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 pathogens—the 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 18–24 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 μl) 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

Test Organisms

Zone of Inhibition (mm)

Wedelia trilobata Tridax parviflora

Escherichia coli 1.83±0.76 0

Bacillus subtilis 0.83±0.29 0.75±0.35

* 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, 43-

51.

Biotechnology Program under the Toxic Substances Control Act (TSCA). (1997, February). Re-

trieved 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 Tradi-

tional Resource Management. (2010). Baguio City, Philippines: Tebtebba Foundation.

Li, Y., & Yi, Z. (2003). Present situation and development of food antistaling agent and preser-

vatives. 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 edi-

tion. 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 , 375-

377.

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

Inhibition zone (mm)

Wedelia trilobata Tridax parviflora

Bacillus subtilis T1= 1

T2= 0.5

T3=1

T1= 1

T2= 0.25

T3=1

Escherichia coli T1= 1

T2= 2

T3=2.5

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).