Feasibility of using Ipil-Ipil (Leucaena leucocephala) Seed Extract as Main Component to Produce Antibacterial Alcohol

An Investigatory Profect presented to the Science and Technology Department

Submitted to: Ms. Ashley Marie Dimanlig Science Adviser

Submitted by: Daniel Torralba Calvin Kyle Narvaez Don Francis Mendoza Enzo Bartolome

I Bl. Nicholas of Gesturi

CHAPTER 1 INTRODUCTION

Background of the Study Alcohols are organic molecules which form a homologous series. It has high market acceptance as medicine. Alcohol has long been widespread used as a solvent of substances intended for human contact or consumption including scents, flavourings, colourings, and medicine. It is also used as a fuel for heat, light, and for combustion engines. It is regarded as a clean fuel. Tropical countries like Philippines, has many species of tropical oil-rich trees like leucaena leucocephala commonly known as Ipil-ipil. Ipil-ipil trees are widely distributed in most localities at low and medium altitudes throughout the Philippines. Ipil-Ipil seeds have been found to contain triterpenoid alcohols1. Statement of the Problem The main problem of this study is to determine the feasibility of producing alcohol from Ipil-ipil seeds (leucaena leucocephala) extract. This study has sub-problems: 1. What will be the density of the extracted alcohol? 2. What will be the boiling point of the extracted alcohol? 3. Is the extracted alcohol soluble in water? 4. Is the extracted alcohol not soluble in salt solution? 5. Will the extracted alcohol have the same pH as that of commercial alcohol?

Antimicrobial and Pharmaceutical Properties of the Seed Oil of Leucaena leucocephala African Journal of Biomedical Research, January 2011

Hypothesis of the Study If the alcohol content of Ipil-Ipil seeds can be extracted economically and in sufficient quantity, then it can be used as alternative source for alcohol. Significance of the Study The potential use of Ipil-Ipil seeds as source for alcohol can help reduce the price of alcohol-based products to the benefit of the general population. Scopes and Limitations The researchers need to determine the physical intensive properties of the extracted alcohol such as density, solubility, pH level, boiling point, color and odor. The researchers need not use advanced test equipment such as gas chromatography or infrared spectroscopy to determine the type of alcohol extracted whether it is methanol, ethanol, propanol or others. If the intensive properties of the extracted alcohol are close to the intensive properties of commercial alcohols, these would already signify that the extracted alcohol is an acceptable antibacterial substitute for commercial alcohols.

Definition of Terms

CHAPTER II RELATED STUDIES AND LITERATURE Alcohols are clear, colourless, flammable mobile liquid that are used as antibacterial and also in antifreeze compounds in lotions and cosmetics, as solvent for shellac, gums and essential oils. Alcohols have a density of 0.785 g/ml up to 0.800 g/ml as the highest. Its boiling point is 25 degrees centigrade, and is soluble in water because alcohols can form hydrogen bonds with water. Alcohols are produced in various ways based on its type. For ethanol alcohol, the method of production is through distillation (see diagram below).

For isopropyl alcohol, the method of production is through the use of a reaction chamber tool. Isopropyl alcohol is produced by combining water and propene in ahydration reaction. There are two routes for the hydration process: indirect hydration via the sulfuric acid process, and direct hydration. The former process, which can use low-quality propene, predominates in

the USA while the latter process, which requires high-purity propene, is more commonly used in Europe2. Indicated below is a sample process for producing Ethyl Alcohol which covers hydrolysis (cooking and cooling of source materials such as starch from grain crops), fermentation and distillation3.

2 3

http://en.wikipedia.org/wiki/Isopropyl_alcohol http://journeytoforever.org/biofuel_library/ethanol_motherearth/meCh1.html

Cinnamyl alcohol is an organic compound that is found in esterified form in storax, balsam Peru and cinnamon leaves. It forms a white crystalline solid when pure, or a yellow oil when even slightly impure. It can be produced by the hydrolysis of storax.4 Hydrolysis is a chemical process in which a water molecule is added to a substance resulting in the split of that substance into two parts. One fragment of the target molecule (or parent molecule) gains a hydrogen ion (H+) from the split water molecule. The other portion of the target molecule collects the hydroxyl group (OH) of the split water molecule. In effect an acid and a base are formed. As an indicator or further proof of alcohol content from Ipil-Ipil seeds, a research study5 showed that Cinnamyl alcohol was isolated from Ipil-Ipil. Leucaena leucocephala cultivar K-636 was used for the studies and it was a selection from Hawaii (Bray et. al. 1998). The cultivar produces erect boles suitable for timber production. For regeneration and transformation experiments, embryo axis of Leucaena leucocephala was used as the ex-plant. Seeds of L. leucocephala were treated with concentrated H2SO4 for 2-3 min and then washed extensively with tap water. The scarified seeds were surface sterilized with 0.1% (w/v) HgCl2 for 10 min followed by five rinses with sterile deionized water. The seeds were soaked in sterile water for two days for imbibing. Embryo was excised from imbibed seeds and was transferred to MS basal medium (Murashige and Skoog, 1962) supplemented with 2% sucrose and 1.5% glucose. One day old excised embryos were used for the transformation experiment.

4 5

http://en.wikipedia.org/wiki/Cinnamyl_alcohol Kulakarni, P (2009) Isolation and Characterization of Cinnamyl Alcohol Dehydrogenase Gene from Leucaena Leucocephala. PhD thesis, National Chemical Laboratory, Pune, India.; http://shodhganga.inflibnet.ac.in/bitstream/10603/2443/8/08_chapter1.pdf http://shodhganga.inflibnet.ac.in/bitstream/10603/2443/9/09_chapter2.pdf

CHAPTER III METHODOLOGY

VARIABLES Independent Variable: Ipil-Ipil Seeds Dependent Variable: Alcohol Constant Variable: Amount of Ipil-Ipil Extracts

INSTRUMENTATION: Reaction Chamber, to distill and to produce the product which will be borrowed on the Science Laboratory or in other schools. Materials for testing: Triple-beam balance, to measure the products mass; Graduated Cylinder, to measure the products volume; Water, to see if the product is soluble with it; Salt Solution; to see if the product is soluble with it; Alcohol lamp, to heat the liquid of the product a to measure its boiling point. RESEARCH PROCEDURE: 1. Creating the Extract 1.1 the cartridge is equilibrated with a non-polar solvent or slightly polar, which wets the surface and penetrates the bonded phase. Then water, or buffer of the same composition as the sample, is typically washed through the column to wet the silica surface.

1.2 the sample is then added to the cartridge. As the sample passes through the stationary phase, the analytes in the sample will interact and retain on the sorbent while the solvent, salts, and other impurities pass through the cartridge. 1.3 After the sample is loaded, the cartridge is washed with buffer or solvent to remove further impurities. Then, the analyte is eluted with a non-polar solvent or a buffer of the appropriate pH. 2. Creating Alcohol: 2.1. prepare the extract, other ingredients excluding the propene ingredient, reaction chamber for the production, and rest of the ingredients in making Alcohol 2.2. place the ingredients into the reaction chamber for fermentation 2.3. gather the fermented liquid which is the alcohol 3. Testing the Product: 3.1. Measuring Density: measure the weight and volume of the product (triple-beam balance for the weight and graduated cylinder for volume) multiply the mass and the volume and subtract the mass of the graduated cylinder 3.2. Measuring the Boiling point: heat the alcohol with the alcohol lamp until it reaches the temperature of 25 degrees centigrade. If it boils, gather the data of its boiling point 3.3. Knowing the Solubility: get a glass/cup, place the water/salt solution pour the alcohol and see if the product mixes with the water/salt solution (for the water, both liquids should be dissolved or unmixed; for salt solutions, it should be mixed