The HAB16R26 endophyte extract was isolated from Psychotria condensa plant which also known as 'Pokok Kura'. Endophytes are microorganism that live in plant and usually can be found in plant tissues. The aim of this study is to evaluate the effect of HAB16R26 endophyte extract as a neuroprotective agent.
The Memantine (positive control), normal saline (negative control) and aqueous extract was intraperitoneally injected (administered on three different acute doses which are 5 mg/kg, 10 mg/kg and 20 mg/kg, and one dose for sub acute dose which is 5 mg/kg) into mice.
The result of this study showed a significant different between the effect of endophyte extract and the control. This finding can be trigger to further study the neuroprotective activity of HAB16R26 endophyte extract.
CHAPTER 1
INTRODUCTION
Background of Study
Neurodegenerative diseases can be defined as a relentlessly progressive loss of the functional and structural integrity of the central nervous system (CNS). It affects over 5 million people in the United State and Europe. This disorder includes Alzheimer's, Parkinson's, stroke, epilepsy, CNS trauma, and amyotrophic lateral sclerosis (Sonkusare et al., 2005). The Alzheimer's disease (AD) is the most common form of dementia diseases. Patient with AD may have trouble remembering things that happen recently. Glutamate toxicity may play a role in the pathogenesis of AD. The disruption of glutamatergic neurotransmission can affects learning and causing memory deficits of AD patients.
Glutamate (amino acid L- glutamate) acts as neurotransmitter in the central nervous systems and plays a major role in synaptic transmission. N-methyl-D-aspartate (NMDA) receptor is a class of glutamate receptor that is involved in learning and memory (Zoladz et al., 2006). NMDA receptors are involved in memory process such as in long-term potentiating (LTP) for triggering learning related plasticity (Cao et al., 2007). When glutamate binds and act on NMDA receptors it can induce the synaptic plasticity, however, it is over stimulated, it can cause neuronal damage, also known as excitotoxicity. The excitoxicity will increase the neuronal calcium overload at NMDA receptors and can cause neurodegenerative disorder such as AD (Cao et al., 2007).
Memantine is a neuroprotective agent and is commonly used to treat Alzheimer's disease. This drug is non-competitive NMDA receptor antagonist which can enhance memory without raising behavioral side effect produced by other NMDA antagonist such as memory and locomotor disturbances (Wise et al., 2007). From the present study done by Tzschantke and Scmidt (1999), it was shown the effect of memantine could improve learning process and memory in mices trained in the radial arm maze (RAM). Extraction from endophyte, HAB16R26 is currently under studies to determine its effect as neuroprotective agent.
Problem Statement
Nowadays, memantine, an uncompetitive NMDA receptor antagonist are used for the treatment of neurogenerative disorders especially for the treatment of Alzheimer's diseases (Wise et al., 2007; Zoladz et al., 2006). However, it produces side effect which is interfere with mild intensity and include hallucinations, confusion, dizziness, headache and tiredness (Sonkusare et al., 2005).
The purpose of the study is to evaluate the effect of HAB16R26 as a neuroprotective agent using the RAM model in mice. The endophyte HAB16R26 may have potential as a next generation of neuroprotective agent with high potency and efficacy and less side effect. The evaluation of neuroprotective activity of HAB16R26 have been done using comparative assessment of the effects of memantine and HAB16R26 by administering both agent in normal adult mices ( Zoladz et al., 2006).
1.3 Objective
The objective of the study is to evaluate the neuroprotective activity of HAB16R26 endophytic extract in mice using the radial arm maze model.
1.4 Research Hypothesis
H0: HAB16R26 extract have neuroprotective activity.
H1: HAB16R26 extract does not have neuroprotective activity.
1.5 Significant of the study
The study will give a better understanding of the effect of HAB16R26 on neuroprotective activity. It may have neuroprotective activity with rapid onset and fewer side effects compared to current available neuroprotective agents or it may not show any side effect at all.
CHAPTER 2
LITERATURE REVIEW
2.1 Brain, Learning and Memory
The brain is the organ that is responsible to mind. The mind is responsible for the basics of thinking, feeling, perceiving, learning and memory. Stanford Encyclopedia of Philosophy define memory as a diverse set of cognitive capacities to retain information and reconstruct past experiences and memory involved in learning process. Canadian Institute of Neurosciences, Mental and Addiction define memory as the ability to remember past experiences and define learning as the process that will modify a subsequent behavior. Learning and memory are closely related because memory is a behavioral change caused by experiences and learning is the process for developing memory.
Memory can be divided by two types, declarative memory and procedural memory. Declarative memory is more focused on events and facts such as date of birth. It involved consciousness. On the other hands, procedural memory is unconsciousness and this memory can be related to the knowledge of rules and procedures. It becomes automatic with repetition (Okano et al., 2000). These types of memory is more to learning skills such as ride a bike and the skills can be improved through practice. The person will learn how to perform a task but have no awareness of how it was learned.
2.2 Glutamate Receptors
The amino acid L- glutamate is the excitatory neurotransmitter in the central nervous system and involved in synaptic transmission via metabotropic and ionotropic receptor (Kew and Kemp 2005; Chaffey and Chazot 2008 ). Both inotropic and metabotropic receptors are distributed on pre- and postsynaptic sites that contribute to neuronal communication and signal processing that involves learning and memory. The synaptic transmission is mediated by three main class of inotropic glutamate receptor, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), kainite, and NMDA receptor (Zoladz et al., 2006; Reidel et al., 2003).
Most AMPA receptor are impermeable to calcium ion Ca2+, therefore it contributes to fast synaptic transmission. However, NMDA receptor slightly different with AMPA receptor, NMDA highly permeable to Ca2+ ions and also contain a voltage - dependent channel binding site of Mg 2+. Higher Ca 2+ permeability on NMDA receptor, the voltage - dependent block by Mg2+ and tends to slow the gating kinetic (Zoladz et al., 2006; Yakuma and Shimoji 1999). These conditions make the NMDA receptor more suitable for mediating plastic changes in the brain, such as the learning process (G. Reidel et al., 2003).
2.3 N-methyl-D-aspartate (NMDA) Receptor
NMDA is subclass of glutamate receptor that is essential for stimulating the LTP of the mammalian brain (Zoladz et al., 2006; Yagi et al., 1998; Lisman et al., 1998). The NMDA receptors are responsible to play a role in learning and memory. The NMDA receptors are heterogonous complexes of essential and modulator subunit. It consists seven subunits, NR1 subunit, four NR2 subunits (NR2A, NR2B, NR2C, and NR2D), and two NR3 (NR3A and NR3 B). Every single subunit provided specific modulators influences on the receptor (Parsons et al., 2007; Moriyoshi 1991). The NR1 subunits is obligatory for NMDA receptor function, and combine with at least one modulatory NR2 (A-D) subunits and frequently a modulatory NR3 (A, B) subunit (Chaffey and Chazot 2008) (Fig 2.1).
Figure 2.1: Schematic of the topology and pharmacological recognition site of NMDA receptors (Parson et al., 2007).
2.3.1 NMDA Receptor Subunit Structure
The basic structure of NMDA receptor consist of a large extracellular N-terminal area followed by the transmembrane domain ( M1,3,4) and a pore-forming domain (M2) and form reentrant loop. The M2 domain contains an asparagines residue in a sequence of three amino acids, glutamine (Q), arginine (R), asparagines (N), (Chaffey and Chazot 2008). The QRN site is important for Ca2+ permieability and Mg 2+ sensitivity. The M3 and M4 domain share a extracellular loop (S2 region) that forms the angonist binding domain with extracellular N-terminal (S1 region) ( Fig.2).
Figure 2.2: Schematic diagram showing the predicted transmembrane topology of an NR1 (A) and NR2 (B) subunits (Chaffey and Chazot 2008)
2.3.2 The NR1 Subunit Structure
The NR1 subunits are responsible for channel formation and integrate the binding site for receptors co angonist glycine. The NR1 gene consists of 22 exons that produce eight distinct splice variants on three sites which is NR1-1a, NR1-1b to NR1-4a and NR1-4b. The splice variant can be identified and characterized by the presence or absence of axon 5 that consists 21 amino acid sequence in the N-terminal region or by differential splicing of axons 21 (C1) and 22(C2) in the C-terminal (Stephenson, 2003).
2.3.3 The NR2 Subunit Structure
To activate the function of NMDA receptor, NR1 must combine with at least one of four modulators NR2 subunit (2A-D), because it is essential for glutamate binding. The structure of NR2 subunits are larger than NR1, the molecular mass for NR2A and NR2B are average of 170 - 180 kDa (Köpke et al., 1993; McBain and Mayer,1994) and for NR2C and NR2D around 150 kDa (Akesson et al., 2000). NR2 subunits consists approximately 70% amino acid sequence homology between NR2A and NR2B subunits and similar degree of homology between the NR2C and NR2D, but the momology will decrease to 55 % between two pairs of subunits (Stephenson, 2003).
2.3.4 The NR3 Subunits Structure
The NR3 subunits are the most recently discovered in NMDA receptor subunit family. NR3 subunit consist NR3A and NR3B, which was identified due to discrepancies between the properties of native and recombinant NMDA receptors. Its contain 27% homology of NR1 and NR2, which is similar to NMDA receptor subunits (Yao and Mayer. 2006). The NR3A was first identified from the rat genome in 1995 (Ciabarra et al., 1995; Sucher et al., 1995). The human NR3 A gene shows 92.7% sequence similar to rat NR3A (Andersson et al., 2001). On the other hand, NR3B was discovered in 2001 in both human and rat genomes (Nishi et al., 2001; Matsuda et al., 2002). It is identified by unique sequence of significant homology to the glutamate receptor ( Nishi et al., 2001).
2.3.5 NMDA Receptor Agonist
The complexity of NMDA receptors arise from the multiple subunits co-assembly. However, to activate the receptors channel, it needs two co-agonists glutamate and glycerin. Without these two co-agonists the NR1 and NR2 subunits fail to produce functional of receptor. At NR1 subunits, glycerin will bind at glycerin-binding site on NR1 subunits (Kuryatov et al., 1994) and NR2 consist glutamate-binding site, and glutamate will bind at this binding site on NR2 ( figure 2.2) (Laube et al., 1998). The agonist (L-glutamate) bind to S1 and S2 regions, the binding will eliciting the conformational changes and folding the proteins and the channel will open.
2.3.6 NMDA Receptor Antagonist
The NMDA receptors play a major role in stimulating transmissions within the CNS (Chaffey and Chazot 2008), but if glutamate is over stimulated (glutamate toxicity) it may causes implication in memory process, dementia and in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease. The over activation of NMDA receptor due to over stimulation of glutamate could be prevented by NMDA antagonist.
The first generation of NMDA antagonist were developed to treat stroke and trauma by either non-selective channel blockers such as phencyclidine (PCP), or competitive antagonists at the agonist glutamate-binding site or at the agonist glycerin-binding site (Chaffey and Chazot 2008). The clinical study shows that NMDA antagonists have a serious side effect on CNS such as psychomimetic effect, impaired learning and memory. Therefore non-competitive modulator are have been chosen to treat the glutamate toxicity, because this modulator is subunit specific, and can be targeted to specific subunit such as NR2B subunit (Chaffey and Chazot 2008).
2.4 Memantine is a Non-Competitive NMDA Receptor Antagonist
Memantine (1-amino-3,5-dimethyladamantane) is an aminoadamantane derivative, and used as low to moderate uncompetitive NMDA receptor antagonist and function as a neuroprotective agent. Memantine also had been used as an antispastic agent for the treatment dementia (More et al., 2008; Tzshentke and Schmidt, 1999). In United States, memantine has been approved for the treatment of Alzheimer's disease, because it enhance memory without eliciting behavioral side effects commonly produced by NMDA receptor antagonists, such as memory and locomotor disturbances ( Wise et al., 2008; Danysz and Parsons, 2003). Memantine can helps to normalize the impairments in synaptic plasticity and cognition that causes by excitotoxic neuronal injury. Memantine are useful to normalized impaired LTP and in vivo study shows that it also increase LTP in aged rat (Frankiewicz and Parsons, 1999; Barnes et al., 1996).
2.4.1 Receptor Binding
Memantine bind at NMDA receptor in human cortex, rat cortex and the CA1 region of hippocampus. The characteristic of memantine as uncompetitive nature of binding site, the inhibition is indirectly vy antagonism at other site of NMDA receptor complex (Parsons et al., 1999).
2.5 Extration of HAB16R26 From Endophytes.
Endophytes are microorganisms (mostly fungi and bacteria) that live in plant and usually can be found in plant tissues. These organisms that colonized living internal tissue of plant, it has shown as a promising sourse of new drug in drug development, but are still poorly exploited. The endophytes produce a plethora of substance that have potential usage in modern medicine, agriculture and industry. The antimycotics, immunosuppressants and anticancer drug that had been discover after isolation, culture, purification, and charactherization of some endophytes (Strobel and Daisy., 2003). The extraction HAB16R26 comes from the isolation of endophytic microorganisms. The endophytes from Psychotria condensa plant which also known as 'Pokok Kura'.
2.5.1 Isolation of Endophytes
By the definition of endophytes, it lives in close affiliation with living plant tissues. To isolate endophytic microorganisms, firstly the selection of interest plant based on its biology, age, endemism. The Psychotria condense plant was selected for this study, and small portion of this plant has harvested (usually, small stem pieces are cut from the plant). The stem piece surface was covered with 70% ethanol until dry under a lamina flow hood. After drying, the inner tissues of stem plant are carefully excised and place on water agar plate and incubation for several days. After incubation, hyphal tips of the fungi are removed and transferred to potato dextrose agar plats ( G.A Strobel, 2003).
CHAPTER 3
MATERIALS AND METHODS
3.1 Preparation of Agar
39 g of Potato Dextrose Agar (PDA) was weighed on analytical balance and mixed with 1L of distilled water in conical flask. The solution was sterilized in autoclaved (Tomy-Japan) at 121oC for 2 hours.
3.2 Preparation of Extract
All the preparation was done in a Biology Safety Cabinet (BSC) Class III (ESCO). The PDA solution was poured into petri dishes and left to solidify. The HAB16R26 strain was cultured into the plates by using an inoculate strip. The plates were then incubated at 28.7oC for two weeks.
3.3 Extraction
Two weeks after incubation, the PDA agar plates with the cultured HAB16R26 endophyte were mashed by spatula and immersed in ethyl acetate. The agar was homogenized until most of the mashed agar was breakdown. The solution was filtered by filter paper and the filtrate was collected. The residue that was left in the conical flask was re-immersed in ethyl acetate and left overnight. After 24 hours, the residue was filtered and the filtrate was collected again the filtrate was dried to remove ethyl acetate by using a rotary evaporator (BUCHI. Rotavapor) and a speed vacuum concentration (Biotron). Then dried extract was then weighed.
3.4 The Animal
Swiss albino male mice weighing 10 - 20 gm were obtained from animal science department, University Putra Malaysia (UPM), the animals were maintained under standard condition with 12 hour light/dark cycle throughout the period of experimentation. The animals were provided with access to food and water. The cages were cleaned weekly.
Figure 3.1: Mice placed in cage with access to food and water.
3.5 Experimental Design
3.5.1 Acute Dose
Three doses (20mg/kg, 10mg/kg and 5mg/kg) of endophytic extract were chosen for the acute dose experiment. The mice were divided into five groups with six mice in each group. The three groups of mice were injected intraperitoneally with the three different doses 5mg/kg, 10mg/kg and 20mg/kg of endophytic extract. The positive control group was injected intraperitoneally (i.p.) with 10mg/kg of memantine. The other group was injected intraperitoneally with 10mg/kg of 0.9% 5 mg/kg normal saline and served as negative control.
3.5.2 Sub-acute Doses
For the sub-acute doses, the mice were divided to three groups with six mice in each group. First group of mice was injected with 5 mg/kg of endophytic extract. The second group of mice was injected with 5 mg/kg of memantine as a positive control group, and for the negative control group, the mice was injected with 5 mg/kg of 0.9% 5 mg/kg normal saline. All the sub-acute groups was injected intraperitoneally daily for three weeks.
3.6 Radial arm procedure
3.6.1 Training phase
Experiments were conducted in a similar way to those described previously (Lichtman and Martin, 1996) with modifications. During the training phase, four arms were randomly selected and blocked its entryways. Each of the remaining four arms was baited with a food pallet, and with a picture of foods as a queue to the mouse placement in the maze. After the mouse enter the four remaining arms and consumed each of the available food pallets, it was removed from the maze and return to the cage.
3.6.2 Test phase
During the test phase, all eight arms were available; however only the previously four opened arms ware baited with food pellet and has a picture as a queue. The number of entries in four arms and duration of time was recorded. For the acute dose group experiment, we evaluated the dose response relationship between extract HAB12R26 with positive and negative control. The sub acute group is to evaluate the relationship between dose and duration of administration.
3.7 Statistical analysis
Each mouse was observed and the number of correct responses and errors committed by each mice to obtain the available food pellets were scored. The data obtained were analyzed statistically and was expressed as mean ± SE. The significance of differences was determined by a Kruskal-Willis non parametric ANOVA test, followed if significant by a Mann-Whitney U-test. A P value less than 0.05 (P < 0.05) were regarded as significant.
CHAPTER 4
RESULT
4.1 Acute dose
Figure 4.1: Effect of HAB16R26 in the right arms entries for acute dose.
Figure 4.1 shows the effect of HAB16R26 endophyte extract on mice memory compared with control by using radial arm maze. The mean average of the right arms entries for 20mg/kg extract is higher when compared to 10mg/kg extract and 10mg/kg memantine. Besides that, the 5 mg/kg extract showed the same effect as 5 mg/kg 5 mg/kg normal saline. The Kruskal-Wallis test revealed a statically significant difference in the right arms entries between five different doses (Dose 1, n = 6: 5 mg/kg 5 mg/kg normal saline, Dose 2, n = 6: 5 mg/kg extract, Dose 3, n = 6: 10 mg/kg extract, Dose 4, n = 6: 20 mg/kg extract, Dose 5, n =6: 10 mg/kg memantine), χ2 (4, n = 30) = 13.982, p < 0.05. The three dose, 10 mg/kg extract, 20 mg/kg extract and 10 mg/kg memantine recorded the higher median score (Md = 2.50) than the other two groups, which both were recorded with median values of 1.50. (Appendix: Table 4.1)
4.2 Mann-Whitney U Test for Acute Group
The statistically significant results from Kruskal Wallis Test do not show the doses are statistically significantly different from one another in acute group. Therefore, the Mann-Whitney U-Test are used to determined the significance between two pair of dose in this group (e.g. between the dose 5 mg/kg 5 mg/kg normal saline and dose of 5 mg/kg extract)
4.2.1 5 mg/kg 5 mg/kg normal saline and 5 mg/kg extract
A Mann-Whitney U test revealed no significant different in the right arms entries of 5 mg/kg 5 mg/kg normal saline (Md = 1.50, n = 6) and 5 mg/kg extract (Md = 1.50, n = 6), U = 18, z = 0, p = 1. (Appendix: Table 4.2)
4.2.2 5 mg/kg 5 mg/kg normal saline and 10mg/kg extract
A Mann-Whitney U test revealed a significant different in the right arms entries of 5 mg/kg 5 mg/kg normal saline (Md = 1.50, n = 6) and 10mg/kg extract (Md = 2.50, n = 6), U = 4.5, z = -2.345, p = 0.019. (Appendix: Table 4.2)
4.2.3 5 mg/kg 5 mg/kg normal saline and 20mg/kg extract
A Mann-Whitney U test revealed a significant different in the right arms entries of 5 mg/kg 5 mg/kg normal saline (Md = 1.50, n = 6) and 20mg/kg extract (Md = 2.50, n = 6), U = 4.5, z = -2.345, p = 0.019. (Appendix: Table 4.2)
4.2.4 5 mg/kg 5 mg/kg normal saline and 10mg/kg memantine
A Mann-Whitney U test revealed a significant different in the right arms entries of 5 mg/kg 5 mg/kg normal saline (Md = 1.50, n = 6) and 10mg/kg memantine (Md = 2.50, n = 6), U = 4.5, z = -2.345, p = 0.019. (Appendix: Table 4.2)
4.2.5 5mg/kg extract and 10mg/kg extract
A Mann-Whitney U test revealed a significant different in the right arms entries of 5mg/kg extract (Md = 1.50, n = 6) and 10mg/kg extract (Md = 2.50, n = 6), U = 4.5, z = -2.345, p = 0.019. (Appendix: Table 4.2)
4.2.6 5mg/kg extract and 20mg/kg extract
A Mann-Whitney U test revealed a significant different in the right arms entries of 5mg/kg extract (Md = 1.50, n = 6) and 20mg/kg extract (Md = 2.50, n = 6), U = 4.5, z = -2.345, p = 0.019. (Appendix: Table 4.3)
4.2.7 5 mg/kg extract and 10 mg/kg memantine
A Mann-Whitney U test revealed a significant different in the right arms entries of 5 mg/kg extract (Md = 1.50, n = 6) and 10 mg/kg memantine (Md = 2.50, n = 6), U = 4.5, z = -2.345, p = 0.019. (Appendix: Table 4.3)
4.2.8 10 mg/kg extract and 20 mg/kg extract
A Mann-Whitney U test revealed no significant different in the right arms entries of 10 mg/kg extract (Md = 2.50, n = 6) and 20 mg/kg extract (Md = 2.50, n = 6), U = 18, z = 0, p > 0.05. (Appendix: Table 4.3)
4.2.9 10 mg/kg extract and 10 mg/kg memantine
A Mann-Whitney U test revealed no significant different in the right arms entries of 10 mg/kg extract (Md = 2.50, n = 6) and 10 mg/kg memantine (Md = 2.50, n = 6), U = 18, z = 0, p > 0.05. (Appendix: Table 4.3)
4.2.10 20 mg/kg extract and 10 mg/kg memantine
A Mann-Whitney U test revealed no significant different in the right arms entries of 20 mg/kg extract (Md = 2.50, n = 6) and 10 mg/kg memantine (Md = 2.50, n = 6), U = 18, z = 0, p > 0.05. (Appendix: Table 4.3)
4.3 Sub Acute administration
Figure 4.2: Effect of HAB16R26 in the right arms entries for sub acute dose.
From the figure above, for the first week of experiment 5 mg/kg extract show the same effect with 5 mg/kg normal saline while being compared to memantine 5mg/kg. For the second week the effect of 5 mg/kg extract increased and slightly shows the same effect of memantine when compared with 5 mg/kg normal saline. The effect of 5 mg/kg extract and memantine 5mg/kg remained constant for week three and week four.
The Kruskal-Wallis test revealed a statically significant difference in the right arms entries between different groups of duration (Group 1, n = 18: week 1, Group 2, n = 18: week 2, Group 3, n = 18: week 3, Group 4, n = 18: week 4), χ2 (3, n = 72) = 14.291, p < 0.05. The all groups share the same median score (Md = 2). (Appendix: Table 4.4). Furthermore, the Kruskal-Wallis test also revealed a statistically significant difference in the right arms entries between different dose group (Group 1, n = 24: 5 mg/kg normal saline, Group 2, n = 24: 5 mg/kg extract, Group 3, n = 24: memantine 5mg/kg), χ2 (2, n = 72) = 7.144, p < 0.05. All groups were recorded to share the same median score (Md = 2). (Appendix: Table 4.5)
4.4 Mann-Whitney U Test for Sub Acute Group
The statistically significant results from Kruskal Wallis Test do not clearly show the duration of administration are statistically significantly different from one another in sub acute group. Therefore, the Mann-Whitney U-Test are used to determined the significance between two different duration in this group.
4.4.1 Week 1 and Week 2
A Mann-Whitney U test revealed significant different in the right arms entries in week 1 (Md = 2, n = 18) and week 2 (Md = 2, n = 18), U = 97, z = -2.340, p < 0.05. (Appendix: Table 4.6)
4.4.2 Week 1 and Week 3
A Mann-Whitney U test revealed significant different in the right arms entries in week 1 (Md = 2, n = 18) and week 3 (Md = 2, n = 18), U = 68, z = -3.239, p < 0.05. (Appendix: Table 4.6)
4.4.3 Week1 and Week 4
A Mann-Whitney U test revealed significant different in the right arms entries in week 1 (Md = 2, n = 18) and week 4 (Md = 2, n = 18), U = 73, z = -3.1, p < 0.05. (Appendix: Table 4.6)
4.4.4 Week 2 and Week 3
A Mann-Whitney U test revealed no significant different in the right arms entries in week 2 (Md = 2, n = 18) and week 3 (Md = 2, n = 18), U = 125, z = -1.298, p > 0.05. (Appendix: Table 4.6)
4.4.5 Week 2 and Week 4
A Mann-Whitney U test revealed no significant different in the right arms entries in week 2 (Md = 2, n = 18) and week 4 (Md = 2, n = 18), U = 132.5, z = -1.045, p > 0.05. (Appendix: Table 4.6)
4.4.6 Week 3 and Week 4
A Mann-Whitney U test revealed no significant different in the right arms entries in week 3 (Md = 2, n = 18) and week 4 (Md = 2, n = 18), U = 154, z = -0.279, p > 0.05. (Appendix: Table 4.6)
4.5 Mann-Whitney U Test for Dose Sub Acute Group
The statistically significant results from Kruskal Wallis Test do not show the doses are statistically significantly different from one another in sub acute group. Therefore, the Mann-Whitney U-Test are used to determined the significance between two pair of dose in this group.
4.5.1 5 mg/kg normal saline and 5 mg/kg extract
A Mann-Whitney U test revealed significant different in the right arms entries of 5 mg/kg normal saline (Md = 2, n = 24) and 5 mg/kg extract (Md = 2, n = 24), U = 198, z = -2.021, p < 0.05. (Appendix: Table 4.7)
4.5.2 5 mg/kg normal saline and memantine 5mg/kg
A Mann-Whitney U test revealed significant different in the right arms entries of 5 mg/kg normal saline (Md = 2, n = 24) and memantine 5mg/kg (Md = 2, n = 24), U = 177, z = -2.532, p < 0.05. (Appendix: Table 4.7)
4.5.3 5 mg/kg extract and memantine 5mg/kg
A Mann-Whitney U test revealed no significant different in the right arms entries of 5 mg/kg extract (Md = 2, n = 24) and 5 mg/kg extract (Md = 2, n = 24), U = 272, z = -0.368, p > 0.05. (Appendix: Table 4.7)
CHAPTER 5
DISCUSSION
5.1 General
Neurodegenerative diseases can be defined as a relentlessly progressive loss of the functional and structural integrity of the central nervous system (CNS). This disorder includes Alzheimer's disease, Parkinson's disease, stroke, epilepsy, CNS trauma, and amyotrophic lateral sclerosis (Sonkusare et al., 2005). AD is the most common form of dementia disease and it is irreversible. The disease progresses slowly by destroying memory and thinking skills and eventually even the ability to carry out the simple tasks.
Memantine is a neuroprotective agent and is commonly used to treat Alzheimer's disease. This drug is a non-competitive NMDA receptor antagonist which can enhance memory (Wise et al., 2007). In another study by Tzschantke and Scmidt (1999), it was shown that memantine could improve learning process and memory in mice trained in the radial arm maze (RAM). Furthermore, memantine has been shown to decrease the error of mice to enter the wrong arm during the test phase in radial arm maze (Wise et al., 2007).The radial arm maze was designed by Olton and Samuelson in 1976 to assess spatial learning and memory in rats or mice. Since it was designed, the maze has been used by researchers interested in studying the spatial learning and memory of animals (Bubreuil et al. 2003).
The objective of this study is to evaluate the neuroprotective activity of HAB16R26 endophyte extract by comparing with memantine as positive control and 5 mg/kg normal saline as negative control.
By comparing the effect of HAB16R26 extract with the negative control in acute dose, the result showed that the different dose of HAB16R26 extract which are 10mg/kg and 20 mg/kg have significant difference for right arm entry, but for dose 5 mg/kg extract there is no significant difference with negative control. From the acute dose result, it is shown that, higher doses of HAB16R26 extract (10mg/kg and 20 mg/kg) , improved memory in mice by reducing the error to enter right arm entry, but lower dose (5mg/kg) showed similar result to with negative control. Therefore, it has no significant difference with negative control.
For sub acute dose, the HAB16R26 extract was compared to two different variables which is the dose and duration of dose administration. The non parametric ANOVA, Kruskal-Wallis test show there is a significant difference in the right arms entries between different duration and with different doses, but the statistically significant results from Kruskal Wallis test did not show the doses are statistically significantly different from the group of subjects. Therefore, the Mann-Whitney U-Test is used to determine the significance between the groups. The Mann-Whitney U test used to compare the week of dose administration, showed that there were significant differences between week one and week two, week three and week four, but there were no significant differences between week two compared to week three and week four, and week three compared to week four.
Mann-Whitney U-Test also showed that there were significant difference between normal saline and different doses of HBA16R26 extract and memantine, but there was no significant difference between dose of extract and memantine.
The result showed that HAB16R26 extract increased memory activities and reduced the error in reentering the wrong arm in the radial arm maze and yielded a similar effect of memantine. Both compounds enhanced memory in the radial arm when given 10 minutes before running the test. Therefore, HAB16R26 extract has an effect of neuroprotective activity.
5.2 Limitations
There are some limitations in this study. Firstly, would be the time. There was not enough time to fully study the effect of HAB16R26 extract in memory by using radial arm maze. Secondly, the study may also be done on rats, porcine, rabbits and other suitable animals to get more reliable data.
The HAB16R26 extract was not enough to do studies on more variable doses. This experiment enabled the preparation of extract only up to 20 mg/kg.
CHAPTER 6
CONCLUSION
HAB16R26 is an endophyte that can be obtained from the local natural resource. This study showed that the extract of this endophytic strain has a significant effect in enhancing memory and learning skill activity of mice in radial arm maze. Therefore, HAB16R26 extract has a potential to be used as neuroprotective agent. However, more studies should be done on the extract in the future to support the current findings since AD is one of the serious neurodegenerative disorders.
