MAKABUHAY EXTRACT (TINOSPORA RUMPHII) AS AN ANTIMICROBIAL AGENT AGAINST STREPTOCOCCUS PYOGENES

PBL 2 GROUP 15 Algabre, Ernest Alvarez, Monsieur Deo Corro, Abegail Joy Jabines, Andressa Padilla, Genesis Lee Pono, Altrexia Marie Rabor, Jovann Frederick Salazar, Ria Vergara, Jovy Louie Anthony Villahermosa, Velissa Yecyes, Cresana

SY 2012-2013

INTRODUCTION

BACKGROUND Medicine is a science that holistically aims for the better health of an individual. With the dawn of conventional, evidence-based medicine, many of these unproven ancient medical theories eventually developed into the alternative medicine that is known today. The high cost of health care and low income levels are prompting Filipinos to choose alternative medicine over conventional medicine. Many Filipinos use herbal medications to supplement conventional medicines because medicinal plants are readily available. Our study investigates the antimicrobial property of Makabuhay, a common vine growing in thickets in the rural areas of the Philippines. The researchers would like to investigate on the effects of Makabuhay against Streptococcus pyogenes. If proven to be effective, the researchers are hoping that this would be applied specifically against superficial skin infections caused by the above-mentioned agent (Streptococcal pyoderma/Impetigo). In previous studies, the plant has been found to have antifungal activity against Aspergillus niger and Trichophyton mentagrophytes, and antibacterial activity against P. aeruginosa and Bacillus subtilis. 1 There is yet no previous study that demonstrates that Makabuhay plant extract has antimicrobial activity against Streptococcus pyogenes.

RATIONALE The use of herbal plants as an alternative management to various diseases is of increasing popularity more so in rural areas all over the country. Among such is the Makabuhay plant, abundant in most forests and claimed to have therapeutic effects on health problems. The result of this study would present additional scientific basis for the folklore on the medicinal uses of Makabuhay. It also aims to inform the lay people and the health professions of the further uses of the Makabuhay extract. This research intends to provide an alternative and cost-effective way of treating skin infections caused by Streptococcus pyogenes, which is one of the leading causes of impetigo (Streptococcal pyoderma)
[5]

. Furthermore, the study could offer an insight for future

researches that aims to investigate Makabuhays effects against other causative agents and diseases.

RESEARCH QUESTION Does Makabuhay plant extract (water extract and methanol extract) have antimicrobial property against stock cultures of Streptococcus pyogenes using disk diffusion method when compared to penicillin and water?

GENERAL OBJECTIVE

To determine whether Makabuhay plant extract (water extract and methanol extract) possessed antimicrobial property against stock cultures of Streptococcus pyogenes using disk diffusion method when compared to penicillin and water

SPECIFIC OBJECTIVES 1. To compare the mean zone of inhibition of stock cultures of Streptococcus pyogenes around discs impregnated with: a. 100% Makabuhay water extract b. 100% Makabuhay ethanol extract c. Penicillin d. Distilled water 2. To compare proportion of Makabuhay impregnated discs which showed zone of inhibition that were: a. Sensitive b. Intermediate c. Specific

REVIEW OF RELATED LITERATURE

Review of Related Literature

Fig. 1 Makabuhay (Tinosphora Rumphii) Makabuhay, belonging to the family Menispermaceae and the genus Tinosphora Rumphi[1], is found in and nearby towns in thickets, in most or all islands of the Philippines. [2] It is a climbing, dioecious vine reaching a height of 4 to 10 meters. The stems are up to 1 centimeter thick and somewhat fleshy, scattered protuberances. The leaves are thin, ovate, 6 to 12 centimeters in length and 7 to 12 centimeters in width.
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The flowers are pale green, and short-pedicelled. The fruit is 7 to 8 millimeters long.
[2]

The aqueous extract is used in the treatment of stomach trouble, indigestion, diarrhea, as a tonic and in the treatment of topical ulcers, parasiticide, and for malaria. Decoction of the stem is considered an effective cure for ordinary and cancerous wounds. [1] In addition, the Filipinos and Malays in general consider this vine as a universal medicine; its common Tagalog name being Makabuhay which means to give life. In powder form, it can be used to treat fevers. A preparation with coconut oil is an effective cure for rheumatism and also the flatulence of children. A decoction of the stem is considered an effective cure if used as a wash for tropical ulcers. [2]

The Disease/bacterial agent Streptococcus Pyogenes is the main human pathogen associated with local/systemic invasion and post-streptococcal immunological disorders. This bacteria is classified as gram positive; its individual cocci are spherical/ovoid and arranged in chains. The members of the chain often have a striking diplococcic appearance while its length varies widely and is conditioned by environmental factors. Its cell wall possesses various proteins, carbohydrates and peptidoglycans. In addition, Streptococcus Pyogenes produces a capsule composed of hyaluronic acid that impedes phagocytosis. Hair-like pili also project through the capsule and are important in the attachment of the bacteria to epithelial surfaces. [3]
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Streptococcus pyogenes is a beta-hemolytic agent which grows in solid media as discoid colonies 1-2 mm in diameter. Energy is obtained principally from glucose utilization whose end product is lactic acid and grows best at a temperature of 37 degrees Celsius as a facultative anaerobe. The Virulence of the bacteria is dictated by the presence of and M protein (appears as hair like projections of the cell wall). Immunity to infection of Streptococcus pyogenes is related to the presence of typespecific antibodies to M protein. Several toxins and enzymes are produced by this bacterium. Streptokinase, also produced by many strains of group A beta-hemolytic streptococci, transforms the plasminogen of human plasma to plasmin, an active proteolytic enzyme that digests fibrin and other proteins. Coupled with

streptodornase, which is also a toxin from the same bacteria, both toxins help liquefy exudates and facilitate removal of pus and nevrotic tissue allowing antimicrobial drugs better access and infected surfaces recover more quickly. [4] Local infection of the superficial layer of the skin is called impetigo, which can be caused by Streptococcus pyogenes infection. This consists of superficial vesicles that break down and eroded areas whose denuded surface is covered with pus and is later encrusted. It spreads by continuity and is highly communicable, especially in hot, humid climates. [5]

The Positive Control/Medicine Mupirocin is derived from fermentation of Pseudomonas fluorescens. It is a topical antibacterial medication that is active against many gram positive and
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selected gram negative bacteria. It has good activity against streptococci pyogenes.
[6]

In topical form, the drug is also bactericidal; however, it has minimal systemic

absorption through intact skin. It is available at a 2% cream and a 2% ointment for dermatological use. Dermatologic preparations are indicated for treatment of traumatic skin lesions and impetigo secondary to infection with Staphylococcus pyogenes or aureus.[6]

Other Related Studies The study on Makabuhay as an Antibacterial agent has gained some attention among the scientific societies today. One such study, made by the Bachelor of Science students of the New Era University, New Era Quezon city, proposed a similar objective to ours, albeit with different methods. This study is about the antibacterial effects of Tinospora rumphii (Makabuhay) leaf crude extract against Escherichia coli and Staphylococcus aureus in varying concentration using the DiskDiffusion Method. Using Chlorampernicol as their positive control, they sought out determine if their plant extracts would be as effective. The results in fact showed that the zones created by the Makabuhay leaf extracts were not as effective as the Chlorampernicol zones in producing zone of inhibition on Escherichia coli and Staphylococcus aureus. Yet despite this, their findings also revealed that Makabuhay leaf extracts both produced zones of inhibition among the two bacterial strains used, that there was a significant difference on the zones of inhibition on each of the

strains, and that different concentrations of Makabuhay leaf extracts produced varied of zones of inhibition on Escherichia coli and Staphylococcus aureus. [7] Yet another study also attested in their findings that Makabuhay extracts had a significant antibacterial effect, although it also was not as effective as their positive control (this time the positive control was Co-trimoxazole) used. This time however some of the approaches were very diverse, one was that not only Makabuhay, but also other leaf extracts were used such as gatas-gatas and silisian. Their sample used consisted of Shigella dysenteriae. Their results indicated that the growth

inhibitory effect of gatas-gatas, makabuhay and silisian against S. dysenteriae varied with gatas-gatas exhibiting greater inhibitory effect among the three extracts used. [7]

CONCEPTUAL FRAMEWORK

100% Makabuhay extract (possible mechanisms of action): -Blockage of bacterial protein production -Inhibition of cell wall synthesis -Cell membrane permeabilization -Inhibition of DNA

Penicillin -Inhibition of synthesis of peptidoglycan cell wall, specifically on the transpeptidation step

Growth of Steptococcus pyogenes Antigenic Structures: 1. M protein 2. T substance 3. Nucleoproteins Toxins and Enzymes 1. Streptokinase 2. Streptodornase 3. Hyaluronidase 4. Pyogenic exotoxins 5. Diphosphopyridi ne nucleotidase 6. Hemolysins

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METHODOLOGY

STUDY DESIGN The design used is the Experimental Design, comparing two proportions from independent samples. The dependent variable is the Microbial growth, while the independent variable is the Makabuhay plant extract.

STUDY SETTING The study will be conducted in the Microbiology Laboratory of Cebu Institute Medicine.

STUDY POPULATION INCLUSION CRITERIA Streptococcus pyogenes

EXCLUSION CRITERIA
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Growth of another organism Synthetic antimicrobials

SAMPLE SIZE COMPUTATION n = P1(1-P1) + P2 (1-P2) (Z/2 + Z)2 (P1-P2)2 P1 = 0.95 P2 = 0.70 Z/2 = 1.36 Z = 0.84 n = 0.95 (1-0.95) + 0.70 (1-0.70) (1.96 + 0.84) 2 (0.95 0.70)2 n = 32.30 or 32

MANEUVERS DATA COLLECTION TOOL Agar plate Autoclave Mupirocin tablets Cotton swab

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Inoculating loop Makabuhay sap Manila papers Mueller Hinton agar with 5% Sheep blood Tongue depressor Ethanol 70% Mortar and pestle Deionized water Distilled water Centrifuge (Orbital Shaker) Whatman paper Beaker Graduated cylinder Methanol Vacuum rotator evaporater Paper punch Wire loop 0.5 MacFarland Standard Vernier caliper Incubator

PREPARATION OF TEST SUBSTANCE Preparation of Plant material

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Leaf and stem samples of Makabuhay plant are harvested, and the samples washed with running water. The plant samples are then sterilized by soaking in ethanol 70% solution for 5 minutes. Rinse them in three (3) separate basins of water and allow drying. Pack them in clean sterile manila papers and label. Oven dry materials at 25C for 5 hours or until dry. Chop and pound to fine powder using mortar and pestle. Preparation of Water Extract Soak sixty (60) grams of ground powder in 300ml sterile distilled and deionised water, at 50C for 1hr. Place in orbital shaker at 100rpm for 24h at 25C. Filter elute and lyophilize into granules. Prepare stock solution by dissolving 0.4g of granules in 20ml deionised sterile distilled water, store at -20C until further use. Preparation of Methanol Extract Soak sixty (60) grams of ground powder in a beaker containing 300ml methanol for 72 hours. Place in orbital shaker at 100rpm for 72h at 25C. Membrane filter the extracts on Whatman paper 1 then concentrate filtrate at 50C using vacuum rotary evaporator. In order to evaporate the methanol used as extraction solvent, oven dry concentrates at 25C until a constant weight is attained. Evaporate excess methanol by further air-drying the paste at 25C. Prepare stock solutions by dissolving 0.4g in 20ml of deionised sterile distilled water. Store at -20C until further use.

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TEST GROUP ALLOCATION PROCEDURE Disc Impregnation Punch Whatman No. 1 filter paper into discs using a paper puncher with a 6mm diameter. Place it in a petri dish and sterilize discs in a hot air oven. Add 10ul of the prepared plant extract to the disc, and dry under sterile condition at room temperature. Antimicrobial Susceptibility Testing Inoculate S. pyogenes pure culture into 5ml NSS using a sterile wire loop. Adjust turbidity by comparing it with a BaSO4 0.5 MacFarland standard. Within 15 minutes of inoculation after adjusting the turbidity of the inoculums, dip a sterile cotton swab into the adjusted suspension. Rotate swab several times and press firmly on the inside wall of the tube above fluid level. The dried surface of MuellerHinton agar with 5% sheep blood is inoculated by simple streaking, 3 times, rotating 60 degrees each time to ensure even distribution of the inoculums. Rim the agar with the swab. Leave the agar for 5 minutes, before applying the impregnated discs. Dispense one each of Penicillin disc, Makabuhay ethanol extract disc, Makabuhay water extract disc. Each disc must be pressed down to ensure complete contact with agar surface, and must be distributed evenly no closer than 24mm from center to center. Invert plates and place in 35C incubator, with a low CO2 atmosphere within 15 minutes after discs are applied. Incubate for 16-18 hours.

DATA MEASUREMENT PROCEDURE

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Examine each plate after 16-18 hours incubation. Measure zones of inhibition to the nearest whole millimeter, including the diameter of the disc using a sliding caliper or ruler. Measure zones from the upper surface of the agar illuminated with reflected light, with the cover removed. The sizes of zones of inhibition are interpreted by referring to Tables 2A through 2I (Zone Diameter Interpretative Standards and Equivalent Minimum Inhibitory Concentration Breakpoints) of the NCCLS M100-S12: Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement. The organisms are reported as either susceptible, intermediate or resistant to the agent that has been tested.

OPERATIONAL DEFINITION Antibacterial activity- the microbe tested (S. pyogenes) will be susceptible to the antibacterial agent used (Makabuhay extract) based on the sizes of the zones of inhibition Kirby-Bauer disk diffusion method- the type of disk diffusion method used in this study Makabuhay vine- a piece of Makabuhay stem with its outer layer removed and thoroughly cleaned Nutrient Agar- a solution containing Beef Extract (0.3%), Peptone (0.5%) and Agar (1.5%) in water

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Zone of Inhibition- the area of the agar plate where the growth of Streptococcus pyogenes is prevented by an antibiotic usually placed on the agar surface Dioecious - Having male and female flowers on separate plants. Pedicel - A small stalk or stalklike part bearing a single flower Catalase positive appereance of gas bubbles after 1ml of hydrogen peroxide is dropped over the bacterial colonies growing on a TSA slant. Urease positive a dark pink color change of the medium (urea broth) duringa urea hydrolysis test.

BUDGET Penicillin disks Plastic Petri dish (100pcs) Ethanol 70% (2bottles) Deionized water (2bottles) Distilled water Methanol Triple distilled water (2bottles) Syringe 10mL (8pcs) Disposable needle G23 (5pcs) Surgical gloves (10pairs) Printing Cost
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P175.00 P650.00 P131.00 P76.00 P69.00 P50.00 P76.00 P80.00 P20.00 P200.00 P150.00

Miscellaneous TOTAL

P300.00 P1977.00

REFERENCES Books and Publications 1. Michael L. Tan (1980) Philippine Medicinal Plants in Common Use, Their Phytochemistry and Pharmacology Revised Edition, pg. 54 2. Dr. Eduardo Quisumbing (1978) Medicinal Plants of the Philippines pg. 300301 3. Nelia P. Cortes Maramba, Et.al, (1982) Guidebook on the Proper use of Medicinal Plants pg. 111 4. Laurence Brunton, Et.al, Goodman and Gilmans The Pharmacological Basis of Therapeutics, 12th edition, p.g 1477 5. Geo F. Brooks, Et.al, (2010) Jawetz, Melnick and Adelburgs Medical Microbiology 25th edition, pg. 185

Internet Sources

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1. http://www.stuartxchange.org/Makabuhay.html 2. http://www.rxlist.com/cipro-drug/clinical-pharmacology.htm 3. http://www.scribd.com/doc/40230002/Antibacterial-Effect-of-MakabuhayAgainst-S-aureus-and-E-coli 4. http://microbewiki.kenyon.edu/index.php/Pseudomonas_aeruginosa

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