band at 535 nm. The sizes and morphologies were influenced by
the BRP sample used in the reaction. FTIR and TGA revealed the involvement of bioactive compounds
from BRP extract or its fractions in the synthesis and stabilization of AuNPs. ­AuNPdichloromethane and
­AuNPhexane exhibited antimicrobial activities against all strains tested, showing their efficacy as
antimicrobial agents to treat infectious diseases. AuNPs showed dose-dependent cytotoxic activity
both in T24 and PC-3 cells. ­AuNPdichloromethane and ­AuNPextract exhibited the highest in vitro cytotoxic
effect. Also, the cytotoxicity of biogenic nanoparticles was induced by mechanisms associated with
apoptosis. The results highlight a potential low-cost green method using Brazilian red propolis to
synthesize AuNPs, which demonstrated significant biological properties.

Metallic nanoparticles can be considered one of the most versatile types of nanoparticles due to their applications
in chemistry, electronics, medicine, and pharmaceutical ­sciences1. Among them, the gold nanoparticles (AuNPs)
stand out for their advantages such as biocompatibility, tunable optical properties, and easily changed surface
­chemistry2,3. Because of these unique physical–chemical properties, the AuNPs are widely used as carriers of
drugs and molecules to improve the diagnosis and treatment of d ­ iseases4,5.
The synthesis of AuNPs through chemical and physical routes has been already well-established. However,
these pathways generally use toxic substances and non-polar solvents, which generate hazardous impacts for the
environment and requires various steps of product purification, resulting in an expensive p ­ rocess6.
For overcoming the challenges related to conventional methods, a biosynthetic route has been proposed in
the ­literature7. The green synthesis uses natural compounds from plants or microorganisms (e.g., fungi, bacteria,
algae) as precursors of the reaction of gold ions r­ eduction8,9. Biosynthesis is considered a simple, low-cost, and
eco-friendly approach since it uses non-toxic solvents, such as ­water10. The production of metallic nanoparticles
using natural sources has already been reported in the literature, showing it is a potential synthetic route that
should be ­explored11,12.
Plant extracts are complex mixtures providing a rich arsenal of molecules with high redox p ­ otential13, such
as flavanones, flavones, flavonols and chalcones, fatty acids, amino acids, terpenoids, aldehydes, and a­ lcohols14.

1
GNanoBio, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenida Do Café S/nº,
Ribeirão Preto, São Paulo 14040‑903, Brazil. 2School of Pharmaceutical Sciences of Ribeirão Preto, University
of São Paulo, São Paulo, Brazil. 3Research Center of Exact and Technological Sciences, UNIFRAN, São Paulo,
Brazil. *email: pmarcato@fcfrp.usp.br

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 1

Vol.:(0123456789)
 www.nature.com/scientificreports/

Furthermore, biogenic synthesis produces large amounts of highly stable nanoparticles with a better-defined
size than some conventional methods since phytochemicals compounds that are used in the reaction also act
as stabilizing a­ gents15,16.
Propolis is a product of bees, known worldwide for its biological properties and used in traditional medi-
cine. Propolis exhibits significant pharmacological activities such as anti-inflammatory17,18, anti-oxidant19,20
and ­antimicrobial21,22. It has also been demonstrated that propolis has anti-proliferative and anti-tumor effects
in vitro and in vivo tumor m ­ odels23–25.
There are several types of propolis with different compositions depending on the region, climate, and extrac-
tion ­season26,27. More than 300 substances have already been identified in different samples of p ­ ropolis28. Some
of the propolis types have been used to produce gold nanoparticles since this natural product presents a high
amount of polyphenolic acids, flavonoids, terpenoids, and other molecules that can reduce A ­ u+3 to ­Au029–31.
Red propolis has been found in countries such as Mexico, Cuba, China and B ­ razil32. Brazilian red propolis
(BRP) is found in northeastern B ­ razil33 and is considered to be a distinct type of propolis since it has some mol-
ecules of pharmacological interest that have not yet been discovered in other kinds of ­propolis34–36.
Several studies about the anticancer activity of red propolis have been described in the l­iterature37–39.
Researchers reported the cytotoxic and anti-proliferative effects of red propolis in cell lines of leukemia and
prostate ­cancer40. Frozza et al.41 reported that red propolis promoted apoptotic effects in human cancer cell lines
through mitochondrial perturbation.
Here, we investigated, for the first time, the use of Brazilian red propolis extract and its fractions in the biosyn-
thesis of gold nanoparticles using an eco-friendly and low-cost method. This study also evaluated the structural
properties of these nanomaterials and their biological activities against microorganisms and cancer cell lines.

Results and discussion

Preparation of Brazilian red propolis extract and its fractions. Brazilian Red Propolis (BRP) dis-
plays different biological activities such as antimicrobial, anticancer, antioxidant, and anti-inflammatory34,35.
These activities are related to its complex chemical composition including liquiritigenin (a), formononetin (b),
vestitol (c), neovestitol (d), medicarpin (e), 7-O-methylvestitol (f), guttiferone E (g) (Fig. 1). These biomolecules
were identified in the chromatographic profile of BRP extract (Table 1). The solid-phase extraction of the red
propolis hydroalcoholic extract left 23.8 g of the hexane fraction (27.3% yield), 54.2 g of the dichloromethane
fraction (62.3% yield), 4.3 g of the ethyl acetate fraction (5%), and 3.6 g of the ethanol fraction (4.1% yield). The
phytocompounds identified by HPLC in each fraction are shown in Table 1.

Biosynthesis of gold nanoparticles. Au(III) has a high reduction potential and can be reduced by phe-
nolic compounds from the natural ­extracts42. In this study, we produced gold nanoparticles (AuNPs) with BRP
extract ­(AuNPextract) and its fractions ­(AuNPhexane, ­AuNPdichloromethane, ­AuNPethyl acetate) using the green synthesis
method. In order to increase the solubility of the compounds present in the extract, the pH of the mixture of
gold solution with the propolis extract was adjusted to 7.0. Moreover, this pH alteration is also involved in the
activation of phytochemical compounds, which facilitates the donation of electrons to the metal, reducing A ­ u3+
042
to ­Au .
The formation of AuNPs was confirmed by the presence of the Surface Plasmon Resonance (SPR) band. This
band occurs on the surface of certain metals on a nanometer s­ cale43. Both the extract and its fractions (hexane,
dichloromethane and ethyl acetate) produced AuNPs, showing a prominent peak at a range of 523–541 nm
(Fig. 2). The optimal extract or fractions concentration to produce AuNPs was also investigated. The high forma-
tion of AuNPs was obtained using 200 μg mL−1 of the extract or its fractions. Moreover, we verified a wider SPR
band and turbidity signs in the AuNPs dispersion when the red propolis extract concentration was increased
twofold (400 μg mL−1) (Fig. 2a).
In consonance with Gatea et al.30 and Roy et al.29, we also observed an increase in the absorbance values related
to the time with a redshift of the SPR band (Fig. 2b–e). All formulations exhibited a similar absorption peaks
profile, indicating a rapid growth of particles. After some time (~ 2 h), the saturation was reached, indicating the
formation of stable n ­ anoparticles29. The acetate and dichloromethane fractions were more efficient to produce
AuNPs since the intensity of SPR bands was higher than the ­others44. This result can be associated with the higher
polarities of dichloromethane and ethyl acetate in comparison with hexane used in the extraction ­process45. The
color of the samples changed from pale yellow to dark red or purple (Fig. 2). The different colors of AuNPs—from
light pink to dark red—are dependent on the size, shape, and structural characteristics of these ­nanoparticles42.

Morphology and diameter distribution. The morphologies and diameter distributions of AuNPs
formed by three fractions and crude extract of BRP were investigated using the transmission electron micros-
copy (TEM) analysis. Figure 3a,c exhibits A ­ uNPextract and A
­ uNPethyl acetate with mostly spherical shapes, whereas
the ­AuNPdichloromethane and A
­ uNPhexane showed a variety of shapes (Fig. 3e,g). In general, the biosynthetic route
produces nanoparticles with different morphology and size as a result of the chemical composition of the
­extract46,47. Hexane fraction is rich in benzophenones (Guttiferone E; Oblongifolin A), while ethyl acetate frac-
tion is rich in flavonoids and isoflavonoids such as liquiritigenin, formononetin, vestitol, and neovestitol. BRP
crude extract and dichloromethane fraction present a similar phytochemical profile (Table 1).
The average size, measured by TEM, was in the range of 8–15 nm for all gold nanoparticles showing a nar-
row particle size distribution (Fig. 3b,d,f,h). The smaller nanoparticles showed spherical shapes, whereas the
larger particles exhibited several geometrical forms, such as triangles, pentagons, hexagons, and rods (Fig. 4a,b).
Similar results were reported by Smitha et al.48, Philip et al.49, and Gosh et al.50, who verified that nanoparticles
size with different geometries were larger than those with spherical morphology due to low quantities of the

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 2

Vol:.(1234567890)
www.nature.com/scientificreports/

Figure 1.  Chemical structures of the main constituents from Brazilian red propolis.

Compounds Retention time BRP extract Hexane fractions Dichloromethane fraction Ethyl acetate fraction
Liquiritigenin 9.89 + − + +
Formononetin 12.90 + − + +
Vestitol 13.17 + − + +
Neovestitol 13.72 + − + +
Medicarpin 14.82 + − + −
7-O-methylvestitol 17.22 + − + −
Guttiferone E 25.69 + + + −
Oblongifolin B 25.96 + + + −

Table 1.  Major phytochemical compounds identified in the BRP extract and its fractions by HPLC.

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 3

Vol.:(0123456789)
 www.nature.com/scientificreports/

a (x) (y)

b c
before aer before aer

d before aer
e before aer

Figure 2.  UV–vis spectra of biosynthesized gold nanoparticles: (a) using different BRP extract concentrations;
(b) ­AuNPextract; (c) ­AuNPethyl acetate; (d) ­AuNPdichloromethane; (e) ­AuNPhexane.

efficient biomolecules responsible for capping and stabilization, leading to the formation of large anisotropic
­nanoparticles49.
The Energy Dispersive X-ray Spectroscopy (EDXS) was performed to gather information about the chemical
composition of samples for elements with atomic numbers (Z) > 3. All AuNPs spectra showed an absorption band
peak of approximately 2.2 keV, which is characteristic of gold a­ bsorption5,51. The HR-TEM images (Fig. 5a–d)
of all AuNPs demonstrated highly ordered planar spacing consistent with the internal spacing of the gold plane.
The ­AuNPhexane showed inter-planer spacing of 1.9 Å, whereas the A ­ uNPethyl acetate and ­AuNPdichloromethane exhib-
ited 2.2 Å of d-spacing, which is in agreement with the (200) and (111) lattice of face-centered cubic (fcc) gold
(JCPDS card ­No 04-0784)52,53. A crystalline structure of the biosynthesized gold nanoparticles was also evidenced
by the SAED pattern (Fig. 5e–h) with circular rings that can be assigned to (111), (200), (220), and (311) Bragg’s
reflection ­planes54.

Fourier‑transform infrared spectroscopy (FTIR) and Thermal gravimetric analysis (TGA). The
relation of phytochemicals compounds involved in the biosynthesis and stabilization of AuNPs was evaluated by
FTIR and TGA. Table 2 shows the main peaks in the FTIR spectra of BRP extract or its fraction, as well as the
AuNPs produced.
The peaks around 1700 cm−1 are distinctive in hexane, and the dichloromethane fractions spectra are associ-
ated with carbonyl groups (C=O). These signals can be related to the major compounds in these fractions such

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 4

Vol:.(1234567890)
www.nature.com/scientificreports/

Figure 3.  TEM images and size distribution histograms of biosynthesized gold nanoparticles: (a,b) ­AuNPextract;
(c,d) ­AuNPethyl acetate; (e,f) ­AuNPdichloromethane; (g,h) ­AuNPhexane.

Figure 4.  TEM images of (a) ­AuNPdichloromethane and (b) ­AuNPhexane showing the different shapes.

as prenylated b­ enzophenones55 and phenolic c­ ompounds56. The peaks at 1600 cm−1 are usually correlated to the
stretching vibration of carboxylate anion –COO−, probably due to the oxidation of polyphenols during ­Au+3
­reduction10,57. These bands show large amounts of alcohol or phenol in plant extracts.
The bands around 3400 cm−1 in the AuNPs dispersions were smaller than the extract FTIR spectra (Fig. 6),
suggesting the oxidation of hydroxyl groups to carbonyl groups. It indicates that the OH groups present in
BRP extract or fractions are the main compounds involved in the reduction of Au i­ ons58. The absorption peaks
measured at 1500 cm−1 are probably due to free NH groups present in proteins. The reduction of this band inten-
sity after the formation of AuNPs indicates that the proteins in the extract were also used for capping AuNPs,
improving its ­stability59. Furthermore, decreased peak intensity at 800 cm−1 suggests the binding between the
C-H group of phenolic acids and ­AuNPs60.
These results explain the role of chemical compounds of the BRP extract in reducing ­Au+3 and stabilizing
­AuNPs65. Also, FTIR spectra results support the idea that biosynthesized nanoparticles are surrounded by a thin
layer of phytomolecules including polyphenols, such as flavonoids and tannins, in addition to terpenoids and
­proteins58. The BRP extract FTIR spectra show intense bands in 3436 cm−1 and 2922 cm−1, before and after the
Au ions reduction (Fig. 6), which corresponds to free O–H bonds and C ­ H2 stretching vibrations, r­ espectively30,59.
The TGA graphs of the BRP extract, fractions, and AuNPs (Fig. 7) showed a steady weight loss in the tem-
perature range of 150–600 °C with a total weight loss of up to 800 °C. Table 3 shows the percentage of weight
loss of the AuNPs. The percentage differences in the weight loss shown in the table are related to the difference in
organic composition present on the AuNP’s surface. It is expected that the weight loss between 100 °C and 200 °C
refers to the evaporation of adsorbed water of the capping ­extract8. The largest weight loss observed in the range
of 250 to 500 °C may be a result of the thin layer burning of organic material surrounding the n ­ anoparticles59.
Also, it is supposed that after 400 °C occurs the degradation of resistant aromatic ­compounds62. At the final of the

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 5

Vol.:(0123456789)
 www.nature.com/scientificreports/

Figure 5.  HR-TEM images and SAED pattern of biosynthesized gold nanoparticles: (a,e) ­AuNPextract; (b,f)
­AuNPethyl acetate; (c,g) ­AuNPdichloromethane; (d,h) ­AuNPhexane.

Bands ­(cm−1)
Peak Extract and fractions AuNPs Possible functional groups References
Ismail et al.59; Park et al.61; Elbagory
1 3436; 3438; 3445; 3436; 3430; 3426; 3426; 3445; Free O–H
et al.62; Gatea et al.30;
2 2922; 2924; 2940; 2921; 2918; 2932; 2921; 2921; CH2 stretching vibrations Benedec et al.10; Liu et al.60; Gatea et al.30;
3 1729; 1713; 1729; 1732; 1729; 1729; 1739; 1689; Carbonyl stretching vibrations Benedec et al., ­201810; Liu et al., 2019;
Benedec et al., 2018; Alexeree et al.,
4 1624; 1623; 1624; 1637; 1605; 1615; 1614; 1622; Carboxylate anion –COO−
­201763;
5 1510; 1510; 1510; 1520; 1510; 1510; 1501; 1462; Free NH groups Ismail et al.59; Leon et al.57
6 Around 1380; CH3 stretching vibrations Elbagory et al.62;
7 Between 1280 and 1155; Aromatic C–O bond stretching Leon et al.57; Elbagory et al.62;
8 Between 890 and 775 C–H bonds in the phenolic rings Liu et al.60; Zhang et al.64

Table 2.  Relation of the main peaks found in FTIR spectra of the BRP extract and its fraction and
biosynthesized AuNPs.

thermal decomposition process, the residue of around 50% refers to the presence of pure AuNPs. These results
indicate the protective effect of the extract compounds on the surface of ­AuNPs66.

Antibacterial and antifungal activities. The MIC and MBC results are shown in Table 4. According
to Aligiannis et al. (2001)67, plant materials with MIC up to 500 μg mL−1 are considered strong inhibitors of
bacterial activity. Moderate inhibition is given by plant extract with MIC values between 600 and 1500 μg mL−1;
whereas MIC above 1600 μg mL−1 is classified as weak inhibition. The BRP crude extract and the fractions
hexane and dichloromethane showed a pronounced antimicrobial effect. No significant antibacterial activity of
ethyl acetate fraction and ­AuNPethyl acetate was observed at the concentrations assessed. The ­AuNPextract showed
only fungicidal activity (MIC = 12.4 µg mL−1 of extract, equivalent to 0.2 × 109 nanoparticles m ­ L−1), whereas the
­AuNPdichloromethane and ­AuNPhexane demonstrated antibacterial and antifungal activity against all tested strains.
­AuNPhexane exhibited the highest activity among the nanoparticles, showing MIC and MBC values similar to
the values presented by the BRP crude extract (MIC = 50.8 and 101.7 µg mL−1 of extract equivalent to 0.5 and
1.9 × 109 particles ­mL−1). Among the strains, C. albicans yeast showed the highest susceptibility, while S. aureus
demonstrated the highest resistance to the AuNPs treatment.
BRP crude extract and its fractions exhibited the highest antimicrobial activity. In fact, the loss of activity of
biosynthesized nanomaterials has been reported in the l­ iterature30,68. This difference might be explained by the
oxidation of some active compounds of plant extracts during the reaction with metals. Several mechanisms have
been proposed for the reduction of metal ions using plant extracts. Several authors suggest that the phenolic
compounds are the main reducing and capping agents involved in the synthesis of metallic n ­ anoparticles69. It
would justify the lower antimicrobial activity of AuNPs, although they present phenolic compounds.

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 6

Vol:.(1234567890)
www.nature.com/scientificreports/

a b

c d

Figure 6.  Fourier transform infrared (FTIR) spectra of the BRP extract (red line) and biosynthesized AuNPs
(black line).

The difference between the antimicrobial activity of each biosynthesized AuNPs can be explained by the
phytochemical composition of each extract and ­fraction70. According to a bio-guided study of Brazilian red
propolis extract and its fractions, the crude extract and hexane fraction showed the highest antimicrobial activity
against microorganisms, including S. aureus and E. coli. This activity can be related to the presence of medicarpin,
elemicin, and ­vestitol71,72. Therefore, the medicarpin and vestitol we quantified in the BRP crude extract might
have contributed to the antimicrobial effect of the BRP extract and ­AuNPdichloromethane.
Furthermore, benzophenones (Guttiferone E and Oblongifolin B) are present in low concentrations in the
BRP crude extract and the dichloromethane fraction, and in high amounts in the hexane extract. Benzophe-
nones are described as effective compounds against bacteria and f­ ungi39,73,74. Also, the activity of A
­ uNPhexane and
­AuNPdichloromethane can be related to the presence of these molecules on the surface of nanoparticles. On the other
hand, ­AuNPextract and A­ uNPethyl acetate showed low or no antimicrobial activity that can be associated with the
low amounts of medicarpin, vestitol, and benzophenones in these extract and fraction. Moreover, ethyl acetate
fraction did not present benzophenones showing the lower antimicrobial activity.
Additionally, the nanoparticle shape can influence its interaction with microorganisms and, conse-
quently, affect its antimicrobial a­ ctivity75,76. Thus, the heterogeneous gold nanoparticle shapes observed in the
­AuNPdichloromethane and ­AuNPhexane dispersion may have influenced its antimicrobial activity.

Cytotoxic activity. The in vitro cytotoxic activity of BRP extract, fractions, and biosynthesized AuNPs were
evaluated in bladder cancer cells (T24) and prostate cancer cells (PC-3) using the Resazurin assay. The resazurin
assay provides a simple, non-toxic and sensitive measurement for the viability of mammalian c­ ells77,78.
Dose-dependent cytotoxic activity was observed in all samples (Fig. 8). The BRP crude extract and its fraction
showed a high cytotoxic effect (Table 5). According to the U.S. National Cancer Institute, extracts with I­ C50 up
to 30 µg mL−1 are classified as potent cytotoxic ­agents79,80. Moreover, BRP crude extract and dichloromethane
fraction exhibited the highest cytotoxic effect (Table 5). BRP crude extract and dichloromethane fraction present
similar phytochemical composition such as formononetin, liquiritigenin, and medicarpin in high concentra-
tion, which are known to exhibit cytotoxic activity in different cancer cells (Table 1)81–83. Ye et al.84 described

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 7

Vol.:(0123456789)
 www.nature.com/scientificreports/

a b

c d

Figure 7.  TGA analysis of BRP crude extract and its fractions as well as the biosynthesized AuNPs.

Sample Weight % up to 200 °C Weight % up to 600 °C Weight % up to 800 °C

AuNPextract < 1.00 58.36 44.34
AuNPethyl acetate 2.97 48.74 49.58
AuNPdichloromethane 2.39 40.87 58.41
AuNPhexane 10.68 47.57 44.24

Table 3.  Weight loss percentage of the AuNPs.

that formononetin inhibited the proliferation of prostate cancer cells (LNCaP and PC-3) and induced apoptosis
through the ERK1/2 MAPK-Bax pathway. Although A ­ uNPextract and A­ uNPdichloromethane presented the same cyto-
toxic profile as their respective precursors (BRP crude extract and its fractions), the A ­ uNPextract exhibited the
highest cytotoxic activity among the nanoparticles (Fig. 8 and Table 5).
The hexane fraction displayed the highest in vitro antitumor effects, with ­IC50 values around 12 µg mL−1
(Table 5), probably due to the presence of benzophenones (Table 1). Novak et al.85 reported that a red propolis
fraction induced tumor regression of melanoma xenografts in mice and related this to a benzophenone’s activity.
On the other hand, the low cytotoxicity of A ­ uNPhexane ­(IC50 of 59.5 µg mL−1 for T24 and 89 µg mL−1 for PC-3)
might be explained by the participation of the benzophenones in the AuNP formation.
Ethyl acetate fraction and A­ uNPethyl acetate showed the least activity among the samples tested. These results
could be related to the absence of compounds such as medicarpin and the benzophenones (present in extract and
hexane fraction), which could have an important role in the anti-cancer activity and the synergism effect between
the molecules. Some studies proposed that the anti-proliferative and cytotoxic effects of propolis against cancer
cells might be correlated to the synergism between properties of several compounds and may not exclusively
­ olecule21.
due to the concentration of a specific m

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 8

Vol:.(1234567890)
www.nature.com/scientificreports/

Results expressed in concentration of extract (µg ­mL−1)

Staphylococcus Streptococcus
aureus Escherichia coli mutans Candida albicans
Samples MIC MBC MIC MBC MIC MBC MIC MBC
AuNPextract > 198.6 > 198.6 > 198.6 > 198.6 > 198.6 > 198.6 12.4 12.4
AuNPethyl acetate > 234.5 > 234.5 > 234.5 > 234.5 > 234.5 > 234.5 > 234.5 > 234.5
AuNPdichloromethane 226.8 226.8 226.8 226.8 56.7 56.7 56.7 113.4
AuNPhexane 101.7 101.7 50.8 50.8 50.8 50.8 6.35 25.4
BRP crude extract 50.0 100.0 50.0 50.0 50.0 50.0 1.56 1.56
Ethyl acetate fraction > 250.0 > 250.0 > 250.0 > 250.0 125.0 125.0 250.0 500.0
Dichloromethane fraction 62.5 125.0 125.0 > 250.0 15.64 31.25 31.25 62.5
Hexane fraction 15.64 31.25 62.5 62.5 7.82 7.82 3.91 15.64

Table 4.  Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) of
biosynthesized gold nanoparticles and BRP crude extract and its fractions against different microorganisms.

Figure 8.  Cytotoxicity assay of extract and its fractions and biosynthesized gold nanoparticle in cells (a,b) PC-3
and (c,d) T24, obtained by resazurin assay.

IC50 (µg
­mL−1)
Samples T24 PC-3
BRP crude extract 22.9 21.8
Ethyl acetate fraction 30.4 39.3
Dichloromethane fraction 22.1 26.1
Hexane fraction 11.6 12.5
AuNPextract 43.1 53.0
AuNPethyl acetate 90.2 –
AuNPdichloromethane 48.5 63.0
AuNPhexane 59.5 89.0

Table 5.  IC50 values of BRP crude extract, and its fractions and biosynthesized gold nanoparticles against T24
and PC-3 cancer cell lines.

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 9

Vol.:(0123456789)
 www.nature.com/scientificreports/

Figure 9.  PC-3 cell viability of (a) biogenic nanoparticles (b) BRP crude extract and its fractions, obtained by
ATP bioluminescence assay.

The difference in the I­ C50 values between PC-3 and T24 cells can be related to the malignant degree of the
cells. T24 cell is derived from transitional cell carcinoma grade II, whereas the PC-3 cell line is derived from bone
metastasis from grade IV adenocarcinoma. Thus, the PC-3 cell is more malignant and resistant to t­ reatment86.
Similar results were verified by Carvalho et al.87. Although all AuNPs were more cytotoxic for T24 cells, some
nanoparticles showed high cytotoxicity to PC-3 cells decreasing the cell viability up to 10% in the highest con-
centration (Fig. 8).
Biogenic gold nanoparticles presented higher ­IC50 than their respectively extract or fraction precursors. These
results can be explained by the mechanism of gold nanoparticles production, since some compounds of extract
or fraction were used to reduce A ­ u3+ to form the metallic nanoparticles in the green s­ ynthesis29. Hence, after
nanoparticles preparation some active molecules of extract or fractions may lose its biological function. Owing
to the complexity of propolis composition, future studies to determine the specific compounds from BRP that
are responsible for gold nanoparticles reduction are demanded.
Although biosynthesized gold nanoparticles did not display a better cytotoxic effect than their precursors,
exhibiting only residual cytotoxicity from BRP active compounds, they still constitute a great advantage compared
with gold nanoparticles synthetized by chemical methods. El Domany et al.88 described that biosynthesized
AuNPs were more cytotoxic than chemically synthesized AuNPs in studies with PC-3, HCT116, and HepG2
tumor cells. Besides, the easy modification of the allows their association with other antitumor drugs and mol-
ecules that can target specifics cells, such as cancer c­ ells89. Researchers have been reported the beneficial effects of
the combination between anticancer agents and p ­ olyphenols90,91, as well as compounds found in B ­ RP92–94. Qiao
et al.95 showed that green tea catechins associated with antitumor drugs are more effective than monotherapy.

Adenosine triphosphate (ATP) bioluminescence assay. Cell culture viability was determined
through quantification of the luminescent signal, produced by transformation of luciferin by luciferase as a
function of intracellular ATP ­concentration96. ATP is the primary source of cellular energy, and its concentration
is related to the number of living c­ ells97. Luminescent ATP detection assay are robust and more sensitive than
MTT or similar assays once the ATP generation depletes as soon as the cell d ­ ies98,99.
The results of ATP tests in PC-3 cells showed that biosynthesized AuNPs and its precursors (crude BRP extract
and its fractions) present the same cytotoxic profile obtained by resazurin colorimetric determination (Fig. 9).
This strain was chosen for the experiment because it is considered more resistant than T24 cells. Thus, as obtained
in the resazurin assay, the hexane fraction was the most cytotoxic between the extract and fractions. Among the
nanoparticles, ­AuNPextract was the most effective in reducing cell viability, followed by ­AuNPdichloromethane. There-
fore, these results confirm the cytotoxic profile previously verified by the resazurin assay.

Flow cytometry. The cell death mechanism by flow cytometry assays demonstrated that BRP crude extract
and its fractions, and biosynthesized AuNPs induced the death mainly by apoptosis. The percentage of apop-
tosis ranged between 44 and 66% related to the total of dead cells for all treatments (Fig. 10). Begnini et al.100
described BRP induced apoptosis in 5637 cells by molecular ways related to the P53 and Bax/Bcl-2. Another

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 10

Vol:.(1234567890)
www.nature.com/scientificreports/

Figure 10.  Percentage of apoptosis in relation of total PC-3 cell death obtained by Flow cytometry.

study conducted by Novak et al.85 demonstrated that BRP components induced cell cycle arrest of B16F10 in
G2/M that triggers the apoptosis pathways activation.
This preliminary evidence has proven that biosynthesized gold nanoparticles using BRP displayed cytotoxic
effects in cancer cells and can be a promising alternative therapeutic agent in cancer treatment.

Conclusion
The Brazilian Red Propolis hydroethanolic extract and its fractions showed a great potential to produce gold
nanoparticles with size range of 8–15 nm. Due to the specific composition of the extract or its fraction, AuNPs
with different morphology were produced. Spherical AuNPs were obtained using BRP crude extract and ethyl
acetate fraction, while dichloromethane and hexane fractions produced AuNPs with different shapes. All AuNPs
showed a crystalline structure with a face-centered cubic (fcc) lattice. FTIR spectroscopy suggested the attach-
ment of bioactive compounds from the Brazilian red propolis extract or its fraction on the surface of AuNPs.
These results were confirmed by antimicrobial and cytotoxic activity of AuNPs produced. ­AuNPextract showed
antifungal activity and high cytotoxicity at low concentrations in bladder and prostate cancer cells. AuNPs
obtained with dichloromethane and hexane fractions displayed high antibacterial and antifungal activities and
cytotoxicity in both cells studied. On the other hand, the nanoparticles prepared with ethyl acetate fraction
did not show antimicrobial activity and exhibited the lowest in vitro cytotoxic effect in the cells evaluated. The
different phytochemical profiles of BRP extract and its fractions enabled the preparation of gold nanoparticles
with distinct properties and interesting for several applications. Therefore, these in vitro results demonstrate the
promising therapeutic applications of the biogenic gold nanoparticles in nanomedicine.

Materials and methods

Materials. Gold (III) chloride trihydrate ­(HAuCl4·3H2O), sodium hydroxide (NaOH), fetal bovine serum
(FBS), penicillin/streptomycin, Resazurin sodium salt, dimethyl sulfoxide (DMSO), trypsin serine protease
enzyme, Trypan Blue solution, tetracycline hydrochloride, and Amphotericin B solution were purchased from
Sigma-Aldrich (Louis, MO, USA). The PC-3 prostate cancer cell line and the T24 bladder cancer cell lines were
purchased from the Cell Bank of Rio de Janeiro (BCRJ). Dulbecco’s Modified Eagle Medium (DMEM), Roswell
Park Memorial Institute medium (RPMI) and BD Brain Heart Infusion (BHI) agar were purchased from Thermo
Fisher Scientific (Waltham, Massachusetts, USA).

Collection of red propolis and preparation of crude extract. Red propolis sample collected in Cana-
vieiras, State of Bahia, Brazil, was supplied by the beekeepers cooperative COAPER (Bahia, Brazil) in April of
2018. For the extraction, the propolis samples were frozen and grinded. Two hundred grams of red propolis were
submitted to maceration with 70% hydroalcoholic ethanol solution in the ratio of 1:10 (w/v); propolis macera-
tion was performed at 30 °C and 120 rpm using a shaker incubator (INNOVA 4300). The extracts obtained were
concentrated under vacuum using a rotary evaporator and then lyophilized to complete dryness.

Fractionation by the liquid–liquid partition of the crude extract of red propolis. The lyophilized
red propolis crude extract (87 g) was subjected to a solid-phase extraction process. The extract was mixed with
200 g of microcrystalline cellulose, and the mixture was transferred to a 13 × 11 cm i.d glass column and submit-
ted to successive extraction with organic solvents in increasing polarities: hexane (2 L), dichloromethane (2 L)
and ethyl acetate (2 L). The fractions obtained were concentrated under vacuum and lyophilized to complete
dryness.

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 11

Vol.:(0123456789)
 www.nature.com/scientificreports/

Biosynthesis of gold nanoparticles. AuNPs were synthesized by mixing a stock solution of tetrachloro-
auric acid trihydrate (0.5 mM) with BRP crude extract solution (200 μg/mL) or its fractions solution (hexane,
acetate and dichloromethane), followed by NaOH addition until pH 7.0. The mixture was stirred for 1 h at
optimized temperature. The gold nanoparticles formation was observed by color changed from pale yellow to
dark red.

Characterization of gold nanoparticles. The formation of gold nanoparticles was verified by the pres-
ence of a surface plasmon resonance (SPR) band, with maximum absorption between 500 and 550 nm, using
UV–Vis spectroscopy. The spectra were collected at different times of formation using a UV–Vis spectropho-
tometer (Implen, Munich, Germany). AuNPs concentration used in the biological analysis was performed by
Nanoparticle tracking analysis (NTA) using a Nanosight NS300 with 488 nm laser (Malvern Instruments). The
morphology, shape, size and elemental composition of AuNPs were analyzed by Transmission Electron Micros-
copy (TEM) and high-resolution TEM FEI TECNAI ­G2 F20 coupled with Energy-dispersive X-ray Spectroscopy
(EDXS) (Thermo Fisher Scientific, USA), operating a beam voltage of 200 keV. The diameter distribution was
obtained using the Image J (NIH, USA) software using the TEM images. The crystalline structure of AuNPs was
assessed by Selected Area Electron Diffraction (SAED). For Fourier-transform infrared spectroscopy (FTIR)
and Thermal gravimetric analysis (TGA), biosynthesized AuNPs dispersion was centrifuged at 15,000 rpm for
60 min at 4 °C. AuNPs pellet was washed three times with deionized water with 50% ethanol to remove the
excess of the extract from the AuNPs solution. The samples were lyophilized, and AuNPs powder or the BRP
crude extract were mixed with potassium bromide (KBr) to obtain pellets. FTIR spectra were recorded using an
IRTracer-100 (Shimadzu, Kyoto, JA) in the range of 4500 to 500 cm−1 with a resolution of 2 cm−1. TGA was car-
ried out using an SDT Q 600 thermal analyzer (TA Instruments, New Castle, DE, USA). The samples were placed
in platinum pans and heated under an inert atmosphere with a rate of 10 °C/min at 900 °C.

Antibacterial and antifungal activities. Antibacterial and antifungal properties of biosynthesized nan-
oparticles were investigated using the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC) methods. The microorganisms used in this study belong to the American Type Culture
Collection (ATCC) and are kept in the collection of the Applied Microbiology Research Laboratory (LaPeMA),
University of Franca (UNIFRAN), under cryopreservation at − 80 °C. Gold nanoparticles were tested against
Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Streptococcus mutans (ATCC 25175),
and Candida albicans (ATCC 28366). MIC was determined by the broth microdilution method, in triplicate, at
the exponential phase of bacterial growth using 96-wells microplates (CLSI 2007)101. The AuNPs samples were
diluted in Brain Infusion Broth (Difco) and different concentrations of the samples (7.3 × 106 to 7.5 × 109 AuNP/
mL) were added to each well. Then, the microorganism (5 × 105 CFU/mL) was added to all wells. Tetracycline
(0.0115 µg mL−1 to 5.9 µg mL−1) was used as a positive control for bacteria and Amphotericin B (0.031 µg mL−1
to 16 µg mL−1) was used as a positive control for yeast. The plates were incubated for 24 h at 37 °C. Afterward,
a resazurin solution (0.02%) was added to determine the microorganism viability. Before Resazurin addition,
an aliquot of the inoculum was removed from each well and seeded on BHI agar supplemented with 5% sheep
blood for the MBC test. The plates were incubated as previously described. MBC was defined as the lowest con-
centration of the sample without microbial growth.

Cytotoxicity assay. The cytotoxic activity of biosynthetic gold nanoparticles was evaluated in human uro-
logic cancer cell lines. T24 bladder cancer and PC-3 prostate cancer cell lines were obtained from the Rio de
Janeiro Cell Bank (BCRJ). T24 cell was cultured in RPMI medium (Sigma-Aldrich, USA) and PC-3 cell was
cultured in DMEM-Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, USA), both containing 1% antibi-
otic (Penicillin–Streptomycin-Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (SBF-GIBCO,
Thermo Fisher Scientific, USA). These cells were maintained in continuous culture under a humid atmosphere
at 37 °C and 5% C ­ O2. T24 or PC-3 cells were seeded in 96-well plates (2 × 104 cells/well) and treated with dif-
ferent concentrations of biosynthesized AuNPs for 24 h. Afterward, the medium containing nanoparticles was
removed and the cells were washed with PBS. Subsequently, a Resazurin solution (25 µg mL−1) was added to each
well and incubated for an additional 4 h. Then, fluorescence was evaluated on a Microplate reader (Synergy HTX
Multi-Mode Microplate Reader, Biotek) using 530 nm and 590 nm as the excitation and emission wavelengths
respectively. The negative control received only medium and the positive control was treated with DMSO 1%.
­IC50 values were determined using the Graph Pad Prism version 8 software (GraphPad Software Inc. San Diego
CA, USA).

Adenosine triphosphate (ATP) bioluminescence assay. Cell viability was assessed using the CellTi-
ter‑Glo Assay kit (Promega Corporation). The kit reagents were prepared according to the manufacturer’s pro-
tocol. PC-3 cells were plated onto a 96-well plate at a density of 20,000 cells per well. The CellTiter‑Glo reagent
was added to the washed cells 24 h after the treatments. The plates were shaken for 10 min at room temperature,
followed by luminescence measurement using absorbance/fluorescence plate reader (Synergy HTX Multi-Mode
Microplate Reader, BioTek).

Flow cytometry. The percentage of apoptosis was determined by flow cytometry using FITC Annexin V
(emission) as the apoptosis marker and FVD eFluor 450 (emission) as the viability marker. PC-3 cells were
seeded in 6-well plates (6 × 105 cell/well) and exposed to a concentration ­(IC50 values) of BRP crude extract and
fractions and biosynthesized AuNPs. After 24 h, the cells were washed twice with phosphate-buffered saline

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 12

Vol:.(1234567890)
www.nature.com/scientificreports/

(PBS) and incubated on ice with the FVD eFluor 450 for 30 min under dark condition. Then, cells were washed
and marked with FITC Annexin V. After incubation with apoptosis marker, samples were acquired using a BD
Accuri flow cytometer (Becton Dickinson). Gating of the viable cells and apoptotic cells was performed using
the BD Accuri software.

Statistical analysis. Statistical analysis was performed using the Prism 8 (GraphPad Software Inc. San
Diego CA, USA) and Origin 2019 version (OriginLab, Northampton, Massachusetts, USA) software with sig-
nificance level of 5% (p < 0.05). Results were analyzed by ANOVA followed by the Tukey post-test and data were
expressed as mean ± standard deviation (SD).

Received: 19 September 2020; Accepted: 2 December 2020

References
1. Murphy, C. J. et al. Anisotropic metal nanoparticles: Synthesis, assembly, and optical applications. J. Phys. Chem. B 109, 13857–
13870 (2005).
2. Sun, Y. et al. Temperature-sensitive gold nanoparticle-coated Pluronic-PLL nanoparticles for drug delivery and chemo-photo-
thermal therapy. Theranostics 7, 4424–4444 (2017).
3. Nguyen, T. T., Mammeri, F. & Ammar, S. Iron oxide and gold based magneto-plasmonic nanostructures for medical applications:
A review. Nanomaterials 8, 149 (2018).
4. Fazal, S. et al. Green synthesis of anisotropic gold nanoparticles for photothermal therapy of cancer. ACS Appl. Mater. Interfaces
6, 8080–8089 (2014).
5. Wang, C. et al. Characterization and antimicrobial application of biosynthesized gold and silver nanoparticles by using Micro-
bacterium resistens. Artif. Cells Nanomed. Biotechnol. 44, 1714–1721 (2016).
6. Khan, M. Z. H., Tareq, F. K., Hossen, M. A. & Roki, M. N. A. M. Green synthesis and characterization of silver nanoparticles
using Coriandrum sativum leaf extract. J. Eng. Sci. Technol. 13, 158–166 (2018).
7. Ovais, M. et al. Multifunctional theranostic applications of biocompatible green-synthesized colloidal nanoparticles. Appl.
Microbiol. Biotechnol. 102, 4393–4408 (2018).
8. Veisi, H., Farokhi, M., Hamelian, M. & Hemmati, S. Green synthesis of Au nanoparticles using an aqueous extract of Stachys
lavandulifolia and their catalytic performance for alkyne/aldehyde/amine A3 coupling reactions. RSC Adv. 8, 38186–38195
(2018).
9. Mukherjee, S. et al. Potential theranostics application of bio-synthesized silver nanoparticles (4-in-1 system). Theranostics 4,
316–335 (2014).
10. Benedec, D. et al. Origanum vulgare mediated green synthesis of biocompatible gold nanoparticles simultaneously possessing
plasmonic, antioxidant and antimicrobial properties. Int. J. Nanomed. 13, 1041–1058 (2018).
11. Francis, S., Joseph, S., Koshy, E. P. & Mathew, B. Green synthesis and characterization of gold and silver nanoparticles using Mus-
saenda glabrata leaf extract and their environmental applications to dye degradation. Environ. Sci. Pollut. Res. 24, 17347–17357
(2017).
12. Medina Cruz, D. et al. Citric juice-mediated synthesis of tellurium nanoparticles with antimicrobial and anticancer properties.
Green Chem. 21, 1982–1998 (2019).
13. Mohammadinejad, R. et al. Plant molecular farming: Production of metallic nanoparticles and therapeutic proteins using green
factories. Green Chem. 21, 1845–1865 (2019).
14. Borase, H. P. et al. Plant extract: A promising biomatrix for ecofriendly, controlled synthesis of silver nanoparticles. Appl. Bio-
chem. Biotechnol. 173, 1–29 (2014).
15. Iravani, S. Green synthesis of metal nanoparticles using plants. Green Chem. 13, 2638–2650 (2011).
16. Mittal, A. K., Chisti, Y. & Banerjee, U. C. Synthesis of metallic nanoparticles using plant extracts. Biotechnol. Adv. 31, 346–356
(2013).
17. Shahinozzaman, M. et al. Anti-inflammatory, anti-diabetic, and anti-Alzheimer’s effects of prenylated flavonoids from Okinawa
propolis: An investigation by experimental and computational studies. Molecules 23, 1–18 (2018).
18. Jabir, M. S., Sulaiman, G. M., Taqi, Z. J. & Li, D. Iraqi propolis increases degradation of IL-1β and NLRC4 by autophagy following
Pseudomonas aeruginosa infection. Microbes Infect. 20, 89–100 (2018).
19. Guzmán-Gutiérrez, S. L. et al. Mexican propolis: A source of antioxidants and anti-inflammatory compounds, and isolation of
a novel chalcone and ε-caprolactone derivative. Molecules 23, 334 (2018).
20. Kocot, J., Kiełczykowska, M., Luchowska-Kocot, D., Kurzepa, J. & Musik, I. Antioxidant potential of propolis, bee pollen, and
royal jelly: Possible medical application. Oxid. Med. Cell. Longev. https​://doi.org/10.1155/2018/70742​09 (2018).
21. Silva, R. P. D. et al. Antioxidant, antimicrobial, antiparasitic, and cytotoxic properties of various Brazilian propolis extracts. PLoS
ONE 12, 1–18 (2017).
22. Xu, X. et al. Chemical compositions of propolis from China and the United States and their antimicrobial activities against
Penicillium notatum. Molecules 24, 3576 (2019).
23. Inoue, K. et al. Anti-tumor effects of water-soluble propolis on a mouse sarcoma cell line in vivo and in vitro. Am. J. Chin. Med.
36, 625–634 (2008).
24. Sulaiman, G. M. et al. Assessing the anti-tumour properties of Iraqi propolis in vitro and in vivo. Food Chem. Toxicol. 50,
1632–1641 (2012).
25. Touzani, S. et al. In vitro evaluation of the potential use of propolis as a multitarget therapeutic product: Physicochemical
properties, chemical composition, and immunomodulatory, antibacterial, and anticancer properties. Biomed Res. Int. https​://
doi.org/10.1155/2019/48363​78 (2019).
26. Barbarić, M. et al. Chemical composition of the ethanolic propolis extracts and its effect on HeLa cells. J. Ethnopharmacol. 135,
772–778 (2011).
27. López, B. G. C., Schmidt, E. M., Eberlin, M. N. & Sawaya, A. C. H. F. Phytochemical markers of different types of red propolis.
Food Chem. 146, 174–180 (2014).
28. Sforcin, J. M. Propolis and the immune system: A review. J. Ethnopharmacol. 113, 1–14 (2007).
29. Roy, N. et al. Biogenic synthesis of Au and Ag nanoparticles by Indian propolis and its constituents. Colloids Surf. B 76, 317–325
(2010).
30. Gatea, F. et al. Antitumour, antimicrobial and catalytic activity of gold nanoparticles synthesized by different pH propolis extracts.
J. Nanopart. Res. 17, 320 (2015).
31. Righi, A. A. et al. Brazilian red propolis: Unreported substances, antioxidant and antimicrobial activities. J. Sci. Food Agric. 91,
2363–2370 (2011).

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 13

Vol.:(0123456789)
 www.nature.com/scientificreports/

32. dos Santos, D. A. et al. Brazilian red propolis extracts: study of chemical composition by ESI-MS/MS (ESI+) and cytotoxic
profiles against colon cancer cell lines. Biotechnol. Res. Innov. 3, 120–130 (2019).
33. Rufatto, L. C. et al. Red propolis: Chemical composition and pharmacological activity. Asian Pac. J. Trop. Biomed. 7, 591–598
(2017).
34. Bueno-Silva, B. et al. Anti-inflammatory and antimicrobial evaluation of neovestitol and vestitol isolated from brazilian red
propolis. J. Agric. Food Chem. 61, 4546–4550 (2013).
35. da Silva Frozza, C. O. et al. Chemical characterization, antioxidant and cytotoxic activities of Brazilian red propolis. Food Chem.
Toxicol. 52, 137–142 (2013).
36. Andrade, J. K. S., Denadai, M., de Oliveira, C. S., Nunes, M. L. & Narain, N. Evaluation of bioactive compounds potential and
antioxidant activity of brown, green and red propolis from Brazilian northeast region. Food Res. Int. 101, 129–138 (2017).
37. Alencar, S. M. et al. Chemical composition and biological activity of a new type of Brazilian propolis: Red propolis. J. Ethnop-
harmacol. 113, 278–283 (2007).
38. de Mendonça, I. C. G. et al. Brazilian red propolis: Phytochemical screening, antioxidant activity and effect against cancer cells.
BMC Complement. Altern. Med. 15, 1–12 (2015).
39. Regueira-Neto, M. S. et al. Antitrypanosomal, antileishmanial and cytotoxic activities of Brazilian red propolis and plant resin
of Dalbergia ecastaphyllum (L.) Taub. Food Chem. Toxicol. 119, 215–221 (2018).
40. De Oliveira Reis, J. H. et al. Evaluation of the antioxidant profile and cytotoxic activity of red propolis extracts from different
regions of northeastern Brazil obtained by conventional and ultrasoundassisted extraction. PLoS ONE 14, 1–27 (2019).
41. Frozza, C. O. S. et al. Antitumor activity of Brazilian red propolis fractions against Hep-2 cancer cell line. Biomed. Pharmacother.
91, 951–963 (2017).
42. Majumdar, R., Bag, B. G. & Ghosh, P. Mimusops elengi bark extract mediated green synthesis of gold nanoparticles and study
of its catalytic activity. Appl. Nanosci. 6, 521–528 (2016).
43. Tang, Y., Zeng, X. & Liang, J. Surface plasmon resonance: An introduction to a surface spectroscopy technique. J. Chem. Educ.
87, 742–746 (2010).
44. Nayef, U. M. & Khudhair, I. M. Synthesis of gold nanoparticles chemically doped with porous silicon for organic vapor sensor
by using photoluminescence. Optik (Stuttg). 154, 398–404 (2018).
45. Wakeel, A., Jan, S. A., Ullah, I., Shinwari, Z. K. & Xu, M. Solvent polarity mediates phytochemical yield and antioxidant capacity
of Isatis tinctoria. PeerJ 2019, 1–19 (2019).
46. Choudhary, M. K., Kataria, J. & Sharma, S. A biomimetic synthesis of stable gold nanoparticles derived from aqueous extract
of Foeniculum vulgare seeds and evaluation of their catalytic activity. Appl. Nanosci. 7, 439–447 (2017).
47. Gangula, A. et al. Catalytic reduction of 4-nitrophenol using biogenic gold and silver nanoparticles derived from breynia rham-
noides. Langmuir 27, 15268–15274 (2011).
48. Smitha, S. L., Philip, D. & Gopchandran, K. G. Green synthesis of gold nanoparticles using Cinnamomum zeylanicum leaf broth.
Spectrochim. Acta A 74, 735–739 (2009).
49. Philip, D. Rapid green synthesis of spherical gold nanoparticles using Mangifera indica leaf. Spectrochim. Acta A 77, 807–810
(2010).
50. Ghosh, S. et al. Gnidia glauca flower extract mediated synthesis of gold nanoparticles and evaluation of its chemocatalytic
potential. J. Nanobiotechnol. 10, 1–9 (2012).
51. Scimeca, M., Bischetti, S., Lamsira, H. K., Bonfiglio, R. & Bonanno, E. Energy dispersive X-ray (EDX) microanalysis: A powerful
tool in biomedical research and diagnosis. Eur. J. Histochem. 62, 89–99 (2018).
52. Lü, X., Song, Y., Zhu, A., Wu, F. & Song, Y. Synthesis of gold nanoparticles using cefopernazone as a stabilizing reagent and its
application. Int. J. Electrochim. Sci. 7, 11236–11245 (2012).
53. Gopalakrishnan, R. & Raghu, K. Biosynthesis and characterization of gold and silver nanoparticles using milk thistle (Silybum
marianum) seed extract. J. Nanosci. 2014, 905404 (2014).
54. Vijayan, S. R. et al. Synthesis and characterization of silver and gold nanoparticles using aqueous extract of seaweed, Turbinaria
conoides, and Their Antimicrofouling Activity. World Sci. J. https​://doi.org/10.1155/2014/93827​2 (2014).
55. Trusheva, B. et al. Bioactive constituents of Brazilian Red Propolis. Evid. Based Complement. Altern. Med 3, 249–254 (2006).
56. Righi, A. A., Negri, G. & Salatino, A. Comparative chemistry of propolis from eight brazilian localities. Evid. Based Complement.
Altern. Med. 2013, 267878 (2013).
57. Rodríguez-león, E. et al. Synthesis of gold nanoparticles using Mimosa tenuiflora extract, assessments of cytotoxicity, cellular
uptake, and catalysis. Nanoscale Res. Lett. 9, 1–16 (2019).
58. Azri, F. A., Selamat, J., Sukor, R. & Yusof, N. A. Etlingera elatior: Mediated synthesis of gold nanoparticles and their application
as electrochemical current enhancer. Molecules 24, 3141 (2019).
59. Ismail, E. H., Saqer, A. M. A., Assirey, E., Naqvi, A. & Okasha, R. M. Successful green synthesis of gold nanoparticles using a
corchorus olitorius extract and their antiproliferative effect in cancer cells. Int. J. Mol. Sci. 19, 1–14 (2018).
60. Kim, S. & Kim, Y. J. Green synthesis of gold nanoparticles using Euphrasia officinalis leaf extract to inhibit lipopolysaccharide-
induced inflammation through NF- κ B and JAK/STAT pathways in RAW 264.7 macrophages.". Int. J. Nanomed. 14, 2945 (2019).
61. Park, S. Y., Yi, E. H., Kim, Y. & Park, G. Anti-neuroinflammatory effects of ephedra sinica stapf extract-capped gold nanoparticles
in microglia. Int. J. Nanomedicine 14, 2861–2877 (2019).
62. Elbagory, A. M., Meyer, M., Cupido, C. N. & Hussein, A. A. Inhibition of bacteria associated with wound infection by biocom-
patible green synthesized gold nanoparticles from south african plant extracts. Nanomaterials https​://doi.org/10.3390/nano7​
12041​7 (2017).
63. Alexeree, S. M. I. et al. Exploiting biosynthetic gold nanoparticles for improving the aqueous solubility of metal-free phthalo-
cyanine as biocompatible PDT agent. Mater. Sci. Eng. C 76, 727–734 (2017).
64. Zhang, P., Wang, P., Yan, L. & Liu, L. Synthesis of gold nanoparticles with Solanum xanthocarpum extract and their in vitro
anticancer potential on nasopharyngeal carcinoma cells. Int. J. Nanomed. 13, 7047–7059 (2018).
65. Foo, Y. Y., Periasamy, V., Kiew, L. V., Kumar, G. G. & Malek, S. N. A. Curcuma mangga-mediated synthesis of gold nanoparticles:
Characterization, stability, cytotoxicity, and blood compatibility. Nanomaterials 7, 123 (2017).
66. Islam, N. U., Amin, R., Shahid, M. & Amin, M. Gummy gold and silver nanoparticles of apricot (Prunus armeniaca) confer high
stability and biological activity. Arab. J. Chem. 12, 3977–3992 (2019).
67. Aligiannis, N., Kalpoutzakis, E., Mitaku, S. & Chinou, I. B. Composition and antimicrobial activity of the essential oils of two
Origanum species. J. Agric. Food Chem. 49, 4168–4170 (2001).
68. Barbosa, V. T. et al. Biogenic synthesis of silver nanoparticles using Brazilian propolis. Biotechnol. Prog. 35, 1–9 (2019).
69. Fierascu, I. et al. Phyto-mediated metallic nano-architectures via Melissa officinalis L.: Synthesis, characterization and biological
properties. Sci. Rep. 7, 1–3 (2017).
70. Joe, M. H. et al. Phytosynthesis of silver and gold nanoparticles using the hot water extract of mixed woodchip powder and their
antibacterial efficacy. J. Nanomater. https​://doi.org/10.1155/2017/87347​58 (2017).
71. Rufatto, L. C. et al. Brazilian red propolis: Chemical composition and antibacterial activity determined using bioguided frac-
tionation. Microbiol. Res. 214, 74–82 (2018).
72. Inui, S. et al. Identification of the phenolic compounds contributing to antibacterial activity in ethanol extracts of Brazilian red
propolis. Nat. Prod. Res. 28, 1293–1296 (2014).

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 14

Vol:.(1234567890)
www.nature.com/scientificreports/

73. Naldoni, F. J. et al. Antimicrobial activity of benzophenones and extracts from the fruits of Garcinia brasiliensis. J. Med. Food
12, 403–407 (2009).
74. Porto, A. L. M. et al. Polyisoprenylated benzophenones from Clusia floral resins. Phytochemistry 55, 755–768 (2000).
75. Tao, C. Antimicrobial activity and toxicity of gold nanoparticles : research progress, challenges and prospects. Lett. Lipid Micro-
biol. 67, 537–543. https​://doi.org/10.1111/lam.13082​ (2018).
76. Penders, J., Stolzoff, M., Hickey, D. J., Andersson, M. & Webster, T. J. Shape-dependent antibacterial effects of non- cytotoxic
gold nanoparticles. Int. J. Nanomed. 12, 2457–2468 (2017).
77. McGaw, L. J., Elgorashi, E. E. & Eloff, J. N. Cytotoxicity of African Medicinal Plants Against Normal Animal and Human Cells.
Toxicological Survey of African Medicinal Plants (Elsevier Inc., Amsterdam, 2014).
78. Kuete, V., Karaosmanoğlu, O. & Sivas, H. Anticancer Activities of African Medicinal Spices and Vegetables 271–297 (Academic
Press, Cambridge, 2017).
79. de Oliveira, P. F. et al. Study of the cytotoxic activity of Styrax camporum extract and its chemical markers, egonol and homoe-
gonol. Cytotechnology 68, 1597–1602 (2016).
80. Itharat, A. et al. In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer. J. Ethnopharmacol. 90,
33–38 (2004).
81. Zhang, J. et al. Formononetin, an isoflavone from Astragalus membranaceus inhibits proliferation and metastasis of ovarian
cancer cells. J. Ethnopharmacol. 221, 91–99 (2018).
82. Kanazawa, M. et al. Isoliquiritigenin inhibits the growth of prostate cancer. Eur. Urol. 43, 580–586 (2003).
83. Gatouillat, G. et al. Medicarpin and millepurpan, two flavonoids isolated from Medicago sativa, induce apoptosis and overcome
multidrug resistance in leukemia P388 cells. Phytomedicine 22, 1186–1194 (2015).
84. Ye, Y. et al. Formononetin-induced apoptosis of human prostate cancer cells through ERK1/2 mitogen-activated protein kinase
inactivation. Horm. Metab. Res. 44, 263–267 (2012).
85. Novak, E. M. et al. Antitumoural activity of Brazilian red propolis fraction enriched with xanthochymol and formononetin: An
in vitro and in vivo study. J. Funct. Foods 11, 91–102 (2014).
86. Lee, Y. J. et al. Maclurin exerts anti-cancer effects on PC-3 human prostate cancer cells via activation of p38 and inhibitions of
JNK, FAK, AKT, and c-Myc signaling pathways. Nutr. Res. 58, 62–71 (2018).
87. de Carvalho, F. M. et al. Brazilian red propolis: Extracts production, physicochemical characterization, and cytotoxicity profile
for antitumor activity. Biomolecules 10, 726 (2020).
88. El Domany, E. B., Essam, T. M., Ahmed, A. E. & Farghali, A. A. Biosynthesis physico-chemical optimization of gold nanoparticles
as anti-cancer and synergetic antimicrobial activity using Pleurotus ostreatus fungus. J. Appl. Pharm. Sci. 8, 119–128 (2018).
89. Goddard, Z. R., Marín, M. J., Russell, D. A. & Searcey, M. Active targeting of gold nanoparticles as cancer therapeutics. Chem.
Soc. Rev. https​://doi.org/10.1039/d0cs0​1121e​ (2020).
90. Cao, J., Han, J., Xiao, H., Qiao, J. & Han, M. Effect of tea polyphenol compounds on anticancer drugs in terms of anti-tumor
activity, toxicology, and pharmacokinetics. Nutrients 8, 762 (2016).
91. Toric, J., Markovic, A. K., Brala, C. J. & Barbaric, M. Anticancer effects of olive oil polyphenols and their combinations with
anticancer drugs. Acta Pharm. Sci. 69, 461–482 (2019).
92. Takara, K. et al. Effects of propolis extract on sensitivity to chemotherapeutic agents in HeLa and resistant sublines. Phyther.
Res. 21, 841–846 (2007).
93. Salim, E. I., Abd El-Magid, A. D., Farara, K. M. & Maria, D. S. M. Antitumoral and antioxidant potential of Egyptian propolis
against the PC-3 prostate cancer cell line. Asian Pac. J. Cancer Prev. 16, 7641–7651 (2015).
94. Oršolić, N. et al. Protective effects of propolis and related polyphenolic/flavonoid compounds against toxicity induced by iri-
notecan. Med. Oncol. 27, 1346–1358 (2010).
95. Qiao, J. et al. Effect of green tea on pharmacokinetics of 5-fluorouracil in rats and pharmacodynamics in human cell lines in vitro.
Food Chem. Toxicol. 49, 1410–1415 (2011).
96. Riss, T., Niles, A. & Minor, L. Cell Viability Assays. Assay Guidance Manual (Eli Lilly & Company and the National Center for
Advancing Translational Science, Bethesda, 2013).
97. Bellamy, W. T. Prediction of response to drug therapy of cancer: A review of in vitro assays. Drugs 44, 690–708 (1992).
98. Maehara, Y., Anai, H., Tamada, R. & Sugimachi, K. The ATP assay is more sensitive than the succinate dehydrogenase inhibition
test for predicting cell viability. Eur. J. Cancer Clin. Oncol. 23, 273–276 (1987).
99. Ahmann, F. R., Garewal, H. S., Schifman, R., Celniker, A. & Rodney, S. Intracellular adenosine triphosphate as a measure of
human tumor cell viability and drug modulated growth. In Vitro Cell. Dev. Biol. 23(7), 474–480 (1987).
100. Begnini, K. R. et al. Brazilian red propolis induces apoptosis-like cell death and decreases migration potential in bladder cancer
cells. Evid.-Based Complement. Altern. Med. 20, 14. https​://doi.org/10.1155/2014/63985​6 (2014).
101. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. CLSI document
M100-S23 (2013).

Acknowledgements
We would like to acknowledge the funding from the São Paulo State Research Support Foundation (FAPESP)
(grants #2018/13465-5 and #2017/04138-8) and Coordination for the Improvement of Higher Education Per-
sonnel (CAPES). This study is part of the National Institute of Science and Technology in Pharmaceutical
Nanotechnology: a transdisciplinary approach INCT-NANOFARMA, which is also supported by FAPESP
(grant #2014/50928-2) and the National Council for Scientific and Technological Development (CNPq, grants
#465687/2014-8).

Author contributions
C.E.A.B., L.B.S., G.V.C–C., S.R.A., R.C.S.V., J.K.B. and P.D.M. conducted the experiments and wrote the manu-
script. T.S.S. conducted the antimicrobial activity experiment with fractions of BRP. All authors reviewed the
manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to P.D.M.
Reprints and permissions information is available at www.nature.com/reprints.

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 15

Vol.:(0123456789)
 www.nature.com/scientificreports/

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/.

© The Author(s) 2021

Scientific Reports | (2021) 11:1974 | https://doi.org/10.1038/s41598-021-81281-w 16

Vol:.(1234567890)