EFFECTIVENESS of VIOLA ODORATA LEAF EXTRACT on MYCOBACTERIUM TUBERCULOSIS

An Undergraduate Thesis Presented to the Faculty of the College of Allied Health Sciences Department of Medical technology Cagayan State University Andrews Campus, Tuguegarao City

In Partial fulfilment of the Requirements for the Degree Bachelor of Science in Medical Technology

by Ethel Vida Mae C. Antonio Rona Joy M. Calimag Nikki Anne B. Garlejo Joimarie S. Liquigan

March, 2012

Approval Sheet This research proposal entitled, EFFECTIVITY of VIOLA ODORATA LEAVES EXTRACT on MYCOBACTERIUM TUBERCULOSIS, is prepared and submitted by Ethel Vida Mae C. Antonio, Rona Joy M. Calimag, Nikki Anne B. Garlejo, and Joimarie S. Liquigan in partial fulfilment of the requirement for the Research Methodology has been examined and is hereby recommended for acceptance and approval for oral examination. ___________________________ Adviser PANEL OF EXAMINERS Approved by the Committee on Oral Examination with a grade of ____________.

________________ Chairman _________________ Member __________________ Member

_________________ English Critic Approved and applied in partial fulfilment of the requirements for the Research Methodology.

Date _______________

_________________ Dean

Table of Contents

Acknowledgement

Chapter 1 THE PROBLEM AND ITS BACKGROUND Introduction Increasing population and poverty are major problems in the country which contribute to the emergence of different diseases having high mortality rate, one of these diseases is Tuberculosis. The increase in population and poverty is not confined not only in one place, but the whole country rendering tuberculosis a common ailment amongst Filipinos. Tuberculosis is one of the leading causes of death in the world. It is a potentially fatal contagious disease that can affect almost any part of the body but is mainly an infection of the lungs. Along with its collaboration with HIV, and the emergence of Multidrug resistant and extensively drug resistant Mycobacterium tuberculosis strains, it continues to be one of the diseases with high mortality rates around the globe. One third of the worlds population is affected with TB. In the World Health Organization Global TB report of 2009, the Philippines rank ninth on the list of 22 highburden tuberculosis (TB) countries in the world. After China, it had the second highest number of cases in the WHO Western Pacific Region in 2007, and TB is the sixth greatest cause of morbidity and mortality in the country. In 2007, approximately one hundred Filipinos died each day from the disease, but significant strides have been made in increasing case detection and treatment. In 2004, the country achieved a TB case detection rate of 72 percent, exceeding WHOs target of 70 percent, and reached seventy five percent in 2007. The DOTS (the internationally recommended strategy for TB

control) treatment success rate reached WHOs target of 85 percent in 1999 and has remained around 88 percent since then. While the national performance levels are already high, many provinces are still below target levels due to various systemic and social factors, including the difficulty of breaking down the stigma of TB, which keeps many of those infected from seeking care. Also, most of the people infected with TB are poor. Since squatter areas, with overcrowding and unventilated and unclean air in their environment, people there are more likely to acquire the infection, adding poverty as one of the factors of the continuous spread of TB. Tuberculosis is a contagious bacterial disease caused by Mycobacterium tuberculosis. Historically, it was then endemic in animals in the Palaeolithic period, long before it ever infected humans. It was not regarded as a major disease then, until the population of the world and unventilated environment increased. It was also then known as consumption in all ages and climates. For example, tuberculosis was the subject of a hymn in a sacred text of India dating from 2,500 BC, while DNA unique to Mycobacterium tuberculosis was identified in lesions from the lung of 1000 year old human remains in Peru. Mycobacterium tuberculosis bacteria can be transmitted via respiratory droplets. Once an individual with TB disease of the lungs or throat coughs, sneezes, speaks, or sings, these respiratory droplets containing the bacteria can stay in the air for several hours, depending on the environment and can be inhaled which can lead to an infection, although close contact is usually necessary for the infection to occur.

In healthy people, infection with Mycobacterium tuberculosis often causes no symptoms, since the persons immune system acts to wall off the bacteria. 15 % to 20% of the people infected with M. Tuberculosis develop the disease. TB will usually occur some years after the initial infection, when the patients immune system breaks down for some reason other than the presence of tuberculosis bacilli within the lung. In a small percentage of infected hosts, the disease becomes systemic, affecting variety of organs. Tuberculosis is treatable with a six month course of antibiotics. Therapy against M. tuberculosis is dependent on the susceptibility of the isolate to various antimicrobial agents. To prevent the selection of resistant mutants, treatment of tuberculosis requires two or three drugs. The most common two drug regimen is Isozianid (INH) and Rifampin administered for 9 months in the case of uncomplicated tuberculosis. However, TB incidences became more serious and complicated when other strains of mycobacterium tuberculosis developed. These are the Multi-drug resistant (MDR) and extensively drug resistant (XDR) strains. Multi-drug resistant Tuberculosis (MDR-TB) is defined as an in vitro resistance of Mycobacterium tuberculosis (MTB) to at least rifampin and isoniazid. Multi-drug resistance (MDR) has become a major concern to control TB particularly in the developing countries. The development of mutations in different genes of mycobacterium leads to drug resistance and subsequent MDR-TB. Management of MDR-TB entails intense chemotherapy for up to 2 years which is very damaging to a patients health due to high levels of drug toxicity.

The emergence of resistance to antimicrobials, though is a natural biological occurrence, has become an important public health issue in many developing countries as the treatment of TB requires the use of more expensive drugs for a longer treatment period, which increases the risk of drug toxicity in patients. There is, therefore, an urgent need for new, inexpensive TB drugs which are more effective and with fewer side effects. Medicinal plants offer a hope for developing alternate medicines for the treatment of TB. In a study of aqueous extracts of ten medicinal plants tested for antibacterial potential against strains of human pathogenic bacteria, Viola odorata was found to be the most effective antibacterial. The present study was done to evaluate in vitro antitubercular activity of Viola odorata. Medicinal and edible, the flowers and leaves of viola are made into syrup used in alternative medicine mainly for respiratory ailments associated with congestion, coughing, and sore throat. Violet plants are considered as anti-inflammatory, anticancer, demulcent, diuretic, emetic, expectorant, and purgative. The flowers contain an odorous principle, blue coloring matter, a glucoside and salicylic acid, a natural aspirin. Elemental analysis showed C, O, Na, Mg, Al, Si, Cl, and Fe in different parts of the plant. The leaves, flowers and roots are parts that can be used. The aim of this study is to evaluate if the antimicrobial activity of Viola odorata encompasses activity against M. Tuberculosis. With the emergence of MDR, an alternative drug which is efficient enough to prevent the development of resistance of the bacteria to antimicrobial drugs is needed as early in the beginning of the infection. MDR is resistant to the first line of drugs against TB. The activity of Viola odorata will be tested along with these drugs for comparison.

Statement of the problem The researchers in their study attempt to ascertain the determination of antimicrobial activity of Viola odorata leaves extract on Medical Technology. Specifically it answers the following questions: 1. What is the profile of the respondents in test of: a. Age b. Sex Male Female

c. Monthly Income d. Profession e. Location Rural Urban

2. What is the medicinal value of the Viola odorata leaves extract as a probable cure to Tuberculosis? 3. What is the level of effectiveness of the Viola odorata leaves extract as an antimicrobial agent? Assumptions The leaf extract of Viola odorata has a medicinal value as an antimicrobial agent against Mycobacterium tuberculosis. Viola odorata has other health benefits as antimicrobial agent.

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Viola odorata as an antitubercular agent provides a cheaper and more effective medicine. Conceptual Framework of the Study This research is guided with the World Health Organization that emphasizes the

need for greater innovation for strategy, diagnostics and new drugs, and universal access to health services, in order to successfully fight tuberculosis. With resistance to current drugs a persistent threat, new, effective drugs for tuberculosis are urgently needed. This research is guided with the World Health Organization that emphasizes the need for greater innovation for strategy, diagnostics and new drugs, and universal access to health services, in order to successfully fight tuberculosis. With resistance to current drugs a persistent threat, new, effective drugs for tuberculosis are urgently needed. Medicinal plants prove to be a successful alternative remedy. Many studies have been conducted involving medicinal plants and their anti-tubercular activity. One example would be the study involving Garlic which was proven successful against Multi drug resistant Mycobacterium tuberculosis and non multi drug resistant Mycobacterium tuberculosis. This study focuses on the Antimicrobial activity of Viola odorata which is the Independent variable of our study and its effectiveness against the bacteria, Mycobacterium tuberculosis in which the microorganism may be resistant or susceptible. The bacterias susceptibility and resistance from the extracts of Viola odorata are our Dependent and Intervening variables respectively.

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Figure 1 Independent Variable Intervening Variable Dependent Variable

Anti-microbial activity of Viola odorata

Resistance of Mycobacterium tuberculosis to Viola odorata

Susceptibility of Mycobacterium tuberculosis to Viola odorata

Figure 1.1 The paradigm illustrating the relationship of the independent, dependent and intervening variables on the Antimicrobial activity of Viola odorata against Multi-Drug Resistant Mycobacterium tuberculosis isolates. Significance of Study The results of this study will benefit the following. Medical Practitioners and Medical Students: The proposed study serves the medical practitioners and medical students as their reference or guide that would help them provide an alternative remedy for their patients infected with Mycobacterium tuberculosis. The Department of Health (DOH): it could give them a more affordable and practical way to offer a cure to people infected with tuberculosis. Future Researchers: it would serve as a basis for them to develop the study, use other plants and improve the methods used.

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Definition of Terms For purpose of clarification, the following terms are hereby defined: Asymptomatic Pulmonary TB Case. It refers to an individual without symptoms of pulmonary TB and is found to have one of the following: a) radiographic abnormalities consistent with PTB and at least one sputum specimen positive for AFB; b) previous chest x-ray normal, current chest x-ray shows abnormalities consistent with PTB, and 3 sputum acid-fast smears are negative; c) previous chest x-ray showed abnormality consistent with PTB. (Dorland) Bacillus Calmette-Gurin (BCG). It is a vaccine for tuberculosis made from a weakened mycobacterium that infects cattle. (Zinsser) Chemosensitization. It refers to the process of altering the susceptibility of a target cell or organism, such that a therapeutic agent, having become ineffective, becomes effective again; observed with antineoplastic and antiparasitic drugs. (MediLexicon) Complement System. It helps or complements the ability of antibodies and phagocytic cells to clear pathogens from an organism. It is part of the immune system called the system that is not adaptable and does not change over the course of an individual's lifetime. (Turgeon) Extra-Pulmonary Tuberculosis. It is tuberculosis affecting organs other than lungs, most frequently pleura, lymph nodes, spine, joints, genitourinary tract, nervous system or abdomen. (Dorland)

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Fluorescence Microscopy. It refers to a method in which a sample is illuminated with light of a wavelength which causes fluorescence in the sample. (Strasinger) Fragment Crystallizable Receptor (Fc Receptor). It is a protein found on the surface of certain cells - including natural killer cells, macrophages, neutrophils, and mast cells - that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc region. (Turgeon) In Vitro. It refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. (Henry) Isoniazid. It is a tuberculostatic antibacterial. Also known as INH (isonicotinic acid hydrazide). Its indications are prescribed for prophylaxis for those who have been exposed to tuberculosis and are used in combination with other agents in the treatment of tuberculosis caused by mycobacterium sensitive to the drug. (Katzung) Latent TB Infection. It refers to individuals who have inhaled the TB bacteria sometime in the past and have been found to have a positive tuberculin skin test and NO clinical, bacteriological or radiographic evidence of active TB are said to have Latent TB Infection (LTI). (Dorland) Mannose Receptor. It is a C-type lectin carbohydrate binding protein primarily present on the surface of macrophages and dendritic cells. It helps recognize pathogens

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that have mannose on their surface, and triggers one pathway of the complement system. (Turgeon) Mantoux Test or the QuantiFERON-TB Gold Test. It is also known as the Mantoux screening test, Tuberculin Sensitivity Test, Pirquet test, or PPD test for Purified Protein Derivative is a diagnostic tool for tuberculosis. (Henry) Mycobacterium tuberculosis. It refers to the complex mycobacterium that is transmitted from person to person and, therefore, is of public health importance. (Henry) Nucleic Acid Amplification Testing. It refers to the direct detection of M. tuberculosis complex should be performed on respiratory specimens from patients with suspected tuberculosis. (Henry) Prophylactic Use of Isoniazid. INH can be given for the prevention as well as the treatment of TB. INH is effective when given daily over a period of six to 12 months to people in high-risk categories. (Katzung)

Pulmonary Tuberculosis. It refers to disease involving the lung parenchyma. It is the most frequent form of the disease, occurring in over 80% of cases. This form of tuberculosis may be infectious. (Dorland) Rifampicin. It is a derivative of rifamycin; an antibacterial and antifungal agent used in the treatment of mycobacterial infections, actinomycosis and histoplasmosis. Its Therapeutic class is Antitubercular. (Katzung)

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Superoxide. By the obsolete name hyperoxide, it is a compound that possesses the superoxide anion with the chemical formula O2. (Champe) Symptomatic Pulmonary TB Case. It refers to an individual with symptoms of pulmonary TB. Symptoms of pulmonary TB include cough for two or more weeks duration and one or more of the following signs and symptoms: fever, sputum expectoration, significant weight loss, hemoptysis or recurrent blood-streaked sputum, and chest and/or back pains not referable to any musculo-skeletal disorders, other symptoms such as chills, fatigue, body malaise, and shortness of breath. (Dorland) SYTOX Green. Dead Cell Stain is a bright, easy-to-use nucleic acid stain for distinguishing dead from live cells in flow cytometry assays. It is excitable at 488 nm with an emission profile similar to FITC, and has been tested in an assortment of cell types. Advantages with SYTOX Green Dead Cell Stain: Bright Signal, Rapid Staining, Validation, Optimization, and Versatility. (Wikipedia) Trafficking Pathway. It undoubtedly evolved alongside cellular

compartmentalization and their complexity reflects the need to maintain at a functional steady state a very dynamic entity, the cell. Tuberculosis. It is a potentially fatal contagious disease that can affect almost any part of the body but is mainly an infection of the lungs. It is caused by a bacterial microorganism, the tubercle bacillus or Mycobacterium tuberculosis. (Henry)

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Chapter 2 REVIEW OF RELATED LITERATURE AND STUDIES Tuberculosis, or TB, is an infectious bacterial disease caused by Mycobacterium tuberculosis, which most commonly affects the lungs. It is transmitted from person to person via droplets from the throat and lungs of people with the active respiratory disease. The microorganism enters the body by inhalation through the lungs. They spread from the initial location in the lungs to other parts of the body via the blood stream, the lymphatic system, via the airways or by direct extension to other organs. In healthy people, infection with Mycobacterium tuberculosis often causes no symptoms, since the person's immune system acts to wall off the bacteria. The symptoms of active TB of the lung are coughing, sometimes with sputum or blood, chest pains, weakness, weight loss, fever and night sweats. Only people who are sick with TB in their lungs are infectious. When infectious people cough, sneeze, talk or spit, they propel TB germs, known as bacilli, into the air. A person needs only to inhale a small number of these to be infected. Left untreated, each person with active TB disease will infect on average between 10 and 15 people every year. But people infected with TB bacilli will not necessarily become sick with the disease. The immune system "walls off" the TB bacilli which, protected by a thick waxy coat, can lie dormant for years. When someone's immune system is weakened, the chances of becoming sick are greater. People with latent TB infection have TB germs in their bodies, but they are not sick because the germs are not active. These people do not have symptoms of TB disease, and they cannot spread the germs to others. However, they may develop TB disease in the future. They are often prescribed treatment to prevent them from developing TB disease.

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People with TB disease are sick from TB germs that are active, meaning that they are multiplying and destroying tissue in their body. They usually have symptoms of TB disease. People with TB disease of the lungs or throat are capable of spreading germs to others. They are prescribed drugs that can treat TB disease. There are two tests that can be used to help detect TB infection. The Mantoux tuberculin skin test is performed by injecting a small amount of fluid (called tuberculin) into the skin in the lower part of the arm. A person given the tuberculin skin test must return within 48 to 72 hours to have a trained health care worker look for a reaction on the arm. A second test is the QuantiFERON-TB Gold test. The QuantiFERON- TB Gold test is a blood test that measures how the patients immune system reacts to the germs that cause TB. A positive tuberculin skin test or QuantiFERON-TB Gold test only tells that a person has been infected with TB germs. It does not tell whether or not the person has progressed to TB disease. Other tests, such as a chest x-ray and a sample of sputum, are needed to see whether the person has TB disease. Bacille Calmette-Gurin (BCG) is a vaccine for TB disease. BCG is used in many countries, but it is not generally recommended in the United States. BCG vaccination does not completely prevent people from getting TB. It may also cause a false positive tuberculin skin test. However, persons who have been vaccinated with BCG can be given a tuberculin skin test or QuantiFERON-TB Gold test. If you have latent TB infection but not TB disease, your doctor may want you to take a drug to kill the TB germs and prevent you from developing TB disease. The

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decision about taking treatment for latent infection will be based on your chances of developing TB disease. Some people are more likely than others to develop TB disease once they have TB infection. This includes people with HIV infection, people who were recently exposed to someone with TB disease, and people with certain medical conditions. Tuberculosis disease can be treated by taking several drugs for 6 to 12 months. It is very important that people who have TB disease finish the medicine, and take the drugs exactly as prescribed. If they stop taking the drugs too soon, they can become sick again; if they do not take the drugs correctly, the germs that are still alive may become resistant to those drugs. TB that is resistant to drugs is harder and more expensive to treat. In some situations, staffs of the local health department meet regularly with patients who have TB to watch them take their medications. This is called directly observed therapy (DOT). DOT helps the patient complete treatment in the least amount of time. Transmission of M. tuberculosis is a recognized risk to patients and HCWs in healthcare facilities. Transmission is most likely to occur from patients who have unrecognized pulmonary or laryngeal TB, are not on effective anti-TB therapy, and have not been placed in TB isolation. Increases in the incidence of TB have been observed in some geographic areas; these increases are related partially to the high risk for TB among immunosuppressed persons, particularly those infected with human immunodeficiency virus (HIV). Transmission of M. tuberculosis to HIV-infected persons is of particular concern because these persons are at high risk for developing active TB if they become infected with the bacteria. Thus, health-care facilities should be particularly alert to the

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need for preventing transmission of M. tuberculosis in settings in which HIV-infected persons work or receive care. Jordao et. al. (2011), in her article on Tuberculosis: New Aspects of an Old Disease, the history of tuberculosis (TB) mixtures with the history of humanity since TB is one of the oldest infectious diseases affecting mankind. Bone TB was identified in 4000 years old skeletons, from Europe and Middle East, as the cause of death, showing that this disease was already a widespread health problem back then. In recorded history, Hippocrates writes about patients with wasting away associated with chest pain and coughing, frequently with blood in sputum. These symptoms allowed Hippocrates to diagnose TB, which at that time was called consumption. The frequency of descriptions of patients with these symptoms indicated that the disease was already well entrenched in ancient times. The interaction of M. tuberculosis with host cells is complex and far from being fully elucidated. The entry of M. tuberculosis into macrophages seems to occur via cholesterolrich domains (rafts) of the plasma membrane, being mediated by receptor binding and phagocytosis. Despite the numerous in vitro studies that clearly identify different receptors involved in M. tuberculosis uptake, mainly by macrophages and dendritic cells, the results obtained in vivo in receptor-deficient animals did not support the in vitro data. In this scenario, it is almost consensual that in vivo mycobacteria uptake is made by multiple receptors, such as C-type lectin receptors, complement receptors and scavenger receptors, rather than by a single receptor-mediated pathway, implying the activation multiple signalling cascades.

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The majority of the in vitro studies indicate that the bacilli favour interaction with complement and mannose receptors, which are benign, because they trigger minimal superoxide production. In contrast, mycobacteria uptake by Fc receptors, which play a minor role in the absence of specific antibodies, would trigger a vigorous host response and would establish a distinct intracellular trafficking pathway. This might explain why virulent mycobacteria avoid internalization by these receptors. However, the majority of experimental data suggest that the receptor type has little impact on intracellular survival of the bacteria. In 2008, Asian Americans (26%) surpassed African Americans (25%) as the second largest racial or ethnic group in the number of TB cases in the U.S.19 Asian Americans represented less than 3 percent of TB cases in U.S.-born persons, but 43 percent of TB cases in foreign-born persons. Four out of the top five countries of origin of birth for foreign-born TB cases were in Asia: Philippines, Vietnam, India and China. During that same year, 69 TB cases were diagnosed in Native Hawaiians and Pacific Islanders. As WHO estimates that the largest number of new TB cases in 2008 occurred in the South-East Asia Region, which accounted for 35% of incident cases globally. However, the estimated incidence rate in sub-Saharan Africa is nearly twice that of the South-East Asia Region with over 350 cases per 100 000 population. An estimated 1.7 million people died from TB in 2009. The highest number of deaths was in the Africa Region.

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In 2008, the estimated per capita TB incidence was stable or falling in all six WHO regions. However, the slow decline in incidence rates per capita is offset by population growth. Consequently, the number of new cases arising each year is still increasing globally in the WHO regions of Africa, the Eastern Mediterranean and South-East Asia. Overall, one-third of the world's population is currently infected with the TB bacillus. 5-10% of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life. People with HIV and TB infection are much more likely to develop TB. Until 50 years ago, there were no medicines to cure TB. Now, strains that are resistant to a single drug have been documented in every country surveyed; what is more, strains of TB resistant to all major anti-TB drugs have emerged. Drug-resistant TB is caused by inconsistent or partial treatment, when patients do not take all their medicines regularly for the required period because they start to feel better, because doctors and health workers prescribe the wrong treatment regimens, or because the drug supply is unreliable. A particularly dangerous form of drug-resistant TB is multidrug-resistant TB (MDR-TB), which is defined as the disease caused by TB bacilli resistant to at least Isoniazid and Rifampicin, the two most powerful anti-TB drugs. The aim was to undertake a comprehensive analysis of ethical issues in TB and to lay the groundwork for the formulation of WHO guidance in order to help governments and other stakeholders to implement TB care and control programmers in an ethical manner.

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Consequently, WHO listed several kinds of TB. This may include pulmonary tuberculosis, Symptomatic pulmonary, asymptomatic pulmonary TB case, Extrapulmonary tuberculosis, and Latent TB Infection. Pulmonary tuberculosis refers to disease involving the lung parenchyma. It is the most frequent form of the disease, occurring in over 80% of cases. This form of tuberculosis may be infectious. Symptomatic pulmonary TB case refers to an individual with symptoms of pulmonary TB and is found to have one of the following: a) at least two sputum specimens positive for acid-fast bacilli (AFB), with or without radiographic abnormalities consistent with PTB; b) one sputum specimen positive for AFB and with radiographic abnormalities consistent with PTB; c) one sputum specimen positive for AFB with sputum culture positive for M. tuberculosis; and d) all three sputum specimens negative for AFB but with radiographic abnormalities consistent with PTB, with no history of antiTB treatment and with a previous normal chest x-ray. Symptoms of pulmonary TB include cough for two or more weeks duration and one or more of the following signs and symptoms: fever, sputum expectoration, significant weight loss, hemoptysis or recurrent blood-streaked sputum, and chest and/or back pains not referable to any musculo-skeletal disorders, other symptoms such as chills, fatigue, body malaise, and shortness of breath. Asymptomatic pulmonary TB case refers to an individual without symptoms of pulmonary TB and is found to have one of the following: a) radiographic abnormalities consistent with PTB and at least one sputum specimen positive for AFB; b) previous

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chest x-ray normal, current chest x-ray shows abnormalities consistent with PTB, and 3 sputum acid-fast smears are negative; c) previous chest x-ray showed abnormality consistent with PTB, current chest x-ray shows progression of radiographic abnormality, and 3 sputum acid-fast smears are negative or N.B. If current CXR shows abnormality consistent with PTB and 3 sputum specimens are negative for AFB, but no previous CXR is available and the patient does not fulfil the criteria for PTB, follow-up CXR and sputum examination should be done at least a month after. Extra-pulmonary tuberculosis is tuberculosis affecting organs other than lungs, most frequently pleura, lymph nodes, spine, joints, genitourinary tract, nervous system or abdomen. Tuberculosis may affect any part of the body. Latent TB Infection Individuals who have inhaled the TB bacteria sometime in the past and have been found to have a positive tuberculin skin test and NO clinical, bacteriological or radiographic evidence of active TB are said to have Latent TB Infection (LTI). Persons with LTI are not infectious to others and the identification of LTI is therefore, NOT a part of the NTP. The NTP prioritizes the identification of infectious pulmonary TB cases. If a private physician wishes to determine if a person has LTI, the cost of the Purified Protein Derivative (PPD) used for tuberculin skin testing must be shouldered by the patient, physician or other entity outside of the NTP. Currently, TB is treated with an initial intensive 2-month regime comprising multiple AntibioticsRifampicin (RIF), Isoniazid (INH), Pyrazinamide (PZA), and Ethambutol (EMB) or Streptomycin (SM)to ensure that mutants resistant to a single drug do not emerge. The next 4 months, only RIF and INH are administered to eliminate

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any persisting tubercle bacilli. INH and RIF, the two most potent antituberculous drugs, kill more than 99% of tubercule bacilli within 2 months of initiation of therapy. Along with these two drugs, PZA, with a high sterilizing effect, appears to act on semi dormant bacilli not affected by any other antitubercular drugs. Using these drugs in conjunction with each other reduces antitubercular therapy from 18 months to 6 months. Therefore, the emergence of strains resistant to either of these drugs causes major concern, as it leaves only drugs that are far less effective, have more toxic side effects, and result in higher death rates, especially among HIV-infected persons. The phrase MDR state in mycobacteriology refers to simultaneous resistance to at least RIF and INH (with or without resistance to other drugs). In the study of Antil et. al.(2011) entitled Evaluation of the Analgesic Activity of Viola odorata aerial parts in rats, the phytochemical screening of Viola odorata revealed the presence of alkaloids, steroids, glycosides, flavonoid, saponin, phenolic compounds, and tannins. Alkaloids, flavonoids, and steroids have been reported to have the ability to inhibit pain perception. Pranting et. al.(2010) had the same study on Viola odorata entitled The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gramnegative bacteria. In this study, they have examined the activity of cyclotides against bacteria. Of the five peptides examined (cyO2, kalata B1, kalata B2, vaby A and vaby D), cyO2 was by far the most potent. Although kalata B2 had some activity against S. aureus, and vaby A and D at concentrations of 3050 mM inhibited the growth of E. coli, their potency was much lower than that observed for cyO2, and these peptides were therefore not studied further. In timekill assays, cyO2 killed all tested Gram-negative bacteria

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including S. enterica serovar Typhimurium LT2 (DA6192), Escherichia coli MG1655 (DA4201), P. Aeruginosa (DA10176) and three K. pneumoniae strains (DA11895, DA11896 and DA15000), while the Gram-positives which includes Staphylococcus aureus (DA7127), Staphylococcus epidermidis (DA10656) and Streptococcus pyogenes (DA7121) were more resistant to treatment. The antibacterial effect of cyO2 was influenced by the composition of the media, with lower activity observed in rich media or media containing salt (data not shown). Similar effects of media components were seen in a study by Tam et al. and are also a common feature among other types of AMPs, which nevertheless are active against bacteria in biological systems. In his conclusion he stated that CyO2 is a cyclotide with potent activity against Gram-negative bacteria. The charged residues in cyO2 are all required for optimum antibacterial activity. In combination with its previously demonstrated cytotoxic activity against cancer cells and the general stability of cyclotides, cyO2 provides a promising scaffold for future drug design. Gerlach et. al. In (2010), stressed on their article entitled Anticancer and Chemosensitizing Abilities of Cycloviolacin O2 from Viola odorata and Psyle Cyclotides from Psychotria leptothyrsa that Cycloviolacin O2 (CyO2), a cyclotide from Viola odorata (Violaceae) has antitumor effects and causes cell death by membrane permeabilization. In breast cancer line, MCF-7 and its drug resistant subline MCF7/ADR, the cytotoxic effects of CyO2 (0.2-10micrometer) were monitored in the presence and absence of doxorubicin (0.1-5 micrometer) using cell proliferation assays to

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establish its chemosensitizing abilities. SYTOX Green assays were performed to verify membrane permeabilization and showed cellular disruption correlates with cyclotide chemosensitization. Fluorescence microscopy studies demonstrated increased cellular internalization of doxorubicin in drug resistant cells when coexposed to CyO2. Interestingly, CyO2 did not produce significant membrane disruption in primary human brain endothelial cells, which suggest cyclotide specificity toward induced pore formation in highly proliferating tumor cells. Furthermore, three novel cyclotides (psyle A, C and E) from Psychotria leptothyrsa (Rubiaceae) were also monitored for cytotoxic activity. The cyclotides displayed potent cytotoxicity (IC50 = 0.39-0.76 mirometer). This studies several cyclotides with robust cytotoxicity that may be promise chemosensitizing agents against drug resistant breast cancer. A correspondence was exemplified by Ireland et. al. (2006) on a Novel Suite of Function and Stability which states that Cyclotides are a fascinating family of plantderived peptides characterized by their head-to tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the cyclic cystine knot, is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities but their insecticidal activities suggest that their primary function is in plant defense. In this study the researchers determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. The researchers identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological

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activities of cyclotides appear to be associated with membrane interactions, we used hemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the cyclic cystine knot motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens. In summary, this work highlights the extensive sequence diversity of cyclotides expressedin V. odorata. Although amino acid sequence variations considerably influenced the activity of the new cyclotides, only minimal changes to the structure and proteolytic stability of the cyclotide framework occurred, owing to the stabilizing influence of the conserved CCK motif. The study reinforces the suggestion that cyclotides have a conserved scaffold onto which a combinatorial array of sequences is displayed for host defense purposes. The new sequence variations observed suggested that the cyclization mechanism is even more robust than previously anticipated and that further diversity in cyclotide sequences can be expected.

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CHAPTER 3 MATERIALS AND METHODS Research Designs Descriptive and experimental design were utilized in the study, particularly, twogroup design. This design is most appropriate because two comparable groups were employed as experimental and control groups. In this study, there were three groups namely, Rifampicin, Isoniazid, and Viola odorata. Rifampicin and Isoniazid were the control groups, and the experimental group was the leaf extract of Viola odorata. Scope and Limitation The proposed study is limited only to the determination of the anti-mycobacterial properties of the leaf extract of Viola odorata. The experiment part of this study deals mainly with the collection and preparation of samples, the bacterial isolates will come from hospitals like Cagayan Valley Medical Center and St. Paul hospital. The Disk diffusion Susceptibility test will be used to test for the activity of the leaf extracts of Violata odorata against Mycobacterium tuberculosis. Only 2 first line drugs will be used for comparison, Isoniazid and Rifampicin. The violet plant to be used will be taken from Benguet and Baguio. Only the leaf extract will be used in the study. It also aims to study the concentration of the maximal antitubercular activity of the plant. The study did not attempt to isolate the main constituents which showed antimicrobial properties.

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The study will be conducted at the Clinical Laboratory of the College of Allied Health Sciences, Cagayan State University, Tuguegarao city, in January 2012 to February 2012. Materials The materials used in this proposed study were Viola odorata leaves (macerated), 50 grams; Viola odorata extract; Mycobacterium tuberculosis isolates; Trypticase soy broth, 5 milliliters; Mueller Hinton agar; and sterile saline. Table 1 Materials used on the Extraction and Disk diffusion Susceptibility Test Materials Viola odorata leaves Viola odorata extract Mycobacterium tuberculosis isolate Trypticase soy broth Mueller Hinton agar Sterile saline 5 milliliters Quantity 50 grams

Equipments and Utensils The equipments and utensils used for the extraction and disk diffusion susceptibility test were as follows: Triple beam balance, Autoclave, Incubator, gas stove, Mortar and Pestle, Erlenmeyer flask, beaker, funnel, Petri dish, stirring rod, test tubes, graduated cylinder, forceps, dropper, cotton swab, wire loop, ruler, filter paper, and filter paper disks.

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Table 2 Equipments and Utensils used on the Extraction and Disk diffusion Susceptibility Test Equipments and Utensils Triple beam balance Autoclave Incubator Gas stove Mortar and pestle Erlenmeyer flask Beaker Funnel Petri dish Stirring rod Test tube Forcep Dropper Cotton swab Wire loop Ruler Filter paper 1 unit 1 unit 1 unit 1 unit 1 unit 2 units 2 units 1 unit 1 unit 1 unit 2 units 1 unit 1 unit 1 unit 1 unit 1 unit 1 unit Quantity

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Filter paper disk Graduated cylinder

3 units 1 unit

Procedure Viola odorata Extract Preparation Fifty grams (g) of Viola odorata leaves will be weighed using a triple beam balance. The leaves will be washed then macerated by using a mortar and pestle and placed on an Erlenmeyer flask. Fifty ml of water will be added and placed on the gas stove in medium fire until it boils. After cooling the extract, the fluid from the leaves are separated using a funnel and a filter paper. Two filter paper discs will be soaked in the extract for 10 minutes and are set aside. Preparing the Agar Media In preparing the agar media, 1.14 g of the medium will be suspended in 30 ml of purified water. It will be heated with frequent agitating while boiling for one minute to completely dissolve the medium. It will be autoclaved at 121C for 15 minutes and cooled at room temperature. Cooled Mueller Hinton Agar will be poured into sterile Petri dishes on a level, horizontal surface to give uniform depth and allowed to cool at room temperature. Preparation of Bacterial Isolates Four to five well-isolated colonies of Mycobacterium tuberculosis will be selected from an agar plate. A wire loop will be heated using an alcohol lamp to prevent or

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remove contaminants. A sterile loop will be used to transfer the growth to a tube containing 4 to 5 mL of trypticase-soy broth. The broth culture will be incubated at 35C. The turbidity of the broth culture is adjusted with sterile saline and read against a white background with contrasting black lines to aid in the visual comparison. Within 15 minutes after adjusting the turbidity of the inoculum suspension, sterile nontoxic swab on an applicator will be dipped into the adjusted suspension. The swab will be rotated several times, pressing firmly on the inside wall of the tube above the fluid level for the excess inoculums to be removed from the swab. Inoculating the Media The mouth of the agar plate will be passed over a flame. The dried surface of the Muller-Hinton agar plate will be inoculated by streaking the swab over the entire sterile agar surface and repeated two more times, and the plate will be rotated 60 each time to ensure an even distribution of inoculum. Applying the Discs The isoniazid, rifampicin disks and the disc will be soaked in the plant extract and placed evenly (no closer than 24 mm from centre to centre) on the surface of the agar plate either using sterile forceps. The plates will be inverted and placed in an incubator at 35C within 15 minutes after disks are applied and the plates will be incubated aerobically (no C02).

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Zone of Inhibition After 16-18 hours of incubation, the plate will be examined and the diameters of the zones of complete inhibition are measured, including the diameter of the disk using a ruler. The different diameters are taken note of and will be referred to a standard.

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Flowchart Figure 2 Illustration showing the steps and procedures in conducting the test of the antimicrobial activity of Viola odorata on Mycobacterium tuberculosis

50 g Violata odorata was weighed and macerated

Leaves are boiled

The fluid of the extract was filtered

Set aside

Filter paper discs were soaked in the extract

Mueller Hinton agar was prepared

Bacterial suspension was prepared

Bacterial isolates were streaked on Mueller Hinton agar

Viola odorata extract discs, isoniazid disc, and rifampicin disc were placed on the Mueller Hinton agar

Inhibition of the discs were measured using a ruler

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Statistical Treatment The t-test assesses whether the means of two groups are statistically different from each other. This analysis is appropriate whenever you want to compare the means of two groups, and especially appropriate as the analysis for the post test-only two-group randomized experimental design.

The formula for the t-test is a ratio. The top part of the ratio is just the difference between the two means or averages. The bottom part is a measure of the variability or dispersion of the scores. This formula is essentially another example of the signal-tonoise metaphor in research: the difference between the means is the signal that, in this case, we think our program or treatment introduced into the data; the bottom part of the formula is a measure of variability that is essentially noise that may make it harder to see the group difference.

The t-value will be positive if the first mean is larger than the second and negative if it is smaller. Once you compute the t-value you have to look it up in a table of significance to test whether the ratio is large enough to say that the difference between the groups is not likely to have been a chance finding. To test the significance, you need to set a risk level (called the alpha level). In most social research, the "rule of thumb" is to set the alpha level at .05. This means that five times out of a hundred you would find a statistically significant difference between the means even if there was none (i.e., by "chance"). You also need to determine the degrees of freedom (df) for the test. In the ttest, the degrees of freedom are the sum of the persons in both groups minus 2. Given the alpha level, the df, and the t-value, you can look the t-value up in a standard table of

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significance (available as an appendix in the back of most statistics texts) to determine whether the t-value is large enough to be significant. If it is, you can conclude that the difference between the means for the two groups is different (even given the variability). Fortunately, statistical computer programs routinely print the significance test results and save you the trouble of looking them up in a table.

The t-test, one-way Analysis of Variance (ANOVA) and a form of regression analysis are mathematically equivalent and would yield identical results.

Training of Panelist Panelist: ______________________________ Date: ____________

The panelists are going to do the following steps for them to know if there is antitubercular activity and the significant difference between Viola odorata leaves extract as the experimental specimen and the Isoniazid and the Rifampicin as the controls for Mycobacterium tuberculosis. Always follow first safety precautions before and after doing the procedures. Violata odorata Extract Preparation Always wash your hands, or treat with an alcohol based before and after Place a 50 g of macerated Violata odorata in an Erlenmeyer flask and add ml of water. Bring to boil. Let the extract be cooled. Separate the fluid from the leaves using funnel and filter paper.

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Soak two filter paper discs in the extract for 10 minutes. Set aside.

Preparing the Agar Media Suspend 1.14 g of the medium in 30 ml of purified water. Heat with frequent agitation and boil for one minute to completely dissolve the medium. Autoclave at 121C for 15 minutes. Cool at room temperature. Pour cooled Muller Hinton Agar into sterile petri dishes on a level, horizontal surface to give uniform depth. Allow to cool at room temperature.

Preparation of Bacterial Isolates Heat the inoculating loop on flame using an alcohol lamp. Inoculate four to five well-isolated colonies of Mycobacterium tuberculosis from an agar plate using a heated inoculating loop. Transfer the growth using an inoculating loop to a tube containing 4-5 ml Trypticase-soy broth. Incubate at35C. Adjust the turbidity of the broth culture with sterile saline. Read the tube against a white background with contrasting black lines to aid in the visual comparison. Dip sterile non-toxic swab or an applicator stick into the adjusted suspension. Rotate the swab several times, pressing firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab.

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Inoculating the Media Pass the mouth of the agar over the flame. Streak the swab over the entire sterile Mueller-Hinton agar surface. Repeat inoculation 2-3 times. Rotate the plate 60 each time to ensure an even distribution of inoculum.

Applying the Discs Heat the forceps and place the Isoniazid, Rifampin and two filter paper discs that were soaked in the extract evenly (no closer than 24 mm from centre to centre) on the surface of the agar plate either using the sterile forceps. Incubate the plates at 35C within 15 minutes after disks are applied. The plates should be incubated aerobically (no C02). After 16 to 18 hours of incubation, examine the plate for the presence of zone of inhibition. Measure the diameter of the zone of inhibition using a Verniers calliper. Refer the different diameter to a standard measurement.