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MANILA SCIENCE HIGH SCHOOL Taft Avenue, Ermita, Manila

INHIBITION OF LEPTOSPIRA CULTURE GROWTH USING Clinopodium douglasii (Yerba Buena) Crude Extract

Nikkole Anne Marie V. Parlade Christine Joy Bernadette R. Angeles Patricia Han Fababeir Allen Belle Pascual Ma. Christina Biol Researchers IV-Newton

Ms. Rovie Pacaldo Research Adviser

INTRODUCTION
Nowadays, Philippines is usually experiencing heavy rains. And of course, if there is rain, there is flood. Leptospirosis, a contagious disease infecting both human and animals, is from an infected animals urine. When mixed with flood, it can affect a lot of human that had contact with the contaminated water. It is a great problem because within just a matter of days, it can cause kidney or liver failure.

Background of the Study

Yerba Buena is a prostrate, smooth , much-branched, usually purplish, strongly aromatic herb, with stems growing up to 40 cm long, with ultimate ascending terminal branches. Leaves are elliptic to oblong-ovate, 1.5 to 4cms long, short-petioled with toothed margins, and rounded or blunt tipped. Flowers are hairy and purplish to bluish, born in axillary head like whorls. Calyx teeth are triangular or lanceolate and hairy; the corolla is also hairy. Introduced by the Spaniards. Widely cultivation to some extent in all parts of the Philippines. Thrives well at high elevations; rarely flowers in lowlands.

In time for the rainy season and tropical storms we experienced, Department of Health (DOH) issued a health advisory to the public to avoid wading in floodwaters due to an increasing number of Leptospirosis cases in the Philippines. He stated that the best way to avoid Leptospirosis is wearing a protective gear such as rubber boots as safety gear against the disease, especially when walking in flooded areas.

The DOH reports that the recorded cases of Leptospirosis from January this year up to 21 May 2011 were 454 cases, yielding a 74.6% higher compared to the same period last year with 260 cases. Thirty-two (32) deaths from the said disease have been recorded this year for the period mentioned, compared to last years 8 mortalities.

Leptospirosis is a potentially fatal infectious disease, especially common in tropical countries with heavy rainfall. Infected rodents and other wild and domestic animals pass the bacteria in their urine.

The bacteria can live for a long time in fresh water, damp soil, vegetation, and mud. Flooding after heavy rainfall helps spread the bacteria in the environment.

Plant yields a volatile oil (0.22%) containing pulegone, menthol, menthene, menthenone and limonene. Study showed the shoot leaf gave the highest yield of oil, 0.62%; while the stems had negligible yield. Menthol was the major component of all the oils. Other oils identified were: B-caryophyllene oxide, a-phellandrene, terpinolene, limonene, menthone and pulegone. Its properties are carminative, stimulant, stomachic, aromatic, antiseptic, antispasmodic, sudorific, emmenagogue. Oil is rubefacient and stimulant. Tops and leaves are carminative. The parts that can be utilized are leaves and stems. Leptospirosis, a potentially deadly disease, is caused by the spirochete Leptospira. Leptospirosis affects both humans and animals, causing a wide range of symptoms in both. In humans, typical symptoms can include fever, headaches, chills, sore muscles, vomiting, jaundice, red eyes, abdominal pain, diarrhea, or rashes. Leptospirosis can become considerably dangerous if not treated, potentially leading to kidney damage, meningitis, liver failure, and respiratory problems. Leptospirosis is typically contracted by humans through water, food, or urine contact with an infected animal (CDC, 2003). It is currently speculated that about one third of infected humans contract Leptospirosis through contact with dogs and about one third from contact with rats (Mar Vista Vet, 2004). Leptospirosis affects animals severely as well. Once in contact with the disease, the organisms spread through the bloodstream then settle in the kidneys to reproduce. In dogs, this leads to fever, loss of appetite, joint pain, nausea, excessive drinking, and jaundice. Organ failure can occur as well, especially in the liver (Mar Vista Vet).

Statement of the problem

The main purpose of this study is to inhibit the growth of Leptospira culture growth using the crude extract from the herbal plant, Clinopodium douglasii (Yerba Buena). It aims to answer the following questions: 1. How effective is the crude extract of Clinopodium douglasii in inhibiting the growth of Leptospira culture in terms of: a. decreasing the population of Leptospira culture? b. duration for the extract to take effort? 2. What are the effects of the crude extract of Clinopodium douglasii to Leptospira culture? 3. What are the components of the crude extract of Clinopodium douglasii that inhibit the growth of Leptospira culture?

HYPOTHESIS
1. The crude extract of Clinopodium douglasii is an effective inhibitor of the growth of Leptospira culture which is determined in 3-7 days. 2. Provided that rats and rodents are irritated with the smell of Clinopodium douglasii, then its crude extract could lessen the population of Leptospira culture 3. The crude extract of Clinopodium douglasii will fail to inhibit the growth of Leptospira culture.

Scope and Limitations

Leptospirosis is a disease that is caused by pathogenic spirochetes of the genus Leptospira. It is considered the most common zoonosis in the world. The researchers are to conduct an experiment by preventing the culture growth of Leptospira. While the Yerba Buena has also been effective to drive away rats and other rodents, because of its strong smell of mint that it emits. Thus, besides its medicinal value, it has long been recognized to have strong commercial demand due to the variety of uses that the plant have. In this research, the researchers will use the Leptospira bacteria and the Yerba Buena. Researchers will test the effect of the medicinal plant could be the prevention of culture growth. It can be proved that it will be an effective preventive plant for leptospirosis.

Significance of the Study

The following will benefit in our study: Obviously, this research study will be a big help to people, especially since nowadays, there are a lot of heavy downpours occurring in the country. The flood that will result from the rain might be a cause of the spread of the bacteria which can infect people by allowing water that has been contaminated by animal urine to come in contact with unhealed breaks in the skin, the eyes, or with the mucous membranes. The research study may help in inhibiting the Leptospira culture growth and stop the spreading of bacteria.

Certain animals such dogs, deer, rabbits, hedgehogs, cows, sheep, raccoons, possums, skunks, and certain marine mammals may be benefited by this study because these animals are considered the secondary hosts because they are able to carry and transmit the disease. These animals might also catch Leptospirosis which can be fatal in their situation.

Other scientists might be studying about Leptospirosis will also benefit from this research. Since they are much more experienced than we are, they can modify and find better ways for applying this kind of study.

The Philippines will also benefit from this research study. It can lower down the trends of Leptospirosis incidents here in the country.

Review of Related Literature

Leptospirosis Leptospirosis is a febrile caused by infection with the bacterium Leptospira interrogans. L. interrogans is sometimes classified as a spirochete because it has a spiral shape. Leptospirosis can be transmitted by many animals such as rats, skunks, opossums, raccoons, foxes, and other vermin. It is transmitted though contact with infected soil or water. People contract the disease by either ingesting contaminated food or water or by broken skin and mucous membrane (eyes, nose, sinuses, mouth) contact with the contaminated water or soil. Leptospirosis symptoms begin from two to 25 days after initial direct exposure to the urine or tissue of an infected animal. This can even occur via contaminated soil or water. Veterinarians, pet shop owners, sewage workers, and farm employees are at particularly high risk. People participating in outdoor sporting activities like canoeing, rafting, hiking, and camping can also come into contact with contaminated water or soil. The illness typically progresses through two phases: The first phase of nonspecific flu-like symptoms includes headaches, muscle aches, eye pain with bright lights, followed by chills and fever. Watering and redness of the eyes occurs and symptoms seem to improve by the fifth to ninth day. The second phase begins after a few days of feeling well. The initial symptoms recur with fever and aching with stiffness of the neck. Some patients develop serious inflammation of the nerves to the eyes, brain, spinal column (meningitis), or other nerves. Right upper area abdominal pain may occur. Less common symptoms relate to disease of the liver, lungs, kidneys, and heart. Leptospirosis associated with liver and kidney disease is called Weil's syndrome and is characterized by yellowing of the eyes (jaundice). Patients with Weil's syndrome can also develop kidney disease and have more serious involvement of the organs affected. In treating leptospirosis antibiotics of choice are penicillin, streptomycin, tetracycline and erythromycin which should be given early in the course of the disease. Intravenous antibiotics may be

required for persons with severe symptoms. Persons with symptoms suggestive of leptospirosis should contact a health care provider as soon as possible. In order to prevent being infected by leptospirosis, you should consider the following: Disease prevention consists of good sanitation. The use of boots and gloves in hazardous places. Protective clothing or footwear should be worn by those exposed to contaminated water or soil because of their job or recreational activities. Rodent control and immunization of farm and pet animals can also minimize the risk of spread. The risk of acquiring leptospirosis can be greatly reduced by not swimming or wading in water that might be contaminated with animal urine. Vaccination of domestic mammals prevents the disease, but cannot avoid the infection, and the shedding of Leptospira in the urine and the consequent human infection. Minimize contact with fresh water and mud that might be contaminated with the urine of infected animals. Minimize contact with fresh water, mud, and vegetation that might be contaminated with the urine of infected animals, especially rodents. If your travel plans might put you at risk for leptospirosis, consider taking antibiotics before and during travel to help prevent infection from shortterm, highrisk exposures. Get rid of rats. Dont touch them with unprotected hands. Consult your vet about the cattle infection. Cover all cuts and broken skin with waterproof plasters before and during work. Wash your hands after handling any animal, or any contaminated clothing or other materials, and always before eating, drinking or smoking.

Leptospirosis cases in the Philippines

Trends in the Philippines:

Leptospira The bacteria have a number of degrees of freedom; when ready to proliferate via binary fission, the bacterium noticeably bends in the place of the future split. Leptospira, both pathogenic and saprophytic, can occupy diverse environments, habitats and life cycles; these bacteria are found throughout the world, except in Antarctica. High humidity and neutral (6.9-7.4) pH are essential for their survival in the environment, with stagnant water reservoirs - bogs, shallow lakes, ponds, puddles, etc. - being the natural habitat for the bacteria. Yerba Buena Yerba Buena (Clinopodiumdouglasii) A small multi- branching aromatic herb commonly known as Peppermint. The leaves are small, elliptical ends with soothed margin. The stem creeps to ground, and develops roots. May also be propagated through cuttings.It has also been effective to drive away rats and other rodents, because of its strong smell of mint that it emits. Thus, besides its medicinal value, it has long been recognized to have strong commercial demand due to the variety of uses that the plant have. Yerba Buena (MenthaSpicata). Genus: Satureja Species: douglasii Variety: None Cultivar:None Common name: Yerba Buena.

Yerba Buena in the Philippines Yerba Buena is cultivated throughout the Philippines, although it does well at higher altitudes. It is grown on Mt. Banahaw. This mint is known as a remedy for weakness of the stomach and for diarrhea. It is valuable as an antiseptic, and has been used in alcohol solutions for asthma. Branched, strongly aromatic herb that stems up to 40 cm long with ascending terminal branches. Leaves are elliptic to oblong-ovate, 1.5 to 2 cms long, short-petioled, toothed margins, rounded or blunt tipped. Hairy purplish to light blue axillary flowers.

Yerba Buena: Scientific name - Clinopodium douglasii and common name - peppermint. It can be used for relieving pain and body aches. It is one of the 10 medicinal plants, which is approved by the Philippine's Department of Health (DOH). Foreign Names of Yerba Buena The plant's most common name, the same in English and Spanish, is an alternate form of the Spanish hierba buena (meaning "good herb"). The name was bestowed by pioneerCatholic priests of Alta California as they settled an area where the plant is native. It was so abundant there that its name was also applied to the settler's town adjacent to Mission San Francisco de Ass. In 1846, the town of Yerba Buena was seized by the United States during the Mexican-American War, and its name was changed in 1847 to San Francisco, after a nearby mission. Three years later, the name was applied to a nearby rocky island; today millions of commuters drive through the tunnel on Yerba Buena Island that connects the spans of the San Francisco Oakland Bay Bridge. Its uses are: for dizziness,headaches, toothaches, flatulence, cough, arthritis.

Methodology Materials
The materials that are needed for the research experiment are Clinopodium douglasii(Yerba Buena). It is used for preventing the culture growth of Leptospira by its crude extract. The crude extract will be applied to the Leptospira bacteria in order to test the effectiveness of this medicinal plant. Another is the, urine of the mus musculus; which is the source of the Leptospira. Where the researchers will test how effective is the Yerba Buena.

Procedure
The Yerba Buena will be collected, after the leaves and stems should be separated. Then, the leaves will be pounded, the stems will be extracted. So, after that procedure the extract will be pured in a test tube. The Leptospira will be cultured. First, mix the agar according to the manufacturer's instructions. Agar is a bit like Jell-o and the ingredients will need to be heated to precise temperatures. Make certain that you have a clean cooking thermometer handy. Then, open a petri dish and pour just enough agar into the dish to cover the bottom completely. Put the lid back on immediately and set the dish aside until the agar firms. Fill each dish the same way, never leaving the top of the dish off any longer than absolutely necessary. Once the agar hardens you may store the dishes in the refrigerator upside down to avoid the possibility of any airborne bacteria getting into the dishes. After, remove the petri dishes from the refrigerator only when you are ready to use them. Open your sterile cotton swab package and rub the cotton swab on a surface you wish to test for bacteria, such as a countertop. Open a petri dish and rub your swab gently across the surface of the agar in an "S" pattern. Close the lid on the petri dish. Then, set the dish in a warm dark cupboard for three to four days. Youll, remove the dish from the cupboard. There is no need to open the dish. Observe the bacteria colonies through the top of the dish. Record their color, shape and anything else distinguishing. Look in a bacteria book or on the Internet for illustrations that match the bacteria you have cultured. And the most important is you should destroy your cultured

bacteria once you have identified it. Pour 1 tbsp. of bleach into the petri dish and then seal the dish closed with packing tape, put the dish in a baggie, seal it and throw away. After all of these procedures the researchers must conduct the data analysis, the testing of the extract and evaluating the results.

Flow Chart
Preparation of Materials and Equipment

Preparation of Yerba Buena

Evaluation of Results

Separating the stems and leaves

Data Analysis

Pounding of the leaves, and extracting the stems

Testing

Putting the extract and the pounded leaves in a test tube.

Destroy your cultured bacteria once you have identified it.

Culturing of Leptospira

Remove the dish from the cupboard. There is no need to open the dish.

Mixing the agar.

Set the dish in a warm dark cupboard for three to four days.

Open a petri dish and pour just enough agar into the dish to cover the bottom completely.

Remove the petri dishes from the refrigerator only when you are ready to use them.

Risk Assessment
In this experiment, the researchers will use the bacteria of Leptospira to determine the effectiveness of Yerba Buena in inhibition of the bacteria. Whereas the bacteria can risk the health or life of the person doing the experiment. So, the researchers must have a supervision of laboratory expert to conduct the experiment.

Disposal
When cleaning a laboratory of bacteria, be sure to properly dispose of materials in specially-marked biohazard containers. If the materials containing the bacteria are nondisposable and are to be used again, you can use a solution of water and bleach or an autoclave to kill bacteria and prevent infection.

Bleach Method 1. Add 1 to 2 milliliters of bleach to each test tube. Allow the bleach to contact the test tube for 15 minutes, then pour the liquid down a drain. 2. Make a water bath containing 10 percent bleach, and soak the instruments for at least one hour. 3. Pour the solution down a drain and allow the instruments to dry completely before re-using. Autoclave Method

1. Place the contaminated instruments in unsealed biohazard bags so the bacteria doesn't leak into the autoclave. Make sure the bags are open so steam can escape. 2. Turn on the autoclave to 15 pounds per square inch (psi) at 121 degrees C for 30 minutes. 3. Allow the biohazard bags to cool. Seal the bags and place in a tightly secured garbage bag. Dispose of the garbage bag.

Bibliography
http://www.filipinoherbshealingwonders.filipinovegetarianrecipe.com/herba-buena.htm http://www.icnhs-herbal-medicine.page.tl/Uses-of-Herba-Buena.htm Source: http://www.buzzle.com/articles/herbal-medicine-in-the-philippines.html Source: http://curbstonevalley.com/blog/?p=6656 Source: http://www.amazines.com/Leptospirosis_related.html http:// http://www.biomedcentral.com/1471-2180/10/31emedicine.medscape.com/article/220563-overview http://www.philippineherbalmedicine.org/ http://www.ehagroup.com/resources/pathogens/leptospira-interrogans/ http://emedicine.medscape.com/article/220563-overview http://www.laspilitas.com/nature-of-california/plants/satureja-douglasii http://medical-dictionary.thefreedictionary.com/leptospirosis http://www.medicinenet.com/leptospirosis/article.htm http://www.aarogya.com/conditions-and-diseases/leptospirosis/3601-prevention-a-treatment-ofleptospirosis.html http://iamjammed.com/health-2/leptospirosis-cases-in-the-philippines-on-the-rise-doh/