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 bodys 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).

The pain medicine treatment, consider of two types of drug which is the nonopioid 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 tigers 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 2

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.

CHAPTER 2

LITERATURE REVIEW

2.1 Pain Pain can be classified as physiologic, which refers to the bodys 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 4

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. 5

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

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);

Table 2.1 A classified of Polypores into following order and families (Hattori et al. ,2007) 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.

2.2.2 Morphology, ecology and reproduction of Lignosus rhinocerus

Figure 2.2 Actual picture of local polypore (Lignosus rhinocerus) flora of Malaysia. Caps (pileus)

Stipetate

Root

Sclerotium (Sclerotial stroma)

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 tigers 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 9

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.

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Table 2.5 Evolutionary names of Lignosus rhinocerus. (Bisby et al., 2007) 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)

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 antiinflammation. 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, 11

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.

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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 2530 g (57 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 7080% 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.

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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 34 weeks at room temperature (27 2 C). The dried sclerotium was then grind into small particles, weighed (250 g) and incubated at 35-40C 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.

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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 0 min 30 min 35 min 60 min

Extract, sc

0.6% acetic acid, at Mice peritoneal cavity

Constriction administration

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: 15

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 l 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 0 min 30 min 35 min 1st phase 45min 2nd phase 60min

Extract, sc

Injecting 25ul of 25% formalin in the subplantar region of the left hind paw

Rest

Figure 3.2 Timeframe review of formalin test

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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 50C hot-plate test by (Wilson et al., 2003). The temperature of the metal plate (Model 7280, Ugo Basile, Italy) will be maintained at 50 0.2C. 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 -ve min 0 min 30 min 1h 2h 3h 4h 5h

Extract, sc Screening For Selection mice 5-10s

Latency to a discomfort reaction (liking of paws or jumping)

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.

17

3.6 Statistical Analysis

The results are presented as mean standard error of mean (SEM). The oneway 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.

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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

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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 dH2O 100 ASA 100 AELR 300 AELR 500 AELR 1000 AELR
a b

Mice

Number of Constriction 39.50 2.35b 16.00 1.55a 22.83 2.48ab 18.83 2.64a 10.50 3.51ab 28.17 4.31
ab

% Inhibition

6 6 6 6 6 6

59.49 42.20 50.33 73.42 28.68

Data differ significantly (p<0.05) when compared with the dH2O-treated group Data differ significantly (p<0.05) when compared with the 100mg/kg ASA-treated

group

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The results shown in Table 4.1 represent the ability of AELR to reduce the number of abdominal constriction in mice. The AELR demonstrated concentrationindependent 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.

21

22

Figure 4.2 Antinociceptive effects of Lignosus rhinocerus assessed by the abdominal constriction test

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 dH2O 100 ASA 100 AELR 300 AELR 500 AELR 1000 AELR
a b

Mice

Early Phase, s 49.30 4.42b 41.94 10.19a 45.73 5.29 36.34 6.25 27.91 14.11ab 42.68 9.99

Late Phase, s 60.44 10.16 b 15.7 17.54 a 56.51 8.24a 39.62 11.00ab 36.62 6.83ab 14.47 11.75b

6 6 6 6 6 6

Data differ significantly (p<0.05) when compared with the dH2O-treated group Data differ significantly (p<0.05) when compared with the 100mg/kg ASA-treated

group

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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.

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25

Figure 4.4 Antinociceptive effects of Lignosus rhinocerus assessed by the formalin test

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.

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Table 4.5 Antinociceptive effect of Lignosus rhinocerus assessed by the hot-plate test Treatme nt mg/kg dH2O 6 7.18
1.15
b

Mice

0 hr

0.5 hr

1 hr

2 hr

3 hr

4 hr

5 hr

5.58
0.53
b

7.0
0.91
b

7.40
0.75
b

9.01
1.15
b

6.92
0.29
b

7.88
0.82
b

100 ASA 100 AELR

7.05
1.49

4.81
0.55
a

6.13 1.19a 10.27

2.61

8.47
1.86
a

10.37
1.22
a

11.85
1.70
a

10.00 1.72a 11.05

2.69

8.28 1.39

11.4
4.55
ab

12.82
1.64
ab

11.9
2.54

11.25
2.58

300 AELR

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

6.55 1.08

5.53
1.75

7.85
2.35

8.20
2.09

7.57
3.32

8.28
0.80

8.35
2.47

1000 AELR
a b

9.13 1.14

11.9 3.22
ab

11.33
3.26
b

12.8
3.15
ab

7.57
3.32

12.43
4.72

11.38
2.09
a

Data differ significantly (p<0.05) when compared with the dH2O-treated group Data differ significantly (p<0.05) when compared with the ASA-treated group

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

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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.

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Figure 4.6 Antinociceptive effects of Lignosus rhinocerus assessed by the hot- plate test

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 centrallymediated 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

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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.

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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.

36

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

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