Chapter 1
The Problem and its Background
Introduction
The body's trillion of cells face dreadful threats, from lack of food up to infection caused by a virus. Another constant danger comes from vicious chemicals called free radicals. Everyone is not safe in the nature of these free radicals. It comes in many shapes, sizes, and chemical configurations. The body normally generates free radicals as the certain byproducts of turning food into energy. But once these atoms are not controlled, damage occurs in different parts of the body, capable of damaging cells and genetic materials.
According to Jessie Szalay, the body is under constant attack from oxidative stress. Oxygen in the body splits into single atoms with unpaired electrons. Electrons like to be in pairs, so these atoms, called free radicals, scavenge the body to seek out other electrons so they can become a pair. This causes damage to cells, proteins and DNA. (Jessie Szalay, 2016)
There is a chief danger that comes from the damage that free radicals can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs. They are capable of attacking the healthy cells of the body causing damage that appears to be a major contributor to aging and diseases like cancer, heart disease, and decline brain function and immune system.
Based on the journal of A.W. Sarini et.al., primary radicals are reduced to nonradical chemical compounds and then converted to oxidize antioxidant radicals by donating the hydrogen radicals. (A.W. Sarini et.al., 2014) There are toxins that induce free radicals into the body such as, cigarette smoke, metals, pollution and high-oxygen atmosphere.
Antioxidants play an important role in the stabilizing or deactivating free radicals before they attack cells. Aside from the fact that antioxidants were normally produced by the body, it is also best to get from a rich diet of fruits and vegetables. According to an article on The Nutritional Source: Live Life Healthy, It's important to seek out foods that have naturally occurring antioxidants to help fight free radicals and to maintain healthy cells in your body. Antioxidants that occur naturally will be more potent and better able to be absorbed into your body. (12 Natural Foods that Fight Free Radicals, 2016)
Ascorbic acid is a widely used free radical scavenger. It plays an important role in the human body. According to Jose Luis Silencio Barrita and Maria del Socorro Santiago Sanchez, ascorbic acid's antioxidant role is useful since it contributes to the maintenance of the vascular system and the reduction of atherogenesis through regulation in collagen synthesis, production of prostacyclin and nitric oxide. In addition to this antioxidant role, the AA has actions at the molecular level because it acts as a cofactor of enzymes such as dopamine hydroxylase (EC 1.14.17.1), influencing neurotransmitter concentration, improves lysosomal protein degradation and mediates consumer monosodium glutamate. (Jose Luis Silencio Barrita and Maria del Socorro Santiago Sanchez, 2013)
There are several studies supporting that seaweeds contain a wealth of special compounds and antioxidants. It showed potential as a radical scavenger and contained total phenolic compound. The Caulerpa lentillifera itself is highly recommended for the formulation of cosmetic products with skin or hair stimulant, antioxidant, moisturizing and revitalizing activities.
Background of the Study
Free radicals are oxygen molecule containing one or more unpaired electrons, making it highly reactive with other molecules. It can chemically interact with cell components (DNA, lipid and protein) stealing their electrons to stabilize. The production of too much free radical can cause oxidative stress to the body. It can lead to many pathophysiological conditions like some neurodegenerative diseases, cancer, heart and blood disorders, lung and gut disorders, skin lesions, autoimmune diseases and so much more. The damage of oxidative stress arises when DNA inside a cell's nucleus is modified and mutates, in which prompts large chain of free radical reactions.
Free radical reactions indeed cause potential serious damage to the cell, to counteract these; antioxidants donate electrons to free radicals, so that it no longer steals the electron pair of healthy cells. Unfortunately, body's internal productions of these antioxidants are not sufficient to combat all free radicals. By increasing dietary intakes rich of antioxidant property and lifestyle modification which includes staying away from population and vices such as alcohol and smoking also support in defending the body from free radicals.
Conceptual Framework
The researchers will be extracting the crude extract of lato (Caulerpa lentillifera) from the family of Caulerpaceae to study its flavonoid content that shall produce the ability to defend the body from free radicals. In order to identify its flavonoid content, the extract will undergo FTIR to further classify and determine it.
To determine its antioxidant property, the researchers will conduct its Total Phenolic Content and DPPH Free Radical Scavenging Assay. For better results Folin-Ciocalteu reagent method, also called the gallic acid equivalence method, is used for its Total Phenolic Content, because it measures antioxidant capacity in vitro. Also DPPH Radical Scavenging Assay will also be perform, which is determined by UV-VIS Spectrophotometer. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical, the odd electron of its nitrogen atom is reduced by receiving a hydrogen atom from antioxidants to the corresponding hydrazine. Also, it is a rapid, inexpensive and widely used method to measure the ability of compounds and evaluate antioxidant activities.
Figure 1. Paradigm of the Study
General Objectives
This study is conducted to determine the antioxidant property from the flavonoid of Lato (Caulerpa lentillifera J. Ag. Family Caulerpaceae).
Specifically, the researchers will seek to fulfill the following:
1. To isolate and determine the yield of the flavonoids from Lato (Caulerpa lentillifera J. Ag. Family Caulerpaceae);
2. To characterize the physico-chemical property of the extracted flavonoid from Lato (Caulerpa lentillifera J. Ag. Family Caulerpaceae);
3. To determine the concentration of flavonoids extracted from Lato (Caulerpa lentillifera J. Ag. Family Caulerpaceae) that will exhibit antioxidant activity; and
4. To compare the antioxidant property between the flavonoids extract and the standard (Ascorbic Acid).
Significance of the Study
The researchers will be extracting the flavonoid content of the Lato (Caulerpa lentillifera J. Ag. Family Caulerpaceae) to support the previous studies about seaweeds for its antioxidant properties.
The study will be a great help for cancer-risk patients, it can be a reference in lowering the danger of developing and threats of having a cancer. The damages caused by free radicals that results to cancer can be prevented the presence of increased levels of antioxidants.
With that, this antioxidant study will also be a huge help to people with high stress levels, with this as a guide, it can be their supportive reference to lower down their stress level and maintain a healthy lifestyle.
While for the public, this will make them aware of the importance of using natural antioxidants in the prevention of degenerative diseases and maintaining good health. It also promotes propagations of seaweed farming for local fisheries in the area.
In addition, the study will be beneficial to open the mind of everyone in looking into substitutes of alternative medicine. For medical practitioners, this will give them another option of medical advice on their patients. The study will widen their list of choices in prescribing medication.
While for medical researcher, especially pharmacists, it will support innovation of new drugs from marine resources, and with that, the study may broaden their knowledge on the components and possible medicinal use of aquatic resources.
For pharmacy students, this will serve as a future reference for them especially on the topics of natural antioxidants. It will also provide recommendations on how to determine the antioxidant property of a substance. While for medical researchers, the study will support innovation of new drugs from marine resources.
Scope and Delimitation
The study will be performing different tests to determine and support its objectives. From the flavonoid content of the Lato extract, it will undergo FTIR to classify the kind of flavonoid the extract contains and to identify the reference standard to compare. After determining the classified flavonoid content and measuring its percentage yield, concentrations are provided. With this, all concentrations will go through two assays to verify its antioxidant property such as Total Phenolic Assay and DPPH Free Radical Scavenging Assay.
In Total Phenolic Assay, the Folic Ciocalteu reagent will be use. While for DPPH or 2,2-diphenyl-1-picrylhydrazyl Free Radical Scavenging Assay, determination of property will be with the help of a UV-VIS Spectrophotometer reading its absorbance and compare it to the ascorbic acid, the reference standard.
Definition of Terms
The researchers defined the following terms so that the medical terms are clear for every reader of the study. All of the following are related to the study of the researchers antioxidant property of the flavonoids from the extract of Lato (Caulerpa lentillifera J. Ag family Caulerpaceae).
Algae. It has long been used to denote all organisms that produce oxygen as a product of photosynthesis. Jawetz et. al., 2013
Anti inflammatory. It is a drug that inhibits physiological response to cell damage (inflammation). Ansel et. al., 2011
Antimicrobial. It is consequently used to include those active compounds prepared synthetically or isolated from higher plants. Most of the clinically used antimicrobial are produced by soil microorganisms or fungi. Trease and Evans, 2009
Antioxidant. Are substances that prevent oxidation of cell components by donating an electron to free radicals. Vanputte et. al., 2013
Antiviral. It is a drug useful in preventing (rather than treating) viral infections. Ansel et. al., 2011
Ascorbic acid. It is prepared synthetically or by extraction from plant materials such as rose hips, blackcurrants and the juice of citrus fruits. Trease and Evans, 2009
Atoms. It is the smallest particle of an element that has the chemical characteristics of that element. Vanputte et. al., 2013
Cancer. It is a disease characterized by a loss in the normal control mechanisms that govern cell survival,proliferation, and differentiation. Katzung et. al., 2012
Cells. Are the basic structural and functional units of organisms, such as plants and animals. Vanputte et. al., 2013
Cell membrane. It is the outermost component of a cell. The cell membrane encloses the cytoplasm and forms the boundary between material inside the cell and material outside. Vanputte et. al., 2013
DNA. Deoxyribonucleic acid is the genetic material of cells, and copies of DNA are transferred from one generation of cells to the next. Vanputte et. al., 2013
Dopamine. It is the immediate precursor in the synthesis of norephinephrine. Endogenous dopamine may have more important effects in regulating sodium excretion and renal function. Katzung et. al., 2012
Enzymes. It usually acts on one substance or class of substances, since it is specific for a particular atomic group or linkage. Trease and Evans, 2009
Ethanol. It is a small water-soluble molecule that is absorbed rapidly from the gastrointestinal tract. It is the primary form of alcohol that has an important place in the history for the past 8000 years. Katzung et. al., 2012
Genetic. It the branch of science that deals with heredity. Vanputte et. al., 2013
Glutathione. It is a tripeptide that is found in most tissues. Vanputte et. al., 2013
Infection. Are acute when a virus first infects a susceptible host. Viral infections are usually self-limiting. Sometimes, however, the virus persists for long periods of time in the host. Jawetz et. al., 2013
Lysosome. It is a loosening, membrane-bound vesicle containing intracellular digestive enzymes. Vanputte et. al., 2013
Microorganisms. It is the product of evolution, the biologic consequence of natural selection operating on a vast array of genetically diverse organisms. It is useful to keep the complexity of natural history in mind before generalizing about microorganisms, the most heterogeneous subset of all living creatures. Jawetz et. al., 2013
Neurotransmitter. It is the chemical that is released by a presynaptic cell into the synaptic cleft and that acts on the postsynaptic cell to cause a response. Vanputte et. al., 2013
Nitric oxide. It is a gaseous signaling molecule that readily diffuses across cell membranes and regulates a wide range of physiologic and pathophysiologic processes including cardiovascular, inflammatory, and neuronal functions. Nitric oxide should not be confused with nitrous oxide, an anesthetic gas, nor with nitrogen dioxide, a toxic pulmonary irritant gas. Katzung et. al., 2012
Prostacyclin. It is synthesized mainly by the vascular endothelium and is powerful vasodilator and inhibitor of platelet aggregation. It is used clinically to treat pulmonary hypertension and portopulmonary hypertension. Katzung et. al., 2012
Protein. It is a large molecule consisting a long sequence of amino acids linked by peptide bonds. Vanputte et. al., 2013
Vitamin C. It is essential for the normal functioning of living cells and is involved in many enzymatic reactions. It is required for the development of cartilage, teeth and bones, for wound healing and for aiding the absorption of iron from the intestine. Trease and Evans, 2009
Vitamin E. Contained in this group are a number of tocopherols, prefixed ''-, ''-, ''-, etc, which are of wide occurrence in plants, being particularly abundant in germ oil of cereals. It is a powerful antioxidant and has an important role in the preservation of the well-being of cells, for sowing their ageing effects and in counteracting the harmful aspects of toxins in the blood and lungs. Trease and Evans, 2009
Virus. It lacks many of the attributes of cells, including the ability to replicate. Only when it infects a cell does a virus acquire the key attribute of a living system reproduction. Jawetz et. al., 2013