Adjuvant effect of green propolis on humoral immune response of
bovines immunized with bovine herpesvirus type 5
Geferson Fischera,b,*, Marlete Brum Cleffb, Luana Alves Dummera,b,
Niraldo Paulinoc, Amarı ´lis Scremin Paulinoc, Camila de Oliveira Vilelab,
Fabrı ´cio Souza Camposb, Tiago Storchb, Gilberto D’Avila Vargasb,
Silvia de Oliveira Hu ¨bnerb, Telmo Vidorb
aCentro de Biotecnologia, Universidade Federal de Pelotas (UFPel), Campus Universita ´rio,
Caixa Postal 354, 96010-900, Pelotas, RS, Brazil
bLaborato ´rio de Virologia e Imunologia, Faculdade de Veterina ´ria, UFPel, Campus Universita ´rio,
Caixa Postal 354, 96010-900, Pelotas, RS, Brazil
cFaculdade de Farma ´cia, Departamento de Pesquisa e Extensa ˜o, Universidade Bandeirante de Sa ˜o Paulo, Sa ˜o Paulo, Brazil
Received 19 October 2006; received in revised form 19 December 2006; accepted 9 January 2007
Despiterecent technological advances invaccine production, mostvaccines depend onthe associationwithadjuvant substances.
In this study, propolis, which has been attracting the attention of researchers due to its bioactive properties, was evaluated as an
immunological adjuvant. The association of 40 mg/dose of an ethanolic extract of green propolis with an inactivated oil vaccine
against bovine herpesvirus type 5 (BoHV-5), resulted in a significant increase (P < 0.01) in the neutralizing antibody levels,
comparing to the bovines that received the same vaccine without propolis. Besides, propolis increased the percentage of animals
with high antibody titers (above 32). Phenolic compounds such as artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) and the
derivatives of cinnamic acid besides other flavonoid substances were abundant in the propolis extract used, and they could be the
main substances with adjuvant action. The effect of the green propolis extract on the humoral immune response can be exploited in
the development of new vaccines.
# 2007 Elsevier B.V. All rights reserved.
Keywords: Green propolis; Immunological adjuvant; Antibodies; Bovines
The recent progress in the development of vaccines
has allowed their use not only as a prophylactic product,
but also in the treatment of cancer, immunological
disorders and chronic infections (Blom and Hilgers,
2004). However, many of them require association with
adjuvant substances which, when combined with an
antigen, increase its immunogenicity, potentiating the
2005; Barr et al., 2006). Besides, immunological
adjuvants can be used to extend the duration of the
immune response or to stimulate mucosal immunity
Even though several substances have been evaluated
regarding their adjuvant capabilities, as a general rule,
Veterinary Immunology and Immunopathology 116 (2007) 79–84
* Corresponding author at: Rua Carlos de Carvalho, 372, Apt. 401,
96030-270, Pelotas, RS, Brazil. Tel.: +55 53 32757498;
fax: +55 53 32757498.
E-mail address: firstname.lastname@example.org (G. Fischer).
0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved.
production of vaccines is still dependent on the use of
aluminum salts (Leclerc, 2003) or oil emulsions,
specific for vaccines of veterinary use (Jansen et al.,
2006). This way, the development of new vaccines will
be highly benefited with the identification of new
substances capable of promoting and directing to a
proper immune response (Cox and Coulter, 1997; Singh
and O’Hagan, 2002).
Oil adjuvants, which are no longer in use for humans
due to their adverse effects (Lindblad, 2000), are still
largely used in vaccines for veterinary use. These oil
emulsions, used specially in association with inacti-
vated antigens (Jansen et al., 2006), potentiate the
immune system through the formation of a deposit at
the inoculation site, with slow and long release of the
antigen (Cox and Coulter, 1997). However, these
inactivated vaccines need periodic revaccinations to
produce an efficient immunological response (Fenner
et al., 1993).
Propolis is a resinous material produced by bees that
displays a variety of biological activities against
viruses, bacteria, fungi, pathogenic protozoa, and also
tumor cells (De Castro, 2001). This natural product also
behaves as anti-hyperalgesic and anti-inflammatory
agent (De Campos et al., 1998). Despite the demonstra-
tion of its immunomodulator activities (Dimov et al.,
1992; Orsi et al., 2000), many of the mechanisms of
action are still unknown.
Chemical studies revealed the complex composi-
tion of propolis, with more than 300 constituents,
including several bioactive phenolic compounds such
as flavonoids and derivatives of hydroxycinnamic
acids (Bankova et al., 2000). These constitutive
characteristics can vary according to the bee species
and the period of the year (Bankova, 2005). Never-
theless, the botanical origin (Bankova et al., 2000;
Bankova, 2005) seems to be the most important factor
to be considered when trying to explain the chemical
variability among different propolis samples. Green
propolis, only found in Brazil, is produced from a
plant commonly known as ‘‘Alecrim do Campo’’
(Baccharis dracunculifolia). This species is not
adapted to the natural conditions of other countries
(Miyataka et al., 1997), which confers to the green
different from the European propolis, produced
predominantly from the exudates of buds of aspen
(Populus sp., Bankova et al., 2000).
The aim of this work was to evaluate the adjuvant
capability of a green propolis extract when associated
with an inactivated oil vaccine against bovine herpes-
virus type 5, through determination of neutralizing
antibody titers, and characterization of the chemical
composition of this extract by high performance liquid
2. Material and methods
2.1. Preparation, botanical and chemical
characterization of the propolis ethanolic extract
The green propolis sample was collected in the
State of Minas Gerais (South-East region of Brazil)
by Nectar Farmace ˆutica Ltda (sample number SBN-
54), and stored at ?20 8C. The ethanolic extract was
prepared as previoulsy described (Paulino et al.,
2002). Briefly, the propolis was ground and macerated
with an extract solution containing absolute ethanol,
using 10 min daily agitation, for 10 days. Then, the
solvent was evaporated and the resulting dried matter
was dissolved in phosphate buffer solution (pH 6.2),
in a final concentration of 40 mg/ml (4%, w/v) and
called GP1. The chemical composition of the green
propolis extract was determined by high performance
liquid chromatography (HPLC), using a Merck-
Hitachi chromatographer (Germany), equiped with
the L-7100 pressure pump and the L-7455 diode array
detector. Separation was carried out in a Lichrochart
125-4 (Merck, Darmstadt, Germany) column as
previously described (Marcucci et al., 2001). The
detection of components was monitored at 280 nm
and standard compounds were co-chromatographed
with the extract. Analysis of the data was carried out
using the Merck-Hitachi D-7000 (Chromatography
Data Station, DAD Manager).
2.2. Vaccines and inoculations
Evaluation of adjuvant properties of the green
propolis extract was made through its association with
an inactivated oil vaccine against bovine herpesvirus
type 5. This virus, supplied by the Virology and
Immunology Laboratory, UFPel (Pelotas, Brazil), was
propagated in Madin Darby Bovine Kidney (MDBK,
ATCC) cell line. After inactivation with bromoethyla-
mine BEI (C2H7Br2N, Merck), in a final concentra-
tion of 0.02 M and pH 7.5, the viral suspension with
titer of 108CCID50/ml (cell culture infections dose
50% ml?1) was emulsified in mineral oil (Marcol 52,
Esso Standart Oil Co.), together with the propolis
In this study, 60 randomly selected Hereford cattle,
male and female, weighting approximately 150 kg
and seronegative for BoHV-5 determined by serum
G. Fischer et al./Veterinary Immunology and Immunopathology 116 (2007) 79–8480
neutralization (House and Baker, 1971), were used. The
animals were kept in pasture rotation together with
other cattle of the farm. They were allocated into three
groups of 20 animals (control, G1 and G2) and
vaccinated intramuscularly (IM) at days 0, 30 and 60.
The volume inoculated was adjusted according to the
concentration of the propolis solution used per dose,
varying from 3 to 5 ml, however the antigen concentra-
tion was constant in all the treatments (108CCID50/ml/
dose). The control group received 3 ml of vaccine
without propolis, the group G1 received 5 ml of the
same vaccine with 20 mg/dose of propolis, whereas
group G2 received 5 ml of this vaccinewith 40 mg/dose
of propolis. The animals were observed daily until the
10th day after each inoculation in order to observe the
occurrence of adverse effects due to vaccination. The
experiment was approved by the UFPel Committee of
Ethics in Animal Experimentation.
For titering neutralizing antibodies against BoHV-5,
individual serum samples were collected 30 days after
each inoculation and stored at ?20 8C. Antibodies were
titered by the serum neutralization method (House and
Baker, 1971). Briefly, each serum was individually
diluted in logarithmic base 2 from 1:2 to 1:256. After
distribution (25 ml) in quadruplicate in polystyrene
plates (TPP), 25 ml of BoHV-5 virus suspension
containing 100 CCID 50% was added. After incubation
for 1 h at 37 8C in an environment with 5% CO2,
approximately 30,000 MDBK cells were added per
well. The microplates were then returned to the
incubator until being read in an inverted microscope
when the 100 CCID 50% was observed in the control
cells. The absence of cytopathic effect was an
indication of the viral neutralization by neutralizing
antibodies. The antibody titer was calculated by
Behrends and Ka ¨rber (Mayr et al., 1982) statistical
method, and it was represented by the highest serum
dilution capable of neutralizing 100 CCID 50% of
2.4. Statistical analysis
Antibody titers were compared using variance
analysis (ANOVA) with repeated measurements. The
(P < 0.05) among the mean of each treatment using the
3.1. Chemical composition of the propolis extract
As can be observed in Table 1, the HPLC analysis of
the green propolis sample utilized in this experiment
showed high levels of the phenolic compounds 3,5-
diprenyl-4-hydroxycinnamic acid (artepillin C), 2,2-
nyl-4-hydroxycinnamic acid, p-coumaric acid, caffeic
acid, ferulic acid, besides cinnamic acid and the
flavonoids pinobanksin and kaempferol. In this sample
of green propolis, the flavonoids corresponded to
22.37% of the dried extract.
3.2. Humoral response
In this work, the adjuvant properties of an ethanolic
extract of green propolis when associated with an
inactivated oil vaccine against BoHV-5 was evaluated.
A dose dependent effect was demonstrated. The
inclusion of 40 mg/dose of this extract in the experi-
mental vaccine increased the humoral immune response
(P < 0.01), measured by antibodies titers, compared to
the control group (vaccine without propolis) (Fig. 1).
Sixty days after the first inoculation, the titer increased
from 35 (negative control, without propolis) to 54 in the
treatment with 40 mg/dose of propolis. This difference
remained the same 90 days after the first inoculation,
when the antibody titers were 43 and 67, respectively.
However, the addition of 20 mg/dose of the propolis
G. Fischer et al./Veterinary Immunology and Immunopathology 116 (2007) 79–8481
Chemical characterization of green propolis determined by high
performance liquid chromatography—HPLC
Component identified in propolisDry extract (mg/g)
acid (derivative 1-9)
acid (artepillin C)
Caffeic acid (derivative 1)
Cinnamic acid derivative
Kaempferol (derivative 1)
Total (mg/g of crude resin)
extract did not result in a statistically significant
alteration, when compared to the control group. Besides
increasing antibody titers of cattle vaccinated with
BoHV-5, the use of 40 mg/dose of green propolis
solution also increased the percentage of animals with
antibody titers equal or higher than 32, especially 30
days after the third vaccination (Table 2).
Adjuvants are used in several types of vaccine
aiming at the optimization of the humoral and/or
cellular responses. However many substances with
adjuvant properties cannot be used in vaccines for
human or veterinary use due to their adverse effects
(Estrada et al., 2000). In this study, conducted in cattle,
no adverse effect was observed due to the association of
propolis with the oil vaccine against BoHV-5.
Due tothe highchemicalcomplexityof propolis, itis
extremely difficult to identify which substances are
responsible for its biological activities. Some research-
besides the combination of natural substances that
confers to propolis its bioactive properties (Kujumgiev
et al., 1999; Ozkul et al., 2005). According to Sforcin
The elements detected in the green propolis extract
utilized in this experiment, determined by HPLC
analysis, show that its botanical origin is the Bacharis
dracunculifolia plant (Marcucci, 1995; Bankova et al.,
2000). In a previous study, multivariate analysis
associating ethanol extracts of different samples with
the levels of bioactivecompounds determined by HPLC
allowed the typing of Brazilian propolis (Marcucci
et al., 2001). The results were similar to the ones found
in this experiment, with predominance of phenolic
compounds and cinnamic acid derivatives. In these
extracts, the flavonoids corresponded to 22.37% of the
dried extract. These substances are known to stimulate
humoral as well as cellular immunity (Havsteen, 2002).
Although the precise mechanism of action remains
unknown, it is possible that the flavonoids stimulate
production of cytokines, particularly interleukin 1 (IL-
1) and IL-2, which have mitogenic action for B and T
lymphocytes (Havsteen, 2002). Artepillin C seems to
increase in the number of auxiliar T lymphocytes
(Kimoto et al., 1998).
The association of different adjuvant substances
aims at combining their properties responsible for the
stimulation of the immune system. Complete Freund
adjuvant, for example, combines the immunomodulator
properties of Mycobacterium tuberculosis with the
activity of the oil emulsion (Cox and Coulter, 1997). In
this experiment, the association of 40 mg/dose of an
ethanolic extract of green propolis whith an inactivated
oil vaccine against BoHV-5 increased the humoral
immune response (P < 0.01), measured by neutralizing
antibodies titers. It is conceivable that the combination
the inoculation site, resulting in a slow and extended
antigen (BoHV-5) and propolis release (Cox and
Coulter, 1997), allowing a constant stimulus of the
immunological system. According to Jansen et al.
(2006), the continuous release of non-replicative
antigens results in extended humoral immunity. The
propolis extract may have acted as an auxiliary adjuvant
G. Fischer et al./Veterinary Immunology and Immunopathology 116 (2007) 79–84 82
Fig. 1. Mean titer ? S.E.M. of neutralizing antibodies (expressed as
reciprocal of the serum dilution) of cattle immunized with an inacti-
vated oil vaccine against BoHV-5 without propolis (control), with
20 mg/dose of an ethanolic extract of green propolis (G1) or 40 mg/
dose of propolis (G2). The titer was determined by the serum
neutralization test, 30 days after the second inoculation (collection
1) or 30 days after the third inoculation (collection 2).**P < 0.01
compared to the control group.
Accumulated percent distribution of neutralizing antibodies titers in
bovines vaccinated with BoHV-5
30 days after second dose30 days after third dose
aTiters expressed by the reciprocal.
bVaccine without propolis.
cVaccine with 20 mg/dose of ethanolic extract of green propolis.
dVaccine with 40 mg/dose of ethanolic extract of green propolis.
substance, potentiating the humoral response triggered
by the antigen associated to the oil. According to
Sforcin et al. (2005), propolis ability in modulating
antibody synthesis is part of its adjuvant action. The
propolis immunostimulating capacity, through an
increase in the immunoglobulin levels has already
been reported in patients with fibrosing alveolitis
(Scheller et al., 1989).
The precise mechanism of action of propolis on cells
from the immune system remains unknown (Ansorge
et al., 2003). However, it is known that artepillin C,
found in large scale in the green propolis sample used,
acts on macrophages stimulating the production of IL-
12 (Sforcin et al., 2002), which potentiates immuno-
globulin production by B cells. Other phenolic
compounds, such as cinnamic acid derivatives found
in the propolis sample used, also induce production and
releasing of cytokines like IL-1, IL-6 and IL-8 by
activated macrophages, stimulating antibody produc-
the increment in the humoral response observed in the
present study.Another hypothesissuggeststhatpropolis
can reduce lipid peroxidation and stimulate the immune
system by means of direct lymphocyte activation
(Kimoto et al., 1998).
Besides increasing humoral immune response of
cattle vaccinated with BoHV-5, the use of 40 mg/dose
of an ethanolic green propolis extract also increased the
percentage of animals with titers higher than 32
(Table 2). According to Lazarowics et al. (1983),
bovines with titer equal or higher than 32 resist
challenge with field virus. This fact is yet more relevant
considering that many farmers do not use vaccines in a
prophylactic way, but only after positive diagnosis of
the disease in the farm, which implies the presence of
the virus in the herd. Even though cellular immunity is
more important in the case of BoHV primary infection,
humoral immunity is more effective in preventing the
resurgence of the disease, once BoHV remains latent in
the animal after infection (Babiuk et al., 1996). As
comparative parameter, the United States Department
80% of vaccinated animals with titer equal toor above 8
Due to the increase of neutralizing antibody titers, in
addition to the percentage of animals with high titers,
further studies in the development of this vaccine can
take into consideration the possibility of increasing the
interval betweenvaccinations, as well as decreasing the
amount of antigen per dose. These approaches, besides
in the handling of the animals.
The data presented in this study showed that an
ethanolic extract of green propolis increased the
potency of the humoral immune response in cattle
when associated with an inactivated oil vaccine against
BoHV-5, showing adjuvant action. Besides the incre-
ment in neutralizing antibody titers, there was an
increase in the percentage of animals with titers higher
than 32. Propolis compounds responsible for the
immunostimulating activities have not yet been
determined, but the flavonoids and other phenolic
compounds, like artepillin C, found in large quantity in
the sample studied, could have been the main
substances with action on the immune system. The
use of ethanolic extract of green propolis can contribute
for the efficacy of inactivated vaccines, acting as an
The authors would like to thank the Conselho
Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico
(CNPq) for financial support; Nectar Pharmaceutical
Ltd., Belo Horizonte, MG, Brazil (for supplying the
propolis); Prof. Jose ´ Vasconcelos Arnoni, for supplying
the animals; Mr. Jose ´ Carlos Ro ¨sler Sandrini and Ms.
Enilda Souza de Oliveira for the technical support.
Ansorge, S., Reinhold, D., Lendeckel, U., 2003. Propolis and some of
its constituents down-regulate DNA synthesis and inflammatory
cytokine production but induce TGF-b1 production of human
immune cells. Z. Naturforsch. 58c, 580–589.
herpesvirus 1 infection. Vet. Microbiol. 53, 31–42.
Bankova, V.S., Castro, S.L., Marcucci, M.C., 2000. Propolis: recent
advances in chemistry and plant origin. Apidologie 31, 3–15.
Bankova, V., 2005. Recent trends and important developments in
Barr, T.A., Carlring, J., Heath, A.W., 2006. Co-stimulatory agonists as
immunological adjuvants. Vaccine 24, 3399–3407.
as novel vaccine adjuvants: effect of the chemical composition.
Vaccine 23, 743–754.
of their modes of action. Vaccine 15, 246–256.
De Campos, R.O., Paulino, N., da Silva, C.H., Scremin, A., Calixto,
J.B., 1998. Anti-hyperalgesic effect of an ethanolic extract of
propolis in mice and rats. J. Pharm. Pharmacol. 50, 1187–1193.
De Castro, S.L., 2001. Propolis: biological and pharmacological
activities. Therapeutic uses of this bee-product. Annu. Rev.
Biomed. Sci. 3, 49–83.
G. Fischer et al./Veterinary Immunology and Immunopathology 116 (2007) 79–84 83
Dimov, V., Ivanovska, N., Bankova, V., Popov, S., 1992. Immuno- Download full-text
modulatory action of propolis: IV. Prophylactic activity against
Gram-negative infections and adjuvant effect of the water-soluble
derivate. Vaccine 10, 817–823.
Estrada, A., Katselis, G.S., Laarveld, B., Barl, B., 2000. Isolation and
evaluation of immunological adjuvant activities of saponins from
Polygala senega L. Comp. Immunol. Microbiol. Infect. Dis. 23,
Fenner, F.J., Gibbs, E.P., Murphy, F.A., Rott, R., Studert, M.J., White,
Havsteen, B.H., 2002. The biochemistry and medical significance of
the flavonoids. Pharmacol. Ther. 96, 67–202.
House, J.A., Baker, J.A., 1971. Bovine herpesvirus IBR-IPV. The
antibody virus neutralization reaction. Cornell Vet. 61, 320–335.
Jansen, T., Hofmans, M.P.M., Theelen, M.J.G., Manders, F., Schijns,
V.E.J.C., 2006. Structure and oil type-based efficacy of emulsion
adjuvants. Vaccine 24, 5400–5405.
Kimoto, T., Arai, S., Kohguchi, M., Aga, M., Nomura, Y., Micallef,
M.J., Kurin, M., Mito, K., 1998. Apoptosis and suppression of
tumor growth by artepillin C extracted from brazilian propolis.
Cancer Detect. Prev. 22, 506–515.
Kujumgiev, A., Tsvetkova, I., Serkedjieva, Yu., Bankova, V., Christov,
R., Popov, S., 1999. Antibacterial, antifungal and antiviral activity
of propolis of different geographic origin. J. Ethnoparmacol. 64,
Lazarowics, M., Steck, F., Ackermann, M., Kihm, U., 1983. Pru ¨fung
Schweiz. Arch. Tierheilk. 125, 797–808.
Leclerc, C., 2003. New approaches in vaccine development. Comp.
Immunol. Microbiol. Infect. Dis. 26, 329–341.
Lindblad, E.B., 2000. Freund’s adjuvants. In: O’Hagan, D.T. (Ed.),
Vaccine Adjuvants. Humana Press, Totowa, NJ, pp. 49–63.
Marcucci, M.C., 1995. Propolis: chemical composition, biological
properties and therapeutic activity. Apidologie 26, 83–99.
Marcucci, M.C., Ferreres, F., Garcı ´a-Viguera, C., Bankova, V.S., De
Castro, S.L., Dantas, A.P., Valente, P.H.M., Paulino, N., 2001.
Phenolic compounds from Brazilian propolis with pharmacologi-
cal activities. J. Ethnopharmacol. 74, 105–112.
Mayr, A., Bachmann, P.A., Bibrack, B.M., Withmann, G., 1982.
Virologische Arbeitsmethoden – Band IV – Sicherheit bei viro-
logischen arbeiten – Biometrische Methoden. Gustav Fischer
Verlag, Stutgart, p. 666.
Miyataka, H., Nishiki, M., Matsumoto, H., Fujimoto,T., Matsuka, M.,
Isobe, A., Satoh, T., 1997. Evaluation of propolis. I: Evaluation of
Brazilian and Chinese propolis by enzymatic and physico-chemi-
cal methods. Biol. Pharm. Bull. 20, 496–501.
Orsi, R.O., Funari, S.R.C., Soares, A.M.V.C., Calvi, S.A., Oliveira,
S.L., Sforcin, J.M., Bankova, V., 2000. Immunomodulatory action
of propolis on macrophage activation. J. Venom. Anim. Toxins 6,
Orsolic, N., Terzic, S., Sver, L., Basic, I., 2005. Polyphenolic com-
pounds from propolis modulate immune responses and increase
host resistence to tumor cells. Food Agric. Immunol. 16, 165–
Ozkul, Y., Silici, S., Eroglu, E., 2005. The anticarcinogenic effect of
propolis in human lymphocytes culture. Phytomedicine 12, 742–
Paulino, N., Scremin, F.M., Raichaski, L.B., Marcucci, M.C., Scre-
min, A., Calixto, J.B., 2002. Mechanisms involved in the relaxant
actionof the ethanolicextract of propolisin theguinea-pig trachea
in-vitro. J. Pharm. Pharmacol. 54, 1–9.
Scheller, S., Owczarek, S., Krol, W., Malinowska, B., Nikodemowicz,
E., Aleksandrowicz, J., 1989. Immunisierungsversuche bei zwei
fallen von alveolitis fibroticans bei abnehmender leistungsfahig-
keit des immunsystems unter anwendung von propolis-athanolex-
trakt(EEP),esberitoxNund eines calcium-magnesium-praparates
(dolomit.). Heikunst 102, 249–255.
Sforcin, J.M., Kanero, R., Funari, S.R.C., 2002. Absence of seasonal
effect on the immunomodulatory action of brazilian propolis on
natural killer activity. J. Venom. Anim. Toxins 8, 19–29.
Sforcin, J.M., Orsi, R.O., Bankova, V., 2005. Effect of propolis, some
isolatedcompoundsand itssourceplanton antibodyproduction.J.
Ethnopharmacol. 98, 301–305.
Singh, M., O’Hagan, D.T., 2002. Recent advances in vaccine adju-
vants. Pharm. Res. 19, 715–728.
Storni, T., Ku ¨ndig, T.M., Senti, G., Johansen, P., 2005. Immunity in
response to particulate antigen-delivery systems.Adv. Drug Deliv.
Rev. 57, 333–355.
USDA, 2005. Animal and Plant Health Inspection Service. USDA,
G. Fischer et al./Veterinary Immunology and Immunopathology 116 (2007) 79–8484