Orthosiphon Stamineus Also Known As Misai Kucing Biology Essay

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Orthosiphon stamineus, also known as Misai Kucing, is belongs to Lamiaceae family and one of the invaluable medicinal plants originated from Southeast Asia. It is also known as Java Tea, Kumis Kucing, Remujung or Cat Whisker's due to the physical characteristics of the plant. O. stamineus were used in variety of applications related to the medicinal purposes and are believed to cure disease such as hypertension, diabetes, urinary disorders, rheumatism, tonsillitis and menstrual disorders [2].

O. stamineus contains several active constituents such as terpenoids, polyphenols, and sterols. The medicinal properties of O. stamineus were attributed by its polyphenols, the most important compound in the leaves which were reported to be effective on reducing oxidative stress by inhibiting the formation of lipid peroxidation products in biological systems [3]. It is already known that the polyphenols in O. stamineus is dominant by caffeic acid.

In this study, the molecular imprint polymer was developing to determine the caffeic acid in Misai Kucing samples. Molecularly imprinted polymer (MIP) is usually obtained by imprinting the polymer on a substrate through a template molecule. This template molecule is the analyte, which produces cavities specific to its detection in a bulk solution. After the extraction of the template molecule, the polymer matrix becomes complementary to the molecule and can rebind it with very high affinity and specificity.

The synthesis of a MIP is based on the polymerization of a certain monomer, containing functional groups complementary to the one present on the target molecule, thus forming adequately strong covalent or non-covalent bonds. A cross-linker is added to co-polymerize with the monomer and produce a rigid polymer network with the desired physicochemical properties. The polymerization takes place in solution, usually initiated by a free radical initiator. After the polymerization is complete the template is removed under mild extraction conditions by using Soxhlet extraction with a polar solvent and the cavities left are complementary to the template in terms of size, shape and functionality, thereby serving as recognition sites for the template used.

In this research, the caffeic acid will be taken as a template to prepare caffeic acid molecular imprint polymer (MIP) biosensor. The caffeic acid imprinted polymer will be immobilized on the Quartz Crystal Microbalance (QCM) sensor for the determination of caffeic acid in misai kucing.

C:\Users\dEyruL\Desktop\misai-kucing-plants.jpg

Figure : Orthosiphon Stamineus

PROBLEM STATEMENT

Caffeic acid is among the major hydroxycinnamic acids present in wine; sinapic acid, which is a potent antioxidant. It has also been identified as one of the active antioxidant. Its conjugates such as chlorogenic and caftaric acids were demonstrated to be more powerful antioxidants in a number of different systems. Caffeic acid and its derivatives are good substrates of polyphenol oxidases, and under certain conditions may undergo oxidation in plant tissues or products of plant origin. The importance of reactive oxygen species (ROS)

and free radicals has attracted increasing attention over the past decade. ROS which include free radicals such as superoxide anion radicals (O2•−), hydroxyl radicals (OH•) and non-free radical species such as H2O2 and singlet oxygen (O2), are various forms of activated oxygen. These molecules exacerbate factors in cellular injury and aging process [10].

Lately, the determination of caffeic acid in plants has been very important in recent research expecially in Misai Kucing. So, there are several physical and chemical methods for the determination of caffeic acid in Misai Kucing and other plants. The most widely used methods for the determination of caffeic acid in plants include various analytical techniques such as spectrophotometer, High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Gas Chromatography Mass Spectrometry (GC-MS), and Liquid Chromatography Mass Spectrometry (LC-MS) and these instruments demand for trained personnel to operate as it is expansive in price. In Misai Kucing also have several other compounds and hence the analysis is difficult. So, a suitable method needs to be developing to determine the caffeic acid in misai kucing samples.

OBJECTIVE

To optimize a suitable cross-linker and functional monomer using computational design and simulation for the preparation of caffeic acid imprinted molecular imprint polymer.

To synthesis MIP based on highest binding energy obtained in molecular modeling.

To immobilized the imprinted polymer on Quartz Crystal Microbalance (QCM) sensor for the determination of caffeic acid in Misai Kucing samples.

LITERATURE REVIEW

Molecular Imprinted Polymer (MIP)

Recently, there have been many active researches on molecular imprint because of the importance in broad range of applications. The need for separation of specific compounds from complex mixtures, industrial or biological, has lead to an increase in the synthesis and use of molecularly imprinted polymers (MIPs). This technology is a rapidly developing technique for the preparation of polymers having specific molecular recognition properties for a given compound [4]. Synthesis of MIP is a relatively straight forward and inexpensive procedure.

The molecularly imprinted polymer is prepared by mixing the template molecule with functional monomers, cross-linking monomers and a radical initiator in a proper solvent [5]. The synthesis of a MIP is based on the polymerization of a certain monomer, containing functional groups complementary to the one present on the target molecule such as methacrylic acid (MAA), 4-vinylpyridine (4-Vpy) and methyl methacrylate (MMA), and thus forming adequately strong covalent or non-covalent bonds. A cross-linker such as ethyl glycol dimethacrylate (EDGMA), trimethylolpropane trimetyacylate (TRIM) any any other example of cross-linker is added to co-polymerize with the monomer and produce a rigid polymer network with the desired physicochemical properties [6].

The polymerization takes place in solution, usually initiated by a free radical initiator [7]. After the polymerization is complete the template is removed under mild extraction conditions such as Soxhlet extraction leaving behind cavities whose size, shape, and chemical functionality complement to the template. These empty cavities can selectively and reversibly rebind molecules identical or similar to the original template.

Functional Monomer

Imprinting components are the most instances reactive monomers which are capable of forming network polymers that are sufficiently stable to maintain a memory of the template. Functional monomers are becoming responsible for the binding interactions in the imprinted binding sites and favor the formation of template. The monomer with functional groups that complement with those found on the template molecule is the one chooses in order to maximize the complex formation and thus imprinting effect [8]. The non-covalent imprinting approach seems to hold more potential for the future of molecular imprinting due to the vast number of compounds, including biological compounds, which are capable of non-covalent interactions with functional monomers.

The most commonly used functional monomer is metacrylic acid (MAA). The strong ionic interactions that MAA can form with basic functional groups on the template, the carboxyl group on this monomer are the best hydrogen bond donor and acceptor. Hydrogen bonding is also the important mechanism of the interaction between the template and functional monomers. Besides, basic functional monomer like triflouromethacrylic acid (TFMAA), vinylpyridine, and vinylimidazole can also participate in the formation of hydrogen bonds and form ionic interactions with acidic template molecules. In this project, MAA is chosen as functional monomer to be synthesis with caffeic acid to develop caffeic acid MIP biosensor.

Cross-linker

The main purpose of the cross-linking monomer is to produce a rigid polymer network with the desired phytochemical [7] and rigidly fix the functional monomer in a place to produce stable binding cavities. In an imprinted polymer, the cross-linker is important in controlling the morphology of the polymer matrix, whether it is gel-type, macroporous or a microgel powder, serves to stabilize the imprinted binding site, and imparts mechanical stability to the polymer matrix [5]. Thus, the cross-linker must be present in very high proportions in the final polymer. Some of the most common cross-linkers are divinylbenzene (DVB), phenylene-diacrylamide, N,N'-Methylene-bisaccrylamide, ethylglycol dimethylcrylate (EDMA) and Trimethylolpropane trimethacrylate (TRIM). In this project, EDMA is chosen as it is rigid cross-linker and presumable to be better imprinted with caffeic acid and MAA.

Computer Design and Simulation

Nowadays, molecular simulation techniques are playing an increasingly important role in the designing and the development of materials for various industrial applications. These simulations are likely to benefit the study of materials by increasing the understanding of the chemical and physical properties at a molecular level and also assisting in the design of new materials and predicting their properties [8]. In this project, the computational design and simulations is chosen in order to design MIP biosensor because it is the cheapest and fastest technique compared to others. Molecular simulations offer a unique perspective on the molecular level processes controlling structural, physical, optical, chemical, mechanical, and transport properties. The software chosen for this computational design is HyperChem 6.0. It will be used to stimulate the polymer properties through molecular modeling and thermodynamics calculations. The ratio of functional monomer to template is determined through this software by comparing the binding energy of each ratio.

Synthesis of MIP

The synthesis of MIP mainly builds up of three main steps which are pre-arrangement, polymerization and removal of template.

Pre-arrangement of the monomers around the template molecule

In the pre-arrangement step, the functional monomers are bind around the template molecule. The chemical structure of the template is used as the starting point for selecting functional monomer candidates especially if the non-covalent approach is followed. Ideally the template molecule should form a stable complex with the print molecule during the pre assembly stage of the polymerization process by obtaining right ratio of synthesis.

There are two kinds of molecular imprinting strategies have been established based on covalent bonds or non-covalent interactions between the template and functional monomers. The non-covalent imprinting approach seems to hold more potential for the future of molecular imprinting due to the vast number of compounds, including biological compounds, which are capable of non-covalent interactions with functional monomers. Therefore, the non covalent imprinting was selected in this project for the preparation of MIP.

Polymerization in the presence of cross-linker

After the pre-arrangement step is complete, the polymerization is done in the presence of cross-linker. The main purpose of the cross-linking monomer is to produce a rigid polymer network with the desired phytochemical [7] and rigidly fix the functional monomer in a place to produce stable binding cavities. Thus, the cross-linker must be present in very high proportions in the final polymer. In this project, bulk polymerization technique was applied due to its simplicity.

Removal of target molecule by extraction process

After the polymerization is complete the template is removed under mild extraction conditions (Soxhlet extraction using a polar solvent) and the cavities left are complementary to the template in terms of size, shape and functionality, thereby serving as recognition sites for the template used. The particles template is washed with an excess amount of 9:1(v/v) MeOH/EtAc [7]. Acetone is used to wash the particles and then dried under vacuum.

Solid phase extraction (Rebinding)

Immobilization of MIP on Quartz Crystal Microbalance (QCM)

METHODOLOGY

Computational Design and Calculation of Energies

Computational design is important in this project in order to predict the interaction of molecular system. The optimization is done for the initial molecular clusters of the simulated monomers and polymers and the binding energy of the molecule is obtained. The molecular structure is constructed using HyperChem 6.0 software.

Synthesis of the Imprinted Polymer

The molecular imprinted polymer is prepared by bulk polymerization technique with the ratio of 1:x:20 for Caffeic acid : MAA : EDMA. The x value is selected after performing simulation using HyperChem software. This simulation technique is actually replaced the conventional of trial and error experiment as a purpose to obtain the stable ratio of monomer to the template or even the selection of suitable monomer in pre-arrangement step.

Pre-arrangement

A non-covalent imprinting method was used for the preparation of caffeic acid imprinted polymers. The MIP is prepared with different ratio of Caffeic acid to MAA from the binding energy obtained from the simulation technique. Caffeic acid and functional monomer (MAA) is dissolved in 10 ml of acetonitrile. Then, EDMA (20 mmol) as a cross-linker and 0.05 g AIBN as an initiator is added to the solution and stirred for 5 minutes. NIP sample is also prepared with the same procedure as MIP but without the presence of caffeic acid.

Polymerization

Bulk polymerization is used for the preparation of MIP and NIP. The prepared solution from pre-arrangement is sonicated and sparged under oxygen-free N2 for 3 minutes. The glass tube is sealed under nitrogen and then placed in a water bath with the temperature at 60OC for 24 hours. After that, the resultant polymer monolith is crushed with mortar and transferred to grinder machine before entering sieving machine. Sample is sieved using automatic sieving machine for 10 minutes to obtain sample that pass through 75µm to 35µm sieve.

Removal of template

In this step, the template which is caffeic acid is removed from the polymer and leaving the cavity inside the MIP. The instrument used for this step is soxhlet extractor with a mixture of methanol/acetic acid (9:1, v/v). The caffeic acid removed is collected in the round bottom flask containing a mixture of methanol-acetic acid during the extraction. When operating soxhlet extractor, MIP and NIP samples is placed inside the cellulose extractor thimble and mixture of acetic acid and methanol is heated to reflux. The solvent vapor travels up on distillation arm and floods into the chamber housing thimble of solid. The condenser ensures that any solvent vapor cools, and drips back down into a chamber housing the sample. The timble is slowly filled with warm solvent and the caffeic acid is dissolved in it. When the soxhlet chamber is almost full, the chamber is automatically emptied by a siphon arm, with the solvent running back to the distillation flask and a cycle was completed. Glass bead is placed to increase the speed in order to accomplish a cycle.

Rebinding of Template

The main purpose of rebinding is to measure the efficiency of the MIP and NIP as recognition element by means that to bind caffeic acid molecule into the cavity created during polymerization. In solid phase extraction (SPE), MIP and NIP act as stationary phase where distilled water acts as mobile phase that pass through the SPE column. The adsorption isotherm can be obtained by adding incremental amounts of template to the polymer, allowing the system to equilibrate and then measuring the concentration of free template in solution. The concentration of caffeic acid inside the polymer is obtained by subtraction of initial concentration with the non-bounded caffeic acid that is collected at the bottom of the cartridge. The concentration of the caffeic acid in remaining free solution is measured by Quatz Crystal Microbalance (QCM).

GANTT CHART

ACTIVITY

SEMESTER 1

SEMESTER 2

Sep

Oct

Nov

Dec

Jan

Feb

Mac

April

May

1-15

16-30

1-15

16-31

1-15

16-30

1-15

16-31

1-15

16-31

1-15

16-28

1-15

16-31

1-15

16-30

1 - 19

20-31

Title Selection

Literature Review

Proposal Writing

Progress Proposal Submission

Proposal Defend

Order Chemical and Equipment

Experiment Started

Progress Thesis Submission

Viva Voce

Final Thesis Submission

MILESTONE

Milestone

Date

Proposal writing, proposal submission and proposal defense

October - November 2012

EXPECTED OUTCOMES

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