[নোঙর 2016] EVALUATION OF ANTI-MICROBIAL PROPERTIES OF COMMON WEEDS : Farhan Ishrak

Abstract
The increasing prevalence and frequency of antibiotic resistance in microbes has resulted in a range of frontline antibiotics to become ineffective and obsolete. Thus, novel antimicrobial compounds must be perpetually developed into antibiotics for clinical usage. In an effort to obtain such compounds from plants, 3 weeds were tested for their antimicrobial properties. The antimicrobial activity of extracts from Phyllanthus urinaria and Peperomia pellucida were evaluated against the Gram positive Staphylococcus aureus and the Gram negative Escherichia coli by using Tetracycline and Kanamycin as standards. Zone of inhibition assays, minimum inhibitory dilution assays and dose response assays were conducted to determine the antibacterial activity levels of the extracts. Kanamycin was shown to be more effective at inhibiting the growth of Escherichia coli while organic fractions from Phyllanthus urinaria and Peperomia pellucida were more effective in inhibiting the growth of Staphylococcus aureus. Hence, these 2 weeds have the potential to be developed into narrow spectrum antibiotics targeting Gram positive bacteria.

INTRODUCTION
Emerging microbial and fungal resistance towards frontline antibiotics is a major healthcare concern, proving to be a challenge for the pharmaceutical and healthcare industry. Even though pharmacological companies have produced a number of novel antibiotics in the last three decades antibiotic resistance in microorganisms had been observed to be increasing, and alarmingly many bacterial strains are multi-drug resistant (MDR). The horizontal and vertical transfer of antibiotic resistance genes occur so rapidly for some bacterial strains that clinical usefulness of the antibiotics is lost within a 5 year period. Hence, the increasing prevalence of MDR microbes and the lack of novel antimicrobial compounds provided the impetus for us to identify new effective therapeutic agents from plants.
While there is a great deal of published data showing the antimicrobial activity of medicinal plants, this has not resulted in the identification of commercially exploitable plant derived antibacterial agents. The problem is that the majority of plant derived antimicrobial compounds generally have higher minimum inhibitory concentrations than bacterial or fungal produced antibiotics, thereby limiting their therapeutic potential. However, it had been established that some crude extracts and pure compounds from plants can increase the effects of antibiotics thus plants represent a key to addressing the problem of increasing antibiotic resistance. Also, weeds are unwanted and invasive plants which thrive in harsh environments. As such, growing them would be easy and large quantities of the desired weed can be obtained in a short period of time. As many weeds and their compounds still remain unknown and unstudied, there are numerous opportunities for the discovery of new plant-derived antimicrobial compounds. Research in this area could provide the basis for identifying and isolating of new therapeutically useful compounds.
Plants had been exploited for their therapeutic properties for millennia; various traditional and folk medicine systems use plant products for the treatment of various infectious diseases. Recent studies were performed on plants to scientifically ascertain their antimicrobial, anti-inflammatory and anticancer properties. In contrast to synthetic drugs, antimicrobial compounds of plant origin are not associated with many side effects, and are deemed to be of low cytotoxicity and so possess immense therapeutic potential to combat and cure numerous infectious diseases.
Plants, especially weeds could be an important source of such antibiotics as they are highly resistant to bacterial and fungal infestations and exhibit much more vigorous growth than crops. Therefore, identifying common weeds with antimicrobial properties can provide the initial screening for broad spectrum anti-bacterial properties. The screen was performed through evaluating the activity of the weed derived fractions on both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).

MATERIALS AND METHODS
Collection and Identification
Phyllanthus urinaria and Peperomia pellucida were collected from a household garden. They were selected based on their rapid growth, exhibiting extreme growth, vigour and adaptation to neglect.
Extraction
The plants were harvested without roots and 40g of green matter from each plant was ground together with 40 ml of deionized water in a domestic blender. The slurry produced was sieved to remove the insoluble plant matter. Following which, the sieved slurry was centrifuged at 4000 revolutions per minute (RPM) for 10 minutes to further sediment the fine insoluble plant matter. The soluble fraction was aspirated into clean 50 ml tubes. Equal volumes of ether were then added to the soluble fraction to extract the organic components. The ether fraction was aspirated from the aqueous fraction and air dried to remove the ether. Both organic and aqueous fractions were then used for antimicrobial screening.

Growth of Bacteria
S. aureus and E. coli were grown in nutrient broth (Oxoid, CM0001) overnight at 37°C, with agitation at 200rpm. The absorbance of the bacteria was measured at the optical density of 600nm (OD600). The bacteria was then diluted to 1/2x, 1/4x, 1/8x and 1/16x concentration and spread on an agar plate. The plates were left in an incubator at 37°C for 24hours. After which, the plates were checked to see which concentration yielded a visible lawn of bacteria. The OD600 value that yields a lawn of bacteria was then interpolated for each species of bacteria.

Antimicrobial Screening
The undiluted aqueous and organic extracts were first evaluated by the disc diffusion method against S. aureus and E. coli. Tetracycline was used as the positive control and the negative controls were ether and water for the organic and aqueous fractions respectively. The bacteria was obtained from Singapore Polytechnic’s microbiology laboratory. The antibacterial activity of the samples was determined by measuring the zone of inhibition radii which are expressed in mm. This screen was performed once on each of the samples (aqueous and organic) to determine which fraction of the plant extract has anti-bacterial activity.

Minimum Inhibitory Dilution
The samples which exhibited antibacterial activities were serially diluted to 5X, 10X, 15X, 20X and 40X in deionised water. The 10 ml of each dilution was then evaluated by the disc diffusion method against S. aureus and E. coli using Kanamycin and deionized water as the positive and negative controls respectively. The antibacterial activity of the samples and controls were determined by measuring the zone of inhibition radii which are expressed in mm. The zones of inhibition were determined by measuring at 3 different radii to obtain an average value. The test was performed for each sample, to determine the dilution factor at which the antibacterial activity is comparable or similar in intensity to Kanamycin.

Dosage Response Assay
Each extract of P. pellucida and P. urinaria was diluted 10X, 50X, 100X, 500X, 1000X and 5000X, and each dilution was tested against E. coli and S. aureus. The bacteria broth was centrifuged at 3000 RPM for 5 minutes and the spent media was replaced with fresh media to obtain an OD600 of approximately 0.4. In each well, 10µl of each diluted extract was added to 90µl of the bacterial broth. Each well was mixed by pipetting up and down multiple times. The plates were then incubated at 37°C overnight before measuring the OD600 in a 96-well plate reader. The reactions were normalized to the no sample controls which were composed of 90ml bacterial broth and 10ml of deionized water. Kanamycin and sodium chloride solution were used as positive and negative controls respectively.
Statistical Calculations
The student’s t-test was used to determine statistical significance. The 2-tailed and unpaired options were chosen to compare the data between the dose response assays.

RESULTS
Extraction
After ether extraction, we obtained 71.4mg of P. urinaria and 115.4mg of P. pellucida organic fractions.
Antimicrobial Activity Screening
In the antimicrobial activity screen, the undiluted aqueous fractions of P. pellucida and P. urinaria did not produce any zones of inhibition for both E. coli and S. aureus. The undiluted organic fractions of P. pellucida and P. urinaria produced extremely huge zones of inhibition for both E. coli and S. aureus which displaced any results from the positive and negative controls on the same plate. The minimum inhibitory dilutions was next determined for the organic fractions on both species of bacteria. The aqueous fractions showed no antibacterial activities and were discarded.

Minimum Inhibitory Dilutions
The organic extracts of P. pellucida, at 10X dilution produced zones of inhibition in E. coli and S. aureus respectively, that were comparable to those of the positive control. For P. urinaria, its organic fraction at 10X dilution, also produced zones of inhibition in E. coli and S. aureus respectively that were comparable to the positive control. The positive control, Kanamycin paper disc was rated at 30µg.

Dosage Response Assays
The organic fractions of both P. pellucida and P. urinaria showed positive results resulting in a sigmoid curve for E. coli and S. aureus.
The P-values for P. pellucida against E. coli and S. aureus when compared with NaCl treatment are 3.0E-10 and 1.8E-04 respectively and when compared with Kanamycin treatment are 1.8E-01 and 1.9E-01 respectively.
The P-values for P. urinaria against E. coli and S. aureus when compared with NaCl treatment are 1.9E-11 and 3.4E-05 respectively and when compared with Kanamycin treatment are 1.0E-01 and 9.3E-02 respectively.
The maximal antibacterial activity for P. pellucida against E. coli was at 500X dilution (66% of bacteria remaining) and the dose response range was from 1000X to 500X dilutions. The maximal antibacterial activity against S. aureus was at 50X dilution (53% of bacteria remaining) and the dose dependent range was from 500X to 100X dilutions.
The maximal antibacterial activity for P. urinaria against E. coli was at 500X (63% of bacteria remaining) and the dose response range was from 1000X to 500X dilutions. The maximal antibacterial activity against S. aureus was at 50x dilution (52%) and the dose response range was from 500X to 100X dilutions.
The positive control Kanamycin was diluted from stock solution of 50mg/ml. When tested against E. coli, the dose response range was from 10µg/ml to 100µg/ml, while for S. aureus the dose dependent range was from 1µg/ml to 10µg/ml. Kanamycin showed maximal antibacterial activity against E. coli at 100µg/ml (52% of bacteria remaining) and against S. aureus at 10µg/ml (80% of bacteria remaining).
The negative control, sodium chloride (NaCl), was diluted from 1M stock solutions, and showed no antibacterial activity against both E. coli and S. aureus.

DISCUSSION
During the extraction process, approximately 70 to 100mg of organic fraction per sample was obtained from 40g of plant matter, equating to a yield of only 0.25%. This result serves to highlight the fact that plant-derived therapeutics constitute a miniscule fraction of plant matter, and industrial scale production would be possible through chemical synthesis rather than post-agricultural distillation.
E. coli and S aureus were chosen as model organisms, representative of Gram negative and Gram positive strains respectively. Clinically, drugs which show activity towards both Gram negative and positive strains of bacteria, are highly desired as they are ‘broad spectrum’, with the ability to act against a large range of bacterial strains.

The initial screen for antibacterial activity, by using the zone of inhibition assays, showed that the undiluted organic fractions of both P. pellucida and P. urinaria could drastically inhibit the growth of both E. coli and S. aureus. And only at 10X dilutions, were the antibacterial activity of the organic fractions moderated downwards and was comparable to that of the Kanamycin standard paper disc which contained 30mg of the antibiotic. Those results indicate that the compounds found in the organic fractions had broad spectrum antibiotic properties, and are most likely lipid soluble macromolecules. This knowledge would aid in the downstream identification of the specific active compound or compounds with antibacterial properties.
In the dose response assays, the raw organic fractions produced interesting results against the Kanamycin standard. In the experiments involving E. coli, Kanamycin readily reduced the bacterial viability down to 40%, while P. pellucida and P. urinaria could only reduce the viability of the bacteria down to 63% and 66% respectively at the minimum dilution factor of 2E-03. However, the differences in antibacterial activity are not statistically significant (p-values=0.18 and 0.1 respectively). In the experiments involving S. aureus, Kanamycin could only reduce the bacterial viability down to 77.1%, even at the highest concentration of 1mg/ml. However, the organic fractions of P. pellucida and P. urinaria could reduce the viability of the bacteria down to 52% and 53% respectively at the minimum dilution factor of 2E-02. Similarly, differences in antibacterial activity are not statistically significant (p-values=1.9E-01 and 9.3E-02 respectively).
These 2 observations indicate that the organic fractions of P. pellucida and P. urinaria were more effective at inhibiting the growth of S. aureus than Kanamycin, while Kanamycin was more effective at inhibiting the growth of E. coli.
The results gathered have shown that P. pellucida and P.urinaria exhibited a certain level of broad spectrum antimicrobial activities when compared with Kanamycin.  Additionally, both samples had a stronger inhibitory effect on the growth of S. aureus as compared to Kanamycin. Therefore, there is great potential for both samples, to be developed into narrow-spectrum antibiotic targeting Gram positive bacteria, especially Methicillin-resistant Staphylococcus aureus (MRSA).
With the verification of antibacterial activity, the outstanding tasks are to determine the specific active compounds in each organic fraction through the use of HPLC or thin layer chromatography, chemically synthesize the active compounds and perform functional analysis on the mechanism of antibacterial activity, i.e. cell wall disruption or protein synthesis disruption.

ACKNOWLEDGEMENTS
I would like to acknowledge and thank Dr Eugene Koh, my research mentor, for his constant guidance and help throughout the course of this project. I would also like to thank my teacher mentor, Ms Verlyn Woo. I would also further thank the staff at Singapore Polytechnic’s Microbiology Labs for the help and assistance they have provided during the project period.

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Farhan Ishraq is a student of Year 6 at NUS High School of Math & Science. He is the son of Baten (18)