The Effects of Propolis in Animals Exposed Oxidative Stress

Abstract

This chapter expresses the effects of propolis on oxidative stress in animals. The term
“stress” was first coined by the endocrinologist Hans Selye (1936) more than 70 years ago to
define the physiological adaptive responses of the organism to emotional or physical threats
(stressors), whether real or perceived (Selye, 1936). Factors causing stress include
physiological factors, such as climate, environment, nutrition, and diseases, and physical
conditions, such as cage density and transport. Under stress, rapid and temporary changes
occur in the body initially; with continuous stress, these are followed by permanent and
irreversible changes (Tatli Seven, 2008). Stress responses are characterized as primary,
secondary and tertiary. The primary stress response is a neuroendocrine response leading to
corticosteroid and catecholamine release. The secondary stress response includes changes in
plasma and tissue ion and metabolite levels induced by neuroendocrine hormones. The
changes in disease resistance, growth, condition factor, and behaviors at a whole organism
level are tertiary responses (Wedemeyer et al., 1990). Finally, a decline in yield and
resistance to diseases may occur. Animals under stress become ill more easily, and excess
medicine may be necessary to maintain health. As a result, drug residues increase in animal
products and threaten public health directly. Stock health and welfare management are key
factors in animal health and food safety. For this reason, stress conditions in animals need to
be examined carefully (Tatli Seven, 2008). Reactive oxygen species (ROS) are chemically
reactive molecules containing oxygen. During times of environmental stress (e.g. ultraviolet
or heat exposure, environmental pollutant), ROS levels can increase dramatically.This may
result in significant damage to cell structures.This cumulates into a situation known as
oxidative stress. This chapter was written to demonstrate the importance of propolis that have effects
antioxidant, antibacterial, antitumor, anti-inflammatory etc. in animals under oxidative
stress.

Full text

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The Effects of Propolis in Animals
Exposed Oxidative Stress
Pinar Tatli Seven1, Seval Yilmaz2,
Ismail Seven3 and Gulizar Tuna Kelestemur4
1University of Firat, Faculty of Veterinary Medicine,
Department of Animal Nutrition and Nutritional Diseases, Elazig,
2University of Firat, Faculty of Veterinary Medicine,
Department of Biochemistry, Elazig,
3University of Firat, Vocation School of Sivrice,
Department of Beekeping, Elazig,
4University of Firat, Faculty of Fisheries,
Department of Aquaculture, Elazig,
Turkey
1. Introduction
This chapter expresses the effects of propolis on oxidative stress in animals. The term
“stress” was first coined by the endocrinologist Hans Selye (1936) more than 70 years ago to
define the physiological adaptive responses of the organism to emotional or physical threats
(stressors), whether real or perceived (Selye, 1936). Factors causing stress include
physiological factors, such as climate, environment, nutrition, and diseases, and physical
conditions, such as cage density and transport. Under stress, rapid and temporary changes
occur in the body initially; with continuous stress, these are followed by permanent and
irreversible changes (Tatli Seven, 2008). Stress responses are characterized as primary,
secondary and tertiary. The primary stress response is a neuroendocrine response leading to
corticosteroid and catecholamine release. The secondary stress response includes changes in
plasma and tissue ion and metabolite levels induced by neuroendocrine hormones. The
changes in disease resistance, growth, condition factor, and behaviors at a whole organism
level are tertiary responses (Wedemeyer et al., 1990). Finally, a decline in yield and
resistance to diseases may occur. Animals under stress become ill more easily, and excess
medicine may be necessary to maintain health. As a result, drug residues increase in animal
products and threaten public health directly. Stock health and welfare management are key
factors in animal health and food safety. For this reason, stress conditions in animals need to
be examined carefully (Tatli Seven, 2008). Reactive oxygen species (ROS) are chemically
reactive molecules containing oxygen. During times of environmental stress (e.g. ultraviolet
or heat exposure, environmental pollutant), ROS levels can increase dramatically.This may
result in significant damage to cell structures.This cumulates into a situation known as
oxidative stress.

Oxidative Stress – Environmental Induction and Dietary Antioxidants
268
This chapter was written to demonstrate the importance of propolis that have effects
antioxidant, antibacterial, antitumor, anti-inflammatory etc. in animals under oxidative
stress.
2. Oxidative stress
Oxidative Stress is a general term used to describe the effect of oxidation in which an
abnormal level of ROS, such as the free radicals (e.g. hydroxyl, nitric acid, superoxide) or the
non-radicals (e.g. hydrogen peroxide (H2O2), lipid peroxide) lead of damage (called
oxidative damage) to specific molecules with consequential injury to cells or tissue.
Oxidative stress is caused by an imbalance between the production of reactive oxygen and a
biological system's ability to readily detoxify the reactive intermediates or easily repair the
resulting damage (Bulger & Helton, 1998). Oxidative stress occurs when the generation of
ROS in a system exceeds the system’s ability to neutralize and eliminate them. The
imbalance can result from a lack of antioxidant capacity caused by disturbance in
production, distribution, or by an over-abundance of ROS from an environmental or
behavioral stressor. This damage can affect a specific molecule or the entire organism. If not
regulated properly, the excess ROS can damage a cell’s lipids, protein or DNA, inhibiting
normal function. Because of this, oxidative stress has been implicated in a growing list of
diseases as well as in the aging process (Sies, 1985).
Fig. 1. Mechanisms of oxidative stress-induced cell damage (Agarwal et al., 2005).

The Effects of Propolis in Animals Exposed Oxidative Stress
269
ROS can impair lipids, proteins, carbohydrates and nucleotides, which are important parts
of cellular constituents, including membranes, enzymes and DNA. Radical damage can be
significant because it can generally proceed as a chain reaction (Chen and Pan, 1996; Wejil et
al., 1997). These radicals can damage cell membranes inducing lipid peroxidation of
polyunsaturated fatty acids in the cell membranes and other complexes (Fang et al., 2002;
Stephan et al., 1995) Malondialdehyde (MDA) is one of the the final products of lipid
peroxidation. The concentration of MDA is the direct evidence of toxic processes caused by
free radicals (Talas & Gulhan, 2009; Tatli Seven et al., 2009). Damaged lipids lead to rigid cell
membranes; oxidized cholesterol often leads to hardening of the arteries and poorly
repaired DNA chains lead to cell mutation (future generation of cells) as implicated in
cancer and aging. Scientific research has established that the root cause of more than seventy
chronic degenerative diseases is due to oxidative stress development, i.e. cell damage
caused by free radicals (Davies, 1995).
Fig. 2. Diseases related to oxidative stress (http://www.enzoprofessional.com/default).
The intensity of oxidative stress is determined by the balance between the rate at which
oxidative damage is induced (input) and the rate at which it is efficiently repaired and
removed (output). The balance provides certain steady-state ROS level (Lushchak, 2011).
The rate at which damage caused is determined by how fast ROS are generated and then
eliminated by endogenous defense agents called antioxidants. The rate at which damage is
removed depends on the efficiency of repair enzymes (Sies, 1985). Detoxification of ROS is
one of the prerequisites of aerobic life, and hence an elaborate antioxidant system has
evolved (Sies, 1991). Antioxidants are agents that scavenge ROS, prevent their formation, or
repair the damage they cause (Halliwell, 1991). Antioxidants are effective because they are
capable to donate their own electrons to free radicals. When a free radical gains the electron
from an antioxidant, it no longer attack the cell and the chain reaction of oxidation is broken.
After donating an electron, an antioxidant becomes a free radical by definition. Antioxidants

Oxidative Stress – Environmental Induction and Dietary Antioxidants
270
in this state are not harmful because they have the ability to accommodate the change in
electrons without becoming reactive. The body has an elaborate antioxidant defense system.
Antioxidants are produced within the body and can also be received from food such as
fruits, vegetables, seeds, nuts, meats, and oil. There are two lines of antioxidant defense
within the cell. The first line, found in the fat-soluble cellular membrane consists of vitamin
E, beta-carotene, and coenzyme Q. Of these, vitamin E is considered the most potent chain
breaking antioxidant within the membrane of the cell (Yilmaz et al., 2006). Inside the cell,
water soluble antioxidant scavengers are present. These include vitamin C, glutathione
(GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT)
(Atessahin et al., 2005).
This complex system consists of GSH, ancillary enzymes (such as glutathione reductase
(GR), glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PD)), metal-
binding proteins (transferrin, ceruloplasmin, and albumin), flavonoids, and urate (Sies, 1991;
Halliwell, 1994). In addition to those antioxidants herbs, such as bilberry, blueberry,
turmeric (curcumin), grape seed or pine bark extracts, licopen, propolis and ginkgo can also
provide powerful antioxidant protection for the body. Thus, there is a delicate balance
between the generation and elimination of oxidant agents, which may be beneficial or
deleterious to the organism. Consuming a wide variety of antioxidant enzymes, vitamins
and minerals may be the best way to provide the body with the most complete protection
against free radical damage (Sies & Masumoto, 1997; Yaralioglu Gurgoze et al., 2005; Yilmaz
et al., 2006).
Fig. 3. The formation of various types of reactive radicals in response to enhanced steady-
state ROS levels related to mucosal damage and antioxidative enzymes involved in
neutralization of free radicals (Czesnikiewicz-Guzik et al., 2007).
In order to protect against lipid peroxidation and oxidative damage, all living organisms
have evolved an interdependent antioxidant system that includes enzymatic and non-
enzymatic components in the liver and erythrocytes. The major antioxidant enzymes are
SOD, CAT, and GPx. GSH, melatonin, ceruloplasmin (Cp), and albumin are non-enzymatic

The Effects of Propolis in Animals Exposed Oxidative Stress
271
antioxidants. Antioxidant enzymes play a vital role in protecting cellular damage from the
harmful effects of ROS. In addition, the stimulation in lipid peroxidation decreases with the
addition of some antioxidant matters (Seven et al., 2010).
Fig. 4. Detoxification of ROS by the primary antioxidant enzymes (bold) (Kahlos, 1999). GR
and G6PD are ancillary enzymes of the antioxidant system.
3. Antioxidant supplements in animal diets
Antioxidant dietary supplement greatly helps in boosting the immune system and thus aids
in preventing the onset of degenerative diseases. Vitamin C antioxidant dietary supplement
is perhaps the most famous form of antioxidant available. Also known as ascorbic acid,
bottles or pills with this antioxidant dietary supplement can be found in any pharmacy or
health food store. Another popular form of antioxidant dietary supplement is vitamin E.
This antioxidant dietary supplement works best when taken with vitamin C as it seems that
both vitamins have synergistic effect when taken in combination. Besides vitamins,
antioxidant dietary supplements may be in the form of botanicals. Antioxidant sources are
rich of the flavonoid derivatives (polyphenols). Antioxidant polyphenols are chemical
compounds that are naturally found in plants. Their function is to hunt down free radicals
and neutralize them. In so doing, they not only prevent free radicals from causing damage
but repair any damages (www.webcontent.com/articles/52/1/Antioxidant-Dietary-
Supplement/Page1.html). Propolis is recently a most important dietary supplement as
antioxidant compound (Tatli Seven, 2008; Tatli Seven et al., 2008; Tatli Seven et al., 2009;
Seven et al., 2011).
3.1 Propolis and its properties
Propolis (bee glue) is an adhesive, dark yellow to brown colored balsam that smells like
resin. It is collected from the buds, leaves and similar parts of trees and other plants like
pine, oak, eucalyptus, poplar, chestnut, and so on by bees and mixed with their wax (Seven
et al., 2010). Propolis is not a new discovery. It has been used for folk medicine and foods
since ancient times in many parts of the world. The use of propolis goes back to ancient
times, at least to 300 BC, and it has been used as a medicine in local and popular medicine in
many parts of the world, both internally and externally. Egyptians, Greeks and Romans
reported the use of propolis for its general healing qualities and for the cure of some lesions
of the skin. Propolis has always been reputed as an anti-inflammatory agent and to heal

Oxidative Stress – Environmental Induction and Dietary Antioxidants
272
sores and ulcers. Ancient Egyptians used it to embalm their dead, and more recently it was
used during the Boer War for healing wounds and tissue regeneration (Ghisalberti, 1979).
However, its use continues today in remedies and personal products, and the list of
preparations and uses is endless. It is still one of the most frequently used remedies in the
Balkan States (Bankova, 2005), and it has only been in the last decades that scientists have
investigated its constituents and biological properties. Propolis is a complex resinous
material that honey bees (Apis mellifera) produce from the exudates of various plants.
Beeswax is derived from the buds and bark of certain trees and other plants. The substance
populus, a favour ingredient, has been confused with propolis. Etymologically, the Greek
word propolis means pro, for or in defense, and polis, the city, that is “defense of the hive”.
Bees use it to seal holes in their honeycombs, smooth out internal walls as well as to cover
carcasses of intruders who died inside the hive in order to avoid their decomposition
(Sforcin, 2007).
Fig. 5. Collecting propolis from plant of honeybee
Fig. 6. The propolis collecting trap
Fig. 7. Raw propolis

The Effects of Propolis in Animals Exposed Oxidative Stress
273
Compounds TB TBA TA TT
Aromatic alcohols
Benzyl alcohol 0.38 0.57 0.19 0.89
Phenyl ethanol 0.66 0.59 0.88 0.83
2-methoxy-4-vinylphenol — 1.74 — 0.24
2-napthalenemethanol 2.18 1.45 0.87 0.30
5-azulenementhanol 0.80 0.04 — —
1-naphtlenemethanol 1.20 0.50 — 1.09
Bisabolol-alpha — 0.20 0.53 0.33
2-phenanthrenol — 0.41 — —
Aromatic acids
Benzoic acid 0.96 1.20 0.53 4.30
Benzenepropanoic acid — — 0.04 —
4-pentenoic acid, 5-phenyl 2.40 — — 0.03
Ferulic acid — 0.60 — 0.12
Caffeic acid 1.20 0.44 0.05 0.61
2-propenoic Acid,3-phenyl 2.23 0.81 1.06 1.53
2-propenoic acid, 3-(4-methoxyphenyl) 1.21 0.39 0.32 0.16
1-phenanthrenecarboxylic acid 0.30 0.21 0.18 0.41
Aromatic aldehydes
Benzaldehyde — — 0.04 —
Cinnamic acid and its esters
Cinnamyl cinnamate 5.28 1.32 0.23 0.86
Benzyl cinamate 0.14 0.45 0.12 0.37
Benzyl benzoate 0.32 0.13 0.05 0.02
Cinnamic acids — — — —
1-3-hydroxy-4-methoxycinnamic acid 0.80 0.80 0.08 0.85
Fatty acids
Lauric acid — 0.07 — —
Myristic acid — 0.04 — 0.03
Palmitic acid 0.22 0.42 0.20 0.21
Oleic acid — 1.10 — 0.47
Stearic acid — 1.26 1.78 0.16
Linoleic acid 0.26 0.37 0.67 0.35
Linear hydrocarbons and their acids
Cyclohexadecane 0.18 0.75 0.10 2.10
Hexadecane — — — —
Nonadecane 0.40 0.18 — —
Octadecane — — 0.11 0.20
Octadecanoic acid 0.41 0.41 — —
Flavanone
Isalpinin 6.17 5.76 4.97 5.04
Pinocembrin 13.61 14.76 7.01 16.26
Pinostropin 13.06 11.45 4.46 2.26
Naringenin 6.20 1.40 0.90 6.20
40,5-dihydroxy-7-methoxyflavanone 1.79 — 0.84 0.69
Chrysin 1.45 2.29 3.11 9.86
3,40,7-trimethoxy flavanone — 0.31 0.12 0.51
Hexadecanol — 0.11 — —
Flavonones
Pinobanksin and its derivatives 4.3 11.5 8.3 7.6
Quercetin and its derivatives 5.1 6.2 9.1 1.1
Galangine and its derivatives 0.9 3.1 3.4 1.6
Apigenin and its derivatives 0.2 3.2 3.8 2.6
The yields of dry propolis extracts were; 44.80% (w/v) for Bartın (TBA), 36.63% (w/v) for Trabzon (TT),
31.58% (w/v) for Bursa (TB) and 20.51% (w/v) for Ankara (TA) using 96% ethanol as solvent.
Table 1. Chemical compositions of ethanol extract of Turkish propolis samples (% of total
ion current) (Uzel et al., 2005)

Oxidative Stress – Environmental Induction and Dietary Antioxidants
274
Propolis has antioxidative, cytostatic, antimutagenic and immunomodulatory properties.
These properties of propolis are based on its rich, flavonoid, phenolic acid and terpenoid
contents (Seven et al., 2010). Propolis antioxidant, antibacterial and antifungal properties,
combined with the fact that several of its constituents are present in food and/or food
additives, and are recognized as Generally recognized as Safe (Burdock, 1998), make it an
attractive candidate as a natural preservative in new food applications. This meets the
demand for natural antioxidants and antimicrobials, fuelled by the increasing consumer
awareness for natural, minimally processed foods with traditional preservatives absent or at
very low concentrations (Han & Park, 1995; Tosi et al., 2007). Most recent studies have
shown that natural preventive compounds have gained popularity day by day as some of
the widely used synthetic pharmaceuticals and therapeutics might have some undesirable
effects. One can think that certain natural food ingredients would be better and safer than
synthetic ones. Many of these compounds, such as plant phenolics, often exhibit antioxidant
activities; therefore the addition of these compounds into food products may be helpful to
the health of consumers and also to the stabilization of food products.
Due to the presence of some of these effective compounds such as flavonoids (flavones and
flavanones), phenolic acids and their esters in propolis and propolis extract, if the positive
physiological properties and the non-toxicity of the propolis sample are proven it could be
used as a mild antioxidant and preservative (Talas & Gulhan, 2009).
Characteristics Mean value
Balsam, % 57
Phenolics, % 28
Flavones and flavonols, % 8
Flavanones and dihydroflavonols, % 6
MIC, μg.mL−1 1 210
1 MIC (Minimum Inhibitory Concentration)
Table 2. Characteristics of poplar propolis samples, based on 114 samples (Popova et al.,
2007)
After administration to mice or to humans, propolis does not appear to have side effects
(Sforcin, 2007). Although few in number, some events of propolis allergy and contact
dermatitis have been informed (Callejo et al., 2001), differently from the common allergy to
honey, which contains allergens obtained flowers. Ethanol and water extracts of propolis
possess antiallergic action, restraining histamine release in rat peritoneal mast cells
(Miyataka et al., 1998). However, in higher concentrations (300 μg/ml), propolis directly
activated mast cells, promoting inflammatory mediator release, what could be linked to
allergic processes in propolis-sensitive individuals (Orsi et al., 2005). In a study (Kashkooli
et al., 2011), to determine the possible toxicity and side effects of propolis, fishes (Rainbow
Trout) were fed on diets containing 0, 0.5, 1.5, 4.5 and 9 g propolis/kg diet for 8 weeks. Their
results showed that all dosages induced no significant alterations in growth parameters and
the levels of total protein, albumin, globulin, low-density lipoprotein cholesterol, high-
density lipoprotein cholesterol, triglycerides and activities of glutamic pyruvic transaminase
(ALT), glutamic oxaloacetic transaminase (AST), alkaline phosphatase (ALP) and lactate
dehydrogenase (LDH), when compared to the control group. On the basis of their findings,
propolis is a non-toxic substance for Rainbow Trout and its long-term administration might

The Effects of Propolis in Animals Exposed Oxidative Stress
275
not have any side effects. Recently, the presence of radioactive particles in propolis samples
was investigated, since these particles may be concentrated in the soil, contaminating the
plants, insects and its products, and, consequently, humans as well. Cesium (Cs137) was not
found in the samples, and only natural radioactive particles such as potassium (K40) and
beryllium (Be7) were found. These data suggested that propolis may be studied as an
environmental pollution indicator in order to understand the soil–plant–bee–propolis chain
(Orsi et al., 2006).
Antioxidative effect of propolis extracts has been reported in different methods including
iodometric method, thiobarbituric acid (TBA) method and free radical scavenging ability
with reduction of radical diphenylpicrylhydrazyl (DPPH) (Mohammadzadeh et al., 2007),
but Mohammadzadeh et al. (2007) reported that the ferric reducing ability of plasma (FRAP)
assay the reagents are inexpensive and simple to prepare, results are fast and reproducible
and the equipment required is of a type commonly found in biochemical laboratories. FRAP
assay is based on ferric to ferrous ion reduction at low pH. In this method the ferric
reducing ability of antioxidant compound is measured. At low pH, ferric-tripyridyltriazine
(Fe+3-TPTZ) complex is reduced to the ferrous (Fe+2) blue colour complex with an absorption
maximum at 593 nm. Test conditions favour reduction of the complex and, thereby, colour
development, provided that an antioxidant is present (Mohammadzadeh et al., 2007).
3.2 The effects of propolis supplementation in animals
3.2.1 Antioxidant effects of propolis
Propolis contains about 300 constituents (Türkez et al., 2010). Latterly, propolis has gained
popularity in connection with oxidative stress (Tatli Seven, 2008; Tatli Seven & Seven, 2008;
Tatli Seven et al., 2008; Tatli Seven et al., 2009) and used widely in healthy drinks and foods
to recuperate health and prevent diseases such as inflammation, heart disease, diabetes and
even cancer (Burdock, 1998; Banskota et al., 2000). Because of such broad spectrum of
biological properties and their different uses, there is a renewed interest in its biological
activities. Several investigations on propolis in Eastern Europe and South America have
showed that flavonoids concentrated in propolis are powerful antioxidants which are
capable to scavenge free radicals (Basnet et al., 1997; Banskota et al., 2000).
Flavonoids of propolis are one of the most important compounds. Flavonoids are thought to
be responsible for many of its biological and pharmacological activities including anticancer,
anti-inflammatory, antimicrobial and antioxidant effects. Active free radicals, together with
other factors are responsible for cellular aging and many conditions such as cardiovascular
diseases, cancer, diabetes, arthritis, heat stress, environmental pollution (Tatli Seven et al.,
2008; Seven et al., 2010; Sforcin & Bankova, 2011). The antioxidant serves as a defensive
factor against free radicals in the body. Enzymes such as SOD, CAT and GPx are the main
system that opposes oxidation. If production free radicals overwhelm the capacity of
enzymatic system, the second line of defense (vitamins) may come to action (Tatli Seven,
2008; Tatli Seven et al., 2009). Such as antioxidants vitamins C and E extinguish free radicals
and become oxidized and non-active (Halliwell, 1994). Flavonoids and various phenolics in
propolis have been appeared to be capable of scavenging free radicals and thereby
defending lipids and other compounds such as vitamin C from being oxidized or destroyed
during oxidative damage (Tatli Seven et al., 2009). Besides, flavonoids inhibit lipid

Oxidative Stress – Environmental Induction and Dietary Antioxidants
276
peroxidation, platelet aggregation, capillary permeability and fragility, and the activity of
enzyme systems including cyclooxygenase (COX) and lipoxygenase (Havsteen, 2002).
Cardio protective effects have also been reported for flavonoids (Celle et al., 2004). Chopra
et al. (1995) reported that doxorubicin-induced cardiomyopathy in rats followed by
treatment with propolis induced a significant reduction of creatine phosphokinase, aspartate
aminotransferase, blood and tissue GSH levels and TBA-reactive substances. It was also
observed a decreased degeneration of cardiac fibers in propolis-treated rats, suggesting that
this effect could be due to flavonoids present in propolis composition (Pinchuk &
Lichtenberg, 2002).
Chyrisin is one of the propolis compounds which is has hepatoprotective and antioxidant
activities in rats (Sathiavelu et al., 2009). Benzoic acid derivate exhibits antioxidant effects
using inhibition assays of luminol luminescence, 2,2-diphenyl-1-picrylhydrazyl, and
lipoperoxidation. Particularly caffeic acid, caffeoylquinic acid and cinnamic acid are
effective O2.- scavenging activity (Christov et al., 2006; Nakajima et al., 2007).
Propolis is effective in neurotoxicity. Kwon et al. (2004) examined the effects of the
antioxidant propolis on seizures induced by kainic acid (KA) in rats. They found that KA
induced increases in the levels of MDA and protein carbonyl, and decreases in the ratio of
GSH/GSSG. In addition, the researchers determined that these changes in oxidative stresses
markers at neuronal degenerations were significantly attenuated by pretreatment with
propolis, and that the neuroprotective effects of propolis appeared to be counteracted by
adenosine receptor antagonists. Their results suggest that the protective effect of propolis
against KA-induced neurotoxic oxidative damage is, at least in part, via adenosine A1
receptor modulation. Thus, they postulate that propolis significantly blocks seizure-induced
neuronal loss by attenuating the impairment of GSH metabolism via, in part, adenosine A1
receptor modulation. The novel antioxidant/anticonvulsant effect could be an important
contribution to extending the neuroprotective potential of propolis.
A recent study (Mannaa et al., 2011), it was found that oral administration of propolis in
epileptic animals which received the anticonvulsant drug valproate, resulted in significant
improvements in the neurotransmitter levels in both hippocampus and in serum. The results
obtained in the mentioned study, regarding the lipid peroxide (LPO) level and total
antioxidant capacity (TAC) in the hippocampus homogenate of epileptic rats showed a
significant increase and a significant decrease in both parameters. This may be explained by
the fact that generalized epilepsy is a chronic disorder characterized by recurrent seizures
which can increase the content of ROS and superoxide generation in the brain. Free radical
generation can induce seizure activity by direct inactivation of glutamine synthase thereby
permitting an abnormal construct of excitatory neurotransmitter glutamic acid (Oliver et al.,
1990). The onset of oxygen-induced convulsions in animals correlated with a decrease in the
cerebral content of neurotransmitter gamma-aminobutyric acid (the main inhibitory
neurotransmitter) because of the inhibition of enzyme glutamate decarboxylase by oxygen
free radicals. Thus, it appears that free radicals may be responsible for the development of
convulsions. The mentioned study showed that propolis improved the effect of valproate on
LPO level towards the normal values (Mannaa et al., 2011).
Brain oxidative injury, resulting from excessive generation of free radicals, likely contribute
to the initiation and progression of epilepsy after brain injury. Therefore, antioxidant

The Effects of Propolis in Animals Exposed Oxidative Stress
277
therapies aimed attenuation of oxidative stress have received considerable attention in the
treatment of epilepsy. The researchers demonstrated that propolis possessed
neuroprotective effects both against neurotoxicity in cell cultures and against ischemic
neuronal damage (Shimazawa et al., 2005). The neuroprotective effects of propolis may be
related to its constituents, such as 3,4-di-O-caffeoylqunic acid, 3,5-di-O-caffeoylqunicacid
and/or p-coumaric acid (Inokuchi et al., 2006).
Propolis effects were analyzed on macrophages of BALB/c mice submitted to
immobilization stress as well as on the histopathological analysis of the thymus, bone
marrow, spleen and adrenal glands. Stressed mice showed higher H2O2 generation by
peritoneal macrophages, and propolis treatment (200 mg/kg) potentiated H2O2 generation
and inhibited nitric oxide production by these cells. Histopathological analysis of stressed
mice showed no alterations in the thymus, bone marrow and adrenal glands, but an increase
in germinal centers in the spleen was seen. Propolis treatment counteracted the alterations
found in the spleen of stressed mice (Missima & Sforcin, 2008).
The chemical content of Turkish propolis was investigated with the focus on protective
effect against alcohol-induced oxidative stress (Kolankaya et al., 2002). The authors declared
that the ethanolic propolis extract of propolis, at dose of 200 mg/kg body weight/day, was
given, by gavage, to male rats for 15 days. It was found that HDL level decreased and LDL
level increased in the alcohol group, while HDL level increased and LDL level decreased in
the alcohol+propolis group. There were decreases in cholesterol and triglyceride levels in
the alcohol+propolis group. Also, there were decreases in activities of serum ALP and AST,
but increases in LDH activity in the propolis treated group compared to the alcohol group.
No toxic effects of Turkish propolis were found, while it caused an increase in HDL level
and a decrease in LDL level. They suggest that these effects are protective against
degenerative diseases and against alcohol-induced oxidative stress via free radicals
(Kolankaya et al., 2002).
Heat stress is an important stressor resulting in the reduced welfare of birds. Heat stress
increased lipid peroxidation as a consequence of increased free radical generation. The rise
of lipid peroxidation increases the MDA level in blood and tissues (Tatli Seven et al., 2009).
Tatli Seven et al. (2009) were found that heat stress-induced oxidative stress was indicated
by increased plasma, liver and muscle MDA levels. Dietary propolis and vitamin C
supplementation significantly decreased plasma, liver and muscle MDA levels. It may be
considered that dietary vitamin C and high dose of propolis attenuated lipid peroxidation.
Besides, 3 g/kg dietary propolis was found to be more effective than dietary vitamin C, on
especially liver and muscle MDA levels. Likewise, Okonenko et al. (1988) reported that
propolis had more pronounced antioxidant action compared to that of vitamin E that has a
similar activity to vitamin C. Living organisms are able to adapt to oxidative stress by
inducing the synthesis of antioxidant enzymes and damage removal/repair enzymes (Tatli
Seven et al., 2009).
Antioxidant enzyme activities such as SOD and CAT under stimulation of lipid
peroxidation may sometimes decrease (Wohaieb & Godin, 1987; Ozkaya et al., 2002) or
increase (Huang et al., 1999; Aliciguzel et al., 2003). The increase of antioxidant enzyme
activities such as SOD, CAT and GSH may be considered as a protective mechanism against
heat-induced free radical production and lipid peroxidation (Tatli Seven et al., 2009).

Oxidative Stress – Environmental Induction and Dietary Antioxidants
278
Exposure of broilers to heat stress resulted in a significant increase in SOD and CAT (Altan
et al., 2003). Moreover, significant differences between enzymes were obtained in
antioxidant enzyme responses to heat treatment. A similar response has been reported in
many human diseases, in which MDA concentrations increased concomitantly with an
increase in antioxidant enzyme activities. McArdle and Jackson (2000) have also
demonstrated a significant increase in free radical production together with an increase in
the expression of antioxidant enzymes during a period of non-damaging exercise in muscle.
These increases in antioxidant enzyme activities have been considered as a protective
response against oxidative stress (Altan et al., 2003). A previous study (Okutan et al., 2005),
investigated the effects of caffeic acid phenethyl ester (CAPE) which is a component of
propolis on lipid peroxidation and antioxidant enzymes in diabetic rat heart. They found
that in untreated diabetic group, the SOD activities and CAT levels have significantly
decreased, while GSH-Px activity was increased in the CAPE-treated diabetic rats compared
to those observed in untreated diabetic rats. The GSH-Px activities in blood, liver and
kidneys of heat stressed birds were significantly reduced, while SOD, CAT and GSH
activities were increased in blood and some tissues. This may be explained by GSH-Px
inhibition at increased free radical levels in tissues (Nakazawa et al., 1996). It can be
concluded that in broilers heat stress induced oxidative stress in blood and tissues. Dietary
propolis decreased lipid peroxidation and regulated antioxidant enzymes activities in the
broilers exposed to heat stress. The protective role of propolis might be related to its
antioxidant effect. Researchers suggest that propolis and especially propolis in dose
supplemented 3 mg/kg diet might be considered to prevent oxidative stress in the broilers
exposed to heat stress (Tatli Seven et al., 2009).
Heavy metal pollution is provoked cardio toxicity, nephrotoxicity and neurotoxicity and
they are show pro-oxidative effects. They demonstrate adverse effects, such as the
production of ROS, disruption of tissue oxidant/antioxidant balance, and alteration of lipid
metabolism (Seven et al., 2010; Türkez et al., 2010). For example, aluminum induced changes
in biochemical parameters, stimulated lipid peroxidation and decreased the activities of the
antioxidant enzymes in plasma and different tissues of male rabbits and rats. Also,
aluminum chloride caused deterioration in sperm quality, enhancement of free radical levels
and alterations in antioxidant enzymes in both in vivo and in vitro. The mechanisms of Al-
induced toxicity may be attributed to the potentiation of Fe+2 oxidationto Fe+3 to cause
oxidative damage (Xie & Yokel, 1996). Türkez et al. (2010) found effectiveness of propolis
(50 mg propolis/kg of body weight (BW)) in modulating the AlCl3 (34 mg AlCl3/kg BW)
was genotoxic and hepatotoxic in liver of rats. AlCl3 significantly increased the amount of
micronucleated hepatocytes ALP, activities of transaminases (AST and ALT) and LDH.
Furthermore, severe pathological damages such as sinusoidal dilatation, congestion of
central vein, lipid accumulation and lymphocyte infiltration were found in liver. On the
contrary, the researchers mentioned that treatment with propolis alone did not cause any
adverse effect on above parameters. The physiological effects of propolis in hepatocytes are
not clear; a hypothesis is that Al-induced genetic damage can be prevented by inductive
effects of propolis on antioxidant capacity. Because the toxic effects of Al appear to be
intervened, at least in part, by free radicals (Abubakar et al., 2003). As known, genetic
damages mainly develops related with oxidative stress. Propolis can be proposed to prevent
Al toxicity as a nutritional supplement or a functional food component (Türkez et al., 2010).

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Seven et al. (2010) investigated the effects of propolis in broilers exposed to lead-induced
oxidative stress. The authors found that the addition of lead increased the plasma MDA
level. The authors found that the addition of propolis significantly decreased blood SOD
activity and the CAT activity of heart tissue. The researchers suggested that propolis (1
g/kg) supplementation in broiler diets might overcome the adverse effects of oxidative
stress induced dietary lead. Antioxidant effects of propolis are based on flavonoids and
CAPE. It was reported that CAPE decreased MDA levels by blocking ROS production as an
antioxidant (Seven et al., 2010).
Propolis supports liver metabolism under oxidative stress. Seven et al. (2010) observed that
dietary propolis significantly decreased the blood triglyceride levels in the lead
supplemented group. According to this finding, the decrease in triglyceride level of propolis
might indicate that the addition of propolis relieved the adverse effects on triglyceride level
of oxidative stress. Seven et al. (2010) suggested that using 1 g/kg of propolis
supplementation in maize soybean meal type broiler diets may attenuate the adverse effects
of oxidative stress on the antioxidant defense system.
Heat stress stimulates lipid peroxidation as a consequence of increased free radical generation.
The increase in lipid peroxidation decreases antioxidant levels such as vitamin C and vitamin
E in tissues (Tatli Seven et al., 2008). Performance of animals in heat stress is decreased (Tatli
Seven, 2008; Tatli Seven et al., 2008; Seven et al., 2011). Antioxidants such as vitamin C,
vitamin E and propolis are used poultry diet because of their anti-stress effects and because
their levels is reduced during heat stress (Tatli Seven, 2008; Tatli Seven et al., 2008). Propolis
prevented negative effects caused by heat stress on performance, digestibility and egg qualities
(Tatli Seven, 2008). The authors reported that supplementation with propolis (5 g/kg diet) was
the most efficient treatment, and increased feed intake and improved hen day egg, egg weight
and digestibility (of dry matter, organic matter, crude protein (CP) and ether extract) in laying
hens. Tatli Seven (2008) explained that the positive results appeared due to palatable and
antioxidant properties of propolis. Especially, effects on performance and digestibility of
propolis dietary supplementation may appear more powerful under stress. Moreover, it was
declared that propolis supplementation increased egg shell thickness and egg shell weight in
heat stressed laying hens. It was due to improved calcium digestibility and absorption
resulting from the acid derivates such as benzoik, 4-hydroxy-benzoic, etc., which are found in
propolis (Haro et al., 2000; Tatli Seven, 2008).
3.2.2 Antimicrobial, anti-inflammatory and antitumor effects of propolis
In addition to antioxidant properties, propolis demonstrate other beneficial effects.
Especially its antibacterial, anti-inflammatory and antitumor effects are very important for
human and animal health, and animal production.
Itavo et al. (2011) indicated that propolis is an alternative to the use of dietary antibiotics.
Propolis has bacteriostatic activity against gram-positive and some gram-negative bacteria.
The action of propolis is likely related to changes in the bioenergetic status, which inhibits
bacterial motility. This is similar to the action of ionophores. The chemical composition of
propolis is complex and variable because it is intrinsically related to the floristic and
ecological composition of the environment visited by the bees. The combination of these
factors affects the pharmacological properties of propolis, which is in fact classified into
different types such as brown, green and red propolis.

Oxidative Stress – Environmental Induction and Dietary Antioxidants
280
Tatli Seven & Seven (2008) reported that propolis stimulated immune system and decreased
mortality rate by improving immunity in broilers. They remarked that propolis
supplementation in poultry diets as alternative to antibiotics may be recommended in
broilers in heat stress conditions.
Epidemiological studies provide evidences that propolis protect humans against cancer, and
also results from animal experiments and in vitro microorganism manipulations showed its
efficiency to reduce pathogenic bacteria. Moreover, propolis showed substantial protection
against cariogenic bacteria and oral pathogens suggesting its valuable clinical use. Kouidhi et
al. (2010) declared that Tunisian propolis presented potential activity against oral Streptococci
causing dental diseases as well as different kind of cancer cell proliferations. Those excellent
activities could be due to specific bioactive compounds of the Tunisian propolis.
Also, propolis is effective on the rumen environment in ruminants. Brodiscou et al. (2000)
determined that propolis affected fermentation and methanogenesis of continuous microbial
culture in ruminants; it decreased protozoa population and raised propionate levels by
10.3%. However, some negative propolis effects could be related to high flavonoid levels
(14.9 g/kg propolis) reported (Itavo et al., 2011). Lambs fed by the diet supplemented with
propolis spent more time resting and less time ruminating. Researchers (Itavo et al., 2011)
explained that this may have had a negative on digestibility because this diet provided the
highest food intake with the worst feed conversion. The higher flavonoid and phenol
content of their diet was probably toxic to the ruminal environment of lambs, which
accelerated food passage through the gastrointestinal tract, causing lower nutrient
absorption and increased dry matter intake, corroborating Van Soest (1994). Rumination rate
was therefore higher in lambs fed propolis because of their increased neutral detergent fiber
intake and reduced rumination time. However, improvement in rumination rate itself was
not enough to raise the productivity of these lambs, which had the lowest weight gain.
Rumination quality is essential to optimize food use, but this was apparently compromised
by the high flavonoid and total phenolic content of propolis, resulting in reduced lamb
performance (Van Soest, 1994).
Propolis also affects bone metabolism (Amany et al., 2008). In the study, it was used orally
fish liver oil and propolis as protective natural products against the effect of the antiepileptic
drug valproate on immunological markers of bone formation in rats. Propolis increased the
bone formation markers and decreases the bone resorption ones. it also increased the
osteoprotegrin and decreased tumor necrosis factor-alpha-α (TNF-α), and NF kappa-B
ligand which inhibited the osteoclastogenesis. The researchers recommended the use of
propolis as a prophylactic treatment for epileptic patients using valproate against the side
effect of valproate on bone.
Propolis is effective in inflammation-related diseases such as rheumatoid arthritis (RA). The
main characteristic of RA is the ongoing damage in arthrosis of cartilage and bone, and at
the same time with a disturbance of immune function. In the context of RA, there exist
neutrophils, activation macrophages, lymphocytes and other elements associated with the
abduction, activation and releasing of cytokine, which is perhaps one of the mechanisms of
RA development (Hu et al., 2005). In the case of RA, the concentration of cytokine derived
from T cells was generally low, whereas that of mononuclear macrophage levels were
significantly higher (Shuyun, 1996). Propolis inhibited the increase of inflammatory medium
and decreased the activation and inducing effects of cytokines, which indicated that both
extracts exhibited the same anti-inflammatory effects (Hu et al., 2005).

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281
Propolis was used in a tumor event (Nada & Ivan, 2003). The tumor was a transplantable
mammary carcinoma of mouse. Metastases in the lung were induced by injection of 2x105
viable tumor cells i.v. and propolis was given intraperitoneally at doses of 50 or 150 mg/kg
before or after tumor cell inoculation. Researchers (Nada & Ivan, 2003) demonstrated that
therapies reduced the number of metastases in the lung and tumor growth was suppressed
significantly by propolis. They commented that it is likely antimetastatic activity of the
propolis is mainly mediated by immunomodulatory activity. Flavonoids in propolis
stimulated macrophages to produce lymphocyte activating factor, a factor relevant for
control of immune cell cooperation.
The observed anti-inflammatory effects of propolis have been attributed to its flavonoid,
phenolic acid and caffeic acid contents. Flavonoids were reported to inhibit the activity of
enzymes involved in the conversion of membrane polyunsaturated fatty acids such as
phospholipase A2, COX and lipoxygenase, to inhibit the release of lysosomal enzymes from
rabbit polymorphonuclear leucocytes and scavenge free radicals. Aqueous extracts of
propolis were formed to have inhibitory effects on dihydrofolate reductase similar to the
well-known non-steroidal anti-inflammatory drugs. CAPE, which is an active component of
propolis extract, was found to inhibit 5-lipoxygenase in micro molar concentrations, and to
block the production of ROS in neutrophils and xanthine/xanthine oxidase system. It was
also believed to contribute to the anti-inflammatory activity of propolis by being both a
lipoxygenase–cyclooxygenase inhibitor and an antioxidant (Onlen et al., 2007).
Sy et al. (2006) investigated the activities of propolis using an OVA-induced asthma
animal model. Mice were immunized and sensitized by exposure to ovalbumin (OVA)
antigen and administered with low (65 mg/kg body weight) and high-dose (325 mg/kg
body weight) propolis water extracts by tube feeding. The serum OVA-specific IgE titer
and cytokine profiles in cultured splenocytes and bronchoalveolar lavage fluids (BALF)
were analyzed. The number of eosinophils in BALF was counted. Here we demonstrate
that propolis extracts can suppress the serum levels of OVA-specific IgE and IgG1, and
airway hyper responsiveness in OVA-sensitized mice. Results suggest that propolis
extracts may be a potential novel therapeutic agent for asthma. CAPE, an anti-
inflammatory component of propolis, is known to be an inhibitor of nuclear factor-kappa
B and significantly reduces the levels of pro-inflammation cytokines (TNF-α and
Interleukin 1, beta) in rats (Fitzpatrick et al., 2001).
CAPE is essential for the anti-inflammatory activity of propolis. Because CAPE derivatives
are more lipophilic, thus easily facilitate their entry into cells (Michaluart et al., 1999). It has
been reported that (Michaluart et al., 1999) CAPE, possesses anti-inflammatory activity by
inhibiting the release of arachidonic acid from cell membrane, suppressing the enzyme
activities of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), and inhibiting the
activation of COX-2 gene expression. Propolis with CAPE and CAPE produce a significant
inhibition of both exudate volume and leukocytes migration induced by carrageenin
injection into the pleural cavity (Borrelli et al., 2002). Furthermore, the observed anti-
inflammatory activity of propolis appears to be due to the presence of flavonoids, especially
galangin. Indeed, this flavonoid has shown to inhibit COX and lipooxygenase activity,
decrease prostaglandin E-2 release and the expression of the inducible isoform of COX- 2
(Gabor & Razga, 1991; Raso et al., 2001).

Oxidative Stress – Environmental Induction and Dietary Antioxidants
282
4. Conclusion
As a result, propolis began to attract the attention of scientists extremely (Table 3). It is used
in researches related to important diseases such as cancer and diabetes. Besides, propolis is
used as diet supplement in poultry researches. Many animal researches’s results showed
that propolis might be relieve the negative effects of oxidative stress. But, new researches
should be made related to propolis.
BWG: Body Weight Gain; DMI: Dry Matter Intake; DWG: Daily Weight Gain; EEP: Ether Extract of
Propolis; EST: Egg Shell Thickness; ESW: Egg Shell Weight; FCR: Feed Convertion Ratio; FI: Feed
Intake; HEP: Hen-day Egg Production; NDFI: Neutral Detergent Fiber Intake; SGR: Specific Growth
Rate; GST: Glutathione S-Transferase; TAC: Total Antioxidant Capacity; BW: Body Weight; CP: Crude
Protein; CAPE: CAT; (+): Significatly Increased; (-): Significatly Decreased.
Table 3. The Effects of Propolis in Animals

The Effects of Propolis in Animals Exposed Oxidative Stress
283
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