See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/236111512

In vitro Anti-cancerous and Anti-microbial Activity of Indian Propolis Nanoparticles

Article · September 2012

DOI: 10.1166/jnd.2013.1004

CITATIONS READS
8 1,043

6 authors, including:

Ramya Chandrasekaran Snima K S

Monash University (Australia) Amrita Vishwa Vidyapeetham
10 PUBLICATIONS   504 CITATIONS    23 PUBLICATIONS   247 CITATIONS   

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Ramya Chandrasekaran on 12 May 2015.

The user has requested enhancement of the downloaded file.

 Article
Journal of
Nanopharmaceutics
and Drug Delivery
Copyright © 2012 American Scientific Publishers
All rights reserved Vol. 1, 1–7, 2012
Printed in the United States of America www.aspbs.com/jnd

In Vitro Anti-Cancerous and Anti-Microbial Activity of

Propolis Nanoparticles
R. Jayakumar∗ , C. Ramya, P. T. Sudheesh Kumar, K. S. Snima, Vinoth-Kumar Lakshmanan,
and Shantikumar V. Nair
Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa
Vidyapeetham University, Kochi 682041, India

Propolis is a naturally available antibiotic and antioxidant in use from the ancient time. This study reports about the
synthesis of propolis nanoparticles and its effectiveness as an anti-microbial and anti-cancerous drug. The propolis
nanoparticles were prepared using simple extraction and reprecipitation method. The prepared nanoparticles were then
characterized using Dynamic light scattering (DLS), Scanning electron microscope (SEM), Atomic force microscopy
(AFM), Fourier transform infrared spectroscopy (FTIR), and UV visible spectroscopy. The anti-microbial activity of the
propolis nanoparticles was tested against Escherichia coli and Staphylococcus aureus and the minimum concentration of
propolis nanoparticles that inhibit bacterial growth was found. The cytotoxicity of propolis nanoparticles against MCF-7,
A375, PC3 and PANC-1 cancer cell lines was tested using MTT assay and the minimal concentration toxic to the cancer
cells were found. The cell uptake studies of propolis nanoparticles on MCF-7 cells demonstrated internalization of the
nanoparticles by the cancer cells. All these studies revealed that propolis nanoparticles could be a good substitute for
the existing materials against cancer.
KEYWORDS: Propolis, Nanoparticles, Antimicrobial, Anticancer, Cell Uptake.

INTRODUCTION gummy. Typically propolis will become liquid at 60 to

Propolis or Bee glue is a brownish waxy product collected
by the honeybee from plant buds, leaves, and exudates.
Propolis, known from ancient times, possesses antimicro-
bial, antioxidant, anti-inflammatory, anaesthetic, hepato-
protective, immunostimulating and cytostatic properties.1–2
The main components of propolis are resins (Flavonoids
and phenolic acid derivatives), waxes, essential oils, pollen
and aromatic balsam.3 Since propolis is of plant origin, the
composition of propolis varies depending upon the geo-
graphical origin and source plant.4 At temperatures of 25
to 45  C propolis is a soft, pliable and very sticky sub-
stance. At less than 15  C, and particularly when frozen
or at near freezing, it becomes hard and brittle. It will
remain brittle after such treatment even at higher tempera-
tures. Above 45  C it will become increasingly sticky and
∗
Author to whom correspondence should be addressed.
Emails: rjayakumar@aims.amrita.edu, jayakumar77@yahoo.com
Received: 2 May 2012
Revised/Accepted: 16 September 2012
70  C, but for some samples the melting point may be as
high as 100  C depending upon the chemical composition.5
The biological activity of propolis is mainly due to
the presence of flavonoids.6 The biochemical effects of
flavonoids can be divided into four categories (1) binding
affinity to biological polymers; (2) binding of heavy metal
ions; (3) catalysis of electron transport; and (4) ability to
scavenge free radicals.7–8 Thus, the antitumoral effect of
propolis was due to the flavonoids inhibiting the incorpo-
ration of thymidine, uridine, and leucine into carcinoma
cells, thus leading to an inhibition of DNA synthesis.9–10
Bees use Propolis to caulk their hives and thus check the
entry of microbes. The antimicrobial properties of propolis
are related to the synergistic effect of its compounds.11–13
Propolis, work against harmful bacteria by affecting the
integrity of cytoplasmic membrane and thus inhibiting
bacterial motility and enzyme activity. Propolis has also
been proven effective against strains of bacteria that resist
chemical antibiotics.14–15
The field of influence of propolis is extremely broad.
It includes cancer, infection of the urinary tract, swelling

J. Nanopharmaceutics Drug Delivery 2012, Vol. 1, No. 1 2167-9312/2012/1/001/007 doi:10.1166/jnd.2012.1004 1

In Vitro Anti-Cancerous and Anti-Microbial Activity of Propolis Nanoparticles
of the throat, gut, open wounds, sinus congestion,
colds, influenza, bronchitis, gastritis, diseases of the ears,
periodontal disease, intestinal infections, ulcers, eczema
eruptions, pneumonia, arthritis, lung disease, stomach
virus, headaches, Parkinson’s disease, bile infections,
sclerosis, circulation deficiencies, warts, conjunctivitis and
hoarseness.16 Monica et al. recently reported the role of
propolis nanoemulsion along with lycopene extract for the
protection of skin.17
MATERIALS AND METHODS
Raw Propolis was purchased from HiTech Natural Prod-
ucts (India) Ltd., Delhi, India. Trypsin-EDTA and Fetal
Bovine Serum (FBS) were obtained from Gibco, Invit-
rogen Corporation. Minimum essential medium (MEM)
was purchased from Sigma aldrich Company. The MCF-7,
PC3, A375 and PANC-1 cell lines were provided by
National Center for Cell Sciences, Pune, India. The chem-
icals were used without further purification.
Preparation of Propolis Nanoparticles
Propolis nanoparticles were prepared as follows; 10 g
of the raw propolis was dissolved in 40 mL of distilled
ethanol and the solution was kept under stirring for 18 h.18
The extracted propolis was then filtered using a filter
paper to remove the impurities. To the filtrate obtained,
approximately 500 mL water was added for reprecipita-
tion of propolis. The solution was then frozen followed
by lyophilization. This process yields pure propolis pow-
der which was then resuspended in water. The suspension
was then bath sonicated for 20–30 min to get the propolis
nanoparticles.
Characterization
The particle size and zeta potential of propolis nanoparticle
was measured by Dynamic light scattering (DLS-
ZP/Particle Sizer Nicomp™ 380 ZLS) taking the average
of 3 measurements. Zeta potential of the propolis nanopar-
ticles was measured on the same day and 2 weeks later of
the preparation. Size of the particles was further confirmed
using SEM (JEOLJSM-6490LA) and AFM (JEOL JSPM-
5200). UV-Visible spectroscopy (SHIMADZU UV-1700)
of propolis nanoparticles and raw propolis were taken.
FT-IR spectral analysis of propolis nanoparticles and raw
propolis was carried out using KBr tablets (1% w/w of
product in KBr) with a resolution of 4 cm−1 and 100
scans per sample on a Perkin Elmer Spectrum RX1 appa-
ratus. The fluorescence spectral analysis of bare propolis
nanoparticles was carried out using a Spectrofluorometer
(HORIBA JOBIN YVON Fluoromax-4).
2 J. Nanopharmaceutics Drug Delivery 1, 1–7, 2012
Jayakumar et al.
Antimicrobial Study
Escherichia Coli (E. coli) and Staphylococcus Aureus
(S. aureus) were used for evaluating the antibacterial activ-
ity of the prepared propolis nanoparticles. Luria-Bertani
broth (LB broth) and Luria-Bertani agar (LB agar) were
used as culturing nutrient sources. E. coli and S. aureus
were inoculated in sterilized LB broth and then incubated
overnight at 37  C in a shaking incubator. The concen-
tration of bacteria was 106 colony forming units per ml
(CFU/mL). Propolis nanoparticles were then added to the
medium and were incubated at 37  C for 24 h. After 24 h,
bacterial viability was measured by serial dilution of the
culture in normal saline, followed by plating on LB agar
plate.19–20
Gram Staining
Gram staining is the most commonly used staining tech-
nique in microbiology. It is based on the ability of bacteria
to retain the primary stain, crystal violet, after the solvent
treatment. Gram positive bacteria have higher peptidogly-
can and lower lipid content in their cell structure while
the gram negative ones have higher lipid content. Iodine
is added as a mordant which forms a crystal violet- iodine
complex thereby fixing the stain in the cell wall. When a
decolorizer, acetone, ethanol or a mixture of both is added,
it leaches away the primary stain in the case of Gram
negative bacteria by removing the lipid layer whereas in
the case of Gram positive bacteria, the cell wall struc-
ture remains intact and thus the bacteria remains stained.
Finally a basic fuchsin stain is applied which will be taken
up by the Gram negative bacteria and they appear pink
where as the Gram positive bacteria remain blue.
Cell Culture
MCF-7 (Human breast cancer cell line), PC3 (Human
prostate cancer cell line), A375 (Human skin cancer cell
line) and PANC-1 (Human pancreatic cancer cell line)
cell lines were purchased from NCCS, Pune. MCF-7 and
PC3 were maintained in MEM and DMEM-F12 medium
respectively. Both A375 and PANC-1 were maintained
in DMEM. The media were supplemented with 10%
Fetal Bovine Serum (FBS) and 100 units/mL of Peni-
cillin/Streptomycin. The cells were incubated in 5% CO2
incubator at 37  C. After reaching confluency, the cells
were used for the studies.
Cytotoxicity Studies
For cytotoxicity experiments cancer cell lines, MCF-7,
PC3, A375 and PANC-1 were seeded with a density
of 8,000 cells/well and 5,000 cells/well in 96 well
plate. MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl-
tetrazolium bromide] assay, a colorimetric test based on
Jayakumar et al. In Vitro Anti-Cancerous and Anti-Microbial Activity of Propolis Nanoparticles

the selective ability of viable cells to reduce the tetra-

zolium component of MTT into purple colored formazan
crystals was used to evaluate cytotoxicity of the prepared
propolis nanoparticles. Four different concentrations of
propolis nanoparticles 100, 50, 10 and 5 g/mL were pre-
pared in media. After 24 h of incubation, each cancer cell
lines were treated with different concentration of nano-
particles. Cells in media without nanoparticles acted as
positive control and cells treated with Triton X-100 as neg-
ative control. The plate which had higher density of cells
was incubated for 24 hrs and the other for 48 h. After treat-
ment, the cells were incubated with 100 L of 0.5 mg/mL
MTT in media for 4 h followed by 1 h incubation with
solubilization buffer (10% v/v Triton X-100, 0.1 N HCl in
Isopropyl alcohol). The optical density of the solution was
measured at a wavelength of 570 nm using a Beckmann
Coulter Elisa plate reader (BioTek Power Wave XS). Trip-
licate samples were analyzed for each experiment. Cell
viability was calculated as

Viability % = Nt/Nc × 100

Nt is the optical density of cells treated with sample and

Nc is the absorbance of untreated cells.
Figure 1. Schematic representation of processes involved in
the synthesis of propolis nanoparticles.
Cell Uptake

Cell uptake studies of the prepared propolis nanoparticles
were studied by fluorescent imaging. For this MCF-7 cells
were seeded on etched cover slips placed in a 24 well
plate with a density of 15,000 cells/well. The plate was
then incubated for 24 h for the cells to grow on the cov-
erslips. Thereafter the media was removed and the wells
were carefully washed with PBS. Then the particles at
a concentration of 10 g/mL was added along with the
media in triplicate to the wells and incubated for 2 h.
After incubation the samples were removed and cover slips
were processed for fluorescent microscopy. The processing
involved washing the cover slips with PBS and fixing the
cells with 3% (v/v) Para Formaldehyde (PFA) followed by
PBS wash. The cover slips were air dried and mounted on
to glass slides with D.P.X. mountant. Then the slides were
dried and viewed with an OLYMPUS DP 71 fluorescent
microscope by providing an excitation wavelength of 460–
490 nm to study the cell uptake. The bright field and flu-
orescence images were obtained at 100X magnifications.
RESULTS AND DISCUSSION
Characterizations of Propolis Nanoparticles
Figure 1 shows the schematic representation of the prepa-
ration procedure and Figure 2(A) shows the particle size of
the prepared propolis nanoparticles. The size distribution
for the nanoparticles was obtained using DLS and the par-
ticles were having size in the range of 80–150 nm. The size
of the prepared nanoparticle was again confirmed by SEM
and AFM. Figure 2(B) shows the SEM images of propo-
lis nanoparticles which showed the size was 50–170 nm
and the morphology is spherical. Figure 2(C) represents
the AFM image (500 nm scale) showing that the particles
were having size in the range of 80–150 nm.
Propolis nanoparticles showed zeta potential value of
+372 ± 2 and +322 ± 3 on the same day and after
2 weeks of the preparation respectively. The obtained data
revealed that propolis nanoparticle possess positive surface
charge and showed zeta potential value above +30 even
after storage for two weeks. This revealed the stability of
the prepared particles in water and the stability of the par-
ticles can be attributed to the positive surface charge which
particle possess and due to this charge, particle tend repel
each other hence reduced the agglomeration.
The FT-IR analysis was performed to find out the struc-
tural modification of raw propolis after being converted
into nanoparticles. In Figure 3(A) we have compared the
FT-IR data of propolis nanoparticles with the raw propo-
lis. It was found that there was no significant change in
the spectrum of propolis nanoparticles when compared
with that of raw propolis. The peaks at 2916, 1618 and
1084 cm−1 were retained in nano propolis as well.
The UV absorption spectrum showed absorption max-
ima at 268 nm both in raw propolis as well as its nanopar-
ticles of propolis (Fig. 3(B)). There was no change in
the absorption maxima and it indicates that the prepared
propolis nanoparticles still contains the same constituents

J. Nanopharmaceutics Drug Delivery 1, 1–7, 2012 3

In Vitro Anti-Cancerous and Anti-Microbial Activity of Propolis Nanoparticles Jayakumar et al.

Figure 2. (A) Particle size analysis of propolis NPs. (B) SEM image of propolis NPs. (C) AFM image of propolis NPs.

Figure 3. (A) FT-IR spectrum of (a) Propolis nanoparticles (b) Raw propolis. (B) UV spectra of (a) Propolis nanoparticle and (b)
Raw propolis. (C) Fluoroscence spectrum of Propolis nanoparticles.

4 J. Nanopharmaceutics Drug Delivery 1, 1–7, 2012

Jayakumar et al. In Vitro Anti-Cancerous and Anti-Microbial Activity of Propolis Nanoparticles

Figure 4. (A) Antimicrobial activity of various concentration

of propolis NPs against S. aureus. (B) Antimicrobial activity of Figure 6. MTT assay of propolis nanoparticles on different
various concentration of propolis NPs against E. coli. cancer cell lines after (A) 24 and (B) 48 hrs incubation.

and it retained its intact structure. In the fluorescence spec-
tral analysis from Figure 3(C), Propolis nanoparticles were
confirmed to exhibit auto fluorescence. The excitation of
propolis nanoparticles was found at 494 nm whereas the
emission was around 549 nm. This data revealed the poten-
tial of this material for imaging of cells in biomedical field.
Antibacterial Activity Evaluation
Propolis nanoparticles, from Figures 4(A) and (B) were
found to show antibacterial activity towards E. coli and
S. aureus at a concentration of 10 g/mL and above. We
have done the antibacterial study of various concentra-
tions of propolis nanoparticle starting from 5 g/mL to
100 g/mL. As the concentration of the nanoparticles was
increased, the survival of both bacteria was decreased. The
mechanism behind the antimicrobial activity is that propo-
lis nanoparticles will bind with the respiratory enzyme
present in the cell wall of bacteria and hence prevent the
respiration. In addition it will cause the cell wall leakage
of bacteria to lead the death of bacteria.21 The propo-
lis nanoparticles proved its antibacterial activity against
gram positive and gram negative strains. The antibacte-
rial activity also confirmed with gram staining (Fig. 5).

Figure 5. Gram staining (A) Bacterial culture of S. aureus without propolis NPs. (B) 15 g/mL of propolis NPs added culture of
S. aureus. (C) 50 g/mL of propolis NPs added culture of S. aureus. (D) 100 g/mL of propolis NPs added culture of S. aureus.
(E) Bacterial culture of E. coli without propolis NPs. (F) 15 g/mL of propolis NPs added culture of E. coli. (G) 50 g/mL of
propolis NPs added culture of E. coli. (H) 100 g/mL of propolis NPs added culture of E. coli.

J. Nanopharmaceutics Drug Delivery 1, 1–7, 2012 5

In Vitro Anti-Cancerous and Anti-Microbial Activity of Propolis Nanoparticles Jayakumar et al.

Figure 7. Cell uptake images (A) Bright field image of MCF-7 cells without propolis NPs. (B) Fluorescence image of MCF-7 cells
without propolis NPs. (C) Bright field image of 10 g/mL of propolis NPs added MCF-7 cells. (D) Fluorescence image of 10 g/mL
of propolis NPs added MCF-7 cells.

It showed the number of bacterial colonies decreased at
higher concentration of propolis. The antibacterial activ-
ity will be helpful for the usage of this material for var-
ious biomedical applications where antimicrobial activity
demands more attention.
Cytotoxicity Studies
Cytotoxicity of propolis nanoparticles was evaluated using
MTT assay as shown in Figures 6(A) and (B). From the
24 h incubation studies it was observed that the particles
at lower concentration (5 g/ml) did not show significant
toxicity towards the cancer cell lines whereas at higher
concentrations (10, 50 and 100 g/mL) the cell viabil-
ity was reduced significantly. Again from 48 h incubation
studies (Fig. 6) the cell viability was further reduced indi-
cating higher extent of cytotoxicity. At highest concentra-
tion (100 g/mL) of propolis nanoparticle approximately
100% cytotoxicity was observed which was comparable
to that of negative control (1% Triton). Thus the propo-
lis nanoparticles showed concentration dependant and time
dependant toxicity towards the different cancer cell lines
confirming its anticancer effects. The anticancerous prop-
erty of propolis is due to the presence of flavonoids that
prevent the cancer cell cycle progression, cell prolifera-
tion and tumor growth, thus inhibiting tumor metastasis
and inducing cell-cycle arrest and apoptosis. This data
revealed the potential of propolis nanoparticles for the can-
cer treatment.
Cell Uptake Analysis
Cellular uptake of nanoparticles was evaluated by visual-
izing the intrinsic fluorescence of propolis using fluores-
cence microscopy (Fig. 7). Control MCF-7 cells without
any exposure to propolis nanoparitcles showed no flu-
orescence (Figs. 7(A) and (B)). MCF-7 cells incubated
with 10 g/mL propolis nanoparticles (Figs. 7(A) and (B))
showed green fluorescence confirming the internalization
of Nps by the cells. The NPs treated cells appeared to be
rounded off so as to undergo apoptosis. This confirms the
uptake of propolis nanoparticles and its anticancer effects.
The positive surface charge of the propolis nanoparticles
caused the cellular uptake of these by the breast cancer
cells. The negative charge of the cells caused the attrac-
tion of these particles towards the cells and them taken
up by the cells. Breast epithelial cells are known to have
negative charge, which could very well interact with the
positively charged propolis nanopartilces, enhancing the
charge based internalization in to the breast cancer cells.
CONCLUSIONS
Propolis nanoparticles were prepared from raw Propolis
through re-precipitation technique and were characterized.

6 J. Nanopharmaceutics Drug Delivery 1, 1–7, 2012

 Jayakumar et al. In Vitro Anti-Cancerous and Anti-Microbial Activity of Propolis Nanoparticles
The particle size was found to be in the range of 80–
150 nm. The antibacterial activity of Propolis nano-
particles was tested against E. coli and S. aureus. Also,
the cytotoxic effect of Propolis nanoparticles against can-
cer cell lines was tested against MCF-7, PC3, A375 and
PANC-1. For a concentration of 10 g/mL and above,
Propolis nanoparticles showed significant antimicrobial
and anticancerous activity and hence can be used as a
potential anti-cancer and anti-microbial drug.
Conflict of Interest
“There is no conflict of interest.”
Acknowledgments: One of the authors R. Jayakumar
is grateful to The Department of Biotechnology (DBT),
Government of India for the financial support, under a
grant of the Nanoscience and Nanotechnology Initiative
program (Ref. No. BT/PR10850/NNT/28/127/2008). The
authors are also thankful to Mr. Sajin. P. Ravi for his help
in SEM analysis.
REFERENCES
1. B. Tosi, A. Domini, C. Romagnoli, and A. Bruni, Antimicrobial
activity of some commercial extracts of propolis prepared with dif-
ferent solvents. Phytotherapy Res. 10, 335 (1996).
2. A. H. Banskota, Y. Tezuka, I. K. Adnyana, E. Ishii, K. Midorikawa,
K. Matsushige, and S. Kadota, Hepatoprotective and anti-
helicobacter pylori activities of constituents from Brazilian propolis.
Phytomedicine 8, 16 (2001).
3. A. E. H. Hegazi, Egyptian propolis: 1-Antimicrobial activity and
chemical composition of Upper Egypt propolis. Z. Naturforsch
56, 82 (2001).
4. S. M. J. Yaghoubi, G. R. Ghorbani, Z. S. Soleimanian, and R. Satari,
Antimicrobial activity of Iranian propolis and its chemical composi-
tion. Daru 15, 1 (2007).
5. H. Katirciolu and H. Mercan. Antimicrobial activity and chemi-
cal compositions of Turkish propolis from different regions. Afr. J.
Biotechnol. 5, 1151 (2006).
6. D. Gruneberger, R. Banerjee, and K. Eisinger, Preferential cytotox-
icity on tumour cells by caffeic acid phenethyl ester isolated from
propolis. Experientia 44, 230 (1988).
7. Z. Kleinrok, Z. Borzeck, S. Scheller, and W. Matuga, Biological
properties and clinical application of propolis. Arzneim.-Forsch. 28,
291 (1978).
J. Nanopharmaceutics Drug Delivery 1, 1–7, 2012
View publication stats
8. G. Coneac, E. Gafitanu, D. I. Hadaruga, N. G. Hadaruga, I. A.
Pinzaru, G. Bandur, L. Ursica, V. Paunescu, and A. Gruia, Flavonoid
Contents of Propolis from the West Side of Romania and Correlation
with the Antioxidant Activity. Chem. Bull. Politehnica University 53,
1 (2008).
9. Y. Akao, H. Maruyama, K. Matsumoto, K. Ohguchi, K. Nishizawa,
T. Sakamato, Y. Araki, S. Mishima, and Y. Nozawa, Cell growth
inhibitory effect of cinnamic acid derivatives from propolis on
human tumor cell lines. Biol. Pharm. Bull. 26, 1057 (2003).
10. S. Kumazawa, T. Hamasaka, and T. Nakayama, Antioxidant activ-
ity of propolis of various geographic origins. Food Chem. 84, 329
(2004).
11. J. M. Grange and R. W. Davey, Antibacterial properties of propolis
(bee glue). J. Royal Soc. Med. 83, 159 (1990).
12. A. Kujumgiev, I. Tsvetkova, Y. Serkedjieva, V. Bankova, R. Christov,
and S. Popov, Antibacterial, antifungal, and antiviral activity of
propolis of different geographic origin. J. Ethnopharmacol. 64, 235
(1999).
13. S. Scheller, S. Dworniczak, K. Waldemar-Klimmek, M. Rajca,
A. Tomczyk, and J. Shani, Synergism between ethanolic extract of
propolis (EEP) and anti-tuberculosis drugs on growth of mycobac-
teria. Z. Naturforsch. 54, 549 (1999).
14. S. Stepanovic, N. Antic, I. Dakic, and M. Svabic-Vlahovic, In vitro
antimicrobial activity of propolis and synergism between propolis
and antimicrobial drugs. Microbiol. Res. 158, 353 (2003).
15. H. N. Hastyar and A. K. H. K. Farhad, Antimicrobial activity
of Propolis collected in different regions of sulaimanu province-
Kurdistan region/Iraq. Duhok University J. 12, 233 (2009).
16. S. Castolda and F. Capasso, Propolis an old remedy used in modern
medicine. Fitoterapia 73, 51 (2002).
17. M. V. Butnariu and C. V. Giuchici, The use of some nanoemulsions
based on aqueous propolis and lycopene extract in the skin’s protec-
tive mechanisms against UVA radiation. J. Nanobiotechnology 9, 1
(2011).
18. S. Woisky, Preparation of water and ethanolic extract of propolis
and evaluation of the preparation. Biosci., Biotechnol., Biochem. 62,
2230 (1998).
19. B. Lyudmila, G. Galina, N. Rossen, D. Sirigan, L. Elena, K. Nikolai,
M. Ivan, and K. Zacharii, Activity of Bulgarian propolis against 94
Helicobacter pylori strains in vitro by agar-well diffusion, agar dilu-
tion and disc diffusion methods. J. Med. Microbiol. 54, 481 (2005).
20. L. Drago, V. E. De, L. Nicola, and M. R. Gismondo, In vitro antimi-
crobial activity of a novel propolis formulation. J. Appl. Microbiol.
103, 1364 (2007).
21. F. A. Santos, E. M. Bastos, M. Uzeda, M. A. Carvalho, L. M. Farias,
E. S. Moreira, and F. C. Braga, Antibacterial activity of Brazilian
propolis and fractions against oral anaerobic bacteria. J. Ethnophar-
macol. 80, 1 (2002).
7