Upload
mohd-safuan-bin-abdul-razak
View
141
Download
2
Embed Size (px)
DESCRIPTION
CHAPTER 1INTRODUCTIONNatural products have become sources of medication to many diseases. Thus, it is most evidence in many countries where it have an importance myth of traditional knowledge or practiced in use a source of natural products such as herbs, tropical plant, local plant and all biological diversity for treatment benefit therein. Some of these practices have also been commercialized and bringing attention internationally. It mean, the study of natural products continue to play an
Citation preview
1
CHAPTER 1
INTRODUCTION
Natural products have become sources of medication to many diseases. Thus,
it is most evidence in many countries where it have an importance myth of
traditional knowledge or practiced in use a source of natural products such as herbs,
tropical plant, local plant and all biological diversity for treatment benefit therein.
Some of these practices have also been commercialized and bringing attention
internationally. It mean, the study of natural products continue to play an important
role in the discovery and development of new pharmaceutical, as starting materials to
produce synthetic drugs or as lead compound which is discover a hidden chemical
diversity designed in biodiversity. Natural products implicate many advantages; the
development in pharmaceutical industry technology has a raise and become more
perfect in holistically purpose of producing a drug, and declares that it may have
many respect and complimentary effect such as used of synthetic drug which is
implicate the stimulation of alleviate to human health problem.
Pain involves an incredibly complicated many of physiochemical responses
leading to the sensitivity of an unpleasant sensation arising from actual or potential
tissue damage. Pain can be classified as physiologic, which refers to the body‟s
protective mechanism to avoid tissue injury, or pathologic, which arises from tissue
injury and inflammation or damage to a portion of the nervous system. Pathologic
pain can be further divided into categories such as nociceptive (peripheral tissue
injury), neuropathic (damage to peripheral nerves or spinal cord), visceral
(stimulation of pain receptors in the thoracic or abdominal viscera), and somatic
(injury to tissues other than viscera, such as bones, joints, muscles and skin). It can
also be defined temporally as acute (arising from a sudden stimulus such as surgery
or trauma) or chronic (persisting beyond the time normally associated with tissue
injury).
2
The pain medicine treatment, consider of two types of drug which is the non-
opioid and opioid drugs. The non-opioid drugs such as paracetamol and aspirin (and
other NSAIDs), are particularly suitable for pain in musculoskeletal conditions,
whereas the opioid analgesics such as Morphine and heroin are more suitable for
moderate to severe pain, particularly of visceral origin (Harvey, 2007). In this study,
the Aspirin was used as a control to offer benchmark activities of comparison
between the natural products selected. Aspirin is indicated for headache, transient
musculoskeletal pain, dysmenorrhoea, pyrexia (Harvey, 2007) and addition the used
of aspirin also benefit to many chronic diseases, however aspirin produces many
side effects and the major one is gastric irritation such as stomach pain, heartburn,
nausea, vomiting and gastrointestinal ulceration (Charles, 2008).
The development of new drugs via natural sources has become important as
well as the need to find new drugs to treat pain and inflammatory disorder with fewer
side effects and have high therapeutics value. This fungus was selected because it has
many traditional claims such as for the treatment of flu, cough, asthma and cancer.
The sclerotium of Lignosus species (“cendawan susu harimau” or “susu rimau” in
Malay language or tiger‟s milk fungus in English) was used to treat consumption and
coughs (Change and Lee, 2004). This mushroom is the most popular wild
mushrooms used for medicinal purposes among the urban population, namely L.
rhinocerus (Chang and Lee, 2005). The used of mushroom to enhance a potential in
pharmacology, it might be surprise and not many scientific articles published on this
mushroom extract and this might be prove to be the single most successful strategy
for the discovery of new drugs. This study is and affordable to increase ability of
fungus function and from phytochemical screening of L. rhinocerus sclerotium
revealed the presence of flavonoid and saponin, which has a potential antinociceptive
and anti-inflammatory property.
This study has completed with a difference methodology which manipulate
the soak method to boiling method and obtain supernatant of hot aqueous extract.
The purpose of change this methodology is due to the influence from soak method,
where mushroom is easily implicating contamination, highly contain of
microorganism, produce a bad smell and in case of sample were rejected by the
system or machine that need hygiene environment. So, the soak method is not a good
way for further investigation by used mushroom as raw material and soak method is
3
not encourage further pharmacology studies. Therefore, boiling method is among the
one of solution which suggests of avoid contamination and it might help to promote
auxiliary study. Although the boiling method was used to assist study so it can be
mean as cooking too, which the heat effect may influence microorganism freely
therein and help of digestive system may easy to function with no side effect.
However, if the supernatant is over expose with environment it will change to
contamination and cause poisoning, because of influence microorganism. Hot
aqueous (supernatant) need highly of care and it should store inside the chiller or
refrigerator, for protected from invade microorganism and only regulate used it when
necessary. More, many of scientific articles published state that the hot aqueous
stimulate a response of some biodiversity activities in pre-clinical test.
Objectives of the study
The specific objectives of this research are:
1. To determine the antinociceptive activity of different concentrations of
Aqueous Extract of Lignosus rhinocerus (AELR) using different
antinociceptive assays.
2. To compare the antinociceptive activities of AELR with Aspirin a standard
drug.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Pain
Pain can be classified as physiologic, which refers to the body‟s protective
mechanism to avoid tissue injury, or pathologic, which arises from tissue injury and
inflammation or damage to a portion of the nervous system. Pathologic pain can be
further divided into categories such as nociceptive (peripheral tissue injury),
neuropathic (damage to peripheral nerves or spinal cord), visceral (stimulation of
pain receptors in the thoracic or abdominal viscera), and somatic (injury to tissues
other than viscera, such as bones, joints, muscles and skin). It can also be defined
temporally as acute (arising from a sudden stimulus such as surgery or trauma) or
chronic (persisting beyond the time normally associated with tissue injury).
Antinociception refers to reduction in pain sensitivity produced within
neurons when an endorphin or similar opium-containing substance opioid combines
with a receptor (Ammirati & Seidl, 2008).
Nociception can be defined as a condition describing nerve fibers, endings, or
pathways that are concerned with the condition of pain (Lindsey, 2003). Nociceptors
are pseudounipolar neurons located in the dorsal root ganglia, which have a
peripheral branch that innervates the somatic and visceral structures, and a central
branch ending in the spinal cord dorsal horn. The main function is to transduction of
noxious stimuli into nerve impulses, and classified according to their stimuli
sensitivity, neurochemical properties, type of peripheral processes, and etc. Central
endings of nociceptors establish synapses with spinal second order neurons, which
can be nociceptive specific or wide dynamic range. Local interneurons and
descending fibres arising from supraspinal structures modulate the nociceptive
information by acting on those neurons or on the central endings of nociceptors.
Projection neurons of the dorsal horn convey the nociceptive information to
5
supraspinal structures, mainly in the brainstem and thalamus. Classically, two main
spinofugal tracts are described that the lateral spino-thalamic tract, which ends
mainly at the lateral nuclei of the thalamus and is related with the sensory
discriminative components of pain, and the medial spinothalamic tract, which ends in
the medial and intralaminar thalamic nuclei and is associated with the affective and
cognitive components of pain (Luise, 2008). Nociceptive information is then relayed
from the lateral thalamus to the somatosensory cortex and from the medial thalamus
to other areas, like the pre-frontal, motor or cingulate cortices. Several brainstem
nuclei also receive noxious input from the spinal cord are involved in the descending
modulation of nociceptive transmission or integration of cardiovascular and pain
responses. Other brainstem nuclei transmit the nociceptive information to the
amygdala and other areas of the limbic system involved in the motivational/affective
components of pain. Nociceptive stimuli activate a widespread neuronal matrix in the
central nervous system, which is responsible for the complex perception of pain.
They may be Aa-fibers, Ad-fibers or C-fibers (Luise, 2008).
2.2. Lignosus rhinocerus.
2.2.1 Description of Lignosus Rhinocerus
According to Malaysian legendry story, L. rhinocerus was believed that this
fungus will only grow on the spillage of tiger milk from the mother tiger during the
breast feeding. The milk that spilled on the ground will produce this fungus
(Anonymous, 2002). It was also believed that this fungus was difficult to find and the
Malaysian believed that only the aborigines (or „orang asli‟) are truly knew about this
fungus habitat and the way to find.
In Malaysia, this species is hard to find. Although this species is not yet extinction
this group of polypores which namely from word polypore mean many pore, it seem
to our mind this species can spread grow well, however, it considered a threatened
species (phase I)-(Anonymous, 2007). The sclerotium L. rhinocerus is sliced and
boiled with other herbs such as “tongkat ali” (Eurycom alongifolia) root, and the
resulting decoction is drunk (Chang & Lee, 2004.). Previously, this species was not
well-known but, the used this species for research now days is being increase.
6
Chinese researchers who had worked on this species for food and nutritional study
declare that the fungus sclerotium contained good dietary fibers (Wong & Cheung,
2005). In addition, (Zainoor et al., 2009) from Faculty of Pharmacy, Liverpool John
Moores University, study about an evaluation of the chemotaxonomy of L.
rhinocerus and reveal the difference distribution of ergot from the young L.
rhinoceus and old L. rhinoceus, this show that exploration of this species toward
enhance the ability of this fungus is being in study. Amazingly, In Malaysia the study
of L. rhinocerus has been ornamental on molecular biology. The Malaysian
researcher who had worked with L. rhinocerus avowed that it successfully translate
the phylogenetic relationships of polyporus and morphologically allied genera by
comparing internal transcribed spacer 1, 2, and 5.8S rDNA sequences (Tan et al.,
2009). In traditional medicine, the L. rhinocerus devoted many traditional claims or
potentially to cure diseases such as asthma, breast cancer, fever, inflammation, cough
and bronchitis. L. rhinocerus become well-known in Malaysia, only after the 4th
Malaysia Prime Minister; Tun Dr. Mahathir Mohammad cured from chronic cough
(Anonymous, 2002).
A species of Lignosus are unusual for polypores because in each case the
fruiting body consists of a cap on a central stem (which occurs in few polypore
genera). L. rhinocerus grows from a sclerotium in the ground, which is even rarer
rather than from wood as is the case with most polypores.
A sclerotium is a mass of compacted hyphae, commonly more or less
spherical in shape and often with a dark, tough outer skin. Sclerotia are produced by
many fungi and range in size from under a millimetre to over 20 centimetres in
diameter. Sclerotia are better able to resist drought and temperature fluctuations and,
therefore, are much longer lived than mycelia. More, its also store lipid,
carbohydrates, and protein until soil conditions are favorable enough for germination
and then form either a new mycelium or a fruiting body (Morton, 2006). A
sclerotium is a resting structure that allows a fungus to sit out the hard times,
analogous but clearly different from a plant bulb or corm. The outer skin protects the
internal hyphal mass from drying out. When conditions improve, the sclerotium may
produce a new mycelium or fruiting bodies (Heino, 2005.). Another species of
7
Lignosus has been found in Papua New Guinea and Africa, and the remaining two
species are confined to Africa.
L. rhinocerus is under the order Polypores and Polypores are commonly
known as conk or bracket fungi. The term Polypore means many tiny holes. This is a
common feature of this solder. More, they nearly all grow on wood, such as trees,
logs, stumps or buried wood and this Polypores all have many tiny holes or pore
under the pileus. They are a non-agaric group of Basidiomycota characterized by
basidiocarps (fruit bodies) with period or other well-develop hymenophore. Many
have leathery, corky, to woody basidiocarps and are more persistent then agaric fungi
field. Polypores usually occur as saprobes on logs, branches and other woody
substrata, but some species are ectomycorrhizal fungi. Polypores were placed in
polyporus and Daedalea (Fries, 1821), in tribe Polypori (Quelet, 1886), and then in
family Polyporaceae. In 1964 scientist suggest that tribe of Polypori are
Polyporaceae, Hymenochaetaceae, Ganodrmataceae, Bondarzewiaceae, and
Fistulinacea (Donk, 1964). Polypores are often difficult to determine even to the
genera. Many of the generic concepts have been repeatedly emended, but there are
still several genera with overlapping of confusing concepts. For time being, the
generic concepts and the key to genera given by (Ryvarden, 1991) the most
comprehensive and widely used. However, in certain cases the polypores are among
the most common, widespread, easily identifiable group of wild mushrooms, with
some edible sepsis and no poisonous one a great group for new muchroom to study.
Hyphal morphology is important in the characterization of polypore genera
(Ryvarden, 1991) there three types of hyphae occur in polypores; generative hyphae,
skeletal hyphae, and binding hyphae. Some poroid genera were scattered in
Coniphoraceae, Corticiaceae, and Thealephoraceae. Recent pyogenetic study
reveals that the Polyporaceae, as used by (Donk, 1964) is phylophyletic, including a
number of well-supported clades and genera with ambiloguous phylogenetic status
(Hibbett & Thorn, 2001). Recently (Hibbett,2006) suggested that the Polyporales as
defined by (Kirk, et al. ,2001);
8
Table 2.1 A classified of Polypores into following order and families (Hattori et al.
,2007)
2.2.2 Morphology, ecology and reproduction of Lignosus rhinocerus
POLYPORALES: Albatrellaceae, Corticiaceae, Fomitopsidaceae,
Ganodermataceae, Gloeophyllaceae, grammothelaceae, Hapalopilaceae,
Meripilaceae, Meruliaaceae, Polyporaceae, Sistotremataceae, and
Steccherinaceae
AGARICALES: Fistulinaceae
BOLETALES: Coniophoraceae
HYMENOCHAETALES: Hymenochaetaceae, and Schizoporaceae;
RUSSULALES: Bondaezewiaceae, and Echinodontiaceae
THELEPHHORALES: Bankeraceae
THEMELLALES: Aporpiaceae. With the following additional order: Atheliales,
Corticiales, Gloeophyllales, and Trechisporales.
9
Figure 2.2 Actual picture of local polypore (Lignosus rhinocerus) flora of Malaysia.
Figure 2.3 A schematic diagram of preliminary polypore (Lignosus rhinocerus) flora
of East Affrica (Ryvarden; (Heino, 2005))
Figure 2.3 shows a picture of tropical polypore of L. rhinocerus. The dotted
line represents the division of the upper level and ground level. The upper level
contains parts of the mushroom known as cap (pileus), and stipetate. One of the
surprising facts is that L. rhinocerus is the most popular wild muchroom used for
medicianal purpose among the urban population, was not widely used as such among
the indigenous people of Peninsular Malaysia. Rather it was more popularly used to
stave hunger especially during the long journey such in hunting and in a cultural
association to ensure bountiful rice harvests (Chang & Lee, 2005).
L. rhinocerus is a fungus or a mushroom which is locally knows as Kulat/
Cendawan Susu Harimau by Malaysians and in English this fungus recognized as
tiger‟s milk fungus. This species is commonly found in Malaysian tropical forest,
where it grows under the soil. L. rhinocerus is a tropical polypore genus and have
four synonym species which is namely as L. rhinocerus, L. sacer, L. goetzii and L.
dimiticus. In Malaysia, the L. rhinocerus is frequently found in Gerik, Perak. In
addition, this species will be found at the forest reserve such as Cameron Highland,
Endau-Rompin, Taman Negara, Pahang and Gerik, Perak-Kelantan, Malaysia. Other
than Malaysia, it can be found in countries such as Papua New Guinea, Borneo,
Philippines, Indonesia, Australia, Sri Lanka and Vanuatu. In the ground level show
the morphology of root and the fruiting body (Basidiocarps) that has grown out from
Caps (pileus)
Stipetate
Root
Sclerotium (Sclerotial stroma)
10
a buried sclerotium. Actually, sclerotium (is also known as sclerotial stroma) where
more or less characteristic form and strictly hypal structure. Hypal elements if all
embedded in the medullar stroma, their presence is merely incidental, and these do
not contribute to the food supply of stroma (Sharma, 2005).
The fungal body may appear simple structure, it is highly adaptable
genetically and physiologically. Not only does a fungus grow locally, but it also
produces prolific spores in a wide range of morphologies, which can be dispersed
long distances on air currents. With this combination of traits, fungi are able to
colonize almost any habitat or organic substrate. As pathogen, they cause diseases in
animal, plant, and other microbes. As beneficial symbionts, they help plant and
animals to grow better or survive longer. They are more abundant than any other
group of microorganism in soil, accumulating between 500 and 5000kg of wet
biomass per hectare (Metting, 1993). L. rhinocerus is placed under Phylum
Basidiomycota, so the probability of their reproduction is asexual. Usually it varies
with many forming chlamydospores or mycelial structures (sclerotia, rhizomorphs,
mycelial strands) and some forming conidia.
2.2.3 Phylogeny and other names of Lignosus rhinocerus.
Table 2.4 Phylogeny of Lignosus Rhinocerus. (Bisby et al. , 2007)
Domain: Eukaryota – (Whittaker & Margulis, 1978)
Kingdom: Fungi – (T.l. Jahn and F.f. Jahn, 1949; R.t. Moore, 1980)
Subkingdom: Dikarya – (D.s. Hibbett Et Al., 2007)
Phylum: Basidiomycota – (H.c. Bold, 1957; R.t. Moore, 1980)
Subphylum: Agaricomycotaina – (Dowell, 2001)
Class: Basidiomycetes – (Dowell, 2001)
Subclass: Agaricomycetidae
Order: Polyporales
Family: Polyporaceae – (Corda, 1839)
Genus: Lignosus
Species: Lignosus Rhinocerus.
11
Table 2.5 Evolutionary names of Lignosus rhinocerus. (Bisby et al., 2007)
2.3 Flavonoids and Saponins
There are not many scientific articles published by used mushroom extract in
pharmacology; however a few potent medicine agents reported such cholesterol
lowering drug (Mevinic acid), immunosuppressive drug, and the drug obtain from
the seclerotia.(Sharma, 2005)
A single plant may contain several flavonoids with distribution being specific
to various parts of the plant. Flavonoids play different roles in each part of the plant.
For instance, because of their attractive colors, flavonols, flavones, and
anthocyanidins are helpful in signaling pollinating insects. Catechins and other
flavonols have astringent qualities that protect the plant by keeping other insects
away. Some flavonoids have UV-absorbing properties and protect the plant from
harmful UV radiation from the sun. (Orrego et al., 1978)
Historically, flavonoids have been described for treating diabetes mellitus,
allergy, cancer, viral infections, headache, stomach and duodenal ulcer, liver
pathology, treatment of thrombopenia (blood coagulation), antinociceptive, and anti-
inflammation. Influence of flavonoid may contribute a prediction of antinociceptive
and anti-inflamatory. Saffron stigma and petal extracts exhibited antinociceptive
effects in chemical pain test as well as acute and/or chronic anti-inflammatory
activity and these effects might be due to their content of flavonoids, tannins,
1. Fomes rhinocerus (Cooke, 1879)
2. Polyporus rhinocerus (Cooke, 1879)
3. Sindalma rhinocerus (Cooke) (Kuntze, 1898)
4. Polyporus sacer var. rhinocerotis (Cooke) (Lloyd,1921)
5. Polystictus rhinocerus (Cooke) (Boedijin, 1940)
6. Microporus rhinocerus (Cooke) (Imazeki, 1952)
7. Lignosus rhinocerus (Cooke) (Ryvarden,1972)
12
anthocyanins, alkaloids, and saponins (Hosseinzadeh and Yiounesi, 2002).They can
bind to enzymes, hormone carriers, and DNA; chelate metal ions such as iron,
copper, zinc, and manganese; catalyze electron transport; and scavenge free radicals.
(Maddrey et al., 1978) Most researchers conclude that the pharmacological effect of
flavonoids is due to their inhibition of certain enzymes, their metal chelating
abilities, and to their antioxidant activity. This finding is expected to have
antinociceptive activity from L. rhinocerus which generally contains the flavonoid
and Saponin. The ability of falvonoid compounds to exhibit antinociceptive activity
(Su et al., 2003).
Saponins are waxy, soap-like substances that exhibit a wide range of
properties and therefore are regarded as important biological compounds and
bioactive compounds known to have anti-inflammatory. Some saponins found in
quinoa act as a natural pesticide for the plant by producing bitter compounds that
deter insects and birds (Reynolds & Derrick, 2009). Saponins are a group of
amorphous colloidal glycodides which is widely distributed in the higher plants. It
has ability to form lasting foam when shaking in aqueous solution. They are
excellent emulsifying agents (modify surface tension). Formerly used as detergents
to replace soap (e.g., quillaia). Saponins are colorless and optical active. They form
colloidal solution with water and are soluble in alcohol and dilute alcohols. Saponins
have haemolytic properties, they precipitate the cholesterol and lethisins that exist in
the membranes of the red blood cells and thus hemoglobin is liberated. So, saponins
are extremely toxic when injected into the blood stream. However, they are not
harmful when taken orally. Saponins are difficult to purify. However, they
precipitated from solutions containing them by the addition of a solution of the
sterol, filtering off the insoluble sterol-saponin compound and boiling it with toluene
which resolves the compound again into sterol (which is soluble in toluene) and
saponin (which is insoluble in toluene). Saponins are generally known to be strong
antibiotics. Saponin is suggested to act as an anti-microbial and anti-feedant.
13
CHAPTER 3
MATERIALS AND METHODS
3.1 Experimental Animal
The present experiments were conducted by using experimental animal male
Imprinting Control Region (ICR) mice weighing 25–30 g (5–7 weeks old) and
Sprague-Dawley male rats weighing 200-250g (5 to 7 weeks old). The animals were
obtained from the Veterinary Animal Unit, Faculty of Pharmacy, Universiti
Teknologi Mara (UiTM), Malaysia. The animals were kept at room temperature 27 ±
2 °C with 70–80% humidity and 12-hour light/darkness cycle in the Animal Holding
Unit for at least 48 hours before use. Food and water were supplied ad libitum up to
the beginning of the experiments. At all times the mice were handled in accordance
with current UiTM guidelines for the care of laboratory animals and the ethical
guidelines for investigations of experimental pain in conscious animals. All
experiments were conducted between 0930 and 1830 hours to minimize the effects of
environmental changes.
3.2 Preparation of Drugs
100 mg/kg acetylsalicylic acid (ASA; Bayer, Singapore), used for the purpose
of comparison, were prepared by dissolving them in water (H2O). Other chemicals
that were being used were acetic acid 0.6% and formalin 5%.
3.3 Mushroom Material
Lignosus rhinocerus or Kulat Susu Harimau was collected at Termeloh,
Pahang, Malaysia in early January 2008.
14
3.4 Preparation of Extract
Preparation of aqueous extract of L. rhinocerus (AELR) was described as
follows. The sclerotium of L. rhinocerus were washed and rinsed with water to
remove all the unwanted particles (dirty) and then air-dried for 3–4 weeks at room
temperature (27 ± 2 °C). The dried sclerotium was then grind into small particles,
weighed (250 g) and incubated at 35-40°C for half an hour. The sample then treated
with distilled water (dH2O) in the ratio of 1: 20 (w/v). This mixture consisted of 250
g of the small particles of L. rhinocerus and 5000 ml of distilled water. This mixture
was then boil until the mixture volume was reached 1000 ml and the supernatant was
collected and filtered using Whatman No. 1 filter paper while the remaining
mushroom residue throw in garbage.
The supernatant obtained was then subjected to freeze-drying for three days,
yielding a crude dried AELR. Based on the amount of crude dried AELR obtained, it
was estimated in 100mg/kg, 300 mg/kg, 500 mg/kg and 1000 mg/kg concentrations
of AELR.
15
3.5 Antinociceptive assays
3.5.1 Abdominal Constriction Test
The peripherally-mediated inflammatory-associated nociceptive assay was
carried out according to the method described by (Zakaria et al., 2006). Groups of 6
mice (n=6) were used for controls and tests. Briefly, the animals were injected
subcutaneously (sc) with dH2O (distilled water), ASA (100mg/kg), or AELR
(100mg/kg, 300mg/kg, 500mg/kg, and 1000mg/kg) followed by intraperitoneal
administration of 0.6% acetic acid (J.T. Baker, USA) 30 min later. The mice were
placed individually into glass beakers and 5min were allowed to elapse. The number
of abdominal constriction produced in this animal was counted for 25 min. The
abdominal constriction resulting from the injection of acetic acid consists of a
contraction of the abdominal region together with a stretching of the hind limbs
(Correa et al., 2006). The number of abdominal constrictions will be counted
cumulatively over the period of 25 min, commencing 5 min following the acetic acid
administration.
Timeframe Review
Figure 3.1 Timeframe review of abdominal constriction test\
Antinociceptive activity will be indicated by the reduction in the mean of the
number of abdominal constrictions in the test groups compared to the control group,
was calculated as the percentage inhibition of abdominal constrictions (percentage of
inhibitory level) using the following formula:
0 min 30 min 35 min 60 min
Extract, sc 0.6% acetic acid, at
Mice peritoneal cavity Constriction administration
16
Mean of (control – test group)/control group x 100%
3.5.2 Formalin test
The formalin test method described by Hunskaar & Hole (1997) was used but
with little alteration. Each rat was injected with 50 µl of 5% formalin to induce pain
in the sub planar region of the left hind paw. Rats were given dH2O orally, ASA (100
mg/kg), AELR concentrations (100mg/kg, 300mg/kg, 500mg/kg and 1000mg/kg) 30
minutes before formalin injection. The rats were individually placed in a translucent
Plexiglass cage inspection chamber. The indicator of pain was measured by licking
and biting of the injected paw during successive period of 5 minutes and continuous
observation until the duration of 30 minutes (Eddy & Leimbach, 1953). The early
phase of nociception, which was measured between 0 to 5 minutes, indicated a
neurogenic type of pain response whereas the late phase of nociception was
measured between 15 to 30 minutes after formalin injection which signifies an
inflammatory type of pain response. The indicator of pain was measured which were
licking and biting of the injected paw during successive period at phase 1 and phase
2.
Timeframe Review
Figure 3.2 Timeframe review of formalin test
0 min 30 min 35 min 45min 60min
1
st phase 2
nd phase
Extract, sc Injecting 25ul
of 25% formalin Rest
in the subplantar
region of the
left hind paw
17
3.5.3 Hot-Plate Test
The centrally-mediated inflammatory-associated nociceptive assay was
carried out according to the method described by (Zakaria et al., 2006). This was
based on the 50°C hot-plate test by (Wilson et al., 2003). The temperature of the
metal plate (Model 7280, Ugo Basile, Italy) will be maintained at 50 ± 0.2°C. Mice
was placed onto the Plexiglas walls on the heated surface to constrain their
locomotion on the plate, and latency to a discomfort reaction (licking of the paws or
jumping) will be recorded at 0, 1, 2, 3, 4 and 5 hrs following sc administration of the
test agents (AELR or Aspirin).
Timeframe Review
Figure 3.3 Timeframe review of Hot-Plate test
The cut-off time of 20 s were chosen to indicate complete analgesia and to
avoid tissue injury. The duration of the latency time to a discomfort of the treated
groups were compared with that of the control groups. One control group of animals
was treated with dH2O while another control group were treated with aspirin (100
mg/kg; sc) which served as a reference drug.
-ve min 0 min 30 min 1h 2h 3h 4h 5h
Extract, sc Latency to a discomfort reaction (liking of
Screening paws or jumping)
For
Selection
mice
5-10s
18
3.6 Statistical Analysis
The results are presented as mean ± standard error of mean (SEM). The one-
way ANOVA test with Dunnett 2-sided post-hoc test was used to analyze and
compare the data obtained for the abdominal constriction test, formalin test, and hot
plate test. The p value of p < 0.05 was set as the limit of significance.
19
CHAPTER 4
RESULTS AND DISCUSSIONS
The antinociceptive and anti-inflammatory properties of AELR were assessed
by using three animal tests which included the abdominal constriction test, formalin
test and hot plate test. The three antinociceptive tests were basically an assessment of
chemical and thermal mediating nociceptive test. The present results demonstrated
the antinociceptive and anti inflammatory properties of AELR when tested on
various animal models. Generally, the results showed that chemically induced
nociceptive pain was inhibited by AELR and this was seen by where the number of
abdominal constriction was reduced by inhibiting or reversing the acetic acid induced
abdominal constriction test. Antinociceptive pain property was also seen in
thermally-induced pain as the latency to discomfort in hot plate test. It was also used
to study and measure the complex reaction to a thermal and non inflammatory
induced nociception effects. Formalin test, demonstrate of inhibition by formalin at
early phase by drugs which acted mainly at central sites, direct activation of
nociceptors by chemical and this suggest that AELR may also have the same
property to inhibit central nociceptive mechanism. The AELR also demonstrated
inhibition effect in the late phase that attributed mainly to the development of an
inflammatory reaction at the site of injection and increased synaptic transmission in
the spinal cord.
The use of aspirin in abdominal constriction was aimed to assess the
peripheral antinociceptive effects of AELR whereas in formalin test it was used for
the assessment towards anti-inflammatory properties. Drug acting centrally, such as
morphine, produced antinociception in both types of assay (Sulaiman et al., 2004)
while drug acting peripherally, such aspirin and indomethacin, produced
20
antinociceptive effect only in the abdominal constriction test ( Seigmund et al.,1957;
Hendershot & Forsaith, 1959)
4.1 Abdominal Constriction Test
The Abdominal constriction test was used to investigate the peripheral
antinociceptive mechanism. Using the acetic acid-induced writhing reaction in mice,
this material has long been used as a screening tool for the assessment of analgesic or
anti-inflammatory properties of new agents, and was described as a typical model for
visceral inflammatory pain (Tjolsen & Hole, 1997). The abdominal constriction test
is generally accepted as the assay for elucidating peripheral antinociceptive (Mat Jais
et al., 1997). It was preferred because of its sensitivity to detect antinociceptive
property was very high where it can even detect the antinociceptive activity that is
not detectable by tail flick assay (Collier et al., 1969). The outcome of this test can
be viewed as the number of abdominal constriction or in the form of percentage of
inhibition. A decrease in the number of abdominal constrictions reflects the
antinociceptive effect, standard drug or extracts being treated.
Table 4.1 Antinociceptive effect of Lignosus rhinocerus assessed by the abdominal
constriction test
Treatment
mg/kg
Mice Number of
Constriction
% Inhibition
dH2O 6 39.50 ± 2.35b
-
100 ASA 6 16.00 ± 1.55a 59.49
100 AELR 6 22.83 ± 2.48ab
42.20
300 AELR 6 18.83 ±2.64a 50.33
500 AELR
1000 AELR
6
6
10.50 ± 3.51ab
28.17 ± 4.31ab
73.42
28.68
a Data differ significantly (p<0.05) when compared with the dH2O-treated group
b Data differ significantly (p<0.05) when compared with the 100mg/kg ASA-treated
group
21
The results shown in Table 4.1 represent the ability of AELR to reduce the
number of abdominal constriction in mice. The AELR demonstrated concentration-
independent antinociceptive activity. From the concentration 100mg/kg until
1000mg/kg, there is an increase the percentage of inhibition. Interestingly, the extract
shows activities that effect comparable and significant to aspirin, a standard drug for
pain relief. At 100mg/kg it show reduce or less effective of antinociceptive activities
but it still in significant. On fact the concentrations 300mg/kg is effective as aspirin
while 500mg/kg is significantly more effective than aspirin. However, increasing the
concentration further to 1000mg/kg does not increase antinociceptive activities but it
less effective. The highest concentration may influence total lost of antinociceptive
.This kind of dose-response relationship is in line with report by (Tripathi, 1994) that
the presence of high concentrations of its active principle can some lead to reduction
of drug effectiveness. This type of relationship also indicates that the concentrations
used in this test have to be within the therapeutic window of AELR in which certain
drug exert their maximum curative effect (Tripathi, 1994). According to Katzung
(1995), the presence of higher concentration of respective extract bioactive
compound can also result in deactivation of the antinociceptive-inducing receptor
within the peritoneal cavity, which lead to the lost in antinociceptive activity of the
AELR at the highest concentration used ( Zakaria et al., 2006).
The results obtained the effect of AELR in inhibiting the abdominal
constriction was concentration independent. Interestingly, it showed significant
(p<0.05) antinociceptive activity with all AELR concentrations; 100mg/kg,
300mg/kg, 500mg/kg and 1000mg/kg of AELR with percent inhibition of 42.20%,
50.33%, 73.42% and 28.68%. Whereas all concentrations AELR caused significant
to 100mg/kg ASA (p>0.05) change in the number of abdominal constriction test.
This can indicate that those concentrations succeed to inhibit or reverse the acetic
acid induced abdominal constriction test. The analgesia activity observed using this
assay involved, at least in part, the local peritoneal cavity. Furthermore, the
expression of peripheral opioid receptor due to inflammation. Figure 4.1 constructing
a form of bar chart to show a view of antinociceptive activities by the number of
constriction.
22
Fig
ure
4.2
Anti
noci
cepti
ve
effe
cts
of
Lig
nosu
s rh
inoce
rus
asse
ssed
by t
he
abdom
inal
const
rict
ion t
est
23
4.2 Formalin Test
Formalin test was recognized as the most used model of pain which yielded
better results compared to other test that based on thermal or mechanical stimuli
(Tjolsen et al., 1992). It also has been regarded as one of the pain model that showed
better representation of clinical pain. Formalin test consisted of a biphasic behavior
reaction which are consisting of the early phase and late phase reaction. The early
phase was the neurogenic phase that lasted for 5 minutes following the injection of
formalin while the late phase was the inflammatory phase that lasted for 15 to 30
minutes after the injection. The first phase resulted from the direct stimulation of
nociceptors which were the A-fibers and C-fibers responsible for the signal
transmission towards the dorsal horn. The late phase referred to as a tonic response in
which inflammatory processes were involved and neurons in the dorsal horns of
spinal cord were triggered.
Table 4.3 Antinociceptive effect of Lignosus rhinocerus assessed by the formalin test
Treatment
mg/kg
Mice Early Phase, s Late Phase, s
dH2O 6 49.30 ± 4.42b
60.44 ± 10.16 b
100 ASA 6 41.94 ± 10.19a 15.7 ± 17.54
a
100 AELR 6 45.73 ± 5.29 56.51± 8.24
a
300 AELR 6 36.34 ± 6.25 39.62 ± 11.00
ab
500 AELR
1000 AELR
6
6
27.91 ± 14.11ab
42.68 ± 9.99
36.62 ± 6.83ab
14.47 ± 11.75b
a Data differ significantly (p<0.05) when compared with the dH2O-treated group
b Data differ significantly (p<0.05) when compared with the 100mg/kg ASA-treated
group
24
The antinociceptive activity of AELR assessed using the formalin test was
resulted that AELR showed a dose-dependent for the late phase and for the early
phase it showed dose independent. Theoretically, increasing the dose of treatment
will result to decrease nociception reaction. Table 4.3 describe at the early phase, the
dosage of 100mg/kg, 300mg/kg and 1000mg/kg AELR concentrations showed
insignificant (p>0.05) of control (aspirin) but, only 500mg/kg AELR show of
significant (p>0.05). This results show that it have more or less similarly like the
abdominal constriction result where it dose independent and it show stimulate toward
a chemical mediator where the reduction of pain was inhibiting or reversing the
formalin. The nociceptive response of AELR at the early phase of formalin test
seemed to result from the inhibition of direct activation of nociceptors by chemical
(Tjolsen et al., 1992). The peripheral mechanism antinociception involve several
mechanisms such as inhibition of prostaglandins released by tissue injury which
hence reduce or reverse the process of sensitization of nociceptors-induced
hyperalgesia. The inhibition of formalin early phase by drugs which acted mainly at
central sites and this can suggest that AELR may also have the same property to
inhibit central nociceptive mechanism (Hunskaar & Hole, 1987). The late phase
shows, all AELR concentrations (100mg/kg, 300mg/kg, 500mg/kg, and 1000mg/kg
AELR) exhibited significant (p<0.05) antinociceptive activity when assessed using
formalin test. Therefore, the AELR demonstrated inhibition effect of 100mg/kg ASA
to the dosage of 100mg/kg, 300mg/kg, and 500mg/kg AELR, more the best
concentration among of three dosage was 100mg/kg AELR because it show the
exhibited of activity which is effective than 100mg/kg ASA. The 1000mg/kg AELR
show it very effective more than aspirin. The late phase result an inhibition of
attributed mainly to the development of an inflammatory reaction at the site of
injection and increased synaptic transmission in the spinal cord. This inhibition
shows that AELR have antinociception and allows anti-inflammatory properties.
Probably, AELR have component that may resemble as aspirin-like drugs which can
inhibit prostaglandins I2 and E2 synthesis, this will explain antinociceptive induced
by inflammatory mediators.The Figure 4.2 plots a chart of antinociceptive activities
by formalin test.
25
Fig
ure
4.4
Anti
noci
cepti
ve
effe
cts
of
Lig
nosu
s rh
inoce
rus
asse
ssed
by t
he
form
alin
tes
t
26
4.3 Hot-Plate Test
The third test was hot-plate test which was used to investigate the central
antinociceptive mechanism (Wilson et al., 2003). It was also used to study and
measure the complex reaction to a thermal and non inflammatory induced
nociception effects. As applying heat as a source of stimuli, hot-plate test is a form of
acute aphasic test. The outcome of this test was the observable responses of paw
licking and jumping responses which indicated the nociceptive signal of the mice.
Mice were used as the experimental animal since a previous study has shown that
mice showed rather constant and stereotype response as compared to rats that will
show more complex behavior upon thermal exposure (Zakaria et al., 2006). Those
complex behavior of rats include sniffing, stamping its feet, starts and stops washing
itself licking both fore and hind paw as well as straightening up comparing the mice
that only show paw licking and jumping responses upon heat exposure that will be
easily identified.
27
Table 4.5 Antinociceptive effect of Lignosus rhinocerus assessed by the hot-plate
test
Above table 4.5 presented, the result was obtained by used the hot plate test
and Figure 4.6 showed that AELR possessors concentration-independent of
antinociceptive activity. The 100mg/kg AELR show significant from beginning
Treatme
nt
mg/kg
Mice 0 hr 0.5 hr 1 hr 2 hr 3 hr 4 hr 5 hr
dH2O 6 7.18 ±
1.15b
5.58 ±
0.53b
7.0 ±
0.91b
7.40 ±
0.75b
9.01±
1.15b
6.92 ±
0.29b
7.88±
0.82b
100
ASA
6 7.05±
1.49
4.81 ±
0.55a
6.13 ±
1.19a
8.47±
1.86a
10.37±
1.22a
11.85±
1.70a
10.00±
1.72a
100
AELR
6 8.28 ±
1.39
11.4 ±
4.55 a b
10.27 ±
2.61
12.82±
1.64 a b
11.9 ±
2.54
11.25 ±
2.58
11.05±
2.69
300
AELR
6 7.01 ±
1.34
7.5 ±
3.12
11.5 ±
6.12 b
8.15 ±
0.46
7.31 ±
1.64
9.95 ±
5.02
7.60 ±
2.66
500
AELR
1000
AELR
6
6
6.55 ±
1.08
9.13 ±
1.14
5.53 ±
1.75
11.9 ±
3.22 a b
7.85 ±
2.35
11.33 ±
3.26 b
8.20 ±
2.09
12.8 ±
3.15 a b
7.57 ±
3.32
7.57 ±
3.32
8.28 ±
0.80
12.43 ±
4.72
8.35 ±
2.47
11.38±
2.09 a
a Data differ significantly (p<0.05) when compared with the dH2O-treated group
b Data differ significantly (p<0.05) when compared with the ASA-treated group
28
toward middle hour than the activities of antociceptive were reduce until end hours,
however it showed mice strongly can sustained of latancy discomfort in long period
of time. Next, the concentration of 300mg/kg AELR shows the activity was
inconsistent where it gave high latency reading as early as 30 minutes to first hour
than it dropped at hour 2 until hour 3 and it back to high latency effect until 4hour
and at the end of experiment graph was reduce. The hot-plate test is to investigate the
central antinociceptive mechanism; it thought to involve the spinal reflex. Heat
stimulation affects mainly the heat-sensitive nociceptors in the skin with no
inflammatory response (Zakaria et al., 2005). The activity of 500mg/kg AELR
concentration showed each hour of latancy time response of mice is insignificant,
however at 30 minutes the graph turn down where it seem to like a control, than it
mild increase slowly until 4 hours. Last, at 5 hours the response switch to reduce.
Increasing dose to high concentration of 1000mg/kg AELR was interestingly
demonstrated significant to 100mg/kg ASA. Where, it shows a mice can highly
sustain on hot-plate during 30 minutes, than suddenly the respond decrease at first
hour however it still in high latency of discomfort time. Interestingly, it shows
consistency of protract during first hour until at time of four but just little scale of
wave. Last slowly turn to reduce its activity until the end of experiment. According
to (Bentley et al,. 1981) the abdominal constriction test can detect analgesia of
compound/dose levels that may inactive in the hot plate test due to its high
sensitivity.
29
Fig
ure
4.6
Anti
noci
cepti
ve
effe
cts
of
Lig
nosu
s rh
inoce
rus
asse
ssed
by t
he
hot-
pla
te t
est
30
CHAPTER 5
CONCLUSION
Thought the three assays, it is show that antinociceptive property of AELR
involved both the central and peripheral antinociceptive mechanisms. The ability of
AELR to inhibit nociception in hot plate and formalin demonstrated analgesic
property with centrally mediated activity. In contrast, the peripheral antinociceptive
property of AELR was demonstrated by the ability to reduce the numbers of
constrictions in abdominal constriction test. The antinociceptive property of AELR
has been assessed by abdominal constriction test, hot plate test, and formalin test. In
the abdominal constriction test, all concentrations of AELR showed significant effect
on the peripheral antinociceptive mechanism. However, the concentrations 300mg/kg
are effective as aspirin while 500mg/kg is significantly more effective than aspirin.
Interestingly in hot plate test AELR gave independent result towards the central
antinociceptive assay. 1000mg/kg showed significant effect on the centrally-
mediated inflammatory-associated nociceptive which stimulate resistance at high
period of time start from beginning. However result showed AELR is mild
antinociceptive effect in formalin test. The best AELR concentration demonstrated
inhibition effect by formalin test is 500mg/kg.These results demonstrate that the
AELR exhibit antinociceptive property via central and peripheral mechanism. Since
AELR showed antinociception in the early and late phase of formalin test which
proved its direct activation of nociceptors by chemical and anti-inflammatory
property. Both antinociceptive and anti-inflammatory of AELR showed
concentration independent and dependent activity. As a conclusion, the aqueous
31
extract of L. rhinocerus is potential source of bioactive compound for the pain relief
drug.
5.1 Recommendation
It may be suggested that further investigate need to be done using method of
anti-inflammatory, for seeking a properties of AELR by using specific assay such as
carrageenan-induced paw edema test, granulomatous tissue induction test, histamine
induced increase in vascular permeability test and cotton pellets test to assess and
prove the claim that AELR have anti-inflammatory properties.
32
REFERENCES
Ammirati J. F., Seidl M. T. 2008. Fungus. Poisonous Mushrooms of the Northern United
States and Canada and other books. msn encarta encycolpedia.
http://encarta.msn.com/encyclopedia_761551534_2/Fungus.html. Accessed on
2008 May 11
Anonymous. 2002, Cough cure 'tiger's milk' is a fungus. The Star, October 4.
Bennett J.W. 1998. Mycotechnology: the role of fungi in biotechnology. Science Direct.
Journal of Biotechnology. Vol. 66, Issue 2-3, pp.101-107.
Bentley GA, Newton SH, Starr SJ. 1981. Br J Phamacol. Evidence for an action of
morphine and the enkephalins on sensory nerve ending in the mouse peritoneum.
73: 325-332.
Brill W.S. 2007, Edible and medicinal wild plants and mushrooms, nature, and ecology.
http://wildmanstevebrill.com/Mushrooms.Folder/Polypore.html Accessed on
2008 October 1
Burkill, I.H. 1966. A Dictionary of the Economic Products of the Malay Peninsula. Vol.
I & II. Kuala Lumpur: Ministry of Agriculture.
Bisby, F.A., Y.R. Roskov, M.A. Ruggiero, T.M. Orrell, L.E. Paglinawan, P.W. Brewer,
N. Bailly, J. van Hertum, 2007. Lignosus rhinocerus spp. Species 2000 & ITIS
Catalogue of Life: 2007 Annual Checklist. Species 2000: Reading, U.K.
http://zipcodezoo.com/Fungi/L/Lignosus_rhinocerus/. Accessed on 2008 August
11
Chang, Y.S. and Lee, S.S. 2004. Utilisation of macrofungi species in Malaysia. Fungal
Diversity: 15: 15-22.
Fayyaz, A., Rafeeq, A.K., and Shaheed, R. 1992. Study of Analgesic and Anti-
Inflammatory Activity From Plant Extracts Of Lactusa Scariola And Artemisia
Absinthium. Journal of Islamic Academy of Sciences 5:2. pp. 111-114.
33
Hattori T., Rashid, N.M.N. and Ujang, S., 2007. Basidiomycota: Diversity of Maylaysian
polypres. In: Malaysian Fungal Diversity (eds E.B.G. Jones, K.D. Hyde and S
Vikineswary) Mushroom Resaerch Center, University of Malaya and Ministry of
Natural Resources and Enviroment, Malaysia: 55-56
Harvey E. A., 2007. Synthetic Drug and Its Side Effects. British National Formulary
www.amazon.com/British-National-Formulary-53-v/dp/0853697310.Accessed
on 2009 January 11
Hattori T. and Lee, S. S., 2003 Community structure of wood- decaying Basidiomycetes
in Pasoh. In Pasoh: ecology of lowland rain forest in Southeast Asia ( eds. T.
Okuda, N. Monokaran, Y Matsumoto, K Niiyama, S.C. Thomas, and P.S.
Ashton) springer, Tokyo, Japan: 161-170
Hibbett, D.S and Thorn R.G., 2001. Basidiomycota: Homobasidiomycetes. In The
mycota VII. Systematics and Evolution. Part B. springer, Berlin, Germany: 121-
168
Hibbett, D.S. 2006. A pylogenetic overview of the Agricomycotina. Mycologia. 98: pp
917-925
Heino, L. 2005. Sclerotia. Australian Fungi Website. Australian National Botanic
Garden. Australian National Herbarium.
http://www.anbg.gov.au/fungi/mycelium.html. Accessed on 2008 June 27
Hunskaar S., Hole K. 1987. The formalin test in mice: dissociation between
inflammatory and non-inflammatory pain. Pain 30, pp103–114.
Hunskaar S, Fasmer OB, and Hole K. 1985. Acetylsalicylic acid, paracetamol and
morphine inhibit behavioral responses to intrathecally administered substance P
or capsaicin. Life Sci 37: 1835–1841.
Hossein, H. and Hani, M.Y. 2002. Antinociceptive and anti-inflammatory effects of
Crocus sativus L.stigma and petal extracts in mice. BioMed Central
Huskaar, S., Fasmer, O.B., and Hole, K. 1985. Formalin test in mice, a useful technique
for evaluating mild analgesics. Journal of Neuroscience Methods, 14: pp. 69-76.
Joseph B.M. 1997. Chapter 6, Fungi. pp: 141-159
Kirk, P.M., Cannon, P.F., David, J.C., Stalpers, J.A. 2001. Dictionary of the fungi. CABI
Publication, Wallingford, UK.
Katzung BG. 1995. Basic and clinical pharmacology (6th ed.) Appleton and Lange,
Connecticut, U.S.A.
Lee, S. S. and Chang, Y.S., 2007.Enthnomycology. In: Malaysian Fungal Diversity (eds
E.B.G. Jones, K.D. Hyde and S Vikineswary) Mushroom Resaerch Center,
University of Malaya and Ministry of Natural Resources and Enviroment,
Malaysia: 307-317
34
Lindsey B., 2003. Concise Medical Dictionary (6th edition). Emerald Group Publishing
Limited. Vol 17, Issue 2
Lee, S. S., Chang, Y.S. and Rashid, N.M.N, 2006, Common Edible Muchrooms of Orang
Asli Communities in Peninsular Malaysia. Forest Research Institute Malaysia,
Kepong.
Luise P., 2008, Noceceptive. www.richardhill.com.au/Nociception Accessed on 2008
October 1
Maddrey, C.; Willis; Boitnott, K.; Bedine, S.; Weber, L.; Fredrick; Mezey, E.; White, R.;
Corticosteriod Therapy of Alcoholic Hepatitis. Gastroenterology 75: 193-199.
1978.
Mat Jais AM, Dambisya YM, Lee TL, 1997. Antinociceptive activity of Channa striatus
(haruan) in mice. J Ethnopharmacol; 57:125–130.
Orrego, H.; Kalant, H.; Israel, Y.; Blake, J.; Medline, A.; Rankin, J.; Armstrong, A.;
Kapur, B.; Effect of Short-Term Therapy with Propylthiouracil in Patients with
Alcoholic Liver Disease. Gastroenterology 76; 105-115. 1978.
Pini LA, Sandrini M, and Vitale G.1996. the antinociceptive action of paracetamol is
associated with changes in the serotonergic system in the rat brain. Eur J
Pharmacol 308: pp31–40.
Pini LA, Vitale G, Ottani A, Sandrini M (1996): Naloxone-reversible antinociception by
paracetamol in the rat. J Pharmacol Exp Ther; 280: pp934–940.
Reynolds & Derrick, 2009. Characterization of a cDNA Encoding β-amyrin Synthase
Involved in Saponin Biosyntheses in Chenopodium quinoa http://orca.byu.edu/Reports/Journals/2006%20Final%20reports/LSCI/Reynolds,
%20Derrick. Accessed on 2009 Febuary 12.
Rashid, N.M.N., Ujang, S. and Noridah A, 2007. Preliminary study in genera of
Malaysian polypores. In: Proceedings 8th
Pacific Rim Bio-Based Composites
Symposium, Kuala Lumpur: 531-533
Su N, Jung Park E, Virgo JS, Graham JG, Carbiess F, Fong HH, Peezzuto JM, Kingorn
AD. Photochemistry 2003. Activitiey-guided isolation of chemical constituens of
Muntingia calabura using a quinine reductase induction assay.63:pp335-241
Sulaiman MR, Somchit MN, Israf DA, Ahmad DA, Ahmad Z, Moin S. 2004. Analgesic
effect of melastoma malbatricum ethanolic extract in mice. Fitoterapia 75, 667-
672.
Sharma P. D., 2005. Mycology. Relate to the seclerotium.; basic info or conceptof
basidiomycota, polyporales, fungi structure, reproductive of asexual, and used of
35
fungi in biotechnology.Alpha Science International Ltd, Oxford. pp2 -25; pp335-
337
Ryvarden, L., 1991. Genera of Polypores Nmen clature and Taxonomy. Fungiflora,
Oslo.
Tripathi KD. 1994. Essential of medical Pharmacology (3rd
ed.) Jaypee Brothers
Medical Publisher Ltd., New Delhi, India.
Wilson SG, Bryant CD, Larivie WR, Olsen MS, Giles BE, Chesler EJ, Mogil JS. 2003.
The heritability of antinociception.II. Pharmacologenetic mediation of three over
the counter analgesic inmice. JPharmacol Exp Ther, 305: 755-764
Zakaria, Z.A., Nelendran M., Pubalan S., Sulaiman, M.R., and Abdullah, F.C. 2006.
Effect of opoid and non-opoid antagonists, pH and enzyme on Corchorus
olitorius antinociceptive in mice. Oriental Parmacy and Experimental Medicine.
6 (3), 186-195
Zakaria, Z.A., Mustapa S., Sulaiman, M.R., Jais, A.M.M., Somchit, M.N., and
Abdullah, F.C. 2005. The antinociceptive action of aqueous extract from
Muntingia calabura leaves: The role of Opioid receptor.. S. Karger AG, Basel,
36
BIODATA OF THE AUTHOR
Mohd Safuan Bin Abdul Razak was born on the July 22nd
, 1987 at Kuala
Lumpur. He received his early education at Sekolah Kebangsaan Tengku Fatimah,
Bentong, Pahang for only a six month. After that his family moved to Kuala Lumpur
and continues his primary study at Sekolah Kebangsaan Taman Tasik, Ampang from
1994 to 1999. In 2000, he entered the secondary school at Sekolah Menengah
Kebangsaan Taman Tasik, Ampang from 2000 until 2004. Where obtain excellent
result in the Penilaian Menengah Rendah(PMR) and Sijil Pelajaran Malaysia (SPM).
In 2005, the author furthered his studies at Universiti Industri Selangor
(UNiSEL), Batang Berjuntai, Selangor. He choose UNiSEL because of it image as a
privet university that encourage independent learning. He obtain Foundation
certificate in year 2006. Than, he was offered a place at Universiti Industri Selangor
(UNiSEL), Shah Alam, Selangor, to pursue for the three-year program degree of
Bachelor of Biotechnology Industry (Hons).
The author is now completing his final year in UNiSEL and wishes to get
involved in drug discovery research by used source of mycology.
37
This thesis was submitted to the Senate of Universiti Industri Selangor and has been
accepted in partial fulfillment of the requirements for the degree of Bachelor of
Biotechnology Industry (Honors). The members of the Supervisory Committee and
examiner are as follows:
Supervisor
FATIMAH CORAZON ABDULLAH, M.Sc.
Senior Lecturer
Faculty of Biotechnology and Life Sciences
Universiti Industri Selangor
Co-Supervisor
ZAINUL AMIRUDDIN ZAKARIA, Ph.D
Senior Lecturer
Faculty of Pharmacy
Universiti Teknologi Mara
Examiner
ROZILAH ALIAS, M.Sc. Lecturer
Faculty of Biotechnology and Life Sciences
Universiti Industri Selangor
MOHD NASIR SAADON, Ph.D
Professor/ Dean
Faculty of Biotechnology and Life Sciences
Universiti Industri Selangor
15th
May, 2009