Antibacterial Activity of Two Common Garden Weeds

Bayating, Lovelynx Kee Espiritu, Joan Camille Herrera, Liezel Lacdao, May

A special project submitted to the Department of Biology College of Science University of the Philippines Baguio

In partial fulfillment of the requirements in the course Biology 120 Department of Biology

October 2011

Abstract Though the number of new antibiotics has increased in the last three decades, resistance to these drugs by microorganisms has increased and the outlook for the use of antimicrobial drugs in the future is still uncertain. Certain actions are being done to reduce this problem such as the control of the use of antibiotics, understanding the genetic mechanisms of resistance, and the continuation of studies to develop new drugs be it synthetic or natural. The main objective of this study is to test the effectivity of the crude extracts of two common garden weeds, Wedelia trilobata and Tridax parviflora against Escherichia coli and Bacillus subtilis. Results showed that Wedelia trilobata has a higher potential of antimicrobial activity against the two bacterial strains as compared to Tridax parviflora.

INTRODUCTION Microbial ubiquity has its desirable social repercussions, especially in the line of food and drug manufacture; however, it also has more lamentable implications, such as microbial pathogenicity and food contamination which eventually leads to spoilage.

The beginnings of microbiology, although lacking with sophisticated and high-tech gadgets, involved the occurrences of diseases, their spread and of course, their epidemiology. Greeks were able to anticipate microbiology by basically observing symptoms and human acts wherein they actually are able to infect other people. Hippocrates made notes such as a person may become ill with the mere touching and passing of cloth or other objects. Thucydides observed that people who had recovered from the plague could take care of plague victims without danger of getting the disease again (Black, 2008). Other advancements in microbiology readily involved the prevention and cure of diseases, such as smallpox. Natural smallpox infection occurred by implantation of variola virus on the oropharyngeal or respiratory mucosa (Plotkin & Orenstein, 1999). Discovery of vaccines and several decontamination processes such as sterilization, pasteurization, etc. were also one of the more important medical breakthroughs.

The process of pasteurization and sterilization, to name a few, were not only contained in the medical field, but expanded to the industrial sector. These processes have become integrated with the production process and helped in prolonging the shelf-life of supposedly perishable goods.

On a historical note, food spoilage and food-borne pathogens has proved to be a problem

with serious repercussions to the economy and society. Pathogens utilize the fat, protein and carbohydrate that are present in the food for their metabolism (Yoga Latha, Darah, Sasidharan, & Jain, 2009). Food may undergo discoloration, exhibit mustiness, deleterious biochemical changes and accumulation of toxic substances. Some species of food-spoilage and food-borne pathogens are capable of producing highly toxic compounds such as aflatoxin, ochratoxin A and cyanogenic glycosides in food, which are harmful to consumer health (Li & Yi, 2003; You, 2006). Given these serious societal repercussions of uncontrolled microbial growth, the necessity of finding antimicrobial substances from plant and animal tissues is quite justified.

Discussions and researches regarding antimicrobial activity usually centralize on the concept of immunity. Immunity, otherwise termed as resistance, basically, is the ability to ward off disease caused by microbes or their products and to protect against environmental agents i.e. pollen, drugs, foods, chemicals and animal dander. The human body has two lines of defenses against invading pathogensthe first line consisting of the skin, mucous membranes and their secretions, and normal microbiota, while the second line consists of phagocytes, inflammation, fever and antimicrobial substances. (Tortora, Funke, & Case, 2010)

In the advent of microbial infection, the body utilizes various defense mechanisms. Generally, there are two types of immunity, namely, innate and adaptive. Innate immunity refers to natal defense, which are relatively more rapid and are omnipresent. On the other hand, adaptive immunity focuses on the specificity of response, once a microbe has successfully breached the innate immunity defense. Thus, innate immunity defense serves to be the immediate response to pathogens and adaptive immunity would kick in at a later time.

Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources; many of these isolations were based on the uses of the agents in traditional medicine. (Doughari, El-mahmood, & Tyoyina, March 2008)

Plant extracts have been studied and were proven to possess antimicrobial effects and have been widely-used in food preservation and medical breakthroughs. (Yoga Latha, Darah, Sasidharan, & Jain, 2009) The Philippines has been classified as one of the 17 mega-diverse countries; ironically, it is also one of the 25 most threatened biologically diverse areas in the world. (Indigenous Peoples, Forests & REDD Plus: Sustaining & Enhancing Forests Through Traditional Resource Management, 2010) However, amidst the environmental threat, local and international scientists have recognized the potential of several plant species for medicinal and nutritional purposes.

According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs. Therefore, such plants should be investigated to better understand their properties, safety and efficacy. (Nascimento, Lacatelli, Freitas, & Silva, 2000)

Wedelia trilobata is a vigorous, creeping, herbaceous groundcover native to tropical America. It grows to 1824 inches high but its height can be reduced and maintained at lower levels by periodic trimming or mowing. Flowering may occur at any height but appears to have the most blossoms when maintained at about 4 inches. Some chemical growth regulators have shown promise in controlling the height of Wedelia. (Hensley, June 1997)

Other members of the genus Wedelia have proven to be used for medicinal purposes, such as W. biflora, wherein its leaves are used to treat cuts, ulcers, sores and varicose veins; the roots on the other hand, once used in a decoction, can be used to ease stomach ache. The phytochemical screening of the root extracts revealed the presence of tannins and flavanoids. Furthermore, it was also proven that ethanolic extracts of the said plant do have an antimicrobial effect. (Yoganandam, Gowri, & Biswas, March 2009)

Tridax parviflora, otherwise known as Galinsoga parviflora, is a soft, erect annual branched herb which can grow up to 60 cm high. It can thrive in sandy, loamy, and clay soils. It will tolerate acidic, neutral and basic conditions and will grow in semi shade to full sun. It requires a moist soil and can be found in cultivated beds, waste places and pavements (Galinsoga parviflora, 2005).

Similarly with Wedelia, Tridax species have wound healing effects, specifically the Tridax procumbens. It is made into a paste and then applied on the fresh cuts (Sharma & Kumar, December 2008-January 2009). Furthermore, in a study involving the said plant, it was established that the plant had a wide range of antimicrobial activity.

Bacillus subtilis is a ubiquitous bacterium commonly recovered from water, soil, air, and decomposing plant residue. The bacterium produces an endospore that allows it to endure extreme conditions of heat and desiccation in the environment. B. subtilis produces a variety of proteases and other enzymes that enable it to degrade a variety of natural substrates and contribute to nutrient cycling. (Biotechnology Program under the Toxic Substances Control Act

(TSCA), 1997) It is not that virulent in humans however, it is still capable of producing enzymes which could disrupt membranes of mammalian cells and may cause allergic reactions to some individuals.

Escherichia coli a consistent inhabitant of the human intestinal tract, and, in actuality, it is the predominant facultative organism in the human GI tract. E. coli has served much more as an indicator of water contamination and fecal pollution. It is not much of a pathogen however, it is a suitable test subject given that it is accessible.

Objectives of the Study The research focuses on investigating the antimicrobial activity of plant extracts from Wedelia trilobata and Tridax parviflora. Specifically, the study aims to:

1. compare the strengths of the antimicrobial activity of W. trilobata and T. parviflora;

and
2. determine which plant extract is more a more effective antimicrobial for Bacillus sub-

tilis and Escherichia coli.

METHODOLOGY

Collection of Plant Material Fresh leaves, stem and flowers of two weeds Tridax parviflora and Wedelia trilobata that are free from disease were collected from La Trinidad, Benguet. The leaves were washed thoroughly 2-3 times with running water and once with distilled water. The leaves were air- dried. Solvent Extraction 20 grams of each species were weighed and made into a slurry through blending. The samples are then filtered. The filtrate was centrifuged at 10,000 x g for 40 minutes. Growth and Maintenance of Test Microorganism for Antibacterial Tests Cultures of Bacillus subtilis (Gram- positive) and Escherichia coli (Gram- negative) were obtained from the laboratory class of Biology 120. The bacteria were maintained on nutrient broth at 370C. Antibacterial Activity The leaf, stem and flower extracts of Wedelia trilobata and Tridax parviflora were tested through the disc diffusion method. Each treatment has three replicates. The bacteria were seeded into the agar by spread plate method (100 l) using the 24- hour culture bacteria in nutrient broths. Filter paper discs were soaked in the extracts and were placed into the seeded plates. The plates were incubated for 24 hours at 370C. Inhibition zones were measured in millimeters.

RESULTS AND DISCUSSION Since the advent of antibiotics in the 1950s, the use of plant derivatives as
antimicrobials has been virtually nonexistent. After a downturn in that pace in recent decades, the pace is again quickening as scientists realize that the effective life span of any antibiotic is limited (Cowan, 1999). The antimicrobial activities of the crude extracts of leaf, stem, flower of W. trilobata and T. parviflora gave different zones of inhibition on the organisms tested (Table 1). The activity of W. trilobata against E.coli was the highest with a mean inhibition zone of 1.83 mm. On the other hand, T. parviflora showed no activity against E.coli. W. trilobata also exhibited a higher activity against B. subtilis with a mean inhibition zone of 0.83 mm as compared to the mean inhibition zone of T. parviflora of 0.7 mm.

Table1. *Antibacterial activity of W. trilobata and T. parviflora crude extracts tested by paper disc method

Zone of Inhibition (mm) Test Organisms Escherichia coli Bacillus subtilis Wedelia trilobata 1.830.76 0.830.29 0 0.750.35 Tridax parviflora

* Values are mean inhibition zone (mm) standard deviation of three replicates

Basing from the results (Figures 1 and 2), it appears that the test microorganisms were not greatly affected by the two crude extracts via water extraction. In the study of Govindappa et al.,(2011) the ethanol extract of leaf, stem and flower of Wedelia trilobata highly inhibited the growth of P. aeruginosa, K.pneumoniae, P. fluorescens, X. oryzae pv. oryzae, X.axanopodis pv. Malvacearum and moderately inhibited the growth of E.coli, C.michiganensis sub sp.

Michiganensis.

Figure1. Effect of the plant crude extracts on E.coli. (a,b,d left) Tridax parviflora has no effect in contrast with the effects of (a,b, c right) Wedelia trilobata.

Figure2. Effect of the plant crude extracts on B.subtilis. (a,b,c left) Tridax parviflora has minimal effects as compared to the effects of Wedelia trilobata (a,b,c right).

The reasons for this could be that all of the identified components from plants active against microorganisms, aromatic or saturated organic compounds, are most often obtained through initial ethanol or methanol extraction (Abu-Shannab et al, 2004). It is also possible that the plant extracts that were used in this study contain antibacterial constituents, just not in sufficient concentrations so as to be effective (Chanda et al, 2006).

Literature Cited Abu- Shanab, B., Adwan, G., Abu-Safiya, D., Adwan, K., Jarrar, N., (2004). Antibacterial activities of some plant extracts utilized in popular medicine in Palestine. Journal of Biology,28, 99-102. Chanda, S.V., Parekh, J., (2007). In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plant. Journal of Biology, 31, 53- 58. Cowan, M.M.,(1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12, 564-582. Govindappa, M., Svarya, N.S., Sadananda, T.S., Chandrappa, C.P., (2011). Antimicrobial, antioxidant and in vitro anti-inflammatory activity of ethanol extract and active phytochemical screening of Wedelia trilobata (L.) Hitchc. Journal of Pharmacognosy and Phytotherapy, 3, 4351. Biotechnology Program under the Toxic Substances Control Act (TSCA). (1997, February). Retrieved October 14, 2011, from EPA: United States Environmental Protection Agency: http://epa.gov/biotech_rule/pubs/fra/fra009.htm

Black, J. G. (2008). Microbiology: Principles and Explorations, 7th edition. 111 River Street, Hoboken, New Jersey 07030-5774: John Wiley & Sons, Inc.

Doughari, J. H., El-mahmood, A. M., & Tyoyina, I. ( March 2008). Antimicrobial activity of leaf extracts of Senna obtisufolia (L). African Journal of Pharmacy and Pharmacology , 7-13.

Galinsoga parviflora. (2005). Retrieved October 14, 2011, from International Environmental Weed Foundation: http://www.iewf.org/weedid/Galinsoga_parviflora.htm

Hensley, D. (June 1997). Wedelia. Hawaii: Cooperative Extension Service College of Tropical Agriculture & Human Resources, University of Hawaii at Manoa.

Indigenous Peoples, Forests & REDD Plus: Sustaining & Enhancing Forests Through Traditional Resource Management. (2010). Baguio City, Philippines: Tebtebba Foundation.

Li, Y., & Yi, Z. (2003). Present situation and development of food antistaling agent and preservatives. J Beijing Institute of Petro-Chem Technol , 18-23. Nascimento, G., Lacatelli, J., Freitas, P., & Silva, G. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J. Microbiol , 886-891.

Plotkin, S., & Orenstein, W. e. (1999). Vaccines, 3rd edition. Philadelphia: Saunders.

Sharma, B., & Kumar, P. (December 2008-January 2009). Extraction and Pharmacological Evaluation of Some Extracts of Tridax procumbens and Capparis decidua. International Journal of Applied Research in Natural Products , 5-12.

Tortora, G. J., Funke, B. R., & Case, C. L. (2010). Microbiology: An Introduction, 10th edition. 1301 Sansome Street, San Francisco, CA 94111, USA: Pearson Education, Inc.

Yoga Latha, L., Darah, I., Sasidharan, S., & Jain, K. (2009). Antimicrobial Activity of Emilia sonchifolia DC., Tridax procumbens L. and Vernonia cinerea L. of Asteracea Family:Potential as Food Preservatives. Mal J Nutr , 223-231.

Yoganandam, G., Gowri, R., & Biswas, D. (March 2009). Evaluation Of Wedelia Biflora (Linn) D.C For Anthelmintic And Antimicrobial Activity. Journal of Pharmacy Research , 375377.

You, X. (2006). Food safety and food additive of antiseptics. Food Sci Technol , 1-4.

APPENDIX

Table2. Inhibition zone (mm) of Wedelia trilobata and Tridax parviflora crude extracts

Test Microorganism Bacillus subtilis

Escherichia coli

Inhibition zone (mm) Wedelia trilobata T1= 1 T2= 0.5 T3=1 T1= 1 T2= 2 T3=2.5

Tridax parviflora T1= 1 T2= 0.25 T3=1 T1= 0 T2= 0 T3=0

Figure3. Two common garden weeds used in this study. (a) Wedelia trilobata and (b) Tridax parviflora.

Figure4. The (a,b,c) leaves, stem and flowers of both the plants were made into a slurry, filtered and centrifuged (d).