CHAPTER 1 INTRODUCTION 1

CHAPTER 1
INTRODUCTION
1.1 Background Study
Galanthamine is a tertiary alkaloid derived from bulbs of Amaryllidaceae family of flowering plants that include daffodils and the common snowdrops. It is a cholinesterase inhibitor and allosteric modulating ligand at nicotinic cholinergic receptors. Nowadays, galanthamine, especially under its brand names Reminyl® and Nivalin®, is commonly used in the treatment of Alzheimer’s disease (AD). Micheal et al (2010) reported that Alzehimer’s disease characterized by Alois Alzheimer in 1907 is a progressive degenerative disorders of the brain. It affect over 20 million individuals worldwide and this number will increase in the future along with the increase number of elderly in the population. According to the chollinergic hypothesis, impairment of the chollinergic function give the critical important in Alzheimer’s Disease especially in brain areas, dealing with memory, learning, behavior and emotional respond.
Ping Jia et al (2008) stated that among all the acetylcholinesterase (AChE) inhibitors in clinic, galanthamine exhibits unique dual mechanism on cholinergic system, not only inhibiting the cholinergic activity but also allosterically modulating nicotinic acetylcholinereceptors (AChER) which will promote the release of acetylcholine. Besides, it is less toxicity compared to tacrine, rivastigmine and donepezil. During the past few years, various galanthmaine derivatives has been synthesized and tested for anticholinesterase activity, structure-activity-relationship (SAR) studies reveal that substitution of nitrogen atom of galanthamine is favourable AChE inhibitory activity because show interaction with peripharal anionic site (PAS).

For this Final Year Project, the researcher synthesis the galanthamine acetate using Novozym435 as a catalyst. Acetic acid react with galanthamine whereby the acetic acid is the most commonly free solvent system that can readily be used. Novozym435 is the enzyme catalyst that able to form peptide bond and speed up the reaction within hours only to give good result.
According to Rao et al (2017) the method on neutralization of galanthamine hydrobromide to galanthamine is by using ammonium hydroxide that will yield about 89% of the product. The galanthamine then will be reacted with other solvent to give new compound under certain condition. The reaction will monitored by thin layer chromatography (TLC).

Miklosi et al (2006) stated that over the past twenty years, the zebrafish (Danio Rerio) has been emerged as a pre-eminent vertebrates model for studying genetics, development, human disease and the screening of therapeutic drugs. A number of favourable attributes including its small size, rapid development and generation time, optical transparency during early development, tractibility in forward genetic screens and genetic similarity to humans rise in popularity to be used in areas of research.

Bertelli et al (2017) explained that zebrafish is a small fresh water teleost fish that has been used to preclinically drug discovery and toxicological investigations. Considering that the zebrafish embryo is an important tool to access the toxicity profile and cholinergic system, the aim of this study is to analyze the toxicity of galanthamine acetate compound using zebrafish embryo as for the biological assay study.

As zebrafish research is to a large degree predicated on consistent production of large numbers of emryos, information of the reproductive biology and behavior of the animals in the wild is of clear for husbandry.
1.2 Objectives
The purpose of this project are synthesizing galanthamine acetate and determine the toxicity of the compound using zebrafish embyros. The objectives of this project are
To synthesize the galanthamine acetate when mixing galanthamine with acetic acid solution using a catalyst Novozym 435.

To characterize the synthesized compound using various spectroscopic method.

To determine the biological assay of the galanthamine derivatives using zebrafish embryo based on the toxicity test.

CHAPTER 2
LITERATURE REVIEW
2.1 Galanthamine (GL)
Bin Dong et al, (2017) stated that (?)-Galanthamine ( HYPERLINK ;http://www.sciencedirect.com/science/article/pii/S0040402017306385; l ;fig1; Fig.?1) belonging to galanthamine-type Amaryllidaceae alkaloid has unique tetracyclic structure and intriguing biological activities. It was found to be a selective, reversible, and competitive acetylcholinesterase (AChE) inhibitor and it also showed allosteric modulation of the neural nicotinic receptors to increase acetylcholine release.
The molecular formula of galanthamine is C17H21NO3 which has average molecular weight 287.354 g/mol. Besides, it is more crystals than benzene and it melts at temperature range of 115-130C. Galanthamine also can be fairly soluble in dichloromethane, tetrahydrofuran and methanol. (Han ep al., 1991). But, the galanthamine not soluble in chloroform and 2-propanol. The chemical structure of galanthamine can be shown in Figure 1:

Figure 1: The chemical structure of (?)-galanthamine (Bin Dong et al, 2017)
Galanthamine hydrobromide salt was used because it is much cheaper than pure galanthamine. Then, Bin Dong et al (2017) describes In 2001, (?)-Galanthamine hydrobromide salt was approved by FDA in the USA for the treatment of early Alzheimer’s disease. A huge amount of efforts have been put into the total synthesis of galanthamine because of its limited supplies from natural sources, high cost of isolation and purification process.. According to Rao et al, (2010) the hydrobromide salt of galanthamine was neutralized with ammonium hydroxide to produce pure galanthamine in 89% yield.
Trinadhachari et al, (2014) reported that HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0957416613005387" l "b0015" Many other synthetic routes for the preparation of (?)-galanthamine have also been reported in the literature. Besides, Cordina et al (2006) explained that there was other techniques that can be used involving multiple and longer steps to produce pure galanthamine. Then, galanthamine act as an intermediate that will combine with other compound.

HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0040402017308347" l "!" NaoshiYamamotoa et al (2017) stated that HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0040402017308347" l "!" (–)-Galanthamine was synthesized from HYPERLINK "http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/naltrexone" o "Learn more about Naltrexone" naltrexone in 18 steps with 3% total yield by overcoming many specific side reactions derived from the 4,5-epoxymorphinan skeleton. The key features are cleavage of the D-ring by the Hofmann elimination and the following the one-pot C9–C10 and C9–14 HYPERLINK "http://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/bond-cleavage" o "Learn more about Bond cleavage" bond cleavages concomitant with the C9 removal by the OsO4–NaIO4 combination reaction. Then, the treatment with HYPERLINK "http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/zinc" o "Learn more about Zinc" zinc powder in HYPERLINK "http://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/acetic-acid" o "Learn more about Acetic acid" acetic acid led to not only removal of the 2,2,2-trichloroethoxycarbonyl (Troc) group, but also reductive amination of the resulting imine to give the desired 7-membered ring.

HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0968089613009577" l "!" HiroyukiKimura et al (2014) explained that the synthetic routes to (±)-galanthamine and (±)-norgalanthamine are synthesized step by step. (±)-3-Hydroxy-N-trifluoroacetyl-N-nornarwedine was converted to triflate by a conventional method, and subsequent reduction with formic acid in the presence of palladium acetate and dppf gave narwedine derivative. The trifluoroacetamide group was cleaved by hydrolysis with methanolic sodium hydroxide to afford the amine. Subsequent diastereoselective reduction with l-selectride® gave (±)-norgalanthamine in good yield about 84%. An alternative N-methylation was attempted using methyl iodide or methyl triflate as the methylating agent. But, the former conditions gave no product, while the latter conditions provided only a low yield of (±)-galanthamine about 33%. The overall yield of (±)-galanthamine was 25%.

HYPERLINK "http://www.sciencedirect.com/science/article/pii/S0957416613005387" l "!" Anand et al (2017) claimed that herein (±)-narwedine was chosen as a key starting material for the synthesis of the galanthamine isomers. This approach involves two critical synthetic stages which are the kinetic dynamic resolution of (±)-narwedine and the reduction of the narwedine isomers by complementary stereoselective reduction reactions to give all four isomers of galanthamine with an enantiomeric purity of more than 97%.

2.2 Synthesis of Galanthamine Derivatives
2.2.1 Chemical catalyst
There are many chemical catalyst used to speed up esterification reaction. Baghwat et al (2012) reported that the esters of low molecular weight such as ethyl acetate and butyl acetate usually have pleasant and fruity odor. Also large volumes of esters are used as solvents. According to a recent review industrial acid-base catalyst, out of the more than 120 process identified over 90% are solid catalyzed. The use to conventional liquid acids such as PTSA, H2SO4, HCL, HF, ALCL3, BF3, NZCL2 and SBF5 involves risks in handling, contaminant disposal and regeneration due to their toxic and corrosive nature. It is required to eliminate the ecologically harmful mineral acids to carry out a a large number of acid-catalyzed industrial process. Therefore, the solid catalyst are gaining attention. They have the proven advantages of heterogenous catalysts, like simplified product isolation, mild reaction condition, high selectivity, ease in recovery and reuse of the catalysts as well as reduction in the generation of wasteful byproduct. The example of solid acid catalyst used is sulfated zincornia catalyst, that highly active in acid catalyzed organic reactions but deactivation of activity occurs by the loss of sulfate ions thereby recycling of the catalyst is limited.

Kale et al (2017) explained that Glycerol esterification with acetic acid is usually performed using homogenous catalysts like sulphuric acid and paratoluene sulphonic acid but these catalysts are hazardous, corrosive and not eco-friendly. Therefore it is desired to replace these mineral acids by strong solid acid catalysts such as sulphated mesoporous silica, heteropolyacids or acidic ion exchange resins like Amberlyst-15.

Shanmugam et al (2004) state that the effort to develop green processes has led to the development of solid acid catalysts to an increase in research activities both in academic and industrial sections. These materials can replace the corrosive liquid acids currently used in many industries. A variety of materials have been used as solid acid catalysts such as clays, zeolites, sulfated metal oxides and heteropolyacids. Each of these materials offers unique properties that can influence the catalytic activity. Among these solid acids, heteropoly compounds are unique in the sense, they can be tuned at atomic/molecular level to exhibit a wide variation on properties like acidity and redox behavior. They behave like mineral acids having the protons in protected environments and exhibit Bronsted acidity, which of several orders of magnitude higher in strength than that of conventional mineral acids.

Table 1: Preparation of galanthamine derivatives using chemical catalyst
Structure of galanthamine derivatives Method/ Reaction condition Chemical catalyst used Yield (%) References
Galanthamine was reacted with bis (2-chloroethyl) phosphoramidic dichloride
triethylamine (TEA) in dry tetrahydrofuran (THF)
NA
Rao et al, 2010
Demethylation of galantamine to norgalantamine was accomplished by a non-classical Polonovski
iron(II) sulfate heptahydrate 74%
Atanasova et al,2009
2.2.2 Enzyme catalyst
Lipases are versatile catalysts. In addition to their natural reaction of fat hydrolysis, lipases catalyze a plethora of other reactions such as esterification, amidation, and transesterification of esters as well as organic carbonates. Moreover, lipases accept a wide variety of substrates while maintaining their regioselectivity and stereoselectivity. Lipases are highly stable even under adverse conditions such as organic solvents, high temperatures, and so forth. Applications of lipases include production of food additives, chiral intermediates, and pharmaceutical products. Among these, synthesis of various chiral intermediates in pharmaceutical industry and cocoa butter substitutes is being commercially exploited currently.

Novozyme435 was used for the present study as a biocatalyst because this enzyme has been confirmed to be the most effective to esterify lactic acid in the previous observations. The influence of substrate concentrations on enzymatic esterification of lactic acid was examined in several hydrophobic ethers and ketones that are miscible with lactic acid (exceptionally, lactic acid was not fully dissolved only in disopropyl ether, but the reaction mixtures using this solvent were completely miscible and monophasic by addition of ethanol under the experimental conditions). The molar ratios of lactic acid to ethanol, used as substrates,were 1:1 or 1:2.
Enzyme-catalyzed esterification acquired increasing attention in many applications, due to the significance of the derived products. More specifically, the lipase-catalyzed esterification reactions attracted research interest during the past decade, due to an increased use of organic esters in biotechnology and the chemical industry (Torres and Castro, 2004). For this reason, esterification by lipases was developed a few decades ago (Okumura et al., 1979) and various microbial lipases have been employed in experiments using either primary or secondary alcohols, or both, free-solvent systems, or organic solvents. Zaks and Klibanov (1988) reported that among the important factors which influence the ester yield are the concentrations of enzyme and substrates, their molar ratio, the reaction pH-value and temperature, the mixing rates, and the water content.

2.3 Biological Assay of Galanthamine on Zebrafish
According to Dubinska-Magiera et. al., (2016) skeletal muscles in zebrafish compromise about 60% of adult body mass so study on toxic effect on muscles are of high relevance. The zebrafish contain the skeletal muscles originate from the paraxial mesoderms which undergoes fregmentation into repititive units called somites. There are three compartments to differentiate during early embryonic development which are dermomyotome, myotome and sclerotome. The source of skeletal muscles of the trunk and pelvic fin muscles comes from myotome. Table 2 summarize how the zebrafish model could be applied in accessing the impact of toxicants and bioactive compounds on neuromuscular system development and functions.
Table 2: Toxicants effect on the development and functioning of zebrafish skeletal muscle.

Toxicant Examples Effect References
Drugs GAL(Galanthamine) Motility impairment induced by myopathy Dubinska et al., 2016
Roy et al (2015) claimed that the zebrafish model has become particularly popular in the laboratory setting given its genetic and embryological similarities to higher order vertebrates including humans. For a toxicological perspective, zebrafish are particularly useful as their development is well characterized and all stages of toxicological accessment can be made ex utero. The organs specific to toxins such as the liver can be seen the toxin conversion at the early stages. These zebrafish can give information that cannot be obtained from other models and knowledge of mechanisms as the development of the toxicity is scarce.
Berteli et al (2017) reported that cholinesterase enzyme have a crucial rules in cholinergic neurotransmission once it regulates the amount of neurotransmitters in synaptic cleft. Acetylcholinesterase (AChE) regulates the transmission of nerve impulses through the synapse by hydrolysis of cholinergic neurotransmitter acetylcholine (ACh) and choline acetate. AChE inhibitors represent the first option pharmacotherapy from initial to moderate treatment of Alzheimer’s Disease (AD). One type of drugs that already registered for the treatment of AD is galanthamine. Zebrafish has been used to pre clinical drug discovery and toxicological investigations.
Hill et al (2005) explained that zebrafish have been used majorly in developmental biology and molecular genetics, also in toxicology and drug discovery has been recognized. To evaluate the toxicity of a chemical, it is essential to identify the endpoints of toxicity and their dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxicodynamics of the chemical. Related to detailed toxicological investigations of a single chemical, there also is a need for high-throughput large-scale screening for toxicity of several hundreds of chemicals at a time. Hence, the zebrafish has numerous attributes.

Table 3: Biological Assay of galanthamine on zebrafish
Biological assay of galanthamine on zebrafish embryos Findings
References
Toxicity
The coagulation of fertilized eggs, lack of somite formation, lack of detachment of the tail-bud from the yolk-sac and lack of heartbeat. Pablo et al., (2017)
IC50
Galanthamine has a dual action mechanism on chollinergic systems whereby inhibits AChE and allosterically modulates nACHR activity Yu Pong et al., (2015)
Neurotoxicity
For AChE inhibition, galanthamine modulates nicotic neurotransmission via allosteric potention of pre- and postsynaptic nAChR. Yu Pong et at., (2015)
CHAPTER 3
MATERIALS AND METHODOLOGY
3.1 Materials
All the reagents and solvents used are commercially available from Sigma-Aldrich. Meanwhile, the immobilised lipase Novozym 435 was purchased from Novozym.com. Table 3 shows the list of the chemicals and reagents used in this research:
Table 3 : Materials
Chemicals Enzyme
Galanthamine hydrobromide Novozym 435
Ethanol Acetic acid Hexane Ethyl acetate Acetone Methanol Silica gel Distilled water 3.2 General flow of experiment
41910110490Enzymatic esterification
Enzymatic esterification

32385269240Galanthamine solution was mixed with acetic acid under optimum condition
Galanthamine solution was mixed with acetic acid under optimum condition

13335323215Galanthamine acetate
Galanthamine acetate

-15240225425Galanthamine acetate structure was purified, identified and charcterized using following analysis:
-TLC,GC, GCMS, FTIR and NMR
Galanthamine acetate structure was purified, identified and charcterized using following analysis:
-TLC,GC, GCMS, FTIR and NMR

3810159385Biological assay of galanthamine acetate on zebrafish
Biological assay of galanthamine acetate on zebrafish

3.2.1 Synthesis of galanthamine acetate
The enzymatic synthesis of this reaction started with 0.100 g of galanthamine was dissolved in 5 ml of ethanol solution in a small beaker. Then, 0.017 g of acetic acid and 0.005 g of Novozym 435 were put into solution. Next, the beaker was covered in water bath at 55? for 24 hours. The product the was cooled and filtered from enzyme. The steps followed with the mixture was dried into 80? using rotary evaporator until white powder of pure galanthamine was produced. The solvent was purified using solvent mixture of acetone: methanol 3:1 ratio to produce high purity yield of ester. The sample then was kept in the bottle for characterization using analytical instruments.

The optimum condition of synthesis galanthamine acetate using Novozym 435 were predicted using optimisation function of the Design-Expert software. The optimum condition that had been proposed from this software were the temperature at 73.24? about 15 hours operation time, 2.00 wt% of enzyme amount and used 3.42:1 substrate molar ratio for the galanthamine:acetic acid. The synthesis of galanthamine acetate was done based on the optimum condition in order to get high percentage yield of the product.

Percentage of conversion was measured by titration with 0.1 M NaOH (aq) solution in the presence of phenolphthalein as an indicator. The colourless solution turned pink as the end point was obtained. The ester produced was expressed as equivalent to the acid conversion. The amount of acid reacted was calculated from the data obtained for the control samples (without enzyme) and test samples (with enzyme). The percentage of conversion of each sample was obtained in triplicate.

Percentage conversion: (Vc?Vs/)Vc×100
Where,
Vs : The volume of NaOH (with Novozym 435) in mL
Vc: The volume of NaOH used (without Novozym 435) in mL
3.3 Purification, Identification and Characterization of Galanthamine Acetate
3.3.1 Thin layer chromatography analysis (TLC)
Thin Layer Chromatography (TLC) is an analytical technique to identify the one or more components of the mixture, monitor the progress of the reaction and check the purity of the sample. This method is the most quick, simple and effective ways to analyze the small samples. The stationary phase is the polar absorbance usually finely alumina or silica particles the absorbent is coated on glass side or plastic sheet to create layer of stationary phase where the silica plates was used. Alumina, when anhydrous, is the more active, that is, it will adsorb substances more strongly. To separate the more polar substrates such as alcohols, carboxylic acids, and amines, the less active adsorbent, Silica Gel,was used. Then, almost all mixtures of solvent can be used as the mobile phase and used ethyl acetate: hexane (3:7). A capillary tube was used to spot the samples 1 cm from the bottom of TLC plates. Next, the plates were slightly dried after a few minutes developed. When the solvents were half eluted 1 cm from the top, the plate was removed.
3.3.2 Column chromatography
Column chromatography is the methods for separation and purification of the organic compounds based on its polarity using hydrobromide salts, HBR and the stationary phase was silica gel. The principle of column chromatography based on differential of adsorption of substance by the adsorbent. The component of mixture was separated, and they form in bands. The component that was weaker absorbed will move faster to the column and get separated first compared to the component that is highly absorbed that will move slower to the column and get separated at last. The galanthamine was subjected to column chromatography eluted with ethyl acetate: hexane (3:7) on column chromatography.
3.3.3 Gas Chromatography
Gas chromatography is an analytical separation techniques used to analyze the volatile substances in the gas phase. In gas chromatography, the components of a sample are dissolved in a solvent and vaporized in order to separate the analytes by distributing the sample between two phases: a stationary phase and a mobile phase. The mobile phase is a chemically inert gas that serves to carry the molecules of the analyte through the heated column. The solvent used for this sample is hexane:ethyl acetate (7:3). The analysis was analyzed by injecting 0.5 ?L of the sample into Shimadzu Gas Cromatography using nitrogrn gas as the inert gas. The total time required for this analysis to be done is 25 minutes.
3.3.4 Gas Chromatography Mass Spectroscopy
Gas Chromatography-Mass Spectroscopy is a combination of both the process of GC and MS. Its purpose is to separate the chemical elements of a certain compound and identify the molecular level component. In the process, the mixture will be heated in order to separate the elements. Once it vaporizes, it passes the column through an inert gas, most likely to be helium, and proceeds to the mass spectroscopy process. Once the vaporized compound proceeds to the mass spectroscopy process, it will be then separated and its components will be identified through the mass of the analyte molecule. The sample that is send to GCMS lab can be in the form of liquid and also in the form of solid. For solid sample, it is diluted with suitable solvent in this case methanol is used as a solvent.
3.3.4 Fourier transform infrared (FTiR)
Fourier Transform Infrared (FTiR) is analytical technique that identify chemical bond in a molecule by producing an infrared absorption spectrum. This technique widely used to identify organic and inorganic materials. It measures the absorption of infrared radiation by the sample material against wavelength. The infrared absorption bands determine the molecular components and structures. Once a material is irradiated with infrared radiation, absorbed IR radiation usually excites molecules into a higher vibrational state. The wavelength of light absorbed by a particular molecule is a function of the energy difference between the at-rest and excited vibrational states. The characteristic of its molecular structure for a given sample depending on the wavelength that is absorbed.
.

3.3.5 Nuclear magnetic resonance (NMR)
This method is the most common method used to determine different component in the sample. The NMR system exposes the sample to a magnetic field and measures the resultant resonant frequency and absorption energy.  This allows for the recording of characteristic NMR spectra that can be used to identify known compounds in the sample. The instrumentation involved are sample holder, permanent magnet, magnetic coils, sweep generator, radio frequency transmitter, radio frequency receiver and read out system. The solvent normally used in which hydrogen replaced by deuterium are carbon tetrachloride, carbon disulphide, deuteriochloroform, hexa deuteriobenzene and deuterium oxide.
A chemical shift is the exact field strength in ppm of a nuclei comes into resonance relative to a reference standard (TMS Electron clouds “shield” nuclei from the external magnetic field causing then to absorb at slightly higher energy Shielding: influence of neighboring functional groups on the electronic structure around a nuclei and consequently the chemical shift of their resonance.
3.4 The biological assay of galanthamine acetate on zebrafish.
When the galanthamine acetate had been characterized, this research continued by studying its toxicity using zebrafish embryos.There are three parameters that had been study which are survival rate and LC50 measurement, body curvature or scoliosis determination and heart rate measurement of zebrafish embryo trated with sample until 96 hours.
For survival rate and LC50 measurement, zebrafish embryo is treated with sample galanthamine acetate (0.05g) and incubated for 24 hours per exposure. Then, the treated embryo is observed under an inverted or streomicroscope after 24 hours per exposure. The acute toxicity endpoint after 24 hour exposure to test solutions is analyzed. Next, the Dead or Coagulated column.which are”1″ is dead or coagulated and “0” is not considered dead or coagulated is observed.

The parameters for the body curvature and heart rate measurement are the zebrafish embryo is treated with sample galanthamine acetate (0.05 g) and incubated for 96 hours per exposure. The treated embryo is observed under an inverted or streomicroscope after 96 hours per exposure. The researcher read the given Acute toxicity endpoint after 96 hours per exposure test solutions. In body curvature measurement. Next, the body curvature was observed with column consists of “1” is severe scoliosis and “0.5” is light scoliosis also “0” is absence of scoliosis. In heart rate measurement, the. researcher observe presence of heart beat and measure the heart rate on no heart beat column consists of “1” is absence of heartbeat and “0” is presence of heartbeat.

The procedure for danio assay acute toxicity kit firstly the embryo is checked under a streomicroscope to ensure it is alive and are at stage of pharyngula (24 hpf). Then the embryo is transferred into 96-well plate using a transfer pipette with one healthy embryo (24 hpf) added to each well. The most of the fluid is carefully removed from around each embryo using micropipette (Figure 2) and the fluid is replaced with 100 ?L of Danio-sprintM Embryo Media containing 0.1% DMSO (untreated embryo). this step is handled carefully and quickly, the embryo do not allow to dry out. This is embryo plate.
The sample is prepared in another 96- well plate. About 150 ?L of embryo media in row B1-12 until H1-H12. Then, 300 ?L of sample with concentration 0.1:9.9 in row A1-A12. After that, a 2-fold serial dilution is made by transferring 150 ?L of the sample from row A until row G. This sample dilution is conducted using a single channel micropipette or multiple channel micropipette (12 channels). this is dilution plate (Figure 3).

Then, 100 ?L of each sample dilution from dilution plate into embryo plate. The total volume in each well of embryo plate is 200 ?L. The treated emrbyo plate is incubated at 28±2?. The embryo is observed under an inverted or stereomicroscope as alive or dead.

The kit contents which are live zebrafish embryo (wild type), Danio-SprintM Embro Media containing 0.1% DMSO, Danio-GripM Mounting Solution, 96-well plate with cover, disposable petri dish, disposable transfer pipettes, micropipette tips and statistical acute toxicity template.

Figure 2: Kit contents

Figure 3: Dilution plate
Dead or coagulated embryos look milky white, opaque and appear dark under microscope, with cellular degeneration visible and its normal structure is unrecognizable totally and partially. The dead embryo is dicarded with the help of the disposable transfer pipettes together with the part of the old medium. This embryos is fragile hence avoid direct contact with the living embryos.

Figure 4: Normal healthy embryo Figure 5 : Dead or coagulated embryo
CHAPTER 4
RESULT ; DISCUSSION
4.1 Synthesis of galanthamine acetate
This research overall about reacting galanthamine with acetic acid to produce galanthamine acetate using Novozym435 as a catalyst.Acetic acid act as solvent free system since the solvent is the most common solvent to be found. The reaction to synthesis galanthamine acetate is as follows:
2165985899795?
?
34709101720850Galanthamine
Galanthamine
7181851638935Acetic acid
Acetic acid

28136851949452737485213360
303276029845Novozym435
Novozym435
173672558420Ethanol
Ethanol

Galanthamine acetate
Figure 6 : Esterificaiton reaction to produce galathamine acetate
4.2 Purification, Identification and Characterization of galanthamine acetate
4.2.1 Thin Layer Chromatography (TLC)
The first thin layer chromatography need was used to determine the presence of galanthamine during the neutralization process of galanthamine hydrobromide to form galanthamine by removing HBr. Ammonium hydroxide is the solution used to react with the HBr. Different compound can be seen on the silica plate depending on their polarity. Next, the second TLC used to determine galanthamine acetate when reacting the pure gaalnthamine with the acetic acid to form galanthamine acetate. Galanthamine acetate is the more polar compared to the other compound, so it was elute slower with lower retention time. Then, acetic acid is less polar than galanthamine acetate hence it will elute faster and give higher retention time. The solvent used as a mobile phase for this sample is hexane:ethyl acetate (3:7) successfully showed peak before and after purification. The silica plate that contain sample was put in the chamber with mobile phase inside it and let it absorb the solvent from the base line until the front line. Then, the silica plate was immersed into KMnO4 and let it dry upon air.

Based on the figure below, the retention time was calculated based on
Rf =distance from based line travelled by solute/distance from base lined travelled by solvent
Rf = a/b
The retention time was calculated for each of the compound which are galanthamine, acetic acid and galanthamine acetate. Before purification, there were two peak that was observed on the silica plate. The highest retention time is 0.72 for the acetic acid and the other one is for galanthamine acetate compound with retention time about 0.36. This mean that the galanthamine not fully react with acetic acid, hence the column chromatography was needed to get pure galanthamine acetate.

4.2.2 Column Chromatography
Column chromatography was used to purify the galanthamine acetate compound. The ratio of the solvent used was ethyl acetate:methanol(3:1). the column was packed and let it 24 hours before pouring the product. The total volume of solvent is 500 ml where 375 ml methanol and 125 ml ethyl acetate. The amount of vial needed can be more than 40 depending on the polarity of the compound.The vials from 1 until 27 started with less polar compound that was eluted first followed by the desired compound, galanthamine acetate which is more polar. The product of galanthamine acetate was collected from vial 25 until 36. next, the researcher used tlc plate to check on the presence of the galanthamine acetate either it is successfully purified or not fully purified.
After purification, there was only one peak on the silica plate at retention time 0.40. It means that column chromatography is the crucial step that need to be done so that the researcher will get pure target product.

4.2.3 Gas Chromatography Mass Spectroscopy (GCMS)
Gas Chromatography Mass Spectroscopy was used to determine the molecular weight of the desired compound. The expected molecular weight of galanthamine acetate is 347.65 g/mol. The GC-2010 was used as an instrument to run the sample. The column oven temperature was 50°C, the injection temperature was 250°C, injection mode was split, the flow control mode was linear velocity, the pressure was 53.5 kPa,
References:
Lopez, B.C., Cerdan, L.E., Medina, A.R., Lopez, E.N., Valverde, E.M., Pena, E.H., Moreno, P.A.G., Grima, E.M. (2015) Production of biodisel from vegetable oil and microalgae by fatty acid extraction and enzymatic esterification. Journal of Bioscience and Bioengineering. Vol 119. No. 6. pp. 706-711.

Ping Jia, Rong Sheng, Jing Zhang, Liang Fang, Qiajun He, Bo Yang, Yongzhou Hu, (2009) Design,synthesis and evaluation of galanthamine derivatives as acetylcholinesterase inhibitors. Europian Journal Medicinal Chemistry, 44 pp. 772-784.

Rao, V.K., Rao, A.J., Reddy, S.S., Raju, C.N., Rao, P.V., Gsosh, S.K.. (2010). Synthesis, spectral characterizaiton and biological evaluation of phosphorylated derivaitves of galanthamine. Europian Journal of Medicinal Chemistry, 45 pp. 203-209.
Sun, S., Hu, B. (2017) A novel method for synthesis of glyceryl monocaffeate by enzymatic esterification and kinetic analysis. Food Chemistry 214. pp. 192-198.

Stergiou, P-Y., Foukis, A., Filippou, M., Koukouritaki, M., Parapouli, M., Theodorou, L.G., Hatziloukas, E., Afendra, A., Pandey, A., Papamichael, E.M. (2013). Advances in lipase catalyzed esterification reactions. Biotechnology Advance, 31 pp. 1846-1859.

Trinadachari, G.N., Kamat, A.G., Babu K.R., Sanasi P.D.,Prabahar K.J. (2014). Stereoselective syntheses of galanthamine and its stereoisomers by complimentary Luche and L-selectride reductions. Tetrahedron:Assymmetry, 25 pp. 117-124.

Anand, P., Singh, B. (2013). A review on cholinesterase inhibitors for Alzehimer disease, 36 pp. 375-399.

Bertelli, P.R., Biegelmeyer, R., Rico, E.P., Klein-Junior, L.C., Toson, N.S.B., Minetto, L., Bordignon, S.A.L., Gasper, A.L., Moura, S., Oliveira, D.L., Henriques, A.T. (2017) Toxicological profile and acetylcholinesterase inhibitory potential of Palicourea deflexa, a source of ?-carboline alkaloids, Part C 201 pp. 44-50.

Rao, V.K., Rao A.J., Reddy, S.S., Raju, C.N., Rao, P.V., Gsosh, P.V. Synthesis, spectral characterization and biological evaluation of phosporylated derivatives of galanthamine, 45 pp. 203-209.
Lawrence, C. (2007) The husbandry of zebrafish (Danio Rerio) : A review, 269 pp. 1-20.

Shangde, S., Bingxue, H. (2017) A novel method for the synthesis of glyceryl monocaffeate by the enzymatic transesterification and kinetic analysis, 214 pp. 192-198