ANTIMICROBIAL EFFECTS OF HONEY AND ITS SPECIFIC ACTIONS ON

CELL WALLS, MEMBRANES AND ENZYMES OF SOME MICROBIAL

PATHOGENS

BY

ATTAH, Friday
MTech/SLS/2017/6703

DEPARTMENT OF MICROBIOLOGY
FEDERAL UNIVERSITY OF TECHNOLOGY
MINNA

JUNE, 2021

i
ANTIMICROBIAL EFFECTS OF HONEY AND ITS SPECIFIC ACTIONS ON
CELL WALLS, MEMBRANES AND ENZYMES OF SOME MICROBIAL
PATHOGENS

ABSTRACT

Antimicrobial agents of plant origin have enormous therapeutic potentials. Honey, which
is a product of plant, is a sugary substance produced by bee from the nectar of flower. It
has been an age long antimicrobial therapy for wounds and burns. The aim of this study
was to determine the antimicrobial effects of honey and its specific actions on cell walls,
membranes and enzymes of the following organisms: Pseudomonas aeruginosa, Bacillus
subtilis, Trichophyton verrucosum. Trichophyton equinum and Escherichia coli using
agar well diffusion method. Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia
coli were tested at 100 %, 80 % and 60 % (v/v) honey concentration and Trichophyton
verrucosum and Trichophyton equinum were tested at 100 % honey concentration only.
Escherichia coli had the highest zone of inhibition of 29.0 mm, 27.0 mm and 19.0 mm at
100 %, 80 % and 60 % respectively followed by Bacillus subtilis, which had 15.0 mm,
1.0 mm and 8.0 mm zones of inhibition while Pseudomonas aeruginosa had 13.7 mm,
11.0 mm and 6.3 mm zones of inhibition as the least. At 100 %, T. verrucosum and T.
equinum had zone of inhibition of 14.0 mm and 17.0 mm respectively. The minimum
inhibitory concentrations recorded for Escherichia. coli, Bacillus subtilis and P.
aeruginosa were 10 %, 80 % and 100 % (v/v) respectively. The minimum bactericidal
concentrations for E. coli and B. subtilis were 20 % and 100 % (v/v) respectively. The
effect of honey on bacterial isolates after incubation for one hour and two hours revealed
that E. coli had 22.0 and 35.3 µg/ml protein leakage; Bacillus subtilis had 31.0 and 49.0
(µg/ml) while Pseudomonas. aeruginosa had 49.7 and 60.0 (µg/ml) respectively. The
result of enzymatic inhibition showed that honey had activity against the cells treated
compared to the control: E. coli had 11.0 and 14.0 (mm); Bacillus. subtilis had 30.0 and
40.0 (mm) while Pseudomonas aeruginosa had 31.7 and 45.0 (mm) for the treated and
untreated cells respectively. The result of this study showed that the honey had a broad
spectrum antimicrobial activities and could be recommended for antibiotics alternative
therapy.

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 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background to the Study

Antimicrobial agents possess immense curative prospects and are very effective in

controlling disease causing organisms (Mahato and Sharma, 2018). Prior to the discovery

of microorganisms, it was generally believed that certain medicinal herbs had curative

properties and, without a doubt, contained what we now refer to as antimicrobial

principles (Mahato and Sharma, 2018). Common infectious diseases have been cured by

the use of plants, and this form part of the customary management of different health

challenges, some of these conventional medicines are still included. The rich sources of

antimicrobial agents are medicinal plants (Jindal and Vashist, 2013).

Medicinal plants are rich sources of antimicrobial agents (Jindal and Vashist, 2013).

According to World Health Organization (WHO), medicinal plants would be the best

source to obtain a variety of drugs and 80 % of world population is dependent on

traditional medicine and a major part of traditional therapies involves the use of plant

extracts or their active constituents. Yet a scientific study to determine their antimicrobial

active compounds is a comparatively new field. (Parmar and Rawat, 2012).

One of the earliest medicines used by traditionalist for both infectious and non-infectious

diseases and was also recommended for management of burns and wounds was honey, a

plant product according to the report of Olatunji et al. (2018).

Honey is a sugary and thick material formed by bees (Olatunji et al., 2018). It is made

up of flower nectars (floral honey) and sweet plant deposits (non-floral honey), as well

as an enzyme secreted by honeybees. These sugary substances are gathered by bees, who

2
then improve them through their own materials before processing them in beehives

(Manyi-Loh et al., 2011; Süerdem and Akyalçin, 2017).

Honey is a popular sweetener, non-toxic, non-irritant and a common household product

(Usman et al., 2015). Ayurveda, an ancient Indian System of Health Care treats honey as

food for health while recommending it as a medicine for some conditions using it

externally as well as orally (Khandal et al., 2010). Ayurveda, which means science of

long life, is believed to have originated over 6000 years ago and was designed to promote

good health and long life rather than to fight disease and was practiced by physicians and

surgeons (called Bheshaja or vaidya) but recently herbal medicine has attracted much

attention as alternative medicines useful for treating or preventing life-style related

disorders (Agyare et al., 2009).

Honey is a well-researched oldest medicine for a variety of pathogenic microbes, as well

as an active antibacterial agent for burn injuries (Brudzynski, 2006). After topically

applied to wounds, osmosis would be expected to draw water from the wound into the

honey helping to dry the infected tissue and reduce bacteria growth (Al-Naama, 2009).

Honey's antibacterial properties have been demonstrated in various reports and clinical

trials against a wide range of microorganisms, including multi-antibiotic drug resistant

strains (Kumarasamy and Mahendran, 2015).

Laboratory studies and clinical trials have shown that honey is an effective broad-

spectrum antimicrobial agent. Honey has been reported to have inhibitory effect on

several bacteria including aerobes and anaerobes, Gram-positive and Gram-negative and

is effective against methicillin resistant Staphylococcus aureus (MRSA), β-hemolytic

Streptococci and vancomycin-resistant enterococci (VRE) (Allen et al., 2000; Kingsley,

2001).

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The use of honey for wound infections treatment dated over 2000 years prior to bacterial

discovery to be the cause of infections (Sushila et al., 2012). Olatunji et al. (2018)

reported that the antimicrobial activity of honey was first recognized in 1892, and this

was then accompanied by detailed studies to further substantiate this argument and to

show factors leading to the activity of antimicrobials.

Natural medicinal products have been used for millennia in the treatment of multiple

ailments (Manyi-Loh, et al., 2011). Although many have been superseded by

conventional pharmaceutical approaches, there is currently, resurgence in interest in the

use of honey and honey products by the populace. This choice of honey for therapeutic

purpose is a branch of medicine called apitherapy (Ghosh and Playford, 2003).

Honey can be classified based on where the bee got resources for honey make up: Floral

and non-floral honeys. Floral honeys can either be unifloral or multifloral, depending

whether the honey collected is from the nectar of the same flower or from nectar of

flowers of various types (Manyi-Loh, et al., 2011). Non floral honey (honey dew) is made

by bees that extract sugars from the living tissues of plants or fruits, and/or scavenge the

excretions of insects (aphids) that tap the veins of higher plants (Subrahmanyam, 2007).

The antimicrobial effects of honey could be bacteriostatic or bactericidal depending on

the concentration that is used. However, such activity has been attributed to certain

factors like high osmolarity (low water activity), acidity (low pH), and hydrogen peroxide

and non-peroxide components (Taormina et al., 2001; Al-Naama, 2009).

Among the possible therapeutic alternatives that are approved, non-toxic and with a wide

range of antimicrobial spectrum of action, honey is considered. This may be a potential

alternative or replacement for antimicrobial agents, but its use is constrained by certain

factors. Inadequate knowledge of antimicrobial properties and lack of adequate

4
information have reduced the clinical applicability of honey (Malik et al., 2010; Mandal

and Mandal, 2011).

In Nigeria, honey is accepted to be important in traditional treatment of respiratory

ailments, skin infections, diarrhoea and other diseases as captured by Eleazu et al. (2013).

There are numerous reports on the physico - chemical, antimicrobial, microbiological and

medicinal properties of honey from many parts of the world, including North America,

Europe, Asia, Australia, and South Africa (Gomes et al., 2010; Mandal and Mandal,

2011; Fahim et al., 2014).

Data on Nigerian honey is limited; however, some physical properties, antibacterial and

chemical properties had been documented from honey in Nigeria (Adebiyi et al., 2004;

Omafuvbe and Akanbi 2009; Anyanwu, 2012; Eleazu et al., 2013; Buba et al., 2013).

Although, the antifugal study on Nigerian honey is minimal, nevertheless, separate study

conducted by Akujobi and Njoku (2010), Anyanwu (2012), Eleazu et al. (2013) and Buba

et al. (2013) indicated that Nigeria honey could kill fungi and also inhibit the growth of

fungi

1.2 Statement of Research Problem

Attention had been drawn nowadays in serious search for antimicrobial compound all

over the world owing to the failure of the already existing antibiotics (Ibrahim and Aliyu,

2015). With the irrational and massive use of antibiotics in underdeveloped and

developing countries, resistance develops and spread beyond human imagination. As a

result, the effectiveness of the antibiotics is reduced (Zakaria, 2015). Rural dwellers and

disadvantaged people who are unable to have enough money to access good health care

services relied on conventional medicines which they are familiar with for their treatment

(Krishnan, 2018).

5
The World Health Organization (WHO) has assessed that up to 80 percent of individuals

in the developing nations rely on local medicinal prescriptions because of their easy

accessibility, wide affordability and cultural familiarity. Indeed, about 40 percent

population of the world's poor have no good hospital, hence, they depend on local

medicinal prescription (Krishnan, 2018).

Clinical acceptability of honey has been slowed down by curtailed knowledge of the

antimicrobial activity and short accurate mechanisms for determine the type of action of

honey and variations of honey (Malik et al., 2010). According to Elijah et al. (2015), the

structural existence of honey may be linked to geographic origin, and the antibacterial

function of floral sources may play a key role. In Nigeria, there is no comprehensive

record that honey from all the States have antimicrobial activity and of course, Nigeria

has different climatic zones

1.3 Justification for the Study

The antimicrobial information of traditional medicine is a panacea for battling antibiotic

resistant microorganisms in the developing nation like Nigeria. Many traditional

medicines had been hawked without antimicrobial authentication of which honey was

part of. Moreover, the specific of action of any antimicrobial agents is very important as

it gives specific information about its activity.

The antimicrobial activities of honey from different countries has been reported in

recognition of the medicinal properties of honey (Olajuyigbe et al., 2017). In Nigeria, the

therapeutic ability and the antibacterial activities of various honey samples widely

marketed for consumption need to be validated. Previous researched works on honey

dwell on the antimicrobial activity

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Therefore, for honey to be recommended as an alternative antimicrobial agent, like other

conventional medicines, there is need for laboratory investigation on the specific action

of honey on bacterial cell and its enzymes.

1.4 Aim and Objectives of the Study

The aim of this study was to determine the antimicrobial effects of honey and its specific

actions on cell walls, membranes and enzymes of some microbial pathogens.

The specific objectives were to:

I Assess the antimicrobial activity of honey.

II Determine the minimum inhibitory concentration (MIC) and minimum

bactericidal concentration (MBC).

III Determine the bioactive component of the honey.

IV Determine the effects of honey on the cell walls, membranes and enzymes of the

test organisms.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Plant as Antimicrobial Agents

2.1.1 Reason for reconsidering plant as antimicrobial agents

Nowadays, the world had observed the amazing success in improvement of technology,

science and medical practices; however, there is futile effort in the control of dramatic

spread of infectious diseases (Abdallah, 2011). Abdallah (2011) reported that the

infectious diseases remain the second leading cause of death worldwide. Though, the

need for new antimicrobial agents is greater than ever. Microbes had been existence in

earth since million years ago, being one of the oldest creatures in this planet.

These microscopic organisms are adapted, developed and survived in this changing

nature throughout the eras, while other advanced huge ancient animals and plants

perished. Though, microbes considered as one of the most adaptive and successive

creatures in nature. These microbes were already-since that time- subjected to antibiotics

produced from other microorganisms such as Penicillium notatum, as example. And they

produce antibiotic resistant mechanisms, naturally (Opal et al, 2000). It is therefore not

shocking that organisms are securely developed against our manufactured and semi-

synthetic antibiotics in the cutting edge time frame. On the other hand, after its arrival on

earth, the battle between man and organisms has begun.

Historical evidence found that 60,000 people lived in Mesopotamia (Iraq) long before,

using a therapeutic plant called Hollyhock (Alcea rosea L.) (Cowan, 1999). Perhaps the

8
best weapon used by old humans against diseases was plants. Man has been using anti-

microbial medications against bacteria since time immemorial. The employment and

development of these medications against microorganisms lingered across civilizations

until the modern era.

Recently, the global problem of the rapid growth of bacterial resistance to

chemotherapeutic agents has led researchers to explore the use of other natural

antimicrobial products, such as medicinal plants (Abdallah, 2011)

2.1.2 Plants: An alternative source for antimicrobial agent.

Traditionally, early civilization regarded herbal plants as the main source of antibiotics

discovery as reported by McChesney et al. (2007). In Mesopotamia, according Newman

et al. (2000), Egyptian had recorded about even hundred drugs mainly of plant origin

between 1500 and 2900 BC. In what is known today as traditional medicine, as stated by

Abdallah (2011), this ancient medical experience has been transferred to us. Until 1990s,

plant roots, leaves and bark contributed about eighty percent of total of all treatments

(McChesney et al., 2007).

Each year, the processing and analysis of herbal plants and its products contribute majorly

to the economic growth in India, both as a source of part-time and full employment. Plant

is the most significant source of drug. Plants had been used for years as drugs in ancient

times. In two distinct fields of health management, medicinal plants are commonly used

worldwide; traditional medicine system and modern medicine system (Mohd et al.,

2012).

So many new drugs have been extracted for thousands of years from plants. Human

beings were completely derived their care services from herbal plant prior to the

discovery of antibiotics (Singh et al., 2008). Herbal plants are known be the gift of nature

to human beings to make well-life free of disease. The diverse tradition of India is a stable

9
and successful source of traditional medicines, many of which are of herbal origin. The

medicinal properties of approximately 45,000 plant species (Grover et al., 2002). People

in olden days gathered knowledge concerning herbal plants and recognized it into herbal

therapeutic categories (Rout et al., 2009). Synthetic analogues are part of drug extracted

from plants as uncovered in recent pharmacopoeia is more than 25 percent (Astin, 1998).

Savant et al. (2014) noted that there are two reasons for clinical microbiologists to be

involved in the subject of extract from plant for antimicrobial. Next, the bioconstituents

in the extracts were used to make antibiotics; some are already being tested in humans.

Alternative types are now being studied, especially plant sources. Second, awareness is

created to many people on the issues with conventional antibiotics being over-prescribed

and misused. Moreover, many individuals are interested in getting more control over their

health care (Savant et al., 2014).

2.1.3 Importance of antimicrobial agents from plant

Thirthy-eight plant-derived flavonoids representing seven distinct structural groups were

checked for antibiotic-resistant bacteria activity by Xu and Lee (2001) using the disc-

diffusion assay and broth dilution assay. Four flavonoids (myricetin, datiscetin,

kaempferol, and quercetin) and two flavones (flavone and luteolin) demonstrated

inhibitory activity against methicillin-resistant Staphylococcus aureus according to the

findings. Myricetin has also been shown to prevent the growth of multidrug-resistant

Burkholderia cepacia, vancomycin-resistant enterococci (VRE), and other medically

important bacteria like Klebsiella pneumoniae and Staphylococcus epidermidis.

Traditional medicinal plants have been proposed by Ortega-Ramirez et al. (2014) and

Seow et al. (2014) as a source of food additives and bioactive compounds, which have

traditionally been used to manage health problems and preventing infection. Therapeutic

plants produce anti-cancer terpenes and phenols, as well as oils derived from plants with

10
pharmacological functions such as antioxidant, anticancer, and antibacterial properties;

use of these drugs as antimicrobial ingredients in food products; synergism between

components affects their effectiveness. One hour of medicinal smoke therapy produced

via wood burning and a combination of odoriferous and medicinal herbs resulted in a 94

percent reduction in bacterial counts according to Nautiyal et al. (2007). After 30 days,

there were no pathogenic bacteria (Corynebacterium urealyticum,

Enterobacteraerogenes, Enterobacter aerogenes, Klebsiella mobilis, Kocuria rosea,

Pseudomonas syringae pv, Persicae, and Staphylococcus lentus) in the open space

indicates that the medicinal smoke treatment is bactericidal. Herbal treatments of the

natural smoke/inhalational approaches to drug delivery are possible with medicinal

smoking.

According to Savant et al. (2014), majority of unexploited element of drugs is

antimicrobials derived from plants. There must be continuous and more discovery of

plant antimicrobials. Antimicrobial substances on plants have huge therapeutic potential.

At the same time, they are successful in treating infectious diseases. In the treatment of

infections, herbal medicine is successful, at the same time; many of the risk factors

associated with naturally derived antibiotics are reduced. Effective, but gentle, they are.

So many plants have tropisms that correspond to human structures.

Phytomedicines have a number of health consequences. Their therapies may also go

beyond the disease's symptomatic care. A good example of this is Hydrastis canadensis.

Hydrastis not only has antimicrobial properties, but it also improves splenic blood flow,

allowing for optimum spleen activity and the development of facilitating compounds

(Savant et al., 2014).

2.1.4 Traditional medicine

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Herbal medication is also known as phytomedicine (Attah et al. 2020). The use of the

following plant part: roots, barks, seeds berries, flower for therapeutic reasons are

referred to herbal drugs. Annuals, biennials and perennials can be medicinal plants.

Annual plants either complete its life span within a year or within 6 Months after they

have fruited and flower. The life cycle of biennial herbal plants is 1 to 2 years

Herbaceous plants have a half-year life cycle, which means they die entirely at the end of

the growing season or after flowering and fruiting. Perennial plants spent many years.

Sometime stem died during the stage of growth but the growth emerges (Krishnan, 2018).

An analysis of annual growth rings in the secondary root xylem will determine the age of

certain herbal plants.

Herbal remedies are becoming a significant trend, and studies show the importance of

plants in preventing and treating. Therapeutically, the data collected scientifically on such

plant derivatives can be used (Gupta, 1994). All over the world, there is a considerable

interest in herbal medicine because herbs contain compounds that are therapeutically

effective and are more safe and ideal for patients than pharmaceutical chemicals (Szabadi,

2006; Ang-Lee et al., 2001).

In the early 19th century, when methods of phytochemical screening first became

available, Parkash et al. (2018) reported that scientists began extracting and manipulating

the active compounds from plants. Chemists began making their own formulation of plant

compounds later, initiating the change towards raw plants to synthetic pharmaceuticals.

The use of herbal medicines has decreased over time in favour of pharmaceutical

products.

Unpurified bioactive compounds are widely used by practitioners of medicinal herbs.

These unpurified plant extracts contain many different constituents. They suggest that

they can work together synergistically in such a way that the impact of the constituents

12
as a whole is greater than the individual components as a whole. Toxicity is often

minimized when whole herbs are used rather than individual components (Vickers et al.,

1999).

Historical and current studies and surveys signify that the Eastern region of the

Mediterranean has been well-known throughout the generations with a rich inventory of

natural medicinal herbs. Arab medicine has contributed greatly to the development of

modern medicine in Europe and remains one of the closest forms of original European

medicine. The rapid increase in consumption of herbal remedies worldwide has been

stimulated by several factors, including the notion that all herbal products are safe and

effective (Saad et al., 2005).

In every area, use of traditional medicine is growing by the day. Alternative therapies are

important to achieve the objective of "health for all," and a traditional medicine program

has been in effect since 1978 (Rojas et al., 2003). In the United States, traditional drugs

are more natural, healthier and safer. Garlic, Echinacea, Ginkgo and many others

products are widely marketed.

As of mid-1998, annual retail sales were close to $4 billion, and medicinal herbs were

used by 12 to 37 percent of US customers, according to recent surveys (Eisenberg et al.,

1998). Fifteen per cent to forty percent of customers have used herbal medicine to treat

many illnesses, according to recent surveys and reports. The cost of prescription drugs

has risen in tandem with a willingness to reuse natural or organic remedies, leading to an

increase with the use of medicinal herbs in the United States over the last 25 years.

Around 70 percent of German doctors recommend herbal medicines to patients (Parkash

et al., 2018).

2.1.5 Synopsis benefits of medicinal plants

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According to the report of Attah et al. (2020), medicinal plants provide people worldwide

with a wide variety of subsistence, cultural and monetary benefits. They provide poor and

disadvantaged people, especially in poor rural areas, with affordable means of primary

health care. Given the fact that medicinal plants have several advantages, including:

I strengthened healthcare delivery services

II Increased guarantee of life

III possibly feasible utilize of the biodiversity and

IV progressed advantage sharing with neighborhood communities

According to Wang et al. (2002), the production capacity from natural sources is 8.5

million tons and the production of cultivated medicinal herbs in 2001-2002 was

estimated at 0.3 million tons.

2.1.6 Reports on the antibacterial activity of medicinal plants

Combination of secondary metabolites present in plants has the beneficial medicinal

effects. These secondary metabolites (steroids, fatty acid resins, alkaloids, hormones,

tannins, are capable of inducing definite physiological effects on health as reported by

Attah et al. (2020).

Nair et al. (2005) screened nine plants for potential antibacterial activity. The plants

screened were Sapindus emarginatus, Hibiscus rosa sinensis, Mirabilis jalapa, Rhoeo

discolor, Nyctanthes arbor-tristis, Colocasia esculenta, Gracilaria corticata, Dictyota

sp. and Pulicaria wightiana. Antibacterial activity was tested against 6 bacterial strains,

Pseudomonas testosteroni, Staphylococcus epidermidis, Klebsiella pneumoniae, Bacillus

subtilis, Proteus morganii and Micrococcus flavus. Two methods, Agar disc diffusion

and Agar disc diffusion, were used to study the antibacterial activity of all these plants.

Pseudomonas testosteroni and Klebsiella pneumonia were the most resistant bacterial

strains. Sapindus emarginatus showed strong activity against the tested bacterial strains.

14
The tube diffusion method was used by Ramasamy and Manoharan (2004) to assess the

antibacterial activity of useful compounds from different solvent extracts of Anosomeles

indica, Blumea lacera, and Melia azadirachta against E. coli, Pseudomonas aeruginosa,

Serratia maraceseuns, and Staphylococcus aureus. Whereas petroleum ether and

aqueous exhibited no effects. Relatively more sensitive were Pseudomonas aeruginosa

and Serratia marcesenes, respectively.

Astal et al. (2005) tested the aqueous extracts of sage and thyme had action against

microorganisms. Antibacterial activity of phenolic extracts of sage and thyme against

Staphylococcus aureus and Enterococcus species has been demonstrated. The ethanolic

extract of parsley was more influenced by Escherichia coli. This extract, on the other

hand, has no discernible effect on the Gram-positive bacteria examined. Antibacterial

activity of synthetic oils of sage, thyme, and parsley against Escherichia coli, Proteus

mirabilis, and Salmonella typhi was not detected. The results revealed that of the ten

microorganisms studied, Staphylococcus aureus was the most susceptible to the majority

of the three plant extracts.

Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis) and Gram negative

bacteria (Pseudomonas aeruginosa and Escherichia coli) were tested against petroleum

ether, benzene ethyl acetate and acetone extract leaves of Galinisoga ciliate. Gram

positive bacteria were more susceptible than gram negative bacteria according to

Poonkothai et al. (2005). It might be concluded that high phenolic content cause toxicity

in microorganisms.

Deshpande et al. (2005) discovered that extracts from Abrus precatorius, Boswellia

serrata, Careya arborea, Emblica officinalis, Syzygium cumini, Woodfordia fruticosa,

and Sphaeranthus indicus had potent antibacterial activity against Gram-positive and

15
Gram-negative bacteria. Only Gram positive bacteria (Bacillus cereus and

Staphylococcus aureus) were used in the experiments against other plant extracts.

According to Tambekatr and Kharate (2005), E. coli, Staphylococcus aureus, Proteus

mirabilis, Salmonella typhi, Enterococcus faecalis, Pseudomonas aeruginosa, and

Yersinia enterocolitica were all inhibited by Ocimum sanctum. Antimicrobial activity

was detected in leaves isolated from different plants, including "Tulsi, Pudina, and

Beet."against Escherichia coli, Staphylococcus aureus, Enterococcus faecalis,

Salmonella typhi, Vibrio cholerae, Proteus mirablis, Pseudomonas aeruginosa and

Yersinia enterocolitica while piper betel showed resistances to treptococcus pneumonia.

2.2 Definition and Description of Honey

Honey is a thick watery substance prepared by bees from the nectar. It was made of water,

glucose, fructose, proteins, vitamins and minerals (Al-Waili and Haq, 2004). It could be

also described as the ordinary sweet substance created by honeybees from the nectar of

blossoms or from the exudations of living parts of plants or excretions of plants sucking

insects on the living parts of plants, which bees gather, convert and mixed with specific

substances of their own, kept and keep in the honeycomb to mature as reported by Elijah

et al. (2015).

Olakunle et al. (2013) reported that Honey predominantly comprises sugar and water.

The sugar content is about 96 – 98 % of honey dry matter, mostly, are simple sugars,

fructose (38.2 %) and glucose (31.3 %), which represents 85 – 95 % of total sugars. These

are ‘‘simple’’ sugars, 6-carbon sugars. Other sugars include disaccharide such as maltose,

sucrose and few oligosaccharides are also present.

Water was ranked second among the component of honey (Olakunle et al., 2013). Its

content is vital, as it affects honey storage. The ultimate water contained in honey

16
attributed to plethora of ecological influences, like temperature plus moisture in the hives

at the time of processing, another thing was environment of the nectar and honey handling

process at the point of harvest and preservation (Peter et al., 2007). Gluconic acid is one

of the 0.57 % organic acids in honey that is a consequence of the use of enzymatic

glucose.

Natural antioxidants are liable for acidity in honey and add to its distinctive taste. Honey

contained small amounts of minerals (0.17 percent) with calcium, copper, iron,

manganese, and phosphorus as the most abundant. There are nitrogen components of

which the enzymes come from the worker bees' salivary liberation. The principal

enzymes present within honey are invertase, amylase, and glucose oxidase (Peter et al.,

2007). Singh et al. (2012) reported that honey also contains numerous other sugar

sources, as well as acids, vitamins, proteins and minerals. In honey, alkaloids,

antraquinone glycosides, cardiac glycosides, flavonoids and reducing compounds were

found (Rakhi et al., 2010).

The colour of honey can vary from nearly colourless to dark brown and its consistency

can be fluid, viscous or partly to entirely crystallized (Elijah et al., 2015). The nectar

origin encountered by the bees, as stated by Elijah et al. (2015), contributes to the

variation of colour, taste and aroma. Honey is commonly marketed for its nutritional

benefits, but was also use since ancient times as a medicinal agent, but recently, clinical

education of honey as therapeutic aid was introduced (Molan, 1999).

Several substances are derived from the secretory glands of honey bees, as well as

different herbal products, alone or even in various combinations. These compounds

included royal jelly, bee wax, propolis, bee pollen, and bee venom (Cornara et al., 2017).

Most of them have been used by humans for dietary and health purposes since ancient

times, according to a study by Cornara et al. (2017).

17
A wide range of scientific research has discovered many biological components in

honeybee products, while numerous results have been devoted to summarizing medicinal

applications and uses as nutritional supplement, cosmetic and pharmaceutical properties

(Viuda-Martos et al., 2008). Steps have been taken to make sure that some of the honey

bee products have been manufactured in clinical environments, but their pharmaceutical

and medical normalization, based on honey variations and plant - based products, is a

challenge due to the high chemical inconsistency. Many compounds were isolated from

honey including pharmaceutical substances indicating the usefulness of honey in

production of antibiotics from plant sources (Cornara et al., 2017).

2.2.1 Types of bee

Honeybees of the genus Apis are social insects which distinguish honey and other

substances of immense human value by their production and storage. Currently, two

domesticated species are known, namely, the western A. niger and the eastern A. niger.

mellifera is the scientific name for a plant that produces honey. A. Cerana, which is found

in South and Southeast Asia, is native to Europe, Asia, and Africa and was imported to

America and Eastern Africa (Cornara et al., 2017). Honeybees were categorized into

stingless bee and sting bee, according to the Rao et al. (2016) study. Stingless honey is

produced from a sweet substance. The honey generated by stingless bees differs in colour,

taste, and viscosity from that produced by Apis bees (honey bees) (Almeida-Muradian et

al., 2014). Honey developed by a variety of stingless bees has powerful antibacterial

effects (Irish et al., 2008; Boorn et al., 2010). According to Rao et al., (2016), this

important bee product has been processed and used in a variety of therapeutic methods,

both in conventional and alternative procedures where honey is collected directly from

the forest, and in the more well-established meliponary as reported by Rao et al. (2016).

18
The honey comb of sting bee and honey pot of stingless bee honey were shown in Figure

2.I

Figure 2.1: A and B: Honey Comb of Sting and Pot of Stingless Bee Honey.

Source: Rao et al. (2016)

2.2.2 Types of honey

Approximately 320 different varieties have been established, depending on some

flower sources (Kaur et al., 2017). The taste, shape, colour and smell of a specific form

of honey depend on the different water sources of the bees visited by the plants and fauna.

In terms of the season in which it is made, existing temperature, precipitation and

changing climate, various kinds of honey are identical. Honey varies from light brown to

dark brown in colour. According to Kaur et al. (2017) review, several popular honey

categories are listed below:

I. Manuka honey- is a common healing agent and acts as a wound antibiotic.

II. Acacia honey- is useful for liver and digestive system cleansing.

III. Buckw heat honey- The colour is darker and made of antioxidants.

IV. Neem honey- is helpful in diabetes or high blood pressure (Kaur et al., 2017).

19
Olatunji et al. (2015) reported that there are mainly two honey types; apiary honey and

forest honey. Apiary honey is made in apiaries by both the honeybees, Apisceranaindica

and Apismellifera, which are collected by the modern extraction process, while forest

honey is manufactured by rock bee, Apisdorsata, or wild A. nests in forests.

Ceranaindica, and are obtained by the crude technique of pressing the comb

(Subrahmanyam, 2007).

Honey can also be graded according to its nectar quality. Floral (sweet deposit from

flower) and non-floral (sweet deposit from plant) honeys are among them (Manyi-Loh et

al., 2011). Honeys can be uni-floral or multi-floral, based on whether the honey is

obtained from the nectar of a single flower or a number of flowers (Manyi-Loh et al.,

2011). Bees that extract sugar from living plant or fruit tissue and/or scavenge insect

excretions (aphids) that tap the veins of plant species are provided by non-floral honey

(honey dew) (Subrahmanyam, 2007).

Another element that has contributed to the colour and shape of honey is volatile

compounds. More than six hundred volatile organic compounds were found in honey. At

standard ambient temperature, volatiles are chemical substances with high vapour

pressure. Honey comprises aldehydes, ketones, acids, alcohols, esters, hydrocarbons, and

cyclic molecules, among other things (Kaskoniene and Venskutonis, 2010). Honey

contains a limited number of volatile organic compounds, but these compounds have an

effect on its color stability; taste, smell, texture, and color are all influenced by the fruits

and trees that bees come across (Manyi-Loh et al., 2011). The majority of volatile organic

compounds are obtained from flowers or nectar sources, while some are produced during

storage and distribution time (Jerkovic et al., 2011).

2.2.3 Production of honey

20
The honey bee (Apismellifera), a high-value nutritious product, is of great significance to

humanity as a pest species of both domestic and commercial crops and a source of honey

(Ratnieks and Carreck, 2010). Honey bee misfortune has led to a far-reaching concern

about nectar bees' near-long-term capacity to provide services due to relationship

generators of bugs and pathogens, introduction of agrochemicals, apicultural bad

management, and the need for genetic discrepancies (Ratnieks and Carreck, 2010). The

quality and nature of honey is affected by a variety of factors, including flower

composition, hive location, bee health, and the temporary impact on native species and

floral morphology (Galimberti et al., 2014).

Honey is produced in a number of ways (pressed, centrifuged, drained, heat analyzed)

and comes in a wide variety of physical forms (comb, chunk, crystallized or granulated,

creamed). Inside a honey bee hive, there are three castes of bees: queens, workers, and

drones according to Saranraj and Sivasakthi (2018). Honey production is possible due to

a combined effort. Honey is made using nectar from fruit trees by honey bees, nectar is a

sugar-rich fluid formed in glands called nectaries (Saranraj and Sivasakthi, 2018). In a

study in Sicula, honey made by black local bees Apismellifera species had approximately

10 times more polyphenolic content and antimicrobial activities than honey produced by

other Apismellifera bee species. (Tenore et al., 2012).

2.3 History of Honey

Honey has been identified since the creation of man as the longest sweeteners ever seen

in the world, though the official timing of origin is far from certain (Nayik et al., 2014).

Honey's use and processing has a long and diverse history (Nayik et al., 2014). This

practice of honey medication had been in ancient prescriptions and present care of wound

for therapeutic purposes is well known. The Egyptian utilized honey for other reason such

as treatment of skin illness, application on wound and embalmment. In the treatment of

21
bruises and sores of the mouth and healed carbuncles and running injuries, Hippocrates

(460-357 BC) found the comfort of honey. Aristotle (384-322 BC) stated that mild honey

was a great balm for sore eyes (Al-Waili, 2003).

Saranraj and Sivasakthi (2018) reported that when there is an identifiable deficiency as a

result of low glucose level inside the body, the old Greeks were thorough in using honey

for rapid vitality recovery: competitors combined honey with water in a major athletic

drinking opportunity. The Babylonian connected honey for the recuperating of eyes and

ears diseases and treatment for topical application (Saranraj and Sivasakthi, 2018).

Honey has been used to treat various diseases since the ancient Egyptians, Assyrians,

Greeks, and Romans used it alone and in supplement form, as well as plants and active

ingredients, to treat burns, injuries, eye infections, and gastrointestinal disorders (Saranraj

and Sivasakthi, 2018).

In order to treat snakebites, fever and laxative, honey is used by various African tribes.

Moreover, as mentioned by Saranraj and Sivasakthi (2018), honey was used by Masai

fighters to gain strength and capacity, most likely due to honey's high calorie content.

2.4 Religious Significance of Honey

Haile et al. (2017) reported that many religious books considered the uses of honey in

different ways: The religion of Islam recommended the use of honey as food and

medicine, and even named an entire chapter in the Holy Qur'an called Surah al-Nahl

which mean chapter of Honeybee. The practice administering honey for therapeutic and

healing purposes was firmly endorsed by the Prophet Muhammad in Hadith.

In Christendom, there are references made to the importance of honey in the scriptures,

these include the Books of Exodus, Psalms, Mathew and Judges. In this Christian sacred

book, the Bible, King Solomon instructed his son. “Eat honey my child, since it is good”

22
as cited by Haile et al. (2017). Honey is accepted by all cultural traditions. Honey is a

beneficial nutritious liquid that has been embraced without reservation by all generations,

traditions, and societies, both ancient and modern, as written in all holy books (Nayik et

al., 2014).

2.5 Antioxidant Property of Honey

Honey has been used for a long time in both health control and household uses, but its

antioxidant possessions have only recently entered public attention. As need for

antioxidant requirements in food increases, honey is accepted as an antioxidant base, as

stated by Abeshu and Geleta (2016). Lack of antioxidants in food induces oxidative

stress, which has led to an unbalanced chemical change between the development of

oxidative stress and our body's normal protective role resulting in cell damage and genetic

composition disruption (Haile et al., 2017).

Deep analysis has been conducted on the molecular mechanisms showing how normal

cells undergo tumour promoter-induced transition to cancer cells. Experiments have

shown, however, that the mitogen-activated protein (MAP) kinase signalling pathways

are activated by different tumour promoters differentially. Survival, growth,

proliferation, apoptosis, cell cycle control, inflammation, and differentiation may be

biological responses (Mohammed and Babiker, 2009).

The antioxidants bind to the signalling pathways and escape the adverse effects indicated

by cancer, cardiovascular disorders, inflammatory diseases, neurological degradation,

tissue repair, bacterial infections and aging as stated by Abeshu and Geleta (2016). Honey

contains major antioxidant activity in the form of glucose oxidase, catalase, ascorbic acid,

flavonoids, phenolic acids, carotenoid derivatives, organic acids, Maillard reaction

products, amino acids, and proteins (Hadjmohammadi and Nazari, 2010). The major

antioxidants in honey are phenols like quercetin, hesperetin, and chyrsin, as well as

23
maillard substances called melanoidins (Hadjmohammadi and Nazari, 2010). The phenol

quercetin precisely attaches and actively prevents the activities of cellular transcription

factors. The suppression of signalling pathways exceeds the mechanism of

phosphorylation and activation, preventing the cellular reactive oxygen species. It also

induces human osteosarcoma cell apoptosis and decreases levels of protein expression in

human fibrosarcoma cells (Abeshu and Geleta, 2016).

2.6 Antibacterial Factors in Honey

Matured honey has a higher proportion of six-carbon sugar, fructose in general, and some

maltose and sucrose, and contains much less than 18 percent water (Paulus et al., 2012).

The excessive sugar concentration, mixed with a small of water, causes osmosis, which

prohibits microorganisms from spoiling the honey. Yeast growth can already result in

only minor concentration of honey, but the amount of sugar of honey is sufficient to

maintain honey's antimicrobial activities if diluted to about 30-40 % (Paulus et al., 2012).

The antibacterial activity at higher dilutions is due to substances other than sugar (Paulus

et al., 2012).

Hydrogen peroxide was recognized as key antimicrobial substances of the honey as from

1960s according to a study by Paulus et al. (2012). The glucose oxidase enzyme, added

by honey bees during honey processing, is activate by little addition of water to honey

and transforms glucose into hydrogen peroxides and gluconic acid. Nevertheless, due to

non-peroxide components, different honeys have substantial antibacterial activity.

Recently, bee defensin-1 and methylglyoxal were recognized in both RS honey and

manuka honey as antimicrobial substances (Adams et al., 2008; Kwakman et al., 2010).

So many studies results showed variability in the sensitivity of low pH to the

antimicrobial properties of honey (usually around 3.2 and 4.5), however it has shown

conclusively that pH has a part to play in this feature (Kwakman et al., 2010). In addition,

24
there are strong indications that additional honey antimicrobial components are present,

the identity of which is not yet established.

2.6.1 Hydrogen peroxide in honey

Glucose oxidase is one of the nutrient enzymes applied to honey by bees. It converts

glucose into hydrogen peroxide and gluconic acid in the presence of oxygen (Bang et al.,

2003). When the concentration of sugar has not yet reached levels capable of stopping

microbial proliferation, hydrogen peroxide is thought to play a role in stopping immature

honey from spoiling. Glucose oxidase is inactivated for the duration of honey ripening,

but re-gains activity on honey dilution. The concentration of H2O2 is high. The

importance of H2O2 to honey's antimicrobial property will be determined by the impact

of inactivating this compound by adding catalase. Dilution of H2O2 reduces the

antimicrobial property of several of these honey samples tested, suggesting the essential

role of H2O2, however after H2O2 inactivation, a significant number of honey maintain

activity (Mundo et al., 2004).

Paulus et al. (2012) reported that the factors known to affect H2O2 accumulation are

inactivation of the H2O2-producing enzyme glucose oxidase by exposure to heat or light

or degradation of H2O2 by honey. It has been suggested that catalase originating from

pollen, nectar, or microorganisms would be responsible for the enzymatic H2O2-

neutralizing activity of honey. As in honey, H2O2 is also a chief antimicrobial defense

system in plant nectar (Carter and Thornburg, 2004) and substantial variation in

accumulation of H2O2 also exists among nectar samples (Hillwig et al., 2010).

Peroxidases are by far the most protein in petunia honey, and the concentration of

hydrogen peroxides in petunia and tobacco honey is indirectly related to the amount of

peroxidase production in those nectars (Gonzalez-Teuber et al., 2009).

25
2.6.2 Methylglyoxal (MGO) in honey

Various honeys have significant non-peroxide antibacterial activity. Manuka honey has

been most expansively subjected to identification of non-peroxide antimicrobial

compounds. This honey is produced from nectar of the manuka tree (Leptospermum

scoparium), a New Zealand indigenous plant known for its non-peroxide antibacterial

activity. Of late, unusually high levels of the antimicrobial compound methylglyoxal

(MGO) have been found in manuka honey (Adams et al., 2008). MGO is derived from

sugars in carbohydrate-containing foods and beverages (Weigel et al., 2004). The high

levels of methylglyoxal in manuka honey, on the other hand, are produced by the

conversion of dihydroxyacetone (DHA), which is contained in extremely high

concentrations in the nectar of Leptospermum scoparium flowers (Adams et al., 2009).

This conversion occurs non-enzymatically and at a slow pace while honey is processed.

In such large quantities, the production of DHA in nectar and its function are unknown

in manuka nectar trees. MGO concentrations in various foods have been estimated to

range from 3-47 mg/kg, whereas manuka honey produces far higher amounts (ranging

from 38 mg/kg to 1,541 mg/kg (0.74-30.0 mM) (Adams et al., 2008).

Based on a clear correlation between MGO levels and the ability of honey to inhibit

Staphylococcus aureus growth (Adams et al., 2008), it has been suggested that MGO is

fully responsible for the non-peroxide antibacterial activity of manuka honey. The

treatment for Staphylococcus aureus has been discontinued, and the treatment for B.

subtilis has been drastically reduced. On the other hand, it had no effect on E. coli and

Pseudomonas aeruginosa.

2.6.3 Osmalarity factor in honey

26
Osmolarity or osmotic concentration is the number of osmoles of a solute per litre of

solution. It is expressed as Osm/L (Erstad, 2003). The surface tension of honey is usually

high due to the high presence of sugar in honey, causing a decrease in water activity,

which gives osmolarity a crucial role in the antimicrobial action of undiluted honey,

because, for example, the development of many species of bacteria is completely subdued

when the moisture content is between 0.94 and 0.99 as reported by Saranraj and

Sivasakthi (2018).

2.6.4 Hydroxymethylfurfuraldehyde in honey

Honey also contains hydroxymethylfurfuraldehyde (HMF) in trace amounts. Although

even fresh honeys have been shown to retain minimal quantities of HMF (Zappala et

al., 2005), which can easily be increased if the honey is kept at mild temperatures, HMF

produced by fructose breakdown in the presence of acid was considered proof of honey

adulteration. As a result, it's critical to keep honey refrigerated or in a cool place to keep

HMF levels low, as HMF is one of the most important factors to consider when it

comes to honey performance and marketing.

2.6.5 Pollen, propolis and royal jelly of honey

Along with nectar, bees gather plant pollen, supplying nutritional protein to the hive. In

honey, there is still pollen present. Pollen from trees and plants pollinated by the wind

is often present in honey (Bruni et al., 2015). Pollen and phenolic compounds are

present in carbohydrates, amino acids, DNA, vitamins, nucleic acids, protein, lipids,

minerals, and flavonoids (Morais et al., 2011).

Bees use propolis, which is made from tree exudates, to cover their nest in a protective

layer against intruders (Viuda-Martos et al., 2008). Propolis contains 50 percent resin, 30

percent wax, 10 % essential oils, 5 % pollen, as well as other organic matter (5 percent).

27
Propolis, phenolic compounds, esters, flavonoids, terpenes, and anthraquinones were

among the 300 compounds present in honey (Bertrams et al., 2013).

Royal jelly is a protein-based fluid developed naturally by glands in the hypopharynx of

worker bees, according to Saranraj and Sivasakthi (2018); it is only for adult bees.

The main proteins of royal jelly are examined and analyzed because protein accounts for

more than half of the dry mass of the jelly (Won et al., 2009). Asthma, high blood

pressure, and allergy symptoms are all treated with royal jelly as a dietary supplement.

The fatty portion is mainly composed of terminally and/or internally hydroxylated

medium-chain fatty acids with terminal mono or dicarboxylic acid functions, which are

either saturated or monounsaturated at two positions. The 10-carbon atoms of royal jelly-

specific trans-10-hydroxy-2-decenoic acid (10-HDA) and 10-hydroxydecanoic acid are

the primary components. There are also small numbers of sterols (Li et al., 2013).

From the first to the third day of their lives, larvae consume this royal jelly before

developing into female workers and male drones, or selected individuals who turn into

queens before the end of the larval period. Adult queens have a special meal every day

of their lives (Fujita et al., 2013). Royal jelly has been used in herbal medicine for

centuries, especially in Asia and Egypt. In the pharmaceutical and cosmetic industries, it

is currently used and marketed as an over-the-counter functional food. Several researches

looked at royal jelly's antimicrobial properties against bacteria, fungi, and viruses, as well

as hypotensive, anti-tumor, anti-hypercholesterolemic, and anti-inflammatory properties

in animal models (Ramadan and Al-Ghamdi, 2012).

2.6.6 Acidity of honey

One of the factors contributing to honey's antimicrobial activity is acidity. It was recently

proposed that honey's antimicrobial properties were due to its acidity.

28
According to Mato et al. (2003), honey contains around thirty organic acids, but the most

abundant is gluconic acid, which is formed by the enzyme glucose oxidase.

2.6.7 Phenolic compounds of honey

Flavonoids: quercetin, pinocembrin, pinobanksin, chrysin, galangin, kaempferol, and

luteolin are the most common phenolic compounds known in honey (Kaskonienė and

Venskutonis, 2010). The aromatic acids play an aromatic ring role in organic acid.

Examples of aromatic acids are phenolic compounds and organic carboxylic acid because

of the presence of a phenolic ring feature. Many plants contained phenolic acids (Pinho

et al., 2014). Plant-specific metabolites, flavonoids which perform more than one

function are also play a crucial role for symbiotic nitrogen fixation, UV filtration and

plant pigmentation according to Dixon and Pasinetti (2010). Two-phenyl-1; 4-

benzopyrone is the essential molecular structure, which is found in plants. Benzoic,

ferulic, gallic, chlorogenic, caffeic, syringic acid, p-coumaric, and ellagic acids are

examples of plant-derived phenolic acids. Phytochemical composition has an impact on

honey's antibacterial activity (Kaskoniene and Venskutonis, 2010). Phenolic compounds

have antibacterial, anti-inflammatory, and antioxidant properties.

2.6.8 Bee defensin-1

Recently, in RS honey, defensin-1 has been recognized as the antimicrobial peptide bee

(Kwakman et al., 2010). This peptide was previously detected in the honeybee head and

thoracic glands of honeybee hemolymph, the insect equivalent of blood (Klaudiny et al.,

2005). In honey bee, Bee defensin-1 has potent activity, but only against Gram-positive

bacteria such as B. Subtilis, S. Aureus, and the P. aenibacillus larvae (P. larvae)

(Kwakman et al., 2010). As part of their innate immune system, invertebrates rely heavily

on antimicrobial peptides to defend against microorganisms. According to Paulus et al.

29
(2012), each one of these AMPs has a separate spectrum of antibacterial properties, and

these peptides cooperatively contain all the most significant microbe groups.

Foulbrood is a debilitating infection affecting larvae of bee in particular in America. The

disease affected the digestive tract of larvae and contributes to significant larval mortality

within the first 48 hours of egg hatching (Genersch, 2010). The presence of defensin-1 in

royal jelly and honey can aid in the protection of bee brood against American Foulbrood,

but this is speculation.

While RS honey of bee defensin-1 was readily detectable, it was not detected in manuka

honey (Kwakman et al., 2011a). The occurrence of defensin-1 in various honeys has not

been extensively studied, and statistical data on the peptide's composition in honey is still

lacking. For six of the 26 honeys, protein-based antibacterial compounds were reported

earlier, but the identification of these proteins wasn't really accompanied (Mundo et al.,

2004). The reported protein-contained antibacterial spectrum of compounds for four of

these honeys strongly matches the bee defensin-1 tested range that is a powerful activity

against Bacillus spp. But, against Staphylococcus aureus, it has no activity.

The hypopharyngeal gland of the honeybee secretes bee defensin-1 (Kwakman et al.,

2010). Bees use hypopharyngeal gland secretions for the manufacture of royal jelly and

honey, as stated by Paulus et al. (2012). In royal jellies and honeys, the quantity of bee

defensin-1 varies greatly (Kwakman et al., 2011b), with this form of peptide completely

absent in certain samples. Since American foulbrood is caused by defensin-1, which is

active against Paenibacillus larvae, it will be interesting to see whether variations in bee

defensin-1 expression are related to honeybee infection susceptibility (Paulus et al.,

2012).

30
2.7 Health Benefit of Honey Intake

2.7.1 Improves haematological parameters

The different health benefits of metabolic precursors on haematological and blood levels

are associated with the regular intake of natural honey: enzymes and minerals (Al-Waili,

2003). It has been shown that the use of natural honey in apitherapy improves anaemic

symptoms and thus benefits patients. One dietary enhancement research reported an

increased haematological advantage in mature experimental rat fed with forest honey

from Nigeria when compared to controls (Ajibola et al., 2007). Improved haemoglobin

concentration; the improvement of haematocrit values and high count in red blood cell

were found in rats that were fed with honey.

Improved haematological parameters and improved immunity in rats were also reported

as a nutritional supplement with 10 % New Zealand forest honey in a comparable study

from another laboratory (Chepulis, 2007). This researcher also documented a higher

lymphocyte count and enhanced phagocytosis by neutrophils in rats fed natural honey

relative to control rats. This aligns with a previous study that verified that prebiotics can

improve immunity (Schley and Field, 2002) and that honey contains oligosaccharides

and other prebiotics (Eteraf-Oskouei and Najafi, 2013).

In a clinical trial in California, human participants given either of two honey treatments

in showed the benefits of haematoprotection and enhanced haematopoiesis (Schramm et

al., 2003). In addition, normal honey has immunoprotective ingredients. According to

Al-Waili and Haq (2004), the oral consumption of Asian polyfloral honey from Al-Theed

31
City, UAE stimulates and increases antibody production during various immune

responses against the T-cells antigens of the thymus- independent and dependent origin.

2.7.2 Boosting of the immune system

Honey can help clear infections by inducing the immune system to attack infection, in

addition to providing direct antibacterial action. The proliferation and activation of

neutrophils by B-lymphocytes and T-lymphocytes in cell culture has been reported to

stimulated by honey (Tonks et al., 2003). A 5.8 kDA portion of manuka honey that

stimulates the development of TNF- via Toll-like receptor in macrophages has recently

been found by Tonks et al. (2007). Honey also supplies glucose, which is required for the

"respiratory blast" of hydrogen peroxide-producing macrophages, which is the primary

component of the microscopic organism action (Molan, 2001).

Furthermore, it provides glucose metabolism substrates, which is a critical pathway for

macrophage power storage, allowing them to operate in environments where oxygen is

scarce, such as damaged tissue and exudates. Because an acid pH inside the phagocytic

vacuole is involved in killing ingested bacteria, the presence of acids in honey aids

macrophages in their bacteria-destroying action. (Molan, 2001).

2.7.3 Anti-inflammatory activity of honey

Melipona marginata is a Brazilian species of endangered stingless bee. With exceptional

physicochemical properties and a special taste, it produces honey. When M. marginata

honey was used in a study, it had anti-inflammatory effects on the human body (Borsato

et al., 2014). Manuka honey's antibacterial activities, as well as its components' anti-

inflammatory characteristics, were documented. By exposing Manuka honey to human

32
monocytes, the development of various inflammatory cytokines was assessed (Tonks et

al., 2003).

According to the findings, honey activated the production of inflammatory cytokines

such as interleukin-1 (IL-1) and IL-6, and also tumor necrosis factor-alpha (TNF-alpha)

through a toll-like 4 (TLR4) receptor-dependent pathway (TNF-). A protein with a

molecular weight of 5.8 kDa contained in Manuka honey has been shown to be important

for stimulating various cytokine forms in human monocytes through the TLR4 pathway

(Tonks et al., 2007).

Animals have been shown to benefit from Tualang honey's anti-inflammatory properties.

Tualang honey was used to treat a chemically induced corneal injury in rabbits and

produced results that were comparable to standard treatment (Bashkaran et al., 2011),

indicating that it has the ability to cure eye problems.

2.7.4 Effect of honey on fertility

Honey seems to have a beneficial effect on fertility and raises hormones linked to fertility

(Rao et al., 2016). In a study conducted of rats exposed to auditory stress, researchers

discovered that 0.2 mL of honey combined with water increased a decrease in fertility

(Rao et al., 2016) Noise is a natural teratogenic element which has a serious impact on

health, reproductive fitness, and reproductive organ function. Honey was found to

increase the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and

testosterone in this study. Vitamin E's potential benefits for these variables have also been

documented (Rao et al., 2016). In many illnesses, conditions and disorders, this tension

plays a critical role. Increased stress levels are also associated with changes in

reproductive health. Tualang honey, given at 1.2 g/kg per day to restraint-stressed

pregnant rats, improved corticosterone levels, pregnancy, and adrenal histomorphometry,

according to one report (Haron et al., 2014).

33
Mosavat et al. (2014) observed that 1 g/kg of honey supplementation had a major

beneficial effect on female rats' altered gonadotropin level. Honey at a dose of 1.2

g/kg/day increased smoke-induced reproductive toxicity in rats, increasing the amount of

active interference and ejaculation, according to other reports. As a result, there is a rise

in fertility and mating rates (Mohammed et al., 2013).

2.7.5 Honey and diabetics

Daily honey consumption lowered the glycemic index relative to sucrose and glucose in

type I diabetic patients and healthy participants, and that honey does not have additional

acute hyperglycemic effects on the isoglycemic volume of bread in type II diabetics at

breakfast (Attah et al., 2021)

Honey has lower glycemic and incremental indices when opposed to glucose and sucrose

in type I diabetic patients (Abdulrhman et al., 2011). In rabbits suffered from drug

induced diabetes, the antihyperglycemic effects of honey were registered. In one

experiment, various honey doses (as low as 5 ml/kg) were found to result in a substantial

reduction in blood glucose and other related parameters (Rao et al., 2016). Honey can be

a healthy sugar replacement for diabetics even in low dosage (5 ml/kg). It has been found

that honey and its ingredients have many long-term health benefits. In one study, honey

resulted in weight gain and blood sugar level reductions (Rao et al., 2016).

Honey contains a lot of fructose, which is a monosaccharide that can boost blood glucose

levels by swallowing it through the mouth. It's also perplexing that scientists and

nutritionists have recommended honey as a dietary supplement for diabetics (Adesoji and

Oluwakemi, 2008).

2.7.6 Anticancer activity of oral honey administration

Among the most significant and dangerous diseases is cancer (Rao et al., 2016). Several

works on honey have been published that it has the possibility to deride and induce

34
angiogenic activity on the effectiveness of honey on different cancers according to Fauzi

et al. (2011), Hawley et al. (2014) and Kustiawan et al. (2014). Research into the

therapeutic potency of honey against cancer cells has shown substantial anti-angiogenic

effects in terms of its durability, viability, and even cell proliferation (Fauzi et al., 2011).

Studies on Malaysian honey have shown that it has anti-cancer, anti-oral, anti-bladder,

and anti-liver activity (Swellam et al., 2003; Baig and Attique, 2014).

Manuka honeys have been confirmed to have cancer-reducing operations. A study on the

impact of Manuka honey on the improvement of symptoms of esophagitis after radiation

has shown its protective impact on heart disease (Rao et al., 2016).

From the therapeutic investigation of Manuka honey on squamous cell carcinoma and

oral injuries, there were notable protective impact and inflammation reduction

respectively (Drain and Fleming, 2015).

2.8 Antimicrobial Activity of Honey

2.8.1 Antibacterial activity

Honey has been shown to be antimicrobial (Dureja et al., 2003). Honey inhibits bacteria

that are gram positive as well as gram negative. Mohapatra et al. (2011) reported that

honey alcohol extracts had an inhibitory effect on all bacteria. Honey has potent

antibacterial properties, including resistance to a wide variety of antibiotics, as well as

pathogenic and non-pathogenic bacteria. Antimicrobial activities may be bactericidal or

bacteriostatic, depending on the concentration used (Manyi-Loh et al., 2011). Gram-

positive bacteria: Staphylococcus aureus, Bacillus subtilis, Bacillus cereus,

Enterococcus faecalis, and Micrococcus luteus, as well as Gram-negative bacteria:

Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi, are all susceptible to

honey extracts of methanol, ethanol, and ethyl acetate in a laboratory environment as

reported by Mohapatra et al. (2011).

35
Honey has bactericidal effect against P. aeuroginosa (Henriques et al., 2011). At

Minimum Inhibitory Concentrations (MIC) of 9.5 % (w/v), and Minimum Bactericidal

Concentration (MBC) of 12 % (w/v), honey exhibits bactericidal effect with a 5 log

reduction estimated within 4hours above. Honey with carbohydrate solutions ≥ 15 %

(v/v) effectively inhibits H. pylori. In-vitro tests of isolates of Campylobacter spp. are

highly susceptible to honey solution (Lin et al., 2009).

According to Hassanein et al. (2010), bacterial isolates from wounds of hospital patients:

Aeromonas schubertii, Haemophilius paraphrohaemlyticus, Micrococcus luteus,

Cellulosimicrobium cellulans, Listonella anguillarum and Acinetobacter baumannii,

exhibit sensitivity to solutions of honey at various concentrations ranging between 25 %

and 40 %.

In many bacterial infections, resistance to clinically administered antibiotics has been

established. Wound infections were eliminated by 4.0-14.8 percent honey using a

medical-grade honey, even with the clinically acquired antibiotics present in the wound

environment. Honey resistance as typical to other antimicrobials was barely induced by

bacteria (Blair et al., 2009).

Honey sensitivity was demonstrated against vancomycin-sensitive enterococci and

methicillin-resistant Staphylococcus aureus isolated from the wound at varying

concentrations according to Cooper et al. (2002). Honey exhibits a synergistic activity

against MRSA (Jenkins and Cooper, 2012), where sub-lethal a concentration (i.e 6

percent w/v) of manuka honey was able to reversed the drug tolerance to oxacilin of

MRSA. By combining with other ingredients, progress was made to boost the

antibacterial efficacy of honey. Medical-grade honey containing antimicrobial peptides

like synthetic bactericidal peptide 2 (BP2) has faster antimicrobial property against

36
Pseudomonas aeruginosa and Staphylococcus epidermidis according to Kwakman et al.

(2011a).

2.8.2 Antiviral effect

Honey's antimicrobial activity against a wide range of microorganisms, including multi-

antibiotic-resistant strains, has been demonstrated in various reviews and clinical trials.

(Kumarasamy and Mahendran, 2015). Antiviral activity against Rubella virus has been

documented in honey and is used topically to treat persistent herpes simplex lesions (Al-

Waili and Haq, 2004).

According to numerous sources, manuka honey has a strong inhibition effect against

influenza virus (Watanabe et al., 2014). As an anti-infective (especially antiviral) action,

due to the hydrogen peroxide, pasture honey is also used. Haile et al. (2017) stated that

raw honey, as opposed to acyclovir, can remove herpes.

2.8.3 Antifungal effects

Honey's antifungal activity was studied, but only a few fungi species were examined. It

has antifungal activity against dermatophytes and can activate human mycosis (Tinea)

(Shariati et al., 2015).

The traditional treatment of fungal diseases is limited and part of the reason for this is

due to the limited range of currently available antifungal medicines and the costly

treatment, especially due to the need for prolonged treatment (Haile et al., 2017). Several

studies have been performed in recent years on the in vitro sensitivity of superficial

mycosis to honey antifungal agents (Jessup et al., 2000). Several studies have looked into

the therapeutic properties of natural honey as well as its antifungal activity (Ji et al.,

2009). Recently, multi-flora honey samples have all been tested for their ability to prevent

37
the growth of 40 yeast strains, including Candida albicans, Candida krusei,

Trichosoporon, and Candida Glabrata (Koc et al., 2009). Uni-floral honey had activity

against Penicillium species is often greater than 10 percent at concentration (Kacaniova

et al., 2011).

2.8.4 Anti-parasite effects

Earthworms (Pheretimaposthuma), tapeworms (Raillietinaspiralis) and roundworms

(Ascaridiagalli) were used to test the anti-parasitic properties of honey (Haile et al.,

2017). For the anti-helmintics assay, different concentrations (100-300 mg/mL) of

sweetener extract have been examined (Haile et al., 2017). The determination of the

worms' paralysis and death time has been confirmed by honey.

The action of natural honey against helminths is due to its acidic pH level (3.2-4.5); this

hinders the growth of helminths and produces an uninhabitable atmosphere for their

growth (Sajid and Kamran, 2012). The result has shown that water extract has vermicidal

activity and as an anthelmintic it has been found to be effective. Higher extract

concentrations have produced paralytic effects much earlier, and all worms have

shortened the time to death. Aqueous extract has demonstrated dose-dependent

anthelmintic activity, providing the shortest amount of time of paralysis and death with

300 mg/ml dilution for all three forms of worms (Prasad et al., 2010).

2.9 Other Microbial Related Use of Honey in Treatment

2.9.1 Dental effect

Oral honey use can have a beneficial impact on tooth decay and oral improvement and is

necessary during teeth operations (Atwa et al., 2014).

In an article published in the Journal of Dental Science, the patients undergoing

convectional treatment with Asian polyfloral honey, as an apitherapeutic agent, reduces

38
tooth extraction pain while also preventing oral infections such as gingivitis and dental

caries (Atwa et al., 2014).

Honey, both unprocessed and refined, has a wide range of antibacterial activity and has

a high potential for minimizing dental caries susceptibility according to Mohapatra et al.

(2011). English et al. (2004) and Atwa et al. (2014) discovered that New Zealand manuka

honey protects against dental plaque and gingivitis, as well as other oral problems, in

addition to its carioprotective properties.

According to Steinberg and colleagues' research, raw honey is either non-cariogenic or

less cariogenic than sugar (Ajibola, 2015). As Ahmadi-Motamayel et al. (2013) and his

colleagues studied Iranian honey's antibacterial properties in apitherapy, they discovered

that it is not only non-cariogenic but also anti-cariogenic (Ahmadi-Motamayel et al.,

2013).

Honey's antimicrobial properties help to prevent dental caries pathogenesis by inhibiting

bacterial growth. Furthermore, raw honey consumption is healthy and does not cause oral

diseases such as gingivitis and periodontal disease, according to one study (English et al.,

2004). Volunteers who chewed New Zealand manuk honey as “honey leather” had highly

significant reductions in mean plaque scores (0.99 reduced to 0.65; P = 0.001) compared

to the control group, indicating a potential therapeutic role for honey in oral health

(English et al., 2004).

Honey's potential to be non-cariogenic may be attributed to the protective effect of its

synergistic constituents (Eteraf-Oskouei and Najafi, 2013).

2.9.2 Gastroenterology

Honey has been confirmed to have effects of preventing and treating gastrointestinal

disorders such as peptic ulcers, gastritis, and gastroenteritis. Honey is a potent inhibitor

39
of the causing agent of peptic ulcers and gastritis, Helicobacter pylori (Osato et al., 1999).

According to Abeshu and Geleta (2016), Honey is natural and will not raise blood-sugar

levels; honey mixed with water is a good curative agent for colic.

The consumption of honey raises the bacterial population of microflora, which is

important for the health of the digestive tract. Honey intake increases the population of

natural flora known as bifidobacteria, according to the paper published by Abeshu and

Geleta (2016), where its constituents have been shown to have a prebiotic effect similar

to fructooligosaccharide (FOS) effects.

2.9.3 Wound healing property of honey

Wound, a body injury that can be cause by abuse, surgery or accident involving

membrane breakage (such as skin) and typically underlying tissue damage. Abeshu and

Geleta (2016) reported that the wound causes tissue damage, blood vessel disturbance,

blood component extravasations and hypoxia. By causing greater pain, irritation and

inconvenience, the production of wound infection has adverse impact on health may lead

to serious sickness or death consequence.

Honey is an example of a naturally occurring remedy that has been used to treat wounds.

Through its restorative tissue growth and epitheliazation impacts, it helps promote faster

healing process with little or no scarring (Al-Waili et al., 2011).

According to Al-Wali et al. (2011), the physical effects of honey, such as acidity and

osmotic effect, as well as its chemical impact, such as hydrogen peroxide antimicrobial

property only at the site of injury, once taken orally, are the primary sources of tissue

repair property. Prostaglandins and nitric oxide play an important part in the healing

process (Abeshu and Geleta, 2016).

40
Honey has proved to be successful in a number of circumstances. Honey was used as a

dressing and applied to the mucous layers of the body cavities with no harmful effects.

Honey has been used without causing any allergic reactions or significant side effects. In

addition, wound odour is easily eliminated, granulation and epithelialization improves,

exudates are decreased in length, and microbe wounds are sterilized (Al-Waili et al.,

2011).

Cut skin grafting is a common technique for protecting skin defects. Infections, delays in

healing, fluid and electrolyte inequalities, scar development, and pain may all occur

during the revitalization of skin removal donor sites. Honey-impregnated gauzes with a

faster epithelization time and a low feeling of pain are comfortable, safe, and easy to use

(Misirlioglu et al., 2003).

Medicated honey bandages are increasing growing recognition and medicinal

applications due to honey's healing effect on different forms of wounds. The Food and

Drug Administration (FDA) has approved Derma Sciences Medihoney® dressing with

active manuka honey to help wound healing by providing a moist environment in 2007

(Al-Waili et al., 2011).

Medihoney® bandages are intended for diabetes patients who have light to moderately

exuding wounds, such as venous or arterial leg ulcers, full or minimum thickness pressure

ulcers/sores, and first and second soft tissue bums (Abeshu and Geleta, 2016).

Tissue repair times were shortened following Medihoney® treatment as compared to

conventional care, according to a study conducted in the United Kingdom (US), and

although the results are not statistically significant, they are clinically significant. Honey

can be used to treat wounds at any point of healing. According to Simon et al. (2006),

antimicrobial honey is useful in treating malignant wounds of different etiologies.

41
This, combined with the ideal tissue regeneration characteristics of maintaining a moist

wound environment (which is required for immediate healing), non-toxicity, anti-

inflammatory activity, deriding procedure, scarring decrease, and re-epithelialization

stimulation, has resurrected honey's use in wound care. Honey is most commonly used

to treat burns on the skin (Al-Waili et al., 2011).

In a total of 104 superficial burn injury cases reported Abeshu and Geleta (2016)

compared the performance of honey as a dressing to silver sulfadiazine gauze dressing.

In the 52 patients treated with honey, 91 percent of the injuries were sterile within 7 days,

and 87 percent of the wounds healed within 15 days. Honey's medical advantages,

according to the report, helped burn burns recover quicker and with less complication

than conventional therapy. Honey's ability to speed up the healing process in wounded

areas, ulcers, and skin burns is due to its hydrophobic nature, hypertonicity, lower pH,

and complex chemical properties.

The stimulatory effects of oral honey administration suggest that a tissue growth factor

may be involved, rather than wound acidification or improved tissue nutrition, which may

promote growth (Al-Waili et al., 2011). Because of its antibacterial properties, honey is

thought to be the most important component in wound healing. According to many

reports, honey, on the other hand, acts directly on skin cells involved in the recovery

process (Majtan et al., 2010; Ranzato et al., 2012).

Numerous studies have been conducted on honey's immunomodulatory properties, which

may explain, at least in part, its ability to speed wound healing. While other researchers

hypothesized that bacterial lipopolysaccharide contamination of honey may cause

immunostimulatory effects, immunoregulatory honey constituents were ruled out. A 5.8

kD a constituent from manuka honey stimulates TNF-a production by macrophages via

toll-like receptors (Tonks et al., 2007).

42
2.10 The Precaution of Using Honey

Honey can be poisoned by the environment, such as pesticides, antibiotics and poisonous

plants, like any other natural food (Bogdanov, 2006). It is known that a few plants

produce nectar that contains toxic substances. Two primary toxin groups relevant to

nectar are diterpenoids and pyrazolidine alkaloids. According to Rao et al. (2016), toxic

cyclic polyhydroxylated hydrocarbons or diterpenoids can be found in plants belonging

to the Ericaceae family's Rhododendron subfamily, such as Rhododendron ponticum.

Consumption of the above-mentioned tainted honey induces symptoms such as dizziness,

nausea, vomiting, sweating, exhaustion, blurred vision, convulsions, and loss of

consciousness, limb parenthesis, heavy perspiration, headache, stomach ache, delirium,

vision weakness, and salivation. Since local beekeepers are aware of these poisonous

plants, honey that may contain poisonous substances is not sold (Bostan et al., 2010).

Honey toxicity from other plants has also been documented (Islam et al., 2014). The

presence of Clostridium botulinum in honey is a health consequence for children (Rao et

al., 2017).

This bacterium's spores can live in honey, but they are incapable of developing toxins.

Therefore, the bacteria spores from honey will live in the stomach of children less than a

year and become toxic in the immature digestive tract leading to disease and even death

(Tanzi and Gabay, 2002). To minimize the risk of honey-borne poisoning in areas where

toxic nectar plants are present, it is recommended that honey be purchased from a market

rather than from individual beekeepers (Edgar et al., 2002).

43
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Source of Honey

Honey was collected from a local bee producer in Unale (6° 53’13.11” N and 6°

44’22.83” E), Ibaji Local Government Area, Kogi State, Nigeria. The location of the

collection area was determined with the aid of Geographical Positioning System (GPS)

and the coordinates were used to generate the map shown in Figure 3.I. This was done in

the Department of Geography Federal University of Technology, Minna, Nigeria. The

sample was collected during the month of February, 2019 in a sterile container and

transported to the Department of Microbiology Laboratory, Federal University of

Technology, Minna, Niger State and stored at 4°C until use.

44
Figure 3.1: Map of Honey Sourced Area.

3.2 Characteristics of the Honey

The quality of the honey was ascertained using the method described by Nwankwo et al.

(2014) and Elijah et al. (2015): (i). Dropping some of the sample onto sand: if it is an

undiluted honey, it will not sink immediately, (ii) Pouring a small quantity into a cup of

water: if undiluted, it will go down to the bottom of the cup without mixing up with the

water except when stirred (iii) Dipping a finger into the honey sample, dropping one or

two drops on the ground: if it is undiluted, it will go down like a thread without breaking.

(iv) Dipping a match stick unto the honey and sticking to it: the matches will be burn if

it is an undiluted honey and the honey will even act as a fuel while the matches is burning.

3.3 Processing of the Honey Sample

45
The honey sample was filtered through sterile muscilin cloth to remove debris, sterilized

at 121°C for 15 minutes, streaked on the nutrient agar plate and incubated at 37°C for

24hrs to verify the presence of microorganism. Thereafter, the honey sample was diluted

into various concentrations with distilled water into the following: 100 (without water

addition), 80 and 60 percent volume by volume for antibacterial susceptibility testing.

3.4 Preparation of Nutrient Agar Medium

Twenty-eight grams of nutrient agar was dissolved in 1L of distilled water according to

the manufacturer’s instruction.

3.5 Test Organisms

Test organisms used were: Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli.

They were collected from the Microbiology Department, Federal University, Minna,

Niger State, Nigeria. The organisms were confirmed using morphological and

biochemical characteristics, and kept at 4oC until the test was conducted.

3.6 Gram Staining

A smear of 24 hours old bacterial culture of all isolates was prepared on a grease free

glass slide. The smear was first air dried and then heat fixed. The heat fixed smear was

then flooded with crystal violet stain for I minute and then washed with distilled water

for 5 seconds. The washed slides were then flooded with Gram's iodine solution for I

minute and then again washed for 5 seconds with distilled water. The slides were then

washed with 95 % ethanol in drop wise manner. After washing with distilled water again

the smear was flooded with safranin for I minute. The stained slides were finally washed

with distilled water, air dried and observed under microscope. All the observation was

done under oil immersion objective.

46
3.7 Catalase Test

The catalase test is biochemical test for aerobic organism that detects the production of

catalase enzymes in an organism. Clean glass slide was used to conduct catalase test.

Emulsion of the isolate was made on a clean glass slide using wire loop followed by

addition of three percent H2O2. Catalase (enzymes) positive will be indicated by

effervescence (Bashua and Oladunmoye, 2017)

3.8 Oxidase Test

The oxidase test is used to analyze the ability of bacteria to produce the enzyme

cytochrome oxidase. This enzyme catalyses the transfer of electrons from cytrochrome c

to molecular oxygen and is usually found in bacteria which uses oxygen as the terminal

electron acceptor during respiration. An 18 hours’ bacteria culture was transferred

aseptically to the filter paper wet with three drops of sterile distilled water. one drop of

oxidase reagent was added and observed. After ten second, the spot the sport for the

development of red colour which indicated was observed and compared with the control,

blue colour indicated oxidase positive (Bashua and Oladunmoye, 2017).

3.9 Starch Hydrolysis Test

Starch agar was prepared by adding 1 % of soluble starch nutrient agar and autoclaved at

121oC for 15mins after which the plates were poured and allowed to gel. The organisms

were then streaked once across the surface of the plates and incubated at 37oC for 24

hours. The plates were then flooded with some quantity of Gram’s iodine. Unhydrolyzed

starch formed blue black colour with the iodine while hydrolyzed starch appeared as a

cleared zone which results from α-amylase activity. Reddish brown zones around the

colony indicated partial hydrolysis of starch (Bashua and Oladunmoye, 2017).

3.10 Triple Sugar Iron Test (TSIA)

47
TSI agar is a modified agar media. It comprises of three main sugars, glucose (0.1 %)

sucrose (1 %) and lactose (1 %) (Kliger, 1917). Besides carbohydrate it contains beef

extract, yeast extract, and peptone which are the source of nitrogen, vitamins and

minerals. Phenol red is the pH indicator. During preparation the tubes containing the

molten agar are angled. The slant is aerobic, while the butt is anaerobic. The tubes were

inoculated by first stabbing at the butt, which was followed by streaking at the slant and

were incubated at 37oC for 24 hours. If any of the carbohydrate is fermented the drop in

pH will cause the medium to change from reddish orange to yellow. A deep red colour

indicates alkalization of the peptones. Sodium thiosulphate in the medium is reduced by

some bacteria to hydrogen sulphide, a colorless gas. The hydrogen sulphide thus upon

reaction with ferric ions in the medium, produce iron sulphide, a black insoluble

precipitate. The bacteria are said to be glucose fermenter if the reaction tube produce an

alkaline slant (red) and an acid butt (yellow), signifying that only glucose is metabolized.

Since, this substrate is present in minimal concentration; the small amount of acid

produced on the slant surface is oxidized rapidly, whereas in the butt, acid reaction is

maintained because of reduced oxygen tension and slower growth of the organism. When

the reaction tube is overlaid with acid, over acid in both the slant (yellow) and the butt

(yellow) then the bacteria are said to be glucose, lactose and / or sucrose fermenters.

When there is no acid produced in both the slant and the tube the bacteria are said to be

glucose, lactose and sucrose non fermenter.

3.11 Preparation of Potato Dextrose Agar Medium

Potato dextrose agar was prepared by adding 39 g of the powder medium into 1000 mL

of distilled water (Bashua and Oladunmoye, 2017). A suspension formed was heated on

the heating mantle with occasional swirling of the flask dissolved. The conical flask was

plugged with cotton wool and wrapped carefully with aluminum foil. This was autoclaved

48
at 121oC for 15 minutes. After sterilization in the autoclave, the medium was allowed to

cool and 1 % of streptomycin was added to inhibit bacteria.

3.12 Identification of Fungi

The identity of the fungi isolate was done by observation of certain morphological

characteristics, macroscopic and microscopic (Bashua and Oladunmoye, 2017). as

follows: On a grease-free slide, a drop of distill water was applied. The teasing needle

was used to picked cultured fungi, teased on the slide and then viewed under the

microscope, using X10 and X40 objective lenses.

3.13 Preparation of Standard Inoculums

The turbidity organism suspension from 18 hours’ bacterial culture were made in sterile

distilled water and compared with the McFarland turbidity standard, until the cloudiness

correspond with the scale number 0.5 standard by visual comparison, which corresponded

to 1.5 x 108 cfu/mL (Odiba et al., 2014).

3.14 Antimicrobial Sensitivity Testing

The following concentration: 100 %, 80 %, and 60 % (v/v) of honey samples were

prepared and used to test against the organisms using agar well diffusion method

(Abalaka et al., 2016). Three well were bored with the aid of 5 mm diameter sterile cork

borer; The well was made at various positions on the plates, equidistantly. Each well was

sealed at the bottom with molten agar to prevent spillage. The sterile medium was seeded

with 0.1 ml of the standard inoculum of the test microorganisms; the inoculum was spread

evenly over the surface of the medium with a sterile swab. A standard cork borer of 5

mm in diameter was use to cut cups (well) at the center of each inoculated medium and

0.1 mL of the honey solutions were introduced separately into each well on the medium,

the plates were incubated at 37ºC for 24 hours for bacteria. The fungi were incubated for

49
four days at room temperature (25ºC ±2ºC) at 100 % honey concentration. The zones of

inhibition were measured with meter rule and recorded.

3.15 Determination of Minimum Inhibitory Concentration (MIC) and Minimum

Bactericidal Concentration (MBC)

The minimum inhibitory concentrations (MICs) were estimated using the tube dilution

method; the lowest concentration of honey with no turbidity was used as the minimum

inhibitory concentration (MIC) (Abalaka et al., 2016). The MIC was determined by

preparing honey concentrations (100 %, 80 %, 60 %, 40 %, 20 % and 10 %, (v/v) in

nutrient broth. Nine mL of each of the concentration was introduced into 10 mL test tube.

This mixture was then inoculated with 0.1mL culture of the test organism standardized

to approximately 1.5 x 108 cfu/mL and was incubated at 37oC for 24 hours. The tubes

were observed to determine the lowest concentration of honey in the test tube with no

turbidity or cloudiness compared with the control. This was taken as the minimum

inhibitory concentration (MIC) (Abalaka et al., 2016; Mba-Omeje et al., 2017)

Minimum bacterial concentration was determined for each set of test tubes used for the

MIC test. A loopful of the broth culture was collected from those test tubes which showed

no growth and was inoculated onto sterile nutrient agar by streaking. MBC was

determined when no visible growth was seen (Mba-Omeje et al., 2017).

3.16 Phytochemical Screening of the Honey

Qualitative tests were carried out on the honey sample using methods described by

Jayashree (2013) and Abalaka et al. (2016).

3.16.1 Test for flavonoids

A portion of honey in each case was heated with 10 mL of ethyl acetate in a test tube

over a steam bath for 3 minutes. The mixture was filtered and 4 mL of the filtrate was

50
shaken with 1 mL of dilute ammonia solution. Yellow coloration was observed thus

indicating the presence of flavonoids.

3.16.2 Test for phenols and tannins

Honey was mixed with 2 mL of 0.1 % ferric chloride (FeCl3), blue-green or black

colouration indicated the presence of phenol and tannins.

3.16.3 Test for saponins

Frothing test: Two mL of the honey was vigorously shaken in the test tube for 2 minutes.

Frothing was observed indicating the presence of saponins.

3.16.4 Test for alkaloid

Alkaloid was tested by adding 2 mL of honey in the test tube contained 5 mL of 1%

aqueous HCI on a steam bath and shaken, two to three drops of solution of picric acid

was added. The preliminary evidence for the presence of alkaloids was formation of a

reddish brown precipitate.

3.16.5 Test for steroids

Steroid test was performed by adding 2 mL of honey into test tube, mixed with 2 ml of

acetic anhydride followed by 2 mL of sulphuric acid. No colour changed from violet to

blue or green in the samples indicated the absence of steroids.

3.16.6 Test phlobatannins

The presence or absence of the phlobatanins was determined by boiling 1 % aqueous

hydrochloric acid with small portion of honey sample; red precipitates indicate the

presence of phlobactanins.

51
3.16.7 Test for cardiac glycosides

Cardiac Glycosides was detected by adding 5 mL of honey into the test tube, mixed with

2 mL of glacial acetic acid and one drop of ferric chloride solution, followed by the

addition of 1 mL concentrated sulphuric acid. Brown ring formed at the interface

indicated deoxysugar characteristics of cardenloides. A violet ring may appear beneath

the brown ring, while in the acetic acid, layer; a greenish ring may also form just gradually

throughout the thin layer.

3.16.8 Test for anthraquinones

Five milligram of honey was shaken with 5 mL chloroform, the chloroform layer was

filtered and 0.5 cm3 of 10 % ammonia was added to the filtrate. The mixture was shaken

thoroughly, the formation of a pink/violet or red, yellow colour in the ammonical phase

indicated the presence of anthraquinones.

3.16.9 Test for reducing sugar (benedict test)

Five milligram of honey was mixed thoroughly with 3 cm3 distilled water and filtered, 3

drops of the filtrate was added to 3 cm3 Benedict reagents and placed in a boiling water

bath for 5 minutes. The formation of a brick red precipitate indicates reducing sugar.

3.17 Specific Actions of Honey on Pathogenic Organisms

3.17.1 Catalase inhibition

Catalase is a widely distributed antioxidant enzyme that breaks down hydrogen peroxide

into water and oxygen. Several pathogens produce catalase to defend themselves against

oxidative stress as well as attacks by hydrogen peroxide, a common weapon used by the

host's immune system.

In order to address these, an assay was developed to measure catalase activity. The assay

uses simple and readily available reagents, namely hydrogen peroxide, Triton X-100, and

52
catalase (Iwase et al., 2013). The underlying principle of this approach is that the oxygen

bubbles generated from the decomposition of hydrogen peroxide by catalase are trapped

by the surfactant Triton X-100. The trapped oxygen bubbles are then visualized as foam,

the test-tube height of which is measured to quantify the catalase activity.

In the present research, 18 – 24 hours of actively growing bacteria was used according to

Iwase et al. (2013) with little modification. One hundred micro milliliter of bacterial

suspension were transferred into 4 mL of honey in the test tube at each respective MIC

concentration (100 %, 80 % and 10 %) and incubated at room temperature (25ºC ±2ºC)

for 3 hours. After the incubation, the cells were harvested by centrifugation at 4000 rmp

for 10 minutes. The pellets were used for quantification of cellular catalase activity. One

hundred micro milliliter bacterial pellet was added in Pyrex test tube 13 mm diameter ×

100 mm height. Subsequently, 0.1 mL of 1 % Triton X-100 and 0.1 mL of undiluted

hydrogen peroxide was added to the solutions and mixed thoroughly and sterile distilled

water which contained the same cell suspension was used as control. Following the

completion of the reaction, the height of oxygen forming foam that remained constant in

the test tube was measured after 5 minutes using a meter rule (ruler) and recorded (Wang

et al. 2015). The enzyme-generated oxygen bubbles trapped by Triton X-100 were

visualized as foam, whose height was estimated.

3.17.2 Protein leakage

An important characteristic of plant extracts is their hydrophobicity which enables them

to attack the lipids of the bacterial cell membrane, disturbing the structures and inducing

more permeability (Kusumaningrum et al., 2014). Leakage of the cytoplasmic membrane

has been analysed by determination of the absorbance of suspension containing cell

materials including proteins that were absorbed at 595 nm (Kusumaningrum et al., 2014).

53
In this study, bacterial cultures were transferred to 9 mL of fresh sterile nutrient broth in

the test tubes and incubated at 37°C for 18 hours. After incubation, 0.1 mL of the actively

growing bacterial cells was suspended in 4 mL of honey at each respective MIC

concentration. These were incubated at room temperature for one hour and two hours.

After incubation, cultures were centrifuged at 4000 rpm for 25 minutes and bacterial

pellets were removed. One ml of Bradford reagent was added to 1 mL of supernatant and

incubated at room temperature (25ºC ±2ºC) for 5 minutes. Then the absorbance of the

supernatant was measured at 595 nm using UV-Vis spectrophotometer (Shimadzu UV-

1800, Japan) for protein leakage determination in each case and the protein estimation

was done using Bovin Serum Albumin (BSA) as standard (Kusumaningrum et al., 2014).

3.17.3 Preparation of Bradford reagents

One hundred milligrams of Coomassie Brilliant Blue G-250 (Sigma-Aldrich) was

dissolved in 50 milliliters of 95 percent ethanol, followed by 100 milliliters of 85 percent

(w/v) phosphoric acid, dilution to 1000 milliliters, and filtration by Whatman paper

number 1

3.18 Data Analysis

The analysis of variance of the values obtained for antibacterial susceptibility, protein

leakage and catalase inhibition analysis were carried out using IBM SPSS Statistic

version 23. All data were expressed as Mean ± Standard Error Mean of triplicate

determinations. Values with the same superscript on the same column are not

significantly different at (P < 0.05). Values with the same subscript on the same row are

not significantly different at (P < 0.05).

54
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 Results

4.1.1: Phytochemical screening of honey

The phytochemical screening of the bioactive component of the honey showed the

presence of saponins, phenols, flavonoids, tannins, reducing sugars, anthraquinones

cardiac glycosides and alkaloids while steroids and phlobactanins were not present as

shown in Table 4.1

55
 Table 4.1: Bioactive Component of Honey

Bioactive components Inference

Saponins +

Phenols +

Akaloids +

Flavonoids +

Taninns +

Reducing sugar +

Cardiac glycosides +

Phlobactanins -

Steroids -

Key: − = Absent and + = Present

4.1.2: Antimicrobial susceptibility test of the honey

The result of the antimicrobial susceptibility test showed E. coli had zone of inhibition of

29.0±0.6mm, 27.0 ±0.6mm and 19.0±0.6mm at 100 %, 80 % and 60 % concentration of

honey respectively, B. subtilis had 15.0±0.6mm, 11. 0±0.6mm and 8.0±0.6mm at 100, 80

and 60 % concentration of honey respectively while P. aeruginosa had 13.7±0.3mm, 11.

0±0.6mm and 6.3±0.3mm at 100, 80 and 60 % concentration of honey respectively. It

showed that E. coli had highest zone of inhibition (29 mm) followed by B. subtilis while

P. aeruginosa had the least zone of inhibition (6.3 mm) showed in Table 4.2.

Table 4.2: Antimicrobial Susceptibility Test (mm) of Honey on the Isolates

56
 Honey Concentration (% v/v) / mean zone of inhibitions (mm)

Bacteria 100 80 60 Ciprofloxacin

E. coli 29.0±0.58c ⃰ b⃰ ⃰ 27.0 ±0.58b⃰ b⃰ ⃰ 19.0±0.58a⃰ b⃰ ⃰ 32.0±0.28d⃰ b⃰⃰ ⃰

B. subtilis 15.0±0.58c⃰ a⃰⃰ ⃰ 11. 0±0.58b⃰ a⃰ ⃰ 8.0±0.58a⃰ a⃰⃰ ⃰ 30.0±0.00d⃰ a⃰ ⃰

P. aeruginosa 13.7±0.33c⃰ a⃰ ⃰ 11. 0±0.58b⃰ a⃰ ⃰ 6.3±0.33a⃰ a⃰⃰ ⃰ 30.0±0.00d⃰ a⃰ ⃰

Results represent Mean ± Standard Error Mean of triplicate determinations. Values with
the same superscript on the same column are not significantly different at (P < 0.05).
Values with the same subscript on the same row are not significantly different at (P <
0.05).

4.1.3: Minimum inhibitory concentration (MIC) and the minimum bactericidal

concentration (MBC)

The result of MIC and MBC are shown in Table 4.3. Escherichia coli had the lowest MIC

at 10 % and MBC at 20 % followed by Bacillus subtilis which had MIC at 80 % and

MBC at 100 % while Pseudomonas aeruginosa had MIC at 100 % without the MBC.

Table 4.3: The MIC and MBC of Honey on the Bacteria Isolates

Honey concentration (% v/v)

Bacteria MIC MBC

Escherichia coli 10 20

Pseudomonas aeruginosa 100 Nil

Bacillus subtilis 80 100

Results represent Mean ± Standard Error Mean of triplicate determinations. Values with
the same superscript on the same column are not significantly different at (P < 0.05).

57
Values with the same subscript on the same row are not significantly different at (P <
0.05).

4.1.4: Catalase inhibition of honey on bacteria isolates

The result of catalase inhibition of honey on tested bacterial isolates after three hours of

incubation at room temperature (25ºC ±2ºC) at respective MIC concentration of honey

and control (untreated) were showed in Table 4. 4. The catalase activity of the untreated

cells were higher than the treated. E. coli had 11.0±0.6 and 14.0±0.6 (mm); B. subtilis

had 30.0±1.1 and 40.0±1.1 (mm) while P. aeruginosa had 31.7±8.5 and 45.0±0.6 (mm)

for the treated and untreated respectively. P. aeruginosa had highest activity for both

treated and untreated cells followed by B. subtilis while E. coli had the least. However,

B. subtilis showed highest catalase inhibition upon treatment compared to other isolates.

Table 4.4: Catalase Inhibition of honey on Bacterial Isolates

Bacteria Treated (mm) Not treated (control) (mm)

E. coli 11.0±0.58a⃰ a⃰⃰ ⃰ 14.0±0.58b⃰ a⃰ ⃰

B. subtilis 30.0±1.16a⃰ b⃰⃰ ⃰ ⃰ 40.0±1.16b⃰⃰ b⃰ ⃰

P. aeruginosa 31.7±8.51a⃰⃰ b⃰⃰ ⃰ 45.0±0.58a⃰ c⃰ ⃰

Results represent Mean ± Standard Error Mean of triplicate determinations. Values with
the same superscript on the same column are not significantly different at (P < 0.05).
Values with the same subscript on the same row are not significantly different at (P <
0.05).

4.1.5: The result of protein leakage activity of honey

The result of protein leaked by the test isolates after incubation at room temperature (25ºC

±2ºC) at respective MIC concentration of honey were recorded as follows: E. coli

58
22.0±0.6 and 35.3±0.9 µg/ml; B. subtilis had 31.0±0.6 and 49.0±0.6 µg/ml while P.

aeruginosa had 49.7±0.9 and 60.0±0.6 µg/ml at 1 hour and two hours’ incubation

respectively were showed in Table 4.5. The leakage of protein was time dependent for

each organism and organism dependent.

Table 4.5: The Protein Leakage (µg/ml) Activity of Honey

Bacteria 1 hour exposure 2 hours exposure

E. coli 22.0±0.58a⃰ a⃰ ⃰ 35.3±0.88b⃰ a⃰⃰ ⃰

B. subtilis 31.0±0.58a⃰ b⃰⃰ ⃰ 49.0±0.58b⃰ b⃰⃰ ⃰

P. aeruginosa 49.7±0.88a⃰ c⃰ ⃰ 60.0±0.58b⃰ c⃰ ⃰

Results represent Mean ± Standard Error Mean of triplicate determinations. Values with
the same superscript on the same column are not significantly different at (P < 0.05).
Values with the same subscript on the same row are not significantly different at (P <
0.05)

59
4.2 Discussion

The result for phytochemical screening of the honey contained some bioactive

compounds which possess good antimicrobial properties on the test isolates used in this

study and therefore justified its aged long medicinal uses for curing purposes. These

bioactive compounds; saponins, phenols, flavonoids, tannins, reducing sugars,

anthraquinones cardiac glycosides and alkaloids were Similar to the results obtained by

Elijah et al. (2015). Also, Nwankwo et al. (2014) in related study also confirmed the

presence of alkaloids, flavonoids, saponins, steroids, reducing sugar and glycosides.

Agbaje et al. (2006) equally attested to this fact that Alkaloids, anthraquinone,

glycosides, cardiac glycosides, flavonoids, saponins, tannins, and reducing compounds

may all be present in honey. The presence of these bioactive compounds were not exactly

the same, which could be as result of nectar source, floral source, climatic factor and soil

nutrients as reported by Elijah et al. (2015). Secondary metabolites like alkaloids,

flavonoids, phenolic compounds may be responsible for various biological activities such

as antioxidants and antimicrobials as reported by Mbah-omeje et al. (2017).

Ali et al. (2018) reported that flavonoids inhibit the activity of enzymes by forming

complexes with bacterial cell walls, extracellular and soluble proteins, more lipophilic

flavonoids disrupt cell wall integrity or microbial membranes at low concentrations.

Saponins facilitated the entry of toxic material or leakage of vital constituents from the

60
cell (Ali et al., 2018). Omojate et al. (2014) reported that phytochemicals have diverse

antimicrobial mechanisms including damaging cell wall and cytoplasmic membrane.

The honey had activity on both Gram positive and Gram negative bacteria used in this

analysis, according to the antimicrobial susceptibility test on the agar well diffusion. The

zones of inhibitions were concentration dependent as reported by Olatunji et al. (2014).

Among the bacterial tested, Escherichia coli had the highest zone of inhibition at all the

various concentration used compared to Pseudomonas aeruginosa and Bacillus subtilis.

Elijah et al. (2015), also recorded highest zone of inhibition for E. coli when compared

to the P. aeruginosa at the same concentration (0.2 mL) of undiluted honey). Similar

result was obtained by Raju and Goli (2013) for E. coli. In their study, E. coli is more

sensitive to honey than Pseudomonas. Olatunji et al. (2015), Who worked on the in-vitro

antimicrobial effect of different honey samples against selected microorganisms

marketed in Abuja, Nigeria, reported that honey have activity against Pseudomonas

aeruginosa, Escherical coli and Bacillus subtilis as reported in this presented study

except one honey sample from one of the five markets that, P. aeruginosa showed

resistant to. Kwakman et al. (2010) reported that honey had bactericidal activity against

Bacillus subtilis, Staphylococcus aureus, Streptococcus epidermidis, Escherichia coli,

and Pseudomonas aeruginosa within 24 hours in the range of 10-40 percent honey

concentration.

The result of the MICs and MBCs showed. E. coli, B. subtilis and P. aeruginosa had MIC

at 10 %, 80 % and 100 % (v/v) concentration respectively. E. coli had the lowest MIC.

Bunza et al. (2019) recorded similar MIC in their work on comparative evaluation of the

antibacterial effects of honey with standard antibiotics on bacterial isolates from wound

infection where E. coli had 20 % MIC and P. aeruginosa had 50 % MIC.

61
E. coli and B. subtilis had 20 % and 100 % MBC respectively while P. aeruginosa had

no MBC. The honey sample used was bacteriostatic to P. aeruginosa. Bunza et al. (2019)

had similar observation where P. mirabilis, which had 100 % MIC had no MBC as

applicable to P. aeruginosa in this present research. Olatunji et al. (2015) also recorded

resistance for P. aeruginosa in some of the honey he used. According to the investigation

by Alemseged et al. (2018) on the antibacterial properties of mixture honey and garlic

(Allium sativum) extracts against respiratory tract infection causing bacteria, they

reported that honey was effective against all tested pathogenic organisms except P.

aeruginosa.

The specific action of honey on enzymes, catalase of the bacterial isolates after 3 hours’

treatment showed that honey inhibited exogenous enzymes, catalase for all the tested

organisms. Wang et al. (2015) recorded similar result when they measured the catalase

activity of S. aureus after treatment with antibiotics, vacomycin and ciprofloxacin.

The bacterial enzymes, catalase, the major weapon used by many aerobic bacteria to

withstand oxidative stress by neutralize H2O2 to water. H2O2 had been the defense used

by immune system to attack bacterial cell (Iwase et al., 2013). For this investigated

honey, the reduced catalase expression could help eliminate P. aerusinosa, B. subtilis and

E. coli infection by reducing the organisms’ ability to cope with oxidative stress. Catalase

inhibition could be one of the specific action of honey against the bacteria.

Quantitatively, the amount of catalase released within the organisms differs. P.

aeruginosa had highest catalase activity followed by B. subtilis while E. coli had the least.

P. aeruginosa, which had higher catalase activity have been reported to have resistance

to honey (Olatunji et al., 2015). This might be the reason for its survival strategy. B.

subtilis had been reported to cause nosocomial infection and opportunistic diseases as

62
reported by Saleh et al. (2014). E. coli which had lower catalase activity was more

susceptible to honey.

The specific actions of honey on bacterial cells were determined by leakage of protein

after bacterial cells have been exposed to honey at their respective MIC concentration

over time. Protein leakages induced were time-depended. The protein leaked after two

hours were higher than the ones after one hour. The increment in protein leakage with

time could be as result of more damage to the cell wall and membrane. This observation

cuts across all the tested organisms used in this experiment. Kusumaningrum et al. (2014)

made similar observation of different values of protein leaked by E. coli and S. aureus in

their experiment. Nadial (2017) equally observed similar leakage difference in potassium

leakage in E. coli and S. aureus where the value of potassium leaked increased with time.

Also, Singh et al. (2016) made similar observation. In their study, they noticed a

progressive protein leakage up to 5 hours after which the leakage decline as a result of

protease, which led to protein degradation. Pseudomonas aeruginosa had higher protein

leakage followed by Bacillus subtilis while Escherichia coli had the least. The observed

lower protein leakage by B. subtilis, gram positive bacteria when compared to gram

negative, P. aeruginosa could be due to the differences in the structural features of the

cell wall, particularly the thickness of the peptidoglycan layer of Gram positive bacteria,

which functions as a protective barrier against antibacterial agents (Yuan et al., 2017).

However, E. coli, which was the most sensitive organism in the study, had lower protein

leakage. May be its sensitivity to honey may not be as a result of cellular leakage.

Protein leakage by all the isolates could be as a result of cytoplasmic membrane damage

which led to cellular leakage of protein. Zakaria (2015) reported similar investigation

when he checked the mechanism of action of honey on pathogenic wound bacterial

strains. From this study, it could be deduced that honey can induce cellular leakage for

63
both gram negative and positive bacteria which may be possible specific actions of honey

against bacterial pathogens.

The antimicrobial activity of honey was equally tested against fungi (Trichophyton

equinum and Trichophyton verricosum) at 100 % concentration. Trichophyton equinum

was more sensistive to honey than Trichophyton verricosum at the same concentration.

Shariati et al. (2015) had similar report in their work on dermatophyte strains from 3

genuses, Trichophyton, Microsporum and Epidemophyton by agar dilution technique

method. These species of fungi are known to be causative agent of dermatophytes as

reported by Shariati et al., (2015). Dermatophytes are groups of hemogene keratinolytic

fungi that have the ability to attack keratinilized tissues of human and animals and cause

dermatophytose infection that is a kind of colonization of fungi on skin (Shariati et al.,

2015).

64
 CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

It was observed that honey sampled from Kogi State, Nigeria had antimicrobial activity

against the tested isolates. The presence of bioactive components like saponins, phenols,

flavonoids, tannins, reducing sugars, anthraquinones cardiac glycosides and alkaloids

may be responsible for its antimicrobial activity. Protein leakage and catalase inhibition

were novel specific actions of honey on microbial pathogens. Once the bacterial cell wall

structural integrity is compromised, the organism in question will be affected, likewise

catalase inhibition. The organism’s ability to withstand stress is impaired, hence it will

either inhibited or died.

Therefore, it can be concluded that this honey could be used to treat diseases cause by

these organisms.

5.2 Recommendations

The following are recommended for profitable use of honey and consideration:

I. Honey should be considered as alternative antimicrobial agents for the treatment

of skin diseases

65
 II. The antibacterial activity of honey should be tested in-vivo.

III. Other mechanism of action of honey like protease inhibition and DNA leakage

should be research into.

IV. The shelf life of honey should be determined

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APPENDIX

Appendix A: Antifungal activity of honey

The results of Antimicrobial activity of honey at 100 % concentration on the selected

fungi were showed in Appendix A. T. equinum is more sensitive than T. verrucosum at

the same concentration

18 17
16
14
Zone of inhibition (mm)

14
12
10
8
6
4
2
0
Trichophyton equinum Trichophyton verrucosum

Figure 3: Anti- fungal activity of honey

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Appendix B: Biochemical characteristics of bacterial isolates

The confirmatory tests were carried out on bacterial isolates and the following bacteria: Escherichia
Coli, Pseudomonas aeruginosa and Bacillus subtilis were confirmed in Table 5

Table 5: Biochemical characteristics of bacterial isolates.

Isolates Shape Gram Catalase Citrate Indole Starch Oxidase Suspected

reaction orgaisms
Hydrolysis

A Rod Negative + _ + _ _ Escherichia

coli

B Rod Negative + + _ _ + Pseudomonas

aeruginosa

C Bacilli Positive + + _ + _ Bacillus

subtilis

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