TABACO NATIONAL HIGH SCHOOL TABACO CITY S.Y.

2011-2012

Utilization of Makahiya extracts as mosquito and filarial nematodes terminator

Proponents:

Louell Jude Que John Bert Onrubia Cyril Bonganay

Year & Section: IV-Einstein

Research Adviser: Mr. Ronaldo Reyes

Abstract
Since mosquitoes have already cleared its name as one of the deadly insects that have ever lived, it would be more practical to focus on how to terminate these insects. And to increase its practicality, available sources are utilized. This study aims to project a cleaner view of killing mosquitoes and to create a treatment for its effect such as filariasis, which involved the characters of the stated insect. Thus, the study also focuses on how to squeeze out the maximum potential of the stated herb that would be the cure to the aging commercial views of people on terminating mosquitoes such as the use of chemical sprays and other pesticides and smoking. These commercial practices have also inflicted the worlds ecological balance which we can say that the root all comes from mosquitoes, so the study concentrated on making an alternative yet effective and eco-friendly, affordable, and available sources of: protection as to the family not to get inflicted by filariasis and other mosquito related epidemics, a model as to other people who used to do the commercial practices and a cleanser as to the earth not to be greatly affected by very harmful chemicals.

Acknowledgement
The study aimed to introduce the combination of the two potentials of a natural, perennial herb makahiya, in order to make an eco-friendly, healthy and practical applications on eliminating mosquitoes and treatment for filariasis. Nevertheless, this study wont be possible without the everlasting love, care and assistance from the researchers family, who supported them in the procurement of the much needed logistics and references, cultivated and propagated the makahiya plants for the study, and uplifted the researcher emotionally when they are taken aback with discouragement. The researcher would never forget to acknowledge Mister Ronaldo Reyes, who extended their invaluable time and patience in explaining the whole course of the research and gave words of encouragement. Furthermore, the researchers would like to thank her classmates for the shared information during casual talk and discussions. Most of all, to God our Father, the source of all strength and wisdom, who gave us the strength and all the hopes and blessing they can receive during the times of their lives, for without His unfaltering love and guidance this study would never be realized.

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Table of Contents
Abstract Acknowledgement CHAPTER I Introduction Background of the study Statement of the problems/objectives Significance of the study Scope and limitations CHAPTER II Review of related literature CHAPTER III Methodology CHAPTER IV Results and Discussion Findings Analysis of Data CHAPTER V Conclusions Recommendations Bibliography .i ii 1 .. 1 .. 1 2 3 .. 4 5 5-8 .9 .. 9 10 10-11 .. 12 .. 12 13 . 13 14 . 14

CHAPTER I - INTRODUCTION

I.

Background of the Study

Developing a mosquito-free environment has already been one of the challenges the Philippines is facing. There are already increasing cases of mosquito epidemics all over the Philippines and this remain as one of the biggest problem the country is struggling with. The large population of mosquitoes is one of the influential factors that trigger to the complication of the different epidemics that arouse from mosquitoes; the other factor is the great distribution of their habitat. There are many naturally growing weeds like makahiya which has unknown power to create a defense mechanism against deadly insects like mosquitoes. Makahiya contains the flavonoid mimosine which has antiproliferative and apoptotic effects. Flavonoids are water-soluble polyphenolic molecules containing 15 carbon atoms. Flavonoids also have the potential to carry out an effect to mosquitoes that would lower down their metabolic processes and immobilize them to increase the probability of death. Thus, in order to create an atmosphere with active flavonoids on air, makahiya is a great deal. The extracts of makahiya contain these flavonoids that could terminate mosquitoes. Therefore, makahiya has also the capacity of inducing an effect to other life forms of mosquito that would help to terminate active filarial nematodes that causes filariasis - an infection caused by mosquito bite. Filariasis is a parasitic disease and is considered an infectious tropical disease, which is caused by thread-like filarial nematodes which are carried by mosquitoes. This disease is one of the terrors in human health system and is a great challenge on the medical institution in the Philippines. Nevertheless, an early prevention is the greatest cure a person can do by just utilizing available sources like makahiya. In order to gain flavonoid, one should extract them from makahiya then utilize the chemical to create a defense mechanism against makahiya. Thus, the chemical an also be used to manufacture a treatment against the infectious tropical disease- filariasis. By doing so, one can already help creating a mosquito free-environment and a healthy human system. 1

II.

Statement of Problem/Objectives
A. Objectives 1. General Objective : a. To manufacture a mosquito and filarial nematodes terminator from the extracts of Makahiya (Mimosa pudica). b. To decrease filariasis cases on human population and decrease the mosquito population using Makahiya flavonoid mimosine. 2. Specific Objective : a. To test the effectivity of Makahiya extracts in terminating mosquitoes and filarial nematodes -which are great factors of filariasis b. To determine the accurate amount of Makahiya extracts needed to maximize the effectivity of terminating mosquitoes and filarial nematodes B. Problems 1. General Problems: a. What are the processes involve in making the mosquito and filarial nematodes terminator using Makahiya as an alternative source to the requirements? b. What is the significant effect of using Makahiya as an alternative source for creating the treatment? 2. Specific Problems: a. What are the parts of Makahiya that have the significant amount of mimosine? b. What extracts are still needed to manufacture the treatment?

III.

Significance of the Study:

Highly educated people living in urban cities and surrounded by modernized hospitals seemed to ignore the importance of herbal medicinal plants like Makahiya (Mimosa pudica), more so those who are well of do not subscribe to any medicinal plants used by ethnic groups. However due to increase and high prices of medicines sold at the drugstores people who belong to the poverty status are compelled to use medicinal plants. This study is about utilizing Makahiya. Makahiya is a naturally growing weed. Therefore, it is one of the towering medicinal plants that are readily available in the Philippines. Due to increasing cases of filariasis and other mosquito related diseases, this study aims to generate a product by utilizing the flavonoid mimosine from Makahiya that can be subjected into transforming into a mosquito and filarial nematodes terminator. To students: The proposed study serves the students as their reference or guide in creating their program. It will also help students taking computer related courses to identify the best programming language to use. To teachers: The proposed study will help teachers to have a deeper understanding to the said programming languages. By this study they will come up with easier and powerful program. To future researcher: The proposed study will benefits and help the future researcher as their guide. The study can also open in development of this study. The study is served as the first step in the discovery of the bioactive compounds of Makahiya leaves. These serve as references for other researchers of the similar study.

IV.

Scope and Limitations

This study provides relevant information regarding the gravity about filariasis and mosquito-related situations in the world as well as in our country. The study also focuses on Makahiya as an alternative source for eliminating mosquitoes and for the treatment of filariasis, an asymptomatic infection caused by mosquitoes, of persons having such disease to avoid further complications on their current problems. The study will be conducted in Tabaco City, Albay with its inhabitants. The study involves a field-work, interviews on the townspeople and observations of their activities and lifestyle that may contribute to their existing problem. The study aims to lower the number of people developing persistent filariasis which can lead to acute manifestations which include Acute Adeno-Lymphangitis (ADL) and Acute Filarial Lymphangitis (AFL). Because of the increasing number of people having filariasis, this study will help them lower their chances of having more complications of acute manifestations and lower the chance of pre-filariasis people on acquiring the said complications.

The study focused on the specialization on how to produce a quality mosquito eliminator and treatment for filariasis, the Makahiya, as the alternative over commercial formulation, is observed conscientiously for proper storage and usage of the product. But, this study is not due for other mosquito-related diseases and is only focused on creating a mosquito-free environment and filariasis-free residents of our country.

CHAPTER II V.

Review of Related Literature

Makahiya is a diffusely spreading, half-woody herb, with branched stems up to 1 meter long, sparingly prickly with numerous deflexed, bristly hairs. Makahiya is also a common weed widely distributed in the Philippines in open, moist, waste places, open grasslands and open thickets, at low and medium altitudes in settled areas, which is introduced from tropical America, and also considered as a Pan tropic weed. The Makahiya Seed contains a toxic alkaloid, mimosine, a non-protein alpha-amino acid, known to cause hair loss and depressed growth in mammals (an unlikely event in humans as this will require unusually large doses).Its roots yield flavonoids, phytosterol, alkaloids, amino acids, tannins, glycoside, fatty acids. Its leaf extract have yielded an adrenaline-like substance.Seeds contain a mucilage composed of d-xylose and d-glucoronic acid, yielding 17% greenish yellow fatty oil.The whole plant contains (1) tubulin with an ability to bind colchicene with its sulfhydryl groups. (2) crocetin dimethyl ester. The chemical properties of Makahiya are considered expectorant, antiasthmatic, analgesic, antispasmodic, alterant, sedative and antidepressant. The roots are bitter, astringent, acrid, alexipharmic, antispasmodic, aphrodisiac, constipating, cooling, diuretic, emetic, febrifuge, resolvent, and vulnerary. Leaves are bitter, sudorific, tonic. Study showed antimicrobial activity against Aspergillus fumigatus, Citrobacter divergens and Klebsiella pneumonia. Study showed intraperitoneal use of Mimosa pudica decoction protected mice against pentylentetrazol and strychnine-induced seizures. Plant extracts showed the plant to be a moderate diuretic, depresses duodenal contractions (similar to atrophine), promotes nerve regeneration and reduce menorrhagia.Study of the methanolic extract exhibited good wound healing activity, an effect attributable to phenol constituents. The investigation showed antivenom activiety in the alcoholic extract of Mimosa pudica, Mucuna pruriens, and Andrographis paniculata. Study on the aqueous extract of dried roots of Mimosa pudica showed significant inhibitory effect on Naja naja and Bangarus caerulus venoms. Of 17 plants screened, only M pudica showed 100% ability in neuralizing venom lethality. Study showed the potential use of M pudica as an antivenom agent of plant origin against five poisonous snake venoms found in Malaysia. Study isolated Fonsecaea from the thorns of M pudica and suggests it could be a natural source of infection for the fungus Fonsecaea pedrosoi. Study showed the dissolution profile from formulation containing mucilage to drug in the proportion of 1:40 was found to be similar to the commercial sustained-release formulation of diclofenac.

Study suggests that M pudica produces antidepressant effect in rats with a profile similar to two tricyclic antidepressants.Mimosine found to be an iron chelator acting on malarial bugs by preventing the replication of cells.Mimosine also causes apoptosis and studied for treating ovarian cancer and other highly vascularized tumors. The Anthelmintic effect of Makahiya (Mimosa pudica) leaves Extract in Native Chicken (Gallus domesticus) naturally infected with Gastro-intestinal Parasites. Study showed the co-administration of Mimosa pudica aqueous extract significantly lowered the level of lipid peroxidation in alcohol-fed mice. Mimosa pudica is one of eight medicinal plants in an Ayuvedic herbal formulation, Ilogen-Excel, showing antihyperglycemic effect in STZ-induced diabetic rats. Study showed Mimosa pudica extract possess nerve-regenerative potential in rats with sciatic nerve injury. In rats with experimentally injured sciatic nerves, nerve regeneration was 30-40% higher in rats treated with M. pudica extract than the hydrocortisone treated group. In a study of alloxan-induced diabetic rats, the ethanolic extract of Mimos pudica showed significant decrease of blood glucose level compared with Metformin as standard drug. Comparative antioxidant, antibacterial and general toxicity studies on extracts of two Bangladeshi medicinal plants, M pudica and M rubicau, showed both exhibited prominent antioxidant property. While M. rubicaulis did not show any antibacterial activity, M pudica displayed considerable bacteriostatic activity against all six bacterial strains tested - B cereus, B subtilis, E coli, ampicillin-resistant E coli, S aureus and P aeruginosa. In an acute toxicity study, a single dose of aqueous extract of 2000 mg KBW showed no clinical signs of toxicity or mortality. Study also showed dose-dependent central and peripheral analgesic property. Chloroform extract exhibited significant hypolipidemic activity. Results suggest that biologically activity phytoconstituents such as flavonoids, glycosides alkoloids may be responsible for the activity. Study showed tannins obtained from M. pudica was better than tannic acid in neutralizing the lethality of N. kaouthia venom in vitro. Results suggest M. pudica has a potential for treating N. Kaouthia snakebites. Mosquitoes Mosquitoes are members of a family of nematocerid flies: the Culicidae (from the Latin culex, genitive culicis meaning midge orgnat). Superficially, mosquitoes resemble crane flies (familyTipulidae) and chironomid flies (family Chironomidae), and as a result casual observers seldom realise that there are important differences between the members of the respective families and also differences between their habits. In particular, many species of female mosquitoes are blood-sucking pests and dangerous vectors of diseases, whereas members of the similar-looking Chironomidae and Tipulidae are not. Over 3,500 species of mosquitoes have already been described from various parts of the world. Some mosquitoes that bite humans routinely act as vectors for a number of infectious diseases affecting millions of people per year. Others that do not routinely bite humans, but are the vectors for animal diseases, may become disastrous agents for zoonosis of new diseases when their habitat is disturbed, for instance by sudden deforestation. 6

Filariasis is a parasitic disease and is considered an infectious tropical disease, that is caused by thread-like nematodes (roundworms) belonging to the super family Filarioidea, also known as "filariae". Eight known filarial nematodes use humans as their definitive hosts. These are divided into three groups according to the niche within the body they occupy: 'lymphatic filariasis', 'subcutaneous filariasis', and 'serous cavity filariasis'. Lymphatic filariasis is caused by the worms Wuchereria bancrofti, Brugia malayi, and Brugia timori. These worms occupy the lymphatic system, including the lymph nodes, and in chronic cases these worms lead to the disease elephantiasis. Subcutaneous filariasis is caused by loa loa (the African eye worm), Mansonella streptocerca, andOnchocerca volvulus. These worms occupy the subcutaneous layer of the skin, in the fat layer. Serous cavity filariasis is caused by the worms Mansonella perstans and Mansonella ozzardi, which occupy the serous cavity of the abdomen. In all cases, the transmitting vectors are blood-feeding arthropods, mainly black flies and mosquitoes. Individuals infected by filarial worms may be described as either "microfilaraemic" or "amicrofilaraemic", depending on whether or not microfilaria can be found in their peripheral blood. Filariasis is diagnosed in microfilaraemic cases primarily through direct observation of microfilaria in the peripheral blood. Occult filariasis is diagnosed in amicrofilaraemic cases based on clinical observations and, in some cases, by finding a circulating antigen in the blood. The most spectacular symptom of lymphatic filariasis is elephantiasisedema with thickening of the skin and underlying tissueswhich was the first disease discovered to be transmitted by mosquito bites. Elephantiasis results when the parasites lodge in the lymphatic system. Elephantiasis affects mainly the lower extremities, while the ears, mucous membranes, and amputation stumps are affected less frequently. However, different species of filarial worms tend to affect different parts of the body: Wuchereria bancrofti can affect the legs, arms, vulva, breasts, and scrotum (causing hydrocele formation), while Brugia timori rarely affects the genitals. Interestingly, those who develop the chronic stages of elephantiasis are usually amicrofilaraemic, and often have adverse immunological reactions to the microfilaria, as well as the adult worms. The subcutaneous worms present with skin rashes, urticarial papules, and arthritis, as well as hyper- and hypopigmentation macules.Onchocerca volvulus manifests itself in the eyes, causing "river blindness" (onchocerciasis), the second-leading cause of blindness in the world. Serous cavity filariasis presents with symptoms similar to subcutaneous filariasis, in addition to abdominal pain, because these worms are also deep tissue dwellers. Filariasis is usually diagnosed by identifying microfilariae on Giemsa stained, thin and thick blood film smears, using the "gold standard" known as the finger prick test. The finger prick test draws blood from the capillaries of the finger tip; larger veins can be used for blood extraction, but strict windows of the time of day must be observed. Blood must be drawn at appropriate times, which reflect the feeding activities of the vector insects. Examples are W. bancrofti, whose vector is a mosquito; night time is the preferred time for blood collection. Loa loa's vector is the deer fly; daytime collection is preferred. This method of diagnosis is only relevant to microfilariae that use the blood as transport from the lungs to the skin. 7

Polymerase chain reaction (PCR) and antigenic assays, which detect circulating filarial antigens, are also available for making the diagnosis. The latter are particularly useful in amicrofilaraemic cases. Spot tests for antigen are far more sensitive, and allow the test to be done any time, rather in the late hours. Lymph node aspirate and chylus fluid may also yield microfilariae. Medical imaging, such as CT or MRI, may reveal "filarial dance sign" in chylus fluid; X-ray tests can show calcified adult worms in lymphatics. The DEC provocation test is performed to obtain satisfying number of parasite in day-time samples. Xenodiagnosis is now obsolete, and eosinophilia is a nonspecific primary sign. Human filarial nematode worms have complicated life cycles, which primarily consists of five stages. After the male and female worms mate, the female gives birth to live microfilariae by the thousands. The microfilariae are taken up by the vector insect (intermediate host) during a blood meal. In the intermediate host, the microfilariae molt and develop into third-stage (infective) larvae. Upon taking another blood meal, the vector insect injects the infectious larvae into the dermis layer of the skin. After about one year, the larvae molt through two more stages, maturing into the adult worms. In 1993, the International Task Force for Disease Eradication declared lymphatic filariaisis to be one of six potentially eradicable diseases.[4]Studies have demonstrated transmission of the infection can be broken when a single dose of combined oral medicines is consistently maintained annually for approximately seven years. With consistent treatment and since the disease needs a human host, the reduction of microfilariae means the disease will not be transmitted, the adult worms will die out, and the cycle will be broken. The strategy for eliminating transmission of lymphatic filariasis is mass distribution of medicines that kill the microfilariae and stop transmission of the parasite by mosquitoes in endemic communities. In sub-Saharan Africa, albendazole (donated by GlaxoSmithKline) is being used withivermectin (donated by Merck & Co.) to treat the disease, whereas elsewhere in the world albendazole is used with diethylcarbamazine. Using a combination of treatments better reduces the number of microfilariae in blood. Avoiding mosquito bites, such as by using insecticidetreated mosquito bed nets, also reduces the transmission of lymphatic filariasis. The recommended treatment for patients outside the United States is albendazole (a broad spectrum anthelmintic) combined withivermectin. A combination of diethylcarbamazine (DEC) and albendazole is also effective. All of these treatments are microfilaricides; they have no effect on the adult worms. In 2003, the common antibiotic doxycycline was suggested for treating elephantiasis. Filarial parasites have symbiotic bacteria in the genus Wolbachia, which live inside the worm and which seem to play a major role in both its reproduction and the development of the disease. Clinical trials in June 2005 by the Liverpool School of Tropical Medicine reported an eight-week course almost completely eliminated microfilaraemia. 8

CHAPTER III

VI.

Methodology
In order to make the desired experimental product, the following requirements are gathered and prepared: 200 grams Makahiya leaves, water, mortar and pestle, kalamansi extracts, filterer/juice extractor, candle wax, jar, 120proof vodka, cooking pan, and a container/molder. The first thing to do is to soak first the Makahiya leaves in water for about 5-10 minutes in order to loosen up/weaken the cellulose of the leaves thus, creating a passage for the oil. After the time being, drain the water and let dry the leaves. Then, crush the leaves using mortar and pestle. Extract the juice from the crushed Makahiya leaves using a filterer or a juice extractor. Then, in a small jar, pour 200 milliliters 120proof vodka and the extracted juice. Cover the jar for about 30 minutes. Then, shake the mixture. Let the vodka settle then, freeze the mixture. After the oil has been freezed, scoop the layer formed on top of the vodka. Prepare for the next step. Heat a cooking pan in a stove. Put the candle wax and let it liquefy. After liquefying the wax, put the extracted oil to the wax and stir. Put out of the fire and let it condense for about 10 minutes. Put the extracted glucose in the container first. Once the temperature has lowered to about 20-25 degrees Celsius, pour into a molder or a container. Finally, preserve the product in a cold temperature.

Treatments and general procedure: To test the effectivity of the alternative product, the researchers planned the experimental set-ups. First, the researchers identified the statistics needed. The following elements were identified: there will be four residential areas to be tested, each having an average population of mosquitoes of 138. The experiment was given the time frame of 8 hours starting at 8:00 p.m. to 4:00 a.m. then; the researchers decided to start the experiment on the month of December and checked every detail in every first day of the week.

CHAPTER IV

VII.

Results and Discussion

The results of the sample analysis yielded consistent data throughout the sample frame. The experimental data on the effect of the Makahiya extracts on the population of the mosquito on the different residential areas yielded the following results:

The effect of Makahiya mosquito terminator on the population of mosquitoes in the different residential areas on the first week of December
Location of the experiment (50 sq. m.) Libod, Malinao Quinale, Tabaco Panal, Tabaco Ziga Building- Boys C. R. (TNHS) Average Population of mosquitoes before after 170 40 130 25 160 40 90 15

The effect of Makahiya mosquito terminator on the population of mosquitoes in the different residential areas on the second week of December
Location of the experiment (50 sq. m.) Libod, Malinao Quinale, Tabaco Panal, Tabaco Ziga Building- Boys C. R. (TNHS) Average Population of mosquitoes before after 170 40 130 25 160 40 90 15

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The effect of Makahiya mosquito terminator on the population of mosquitoes in the different residential areas on the third week of December
Location of the experiment (50 sq. m.) Libod, Malinao Quinale, Tabaco Panal, Tabaco Ziga Building- Boys C. R. (TNHS) Average Population of mosquitoes before after 170 40 130 25 160 40 90 15

The effect of Makahiya mosquito terminator on the population of mosquitoes in the different residential areas on the last week of December
Location of the experiment (50 sq. m.) Libod, Malinao Quinale, Tabaco Panal, Tabaco Ziga Building- Boys C. R. (TNHS) Average Population of mosquitoes before after 170 40 130 25 160 40 90 15

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VIII.

Findings

Analysis of Data

Preparation of the product was easy as to so many cases and conditions met during the experiments, Makahiya showed a great potential in a form of a candle as a terminator of mosquito insects. Thus, the production also of a treatment for filariasis has come to an additional advantage for it would be produced after the mosquito terminator was already used up. As stated from the earlier mentioned data, the insecticidal property of Makahiya has reached a more than average percentage of the total potential compared to other commercial and chemical sprays to kill mosquitoes. From the stated experimental set-ups on specific conditions and locations, the Makahiya showed a magnificent potential as explicitly informed through the numbers of mosquitoes left and mosquitoes that died. The experiment showed that one candle would be able to kill at least 75% of the total population of mosquitoes.

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CHAPTER V

Conclusions
It would be clear to all then that the utilization of Makahiya as mosquito and filarial nematodes terminator has its availability at its peak, that the Makahiya has 100% chance to be found and to be utilized easily due to its abundances. Thus, creating the product was simple so it is a great advantage to people at rural places since they are more prone to mosquito attacks than at urban places, though the effect or the damage that can be caused by an urban mosquito is greater than that of the earlier stated. The production of the product also involved many processes such as extracting its juice to squeeze out the oil or mimosine from Makahiya. Though it was easy to make the product, careful handling of the extracts should be observed, for the chemical from the Makahiya has other chemical components such as toxins that are harmful to the human body system, thus, the product should still be kept and preserved out of reach of children.

The product has 89%-95% efficiency that could clearly state that the Makahiya has a great potential on creating a great divergence on the ecological balance because at least 5% only of the gases expelled by the product mixes with the atmosphere thereby concluded to have a great potential clearing numerous number of mosquitoes within the activated time of diffusion.

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Recommendations

Additional recommended research involving an analysis on the potential effect of Makahiya extracts on the population of the mosquitoes and on its effect on the filarial nematodes carried-out by the adult mosquitoes that causes a more complex illness called filariasis would include a point source investigation on the effect of the chemical composition of the Makahiya extracts to the immunity of mosquitoes. A point source investigation could be as simple as a series of one time profiles to seek out the likely sources of the implications of Makahiya on a particular insect such as on a mosquito. A more detailed approach might involve a series of profiles to determine the hot spots and follow up with a network of regular interval monitoring stations and periodic profiles.

Bibliography
http://www.freesciencefairproject.com/requirements.htm http://www.stuartxchange.com/Makahiya.html http://tl.wikipedia.org/wiki/Makahiya http://en.wikipedia.org/wiki/Mosquito http://en.wikipedia.org/wiki/Filariasis

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