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Characterization of Honey Bee by Product; Propolis as an
Antimicrobial and Nutraceutical Agent in Food Applications
By
MUHAMMAD SHAHBAZ
M.Sc. (Hons.) Food Technology
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in
Food Technology
National Institute of Food Science and Technology
Faculty of Food, Nutrition and Home Sciences
University of Agriculture Faisalabad
2015
iii
DECLARATION
I hereby declare that the contents of the thesis “Characterization of honey bee by product;
propolis as an antimicrobial and nutraceutical agent in food applications" are creation of my
own research and no part has been copied from any published source (except the references,
standard mathematical or geometrical models/equations/formulae/protocols etc.). I further
declare that this work has not been submitted for award of any other diploma/degree. The
University may take action if information provided is found inaccurate at any stage. (In case
of default the scholar will be proceeded against as per HEC plagiarism policy).
Muhammad Shahbaz
2003-ag-1658
iv
To
The Controller of Examinations,
University of Agriculture,
Faisalabad.
We, the Supervisory Committee, certify that the contents and form of this thesis submitted by
Mr. MUHAMMAD SHAHBAZ, Reg. No. 2003-ag-1658 have been found satisfactory, and
recommend that it be processed for evaluation by the external examiner(s) for the award of
degree.
SUPERVISORY COMMITTEE:
Chairman:
(Prof. Dr. Tahir Zahoor)
Member:
(Dr. Muhammad Atif Randhawa)
Member:
(Dr. Haq Nawaz)
v
Oh, Allah Almighty open our eyes,
To see what is beautiful,
Our minds to know what is true,
Our heart to love what is Allah
My Worthy
Supervisor
His words of inspiration and encouragement in pursuit of excellence, still linger on.
vi
ACKNOWLEDGEMENT
I feel myself inept to regard the Highness of Almighty ALLAH, my words have lost their
expressions, knowledge is lacking and lexis scarce to express gratitude in the rightful manner
to the blessings and support of ALLAH Almighty who flourished my ambitions and helped
me to attain goals. I present my humble gratitude from the deep sense of heart to the Holy
Prophet MUHAMMAD (Peace Be Upon Him) that without him the life would have been
worthless.
I want to pay my thanks to Higher Education Commission (HEC), Pakistan for its support
and encouragement to complete my work. I am grateful to HEC for providing me financial
assistance in the form of indigenous scholarship and an esteemed opportunity of six month
foreign training (IRSIP) to expand my erudite exposure.
I am indebted to the sincere contribution of my honourable supervisor Prof. Dr. Tahir
Zahoor, National Institute of Food Science and Technology, University of Agriculture,
Faisalabad for his diligent cooperation and scrupulous support during the entire degree
program. I want to say thanks to my committee members Dr. Muhammad Atif Randhawa and
Dr. Haq Nawaz for their support and research oriented counselling. I am also obliged to Dr.
Kirsten Brandt, senior lecturer, School of Agriculture, Food and Rural Development,
Newcastle University, Newcastle Upon Tyne, UK for her tremendous support to carry out
studies in her laboratories. Lastly, I am grateful to my family and friends for their consistent
care and encouragement.
vii
CONTENTS
Sr #
Title Page #
1 INTRODUCTION
1
2 REVIEW OF LITERATURE
2.1. Bee propolis: An overview
2.2. Polyphenols: Bioactive moieties
2.2.1 Phenolic acids
2.2.2 Flavonoids
2.3. Natural antimicrobial agent; A new millennia perspective
2.4. Functional and nutraceutical foods; A concept of modern era
2.5. Metabolic syndromes and propolis
2.6. Oxidative stress management
2.7. Novel approach against hyperglycemia
2.8. Hyporcholesterolemic perspective of propolis
6
6
8
9
10
12
17
18
20
22
25
3 MATERIALS AND METHODS
3.1. Materials
3.2. Characterization of Honey Bee propolis
3.2.1. Compositional analysis
3.2.1.1. Moisture content
28
28
28
28
29
viii
3.2.1.2. Crude protein
3.2.1.3. Crude Fat
3.2.1.4. Crude fibre
3.2.1.5. Total ash
3.2.1.6. Nitrogen free extracts (NFE)
3.2.2. Mineral Analysis
3.3. Preparation of Propolis Extracts
3.4. Analysis of propolis extracts
3.4.1 Total Polyphenol contents (TPC)
3.4.2 Free radical scavenging activity of bee propolis (DPPH assay)
3.4.3. Antioxidant activity by β-carotene system of propolis extracts
3.5. High performance liquid chromatography (HPLC) analysis of
propolis extracts
3.6. Evaluation of antimicrobial potential of bee propolis
3.6.1. Antibacterial activity
3.6.2. Preparation of inoculum
3.6.3. Disc Diffusion assay
3.6.4. Determination of minimum inhibitory concentration
3.7. Product development
3.7.1. Functional/nutraceutical drink
3.7.2. Physicochemical analysis of functional drinks
29
29
29
29
29
29
30
31
31
31
31
32
32
32
32
33
33
33
33
34
ix
3.7.2.1. pH
3.7.2.2 Total acidity
3.7.2.3. Total Soluble Solids
3.7.2.4. Sensory evaluation
3.8. Efficacy studies
3.8.1. Study-I: Normal diet
3.8.2. Study II: High sucrose diet
3.8.3. Study-III: High cholesterol diet
3.8.4. Feed and drink intake
3.8.5. Body weight
3.8.6. Serum lipid profile
3.8.6.1. Total Cholesterol
3.8.6.2. High density lipoprotein
3.8.6.3. Low density lipoprotein
3.8.6.4. Triglycerides
3.8.7. Serum glucose and insulin levels
3.8.8. Liver functioning tests
3.8.9. Kidney functioning tests
3.9. Statistical analysis
34
34
34
34
35
35
36
36
37
37
37
37
37
37
37
37
38
38
38
4 RESULTS & DISCUSSION 39
x
4.1. Compositional analysis of bee propolis
4.2. Mineral contents of propolis
4.3. Analysis of honey bee propolis extracts
4.3.1. Total polyphenol contents (TPC) mg/g Gallic Acid Equivalent
(GAE)
4.3.2. Free radical scavenging activity and Antioxidant potential
4.3.3. Quantification of bioactive compounds through HPLC
4.4. Evaluation of Antimicrobial potential of propolis
4.4.1. Antimicrobial activity against E. coli ATCC-35218
4.4.2. Antimicrobial activity against B. subtilis ATCC-6633
4.4.3. Antimicrobial activity against S. aureus ATCC-25923
4.5. Functional drink analysis
4.8.1. Physicochemical analysis
4.6. Sensory evaluation
4.7. Bio-evaluation studies
4.7.1. Feed intake
4.7.2. Drink intake
4.7.3. Body weight
4.7.4. Blood Cholesterol
4.7.5. High density lipoprotein (HDL)
4.7.6. Low density lipoprotein (LDL)
39
41
41
41
45
47
50
50
52
52
57
57
62
66
67
68
74
77
80
85
xi
4.7.7. Triglycerides
4.7.8. Blood Glucose Level
4.7.9. Plasma Insulin concentration
4.7.10. Liver function tests
4.7.10.1. Aspartate aminotransferase (AST)
4.7.10.2. Alanine transaminase (ALT)
4.7.10.3. Alkaline phosphatase (ALP)
4.10.11. Renal functioning Tests
4.7.11.1. Urea
4.7.11.2. Creatinine
89
93
97
101
101
101
105
106
106
106
5 SUMMARY
110
6 RECOMMENDATIONS
114
7 LITERATURE CITED
115
8 APPENDICES
144
xii
List of Tables
Sr.No. Title Page
no.
3.1 Treatment plan of propolis extract 30
3.2 Treatments used for the preparation of functional drinks 34
3.3 Different studies conducted during efficacy trials 36
3.4 Diets and functional drinks plan 36
4.1 Compositional analysis of bee propolis
40
4.2 Mineral contents of bee propolis
42
4.3 Mean square values for the effect of treatments on the antioxidant indices
of the extracts
44
4.4 Mean values for the effect of treatments on TPC, free radical scavenging
activity and antioxidant potential
44
4.5 Polyphenols quantification of extracts through HPLC (mg/Kg)
49
4.6 Mean values showing effect of propolis on zone of inhibition (mm) extent
against Escherichia coli
51
4.7 Mean values showing effect of propolis extract on Minimum Inhibitory
Concentration (MIC) against Escherichia coli
51
4.8 Mean values showing effect of propolis extract on zone inhibition extent 53
xiii
against Bacillus subtilis (mm)
4.9 Mean values showing effect of propolis extract on Minimum Inhibitory
Concentration (MIC) against Bacillus subtilis (µg/mL)
53
4.10 Mean values showing effect of propolis extract on zone Inhibition extent
against Staphylococcus aureus (mm)
54
4.11 Mean values showing effect of propolis extract on Minimum Inhibitory
Concentration (MIC) against Staphylococcus aureus (µg/mL)
54
4.12 Mean square values for the effect of treatments on the acidity, pH and TSS
of the functional drinks
59
4.13 Mean values for the effect of treatments and storage on the acidity (%) of
the functional drinks
59
4.14 Mean values for the effect of treatments and storage on the pH of the
drinks
60
4.15 Mean values for the effect of treatments and storage on the TSS (%) of the
drinks
60
4.16 Mean square values for the effect of treatments and storage on the sensory
attributes of the drinks
63
4.17 Mean values for the effect of treatments and storage on the color of
functional drinks
63
4.18 Mean values for the effect of treatments and storage on the flavor of the
functional drinks
64
4.19 Mean values for the effect of treatments and storage on sweetness of the
functional drinks
64
4.20 Mean values for the effect of treatments and storage on the sourness of the 65
xiv
functional drinks
4.21 Mean values for the effect of treatments and storage on the overall
acceptability of the functional drinks
65
4.22 Mean square values for the effect of treatments and study period (weeks)
on feed intake of animals (g/rat/day)
70
4.23 Mean square values for the effect of treatments and study period (weeks)
on drink intake of animals (mL/rat/day)
72
4.24 Mean square values for the effect of treatments and study period (weeks)
on the body weight of animals (g/rat)
75
4.25 Mean values for the effect of treatments and study weeks on the body
weight of animals at 8th
week in different studies (g/rat)
75
4.26 Mean square values for the effect of treatments on the cholesterol (mg/dL)
79
4.27 Mean values for the effect of treatments on the cholesterol (mg/dL)
79
4.28 Mean square values for the effect of treatments on the HDL (mg/dL)
82
4.29 Mean values for the effect of treatments on the HDL (mg/dL)
82
4.30 Mean square values for the effect of treatments on the LDL (mg/dL)
86
4.31 Mean values for the effect of treatments on the LDL (mg/dL) 86
xv
4.32 Mean square values for the effect of treatments on the Triglycerides
(mg/dL)
90
4.33 Mean values for the effect of treatments on the Triglycerides (mg/dL)
90
4.34 Mean square values for the effect of treatments on the blood glucose
(mg/dL)
94
4.35 Mean values for the effect of treatments on the blood glucose (mg/dL)
94
4.36 Mean square values for the effect of treatments on the blood plasma
insulin (µm/mL)
98
4.37 Mean values for the effect of treatments on the plasma insulin (µm/mL)
98
4.38 Mean square values for the effect of treatments on AST (IU/L)
102
4.39 Mean values for the effect of treatments on the AST (IU/L)
102
4.40 Mean square values for the effect of treatments on the ALT (IU/L)
103
4.41 Mean values for the effect of treatments on the ALT (IU/L)
103
xvi
4.42 Mean square values for the effect of treatments on the ALP (IU/L)
104
4.43 Mean values for the effect of treatments on the ALP (IU/L)
104
4.44 Mean square values for the effect of treatments on the urea (mg/dL)
108
4.45 Mean values for the effect of treatments on the urea (mg/dL)
108
4.46 Mean square values for the effect of treatments on the creatinine (mg/dL)
109
4.47 Mean values for the effect of treatments on the creatinine (mg/dL)
109
xvii
List of Figures
Sr.No. Title Page
no.
2.1 Some important phenolic acids associated with honey bee propolis
10
2.2 Some important flavonoids associated with honey bee propolis
12
4.1 Feed intake during study-I, II, III (g/rat/day)
71
4.2 Drink intake during study-I, II, III (mL/rat/day)
73
4.3 Body weight during study-I, II, III (g/rat)
76
4.4 Effect of treatments on percent cholesterol reduction in animal
models
81
4.5 Effect of treatments on percent increase in plasma HDL in animal
models
83
4.6 Effect of treatments on percent reduction in plasma LDL level in
animal models
87
4.7 Effect of treatments on percent reduction in Triglyceride level in
animal models
91
4.8 Effect of treatments on percent reduction in Blood Glucose level in
animal models
95
4.9 Effect of treatments on percent increase in plasma insulin in animal
models
99
xviii
List of Appendices
Sr.No. Title Page
no.
i Performa for sensory evaluation of functional drink
144
ii Composition of diet used during efficacy study
145
iii Composition of vitamin mixture
146
iv Composition of salt mixture
147
xix
Abstract
In present study, locally available honey bee propolis was characterized for its various
physicochemical, antimicrobial properties and nutraceutical behaviour using animal
modelling system for food applications. The compositional analysis depicted a higher
content of ether extract along with mineral elements (K, Mg, Na, Ca, Fe, Zn, Cu, Mn) in
variable amounts. Propolis extract using ethanol and methanol (65%, 80% & 95%) and water
quantified for total phenolic contents (TPC) and characterized using HPLC. Antioxidant
(DPPH activity, Beta-carotene assay) potential of the extracts was also evaluated. Ethanol
extract (65%) propolis exhibited higher total polyphenols (327.30±14.89mg/gGAE) and
better antioxidant potential (60.59±4.38%) and indicated maximum zone of inhibition against
Escherichia coli (22.19±0.61mm) followed by Bacillus subtilis (26.37±0.31mm) and
Staphylococcus aureus (29.18±1.13mm) followed by methanol extract (65%) for the same
parameters. Functional propolis based drinks remained acceptable for two months of storage
period when subjected to compositional analyses and sensory evaluation. Bio-evaluation
based on the composition of diets given to rats: [study-I (normal diet), study-II (high sucrose
diet), study-III (high cholesterol diet)] exhibited considerable increase in feed, drink intake
and weight gain whereas, decreased hypercholesterolemia and hyperglycaemia. A
pronounced decrease (p≤0.05) in serum glucose concentration and increase (p≤0.05) in
insulin level was noticed in the ethanol extract drink while keeping liver and kidney
functioning tests within normal values. It is deduced from the present exploration that locally
available propolis possess significant antimicrobial properties against foodborne pathogens,
antioxidant potential and hence may be considered for food applications during food product
development to encourage diet based therapies ultimately promoting health management.
1
CHAPTER 1
INTRODUCTION
Phytochemicals are common in the food of human beings since primitive times to
fight against diseases as most of the medicines were derived from plants. In modern era, diet
based therapy has been revitalized globally and people are adopting the natural materials as
an intervention against various physiological ailments. Similarly, increased demand of safe
food has opened new horizons for the identification, development and utilization of natural
antimicrobial agents to inhibit food spoilage and associated health claims. The use of
bioactive compounds from plants in this regime is becoming popular not only in developed
but also in the developing countries for their complex chemical nature, antimicrobial
behaviour, health care and safe status for human consumption (Potawale et al., 2008;
Gutierrez et al., 2008).
In human body, significance of cellular oxidation is well documented as the oxidative
metabolism is vital for the survival of cells; resulting in the production of free radicals and
reactive oxygen species (ROS) causing oxidative damages. Excessive production of free
radicals inhibit the activities of some protective enzymes like superoxide dismutase and
catalase, causing destructive effect on cellular components by oxidizing the lipids, proteins
and deoxyribonucleic acid (DNA). The condition ultimately affects the cellular respiration
(Winrow et al., 1993; Bauer et al., 1999; Bae et al., 1999).
During the last few years, scientific investigations have proposed several modules
through diet based regime to prevent life threatening disorders of obesity,
hypercholesterolemia and diabetes etc. Among these strategies, a promising tool is the use of
functional foods which in addition to improvement in consumer‟s health and wellness also
reduces disease risk and ultimate medication cost in general (Shahidi, 2009). According to
the American Dietetic Association (2009), functional foods are enriched products that can be
used as conventional foods. In this context, functional beverages like juices, tea, fortified
water and dairy products are gaining good repute for satisfying the consumers quench and
providing additional health benefits. Likewise utilization of natural sources as food
preservatives for the provision of safe food is a dire need of modern world because of
minimum process requirements to ensure microbial safety without interfering food
2
ingredients. Such a type of intervention is considered as novel approach in food processing
and manufacturing that provides nutritionally well accepted and minimally processed food
for the safer usage (Juneja et al., 2012).
Propolis, a naturally resinous and strongly adhesive substance, produced by honey
bees (Apis mellifera L.) from leaves, buds and plant exudates. Bees collect propolis from
plants and muddle up with their secretions, metabolites, pollens, waxes and numerous
valuable enzymes to make it suitable for ultimate usage. The term “propolis” is derived from
two Greek words “pro” and “polis”. “Pro” means in front and “polis” means city, a substance
found in front of hives for defence (Toreti et al., 2013). It owes the characteristics of both
plants and animals origin because honey bees collect secretions from plants exudates and
convert them into propolis within their bodies. Bees utilize this product to seal cracks and
fissures in the hives, to smoothen internal walls of the comb, to check the entry of foreign
intruders and to protect their colonies from various diseases (Stan et al., 2013). Propolis is
being used as a part of folk medicine in various parts of world for its numerous biological
applications. Western countries have adopted propolis for its therapeutic role with
remarkable outcomes. Now a day, propolis has become popular form of alternative medicine
with 700-800 tons/year global consumption (Chan et al., 2012).
Bee propolis is usually a dark green to dark brown waxy product having variable
chemical nature which is exclusively dependent on climate, botanical origin, geographical
location and nature of the queen bee in the colony. Chemically, it is composed of plant resins
(40-55%), bee wax and fatty acids (20–35%), bee pollens (about 5%), essential oils (about
10%) and other organic and inorganic substances. Propolis contains a number of important
mineral elements like magnesium (Mg), sodium (Na), copper (Cu), zinc (Zn), manganese
(Mn), iodine (I), iron (Fe) in its composition. It also contains appreciable amount of vitamins
B1, B2, B6, as well as vitamin C and vitamin E (Wieckiewicz et al., 2013). In resinous
substances, more than 300 compounds of different categories have been identified so far.
These include flavonoids, polyphenols, phenolic acids, esters, terpens, phenolic aldehyde,
ketones and sterols. However, polyphenols and flavonoids are the most active constituents of
propolis and responsible for its biological properties (Sulaiman et al., 2011). Abundance of
these compounds such as pinocambrin, querecetin, rutin, apigenin, myricetin, galangin,
3
kaempferol, chrysin and other polyphenols contents serve as a marker for propolis (Cai et al.,
2012).
During the last three decades, propolis has become a subject of great interest among
the researchers throughout the world because of its natural origin and health promoting
characteristic behaviour. There are 12 different classes of propolis found in Brazil. The most
popular one is “green propolis” and is broadly studied as a supplement in food and beverages
in most of the western countries (Frozza et al., 2013). China, Brazil, New Zealand and Japan
produce and export propolis on a large scale. China is the largest producer of propolis in the
world by producing about 350 tons of propolis each year and accounts for 80% of total world
propolis production (Cai et al., 2012).
Propolis possesses a wide spectrum of properties including anti-oxidative, anti-
inflammatory, immunomodulatory, anticancer, antibacterial, antiviral, antifungal and
antiparasitic effects. Although, it is a mixture of various organic and inorganic substances but
most of the biological properties are because of its flavonoids, phenolic acids and their
derivatives (Petelinc et al., 2013). Flavonoids, the secondary metabolites of plants are
classified in to six various classes. This classification is based upon their chemistry and
structure including flavanones, anthocyanins, isoflavones, flavonols, flavones and flavanols.
Most of these classes of flavonoids are associated with bee propolis and its associated
properties (Volpi and Bergonzini, 2006; Dimpfel et al., 2007).
Honey bee propolis being rich in polyphenols and flavonoids has great potential as a
natural antimicrobial agent against pathogenic microorganisms without causing any adverse
action. It inhibits the growth, division and even enzyme activity of bacteria to lessen their
effects on biological systems (Zeighampour et al., 2013). Both Gram positive and Gram
negative bacteria are susceptible to propolis extract. Propolis can retard the development of
biofilm formation among different pathogen domains of microorganisms. Listeria spp.,
Staphylococcus spp., Streptococcus spp., Bacillus spp., Escherichia coli and Pseudomonas
specie are the group of bacteria for which propolis possessed strong antimicrobial activity
(Stan et al., 2013). Different researchers has explored the antibacterial and antifungal
activity of propolis against various food spoilage organisms and found that phenolic
substances of propolis are responsible for antimicrobial behaviour (Wojtyczka et al., 2013).
4
Propolis and its extract are considered as the best alternate natural food preservatives
for the preservation of fruit juices. It is preferable to use against yeast and fungi to extend the
shelf life of fruit juices (Koc et al., 2007). Application of propolis extract in fruit reduces the
fungal attack, retards water loss, fixes the colour, maintains quality attributes and inhibits the
postharvest changes and microbial load during storage and transit (Ozdemir et al., 2010).
Ethanol extract of propolis has strong inhibitory effect on the growth of coliform bacteria in
meat and meat based product. Substances found in propolis are generally recognized as safe
(GRAS) and considered typical constituents of food and food products as they are good
alternative to chemical preservatives for perishable food commodities (Tosi et al., 2007).
The effects of bioactive compounds from natural sources have provoked a new
horizon of polyphenols and flavonoids. Their behavior against disease management is being
explored due to their action against oxidation of lipids, degeneration of proteins and nucleic
acids offered by free radicals and ROS (Cirico and Omaye, 2006). Human body contains a
number of endogenous antioxidant substances. But there is a great need of exogenous
antioxidants to appease the free radicals (Maeta et al., 2007). Antioxidants protect cells from
free radicals damage to mitigate the oxidative stress (Pal et al., 2012). Propolis is regarded as
a good source of natural antioxidants because of its oxygen scavenging ability which makes
it best to serve as a safeguard in the management of oxidative stress in human body. As for as
mechanism of action of propolis is concerned, it is associated with the reducing properties,
acts as a donor of hydrogen and metal binding agent to eliminate the ROS (Gulcin et al.,
2010). Propolis polyphenols impart strong inhibitory action against various physiological
disorders by upsetting the oxidative chain reaction, protecting membranes of cells from
damage thus retards the onset of different metabolic syndromes (Daleprane and Abdalla,
2013).
Diabetes mellitus is a chronic metabolic disorder, marked as a third biggest killer of
human beings throughout the world after the cardiovascular and oncogenic diseases.
Presently, Pakistan is one of the major affected countries with reference to diabetes and other
lifestyle related disorders. Pakistan has been ranked at 6th
number in the world regarding
diabetic patients and is being expected further rise up to 13.85 million in 2030 (WHO, 2006).
Now a day, management of diabetes through diet possessing drug treatment is a prime
concern of patients and physicians. Modern studies have revealed a pivotal link in the
5
consumption of propolis and reduction of serum glucose in various animal modelling.
Propolis extract maintains the nature, function and conformational integrity of β-cells of
pancreas by stimulating immune system and altering glucose metabolism to attenuate the
hyperglycaemia (Orsolic et al., 2013).
Low density lipoprotein (LDL) and high blood cholesterol are the leading factors in
the onset of various blood vascular diseases like atherosclerosis and coronary heart diseases
(CHD). The regulations of blood cholesterol level play a key role in the management of
several atherogenic maladies (Abeywickrama et al., 2011). Bioactive components found in
propolis are considered beneficial as a diet related strategy in the modulation of cholesterol
level and dyslipidaemia. It inhibits the hyperlipidaemia by regulating the expression of
genome responsible for enzyme synthesis involved in the lipid metabolism (Ichi et al., 2009).
Moderate intake of propolis causes substantial change in serum lipid profile by monitoring
the blood cholesterol and improving the high density lipoprotein (HDL) to low density
lipoprotein (LDL) ratio (Li et al., 2012).
In Pakistan, many apiaries are found throughout the country those are producing huge
amount of honey. Alongside honey production, a massive quantity of propolis is produced
which is not yet being processed in any way and discarded as a waste. Keeping in view the
significance of propolis and its constituents, the current study was hypothesised as the
propolis is a multiple complex with strong antimicrobial, antioxidant and nutraceutical
mediator in food formulations as an ingredient. The effort were made to explore the nature
of locally available propolis as a useful product/ingredient from bee hive which may be
utilized by the food manufacturing industries for healthy life at the same time minimizing
health care cost. Optimize conditions for extraction and characterization of bee propolis,
antimicrobial behaviour against certain foodborne pathogens were also evaluated. Finally,
bio-evaluation of the developed functional drink thus produced containing propolis extract
was studied against certain metabolic disorders like hyperglycaemia and
hypercholesterolemia. It was, therefore, the specific objectives to be attained during the
present study were:
1. To characterize the honey bee propolis
2. To elucidate the antimicrobial activity of propolis extract
3. To evaluate the nutraceutical potential of bee propolis
6
CHAPTER 2
REVIEW OF LITERATURE
Bioactive compounds isolated from propolis are rich in phenolics and flavonoids;
possess strong antimicrobial activity against food borne pathogens. Along with this property,
they also act as functional ingredient to combat physiological problems like hyperglycemia,
hypercholesterolemia and various oncogenic disorders due to their antioxidant, anti-
inflammatory and cytotoxic effects. The present research work was conducted to explore the
chemical composition of locally found propolis with special reference to polyphenols,
antimicrobial potential and its therapeutic role/nutraceutical against selected diet related
disorders including hyperglycemia and hypercholesterolemia. The literature about various
aspects of the present study has been piled up under following aspects:
2.1 Bee propolis: An overview
2.2 Polyphenols: Bioactive moieties
2.2.1 Phenolic acids
2.2.2 Flavonoids
2.3 Natural antimicrobial agent; a new millennia perspective
2.4 Functional and nutraceutical foods; a concept of modern era
2.5 Metabolic syndromes and propolis
2.6.1 Oxidative stress management
2.6.2 Novel approach against Hyperglycemia
2.6.3 Hyporcholesterolemic perspective of propolis
2.1. Bee propolis; An overview
Allah Almighty said in Holy Quran about bees;
“And your Lord inspired the bee, saying: “Take you habitations in the mountains and in the
trees and in that what they erect; Then eat of all fruits, and follow the ways of your Lord,
7
made easy [for you]. There comes drinks from their bellies, of varying colour wherein is
healing for mankind. Verily, in this is indeed a sign for people who think.”
(Al-Hilali and Khan, 1993)
Propolis, or "beeglue," is a well-known substance that beekeepers found in their hives
along with honey, royal jelly and pollens. It is a resinous and strongly adhesive substance,
collected by bees (Apis mellifera L.) from buds, tree leaves and flowers. Honey bees mixed
plant secretions with their pollen and self-secreting enzymes to produce propolis. Bees use
propolis as a general-purpose sealer, to soften the hives, to protect internal walls of the comb
and to stop the entry of foreign intruders into bee colonies (Bankova et al., 2000). The term
propolis was derived from Greek words; pro means “before” and polis means “city”, thus
bees utilize propoli to protect hive from microbial contamination and foreign invaders (Chan
et al., 2012). Propolis is a product of bee hive with complex chemical nature. During its
production bees use wax and plant exudates with their 13-glicosidase enzyme from saliva
which hydrolyzes polyphenols glycosides to flavonoid aglycones. Like honey, bee pollens
and royal jelly, propolis has tremendous therapeutic and biological properties. However
propolis, due to its complex chemical nature and suitability for human consumption has been
explored as a natural product for usage in food and medicine since the last four decades
(Ramos and Miranda, 2007).
Avicenna has described in his famous description “The Canon Medical Science” that
honey bees produce a special kind of product for the protection of hive which is different
from comb wall and specialized for its structure and function called propolis (Lotfy, 2006).
In the ancient times, propolis was well known to the priests who had developed medicine
from propolis and utilize its components for preserving bodies. With the passage of time use
of propolis acquired much popularity among Egyptians, Arabs, Greeks, and many other
civilizations of the world. Its practice continues today as a potential remedy against various
disorders and marketed in pure form, extracted solution, pooled with natural substances in
cosmetics, medicine and various food products. It is accredited as an integral part of healthy
foods, beverages and drinks, its annual consumption almost reaches up to 700-800 tons/year
around the world (Orsolic and Basic, 2006; Moreira et al., 2008; Chan et al., 2012 and Yuan
et al., 2013).
8
Chemical constituents and biological activities of propolis are highly dependent on
the climate, geographical location, plants species, exudates nature and genetics of queen bee
in the hive (Gregoris and Stevanato, 2010). Generally, propolis contains 55 % balsams and
resins, 25-30% waxes, 10-15% etheric and aromatic oils, 5% pollens and other compounds of
organic and inorganic nature. It also contains minerals (Mg, Ca, I, K, Na, Cu, Zn, Mn and
Fe), vitamins (B1, B2, B6, Vit. C and Vit.E) and fatty acids. Propolis also possessed enzymes
of bee metabolism including succinic dehydrogenase, glucose-6-phosphatase, adenosine
triphosphatase and acid phosphatase (Zeighampour et al., 2013). More than 180 different
compounds have been identified so far in the resinous part of propolis. Among them major
constituents are polyphenols and flavonoids that comprised 10-15% and dominated by
flavonoids, phenolic acid and their derivatives (Castaldo and Capasso, 2002). During the last
three decades various kinds of propolis have been recognized from different parts of the
world. There are 12 different groups of propolis which have been categorised on the basis of
physicochemical composition and phytogeographical locations. Green propolis, the most
popular form in Brazil and has been explored on a large scale as constituent of food and
beverages. Since 2007, red propolis from Europe and America has become a subject of great
interest for researchers throughout the world. It is different from other forms of propolis in its
composition and found more effective in terms of biological properties than other forms of
propolis (Frozza et al., 2013).
Biological properties including antimicrobial, antioxidant and anti-inflammatory of
propolis attributed due to chemical its constituents. Propolis is gaining popularity for its
usage in health drinks and beverages to improve general health and particular to prevent
cardiovascular, diabetes and various oncogenic disorders (Mohammadzadeh et al., 2007).
Modern research explored that propolis has immunostimulatory effect on human body by
producing immunoglobulins and activating B and T lymphocytes. Propolis and its
bioflavonoids considered as food supplement to fight against diseases by enhancing the
working of immune system (Sforcin, 2007). The consumption of propolis extract inhibits
differentiation of cancerous cells and induces apoptosis to suppress the onset of carcinoma.
In cancerous granulocytes propolis retards their growth and multiplication by inducing
changes in nucleic acid and other biomolecules to stop the progression of disease (Mishima
et al., 2005). Isla et al. (2001) revealed that oxidative stress has a direct effect on the
9
development of various disorders whereas intake of propolis play a significant role for
inactivation of free radicals and reactive oxygen species (ROS) to protect serum lipids from
oxidation.
2.2. Polyphenols: Bioactive moieties
Polyphenols are secondary metabolic products of plants. They are abundantly found
in fruits and vegetables for protection against pathogens invasion and ultraviolet radiations.
Almost 8,000 polyphenols have been identified from various botanical sources and they have
same basic structural organization of sugar linked with hydroxyl group and aromatic carbon
but differ in their substitution groups. The classification of these bioactive moieties is largely
based upon their function, biological potential and nature of phenol ring in the structure. The
diverse configuration of polyphenols plays an integral role in the complexity, vast variety and
properties of these compounds (Pandey and Syed, 2009). Polyphenols have immense
significance among the researchers due to their role in human diet. They play antioxidant role
to protect body from oxidative damages caused by free radicals and reactive oxygen species
(ROS). Particularly, they help to combat various atherogenic and degenerative ailments in
several ways (Tsao, 2010). Plant phenolics serve as natural antioxidants for the biological
systems as they hinder the oxidation reactions in different ways by inhibiting initiation,
propagation and termination stages of oxidative reactions. Hydroxyl group of these
compounds is an excellent hydrogen donor for the quenching free radicals including reactive
nitrogen species (RNS) and Reactive oxygen species (ROS). As natural antioxidants
polyphenols able to chelate various metals, responsible for the production of free radicals
(Pereira et al., 2009). Plant polyphenols have been categorized into phenolic acids including
gallic acid, coumaric acid, feraulic acid and caffeic acid, flavonoids; flavanols, flavonols,
flavones, flavanones, isoflavones, anthocyanins and stilbenes which contain lignans and its
polymeric forms (Han et al., 2007).
2.2.1. Phenolic acids
Phenolic acids are the organic compounds with phenol and carboxylic acid functional
group in their organization. They have been divided into two groups, first the derivatives of
cinnamic acids and the compounds of benzoic acids (Robbin, 2003). Hydroxycinnamicds are
common types of phenolic acids and that associated with various foodstuffs. Feraulic acid,
coumaric acid, caffeic acid sinapic acid, gallic acid and p-coumaric acid are the common
10
form of phenolic acids in food systems for the protection and longevity of food items during
storage and transportation (Tsao, 2010). Chemical structure of some of the important
phenolic acids related to bee propolis;
gallic acid caffeic acid
vanilic acid chloroogenic acid
syringic acid m-coumeric acid
Figure: 2.1. Some important phenolic acids associated with honey bee propolis
11
2.2.2. Flavonoids
Flavonoids are secondary metabolites of plants with low molecular weight that play
important function in human diet. They are good chelators of free radicles thus inhibit the
degradation of various biomolecules by induced by oxidative stress. They have central flavan
nucleus as a structural unit. More than 4000 different forms of flavonoids have been isolated
from various natural sources. All of these have same nuclear pattern but differ in their
complexity due to nature and position of substitution groups (Heim et al., 2002). Flavonoids
occur throughout natural products and distributed in appreciable amount in both plants and
animals products. Among these products, seed, nuts, fruits, honey and propolis are the
products of plants and animal origin respectively with polyphenols and flavonoids. Dietary
intake of flavonoids ranges between 1-2 g/day and it was observed by previous researchers
that average intake of flavonoids was 23mg/day protect the body from damages induced by
oxidative reactions (Sandhar et al., 2011).
Flavonoids are beneficial for human health due to their antioxidant and free radicals
scavenging activities. By virtue of their ability; they inhibit peroxidation of LDL which leads
to protect against dyslipidaemia and associated disorders (Kondo et al., 1996 and Mazur et
al., 1999). High intake of flavonoids predicted marked reduction in mortality due to coronary
heart diseases thus lowers the risk of myocardial infarction and cardiovascular problems by
38% in older peoples (Hertog et al., 1993). According to modern investigations regular
intake of flavonoids provide protection against various life threatening disorder including
arteriosclerosis, Alzheimer‟s disease and even cancer. They are helpful in maintaining the
blood cholesterol and protect the body against ultraviolet radiations promoted oxidative
reactions. Due to multiple benefits of flavonoids there is a great demand for utilization of
these substances as food additives in food industry. Recently, they are produced and
marketed commercially in various forms and products throughout the world (Sisa et al.,
2010).
Depending upon chemical nature, flavonoids have been categorized into various
groups as (a) anthocyanins, comprising on anthocyanidin and glycosylates, abundantly found
in colourful parts of plants; (b) group of colorless compounds of anthoxanthans containing
several classes including flavonols, flavones, flavanols, flavans, isoflavanols and their
12
derivatives. Quercetin, fisetin, kampferol and myricetin are the examples of flavonoids
commonly found in various plant species (Han et al., 2007).
Chang et al. (2002) investigated the flavonoid contents of propolis as a key ingredient
for its biological functions. They examined twelve different commercially available products
of propolis and deduced that total contents of flavonoids ranges upto 7% in liquid products
whereas propolis powder contains 2.97 to 22.73% flavonoids. Propolis possesses numerous
biological functions due to rich chemistry of flavonoids and their derivatives. Antioxidant,
anti-inflammatory and antitumoral activities of propolis are associated with flavonoids
present in its composition. Rutin, pinocambrin, kaempferol, quercetin, gallangin, pinobanksin
and chrysin etc. are flavonoids naturally associated with propolis that make it suitable for
safe usage to promote general health (Coneac et al., 2008).
Rutin Kaempferol
Quercetin Myrcetin
13
Chrysin Pinocambrin
Pinobankson Gingerol
Figure: 2.2. Some important flavonoids associated with honey bee propolis
2.3. Natural antimicrobial agent; A new millennia perspective
Food spoilage caused by microorganism leads deterioration of food commodities. The
food substances can be preserved for longer period of time when basic causes of spoilage
controlled. Food preservation methods solely depend upon the nature of product and
causative agents associated with spoilage. Preservation of high moisture food like meat and
meat based food items may be accomplished by low temperature for some time but rendered
to microbial contamination when expose to environmental conditions. The antimicrobial,
antifungal and antioxidant properties of propolis offer a wide scope for its utilization in food
technology; unlike some orthodox preservatives its residues play a beneficial role in human
health also. Chemical food preservatives have been used for centuries to prevent bacterial
and fungal spoilage of foods. Among them Potassium sorbates, Benzoate of Sodium or
14
Potassium, and their mixtures are preservatives with broad-spectrum activity against
microorganisms. They have been generally recognized as safe and well accepted worldwide.
The application of naturally occurring chemical substances with antimicrobial properties in
food products may serve as an alternate approach for food preservation and in different parts
of the world (Fleet, 1992).
During the last 50 years, many natural substances have been explored to combat
infectious microorganism. In this regards plants secondary metabolites like polyphenols and
flavonoids have revolutionized the concept of natural antimicrobial agents. Drug resistance,
chemical nature, metabolic residues and high cost of synthetic products are leading concerns
responsible for innovation and development of natural antimicrobial products for future use
(Demain, 2009). Moreover bioactive components extracted from natural sources are
generally regarded as safe and represent a source to combat various pathogens in natural
way. Similarly antimicrobial behavior of propolis has been exploited on large scale during
recent years and attracted much attention due to its peculiar chemical nature. Propolis
considered a substance with potential activity against different food spoilage agents and
pathogens especially bacteria and fungi (Yang et al., 2010).
Spices, herbs and essential oils from different plant sources have been added to food
primarily as flavoring agents but also represent broad range of antimicrobial activities to
increase shelf life of food commodities (Palou et al., 2002). Ali et al. (2010) considered
propolis is one of the natural antioxidants from honey bee which enhance the storage life of
meat sausage by hindering the bacterial load. It could be safe to use as natural replacer of
chemical preservatives and have been explored in developed countries for its applications in
different food systems. Propolis serve as a good natural preservative and flavor enhancer in
meat and meat based products and contribute a lot in promotion of human health (Han et al.,
2001). It is proven that propolis has an inhibitory effect on growth of mold and yeast in fruit
juices. Consequently, it extends shelf life of fruit juices with good organoleptic properties
(Koc et al., 2007). Ozdemir et al. (2010) concluded that grapefruits treated with ethanol
extract of propolis can preserved by hindering fungal decay. The use of propolis extract has
positive effect on postharvest quality attributes of fruits including green bottom, skin colour
and weight loss thus propolis can be used as a medium to reduce postharvest changes in the
fruits.
15
Propolis has become popular in traditional medicine as food supplementary material
in different parts of world (Pereira et al., 2009). Propolis, commercially available resinous
product contains polyphenols, flavonoids and many other organic compounds responsible for
antibacterial, antiviral, antitumor, anti-inflammatory and anticancer activities (Sforcin et al.,
2000, Kimoto et al., 2001, Banskota et al., 2002, , Murad et al., 2002, , Zhou et al., 2009).
On the other hand, Kedzia et al. (1990) reported that mechanism of antimicrobial activity of
propolis is complicated and could be attributed to synergism between flavonoids
hydroxyacids and sesquiterpenes. Bacteria responsible for diseases in humans belong to G-
negative and G-positive domains, propolis extract has remarkable role to inhibit their growth
and multiplication due to vast spectra of polyphenols (Choi et al., 2006). Silva et al. (2012)
described the role of bee propolis against different groups of bacteria including both G-
positive and G-negative domain. Pathogens related to these genera can be distinguished as
M. tuberculosis, S. aureus, Enterococcus spp., B. cereus, Pseudomonas spp., Shigella spp.
and Salmonella spp.
The antimicrobial role of propolis against different strains of microorganisms widely
explored and cleared that ethanol extract of propolis in concentrations from 0.4 to 14.0% (%
v:v) found more effective in inhibiting the growth of microorganisms particularly bacteria. It
is deduced from previous investigations that G-positive bacteria is inhibited by low
concentrations (0.4%) whereas G-negative bacteria needs higher concentration to suppress
their activities (4.5 to 8.0%). Furthemore, no significant difference was seen in activity due to
seasonal variations in growth curve of microorganisms (Sforcin et al., 2000). Ugur and
Arslan (2004) examined the antibacterial activity of different extract of propolis collected
from Turkey. They demonstrated that antibacterial activity is highly influenced due to sample
concentration, dosage rate and solvent used for extraction in each case and could enhanced
with increase in dosage. They also reported that most sensitive organism to propolis is S.
sonnei in the G-negative group and S. mutans in the G-positive. In another investigation
conducted by Tosi et al. (2007) observed that propolis extract upto concentration of 14mg/ml
has inhibitory effect against rod shape E. coli and considered safe for human consumption at
this level and could be incorporated in different food products as a food preservative.
Wojtyczka et al. (2013) explored the antimicrobial activity of ethanol extract of propolis
against S. epidermidis and documented that propolis reduces growth and biofilm formation in
16
S. epidermidis. Rahman et al. (2010) investigated the role of temperature and pH on behavior
of propolis extract against S. aureus. The variation in pH and temperature significantly affect
antibacterial action of ethanol extract of propolis (EEP) against S. aureus. At the end of study
they depicted that optimum pH and temperature required for extract preparation.
Similarly, Propolis extract using different solvents at different pH values imparted
different antibacterial action. As comparison ether extract of propolis showed stronger
inhibitory outcome on growth of S. aureus cultures at pH 6, 7 and 8 whereas toluol extract of
propolis exhibited inhibitory effect on B. cereus in cultures at pH 6 and 8. Propolis use could
be increased to make it more operative as medicine of choice against pathogenic bacteria
alone or together with other antibiotics depending upon the nature of pathogens (Ivancajic et
al., 2010). According to another experiment conducted by Hendi et al. (2011) proved that
ethanol extract of propolis is more active and effective against bacteria among other extracts
prepared during study. Likewise, Benhanifia et al. (2013) reported correlation between total
flavonoids found in propolis and antimicrobial activity and marked a direct relation between
them. Their findings explored propolis as a good antimicrobial and antioxidant agent which
could be an alternative to other food additives and supplements for application in food
technology.
Pathogenic fungi cause numerous economic losses in production and post-harvest
handling of fruits and vegetables. Fungicide application to control these pathogens is still
controversial due to ustainability of ecosystem; there is a dire need to find a link between
risks of fungal deterioration of crops and protection of ecosystems (Wightwick et al., 2010).
Recently, losses due to fungal attacks on crops approaching 12% of the total world
production and it is much higher in the developing countries which is a serious threat for
food supply chain around the world (El-Shafei et al., 2010). Genetic variation, loss of
efficacy and resistance to chemicals in fungi lead us to adopt natural products from plants
and animal sources like propolis to control diseases and spoilage of horticultural crops, as a
promising alternate to man-made fungicides due to long lasting after effects of these agents
on environment (Ordonez et al., 2011).
Curifuta et al. (2012) investigated role of ethanolic extract of propolis against
different fungal strains and depicted that fungal growth retarded effectively that promote
safety and longevity of fruits and vegetables. Similarly, propolis extract play important role
17
against different yeasts and marked that propolis not only fungistatic but also fungicidal in
nature. Furthermore they deduce that Trichosporon spp. is the most susceptible and C.
tropicalis resistant yeast among all the selected genera (Oliveira et al., 2006). Kacaniova et
al. (2012) explored antifungal potential of propolis extract using ethanol and methanol
against three fungal strains, A. niger, A. fumigatus, A. flavus and seven different yeasts R.
mucilaginosa, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, G. candidum and C.
albicans. They confirmed that Aspergillus. fumigatus is the most sensitive fungal and
C.glabrata is the most sensitive yeast strain for 70% alcoholic extract of propolis. Likewise,
Temiz et al. (2013) examined ten different propolis samples collected from various regions
of Turkey against two most mycotoxin producing fungi, A. versicolor and P.
aurantiogriseum. Antifungal activity of ethanol extract of propolis were determined at three
different concentrations levels, 1%, 5% and 10% (v/v) using Potato Dextrose Agar (PDA).
Ethanol extract at 10% concentration indicated 100% inhibition of both fungal strains and
found variable at 1% and 5%. Fruit juices are susceptible to spoilage caused by yeasts and
moulds. The role of propolis in orange, apple, mandarin and white grape juice preservation
was assessed and observed that concentration ranging from 0.01 to 0.375mg/mL retards yeast
growth effectively at room temperature. Minimum inhibitory concentration (MIC) of
propolis extract determined against yeast were 0.02–0.375, 0.04–0.375, 0.01–0.185 and
0.02–0.185mg/mL in mandarin, apple, orange and white grape juices respectively in
comparison to sodium benzoates (Koc et al., 2007). Application of ethanol extract of green
propolis as disinfectant on embryonated eggs showed antifungal effect without any change in
nature of eggs and found effective alternate for incubation of eggs in place of formaldehyde
(Vilela et al., 2012).
Propolis has potential to inhibit the growth of molds on fruits. Polyphenols,
compounds associated with propolis showed this characteristic and strong activity against
other spoilage agents. Pinocambrin, the leading compound from propolis has potent activity
against fungi and molds. Application of pinocambrin reduced growth of molds, enhance
freshness of fruits and maintain natural color for a longer period of time (Peng et al., 2012).
In a study, Yang et al. (2010) investigated inhibitory role of propolis against two different
strains of moulds that commonly contaminate citrus fruit. The use of propolis, considered
better choice in citrus fruit to control the attack of pathogens during storage. They found that
18
P. digitatum and P. italicum ; blue and green moulds of citrus fruits effectively controlled by
the application of propolis extract on fruits especially in citrus and considered as better
option to synthetic antifungal compounds. Silici and Karaman, (2013) studied shelf life of
apple juice while using propolis as preservative in different concentrations (0.1,1 and
2mg/mL) and deduced that 2mg/mL showed best results to reduce the patulin production.
Futhremore they regarded propolis as best alternate to chemical preservatives against moulds
in fruit based beverages.
2.4. Functional and nutraceutical foods; Concept of modern era
The role of diet and nutrition is imperative for human health to manage physiological
disorders throughout life. The link of food and health modified daily intake during the last
few years. Presently, food is not a source of nutrients for growth and maintenance but also
responsible for good health and immune response due that exerted much attention of
consumers towards therapeutic role of food. This concept explored the idea of functional and
nutraceutical foods as the commodities which possessed health promoting benefits along
with regular body nourishment thus lowering the risk of various disorders (Henson et al.,
2008).
Functional foods, a new trend in human nutrition was presented in Asia (Japan) in the
80‟s and referred as foods with some physiological role in spite of nutrient requirement of the
body. In Japan, more than two hundred food items marked as functional foods and known as
Food for Special Health Use (FOSHU). Among the developing countries, such food products
have gained popularity and developing huge market due to consumer concern and easy
availability (Rajasekaran and Kalaivani, 2011; Serafini et al., 2012). Several domains
demonstrated relation between health and disease attenuation with special reference to
certain food constituents. In 1989 US Foundation for Innovation in Medicine has explored
the idea of nutraceuticals as food or any ingredient of food that associated with disease
prevention and health promotion (Alissa and Ferns, 2012).
Phytochemicals are diverse in their use as functional ingredient in food products since
the last few decades and becoming more popular across the world. Nutritionists considered
these bioactive components a natural tool for the promotion of health because of their natural
origin and safe use. Phytochemicals are good antioxidants containing compounds of different
nature like polyphenols, flavonoids, fatty acids, essential oils, sulphurous compounds, pectic
19
substances, minerals and fibres. These are abundantly found in food of plant origin and
associated with innate therapeutic behaviour against different maladies (Basu et al., 2007;
American Dietetic Association, 2009). Various botanical sources have been explored for
their antioxidant potential to mitigate several metabolic syndromes. Members of family
Alliaceae enhance active immunity due to promotion of glutathione redox process. They
regarded as good antioxidants, antimicrobials, antioncogenic and immunomodulating agent.
Similarly polyphenols, flavonoids and anthocyanins strengthen body against bacterial, viral,
allergens and mutagenic factors (Yi et al., 2005; Hwang et al., 2012). Nutraceuticals and
functional foods are being used in modern world to address various age relate abnormalities
due to their prophylactic action. Several health claims including hyperglycemia,
hyperlipidemia and obesity can be treated by diet management. Cardiovascular ailments,
degenerative disorders and atherogenesis may be prohibited by regular intake of food
products containing polyphenols, tocopherols, resveratrol and ascorbic acid. Many studies
have explored the correlation between consumption of fruits and vegetables and attenuation
of hypertensive, atherosclerotic and hypoglycemic conditions. Nutraceutical also play a key
role in the prevention of ulcer and renal problems due to their antioxidant properties (Betoret
et al., 2011). Fruit drinks and beverages contain plenty of phenolics, carotenes, carotenoids,
ascorbic acid as active ingredients (Beceanu, 2008).
Modern research reveals the functional and nutarceutical role of propolis against
various maladies. Propolis, due to its rich phenolic profile used in health drinks and
beverages to combat various physiological disorders including diabetes, dyslipidemia and
even cancer around the world (Kang et al., 2010). Propolis has potential antioxidative
properties therefore holds high status in treating diseases in natural way. Similarly, propolis
is a natural potent for cardiac diseases and reduces serum cholesterol by promoting anti-
inflammatory and antiproliferative response of body. These features encouraged the role of
propolis as a subject of great interest against cardiac diseases, arteriosclerosis, diabetes,
dyslipidemia and even oncogenic disorders (Daleprane et al., 2012). Currently, functional
drinks and beverages are leading trend in regards to nutraceutical foods. New techniques
have been employed to isolate constituents from propolis for their role in food industry as
functional ingredients to enhance human health and immune response.
20
2.5. Metabolic syndromes and propolis
Diet inequity leads to hoards a number of elements that cause numerous metabolic
disorders. Abnormal lipid profile and blood glucose level considered serious threat for onset
atherogenic problems including hyperlipidemia and hyperglycemia. During the year 2000, in
United States (US), 47 million peoples suffered from different metabolic disorders (Ervin,
2009). According to international diabetes federation (IDF), obesity, abnormal lipid profile
and disturbance in glycemic index are leading factors associated with physiological
abnormalities. Obesity and high level of cholesterol produced deleterious effects on liver
cells and enhance the progression of various chronic diseases (Sargin et al., 2003). The
increased production of free radicals than available antioxidants found undesirable that
caused abnormal functioning of lipids and glucose moieties. Ultimately, these changes affect
normal insulin homeostasis leading to diabetes and hypercholesterolemia (Bursill et al., 2007
and Basu et al., 2010).
Functional and nutraceutical foods are enriched with natural antioxidants; that
considered prototypes to combat atherogenic disorders and illuminated a new regimen of
food as medicine. Bearing in minds about the functional and nutraceutical foods has become
an emerging food industry with billions of dollars incentive in modern world (Colonna et al.,
2008 and Martin-Moreno et al., 2008). Previous studies proved that chances of metabolic
abnormalities have been reduced up to 40% by implication of diet interventions and changing
lifestyle (Farah, 2005, Barta et al, 2006, Divisi et al., 2006 and Nies et al., 2006).
Propolis has strong inhibitory role on free radical production that prevents the
incidence of liver and kidney problems induced by diabetes. Chinese propolis reduced
glycaemic index significantly that increased superoxide dismutase activity in blood serum.
Similarly, Brazilian propolis involved in increased production of SOD, reduced level of
malonaldehyde and inhibits the activity of nitric synthetase. Exposure of propolis increased
glutathione peroxide activity in renal and hepatic tissues which is responsible to lower
hepatorenal damage by inhibiting lipid peroxidation and promoting antioxidative enzyme
activity (Zhu et al., 2010). Orsolic et al. (2013) explored that propolis intake ameliorate
toxicity induced by alloxan and lessen hepatotoxicity induced by oxidative stress. Propolis
has direct inhibitory action on diabetes because of its antioxidative properties and could be
dietary supplement to monitor various pathophysiological processes involved in progression
21
of renal and hepatic tissues destruction. Recent investigations proposed by Nakamura et al.
(2013) indicate that oral intake of propolis extract concentration of 50mg/kg attenuates liver
damage induced by α-naphthylisothiocyanate (ANIT) while higher concentration (250mg/kg)
impart lesser action. Furthermore they deduced that propolis protects liver cells effectively
than vitamin E antioxidant.
Ichi et al. (2009) conducted a study on wistar rats and found that administration of
0.5% propolis with regular diet improves fat index by modulating dyslipidaemia. Propolis
altered the role of peroxisome proliferater-activated receptor α (PPARα) protein associated
with lipid metabolism. Ahmed et al. (2012) explored antiinflamatory potential of propolis
induced by thioacetamide. Laboratory rats were given oral dose of 100mg/kg body weight of
aqueous extract of propolis (AEP) and oil extract of propolis (OEP) for eight weeks and
observed significant reduction in toxic effects of thioacetamide. It was noticed that AEP
responds better than OEP in antiinflamatory and hepatotoxicity to maintain normal hepatic
physiology. Recent studies indicated caffeic acid phenyl ester (CAPE) involved in various
biological activities associated with propolis. CAPE play immense role as antitumor and
antinflamatory agent in cellular systems; also gives protection to cartilaginous tissues
damaged by inflammatory responses (Cardile et al., 2003). Propolis has become popular
among pharmaceutics and is being marketed in the form of capsules, tinctures, food items,
healthy drinks and beverages to enhance human health immune response against atherogenic
diseases (Newairy and Abdou, 2013). Franchi et al. (2012) investigated cytotoxic role of
propolis on human leukaemia cell line. During study cell lines were placed in incubator
containing propolis solution for 48 hours and data were recorded for each cell line at
different concentrations. It was inferred that red propolis showed more activity against than
green propolis that possessed certain inhibitory agents having cytotoxic activities against
leukaemia cells.
2.6. Oxidative stress management
The production of reactive oxygen species (ROS) as superoxide anion, hydrogen
peroxide and reactive hydroxyl ions with reactive nitrogen species ( RNS) during metabolism
monitored by antioxidant enzymes (Metodiewa and Koska, 2000 and Powers et al., 2011).
Uneven production of ROS and RNS caused amassing of these species in cells result in
oxidative stress. Oxidative stress caused damage to biomolecules especially proteins, nucleic
22
acids and lipids (Cooke et al., 2003 and Fialkow et al., 2007). The onset of chronic
atherogenic diseases like cardiovascular ailments, osteoporosis, diabetes and cancer initiated
by oxidative stress (Ratnam et al., 2006). Sedentary lifestyle, smoking, depression and
certain environmental factors enhanced generation of ROS that inhibit activation of nitric
oxide and caused endothelial dependent vasodilation which promotes cellular degradation
(Weisburger, 2002; Espin et al., 2007 and Migliore and Coppede, 2009). Propolis has strong
antioxidant potential responsible for scavenging free radicals thus prevent degradation of
cellular components. Propolis also decreased level of oxide of nitrogen and hydrogen in cells
which involve for its chemopreventive actions and other biological activities (Kolankaya et
al., 2002 and Tan-No et al., (2006).
The oxidative stress is responsible to generate allylic hydrogen atoms which start
lipids oxidation rapidly. In response, white blood cells generate compounds that caused
cellular damage. In such conditions body produces enzymes including glutathione peroxidase
(GSH-Px) and superoxide dismutase (SOD) to suppress these changes. Superoxide dismutase
transformed free singlet oxygen to hydrogen peroxides thus glutathione peroxidase with
catalase changed hydrogen peroxide to water. Such enzymes are responsible for protection of
body against free radicals. Due to excessive production of reactive oxygen species,
mechanism may interrupt leading to deleterious implications and necrosis. Propolis
considered potent agent to combat oxidative stress by alleviating the generation of free
radicals (Erdman et al., 2009 and Zhu et al., 2010). Honey bee propolis preparations found
suitable to mitigate tissue damage by eliminating ROS and free radicals (Orsolic et al.,
2013).
Propolis has variety of compounds with potential inhibitory effect on oxidative stress.
Previously different studies explored its composition with spectra of polyphenols. It was
observed that caffeic acid phenyl ester (CAPE) regarded major component of propolis that
inhibits production of ROS in biological systems (Hosnuter et al., 2004). Commonly CAPE
serves an anticancerous and antiinflamatory substance of propolis. It was documented that
propolis impedes low density lipoprotein oxidation and nitration of organic compounds like
proteins. Furthermore, in animal soft tissues of endothelium, CAPE overwhelms the activity
of NADPH oxidase and increase the expression of NOS (Silva et al., 2011). Propolis
increased antioxidant potential of living tissues that lowers lipids peroxidation closely
23
associated with onset of cardiovascular disorders. Similarly, in another study propolis from
Turkey found effective against nucleic acid damage caused by hydrogen peroxide in
fibroblast cells (Jasprica et al., 2007, Zhao et al., 2009, Kart et al., 2009, Tekin et al., 2009
and Aliyazicioglu et al., 2011). The antioxidant role of polyphenols of Turkish propolis
inhibits hydrogen peroxide induced changes in DNA that leads towards chemo-preventive
actions of propolis in animal tissues. Likewise red propolis prevents liver dysfunction caused
by alcohol consumption that enlightens the role of propolis as natural antioxidant agent
(Remirez et al., 1997). Propolis also retards white blood cells destruction by inhibiting
tumour necrosis factor or nuclear factor kappa B (TNF/NF-𝜅B) process and glutathione
(GSH) activity (Claus et al., 2000 and Pascual et al., 1994). Furthermore, it was noticed that
propolis decreased chemical changes induced by 1, 2-dimethlyhydrazine (DMH) in genetic
material (DNA) of colon cells (Lima et al., 2005).
Modern research explored the preventive effect of propolis against serum lipid
oxidation caused mineral elements. Propolis disrupted oxidation reaction at different steps
including initiation and propagation of oxidative reactions (Isla et al., 2001). In another study
it was proved that intake of propolis lessened the concentration of malondialdehyde in
plasma and increased the activity of antioxidative enzymes cessesto alter the red blood cells
processes (Jasprica et al., 2007 and Righi et al., 2011). It was cleared from previous
invetigations that propolis collected from different geological areas bears different
composition and antioxidant status. Bioactive compounds associated with propolis influenced
variety of physiological pathways to inhibit the onset of ailments. Antioxidative potential of
propolis explored its pivotal role in oxidative stress management due to its bioflavonoids.
2.6.1. Novel approach against hyperglycaemia
Diabetes mellitus is a metabolic disorder caused by abnormality of one of the
endocrine gland. Generally diabetes spread due to insufficient insulin production, its
malfunction or both depending upon the onset of disease. Low level of insulin also disturbs
the metabolism of various biomolecules including proteins, fats and carbohydrate. There are
200 million peoples affected around the globe in the year 2010 and this number might be
projected to 300 million in coming years by 2025 (Bastaki, 2005). Diabetes is characterized
by hyperglycaemia and glycosuria due to insufficient production of insulin. High circulating
blood glucose reduced the absorption of blood sugar by body tissues and promotes the
24
release of glucose from hepatic tissues. Furthermore, glycosuria occurred due to excessive
uptake of glucose by renal tissues from blood. Diabetes mellitus induces a number of
abnormal physiological interactions associated resulting in degeneration of nerve tissues that
leads towards diabetic neuropathy. Excessive production of free radicals imparted a key role
in progression of nephropathy associated with diabetes. Different research groups utilize
streptozotocin (STZ) to induce diabetes among experimental organisms to measure the extent
of antidiabetic products (Sforcin and Bankova 2011).
Propolis has strong hypoglycemic potential due to rich polyphenol chemistry and help
to protect islets of pancreas to improve function of β-tissues of pancreas. Propolis helps in
prevention of diabetes and associated complications due to change in glucose absorption
followed by carbohydrate uptake. (Matsui et al., 2004 and El-Sayed et al., 2009). Matsushige
et al. (1996) explored that aqueous extract of propolis in a dose of 200mg/kg exert strong
preventive action on β-cells of pancreas by hindering the production of 1L-1β and inhibiting
the NO activity. According to Fuliang et al. (2005) regular administration of propolis extract
up to seven weeks to laboratory animals in which diabetes induced by STZ; there was a
decrease in the glycemic index along with modulation of oxidative stress to inhibit
peroxidation induced by diabetes. Zamami et al. (2007) conducted study on rats fed on high
fructose diet and inferred that intake of propolis in a dose of 100-300mg/kg of body weight
for two months considerably alter insulin level and reduced body weight without interrupting
level blood glucose. Diabetic nephropathy regarded concern of physicians during prevalence
of diabetes, while intake of propolis up to 300mg/kg retarded progression of renal
dysfunction by reducing load of free radicals (Abo-Salem et al., 2009). The lesion formation
at injured site considered major problem during diabetes that can be treated with propolis.
Propolis induced regeneration of tissues in diabetic wounds due to epithelial cell
differentiation at the site of wounds. Propolis can be used for topical application in diabetic
patients to treat wounds and leisions (McLennan et al., 2008). Abnormal production of ROS
and cytokines due to inflammatory response caused destruction of pancreatic cells, while
uptake of alcoholic extract of propolis improves many histological, physiological and
biochemical processes in animals infected with pancreatitis (Buyukberber et al., 2009).
Ethanol extract of propolis reduced plasma sugar level in diabetic animals induced by
alloxan. It also imparts beneficial action on lowering blood glucose concentration in humans
25
affected by diabetes type 2 disease. A number of studies explored that bioactive components
of propolis reduced blood glucose level and modulate lipid metabolism interrupted by
oxidative stress. Ultimately, it plays a promising role in the management of diabetes and
associated complications by inhibiting intestinal maltase activity. Propolis could be an active
functional food or supplement to reduce insulin resistance for managing hyperglycemia (Al-
Hariri, 2011). Li et al. (2012) conducted a study on STZ induced type 2 diabetic animals.
They inferred that oral administration of encapsulated propolis for 10 weeks showed
significant reduction in blood glucose by increasing the insulin sensitivity. Propolis also
modulates lipid functioning without affecting body weight and other physiological pathways.
Recently, Al-Hariri et al. (2011) investigated propolis with insulin hormone attenuate
hyperglycaemia in diabetic rats. During experiments STZ used for induction of diabetes,
animals were sacrificed. Different parameters were recorded including bone density, minerals
(calcium, phosphorus, magnesium) and fasting blood glucose to identify the role of propolis
and insulin on hyperglycaemia. They concluded that propolis and insulin exert positive effect
on pancreatic islets activity, oxidative stress, glucose concentration and osteopathy in
animals.
Caffeic acid phenyl ester (CAPE) , major chemical constitute of propolis reduced
level of melondialdehyde (MDA) and promote the level of superoxide dismutase (SOD) and
glutathione peroxidase (GSH-Px) in diabetic animals to prevent the complications of
oxidative stress on cardiac tissues induced by diabetes (Rahimi et al., 2005). CAPE proved a
safe approach to treat oxidative damage due to diabetes mellitus as it has no harms on normal
body. Previous studies revealed that administration of CAPE at 10µM concentration stop the
generation of ROS in blood cells especially cells involved in body defence ultimately help to
manage post diabetic effects on body and liver cells (Yilmaz et al., 2004). Matsushige et al.
(1996) conducted a research trial on artificially induced diabetic rats grou and demonstrated
that aqueous extract of propolis inhibit destruction of β-cells of pancreas and its effect found
similar to nicotinamide. Water extract of propolis prevent the production of interleukin 1-β
(IL-1β) from white blood cells. Inhibition of free radicals production with suppression of IL-
1β and nitric oxide synthase activity play significant role in suppression of toxicity induced
by STZ in diabetic animals.
26
Matsui et al. (2004) isolated 3,4,5-tri-caffeoylquinic acid (tri-CQA) from Brazilian
propolis as potent agent for hypoglycaemic activities. They confirmed that propolis has
remarkable action on inhibition of maltase activity preferably than sucrase in intestine in
order to modulate postprandial blood sugar. The 3,4,5-tri-CQA also involve in homeostasis
due to modulation of different physiological processes thus exert positive effect on reduction
of blood glucose level through retarding maltase activity to manage of noninsulin dependent
diabetes mellitus. Kang et al. (2010) concluded that propolis may be used as functional
ingredient in food and drinks to improve health and attenuated the diabetes and its
complications. According to their findings propolis exerted antagonistic action on insulin
resistance diabetes due to inhibition of gluconeogenic gene expression, glucose-6-
phosphatase activity, influencing tyrosine phosphorylation of glycogen synthase kinase-α
(GSK- α) and glycogen synthase kinase-β (GSK- β) . Similarly Aoi et al. (2013) carried out
study on otsuka long evans tokushima (OLEFT) rats modelling. Ten weeks old animals were
divided into three categories and fed with 0.1% and 0.5% propolis diet to explore the role of
propolis on insulin sensitivity and hypertension. After a period of eight weeks, animals
sacrificed and blood parameters; plasma insulin, blood glucose, interstitial fluid pH etc. were
assessed to explain the effect of propolis diet. They found significant reduction in blood
glucose concentration that linked with increased insulin activity in propolis diet groups with
reference to control. Propolis reduced systolic pressure without influencing aldosterone
concentration and averted the chemistry of tissue fluid in liver and skeletal tissues by
changing sodium absorption thus it would be a natural treatment to address diabetes and
cardiovascular problems.
2.6.2. Hypocholesterolaemic perspective of propolis
Cholesterol is an important hydrophobic compound in various biomolecules involved
in many metabolic pathways of human body. Four different types of lipoproteins; very low
density lipoprotein (VLDL), chylomicron, low density lipoprotein (LDL) and high density
lipoproteins (HDL) required cholesterol for their transportation in bold plasma. Various
atherogenic diseases like coronary heart disease, hypertension, cardiovascular ailments,
stroke and heart attack are associated with elevated cholesterol level (Chan et al., 2012). A
number of cardiac problems are associated with high level of blood cholesterol.
Accumulation of lipid in blood vessels caused atherosclerosis that occludes normal blood
27
flow. During hypercholestolaemia and atherosclerosis, elastic nature of arterial endothelium
impaired that promotes endothelial dysfunction. Peroxidation of LDL and systemic
inflammation influenced the endothelial membrane damage which considered a major risk
factor in coronary heart diseases (Le and Walter, 2007).
Since last few years, much attention has been paid to natural bioflavonoids to explore
their potential against hyperlipidemia. Previous studies advocated the role of polyphenols and
flavonoids to attenuate the condition of abnormal blood lipid profile. Blood vascular diseases
considered leading cause of increase death rate due to poor eating habits and sedentary life
style. High cholesterol and LDL are important risk factors to facilitate the progression of
atherosclerosis. However, many investigations indicated antagonistic relation between
dietary polyphenols and cardiac ailments (Verschuren et al., 2011). Atherosclerosis, due to
dyslipidemia is a leading concern of morbidity and death rate in both developed and
developing communities. It was assumed that atherosclerosis affects more number of peoples
in comparison to cancer. Propolis inhibits the oxidation of LDL to prevent cardiac tissue
damage due to their anti-oxidative potential. Additionally, propolis bioactive compounds
possess potential to grab endothelial deformities and other blood vascular problems (Purohit
et al., 2012).
According to Fang et al. (2013) propolis extract possessed potential against
atherosclerotic lesion induced by high fat diet. Propolis involved in cholesterol modulation,
inhibit inflammatory response of endothelial tissues and protect blood vessels to stop the
phenomenon of atherosclerosis. Similarly, Yousef et al. (2005) demonstrated that
bioflavonoids inhibit the onset of cardiovascular and heart diseases due to lowering plasma
lipids and inhibition oxidation due to excessive ROS. Quercetin, an important flavonoid
found of propolis and regarded key element to reduce oxidation of LDL and helps to lower
bad cholesterol level plasma (Frankel et al., 1998). Lipids peroxidation involved in
progression of pathophysiology of blood vascular disorders leads to chronic atherosclerosis.
The removal of lipid peroxyl radicals by natural polyphenols inhibits atherosclerotic effects.
Polyphenols also reduced oxidation of lipoproteins and suppress the production of oxidative
products of lipids in plasma thus reduces the risk of blood vascular diseases (Stocker and
Keany, 2004). Humans absorb natural antioxidants from daily diet to inhibit LDL oxidation
and protection from onset of atherogenic disorders (Kamiya, et al., 2004).
28
Propolis is a complex resinous product imitative originated from botanical source,
considered a potent and safe produce for hypocholesterolemic activity. Nowadays, propolis is
being explored as a drug of choice from nature against dyslipidaemia and other oxidative
stress conditions to promot human health (Orsolic and Basic, 2006). Previously, Kolankaya
et al. (2002) examined the role of propolis in laboratory against alcohol induced oxidative
stress and other blood indicators. During the study animals were fed with 200mg/Kg of body
weight per day with ethanol extract of propolis for 15 days. At the end of experiment various
blood parameters including HDL, LDL and different enzyme concentration were evaluated
and compared with control group. They observed that propolis extract induced retardation in
LDL level, triglyceride contents and total cholesterol level. Furthermore they examined
increased in HDL concentration as well as in concentration of various antioxidative enzymes.
Similarly, Mani et al. (2006) demonstrated different levels of popolis to explain their effect
on body tissues of animals. They concluded that regular uptake of 1mgKg-1
day-1
of propolis
does not induce any negative change in living organisms but promoted balance in blood lipid
chemistry to lower the chance of myocardiopathy. Castaldo and Capasoo (2002) explored
the effect of propolis on lipid profile and inferred that intake of propolis modulate lipid
metabolism by influencing the metabolic pathways and lowers LDL, VLDL, triglycerides
with considerable increase in concentration of HDL. Furthermore, they observed that daily
intake of plant bioactive compounds like flavonoids from natural sources including propolis
lowers LDL-HDL cholesterol level by promoting the level of HDL cholesterol (Kurowska
and Manthey, 2004). Similarly, Fernandes et al. (2006) carried out a research on rabbits to
explore the role of propolis against atherogenic diseases induced by hypercholesterolemia.
During the study hyperlipidaemia induced by atherogenic diet in experimental rabbits and
found increased concentration of total cholesterol, LDL, VLDL and triglyceride level.
Animals were fed with ethanol extract of propolis (EEP) in a dose of 100mg/Kg/day for 56
days. At the end of study, they observed that EEP significantly reduced the level of
triglycerides, total cholesterol, LDLs while increased the concentration of HDLs. They
concluded that EEP possessed protective role against dyslipidaemia and hepatotoxicity
induced by atherogenic diet. In another study conducted by Kolankaya et al. (2002)
investigated the protective role of Turkish propolis against alcohol induced oxidative stress.
Experimental animals were given propolis in 200mg/Kg body weight per day for 15 days. At
29
the end of study they observed significant reduction in LDLs, total cholesterol, triglyceride
level, whereas sharp increase in HDL was observed among animals those were treated with
propolis in comparison to control group.
30
Chapter 3
MATERIAL AND METHODS
The present study was carried out in the Postgraduate Research Laboratories of
National Institute of Food Science and Technology (NIFSAT), Department of
Biochemistry, University of Agriculture Faisalabad (UAF) and School of Agriculture,
Food & Rural Development, Newcastle University, Newcastle Upon Tyne, United
Kingdom. Characterization of honey bee propolis, collected from surrounding areas of
Faisalabad region, was done for its chemical profiling, antioxidant and antimicrobial
status. Its functional and therapeutic role was elucidated by developing functional drink
and using animal modelling system. The materials used and procedure followed during
the entire study is stated herein;
3.1. Materials
Raw bee propolis was collected in collaboration with Department of Entomology,
UAF. Analytical reagents, HPLC grade chemicals and standards were purchased from
Merck (Merck KGaA, Darmstadt, Germany) and Sigma-Aldrich (Sigma-Aldrich Tokyo,
Japan). Selected pathogenic bacterial strains (Eschericia coli ATCC-35218,
Staphylococcus aureus ATCC-6633, Bacillus subtilis ATCC-25923) for antimicrobial
perspective were obtained from Department of Biochemistry, UAF. Laboratory animals
(Sprague Dawly rats) used for in-vivo studies were purchased from National Institute of
health Islamabad and kept in animal room of NIFSAT. For the estimation of bioassays,
the diagnostic kits were purchased from Sigma-Aldrich, Bioassay (Bioassays Chemical
Co. Germany) and Cayman Chemicals (Cayman Europe, Estonia
3.2. Characterization of Honey Bee Propolis
Honey bee propolis was subjected to estimation of various quality attributes
including proximate composition, mineral contents, polyphenols contents and
characterization. The detail is given as follows;
3.2.1. Compositional analysis
The Propolis was analysed for its proximate composition under standard
conditions for the following parameters and results were taken as means of triplicates.
31
3.2.1.1. Moisture content
Moisture contents of bee propolis were determined by drying the (5g) sample in
Air Forced Draft Oven (Model: DO-1-30/02, PCSIR, Pakistan) at 105±5°C till constant
weight according to AACC (2000) Method No. 44-15A.
3.2.1.2. Crude protein
Crude protein was estimated by using Kjeltech Apparatus (Model: D-40599, Behr
Labor Technik, Gmbh-Germany) as specified in AACC (2000) Method No. 46-30.
3.2.1.3. Crude Fat
Soxtec System (Model:H-2 1045 Extraction Unit, Hoganas, Sweden) was used for
the estimation of crude fat (ether extract) using petroleum ether as solvent according to
protocol of AACC (2000) Method No. 30-25.
3.2.1.4. Crude fibre
Fat free samples were subjected to crude fibre estimation by digesting sample with
1.25% H2SO4 and 1.25% NaOH solution using Labconco Fibertech (Labconco
Corporation Kansas, USA) as described in AACC (2000) Method No.32-10.
3.2.1.5. Total ash
Ash in each dry sample was estimated by direct incineration in Muffle Furnace
(MF-1/02, PCSIR, Pakistan) at 550°C after charring till greyish white residue (AACC,
2000; Method No. 08-01).
3.2.1.6. Nitrogen free extracts (NFE)
NFE was calculated using the following expression by subtraction:
NFE % = 100 – (Moisture + CP + CF + CF + Ash)
CP = Crude protein
CF = Crude fat
CF = Crude Fibre
3.2.2. Mineral Analysis
Bee propolis explored for mineral contents using wet digestion principle under the
guidelines described in AOAC (2006). Flame Photometer-410 (Sherwood Scientific Ltd.,
Cambridge) and Atomic Absorption Spectrophotometer (Varian AA240, Australia) was
used for quantification of different minerals according to sensitivity of the detectors.
32
3.3. Preparation of Propolis Extracts
Honey bee propolis was subjected for preparation of propolis extracts following
procedure of Yaghoubi et al. (2007) and Christov et al. (2006) with some modifications.
Different solvent namely, water, ethanol (three levels; 95%, 80%, 65%) and methanol
(three levels; 95%, 80%, 65%) were used under dark conditions for the preparation of
propolis extracts (Table 3.1). The extracts thus obtained filtered through vacuum
filtration technique and centrifuged at 3000rpm for 15 min. The supernatant obtained was
placed at -20°C for 24h to remove waxy residues and concentrated through rotary
evaporator (EYELA, N-N series, Japan) under reduced pressure at 40°C to recover
solvent.
Table. 3.1. Treatment plan of propolis extract
Treatments Extraction Plan
T1 Water Extract of propolis
T2 Ethanol (95%) Extract of propolis
T3 Ethanol (80%) Extract of propolis
T4 Ethanol (65%) Extract of propolis
T5 Methanol (95%) extract of propolis
T6 Methanol (80%) extract of propolis
T7 Methanol (65%) extract of propolis
33
3.4. Analysis of propolis extracts
3.4.1 Total Polyphenol contents (TPC)
Spectrophotometric technique was applied to measure total phenolic contents of
resultant propolis extracts using Folin-Ciocalteu reagent according to procedure adopted by
Singleton et al. (1999). According to this method 125 μL of extract was mixed with 125 μL
of Folin- Ciocalteau reagent followed by the addition of 500 μL water. The mixture was
allowed to stay under dark for 5 min at room temperature. 4.5mL of 7% sodium bicarbonate
solution was added to the mixture and placed again in dark for 90 min. and afterwards
absorbance was checked using Spectrophotometer (UV/Vis, CECIL CE7200) at 765nm
against control (without sample). Total polyphenols of each sample was calculated using
standard curve and results were expressed as gallic acid equivalent (mg/gGAE).
3.4.2 Free radical scavenging activity of bee propolis (DPPH assay)
Free radical scavenging activity of propolis extract was assessed using 2,2-diphenyl-
1-picrylhydrazyl (DPPH) assay as described by Brand-Williams et al. (1995). Four mL of
each extract was taken in test tubes, 1mL of DPPH solution was added in each test tube and
was incubated at room temperature for 30 min. Absorbance of sample mixture was observed
at 520 nm through spectrophotometer (CECIL CE7200). The percent inhibition of DPPH by
each extract was calculated using the following equation as;
Reduction of absorbance (%) = [(AB - AA) / AB] × 100
Where,
AB = absorbance of blank sample (t = 0 min)
AA = absorbance of tested extract solution (t = 30 min)
3.4.3. Antioxidant activity by β-carotene system of propolis extracts
In-vitro antioxidant potential of each extract (samples) was examined using β-
carotene and linoleic acid oxidation system as specified by (Taga et al., 1984). Two mg of -
carotene was mixed with 20mL chloroform, 40mg linoleic acid and 400mg of tween20 to
form an emulsion. The resultant emulsion heated in water bath to remove chloroform. Three
mL of resultant emulsion was added to 0.10 mL of sample and incubated in water bath for
120 min. β-carotene oxidation in each sample was measured through spectrophotometer at
470 nm. Antioxidant activity (AA) was expressed as percent inhibition in relation to control.
34
AA= Degradation rate of control- Degradation rate of sample × 100
Degradation rate of control
3.5. High performance liquid chromatography (HPLC) analysis of propolis
extracts
Bee propolis extracts were subjected for polyphenols characterization through HPLC
following the protocol as designed by Ahn et al. (2007) with some modifications.
Schimadzu, series 10 A, Japan, HPLC system equipped with C18 column (250 mm x 4.6
mm, 5.0 μm particle size) was used for analysis. A 10µL of sample was injected using
manual injection and column at adjusted temperature at 40°C throughout the analysis.
During phenolic quantification mobile phase was used as water and acetic acid as 94:6%
(A) and 100% acetonitrile (B) by maintaining gradient elusion and flow rate of 1ml/min
followed by passing through UV/Vis detector (wavelength at 280 nm).
3.6. Evaluation of antimicrobial potential of bee propolis
The propolis extract samples were individually tested against food borne pathogenic
bacteria (E. coli ATCC-35218, S. aureus ATCC-6633, B. subtilis ATCC-25923) obtained
from Department Biochemistry, UAF. Primary screening was done through disc diffusion
method and secondary screening to determine minimum inhibitory concentration of each
extract according to protocol as specified by kartal et al., (2003) and National Committee for
Clinical Laboratory Standards (NCCL), (1985) with some modifications in reference to
laboratory conditions. The detail experiment procedure followed is stated herein under
various sub-headings;
3.6.1. Antibacterial activity
Three different strains of pathogenic bacteria; E. coli ATCC-35218, S. aureus
ATCC-6633, B. subtilis ATCC-25923 were used to study the antibacterial activity of the
propolis extracts during the experiments.
3.6.2. Preparation of inoculum
Cultures of S. aureus, B. subtilis, and E. coli were prepared in nutrient broth,
incubated at 37°C with continuous shaking for 24 hours and desired growth was maintained
at 1×105-6
cfu/mL of bacteria. The prepared culture of desired concentration was placed in
refrigerator at 2-8°C until further use (Dhale and Markandeya, 2011).
35
3.6.3. Disc Diffusion assay
For primary screening disc diffusion method used to assess antimicrobial activity of
propolis extracts. Nutrient agar (Oxoid, Japan) was prepared by dissolving 37g of nutrient
agar in one litre of distilled water, autoclaved at 121°C for 15 min with all the materials used
for the assay. Approximately 15-20 mL of media transferred in each petri plate and allowed
to solidify under laminar air flow cabinet (Culture hood) with addition of 100 µL of
inoculum for desired growth of respective strain on the petri plates. 10 mm discs of wicks
sheets were prepared, autoclaved, soaked in 100 µL of 12mg/mL of each extract and placed
on petri plates using rifampicin as positive control. Prepared discs were placed on petri plates
on marked site by maintaining suitable distance for the development of proper zone of
inhibition. After administration of extract soaked discs the petri plates were incubated at
37°C for 24 hours and zone of inhibition was recorded in millimetres.
3.6.4. Determination of minimum inhibitory concentration
The minimum inhibitory concentration defined as the lowest concentration able to
inhibit any visible bacterial growth and assessed separately for each propolis extract. The
minimum inhibitory concentration (MIC) of extracts was determined spectrophotometrically.
Broth micro-dilution method was used to determine MIC values according to method stated
by Mehmood et al. (2012). This method includes 96 well microtitre plate containing Muller
Hinton (MH) broth medium used for spectrophotometric assay. Each extract was used at
concentration of 12mg/ml and diluted periodically in wells to determine the minimum
inhibitory concentration value for each extract. Thereafter 10μl inoculum was added to each
well containing nutrient broth and standard drug (Rifampicin) was used as positive control.
The microtiter plates were incubated at 37°C for 24 hours and 10µL of resazurin solution (an
indicator) added in each well and absorbance was recorded at 620nm using ELISA reader.
3.7. Product development
3.7.1. Functional/nutraceutical drink
Functional drink (Nutra-1) was developed using two selected treatments (Ethanol and
Methanol 65% each) out of other treatments on the basis of in-vitro antioxidant potential,
HPLC characterization and quantification. The major ingredients used for functional drink
include aspartame, citric acid, sodium benzoate, carboxy methyl cellulose (CMC), food grade
colour and flavour along with propolis extract of selected treatments at the dose rate of 400
36
mg/500mL of functional drink. Due to more prevalence of hot summer season in the country,
Nutra-1 was prepared with added extracts as it could be consumed trough out the year.
Table 3.2. Treatments used for the preparation of functional drinks
Treatments Description
To Drink prepared without propolis extract
T1 Functional drink prepared with ethanol extract of propolis
T2 Functional drink prepared with methanol extract of propolis
T0= Drink prepared without propolis extract
T1= Functional drink prepared with ethanol extract of propolis
T2= Functional drink prepared with methanol of extract of propolis
3.7.2. Physicochemical analysis of functional drinks
Functional drinks were analysed for their physicochemical and sensory attributes at
different intervals during storage.
3.7.2.1. pH
pH was recorded directly through calibrated pH meter (InoLab 720, Germany)
according to method described in AOAC, 2006.
3.7.2.2 Total acidity
Total acidity of functional drink samples was determined by titrating the sample
against 0.1N sodium hydroxide solution to persistent pink colour following protocols of
AOAC, 2006.
3.7.2.3. Total Soluble Solids
Total soluble solids (TSS) were estimated by digital refractometer (Koyo Hoto,
Model No. RA-360, Japan) and results were expressed as percent soluble solids (°Brix).
3.7.2.4. Sensory evaluation
Functional drinks prepared with treatments (To, T1, T2) were evaluated for sensory
attributes and consumer acceptability by panel of judges from NIFSAT, UAF using 9 point
hedonic scale (9= extremely like; 1= extremely dislike) as specified by Meilgaard et al.
(2007). On the day of evaluation, drinks were presented to panelists in transparent glasses
37
labelled with random codes. The panelists were provided with water and unsalted crackers to
neutralize their mouth feel between samples testing. Samples were presented to panellists
randomly and were asked to rate their acceptance by giving score for selected parameters that
included colour, flavour, sweetness, sourness and overall acceptability (Appendix-1) at
different days during the period of storage.
3.8. Efficacy studies
For the elucidation of therapeutic role of propolis, ninety male Sprague Dawly rats
were purchased from National Institute of Health (NIH), Islamabad and kept in animal room
of NIFSAT with maintenance of proper light and dark period and air circulation. During
three different types of studies, animals were fed with normal diet, high sucrose diet and high
cholesterol diet to explore therapeutic role of functional drinks prepared with propolis extract
on the selected blood indicators including serum lipid profile, blood glucose level, insulin
levels, liver function test and kidney function test. Three different studies were carried out
separately whilst the results of all studies expressed collectively for better understanding.
3.8.1. Study-I: Normal diet
Rats were acclimatized by feeding basal diet for one week. Initially efficacy trials
were conducted in rats fed on normal diet. For the purpose, thirty Sprague Dawley rats were
randomly divided into three groups, of ten each. The functional drink prepared from the
selected treatments was provided for a period of 8 weeks. The experimental diet (Appendix-
II) comprised of corn oil (10%), protein (10%), corn starch (66%) and cellulose (10%),
mineral (3%) and vitamin mixture (1%) with simultaneous intake of functional drinks. The
temperature (23±2ºC) and relative humidity (55±5%) were maintained throughout the
experiment with 12 hours light-dark period. Feed & drink intake was recorded daily whilst
body weight on weekly basis throughout the experiment. After eight weeks of study,
overnight fasted rats were sacrificed and blood samples were collected through cardiac
puncture; EDTA coated tubes employed for serum collection to perform various assays.
Following similar approach, two other studies were conducted to determine the impact of
functional drinks against respective diets i.e. high sucrose and high cholesterol in separate
animal modelling system.
38
3.8.2. Study II: High sucrose diet
In study-II, high sucrose diet containing 40% sucrose was given to normal rats to
induce Hyperglycaemia. At the same time, effect of functional drinks on induced traits in
respective groups of rats was assessed by testing blood glucose and serum insulin level.
3.8.3. Study-III: High cholesterol diet
In study-III, high cholesterol diet containing 1% of cholesterol was fed to normal rats
to raise their lipid profile i.e. cholesterol, high density lipoprotein (HDL), low density
lipoprotein (LDL) and triglycerides concentrations. The functional drinks were also provided
to rats groups simultaneously to synchronize their effect on the respective blood parameter.
Table.3.3. Different studies conducted in efficacy trials
Study-I Normal diet
Study-II High sucrose diet
Study-III High cholesterol diet
The outlines of these studies are here in:
Table.3.4. Diets and functional drinks plan
Studies
Normal diet High Sucrose Diet High Cholesterol Diet
Control rats Diabetic rat Hypercholesterolemic
rats
Groups 1 2 3 1 2 3 1 2 3
Drinks To T1 T2 To T1 T2 To T1 T2
T0: Drink prepared without propolis extract
T1: Functional drink prepared with ethanol extract of propolis
T2: Functional drink prepared with methanol extract of propolis
39
The following parameters were recorded separately in all rat modelling studies.
3.8.4. Feed and drink intake
Net feed intake of each group was measured daily by excluding spilled diet from the
total diet during the entire study period (Wolf and Weidbrode, 2003). The functional drink
intake of each group was also recorded daily by monitoring the difference in graduated
bottles.
3.8.5. Body weight
Gain in body weight of experimental groups was measured weekly throughout the
study period to monitor the effect of functional drink and study weeks on body weight.
3.8.6. Serum lipid profile
Serum lipid profile including cholesterol, high density lipoproteins, low density
lipoproteins and triglycerides were measured according to their respective protocols. The
detail of each is given below:
3.8.6.1. Total Cholesterol
Serum cholesterol level was determined using CHOD-PAP method following the
protocol of Stockbridge et al. (1989).
3.8.6.2. High density lipoprotein
High density lipoprotein (HDL) was measured by HDL Cholesterol precipitant
method as mentioned by Assmann (1979).
3.8.6.3. Low density lipoprotein
Serum samples were analysed for low density lipoproteins (LDL) following the
procedure of McNamara et al. (1990).
3.8.6.4. Triglycerides
Total triglycerides in serum samples were determined by liquid triglycerides (GPO-
PAP) method as outlined by Annoni et al. (1982).
3.8.7. Serum glucose and insulin levels
In each study, rats serum samples were evaluated for glucose concentration by GOD-
PAP method as described by Thomas and Labor (1992) whereas insulin level was assessed
following the method of Besch (1987).
40
3.8.8. Liver functioning tests
Liver function tests including aspartate aminotransferase (AST), alanine
aminotransferase (ALT), and alkaline phosphatase (ALP) were assessed. Levels of AST and
ALT were measured by the dinitrophenylhydrazene (DNPH) method using Sigma Kits 59-50
and 58-50 respectively and ALP by Alkaline Phosphates–DGKC method (Thomas, 1998;
Moss and Henderson, 1999).
3.8.9. Kidney functioning tests
The blood samples were subjected to urea and creatinine analysis by GLDH-method
and Jaffe-method respectively through commercial kits to examine the renal functionality in
all study groups (Jacobs et al., 1996; Thomas, 1998).
3.9. Statistical analysis
Completely Randomized Design (CRD) was applied and resultant data was subjected
to statistical analysis using suitable statistical package. Analysis of variance technique
(ANOVA) was used to determine the level of significance (Steel et al., 1997).
41
CHAPTER 4
RESULTS AND DISCUSSION
Bioactive compounds from plants and animals provide multidisciplinary actions
against various pathogens to retard their growth and reproductive behavior as well as provide
general health benefits in various ways. In this reference honey bee propolis is an important
natural product holding potential characteristics to retards the growth of food borne
pathogens and helps to attenuate various diet related maladies including hyperglycemia and
hypercholesterolemia etc. In the present study, locally available propolis was analyzed for its
composition, polyphenol extraction and characterization along with its in-vitro antioxidant
properties and antimicrobial potential against selected food borne pathogens. The therapeutic
role of propolis was highlighted by developing functional drinks that further utilize in
bioefficacy study to elucidate its nutraceutical role against selected metabolic disorders. The
results of present study for various parameters are discussed under following headings;
4.1. Compositional analysis of bee propolis
Physicochemical analysis of experimental material is a mandatory step for the
evaluation of component of interest. In this concern, proximate composition is an important
factor for estimation about the quality of raw material. Raw bee propolis was subjected for
the analysis of various compositional attributes and observed that moisture content, crude
protein, crude fat, crude fiber, ash content and nitrogen free extract (NFE) was found to
2.22±0.14, 1.84±0.09, 85.59±0.87, 0.31±0.08, 1.03±0.04 and 9.01±0.05% respectively
(Table: 4.1). The results pertaining to proximate compositions of bee propolis during the
present study were in accordance with the previous findings of Kim et al., (2002), who
investigated the composition of propolis for moisture contents, crude protein, crude fat, ash
contents and carbohydrate contents as 3.00, 0.70, 90.90, 0.20, and 5.30% respectively. Jeong
et al. (2003) explored the proximate composition of bee propolis from different regions and
described as crude fat (86.40%), crude protein (2.71%), crude fiber (0.20%), ash (1.05%) and
NFE (7.32%). Likewise Song and Gil (2002) examined the composition of bee propolis
collected from falsacacia and chestnut tree and found that propolis comprised of moisture
content (3.60-3.90%), crude lipids/fats (81.1-86.9%), crude protein (2.0-2.50%), crude fiber
(3.50-4.00%) and ash (1.10-1.50%).
42
Table: 4.1. Compositional analysis of bee propolis
Components Quantity (%)
Moisture 2.22±0.14
Crude fat 85.59±0.87
Crude protein 1.84±0.09
Crude fiber 0.31±0.08
Ash 1.03±0.04
NFE 9.01±0.05
Values are expressed as means of triplicates ± standard deviation
NFE= Nitrogen Free Extract
43
The source of propolis is plant exudates that vary from plant to plant and region to region.
The plant species found in Brazil and China are different from Pakistan that may be
attributed to climate and geographical variations which is critical in determining the
composition of propolis. Therefore the results regarding physicochemical composition of
locally available propolis are somewhat different from other regions due to the
environmental, seasonal and phytogeographical variations found in Pakistan (Daleprane et
al., 2012).
4.2. Mineral contents of propolis
Bee propolis is a natural product of versatile nature having polyphenols and
flavonoids along with a number of mineral elements including iron, zinc, copper, magnesium
and manganese as well as varied amount of vitamins B1, B2, E and Vit.C also associated with
propolis (Jean 1988, Atiasov et al., 1990, Liviu-Alexandru, 1997). Mineral composition of
bee propolis during the current study presented in Table: 4.2. The mineral profile was
consisted of calcium (10.53±0.80mg/Kg), potassium (52.10±2.90mg/Kg), sodium
(11.33±0.91mg/Kg), magnesium (32.13±2.30mg/Kg), iron (29.3±1.70mg/Kg), zinc
(3.59±0.23mg/Kg), copper (1.50±0.10mg/Kg) and manganese (0.67±0.03mg/Kg). The
results obtained are in comparison to previous findings of Nicolae and Tatiana (2007), who
investigated micro and macro elemental composition of propolis collected from different
regions of Maldovia. They observed that propolis possessed zinc (39.10-98.60mg/Kg),
manganese (8.40-14.60mg/Kg), copper (2.40-13.70mg/Kg), calcium (1.00-4.27mg/Kg),
magnesium (179.0-255.80mg/Kg), iron (407.50-531.10mg/Kg), sodium (3.13-5.94mg/Kg)
and potassium (0.75-1.00g/Kg). Similarly, Bonvehi and Bermejo (2013) observed various
inorganic elements in different samples of propolis and the values for the measured elements
were ranged as 93–225mg/kg 1773-6683mg/kg, 735–4790, 301–1405, 301–1405, 163–1,364,
312–1270mg/kg for sodium, calcium, potassium, magnesium, zinc and iron respectively that
were in agreement with present outcomes. They used spectrometric procedure and depicted
that variation in mineral composition of propolis attributed due to climate, plant origin and
nature of bee in the hive and similar effect related to variation in mineral profile obtained in
this manuscript.
4.3. Analysis of honey bee propolis extracts
4.3.1. Total polyphenol contents (TPC) mg/g Gallic Acid Equivalent (GAE)
44
Table: 4.2. Mineral contents of bee propolis
Minerals (mg/Kg)
Calcium 10.53±0.8
Potassium 52.10±2.9
Sodium 11.33±0.91
Magnesium 32.13± 2.3
Iron 29.3±1.7
Zinc 3.59±0.23
Copper 1.50±0.1
Manganese 0.67±0.03
Values are expressed as means of triplicates (n=3) ± standard deviation
45
Phytochemicals are the chemical substances those exhibits a potential role for the modulation
of metabolic processes of human body thus helps in the prevention of different degenerative
and atherogenic disorders (Tripoli et al., 2007). Polyphenols obtained from botanical sources
are associated with numerous biological properties including anti-inflammatory, anti-radical,
anti-oxidant and antimicrobial activities those were found in bee propolis. Plant phenolics;
the important class of polyphenols are plant secondary metabolites considered pivotal for
their antioxidant potential and usefulness in combating different maladies (Slade et al., 2005;
Williams et al., 2004).
The measurement of TPC is a good index for the antioxidant, antiradical and free
radical scavenging activities. The statistical values for the total phenolic contents of propolis
extracts (ethanol, methanol and water treated propolis) showed that treatments imparted a
highly significant effect on the on TPC values as presented in Table: 4.3 whereas the mean
values pertaining to TPC are shown in Table: 4.4. Different treatments were evaluated and
result showed the highest value of 327.30±14.89 mg/gGAE in ethanol extract (T4) followed
by 292.10± 15.27 mg/gGAE in methanol extract (T7) whereas minimum value 134.02±12.67
mg/gGAE was obtained in aqueous extract (T1). However, the overall values for TPC with
respect to other treatments was recorded as 239.10± 13.23, 251.71±22.43, 225.88±17.63 and
246.87± 19.37 mg/g GAE for T2 (95% ethanol extract), T3 (80% ethanol extract), T5 (95%
methanol) and T6 (80% methanol) accordingly.
The findings of present study regarding TPC values are in close conformity to the
earlier findings of Ahn et al. (2007), who examined the different samples of propolis from
different regions of China and noticed that the value of TPC ranged between 42.90±0.80-
302±4.30 mg/gGAE in alcoholic extract of propolis collected from various regions of china
which is at par to the highest level however variation among the results may be of climate,
geographical and botanical source dependant. They further stated that disparity among the
results of the study regarding various samples from different regions appeared due to
seasonal variation, collecting time and area of collection of propolis samples. Previously,
Kumazawa et al. (2004) investigated the polyphenol contents of ethanol extract of propolis
from different regions of Europe and China, after the evaluation of samples they inferred that
TPC ranged between 200 to 300 mg/gGAE in different samples of Europe and China.
46
Table: 4.3. Mean squares for the effect of treatments on the antioxidant indices of propolis extracts
SOV df TPC DPPH β-Carotene
Treatments 6 9342.99** 261.90** 136.89**
Error 14 241.22 15.89 11.95
Total 20
**= Highly significant
Table: 4.4. Mean values for the effect of treatments on TPC, free radical scavenging activity and antioxidant activity
Treatments Solvent TPC (mg/g GAE) DPPH Value (%) Β-Carotene assay (%)
T1 Water 134.02±12.67d
44.73±3.32d
39.21±2.83d
T2 Ethanol (95%) 239.10± 13.23c
61.26±4.58bc 50.14±3.37bc
T3 Ethanol (80%) 251.71±22.43c 66.93±3.62ab 49.94±2.86ab
T4 Ethanol (65%) 327.30±14.89a 73.18±4.43a 60.59±4.38a
T5 Methanol (95%) 225.88±17.63c 59.01±2.81c 48.70±2.52c
T6 Methanol (80%) 246.87± 19.37c 60.51±3.37bc 50.43±3.31bc
T7 Methanol (65%) 292.10± 15.27b
70.06±5.28a 57.01±4.38a
Values are expressed as means of triplicates ± standard deviation. T1: water extract T2: 95% ethanol extract, T3: 80% ethanol extract,
T4: 65% ethanol extract, T5: 95% methanol extract, T6: 80% methanol extract, T7: 65% methanol
47
Similarly, Choi et al. (2006) while studying the antioxidant and antimicrobial activities of
propolis from several regions of Korea observed the phenolic contents of propolis extracts
and their findings revealed that TPC values ranged from 120.07±3.5 to 212.7±7.4 mg/gGAE
but the results were found different from those of Europe and Brazil samples because of their
geographical variation and phytodiversity of the area.
4.3.2. Free radical scavenging activity and Antioxidant potential
The free radical scavenging activity using 2,2-diphenyl-1-picryhydrazyl (DPPH)
reagents is a widely accepted and supportive feature for the estimation of antioxidant
potential of natural extracts. The mean square values for antioxidant indices of propolis
extracts (Table: 4.3) indicated that solvent concentration imparted a highly significant effect
on the free radical scavenging activity and antioxidant indices of the extracts.
The mean values for DPPH percent inhibition was as for T1 (44.73±3.32%), T2
(61.26±4.58%), T3 (66.93±3.62), T4 (73.18±4.43%), T5 (59.01±2.81%), T6 (60.51±3.37%)
and T7 (70.06±5.28%) were obtained. Similarly the antioxidant potential values for
treatments T1, T2, T3, T4, T5, T6 and T7 were recorded as 39.21±2.83, 50.14±3.37,
49.94±2.86, 60.59±4.38, 48.70±2.52, 50.43±3.31 and 57.01±4.38% respectively. The highest
values for DPPH activity (73.18±4.43%) was observed in T4 followed by T7 (70.06±5.28%)
whereas minimum value for free radical scavenging activity (44.73±3.32) was noticed in T1.
Likewise, maximum antioxidant potential value (60.59±4.38%) was examined in ethanol
extract (T4) followed by methanol extract (T7) as 57.01±4.38 percent whereas the minimum
antioxidant potential value (39.21±2.83%) was observed in T1 as represented in Table: 4.4.
The radical quenching capacity of polyphenols is mainly due to hydrogen transferring
ability to free radicals and reactive oxygen specie produced during oxidative reaction
reactions (Teixeira et al., 2010). The results pertaining to free radical scavenging activity
during the current study are supported with the earlier outcomes of Sulaiman et al. (2011),
who demonstrated the free radical scavenging activity of propolis samples is correlated with
the presence of phenolic compounds in particular to its flavonoids contents those are most
effective anti-oxidative compounds of propolis. In another study conducted by Christov et al.
(2006) determined the DPPH radical scavenging activity of the two bee propolis samples
from two different regions of Canada and demonstrated that propolis extracts possess
significant free radical scavenging activity. At the end of study they deduced that free radical
48
quenching activity of propolis is in direct correlation with its total phenolic contents.
Previously, Choi et al. (2006) assessed DPPH inhibition of the ethanol extracts of propolis
from different origins by 35- 95% inhibition and observed that polyphenols are the active
components of propolis responsible for the quenching of free radicals. Afterwards, Ahn et al.
(2007) reported the phenolic composition and antioxidant status of various samples of
ethanol extract of propolis collected from different regions of China and found that propolis
samples from different origins possess different free radical scavenging activities, while
studying the antioxidant activity and constituents of propolis collected in various areas of
China they depicted that the samples from Neimongol, Hebei and Hubei showed highest
DPPH free radical-scavenging activity as 70% as compared to control. In another study
conducted by Moreira et al. (2008) exhibited the DPPH radical inhibition of propolis samples
collected from Bornes and Fundo (two regions of Portugal). After analysing the propolis
extract they deduced that the radical scavenging activity is concentration and region
dependant and Bornes samples showed inhibition activity from 33-94% whereas, Fundo
samples represented 18-57% inhibition activity.
Likewise, Kumazawa et al. (2004) observed antioxidant activity of propolis extract
using linoleic acid oxidation system and inferred that antioxidant activity is related to their
flavonoids contents as they are the most effective components of propolis. In a similar way
Choi et al. (2006) examined the different propolis samples from various regions of Korea for
their biological properties and observed that extract of propolis collected from Yeosu and
Cheorwon showed more antioxidant activity as compared to Brazilian propolis as a function
of polyphenol and flavonoids contents. Recently, Gregoris and Stevanato (2010) reported the
antioxidant activity of various components derived from propolis. Higher values for lipid
peroxidation using linoleic acid were observed for caffeic acids, quercetin, galangin and
kaempferol and three to six times lower values were noticed for apigenin, naringin, chrysin
and pinocambrin. However caffeic acid phenyl ester, a principle component in propolis
extract showed highest antioxidant activities as compared to other phenolic acids and
flavonoids found in propolis. Orsolic et al. (2012) examined the antioxidant status different
extracts of propolis using β-carotene degradation system concluded that extracts prepared
with different solvent showed varied antioxidant potential. They further depicted that water
soluble derivatives of propolis (WSDP) represented higher antioxidant potential in
49
comparison to ethanol extract of propolis (EEP) which is in contrast to previous findings in
which EEP showed high antioxidant status, the variation may be attributed to method used
for extract preparation and nature of bioactive components found during the study.
Furthermore, different solvents have different affinity for particular bioactive molecule.
Ethanol may dissolve maximum bioactive molecule thus showing higher activity as
compared to other solvent used during study. The difference in the results for free radical
scavenging activity is therefore, mainly due to variation in the amount of phenolic contents
which arises due to difference in nature and polarity of the solvents used for extraction.
Spigno et al. (2007) inferred that phenolic contents yield of the natural substances increased
with ethanol by adding water from 10% to 30% and remained constant for water
concentration upto 60% in ethanol. The polarity difference among the solvent provides
solubility difference. The solvent giving higher yield for antioxidants shows better properties
than the other. Similarly, water with difference in polarity in comparison to ethanol and
methanol shows variation. The binary solution of aqueous organic compounds exhibited
better phenolic contents, indicating good antioxidant indices. Therefore the extracts with
higher proportions of polyphenols showed more free radical scavenging activities and
antioxidant properties those are compareable with results of the present study (Sultana et al.,
2009)
4.3.3. Quantification of bioactive compounds through HPLC
High performance liquid chromatography (HPLC) is an ideal procedure for the
characterization and quantification of polyphenols and flavonoids from the natural extracts.
The various treatments were subjected to assess the bioactive components of extracts and
mean values pertaining to results of these bioactive compounds from propolis extract
represented the effect of solvent concentration on the extraction and quantification of
polyphenols (Table: 4.5).
In present study Caffeic acid recorded as 8.21±0.06, 21.66±2.10, 5.33±0.04,
4.13±0.80mg/kg and 6.39±0.02mg/Kg in different treatments of T1, T4, T5, T6 and T7
respectively whereas, gallic acid was noted as 0.21±0.01, 13.77±1.29, 0.75±0.06 and
1.06±0.80mg/Kg in T1, T4, T5 and T6 accordingly whereas, in rest of the treatments gallic
acid was not detected. Similarly, p-Coumaric acid a principle phenolic acid of propolis was
observed as 6.65±0.04, 12.31±0.09, 2.86±0.01, 6.27±0.01, 14.98±1.21 and 11.76±0.03mg/Kg
50
in T1, T2, T3, T4, T5 and T7 respectively. Likewise m-Coumaric acid was investigated as
8.57±0.30, 1.33±0.08 and 13.57±0.30mg/Kg in T1, T3 and T7 accordingly; however in
remaining treatments m-Coumaric acid was not detected. Regarding vanillic acid
concentration, it was noticed in T3, T4, T5 and T6 as 19.53±1.06, 26.81±2.18, 12.67±1.21 and
21.69±1.73mg/Kg accordingly whereas; in other treatments the concentration of vanillic acid
was not observed. Considering the concentration of syringic acid; T7 exhibited maximum
concentration of syringic acid as 23.36±0.02mg/Kg and minimum value 2.36±0.16mg/Kg
was recorded in T3 whereas, 3.82±0.02mg/Kg and 20.43±1.89mg/Kg (syringic acid) was also
noticed in T1 and T4 respectively. In a similar way the concentration of chlorogenic acid was
observed as 7.45±0.48, 19.15±0.48, 6.33±0.42 and 31.80±2.56mg/Kg in T1, T2, T6 and T7
accordingly whilst in rest of treatments chromogenic acid was not detected. Feraulic acid
quantification was noticed as 4.74±0.19, 5.86±0.47, 5.40±0.38 and 24.32±1.89mg/Kg in T2,
T3, T5 and T7 respectively whereas; in other treatments it was not detected. However, 4-
Hydroxy benzoic acid was observed only in T4 and measured as 43.17±2.89mg/Kg while in
rest of the treatments it was not observed.
The results of present study about the polyphenol contents of propolis extracts were
comparable with previous findings of Tosi et al. (2007), they observed different propolis
samples and their ethanol extracts from Argentina and found coumaric acids and syringic
acid in concentrations as 0.05 to 2.1% and 0.59 to 12.1% respectively in all their tested
samples. Similarly Christov et al. (2006) determined the feraulic acid, caffeic acid and p-
coumaric acid as 1.00 to 3 to 10%, 0.8% and 3.4 to 18.8% in ethanol extract of propolis from
different regions of Canada. Kumazawa et al. (2004) narrated the amount of caffeic acid and
p-coumaric acid by 0.2 to 7.2mg/g and 0.9 to 27.4mg/g respectively in different extracts of
propolis. Mello et al. (2010) indicated the contents of feraulic acid and caffeic acid by 0.92 to
1.56µg/mL and 0.25 to 1.04µg/mL in various mixed samples of Brazil and noticed the
variations as a function of climate and geographic distribution and their difference among
results of the present study may be attributed as a function of climate variation and
geographical location as composition of propolis is highly influenced by these variations.
51
Table: 4.5. Polyphenols quantification of extracts through HPLC (mg/Kg)
Polyphenol T1 T2 T3 T4 T5 T6 T7
Caffeic acid ND 8.21±0.06 ND 21.66±2.10 5.33±0.04 4.13±0.80 6.39±0.02
Gallic acid 0.21±0.01 ND ND 13.77±1.29 0.75±0.06 1.06±0.80 ND
p-Coumaric
acid
6.65±0.04 12.31±0.09 2.86±0.01 6.27±0.01 14.98±1.21 ND 11.76±0.03
m-Coumaric
acid
8.57±0.30 ND 1.33±0.08 ND ND ND 13.57±0.30
Vanillic acid ND ND 19.53±1.06 26.81±2.18 12.67±1.21 21.69±1.73 ND
Syringic acid 3.82±0.02 ND 2.36±0.16 20.43±1.89 ND ND 23.36±0.02
Chlorogenic
acid
7.45±0.48 19.15±0.48 ND ND ND 6.33±0.42 31.80±2.56
Feraulic acid ND 4.74±0.19 5.86±0.47 ND 5.40±0.38 ND 24.32±1.89
4-Hydroxy
benzoic acid
ND ND ND 43.17±2.89 ND ND ND
Values are expressed as means of triplicates ± standard deviation
ND= Not detected
T1: water extract T2: 95% ethanol extract, T3: 80% ethanol extract,
T4: 65% ethanol extract, T5: 95% methanol extract,
T6: 80% methanol extract, T7: 65% methanol
52
4.4. Evaluation of Antimicrobial potential of propolis
The bioactive components from botanical sources are always being for the benefits of
human society to combat maladies since many decades. Different kind of plants and their
products containing bioactive compounds are used worldwide as a source of natural
antibiotics. Nowadays a variety of plant products has been exploited for their safe use with
none or less damaging actions on environment and biological systems and to the end user
(Thomas et al., 1998). The pathogens utilized in the study with respect to antimicrobial
activity of proplis were E. coli ATCC-35218, B. subtilis ATCC-6633 and S. aureus ATCC-
25923 that cause spoilage in vegetables, meat and rice and their products. In Pakistan, food
safety issues are alarming and there is a big gap to conduct scientific studies for antimicrobial
and nutraceutical therapy based and safe diet. The stakeholders on the other way also need to
know about the innovative technologies to overcome the problem. The food borne pathogen
strains, therefore selected for the current study were E. coli ATCC-35218, B. subtilis ATCC-
6633 and S. aureus ATCC-25923.
4.4.1. Antimicrobial activity against E. coli ATCC-35218
Honey bee propolis extract with various solvents (ethanol and methanol) was studied
to judge the antimicrobial behavior against E. coli. The results are presented in Table 4.6 to
4.11 for zone of inhibition (ZI) and minimum inhibitory concentration (MIC) for their
respective results. The mean values (Table: 4.6) for the zone of inhibition using disc
diffusion assay against E. coli exhibited 14.42±0.89, 17.68±0.32, 22.19±0.61, 16.13±0.29,
18.73±0.39 and 19.89±0.43 millimeters (mm) for, T2, T3, T4, T5, T6 and T7 respectively.
Minimum zone of inhibition was observed in aqueous extract T1 (8.72±0.51mm) whereas,
the positive control (Rifampicin) showed 34.71±0.58 millimeters zone of inhibition. Mean
values for minimum inhibitory concentration (MIC) against E. coli were 855.68±12.46,
784.32±9.27, 626.83±13.91, 731.29±10.43, 693.72±8.61 and 645.83±11.45µg/mL for T2, T3,
T4, T5, T6, T7 respectively whereas, water extract showed higher MIC values in comparison
to positive control which was 164.67±8.13µg/mL (Table: 4.7).
53
Table: 4.6. Mean values showing effect of propolis extract on zone of inhibition (mm)
against E. coli
Treatments Zone of inhibition
T1 8.72±0.51g
T2 14.42±0.89f
T3 17.68±0.32d
T4 22.19±0.61b
T5 16.73±0.39e
T6 18.13±0.29d
T7 19.89±0.43c
Positive control (Rifampicin) 34.71±0.58a
Mean values showing different letters differs significantly (p<0.05). T1: water extract T2:
95% ethanol extract, T3: 80% ethanol extract, T4: 65% ethanol extract, T5: 95% methanol
extract, T6: 80% methanol extract, T7: 65% methanol
Table: 4.7. Mean values showing effect of propolis extract on Minimum Inhibitory
Concentration (MIC) against E. coli (µg/mL)
Treatments Minimum inhibitory concentration
T1 1273.4±11.61a
T2 855.68±12.46b
T3 784.32±9.27c
T4 626.83±13.91g
T5 731.29±10.43d
T6 693.72±8.61e
T7 645.83±11.45f
Positive control (Rifampicin) 164.67±8.13h
Mean values showing different letters differs significantly (p<0.05). T1: water extract T2:
95% ethanol extract, T3: 80% ethanol extract, T4: 65% ethanol extract, T5: 95% methanol
extract, T6: 80% methanol extract, T7: 65% methanol.
54
4.4.2. Antimicrobial activity against B. subtilis ATCC-6633
The results pertaining to disc diffusion assay against B. subtilis (Table: 4.8) showed
antimicrobial activity of propolis extracts for zone of inhibition as 18.89±1.04, 17.72±0.76,
26.37±1.13, 15.39±0.51, 19.27±1.07 and 20.37±0.63 mm for T2, T3, T4, T5, T6 and T7
respectively. The minimum zone of inhibition was observed in T1 (water extract) exhibiting
11.72±0.38 mm however, positive control (Rifampicin) showed detrimental zone of
41.19±1.42 millimeters.
Likewise, the MIC values for the propolis extracts of T2, T3, T4, T5, T6 and T7
revealed values of 648.37±11.42, 469.28±13.68, 286.67±9.56, 432.32±14.73, 373.89±12.38
and 318.43±9.69µg/mL respectively for inhibiting the bacteria. The positive control in
comparison to the treated samples inhibited the same bacteria with concentration of
142.33±10.87µg/mL (Table: 4.9).
4.4.3. Antimicrobial activity against S. aureus ATCC-25923
The results regarding zone of inhibition extent of the propolis extracts against S.
aureus are presented in Table: 4.10. The mean values for zone of inhibition with treatments
T1, T2, T3, T4, T5, T6 and T7 were noticed as 12.43±0.86, 16.36±0.13, 18.19±0.42,
26.37±0.31, 20.82±0.54, 19.46±0.42 and 23.58±0.28 millimeters respectively against S.
aureus, however, positive control (Rifampicin) showed highest inhibition zone showing
46.78±1.53millimeters.
In a same manner the MIC of the extracts against S. aureus were examined as
438.63±16.52, 372.89±9.23, 225.58±10.37, 338.71±14.64, 309.24±13.69,
289.73±12.71µg/mL in response to T2, T3, T4, T5, T6 and T7 respectively whereas water
extracts (T1) showed least inhibitory effect and higher MIC values in comparison to positive
control (106.88±7.16µg/mL) against S. aureus (Table: 4.11).
55
Table: 4.8. Mean values showing effect of propolis extract on zone inhibition extent
against B. subtilis (mm)
Treatments Zone of inhibition
T1 11.72±0.38h
T2 18.89±1.04e
T3 17.72±0.76f
T4 26.37±1.13b
T5 15.39±0.51g
T6 19.27±1.07d
T7 20.37±0.63c
Positive control (Rifampicin) 41.19±1.42a
Mean values showing different letters differs significantly (p<0.05). T1: water extract T2:
95% ethanol extract, T3: 80% ethanol extract, T4: 65% ethanol extract, T5: 95% methanol
extract, T6: 80% methanol extract, T7: 65% methanol
Table: 4.9. Mean values showing effect of propolis extract on Minimum Inhibitory
Concentration (MIC) against B. subtilis (µg/mL)
Treatments Minimum inhibitory concentration
T1 1244.4±14.61a
T2 648.37±11.42b
T3 469.28±13.68c
T4 286.67±9.56g
T5 432.32±14.73d
T6 373.89±12.38e
T7 318.43±9.69f
Positive control (Rifampicin) 142.33±10.87h
Mean values showing different letters differs significantly (p<0.05). T1: water extract T2:
95% ethanol extract, T3: 80% ethanol extract, T4: 65% ethanol extract, T5: 95% methanol
extract, T6: 80% methanol extract, T7: 65% methanol
56
Table: 4.10. Mean values showing effect of propolis extract on zone Inhibition extent
against S. aureus (mm)
Treatments Zone of inhibition
T1 12.43±0.86h
T2 16.36±0.13g
T3 18.19±0.42f
T4 29.18±1.19b
T5 20.82±0.54d
T6 19.46±0.42e
T7 23.58±0.28c
Positive control (Rifampicin) 46.78±1.53a
Mean values showing different letters differs significantly (p<0.05). T1: water extract T2:
95% ethanol extract, T3: 80% ethanol extract, T4: 65% ethanol extract, T5: 95% methanol
extract, T6: 80% methanol extract, T7: 65% methanol
Table: 4.11. Mean values showing effect of propolis extract on Minimum Inhibitory
Concentration (MIC) against S. aureus (µg/mL)
Treatments Minimum inhibitory concentration
T1 1123.4±9.61a
T2 438.63±16.52b
T3 372.89±9.23c
T4 225.58±10.37g
T5 338.71±14.64d
T6 309.24±13.69e
T7 289.73±12.71f
Positive control (Rifampicin) 106.88±7.16h
Mean values showing different letters differs significantly (p<0.05). T1: water extract T2:
95% ethanol extract, T3: 80% ethanol extract, T4: 65% ethanol extract, T5: 95% methanol
extract, T6: 80% methanol extract, T7: 65% methanol
57
According to a number of investigations, propolis inhibits the bacterial growth by a
number of ways including disintegration of cytoplasmic matrix, breakdown of cell wall and
cell membrane structure and retarding protein synthesis thus leads to lysis of the cell.
Moreover, it is proven fact that ethanol extract of propolis with active phenolic constituents
associated with chemistry of propolis affect the membrane potential which increase the
movement of ions and charged particles along the bacterial membrane leading to degradation
of bacterial cell (Takasi et al., 1994 and Mirzoeva et al., 1997).
Bee propolis is a resinous product that exhibits a number of biological,
pharmacological and antimicrobial properties and is being investigated worldwide against
pathogens (Cunha et al., 2013). Previously, Tosi et al. (2007) conducted a study to elucidate
the nature of propolis samples from Argentine against E. coli and found that ethanol extract
of propolis successfully retard the growth of E. coli and could be used as a natural food
preservative to inhibit the bacterial spoilage of perishable food commodities. Likewise Stan
et al., 2013 carried out research to examine the inhibitory effect of propolis solution on
biofilm formation in both gram positive and gram negative bacteria and confirmed the
antimicrobial and antibiofilm role of Romanian propolis. They explored that Romanian
propolis inhibit the biofilm formation in S. aureus and considered a subject of great interest
and research in coming era as a natural tool to combat bacteria. Similarly, in another study
conducted by Naher et al. (2014) explored that propolis exhibits antimicrobial potential
against gram positive and gram negative whereas among the gram positive domain S. aureus
was found to be more sensitive against ethanol extract of propolis. Zeighampour et al. (2013)
determined the antibacterial activity of 70% ethanol extract of propolis against S. aureus and
P. aeruginosa by well diffusion method and found that ethanol extract of propolis was
effective against S. aureus showing a zone of inhibition of 17.66±0.47 mm whereas 7 mm
zone of inhibition was observed for P. aeruginosa. They confirmed that bee propolis
collected from Isfahan showed more activity against S. aureus than P. aeruginosa. Similarly
in another study Lu et al. (2005) examined the antimicrobial perspective of propolis samples
collected from different regions of Taiwan and different time of the year and inferred that
antimicrobial activity affected with the season of collection, area of collection, temperature
and pH of the media used during the study. However, propolis samples showed intensive
activity against S. aureus which is varied with the change in pH and temperature. Bee
58
propolis has nontoxic and natural origin and has potential use against to retards the growth of
food spoilage microorganisms and considered an efficient antimicrobial and antioxidant
component which could be incorporated in food based system to control microflora
particularly in fermented products for the inhibition of pathogenic bacteria (Kalogeropoulos
et al., 2009). Previously the antimicrobial activity of propolis extracts against S. aureus was
identified from Mangolia, Albania, Egypt and Brazil and results relating to zone of inhibition
were recorded as 24.0, 21.8, 24.3, and 21.8 mm respectively those are in close association
with the present exploration (Kujumgiev et al., 1999).
In another study conducted by Yaghoubi et al. (2007) identified the antimicrobial
potential of bee propolis from Iran using disc diffusion methods. Ethanol extract of propolis
showed a significant inhibitory effect on the growth of bacteria and fungi, they deduced that
antimicrobial activity of propolis is linked with the polyphenols and flavonoids content of
propolis whereas extracts represent more activity against Gram positive bacteria than the
gram negative bacteria. In another study conducted by Dizaji et al. (2008) explored the role
of propolis against different group of bacteria and fungi. They depicted that propolis from
Iran imparted strong potential against gram positive bacteria as in particular against S. aureus
as a function of phenolic contents of propolis.
Recently, Rahman et al. (2010) conducted an experiment to explore the antimicrobial
behavior of propolis and honey against gram positive and gram negative kind of bacteria
including S. aureus and E. coli through disc diffusion assay. They observed a significant
growth inhibition in S.aureus as compared to E. coli and confirmed that propolis extract
showed more response against gram positive bacteria than gram negative bacteria. A number
of studies indicated that antimicrobial activity of propolis is mainly due to certain bioactive
compounds like caffeic acid, cinnamic acid, benzoic acid, quercetin, galangin and
pinocambrin found in propolis which is in conformity to the present study. As a mechanistic
approach such components mainly destruct the cell wall structure and membrane of the cell
leading a retarding effect on the growth of microorganisms (Marcucci, 1995; Cook and
Samman, 1996; Mirzoeva et al., 1997; Gatto et al., 2002). As a whole, in our experiment
propolis extracts showed more activity against S. aureus followed by B. subtilis and E. coli
however ethanol extract showed maximum zone of inhibition (22.19±0.61, 26.37±1.13 and
59
29.18±1.19mm) for E. coli, B. subtilis and S. aureus respectively followed by methanol
extracts.
In mechanistic approach propolis inhibit growth of bacteria by a number means
especially by altering the permeability of inner membrane causing dissipation in membrane
potential and inhibiting cell motility. As a structural difference G-positive bacteria are more
permeable than G-negative bacteria so constituents of propolis penetrate more in G-positive
so retard their growth effectively in comparison to G-negative bacteria as reported in the
present study (Carvalho et al., 2015). The results for antimicrobial activity of propolis against
selected microbes have been compared with the rifampicin which is a proven antimicrobial
agent. Rifampicin used as positive control shwed more activity against seleceted
microorganisms but the results of other extracts are in close conformity. However ethanol
extract (65%) of propolis imparted higher values among other extracts those were in the close
association of positive control which showed that ethanol extract could be the best natural
alternate to chemical antimicrobial agent.
4.5. Functional drink analysis
4.8.1. Physicochemical analysis
The statistical values regarding physicochemical attributes of the functional drinks
showed significant effect of treatments and storage days, whereas their interaction
(treatments x storage) was found to be non-significant for acidity and pH. Similarly, a non-
significant effect of treatments, storage days and their interaction was noticed in total soluble
solids (TSS) of the functional drinks (Table: 4.12).
At the initial day („0‟ day) the mean values for acidity of the functional drinks were
recorded as 0.146±0.002, 0.147±0.007 and 0.153±0.005 in drink prepared without propolis
extract (To), with ethanol extract of propolis (T1) and with methanol extract of propolis (T2)
respectively. Functional drink after 45 days of storage at room temperature showed acidity
as 0.170±0.007, 0.174±0.006, 0.179±0.005 for To, T1 and T2 respectively. It was observed
that acidity of the drinks was increased during the storage period from 0.146±0.002 to
0.170±0.007, 0.147±0.007 to 0.174±0.006 and 0.153±0.005 to 0.179±0.007 for control To, T1
and T2 respectively (Table: 4.13). Likewise the mean values for pH of the functional drink at
zero day storage was 4.64±0.06, 4.76±0.04 and 4.87±0.01 for control (To), drink with ethanol
extract (T1) and drink with methanol extract of propolis (T2) respectively whereas, the pH
60
values on the 45 day of storage was 4.28±0.07, 4.39±0.06 and 4.48±0.02 for To, T1 and T2
accordingly. A reduction in pH values for the drinks during storage was noticed from
4.64±0.06 to 4.28±0.07, 4.76±0.04 to 4.39±0.06 and 4.87±0.01 to 4.48±0.02 for the
treatments To, T1, and T2 accordingly (Table: 4.14).
The mean values regarding total soluble solids (TSS) depicted a non-significant effect of
storage, treatments and their interaction. The TSS was found to be 1.62±0.03, 1.68±0.06 and
1.63±0.04 for the To, T1 and T2 on treated samples respectively. However, the values
examined after 45th
day of storage were found as 1.73±0.02, 1.79± 0.01 and 1.75±0.03 for
control (To), drink with ethanol extract (T1) and drink with methanol extract (T2) respectively
(Table: 4.15).
During storage change in pH, acidity and total soluble solids (TSS) during storage are
the indicators for the acceptance of the product. Normally, acidity of the juice or drink
increased whereas pH decreased whilst TSS of the drink not affected as a function of storage
(Castellari et al., 2000; Lee et al., 2009). Previously, King et al. (2007) observed the total
soluble solids of the beverages. They depicted that apple diet drink prepared with addition of
artificial sweetener contained an average of 1.4 Brix total soluble solids. In a same manner,
the outcomes of present exploration are supported by the investigations of González-Molina
et al. (2009), who explored the nature of pomegranate and apple juices and their blending
and confirmed a non-significant effect of storage on the total soluble solids of the product.
The current studies indicated that the results for acidity and pH of the functional
drinks are in agreement to those of work done by of Ahmed et al. (2012), who noticed an
inversely proportional relation between pH and acidity during storage period. They further
explained that acidity of functional drinks significantly increases with the passage of time
and treatments do not impart momentous change on acidity which, although in contradiction
to our results for treatment effect in particular to acidity but the reason could be due to nature
of the extract. In another study conducted by Klimczak et al. (2007) it is concluded that the
behavior of orange juice during storage exhibited a decrease in pH and increase in acidity
and decrease in pH is linked with increase in acidity of the juice. Likewise the findings of
current study for acidity and pH are also in line with the observations of Murtaza et al.
(2004), who examined physicochemical attributes of fruit drink during 90 days of storage and
61
Table: 4.12. Mean square values for the effect of treatments on acidity, pH and TSS of
functional drink
*=Significant
**= Highly significant
NS= Non significant
Table: 4.13. Mean values for the effect of treatments and storage on Acidity (%) of
functional drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
SOV df Acidity pH TSS
Treatments (A) 2 0.0012* 0.267* 0.001NS
Days (B) 3 2.577* 0.205* 0.028 NS
A x B 6 3.251 NS
0.173 NS
4.301 NS
Error 24 2.123 0.001 2.349
Total 35
Treatments
Storage Intervals T0 T1 T2 Mean
0 0.146±0.002 0.147±0.007 0.153±0.004 0.149d
15 0.155±0.001 0.158±0.003 0.165±0.006 0.160c
30 0.162±0.005 0.164±0.001 0.167±0.008 0.165b
45 0.170±0.007 0.174±0.006
0.179±0.005 0.175a
Mean 0.159c 0.161b 0.167a
62
Table: 4.14. Mean values for the effect of treatments and storage on the pH values of
functional drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
Table: 4.15. Mean values for the effect of treatments and storage on Total Soluble
Solids (%) of functional drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
Treatments
Storage Intervals T0 T1 T2 Mean
0 4.64±0.06 4.76±0.04 4.87±0.01 4.75±0.06a
15 4.53±0.03 4.61±0.05 4.70±0.03 4.61±0.04b
30 4.44±0.01 4.51±0.08 4.65±0.01 4.53±0.02c
45
4.28±0.07
4.39±0.06
4.48±0.02 4.38±0.03d
Mean 4.47±0.03c
4.56±0.01b 4.67±0.07a
Treatments
Storage Intervals T0 T1 T2 Mean
0 1.62±0.03 1.68±0.06 1.63±0.04 1.64±0.03
15 1.65±0.01 1.70±0.04 1.66±0.02 1.67±0.07
30 1.71±0.03 1.74±0.05 1.68±0.06 1.71±0.01
45 1.73±0.02
1.79±0.01
1.75.±0.03 1.75±0.05
Mean 1.67±0.04 1.72±0.01 1.68±0.05
63
noticed a significant increase in acidity with sharp decline in pH. Coda et al. (2012)
determined the variation in physicochemical attributes of drinks while conducting a study on
Yogurt-like beverages made of a mixture of cereals, soy and grape must. They concluded that
the nature of yoghurt based drink prepared with addition of fruits and cereals influenced by
the change in acidity during storage. They also depicted an inverse link between acidity and
pH of the product. Our results are supported by the various studies that increase in acidity of
drinks is due to the breakdown of citric acid along with acidic features of artificial
sweeteners in replacement of sugar to develop therapeutic drinks (Ahmed et al.,2008).
Recently, Kausar et al. (2012) prepared a cucumber-melon based functional drink and
studied the shelf life for 4 months of storage period concluding a significant increase in
acidity from 0.44 to 0.51% and decrease in pH from 4.89 to 4.77 as an effect of storage.
Mishra et al. (2012) investigated pH, acidity and total soluble solids of lemon based drink
with added vitamin C during storage of the product and observed sharp increase in acidity
from 0.49 to 0.81 as a function of storage whilst a significant decrease in pH values was
observed from 4.05 to 3.80 as a part of increased acidity. They deduced that these variations
in acidity and pH values of the drinks were due to the degradations of different organic acids
found in lemon based drink.
In another study conducted by Akhtar et al. (2010) examined the effect of storage on
the physicochemical behavior of mango pulp with special reference to acidity and pH during
three months of storage. During storage period a gradual decrease in pH was noticed with
substantial increase in acidity and the values ranged from 0.52 to 0.69 and 3.94 to 3.78 for
acidity and pH of the mango pulp accordingly. They depicted that increase in acidity referred
due to the formation of weak acids and their respective salts. Moreover the decrease in pH
and increase in acidity is also attributed to the production of acidic components as a result of
polysaccharide degradation and products of oxidation of reducing sugars. Additionally, the
formation of pectic breakdown products especially uronic acid may also be one of the
important phenomenon for the change in the said parameters.
On overall basis it is concluded that acidity in functional drinks was increased and pH
was decreased as an effect of storage days and treatments whereas interaction did not affect
the two parameters as supported by various workers stated above based on their respective
research findings.
64
4.6. Sensory evaluation
Sensory evaluation on 9-point hedonic scale is an inexorable for the acceptance and
rejection of a produce for end usage. A very good sensory score about developed product
ensure consumers likeness and confidence for its marketability. The functional drinks
prepared with propolis extract were subjected to different sensory characteristics of color,
flavor, sweetness, sourness and overall acceptability during the storage period.
It is evident from the statistical analysis shown in (Table: 4.16) that treatment, storage
intervals and their interaction (Treatments x Days) imparted a non-significant effect on the
sensory attributes of the functional drinks. Physical appearance of a product is a preliminary
sensory criterion for the selection or rejection of a product by the consumers.
The mean values for color of the functional drinks on the first day of drink development were
observed as 7.62±0.04, 8.14±0.04 and 7.76±0.01 whereas storage data after 45th
days was
recorded as 7.45±0.01, 7.23±0.06 and 7.60±0.07 in T0, T1 and T2 accordingly. It was
observed that score for color of the functional drinks varies from 7.62±0.04 to 7.45±0.01,
8.14±0.04 to 7.23±0.06 and 7.76±0.01 to 7.60±0.07 in control (To), ethanol extract (T1) and
methanol extract (T2) accordingly as a function of storage (Table: 4.17) which was at par due
to non-significant effect.
The results pertaining to flavor of the functional drinks shown in (Table: 4.18)
indicated maximum score as 7.71±0.05 for T1 whereas minimum value for the flavor was
noticed in T0 as 7.56±0.07 on the initial day of study. The overall mean value for the flavor
of on first day of drink development were recorded as 7.56±0.07, 7.71±0.05 and 7.67±0.09 in
T0, T1 and T2 accordingly whereas the values for factors were as 7.34±0.02, 7.53±0.06 and
7.52±0.03 after 45th
day of storage with T0, T1 and T2 accordingly. It was observed that a
slight decline in flavor occurred during storage but was found as non-significant statistically
exhibiting the values ranging from 7.56±0.07 to 7.34±0.02, 7.71±0.05 to 7.53±0.06 and
7.67±0.09 to 7.52±0.03 with T0, T1 and T2 treated functional drinks.
Results regarding sweetness and sourness showed a non-substantial effect of
treatments as presented in Table: 4.19 & 4.20. Mean values recorded for sweetness and
sourness at 0 day were 7.70±0.07 & 7.58±0.03, 8.10±0.04 & 7.69±0.09 and 7.86±0.09 &
7.45±0.05 accordingly whereas, the values for sweetness and sourness examined after 45th
65
Table: 4.16. Mean squares for the effect of treatments and storage on the sensory attributes of functional drink
NS= Non significant
Table: 4.17. Mean values for the effect of treatments and storage on color of drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
SOV df color Flavor sourness sweetness Overall acceptability
Treatments (A) 2 0.5852NS
0.5689NS
0.54141NS
0.60129NS
0.58447NS
Days (B) 3 0.5055NS
0.5055NS
0.1816NS
0.02775NS
0.03999NS
A x B 6 0.00061NS
0.00081NS
0.00042 NS
0.00091NS
0.00031NS
Error 24 0.00480 0.00468 0.0044 0.00494 0.00480
Total 35
Treatments
Storage intervals T0 T1 T2 Mean
0 7.62±0.04 8.14±0.03 7.76±0.01 7.84±0.05
15 7.55±0.02 7.94±0.07 7.72±0.06 7.73±0.02
30 7.50±0.04 7.29±0.05 7.65±0.03 7.48±0.03
45 7.45±0.01 7.23±0.06 7.60±0.07 7.42±0.06
Mean 7.53±0.03 7.65±0.01 7.68±0.07
66
Table: 4.18. Mean values for the effect of treatments and storage on flavor of functional
drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
Table: 4.19. Mean values for the effect of treatments and storage on sweetness of
functional drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
Treatments
Storage intervals T0 T1 T2 Mean
0 7.56±0.07 7.71±0.05 7.67±0.09 7.64±0.06
15 7.45±0.03 7.68±0.03 7.60±0.01 7.57±0.01
30 7.42±0.05 7.63±0.01 7.55±0.04 7.53±0.03
45 7.34±0.02
7.53±0.06 7.52±0.03
7.46±0.04
Mean 7.44±0.05 7.61±0.02 7.58±0.04
Treatments
Storage Intervals T0 T1 T2 Mean
0 7.70±0.07 8.10±0.04 7.86±0.09 7.88±0.01
15 7.66±0.03 7.84±0.02 7.78±0.07 7.76±0.05
30 7.54±0.01 7.79±0.01 7.74±0.05 7.69±0.03
45 7.58±0.04
7.72±0.05 7.68±0.09
7.66±0.02
Mean 7.62±0.01 7.86±0.06 7.76±0.03
67
Table: 4.20. Mean values for the effect of treatments and storage on sourness of
functional drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
Table: 4.21. Mean values for the effect of treatments and storage on overall
acceptability of functional drink
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
Treatments
Storage intervals T0 T1 T2 Mean
0 7.58±0.03 7.69±0.09 7.45±0.05 7.57±0.03
15 7.43±0.06 7.61±0.08 7.41±0.05 7.48±0.07
30 7.35±0.04 7.56±0.01 7.33±0.03 7.41±0.04
45 7.28±0.05
7.47±0.02
7.29±0.06 7.34±0.02
Mean 7.41±0.01 7.58±0.04 7.33±0.03
Treatments
Storage Intervals T0 T1 T2 Mean
0 7.67±0.06 7.92±0.06 7.76±0.04 7.78±0.05
15 7.59±0.03 7.83±0.02 7.68±0.05 7.70±0.03
30 7.54±0.07 7.76±0.09 7.57±0.08 7.62±0.01
45 7.48±0.06
7.69±0.04 7.49±0.05
7.55±0.02
Mean 7.57±0.05 7.80±0.06 7.62±0.02
68
days of storage were 7.58±0.04 & 7.28±0.05, 7.72± 0.05 & 7.47±0.02 and 7.68±0.09 &
7.29±0.06 for T0, T1 and T2 treated drink accordingly.
The overall mean values recorded for overall acceptability for functional drinks with
(T0, T1 and T2) was found as 7.57±0.05, 7.80±0.06 and 7.62±0.02 respectively. Nonetheless,
storage period represented a non- substantial decrease in overall acceptability values from
7.78±0.05 to 7.55±0.02 at 0 and 45th
day of storage accordingly (Table: 4.21).
The findings of present study about sensory attributes of functional drinks are in
harmony with the previous outcomes of Ahmed et al. (2012) who examined the sensory
characteristics of polyphenol based functional drinks due to treatments and storage period
and they deduced a non-momentous decline in different sensory traits like color, flavor,
soreness and overall acceptability of the polyphenols based diet drink. Mishra et al. (2012)
studied on the development of Vitamin C rich value added beverage and depicted non-
substantial variations in flavor and overall acceptability of vitamin C supplemented
functional drink during the prescribed storage period. Whereas, aignificant changes in color
were observed due to different treatments and storage period. One of their peers, Bekhit et al.
(2011) examined the sensory profile of functional drink prepared with polyphenols in
different treatments of bioactive compounds and noticed a non-momentous change within the
treatments. They concluded that polyphenols impart color variation to product because of
different color imparting bodies associated with plant polyphenols.
In the present study, addition of propolis extract for the preparation of functional
drink did not indicate any opposing effect on the sensory response. The addition of food
grade color during functional drink preparation mask the color of active ingredients, thus no
adverse response was noticed by the panelist for the functional drink prepared. During
storage period a mild decrease in the sensory attributes were noticed however, the scores
were remained within the acceptable limits. Therefore, the sensory response of the drinks
was in close conformity to control hence depicting their for end usage and for further in-vivo
study trials.
4.7. Bio-evaluation studies
In-vivo study was designed to investigate the nutraceutical potential of honey bee
propolis against selected lifestyle related maladies through animal modeling (Sprague
Dawley rats). The efficacy study was carried out on animals instead of humans because of
69
controlled condition for the diet and environmental factors for feasible management of the
experiments. During the current study, each experiment was comprised of three different
experimental models with different types of diet plan. In study-I, normal diet was used
whereas in study-II and study-III high sucrose and high cholesterol diets were respectively
supplied to animals along with regular intake of functional drinks (To, T 1 and T2). The
biological evaluation for the effect on blood lipid profile (cholesterol, HDL, LDL and
triglycerides level), blood glucose and insulin was carried out on disease free animals those
were provided with calculated amount of feed and drink for the respective study and at the
end of experiment data for the said parameters was compared with control to find out the
effect of treatments. The results are discussed for each parameter as stated under the
following sub-headings.
4.7.1. Feed intake
The mean square values for feed intake (Table: 4.22) depicted the significant effect of
treatments and time interval on the feed intake in all studies (I, II and III) during the
consecutive study trials. The consumption of feed increased with time interval. During 1st
week of study-I the values for feed intake was found as 15.25±0.63, 14.63±0.37 and
15.21±0.41 g/rat/day on average basis in To, T1 and T2 groups respectively that increased to
20.83±0.91, 18.52±0.58 and 19.27±0.81 g/rat/ day correspondingly at the 8th
week of study.
Similarly during trial-2, an increasing trend in feed intake was noticed in all study groups and
the mean values for the feed intake during 1st week of study were recorded as 14.95±0.43,
15.01±0.73 and 15.12±0.84 g/rat/day for the To, T1 and T2 groups which was substantially
increased as 20.59±1.03, 18.69±0.94 and 19.31±1.02 g/rat/day on average basis at the
termination of study for respective study groups. The trend of increased feed intake depicted
in figure 4.1 for the two trials of study-I. During study-II (High sucrose diet) the values for
feed intake in 1st week was recorded as 16.96±0.72, 16.21±0.64 and 16.35±0.93 g/rat/day for
To, T1 and T2 groups that was gradually increased with the passage of time as 23.16±1.05,
22.21±0.93 and 22.35±1.03 g/rat/day on average basis of the 8 weeks of study in all study
groups (To, T1, T2). Likewise in trial-2 the mean values for feed intake during the initiation of
study for To, T1 and T2 were 16.25±0.72, 16.23±0.31, 16.45±0.51 g/rat/day that was
increased up to 22.30±0.84, 22.23±0.67 and 22.45±1.04 g/rat/day on average basis at the
termination of 8th
week of study in all groups accordingly (Figure: 4.1).
70
During study-III (High cholesterol diet) the recorded values for the feed intake in the
1st week for To, T1 and T2 groups during trial-1 were 15.99±0.71, 15.65±0.62 and 15.89±0.34
g/rat/day respectively and were increased successively upto 21.99±1.04, 20.98±0.93 and
21.10±1.07 g/rat/day as function of time for To, T1 and T2 groups correspondingly. During
the subsequent trial 2 the observed values for feed intake in 1st week were as 16.01±0.37,
15.75±0.62 and 15.83±0.73 g/rat/ day for To, T1 and T2 groups respectively later on,
gradually uplifted upto 21.59±0.97, 20.90±0.83 and 21.14±0.94 g/rat/day on average basis
for all study groups accordingly at the end of study (Figure: 4.1).
The results regarding the feed intake of animals in present exploration are supported
by the earlier findings of Kwon et al. (2001) who noticed that propolis consumption by the
laboratory animal increases the feed efficiency ratio but there is no substantial difference for
feed consumption among the different experimental groups as concluded by their experiment
whereas in this manuscript we found the substantial difference in the groups as far as feed
consumption is concerned but it confirmed that feed consumption increases with passage of
time regardless of significance or non-significance. Galal et al. (2008) noticed an effect of
dietary propolis on the productive and immune response of animals supplied with propolis
upto 54 weeks of the study. They explicated a sharp increase in the feed consumption of the
animals as a function of propolis consumption as compared to control group, not supplied
with propolis and similar findings are obtained in the results hence supported by the previous
workers.
4.7.2. Drink intake
The statistical values for the drink intake (Table: 4.23) depicted a non-significant effect of
treatments whereas time interval imparted a significant differences during the the study
period depicted in Figure: 4.2 showing increasing trend with time intervals.
The mean values for drink intake (Figure: 4.2) at the start of study-I in both consecutive trial-
1 & 2 were observed as 20.12±1.06 & 19.96±1.03, 19.95±0.93 & 19.85±0.89 and 19.99±1.04
& 20.01±1.02 mL/rat/day for To, T1 and T2 treated groups respectively that was found to
26.64±1.06 & 26.48±1.08, 26.47±0.76 & 26.37±0.83 and 26.51±0.63 & 26.53±0.82
mL/rat/day in To, T1 and T2 treated groups accordingly. The similar increasing trend in drink
intake of animals was also noticed during the study-II. The mean values for drink intake in
the 1st week of study for To, T1 and T2 groups were observed as 22.36±0.73, 22.01±0.48 and
71
22.12±0.92 mL/rat/day with gradual increase with the passage of time as indicated with
respective figure 4.2. The mean values for the drink intake by the animals was found as
28.42±0.72, 27.97±0.61 and 28.17±0.38 mL/rat/day with To, T1 and T2 respectively at the
end of study period. Similarly in the beginning of the trial-2 the recorded values for drink
intake were as 22.99±0.64, 21.96±0.83 and 22.19±0.96 mL/rat/day for To, T1 and T2 treated
groups correspondingly that was raised as 28.32±1.06, 27.97±1.06 and 28.22±1.05
mL/rat/day at the end of study for all study groups respectively. An increasing trend in the
drink intake values for study-III (trial-1) for To, T1 and T2 in the 1st
week were noticed as
21.36±0.76, 20.31±0.93 and 20.75±0.82 mL/rat/day that increased to 26.37±0.51, 26.24±0.84
and 26.34±0.48 mL/rat/day accordingly. Similar trend was observed in trial-2, the mean
values for the said parameter were recorded as 20.12±0.74, 20.69±0.83 and 21.22±0.78
mL/rat/day for To, T1 and T2 groups accordingly and an increasing trend was examined with
the passage of time with values of 26.22±0.95, 26.33±0.71 and 27.12±0.64 mL/rat/day for
To, T1 and T2 groups respectively.
In the present study a non-substantial effect of treatments was observed in drink
intake during bio-evaluation trials. The similar findings have also been observed regarding
polyphenol/nutraceutical based functional drink intake by animals by different researchers.
They observed non-significant effect of functional drink while conducting their respective
research which supports the findings observed in our study (Kuo et al., 2005; Alshatwi et al.,
2011 and Uchiyama et al., 2011). The results pertaining to drink intake during study period
depicted in our results are in harmony with the previous investigations of Kim et al. (2012),
they noticed a non-significant differences in wine consumption containing polyphenols
during animal modeling system. Likewise the investigations of Edward et al. (2008)
explicated the link between thirst response and fluid homeostatic level which enhance the
need for more drink intake. Furthermore, the findings of the Jian et al. (2008) and Cohen
(2002) are also in accordance the efficacy trials.
72
Table: 4.22. Mean squares for the effect of treatments and study period (weeks) on feed intake of animals (g/day/rat)
SOV df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 3.431* 5.231* 2.022* 3.052* 1.231* 1.251*
Weeks
7
21.221*
23.161*
12.36*
15.120*
9.34*
9.361*
Error
14
0.221
0.321
0.142
0.221
0.214
0.159
*=Significant
**=Highly significant
NS=Non significant
73
Study-I
Study-II
Study-III
Figur:4.1. Feed intake during study-I,II and III (g/rat/day)
14
15
16
17
18
19
20
21
22
week1 week2 week3 week4 week5 week6 week7 week8
g/ra
t/d
ay
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
16
17
18
19
20
21
22
23
24
week1 week2 week3 week4 week5 week6 week7 week8
g/ra
t/d
ay
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
1516171819202122232425
week1 week2 week3 week4 week5 week6 week7 week8
g/ra
t/d
ay
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
74
Table: 4.23. Mean squares for the effect of treatments and study periods (weeks) on drink intake of animals (mL/day/rat)
*=Significant
**=Highly significant
NS=Non significant
SOV Df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 0.445 NS
0.550 NS
0.326 NS
0.432 NS
0.631 NS
0.772 NS
Weeks
7
6.365*
7.632*
9.326*
10.231*
9.631*
6.321*
Error
14
0.231
0.401
0.342
0.201
0.241
0.278
75
Study-I
Study-II
Study-III
Figure:4.2. Drink intake during study-I, II and III (mL/rat/day)
18
19
20
21
22
23
24
25
26
27
28
week1 week2 week3 week4 week5 week6 week7 week8
ml/
rat/
day
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
20
21
22
23
24
25
26
27
28
29
30
week1 week2 week3 week4 week5 week6 week7 week8
ml/
rat/
day
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
18
19
20
21
22
23
24
25
26
27
28
week1 week2 week3 week4 week5 week6 week7 week8
ml/
rat/
day
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
76
4.7.3. Body weight
The mean square values for the effect of treatments on body weight (Table: 4.24)
depicted that treatments imparted a non-significant effect on the body weight of rats whereas
time interval was to significant (p≤0.05). The trend in an increase in the body weight as an
effect of treatment and time interval is presented in figure 4.3. Likewise the mean values
(Table: 4.25) for the body weight of rats in study-I (trial-1) at the beginning of study were
observed as 131±5.67, 130±4.89 and 129±6.27 g/rat on average basis for the To, T1 and T2
groups respectively that was gradually increased during the study period and at the end of
time period the values were 230±5.43, 222±6.39 and 226±7.13 g/rat on average basis for T0,
T1 and T2 groups correspondingly. In the trial-2 (study-I) the recorded values for body weight
of rats during first week for To, T1 and T2 groups are 133±7.52, 132±4.56 and 127±8.31 g/rat
on average basis that increased with the passage of time upto 236±6.43, 220±5.74 and
224±7.49 g/rat on average basis at the 8th
week of study for To, T1 and T2 groups accordingly.
The recorded body for the animals at the beginning of study-II (trial-1 & 2) was 133±6.34 &
131±7.21, 129±5.38 & 131±5.74 and 127±8.51 & 130±6.93 g/rat on average basis for the To,
T1 and T2 groups accordingly that increased to 260±9.75 & 255±8.56, 235±7.63 & 238±5.78
and 240±7.38 & 243±9.61 g/rat on average basis at final stage of time for To, T1 and T2
groups correspondingly. Study-III (trial-1) exhibited body weight for the To, T1 and T2 in the
1st week as 134±5.43, 131±7.51 and 129±4.91 g/rat on average basis respectively which
increased as a function of time interval exhibiting the values at the 8th
week as 241±6.41,
226±5.48 and 230±6.78 g/rat on average basis for To, T1 and T2 accordingly. Whereas, trial 2
showed mean values for body weight at the beginning as 132±6.83, 130±9.51 and 131±4.89
g/rat on average basis in To, T1 and T2 respectively that gradually increased with the passage
of time (245±9.13, 228±7.83 and 231±8.21 g/rat/day on average basis for the To, T1 and T2).
The results for the parameter under discussion in present studies are supported by the
previous findings of Haro et al. (2000), who suggested that the addition of propolis and
pollen to diet of animals pronounced a positive effect on the body weight of animals as a
function of dietary interventions.
77
Table: 4.24. Mean squares for the effect of treatments and study period (weeks) on body
weight of animals (g/rat/week)
*=Significant
**=Highly significant
NS=Non significant
Table: 4.25. Mean values for the body weight of animals at 8th
week in different studies
(g/rat)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
230±5.43
236±6.43
222±6.39
220±5.74
226±7.13
224±7.49
Study II
(Trial 1)
(Trial 2)
260±9.75
255±8.56
235±7.63
238±5.78
240±7.38
243±9.61
Study III
(Trial 1)
(Trial 2)
241±6.41a
245±9.13a
226±5.48b
228±7.83b
230±6.78b
231±8.21b
Study I : Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
SOV df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 232.63* 221.36 * 632.01
** 702.12** 1002.63** 891.71
**
Weeks
7
3021.01*
2631.5*
1986.21**
4012.9**
5023.2**
63211**
Error 14 31.251 73.02 30.23 36.06 12.21
21.03
78
Study-I
Study-II
Study-III
Figure: 4.3. Body weight during study-I, II and III (g/rat/week)
100
150
200
250
week1 week2 week3 week4 week5 week6 week7 week8
g/ra
t/w
ee
k
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
100
150
200
250
week1 week2 week3 week4 week5 week6 week7 week8
g/ra
t/w
ee
k
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
100
150
200
250
week1 week2 week3 week4 week5 week6 week7 week8
g/ra
t/w
ee
k
to(y1)
to(y2)
t1(y1)
t1(y2)
t2(y1)
t2(y2)
79
In another study Denli et al. (2005) conducted an experiment on the animals to assess the
effect of propolis extract on the growth performance and other biochemical parameters of the
body treated with 0.5, 1.00 and 1.50 g/Kg of propolis. Resultantly, a significant inclination in
growth performance of the animals was noticed with increase in body weight and other
general response of the body. Abdulbasit et al. (2013) explored the effect of propolis on
glycemic and blood profile of alloxan induced diabetic rats and observed that consumption of
propolis in alloxan treated groups imparted a sharp increase in body weight up to of 11% as
compared to group which is not supplied with propolis.
4.7.4. Blood Cholesterol
Cholesterol is an important type of lipid associated with numerous cellular parts of
animal tissues. The source of cholesterol in animal bodies is mainly from de novo synthesis
in the liver cells as well as from the endocytic uptake of plasma low density lipoprotein
(LDL) through receptor mediated process (Fuhrman et al., 1997; Ma et al., 2010).
Cardiovascular complications are the leading cause of mortality in the world due to poor
eating habits and sedentary life pattern. In particular, high cholesterol level is the major
factor in the progression of atherosclerosis and other cardiovascular problems (Verschuren et
al., 2011).
The statistical results pertaining to blood cholesterol level as presented in Table: 4.26
indicated that treatments showed a non-significant effect on cholesterol in study-I whilst,
significant and highly significant effects were noticed in study-II & study-III respectively. In
study-I (trial-1), maximum serum cholesterol was measured in To (83.61±5.42 mg/dL)
followed by T2 (82.20±5.57 mg/dL) group whereas minimum level was observed in T1
(81.11±6.14 mg/dL). Means for serum cholesterol in study-II showed maximum value for To
(98.68±8.51 mg/dL) that reduced to 91.34±7.04 and 92.75±7.12 mg/dL in T1 and T2 treated
groups respectively. In study-III, maximum serum cholesterol value 152.1±8.69 mg/dL was
measured in To which was significantly reduced to 143.61±5.48mg/dL in T2 whilst lowest
value was observed in T1 as 136.51±9.21mg/dL. Reducing trend in cholesterol level was
observed during trial-2 in all studies (study-I, study-II and study-III). In study-I, the To
showed the highest value (81.19±6.01 mg/dL) of cholesterol that reduced significantly in T1
(78.75±5.21 mg/dL) as a function of functional/nutraceutical drink intake. In study-II,
cholesterol reduction was observed in a similar fashion from maximum to minimum value of
80
100.89±6.25mg/dL to 93.89±6.18mg/dL in To and T1 administered group respectively.
Whilst, in study-III maximum reduction in cholesterol was observed in T1 as compared to
control T0 from 156.01±11.13mg/dL to 140.98±7.38mg/dL respectively (Table: 4.27). It is
evident from the figure: 4.4 that T1 produced maximum reduction in serum cholesterol
followed by T2 in relation to control To. In study-I during trial-1 and trial-2 the treatments T1
and T2 revealed 2.99 & 3.01% and 1.69 & 2.21% reduction in cholesterol respectively as
compared to control. In the same way during study-II in trial-1 and trial-2 maximum and
minimum decrease in serum cholesterol was noticed in T1 (6.63 & 6.01%) and T2 (5.36 &
4.99%) as compared to control. Likewise, the results regarding percent reduction in
cholesterol for T1 and T2 treated groups as compared to control during study-III were
indicated as 10.25 & 9.63% and 5.59 & 5.21% during trial-1 and trial-2 of the study
accordingly. The trend regarding cholesterol reduction in the current study is in line with
previous findings of Abo-Saleem et al. (2009), they observed a significant decrease in total
cholesterol values of sprauge dawly rats group as compared to control group. The animals
were provided with propolis extract orally with a dose of 100 to 300 mg/Kg of body weight
for fourty days and substantial reduction in cholesterol was noticed due to propolis
consumption. Similarly Ichi et al. (2009) also found reduction in cholesterol in propolis fed
animals as compared to control group. In their study, during eight weeks, the rats were fed
with high fat diet along with low and high dose in a concentration 0.05 and 0.5% of propolis
respectively. During the study a significant reduction in cholesterol was observed in propolis
treated group of animals, however they concluded that propolis contains certain active
compounds which are responsible to alter the gene expression involving lipid metabolism.
Similarly, previous investigations of Ahmad et al. (2013) are in harmony with the findings of
present study; who observed an inverse link between the consumption of propolis extract and
serum cholesterol level. They administered rats with propolis extract in combination with the
diet with a dose of 100mg/Kg of body weight for 8 weeks. Likewise, recent observations of
Saleh (2012) are in agreement with the outcomes of present study; they found a transposed
link between propolis extract and level of serum cholesterol when they studied rats for a
period of six weeks with daily provision of propolis extract along with experimental diet and
concluded that provision of propolis extract reduce blood cholesterol level and improve
blood lipid profile significantly as compared to normal group. According to recent findings
81
Table: 4.26. Mean squares for the effect of treatments on cholesterol (mg/dL)
SOV df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 4.54NS
4.04NS
99.86*
89.76*
472.41** 430.06**
Error
Total
27
29
6.78
4.91
6.73
7.08
15.65
16.62
*=Significant
**= Highly significant
NS=Non significant
Table: 4.27. Mean values for the effect of treatments on cholesterol (mg/dL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
83.61±5.42
81.19±6.01
81.11±6.14
78.75±5.21
82.20±5.57
79.40±6.13
Study II
(Trial 1)
(Trial 2)
98.68±8.51a
100.89±6.25a
91.34±7.04b
93.89±6.18 b
92.75±7.12ab
95.01±8.43ab
Study III
(Trial 1)
(Trial 2)
152.1±8.69 a
156.01±11.13a
136.51±9.21c
140.98±7.38c
143.61±5.48b
147.88±6.23b
Study I : Normal diet
Study II : High sucrose diet
Study III: High cholesterol diet
T0 : Drink without propolis extract
T1 : Drink prepared with ethanol extract of propolis
T2 : Drink prepared with methanol extract of propolis
82
of Daleprane et al. (2012), propolis impart protective action against atherosclerosis,
modulate blood lipid profile by reducing the pro-inflamatory cytokines and chemokines and
effect was due to influencing mRNA and regulating genes expression those involve in the
pathomechnism of atherosclerosis including as MCP-1. Previously, one of the researchers
group, Castaldo and Capasso (2002) conducted studies and found that propolis exhibits a
strong ameliorative action against dyslipidemia and reduce the cholesterol level in a dose
dependent manner which is in agreement with present study. The behavior of decline in
cholesterol level observed in present work are also in accordance with Nirala et al. (2008)
who conducted several experiments on the bee propolis and its extracts to find out its
therapeutic action against hyperlipidemia. According to their investigation propolis has
strong modulating action on abnormal blood lipid profile by lowering total cholesterol value
and improving level of high density lipoprotein cholesterol (HDL-C). The consumption of
propolis modulates the metabolic pathways of lipid and lipoproteins thus producing a
significant lowering effect on the blood cholesterol level in the laboratory animals.
Furthermore, propolis being rich in phenolic acids and flavonoids retards the synthesis of
triglycerides in liver tissues for the mitigation of blood lipid abnormalities (Fuliang et al.,
2005; Li et al., 2012). Recently, Daleprane et al. (2012) conducted studies on animal
modelling system and observed that administration of propolis to hypercholesterolemic mice
retards the level of cholesterol and promote the production of HDL. They concluded that
propolis regulates ABCA1 gene expression for lowering blood cholesterol level. The regular
intake of propolis substantially improves blood lipid profile by modulating the concentration
of HDL and LDL.
4.7.5. High density lipoprotein (HDL)
Blood cholesterol mainly comprises of low density lipoproteins (LDL) and high
density lipoproteins (HDL). The proportion of both is a good biomarker for the diagnosis of
abnormal blood lipid profile. HDL considered as good cholesterol and its proportional higher
concentration as compared to LDL is marked as good for health and normal blood lipid
profile.
83
Study I: Normal diet; Study II: High sucrose diet; Study III: High cholesterol diet
T1= Functional drink prepared with ethanol extract of propolis
T2 = Functional drink prepared with methanol extract of propolis
Figure: 4.4. Effect of treatments on percent cholesterol reduction in animal models
2.99 3.01
6.63
6.01
10.25
9.63
1.69
2.21
5.36 4.99
5.59 5.21
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Study1(Tr 1) Study 1(Tr 2) Study 2(Tr 1) Study 2(Tr 2) Study 3( Tr 1) Study 3(Tr 2)
% D
ecr
eas
e in
ch
ole
ste
rol
Treatments
T1
T2
84
Table: 4.28. Mean square values for the effect of treatments on HDL (mg/dL)
SOV df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments
2
4.76NS
5.70NS
14.84*
14.82*
35.50**
40.80**
Error
27
0.91
1.00
1.44
1.44
2.87
3.03
Total
29
*= Significant
**= Highly significant
NS= Non-significant
Table: 4.29. Mean values for the effect of treatments on HDL (mg/dL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
33.63±2.34
35.26±2.87
34.31±2.54
36.04±1.87
34.27±2.81
35.97±1.17
Study II
(Trial 1)
(Trial 2)
41.89±3.63b
42.01±2.74b
43.62±3.41a
43.78±2.68 a
42.66±4.24ab
42.83±3.56ab
Study III
(Trial 1)
(Trial 2)
58.69±4.29b
60.22±5.13b
61.33 ±4.79 a
63.14±5.32a
60.46±5.41a
62.02±4.29a
Study I: Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2 : Drink prepared with methanol extract of propolis
85
Study I: Normal diet; Study II: High sucrose diet; Study III: High cholesterol diet
T1= Functional drink prepared with ethanol extract of propolis
T2 = Functional drink prepared with methanol extract of propolis
Figure:4.5. Effect of treatments on percent increase in plasma HDL in animal models
2.02 2.22
4.12 4.21
4.49
4.85
1.90 2.01
1.83 1.96
3.01 2.99
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Study1(Tr 1) Study 1(Tr 2) Study 2(Tr 1) Study 2(Tr 2) Study 3( Tr 1) Study 3(Tr 2)
% I
ncr
ease
in H
DL
Treatments
T1
T2
86
The statistical values concerning for the effect of treatments on the HDL level as presented in
Table: 4.28 indicated a non-momentous effect of functional drinks in study-I whereas a
significant and highly significant reduction was noticed during the study-II and study-III
respectively in both trials. The trend of percent HDL increases in animal models represented
in figure 4.5. The results (mean values) for HDL in study-I (normal diet), trial-1 showed the
highest value in To group as 33.63±2.34mg/dL which was enhanced upto 34.31±2.54 and
34.27±2.81 mg/dL with the effect of T1 and T2 respectively. The similar behavior of
functional drinks (treatments) was noticed in trial-2 where HDL increased in T1 and T2 as
compared to To group treated with drink. The mean values for HDL were recorded as
35.26±2.87, 36.04±1.87 and 35.97±1.17mg/dL for To, T1 and T2 groups respectively. Whilst,
in study-II (trial-1) minimum HDL value 41.89±3.63mg/dL was observed in To that
significantly increased up to 43.62±3.41 and 42.66± 4.24mg/dL in T1 and T2 treated groups
respectively. Similarly during the following trial momentously enhancement in the level of
HDL was observed from 42.01±2.74 to 43.78±2.68mg/dL in T1 followed by T2 from
42.01±2.74 to 42.83±3.56 mg/dL as a result of functional drinks (containing propolis extract)
intake. Likewise mean HDL level for To group in study-III during trial-1 and trial-2 was
measured as 58.69±4.29 & 60.22±5.13mg/dL correspondingly that increased considerably
(p≤0.05) to 61.33±4.79 & 63.14±5.32mg/dL in T1 group in corresponding study trials.
Functional drink containing methanol extract of propolis (T2) showed increasing trend for the
said parameter from 58.69±4.29 to 60.46±5.41 mg/dL and 60.22±5.13 to 62.02±4.29mg/dL
in consecutive two study trials (Table: 4.29). It is clear from (Figure: 4.5) that treatments T1
and T2 during study-I led to non-significant increase in the HDL concentration as compared
to control whereas, in study-II & study-III a considerable surge was observed in T1 (4.12 &
4.21% and 1.83 & 1.96%) and T2 (4.49 & 4.85% and 3.01 & 2.99 %) accordingly when
compared with animals fed on normal diet.
The presence of HDL and its major constituent protein apolipoprotein A-1 (ApoA-1)
are involved in the outflow of cholesterol from the foam cell found in artery walls and
facilitate its transportation towards liver for further metabolism and removal (Fielding and
Fielding 1995; Panagotopulos et al., 2002). Previous scientific explorations have revealed a
negative correlation between propolis intake and blood lipid abnormalities as it involves in
the lowering of LDL oxidation and upshooting HDL concentration among different
87
hyperglycemic and hyperlipidemic animal modeling studies (Bogdanov, 2011). The
substantial improvement in the HDL concentration in present study are in agreement with the
earlier outcomes of Fuliang et al. (2005), who mentioned that regular administration of
propolis extracts to animals with induced diabetes cause a momentous increase in HDL level
and reduction in LDL level as a function of propolis chemical constituents. During present
study effect of functional drink prepared with the addition of propolis extract on rats HDL
level is supported by the previous findings of Newairy et al. (2009), they carried out a study
on animals and observed an increase in HDL concentration whereas a remarkable decrease
was noticed in LDL, triglycerides and total cholesterol in propolis fed group of animals as
compared to control. The trend shown in the results of present research work are in harmony
and could be supported with earlier investigations of Kolankaya et al. (2002) who inferred
from their experiments on rats induced by certain biochemical and haematological changes
due to alcohol consumption. They noticed that alcohol can induce abnormal changes in blood
lipid profile especially by decreasing HDL and elevating LDL whilst, intake of propolis
extracts modify blood dyslipidaemia by increasing HDL and decreasing LDL, triglycerides
and total cholesterol. Likewise behavior of functional drinks containing propolis extract on
blood HDL concentration is in synchronization with outcomes of Mani et al. (2006), who
conducted a study on laboratory animals to examine the effect of propolis consumption on
biochemical profile of animals. They administered different treatments of propolis (1, 3 and 6
mg/kg/day) and its water extract with different solvents at the dose of 1 mg/kg/day to
experimental organisms for 30 days and 90 days respectively. At the last, they noticed that
there is no negative correlation of propolis and extract of propolis consumption on
biochemical profile of animals. Whereas, administration of propolis dosage is helpful to
maintain normal level of HDL, LDL, triglyceride and cholesterol in comparison to control
group those were not provided with dosage of propolis.
4.7.6. Low density lipoprotein (LDL)
The food containing high content of saturated fatty acids and cholesterol enhance the level of
LDL-cholesterol in blood plasma (NIH, 2002). High cholesterol level not only increase the risk of
heart diseases but also considered as responsible for the production of lipid peroxidation products
leading to the onset of atherosclerosis (Cox and Cohen, 1996; Itabe, 2003).
88
Table: 4.30. Mean square values for the effect of treatments on LDL (mg/dL)
SOV df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 1.51NS
1.76NS
40.73** 32.22** 128.82** 119.36**
Error
27
0.63
0.66
1.44
1.60
2.14
2.14
Total
29
*=Significant
**= Highly significant
NS= Non significant
Table: 4.31. Mean values for the effect of treatments on LDL (mg/dL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
29.33±2.32
30.18±2.78
28.16±2.14
28.92±1.27
28.68±2.58
29.27±1.17
Study II
0(Trial 1)
(Trial 2)
45.96±3.58a
48.36±4.13a
41.38±3.43c
44.16±2.18c
43.67±3.21 b
45.79±4.56b
Study III
(Trial 1)
(Trial 2)
63.20±5.29a
58.12±4.13a
56.10 ±4.21c
50.42±3.32c
58.06±5.48b
52.68±4.59b
Study I : Normal diet
Study II : High sucrose diet
Study III: High cholesterol diet
T0 : Drink without propolis extract
T1 : Drink prepared with ethanol extract of propolis
T2 : Drink prepared with methanol extract of propolis
89
Study I: Normal diet; Study II: High sucrose diet; Study III: High cholesterol diet
T1= Functional drink prepared with ethanol extract of propolis
T2 = Functional drink prepared with methanol extract of propolis
Figure: 4.6. Effect of treatments on percent reduction in plasma LDL level in animal models
3.99 4.16
9.96
8.69
11.23
13.25
2.22
3.01
4.99 5.32
8.14
9.36
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Study1(Tr 1) Study 1(Tr 2) Study 2(Tr 1) Study 2(Tr 2) Study 3( Tr 1) Study 3(Tr 2)
% d
ecre
ase
in L
DL
Treatments
T1
T2
90
The statistical values in our results showed non-significant difference on LDL in study-I
whereas, highly significant effect of treatments (functional drink) was noticed in the study-II
and study-III (Table: 4.30). The trend of percent LDL reduction in the animal study is
presented in figure 4.6. The means values (Table: 4.31) of LDL level in study-I (trial-1)
showed maximum level 29.33±2.32mg/dL in To which was non-substantially reduced to
28.16±2.14 and 28.68±2.58mg/dL in animals treated T1 (functional drink prepared with
ethanol extract of propolis) and T2 (functional drink prepared with methanol extract of
propolis) respectively. Trial-2 of the same study showed maximum LDL value was exhibited
by To (30.18±2.78mg/dL) that although reduced in T1 (28.92±1.27mg/dL) and T2
(29.27±1.173mg/dL) but at par with the control group i.e non-significant. During study-II
(trial-1), highest value for LDL concentration observed in To (45.96±3.58mg/dL) that was
substantially reduced in T1 (41.38±3.43mg/dL) and T2 (43.67±3.21mg/dL). Trial-2, also
exhibited maximum value in TO (48.36±4.13mg/dL) with highly significant reduction in T1
(44.16±2.18mg/dL) and T2 (45.79±4.56mg/dL) explicating the effect of functional drinks.
Similarly in study-III (trial-1), mean value for LDL was recorded as 63.20±5.29mg/dL for To
treated group, whereas T1 & T2 imparted a highly significant decreased in LDL
concentration. The values for LDL level observed during the two trials were 56.10±4.21 and
58.06±5.48mg/dL for T1 and T2 respectively. In a same sequence, during trial-2 highest value
for LDL level as 58.12±4.13mg/dL was observed in T0 which was substantially decreased in
T1 and T2 treated groups as 50.42±3.32 and 52.68±4.59mg/dL correspondingly.
The results regarding percent reduction in LDL due to functional drink (Figure:4.6):
indicated that 3.99 & 4.16% and 2.22 & 3.01% reduction in LDL level was observed in T1
and T2 groups as compared to control in study-I, trial-1 & 2. However, in study-II (trial-1 &
2) percent reduction in LDL level was 9.96 & 8.69% and 4.99 & 5.32% in T1 and T2 groups
respectively as a result of functional drink intake. Study-III (trial-1 & 2) showed 11.23 &
13.25% and 8.14 & 9.36% decrease in LDL in T1 and T2 groups accordingly in comparison to
control.
The trend in LDL reduction in our study is in close association with the previous
observations of Nader et al. (2010), who conducted an experiment and found that addition of
1% cholesterol in the diet of animals increased the level of total cholesterol, triglycerides
level and low density lipoprotein cholesterol at a significant manner. They demonstrated that
91
consumption of propolis extracts reduced the effect of high cholesterol diet by lowering total
cholesterol and LDL values. Intake of propolis improves the dyslipidemic profile by
modulating the metabolism of blood lipid moieties. According to Weisburger and Chung,
(2002), reactive oxygen species and free radicals are responsible for the oxidation of LDL
which is associated with abnormal blood lipid profile and hypercholesterolemia. Many
natural substances with antioxidant properties including polyphenols can inhibit the
development of atherosclerotic lesions formation which is in agreement with the findings of
present study.
4.7.7. Triglycerides
The statistical values for the effect of treatments (functional drinks prepared with addition of
propolis extract) on triglyceride level (Table: 4.32) showed a non-significant effect in study-I
whereas a considerable differences were measured in study-II & III which is depicted in
figure 4.7. The mean values (Table: 4.33) of triglyceride level in study-I showed maximum
value in To (67.25±4.42 & 70.12±6.08mg/dL) with decreasing trend but non-momentously in
T1 (65.69±5.14 & 68.23±4.27 mg/dL) and T2 (66.57±5.58 & 68.72±4.13mg/dL) in the study
trials. During study-II, level of triglyceride was measured as 75.96±5.41 & 78.96±6.23
mg/dL, 71.76±6.43 & 74.23±5.18 and 73.52±6.21 & 75.81±5.56 mg/dL in treatments To, T1
and T2 in consecutive trial-1 and trial-2 accordingly. In study-III, triglyceride level raised to
99.36±7.69 & 96.35±9.13mg/dL in To group treated as control in both trial-1 and trial-2
respectively. Functional drink prepared with added propolis extracts showed decreasing trend
of triglycerides upto 90.46±8.21 & 89.90±7.32mg/dL and 95.88±7.48 & 92.49±6.59 mg/dL
in T1 and T2 groups correspondingly in the experiments.
It is evident from (Fig:4.7) that treatment; T1 and T2 caused 2.32 & 2.69% and 1.01 &
1.99% suppression in triglyceride level as compared to control in study-I. Similarly, during
study-II, functional drinks (T1 and T2) resulted 6.32 & 5.99% and 3.21 & 3.99% reduction
intriglyceride level in both study trial as compared to control group. Similarly, in study-III,
8.96 & 6.69% and 3.50 & 4.01% decreased triglyceride level was observed in T1 and T2
treated groups respectively depicting more beneficial effect of T1 in comparison T2.
92
Table: 4.32. Mean square values for the effect of treatments on Triglycerides (mg/dL)
SOV df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments
2
2.10NS
1.92NS
25.22**
33.73**
56.64**
66.89**
Error 27 3.43 3.68 4.16 4.45 6.89 6.58
Total
29
*=Significant
**= Highly significant
NS=Non significant
Table: 4.33. Mean values for the effect of treatments on Triglycerides (mg/dL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
67.25±4.42
70.12±6.08
65.69±5.14
68.23±4.27
66.57±5.58
68.72±4.13
Study II
(Trial 1)
(Trial 2)
75.96±5.41a
78.96±6.23a
71.76±6.43b
74.23±5.18 b
73.52±6.21ab
75.81±5.56ab
Study III
(Trial 1)
(Trial 2)
99.36±7.69 a
96.35±9.13a
90.46±8.21c
89.90±7.32c
95.88±7.48b
92.49±6.59b
Study I : Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0 : Drink without propolis extract
T1 : Drink prepared with ethanol extract of propolis
T2 : Drink prepared with methanol extract of propolis
93
Study I: Normal diet; Study II: High sucrose diet; Study III: High cholesterol diet
T1= Functional drink prepared with ethanol extract of propolis
T2 = Functional drink prepared with methanol extract of propolis
Figure:4.7. Effect of treatments on percent reduction in Triglyceride level in animal models
2.32 2.69
6.32 5.99
8.96
6.69
1.01
1.99
3.21
3.99
3.50
4.01
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Study1(Tr 1) Study 1(Tr 2) Study 2(Tr 1) Study 2(Tr 2) Study 3( Tr 1) Study 3(Tr 2)
% D
ecre
ase
in t
rigly
ceri
des
Treatments
T1
T2
94
The higher triglyceride concentration in blood involved in the establishment of different
cardiovascular complications leading to cause atherogenic state because of increased level of
LDL or decreased HDL level (Gotto, 1998). The trend in triglyceride reduction during the
current study is supported by investigations of Fang et al. (2013); who conducted a study on
eight weeks old mice, supplied with high fat diet. They administered ethanol extract of
propolis to animals with a dose of 160 mg/kg/day for 14 weeks along with high fat diet and at
the end of study a significant reduction in triglyceride, total cholesterol and LDL level was
observed. They further inferred that propolis reduces the risk of atherosclerosis due to its
bioactive compounds e.g caffeic acid phenyl ester (CAPE). Previously, Koya-miyata et al.
(2009), examined hypolipidemic role of propolis extract on high fat diet induced obese
mouse. During the study, animals were supplied with propolis extract (5mg/kg and 50mg/kg)
twice a day for 10 days and observed that propolis extract caused a significant reduction in
triglyceride concentration. Likewise, findings of Nader et al. (2010) are in association with
present study who conducted experiments using high cholesterol diet to rabbits for the
induction of hyperlipidaemia. At the same time ethanol extract of propolis (75mg/Kg body
weight) were administered to animals for four weeks. At the termination of study, they
deduced that propolis reduced triglycerides, total cholesterol and LDL level significantly thus
exhibiting the potential to mitigate atherosclerosis. The results pertaining to triglycerides
reduction are also in conformity with the investigations of Hegazi et al. (2004) and Talas &
Gulhan (2009); who concluded that propolis flavonoids, phenolic acids and their esters are
the key factors to produce decreasing effect on serum triglycerides and total cholesterol
which could be related to findings in this manuscript. Furthermore, they stated that
polyphenols are the chief constituents of propolis having a direct effect on the suppression of
triglyceride and cholesterol (Eraslan et al., 2007). According to earlier observations of
Weisburger and Chung (2002), it is inferred that polyphenols intake has an inverse role on
the development of atherosclerotic lesion formation due to hypercholesterolemia and these
outcomes are in strong association with the present study. Likewise, Alves et al. (2008)
reported that propolis avert the metabolic pathways involved in lipid metabolism to address
dyslipidaemia. The constituents of propolis affect directly on liver cell as well as impart an
indirect effect on thyroid hormones to overcome abnormal production of lipid components
including triglycerides.
95
Bioflavonoids are important natural compounds to inhibit the onset of cardiovascular
and heart diseases by lowering the plasma lipids and stopping their oxidation due to
excessive ROS. Quercetin is the important flavonoid in propolis, a key element to reduce the
oxidation of LDL. Mnay of polyphenols including Caffeic acid, p-Coumaric acid and
Feraulic acid etc. supposed to be responsible for removal of lipids peroxyl radicals thus
inhibit the problem associated with dyslipidaemia which is reported in the current study
(Yousef et al., 2005; Frankel et al., 1998; Stocker and Keany, 2004). Propolis extract used
during current study contains wide spectrum of bioflavonoids those inhibit the onset of
abnormal lipid metabolism by retarding the production of free radicals and lipid
peroxidation. It may deduce from the above discussion that functional drinks prepared using
propolis extract have pronounced affect against lipid related maladies especially LDL and
high cholesterol levels. However, they showed more reduction in hypercholesterolemic and
hyperglycemic conditions. It is worth mentioning here that drink prepared with ethanol
extract of propolis is more effectual in managing the hyperlipidemic situation in comparison
with drink prepared with methanol extract of propolis. Due to rich phytochemistry of
bioactive compounds propolis based functional drink can be used for combating the diet
related disorders with special to hypercholesterolemia.
4.7.8. Blood Glucose Level
The Mean square values represent a non-significant effect of treatments on blood
glucose level in study-I whereas, a highly significant effect on blood glucose level was
observed in study-II and significant in study-III (Table: 4.34). The results pertaining to blood
glucose level during study-I To, T1 and T2 treated groups showed blood glucose values as
89.63±6.24, 85.94±6.39 and 86.78±7.18 mg/dL and 90.12±8.16, 86.52±7.21 and 87.22±5.43
mg/dL for To, T1 and T2 treated groups in trial 1 and trial 2 accordingly. Study-II (high
sucrose diet) group (trial 1 & 2) exhibited highest glucose concentration as 140.25±12.19 &
147.96±13.47 mg/dL in To group that was highly significantly reduced in consecutive study
trials for T1 (125.87±17.58 & 131.65±19.73mg/dL) followed by T2 (130.26±18.14 &
137.62±14.57 mg/dL). Similarly in study-III (trial 1 & 2), To group exhibited maximum
blood glucose values as 101.23±9.53 & 103.63±11.36mg/dL, respectively that was
momentously decreased to 94.16±8.76 & 95.80±7.45mg/dL and 94.76±8.41 & 97.42±6.28
96
Table: 4.34. Mean square values for effect of treatments on blood glucose (mg/dL)
SOV Df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 83.73NS
14.23NS
412.35** 532.85** 99.12* 113.29*
Error 27 6.15 5.95 13.11 14.50 7.09 7.42
Total
29
*= Significant
**= Highly significant
NS= Non Significant
Table: 4.35. Mean values for the effect of treatments on blood glucose (mg/dL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
89.63±6.24
90.12±8.16
85.94±6.39
86.52±7.21
86.78±7.18
87.22±5.43
Study II
(Trial 1)
(Trial 2)
140.25±12.19a
147.96±13.47a
125.87±17.58c
131.65±19.73c
130.26±18.14b
137.62±14.57b
Study III
(Trial 1)
(Trial 2)
101.23±9.53a
103.63±11.36a
94.16 ±8.76b
95.80±7.45b
94.76±8.41b
97.42±6.28b
Study I: Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
97
Study I: Normal diet; Study II: High sucrose diet; Study III: High cholesterol diet
T1= Functional drink prepared with ethanol extract of propolis
T2 = Functional drink prepared with methanol extract of propolis
Figure.4.8: Effect of treatments on percent reduction in Blood Glucose level in animal models
4.12 3.99
10.25
11.02
6.98
7.56
3.18 3.22
7.12 6.99
6.21 5.99
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Study1(Tr 1) Study 1(Tr 2) Study 2(Tr 1) Study 2(Tr 2) Study 3( Tr 1) Study 3(Tr 2)
T1
T2
98
mg/dL in T1 and T2 groups. The percent reduction in blood glucose level as compared to
control group as shown in figure: 4.8 determined that T1 and T2 imparted 4.12 & 3.99 and
3.18 & 3.22% decrease in blood glucose in study-I. In a same way during study-II showed
decrease in blood glucose as 10.25 & 11.02% and 7.12 & 6.99% respectively in animal
groups treated with T1 and T2 group when trial 1 & 2 were done in accordance. However,
during study-III (trial 1 & 2) the percent reduction in blood glucose was observed as 6.98 &
7.56% and 6.21 & 5.99% in T1 and T2 treated groups respectively.
In diabetes, metabolism of carbohydrates and lipids is improperly regulated due to
abnormalities in insulin production and sensitivity which leads to increased fasting and
postprandial blood glucose level and the condition over a long period of time caused
hyperglycemia which ultimately is converted to diabetes mellitus (Tiwari & Rao, 2002;
Sailaja et al., 2003). The results of present study with respect to glucose concentration are in
line with earlier findings of Li et al. (2012) who conducted a study trial on laboratory
animals in which hyperglycemia was induced by injecting streptozotocin. The animals were
provided with encapsulated propolis extracts (50, 100 and 200 mg/Kg) for 10 weeks. At the
termination of study they found that consumption of propolis significantly improves the
hyperglycemia by modulating the blood glucose concentration and deduced that this
alleviation in hyperglycemic condition is due to improvement in insulin sensitivity. The
findings of Al-Hariri et al. (2011) are in agreement with the present exploration; they
documented a significant decrease in diabetic rats consuming propolis and further
demonstrated that propolis manages glucose abnormalities due to various mechanistic
approaches including metabolic alterations, diminishing the uptake of glucose by liver while
promoting the utilization by the peripheral tissues. It was also depicted that propolis involves
in the upshoooting of insulin sensitivity and lowering the glucagon index in streptozotocin
induced diabetic rats. Previously, Abo-Salem et al. (2009) also documented hypoglycemic
role of propolis in a dose dependant manner while inducing hyperglycaemia in rats and
simultaneously supplied with ethanol extract of propolis (100, 200, 300 mg/Kg) for 40 days.
At the end of study, they observed a strong antioxidant role of propolis to ameliorate the
oxidative stress associated with diabetes/hyperglycaemia. They concluded that 300 mg/Kg of
propolis extract improves the blood glucose concentration up to normal level by altering the
metabolic pathways linked with glucose absorption and utilization. The conclusion of
99
Burdock, (1998) is also in accordance with present investigation that propolis has potential to
treat the diabetes/ hyperglycemia by promoting the regeneration of damaged pancreatic cells
and clearly regarded as a therapeutic adjuvant for the amelioration of diabetic syndrome.
Recently, Abdulbasit et al. (2013) conducted a study to explore the behaviour of Nigerian
propolis against hyperglycaemia and associated oxidative stress in diabetic rats modelling.
They administered the propolis (150, 200, 300 mg/Kg) among different groups and found
that Nigerian propolis possess hypoglycaemic activity and therefore associated with the
amelioration of oxidative stress induced by hyperglycaemia/diabetes.
4.7.9. Plasma Insulin concentration
The statistical values presented in (Table: 4.36) elucidated a non-significant effect of
treatments on plasma insulin level in study-I whereas, insulin value was affected highly
significantly in study-II and significantly affected study-III due to treatments.
The mean values (Table: 4.37) for insulin level in study-I were observed as 7.21±0.34
& 7.96±0.27, 7.38±0.27 & 8.14±0.69 and 7.35±0.81 & 8.12±0.17 µU/mL in To, T1 and T2
groups, respectively during both trial-1 and trial-2. However, in study-II (trial-1 & 2), lowest
value of insulin level was examined in To as 9.12±0.24 & 9.91±0.56 µU/mL which was
uplifted highly significantly to 11.80±0.73 & 11.40±0.42 and 11.03±0.37 & 10.65±0.54
µU/mL in T1, and T2 treated groups accordingly due to consumption of functional drink.
Likewise in study-III, To showed minimum insulin level (8.88±0.29 & 9.63±0.13µU/mL)
that was substantially increased with T1 and T2 groups (11.49±1.41 & 11.07±1.29; 9.28±0.79
& 10.05±1.02 µU/mL) in trial-1 and trial-2 as a function of functional drink consumption.
It is clear from (Figure: 4.9) that in study-I ( trial-1 & 2) drinks T1 and T2 imparted
2.36 & 2.21 and 1.99 & 2.01% increase in insulin level respectively as compared to control.
Similarly in study-II during trial-1 and trial-2 increased in insulin value was recorded as 6.99
& 7.01% and 4.21 & 3.96% for T1 and T2 groups accordingly as a function of propolis based
functional drink. Study-III showed maximum increase in insulin level in T1 (4.56 & 4.36%)
followed by T2 (2.75 & 2.89%) in trial-1 and trial-2 respectively.
The outcomes of the present study are supported by the recent findings of Ho et al.
(2013), who conducted a study on the laboratory animals to assess the hypoglycemic activity
of Caffeic acid phenyl amide (CAPA), a compound derived from propolis extract. They
observed that CAPA induces hypoglycemic action and increase the concentration of insulin
100
Table: 4.36. Mean square values for the effect of treatments on plasma insulin (µm/mL)
SOV Df
Study I
(Normal diet)
Study II
(High sucrose diet)
Study III
(High cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 1.98NS
1.29NS
19.64** 15.98** 22.44* 16.83*
Error 27 0.11 0.05 0.06 0.04 0.08 0.03
Total
29
*= Significant
**= Highly significant
NS= Non Significant
Table: 4.37. Mean values for the effect of treatments on plasma insulin (µm/mL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
7.21±0.34
7.96±0.27
7.38±0.27
8.14±0.69
7.35±0.81
8.12±0.17
Study II
(Trial 1)
(Trial 2)
9.12±0.24b
9.91±0.56b
11.80 ±0.73a
11.40±0.42a
11.03±1.13a
10.65±0.74a
Study III
(Trial 1)
(Trial 2)
8.88±0.29c
9.63±0.13c
11.49±1.41a
11.07±1.29a
9.28±0.79b
10.05±1.32b
Study I: Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
101
Study I: Normal diet; Study II: High sucrose diet; Study III: High cholesterol diet
T1= Functional drink prepared with ethanol extract of propolis
T2 = Functional drink prepared with methanol extract of propolis
Figure: 4.9. Effect of treatments on percent increase in plasma insulin in animal models
2.36 2.21
6.99 7.01
4.56 4.36
1.99 2.01
4.21 3.96
2.75 2.89
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Study1(Tr 1) Study 1(Tr 2) Study 2(Tr 1) Study 2(Tr 2) Study 3( Tr 1) Study 3(Tr 2)
% i
ncr
ease
in
in
suli
n
Treatments
T1
T2
102
significantly from 7.7±1.0μIU/mL to 14.9±3.4μIU/mL in the group of animals treated
with CAPA intake. Previously, Zamami et al. (2007) explored the role of Brazilian
propolis extract on the development of insulin resistance by observing the effect of
propolis extracts on fructose drinking rats. The Wistar rats were fed with 15%
fructose solution along with drinking water to induce hyperglycemia with
simultaneous intake of 100-300mg/kg propolis extract and a sharp improvement in
insulin sensitivity was observed. They concluded that propolis could be an effective
functional food to retard the insulin resistance. Likewise, Kang et al. (2010) reported
the impact of propolis on the insulin resistance diabetes and suggested that extract of
propolis has strong antidiabetic properties and may be used to improve the insulin
resistance diabetes complications. The proposed route of action of propolis in insulin
improvement is suppressing the expression of gluconeogenic genes G6Pase and
inhibiting the Tyrosin phosphorylation of GSK3α/β. Similarly, Li et al. (2012)
conducted a trial on sprauge dawly male rats to elucidate the effect of encapsulated
propolis on the blood glycemic, lipid metabolism and insulin resistance in diabetic
group of animals. They suggested that encapsulated propolis improves the blood
glucose profile and improve the insulin sensitivity by upshooting the insulin and
cellular interaction.
As far as mechanism is concerened polyphenols associated with propolis in
the current study are involved in the management of diabetes and associated health
problems by inhibition of melondialdehyde (MDA) as well as by producing
superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). CAPE one of the
important bioflavonoids of propolis, retards the oxidative damage caused by diabetes
mellitus by inhibiting the genesis of ROS that lowers the post diabetic effects on body
tissue and liver cells. The active ingrediants of propolis like polyphenols and
flavonoids, reported in the present study inhibit the destruction of β-cells of pancreas.
They prevent the production of interleukin 1-β (IL-1β) from human white blood cells,
inhibit free radicals production and suppress IL-1β and nitric oxide synthase activity
which play a significant role in the the management of diabetes as examined dusring
the current study(Rahimi et al., 2005; Yilmaz et al., 2004; Matsushige et al.,1996). It
is inferred from the present discussion that bioactive compounds of locally available
propolis are effectual in the treatment of blood glucose related abnormalities.
Considering the importance of natural ingredients of propolis, it can be used as an
103
alternate approach in diet based therapies to cope the progression of hyperglycemia
and related complications.
4.7.10. Liver function tests
Liver function tests were performed for safety concerns comprised of alanine
transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP).
4.7.10.1. Aspartate aminotransferase (AST)
The Mean square values for the effect of treatments on AST (Table: 4.38)
showed that AST was affected non-significantly in study-I whereas, substantial
influence was noticed in study-II and study-III. The mean values (Table: 4.39) for To,
T1 and T2 groups during study-I were recorded as 103.4±8.42, 99.25±7.18 and
100.28±9.54 IU/L respectively. Whereas, in study-II, the means for AST showed
maximum value in To (138.98±8.58 IU/L) as compared to T1 (129.76±7.59IU/L) and
T2 (131.47±9.23 IU/L). Likewise, in in study-III maximum value for AST was
measured in To (116.45±7.52 IU/L) which was substantially reduced in T1
(107.54±6.28IU/L) and T2 (111.65±8.97 IU/L) respectively. Trial-2 exhibited AST
values for To in study-I, II and III 105.23±7.01IU/L, 101.06±8.23IU/L and
102.08±6.74IU/L respectively. Similarly, in study-II the maximum value was noticed
in To group (140.13±10.25IU/L) which was substantially reduced in T1
(131.77±9.48IU/L) and T2 (135.57±10.37IU/L). Likewise, the AST values measured
during study-III for the To, T1 and T2 groups were 115.52±9.16IU/L, 107.79±9.68
IU/L and 110.76±9.24 respectively.
4.7.10.2. Alanine transaminase (ALT)
It is obvious from the mean square values that treatments (functional drinks) showed
significant effect on serum ALT level in all study groups except for study-I (Table:
4.40). The observed mean values of ALT for To, T1 and T2 during study-I were
46.47±3.43, 44.60±3.32 and 44.97±3.53 IU/L, respectively. However in study-II, the
highest value for ALT level was noticed in To (57.98±3.52 IU/L) which was
substantially decreased in T1 (55.07±2.89IU/L) and T2 (56.10±3.28IU/L) groups as a
result of functional drink consumption. In a same manner during study-III, the
maximum ALT level as 47.65±3.16IU/L was recorded in To group that significantly
reduced to 44.49±4.34 and 45.68±2.82 IU/L in T1 (functional drink containing ethanol
extract of propolis) and T2 (functional drink containing methanol extract of propolis)
groups, correspondingly. Likewise a similar trend was observed in trial-2, where the
maximum values for ALT was noticed in TO 47.98±4.15, 58.98±4.13 and 45.67±4.27
104
Table: 4.38. Mean square values for the effect of treatments on AST (IU/L)
SOV df
Study I
(Normal diet)
Study II
(High Sucrose diet)
Study III
(High Cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatment
s 2 17.88
NS 2.99
NS 134.99* 143.28* 15.37* 13.04*
Error 27 15.39 15.46 15.36 12.93 115.91 114.23
Total 29
*= Significant
**= Highly significant
NS=Non Significant
Table: 4.39. Mean values for the effect of treatments on AST (IU/L)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
103.4±8.42
105.23±7.01
99.25±7.18
101.06±8.23
100.28±11.54
102.08±13.18
Study II
(Trial 1)
(Trial 2)
138.98±8.58a
140.13±10.25a
129.76±7.59c
131.77±9.48c
131.47±9.23b
135.57±10.37b
Study III
(Trial 1)
(Trial 2)
116.45±13.67a
115.52±9.16 a
107.54±12.47c
107.79±9.68c
111.65±13.21b
110.76±9.24b
Study I : Normal diet
Study II : High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2 : Drink prepared methanol extract of propolis
105
Table: 4.40. Mean square values for the effect of treatments on ALT (IU/L)
SOV Df
Study I
(Normal diet)
Study II
(High Sucrose diet)
Study III
(High Cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatment
s 2 1.67
NS 3.33
NS 136.41* 82.23* 96.48* 91.72*
Error 27 6.90 7.41 27.81 25.28 9.98
9.54
Total 29
*=Significant
**= Highly significant
NS=Non Significant
Table: 4.41. Mean values for the effect of treatments on ALT (IU/L)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
46.47±3.43
47.98±4.15
44.60±3.32
46.01±4.21
44.97±3.53
46.51±4.31
Study II
(Trial 1)
(Trial 2)
57.98±3.52a
58.98±4.13a
55.07±2.89c
55.94±3.57c
56.10±3.28b
56.82±3.39b
Study III
(Trial 1)
(Trial 2)
47.65± 3.16a
45.67±4.27a
44.49±4.34c
42.90±2.79c
45.68±2.82b
43.83±4.29b
Study I : Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared methanol extract of propolis
106
Table: 4.42. Mean square values for the effect of treatments on ALP(IU/L)
SOV df
Study I
(Normal diet)
Study II
(High Sucrose diet)
Study III
(High Cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 128.18NS
121.34NS
2558.86* 4078.03* 12086.4* 14502.8*
Error 27 26.37 27.89 47.44 42.25 673.5 655.0
Total 29
*=Significant
**= Highly significant
NS=Non Significant
Table: 4.43. Mean values for the effect of treatments on ALP (IU/L)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
144.56±8.42
151.12±13.01
138.83±9.14
145.06±11.21
141.33±11.57
147.34±8.13
Study II
(Trial 1)
(Trial 2)
212.67±18.51a
209.34±13.25a
204.12±12.04c
200.69±15.18c
205.56±12.18b
202.45±11.43b
Study III
(Trial 1)
(Trial 2)
223.45±17.69a
217.17±14.13a
212.25±13.21c
204.89±15.38c
216.23±13.48b
208.57±17.23b
Study I : Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared ethanol extract of propolis
T2: Drink prepared methanol extract of propolis
107
IU/L in study-I, II and III respectively whereas a significant reduction in ALT level
was observed in T1 (46.01±4.21, 55.94±3.57 and 42.90±2.79IU/L) and T2
(46.51±4.31, 56.82±3.39 and 43.83±4.29IU/L) groups respectively (Table: 4.41).
4.7.10.3. Alkaline phosphatase (ALP)
The statistical values for the effect of treatments on ALP level showed a
substantial change in all study groups except for study-I as represented in Table: 4.42.
Similarly the mean values for ALP in stud- I (trial-1) were 144.56±8.42, 138.83±9.14
and 141.33±11.57 IU/L in To, T1 and T2 treated groups accordingly. Nonetheless
during study-II, maximum value was noticed in To (212.67±18.51 IU/L) which was
reduced in T1 (204.12±12.04IU/L) and T2 (205.56±12.18IU/L) groups subsequently.
Likewise the mean ALP values recorded during study-III were 223.45±17.69,
212.25±13.21 and 216.23±13.48 IU/L in To, T1 and T2 treated groups respectively. In
a same manner, a similar decreasing trend was observed during the subsequent trial in
which maximum value for ALP level was examined in To (151.12±13.01,
209.34±13.25 and 217.17±14.13IU/L) for study-I, study-II and study-III respectively
which was reduced in T1 (145.06±11.21, 200.69±15.81 and 204.89±15.38IU/L) and
T2 (147.34±8.13, 202.45±11.43 and 208.57±17.23IU/L) in study-I, study-II and study-
III accordingly (Table: 4.43).
The numerous studies indicated that high cholesterol and high sucrose intake
are the leading cause for the hepatotoxicity and other related problems. The diets with
excess of cholesterol and sugar are responsible for the generation of reactive oxygen
species (ROS) that interacts with unsaturated fatty acids of cellular membrane thus
causing to structural and functional impairment of body tissues. The movement of
AST and ALT from liver to plasma during hyperglycemia and hypercholesterolemia
conditions indicates liver malfunctioning. The role of propolis for the suppression of
increased level of ALT and AST has been demonstrated from different animal
modeling systems due to its strong antioxidant, anti-inflammatory and
hepatoprotective properties (McEneny et al., 2012; Kolankaya et al., 2002).
Previously, Saleh (2012) conducted a study on the laboratory animals to explore the
role of propolis extract in liver hepatotoxicity and confirmed that intake of propolis
extract decrease the ALT, AST and ALP values at a significant level as compared to
control those were not supplied with propolis extract. Recently, Abdelsameea et al.
(2013) reported the hepatoprotective role of propolis in albino rats those were induced
108
by atorvastatin hepatotoxicity and observed that atorvastatin upto 80mg/Kg of the
body weight significantly enhance the level of ALT and AST in a dose dependent
manner. Whereas administration of propolis to laboratory animals with abnormal liver
function in a dose rate of 50-100 mg/Kg of body weight substantially lowers the level
of ALT and AST thus promoting normal liver physiology. The findings of present
study are in harmony with the previous findings of Turkez et al. (2010), who
conducted a study to elucidate the protective role of propolis against liver toxicity.
They observed the effect of propolis consumption on the liver function among
animals treated with aluminum chloride and found that propolis significantly lowers
the serum levels of AST, ALP and ALT as compared to control group. Likewise
Abdulbasit et al. (2013) reported a marked reduction in serum ALT concentration in
diabetic rats treated with Nigerian propolis. During the study, animals were provided
propolis in a dose of 200-300mg/Kg body weight for 28 days. At the end of
experiment they examined that propolis exhibited antihyperglycemic activity by
reducing blood glucose level and also retards oxidative stress induced by
hyperglycemia by lowering the serum ALT level remarkably.
4.10.11. Renal functioning Tests
Renal functioning tests with special reference to serum urea and creatinine
level were performed to assess the renal safety status of functional drinks prepared
with addition of propolis extracts.
4.7.11.1. Urea
The statistical values (trial-1 & 2) for the effect of treatments on blood urea
level showed a non-significant effect in study-I whereas a significant effect was
noticed in study-II study-III (Table: 4.44). The mean values for blood urea (Table:
4.45) in study-I (trial-1 & 2) were found as 22.01±1.87 & 23.56±1.34mg/dL,
21.66±1.63 & 23.20±1.74mg/dL and 21.75±1.72 & 23.23±1.25mg/dL in To, T1 and T2
treated groups accordingly. Study-II (trial-1 & 2) for the To, T1 and T2 groups
exhibited 28.98±2.08 & 26.54±1.89mg/dL, 27.81±2.38 & 25.38±2.62 mg/dL and
27.92±1.83 & 25.56±1.94mg/dL accorodingly. However in study-III (trial-1 & 2)
functional drinks imparted a significant effect on the blood urea level. The mean
values were noticed as 33.44±2.11 & 33.12±2.26mg/dL, 31.91±2.17 &
31.37±2.23mg/dL and 32.10±2.43 & 31.79±2.12mg/dL in To, T1 and T2 treated
groups correspondingly.
109
4.7.11.2. Creatinine
The mean square values (trial-1 & 2) for the effect of treatments on the serum
creatinine level (Table: 4.46) depicted a non-momentous effect of functional drinks in
study-I whilst a momentous reduction was noticed in study-II and study-III. The mean
values for the effect of functional drinks intake (treatments) on serum creatinine level
in study-I (trial-1 & 2) were recorded as 0.82±0.03 & 0.84±0.01mg/dL, 0.80±0.01 &
0.82±0.02mg/dL and 0.80±0.02 & 0.82± 0.05mg/dL for the To, T1 and T2 treated
groups accordingly. Whereas, the mean values for serum creatinine during study-II
were measured as 0.99±0.02 & 0.98±0.01, 0.94±0.01 & 0.93±0.05 and 0.96±0.03 &
0.94±0.01mg/dL for To, T1 and T2 treated groups accordingly in both study trials.
Similarly treatments imparted a retarding effect during study-III (trial-1 & 2) on
serum creatinine concentration and mean values were observed as 1.06±0.01 &
1.03±0.02, 1.01±0.03 & 0.97±0.04 and 1.02±0.03 & 0.99±0.04mg/dL in T0, T1 and T2
treated groups respectively (Table: 4.47).
In human body kidney performs numerous important functions including
regulation of blood pressure, electrolyte balance and removal of toxins from the body
via urine (Garcia et al., 2012). The abnormal functioning of renal system is more
prevalent in patients suffering from diabetes, high blood pressure and cardiovascular
ailments (Chauhan and Vaid, 2009). Propolis has been recognized safe for the kidney
as it improves the functionality of renal system by managing the urea and creatinine
level in blood (Al-Qayim and Mashi, 2014). The results pertaining to urea and
creatinine level during the present study are in harmony with the earlier findings of
Abo-Saleem et al. (2009), who investigated a sharp decline in urea and creatinine
level in in diabetic induced rat model as a result of propolis consumption in a dose
dependent manner. Similarly, in another experiment Oktem et al. (2005) elucidated a
positive concern of propolis bioactive constituent like caffeic acid phenyl ester in the
management of renal tubule system and oxidative stress thus improve the
functionality of renal system by modulating the antioxidant defence system of body.
It is obvious from the above discussion that functional drinks intake do not
produce any unhealthy effect on liver and kidney function of experimental organisms.
Propolis based drinks are effectual in managing the hyperglycemic and
hyperlipidemic conditions without causing any harm to liver and renal physiology.
110
Table: 4.44. Mean square values for effect of treatments on urea (mg/dL)
SOV df
Study I
(Normal diet)
Study II
(High Sucrose diet)
Study III
(High Cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 0.99NS
0.08NS
77.48* 58.19* 37.04* 52.93*
Error 27 0.39 0.47 10.22 10.00 3.46
2.93
Total
29
*=Significant
**= Highly significant
NS=Non-Significant
Table: 4.45. Effect of functional drinks on serum urea (mg/dL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
22.01±1.87
23.56±1.34
21.66±1.63
23.20±1.74
21.75±1.72
23.23±1.25
Study II
(Trial 1)
(Trial 2)
28.98±2.08a
26.54±1.89a
27.81±2.38b
25.38±2.62b
27.92±1.83ab
25.56±1.94ab
Study III
(Trial 1)
(Trial 2)
33.44±2.11a
33.12±2.26a
31.91±2.17c
31.37±2.23c
32.10±2.43b
31.79±2.12b
Study I: Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
111
Table: 4.46. Mean squares for effect of treatments on cretinine (mg/dL)
SOV df
Study I
(Normal diet)
Study II
(High Sucrose diet)
Study III
(High Cholesterol
diet)
Trial 1 Trial 2 Trial 1 Trial 2 Trial 1 Trial 2
Treatments 2 0.002NS
9.14NS
0.05* 0.034* 0.002* 0.001*
Error 27 0.0005 6.11 0.008 0.007 0.003
0.003
Total 29
*=Significant
**= Highly significant
NS=Non-Significant
Table: 4.47. Mean values for the effect of treatments on the creatinine (mg/dL)
Studies Treatments
T0 T1 T2
Study I
(Trial 1)
(Trial 2)
0.82±0.03
0.84±0.01
0.80±0.01
0.82±0.02
0.80±0.02
0.82±0.05
Study II
(Trial 1)
(Trial 2)
0.99±0.02a
0.98±0.01a
0.94±0.01c
0.93±0.05c
0.96±0.03b
0.94±0.01b
Study III
(Trial 1)
(Trial 2)
1.06±0.01a
1.03±0.02a
1.01±0.03c
0.97±0.04c
1.02±0.03b
0.99±0.04b
Study I: Normal diet
Study II: High sucrose diet
Study III: High cholesterol diet
T0: Drink without propolis extract
T1: Drink prepared with ethanol extract of propolis
T2: Drink prepared with methanol extract of propolis
112
CHAPTER 5
SUMMARY
Bioactive moieties from natural sources are gaining attention by the
consumers and food processors owing to their antimicrobial & therapeutic properties.
These compounds play a significant role in the preservation of food products by
hindering the growth of food spoilage agents as well as have a curative role in
managing the lifestyle related maladies. In this context, honey bee propolis has strong
potential to address various disorders due to its distinct flavonoids and phenolic
contents. The present study was an effort to explore the composition of locally
available propolis for its antimicrobial behavior and nutraceutical worth against
metabolic syndromes including hyperglycemia and hyperlipidemia.
Proximate composition of propolis depicted moisture, crude fat, crude protein,
crude fiber, ash and NFE as 2.22±0.14%, 85.95±0.87%, 1.83±0.09%, 0.31±0.08%,
1.03±0.04% and 9.01±0.05%, respectively. Mineral elements including calcium,
potassium, sodium, magnesium, iron, zinc, copper and manganese were recorded as
0.53±0.8mg/Kg, 52.10±2.9, 11.33±0.91, 32.13±2.3, 29.3±1.7, 0.59±0.23, 1.50±0.1
and 0.67±0.03mg/Kg respectively. Aqueous extract, ethanol (65%, 80% & 95%) and
methanol (65%, 80% & 95%) extracts were prepared and subjected for
characterization of bioactive compounds and their antioxidant status was examined.
Among all the solvents ethanol exhibited highest score for total phenolic contents
(TPC), DPPH (2,2 diphenyl-1-picrylhydrazyl) activity and antioxidant potential
whereas minimum values were observed for aqueous extract. HPLC analysis showed
maximum polyphenols in ethanol extract (65%) and among those 4-Hydroxy benzoic
(43.17±2.89 mg/Kg) was noticed in highest concentration. Regarding antimicrobial
activity maximum zone of inhibition (29.18±1.13mm) against S. aureus was found for
ethanol extract followed by methanol extract (23.58±0.28mm) depicting that G-
positive bacteria are more susceptible to locally available propolis.
After in-vitro evaluation, two best treatments were chosen for the development
of functional drinks and subjected for physicochemical and sensory attributes. The
results showed that storage period did not impart any deleterious effect on the said
parameter of functional drinks thus suitable for bioevaluation study. Three types of
studies i.e. study-I (normal diet), study-II (high sucrose diet) and study-III (high
113
cholesterol diet) with simultaneous provision of respective functional drinks were
designed to elucidate nutraceutical aspects of propolis. The results regarding body
weight, feed and drink intake showed considerable increase in all groups of animals
but more pronounced effect was observed in control group fed with normal diet.
The provision of functional drinks showed significant reduction in cholesterol
level. Considering the results, T1 showed maximum reduction (10.25%) followed by
T2 (5.59%) in study-III while in study II 6.63% and 5.36% reduction was observed in
T1 and T2 treated groups and similar decreasing trend was noticed in the subsequent
trial. A significant change in LDL level was observed due to functional drinks in all
studies except study-I. The mean square values concerning the effect of treatments on
HDL level depicted non-momentous change on HDL in study-I whereas a significant
increase in HDL was noticed in study-II and study-III as a result of consumption of
functional drinks. During study-II, HDL level increased as 4.12% and 4.21% in T1
and T2 treated groups. Likewise in high cholesterol diet group T1 imparted 4.49 &
4.85% increase in HDL whereas T2 showed 3.01 & 2.99% increase in comparison to
control. Likewise, triglycerides level in study-II and III was decreased as 6.32 &
8.96% and 3.21% & 3.50% in T1 and T2 treated groups whereas during trial-2
decrease in triglycerides level was noticed as 5.99 & 6.69% and 3.99 & 4.01%.
Similarly the results regarding glucose concentration represent the non-
significant effect of treatments in study-I whereas significant variation was observed
in study-II and study III. Considering percent decrease in blood glucose in study-II
found as 10.25% and 7.12% in T1 and T2 treated groups due to result of drink intake.
In a same way during study-III, maximum decline 6.98% was observed in T1 followed
by T2 (6.21%) and similar trend was noticed during consecutive study trial. Likewise
statistical values for change in plasma insulin showed non-significant change in
study-I whereas, insulin values affected significantly in study-II and study-III as a
result of consumption of functional drinks. The treatments (T1 and T2) imparted
2.36% and 1.99% increase in insulin level in study-I whereas in study-II percent
increase in insulin values were examined as 6.99 % and 4.21% for T1 and T2. In study-
III, maximum increase in insulin level was found in T1 (4.56%) followed by T2
(2.75%) and similar trend was noticed in trial-2. Furthermore, safety evaluation of
developed drinks with respect to liver function and kidney function tests showed
results within acceptable limits.
114
In the nutshell, locally available propolis has different beneficial properties
especially antimicrobial and therapeutic owing to the its rich polyphenolic profile. It is
also concluded that the utilization of propolis is helpful to mitigate the life style
related disorders especially hypercholesterolemia, hyperglycemia and other metabolic
syndromes. Moreover, Propolis extracts prepared using ethanol and methanol showed
more responses in comparison to aqueous extract whereas extract prepared with 65%
ethanol found good in all concerns and in particular to attenuate hyperglycemia and
hyperlipidemia. Conclusively, propolis is effective in controlling food borne
pathogens and combating physiological disturbances and could be used as novel
ingredient in diet based therapies in various food applications.
RECOMMENDATIONS
A country wide campaign should start to produce propolis on commercial
scale to support the economy and foreign exchange.
In developing economies with special references to Pakistan propolis as
bioactive waste must be converted into useful products and ultimately as food
ingredient through academia industry linkages.
Clinical trials be planned to elucidate various health claims associated with
propolis.
Use of bee propolis as a natural food preservative may be encouraged in food
industry based on specific foods product.
Propolis derived nutraceuticals may be added in diet based therapies to address
hyperglycemia and dyslipidemia.
Further studies should be carried out to develop modern techniques for the
efficient production and utilization of propolis in food industries.
Implications and awareness of diet based remedies should be made among the
peoples through media communications and effective outreach programs.
Government should formulate policies in order to enhance the utilization of
such valuable waste material in all concerned quarters.
Impact of this research on food industry:
The findings of this study are beneficiary for farmer community and industries
involved in food processing in order to develop bee keeping more profitable industry
with respect to propolis production. The production of propolis on industrial scale
generates high value foreign exchange that ultimately promotes utility of this valuable
115
waste in a number of ways. The propolis sample used during current study possessed
significant amount of bioflavonoids that can be used as alternate medicine source to
treat different diet related maladies. The beneficial aspects of propolis attributed many
useful impacts by:
Propolis possessed appreciable amount of bioactive moieties responsible for
antimicrobial and antioxidant activity
Incorporation of propolis ingredients as natural preservatives promotes shelf
life of various food items that can be used in place of chemical preservatives
The use of propolis and its components will reduce the risk of various
maladies ultimateltely reduce the cost of medication
Future Research Directions:
Polyphenols and flavonoids of propolis have potential as antimicrobial and
nutraceutical agents which serve as part of functional foods. The isolation of
polyphenols of propolis should start to identify the nature, chemistry and functional
properties of such valuable compounds. Natural ingredients of propolis possessed
strong clinical implications that should be confirmed through various clinical trials.
Much of the work has been done in this regards however number of issues concerned
with propolis should be highlighted and addressed in a novel and scientific way to
establish scientific and industrial standards for the production and utilization of
propolis as valuable natural product. The quality of propolis further analyzed through
various modern techniques for novel bioactive moieties. The sharing of information
between scientific research and farmer community should be planned to develop
linkages for the production and utilization of propolis
Limitations of the present study:
Although physicochemical, antioxidant, antimicrobial and nutraceutical value
of the locally available propolis has been studied during the current study.
Further standardization regarding propolis bioactive compounds having
antimicrobial and nutraceutical role could not isolate as individual components
that should start in new study trials.
Bio-mechanistic study of propolis regarding its hypocholesterolemic and
hypoglycemic activity has not studied during current study that should be
planned in future to examine role propolis in human body.
116
Antimicrobial behaviour of locally avai lable propolis studied against
pathogenic bacteria but should be planned ti investigate its role against
pathogenic fungi and other spoiling agents.
In future collaboration is needed with any foreign or local funding agency to
develop the production technology of propolis on commercial scale using local
infrastructure.
Future Studies:
Modern lifestyle enhanced different athrogenic disorders that can be managed
by monitoring the daily intake. The use of functional and nutraceutical foods is the
concept of modern era. Propolis should be explored on industrial scale as source of
various nutraceutical. Modern technologies should develop to identify and isolate
individual polyphenol associated with propolis and their possible health claim should
address through clinical trials.
117
LITERATURE CITED
AACC. 2000. Approved Methods of American Association of Cereal Chemists. The
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APPENDICES Appendix-I
Performa for sensory evaluation Functional drink
Name of the judge………………………………….
Date……………..
Character T0 T1 T2
Color
Flavor
Sweetness
Sourness
Overall acceptability
Signature………………
……
INSTRUCTIONS
Take a sample of Functional drink and mark for color, flavor, sweetness, sourness and
overall acceptability using the following 9-point Hedonic Scale:
Extremely poor 1
Very poor 2
Poor 3
Below fair above poor 4
Fair 5
Below good above fair 6
Good 7
Very good 8
Excellent 9
Note:
1. Take a sample of functional drink and mark for color, flavor etc.
2. Rinse mouth with water before proceeding to the new sample
3. Make comparison among the samples and record the reading.
4. Don't change the sequence of samples.
143
Appendix-II
Composition of diet used during efficacy study
Ingredients (%) Normal diet High sucrose diet High cholesterol
diet
Corn oil 10 10 10
Corn starch 66 26 64.5
Casein 10 10 10
Cellulose 10 10 10
Salt mixture 3 3 3
Vitamins 1 1 1
Sucrose - 40 -
Cholesterol - - 1.5
144
Appendix-III
Composition of Vitamin Mixture
Thiamine hydrocholoride 0.060
Riboflavin 0.200
Pyridoxine hydrochloride 0.040
Calcium pentothenate 1.200
Nicotinic acid 4.000
Inositol 4.000
p-aminobenzoic acid 12.000
Biotin 0.040
Folic acid 0.040
Cyanobacterium 0.001
Choline chloride 12.000
Maize starch 966.419
1000
145
Appendix-IV
Composition of salt mixture
Calcium citrate
308.2
Ca(H2PO4)2H2O
112.8
H2HPO4
218.7
HCl
124.7
NaCl
77.0
CaCO3
68.5
3MgCO3.Mg(OH)2.3H2O
35.1
MgSO4 anhydrous
38.3
Ferric ammonium citrate
CuSO4.5H2O
NaF
MnSO4.2H2O
KAL(SO4)2.12H2O
KI
91.41
5.98
0.76
1.07
0.54
0.24
16.7
100.00 1000.00
