CHAPTER ONE

INTRODUCTION

1.1 Background of Study

Skin is the soft outer tissue covering the vertebrates and also the largest
organ that plays important biological roles in the body. Skin is the first line of
defense from external factors. Severely damaged skin may heal by forming scar
tissue. However, this is sometimes discolored and depigmented and it also takes
longer time to heal (Lien et al., 2015). Accelerating wound healing and increasing
the quality and speed of healing is very important to prevent further damage to the
skin. When the skin is damaged, the hair around the damaged skin will be lost and
as the worst cases, a longer time is needed for it to regenerate.
Honey is a natural sweet substance that is produced by honeybees from the
nectar of the blossomed flowers either unifloral or multifloral. It is super saturated
sugar solution which is rich in proteins, minerals, vitamins, organic acids and
polyphenols (Saba et al., 2013). Honey has been used as a traditional natural
therapeutic agent to boost up the immune system and defense against diseases. It
contains high nutritional values and prophylactic medical value (Jing, 2016).
Recently stingless bee has grabbed the interest of researcher due to its
higher nutritional values as compared to ordinary honey. According to Rintos
(2014), a Borneo Post Reporter, stated that having stingless bee honey regularly
can promote anti-aging, enhance immune system and libido, fight against bacteria
and treat sore throat, coughs and colds. It is also used as antiseptic and therapeutic
agent to sooth pain, promote healing, relieve cough and also effective in curing
burns, carbuncle, boils and diabetic wound.

1.2 Problem Statement

Oskouei and Najafi (2013) found that the most effective uses of honey is
found in wound healing and nearly all type of wounds are found to be responsive

1
to honey therapy. However, study on Trigona itama honey potential on wound
healing are still lacking.
Nowadays, instead of using beneficial natural resources available to cure
wound there were excessive usage of chemicals in medicines that may lead to
some negative effects to the body (Wild et al., 2010). Thus, this study is going to
identify the effect of Trigona honey on wound healing due to the lack of research
on this type of honey.

1.3 Significance of Study

Nowadays, there are so many diseases that may prevent the process of
wound healing such as diabetes and obesity. For example, person with obesity is
more likely to develop ulcers cause by pressure to the skin and they tend to
experience delayed wound healing. In fact, there are several factors that play
crucial roles in the wound healing process. The factors include nutrition, age,
hygiene, and also wound type. Some of these obstacles are under the control of
the patient or clinician. Consuming traditional treatment may help in reducing the
impact of facing problem such as wound healing. Since we live in modern era
with the aids of technologies, trigona honey can be identify as one of the
traditional treatment to overcome the problem above. Moreover, by using trigona
honey, it will help us to live healthier as its contains 100% organic substances and
free from harmful chemicals, and because of that, trigona honey is very
recommended to aid in wound healing.

1.4 Objectives of Study

The objectives of the study are:

1. To determine the purity of Trigona itama honey through
physicochemical test.
2. To test the wound healing effect of Trigona itama honey on mice.

2
 CHAPTER TWO
LITERATURE REVIEW

2.1 Wound

Wound is an injury that damages the skin or other body tissues that include
cuts, scrapes, scratches, and punctured skin. They often happen because of an
accident, but surgery, sutures, and stitches (Stubblefield, 2016). According to
Deer and Stewart (2017), the natural process of wound healing is initiated when
the skin is broken.
Wound is separated into two which are open and closed wound. Open
wound occurs when the protective body surface which is the skin or mucous
membranes has been broken, permitting the foreign material to enter the tissues.
As example which stated in the Encyclopaedia Britannica (2017), a needle or a
sharp knife that passes through the tissues with ease, dividing them cleanly or
separating them, will produce relatively little damage except to those tissues
directly in its course, and, indeed, unless an important structure is injured, the
wounds caused are seldom serious.
In contrast, closed wound which involved the damaged tissues are not
exposed to the exterior, and the repair process can take place without the
interference that contamination brings, in greater or lesser degree. For example, a
hammer blows to the side of the head may severely bruise the scalp or, delivered
with equal force but directed in a slightly different way, may cause extensive
damage to the base of the skull (Encyclopaedia Britannica, 2017).

2.1.1 Wound Healing

Wound healing is an outstanding biological process for the

restoration of the integrity of the skin after injury (Jackson et al., 2012). It
can be divided into 4 overlapping phases, which are haemostasis,
inflammation, proliferation, and maturation (Simon and Moutran, 2016).

3
 All four phases must occur in the proper sequence and time frame in order
for a wound to heal successfully (Guo and DiPietro, 2010).

The first phase is haemostasis, where platelets play a role by

aggregating in the beginning of injury, as well as by releasing cytokines,
chemokines, and hormones. Chemokines released by platelet activation
attract inflammatory cells to the area, leading to the next phase in the
healing process (Simon, 2016). The inflammatory phase of the wound
healing cascade gets activated during the coagulation phase and can
roughly be divided into an early phase with neutrophil recruitment and a
late phase with the appearance and transformation of monocytes (Reinke
and Sorg, 2017). Next, the proliferative phase where involves the
formation of granulation tissue which occurs 3-5 days following injury and
the wound begins to contract (Simon, 2016). The main focus of the healing
process in this phase lies on the recovering of the wound surface (Reinke
and Sorg, 2017). Lastly is the maturation phase that involves the
replacement of granulation with scar tissue (Rieger et al., 2013).

2.2 Trigona Honey

2.2.1 Background of Trigona Honey

Trigona honey is commonly known as madu kelulut produced by

stingless bee, Trigona spp. that is one of two highly eusocial bee (Eardley
et al., 2012). Honey is characterised by its complex composition such as
origin of the raw material as nectar, edaphoclimatic conditions, the
available floral source, the storage condition and the bee species (Maria et
al., 2015). Honey had varied colour and flavour depending on the nectar
sources or the blossom encountered by the bees. The range of honey
colour available from colourless to dark brown, meanwhile the flavours is
from mild to strong. It is affected by the location of honey bees buzzed.
Honey that has stronger in taste is dark-coloured honey and the light-
coloured honey is milder (National Honey Board, 2010).

4
 Honey is mostly formed of hydrocarbons (75%) as sugars, maltose,
sucrose, glucose and fructose; water (20%); minerals such as copper,
magnesium, calcium, phosphorus and others. However, most of these
sugars are not found in nectar as there are only formed by the bees during
the ripening and its storage (Soud, 2012) Ways to distuinguish honey by
its species of bee of Apis spp (honey bees) and Trigona spp (stinglees bee)
are based on distinctive differences in chemical compositions and minerals
contents. The major elements raw in honey samples were potassium and
sodium at average of 904.9 and 617.6mg/kg, respectively. Honey that
contained more protein, 0.85g/100g and less total sugar off fructose and
glucose, 24.99g/100g is honey from stingless bee (Yusof et al., 2017).
An associated microbiota presented by stingless bees suspected to
be responsible for transforming pollen to bee bread or the formation of
honey. These microorganisms play an important role on the maturation of
honey and biochemical modification of stored honey. Relatively, the
bacteria in the nest avoid themselves from competitors (Morais et al.,
2012). As shown in figure 2.1, trigona honey produced by various species
of stingless bee such as Lepidotrigona, Hetrotirogona,Tetragonula melina,
Geniotrigona thoracica, Tetragonula zucchi, Tetragonula melanochepala,
Homotrigona spp, Lepidotrigona latipes, Tetragonula atripes (Rasmussen,
2013).

Figure 2.1 Species of stingless bee

Source: Jalil (2012)

5
 The most commonly visited plants of stingless bee were of
herbaceous habits, white or yellowish colour flowers, small-sized flowers
and moderate mass-flowering degree. The nesting substrate of stingless
bee honey that most commonly found was on large living tree trunks and
base of the trunks (Vossler, 2012). The bees will pass the nectar to other
bees as they return to hive (Figure 2.2) by transfering the liquid in the
other bees’ mouth before converted it into honey through evaporation
process (Palermo, 2013). According to MARDI, stingless bees were not
choosen in building colony hive. As a result, the production of honey will
increase because an artificial hive is easy to build and manipulate the
colony. Resin was used by trigona bees to build their nest which is
function as nest maintenance and defence. They harvest resins from other
plant species and bring them to their colony before it been utilized as
propolis. Propolis is anapicultural term for the resins when used by bees
within the hive (Finstrom, 2010). The resin was used by certain plant to
attract bees (Leonhardt et al., 2011). Trigona honey was suggested to be
more effective than honey bee honey to treat infectious disease
(Andualem, 2014). It is very beneficial for medication.

Figure 2.2 Trigona hive

Source: TLH Production Industries (2010)

6
2.2.2 Benefits of Trigona Honey

Department of Pathology, Universiti Sains Malaysia (2012) stated

that trigona honey acts as a natural immune booster, natural anti-
inflammatory agent, natural antimicrobial agent and natural promoter for
healing the wound. Endogenous bacteria in honey bees have a specific
immune stimulation. Oral intake of honey is an addition of antibody
production in primary and secondary immune responses (Hayati, 2012).
As immune responses are costly, this specificity may be adaptive for
saving energy and negative side effects on the host development or
survival (Janashia et al., 2016).
Honey also said to be a natural anti-inflammatory agent.
Inflammatory responses are beneficial but at times, chronic inflammatory
responses are not good to health (Hayati, 2012). As an example, infant
who suffers diaper dermatitis can be healed after the application of a
mixture containing honey, olive oil and beeswax after 7 days . Besides
that, honey offers broad spectrum antimicrobial properties and promotes
rapid wound healing (Hayati, 2012). The antibacterial potential of honey
has been considered the exclusive criterion for its wound healing
properties. Honey and its components are able to either stimulate or inhibit
the release of certain cytokines from human monocytes and macrophages,
depending on wound condition (Hayati, 2012). The activation of honey-
induced will speed up the process of healing. Moreover, honey contains
various kinds of phytochemicals with high phenolic and flavonoid content
which contribute to its high antioxidant activity (Hayati, 2012). Agent that
has strong antioxidant property plays a significant role in preventing the
formation of cancers potential such as free radicals and oxidative stress.
Phenolic acids and polyphenols were phytochemicals available in honey.
Variants of polyphenol in honey are reported to have anti-proliferative
property against several types of cancer (Hayati, 2012). Nowadays, there

7
are several product of honey had been commercial such as Golden B drop
by Dr. Azlan and Madu Kelulut Asli from Syamille Agro Farm.

2.2.3 Chemical Composition in Trigona Honey

Honey’s antibacterial properties and its effects on wound healing

have been thoroughly investigated (Waili et al., 2011). A systematic
database research, 241 compounds were identified in trigona honey for the
first time between 2000 and 2012 and they belong to such diverse
chemical classes such as proteins and amino acids, vitamins, enzymes,
minerals and other components. Honey and propolis have anti-
inflammatory and anti-bacterial activities, its natural compounds are useful
for diseases associated with inflammatory process. Honey produced from
trigona stingless bee has antimicrobial activity and effective against
Staphilococcus aureus, Pseudomonas and Candida albicans.
Antioxidants activity of honey are correlated with polyphenols and
vitamins content as total Phenols, Flavonoids, vitamin C and Vitamin E
(Usman et al., 2013). Trigona honey has been reported to have inhibitory
effect on around 60 species of bacteria, some species of fungi and viruses.
Antioxidant capacity of honey is important in many diseases conditions
and is due to a wide range of compounds including phenolics, peptides,
organic acids and enzymes. Honey has also been used in some
gastrointestinal cardiovascular, inflammotary and neoplastic states. This
review covers the composition, physicochemical properties and the most
important uses of natural honey in human diseases. The wide application
of trigona honey in modern medicine has drawn growing attention to its
chemical composition.

2.2.3.1 Sugars

Sugar is the major constituent in honey and it accounts for

95-99% of honey dry matter. Honey produced by stingless bee

8
species are rich in maltose and show slightly higher values of
turanose, erlose and trehalose (Souza, 2015). Maltose is so
important to our body. Our body can absorb maltose, which later be
broken into individual glucose molecules and then be used as
energy. The sugars present in honey are also used for some
viscocity, and hygroscopicity (Kamal and Klein, 2011).

2.2.3.2 Water

Water is the second main component in honey whereby it

constitutes approximately 17% of the honey. The water content of
honey is inversely proportionate to the sugar content of honey
(Oskouei and Najafi, 2013). The water of honey of stingless bee
varies according to the species and to the area where it is produced,
complicates the establishment of a quality standard for these
honeys. (Mariens, 2011).

2.2.3.3 Flavonoids

As a major constituents of trigona honey, flavonoids

contribute greatly to the pharmacological activities of trigona
honey (Bankova, 2011). The quantity of flavonoids is used as a
criterion to evaluate the quality of temperate trigona honey.
Flavonoids have a broad spectrum of biological properties, such as
anti-bacterial, antiviral and anti-inflammatory effects. So it is very
good in treating wound. According to the chemical structure,
flavonoids in trigona honey are classified into flavones, flavonols,
flavanones, flavonols, flavanones, flavanonols, chalcones,
dihydrochalcones, isoflavones, isodihydroflavones, flavans,
isoflavans and neoflavanoids (Bankova, 2011).

2.2.3.4 Terpenoids

9
 Although represents only 10% of the trigona honey
constituents, they account for the characteristics resinous odor and
contribute to the pharmacological effects of trigona honey. As the
major compounds among the volatile substances, terpenoids play
an important role in distinguishing premium trigona honey from
inferior or fake honey and they exhibit antioxidant, antimicrobial
and other biological activities (Bankova, 2011).

2.2.3.5 Low pH and phenol

Low pH is beneficial in preventing of microbial growth.

About 0.57% of honey is made up of organic acid which are
derived from sugars in the honey. This organic acid contributes
largely to the acidity of honey and hence, honey usually has low
pH (Oskuei and Najafi, 2013). pH and nutrient content of trigona
honey has potential benefits for prevention and treatment of many
kind of diseases as well as in treating wound, scar fader and hair
growth (Rahardjo, 2016).

2.4.3.6 Minerals and Trace Elements

Although it is a rare occasion, several studies have shown

that trigona honey contains minerals such as magnesium, calcium,
potassium and zinc. Mineral content of trigona honey is affected by
seasonal variations. Some studies have shown different results
concerning the highest mineral content of honey, sometimes
calcium, potassium or sodium becomes the highest mineral content
of honey while metals such as zinc always has low level
(Moniruzzaman et al.,2014).

10
 CHAPTER THREE
METHODOLOGY

3.1 Materials

Trigona honey (as a raw material), Chloroform, Aloe Vera.

3.2 Physicochemical Analysis of Trigona Honey

3.2.1 Moisture Test

The preparation of trigona honey for physicochemical analysis for

moisture was performed according to Gebremariam et al., (2014) with
slightly modification. 5.0 gram of trigona honey was weighed and placed
into a small beaker. Then, the beaker was put into a vacuum oven and heat
for four hours with a temperature of 105°C. The percentage of the
moisture was calculated by using this formula:

mass of wet sample - mass of dried sample

% Moisture: x 100
mass of wet sample

3.2.2 Ash Test

The preparation of trigona honey for physicochemical analysis for

ash was performed by referring to Gebremariam et al., (2013). Firstly, 5.0
gram of trigona honey was weighed and placed into a crucible.
Afterwards, the crucible was placed in the flame of Bunsen burner for a
while until the sample dry and smokeless. After that, the sample was kept
into the preheated furnace for at least 4 hours with a temperature of 600°C.

11
 The sample was taken out from the furnace after 4 hours and then it was
put into a desiccator until the sample can be handle and weight. The ash
content was identified by using this formula:

(mass of ash+crucible)-(mass of empty crucible)

% Ash content: x 100
mass of sample

3.2.3 Ph Test

A method proposed by Sarwar et al., (2014) was used for a

preparation of physicochemical analysis for pH. First of all, 10.0 gram of
pure trigona honey was taken and dilute until 75 ml in a 100 ml of
volumetric flask. The solution was stir until it was mixed homogenously
and the pH meter was inserted into the solution to read the pH.

3.3 Efficacy of Trigona Honey in Accelerating the Healing of Full

Thickness Wound of Skin in Mice.

The sample that was used in this experiment were mice as referred to
Ghaderi et al., (2010). The total of eighteen mice aged six weeks old were
obtained from the Malaysia Research Institute. The mice were grouped into three
parts which were classified into control group, aloe Vera treated group and trigona
honey treated group respectively. Each parts consist of two cages with a
maximum of three mice per cage. As referred to Orasan et al., (2016), the mice
were acclimatized at the experimental room with temperature of 23°C. The food,
in the form of pallet, were given to the mice is the same for each cage and their
amount was 10 pallets for each cage per day. Under general chloroform, the back
of the skin of the mice in the group was incised to 1.5 cm. The control group were
left untreated, whereas the positive control group received daily topical
application of aloe Vera and honey-treated group received daily topical
application of natural trigona honey. The wound on the mice were observed every

12
day for 10 days and the honey and aloe Vera were applied. The length and width
of the wound were measured by using a plastic ruler.

3.4 Flowchart

Collection of materials

Sample preparation

Physicochemical analysis

Efficacy test of Trigona itama honey on mice.

Data Analysis

Figure 3.1: Flowchart of the study

13
 CHAPTER FOUR
RESULTS AND DISCUSSION

4.1 Physicochemical Analysis of Trigona Honey

Table 4.1: Comparison of Physicochemical Analysis

Parameter Average result Standard (MARDI)
Moisture 26.11% 23.5 – 40.5%
Ash 0.3192% <0.5%
pH 3.22 3.70 – 4.00 (common honey)

4.1.1 Moisture Test

The water or moisture content of honey is a measure of its stability

and resistance to fermentation. The standard moisture content
recommended by MARDI is in range 23.5 – 40.5%.

Table 4.2: Moisture Test Data

Mass of Mass of Mass of Mass of Beaker
Beaker Empty Empty Beaker Sample, + Dried
Beaker, (g) + Sample, (g) (g) Sample, (g)
A 25.1395 30.2017 5.0622 28.8843
B 25.2835 30.3333 5.0498 29.0173
C 24.7170 29.7899 5.0729 28.4583
Average 25.0467 30.1083 5.0616 28.7866

mass of empty beaker - (mass of beaker + dried sample)

% Moisture : x100
mass of empty beaker -(mass of beaker + sample)
5.0616 - 3.7399
: x 100
5.0616

14
 : 26.11%
The percentage of moisture of Trigona itama honey in this study is
26.11% which is in standard. The percentage of moisture is obtained from
the calculation above. The average reading of three data were taken. In
general, high amount of water causes the honey to ferment, to spoil and to
lose flavor, with ensuring honey quality loss. Honey moisture content
depends on the environmental conditions and the manipulation from
beekeepers at the harvest period, and it can vary from year to year. High
moisture content could accelerate crystallization in certain types of honey
and increase its water activity to values where certain yeasts could grow
(Gembremariam and Brhane, 2014).

4.1.2 Ash Test

In order to determine the quality of botanical and geographical of

the honey, ash content is one of the criteria. The purpose of ash test is to
determine the amount of minerals that can determine physiochemical
properties of honey, as well as retard the growth of microorganisms (Dairy
Food, 2010).

Table 4.3: Ash Test Data

Mass of Empty Mass of Mass of Crucible +
Beaker
Crucible, (g) Sample, (g) Ash of Sample, (g)
A 34.9418 5.0088 34.9495
B 37.6329 5.0362 37.6415
C 34.5658 5.0861 34.5782
Average 35.7056 5.0437 35.7217

(mass of ash+crucible) - (mass of empty crucible)

% Ash content : x 100
mass of sample
(35.7217) - (35.7056)
: x 100
5.0437

15
 : 0.3192 %

Figure 4.1 Result of Ash Test

The percentage of ash obtained is 0.3192% by using the formula as

above. The average of three data were also taken. All analysis results for
ash contents were within the internationally acceptable range of < 0.6%,
(Gebremariam and Brhane, 2014). However, it is lower than the value that
has been recommended by MARDI which is 0.5%.

4.1.3 pH Test Data

The pH of the trigona honey that were obtained in this study was
on average of 3.22. As stated in International Journal of Sciences, the
standard pH of trigona honey is 3.34 (Usman et al., 2016). The result
obtained is around the standard value. Based on the pH, it is proven that
the honey is acidic. The acidity of honey is due to the presence of organic
acid such as gluconic acid and inorganic ion such as phosphate and
chloride (Hemalatha and Satyanarayana, 2017). This is why the taste of
trigona itama honey is slightly sour and less sweet due to the acidic nature

16
 of honey (Khalil et al. 2012). According to Gebremariam and Brahne
(2014), honey is considered as a buffer which means its pH value will not
change by the addition of small quantity of acid and bases. The buffer
characteristics shown in honey is due to the content of phosphate,
carbonates and other minerals salts. This had been observed in honey
samples and may have attributed to the presence of different acid found in
different floral type (Gebremariam and Brahne, 2014).

4.2 Efficacy of Trigona Honey in Accelerating the Healing of Full

Thickness Wound of Skin in Mice.

In the present work, the trigona honey was tested on mice’s wound and
was comparatively analysed with aloe Vera gel as positive control and negative
control which is the mice was left untreated. Despite some differences, both
trigona honey and aloe Vera gel gave similar results at the end of the analysed
period. The back dermal of mice was cut to 1.5cm to observe the healing of the
wound (Ghaderi et al., 2010).

Table 4.4: Picture of Mice Wound

Day Trigona Honey Positive Control Negative Control

17
3

10

18
Table 4.5: Width and Length of Mice Wound
POSITIVE NEGATIVE
TRIGONA HONEY
DAY CONTROL CONTROL
WIDTH LENGTH WIDTH LENGTH WIDTH LENGTH
1 0.5 1.50 0.50 1.50 0.50 1.50
2 0.5 1.40 0.50 1.50 0.50 1.50
3 0.30 1.30 0.50 1.50 0.50 1.50
4 0.30 1.00 0.40 1.40 0.40 1.50
5 0.20 0.90 0.40 1.40 0.40 1.40
6 0.20 0.90 0.40 1.30 0.40 1.30
7 0.10 0.80 0.30 1.20 0.40 1.20
8 0.10 0.70 0.20 1.00 0.30 1.20
9 0.10 0.60 0.20 0.90 0.30 0.90
10 0.10 0.40 0.20 0.70 0.30 0.90

Width of Wound Against Days

0.6
0.5
Width, (cm)

0.4
0.3
0.2
0.1
0
1 2 3 4 5 6 7 8 9 10
Trigona Honey 0.5 0.5 0.3 0.3 0.2 0.2 0.1 0.1 0.1 0.1
Positive Control 0.5 0.5 0.5 0.4 0.4 0.4 0.3 0.2 0.2 0.2
Negative Control 0.5 0.5 0.5 0.4 0.4 0.4 0.4 0.3 0.3 0.3

19
 Length against Day
1.6
1.4
1.2
Length, (cm)

1
0.8
0.6
0.4
0.2
0
1 2 3 4 5 6 7 8 9 10
Trigona Honey 1.5 1.4 1.3 1 0.9 0.9 0.8 0.7 0.6 0.4
Positive Control 1.5 1.5 1.5 1.4 1.4 1.3 1.2 1 0.9 0.7
Negative Control 1.5 1.5 1.5 1.5 1.4 1.3 1.2 1.2 0.9 0.9

Regarding the healing process, the mice was observed for 10 days. The
reading taken was on average of six mice for each group. For the first two days of
observation, there were no changes observed for the wound length. The healing
process on the third day shows a slight change for the length of the wound that
was tested with trigona honey which was 1.30 cm meanwhile the result of positive
and negative control remains unchanged. On the fourth day, the mice wound
lengths were 1.00 cm and 1.40 cm for trigona honey and positive control
respectively. Meanwhile, there was still no changes for the negative control mice.
However, on the fifth day, the negative control mice showed a positive result. Its
wound length was 1.40 cm while for mice that was treated with trigona honey was
0.90 and positive control were remain unchaged. The observation was continued
to the sixth day. The results showed that wound length of mice for trigona honey
were still 0.90 cm, positive control were 1.30 cm and the negative control mice
were 1.30 cm. However, most of the negative control mice were found dead on
the next day.
The negative control shows the highest death rate. This was probably
because it might be infected with microorganisms that will delay the process of
healing. Microorganisms exist inside all wounds but the majority of them do not

20
infect the wound, and the wound will heal eventually. This situation happens
when the host’s immune system and the bioburden of the wound are in a state of
equilibrium. Bioburden is a condition where an object is contaminated by a
number of bacteria (Korting et al.,2011). The negative control mice that were left
untreated do not receive any treatment during the healing process.
The wound length decreased for both trigona honey and positive control on
the seventh day. For the mice that were treated with trigona honey, the wound
lengths were 0.80 cm and the mice that were treated with aloe Vera gel was 1.20
cm. At the ninth and tenth day after the incision, the length of the wound treated
with trigona honey and aloe Vera gel had shown a positive result.
Trigona honey shows the best result by becoming the fastest to heal the
wound. It might be due to the flavonoids content which is the major constituents
of trigona honey. Flavonoids have a broad spectrum of biological properties, such
as anti-bacterial, anti-viral and anti-inflammatory effects. According to the
chemical structure, flavonoids in trigona honey are classified into flavones,
flavonols, flavanones, flavanonols, chalcones, dihydrochalcones, isoflavones,
isodihydroflavones, flavans, isoflavans and neoflavanoids. Besides that, it
contains terpenoids that exhibit antioxidant, antimicrobial and other biological
activities (Bankova,2011). pH and nutrient content of trigona honey has potential
benefits for prevention and treatment of many kind of diseases as well as in
treating wound(Rahardjo,2016). So, it is very good in treating wound.
Meanwhile, aloe Vera gel was used as positive control because it contains
enzyme such as aliiase, alkaline phosphatase, amylase, bradykinase,
carboxypeptidase, catalase, cellulose, lipase and peroxidase. Bradykinase helps to
reduce excessive inflammation when applied to skin topically. Besides, aloe Vera
contains anthraquinones that provides 12 anthraquinones, which are phenolic
compounds traditionally known as laxatives. Aloin andemodin act as analgesics,
anti-bacterials and antivirals. It also contains auxins and gibberellins hormones
that help in wound healing and have anti-inflammatory action. Moreover, fatty
acid in the aloe Vera provides steroids; cholesterol, β-sisosterol and lupeol. All
these have anti-inflammatory action and lupeol also possesses antiseptic and
analgesic properties (Surjeshe et al.,2010).

21
 CHAPTER FIVE
CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

In conclusion, the purity of Trigona itama honey was determined through

physicochemical test. Physicochemical test included the moisture, ash and pH
test. For the moisture test, the percentage of moisture for Trigona itama honey is
26.11%. while, the percentage of ash obtained is 0.3192% and for pH test, the pH
is 3.22. Based on the results obtained, it can be concluded that the honey used is
pure.
In addition, it is important to take a serious note concerning wound care
and healing agents due to the nature of the wound healing process which is highly
complex and highly exposed to external infection. Based on the study conducted,
it is proven that stingless bee honey can be used as wound healing agent.
Historically, the application of honey to the wound has been used since ancient
times; therefore, it is essential to understand its pharmacological action towards
the physiology of the wounded skin in order to optimise the healing rate. Stingless
bee honey has a lot of similarities with other honey in terms of its bioactive
components, but the efficacy of the components has yet to be discovered.

5.2 RECOMMENDATION

After conducting this study, to ensure a better result, it is

recommended that the duration of the observation should be

increased by another week. This was in order to obtain a better

result and also might be able to make further research related to

scar fader and hair growth. To further explore the benefits of

Trigona honey, therefore, a more systematic research is needed to

22
provide comprehensive scientific evidence for its used as well as to

clarify any doubt and false acclaim. The potent biological activities

of stingless bee honey may create a new therapeutic choice from

the current honey and represent an interesting advance in the

search for promising applications in the pharmaceutical industry

for the wound healing, scar fader and hair growth area. The

depilation method is the deliberate removal of body hair commonly

by shave. This experiment of scar fader can be further by

increasing the duration of the observation while the hair growth

experiment will be carry out by using some specific areas on body

of the mice to shave .As referred to Orasan et al., (2016), the mice

must be acclimatize at the experimental room with temperature of

23°C as the temperature influence the physical factors for the hair

regrowth process. Then, maximum of 2 mice per cage are place to

avoid licking with access to standard laboratory diet and water.

The mice will be observed day by day until the hair growth.

23
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APPENDIX A
Incision of Mice

29
 AUTHOR’S PROFILE

Aqilah Binti Anuar currently studying her diploma in Science at the

Faculty of Applied Sciences, Univerisiti Teknologi MARA.

Lyana Fasihah Binti Ahmad Sobri currently studying her diploma in

Science at the Faculty of Applied Sciences, Univerisiti Teknologi MARA.

30
 Nor Syafikah binti Mohd Nasir currently studying her diploma in Science
at the Faculty of Applied Sciences, Univerisiti Teknologi MARA.

Nur Atiqah Farzana binti Zaini currently studying her diploma in Science
at the Faculty of Applied Sciences, Univerisiti Teknologi MARA.

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 Nurul Najwa binti Mohamed Redzuan currently studying her diploma in
Science at the Faculty of Applied Sciences, Univerisiti Teknologi MARA.

32