IN VITRO ANTIBACTERIAL ACTIVITY OF CRUDE ETHANOLIC EXTRACT FROM THE LEAVES OF LIPOTE

IN VITRO ANTIBACTERIAL ACTIVITY OF CRUDE ETHANOLIC EXTRACT FROM THE LEAVES OF LIPOTE (Syzygium polycephaloides)
AGAINST Mycobacterium tuberculosis
A Thesis Proposal
Presented to
The Faculty of the College of Pharmacy
Manila Central University
In Partial Fulfilment
of the Requirements for the Degree
Bachelor of Science in Pharmacy
Jasmine P. Camacho
Samantha Joy N. Valenzuela
October 2018
5120640-59880500CHAPTER 1
THE PROBLEM AND ITS BACKGROUND
Introduction
Tuberculosis (TB) is a communicable disease which is one of the major causes of death worldwide. Ninety-five percent (95%) of deaths occur in under developed and developing countries according to World Health Organization (WHO, 2017). In 2015, there were an estimated 10.4 million new TB cases worldwide of which 6.9 million (56%) were among men, 3.5 million (34%) among women and 1.0 million (10%) among children (WHO, 2016). In the Philippines, Tuberculosis is the eight leading cause of morbidity and mortality (DOH, 2016). The country is the 4th out of the 27 high Multidrug-resistant tuberculosis (MDR-TB) burdened countries accounting for 90% of the global burden of MDR-TB and is seventh among the 22 high TB burden countries (WHO, 2014).
Tuberculosis is caused by Mycobacterium tuberculosis that generally affects the lungs. It is a rod shaped acid fast bacteria that is usually transmitted by inhalation of contagious droplets caused by coughing, laughing or talking (Gladwin and Trattler, 2016). It is very important for people who have TB to finish the medication, and to take the drugs exactly as prescribed so that they will be treated (Center for Disease Control and Prevention (CDC), 2016).
Anti-tuberculosis (anti-TB) drugs consist of two groups: essential or first line drugs which are used for the treatment of TB patients with susceptible Mycobacterium tuberculosis and reserve or second-line anti-TB drugs used for the treatment of MDR-TB (Pankaj et al, 2015). The first line anti-TB drugs includes rifampicin, isoniazid, pyrazinamide and ethambutol (CDC, 2016). Second line anti-TB drugs consist of various drugs including ethionamide, capreomycin, cycloserine and aminosalicylic acid (Katzung et al, 2012).
The first line drugs are the primary choice of treatment for TB, however the bacteria that causes TB can develop resistance to these antimicrobial drugs (WHO, 2017). An organism resistant to at least Isoniazid and Rifampicin, the two most potent TB drugs can cause MDR-TB (CDC, 2012). Inadequate TB treatment, lack of adherence to TB treatment, and the use of poor quality TB drugs can result in the development of MDR-TB (United States Agency for International Development, 2017). In other countries, MDR-TB is becoming difficult to treat because of limited, expensive treatment options, also some medicines recommended by physicians are not always available and patients expense many adverse effects from the drugs (WHO, 2017).

In light of the continuous development of resistance in diseases with existing drugs, one strategy employed is the development of herbal medicines (Bagla, 2017). Herbal medicine can offer hope for the development of alternate medicines for the treatment of tuberculosis (Tijjani, 2013). One of the greatest benefits associated with herbal medicine is the less existence of side effects. They also have an advantage of being very cheap in comparison to the conventional form of medication (Tian, 2016). Medicinal plants and their derivatives are natural resources that play critical roles in modern drug development (Shakya, 2016).

Lipote (Syzygium polycephaloides) is a syzygium specie that is endemic in the Philippines. It is a small to medium-sized tree found in primary forests at low and medium altitude. Lipote is occasionally cultivated for its edible fruit (Jansen et al, 2016; Stuart, 2013). According to a study, lipote has high total flavonoid content, and flavonoids are known as an antibacterial agent against a wide range of pathogenic microorganisms (Santiago et al, 2007; Xie et al, 2014). Thus, the researchers will subject the susceptibility of Mycobacterium tuberculosis to lipote leaves because of its known flavonoid content and to observe if it really has antibacterial activity.
Background of the Study
Tuberculosis remains to be a threat worldwide despite being a curable disease. There is a set of drugs used to treat TB but there are strains resistant to the first line treatment. The strains resistant to the treatment are a problem as they are harder to kill. (CDC, 2016).This means that new antibacterial drugs used to treat TB must be developed.
The use of herbals is becoming more popular as they are seen as more healthy and natural than synthetic drugs. Their use as both supplements and medicines has been rising to about 80% worldwide. Despite that, not all are proven effective (Ekor, 2013). Because one of the factors MDR-TB is patient incompliance, this study will also help provide a possible anti-TB alternative to patients who cannot properly comply with the different requirements when drinking these drugs and prefers using herbals, not only the patients who have grown resistant to the common treatment.
Lipote (Syzygium polycephaloides) will be used in this study as it is a native plant in the Philippines and despite their potential as a source of drugs, lipote does not have a lot of studies. Based on an early study conducted by Chacon, (as cited by Stuart, 2013) lipote has shown antibacterial activity against the gram-positive bacteria, Staphylococcus aureus. Previous research has shown that lipote contains flavonoids and flavonoids are said to have antibacterial activity (Ochoco, 2011; Xie, 2014). With this, this study will determine the susceptibility of In vitro antibacterial activity of the bacteria Mycobacterium tuberculosis H37Rv strain to crude ethanolic extract from lipote.

Statement of the Problem
This study aimed to determine the in vitro antibacterial activity of crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides) against Mycobacterium tuberculosis. Thus, it will seek to answer the following questions:
What is the total crude ethanolic extractive from the leaves of lipote (Syzygium polycephaloides)?
What are the physicochemical properties of the crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides)?
Does it possess in vitro antibacterial activity against Mycobacterium tuberculosis?
Is there a significant difference in the antibacterial activity of lipote (Syzygium polycephaloides) extract and the following comparator drugs:
a) Rifampicin,
b) Isoniazid,
c) Pyrazinamide,
d) Ethambutol?
Hypothesis
Null Hypothesis (H0): There is no significant difference between the antibacterial activities of the crude ethanolic extract from the leaves of lipote and the reference drugs, Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol.

Alternative Hypothesis (Ha): There is a significant difference between the antibacterial activities of the crude ethanolic extract from the leaves of lipote and the reference drugs, Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol.

Scope and Limitations of the Study
Lipote (Syzygium polycephaloides) leaves were collected in Los Baños, Laguna on November 2017; it was authenticated at the Institute of Biology, University of the Philippines Diliman. The plant sample was dried, ground, and macerated with 95% ethanol for 72 hours. It was placed in the rotatory evaporator at the University of the Philippines Diliman to remove the ethanol present in the crude extract. Then, the total crude ethanolic extractives from the leaves of lipote was calculated. The physical and chemical properties of the obtained crude ethanolic extract of lipote was determined. The determination of physical properties includes organoleptic evaluation, solubility, and pH determination; the determination of chemical properties includes tests for the presence of the desired constituent and other tests. The crude ethanolic extract of lipote was submitted at Philippine General Hospital – Medical Research Laboratory (PGH-MRL) together with the desired concentrations and storage condition of the extract. Susceptibility testing of the crude ethanolic extract of lipote has been done by PGH-MRL. The lipote extract was compared to rifampicin, isoniazid, pyrazinamide, and ethambutol using Analysis of Variance (ANOVA). The isolation of specific constituent and formulation of the dosage form was not included in this study.
Significance of the Study
This study was conducted to identify the susceptibility of lipote against Mycobacterium tuberculosis that would help to treat tuberculosis patients of some information that will be beneficial to the following:
The TB patients, as this study helped patients who have developed resistance to antituberculosis drugs by providing a potential alternative treatment.
The Pharmacists, as this study was able to help them have an additional knowledge about the therapeutic use of lipote (Syzygium polycephaloides) and its potential antibacterial action against Mycobacterium tuberculosis.
The Researchers, as this study was able to help them have knowledge about Tuberculosis and Lipote (Syzygium polycephaloides).

The Future Researchers, as this study have introduced the antibacterial activity of lipote (Syzygium polycephaloides) against Mycobacterium tuberculosis.

Definition of Terms
The following terms are defined to guide the readers throughout the study:
Antibacterial is an agent that interferes with the growth and reproduction of bacteria (Alliance for the Prudent Use of Antibiotics, 2014). In this study, in vitro method will be used in testing the antibacterial activity against Mycobacterium tuberculosis.
Crude ethanolic extract is a preparation of dried plant with the use of ethanol (Torio, 2014). In this study, the crude ethanolic extract will be prepared by maceration of the dried and ground lipote leaves with 95% ethanol.

In vitro: tests done in a laboratory setting on biological components that have been extracted from a living organism (Keiser, 2016).
Lipote (Syzygium polycephaloides) is a plant that belong to the syzygium specie and is cultivated in the Philippines because of its edible fruits (Jansen et al, 2016).

Mycobacterium tuberculosis is an acid-fast obligate aerobe bacillus which causes tuberculosis (Gladwin, M. et al, 2016). In this study, Mycobacterium tuberculosis H37Rv strain will be used to test for the antibacterial action of the leaves of lipote.

Susceptibility testing is used to determine which antibacterials will inhibit the growth of the bacteria causing an infection (Tortora et al, 2014).

5194935-68262500CHAPTER 2
REVIEW OF RELATED LITERATURE
This chapter presents available literature and studies about the plant materials and subjects in the investigation of in vitro antibacterial activity of lipote (Syzygium polycephaloides) against Mycobacterium tuberculosis.
Lipote (Syzygium polycephaloides, Fam. Myrtaceae)
Lipote (Syzygium polycephaloides) also known as bahag, baligang, or igot belongs to the syzygium species and can be found in the Philippines. It occurs wild and is cultivated because of its edible fruits. It is an evergreen tree that is found in forests at low and medium altitudes (Jansen et al, 2016; Stuart, 2013). Plate 1 shows the image of the leaves of lipote (Syzygium polycephaloides).

902335109855Plate 1: Leaves of Lipote (Syzygium polycephaloides)
00Plate 1: Leaves of Lipote (Syzygium polycephaloides)

Lipote (Syzygium polycephaloides) is a small to medium-sized tree that can grow up to 14m tall having oblong leaves and acute, white clustered flowers and mature twigs. The fruits are in compact clusters; round, dark red to black when mature, dry, and slightly acidic. It can be found in primary forests at low and medium altitudes (Barreveld, 2014). The flowering and fruiting season of the tree happens in between May and June (Jansen et al, 2016).

Constituents and Uses of Lipote (Syzygium polycephaloides)
Lipote is known to have various constituents, one example is the fruits which are rich in Vitamin C, and these are edible and can be made into a preserves, jelly, wine, pickles, and beverages. It also contains high total phenolic and flavonoid content and is said to have antioxidant and antihypertensive activity. Some studies also suggest that lipote has anti-inflammatory, antibacterial and anti-tumor properties. It is traditionally used for diabetes, hypertension and high cholesterol. Ifugaos uses the sour fruit for treating coughs and leaf decoction for hypertension (Santiago et al, 2007; Stuart, 2013).

Studies on Lipote (Syzygium polycephaloides)
In an early study conducted by Chacon (as cited by Stuart, 2013), it was shown that the crude alcoholic extract of the bark of lipote yielded active constituents, inorganic salts, carbohydrates, and proteins. The study also showed that the lipote extract has antibacterial (Staphylococcus aureus) and anti-inflammatory activity in female rats (Stuart, 2013). In another study by Santiago et al in 2007 about antioxidant activities, flavonol and flavanol content of selected southeast Asian Indigenous Fruits, lipote showed 73.7% inhibition of linoleic acid peroxidation and the antioxidant flavonoids, catechin, epicatechin, quercetin and myricetin was also present in lipote.
Flavonoids have shown antibacterial activity against Mycobacterium tuberculosis based on various studies. On the study conducted by Mativandlela in 2009, flavonoids from Galeniaafricana has shown antituberculosis activity against Mycobacterium tuberculosis. It showed synergistic activity with Isoniazid, indicating the possibility of its mechanism being similar to Isoniazid. Another study by Villaume in 2017, natural and synthetic flavonoids were proven to be potent Mycobacterium tuberculosisuridine 5?-diphosphate galactopyranose mutase (UGM) inhibitors. UGM is an essential biocatalyst involved in the cell wall biosynthesis of Mycobacterium tuberculosis. The study reported the first natural products that act as an inhibitor of UGM.

Tuberculosis
Tuberculosis (TB) is a contagious disease that is usually transmitted from person to person through air. It is caused by the bacteria Mycobacterium tuberculosis. Tuberculosis causes two types of illness, latent or active. TB is latent when the body’s immune system forms a wall around the TB bacteria so they cannot spread to others. A latent TB person has no symptoms, do not feel sick and cannot spread the TB bacteria to others. People can have latent TB for long periods of time. If a person with latent TB does not get treatment, the TB bacteria can “activate” and cause disease (Rhode Island Department of Health (RIDOH), 2012). Active TB is when the body cannot adequately fight the TB bacteria and the person has symptoms includes cough of three weeks or more, weight loss, loss of appetite, coughing up blood or mucus, weakness, fever and night sweats (NIH, 2016).

Throughout the years, there has been a positive progress with the fight against Tuberculosis however, an estimated 1.5 million people still die of Tuberculosis each year (Philippine Council for Health Research and Development (PCHRD), 2015). Tuberculosis still remains to be a major worldwide problem to this day that causes ill-health to people each year that in 2015, it ranked higher than Human Immunodeficiency Virus/ Acquired Immunodeficiency Syndrome (HIV/AIDS) as one of the leading causes of death from an infectious disease (WHO, 2016). Philippines saw 14,000 deaths caused by TB and at the same year, there were approximately 324,000 new TB patients. Philippines also recorded 17,000 cases of MDR-TB patients due to inappropriate treatment in the unengaged private sector. Tuberculosis is prevalent among the elderly, smoker, urban poor and the immunocompromised such as those living with HIV, diabetes and malnutrition (Western Pacific Region WHO, (2016).

Mycobacterium tuberculosis
Mycobacterium tuberculosis (MTB) is a rod-shaped acid fact bacterium, causing tuberculosis. It is an aerobe that is 2-4 micrometers in length 0.25-0.5 micrometers in width that commonly infects the lungs where oxygen is abundant. Due to MTB’s hydrophobic lipid nature, the colonies that form lump together resulting in clumped colonies on agar and floating blobs on liquid media. Middlebrook’s medium, an agar-based medium and Lowenstein-Jensen medium which is an egg-based medium are the two media that is used in growing Mycobacterium tuberculosis. It is slow growing, taking up to 6 weeks for visible growth.

Mycobacterium tuberculosis is structurally a gram-positive bacterium containing peptidoglycan (murein) in their cell wall. However, it also shares properties with gram-negative organisms such as not retaining the Gram stain, if a Gram stain is performed on MTB, it stains very weakly or not at all (Gladwin et al, 2016; Todar, 2008).
Transmission and Pathogenesis of Tuberculosis
Mycobacterium tuberculosis is transmitted through droplet nuclei, an airborne particle that is about one to five (1-5) microns in diameter and can remain in the air for several hours, depending on the environment. It can be generated when a person has pulmonary or laryngeal TB disease cough. Transmission begins with the inhalation of infectious aerosols containing Mycobacterium tuberculosis. This generally occurs indoors, since ventilation removes droplet nuclei from a contaminated space and direct sunlight rapidly kills Mycobacterium tuberculosis.
When a person inhales droplet nuclei with tubercle bacilli, it reaches the alveoli of the lungs and this is when infection occurs. The alveolar macrophage ingests their tubercle bacilli and the majority are destroyed or inhibited. These macrophages can either die or live; if it dies, a small number may multiply intracellular and released. If it is alive it may spread through the bloodstream to different areas of the body including those areas in which TB disease usually develops: regional lymph nodes, the apex of the lung, kidneys, brain, and bone (CDC, 2013).
First line Antituberculosis Drugs
The first line or essential antituberculosis drugs are the primary choices in treating tuberculosis. It includes rifampicin, isoniazid, pyrazinamide, and ethambutol.

Rifampicin
Rifampicin is an addition to the cocktail drug treatment of tuberculosis combined with isoniazid, pyrazinamide, and ethambutol (Firdaus, 2013). It is from Streptomyces mediterranei and is a synthetic derivative of Rifamycin (Katzung et al, 2012).
Rifampicin is known as the most potent drug for the treatment of tuberculosis. Rifampicin works by binding to ? subunit of the bacterial DNA-dependent RNA polymerase and thereby inhibits RNA synthesis. In vitro, it is active against gram-positive and gram-negative cocci, chlamydiae, mycobacteria, and some enteric bacteria. It imparts a harmless orange color body secretions (urine, sweat, and tears) (Katzung et al, 2012).

Isoniazid
Isoniazid is chemically known as isonicotinyl hydrazine or isonicotinic acid hydrazide. It is freely soluble in water, slightly soluble in chloroform and ether, sparingly soluble in alcohol. It is physically described as an odorless, colorless or white crystalline powder or as white crystals.

Isoniazid is used in combination with the other antituberculosis drugs and is also said to be the potent drug along with Rifampicin for the treatment of tuberculosis. It works by inhibiting the synthesis of mycolic acids, which are essential components of the mycobacterial cell wall. Against actively growing intracellular and extracellular Mycobacterium tuberculosis organisms Isoniazid at therapeutic level is known to be bactericidal. The most common adverse effect of isoniazid is peripheral neuritis due to the competition of isoniazid with pyridoxal phosphate for the enzyme apotryptophanase (Katzung et al, 2012).

Pyrazinamide
Pyrazinamide is another first line drug in combination with rifampicin, isoniazid, and ethambutol which is a relative of nicotinamide. It is stable and slightly soluble in water and is active at neutral pH. It is converted to pyrazinoic acid, which is the active form of the drug, by mycobacterial pyrazinamidase. The specific target is not known, but pyrazinoic acid disrupts mycobacterial cell membrane metabolism and transport functions. The major adverse effects of pyrazinamide are hepatotoxicity, nausea, vomiting, hyperuricemia, and drug fever (Katzung et al, 2012; Ontario HIV Pharmacy Professional Specialty Group, 2009).

Ethambutol
Ethambutol is a synthetic, water soluble, heat stable compound. It is generally used in combination with the other drugs to treat tuberculosis. In vitro, ethambutol inhibits susceptible strains of Mycobacterium tuberculosis and other mycobacteria. Ethambutol works by inhibiting mycobacterial arabinosyl transferases which are involved in the polymerization reaction of arabinoglycan, an essential component of the mycobacterial cell wall. The most common adverse effect of ethambutol is retrobulbar neuritis, resulting in loss of visual acuity and red green color blindness (Ontario HIV Pharmacy Professional Specialty Group, 2009; Katzung et al, 2012).
The summary of the different mechanism of action of the four (4) first line anti-tuberculosis drugs, Isoniazid, Rifampicin, Pyrazinamide, and Ethambutol are shown in Table 1.
Table 1. Different Mode of Action of First Line Anti-tuberculosis Drugs
DIFFERENT MODE OF ACTION OF FIRST LINE ANTI-TUBERCULOSIS DRUGS
Isoniazid Inhibits synthesis of mycolic acids, an essential components of mycobacterial cell walls.

Rifampicin Inhibits DNA-dependent RNA polymerase, thereby blocking production of RNA.

Pyrazinamide It is converted to the active pyrazinoic acid under acidic conditions in macrophage lysosomes.

Ethambutol Inhibits mycobacterial arabinosyl transferases.

Table 2 shows Isoniazid-Rifampin combination administered for nine (9) months. The addition of Pyrazinamide to Isoniazid and Rifampin combination for the first two (2) months allows the total duration of therapy to be reduced to six (6) months.
Table 2. Recommended duration of therapy for tuberculosis
Regimen ( in Approximate Order of Preference ) Duration in Months
Isoniazid, Rifampin, Pyrazinamide 6
Isoniazid, Rifampin 9
Rifampin, Ethambutol, Pyrazinamide 6
Rifampin, Ethambutol 12
Isoniazid, Ethambutol 18
5097259-58356500CHAPTER 3
METHODOLOGY
-26670017278350This chapter presents the processes, methods and procedures used for gathering of data used in the study of antibacterial activity of crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides) against Mycobacterium tuberculosis. Figure 1 shows the different processes that was done in the study.

Figure 1: Flowchart of Methodology
1. Authentication, Collection, and Preparation of the Plant Sample
1.1 Collection of Plant Material
Five hundred (500) grams of fresh leaves of lipote (Syzygium polycephaloides) was collected from Los Baños, Laguna on November. The plant sample was washed with tap water to eliminate other extraneous matters and rinsed with distilled water to minimize contamination of the plant extract for extraction procedure.

1.2 Authentication of Plant Sample
The leaves of lipote (Syzygium polycephaloides) was submitted to the Institute of Biology, University of the Philippines Diliman for authentication of the plant.

1.3 Preparation of the Crude Ethanolic Extract from the leaves of Lipote (Syzygium polycephaloides) (Garcia, 2016)
The rinsed plant sample was air dried in a shaded area for two weeks before drying in the oven. The dried plant sample was powdered using grinding apparatus and was prepared for extraction. The plant sample was macerated for 72 hours using 95% ethanol. The extract was filtered with a Buchner funnel fitted with Whatman No. 1 filter paper. The remaining ethanol was evaporated using rotary evaporator operating under reduced pressure. The extract was stored at temperature -20°C (Celsius). The extract was weighed and the total crude extractives was calculated as follows:
% Total Crude Extractive = Weight of the extract x 100
Weight of the sample 2. Physicochemical Properties of the Crude Ethanolic Extract from the leaves of Lipote (Syzygium polycephaloides) (Torio, 2014)
2.1 Physical Properties
2.1.1 Organoleptic Properties. The physical appearance, texture and odor of crude ethanolic extract was recorded.

2.1.2 Solubility. One (1) mL of the crude ethanolic extract of plant sample was placed in a test tube for determining the solubility in different solvents: water (hot and cold), ethanol, ether, carbon tetrachloride and chloroform.
2.1.3 pH Determination. Enough amount of crude ethanolic extract of plant sample was dissolved in water and the pH was determined using pH meter.
2.2 Chemical Properties
2.2.1 Phytochemical Analysis
The phytochemical analysis was conducted to the ethanolic extract from the leaves of lipote (Syzygium polycephaloides) to determine the presence and absence of constituents.

2.2.1.1 Test for Carbohydrates
a.) Molisch Test. A few drops of alpha-naphthol was added to 1 ml of ethanolic extract and then concentrated sulfuric acid was added to form a layer below the solution.

VPR – A purple layer below the solution illustrates a positive result
b.) Fehling’s Test. An equal amount of Fehling’s A and B was mixed, a few drops of the mixture was added to 1 ml of the ethanolic extract and warmed in a water bath.

VPR – A brick red precipitate indicates the presence of reducing sugars.

c.) Seliwanoff’s Test. The crystal of resorcinol was added to 1 ml of the ethanolic extract and was warmed on a water bath with an equal volume of concentrated hydrochloric acid.

VPR – A rose color is produced if ketone is present.

d.) Keller Killiani Test for Deoxysugars. An acetic acid with traces of ferric chloride was added to the ethanolic extract then transferred to the surface of concentrated sulfuric acid.

VPR – The presence of deoxysugars shows a reddish brown solution which gradually turns to blue
2.2.1.2 Test for Alkaloids
Preliminary tests. About 10 ml of the ethanolic extract from the leaves of lipote (Syzygium polycephaloides) was treated with 2M hydrochloric acid. About 1 g of sodium chloride was added, stirred and filtered. The residue was washed with enough 2M hydrochloric acid to bring the filtrate to a volume of 10 ml. About 1 ml of the filtrate was taken and tested with 2 to 3 drops of the following reagents:
a.) Mayer’s reagent (Mercuric-Potassium Iodide TS)
VPR – White precipitate
b.) Valser’s reagent (Mercuric Iodide TS)
VPR – Reddish brown precipitate
c.) Wagner’s reagent (Iodine test solution)
VPR – Reddish brown precipitate
d.) Dragendorff’s reagent (Bismuth Potassium Iodide TS)
VPR – Orange precipitate
2.2.1.3 Test for Flavonoids
a.) Wilstater “Cyanidin” Test. A small piece of magnesium and few drops of hydrochloric acid was added to the crude ethanolic extract.

VPR – Colors ranging from orange to red, to crimson and magenta and occasionally to green or blue.

b.) Batesmith and Metcalf Test. A portion of the crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides) was treated with 0.5 ml of concentrated hydrochloric acid and heated for 15 minutes in a water bath.

VPR – A formation of a strong red or violet color indicates the presence of leucoanthocyanins.

c.) Modified Borntrager’s Test. A portion of the crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides) equal to 3 grams of the sample was evaporated to dryness over water bath. 10 ml of potassium hydroxide and 1 ml of 5% hydrogen peroxide was added. It was heated for 10 minutes, then filtered. The resulting filtrate was acidified with glacial acetic acid, and then extracted twice with 5 ml of benzene. The benzene extracts was combined and the resulting benzene layer was divided into two portions. One portion was used as a control and the other portion is to be alkalinified with ammonia and compared with the control.
VPR – A pink color is developed confirming the presence of flavonoids.

2.2.1.4 Test for Glycosides
To two (2) mL of extract, a few drops of lead acetate T.S was added. Precipitation shows the presence of glycosides, mucons, tannins and proteins. Addition of more lead acetate was required for complete precipitation. The mixture was filtered and lead subacetate T.S was added to the filtrate until the liquid became neutral or weakly alkaline.
VPR – Precipitation or turbidity indicates the presence of glycosides.

2.2.1.5 Test for Plant Acids
a.) Sodium Carbonate Test. A few ml of sodium carbonate T.S. was added to the ethanolic extract.

VPR – The formation of a stable and dense froth indicates the presence of free acids (stearic, diterpene, triterpene and dicarboxylic acids).
2.2.1.6 Test for Saponins
Differentiation of Saponins. About 10 grams of the plant material was slowly boiled in 30 ml of alcohol for one hour in an Erlenmeyer flask covered with a funnel in an upright position. The alcohol extract was filtered then the alcohol layer was evaporated to dry in a water bath. The residue was defatted by taking up 6 ml of hexane and water (2:1). Then, the upper hexane layer was piped out. It was repeated until most of the colored pigments has been removed. The hexane extract was discarded. A 10 ml of chloroform was added to the ethanolic layer and was shaken gently. The collected chloroform layer was dried by filtering the mixture through 100 mg of anhydrous sodium sulfate held over dry filter paper. The collected filtrate was divided into 3 clean, dry test tubes. One test sample was used as the control.

a. Liebermann Burchard Test. Three drops of acetic anhydride was added to the first test tube then added with concentrated sulfuric acid. It was mixed gently and was observed within a period of one hour for color changes.
VPR – A color range from blue to green, red, pink, purple or violet indicates the presence of unsaturated sterols and triterpenes.
b. Salkowski Test. The second test tube was held at 45o angle then added with 2 ml of concentrated sulfuric acid by allowing it to run down inside the test tube. Any immediate color changes was noted at the junction of the extract and sulfuric acid and the extract was mixed gently and was observed for any color changes over an hour.

VPR – A cherry red / reddish brown coloration is indicative of the presence of terpenoids.

2.2.1.7 Test for Tannins
a.) Ferric Chloride Test. Two (2) ml of crude ethanolic extract was placed in a test tube and 3 drops of Ferric Chloride test solution was added.
VPR – Formation of bluish black color indicates the presence of hydrolysable tannins including polyphenols.
b.) Gelatin Test. A portion of the filtrate was treated by the 1% gelatin salt solution.
VPR – Formation of a jelly precipitate confirms the test.

3. Susceptibility Test of Mycobacterium tuberculosis strain to crude ethanolic extract from the leaves of Lipote (Syzygium polycephaloides)
3.1 Test Organism
Mycobacterium tuberculosis H37Rv was obtained from the Philippine General Hospital – Medical Research Laboratory (PGH-MRL). This strain was used in the evaluation of the susceptibility test of Mycobacterium tuberculosis to crude ethanolic extract from the leaves of lipote (Syzigium polycephaloides).

3.2 Preparation of Inoculum of H37Rv
Freshly grown standard strain of Mycobacterium tuberculosis H37Rv strain was transferred to a tube of Middlebrook 7H9 broth with sterile beads to homogenize. Using a densitometer, the inoculum was compared to Mac ferland standard 1.0. In sterile distilled water a further dilution of the adjusted inoculum to 10-2 and 10-4 was made.

3.3 Preparation of the 7H10 agar
The middle brook 7H10 agar was prepared according to package instruction. Middle brook 7H10 was dissolved in sterile distilled and was autoclaved at 121 psi for 15 minutes. Middle brook Oleic Dextrose Catalase (OADC) was added after cooling the agar at 56oC.

3.4 Preparation of extract into agar plates
Each plate was labelled with different increasing concentration of 1 mg, 3 mg, 5 mg, 10 mg and 20 mg that was used in the susceptibility test. Each concentration was delivered 1.0ml each quadrant and overlay with 5 mL of the prepared Middle brook 7H10 agar. All test concentrations were delivered 0.1 mL of the standardized inoculum in all quadrants including a control quadrant without the extract. The plate were incubated at 37oC for 3 weeks.
3.5 In Vitro Antibacterial testing of Crude ethanolic extract of Lipote (Syzygium polycephaloides) and Comparator drugs (Mlozi et al, 2017)
The crude ethanolic extract of leaves of lipote (Syzygium polycephaloides) was submitted at Philippine General Hospital – Medical Research Laboratory (PGH-MRL) together with specified storage condition of the extract which is -20°C (Celsius) and desired concentrations: 1 mg, 3 mg, 5 mg, 10 mg and 20 mg. In vitro antibacterial testing of the crude ethanolic extract of leaves of lipote was conducted using the comparator drugs: Rifampicin, Isoniazid, Ethambutol and Pyrazinamide using the same five increasing concentrations. Comparability testing was done by PGH-MRL according to the following procedures.

4. Statistical Analysis
One-Way Analysis of variance (ANOVA) was used to find out if there is a significant difference between the antibacterial activity of the crude extract from the leaves of lipote and the four comparator drugs: Rifampicin, Isoniazid, Pyrazinamide and Ethambutol.
The dependent variable which is expected to change upon manipulation of another variable is the bacterial strain Mycobacterium tuberculosis H37Rv strain. On the other hand, the independent variable are the variables being manipulated and have a possibility of having an effect on the dependent variable, these includes the crude ethanolic extract from the leaves of lipote with its different concentrations: 1 mg, 3 mg, 5 mg, 10 mg, and 20 mg.

5192973-532263CHAPTER 4
PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA
This chapter presents the results, analysis and interpretation of data collected in the determination of the in vitro antibacterial activity of the crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides) against Mycobacterium tuberculosis.

1. Authentication, Collection, and Preparation of the Plant Sample
1.1 Collection of Plant Material
The leaves of lipote were collected from Los Baños, Laguna by handpicking. The collection site was a plant nursery located near the foot of a mountain.

1.2 Authentication of Plant Sample
The leaves was authenticated and verified by the Institute of Biology of University of the Philippines Diliman. It was identified to be lipote with the scientific name Syzygium polycephaloides of the family Myrtaceae. The authentication is presented in Appendix.

1.3 Preparation of the Crude Ethanolic Extract from the leaves of Lipote (Syzygium polycephaloides)
The leaves of lipote was extracted by maceration using 95% ethanol as extracting solvent. The percentage yield of the total crude ethanolic extract was presented in Table 3.

Table 3. Percentage Yield of the Total Crude Ethanolic Extract
Weight of Extract Weight of Sample Percentage Yield
50.14 g 236.88 g 21.17 %
2. Physicochemical Properties of the Crude Ethanolic Extract from the Leaves of Lipote (Syzygium polycephaloides)
2.1 Physical Properties
2.1.1 Organoleptic Properties
The crude ethanolic extract from the leaves of lipote was subjected to organoleptic analysis and the following results were obtained (Table 4):
Table 4. Organoleptic Properties of the Crude Ethanolic Extract from the leaves of Lipote
Organoleptic Properties Results
Color Green
Texture Non-viscous
Odor Aromatic odor
Physical Appearance Liquid
The crude ethanolic extract obtained from the leaves of lipote was a green, non-viscous liquid that has an aromatic odor.

2.1.3 pH Determination
Using pH meter the pH of the crude ethanolic extract was determined to be weakly acidic with a pH of 5.40.
2.1.2 Solubility
The crude ethanolic extract obtained from the leaves of lipote was found to be miscible in different solvents, Table 5 shows the result for thr solubility testing.

Table 5. Solubility of the Crude Ethanolic Extract from the leaves of Lipote
Solvent Crude Ethanolic Extract
Hot Water Miscible
Cold Water Miscible
Ethanol Miscible
Ether Immiscible
Carbon Tetrachloride Immiscible
Chloroform Miscible
It has been observed that the crude ethanolic extract from the leaves of lipote are miscible in hot and cold water, ethanol and chloroform and immiscible inorganic solvents like ether and carbon tetrachloride.

2.2 Chemical Properties
2.2.1 Phytochemical Screening
The crude ethanolic extract from the leaves of lipote was subjected to phyrochemical analysis to determine the presence and absence of constituents. Table 6 presents the results of the phytochemical analysis.

Table 6. Phytochemical Analysis
Constituents Tests Positive Result Actual Result Interpretation
Carbohydrates Molisch Test Violet ring at the junction of the two liquid No appearance of violet ring (-)
Fehling’s Test Brick red precipitate Brick red precipitate (+)
Seliwanoff’s Test Rose color Red color (-)
Keller Killiani Test Reddish brown solution Green color solution (-)
Alkaloids Mayer’s reagent White precipitate No precipitate (-)
Valser’s reagent Reddish brown precipitate No precipitate (-)
Wagner’s reagent Reddish brown precipitate Reddish brown precipitate (+)
Dragendorff’s reagent Orange precipitate Orange precipitate (+)
Flavonoids Wilstater “Cyanidin” Test Orange to red, to crimson and magenta, and to green or blue Green color solution (+)
Batesmith and Metcalf Test Strong red or violet color Green color solution (-)
Modified Borntrager’s Test Pink color (+)
Glycosides Precipitation or turbidity Turbidity (+)
Plant Acids Sodium Carbonate Test Froth formation No formation of froth (-)
Saponins Liebermann Burchard Test Blue to green, red, pink, purple or violet (+)
Salkowski Test Cherry red or Reddish brown (-)
Tannins Ferric Chloride Test Blue-black color or Brownish-green color Blue-black color (+)
Gelatin Test Jelly precipitate Jelly precipitate (+)
Table 6. It shows that the crude ethanolic extract from the leaves of Lipote have presence of Alkaloids, Flavonoids, Glycosides, and Tannins.

3. Susceptibility Test of Mycobacterium tuberculosis strain to crude ethanolic extract from the leaves of Lipote (Syzygium polycephaloides)
Figure 2. Raw Data of the Susceptibility Test of Mycobacterium tuberculosis H37Rv strain
Table 7. Results of the Susceptibility Test of Mycobacterium tuberculosis H37Rv strain
EXTRACT CONCENTRATION
(mg/ml) RESULT
0.2 mg/mL Resistant
0.6 mg/mL Resistant
1.0 mg/mL Resistant
2.0 mg/mL Resistant
4.0 mg/mL Resistant
3.1 Interpretation of Results
All control quadrants should have no growth. However, all quadrants with extract showed growth and were read as resistant. This means that the crude ethanolic extract does not possess antibacterial activity.

4975225-59499500CHAPTER 5
SUMMARY, CONCLUSION AND RECOMMENDATION
This chapter presents the summary and conclusion of the study that aims to determine the in vitro antibacterial activity of crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides) against Mycobacterium tuberculosis.

Statement of the Problem
This study aimed to determine the in vitro antibacterial activity of crude ethanolic extract from the leaves of lipote (Syzygium polycephaloides) against Mycobacterium tuberculosis. Thus, it will seek to answer the following questions:
1. What is the total crude ethanolic extractive from the leaves of lipote (Syzygium polycephaloides)?
2. What are the physicochemical properties of the crude ethanolic extract from the leaves of Lipote (Syzygium polycephaloides)?
3. Does it possess in vitro antibacterial activity against Mycobacterium tuberculosis?
Summary of Findings
After conducting this study, the following results were obtained:
1. The crude ethanolic extract obtained from the leaves of lipote (Syzigium polycephaloides) yielded 21.17%
2. The crude ethanolic extract from the leaves of lipote are miscible in hot and cold water, ethanol and chloroform and immiscible in ether and carbon tetrachloride.

The phytochemical constituent that is present in the extract lipote are Alkaloids, Flavonoids, Glycosides, Plant Acids, Saponins and Tannins.

3. Based on the analysis conducted by Philippine General Hospital regarding the antibacterial activity of the crude ethanolic extract from the leaves of lipote, a concentration as high 4mg/ml does not have any antibacterial activity against Mycobacterium tuberculosis.
Conclusion
Based on the findings of the study, crude ethanolic extract from the leaves of lipote does not have any antibacterial activity against Mycobacterium tuberculosis.

Recommendation
These are the following recommendations for the future researchers in expanding the investigation and experimentation using the lipote.

1. The researchers recommend the use of different solvents for the leaf extract.
2. Use a different and a higher concentration of the crude extract.
3. Conduct the antibacterial activity testing in other forms of bacteria.

4. Test for the antibacterial activity of the extract through in vivo testing.
5. If susceptible, the researchers recommend the formulation of dosage form for the extract.

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APPENDIX A
-15748036322000CERTIFICATE OF AUTHENTICATION
APPENDIX B
15093959144000
15078986409122pH Determination
pH Determination
104775035731450016246052776855Maceration
Maceration

APPENDIX C
PLANT EXTRACT SUSCEPTIBILITY TESTING
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